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

TDP-43 induces p53-mediated cell death of cortical progenitors and immature neurons

TAR DNA-binding protein 43 (TDP-43) is a key player in neurodegenerative diseases including frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Accumulation of TDP-43 is associated with neuronal death in the brain. How increased and disease-causing mutant forms of TDP-43 induce cell death remains unclear. Here we addressed the role of TDP-43 during neural development and show that reduced TDP-43 causes defects in neural stem/progenitor cell proliferation but not cell death. However, overexpression of wild type and TDP-43A315T proteins induce p53-dependent apoptosis of neural stem/progenitors and human induced pluripotent cell (iPS)-derived immature cortical neurons. We show that TDP-43 induces expression of the proapoptotic BH3-only genes Bbc3 and Bax, and that p53 inhibition rescues TDP-43 induced cell death of embryonic mouse, and human cortical neurons, including those derived from TDP-43G298S ALS patient iPS cells. Hence, an increase in wild type and mutant TDP-43 induces p53-dependent cell death in neural progenitors developing neurons and this can be rescued. These findings may have important implications for accumulated or mutant TDP-43 induced neurodegenerative diseases

TDP-43-Mediated Neuron Loss In Vivo Requires RNA-Binding Activity

Alteration and/or mutations of the ribonucleoprotein TDP-43 have been firmly linked to human neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). The relative impacts of TDP-43 alteration, mutation, or inherent protein function on neural integrity, however, remain less clear—a situation confounded by conflicting reports based on transient and/or random-insertion transgenic expression. We therefore performed a stringent comparative investigation of impacts of these TDP-43 modifications on neural integrity in vivo. To achieve this, we systematically screened ALS/FTLD-associated and synthetic TDP-43 isoforms via same-site gene insertion and neural expression in Drosophila; followed by transposon-based motor neuron-specific transgenesis in a chick vertebrate system. Using this bi-systemic approach we uncovered a requirement of inherent TDP-43 RNA-binding function—but not ALS/FTLD-linked mutation, mislocalization, or truncation—for TDP-43-mediated neurotoxicity in vivo

Aberrant Localization of FUS and TDP43 Is Associated with Misfolding of SOD1 in Amyotrophic Lateral Sclerosis

Background: Amyotrophic lateral sclerosis (ALS) is incurable and characterized by progressive paralysis of the muscles of the limbs, speech and swallowing, and respiration due to the progressive degeneration of voluntary motor neurons. Clinically indistinguishable ALS can be caused by genetic mutations of Cu/Zn superoxide dismutase (SOD1), TAR-DNA binding protein 43 (TDP43), or fused in sarcoma/translocated in liposarcoma (FUS/TLS), or can occur in the absence of known mutation as sporadic disease. In this study, we tested the hypothesis that FUS/TLS and TDP43 gain new pathogenic functions upon aberrant accumulation in the cytosol that directly or indirectly include misfolding of SOD1. Methodology/Principal Findings: Patient spinal cord necropsy immunohistochemistry with SOD1 misfolding-specific antibodies revealed misfolded SOD1 in perikarya and motor axons of SOD1-familial ALS (SOD1-FALS), and in motor axons of R521C-FUS FALS and sporadic ALS (SALS) with cytoplasmic TDP43 inclusions. SOD1 misfolding and oxidation was also detected using immunocytochemistry and quantitative immunoprecipitation of human neuroblastoma SH-SY5Y cells as well as cultured murine spinal neural cells transgenic for human wtSOD1, which were transiently transfected with human cytosolic mutant FUS or TDP43, or wtTDP43. Conclusion/Significance: We conclude that cytosolic mislocalization of FUS or TDP43 in vitro and ALS in vivo may kindle wtSOD1 misfolding in non-SOD1 FALS and SALS. The lack of immunohistochemical compartmental co-localization o

Longitudinal Imaging and Analysis of Neurons Expressing Polyglutamine-Expanded Proteins

