Caspase-Mediated Cleavage of Glial Fibrillary Acidic Protein within Degenerating Astrocytes of the Alzheimer's Disease Brain

Abstract

Recent studies demonstrate roles for activation of caspases and cleavage of cellular proteins within neurons of the Alzheimer's disease (AD) brain. To determine whether a similar role for caspases also occurs within glial cells in AD, we designed a site-directed caspase-cleavage antibody specific to glial fibrillary acidic protein (GFAP), a cytoskeleton protein specifically expressed in astrocytes. In vitro characterization of this antibody using both a cell-free system and a cell model system of apoptosis demonstrated that the antibody (termed GFAP caspase-cleavage product antibody or GFAP-CCP Ab) immunolabeled the predicted caspase-cleavage fragment, but not full-length GFAP, by Western blot analysis. To determine whether caspases cleave GFAP in vivo, tissue sections from control and AD brains were examined by immunohistochemistry using the GFAP-CCP Ab. Two prominent features of staining were evident: immunolabeling of degenerating astrocytes in proximity to blood vessels and staining within plaque-rich regions of the AD brain. Furthermore, co-localization of the GFAP-CCP Ab and an antibody specific to active caspase-3 was demonstrated within damaged astrocytes of the AD brain. These data suggest that the activation of caspases and cleavage of cellular proteins such as GFAP may contribute to astrocyte injury and damage in the AD brain.

Caspase‐3 activation in astrocytes following postnatal excitotoxic damage correlates with cytoskeletal remodeling but not with cell death or proliferation

“…For double fluorescent labeling, sections were processed for cleaved caspase-3 labeling as described earlier but using a Cy3-conjugated anti-rabbit secondary antibody (1:1,000; PA4300V, Amersham Pharmacia). Afterwards, sections were incubated overnight at 4°C and 1 h at room temperature in either monoclonal mouse anti-NeuN (1:1,000; MAB377, Chemicon); polyclonal rabbit anti-GFAP (1:1,800; Z-0224, Dakopatts); monoclonal mouse anti-vimentin (1:1,000; M0725, Dakopatts); monoclonal mouse anti-APC (CC-1) (1:500, OP80, Calbiochem); monoclonal mouse anti-BrdU (1:80, B5002, Dakopatts); or polyclonal rabbit anti-CCP-GFAP (Mouser et al, 2006) diluted in BB. Sections were then washed in TBS 1 1% triton X-100 and incubated for 1 h at room temperature with either Cy2-conjugated anti-rabbit secondary antibody (1:1,000; PA-42004, Amersham Pharmacia) or Cy2-conjugated anti-mouse secondary antibody (1:1,000; PA42002, Amersham Pharmacia).…”

“…wiley.com. ] ments by using a recently described site-directed caspase-cleavage antibody specific to GFAP, termed CCP-GFAP (Mouser et al, 2006). CCP-GFAP immunostaining was located within the lesion core from 10 h until the last survival time examined, peaking at day 1.…”

“…Decreased GFAP immunoreactivity induced by excitotoxicity and ischemia within the lesion core during the first 24 h postlesion is a well characterized event, both in the immature (Acarin et al, 1999b), adult (Dihne et al, 2001), and aged brain (Castillo-Ruiz et al, 2007). As caspasegenerated GFAP fragments are not recognized by commonly used GFAP antibodies directed to the full length molecule (Mouser et al, 2006), caspase-mediated cleavage of GFAP could, at least in part, explain the decrease in GFAP immunoreactivity observed in the lesion core at early times in several injury paradigms. This decrease in GFAP, together with the existence of astrocytes showing low GFAP content in gray matter (Walz, 2000), obviously leads an underestimation of the number of GFAP-positive astrocytes showing caspase-3 at early times (see Fig.…”

“…Similarly, in vitro studies have also suggested that caspase-3 proteolytic activity plays a crucial role in excitotoxin-induced neuronal apoptosis (Allen et al, 1999;Du et al, 1997;Tenneti and Lipton, 2000), although caspase-3 activation and DNA fragmentation often do not co-localize within the same cell at the same time (Brecht et al, 2001). In addition, expression of caspase-3 has been described in oligodendrocytes (Beer et al, 2000;Nottingham and Springer, 2003) and astrocytes following CNS damage (Beer et al, 2000;Benjelloun et al, 2003;Mouser et al, 2006;Narkilahti et al, 2003;Su et al, 2000).…”

Neuroprotective roles of the P2Y2 receptor

“…In CNS injury, the upregulation and activation of MMPs, a known component of the P2Y 2 R signaling pathway [38], can be either beneficial or detrimental depending on the length of time after the injury, the profile of the inflammatory cells at the injury site, and the substrates present [201]. During neuroinflammation, astrocytes undergo morphological and functional changes (i.e., reactive gliosis) characterized by hypertrophy, proliferation, upregulation of the intermediate filament protein glial fibrillary acidic protein, accumulation of activated glial cells around plaques, adhesion of cells to Aβ peptides, internalization and degradation of Aβ peptides by activated glial cells, expression of proteinases required for Aβ peptide catabolism, production of arachidonic acid and related proinflammatory substances in the vicinity of plaques, and regulation of regenerative processes in the brain [202][203][204][205][206]. Activated glia have been shown to produce neurotrophic factors [207,208] and stimulate neuronal outgrowth during development and repair of damaged brain cells in the adult [209].…”