Refine
Has Fulltext
- yes (20)
Is part of the Bibliography
- yes (20)
Year of publication
Document Type
- Doctoral Thesis (20) (remove)
Language
- English (20) (remove)
Keywords
- Motoneuron (7)
- Spinale Muskelatrophie (4)
- Ciliary neurotrophic factor (2)
- Drosophila melanogaster (2)
- Entzündung (2)
- Ionenkanal (2)
- Nociceptor (2)
- Phospholipide (2)
- Signaltransduktion (2)
- Spinal Muscular Atrophy (2)
Institute
- Institut für Klinische Neurobiologie (20) (remove)
In the face of threat, animals react with a defensive reaction to avoid or reduce harm. This defensive reaction encompasses apart from behavioral changes also physiological, analgetic, and endocrine adaptations. Nonetheless, most animal studies on fear and anxiety are based on behavioral observations only, disregarding other aspects of the defensive reaction, or integrating their inter-related dynamics only insufficiently. The first part of this thesis aimed in characterizing patterned associations of behavioral and physiological responses, termed integrated defensive states. Analyzing cardiac and behavioral responses in mice undergoing multiple fear and anxiety paradigms revealed a complex and dynamic interaction of those readouts on both, short and long timescales. Microstates, stereotypical combinations of i.e. freezing and decelerating heart rates, are short-lasting and were, in turn, shown to be influenced by slow acting macrostate changes. One of those higher order macrostates, called `rigidity`, was defined as a latent process that constrains the range of momentary displayed heart rate values. Furthermore, integrated defensive states were found to be highly dependent on the cue and the context the animals are confronted with. Importantly, same behavioral observations, i.e. freezing, were associated with distinct cardiac responses, highlighting the importance of multivariate analysis of integrated defensive states. Defensive states are orchestrated by the brain, which has evolved evolutionary conserved survival circuits. A central brain area of these circuits is the periaqueductal gray (PAG) in the midbrain. It plays a pivotal role in mediating defensive states, as it receives signals about external and internal information from multiple brain regions and sends information to both, higher order brain areas as well as to the brainstem ultimately causing the execution of threat responses. In the second part of this thesis, different neuronal circuit elements in the PAG were optically manipulated in order to gain mechanistic insight into the defense network in the brain underlying the previously delineated cardio-behavioral defensive states. Optical activation of glutamatergic PAG neurons evoked heterogeneous, light-intensity dependent responses. However, a further molecular restriction of the glutamatergic neuronal population targeting only Chx10+ neurons, led to a cardio-behavioral state that resembled spontaneous freezing-bradycardia bouts.
In summary, this thesis presents a multivariate description of defensive states, which includes the complex interaction of cardiac and behavioral responses on different timescales and, furthermore, functionally dissects different excitatory and inhibitory PAG circuit elements mediating these defensive states.
Motor neuron diseases (MNDs) encompass a variety of clinically and genetically heterogeneous disorders, which lead to the degeneration of motor neurons (MNs) and impaired motor functions. MNs coordinate and control movement by transmitting their signal to a target muscle cell. The synaptic endings of the MN axon and the contact site of the muscle cell thereby form the presynaptic and postsynaptic structures of the neuromuscular junction (NMJ). In MNDs, synaptic dysfunction and synapse elimination precede MN loss suggesting that the NMJ is an early target in the pathophysiological cascade leading to MN death. In this study, we established new experimental strategies to analyze human MNDs by patient derived induced pluripotent stem cells (iPSCs) and investigated pathophysiological mechanisms in two different MNDs.
To study human MNDs, specialized cell culture systems that enable the connection of MNs to their target muscle cells are required to allow the formation of NMJs. In the first part of this study, we established and validated a human neuromuscular co-culture system consisting of iPSC derived MNs and 3D skeletal muscle tissue derived from myoblasts. We generated 3D muscle tissue by culturing primary myoblasts in a defined extracellular matrix in self-microfabricated silicone dishes that support the 3D tissue formation. Subsequently, iPSCs from healthy donors and iPSCs from patients with the progressive MND Amyotrophic Lateral Sclerosis (ALS) were differentiated into MNs and used for 3D neuromuscular co-cultures. Using a combination of immunohistochemistry, calcium imaging, and pharmacological stimulations, we characterized and confirmed the functionality 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 ALS and found a decrease in neuromuscular coupling, muscle contraction, and axonal outgrowth in co-cultures with MNs harboring ALS-linked superoxide dismutase 1 (SOD1) mutation. In summary, this co-culture system presents a human model for MNDs that can recapitulate aspects of ALS pathophysiology.
