Characterization of endotoxin free protein production of brain-derived neurotrophic factor (BDNF) for the study of Parkinson model in SH-SY5Y differentiated cells.

Human neuronal cells are a more appropriate cell model for neurological disease studies such as Alzheimer and Parkinson's disease. SH-SY5Y neuroblastoma cells have been widely used for differentiation into a mature neuronal cell phenotype. The cellular differentiation process begins with retinoic acid incubation, followed by incubation with brain-derived neurotrophic factor (BDNF), a recombinant protein produced in E. coli cells. Endotoxin or lipopolysaccharide (LPS) is the major component of the outer membrane of bacterial cells that triggers the activation of pro-inflammatory cytokines and ultimately cell death. Consequently, any endotoxin contamination of the recombinant BDNF used for cell culture experiments would impact on data interpretation. Therefore, in this study, we expressed the BDNF recombinant protein in bacterial endotoxin-free cells that were engineered to modify the oligosaccharide chain of LPS rendering the LPS unable to trigger the immune response of human cells. The expression of DCX and MAP-2 in differentiated cells indicate that in-house and commercial BDNF are equally effective in inducing differentiation. This suggests that our in-house BDNF protein can be used to differentiate SH-SY5Y neuroblastoma cells without the need for an endotoxin removal step.

Pinin protects astrocytes from cell death after acute ischemic stroke via maintenance of mitochondrial anti-apoptotic and bioenergetics functions.

BACKGROUND: Stroke is the second most common cause of deaths worldwide. After an ischemic stroke, the proliferated reactive astrocytes in the peri-infarct areas play a beneficial role in neuronal survival. As such, astrocytes have gradually become a target for neuroprotection in stroke. The present study assessed the hypothesis that Pinin (Pnn), originally identified as a nuclear and desmosome-associated protein and is now known to possess anti-apoptotic capacity, protects astrocytes from cell death after ischemic stroke and delineated the underlying mechanisms. METHODS: In in vivo experiments, adult male Sprague-Dawley rats (12-week old) were used to induce acute focal cerebral ischemia employing the middle cerebral artery occlusion (MCAO) method. In in vitro experiments, postnatal day 1 (P1) Sprague-Dawley rat pups were used to prepare cultures of primary astrocytes. Oxygen-glucose deprivation (OGD) and re-oxygenation (OGD/R) procedures were employed to mimic the hypoxic-ischemic condition of stroke in those astrocytes. RESULTS: We found in the peri-infarct area of the ipsilateral cortex and striatum in Sprague-Dawley rats after transient MCAO an increase in Pnn expression that correlated positively with the time-course of infarction as detected by T2-weighted imaging and triphenyltetrazolium chloride staining, augmented number of reactive astrocytes that double-labelled with Pnn as determined by immunofluorescence, and enhanced cytotoxic edema as revealed by diffusion weighted imaging; but mirrored the decreased cleaved caspase-3 as measured by western blot. In an OGD and OGD/R model using primary cultured astrocytes, treatment with Pnn siRNA doubled the chance of surviving astrocytes to manifest cell death as revealed by flow cytometry, and blunted activated ERK signaling, reduced Bcl-2 expression and augmented cleaved caspase 3 detected by western blot in the normoxia, OGD or OGD/R group. Gene-knockdown of Pnn also impeded the reversal from decline in cell viability, elevation in lactate dehydrogenase leakage and decrease in ATP production in the OGD/R group. CONCLUSION: We conclude that the endogenous Pnn participates in neuroprotection after acute ischemic stroke by preserving the viability of astrocytes that survived the ischemic challenge via maintenance of mitochondrial anti-apoptotic and bioenergetics functions.

Comparing the Effects of Melatonin with Caloric Restriction in the Hippocampus of Aging Mice: Involvement of Sirtuin1 and the FOXOs Pathway.

