Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative condition that affects corticospinal and spinal motor neurons and leads to death within 30 months of symptom onset in half of all cases. It remains incurable and treatment is supportive. The genetic and molecular understanding of ALS has gone through a rapid expansion in recent years, notably with the discoveries of TDP-43, a heterogeneous ribonucleoprotein as a major component of neuronal inclusions in ALS, as well as the discovery of the C9orf72 hexanucleotide expansion as the most common genetic cause of this disease. This first part of this thesis addresses the question of which of the various pathological hallmarks of the C9orf72 Hexanucleotide Repeat Expansion (HRE) in autopsy material correlates best with the clinical presentation. The main finding is that TDP-43 distribution, rather than C9orf72 RNA foci or dipeptide aggregation in the brain, corresponds best with the areas relevant to the clinical subtype of ALS-FTD. Subsequently the role of TDP-43 was investigated in induced pluripotent stem cell derived motor neurons, and no evidence of the hallmarks of TDP-43 dysfunction, were seen in this model of the disease. No mislocalisation is found on immunofluorescence, and biochemical analysis shows no differences in insoluble species between the patient and control cell lines. In the final section, RNA sequencing was used to study the transcriptome of a BAC transgenic mouse carrying a human M337V transgene expressed at low levels, to identify early presymptomatic differences in gene expression. Interestingly, no changes were found in genes known to be associated with ALS through mutations, and the constitutive nuclear functions of TDP-43 in the regulation of splicing was maintained, prior to the emergence of a clinical phenotype in the mouse. This favours a gain of function mechanism for TDP-43 mutations in ALS.

Old-age hippocampal sclerosis (HS), characterised by severe neuron loss in hippocampal CA1, is a poorly understood cause of dementia. At present no objective pathological HS criteria exist. In life HS is commonly diagnosed as Alzheimer's disease. HS aetiology is unclear, although it has been associated with both ischaemia and TAR-DNA-binding protein-43 (TDP-43)-related neurodegeneration. Variations in genes GRN, TMEM106B and ABCC9 are proposed as HS risk factors. The aim of this thesis was to investigate epidemiological, clinical, pathological and genetic characteristics of HS in older European populations. 976 brains donated for the Cambridge City over-75s Cohort, the Cognitive Function and Ageing Study and the Finnish Vantaa 85+ study were available for evaluation -including bilateral hippocampi from 302 individuals. A protocol capturing the extent and severity of hippocampal neuron loss was developed, establishing objective HS diagnosis criteria and allowing observation of distinct neuron loss patterns associated with ischaemia and neurodegeneration. 71 HS cases (overall prevalence: 7.3%) were identified. HS was significantly associated with an advanced age at death as well as dementia at the end of life. Neuropsychological and cardiovascular characteristics were similar between HS and AD, except for a longer duration of dementia and more disability in HS. HS was not associated with neurofibrillary tangles, amyloid plaques, or vascular pathologies, but all HS cases evaluated for TDP-43 showed neuronal inclusions in the hippocampal dentate and a high frequency of other glial, neuronal and neurite TDP-43 pathologies. GRN and TMEM106B but not ABCC9 variations were linked to HS. A moderating effect of TDP-43 on this association was detected. HS presented pathologically similarly to frontotemporal dementia cases with TDP-43 (FTLD-TDP) caused by mutations in GRN, but differed from other FTLD-TDP subtypes. Results of this thesis reveal the importance of HS in the oldest old in the population, the key role of TDP-43, as well as providing robust methods to capture HS characteristics for an area that has been under-researched but is clearly vital to understanding dementia in the oldest old.