Fetal oxidative stress mechanisms of neurodevelopmental deficits and exacerbation by ethanol and methamphetamine

In utero exposure of mouse progeny to alcohol (ethanol, EtOH) and methamphetamine (METH) causes substantial postnatal neurodevelopmental deficits. One emerging pathogenic mechanism underlying these deficits involves fetal brain production of reactive oxygen species (ROS) that alter signal transduction, and/or oxidatively damage cellular macromolecules like lipids, proteins, and DNA, the latter leading to altered gene expression, likely via non‐mutagenic mechanisms. Even physiological levels of fetal ROS production can be pathogenic in biochemically predisposed progeny, and ROS formation can be enhanced by drugs like EtOH and METH, via activation/induction of ROS‐producing NADPH oxidases (NOX), drug bioactivation to free radical intermediates by prostaglandin H synthases (PHS), and other mechanisms. Antioxidative enzymes, like catalase in the fetal brain, while low, provide critical protection. Oxidatively damaged DNA is normally rapidly repaired, and fetal deficiencies in several DNA repair proteins, including oxoguanine glycosylase 1 (OGG1) and breast cancer protein 1 (BRCA1), enhance the risk of drug‐initiated postnatal neurodevelopmental deficits, and in some cases deficits in untreated progeny, the latter of which may be relevant to conditions like autism spectrum disorders (ASD). Risk is further regulated by fetal nuclear factor erythroid 2‐related factor 2 (Nrf2), a ROS‐sensing protein that upregulates an array of proteins, including antioxidative enzymes and DNA repair proteins. Imbalances between conceptal pathways for ROS formation, versus those for ROS detoxification and DNA repair, are important determinants of risk. Birth Defects Research (Part C) 108:108–130, 2016. © 2016 Wiley Periodicals, Inc.     

An embryoprotective role for glucose-6-phosphate dehydrogenase in developmental oxidative stress and chemical teratogenesis.

The primary recognized health risk from common deficiencies in glucose-6-phosphate dehydrogenase (G6PD), a cytoprotective enzyme for oxidative stress, is red blood cell hemolysis. Here we show that litters from untreated pregnant mutant mice with a hereditary G6PD deficiency had increased prenatal (fetal resorptions) and postnatal death. When treated with the anticonvulsant drug phenytoin, a human teratogen that is commonly used in pregnant women and causes embryonic oxidative stress, G6PD-deficient dams had higher embryonic DNA oxidation and more fetal death and birth defects. The reported G6PD gene mutation was confirmed and used to genotype fetal resorptions, which were primarily G6PD deficient. This is the first evidence that G6PD is a developmentally critical cytoprotective enzyme for both endogenous and xenobiotic-initiated embryopathic oxidative stress and DNA damage. G6PD deficiencies accordingly may have a broader biological relevance as important determinants of infertility, in utero and postnatal death, and teratogenesis.     

Melatonin attenuates methamphetamine-induced reduction of tyrosine hydroxylase, synaptophysin and growth-associated protein-43 levels in the neonatal rat brain

Methamphetamine (METH) is a most commonly abused drug which damages nerve terminals by causing formation of reactive oxygen species (ROS), apoptosis, and finally neuronal damage. Fetal exposure to neurotoxic METH causes significant behavioral effects. The developing fetus is substantially deficient in most antioxidative enzymes, and may therefore be at high risk from both endogenous and drug-enhanced oxidative stress. Little is known about the effects of METH on vesicular proteins such as synaptophysin and growth-associated protein 43 (GAP-43) in the immature brain. The present study attempted to investigate the effects of METH-induced neurotoxicity in the dopaminergic system of the neonatal rat brain. Neonatal rats were subcutaneously exposed to 5–10 mg/kg METH daily from postnatal day 4–10 for 7 consecutive days. The results showed that tyrosine hydroxylase enzyme levels were significantly decreased in the dorsal striatum, prefrontal cortex, nucleus accumbens and substantia nigra, synaptophysin levels decreased in the striatum and prefrontal cortex and growth-associated protein-43 (GAP-43) levels significantly decreased in the nucleus accumbens of neonatal rats. Pretreatment with 2 mg/kg melatonin 30 min prior to METH administration prevented METH-induced reduction in tyrosine hydroxylase, synaptophysin and growth-associated protein-43 protein levels in different brain regions. These results suggest that melatonin provides a protective effect against METH-induced nerve terminal degeneration in the immature rat brain probably via its antioxidant properties.     

