Multiple Sclerosis-Associated hnRNPA1 Mutations Alter hnRNPA1 Dynamics and Influence Stress Granule Formation.

Joseph-Patrick W E Clarke,David E Kim,Michael C Levin,Hannah E Salapa,Catherine Hutchinson,Patricia A Thibault

doi:10.3390/ijms22062909

Joseph-Patrick W E Clarke, David E Kim + Show 4 more

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https://doi.org/10.3390/ijms22062909

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Abstract

Evidence indicates that dysfunctional heterogeneous ribonucleoprotein A1 (hnRNPA1; A1) contributes to the pathogenesis of neurodegeneration in multiple sclerosis. Understanding molecular mechanisms of neurodegeneration in multiple sclerosis may result in novel therapies that attenuate neurodegeneration, thereby improving the lives of MS patients with multiple sclerosis. Using an in vitro, blue light induced, optogenetic protein expression system containing the optogene Cryptochrome 2 and a fluorescent mCherry reporter, we examined the effects of multiple sclerosis-associated somatic A1 mutations (P275S and F281L) in A1 localization, cluster kinetics and stress granule formation in real-time. We show that A1 mutations caused cytoplasmic mislocalization, and significantly altered the kinetics of A1 cluster formation/dissociation, and the quantity and size of clusters. A1 mutations also caused stress granule formation to occur more quickly and frequently in response to blue light stimulation. This study establishes a live cell optogenetics imaging system to probe localization and association characteristics of A1. It also demonstrates that somatic mutations in A1 alter its function and promote stress granule formation, which supports the hypothesis that A1 dysfunction may exacerbate neurodegeneration in multiple sclerosis.

Highlights

Multiple sclerosis (MS) is an autoimmune disease with a significant neurodegenerative component characterized by inflammation-mediated demyelination of neuronal axons, and subsequent neurodegenerative axonal and neuronal cell loss [1]
To help further understand A1 molecular mechanisms and establish methodology to study A1 mutant species in vitro and their effects on A1 dysfunction, we developed an optogenetic system that induces A1 mislocalization and aggregation and recapitulates pathological features of A1 observed in central nervous system (CNS) tissue from MS patients and animal models of disease [28,29,31]
Studies have utilized optogenetics to focus upon other RNA binding protein (RBP) (e.g., TAR DNA-binding protein 43 (TDP-43), G3BP1) whose selfassociation properties are associated with neurodegenerative diseases, but prior to this study none have been developed to study full-length wild-type A1, nor to describe the effects of MS-associated A1 mutations [50,58,59,65]

Summary

Introduction

Multiple sclerosis (MS) is an autoimmune disease with a significant neurodegenerative component characterized by inflammation-mediated demyelination of neuronal axons, and subsequent neurodegenerative axonal and neuronal cell loss [1]. Current research posits a multitude of molecular mechanisms underlying MS-associated neurodegeneration, many of which likely interact to initiate and exacerbate each other. Studies have shown that mitochondrial injury, microglial activation, reactive oxygen species, and apoptosis contribute to axonal injury [2,3,4,5,6,7,8]. Other studies implicate increased energy demands and the reduction of ATP production in axons, resulting in ‘virtual hypoxia’ [9,10,11]. Neurodegeneration can be driven by inflammatory cytokines, autoantibodies, a lack of trophic support from myelin, glutamate toxicity, endoplasmic stress, altered iron homeostasis, and abnormal sodium channel expression on, and calcium accumulation within, damaged axons [12,13,14,15,16,17,18,19,20,21,22]. Our lab and others have demonstrated a substantial role for RNA binding protein (RBP) dysfunction, including that of heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1, or A), in the pathogenesis of MS and relevant MS models [9,23,24,25,26,27,28,29,30,31]

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