Filtern
Volltext vorhanden
- ja (152)
Gehört zur Bibliographie
- ja (152)
Erscheinungsjahr
Dokumenttyp
Schlagworte
- Motoneuron (16)
- Spinale Muskelatrophie (11)
- BDNF (8)
- glycine receptor (8)
- spinal muscular atrophy (6)
- Actin (5)
- CNTF (5)
- SMN (5)
- hyperekplexia (5)
- motoneurons (5)
Institut
- Institut für Klinische Neurobiologie (152) (entfernen)
The purpose of the experiments reported is to provide an unambiguous demonstration that embryonie skeletal muscle contains factors that act directly on embryonie spinal motor neurons both to support their survival and to stimulate the outgrowth of neurites. Cells of lumbar and brachial ventral spinal cords from 6-day-old chick embryos were separated by centrifugation in a two-step metrizamide gradient, and a motor neuron enriched fraction was obtained. Motor neurons were identified by retrogradely labeling with rhodamine isothiocyanate, and were enriched fourfold in the motor neuron fraction relative to unfractionated cells. In culture, the isolated motor neurons died within 3-4 days unless they were supplemented with embryonie chick skeletal muscle extract. Two functionally distinct entities separable by ammonium sulfate precipitation were responsible for the effects of muscle extracts on motor neurons. The 0-25% ammonium sulfate precipitate contained molecules that alone bad no effect on neuronal survival but when bound to polyornithine-coated culture substrata, stimulated neurite outgrowth and potentiated the survival activity present in muscle. Most of this activity was due to a laminin-like molecule being immunoprecipitated with antisera against laminin, and immunoblotting demonstrated the presence of both the A and B chains of laminin. A long-term survival activity resided in the 25-70% ammonium sulfate fraction, and its apparent total and specific activities were strongly dependent on the culture substrate. In contrast to the motor neurons, the cells from the other metrizamide fraction (including neuronal cells) could be kept in culture for a prolonged time without addition of exogenous factor(s).
We have been studying a population of bipotential glial progenitor cells in the perinatal rat optic nerve and brain in an attempt to understand how cells choose between alternative fates in the developing mammalian central nervous system (CNS). This cell population gives rise initially to oligodendrocytes and then to type-2 astrocytes1 both of which apparently collaborate in sheathing axons in the CNS2,3. In vitro studies suggest that oligodendrocyte differentiation is the constitutive pathway of development for the oligodendrocyte-type-2-astrocyte (O-2A) progenitor cell4,5, whereas type-2 astrocyte differentiation depends on a specific inducing protein6. This protein is present in the developing optic nerve when type-2 astrocytes are differentiating and can induce 0-2A progenitor cells in vitro to express glial fibrillary acidic protein (GFAP)6, a marker of astrocyte differentiation7. Here we show that the type-2-astrocyte-inducing protein is similar or identical to ciliary neutrotrophic factor (CNTF)8,9, which promotes the survival of some types of peripheral neurons in vitro8, including ciliary ganglion neurons8,10. This suggests that CNTF, in addition to its effect on neurons, may be responsible for triggering type-2 astrocyte differentiation in the developing CNS.
The ability of nerve growth factor to cause rapid activation of the Na+K+ pump of its responsive cells was examined by measuring the uptake of 86Rb+. A significant increase in 86Rb+ uptake in Ea chick dorsal root ganglion sensory neurons after NGF treatment was seen only if the cells had been damaged during the preparation procedure. Such damaged cells could not survive in culture in the presence of NGF, and undamaged cells that did survive in response to NGF exhibited no increased 86Rb+ uptake rate. Furthermore, cultured calf adrenal medullary cells did not show an increase in 86Rb+ uptake after treatment with NGF, although these cells respond to NGF with an increased synthesis of catecholaminergic enzymes. These results are incompatible with the hypothesis that the mechanism of action of NGF that promotes neuronal survival and enzyme induction results from an initial stimulation of the Na+K+ pump.
At early developmental stages (embryonic day 7, E7), chick paravertebral sympathetic ganglia contain a cell population that divides in culture while expressing various neuronal properties. In an attempt to identify factors that control neuronal proliferation, we found that ciliary neurotrophic factor (CNTF) specifically inhibits the proliferation of those cells expressing neuronal markers. In addition, CNTF affects the differentiation of sympathetic ganglion cells by inducing the expression of vasoactive intestinal peptide immunoreactivity (VIP-IR). After 1 day in culture, tyrosine hydroxylase immunoreactivity (TH-I R) was expressed by about 86% of the cells whereas VIP-IR was virtually absent. In the presence of CNTF, 50%-60% of the cells expressed VIP-IR after 4 days in culture; however, none of the cells expressed VIP-IR in the absence of CNTF. These results, and the demonstration of cells that express both VIP and TH-IR, indicate that VIP is induced in cells that initially express tyrosine hydroxylase. The findings suggest a potential role for CNTF as a factor affecting the proliferation and differentiation of developing sympathetic neurons.
Although evidence obtained with the PC12 cell line has suggested a role for the ras oncogene proteins in the signal transduction of nerve growth factor-mediated fiber outgrowth, little is known about the signal transduction mechanisms involved in the neuronal response to neurotrophic factors in nontransformed cells. We report here that the oncogene protein T24-ras, when introduced into the cytoplasm of freshly dissociated chick embryonic neurons, promotes the in vitro survival and neurite outgrowth of nerve growth factor-responsive dorsal rootganglion neurons, brain-derived neurotrophic factor-responsive nodose ganglion neurons, and ciliary neuronotrophic factor-responsive ciliary ganglion neurons. The proto-oncogene product c-Ha-ras also promotes neuronal survival, albeit less strongly. No effect could be observed with truncated counterparts of T24-ras and c-Ha-ras lacking the 23 C-terminal amino acids including the membrane-an-choring, palmityl-accepting cysteine. These results sug-gest a generalized involvement of ras or ras-like proteins in the intracellular signal transduction pathway for neurotrophic factors.
CILIARY neurotrophic factor (CNTF) was originally characterized as a survival factor for chick ciliary neurons in vitro. More recently, it was shown to promote the survival of a variety of otherneuronal cell types and to affect the differentiation of E7 chick sympathetic neurons by inhibiting their proliferation and by inducing the expression of yasoactiYe intestinal peptide immunoreactiyity (VIP-IR). In cultures of dissociated sympathetic neurons from newborn rats, CNTF induces cholinergic differentiation as shown by increased levels of choline acetyltransferase (ChAT.
Ciliary neurotrophic factor (CNTF) influences the levels of choline acetyltransferase (ChAT) and tyrosine hydroxylase (TH) in cultures of dissociated sYmpathetic neurons from newborn rats. In the presence of CNTF both the total and specific activity of ChAT was increased 7 d after culture by 15- and 18-fold, respectively, as compared to cultures kept in the absence of CNTF. Between 3 and 21 d in culture in the presence of CNTF . the total ChAT activity increased by a factor of >100. Immunotitration demonstrated that the elevated ChAT levels were due to an increased number of enzyme molecules. In contrast to the increase in ChAT levels, the total and specific activity levels' of TH were decreased by 42 and 36 %, respectively, after 7 d in culture. Half-maximal effects for both ChAT increase and TH decrease were obtained at CNTF concentrations of rvO.6 ng and maximal levels were reached at I ng of CNTF per milliliter of medium. The effect of CNTF on TH and ChAT levels were seen in serum-containing medium as well as in serum-free medium. CNTF was shown to have only a small effect on the long-term s.urviVal of rat sympathetic neurons. We therefore concluded that the effects of CNTF on ChAT and TH are not due to selective survival of cells that acquire cholinergic traits in vitro, but are rather due to the induction of cholinergic differentiation of noradrenergic sympathetic neurons.
The period of natural cell death in the development of rodent motor neurons is followed by a period of sensitivity to axonal injury1-3. In the rat this early postnatal period of vulnerability coincides with that of very low ciliary neurotrophic factor (CNTF) levels in the sciatic nerve before CNTF increases to the high, adult levels4. The developmental time course of CNTF expression, its regional tissue distribution and its cytosolic localization (as suggested by its primary structure)4*5 favour a role for CNTF as a lesion factor rather than a target-derived neurotrophic molecule like nerve growth factor. Nevertheless CNTF exhibits neurotrophic activity in vitro on different populations of embryonic neurons6. To determine whether the vulnerability of motor neurons to axotomy in the early postnatal phase is due to insufficient availability of CNTF, we transected the axons of newborn rat motor neurons and demonstrated that iocal application of CNTF prevents the degeneration of the corresponding cell bodies.
0-2A progenitor cells give rise to both oligodendrocytes and type-2 astrocytes in vitro. Whereas oligodendrocyte differentiation occurs constitutively, type-2 astrocyte differentiation requires extracellular signals, one of which is thought to be ciliary neurotrophic factor (CNTF). CNTF, however, is insufficient by itself to induce the development of stable type-2 astrocytes. In this report we show the following: (a) that molecules associated with the extracellular matrix (ECM) cooperate with CNTF to induce stable type-2 astrocyte differentiation in serumfree cultures. The combination of CNTF and the ECM-associated molecules thus mimics the effect of FCS, which has been shown previously to induce stable type-2 astrocyte differentiation in vitro. (b) Both the ECM-associated molecules and CNTF act directly on 0-2A progenitor cells and can induce them to differentiate prematurely into type-2 astrocytes. (c) ECM-associated molecules also inhibit oligodendrocyte differentiation, even in the absence of CNTF, but this inhibition is not sufficient on its own to induce type-2 astrocyte differentiation. (d) Whereas the effect of ECM on oligodendrocyte differentiation is mimicked by basic fibroblast growth factor (bFGF), the effect of ECM on type-2 astrocyte differentiation is not. (e) The ECM-associated molecules that are responsible for inhibitin~ oligodendrocyte differentiation and for cooperating with CNTF to induce type-2 astrocyte differentiation are made by non-glial cells in vitro. (f) Molecules that have these activities and bind to ECM are present in the optic nerve at the time type-2 astrocytes are thought to be developing.
Ciliary neurotrophic factor (CNTF) is a potent survival molecule for a variety of embryonic neurons in culture. The developmental expression of CNTF occurs clearly after the time period of the physiological cell death of CNTF-responsive neurons. This, together with the sites of expression, excludes CNTF as a target-derived neuronal survival factor, at least in rodents. However, CNTF also participates in the induction of type 2 astrocyte differentiation in vitro. Here we demonstrate that the time course of the expression of CNTF-mRNA and protein in the rat optic nerve (as evaluated by quantitative Northern blot analysis and biological activity, respectively) is compatible with such a glial differentiation function of CNTF in vivo. We also show that the type 2 astrocyte-inducing- activity previously demonstrated in optic nerve extract can be precipitated by an antiserum against CNTF. Immunohistochemical analysis of astrocytes in vitro and in vivo demonstrates that the expression of CNTF is confined to a subpopulation of type 1 astrocytes. The olfactory bulb of adult rats has comparably high levels of CNTF to the optic nerve, and here again, CNTF-immunoreactivity is localized in a subpopulation of astrocytes. However, the postnatal expression of CNTF in the olfactory bulb occurs later than in the optic nerve. In other brain regions both CNTF-mRNA and protein levels are much lower.
We have demonstrated that the extensive degeneration of motoneurons in the rat facial nucleus after transection of the facial nerve in newborn rats can be prevented by local ciliary neurotrophic factor (CNTF) administration. CNTF differs distinctly from known neurotrophic molecules such as NGF, BDNF and NT-3 in both its molecular characteristics (CNTF is a cytosolic rather than a secretory molecule) and its broad spectrum of biological activities. CNTF is expressed selectively by Schwann cells and astrocytes of the peripheral and central nervous system, respectively, but not by target tissues of the great variety of CNTF -responsive neurons. CNTF mRNA is not detectable by Northern blot or PCR analysis during embryonic development and immediately after birth. However, during the second post-natal week, a more than 30-fold increase in CNTF mRNA and pro tein occurs in the sciatic nerve. Since the period of low CNTF levels in peripheral nerves coincides with that of high vulnerability of motoneurons (i.e. axonallesion results in degeneration of motoneuron cell bodies), insufficient availability of CNTF may be the reason for the rate of lesioninduced cell death of early post-natal motoneurons. Highly enriched embryonic chick motoneurons in culture are supported at survival rates higher than 60% by CNTF, even in single cell cultures, indicating that CNTF acts directly on motoneurons. In contrast to CNTF, the members of the neurotrophin gene family (NGF, BDNF and NT-3) do not support the survival of motoneurons in culture. However, aFGF and bFGF show distinct survival activities which are additive to those of CNTF, resulting in the survival of virtually all motoneurons cultured in the presence of CNTF and bFGF.
The cDNA for ciliary neurotrophic factor (CNTF), a polypeptide involved in the survival of motoneurons in mammals, has recently been cloned (Stöckli et al., Nature, 342, 920 - 923, 1989; Lin et al. Science, 246, 1023 - 1025, 1989). We have now localized the corresponding gene Cntf to chromosome 19 in the mouse, using an interspecific cross between Mus spretus and Mus musculus domesticus. The latter was carrying the gene wobbler (wr) for spinal muscular atrophy. DNA was prepared from backcross individuals and typed for the segregation of species-specific Cntf restriction fragments in relation to DNA markers of known chromosomal location. The M.spretus allele of Cntf cosegregated with chromosome 19 markers and mapped closely to Ly-1, to a region of mouse chromosome 19 with conserved synteny to human chromosome 11q. Cntf is not linked to wr, and the expression of CNTF mRNA and protein appears close to normal in facial and sciatic nerves, of affected (wr/wr) mice, suggesting that motoneuron degeneration of wobbler mice has its origin in defects other than reduced CNTF expression.
Ciliary neurotrophic factor (CNTF) is expressed in high quantities in Schwann cells of peripheral nerves during postnatal development of the rat. The absence of a hydrophobic leader sequence and the immunohistochemical localization of CNTF within the cytoplasm of these cells indicate that the factor might not be available to responsive neurons under physiological conditions. However, CNTF supports the survival of a variety of embryonic neurons, including spinal motoneurons in culture. Moreover we have recently demonstrated that the exogenous application of CNTF protein to the lesioned facial nerve of the newborn rat rescued these motoneurons from cell death. These results indicate that CNTF might indeed play a major role in assisting the survival of lesioned neurons in the adult peripheral nervous system. Here we demonstrate that the CNTF mRNA and protein levels and the manner in which they are regulated are compatible with such a function in lesioned peripheral neurons. In particular, immunohistochemical analysis showed significant quantities of CNTF at extracellular sites after sciatic nerve lesion. Western blots and determination of CNTF biological activity of the same nerve segments indicate that extracellular CNTF seems to be biologically active. After nerve lesion CNTF mRNA levels were reduced to <5 % in distal regions of the sciatic nerve whereas CNTF bioactivity decreased to only one third of the original before-lesion levels. A gradual reincrease in Schwann cells occurred concomitant with regeneration.
CILIARY neurotrophic factor (CNTF) supports the survival of embryonic motor neurons in vitro and in vivo and prevents lesion-mediated degeneration of rat motor neuron~ during early post-natal stages. Here we report that CNTF greatly reduces all the functional and morphological changes in pmnlpmn mice5, an autosomal recessive mutant leading to progressive caudo-cranial motor neuron degeneration. The first manifestations of progressive motor neuronopathy in homozygous pmnl pmn mice become apparent in the hind limbs at the end of the third post-natal week and all the mice die up to 6 or 7 weeks after birth from respiratory paralysis. Treatment with CNTF prolongs- survival- and greatly Impoves motor function of these mice. Moreover, morphological manifestations, such as loss of motor axons in the phrenic nerve and degeneration of facial motor neurons, were greatly reduced by CNTF, although the treatment did not start until the first symptoms of the disease had already become apparent and substantial degenerative changes were already present. The protective and restorative effects of CNTF in this mouse mutant give new perspectives for the treatment of human degenerative motor neuron diseases with CNTF.
Motoneurons innervating the skeletal musculature were among the first neurons shown to require the presence of their target cells to develop appropriatelyl,2. But the characterization of molecules allowing motoneuron survival has been difficult. Ciliary neurotrophic factor prevents the death of motoneurons3-6, but its gene is not expressed during development7. Although the presence of a neurotrophin receptor on developing motoneurons8-1O has suggested a role for neurotrophins, none could be shown to promote motoneuron survival in vitro3. We report here that brainderived neurotrophic factor can prevent the death of axotomized motoneurons in newborn rats, suggesting a role for this neurotrophin for motoneuron survival in vivo.
More on motor neurons
(1992)
Motoneuron diseases represent a m&jor challenge to modern neurology, yet their clinical manifestations ware first described more than hundred years ago, and despite many studies the etiology of these diseases ramd,ns obscure with no effective treatments having been reported. Although progress has been made in establishing genetic linkage in the rare inherited for.ms of these diseases such as familial amyotrophic lateral scleriosisl , spinal mDscular atrophy and X-linked bulbo-spinal-mDscular atrophy, this new information has not yet affected therapeutic techniques. During the last few years several important steps have been taken concerning the physiological mechanisms involved in motoneuron survival during development, after lesion and in animal models of degenerative diseases, the molecular clOning of several new neurotrophic factors (brain-derived neurotrophic factor (BDNP), neurotrophin-3 and-4 (NT-3 and NT-4) and ciliary neurotrophic factor (CNTP)); the identification of a gene family of receptor molecules for same of these factors, progress in the understanding of the effects of polypeptide growth factors on muscle cell differentiation, neuronal sprouting (insulin-like growth factor-I and -11 (IGF-I and IGF-II), and in vitro motoneuronal survival (CNTF, IGF-I and -II and basic FGF). These findings have raised new hopes in that they could lead to a better understanding of the pathophysiological processes underlying these diseases, and that the pharmacological use of same of these newly characterized neurotrophic factors could present new possibilities for the treatment of these diseases.
CNTF is a cytosolic molecule expressed postnatally in myelinating Schwann cells and in a subpopulation of astrocytes. Although CNTF administration prevents lesion-mediated and genetically determined motor neuron degeneration, its physiological function remained elusive. Here it is reported that abolition of CNTF gene expression by homologous recombination results in a progressive atrophy and loss of motor neurons in adult mice, which is functionally reflected by a small but significant reduction in muscle strength.
The structure of the rat ciliary neurotrophic factor (CNTF) gene and the regulation ofCNTF mRNA levels in cultured glial cells were investigated. The rat mRNA is encoded by a simple two-exon transcription unit. Sequence analysis of the region upstream of the transcription start-site did not reveal a typical TATA-box consensus sequence. Low levels of CNTF mRNA were detected in cultured Schwann cells, and CNTF mRNA was not increased by a variety of treatments. Three-week-old astrocyteenriched cell cultures from new-born rat brain contained easily detectable CNTF mRNA. In astrocyte-enriched cultures, upregulation of CNTF mRNA levels was observed after treatment with IFN-gamma. CNTF mRNA levels were down-regulated in these cells by treatments that elevate intracellular cyclic AMP and by members of the fibroblast growth factor (FGF) family. The implications of these results for potential in vivo functions of CNTF are discussed.
We examined the potential role of fibroblast growth factor 5 (FGF-5) as a target-derived trophic factor for spinal motoneurons. Northern analysis of total RNA from rat skeletal muscle revealed an FGF-5 mRNA transcript both during the period of embryonic motoneuron death and in the adult. Recombinant human FGF-5 supported the survival of highly enriched cultures of embryonic chick motoneurons. Significant proportions of the motoneuron survival activity of rat skeletal muscle extracts could be immunoprecipitated using an antiserum to FGF-5. The immunoprecipitable activity was present in soluble and matrix-bound forms in embryonic muscle, but bound exclusively to the extracellular matrix in adult muscle. These results, along with the secretory nature of FGF-5, suggest that FGF-5 may act as a target-derived trophic factor for motoneurons.
Motoneurons played an essential role in establishing the concept of target-mediated support of innervating neurons. However, it took several decades until molecules were identined which trophically support motoneurons in vitro and in vivo. The most potent molecule identined so far is ciliary neurotrophic factor (CNTF). It is expressed as a cytosolic molecule in myelinating Schwann cells rather than in skeletal muscle in the postnatal period and therefore does not qualify as a target-derived neurotrophic factor regulating motoneuron survival during embryonic development. However, the inactivation of CNTF by gene targeting experiments results in progressive atrophy and degeneration of motoneurons, demonstrating that CNTF plays an essential role as a maintenance factor for motoneurons postnatally. Secretory molecules which are expressed in skeletal muscle during embryonic development and which support motoneurons in culture and partially also in vivo include members of the NGF gene family (BDNF, NT-3, NT-4/S) , FGF-S, IGF-I, and UF. The evaluation of the physiological importance of these molecules is under investigation.
