Generation and characterization of monoclonal antibodies against pathologically phosphorylated TDP-43.

Inclusions containing TAR DNA binding protein 43 (TDP-43) are a pathological hallmark of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). One of the disease-specific features of TDP-43 inclusions is the aberrant phosphorylation of TDP-43 at serines 409/410 (pS409/410). Here, we developed rabbit monoclonal antibodies (mAbs) that specifically detect pS409/410-TDP-43 in multiple model systems and FTD/ALS patient samples. Specifically, we identified three mAbs (26H10, 2E9 and 23A1) from spleen B cell clones that exhibit high specificity and sensitivity to pS409/410-TDP-43 peptides in an ELISA assay. Biochemical analyses revealed that pS409/410 of recombinant TDP-43 and of exogenous 25 kDa TDP-43 C-terminal fragments in cultured HEK293T cells are detected by all three mAbs. Moreover, the mAbs detect pS409/410-positive TDP-43 inclusions in the brains of FTD/ALS patients and mouse models of TDP-43 proteinopathy by immunohistochemistry. Our findings indicate that these mAbs are a valuable resource for investigating TDP-43 pathology both in vitro and in vivo.

TMEM106B core deposition associates with TDP-43 pathology and is increased in risk SNP carriers for frontotemporal dementia.

Genetic variation at the transmembrane protein 106B gene (TMEM106B) has been linked to risk of frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) through an unknown mechanism. We found that presence of the TMEM106B rs3173615 protective genotype was associated with longer survival after symptom onset in a postmortem FTLD-TDP cohort, suggesting a slower disease course. The seminal discovery that filaments derived from TMEM106B is a common feature in aging and, across a range of neurodegenerative disorders, suggests that genetic variants in TMEM106B could modulate disease risk and progression through modulating TMEM106B aggregation. To explore this possibility and assess the pathological relevance of TMEM106B accumulation, we generated a new antibody targeting the TMEM106B filament core sequence. Analysis of postmortem samples revealed that the TMEM106B rs3173615 risk allele was associated with higher TMEM106B core accumulation in patients with FTLD-TDP. In contrast, minimal TMEM106B core deposition was detected in carriers of the protective allele. Although the abundance of monomeric full-length TMEM106B was unchanged, carriers of the protective genotype exhibited an increase in dimeric full-length TMEM106B. Increased TMEM106B core deposition was also associated with enhanced TDP-43 dysfunction, and interactome data suggested a role for TMEM106B core filaments in impaired RNA transport, local translation, and endolysosomal function in FTLD-TDP. Overall, these findings suggest that prevention of TMEM106B core accumulation is central to the mechanism by which the TMEM106B protective haplotype reduces disease risk and slows progression.

The endolysosomal pathway and ALS/FTD.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are considered to be part of a disease spectrum that is associated with causative mutations and risk variants in a wide range of genes. Mounting evidence indicates that several of these genes are linked to the endolysosomal system, highlighting the importance of this pathway in ALS/FTD. Although many studies have focused on how disruption of this pathway impacts on autophagy, recent findings reveal that this may not be the whole picture: specifically, disrupting autophagy may not be sufficient to induce disease, whereas disrupting the endolysosomal system could represent a crucial pathogenic driver. In this review we discuss the connections between ALS/FTD and the endolysosomal system, including a breakdown of how disease-associated genes are implicated in this pathway. We also explore the potential downstream consequences of disrupting endolysosomal activity in the brain, outside of an effect on autophagy.

Two FTD-ALS genes converge on the endosomal pathway to induce TDP-43 pathology and degeneration.