In the second part of this study, we identified an impaired unconventional protein secretion (UPS) of Sod1 as pathological mechanisms in Pleckstrin homology domain-containing family G member 5 (Plekhg5)-associated MND. Sod1 is a leaderless cytosolic protein which is secreted in an autophagy-dependent manner. We found that Plekhg5 depletion in primary MNs and NSC34 cells leads to an impaired secretion of wildtype Sod1, indicating that Plekhg5 drives the UPS of Sod1 in vitro. By interfering with different steps during the biogenesis of autophagosomes, we could show that Plekhg5-regulated Sod1 secretion is determined by autophagy. To analyze our findings in a clinically more relevant model we utilized human iPSC MNs from healthy donors and ALS patients with SOD1 mutations. We observed reduced SOD1 secretion in ALS MNs which coincides with reduced protein expression of PLEKHG5 compared to healthy and isogenic control MNs. To confirm this correlation, we depleted PLEKHG5 in control MNs and found reduced extracellular SOD1 levels, implying that SOD1 secretion depends on PLEKHG5. In summary, we found that Plekh5 regulates the UPS of Sod1 in mouse and human MNs and that Sod1 secretion occurs in an autophagy dependent manner. Our data shows an unreported mechanistic link between two MND-associated proteins.
Plexus injury often occurs after motor vehicle accidents and results in lifelong disability with severe neuropathic pain. Surgical treatment can partially restore motor functions, but sensory loss and neuropathic pain persist. Regenerative medicine concepts, such as cell replacement therapies for restoring dorsal root ganglia (DRG) function, set high expectations. However, up to now, it is unclear which DRG cell types are affected by nerve injury and can be targeted in regenerative medicine approaches.
This study followed the hypothesis that satellite glial cells (SGCs) might be a suitable endogenous cell source for regenerative medicine concepts in the DRG. SGCs originate from the same neural crest-derived cell lineage as sensory neurons, making them attractive for neural repair strategies in the peripheral nervous system. Our hypothesis was investigated on three levels of experimentation. First, we asked whether adult SGCs have the potential of sensory neuron precursors and can be reprogrammed into sensory neurons in vitro. We found that adult mouse DRG harbor SGC-like cells that can still dedifferentiate into progenitor-like cells. Surprisingly, expression of the early developmental transcription factors Neurog1 and Neurog2 was sufficient to induce neuronal and glial cell phenotypes. In the presence of nerve growth factor, induced neurons developed a nociceptor-like phenotype expressing functional nociceptor markers, such as the ion channels TrpA1, TrpV1 and NaV1.9. In a second set of experiments, we used a rat model for peripheral nerve injury to look for changes in the DRG cell composition. Using an unbiased deep learning-based approach for cell analysis, we found that cellular plasticity responses after nerve injury activate SGCs in the whole DRG. However, neither injury-induced neuronal death nor gliosis was observed. Finally, we asked whether a severe nerve injury changed the cell composition in the human DRG. For this, a cohort of 13 patients with brachial plexus injury was investigated. Surprisingly, in about half of all patients, the injury-affected DRG showed no characteristic DRG tissue. The complete entity of neurons, satellite cells, and axons was lost and fully replaced by mesodermal/connective tissue. In the other half of the patients, the basic cellular entity of the DRG was well preserved. Objective deep learning-based analysis of large-scale bioimages of the “intact” DRG showed no loss of neurons and no signs of gliosis.
