Sex and APOE Genotype Alter the Basal and Induced Inflammatory States of Primary Astrocytes from Humanized Targeted Replacement Mice.

Apolipoprotein E4 (APOE4) genotype and sex are significant risk factors for Alzheimer's disease (AD), with females demonstrating increased risk modulated by APOE genotype. APOE is predominantly expressed in astrocytes, however, there is a lack of comprehensive assessments of sex differences in astrocytes stratified by APOE genotype. Here, we examined the response of mixed-sex and sex-specific neonatal APOE3 and APOE4 primary mouse astrocytes (PMA) to a cytokine mix of IL1b, TNFa, and IFNg. Pro-inflammatory and anti-inflammatory cytokine profiles were assessed by qRT-PCR and Meso Scale Discovery multiplex assay. Mixed-sex APOE4 PMA were found to have higher basal messenger RNA expression of several pro-inflammatory cytokines including Il6, Tnfa, Il1b, Mcp1, Mip1a, and Nos2 compared to APOE3 PMA, which was accompanied by increased levels of these secreted cytokines. In sex-specific cultures, basal expression of Il1b, Il6, and Nos2 was 1.5 to 2.5 fold higher in APOE4 female PMA compared to APOE4 males, with both being higher than APOE3 PMA. Similar results were found for secreted levels of these cytokines. Together, these findings indicate that APOE4 genotype and female sex, contribute to a greater inflammatory response in primary astrocytes and these data may provide a framework for investigating the mechanisms contributing to genotype and sex differences in AD-related neuroinflammation.

Sex and APOE Genotype Alter the Basal and Induced Inflammatory States of Primary Microglia from APOE Targeted Replacement Mice.

The sex and APOE4 genotype are significant risk factors for Alzheimer's disease (AD); however, the mechanism(s) responsible for this interaction are still a matter of debate. Here, we assess the responses of mixed-sex and sex-specific APOE3 and APOE4 primary microglia (PMG) to lipopolysaccharide and interferon-gamma. In our investigation, inflammatory cytokine profiles were assessed by qPCR and multiplex ELISA assays. Mixed-sex APOE4 PMG exhibited higher basal mRNA expression and secreted levels of TNFa and IL1b. In sex-specific cultures, basal expression and secreted levels of IL1b, TNFa, IL6, and NOS2 were 2−3 fold higher in APOE4 female PMG compared to APOE4 males, with both higher than APOE3 cells. Following an inflammatory stimulus, the expression of pro-inflammatory cytokines and the secreted cytokine level were upregulated in the order E4 female > E4 male > E3 female > E3 male in sex-specific cultures. These data indicate that the APOE4 genotype and female sex together contribute to a greater inflammatory response in PMG isolated from targeted replacement humanized APOE mice. These data are consistent with clinical data and indicate that sex-specific PMG may provide a platform for exploring mechanisms of genotype and sex differences in AD related to neuroinflammation and neurodegeneration.

Effects of DDT on Amyloid Precursor Protein Levels and Amyloid Beta Pathology: Mechanistic Links to Alzheimer's Disease Risk.

BACKGROUND: The interaction of aging-related, genetic, and environmental factors is thought to contribute to the etiology of late-onset, sporadic Alzheimer's disease (AD). We previously reported that serum levels of p,p'-dichlorodiphenyldichloroethylene (DDE), a long-lasting metabolite of the organochlorine pesticide dichlorodiphenyltrichloroethane (DDT), were significantly elevated in patients with AD and associated with the risk of AD diagnosis. However, the mechanism by which DDT may contribute to AD pathogenesis is unknown. OBJECTIVES: This study sought to assess effects of DDT exposure on the amyloid pathway in multiple in vitro and in vivo models. METHODS: Cultured cells (SH-SY5Y and primary neurons), transgenic flies overexpressing amyloid beta (Aß), and C57BL/6J and 3xTG-AD mice were treated with DDT to assess impacts on the amyloid pathway. Real time quantitative polymerase chain reaction, multiplex assay, western immunoblotting and immunohistochemical methods were used to assess the effects of DDT on amyloid precursor protein (APP) and other contributors to amyloid processing and deposition. RESULTS: Exposure to DDT revealed significantly higher APP mRNA and protein levels in immortalized and primary neurons, as well as in wild-type and AD-models. This was accompanied by higher levels of secreted Aß in SH-SY5Y cells, an effect abolished by the sodium channel antagonist tetrodotoxin. Transgenic flies and 3xTG-AD mice had more Aß pathology following DDT exposure. Furthermore, loss of the synaptic markers synaptophysin and PSD95 were observed in the cortex of the brains of 3xTG-AD mice. DISCUSSION: Sporadic Alzheimer's disease risk involves contributions from genetic and environmental factors. Here, we used multiple model systems, including primary neurons, transgenic flies, and mice to demonstrate the effects of DDT on APP and its pathological product Aß. These data, combined with our previous epidemiological findings, provide a mechanistic framework by which DDT exposure may contribute to increased risk of AD by impacting the amyloid pathway. https://doi.org/10.1289/EHP10576.

