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The signals that coordinate and control movement in vertebrates are transmitted from motoneurons (MNs) to their target muscle cells at neuromuscular junctions (NMJs). Human NMJs display unique structural and physiological features, which make them vulnerable to pathological processes. NMJs are an early target in the pathology of motoneuron diseases (MND). Synaptic dysfunction and synapse elimination precede MN loss suggesting that the NMJ is the starting point of the pathophysiological cascade leading to MN death. Therefore, the study of human MNs in health and disease requires cell culture systems that enable the connection to their target muscle cells for NMJ formation. Here, we present a human neuromuscular co-culture system consisting of induced pluripotent stem cell (iPSC)-derived MNs and 3D skeletal muscle tissue derived from myoblasts. We used self-microfabricated silicone dishes combined with Velcro hooks to support the formation of 3D muscle tissue in a defined extracellular matrix, which enhances NMJ function and maturity. Using a combination of immunohistochemistry, calcium imaging, and pharmacological stimulations, we characterized and confirmed the function of the 3D muscle tissue and the 3D neuromuscular co-cultures. Finally, we applied this system as an in vitro model to study the pathophysiology of Amyotrophic Lateral Sclerosis (ALS) and found a decrease in neuromuscular coupling and muscle contraction in co-cultures with MNs harboring ALS-linked SOD1 mutation. In summary, the human 3D neuromuscular cell culture system presented here recapitulates aspects of human physiology in a controlled in vitro setting and is suitable for modeling of MND.
The amyotrophic lateral sclerosis (ALS) neurodegenerative disorder has been associated with multiple genetic lesions, including mutations in the gene for fused in sarcoma (FUS), a nuclear-localized RNA/DNA-binding protein. Neuronal expression of the pathological form of FUS proteins in Caenorhabditis elegans results in mislocalization and aggregation of FUS in the cytoplasm, and leads to impairment of motility. However, the mechanisms by which the mutant FUS disrupts neuronal health and function remain unclear. Here we investigated the impact of ALS-associated FUS on motor neuron health using correlative light and electron microscopy, electron tomography, and electrophysiology. We show that ectopic expression of wild-type or ALS-associated human FUS impairs synaptic vesicle docking at neuromuscular junctions. ALS-associated FUS led to the emergence of a population of large, electron-dense, and filament-filled endosomes. Electrophysiological recording revealed reduced transmission from motor neurons to muscles. Together, these results suggest a pathological effect of ALS-causing FUS at synaptic structure and function organization.
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.
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.
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.
Inflammation and dysregulation of the immune system are hallmarks of several neurodegenerative diseases. An activated immune response is considered to be the cause of myelin breakdown in demyelinating disorders. In the peripheral nervous system (PNS), myelin can be degraded in an autophagy-dependent manner directly by Schwann cells or by macrophages, which are modulated by T-lymphocytes. Here, we show that the NF-κB activator Pleckstrin homology containing family member 5 (Plekhg5) is involved in the regulation of both Schwann cell autophagy and recruitment of T-lymphocytes in peripheral nerves during motoneuron disease. Plekhg5-deficient mice show defective axon/Schwann cell units characterized by myelin infoldings in peripheral nerves. Even at late stages, Plekhg5-deficient mice do not show any signs of demyelination and inflammation. Using RNAseq, we identified a transcriptional signature for an impaired immune response in sciatic nerves, which manifested in a reduced number of CD4\(^+\) and CD8\(^+\) T-cells. These findings identify Plekhg5 as a promising target to impede myelin breakdown in demyelinating PNS disorders.
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.
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.
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.
Gene expression requires tight coordination of the molecular machineries that mediate transcription and splicing. While the interplay between transcription kinetics and spliceosome fidelity has been investigated before, less is known about mechanisms regulating the assembly of the spliceosomal machinery in response to transcription changes. Here, we report an association of the Smn complex, which mediates spliceosomal snRNP biogenesis, with the 7SK complex involved in transcriptional regulation. We found that Smn interacts with the 7SK core components Larp7 and Mepce and specifically associates with 7SK subcomplexes containing hnRNP R. The association between Smn and 7SK complexes is enhanced upon transcriptional inhibition leading to reduced production of snRNPs. Taken together, our findings reveal a functional association of Smn and 7SK complexes that is governed by global changes in transcription. Thus, in addition to its canonical nuclear role in transcriptional regulation, 7SK has cytosolic functions in fine-tuning spliceosome production according to transcriptional demand.