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder in which an RNA-binding protein, TDP-43, mislocalizes and pathologically accumulates from its normal nuclear locale to the cytosol. Given that the subcellular localization and expression of TDP-43 is tightly regulated, we posit that identifying novel interactors of wild-type and mutant TDP-43 could reveal insight into networks involved in regulating its localization, ultimately driving neurodegeneration in ALS. Using CRISPR/Cas9, our lab previously generated knock-in cell lines expressing GFP in the endogenous TARDBP locus (encoding for TDP-43) for both wildtype (WT) and an ALS-causing mutant (Q331K). We have shown that the Q331K mutation causes loss-of-function and mislocalization of TDP-43. I performed immunoprecipitation coupled to mass spectrometry (IP-MS) on this cell model to elucidate interactors of WT- and Q331K- TDP-43. Our data show that there is an overall loss of TDP-43 interactors in cells with the TDP-43Q331K mutation. By setting statistical cut-offs for significance, we identified 34 shared and 12 unique interactors of TDP-43WT. We used bioinformatic approaches to identify enriched pathways and literature searches to look for interactors relevant to TDP-43 and ALS pathobiology. Our shortlist of 14 candidates for validation included proteins involved in the nuclear mRNA export pathways, RNA binding proteins and proteins identified in other interactome studies and TDP-43 based screens. Using orthogonal approaches, we show evidence of robust interaction of four top hits (PABPC1, HNRNPC, DDX39b and ELAVL1) with TDP-43WT, and a significant decrease in the degree of interaction of HNRNPC, DDX39b and ELAVL1 with TDP-43Q331K. Importantly, this decrease in interaction was only observed at the endogenous level, highlighting the importance of maintaining the steady state levels of TDP-43 in the cell for these assays. We characterized the effects of knockdown and overexpression of these four hits using protein-specific overexpression constructs and shRNAs and observed a significant increase in TDP-43 nuclear localization upon knockdown of these four hits, suggesting that there is a functional effect associated with hit knockdown. Overexpression or knockdown of the top hits in a splicing assay did not identify significant changes in TDP-43’s splicing or RNA binding abilities, suggesting that these hits do not affect splicing function in our hit characterization studies. Using this novel experimental tool and unbiased screen, we identified alterations in TDP-43 protein-protein interactions in the context of ALS and have generated tools to characterize their roles in cellular functions using knockdown and overexpression approaches. Together with the knock-in cells, these tools will allow us to gain insight into pathways involved in driving neurodegeneration, in the context of ALS.

INTRODUÇÃO: A proteína TDP-43 (Transactive DNA-binding protein 43) é o principal agregado proteico anormal verificado em casos de Degeneração Lobar Frontotemporal (DLFT) e Esclerose Lateral Amiotrófica (ELA). Apesar dos avanços na área, o papel da TDP-43 no envelhecimento normal ainda é pouco compreendido. A falta de indivíduos controles em estudos de associação clinicopatológica com tecido cerebral humano é resultado do alto custo da prática de autópsia mundialmente. O que é um diferencial do Brasil, onde essa prática é obrigatória em casos de morte natural sem causa definida. O Banco de Encéfalos Humanos do Grupo de Estudos em Envelhecimento Cerebral (BEHGEEC) possui grande número de encéfalos de sujeitos sem déficit cognitivo com alta miscigenação étnica e variação de graus de escolaridade. O que possibilita estudos mais aprofundados sobre envelhecimento cerebral humano não-patológico. OBJETIVOS: (1) investigar de maneira sistemática a distribuição dos achados neuropatológicos da TDP-43 no envelhecimento humano normal; (2) caracterizar a distribuição dessa proteína em diferentes áreas encefálicas; (3) explorar associação de características, clínicas, sociodemográficas ou neuropatológicas com o aparecimento das inclusões de TDP-43. MÉTODOS: Foram incluídos participantes com idade acima de 50 anos e classificados clinicamente e anatomopatologicamente como normais. Reação imunoistoquímica contra a conformação anormal da TDP-43 foi realizada em três regiões cerebrais de interesse córtex temporal, amígdala e hipocampo. RESULTADOS: Os agregados de TDP-43 estavam presentes em 10,5% dos indivíduos estudados (n=323). A região mais frequentemente acometida foi a amígdala (85,3% dos casos). O acúmulo de TDP-43 foi associado à idade e raça (p=0,002). Análise de regressão logística demonstrou que indivíduos da raça asiática possuem maior chance de apresentar os agregados de TDP-43 do que caucasianos, independente dos fatores gênero, idade, estágio de Braak para emaranhados neurofibrilares e escolaridade (odds ratio=3,5: intervalo de confiança: 1,41-8,69, p=0,007). CONCLUSÕES: Os resultados sugerem que o acúmulo anormal de TDP-43 aumenta com o avanço