In Silico Analysis of the Antagonist Effect of Enoxaparin on the ApoE4-Amyloid-Beta (Aß) Complex at Different pH Conditions.

Apolipoprotein E4 (ApoE4) is thought to increase the risk of developing Alzheimer's disease. Several studies have shown that ApoE4-Amyloid ß (Aß) interactions can increment amyloid depositions in the brain and that this can be augmented at low pH values. On the other hand, experimental studies in transgenic mouse models have shown that treatment with enoxaparin significantly reduces cortical Aß levels, as well as decreases the number of activated astrocytes around Aß plaques. However, the interactions between enoxaparin and the ApoE4-Aß proteins have been poorly explored. In this work, we combine molecular dynamics simulations, molecular docking, and binding free energy calculations to elucidate the molecular properties of the ApoE4-Aß interactions and the competitive binding affinity of the enoxaparin on the ApoE4 binding sites. In addition, we investigated the effect of the environmental pH levels on those interactions. Our results showed that under different pH conditions, the closed form of the ApoE4 protein, in which the C-terminal domain folds into the protein, remains stabilized by a network of hydrogen bonds. This closed conformation allowed the generation of six different ApoE4-Aß interaction sites, which were energetically favorable. Systems at pH5 and 6 showed the highest energetic affinity. The enoxaparin molecule was found to have a strong energetic affinity for ApoE4-interacting sites and thus can neutralize or disrupt ApoE4-Aß complex formation.

Vascular smooth muscle cell dysfunction contribute to neuroinflammation and Tau hyperphosphorylation in Alzheimer disease.

Despite the emerging evidence implying early vascular contributions to neurodegenerative syndromes, the role of vascular smooth muscle cells (VSMCs) in the pathogenesis of Alzheimer disease (AD) is still not well understood. Herein, we show that VSMCs in brains of patients with AD and animal models of the disease are deficient in multiple VSMC contractile markers which correlated with Tau accumulation in brain arterioles. Ex vivo and in vitro experiments demonstrated that VSMCs undergo dramatic phenotypic transitions under AD-like conditions, adopting pro-inflammatory phenotypes. Notably, these changes coincided with Tau hyperphosphorylation at residues Y18, T205, and S262. We also observed that VSMC dysfunction occurred in an age-dependent manner and that expression of Sm22α protein was inversely correlated with CD68 and Tau expression in brain arterioles of the 3xTg-AD and 5xFAD mice. Together, these findings further support the contribution of dysfunctional VSMCs in AD pathogenesis and nominate VSMCs as a potential therapeutic target in AD.

Diagnosis of Alzheimer's Disease in Developed and Developing Countries: Systematic Review and Meta-Analysis of Diagnostic Test Accuracy.

BACKGROUND: The present systematic review and meta-analysis of diagnostic test accuracy summarizes the last three decades in advances on diagnosis of Alzheimer's disease (AD) in developed and developing countries. OBJECTIVE: To determine the accuracy of biomarkers in diagnostic tools in AD, for example, cerebrospinal fluid, positron emission tomography (PET), and magnetic resonance imaging (MRI), etc. METHODS: The authors searched PubMed for published studies from 1990 to April 2020 on AD diagnostic biomarkers. 84 published studies were pooled and analyzed in this meta-analysis and diagnostic accuracy was compared by summary receiver operating characteristic statistics. RESULTS: Overall, 84 studies met the criteria and were included in a meta-analysis. For EEG, the sensitivity ranged from 67 to 98%, with a median of 80%, 95% CI [75, 91], tau-PET diagnosis sensitivity ranged from 76 to 97%, with a median of 94%, 95% CI [76, 97]; and MRI sensitivity ranged from 41 to 99%, with a median of 84%, 95% CI [81, 87]. Our results showed that tau-PET diagnosis had higher performance as compared to other diagnostic methods in this meta-analysis. CONCLUSION: Our findings showed an important discrepancy in diagnostic data for AD between developed and developing countries, which can impact global prevalence estimation and management of AD. Also, our analysis found a better performance for the tau-PET diagnostic over other methods to diagnose AD patients, but the expense of tau-PET scan seems to be the limiting factor in the diagnosis of AD in developing countries such as those found in Asia, Africa, and Latin America.

Early life alcohol exposure primes hypothalamic microglia to later-life hypersensitivity to immune stress: possible epigenetic mechanism.

