Requirements for stress granule recruitment of fused in sarcoma (FUS) and TAR DNA-binding protein of 43 kDa (TDP-43)

Cytoplasmic inclusions containing TAR DNA-binding protein of 43 kDa (TDP-43) or Fused in sarcoma (FUS) are a hallmark of amyotrophic lateral sclerosis (ALS) and several subtypes of frontotemporal lobar degeneration (FTLD). FUS-positive inclusions in FTLD and ALS patients are consistently co-labeled with stress granule (SG) marker proteins. Whether TDP-43 inclusions contain SG markers is currently still debated. We determined the requirements for SG recruitment of FUS and TDP-43 and found that cytoplasmic mislocalization is a common prerequisite for SG recruitment of FUS and TDP-43. For FUS, the arginine-glycine-glycine zinc finger domain, which is the protein's main RNA binding domain, is most important for SG recruitment, whereas the glycine-rich domain and RNA recognition motif (RRM) domain have a minor contribution and the glutamine-rich domain is dispensable. For TDP-43, both the RRM1 and the C-terminal glycine-rich domain are required for SG localization. ALS-associated point mutations located in the glycine-rich domain of TDP-43 do not affect SG recruitment. Interestingly, a 25-kDa C-terminal fragment of TDP-43, which is enriched in FTLD/ALS cortical inclusions but not spinal cord inclusions, fails to be recruited into SG. Consistently, inclusions in the cortex of FTLD patients, which are enriched for C-terminal fragments, are not co-labeled with the SG marker poly(A)-binding protein 1 (PABP-1), whereas inclusions in spinal cord, which contain full-length TDP-43, are frequently positive for this marker protein.     

Identification of neuronal RNA targets of TDP-43-containing ribonucleoprotein complexes

TAR DNA-binding protein 43 (TDP-43) is associated with a spectrum of neurodegenerative diseases. Although TDP-43 resembles heterogeneous nuclear ribonucleoproteins, its RNA targets and physiological protein partners remain unknown. Here we identify RNA targets of TDP-43 from cortical neurons by RNA immunoprecipitation followed by deep sequencing (RIP-seq). The canonical TDP-43 binding site (TG)(n) is 55.1-fold enriched, and moreover, a variant with adenine in the middle, (TG)(n)TA(TG)(m), is highly abundant among reads in our TDP-43 RIP-seq library. TDP-43 RNA targets can be divided into three different groups: those primarily binding in introns, in exons, and across both introns and exons. TDP-43 RNA targets are particularly enriched for Gene Ontology terms related to synaptic function, RNA metabolism, and neuronal development. Furthermore, TDP-43 binds to a number of RNAs encoding for proteins implicated in neurodegeneration, including TDP-43 itself, FUS/TLS, progranulin, Tau, and ataxin 1 and -2. We also identify 25 proteins that co-purify with TDP-43 from rodent brain nuclear extracts. Prominent among them are nuclear proteins involved in pre-mRNA splicing and RNA stability and transport. Also notable are two neuron-enriched proteins, methyl CpG-binding protein 2 and polypyrimidine tract-binding protein 2 (PTBP2). A PTBP2 consensus RNA binding motif is enriched in the TDP-43 RIP-seq library, suggesting that PTBP2 may co-regulate TDP-43 RNA targets. This work thus reveals the protein and RNA components of the TDP-43-containing ribonucleoprotein complexes and provides a framework for understanding how dysregulation of TDP-43 in RNA metabolism contributes to neurodegeneration.     

Targeted Depletion of TDP-43 Expression in the Spinal Cord Motor Neurons Leads to the Development of Amyotrophic Lateral Sclerosis-like Phenotypes in Mice

ALS, or amyotrophic lateral sclerosis, is a progressive and fatal motor neuron disease with no effective medicine. Importantly, the majority of the ALS cases are with TDP-43 proteinopathies characterized with TDP-43-positive, ubiquitin-positive inclusions (UBIs) in the cytosol. However, the role of the mismetabolism of TDP-43 in the pathogenesis of ALS with TDP-43 proteinopathies is unclear. Using the conditional mouse gene targeting approach, we show that mice with inactivation of the Tardbp gene in the spinal cord motor neurons (HB9:Cre-Tardbp(lx/-)) exhibit progressive and male-dominant development of ALS-related phenotypes including kyphosis, motor dysfunctions, muscle weakness/atrophy, motor neuron loss, and astrocytosis in the spinal cord. Significantly, ubiquitinated proteins accumulate in the TDP-43-depleted motor neurons of the spinal cords of HB9:Cre-Tardbp(lx/-) mice with the ALS phenotypes. This study not only establishes an important role of TDP-43 in the long term survival and functioning of the mammalian spinal cord motor neurons, but also establishes that loss of TDP-43 function could be one major cause for neurodegeneration in ALS with TDP-43 proteinopathies.     

