The neuromuscular junction (NMJ), the synapse formed between lower motor neuron and skeletal muscle fibre, is known to be a target in a number of neurodegenerative conditions, including motor neuron disease (MND). Located in an accessible part of the peripheral nervous system, the NMJ can be used as a ‘model synapse’ in the context of ‘connectomics’ – the study of synaptic connectivity throughout the nervous system as a whole. Although the NMJ has been studied in a number of species, relatively little is known about its structure in humans, complicating the translation of animal models of disease to the human condition. Described here is the first detailed cellular and molecular characterization of the human NMJ. A standardized methodology for comparative morphometric analysis of NMJs was developed and validated (‘NMJ-morph’). NMJ-morph was used to generate baseline data for 2160 NMJs from a single litter of wild type mice, representing 9 distinct muscles across 3 body regions. Principal components analysis (PCA) revealed synaptic size and fragmentation to be the key determinants of synaptic variability. Correlation data revealed the pre-synaptic cell (motor neuron) to be a stronger predictor of synaptic morphology than the post-synaptic cell (muscle fibre). Other factors influencing synaptic variability were in a clear hierarchy: muscle identity accounted for more variation in synaptic form than animal identity, with side having no effect. Human tissue was obtained from 20 patients (aged 34 to 92 years) undergoing lower limb amputation, primarily for the complications of peripheral vascular disease (PVD). Muscle samples were harvested from non-pathological regions of the surgical discard tissue. 2860 human NMJs were analyzed from 4 distinct muscles (extensor digitorum longus, soleus, peroneus longus and peroneus brevis), and compared with equivalent NMJs from wild type mice. Human NMJs displayed unique morphological characteristics, including small size, thin axons, rudimentary nerve terminals and distinctive ‘nummular’ endplates, all of which distinguished them from equivalent mouse NMJs. The previous notion of partial occupancy in human NMJs was disproved. As in mice, the pre-synaptic cell was shown to correlate more strongly with NMJ morphology; in contrast to mice, the human NMJ was found to be relatively stable throughout its 90+ year lifespan. In support of the tissue harvesting procedure, patient co-morbidities (diabetes mellitus and vascular disease) did not significantly impact NMJ morphology. Super-resolution imaging of the NMJ revealed significant differences in the functional architecture of human and mouse active zones. Despite the smaller synaptic size in humans, the total quantity of active zone material was conserved between the species, suggesting a homeostatic mechanism to preserve effective neurotransmission. Parallel proteomic profiling demonstrated further species-specific differences in the broader molecular composition of the NMJ. The cellular and molecular anatomy of the human NMJ is fundamentally different to that of other mammalian species. These differences must be taken into account when translating animal models of disease to the human condition.

Mounting evidence suggests that synaptic connections are early pathological targets in many neurodegenerative diseases, including motor neuron disease. A better understanding of synaptic pathology is therefore likely to be critical in order to develop effective therapeutic strategies. Spinal muscular atrophy (SMA) is a common autosomal recessive childhood form of motor neuron disease. Previous studies have highlighted nerve- and muscle-specific events in SMA, including atrophy of muscle fibres and postsynaptic motor endplates, loss of lower motor neuron cell bodies and denervation of neuromuscular junctions caused by loss of pre-synaptic inputs. Here I have undertaken a detailed morphological investigation of neuromuscular synaptic pathology in the Smn-/- ;SMN2 and Smn-/-;SMN2;Δ7 mouse models of SMA. Results imply that synaptic degeneration is an early and significant event in SMA, with progressive denervation and neurofilament accumulation being present at early symptomatic time points. I have identified selectively vulnerable motor units, which appear to conform to a distinct developmental subtype compared to more stable motor units. I have also identified significant postsynaptic atrophy which does no correlate with pre-synaptic denervation, suggesting that there is a requirement for Smn in both muscle and nerve and pathological events can occur in both tissues independently. Rigorous investigation of lower motor neuron development, connectivity and gene expression at pre-symptomatic time points revealed developmental abnormalities do not underlie neuromuscular vulnerability in SMA. Equivalent gene expression analysis at end-stage time points has implicated growth factor signalling and extracellular matrix integrity in SMA pathology. Using an alternative model of early onset neurodegeneration, I provide evidence that the processes regulating morphologically distinct types of synaptic degeneration are also mechanistically distinct. In summary, in this work I highlight the importance and incidence of synaptic pathology in mouse models of spinal muscular atrophy and provide mechanistic insight into the processes regulating neurodegeneration.

