Abstract
Reactive gliosis is an early pathological feature common to most neurodegenerative diseases, yet its regulation and impact remain poorly understood. Normally astrocytes maintain a critical homeostatic balance. After stress or injury they undergo rapid parainflammatory activation, characterized by hypertrophy, and increased polymerization of type III intermediate filaments (IFs), particularly glial fibrillary acidic protein and vimentin. However, the consequences of IF dynamics in the adult CNS remains unclear, and no pharmacologic tools have been available to target this mechanism in vivo. The mammalian retina is an accessible model to study the regulation of astrocyte stress responses, and their influence on retinal neuronal homeostasis. In particular, our work and others have implicated p38 mitogen-activated protein kinase (MAPK) signaling as a key regulator of glutamate recycling, antioxidant activity and cytokine secretion by astrocytes and related Müller glia, with potent influences on neighboring neurons. Here we report experiments with the small molecule inhibitor, withaferin A (WFA), to specifically block type III IF dynamics in vivo. WFA was administered in a model of metabolic retinal injury induced by kainic acid, and in combination with a recent model of debridement-induced astrocyte reactivity. We show that WFA specifically targets IFs and reduces astrocyte and Müller glial reactivity in vivo. Inhibition of glial IF polymerization blocked p38 MAPK-dependent secretion of TNF-α, resulting in markedly reduced neuronal apoptosis. To our knowledge this is the first study to demonstrate that pharmacologic inhibition of IF dynamics in reactive glia protects neurons in vivo.
Highlights
Astrocyte reactivity is an early pathological feature common to most neurodegenerative diseases, yet its regulation and impact remains poorly understood
In order to study the influence of intermediate filaments (IFs) dynamics on this model, we first tested the efficacy of Withaferin A (WFA) and the selective p38α and β-mitogen-activated protein kinase (MAPK) inhibitor SB203580 (SB)
WFA treatment resulted in a corresponding marked reduction in inner retinal apoptosis in the ganglion cell layer (GCL) and inner nuclear layer (INL) compared with control mice (Figure 1f)
Summary
Astrocyte reactivity (reactive gliosis) is an early pathological feature common to most neurodegenerative diseases, yet its regulation and impact remains poorly understood. Withaferin A (WFA) is a small molecule withanolide that is a potent and specific inhibitor of type III intermediate filament dynamics.[16,17,18] Its activity has been most closely studied with respect to vimentin rearrangement and phosphorylation in the context of angiogenesis, fibrosis and cancer, through downstream effects on inflammatory signaling and cell proliferation.[19,20,21,22,23,24] Interestingly, WFA has been reported to regulate vimentin-mediated activation of MAPKs in a context dependent manner, as well as NFκB.[25,26] Recently BargagnaMohan et al.[27] reported that, in addition to vimentin, WFA binds covalently to GFAP at cysteine 294 In these studies WFA impaired GFAP filament assembly and polymerization in cultured astrocytes, and in vivo in retinal astrocytes and related Müller glia in a model of injury-induced gliosis.[27] WFA presents a novel tool to test the pharmacologic blockade of intermediate filament remodeling during gliosis. We have previously used the retina as a uniquely accessible model to study the regulation of astrocyte stress responses, and their influence on retinal neuronal survival.[28,29,30] In the human and rodent eye retinal ganglion cells (RGCs) and amacrine cells of the inner retina maintain a delicate homeostatic balance and are vulnerable to excitotoxic and metabolic damage, mediated in part through non-cell autonomous interactions with neighboring glia.[31,32,33,34] In addition, our work and others has implicated signaling through p38 MAPKs as key regulators of Received 14.12.15; revised 25.7.16; accepted 29.7.16; Edited by A Yaron glutamate recycling, antioxidant activity, and cytokine secretion in neighboring stress-activated retinal astrocytes and Müller glia.[29,35,36,37] Here we take advantage of a model of induced retinal astrocyte reactivity to establish whether WFA, and the selective p38 MAPK inhibitor SB203580 (SB), affect neuronal apoptosis in a mouse model of excitotoxic injury
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