Cocaine Modulates the Neuronal Endosomal System and Extracellular Vesicles in a Sex-Dependent Manner
In multiple neurodevelopmental and neurodegenerative disorders, endosomal changes correlate with changes in exosomes. We examined this linkage in the brain of mice that received cocaine injections for two weeks starting at 2.5Â months of age. Cocaine caused a decrease in the number of both neuronal early and late endosomes and exosomes in the brains of male but not female mice. The response to cocaine in ovariectomized females mirrored male, demonstrating that these sex-differences in response to cocaine are driven by hormonal differences. Moreover, cocaine increased the amount of Î±-synuclein per exosome in the brain of females but did not affect exosomal Î±-synuclein content in the brain of males, a sex-difference eliminated by ovariectomy. Enhanced packaging of Î±-synuclein into female brain exosomes with the potential for propagation of pathology throughout the brain suggests a mechanism for the different response of females to chronic cocaine exposure as compared to males.
Cocaine Induces Sex-Associated Changes in Lipid Profiles of Brain Extracellular Vesicles
Cocaine is a highly addictive stimulant with diverse effects on physiology. Recent studies indicate the involvement of extracellular vesicles (EVs) secreted by neural cells in the cocaine addiction process. It is hypothesized that cocaine affects secretion levels of EVs and their cargos, resulting in modulation of synaptic transmission and plasticity related to addiction physiology and pathology. Lipids present in EVs are important for EV formation and for intercellular lipid exchange that may trigger physiological and pathological responses, including neuroplasticity, neurotoxicity, and neuroinflammation. Specific lipids are highly enriched in EVs compared to parent cells, and recent studies suggest the involvement of various lipids in drug-induced synaptic plasticity during the development and maintenance of addiction processes. Therefore, we examined interstitial small EVs isolated from the brain of mice treated with either saline or cocaine, focusing on the effects of cocaine on the lipid composition of EVs. We demonstrate that 12Â days of noncontingent repeated cocaine (10Â mg/kg) injections to mice, which induce locomotor sensitization, cause lipid composition changes in brain EVs of male mice as compared with saline-injected controls. The most prominent change is the elevation of GD1a ganglioside in brain EVs of males. However, cocaine does not affect the EV lipid profiles of the brain in female mice. Understanding the relationship between lipid composition in EVs and vulnerability to cocaine addiction may provide insight into novel targets for therapies for addiction.
Neonatal ethanol causes profound reduction of cholinergic cell number in the basal forebrain of adult animals
In animal models that mimic human third-trimester fetal development, ethanol causes substantial cellular apoptosis in the brain, but for most brain structures the extent of permanent neuron loss that persists into adulthood is unknown. We injected ethanol into C57BL/6J mouse pups at postnatal day 7 (P7) to model human late-gestation ethanol toxicity, and then used stereological methods to investigate adult cell numbers in several subcortical neurotransmitter systems that project extensively in the forebrain to regulate arousal states. Ethanol treatment caused especially large reductions (34-42%) in the cholinergic cells of the basal forebrain, including cholinergic cells in the medial septal/vertical diagonal band (Ch1/Ch2) and in the horizontal diagonal band/substantia innominata/nucleus basalis (Ch3/Ch4) nuclei. Cell loss was also present in non-cholinergic basal forebrain cells, as demonstrated by 34% reduction of parvalbumin immunolabeled GABA cells and 25% reduction of total Nissl-stained neurons in the Ch1/Ch2 region. In contrast, cholinergic cells in the striatum were reduced only 12% by ethanol, and those of the brainstem pedunculopontine/lateral dorsal tegmental nuclei (Ch5/Ch6) were not significantly reduced. Similarly, ethanol did not significantly reduce dopamine cells of the ventral tegmental area/substantia nigra or serotonin cells in the in the dorsal raphe nucleus. Orexin (hypocretin) cells in the hypothalamus showed a modest reduction (14%). Our findings indicate that the basal forebrain is especially vulnerable to alcohol exposure in the late gestational period. Reduction of cholinergic and GABAergic projection neurons from the basal forebrain that regulate forebrain arousal may contribute to the behavioral and cognitive deficits associated with neonatal ethanol exposure.
