@phdthesis{Ji2022,
  author    = {Ji, Changhe},
  title     = {The role of 7SK noncoding RNA in development and function of motoneurons},
  doi       = {10.25972/OPUS-22463},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-224638},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {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.},
  subject      = {Spliceosome},
  language  = {en}
}
@phdthesis{SoaresMachado2019,
  author    = {Soares Machado, J{\´e}ssica},
  title     = {Dosimetry-based Assessment of Radiation-associated Cancer risk for \(^9\)\(^9\)\(^m\)Tc-MAG3 Scans in Infants and Optimization of Administered Activities for \(^6\)\(^8\)Ga-labelled Peptides in Children and Adolescents},
  doi       = {10.25972/OPUS-19264},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-192640},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {In 2006, 0.18 Mio pediatric nuclear medicine diagnostic exams were performed worldwide. However, for most of the radiopharmaceuticals used data on biokinetics and, as a consequence on dosimetry, are missing or have not been made publicly available. Therefore, most of the dosimetry assessments presented today for diagnostic agents in children and adolescents rely on the biokinetics data of adults. Even for one of the most common nuclear medicine exams for this patient group, renal scintigraphy with 99mTc-MAG3 for assessing renal function measured data on biokinetics is available only from a study performed on four children of different ages. In particular, renal scans are among the most frequent exams performed on infants and toddlers. Due to the young age, this patient group can be classified as a risk group with a higher probability of developing stochastic radiation effects compared to adults. As there are only limited data on biokinetics and dosimetry in this patient group, the aim of this study is to reassess the dosimetry and the associated radiation risk for a larger number of infants undergoing 99mTc-MAG3 renal scans based on a retrospective analysis of existing patient data. Data were collected retrospectively from 34 patients younger than 20 months with normal (20 patients) and abnormal renal function (14 patients) undergoing 99mTc-MAG3 scans. The patient-specific organ activity was estimated based on a retrospective calibration which was performed based on a set of two 3D-printed infant kidneys (newborns: 8.6 ml; 1-year-old: 23.4 ml) filled with known activities. Both phantoms were scanned at different positions along the anteroposterior axis inside a water phantom, providing depth- and size-dependent attenuation correction factors for planar imaging. Time-activity curves were determined by drawing kidney, bladder, and whole body regions-of-interest for each patient, and subsequently applying the calibration factor for conversion of counts to activity. Patient-specific time-integrated activity coefficients were obtained by integrating the organ-specific time-activity curves. Absorbed and effective dose coefficients for each patient were assessed with OLINDA/EXM for the provided newborn and 1-year-old phantom. Based on absorbed dose values, the radiation risk estimation was performed individually for each of the 34 patients with the National Cancer Institute's Radiation Risk Assessment Tool. The patients' organ-specific mean absorbed dose coefficients for the patients with normal renal function were 0.04±0.03 mGy/MBq for the kidneys and 0.27±0.24 mGy/MBq for the bladder. This resulted in a mean effective dose coefficient of 0.02±0.02 mSv/MBq. Based on the dosimetry results, the evaluation of the excess lifetime risk (ELR) for the development of radiation-induced cancer showed that the group of newborns has an ELR of 16.8 per 100,000 persons, which is higher in comparison with the 1-year-old group with an ELR of 14.7 per 100,000 persons. With regard to the 14 patients with abnormal renal function, the mean values for the organ absorbed dose coefficients for the patients were: 0.40±0.34 mGy/MBq for the kidneys and 0.46±0.37 mGy/MBq for the bladder. The corresponding effective dose coefficients (mSv/MBq) was: 0.05±0.02 mSv/MBq. The mean ELR (per 100,000 persons) for developing cancer from radiation exposure for patients with abnormal renal function was 29.2±18.7 per 100,000 persons. As a result, the radiation-associated stochastic risk increases with the organ doses, taking age- and gender-specific influences into account. Overall, the lifetime radiation risk associated with the 99mTc-MAG3 scans is very low in comparison to the general population risk for developing cancer. Furthermore, due to the increasing demand for PET-scans in children and adolescents with 68Ga-labelled peptides, in this work published data sets for those compounds were analyzed to derive recommendations for the administered activities in children and adolescents. The recommendation for the activities to be administered were based on the weight-independent effective dose model, proposed by the EANM Pediatric Dosage Card for application in pediatric nuclear medicine. The aim was to derive recommendations on administered activities for obtaining age-independent effective doses. Consequently, the corresponding weight-dependent effective dose coefficients were rescaled according to