Drosophila melanogaster is an excellent model system to identify genes involved in synaptic growth and function. In Drosophila, the Serrate (Ser) gene encodes a transmembrane protein that is a ligand for Notch receptor. Several previous studies implicated a role for Serrate in normal wing development and patterning. In this study, I demonstrate that Serrate is required for normal synaptic growth and function. I characterized the phenotype of a Serrate mutation (serB936) that was identified by an EMS-induced genetic screen aimed at identifying novel genes that play a role in synaptic growth and function. Co-localization studies show that Serrate protein is expressed at both the pre- and postsynaptic side of larval neuromuscular junctions (NMJs). Mutations in ser impair synaptic transmission at larval NMJs. This defect is entirely presynaptic, as nerve-evoked excitatory junction potentials (EJP) and quantal content (QC) of neurotransmitter release are significantly reduced when compared to wild-type control. Further, mutations in ser also alter the growth of the NMJ and the underlying muscle. Mutations in ser significantly reduce the size of larval body wall muscles (length and surface area) as well as the number and size of synaptic boutons, and the number of secondary axonal branches. Ubiquitous or muscle-specific expression of normal Serrate in serB936 mutants restores a normal muscle size but not a normal size and structure of the innervating NMJ. However, expression of normal Serrate in the motor axon restores a normal number of synaptic boutons and secondary branches at serB936 mutant NMJs. In addition, it restores normal neurotransmitter release. These data suggest that Serrate protein is required presynaptically for normal synaptic growth and function. Interestingly, overexpression of Serrate in a wild type background resulted in similar phenotypes than to those of loss-of-function mutants. In conclusion, these data suggest a new functional role for Serrate in synaptic growth and function.

abstract: The Erk/MAPK pathway plays a major role in cell growth, differentiation, and survival. Genetic mutations that cause dysregulation in this pathway can result in the development of Rasopathies, a group of several different syndromes including Noonan Syndrome, Costello Syndrome, and Neurofibromatosis Type-1. Since these mutations are germline and affect all cell types it is hard to differentiate the role that Erk/MAPK plays in each cell type. Previous research has shown that individual cell types utilize the Erk/MAPK pathway in different ways. For example, the morphological development of lower motor neuron axonal projections is Erk/MAPK-independent during embryogenesis, while nociceptive neuron projections require Erk/MAPK to innervate epidermal targets. Here, we tested whether Erk/MAPK played a role in the postnatal development of lower motor neurons during crucial periods of activity-dependent circuit modifications. We have generated Cre-dependent conditional Erk/MAPK mutant mice that exhibit either loss or gain of Erk/MAPK signaling specifically in ChAT:Cre expressing lower motor neurons. Importantly, we found that Erk/MAPK is necessary for the development of neuromuscular junction morphology by the second postnatal week. In contrast, we were unable to detect a significant difference in lower motor neuron development in Erk/MAPK gain-of-function mice. The data suggests that Erk/MAPK plays an important role in postnatal lower motor neuron development by regulating the morphological maturation of the neuromuscular junction. / Dissertation/Thesis / Masters Thesis Biology 2017

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Neurons are specialized cells that communicate via electrical and chemical signaling. It is well-known that homeostatic mechanisms exist to potentiate neuronal output when activity falls. Likewise, while neurons rely on excitable states to function, these same excitable states must be kept in check for stable function. However, the identity of molecular factors and pathways regulating these pathways remain elusive. Chapter 2 of this thesis reports the findings from an RNA interference- and electrophysiology-based screen to identify factors necessary for the long-term maintenance of homeostatic synaptic potentiation. Data is reported to resolve a long-standing question as to the role of presynaptic Cav2-type channels in homeostatic synaptic potentiation at the Drosophila NMJ. It is shown that reduction in Cav2 channel expression and resultant activity is not sufficient to occlude homeostatic potentiation. Thus, the homeostatic block of a amino-acid substituted Cav2-type calcium channel (cacS) channel is presumed to be due to loss of a specific signaling or binding activity, but not due to overall diminishment in channel function. It is also reported that both Drosophila homologs of phospholipase Cβ (PLCβ) and its putative activator Gαq were found to be necessary for a scaling up of neurotransmitter release upon genetic ablation of glutamate receptors. These factors are canonically involved in the activation of intracellular calcium stores through the inositol trisphosphate receptor (IP3R) and the closely related ryanodine receptor (RyR). Likewise, the Drosophila homolog of Cysteine String Protein (Csp) is identified as important for long-term homeostatic potentiation. CSP has also been reported to be involved in regulation of intracellular calcium. PLCβ, Gαq, and CSP are also known to regulate Cav2-type channels directly, and this possibility, as well as others, are discussed as mechanisms underlying their roles in homeostatic potentiation. Chapter 3 of this thesis reports the extended findings from expression of a gain-of-function Cav2-type channel. The Cav2.1 channel in humans is known to cause a dominant, heritable form of migraine called familial hemiplegic migraine (FHM). Two amino-acid substitutions causative for migraine were cloned into their analogous residues of the Drosophila Cav2 homolog. Expression of these migraine-modeled channels gave rise to several forms of hyperexcitability. Hyperexcitability defects included abnormal evoked waveforms, generation of spontaneous action potential-like events, and multi-quantal release. It is shown that these forms of hyperexcitability can be mitigated through targeted down-regulation of the PLCβ-IP3R-RyR intracellular signaling pathway. Chapter 4 presents an extended discussion as to the roles for presynaptic calcium channels, PLCβ, and CSP in homeostatic synaptic potentiation, and the mechanism underlying hyperexcitability downstream of gain-of-function Cav2-type channels. The proposed model aims to bridge the involvement of the PLCβ pathway in both homeostatic potentiation and neuronal excitability. Last, the implications for these findings on human disease conditions are elucidated.

