KCa3.1 upregulation preserves endothelium‐dependent vasorelaxation during aging and oxidative stress

Endothelial oxidative stress develops with aging and reactive oxygen species impair endothelium‐dependent relaxation (EDR) by decreasing nitric oxide (NO) availability. Endothelial K_Ca3.1, which contributes to EDR, is upregulated by H₂O₂. We investigated whether K_Ca3.1 upregulation compensates for diminished EDR to NO during aging‐related oxidative stress. Previous studies identified that the levels of ceramide synthase 5 (CerS5), sphingosine, and sphingosine 1‐phosphate were increased in aged wild‐type and CerS2 mice. In primary mouse aortic endothelial cells (MAECs) from aged wild‐type and CerS2 null mice, superoxide dismutase (SOD) was upregulated, and catalase and glutathione peroxidase 1 (GPX1) were downregulated, when compared to MAECs from young and age‐matched wild‐type mice. Increased H₂O₂ levels induced Fyn and extracellular signal‐regulated kinases (ERKs) phosphorylation and K_Ca3.1 upregulation. Catalase/GPX1 double knockout (catalase^?/?/GPX1^?/?) upregulated K_Ca3.1 in MAECs. NO production was decreased in aged wild‐type, CerS2 null, and catalase^?/?/GPX1^?/? MAECs. However, K_Ca3.1 activation‐induced, N^G‐nitro‐l ‐arginine‐, and indomethacin‐resistant EDR was increased without a change in acetylcholine‐induced EDR in aortic rings from aged wild‐type, CerS2 null, and catalase^?/?/GPX1^?/? mice. CerS5 transfection or exogenous application of sphingosine or sphingosine 1‐phosphate induced similar changes in levels of the antioxidant enzymes and upregulated K_Ca3.1. Our findings suggest that, during aging‐related oxidative stress, SOD upregulation and downregulation of catalase and GPX1, which occur upon altering the sphingolipid composition or acyl chain length, generate H₂O₂ and thereby upregulate K_Ca3.1 expression and function via a H₂O₂/Fyn‐mediated pathway. Altogether, enhanced K_Ca3.1 activity may compensate for decreased NO signaling during vascular aging.     

Mitochondrial PKC‐ε deficiency promotes I/R‐mediated myocardial injury via GSK3β‐dependent mitochondrial permeability transition pore opening

Mitochondrial fission is critically involved in cardiomyocyte apoptosis, which has been considered as one of the leading causes of ischaemia/reperfusion (I/R)‐induced myocardial injury. In our previous works, we demonstrate that aldehyde dehydrogenase‐2 (ALDH2) deficiency aggravates cardiomyocyte apoptosis and cardiac dysfunction. The aim of this study was to elucidate whether ALDH2 deficiency promotes mitochondrial injury and cardiomyocyte death in response to I/R stress and the underlying mechanism. I/R injury was induced by aortic cross‐clamping for 45 min. followed by unclamping for 24 hrs in ALDH2 knockout (ALDH2^?/?) and wild‐type (WT) mice. Then myocardial infarct size, cell apoptosis and cardiac function were examined. The protein kinase C (PKC) isoform expressions and their mitochondrial translocation, the activity of dynamin‐related protein 1 (Drp1), caspase9 and caspase3 were determined by Western blot. The effects of N‐acetylcysteine (NAC) or PKC‐δ shRNA treatment on glycogen synthase kinase‐3β (GSK‐3β) activity and mitochondrial permeability transition pore (mPTP) opening were also detected. The results showed that ALDH2^?/? mice exhibited increased myocardial infarct size and cardiomyocyte apoptosis, enhanced levels of cleaved caspase9, caspase3 and phosphorylated Drp1. Mitochondrial PKC‐ε translocation was lower in ALDH2^?/? mice than in WT mice, and PKC‐δ was the opposite. Further data showed that mitochondrial PKC isoform ratio was regulated by cellular reactive oxygen species (ROS) level, which could be reversed by NAC pre‐treatment under I/R injury. In addition, PKC‐ε inhibition caused activation of caspase9, caspase3 and Drp1Ser⁶¹⁶ in response to I/R stress. Importantly, expression of phosphorylated GSK‐3β (inactive form) was lower in ALDH2^?/? mice than in WT mice, and both were increased by NAC pre‐treatment. I/R‐induced mitochondrial translocation of GSK‐3β was inhibited by PKC‐δ shRNA or NAC pre‐treatment. In addition, mitochondrial membrane potential (?Ψ_m) was reduced in ALDH2^?/? mice after I/R, which was partly reversed by the GSK‐3β inhibitor (SB216763) or PKC‐δ shRNA. Collectively, our data provide the evidence that abnormal PKC‐ε/PKC‐δ ratio promotes the activation of Drp1 signalling, caspase cascades and GSK‐3β‐dependent mPTP opening, which results in mitochondrial injury‐triggered cardiomyocyte apoptosis and myocardial dysfuction in ALDH2^?/? mice following I/R stress.     