It has been suggested that age-related neurodegeneration might be associated with neuropeptide Y (NPY); sirtuin1 (SIRT1) and forkhead box transcription factors O subfamily (FOXOs) pathways. Melatonin, a hormone mainly secreted by the pineal gland, is another anti-aging agent associated with the SIRT1-FOXOs pathway. This study aimed to compare the effects of melatonin (Mel) and caloric restriction (CR) on the expression of Sirt1, FoxO1, FoxO3a and FOXOs target genes in the aging mouse hippocampus. Neuropeptide Y-knockout (NpyKO) and wild-type (WT) male mice aged 19 months were previously treated either with food ad libitum or CR for 16 months. WT old animals were divided into four groups: control, CR, Mel and CR+Mel treated groups. The Mel and CR+Mel were treated with melatonin 10 mg/kg, daily, subcutaneously for 7 consecutive days. Mel treatment upregulated the mRNA expression of Sirt1, FOXOs (FoxO1 and FoxO3a) target genes that regulated the cell cycle [e.g., cyclin-dependent kinase inhibitor 1B (p27)], Wingless and INT-1 (Wnt1) and inducible signaling pathway protein 1 (Wisp1) in the aged mouse hippocampus. CR treatment also showed the similar actions. However, the mRNA expression of Sirt1, FoxO1, FoxO3a, p27 or Wisp1 did not alter in the CR+Mel group when compared with CR or Mel group. Melatonin could not produce any additive effect on the CR treatment group, suggesting that both treatments mimicked the effect, possibly via the same pathway. NPY which mediates physiological adaptations to energy deficits is an essential link between CR and longevity in mice. In order to focus on the role of Npy in mediating the effects of melatonin, the gene expression between NpyKO and WT male mice were compared. Our data showed that, in the absence of Npy, melatonin could not mediate effects on those gene expressions, suggesting that Npy was required for melatonin to mediate the effect, possibly, on life extension.

Effects of melatonin on severe crush spinal cord injury-induced reactive astrocyte and scar formation.

The present work aimed at analyzing the effects of melatonin on scar formation after spinal cord injury (SCI). Upregulation of reactive astrocyte under SCI pathological conditions has been presented in several studies. It has been proved that the crucial factor in triggering this upregulation is proinflammatory cytokines. Moreover, scar formation is an important barrier to axonal regeneration through the lesion area. Melatonin plays an important role in reducing inflammation, but its effects on scar formation in the injured spinal cord remain unknown. Hence, we used the model of severe crush injury in mice to investigate the effects of melatonin on scar formation. Mice were randomly separated into four groups; SCI, SCI+Melatonin 1 (single dose), SCI+Melatonin 14 (14 daily doses), and control. Melatonin was administered by intraperitoneal injection (10 mg/kg) after injury. Immunohistochemical analysis, Western blot, and behavioral evaluation were used to explore the effects of melatonin after SCI for 14 days. The melatonin-treated mice presented higher expression of neuronal markers (P < 0.001). Remarkably, the inflammatory response appeared to be greatly reduced in the SCI+Melatonin 14 group (P < 0.001), which also displayed less scar formation (P < 0.05). These findings suggest that melatonin inhibits scar formation by acting on inflammatory cytokines after SCI. Overall, our results suggest that melatonin is a promising treatment strategy after SCI that deserves further investigation. © 2016 Wiley Periodicals, Inc.

Melatonin regulates the transcription of ßAPP-cleaving secretases mediated through melatonin receptors in human neuroblastoma SH-SY5Y cells.

Melatonin is involved in the control of various physiological functions, such as sleep, cell growth and free radical scavenging. The ability of melatonin to behave as an antioxidant, together with the fact that the Alzheimer-related amyloid ß-peptide (Aß) triggers oxidative stress through hydroxyl radical-induced cell death, suggests that melatonin could reduce Alzheimer's pathology. Although the exact etiology of Alzheimer's disease (AD) remains to be established, excess Aß is believed to be the primary contributor to the dysfunction and degeneration of neurons that occurs in AD. Aß peptides are produced via the sequential cleavage of ß-secretase ß-site APP-cleaving enzyme 1 (BACE1) and γ-secretase (PS1/PS2), while α-secretase (ADAM10) prevents the production of Aß peptides. We hypothesized that melatonin could inhibit BACE1 and PS1/PS2 and enhance ADAM10 expression. Using the human neuronal SH-SY5Y cell line, we found that melatonin inhibited BACE1 and PS1 and activated ADAM10 mRNA level and protein expression in a concentration-dependent manner and mediated via melatonin G protein-coupled receptors. Melatonin inhibits BACE1 and PS1 protein expressions through the attenuation of nuclear factor-κB phosphorylation (pNF-κB). Moreover, melatonin reduced BACE1 promoter transactivation and consequently downregulated ß-secretase catalytic activity. The present data show that melatonin is not only a potential regulator of ß/γ-secretase but also an activator of α-secretase expression through the activation of protein kinase C, thereby favoring the nonamyloidogenic pathway over the amyloidogenic pathway. Altogether, our findings suggest that melatonin may be a potential therapeutic agent for reducing the risk of AD in humans.