the formalism of the EANM dosage card, to determine the radiopharmaceutical class of 68Ga-labeled peptides ("multiples"), and to calculate the baseline activities based on the biokinetics of these compounds and an upper limit of the administered activity of 185 MBq for an adult. Analogous to 18F-fluoride, a minimum activity of 14 MBq is recommended. As a result, for those pediatric nuclear medicine applications involving 68Ga-labeled peptides, new values for the EANM dosage card were proposed and implemented based on the results derived in this work. Overall, despite the low additional radiation-related cancer risk, all efforts should be undertaken to optimize administered activities in children and adolescents for obtaining sufficient diagnostic information with minimal associated radiation risk.},
  subject      = {Biokinetics},
  language  = {en}
}
@phdthesis{Sasi2020,
  author    = {Sasi, Manju},
  title     = {A mouse model for genetic deletion of presynaptic BDNF from adult hippocampal mossy fiber terminals},
  doi       = {10.25972/OPUS-18625},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-186250},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Brain-derived neurotrophic factor (BDNF) is a modulator and mediator of structural and functional plasticity at synapses in the central nervous system. Despite our profound knowledge about the synaptic function of BDNF at synapses, it is still controversially discussed whether synaptic BDNF acts primarily from pre- or postsynaptic sites. In the central nervous system, several studies show that mossy fiber (MF) projections formed by hippocampal granule neurons store the highest amount of BDNF. However, immunofluorescence and RNA labelling studies suggest that MF BDNF is primarily produced by granule neurons. Multiple other studies prefer the view that BDNF is primarily produced by postsynaptic neurons such as CA3 pyramidal neurons. Here, we question whether the BDNF, which is stored in the mossy fiber synapse, is primarily produced by granule neurons or whether by other cells in the MF-CA3 microcircuit. After standardization of immunolabelling of BDNF, confocal imaging confirmed the localization of BDNF in presynaptic MF terminals. This anterograde location of synaptic BDNF was also found in distinct regions of the fear and anxiety circuit, namely in the oval nucleus of the bed nucleus stria terminals (ovBNST) and in the central amygdala. To find out whether the presynaptic BDNF location is due to protein translation in the corresponding presynaptic dentate gyrus (DG) granule neuron, we developed and characterized a mouse model that exhibits BDNF deletion specifically from adult DG granule neurons. In this mouse model, loss of presynaptic BDNF immunoreactivity correlated with the specific Creactivity in granule neurons, thus confirming that MF BDNF is principally released by granule neurons. After BDNF deletion from granule neurons, we observed more immature neurons with widely arborized dendritic trees. This indicated that local BDNF deletion also affects the local adult neurogenesis, albeit Cre-mediated BDNF deletion only occur in adult granule neurons. Since BDNF is a master regulator of structural synaptic plasticity, it was questioned whether it is possible to visualize presynaptic, synapse-specific, structural plasticity in mossy fiber synapses. It was established that a combination of Cre-techniques together with targeting of GFP to membranes with the help of palmitoylation / myristoylation anchors was able to distinctly outline the synaptic structure of the BDNF-containing MF synapse. In summary, the mouse model characterized in here is suited to investigate the synaptic signalling function of presynaptic BDNF at the mossy fiber terminal, a model synapse to investigate microcircuit information processing from molecule to behaviour.},
  subject      = {Wachstumsfaktor},
  language  = {en}
}
@phdthesis{Schmitt2017,
  author    = {Schmitt, Dominique},
  title     = {Initial characterization of mouse Syap1 in the nervous system: Search for interaction partners, effects of gene knockdown and knockout, and tissue distribution with focus on the adult brain},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-147319},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {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.},
  subject      = {Synapse},
  language  = {en}
}
@phdthesis{Moradi2017,
  author    = {Moradi, Mehri},
  title     = {Differential roles of α-, β- and γ-actin isoforms in regulation of cytoskeletal dynamics and stability during axon elongation and collateral branch formation in motoneurons},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-147453},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {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.},
  subject      = {Motoneuron},
  language  = {en}
}
@phdthesis{Sivadasan2016,
  author    = {Sivadasan, Rajeeve},