The ATM cofactor ATMIN protects against oxidative stress and accumulation of DNA damage in the aging brain

Progressive accumulation of DNA damage is causally involved in cellular senescence and organismal aging. The DNA damage kinase ATM plays a central role in maintaining genomic stability. ATM mutations cause the genetic disorder ataxia telangiectasia, which is primarily characterized by progressive neurodegeneration and cancer susceptibility. Although the importance of ATM function to protect against oxidative DNA damage and during aging is well described, the mechanism of ATM activation by these stimuli is not known. Here we identify ATM interactor (ATMIN) as an essential component of the ATM signaling pathway in response to oxidative stress and aging. Embryos lacking ATMIN (atmin(Δ/Δ)) died in utero and showed increased numbers of cells positive for phosphorylated histone H2aX, indicative of increased DNA damage. atmin(Δ/Δ) mouse embryonic fibroblasts accumulated DNA damage and prematurely entered senescence when cultured at atmospheric oxygen levels (20%), but this defect was rescued by addition of an antioxidant and also by culturing cells at physiological oxygen levels (3%). In response to acute oxidative stress, atmin(Δ/Δ) mouse embryonic fibroblasts showed slightly lower levels of ATM phosphorylation and reduced ATM substrate phosphorylation. Conditional deletion of ATMIN in the murine nervous system (atmin(ΔN)) resulted in reduced numbers of dopaminergic neurons, as does ATM deficiency. ATM activity was observed in old, but not in young, control mice, but aging-induced ATM signaling was impaired by ATMIN deficiency. Consequently, old atmin(ΔN) mice showed accumulation of DNA damage in the cortex accompanied by gliosis, resulting in increased mortality of aging mutant mice. These results suggest that ATMIN mediates ATM activation by oxidative stress, and thereby ATMIN protects the aging brain by preventing accumulation of DNA damage.     

The free radical spin trapping agent phenylbutylnitrone reduces fetal brain DNA oxidation and postnatal cognitive deficits caused by in utero exposure to a non-structurally teratogenic dose of ethanol: A role for oxidative stress

Reactive oxygen species (ROS), although implicated in morphological birth defects caused by ethanol (EtOH) during pregnancy, have not been directly linked to its behavioral deficits. To determine this, a pathogenic oxidative DNA lesion was measured in fetal brain, and a passive avoidance learning test was assessed postnatally in the progeny of CD-1 mice treated once on gestational day 17 with 4 g/kg EtOH or its saline vehicle, with or without pretreatment with the free radical spin trapping agent α-phenyl-N-tert-butylnitrone (PBN; 40 mg/kg). EtOH-exposed CD-1 progeny, unlike C57BL/6 progeny, had no morphological birth defects, but exhibited a learning deficit at 12 weeks of age (p<0.001), which continued to 16 weeks in males (p<0.01). Peak blood EtOH concentrations were 2.5-fold higher in C57BL/6 mice compared to CD-1 mice given the same dose. PBN pretreatment of CD-1 dams blocked both EtOH-initiated DNA oxidation in fetal brain (p<0.05) and postnatal learning deficits (p<0.01), providing the first direct evidence for ROS in the mechanism of EtOH-initiated neurodevelopmental deficits.     

