Tay-Sachs disease and its related disorders (GM2 gangliosidoses) are neurodegenerative diseases caused by the excessive accumulation of ganglioside GM2, an otherwise non-toxic plasma membrane component, in the lysosomes of cells of the central nervous system. The accumulation of ganglioside GM2 is the result of a defect in the gene encoding the α-subunit of β-hexosaminidase A (Hex A), an acid hydrolase responsible for the metabolism of gangloside GM2 in the lysosome. Though a debilitating disease in humans, Tay-Sachs model mice (𝘏𝘦𝘹𝘢-/-) escape symptoms by the action of lysosomal sialidase, which is expressed in mice at levels sufficient to metabolize ganglioside GM2 and effectively "bypass" Hex A deficiency. In an attempt to understand why a lysosomal sialidase-mediated bypass of Hex A deficiency is not observed in humans, we cloned ~ 2.9 kb of the human lysosomal sialdiase promoter and began characterization of the regulatory machinery that determines its activity. The transcription factor CDP (CCAAT -Displacement Protein) and truncations thereof were found to have a clear and consistent effect on promoter activity 𝘪𝘯 𝘷𝘪𝘵𝘳𝘰, with the truncation CDP⁸³¹⁻¹⁵⁰⁵ resulting in a near 50-fold increase in activity. Adenovirus-mediated gene transfer of CDP⁸³¹⁻¹⁵⁰⁵ into CRB/TSD cells, a human Tay-Sachs neuroglia cell line, resulted in elevated lysosomal sialidase activity and a decrease in ganglioside GM2 stores. These results suggest that the regulatory machinery responsible for lysosomal sialidase expression may be manipulated in such a way as to "activate" a sialidase-mediated bypass of Hex A deficiency in human Tay-Sachs cells. Thus, induction of lysosomal sialidase may have a potential therapeutic benefit in human Tay-Sachs disease and other Hex A-deficient GM2 gangliosidoses. / Thesis / Master of Science (MS)

Este estudo descreve a análise molecular de pacientes da doença de Gaucher (DG) e Tay-Sachs (DTS) no Brasil. Foram estudados nove casos de formas clássicas da DTS que mostraram uma prevalência da mutação IVS7+1g>c, já descrita em pacientes Portugueses e dez casos das variantes de início juvenil e tardio da DTS, mostrando heterogeneidade genética. Nos casos da variante B1, percebeu-se uma maior incidência da mutação R178H, também descrita previamente em pacientes Portugueses. A presença das mesmas mutações nos casos Brasileiros e Portugueses se deve provavelmente à ancestralidade comum. Uma família com quatro pacientes da variante de início tardio da DTS mostrou uma extensa variabilidade clínica intrafamilial e identificou relevantes aspectos do diagnóstico e das implicações dos programas de triagem populacional. A análise por RFLP de nove mutações causadoras da DG, em 262 pacientes permitiu detectar 76% das alterações e mostrou uma prevalência das mutações N370S e L444P, similar à descrita em diversas outras populações. Os pacientes com variantes neuronopáticas da doença apresentaram uma alta freqüência da mutação G377S, que também é encontrada em pacientes Portugueses. Os pacientes apresentando a G377S indicaram a existência de um provável mecanismo de efeito de dose alélica para essa mutação. Foi observada uma alta freqüência de alelos resultantes da recombinação do gene GBA com seu pseudogene. Diversas outras relações genótipo-fenótipo puderam ser verificadas, mostrando uma baixa penetrância do genótipo N370S/N370S e corroborando a importância do diagnóstico molecular da DG, devido a seu valor preditivo. A análise de mutações raras no gene GBA usando as técnicas de RFLP, dHPLC e seqüenciamento de DNA possibilitou detectar mutações em 84% dos alelos de 54 pacientes. Foram identificadas 14 novas mutações causadoras da DG. Diversas relações genótipo-fenótipo puderam ser verificadas, conferindo valor preditivo para a detecção dessas mutações. Por fim, a análise da associação da DG e da DP permitiu encontrar uma freqüência significativamente maior de portadores das principais mutações no gene GBA em pacientes parkinsonianos (2/65=3%), com aparecimento precoce da doença, comparados a um grupo controle de 267 indivíduos. Esse trabalho fornece nova evidência de que mutações no gene GBA são um raro, mas consistente fator de risco para a DP. / This study describes the molecular diagnosis of Gaucher (GD) and Tay-Sachs disease (TSD) patients in Brazil. Nine cases of the classic