The role of TGF‐β1/Smad2/3 pathway in platelet‐rich plasma in retarding intervertebral disc degeneration

Recent studies have suggested that platelet‐rich plasma (PRP) injections are an effective way to retard intervertebral disc degeneration, but the mechanism of action is unclear. Activated platelets release some growth factors, such as transforming growth factor‐β1 (TGF‐β1), which positively modulate the extracellular matrix of nucleus pulposus cells. The purpose of this study was to explore the mechanism underlying the PRP‐mediated inhibition of intervertebral disc degeneration. In an in vitro study, we found that the proliferation of nucleus pulposus cells was greatly enhanced with 2.5% PRP treatment. The TGF‐β1 concentration was much higher after PRP treatment. PRP administration effectively increased the collagen II, aggrecan and sox‐9 mRNA levels and decreased collagen X levels. However, Western blotting demonstrated that specifically inhibiting TGF‐β1 signalling could significantly prevent nucleus pulpous cellular expression of Smad2/3 and matrix protein. In a rabbit study, magnetic resonance imaging revealed significant recovery signal intensity in the intervertebral discs of the PRP injection group compared with the very low signal intensity in the control groups. Histologically, the PRP plus inhibitor injection group had significantly lower expression levels of Smad2/3 and collagen II than the PRP group. These results demonstrated that a high TGF‐β1 content in the platelets retarded disc degeneration in vitro and in vivo. Inhibiting the TGF‐β1/Smad2/3 pathway could prevent this recovery by inactivating Smad2/3 and down‐regulating the extracellular matrix. Therefore, the TGF‐β1/Smad2/3 pathway might play a critical role in the ability of PRP to retard intervertebral disc degeneration.     

Gender‐dependent regulation of G‐protein‐gated inwardly rectifying potassium current in dorsal raphe neurons in knock‐out mice devoid of the 5‐hydroxytryptamine transporter

Agonists at G‐protein‐coupled receptors in neurons of the dorsal raphe nucleus (DRN) of knock‐out mice devoid of the serotonin transporter (5‐HTT^?/?) exhibit lower efficacy to inhibit cellular discharge than in wild‐type counterparts. Using patch‐clamp whole‐cell recordings, we found that a G‐protein‐gated inwardly rectifying potassium (GIRK) current is involved in the inhibition of spike discharge induced by 5‐HT_1A agonists (5‐carboxamidotryptamine (5‐CT) and (±)‐2‐dipropylamino‐8‐hydroxy‐1,2,3,4‐tetrahydronaphthalene hydrobromide (8‐OH‐DPAT); 50 nM–30 μM) in both wild‐type and 5‐HTT^?/? female and male mice. These effects were mimicked by 5′‐guanylyl‐imido‐diphosphate (Gpp(NH)p; 400 μM) dialysis into cells with differences between genders. The 5‐HTT^?/? knock‐out mutation reduced the current density induced by Gpp(NH)p in females but not in males. These data suggest that the decreased response of 5‐HT_1A receptors to agonists in 5‐HTT^?/? mutants reflects notably alteration in the coupling between G‐proteins and GIRK channels in females but not in males. Accordingly, gender differences in central 5‐HT neurotransmission appear to depend—at least in part—on sex‐related variations in corresponding receptor‐G protein signaling mechanisms. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Progesterone modulation of α5 nAChR subunits influences anxiety‐related behavior during estrus cycle