This study suggests that concepts for regenerative medicine for restoring DRG function need at least two translational research directions: reafferentation of existing DRG units or full replacement of the entire multicellular DRG structure. For DRG replacement, SGCs of the adult DRG are an attractive endogenous cell source, as the multicellular DRG units could possibly be rebuilt by transdifferentiating neural crest-derived sensory progenitor cells into peripheral sensory neurons and glial cells using Neurog1 and Neurog2.
In highly polarized neurons, endoplasmic reticulum (ER) forms a dynamic and continuous network in axons that plays important roles in lipid synthesis, Ca2+ homeostasis and the maintenance of synapses. However, the mechanisms underlying the regulation of axonal ER dynamics and its function in regulation of local translation still remain elusive. In the course of my thesis, I investigated the fast dynamic movements of ER and ribosomes in the growth cone of wildtype motoneurons as well as motoneurons from a mouse model of Spinal Muscular Atrophy (SMA), in response to Brain-derived neurotrophic factor (BDNF) stimulation. Live cell imaging data show that ER extends into axonal growth cone filopodia along actin filaments and disruption of actin cytoskeleton by cytochalasin D treatment impairs the dynamic movement of ER in the axonal filopodia. In contrast to filopodia, ER movements in the growth cone core seem to depend on coordinated actions of the actin and microtubule cytoskeleton. Myosin VI is especially required for ER movements into filopodia and drebrin A mediates actin/microtubule coordinated ER dynamics. Furthermore, we found that BDNF/TrkB signaling induces assembly of 80S ribosomes in growth cones on a time scale of seconds. Activated ribosomes relocate to the presynaptic ER and undergo local translation. These findings describe the dynamic interaction between ER and ribosomes during local translation and identify a novel potential function for the presynaptic ER in intra-axonal synthesis of transmembrane proteins such as the α-1β subunit of N-type Ca2+ channels in motoneurons. In addition, we demonstrate that in Smn-deficient motoneurons, ER dynamic movements are impaired in axonal growth cones that seems to be due to impaired actin cytoskeleton. Interestingly, ribosomes fail to undergo rapid structural changes in Smn-deficient growth cones and do not associate to ER in response to BDNF. Thus, aberrant ER dynamics and ribosome response to extracellular stimuli could affect axonal growth and presynaptic function and maintenance, thereby contributing to the pathology of SMA.
Chronic pain conditions are a major reason for the utilization of the health care system. Inflammatory pain states can persist facilitated by peripheral sensitization of nociceptors. The voltage-gated sodium channel 1.9 (NaV1.9) is an important regulator of neuronal excitability and is involved in inflammation-induced pain hypersensitivity. Recently, oxidized 1-palmitoyl-2-arachidonoyl-sn-glycerol-3-phosphatidylcholine (OxPAPC) was identified as a mediator of acute inflammatory pain and persistent hyperalgesia, suggesting an involvement in proalgesic cascades and peripheral sensitization. Peripheral sensitization implies an increase in neuronal excitability. This thesis aims to characterize spontaneous calcium activity in neuronal compartments as a proxy to investigate neuronal excitability, making use of the computational tool Neural Activity Cubic (NA3). NA3 allows automated calcium activity event detection of signal-close-to-noise calcium activity and evaluation of neuronal activity states. Additionally, the influence of OxPAPC and NaV1.9 on the excitability of murine dorsal root ganglion (DRG) neurons and the effect of OxPAPC on the response of DRG neurons towards other inflammatory mediators (prostaglandin E2, histamine, and bradykinin) is investigated. Using calcium imaging, the presence of spontaneous calcium activity in murine DRG neurons was established. NA3 was used to quantify this spontaneous calcium activity, which revealed decreased activity counts in axons and somata of NaV1.9 knockout (KO) neurons compared to wildtype (WT). Incubation of WT DRG neurons with OxPAPC before calcium imaging did not show altered activity counts compared to controls. OxPAPC incubation also did not modify the response of DRG neurons treated with inflammatory mediators. However, the variance ratio computed by NA3 conclusively allowed to determine neuronal activity states. In conclusion, my findings indicate an important function of NaV1.9 in determining the neuronal excitability of DRG neurons in resting states. OxPAPC exposition does not influence neuronal excitability nor sensitizes neurons for other inflammatory mediators. This evidence reduces the primary mechanism of OxPAPC-induced hyperalgesia to acute effects. Importantly, it was possible to establish an approach for unbiased excitability quantification of DRG neurons by calcium activity event detection and calcium trace variance analysis by NA3. It was possible to show that signal-close-to-noise calcium activity reflects neuronal excitability states.
