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Background: Mild traumatic brain injury (mTBI) generally resolves within weeks. However, 15-30% of patients present persistent pathological and neurobehavioral sequelae that negatively affect their quality of life. Hyperhomocysteinemia (HHCY) is a neurotoxic condition derived from homocysteine accumulation above 15 μM. HHCY can occur in diverse stressful situations, including those sustained by U.S. active-duty service members on the battlefield or during routine combat practice. Mild-TBI accounts for more than 80% of all TBI cases, and HHCY exists in 5-7% of the general population. We recently reported that moderate HHCY exacerbates mTBI-induced cortical injury pathophysiology, including increased oxidative stress. Several studies have demonstrated hippocampus vulnerability to oxidative stress and its downstream effects on inflammation and cell death., Objective: This study aimed to assess the deleterious impact of HHCY on mTBI-associated hippocampal pathological changes. We tested the hypothesis that moderate HHCY aggravates mTBI-induced hippocampal pathological changes., Methods: HHCY was induced in adult male Sprague-Dawley rats with a high methionine dose. Rats were then subjected to mTBI by controlled cortical impact under sustained HHCY. Blood plasma was assessed for homocysteine levels and brain tissue for markers of oxidative stress, blood-brain barrier integrity, and cell death. Endothelial cell ultrastructure was assessed by Electron Microscopy and working memory performance using the Y maze test., Results: HHCY increased the hippocampal expression of nitrotyrosine in astroglial cells and decreased tight junction protein occludin levels associated with the enlargement of the endothelial cell nucleus. Furthermore, HHCY altered the expression of apoptosis-regulating proteins α-ii spectrin hydrolysis, ERK1/2, and AKT phosphorylation, mirrored by exacerbated mTBI-related hippocampal neuronal loss and working memory deficits., Conclusion: Our findings indicate that HHCY is an epigenetic factor that modulates mTBI pathological progression in the hippocampus and represents a putative therapeutic target for mitigating such physiological stressors that increase severity., Competing Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2023.)

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality among military service members and civilians in the United States. Despite significant advances in the understanding of TBI pathophysiology, several clinical reports indicate that multiple genetic and epigenetic factors can influence outcome. Homocysteine (HCY) is a non-proteinogenic amino acid, the catabolism of which can be dysregulated by stress, lifestyle, aging, or genetic abnormalities leading to hyperhomocysteinemia (HHCY). HHCY is a neurotoxic condition and a risk factor for multiple neurological and cardiovascular disorders that occurs when HCY levels is clinically > 15 µM. Although the deleterious impact of HHCY has been studied in human and animal models of neurological disorders such as stroke, Alzheimer's disease and Parkinson's disease, it has not been addressed in TBI models. This study tested the hypothesis that HHCY has detrimental effects on TBI pathophysiology. Moderate HHCY was induced in adult male Sprague Dawley rats via daily administration of methionine followed by impact-induced traumatic brain injury. In this model, HHCY increased oxidative stress, upregulated expression of proteins that promote blood coagulation, exacerbated TBI-associated blood-brain barrier dysfunction and promoted the infiltration of inflammatory cells into the cortex. We also observed an increase of brain injury-induced lesion size and aggravated anxiety-like behavior. These findings show that moderate HHCY exacerbates TBI outcomes and suggest that HCY catabolic dysregulation may be a significant biological variable that could contribute to TBI pathophysiology heterogeneity.

Background: United States service members injured in combat theatre are often aeromedically evacuated within a few days to regional military hospitals. Animal and epidemiological research indicates that early exposure to flight hypobaria may worsen brain and other injuries. The mechanisms by which secondary exposure to hypobaria worsen trauma outcomes are not well elucidated. This study tested the hypothesis that hypobaria-induced oxidative stress and associated changes in homocysteine levels play a role in traumatic brain injury (TBI) pathological progression caused by hypobaria., Methods: Male Sprague Dawley rats were exposed to a 6 h hypobaria 24 h after mild TBI by the controlled cortical impact. Plasma and brain tissues were assessed for homocysteine levels, oxidative stress markers or glutathione metabolism, and behavioral deficits post-injury in the absence and presence of hypobaria exposure., Results: We found that hypobaria after TBI increased oxidative stress markers, altered homocysteine metabolism, and promoted glutathione oxidation. Increased glutathione metabolism was driven by differential upregulation of glutathione metabolizing genes. These changes correlated with increased anxiety-like behavior., Conclusion: These data provide evidence that hypobaria exposure after TBI increases oxidative stress and alters homocysteine elimination likely through enhanced glutathione metabolism. This pathway may represent a compensatory mechanism to attenuate free radical formation. Thus, hypobaria-induced enhancement of glutathione metabolism represents a potential therapeutic target for TBI management., Competing Interests: Declaration of conflicting interests:The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2021.)

