Abstract: Optogenetics Defines RNA Binding Protein Dysfunction in a Model of Neurodegeneration in Multiple Sclerosis (MS). (2021 Annual Meeting of the Consortium of Multiple Sclerosis Centers)

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Optogenetics Defines RNA Binding Protein Dysfunction in a Model of Neurodegeneration in Multiple Sclerosis (MS).

Monday, October 25, 2021: 2:05 PM

Gatlin A3/A4 (Rosen Shingle Creek)

Joseph-Patrick W.E. Clarke, Ph.D. , College of Medicine, University of Saskatchewan, Saskatoon, SK, Canada

Patricia A Thibault, Ph.D. , College of Medicine, University of Saskatchewan, Saskatoon, SK, Canada

Hannah E Salapa, PhD , Anatomy, Physiology and Pharmacology, University of Saskatchewan, Saskatoon, SK, Canada

David E Kim, B.Sc. , College of Medicine, University of Saskatchewan, Saskatoon, SK, Canada

Catherine Hutchinson, B.Sc. M.Sc. , Office of the Saskatchewan MS Clinical Research Chair, University of Saskatchewan, Saskatoon, SK, Canada

Michael Levin, FRCPC , Office of the Saskatchewan Multiple Sclerosis Clinical Research Chair, University of Saskatchewan, Saskatoon, SK, Canada

Background: Evidence indicates that neurodegeneration (NDG) is a prominent feature and the primary cause of disability in MS. Yet, knowledge of the molecular mechanisms of NDG in MS, as well as treatment options for NDG are lacking. Data from our lab indicates that dysfunction of the RNA binding protein (RBP) heterogeneous ribonucleoprotein A1 (A1) may contribute to MS pathogenesis.

Objectives: To examine A1 dysfunction, we utilized cutting-edge optogenetics to examine how somatic (acquired) MS-associated genetic mutations in A1 cause its molecular dysregulation in the pathogenesis of NDG in a cellular model of MS.

Methods: Reversible, blue light (BL) stimulated, optogenetic A1 protein expression plasmids, containing wild-type (WT) and mutant A1 (p.P275S and p.F281L) tagged with Cryptochrome 2 (Cry2) and mCherry, were used to examine the effects of mutations on protein dynamics in real-time. We established an in vitro optogenetic paradigm of A1 dysfunction in HEK293T cells and analyzed how mutations affect A1 cellular localization, cluster kinetics and stress granule (SG) formation – a marker of NDG.

Results: Using BL stimulation followed by a period of recovery (imitating an MS relapse), revealed that MS-associated A1 mutations p.P275S and p.F281L caused significant A1 cytoplasmic mislocalization compared to WT (cytoplasmic/nuclear localization ratio: p.P275S=1.14; p.F281L=0.85; WT=0.59). The kinetics of cytoplasmic cluster formation [half-maximal formation time (KA_1/2) (minutes): p.P275S=40; p.F281L=42; WT=55] and dissociation of A1 [half-maximal dissociation time (KD_1/2) (minutes): p.P275S=11; p.F281L=21; WT=18] were signiﬁcantly altered with A1 mutations. A1 mutations altered the quantity (clusters/cell: p.P275S=3.2; p.F281L=2.1; WT=3.4) and size [average cluster size (µm²): p.P275S=0.49; p.F281L=0.37; WT=0.24] of A1 clusters. A1 mutations also caused SG formation to occur more quickly [KA_1/2 (minutes): p.P275S=55; p.F281L=51; WT=73] and frequently (fold change of cells with SG: p.P275S=1.6; p.F281L=2.3; WT=1.1).

Conclusions: This study presents evidence that mutations in A1 promote A1 mislocalization, self-association clustering, altered RBP function, and cell stress leading to SG formation. These results indicate a potential link between A1 protein dysfunction and NDG in MS pathogenesis and may allow us to develop therapies that attenuate ND and inhibit disability in MS.

7570 Optogenetics Defines RNA Binding Protein Dysfunction in a Model of Neurodegeneration in Multiple Sclerosis (MS).

7570
Optogenetics Defines RNA Binding Protein Dysfunction in a Model of Neurodegeneration in Multiple Sclerosis (MS).