Selective neuronal loss in response to loss or dysfunction of a ubiquitously expressed protein is a hallmark of neurodegenerative disease. Proximal spinal muscular atrophy (SMA) is caused by homozygous loss of the ubiquitously expressed survival motor neuron 1 (SMN1) gene, resulting in progressive neuromuscular weakness that eventually leads to flaccid paralysis and death from respiratory failure by two years of age in the most severely affected patients. Despite widespread motor neuron loss, certain motor pools are clinically spared. Type I SMA patients exhibit intercostal recession in conjunction with diaphragmatic sparing that produces a characteristic “bell-shaped chest.” Additionally, patients retain extraocular and external sphincter function, even in late disease stages. In order to fully define this differential vulnerability, I performed an extensive characterization of neuromuscular autopsies from Type I SMA patients and age-matched control patients. I found highly divergent degrees of motor unit degeneration, even within individual cranial nerves or a select anatomical region such as the neck. Remarkably, the diaphragm in a Type I SMA patient kept alive on life support for 17 years was still relatively preserved, despite virtually complete fibro-fatty infiltration in other muscles. Extraocular functions were also normal in this patient. These findings suggest that the molecular determinants of SMA-resistance provide indefinite protection against low SMN protein. Thus, identification and modulation of these genes and pathways represents a promising potential therapeutic strategy. Remarkably, this exquisite pattern of selectivity was preserved in the SMNΔ7 mouse, a widely used SMA mouse model. This suggests that the molecular determinants of differential vulnerability are conserved between mouse and human. Given the high degree of diversity between motor pools, I performed a comparative transcriptional microarray between multiple SMA-vulnerable and –resistant motor pools in healthy mice. This analysis revealed a small number of candidate therapeutic genes that segregate closely with vulnerability. I present a series of preliminary studies evaluating these targets in the SMNΔ7 mouse. Ongoing and future studies combine pharmacological, viral, and genetic approaches to modulate these candidate targets in the SMNΔ7 mouse and assess for improvements in neuromuscular pathology. Given the remarkable preservation of select motor pools in SMA patients, changing expression levels of the candidate targets I have identified may provide substantial clinical benefit.

Neuronal circuit perturbations are emerging as important determinants in the pathogenesis of neurodegenerative diseases, including Alzheimer’s disease, Huntington’s disease, and spinal muscular atrophy (SMA). SMA is a motor neuron disease caused by deficiency in the ubiquitously expressed survival motor neuron (SMN) protein. The hallmarks of SMA include loss of motor neurons, muscle atrophy, and abnormal postural reflexes. Although cell-autonomous mechanisms of motor neuron death have received much attention, recent studies in animal models of SMA revealed that motor circuit deficits resulting from early impairment of synaptic function and sensory-motor connectivity precede motor neuron death. It remains to be established whether motor circuit dysfunction is a consequence of SMN-deficiency in the motor neuron or SMN-dependent alterations in the activity of premotor neurons. Here I sought to address these outstanding issues through the development and characterization of a simplified in vitro model of the motor circuit based on the use of embryonic stem cell-derived motor neurons and interneurons. I found that SMN deficiency caused death of motor neurons in co-culture with other neurons as well as in isolation, demonstrating the cell autonomous origin of this defect. SMN requirement for motor neuron function was investigated using intracellular patch clamp recordings to measure both passive and active membrane properties. Remarkably, SMN deficiency induced hyperexcitability of motor neurons only when they are cultured in the presence of interneurons but not in isolation, providing initial evidence that SMN deficiency induces motor neuron hyperexcitability in a non-cell autonomous manner and that dysfunction and death of motor neurons are uncoupled. To determine the role of SMN-dependent interneuron dysfunction on motor neuron hyperexcitability, the effect of selective SMN depletion in either motor neurons or interneurons was investigated. Importantly, I found that SMN-deficient motor neurons cultured in the presence of wild type interneurons are not hyperexcitable, while the presence of SMN-deficient interneurons is necessary and sufficient to elicit hyperexcitability of wild type motor neurons. Therefore, in the context of SMN deficiency, increased excitability of motor neurons is a homeostatic response to interneuron dysfunction. Although the exact mechanism is currently unknown, reduced glutamatergic drive appears to play a role since glutamatergic receptor blockers phenocopied SMN deficiency in inducing motor neuron hyperexcitability but not neuronal death. Moreover, SMN deficiency caused reduction of excitatory VGluT2 synapses on motor neurons. In addition to changes in intrinsic membrane properties, SMN deficiency caused severe reduction in the spontaneous activity and firing pattern of motor neurons. However, in contrast to death and hyperexcitability, SMN-dependent deficits in both motor neurons and interneurons appear to underlie this complex phenotype. The findings presented in this study validate the use of in vitro models to study SMA disease mechanisms and shed new light on the cellular basis of motor circuit dysfunction induced by SMN deficiency that can have predictive value in vivo.