Misfolded proteins have been implicated in most of the major neurodegenerative diseases, and identifying drugs and pathways that protect neurons from the toxicity of misfolded proteins is of paramount importance. We invented a form of automated imaging and analysis called robotic microscopy that is well suited to the study of neurodegeneration. It enables the monitoring of large cohorts of individual neurons over their lifetimes as they undergo neurodegeneration. With automated analysis, multiple endpoints in neurons can be measured, including survival. Statistical approaches, typically reserved for engineering and clinical medicine, can be applied to these data in an unbiased fashion to discover whether factors contribute positively or negatively to neuronal fate and to quantify the importance of their contribution. Ultimately, multivariate dynamic models can be constructed from these data, which can provide a systems-level understanding of the neurodegenerative disease process and guide the rationale for the development of therapies

TDP-43 Identified from a Genome Wide RNAi Screen for SOD1 Regulators

Amyotrophic Lateral Sclerosis (ALS) is a late-onset, progressive neurodegenerative disease affecting motor neurons in the brain stem and spinal cord leading to loss of voluntary muscular function and ultimately, death due to respiratory failure. A subset of ALS cases are familial and associated with mutations in superoxide dismutase 1 (SOD1) that destabilize the protein and predispose it to aggregation. In spite of the fact that sporadic and familial forms of ALS share many common patho-physiological features, the mechanistic relationship between SOD1-associated and sporadic forms of the disease if any, is not well understood. To better understand any molecular connections, a cell-based protein folding assay was employed to screen a whole genome RNAi library for genes that regulate levels of soluble SOD1. Statistically significant hits that modulate SOD1 levels, when analyzed by pathway analysis revealed a highly ranked network containing TAR DNA binging protein (TDP-43), a major component of aggregates characteristic of sporadic ALS. Biochemical experiments confirmed the action of TDP-43 on SOD1. These results highlight an unexpected relationship between TDP-43 and SOD1 which may have implications in disease pathogenesis

Tar DNA Binding Protein-43 (TDP-43) Associates with Stress Granules: Analysis of Cultured Cells and Pathological Brain Tissue

Tar DNA Binding Protein-43 (TDP-43) is a principle component of inclusions in many cases of frontotemporal lobar degeneration (FTLD-U) and amyotrophic lateral sclerosis (ALS). TDP-43 resides predominantly in the nucleus, but in affected areas of ALS and FTLD-U central nervous system, TDP-43 is aberrantly processed and forms cytoplasmic inclusions. The mechanisms governing TDP-43 inclusion formation are poorly understood. Increasing evidence indicates that TDP-43 regulates mRNA metabolism by interacting with mRNA binding proteins that are known to associate with RNA granules. Here we show that TDP-43 can be induced to form inclusions in cell culture and that most TDP-43 inclusions co-localize with SGs. SGs are cytoplasmic RNA granules that consist of mixed protein - RNA complexes. Under stressful conditions SGs are generated by the reversible aggregation of prion-like proteins, such as TIA-1, to regulate mRNA metabolism and protein translation. We also show that disease-linked mutations in TDP-43 increased TDP-43 inclusion formation in response to stressful stimuli. Biochemical studies demonstrated that the increased TDP-43 inclusion formation is associated with accumulation of TDP-43 detergent insoluble complexes. TDP-43 associates with SG by interacting with SG proteins, such as TIA-1, via direct protein-protein interactions, as well as RNA-dependent interactions. The signaling pathway that regulates SGs formation also modulates TDP-43 inclusion formation. We observed that inclusion formation mediated by WT or mutant TDP-43 can be suppressed by treatment with translational inhibitors that suppress or reverse SG formation. Finally, using Sudan black to quench endogenous autofluorescence, we also demonstrate that TDP-43 positive-inclusions in pathological CNS tissue co-localize with multiple protein markers of stress granules, including TIA-1 and eIF3. These data provide support for accumulating evidence that TDP-43 participates in the SG pathway

In Vivo Generation of Neurotoxic Prion Protein: Role for Hsp70 in Accumulation of Misfolded Isoforms

