A mis-sense point mutation in the human VAPB gene is associated with a familial form of motor neuron disease that has been classified as Amyotrophic Lateral Sclerosis type VIII. Affected individuals suffer from a spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS) or an atypical slowly progressing form of ALS. Mammals have two homologous VAP genes, vapA and vapB. VAPA and VAPB share 76% similar or identical amino acid residues; both are COOHterminally anchored membrane proteins enriched on the endoplasmic reticulum. Several functions have been ascribed to VAP proteins including membrane trafficking, cytoskeleton association and membrane docking interactions for cytoplasmic factors. It is shown here that VAPA and VAPB are expressed in tissues throughout the body but at different levels, and that they are present in overlapping but distinct regions of the endoplasmic reticulum. The disease-associated mutation in VAPB, VAPB (P56S) is within a highly conserved N-terminal region of the protein that shares extensive structural homology with the major sperm protein (MSP) from nematodes. The MSP domain of VAPA and VAPB is found to interact with the ERlocalized transcription factor ATF6. Over expression of VAPB or VAPB (P56S) attenuates the activity of ATF6-regulated transcription and the mutant protein VAPB (P56S) appears to be a more potent inhibitor of ATF6 activity. Moreover VAP proteins affect the activity of XBP1 and BiP promoter elements, two major components of the Unfolded Protein Response (UPR) of the Endoplasmic Reticulum and the different domains of VAPB have a differential effect on UPR regulation. Finally, over expression of the MSP domain of VAPB leads to cell death via apoptosis, while overexpression of other VAPB domains renders cells more susceptible to apoptotic death after ER stress. The data presented in this thesis indicate that VAP proteins interact directly with components of ER homeostatic and stress signalling systems and may therefore be parts of a previously unidentified regulatory pathway. The mis-function of such regulatory systems may contribute to the pathological mechanisms of degenerative motor neuron disease.

A Esclerose Lateral Amiotrófica é uma doença neurodegenerativa que afeta seletivamente neurônios motores. A maior parte dos casos de ELA (90%) é esporádica. Para os casos familiais, mais de vinte genes já foram associados. Diversos mecanismos estão envolvidos na patogênese, entre eles o estresse oxidativo, proteostase e agregação, excitoxicidade, tráfego intracelular, entre outros. A mutação P56S na proteína VAPB está associada à ELA8. A VAPB é uma proteína de membrana do retículo endoplasmático e está possivelmente envolvida em diversas funções celulares, dentre elas tráfego intracelular, interação retículo endoplasmático-aparelho de Golgi e UPR. Sabendo que mutações no gene que codifica VAPB resultam em ELA e que indivíduos com a mesma mutação neste gene podem apresentar quadros clínicos bastante diferentes, propõe-se estudar a suscetibilidade ao estresse oxidativo e ao estresse do retículo endoplasmático e as possíveis vias de degradação das proteínas mutantes como fatores subjacentes a essa heterogeneidade clínica. Desta forma, objetivou-se realizar uma análise integrada de ELA8, buscando compreender os mecanismos moleculares envolvidos na doença, utilizando a levedura Saccharomyces cerevisiae como modelo para estudo. Foram obtidas diferentes linhagens BY4741 de S. cerevisiae, expressando os genes de VAPBWT e VAPBP56S ou o plasmídeo vazio (que foi utilizado como controle em todos os experimentos) sob controle do promotor GAL1. Foram avaliadas as viabilidades das células expressando as proteínas humanas, a sua localização celular e possível formação de agregados. Os resultados mostram que a expressão da proteína VAPBP56S é tóxica e leva à formação de agregados dispersos nas células, enquanto a expressão da proteína selvagem se concentra no retículo endoplasmático e não altera significativamente a viabilidade das células. Scs2 é a proteína de levedura homóloga a VAPB, e a deleção do gene