A esclerose lateral amiotrófica (ELA) é uma doença neurodegenerativa que afeta os neurônios motores levando a atrofia muscular e morte por insuficiência respiratória. Esta patologia se manifesta de forma esporádica ou familiar, que são indistinguíveis clinicamente. Mutações na enzima antioxidante superóxido dismutase 1 (hSod1) respondem por aproximadamente 20% dos casos familiares de ELA. Além disso, o caráter autossômico dominante destas mutações revela que a hSod1 adquire propriedades tóxicas aos neurônios motores. Atualmente, duas hipóteses não mutuamente excludentes existem para explicar o caráter tóxico das mutantes da hSod1 relacionadas à ELA. A primeira refere-se à produção de oxidantes pela atividade peroxidásica exacerbada das mutantes contribuindo para o estresse oxidativo observado em ELA. A segunda refere-se à agregação de proteínas como ocorre em outras doenças neurodegenerativas. Digno de nota, o radical carbonato produzido na atividade peroxidásica da hSod1 causa a formação de um dímero covalente da proteína análogo a uma espécie de hSod1 frequentemente detectada em modelos experimentais e pacientes da doença e associada à propriedade tóxica das mutantes. Desta forma, o presente trabalho buscou esclarecer o mecanismo de produção do radical carbonato pela hSod1, bem como caracterizar o dímero covalente da proteína para posterior estudo de sua formação em um modelo de ELA em ratos que superexpressam a mutante G93A da hSod1. Os estudos cinéticos da variação do pH sobre os efeitos de bicarbonato/CO2, nitrito e formato na atividade peroxidásica da hSod1, medidos pelo consumo de peróxido de hidrogênio e produção de radical, permitiram excluir o mecanismo de Fenton para explicar o ciclo peroxidativo da enzima em tampão bicarbonato em favor de outros intermediários reativos. Já, os experimentos de 13C RMN, modelagem molecular e cinética de fluxo interrompido com mistura assimétrica demonstraram que o ânion peroxomonocarbonato constitui o precursor do radical carbonato produzido pela hSod1. A caracterização do dímero covalente da hSod1 por proteólise com tripsina seguida de análise por HPLC/UV-vis e HPLC/ESI-MS identificou um peptídeo característico do dímero covalente da hSod1. A digestão enzimática em H2 18O demonstrou de forma inequívoca a natureza dímerica deste peptídeo pela marcação da extremidade C-terminal. Ainda, o sequenciamento do peptídeo dimérico por MS/MS revelou a estrutura primária ESNGPVKVW(ESNGPVKVWGSIK)GSIK, na qual as cadeias polipeptídicas estão ligadas através de um aduto de ditriptofano composto por resíduos Trp32 da proteína. Por fim, este peptídeo dimérico pode ser empregado como marcador bioquímico específico para o estudo do dímero covalente da hSod1 in vivo. A análise do extrato de proteínas das medulas dos ratos modelo de ELA identificou quinze candidatos a dímero covalente da hSod1 por Western-blot, sendo que dois deles foram excluídos por espectrometria de massa, pois tiveram o resíduo Trp32 identificado. O peptídeo ESNGPVKVW(ESNGPVKVWGSIK)GSIK não foi observado, porém as treze espécies restantes permanecem candidatas e deverão ser reexaminadas em trabalhos que darão sequência a esta tese de doutorado. Em suma, o peroxomonocarbonato constitui o intermediário na produção do radical carbonato pela hSod1 e o peptídeo ESNGPVKVW(ESNGPVKVWGSIK)GSIK uma ferramenta importante no estudo da agregação covalente da hSod1 em ELA. / Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of motors neurons that causes muscle atrophy, weakness, and death by respiratory failure. This pathology occurs in both sporadic and familiar forms that are clinically indistinguishable. Mutations in the antioxidant enzyme superoxide dismutase 1 (hSod1) respond to about 20% of the familiar cases of ALS. Besides, the autosomal dominant nature of these hSod1-associated ALS suggests that the mutants gain toxic properties to motor neurons. Currently, two hypotheses exist to explain the toxicity of hSod1 mutants but they do not exclude each other. The first one is related to the production of oxidants by the increased peroxidase activity of the ALS-linked mutants that could contribute to the oxidative stress reported in ALS. The second refers to protein aggregation as proposed in other neurodegenerative diseases. Noteworthy, the carbonate radical produced during hSod1 peroxidase activity leads to the formation of a covalent dimer of the protein similar to a hSod1 species often detected in experimental models and patients of the disease and implicated in the toxic properties of hSod1 mutants. Thus, the present work aimed to determine the mechanism of carbonate radical production by hSod1 and to characterize the covalent dimer of the