Many victims of blast traumatic brain injury (TBI) are occupants of vehicles targeted by land mines. A rat model of under-vehicle blast TBI was used to test the hypothesis that the ensuing neuropathology and altered behavior are mitigated by vehicle frame designs that dramatically reduce blast-induced acceleration (G force). Male rats were restrained on an aluminum platform that was accelerated vertically at up to 2850g, in response to detonation of an explosive positioned under a second platform in contact with the top via different structures. The presence of elastomeric, polyurea-coated aluminum cylinders between the platforms reduced acceleration by 80% to 550g compared with 2350g with uncoated cylinders. Moreover, 67% of rats exposed to 2850g, and 20% of those exposed to 2350g died immediately after blast, whereas all rats subjected to 550g blast survived. Assays for working memory (Y maze) and anxiety (Plus maze) were conducted for up to 28 days. Rats were euthanized at 24 h or 29 days, and their brains were used for histopathology and neurochemical measurements. Rats exposed to 2350g blasts exhibited increased cleaved caspase-3 immunoreactive neurons in the hippocampus. There was also increased vascular immunoglobulin (Ig)G effusion and F4/80 immunopositive macrophages/microglia. Blast exposure reduced hippocampal levels of synaptic proteins Bassoon and Homer-1, which were associated with impaired performance in the Y maze and the Plus maze tests. These changes observed after 2350g blasts were reduced or eliminated with the use of polyurea-coated cylinders. Such advances in vehicle designs should aid in the development of the next generation of blast-resistant vehicles.

Many victims of blast-induced traumatic brain injury are occupants of military vehicles targeted by land mines. Recently improved vehicle designs protect these individuals against blast overpressure, leaving acceleration as the main force potentially responsible for brain injury. We recently developed a unique rat model of under-vehicle blast-induced hyperacceleration where exposure to acceleration as low as 50G force results in histopathological evidence of diffuse axonal injury and astrocyte activation, with no evidence of neuronal cell death. This study investigated the effects of much higher blast-induced accelerations (1200 to 2800G) on neuronal cell death, neuro-inflammation, behavioral deficits and mortality. Adult male rats were subjected to this range of accelerations, in the absence of exposure to blast overpressure, and evaluated over 28days for working memory (Y maze) and anxiety (elevated plus maze). In addition, brains obtained from rats at one and seven days post-injury were used for neuropathology and neurochemical assays. Sixty seven percent of rats died soon after being subjected to blasts resulting in 2800G acceleration. All rats exposed to 2400G acceleration survived and exhibited transient deficits in working memory and long-term anxiety like behaviors, while those exposed to 1200 acceleration G force only demonstrated increased anxiety. Behavioral deficits were associated with acute microglia/macrophage activation, increased hippocampal neuronal death, and reduced levels of tight junction- and synapse- associated proteins. Taken together, these results suggest that exposure of rats to high underbody blast-induced G forces results in neurologic injury accompanied by neuronal apoptosis, neuroinflammation and evidence for neurosynaptic alterations., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Traumatic brain injury (TBI) causes microglial activation and related neurotoxicity that contributes to chronic neurodegeneration and loss of neurological function. Selective activation of metabotropic glutamate receptor 5 (mGluR5) by the orthosteric agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG), is neuroprotective in experimental models of TBI, and has potent anti-inflammatory effects in vitro. However, the therapeutic potential of CHPG is limited due to its relatively weak potency and brain permeability. Highly potent, selective and brain penetrant mGluR5 positive allosteric modulators (PAMs) have been developed and show promise as therapeutic agents. We evaluated the therapeutic potential of a novel mGluR5 PAM, VU0360172, after controlled cortical impact (CCI) in mice. Vehicle, VU0360172, or VU0360172 plus mGluR5 antagonist (MTEP), were administered systemically to CCI mice at 3 h post-injury; lesion volume, hippocampal neurodegeneration, microglial activation, and functional recovery were assessed through 28 days post-injury. Anti-inflammatory effects of VU0360172 were also examined in vitro using BV2 and primary microglia. VU0360172 treatment significantly reduced the lesion, attenuated hippocampal neurodegeneration, and improved motor function recovery after CCI. Effects were mediated by mGluR5 as co-administration of MTEP blocked the protective effects of VU0360172. VU0360172 significantly reduced CD68 and NOX2 expression in activated microglia in the cortex at 28 days post-injury, and also suppressed pro-inflammatory signaling pathways in BV2 and primary microglia. In addition, VU0360172 treatment shifted the balance between M1/M2 microglial activation states towards an M2 pro-repair phenotype. This study demonstrates that VU0360172 confers neuroprotection after experimental TBI, and suggests that mGluR5 PAMs may be promising therapeutic agents for head injury.

