Modulation of Oxidative Stress and Neuroinflammation by Cannabidiol (CBD): Promising Targets for the Treatment of Alzheimer's Disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disease and the most common form of dementia globally. Although the direct cause of AD remains under debate, neuroinflammation and oxidative stress are critical components in its pathogenesis and progression. As a result, compounds like cannabidiol (CBD) are being increasingly investigated for their ability to provide antioxidant and anti-inflammatory neuroprotection. CBD is the primary non-psychotropic phytocannabinoid derived from Cannabis sativa. It has been found to provide beneficial outcomes in a variety of medical conditions and is gaining increasing attention for its potential therapeutic application in AD. CBD is not psychoactive and its lipophilic nature allows its rapid distribution throughout the body, including across the blood-brain barrier (BBB). CBD also possesses anti-inflammatory, antioxidant, and neuroprotective properties, making it a viable candidate for AD treatment. This review outlines CBD's mechanism of action, the role of oxidative stress and neuroinflammation in AD, and the effectiveness and limitations of CBD in preclinical models of AD.

Exploring Rosiglitazone's Potential to Treat Alzheimer's Disease through the Modulation of Brain-Derived Neurotrophic Factor.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that debilitates over 55 million individuals worldwide. Currently, treatments manage and alleviate its symptoms; however, there is still a need to find a therapy that prevents or halts disease progression. Since AD has been labeled as "type 3 diabetes" due to its similarity in pathological hallmarks, molecular pathways, and comorbidity with type 2 diabetes mellitus (T2DM), there is growing interest in using anti-diabetic drugs for its treatment. Rosiglitazone (RSG) is a peroxisome proliferator-activated receptor-gamma agonist that reduces hyperglycemia and hyperinsulinemia and improves insulin signaling. In cellular and rodent models of T2DM-associated cognitive decline and AD, RSG has been reported to improve cognitive impairment and reverse AD-like pathology; however, results from human clinical trials remain consistently unsuccessful. RSG has also been reported to modulate the expression of brain-derived neurotrophic factor (BDNF), a protein that regulates neuroplasticity and energy homeostasis and is implicated in both AD and T2DM. The present review investigates RSG's limitations and potential therapeutic benefits in pre-clinical models of AD through its modulation of BDNF expression.

Sex Differences in the Level of Homocysteine in Alzheimer's Disease and Parkinson's Disease Patients: A Meta-Analysis.

Although recent studies suggest homocysteine (Hcy) is an independent risk factor for neurodegenerative disorders, little is known about sex differences in the levels of Hcy. In this study, we conducted a comparative meta-analysis to investigate sex differences in the levels of Hcy in both Alzheimer's disease (AD) and Parkinson's disease (PD) patients. Reports of Hcy stratified by sex in both AD and PD patients were obtained from electronic databases. From the initial 1595 records, 921 were assessed for eligibility, of which 16 sufficiently reported sex differences. Standardized mean difference (SMDs) using random effects together with tests of heterogeneity and quality assessment were applied in this meta-analysis. Data from 3082 diagnosed patients (1162 males and 1920 females) were included. There were statistically significant differences in the levels of Hcy between sexes in AD and PD patients, with an SMD of 0.291 [0.17, 0.41], p < 0.05, 95% CI, with higher Hcy levels detected in males. Subgroup comparisons did not find a statistically significant difference in the levels of Hcy between AD and PD patients. The overall risk of bias for the analyzed studies was low, with some moderate risk of bias across select domains. This meta-analysis determined that compared to females, males with either AD or PD have higher levels of Hcy. These findings suggest that Hcy could be a useful biomarker for predicting neurodegenerative diseases in males; however, further studies are needed to confirm the clinical utility of this suggestion.

Naturally Occurring Antioxidant Therapy in Alzheimer's Disease.