protein in vitro followed by the study of covalent aggregates of hSod1 in a rat model of ALS that overexpresses the G93A mutant of the protein. The kinetic studies of the effect of bicarbonate/CO2, nitrite and formate in the peroxidase activity of hSod1 at various pH, measured by hydrogen peroxide consumption and radical production, permitted to exclude the Fenton mechanism to explain the enzyme peroxidative cycle in bicarbonate buffer in favor of other reactive intermediates. Furthermore, 13C NMR, molecular docking and stopped-flow experiments with asymmetric mixing demonstrated that the anion peroxomonocarbonate is the precursor of the carbonate radical produced by hSod1. The characterization of hSod1 covalent dimer by proteolysis with trypsin followed by HPLC/UV-vis and HPLC/ESI-MS analysis identified a peptide characteristic of the covalent dimer of the protein. The enzymatic digestion in H2 18 O irrefutably demonstrated the dimeric nature of this peptide because of the C-terminal labeling with oxygen-18 isotopes. In addition, sequencing of the dimeric peptide by MS/MS determined the primary structure ESNGPVKVW(ESNGPVKVWGSIK)GSIK, in which the polipeptide chains are crosslinked through a ditryptophan adduct formed by a covalent bond between the Trp32 residues of each subunit. So, this dimeric peptide can be employed as a biochemical marker for studying the hSod1 covalent dimer in vivo. The analysis of protein extracts from the spinal cord of the rat model of ALS by Western-blot identified fifteen candidates to hSod1 covalent dimer, but two of them were excluded by mass spectrometry analysis that identified unmodified Trp32 residues. Moreover, neither the dimeric peptide nor the Trp32 residue were observed in the remaining species. Therefore, these thirteen candidates must be reexamined in subsequent studies. In conclusion, the anion peroxomonocarbonate is the key intermediate in the production of the carbonate radical by hSod1 and the dimeric peptide constitutes a specific tool to study hSod1 covalent aggregation in ALS

A Esclerose Lateral Amiotrófica (ELA) é a doença neurodegenerativa do neurônio motor que acomete indivíduos adultos e promove a perda progressiva das funções motoras. A evolução é rápida (2 a 5 anos) e culmina na morte por complicações e falência dos músculos respiratórios. Descrições recentes sugerem a contribuição de tipos celulares não neuronais, particularmente o astrócito e a microglia, para a morte do neurônio motor. O camundongo transgênico SOD1G93A, que carrega a SOD1 humana mutada, foi utilizado neste trabalho. Estudos comportamentais apontaram alterações motoras importantes no animal transgênico a partir de 90 dias de vida e permitiram selecionar, então, as idades pré-sintomáticas de 40 dias e 80 dias para os estudos moleculares. A análise da expressão gênica nos animais transgênicos e selvagens destas duas idades foi realizada por microarray utilizando-se a plataforma que contém o genoma completo do camundongo e detectou 492 e 1105 transcritos diferencialmente expressos nos animais de 40 e 80 dias, respectivamente. Estes resultados foram validados por PCR quantitativa (qPCR). As análises bioinformáticas dos resultados identificaram 17 e 11 vias moleculares super-representadas nas idades de 40 dias e 80 dias, respectivamente. Destas, as vias endocitose, sinapse glutamatérgica, proteólise mediada por ubiquitina, via de sinalização de quimiocina, fosforilação oxidativa, processamento e apresentação de antígeno e junção oclusiva foram comuns a ambas as idades. Ainda, as vias sinapse glutamatérgica e fagossomo foram sugeridas como potencialmente mais importantes em animais transgênicos de 40 dias e 80 dias, respectivamente. Transcritos específicos foram analisados em amostras enriquecidas de células (astrócito, microglia e neurônio motor) microdissecadas a laser do corno anterior da medula espinal dos animais. Os transcritos Cxcr4, Slc1a2 e Ube2i foram avaliados por qPCR nas amostras enriquecidas de astrócitos dos animais de 40 dias, enquanto que Cxcr4 e Slc17a6 foram avaliados nas amostras de neurônios motores dos animais desta idade. Cxcr4 apresentou expressão diminuída nos astrócitos transgênicos e aumentada nos neurônios destes animais. Slc1a2, Ube2i e Slc17a6 estavam aumentados nos tipos celulares estudados nos animais transgênicos. Tap2 e Tuba1a foram avaliados nas amostras enriquecidas de microglias dos animais de 80 dias e mostraram-se aumentados nas amostras dos transgênicos. Finalmente, Akt1 apresentou expressão diminuída nos neurônios motores microdissecados dos animais transgênicos em comparação aos selvagens. Os resultados sugerem que alterações na sinalização glutamatérgica podem exercer papel