Leptin protects against 6-hydroxydopamine-induced dopaminergic cell death via mitogen-activated protein kinase signaling

The death of midbrain dopaminergic neurons in sporadic Parkinson disease is of unknown etiology but may involve altered growth factor signaling. The present study showed that leptin, a centrally acting hormone secreted by adipocytes, rescued dopaminergic neurons, reversed behavioral asymmetry, and restored striatal catecholamine levels in the unilateral 6-hydroxydopamine (6-OHDA) mouse model of dopaminergic cell death. In vitro studies using the murine dopaminergic cell line MN9D showed that leptin attenuated 6-OHDA-induced apoptotic markers, including caspase-9 and caspase-3 activation, internucleosomal DNA fragmentation, and cytochrome c release. ERK1/2 phosphorylation (pERK1/2) was found to be critical for mediating leptin-induced neuroprotection, because inhibition of the MEK pathway blocked both the pERK1/2 response and the pro-survival effect of leptin in cultures. Knockdown of the downstream messengers JAK2 or GRB2 precluded leptin-induced pERK1/2 activation and neuroprotection. Leptin/pERK1/2 signaling involved phosphorylation and nuclear localization of CREB (pCREB), a well known survival factor for dopaminergic neurons. Leptin induced a marked MEK-dependent increase in pCREB that was essential for neuroprotection following 6-OHDA toxicity. Transfection of a dominant negative MEK protein abolished leptin-enhanced pCREB formation, whereas a dominant negative CREB or decoy oligonucleotide diminished both pCREB binding to its target DNA sequence and MN9D survival against 6-OHDA toxicity. Moreover, in the substantia nigra of mice, leptin treatment increased the levels of pERK1/2, pCREB, and the downstream gene product BDNF, which were reversed by the MEK inhibitor PD98059. Collectively, these data provide evidence that leptin prevents the degeneration of dopaminergic neurons by 6-OHDA and may prove useful in the treatment of Parkinson disease.     

Deprenyl and the relationship between its effects on spatial memory, oxidant stress and hippocampal neurons in aged male rats

Oxidative stress may play a major role in the aging process and associated cognitive decline. Therefore, antioxidant treatment may alleviate age-related impairment in spatial memory. Cognitive impairment could also involve the age-related morphological alterations of the hippocampal formation. The aim of this study was to examine the relationship between the effects of deprenyl, an irreversible monoamine-oxidase B inhibitor, on spatial memory by oxidant stress and on the total number of neurons in the hippocampus CA1 region of aged male rats. In this study, 24-month-old male rats were used. Rats were divided into control and experimental groups which received an injection of deprenyl for 21 days. Learning experiments were performed for six days in the Morris water maze. Spatial learning was significantly better in deprenyl-treated rats compared to saline-treated rats. Deprenyl treatment elicited a significant decrease of lipid peroxidation in the prefrontal cortex, striatum and hippocampus regions and a significant increase of glutathione peroxidase activity in the prefrontal cortex and hippocampus. It was observed that deprenyl had no effect on superoxide dismutase activity. The total number of neurons in the hippocampus CA1 region was significantly higher in the deprenyl group than in the control group. In conclusion, we demonstrated that deprenyl increases spatial memory performance in aged male rats and this increase may be related to suppression of lipid peroxidation and alleviation of the age-related decrease of the number of neurons in the hippocampus. The results of such studies may be useful in pharmacological alleviation of the aging process.     

