Nerve signals in the form of action potentials are relayed between neurons through specialized connections called synapses via neurotransmitter released from synaptic vesicles. The release process is Ca2+ dependent, and relies on fusion of neurotransmitter filled synaptic vesicle with the presynaptic membrane. During high frequency stimulation, the amount of vesicle release increases at some synapses (e.g., frog neuromuscular junction (NMJ)), a process known as short-term plasticity. Due to the micron scale size of the presynaptic active zone where vesicle fusion takes place, experimentally study is often difficult. Thus, computational modeling can provide important insight into the mechanism of synaptic vesicle release at active zones. In the first part of my thesis, I used the frog NMJ as a model synapse for computer simulation studies aimed as testing various mechanistic hypotheses proposed to underlie short-term plasticity. Building off a recently reported excess-bindingsite model of synaptic vesicle release at the frog NMJ (Dittrich et al., 2013), I have investigated several mechanisms of short-term facilitation at the frog NMJ. My studies placed constraints on previously proposed mechanistic models, and concluded that the presence of a second calcium sensor protein on synaptic vesicles distinct from synaptotagmin, can explain known properties of facilitation observed at the frog NMJ. In addition, I was able to identify a second facilitation mechanism, which relied on the persistent binding of calcium bound synaptotagmin molecules to lipids of the presynaptic membrane. In the second part of my thesis, I investigated the structure function relationship at active zones, with the hypothesis that active zones are organized from the same basic synaptic building block consisting of a docked vesicle and a small number of closely associated voltage-gated-calcium-channels (VGCCs). To test this hypothesis, I constructed a vesicle release model of the mouse NMJ by reassembling frog NMJ model building blocks based on electron-microscopy imaging data. These two models successfully predicted the functional divergence between frog and mouse NMJ in terms of average vesicle release and short-term plasticity. In the meanwhile, I found that frog NMJ loses facilitation when VGCCs were systematically removed from active zone. By tracking Ca2+ ions from each individual VGCCs, I further show how the difference in short-term plasticity between frog and mouse NMJ may rise from their distinct release building block assemblies. In summary, I have developed a stochastic computer model of synaptic transmission, which not only shed light on the underlying mechanisms of short-term plasticity, but was also proved powerful in understanding structural and functional relationships at synaptic active zones.

Spinal Muscular Atrophy (SMA) is a childhood form of motor neuron disease that causes a progressive paralysis that, in its most severe form, results in death before two years of age. There is currently no cure or treatment for SMA. SMA is caused by a reduction in levels of Survival Motor Neuron (SMN) protein, which results in the degeneration of lower motor neurons. This degeneration is first observed at the neuromuscular junction (NMJ), where pre-synaptic nerve terminals belonging to the motor neuron become dysfunctional and degenerate during the early stages of disease. Several previous studies have shown that the some populations of motor neurons appear to have a resistance to SMA pathology, while other neighbouring populations are vulnerable. In this study, we attempted to elucidate the cause of this vulnerability spectrum. Initially, we characterised the relative vulnerability of ten different motor unit pools in an established mouse model of severe SMA and attempted to correlate these vulnerabilities with quantified aspects of motor unit morphology. From this study, no significant correlation could be found with any aspect of motor unit morphology examined, suggesting that morphological parameters of motor neurons do no influence their relative susceptibility. We then attempted to identify changes in basal gene expression between protected and vulnerable pools of motor units using microarray analysis. Motor unit pools were labelled using a retrograde tracer injected into muscles that had previously been identified as having highly vulnerable or resistant motor units. Labelled motor neuron cell bodies were then isolated from the spinal cord using laser capture micro-dissection and RNA was extracted for microarray analysis. From this study, we identified several molecular pathways and individual genes whose expression levels compared the gene expression profiles of vulnerable and resistant motor units. Thus, molecular differences between motor neuron pools likely underlie their relative vulnerability to degeneration in SMA. Lastly, we attempted to identify a novel peptide that could be used to label synapses, including neuromuscular junctions, in vivo. This would allow us to non-invasively visualise degenerating NMJs and other synapses in human patients without the need for a biopsy. Such a tool would be extremely valuable in assessing the effectiveness of drug trials, both in human patients and animal models, and may also contribute to earlier diagnosis of motor neuron disorders. To identify a potentially suitable peptide, we used a phage display library and panned for peptides that specifically bound to the outer surface of synapses using synaptosome preparations. From this panning we successfully enriched two peptides, the sequences of which were used to manufacture fluorescently tagged peptides.