Traumatic brain injury (TBI) is the leading cause of death in young adults in the United States, but there is still no effective agent for treatment. N-arachidonoylethanolamine (anandamide, AEA) is a major endocannabinoid in the brain. Its increase after brain injury is believed to be protective. However, the compensatory role of AEA is transient due to its rapid hydrolysis by the fatty acid amide hydrolase (FAAH). Thus, inhibition of FAAH can boost the endogenous levels of AEA and prolong its protective effect. Using a TBI mouse model, we found that post-injury chronic treatment with PF3845, a selective and potent FAAH inhibitor, reversed TBI-induced impairments in fine motor movement, hippocampus dependent working memory and anxiety-like behavior. Treatment with PF3845 inactivated FAAH activity and enhanced the AEA levels in the brain. It reduced neurodegeneration in the dentate gyrus, and up-regulated the expression of Bcl-2 and Hsp70/72 in both cortex and hippocampus. PF3845 also suppressed the increased production of amyloid precursor protein, prevented dendritic loss and restored the levels of synaptophysin in the ipsilateral dentate gyrus. Furthermore, PF3845 suppressed the expression of inducible nitric oxide synthase and cyclooxygenase-2 and enhanced the expression of arginase-1 post-TBI, suggesting a shift of microglia/macrophages from M1 to M2 phenotype. The effects of PF3845 on TBI-induced behavioral deficits and neurodegeneration were mediated by activation of cannabinoid type 1 and 2 receptors and might be attributable to the phosphorylation of ERK1/2 and AKT. These results suggest that selective inhibition of FAAH is likely to be beneficial for TBI treatment., (Published by Elsevier Ltd.)

2-arachidonylglycerol (2-AG) is the most abundant endocannabinoid in the central nervous system and is elevated after brain injury. Because of its rapid hydrolysis, however, the compensatory and neuroprotective effect of 2-AG is short-lived. Although inhibition of monoacylglycerol lipase, a principal enzyme for 2-AG degradation, causes a robust increase of brain levels of 2-AG, it also leads to cannabinoid receptor desensitization and behavioral tolerance. Alpha/beta hydrolase domain 6 (ABHD6) is a novel 2-AG hydrolytic enzyme that accounts for a small portion of 2-AG hydrolysis, but its inhibition is believed to elevate the levels of 2-AG within the therapeutic window without causing side effect. Using a mouse model of traumatic brain injury (TBI), we found that post-insult chronic treatment with a selective ABHD6 inhibitor WWL70 improved motor coordination and working memory performance. WWL70 treatment reduced lesion volume in the cortex and neurodegeneration in the dendate gyrus. It also suppressed the expression of inducible nitric oxide synthase and cyclooxygenase-2 and enhanced the expression of arginase-1 in the ipsilateral cortex at 3 and 7 days post-TBI, suggesting microglia/macrophages shifted from M1 to M2 phenotypes after treatment. The blood-brain barrier dysfunction at 3 and 7 days post-TBI was dramatically reduced. Furthermore, the beneficial effects of WWL70 involved up-regulation and activation of cannabinoid type 1 and type 2 receptors and were attributable to the phosphorylation of the extracellular signal regulated kinase and the serine/threonine protein kinase AKT. This study indicates that the fine-tuning of 2-AG signaling by modulating ABHD6 activity can exert anti-inflammatory and neuroprotective effects in TBI.