In the central nervous system, excitatory and inhibitory signal transduction processes are mediated by presynaptic release of neurotransmitters, which bind to postsynaptic receptors. Glycine receptors (GlyRs) and GABAA receptors (GABAARs) are ligand-gated ion channels that enable synaptic inhibition. One part of the present thesis elucidated the role of the GlyRα1 β8 β9 loop in receptor expression, localization, and function by means of amino acid substitutions at residue Q177. This residue is underlying a startle disease phenotype in the spontaneous mouse model shaky and affected homozygous animals are dying 4-6 weeks after birth. The residue is located in the β8 β9 loop and thus part of the signal transduction unit essential for proper ion channel function. Moreover, residue Q177 is involved in a hydrogen network important for ligand binding. We observed no difference in ion channel trafficking to the cellular membrane for GlyRα1Q177 variants. However, electrophysiological measurements demonstrated reduced glycine, taurine, and β alanine potency in comparison to the wildtype protein. Modeling revealed that some GlyRα1Q177 variants disrupt the hydrogen network around residue Q177. The largest alterations were observed for the Q177R variant, which displayed similar effects as the Q177K mutation present in shaky mice. Exchange with structurally related amino acids to the original glutamine preserved the hydrogen bond network. Our results underlined the importance of the GlyR β8 β9 loop for proper ion channel gating.
GlyRs as well as GABAARs can be modulated by numerous allosteric substances. Recently, we focused on monoterpenes from plant extracts and showed positive allosteric modulation of GABAARs. Here, we focused on the effect of 11 sesquiterpenes and sesquiterpenoids (SQTs) on GABAARs. SQTs are compounds naturally occurring in plants. We tested SQTs of the volatile fractions of hop and chamomile, including their secondary metabolites generated during digestion. Using the patch-clamp technique on transfected cells and neurons, we were able to observe significant GABAAR modulation by some of the compounds analyzed. Furthermore, a possible binding mechanism of SQTs to the neurosteroid binding site of the GABAAR was revealed by modeling and docking studies. We successfully demonstrated GABAAR modulation by SQTs and their secondary metabolites.
The second part of the thesis investigated three-dimensional (3D) in vitro cell culture models which are becoming more and more important in different part of natural sciences. The third dimension allows developing of complex models closer to the natural environment of cells, but also requires materials with mechanical and biological properties comparable to the native tissue of the encapsulated cells. This is especially challenging for 3D in vitro cultures of primary neurons and astrocytes as the brain is one of the softest tissues found in the body. Ultra-soft matrices that mimic the neuronal in vivo environment are difficult to handle. We have overcome these challenges using fiber scaffolds created by melt electrowriting to reinforce ultra-soft matrigel. Hence, the scaffolds enabled proper handling of the whole composites and thus structural and functional characterizations requiring movement of the composites to different experimental setups. Using these scaffold-matrigel composites, we successfully established methods necessary for the characterization of neuronal network formation. Before starting with neurons, a mouse fibroblast cell line was seeded in scaffold-matrigel composites and transfected with the GlyR. 3D cultured cells displayed high viability, could be immunocytochemically stained, and electrophysiologically analyzed.
In a follow-up study, primary mouse cortical neurons in fiber-reinforced matrigel were grown for up to 21 days in vitro. Neurons displayed high viability, and quantification of neurite lengths and synapse density revealed a fully formed neuronal network already after 7 days in 3D culture. Calcium imaging and patch clamp experiments demonstrated spontaneous network activity, functional voltage-gated sodium channels as well as action potential firing. By combining ultra-soft hydrogels with fiber scaffolds, we successfully created a cell culture model suitable for future work in the context of cell-cell interactions between primary cells of the brain and tumor cells, which will help to elucidate the molecular pathology of aggressive brain tumors and possibly other disease mechanisms.
