Muhammad M. Hossain

Elevated serum pesticide levels and risk for Alzheimer disease.

IMPORTANCE The causes of late-onset Alzheimer disease (AD) are not yet understood but likely include a combination of genetic, environmental, and lifestyle factors. Limited epidemiological studies suggest that occupational pesticide exposures are associated with AD. Previously, we reported that serum levels of dichlorodiphenyldichloroethylene (DDE), the metabolite of the pesticide dichlorodiphenyltrichloroethane (DDT), were elevated in a small number of patients with AD (n=20). OBJECTIVE To evaluate the association between serum levels of DDE and AD and whether the apolipoprotein E (APOE) genotype modifies the association. DESIGN, SETTING, AND PARTICIPANTS A case-control study consisting of existing samples from patients with AD and control participants from the Emory University Alzheimers Disease Research Center and the University of Texas Southwestern Medical Schools Alzheimers Disease Center. Serum levels of DDE were measured in 79 control and 86 AD cases. MAIN OUTCOMES AND MEASURES Serum DDE levels, AD diagnosis, severity of AD measured by the Mini-Mental State Examination score, and interaction with APOE4 status. RESULTS Levels of DDE were 3.8-fold higher in the serum of those with AD (mean [SEM], 2.64 [0.35] ng/mg cholesterol) when compared with control participants (mean [SEM], 0.69 [0.1] ng/mg cholesterol; P < .001). The highest tertile of DDE levels was associated with an odds ratio of 4.18 for increased risk for AD (95% CI, 2.54-5.82; P < .001) and lower Mini-Mental State Examination scores (-1.605; range, -3.095 to -0.114; P < .0001). The Mini-Mental State Examination scores in the highest tertile of DDE were -1.753 points lower in the subpopulation carrying an APOE ε4 allele compared with those carrying an APOE ε3 allele (P interaction = .04). Serum levels of DDE were highly correlated with brain levels of DDE (ρ = 0.95). Exposure of human neuroblastoma cells to DDT or DDE increased levels of amyloid precursor protein. CONCLUSIONS AND RELEVANCE Elevated serum DDE levels are associated with an increased risk for AD and carriers of an APOE4 ε4 allele may be more susceptible to the effects of DDE. Both DDT and DDE increase amyloid precursor protein levels, providing mechanistic plausibility for the association of DDE exposure with AD. Identifying people who have elevated levels of DDE and carry an APOE ε4 allele may lead to early identification of some cases of AD.

Mechanism of Pyrethroid Pesticide–Induced Apoptosis: Role of Calpain and the ER Stress Pathway

Exposure to the pyrethroid pesticide deltamethrin has been demonstrated to cause apoptosis both in vitro and in vivo. However, the molecular pathways leading to deltamethrin-induced apoptosis have not been established. To identify these pathways, SK-N-AS neuroblastoma cells were exposed to deltamethrin (100 nM-5 μM) for 24-48 h. Deltamethrin produced a time- and dose-dependent increase (21-300%) in DNA fragmentation, an indicator of apoptosis. Data demonstrate that the initiation of DNA fragmentation resulted from interaction of deltamethrin with Na⁺ channels and consequent calcium influx, as tetrodotoxin and the intracellular Ca²⁺ chelator BAPTA-AM completely prevented apoptosis. DNA fragmentation was accompanied by increased caspase-9 and -3 activities and was abolished by specific caspase-9 and -3 inhibitors. However, deltamethrin did not increase cytosolic cytochrome c levels, indicating that the mitochondrial pathway was likely not involved. Additional studies demonstrated that deltamethrin exposure activated caspase-12 activity and that pharmacological inhibition and siRNA knockdown of calpain prevented deltamethrin-induced DNA fragmentation, thus indicating a role for the endoplasmic reticulum (ER) stress pathway. This was confirmed by the observation that inhibition of eIF2α abolished deltamethrin-induced DNA fragmentation. Together, these data demonstrate that deltamethrin causes apoptosis through its interaction with Na⁺ channels, leading to calcium overload and activation of the ER stress pathway. Because ER stress and the subsequent unfolded protein response have been observed in a number of neurodegenerative diseases, these data provide mechanistic information by which high-level exposure to pyrethroids may contribute to neurodegeneration.