infantile form of TSD were analyzed disclosing a prevalence of the IVS7+1g>c mutation, described previously in Portuguese patients. Ten cases of juvenile and late-onset TSD forms were diagnosed showing genetic heterogeneity. Among the B1 variant cases, there was a predomenance of mutation R178H that was also associated to Portuguese ancestry. The presence of the same mutations in Brazilian and Portuguese cases are probably due to common ancestry. A family with 4 affected patients of late onset TSD showed and extensive intrafamilial clinical variability, highlighting relevant characteristics of diagnosis and implications of heterozygote screening programs. Among 263 GD patients, the detection of nine mutations by RFLP revealed 76% of the mutant alleles and a preponderance of N370S and L444P, similar to other populations. The type 3 neuronopathic patients presented a high frequency of mutation G377S, that is also described among Portuguese cases. The patients with G377S indicated the possibility of an allele dose effect for this mutation. Recombinant alleles, presenting pseudogene mutations were detected at a high frequency. Several genotype-phenotype correlations could be verified, highlighting a low penetrance of genotype N370S/N370S and corroborating the importance of molecular diagnosis in GD cases, due to its predictive value. The search for rare mutations at the GBA gene, using dHPLC and DNA sequencing after RFLP analysis, allowed the detection of 84% of the alleles among 54 patients. Fourteen new GD causing mutations were described. Several genotype-phenotype correlations could be established, confering prective value to the identification of these mutations. Finally, the study of the association of GD and Parkinson disease (PD) lead to the detection of a significant increase in the frequency of GBA mutations carriers, among 65 PD patients (2/65=3%) with earlier disease onset compared to a control group (n=267 individuals). This work confers further evidence for the fact that GBA mutations are a rare but consistent risk factor for PD.

A doença de Tay-Sachs (DTS) é uma doença neurodegenerativa, de herança autossômica recessiva, que se manifesta a partir do sexto mês de vida. Crianças afetadas desenvolvem degeneração física e mental intensa, levando à morte até os 5 anos de idade. Não há atualmente cura ou tratamento disponível. Na população judaica, 1 em cada 31 indivíduos é portador da DTS, e a incidência da doença (aproximadamente 1 em cada 4.000 nascimentos) é cerca de 100 vezes maior nesta do que em outras populações. O advento do diagnóstico pré-natal para a DTS e o concomitante desenvolvimento de programas de detecção e orientação de heterozigotos da DTS em populações de judeus Ashkenazitas, iniciados em massa desde 1970, levaram a uma diminuição de 90% da incidência da doença nesta população. Estes programas são realizados em Israel, EUA e no Canadá, na população francocanadense. Três mutações no gene HEXA, codificador da sub-unidade ? da enzima hexosaminidase A, são responsáveis por 98% dos casos da DTS na população judaica Ashkenazita. Esse fato possibilita a utilização de um teste de DNA para a identificação de portadores da DTS nesta população. Segundo o censo do IBGE de 1991, a população judaica no Brasil é de 86.416 indivíduos. Este trabalho visou analisar a necessidade e a aceitação de um programa de detecção e orientação de portadores da DTS nesta população. Em particular, procurou-se (1) estabelecer a freqüência das 3 principais mutações causadoras da DTS na população judaica brasileira; (2) avaliar a reação de indivíduos desta população à proposta de um programa de triagem de portadores da DTS, com o objetivo de orientação para casais em risco de terem crianças afetadas. Este estudo foi realizado em escolas judaicas de ensino médio, em São Paulo e no Rio de Janeiro, seguindo o modelo canadense. Dentre 581 alunos (>=16 anos) que assistiram palestras informativas, 404 participaram da pesquisa, indicando uma taxa de participação de 70%. De acordo com os dados dos formulários de consentimento, aproximadamente 65% dos cromossomos analisados eram de origem judaica Ashkenazita. A análise das 3 mutações comuns no gene HEXA nestes 404 indivíduos detectou 8 portadores da DTS (7 da mutação InsTATC1278 e 1 de IVS12+1). Assim, a freqüência de portadores na