  title     = {The role of RNA binding proteins in motoneuron diseases},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-141907},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {Motoneuron diseases form a heterogeneous group of pathologies characterized by the progressive degeneration of motoneurons. More and more genetic factors associated with motoneuron diseases encode proteins that have a function in RNA metabolism, suggesting that disturbed RNA metabolism could be a common underlying problem in several, perhaps all, forms of motoneuron diseases. Recent results suggest that SMN interacts with hnRNP R and TDP-43 in neuronal processes, which are not part of the classical SMN complex. This point to an additional function of SMN, which could contribute to the high vulnerability of spinal motoneurons in spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). The current study elucidates functional links between SMN, the causative factor of SMA (spinal muscular atrophy), hnRNP R, and TDP-43, a genetic factor in ALS (amyotrophic lateral sclerosis). In order to characterize the functional interaction of SMN with hnRNP R and TDP-43, we produced recombinant proteins and investigated their interaction by co-immunoprecipitation. These proteins bind directly to each other, indicating that no other co-factors are needed for this interaction. SMN potentiates the ability of hnRNP R and TDP-43 to bind to ß-actin mRNA. Depletion of SMN alters the subcellular distribution of hnRNP R in motoneurons both in SMN-knockdown motoneurons and SMA mutant mouse (delta7 SMA). 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 and ALS. ALS and FTLD (frontotemporal lobar degeneration) are linked by several lines of evidence with respect to clinical and pathological characteristics. Both sporadic and familial forms are a feature of the ALS-FTLD spectrum, with numerous genes having been associated with these pathological conditions. Both diseases are characterized by the pathological cellular aggregation of proteins. Interestingly, some of these proteins such as TDP-43 and FUS have also common relations not only with ALS-FTLD but also with SMA. Intronic hexanucleotide expansions in C9ORF72 are common in ALS and FTLD but it is unknown whether loss of function, toxicity by the expanded RNA or dipeptides from non ATG-initiated translation is responsible for the pathophysiology. This study tries to characterize the cellular function of C9ORF72 protein. To address this, lentiviral based knockdown and overexpression of C9ORF72 was used in isolated mouse motoneurons. The results clearly show that survival of these motoneurons was not affected by altered C9ORF72 levels, whereas adverse effects on axon growth and growth cone size became apparent after C9ORF72 suppression. Determining the protein interactome revealed several proteins in complexes with C9ORF72. Interestingly, C9ORF72 is present in a complex with cofilin and other actin binding proteins that modulate actin dynamics. These interactions were confirmed both by co-precipitation analyses and in particular by functional studies showing altered actin dynamics in motoneurons with reduced levels of C9ORF72. Importantly, the phosphorylation of cofilin is enhanced in C9ORF72 depleted motoneurons and patient derived lymphoblastoid cells with reduced C9ORF72 levels. These findings indicate that C9ORF72 regulates axonal actin dynamics and the loss of this function could contribute to disease pathomechanisms in ALS and FTLD.},
  subject      = {Motoneuron},
  language  = {en}
}
@phdthesis{RuedtvonCollenberg2021,
  author    = {R{\"u}dt von Collenberg, Cora Freifrau},
  title     = {The role of Ciliary Neurotrophic Factor in hippocampal synaptic plasticity and learning},
  doi       = {10.25972/OPUS-20664},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-206646},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Ciliary neurotrophic factor (Cntf) acts as a differentiation and survival factor for different types of neurons and glial cells. It is expressed by peripheral Schwann cells and astrocytes in the central nervous system and mediates its effects via a receptor complex involving CntfRα, LifRß and gp130, leading to downstream activation of Stat3. Recent studies by our group have shown that Cntf modulates neuronal microtubule dynamics via Stat3/stathmin interaction. In a mouse model for motor neuron disease, i.e. pmn, Cntf is able to rescue axonal degeneration through Stat3/stathmin signaling. While these findings suggest a role of Cntf in controlling axonal functions in the neuromuscular system, additional data indicate that Cntf might also play a role in synaptic plasticity in the hippocampus. Electrophysiological recordings in hippocampal organotypic cultures and acute slices revealed a deficit in long-term potentiation (LTP) in Cntf -/- mice. This deficit was rescued by 24 h stimulation with Cntf, combined with an acute application of Cntf during LTP-measurements indicating that Cntf is both necessary and sufficient for hippocampal LTP, and possibly synaptic plasticity. Therefore, Cntf knockout mice were investigated to elucidate this possible role of Cntf in hippocampal LTP and synaptic plasticity. First, we validated the presence of Cntf in the target tissue: in the hippocampus, Cntf was localized in Gfap-positive astrocytes surrounding small blood vessels in the fissure and in meningeal areas close to the dentate gyrus. Laser micro-dissection and qPCR analysis showed a similar distribution of Cntf-coding mRNA validating the obtained immunofluorescent results. Despite the strong LTP deficit in organotypic