Rodrigo Portes Ureshino

Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) Regulates Autophagy in Cultured Astrocytes

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca2+-mobilizing messenger that in many cells releases Ca2+ from the endolysosomal system. Recent studies have shown that NAADP-induced Ca2+ mobilization is mediated by the two-pore channels (TPCs). Whether NAADP acts as a messenger in astrocytes is unclear, and downstream functional consequences have yet to be defined. Here, we show that intracellular delivery of NAADP evokes Ca2+ signals from acidic organelles in rat astrocytes and that these signals are potentiated upon overexpression of TPCs. We also show that NAADP increases acidic vesicular organelle formation and levels of the autophagic markers, LC3II and beclin-1. NAADP-mediated increases in LC3II levels were reduced in cells expressing a dominant-negative TPC2 construct. Our data provide evidence that NAADP-evoked Ca2+ signals mediated by TPCs regulate autophagy.

Calcium and cell death signaling in neurodegeneration and aging

Transient increase in cytosolic (Cac2+) and mitochondrial Ca2+ (Ca m2+) are essential elements in the control of many physiological processes. However, sustained increases in Ca c2+ and Ca m2+ may contribute to oxidative stress and cell death. Several events are related to the increase in Ca m2+, including regulation and activation of a number of Ca2+ dependent enzymes, such as phospholipases, proteases and nucleases. Mitochondria and endoplasmic reticulum (ER) play pivotal roles in the maintenance of intracellular Ca2+ homeostasis and regulation of cell death. Several lines of evidence have shown that, in the presence of some apoptotic stimuli, the activation of mitochondrial processes may lead to the release of cytochrome c followed by the activation of caspases, nuclear fragmentation and apoptotic cell death. The aim of this review was to show how changes in calcium signaling can be related to the apoptotic cell death induction. Calcium homeostasis was also shown to be an important mechanism involved in neurodegenerative and aging processes.

Calcium signaling alterations, oxidative stress, and autophagy in aging.

SIGNIFICANCE Aging is a multi-factorial process that may be associated with several functional and structural deficits which can evolve into degenerative diseases. In this review, we present data that may depict an expanded view of molecular aging theories, beginning with the idea that reactive oxygen species (ROS) are the major effectors in this process. In addition, we have correlated the importance of autophagy as a neuroprotective mechanism and discussed a link between age-related molecules, Ca(2+) signaling, and oxidative stress. RECENT ADVANCES There is evidence suggesting that alterations in Ca(2+) homeostasis, including mitochondrial Ca(2+) overload and alterations in electron transport chain (ETC) complexes, which increase cell vulnerability, are linked to oxidative stress in aging. As much as Ca(2+) signaling is altered in aged cells, excess ROS can be produced due to an ineffective coupling of mitochondrial respiration. Damaged mitochondria might not be removed by the macroautophagic system, which is hampered in aging by lipofuscin accumulation, boosting ROS generation, damaging DNA, and, ultimately, leading to apoptosis. CRITICAL ISSUES This process can lead to altered protein expression (such as p53, Sirt1, and IGF-1) and progress to cell death. This cycle can lead to increased cell vulnerability in aging and contribute to an increased susceptibility to degenerative processes. FUTURE DIRECTIONS A better understanding of Ca(2+) signaling and molecular aging alterations is important for preventing apoptosis in age-related diseases. In addition, caloric restriction, resveratrol and autophagy modulation appear to be predominantly cytoprotective, and further studies of this process are promising in age-related disease therapeutics.

DIFFERENTIAL PROLIFERATIVE RESPONSE OF THE VENTRAL PROSTATE AND SEMINAL VESICLE TO TESTOSTERONE REPLACEMENT

We have investigated epithelial cell proliferation and the rate of glandular recovery of the ventral prostate (VP) and seminal vesicle (SV) promoted by testosterone replacement (TR) in castration‐induced regressed glands. Adult male Wistar rats were castrated and, after 21 days, they were treated with testosterone propionate (4 mg/kg/day). Intact (CT) and castrated rats without TR (CS) were also analysed. VP and SV were processed for histochemistry, morphometric—stereological analysis and immunocytochemistry to determine the PCNA index (PI). After 10 days of TR, the VP weight reached ∼72% of the CT values, while the SV weight exceeded ∼17% of the CT values. By the third day of TR, VP and SV presented a mean PI of 34% and 94% for distal region and 14% and 22% for proximal region, respectively. SV also had more luminal cells PCNA‐positive than VP, mainly in the distal region. The PI values fell on days 5, 7 and 10, but were still higher than CT. These findings indicate that epithelial cells from involuted SV are more responsive to TR than those from VP when stimulated to proliferate and replace the luminal cell population, suggesting a different mechanism regulating cell proliferation in response to androgenic stimuli.

