The Stroke Preclinical Assessment Network: Rationale, Design, Feasibility, and Stage 1 Results.

Cerebral ischemia and reperfusion initiate cellular events in brain that lead to neurological disability. Investigating these cellular events provides ample targets for developing new treatments. Despite considerable work, no such therapy has translated into successful stroke treatment. Among other issues-such as incomplete mechanistic knowledge and faulty clinical trial design-a key contributor to prior translational failures may be insufficient scientific rigor during preclinical assessment: nonblinded outcome assessment; missing randomization; inappropriate sample sizes; and preclinical assessments in young male animals that ignore relevant biological variables, such as age, sex, and relevant comorbid diseases. Promising results are rarely replicated in multiple laboratories. We sought to address some of these issues with rigorous assessment of candidate treatments across 6 independent research laboratories. The Stroke Preclinical Assessment Network (SPAN) implements state-of-the-art experimental design to test the hypothesis that rigorous preclinical assessment can successfully reduce or eliminate common sources of bias in choosing treatments for evaluation in clinical studies. SPAN is a randomized, placebo-controlled, blinded, multilaboratory trial using a multi-arm multi-stage protocol to select one or more putative stroke treatments with an implied high likelihood of success in human clinical stroke trials. The first stage of SPAN implemented procedural standardization and experimental rigor. All participating research laboratories performed middle cerebral artery occlusion surgery adhering to a common protocol and rapidly enrolled 913 mice in the first of 4 planned stages with excellent protocol adherence, remarkable data completion and low rates of subject loss. SPAN stage 1 successfully implemented treatment masking, randomization, prerandomization inclusion/exclusion criteria, and blinded assessment to exclude bias. Our data suggest that a large, multilaboratory, preclinical assessment effort to reduce known sources of bias is feasible and practical. Subsequent SPAN stages will evaluate candidate treatments for potential success in future stroke clinical trials using aged animals and animals with comorbid conditions.

Carbon monoxide attenuates vasospasm and improves neurobehavioral function after subarachnoid hemorrhage.

Subarachnoid hemorrhage (SAH) is a devastating form of hemorrhagic stroke and is a serious medical condition caused by bleeding usually due to a ruptured aneurysm. Oxidative stress and inflammation from hemoglobin and heme released from lysed red blood cells are some postulated causes of vasospasm during SAH, which could lead to delayed cerebral ischemia. At low amounts, carbon monoxide (CO) gas may be neuroprotective through anti-inflammation, anti-cell death, and restoration of normal blood flow. Hence, this study focuses on a noninvasive strategy to treat SAH by using CO as a therapeutic medical gas. Mice were treated with 250 ppm CO or air for 1h started at 2h after SAH. Various anatomical and functional outcomes were monitored at 1 and 7d after SAH. CO decreased neurological deficit score (47.4 ±â€¯10.5%) and increased activity (30.0 ±â€¯9.1%) and stereotypic counts (261.5 ±â€¯62.1%) at 7d. There was a significant increase in lumen area/wall thickness ratio in the middle cerebral artery (173.5 ±â€¯19.3%), which tended to increase in the anterior cerebral artery (25.5 ±â€¯4.3%) at 7d. This is the first report to demonstrate that CO minimizes delayed SAH-induced neurobehavioral deficits, which suggests that post-treatment with CO gas or CO-donors can be further tested as a potential therapy against SAH.

Tetrahydrocurcumin ameliorates homocysteine-mediated mitochondrial remodeling in brain endothelial cells.