correspondente gera linhagens auxotróficas para inositol. VAPBWT e VAPBP56S foram expressas em linhagem nocaute para o gene Scs2, a fim de analisar se os homólogos humanos complementam a auxotrofia a inositol. Observou-se que a linhagem expressando a proteína selvagem é capaz de restaurar o fenótipo selvagem e a linhagem expressando a proteína mutante não. Para avaliar os efeitos de estresse oxidativo nas linhagens BY4741, foram determinados: a viabilidade e sensibilidade das linhagens sob condições de estresse induzido por H2O2, a razão GSH/GSSG e a produção de H2O2 por mitocôndrias, além da viabilidade após tratamento com o antioxidante N-acetil-L-cisteína. De modo geral foi verificado que a linhagem expressando a proteína mutante é discretamente mais sensível ao tratamento com H2O2, possui menor razão GSH/GSSG, e produz mais H2O2 em mitocôndrias isoladas. Em conjunto, estes dados sugerem alteração no metabolismo redox a partir da expressão de VAPBP56S. Os efeitos da inibição do proteassomo (ΔPdr5 + MG132) e da autofagia (ΔAtg8) foram avaliados em ensaios de viabilidade e degradação proteica. Verificou-se que a inibição do proteassomo tem maior efeito sobre a viabilidade das linhagens expressando VAPBWT e diminui a degradação desta proteína. A inibição da autofagia, ao contrário, afeta mais a linhagem expressando VAPBP56S. A atividade do proteassomo, a ubiquitinação de proteínas e os níveis de autofagia também foram avaliados, sendo verificado que há maior expressão de subunidades do proteassomo nas linhagens expressando ambas as proteínas. Na linhagem expressando VAPBWT, observou-se maior atividade do proteassomo e uma diminuição no pool de proteínas ubiquitinadas, de acordo com a maior expressão de subunidades do proteassomo. Na linhagem expressando VAPBP56S, ao contrário, há diminuição da atividade do proteassomo e acúmulo de proteínas ubiquitinadas, sugerindo uma inibição do proteassomo. Por meio do monitoramento da fusão GFP-Atg8 foi verificada a maior formação de autofagossomos nas linhagens expressando VAPBP56S, o que sugere maiores níveis de autofagia. Foi avaliada a viabilidade das células sob o efeito do aumento da expressão de Tsa1, uma peroxirredoxina com capacidade de recrutar chaperonas para agregados de forma redox dependente. Observou-se que esta proteína é capaz de atenuar a toxicidade de VAPBP56S, especialmente no ensaio de diluição seriada. Por fim foram verificados os níveis de marcadores de estresse do retículo endoplasmático, Pdi1, Ero1, Lhs1 e Kar2, e de UPR, SHac1, e foi visto que a expressão da proteína mutante alterou todos estes indicadores. Os dados em conjunto sugerem alterações no metabolismo redox e na proteostase resultantes da expressão de VAPBP56S / Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease that affects motor neurons. The majority of ALS cases (90%) are sporadic. More than twenty genes have been associated with familial cases. Several mechanisms are involved in ALS pathogenesis, including oxidative stress, proteostasis and aggregation, excitotoxicity, intracellular trafficking, and others. The P56S mutation in the protein VAPB is associated with ALS8. VAPB is a membrane protein of the endoplasmic reticulum that is possibly involved in diverse cellular functions, including intracellular trafficking, interaction endoplasmic reticulum-Golgi and UPR. Knowing that mutations in the gene encoding VAPB result in ALS and that individuals with the same mutation in this gene can show different clinical conditions, we aimed to analyze the susceptibility to oxidative and endoplasmic reticulum stresses and protein degradation pathways as factors underlying this clinical heterogeneity. Thus, the objective was to perform an integrated analysis of ALS8, trying to understand the molecular mechanisms involved in the disease using, for this purpose, budding yeast Saccharomyces cerevisiae was employed as a model for this study. Different strains of S. cerevisiae containing the gene VAPBWT or VAPBP56S or the empty plasmid were obtained. BY4741 strains were evaluated for their viability when expressing