TAR DNA-binding Protein 43 Aggregation Research Articles

Liquid-Liquid Phase Separation Enhances TDP-43 LCD Aggregation but Delays Seeded Aggregation.

Aggregates of TAR DNA-binding protein (TDP-43) are a hallmark of several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). Although TDP-43 aggregates are an undisputed pathological species at the end stage of these diseases, the molecular changes underlying the initiation of aggregation are not fully understood. The aim of this study was to investigate how phase separation affects self-aggregation and aggregation seeded by pre-formed aggregates of either the low-complexity domain (LCD) or its short aggregation-promoting regions (APRs). By systematically varying the physicochemical conditions, we observed that liquid–liquid phase separation (LLPS) promotes spontaneous aggregation. However, we noticed less efficient seeded aggregation in phase separating conditions. By analyzing a broad range of conditions using the Hofmeister series of buffers, we confirmed that stabilizing hydrophobic interactions prevail over destabilizing electrostatic forces. RNA affected the cooperativity between LLPS and aggregation in a “reentrant” fashion, having the strongest positive effect at intermediate concentrations. Altogether, we conclude that conditions which favor LLPS enhance the subsequent aggregation of the TDP-43 LCD with complex dependence, but also negatively affect seeding kinetics.

Triad of TDP43 control in neurodegeneration: autoregulation, localization and aggregation.

Cytoplasmic aggregation of TAR DNA-binding protein 43 (TDP43; also known as TARDBP or TDP-43) is a key pathological feature of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). TDP43 typically resides in the nucleus but can shuttle between the nucleus and the cytoplasm to exert its multiple functions, which include regulation of the splicing, trafficking and stabilization of RNA. Cytoplasmic mislocalization and nuclear loss of TDP43 have both been associated with ALS and FTD, suggesting that calibrated levels and correct localization of TDP43 - achieved through an autoregulatory loop and tightly controlled nucleocytoplasmic transport - safeguard its normal function. Furthermore, TDP43 can undergo phase transitions, including its dispersion into liquid droplets and its accumulation into irreversible cytoplasmic aggregates. Thus, autoregulation, nucleocytoplasmic transport and phase transition are all part of an intrinsic control system regulating the physiological levels and localization of TDP43, and together are essential for the cellular homeostasis that is affected in neurodegenerative disease.

Monoclonal full-length antibody against TAR DNA binding protein 43 reduces related proteinopathy in neurons.

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), 2 incurable neurodegenerative disorders, share the same pathological hallmark named TDP43 (TAR DNA binding protein 43) proteinopathy. This event is characterized by a consistent cytoplasmic mislocalization and aggregation of the protein TDP43, which loses its physiological properties, leading neurons to death. Antibody-based approaches are now emerging interventions in the field of neurodegenerative disorders. Here, we tested the target specificity, in vivo distribution, and therapeutic efficacy of a monoclonal full-length antibody, named E6, in TDP43-related conditions. We observed that the antibody recognizes specifically the cytoplasmic fraction of TDP43. We demonstrated its ability in targeting large neurons in the spinal cord of mice and in reducing TDP43 mislocalization and NF-κB activation. We also recognized the proteasome as well as the lysosome machineries as possible mechanisms used by the antibody to reduce TDP43 proteinopathy. To our knowledge, this is the first report showing the therapeutic efficacy and feasibility of a full-length antibody against TDP43 in reducing TDP43 proteinopathy in spinal neurons of an ALS/FTLD mouse model.

Stathmin-2: adding another piece to the puzzle of TDP-43 proteinopathies and neurodegeneration.

