Phenylbutyric Acid Rescues Endoplasmic Reticulum Stress-Induced Suppression of APP Proteolysis and Prevents Apoptosis in Neuronal Cells

BACKGROUND: The familial and sporadic forms of Alzheimer's disease (AD) have an identical pathology with a severe disparity in the time of onset [1]. The pathological similarity suggests that epigenetic processes may phenocopy the Familial Alzheimer's disease (FAD) mutations within sporadic AD. Numerous groups have demonstrated that FAD mutations in presenilin result in 'loss of function' of gamma-secretase mediated APP cleavage [2], [3], [4], [5]. Accordingly, ER stress is prominent within the pathologically impacted brain regions in AD patients [6] and is reported to inhibit APP trafficking through the secretory pathway [7], [8]. As the maturation of APP and the cleaving secretases requires trafficking through the secretory pathway [9], [10], [11], we hypothesized that ER stress may block trafficking requisite for normal levels of APP cleavage and that the small molecular chaperone 4-phenylbutyrate (PBA) may rescue the proteolytic deficit. METHODOLOGY/PRINCIPAL FINDINGS: The APP-Gal4VP16/Gal4-reporter screen was stably incorporated into neuroblastoma cells in order to assay gamma-secretase mediated APP proteolysis under normal and pharmacologically induced ER stress conditions. Three unrelated pharmacological agents (tunicamycin, thapsigargin and brefeldin A) all repressed APP proteolysis in parallel with activation of unfolded protein response (UPR) signaling-a biochemical marker of ER stress. Co-treatment of the gamma-secretase reporter cells with PBA blocked the repressive effects of tunicamycin and thapsigargin upon APP proteolysis, UPR activation, and apoptosis. In unstressed cells, PBA stimulated gamma-secretase mediated cleavage of APP by 8-10 fold, in the absence of any significant effects upon amyloid production, by promoting APP trafficking through the secretory pathway and the stimulation of the non-pathogenic alpha/gamma-cleavage. CONCLUSIONS/SIGNIFICANCE: ER stress represses gamma-secretase mediated APP proteolysis, which replicates some of the proteolytic deficits associated with the FAD mutations. The small molecular chaperone PBA can reverse ER stress induced effects upon APP proteolysis, trafficking and cellular viability. Pharmaceutical agents, such as PBA, that stimulate alpha/gamma-cleavage of APP by modifying intracellular trafficking should be explored as AD therapeutics

Disruption of Protein Kinase A in Mice Enhances Healthy Aging

Mutations that cause a reduction in protein kinase A (PKA) activity have been shown to extend lifespan in yeast. Loss of function of mammalian RIIβ, a regulatory subunit of PKA expressed in brain and adipose tissue, results in mice that are lean and insulin sensitive. It was therefore hypothesized that RIIB null (RIIβ−/−) mice would express anti-aging phenotypes. We conducted lifespan studies using 40 mutant and 40 wild type (WT) littermates of equal gender numbers and found that both the median and maximum lifespans were significantly increased in mutant males compared to WT littermates. The median lifespan was increased from 884 days to 1005 days (p = 0.006 as determined by the log rank test) and the 80% lifespan (defined here as 80% deaths) was increased from 941 days to 1073 days (p = 0.004 as determined by the Wang-Allison test). There was no difference in either median or 80% lifespan in female genotypes. WT mice of both genders became increasingly obese with age, while mutant mice maintained their lean phenotype into old age. Adiposity was found to correlate with lifespan for males only. 50% of male mice between 30 and 35 g, corresponding to about 5% body fat, for either genotype lived over 1000 days. No male mouse outside of this weight range achieved this lifespan. During their last month of life, WT mice began losing weight (a total of 8% and 15% of body weight was lost for males and females, respectively), but RIIβ−/− male mice maintained their lean body mass to end of life. This attenuation of decline was not seen in female mutant mice. Old male mutant mice were insulin sensitive throughout their life. Both genders showed modestly lower blood glucose levels in old mutants compared to WT. Male mutants were also resistant to age-induced fatty liver. Pathological assessment of tissues from end of life male mutant mice showed a decrease in tumor incidence, decreased severity of renal lesions, and a trend towards a decrease in age-related cardiac pathology. These findings help establish the highly conserved nature of PKA and suggest that disruption of PKA affects physiological mechanisms known to be associated with healthy aging

Deletion of P58^IPK, the cellular inhibitor of the protein kinases PKR and PERK, causes bone changes and joint degeneration in mice