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.
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.
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.
Objective
Alzheimer’s disease (AD) is a growing challenge worldwide, which is why the search for early-onset predictors must be focused as soon as possible. Longitudinal studies that investigate courses of neuropsychological and other variables screen for such predictors correlated to mild cognitive impairment (MCI). However, one often neglected issue in analyses of such studies is measurement invariance (MI), which is often assumed but not tested for. This study uses the absence of MI (non-MI) and latent factor scores instead of composite variables to assess properties of cognitive domains, compensation mechanisms, and their predictability to establish a method for a more comprehensive understanding of pathological cognitive decline.
Methods
An exploratory factor analysis (EFA) and a set of increasingly restricted confirmatory factor analyses (CFAs) were conducted to find latent factors, compared them with the composite approach, and to test for longitudinal (partial-)MI in a neuropsychiatric test battery, consisting of 14 test variables. A total of 330 elderly (mean age: 73.78 ± 1.52 years at baseline) were analyzed two times (3 years apart).
Results
EFA revealed a four-factor model representing declarative memory, attention, working memory, and visual–spatial processing. Based on CFA, an accurate model was estimated across both measurement timepoints. Partial non-MI was found for parameters such as loadings, test- and latent factor intercepts as well as latent factor variances. The latent factor approach was preferable to the composite approach.
Conclusion
The overall assessment of non-MI latent factors may pose a possible target for this field of research. Hence, the non-MI of variances indicated variables that are especially suited for the prediction of pathological cognitive decline, while non-MI of intercepts indicated general aging-related decline. As a result, the sole assessment of MI may help distinguish pathological from normative aging processes and additionally may reveal compensatory neuropsychological mechanisms.
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.
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.
Neurons critically rely on the functions of RNA-binding proteins to maintain their polarity and resistance to neurotoxic stress. HnRNP R has a diverse range of post-transcriptional regulatory functions and is important for neuronal development by regulating axon growth. Hnrnpr pre-mRNA undergoes alternative splicing giving rise to a full-length protein and a shorter isoform lacking its N-terminal acidic domain. To investigate functions selectively associated with the full-length hnRNP R isoform, we generated a Hnrnpr knockout mouse (Hnrnpr\(^{tm1a/tm1a}\)) in which expression of full-length hnRNP R was abolished while production of the truncated hnRNP R isoform was retained. Motoneurons cultured from Hnrnpr\(^{tm1a/tm1a}\) mice did not show any axonal growth defects but exhibited enhanced accumulation of double-strand breaks and an impaired DNA damage response upon exposure to genotoxic agents. Proteomic analysis of the hnRNP R interactome revealed the multifunctional protein Yb1 as a top interactor. Yb1-depleted motoneurons were defective in DNA damage repair. We show that Yb1 is recruited to chromatin upon DNA damage where it interacts with gamma-H2AX, a mechanism that is dependent on full-length hnRNP R. Our findings thus suggest a novel role of hnRNP R in maintaining genomic integrity and highlight the function of its N-terminal acidic domain in this context.