Smokers often report an anxiolytic effect of cigarettes. In addition, stress‐related disorders such as anxiety, post‐traumatic stress syndrome and depression are often associated with chronic nicotine use. To study the role of the α5 nicotinic acetylcholine receptor subunit in anxiety‐related responses, control and α5 subunit null mice (α5^?/?) were subjected to the open field activity (OFA), light–dark box (LDB) and elevated plus maze (EPM) tests. In the OFA and LDB, α5^?/? behaved like wild‐type controls. In the EPM, female α5^?/? mice displayed an anxiolytic‐like phenotype, while male α5^?/? mice were undistinguishable from littermate controls. We studied the hypothalamus–pituitary–adrenal axis by measuring plasma corticosterone and hypothalamic corticotropin‐releasing factor. Consistent with an anxiolytic‐like phenotype, female α5^?/? mice displayed lower basal corticosterone levels. To test whether gonadal steroids regulate the expression of α5, we treated cultured NTera 2 cells with progesterone and found that α5 protein levels were upregulated. In addition, brain levels of α5 mRNA increased upon progesterone injection into ovariectomized wild‐type females. Finally, we tested anxiety levels in the EPM during the estrous cycle. The estrus phase (when progesterone levels are low) is anxiolytic‐like in wild‐type mice, but no cycle‐dependent fluctuations in anxiety levels were found in α5^?/? females. Thus, α5‐containing neuronal nicotinic acetylcholine receptors may be mediators of anxiogenic responses, and progesterone‐dependent modulation of α5 expression may contribute to fluctuations in anxiety levels during the ovarian cycle.     

Knockout of SOD1 promotes conversion of selenocysteine to dehydroalanine in murine hepatic GPX1 protein

Se-dependent glutathione peroxidase-1 (GPX1) and Cu,Zn-superoxide dismutase (SOD1) are two major intracellular antioxidant enzymes. The purpose of this study was to elucidate the biochemical mechanisms for the 40% loss of hepatic GPX1 activity in SOD1^−/− mice. Compared with the wild type (WT), the SOD1^−/− mice showed no change in the total amount of GPX1 protein. However, their total enzyme protein exhibited 31 and 38% decreases (P < 0.05) in the apparent k_cat for hydrogen peroxide and tert-butylperoxide (at 2 mM GSH), respectively. Most striking, mass spectrometry revealed two chemical forms of the 47th residue of GPX1: the projected native selenocysteine (Sec) and the Se-lacking dehydroalanine (DHA). The hepatic GPX1 protein of the SOD1^−/− mice contained 38% less Sec and 77% more DHA than that of WT and showed aggravated dissociation of the tetramer structure. In conclusion, knockout of SOD1 elevated the conversion of Sec to DHA in the active site of hepatic GPX1, leading to proportional decreases in the apparent k_cat and activity of the enzyme protein as a whole. Our data reveal a structural and kinetic mechanism for the in vivo functional dependence of GPX1 on SOD1 in mammals and provide a novel mass spectrometric method for the assay of oxidative modification of the GPX1 protein.     

Dysregulation of the Bmi‐1/p16Ink4a pathway provokes an aging‐associated decline of submandibular gland function

Bmi‐1 prevents stem cell aging, at least partly, by blocking expression of the cyclin‐dependent kinase inhibitor p16^Ink4a. Therefore, dysregulation of the Bmi‐1/p16^Ink4a pathway is considered key to the loss of tissue homeostasis and development of associated degenerative diseases during aging. However, because Bmi‐1 knockout (KO) mice die within 20 weeks after birth, it is difficult to determine exactly where and when dysregulation of the Bmi‐1/p16^Ink4a pathway occurs during aging in vivo. Using real‐time in vivo imaging of p16^Ink4a expression in Bmi‐1‐KO mice, we uncovered a novel function of the Bmi‐1/p16^Ink4a pathway in controlling homeostasis of the submandibular glands (SMGs), which secrete saliva into the oral cavity. This pathway is dysregulated during aging in vivo, leading to induction of p16^Ink4a expression and subsequent declined SMG function. These findings will advance our understanding of the molecular mechanisms underlying the aging‐related decline of SMG function and associated salivary gland hypofunction, which is particularly problematic among the elderly.     