human proteins, the subcellular localization of these proteins and the ability to form aggregates. The results show that the expression of the mutant protein is toxic and leads to the formation of disperse aggregates in the cell, while the expression of the wild-type protein is concentrated in the endoplasmic reticulum and does not alter cell viability. Scs2 is the yeast homologue of VAPB and deletion of the corresponding gene renders cells auxotrophic for inositol. Therefore, VAPBWT and VAPBP56S genes were expressed in Δscs2 cells to evaluate their ability to complement the inositol auxotrophy. The strain expressing the wild-type protein was able to restore the wild type phenotype, while the strain expressing the mutant protein was not. To evaluate oxidative stress in BY4741 strains expressing human proteins, it was determined: viabilities and sensitivities to stress induced by H2O2; GSH/GSSG ratio and H2O2 production in the mitochondria, as well as viabilities after treatment of cells with N-acetyl-L-cysteine. In general, it was found that the strain expressing the mutant protein is slightly sensitive to treatment with H2O2, had minor GSH/GSSG ratio, which indicates more oxidative cellular environment, and has a major production of H2O2 in isolated mitochondria. Together, these data suggest important changes in the redox metabolism associated with VAPBP56Sexpression. The effects of inhibition of the proteasome (ΔPdr5 + MG132) and autophagy (ΔAtg8) were evaluated through viability assays and protein degradation. Inhibition of the proteasome had a greater effect on the viability of strains expressing VAPBWT and decreased the degradation of this protein. Inhibition of autophagy, in contrast, mainly affected the strain expressing VAPBP56S. The activity of the proteasome, protein ubiquitilation and autophagy levels were evaluated in BY4741 strains expressing human proteins. We found an increased expression of proteasome subunits in the strains expressing both proteins, which lead to an increased activity of proteasome in VAPBWT strain and a decrease in the pool of ubiquitilated proteins. In strain expressing VAPBP56S instead, there is a reduced proteasome activity and accumulation of ubiquitilated proteins. By monitoring the GFP-Atg8 fusion, it was verified that the formation of autophagosomes was increased in strains expressing VAPBP56S, suggesting higher levels of autophagy. The effect of Tsa1 expression, a peroxiredoxin capable to recruit chaperone to aggregates, on cell viability was evaluated and it was observed that this protein was able to attenuate the toxicity of VAPBP56S. Finally the levels of endoplasmic reticulum stress markers, Pdi1, Ero1, Lhs1 and Kar2, and the UPR marker, SHac1, were checked and it was found that the expression of the mutant protein is able to change all these indicators. Taken together, our data suggest changes in the redox metabolism and proteostasis linked to VAPBP56S expression

A Esclerose Lateral Amiotrófica (ELA) é uma doença crônica, progressiva e neurodegenerativa causada pela morte dos neurônios motores. O diagnóstico destes pacientes pode levar até 12 meses para acontecer, sendo que estes vão à óbito entre 3-5 anos do início dos sintomas. Há, porém, grande variabilidade de quadro clínico, com alguns pacientes falecendo com menos de 1 ano do início dos primeiros sinais, e outros que sobrevivem por décadas. A identificação da ELA8, causada por uma mutação missense no gene VAPB (c.C166T, p.P56S), tem contribuído significativamente com o conhecimento dos mecanismos moleculares por trás da ELA. A literatura recente tem evidenciado que a diminuição dos níveis de VAPB está presente em modelos celulares e murinos da doença, e também em amostras de pacientes, sugerindo que esta proteína teria papel central na doença e uma contribuição significativa para a morte dos neurônios motores. O presente trabalho buscou três objetivos principais: (1) o diagnóstico molecular através de um painel de sequenciamento de nova geração que inclui os genes SOD1, FUS, TARDBP, SETX, SPG11, FIG4 e VAPB; (2) a avaliação dos