<p>RanBPM is a conserved putative scaffold protein of unknown function. Loss-of-function in <em>RanBPM</em> leads to pleiotropic phenotypes such as reduced locomotion, decreased size and larval lethality in the <em>Drosophila melanogaster</em>.</p> <p><em>dRanBPM</em> mutants have decreased branching and boutons at the neuromuscular junction, which may contribute to their locomotory defect. To investigate if dRanBPM is involved in controlling synaptic architecture at the neuromuscular junction, levels of two cytoskeletal proteins, Futsch and profilin, were assessed in <em>dRanBPM</em> mutants.</p> <p>Due to time constraints, immunoblots for Futsch were not fully optimized for protein measurement. Immunoblots for profilin, on the other hand, were successfully carried out. However, results from the reproduction of a blot demonstrating the negative regulation of <em>Drosophila</em> FMRP on profilin did not agree with that of the literature. In addition, results from an epistatic experiment demonstrated that profilin levels were not affected in FMRP deficient flies when compared to those with additional decrease in dRanBPM function.</p> <p>Targeted expression of <em>dRanBPM</em> to neurosecretory cells is able to rescue size and lethality of <em>dRanBPM</em> mutants, suggesting a common pathway through which both phenotypes operate is disrupted in these mutants. Activation of the insulin signaling pathway was indeed found to be downregulated in <em>dRanBPM</em> mutants. A longevity assay was alternatively carried out to demonstrate decreased insulin pathway activation in <em>dRanBPM</em> mutants. Unfortunately, due to inappropriate controls used for this experiment, no conclusive points can be made. Together, these findings contribute to the knowledge that RanBPM plays and to designing future experiments to test for RanBPM function.</p> / Master of Science (MSc)

Les protéines associées aux microtubules (MAP) de structures, telles que celles appartenant à la famille des MAP1 sont connues pour contrôler la stabilité et la dynamique des microtubules (MTs). Elles sont aussi connues pour interagir avec des protéines post-synaptiques telles que les récepteurs GABAergique ou glutamatergique. Cependant, leur rôle pré-synaptique dans la libération de neurotransmetteurs a été très peu étudié. Dans cette thèse, j'utilise l'avantage du modèle Drosophila melanogaster dans lequel il n'y a qu'un seul homologue des MAP1 des vertébrés, nommé Futsch. J'ai étudié la fonction de Futsch à la jonction neuromusculaire (JNM) de larve, où cette protéine n'est trouvée que dans la partie pré-synaptique. Ici, j'ai montré qu'en plus de sa fonction connue sur la morphologie de la JNM (Roos et al., 2000; Gogel et al., 2006), Futsch est également important pour la physiologie de la JNM, par le contrôle de la libération de neurotransmetteurs ainsi que de la densité des zones actives (ZAs). J'ai montré que l'effet physiologique de Futsch n'est pas la conséquence de l'altération du cytosquelette de MTs ou d'un défaut de transport axonal, mais doit être la conséquence d'un effet local de Futsch à la terminaison synaptique. J'ai utilisé la microscopie d'éclairage structuré 3D (3D-SIM) pour étudier plus précisément la localisation de Futsch et des MTs au niveau de la ZA. Futsch et les MTs se trouvent presque toujours à proximité des ZAs, avec Futsch en position intermédiaire entre les MTs et les ZAs. En utilisant la technique de « proximity ligation assays », j'ai aussi démontré la proximité fonctionnelle de Futsch avec Bruchpilot un composant de la ZA, ce qui n'est pas le cas des MTs. En conclusion, mes données sont en faveur d'un modèle pour lequel Futsch stabilise localement les ZAs, en renforçant leur lien avec le cytosquelette de MTs sous-jacent. / Structural microtubule associated proteins like those belonging to the MAP1 family are known to control the stability and dynamics of microtubules (MTs). They are also known to interact with postsynaptic proteins like GABA or glutamate receptors. However, their presynaptic role in neurotransmitter release was barely studied. Here, we took advantage of the Drosophila model in which there is only one MAP1 homologue, called Futsch. We studied the function of Futsch at the larval neuromuscular junction (NMJ), where this protein is found presynaptically only. Here, we show that, in addition to its known function on NMJ morphology (Roos et al., 2000; Gogel et al., 2006), Futsch is also important for NMJ physiology, by controlling neurotransmitter release as well as active zone density. We show that this physiological effect of Futsch is not the consequence of disrupted microtubule bundle and disrupted axonal transport, but must be the consequence of a local effect of Futsch at the synaptic terminal. We used 3D-Structured Illumination Microscopy (3D-SIM) to further study the localization of Futsch and MTs with respect to active zones. Both Futsch and MTs are almost systematically present in close proximity active zones, with Futsch being localized in-between MTs and active zones. Using proximity ligation assays, we further demonstrated the functional proximity of Futsch, but not MTs, with the active zone component Bruchpilot. Altogether our data are in favor of a model by which Futsch locally stabilizes active zones, by reinforcing their link with the underlying MT cytoskeleton.

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