Fungal-derived semiochemical 1-octen-3-ol disrupts dopamine packaging and causes neurodegeneration

Significance Poor air quality from fungal growth in water-damaged, moldy buildings/residences is correlated with a negative impact on human health. The volatile organic compound 1-octen-3-ol is commonly emitted by molds and is responsible for much of the distinctive moldy odor associated with fungal colonization. Using a Drosophila model, we demonstrate via genetic, biochemical, and immunological studies that 1-octen-3-ol causes dopamine neuron degeneration through disruption of dopamine handling. These data demonstrate that 1-octen-3-ol exerts toxicity via disruption of dopamine homeostasis and may represent a naturally occurring environmental agent involved in parkinsonism. Moreover, it provides possible insights into reported movement disorders associated with human exposure to fungi and their volatile organic compounds. Parkinson disease (PD) is the most common movement disorder and, although the exact causes are unknown, recent epidemiological and experimental studies indicate that several environmental agents may be significant risk factors. To date, these suspected environmental risk factors have been man-made chemicals. In this report, we demonstrate via genetic, biochemical, and immunological studies that the common volatile fungal semiochemical 1-octen-3-ol reduces dopamine levels and causes dopamine neuron degeneration in Drosophila melanogaster. Overexpression of the vesicular monoamine transporter (VMAT) rescued the dopamine toxicity and neurodegeneration, whereas mutations decreasing VMAT and tyrosine hydroxylase exacerbated toxicity. Furthermore, 1-octen-3-ol also inhibited uptake of dopamine in human cell lines expressing the human plasma membrane dopamine transporter (DAT) and human VMAT ortholog, VMAT2. These data demonstrate that 1-octen-3-ol exerts toxicity via disruption of dopamine homeostasis and may represent a naturally occurring environmental agent involved in parkinsonism.

Inflammatory Regulation of ATP Binding Cassette Efflux Transporter Expression and Function in Microglia

ATP-binding cassette (ABC) efflux transporters, including multidrug resistance protein 1 (Mdr1), breast cancer resistance protein (Bcrp), and multidrug resistance-associated proteins (Mrps) extrude chemicals from the brain. Although ABC transporters are critical for blood-brain barrier integrity, less attention has been placed on the regulation of these proteins in brain parenchymal cells such as microglia. Prior studies demonstrate that inflammation after lipopolysaccharide (LPS) treatment alters transporter expression in the livers of mice. Here, we sought to determine the effects of inflammation on the expression and function of transporters in microglia. To test this, the expression and function of ABC efflux transport proteins were quantified in mouse BV-2 microglial cells in response to activation with LPS. Intracellular retention of fluorescent rhodamine 123, Hoechst 33342, and calcein acetoxymethyl ester was increased in LPS-treated microglia, suggesting that the functions of Mdr1, Bcrp, and Mrps were decreased, respectively. LPS reduced Mdr1, Bcrp, and Mrp4 mRNA and protein expression between 40 and 70%. Conversely, LPS increased expression of Mrp1 and Mrp5 mRNA and protein. Immunofluorescent staining confirmed reduced Bcrp and Mrp4 and elevated Mrp1 and Mrp5 protein in activated microglia. Pharmacological inhibition of nuclear factor κB (NF-κB) transcriptional signaling attenuated down-regulation of Mdr1a mRNA and potentiated up-regulation of Mrp5 mRNA in LPS-treated cells. Together, these data suggest that LPS stimulates microglia and impairs efflux of prototypical ABC transporter substrates by altering mRNA and protein expression, in part through NF-κB signaling. Decreased transporter efflux function in microglia may lead to the retention of toxic chemicals and aberrant cell-cell communication during neuroinflammation.

Prenatal cigarette smoke exposure causes hyperactivity and aggressive behavior: role of altered catecholamines and BDNF.

