Synapses are the communication junctions of the nervous system and contain protein machinery necessary for cognitive functions such as learning and memory. Postsynaptic density protein-95 (PSD-95) is a key scaffolding molecule at the PSD of synapses, yet its sub-synaptic organisation in the mammalian brain remains poorly understood. This thesis presents the use of genetically labelled PSD-95 with super-resolution imaging to resolve its nano-architecture in the mouse brain. To visualize PSD-95, two knock-in mouse lines were generated where the fluorescent proteins eGFP or mEos2 was fused to the carboxyl terminus of the endogenous PSD- 95 protein (PSD-95-eGFP or PSD-95-mEos2). Methods were developed by which fixed tissue sections of PSD-95-eGFP mice were examined using gated-stimulated emission depletion (g-STED) microscopy and PSD-95-mEos2 sections were examined with photoactivatable localisation microscopy (PALM) and quantitative image analysis was developed for both methods. From these platforms it was demonstrated that PSD-95 has a two tiered organisation: it is assembled into nanoclusters (NCs) approximately 140 nm diameter, which form part of the greater envelope of the PSD within synapses. Synapse subtypes were observed as characterised by the number of NCs per PSD. Using double colour g- STED microscopy. It was then asked whether PSD-95 nano-architecture remained the same across different sub-regions of the brain. A survey of PSD-95 was performed from seven different sub-regions of the hippocampus, quantifying ~110,000 NCs within ~70,000 PSDs from across the two super-resolution platforms. It was found that synapses displayed structural diversity both within and between different brain subregions as a function of the number of NCs per PSD. PSD-95 NCs were structurally conserved across the hippocampus, but showed molecular diversity in the abundance of PSD-95 molecules within. The findings of this thesis are: 1) genetic labelling of endogenous proteins combined with super-resolution microscopy is a powerful tool to study synaptic protein organisation in tissue. 2) Synaptic structural diversity in the brain is underlined by the number of PSD-95 NC units per synapse 3) PSD-95 NCs are structurally conserved but molecularly diverse synaptic units of synapses throughout the brain. These findings suggest that cognitive processing at the synapse is based upon a conserved, fundamental, molecular architecture.

Synapses are specialised contacts between neurons. At postsynaptic terminals of glutamatergic synapses, protein complexes process and transmit the information received from the presynaptic terminal. Scaffolding proteins, among which members of the disc large homologue (DLG) family are the most abundant, assemble the molecular machinery in the postsynaptic terminal. Recently, two members of the DLG family, postsynaptic density protein 95 (PSD95) and synapse associated protein 102 (SAP102), have been shown to form different types of complexes, thus giving the synapse different signalling capabilities. However, the spatial distribution of these synaptic markers in different synapses remains elusive due to technical challenges. This thesis presents the first applications of a new method, the Genes to Cognition Synaptome Mapping pipeline (G2CSynMapp), to map individual synapses at the whole-brain level, in a quantitative and unbiased manner. This method was used to generate PSD95 and SAP102 synaptome maps – i.e. comprehensive maps of PSD95 and SAP102 positive synapses – in the mouse brain and to achieve three aims: i) characterise PSD95 and SAP102 synapse diversity, ii) measure the trajectory of PSD95 and SAP102 synapse changes during the postnatal lifespan and iii) determine whether PSD95 synaptome is reorganised by mutation. First, I have used G2CSynMapp to generate the first synaptome maps in the adult mouse brain. This reference map of PSD95 and SAP102 positive synapses revealed a highly organised distribution pattern of glutamatergic synapses between anatomical regions. Moreover, it uncovered that synapse populations are very diverse within anatomical regions and can form patches, gradients and input-specific glomeruli. Second, the trajectories of PSD95 and SAP102 synaptomes were mapped across the mouse postnatal lifespan. At birth, synapse densities are low and increase rapidly during the first month of life. During ageing, the density of SAP102 and PSD95 positive synapses decrease gradually. Interestingly, different anatomical regions show different trajectories of synapse density and parameters across the lifespan. Moreover, the packing of PSD95 and SAP102 at synapses have specific pattern of changes. Third, the PSD95 synaptome was found to be reorganised differently in two disease models, PSD93 and SAP102 knock-out mice. In humans, mutations in the genes encoding PSD93 or SAP102 have been involved in schizophrenia and mental retardation, respectively. Of particular interest, opposite changes were identified in the neocortex of the two mutant lines that are reminiscent of their inverse behavioural phenotypes.