Immunity of Fetal Mice to Prenatal Administration of the Dopaminergic Neurotoxin 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine

Abstract: Subcutaneous injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) HC1 (25 mg/kg) in pregnant female mice at the 17th day of gestation markedly depleted striatal dopamine (DA) concentrations in the mothers 24 h later and at 24 h and 28 days after delivery. By contrast, in the offspring of the female mice exposed to MPTP during pregnancy, fetal brain DA concentrations at 24 h after injection and at 24 h after birth and striatal DA levels at 14 and 28 days postnatally were unaffected and identical to those in age-matched controls. The postnatal ontogenesis of striatal DA levels was identical in offspring of control vehicle- and MPTP-treated pregnant mice. Also, prenatal challenge with MPTP did not make nigrostriatal DA neurons more vulnerable to a second postnatal treatment with the toxin. Striatal DA depletions were identical in 6-week-old mice given MPTP, whether they were exposed to MPTP or to vehicle in utero. Monoamine oxidase (EC 1.4.3.4; MAO) type B activity was extremely low in the fetal brain and, relatively, much lower than that of MAO-A. Prenatal MPTP administration reduced maternal striatal and also embryonal brain MAO-B activity at 24 h post treatment but did not alter the normal postnatal development of striatal MAO-A and -B activities in the offspring. Study suggests that resistance of fetal DA neurons to the DA-depleting effect of MPTP may be due, at least in part, to an absence in the embryonal brain of adequately developed MAO-B activity required for the conversion of MPTP to its toxic metabolite, 1-methyl-4-phenylpyridinium ion.     

Parallel increases in lipid and protein oxidative markers in several mouse brain regions after methamphetamine treatment

The neurotoxic actions of methamphetamine (METH) may be mediated in part by reactive oxygen species (ROS). Methamphetamine administration leads to increases in ROS formation and lipid peroxidation in rodent brain; however, the extent to which proteins may be modified or whether affected brain regions exhibit similar elevations of lipid and protein oxidative markers have not been investigated. In this study we measured concentrations of TBARs, protein carbonyls and monoamines in various mouse brain regions at 4 h and 24 h after the last of four injections of METH (10 mg/kg/injection q 2 h). Substantial increases in TBARs and protein carbonyls were observed in the striatum and hippocampus but not the frontal cortex nor the cerebellum of METH-treated mice. Furthermore, lipid and protein oxidative markers were highly correlated within each brain region. In the hippocampus and striatum elevations in oxidative markers were significantly greater at 24 h than at 4 h. Monoamine levels were maximally reduced within 4 h (striatal dopamine [DA] by 95% and serotonin [5-HT] in striatum, cortex and hippocampus by 60-90%). These decrements persisted for 7 days after METH, indicating effects reflective of nerve terminal damage. Interestingly, NE was only transiently depleted in the brain regions investigated (hippocampus and cortex), suggesting a pharmacological and non-toxic action of METH on the noradrenergic nerve terminals. This study provides the first evidence for concurrent formation of lipid and protein markers of oxidative stress in several brain regions of mice that are severely affected by large neurotoxic doses of METH. Moreover, the differential time course for monoamine depletion and the elevations in oxidative markers indicate that the source of oxidative stress is not derived directly from DA or 5HT oxidation.     

Chronic exposure to corticosterone enhances the neuroinflammatory and neurotoxic responses to methamphetamine

J. Neurochem. (2012) 122, 995-1009. ABSTRACT: Up-regulation of proinflammatory cytokines and chemokines in brain ("neuroinflammation") accompanies neurological disease and neurotoxicity. Previously, we documented a striatal neuroinflammatory response to acute administration of a neurotoxic dose of methamphetamine (METH), i.e. one associated with evidence of dopaminergic terminal damage and activation of microglia and astroglia. When we used minocycline to suppress METH-induced neuroinflammation, indices of dopaminergic neurotoxicity were not affected, but suppression of neuroinflammation was incomplete. Here, we administered the classic anti-inflammatory glucocorticoid, corticosterone (CORT), in an attempt to completely suppress METH-related neuroinflammation. METH alone caused large increases in striatal proinflammatory cytokine/chemokine mRNA and subsequent astrocytic hypertrophy, microglial activation, and dopaminergic nerve terminal damage. Pre-treatment of mice with acute CORT failed to prevent neuroinflammatory responses to METH. Surprisingly, when mice were pre-treated with chronic CORT in the drinking water, an enhanced striatal neuroinflammatory response to METH was observed, an effect that was accompanied by enhanced METH-induced astrogliosis and dopaminergic neurotoxicity. Chronic CORT pre-treatment also sensitized frontal cortex and hippocampus to mount a neuroinflammatory response to METH. Because the levels of chronic CORT used are associated with high physiological stress, our data suggest that chronic CORT therapy or sustained physiological stress may sensitize the neuroinflammatory and neurotoxicity responses to METH.     