Although traumatic brain injury (TBI) is recognized as one of the leading causes of death from trauma to the central nervous system (CNS), no known treatment effectively mitigates its effects. Lithium, a primary drug for the treatment of bipolar disorder, has been known to have neuroprotective effects in various neurodegenerative conditions such as stroke. Until this study, however, it has not been investigated as a post-insult treatment for TBI. To evaluate whether lithium could have beneficial effects following TBI, lithium at a dose of 1.5 mEq/kg was administered after injury. Assessed at 3 days and 3 weeks post-injury using hematoxylin and eosin staining, lithium treatment was found to reduce lesion volume. Lithium at doses of 2.0 and 3.0 mEq/kg also significantly reduced lesion volume at 3 days after injury, and the therapeutic window was at least 3 h post-injury. TBI-induced neuronal death, microglial activation, and cyclooxygenase-2 induction were all attenuated by lithium at 3 days after injury. In addition, lithium treatment reduced TBI-induced matrix metalloproteinase-9 expression and preserved the integrity of the blood-brain barrier. As for behavioral outcomes, lithium treatment reduced anxiety-like behavior in an open-field test, and improved short- and long-term motor coordination in rotarod and beam-walk tests. Lithium robustly increased serine phosphorylation of glycogen synthase kinase-3β (GSK-3β), suggesting that the underlying mechanisms responsible for lithium's protective effects are triggered by increasing phosphorylation of this kinase and thereby inhibiting its activity. Our results support the notion that lithium has heretofore unrecognized capacity to mitigate the neurodegenerative effects and improve functional outcomes in TBI.

Peroxynitrite has been suggested to be the potent oxidant causing toxicity to neurons and oligodendrocytes (OLs). Our previous studies have illustrated that intracellular zinc liberation contributes to peroxynitrite toxicity to mature OLs. In this study, we further investigated the signaling pathways involved in this event and identified protein kinase C (PKC) as an important early signaling molecule. We found that a non-selective PKC inhibitor bisindolylmaleimide-1 blocked OL toxicity induced by a peroxynitrite generator SIN-1 and exogenous zinc. The protective effects were due to its inhibition on ERK1/2 phosphorylation and ROS generation. The same phenomenon was also observed in OLs following prolonged treatment with phorbol 12 myristate 13 acetate (PMA), which downregulates the conventional and the novel PKC isoforms (cPKCs and nPKCs). To determine the role of specific PKC isoforms, we found that a specific nPKC inhibitor rottlerin significantly reduced SIN-1- or zinc-induced toxicity, whereas Go6976, a cPKC inhibitor, reduced OL toxicity triggered by zinc, but not by SIN-1 at high concentrations. Rottlerin was more potent than Go6976 to attenuate ERK1/2 phosphorylation and ROS generation induced by SIN-1 or zinc. Surprisingly, zinc only induced phosphorylation of PKCθ, but not PKCδ. Knockdown of PKCθ using lentiviral shRNA attenuated SIN-1- or zinc-induced toxicity. These results suggest that PKCθ might be the major PKC isoform involved in peroxynitrite and zinc toxicity to mature OLs, and provide a rationale for development of specific inhibitors of PKCθ in the treatment of multiple sclerosis and other neurodegenerative diseases, in which peroxynitrite formation plays a pathogenic role., (Published by Elsevier Inc.)

