C-terminal frameshift variant of TDP-43 with pronounced aggregation-propensity causes rimmed vacuole myopathy but not ALS/FTD

Acta Neuropathologica https://doi.org/10.1007/s00401-023-02565-1 ORIGINAL PAPER C‑terminal frameshift variant of TDP‑43 with pronounced aggregation‑propensity causes rimmed vacuole myopathy but not ALS/FTD Pedro Ervilha Pereira1,2 · Nika Schuermans1,2 · Antoon Meylemans3,4 · Pontus LeBlanc1,2 · Lauren Versluys1,2 · Katie E. Copley5,6 · Jack D. Rubien5 · Christopher Altheimer7 · Myra Peetermans1,2 · Elke Debackere1,2 · Olivier Vanakker1,2 · Sandra Janssens1,2 · Jonathan Baets8,9,10 · Kristof Verhoeven3,11 · Martin Lammens12 · Sofie Symoens1,2 · Boel De Paepe3,4 · Sami J. Barmada7 · James Shorter5,6 · Jan L. De Bleecker3,4 · Elke Bogaert1,2 · Bart Dermaut1,2 Received: 29 December 2022 / Revised: 15 March 2023 / Accepted: 17 March 2023 © The Author(s) 2023 Abstract Neuronal TDP-43-positive inclusions are neuropathological hallmark lesions in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Pathogenic missense variants in TARDBP, the gene encoding TDP-43, can cause ALS and cluster in the C-terminal prion-like domain (PrLD), where they modulate the liquid condensation and aggregation properties of the protein. TDP-43-positive inclusions are also found in rimmed vacuole myopathies, including sporadic inclusion body myositis, but myopathy-causing TDP-43 variants have not been reported. Using genome-wide linkage analysis and whole exome sequencing in an extended five-generation family with an autosomal dominant rimmed vacuole myopathy, we identified a conclusively linked frameshift mutation in TDP-43 producing a C-terminally altered PrLD (TDP-43p.Trp385IlefsTer10) (maximum multipoint LOD-score 3.61). Patient-derived muscle biopsies showed TDP-43-positive sarcoplasmic inclusions, accumulation of autophagosomes and transcriptomes with abnormally spliced sarcomeric genes (including TTN and NEB) and increased expression of muscle regeneration genes. In vitro phase separation assays demonstrated that TDP-43Trp385IlefsTer10 does not form liquid-like condensates and readily forms solid-like fibrils indicating increased aggregation propensity compared to wild-type TDP-43. In Drosophila TDP-43p.Trp385IlefsTer10 behaved as a partial loss-of-function allele as it was able to rescue the TBPH (fly ortholog of TARDBP) neurodevelopmental lethal null phenotype while showing strongly reduced toxic gain-of-function properties upon overexpression. Accordingly, TDP-43p.Trp385IlefsTer10 showed reduced toxicity in a primary rat neuron disease model. Together, these genetic, pathological, in vitro and in vivo results demonstrate that TDP43p.Trp385IlefsTer10 is an aggregation-prone partial loss-of-function variant that causes autosomal dominant vacuolar myopathy but not ALS/FTD. Our study genetically links TDP-43 proteinopathy to myodegeneration, and reveals a tissue-specific role of the PrLD in directing pathology. Keywords TDP-43 · Myopathy · ALS/FTD · Genetics · Drosophila · Phase separation Introduction Pedro Ervilha Pereira and Nika Schuermans have contributed equally. Elke Bogaert and Bart Dermaut have contributed equally as co-last authors. * Elke Bogaert * Bart Dermaut Extended author information available on the last page of the article TDP-43, encoded by TARDBP, is an evolutionary highly conserved and ubiquitously expressed RNA- and DNAbinding protein (reviewed in [57]). It is involved in multiple pathways of RNA metabolism, including pre-mRNA splicing, RNA transport, mRNA stability, miRNA processing and stress granule assembly. It is predominantly localized in the nucleus but also shuttles to the cytoplasm. The protein contains a nuclear localization signal (NLS) within its N-terminal domain (NTD), two RNA recognition motifs 13 Vol.:(0123456789) Acta Neuropathologica (RMM1, RMM2) and a C-terminal region (aa.272-414), which is highly disordered and forms a low complexity domain (LCD) that is similar to that of prion-like domains (PrLD) of yeast proteins [23, 44]. The PrLD of TDP-43 is responsible for protein–protein interactions and is needed for the proper functioning of the protein through the process of liquid–liquid phase separation (LLPS) [21]. Nearly all cases of amyotrophic lateral sclerosis (ALS) and ~ 45% of frontotemporal dementia (FTD) patients are neuropathologically characterized by neuronal cytoplasmic TDP-43 inclusions accompanied by a nuclear depletion of TDP-43 [1, 42]. Pathogenic missense variants in TARDBP [15, 26, 53], which cluster in the PrLD, account for ~ 4% of familial ALS cases and < 1% of sporadic ALS cases [27]. These findings indicate that the PrLD of TDP-43 is crucially involved in ALS/FTD pathogenesis and that altered LLPS-related physical–chemical properties of this region, which are essential for the formation of TDP-43-associated stress granules and RNA binding, could lead to the irreversible formation of solid aggregates [21, 57]. Interestingly, a cryo-electron microscopy study of TDP-43 filaments from brain cortices of ALS/FTD patients revealed an amyloid-like structure of which the filament core spans aa. 282-360 in the PrLD and adopts a double-spiral-shaped fold [4]. Although such filaments are generally assumed to be neurotoxic [32], the exact pathogenic roles of insoluble aggregates and liquid-like condensates are still unclear and under debate [25]. Based on an unbiased deep mutagenesis study in yeast, Bolognesi et al. suggested that TDP-43 aggregates are protective, and that cellular toxicity is caused by liquid-like TDP-43 condensates [8]. Therefore, whether TDP-43-mediated neuronal cell death results from a toxic gain-of-function of cytoplasmic TDP-43, as either aggregates or liquid-like droplets, and/or a loss of its normal function due to nuclear depletion remains unknown [11]. Although TDP-43 aggregates are a typical cellular hallmark of neurodegeneration, its loss-of-function has been associated with myodegeneration in zebrafish [50] and Drosophila [16, 36]. In humans, accumulation of TDP-43 is also frequently observed in myopathies with rimmed vacuoles [31, 58]. Rimmed vacuoles are a common feature of myopathies in which autophagic impairment plays a crucial pathogenic role [38]. Under the light microscope, rimmed vacuoles appear as empty spaces in the sarcoplasm surrounded by a rim of basophilic granular material. However, their content can be identified in transmission electron microscopy (TEM) preparations as filamentous protein inclusions surrounded by a rim of autophagic material. TDP-43-positive rimmed vacuole myopathies [31] include sporadic inclusion body myositis (sIBM) [10], hereditary inclusion body myopathy with early-onset Paget disease and FTD (caused by heterozygous mutations in VCP) [41, 60, 61], inclusion body myopathy associated with multisystem proteinopathy 13 (caused by heterozygous mutations in HNRNPA2B1 and HNRNPA1) [28], distal myopathy with rimmed vacuoles (caused by bi-allelic mutations in GNE) [19] and oculopharyngeal muscular dystrophies (caused by heterozygous mutations in PABP (...truncated)