Autism spectrum disorders (ASD) comprise a heterogeneous group of neurodevelopmental disorders characterized by complex genetic etiology. Mutations in human APBA2, which encodes for the neuronal adaptor protein Mint2, have been genetically linked to ASD patients. APBA2 maps to the distal portion of chromosome 15q13.1, a region commonly deleted in Prader-Willi and Angelman neurodevelopmental disorders and duplicated in cases of autism, making APBA2 an attractive candidate gene associated with autism. Seven novel nonsynonymous coding variants in APBA2 in ASD subjects have been identified, five of which were predicted to affect protein function; however, they have not been examined functionally. Mint2 belongs to a family of neuronal adaptor proteins that are important for synaptic function. Mint2 interacts directly with the cell adhesion protein neurexin-1α, as part of a multi-protein complex that acts as a facilitator of neurotransmitter release. Together, these data suggest that Mint2 plays an important role in neuronal function, and sequence variations in Mint2 may alter neuronal dysfunction associated with ASD. This thesis examines a point mutation in Mint2, which changes a conserved asparagine residue to a serine (N723S) in the second PDZ domain of Mint2, which binds to neurexin-1α. We found the Mint2 N723S mutation did not affect the binding to neurexin-1α; however, it dramatically altered neurexin-1α stabilization and trafficking in HEK293T cells. While Mint2 wild type greatly increased neurexin-1α at the membrane, Mint2 N723S showed a decreased membrane level of neurexin-1α, indicating the steady-state surface expression of neurexin is affected by the Mint2 N723S mutation. Also, we found that Mint2 N723S decreased neurexin localization in axons and the presynaptic terminal in neurons, which correlated with a decrease in synaptogenesis and miniature event frequency in excitatory synapses in neurons. Together, these results suggest that Mint2 N723S leads to dysfunction in neuronal development, in part due to alterations in intracellular neurexin trafficking and altered synaptic function of Mint2, as potential mechanisms that contribute to ASD pathogenesis. / 2018-11-01T00:00:00Z

During my PhD I studied two principal biological aspects employing Drosophila melanogaster. Therefore, this study is divided into Part I and II. Part I: Bruchpilot and Complexin interact to regulate synaptic vesicle tethering to the active zone cytomatrix At the presynaptic active zone (AZ) synaptic vesicles (SVs) are often physically linked to an electron-dense cytomatrix – a process referred to as “SV tethering”. This process serves to concentrate SVs in close proximity to their release sites before contacting the SNARE complex for subsequent fusion (Hallermann and Silver, 2013). In Drosophila, the AZ protein Bruchpilot (BRP) is part of the proteinous cytomatrix at which SVs accumulate (Kittel et al., 2006b; Wagh et al., 2006; Fouquet et al., 2009). Intriguingly, truncation of only 1% of the C-terminal region of BRP results in a severe defect in SV tethering to this AZ scaffold (hence named brpnude; Hallermann et al., 2010b). Consistent with these findings, cell-specific overexpression of a C-terminal BRP fragment, named mBRPC-tip (corresponds to 1% absent in brpnude; m = mobile) phenocopied the brpnude mutant in behavioral and functional experiments. These data indicate that mBRPC-tip suffices to saturate putative SV binding sites, which induced a functional tethering deficit at motoneuronal AZs. However, the molecular identity of the BRP complement to tether SVs to the presynaptic AZ scaffold remains unknown. Moreover, within larval motoneurons membrane-attached C-terminal portions of BRP were sufficient to tether SVs to sites outside of the AZ. Based on this finding a genetic screen was designed to identify BRP interactors in vivo. This screen identified Complexin (CPX), which is known to inhibit spontaneous SV fusion and to enhance stimulus evoked