Inhibition of phospholipase A(2) (PLA(2)) has recently been found to attenuate the pathogenesis of experimental autoimmune encephalomyelitis (EAE), a commonly used animal model of multiple sclerosis (MS). However, the protective mechanisms that underlie PLA(2) inhibition are still not well understood. In this study, we found that cytosolic PLA(2) (cPLA(2)) was highly expressed in infiltrating lymphocytes and macrophages/microglia in mouse spinal cord white matter. Although cPLA(2) is also expressed in spinal cord neurons and oligodendrocytes, there were no differences observed in these cell types between EAE and control animals. Arachidonyl trifluoromethyl ketone (AACOCF3), a cPLA(2) inhibitor, significantly reduced the clinical symptoms and inhibited the body weight loss typically found in EAE mice. AACOCF3 also attenuated the loss of mature, myelin producing, oligodendrocytes, and axonal damage in the spinal cord white matter. Nitrotyrosine immunoreactivity, an indicator of peroxynitrite formation, was dramatically increased in EAE mice and attenuated by treatment with AACOCF3. These protective effects were not evident when AA861, an inhibitor of lipoxygenase, was used. In primary cultures of microglia, lipopolysaccharide (LPS) induced an upregulation of cPLA(2), inducible nitric oxide synthase (iNOS) and components of the NADPH oxidase complex, p47phox and p67phox. AACOCF3 significantly attenuated iNOS induction, nitric oxide production and the generation of reactive oxygen species in reactive microglia. Similar to the decomposition catalyst of peroxynitrite, AACOCF3 also blocked oligodendrocyte toxicity induced by reactive microglia. These results suggest that AACOCF3 may prevent oligodendrocyte loss in EAE by attenuating peroxynitrite formation in the spinal cord white matter., (Published by Elsevier Inc.)

In this study intestinal and blood-brain barrier (BBB) permeability of ginkgolides A, B, C, J and bilobalide, isolated from Ginkgo biloba (Ginkgoaceae), was evaluated in Caco-2 and MDR1-MDCK cell monolayer models. The transport was examined for 2 hours in both absorptive and secretory directions. Quantitation was performed by UPLC-MS. In the Caco-2 model, each compound (100 microM) displayed a pH-dependent transport in the absorptive direction. A low permeability of ginkgolides was observed across the MDR1-MDCK model in the absorptive direction. An efflux was observed for all compounds in both the models. The efflux ratio was much higher in the MDR1-MDCK cell model (> 10) compared to the Caco-2 cell model (1.5-3.6). In comparison to ginkgolides, the permeability of bilobalide was much higher across the Caco-2 monolayer in both directions. However, a poor transport of bilobalide was observed in the MDR1-MDCK model in the absorptive direction. A high efflux was observed for all compounds in the mixture form as compared to their isolated forms. In rats, a single dose of bilobalide (8 mg/kg) administered intravenously resulted in a significant level of bilobalide in both plasma and brain. A brain-to-plasma partition coefficient of 0.56 at 120 min indicated its possibility of brain uptake., (Georg Thieme Verlag KG Stuttgart.New York.)

Apolipoprotein E4 (ApoE4) is a risk factor for Alzheimer's disease (AD). Whether this risk arises from a deficient function of E4 or the lack of protection provided by E2 or E3 is unclear. Previous studies demonstrate that deprivation of folate and vitamin E, coupled with dietary iron as a pro-oxidant, for 1 month displayed increased presenilin 1 (PS-1) expression, gamma-secretase, and Abeta generation in mice lacking ApoE (ApoE-/- mice). While ApoE-/- mice are a model for ApoE deficiency, they may not reflect the entire range of consequences of E4 expression. We therefore compared herein the impact of the above deficient diet on mice expressing human E2, E3, or E4. As folate deficiency is accompanied by a decrease in the major methyl donor, S-adenosyl methionine (SAM), additional mice received the deficient diet plus SAM. E2 was more protective than murine ApoE or E3 and E4. Surprisingly, PS-1 and gamma-secretase were over-expressed in E3 to the same extent as in E4 even under a complete diet, and were not alleviated by SAM supplementation. Abeta increased only in E4 mice maintained under the complete diet, and was alleviated by SAM supplementation. These findings suggest dietary compromise can potentiate latent risk factors for AD.

