Pathogenesis – Neurodegeneration Poster Presentation

P0990 - Pro-inflammatory cytokines and autoantibodies induce dysfunctional RNA binding protein biology in primary cortical neurons (ID 1518)

Speakers
  • M. Li
Authors
  • M. Li
  • R. Hamilton
  • H. Salapa
  • M. Levin
Presentation Number
P0990
Presentation Topic
Pathogenesis – Neurodegeneration

Abstract

Background

Dysfunctional RNA binding proteins (RBPs), including heterogeneous nuclear ribonucleoprotein A1 (A1), have been suggested to play a role in neurodegeneration in multiple sclerosis (MS). Features of dysfunctional RBPs include a triad of molecular changes comprised of [i] stress granule (SG) formation, [ii] mislocalization of the RBP from its homeostatic nuclear location to the cytoplasm, and [iii] altered RNA metabolism. Previous studies have demonstrated that MS patients make antibodies to A1. The administration of anti-A1 antibodies to experimental autoimmune encephalomyelitis (EAE) mice exacerbated disease and induced the triad of molecular changes. Furthermore, in EAE mice, increased neuronal A1 mislocalization was observed in areas of the spinal cord with more inflammatory cytokine-producing T-cells. These data suggest a relationship between inflammation and dysfunctional RBPs.

Objectives

We hypothesized that the pro-inflammatory cytokines interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) and anti-A1 antibodies would induce dysfunctional RBP biology in primary cortical neurons.

Methods

Primary cortical neurons were isolated from C57BL/6 female mice. For cytokine experiments, neurons were treated for 24 hours with either IFNγ or TNFα at varying concentrations (0.625 μg/mL, 1.25 μg/mL, 2.5 μg/mL). For antibody experiments, neurons were treated for 24 hours with fluorescently conjugated IgG isotype control or anti-A1 antibodies at varying concentrations (5 μg/mL, 10 μg/mL, 20 μg/mL). Following treatments, neurons were fixed and immunostained for endogenous A1 localization, SG formation, and beta III tubulin to assess changes in neurites (as a marker of neurodegeneration).

Results

Neurons treated with pro-inflammatory cytokines exhibited increased SG formation (****p<0.0001), increased A1 mislocalization (*p<0.0156), and decreased neurite length (****p<0.0001) compared to untreated controls. Neurons treated with anti-A1 antibodies also showed an increase in the number of neurons with SGs (*p<0.043), increased A1 mislocalization, and decreased neurite length (****p<0.0001) as compared to IgG and untreated controls.

Conclusions

These experiments suggest that pro-inflammatory cytokines and anti-A1 antibodies, both characteristics of the inflammatory response in MS, induce RBP dysfunction, including A1 mislocalization and SG formation, in primary cortical neurons, subsequently leading to neurodegeneration.

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