níveis de RNAm de VAPA, VAPB e EPHA4 em pacientes de ELA8, controles familiares e outros pacientes de ELA, com o intuito de investigar possíveis papéis destes genes na doença; e por fim, (3) o desenvolvimento de um ensaio quantitativo para as proteínas VAPA, VAPB e VAPC baseado em cromatografia líquida acoplada à espectrometria de massas em tandem (LC-MS/MS), para a posterior avaliação de VAPB como possível biomarcador em ELA, e de suas isoformas VAPA e VAPC como modificadores da doença. Para a análise genômica, foram avaliados 67 pacientes, sendo que 31 (ou 46%) apresentaram a mutação c.C166T em VAPB; 4 pacientes (6%) em SOD1, sendo que um destes apresentou uma mutação também em FIG4; 1 paciente (1.5%) foi identificado uma mutação patogênica em FUS; outro, duas mutações deletérias em trans em SPG11. Os níveis de RNAm de VAPB, VAPA e EPHA4 não são estatisticamente distintos entre pacientes e controles; porém, os níveis de EPHA4 estavam significativamente elevados em dois pacientes de início bulbar da doença. Para o desenvolvimento do método quantitativo por LC-MS/MS, foram escolhidos 8 peptídeos inequívocos para análise, estabelecidos dos parâmetros de corrida, e desenvolvidos dois padrões internos (linhagens SILAC e VAPB recombinante) para a quantificação. Esta ferramenta desenvolvida poderá auxiliar não apenas os estudos moleculares que envolvem os mecanismos por trás ELA8, responsável por uma elevada taxa dos casos familiais brasileiros, mas também poderá determinar o potencial de VAPB como biomarcador para Esclerose Lateral Amiotrófica / Amyotrophic Lateral Sclerosis is a chronic, progressive neurodegenerative disorder caused by the death of motor neurons. Diagnosis can take up to 12 months, with no molecular marker to expedite this process. In this scenario, patients die within 3 to 5 years of symptom onset, although a large clinical variability is seen, with severe patients dying less than one year after onset, and others surviving for decades. The identification of ALS8, caused by a missense mutation in the VAPB gene (c.C166T; p.P56S), has contributed significantly to the knowledge of molecular mechanisms behind ALS. Recent literature has evidenced that the decrease of VAPB levels is present in cellular and murine models, and also in patient samples, suggesting a central role in motor neuron death in ALS. The present work sought three main objectives: (1) a molecular diagnosis through a NGS sequencing panel including the SOD1, FUS, TARDBP, SETX, SPG11, FIG4 and VAPB genes; (2) analyze the expression levels of VAPA, VAPB and EPHA4 in patients, family controls and other forms of ALS, in order to investigate their possible roles in ALS8; and (3) the development of a targeted quantitative mass spectrometry based assay, gold standard in protein quantification due to its precision and sensitivity, for the VAPA, VAPB and VAPC proteins, seeking the analysis of VAPB as a potential biomarker in ALS and of its isoform\'s potential roles as modifiers in the disease. The genomic analyses revealed that out of 67 patients, 31 presented the ALS8 mutation in VAPB, 4 patients (6%) presented a mutation in SOD1, with one patient carrying a second mutation in FIG4; 1 (1.5%) patient was identified with a pathogenic mutation in FUS; and another presented two pathogenic mutations in trans in the SPG11 gene. Thus, we were able to diagnose over half of the patients included in this study with a panel of only 7 genes. VAPB, VAPA and EPHA4 mRNA levels are not statistically different between patients and controls; however, EPHA4 was shown to be highly elevated in two bulbar-onset non-ALS8 patients. For the development of the LC-MS/MS targeted assay, 8 surrogate peptides were chosen for analysis, run parameters were established, and two internal standards for quantification were developed (SILAC cell lines and recombinant VAPB). This tool will prove to be useful not only towards elucidating the molecular mechanisms behind ALS8, one of the most prevalent forms of familial ALS in Brazil, but also to determine VAPB\'s potential as a biomarker for ALS