cultures, in vivo behavior of Cntf -/- mice regarding hippocampus-dependent learning and anxiety-related paradigms was largely inconspicuous. However, western blot analysis of hippocampal organotypic cultures revealed a significant reduction of pStat3 levels in Cntf -/- cultures under baseline conditions, which in turn were elevated upon Cntf stimulation. In order to resolve and examine synaptic structures we turned to in vitro analysis of cultured hippocampal neurons which indicated that pStat3 is predominantly located in the presynapse. In line with these findings, presynapses of Cntf -/- cultures were reduced in size and when in contact to astrocytes, contained less pStat3 immunoreactivity compared to presynapses in wildtype cultures. In conclusion, our findings hypothesize that despite of a largely inconspicuous behavioral phenotype of Cntf -/- mice, Cntf appears to have an influence on pStat3 levels at hippocampal synapses. In a next step these two key questions need to be addressed experimentally: 1) is there a compensatory mechanism by members of the Cntf family, possibly downstream of pStat3, which explains the in vivo behavioral results of Cntf -/- mice and can likewise account for the largely inconspicuous phenotype in CNTF-deficient humans? 2) How exactly does Cntf influence LTP through Stat3 signaling? To unravel the underlying mechanism further experiments should therefore investigate whether microtubule dynamics downstream of Stat3 and stathmin signaling are involved in the Cntf-induced modulation of hippocampal synaptic plasticity, similar to as it was shown in motoneurons.},
  subject      = {Hippocampus},
  language  = {en}
}
@phdthesis{Balk2020,
  author    = {Balk, Stefanie Margarete},
  title     = {Der Einfluss des Kalziumkanalagonisten R-Roscovitine auf die zellul{\"a}re Differenzierung von Motoneuronen eines Mausmodells f{\"u}r Spinale Muskelatrophie Typ 1 (SMA)},
  doi       = {10.25972/OPUS-18986},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-189861},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Die spinale Muskelatrophie (SMA) ist eine monogenetische Erkrankung, bei der es durch den Verlust des SMN Proteins zur Degeneration der α-Motoneurone im R{\"u}ckenmark kommt. Abh{\"a}ngig vom Schweregrad zeigen die Patienten bereits innerhalb der ersten Lebensmonate ausgepr{\"a}gte L{\"a}hmungen der Skelettmuskulatur und eine Zwerchfellparese einhergehend mit einer reduzierten Lebenserwartung. Mithilfe von Mausmodellen f{\"u}r die SMA konnte gezeigt werden, dass der Motoneuronenverlust bei Smn-defizienten M{\"a}usen mit St{\"o}rungen der Neurotransmission an der motorischen Endplatte und mit Differenzierungsst{\"o}rungen der Motoneurone einhergeht. Die Differenzierungs-st{\"o}rungen prim{\"a}rer Smn-defizienter Motoneurone sind eng gekoppelt mit einer verminderten Clusterbildung spannungsabh{\"a}ngiger Kalziumkan{\"a}le im distalen axonalen Bereich. Dies wiederum f{\"u}hrt zu einer verminderten Frequenz spontaner Kalziumeinstr{\"o}me am Axonterminus und hat eine ver{\"a}nderte axonale Elongation zur Folge. Es wurden folgende Aspekte in Bezug auf die Verst{\"a}rkung und die Induktion spontaner Kalziumeinstr{\"o}me in Mausmodellen f{\"u}r spinale Muskelatrophien in dieser Arbeit adressiert: 1) Lassen sich spontane Kalziumeinstr{\"o}me in Smn-defizienten Motoneuronen durch die externe Applikation von Kalziumkanalagonisten verst{\"a}rken? 2) Sind spontane Kalziumeinstr{\"o}me in prim{\"a}ren Motoneuronen durch den Brain-derived-neurotrophic-factor (BDNF) induzierbar? 3) Zeigen prim{\"a}re Motoneurone eines Mausmodells f{\"u}r spinale Muskelatrophie mit Ateminsuffizienz Typ 1 (SMARD1) ebenfalls ver{\"a}nderte Kalziumtransienten? Die Ergebnisse meiner Arbeit zeigen, dass durch den Kalziumkanalagonisten R-Roscovitine die Frequenz der spontanen Kalziumeinstr{\"o}me im distalen Axon von Smn-defizienten Motoneuronen signifikant erh{\"o}ht wird. Dies hat wiederum einen regulierenden Effekt auf die Differenzierung der SMA Motoneurone zur Folge. Smn-defiziente Motoneurone zeigen somit keine Unterschiede mehr in Bezug auf Axonl{\"a}ngen und Wachstumskegelfl{\"a}chen im Vergleich zu Kontrollzellen. F{\"u}r R- 10 Roscovitine ist neben der agonistischen Wirkung am Kalziumkanal auch ein inhibitorischer Effekt auf die Cyclin-abh{\"a}ngige Kinase 5 beschrieben. Es konnte jedoch gezeigt werden, dass die erh{\"o}hten Kalziumtransienten unter der Behandlung mit R-Roscovitine durch eine direkte Bindung an die Cav2 Kalziumkan{\"a}le verursacht werden und nicht durch eine Cdk5 Blockade. Daf{\"u}r spricht die schnelle und reversible Wirkung von R-Roscovitine, sowie die Aufhebung des R-Roscovitines Effekts bei gleichzeitiger Gabe des Cav2.2 Antagonisten ω-Conotoxin MVIIC. Der zweite Aspekt dieser Arbeit behandelt den Einfluss der neurotrophen Faktoren BDNF, CNTF und GDNF auf die Kalziumtransienten am Wachstumskegel wildtypischer Motoneurone. Der Vergleich der neurotrophen Faktoren zeigt, dass nur BDNF eine induzierende Wirkung auf spontane Kalziumtransienten am Wachstumskegel hat. Der letzte Abschnitt dieser Arbeit besch{\"a}ftigt sich mit den Kalziumtransienten bei Motoneuronen aus dem Nmd2J (SMARD1) Mausmodell. Die SMARD1 gilt als eigenst{\"a}ndige Form der spinalen Muskelatrophien mit unterschiedlicher Genetik und unterschiedlichen klinischen Merkmalen. Die Motoneurone weisen in Bezug auf die Kalziumtransienten keine Unterschiede zwischen Wildtyp und Nmd2J Mutante auf. Es ergibt sich somit kein Hinweis darauf, dass die Degeneration der Motoneurone bei der SMARD1 von einer St{\"o}rung der Kalziumhom{\"o}ostase im distalen axonalen Bereich ausgeht.},