The survival and functional maintenance of spinal motoneurons, both during the period of developmental cell death and in adulthood, have been shown to be dependent on trophic factors. In vitro experiments have previously been used to identify several survival factors for motoneurons, including CNTF, UF, and members of the neurotrophin, FGF, and IGF gene families. Some of these factors have also been shown to be active in vivo, either on chick motoneurons during embryonic development or on lesioned facial and spinal motoneurons of the newborn rat. Here we demonstrate that lesioned newborn rat facial motoneurons can be rescued by NT-4/5, IGF-I, and UF. Furthermore, in contrast to chick motoneurons, the survival of isolated embryonic rat motoneurons can be maintained by the neurotrophins BDNF, NT-3, and NT-4/5. IGF-I and FGF-5 were also active in this system, each supporting more than 50% of the originally plated neurons. The responsiveness of motoneurons to multiple factors in vitro and in vivo suggests that motoneuron survival and function are regulated by the coordinated actions of members of different gene families.
We showed previously that oligodendrocytes and their precursors require continuous signalling by protein trophic factors to avoid programmed cell death in culture. Here we show that three classes of such trophic factors promote oligodendrocyte survival in vitro: (1) insulin and insulin-like growth factors (IGFs), (2) neurotrophins, particularly neurotrophin-3 (NT -3), and (3) ciliary-neurotrophic factor (CNTF), leukemia inhibitory factor (LIF) and interleukin 6 (IL-6). A single factor, or combinations of factors within the same class, promote only short-term survival of oligodendrocytes and their precursors, while combinations of factors from different classes promote survival additively. Long-term survival of oligodendrocytes in vitro requires at least one factor from each class, suggesting that multiple signals may be required for long-term oligodendrocyte survival in vivo. We also show that CNTF promotes oligodendrocyte survival in vivo, that platelet-derived growth factor (PDGF) can promote the survival of oligodendrocyte precursors in vitro by acting on a novel, very high affinity PDGF receptor, and that, in addition to its effect on survival, NT-3 is a potent mitogen for oligodendrocyte precursor cells.
Spinal motoneurons innervating skeletal muscle were amongst the first neurons shown to require the presence of their target cells to develop appropriately. Isolated embryonie chick and rat motoneurons have been used to identify neurotrophic factors and cytokines capable of supporting the survival of developing motoneurons. Such factors include ciliary neurotrophic factor (CNTF), which is present physiologically in high amounts in myelinating Schwann cells of peripheral nerves, and brain-derived neurotrophic factor (BDNF) which is synthesized in skeletal muscle and, after peripheral nerve lesion. in Schwann cells. These factors have been further analyzed for their physiological significance in maintaining motoneuron function in vivo, and for their potential therapeutic usefulness in degenerative motoneuron disease. Both CNTF and BDNF are capable of rescuing injured facial motoneurons in newbom rats. Furthermore, CNTF prolongs survival and improves motor function of pmn mice, an animal model for degenerative motoneuron disease, by preventing degeneration of motoneuron axons and somata. Thus treatment of human motoneuron disease with neurotrophic factors should be possible, provided that rational means for application of these factors can be established considering also the appearance of potential side effects.
Ciliary Neurotrophic Factor
(1994)
Interleukin-6, leukemia inhibitory factor, oncostatin M. Interleukin-11, and cilialy neurotrophic factor bind to receptor complexes that share the signal transducer gp130. Upon binding, the ligands rapidly activate DNA binding of acute-phase response factor (APRF), a protein antigenicaly relaled to the p91 subunit of the interferon-stimulated gene factor-(ISGF-3a). These cytokines caused tyrosine phosphorylation of APRF and ISGF-3a p91. Protein kinases of the Jak family were also rapidly tyrosine phosphorylated, and both APRF and Jak1 associated with gp130. These data indicate that Jak family protein kinases may participate in IL-6 signaling and that APRF may be activated in a complex with gp130.
D EVELOPME'iT and maintenance of the mammalian nervous system is dependent upon neurotrophic cytokines. One class of neurotrophic factor acts through rcccptor complexes involving the lowaffinity leukaemia inhibitor y faclor receptor subunit (LlF-R). Members of this fa mily of cytokines, such as ciliary neurotrophic factor (CNTF) and leukaemia inhibitory factor (LIF), have profound effects on the survival and maintenance of motor neurons, Recently it was reported that mice lacking LlF-R die shortly after birth unlike mice lacking CNTF or LIF which are viable. Here we describe histopathological analyses of lifr mutants tha t reveal a loss > 35% of facia l motor neurons, 40% of spinal motor neurons and 50% of neurons in the nucleus ambiguus. These findings point to the existence of a ligand for LIF-R tha t is required for the normal development of motor neurons in both brainstem nuclei and spinal cord.
The transmission of proliferative and developmental signals from activated cell-surface receptors to initiation of cellular responses in the nucleus is synergically controlled by the coordinated action of a diverse set of intracellular signalling proteins. The Ras/Raf/MEK/MAPK signalling pathway has been shown to control the expression of genes which are crucial for the physiological regulation of cell proliferation, differentiation and apoptosis. Within this signalling cascade, the Raf protein family of serine/threonine kinases serves as a central intermediate which connects to many of other signal transduction pathways. To elucidate the signalling functions of the different Raf kinases in motoneurons during development, the expression, distribution and subcellular localization of Rafs in the spinal cord and the facial nucleus in brainstem of mice at various embryonic and postnatal stages were investigated. Moreover, we have investigated the intracellular redistribution of Raf molecules in isolated motoneurons from 13 or 14 day old mouse embryos, after addition or withdrawal of neurotrophic factors to induce Raf kinases activation in vitro. Furthermore, in order to investigate the potential anti-apoptotic function of Raf kinases on motoneurons, we isolated motoneurons from B-raf-/- and c-raf-1-/- mouse embryos and analysed the survival and differentiation effects of neurotrophic factors in motoneurons lacking B-Raf and c-Raf-1. We provide evidence here that all three Raf kinases are expressed in mouse spinal motoneurons. Their expression increases during the period of naturally occurring cell death of motoneurons. In sections of embryonic and postnatal spinal cord, motoneurons express exclusively B-Raf and c-Raf-1, but not A-Raf, and subcellularly Raf kinases are obviously colocalized with mitochondria. In isolated motoneurons, most of the B-Raf or c-Raf-1 immunoreactivity is located in the perinuclear space but also in the nucleus, especially after activation by addition of CNTF and BDNF in vitro. We found that c-Raf-1 translocation from the cytosol into the nucleus of motoneurons after its activation by neurotrophic factors is a distinct event. As a central finding of our study, we observed that the viability of isolated motoneurons from B-raf but not c-raf-1 knockout mice is lost even in the presence of CNTF and other neurotrophic factors. This indicates that B-Raf but not c-Raf-1, which is still present in B-raf deficient motoneurons, plays a crucial role in mediating the survival effect of neurotrophic factors during development. In order to prove that B-Raf is an essential player in this scenario, we have re-expressed B-Raf in mutant sensory and motor neurons by transfection. The motoneurons and the sensory neurons from B-raf knockout mouse which were transfected with exogenous B-raf gene revealed the same viability in the presence of neurotrophic factors as primary neurons from wild-type mice. Our results suggest that Raf kinases have important signalling functions in motoneurons in mouse CNS. In vitro, activation causes redistribution of Raf protein kinases, particularly for c-Raf-1, from motoneuronal cytoplasm into the nucleus. This redistribution of c-Raf-1, however, is not necessary for the survival effect of neurotrophic factors, given that B-raf-/- motor and sensory neurons can not survive despite the presence of c-Raf-1. We hypothesize that c-Raf-1 nuclear translocation may play a direct role in transcriptional regulation as a consequence of neurotrophic factor induced phosphorylation and activation of c-Raf-1 in motoneurons. Moreover, the identification of target genes for nuclear translocated c-Raf-1 and of specific cellular functions initiated by this mechanism awaits its characterization.
Genetische Inaktivierung des somatischen Cytochrom C Gens der Maus Cytochrom C wurde als ein Interaktionspartner im Apoptosom beschrieben. Ziel dieses Projektes war es, die Rolle von Cytochrom C bei der Apoptose von Nervenzellen in vivo durch genetische Inaktivierung in der Maus zu untersuchen. Die homozygote Deletion des Cytochrom C Gens führt jedoch zu einem sehr frühen Entwicklungsdefekt: Schon am 8. Embryonaltag findet man nur noch Embryonen ohne erkennbare Körperachse. Im weiteren wurden daher heterozygote Tiere untersucht, die in bestimmten Geweben, wie Gehirn und Rückenmark, eine Reduktion der Menge von Cytochrom C aufweisen. Am ersten Tag nach der Geburt konnten keine Unterschiede zwischen Tieren mit einem oder zwei Cytochrom C Genen in Bezug die Anzahl von Motoneuronen gefunden werden. Auch nach perinataler Fazialisläsion war die Rate des Zelltods bei Tieren mit heterozygoter Deletion des Cytochrom C Gens unverändert. In vitro zeigte sich jedoch eine erhöhte Resitenz von Motoneuronen gegenüber Fas-induzierter Apoptose. Bei der Analyse der Apoptose von Thymozyten zeigte sich ein Trend, der eine kleine, aber reproduzierbare Verzögerung einer späten Zelltodphase nach UV-induzierter Apoptose nahelegt. Erste Experimente deuten außerdem auf einen Effekt der Cytochrom C Gendosis auf den Verlauf einer Experimentellen Autoimmunencephalitis (EAE) hin. Charakterisierung der NFL-Cre Maus Die zelltypspezifische Genablation mit dem Cre/loxP System umgeht einige der größten Probleme der klassischen Methode der Geninaktivierung in Mäusen, indem nur in bestimmten Geweben oder Zelltypen, eventuell sogar nur ab einem bestimmten Zeitpunkt, ein Gen gezielt ausgeschaltet werden kann. Allerdings hängt das Cre/loxP System von der Verfügbarkeit von brauchbaren Cre-transgenen Mauslinien mit entsprechenden Expressionsmustern und –kinetiken ab. Wir haben eine transgene Mauslinie etabliert und analysiert, die die Cre Rekombinase unter der Kontrolle des humanen Neurofilament-L Promotors exprimiert. Das Expressionsmuster von Cre wurde in mehreren Geweben mit RT-PCR und durch Verkreuzung mit einer Reportergenmaus untersucht. Im Gehirn wurden Cre exprimierende Zelltypen mit in-situ Hybridisierung, Immunhistochemie und wiederum mit Hilfe der Reportermaus identifiziert. Dabei zeigte sich eine spezifische Cre Expression in bestimmten Neuronpopulationen wie hippocampalen Pyramidenzellen und spinalen und cranialen Motoneuronen. Unsere NFL-Cre Maus besitzt einige Eigenschaften, die bisher publizierte Cre-Linien nicht aufweisen, so z.B.eine starke Cre Expression in hippocampalen Pyramidenzellen, aber nicht in Körnerzellen des Gyrus dentatus; Expression in cortikalen Pyramidenzellen, aber keine Expression im Striatum; Expression in zerebellären Purkinje-, aber nicht Körnerzellen; sowie die Expression in spinalen und cranialen Motoneuronen, aber nicht in angrenzenden Interneuronen. Die Rolle von Stat3 für das Überleben von Motoneuronen Die Mitglieder der CNTF/LIF/Cardiotrophin Genfamilie sind potente Überlebensfaktoren für embryonale und lädierte Motoneurone sowohl in vitro als auch in vivo. Diese Faktoren binden an Rezeptorkomplexe, die gp130 und LIFR als signaltransduzierende Komponenten enthalten. Im Gegensatz zu den Rezeptoren für andere neurotrophe Faktoren, führt die Aktivierung von gp130 und LIFR zur Phosphorylierung und Aktivierung des Transkriptionsfaktors Stat3. Es war aber zu Beginn dieser Arbeiten unklar, ob die Aktivierung von Stat3 für den Überlebenseffekt der neuropoietischen Zytokine notwendig ist. Um diese Frage zu beantworten, wurde Stat3 in Motoneuronen mit Hilfe des Cre/loxP Systems konditional inaktiviert. Stat3 ist nicht für das Überleben embryonaler Motoneurone essentiell, obwohl man in vitro eine Verschiebung der Dosis-Wirkungskurve für CNTF findet. In vivo hingegen kann kein erhöhter Zelltod von Motoneuronen nachgewiesen werden. Im Gegensatz dazu, kommt es bei adulten Tieren mit Inaktivierung von Stat3 in Motoneuronen zu einem erhöhten Zelltod nach Fazialisläsion. Diese Neurone können wiederum durch die Applikation neurotropher Faktoren, einschließlich CNTF, gerettet werden. Durch semiquantitative RT-PCR kann man zeigen, daß Stat3-regulierte Gene, deren Expression nach Nervenläsion induziert wird, in Neuronen mit Inaktivierung von Stat3 weniger stark exprimiert werden. Zu diesen Genen gehören Reg-2, ein Mitogen für Schwannzellen, das von regenerierenden Neuronen exprimiert wird, und Bcl-xL, ein Gen, welches direkt in die Apoptoseregulation eingreift. Diese Daten zeigen, daß Stat3 Aktivierung eine essentielle Rolle für das Überleben nach Läsion von postnatalen Motoneuronen spielt, aber nicht während der Embryonalentwicklung. Das bedeutet, daß die Signalwege ein und desselben neurotrophen Faktors sich während der Entwicklung und reifung des Organismus verändern können.
Spinale Muskelatrophie (SMA), die häufigste autosomal rezessive neuromuskuläre Erkrankung bei Kindern und jungen Erwachsenen, wird durch Mutationen in der telomeren Kopie des survival motor neuron (SMN1) Gens auf dem humanen Chromosom 5 verursacht. Anders als bei Mäusen, welche nur ein Smn Gen haben, gibt es beim Menschen eine zweite Kopie (SMN2). Das Genprodukt dieser zweiten Kopie wird am C-Terminus bevorzugt alternativ gespleißt. Es bringt nur eine kleine Menge des vollständigen SMN Proteins hervor. Der Grund, warum eine reduzierte Menge des ubiquitär exprimierten SMN Proteins speziell zu einer Motorneuronendegeneration führt, ohne andere Zelltypen gleichermaßen zu betreffen ist noch immer nicht bekannt. Mit Hilfe der Yeast-Two-Hybrid Technik wurden die beiden heterogenen nukleären Ribonukleoproteine hnRNP-R und hnRNP-Q als neue SMN-bindende Proteine identifiziert. Diese beiden hochhomologen Proteine waren bereits bekannt und stehen in Verbindung mit dem RNA Metabolismus, im Speziellen: Editing, Transport und Spleißing. hnRNP-R und -Q interagieren mit Wildtyp Smn, aber nicht mit trunkierten oder mutierten Smn Formen, welche in SMA-Patienten gefunden wurden. Beide Proteine werden in den meisten Geweben exprimiert. Im Rückenmark von Mäusen ist die stärkste Expression am neunzehnten embryonalen Tag zu beobachten. Interessanterweise ist hnRNP-R hauptsächlich in den Axonen von Motoneuronen zu finden und kolokalisiert dort mit Smn. Im Mausmodell für die SMA konnte gezeigt werden, dass sich die Motoneurone von erkrankten Mäusen hinsichtlich der Morphologie ihrer Neuriten von solchen aus Wildtyp Mäusen unterscheiden. Werden hnRNP-R oder hnRNP-Q in kultivierten Nervenzellen exprimiert, so fördern sie das Wachstum von Neuriten. Bei SMA-Patienten ohne Mutation im SMN Gen konnte allerdings weder Mutation noch Deletion in hnRNP-R oder hnRNP-Q nachgewiesen werden. Die Ergebnisse dieser Arbeit können entscheidend zu einem besseren Verständnis der motoneuronen spezifischen Funktion von Smn bei der SMA beitragen.
Der neurotrophe Wachstumsfaktor BDNF gehört neben NGF, NT-3 und NT-4/5 zur Familie der Neurotrophine. Er spielt eine wichtige Rolle für Überleben und Differenzierung von Nervenzellen und ist insbesondere auch verantwortlich für die Regulation synaptischer Plastizität. Besonders im Hippocampus, dem Ort der höchsten Expression von BDNF im adulten Gehirn, wirkt BDNF bei den Vorgängen von Lernen und Gedächtnis mit, welches als Phänomen der LTP untersucht werden kann. Bisher ist eine Lokalisation von BDNF-Protein mittels Immunfluoreszenz-Techniken im Gehirn der Maus oder Ratte nur sehr schwer gelungen. In den meisten Arbeiten gelang die Lokalisation von BDNF über den Nachweis von mRNA oder im Western Blot, die Gruppe von Conner et al. konnte einen qualitativen Nachweis von BDNF-Protein mittels eines eigens hergestellten Antiserums erbringen (Conner et al. 1997). Das Ziel dieser Arbeit war die Entwicklung eines Antiserums gegen BDNF zur subzellulären Lokalisation mittels Immunhistochemie. Durch die Verwendung von Immunfluoreszenz-gekoppelten Sekundärantikörpern sollte zum einen eine quantitative Bestimmung von BDNF möglich sein, zum anderen sollte durch die Möglichkeit einer nahezu dreidimensionalen Darstellung des Gewebes mittels Vibratomschnitten auch eine Aussage über eine genauere Lokalisation von BDNF gemacht werden können. Um den immunhistochemischen Nachweis von BDNF-Protein im Hippocampus der Maus mittels Immunfluoreszenz führen zu können, wurde zunächst ein geeignetes Anti-serum benötigt. Zwei zu Vergleichszwecken ausgetestete kommerzielle Antikörper zeigten keine Färbung. Nach dem Vorbild zweier Arbeitsgruppen (Yan et al. 1997b und Conner et al. 1996, 1997) wurde ein Antiserum gegen humanes rekombinantes BDNF in Kaninchen hergestellt. Das Antiserum erhielt den Namen „BDNF RabbitB“. Die Spezifität des Antiserums wurde mittels Western Blot und in der Zellkultur anhand von Hühnchen-DRGs überprüft. Im Western Blot zeigte das Antiserum eine spezifische Anfärbung von rekombinantem BDNF sowie im Hippocampus-Proteinextrakt. In der Kontrolle mit Präimmunserum zeigte sich keine Anfärbung. In der Zellkultur mit Hühnchen-DRGs konnte eine blockierende Wirkung des Antiserums in Gegenwart von BDNF als neurotrophem Wachstumsfaktor im Zellkulturmedium nachgewiesen werden, es zeigte sich eine signifikante Reduktion des Überlebens von Zellen bei einer Verdünnung des Antiserums von 1:1.000. Das Präimmunserum zeigte keine Wirkung. Eine Kreuzreaktivität mit NGF als strukturähnlichem Protein konnte ausgeschlossen werden, da das Antiserum in Gegenwart von NGF im Kulturmedium keine Wirkung zeigte. Anschließend galt es, die Methoden für die Immunhistochemie mit diesem Antiserum zu optimieren, da es Hinweise gab, daß gerade die Immunhistochemie neurotropher Faktoren sehr sensibel auf verschiedene Methoden reagiert. Daher wurden sowohl die Fixierungsmethode, unterschiedliche Gewebeschnitte, verschiedene Puffersysteme und immunhistochemische Färbemethoden untersucht und verglichen. Die Standard-Fixierungsmethode mit Phosphat-Puffer, modifiziert nach der Methode nach Yan et al. 1997b mit maximal 2 h Nachfixierung stellte sich als beste Methode heraus. Eine Kombination zweier verschiedener Puffer (TBS und PB) innerhalb der Fixierung ist ungünstig. Daher sollte innerhalb einer Methode immer bei einem Puffersystem geblieben werden, wobei hier insgesamt bei dem Vergleich von PBS, TBS und TRIS-Puffer sowohl in der Fixierung als auch in der Färbemethode dem Phosphat-Puffer der Vorzug gegeben wird, welches auch das Standard-System darstellt. Bei den Gewebeschnitten sind, wie ursprünglich geplant Vibratomschnitte zu bevorzugen. Insgesamt konnten jedoch mögliche Ursachen für die Anfälligkeit der BDNF-Immunreaktivität bei Fixierungs- und Färbemethoden hier nicht abschließend erklärt werden. Problematisch war die ausgeprägte Hintergrundfärbung des Antiserums v.a. in der Immunhistochemie, die nicht ausreichend behoben werden konnte. Insofern sollte das Antiserum für die Verwendung bei immunhistochemischen Färbungen noch weiter optimiert werden. Für die Verwendung in der Zellkultur ist das Antiserum auf Grund seiner BDNF-blockierenden Eigenschaften gut einsetzbar. Im Western Blot sollte „BDNF RabbitB“ in einer Verdünnung von 1:5.000, in Zellkultur mit 1:1.000 und in der Immunhistochemie mit Vibratomschnitten mit 1:2.000 eingesetzt werden.
