The contribution of activated astrocytes to Aβ production: Implications for Alzheimer's disease pathogenesis

Tracy O'Connor,Jie Zhao,Robert Vassar

doi:10.1186/1742-2094-8-150

Abstract

Backgroundβ-Amyloid (Aβ) plays a central role in Alzheimer's disease (AD) pathogenesis. Neurons are major sources of Aβ in the brain. However, astrocytes outnumber neurons by at least five-fold. Thus, even a small level of astrocytic Aβ production could make a significant contribution to Aβ burden in AD. Moreover, activated astrocytes may increase Aβ generation. β-Site APP cleaving enzyme 1 (BACE1) cleavage of amyloid precursor protein (APP) initiates Aβ production. Here, we explored whether pro-inflammatory cytokines or Aβ42 would increase astrocytic levels of BACE1, APP, and β-secretase processing, implying a feed-forward mechanism of astrocytic Aβ production.MethodsMouse primary astrocytes were treated with combinations of LPS, TNF-α, IFN-γ, and IL-1β and analyzed by immunoblot and ELISA for endogenous BACE1, APP, and secreted Aβ40 levels. Inhibition of JAK and iNOS signaling in TNF-α+IFN-γ-stimulated astrocytes was also analyzed. In addition, C57BL/6J or Tg2576 mouse astrocytes were treated with oligomeric or fibrillar Aβ42 and analyzed by immunoblot for levels of BACE1, APP, and APPsβsw. Astrocytic BACE1 and APP mRNA levels were measured by TaqMan RT-PCR.ResultsTNF-α+IFN-γ stimulation significantly increased levels of astrocytic BACE1, APP, and secreted Aβ40. BACE1 and APP elevations were post-transcriptional at early time-points, but became transcriptional with longer TNF-α+IFN-γ treatment. Despite a ~4-fold increase in astrocytic BACE1 protein level following TNF-α+IFN-γ stimulation, BACE1 mRNA level was significantly decreased suggesting a post-transcriptional mechanism. Inhibition of iNOS and JAK did not reduce TNF-α+IFN-γ-stimulated elevation of astrocytic BACE1, APP, and Aβ40, except that JAK inhibition blocked the APP increase. Finally, oligomeric and fibrillar Aβ42 dramatically increased levels of astrocytic BACE1, APP, and APPsβsw through transcriptional mechanisms, at least in part.ConclusionsCytokines including TNF-α+IFN-γ increase levels of endogenous BACE1, APP, and Aβ and stimulate amyloidogenic APP processing in astrocytes. Oligomeric and fibrillar Aβ42 also increase levels of astrocytic BACE1, APP, and β-secretase processing. Together, our results suggest a cytokine- and Aβ42-driven feed-forward mechanism that promotes astrocytic Aβ production. Given that astrocytes greatly outnumber neurons, activated astrocytes may represent significant sources of Aβ during neuroinflammation in AD.

Highlights

The neuropathology of Alzheimer’s disease (AD) is characterized by the development of extracellular deposits of senile amyloid plaques that are mainly composed of the b-amyloid peptide (Ab)
Pro-inflammatory cytokine combinations increase astrocytic b-Site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1), APP, and Ab To investigate whether activated astrocytes increase amyloidogenic APP processing under pro-inflammatory conditions, we treated primary astrocytes cultured from neonatal C57BL/6J mouse pups with pro-inflammatory agents LPS, tumor necrosis factor-a (TNF-a), IL-1b, and IFN-g, both individually and in the combinations LPS+IFN-g, TNF-a+IFN-g, TNF-a+IL-1b+IFN-g
After astrocyte cultures were treated for 24, 48, and 96 h, cell lysates were prepared for immunoblot analysis of BACE1, APP, and activation markers iNOS and pro-IL-1b, and conditioned media was harvested for mouse Ab40 measurement

Summary

Introduction

The neuropathology of Alzheimer’s disease (AD) is characterized by the development of extracellular deposits of senile amyloid plaques that are mainly composed of the b-amyloid peptide (Ab). The resulting neuroinflammatory processes usually involve the release from activated glia of a number of potentially neurotoxic molecules, including reactive oxygen species, nitric oxide, and pro-inflammatory chemokines and cytokines such as interleukin-1b (IL-1b), tumor necrosis factor-a (TNF-a), and interferon-g (IFN-g). Excessive levels of these mediators are apt to induce neuronal damage through a variety of mechanisms in AD and other neurodegenerative disorders [20]. The inflammatory processes in AD have been well studied, the amyloidogenic potential of glial cells under pro-inflammatory conditions and the mechanisms involved have been relatively unexplored

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