SV release (Huntwork and Littleton, 2007; Cho et al., 2010; Martin et al., 2011). However, so far CPX has not been associated with a function upstream of priming/docking and release of SVs. This work provides morphological and functional evidence, which suggests that CPX promotes recruitment of SVs to the AZ and thereby curtails synaptic short-term depression. Together, the presented findings indicate a functional interaction between BRP and CPX at Drosophila AZs. Part II: The Adhesion-GPCR Latrophilin/CIRL shapes mechanosensation The calcium independent receptor of α-latrotoxin (CIRL), also named Latrophilin, represents a prototypic Adhesion class G-protein coupled-receptor (aGPCR). Initially, Latrophilin was identified based on its capacity to bind the α-component of latrotoxin (α-LTX; Davletov et al., 1996; Krasnoperov et al., 1996), which triggers massive exocytotic activity from neurons of the peripheral nervous system (Scheer et al., 1984; Umbach et al., 1998; Orlova et al., 2000). As a result Latrophilin is considered to play a role in synaptic transmission. Later on, Latrophilins have been associated with other biological processes including tissue polarity (Langenhan et al., 2009), fertility (Prömel et al., 2012) and synaptogenesis (Silva et al., 2011). However, thus far its subcellular localization and the identity of endogenous ligands, two aspects crucial for the comprehension of Latrophilin’s in vivo function, remain enigmatic. Drosophila contains only one latrophilin homolog, named dCirl, whose function has not been investigated thus far. This study demonstrates abundant dCirl expression throughout the nervous system of Drosophila larvae. dCirlKO animals are viable and display no defects in development and neuronal differentiation. However, dCirl appears to influence the dimension of the postsynaptic sub-synaptic reticulum (SSR), which was accompanied by an increase in the postsynaptic Discs-large abundance (DLG). In contrast, morphological and functional properties of presynaptic motoneurons were not compromised by the removal of dCirl. Instead, dCirl is required for the perception of mechanical challenges (acoustic-, tactile- and proprioceptive stimuli) through specialized mechanosensory devices, chordotonal organs (Eberl, 1999). The data indicate that dCirl modulates the sensitivity of chordotonal neurons towards mechanical stimulation and thereby adjusts their input-output relation. Genetic interaction analyses suggest that adaption of the molecular mechanotransduction machinery by dCirl may underlie this process. Together, these results uncover an unexpected function of Latrophilin/dCIRL in mechanosensation and imply general modulatory roles of aGPCR in mechanoception. / In dieser These wurden zwei grundlegende biologische Aspekte mittels Drosophila melanogaster untersucht, weshalb diese in zwei Teile gegliedert ist. TeiL I: Die Interaktion von Bruchpilot und Complexin vermittelt die Anbindung von synaptischen Vesikeln an die Zytomatrix der aktiven Zone Oft findet man an aktiven Zonen (AZ) von Präsynapsen elektronendichte Matrices, welche meist in physischem Kontakt mit synaptischen Vesikeln (SV) stehen. Dieser als „SV Tethering“ bezeichnete Prozess dient der Anreicherung SV in der unmittelbaren Nähe ihrer Freisetzungszonen, noch bevor diese mit dem SNARE Komplex interagieren, um mit der präsynapti-schen Plasmamembran zu fusionieren (Hallermann und Silver, 2013). In der Taufliege Drosophila melanogaster bildet das AZ Protein Bruchpilot (BRP) Protrusionen, um welche SV akkumulieren (Kittel et al., 2006b; Wagh et al., 2006; Fouquet et al., 2009). Interessan-terweise resultiert bereits eine minimale Verkürzung von BRP (1% der Gesamtlänge) am C-terminalen Ende in einem schwerwiegenden Anbindedefekt von SV, der mit einem Funkti-onsverlust dieser Synapsen einhergeht (brpnude; Hallermann et al., 2010b). Entsprechend diesem Vorbefund resultierte die gewebespezifische Überexpression eines C-terminalen BRP Fragments - mBRPC-tip (entspricht dem fehlenden Fragment der brpnude Mu-tante; m = mobil) - sowohl in Verhaltens- als auch funktionellen Analysen in einer Phänoko-pie der brpnude Mutante. Dies deutet daraufhin, dass mBRPC-tip vermeintliche vesikuläre Interaktionspartner blockiert und so die Anreicherung von SV an motoneuronalen AZ verhindert, was ähnlich wie in brpnude Mutanten zu einem funktionellen Tethering-Defekt führt. Die molekulare Identität eines BRP Partners zur Anreicherung von SV an der Zytomatrix der AZ wurde bisher nicht beschrieben. Weiterhin zeigt diese Arbeit, dass membrangebundene C-terminale BRP Anteile genügen, um SV an Positionen außerhalb von AZ zu binden. Basierend auf diesem Befund wurde ein gene-tischer in vivo Screen zur Identifikation von BRP Interaktoren entwickelt. Dieser Screen identifizierte Complexin (CPX), ein Protein, dessen hemmende beziehungsweise fördernde Wirkung auf die spontane und reizinduzierte Vesikelfusion bekannt ist (Huntwork und Littleton, 2007; Cho et al., 2010; Martin et al., 2011). CPX wurde bisher nicht mit einer Funktion ober-halb von Vesikelpriming und -fusion in Verbindung gebracht. Diese Studie dokumentiert strukturelle und funktionelle Hinweise, die darauf hindeuten, dass CPX mit BRP interagiert, um Vesikelakkumulation an AZ zu fördern und dadurch synaptischer Kurzzeit-Depression entgegen zu wirken. Teil II: Adhäsions-GPCR Latrophilin/CIRL moduliert die Wahrnehmung mechanischer Reize Der Kalzium-unabhängige Rezeptor für α-Latrotoxin (CIRL), oder Latrophilin, ist ein prototypischer Rezeptor der Adhäsions G-Protein gekoppelten Klasse (aGPCR). Identifiziert wurde Latrophilin ursprünglich aufgrund seiner Fähigkeit die α-Komponente von Latrotoxin (α-LTX) zu binden (Davletov et al., 1996; Krasnoperov et al., 1996), welches seine Wirkung am peripheren Nervensystem entfaltet und dort übermäßige Transmitterausschüttung an neuronalen Endigungen induziert (Scheer et al., 1984; Umbach et al., 1998; Orlova et al., 2000). Basierend auf diesem Effekt wurde Latrophilin eine Rolle bei der synaptischen Transmission zugesprochen. Später wurden Latrophiline mit weiteren biologischen Prozessen in Zusammenhang gebracht, darunter Gewebepolarität (Langenhan et al., 2009), Fertilität (Prömel et al., 2012) und Synaptogenese (Silva et al., 2011). Allerdings blieb sowohl die subzelluläre Lokalisation als auch die Identität endogener Liganden, zwei Schlüsselaspekte im Verständnis der in vivo Funktion von Latrophilinen bisher rätselhaft. Drosophila besitzt lediglich ein latrophilin Homolog, dCirl, dessen Funktion bisher nicht untersucht wurde. Diese Arbeit zeigt, dass dCirl in weiten Teilen des larvalen Nervensystems von Drosophila exprimiert ist. dCirl knock-out Mutanten sind lebensfähig und weisen keine Störungen in der Entwicklung und neuronalen Differenzierung auf. Allerdings schien dCirl Einfluss auf die Ausdehnung des postsynaptischen subsynaptischen Retikulums (SSR) zu nehmen, was mit einer erhöhten Menge an Discs-large (DLG) assoziiert war. Die morphologischen und funktionellen Eigenschaften präsynaptischer Motoneurone der Fliegenlarve hingegen, waren durch den Verlust von dCirl funktionell weitestgehend unbeeinträchtigt. Vielmehr ist dCirl notwendig für die Wahrnehmung mechanischer Reize (akustische-, taktile und propriozeptive) durch spezialisierte Vorrichtungen - Chordotonalorgane (Eberl, 1999). Die Befunde deuten daraufhin, dass dCirl die Sensitivität der Chordotonalneurone gegenüber mechanischen Reizen moduliert und dadurch das Input-Output Verhältnis einstellt. Adaptation der molekularen Mechanotransduktionsmaschinerie durch dCirl könnte die molekulare Grundlage für diesen Prozess darstellen, eine Hypothese die durch genetische Interaktionsanalysen gestützt wird. Schlussfolglich enthüllen die experimentellen Befunde dieser These eine unerwartete Funktion von Latrophilin/dCirl bei der Mechanoperzeption und implizieren eine generelle modula-torische Rolle für aGPCR bei der Wahrnehmung mechanischer Reize.