Homocysteine (Hcy) causes endothelial dysfunction by inducing oxidative stress in most neurodegenerative disorders. This dysfunction is highly correlated with mitochondrial dynamics such as fusion and fission. However, there are no strategies to prevent Hcy-induced mitochondrial remodeling. Tetrahydrocurcumin (THC) is an anti-inflammatory and anti-oxidant compound. We hypothesized that THC may ameliorates Hcy-induced mitochondria remodeling in mouse brain endothelial cells (bEnd3) cells. bEnd3 cells were exposed to Hcy treatment in the presence or absence of THC. Cell viability and autophagic cell death were measured with MTT and MDC staining assay. Reactive oxygen species (ROS) production was determined using DCFH-DA staining by confocal microscopy. Autophagy flux was assessed using a conventional GFP-microtubule-associated protein 1 light chain 3 (LC3) dot assay. Interaction of phagophore marker LC-3 with mitochondrial receptor NIX was observed by confocal imaging. Mitochondrial fusion and fission were evaluated by western blot and RT-PCR. Our results demonstrated that Hcy resulted in cell toxicity in a dose-dependent manner and supplementation of THC prevented the detrimental effects of Hcy on cell survival. Furthermore, Hcy also upregulated fission marker (DRP-1), fusion marker (Mfn2), and autophagy marker (LC-3). Finally, we observed that Hcy activated mitochondrial specific phagophore marker (LC-3) and co-localized with the mitochondrial receptor NIX, as viewed by confocal microscopy. Pretreatment of bEnd3 with THC (15 µM) ameliorated Hcy-induced oxidative damage, mitochondrial fission/fusion, and mitophagy. Our studies strongly suggest that THC has beneficial effects on mitochondrial remodeling and could be developed as a potential therapeutic agent against hyperhomocysteinemia (HHcy) induced mitochondrial dysfunction.

High Methionine Diet Poses Cardiac Threat: A Molecular Insight.

High methionine diet (HMD) for example red meat which includes lamb, beef, pork can pose cardiac threat and vascular dysfunction but the mechanisms are unclear. We hypothesize that a diet rich in methionine can malfunction the cardiovascular system in three ways: (1) by augmenting oxidative stress; (2) by inflammatory manifestations; and (3) by matrix/vascular remodeling. To test this hypothesis we used four groups of mice: (1) WT; (2) WT + methionine; (3) CBS(+/-) ; (4) CBS(+/-) +methionine. We observed high oxidative stress in mice fed with methionine which was even higher in CBS(+/-) and CBS(+/-) +methionine. Higher oxidative stress was indicated by high levels of SOD-1 in methionine fed mouse hearts whereas IL-1ß, IL-6, TNFα, and TLR4 showed high inflammatory manifestations. The upregulated levels of eNOS/iNOS and upregulated levels of MMP2/MMP9 along with high collagen deposition indicated vascular and matrix remodeling in methionine fed mouse. We evaluated the cardiac function which was dysregulated in the mice fed with HMD. These mice had decreased ejection fraction and left ventricular dysfunction which subsequently leads to adverse cardiac remodeling. In conclusion, our study clearly shows that HMD poses a cardiac threat by increasing oxidative stress, inflammatory manifestations, matrix/vascular remodeling, and decreased cardiac function.

Homocysteine, Alcoholism, and Its Potential Epigenetic Mechanism.

Alcohol is the most socially accepted addictive drug. Alcohol consumption is associated with some health problems such as neurological, cognitive, behavioral deficits, cancer, heart, and liver disease. Mechanisms of alcohol-induced toxicity are presently not yet clear. One of the mechanisms underlying alcohol toxicity has to do with its interaction with amino acid homocysteine (Hcy), which has been linked with brain neurotoxicity. Elevated Hcy impairs with various physiological mechanisms in the body, especially metabolic pathways. Hcy metabolism is predominantly controlled by epigenetic regulation such as DNA methylation, histone modifications, and acetylation. An alteration in these processes leads to epigenetic modification. Therefore, in this review, we summarize the role of Hcy metabolism abnormalities in alcohol-induced toxicity with epigenetic adaptation and their influences on cerebrovascular pathology.

Hydrogen Sulfide Epigenetically Attenuates Homocysteine-Induced Mitochondrial Toxicity Mediated Through NMDA Receptor in Mouse Brain Endothelial (bEnd3) Cells.