Cytoplasmic aggregated proteins are a common neuropathological feature of neurodegenerative diseases. Cytoplasmic mislocalization and aggregation of TAR-DNA binding protein 43 (TDP-43) is found in the majority of patients with amyotrophic lateral sclerosis (ALS) and in approximately 50% of patients dying of frontotemporal lobar degeneration (FTLD). In this issue of the JCI, Prudencio, Humphrey, Pickles, and colleagues investigated the relationship of TDP-43 pathology with the loss of stathmin-2 (STMN2), an essential protein for axonal growth and maintenance. Comparing genetic, cellular, and neuropathological data from patients with TDP-43 proteinopathies (ALS, ALS-frontotemporal dementia [ALS-FTD], and FTLD-TDP-43 [FTLD-TDP]) with data from patients with non-TDP-related neurodegenerations, they demonstrate a direct relationship between TDP-43 pathology and STMN2 reduction. Loss of the normal transcription suppressor function of TDP-43 allowed transcription of an early termination cryptic axon, resulting in truncated, nonfunctional mRNA. The authors suggest that measurement of truncated STMN2 mRNA could be a biomarker for discerning TDP proteinopathies from other pathologies.

Aggresome formation and liquid\u2013liquid phase separation independently induce cytoplasmic aggregation of TAR DNA-binding protein 43

Cytoplasmic inclusion of TAR DNA-binding protein 43 (TDP-43) is a pathological hallmark of amyotrophic lateral sclerosis (ALS) and a subtype of frontotemporal lobar degeneration (FTLD). Recent studies have suggested that the formation of cytoplasmic TDP-43 aggregates is dependent on a liquid–liquid phase separation (LLPS) mechanism. However, it is unclear whether TDP-43 pathology is induced through a single intracellular mechanism such as LLPS. To identify intracellular mechanisms responsible for TDP-43 aggregation, we established a TDP-43 aggregation screening system using a cultured neuronal cell line stably expressing EGFP-fused TDP-43 and a mammalian expression library of the inherited ALS/FTLD causative genes, and performed a screening. We found that microtubule-related proteins (MRPs) and RNA-binding proteins (RBPs) co-aggregated with TDP-43. MRPs and RBPs sequestered TDP-43 into the cytoplasmic aggregates through distinct mechanisms, such as microtubules and LLPS, respectively. The MRPs-induced TDP-43 aggregates were co-localized with aggresomal markers and dependent on histone deacetylase 6 (HDAC6), suggesting that aggresome formation induced the co-aggregation. However, the MRPs-induced aggregates were not affected by 1,6-hexanediol, an LLPS inhibitor. On the other hand, the RBPs-induced TDP-43 aggregates were sensitive to 1,6-hexanediol, but not dependent on microtubules or HDAC6. In sporadic ALS patients, approximately half of skein-like TDP-43 inclusions were co-localized with HDAC6, but round and granular type inclusion were not. Moreover, HDAC6-positive and HDAC6-negative inclusions were found in the same ALS patient, suggesting that the two distinct pathways are both involved in TDP-43 pathology. Our findings suggest that at least two distinct pathways (i.e., aggresome formation and LLPS) are involved in inducing the TDP-43 pathologies.

Stress Granule Assembly Can Facilitate but Is Not Required for TDP-43 Cytoplasmic Aggregation.

Stress granules (SGs) are hypothesized to facilitate TAR DNA-binding protein 43 (TDP-43) cytoplasmic mislocalization and aggregation, which may underly amyotrophic lateral sclerosis pathology. However, much data for this hypothesis is indirect. Additionally, whether P-bodies (PBs; related mRNA-protein granules) affect TDP-43 phenotypes is unclear. Here, we determine that induction of TDP-43 expression in yeast results in the accumulation of SG-like foci that in >90% of cases become the sites where TDP-43 cytoplasmic foci first appear. Later, TDP-43 foci associate less with SGs and more with PBs, though independent TDP-43 foci also accumulate. However, depleting or over-expressing yeast SG and PB proteins reveals no consistent trend between SG or PB assembly and TDP-43 foci formation, toxicity or protein abundance. In human cells, immunostaining endogenous TDP-43 with different TDP-43 antibodies reveals distinct localization and aggregation behaviors. Following acute arsenite stress, all phospho-TDP-43 foci colocalize with SGs. Finally, formation of TDP-43 cytoplasmic foci following low-dose chronic arsenite stress is impaired, but not completely blocked, in G3BP1/2ΔΔ cells. Collectively, our data suggest that SG and PB assembly may facilitate TDP-43 cytoplasmic localization and aggregation but are likely not essential for these events.