Frontotemporal dementia and amyotrophic lateral sclerosis (FTD-ALS) are associated with both a repeat expansion in the C9orf72 gene and mutations in the TANK-binding kinase 1 (TBK1) gene. We found that TBK1 is phosphorylated in response to C9orf72 poly(Gly-Ala) [poly(GA)] aggregation and sequestered into inclusions, which leads to a loss of TBK1 activity and contributes to neurodegeneration. When we reduced TBK1 activity using a TBK1-R228H (Arg228→His) mutation in mice, poly(GA)-induced phenotypes were exacerbated. These phenotypes included an increase in TAR DNA binding protein 43 (TDP-43) pathology and the accumulation of defective endosomes in poly(GA)-positive neurons. Inhibiting the endosomal pathway induced TDP-43 aggregation, which highlights the importance of this pathway and TBK1 activity in pathogenesis. This interplay between C9orf72, TBK1, and TDP-43 connects three different facets of FTD-ALS into one coherent pathway.

Plasma PolyQ-ATXN3 Levels Associate With Cerebellar Degeneration and Behavioral Abnormalities in a New AAV-Based SCA3 Mouse Model.

Spinocerebellar ataxia type 3 (SCA3) is a dominantly inherited cerebellar ataxia caused by the expansion of a polyglutamine (polyQ) repeat in the gene encoding ATXN3. The polyQ expansion induces protein inclusion formation in the neurons of patients and results in neuronal degeneration in the cerebellum and other brain regions. We used adeno-associated virus (AAV) technology to develop a new mouse model of SCA3 that recapitulates several features of the human disease, including locomotor defects, cerebellar-specific neuronal loss, polyQ-expanded ATXN3 inclusions, and TDP-43 pathology. We also found that neurofilament light is elevated in the cerebrospinal fluid (CSF) of the SCA3 animals, and the expanded polyQ-ATXN3 protein can be detected in the plasma. Interestingly, the levels of polyQ-ATXN3 in plasma correlated with measures of cerebellar degeneration and locomotor deficits in 6-month-old SCA3 mice, supporting the hypothesis that this factor could act as a biomarker for SCA3.

Modelling amyotrophic lateral sclerosis in rodents.

The efficient study of human disease requires the proper tools, one of the most crucial of which is an accurate animal model that faithfully recapitulates the human condition. The study of amyotrophic lateral sclerosis (ALS) is no exception. Although the majority of ALS cases are considered sporadic, most animal models of this disease rely on genetic mutations identified in familial cases. Over the past decade, the number of genes associated with ALS has risen dramatically and, with each new genetic variant, there is a drive to develop associated animal models. Rodent models are of particular importance as they allow for the study of ALS in the context of a living mammal with a comparable CNS. Such models not only help to verify the pathogenicity of novel mutations but also provide critical insight into disease mechanisms and are crucial for the testing of new therapeutics. In this Review, we aim to summarize the full spectrum of ALS rodent models developed to date.

Microglial lysosome dysfunction contributes to white matter pathology and TDP-43 proteinopathy in GRN-associated FTD.

Loss-of-function mutations in the progranulin gene (GRN), which encodes progranulin (PGRN), are a major cause of frontotemporal dementia (FTD). GRN-associated FTD is characterized by TDP-43 inclusions and neuroinflammation, but how PGRN loss causes disease remains elusive. We show that Grn knockout (KO) mice have increased microgliosis in white matter and an accumulation of myelin debris in microglial lysosomes in the same regions. Accumulation of myelin debris is also observed in white matter of patients with GRN-associated FTD. In addition, our findings also suggest that PGRN insufficiency in microglia leads to impaired lysosomal-mediated clearance of myelin debris. Finally, Grn KO mice that are deficient in cathepsin D (Ctsd), a key lysosomal enzyme, have augmented myelin debris and increased neuronal TDP-43 pathology. Together, our data strongly imply that PGRN loss affects microglial activation and lysosomal function, resulting in the accumulation of myelin debris and contributing to TDP-43 pathology.

HDAC6 Interacts With Poly (GA) and Modulates its Accumulation in c9FTD/ALS.

