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Repeat expansion in C9orf72 causes amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Expanded sense and antisense repeat RNA transcripts in C9orf72 are translated into five dipeptide-repeat proteins (DPRs) in an AUG-independent manner. We previously identified the heterogeneous ribonucleoprotein (hnRNP) A3 as an interactor of the sense repeat RNA that reduces its translation into DPRs. Furthermore, we found that hnRNPA3 is depleted from the nucleus and partially mislocalized to cytoplasmic poly-GA inclusions in C9orf72 patients, suggesting that poly-GA sequesters hnRNPA3 within the cytoplasm. We now demonstrate that hnRNPA3 also binds to the antisense repeat RNA. Both DPR production and deposition from sense and antisense RNA repeats are increased upon hnRNPA3 reduction. All DPRs induced DNA double strand breaks (DSB), which was further enhanced upon reduction of hnRNPA3. Poly-glycine–arginine and poly-proline-arginine increased foci formed by phosphorylated Ataxia Telangiectasia Mutated (pATM), a major sensor of DSBs, whereas poly-glycine–alanine (poly-GA) evoked a reduction of pATM foci. In dentate gyri of C9orf72 patients, lower nuclear hnRNPA3 levels were associated with increased DNA damage. Moreover, enhanced poly-GA deposition correlated with reduced pATM foci. Since cytoplasmic pATM deposits partially colocalized with poly-GA deposits, these results suggest that poly-GA, the most frequent DPR observed in C9orf72 patients, differentially causes DNA damage and that poly-GA selectively sequesters pATM in the cytoplasm inhibiting its recruitment to sites of DNA damage. Thus, mislocalization of nuclear hnRNPA3 caused by poly-GA leads to increased poly-GA production, which partially depletes pATM, and consequently enhances DSB.

The abnormal assembly of TAR DNA-binding protein 43 (TDP-43) in neuronal and glial cells characterizes nearly all cases of amyotrophic lateral sclerosis (ALS) and around half of cases of frontotemporal lobar degeneration (FTLD) 1 , 2 . A causal role for TDP-43 assembly in neurodegeneration is evidenced by dominantly inherited missense mutations in TARDBP , the gene encoding TDP-43, that promote assembly and give rise to ALS and FTLD 3 – 7 . At least four types (A–D) of FTLD with TDP-43 pathology (FTLD-TDP) are defined by distinct brain distributions of assembled TDP-43 and are associated with different clinical presentations of frontotemporal dementia 8 . We previously showed, using cryo-electron microscopy, that TDP-43 assembles into amyloid filaments in ALS and type B FTLD-TDP 9 . However, the structures of assembled TDP-43 in FTLD without ALS remained unknown. Here we report the cryo-electron microscopy structures of assembled TDP-43 from the brains of three individuals with the most common type of FTLD-TDP, type A. TDP-43 formed amyloid filaments with a new fold that was the same across individuals, indicating that this fold may characterize type A FTLD-TDP. The fold resembles a chevron badge and is unlike the double-spiral-shaped fold of ALS and type B FTLD-TDP, establishing that distinct filament folds of TDP-43 characterize different neurodegenerative conditions. The structures, in combination with mass spectrometry, led to the identification of two new post-translational modifications of assembled TDP-43, citrullination and monomethylation of R293, and indicate that they may facilitate filament formation and observed structural variation in individual filaments. The structures of TDP-43 filaments from type A FTLD-TDP will guide mechanistic studies of TDP-43 assembly, as well as the development of diagnostic and therapeutic compounds for TDP-43 proteinopathies. Cryo-electron microscopy structures and mass spectrometry analyses show that TAR DNA-binding protein 43 (TDP-43) forms amyloid filaments with a distinct fold in type A frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP) compared with TDP-43 filaments in type B FTLD-TDP and amyotrophic lateral sclerosis.

Frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) can typically be categorized into one of four distinct histopathologic patterns of TDP-43 pathology, types A to D. The strength of this histopathologic classification lies in the association between FTLD-TDP subtypes and various clinical and genetic features of disease. Seven cases of FTLD-TDP were identified here which were difficult to classify based on existing pathologic criteria. Distinct features common to these cases included TDP-43 aggregates over a wide neuroanatomic distribution comprised of granulofilamentous neuronal inclusions, abundant grains, and oligodendroglial inclusions. TDP-43 aggregates were phosphorylated and associated with loss of normal nuclear TDP-43 protein (nuclear clearance) but were negative for ubiquitin. Biochemical analysis confirmed the presence of insoluble and phosphorylated TDP-43 and also revealed a distinct pattern of TDP-43 C-terminal fragments relative to other FTLD-TDP subtypes. Finally, these cases were uniformly associated with a very rapid clinical course culminating in death within ~3 years of disease onset. We suggest that these cases may represent a unique clinicopathologic subtype of FTLD-TDP which we provisionally call “type E.” The immature appearance of TDP-43 aggregates, widespread distribution, uniform biochemical profile and rapid clinical course highlights the clinical and pathologic variability within FTLD-TDP, and raises the possibility that type E neuropathology is the sequelae of a particularly virulent strain of TDP-43 proteinopathy.

Expansion of a GGGGCC hexanucleotide repeat upstream of the C9orf72 coding region is the most common cause of familial frontotemporal lobar degeneration and amyotrophic lateral sclerosis (FTLD/ALS), but the pathomechanisms involved are unknown. As in other FTLD/ALS variants, characteristic intracellular inclusions of misfolded proteins define C9orf72 pathology, but the core proteins of the majority of inclusions are still unknown. Here, we found that most of these characteristic inclusions contain poly-(Gly-Ala) and, to a lesser extent, poly-(Gly-Pro) and poly-(Gly-Arg) dipeptide-repeat proteins presumably generated by non-ATG—initiated translation from the expanded GGGGCC repeat in three reading frames. These findings directly link the FTLD/ALS-associated genetic mutation to the predominant pathology in patients with C9orf72 hexanucleotide expansion.

We examined regional distribution patterns of phosphorylated 43-kDa TAR DNA-binding protein (pTDP-43) intraneuronal inclusions in frontotemporal lobar degeneration (FTLD). Immunohistochemistry was performed on 70 μm sections from FTLD-TDP autopsy cases ( n  = 39) presenting with behavioral variant frontotemporal dementia. Two main types of cortical pTDP-43 pathology emerged, characterized by either predominantly perikaryal pTDP-43 inclusions (cytoplasmic type, cFTLD) or long aggregates in dendrites (neuritic type, nFTLD). Cortical involvement in nFTLD was extensive and frequently reached occipital areas, whereas cases with cFTLD often involved bulbar somatomotor neurons and the spinal cord. We observed four patterns indicative of potentially sequential dissemination of pTDP-43: cases with the lowest burden of pathology (pattern I) were characterized by widespread pTDP-43 lesions in the orbital gyri, gyrus rectus, and amygdala. With increasing burden of pathology (pattern II) pTDP-43 lesions emerged in the middle frontal and anterior cingulate gyrus as well as in anteromedial temporal lobe areas, the superior and medial temporal gyri, striatum, red nucleus, thalamus, and precerebellar nuclei. More advanced cases showed a third pattern (III) with involvement of the motor cortex, bulbar somatomotor neurons, and the spinal cord anterior horn, whereas cases with the highest burden of pathology (pattern IV) were characterized by pTDP-43 lesions in the visual cortex. We interpret the four neuropathological patterns in bvFTD to be consistent with the hypothesis that pTDP-43 pathology can spread sequentially and may propagate along axonal pathways.