Loss of synapses has been correlated with dementia in Alzheimer's disease (AD) as an early event during the disease progression. Hence, synaptogenesis and neurogenesis in adulthood could serve as a therapeutic target for the prevention and treatment of AD. Recently, we have demonstrated enhanced hippocampal neurogenesis by oral administration of Ginkgo biloba extract (EGb 761) to a mouse model of AD. This study aims to identify the constituents that contribute to EGb 761-induced neurogenesis. Among the constituents tested, bilobalide and quercetin significantly increased cell proliferation in the hippocampal neurons in a dose-dependent manner. Bilobalide and quercetin also enhanced phosphorylation of cyclic-AMP Response Element Binding Protein (CREB) in these cells, and elevated the levels of pCREB and, brain-derived neurotrophic factor in mice brain. Immunofluorescence staining of synaptic markers shows remarkable dendritic processes in hippocampal neurons treated with either quercetin or bilobalide. Furthermore, both constituents restored amyloid-beta oligomers (also known as ADDL)-induced synaptic loss and phosphorylation of CREB. The present findings suggest that enhanced neurogenesis and synaptogenesis by bilobalide and quercetin may share a common final signaling pathway mediated by phosphorylation of CREB. Despite a recent report showing that EGb 761 was insufficient in prevent dementia, its constituents still warrant future investigation.

When maintained on a folate-deficient, iron-rich diet, transgenic mice lacking in apolipoprotein E (ApoE-/- mice) demonstrate impaired activity of glutathione S-transferase (GST), resulting in increased oxidative species within brain tissue despite abnormally high levels of glutathione. These mice also exhibit reduced levels of S-adenosyl methionine (SAM) and increased levels of its hydrolysis product S-adenosyl homocysteine, which inhibits SAM usage. Supplementation of the above diet with SAM restored GST activity and eliminated reactive oxygen species at the expense of stockpiled glutathione, suggesting that one or more SAM-dependent reactions were required to maintain GST activity. We examined herein the impact of SAM on GST activity using a cell-free assay. SAM stimulated GST activity in a dose-response manner when added to homogenates derived from the above ApoE-/- mice. SAM also increased activity of purified rat liver GST and recombinant GST. Filtering of SAM through a 4 kDa cutoff and systematic withholding of reaction components eliminated the possibility of any additional contaminating enzyme. These findings confirm that SAM can exert a direct effect on GST activity. Since Alzheimer's disease is accompanied by reduced GST activity, diminished SAM and increased SAH, these findings underscore the critical role of SAM in maintenance of neuronal health.

Folate deficiency is associated with increase in homocysteine levels. Abnormal plasma levels of that neurotoxic nonproteinogenic amino acid is implicated in many pathological conditions including cardiovascular diseases, neural tube defects, and is now recognized as a risk factor in Alzheimer's disease (AD) dementia. Homocysteine elimination is regulated by two metabolic pathways, namely, the transmethylation and the transsulfuration pathways. Its elimination via these two metabolic pathways is modulated by folate, a member of the B-vitamin family. Folate provides, via its metabolic end product 5-methyltetrahydrofolate, a methyl group that is used to reconvert homocysteine back to methionine through the transmethylation pathway. The efficiency of folate metabolism has an impact on the availability of S-adenosylmethionine, a compound that is known to activate homocysteine flux through the transsulfuration pathway and is necessary for utilization of a downstream antioxidant called glutathione under the catalysis of glutathione S-transferase enzyme. In this review, we will explore the impact of folate deprivation on the regulation of the methionine cycle and exhaustively describe different biochemical reactions that are implicated in the regulation of homocysteine elimination and that folate deficiency influences in AD neuropathology.

Standardized Ginkgo biloba extract EGb 761 exhibits beneficial effects to patients with Alzheimer's disease (AD). It was previously demonstrated that EGb 761 inhibits amyloid beta (Abeta) oligomerization in vitro, protects neuronal cells against Abeta toxicity, and improves cognitive defects in a mouse model of AD (Tg 2576). In this study, the neurogenic potential of EGb 761 and its effect on cAMP response element binding protein (CREB) were examined in a double transgenic mouse model (TgAPP/PS1). EGb 761 significantly increases cell proliferation in the hippocampus of both young (6 months) and old (22 months) TgAPP/PS1 mice, and the total number of neuronal precursor cells in vitro in a dose-dependent manner. Furthermore, Abeta oligomers inhibit phosphorylation of CREB and cell proliferation in the hippocampus of TgAPP/PS1 mice. Administration of EGb 761 reduces Abeta oligomers and restores CREB phosphorylation in the hippocampus of these mice. The present findings suggest that 1) enhanced neurogenesis by EGb 761 may be mediated by activation of CREB, 2) stimulation of neurogenesis by EGb 761 may contribute to its beneficial effects in AD patients and improved cognitive functions in the mouse model of AD, and 3) EGb 761 has therapeutic potential for the prevention and improved treatment of AD.