Cooperative effects of SIRT1 and SIRT2 on APP acetylation

Alzheimer's disease (AD) is an age-related neurodegenerative disorder characterized by amyloid-β (Aβ) deposition and neurofibrillary tangles. Although the NAD⁺-dependent deacetylases SIRT1 and SIRT2 play pivotal roles in age-related diseases, their cooperative effects in AD have not yet been elucidated. Here, we report that the SIRT2:SIRT1 ratio is elevated in the brains of aging mice and in the AD mouse models. In HT22 mouse hippocampal neuronal cells, Aβ challenge correlates with decreased SIRT1 expression, while SIRT2 expression is increased. Overexpression of SIRT1 prevents Aβ-induced neurotoxicity. We find that SIRT1 impedes SIRT2-mediated APP deacetylation by inhibiting the binding of SIRT2 to APP. Deletion of SIRT1 reduces APP recycling back to the cell surface and promotes APP transiting toward the endosome, thus contributing to the amyloidogenic processing of APP. Our findings define a mechanism for neuroprotection by SIRT1 through suppression of SIRT2 deacetylation, and provide a promising avenue for therapeutic intervention of AD.     

Pyrroloquinoline quinone rescues hippocampal neurons from glutamate-induced cell death through activation of Nrf2 and up-regulation of antioxidant genes

Pyrroloquinoline quinone (PQQ) has been shown to protect primary cultured hippocampal neurons from glutamate-induced cell apoptosis by scavenging reactive oxygen species (ROS) and activating phosphatidylinositol-3-kinase (PI3K)/Akt signaling. We investigated the downstream pathways of PI3K/Akt involved in PQQ protection of glutamate-injured hippocampal neurons. Western blot analysis indicated that PQQ treatment following glutamate stimulation triggers phosphorylation of glycogen synthase kinase 3β, accompanied by maintenance of Akt activation. Immunostaining and quantitative RT-PCR revealed that PQQ treatment promotes nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2), and up-regulates mRNA expression of Nrf2 and the antioxidant enzyme genes, heme oxygenase-1 and glutamate cysteine ligase catalytic in glutamate-injured hippocampal neurons; this is a process dependent on the PI3K/Akt pathway, as evidenced by blocking experiments with PI3K inhibitors. In addition, increased ROS production and decreased glutathione levels in glutamate-injured hippocampal neurons were found to be reduced by PQQ treatment. Collectively, our findings suggest that PQQ exerts neuroprotective activity, possibly through PI3K/Akt-dependent activation of Nrf2 and up-regulation of antioxidant genes. However, the ability of PQQ to scavenge ROS was not totally regulated by PI3K/Akt signaling; possibly it is governed by other mechanisms.     

Alterations in intrinsic neuronal excitability during normal aging

Normal aging subjects, including humans, have difficulty learning hippocampus-dependent tasks. For example, at least 50% of normal aging rabbits and rats fail to meet a learning criterion in trace eyeblink conditioning. Many factors may contribute to this age-related learning impairment. An important cause is the reduced intrinsic excitability observed in hippocampal pyramidal neurons from normal aging subjects, as reflected by an enlarged postburst afterhyperpolarization (AHP) and an increased spike-frequency adaptation (accommodation). In this review, we will focus on the alterations in the AHP and accommodation during learning and normal aging. We propose that age-related increases in the postburst AHP and accommodation in hippocampal pyramidal neurons play an integral role in the learning impairment observed in normal aging subjects.     

Estrogen rapidly modulates mustard oil-induced visceral hypersensitivity in conscious female rats: A role of CREB phosphorylation in spinal dorsal horn neurons

This study investigated the effect of sex hormones on mustard oil (MO)-induced visceral hypersensitivity in female rats and analyzed possible involved signaling pathways. Female rats, either intact or ovariectomized (OVX), were prepared for abdominal muscle electromyography in response to colorectal distension after intracolonic instillation of MO. The effect of MO intracolonic sensitization was evaluated in intact rats, OVX rats, and OVX rats pretreated with a single injection of 17beta-estradiol (E), progesterone (P), E+P, or vehicle. cAMP-responsive element-binding protein (CREB) and phosphorylated CREB (pCREB) were detected in the superficial dorsal horn of L6 and S1 in MO or mineral oil-treated OVX rats with/without colorectal distension and estrogen replacement. The distal colorectum was removed for histological evaluation of inflammatory severity in MO-treated intact or OVX rats. The MO-treated rats had significantly higher visceromotor reflex than controls (enhanced visceral hypersensitivity), whereas OVX eliminated this hypersensitivity. After a single injection of E or E+P, the rats rapidly restored MO-induced visceral hypersensitivity within 2 h. Estrogen also rapidly induced a dose-dependent increase in pCREB expression in the superficial dorsal horn neurons in MO-treated, but not mineral oil-treated, OVX rats. The present study suggests that estrogen can rapidly modulate visceral hypersensitivity induced by MO intracolonic instillation in conscious female rats, which may involve spinal activation of the cAMP response element-mediated gene induction pathway.     