amostra foi de 1 em cada 51 indivíduos. Considerando somente os cromossomos de origem judaica Ashkenazita, nossos dados indicam que a freqüência de portadores da DTS na amostra é de 1 em cada 33 indivíduos. As freqüências encontradas são equivalentes àquelas descritas em outras populações judaicas (P>0,05). Baseando-se nestes dados, concluí-se que justifica-se a implementação de um programa de identificação e orientação de portadores da DTS na população judaica brasileira. A contemplação dos diversos aspectos éticos envolvidos são parte essencial deste programa. / Tay-Sachs disease (TSD) is an autossomal recessive disease of lysossome storage characterized by progressive nervous degeneration. Children affected by TSD manifest first symptoms around 6 months and die before 5 years of age. TSD is caused by mutations in the HEXA gene, coding for the ? subunit of the hexosaminidase A enzyme. In the absence of the enzyme, its substrate, GM2 ganglioside, accumulates in the neurons of the central nervous cortex. Late onset TSD (chronic form) is a rare variant phenotype with appearance of first symptoms during the second or third decade of life. In the Ashkenazi jewish population, 1 in every 31 individuals is a TSD carrier. The disease incidence (1 in every 4.000 newborns) is 100 times higher in this population. The advent of pre-natal diagnosis for TSD and the development of preventive screening programs massively adopted by Ashkenazi jewish populations led to a 90% decrease in the disease incidence in this group. Three mutations in the HEXA gene are responsible for 98% of the disease incidence in Ashkenazi jews. This lead to the establishment of a DNA test for detection of TSD carriers in this population. The brazilian Ashkenazi jewish populations is around 100.000 individuals. This work aimed to establish the need and the acceptance of a screening program for this population. Specifically, it established the frequency of the 3 most common TSD mutations in a sample of the brazilian jewish population and evaluated the reaction of the community to the offer of a preventive program. This work was undertaken in jewish senior high-school students, in São Paulo and Rio de Janeiro, following the canadian preventive model. From 581 students (>=16 years old) that attended educational sessions, 404 volunteered to the test, indicating a 70% participation rate. According to the data of the consent form, approximately 65% of the chromosomes were associated to Ashkenazi jewish ancestry. The molecular analysis of the 3 most common mutations in the HEXA gene from the 404 participants detected 8 carriers (7 of InsTATC1278 and 1 of IVS12+1), thus indicating a carrier frequency of 1/51. In the Ashkenazi fraction, the estimated carrier frequency is 1 in 33 individuals. Both frequencies are similar to those described for other jewish communities (P>0,05). Based in this findings, it was concluded that the implementation of a screening program for TSD in the brazilian jewish population is feasible. The ethical aspects involved are an essential part of such a program.

Este estudo descreve a análise molecular de pacientes da doença de Gaucher (DG) e Tay-Sachs (DTS) no Brasil. Foram estudados nove casos de formas clássicas da DTS que mostraram uma prevalência da mutação IVS7+1g>c, já descrita em pacientes Portugueses e dez casos das variantes de início juvenil e tardio da DTS, mostrando heterogeneidade genética. Nos casos da variante B1, percebeu-se uma maior incidência da mutação R178H, também descrita previamente em pacientes Portugueses. A presença das mesmas mutações nos casos Brasileiros e Portugueses se deve provavelmente à ancestralidade comum. Uma família com quatro pacientes da variante de início tardio da DTS mostrou uma extensa variabilidade clínica intrafamilial e identificou relevantes aspectos do diagnóstico e das implicações dos programas de triagem populacional. A análise por RFLP de nove mutações causadoras da DG, em 262 pacientes permitiu detectar 76% das alterações e mostrou uma prevalência das mutações N370S e L444P, similar à descrita em diversas outras populações. Os pacientes com variantes neuronopáticas da doença apresentaram uma alta freqüência da mutação G377S, que também é encontrada em pacientes Portugueses. Os pacientes apresentando a G377S indicaram a existência de um