A Esclerose Lateral Amiotrófica (ELA) é uma doença crônica, progressiva e neurodegenerativa causada pela morte dos neurônios motores. O diagnóstico destes pacientes pode levar até 12 meses para acontecer, sendo que estes vão à óbito entre 3-5 anos do início dos sintomas. Há, porém, grande variabilidade de quadro clínico, com alguns pacientes falecendo com menos de 1 ano do início dos primeiros sinais, e outros que sobrevivem por décadas. A identificação da ELA8, causada por uma mutação missense no gene VAPB (c.C166T, p.P56S), tem contribuído significativamente com o conhecimento dos mecanismos moleculares por trás da ELA. A literatura recente tem evidenciado que a diminuição dos níveis de VAPB está presente em modelos celulares e murinos da doença, e também em amostras de pacientes, sugerindo que esta proteína teria papel central na doença e uma contribuição significativa para a morte dos neurônios motores. O presente trabalho buscou três objetivos principais: (1) o diagnóstico molecular através de um painel de sequenciamento de nova geração que inclui os genes SOD1, FUS, TARDBP, SETX, SPG11, FIG4 e VAPB; (2) a avaliação dos níveis de RNAm de VAPA, VAPB e EPHA4 em pacientes de ELA8, controles familiares e outros pacientes de ELA, com o intuito de investigar possíveis papéis destes genes na doença; e por fim, (3) o desenvolvimento de um ensaio quantitativo para as proteínas VAPA, VAPB e VAPC baseado em cromatografia líquida acoplada à espectrometria de massas em tandem (LC-MS/MS), para a posterior avaliação de VAPB como possível biomarcador em ELA, e de suas isoformas VAPA e VAPC como modificadores da doença. Para a análise genômica, foram avaliados 67 pacientes, sendo que 31 (ou 46%) apresentaram a mutação c.C166T em VAPB; 4 pacientes (6%) em SOD1, sendo que um destes apresentou uma mutação também em FIG4; 1 paciente (1.5%) foi identificado uma mutação patogênica em FUS; outro, duas mutações deletérias em trans em SPG11. Os níveis de RNAm de VAPB, VAPA e EPHA4 não são estatisticamente distintos entre pacientes e controles; porém, os níveis de EPHA4 estavam significativamente elevados em dois pacientes de início bulbar da doença. Para o desenvolvimento do método quantitativo por LC-MS/MS, foram escolhidos 8 peptídeos inequívocos para análise, estabelecidos dos parâmetros de corrida, e desenvolvidos dois padrões internos (linhagens SILAC e VAPB recombinante) para a quantificação. Esta ferramenta desenvolvida poderá auxiliar não apenas os estudos moleculares que envolvem os mecanismos por trás ELA8, responsável por uma elevada taxa dos casos familiais brasileiros, mas também poderá determinar o potencial de VAPB como biomarcador para Esclerose Lateral Amiotrófica / Amyotrophic Lateral Sclerosis is a chronic, progressive neurodegenerative disorder caused by the death of motor neurons. Diagnosis can take up to 12 months, with no molecular marker to expedite this process. In this scenario, patients die within 3 to 5 years of symptom onset, although a large clinical variability is seen, with severe patients dying less than one year after onset, and others surviving for decades. The identification of ALS8, caused by a missense mutation in the VAPB gene (c.C166T; p.P56S), has contributed significantly to the knowledge of molecular mechanisms behind ALS. Recent literature has evidenced that the decrease of VAPB levels is present in cellular and murine models, and also in patient samples, suggesting a central role in motor neuron death in ALS. The present work sought three main objectives: (1) a molecular diagnosis through a NGS sequencing panel including the SOD1, FUS, TARDBP, SETX, SPG11, FIG4 and VAPB genes; (2) analyze the expression levels of VAPA, VAPB and EPHA4 in patients, family controls and other forms of ALS, in order to investigate their possible roles in ALS8; and (3) the development of a targeted quantitative mass spectrometry based assay, gold standard in protein quantification due to its precision and sensitivity, for the VAPA, VAPB and VAPC proteins, seeking the analysis of VAPB as a potential biomarker in ALS and of its