Amyloid-like aggregation and fibril core determination of TDP-43 C-terminal domain

Cytoplasmic inclusion of TAR DNA-binding protein 43 (TDP-43) is a hallmark of most ALS (amyotrophic lateral sclerosis) and FTLD (Frontotemporal dementia), yet the aggregation of TDP-43 remains unclear. In this study, we proved the existence of amyloid-like structures of C-terminal domain of TDP-43 (TDP-C) in bacterial inclusion bodies (IBs), and obtained a homogenous fibril sample by seeding from the components of aggregated TDP-C in Escherichiacoli IBs. The results from solid-state NMR spectroscopy suggest that the homogenous fibrils were seeded from a tiny amount of aggregated TDP-C compositions in IBs; the structure characteristics of the rigid fibril core are identified of β-rich structures, and show subtle relativity with the hydrophobicity of residues. Our study here provides a further understanding of TDP-43 protein aggregation and fibrillation.

Hypusination of Eif5a regulates cytoplasmic TDP-43 aggregation and accumulation in a stress-induced cellular model

TAR DNA-binding protein 43 (TDP-43) is a nuclear RNA/DNA binding protein involved in mRNA metabolism. Aberrant mislocalization to the cytoplasm and formation of phosphorylated/aggregated TDP-43 inclusions remains the hallmark pathology in a spectrum of neurodegenerative diseases, including frontotemporal disorders and Alzheimer's disease. Eukaryotic Translation Initiation Factor 5A undergoes a unique post-translation modification of lysine to hypusine (K50), which determines eIF5A binding partners. We used a sodium arsenite-induced cellular stress model to investigate the role of hypusinated eIF5A (eIF5AHypK50) in governing TDP-43 cytoplasmic mislocalization and accumulation in stress granule. Our proteomics and functional data provide evidence that eIF5A interacts with TDP-43 in a hypusine-dependent manner. Additionally, we showed that following stress TDP-43 interactions with eIF5AHypK50 were induced both in the cytoplasm and stress granules. Pharmacological reduction of hypusination or mutations of lysine residues within the hypusine loop decreased phosphorylated and insoluble TDP-43 levels. The proteomic and biochemical analysis also identified nuclear pore complex importins KPNA1/2, KPNB1, and RanGTP as interacting partners of eIF5AHypK50. These findings are the first to provide a novel pathway and potential therapeutic targets that require further investigation in models of TDP-43 proteinopathies.

TAR DNA-Binding Protein 43 is Cleaved by the Protease 3C of Enterovirus A71.

Enterovirus A71 (EV-A71) is one of the etiological pathogens leading to hand, foot, and mouth disease (HFMD), which can cause severe neurological complications. The neuropathogenesis of EV-A71 infection is not well understood. The mislocalization and aggregation of TAR DNA-binding protein 43 (TDP-43) is the pathological hallmark of amyotrophic lateral sclerosis (ALS). However, whether TDP-43 was impacted by EV-A71 infection is unknown. This study demonstrated that TDP-43 was cleaved during EV-A71 infection. The cleavage of TDP-43 requires EV-A71 replication rather than the activated caspases due to viral infection. TDP-43 is cleaved by viral protease 3C between the residues 331Q and 332S, while mutated TDP-43 (Q331A) was not cleaved. In addition, mutated 3C which lacks the protease activity failed to induce TDP-43 cleavage. We also found that TDP-43 was translocated from the nucleus to the cytoplasm, and the mislocalization of TDP-43 was induced by viral protease 2A rather than 3C. Taken together, we demonstrated that TDP-43 was cleaved by viral protease and translocated to the cytoplasm during EV-A71 infection, implicating the possible involvement of TDP-43 in the pathogenesis of EV-A71infection.