da idade e que a amígdala é a região cerebral mais susceptível ao acúmulo desse tipo de inclusão. Como os idosos estudados são cognitivamente normais, sugerimos que indivíduos asiáticos estão protegidos contra os efeitos deletérios da proteína TDP-43 no cérebro. Futuros estudos de associação clinicopatológica e genética em populações de diferentes raças poderão contribuir para identificação de possíveis fatores de proteção contra a expressão clínica dos achados da proteína TDP-43 / BACKGROUND: Transactive DNA-binding protein 43 (TDP-43) is the major abnormal aggregate present in Frontotemporal Lobar Degeneration (FTLD) and Amyotrophic Lateral Slcerosis (ALS). Although all the efforts in research in this field, the role played by TDP-43 in normal aging is still unknown. The lack of normal controls in studies focusing on clinicopathological associations is a result of the high cost of autopsy practice worldwide. In Brazil, autopsy is mandatory by law in cases without a certificate. The Brain Bank of the Brazilian Aging Study Group (BBBASG) comprises a large number of cognitively normal elderly subjects highly ethnically admixtured and with broad education attainment. This allow us to further study the non-pathological process of the human aging brain. GOALS: (1) sistematically investigate the distrubution of neuropathological findings of TDP-43 in the normal human brain; (2) characterize the distribution of theses findings in different brain regions; (3) explore clinical, sociodemographics or neuropathological variables that could be associated with TDP-43 inclusion outcome. METHODS: We included participants over 50 years old previously classified cognitively and neuropathologically as normals. Imunnohistochemistry against abnormal form of TDP-43 was performed in three brain regions: temporal cortex, hippocampal formation and amygdala. RESULTS: TDP-43 aggregates were present in 10,5% of the study subjects (n=323). Amygdala was the most frequently affected brain region (85.3% of the cases). TDP-43 accumulation was associated with age at death and race (p=0.002). Logistic regression analysis showed that asians older adults have higher odds of presenting TDP-43 inclusions than caucasians, regardless of gender, age, Braak stage for neurofibrilarly tangles and education attainment (OD=3.5, CI: 1.41-8.69, p=0.007). CONCLUSIONS: Our results suggest that TDP-43 abnormal accumulation increases along aging process and the amygdala is the brain region most susceptible to these alterations. Because we studied cognitively normal elderlies, we suggest that Asians older adults are protected against deleterious effects of TDP-43 in the brain. Future clinicopathological and genetic studies in populations dwelling in different ethnical backgrounds may identify possible protecting factors against the clinical expression of TDP-43 neuropathological findings

INTRODUÇÃO: A proteína TDP-43 (Transactive DNA-binding protein 43) é o principal agregado proteico anormal verificado em casos de Degeneração Lobar Frontotemporal (DLFT) e Esclerose Lateral Amiotrófica (ELA). Apesar dos avanços na área, o papel da TDP-43 no envelhecimento normal ainda é pouco compreendido. A falta de indivíduos controles em estudos de associação clinicopatológica com tecido cerebral humano é resultado do alto custo da prática de autópsia mundialmente. O que é um diferencial do Brasil, onde essa prática é obrigatória em casos de morte natural sem causa definida. O Banco de Encéfalos Humanos do Grupo de Estudos em Envelhecimento Cerebral (BEHGEEC) possui grande número de encéfalos de sujeitos sem déficit cognitivo com alta miscigenação étnica e variação de graus de escolaridade. O que possibilita estudos mais aprofundados sobre envelhecimento cerebral humano não-patológico. OBJETIVOS: (1) investigar de maneira sistemática a distribuição dos achados neuropatológicos da TDP-43 no envelhecimento humano normal; (2) caracterizar a distribuição dessa proteína em diferentes áreas encefálicas; (3) explorar associação de características, clínicas, sociodemográficas ou neuropatológicas com o aparecimento das inclusões de TDP-43. MÉTODOS: Foram incluídos participantes com idade acima de 50 anos e classificados clinicamente e anatomopatologicamente como normais. Reação imunoistoquímica contra a conformação anormal da TDP-43 foi realizada em três regiões cerebrais de interesse córtex temporal, amígdala e hipocampo. RESULTADOS: Os agregados de TDP-43 estavam presentes em 10,5% dos indivíduos estudados (n=323). A região mais frequentemente acometida foi a amígdala (85,3% dos casos). O