Neurotrophe Faktoren haben ein breites Aufgabenfeld und spielen eine wichtige Rolle als Überlebensfaktoren embryonaler Neurone, bei Proliferation und Differenzierung im Nervensystem sowie als Modulatoren synaptischer Plastizität. Im ersten Themenkomplex der vorliegenden Arbeit wurden neurotrophe Faktoren als Modulatoren synaptischer Plastizität und ihr Einfluß auf die BDNF-Regulation im Hippocampus untersucht. Dabei wurde zunächst das selbsthergestellte polyclonale BDNF-Immunserum für die Anwendung in der Immunhistochemie und im Western Blot optimiert, doch es konnten bezüglich BDNF keine Veränderungen in Hippocampi CNTF-defizienter Mäuse gegenüber Wildtyp-Tieren festgestellt werden. Die Ergebnisse der Voruntersuchungen, die im Hippocampus CNTF-defizienter Tiere verminderte BDNF-Level gezeigt hatten, konnten somit nicht verifiziert werden. Im Rahmen dieser Arbeit wurde an CNTF-defizienten Mäusen eine eingeschränkte LTP und LTD nachgewiesen. Zum besseren Verständnis der – laut LTP-Untersuchungen – veränderten Situation an der hippocampalen CA1-Synapse bei CNTF-defizienten Tieren wurden elektronenmikroskopische Bilder dieser Region angefertigt, deren Auswertung keine augenscheinlichen Unterschiede ergab. Im Stratum radiatum der CA1-Region war zudem keine spezifische CNTF-Färbung nachweisbar. Zur Klärung der Frage, ob es IGF-vermittelt nach Training zu hippocampaler BDNF-Hochregulation kommt, wurden Laufradexperimente mit wildtypischen und konditionalen IGF1-Rezeptor-knockout Mäusen durchgeführt und die jeweiligen BDNF-Level untersucht. Dabei wurde BDNF durch Laufradtraining in beiden Genotypen in ähnlichem Maße hochreguliert, was für alternative Wege der BDNF-Hochregulation spricht. Der zweite Themenkomplex befasste sich mit dem Einfluß neurotropher Faktoren auf die Proliferation und Differenzierung in Hippocampus und Cortex. BrdU-Inkorporationsexperimenten zeigten in der Körnerzellschicht des Gyrus dentatus gesteigerte Proliferationsraten bei CNTF-defizienten und CNTF&LIF-defizienten Mäusen, wobei LIF-defiziente Tiere keine veränderten Proliferationsraten zeigten. Untersuchungen an Kulturen cortikaler Vorläuferzellen bestätigten die Hypothese, wonach cortikale Vorläuferzellen zunächst Neurone bilden, die einen Faktor sezernieren, der auf die cortikalen Vorläuferzellen wirkt und sie zur Bildung von Astrozyten veranlasst. Es konnte gezeigt werden, dass CT-1 der Hypothese folgend in vitro und in vivo für die Einleitung der Astrozytogenese im Cortex verantwortlich ist.
Bag-1 Defizienz in Mäusen führt in der embryonalen Entwicklung zu einem lethalen Phänotyp mit schweren Defekten in Nervensystem und Leber. Neben der Expression der Inhibitor of Apoptosis Proteinen (IAP), ist ein Komplex aus Akt, Hsp70, Bag-1 und B-Raf für die Phosphorylierung eines spezifischen AS-Restes des pro-apoptotischen Proteins Bad für das Überleben wichtig (Gotz und Wiese et al., 2005). Das Ziel dieser Arbeit bestand darin, die Funktionen der Maus Bag-1 Isoformen in der neuronalen Entwicklung anhand von in vitro Modellen näher zu charakterisieren. Überexpression von Bag-1S und Bag-1L in PC12 Zellen zeigte, dass Bag-1S im Zytoplasma und im Zellkern exprimiert wird, Bag-1L nur im Zellkern. Eine eingeführte Punktmutation, die die Interaktion mit Hsp70 verhindert, führte zu einer zytoplasmatischen Expression von Bag-1Sm. Die Mutante Bag-1Lm blieb nukleär lokalisiert. Überexpression von Bag-1S führte zu einer Reduktion der Neuritenlänge. Bag-1L und die mutanten Isoformen zeigten diesen Effekt nicht. Der inhibierende Einfluss von Bag-1S auf das Neuritenwachstum ist bislang spekulativ. Die Regulation erfolgt vermutlich über den Komplex Bag-1, Hsp70, Akt und B-Raf. Die Analyse von Bag-1 - /- Neuralen Stammzellen zeigte im Vergleich zu Bag-1 +/+ Neuralen Stammzellen eine erhöhte Apoptose. Wurde durch Vireninfektion Bag-1S oder Bag-1L zurück in die Zellen gebracht, waren diese wieder in der Lage zu überleben. Die Mutanten zeigten diese Effekte nicht, so dass Hsp70 ein notwendiger Interaktionspartner für die überlebensfördernde Wirkung von Bag-1 ist. Bag-1 -/- Neurale Stammzellen zeigten außerdem gliale Differenzierungsdefekte, die nicht durch eine Rückführung der Isoformen gerettet werden konnten. Zusätzliche Experimente, die Neurale Stammzellen gezielt in die gliale Differenzierung durch Gabe von CNTF oder LIF leiten, zeigten, dass Bag-1 -/- Neurale Stammzellen durchaus in der Lage sind, gliale Zellen zu bilden. Bag-1 gilt auch als Modulator nukleärer Rezeptoren, wie dem Glucocorticoid-Rezeptor (GR). Die Kotransfektion der Bag-1 Isoformen mit GR-GFP, zeigte Änderungen des Expressionsmusters bei Bag-1L und Bag-1Lm, jedoch nicht bei Bag-1S und Bag-1Sm. Die Etablierung einer Methode zur in vivo Analyse von Glucocorticoid-Signalwegen in der Neuroneogenese von adulten Mäusen, war in ihren ersten Ansätzen erfolgreich, so dass diese nach einigen Optimierungen für weitere Analysen genutzt werden kann.
Aufklärung des Pathomechanismus bei der pmn-Mausmutante, einem Mausmodell für Motoneuronerkrankungen
(2007)
Die pmn-Maus dient als Modell für degenerative Motoneuronerkrankungen: Während heterozygote Mäuse klinisch unauffällig sind, entwickeln homozygote einige Anzeichen, wie man sie auch bei humanen Motoneuronerkrankungen findet. Ab der 2. postnatalen Woche weisen sie eine progrediente Schwäche der Hinterläufe auf. Innerhalb kurzer Zeit sind auch andere Muskelgruppen betroffen, was zwischen der 4. und 6. postnatalen Woche zum Tod durch Atemversagen führt. Verantwortlich für die Erkrankung der pmn-Mäuse ist eine Punktmutation im Tubulin-spezifischen Chaperon E (tbce) Gen, die zu einem Aminosäureaustausch an einer evolutionär konservierten Aminosäure im TBCE-Protein führt. TBCE wird ubiquitär exprimiert und spielt eine Rolle bei der Assemblierung der Mikrotubuli. Phänotypisch sind von der Mutation spezifisch Motoneurone betroffen. Nach der Herstellung und Charakterisierung eines Antiserums gegen TBCE war es möglich, nach Unterschieden zwischen pmn-mutierten und wildtypischen Motoneuronen hinsichtlich der Stabilität und der subzellulären Lokalisation des TBCE Proteins zu suchen. Western Blot Analysen mit Rückenmarkslysaten von vier Wochen alten pmn-Mäusen zeigen eine deutliche Reduktion der TBCE-Expression. Mittels Immunfluoreszenz waren in isolierten embryonalen Motoneuronen indes keine Unterschiede hinsichtlich der Expressionsstärke und der subzellulären Lokalisation festzustellen. Das TBCE-Protein wird überwiegend im Zellsoma exprimiert und befindet sich dort im Golgi-Apparat und an den Centrosomen, die als Generatoren der axonalen Mikrotubuli angesehen werden. Obwohl mittels Immunfluoreszenz zu diesem Zeitpunkt keine Unterschiede detektierbar sind, weisen die pmn-mutierten Motoneurone nach sieben Tagen in Kultur einige axonale Pathologien auf, wenn sie in Gegenwart des neurotrophen Faktors BDNF kultiviert werden: Das Längenwachstum der Axone ist deutlich reduziert und entlang der Axone finden sich zahlreiche axonale Schwellungen mit Proteinaggregaten. Elektronenmikroskopisch findet sich eine Reduktion der Mikrotubulianzahl im proximalen Axonabschnitt, während die medialen und distalen Teile eine unveränderte Anzahl an Mikrotubuli aufweisen. Parallel findet sich in allen Axonabschnitten der pmn-mutierten Motoneurone eine deutliche Zunahme an Neurofilamenten. Neben den morphologischen Veränderungen weisen die Motoneurone aus pmn-Mäusen zu diesem Zeitpunkt auch eine Störung im axonalen Transport der Mitochondrien auf, die in den Axonen saltatorisch und bidirektional entlang von Mikrotubuli transportiert werden, auf. So ist die Anzahl stationärer Mitochondrien in pmn-mutierten Motoneuronen signifikant erhöht, während die Anzahl an transportierten Mitochondrien und deren maximale Transportgeschwindigkeit reduziert ist. Die morphologischen Veränderungen und die Störungen im axonalen Transport können kompensiert werden, wenn die pmn-mutierten Motoneurone statt mit BDNF mit dem neurotrophen Faktor CNTF kultiviert werden. Die Effekte von CNTF auf das Längenwachstum der Axone ist STAT3 vermittelt, da pmn-mutierte Motoneurone mit einer STAT3-Defizienz keine Reaktion mehr auf die Gabe von CNTF zeigen. Da STAT3 direkt mit Stathmin interagieren kann und dessen destabilisierende Wirkung auf Mikrotubuli dadurch verhindert, wurde angenommen, dass die STAT3 vermittelten CNTF Effekte auf eine lokale Wirkung von STAT3 in Axonen zurückzuführen ist. Diese Hypothese wird dadurch gestützt, dass die Herunterregulation der Stathmin Expression in pmn-mutierten Motoneuronen den gleichen Effekt auf das Längenwachstum zeigt, wie eine CNTF Gabe während der Kultivierung.
Der adoptive Transfer myelinspezifischer, enzephalithogener T-Lymphozyten führt bei Lewis-Ratten zu einer monophasisch verlaufenden Enzephalomyelitis (AT-EAE). Das Tiermodell AT-EAE ist gut geeignet, um die Transmigration von Lymphozyten über die Blut-Hirn-Schranke (BHS) ins Hirngewebe zu untersuchen. Der Einwanderung aktivierter Lymphozyten in das ZNS-Parenchym geht eine komplexe Kaskade von Zell-Zell-Interaktionen zwischen Lymphozyten und Endothel der BHS voraus. Die endothelialen Adhäsionsmoleküle Intercellular Adhesion Molecule 1 (ICAM-1) und Vascular Adhesion Molecule 1 (VCAM-1) sind entscheidend an diesem Prozess beteiligt. Mit einer kürzlich entwickelten, ultraschallbasierte molekularen Bildgebung und Quantifizierung ist die sequentielle Messung der Moleküle ICAM-1 und VCAM-1 im Verlauf der AT-EAE am lebenden Tier möglich. Schon vor dem Einsetzen der ersten klinischen Symptomatik zeigte sich bei den Versuchstieren ein Anstieg der Expression der Zelladhäsionsmoleküle ICAM-1- und VCAM-1.Diese Expression persistierte unerwartet über das Maximum der klinischen Symptomatik hinaus und bis in die Phasen der frühen Remission. Immunhistochemische Färbungen von Gehirn und Rückenmark bestätigten diese Expressionskinetik in situ. Darüber hinaus konnte histologisch und durchflusszytometrisch eine Persistenz CD4-positiver Lymphozyten in der frühen Remissionphase nachgewiesen werden. Hier war vor allem ein Anstieg der CD4- und FoxP3- positiven regulatorischen T-Zellen in der CD4 Subpopulation festzustellen. Diesen Zellen wird eine wichtige regulatorische Bedeutung für die Beendigung von Entzündungsreaktionen zugeschrieben. Ein experimentellen Beleg dafür, dass regulatorische Zellen in den Phasen der Remission die selben Migrationswege wie proinflammatorische Zellen nutzen, ergab sich durch die Blockade von ICAM-1 mit hohen Dosen eines monoklonalen Antikörpers. Wurde dieser AK in der Progressionsphase der Erkrankung gegeben, resultierte dies in einer signifikanten Reduktion der klinischen Symptomatik. Im Gegensatz dazu führte die spätere Gabe des Antikörpers in der frühen Remission zu einer signifikanten Verschlechterung des Krankheitverlaufes. In Zusammenschau legen diese Ergebnisse die Hypothese nahe, dass Adhäsionsmoleküle wie ICAM-1 nicht nur an der Einwanderung pathogener proinflammatorischer Zellen entscheidend beteiligt sind, sondern dass sie auch die Einwanderung antiinflammorischer und regulatorischer Zellen in das ZNS ermöglichen, die für eine Abschwächung der Gewebsentzündung und Zerstörung wichtig sind. Therapeutische Intervention an der BHS sind auf dem Boden dieser Erkenntnisse wahrscheinlich stadienabhängig wirksam und könnten bei falschem Einsatz mehr schaden als nutzen. Molekulare Bildgebungstechniken, wie hier paradigmatisch für die. ultraschallbasierten SPAQ-Technologie gezeigt, werden deshalb in Zukunft für die Bestimmung der geeigneten Phase einer entzündlichen ZNS Erkrankung und damit den geeigneten Zeitpunkt für eine therapeutische Intervention großes Potential erlangen.
Die proximale spinale Muskelatrophie (SMA) stellt eine der häufigsten erblichen Ursachen für den Tod im Kindesalter dar. Die Patienten leiden unter symmetrischer, langsam progredienter Muskelschwäche und in schweren Fällen auch an sensiblen Ausfällen. Die neurodegenerative Erkrankung wird autosomal-rezessiv durch Deletion bzw. Mutationen des SMN1-Gens (survival motor neuron 1-Gens) auf Chromosom 5q13 vererbt. Das SMN-Protein wird ubiquitär exprimiert und findet sich in allen untersuchten Geweben in einem Multiproteinkomplex, dem sogenannten SMN-Komplex, der die Zusammenlagerung von spleißosomalen Komplexen koordiniert. Die Funktion solcher Komplexe ist für alle Zelltypen essentiell. Deshalb stellt sich die Frage, welcher Pathomechanismus für die Erkrankung SMA verantwortlich ist. Die vorliegende Arbeit zeigt, dass die Überlebensraten der Smn–/–;SMN2-Motoneurone 14 Tage alter Mausembryonen gegenüber Smn+/+;SMN2-Motoneuronen (Kontrollen) nicht reduziert waren. Bei der morphologischen Untersuchung der Zellen zum gleichen Entwicklungszeitpunkt zeigten sich jedoch deutliche Unterschiede. Die Axonlängen der Smn-defizienten Motoneurone waren gegenüber Kontrollen signifikant verringert. Das Dendritenwachstum war nicht beeinträchtigt. Die Untersuchung der Wachstumskegel ergab bei den Smn–/–;SMN2 Motoneuronen eine signifikante Verminderung der Fläche gegenüber Kontrollen. Weiterhin zeigten sich Defekte im Zytoskelett. In den Motoneuronen von Kontrolltieren fand sich eine Anreicherung von beta-Aktin in perinukleären Kompartimenten sowie besonders stark in den Wachstumskegeln. Die beta-Aktin-Anreicherung nahm im Verlauf des Axons zu. In Smn–/–;SMN2-Motoneuronen war keine Anreicherung im distalen Axon oder in den Wachstumskegeln detektierbar. Eine gleichartige Verteilungsstörung fand sich für das SMN-Interaktionsprotein hnRNP R (heterogenous nuclear ribonucleoprotein R) und, wie andere Arbeiten zeigen konnten, auch für die beta-Aktin-mRNA, die spezifisch an hnRNP R bindet. In gleicher Weise wurden auch Veränderungen in den sensorischen Neuronen aus den Hinterwurzelganglien 14 Tage alter Mausembryonen untersucht. Bei Smn–/–;SMN2-Mäusen war die Neuritenlänge sensorischer Neurone im Vergleich zur Kontrolle gering, jedoch signifikant verkürzt und die Fläche der Wachstumskegel hochsignifikant verringert. Im Smn–/–;SMN2 Mausmodell für eine schwere Form der SMA fanden sich in den sensorischen Nervenzellen im Vergleich zu den Motoneuronen geringer ausgeprägte, jedoch gleichartige Veränderungen, was auf einen ähnlichen Pathomechanismus in beiden Zelltypen hinweist.
Differenzielle Wirkungen neurotropher Faktoren auf das Axon-und Dendritenwachstum von Motoneuronen
(2008)
In der vorliegenden Dissertation wurde die subzelluläre Lokalisation der Rezeptoren für die neurotrophen Faktoren BDNF, CNTF und GDNF in primären embryonalen und adulten Motoneuronen erstmalig genau charakterisiert. Die Rezeptoruntereinheiten des BDNF und CNTF Rezeptors, TrkB, p-TrkB, gp130 und p-Stat3, sind im Perikaryon, in Dendriten, im Axon und an den Axonterminalen bzw. Wachstumskegeln von Motoneuronen lokalisiert. Dabei sind die nativen Formen (TrkB, gp130) im Axon überwiegend membranständig, die aktivierten Formen (p-TrkB, p-Stat3) überwiegend im Inneren des Axons lokalisiert. Demgegenüber sind die Rezeptoruntereinheiten des GDNF Rezeptors, Ret und p-Ret, besonders stark in den Dendriten exprimiert. Auch im Perikaryon und an der neuromuskulären Endplatte sind Ret und p-Ret lokalisiert, nicht jedoch im Axon. Im zweiten Teil der Arbeit wurde das durch neurotrophe Faktoren bedingte Neuritenwachstum genau quantifiziert. Dabei wurde zwischen einer Stimulation des Axon- bzw. des Dendritenwachstums differenziert. Die mit GDNF behandelten Dendriten werden etwa doppelt so lang wie die Dendriten, der mit BDNF oder CNTF behandelten Motoneurone. GDNF ist somit ein potenter Stimulator des Dendritenwachstums bei isolierten primären Motoneuronen. Dieser Befund korreliert gut mit der starken Expression von Ret und p-Ret in den Dendriten. Des Weiteren wurde eine Analyse der Interaktion der neurotrophen Faktoren mit dem glutamatergen AMPA Rezeptor in Hinblick auf das Neuritenwachstum durchgeführt. Dabei zeigte sich, dass die Interaktion zwischen neurotrophen Faktoren und dem AMPA Rezeptor besonders für das Dendritenwachstum von Bedeutung ist. Die klinische Bedeutung neurotropher Faktoren und deren Rezeptoren wird im dritten Teil der Arbeit dargestellt. Die pmn Maus ist ein Mausmodell für humane degenerative Erkrankungen des Motoneurons, wie der ALS und der SMA. Pmn Motoneurone, die mit BDNF oder GDNF kultiviert werden, weisen den charakteristischen axonalen Wachstumsdefekt der pmn Motoneurone auf und werden nur etwa halb so lang wie gesunde Kontrollmotoneurone. Bemerkenswerterweise führt die Behandlung der pmn Motoneurone mit CNTF zu einer kompletten Remission des axonalen Wachstumsdefekts, so dass die Axone eine normale Axonlänge erreichen. Auch die Anzahl der pathologischen axonalen Schwellungen werden in vitro durch CNTF stark reduziert. CNTF scheint demnach der interessanteste neurotrophe Faktor für eine Behandlung degenerativer Motoneuronerkrankungen zu sein.
