Sami J. Barmada

Cytoplasmic Mislocalization of TDP-43 Is Toxic to Neurons and Enhanced by a Mutation Associated with Familial Amyotrophic Lateral Sclerosis

Mutations in the gene encoding TDP-43—the major protein component of neuronal aggregates characteristic of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) with ubiquitin-positive inclusion bodies—have been linked to familial forms of both disorders. Aggregates of TDP-43 in cortical and spinal motorneurons in ALS, or in neurons of the frontal and temporal cortices in FTLD, are closely linked to neuron loss and atrophy in these areas. However, the mechanism by which TDP-43 mutations lead to neurodegeneration is unclear. To investigate the pathogenic role of TDP-43 mutations, we established a model of TDP-43 proteinopathies by expressing fluorescently tagged wild-type and mutant TDP-43 in primary rat cortical neurons. Expression of mutant TDP-43 was toxic to neurons, and mutant-specific toxicity was associated with increased cytoplasmic mislocalization of TDP-43. Inclusion bodies were not necessary for the toxicity and did not affect the risk of cell death. Cellular survival was unaffected by the total amount of exogenous TDP-43 in the nucleus, but the amount of cytoplasmic TDP-43 was a strong and independent predictor of neuronal death. These results suggest that mutant TDP-43 is mislocalized to the cytoplasm, where it exhibits a toxic gain-of-function and induces cell death.

Mutant induced pluripotent stem cell lines recapitulate aspects of TDP-43 proteinopathies and reveal cell-specific vulnerability

Transactive response DNA-binding (TDP-43) 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.

Astrocyte pathology and the absence of non-cell autonomy in an induced pluripotent stem cell model of TDP-43 proteinopathy.

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 DNA-binding protein (TDP-43) proteinopathies. We generated functional astroglia from human induced pluripotent stem cells carrying an ALS-causing 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.

Autophagy induction enhances TDP43 turnover and survival in neuronal ALS models

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) have distinct clinical features but a common pathology—cytoplasmic inclusions rich in TDP43. Rare TDP43 mutations cause ALS or FTD, but abnormal TDP43 levels and localization may cause disease even if TDP43 lacks a mutation. Here we showed that individual neurons vary in their ability to clear TDP43 and are exquisitely sensitive to TDP43 levels. To measure TDP43 clearance, we developed and validated a single-cell optical method that overcomes the confounding effects of aggregation and toxicity, and discovered that pathogenic mutations significantly shorten TDP43 half-life. Novel compounds that stimulate autophagy improved TDP43 clearance and localization, and enhanced survival in primary murine neurons and in human stem cell–derived neurons and astrocytes harboring mutant TDP43. These findings indicate that the levels and localization of TDP43 critically determine neurotoxicity and show that autophagy induction mitigates neurodegeneration by acting directly on TDP43 clearance.

ALS-associated mutation FUS-R521C causes DNA damage and RNA splicing defects

Autosomal dominant mutations of the RNA/DNA binding protein FUS are linked to familial amyotrophic lateral sclerosis (FALS); however, it is not clear how FUS mutations cause neurodegeneration. Using transgenic mice expressing a common FALS-associated FUS mutation (FUS-R521C mice), we found that mutant FUS proteins formed a stable complex with WT FUS proteins and interfered with the normal interactions between FUS and histone deacetylase 1 (HDAC1). Consequently, FUS-R521C mice exhibited evidence of DNA damage as well as profound dendritic and synaptic phenotypes in brain and spinal cord. To provide insights into these defects, we screened neural genes for nucleotide oxidation and identified brain-derived neurotrophic factor (Bdnf) as a target of FUS-R521C-associated DNA damage and RNA splicing defects in mice. Compared with WT FUS, mutant FUS-R521C proteins formed a more stable complex with Bdnf RNA in electrophoretic mobility shift assays. Stabilization of the FUS/Bdnf RNA complex contributed to Bdnf splicing defects and impaired BDNF signaling through receptor TrkB. Exogenous BDNF only partially restored dendrite phenotype in FUS-R521C neurons, suggesting that BDNF-independent mechanisms may contribute to the defects in these neurons. Indeed, RNA-seq analyses of FUS-R521C spinal cords revealed additional transcription and splicing defects in genes that regulate dendritic growth and synaptic functions. Together, our results provide insight into how gain-of-function FUS mutations affect critical neuronal functions.

Inhibition of RNA lariat debranching enzyme suppresses TDP-43 toxicity in ALS disease models

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease primarily affecting motor neurons. Mutations in the gene encoding TDP-43 cause some forms of the disease, and cytoplasmic TDP-43 aggregates accumulate in degenerating neurons of most individuals with ALS. Thus, strategies aimed at targeting the toxicity of cytoplasmic TDP-43 aggregates may be effective. Here, we report results from two genome-wide loss-of-function TDP-43 toxicity suppressor screens in yeast. The strongest suppressor of TDP-43 toxicity was deletion of DBR1, which encodes an RNA lariat debranching enzyme. We show that, in the absence of Dbr1 enzymatic activity, intronic lariats accumulate in the cytoplasm and likely act as decoys to sequester TDP-43, preventing it from interfering with essential cellular RNAs and RNA-binding proteins. Knockdown of Dbr1 in a human neuronal cell line or in primary rat neurons is also sufficient to rescue TDP-43 toxicity. Our findings provide insight into TDP-43–mediated cytotoxicity and suggest that decreasing Dbr1 activity could be a potential therapeutic approach for ALS.