Epigenetic Regulation of the Ontogenic Expression of the Dopamine Transporter.

The dopamine transporter (DAT) is a plasma membrane transport protein responsible for regulating the duration and intensity of dopaminergic signaling. Altered expression of DAT is linked to neurodevelopmental disorders, including attention deficit hyperactivity disorder and autism spectrum disorder, and is shown to contribute to the response of psychotropic drugs and neurotoxicants. Although the postnatal levels of DAT have been characterized, there are few data regarding the mechanisms that regulate postnatal DAT expression. Here, we examine the ontogeny of DAT mRNA from postnatal days 0 to 182 in the rat brain and define a role for epigenetic mechanisms regulating DAT expression. DAT mRNA and protein significantly increased between PND 0 and 6 months in rat midbrain and striatum, respectively. The epigenetic modifiers Dnmt1, Dnmt3a, Dnmt3b, and Hdac2 demonstrated age associated decreases in mRNA expression whereas Hdac5 and Hdac8 showed increased mRNA expression with age. Chromatin immunoprecipitation studies revealed increased protein enrichment of acetylated histone 3 at lysines 9 and 14 and the dopaminergic transcription factors Nurr1 and Pitx3 within the DAT promoter in an age-related manner. Together these studies provide evidence for the role of epigenetic modifications in the regulation of DAT during development. The identification of these mechanisms may contribute to potential therapeutic interventions aimed at neurodevelopmental disorders of the dopaminergic system.

Gene-environment interactions in Alzheimer's disease: A potential path to precision medicine.

Alzheimer's disease (AD) is the leading cause of dementia in the United States and afflicts >5.7 million Americans in 2018. Therapeutic options remain extremely limited to those that are symptom targeting, while no drugs have been approved for the modification or reversal of the disease itself. Risk factors for AD including aging, the female sex, as well as carrying an APOE4 genotype. These risk factors have been extensively examined in the literature, while less attention has been paid to modifiable risk factors, including lifestyle, and environmental risk factors such as exposures to air pollution and pesticides. This review highlights the most recent data on risk factors in AD and identifies gene by environment interactions that have been investigated. It also provides a suggested framework for a personalized therapeutic approach to AD, by combining genetic, environmental and lifestyle risk factors. Understanding modifiable risk factors and their interaction with non-modifiable factors (age, susceptibility alleles, and sex) is paramount for designing personalized therapeutic interventions.

Pomegranate ellagitannin-gut microbial-derived metabolites, urolithins, inhibit neuroinflammation in vitro.

OBJECTIVES: Urolithins, ellagitannin-gut microbial-derived metabolites, have been reported to mediate pomegranate's neuroprotective effects against Alzheimer's disease (AD), but there are limited data on their effects against neuroinflammation. Herein, we: (1) evaluated whether urolithins (urolithins A and B and their methylated derivatives) attenuate neuroinflammation in murine BV-2 microglia and human SH-SY5Y neurons, and (2) evaluated hippocampus of transgenic AD (R1.40) mice administered a pomegranate extract (PE; 100 or 200 mg/kg/day for 3 weeks) for inflammatory biomarkers. METHODS: Effects of urolithins (10 µM) on inflammatory biomarkers were evaluated in lipopolysaccharide (LPS)-stimulated BV-2 microglia. In a non-contact co-culture cell model, SH-SY5Y cell viability was assessed after exposure to media collected from LPS-BV-2 cells treated with or without urolithins. Effects of urolithins on apoptosis and caspase 3/7 and 9 release from H2O2-induced oxidative stress of BV-2 and SH-SY5Y cells were assessed. Hippocampal tissues of vehicle and PE-treated transgenic R1.40 mice were evaluated for gene expression of inflammatory biomarkers by qRT-PCR. RESULTS: Urolithins decreased media levels of nitric oxide, interleukin 6 (IL-6), prostaglandin E2, and tumor necrosis factor alpha from LPS-BV-2 microglia. In the co-culture cell model, media from LPS-BV-2 cells treated with urolithins preserved SH-SY5Y cell viability greater than media from cells treated without urolithins. Urolithins mitigated apoptosis and caspase 3/7 and 9 release from H2O2-induced oxidative stress of BV-2 and SH-SY5Y cells. While not statistically significant, inflammatory biomarkers (TNF-α, COX-2, IL-1, and IL-6) appeared to follow a decreasing trend in the hippocampus of high-dose PE-treated animals compared to controls. DISCUSSION: The attenuation of neuroinflammation by urolithins may contribute, in part, toward pomegranate's neuroprotective effects against AD.