Bmi1 regulate tooth and mandible development by inhibiting p16 signal pathway

To determine whether the deletion of p16 can correct tooth and mandible growth retardation caused by Bmi1 deficiency, we compared the tooth and mandible phenotypes of homozygous p16-deficient (p16^−/−) mice, homozygous Bmi1-deficient (Bmi1^−/−) mice, double homozygous Bmi1 and p16-deficient (Bmi1^−/−p16^−/−) mice to those of their wild-type littermates at 4 weeks of age by radiograph, histochemistry and immunohistochemistry. Results showed that compared to Bmi1^−/− mice, the dental mineral density, dental volume and dentin sialoprotein immunopositive areas were increased, whereas the ratio of the predentin area to total dentin area and that of biglycan immunopositive area to dentin area were decreased in Bmi1^−/−p16^−/− mice. These results indicate that the deletion of p16 can improve tooth development in Bmi1 knockout mice. Compared to Bmi1^−/− mice, the mandible mineral density, cortical thickness, alveolar bone volume, osteoblast number and activity, alkaline phosphatase positive area were all increased significantly in Bmi1^−/−p16^−/− mice. These results indicate that the deletion of p16 can improve mandible growth in Bmi1 knockout mice. Furthermore, the protein expression levels of cyclin D, CDK4 and p53 were increased significantly in p16^−/− mice compared with those from wild-type mice; the protein expression levels of cyclin D and CDK4 were decreased significantly, whereas those of p27 and p53 were increased significantly in Bmi1^−/− mice; these parameters were partly rescued in Bmi1^−/−p16^−/− mice compared with those from Bmi1^−/− mice. Therefore, our results indicate that Bmi1 plays roles in regulating tooth and mandible development by inhibiting p16 signal pathway which initiated entry into cell cycle.     

Role of nitric oxide and nuclear factor-κB in the CYP2E1 potentiation of tumor necrosis factor α hepatotoxicity in mice

Induction of CYP2E1 by pyrazole (PY) potentiated the hepatotoxicity induced by TNFα in mice. We evaluated the role of nitrosative and oxidative stress and the NF-κB activation pathway in this liver injury. The iNOS inhibitor N-(3-aminomethyl)benzylacetamindine (1400W) or the antioxidant N-acetyl-l-cysteine (NAC) prevented this liver injury. TNFα plus PY treatment triggered radical stress in the liver with increased lipid peroxidation and decreased glutathione and caused mitochondrial damage as reflected by elevated membrane swelling and cytochrome c release. The radical stress and mitochondrial damage were prevented by 1400W and NAC. TNFα plus PY treatment elevated 3-nitrotyrosine adduct formation and induced NOS2 in the liver; 1400W and NAC blocked these changes. A lower extent of liver injury and oxidative stress was found in NOS2^?/? mice treated with TNFα plus PY compared with wild-type controls. Neither 1400W nor NAC modified CYP2E1 activity or protein. Activation of JNK and p38MAPK was weaker in TNFα plus PY-treated NOS2^?/? mice and 1400W and NAC blocked the activation of JNK and p38MAPK in wild-type mice. IKKα/β protein levels were decreased by TNFα plus PY treatment, whereas IκBα and IκBβ protein levels were elevated compared with saline, PY, or TNFα alone. NF-κB DNA binding activity was increased by TNFα alone but lowered by TNFα plus PY. All these changes were blocked by 1400W and NAC. NF-κB activation products such as Bcl-2, Bcl-X_L, cFLIP_S, cFLIP_L, and Mn-SOD were reduced by TNFα plus PY and restored by 1400W or NAC. We conclude that TNFα plus CYP2E1 induces oxidative/nitrosative stress, which plays a role in the activation of JNK or p38MAPK and mitochondrial damage. These effects combine with the blunting of the NF-κB activation pathways and the synthesis of protective factors to cause liver injury.     