Growing evidence has shown that developmental alcohol exposure induces central nervous system inflammation and microglia activation, which may contribute to long-term health conditions, such as fetal alcohol spectrum disorders. These studies sought to investigate whether neonatal alcohol exposure during postnatal days (PND) 2-6 in rats (third trimester human equivalent) leads to long-term disruption of the neuroimmune response by microglia. Exposure to neonatal alcohol resulted in acute increases in activation and inflammatory gene expression in hypothalamic microglia including tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6). Adults with neonatal alcohol pre-exposure (alcohol fed; AF) animals showed an exaggerated peripheral stress hormonal response to an immune challenge (lipopolysaccharides; LPS). In addition, there were significantly more microglia present in the hypothalamus of adult AF animals, and their hypothalamic microglia showed more cluster of differentiation molecule 11b (Cd11b) activation, TNF-α expression, and IL-6 expression in response to LPS. Interestingly, blocking microglia activation with minocycline treatment during PND 2-6 alcohol exposure ameliorated the hormonal and microglial hypersensitivity to LPS in AF adult animals. Investigation of possible epigenetic programming mechanisms by alcohol revealed neonatal alcohol decreased several repressive regulators of transcription in hypothalamic microglia, while concomitantly increasing histone H3 acetyl lysine 9 (H3K9ac) enrichment at TNF-α and IL-6 promoter regions. Importantly, adult hypothalamic microglia from AF animals showed enduring increases in H3K9ac enrichment of TNF-α and IL-6 promoters both at baseline and after LPS exposure, suggesting a possible epigenetic mechanism for the long-term immune disruption due to hypothalamic microglial priming.

Current Status of Stem Cell Treatment for Type I Diabetes Mellitus.

BACKGROUND: Diabetes mellitus is a major health concern in current scenario which has been found to affect people of almost all ages. The disease has huge impact on global health; therefore, alternate methods apart from insulin injection are being explored to cure diabetes. Therefore, this review mainly focuses on the current status and therapeutic potential of stem cells mainly mesenchymal stem cells (MSCs) for Type 1 diabetes mellitus in preclinical animal models as well as humans. METHODS: Current treatment for Type 1 diabetes mellitus mainly includes use of insulin which has its own limitations and also the underlying mechanism of diseases is still not explored. Therefore, alternate methods to cure diabetes are being explored. Stem cells are being investigated as an alternative therapy for treatment of various diseases including diabetes. Few preclinical studies have also been conducted using undifferentiated MSCs as well as in vitro MSCs differentiated into ß islet cells. RESULTS: These stem cell transplant studies have highlighted the benefits of MSCs, which have shown promising results. Few human trials using stem cells have also affirmed the potential of these cells in alleviating the symptoms. CONCLUSION: Stem cell transplantation may prove to be a safe and effective treatment for patients with Type 1 diabetes mellitus.

Mu-opioid receptor and delta-opioid receptor differentially regulate microglial inflammatory response to control proopiomelanocortin neuronal apoptosis in the hypothalamus: effects of neonatal alcohol.

BACKGROUND: Opioid receptors are known to control neurotransmission of various peptidergic neurons, but their potential role in regulation of microglia and neuronal cell communications is unknown. We investigated the role of mu-opioid receptors (MOR) and delta-opioid receptors (DOR) on microglia in the regulation of apoptosis in proopiomelanocortin (POMC) neurons induced by neonatal ethanol in the hypothalamus. METHODS: Neonatal rat pups were fed a milk formula containing ethanol or control diets between postnatal days 2-6. Some of the alcohol-fed rats additionally received pretreatment of a microglia activation blocker minocycline. Two hours after the last feeding, some of the pups were sacrificed and processed for histochemical detection of microglial cell functions or confocal microscopy for detection of cellular physical interaction or used for gene and protein expression analysis. The rest of the pups were dissected for microglia separation by differential gradient centrifugation and characterization by measuring production of various activation markers and cytokines. In addition, primary cultures of microglial cells were prepared using hypothalamic tissues of neonatal rats and used for determination of cytokine production/secretion and apoptotic activity of neurons. RESULTS: In the hypothalamus, neonatal alcohol feeding elevated cytokine receptor levels, increased the number of microglial cells with amoeboid-type circularity, enhanced POMC and microglial cell physical interaction, and decreased POMC cell numbers. Minocycline reversed these cellular effects of alcohol. Alcohol feeding also increased levels of microglia MOR protein and pro-inflammatory signaling molecules in the hypothalamus, and MOR receptor antagonist naltrexone prevented these effects of alcohol. In primary cultures of hypothalamic microglia, both MOR agonist [D-Ala 2, N-MePhe 4, Gly-ol]-enkephalin (DAMGO) and ethanol increased microglial cellular levels and secretion of pro-inflammatory cell signaling proteins. However, a DOR agonist [D-Pen2,5]enkephalin (DPDPE) increased microglial secretion of anti-inflammatory cytokines and suppressed ethanol's ability to increase microglial production of inflammatory signaling proteins and secretion of pro-inflammatory cytokines. In addition, MOR-activated inflammation promoted while DOR-suppressed inflammation inhibited the apoptotic effect of ethanol on POMC neurons. CONCLUSIONS: These results suggest that ethanol's neurotoxic action on POMC neurons results from MOR-activated neuroinflammatory signaling. Additionally, these results identify a protective effect of a DOR agonist against the pro-inflammatory and neurotoxic action of ethanol.