  subject      = {Spinal muscular atrophy (DLC)},
  language  = {de}
}
@phdthesis{Drexl2018,
  author    = {Drexl, Hans Henning},
  title     = {Der Einfluss von R-Roscovitine und Valproat auf das Wachstums- und pr{\"a}synaptische Differenzierungsverhalten SMN-defizienter Motoneurone},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-171696},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Die spinale Muskelatrophie ist eine monogenetische Erkrankung, die bereits im Kindesalter aufgrund von Motoneurondegeneration zu Muskelatrophie f{\"u}hrt und nicht selten einen t{\"o}dlichen Verlauf nimmt. Ursache der Erkrankung ist ein Mangel an SMN-Protein. Der hierf{\"u}r verantwortliche Verlust des SMN1-Gens kann durch das SMN2-Gen aufgrund eines gest{\"o}rten Spleißprozesses am Exon 7 nicht kompensiert werden. Neben Aufgaben in der RNA-Prozessierung wird das SMN-Protein f{\"u}r den axonalen Transport von Ribonucleinpartikeln in Motoneuronen ben{\"o}tigt, was bei der SMA zu pathologischem Wachstum, Differenzierung und Funktion der Motoraxone f{\"u}hrt. Im Rahmen dieser Arbeit wurden kultivierte Motoneurone aus einem Mausmodell f{\"u}r die SMA Typ I (Genotyp Smn-/-;SMN2) mit zwei unterschiedlichen Substanzen behandelt und deren Wirkungen auf das pr{\"a}synaptische Differenzierungsverhalten der Motoneurone verglichen: R-Roscovitine, ein Agonist/Modulator spannungsabh{\"a}ngiger N-Typ- und P/Q-Typ-Kalziumkan{\"a}le, welcher zudem eine CDK-inhibierende Wirkung besitzt, sowie Valproat, ein HDAC-Inhibitor, der eine stimulierende Wirkung auf die SMN-Transkription hat. Es zeigte sich, dass R-Roscovitine in der Lage ist, das pathologische Wachstums- und pr{\"a}synaptische Differenzierungsverhalten der Smn-defizienten Motoneurone zu normalisieren, ohne hierbei Einfluss auf die erniedrigte Menge an Smn-Protein zu nehmen. Die Behandlung mit Valproat beeinflusst hingegen weder die Menge an Smn-Protein, noch die pathologische Differenzierung der Wachstumskegel Smn-defizienter Motoneurone. Erkl{\"a}ren lassen sich diese Effekte in erster Linie durch den Agonismus an spannungsabh{\"a}ngigen Kalziumkan{\"a}len durch R-Roscovitine. Durch vermehrten Kalziumeinstrom kommt es zur Normalisierung von Struktur und Funktion der Wachstumskegel. Ein CDK-vermittelter Effekt scheint unwahrscheinlich. Obgleich die genauen Vorg{\"a}nge noch nicht verstanden sind, zeigen diese Ergebnisse, dass sich Smn-defiziente Motoneurone normal entwickeln k{\"o}nnen, wenn die hierf{\"u}r erforderlichen kalziumabh{\"a}ngigen pr{\"a}synaptischen Differenzierungssignale korrekt ausgel{\"o}st werden. Bei weiterer Erforschung sind Therapeutika denkbar, die in Zukunft die {\"u}berwiegend genetisch orientierten Therapieans{\"a}tze zur Hochregulation der SMN-Expression bei SMA-Patienten {\"u}ber einen von der Genetik unabh{\"a}ngigen Wirkmechanismus unterst{\"u}tzen k{\"o}nnen.},
  subject      = {Spinale Muskelatrophie},
  language  = {de}
}
@phdthesis{Frank2015,
  author    = {Frank, Nicolas Clemens},
  title     = {Lokale axonale Wirkungen der CNTF-STAT3 Signalkaskade in Motoneuronen der pmn Maus - einem Mausmodel f{\"u}r die Amyotrophe Lateralsklerose},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-121065},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {1. Zusammenfassung W{\"a}hrend der Embryogenese und nach Verletzungen von Nerven regulieren neurotrophe Faktoren Signalwege f{\"u}r Apoptose, Differenzierung, Wachstum und Regeneration von Neuronen. In vivo Experimente an neugeborenen Nagern haben gezeigt, dass der Verlust von Motoneuronen nach peripherer Nervenl{\"a}sion durch die Behandlung mit GDNF, BDNF, und CNTF reduziert werden kann In der pmn-Mausmutante, einem Modell f{\"u}r die Amyotrophe Lateralsklerose, f{\"u}hrt die Gabe von CNTF, nicht aber von GDNF zu einem verz{\"o}gerten Krankheitsbeginn und einem verlangsamten Fortschreiten der Motoneuronendegeneration. Ausl{\"o}ser der Motoneuronendegeneration in der pmn-Maus ist eine Mutation im Tubulin spezifischen Chaperon E (Tbce) Gen, das f{\"u}r eines von f{\"u}nf Tubulin spezifischen Chaperonen (TBCA-TBCE) kodiert und an der Bildung von -Tubulinheterodimeren beteiligt ist. Diese Arbeit sollte dazu beitragen, die CNTF-induzierten Signalwege zu entschl{\"u}sseln, die sich lindernd auf den progredienten Verlauf der Motoneuronendegeneration in der pmn-Maus auswirken. Prim{\"a}re pmn mutierte Motoneurone zeigen ein reduziertes Axonwachstum und eine erh{\"o}hte Anzahl axonaler Schwellungen mit einer anomalen H{\"a}ufung von Mitochondrien - ein fr{\"u}hes Erkennungsmerkmal bei ALS-Patienten. Die Applikation von CNTF nicht aber von BDNF oder GDNF, kann in vitro die beobachteten Wachstumsdefekte und das bidirektionale axonale Transportdefizit in pmn mutierten Motoneurone verhindern. Aus {\"a}lteren Untersuchungen war bekannt, dass CNTF {\"u}ber den dreiteiligen transmembranen Rezeptorkomplex, bestehend aus CNTFR, LIFR und gp130, Januskinasen