Mutant TDP-43 Expression Triggers TDP-43 Pathology and Cell Autonomous Effects on Primary Astrocytes: Implications for Non-cell Autonomous Pathology in ALS.

Motor neuron degeneration in amyotrophic lateral sclerosis (ALS) caused by mutations in superoxide dismutase 1 (SOD1) is partly non-cell autonomous, involving cellular dysfunction of astrocytes. Whether non-cell autonomous effects occur in other forms of ALS, such as TAR DNA binding protein 43 (TDP-43)-related disease, remains unclear. Here, we characterised the impact of mutant TDP-43 expression on primary astrocytes derived from transgenic TDP-43A315T mice. Mutant TDP-43 astrocytes revealed evidence for TDP-43 pathology, shown by cytoplasmic TDP-43 inclusions and accumulation in insoluble cell fractions which was exacerbated by proteasomal inhibition. L-glutamate uptake, measured using an [3H]D-aspartate assay, was impaired in mutant TDP-43 astrocytes, while ATP accumulation was abnormal, suggesting mutant TDP-43 induced astrocytic dysfunction. Astrocyte activation coupled with spinal and cortical motor neuron loss in transgenic TDP-43A315T mice could imply non-cell autonomous effects of astrocytes in vivo. These data demonstrate mutant TDP-43-mediated cell autonomous effects on astrocytes that may contribute to motor neuron pathology in ALS.

Release of Motor Neuron Exosomes Containing TDP-43 and mTNF-α Near the Neuromuscular Junction Induces Skeletal Muscle Atrophy and Reduced Contractility in a 3D Human Model of ALS

Amyotrophic lateral sclerosis (ALS) is a spectrum of fatal neurodegenerative diseases altering upper and lower motor neuron (MN) and skeletal muscle (SkM) function. Disrupted communication between MN and SkM is believed to be driven by both inflammatory signaling and accumulation of potentially toxic intracellular protein aggregates of TDP-43 (TAR DNA binding protein 43). Here, we examine early events in ALS using a novel 3D model comparing motor unit function in healthy and ALS (OPTNE478G and TARDBPG298S) human iPSC-derived MN in co-culture with healthy iPSC-SkM. We show that in ALS-MN, OPTN inflammatory signaling through NF-κB drives accumulation of mutant TDP-43 to disrupt neuronal function. ALS-MN can transfer inflammatory mTNF-α and TDP-43 aggregates directly to SkM via axonal transport and induce peri-neuromsucular junction (NMJ) release of CD63-positive exosomes leading to cell death, atrophy, and reduced contractile force in SkM. Importantly, SkM pathogenesis could be improved by increasing ALS-MN autophagy which decreases both accumulation of TDP-43 aggregates and pathogenic exosome release. Our results provide the first mechanistic insight into how MN within the spinal cord can propagate pathologic signals to SkM at anatomically distinct sites through axonally transported exosomes and suggest that enhancing MN autophagy could serve as a viable intervention in early ALS.

Riluzole Exhibits No Therapeutic Efficacy on a Transgenic Rat model of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurological disorder clinically characterized by motor system dysfunction, with intraneuronal accumulation of the TAR DNAbinding protein 43 (TDP-43) being a pathological hallmark. Riluzole is a primarily prescribed medicine for ALS patients, while its therapeutical efficacy appears limited. TDP-43 transgenic mice are existing animal models for mechanistic/translational research into ALS. We developed a transgenic rat model of ALS expressing a mutant human TDP-43 transgene (TDP-43M337V) and evaluated the therapeutic effect of Riluzole on this model. Relative to control, rats with TDP-43M337V expression promoted by the neurofilament heavy subunit (NEF) gene or specifically in motor neurons promoted by the choline acetyltransferase (ChAT) gene showed progressive worsening of mobility and grip strength, along with loss of motor neurons, microglial activation, and intraneuronal accumulation of TDP-43 and ubiquitin aggregations in the spinal cord. Compared to vehicle control, intragastric administration of Riluzole (30 mg/kg/d) did not mitigate the behavioral deficits nor alter the neuropathologies in the transgenics. These findings indicate that transgenic rats recapitulate the basic neurological and neuropathological characteristics of human ALS, while Riluzole treatment can not halt the development of the behavioral and histopathological phenotypes in this new transgenic rodent model of ALS.