The nervous system is a dynamic structure. Both during development and in the adult, synapses display activity-dependent plasticity which can modify their structure and function. In the neonate, activity influences the stability of functional connections between the muscle and nerve. In adults, the process of neurotransmitter release and the structure of the postsynaptic muscle can also be altered by external stimuli such as exercise. It is important to understand this plasticity of the neuromuscular system, the ways in which it can be modified, and its relationship to the maintenance or degeneration of synapses. After injury, peripheral nerve undergoes Wallerian Degeneration, during which the connections between axons and muscle fibres are lost, followed by the fragmentation of the nerve itself. The primary goal of this thesis was to determine whether activity modulates this process; that is, whether enhancing or reducing neuromuscular activity creates a susceptibility to degeneration or alternatively provides any protection against it. Developing greater understanding of this process is essential in relation to neurodegenerative disorders in which the benefits of activity, in the form of exercise, are controversial. Using Wlds mice, in which synaptic degeneration occurs approximately ten times more slowly than normal after nerve injury, I investigated the influence of both decreased (tetrodotoxin induced paralysis) and increased (voluntary wheel running) activity in vivo on this process. Paralysis prior to axotomy resulted in a significant increase in the rate of synapse degeneration. Using a novel method of repeatedly visualising degenerating synapses and axons in vivo I also established that this effect was specific to the synapse, as it did not affect the degeneration of axons. In contrast, voluntary wheel running had no effect on the rate of either axonal or neuromuscular synapse degeneration, but induced a slight modification of neuromuscular transmission. To provide a more stringent test I developed a novel assay based on overnight, ex vivo incubation of nerve-muscle preparations at 32°C. I first demonstrated that this procedure separates the different degeneration time courses for neuromuscular synapse degeneration in wild-type and Wlds preparations. I then extended the study to investigate further ways of modulating synaptic degeneration. First, I tested the effects of electrical stimulation. Intermittent high frequency (100Hz) stimulation reduced the level of protection. Finally, I tested the effects of NAMPT enzymatic inhibitor FK866 on synaptic degeneration. Interestingly, the synaptic protection observed in Wlds muscles was enhanced in the presence of FK866. The results of my findings are relevant to understanding the plasticity of synapses and its relationship to degeneration. Together, these studies highlight the potential of genetic and epigenetic factors, including activity, to regulate neuromuscular synapse degeneration. My study also provides proof of concept for a novel organotypic culture system in which to identify pharmacological modulators of synaptic degeneration that could form part of a second-line screen for neuroprotective compounds or phenotypes. My findings may be viewed in the wide context of neurodegenerative disease, since synaptic use or disuse is widely thought to influence susceptibility, onset and progression in such disorders.

Understanding how different regions of the central nervous system (CNS) are affected by genetic insults is critical to advancing the study of CNS pathologies. The cerebellum is one such region which is disproportionately hypoplastic in the majority of cases of CASK gene mutation in humans. CASK is an enigmatic multi-domain scaffolding protein which plays a vital role in organizing protein complexes at the pre-synapse through interactions with both active zone proteins and trans-synaptic adhesion molecules such as liprins-α and neurexins. Mutations in the X-linked CASK gene in humans are largely post-natally lethal in the hemizygous condition and result in microcephaly with pontine and cerebellar hypoplasia (PCH) and also optic nerve hypoplasia (ONH) in heterozygous mutations. Herein, I used various molecular and genetic strategies to uncover the role of the CASK protein in brain function and pathogenesis of cerebellar hypoplasia associated with CASK mutations/deletions. First, using the face- and construct-validated heterozygous CASK knockout (Cask+/-) murine model, I conducted bulk RNA-sequencing and proteomics experiments from whole brain lysates to uncover changes in the Cask+/- brain. RNA-sequencing revealed the majority of changes to be broadly categorized into metabolic, nuclear, synaptic, and extracellular-matrix associated transcripts. Proteomics revealed the majority of changes cluster as synaptic proteins, metabolic proteins, and ribosomal subunits. Thus, absence of CASK in half of brain cells seems to affect synaptic protein content, cell metabolism, and protein homeostasis. Extending these observations, I conducted GFP-trap immunoprecipitation followed by tandem mass spectroscopy to reveal protein complexes in which CASK participates. Commensurate with proteomic changes, CASK was found to complex with synaptic proteins, metabolic proteins, cytoskeletal elements, ribosomal subunits, and protein folding machinery. Next, in order to investigate the pathogenesis of CASK-linked cerebellar hypoplasia, I utilized a human case of early truncation wherein the 27th arginine of CASK is converted to a