Beta-endorphin cell therapy for cancer prevention.

ß-Endorphin (BEP)-producing neuron in the hypothalamus plays a key role in bringing the stress axis to a state of homeostasis and maintaining body immune defense system. Long-term delivery of BEP to obtain beneficial effect on chemoprevention is challenging, as the peptides rapidly develop tolerance. Using rats as animal models, we show here that transplantation of BEP neurons into the hypothalamus suppressed carcinogens- and hormone-induced cancers in various tissues and prevented growth and metastasis of established tumors via activation of innate immune functions. In addition, we show that intracerebroventricular administration of nanosphere-attached dibutyryl cyclic adenosine monophosphate (dbcAMP) increased the number of BEP neurons in the hypothalamus, reduced the stress response, enhanced the innate immune function, and prevented tumor cell growth, progression, and metastasis. BEP neuronal supplementation did not produce any deleterious effects on general health but was beneficial in suppressing age-induced alterations in physical activity, metabolic, and immune functions. We conclude that the neuroimmune system has significant control over cancer growth and progression, and that activation of the neuroimmune system via BEP neuronal supplementation/induction may have therapeutic value for cancer prevention and improvement of general health.

Perillyl alcohol improves functional and histological outcomes against ischemia-reperfusion injury by attenuation of oxidative stress and repression of COX-2, NOS-2 and NF-κB in middle cerebral artery occlusion rats.

Perillyl alcohol (PA) is a monoterpene found in essential oils of mints, cherries, citreous fruits and lemon grass, reported to have antioxidant and anti-inflammatory properties. However, the role of PA in stroke is still illusive. Since oxidative stress and inflammation play a pivotal role in ischemia-reperfusion (I-R) injury, this study was designed to elucidate the potential effects of PA against I-R induced pathology in rat׳s brain. Middle cerebral artery occlusion (MCAO) for 2h followed by 22h reperfusion in Wistar male rats (250-280g, 14-16 weeks old) induced the behavioral and histological alterations along with exhausted antioxidant status and enhanced inflammatory mediators. However, PA administration (25, 50 and 100mg/kg b.wt orally once daily for 7 days) prior to MCAO significantly attenuated neurological deficits related to flexion test and spontaneous motor activity, improved grip strength and motor coordination in a dose dependent manner. PA treatment also inhibited oxidative stress in MCAO rats as evident from decreased lipid peroxidation and augmented level of reduced glutathione and restored activities of catalase, glutathione peroxidase, and glutathione reductase and thus, reduced infarct volume and protected the brain histology after I-R injury. Furthermore, PA markedly suppressed the level of proinflammatory cytokines (IL-1ß, TNF α and IL-6) and down regulated expressions of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (NOS-2) and nuclear factor κB (NF-κB) in MCAO group. In conclusion, PA mediates neuroprotection against I-R injury via mitigation of oxidative stress and inflammation and thus, may be a good therapeutic approach in stroke prone patient.

1,8-cineole (eucalyptol) mitigates inflammation in amyloid Beta toxicated PC12 cells: relevance to Alzheimer's disease.

Inflammatory process has a fundamental role in the pathogenesis of Alzheimer's disease and insoluble amyloid beta deposits and neurofibrillary tangles provide the obvious stimuli for inflammation. The present study demonstrate the effect of pretreatment of 1,8-cineole (Cin) on inflammation induced by Aß(25-35) in differentiated PC12 cells. The cells were treated with Cin at different doses for 24 h and then replaced by media containing Aß(25-35) for another 24 h. The cell viability was decreased in Aß(25-35) treated cells which was significantly restored by Cin pretreatment. Cin successfully reduced the mitochondrial membrane potential, ROS and NO levels in Aß(25-35) treated cells. Cin also lowered the levels of proinflammatory cytokines TNF-α, IL-1ß and IL-6 in Aß(25-35) treated cells. Moreover, Cin also succeeded in lowering the expression of NOS-2, COX-2 and NF-κB. This study suggests the protective effects of Cin on inflammation and provides additional evidence for its potential beneficial use in therapy as an anti-inflammatory agent in neurodegenerative disease.

