Unmasking the skiptic task of TDP‐43

The mechanism by which mutations in TAR DNA‐binding protein 43 (TDP‐43) cause neurodegeneration remains incompletely understood. In this issue of The EMBO Journal, Fratta et al ( 2018 ) describe how a point mutation in the C‐terminal low complexity domain of TDP‐43 leads to the skipping of otherwise constitutively conserved exons. In vivo, this mutation triggers late‐onset progressive neuromuscular disturbances, as seen in amyotrophic lateral sclerosis (ALS), suggesting that TDP‐43 splicing gain‐of‐function contributes to ALS pathogenesis.     

Disease‐linked TDP‐43 hyperphosphorylation suppresses TDP‐43 condensation and aggregation

Post‐translational modifications (PTMs) have emerged as key modulators of protein phase separation and have been linked to protein aggregation in neurodegenerative disorders. The major aggregating protein in amyotrophic lateral sclerosis and frontotemporal dementia, the RNA‐binding protein TAR DNA‐binding protein (TDP‐43), is hyperphosphorylated in disease on several C‐terminal serine residues, a process generally believed to promote TDP‐43 aggregation. Here, we however find that Casein kinase 1δ‐mediated TDP‐43 hyperphosphorylation or C‐terminal phosphomimetic mutations reduce TDP‐43 phase separation and aggregation, and instead render TDP‐43 condensates more liquid‐like and dynamic. Multi‐scale molecular dynamics simulations reveal reduced homotypic interactions of TDP‐43 low‐complexity domains through enhanced solvation of phosphomimetic residues. Cellular experiments show that phosphomimetic substitutions do not affect nuclear import or RNA regulatory functions of TDP‐43, but suppress accumulation of TDP‐43 in membrane‐less organelles and promote its solubility in neurons. We speculate that TDP‐43 hyperphosphorylation may be a protective cellular response to counteract TDP‐43 aggregation.     

Profiling the genes affected by pathogenic TDP‐43 in astrocytes

Mutation in TAR DNA binding protein 43 (TDP‐43) is a causative factor of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Neurodegeneration may not require the presence of pathogenic TDP‐43 in all types of relevant cells. Rather, expression of pathogenic TDP‐43 in neurons or astrocytes alone is sufficient to cause cell‐autonomous or non‐cell‐autonomous neuron death in transgenic rats. How pathogenic TDP‐43 in astrocytes causes non‐cell‐autonomous neuron death, however, is not clear. Here, we examined the effect of pathogenic TDP‐43 on gene expression in astrocytes. Microarray assay revealed that pathogenic TDP‐43 in astrocytes preferentially altered expression of the genes encoding secretory proteins. Whereas neurotrophic genes were down‐regulated, neurotoxic genes were up‐regulated. Representative genes Lcn2 and chitinase‐3‐like protein 1 were markedly up‐regulated in astrocytes from primary culture and intact transgenic rats. Furthermore, synthetic chitinase‐3‐like protein 1 induced neuron death in a dose‐dependent manner. Our results suggest that TDP‐43 pathogenesis is associated with the simultaneous induction of multiple neurotoxic genes in astrocytes, which may synergistically produce adverse effects on neuronal survival and contribute to non‐cell‐autonomous neuron death.

     

TDP‐43 associates with stalled ribosomes and contributes to cell survival during cellular stress

TAR DNA‐binding protein 43 (TDP‐43) has emerged as an important contributor to amyotrophic lateral sclerosis and frontotemporal lobar degeneration. To understand the physiological roles of TDP‐43 in the complex translational regulation mechanisms, we exposed cultured cells to oxidative stress induced by sodium arsenite (ARS) for different periods of time, leading to non‐lethal or sublethal injury. Polysome profile analysis revealed that ARS‐induced stress caused the association of TDP‐43 with stalled ribosomes via binding to mRNA, which was not found under the steady‐state condition. When the cells were exposed to short‐term/non‐lethal stress, TDP‐43 associating with ribosomes localized to stress granules (SGs); this association was transient because it was immediately dissolved by the removal of the stress. In contrast, when the cells were exposed to long‐term/sublethal stress, TDP‐43 was excluded from SGs and shifted to the heavy fractions independent of any binding to mRNA. In these severely stressed cells, biochemical alterations of TDP‐43, such as increased insolubility and disulfide bond formation, were irreversible. TDP‐43 was finally phosphorylated via the ARS‐induced c‐jun N‐terminal kinase pathway. In TDP‐43‐silenced cells, stalled mRNA and poly (A)⁺ RNA stability was disturbed and cytotoxicity increased under sublethal stress. Thus, TDP‐43 associates with stalled ribosomes and contributes to cell survival during cellular stress.     