Melatonin regulates aging and neurodegeneration through energy metabolism, epigenetics, autophagy and circadian rhythm pathways.

Brain aging is linked to certain types of neurodegenerative diseases and identifying new therapeutic targets has become critical. Melatonin, a pineal hormone, associates with molecules and signaling pathways that sense and influence energy metabolism, autophagy, and circadian rhythms, including insulin-like growth factor 1 (IGF-1), Forkhead box O (FoxOs), sirtuins and mammalian target of rapamycin (mTOR) signaling pathways. This review summarizes the current understanding of how melatonin, together with molecular, cellular and systemic energy metabolisms, regulates epigenetic processes in the neurons. This information will lead to a greater understanding of molecular epigenetic aging of the brain and anti-aging mechanisms to increase lifespan under healthy conditions.

Effects of mitragynine on viability, proliferation, and migration of C6 rat glioma, SH-SY5Y human neuroblastoma, and HT22 immortalized mouse hippocampal neuron cell lines.

Mitragynine (MG) is an indole alkaloid found in the extract of Mitragyna speciosa Korth native to Southeast Asia. Although MG is known for its pain-relieving and psychoactive effects, reports have suggested that it has therapeutic potential against neoplasms and psychiatric disorders. However, no evidence currently exists to support the effect of MG on brain tumors. This study aimed to investigate the antitumor effects of MG in C6 rat glioma and SH-SY5Y human neuroblastoma tumor cell lines compared with those in the non-tumor HT22 mouse hippocampal neuronal cell line. MTT assay for cell viability, clonogenic and wound healing assays for cell migration, Hoechst 33342/propidium iodide staining for nuclear morphology, and cell cycle distribution using flow cytometry were performed. MG at 125.47 µM (50 µg/ml) significantly reduced the viability of all cell lines, and the clonogenicity of C6 glioma cells began decreasing at 75.28 µM (30 µg/ml) of MG. Cell migration was inhibited in C6 and HT22 cells treated with 75.28 µM (30 µg/ml) of MG. Apoptotic nuclear condensation and fragmentation were observed in all cell lines treated with 125.47 µM (50 µg/ml) MG, whereas late-phase apoptotic cells were predominant in the group treated with 250.94 µM (100 µg/ml) of MG. The cell cycle assay results suggest that MG arrested the S phase in the C6 cell line and the G2/M phase in the HT22 cell lines. This study showed that MG induces cell death and cell cycle arrest, disrupting cell migration and reducing the clonogenicity of brain tumor cells.

Neuronal Pnn Deficiency Increases Oxidative Stress and Exacerbates Cerebral Ischemia/Reperfusion Injury in Mice.

Cerebral stroke remains one of the leading causes of death worldwide. Ischemic stroke caused by the sudden loss of blood flow in brain is the major type of cerebral stroke. In addition to necrotic cell death in the ischemic core region, neuronal apoptosis is usually observed in the ischemic penumbra. Pnn, a multi-functional protein, participates in cellular proliferation, migration, differentiation, apoptosis as well as cell-cell interaction through its abilities in regulating gene transcription and mRNA processing. Our recent studies have demonstrated that Pnn has a cell type-specific distribution manner in neural cells under ischemic injury and plays a protective role in astrocytes against ischemic stress. In this study, we generated an inducible neuron-specific Pnn deficiency mouse model to further investigate the physiological role of Pnn in neurons. To directly examine the role of neuronal Pnn in ischemic stress, four weeks after induction of Pnn deficiency in neurons, middle cerebral artery occlusion (MCAO) was applied to induce cerebral ischemia/reperfusion in mice. In the cerebrum and hippocampus with neuronal Pnn depletion, the expression of SRSF2, a mRNA splicing regulator, was increased, while the expression of SRSF1, a functional antagonist of SRSF2, was reduced. Expression levels of ROS generators (NOX-1 and NOX-2) and antioxidant proteins (GR, HO-1, NQO-1) were upregulated in brain tissue with loss of neuronal Pnn, echoing an increase in oxidized proteins in cortical and hippocampal neurons. Furthermore, the expression of DNA damage marker, p53bp1, was found in the choroid plexus of mice with neuronal Pnn depletion. In mice with MCAO, compared to wild type mice, both increased cerebral infarcted area and elevated expressions of proapoptotic proteins were found in mice with neuronal Pnn depletion. In conclusion, Pnn deficiency increases oxidative stress in neurons and exacerbates cerebral ischemia/reperfusion injury in mice.