It is estimated that the prevalence rate of Alzheimer's disease (AD) will double by the year 2040. Although currently available treatments help with symptom management, they do not prevent, delay the progression of, or cure the disease. Interestingly, a shared characteristic of AD and other neurodegenerative diseases and disorders is oxidative stress. Despite profound evidence supporting the role of oxidative stress in the pathogenesis and progression of AD, none of the currently available treatment options address oxidative stress. Recently, attention has been placed on the use of antioxidants to mitigate the effects of oxidative stress in the central nervous system. In preclinical studies utilizing cellular and animal models, natural antioxidants showed therapeutic promise when administered alone or in combination with other compounds. More recently, the concept of combination antioxidant therapy has been explored as a novel approach to preventing and treating neurodegenerative conditions that present with oxidative stress as a contributing factor. In this review, the relationship between oxidative stress and AD pathology and the neuroprotective role of natural antioxidants from natural sources are discussed. Additionally, the therapeutic potential of natural antioxidants as preventatives and/or treatment for AD is examined, with special attention paid to natural antioxidant combinations and conjugates that are currently being investigated in human clinical trials.

VANL-100 Attenuates Beta-Amyloid-Induced Toxicity in SH-SY5Y Cells.

Antioxidants are being explored as novel therapeutics for the treatment of neurodegenerative diseases such as Alzheimer's disease (AD) through strategies such as chemically linking antioxidants to synthesize novel co-drugs. The main objective of this study was to assess the cytoprotective effects of the novel antioxidant compound VANL-100 in a cellular model of beta-amyloid (Aß)-induced toxicity. The cytotoxic effects of Aß in the presence and absence of all antioxidant compounds were measured using the 3-(4,5-dimethylthiazol-2-yl)2-5-diphenyl-2H-tetrazolium bromide (MTT) assay in SH-SY5Y cells in both pre-treatment and co-treatment experiments. In pre-treatment experiments, VANL-100, or one of its parent compounds, naringenin (NAR), alpha-lipoic acid (ALA), or naringenin + alpha-lipoic acid (NAR + ALA), was administrated 24 h prior to an additional 24-h incubation with 20 µM non-fibril or fibril Aß25-35. Co-treatment experiments consisted of simultaneous treatment with Aß and antioxidants. Pre-treatment and co-treatment with VANL-100 significantly attenuated Aß-induced cell death. There were no significant differences between the protective effects of VANL-100, NAR, ALA, and NAR + ALA with either form of Aß, or in the effect of VANL-100 between 24-h pre-treatment and co-treatment. These results demonstrate that the novel co-drug VANL-100 is capable of eliciting cytoprotective effects against Aß-induced toxicity.

Dissociable cognitive impairments in two strains of transgenic Alzheimer's disease mice revealed by a battery of object-based tests.

Object recognition tasks detect cognitive deficits in transgenic Alzheimer's disease (AD) mouse models. Object recognition, however, is not a unitary process, and there are many uncharacterized facets of object processing with relevance to AD. We therefore systematically evaluated object processing in 5xFAD and 3xTG AD mice to clarify the nature of object recognition-related deficits. Twelve-month-old male and female 5xFAD and 3xTG mice were assessed on tasks for object identity recognition, spatial recognition, and multisensory object perception. Memory and multisensory perceptual impairments were observed, with interesting dissociations between transgenic AD strains and sex that paralleled neuropathological changes. Overreliance on the widespread "object recognition" task threatens to slow discovery of potentially significant and clinically relevant behavioural effects related to this multifaceted cognitive function. The current results support the use of carefully designed object-based test batteries to clarify the relationship between "object recognition" impairments and specific aspects of AD pathology in rodent models.

Neurosteroid metabolites of testosterone and progesterone differentially inhibit ERK phosphorylation induced by amyloid ß in SH-SY5Y cells and primary cortical neurons.