isoform\'s potential roles as modifiers in the disease. The genomic analyses revealed that out of 67 patients, 31 presented the ALS8 mutation in VAPB, 4 patients (6%) presented a mutation in SOD1, with one patient carrying a second mutation in FIG4; 1 (1.5%) patient was identified with a pathogenic mutation in FUS; and another presented two pathogenic mutations in trans in the SPG11 gene. Thus, we were able to diagnose over half of the patients included in this study with a panel of only 7 genes. VAPB, VAPA and EPHA4 mRNA levels are not statistically different between patients and controls; however, EPHA4 was shown to be highly elevated in two bulbar-onset non-ALS8 patients. For the development of the LC-MS/MS targeted assay, 8 surrogate peptides were chosen for analysis, run parameters were established, and two internal standards for quantification were developed (SILAC cell lines and recombinant VAPB). This tool will prove to be useful not only towards elucidating the molecular mechanisms behind ALS8, one of the most prevalent forms of familial ALS in Brazil, but also to determine VAPB\'s potential as a biomarker for ALS

A P56S mutation in the VAPB MSP domain is linked to adult-onset amyotrophic lateral sclerosis 8. The objective of this study is to characterize the functional role of VAPB in transport of NE and NPC proteins from the ER to the NE. Over-expression of VAPB-P56S blocked the transport of nucleoporins (Nups) and NE proteins, resulting in their sequestration in dilated cytoplasmic membranes. Simultaneous overexpression of the FFAT motif (two phenylalanines in an acidic track) antagonizes mutant VAPB effects and restores transport to the NE. VAPB function is required for transport to the NE because knockdown of endogenous VAPB recapitulates this phenotype. Moreover, the compartment in which Nups and NE proteins are sequestered and retained was identified as ER-Golgi intermediate compartment (ERGIC). Moreover, a defect in the transport of NE and NPC proteins attenuates nucleocytoplasmic shuttling of the glucocorticoid receptor (GR). Further, VAPB-P56S which is only soluble in SDS was solubilized in the Triton-X-100 fraction similar to VAPB-WT upon co-transfection with the FFAT motif suggesting that FFAT interacts with the insoluble VAPB-P56S protein changing its biophysical properties.

La SLA est une maladie neurodégénérative se caractérisant par la perte progressive et sélective des neurones moteurs. une mutation dans le gène codant pour VAPB a été associée avec la SLA. VAPB est une protéine du réticulum endoplasmique (RE) qui jouerait un rôle dans le transport de protéines et dans la réponse du RE aux protéines mal repliées. J'ai utilisé la technique de transfert viral de gène pour induire l'expression de VAPB humaine (hVAP-Bwt et hVAP-BP56S) dans les neurones moteurs in vitro. Nous avons observé que hVAPBP56S forme des agrégats cytoplasmiques et que la surexpression de hVAPBwt et hVAPBp56s induit la mort des neurones via un mécanisme dépendant du RE et du calcium. Dans les cellules Cos-7, l'expression de hVAP-Bwt et hVAP-Bp(-s bloque l'activité du protéasome via l'activation d'un stress du RE et la séquestration de la sous-unité 20S. Enfin, nous avons développé des souris transgéniques hVAPBp56s mais elles ne développent aucun phénotype moteur / ALS is a neurodegenerative disease characterized by a selective loss of motor neurons. A mutation in VAPB protein has been associated with ALS. VAPB, an endoplamic reticulum (ER) resident protein is proposed to play a role in protein transport and in the unfolded protein response. To manipulate VAPB (hVAPBwt and hVAPBp56s) expression in motor neurons in vitro, I used the viral gene transfer technology. hVAPBp56s induces selective motor neuron death which involved an ER-related pathway dependent on calcium signals. Studies on Cos-7 cells showed that hVAPBwt and hVAPBp56s impair the proteasome activity through the activation of ER stress and the sequestration of the 20S subnit. Moreover, we developed transgenic mice overxpressing hVAPBp56s which do not display any motor disorder.