Structural heterogeneity in RNA recognition motif 2 (RRM2) of TAR DNA-binding protein 43 (TDP-43): clue to amyotrophic lateral sclerosis

Aberrant misfolding and aggregation of TAR DNA-binding protein 43 (TDP-43) and its fragments have been implicated in amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases. Within the protein, the second RNA recognition motif (RRM2) has recently been demonstrated to be a major contributor towards aggregation and the resultant toxicity. However, the physicochemical mechanism of its misfolding from the functional folded state is poorly understood. In the present work, we have used a cumulative ∼2µs of molecular dynamics (MD) simulation to study the structural and thermodynamic characteristics of different unfolded intermediates of RRM2 domain of TDP-43. In 6 M GdmCl at 400 K, at RMSD around 1.5 nm, part of the secondary structure i.e. helix still does not melt without significant change in solvent accessibility and intra-protein hydrogen bonds. However, hydrophobic contacts disrupt significantly suggesting that unfolding proceeds through disruption of hydrophobic core of the protein.The temperature dependent free-energy landscapes (FELs) reveal the presence of multiple metastable intermediate states stabilized by hydrophobic (ILV) contacts and hydrogen bonds. These conformational states have all the native helices intact with significant loss of β-sheets. These partially unfolded states are quite compact and characterized by the exposure of aggregation-prone β-sheets, suggesting the increased aggregation propensity of the partially unfolded states. Our results will thus serve to uncover the structural properties of partially unfolded intermediate states that drive TDP-43 misfolding and aggregation. Elucidating the structural characterization of the misfolding and aggregation prone intermediate states of TDP-43 are important to understand its role in ALS and other neurodegenerative diseases.Communicated by Ramaswamy H. Sarma

Multiple distinct pathways lead to hyperubiquitylated insoluble TDP-43 protein independent of its translocation into stress granules

Multiple distinct pathways lead to hyperubiquitylated insoluble TDP-43 protein independent of its translocation into stress granules

The Gypsy Endogenous Retrovirus Drives Non-Cell-Autonomous Propagation in a Drosophila TDP-43 Model of Neurodegeneration.

A hallmark of neurodegenerative disease is focal onset of pathological protein aggregation, followed by progressive spread of pathology to connected brain regions. In amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), pathology is often associated with aggregation of TAR DNA-binding protein 43 (TDP-43). Although aggregated TDP-43 protein moves between cells, it is not clear whether and how this movement propagates the degeneration. Here, we have established a Drosophila model of human TDP-43 in which we initiated toxic expression of human TDP-43 focally within small groups of glial cells. We found that this focal onset kills adjacent neurons. Surprisingly, we show that this spreading death is caused by an endogenous retrovirus within the glia, which leads to DNA damage and death in adjacent neurons. These findings suggest a possible mechanism by which human retroviruses such as HERV-K might contribute to TDP-43-mediated propagation of neurodegeneration.

Characterizing TDP-43307-319 Oligomeric Assembly: Mechanistic and Structural Implications Involved in the Etiology of Amyotrophic Lateral Sclerosis.