provável mecanismo de efeito de dose alélica para essa mutação. Foi observada uma alta freqüência de alelos resultantes da recombinação do gene GBA com seu pseudogene. Diversas outras relações genótipo-fenótipo puderam ser verificadas, mostrando uma baixa penetrância do genótipo N370S/N370S e corroborando a importância do diagnóstico molecular da DG, devido a seu valor preditivo. A análise de mutações raras no gene GBA usando as técnicas de RFLP, dHPLC e seqüenciamento de DNA possibilitou detectar mutações em 84% dos alelos de 54 pacientes. Foram identificadas 14 novas mutações causadoras da DG. Diversas relações genótipo-fenótipo puderam ser verificadas, conferindo valor preditivo para a detecção dessas mutações. Por fim, a análise da associação da DG e da DP permitiu encontrar uma freqüência significativamente maior de portadores das principais mutações no gene GBA em pacientes parkinsonianos (2/65=3%), com aparecimento precoce da doença, comparados a um grupo controle de 267 indivíduos. Esse trabalho fornece nova evidência de que mutações no gene GBA são um raro, mas consistente fator de risco para a DP. / This study describes the molecular diagnosis of Gaucher (GD) and Tay-Sachs disease (TSD) patients in Brazil. Nine cases of the classic infantile form of TSD were analyzed disclosing a prevalence of the IVS7+1g>c mutation, described previously in Portuguese patients. Ten cases of juvenile and late-onset TSD forms were diagnosed showing genetic heterogeneity. Among the B1 variant cases, there was a predomenance of mutation R178H that was also associated to Portuguese ancestry. The presence of the same mutations in Brazilian and Portuguese cases are probably due to common ancestry. A family with 4 affected patients of late onset TSD showed and extensive intrafamilial clinical variability, highlighting relevant characteristics of diagnosis and implications of heterozygote screening programs. Among 263 GD patients, the detection of nine mutations by RFLP revealed 76% of the mutant alleles and a preponderance of N370S and L444P, similar to other populations. The type 3 neuronopathic patients presented a high frequency of mutation G377S, that is also described among Portuguese cases. The patients with G377S indicated the possibility of an allele dose effect for this mutation. Recombinant alleles, presenting pseudogene mutations were detected at a high frequency. Several genotype-phenotype correlations could be verified, highlighting a low penetrance of genotype N370S/N370S and corroborating the importance of molecular diagnosis in GD cases, due to its predictive value. The search for rare mutations at the GBA gene, using dHPLC and DNA sequencing after RFLP analysis, allowed the detection of 84% of the alleles among 54 patients. Fourteen new GD causing mutations were described. Several genotype-phenotype correlations could be established, confering prective value to the identification of these mutations. Finally, the study of the association of GD and Parkinson disease (PD) lead to the detection of a significant increase in the frequency of GBA mutations carriers, among 65 PD patients (2/65=3%) with earlier disease onset compared to a control group (n=267 individuals). This work confers further evidence for the fact that GBA mutations are a rare but consistent risk factor for PD.

A doença de Tay-Sachs (DTS) é uma doença neurodegenerativa, de herança autossômica recessiva, que se manifesta a partir do sexto mês de vida. Crianças afetadas desenvolvem degeneração física e mental intensa, levando à morte até os 5 anos de idade. Não há atualmente cura ou tratamento disponível. Na população judaica, 1 em cada 31 indivíduos é portador da DTS, e a incidência da doença (aproximadamente 1 em cada 4.000 nascimentos) é cerca de 100 vezes maior nesta do que em outras populações. O advento do diagnóstico pré-natal para a DTS e o concomitante desenvolvimento de programas de detecção e orientação de heterozigotos da DTS em populações de judeus Ashkenazitas, iniciados em massa desde 1970, levaram a uma diminuição de 90% da incidência da doença nesta população. Estes programas são realizados em Israel, EUA e no Canadá, na população francocanadense. Três mutações no gene HEXA, codificador da sub-unidade ? da enzima hexosaminidase A, são responsáveis por 98% dos casos da DTS na população judaica