acúmulo de TDP-43 foi associado à idade e raça (p=0,002). Análise de regressão logística demonstrou que indivíduos da raça asiática possuem maior chance de apresentar os agregados de TDP-43 do que caucasianos, independente dos fatores gênero, idade, estágio de Braak para emaranhados neurofibrilares e escolaridade (odds ratio=3,5: intervalo de confiança: 1,41-8,69, p=0,007). CONCLUSÕES: Os resultados sugerem que o acúmulo anormal de TDP-43 aumenta com o avanço da idade e que a amígdala é a região cerebral mais susceptível ao acúmulo desse tipo de inclusão. Como os idosos estudados são cognitivamente normais, sugerimos que indivíduos asiáticos estão protegidos contra os efeitos deletérios da proteína TDP-43 no cérebro. Futuros estudos de associação clinicopatológica e genética em populações de diferentes raças poderão contribuir para identificação de possíveis fatores de proteção contra a expressão clínica dos achados da proteína TDP-43 / BACKGROUND: Transactive DNA-binding protein 43 (TDP-43) is the major abnormal aggregate present in Frontotemporal Lobar Degeneration (FTLD) and Amyotrophic Lateral Slcerosis (ALS). Although all the efforts in research in this field, the role played by TDP-43 in normal aging is still unknown. The lack of normal controls in studies focusing on clinicopathological associations is a result of the high cost of autopsy practice worldwide. In Brazil, autopsy is mandatory by law in cases without a certificate. The Brain Bank of the Brazilian Aging Study Group (BBBASG) comprises a large number of cognitively normal elderly subjects highly ethnically admixtured and with broad education attainment. This allow us to further study the non-pathological process of the human aging brain. GOALS: (1) sistematically investigate the distrubution of neuropathological findings of TDP-43 in the normal human brain; (2) characterize the distribution of theses findings in different brain regions; (3) explore clinical, sociodemographics or neuropathological variables that could be associated with TDP-43 inclusion outcome. METHODS: We included participants over 50 years old previously classified cognitively and neuropathologically as normals. Imunnohistochemistry against abnormal form of TDP-43 was performed in three brain regions: temporal cortex, hippocampal formation and amygdala. RESULTS: TDP-43 aggregates were present in 10,5% of the study subjects (n=323). Amygdala was the most frequently affected brain region (85.3% of the cases). TDP-43 accumulation was associated with age at death and race (p=0.002). Logistic regression analysis showed that asians older adults have higher odds of presenting TDP-43 inclusions than caucasians, regardless of gender, age, Braak stage for neurofibrilarly tangles and education attainment (OD=3.5, CI: 1.41-8.69, p=0.007). CONCLUSIONS: Our results suggest that TDP-43 abnormal accumulation increases along aging process and the amygdala is the brain region most susceptible to these alterations. Because we studied cognitively normal elderlies, we suggest that Asians older adults are protected against deleterious effects of TDP-43 in the brain. Future clinicopathological and genetic studies in populations dwelling in different ethnical backgrounds may identify possible protecting factors against the clinical expression of TDP-43 neuropathological findings

Neurodegenerative Erkrankungen (NDs) wie Alzheimer (AD), Parkinson (PD), und amyotrophe lateral Sklerose (ALS) sind Hirnerkrankungen, die durch unlösliche Proteinaggregate in Neuronen oder im Extrazellularraum charakterisiert sind. In dieser Arbeit habe ich für verschiede bekannte und vorhergesagte neurodegenerative Krankheitsproteine (NDPs) Proteininteraktionsnetzwerke erstellt, um mögliche gemeinsame Krankheitsmechanismen genauer zu studieren. Mit Hilfe eines automatisierten Hefe-Zwei-Hybrid-Systems (Y2H) konnte ich 18.663 Protein-Protein-Interaktionen (PPIs) für 449 wildtyp und 22 mutierte Proteine identifizieren. Eine genaue funktionelle Analyse der Interaktionspartner von korrespondierenden wildtyp und mutierten Proteinen ergab deutliche Unterschiede zum einen im Fall von allen untersuchten Proteinen und insbesondere im Fall vom ALS Krankheitsprotein TDP-43. Die identifizierten PPIs wurden außerdem verwendet um krankheitsspezifische Netzwerke zu erstellen und um Proteine zu identifizieren, die mit mehreren NDPs verbunden sind. Ich habe auf diese Weise vier Proteine (APP, IQSEC1, ZNF179 und ZMAT2) gefunden, die mit bekannten NDPs with Huntingtin, TDP-43, Parkin und Ataxin-1 interagieren und so fünf verschiedene NDs