The Adipose Tissue Phenotype of Hormone‐Sensitive Lipase Deficiency in Mice

Objective: To directly ascertain the physiological roles in adipocytes of hormone‐sensitive lipase (HSL; E.C. 3.1.1.3), a multifunctional hydrolase that can mediate triacylglycerol cleavage in adipocytes. Research Methods and Procedures: We performed constitutive gene targeting of the mouse HSL gene (Lipe), subsequently studied the adipose tissue phenotype clinically and histologically, and measured lipolysis in isolated adipocytes. Results: Homozygous HSL^?/? mice have no detectable HSL peptide or cholesteryl esterase activity in adipose tissue, and heterozygous mice have intermediate levels with respect to wild‐type and deficient littermates. HSL‐deficient mice have normal body weight but reduced abdominal fat mass compared with normal littermates. Histologically, both white and brown adipose tissues in HSL^?/? mice show marked heterogeneity in cell size, with markedly enlarged adipocytes juxtaposed to cells of normal morphology. In isolated HSL^?/? adipocytes, lipolysis is not significantly increased by β₃‐adrenergic stimulation, but under basal conditions in the absence of added catecholamines, the lipolytic rate of isolated HSL^?/? adipocytes is at least as high as that of cells from normal controls. Cold tolerance during a 48‐hour period at 4 °C was similar in HSL^?/? mice and controls. Overnight fasting was well‐tolerated clinically by HSL^?/? mice, but after fasting, liver triglyceride content was significantly lower in HSL^?/? mice compared with wild‐type controls. Conclusions: In isolated fat cells, the lipolytic rate after β‐adrenergic stimulation is mainly dependent on HSL. However, the observation of a normal rate of lipolysis in unstimulated HSL^?/? adipocytes suggests that HSL‐independent lipolytic pathway(s) exist in fat. Physiologically, HSL deficiency in mice has a modest effect under normal fed conditions and is compatible with normal maintenance of core body temperature during cold stress. However, the lipolytic response to overnight fasting is subnormal.     

Tissue specificity of senescent cell accumulation during physiologic and accelerated aging of mice

Senescent cells accumulate with age in vertebrates and promote aging largely through their senescence‐associated secretory phenotype (SASP). Many types of stress induce senescence, including genotoxic stress. ERCC1‐XPF is a DNA repair endonuclease required for multiple DNA repair mechanisms that protect the nuclear genome. Humans or mice with reduced expression of this enzyme age rapidly due to increased levels of spontaneous, genotoxic stress. Here, we asked whether this corresponds to an increased level of senescent cells. p16^Ink4a and p21^Cip1 mRNA were increased ~15‐fold in peripheral lymphocytes from 4‐ to 5‐month‐old Ercc1^?/? and 2.5‐year‐old wild‐type (WT) mice, suggesting that these animals exhibit a similar biological age. p16^Ink4a and p21^Cip1 mRNA were elevated in 10 of 13 tissues analyzed from 4‐ to 5‐month‐old Ercc1^?/? mice, indicating where endogenous DNA damage drives senescence in vivo. Aged WT mice had similar increases of p16^Ink4a and p21^Cip1 mRNA in the same 10 tissues as the mutant mice. Senescence‐associated β–galactosidase activity and p21^Cip1 protein also were increased in tissues of the progeroid and aged mice, while Lamin B1 mRNA and protein levels were diminished. In Ercc1^?/Δ mice with a p16^Ink4a luciferase reporter, bioluminescence rose steadily with age, particularly in lung, thymus, and pancreas. These data illustrate where senescence occurs with natural and accelerated aging in mice and the relative extent of senescence among tissues. Interestingly, senescence was greater in male mice until the end of life. The similarities between Ercc1^?/? and aged WT mice support the conclusion that the DNA repair‐deficient mice accurately model the age‐related accumulation of senescent cells, albeit six‐times faster.     

Altered synaptic plasticity and behavioral abnormalities in CNGA3‐deficient mice

The role of the cyclic nucleotide‐gated (CNG) channel CNGA3 is well established in cone photoreceptors and guanylyl cyclase‐D‐expressing olfactory neurons. To assess a potential function of CNGA3 in the mouse amygdala and hippocampus, we examined synaptic plasticity and performed a comparative analysis of spatial learning, fear conditioning and step‐down avoidance in wild‐type mice and CNGA3 null mutants (CNGA3^?/?). CNGA3^?/? mice showed normal basal synaptic transmission in the amygdala and the hippocampus. However, cornu Ammonis (CA1) hippocampal long‐term potentiation (LTP) induced by a strong tetanus was significantly enhanced in CNGA3^?/? mice as compared with their wild‐type littermates. Unlike in the hippocampus, LTP was not significantly altered in the amygdala of CNGA3^?/? mice. Enhanced hippocampal LTP did not coincide with changes in hippocampus‐dependent learning, as both wild‐type and mutant mice showed a similar performance in water maze tasks and contextual fear conditioning, except for a trend toward higher step‐down latencies in a passive avoidance task. In contrast, CNGA3^?/? mice showed markedly reduced freezing to the conditioned tone in the amygdala‐dependent cued fear conditioning task. In conclusion, our study adds a new entry on the list of physiological functions of the CNGA3 channel. Despite the dissociation between physiological and behavioral parameters, our data describe a so far unrecognized role of CNGA3 in modulation of hippocampal plasticity and amydgala‐dependent fear memory.     