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.

No description available.

Hu Xiaoling. / "May 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references. / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

To experience the world, we depend on the ability of our brains to process information. Problems can occur when communication between neurons is not regulated, and a significant enough loss of stability could lead to conditions such as migraine and epilepsy. Homeostatic plasticity is thought to constrain activity within physiologically useful ranges. Our lab uses the fruit fly neuromuscular junction as a model synapse to study homeostatic plasticity. Homeostatic potentiation and homeostatic depression are two forms of homeostatic synaptic plasticity. Expression of a dominant negative glutamate receptor subunit in the muscle impairs its sensitivity to glutamate and triggers an increase in the number of vesicles released per evoked potential, or quantal content. This increase in quantal content is called homeostatic potentiation. We found that homeostatic potentiation is a reversible process: quantal content returns to normal levels when expression of the dominant negative ceases. We additionally found that homeostatic potentiation can be ablated at high temperature. Overexpression of the Vesicular Glutamate transporter (VGlut) causes an increase in the amplitude of spontaneous events, leading to a corresponding decrease in quantal content, called homeostatic depression. It is unknown to what degree homeostatic potentiation and homeostatic depression may share regulatory machinery. We screened genes required for homeostatic potentiation in the neuron for additional roles in homeostatic depression. We found that certain genes involved in calcium regulation, such as the IP3 receptor and ryanodine receptor, showed a substantial decrease in evoked potential amplitude in a VGlut overexpression background.

In skeletal muscle acetylcholinesterase AChE is highly expressed at sites of nerve-muscle contact where it is regulated at both the transcriptional and post-transcriptional levels. Scientists have elucidated many aspects of synaptic AChE structure, function, and localization during the past 80 years. However our understanding of the molecular mechanisms underlying its regulation is incomplete, but it appears to involve both translational and post-translational events as well. We found that Pumilio-2 (PUM2), an RNA binding translational repressor, is highly localized at the neuromuscular junction where AChE mRNA concentrates and that PUM2 binds to the AChE transcripts when immoprecipitation studies were performed. A direct binding between a recombinant PUM2-HD and the Pumilio Binding Site (PBE) in a segment of the AChE 3’UTR was demonstrated by Gel shift assays. Transfecting skeletal muscle cells with shRNAs specific for PUM2 upregulated AChE expression, whereas overexpression of PUM2 decreased AChE activity. We conclude that PUM2 binds to AChE mRNA and regulates AChE expression translationally at the neuromuscular synapse. We found that PUM2 is regulated by the motor nerve suggesting a trans-synaptic mechanism for locally regulating translation of specific synaptic proteins involved in modulating synaptic transmission, analogous to CNS synapses. PUM2 expression is critically important in many cell types, virtually nothing is known about the regulation of PUM2 expression itself. Analyzing the PUM2 mRNA 3’UTR we found fifteen possible PBEs in the 3 Kb 3’ UTR. We show that PUM2 binds in vivo to its own mRNA. Overexpression of PUM2 in several cell types transfected with a green fluorescent protein (GFP) reporter construct linked to the full length PUM2 3’UTR (GFP-PUM2-3’UTRFL) suppresses GFP expression suggesting that PUM2 downregulates its own expression by binding to its own 3’UTR. Mutations of the first five PBEs yield the expression of the reporter gene indicating that at least one PBE is functional in the autoregulation of PUM2. These observations suggest a novel model for the localized regulation of protein translation through a negative feedback loop. Much is known about PUM2 as a translational regulative protein but little is known about PUM2 cell localization and possible mechanism of translational regulation. In this work we found PUM2 to be highly localized to the cell rough endoplasmic reticulum and that PUM2 is associated with ribosomal RNA. In addition, we found that the GFP protein itself, together with its mRNA and ribosomal RNA (rRNA), were localized in the PUM2 positive complexes when GFP-PUM2-3’UTRFL was transfected into muscle cells. These observations further suggest a mechanism of regulation where translation of the protein occurs but the protein remains associated with the ribonucleoprotein complex, possibly to be transported together with its mRNA to specific domains inside the cell. Thus when needed, more protein is produced in those specific cell regions.

