Protective paraspeckle hyper-assembly downstream of TDP-43 loss of function in amyotrophic lateral sclerosis

Abstract

BackgroundParaspeckles are subnuclear bodies assembled on a long non-coding RNA (lncRNA) NEAT1. Their enhanced formation in spinal neurons of sporadic amyotrophic lateral sclerosis (ALS) patients has been reported but underlying mechanisms are unknown. The majority of ALS cases are characterized by TDP-43 proteinopathy. In current study we aimed to establish whether and how TDP-43 pathology may augment paraspeckle assembly.MethodsParaspeckle formation in human samples was analysed by RNA-FISH and laser capture microdissection followed by qRT-PCR. Mechanistic studies were performed in stable cell lines, mouse primary neurons and human embryonic stem cell-derived neurons. Loss and gain of function for TDP-43 and other microRNA pathway factors were modelled by siRNA-mediated knockdown and protein overexpression.ResultsWe show that de novo paraspeckle assembly in spinal neurons and glial cells is a hallmark of both sporadic and familial ALS with TDP-43 pathology. Mechanistically, loss of TDP-43 but not its cytoplasmic accumulation or aggregation augments paraspeckle assembly in cultured cells. TDP-43 is a component of the microRNA machinery, and recently, paraspeckles have been shown to regulate pri-miRNA processing. Consistently, downregulation of core protein components of the miRNA pathway also promotes paraspeckle assembly. In addition, depletion of these proteins or TDP-43 results in accumulation of endogenous dsRNA and activation of type I interferon response which also stimulates paraspeckle formation. We demonstrate that human or mouse neurons in vitro lack paraspeckles, but a synthetic dsRNA is able to trigger their de novo formation. Finally, paraspeckles are protective in cells with compromised microRNA/dsRNA metabolism, and their assembly can be promoted by a small-molecule microRNA enhancer.ConclusionsOur study establishes possible mechanisms behind paraspeckle hyper-assembly in ALS and suggests their utility as therapeutic targets in ALS and other diseases with abnormal metabolism of microRNA and dsRNA.Electronic supplementary materialThe online version of this article (10.1186/s13024-018-0263-7) contains supplementary material, which is available to authorized users.

Cross-Regulation Between TDP-43 and Paraspeckles Promotes Pluripotency-Differentiation Transition

“…Beyond the mechanisms of early development, our findings are likely also relevant also for other types of cell fate transitions because both TDP-43 and NEAT1 are broadly expressed (Shelkovnikova et al, 2018). Moreover, amyotrophic lateral sclerosis (ALS)-causing mutations in TDP-43 affect its phase separation Figures S6B and S6C], n = 7 for Neat1DpA [2 are shown in Figure S6D], and n = 3 for parental control mESC [2 are shown in Figure S6E]).…”

Stress granules regulate stress-induced paraspeckle assembly

“…SGs are believed to play a general prosurvival role (Arimoto et al, 2008;Takahashi et al, 2013). Similarly, PSs have been shown to promote viability in cells subjected to proteasome inhibition, heat shock, and foreign dsRNA (Hirose et al, 2014;Lellahi et al, 2018;Shelkovnikova et al, 2018). It is tempting to speculate that the protective role of SGs may be at least partially mediated by PSs.…”

“…This overlap between PS and SG proteomes was a somewhat surprising finding given that the majority of PSPs are predominantly nuclear proteins. Among SG-recruited PSPs, 13 are either essential or important for PS assembly and 8 of them regulate levels of NEAT1 isoforms (Banerjee et al, 2017;Naganuma et al, 2012;Shelkovnikova et al, 2018; Fig. 2 B).…”

“…Intense interest in the biology of RNP granules in the past decade has followed the discovery of their tight connection to neurodegenerative diseases, primarily amyotrophic lateral sclerosis (ALS). There is substantial genetic and experimental evidence on the involvement of disturbed SG metabolism in ALS (Li et al, 2013), and the link between PSs and ALS pathogenesis is also emerging Nishimoto et al, 2013;Shelkovnikova et al, 2014Shelkovnikova et al, , 2018. affinity purification, sorting, and proximity labeling, have allowed characterization of the proteome of cytoplasmic RNP granules such as SGs and P-bodies (Hubstenberger et al, 2017;Jain et al, 2016;Markmiller et al, 2018).…”

Neuro‐Cells therapy improves motor outcomes and suppresses inflammation during experimental syndrome of amyotrophic lateral sclerosis in mice

“…However, the most recent studies have revealed that the expression of mutant FUS leads to stress‐mediated induction of chaperones, decreased expression of ion channels and transporters essential for synaptic function, and reduced synaptic activity without the loss of nuclear FUS or its cytoplasmic aggregation . The nuclear effects of FUS also seem to result in impairment of the function of paraspeckles, granules in the nuclear interchromatin space that are assembled on a scaffold long noncoding RNA (lncRNA) NEAT1 . This results in aberrant microRNA biogenesis, apoptotic processes, oxidative stress, and mitochondrial dysfunction contributing to neurodegenerative processes .…”