stop codon. Immunohistochemical analysis of this brain revealed an upregulation of glial fibrillary acidic protein, a common marker for degenerative cell death. To mechanistically test the hypothesis that cerebellar hypoplasia results from cell death rather than developmental failure, I created a murine model wherein CASK is deleted from the majority of cerebellar cells post-development using Cre recombinase driven by the Calb2 promoter. Deleting CASK from all cerebellar granule neurons post-migration indeed leads to degeneration of the cerebellum via massive depletion of granule cells while sparing Purkinje cells. Overall, the cerebellum shrinks by approximately half in cross-sectional area and degeneration is accompanied by a collapsing of the molecular layer and of Purkinje cell dendrites. In addition, cerebellar degeneration presents with a profound locomotor ataxia. In conclusion, CASK seems to be affecting brain energy homeostasis and synaptic connections via interactions with metabolic proteins, synaptic proteins, and protein homeostatic elements. Further, alterations in brain volume associated with CASK-linked disorders is the result of degenerative cell death rather than developmental failure as previously posited. / Doctor of Philosophy / One of the main challenges facing modern neuroscience is the question of how constitutive mutations in genes present in every cell can cause different effects on different parts of the brain. CASK is one such gene which is expressed in every cell of the brain and, when mutated, typically results in an overall smaller brain volume. However, the cerebellum is one region of the brain involved in motor coordination which is disproportionately smaller than the rest of the brain. Through this gene, I investigate here two questions principally: (1) what is the role of the CASK protein in cells? And (2) how is the cerebellum differentially affected? Firstly, I conduct a molecular investigation into what changes in the brain of a mouse model of CASK deletion which recapitulates the majority of human cases found in girls. This genetic model results in half of cells in the body lacking CASK and leads to smaller brain volume with disproportionate reduction in cerebellum size, as in the human subjects. Using a variety of molecular and biochemical methods, I uncover that several classes of proteins are changed in this brain, primarily those associated metabolism and cell-to-cell communication. Further, my experiments indicate that CASK interacts with many of these proteins. Next, I use human cases as well as a novel mouse model to uncover the trajectory of CASK-linked reduction in cerebellar size. The human case indicates molecular signatures of cell death, a surprising finding given that CASK-linked disorders are thought to result from developmental failure. Investigating this mechanistically in a mouse model, I uncover that when CASK is deleted after development, cerebellar cells still die and the cerebellum actually shrinks. Thus, my work herein elucidates potential roles for the CASK molecule in cells and shows, for the first time, that CASK-linked cerebellar size diminishment is degenerative in nature rather than developmental. This degeneration of the cerebellum occurs very early on in infancy and so was missed until now. The most important implication is that a degenerative process could be halted with therapies other than relying exclusively on genetic therapies.

Intracellular Ca2+ ions are highly regulated in animal cells for them to function normally. Since the tight regulation of [Ca2+]i is so ubiquitous among cells, it is not surprising that altered function in [Ca2+]i regulation is associated with a myriad of disease states in humans. This is particularly evident in pacing myocytes and nerve terminals related to synaptic transmission. A common thread through this dissertation is on the role of three regulators proteins that are common to many cell types. These are the plasmalemmal Na+/Ca2+ exchanger (NCX), the Ca2+-ATPase (PMCA) and the SERCA on the endoplasmic reticulum. In chapter 1 a historical overview is provided on how the understanding in the importance of Ca2+ came about. In Chapter 2, I address indirectly the function of residual [Ca2+]i on the efficacy of synaptic transmission by quantal analysis but also develop novel means of assessing quantal analysis to assign a n and p value to particular synapses. Chapters 3 and 4 address the role of the three Ca2+ regulator proteins in short bursts of synaptic transmission related to short-term facilitation or depression depending on the type of neuromuscular junction (NMJ). Two key model NMJs I used were the crayfish (Chapter 3) and the larval Drosophila (Chapter 4). For comparative purposes in investigating the role of the three proteins in [Ca2+]i regulation, I used the Drosophila larval heart preparation (Chapter 5). Throughout these studies, I used various pharmacological and ionic approaches to compromise the function of these Ca2+ channels. The results were unexpected in some cases due to non-specific effects of the pharmacological agent or ionic manipulations. In addition, a mutational line of Drosophila was used to asses SERCA function, but the results at the NMJ were not as expected. However, results with the mutation on the function of the heart were promising. The significance of these studies stresses that multiple approaches to compromise channels is warranted and the findings should be beneficial for future investigators to advance in mechanistic studies.