In mammals, a major fraction of the genome is transcribed as non-coding RNAs. An increasing amount of evidence has accumulated showing that non-coding RNAs play important roles both for normal cell function and in disease processes such as cancer or neurodegeneration. Interpreting the functions of non-coding RNAs and the molecular mechanisms through which they act is one of the most important challenges facing RNA biology today.
In my Ph.D. thesis, I have been investigating the role of 7SK, one of the most abundant non-coding RNAs, in the development and function of motoneurons. 7SK is a highly structured 331 nt RNA transcribed by RNA polymerase III. It forms four stem-loop (SL) structures that serve as binding sites for different proteins. Larp7 binds to SL4 and protects the 3' end from exonucleolytic degradation. SL1 serves as a binding site for HEXIM1, which recruits the pTEFb complex composed of CDK9 and cyclin T1. pTEFb has a stimulatory role for transcription and is regulated through sequestration by 7SK. More recently, a number of heterogeneous nuclear ribonucleoproteins (hnRNPs) have been identified as 7SK interactors. One of these is hnRNP R, which has been shown to have a role in motoneuron development by regulating axon growth. Taken together, 7SK’s function involves interactions with RNA binding proteins, and different RNA binding proteins interact with different regions of 7SK, such that 7SK can be considered as a hub for recruitment and release of different proteins. The questions I have addressed during my Ph.D. are as follows: 1) which region of 7SK interacts with hnRNP R, a main interactor of 7SK? 2) What effects occur in motoneurons after the protein binding sites of 7SK are abolished? 3) Are there additional 7SK binding proteins that regulate the functions of the 7SK RNP?
Using in vitro and in vivo experiments, I found that hnRNP R binds both the SL1 and SL3 region of 7SK, and also that pTEFb cannot be recruited after deleting the SL1 region but is able to bind to a 7SK mutant with deletion of SL3. In order to answer the question of how the 7SK mutations affect axon outgrowth and elongation in mouse primary motoneurons, we proceeded to conduct rescue experiments in motoneurons by using lentiviral vectors. The constructs were designed to express 7SK deletion mutants under the mouse U6 promoter and at the same time to drive expression of a 7SK shRNA from an H1 promoter for the depletion of endogenous 7SK. Using this system we found that 7SK mutants harboring deletions of either SL1 or SL3 could not rescue the axon growth defect of 7SK-depleted motoneurons suggesting that 7SK/hnRNP R complexes are integral for this process.
In order to identify novel 7SK binding proteins and investigate their functions, I proceeded to conduct pull-down experiments by using a biotinylated RNA antisense oligonucleotide that targets the U17-C33 region of 7SK thereby purifying endogenous 7SK complexes. Following mass spectrometry of purified 7SK complexes, we identified a number of novel 7SK interactors. Among these is the Smn complex. Deficiency of the Smn complex causes the motoneuron disease spinal muscular atrophy (SMA) characterized by loss of lower motoneurons in the spinal cord. Smn has previously been shown to interact with hnRNP R. Accordingly, we found Smn as part of 7SK/hnRNP R complexes. These proteomics data suggest that 7SK potentially plays important roles in different signaling pathways in addition to transcription.
Motoneurons are highly compartmentalized cells with very long extensions that separate their nerve terminals from cell bodies. To maintain their extensive morphological complexity and protect their cellular integrity from neurotoxic stresses, neurons rely on the functions of RNA-binding proteins. One such protein is hnRNP R, a multifunctional protein with a plethora of roles related to RNA metabolism that comes into play in the nervous system. hnRNP R is localized mainly in the nucleus but also exists in the cytoplasm and axons of motoneurons. Increasing in vitro evidence indicates a potential function of hnRNP R in the development and maintenance of motoneurons by regulating axon growth and axonal RNA transport. Additionally, hnRNP R interacts with several proteins involved in motoneuron diseases. Hnrnpr pre-mRNA undergoes alternative splicing to produce transcripts encoding two protein isoforms: a full-length protein (hnRNP R-FL) and a shorter form lacking the N-terminal acidic domain (hnRNP R-ΔN). While the neuronal defects produced by total hnRNP R depletion have been investigated before, the contribution of individual isoforms towards such functions has remained mostly unknown.