Gonadal steroid hormones exert neurotrophic and neuroprotective effects on the brain. Recent work suggests potential neuroprotective roles for the 3α-hydroxy, 5α-reduced metabolites of these hormones. Two such metabolites are 5α-androstane-3α,17ß-diol (3α-diol) and 5α-pregnan-3α-ol-20-one (allopregnanolone; Allo), which may contribute to the overall protection conferred by their precursors (testosterone and progesterone, respectively) through mechanisms including potentiation of gamma-aminobutyric acid (GABA)A receptor (GABAAR) activity. We have previously demonstrated that physiological concentrations of 3α-diol inhibit prolonged phosphorylation of extracellular signal-regulated kinase (ERK) and the associated neurotoxicity resulting from amyloid ß peptide 1-42 (Aß42) exposure in vitro. In the present study, we sought to characterize the GABAAR-dependency of 3α-diol's effects, compared to those of Allo, in SH-SY5Y human female neuroblastoma cells and primary cortical neurons isolated from postnatal day 0-1 mice. Both 3α-diol and Allo prevented Aß42-mediated ERK phosphorylation in SH-SY5Y cells, with substantially different concentration requirements (10 nM for 3α-diol, 100 nM for Allo). Pharmacological inhibition of GABAAR with picrotoxin did not prevent this effect, indicating that neurosteroid-mediated ERK inhibition in SH-SY5Y cells may be GABAAR-independent. While 10 nM and 100 nM concentrations of both neurosteroids inhibited ERK phosphorylation induced by Aß42 in primary cortical neurons, which have high expression levels of GABAARs, only the effects of Allo were significantly inhibited by picrotoxin. These results suggest that neurosteroid metabolites of testosterone and progesterone may contribute to neuroprotection by suppressing ERK phosphorylation through both GABAAR-dependent and -independent mechanisms.

Map kinase and PKC signaling pathways modulate NGF-mediated apoE transcription.

The present study assessed the mechanisms by which nerve growth factor (NGF) increased the level of apolipoprotein E (apoE) in PC12 cells. NGF (50ng/mL) significantly increased apoE protein levels following 72h of treatment. Similarly NGF increased luciferase activity in cells transfected with a luciferase reporter construct containing a 500bp fragment of the apoE promoter, indicating NGF-induced apoE expression is regulated, at least in part, at the level of transcription. The non-selective nitric oxide synthase (NOS) inhibitor N(É·)-nitro-L-arginine methylester (L-NAME; 20mM) did not attenuate the NGF-mediated increase in luciferase activity, while the inducible NOS inhibitor s-methylisothiourea (S-MIU; 2mM) partially attenuated this action of NGF. Inhibition of MAP kinase activation with 50µM U0126 or pre-treatment with the PKC inhibitor bisindolylmaleimide 1 (BIS-1; 10µM) prevented the NGF-mediated activation of the apoE promoter. Pre-treatment with the phospholipase C (PLC) inhibitor U73122 (5µM) partially inhibited the NGF-induced increase in luciferase activity while the Akt inhibitor LY294002 (10µM) had no effect. These data suggest NGF-induced apoE transcription requires MAP kinase and PKC activation and that these TrkA signaling pathways may be modulated by NO.

Differential contributions of de novo and maintenance DNA methyltransferases to object memory processing in the rat hippocampus and perirhinal cortex--a double dissociation.

Epigenetic mechanisms are increasingly acknowledged as major players in memory formation. Specifically, DNA methylation is necessary for the formation of long-term memory in various brain regions, including the hippocampus (HPC); however, its role in the perirhinal cortex (PRh), a structure critical for object memory, has not been characterized. Moreover, the mnemonic effects of selective DNA methyltransferase (DNMT) inhibition have not yet been investigated systematically, despite distinct roles for de novo (DNMT3a, 3b) and maintenance (DNMT1) methyltransferases. Consequently, we assessed the effects of various DNMT inhibitors within the HPC and PRh of rats using the object-in-place paradigm, which requires both brain regions. The non-nucleoside DNA methyltransferase inhibitor RG-108 impaired long-term object-in-place memory in both regions. Furthermore, intracranial administration of Accell short-interference RNA sequences to inhibit the expression of individual DNMTs implicated DNMT3a and DNMT1 in the HPC and PRh effects, respectively. mRNA expression analyses revealed a complementary pattern of results, as only de novo DNMT3a and DNMT3b mRNA was upregulated in the HPC (dentate gyrus) following object-in-place learning, whereas DNMT1 mRNA was selectively upregulated in the PRh. These results reinforce the established functional double dissociation between the HPC and PRh and imply the operation of different epigenetic mechanisms in brain regions dedicated to long-term memory processing for different types of information.