Previously we have shown that homocysteine (Hcy) caused oxidative stress and altered mitochondrial function. Hydrogen sulfide (H2S) has potent anti-inflammatory, anti-oxidative, and anti-apoptotic effects. Therefore, in the present study we examined whether H2S ameliorates Hcy-induced mitochondrial toxicity which led to endothelial dysfunction in part, by epigenetic alterations in mouse brain endothelial cells (bEnd3). The bEnd3 cells were exposed to 100 µM Hcy treatment in the presence or absence of 30 µM NaHS (donor of H2S) for 24 h. Hcy-activate NMDA receptor and induced mitochondrial toxicity by increased levels of Ca(2+), NADPH-oxidase-4 (NOX-4) expression, mitochondrial dehydrogenase activity and decreased the level of nitrate, superoxide dismutase (SOD-2) expression, mitochondria membrane potentials, ATP production. To confirm the role of epigenetic, 5'-azacitidine (an epigenetic modulator) treatment was given to the cells. Pretreatment with NaHS (30 µM) attenuated the Hcy-induced increased expression of DNMT1, DNMT3a, Ca(2+), and decreased expression of DNMT3b in bEND3 cells. Furthermore, NaHS treatment also mitigated mitochondrial oxidative stress (NOX4, ROS, and NO) and restored ATP that indicates its protective effects against mitochondrial toxicity. Additional, NaHS significantly alleviated Hcy-induced LC3-I/II, CSE, Atg3/7, and low p62 expression which confirm its effect on mitophagy. Likewise, NaHS also restored level of eNOS, CD31, VE-cadherin and ET-1 and maintains endothelial function in Hcy treated cells. Molecular inhibition of NMDA receptor by using small interfering RNA showed protective effect whereas inhibition of H2S production by propargylglycine (PG) (inhibitor of enzyme CSE) showed mitotoxic effect. Taken together, results demonstrate that, administration of H2S protected the cells from HHcy-induced mitochondrial toxicity and endothelial dysfunction.

Epigenetic impact of curcumin on stroke prevention.

The epigenetic impact of curcumin in stroke and neurodegenerative disorders is curiosity-arousing. It is derived from Curcuma longa (spice), possesses anti-oxidative, anti-inflammatory, anti-lipidemic, neuro-protective and recently shown to exhibit epigenetic modulatory properties. Epigenetic studies include DNA methylation, histone modifications and RNA-based mechanisms which regulate gene expression without altering nucleotide sequences. Curcumin has been shown to affect cancer by altering epigenetic changes but its role as an epigenetic agent in cerebral stroke has not been much explored. Although curcumin possesses remarkable medicinal properties, the bioavailability of curcumin has limited its success in epigenetic studies and clinical trials. The present review is therefore designed to look into epigenetic mechanisms that could be induced with curcumin during stroke, along with its molecular designing with different moieties that may increase its bioavailability. Curcumin has been shown to be encapsulated in exosomes, nano-vesicles (<200 nm), thereby showing its therapeutic effects in brain diseases. Curcumin delivered through nanoparticles has been shown to be neuroregenerative but the use of nanoparticles in brain has limitations. Hence, curcumin-encapsulated exosomes along with curcumin-primed exosomes (exosomes released by curcumin-treated cells) are much needed to be explored to broadly look into their use as a novel therapy for stroke.

Mitochondrial epigenetics in bone remodeling during hyperhomocysteinemia.

Increased levels of homocysteine (Hcy), known as hyperhomocysteinemia (HHcy), is an independent risk factor of various diseases. Clinical studies report that people born with severe HHcy develop skeletal malformations with weaker bone. Studies also report that altered mitochondrial dynamics and altered epigenetics contribute to weaker bones and bone diseases. Although Hcy-induced mitochondrial dysfunction has been shown to affect bone metabolism, the role of mitochondrial epigenetics (mito-epigenetics) has not been studied in bones. The epigenetics in mitochondria is interesting as the mitochondrial genome size is small (16 kb) with fewer CpG, and without histones and introns. Recently, fascinating works on epigenetics along with the discovery of histone-like proteins in mitochondria are giving exciting areas for novel studies on mitochondria epigenetics. There are mutual cause and effect relationships between bone, mitochondria, Hcy, and epigenetics, but unfortunately, studies are lacking that describe the involvement of all these together in bone disease progression. This review describes the reciprocal relationships and mechanisms of Hcy-bone-mitochondria-epigenetics along with a short discussion of techniques which could be employed to assess Hcy-induced anomaly in bone, mediated through alterations in mito-epigenetics.

The time dimension to stroke: Circadian effects on stroke outcomes and mechanisms.