In this study, we showed that while both isoforms are expressed across multiple tissues, the full-length isoform is particularly abundant in the nervous system. We generated a mouse model for selective knockout of the full-length hnRNP R isoform (Hnrnprtm1a/tm1a) and found that the hnRNP R-∆N isoform remains expressed in these mice and is upregulated in a compensatory post-transcriptional process. We found that the truncated isoform is sufficient to support subcellular RNA transport related to axon growth in primary motoneurons. However, Hnrnprtm1a/tm1a mice show defects in DNA damage repair after exposure to γ-irradiation and etoposide. Knock down of both hnRNP R isoforms showed a similar extent of DNA damage as for motoneurons depleted of just full-length hnRNP R. Rescue experiments showed that expression of full-length hnRNP R but not of hnRNP R-ΔN can restore DNA damage repair when endogenous hnRNP R is depleted. By performing subcellular fractionation, we found that hnRNP R associates with chromatin independently from its association with pre-mRNA. Interestingly, we show that hnRNP R interacts with phosphorylated histone H2AX (γ-H2AX), following DNA damage. Proteomics analysis identifies the multifunctional protein Y-box binding protein 1 (Yb1) as one of the top interacting partners of hnRNP R. Similar to loss of full-length hnRNP R, DNA damage repair was impaired upon knockdown of Yb1 in motoneurons. Finally, we show that following exposure to γ-irradiation, Yb1 is recruited to the chromatin where it interacts with γ-H2AX, a mechanism that is dependent on the full-length hnRNP R.
Taken together, this study describes a novel function of the full-length isoform of hnRNP R in maintaining the genomic integrity of motoneurons and provides new mechanistic insights into its function in DNA damage response.
Investigations into the Pathogenic Antibody-Antigen-Interference of Glycine Receptor Autoantibodies
(2021)
Anti-glycine receptor (GlyR) autoantibodies belong to the novel group of autoantibodies that target neuronal cell-surface antigens (NCS), which are accompanied with various neurologic and neuropsychiatric conditions. The inhibitory ionotropic GlyR is one of the major inhibitory neurotransmitter receptors and therefore involved in maintaining homeostasis of neuronal excitation levels at brain stem and spinal cord. Anti-GlyR autoantibodies are associated with progressive encephalomyelitis with rigidity and myoclonus or stiff person syndrome. These neuromotor disorders are characterized by exaggerated startle, muscle stiffness, and painful spasms, leading to immobility and fatal outcome in some cases. It was hypothesized that imbalance of motoneuronal inhibition by functional impairment of GlyR and receptor internalization are direct consequences of antibody-antigen interference. Here, serum samples of four patients were tested for anti-GlyR autoantibodies and were used for the analysis of the functional impact on the electrophysiological properties of recombinant GlyRs, transiently expressed in HEK293 cells. Furthermore, the recognition pattern of anti- GlyR autoantibodies to human, zebrafish and chimeric GlyRα1 located the epitope to the far N-terminal region. The pathogenicity of anti-GlyR autoantibodies and thereby the autoimmunologic etiology of the disease was confirmed by passive transfer of patient serum to zebrafish (Danio rerio) larvae, that yielded an abnormal escape response – a brain stem reflex that corresponds to the exaggerated startle of afflicted patients. The phenotype was accompanied by profound reduction of GlyR clusters in spinal cord cryosections of treated zebrafish larvae. Together, these novel insights into the pathogenicity of GlyR autoantibodies confirm the concept of a novel neurologic autoimmune disease and might contribute to the development of innovative therapeutic strategies.