This thesis describes the development of an systems biology method to study transcriptional programs that are activated during early and late phases of cell-fusion mediated reprogramming, as well as an implementation of systems-level analysis using reverse-engineered regulatory networks to study CNS disorders like Alcohol Addiction, and neurodegenerative disorders like Alzheimer's Disease (AD), and Parkinson's Disease (PD). The results will show an unprecedented view into the mechanisms underlying complex processes and diseases, and will demonstrate the predictive power of these methodologies that extended far beyond their original contexts.

Polyglutamine (polyQ) diseases describe a group of late-onset progressive neurodegenerative disorders which are caused by the CAG triplet repeat expansion in the coding region of disease genes. Such expansions result in expanded polyQ tracts in the disease proteins which confer neurotoxicity. To date, nine such diseases are reported including Huntington's disease and several types of spinocerebellar ataxias. Misfolding of polyQ proteins and formation of intracellular SDS-insoluble protein aggregates are closely associated with the toxicity of these diseases. In particular, impairment of the ubiquitin-proteasome system (UPS) which is responsible for protein degradation has been observed in polyQ diseases. Recently, ubiquitin ligases, which govern substrate specificity of the UPS, have gained huge attention in polyQ disease pathogenesis studies. In humans, cullin (Cul) proteins, including Cul1, 2, 3, 4 & 5, are integral components of a group of ubiquitin ligases called cullin- RING ubiquitin ligases (CRLs). Each CRL displays distinct substrate specificity through specific substrate receptors. Cullin proteins are evolutionarily conserved and Cul orthologues are found in the Drosophila genome. In the present study, it was found that individual Culs displayed distinct effects on polyQ pathogenesis in Drosophila polyQ models. Particularly, it was found that Cul1 modulated polyQ-induced toxic phenotype. This modification was accompanied with an alteration in the ubiquitination level and SDS-solubility properties of expanded polyQ protein. Through genetic interaction studies and biochemical analyses, it is suggested that Cul1-based CRL specifically targets SDS-insoluble species of expanded polyQ protein for ubiquitination via selective recognition by CG2010 substrate receptor. On the other hand, it was found that expanded polyQ protein induced accumulation of CRL substrates in cells. Current data support a hypothesis that polyQ protein would impair the ubiquitin ligase activity of CRLs upon expansion of the polyQ domain, through interfering with neddylation of cullin and other uncharacterized mechanisms. Taken together, the present study identifies Cul1-CRL as a novel E3 ligase that modifies polyQ toxicity through modulating ubiquitination of expanded polyQ protein, and demonstrates a pathological mechanism by expanded polyQ protein through impairing CRL activity. These findings would lead to a better understanding of polyQ pathogenesis and give insights on developing treatments against polyQ diseases. / Wong, Kam Yan. / Adviser: Ho-Yin Edwin Chan. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 260-273). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Polyglutamine disease is a class of neurodegenerative diseases, which is manifested by the atrophy of nervous system that results in dementia and/or motor dysfunction. The major pathological characteristics include progressive loss of neuronal cells as well as the appearance of insoluble nuclear inclusions in degenerating neuronal cells. Polyglutamine disease is caused by CAG triplet expansion in the genome. When translated, such expansion leads to the formation of expanded polyglutamine domain within the respective disease proteins and promotes abnormal protein conformational changes. Owing to their misfolded nature, the expanded polyglutamine proteins form insoluble nuclear inclusions. These insoluble nuclear inclusions are heterogeneous in nature, in which polyglutamine protein and molecular chaperones are the recruited components. All eukaryotic cells express molecular chaperones which function to mediate the proper folding of proteins. The recruitment of molecular chaperones into nuclear inclusions that contain misfolded triplet-expanded proteins strongly suggests the involvement of molecular chaperones in polyglutamine disease progression. It has been shown that over-expression of molecular chaperones in polyglutamine disease models can lead to a suppression of polyglutamine toxicity and a concomitant increase in the solubility of disease proteins, i.e. the solubility of polyglutamine disease protein is related to its toxicity. Intrigued by these observations, I aimed at elucidating the mechanism of polyglutamine disease pathogenesis by first studying the cellular regulation of endogenous chaperone expression in neurodegeneration in a transgenic Drosophila model of polyglutamine disease. A biphasic regulation of Hsp70 expression was observed, which the regulation was at the transcription level. Moreover, over-expression of Hsp70 could alter the endogenous Hsp70 protein and mRNA level of polyglutamine disease fly model. The study may help the understanding of how the chaperone expression is regulated under the effects of polyglutamine expression and thus to find out the mechanism of pathogenesis. In addition, cellular proteins that change in solubility other than disease protein will also be identified. Small heat shock proteins, glutathione S transferase and alpha 4 proteasome subunit, etc., showed change in solubility or expression by 2D gel electrophoresis analysis. Identifying the proteins that change in solubility or expression may help the finding of the interplay of proteins and thus the pathways involve in the mechanism of polyglutamine disease pathogenesis. Understanding pathogenic pathways can give ideas on how polyglutamine lead to the disease, up- or down-regulation of those protein interplays may provide direction and therapeutic candidates to suppress polyglutamine disease. / Huen Ngar Yee. / "September 2006." / Advisers: Ho Yin Chan; Siu Kai Kong. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1465. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 134-146). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Abstract not available

