Expression and activation of caspase-6 in human fetal and adult tissues.

Caspase-6 is an effector caspase that has not been investigated thoroughly despite the fact that Caspase-6 is strongly activated in Alzheimer disease brains. To understand the full physiological impact of Caspase-6 in humans, we investigated Caspase-6 expression. We performed western blot analyses to detect the pro-Caspase-6 and its active p20 subunit in fetal and adult lung, kidney, brain, spleen, muscle, stomach, colon, heart, liver, skin, and adrenals tissues. The levels were semi-quantitated by densitometry. The results show a ubiquitous expression of Caspase-6 in most fetal tissues with the lowest levels in the brain and the highest levels in the gastrointestinal system. Caspase-6 active p20 subunits were only detected in fetal stomach. Immunohistochemical analysis of a human fetal embryo showed active Caspase-6 positive apoptotic cells in the dorsal root ganglion, liver, lung, kidney, ovary, skeletal muscle and the intestine. In the adult tissues, the levels of Caspase-6 were lower than in fetal tissues but remained high in the colon, stomach, lung, kidney and liver. Immunohistological analyses revealed that active Caspase-6 was abundant in goblet cells and epithelial cells sloughing off the intestinal lining of the adult colon. These results suggest that Caspase-6 is likely important in most tissues during early development but is less involved in adult tissues. The low levels of Caspase-6 in fetal and adult brain indicate that increased expression as observed in Alzheimer Disease is a pathological condition. Lastly, the high levels of Caspase-6 in the gastrointestinal system indicate a potential specific function of Caspase-6 in these tissues

Inflammation-induced tumorigenesis in mouse colon is caspase-6 independent.

Caspases play an important role in maintaining tissue homeostasis. Active Caspase-6 (Casp6) is considered a novel therapeutic target against Alzheimer disease (AD) since it is present in AD pathological brain lesions, associated with age-dependent cognitive decline, and causes age-dependent cognitive impairment in the mouse brain. However, active Casp6 is highly expressed and activated in normal human colon epithelial cells raising concerns that inhibiting Casp6 in AD may promote colon carcinogenesis. Furthermore, others have reported rare mutations of Casp6 in human colorectal cancers and an effect of Casp6 on apoptosis and metastasis of colon cancer cell lines. Here, we investigated the role of Casp6 in inflammation-associated azoxymethane/dextran sulfate sodium (AOM/DSS) colon cancer in Casp6-overexpressing and -deficient mice. In wild-type mice, AOM/DSS-induced tumors had significantly higher Casp6 mRNA, protein and activity levels compared to normal adjacent colon tissues. Increased human Casp6 or absence of Casp6 expression in mice colon epithelial cells did not change colonic tumor multiplicity, burden or distribution. Nevertheless, the incidence of hyperplasia was slightly reduced in human Casp6-overexpressing colons and increased in Casp6 null colons. Overexpression of Casp6 did not affect the grade of the tumors while all tumors in heterozygous or homozygous Casp6 null colons were high grade compared to only 50% high grade in wild-type mice. Casp6 levels did not alter cellular proliferation and apoptosis. These results suggest that Casp6 is unlikely to be involved in colitis-associated tumors

Activation of caspase-6 in aging and mild cognitive impairment

Active caspase-6 (Csp6) and Tau cleaved by Csp6 (TauΔCsp6) are abundant in neuritic plaques (NPs), neuropil threads (NPTs), and neurofibrillary tangles (NFTs) in end-stage Alzheimer\u27s disease (AD) (Guo H, Albrecht S, Bourdeau M, Petzke T, Bergeron C, LeBlanc AC: Active caspase-6 and caspase-6 cleaved Tau in neuropil threads, neuritic plaques and neurofibrillary tangles of Alzheimer\u27s disease. Am J Pathol 2004, 165:523-531). The goal of this study was to determine whether active Csp6 is present in young and aged noncognitively impaired (NCI); aged mild cognitively impaired (MCI); and aged mild, moderate, severe, and very severe AD individuals. Csp6 activity was assessed with anti-p20Csp6 and TauΔCsp6 immunoreactivity. Active Csp6 is present in NFTs, NPTs, and NPs at all stages of AD. Active Csp6 is present in NFTs of all MCI cases and present in NPTs and NPs of some MCI cases. Active Csp6 is present in NFTs and NPTs of all NCI cases but is absent in younger cases. The level of TauΔCsp6-positive NFTs and NPTs correlates inversely with global cognitive scores in NCI individuals. Therefore, Csp6 activity can occur with aging in the absence of AD and is always associated with clinical and pathological features of confirmed AD cases. Given the ability of active Csp6 to increase amyloid-β peptide production and cleave Tau and several synaptic proteins (LeBlanc AC, Liu H, Goodyer C, Bergeron C, Hammond J: Caspase-6 role in apoptosis of human neurons, amyloidogenesis and Alzheimer\u27s disease. J Biol Chem 1999, 274:23426-23436; Petzke TL, Rousselet E, Goodyer C, LeBlanc AC: Substrates of caspase-6 in human primary neurons: a proteomic study. Program No. 80.9. 2005 Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience. Online), we suggest that active Csp6 could be an early instigator of neuronal dysfunction. Copyright © American Society for Investigative Pathology