Aggregation of TAR DNA-binding protein of 43 kDa (TDP-43) is a salient feature of amyotrophic lateral sclerosis (ALS), a debilitating neurodegenerative disorder affecting over 200 000 people worldwide. The protein undergoes both functional and pathogenic aggregation; the latter is irreversible and hypothesized to produce soluble oligomers that are toxic to neurons in addition to inclusions made of stable fibrous deposits. Despite progress made toward identifying disease-related proteins, the underlying pathogenic mechanism associated with these toxic oligomers remains elusive. Utilizing a multimodal approach that combines several measurement techniques (circular dichroism (CD), thioflavin T spectroscopy (ThT), Fourier transform infrared spectroscopy (FTIR)) and high spatial resolution imaging tools (electron microscopy (EM) and atomic force microscopy (AFM)), with soft ion mobility mass spectrometry (IM-MS) and atomistic molecular dynamics (MD) simulations, we explore the oligomerization mechanisms, structures, and assembly pathways of TDP-43307-319. This fragment is both amyloidogenic and toxic and is within the glycine-rich C-terminal domain essential for both toxicity and aggregation of the full-length protein. In addition to the wild-type peptide, two ALS-related mutants (A315T and A315E) and a non-axon-toxic mutant (G314V) were investigated to determine how mutations affect the oligomerization of TDP-43307-319 and structures of toxic oligomers. The results of our study provide new insights into how ALS-related mutants, A315T and A315E, accelerate or alter the pathogenic mechanism and highlight the role of an internal glycine, G314, in maintaining efficient packing known to be critical for functional oligomer assembly. More importantly, our data demonstrate that G314 plays a vital role in TDP-43 assembly and prevents cytotoxicity via its unique aversion to oligomers larger than trimer. Our observation is consistent with previous studies showing that G314V mutation of the full-length TDP-43 induced remediation of both axonotoxicity and neuronal apoptosis. Our findings reveal a distinct aggregation mechanism for each peptide and elucidate oligomeric species and possible structures that may be involved in the pathology of ALS.

Endoplasmic Reticulum Stress Signalling Induces Casein Kinase 1-Dependent Formation of Cytosolic TDP-43 Inclusions in Motor Neuron-Like Cells

Motor neuron disease (MND) is a progressive neurodegenerative disease with no effective treatment. One of the principal pathological hallmarks is the deposition of TAR DNA binding protein 43 (TDP-43) in cytoplasmic inclusions. TDP-43 aggregation occurs in both familial and sporadic MND; however, the mechanism of endogenous TDP-43 aggregation in disease is incompletely understood. This study focused on the induction of cytoplasmic accumulation of endogenous TDP-43 in the motor neuronal cell line NSC-34. The endoplasmic reticulum (ER) stressor tunicamycin induced casein kinase 1 (CK1)-dependent cytoplasmic accumulation of endogenous TDP-43 in differentiated NSC-34 cells, as seen by immunocytochemistry. Immunoblotting showed that induction of ER stress had no effect on abundance of TDP-43 or phosphorylated TDP-43 in the NP-40/RIPA soluble fraction. However, there were significant increases in abundance of TDP-43 and phosphorylated TDP-43 in the NP-40/RIPA-insoluble, urea-soluble fraction, including high molecular weight species. In all cases, these increases were lowered by CK1 inhibition. Thus ER stress signalling, as induced by tunicamycin, causes CK1-dependent phosphorylation of TDP-43 and its consequent cytosolic accumulation.

Detection of TAR DNA-binding protein 43 (TDP-43) oligomers as initial intermediate species during aggregate formation

Aggregates of the RNA-binding protein TDP-43 (TAR DNA-binding protein) are a hallmark of the overlapping neurodegenerative disorders amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. The process of TDP-43 aggregation remains poorly understood, and whether it includes formation of intermediate complexes is unknown. Here, we analyzed aggregates derived from purified TDP-43 under semidenaturing conditions, identifying distinct oligomeric complexes at the initial time points before the formation of large aggregates. We found that this early oligomerization stage is primarily driven by TDP-43's RNA-binding region. Specific binding to GU-rich RNA strongly inhibited both TDP-43 oligomerization and aggregation, suggesting that RNA interactions are critical for maintaining TDP-43 solubility. Moreover, we analyzed TDP-43 liquid-liquid phase separation and detected similar detergent-resistant oligomers upon maturation of liquid droplets into solid-like fibrils. These results strongly suggest that the oligomers form during the early steps of TDP-43 misfolding. Importantly, the ALS-linked TDP-43 mutations A315T and M337V significantly accelerate aggregation, rapidly decreasing the monomeric population and shortening the oligomeric phase. We also show that aggregates generated from purified TDP-43 seed intracellular aggregation detected by established TDP-43 pathology markers. Remarkably, cytoplasmic aggregate seeding was detected earlier for the A315T and M337V variants and was 50% more widespread than for WT TDP-43 aggregates. We provide evidence for an initial step of TDP-43 self-assembly into intermediate oligomeric complexes, whereby these complexes may provide a scaffold for aggregation. This process is altered by ALS-linked mutations, underscoring the role of perturbations in TDP-43 homeostasis in protein aggregation and ALS-FTD pathogenesis.