Graduation date: 1998

The word "Prion" refers to self-perpetuating protein aggregates that cause neurodegenerative diseases in mammals. It is a protein isoform that has undergone a conformational change which converts the normal form of the protein into the infectious form with the same amino acid sequence. Yeast [PSI+] prion is the prion isoform of Sup35 protein, a translation termination factor eRF3. It has been suggested that prion [PSI+] is controlled by the ensemble of chaperones with Hsp104 playing the major role. The previous work performed in the Chernoffs lab showed that the defective GET pathway caused by get led to the defect in [PSI+] curing by excess Hsp104. The GET pathway is a system responsible for transporting newly synthesized TA-protein to the ER membrane, and the components which have been proven to be involved in this pathway include: Get1, Get2, Get3, Get4, Get5 and Sgt2. In this study we describe the mechanism underlying the effect of the defective GET pathway on [PSI+]. We demonstrate that Sgt2, one of the components of GET pathway, interacts with Sup35 in both [PSI+] and [psi-] strains through its prion domain. Overproduction of Sgt2 and Hsp70-Ssa is triggered by the defective GET pathway and leads to the protection of [PSI+] aggregates from curing by excess Hsp104. We show that the direct interaction between Sgt2 and Hsp70-Ssa is not required for this protective effect.

This dissertation presents the development of a multielectrode microcompartment platform for understanding signal transduction in the nervous system. The design and fabrication of the system and the characterization of the system for pharmacological and electrophysiological measurements of cultured neurons is presented in this work. The electrophysiological activity of cultured dorsal root ganglion (DRG) neurons and cortical neurons is shown on the MEA substrate. These recordings were measured and tied to the toxicological effects of the chemotherapeutic drug vincristine on DRGs. Conventional electrophysiological recordings (via a patch micropipette) are made routinely to record action potentials and ion channel activity in neurons. Moreover, Campenot chambers (traditional compartmented culture systems) have been used for the last thirty years to study the selective application of drugs to neurons. Both of these techniques are useful and well established; however they have their limitations. For instance, Campenot chambers cannot be used very well for small processs-producing neurons, since the barriers are difficult to tranverse. Moreover, conventional patch recordings are labor-intensive, especially when more than one microelectrode needs to be positioned. The developed system is composed of a two compartment divider, each compartment capable of housing axons or cell bodies. Underneath the divider, the substrate has 60 electrodes, arranged in several lines to accommodate several different neurite tracks. Neurons can be stimulated and their activity can be recorded in both of the compartments. The neurotoxin and chemotherapeutic drug vincristine was tested in the system on the DRGs. The drug caused length-dependent axonal degeneration in the DRGs when applied locally. Moreover, electrophysiological activity in both compartments showed that only the activity in the axonal compartment was affected, leading us to believe that the mechanism behind the degeneration is localized to the distal axon.

No description available.