Clinical manifestation of Alzheimer's disease may depend upon interaction among its risk factors. Apolipoprotein E-deficient mice undergo oxidative damage and cognitive impairment when deprived of folate. We demonstrate herein that these mice were depleted in the methyl donor S-adenosyl methionine (SAM), which inhibited glutathione S-transferase, since this enzyme requires methylation of oxidative species prior to glutathione-dependent reduction. Dietary supplementation with SAM alleviated neuropathology. Since SAM deficiency promotes presenilin-1 overexpression, which increases gamma-secretase expression and Abeta generation, these findings directly link nutritional deficiency and genetic risk factors, and support supplementation with SAM for Alzheimer's therapy.

Homocysteine is a neurotoxic non-proteinogenic amino acid, an abnormal increase of which in plasma has been implicated in many pathological conditions including cardiovascular diseases, neural tube defects and is now recognized and Alzheimer's disease. Homocysteine elimination is regulated by the transmethylation and the transsulfuration pathways and is modulated by folate, a member of the B-vitamin family. A metabolic product of folate, 5 methyltetrahydrofolate, provides a methyl group that is used to reconvert homocysteine back to methionine through the transmethylation pathway. The efficiency of folate metabolism has an impact on the availability of S-adenosylmethionine (SAM), a compound that is known to activate homocysteine flux through the transsulfuration pathway. SAM is also necessary for utilization of the antioxidant glutathione via glutathione S-transferase. In this review, I will elaborate on different biochemical reactions that are implicated in the regulation of homocysteine elimination through the transmethylation and the transsulfuration pathways and on various consequences of folate deficiency on homocysteine metabolism.

Folate deficiency increases neuronal oxidative damage and potentiates the deleterious effects of apolipoprotein E (ApoE) deficiency. Mice lacking ApoE (ApoE -/- mice) upregulate the expression and activity of another enzyme, glutathione synthase (GS), when deprived of folate, in an apparent attempt to compensate for increased oxidative damage. Herein, we examined the influence of ApoE and folate deficiency on expression and activity of several enzymes of the methionine cycle. Expression and activity of methylene tetrahydrofolate reductase was increased in the order ApoE +/+ < ApoE +/- < ApoE -/- in response to folate deprivation. Expression of cystathione beta synthase followed a similar pattern. By contrast, expression and activity of methionine synthase decreased following folate deprivation in the order ApoE +/+ < ApoE +/- < ApoE -/-. These studies demonstrate that folate deficiency induces compensatory regulation of methionine cycle genes, and that these effects are potentiated by ApoE deficiency in a gene-dosage manner. They further support the notion that latent genetic deficiencies, including those of methionine cycle, may contribute to Alzheimer's disease, especially in concert with age-related nutritional deficiencies.

Oxidative stress contributes to age-related cognitive decline. In some instances, consumption of fruits and vegetables rich in antioxidant can provide superior protection than supplementation with purified antioxidants. Our prior studies have shown that supplementation with apple juice concentrate (AJC) alleviates oxidative damage and cognitive decline in a transgenic murine model compromised in endogenous antioxidant potential when challenged with a vitamin-deficient, oxidative stress-promoting diet. Herein, we demonstrate that AJC, administered in drinking water, is neuroprotective in normal, aged mice. Normal mice aged either 9-10 months or 2-2.5 years were maintained for 1 month on a complete diet or a diet lacking folate and vitamin E and containing iron as a pro-oxidant, after which oxidative damage was assayed by thiobarbituric acid-reactive substances and cognitive decline as assayed by performance in a standard Y-maze. Mice 9-12 months of age were unaffected by the deficient diet, while older mice demonstrated statistically-increased oxidative damage and poorer performance in a Y maze test. Supplementation with AJC prevented these neurodegenerative effects. These data are consistent with normal aged individuals being susceptible to neurodegeneration following dietary compromise such as folate deficiency, and a hastened onset of neurodegeneration in those individuals harboring a genetic risk factor such as ApoE deficiency. These findings also support the efficacy of antioxidant supplementation, including consumption of antioxidant-rich foods such as apples, in preventing the decline in cognitive performance that accompanies normal aging.

