Probes for LONG

Polyglutamine repeat (polyQ) diseases are a class of neurodegenerative disorders caused by CAG repeat expansion. There are diverse cellular mechanisms behind the pathogenesis of polyQ disorders, including transcriptional dysregulation. Interestingly, we find that levels of the long isoform of nuclear paraspeckle assembly transcript 1(NEAT1L) are elevated in the brains of mouse models of spinocerebellar ataxia types 1, 2, 7, and Huntington's disease (HD). Neat1L was also elevated in differentiated striatal neurons derived from HD knock in mice and in HD patient brains. The elevation was mutant Huntingtin (mHTT) dependent, as knockdown of mHTT in vitro and in vivo restored NEAT1L to normal levels. In additional studies, we found that NEAT1L is repressed by MeCP2 by RNA-protein interaction, but not by occupancy of MeCP2 at the NEAT1L promoter. We also found that NEAT1L overexpression protects from mHTT-induced cytotoxicity, while reduced NEAT1L levels enhance mHTT-dependent toxicity. Gene set enrichment analysis of previously-published RNA-seq data from NEAT1-null mouse embryonic fibroblasts and cells derived from HD patients show that loss of NEAT1L impairs multiple cellular functions, including pathways involved in cell proliferation and development. Intriguingly, the genes dysregulated in HD human brain samples overlap with pathways affected by a reduction in NEAT1, confirming the correlation of NEAT1L and HD-induced perturbations. Cumulatively, the role of NEAT1L in polyQ disease model systems and human tissues suggests that NEAT1L may play a protective role in CAG-repeat expansion diseases.

Abstract

BACKGROUND:

Paraspeckles 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.

METHODS:

Paraspeckle 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.

RESULTS:

We 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.

CONCLUSIONS:

Our 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.