The abnormal aggregation of TAR DNA-binding protein 43 kDa (TDP-43) in neurons and glia is the defining pathological hallmark of the neurodegenerative disease amyotrophic lateral sclerosis (ALS) and multiple forms of frontotemporal lobar degeneration (FTLD) 1 , 2 . It is also common in other diseases, including Alzheimer’s and Parkinson’s. No disease-modifying therapies exist for these conditions and early diagnosis is not possible. The structures of pathological TDP-43 aggregates are unknown. Here we used cryo-electron microscopy to determine the structures of aggregated TDP-43 in the frontal and motor cortices of an individual who had ALS with FTLD and from the frontal cortex of a second individual with the same diagnosis. An identical amyloid-like filament structure comprising a single protofilament was found in both brain regions and individuals. The ordered filament core spans residues 282–360 in the TDP-43 low-complexity domain and adopts a previously undescribed double-spiral-shaped fold, which shows no similarity to those of TDP-43 filaments formed in vitro 3 , 4 . An abundance of glycine and neutral polar residues facilitates numerous turns and restricts β-strand length, which results in an absence of β-sheet stacking that is associated with cross-β amyloid structure. An uneven distribution of residues gives rise to structurally and chemically distinct surfaces that face external densities and suggest possible ligand-binding sites. This work enhances our understanding of the molecular pathogenesis of ALS and FTLD and informs the development of diagnostic and therapeutic agents that target aggregated TDP-43. Cryo-electron microscopy of aggregated TDP-43 from postmortem brain tissue of individuals who had ALS with FTLD reveals a filament structure with distinct features to other neuropathological protein filaments, such as those of tau and α-synuclein.

Mutations in or dys-regulation of the TDP-43 gene have been associated with TDP-43 proteinopathy, a spectrum of neurodegenerative diseases including Frontotemporal Lobar Degeneration (FTLD) and Amyotrophic Lateral Sclerosis (ALS). The underlying molecular and cellular defects, however, remain unclear. Here, we report a systematic study combining analyses of patient brain samples with cellular and animal models for TDP-43 proteinopathy. Electron microscopy (EM) analyses of patient samples revealed prominent mitochondrial impairment, including abnormal cristae and a loss of cristae; these ultrastructural changes were consistently observed in both cellular and animal models of TDP-43 proteinopathy. In these models, increased TDP-43 expression induced mitochondrial dysfunction, including decreased mitochondrial membrane potential and elevated production of reactive oxygen species (ROS). TDP-43 expression suppressed mitochondrial complex I activity and reduced mitochondrial ATP synthesis. Importantly, TDP-43 activated the mitochondrial unfolded protein response (UPRmt) in both cellular and animal models. Down-regulating mitochondrial protease LonP1 increased mitochondrial TDP-43 levels and exacerbated TDP-43-induced mitochondrial damage as well as neurodegeneration. Together, our results demonstrate that TDP-43 induced mitochondrial impairment is a critical aspect in TDP-43 proteinopathy. Our work has not only uncovered a previously unknown role of LonP1 in regulating mitochondrial TDP-43 levels, but also advanced our understanding of the pathogenic mechanisms for TDP-43 proteinopathy. Our study suggests that blocking or reversing mitochondrial damage may provide a potential therapeutic approach to these devastating diseases.