Die Spinale Muskelatrophie (SMA) ist mit einer Inzidenz von 1:6000 die zweithäufigste autosomal-rezessive Erbkrankheit im frühen Kindesalter. Die durch den Verlust des SMN- (survival of motoneuron) Gens reduzierte SMN Protein Expression führt zu einer Degeneration der spinalen Motoneurone mit proximal betonter Muskelschwäche. Deshalb zielen erste Therapieversuche darauf ab, diese zu erhöhen. Es war gezeigt worden, dass durch den Einsatz von Histon Deacetylase Inhibitoren (HDAC) in neuronalen Kontroll Zellen und in nicht neuronalen Zellen von SMA Patienten die SMN Protein Expression signifikant gesteigert werden konnte. In der vorgelegten Arbeit wurde untersucht, ob die HDAC Inhibitoren Valproat, SAHA und FK228 Einfluss auf die SMN Protein Expression in kortikalen neuronalen Vorläuferzellen (NSC), auf primär embryonale Fibroblasten (PMEF) und auf die morphologischen Veränderungen in primär kultivierten embryonalen Motoneuronen eines Mausmodells der SMA haben. Es konnte eine signifikante Steigerung der SMN Protein Expression durch den Einsatz von Valproat und FK228 in kortikalen neuronalen Vorläuferzellen nachgewiesen werden. Es ergab sich jedoch kein Einfluss auf die SMN Protein Expression in primär embryonalen Fibroblasten. Bei NSCs und primär kultivierten embryonalen Motoneuronen wirkten die HDAC Inhibitoren Valproat und FK228 konzentrationsabhängig toxisch auf das Überleben, die Länge der Axone und die Größe der Wachstumskegel. Es konnte kein positiver Einfluss auf die morphologischen Veränderungen des Mausmodells gesehen werden. Zusammenfassend zeigte sich in der vorgelegten Arbeit ein positiver Effekt auf die SMN Protein Expression durch den Einsatz von HDAC Inhibitoren, der jedoch mit einem toxischen Effekt auf die behandelten neuronalen Zellen einherging.
Die Spinale Muskelatrophie (SMA) ist eine häufige autosomal-rezessiv vererbte Erkrankung des motorischen Nervensystems bei Kindern. Ursache der Degeneration von spinalen Motoneuronen ist der homozygote Verlust des SMN- (survival of motoneuron) Gens und ein dadurch bedingter Mangel an SMN-Protein. Untersuchungen an Motoneuronen von Smn-defizienten Mäusen ergaben Störungen des axonalen Längenwachstums aufgrund einer Fehlverteilung des Zytoskelettproteins beta-Aktin und seiner mRNA in den Axonterminalen. Das Axonwachstum wird durch Aktin-Polymerisierung im Wachstumskegel gesteuert. beta-Aktin-mRNA findet sich auch in Axonen, und die lokale Proteinsynthese kann durch neuronale Aktivierung gesteigert werden. Das SMN-Protein ist am axonalen Transport von beta-Aktin beteiligt. In der vorliegenden Arbeit ergaben Western Blot-Analysen in neuralen Stammzellen (NSC) sowie spinalen Motoneuronen in vitro eine Steigerung der SMN-Proteinexpression durch 8-CPT-cAMP. Zur Untersuchung der Auswirkungen der erhöhten SMN-Proteinmenge auf die Pathologie der Motoneurone wurde ein in-vitro-Assay entwickelt, mit dessen Hilfe gezeigt werden konnte, dass eine Behandlung mit 100 µM 8-CPT-cAMP die axonalen Veränderungen isolierter embryonaler Smn-defizienter Motoneurone kompensieren kann. Motoneurone von 14 Tage alten Smn-defizienten und Kontroll-Mausembryonen wurden über sieben Tage hinweg auf einer Matrix aus Poly-Ornithin und Laminin-111 bzw. Laminin-121/221 kultiviert und mit 100µM cAMP und neurotrophen Faktoren behandelt. Nach Fixierung wurden die Zellen mit Antikörpern gegen Islet-1/2, tau und beta-Aktin gefärbt, mit Hilfe eines konfokalen Mikroskops fotografiert und digital vermessen. 8-CPT-cAMP erhöht den beta-Aktin-Gehalt in den axonalen Wachstumskegeln von Smn-defizienten Motoneuronen. Die Größe der Wachstumskegel nimmt durch die Behandlung um das 2-3fache zu und erreicht normale Werte. Auf Laminin-111 bleibt das Längenwachstum der Axone durch 100µM 8-CPT-cAMP unbeeinflusst, auf Laminin-121/221 wird das Längenwachstum normalisiert. Die beta-Aktin-Verteilung innerhalb der Axone und Wachstumskegel von Smn-defizienten Motoneuronen erscheint durch die cAMP-Behandlung nahezu normalisiert. Die Wiederherstellung der beta-Aktin-Verteilung in Wachstumskegeln durch cAMP kann große Auswirkungen auf die Funktionalität der Motoneurone haben. Die Ergebnisse sind möglicherweise ein erster Schritt auf dem Weg zu einer Therapie für die Spinale Muskelatrophie.
Die proximale spinale Muskelatrophie (SMA) ist eine autosomal rezessive Erb-krankheit, welche durch fortschreitende Muskelatrophie mit Betonung der pro-ximalen Extremitäten, sowie zunehmende motorische Lähmungen charakterisiert wird. Bedingt wird diese neurodegenerative Erkrankung durch Mutation bzw. Deletion des SMN1-Gens auf Chromosom 5q13. Dies führt zu reduzierten Mengen des ubiquitär exprimierten SMN-Proteins, da der Verlust des SMN1-Gens nicht durch das noch verbleibende SMN2-Gen kompensiert werden kann. Die SMN-Promotor-Region enthält ein CRE II bindendes Element, welches Effekte von zyklischem Adenosinmonophosphat (cAMP) vermittelt und so die SMN-Transkription in untersuchten Zellen stimuliert. Ausgehend von diesem Befund stellte sich die Frage, ob cAMP dem Mangel an volllängen SMN bei der SMA entgegen wirkt. Daher wurden für diese Dissertation neurosphärenbildende kortikale Vorläuferzellen und primär kultivierte Motoneuronen von Smn+/+; SMN2- und Smn–/–;SMN2-Mausembryonen untersucht, um zu klären, ob die cAMP-Behandlung der Zellen zu einer Hochregulierung des SMN2-Transkripts führt, und durch die resultierende Erhöhung des SMN-Proteingehalts morphologische und funktionelle Defekte kompensiert werden. Die Untersuchung zeigte eine signifikante Zunahme des SMN2-Transkriptgehalts in Anwesenheit von cAMP. Dadurch kam es zu einem Anstieg der SMN-Proteinmenge im Soma, Axon und Wachstumskegel von Smn–/–;SMN2-Motoneuronen. Die Verteilungs-störung des SMN-Interaktionspartners hnRNP R mit fehlender kontrolltypischer Anreicherung im distalen Axon und Wachstumskegel von Smn–/–;SMN2-Motoneuronen wurde ebenfalls durch cAMP kompensiert. Smn-defiziente Mo-toneurone zeigten im Vergleich zu Kontrollzellen kleinere Wachstumskegel sowie ein Defizit an β-Aktin im distalen Axon. Zudem fehlte in Smn–/–;SMN2-Motoneuronen die bei Kontrollen ausgeprägte Zusammenlagerung von N-Typ spezifischen Ca2+-Kanälen in der Präsynapse, die nach Kontakt mit der β2-Kette des endplattenspezifischen Laminin-221 spontan öffnen und so einen in-trazellulären Kalziumanstieg bewirken, wodurch es zu Erregbarkeitsstörungen und Axonelongationsdefekten bei Smn-defizienten Motoneuronen kommt. Die Behandlung der Smn-defizienten Motoneuronen mit cAMP führte zur Vergrößerung der Wachstumskegelfläche und zu einer im Verlauf des Axons zunehmenden Anfärbung mit β-Aktin. Außerdem kam es zu einer Erhöhung der Menge an Cav2.2-Kanalprotein in den Wachstumskegeln Smn-defizienter Motoneurone, was mit einer erhöhten Erregbarkeit korrelierte und zu einer Normalisierung der Axonlänge von Smn–/–;SMN2-Motoneuronen auf Laminin-221 führte. Die Ergebnisse dieser Arbeit lassen die Vermutung zu, dass Smn-defiziente Motoneurone in vivo Defekte im präsynaptischen Bereich der Motorendplatte aufweisen. In Zukunft können mit dem beschriebenen in vitro Assay weitere Substanzen, welche die SMN2-Traskription stimulieren, auf ihr kompensatorisches Potential getestet werden.
Die Rolle der Hirnrinde als Zentrum komplexer Funktionen wie Lernen und Ge-dächtnis wird nicht zuletzt durch deren komplexe, in Schichten organisierte Architek-tur ermöglicht. Von entscheidender Bedeutung ist die präzise Positionierung von Nervenzellen, die im Laufe der Embryonalentwicklung in der Ventrikularzone (VZ) geboren werden und anschließend in radialer Richtung zu ihrem Bestimmungsort wandern. Die Funktion des Neurotrophin-Rezeptors TrkB an der Entwicklung des zerebralen Kortex war Gegenstand dieser Arbeit. Am Tag 12,5 der Embryonalentwicklung konnte die Expression von TrkB so-wohl in den Zellen der VZ als auch in neu geborenen Neuronen der Präplatte nach-gewiesen werden. Die Phosphorylierung des Rezeptors erfolgte dabei unabhängig von den beiden Liganden BDNF und NT-3. Ebenso führten BDNF oder NT-3 zu keiner zellulären Antwort in isolierten kortikalen Vorläuferzellen, wohingegen die Stimulation mit EGF eine Phosphorylierung von TrkB an der PLCγ- und der Shc-Bindungsstelle hervorrief. Durch pharmakologische Inhibition und die Überexpression dominant negativer Src-Mutanten konnte die Beteiligung des EGF-Rezeptors und zweier neuronal exprimierter Src-Kinasen, cSrc und Fyn, an dieser Transaktivierung von TrkB durch EGF gezeigt werden. Durch die Zugabe von EGF kam es im Zuge der Aktivierung von TrkB auch zur Umverteilung des Rezeptors von intrazellulären Kompartimenten zur Zellmem-bran. Die Retention des Rezeptors im Zytoplasma wurde über post-translationelle Modifikation reguliert. Die Verhinderung von N-Glykosylierung durch Tunicamycin-Behandlung kortikaler Vorläuferzellen führte zur Exposition von TrkB an der Zellober-fläche und konnte so Responsivität gegenüber BDNF herstellen. Die physiologische Bedeutung einer Transaktivierung von TrkB durch EGF wurde durch das Fehlen der TrkB-Aktivierung in EGFR KO-Mäusen am Embryonal-tag 12,5 gezeigt. Dies hatte eine fehlerhafte Positionierung kortikaler Nervenzellen zum Zeitpunkt E15,5 zur Folge. Anhand eines Migrationsassays konnte schließlich gezeigt werden, dass die EGF-induzierte Wanderung kortikaler Vorläuferzellen in vitro mit einer asymmetrischen Translokation von TrkB einhergeht. Über die Transaktivierung von TrkB in frühen Phasen der Kortexentwicklung spielt EGF eine wichtige Rolle bei der Induktion neuronaler Differenzierung und ist an der Regulation der Wanderung postmitotischer Neurone in der Hirnrinde beteiligt.
In dieser Arbeit sollte die Funktion der Ribosomalen S6 Kinase 2 (RSK2) auf neuronaler Ebene untersucht werden. Dahingehend gab es, z.B. auf Grund der Phänotypen von Fliegen und Mäusen mit Mutationen im entsprechenden Gen oder von Patienten mit Coffin-Lowry-Syndrom (CLS) nur Vermutungen. Es bestand letztlich die Hoffnung, einen Beitrag zur Aufklärung der Pathophysiologie des CLS zu leisten. Es stellte sich auf Grund von Experimenten sowohl in vivo als auch in vitro in verschiedenen Modellsystemen in dieser Arbeit heraus, daß RSK2 einen negativen Einfluß auf das Neuriten- und Synapsenwachstum hat. In kultivierten Motoneuronen führte der KO von RSK2 zu längeren Axonen und die Überexpression eines konstitutiv aktiven RSK2-Konstrukts zu kürzeren Axonen. In PC12-Zellen führte die Expression von konstitutiv aktiven RSK2 Konstrukten zur Verkürzung der Neuriten und die Expression eines Kinase-inaktiven RSK2 Konstrukts zu längeren Neuriten. In vivo war die neuromuskuläre Synapse bei RSK2-KO Mäusen vergrößert und hatte bei Drosophila rsk Mutanten mehr Boutons. Das RSK2-Protein ist in Motoneuronen der Maus und in überexprimierter Form in den Boutons der neuromuskulären Synapse bei Drosophila nachweisbar. Damit wurde zum ersten Mal die Funktion von RSK2 auf neuronaler Ebene beschrieben. Bezüglich des Mechanismus, wie RSK2 das Nervenwachstum beeinflußt gab es deutliche Hinweise, die dafür sprechen, daß RSK2 dies über eine in der Literatur schon häufiger beschriebene Hemmung der MAPK ERK1/2 erreicht. Für diese Hypothese spricht die Tatsache, daß die ERK-Phosphorylierung in murinen Motoneuronen und im Rückenmark embryonaler Mäuse der RSK2-Mutante erhöht ist und der Axonwachstumsdefekt durch eine Hemmung von MEK/ERK behoben werden kann. Auch ist die ERK-Phosphorylierung an der murinen Muskel-Endplatte in der Mutante erhöht. Zudem zeigen genetische Epistasis-Experimente in Drosophila, daß RSK die Bouton-Zahl über ERK/RL hemmt. RSK scheint also in Drosophila von der Funktion her der RSK2-Isoform in Wirbeltieren sehr ähnlich zu sein. Ein weiteres wichtiges Ergebnis ist die Beobachtung, daß RSK2 bei Motoneuronen keinen wesentlichen Einfluß auf das Überleben der Zellen in Gegenwart neurotropher Faktoren hat. Möglicherweise spielen hier redundante Funktionen der RSK Familienmitglieder eine Rolle. Ein bislang unerklärter Befund ist die reduzierte Frequenz spontaner Depolarisationen bzw. damit einhergehender Ca2+ Einströme bei RSK2-KO Motoneuronen in Zellkultur. Die Häufigkeit und Dichte von Ca2+-Kanälen und aktive Zonen Proteinen war in Motoneuronen nicht von der Anwesenheit des RSK2-Proteins abhängig. Im Hippocampus konnte außerdem das RSK2-Protein präsynaptisch in den Moosfaser-Boutons der CA3 Region nachgewiesen werden. Es befindet sich auch in den Pyramidenzellen, aber nicht in den Pyramidenzell-Dendriten in CA3. Bezüglich der Bedeutung dieser Befunde für die Aufklärung der Pathologie des CLS ist zu folgern, daß der neuro-psychologische Phänotyp bei CLS Patienten wahrscheinlich nicht durch reduziertes Überleben von Neuronen, sondern eher durch disinhibiertes Axonwachstum oder Synapsenwachstum bedingt ist. Dies kann grob sowohl für die peripheren als auch die zentralen Defekte gelten, denn die Synapsen im ZNS und am Muskel sind in ihrer molekularen Ausstattung z.B. im Bereich der Vesikel, der aktiven Zonen oder der Transmitterausschüttung sehr ähnlich. Weiterhin könnte eine veränderte synaptische Plastizität u.a. an der Moosfaser-Pyramidenzell-Synapse in der CA3 Region des Hippocampus eine Rolle bei den kognitiven und mnestischen Einschränkungen der Patienten spielen. Die Entdeckung, daß aktiviertes ERK bei den beobachteten Effekten eine Rolle spielt kann für die Entwicklung von Therapiestrategien eine wertvolle Erkenntnis sein.
In this study I investigate the role of Schwann cell and axon-derived trophic signals as modifiers of axonal integrity and sprouting in motoneuron disease and diabetic neuropathy (DNP). The first part of this thesis focuses on the role of the Schwann-cell-derived ciliary neurotrophic factor (CNTF) for compensatory sprouting in a mouse model for mild spinal muscular atrophy (SMA). In the second part, the role of the insulin-like growth factor 1 (IGF-1) and its binding protein 5 (IGFBP-5) is examined in the peripheral nerves of patients with DNP and in two corresponding mouse models. Proximal SMA is caused by homozygous loss or mutation of the SMN1 gene on human chromosome 5. The different forms of SMA can be divided into four groups, depending on the levels of SMN protein produced from a second SMN gene (SMN2) and the severity of the disease. Patients with milder forms of the disease, type III and type IV SMA, normally reach adulthood and regularly show enlargement of motor units, signifying the reinnervation of denervated muscle fibers. However, the underlying mechanisms are not understood. Smn+/- mice, a model of type III/IV SMA, are phenotypically normal, but they reveal progressive loss of motor neurons and denervation of motor endplates starting at 4 weeks of age. The progressive loss of spinal motor neurons reaches 50% at 12 months but muscle strength is not reduced. The first evidence for axonal sprouting as a compensatory mechanism in these animals was the more than 2-fold increase in amplitude of single motor unit action potentials (SMUAP) in the gastrocnemius muscle. Confocal analysis confirmed pronounced sprouting of innervating motor axons. As CNTF is highly expressed in Schwann cells and known to be involved in sprouting, its role for this compensatory sprouting response and the maintenance of muscle strength in Smn+/- mice was investigated. Deletion of CNTF in this mouse model results in reduced sprouting and decline of muscle strength in Smn+/- Cntf-/- mice. These findings indicate that CNTF is necessary for a sprouting response and thus enhances the size of motor units in skeletal muscles of Smn+/- mice. DNP afflicting motor and sensory nerve fibers is a major complication in diabetes mellitus. The underlying cellular mechanisms of motor axon degeneration are poorly understood. IGFBP-5, an inhibitory binding protein for IGF-1, is highly upregulated in peripheral nerves in patients with DNP. The study investigates the pathogenic relevance of this finding in transgenic mice overexpressing IGFBP-5 in motor axons. These mice develop motor axonopathy similar to that seen in DNP. Motor axon degeneration is also observed in mice in which the IGF-1 receptor (IGF-1R) was conditionally depleted in motoneurons, indicating that reduced activity of IGF-1 on IGF-1R in motoneurons is responsible for the observed effect. These data provide evidence that elevated expression of IGFBP-5 in diabetic nerves reduces the availability of IGF-1 for IGF-1R on motor axons leading to progressive neurodegeneration, and thus offers novel treatment strategies.
Zusammenfassend konnte durch unsere Daten die eingangs gestellte Hypothese, dass Patienten mit SFN eine lokal und systemisch erhöhte Expression pro-inflammatorischer und algetischer Zytokine haben, auf lokaler Ebene bei der Untergruppe mit LD-SFN bestätigt werden. Bei der Untergruppe mit NLD-SFN waren keine Unterschiede bei den Zytokinexpressionen zwischen proximalen und distalen Hautbiopsien im Vergleich zu Kontrollprobanden nachweisbar. Zudem zeigten sich deutliche Unterschiede bei den Quotienten der IENFD zwischen beiden Untergruppen. Dies legt die Vermutung nahe, dass die Unterteilung in LD-SFN und NLD-SFN klinisch bedeutsam und ein möglicher Grundstein für das Verständnis der pathophysiologischen Mechanismen der SFN sein könnte. Hieraus könnten sich Fortschritte in der Diagnostik ergeben und gezielte symptomatische und vielleicht sogar kausale Therapien auf lokaler Ebene bei der SFN entwickeln.