Aberrant Assembly of RNA Recognition Motif 1 Links to Pathogenic Conversion of TAR DNA-binding Protein of 43 kDa (TDP-43)

Aggregation of TAR DNA-binding protein of 43 kDa (TDP-43) is a pathological signature of amyotrophic lateral sclerosis (ALS). Although accumulating evidence suggests the involvement of RNA recognition motifs (RRMs) in TDP-43 proteinopathy, it remains unclear how native TDP-43 is converted to pathogenic forms. To elucidate the role of homeostasis of RRM1 structure in ALS pathogenesis, conformations of RRM1 under high pressure were monitored by NMR. We first found that RRM1 was prone to aggregation and had three regions showing stable chemical shifts during misfolding. Moreover, mass spectrometric analysis of aggregated RRM1 revealed that one of the regions was located on protease-resistant β-strands containing two cysteines (Cys-173 and Cys-175), indicating that this region served as a core assembly interface in RRM1 aggregation. Although a fraction of RRM1 aggregates comprised disulfide-bonded oligomers, the substitution of cysteine(s) to serine(s) (C/S) resulted in unexpected acceleration of amyloid fibrils of RRM1 and disulfide-independent aggregate formation of full-length TDP-43. Notably, TDP-43 aggregates with RRM1-C/S required the C terminus, and replicated cytopathologies of ALS, including mislocalization, impaired RNA splicing, ubiquitination, phosphorylation, and motor neuron toxicity. Furthermore, RRM1-C/S accentuated inclusions of familial ALS-linked TDP-43 mutants in the C terminus. The relevance of RRM1-C/S-induced TDP-43 aggregates in ALS pathogenesis was verified by immunolabeling of inclusions of ALS patients and cultured cells overexpressing the RRM1-C/S TDP-43 with antibody targeting misfolding-relevant regions. Our results indicate that cysteines in RRM1 crucially govern the conformation of TDP-43, and aberrant self-assembly of RRM1 at amyloidogenic regions contributes to pathogenic conversion of TDP-43 in ALS.     

A "two-hit" hypothesis for inclusion formation by carboxyl-terminal fragments of TDP-43 protein linked to RNA depletion and impaired microtubule-dependent transport

Carboxyl-terminal fragments (CTFs) of TDP-43 aggregate to form the diagnostic signature inclusions of frontotemporal lobar degeneration and amyotrophic lateral sclerosis, but the biological significance of these CTFs and how they are generated remain enigmatic. To address these issues, we engineered mammalian cells with an inducible tobacco etch virus (TEV) protease that cleaves TDP-43 containing a TEV cleavage site. Regions of TDP-43 flanking the second RNA recognition motif (RRM2) are efficiently cleaved by TEV, whereas sites within this domain are more resistant to cleavage. CTFs containing RRM2 generated from de novo cleavage of nuclear TDP-43 are transported to the cytoplasm and efficiently cleared, indicating that cleavage alone is not sufficient to initiate CTF aggregation. However, CTFs rapidly aggregated into stable cytoplasmic inclusions following de novo cleavage when dynein-mediated microtubule transport was disrupted, RNA was depleted, or natively misfolded CTFs were introduced into these cells. Our data support a "two-hit" mechanism of CTF aggregation dependent on TDP-43 cleavage.     

Folding of the RNA Recognition Motif (RRM) Domains of the Amyotrophic Lateral Sclerosis (ALS)-linked Protein TDP-43 Reveals an Intermediate State

Pathological alteration of TDP-43 (TAR DNA-binding protein-43), a protein involved in various RNA-mediated processes, is a hallmark feature of the neurodegenerative diseases amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Fragments of TDP-43, composed of the second RNA recognition motif (RRM2) and the disordered C terminus, have been observed in cytoplasmic inclusions in sporadic amyotrophic lateral sclerosis cases, suggesting that conformational changes involving RRM2 together with the disordered C terminus play a role in aggregation and toxicity. The biophysical data collected by CD and fluorescence spectroscopies reveal a three-state equilibrium unfolding model for RRM2, with a partially folded intermediate state that is not observed in RRM1. Strikingly, a portion of RRM2 beginning at position 208, which mimics a cleavage site observed in patient tissues, increases the population of this intermediate state. Mutually stabilizing interactions between the domains in the tethered RRM1 and RRM2 construct reduce the population of the intermediate state and enhance DNA/RNA binding. Despite the high sequence homology of the two domains, a network of large hydrophobic residues in RRM2 provides a possible explanation for the increased stability of RRM2 compared with RRM1. The cluster analysis suggests that the intermediate state may play a functional role by enhancing access to the nuclear export signal contained within its sequence. The intermediate state may also serve as a molecular hazard linking productive folding and function with pathological misfolding and aggregation that may contribute to disease.     

The Legs at odd angles (Loa) Mutation in Cytoplasmic Dynein Ameliorates Mitochondrial Function in SOD1G93A Mouse Model for Motor Neuron Disease

Amyotrophic lateral sclerosis (ALS) is a debilitating and fatal late-onset neurodegenerative disease. Familial cases of ALS (FALS) constitute ∼10% of all ALS cases, and mutant superoxide dismutase 1 (SOD1) is found in 15–20% of FALS. SOD1 mutations confer a toxic gain of unknown function to the protein that specifically targets the motor neurons in the cortex and the spinal cord. We have previously shown that the autosomal dominant Legs at odd angles (Loa) mutation in cytoplasmic dynein heavy chain (Dync1h1) delays disease onset and extends the life span of transgenic mice harboring human mutant SOD1^G93A. In this study we provide evidence that despite the lack of direct interactions between mutant SOD1 and either mutant or wild-type cytoplasmic dynein, the Loa mutation confers significant reductions in the amount of mutant SOD1 protein in the mitochondrial matrix. Moreover, we show that the Loa mutation ameliorates defects in mitochondrial respiration and membrane potential observed in SOD1^G93A motor neuron mitochondria. These data suggest that the Loa mutation reduces the vulnerability of mitochondria to the toxic effects of mutant SOD1, leading to improved mitochondrial function in SOD1^G93A motor neurons.