Methamphetamine oxidatively damages parkin and decreases the activity of 26S proteasome in vivo

Methamphetamine (METH) is toxic to dopaminergic (DAergic) terminals in animals and humans. An early event in METH neurotoxicity is an oxidative stress followed by damage to proteins and lipids. The removal of damaged proteins is accomplished by the ubiquitin-proteasome system (UPS) and the impairment of this system can cause neurodegeneration. Whether dysfunction of the UPS contributes to METH toxicity to DAergic terminals has not been determined. The present investigation examined the effects of METH on functions of parkin and proteasome in rat striatal synaptosomes. METH rapidly modified parkin via conjugation with 4-hydroxy-2-nonenal (4-HNE) to decrease parkin levels and decreased the activity of the 26S proteasome while simultaneously increasing chymotrypsin-like activity and 20S proteasome levels. Prior injections of vitamin E diminished METH-induced changes to parkin and the 26S proteasome as well as long-term decreases in DA and its metabolites' concentrations in striatal tissue. These results suggest that METH causes lipid peroxidation-mediated damage to parkin and the 26S proteasome. As the changes in parkin and 26S occur before the sustained deficits in DAergic markers, an early loss of UPS function may be important in mediating the long-term degeneration of striatal DAergic terminals via toxic accumulation of parkin substrates and damaged proteins.     

Decreased level of Nurr1 in heterozygous young adult mice leads to exacerbated acute and long-term toxicity after repeated methamphetamine exposure

The abuse of psychostimulants, such as methamphetamine (METH), is prevalent in young adults and could lead to long-term adaptations in the midbrain dopamine system in abstinent human METH abusers. Nurr1 is a gene that is critical for the survival and maintenance of dopaminergic neurons and has been implicated in dopaminergic neuron related disorders. In this study, we examined the synergistic effects of repeated early exposure to methamphetamine in adolescence and reduction in Nurr1 gene levels. METH binge exposure in adolescence led to greater damage in the nigrostrial dopaminergic system when mice were exposed to METH binge later in life, suggesting a long-term adverse effect on the dopaminergic system. Compared to naïve mice that received METH binge treatment for the first time, mice pretreated with METH in adolescence showed a greater loss of tyrosine hydroxylase (TH) immunoreactivity in striatum, loss of THir fibers in the substantia nigra reticulata (SNr) as well as decreased dopamine transporter (DAT) level and compromised DA clearance in striatum. These effects were further exacerbated in Nurr1 heterozygous mice. Our data suggest that a prolonged adverse effect exists following adolescent METH binge exposure which may lead to greater damage to the dopaminergic system when exposed to repeated METH later in life. Furthermore, our data support that Nurr1 mutations or deficiency could be a potential genetic predisposition which may lead to higher vulnerability in some individuals.     

Primary cilia in the postnatal brain: Subcellular compartments for organizing neuromodulatory signaling

Primary cilia have well characterized roles in early brain development, relaying signals critical for neurogenesis and brain formation during embryonic stages. Less understood are the contributions of cilia-mediated signaling to postnatal brain function. Several cilia-localized receptors that bind neuropeptides and neurotransmitters endogenous to the brain have been identified in adult neurons, but the functional significance of signaling through these cilia-localized receptors is largely unexplored. Ciliopathic disorders in humans often manifest with neurodevelopmental abnormalities and cognitive deficits. Intriguingly, recent research has also linked several neuropsychiatric disorders and neurodegenerative diseases to ciliary dysfunction. This review summarizes recent evidence suggesting that cilia signaling may dynamically regulate postnatal neuronal physiology and connectivity, and highlights possible links among cilia, neuronal circuitry, neuron survival, and neurological disorders.     