The aberrant translation of a repeat expansion in chromosome 9 open reading frame 72 (C9orf72), the most common cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), results in the accumulation of toxic dipeptide repeat (DPR) proteins in the central nervous system We have found that, among the sense DPR proteins, HDAC6 specifically interacts with the poly (GA) and co-localizes with inclusions in both patient tissue and a mouse model of this disease (c9FTD/ALS). Overexpression of HDAC6 increased poly (GA) levels in cultured cells independently of HDAC6 deacetylase activity, suggesting that HDAC6 can modulate poly (GA) pathology through a mechanism that depends upon their physical interaction. Moreover, decreasing HDAC6 expression by stereotaxic injection of antisense oligonucleotides significantly reduced the number of poly (GA) inclusions in c9FTD/ALS mice. These findings suggest that pharmacologically reducing HDAC6 levels could be of therapeutic value in c9FTD/ALS.

Hexanucleotide Repeat Expansions in c9FTD/ALS and SCA36 Confer Selective Patterns of Neurodegeneration In Vivo.

A G4C2 hexanucleotide repeat expansion in an intron of C9orf72 is the most common cause of frontal temporal dementia and amyotrophic lateral sclerosis (c9FTD/ALS). A remarkably similar intronic TG3C2 repeat expansion is associated with spinocerebellar ataxia 36 (SCA36). Both expansions are widely expressed, form RNA foci, and can undergo repeat-associated non-ATG (RAN) translation to form similar dipeptide repeat proteins (DPRs). Yet, these diseases result in the degeneration of distinct subsets of neurons. We show that the expression of these repeat expansions in mice is sufficient to recapitulate the unique features of each disease, including this selective neuronal vulnerability. Furthermore, only the G4C2 repeat induces the formation of aberrant stress granules and pTDP-43 inclusions. Overall, our results demonstrate that the pathomechanisms responsible for each disease are intrinsic to the individual repeat sequence, highlighting the importance of sequence-specific RNA-mediated toxicity in each disorder.

Chimeric Peptide Species Contribute to Divergent Dipeptide Repeat Pathology in c9ALS/FTD and SCA36.

GGGGCC hexanucleotide repeat expansions (HREs) in C9orf72 cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) and lead to the production of aggregating dipeptide repeat proteins (DPRs) via repeat associated non-AUG (RAN) translation. Here, we show the similar intronic GGCCTG HREs that causes spinocerebellar ataxia type 36 (SCA36) is also translated into DPRs, including poly(GP) and poly(PR). We demonstrate that poly(GP) is more abundant in SCA36 compared to c9ALS/FTD patient tissue due to canonical AUG-mediated translation from intron-retained GGCCTG repeat RNAs. However, the frequency of the antisense RAN translation product poly(PR) is comparable between c9ALS/FTD and SCA36 patient samples. Interestingly, in SCA36 patient tissue, poly(GP) exists as a soluble species, and no TDP-43 pathology is present. We show that aggregate-prone chimeric DPR (cDPR) species underlie the divergent DPR pathology between c9ALS/FTD and SCA36. These findings reveal key differences in translation, solubility, and protein aggregation of DPRs between c9ALS/FTD and SCA36.

Insights into the pathogenic mechanisms of Chromosome 9 open reading frame 72 (C9orf72) repeat expansions.

