RNA Nanotherapeutics for the Amelioration of Astroglial Reactivity.

Jayden A Smith,Farzin Haque,Alice Braga,Silvia Basilico,Clara Alfaro-Cervello,Peixuan Guo,Jeroen Verheyen,Luca Peruzzotti-Jametti,Stefano Pluchino,Sara Bandiera,Dan Shu

doi:10.1016/j.omtn.2017.11.008

Abstract

In response to injuries to the CNS, astrocytes enter a reactive state known as astrogliosis, which is believed to be deleterious in some contexts. Activated astrocytes overexpress intermediate filaments including glial fibrillary acidic protein (GFAP) and vimentin (Vim), resulting in entangled cells that inhibit neurite growth and functional recovery. Reactive astrocytes also secrete inflammatory molecules such as Lipocalin 2 (Lcn2), which perpetuate reactivity and adversely affect other cells of the CNS. Herein, we report proof-of-concept use of the packaging RNA (pRNA)-derived three-way junction (3WJ) motif as a platform for the delivery of siRNAs to downregulate such reactivity-associated genes. In vitro, siRNA-3WJs induced a significant knockdown of Gfap, Vim, and Lcn2 in a model of astroglial activation, with a concomitant reduction in protein expression. Knockdown of Lcn2 also led to reduced protein secretion from reactive astroglial cells, significantly impeding the perpetuation of inflammation in otherwise quiescent astrocytes. Intralesional injection of anti-Lcn2-3WJs in mice with contusion spinal cord injury led to knockdown of Lcn2 at mRNA and protein levels in vivo. Our results provide evidence for siRNA-3WJs as a promising platform for ameliorating astroglial reactivity, with significant potential for further functionalization and adaptation for therapeutic applications in the CNS.

Highlights

Astrocytes are abundant and highly heterogeneous cells that provide a variety of essential support and regulatory functions within the CNS.[1,2] In response to CNS pathologies such as spinal cord injury (SCI), stroke, multiple sclerosis (MS), and Alzheimer’s disease, astrocytes show changes in gene expression, cellular structure, and function
Astrocytes Cultured in Low-FBS Medium Enter a Reactive State When Exposed to Lipopolysaccharide + Interferon-g Differentiation of neural stem cells (NSCs) into astrocytes was induced by the presence of fetal bovine serum (FBS), a well-established technique,[45,46,47] with 15 days in vitro (DIV) chosen as a time point at which cultured astroglial cells had achieved a desirable level of maturity.[47]
These morphological differences, coupled with the significantly less intense glial fibrillary acidic protein (GFAP) and Vim expression observed in low-FBS astrocytes, suggested a strong basal reactivity in high-FBS astrocytes

Summary

Introduction

Astrocytes are abundant and highly heterogeneous cells that provide a variety of essential support and regulatory functions within the CNS.[1,2] In response to CNS pathologies such as spinal cord injury (SCI), stroke, multiple sclerosis (MS), and Alzheimer’s disease, astrocytes show changes in gene expression, cellular structure, and function. Such responses are commonly referred to as astrogliosis.[3,4,5,6].

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Conclusion