miteinander verbinden. Die Reduktion der mRNA Expression von IQSEC1, ZNF179 oder ZMAT2 mit Hilfe von siRNA führte zu einer Verstärkung von pathogenen Mechanismen wie der Aggregation von mutiertem Huntingtin und TDP-43 sowie der Hyperphosphorylierung des Proteins Tau. Außerdem habe ich 22 Proteine entdeckt, die die Aggregation von TDP-43 deutlich verändern und außerdem Mitglieder in sieben vorhergesagten Proteinkomplexen sind. Die Proteinkomplexe habe ich durch Kombination von Interaktionsdaten und Daten eines siRNA Screenings vorhergesagt. Zusätzlich habe ich herausgefunden, dass die Proteine eines vorhergesagten Komplexes, nämlich HDAC1, pRB, HP1, BRG1 und c-MYC, die Aggregation von TDP-43 durch Veränderung von dessen Genexpression beeinflussen. / Neurodegenerative diseases (NDs) such as Alzheimer’s disease (AD), Parkinson’s disease (PD) or amyotrophic lateral sclerosis (ALS) are progressive brain disorders characterized by the accumulation of insoluble protein aggregates in neuronal cells or the extracellular space of patient brains. To elucidate potential common pathological mechanisms in different NDs, I created comprehensive interaction networks for various known and predicted neurodegenerative disease proteins (NDPs). I identified 18,663 protein-protein interactions (PPIs) for 449 bioinformatically selected wild-type target proteins and 22 mutant variants of 11 known NDPs by using an automated yeast two-hybrid (Y2H) system. The functional analysis of the interaction partners of corresponding wild-type and mutant NDPs revealed strong differences in the case of all 11 NDPs and especially for the ALS protein TDP-43. The identified PPIs were used to generate networks for individual NDs such as AD or PD and to identify proteins that are connected to multiple NDPs. For example, I found that five neurodegenerative diseases are connected by four proteins (APP, ZMAT2, ZNF179 and IQSEC1) that link known NDPs such as huntingtin, TDP-43, parkin, ataxin-1 and SOD1. Analysis of publicly available gene expression data suggested that the mRNA expression of the four proteins is abnormally altered in brains of ND patients. Moreover, the knock-down of IQSEC1, ZNF179 or ZMAT2 aggravates pathogenic disease mechanisms such as aggregation of mutant huntingtin or TDP-43 as well as hyperphosphorylation of tau. Additionally, I identified 22 modifiers of TDP-43 aggregation, which are members in 7 protein complexes. These complexes were predicted based on combined data from PPI as well as siRNA screenings. Finally, I found that the proteins HDAC1, pRB, HP1, BRG1 and c-MYC, which form one of the predicted complexes, influence TDP-43 aggregation by altering its mRNA expression.

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease which currently lacks rapid and definitive diagnostic tests. Recently identified neuron specific splice variant molecules, Per28 and NFL-60, have been shown to contain unique epitopes and to have altered levels of expression in ALS patients. It is believed that these factors make Per28 and NFL-60 excellent candidate biomarkers for the ALS disease state. In this study, we attempted to develop ELISA assays directed against Per28 and NFL-60, as well as a generalized guideline for splice variant ELISA development, which could be used in a clinical setting. Limitations in currently identified antibodies to the splice variants allowed only for the completion of a Per28 ELISA, which lacked the sensitivity for clinical relevance. This assay creation process, however, did produce a guideline for similar ELISA development, which should allow for the more expeditious creation future ELISA.

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease which currently lacks rapid and definitive diagnostic tests. Recently identified neuron specific splice variant molecules, Per28 and NFL-60, have been shown to contain unique epitopes and to have altered levels of expression in ALS patients. It is believed that these factors make Per28 and NFL-60 excellent candidate biomarkers for the ALS disease state. In this study, we attempted to develop ELISA assays directed against Per28 and NFL-60, as well as a generalized guideline for splice variant ELISA development, which could be used in a clinical setting. Limitations in currently identified antibodies to the splice variants allowed only for the completion of a Per28 ELISA, which lacked the sensitivity for clinical relevance. This assay creation process, however, did produce a guideline for similar ELISA development, which should allow for the more expeditious creation future ELISA.