The identification of a hexanucleotide repeat expansion in a non-coding region of C9orf72 as a major cause of both frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) drastically changed the field of research on both of these conditions. Yet, despite the vast amount of work aimed at elucidating the molecular mechanisms underlying the role of this repeat in disease, the exact pathomechanisms are still unclear. A reduction in the expression of the C9orf72 gene is observed in patients, but a gain-of-function model is now preferred. The hexanucleotide repeat expansion forms RNA foci in the central nervous system (CNS) of repeat-positive FTD and ALS patients, and these foci are believed to sequester RNA-binding proteins (RBPs) and impair their function in RNA processing. At the same time, the repeat undergoes repeat-associated non-ATG translation to produce dipeptide repeat proteins that also form inclusions in the patient CNS. Studies from cells and flies suggest that these proteins may also be an important factor in the disease. Finally, the hexanucleotide repeat also induces the mislocalization and aggregation of TAR DNA-binding protein 43 (TDP-43) through an as yet unknown mechanism. This review covers the different potential pathogenic factors that have been put forth for C9orf72-repeat-associated FTD and ALS (C9-FTD/ALS), while highlighting some remaining questions. A repeat expansion in C9orf72 is a common cause of both frontal temporal dementia and amyotrophic lateral sclerosis. Although there is a decrease in C9orf72 expression in patients, this repeat is believed to induce disease primarily through an unknown gain-of-function mechanism involving the RNA, repeat-associated non-AUG translation, or both. This review summarizes and discusses current knowledge on C9orf72 repeat-associated pathophysiology. This article is part of the Frontotemporal Dementia special issue.

Nemo-like kinase is a novel regulator of spinal and bulbar muscular atrophy.

Spinal and bulbar muscular atrophy (SBMA) is a progressive neuromuscular disease caused by polyglutamine expansion in the androgen receptor (AR) protein. Despite extensive research, the exact pathogenic mechanisms underlying SBMA remain elusive. In this study, we present evidence that Nemo-like kinase (NLK) promotes disease pathogenesis across multiple SBMA model systems. Most remarkably, loss of one copy of Nlk rescues SBMA phenotypes in mice, including extending lifespan. We also investigated the molecular mechanisms by which NLK exerts its effects in SBMA. Specifically, we have found that NLK can phosphorylate the mutant polyglutamine-expanded AR, enhance its aggregation, and promote AR-dependent gene transcription by regulating AR-cofactor interactions. Furthermore, NLK modulates the toxicity of a mutant AR fragment via a mechanism that is independent of AR-mediated gene transcription. Our findings uncover a crucial role for NLK in controlling SBMA toxicity and reveal a novel avenue for therapy development in SBMA.

Aggregation formation in the polyglutamine diseases: protection at a cost?

Mutant protein aggregation is a hallmark of many neurodegenerative diseases, including the polyglutamine disorders. Although the correlation between aggregation formation and disease pathology originally suggested that the visible inclusions seen in patient tissue might directly contribute to pathology, additional studies failed to confirm this hypothesis. Current opinion in the field of polyglutamine disease research now favors a model in which large inclusions are cytoprotective and smaller oligomers or misfolded monomers underlie pathogenesis. Nonetheless, therapies aimed at reducing or preventing aggregation show promise. This review outlines the debate about the role of aggregation in the polyglutamine diseases as it has unfolded in the literature and concludes with a brief discussion on the manipulation of aggregation formation and clearance mechanisms as a means of therapeutic intervention.

Polyglutamine disease toxicity is regulated by Nemo-like kinase in spinocerebellar ataxia type 1.

Polyglutamine diseases are dominantly inherited neurodegenerative diseases caused by an expansion of a CAG trinucleotide repeat encoding a glutamine tract in the respective disease-causing proteins. Extensive studies have been performed to unravel disease pathogenesis and to develop therapeutics. Here, we report on several lines of evidence demonstrating that Nemo-like kinase (NLK) is a key molecule modulating disease toxicity in spinocerebellar ataxia type 1 (SCA1), a disease caused by a polyglutamine expansion in the protein ATAXIN1 (ATXN1). Specifically, we show that NLK, a serine/threonine kinase that interacts with ATXN1, modulates disease phenotypes of polyglutamine-expanded ATXN1 in a Drosophila model of SCA1. Importantly, the effect of NLK on SCA1 pathology is dependent upon NLK's enzymatic activity. Consistent with this, reduced Nlk expression suppresses the behavioral and neuropathological phenotypes in SCA1 knock-in mice. These data clearly indicate that either reducing NLK enzymatic activity or decreasing NLK expression levels can have beneficial effects against the toxicity induced by polyglutamine-expanded ATXN1.