Bei Yeast Two-Hybrid Untersuchungen wurde in unserer Arbeitsgruppe das RNA-Bindungsprotein hnRNP-R als Interaktionspartner von SMN gefunden und es konnte gezeigt werden, dass hnRNP-R mit SMN in Axonen von primären Motoneuronen kolokalisiert (Rossoll et al., 2002). hnRNP-R assoziiert mit der β-Aktin mRNA und nach Überexpression kommt es zu einer Akkumulation von β-Aktin in den Wachstumskegeln von neuronalen Zellen, sowie zu verstärktem Neuritenwachstum bei PC12 Zellen. Wird die SMN-Bindungsdomäne von hnRNP-R deletiert, ist dieser Effekt stark reduziert (Rossoll et al., 2003). Auf diesen in vitro Befunden ist die Hypothese begründet, dass hnRNP-R an der Translokation der β-Aktin mRNA in die Wachstumskegel von neuronalen Zellen beteiligt ist. Deshalb wurde im Rahmen dieser Arbeit die Rolle von hnRNP-R bei der Entwicklung in Neuronen des Nervensystems näher untersucht. Dazu wurden Zebrafisch Embryonen als in vivo Modellsystem für Morpholino vermittelte Knockdown Untersuchungen gewählt. Zunächst wurde ein gegen murines Protein hergestelltes hnRNP-R Antiserum charakterisiert und gezeigt, dass es das Zebrafisch Protein spezifisch erkennt. Dieses Antiserum wurde in Western Blot Analysen verwendet um den hnRNP-R Knockdown in Zebrafisch Embryonen zu verifizieren. Bei den hnRNP-R Morpholino injizierten Embryonen konnten dosisabhängig axonale Veränderungen beobachtet werden. Diese Veränderungen stimmen mit einem Krankheitsmodell für SMA im Zebrafisch überein. Es konnte gezeigt werden, dass das Überleben primärer Motoneurone in Zebrafisch Embryonen nicht beeinträchtigt ist und dass andere neuronale Zellen keine signifikante Beeinflussung durch einen hnRNP-R Knockdown erfahren. Um die Spezifität des axonalen Phänotyps, der durch hnRNP-R Knockdown hervorgerufen wurde zu belegen, wurde mit muriner hnRNP-R mRNA ein Rescue-Experiment durchgeführt. Es konnte gezeigt werden, dass dabei der axonale Phänotyp weitestgehend wieder aufgehoben wurde. Parallel zu den Zebrafisch Experimenten wurde ein hnRNP-R Knockout Konstrukt mittels homologer Rekombination in Escherichia coli hergestellt und in murine embryonale Stammzellen elektroporiert. Die Charakterisierung einer hnRNP-R Knockout Maus könnte weitere bedeutende Einsichten in die in vivo Funktionen von hnRNP-R bei der Embryonalentwicklung und speziell der Entwicklung von Motoneuronen gewähren. Um der Frage nach zu gehen, welche mRNAs in Wachstumskegeln von Axonen primärer Maus Motoneuronen zu finden sind oder durch Transportprozesse lokal akkumuliert sind,wurden Versuche unternommen, um mittels Laser-Mikrodissektion einzelne Wachstumskegel von Motoneuronen für Untersuchungen der enthaltenen mRNAs zu gewinnen. Erstmalig ist es im Rahmen dieser Arbeit gelungen, kompartimentalisierte Kulturen von primären Motoneuronen der Maus zu etablieren. Damit wurde die Grundlage geschaffen, um RNA-Profile von distalen Zellkompartimenten wie den Axonen und Wachstumskegeln zu bestimmen.
Structural and functional modifications of synaptic connections (“synaptic plasticity”) are believed to mediate learning and memory processes. Thus, molecular mechanisms of how synapses assemble in both structural and functional terms are relevant for our understanding of neuronal development as well as the processes of learning and memory. Synapses form by an asymmetric association of highly specialized membrane domains: at the presynaptic active zone transmitter filled vesicles fuse, while transmitter receptors at the opposite postsynaptic density sense this signal. By genetic analysis, matrix proteins of active zones from various families have been shown to be important for fast vesicle fusion, and were suggested to contribute to synapse stability and assembly. The Sigrist lab in collaboration with the Buchner lab previously had shown that the large scaffold protein Bruchpilot (Brp) is essential for both the structural and functional integrity of active zones and for synaptic plasticity in Drosophila melanogaster. The work described in this thesis investigated several candidate proteins which appear to be involved in preand postsynaptic function, as summarized in the following: (1) DREP-2 (DEF45 related protein-2) had been found by co-immunoprecipitations with anti-Brp antibodies by Dr. Manuela Schmidt (unpublished data). Mutants and antibodies for the further study of DREP- 2 were generated in this thesis. Yeast two hybrid results suggest that DREP-2 might interact with dynein light chain 2, while in vivo imaging indicates that DREP-2 might be involved in bidirectional axonal transport. (2) Coimmunoprecipitation and pull down experiments suggested that the ARFGAP [ADP-ribosylation factor (ARF)-directed GTPase activating protein (GAP)] protein GIT (G-protein coupled receptor kinase interacting protein) could interact with the endocytosis associated molecule Stoned B (StnB). Mutants in the dgit gene showed an accumulation of large size vesicles, membrane intermediates and decreased vesicle density at the 3rd instar larval neuromuscular junction (NMJ) by electron microscopy (EM). The phenotypes accumulation of large size vesicles and membrane intermediates could be rescued partially by expression of Drosophila GIT (DGIT) or human GIT in dgit mutant background. Furthermore, by immunofluorescence the dgit mutant shows specifically decreased levels of StnB, which could be restored partially by the expression of DGIT. These results strongly support the suggestion that DGIT interacts with StnB, which is involved in the regulation of vesicle size, endocytosis or recycling of synaptic vesicles (SVs). Furthermore, the dgit mutants also showed signs of a mislocalization of the presynaptic protein Brp relative to the postsynaptic protein GluRIID, which could be rescued by expression of DGIT or human GIT in the dgit mutant background, but not by StnB. These results suggest that GIT on one hand executes roles in the regulation of synaptic vesicle endocytosis, but potentially also has structural roles for synapse assembly (3) Djm-1 is a candidate locus to mediate mental retardation in human patients when it is mutated. As a first step towards an understanding of the mechanistic role of DJM-1, Drosophila genetics were used to address DJM-1 function. So far, however, the djm-1 mutant generated in this thesis did not show a nervous system phenotype.
The Wuerzburg Hybridoma Library against the Drosophila brain represents a collection of around 200 monoclonal antibodies that bind to specific structures in the Drosophila brain. Here we describe the immunohistochemical staining patterns, the Western blot signals of one- and two-dimensional electrophoretic separation, and the mass spectrometric characterization of the target protein candidates recognized by the monoclonal antibodies aa2 and ab52 from the library. Analysis of a mutant of a candidate gene identified the Drosophila homolog of the Epidermal growth factor receptor Pathway Substrate clone 15 (Eps15) as the antigen for these two antibodies.
Background
We aimed to accurately estimate the frequency of a hexanucleotide repeat expansion in C9orf72 that has been associated with a large proportion of cases of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD).
Methods
We screened 4448 patients diagnosed with ALS (El Escorial criteria) and 1425 patients with FTD (Lund-Manchester criteria) from 17 regions worldwide for the GGGGCC hexanucleotide expansion using a repeat-primed PCR assay. We assessed familial disease status on the basis of self-reported family history of similar neurodegenerative diseases at the time of sample collection. We compared haplotype data for 262 patients carrying the expansion with the known Finnish founder risk haplotype across the chromosomal locus. We calculated age-related penetrance using the Kaplan-Meier method with data for 603 individuals with the expansion.
Findings
In patients with sporadic ALS, we identified the repeat expansion in 236 (7·0%) of 3377 white individuals from the USA, Europe, and Australia, two (4·1%) of 49 black individuals from the USA, and six (8·3%) of 72 Hispanic individuals from the USA. The mutation was present in 217 (39·3%) of 552 white individuals with familial ALS from Europe and the USA. 59 (6·0%) of 981 white Europeans with sporadic FTD had the mutation, as did 99 (24·8%) of 400 white Europeans with familial FTD. Data for other ethnic groups were sparse, but we identified one Asian patient with familial ALS (from 20 assessed) and two with familial FTD (from three assessed) who carried the mutation. The mutation was not carried by the three Native Americans or 360 patients from Asia or the Pacific Islands with sporadic ALS who were tested, or by 41 Asian patients with sporadic FTD. All patients with the repeat expansion had (partly or fully) the founder haplotype, suggesting a one-off expansion occurring about 1500 years ago. The pathogenic expansion was non-penetrant in individuals younger than 35 years, 50% penetrant by 58 years, and almost fully penetrant by 80 years.
Interpretation
A common Mendelian genetic lesion in C9orf72 is implicated in many cases of sporadic and familial ALS and FTD. Testing for this pathogenic expansion should be considered in the management and genetic counselling of patients with these fatal neurodegenerative diseases.
Defects in glycinergic inhibition result in a complex neuromotor disorder in humans known as hyperekplexia (OMIM 149400) with similar phenotypes in rodents characterized by an exaggerated startle reflex and hypertonia. Analogous to genetic defects in humans single point mutations, microdeletions, or insertions in the Glra1 gene but also in the Glrb gene underlie the pathology in mice. The mutations either localized in the (spasmodic, oscillator, cincinnati, Nmf11) or the (spastic) subunit of the glycine receptor (GlyR) are much less tolerated in mice than in humans, leaving the question for the existence of different regulatory elements of the pathomechanisms in humans and rodents. In addition to the spontaneous mutations, new insights into understanding of the regulatory pathways in hyperekplexia or glycine encephalopathy arose from the constantly increasing number of knock-out as well as knock-in mutants of GlyRs. Over the last five years, various efforts using in vivo whole cell recordings provided a detailed analysis of the kinetic parameters underlying glycinergic dysfunction. Presynaptic compensation as well as postsynaptic compensatory mechanisms in these mice by other GlyR subunits or GABA(A) receptors, and the role of extra-synaptic GlyRs is still a matter of debate. A recent study on the mouse mutant oscillator displayed a novel aspect for compensation of functionality by complementation of receptor domains that fold independently. This review focuses on defects in glycinergic neurotransmission in mice discussed with the background of human hyperekplexia en route to strategies of compensation.
Leukemia inhibitory factor (LIF) and Ciliary Neurotrophic factor (CNTF) are members of the interleukin-6 family of cytokines, defined by use of the gp130 molecule as an obligate receptor. In the murine experimental autoimmune encephalomyelitis (EAE) model, antagonism of LIF and genetic deletion of CNTF worsen disease. The potential mechanism of action of these cytokines in EAE is complex, as gp130 is expressed by all neural cells, and could involve immuno-modulation, reduction of oligodendrocyte injury, neuronal protection, or a combination of these actions. In this study we aim to investigate whether the beneficial effects of CNTF/LIF signalling in EAE are associated with axonal protection; and whether this requires signalling through oligodendrocytes. We induced MOG\(_{35-55}\) EAE in CNTF, LIF and double knockout mice. On a CNTF null background, LIF knockout was associated with increased EAE severity (EAE grade 2.1\(\pm\)0.14 vs 2.6\(\pm\)0.19; P<0.05). These mice also showed increased axonal damage relative to LIF heterozygous mice, as indicated by decreased optic nerve parallel diffusivity on MRI (1540\(\pm\)207 \(\mu\)m\(^2\)-/s vs 1310\(\pm\)175 \(\mu\)m\(^2\)-/s; P<0.05), and optic nerve (-12.5%) and spinal cord (-16%) axon densities; and increased serum neurofilament-H levels (2.5 fold increase). No differences in inflammatory cell numbers or peripheral auto-immune T-cell priming were evident. Oligodendrocyte-targeted gp130 knockout mice showed that disruption of CNTF/LIF signalling in these cells has no effect on acute EAE severity. These studies demonstrate that endogenous CNTF and LIF act centrally to protect axons from acute inflammatory destruction via an oligodendrocyte-independent mechanism.
Cell-autonomous axon growth of young motoneurons is triggered by a voltage-gated sodium channel
(2013)
Spontaneous electrical activity preceding synapse formation contributes to the precise regulation of neuronal development. Examining the origins of spontaneous activity revealed roles for neurotransmitters that depolarize neurons and activate ion channels. Recently, we identified a new molecular mechanism underlying fluctuations in spontaneous neuronal excitability. We found that embryonic motoneurons with a genetic loss of the low-threshold sodium channel Na\(_V\)1.9 show fewer fluctuations in intracellular calcium in axonal compartments and growth cones than wild-type littermates. As a consequence, axon growth of Na\(_V\)1.9-deficient motoneurons in cell culture is drastically reduced while dendritic growth and cell survival are not affected. Interestingly, Na\(_V\)1.9 function is observed under conditions that would hardly allow a ligand- or neurotransmitter-dependent depolarization. Thus, Na\(_V\)1.9 may serve as a cell-autonomous trigger for neuronal excitation. In this addendum, we discuss a model for the interplay between cell-autonomous local neuronal activity and local cytoskeleton dynamics in growth cone function.
Ablation of BRaf Impairs Neuronal Differentiation in the Postnatal Hippocampus and Cerebellum
(2013)
This study focuses on the role of the kinase BRaf in postnatal brain development. Mice expressing truncated, non-functional BRaf in neural stem cell-derived brain tissue demonstrate alterations in the cerebellum, with decreased sizes and fuzzy borders of the glomeruli in the granule cell layer. In addition we observed reduced numbers and misplaced ectopic Purkinje cells that showed an altered structure of their dendritic arborizations in the hippocampus, while the overall cornus ammonis architecture appeared to be unchanged. In male mice lacking BRaf in the hippocampus the size of the granule cell layer was normal at postnatal day 12 (P12) but diminished at P21, as compared to control littermates. This defect was caused by a reduced ability of dentate gyrus progenitor cells to differentiate into NeuN positive granule cell neurons. In vitro cell culture of P0/P1 hippocampal cells revealed that BRaf deficient cells were impaired in their ability to form microtubule-associated protein 2 positive neurons. Together with the alterations in behaviour, such as autoaggression and loss of balance fitness, these observations indicate that in the absence of BRaf all neuronal cellular structures develop, but neuronal circuits in the cerebellum and hippocampus are partially disturbed besides impaired neuronal generation in both structures.
Cerebellar granule cell (CGC) apoptosis by trophic/potassium (TK) deprivation is a model of election to study the interplay of pro-apoptotic and pro-survival signaling pathways in neuronal cell death. In this model, the c-Jun N-terminal kinase (JNK) induces pro-apoptotic genes through the c-Jun/activator protein 1 (AP-1) transcription factor. On the other side, a survival pathway initiated by lithium leads to repression of pro-apoptotic c-Jun/AP-1 target genes without interfering with JNK activity. Yet, the mechanism by which lithium inhibits c-Jun activity remains to be elucidated. Here, we used this model system to study the regulation and function of site-specific c-Jun phosphorylation at the S63 and T91/T93 JNK sites in neuronal cell death. We found that TK-deprivation led to c-Jun multiphosphorylation at all three JNK sites. However, immunofluorescence analysis of c-Jun phosphorylation at single cell level revealed that the S63 site was phosphorylated in all c-Jun-expressing cells, whereas the response of T91/T93 phosphorylation was more sensitive, mirroring the switch-like apoptotic response of CGCs. Conversely, lithium prevented T91T93 phosphorylation and cell death without affecting the S63 site, suggesting that T91T93 phosphorylation triggers c-Jun pro-apoptotic activity. Accordingly, a c-Jun mutant lacking the T95 priming site for T91/93 phosphorylation protected CGCs from apoptosis, whereas it was able to induce neurite outgrowth in PC12 cells. Vice versa, a c-Jun mutant bearing aspartate substitution of T95 overwhelmed lithium-mediate protection of CGCs from TK-deprivation, validating that inhibition of T91/T93/T95 phosphorylation underlies the effect of lithium on cell death. Mass spectrometry analysis confirmed multiphosphorylation of c-Jun at T91/T93/T95 in cells. Moreover, JNK phosphorylated recombinant c-Jun at T91/T93 in a T95-dependent manner. On the basis of our results, we propose that T91/T93/T95 multiphosphorylation of c-Jun functions as a sensitivity amplifier of the JNK cascade, setting the threshold for c-Jun pro-apoptotic activity in neuronal cells.
Several novel synaptic proteins have been identified by monoclonal antibodies (mAbs) of the Würzburg hybridoma library generated against homogenized Drosophila brains, e.g. cysteine string protein, synapse-associated protein of 47 kDa, and Bruchpilot. However, at present no routine technique exists to identify the antigens of mAbs of our library that label only a small number of cells in the brain. Yet these antibodies can be used to reproducibly label and thereby identify these cells by immunohistochemical staining. Here we describe the staining patterns in the Drosophila brain for ten mAbs of the Würzburg hybridoma library. Besides revealing the neuroanatomical structure and distribution of ten different sets of cells we compare the staining patterns with those of antibodies against known antigens and GFP expression patterns driven by selected Gal4 lines employing regulatory sequences of neuronal genes. We present examples where our antibodies apparently stain the same cells in different Gal4 lines suggesting that the corresponding regulatory sequences can be exploited by the split-Gal4 technique for transgene expression exclusively in these cells. The detection of Gal4 expression in cells labeled by mAbs may also help in the identification of the antigens recognized by the antibodies which then in addition to their value for neuroanatomy will represent important tools for the characterization of the antigens. Implications and future strategies for the identification of the antigens are discussed.
Neurotrophic factor signaling modulates differentiation, axon growth and maintenance, synaptic plasticity and regeneration of neurons after injury. Ciliary neurotrophic factor (CNTF), a Schwann cell derived neurotrophic factor, has an exclusive role in axon maintenance, sprouting and synaptic preservation. CNTF, but not GDNF, has been shown to alleviate motoneuron degeneration in pmn mutant mice carrying a missense mutation in Tbce gene, a model for Amyotrophic Lateral Sclerosis (ALS). This current study elucidates the distinct signaling mechanism by which CNTF rescues the axonal degeneration in pmn mutant mice. ...
Learning and memory is considered to require synaptic plasticity at presynaptic specializations of neurons. Kenyon cells are the intrinsic neurons of the primary olfactory learning center in the brain of arthropods – the mushroom body neuropils. An olfactory mushroom body memory trace is supposed to be located at the presynapses of Kenyon cells. In the calyx, a sub-compartment of the mushroom bodies, Kenyon cell dendrites receive olfactory input provided via projection neurons. Their output synapses, however, were thought to reside exclusively along their axonal projections outside the calyx, in the mushroom body lobes. By means of high-resolution imaging and with novel transgenic tools, we showed that the calyx of the fruit fly Drosophila melanogaster also comprised Kenyon cell presynapses. At these presynapses, synaptic vesicles were present, which were capable of neurotransmitter release upon stimulation. In addition, the newly identified Kenyon cell presynapses shared similarities with most other presynapses: their active zones, the sites of vesicle fusion, contained the proteins Bruchpilot and Syd-1. These proteins are part of the cytomatrix at the active zone, a scaffold controlling synaptic vesicle endo- and exocytosis. Kenyon cell presynapses were present in γ- and α/β-type KCs but not in α/β-type Kenyon cells.
The newly identified Kenyon cell derived presynapses in the calyx are candidate sites for an olfactory associative memory trace. We hypothesize that, as in mammals, recurrent neuronal activity might operate for memory retrieval in the fly olfactory system.
Moreover, we present evidence for structural synaptic plasticity in the mushroom body calyx. This is the first demonstration of synaptic plasticity in the central nervous system of Drosophila melanogaster. The volume of the mushroom body calyx can change according to changes in the environment. Also size and numbers of microglomeruli - sub-structures of the calyx, at which projection neurons contact Kenyon cells – can change. We investigated the synapses within the microglomeruli in detail by using new transgenic tools for visualizing presynaptic active zones and postsynaptic densities. Here, we could show, by disruption of the projection neuron - Kenyon cell circuit, that synapses of microglomeruli were subject to activity-dependent synaptic plasticity. Projection neurons that could not generate action potentials compensated their functional limitation by increasing the number of active zones per microglomerulus. Moreover, they built more and enlarged microglomeruli. Our data provide clear evidence for an activity-induced, structural synaptic plasticity as well as for the activity-induced reorganization of the olfactory circuitry in the mushroom body calyx.
The fatal neurodegenerative disorders amyotrophic lateral sclerosis and spinal muscular atrophy are, respectively, the most common motoneuron disease and genetic cause of infant death. Various in vitro model systems have been established to investigate motoneuron disease mechanisms, in particular immortalized cell lines and primary neurons. Using quantitative mass-spectrometry-based proteomics, we compared the proteomes of primary motoneurons to motoneuron-like cell lines NSC-34 and N2a, as well as to non-neuronal control cells, at a depth of 10,000 proteins. We used this resource to evaluate the suitability of murine in vitro model systems for cell biological and biochemical analysis of motoneuron disease mechanisms. Individual protein and pathway analysis indicated substantial differences between motoneuron-like cell lines and primary motoneurons, especially for proteins involved in differentiation, cytoskeleton, and receptor signaling, whereas common metabolic pathways were more similar. The proteins associated with amyotrophic lateral sclerosis also showed distinct differences between cell lines and primary motoneurons, providing a molecular basis for understanding fundamental alterations between cell lines and neurons with respect to neuronal pathways with relevance for disease mechanisms. Our study provides a proteomics resource for motoneuron research and presents a paradigm of how mass-spectrometry-based proteomics can be used to evaluate disease model systems.
