Inhibitors of ORAI1 Prevent Cytosolic Calcium-Associated Injury of Human Pancreatic Acinar Cells and Acute Pancreatitis in 3 Mouse Models.

BACKGROUND & AIMS: Sustained activation of the cytosolic calcium concentration induces injury to pancreatic acinar cells and necrosis. The calcium release-activated calcium modulator ORAI1 is the most abundant Ca(2+) entry channel in pancreatic acinar cells; it sustains calcium overload in mice exposed to toxins that induce pancreatitis. We investigated the roles of ORAI1 in pancreatic acinar cell injury and the development of acute pancreatitis in mice. METHODS: Mouse and human acinar cells, as well as HEK 293 cells transfected to express human ORAI1 with human stromal interaction molecule 1, were hyperstimulated or incubated with human bile acid, thapsigargin, or cyclopiazonic acid to induce calcium entry. GSK-7975A or CM_128 were added to some cells, which were analyzed by confocal and video microscopy and patch clamp recordings. Acute pancreatitis was induced in C57BL/6J mice by ductal injection of taurolithocholic acid 3-sulfate or intravenous' administration of cerulein or ethanol and palmitoleic acid. Some mice then were given GSK-7975A or CM_128, which inhibit ORAI1, at different time points to assess local and systemic effects. RESULTS: GSK-7975A and CM_128 each separately inhibited toxin-induced activation of ORAI1 and/or activation of Ca(2+) currents after Ca(2+) release, in a concentration-dependent manner, in mouse and human pancreatic acinar cells (inhibition >90% of the levels observed in control cells). The ORAI1 inhibitors also prevented activation of the necrotic cell death pathway in mouse and human pancreatic acinar cells. GSK-7975A and CM_128 each inhibited all local and systemic features of acute pancreatitis in all 3 models, in dose- and time-dependent manners. The agents were significantly more effective, in a range of parameters, when given at 1 vs 6 hours after induction of pancreatitis. CONCLUSIONS: Cytosolic calcium overload, mediated via ORAI1, contributes to the pathogenesis of acute pancreatitis. ORAI1 inhibitors might be developed for the treatment of patients with pancreatitis.

STIM1, an essential and conserved component of store-operated Ca2+ channel function.

Store-operated Ca2+ (SOC) channels regulate many cellular processes, but the underlying molecular components are not well defined. Using an RNA interference (RNAi)-based screen to identify genes that alter thapsigargin (TG)-dependent Ca2+ entry, we discovered a required and conserved role of Stim in SOC influx. RNAi-mediated knockdown of Stim in Drosophila S2 cells significantly reduced TG-dependent Ca2+ entry. Patch-clamp recording revealed nearly complete suppression of the Drosophila Ca2+ release-activated Ca2+ (CRAC) current that has biophysical characteristics similar to CRAC current in human T cells. Similarly, knockdown of the human homologue STIM1 significantly reduced CRAC channel activity in Jurkat T cells. RNAi-mediated knockdown of STIM1 inhibited TG- or agonist-dependent Ca2+ entry in HEK293 or SH-SY5Y cells. Conversely, overexpression of STIM1 in HEK293 cells modestly enhanced TG-induced Ca2+ entry. We propose that STIM1, a ubiquitously expressed protein that is conserved from Drosophila to mammalian cells, plays an essential role in SOC influx and may be a common component of SOC and CRAC channels.

Family-based association between Alzheimer's disease and variants in UBQLN1.

BACKGROUND: Recent analyses suggest that the known Alzheimer's disease genes account for less than half the genetic variance in this disease. The gene encoding ubiquilin 1 (UBQLN1) is one of several candidate genes for Alzheimer's disease located near a well-established linkage peak on chromosome 9q22. METHODS: We evaluated 19 single-nucleotide polymorphisms in three genes within the chromosome 9q linkage region in 437 multiplex families with Alzheimer's disease from the National Institute of Mental Health (NIMH) sample (1439 subjects). We then tested the single-nucleotide polymorphisms showing a positive result in an independently identified set of 217 sibships discordant for Alzheimer's disease (Consortium on Alzheimer's Genetics [CAG] sample; 489 subjects) and assessed the functional effect of an implicated single-nucleotide polymorphism in brain tissue from 25 patients with Alzheimer's disease and 17 controls. RESULTS: In the NIMH sample, we observed a significant association between Alzheimer's disease and various single-nucleotide polymorphisms in UBQLN1. We confirmed these associations in the CAG sample. The risk-conferring haplotype in both samples was defined by a single intronic single-nucleotide polymorphism located downstream of exon 8. The risk allele was associated with a dose-dependent increase in an alternatively spliced UBQLN1 (lacking exon 8) transcript in RNA extracted from brain samples of patients with Alzheimer's disease. CONCLUSIONS: Our findings suggest that genetic variants in UBQLN1 on chromosome 9q22 substantially increase the risk of Alzheimer's disease, possibly by influencing alternative splicing of this gene in the brain.

Is alpha-T catenin (VR22) an Alzheimer's disease risk gene?