Proteomic profiling reveals neuronal ion channel dysregulation and cellular responses to DNA damage-induced cell cycle arrest and senescence in human neuroblastoma SH-SY5Y cells exposed to cypermethrin.

Cypermethrin (CYP), a synthetic pyrethroid of class II, is widely used as a pesticide worldwide. The primary target of cypermethrin is a voltage-gated sodium channel. The neurotoxicity of CYP has been extensively studied in terms of affecting neuronal development, increasing cellular oxidative stress, and apoptosis. However, little is known about how it affects the expression of channel proteins involved in synaptic transmission, as well as the effects of cypermethrin on DNA damage and cell cycle processes. We found that the ligand and voltage-gated calcium channels and proteins involved in synaptic transmission including NMDA 1 receptor subunit, alpha 1A-voltage-dependent calcium channel, synaptotagmin-17, and synaptojanin-2 were downregulated in CYP-treated cells. After 48 h of CYP exposure, cell viability was reduced with flattened and enlarged morphology. The levels of 23 proteins regulating cell cycle processes were altered in CYP-treated cells, according to a proteomic study. The cell cycle analysis showed elevated G0/G1 cell cycle arrest and DNA fragmentation at the sub-G0 stage after CYP exposure. CYP treatment also increased senescence-associated ß-galactosidase positive cells, DNA damage, and apoptotic markers. Taken together, the current study showed that cypermethrin exposure caused DNA damage and hastened cellular senescence and apoptosis via disrupting cell cycle regulation. In addition, despite its primary target sodium channel, CYP might cause synaptic dysfunction via the downregulation of synaptic proteins and dysregulation of synapse-associated ion channels.

Spilanthes acmella Murr. ameliorates chronic stress through improving mitochondrial function in chronic restraint stress rats.

Chronic stress is a risk factor for the development of psychiatric illnesses through impairment of the ability to appropriately regulate physiological and behavioral responses, but the molecular events that lead to damage of hippocampal neurons remain unclear. The medicinal herb Spilanthes acmella Murr. has been used as a traditional medicine for various diseases and its extracts exhibit antioxidant activity. The present study explored the molecular signals of mitochondrial dynamics and investigated the beneficial effects of S. acmella Murr. An ethyl acetate extract of this plant was used to assess mitochondrial dynamics in response to chronic restraint stress (CRS) in male Sprague-Dawley rats. The results demonstrated that the S. acmella Murr. extract reduced the expression of mitochondrial fission protein but induced HSP60, MnSOD and ATPsynthase in the hippocampus of the CRS rats. In addition, S. acmella Murr. extract reversed depressive symptoms in the forced swim test. Our findings suggested that S. acmella Murr. extract provides a potential treatment of chronic stress, and that the mechanism is associated with the alleviation of neuronal injury and maintenance of mitochondrial function.

Melatonin ameliorates methamphetamine-induced cognitive impairments by inhibiting neuroinflammation via suppression of the TLR4/MyD88/NFκB signaling pathway in the mouse hippocampus.

Methamphetamine (METH) is a highly addictive psychostimulant that causes significant health issues due to high prevalence of its illegal use. Chronic use of METH is associated with cognitive impairments in both human and animal studies, but the underlying mechanism remains unclear. METH-induced neuroinflammation is, potentially, one of the factors that causes cognitive impairments. Therefore, the present study aimed to assess whether melatonin could provide protection against inflammation, in a manner comparable to the anti-inflammatory agent, minocycline, with consequent improvements of METH-induced cognitive impairments and associated abnormalities in the mouse hippocampus. Results from the Morris water maze (MWM) test and the novel object recognition test (NORT) showed that melatonin given after METH injections could ameliorate both METH-induced spatial and recognition memory impairments. These memory impairments are associated with changes in the neuroinflammatory profiles, including IL-6, IL-1ß, and TNF-α, both in the blood serum and hippocampus of adult mice. METH-treated mice also exhibited reactive astrocytes and activated microglia in the hippocampus. METH-induced activation of glial cells is associated with the activation of the TLR4/MyD88/NFκB signaling pathway. Moreover, melatonin administration led to recovery of these METH-induced markers to control levels. Thus, we conclude that melatonin could potentially be used as a cognitive enhancer and anti-inflammatory agent in the treatment of METH use disorder in humans.