O‐GlcNAcylation of TDP‐43 suppresses proteinopathies and promotes TDP‐43’s mRNA splicing activity

Pathological TDP‐43 aggregation is characteristic of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD‐TDP); however, how TDP‐43 aggregation and function are regulated remain poorly understood. Here, we show that O‐GlcNAc transferase OGT‐mediated O‐GlcNAcylation of TDP‐43 suppresses ALS‐associated proteinopathies and promotes TDP‐43''s splicing function. Biochemical and cell‐based assays indicate that OGT''s catalytic activity suppresses TDP‐43 aggregation and hyperphosphorylation, whereas abolishment of TDP‐43 O‐GlcNAcylation impairs its RNA splicing activity. We further show that TDP‐43 mutations in the O‐GlcNAcylation sites improve locomotion defects of larvae and adult flies and extend adult life spans, following TDP‐43 overexpression in Drosophila motor neurons. We finally demonstrate that O‐GlcNAcylation of TDP‐43 promotes proper splicing of many mRNAs, including STMN2, which is required for normal axonal outgrowth and regeneration. Our findings suggest that O‐GlcNAcylation might be a target for the treatment of TDP‐43‐linked pathogenesis.     

Parkin reverses TDP‐43‐induced cell death and failure of amino acid homeostasis

The E3 ubiquitin ligase Parkin plays a central role in the pathogenesis of many neurodegenerative diseases. Parkin promotes specific ubiquitination and affects the localization of transactivation response DNA‐binding protein 43 (TDP‐43), which controls the translation of thousands of mRNAs. Here we tested the effects of lentiviral Parkin and TDP‐43 expression on amino acid metabolism in the rat motor cortex using high frequency ¹³C NMR spectroscopy. TDP‐43 expression increased glutamate levels, decreased the levels of other amino acids, including glutamine, aspartate, leucine and isoleucine, and impaired mitochondrial tricarboxylic acid cycle. TDP‐43 induced lactate accumulation and altered the balance between excitatory (glutamate) and inhibitory (GABA) neurotransmitters. Parkin restored amino acid levels, neurotransmitter balance and tricarboxylic acid cycle metabolism, rescuing neurons from TDP‐43‐induced apoptotic death. Furthermore, TDP‐43 expression led to an increase in 4E‐BP levels, perhaps altering translational control and deregulating amino acid synthesis; while Parkin reversed the effects of TDP‐43 on the 4E‐BP signaling pathway. Taken together, these data suggest that Parkin may affect TDP‐43 localization and mitigate its effects on 4E‐BP signaling and loss of amino acid homeostasis.

     

TDP‐43 loss of function increases TFEB activity and blocks autophagosome–lysosome fusion

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that is characterized by selective loss of motor neurons in brain and spinal cord. TAR DNA‐binding protein 43 (TDP‐43) was identified as a major component of disease pathogenesis in ALS, frontotemporal lobar degeneration (FTLD), and other neurodegenerative disease. Despite the fact that TDP‐43 is a multi‐functional protein involved in RNA processing and a large number of TDP‐43 RNA targets have been discovered, the initial toxic effect and the pathogenic mechanism underlying TDP‐43‐linked neurodegeneration remain elusive. In this study, we found that loss of TDP‐43 strongly induced a nuclear translocation of TFEB, the master regulator of lysosomal biogenesis and autophagy, through targeting the mTORC1 key component raptor. This regulation in turn enhanced global gene expressions in the autophagy–lysosome pathway (ALP) and increased autophagosomal and lysosomal biogenesis. However, loss of TDP‐43 also impaired the fusion of autophagosomes with lysosomes through dynactin 1 downregulation, leading to accumulation of immature autophagic vesicles and overwhelmed ALP function. Importantly, inhibition of mTORC1 signaling by rapamycin treatment aggravated the neurodegenerative phenotype in a TDP‐43‐depleted Drosophila model, whereas activation of mTORC1 signaling by PA treatment ameliorated the neurodegenerative phenotype. Taken together, our data indicate that impaired mTORC1 signaling and influenced ALP may contribute to TDP‐43‐mediated neurodegeneration.     