The ability of neurons to dynamically regulate their response to changing inputs is essential for the correct development and function of a nervous system capable of learning and memory. The post synaptic compartment of excitatory synapses contains a dense proteinaceous complex of molecules that link excitatory glutamatergic neurotransmission to downstream signalling pathways that ultimately result in modification of the synapse. One of the most abundant of such postsynaptic signalling molecules, synaptic GTPase activation protein, SynGAP, represents a key signalling link between the activation of the NMDA sensitive glutamate receptor to outcomes such as the structural rearrangement of synaptic sites and altered synaptic content of AMPA type glutamate receptors, molecular processes that underly learning and memory. The primary finding of this thesis is that different isoforms of SynGAP, which varies at it N terminus through alternative transcription start sites and at its C terminus through alternative splicing, can differentially affect the function of the synapse. Using primary murine neuronal cultures we show that despite being crucial for the survival of the mouse the absence of SynGAP does not effect mean dendritic spine morphology and density or miniature excitiatory post synaptic currents under a range of experimental conditions (days in vitro 10 – 14, with and without serum, high and low cell plating density). In order to examine the effects of different SynGAP isoforms we cloned two full length transcripts (SynGAP A-alpha-2 and SynGAP Ealpha- 1) which were used to construct a range of isoforms. Whole cell patch clamping of SynGAP transfected neurons revealed that the post synaptic expression of SynGAPs which terminate as an alpha-1 isoform can lead to the elimination of mEPSCs, while isoforms that terminate as an alpha-2 isoform can lead to synaptic strengthening. The magnitude of the effect in both cases is determined by the identity of the N terminus of the protein; SynGAP A-alpha-1 has the largest synaptic weakening effect and SynGAP B-and C alpha-2 strenghten the synapse. The changes in miniature electrophysiological properties are not mirrored by changes in dendritic spine morphology, whole cell AMPA/NMDA currents, or synaptic responsiveness to stimulation suggesting an undefined novel mechanism of action. SynGAPs A, B and C appear to be under the control of different promoters which are differentially regulated by development and synaptic activity, thus the differential function of SynGAP N and C terminal combinations could play a part in the activity dependent regulation of synaptic strength.

No description available.

Astrocytes, the most abundant glial cell type in the brain, undergo a number of roles in brain physiology; among them, the energetic support of neurons is the best characterized. Contained within astrocytes is the brain’s obligate energy store, glycogen. Through glycogenolysis, glycogen, a storage form of glucose, is converted to pyruvate that is further reduced to lactate and transferred to neurons as an energy source via MCTs. Glycogen is a multi-branched polysaccharide synthesized from the glucose uptaken in astrocytes. It has been shown that glycogen accumulates with age and contributes to the physiological ageing process in the brain. In this study, we compared glycogen distribution between young adults and geriatric mice to understand the energy consumption of synaptic terminals during ageing using computational tools. We segmented and densely reconstructed neuropil and glycogen granules within six (three 4 month old old and three 24 month old) volumes of Layer 1 somatosensory cortex mice brains from FIB-SEM stacks, using a combination of semi-automated and manual tools, ilastik and TrakEM2. Finally, the 3D visualization software, Blender, was used to analyze the dataset using the DBSCAN and KDTree Nearest neighbor algorithms to study the distribution of glycogen granules compared to synapses, using a plugin that was developed for this purpose. The Nearest Neighbors and clustering results of 6 datasets show that glycogen clusters around excitatory synapses more than inhibitory synapses and that, in general, glycogen is found around axonal boutons more than dendritic spines. There was no significant accumulation of glycogen with ageing within our admittedly small dataset. However, there was a homogenization of glycogen distribution with age and that is consistent with published literature. We conclude that glycogen distribution in the brain is not a random process but follows a function distribution.