The circadian system is widely involved in the various pathological outcomes affected by time dimension changes. In the brain, the master circadian clock, also known as the "pacemaker," is present in the hypothalamus's suprachiasmatic nucleus (SCN). The SCN consists of molecular circadian clocks that operate in each neuron and other brain cells. These circadian mechanisms are controlled by the transcription and translation of specific genes such as the clock circadian regulator (Clock) and brain and muscle ARNT-Like 1 (Bmal1). Period (Per1-3) and cryptochrome (Cry1 and 2) negatively feedback and regulate the clock genes. Variations in the circadian cycle and these clock genes can affect stroke outcomes. Studies suggest that the peak stroke occurs in the morning after patients awaken from sleep, while stroke severity and poor outcomes worsen at midnight. The main risk factor associated with stroke is high blood pressure (hypertension). Blood pressure usually dips by 15-20% during sleep, but many hypertensives do not display this normal dipping pattern and are non-dippers. A sleep blood pressure is the primary determinant of stroke risk. This article discusses the possible mechanism associated with circadian rhythm and stroke outcomes.

Time Dimension Influences Severity of Stroke and Heightened Immune Response in Mice.

Ischemic stroke is caused by obstructed cerebral blood flow, which results in neurological injury and poor outcomes. Pro-inflammatory signaling from both residential and infiltrating immune cells potentiates cerebral injury and worsens patient outcomes after stroke. While the occurrence of a stroke exhibits a time-of-day-dependent pattern, it remains unclear whether disrupted circadian rhythms modulate post-stroke immunity. In this study, we hypothesized that stroke timing differentially affects immune activation in mice. Following middle cerebral artery occlusion (MCAO), circadian genes BMAL1, CLOCK, Cry1, and Cry2 elevated at ZT06, with a significant difference between ZT06 and ZT18. Conversely, expression of the negative limb circadian clock gene, Per1, decreased at ZT06 and ZT18 in stroke mice compared to sham. Paralleling these circadian gene expression changes, we observed a significant increase in TNF-α and a decrease in IL-10 expression at 48 h post-MCAO, when the procedure was performed at ZT06 (MCAO-ZT6), which corresponds to a deep sleep period, as compared to when the stroke was induced at ZT12 (MCAO-ZT12), ZT18 (MCAO-ZT18), or ZT0 (MCAO-ZT12). Similarly, increased pro-inflammatory, decreased anti-inflammatory monocytes, and increased NLRP3 were observed in blood, while changes in the expression of CD11b and Iba1 were noted within brain tissue at 48 h of MCAO-ZT06, as compared to MCAO-ZT18. Consistent with the increased immune response, infarct volume and sensorimotor deficits were greater in MCAO-ZT06 mice compared to MCAO-ZT18 mice at 48 h. Finally, we found reduced weight and length of the spleen while splenocytes showed significant time-dependent changes in Tregs, Bregs, and monocytes in MCAO-ZT06 mice. Taken together, this study demonstrates that circulating and splenic immune responses following ischemic stroke exhibit a circadian expression pattern which may contribute to time-of-day-dependent stroke outcomes.

Preclinical evaluation of circadian rhythm in ischemic stroke outcomes.

Stroke is a leading cause of disability and death worldwide. There is evidence that there is a circadian rhythm in stroke with peak occurrence in the morning (6 to 10 am). However, it is not clear if the size of infarcts and the outcome of stroke also varies during the 24-hour period. We hypothesized that the size of cerebral infarct and outcome from stroke would show circadian variation in a mouse suture occlusion model. Seven to eight-month-old C57BL/6J (n =10-12 mice/group) mice were randomly assigned to undergo middle cerebral artery occlusion (MCAO) for 60 minutes at different time points during the 24h day following zeitgeber time at ZT0, ZT6, ZT12, and Z18. Cerebral blood flow was monitored by Laser Speckle Contrast Imaging at baseline after occlusion, and again at 24h post-occlusion. Neurological deficit was observed by using Bederson score at 24h and 48h. The corner test was used to detect unilateral abnormalities in sensory and motor functions in the stroke mice at 48h. To estimate brain infarction, 2,3,5-tryphenyltetrazolium chloride staining was performed 48h after stroke and the infarct area was quantified using NIH-Image J software. We did not find a significant difference in cerebral blood flow at any time point. There was a significant decrease in neurological deficit as assessed using the Bederson Score from 24h (1.82 ± 1.11) to 48h (1.10 ± 0.12) in the ZT18 (midnight) period (p = 0.0025), however there were no differences between groups at 48h. In the corner test, we found right turn preference significantly higher (p = 0.0348) at noon/ZT06 (9.5 ± 1.06) compared to the fully awake (5.5 ± 4.06) (midnight, ZT18) period and ZT0 (6 am, 4.8 ± 0.97, p = 0.0087). Similarly, the infarction volume was significantly higher (p = 0.0220) during the sleep (ZT06, noon) period (35.22 ± 20.77) than when the ischemic mice were fully awake during the midnight/ZT18 period (15.68 ± 7.54). This is the first report demonstrating that mice have larger infarcts and worse short-term outcomes during their sleep period (noon/ZT06) than during their awake period (midnight/ZT18).

