Author Of 1 Presentation
P0934 - A novel experimental model to assess the contribution of heterogeneous nuclear ribonucleoprotein A1 (A1) mutations to neurodegeneration in MS. (ID 1500)
Abstract
Background
Current evidence indicates that neurodegeneration (NDG) is a prominent feature in the pathogenesis of MS, and the primary cause of disability in MS patients. Yet, knowledge of the molecular mechanisms of NDG in MS, as well as treatment options to prevent or reverse NDG, is lacking. Exploration of perturbed molecular mechanisms in MS may allow us to develop therapies that attenuate NDG and in turn, inhibit disability, and improve long-term quality of life of persons living with MS
Objectives
To characterize somatic (acquired) MS genetic mutations in A1 that cause molecular dysregulation of A1, using innovative cutting-edge optogenetic technology, and elucidate the role of dysregulated A1 in the pathogenesis of NDG in a model of MS.
Methods
We created optogenetic A1 protein expression constructs containing wildtype (WT) and mutant A1, tagged with both the optogene Cryptochrome 2 (Cry2) and mCherry. Cry2 is an optogenetic protein that self-clusters in response to blue light (BL) stimulation, and reverses when BL is turned off, therefore allowing real-time examination of protein clustering kinetics. We established an in vitro optogenetic paradigm of A1 dysfunction in HEK293T cells and gathered evidence of how select mutations affect A1 cellular localization and function.
Results
Using a chronic, single BL stimulus followed by a steady period of recovery (imitating chronic environmental cell stress), our data showed that the MS associated A1 mutations pP275S and pF281L increased the kinetics of both cytoplasmic cluster formation [17 and 32 minutes, respectively, compared to 51 minutes for WT (p < 0.0001)] and clearance of A1 [22 and 26 minutes, respectively, compared to 19 minutes for WT (p < 0.05)]. A1 clusters also decreased in quantity [clusters/cell: pF281L=2.1; pP275S=3.2; WT=3.4 (p < 0.05)] and increased in size [average cluster size (µm2): pF281L=0.37; pP275S=0.49; WT=0.24 (p < 0.001)], compared to WT. These data demonstrate changes in the molecular function of mutant A1 compared to WT, indicative of A1 dysfunction.
Conclusions
Using an in vitro optogenetic approach, this study presents evidence that somatic MS genetic mutations in A1, found in MS patient tissue, promotes A1 mislocalization and self-association, leading to protein dysfunction that may have an effect on NDG in MS pathogenesis.