Peripheral nerve injury (PNI) is common and despite modern microsurgical techniques of repair, functional restoration is always incomplete. This results in impaired sensation and reduced motor function alongside pain and cold intolerance. Traumatic PNI are often associated with loss of nerve tissue, creating a gap, and direct repair of the two damaged nerve stumps is not possible. These types of injuries are reconstructed using autologous nerve grafts but this is far from ideal since it necessitates the sacrifice of a functional nerve from elsewhere in the body. Chronic muscle atrophy because of the prolonged delay in nerve regeneration across gaps is a significant impediment to an optimal functional recovery.   Tissue engineering and regenerative medicine approaches to nerve repair might one day replace the need for autologous nerve grafts. This thesis investigates the effects of adipose derived stem cells (ASC) on nerve regeneration and muscle recovery by using the stem cells for intramuscular injection and combined with a biomaterial, poly-3-hydroxybutyrate (PHB), to create a bioengineered artificial nerve repair construct.  The mechanisms of interaction between the stem cells and neuromuscular system cells were investigated and with a view to translating this work into clinical practice, an optimal source of cells was investigated from human donors.   It was hypothesized that injecting regenerative cells into muscle would reduce nerve injury induced muscle atrophy. A rat sciatic nerve lesion was performed and three different types of cells were injected into the denervated gastrocnemius muscle; either (1) undifferentiated ASC, (2) ASC induced to a ‘Schwann cell-like’ phenotype (dASC) or (3) primary Schwann cells. Nerves were either repaired by direct end-end suture or capped to prevent muscle reinnervation. One month later, functionality was measured using a walking track test, and muscle atrophy was assessed by examining muscle weight and histology. The Schwann cells and dASC groups showed significantly better scores on functional tests when compared with control injections of growth medium alone. Muscle weight and histology were also significantly improved in the cell groups in comparison with the control group.   PHB strips seeded with either primary Schwann cells or dASC suspended in a fibrin glue matrix were used to bridge a 10mm rat sciatic nerve gap. After 12 weeks, functional and morphological analysis (walking track test, electromyography, muscle weight and muscle and nerve histology) was performed. The results showed significantly better functional results for the PHB strips seeded with cells versus the control group with fibrin matrix only. This correlated with less muscle atrophy and greater distal axon myelination in the cell groups.   To further optimize the nerve regeneration and muscle recovery, the nerve gap lesion was repaired by treatment with the bioengineered constructs seeded with dASC or nerve autograft in combination with stem cell injection in the muscle. After 6 weeks, the best results were obtained in the nerve graft group combined with intramuscular dASC injection which showed significantly less atrophy than the other groups. The results also showed that using the stem cells in a matrix on a PHB strip in combination with intramuscular injections could significantly reduce muscle atrophy.   In vitro experiments showed that dASC expressed a wide range of neurotrophic and myogenic factors including BDNF, VEGF-A, IGF-1 and HGF. Stem cell conditioned medium enhanced the proliferation of myoblast cell lines and primary Schwann cells. Various signaling pathways (PKA, MAP kinase) were involved in these effects dependent on the cell type investigated. Furthermore, in direct co-culture with myoblast cells, a small population of the cells fused together to form myotube-like structures and expressed myogenic markers.   Human ASC were isolated from the deep and superficial layers of abdominal fat tissue obtained during abdominoplasty procedures.  Cells from the superficial layer proliferated significantly faster than those from the deep layer. Superficial layer ASC induced significantly enhanced neurite outgrowth from neuronal cell lines when compared with the deep layer cells.  However, RT-PCR and ELISA analysis showed that ASC isolated from both layers expressed similar levels of the neurotrophic factors NGF, BDNF and GDNF.   In summary, these results show that stem cell therapy at both levels (the nerve lesion site and in the target denervated muscle) offers a promising approach for clinical application for treatment of peripheral nerve lesions. The bioengineered artificial nerve construct, combining PHB strip with cells, also provides a beneficial environment for nerve regeneration. Many of the benefits of the ASC are likely to be mediated through their secretome, a rich source of neurotrophic and myogenic factors. Thus adipose tissue contains a pool of regenerative stem cells which have significant potential application to tissue engineering and regenerative medicine for nerve repair.

Dissertation (Ph.D.).--University of Texas Graduate School of Biomedical Sciences at San Antonio, 2008. / Vita. Includes bibliographical references.

Thesis (doctoral)--Lund University, 1992. / Added t.p. with thesis statement inserted.