La sclérose latérale amyotrophique (SLA) est une maladie neurodégénérative fatale caractérisée par une perte progressive et sélective des neurones moteurs. La SLA est incurable et il n’existe aucun traitement efficace pour les personnes atteintes de cette maladie. Environ 90% des cas sont sporadiques tandis que 10% sont familiaux, et les patients décèdent généralement deux à cinq ans après l'apparition des premiers symptômes. De nombreuses anomalies génétiques sont associées à la SLA, incluant des mutations dans les protéines FUS, C9orf72, SOD-1 et TDP-43. Le laboratoire a développé un modèle transgénique de Caenorhabditis elegans surexprimant la protéine humaine mutante TDP-43(Q331K) dans les neurones moteurs GABAergiques. Nous avons également obtenu par mutagénèse et CRISPR-Cas9 des modèles physiologiquement représentatifs du nématode basés sur des mutations dans tdp-1, l'orthologue de TARDBP chez le C. elegans. L'objectif est de caractériser ces modèles et de déterminer s'ils peuvent récapituler certains aspects phénotypiques clés de la SLA, tels que des déficits moteurs et une neurodégénérescence dépendante de l'âge générant une paralysie. L’hypothèse est que le modèle TDP-1 pourra refléter plus précisément l’expression physiologique du gène dans la maladie humaine grâce à la mutation dans un gène endogène, l’absence de surexpression et l’expression ubiquitaire de la protéine TDP-1. Les résultats montrent que les modèles TDP-43/TDP-1 ont des déficits moteurs, une transmission synaptique altérée et une neurodégénérescence liée à l’âge. Cependant, seule la mutation dans TDP-43 semble avoir un effet sur la durée de vie. Ces modèles procurent différentes expressions physiologiques des protéines mutantes et donc, des phénotypes de niveaux d'intensité variables. Ils constitueront des outils utiles pour élucider de nouveaux mécanismes pathogéniques de la SLA ainsi que de bons candidats pour le criblage de médicaments et le développement de stratégies thérapeutiques. / Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by a progressive and selective loss of motor neurons. ALS is incurable and there are no effective treatments available for people living with the disease. About 90% of the cases are sporadic whereas 10% are familial, and patients usually die two to five years after symptom onset. Many gene defects are associated with ALS, including mutations in genes encoding FUS, C9orf72, SOD-1 and TDP-43 proteins. We have developed a transgenic Caenorhabditis elegans model expressing human mutant TDP-43(Q331K) in GABAergic motor neurons. We have also obtained by mutagenesis and CRISPR-Cas9 physiologically accurate models based on mutations in tdp-1, the C. elegans ortholog of TARDBP. Our objective is to characterize these models and determine if they can recapitulate key aspects of the disease such as motor deficits and age-dependent neurodegeneration causing paralysis. We believe that the TDP-1 model will reflect more precisely the physiological expression of the gene in the human disease because of its mutation in an endogenous gene, the absence of overexpression and ubiquitous protein expression. Our results show that both TDP-43 and TDP-1 models have motor deficits, synaptic transmission impairments and age-dependent neurodegeneration. However, only the TDP-43 mutation seems to have an effect on lifespan. These models provide different physiological expression of mutant proteins and thus phenotypes of varying intensity levels. They will be useful tools to elucidate new pathogenic mechanisms of ALS as well as being good candidates for drug screening and developing therapeutic strategies.

Amyotrophic Lateral Sclerosis (ALS) is an incurable late onset neurodegenerative disorder characterised by the specific loss of motor neurones (MNs). It has been recently demonstrated that Transactive response DNA-binding protein (TDP-43) is the dominant disease protein in both ALS and a sub-group of frontotemporal lobar degeneration (FTLDTDP). Moreover, the identification of TARDBP mutations in familial ALS confirms a mechanistic link between the observed mis-accumulation of TDP-43 and neurodegeneration but also provides an opportunity to establish an in vitro platform to model these diseases, based on patient-derived induced pluripotent stem cells (iPSCs). This study presents the optimization of an iPSC-based platform to study the consequences of TDP-43 M337V mutation in human functional populations of MNs and astrocytes in isolation as well as in co-culture. To develop this platform, two protocols to differentiate patient-derived iPSCs into functional MNs and astrocytes were first optimized, and the obtained cellular populations were then used to characterize the behaviour of mutant TDP-43 and its effect on the different cell types. This study show that it is possible to use iPSC-based platforms to recapitulate in vitro key aspects of TDP-43 proteinopathies such as MN cell autonomous toxicity and TDP-43 accumulation, but they can also be used to highlight previously unrecognised disease specific mechanisms and to test novel therapeutic approaches. Moreover, by performing co-culture experiments it was possible to evaluate the effects of M337V astrocytes on the survival of wild-type and M337V TDP-43 motor neurons, showing that mutant TDP-43 astrocytes do not adversely affect survival of co-cultured neurons. This iPSC-based platform represents an in vitro model to study both the effect of somatic mutations on isolated patient-specific cultures, but also to investigate cellular autonomy and neurodegeneration in the context of TDP-43 proteinopathies.