Ashkenazita. Esse fato possibilita a utilização de um teste de DNA para a identificação de portadores da DTS nesta população. Segundo o censo do IBGE de 1991, a população judaica no Brasil é de 86.416 indivíduos. Este trabalho visou analisar a necessidade e a aceitação de um programa de detecção e orientação de portadores da DTS nesta população. Em particular, procurou-se (1) estabelecer a freqüência das 3 principais mutações causadoras da DTS na população judaica brasileira; (2) avaliar a reação de indivíduos desta população à proposta de um programa de triagem de portadores da DTS, com o objetivo de orientação para casais em risco de terem crianças afetadas. Este estudo foi realizado em escolas judaicas de ensino médio, em São Paulo e no Rio de Janeiro, seguindo o modelo canadense. Dentre 581 alunos (>=16 anos) que assistiram palestras informativas, 404 participaram da pesquisa, indicando uma taxa de participação de 70%. De acordo com os dados dos formulários de consentimento, aproximadamente 65% dos cromossomos analisados eram de origem judaica Ashkenazita. A análise das 3 mutações comuns no gene HEXA nestes 404 indivíduos detectou 8 portadores da DTS (7 da mutação InsTATC1278 e 1 de IVS12+1). Assim, a freqüência de portadores na amostra foi de 1 em cada 51 indivíduos. Considerando somente os cromossomos de origem judaica Ashkenazita, nossos dados indicam que a freqüência de portadores da DTS na amostra é de 1 em cada 33 indivíduos. As freqüências encontradas são equivalentes àquelas descritas em outras populações judaicas (P>0,05). Baseando-se nestes dados, concluí-se que justifica-se a implementação de um programa de identificação e orientação de portadores da DTS na população judaica brasileira. A contemplação dos diversos aspectos éticos envolvidos são parte essencial deste programa. / Tay-Sachs disease (TSD) is an autossomal recessive disease of lysossome storage characterized by progressive nervous degeneration. Children affected by TSD manifest first symptoms around 6 months and die before 5 years of age. TSD is caused by mutations in the HEXA gene, coding for the ? subunit of the hexosaminidase A enzyme. In the absence of the enzyme, its substrate, GM2 ganglioside, accumulates in the neurons of the central nervous cortex. Late onset TSD (chronic form) is a rare variant phenotype with appearance of first symptoms during the second or third decade of life. In the Ashkenazi jewish population, 1 in every 31 individuals is a TSD carrier. The disease incidence (1 in every 4.000 newborns) is 100 times higher in this population. The advent of pre-natal diagnosis for TSD and the development of preventive screening programs massively adopted by Ashkenazi jewish populations led to a 90% decrease in the disease incidence in this group. Three mutations in the HEXA gene are responsible for 98% of the disease incidence in Ashkenazi jews. This lead to the establishment of a DNA test for detection of TSD carriers in this population. The brazilian Ashkenazi jewish populations is around 100.000 individuals. This work aimed to establish the need and the acceptance of a screening program for this population. Specifically, it established the frequency of the 3 most common TSD mutations in a sample of the brazilian jewish population and evaluated the reaction of the community to the offer of a preventive program. This work was undertaken in jewish senior high-school students, in São Paulo and Rio de Janeiro, following the canadian preventive model. From 581 students (>=16 years old) that attended educational sessions, 404 volunteered to the test, indicating a 70% participation rate. According to the data of the consent form, approximately 65% of the chromosomes were associated to Ashkenazi jewish ancestry. The molecular analysis of the 3 most common mutations in the HEXA gene from the 404 participants detected 8 carriers (7 of InsTATC1278 and 1 of IVS12+1), thus indicating a carrier frequency of 1/51. In the Ashkenazi fraction, the estimated carrier frequency is 1 in 33 individuals. Both frequencies are similar to those described for other jewish communities (P>0,05). Based in this findings, it was concluded that the implementation of a screening program for TSD in the brazilian jewish population is feasible. The ethical aspects involved are an essential part of such a program.