Objective: Protein kinase-like endoplasmic reticulum kinase (PERK) and protein kinase R (PKR) are implicated in endoplasmic reticulum stress-induced arthritis and pro-inflammatory cytokine-mediated cartilage degradation in vitro, respectively. We determined whether knockout of the cellular inhibitor of PERK and PKR, P58IPK causes joint degeneration in vivo and whether these molecules are activated in human osteoarthritis (OA). Materials and Methods: Sections of knee joints from P58IPK-null and wild-type mice aged 12–13 and 23–25 months were stained with toluidine blue and scored for degeneration using the osteoarthritis research society international (OARSI) system. Bone changes were assessed by radiology and high-resolution micro-computed tomography of hind limbs. Sections from the medial tibial plateaus of two human knees, removed in total knee replacement surgery for OA, were immunolabelled for phosphorylated PERK and PKR and P58IPK. Results: Knockout mice exhibited narrower tibiae (p = 0.0031) and smaller epiphyses in tibiae (p = 0.0004) and femora (p = 0.0214). Older knockout mice had reduced total volume inside the femoral periosteal envelope (p = 0.023), reduced tibial (p = 0.03), and femoral (p = 0.0012) bone volumes (BV) and reduced femoral BV fraction (p = 0.025). Compared with wild-types, younger P58IPK-null mice had increased OARSI scores in medial femoral condyles (p = 0.035). Thirty four percent of null mice displayed severe joint degeneration with complete articular cartilage loss from the medial compartment and heterotopic chondro-osseous tissue in the medial joint capsule. Phosphorylated PERK and PKR were localized throughout human osteoarthritic tibial plateaus but, in particular, in areas exhibiting the most degeneration. There was limited expression of P58IPK. Conclusion: This study is the first to reveal a critical role for P58IPK in maintaining joint integrity in vivo, implicating the PKR and PERK stress signaling pathways in bony changes underlying the pathogenesis of joint degeneration

XRCC1 haploinsufficiency in mice has little effect on aging, but adversely modifies exposure-dependent susceptibility

Oxidative DNA damage plays a role in disease development and the aging process. A prominent participant in orchestrating the repair of oxidative DNA damage, particularly single-strand breaks, is the scaffold protein XRCC1. A series of chronological and biological aging parameters in XRCC1 heterozygous (HZ) mice were examined. HZ and wild-type (WT) C57BL/6 mice exhibit a similar median lifespan of ~26 months and a nearly identical maximal life expectancy of ~37 months. However, a number of HZ animals (7 of 92) showed a propensity for abdominal organ rupture, which may stem from developmental abnormalities given the prominent role of XRCC1 in endoderm and mesoderm formation. For other end-points evaluated—weight, fat composition, blood chemistries, condition of major organs, tissues and relevant cell types, behavior, brain volume and function, and chromosome and telomere integrity—HZ mice exhibited by-and-large a normal phenotype. Treatment of animals with the alkylating agent azoxymethane resulted in both liver toxicity and an increased incidence of precancerous lesions in the colon of HZ mice. Our study indicates that XRCC1 haploinsufficiency in mammals has little effect on chronological longevity and many key biological markers of aging in the absence of environmental challenges, but may adversely affect normal animal development or increase disease susceptibility to a relevant genotoxic exposure

XRCC1 haploinsufficiency in mice has little effect on aging, but adversely modifies exposure-dependent susceptibility

Oxidative DNA damage plays a role in disease development and the aging process. A prominent participant in orchestrating the repair of oxidative DNA damage, particularly single-strand breaks, is the scaffold protein XRCC1. A series of chronological and biological aging parameters in XRCC1 heterozygous (HZ) mice were examined. HZ and wild-type (WT) C57BL/6 mice exhibit a similar median lifespan of ~26 months and a nearly identical maximal life expectancy of ~37 months. However, a number of HZ animals (7 of 92) showed a propensity for abdominal organ rupture, which may stem from developmental abnormalities given the prominent role of XRCC1 in endoderm and mesoderm formation. For other end-points evaluated—weight, fat composition, blood chemistries, condition of major organs, tissues and relevant cell types, behavior, brain volume and function, and chromosome and telomere integrity—HZ mice exhibited by-and-large a normal phenotype. Treatment of animals with the alkylating agent azoxymethane resulted in both liver toxicity and an increased incidence of precancerous lesions in the colon of HZ mice. Our study indicates that XRCC1 haploinsufficiency in mammals has little effect on chronological longevity and many key biological markers of aging in the absence of environmental challenges, but may adversely affect normal animal development or increase disease susceptibility to a relevant genotoxic exposure