Histone acetylation maps in aged mice developmentally exposed to lead: epigenetic drift and Alzheimer-related genes.

AIM: Early life exposure to lead (Pb) has been shown to increase late life biomarkers involved in Alzheimer's disease (AD) pathology. Here, we tested the hypothesis that latent over expression of AD-related genes may be regulated through histone activation pathways. METHODS: Chromatin immunoprecipitation sequencing was used to map the histone activation mark (H3K9Ac) to the mouse genome in developmentally Pb exposed mice on postnatal days 20, 270 and 700. RESULTS: Exposure to Pb resulted in a global downregulation of H3K9Ac across the lifespan; except in genes associated with the Alzheimer pathway. DISCUSSION: Early life exposure to Pb results in an epigenetic drift in H3K9Ac consistent with latent global gene repression. Alzheimer-related genes do not follow this trend.

Tolfenamic Acid: A Modifier of the Tau Protein and its Role in Cognition and Tauopathy.

BACKGROUND: Tangles are deposits of hyperphosphorylated tau, which are found in multiple neurodegenerative disorders that are referred to as tauopathies, of which Alzheimer's disease (AD) is the most common. Tauopathies are clinically characterized by dementia and share common cortical lesions composed of aggregates of the protein tau. OBJECTIVE: In this study, we explored the therapeutic potential of tolfenamic acid (TA), in modifying disease processes in a transgenic animal model that carries the human tau gene (hTau). METHODS: Behavioral tests, Western blotting and Immunohistochemical analysis were used to demonstrate the efficacy of TA. RESULTS: Treatment of TA improved improving spatial learning deficits and memory impairments in young and aged hTau mice. Western blot analysis of the hTau protein revealed reductions in total tau as well as in sitespecific hyperphosphorylation of tau in response to TA administration. Immunohistochemical analysis for phosphorylated tau protein revealed reduced staining in the frontal cortex, hippocampus, and striatum in animals treated with TA. CONCLUSION: TA holds the potential as a disease-modifying agent for the treatment of tauopathies including AD.

Lead exposure and tau hyperphosphorylation: An in vitro study.

The presence of fibrillary lesions, which are mainly composed of the microtubule associated protein tau (MAPT) in neurons, has gained immense recognition due to their presence in numerous neurodegenerative diseases, including Alzheimer's disease (AD). Dysregulation of tau is related with its altered site-specific phosphorylation which is followed by tau polymerization, neuronal dysfunction and death. Previous reports by us suggest that molecular alterations favor abundant tau phosphorylation and immunoreactivity in the frontal cortex of aged primates and rodents with past exposure to lead (Pb). Here we report the involvement of Pb-induced alterations in tau and hyperphosphorylation of tau in differentiated Human Neuroblastoma SH-SY5Y cells exposed to a series of Pb concentrations (5-100µM) for 48h. These cells were analyzed for the protein expression of total tau, site-specific tau hyperphosphorylation, cyclin dependent kinase 5 (CDK5) and p35/p25 at selected time points (24-144h), after Pb exposure had ceased. Western blot analysis revealed aberrant tau levels as well as site-specific tau hyperphosphorylation accompanied by elevated CDK5 levels and altered protein ratio of p35/p25 particularly at 72 and 144h. These changes provide additional evidence that neurodegenerative events are subject to environmental influences.

Valproate increases dopamine transporter expression through histone acetylation and enhanced promoter binding of Nurr1.