Melatonin reverts methamphetamine-induced learning and memory impairments and hippocampal alterations in mice.

AIMS: Methamphetamine (METH) has become a major public health problem because of its abuse and profound neurotoxic effects, causing alterations in brain structure and function, and impairing cognitive functions, including attention, decision making, emotional memory, and working memory. This study aimed to determine whether melatonin (MEL), the circadian-control hormone, which has roles beyond circadian rhythm regulation, could restore METH-induced cognitive and neuronal impairment. MAIN METHODS: Mice were treated with either METH (1 mg/kg) or saline for 7 days, followed by MEL (10 mg/kg) or saline for another 14 days. The Morris water maze (MWM) test was performed one day after the last saline or MEL injection. The hippocampal neuronal density, synaptic density, and receptors involved in learning and memory, along with downstream signaling molecules (NMDA receptor subunits GluN2A, GluN2B, and CaMKII) were investigated by immunoblotting. KEY FINDINGS: METH administration significantly extended escape latency in learning phase and reduced the number of target crossings in memory test-phase as well as decreased the expression of BDNF, NMDA receptors, TrkB receptors, CaMKII, ßIII tubulin, and synaptophysin. MEL treatment significantly ameliorated METH-induced increased escape latency, decreased the number of target crossings and decreased expression of BDNF, NMDA receptors, TrkB receptors, CaMKII, ßIII tubulin and synaptophysin. SIGNIFICANCE: METH administration impairs learning and memory in mice, and MEL administration restores METH-induced neuronal impairments which is probably through the changes in BDNF, NMDA receptors, TrkB receptors, CaMKII, ßIII tubulin and synaptophysin. Therefore, MEL is potentially an innovative and promising treatment for learning and memory impairment of humans.

Expression and Distribution Pattern of Pnn in Ischemic Cerebral Cortex and Cultured Neural Cells Exposed to Oxygen-Glucose Deprivation.

Pinin (Pnn), a multifunctional protein, participates in embryonic development as well as in cellular apoptosis, proliferation, and migration through regulating mRNA alternative splicing and gene transcription. Previous studies have shown that Pnn plays important roles in neural system development and the expression level of Pnn in astrocytes is altered by ischemic stress and associated with cellular apoptosis. In the present study, we further utilized primary cultured rat neurons and astrocytes with oxygen-glucose deprivation (OGD) and a mouse model with middle cerebral artery occlusion (MCAO)-induced ischemic stroke to examine the effect of ischemic stress on Pnn expression and distribution in different types of neural cells. Under normoxia, Pnn is mainly localized in the nuclear speckle of primary cultured neurons. The expression level of Pnn was increased after the OGD treatment and then decreased in the reoxygenation period. Moreover, the cytoplasmic expression of Pnn was observed in neurons with OGD and reoxygenation (OGD/R). Unlike that in neurons, the Pnn expression in astrocytes was decreased after OGD treatment and then gradually increased during the reoxygenation period. Of interest, the nuclear-cytoplasmic translocation of Pnn was not observed in astrocytes with OGD/R. In the MCAO mouse model, the neuronal expression of Pnn in the peri-ischemic region was reduced by three days post induction of ischemic stroke. However, the Pnn expression in astrocytes was not altered. Moreover, the nuclear speckle distribution of Pnn in neurons was also diminished following ischemic stroke. In conclusion, the Pnn expression and distribution after OGD and during reoxygenation showed distinct manners in neurons and astrocytes, implying that Pnn may play different roles in different types of neural cells in the stress response to ischemic injury.

The induction of Neuron-Glial2 (NG2) expressing cells in methamphetamine toxicity-induced neuroinflammation in rat brain are averted by melatonin.