Smoking during pregnancy is associated with a variety of untoward effects on the offspring. However, recent epidemiological studies have brought into question whether the association between neurobehavioral deficits and maternal smoking is causal. We utilized an animal model of maternal smoking to determine the effects of prenatal cigarette smoke (CS) exposure on neurobehavioral development. Pregnant mice were exposed to either filtered air or mainstream CS from gestation day (GD) 4 to parturition for 4h/d and 5d/wk, with each exposure producing maternal plasma concentration of cotinine equivalent to smoking <1 pack of cigarettes per day (25ng/ml plasma cotinine level). Pups were weaned at postnatal day (PND) 21 and behavior was assessed at 4weeks of age and again at 4-6months of age. Male, but not female, offspring of CS-exposed dams demonstrated a significant increase in locomotor activity during adolescence and adulthood that was ameliorated by methylphenidate treatment. Additionally, male offspring exhibited increased aggression, as evidenced by decreased latency to attack and number of attacks in a resident-intruder task. These behavioral abnormalities were accompanied by a significant decrease in striatal and cortical dopamine and serotonin and a significant reduction in brain-derived neurotrophic factor (BDNF) mRNA and protein. Taken in concert, these data demonstrate that prenatal exposure to CS produces behavioral alterations in mice that are similar to those observed in epidemiological studies linking maternal smoking to neurodevelopmental disorders. Further, these data also suggest a role for monaminergic and BDNF alterations in these effects.

Developmental pesticide exposure reproduces features of attention deficit hyperactivity disorder.

Attention‐deficit hyperactivity disorder (ADHD) is estimated to affect 8‐12% of school‐age children worldwide. ADHD is a complex disorder with significant genetic contributions. However, no single gene has been linked to a significant percentage of cases, suggesting that environmental factors may contribute to ADHD. Here, we used behavioral, molecular, and neurochemical techniques to characterize the effects of developmental exposure to the pyrethroid pesticide deltamethrin. We also used epidemiologic methods to determine whether there is an association between pyrethroid exposure and diagnosis of ADHD. Mice exposed to the pyrethroid pesticide deltamethrin during development exhibit several features reminiscent of ADHD, including elevated dopamine transporter (DAT) levels, hyperactivity, working memory and attention deficits, and impulsive‐like behavior. Increased DAT and D1 dopamine receptor levels appear to be responsible for the behavioral deficits. Epidemiologic data reveal that children aged 6‐15 with detectable levels of pyrethroid metabolites in their urine were more than twice as likely to be diagnosed with ADHD. Our epidemiologic finding, combined with the recapitulation of ADHD behavior in pesticide‐treated mice, provides a mechanistic basis to suggest that developmental pyrethroid exposure is a risk factor for ADHD.—Richardson, J. R., Taylor, M. M., Shalat, S. L., Guillot III, T. S., Caudle, W. M., Hossain, M. M., Mathews, T. A., Jones, S. R., Cory‐Slechta, D. A., Miller, G. W. Developmental pesticide exposure reproduces features of attention deficit hyperactivity disorder. FASEB J. 29, 1960‐1972 (2015). www.fasebj.org

Microglial Ion Channels as Potential Targets for Neuroprotection in Parkinson’s Disease

Parkinsons disease (PD) is a chronic, degenerative neurological disorder that is estimated to affect at least 1 million individuals in the USA and over 10 million worldwide. It is thought that the loss of neurons and development of inclusion bodies occur gradually over decades until they progress to the point where ~60% of the dopamine neurons are lost and patients present with motor dysfunction. At present, it is not clear what causes this progression, and there are no current therapies that have been successful in preventing PD progression. Although there are many hypotheses regarding the mechanism of PD progression, neuroinflammation may be a major contributor to PD pathogenesis. Indeed, activated microglia and subsequent neuroinflammation have been consistently associated with the pathogenesis of PD. Thus, interference with this process could provide a means of neuroprotection in PD. This review will discuss the potential of targeting microglia to reduce neuroinflammation in PD. Further, we discuss the potential of microglial ion channels to serve as novel targets for neuroprotection in PD.

Coordinated role of voltage-gated sodium channels and the Na+/H+ exchanger in sustaining microglial activation during inflammation.