Phosphorylated and aggregated TDP-43 with seeding properties are induced upon mutant Huntingtin (mHtt) polyglutamine expression in human cellular models.

The Tar DNA-Binding Protein 43 (TDP-43) and its phosphorylated isoform (pTDP-43) are the major components associated with ubiquitin positive/Tau-negative inclusions found in neurons and glial cells of patients suffering of amyotrophic lateral sclerosis (ALS) or frontotemporal lobar degeneration-TDP-43 (FTLD-TDP). Many studies have revealed that TDP-43 is also in the protein inclusions associated with neurodegenerative conditions other than ALS and FTLD-TDP, thus suggesting that this protein may be involved in the pathogenesis of a variety of neurological disorders. In brains of Huntington-affected patients, pTDP-43 aggregates were shown to co-localize with mutant Huntingtin (mHtt) inclusions. Here, we show that expression of mHtt carrying 80-97 polyglutamines repeats in human cell cultures induces the aggregation and the phosphorylation of endogenous TDP-43, whereas non-pathological Htt with 25 polyglutamines repeats has no effect. Mutant Htt aggregation precedes accumulation of pTDP-43 and pTDP-43 co-localizes with mHtt inclusions reminding what it was previously described in brains of Huntington-affected patients. Detergent-insoluble fractions from cells expressing mHtt and containing mHtt-pTDP-43 co-aggregates can function as seeds for further TDP-43 aggregation in human cell culture. The human cellular prion protein PrPC was previously identified as a negative modulator of mHtt aggregation; here, we show that PrPC-mediated reduction of mHtt aggregation is tightly correlated with a decrease of TDP-43 aggregation and phosphorylation, thus confirming the close relationships between TDP-43 and mHtt.

Aggregation of the nucleic acid–binding protein TDP-43 occurs via distinct routes that are coordinated with stress granule formation

TAR DNA-binding protein 43 (TDP-43) is a nucleic acid-binding protein, and its aggregation represents the defining pathology in amyotrophic lateral sclerosis (ALS) and related proteinopathies. Recent studies implicate cytoplasmic stress granules (SGs) as hubs that may facilitate TDP-43 aggregation. Here, using cellular fractionation, biochemical analyses, and histological assays, we show that TDP-43 targeted to the cytoplasm has multiple fates. Whereas a TDP-43 subpopulation is indeed recruited to SGs, mature aggregated TDP-43, produced with aggregate-prone TDP-43 variants or exposure to oxidative stress, generates distinct TDP-43 inclusions that are surprisingly devoid of SGs. Consistent with this observation, we found that SG components are predominantly excluded from TDP-43 pathology in motor neurons from individuals with ALS. We generated de novo SGs by expressing the fragile X protein (FMRP) and found that rather than directly engaging TDP-43 aggregates, SGs can sequester the proteostasis factor histone deacetylase 6 (HDAC6) and thereby impede TDP-43 clearance from cells. These findings indicate that SGs form distinct cytoplasmic structures that can indirectly enhance TDP-43 aggregation. Therapeutic approaches that inhibit SG formation may therefore be effective at suppressing TDP-43-mediated toxicity in patients with ALS and related TDP-43 proteinopathies.