The voltage‐gated calcium channel blocker lomerizine is neuroprotective in motor neurons expressing mutant SOD1, but not TDP‐43

Excitotoxicity and disruption of Ca²⁺ homeostasis have been implicated in amyotrophic lateral sclerosis (ALS) and limiting Ca²⁺ entry is protective in models of ALS caused by mutation of SOD1. Lomerizine, an antagonist of L‐ and T‐type voltage‐gated calcium channels and transient receptor potential channel 5 transient receptor potential channels, is well tolerated clinically, making it a potential therapeutic candidate. Lomerizine reduced glutamate excitotoxicity in cultured motor neurons by reducing the accumulation of cytoplasmic Ca²⁺ and protected motor neurons against multiple measures of mutant SOD1 toxicity: Ca²⁺ overload, impaired mitochondrial trafficking, mitochondrial fragmentation, formation of mutant SOD1 inclusions, and loss of viability. To assess the utility of lomerizine in other forms of ALS, calcium homeostasis was evaluated in culture models of disease because of mutations in the RNA‐binding proteins transactive response DNA‐binding protein 43 (TDP‐43) and Fused in Sarcoma (FUS). Calcium did not play the same role in the toxicity of these mutant proteins as with mutant SOD1 and lomerizine failed to prevent cytoplasmic accumulation of mutant TDP‐43, a hallmark of its pathology. These experiments point to differences in the pathogenic pathways between types of ALS and show the utility of primary culture models in comparing those mechanisms and effectiveness of therapeutic strategies.

     

A single N‐terminal phosphomimic disrupts TDP‐43 polymerization,phase separation,and RNA splicing

TDP‐43 is an RNA‐binding protein active in splicing that concentrates into membraneless ribonucleoprotein granules and forms aggregates in amyotrophic lateral sclerosis (ALS) and Alzheimer's disease. Although best known for its predominantly disordered C‐terminal domain which mediates ALS inclusions, TDP‐43 has a globular N‐terminal domain (NTD). Here, we show that TDP‐43 NTD assembles into head‐to‐tail linear chains and that phosphomimetic substitution at S48 disrupts TDP‐43 polymeric assembly, discourages liquid–liquid phase separation (LLPS) in vitro, fluidizes liquid–liquid phase separated nuclear TDP‐43 reporter constructs in cells, and disrupts RNA splicing activity. Finally, we present the solution NMR structure of a head‐to‐tail NTD dimer comprised of two engineered variants that allow saturation of the native polymerization interface while disrupting higher‐order polymerization. These data provide structural detail for the established mechanistic role of the well‐folded TDP‐43 NTD in splicing and link this function to LLPS. In addition, the fusion‐tag solubilized, recombinant form of TDP‐43 full‐length protein developed here will enable future phase separation and in vitro biochemical assays on TDP‐43 function and interactions that have been hampered in the past by TDP‐43 aggregation.     

Expression of ALS‐linked TDP‐43 mutant in astrocytes causes non‐cell‐autonomous motor neuron death in rats

Mutation of Tar DNA‐binding protein 43 (TDP‐43) is linked to amyotrophic lateral sclerosis. Although astrocytes have important roles in neuron function and survival, their potential contribution to TDP‐43 pathogenesis is unclear. Here, we created novel lines of transgenic rats that express a mutant form of human TDP‐43 (M337V substitution) restricted to astrocytes. Selective expression of mutant TDP‐43 in astrocytes caused a progressive loss of motor neurons and the denervation atrophy of skeletal muscles, resulting in progressive paralysis. The spinal cord of transgenic rats also exhibited a progressive depletion of the astroglial glutamate transporters GLT‐1 and GLAST. Astrocytic expression of mutant TDP‐43 led to activation of astrocytes and microglia, with an induction of the neurotoxic factor Lcn2 in reactive astrocytes that was independent of TDP‐43 expression. These results indicate that mutant TDP‐43 in astrocytes is sufficient to cause non‐cell‐autonomous death of motor neurons. This motor neuron death likely involves deficiency in neuroprotective genes and induction of neurotoxic genes in astrocytes.     