A P56S mutation in the VAPB MSP domain is linked to adult-onset amyotrophic lateral sclerosis 8. The objective of this study is to characterize the functional role of VAPB in transport of NE and NPC proteins from the ER to the NE. Over-expression of VAPB-P56S blocked the transport of nucleoporins (Nups) and NE proteins, resulting in their sequestration in dilated cytoplasmic membranes. Simultaneous overexpression of the FFAT motif (two phenylalanines in an acidic track) antagonizes mutant VAPB effects and restores transport to the NE. VAPB function is required for transport to the NE because knockdown of endogenous VAPB recapitulates this phenotype. Moreover, the compartment in which Nups and NE proteins are sequestered and retained was identified as ER-Golgi intermediate compartment (ERGIC). Moreover, a defect in the transport of NE and NPC proteins attenuates nucleocytoplasmic shuttling of the glucocorticoid receptor (GR). Further, VAPB-P56S which is only soluble in SDS was solubilized in the Triton-X-100 fraction similar to VAPB-WT upon co-transfection with the FFAT motif suggesting that FFAT interacts with the insoluble VAPB-P56S protein changing its biophysical properties.

A P56S mutation in the VAPB MSP domain is linked to adult-onset amyotrophic lateral sclerosis 8. The objective of this study is to characterize the functional role of VAPB in transport of NE and NPC proteins from the ER to the NE. Over-expression of VAPB-P56S blocked the transport of nucleoporins (Nups) and NE proteins, resulting in their sequestration in dilated cytoplasmic membranes. Simultaneous overexpression of the FFAT motif (two phenylalanines in an acidic track) antagonizes mutant VAPB effects and restores transport to the NE. VAPB function is required for transport to the NE because knockdown of endogenous VAPB recapitulates this phenotype. Moreover, the compartment in which Nups and NE proteins are sequestered and retained was identified as ER-Golgi intermediate compartment (ERGIC). Moreover, a defect in the transport of NE and NPC proteins attenuates nucleocytoplasmic shuttling of the glucocorticoid receptor (GR). Further, VAPB-P56S which is only soluble in SDS was solubilized in the Triton-X-100 fraction similar to VAPB-WT upon co-transfection with the FFAT motif suggesting that FFAT interacts with the insoluble VAPB-P56S protein changing its biophysical properties.

Amyotrophic Lateral Sclerosis (ALS) is a fatal, late-onset, progressive neurodegenerative disease. A familial form of ALS, autosomal dominant ALS8, is characterized by a mutation in an ER membrane protein, VAPB. To characterize the role of VAPB in motor neurons, two C. elegans models were generated: one expressing human VAPB-P56S and another with the knockdown of C. elegans VAPB ortholog, VPR-1. Overexpression of human VAPB in DA neurons caused backward locomotion defects, enhanced vulnerability to oxidative stress and premature neuronal death. Knockdown of vpr-1 in C. elegans recapitulated the loss of protein function believed to be associated with human cases of ALS8. It caused backward locomotion defects, such as uncoordination and slowed rates of movement, as well as age-dependent motor neuronal death. In both models, DA6 and DA7 were the most vulnerable motor neurons. Because of the unexpected developmental defects associated with the VAPB transgenic model, the knockdown of vpr-1 may be a better model to recapitulate the human disease. This model provides further support that ALS8 pathogenesis is due to a loss of VAPB protein function and can also be used to test drugs or treatments that may delay the onset of neuronal death.

No description available.