Survival motor neuron (SMN) is an essential and ubiquitously expressed protein that participates in several aspects of RNA metabolism. SMN deficiency causes a devastating motor neuron disease called spinal muscular atrophy (SMA). SMN forms the core of a protein complex localized at the cytoplasm and nuclear gems and that catalyzes spliceosomal snRNP particle synthesis. In cultured motor neurons, SMN is also present in dendrites and axons, and forms part of the ribonucleoprotein transport granules implicated in mRNA trafficking and local translation. Nevertheless, the distribution, regulation, and role of SMN at the axons and presynaptic motor terminals in vivo are still unclear. By using conventional confocal microscopy and STED super-resolution nanoscopy, we found that SMN appears in the form of granules distributed along motor axons at nerve terminals. Our fluorescence in situ hybridization and electron microscopy studies also confirmed the presence of β-actin mRNA, ribosomes, and polysomes in the presynaptic motor terminal, key elements of the protein synthesis machinery involved in local translation in this compartment. SMN granules co-localize with the microtubule-associated protein 1B (MAP1B) and neurofilaments, suggesting that the cytoskeleton participates in transporting and positioning the granules. We also found that, while SMN granules are physiologically downregulated at the presynaptic element during the period of postnatal maturation in wild-type (non-transgenic) mice, they accumulate in areas of neurofilament aggregation in SMA mice, suggesting that the high expression of SMN at the NMJ, together with the cytoskeletal defects, contribute to impairing the bi-directional traffic of proteins and organelles between the axon and the presynaptic terminal.
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.
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.
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.
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).
Background: Axonal degeneration and defects in neuromuscular neurotransmission represent a pathological hallmark
in spinal muscular atrophy (SMA) and other forms of motoneuron disease. These pathological changes do not
only base on altered axonal and presynaptic architecture, but also on alterations in dynamic movements of organelles
and subcellular structures that are not necessarily reflected by static histopathological changes. The dynamic interplay
between the axonal endoplasmic reticulum (ER) and ribosomes is essential for stimulus-induced local translation
in motor axons and presynaptic terminals. However, it remains enigmatic whether the ER and ribosome crosstalk is
impaired in the presynaptic compartment of motoneurons with Smn (survival of motor neuron) deficiency that could
contribute to axonopathy and presynaptic dysfunction in SMA.
Methods: Using super-resolution microscopy, proximity ligation assay (PLA) and live imaging of cultured motoneurons
from a mouse model of SMA, we investigated the dynamics of the axonal ER and ribosome distribution and
activation.
Results: We observed that the dynamic remodeling of ER was impaired in axon terminals of Smn-deficient motoneurons.
In addition, in axon terminals of Smn-deficient motoneurons, ribosomes failed to respond to the brain-derived
neurotrophic factor stimulation, and did not undergo rapid association with the axonal ER in response to extracellular
stimuli.
Conclusions: These findings implicate impaired dynamic interplay between the ribosomes and ER in axon terminals
of motoneurons as a contributor to the pathophysiology of SMA and possibly also other motoneuron diseases.
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects 1 in ~350 individuals. Genetic association studies have established ALS as a multifactorial disease with heritability estimated at ~61%, and recent studies show a prominent role for rare variation in its genetic architecture. To identify rare variants associated with disease onset we performed exome array genotyping in 4,244 cases and 3,106 controls from European cohorts. In this largest exome-wide study of rare variants in ALS to date, we performed single-variant association testing, gene-based burden, and exome-wide individual set-unique burden (ISUB) testing to identify single or aggregated rare variation that modifies disease risk. In single-variant testing no variants reached exome-wide significance, likely due to limited statistical power. Gene-based burden testing of rare non-synonymous and loss-of-function variants showed NEK1 as the top associated gene. ISUB analysis did not show an increased exome-wide burden of deleterious variants in patients, possibly suggesting a more region-specific role for rare variation. Complete summary statistics are released publicly. This study did not implicate new risk loci, emphasizing the immediate need for future large-scale collaborations in ALS that will expand available sample sizes, increase genome coverage, and improve our ability to detect rare variants associated to ALS.
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.