Neuron-Glial2 (NG2) expressing cells are described as the oligodendrocyte precursor cells in the brain. This study aimed to investigate the possible involvement of NG2 cells under the methamphetamine (METH)-induced neurotoxicity and neuroprotective capacity of melatonin. The results showed that the levels of NG2 in rat brain gradually increase from postnatal day 0 to postnatal day 8 and then the lower levels of NG2 are shown in adults. In adult rats, the levels of NG2 and COX-2 in the brain were significantly increased in lipopolysaccharide treatment. Pretreatment of 10 mg/kg melatonin prior to treating with METH was able to reduce an increase in the levels of NG2 and activation in astrocyte and microglia. These findings would extend the contribution of NG2 expressing cells in the adult brain during pathological conditions such as neuroinflammation.

Amphetamine-induced changes in dopamine receptors in early postnatal rat brain.

Amphetamines are among the most widely abused drugs. The user population includes a large proportion of women of child-bearing age. The early ontogeny of the axons in the neocortex and other neural structures positions them to influence the development and connectivity of non-aminergic dendrites and axons in these structures. A cascade of abnormalities in neural circuitry may result from the effects of amphetamines on the dopaminergic system. An attempt has been made to investigate the possible changes in the dopaminergic system in neonatal rats (a human third trimester equivalent model) following chronic D-amphetamine exposure. Neonatal rats were administered 5-15 mg/kg D-amphetamine subcutaneously daily from postnatal day 4 to day 10. Several parameters related to the dopaminergic system were measured. The results showed that tyrosine hydroxylase enzyme levels were significantly decreased in the prefrontal cortex, dorsal striatum and nucleus accumbens. Dopamine D1 receptor (DRD1) levels increased in the dorsal striatum whereas dopamine D2 receptor (DRD2) levels significantly decreased in both the prefrontal cortex and the dorsal striatum but significantly increased in the nucleus accumbens. In order to investigate whether these changes occurred at the transcriptional level, DRD1 and DRD2 mRNAs were detected. The results showed that DRD1 mRNA levels were significantly increased in the dorsal striatum whereas DRD2 mRNA levels were significantly increased in all three brain regions. These results indicate that early D-amphetamine exposure altered the dopaminergic system in the developing rat brain. This change may lead to abnormal perinatal stimulation that may yield long-term consequences.

Melatonin attenuates methamphetamine-induced deactivation of the mammalian target of rapamycin signaling to induce autophagy in SK-N-SH cells.

Methamphetamine (METH) is a commonly abused drug that damages nerve terminals by causing reactive oxygen species (ROS) formation, apoptosis, and neuronal damage. Autophagy, a type of programmed cell death independent of apoptosis, is negatively regulated by the mammalian target of the rapamycin (mTOR) signaling pathway. It is not known, however, whether autophagy is involved in METH-induced neurotoxicity. Therefore, we investigated the effect of METH on autophagy and its upstream regulator, the mTOR signaling pathway. Using the SK-N-SH dopaminergic cell line, we found that METH induces the expression of LC3-II, a protein associated with the autophagosome membrane, in a dose-dependent manner. Moreover, METH inhibits the phosphorylation of mTOR and the action of its downstream target, the eukaryotic initiation factor (eIF)4E-binding protein, 4EBP1. Melatonin, a major secretory product of pineal, is a potent naturally produced antioxidant that acts through various mechanisms to ameliorate the toxic effects of ROS. We found that a pretreatment with melatonin enhances mTOR activity and 4EBP1 phosphorylation and protects against the formation of LC3-II in SK-N-SH cells exposed to METH. This work demonstrates a novel role for melatonin as a neuroprotective agent against METH.

Diverse roles of mitochondria in ischemic stroke.

Stroke is the leading cause of adult disability and mortality in most developing and developed countries. The current best practices for patients with acute ischemic stroke include intravenous tissue plasminogen activator and endovascular thrombectomy for large-vessel occlusion to improve clinical outcomes. However, only a limited portion of patients receive thrombolytic therapy or endovascular treatment because the therapeutic time window after ischemic stroke is narrow. To address the current shortage of stroke management approaches, it is critical to identify new potential therapeutic targets. The mitochondrion is an often overlooked target for the clinical treatment of stroke. Early studies of mitochondria focused on their bioenergetic role; however, these organelles are now known to be important in a wide range of cellular functions and signaling events. This review aims to summarize the current knowledge on the mitochondrial molecular mechanisms underlying cerebral ischemia and involved in reactive oxygen species generation and scavenging, electron transport chain dysfunction, apoptosis, mitochondrial dynamics and biogenesis, and inflammation. A better understanding of the roles of mitochondria in ischemia-related neuronal death and protection may provide a rationale for the development of innovative therapeutic regimens for ischemic stroke and other stroke syndromes.