Nerve growth factor-mediated regulation of low density lipoprotein receptor-related protein promoter activation.

The mechanisms by which nerve growth factor (NGF) increases the level of low density lipoprotein receptor-related protein (LRP1) are not known. Administration of nitric oxide synthase (NOS) inhibitors modulates several of the neurotrophic actions of NGF, including TrkA signalling pathway activation, increases in gene expression and neurite outgrowth. The present study investigated whether NGF regulates the transcription of LRP1 as well as the role of NO and the individual TrkA signalling pathways in this action of NGF. PC12 cells were transfected with luciferase reporter constructs containing various sized fragments of the LRP1 promoter and treated with NGF (50 ng/mL) to establish whether NGF altered LRP transcription. NGF significantly increased luciferase activity in all LRP1 promoter construct-transfected cells with the NGF-responsive region of the promoter identified to be present in the first 1000 bp. The non-selective NOS inhibitor N(ω)-nitro-L-arginine methylester (L-NAME; 20 mM) had no effect on the NGF-mediated increase in luciferase activity, while the inducible NOS selective inhibitor s-methylisothiourea (S-MIU; 2 mM) attenuated the NGF-induced activation of the LRP1 promoter. Pretreatment of PC12 cells with 10 µM bisindolylmaleimide 1 (BIS-1) prevented the NGF-mediated increase in LRP1 promoter activation while 50 µM U0126 partially inhibited this response. In combination with S-MIU, all of the TrkA signalling pathway inhibitors blocked the ability of NGF to increase LRP1 transcription. These data suggest the NGF-mediated increase in LRP1 levels occurs, at least in part, at the level of transcription and that NO and the TrkA signalling pathways cooperate in the modulation of LRP1 transcription.

Nitric oxide and histone deacetylases modulate cocaine-induced mu-opioid receptor levels in PC12 cells.

BACKGROUND: Cocaine exposure has been reported to alter central µ-opioid receptor (MOR) expression in vivo. The present study employed an in vitro cellular model to explore possible mechanisms that may be involved in this action of cocaine. METHODS: To assess the effects of cocaine on MOR levels, two treatment regimens were tested in PC12 cells: single continuous or multiple intermittent. MOR protein levels were assessed by western blot analysis and quantitative PCR was used to determine relative MOR mRNA expression levels. To evaluate the role of nitric oxide (NO) and histone acetylation in cocaine-induced MOR expression, cells were pre-treated with the NO synthase inhibitor Nω-nitro-L-arginine methylester (L-NAME) or the non-selective histone acetyltransferase inhibitor curcumin. RESULTS: Both cocaine treatment regimens significantly increased MOR protein levels and protein stability, but only multiple intermittent treatments increased MOR mRNA levels as well as c-fos mRNA levels and activator protein 1 binding activity. Both regimens increased NO production, and pre-treatment with L-NAME prevented cocaine-induced increases in MOR protein and mRNA levels. Single and multiple cocaine treatment regimens inhibited histone deacetylase activity, and pre-treatment with curcumin prevented cocaine-induced up-regulation of MOR protein expression. CONCLUSIONS: In the PC12 cell model, both NO and histone deacetylase activity regulate cocaine-induced MOR expression at both the transcriptional and post-transcriptional levels. Based on these novel findings, it is hypothesized that epigenetic mechanisms are implicated in cocaine's action on MOR expression in neurons.