Modulation of NADP(+)-dependent isocitrate dehydrogenase in aging

NADPH is an important cofactor in many biosynthesis pathways and the regeneration of reduced glutathione, critically important in cellular defense against oxidative damage. It is mainly produced by glucose-6-phosphate dehydrogenase, malic enzyme, and NADP(+)-specific isocitrate dehydrogenases (ICDHs). Here, we investigated age-related changes in ICDH activity and protein expression in IMR-90 human diploid fibroblast cells and tissues from Fischer 344 rats. We found that in IMR-90 cells the activity of cytosolic ICDH (IDPc) gradually increased with age up to the 46-48 population doubling level (PDL) and then gradually decreased at later PDL. 2',7'-Dichloro-fluorescein fluorescence which reflects intracellular ROS generation was increased with aging in IMR-90 cells. In ad libitum-fed rats, we noted age-related, tissue-specific modulations of IDPc and mitochondrial ICDH (IDPm) activities and protein expression in the liver, kidney and testes. In contrast, ICDH activities and protein expression were not significantly modulated in diet-restricted rats. These data suggest that modulation of ICDH is an age-dependent and a tissue-specific phenomenon.     

Thyroid hormones protect astrocytes from morphine-induced apoptosis by regulating nitric oxide and pERK 1/2 pathways

Unlike neurons and various other non-neuronal cells, astrocytes have been reported to be resistant to morphine induced cytotoxicity. The present work demonstrates that primary cultures of astrocytes are also sensitive to morphine toxicity depending upon the thyroidal status of the culture medium. Chronic morphine treatment of astrocytes, cultured under thyroid hormone (TH)-deficient conditions, induced apoptotic cell death which was characterized by nuclear condensation, DNA fragmentation and activation of caspase-3 like enzymes. Cell death was accompanied with increase in nNOS level, nitration of cellular proteins and down regulation of pAKT level. Phosphorylation of ERK1/2 showed a biphasic response, an initial induction followed by sustained decline during chronic morphine treatment and the initial induction of pERK1/2 level appeared to be critical for apoptosis in the cells. Interestingly, supplementation with normal levels of TH to cells attenuated morphine-induced apoptosis as well as the biphasic response of pERK1/2 in the astrocytes. However, in the presence of glutathione synthetase inhibitor L-buthionine-S,R-sulfoximine, TH failed to protect astrocytes. Overall, the study demonstrates a possible signaling mechanism of morphine induced toxicity to cells and suggests that alteration of glutathione homeostasis by TH protect astrocytes from morphine by regulating NO and pERK1/2 pathways in the cells.     

Blood–brain barrier dysfunction promotes astrocyte senescence through albumin-induced TGFβ signaling activation