Persistent neuroinflammation and microglial activation play an integral role in the pathogenesis of many neurological disorders. We investigated the role of voltage-gated sodium channels (VGSC) and Na(+)/H(+) exchangers (NHE) in the activation of immortalized microglial cells (BV-2) after lipopolysaccharide (LPS) exposure. LPS (10 and 100 ng/ml) caused a dose- and time-dependent accumulation of intracellular sodium [(Na(+))i] in BV-2 cells. Pre-treatment of cells with the VGSC antagonist tetrodotoxin (TTX, 1 μM) abolished short-term Na(+) influx, but was unable to prevent the accumulation of (Na(+))i observed at 6 and 24h after LPS exposure. The NHE inhibitor cariporide (1 μM) significantly reduced accumulation of (Na(+))i 6 and 24h after LPS exposure. Furthermore, LPS increased the mRNA expression and protein level of NHE-1 in a dose- and time-dependent manner, which was significantly reduced after co-treatment with TTX and/or cariporide. LPS increased production of TNF-α, ROS, and H2O2 and expression of gp91(phox), an active subunit of NADPH oxidase, in a dose- and time-dependent manner, which was significantly reduced by TTX or TTX+cariporide. Collectively, these data demonstrate a closely-linked temporal relationship between VGSC and NHE-1 in regulating function in activated microglia, which may provide avenues for therapeutic interventions aimed at reducing neuroinflammation.

Hippocampal ER Stress and Learning Deficits Following Repeated Pyrethroid Exposure

Endoplasmic reticulum (ER) stress is implicated as a significant contributor to neurodegeneration and cognitive dysfunction. Previously, we reported that the widely used pyrethroid pesticide deltamethrin causes ER stress-mediated apoptosis in SK-N-AS neuroblastoma cells. Whether or not this occurs in vivo remains unknown. Here, we demonstrate that repeated deltamethrin exposure (3 mg/kg every 3 days for 60 days) causes hippocampal ER stress and learning deficits in adult mice. Repeated exposure to deltamethrin caused ER stress in the hippocampus as indicated by increased levels of C/EBP-homologous protein (131%) and glucose-regulated protein 78 (96%). This was accompanied by increased levels of caspase-12 (110%) and activated caspase-3 (50%). To determine whether these effects resulted in learning deficits, hippocampal-dependent learning was evaluated using the Morris water maze. Deltamethrin-treated animals exhibited profound deficits in the acquisition of learning. We also found that deltamethrin exposure resulted in decreased BrdU-positive cells (37%) in the dentate gyrus of the hippocampus, suggesting potential impairment of hippocampal neurogenesis. Collectively, these results demonstrate that repeated deltamethrin exposure leads to ER stress, apoptotic cell death in the hippocampus, and deficits in hippocampal precursor proliferation, which is associated with learning deficits.

Acute Effects of Pyrethroids on Serotonin Release in the Striatum of Awake Rats: An In Vivo Microdialysis Study

The present study examined the acute neurotoxic effects of three different pyrethroids, allethrin, cyhalothrin, and deltamethrin on the release of serotonin (5‐HT) and its metabolite 5‐hydroxyindoleacetic acid (5‐HIAA) in the striatum of conscious rats using microdialysis. Allethrin 10 mg/kg reduced extracellular levels of 5‐HT to 46%, whereas 20 and 60 mg/kg increased the release to 177% and 243% of baseline, respectively. Cyhalothrin increased 5‐HT release to 145–204% and deltamethrin decreased to 58–32% of baseline in a dose‐dependent manner. None of the pyrethroids tested altered extracellular levels of 5‐HIAA. Local infusion of the voltage‐gated sodium channel antagonist tetrodotoxin (TTX) into striatum completely prevented the effects of allethrin, cyhalothrin, and deltamethrin (10 and 20 mg/kg) on 5‐HT release. The effect of deltamethrin at 60 mg/kg was completely abolished by striatal infusion of nimodipine (L‐type Ca++ channel antagonist) with TTX. These findings suggest that pyrethroids disrupt the serotonergic neurotransmission in striatum in a dose‐related manner with Na+ and Ca2+ channel‐dependent mechanisms.