Hereditary Inclusion Body Myopathy-Linked p97/VCP Mutations in the NH2 Domain and the D1 Ring Modulate p97/VCP ATPase Activity and D2 Ring Conformation▿

Hereditary inclusion body myopathy associated with early-onset Paget disease of bone and frontotemporal dementia (hIBMPFTD) is a degenerative disorder caused by single substitutions in highly conserved residues of p97/VCP. All mutations identified thus far cluster within the NH2 domain or the D1 ring, which are both required for communicating conformational changes to adaptor protein complexes. In this study, biochemical approaches were used to identify the consequences of the mutations R155P and A232E on p97/VCP structure. Assessment of p97/VCP oligomerization revealed that p97R155P and p97A232E formed hexameric ring-shaped structures of ∼600 kDa. p97R155P and p97A232E exhibited an ∼3-fold increase in ATPase activity compared to wild-type p97 (p97WT) and displayed increased sensitivity to heat-induced upregulation of ATPase activity. Protein fluorescence analysis provided evidence for conformational differences in the D2 rings of both hIBMPFTD mutants. Furthermore, both mutations increased the proteolytic susceptibility of the D2 ring. The solution structures of all p97/VCP proteins revealed a didispersed distribution of a predominant hexameric population and a minor population of large-diameter complexes. ATP binding significantly increased the abundance of large-diameter complexes for p97R155P and p97A232E, but not p97WT or the ATP-binding mutant p97K524A. Therefore, we propose that hIBMPFTD p97/VCP mutants p97R155P and p97A232E possess structural defects that may compromise the mechanism of p97/VCP activity within large multiprotein complexes

Loss of Anti-Bax Function in Gerstmann-Sträussler- Scheinker Syndrome-Associated Prion Protein Mutants

Previously, we have shown the loss of anti-Bax function in Creutzfeldt Jakob disease (CJD)-associated prion protein (PrP) mutants that are unable to generate cytosolic PrP (CyPrP). To determine if the anti-Bax function of PrP modulates the manifestation of prion diseases, we further investigated the anti-Bax function of eight familial Gerstmann-Sträussler-Scheinker Syndrome (GSS)-associated PrP mutants. These PrP mutants contained their respective methionine ( M) or valine ( V) at codon 129. All of the mutants lost their ability to prevent Bax-mediated chromatin condensation or DNA fragmentation in primary human neurons. In the breast carcinoma MCF-7 cells, the F198S V, D202N V, P102L V and Q217R V retained, whereas the P102L M, P105L V, Y145stop M and Q212P M PrP mutants lost their ability to inhibit Bax-mediated condensed chromatin. The inhibition of Bax-mediated condensed chromatin depended on the ability of the mutants to generate cytosolic PrP. However, except for the P102L V, none of the mutants significantly inhibited Bax-mediated caspase activation. These results show that the cytosolic PrP generated from the GSS mutants is not as efficient as wild type PrP in inhibiting Bax-mediated cell death. Furthermore, these results indicate that the anti-Bax function is also disrupted in GSSassociated PrP mutants and is not associated with the difference between CJD and GSS

Steady state levels of Casp6, Casp1, and Casp3 in 8 different human fetal stomachs.

<p>Representative western blots containing 100 µg total protein/lane from fetal stomachs of different developmental ages with <b>A</b>. Neomarker anti-Casp6 antisera (top panel), neoepitope 10630 antisera (second panel), and β-Actin antibody (third panel), <b>B. Micrograph of human fetal stomach stained with 10630 anti-active Casp6 antiserum. C& D.</b> Western blot of (<b>C</b>) anti-Casp1 antisera (ProCasp1), neoepitope antisera to the p20 subunit of Casp1 (p20Casp1) and β-Actin antibody, and (<b>D</b>) anti-Casp3 antisera (ProCasp3), neoepitope antisera to the p20 subunit of Casp3 (p20Casp3) and β-Actin antibody.</p

Micrographs of immunohistochemical analyses of fetal tissues with 1277 neoepitope antisera detecting the active p20 subunit of Casp6.

<p>A. At low power (original magnification, 100x), immunopositive cells (arrows) in the dorsal root ganglion (DRG), in the anterior horn (AH), and in the dorsal root entry zone (DREZ) of the spinal cord white matter (WM). B. High power view of dorsal root ganglion (DRG: original magnification, 400×) indicating mitotic activity (solid arrowhead), features of early neuronal differentiation (open arrowheads) and several apoptotic immunopositive cells (arrows). C. High power view of anterior horn (AH: original magnification, 400×) indicating dividing cells (solid arrow heads), early maturation of cells (open arrowheads), and an immunopositive apoptotic precursor cell (arrow). WM; white matter). D. High power view of dorsal spinal cord (original magnification, 400×). Immunopositive finely granular (synaptic) pattern in the dorsal root entry zone (DREZ) of the spinal cord white matter (WM). DRG, dorsal root ganglion; DR, dorsal root; PH, posterior horn). E. Extensive extramedullary hematopoiesis (EMH) in the sinusoids that separate the cords of hepatocytes (HC) in the liver (original magnification 400×). In the hematopoietic islands, there are many immunopositive apoptotic cells (arrows).</p