Prion diseases are incurable neurodegenerative disorders in which the normal cellular prion protein (PrPC) converts into a misfolded isoform (PrPSc) with unique biochemical and structural properties that correlate with disease. In humans, prion disorders, such as Creutzfeldt-Jakob disease, present typically with a sporadic origin, where unknown mechanisms lead to the spontaneous misfolding and deposition of wild type PrP. To shed light on how wild-type PrP undergoes conformational changes and which are the cellular components involved in this process, we analyzed the dynamics of wild-type PrP from hamster in transgenic flies. In young flies, PrP demonstrates properties of the benign PrPC; in older flies, PrP misfolds, acquires biochemical and structural properties of PrPSc, and induces spongiform degeneration of brain neurons. Aged flies accumulate insoluble PrP that resists high concentrations of denaturing agents and contains PrPSc-specific conformational epitopes. In contrast to PrPSc from mammals, PrP is proteinase-sensitive in flies. Thus, wild-type PrP rapidly converts in vivo into a neurotoxic, protease-sensitive isoform distinct from prototypical PrPSc. Next, we investigated the role of molecular chaperones in PrP misfolding in vivo. Remarkably, Hsp70 prevents the accumulation of PrPSc-like conformers and protects against PrP-dependent neurodegeneration. This protective activity involves the direct interaction between Hsp70 and PrP, which may occur in active membrane microdomains such as lipid rafts, where we detected Hsp70. These results highlight the ability of wild-type PrP to spontaneously convert in vivo into a protease-sensitive isoform that is neurotoxic, supporting the idea that protease-resistant PrPSc is not required for pathology. Moreover, we identify a new role for Hsp70 in the accumulation of misfolded PrP. Overall, we provide new insight into the mechanisms of spontaneous accumulation of neurotoxic PrP and uncover the potential therapeutic role of Hsp70 in treating these devastating disorders

Cell Type-Specific Neuroprotective Activity of Untranslocated Prion Protein

Background: A key pathogenic role in prion diseases was proposed for a cytosolic form of the prion protein (PrP). However, it is not clear how cytosolic PrP localization influences neuronal viability, with either cytotoxic or anti-apoptotic effects reported in different studies. The cellular mechanism by which PrP is delivered to the cytosol of neurons is also debated, and either retrograde transport from the endoplasmic reticulum or inefficient translocation during biosynthesis has been proposed. We investigated cytosolic PrP biogenesis and effect on cell viability in primary neuronal cultures from different mouse brain regions. Principal Findings: Mild proteasome inhibition induced accumulation of an untranslocated form of cytosolic PrP in cortical and hippocampal cells, but not in cerebellar granules. A cyclopeptolide that interferes with the correct insertion of the PrP signal sequence into the translocon increased the amount of untranslocated PrP in cortical and hippocampal cells, and induced its synthesis in cerebellar neurons. Untranslocated PrP boosted the resistance of cortical and hippocampal neurons to apoptotic insults but had no effect on cerebellar cells. Significance: These results indicate cell type-dependent differences in the efficiency of PrP translocation, and argue that cytosolic PrP targeting might serve a physiological neuroprotective function

Hyperphosphorylation as a Defense Mechanism to Reduce TDP-43 Aggregation

Several neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U) are characterized by inclusion bodies formed by TDP-43 (TDP). We established cell and transgenic Drosophila models expressing TDP carboxyl terminal fragment (ND251 and ND207), which developed aggregates recapitulating important features of TDP inclusions in ALS/FTLD-U, including hyperphosphorylation at previously reported serine403,404,409,410 residues, polyubiquitination and colocalization with optineurin. These models were used to address the pathogenic role of hyperphosphorylation in ALS/FTLD-U. We demonstrated that hyperphosphorylation and ubiquitination occurred temporally later than aggregation in cells. Expression of CK2α which phosphorylated TDP decreased the aggregation propensity of ND251 or ND207; this effect could be blocked by CK2 inhibitor DMAT. Mutation of serines379,403,404,409,410 to alanines (S5A) to eliminate phosphorylation increased the aggregation propensity and number of aggregates of TDP, but mutation to aspartic acids (S5D) or glutamic acids (S5E) to simulate hyperphosphorylation had the opposite effect. Functionally, ND251 or ND207 aggregates decreased the number of neurites of Neuro2a cells induced by retinoic acid or number of cells by MTT assay. S5A mutation aggravated, but S5E mutation alleviated these cytotoxic effects of aggregates. Finally, ND251 or ND251S5A developed aggregates in neurons, and salivary gland of transgenic Drosophila, but ND251S5E did not. Taken together, our data indicate that hyperphosphorylation may represent a compensatory defense mechanism to stop or prevent pathogenic TDP from aggregation. Therefore, enhancement of phosphorylation may serve as an effective therapeutic strategy against ALS/FTLD-U