Alterations in Calcium Signaling and a Decrease in Bcl-2 Expression: Possible Correlation With Apoptosis in Aged Striatum

Aging is a multifaceted process associated with various functional and structural deficits that might be evolved in degenerative diseases. It has been shown that neurodegenerative disorders are associated with alterations in Ca2+ homeostasis. Thus, in the present work, we have investigated Ca2+ signaling and apoptosis in aged striatum. Our results show that glutamate and NMDA evoke a greater Ca2+ rise in striatum slices from aged animals. However, this difference is not present when glutamate is tested in the absence of external Ca2+. Immunostaining of glutamate receptors shows that only NMDA receptors (NR1) are increased in the striatum of aged rats. Increases in mitochondrial Ca2+ content and in the reactive oxygen species levels were also observed in aged animals, which could be associated with tissue vulnerability. In addition, a decrease in the Bcl‐2 protein expression and an enhancement in apoptosis were also present in aged striatum. Together the results indicate that, in aged animals, alterations in Ca2+ handling coupled to an increase in ROS accumulation and a decrease in the prosurvival protein Bcl‐2 may contribute to apoptosis induction and cell death in rat striatum.

The Role of Mitochondrial Function in Glutamate-Dependent Metabolism in Neuronal Cells

Glutamate is an important neurotransmitter in neurons and glial cells and it is one of the keys to the neuron-glial interaction in the brain. Glutamate transmission is strongly dependent on calcium homeostasis and on mitochondrial function. In the present work we presented several aspects related to the role of mitochondria in glutamate signaling and in brain diseases. We focused on glutamateinduced calcium signaling and its relation to the organelle dysfunction with cell death processes. In addition, we have discussed how alterations in this pathway may lead or aggravate a variety of neurodegenerative diseases. We compiled information on how mitochondria can influence cell fate during glutamate stimulation and calcium signaling. These organelles play a pivotal role in neuron and glial exchange, in synaptic plasticity and several pathological conditions related to Aging, Alzheimers, Parkinsons and Huntingtons diseases. We have also presented autophagy as a mechanism activated during mitochondrial dysfunction which may function as a protective mechanism during injury. Furthermore, some new perspectives and approaches to treat these neurodegenerative diseases are offered and evaluated.

Effects of 17β-estradiol replacement on the apoptotic effects caused by ovariectomy in the rat hippocampus

AIMS The aim of the present study was to investigate the effects of different periods of ovariectomy and 17beta-estradiol replacement on apoptotic cell death and expression of members of the Bcl-2 family in the rat hippocampus. MAIN METHODS Hippocampi were obtained from rats in proestrus, ovariectomized (15 days, 21 days and 36 days), ovariectomized for 15 days and then treated with 17beta-estradiol for 7 or 21 days, and rats ovariectomized and immediately treated with 17beta-estradiol for 21 days. The expression of Bcl-2 and Bax and the number of apoptotic cells were determined. KEY FINDINGS Ovariectomy decreased Bcl-2 expression and increased Bax expression and the number of apoptotic cells. Replacement with 17beta-estradiol (21 days) throughout the post-ovariectomy period reduced the number of apoptotic cells to the control levels, and prevented the effects of ovariectomy on Bax expression, but only partially restored the Bcl-2 expression. After 15 days of ovariectomy, the replacement with 17beta-estradiol for 21 days, but not for 7 days, restored the Bcl-2 and Bax expression and the percentage of apoptotic cells to the levels found in the proestrus control. SIGNIFICANCE The present results show that a physiological concentration of 17beta-estradiol may help maintain long-term neuronal viability by regulating the expression of members of the Bcl-2 family. Even after a period of hormonal deprivation, treatment with 17beta-estradiol is able to restore the expression of Bax and Bcl-2 to control levels, but the duration of the treatment is a key factor to obtain the desired effect. These data provide new understanding into the mechanisms contributing to the neuroprotective action of estrogen.