Long-term administration of melatonin attenuates neuroinflammation in the aged mouse brain.

Aging is often accompanied by a decline in cognitive function in conjunction with a variety of neurobiological changes, including neuroinflammation. Melatonin is a key endogenous indoleamine secreted by the pineal gland that plays a crucial role in the regulation of circadian rhythms, is a potent free radical scavenger, has anti-inflammatory activity and serves numerous other functions. However, the role of melatonin in sterile inflammation in the brain has not been fully investigated. In the present study, we investigated the neuroinflammation status in aged mouse brains. The results showed that the protein levels of integrin αM (CD11b), glial fibrillary acidic protein (GFAP), the major pro-inflammatory cytokines (interleukin-1 beta [IL-1ß], interleukin-6 [IL-6], and tumor necrosis factor alpha [TNF-α]) and phosphor-nuclear factor kappa B (pNFκB) were significantly increased, while N-methyl-D-aspartate (NMDA) receptor subunits NR2A and NR2B, Ca2+/calmodulin-dependent protein kinase II (CaMKII), and brain-derived neurotrophic factor (BDNF) were down-regulated in the hippocampus and prefrontal cortex (PFC) of 22-months-old (aged) mice compared with 2-months-old (young adult) mice. Melatonin was administered in the drinking water to a cohort of the aged mice at a dose of 10 mg/kg/day, beginning at an age of 16 months for 6 months. Our results revealed that melatonin significantly attenuated the alterations in these protein levels. The present study suggests an advantageous role for melatonin in anti-inflammation, and this may lead to the prevention of memory impairment in aging.

Evaluation of Autonomic Nervous System, Saliva Cortisol Levels, and Cognitive Function in Major Depressive Disorder Patients.

Major depressive disorder (MDD) is associated with changes in autonomic nervous system (ANS) and cognitive impairment. Heart rate variability (HRV) and Pulse pressure (PP) parameters reflect influences of the sympathetic and parasympathetic nervous system. Cortisol exerts its greatest effect on the hippocampus, a brain area closely related to cognitive function. This study aims to examine the effect of HRV, PPG, salivary cortisol levels, and cognitive function in MDD patients by using noninvasive techniques. We have recruited MDD patients, diagnosed based on DSM-V-TR criteria compared with healthy control subjects. Their HRV and PP were measured by electrocardiogram (ECG) and photoplethysmography (PPG). Salivary cortisol levels were collected and measured on the same day. MDD patients exhibited elevated values of mean HR, standard deviation of HR (SDHR), low frequency (LF) power, low frequency/high frequency (LF/HF) ratio, mean PP, standard deviation of pulse pressure (SDPP), and salivary cortisol levels. Simultaneously, they displayed lower values of mean of R-R intervals (mean NN), standard deviation of R-R intervals (SDNN), high frequency (HF) power, and WCST scores. Results have shown that the ANS of MDD patients were dominated by the sympathetic activity and that they have cognitive deficits especially in the domain of executive functioning.

Role of Melatonin in Reducing Amphetamine-Induced Degeneration in Substantia Nigra of Rats via Calpain and Calpastatin Interaction.

Excessive intracellular calcium levels induce calpain activation, thereby triggering the cell death cascade. Several lines of evidence have demonstrated the neuroprotective role of the overexpression of calpain inhibitor, calpastatin. In this study, amphetamine-induced degeneration in the substantia nigra of rats was determined by evaluating the decrease in the levels of tyrosine hydroxylase phosphorylation. Amphetamine significantly decreased calpastatin levels but increased calpain levels. An induction in calpain activity was demonstrated by an increase in the formation of calpain spectrin breakdown products. The deleterious effects of amphetamine exposure were diminished in rats by pretreatment with melatonin. In addition, the effect of melatonin on calpastatin expression was investigated in human neuroblastoma SH-SY5Y cells. Melatonin was able to increase the calpastatin levels, and this effect could be blocked by luzindole, a melatonin receptor antagonist. These results demonstrate the neuroprotective ability of melatonin and its role in inducing calpastatin expression via a receptor-dependent pathway.