Blood–brain barrier dysfunction (BBBD) and accumulation of senescent astrocytes occur during brain aging and contribute to neuroinflammation and disease. Here, we explored the relationship between these two age-related events, hypothesizing that chronic hippocampal exposure to the blood-borne protein serum albumin could induce stress-induced premature senescence (SIPS) in astrocytes via transforming growth factor beta 1 (TGFβ) signaling. We found that 1 week of albumin exposure significantly increased TGFβ signaling and senescence marker expression in cultured rat hippocampal astrocytes. These changes were preventable by pharmacological inhibition of the type I TGFβ receptor (TGFβR) ALK5. To study these effects in vivo, we utilized an animal model of BBBD in which albumin was continuously infused into the lateral ventricles of adult mice. Consistent with our in vitro results, 1 week of albumin infusion significantly increased TGFβ signaling activation and the burden of senescent astrocytes in hippocampal tissue. Pharmacological inhibition of ALK5 TGFβR or conditional genetic knockdown of astrocytic TGFβR prior to albumin infusion was sufficient to prevent albumin-induced astrocyte senescence. Together, these results establish a link between TGFβ signaling activation and astrocyte senescence and suggest that prolonged exposure to serum albumin due to BBBD can trigger these phenotypic changes.     

Pivotal role of RanBP9 in integrin-dependent focal adhesion signaling and assembly

Accumulation of the amyloid β (Aβ) peptide derived from the amyloid precursor protein (APP) plays a central role in the pathogenesis of Alzheimer's disease (AD). We previously reported that the scaffolding protein RanBP9 is markedly increased in AD brains and promotes Aβ generation by scaffolding APP/BACE1/LRP complexes together and accelerating APP endocytosis. Because APP, LRP, and RanBP9 all physically interact with β-integrins, we investigated whether RanBP9 alters integrin-dependent cell adhesion and focal adhesion signaling. Here, we show that RanBP9 overexpression dramatically disrupts integrin-dependent cell attachment and spreading in NIH3T3 and hippocampus-derived HT22 cells, concomitant with strongly decreased Pyk2/paxillin signaling and talin/vinculin localization in focal adhesion complexes. Conversely, RanBP9 knockdown robustly promotes cell attachment, spreading, and focal adhesion signaling and assembly. Cell surface biotinylation and endocytosis assays reveal that RanBP9 overexpression and RanBP9 siRNA potently reduces and increases surface β1-integrin and LRP by accelerating and inhibiting their endocytosis, respectively. Primary hippocampal neurons derived from RanBP9-transgenic mice also demonstrate severely reduced levels of surface β1-integrin, LRP, and APP, as well as neurite arborization. Therefore, these data indicate that RanBP9 simultaneously inhibits cell-adhesive processes and enhances Aβ generation by accelerating APP, LRP, and β1-integrin endocytosis.     

Aging impact on amyloid precursor protein neuronal trafficking

Neurons live a lifetime. Neuronal aging may increase the risk of Alzheimer's disease. How does neuronal membrane trafficking maintain synapse function during aging? In the normal aged brain, intraneuronal beta-amyloid (Aβ) accumulates without Alzheimer's disease mutations or risk variants. However, do changes with neuronal aging potentiate Aβ accumulation? We reviewed the membrane trafficking of the amyloid precursor protein in neurons and highlighted its importance in Aβ production. Importantly, we reviewed the evidence supporting the impact of aging on neuronal membrane trafficking, APP processing, and consequently Aβ production. Dissecting the molecular regulators of APP trafficking during neuronal aging is required to identify strategies to delay synaptic decline and protect from Alzheimer's disease.     

TGF-β1–ROS–ATM–CREB signaling axis in macrophage mediated migration of human breast cancer MCF7 cells

Macrophages in the tumor microenvironment play an important role in tumor cell survival. They influence the tumor cell to proliferate, invade into surrounding normal tissues and metastasize to local and distant sites. In this study, we evaluated the effect of conditioned medium from monocytes and macrophages on growth and migration of breast cancer cells. Macrophage conditioned medium (MϕCM) containing elevated levels of cytokines TNF-α, IL-1β and IL-6 had a differential effect on non-invasive (MCF7) and highly invasive (MDA-MB-231) breast cancer cell lines. MϕCM induced the secretion of TGF-β1 in MCF7 cells. This was associated with apoptosis in a fraction of cells and generation of reactive oxygen and nitrogen species (ROS and RNS) and DNA damage in the remaining cells. This, in turn, increased expression of cAMP response element binding protein (CREB) and vimentin resulting in migration of cells. These effects were inhibited by neutralization of TNF-α, IL-1β and IL-6, inhibition of ROS and RNS, DNA damage and siRNA mediated knockdown of ATM. In contrast, MDA-MB-231 cells which had higher basal levels of pCREB were not affected by MϕCM. In summary, we have found that pro-inflammatory cytokines secreted by macrophages induce TGF-β1 in tumor cells, which activate pCREB signaling, epithelial–mesenchymal-transition (EMT) responses and enhanced migration.     