Aquaporin‐4 deficiency reduces TGF‐β1 in mouse midbrains and exacerbates pathology in experimental Parkinson's disease

Aquaporin‐4 (AQP4), the main water‐selective membrane transport protein in the brain, is localized to the astrocyte plasma membrane. Following the establishment of a 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐induced Parkinson's disease (PD) model, AQP4‐deficient (AQP4^?/?) mice displayed significantly stronger microglial inflammatory responses and remarkably greater losses of tyrosine hydroxylase (TH⁺)‐positive neurons than did wild‐type AQP4 (AQP4^+/+) controls. Microglia are the most important immune cells that mediate immune inflammation in PD. However, recently, few studies have reported why AQP4 deficiency results in more severe hypermicrogliosis and neuronal damage after MPTP treatment. In this study, transforming growth factor‐β1 (TGF‐β1), a key suppressive cytokine in PD onset and development, failed to increase in the midbrain and peripheral blood of AQP4^?/? mice after MPTP treatment. Furthermore, the lower level of TGF‐β1 in AQP4^?/? mice partially resulted from impairment of its generation by astrocytes; reduced TGF‐β1 may partially contribute to the uncontrolled microglial inflammatory responses and subsequent severe loss of TH⁺ neurons in AQP4^?/? mice after MPTP treatment. Our study provides not only a better understanding of both aetiological and pathogenical factors implicated in the neurodegenerative mechanism of PD but also a possible approach to developing new treatments for PD via intervention in AQP4‐mediated immune regulation.     

Protective role of mouse mast cell tryptase Mcpt6 in melanoma

Tryptase‐positive mast cells populate melanomas, but it is not known whether tryptase impacts on melanoma progression. Here we addressed this and show that melanoma growth is significantly higher in tryptase‐deficient (Mcpt6^?/?) versus wild‐type mice. Histochemical analysis showed that mast cells were frequent in the tumor stroma of both wild‐type and Mcpt6^?/? mice, and also revealed their presence within the tumor parenchyma. Confocal microscopy analysis revealed that tryptase was taken up by the tumor cells. Further, tryptase‐positive granules were released from mast cells and were widely distributed within the tumor tissue, suggesting that tryptase could impact on the tumor microenvironment. Indeed, gene expression analysis showed that the absence of Mcpt6 caused decreased expression of numerous genes, including Cxcl9, Tgtp2, and Gbp10, while the expression of 5p‐miR3098 was enhanced. The levels of CXCL9 were lower in serum from Mcpt6^?/? versus wild‐type mice. In further support of a functional impact of tryptase on melanoma, recombinant tryptase (Mcpt6) was taken up by cultured melanoma cells and caused reduced proliferation. Altogether, our results indicate a protective role of mast cell tryptase in melanoma growth.     

Knockout of receptor for advanced glycation end‐products attenuates age‐related renal lesions