Mitovesicles are a novel population of extracellular vesicles of mitochondrial origin altered in Down syndrome
Mitochondrial dysfunction is an established hallmark of aging and neurodegenerative disorders such as Down syndrome (DS) and Alzheimer's disease (AD). Using a high-resolution density gradient separation of extracellular vesicles (EVs) isolated from murine and human DS and diploid control brains, we identify and characterize a previously unknown population of double-membraned EVs containing multiple mitochondrial proteins distinct from previously described EV subtypes, including microvesicles and exosomes. We term these newly identified mitochondria-derived EVs "mitovesicles." We demonstrate that brain-derived mitovesicles contain a specific subset of mitochondrial constituents and that their levels and cargo are altered during pathophysiological processes where mitochondrial dysfunction occurs, including in DS. The development of a method for the selective isolation of mitovesicles paves the way for the characterization in vivo of biological processes connecting EV biology and mitochondria dynamics and for innovative therapeutic and diagnostic strategies.
Post-exposure environment modulates long-term developmental ethanol effects on behavior, neuroanatomy, and cortical oscillations
Developmental exposure to ethanol has a wide range of anatomical, cellular, physiological and behavioral impacts that can last throughout life. In humans, this cluster of effects is termed fetal alcohol spectrum disorder and is highly prevalent in western cultures. The ultimate expression of the effects of developmental ethanol exposure however can be influenced by post-exposure experience. Here we examined the effects of developmental binge exposure to ethanol (postnatal day 7) in C57BL/6By mice on a specific cohort of inter-related long-term outcomes including contextual memory, hippocampal parvalbumin-expressing neuron density, frontal cortex oscillations related to sleep-wake cycling including delta oscillation amplitude and sleep spindle density, and home-cage behavioral activity. When assessed in adults that were raised in standard housing, all of these factors were altered by early ethanol exposure compared to saline controls except home-cage activity. However, exposure to an enriched environment and exercise from weaning to postnatal day 90 reversed most of these ethanol-induced impairments including memory, CA1 but not dentate gyrus PV+ cell density, delta oscillations and sleep spindles, and enhanced home-cage behavioral activity in Saline- but not EtOH-treated mice. The results are discussed in terms of the inter-dependence of diverse developmental ethanol outcomes and potential mechanisms of post-exposure experiences to regulate those outcomes.
Involvement of AMP-activated protein kinase in neuroinflammation and neurodegeneration in the adult and developing brain
Microglial activation followed by neuroinflammation is a defense mechanism of the brain to eliminate harmful endogenous and exogenous materials including pathogens and damaged tissues, while excessive or chronic neuroinflammation may cause or exacerbate neurodegeneration observed in brain injuries and neurodegenerative diseases. Depending on conditions/environments during activation, microglia acquire distinct phenotypes, such as pro-inflammatory, anti-inflammatory, and disease-associated phenotypes, and show their ability to phagocytose various objects and produce pro-and anti-inflammatory mediators. Prevention of excessive inflammation by regulating the microglia's pro/anti-inflammatory balance is important for alleviating progression of brain injuries and diseases. Among many factors involved in the regulation of microglial phenotypes, cellular energy status plays an important role. Adenosine monophosphate-activated protein kinase (AMPK), which serves as a master sensor and regulator of energy balance, is considered a candidate molecule. Accumulating evidence from adult rodent studies indicates that AMPK activation promotes anti-inflammatory responses in microglia exposed to danger signals or various stressors mainly through inhibition of the nuclear factor ÎºB (NF-ÎºB) signaling and activation of the nuclear factor erythroid-2-related factor-2 (Nrf2) pathway. However, AMPK activation in neurons exposed to stressors/insults may exacerbate neuronal damage if AMPK activation is excessive or prolonged. While AMPK affects microglial activation states and neuronal cell survival rates in both the adult and the developing brain, studies in the developing brain are still scarce, even though activated AMPK is highly expressed especially in the neonatal brain. More in depth studies in the developing brain are important, because neuroinflammation/neurodegeneration occurred during development can result in long-lasting brain damage.
Effects of neonatal ethanol on cerebral cortex development through adolescence
Neonatal brain lesions cause deficits in structure and function of the cerebral cortex that sometimes are not fully expressed until adolescence. To better understand the onset and persistence of changes caused by postnatal day 7 (P7) ethanol treatment, we examined neocortical cell numbers, volume, surface area and thickness from neonatal to post-adolescent ages. In control mice, total neuron number decreased from P8 to reach approximately stable levels at about P30, as expected from normal programmed cell death. Cortical thickness reached adult levels by P14, but cortical volume and surface area continued to increase from juvenile (P20-30) to post-adolescent (P54-93) ages. P7 ethanol caused a reduction of total neurons by P14, but this deficit was transient, with later ages having only small and non-significant reductions. Previous studies also reported transient neuron loss after neonatal lesions that might be partially explained by an acute acceleration of normally occurring programmed cell death. GABAergic neurons expressing parvalbumin, calretinin, or somatostatin were reduced by P14, but unlike total neurons the reductions persisted or increased in later ages. Cortical volume, surface area and thickness were also reduced by P7 ethanol. Cortical volume showed evidence of a transient reduction at P14, and then was reduced again in post-adolescent ages. The results show a developmental sequence of neonatal ethanol effects. By juvenile ages the cortex overcomes the P14 deficit of total neurons, whereas P14 GABA cell deficits persist. Cortical volume reductions were present at P14, and again in post-adolescent ages.