aktiviert, die STAT3 an Tyrosin 705 phosphorylieren (pSTAT3Y705). Ich konnte beobachten, dass axonales fluoreszenzmarkiertes pSTAT3Y705 nach CNTF-Gabe nicht retrograd in den Nukleus transportiert wird. Stattdessen f{\"u}hrt die CNTF-induzierte Phosphorylierung von STAT3 an Tyrosin 705 zu einer transkriptionsunabh{\"a}ngigen lokalen Reaktion im Axon. Diese pSTAT3Y705 abh{\"a}ngige Reaktion ist notwendig und ausreichend, um das reduzierte Axonwachstum pmn mutierter Motoneurone zu beheben. Wie die Kombination einer CNTF Behandlung mit dem shRNA vermittelten knock-down von Stathmin in pmn mutierten Motoneuronen zeigt, zielt die CNTF-STAT3 Signalkaskade auf die Stabilisierung axonaler Mikrotubuli ab und wirkt sich positiv auf die anterograde und retrograde Mobilit{\"a}t von axonalen Mitochondrien aus. Interessanter Weise konnte ich außerdem feststellen, dass eine akute Gabe von CNTF das mitochondriale Membranpotential in Axonen prim{\"a}rer pmn mutierter und wildtypischer Motoneurone erh{\"o}ht und einen Anstieg von ATP ausl{\"o}st. Meine Beobachtungen legen nahe, dass CNTF unerwarteter Weise auch eine transiente Phosphorylierung an STAT3 Serin 727 (pSTAT3S727) ausl{\"o}st, die zur anschließenden Translokation von pSTAT3S727 in Mitochondrien f{\"u}hrt. Diese Ergebnisse zeigen, dass STAT3 mehrere lokale Ziele im Axon besitzt, n{\"a}mlich axonale Mikrotubuli und Mitochondrien.},
  subject      = {Motoneuron},
  language  = {de}
}
@phdthesis{Saal2017,
  author    = {Saal, Lena},
  title     = {Whole transcriptome profiling of compartmentalized motoneurons},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-140006},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {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.},
  subject      = {Axon},
  language  = {en}
}
@phdthesis{Yadav2016,
  author    = {Yadav, Preeti},
  title     = {Studying Neuronal Cytoskeleton Defects and Synaptic Defects in Mouse Model of Amyotrophic Lateral Sclerosis and Spinal Muscular Atrophy},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-138093},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {Amyotrophic lateral sclerosis and spinal muscular atrophy are the two most common motoneuron diseases. Both are characterized by destabilization of axon terminals, axon degeneration and alterations in neuronal cytoskeleton. Accumulation of neurofilaments has been observed in several neurodegenerative diseases but the mechanisms how elevated neurofilament levels destabilize axons are unknown so far. Here, I show that increased neurofilament expression in motor nerves of pmn mutant mice causes disturbed microtubule dynamics. Depletion of neurofilament by Nefl knockout increases the number and regrowth of microtubules in pmn mutant motoneurons and restores axon elongation. This effect is mediated by interaction of neurofilament with the stathmin complex. Depletion of neurofilament increases stathmin-Stat3 interaction and stabilizes the microtubules. Consequently, the axonal maintenance is improved and the pmn mutant mice survive longer. We propose that this mechanism could also be relevant for other neurodegenerative diseases in which neurofilament accumulation is a prominent feature. Next, using Smn-/-;SMN2 mouse as a model, the molecular mechanism behind synapse loss in SMA is studied. SMA is characterized by degeneration of lower α-motoneurons in spinal cord; however, how reduction of ubiquitously expressed SMN leads to MN-specific degeneration remains unclear. SMN is involved in pre-mRNA splicing (Pellizzoni, Kataoka et al. 1998) and its deficiency in SMA affects the splicing machinery. Neuromuscular junction denervation precedes neurodegeneration in SMA. However, there is no evidence of a link between aberrant splicing of transcripts downstream of Smn and reduced presynaptic axon excitability observed in SMA. In this study, we observed that expression and splicing of Nrxn2, that encodes a presynaptic protein is affected in the SMA mouse and that Nrxn2 could be a candidate that relates aberrant splicing to synaptic motoneuron defects in SMA.},
  subject      = {Neurofilament},
  language  = {en}
}
@phdthesis{BlancoRedondo2014,
  author    = {Blanco Redondo, Beatriz},
  title     = {Studies of synapsin phosphorylation and characterization of monoclonal antibodies from the W{\"u}rzburg Hybridoma Library in Drosophila melanogaster},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-93766},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {Synapsins are conserved synapse-associated hosphoproteins involved in the fine regulation of neurotransmitter release. The aim of the present project is to study the phosphorylation of synapsins and the distribution of phospho-synapsin in the brain of Drosophila melanogaster. Three antibodies served as important tools in this work, a monoclonal antibody (3C11/α-Syn) that recognizes all known synapsin isoforms and two antisera against phosphorylated synapsin peptides (antiserum PSyn(S6) against phospho-serine 6 and antiserum PSyn(S464) against phospho-serine 464). These antisera were recently generated in collaboration with Bertram Gerber and Eurogentec. ...},
  subject      = {Synapsine},
  language  = {en}
}
@phdthesis{Andreska2021,
  author    = {Andreska, Thomas},