Pro‐aging effects of endogenous advanced glycation end‐products (AGEs) have been reported, and there is increasing interest in the pro‐inflammatory and ‐fibrotic effects of their binding to RAGE (the main AGE receptor). The role of dietary AGEs in aging remains ill‐defined, but the predominantly renal accumulation of dietary carboxymethyllysine (CML) suggests the kidneys may be particularly affected. We studied the impact of RAGE invalidation and a CML‐enriched diet on renal aging. Two‐month‐old male, wild‐type (WT) and RAGE^?/? C57Bl/6 mice were fed a control or a CML‐enriched diet (200 μg CML/g_food) for 18 months. Compared to controls, we observed higher CML levels in the kidneys of both CML WT and CML RAGE^?/? mice, with a predominantly tubular localization. The CML‐rich diet had no significant impact on the studied renal parameters, whereby only a trend to worsening glomerular sclerosis was detected. Irrespective of diet, RAGE^?/? mice were significantly protected against nephrosclerosis lesions (hyalinosis, tubular atrophy, fibrosis and glomerular sclerosis) and renal senile apolipoprotein A‐II (ApoA‐II) amyloidosis (p < 0.001). A positive linear correlation between sclerosis score and ApoA‐II amyloidosis score (r = 0.92) was observed. Compared with old WT mice, old RAGE^?/? mice exhibited lower expression of inflammation markers and activation of AKT, and greater expression of Sod2 and SIRT1. Overall, nephrosclerosis lesions and senile amyloidosis were significantly reduced in RAGE^?/? mice, indicating a protective effect of RAGE deletion with respect to renal aging. This could be due to reduced inflammation and oxidative stress in RAGE^?/? mice, suggesting RAGE is an important receptor in so‐called inflamm‐aging.     

Endothelial nitric oxide deficiency promotes Alzheimer's disease pathology

Aging and the presence of cerebrovascular disease are associated with increased incidence of Alzheimer's disease. A common feature of aging and cerebrovascular disease is decreased endothelial nitric oxide (NO). We studied the effect of a loss of endothelium derived NO on amyloid precursor protein (APP) related phenotype in late middle aged (LMA) (14–15 month) endothelial nitric oxide synthase deficient (eNOS^?/?) mice. APP, β‐site APP cleaving enzyme (BACE) 1, and amyloid beta (Aβ) levels were significantly higher in the brains of LMA eNOS^?/? mice as compared with LMA wild‐type controls. APP and Aβ_1‐40 were increased in hippocampal tissue of eNOS^?/? mice as compared with wild‐type mice. LMA eNOS^?/? mice displayed an increased inflammatory phenotype as compared with LMA wild‐type mice. Importantly, LMA eNOS^?/? mice performed worse in a radial arm maze test of spatial learning and memory as compared with LMA wild‐type mice. These data suggest that chronic loss of endothelial NO may be an important contributor to both Aβ related pathology and cognitive decline.

     

Heat shock factor 1 deficiency via its downstream target gene αB‐crystallin (Hspb5) impairs p53 degradation

Heat shock factor Hsf1 regulates the stress‐inducibility of heat shock proteins (Hsps) or molecular chaperones. One of the functions attributed to Hsps is their participation in folding and degradation of proteins. We recently showed that hsf1^?/? cells accumulate ubiquitinated proteins. However, a direct role for Hsf1 in stability of specific proteins such as p53 has not been elucidated. We present evidence that cells deficient in hsf1 accumulate wild‐type p53 protein. We further show that hsf1^?/? cells express lower levels of αB‐crystallin and cells deficient in αB‐crystallin also accumulate p53 protein. Reports indicate that αB‐crystallin binds to Fbx4 ubiquitin ligase, and they target cyclin D1 for degradation through a pathway involving the SCF (Skp1‐Cul1‐F‐box) complex. Towards determining a mechanism for p53 degradation involving αB‐crystallin and Hsf1, we have found that ectopic expression of Fbx4 in wild‐type mouse embryo fibroblasts (MEFs) expressing mutant p53 (p53R175H) leads to increase in its degradation, while MEFs deficient in hsf1 or αBcry are defective in degradation of this p53 protein. In addition, immunoprecipitated p53R175H from wild‐type MEFs is able to pull‐down both αB‐crystallin and Fbx4. Finally, immunoprecipitated wild‐type p53 from doxorubicin treated U2OS cells can pull‐down endogenous αB‐crystallin and Fbx4. These results indicate that hsf1‐ and αBcry‐deficient cells accumulate p53 due to reduced levels of αB‐crystallin in these cells. Elevated levels of p53 in hsf1‐ and αBcry‐deficient cells lead to their increased sensitivity to DNA damaging agents. These data reveal a novel mechanism for protein degradation through Hsf1 and αB‐crystallin. J. Cell. Biochem. 107: 504–515, 2009. © 2009 Wiley‐Liss, Inc.