Apoptosis induced by Aβ25-35 peptide is Ca(2+) -IP3 signaling-dependent in murine astrocytes.

Although the accumulation of the neurotoxic peptide β‐amyloid (Aβ) in the central nervous system is a hallmark of Alzheimers disease, whether Aβ acts in astrocytes is unclear, and downstream functional consequences have yet to be defined. Here, we show that cytosolic Ca2+ dysregulation, induced by a neurotoxic fragment (Aβ25–35), caused apoptosis in a concentration‐dependent manner, leading to cytoplasmic Ca2+ mobilization from extra‐ and intracellular sources, mainly from the endoplasmic reticulum (ER) via IP3 receptor activation. This mechanism was related to Aβ‐mediated apoptosis by the intrinsic pathway because the expression of pro‐apoptotic Bax was accompanied by its translocation in cells transfected with GFP‐Bax. Aβ‐mediated apoptosis was reduced by BAPTA‐AM, a fast Ca2+ chelator, indicating that an increase in intracellular Ca2+ was involved in cell death. Interestingly, the Bax translocation was dependent on Ca2+ mobilization from IP3 receptors because pre‐incubation with xestospongin C, a selective IP3 receptor inhibitor, abolished this response. Taken together, these results provide evidence that Aβ dysregulation of Ca2+ homeostasis induces ER depletion of Ca2+ stores and leads to apoptosis; this mechanism plays a significant role in Aβ apoptotic cell death and might be a new target for neurodegeneration treatments.

Effect of chronic sleep restriction and aging on calcium signaling and apoptosis in the hippocampus of young and aged animals

Aging leads to progressive deterioration of physiological function and diminished responses to environmental stress. Organic and functional alterations are frequently observed in elderly subjects. Although chronic sleep loss is observed during senescence, little is known about the impact of insufficient sleep on cellular function in aging neurons. Disruption of neuronal calcium (Ca²⁺) signaling is related to impaired neuronal function and cell death. It has been hypothesized that sleep deprivation may compromise neuronal stability and induce cell death in young neurons; however, it is necessary to evaluate the impact of aging on this process. Therefore, the aim of this study was to evaluate the effects of chronic sleep restriction (CSR) on Ca²⁺ signaling and cell death in the hippocampus of young and aged animals. We found that glutamate and carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) induced a greater elevation in cytosolic Ca²⁺ ([Ca²⁺](c)) in hippocampal slices from aged rats subjected to CSR compared to age-matched controls. Interestingly, aged-matched controls showed a reduced Ca²⁺ response to glutamate and FCCP, relative to both CSR and control young animals. Apoptotic nuclei were observed in aged rats from both treatment groups; however, the profile of apoptotic nuclei in aged CSR rats was highly variable. Bax and Bcl-2 protein expression did not change with aging in the CSR groups. Our study indicates that aging promotes changes in Ca²⁺ signaling, which may also be affected by CSR. These age-dependent changes in Ca²⁺ signaling may increase cellular vulnerability during CSR and contribute to Ca²⁺ signaling dysregulation, which may ultimately induce cell death.

Bcl-xL inhibits Bax-induced alterations in mitochondrial respiration and calcium release

Apoptosis is a natural cell elimination process involved in a number of physiological and pathological events. This process can be regulated by members of the Bcl-2 family. Bax, a pro-apoptotic member of this family, accelerates cell death, while the pro-survival member, Bcl-x(L), can antagonize the pro-apoptotic function of Bax to promote cell survival. In the present study, we have evaluated the effect of Bcl-x(L) on Bax-induced alterations in mitochondrial respiration and calcium release. We found that in primary cultured astrocytes, recombinant Bcl-x(L) is able to antagonize Bax-induced decrease in mitochondrial respiration and increase in mitochondrial calcium release. In addition, we found that Bcl-x(L) can lower the calcium store in the endoplasmic reticulum, thus limiting potential calcium flux induced by apoptosis. This regulation of calcium flux by Bcl-x(L) may represent an important mechanism by which this protein promotes cell survival.