  title     = {Effects of dopamine on BDNF / TrkB mediated signaling and plasticity on cortico-striatal synapses},
  doi       = {10.25972/OPUS-17431},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-174317},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Progressive loss of voluntary movement control is the central symptom of Parkinson's disease (PD). Even today, we are not yet able to cure PD. This is mainly due to a lack of understanding the mechanisms of movement control, network activity and plasticity in motor circuits, in particular between the cerebral cortex and the striatum. Brain-derived neurotrophic factor (BDNF) has emerged as one of the most important factors for the development and survival of neurons, as well as for synaptic plasticity. It is thus an important target for the development of new therapeutic strategies against neurodegenerative diseases. Together with its receptor, the Tropomyosin receptor kinase B (TrkB), it is critically involved in development and function of the striatum. Nevertheless, little is known about the localization of BDNF within presynaptic terminals in the striatum, as well as the types of neurons that produce BDNF in the cerebral cortex. Furthermore, the influence of midbrain derived dopamine on the control of BDNF / TrkB interaction in striatal medium spiny neurons (MSNs) remains elusive so far. Dopamine, however, appears to play an important role, as its absence leads to drastic changes in striatal synaptic plasticity. This suggests that dopamine could regulate synaptic activity in the striatum via modulation of BDNF / TrkB function. To answer these questions, we have developed a sensitive and reliable protocol for the immunohistochemical detection of endogenous BDNF. We find that the majority of striatal BDNF is provided by glutamatergic, cortex derived afferents and not dopaminergic inputs from the midbrain. In fact, we found BDNF in cell bodies of neurons in layers II-III and V of the primary and secondary motor cortex as well as layer V of the somatosensory cortex. These are the brain areas that send dense projections to the dorsolateral striatum for control of voluntary movement. Furthermore, we could show that these projection neurons significantly downregulate the expression of BDNF during the juvenile development of mice between 3 and 12 weeks. In parallel, we found a modulatory effect of dopamine on the translocation of TrkB to the cell surface in postsynaptic striatal Medium Spiny Neurons (MSNs). In MSNs of the direct pathway (dMSNs), which express dopamine receptor 1 (DRD1), we observed the formation of TrkB aggregates in the 6-hydroxydopamine (6-OHDA) model of PD. This suggests that DRD1 activity controls TrkB surface expression in these neurons. In contrast, we found that DRD2 activation has opposite effects in MSNs of the indirect pathway (iMSNs). Activation of DRD2 promotes a rapid decrease in TrkB surface expression which was reversible and depended on cAMP. In parallel, stimulation of DRD2 led to induction of phospho-TrkB (pTrkB). This effect was significantly slower than the effect on TrkB surface expression and indicates that TrkB is transactivated by DRD2. Together, our data provide evidence that dopamine triggers dual modes of plasticity on striatal MSNs by acting on TrkB surface expression in DRD1 and DRD2 expressing MSNs. This surface expression of the receptor is crucial for the binding of BDNF, which is released from corticostriatal afferents. This leads to the induction of TrkB-mediated downstream signal transduction cascades and long-term potentiation (LTP). Therefore, the dopamine-mediated translocation of TrkB could be a mediator that modulates the balance between dopaminergic and glutamatergic signaling to allow synaptic plasticity in a spatiotemporal manner. This information and the fact that TrkB is segregated to persistent aggregates in PD could help to improve our understanding of voluntary movement control and to develop new therapeutic strategies beyond those focusing on dopaminergic supply.},
  subject      = {Brain-derived neurotrophic factor},
  language  = {en}
}
@phdthesis{Juergens2020,
  author    = {J{\"u}rgens, Lukas Julian Christoph},
  title     = {Spatio-temporale Distribution der Tubuline und Tubulin spezifischen Chaperone im sensorischen Epithel der murinen Cochlea},
  doi       = {10.25972/OPUS-20649},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-206498},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Die f{\"u}nf Tubulin-bindenden Kofaktoren (TBC) sind an der Tubulinsynthese und der Bildung von Mikrotubuli beteiligt. Ihre Bedeutung wird durch verschiedene Krankheiten und Syndrome hervorgehoben, die durch Funktionsst{\"o}rungen oder Mutationen dieser Proteine verursacht werden. Posttranslationale Modifikationen (PTMs) von Tubulin f{\"o}rdern verschiedene Eigenschaften, einschließlich stabilit{\"a}tsf{\"o}rdernder Subpopulationen von Tubulin. Die zell- und zeitspezifische Verteilung der PTMs ist bisher nur im Corti-Organ bei Gerbils untersucht worden. Ziel der vorliegenden Studie war es, die zelltyp- und zeitspezifischen Expressionsmuster von TBC-Proteinen und PTMs erstmals in der murinen Cochlea {\"u}ber mehrere Entwicklungsstadien hinweg zu untersuchen. Dazu wurden murine Cochleae im postnatalen (P) Alter P1, P7 und P14 mittels Immunfluoreszenzanalyse untersucht. Die Untersuchungen zeigten mehrere erhebliche Interspezies-Unterschiede in der Verteilung der PTMs zwischen Gerbil und Maus. Dar{\"u}ber hinaus ist dies die erste Studie, die die r{\"a}umlich-zeitliche Verteilung von TBCs in einem Gewebe beschreibt, das ein volatiles Expressionsmuster aufweist. Die Expressionsanalyse von TBC-Proteinen und PTMs des Tubulins zeigt, dass diese Proteine eine wichtige Rolle bei der physiologischen Entwicklung der Cochlea spielen und f{\"u}r das H{\"o}ren essentiell sein k{\"o}nnten.},
