Amyloid-β peptide activates cultured astrocytes: morphological alterations, cytokine induction and nitric oxide release

Abstract

A common feature of many neurodegenerative disorders is an abundance of activated glial cells (astrocytes and microglia). In Alzheimer's disease (AD), activated astrocytes are in close apposition to and surrounding the amyloid plaques. The mechanisms by which the astrocytes become activated in AD and the consequences of reactive astrocytosis to disease progression are not known. We examined the possibility that the amyloid-β (Aβ) peptide, a major constituent of the amyloid plaque, could act as a stimulus leading to activation. We found that treatment of rat cortical astrocyte cultures with aggregated Aβ 1–42 peptide induces activation, as assessed by reactive morphological changes and upregulation of selective glial mRNA and proteins, such as the inflammatory cytokine interleukin-1β. Aβ also stimulates inducible nitric oxide synthase (iNOS) mRNA levels and nitric oxide (NO) release. Aβ 1–42, a major form of amyloid associated with neurotoxicity, activated astrocytes in a time- and dose-dependent manner, whereas a scrambled Aβ 1–42 sequence or Aβ 17–42 had little or no effect. We also determined that the Aβ activity can be found in a supernatant fraction containing soluble Aβ oligomers. Our data suggest that Aβ plays a role in the reactive astrocytosis of AD and that the inflammatory response induced upon glial activation is a critical component of the neurodegenerative process.

Introduction

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a progressive cognitive decline resulting from selective neuronal dysfunction, synaptic loss, and neuronal cell death. The well-studied neuropathological features of AD include: loss of neurons; formation of intra-neuronal neurofibrillary tangles composed of paired helical filaments of the cytoskeletal protein tau; and extracellular plaques composed primarily of diffuse or compacted deposits of amyloid-β (Aβ) aggregates, with or without a component of dystrophic neurites 41, 46. What has been less well appreciated are the characteristic changes seen in glial cells in AD, and the role that these cells may play in the neurodegenerative cascade leading to Alzheimer dementia. One common feature of a variety of neurodegenerative disorders is the presence of large numbers of activated astrocytes and microglia (gliosis). Glial activation involves morphological changes (more spherical cell soma, hypertrophy of nuclei, appearance of extensive cellular processes) and changes in expression of a large number of proteins (for review, see Ref. [9]). In AD, activated astrocytes surround the neuritic shell of the amyloid plaque, and activated microglia are near the center of the neuritic shell adjacent to the amyloid core (for reviews, see Refs. 14, 28).

There are a number of stimuli that lead to glial activation. One of the best recognized inducers of glial activation is neuronal dysfunction or injury. For example, upon traumatic brain injury or experimentally induced central nervous system lesions, there is a rapid and vigorous glial activation response which is transient and reversible. However, in a number of diseases, glial cells remain in a chronic state of activation. For example, gliosis is a common feature of a number of neurodegenerative conditions, including sustained brain trauma, vascular insufficiency, AIDS, scrapie, Down syndrome, Pick's disease, and AD (for reviews, see Refs. 14, 28, 30).

The role of glial activation in AD is not known, although reactive glia have been reported to be associated with amyloid plaques at relatively early stages of the disease before the appearance of neuritic plaque components 6, 15, 29. In addition, several cytokines and inflammatory mediators produced by activated glia have the potential to initiate or exacerbate the progression of neuropathology (for reviews, see Refs. 3, 6, 14, 26, 33). Therefore, understanding the mechanisms by which glia become reactive and deciphering the regulatory pathways involved are important to begin to elucidate the contribution of glia to the neurodegenerative progression in AD. The factors responsible for inducing and maintaining the glial activation state in AD are unknown. However, because of the proximity of reactive glia to the amyloid plaques in AD, one possibility is that Aβ is involved in this process. There are numerous studies demonstrating that Aβ can be directly neurotoxic and can increase neuronal susceptibility to other toxic agents (for reviews, see Refs. 5, 48). However, the actions of Aβ on glia, the supramolecular forms of Aβ that affect glia, and the glial responses to Aβ exposure are less well studied.

As an initial approach to defining the interactions between Aβ and glia, we examined the effects of synthetic Aβ peptides on cultured astrocytes. We tested different Aβ peptides (Aβ 1–42, Aβ 17–42, and a scrambled Aβ 1–42) aggregated under a variety of conditions. We report here that Aβ 1–42 induces a robust astrocyte activation, as evidenced by morphological changes, upregulation of the cytokine interleukin-1β (IL-1β) mRNA, and stimulation of inducible nitric oxide synthase (iNOS) mRNA and nitric oxide (NO) release. We also report that a supernatant fraction containing globular Aβ oligomers is biologically active.