Muscle atrophy and diaphragmatic palsy are the clinical characteristics of spinal muscular atrophy with respiratory distress type 1 (SMARD1), and are well represented in the neuromuscular degeneration \((Nmd^{2J})\) mouse, modeling the juvenile form of SMARD1. Both in humans and mice mutations in the IGHMBP2 gene lead to motoneuron degeneration. We could previously demonstrate that treatment with a polyethylene glycol-coupled variant of IGF1 (PEG-IGF1) improves motor functions accompanied by reduced fiber degeneration in the gastrocnemius muscle and the diaphragm, but has no beneficial effect on motoneuron survival. These data raised the question which cell autonomous disease mechanisms contribute to dysfunction and loss of Ighmbp2-deficient motoneurons. An analysis of primary Ighmbp2-deficient motoneurons exhibited differentiation deficits such as reduced spontaneous \(Ca^{2+}\) transients and altered axon elongation, which was not compensated by PEG-IGF1. This points to an IGF1 independent mechanism of motoneuron degeneration that deserves treatment approaches in addition to IGF1.
In the mammalian brain, the neurotrophin brain-derived neurotrophic factor (BDNF) has emerged as a key factor for synaptic refinement, plasticity and learning. Although BDNF-induced signaling cascades are well known, the spatial aspects of the synaptic BDNF localization remained unclear. Recent data provide strong evidence for an exclusive presynaptic location and anterograde secretion of endogenous BDNF at synapses of the hippocampal circuit. In contrast, various studies using BDNF overexpression in cultured hippocampal neurons support the idea that postsynaptic elements and other dendritic structures are the preferential sites of BDNF localization and release. In this study we used rigorously tested anti-BDNF antibodies and achieved a dense labeling of endogenous BDNF close to synapses. Confocal microscopy showed natural BDNF close to many, but not all glutamatergic synapses, while neither GABAergic synapses nor postsynaptic structures carried a typical synaptic BDNF label. To visualize the BDNF distribution within the fine structure of synapses, we implemented super resolution fluorescence imaging by direct stochastic optical reconstruction microscopy (dSTORM). Two-color dSTORM images of neurites were acquired with a spatial resolution of ~20 nm. At this resolution, the synaptic scaffold proteins Bassoon and Homer exhibit hallmarks of mature synapses and form juxtaposed bars, separated by a synaptic cleft. BDNF imaging signals form granule-like clusters with a mean size of ~60 nm and are preferentially found within the fine structure of the glutamatergic presynapse. Individual glutamatergic presynapses carried up to 90% of the synaptic BDNF immunoreactivity, and only a minor fraction of BDNF molecules was found close to the postsynaptic bars. Our data proof that hippocampal neurons are able to enrich and store high amounts of BDNF in small granules within the mature glutamatergic presynapse, at a principle site of synaptic plasticity.
TrkB mediates the effects of brain-derived neurotrophic factor (BDNF) in neuronal and nonnneuronal cells. Based on recent reports that TrkB can also be transactivated through epidermal growth-factor receptor (EGFR) signaling and thus regulates migration of early neurons, we investigated the role of TrkB in migration of lung tumor cells. Early metastasis remains a major challenge in the clinical management of non-small cell lung cancer (NSCLC). TrkB receptor signaling is associated with metastasis and poor patient prognosis in NSCLC. Expression of this receptor in A549 cells and in another adenocarcinoma cell line, NCI-H441, promoted enhanced migratory capacity in wound healing assays in the presence of the TrkB ligand BDNF. Furthermore, TrkB expression in A549 cells potentiated the stimulatory effect of EGF in wound healing and in Boyden chamber migration experiments. Consistent with a potential loss of cell polarity upon TrkB expression, cell dispersal and de-clustering was induced in A549 cells independently of exogeneous BDNF. Morphological transformation involved extensive cytoskeletal changes, reduced E-cadherin expression and suppression of E-cadherin expression on the cell surface in TrkB expressing tumor cells. This function depended on MEK and Akt kinase activity but was independent of Src. These data indicate that TrkB expression in lung adenoma cells is an early step in tumor cell dissemination, and thus could represent a target for therapy development.
Background: Localization of mRNAs encoding cytoskeletal or signaling proteins to neuronal processes is known to contribute to axon growth, synaptic differentiation and plasticity. In addition, a still increasing spectrum of mRNAs has been demonstrated to be localized under different conditions and developing stages thus reflecting a highly regulated mechanism and a role of mRNA localization in a broad range of cellular processes.
Results: Applying fluorescence in-situ-hybridization with specific riboprobes on cultured neurons and nervous tissue sections, we investigated whether the mRNAs for two metabolic enzymes, namely glycogen synthase (GS) and glycogen phosphorylase (GP), the key enzymes of glycogen metabolism, may also be targeted to neuronal processes. If it were so, this might contribute to clarify the so far enigmatic role of neuronal glycogen. We found that the mRNAs for both enzymes are localized to axonal and dendritic processes in cultured lumbar spinal motoneurons, but not in cultured trigeminal neurons. In cultured cortical neurons which do not store glycogen but nevertheless express glycogen synthase, the GS mRNA is also subject to axonal and dendritic localization. In spinal motoneurons and trigeminal neurons in situ, however, the mRNAs could only be demonstrated in the neuronal somata but not in the nerves.
Conclusions: We could demonstrate that the mRNAs for major enzymes of neural energy metabolism can be localized to neuronal processes. The heterogeneous pattern of mRNA localization in different culture types and developmental stages stresses that mRNA localization is a versatile mechanism for the fine-tuning of cellular events. Our findings suggest that mRNA localization for enzymes of glycogen metabolism could allow adaptation to spatial and temporal energy demands in neuronal events like growth, repair and synaptic transmission.
Spinal muscular atrophy (SMA) is caused by deficiency of the ubiquitously expressed survival motoneuron (SMN) protein. SMN is crucial component of a complex for the assembly of spliceosomal small nuclear ribonucleoprotein (snRNP) particles. Other cellular functions of SMN are less characterized so far. SMA predominantly affects lower motoneurons, but the cellular basis for this relative specificity is still unknown. In contrast to nonneuronal cells where the protein is mainly localized in perinuclear regions and the nucleus, Smn is also present in dendrites, axons and axonal growth cones of isolated motoneurons in vitro. However, this distribution has not been shown in vivo and it is not clear whether Smn and hnRNP R are also present in presynaptic axon terminals of motoneurons in postnatal mice. Smn also associates with components not included in the classical SMN complex like RNA-binding proteins FUS, TDP43, HuD and hnRNP R which are involved in RNA processing, subcellular localization and translation. We show here that Smn and hnRNP R are present in presynaptic compartments at neuromuscular endplates of embryonic and postnatal mice. Smn and hnRNP R are localized in close proximity to each other in axons and axon terminals both in vitro and in vivo. We also provide new evidence for a direct interaction of Smn and hnRNP R in vitro and in vivo, particularly in the cytosol of motoneurons. These data point to functions of SMN beyond snRNP assembly which could be crucial for recruitment and transport of RNA particles into axons and axon terminals, a mechanism which may contribute to SMA pathogenesis.
Introduction
The neuronal ceroid lipofuscinoses constitute a group of fatal inherited lysosomal storage diseases that manifest in profound neurodegeneration in the CNS. Visual impairment usually is an early symptom and selective degeneration of retinal neurons has been described in patients suffering from distinct disease subtypes. We have previously demonstrated that palmitoyl protein thioesterase 1 deficient (Ppt1-/-) mice, a model of the infantile disease subtype, exhibit progressive axonal degeneration in the optic nerve and loss of retinal ganglion cells, faithfully reflecting disease severity in the CNS. Here we performed spectral domain optical coherence tomography (OCT) in Ppt1-/- and ceroid lipofuscinosis neuronal 3 deficient (Cln3-/-) mice, which are models of infantile and juvenile neuronal ceroid lipofuscinosis, respectively, in order to establish a non-invasive method to assess retinal alterations and monitor disease severity in vivo.
Results
Blue laser autofluorescence imaging revealed increased accumulation of autofluorescent storage material in the inner retinae of 7-month-old Ppt1-/- and of 16-month-old Cln3-/- mice in comparison with age-matched control littermates. Additionally, optical coherence tomography demonstrated reduced thickness of retinae in knockout mice in comparison with age-matched control littermates. High resolution scans and manual measurements allowed for separation of different retinal composite layers and revealed a thinning of layers in the inner retinae of both mouse models at distinct ages. OCT measurements correlated well with subsequent histological analysis of the same retinae.
Conclusions
These results demonstrate the feasibility of OCT to assess neurodegenerative disease severity in mouse models of neuronal ceroid lipofuscinosis and might have important implications for diagnostic evaluation of disease progression and therapeutic efficacy in patients. Moreover, the non-invasive method allows for longitudinal studies in experimental models, reducing the number of animals used for research.
In dorsal root ganglia (DRG) neurons TRESK channels constitute a major current component of the standing outward current IK\(_{SO}\). A prominent physiological role of TRESK has been attributed to pain sensation. During inflammation mediators of pain e.g. lysophosphatidic acid (LPA) are released and modulate nociception. We demonstrate co-expression of TRESK and LPA receptors in DRG neurons. Heterologous expression of TRESK and LPA receptors in Xenopus oocytes revealed augmentation of basal K\(^{+}\) currents upon LPA application. In DRG neurons nociception can result from TRPV\(_{1}\) activation by capsaicin or LPA. Upon co-expression in Xenopus oocytes LPA simultaneously increased both depolarising TRPV\(_{1}\) and hyperpolarising TRESK currents. Patch-clamp recordings in cultured DRG neurons from TRESK[wt] mice displayed increased IK\(_{SO}\) after application of LPA whereas under these conditions IK\(_{SO}\) in neurons from TRESK[ko] mice remained unaltered. Under current-clamp conditions LPA application differentially modulated excitability in these genotypes upon depolarising pulses. Spike frequency was attenuated in TRESK[wt] neurons and, in contrast, augmented in TRESK[ko] neurons. Accordingly, excitation of nociceptive neurons by LPA is balanced by co-activation of TRESK channels. Hence excitation of sensory neurons is strongly controlled by the activity of TRESK channels, which therefore are good candidates for the treatment of pain disorders.
Most RNAs within polarized cells such as neurons are sorted subcellularly in a coordinated manner. Despite advances in the development of methods for profiling polyadenylated RNAs from small amounts of input RNA, techniques for profiling coding and non-coding RNAs simultaneously are not well established. Here, we optimized a transcriptome profiling method based on double-random priming and applied it to serially diluted total RNA down to 10 pg. Read counts of expressed genes were robustly correlated between replicates, indicating that the method is both reproducible and scalable. Our transcriptome profiling method detected both coding and long non-coding RNAs sized >300 bases. Compared to total RNAseq using a conventional approach our protocol detected 70% more genes due to reduced capture of ribosomal RNAs. We used our method to analyze the RNA composition of compartmentalized motoneurons. The somatodendritic compartment was enriched for transcripts with post-synaptic functions as well as for certain nuclear non-coding RNAs such as 7SK. In axons, transcripts related to translation were enriched including the cytoplasmic non-coding RNA 7SL. Our profiling method can be applied to a wide range of investigations including perturbations of subcellular transcriptomes in neurodegenerative diseases and investigations of microdissected tissue samples such as anatomically defined fiber tracts.
1. Zusammenfassung
Während der Embryogenese und nach Verletzungen von Nerven regulieren neurotrophe Faktoren Signalwege für Apoptose, Differenzierung, Wachstum und Regeneration von Neuronen. In vivo Experimente an neugeborenen Nagern haben gezeigt, dass der Verlust von Motoneuronen nach peripherer Nervenläsion durch die Behandlung mit GDNF, BDNF, und CNTF reduziert werden kann In der pmn-Mausmutante, einem Modell für die Amyotrophe Lateralsklerose, führt die Gabe von CNTF, nicht aber von GDNF zu einem verzögerten Krankheitsbeginn und einem verlangsamten Fortschreiten der Motoneuronendegeneration. Auslöser der Motoneuronendegeneration in der pmn-Maus ist eine Mutation im Tubulin spezifischen Chaperon E (Tbce) Gen, das für eines von fünf Tubulin spezifischen Chaperonen (TBCA-TBCE) kodiert und an der Bildung von -Tubulinheterodimeren beteiligt ist. Diese Arbeit sollte dazu beitragen, die CNTF-induzierten Signalwege zu entschlüsseln, die sich lindernd auf den progredienten Verlauf der Motoneuronendegeneration in der pmn-Maus auswirken.
Primäre pmn mutierte Motoneurone zeigen ein reduziertes Axonwachstum und eine erhöhte Anzahl axonaler Schwellungen mit einer anomalen Häufung von Mitochondrien - ein frühes Erkennungsmerkmal bei ALS-Patienten. Die Applikation von CNTF nicht aber von BDNF oder GDNF, kann in vitro die beobachteten Wachstumsdefekte und das bidirektionale axonale Transportdefizit in pmn mutierten Motoneurone verhindern.
Aus älteren Untersuchungen war bekannt, dass CNTF über den dreiteiligen transmembranen Rezeptorkomplex, bestehend aus CNTFR, LIFR und gp130, Januskinasen aktiviert, die STAT3 an Tyrosin 705 phosphorylieren (pSTAT3Y705). Ich konnte beobachten, dass axonales fluoreszenzmarkiertes pSTAT3Y705 nach CNTF-Gabe nicht retrograd in den Nukleus transportiert wird. Stattdessen führt die CNTF-induzierte Phosphorylierung von STAT3 an Tyrosin 705 zu einer transkriptionsunabhängigen lokalen Reaktion im Axon. Diese pSTAT3Y705 abhängige Reaktion ist notwendig und ausreichend, um das reduzierte Axonwachstum pmn mutierter Motoneurone zu beheben. Wie die Kombination einer CNTF Behandlung mit dem shRNA vermittelten knock-down von Stathmin in pmn mutierten Motoneuronen zeigt, zielt die CNTF-STAT3 Signalkaskade auf die Stabilisierung axonaler Mikrotubuli ab und wirkt sich positiv auf die anterograde und retrograde Mobilität von axonalen Mitochondrien aus.
Interessanter Weise konnte ich außerdem feststellen, dass eine akute Gabe von CNTF das mitochondriale Membranpotential in Axonen primärer pmn mutierter und wildtypischer
Motoneurone erhöht und einen Anstieg von ATP auslöst. Meine Beobachtungen legen nahe, dass CNTF unerwarteter Weise auch eine transiente Phosphorylierung an STAT3 Serin 727 (pSTAT3S727) auslöst, die zur anschließenden Translokation von pSTAT3S727 in Mitochondrien führt. Diese Ergebnisse zeigen, dass STAT3 mehrere lokale Ziele im Axon besitzt, nämlich axonale Mikrotubuli und Mitochondrien.
Ligand-binding of Cys-loop receptors is determined by N-terminal extracellular loop structures from the plus as well as from the minus side of two adjacent subunits in the pentameric receptor complex. An aromatic residue in loop B of the glycine receptor (GIyR) undergoes direct interaction with the incoming ligand via a cation-π interaction. Recently, we showed that mutated residues in loop B identified from human patients suffering from hyperekplexia disturb ligand-binding. Here, we exchanged the affected human residues by amino acids found in related members of the Cys-loop receptor family to determine the effects of side chain volume for ion channel properties. GIyR variants were characterized in vitro following transfection into cell lines in order to analyze protein expression, trafficking, degradation and ion channel function. GIyR α1 G160 mutations significantly decrease glycine potency arguing for a positional effect on neighboring aromatic residues and consequently glycine-binding within the ligand-binding pocket. Disturbed glycinergic inhibition due to T162 α1 mutations is an additive effect of affected biogenesis and structural changes within the ligand-binding site. Protein trafficking from the ER toward the ER-Golgi intermediate compartment, the secretory Golgi pathways and finally the cell surface is largely diminished, but still sufficient to deliver ion channels that are functional at least at high glycine concentrations. The majority of T162 mutant protein accumulates in the ER and is delivered to ER-associated proteasomal degradation. Hence, G160 is an important determinant during glycine binding. In contrast, 1162 affects primarily receptor biogenesis whereas exchanges in functionality are secondary effects thereof.
Der zur Familie der pentameren ligandengesteuerten Ionenkanäle zugehörige Glycinrezeptor (GlyR) ist ein wichtiger Vermittler synaptischer Inhibition im Zentralnervensystem von Säugetieren. GlyR-Mutationen führen zur neurologischen Bewegungsstörung Hyperekplexie. Aufgrund fehlender struktureller Daten ist die intrazelluläre Loop-Struktur zwischen den Transmembransegmenten 3 und 4 (TM3-4 Loop) eine weitgehend unerforschte Domäne des GlyR. Innerhalb dieser Domäne wurden Rezeptortrunkierungen sowie Punktmutationen identifiziert. Rezeptortrunkierung geht mit Funktionslosigkeit einher, welche jedoch durch Koexpression des fehlenden Sequenzabschnitts zum Teil wiederhergestellt werden kann. Innerhalb dieser Arbeit wurde die Interaktion zwischen trunkierten, funktionslosen GlyR und sukzessiv verkürzten Komplementationskonstrukten untersucht. Dabei wurden als Minimaldomänen für die Interaktion das C-terminalen basische Motive des TM3-4 Loops, die TM4 sowie der extrazelluläre C-Terminus identifiziert. Die Rückkreuzung transgener Mäuse, die das Komplementationskonstrukt iD-TM4 unter Kontrolle des GlyR-Promotors exprimierten, mit der oscillator-Maus spdot, die einen trunkierten GlyR exprimiert und 3 Wochen nach der Geburt verstirbt, hatte aufgrund fehlender Proteinexpression keinen Effekt auf die Letalität der Mutation. Des Weiteren wurde die Bedeutsamkeit der Integrität beider basischer Motive 316RFRRKRR322 und 385KKIDKISR392 im TM3-4 Loop in Kombination mit der Loop-Länge für die Funktionalität und das Desensitisierungsverhalten des humanen GlyRα1 anhand von chimären Rezeptoren identifiziert. Eine bisher unbekannte Patientenmutation P366L innerhalb des TM3-4 Loops wurde mit molekularbiologischen, biochemischen und elektrophysiologischen Methoden charakterisiert. Es wurde gezeigt, dass die mutierten Rezeptorkomplexe in vitro deutlich reduzierte Glycin-induzierte Maximalströme sowie eine beschleunigte Schließkinetik aufweisen. P366L hat im Gegensatz zu bereits charakterisierten Hyperekplexiemutationen innerhalb des TM3-4 Loops keinen Einfluss auf die Biogenese des Rezeptors. P366 ist Teil einer möglichen Poly-Prolin-Helix, die eine Erkennungssequenz für SH3-Domänen darstellt. Ein potenzieller Interaktionspartner des TM3-4 Loops des GlyRα1 ist Collybistin, welches eine wichtige Rolle bei der synaptischen Rezeptorintegration spielt und die Verbindung zum Zytoskelett vermittelt. An der inhibitorischen Synapse verursacht P366L durch die Reduzierung postsynaptischer Chloridströme, das beschleunigte Desensitisierungsverhalten des GlyRα1 sowie ein verändertes Interaktionsmotiv Störungen der glycinergen Transmission, die zur Ausprägung phänotypischer Symptome der Hyperekplexie führen.
Synapse-associated protein 1 (Syap1/BSTA) is the mammalian homologue of Sap47 (synapse-associated protein of 47 kDa) in Drosophila. Sap47 null mutant larvae show reduced short-term synaptic plasticity and a defect in associative behavioral plasticity. In cultured adipocytes, Syap1 functions as part of a complex that phosphorylates protein kinase B alpha/Akt1 (Akt1) at Ser\(^{473}\) and promotes differentiation. The role of Syap1 in the vertebrate nervous system is unknown. Here, we generated a Syap1 knock-out mouse and show that lack of Syap1 is compatible with viability and fertility. Adult knock-out mice show no overt defects in brain morphology. In wild-type brain, Syap1 is found widely distributed in synaptic neuropil, notably in regions rich in glutamatergic synapses, but also in perinuclear structures associated with the Golgi apparatus of specific groups of neuronal cell bodies. In cultured motoneurons, Syap1 is located in axons and growth cones and is enriched in a perinuclear region partially overlapping with Golgi markers. We studied in detail the influence of Syap1 knockdown and knockout on structure and development of these cells. Importantly, Syap1 knockout does not affect motoneuron survival or axon growth. Unexpectedly, neither knockdown nor knockout of Syap1 in cultured motoneurons is associated with reduced Ser\(^{473}\) or Thr\(^{308}\) phosphorylation of Akt. Our findings demonstrate a widespread expression of Syap1 in the mouse central nervous system with regionally specific distribution patterns as illustrated in particular for olfactory bulb, hippocampus, and cerebellum.