Effect of Experimental Ischemic Stroke and PGE2 EP1 Selective Antagonism in Alzheimer's Disease Mouse Models.

BACKGROUND: Neuroinflammation has been recognized as an important factor in the pathogenesis of Alzheimer's disease (AD). One of the most recognized pathways in mediating neuroinflammation is the prostaglandin E2-EP1 receptor pathway. OBJECTIVE: Here, we examined the efficacy of the selective EP1 antagonist ONO-8713 in limiting amyloid-ß (Aß), lesion volumes, and behavioral indexes in AD mouse models after ischemic stroke. METHODS: Transgenic APP/PS1, 3xTgAD, and wildtype (WT) mice were subjected to permanent distal middle cerebral artery occlusion (pdMCAO) and sham surgeries. Functional outcomes, memory, anatomical outcomes, and Aß concentrations were assessed 14 days after surgery. RESULTS: pdMCAO resulted in significant deterioration in functional and anatomical outcomes in the transgenic mice compared with the WT mice. No relevant differences were observed in the behavioral tests when comparing the ONO-8713 and vehicle-treated groups. Significantly lower cavitation (p = 0.0373) and percent tissue loss (p = 0.0247) were observed in APP/PS1 + ONO-8713 mice compared with the WT + ONO-8713 mice. However, the percent tissue injury was significantly higher in APP/PS1 + ONO-8713 mice compared with the WT + ONO-8713 group (p = 0.0373). Percent tissue loss was also significantly lower in the 3xTgAD + ONO-8713 mice than in the WT + ONO-8713 mice (p = 0.0185). ONO-8713 treatment also attenuated cortical microgliosis in APP/PS1 mice as compared with the vehicle (p = 0.0079); however, no differences were observed in astrogliosis across the groups. Finally, APP/PS1 mice presented with characteristic Aß load in the cortex while 3xTgAD mice exhibited very low Aß levels. CONCLUSION: In conclusion, under the experimental conditions, EP1 receptor antagonist ONO-8713 showed modest benefits in anatomical outcomes after stroke, mainly in APP/PS1 mice.

A high methionine, low folate and vitamin B6/B12 containing diet can be associated with memory loss by epigenetic silencing of netrin-1.

Memory-epigenetics which is the loss of memory due to epigenetic modifications can be due to the silencing of genes involved in cognitive functions and this is the basis of the current study. We hypothesize that a diet containing high methionine and low vitamins can lead to memory impairment by increasing global DNA methylation and therefore, silencing the netrin-1 gene, which encodes the glycoprotein involved in neurogenesis, axonal guidance and maintenance of the synaptic plasticity. Wild type (C57BL/6J) mice were fed with a diet containing excess methionine (1.2%), low-folate (0.08 mg/kg), vitamin B6 (0.01 mg/kg), and B12 (10.4 mg/kg) for 6 weeks. Mice were examined weekly for the long-term memory function, using a passive avoidance test, which determined loss of fear-motivated long-term memory starting from the fourth week of diet. Similarly, an increase in brain %5-methyl cytosine was observed starting from the 4th week of diet in mice. Mice fed with a high methionine, low folate and vitamins containing diet showed a decrease in netrin-1 protein expression and an increase in netrin-1 gene promotor methylation, as determined by methylation-sensitive restriction enzyme-polymerase chain reaction analysis. The increase in methylation of netrin-1 gene was validated by high-resolution melting and sequencing analysis. Furthermore, the association of netrin-1 with memory was established by administering netrin that considerably restored long-term fear motivated memory. Taken together, these results suggest that a diet rich in methionine and lacking in folate and vitamin B6/B12 can induce defects in learning and memory. Furthermore, the data indicates that decrease in netrin-1 expression due to hyper-methylation of its gene can be associated with memory loss. The animal procedures were approved by the Institutional Animal Care and Use Committee, University of Louisville, USA (No. A3586-01) on February 2, 2018.