TOM40 Mediates Mitochondrial Dysfunction Induced by α-Synuclein Accumulation in Parkinson’s Disease

Alpha-synuclein (α-Syn) accumulation/aggregation and mitochondrial dysfunction play prominent roles in the pathology of Parkinson’s disease. We have previously shown that postmortem human dopaminergic neurons from PD brains accumulate high levels of mitochondrial DNA (mtDNA) deletions. We now addressed the question, whether alterations in a component of the mitochondrial import machinery -TOM40- might contribute to the mitochondrial dysfunction and damage in PD. For this purpose, we studied levels of TOM40, mtDNA deletions, oxidative damage, energy production, and complexes of the respiratory chain in brain homogenates as well as in single neurons, using laser-capture-microdissection in transgenic mice overexpressing human wildtype α-Syn. Additionally, we used lentivirus-mediated stereotactic delivery of a component of this import machinery into mouse brain as a novel therapeutic strategy. We report here that TOM40 is significantly reduced in the brain of PD patients and in α-Syn transgenic mice. TOM40 deficits were associated with increased mtDNA deletions and oxidative DNA damage, and with decreased energy production and altered levels of complex I proteins in α-Syn transgenic mice. Lentiviral-mediated overexpression of Tom40 in α-Syn-transgenic mice brains ameliorated energy deficits as well as oxidative burden. Our results suggest that alterations in the mitochondrial protein transport machinery might contribute to mitochondrial impairment in α-Synucleinopathies.     

Developmental cell death in the liver and newborn lethality of Ku86 deficient mice suppressed by antioxidant N-acetyl-cysteine

Repair of DNA double-strand breaks (DSBs) is essential for genome integrity and cell survival. Ku86 is involved in the repair of DNA DSBs by non-homologous end joining (NHEJ). Mice deficient in Ku86 show growth retardation, dwarfism, premature aging, and immunodeficiency. In this study, we observed severely compromised survival of Ku86(-/-) mice, such that most Ku86(-/-) mice died within the first postnatal weeks and only 1.5% of the expected 25% from heterozygous crosses survived for 1 month. Since post-mortem analysis was not possible due to parental cannibalism, histopathological examination was performed on Ku86(-/-) fetuses to assess possible causes of newborn death. Eighty percent and 75% of Ku86(-/-) fetuses exhibited apoptosis and necrosis in the liver, while only 20% and 10% of Ku86(+/+) littermates had apoptosis and necrosis, respectively. In addition, the severity of liver damage was significantly higher in Ku86(-/-) fetuses. Developmental liver damage may have led to postnatal lethality because the fetal liver with pre-existing injury may not be able to undergo transformation from a lymphohematopoietic to an indispensable metabolic organ. Free radicals can cause chromosomal breaks and lead to cell death. We postulated that endogenous oxidative stress might be involved in the resulting liver damage and animal lethality in Ku86(-/-) mice deficient in DNA DSB repair. This hypothesis was tested by treating Ku86(-/-) mice with the well known free radical scavenger, thiol antioxidant N-acetyl-cysteine (NAC), during embryonic development. We found that a significantly higher percentage, 7.7% of NAC treated Ku86(-/-) offspring versus 1.5% untreated Ku86(-/-) mice were alive at 1 month of age. In addition, the incidence of liver necrosis decreased by 21% and the severity of necrosis significantly reduced. Thus, Ku86 deficiency results in severe developmental liver damage and newborn lethality associated with oxidative stress.     

Effects of advanced paternal age on trajectories of social behavior in offspring

Our study is the first investigation of the effects of advanced paternal age (APA) on the developmental trajectory of social behavior in rodent offspring. Given the strong epidemiological association between APA and sexually dimorphic neurodevelopmental disorders that are characterized by abnormalities in social behavior (autism, schizophrenia), we assessed sociability in male and female inbred mice (C57BL/6J) across postnatal development (N = 104) in relation to paternal age. We found differences in early social behavior in both male and female offspring of older breeders, with differences in this social domain persisting into adulthood in males only. We showed that these social deficits were not present in the fathers of these offspring, confirming a de novo origin of an altered social trajectory in the offspring generation. Our results, highly novel in rodent research, support the epidemiological observations in humans and provide evidence for a causal link between APA, age‐related changes in the paternal sperm DNA and neurodevelopmental disorders in their offspring.     