High-resolution ultrasonography (HRUS) is an emerging new tool in the investigation of peripheral nerves. We set out to assess the utility of HRUS performed at lower extremity nerves in peripheral neuropathies. Nerves of 26 patients with polyneuropathies of different etiologies and 26 controls were investigated using HRUS. Patients underwent clinical, laboratory, electrophysiological assessment, and a diagnostic sural nerve biopsy as part of the routine work-up. HRUS was performed at the sural, tibial, and the common, superficial, and deep peroneal nerves. The superficial peroneal nerve longitudinal diameter (LD) distinguished best between the groups: patients with immune-mediated neuropathies (n = 13, including six with histology-proven vasculitic neuropathy) had larger LD compared to patients with non-immune-mediated neuropathies (p < 0.05) and to controls (p < 0.001). Among all subgroups, patients with vasculitic neuropathy showed the largest superficial peroneal nerve LD (p < 0.001) and had a larger sural nerve cross-sectional area when compared with disease controls (p < 0.001). Enlargement of the superficial peroneal and sural nerves as detected by HRUS may be a useful additional finding in the differential diagnosis of vasculitic and other immune-mediated neuropathies.
Synaptopathies: synaptic dysfunction in neurological disorders - a review from students to students
(2016)
Synapses are essential components of neurons and allow information to travel coordinately throughout the nervous system to adjust behavior to environmental stimuli and to control body functions, memories, and emotions. Thus, optimal synaptic communication is required for proper brain physiology, and slight perturbations of synapse function can lead to brain disorders. In fact, increasing evidence has demonstrated the relevance of synapse dysfunction as a major determinant of many neurological diseases. This notion has led to the concept of synaptopathies as brain diseases with synapse defects as shared pathogenic features. In this review, which was initiated at the 13th International Society for Neurochemistry Advanced School, we discuss basic concepts of synapse structure and function, and provide a critical view of how aberrant synapse physiology may contribute to neurodevelopmental disorders (autism, Down syndrome, startle disease, and epilepsy) as well as neurodegenerative disorders (Alzheimer and Parkinson disease). We finally discuss the appropriateness and potential implications of gathering synapse diseases under a single term. Understanding common causes and intrinsic differences in disease-associated synaptic dysfunction could offer novel clues toward synapse-based therapeutic intervention for neurological and neuropsychiatric disorders. In this Review, which was initiated at the 13th International Society for Neurochemistry (ISN) Advanced School, we discuss basic concepts of synapse structure and function, and provide a critical view of how aberrant synapse physiology may contribute to neurodevelopmental (autism, Down syndrome, startle disease, and epilepsy) as well as neurodegenerative disorders (Alzheimer's and Parkinson's diseases), gathered together under the term of synaptopathies. Read the Editorial Highlight for this article on page .
Animal models point towards a key role of brain-derived neurotrophic factor (BDNF), insulin-like growth factor-I (IGF-I) and vascular endothelial growth factor (VEGF) in mediating exercise-induced structural and functional changes in the hippocampus. Recently, also platelet derived growth factor-C (PDGF-C) has been shown to promote blood vessel growth and neuronal survival. Moreover, reductions of these neurotrophic and angiogenic factors in old age have been related to hippocampal atrophy, decreased vascularization and cognitive decline. In a 3-month aerobic exercise study, forty healthy older humans (60 to 77 years) were pseudo-randomly assigned to either an aerobic exercise group (indoor treadmill, n = 21) or to a control group (indoor progressive-muscle relaxation/stretching, n = 19). As reported recently, we found evidence for fitness-related perfusion changes of the aged human hippocampus that were closely linked to changes in episodic memory function. Here, we test whether peripheral levels of BDNF, IGF-I, VEGF or PDGF-C are related to changes in hippocampal blood flow, volume and memory performance. Growth factor levels were not significantly affected by exercise, and their changes were not related to changes in fitness or perfusion. However, changes in IGF-I levels were positively correlated with hippocampal volume changes (derived by manual volumetry and voxel-based morphometry) and late verbal recall performance, a relationship that seemed to be independent of fitness, perfusion or their changes over time. These preliminary findings link IGF-I levels to hippocampal volume changes and putatively hippocampus-dependent memory changes that seem to occur over time independently of exercise. We discuss methodological shortcomings of our study and potential differences in the temporal dynamics of how IGF-1, VEGF and BDNF may be affected by exercise and to what extent these differences may have led to the negative findings reported here.
Physical exercise can convey a protective effect against cognitive decline in ageing and Alzheimer’s disease. While the long-term health-promoting and protective effects of exercise are encouraging, it’s potential to induce neuronal and vascular plasticity in the ageing brain is still poorly understood. It remains unclear whether exercise slows the trajectory of normal ageing by modifying vascular and metabolic risk factors and/or consistently boosts brain function by inducing structural and neurochemical changes in the hippocampus and related medial temporal lobe circuitry—brain areas that are important for learning and memory. Hence, it remains to be established to what extent exercise interventions in old age can improve brain plasticity above and beyond preservation of function. Existing data suggest that exercise trials aiming for improvement and preservation may require different outcome measures and that the balance between the two may depend on exercise intensity and duration, the presence of preclinical Alzheimer’s disease pathology, vascular and metabolic risk factors and genetic variability.
The family of Cys-loop receptors (CLRs) shares a high degree of homology and sequence identity. The overall structural elements are highly conserved with a large extracellular domain (ECD) harboring an α-helix and 10 β-sheets. Following the ECD, four transmembrane domains (TMD) are connected by intracellular and extracellular loop structures. Except the TM3–4 loop, their length comprises 7–14 residues. The TM3–4 loop forms the largest part of the intracellular domain (ICD) and exhibits the most variable region between all CLRs. The ICD is defined by the TM3–4 loop together with the TM1–2 loop preceding the ion channel pore. During the last decade, crystallization approaches were successful for some members of the CLR family. To allow crystallization, the intracellular loop was in most structures replaced by a short linker present in prokaryotic CLRs. Therefore, no structural information about the large TM3–4 loop of CLRs including the glycine receptors (GlyRs) is available except for some basic stretches close to TM3 and TM4. The intracellular loop has been intensively studied with regard to functional aspects including desensitization, modulation of channel physiology by pharmacological substances, posttranslational modifications, and motifs important for trafficking. Furthermore, the ICD interacts with scaffold proteins enabling inhibitory synapse formation. This review focuses on attempts to define structural and functional elements within the ICD of GlyRs discussed with the background of protein-protein interactions and functional channel formation in the absence of the TM3–4 loop.
Synapsin is an evolutionarily conserved presynaptic phosphoprotein. It is encoded by only one gene in the Drosophila genome and is expressed throughout the nervous system. It regulates the balance between reserve and releasable vesicles, is required to maintain transmission upon heavy demand, and is essential for proper memory function at the behavioral level. Task-relevant sensorimotor functions, however, remain intact in the absence of Synapsin. Using an odor-sugar reward associative learning paradigm in larval Drosophila, we show that memory scores in mutants lacking Synapsin (syn\(^{97}\)) are lower than in wild-type animals only when more salient, higher concentrations of odor or of the sugar reward are used. Furthermore, we show that Synapsin is selectively required for larval short-term memory. Thus, without Synapsin Drosophila larvae can learn and remember, but Synapsin is required to form memories that match in strength to event salience-in particular to a high saliency of odors, of rewards, or the salient recency of an event. We further show that the residual memory scores upon a lack of Synapsin are not further decreased by an additional lack of the Sap47 protein. In combination with mass spectrometry data showing an up-regulated phosphorylation of Synapsin in the larval nervous system upon a lack of Sap47, this is suggestive of a functional interdependence of Synapsin and Sap47.
Neurofilament depletion improves microtubule dynamics via modulation of Stat3/stathmin signaling
(2016)
In neurons, microtubules form a dense array within axons, and the stability and function of this microtubule network is modulated by neurofilaments. Accumulation of neurofilaments has been observed in several forms of neurodegenerative diseases, but the mechanisms how elevated neurofilament levels destabilize axons are unknown so far. Here, we show that increased neurofilament expression in motor nerves of pmn mutant mice, a model of motoneuron disease, causes disturbed microtubule dynamics. The disease is caused by a point mutation in the tubulin-specific chaperone E (Tbce) gene, leading to an exchange of the most C-terminal amino acid tryptophan to glycine. As a consequence, the TBCE protein becomes instable which then results in destabilization of axonal microtubules and defects in axonal transport, in particular in motoneurons. Depletion of neurofilament increases the number and regrowth of microtubules in pmn mutant motoneurons and restores axon elongation. This effect is mediated by interaction of neurofilament with the stathmin complex. Accumulating neurofilaments associate with stathmin in axons of pmn mutant motoneurons. Depletion of neurofilament by Nefl knockout increases Stat3-stathmin interaction and stabilizes the microtubules in pmn mutant motoneurons. Consequently, counteracting enhanced neurofilament expression improves axonal maintenance and prolongs survival of pmn mutant mice. We propose that this mechanism could also be relevant for other neurodegenerative diseases in which neurofilament accumulation and loss of microtubules are prominent features.
The synapse-associated protein of 47 kDa (Sap47) in Drosophila melanogaster is the founding member of a phylogenetically conserved protein family of hitherto unknown molecular function. Sap47 is localized throughout the entire neuropil of adult and larval brains and closely associated with glutamatergic presynaptic vesicles of larval motoneurons. Flies lacking the protein are viable and fertile and do not exhibit gross structural or marked behavioral deficiencies indicating that Sap47 is dispensable for basic synaptic function, or that its function is compensated by other related proteins.
Syap1 - the mammalian homologue of Sap47 - was reported to play an essential role in Akt1 phosphorylation in various non-neuronal cells by promoting the association of mTORC2 with Akt1 which is critical for the downstream signaling cascade for adipogenesis. The function of Syap1 in the vertebrate nervous system, however, is unknown so far.
The present study provides a first description of the subcellular localization of mouse Syap1 in cultured motoneurons as well as in selected structures of the adult mouse nervous system and reports initial functional experiments. Preceding all descriptive experiments, commercially available Syap1 antibodies were tested for their specificity and suitability for this study. One antibody raised against the human protein was found to recognize specifically both the human and murine Syap1 protein, providing an indispensable tool for biochemical, immunocytochemical and immunohistochemical studies.
In the course of this work, a Syap1 knockout mouse was established and investigated. These mice are viable and fertile and do not show obvious changes in morphology or phenotype. As observed for Sap47 in flies, Syap1 is widely distributed in the synaptic neuropil, particularly in regions rich in glutamatergic synapses but it was also detected at perinuclear Golgi-associated sites in certain groups of neuronal somata. In motoneurons the protein is especially observed in similar perinuclear structures, partially overlapping with Golgi markers and in axons, dendrites and axonal growth cones. Biochemical and immunohistochemical analyses showed widespread Syap1 expression in the central nervous system with regionally distinct distribution patterns in cerebellum, hippocampus or olfactory bulb. Besides its expression in neurons, Syap1 is also detected in non-neuronal tissue e.g. liver, kidney and muscle tissue. In contrast, non-neuronal cells in the brain lack the typical perinuclear accumulation.
First functional studies with cultured primary motoneurons on developmental, structural and functional aspects reveal no influence of Syap1 depletion on survival and morphological features such as axon length or dendritic length. Contrary to expectations, in neuronal tissues or cultured motoneurons a reduction of Akt phosphorylation at Ser473 or Thr308 was not detected after Syap1 knockdown or knockout.
In highly polarized cells like neurons, cytoskeleton dynamics play a crucial role in establishing neuronal connections during development and are required for adult plasticity. Actin turnover is particularly important for neurite growth, axon path finding, branching and synaptogenesis. Motoneurons establish several thousand branches that innervate neuromuscular synapses (NMJs). Axonal branching and terminal arborization are fundamental events during the establishment of synapses in motor endplates. Branching process is triggered by the assembly of actin filaments along the axon shaft giving rise to filopodia formation. The unique contribution of the three actin isoforms, α-, β- and γ-actin, in filopodia stability and dynamics during this process is not well characterized. Here, we performed high resolution in situ hybridization and qRT-PCR and showed that in primary mouse motoneurons α-, β- and γ-actin isoforms are expressed and their transcripts are translocated into axons. Using FRAP experiments, we showed that transcripts for α-, β- and γ-actin become locally translated in axonal growth cones and translation hot spots of the axonal branch points. Using live cell imaging, we showed that shRNA depletion of α-actin reduces dynamics of axonal filopodia which correlates with reduced number of collateral branches and impairs axon elongation. Depletion of β-actin correlates with reduced dynamics of growth cone filopoida, disturbs axon elongation and impairs presynaptic differentiation. Also, depletion of γ-actin impairs axonal growth and decreases axonal filopodia dynamics. These findings implicate that actin isoforms accomplish unique functions during development of motor axons. Depletions of β- and γ-actin lead to compensatory upregulation of other two isoforms. Consistent with this, total actin levels remain unaltered and F-actin polymerization capacity is preserved. After the knockdown of either α- or γ-actin, the levels of β-actin increase in the G-actin pool indicating that polymerization and stability of β-actin filaments depend on α- or γ-actin. This study provides evidence both for unique and overlapping function of actin isoforms in motoneuron growth and differentiation. In the soma of developing motoneurons, actin isoforms act redundantly and thus could compensate for each other’s loss. In the axon, α-, β- and γ-actin accomplish specific functions, i.e. β-actin regulates axon elongation and plasticity and α- and γ-actin regulate axonal branching.
Furthermore, we show that both axonal transport and local translation of α-, β- and γ-actin isoforms are impaired in Smn knockout motoneurons, indicating a role for Smn protein in RNA granule assembly and local translation of these actin isoforms in primary mouse motoneurons.
Spinal muscular atrophy and amyotrophic lateral sclerosis are the two most common devastating motoneuron diseases. The mechanisms leading to motoneuron degeneration are not resolved so far, although different hypotheses have been built on existing data. One possible mechanism is disturbed axonal transport of RNAs in the affected motoneurons. The underlying question of this study was therefore to characterize changes in transcript levels of distinct RNAs in cell culture models of spinal muscular atrophy and amyotrophic lateral sclerosis, especially in the axonal compartment of primary motoneurons.
To investigate this in detail we first established compartmentalized cultures of Primary mouse motoneurons. Subsequently, total RNA of both compartments was extracted
separately and either linearly amplified and subjected to microarray profiling or whole transcriptome amplification followed by RNA-Sequencing was performed. To make
the whole transcriptome amplification method suitable for compartmentalized cultures, we adapted a double-random priming strategy. First, we applied this method
for initial optimization onto serial dilutions of spinal cord RNA and later on to the compartmentalized motoneurons.
Analysis of the data obtained from wildtype cultures already revealed interesting results. First, the RNA composition of axons turned out to be highly similar to the somatodendritic compartment. Second, axons seem to be particularly enriched for transcripts related to protein synthesis and energy production. In a next step we
repeated the experiments by using knockdown cultures. The proteins depleted hereby are Smn, Tdp-43 and hnRNP R. Another experiment was performed by knocking down the non-coding RNA 7SK, the main interacting RNA of hnRNP R.
Depletion of Smn led to a vast number of deregulated transcripts in the axonal and somatodendritic compartment. Transcripts downregulated in the axons upon Smn depletion were especially enriched for GOterms related to RNA processing and encode proteins located in neuron projections including axons and growth cones.
Strinkingly, among the upregulated transcripts in the somatodendritic compartment we mainly found MHC class I transcripts suggesting a potential neuroprotective role.
In contrast, although knockdown of Tdp-43 also revealed a large number of downregulated transcripts in the axonal compartment, these transcripts were mainly
associated with functions in transcriptional regulation and RNA splicing. For the hnRNP R knockdown our results were again different. Here, we observed
downregulated transcripts in the axonal compartment mainly associated with regulation of synaptic transmission and nerve impulses. Interestingly, a comparison between deregulated transcripts in the axonal compartment of both hnRNP R and 7SK knockdown presented a significant overlap of several transcripts suggesting
some common mechanism for both knockdowns.
Thus, our data indicate that a loss of disease-associated proteins involved in axonal RNA transport causes distinct transcriptome alterations in motor axons.
Background: During early prenatal stages of brain development, serotonin (5-HT)-specific neurons migrate through somal translocation to form the raphe nuclei and subsequently begin to project to their target regions. The rostral cluster of cells, comprising the median and dorsal raphe (DR), innervates anterior regions of the brain, including the prefrontal cortex. Differential analysis of the mouse 5-HT system transcriptome identified enrichment of cell adhesion molecules in 5-HT neurons of the DR. One of these molecules, cadherin-13 (Cdh13) has been shown to play a role in cell migration, axon pathfinding, and synaptogenesis. This study aimed to investigate the contribution of Cdh13 to the development of the murine brain 5-HT system.
Methods: For detection of Cdh13 and components of the 5-HT system at different embryonic developmental stages of the mouse brain, we employed immunofluorescence protocols and imaging techniques, including epifluorescence, confocal and structured illumination microscopy. The consequence of CDH13 loss-of-function mutations on brain 5-HT system development was explored in a mouse model of Cdh13 deficiency.
Results: Our data show that in murine embryonic brain Cdh13 is strongly expressed on 5-HT specific neurons of the DR and in radial glial cells (RGCs), which are critically involved in regulation of neuronal migration. We observed that 5-HT neurons are intertwined with these RGCs, suggesting that these neurons undergo RGC-guided migration. Cdh13 is present at points of intersection between these two cell types. Compared to wildtype controls, Cdh13-deficient mice display increased cell densities in the DR at embryonic stages E13.5, E17.5, and adulthood, and higher serotonergic innervation of the prefrontal cortex at E17.5.
Conclusion: Our findings provide evidence for a role of CDH13 in the development of the serotonergic system in early embryonic stages. Specifically, we indicate that Cdh13 deficiency affects the cell density of the developing DR and the posterior innervation of the prefrontal cortex (PFC), and therefore might be involved in the migration, axonal outgrowth and terminal target finding of DR 5-HT neurons. Dysregulation of CDH13 expression may thus contribute to alterations in this system of neurotransmission, impacting cognitive function, which is frequently impaired in neurodevelopmental disorders including attention-deficit/hyperactivity and autism spectrum disorders.
Phospholipids occurring in cell membranes and lipoproteins are converted into oxidized phospholipids (OxPL) by oxidative stress promoting atherosclerotic plaque formation. Here, OxPL were characterized as novel targets in acute and chronic inflammatory pain. Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) and its derivatives were identified in inflamed tissue by mass spectrometry and binding assays. They elicited calcium influx, hyperalgesia and induced pro-nociceptive peptide release. Genetic, pharmacological and mass spectrometric evidence in vivo as well as in vitro confirmed the role of transient receptor potential channels (TRPA1 and TRPV1) as OxPAPC targets. Treatment with the monoclonal antibody E06 or with apolipoprotein A-I mimetic peptide D-4F, capturing OxPAPC in atherosclerosis, prevented inflammatory hyperalgesia, and in vitro TRPA1 activation. Administration of D-4F or E06 to rats profoundly ameliorated mechanical hyperalgesia and inflammation in collagen-induced arthritis. These data reveal a clinically relevant role for OxPAPC in inflammation offering therapy for acute and chronic inflammatory pain treatment by scavenging OxPAPC.