Effects of nerve growth factor and nitric oxide synthase inhibitors on amyloid precursor protein mRNA levels and protein stability.

We determined previously that nitric oxide (NO) modulates the nerve growth factor (NGF)-mediated increases in amyloid precursor protein (APP) levels in PC12 cells. To elucidate potential mechanisms, the effects of NGF and NO synthase (NOS) inhibitors on APP mRNA levels and protein stability were evaluated. Surprisingly, treatment of PC12 cells with NGF resulted in decreased levels of APP695 and APP751/770 mRNA. Therefore, the effect of NGF on APP protein stability was examined using the translation inhibitor, cycloheximide. Under these conditions, NGF did not alter the rate of APP degradation, suggesting that NGF may be enhancing the translation rate of APP. Since NOS inhibitors attenuate the NGF-mediated increase in APP levels, their effect on APP mRNA levels and protein stability was also assessed. S-methylisothiourea (S-MIU), selective for inducible NOS, decreased both APP695 and APP751/770 mRNA levels while the non-selective NOS inhibitor, N(ω)-nitro-L-arginine methylester (L-NAME) had no effect. In both control and NGF-treated PC12 cells, S-MIU increased the half-life of APP, with the greatest effect observed with the APP695 isoform. Based on these data we propose that in PC12 cells, NGF increases APP levels through enhanced translation rate and that NO, which modulates the NGF-induced increase in APP protein, also regulates APP mRNA levels and could play a role in APP processing.

Nitric oxide synthase inhibitors modulate nerve growth factor-mediated regulation of amyloid precursor protein expression in PC12 cells.

Nerve growth factor (NGF) can regulate nitric oxide synthase (NOS) expression and nitric oxide (NO) can modulate NGF-mediated neurotrophic responses. In this study, the role of NO in NGF-stimulated amyloid precursor protein (APP) levels was studied. PC12 cells were treated with either the non-selective NOS inhibitor N(omega)-nitro-L-arginine methylester (L-NAME) or the inducible NOS selective inhibitor s-methylisothiourea (S-MIU), and the effect on NGF-mediated increases in APP expression was determined. NGF significantly increased total APP protein levels following 96 h of treatment and this increase was prevented in cells pre-treated with S-MIU. Pre-treatment of cells with actinomycin D also blocked this NGF-mediated induction of APP, indicating de novo protein synthesis is necessary. Treatment with NGF increased APP promoter activity; however, this increase was only partially inhibited by pre-treatment with S-MIU and was increased in the presence of L-NAME. This suggests that NO may be modulating other aspects of APP expression in addition to transcription. Inhibition of NGF signaling pathways was also investigated using inhibitors of mitogen-activated protein (MAP) kinase (U0126), Akt (LY294002) and protein kinase C (PKC; U73122 and bisindolylmaleimide 1 (BIS-1)) activation. Inhibition of each of these pathways prevented NGF-mediated increases in APP protein expression; however, only BIS-1 attenuated NGF-mediated increases in promoter activation. This study indicates that NO is involved in the NGF-mediated regulation of APP, in part at the level of APP transcription and could involve the modulation of NGF signal transduction pathways.

Modulation of nerve growth factor-induced activation of MAP kinase in PC12 cells by inhibitors of nitric oxide synthase.