Delayed administration of zingerone mitigates the behavioral and histological alteration via repression of oxidative stress and intrinsic programmed cell death in focal transient ischemic rats.

The neuronal mitochondria succumb to ischemia-reperfusion injury and release huge amount of reactive oxygen species and ultimately lead the neurons to intrinsic pathway of programmed cell death (iPCD). The present study was undertaken to elucidate the ischemia-reperfusion-induced oxidative stress and molecular events in iPCD 24 h post ischemia-reperfusion injury and plausible mitigation by zingerone, a potent antioxidant of ginger rhizome. The right middle cerebral artery was occluded for 2 h followed by reperfusion for 22 hours. A maximum infarct volume (43.29%) and mitochondrial injury (56.99%) was observed in middle cerebral artery occlusion (MCAO) group. However, zingerone administration (50 and 100 mg/kg b.wt. orally twice) at 5 h and 12 h from initiation of MCAO showed a significant reduction in infarct volume and mitochondrial injury (p<0.001). Zingerone treatment significantly improved behavioral outputs (p<0.05) and histological architecture (p<0.001) by reducing lipid peroxidation (p<0.01), augmenting the reduced glutathione content (p<0.01) and restoring Na(+)-K(+) ATPase and superoxide dismutase activities (p<0.01) in MCAO brain. Zingerone successfully reduced the caspase-3 and -9 activities in MCAO group (p<0.05) and succeeded in lowering the expressions of pro-apoptotic proteins - Apaf-1 and Bax (p<0.001). The present study suggests that zingerone is a potent antioxidant that salvaged the ischemic penumbral zone neurons by inhibiting iPCD and oxidative stress.

Anti-apoptotic and anti-inflammatory effect of Piperine on 6-OHDA induced Parkinson's rat model.

In the present study, we examined the molecular mechanism by which Piperine (bioactive compound of Piper nigrum) inhibits neuronal cell apoptosis. We further investigated the anti-inflammatory effect of Piperine on 6-OHDA induced Parkinson's disease. Consistent with its antioxidant properties, Piperine (10 mg/kg bwt) reduced 6-OHDA-induced lipid peroxidation and stimulated glutathione levels in striatum of rats. Furthermore, Piperine treatment diminished cytochrome-c release from mitochondria and reduced caspase-3 and caspase-9 activation induced by 6-OHDA. Treatment with Piperine markedly inhibited poly(ADP-ribose) polymerase activation, pro-apoptotic Bax levels and elevation of Bcl-2 levels. Piperine reduces contralateral rotations induced by apomorphine. Further narrow beam test and rotarod also showed improvement in motor coordination and balance behavior in rats treated with Piperine. In addition Piperine depletes inflammatory markers, TNF-α and IL-1ß in 6-OHDA-induced Parkinson's rats. We propose that, in addition to its antioxidant properties Piperine exerts a protective effect via anti-apoptotic and anti-inflammatory mechanism on 6-OHDA induced Parkinson's disease.

Azadirachta indica mitigates behavioral impairments, oxidative damage, histological alterations and apoptosis in focal cerebral ischemia-reperfusion model of rats.

Azadirachta indica Linn. (Meliaceae) has been used from ancient times as a remedy for various ailments. The present study was designed to investigate the antioxidant and anti-apoptotic properties of A. indica seed extract (ASE) in transient middle cerebral artery occlusion (MCAO) rat model. Antioxidant potential of ASE was determined in vitro. Further, ASE was evaluated against neurological deficits, histological alterations (TTC, CV and H&E) and oxidative damage (TBARS, GSH and nitrite) in MCAO rats. Moreover, caspase-3 and -9 were analyzed to evaluate the anti-apoptotic activity of ASE. ASE has shown potent in vitro reducing power (126.2 mg AsAE/g extract) and free radical scavenging activities (DPPH 171.0 and NO 176.0 µg/ml). Furthermore, ASE inhibited oxidative stress and decreased the activities of caspase-3 (26.7 %, p < 0.05) and caspase-9 (31.2 %, p < 0.01) thus, reduced neuronal loss in MCAO rats. Our data revealed that ASE has potent antioxidant and anti-apoptotic properties, and may be explored for its active constituents against neurodegenerative diseases.

Centella asiatica attenuates the neurobehavioral, neurochemical and histological changes in transient focal middle cerebral artery occlusion rats.

