In vitro prion-like behaviour of TDP-43 in ALS

Abstract

Amyotrophic lateral sclerosis (ALS) is the most common form of motor neuron disease (MND), and > 95% of familial and sporadic cases involve the deposition of insoluble aggregated, phosphorylated and cleaved TDP-43 protein. Accumulating clinical and biological evidence now indicates that ALS bears a number of similarities to the prion diseases, with TDP-43 acting as a misfolded ‘prion-like’ protein demonstrating similar underlying pathobiology. Here we systematically address the hypothesis that ALS is a prion-like disorder. First we demonstrate that TDP-43 demonstrates seeded polymerisation in vitro directly from both ALS brain and spinal cord. We next show that the seeding of TDP-43 results in the formation of characteristic insoluble, aggregated, and phosphorylated TDP-43 pathology that directly recapitulates the morphological diversity of TDP-43 inclusions detected in ALS patient CNS tissue. We next demonstrate that this reaction can be serially propagated to produce increasing amounts of phosphorylated TDP-43 pathology, and that aggregates can spread from cell to cell in an analogous fashion to that seen in the prion diseases. Finally, we reproduced our findings in a murine motor neuron-like cell line (NSC-34), where the seeding of TDP-43 induces the formation of TDP-43 oligomers and reduced cell viability. These findings may guide therapeutic strategies in this rapidly progressive and invariably fatal disease.

Nucleo-cytoplasmic transport of TDP-43 studied in real time: impaired microglia function leads to axonal spreading of TDP-43 in degenerating motor neurons

“…In vitro, the intercellular transfer of TDP-43 has been demonstrated [21, 47, 57, 67]. However, whether this also occurs in vivo remains an open question.…”

“…This was mediated by the packaging of TDP-43 into microvesicles or exosomes, which were more effective at transmission than free protein. Linking this phenomenon to clinical observations, when cell culture models were seeded with phosphorylated TDP-43 +ve insoluble fractions from ALS and FTLD patient brain or spinal cord, there was a significant increase in intracellular endogenous TDP-43 aggregation and subsequent cell death [44, 57]. To follow on from this we examined how motor neuron injury and subsequent degeneration mobilises TDP-43 in vivo and may contribute to pathogenicity in ALS.…”

Does Sporadic Amyotrophic Lateral Sclerosis Spread via Axonal Connectivities?

“…The assumption that cortical neurons (Betz cells) are the first to become involved followed by the α-motor neurons, tends to favor Charcot's theory [35], namely, that ALS originates in the lateral funiculus (i. e., corticospinal tract, where the axons of the Betz cells are located) and only then affects the α-motor neurons in the anterior horn of the spinal cord [36]. The sources of the very first malfunctions within the protein TDP-43 in Betz cells are incompletely understood; but, it is conceivable that the further spread of the pathology to interconnected neurons takes place by means of the phylogenetically 'new' or recent monosynaptic contacts [9,29,32,[37][38][39][40][41]. Whether the same holds true not only for corticobulbar and corticospinal projections but also for corticorubral and corticostriatal connectivities is currently an open question.…”

Flow cytometric measurement of the cellular propagation of TDP-43 aggregation

“…9 Similarly, recent work has also observed that TDP-43 aggregates from ALS brain and spinal cord can seed cellular TDP-43 aggregation after protein transfection, and that aggregates can move cell to cell in a co-culture system using HEK293 cells. 10 An additional study presented evidence to suggest that TDP-43 can transfer between HEK293 cells and primary cortical mouse neurons and interact with endogenous TDP-43 in recipient cells. 11 More recently, it was observed that TDP-43 fibrils formed from short C-terminal derived peptides triggered the seeddependent aggregation of wild type TDP-43 (TDP-43 WT ) or TDP-43 lacking a nuclear localization signal in SH-SY5Y cells resulting in different peptides sequences of TDP-43 producing fibrils with distinct biochemical properties reminiscent of prion strains.…”