  subject      = {Mikrotubulus},
  language  = {de}
}
@phdthesis{Hennlein2023,
  author    = {Hennlein, Luisa},
  title     = {Plastin 3 rescues defective cell surface translocation and activation of TrkB in mouse models for spinal muscular atrophy},
  publisher = {Journal of Cell Biology},
  doi       = {10.25972/OPUS-29879},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-298793},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Spinal muscular atrophy (SMA) is a genetic pediatric condition that affects lower motoneurons leading to their degeneration and muscle weakness. It is caused by homozygous loss or mutations in the Survival Motor Neuron 1 (SMN1) gene; however, the pathomechanism leading to motoneuron degeneration is not fully resolved. Cultured embryonic SMA motoneurons display axon elongation and differentiation defects accompanied by collapsed growth cones with a disturbed actin cytoskeleton. Intriguingly, motoneurons cultured from mice deficient for the Tropomyosin-kinase receptor B (TrkB), exhibit similar pathological features. Thus, the question arises whether SMA motoneurons suffer from defective Brain-derived neurotrophic factor (BDNF)/TrkB signaling and whether there is a link to the disturbed actin cytoskeleton. In the recent years, modifier genes such as Plastin 3 (PLS3) were shown to beneficially interfere with SMA pathology. Nevertheless, the mechanism of how the actin-bundler PLS3 counteracts SMN deficiency is not well understood. In this study, we investigated TrkB localization and its activation in cultured SMA motoneurons and neuromuscular junctions (NMJs). While TrkB levels are only mildly affected locally in axon terminals, BDNF-mediated TrkB phosphorylation was massively disturbed. The activity-dependent TrkB translocation to the cell surface and its activation via BDNF were shown to be Pls3-dependent processes, that can be abolished by knockdown of Pls3. In contrast, PLS3 overexpression in SMA motoneurons rescued the defects on morphological and functional level. In particular, the relocation of TrkB to the cell surface after BDNF-induced internalization is disturbed in SMA, which is based on an actin-dependent TrkB translocation defect from intracellular stores. Lastly, AAV9-mediated PLS3 overexpression in vivo in neonatal SMA mice provided further evidence for the capacity of PLS3 to modulate actin dynamics necessary for accurate BDNF/TrkB signaling. In conclusion, we provide a novel role for PLS3 in mediating proper alignment of transmembrane proteins as prerequisite for their appropriate functioning. Hence, PLS3 is required for a key process indispensable for the development and function of motoneurons even beyond the context of SMA.},
  subject      = {Spinale Muskelatrophie},
  language  = {en}
}
@phdthesis{Ghanawi2022,
  author    = {Ghanawi, Hanaa},
  title     = {Loss of full-length hnRNP R isoform impairs DNA damage response in motoneurons by inhibiting Yb1 recruitment to Chromatin},
  doi       = {10.25972/OPUS-25849},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-258492},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {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.},
  language  = {en}
}
@phdthesis{Deng2023,
  author    = {Deng, Chunchu},
  title     = {Dynamic remodeling of endoplasmic reticulum and ribosomes in axon terminals of wildtype and Spinal Muscular Atrophy motoneurons},
  doi       = {10.25972/OPUS-26495},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-264954},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {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.},
  subject      = {Motoneuron},
  language  = {en}
}
@phdthesis{Mueller2023,
  author    = {M{\"u}ller, Erich-Engelbert},
  title     = {Der Einfluss des Ciliary Neurotrophic Factor (CNTF) auf die mikroskopische Anatomie des Sehnervs und der Retina im Mausmodell},
  doi       = {10.25972/OPUS-33010},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-330108},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Der Einfluss des Ciliary Neurotrophic Factor (CNTF) auf die mikroskopische Anatomie des Sehnervs und der Retina wurde im Mausmodell untersucht. Unter Verwendung von Immunhistochemie, konfokaler Lasermikroskopie und Elektronenmikroskopie wurde untersucht, inwieweit eine CNTF-Defizienz zu degenerativen Ver{\"a}nderungen in Sehnerv und Retina von insbesondere adulten M{\"a}usen f{\"u}hrt. Hinsichtlich der verschiedenen untersuchten Parameter, einschließlich der Myelinisierung des Sehnervs und der retinalen Schichtung, konnten keine signifikanten Unterschiede zwischen CNTF-defizienten und Wild-Typ-M{\"a}usen festgestellt werden.},
  subject      = {Sehnerv},
  language  = {de}
}