A Comparison of Pathophysiology in Humans and Rodent Models of Subarachnoid Hemorrhage.

Non-traumatic subarachnoid hemorrhage (SAH) affects an estimated 30,000 people each year in the United States, with an overall mortality of ~30%. Most cases of SAH result from a ruptured intracranial aneurysm, require long hospital stays, and result in significant disability and high fatality. Early brain injury (EBI) and delayed cerebral vasospasm (CV) have been implicated as leading causes of morbidity and mortality in these patients, necessitating intense focus on developing preclinical animal models that replicate clinical SAH complete with delayed CV. Despite the variety of animal models currently available, translation of findings from rodent models to clinical trials has proven especially difficult. While the explanation for this lack of translation is unclear, possibilities include the lack of standardized practices and poor replication of human pathophysiology, such as delayed cerebral vasospasm and ischemia, in rodent models of SAH. In this review, we summarize the different approaches to simulating SAH in rodents, in particular elucidating the key pathophysiology of the various methods and models. Ultimately, we suggest the development of standardized model of rodent SAH that better replicates human pathophysiology for moving forward with translational research.

Hydrogen Sulfide Ameliorates Homocysteine-Induced Alzheimer's Disease-Like Pathology, Blood-Brain Barrier Disruption, and Synaptic Disorder.

Elevated plasma total homocysteine (Hcy) level is associated with an increased risk of Alzheimer's disease (AD). During transsulfuration pathways, Hcy is metabolized into hydrogen sulfide (H2S), which is a synaptic modulator, as well as a neuro-protective agent. However, the role of hydrogen sulfide, as well as N-methyl-D-aspartate receptor (NMDAR) activation, in hyperhomocysteinemia (HHcy) induced blood-brain barrier (BBB) disruption and synaptic dysfunction, leading to AD pathology is not clear. Therefore, we hypothesized that the inhibition of neuronal NMDA-R by H2S and MK801 mitigate the Hcy-induced BBB disruption and synapse dysfunction, in part by decreasing neuronal matrix degradation. Hcy intracerebral (IC) treatment significantly impaired cerebral blood flow (CBF), and cerebral circulation and memory function. Hcy treatment also decreases the expression of cystathionine-ß-synthase (CBS) and cystathionine-γ-lyase (CSE) in the brain along with increased expression of NMDA-R (NR1) and synaptosomal Ca(2+) indicating excitotoxicity. Additionally, we found that Hcy treatment increased protein and mRNA expression of intracellular adhesion molecule 1 (ICAM-1), matrix metalloproteinase (MMP)-2, and MMP-9 and also increased MMP-2 and MMP-9 activity in the brain. The increased expression of ICAM-1, glial fibrillary acidic protein (GFAP), and the decreased expression of vascular endothelial (VE)-cadherin and claudin-5 indicates BBB disruption and vascular inflammation. Moreover, we also found decreased expression of microtubule-associated protein 2 (MAP-2), postsynaptic density protein 95 (PSD-95), synapse-associated protein 97 (SAP-97), synaptosomal-associated protein 25 (SNAP-25), synaptophysin, and brain-derived neurotrophic factor (BDNF) showing synapse dysfunction in the hippocampus. Furthermore, NaHS and MK801 treatment ameliorates BBB disruption, CBF, and synapse functions in the mice brain. These results demonstrate a neuro-protective effect of H2S over Hcy-induced cerebrovascular pathology through the NMDA receptor. Our present study clearly signifies the therapeutic ramifications of H2S for cerebrovascular diseases such as Alzheimer's disease. Graphical Abstract ᅟ.