essencial em fases pré-sintomáticas mais precoces da doença (40 dias), enquanto que em fases pré-clínicas mais próximas ao aparecimento dos sintomas (80 dias), as respostas mais importantes parecem estar relacionadas à neuroimmunomodulação. Dessa forma, este trabalho aponta para novas perspectivas para o estudo da ELA / Amyotrophic Lateral Sclerosis (ALS) is an adult onset motor neuron neurodegenerative disease that leads to the progressive loss of muscular functions. It is a fast progression disorder (2 to 5 years) culminating in death by respiratory failure. Recent findings suggest that non neuronal cell types, especially astrocytes and microglia, might contribute to the neuronal death. The transgenic mouse SOD1G93A, carring human mutant SOD1, was used in this study. Behavioral studies pointed to the onset of the clinical symptoms occurring at 90 days in the animal model, thus, allowing the selection of the pre-symptomatic ages of 40 and 80 days to the molecular studies. Gene expression analysis of transgenic mice and their non-transgenic littermates at those ages was performed by using a microarray platform containing the whole mouse genome and has detected 492 and 1105 differentially expressed genes at 40 days and 80 days old mice, respectively. These results were validated by quantitative PCR (qPCR). Bioinformatic analysis of the results identified 17 and 11 over-represented molecular pathways at 40 days and 80 days, respectively. Of these, endocytosis, glutamatergic synapse, ubiquitin-mediated proteolysis, chemokine signaling pathway, oxidative phosphorylation, antigen processing and presentation and also tight junction were common to both ages. Furthermore, glutamatergic synapse and fagosome were suggested as potentially more important at 40 and 80 days, respectively. Specific transcripts were analyzed on enriched samples of cells (astrocytes, microglia and motor neuron) obtained by laser microdissection from the ventral horn of mouse spinal cord. The transcripts Cxcr4, Slc1a2 and Ube2i were evaluated by qPCR in enriched samples of astrocytes of the 40 days old mice, and Cxcr4 and Slc17a6 were analyzed in motor neuron samples at this age. Cxcr4 has been found decreased in astrocytes from transgenic mice and increased in the motor neurons of these animals. Slc1a2, Ube2i and Slc17a6 have increased in the cell type in which they were evaluated in the transgenic mice. Tap2 and Tuba1a were evaluated at microglia enriched samples of 80 days old mice and were found to be increased. Finally, Akt1 has decreased in enriched samples of motor neurons from 80 days old mice. The results suggest that glutamatergic signaling might play essential role in early stages of the disease (40 days), while in phases closer to the appearance of the symptoms (80 days), the neuroimmunomodulation takes place. Thus, this study points to new perspectives for ALS study

La technologie de reprogrammation de cellules somatiques en cellules souches pluripotentes induites (iPS) offre aujourd’hui l’opportunité de modéliser des maladies neurodégénératives et d’étudier des neurones de patients. Nous avons utilisé cette technologie pour générer deux modèles de maladies neurodégénératives : la mucopolysaccharidose de type IIIB (MPSIIIB) et la forme ALS2 de la sclérose latérale amyotrophique (SLA). Dans le modèle MPSIIIB, nous avons montré que les iPS et les neurones de patients présentaient des défauts caractéristiques de la pathologie telle que l’accumulation de vésicules de surcharge. Des altérations de l’appareil de Golgi dans ces cellules ont également été mises en évidence. Une analyse du transcriptome de précurseurs neuraux MPSIIIB a montré des modifications transcriptionnelles touchant notamment des gènes impliqués dans les interactions de la cellule avec la matrice extracellulaire. Ainsi, dans une seconde étude, des altérations de la migration et de l’orientation de cellules de souris mutantes MPSIIIB ou de patients ont été démontrées. Ces altérations pourraient être responsables des perturbations de la neurogénèse et de la neuritogénèse chez les enfants malades. Dans le modèle SLA/ALS2, nous avons montré que les neurones de patients présentaient des défauts incluant une diminution de la surface des endosomes et des anomalies de la croissance neuritique. Alors qu’il n’existait jusqu’alors aucun modèle cellulaire pertinent reproduisant cette maladie, ce modèle permettra à présent d’étudier les processus physiopathologiques impliqués dans la maladie. En conclusion, la génération de cellules iPS permet de modéliser des maladies neurodégénératives et d’étudier les processus physiopathologiques qui sont associés sur des neurones humains en culture. Ces modèles