Nerve growth factor (NGF) increases expression of nitric oxide synthase (NOS) isozymes leading to enhanced production of nitric oxide (NO). NOS inhibitors attenuate NGF-mediated increases in cholinergic gene expression and neurite outgrowth. Mechanisms underlying this are unknown, but the mitogen-activated protein (MAP) kinase pathway plays an important role in NGF signaling. Like NGF, NO donors activate Ras leading to phosphorylation of MAP kinase. The present study investigated the role of NO in NGF-mediated activation of MAP kinase in PC12 cells. Cells were treated with 50 ng/mL NGF to establish the temporal pattern for rapid and sustained activation phases of MAP kinase kinase (MEK)-1/2 and p42/p44-MAP kinase. Subsequently, cells were pretreated with NOS inhibitors Nomega-nitro-L-arginine methylester and s-methylisothiourea and exposed to NGF for up to 24 h. NGF-induced activation of MEK-1/2 and p42/p44-MAP kinase was not dependent on NO, but sustained phosphorylation of MAP kinase was modulated by NO. This modulation did not occur at the level of Ras-Raf-MEK signaling or require activation of cGMP/PKG pathway. NOS inhibitors did not affect NGF-mediated phosphorylation of MEK. Expression of constitutively active-MEKK1 in cells led to phosphorylation of p42/p44-MAP kinase and robust neurite outgrowth; constitutively active-MKK1 also caused differentiation with neurite extension. NOS inhibitor treatment of cells expressing constitutively active kinases did not affect MAP kinase activation, but neurite outgrowth was attenuated. NOS inhibitors did not alter NGF-mediated nuclear translocation of phospho-MAP kinase, but phosphorylated kinases disappeared more rapidly from NOS inhibitor-treated cells suggesting greater phosphatase activity and termination of sustained activation of MAP kinase.

Inhibitors of nitric oxide synthase attenuate nerve growth factor-mediated increases in choline acetyltransferase expression in PC12 cells.

NGF can regulate nitric oxide synthase (NOS) expression and nitric oxide (NO) can modulate NGF-mediated neurotrophic responses. To investigate the role of NO in NGF-activated expression of cholinergic phenotype, PC12 cells were treated with either the nonselective NOS inhibitor L-NAME (N (omega)-nitro-L-arginine methylester) or the inducible NOS selective inhibitor MIU (s-methylisothiourea), and the effect on NGF-stimulated ChAT mRNA levels and ChAT specific activity was determined. NGF increased steady-state levels of mRNA and protein for both inducible and constitutive isozymes of NOS in PC12 cells, and led to enhanced NOS activity and NO production. MIU and, to a lesser extent, L-NAME blocked neurite outgrowth in nerve growth factor (NGF)-treated PC12 cells. Both L-NAME and MIU attenuated NGF-mediated increases in choline transferase (ChAT)-specific activity and prevented the increase in expression of ChAT mRNA normally produced by NGF treatment of PC12 cells. The present study indicates that NO may be involved in the modulation of signal transduction pathways by which NGF leads to increased ChAT gene expression in PC12 cells.

PC12nnr5 cells expressing TrkA receptors undergo morphological but not cholinergic phenotypic differentiation in response to nerve growth factor.

We investigated mechanisms underlying nerve growth factor-mediated morphological differentiation and expression of cholinergic neuronal phenotype. In PC12, but not PC12nnr5 cells, nerve growth factor induces neurite-like outgrowths and enhances cholinergic phenotype; stable expression of TrkA receptors in nnr5 cells (called B5P cells) restores morphological differentiation but not expression of choline acetyltransferase. Transfection with an AP-1 luciferase reporter gene revealed that PC12 but not B5P cells expressed nerve growth factor-induced functional AP-1 activity. RT-PCR analysis of nerve growth factor-mediated changes in AP-1 transcription factors showed rapid increases in c-fos and junB mRNA in PC12 and B5P cells, while increases in c-jun were small. Using DNA-protein gel shift assays we determined that nerve growth factor stimulates AP-1 binding in both PC12 and B5P cells, and identified c-Fos, FosB, Fra-1, Fra-2, c-Jun, JunB and JunD in AP-1 complexes. In Fos/Jun functional luciferase reporter assays, nerve growth factor stimulated phosphorylation of c-Fos in both PC12 and B5P cells, but phosphorylation of c-Jun only in PC12, and not in B5P cells. These data indicate that mechanisms relating to AP-1 transcription factor complexes underlying nerve growth factor-mediated enhancement of cholinergic gene expression may differ from those required for morphological differentiation.