Frontotemporal lobar degeneration (FTLD) causes frontotemporal dementia (FTD), the most common form of dementia after Alzheimer’s disease, and is often also associated with motor disorders 1 . The pathological hallmarks of FTLD are neuronal inclusions of specific, abnormally assembled proteins 2 . In the majority of cases the inclusions contain amyloid filament assemblies of TAR DNA-binding protein 43 (TDP-43) or tau, with distinct filament structures characterizing different FTLD subtypes 3 , 4 . The presence of amyloid filaments and their identities and structures in the remaining approximately 10% of FTLD cases are unknown but are widely believed to be composed of the protein fused in sarcoma (FUS, also known as translocated in liposarcoma). As such, these cases are commonly referred to as FTLD–FUS. Here we used cryogenic electron microscopy (cryo-EM) to determine the structures of amyloid filaments extracted from the prefrontal and temporal cortices of four individuals with FTLD–FUS. Surprisingly, we found abundant amyloid filaments of the FUS homologue TATA-binding protein-associated factor 15 (TAF15, also known as TATA-binding protein-associated factor 2N) rather than of FUS itself. The filament fold is formed from residues 7–99 in the low-complexity domain (LCD) of TAF15 and was identical between individuals. Furthermore, we found TAF15 filaments with the same fold in the motor cortex and brainstem of two of the individuals, both showing upper and lower motor neuron pathology. The formation of TAF15 amyloid filaments with a characteristic fold in FTLD establishes TAF15 proteinopathy in neurodegenerative disease. The structure of TAF15 amyloid filaments provides a basis for the development of model systems of neurodegenerative disease, as well as for the design of diagnostic and therapeutic tools targeting TAF15 proteinopathy. Cryogenic electron microscopy structures of amyloid filaments extracted from patient brains reveal that the protein TAF15 forms filaments that characterize certain cases of frontotemporal lobar degeneration.

Frontotemporal lobar degeneration (FTLD) is the third most common neurodegenerative condition after Alzheimer’s and Parkinson’s diseases 1 . FTLD typically presents in 45 to 64 year olds with behavioural changes or progressive decline of language skills 2 . The subtype FTLD-TDP is characterized by certain clinical symptoms and pathological neuronal inclusions with TAR DNA-binding protein (TDP-43) immunoreactivity 3 . Here we extracted amyloid fibrils from brains of four patients representing four of the five FTLD-TDP subclasses, and determined their structures by cryo-electron microscopy. Unexpectedly, all amyloid fibrils examined were composed of a 135-residue carboxy-terminal fragment of transmembrane protein 106B (TMEM106B), a lysosomal membrane protein previously implicated as a genetic risk factor for FTLD-TDP 4 . In addition to TMEM106B fibrils, we detected abundant non-fibrillar aggregated TDP-43 by immunogold labelling. Our observations confirm that FTLD-TDP is associated with amyloid fibrils, and that the fibrils are formed by TMEM106B rather than TDP-43. Amyloid fibrils extracted from brains of patients with frontotemporal lobar degeneration with TAR DNA-binding-protein immunoreactivity (FTLD-TDP) are made up of transmembrane protein 106B.

The stereotypical distribution of TAR DNA-binding 43 protein (TDP-43) aggregates in frontotemporal lobar degeneration (FTLD-TDP) suggests that pathological TDP-43 spreads throughout the brain via cell-to-cell transmission and correlates with disease progression, but no in vivo experimental data support this hypothesis. We first develop a doxycycline-inducible cell line expressing GFP-tagged cytoplasmic TDP-43 protein (iGFP-NLSm) as a cell-based system to screen and identify seeding activity of human brain-derived pathological TDP-43 isolated from sporadic FTLD-TDP and familial cases with Granulin (FTLD-TDP-GRN) or C9orf72 repeat expansion mutations (FTLD-TDP-C9+). We demonstrate that intracerebral injections of biologically active pathogenic FTLD-TDP seeds into transgenic mice expressing cytoplasmic human TDP-43 (lines CamKIIa-hTDP-43 NLSm , rNLS8, and CamKIIa-208) and non-transgenic mice led to the induction of de-novo TDP-43 pathology. Moreover, TDP-43 pathology progressively spreads throughout the brain in a time-dependent manner via the neuroanatomic connectome. Our study suggests that the progression of FTLD-TDP reflects the templated cell-to-cell transneuronal spread of pathological TDP-43. Cell-to-cell transmission of TDP43 occurs in cell cultures and may contribute to pathological TDP43 propagation in FTLD-TDP. In this study, the authors demonstrate using mouse models that a single intracerebral injection of human brain-derived pathological TDP43 from FTLD-TDP cases initiates the process of seeding and spreading of TDP43 pathology in a spatio-temporal dependent manner in the brain.