The dopamine transporter (DAT) is the key regulator of dopaminergic transmission and is a target of several xenobiotics, including pesticides and pharmacological agents. Previously, we identified a prominent role for histone deacetylases in the regulation of DAT expression. Here, we utilized a rat dopaminergic cell line (N27) to probe the responsiveness of DAT mRNA expression to inhibitors of histone acetylation. Inhibition of histone deacetylases (HDACs) by valproate, butyrate and Trichostatin A led to a 3-10-fold increase in DAT mRNA expression, a 50% increase in protein levels, which were accompanied by increased H3 acetylation levels. To confirm the mechanism of valproate-mediated increase in DAT mRNA, chromatin immunoprecipitation (ChIP) assays were used and demonstrated a significant increase in enrichment of acetylation of histone 3 on lysines 9 and 14 (H3K9/K14ac) in the DAT promoter. Expression of Nurr1 and Pitx3, key regulators of DAT expression, were increased following valproate treatment and Nurr1 binding was enriched in the DAT promoter. Together, these results indicate that histone acetylation and subsequent enhancement of transcription factor binding are plausible mechanisms for DAT regulation by valproate and, perhaps, by other xenobiotics.

Developmental lead exposure and lifespan alterations in epigenetic regulators and their correspondence to biomarkers of Alzheimer's disease.

INTRODUCTION: Early life lead (Pb) exposure results in a latent increase in Alzheimer's disease (AD)-related proteins, and cognitive deficits late in life in both rodents and primates. This study was conducted to investigate if these late life changes were accompanied by epigenetic alterations. METHODS: Western blot analysis and RT-PCR were used to measure Deoxyribonucleic acid methylation regulators (DNMT1, DNMT3a, MeCP2, MAT2A) and histone proteins (H3K9Ac, H3K4me2, H3K27me3). RESULTS: Cerebral levels of DNMT1 and MeCP2 were significantly reduced in mice exposed to Pb early in life, whereas the expression of DNMT3a was not altered. Levels of MAT2a were increased in the Pb-exposed mice across the lifespan. H3K9Ac and H3K4me2, involved in gene activation, were decreased, whereas the repressive mark H3K27me3 was elevated. DISCUSSION: Epigenetic modifiers are affected by the developmental exposure to Pb and may play a role in mediating the latent increases in AD-related proteins in the brain.

Consequences of lead exposure, and it's emerging role as an epigenetic modifier in the aging brain.

Lead exposure has primarily been a concern during development in young children and little attention has been paid to exposure outcomes as these children age, or even to exposures in adulthood. Childhood exposures have long term consequences, and adults who have been exposed to lead as children show a host of cognitive deficits. Lead has also been shown to induce latent changes in the aging brain, and has been implicated in the pathogenesis of neurodegenerative diseases, particularly Alzheimer's Disease, and Parkinson's. Recent research has shown that lead has the ability to alter DNA methylation, histone modifications, and miRNA expression in experimental models, and in humans. These findings implicate epigenetics in lead induced toxicity, and long term changes in individuals. Epigenetic modification could potentially provide us a mechanism by which the environment, and toxic exposures contribute to the increased susceptibility of adult neurodegenerative disease.

Pomegranate extract modulates processing of amyloid-ß precursor protein in an aged Alzheimer's disease animal model.

Accumulating research supports the neuroprotective effects of pomegranate (Punica granatum) juice and extracts against Alzheimer's disease (AD) but there is limited data available in animal models. Here we investigated the effects of a standardized pomegranate extract (PE) on AD pathology in an aged transgenic AD animal model (R1.40).The mice (age 24-30 months) received either PE (at 100 and 200 mg/kg) or a control solution daily for three weeks, and were evaluated in the Morris water maze and the Y-maze for improvements in spatial long-term and working memory functions. Cortical amyloid-ß precursor protein (APP) and amyloid-ß (Aß) levels, along with other relevant biomarkers for AD, were measured in brain tissues. PE did not improve cognitive performance of the mice, but altered levels and ratio of the Aß42 and Aß40 peptides which would favor a diminution in AD pathogenesis. Further analysis revealed that this reversal could be the product of the modification of γ-secretase enzyme activity, the enzyme involved in the generation of these Aß isoforms. Our findings support a specific anti-amyloidogenic mechanism of a pomegranate extract in this aged AD animal model.