cellulaires pourraient permettre dans un avenir proche de réaliser des criblages de molécules à visée thérapeutique / Reprogramming technology of somatic cells in induced pluripotent stem cells (iPS) now offers the opportunity to model neurodegenerative diseases and to study patient’s neurons. We used this technology for generating two models of neurodegenerative diseases: the muccopolysaccharidosis type IIIB (MPSIIIB) and the ALS2 form of amyotrophic lateral sclerosis (ALS). In the MPSIIIB model, we have shown that iPS and neurons of patients had characteristic defects of the disease such as the accumulation of storage vesicles. Alterations of the Golgi apparatus in these cells were also highlighted. Transcriptome analysis of MPSIIIB neural precursors showed transcriptional changes involving particularly genes implicated in cell-extracellular matrix interactions. Thus, in a subsequent study, alterations of migration and orientation of MPSIIIB mutant mouse cells and MPSIIIB patients’ cells have been demonstrated. These alterations may be responsible for the disruption of neurogenesis and neuritogenesis in sick children. In the ALS2 model, we have shown that patients’ neurons had defects including decreased endosomes’ surface and abnormal neurite outgrowth. As there was previously no relevant cellular model reproducing the disease, this model will now allow the study of physiopathological processes involved in the disease. In conclusion, the generation of iPS cells allows to model neurodegenerative diseases and to study associated physiopathological processes on cultured human neurons. These cell models could allow in the near future the screening of molecules of potential therapeutical interest

No description available.

Cerca de 20 % dos casos familiares de esclerose lateral amiotrófica (ELAf) são causados por mutações na enzima Cu,Zn-superóxido dismutase (SOD1). Inicialmente se supôs que as enzimas mutantes teriam a atividade SOD comprometida, entretanto isto não foi comprovado. Atualmente, considera-se que as enzimas mutantes adquiram propriedades tóxicas. Quais seriam estas propriedades e como levariam à degeneração do neurônio motor são questões ainda não respondidas. Neste trabalho, comparamos neuroblastomas humanos transfectados com SOD1 G93A associada à ELAf (SH-SY5YG93A), e SOD1 selvagem (SH-SY5YWT) com células parentais (SH-SY5Y) em relação ao crescimento, viabilidade, produção basal de oxidantes, atividades SOD e peroxidásica e modificações estruturais da SOD. As células transfectadas apresentaram aumento na taxa de crescimento e na produção basal de oxidantes. As células SH-SY5YWT e SH-SY5YG93A mantiveram a expressão de SOD1 e atividade consistente com o aumento esperado de duas vezes, em estágios iniciais de cultura. A atividade peroxidásica do homogenato da célula SH-SY5YG93A foi maior. Após quatro semanas, a linhagem SH-SY5YG93A manteve a expressão de SOD1, mas as atividades dismutásica e peroxidásica diminuíram. A expressão de SOD1 aumentou a proporção de formas alteradas de SOD1, como enzima reduzida, multímeros formados por ponte dissulfeto e formas insolúveis em detergente, particularmente na linhagem SH-SY5YG93A. Entre estas formas insolúveis, identificamos um dímero covalente de SOD. Estas formas alteradas provavelmente são responsáveis pela ativação do proteassomo e estresse do retículo endoplasmático, verificados nas células transfectadas. Concluindo, a superexpressão da SOD1 foi suficiente para elevar as formas imaturas e oligomerizadas de SOD1 e a oxidação basal, e a mutação G93A ressaltou estes processos. / Some familial ALS (fALS) are caused by mutations in the Cu,Zn-superoxide dismutase enzyme (SOD1). It was thought that the mutated enzymes would have impaired SOD activity, but this has not been corroborated so far. Presently, it is more accepted that the mutated enzymes acquire a new toxic function. What this new toxic function is and how it relates to the degeneration of motor neurons remains debatable. Here, we compared human neuroblastoma cells transfected with fALS mutant G93A (SH-SY5YG93A) or wild-type SOD1 (SH-SY5YWT) with parent cells (SH-SY5Y) in regard to growth, viability, basal oxidant production, SOD and peroxidase activities, and SOD forms. Transfected cells presented increased growth rate and basal oxidant production. SH-SY5YWT and SH-SY5YG93A cells in early culture stage showed SOD expression and activity consistent with the expected two-fold increase; SH-SY5YWT homogenates showed increased peroxidase activity. After four weeks, SH-SY5YG93A maintained SOD1 expression levels but peroxidase and dismutase activities were lower. SOD1 expression increased the levels of altered SOD1 forms such as the reduced enzyme, disulfide multimers and detergent-insoluble forms, particularly in SH-SY5YG93A cells. Among the insoluble forms a covalent SOD dimer was identified. These altered SOD forms are probably responsible for proteasome activation and endoplasmatic reticulum stress response verified in transfected cells. In conclusion, SOD1 over-expression was sufficient to increase intracellular immature and oligomerized SOD1 forms and basal oxidation and the G93A mutation enhanced these processes.

