Glial proliferation and activation are associated with disease progression in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia. In this study, we describe a unique platform to address the question of cell autonomy in transactive response DNAbinding protein (TDP-43) proteinopathies. We generated functional astroglia from human induced pluripotent stem cells carrying an ALScausing TDP-43 mutation and show that mutant astrocytes exhibit increased levels of TDP-43, subcellular mislocalization of TDP-43, and decreased cell survival. We then performed coculture experiments 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 cocultured neurons. These observations reveal a significant and previously unrecognized glial cell-autonomous pathological phenotype associated with a pathogenic mutation in TDP-43 and show that TDP-43 proteinopathies do not display an astrocyte non-cell-autonomous component in cell culture, as previously described for SOD1 ALS. This study highlights the utility of induced pluripotent stem cell-based in vitro disease models to investigate mechanisms of disease in ALS and other TDP-43 proteinopathies.glia | motor neuron disease | disease modeling T ransactive response DNA-binding protein (TDP-43) is the major component of ubiquitinated cytoplasmic and nuclear inclusions in neurons and astroglia in amyotrophic lateral sclerosis (ALS) and a subgroup of frontotemporal lobar degeneration (FTLD-TDP) (1-3). These pathological hallmarks provide a unifying description of a range of conditions defined as TDP-43 proteinopathies (4). At present, >30 mutations in the TDP-43 gene (TARDBP) have been linked to familial ALS (fALS) (5), strongly suggesting a causative role for TDP-43 in the pathogenesis of ALS.Accumulating evidence from experimental systems implicating non-cell-autonomous mechanisms in ALS has highlighted the importance of the glial cellular environment to motor neuron (MN) degeneration (1,3,(6)(7)(8)(9). In vivo rodent models of ALS with lineage-specific SOD1 expression have particularly influenced our understanding of the nonneuronal contribution to disease progression. Glial expression of mutant SOD1 cannot initiate MN disease on its own, but is necessary for disease progression (6, 7). Furthermore, astrogliosis precedes MN degeneration in some animal models and is a dominant feature of all human ALS pathology (4, 6, 10). Collectively, these observations highlight the need to better understand the nature of astroglial pathology in ALS. Combining developmental neurobiological principles of cell fate determination with human induced pluripotent stem cell (iPSC) lines derived from patients carrying ALS disease-causing mutations may provide important insights into astroglia pathology.We recently generated human MNs from iPSC lines derived from a fALS patient and demonstrated that the M337V TDP-43 mutation confers cell-autonomous toxicity to MNs (11). More...

Transactive response protein is the dominant disease protein in amyotrophic lateral sclerosis (ALS) and a subgroup of frontotemporal lobar degeneration (FTLD-TDP). Identification of mutations in the gene encoding TDP-43 (TARDBP) in familial ALS confirms a mechanistic link between misaccumulation of TDP-43 and neurodegeneration and provides an opportunity to study TDP-43 proteinopathies in human neurons generated from patient fibroblasts by using induced pluripotent stem cells (iPSCs). Here, we report the generation of iPSCs that carry the TDP-43 M337V mutation and their differentiation into neurons and functional motor neurons. Mutant neurons had elevated levels of soluble and detergent-resistant TDP-43 protein, decreased survival in longitudinal studies, and increased vulnerability to antagonism of the PI3K pathway. We conclude that expression of physiological levels of TDP-43 in human neurons is sufficient to reveal a mutation-specific cell-autonomous phenotype and strongly supports this approach for the study of disease mechanisms and for drug screening. Several in vitro and in vivo models established the toxicity of ALS-associated TDP-43 mutations, although the underlying mechanism is unclear (9, 10). Most cellular and animal models of ALS and FTLD-TDP pathogenesis involve overexpression of TDP-43 in nonneuronal or nonhuman cells and cannot be used to investigate the selective vulnerability of neurons or key molecular events that are unique to human cells. By contrast, induced pluripotent stem cells (iPSCs) (11-14) coupled with defined in vitro differentiation protocols (15-20) offer a model system to investigate disease mechanisms in a more physiological context. Here, we report the pathological effects of endogenous mutant TDP-43 in iPSC-derived human neurons from an ALS patient carrying the M337V mutation.

Mutations in C9ORF72 are the most common cause of familial amyotrophic lateral sclerosis (ALS). Here, through a combination of RNA-Seq and electrophysiological studies on induced pluripotent stem cell (iPSC)-derived motor neurons (MNs), we show that increased expression of GluA1 AMPA receptor (AMPAR) subunit occurs in MNs with C9ORF72 mutations that leads to increased Ca2+-permeable AMPAR expression and results in enhanced selective MN vulnerability to excitotoxicity. These deficits are not found in iPSC-derived cortical neurons and are abolished by CRISPR/Cas9-mediated correction of the C9ORF72 repeat expansion in MNs. We also demonstrate that MN-specific dysregulation of AMPAR expression is also present in C9ORF72 patient post-mortem material. We therefore present multiple lines of evidence for the specific upregulation of GluA1 subunits in human mutant C9ORF72 MNs that could lead to a potential pathogenic excitotoxic mechanism in ALS.

Mutations in C9orf72 are the most common genetic cause of amyotrophic lateral sclerosis (ALS). Accumulating evidence implicates astrocytes as important non‐cell autonomous contributors to ALS pathogenesis, although the potential deleterious effects of astrocytes on the function of motor neurons remains to be determined in a completely humanized model of C9orf72‐mediated ALS. Here, we use a human iPSC‐based model to study the cell autonomous and non‐autonomous consequences of mutant C9orf72 expression by astrocytes. We show that mutant astrocytes both recapitulate key aspects of C9orf72‐related ALS pathology and, upon co‐culture, cause motor neurons to undergo a progressive loss of action potential output due to decreases in the magnitude of voltage‐activated Na+ and K+ currents. Importantly, CRISPR/Cas‐9 mediated excision of the C9orf72 repeat expansion reverses these phenotypes, confirming that the C9orf72 mutation is responsible for both cell‐autonomous astrocyte pathology and non‐cell autonomous motor neuron pathophysiology.