Streptozotocin Intracerebroventricular-Induced Neurotoxicity and Brain Insulin Resistance: a Therapeutic Intervention for Treatment of Sporadic Alzheimer's Disease (sAD)-Like Pathology.

Alzheimer's disease (AD) is a neurodegenerative disorder that is remarkably characterized by pathological hallmarks which include amyloid plaques, neurofibrillary tangles, neuronal loss, and progressive cognitive loss. Several well-known genetic mutations which are being used for the development of a transgenic model of AD lead to an early onset familial AD (fAD)-like condition. However, these settings are only reasons for a small percentage of the total AD cases. The large majorities of AD cases are considered as a sporadic in origin and are less influenced by a single mutation of a gene. The etiology of sporadic Alzheimer's disease (sAD) remains unclear, but numerous risk factors have been identified that increase the chance of developing AD. Among these risk factors are insulin desensitization/resistance state, oxidative stress, neuroinflammation, synapse dysfunction, tau hyperphosphorylation, and deposition of Aß in the brain. Subsequently, these risk factors lead to development of sAD. However, the underlying molecular mechanism is not so clear. Streptozotocin (STZ) produces similar characteristic pathology of sAD such as altered glucose metabolism, insulin signaling, synaptic dysfunction, protein kinases such as protein kinase B/C, glycogen synthase-3ß (GSK-3ß) activation, tau hyperphosphorylation, Aß deposition, and neuronal apoptosis. Further, STZ also leads to inhibition of Akt/PKB, insulin receptor (IR) signaling molecule, and insulin resistance in brain. These alterations mediated by STZ can be used to explore the underlying molecular and pathophysiological mechanism of AD (especially sAD) and their therapeutic intervention for drug development against AD pathology.

Mechanism of Oxidative Stress and Synapse Dysfunction in the Pathogenesis of Alzheimer's Disease: Understanding the Therapeutics Strategies.

Synapses are formed by interneuronal connections that permit a neuronal cell to pass an electrical or chemical signal to another cell. This passage usually gets damaged or lost in most of the neurodegenerative diseases. It is widely believed that the synaptic dysfunction and synapse loss contribute to the cognitive deficits in patients with Alzheimer's disease (AD). Although pathological hallmarks of AD are senile plaques, neurofibrillary tangles, and neuronal degeneration which are associated with increased oxidative stress, synaptic loss is an early event in the pathogenesis of AD. The involvement of major kinases such as mitogen-activated protein kinase (MAPK), extracellular receptor kinase (ERK), calmodulin-dependent protein kinase (CaMKII), glycogen synthase-3ß (GSK-3ß), cAMP response element-binding protein (CREB), and calcineurin is dynamically associated with oxidative stress-mediated abnormal hyperphosphorylation of tau and suggests that alteration of these kinases could exclusively be involved in the pathogenesis of AD. N-methyl-D-aspartate (NMDA) receptor (NMDAR) activation and beta amyloid (Aß) toxicity alter the synapse function, which is also associated with protein phosphatase (PP) inhibition and tau hyperphosphorylation (two main events of AD). However, the involvement of oxidative stress in synapse dysfunction is poorly understood. Oxidative stress and free radical generation in the brain along with excitotoxicity leads to neuronal cell death. It is inferred from several studies that excitotoxicity, free radical generation, and altered synaptic function encouraged by oxidative stress are associated with AD pathology. NMDARs maintain neuronal excitability, Ca(2+) influx, and memory formation through mechanisms of synaptic plasticity. Recently, we have reported the mechanism of the synapse redox stress associated with NMDARs altered expression. We suggest that oxidative stress mediated through NMDAR and their interaction with other molecules might be a driving force for tau hyperphosphorylation and synapse dysfunction. Thus, understanding the oxidative stress mechanism and degenerating synapses is crucial for the development of therapeutic strategies designed to prevent AD pathogenesis.

Curcumin-loaded embryonic stem cell exosomes restored neurovascular unit following ischemia-reperfusion injury.