Proteostasis of polyglutamine varies among neurons and predicts neurodegeneration

In polyglutamine (polyQ) diseases, only certain neurons die, despite widespread expression of the offending protein. PolyQ expansion may induce neurodegeneration by impairing proteostasis, but protein aggregation and toxicity tend to confound conventional measurements of protein stability. Here, we used optical pulse labeling to measure effects of polyQ expansions on the mean lifetime of a fragment of huntingtin, the protein that causes Huntingtons disease, in living neurons. We show that polyQ expansion reduced the mean lifetime of mutant huntingtin within a given neuron and that the mean lifetime varied among neurons, indicating differences in their capacity to clear the polypeptide. We found that neuronal longevity is predicted by the mean lifetime of huntingtin, as cortical neurons cleared mutant huntingtin faster and lived longer than striatal neurons. Thus, cell type-specific differences in turnover capacity may contribute to cellular susceptibility to toxic proteins, and efforts to bolster proteostasis in Huntingtons disease, such as protein clearance, could be neuroprotective.

Visualization of Prion Infection in Transgenic Mice Expressing Green Fluorescent Protein-Tagged Prion Protein

Tg(PrP-EGFP) mice express an enhanced green fluorescent protein (EGFP)-tagged version of the prion protein (PrP) that behaves like endogenous PrP in terms of its posttranslational processing, anatomical localization, and functional activity. In this study, we describe experiments in which Tg(PrP-EGFP) mice were inoculated intracerebrally with scrapie prions. Although PrP-EGFP was incapable of sustaining prion infection in Tg(PrP-EGFP)/Prn-p0/0 mice, it acted as a dominant-negative inhibitor that bound to, and fluorescently marked, deposits of PrPSc generated from endogenous PrP in Tg(PrP-EGFP)/Prn-p+/+ mice. Scrapie infection of these latter animals caused a progressive accumulation of fluorescent PrP-EGFP aggregates in neuropil, axons, and prominently in the Golgi apparatus of neurons. Our results provide an entirely new picture of PrPSc localization during the course of prion infection, and they identify for the first time intracellular sites of PrPSc formation that are not well visualized with conventional immunohistochemical techniques.

N-Terminally Deleted Forms of the Prion Protein Activate Both Bax-Dependent and Bax-Independent Neurotoxic Pathways

Transgenic (Tg) mice expressing prion protein (PrP) with a deletion of the flexible, N-terminal tail encompassing residues 32–134 spontaneously develop ataxia, degeneration of cerebellar granule cells, and vacuolation of white matter in the brain and spinal cord, resulting in death by 3 months of age. These abnormalities are completely abrogated by coexpression of wild-type PrP from a single copy of the endogenous Prn-p gene. A similar but much more severe phenotype is seen in transgenic mice expressing PrP deleted for a conserved block of 21 amino acids (residues 105–125) within the N-terminal tail. The latter animals die within 1 week of birth in the absence of endogenous PrP, and fivefold overexpression of wild-type PrP is required to delay death beyond 1 year. To define the cellular pathways mediating the neurotoxicity of PrPΔ32–134 and PrPΔ105–125, we analyzed the effect of genetically deleting the proapoptotic protein Bax in mice expressing these neurotoxic forms of PrP. We find that Bax deletion in Tg(PrPΔ32–134) mice delays the development of clinical illness and slows apoptosis of cerebellar granule cells but has no effect on white matter degeneration. In contrast, Bax deletion has no effect on the clinical or neuropathological phenotype of Tg(Δ105–125) mice. Our results indicate that Bax-related pathways mediate the initial neurotoxic actions of PrPΔ32–134 but that neurodegeneration induced by this protein as well as by PrPΔ105–125 also involves Bax-independent pathways.

Pathogenic TARDBP mutations in amyotrophic lateral sclerosis and frontotemporal dementia: disease-associated pathways.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are late-onset neurodegenerative disorders that are associated with mutations in the TARDBP gene. The product of this gene, TDP-43, has also been identified as the main component of the intracellular inclusions typical of most cases of ALS and FTD. Recent evidence suggests that TDP-43 is essential for proper development and involved in several fundamental cellular processes, including gene transcription, RNA processing, and the spatial regulation of mRNA translation. Pathogenic TARDBP mutations that impair TDP-43 function could therefore be related to neuronal degeneration in ALS and FTD. Conversely, cellular and animal studies have shown that pathogenic TARDBP mutations induce neuronal toxicity through mislocalization or elevated concentrations of TDP-43, consistent with a gain-of-function mechanism. In this review, we focus on the physiologic functions of TDP-43 within the central nervous system and discuss how these functions may be perturbed or pathologically altered by disease-associated mutations.