Chronic 1alpha,25-(OH)2 vitamin D3 treatment reduces Ca2+ -mediated hippocampal biomarkers of aging

Aging in the hippocampus of several species is characterized by alterations in multiple Ca(2+)-mediated processes, including an increase in L-type voltage-gated Ca(2+) channel (L-VGCC) current, an enhanced Ca(2+)-dependent slow afterhyperpolarization (AHP), impaired synaptic plasticity and elevated Ca(2+) transients. Previously, we found that 1alpha,25-dihydoxyvitamin D(3) (1,25VitD), a major Ca(2+) regulating hormone, down-regulates L-VGCC expression in cultured hippocampal neurons. Here, we tested whether in vivo treatment of aged F344 rats with 1,25VitD would reverse some of the Ca(2+) -mediated biomarkers of aging seen in hippocampal CA1 neurons. As previously reported, L-VGCC currents and the AHP were larger in aged than in young neurons. Treatment with 1,25VitD over 7 days decreased L-VGCC activity in aged rats, as well as the age-related increase in AHP amplitude and duration. In addition, reduced L-VGCC activity was correlated with reduced AHPs in the same animals. These data provide direct evidence that 1,25VitD can regulate multiple Ca(2+)-dependent processes in neurons, with particular impact on reducing age-related changes associated with Ca(2+) dysregulation. Thus, these results may have therapeutic implications and suggest that 1,25VitD, often taken to maintain bone health, may also retard some consequences of brain aging.     

Reduction in neuronal L-type calcium channel activity in a double knock-in mouse model of Alzheimer's disease

Increased function of neuronal L-type voltage-sensitive Ca(2+) channels (L-VSCCs) is strongly linked to impaired memory and altered hippocampal synaptic plasticity in aged rats. However, no studies have directly assessed L-VSCC function in any of the common mouse models of Alzheimer's disease where neurologic deficits are typically more robust. Here, we used cell-attached patch-clamp recording techniques to measure L-VSCC activity in CA1 pyramidal neurons of partially dissociated hippocampal "zipper" slices prepared from 14-month-old wild-type mice and memory-impaired APP/PS1 double knock-in mice. Surprisingly, the functional channel density of L-VSCCs was significantly reduced in the APP/PS1 group. No differences in voltage dependency and unitary conductance of L-VSCCs were observed. The results suggest that mechanisms for Ca(2+) dysregulation can differ substantially between animal models of normal aging and models of pathological aging.     

Mitochondrial longevity pathways

Average lifespan has increased over the last centuries, as a consequence of medical and environmental factors, but maximal life span remains unchanged. Better understanding of the underlying mechanisms of aging and determinants of life span will help to reduce age-related morbidity and facilitate healthy aging. Extension of maximal life span is currently possible in animal models with measures such as genetic manipulations and caloric restriction (CR). CR appears to prolong life by reducing oxidative damage. Reactive oxygen species (ROS) have been proposed to cause deleterious effects on DNA, proteins, and lipids, and generation of these highly reactive molecules takes place in the mitochondria. But ROS is positively implicated in cellular stress defense mechanisms and formation of ROS a highly regulated process controlled by a complex network of intracellular signaling pathways. There are endogenous anti-oxidant defense systems that have the potential to partially counteract ROS impact. In this review, we will describe pathways contributing to the regulation of the age-related decline in mitochondrial function and their impact on longevity. This article is part of a Special Issue entitled Mitochondria: the deadly organelle.     