Oxidative stress is an early neurodegenerative insult in Alzheimer's disease (AD). Antioxidant mechanisms, including elements of the glutathione (GSH) pathway, undergo at least a transient compensatory increase that is apparently insufficient due to continued oxidative damage during disease progression. Mice deficient in apolipoprotein E, which provide a model for some aspects of AD, undergo increased oxidative damage to brain tissue and cognitive decline when maintained on a folate-free diet, despite a compensatory increase in glutathione synthase transcription and activity as well as increased levels of GSH. Dietary supplementation with N-acetyl cysteine (1 g/kg diet), a cell-permeant antioxidant and GSH precursor, alleviated oxidative damage and cognitive decline, and restored glutathione synthase and GSH levels in ApoE-deficient mice deprived of folate to those of normal mice maintained in the presence of folate. These data support the administration of antioxidant precursors to buffer oxidative damage in neurodegenerative disorders.

Oxidative stress is a major contributing factor in neurodegeneration and can arise from dietary, environmental, and genetic sources. Here we examine the separate and combined impact of deprivation of folate and vitamin E, coupled with dietary iron as a prooxidant, on normal mice and transgenic mice lacking apolipoprotein E (ApoE-/- mice). Both mouse strains exhibited increased levels of glutathione when deprived of folate and vitamin E, but a substantial further increase was observed in ApoE-/- mice. To determine the mechanism(s) underlying this increase, we quantified transcription and activity of glutathione synthase (GS). Both normal and ApoE-/- mice demonstrated increased GS activity when deprived of folate and vitamin E. However, transcription was increased only in ApoE-/- mice deprived of folate and vitamin E. These findings demonstrate that deficiency in one gene can result in compensatory up-regulation in a second relevant gene and, furthermore, indicate that compensation for oxidative stress can occur in brain tissue at epigenetic and genetic levels depending on the nature and/or extent of oxidative stress., (Copyright 2004 Wiley-Liss, Inc.)

Folate deprivation induces neurotoxicity that is potentiated by additional nutritional and genetic deficiencies including vitamin E and apolipoprotein E deficiency. These deficiencies collectively induce oxidative damage, cognitive impairment, and compensatory alteration in glutathione generation. Treatment with agents that regulate distinct portions of the methionine cycle, including the S-adenosyl homocysteine hydrolase inhibitor, 3-deaza adenosine, the methyl donor S-adenosyl methionine, and the antioxidant N-acetyl cysteine, provide neuroprotection against various aspects of neurotoxicity in normal and apolipoprotein E-deficient mice and in cultured neuronal cells deprived of dietary folate and vitamin E and subjected to iron overload. Here it is demonstrated that simultaneous treatment with these agents provide superior neuroprotection by alleviating individual and overlapping neurotoxic consequences. These findings support combinatorial treatments with agents that compensate for differential insults in age-related neurodegenerative disorders.

Increased oxidative stress, which can arise from dietary, environmental and/or genetic sources, contributes to the decline in cognitive performance during normal aging and in neurodegenerative conditions such as Alzheimer's disease. Supplementation with fruits and vegetables that are high in antioxidant potential can compensate for dietary and/or genetic deficiencies that promote increased oxidative stress. We have recently demonstrated that apple juice concentrate (AJC) prevents the increase in oxidative damage to brain tissue and decline in cognitive performance observed when transgenic mice lacking apolipoprotein E (ApoE-/-) are maintained on a vitamin-deficient diet and challenged with excess iron (included in the diet as a pro-oxidant). However, the mechanism by which AJC provided neuroprotection was not conclusively determined. Herein, we demonstrate that supplementation with AJC also prevents the compensatory increases in glutathione synthase transcription and activity that otherwise accompany maintenance of ApoE-/- mice on this vitamin-free diet in the presence of iron. Inclusion of the equivalent composition and concentration of sugars of AJC did not prevent these increases. These findings provide further evidence that the antioxidant potential of AJC can compensate for dietary and genetic deficiencies that otherwise promote neurodegeneration.