Keeping the balance: the noncoding RNA 7SK as a master regulator for neuron development and function
(2021)
The noncoding RNA 7SK is a critical regulator of transcription by adjusting the activity of the kinase complex P-TEFb. Release of P-TEFb from 7SK stimulates transcription at many genes by promoting productive elongation. Conversely, P-TEFb sequestration by 7SK inhibits transcription. Recent studies have shown that 7SK functions are particularly important for neuron development and maintenance and it can thus be hypothesized that 7SK is at the center of many signaling pathways contributing to neuron function. 7SK activates neuronal gene expression programs that are key for terminal differentiation of neurons. Proteomics studies revealed a complex protein interactome of 7SK that includes several RNA-binding proteins. Some of these novel 7SK subcomplexes exert non-canonical cytosolic functions in neurons by regulating axonal mRNA transport and fine-tuning spliceosome production in response to transcription alterations. Thus, a picture emerges according to which 7SK acts as a multi-functional RNA scaffold that is integral for neuron homeostasis.
Protein inclusions containing the RNA-binding protein TDP-43 are a pathological hallmark of amyotrophic lateral sclerosis and other neurodegenerative disorders. The loss of TDP-43 function that is associated with these inclusions affects post-transcriptional processing of RNAs in multiple ways including pre-mRNA splicing, nucleocytoplasmic transport, modulation of mRNA stability and translation. In contrast, less is known about the role of TDP-43 in axonal RNA metabolism in motoneurons. Here we show that depletion of Tdp-43 in primary motoneurons affects axon growth. This defect is accompanied by subcellular transcriptome alterations in the axonal and somatodendritic compartment. The axonal localization of transcripts encoding components of the cytoskeleton, the translational machinery and transcripts involved in mitochondrial energy metabolism were particularly affected by loss of Tdp-43. Accordingly, we observed reduced protein synthesis and disturbed mitochondrial functions in axons of Tdp-43-depleted motoneurons. Treatment with nicotinamide rescued the axon growth defect associated with loss of Tdp-43. These results show that Tdp-43 depletion in motoneurons affects several pathways integral to axon health indicating that loss of TDP-43 function could thus make a major contribution to axonal pathomechanisms in ALS.
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.
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.
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.
Highlights
• Dopamine receptor-1 activation induces TrkB cell-surface expression in striatal neurons
• Dopaminergic deficits cause TrkB accumulation and clustering in the ER
• TrkB clusters colocalize with cargo receptor SORCS-2 in direct pathway striatal neurons
• Intracellular TrkB clusters fail to fuse with lysosomes after dopamine depletion
Summary
Disturbed motor control is a hallmark of Parkinson’s disease (PD). Cortico-striatal synapses play a central role in motor learning and adaption, and brain-derived neurotrophic factor (BDNF) from cortico-striatal afferents modulates their plasticity via TrkB in striatal medium spiny projection neurons (SPNs). We studied the role of dopamine in modulating the sensitivity of direct pathway SPNs (dSPNs) to BDNF in cultures of fluorescence-activated cell sorting (FACS)-enriched D1-expressing SPNs and 6-hydroxydopamine (6-OHDA)-treated rats. DRD1 activation causes enhanced TrkB translocation to the cell surface and increased sensitivity for BDNF. In contrast, dopamine depletion in cultured dSPN neurons, 6-OHDA-treated rats, and postmortem brain of patients with PD reduces BDNF responsiveness and causes formation of intracellular TrkB clusters. These clusters associate with sortilin related VPS10 domain containing receptor 2 (SORCS-2) in multivesicular-like structures, which apparently protects them from lysosomal degradation. Thus, impaired TrkB processing might contribute to disturbed motor function in PD.
Neurotrophin signaling via receptor tyrosine kinases is essential for the development and function of the nervous system in vertebrates. TrkB activation and signaling show substantial differences to other receptor tyrosine kinases of the Trk family that mediate the responses to nerve growth factor and neurotrophin-3. Growing evidence suggests that TrkB cell surface expression is highly regulated and determines the sensitivity of neurons to brain-derived neurotrophic factor (BDNF). This translocation of TrkB depends on co-factors and modulators of cAMP levels, N-glycosylation, and receptor transactivation. This process can occur in very short time periods and the resulting rapid modulation of target cell sensitivity to BDNF could represent a mechanism for fine-tuning of synaptic plasticity and communication in complex neuronal networks. This review focuses on those modulatory mechanisms in neurons that regulate responsiveness to BDNF via control of TrkB surface expression.