Spontaneous Ca\(^{2+}\) transients and actin dynamics in primary motoneurons correspond to cellular differentiation such as axon elongation and growth cone formation. Brain-derived neurotrophic factor (BDNF) and its receptor trkB support both motoneuron survival and synaptic differentiation. However, in motoneurons effects of BDNF/trkB signaling on spontaneous Ca\(^{2+}\) influx and actin dynamics at axonal growth cones are not fully unraveled. In our study we addressed the question how neurotrophic factor signaling corresponds to cell autonomous excitability and growth cone formation. Primary motoneurons from mouse embryos were cultured on the synapse specific, β2-chain containing laminin isoform (221) regulating axon elongation through spontaneous Ca\(^{2+}\) transients that are in turn induced by enhanced clustering of N-type specific voltage-gated Ca\(^{2+}\) channels (Ca\(_{v}\)2.2) in axonal growth cones. TrkB-deficient (trkBTK\(^{-/-}\)) mouse motoneurons which express no full-length trkB receptor and wildtype motoneurons cultured without BDNF exhibited reduced spontaneous Ca\(^{2+}\) transients that corresponded to altered axon elongation and defects in growth cone morphology which was accompanied by changes in the local actin cytoskeleton. Vice versa, the acute application of BDNF resulted in the induction of spontaneous Ca\(^{2+}\) transients and Ca\(_{v}\)2.2 clustering in motor growth cones, as well as the activation of trkB downstream signaling cascades which promoted the stabilization of β-actin via the LIM kinase pathway and phosphorylation of profilin at Tyr129. Finally, we identified a mutual regulation of neuronal excitability and actin dynamics in axonal growth cones of embryonic motoneurons cultured on laminin-221/211. Impaired excitability resulted in dysregulated axon extension and local actin cytoskeleton, whereas upon β-actin knockdown Ca\(_{v}\)2.2 clustering was affected. We conclude from our data that in embryonic motoneurons BDNF/trkB signaling contributes to axon elongation and growth cone formation through changes in the local actin cytoskeleton accompanied by increased Ca\(_{v}\)2.2 clustering and local calcium transients. These findings may help to explore cellular mechanisms which might be dysregulated during maturation of embryonic motoneurons leading to motoneuron disease.
Terpenoids are major subcomponents in aroma substances which harbor sedative physiological potential. We have demonstrated that various monoterpenoids such as the acyclic linalool enhance GABAergic currents in an allosteric manner in vitro upon overexpression of inhibitory α1β2 GABA\(_{A}\) receptors in various expression systems. However, in plants or humans, i.e., following intake via inhalation or ingestion, linalool undergoes metabolic modifications including oxygenation and acetylation, which may affect the modulatory efficacy of the generated linalool derivatives. Here, we analyzed the modulatory potential of linalool derivatives at α1β2γ2 GABA\(_{A}\) receptors upon transient overexpression. Following receptor expression control, electrophysiological recordings in a whole cell configuration were used to determine the chloride influx upon co-application of GABA EC\(_{10-30}\) together with the modulatory substance. Our results show that only oxygenated linalool metabolites at carbon 8 positively affect GABAergic currents whereas derivatives hydroxylated or carboxylated at carbon 8 were rather ineffective. Acetylated linalool derivatives resulted in non-significant changes of GABAergic currents. We can conclude that metabolism of linalool reduces its positive allosteric potential at GABAA receptors compared to the significant potentiation effects of the parent molecule linalool itself.
Autophagy-mediated degradation of synaptic components maintains synaptic homeostasis but also constitutes a mechanism of neurodegeneration. It is unclear how autophagy of synaptic vesicles and components of presynaptic active zones is regulated. Here, we show that Pleckstrin homology containing family member 5 (Plekhg5) modulates autophagy of synaptic vesicles in axon terminals of motoneurons via its function as a guanine exchange factor for Rab26, a small GTPase that specifically directs synaptic vesicles to preautophagosomal structures. Plekhg5 gene inactivation in mice results in a late-onset motoneuron disease, characterized by degeneration of axon terminals. Plekhg5-depleted cultured motoneurons show defective axon growth and impaired autophagy of synaptic vesicles, which can be rescued by constitutively active Rab26. These findings define a mechanism for regulating autophagy in neurons that specifically targets synaptic vesicles. Disruption of this mechanism may contribute to the pathophysiology of several forms of motoneuron disease.
Die spinale Muskelatrophie ist eine monogenetische Erkrankung, die bereits im Kindesalter aufgrund von Motoneurondegeneration zu Muskelatrophie führt und nicht selten einen tödlichen Verlauf nimmt. Ursache der Erkrankung ist ein Mangel an SMN-Protein. Der hierfür verantwortliche Verlust des SMN1-Gens kann durch das SMN2-Gen aufgrund eines gestörten Spleißprozesses am Exon 7 nicht kompensiert werden. Neben Aufgaben in der RNA-Prozessierung wird das SMN-Protein für den axonalen Transport von Ribonucleinpartikeln in Motoneuronen benötigt, was bei der SMA zu pathologischem Wachstum, Differenzierung und Funktion der Motoraxone führt. Im Rahmen dieser Arbeit wurden kultivierte Motoneurone aus einem Mausmodell für die SMA Typ I (Genotyp Smn-/-;SMN2) mit zwei unterschiedlichen Substanzen behandelt und deren Wirkungen auf das präsynaptische Differenzierungsverhalten der Motoneurone verglichen: R-Roscovitine, ein Agonist/Modulator spannungsabhängiger N-Typ- und P/Q-Typ-Kalziumkanäle, welcher zudem eine CDK-inhibierende Wirkung besitzt, sowie Valproat, ein HDAC-Inhibitor, der eine stimulierende Wirkung auf die SMN-Transkription hat. Es zeigte sich, dass R-Roscovitine in der Lage ist, das pathologische Wachstums- und präsynaptische Differenzierungsverhalten der Smn-defizienten Motoneurone zu normalisieren, ohne hierbei Einfluss auf die erniedrigte Menge an Smn-Protein zu nehmen. Die Behandlung mit Valproat beeinflusst hingegen weder die Menge an Smn-Protein, noch die pathologische Differenzierung der Wachstumskegel Smn-defizienter Motoneurone. Erklären lassen sich diese Effekte in erster Linie durch den Agonismus an spannungsabhängigen Kalziumkanälen durch R-Roscovitine. Durch vermehrten Kalziumeinstrom kommt es zur Normalisierung von Struktur und Funktion der Wachstumskegel. Ein CDK-vermittelter Effekt scheint unwahrscheinlich. Obgleich die genauen Vorgänge noch nicht verstanden sind, zeigen diese Ergebnisse, dass sich Smn-defiziente Motoneurone normal entwickeln können, wenn die hierfür erforderlichen kalziumabhängigen präsynaptischen Differenzierungssignale korrekt ausgelöst werden. Bei weiterer Erforschung sind Therapeutika denkbar, die in Zukunft die überwiegend genetisch orientierten Therapieansätze zur Hochregulation der SMN-Expression bei SMA-Patienten über einen von der Genetik unabhängigen Wirkmechanismus unterstützen können.
Ionotropic glycine receptors (GlyRs) enable fast synaptic neurotransmission in the adult spinal cord and brainstem. The inhibitory GlyR is a transmembrane glycinegated chloride channel. The immature GlyR protein undergoes various processing steps, e.g., folding, assembly, and maturation while traveling from the endoplasmic reticulum to and through the Golgi apparatus, where post-translational modifications, e.g., glycosylation occur. The mature receptors are forward transported via microtubules to the cellular surface and inserted into neuronal membranes followed by synaptic clustering. The normal life cycle of a receptor protein includes further processes like internalization, recycling, and degradation. Defects in GlyR life cycle, e.g., impaired protein maturation and degradation have been demonstrated to underlie pathological mechanisms of various neurological diseases. The neurological disorder startle disease is caused by glycinergic dysfunction mainly due to missense mutations in genes encoding GlyR subunits (GLRA1 and GLRB). In vitro studies have shown that most recessive forms of startle disease are associated with impaired receptor biogenesis. Another neurological disease with a phenotype similar to startle disease is a special form of stiff-person syndrome (SPS), which is most probably due to the development of GlyR autoantibodies. Binding of GlyR autoantibodies leads to enhanced receptor internalization. Here we focus on the normal life cycle of GlyRs concentrating on assembly and maturation, receptor trafficking, post-synaptic integration and clustering, and GlyR internalization/recycling/degradation. Furthermore, this review highlights findings on impairment of these processes under disease conditions such as disturbed neuronal ER-Golgi trafficking as the major pathomechanism for recessive forms of human startle disease. In SPS, enhanced receptor internalization upon autoantibody binding to the GlyR has been shown to underlie the human pathology. In addition, we discuss how the existing mouse models of startle disease increased our current knowledge of GlyR trafficking routes and function. This review further illuminates receptor trafficking of GlyR variants originally identified in startle disease patients and explains changes in the life cycle of GlyRs in patients with SPS with respect to structural and functional consequences at the receptor level.
Mutations in GlyR α1 or β subunit genes in humans and rodents lead to severe startle disease characterized by rigidity, massive stiffness and excessive startle responses upon unexpected tactile or acoustic stimuli. The recently characterized startle disease mouse mutant shaky carries a missense mutation (Q177K) in the β8-β9 loop within the large extracellular N-terminal domain of the GlyR α1 subunit. This results in a disrupted hydrogen bond network around K177 and faster GlyR decay times. Symptoms in mice start at postnatal day 14 and increase until premature death of homozygous shaky mice around 4–6 weeks after birth. Here we investigate the in vivo functional effects of the Q177K mutation using behavioral analysis coupled to protein biochemistry and functional assays. Western blot analysis revealed GlyR α1 subunit expression in wild-type and shaky animals around postnatal day 7, a week before symptoms in mutant mice become obvious. Before 2 weeks of age, homozygous shaky mice appeared healthy and showed no changes in body weight. However, analysis of gait and hind-limb clasping revealed that motor coordination was already impaired. Motor coordination and the activity pattern at P28 improved significantly upon diazepam treatment, a pharmacotherapy used in human startle disease. To investigate whether functional deficits in glycinergic neurotransmission are present prior to phenotypic onset, we performed whole-cell recordings from hypoglossal motoneurons (HMs) in brain stem slices from wild-type and shaky mice at different postnatal stages. Shaky homozygotes showed a decline in mIPSC amplitude and frequency at P9-P13, progressing to significant reductions in mIPSC amplitude and decay time at P18-24 compared to wild-type littermates. Extrasynaptic GlyRs recorded by bath-application of glycine also revealed reduced current amplitudes in shaky mice compared to wild-type neurons, suggesting that presynaptic GlyR function is also impaired. Thus, a distinct, but behaviorally ineffective impairment of glycinergic synapses precedes the symptoms onset in shaky mice. These findings extend our current knowledge on startle disease in the shaky mouse model in that they demonstrate how the progression of GlyR dysfunction causes, with a delay of about 1 week, the appearance of disease symptoms.
Neuste Studien haben ergeben, dass Asc-1 Knock-out Mäuse aufgrund einer verminderten intrazellulären Glycinkonzentration in synaptischen Boutons im Gehirn, einen Hyperekplexie-ähnlichen Phänotyp entwickeln. Aufgrund nicht vollständig geklärter Ursachen für die Entstehung des Krankheitsbildes der Hyperekplexie beim Menschen, wurde eine Kohorte von 51 Patienten zusammengetragen, um vor dem Hintergrund der Forschungsergebnisse zu Asc-1 im Tiermodell, das kodierende Gen beim Menschen SLC7A10 als mögliches Kandidatengen auf Sequenzalterationen zu untersuchen. Hierfür wurde aus Vollblut der an Hyperekplexie erkrankten Patienten genomische DNA isoliert, um mittels PCR und anschließendem Screening der Sequenzen, Mutationen innerhalb funktionell wichtiger Bereiche des Gens zu eruieren. Neben weiteren Sequenzunterschieden, die meist in Introns gefunden wurden, wurde die codierende Mutation G307R innerhalb von Exon 7 identifiziert, die letztendlich der Grund für eine Versuchsreihe war, um zu hinterfragen, ob dieser Aminosäureaustausch in der Proteinsequenz funktionelle Konsequenzen zur Folge hat. HEK293-Zellen wurden mit dem zuvor hergestellten Klon G307R transfiziert, um über Biotinylierung, immuncytochemische Färbungen und funktionelle Untersuchungen die Aktivität des Transporters zu beurteilen. Hier zeigte sich ein Funktionsverlust von über 95 %, bei uneingeschränkter Oberflächenexpression. ASC-1 bestätigt sich damit als neue Ursache in der Ausprägung von Hyperekplexie. Ferner können Zusammenhänge mit geistiger Retardierung und eingeschränkter neuronaler Plastizität bestehen.
Non-steroidal antiinflammatory drugs are most commonly used for inflammatory and postoperative pain. But they lack effectiveness and specificity, leading to severe side effects, like gastric ulcers, asthma and severe bleeding. Oxidized 1-palmitoyl-2-arachinidonoyl-sn-glycero-3-phosphocholine (OxPAPC) plays an important role in inflammatory pain. PAPC is a common phosphatidylcholine of membranes, which can be oxidized by reactive oxygen species. In preliminary experiments, our group found that local injection of OxPAPC in rat paws induces hyperalgesia.
In this study we examined the effect of OxPAPC on transient receptor potential A1 (TRPA1), an ion channel expressed in C-fiber neurons. Furthermore, we investigated if intracellular cysteine residues of TRPA1 were necessary for agonist-channel-interactions and if a subsequent TRPA1 activation could be prevented by OxPAPC scavengers.
To answer these questions, we performed calcium imaging using HEK-293 cells stably expressing hTRPA1, or transiently expressing the triple mutant channel hTRPA1-3C and naïve DRG neurons. Cells were incubated with the ratiometric, fluorescent dye Fura-2/AM and stimulated with OxPAPC. The change of light emission after excitation with 340 and 380 nm wavelengths allowed conclusions regarding changes of intracellular calcium concentrations after TRPA1 activation.
In our investigation we proved evidence that OxPAPC activates TRPA1, which caused a flow of calcium ions into the cytoplasm. The TRPA1-specific channel blocker HC-030031 eliminated this agonist-induced response. TRPA1-3C was not completely sensitive to OxPAPC. The peptide D-4F and the monoclonal antibody E06 neutralized OxPAPC-induced TRPA1 activation.
In this work, the importance of OxPAPC as a key mediator of inflammatory pain and as a promising target for drug design is highlighted. Our results indicate that TRPA1 activation by OxPAPC involves cysteine-dependent mechanisms, but there are other, cysteine-independent activation mechanisms as well. Potential pharmaceuticals for the treatment of inflammatory pain are D-4F and E06, whose efficiency has recently been confirmed in the animal model by our research group.
Für nozizeptive Wundschmerzen ist der Transient Receptor Potential Channel (TRP) vermittelte Kalziumeinstrom unerlässlich. Reaktive Sauerstoffspezies (ROS) und deren Oxidationsprodukte wie 4-Hydroxynonenal (4-HNE) aktivieren das Ankyrin-1-Homolog TRPA1 in vivo und in vitro.
Die in dieser Studie durchgeführten Kalzium-Imaging Experimente wurden an stabil mit TRPA1 und TRPV1 transfizierten HEK-293-Zellen und spinalen Hinterwurzelganglien durchgeführt, um die mechanistischen Zusammenhänge der nozizeptiven Schmerzentstehung bei entzündlichen Wundschmerzen besser zu verstehen.
E06, ein monoklonaler Autoantikörper (mAb) gegen oxidiertes Phosphatidylcholin (OxPC) und D-4F, ein mimetisches Peptid des Strukturproteins Apolipoprotein A-I aus dem high density lipoprotein (HDL), wurden bisher als diagnostischer Marker bei Atherosklerose eingesetzt.
In den durchgeführten Experimenten reduzierten E06 mAb und D-4F den durch Lipidperoxidationsprodukte (OxPL) wie 4-HNE und reaktive Sauerstoffspezies wie H2O2 verursachten TRPA1-vermittelten Kalziumeinfluss in vitro.
Darüber hinaus zeigte sich, dass weder E06 mAb noch D-4F eine Kalziumeinstromrelevante Interaktion mit dem Transient Receptor Potential Channel Vanillin 1 (TRPV1)-Aktivator Capsaicin oder dem TRPV1-Kanal aufweisen.
E06 mAb und ApoA-I mimetisches Peptid D-4F erscheinen deshalb als zwei vielversprechende Substanzen, um den inflammatorischen Wundschmerz zu verringern. Deshalb sind sie auch als potentielles Analgetikum für eine nebenwirkungsärmere, lokale Schmerzbekämpfung vielversprechend.
2D electrophysiology is often used to determine the electrical properties of neurons, while in the brain, neurons form extensive 3D networks. Thus, performing electrophysiology in a 3D environment provides a closer situation to the physiological condition and serves as a useful tool for various applications in the field of neuroscience. In this study, we established 3D electrophysiology within a fiber-reinforced matrix to enable fast readouts from transfected cells, which are often used as model systems for 2D electrophysiology. Using melt electrowriting (MEW) of scaffolds to reinforce Matrigel, we performed 3D electrophysiology on a glycine receptor-transfected Ltk-11 mouse fibroblast cell line. The glycine receptor is an inhibitory ion channel associated when mutated with impaired neuromotor behaviour. The average thickness of the MEW scaffold was 141.4 ± 5.7µm, using 9.7 ± 0.2µm diameter fibers, and square pore spacings of 100 µm, 200 µm and 400 µm. We demonstrate, for the first time, the electrophysiological characterization of glycine receptor-transfected cells with respect to agonist efficacy and potency in a 3D matrix. With the MEW scaffold reinforcement not interfering with the electrophysiology measurement, this approach can now be further adapted and developed for different kinds of neuronal cultures to study and understand pathological mechanisms under disease conditions.
Dopaminergic neurons in the brain of the Drosophila larva play a key role in mediating reward information to the mushroom bodies during appetitive olfactory learning and memory. Using optogenetic activation of Kenyon cells we provide evidence that recurrent signaling exists between Kenyon cells and dopaminergic neurons of the primary protocerebral anterior (pPAM) cluster. Optogenetic activation of Kenyon cells paired with odor stimulation is sufficient to induce appetitive memory. Simultaneous impairment of the dopaminergic pPAM neurons abolishes appetitive memory expression. Thus, we argue that dopaminergic pPAM neurons mediate reward information to the Kenyon cells, and in turn receive feedback from Kenyon cells. We further show that this feedback signaling is dependent on short neuropeptide F, but not on acetylcholine known to be important for odor-shock memories in adult flies. Our data suggest that recurrent signaling routes within the larval mushroom body circuitry may represent a mechanism subserving memory stabilization.
The nervous system is shielded by special barriers. Nerve injury results in blood–nerve barrier breakdown with downregulation of certain tight junction proteins accompanying the painful neuropathic phenotype. The dorsal root ganglion (DRG) consists of a neuron-rich region (NRR, somata of somatosensory and nociceptive neurons) and a fibre-rich region (FRR), and their putative epi-/perineurium (EPN). Here, we analysed blood–DRG barrier (BDB) properties in these physiologically distinct regions in Wistar rats after chronic constriction injury (CCI). Cldn5, Cldn12, and Tjp1 (rats) mRNA were downregulated 1 week after traumatic nerve injury. Claudin-1 immunoreactivity (IR) found in the EPN, claudin-19-IR in the FRR, and ZO-1-IR in FRR-EPN were unaltered after CCI. However, laser-assisted, vessel specific qPCR, and IR studies confirmed a significant loss of claudin-5 in the NRR. The NRR was three-times more permeable compared to the FRR for high and low molecular weight markers. NRR permeability was not further increased 1-week after CCI, but significantly more CD68\(^+\) macrophages had migrated into the NRR. In summary, NRR and FRR are different in naïve rats. Short-term traumatic nerve injury leaves the already highly permeable BDB in the NRR unaltered for small and large molecules. Claudin-5 is downregulated in the NRR. This could facilitate macrophage invasion, and thereby neuronal sensitisation and hyperalgesia. Targeting the stabilisation of claudin-5 in microvessels and the BDB barrier could be a future approach for neuropathic pain therapy.
Objective
To identify and characterize patients with autoantibodies against different neurofascin (NF) isoforms.
Methods
Screening of a large cohort of patient sera for anti-NF autoantibodies by ELISA and further characterization by cell-based assays, epitope mapping, and complement binding assays.
Results
Two different clinical phenotypes became apparent in this study: The well-known clinical picture of subacute-onset severe sensorimotor neuropathy with tremor that is known to be associated with IgG4 autoantibodies against the paranodal isoform NF-155 was found in 2 patients. The second phenotype with a dramatic course of disease with tetraplegia and almost locked-in syndrome was associated with IgG3 autoantibodies against nodal and paranodal isoforms of NF in 3 patients. The epitope against which these autoantibodies were directed in this second phenotype was the common Ig domain found in all 3 NF isoforms. In contrast, anti–NF-155 IgG4 were directed against the NF-155–specific Fn3Fn4 domain. The description of a second phenotype of anti–NF-associated neuropathy is in line with some case reports of similar patients that were published in the last year.
Conclusions
Our results indicate that anti–pan-NF-associated neuropathy differs from anti–NF-155-associated neuropathy, and epitope and subclass play a major role in the pathogenesis and severity of anti–NF-associated neuropathy and should be determined to correctly classify patients, also in respect to possible differences in therapeutic response.