BACKGROUND: Recently, conflicting reports have been published on the potential role of genetic variants in the alpha-T catenin gene (VR22; CTNNA3) on the risk for Alzheimer's disease. In these papers, evidence for association is mostly observed in multiplex families with Alzheimer's disease, whereas case-control samples of sporadic Alzheimer's disease are predominantly negative. METHODS: After sequencing VR22 in multiplex families with Alzheimer's disease linked to chromosome 10q21, we identified a novel non-synonymous (Ser596Asn; rs4548513) single nucleotide polymorphism (SNP). This and four non-coding SNPs were assessed in two independent samples of families with Alzheimer's disease, one with 1439 subjects from 437 multiplex families with Alzheimer's disease and the other with 489 subjects from 217 discordant sibships. RESULTS: A weak association with the Ser596Asn SNP in the multiplex sample, predominantly in families with late-onset Alzheimer's disease (p = 0.02), was observed. However, this association does not seem to contribute substantially to the chromosome 10 Alzheimer's disease linkage signal that we and others have reported previously. No evidence was found of association with any of the four additional SNPs tested in the multiplex families with Alzheimer's disease. Finally, the Ser596Asn change was not associated with the risk for Alzheimer's disease in the independent discordant sibship sample. CONCLUSIONS: This is the first study to report evidence of an association between a potentially functional, non-synonymous SNP in VR22 and the risk for Alzheimer's disease. As the underlying effects are probably small, and are only seen in families with multiple affected members, the population-wide significance of this finding remains to be determined.

High-throughput assessment of mitochondrial membrane potential in situ using fluorescence resonance energy transfer.

Mitochondrial dysfunction causes dozens of debilitating diseases, and is implicated in the etiology of type 2 diabetes, Parkinson's, and Alzheimer's diseases, among others. However, development of mitochondrially targeted therapeutic agents has been impeded by the lack of high-throughput screening techniques that are capable of distinguishing in intact cells the mitochondrial membrane potential (deltapsi(m)) from the plasma membrane potential, (deltapsi(p)). We report here a fluorescence resonance energy transfer (FRET) assay that specifically monitors deltapsi(m) that is not confounded by background signal arising from potentiometric dye responding to deltapsi(p). The technique relies on energy transfer between nonyl acridine orange (NAO), which stains diphosphatidyl glycerol (cardiolipin) that is indigenous to the inner mitochondrial membrane, and tetramethylrhodamine methyl ester (TMR), a potentiometric dye that is sequestered by mitochondria as a Nernstian function of deltapsi(m) and concentration. FRET occurs only when both dyes co-localize to the mitochondria, and results in quenching of NAO emission by TMR in proportion to deltapsi(m). Validation studies using compounds with well-characterized mitochondrial effects, including oligomycin, CCCP+, bongkrekic acid, cyclosporin A, nigericin, ADP, and ruthenium red, demonstrate that the FRET-based deltapsi(m) assay responds in accord with the known pharmacology. Validation studies assessing the suitability of the technique for high-throughput compound screening indicate that the assay provides a sensitive and robust assessment not only of mitochondrial integrity in situ, but also, when used in conjunction with agents such as cyclosporin A, an indicator of permeability transition.

Modulation of gamma-secretase reduces beta-amyloid deposition in a transgenic mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is characterized pathologically by the abundance of senile plaques and neurofibrillary tangles in the brain. We synthesized over 1200 novel gamma-secretase modulator (GSM) compounds that reduced Abeta(42) levels without inhibiting epsilon-site cleavage of APP and Notch, the generation of the APP and Notch intracellular domains, respectively. These compounds also reduced Abeta(40) levels while concomitantly elevating levels of Abeta(38) and Abeta(37). Immobilization of a potent GSM onto an agarose matrix quantitatively recovered Pen-2 and to a lesser degree PS-1 NTFs from cellular extracts. Moreover, oral administration (once daily) of another potent GSM to Tg 2576 transgenic AD mice displayed dose-responsive lowering of plasma and brain Abeta(42); chronic daily administration led to significant reductions in both diffuse and neuritic plaques. These effects were observed in the absence of Notch-related changes (e.g., intestinal proliferation of goblet cells), which are commonly associated with repeated exposure to functional gamma-secretase inhibitors (GSIs).

Genetic association of Alzheimer's disease with multiple polymorphisms in alpha-2-macroglobulin.

Alpha-2-Macroglobulin (A2M) is a highly plausible candidate gene for Alzheimer's disease (AD) in a region of chromosome 12 that has numerous independent reports of genetic linkage. We previously reported that a 5 bp deletion in A2M was associated with AD in a subset of the National Institute of Health (NIMH) Genetics Initiative AD family sample. Efforts to replicate this association finding in case - control samples have been largely negative, while those in family samples have been more positive. We hypothesized that variable findings regarding this deletion, along with variable reports of association with V1000I, another polymorphism in the gene, result from linkage disequilibrium in the area as well as ascertainment differences between family-based and case-control studies. Thus, we resequenced the A2M locus to identify novel polymorphisms to test for genetic association with AD. We identified seven novel polymorphisms and tested them in the full NIMH sample of 1439 individuals in 437 families. We found significant genetic association of the 5 bp deletion and two novel polymorphisms with AD. Substantial linkage disequilibrium was detected across the gene as a whole, and haplotype analysis also showed significant association between AD and groups of A2M polymorphisms. Several of these polymorphisms and haplotypes remain significantly associated with AD even after correction for multiple testing. Taken together, these findings, and the positive reports in other family-based studies, continue to support a potential role for A2M or a nearby gene in AD. However, the negative case - control studies suggest that any underlying pathogenic polymorphisms have a modest effect, and may operate primarily among individuals with a family history of AD.