Centella asiatica has been used as psychoactive and antioxidant herbal medicine since ancient time. The present study was design to evaluate the preventive role of ethanolic extract of C. asiatica in middle cerebral artery occlusion (MCAO) in rats. Male Wistar rats were gavaged orally with C. asiatica extract (100, 200 and 300 mg/kg body weight once daily) for 21 days and thereafter subjected to right MCAO for 2 h followed by 22-h reperfusion. Brain injury was evaluated by 2,3,5-triphenyltetrazolium chloride and hematoxylin and eosin staining. Behavioural outcomes as neurological deficit, rota rod test, and grip strength were assessed. In addition, lipid peroxidation, enzymatic and non enzymatic antioxidants were analyzed to assess the oxidative stress. Our results revealed that C. asiatica administration greatly improved neurobehavioral activity and diminished infarction volume along with the restored histological morphology of brain in MCAO rats. Furthermore, supplementation with this extract to MCAO group has reduced the level of thiobarbituric acid reactive species, restored glutathione content and augmented the activities of antioxidant enzymes-catalase, glutathione peroxidase, glutathione reductase, glutathione-S-transferase and superoxide dismutase in a dose-dependent manner in ischemic rats. The remarkable antioxidant activity of C. asiatica may be attributed to its bioactive triterpenes, asiatic acid, asiaticoside, madecassic acid and madecosside and may be translated to clinical level for prevention of ischemic stroke.

Piperine suppresses cerebral ischemia-reperfusion-induced inflammation through the repression of COX-2, NOS-2, and NF-κB in middle cerebral artery occlusion rat model.

The pathophysiological mechanisms leading to neuronal injury in middle cerebral artery occlusion (MCAO) model of cerebral stroke are complex and multifactorial that form the bases of behavioral deficits and inflammation mediated damage. The present study demonstrates the effect of piperine pretreatment (10 mg/kg b wt, once daily p.o. for 15 days) on cerebral ischemia-induced inflammation in male Wistar rats. The right middle cerebral artery was occluded for 2 h followed by reperfusion for 22 h. A maximum infarct volume (57.80 %) was observed in ischemic MCAO group. However, piperine administration prior to ischemia showed a significant reduction in infarct volume (28.29 %; p < 0.05) and neuronal loss (12.72 %; p < 0.01). As a result of piperine pretreatment, a significant improvement in behavioral outputs of MCAO rats (p < 0.05-0.01) was observed. Piperine successfully reduced the level of proinflammatory cytokines IL-1ß, IL-6 and TNF-α, in ischemic group (p < 0.01). Ischemic group brain has shown edematous morphology with vacuolated architecture and pyknotic nuclei in H & E staining which was successfully ameliorated by piperine administration. Moreover, piperine also succeeded in lowering the expression of COX-2, NOS-2, and NF-κB (p < 0.01). Both cytosolic and nuclear NF-κB were down-regulated in ischemic group pre-administered with piperine (p < 0.01). The present study suggests that piperine is able to salvage the ischemic penumbral zone neurons by virtue of its anti-inflammatory property, thereby limiting ischemic cell death.

Synergistic effect of selenium and melatonin on neuroprotection in cerebral ischemia in rats.

The synergistic scavenger effects of selenium and melatonin collectively we called Se-Mel was studied on the prevention of neuronal injury induced by ischemia/reperfusion. Male Wistar rats were treated with sodium selenite (0.1 mg/kg, i.p.) and melatonin (10 mg/kg, i.p.) 30 min before the middle carotid artery occlusion (MCAO) and immediately after MCAO to male Wistar rats and was continued for 3 days once daily at the interval of 24 h. Behavioral activity (spontaneous motor activity and motor deficit) was improved in Se-Mel-treated rats as compared to MCAO group rats. The level of glutathione and the activity of antioxidant enzymes was depleted significantly while the content of thiobarbituric acid reactive substances, protein carbonyl, and nitric oxide radical (NO(·)) was increased significantly in MCAO group. Systemic administration of Se-Mel ameliorated oxidative stress and improves ischemia/reperfusion-induced focal cerebral ischemia. Se-Mel also inhibited inducible nitric oxide synthase expression in Se-Mel+MCAO group as compared to MCAO group rats. Thus, Se-Mel has shown an excellent neuroprotective effect against ischemia/reperfusion injury through an anti-ischemic pathway. In conclusion, we demonstrated that the pretreatment with Se-Mel at the onset of reperfusion, reduced post-ischemic damage, and improved neurological outcome following transient focal cerebral ischemia in male Wistar rat.