The major beta-hexosaminidase isozymes in humans are Hex A (deficient in Tay-Sachs disease, TSD), an alphabeta heterodimer and Hex B (deficient in Sandhoff disease) a betabeta homodimer. Hex S, the alphaalpha homodimer is physiologically unstable. Mature alpha and beta subunits share 60% sequence identity. The beta subunit active site hydrolyzes neutral substrates. The alpha subunit active site hydrolyzes neutral (4MUG) and charged substrates (4MUGS, GM2 ganglioside). Only Hex A hydrolyzes the natural substrate, GM2 ganglioside, in the presence of the GM2 activator protein (AP). / We investigated regions of the alpha and beta subunits involved in AP binding, subunit dimerization, and substrate specificity. We constructed four chimeric cDNAs: alpha1--259beta292--544 , alpha1--118beta152--544, beta 1--418alpha387--529, and beta1--151 alpha119--259beta292--544 (subscripts refer to amino acid residues). Chimeric cDNAs were expressed in a TSD neuroglial cell line, which produces no endogenous alpha subunits. The chimeric isozymes were chromatofocused and assayed for hydrolysis of (a) 4MUG, (b) 4MUGS and (c) GM2 ganglioside. / Transfection of the cDNA constructs lead to expression of homodimeric and heterodimeric chimeric proteins, albeit at lower yields than transfection of wild alpha-cDNA. All of the chimeric proteins hydrolyzed 4MUG but none were active towards 4MUGS or GM2 ganglioside. These results suggest that (a) all constructs contained sufficient information to form both heterodimeric and homodimeric chimeric proteins, (b) the chimeras lacked the alpha-subunit sequence necessary for the hydrolysis of charged substrates.

No description available.

<p> The objective is to investigate the molecular mechanisms leading to neurodegeneration in Tay Sachs disease, a lysosomal storage disorder caused by a deficiency in the enzyme hexosaminidase A. This was accomplished using microarray analysis of normal and Tay Sachs neuroglia. Microarray data analysis was performed using Onto Express, PANTHER software, and cluster analysis. The expression levels of selected genes were validated using Real Time PCR. To establish a physiological relationship between GM2 accumulation and the expression of these genes, their expression was assessed after treatment with an inhibitor of ganglioside synthesis, nbutyldeoxynojirimycin (NBDNJ). Neuronal pentraxin 1 (NPTX1), potassium channel, subfamily K, member 2 (KCNK2), and prostaglandin synthase 1 (PTGS 1) were found to be upregulated in Tay Sachs neuroglia while heme oxygenase (HMOX1) was downregulated. The mRNA levels ofNPTX1, KCNK2, PTGS1, and HMOX1 all reverted to normal levels in response to ganglioside synthesis inhibitor. Pentraxin RNA levels were also reduced in response to sialidase overexpression, another method of GM2 reduction. Pentraxin protein levels were also increased in Tay Sachs cells in response to u-amino-3-hydroxy-5-methylisoxazole-4- propionic acid (AMPA), and were attenuated upon treatment with either NBDNJ or AMPA antagonist 2,3-dihydroxy-6-nitro-7- sulfamoyl-benzo [f]quinoxaline-2,3-dione (NBQX). Strong colocalization was seen between NPTX1 and the AMPA receptor as well as the glutamate transporter EAACI. AMP A receptor function was also enhanced as illustrated by an increase in calcium influx upon stimulation. This stimulation also resulted in increased apoptosis during AMPA receptor stimulation. In this study, our genetic profiling experiment led to the identification of NPTXl as a marker of pathogenesis in Tay Sachs Cells. The role of NPTXl in excitotoxicity implicates the latter in disease mechanism associated with Tay Sachs disease. Furthermore, this study provides evidence that TSD cells undergo programmed cell death in response to increased intracellular calcium as a result of increased glutamate receptor stimulation. </p> / Thesis / Master of Science (MSc)

Lysosomal storage disorders are a group of rare neurodegenerative diseases that are collectively common, sharing many aspects with other neurodegenerative disorders, including substrate build-up and neuroinflammation. The GM2 Gangliosidoses, Tay-Sachs disease and Sandhoff disease, are pathologically overlapping lysosomal storage disorders, with high prevalence within specific ethnicities. Their effects are neurologically devastating and often fatal at young ages. Current treatments only slow or stall an inevitable decline in health. Novel treatment targets are needed for these disorders, and others with similar pathologies. In these works we demonstrate the negative effect the inflammatory cytokine tumour necrosis factor-alpha has on survival of a model of Sandhoff disease. We demonstrate its role in the upregulation of astrogliosis, and apoptosis, and we present evidence that this effect on astrogliosis