7 research outputs found
Additional file 2: Figure S2. of Spatio-temporal activation of caspase-8 in myeloid cells upon ischemic stroke
Positive controls for immunohistochemistry antibodies. Antibodies detecting cleaved caspase-8 and cleaved caspase-3 showed positive signal in sections from human colon tissue. Antibody detecting CD68 showed positive signal in sections from human lymph node. Scale bar represents 100Â Îźm. (TIF 1191Â kb
<i>Caspase-8</i>, association with Alzheimer’s Disease and functional analysis of rare variants
<div><p>The accumulation of amyloid beta (Aβ) peptide (Amyloid cascade hypothesis), an APP protein cleavage product, is a leading hypothesis in the etiology of Alzheimer's disease (AD). In order to identify additional AD risk genes, we performed targeted sequencing and rare variant burden association study for nine candidate genes involved in the amyloid metabolism in 1886 AD cases and 1700 controls. We identified a significant variant burden association for the gene encoding caspase-8, <i>CASP8</i> (p = 8.6x10<sup>-5</sup>). For two CASP8 variants, p.K148R and p.I298V, the association remained significant in a combined sample of 10,820 cases and 8,881 controls. For both variants we performed bioinformatics structural, expression and enzymatic activity studies and obtained evidence for loss of function effects. In addition to their role in amyloid processing, caspase-8 and its downstream effector caspase-3 are involved in synaptic plasticity, learning, memory and control of microglia pro-inflammatory activation and associated neurotoxicity, indicating additional mechanisms that might contribute to AD. As caspase inhibition has been proposed as a mechanism for AD treatment, our finding that AD-associated CASP8 variants reduce caspase function calls for caution and is an impetus for further studies on the role of caspases in AD and other neurodegenerative diseases.</p></div
Age and ApoE4 genotypes for subjects in discovery sample.
<p>Age and ApoE4 genotypes for subjects in discovery sample.</p
Caspase-8 modeling and expression.
<p>(<b>A</b>) Schematic illustration of pro-caspase-8 protein and its p43, p18 and p10 fragments resulting from proteolytic processing and activation. (<b>B</b>) Protein folding (top) and 3D model (bottom) of caspase-8 DED<sub>2</sub> (left side) and p18 domain (right side). The K<sup>148</sup> and I<sup>298</sup> variants are depicted in red color. F<sup>122</sup> and L<sup>123</sup> of the hydrophobic FL motif within the DED<sub>2</sub> is shown in purple, and critical H<sup>317</sup> and C<sup>360</sup> active site residues within the p18 domain are in green. (<b>C</b>) SK-N-BE(2) cells were transfected with expression vectors encoding WT-, K148R-, or I298V-caspase-8 and mock as control. Corresponding immunoblot analysis, 24 h post-transfection, indicating the expression levels for pro-caspase-8 and its p43, p18 and p10 fragments. For the LOAD caspase-8 variant, two clones (<i>a</i> and <i>b</i>) are presented.(<b>D</b>) Representative confocal images of SK-N-BE(2) cells transfected as described in panel C. The cleaved caspase-8 is labeled red and Hoechst counterstained nuclei are blue. Images were taken 24 h after transfection.</p
Variant count and association of <i>CASP8</i> K148R and I298V.
<p>Variant count and association of <i>CASP8</i> K148R and I298V.</p
Caspase-8 enzymatic activity.
<p>SK-N-BE(2) cells were transfected with expression vectors encoding WT-, K148R-, or I298V-caspase-8 and mock as control. (A) Caspase-8 (LETDase) and (B) Caspase-3-like (DEVDase) activities were measured 24 h post-transfection. Data are presented as fold over mock untreated. Statistics and error bars: mean±s.d. n = 8 of biological replicates. Data was analyzed as comparison to Caspase-8 WT using two-sided student’s t-test. *P< 0.05; **P< 0.01 and ***P< 0.001.</p
Coverage, aggregated variant count and variant-burden test of association in discovery sample.
<p>Coverage, aggregated variant count and variant-burden test of association in discovery sample.</p