Role of hippocalcin in mediating Aβ toxicity

Alzheimer's disease (AD) is the most common cause of dementia, and amyloid-β (Aβ) plaques and tau-containing tangles are its histopathological hallmark lesions. These do not occur at random; rather, the neurodegenerative process is stereotyped in that it is initiated in the entorhinal cortex and hippocampal formation. Interestingly, it is the latter brain area where the calcium-sensing enzyme hippocalcin is highly expressed. Because calcium deregulation is a well-established pathomechanism in AD, we aimed to address the putative role of hippocalcin in human AD brain and transgenic mouse models. We found that hippocalcin levels are increased in human AD brain and in Aβ plaque-forming APP23 transgenic mice compared to controls. To determine the role of hippocalcin in Aβ toxicity, we treated primary cultures derived from hippocalcin knockout (HC KO) mice with Aβ and found them to be more susceptible to Aβ toxicity than controls. Likewise, treatment with either thapsigargin or ionomycin, both known to deregulate intracellular calcium levels, caused an increased toxicity in hippocampal neurons from HC KO mice compared to wild-type. We found further that mitochondrial complex I activity increased from 3 to 6months in hippocampal mitochondria from wild-type and HC KO mice, but that the latter exhibited a significantly stronger aging phenotype than wild-type. Aβ treatment induced significant toxicity on hippocampal mitochondria from HC KO mice already at 3months of age, while wild-type mitochondria were spared. Our data suggest that hippocalcin has a neuroprotective role in AD, presenting it as a putative biomarker.     

Modulation of NADP+-dependent isocitrate dehydrogenase in aging

Abstract

NADPH is an important cofactor in many biosynthesis pathways and the regeneration of reduced glutathione, critically important in cellular defense against oxidative damage. It is mainly produced by glucose-6-phosphate dehydrogenase, malic enzyme, and NADP⁺-specific isocitrate dehydrogenases (ICDHs). Here, we investigated age-related changes in ICDH activity and protein expression in IMR-90 human diploid fibroblast cells and tissues from Fischer 344 rats. We found that in IMR-90 cells the activity of cytosolic ICDH (IDPc) gradually increased with age up to the 46–48 population doubling level (PDL) and then gradually decreased at later PDL. 2′,7′-Dichloro-fluorescein fluorescence which reflects intracellular ROS generation was increased with aging in IMR-90 cells. In ad libitum-fed rats, we noted age-related, tissue-specific modulations of IDPc and mitochondrial ICDH (IDPm) activities and protein expression in the liver, kidney and testes. In contrast, ICDH activities and protein expression were not significantly modulated in diet-restricted rats. These data suggest that modulation of ICDH is an age-dependent and a tissue-specific phenomenon.     

MicroRNA-134/CREB/pCREB通路在癫痫中的表达及意义

目的探讨miR-134、CREB、pCREB在癫痫大鼠海马及难治性癫痫患者颞叶脑组织中的表达及意义。方法难治性癫痫患者及非癫痫对照组颞叶组织、氯化锂-匹罗卡品癫痫大鼠及空白对照组海马组织中,应用实时荧光定量PCR技术检测microRNA-134（miR-134）的表达,用Western blot方法检测CREB及p CREB的表达,用免疫组织化学方法检测人脑颞叶皮质及大鼠海马区CREB、p CREB的表达。结果与对照组相比miR-134表达在难治性癫痫患者中明显降低（P〈0.05）,在癫痫模型组中点燃后3、7、14、60 d明显降低（P〈0.05）,1 d与30 d表达降低较对照组差异无显著性（P〉0.05）;癫痫模型组CREB在3、7、14、60 d时间点明显升高（P〈0.05）、pCREB各时间点表达均高于空白对照组（P〈0.05）。结论难治性癫痫患者颞叶皮质及癫痫动物海马中miR-134表达下降,CREB、pCREB表达升高,提示其可能在癫痫发生发展机制中起重要作用。