occurs through an upregulation of the JAK-2/STAT3 pathway. Though fruitful, a singular focus on inflammation/gliosis in these diseases has left a vacuum in the research into neuron specific molecular processes. We observe the development of inflammation, astrogliosis and neuronal processes in our model, and demonstrate a bi-phasic disease progression, in which early onset microgliosis precedes terminal astrogliosis, apoptosis, and a decline in excitatory glutamate receptors, suggesting neuron-specific malfunction. Furthermore, we show that knockout of the synaptic protein neuronal pentraxin 1 retards neurodegeneration and extends the lifespan of Sandhoff disease mice, independent of inflammation or astrogliosis. Through electrophysiology, we provide evidence of dysregulation of glutamate receptors in Sandhoff disease, and show that knockout of neuronal pentraxin 1 provides rescue from this dysregulation. This work expands on research into gliosis in GM2 gangliosidoses, presents the finding of a novel protein isoform, and presents a new focus on non-glial disease mechanisms and treatments for these and other neurodegenerative disorders. / Thesis / Doctor of Philosophy (PhD) / Lysosomal storage disorders are a group of neurological diseases that are debilitating, and often fatal at a young age. Two diseases of this group- Tay-Sachs disease and Sandhoff disease – are similar in their causes and symptoms. Current treatments for these diseases only slow or stall an inevitable decline in health. New targets for treatment are required, and we provide data suggesting several proteins that may fit this criterion. We also provide evidence of the discovery of a new form of one of these proteins, which is found in high levels in the disease, indicating it may be important in these and other neurodegenerative diseases. Finally, we provide findings indicating that a certain cell type, which is largely ignored in current research for these diseases, may be important in the disease progress. These findings increase our knowledge of Tay-Sachs disease and Sandhoff disease, and open new avenues for medicinal intervention.

<p>GM2 gangliosidoses encompass a group of chronic neurodegenerative disorders characterized by metabolic defects in ganglioside catabolism and marked intralysosomal accumulation of GM2 in central nervous system (CNS)-resident neurons. Included in this group are Tay-Sachs and Sandhoff disease. Human cases of Tay-Sachs and Sandhoff disease present with devastating neurological deterioration; however, murine models display drastically divergent phenotypes. Tay-Sachs mice avoid pathology via a sialidase-mediated bypass of β-hexosaminidase A (HEXA) deficiency, though the precise mechanism of avoidance is not fully elucidated. The following work aimed to: i) determine if the murine sialidase-mediated bypass could be potentiated in human cells, and ii) help clarify the mechanism of disease avoidance in Tay-Sachs animals.</p> <p>Adenoviral overexpression of truncated CCAAT displacement protein (CDP^831-1505) in human Tay-Sachs neuroglia augmented neuraminidase 1/lysosomal sialidase (NEU1) protein levels, which reduced intralysosomal GM2 accumulations. Chromatin immunoprecipitation revealed binding of CDP^831-1505 to the human <em>NEU1</em> promoter in Tay-Sachs neuroglia. These results provide mechanistic and functional evidence supporting therapeutic exploitation of <em>NEU1</em> for Tay-Sachs disease.</p> <p>Comparison of immunological responses of bone marrow-derived macrophages (BMDMs) to pathogen associated molecular patterns (PAMPs) or GM2 demonstrated that Sandhoff macrophages secrete increased TNF and reduced IL-10 following lipopolysaccharide stimulation. GM2 treatment failed to stimulate an immune response. Such behaviour occurred in the absence of clearly observable intralysosomal ganglioside accumulations. Altered LAMP2 protein size, potentially due to aberrant glycosylation, is hypothesized to disrupt autophagosomal/lysosomal fusion. Subsequent autophagosomal accumulation could result in inherent macrophage hypersensitivity and immunologic irritability. Downstream interleukin-10 (IL-10)/signal transducer and activator of transcription 3 (Stat3) axis, mitogen activated protein kinase (MAPK), and glycogen synthase kinase 3-beta (GSK3β) signaling pathways were affected in Sandhoff BMDMs. These data indicate inherent differences in immunological responses of BMDMs from Sandhoff mice, presumably related to their β-hexosaminidase B (HEXB) deficiency.</p> <p>Data presented here provides evidence to suggest a paradigm shift in the neurodegenerative model of Tay-Sachs and Sandhoff Diseases towards one that places immune cells as an initiating factor for widespread neuroinflammation.</p> / Master of Science (MSc)