The tropomysin receptor kinase B (TrkB), the receptor for the neurotrophin brain-derived neurotrophic factor (BDNF), plays an important role in neuronal survival, neuronal differentiation, and cellular plasticity. Conventionally, TrkB activation is induced by binding of BDNF at extracellular sites and subsequent dimerization of receptor monomers. Classical Trk signaling concepts have failed to explain ligand-independent signaling of intracellular TrkB or oncogenic NTRK-fusion proteins. The intracellular activation domain of TrkB consists of a tyrosine kinase core, with three tyrosine (Y) residues at positions 701, 705 and 706, that catalyzes the phosphorylation reaction between ATPγ and tyrosine. The release of cisautoinhibition of the kinase domain activates the kinase domain and tyrosine residues outside of the catalytic domain become phosphorylated. The aim of this study was to find out how ligand-independent activation of TrkB is brought about. With the help of phosphorylation mutants of TrkB, it has been found that a high, local abundance of the receptor is sufficient to activate TrkB in a ligand-independent manner. This self-activation of TrkB was blocked when either the ATP-binding site or Y705 in the core domain was mutated. The vast majority of this self-active TrkB was found at intracellular locations and was preferentially seen in roundish cells, lacking filopodia. Live cell imaging of actin dynamics showed that self-active TrkB changed the cellular morphology by reducing actin filopodia formation. Signaling cascade analysis confirmed that self-active TrkB is a powerful activator of focal adhesion kinase (FAK). This might be the reason why self-active TrkB is able to disrupt actin filopodia formation. The signaling axis from Y705 to FAK could be mimicked by expression of the soluble, cytosolic TrkB kinase domain. However, the signaling pathway was inactive, when the TrkB kinase domain was targeted to the plasmamembrane with the help of artificial myristoylation membrane anchors. A cancer-related intracellular NTRK2-fusion protein (SQSTM1-NTRK2) also underwent constitutive kinase activation. In glioblastoma-like U87MG cells, self-active TrkB kinase reduced cell migration. These constitutive signaling pathways could be fully blocked within minutes by clinically approved, anti-tumorigenic Trk inhibitors. Moreover, this study found evidences for constitutively active, intracellular TrkB in tissue of human grade IV glioblastoma. In conclusion, the data provide an explanation and biological function for selfactive, constitutive TrkB kinase domain signaling, in the absence of a ligand.
Significant advances in fluorescence imaging techniques enable life scientists today to gain insights into biological systems at an unprecedented scale. The interpretation of image features in such bioimage datasets and their subsequent quantitative analysis is referred to as bioimage analysis. A substantial proportion of bioimage analyses is still performed manually by a human expert - a tedious process that is long known to be subjective. Particularly in tasks that require the annotation of image features with a low signal-to-noise ratio, like in fluorescence images of tissue samples, the inter-rater agreement drops. However, like any other scientific analysis, also bioimage analysis has to meet the general quality criteria of quantitative research, which are objectivity, reliability, and validity. Thus, the automation of bioimage analysis with computer-aided approaches is highly desirable. Albeit conventional hard-coded algorithms are fully unbiased, a human user has to set its respective feature extraction parameters. Thus, also these approaches can be considered subjective.
Recently, deep learning (DL) has enabled impressive advances in computer vision research. The predominant difference between DL and conventional algorithms is the capability of DL models to learn the respective task on base of an annotated training dataset, instead of following user-defined rules for feature extraction. This thesis hypothesized that DL can be used to increase the objectivity, reliability, and validity of bioimage analyses, thus going beyond mere automation. However, in absence of ground truth annotations, DL models have to be trained on manual and thus subjective annotations, which could cause the model to incorporate such a bias. Moreover, model training is stochastic and even training on the same data could result in models with divergent outputs. Consequently, both the training on subjective annotations and the model-to-model variability could impair the quality of DL-based bioimage analyses. This thesis systematically assessed the impacts of these two limitations experimentally by analyzing fluorescence signals of a protein called cFOS in mouse brain sections. Since the abundance of cFOS correlates with mouse behavior, behavioral analyses could be used for cross-validation of the bioimage analysis results. Furthermore, this thesis showed that pooling the input of multiple human experts during model training and integration of multiple trained models in a model ensemble can mitigate the impact of these limitations. In summary, the present study establishes guidelines for how DL can be used to increase the general quality of bioimage analyses.
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.
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.
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.
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.
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 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.