Exposure to bisphenol A during embryonic/fetal life and infancy increases oxidative injury and causes underdevelopment of the brain and testis in mice

We investigated the modifications in endogenous antioxidant capacity and oxidative damage in the brain, liver, kidney and testis in mice exposed to bisphenol A (BPA), an environmental endocrine disrupter. Mice were exposed to BPA throughout embryonic/fetal life and during lactation by feeding their pregnant/lactating mothers BPA at 5 or 10 microg per milliliter of drinking water. At the age of four weeks, male mice were sacrificed. Exposure to BPA increased the activity of catalase and glutathione peroxidase in the liver and kidney, respectively. It also increased thiobarbituric acid-reactive substances in the brain, kidney and testis, and decreased the wet weight of the brain, kidney and testis. Our results suggest that exposure to BPA throughout embryonic/fetal life and during infancy induces tissue oxidative stress and peroxidation, ultimately leading to underdevelopment of the brain, kidney and testis.     

The effects of prenatal and postnatal environmental interaction: Prenatal environmental adaptation hypothesis

Adverse antenatal maternal environments during pregnancy influence fetal development that consequently increases risks of mental health problems including psychiatric disorders in offspring. Therefore, behavioral and brain alterations caused by adverse prenatal environmental conditions are generally considered as deficits. In this article, we propose a novel hypothesis, along with summarizing a body of literatures supporting it, that fetal neurodevelopmental alterations, particularly synaptic network changes occurring in the prefrontal cortex, associated with adverse prenatal environmental conditions may be adaptation to cope with expected severe postnatal environments, and therefore, psychiatric disorders may be able to be understood as adaptive strategies against severe environmental conditions through evolution. It is hoped that the hypothesis presented in this article stimulates and opens a new venue on research toward understanding of biological mechanisms and therapeutic treatments of psychiatric disorders.     

Diesel exhaust particles cause increased levels of DNA deletions after transplacental exposure in mice

Diesel exhaust particles (DEP) are a major source of air-borne pollution and are linked to increased risk of disease including lung cancer. Here we investigated effects of exposure to DEP on the frequency of DNA deletions, levels of oxidative DNA damage and DNA adduct formation during embryonic development in mice. Pregnant dams were orally exposed to various doses of DEP (500, 250, 125, 62.5, 31.25 mg/kg/day) at embryonic days 10.5–15.5. We determined the frequency of 70 kb DNA deletions spanning exons 6–18 at the p^un allele that results in black-pigmented spots in the unpigmented retinal pigment epithelium in the eyes of p^un/p^un offspring mice. DEP caused a significant increase in the frequency of DNA deletions. Levels of 8-OH deoxyguanosine indicating oxidative DNA damage were within the limits of the unexposed mouse embryos. ³³P post-labeling analysis revealed very low levels of DNA adducts in the embryo tissue. Thus, transplacental exposure to DEP resulted in a significant increase in the frequency of DNA deletions in the mouse fetus and such genetic alterations in the offspring may have pathological consequences later in life.     

Intrauterine infection/inflammation during pregnancy and offspring brain damages: Possible mechanisms involved

Intrauterine infection is considered as one of the major maternal insults during pregnancy. Intrauterine infection during pregnancy could lead to brain damage of the developmental fetus and offspring. Effects on the fetal, newborn, and adult central nervous system (CNS) may include signs of neurological problems, developmental abnormalities and delays, and intellectual deficits. However, the mechanisms or pathophysiology that leads to permanent brain damage during development are complex and not fully understood. This damage may affect morphogenic and behavioral phenotypes of the developed offspring, and that mice brain damage could be mediated through a final common pathway, which includes over-stimulation of excitatory amino acid receptor, over-production of vascularization/angiogenesis, pro-inflammatory cytokines, neurotrophic factors and apoptotic-inducing factors.