No description available.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a loss of voluntary movement over time, leading to paralysis and death. While 10% of ALS cases are inherited or familial (FALS), the majority of cases (90%) are sporadic (SALS) with unknown etiology. Approximately 20% of FALS cases are genetically linked to a mutation in the anti-oxidizing enzyme, superoxide dismutase (SOD1). SALS and FALS are clinically indistinguishable, suggesting a common pathogenic mechanism exists for both types. Since such a large number of genetic mutations in SOD1 result in FALS (>170), it is reasonable to suspect that non-genetic modifications to SOD1 induce structural perturbations that result in ALS pathology as well. In fact, misfolded SOD1 lacking any genetic mutation was identified in end stage spinal cord tissues of SALS patients using misfolded SOD1-specific antibodies. In addition, this misfolded WT SOD1 found in SALS tissue inhibits axonal transport in vitro, supporting the notion that misfolded WT SOD1 exhibits toxic properties like that of FALS-linked SOD1. Indeed, aberrant post-translational modifications, such as oxidation, cause WT SOD1 to mimic the toxic properties of FALS-linked mutant SOD1. Based on these data, I hypothesize that modified, misfolded forms of WT SOD1 contribute to SALS disease progression in a manner similar to FALS linked mutant SOD1 in FALS. The work presented in this dissertation supports this hypothesis. Specifically, one common misfolded form of SOD1 is defined and exposure of this toxic region is shown to enhance SOD1 toxicity. Preventing exposure, or perhaps stabilization, of this “toxic” region is a potential therapeutic target for a subset of both familial and sporadic ALS patients. Further, the possibility of exploiting this misfolded SOD1 species as a biomarker is explored. For example, an over-oxidized SOD1 species was identified in peripheral blood mononuclear cells (PBMCs) from SALS patients that is reduced in controls. Moreover, 2-dimensional gel electrophoresis revealed a more negatively charged species of SOD1 in PBMCs of healthy controls greatly reduced in SALS patients. This species is hypothesized to be involved in the degradation of SOD1, further implicating both misfolded SOD1 and altered protein homeostasis in ALS pathogenesis.

Cu, Zn superoxide dismutase (SOD1) is a dimeric, β-sandwich, metalloenzyme responsible for the dismutation of superoxide. Mutations covering nearly 50% of the amino acid sequence of SOD1 have been found to acquire a toxic gain-of-function leading to amyotrophic lateral sclerosis. A hallmark of this disease is the presence of insoluble aggregates containing SOD1 found in the brain and spinal cord. While it is unclear how these aggregates or smaller, precursor oligomeric species may be the source of the toxicity, mutations leading to increased populations of unstable, partially folded species along the folding pathway of SOD1 may be responsible for seeding and propagating aggregation. In an effort to determine the responsible species, we have systematically characterized the stability and folding kinetics of five well studied ALS variants: A4V, L38V, G93A, L106V and S134N. The effect of the amino acid substitutions was determined on a variety of different constructs characterizing the various post-translational maturation steps of SOD1: folding, disulfide bond formation and Zn binding. Zn was found to bind progressively tighter along the folding pathway of SOD1, minimizing populations of monomeric species. In contrast, ALS variants were found to have the greatest perturbation in the equilibrium populations of the folded and unfolded state for the most immature, disulfide-reduced metal-free SOD1. In this species, at physiological temperature, four out of five ALS variants were >50% unfolded. Finally the energetic barriers in the folding and unfolding reaction were studied to investigate the unusually slow folding of SOD1. These results reveal that both unfolding and refolding are dominated by enthalpic barriers which may be explained by the desolvation of the chain and provide insights into the role of sequence in governing the folding pathway and rate.

Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease is a fatal neurodegenerative disease. ALS is typically adult onset and is characterized by rapidly progressive loss of both upper and lower motor neurons that leads to death usually within 3-5 years. About 90% of all the cases are sporadic with no family history while the remaining 10% are familial cases with mutations in several genes including SOD1, FUS/TLS, TDP43 and C9ORF72. FUS/TLS (Fused in Sarcoma/Translocated in Liposarcoma or FUS) is an RNA/DNA binding protein that is involved in multiple cellular functions including DNA damage repair, transcription, mRNA splicing, RNA transport and stress response. More than 40 mutations have now been identified in FUS that account for about 5% of all the familial cases of ALS. However, the exact mechanism by which FUS causes ALS is unknown. While significant progress has been made in understanding the disease mechanism and identifying therapeutic strategies, several questions still remain largely unknown. The work presented here aims at understanding the normal functions of FUS as well as the pathogenic mechanisms by which it leads to disease. Several studies showed the association of mutant-FUS with structures made up of RNA and proteins, called stress granules that form under various stress conditions. However, little is known about the role of endogenous FUS under stress conditions. I have shown that under hyperosmolar conditions, the predominantly nuclear FUS translocates into the cytoplasm and incorporates into stress granules. The response is specific to hyperosmolar stress because FUS remains nuclear under other stress conditions tested, such as oxidative stress, ER stress and heat shock. The response of FUS is rapid, and cells with reduced FUS levels are susceptible to the hyperosmolar stress, indicating a pro-survival role for FUS. In addition to investigating the functions of endogenous wild-type (WT) FUS, the work presented also focuses on identifying the pathogenic mechanism(s) of FUS variants. Using various biochemical techniques, I have shown that ALS-causing FUS variants are misfolded compared to the WT protein. Furthermore, in a squid axoplasm based vesicle motility assay, the FUS variants inhibit fast axonal transport (FAT) in a p38 MAPK dependent manner, indicating a role for the kinase in mutant-FUS mediated disease pathogenesis. Analysis of human ALS patient samples indicates higher levels of total and phospho p38, supporting the notion that aberrant regulation of p38 MAPK is involved in ALS. The results presented in this dissertation 1) support a novel prosurvival role for FUS under hyperosmolar stress conditions and, 2) demonstrate that protein misfolding and aberrant kinase activation contribute to ALS pathogenesis by FUS variants.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by loss of motor neurons, resulting in progressive muscle weakness, atrophy, paralysis and death within five years of diagnosis. About ten percent of cases are inherited, of which twenty percent are due to mutations in the superoxide dismutase 1 (SOD1) gene. Since the only FDA approved ALS drug prolongs survival by just a few months, new therapies for this disease are needed. Experiments in transgenic ALS mouse models have shown that decreasing levels of mutant SOD1 protein alters and in some cases entirely prevents disease progression. We explored this potential therapeutic approach by using a single stranded AAV9 vector encoding an artificial microRNA against human SOD1 injected bilaterally into the cerebral lateral ventricles of neonatal SOD1G93A mice. This therapy extended median survival from 135 to 206 days (a 50% increase) and delayed hind limb paralysis. Animals remained ambulatory until endpoint, as defined by a sharp drop in body weight. Treated animals had a reduction of mutant human SOD1 mRNA levels in upper and lower motor neurons. As compared to untreated SOD1G93A mice, the AAV9 treated mice also had significant improvements in multiple parameters including the number of motor neurons, diameter of ventral root axons, and degree of neuroinflammation in the spinal cord. These studies clearly show that an AAV9-delivered artificial microRNA is a translatable therapeutic approach for ALS.