XBP1 depletion precedes ubiquitin aggregation and Golgi fragmentation in TDP‐43 transgenic rats

Protein inclusion is a prominent feature of neurodegenerative diseases including frontotemporal lobar degeneration (FTLD) that is characterized by the presence of ubiquitinated TDP‐43 inclusion. Presence of protein inclusions indicates an interruption to protein degradation machinery or the overload of misfolded proteins. In response to the increase in misfolded proteins, cells usually initiate a mechanism called unfolded protein response (UPR) to reduce misfolded proteins in the lumen of endoplasmic reticules. Here, we examined the effects of mutant TDP‐43 on the UPR in transgenic rats that express mutant human TDP‐43 restrictedly in the neurons of the forebrain. Over‐expression of mutant TDP‐43 in rats caused prominent aggregation of ubiquitin and remarkable fragmentation of Golgi complexes prior to neuronal loss. While ubiquitin aggregates and Golgi fragments were accumulating, neurons expressing mutant TDP‐43 failed to up‐regulate chaperones residing in the endoplasmic reticules and failed to initiate the UPR. Prior to ubiquitin aggregation and Golgi fragmentation, neurons were depleted of X‐box‐binding protein 1 (XBP1), a key player of UPR machinery. Although it remains to determine how mutation of TDP‐43 leads to the failure of the UPR, our data demonstrate that failure of the UPR is implicated in TDP‐43 pathogenesis.     

Mice with endogenous TDP‐43 mutations exhibit gain of splicing function and characteristics of amyotrophic lateral sclerosis

TDP‐43 (encoded by the gene TARDBP) is an RNA binding protein central to the pathogenesis of amyotrophic lateral sclerosis (ALS). However, how TARDBP mutations trigger pathogenesis remains unknown. Here, we use novel mouse mutants carrying point mutations in endogenous Tardbp to dissect TDP‐43 function at physiological levels both in vitro and in vivo. Interestingly, we find that mutations within the C‐terminal domain of TDP‐43 lead to a gain of splicing function. Using two different strains, we are able to separate TDP‐43 loss‐ and gain‐of‐function effects. TDP‐43 gain‐of‐function effects in these mice reveal a novel category of splicing events controlled by TDP‐43, referred to as “skiptic” exons, in which skipping of constitutive exons causes changes in gene expression. In vivo, this gain‐of‐function mutation in endogenous Tardbp causes an adult‐onset neuromuscular phenotype accompanied by motor neuron loss and neurodegenerative changes. Furthermore, we have validated the splicing gain‐of‐function and skiptic exons in ALS patient‐derived cells. Our findings provide a novel pathogenic mechanism and highlight how TDP‐43 gain of function and loss of function affect RNA processing differently, suggesting they may act at different disease stages.     

Substantial differences in bias between single‐digest and double‐digest RAD‐seq libraries: A case study

The trade‐offs of using single‐digest vs. double‐digest restriction site‐associated DNA sequencing (RAD‐seq) protocols have been widely discussed. However, no direct empirical comparisons of the two methods have been conducted. Here, we sampled a single population of Gulf pipefish (Syngnathus scovelli) and genotyped 444 individuals using RAD‐seq. Sixty individuals were subjected to single‐digest RAD‐seq (sdRAD‐seq), and the remaining 384 individuals were genotyped using a double‐digest RAD‐seq (ddRAD‐seq) protocol. We analysed the resulting Illumina sequencing data and compared the two genotyping methods when reads were analysed either together or separately. Coverage statistics, observed heterozygosity, and allele frequencies differed significantly between the two protocols, as did the results of selection components analysis. We also performed an in silico digestion of the Gulf pipefish genome and modelled five major sources of bias: PCR duplicates, polymorphic restriction sites, shearing bias, asymmetric sampling (i.e., genotyping fewer individuals with sdRAD‐seq than with ddRAD‐seq) and higher major allele frequencies. This combination of approaches allowed us to determine that polymorphic restriction sites, an asymmetric sampling scheme, mean allele frequencies and to some extent PCR duplicates all contribute to different estimates of allele frequencies between samples genotyped using sdRAD‐seq versus ddRAD‐seq. Our finding that sdRAD‐seq and ddRAD‐seq can result in different allele frequencies has implications for comparisons across studies and techniques that endeavour to identify genomewide signatures of evolutionary processes in natural populations.