We tested whether the combined nano-formulation, prepared with curcumin (anti-inflammatory and neuroprotective molecule) and embryonic stem cell exosomes (MESC-exocur), restored neurovascular loss following an ischemia reperfusion (IR) injury in mice. IR-injury was created in 8-10 weeks old mice and divided into two groups. Out of two IR-injured groups, one group received intranasal administration of MESC-exocur for 7days. Similarly, two sham groups were made and one group received MESC-exocur treatment. The study determined that MESC-exocur treatment reduced neurological score, infarct volume and edema following IR-injury. As compared to untreated IR group, MESC-exocur treated-IR group showed reduced inflammation and N-methyl-d-aspartate receptor expression. Treatment of MESC-exocur also reduced astrocytic GFAP expression and alleviated the expression of NeuN positive neurons in IR-injured mice. In addition, MESC-exocur treatment restored vascular endothelial tight (claudin-5 and occludin) and adherent (VE-cadherin) junction proteins in IR-injured mice as compared to untreated IR-injured mice. These results suggest that combining the potentials of embryonic stem cell exosomes and curcumin can help neurovascular restoration following ischemia-reperfusion injury in mice.

Mechanism of synapse redox stress in Okadaic acid (ICV) induced memory impairment: Role of NMDA receptor.

The N-methyl-D-aspartate (NMDA) receptor is a subtype of ionotropic glutamate receptor that is involved in synaptic mechanisms of learning and memory, and mediates excitotoxic neuronal injury. In this study, we tested the hypothesis that NMDA receptor subunit gene expression is altered in cortex and hippocampus of OKA induced memory impairment. Therefore in the present study, we checked the effect of OKA (ICV) on NMDA receptor regulation and synapse function. The memory function anomalies and synaptosomal calcium ion (Ca(2+)) level were increased in OKA treated rats brain; which was further protected by MK801 (0.05mg/kg. i.p) treatment daily for 13days. To elucidate the involvement of NMDA receptor, we estimated NR1, NR2A and NR2B (subunits) expression in rat brain. Results showed that expression of NR1 and NR2B were significantly increased, but expression of NR2A had no significant change in OKA treated rat brain. We also observed decrease in synapsin-1 mRNA and protein expression which indicates synapse dysfunction. In addition, we detected an increase in MDA and nitrite levels and a decrease in GSH level in synapse preparation which indicates synapse altered redox stress. Moreover, neuronal loss was also confirmed by nissl staining in periventricular cortex and hippocampus. Altered level of oxidative stress markers along with neuronal loss confirmed neurotoxicity. Further, MK801 treatment restored the level of NR1, NR2B and synapsin-1 expression, and protected from neuronal loss and synapse redox stress. In conclusion, Okadaic acid (OKA) induced expression of NR1 and NR2B deteriorates synapse function in rat brain which was confirmed by the neuroprotective effect of MK801.

Astrocyte mediated MMP-9 activation in the synapse dysfunction: An implication in Alzheimer disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that occurs due to spasms of the neurons, resulting in loss of memory and behavioral changes. In particular, synaptic loss has been described as an early event in the pathogenesis of AD. The increasing evidences have suggested the role of many matrix metalloproteinase (MMPs) in central nervous system (CNS) pathology. Many studies showed that MMPs enzymes are important for the pathophysiological process during Alzheimer's disease (AD). It is usually believed that the synaptic dysfunction and synapse loss contribute to the cognitive deficits of patients with AD. Cerebrovascular events such as blood-brain barrier (BBB) disruption lead to neuronal damage as well as neuroinflammation. BBB dysfunctions are observed at an early post injury time point, and are associated with activation of proteases, such as MMPs especially MMP-9 which is actively engage in a neuronal injury in the most of the neurodegenerative disorders. BBB opening is accompanied by astrocytic activation, BBB injury and dysregulation of cerebral blood flow. Activated MMPs disrupt neurovascular unit (NVU) which may starve the neurons and affect the synapse function by altering synaptic plasticity and ultimately lead to cognitive decline. However, how MMPs implicated in synaptic dysfunction what are the mechanism associated with this disparity needs to discuss for better understanding the role of MMP-9 in pathogenesis of AD. In this review, we focused on the role of astrocytes and MMP-9 in synaptic dysfunction. We also, underlined possible pharmacological strategies for drug development that might offer more insight into the pathogenesis of cerebrovascular disease such as stroke and Vascular dementia.