Accumulation of abnormally phosphorylated TDP-43 (pTDP-43) is the main pathology in affected neurons of patients with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Morphological diversity and neuroanatomical distribution of pTDP-43 accumulations allowed classification of FTD cases into at least four different subtypes, which correlate with clinical presentations and genetic causes. To understand the molecular basis of this heterogeneity, we developed SarkoSpin, a new method for extremely pure biochemical isolation of pathological TDP-43. Combining SarkoSpin with mass spectrometry, we revealed proteins beyond TDP-43, which become abnormally insoluble in a disease subtype-specific manner. We show that pTDP-43 extracted from disease brain forms large and stable assemblies of distinct densities and morphologies that correlate with disease subtypes. Importantly, biochemically extracted pTDP-43 assemblies displayed differential neurotoxicity and seeding that correlated with disease duration of FTLD patients. Our data indicate that disease heterogeneity may originate from alternate pathological TDP-43 conformations, reminiscent of prion strains. 5 developed SarkoSpin, a novel and simple extraction method for physical separation of pathological TDP-43 from more than 99% of total protein mass including the extreme bulk of physiological, monomeric and oligomeric 11 TDP-43. Using SarkoSpin on brain cortical samples from over 80 patients, we found that TDP-43 forms large and buoyant assemblies of distinct densities, polyubiquitination levels and morphologies that correlate with specific neuropathological classifications. Importantly, coupling SarkoSpin with mass spectrometry, we illustrate that a specific subset of proteins, beyond TDP-43, become insoluble in each disease subtype. These proteins are rarely co-aggregated with pTDP-43 and most likely represent a downstream effect of TDP-43 pathology. One of these proteins depicts a distinct astrocytic reaction discriminating FTLD-TDP-A from FTLD-TDP-C patients, illustrating divergent pathogenic mechanisms within these two disease subtypes. Most importantly, we show evidence that SarkoSpin extracted pTDP-43 assemblies exhibit cytotoxicity and protein seeding ability. Remarkably, pathological aggregates extracted from FTLD-TDP-A were significantly more cyto-and neurotoxic than those extracted from FTLD-TDP-C, thereby correlating with the significant difference in disease duration between these two subtypes. Collectively, our data demonstrate that ALS and FTLD heterogeneity is consistently reflected in the biochemical, neurotoxic and seeding properties of TDP-43 and the associated insoluble proteome. We propose that alternative TDP-43 pathological conformations may underlie the diversity of TDP-43 proteinopathies, reminiscent of prion strains 33,34. Results Summary of patient cohort and characterization of FTLD-TDP-A and FTLD-TDP-C cases Brain cortical samples from over 80 patients, including control patients with no apparent CNS pathology or with non-TDP...