Caspase-cleaved Fragment Research Articles

Amyloid beta protein toxicity mediated by the formation of amyloid-beta protein precursor complexes.

The amyloid-beta protein precursor, a type 1 transmembrane protein, gives rise to the amyloid beta-protein, a neurotoxic peptide postulated to be involved in the pathogenesis of Alzheimer's disease. Here, we show that soluble amyloid beta protein accelerates amyloid precursor protein complex formation, a process that contributes to neuronal cell death. The mechanism of cell death involves the recruitment of caspase-8 to the complex, followed by intracytoplasmic caspase cleavage of amyloid precursor protein. In vivo, the levels of soluble amyloid beta protein correlated with caspase-cleaved fragments of the amyloid precursor protein in brains of Alzheimer's disease subjects. These findings suggest that soluble amyloid beta protein-induced multimerization of the amyloid precursor protein may be another mechanism by which amyloid beta protein contributes to synapse loss and neuronal cell death seen in Alzheimer's disease.

Tumor Necrosis Factor Alpha and Apoptosis in Helicobacter pylori Related Progressive Gastric Damage: A Possible Mechanism of Immune System Involvement in Epithelial Turnover Regulation

Helicobacter pylori (HP) related inflammation is mediated by tumour necrosis factor alpha (TNFα), which “in vitro” increases epithelial apoptosis in response to infection. In the early stages of HP gastritis, a raised epithelial apoptosis occurs; this phenomenon becomes less evident with progression towards intestinal metaplasia. Aim of our study was to analyze “in vivo” mucosal TNFα in relation to epithelial apoptosis in the progression of HP related histological damage. Antral biopsies from 20 HP positive patients were retrospectively studied: 10 with and 10 without intestinal metaplasia (IM and CG group respectively); samples of 10 dyspeptics with normal HP negative stomach (N) were used as control. The following parameters were evaluated by immunohistochemistry: 85 kDa caspase-cleaved fragment (p85) of human poly (ADP-ribose) polymerase (PARP) labelling index (LI) as marker of apoptosis and TNFα LI in stromal cells as marker of inflammatory response. Both epithelial apoptosis and mucosal TNFα expression were higher in chronic active gastritis compared to intestinal metaplasia and controls (PARP and TNFα LI: CG>IM>N; ANOVA & Student-Neumann-Keuls; p<0.05 and p<0.01, respectively). Pearson' s coefficient showed a significant correlation between PARP and TNFα LI in IM and CG groups. Our data show that mucosal TNFα, similarly to what suggested “in vitro”, may be related “in vivo” to epithelial apoptosis thus suggesting a possible mechanism for immune system involvement in the control of gastric epithelial turnover.

Apoptosis and microglial activation in influenza encephalopathy.

During influenza epidemics in Japan, the number of children with acute encephalopathies and encephalitis has recently increased. Although the pathophysiologies remain unclear, there is usually brain edema with evidence of damage to the blood-brain-barrier (BBB). We investigated the glial reaction and apoptosis in brains of eight such cases comprising two of acute necrotizing encephalopathy and six of influenza encephalopathy, and compared the results with those in five control brains. Apoptosis, evidenced by chromatin condensation and fragmentation in hematoxylin sections, in situ end labeling of fragmented DNA (TUNEL) and DNA laddering, was observed in neurons and glial cells in four brains with influenza encephalopathy. In the TUNEL-positive brains, the increase in microglia was greater than in the TUNEL-negative brains. Immunoreactivity for active-caspase 3, demonstrated by immunohistochemistry, and the overexpression of a caspase-cleaved fragment of poly(ADP-ribose) polymerase, demonstrated by Western blotting, indicated that activation of caspase 3 is involved in the apoptotic pathway in the brains of influenza encephalopathy cases. Apoptosis or specific pathological processes that cause apoptosis may give rise to aggravated encephalopathy.

Caspase-9 Activation and Caspase Cleavage of tau in the Alzheimer's Disease Brain

Accumulating evidence supports a role for the activation of proteolytic enzymes, caspases, in the Alzheimer's disease (AD) brain. Neurons committed to apoptosis may do so through a mitochondrial pathway employing caspase-9 or through an alternative, receptor-mediated pathway involving caspase-8. Considering the role of mitochondrial dysfunction in AD, we examined the possible activation of caspase-9 in the AD brain using an antibody that recognizes the active fragments of caspase-9, but not the full-length proform of the enzyme. In vivo immunohistochemical analysis demonstrated little caspase-9 activation in the majority of hippocampal brain sections from control brains. However, labeling of neurons as well as dystrophic neurites within plaque regions was observed in all AD hippocampal brain sections examined. In addition, active caspase-9 was colocalized with active caspase-8 and the accumulation of caspase-3-cleavage products of fodrin. The activation of caspase-9 was also observed in neurons positive for oxidative damage to DNA/RNA. A quantitative analysis indicates that as the number of neurons containing neurofibrillary tangles (NFTs) increases, the extent of caspase-9 activation decreases, supporting the idea that caspase-9 activation may precede NFT formation. In addition, a site-directed caspase-cleavage antibody was designed to the amino-terminal caspase-3 consensus cleavage site located in tau, and shown to be an effective marker for caspase-cleaved fragments of tau in vitro. Analysis with this antibody using age-matched control or AD brain sections demonstrated no staining in control brains while widespread labeling of NFTs, neuropil threads, and dystrophic neurites was observed in AD sections. Taken together, these results demonstrate the activation of caspases and cleavage of tau in the AD brain, events which may precede and lead to the formation of NFTs.

Cleavage and phosphorylation of XRCC4 protein induced by X-irradiation

We report the p35 and p60 forms of XRCC4 protein, appearing in human leukemia MOLT-4 or U937 cells following X-irradiation or hyperthermia. p35 appeared in conjunction with the cleavage of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and the fragmentation of internucleosomal DNA, and was suppressed by Ac-DEVD-CHO. p35 was also produced in vitro by treating MOLT-4 cell lysate with recombinant caspases, suggesting that p35 was a caspase-cleaved fragment of XRCC4 in apoptotic cell death. p60 was sensitive to treatment with phosphatase or wortmannin and was undetectable in M059J cells deficient in DNA-PKcs. However, p60 was found in ataxia-telangiectasia cells after irradiation. These results indicated p60 as a phosphorylated form of XRCC4, requiring DNA-PKcs but not ataxia-telangiectasia mutated (ATM).