Neonatal Ethanol Disturbs the Normal Maturation of Parvalbumin Interneurons Surrounded by Subsets of Perineuronal Nets in the Cerebral Cortex: Partial Reversal by Lithium
Reduction in parvalbumin-positive (PV+) interneurons is observed in adult mice exposed to ethanol at postnatal day 7 (P7), a late gestation fetal alcohol spectrum disorder model. To evaluate whether PV+ cells are lost, or PV expression is reduced, we quantified PV+ and associated perineuronal net (PNN)+ cell densities in barrel cortex. While PNN+ cell density was not reduced by P7 ethanol, PV cell density decreased by 25% at P90 with no decrease at P14. PNN+ cells in controls were virtually all PV+, whereas more than 20% lacked PV in ethanol-treated adult animals. P7 ethanol caused immediate apoptosis in 10% of GFP+ cells in G42 mice, which express GFP in a subset of PV+ cells, and GFP+ cell density decreased by 60% at P90 without reduction at P14. The ethanol effect on PV+ cell density was attenuated by lithium treatment at P7 or at P14-28. Thus, reduced PV+ cell density may be caused by disrupted cell maturation, in addition to acute apoptosis. This effect may be regionally specific: in the dentate gyrus, P7 ethanol reduced PV+ cell density by 70% at P14 and both PV+ and PNN+ cell densities by 50% at P90, and delayed lithium did not alleviate ethanol's effect.
Apolipoprotein E4 genotype compromises brain exosome production
In addition to being the greatest genetic risk factor for Alzheimer's disease, expression of the É›4 allele of apolipoprotein E can lead to cognitive decline during ageing that is independent of Alzheimer's amyloid-Î² and tau pathology. In human post-mortem tissue and mouse models humanized for apolipoprotein E, we examined the impact of apolipoprotein E4 expression on brain exosomes, vesicles that are produced within and secreted from late-endocytic multivesicular bodies. Compared to humans or mice homozygous for the risk-neutral É›3 allele we show that the É›4 allele, whether homozygous or heterozygous with an É›3 allele, drives lower exosome levels in the brain extracellular space. In mice, we show that the apolipoprotein E4-driven change in brain exosome levels is age-dependent: while not present at age 6 months, it is detectable at 12 months of age. Expression levels of the exosome pathway regulators tumor susceptibility gene 101 (TSG101) and Ras-related protein Rab35 (RAB35) were found to be reduced in the brain at the protein and mRNA levels, arguing that apolipoprotein E4 genotype leads to a downregulation of exosome biosynthesis and release. Compromised exosome production is likely to have adverse effects, including diminishing a cell's ability to eliminate materials from the endosomal-lysosomal system. This reduction in brain exosome levels in 12-month-old apolipoprotein E4 mice occurs earlier than our previously reported brain endosomal pathway changes, arguing that an apolipoprotein E4-driven failure in exosome production plays a primary role in endosomal and lysosomal deficits that occur in apolipoprotein E4 mouse and human brains. Disruption of these interdependent endosomal-exosomal-lysosomal systems in apolipoprotein E4-expressing individuals may contribute to amyloidogenic amyloid-Î² precursor protein processing, compromise trophic signalling and synaptic function, and interfere with a neuron's ability to degrade material, all of which are events that lead to neuronal vulnerability and higher risk of Alzheimer's disease development. Together, these data suggest that exosome pathway dysfunction is a previously unappreciated component of the brain pathologies that occur as a result of apolipoprotein E4 expression.
Developmental Ethanol-Induced Sleep Fragmentation, Behavioral Hyperactivity, Cognitive Impairment and Parvalbumin Cell Loss are Prevented by Lithium Co-treatment
Developmental ethanol exposure is a well-known cause of lifelong cognitive deficits, behavioral hyperactivity, emotional dysregulation, and more. In healthy adults, sleep is thought to have a critical involvement in each of these processes. Our previous work has demonstrated that some aspects of cognitive impairment in adult mice exposed at postnatal day 7 (P7) to ethanol (EtOH) correlate with slow-wave sleep (SWS) fragmentation (Wilson et al., 2016). We and others have also previously demonstrated that co-treatment with LiCl on the day of EtOH exposure prevents many of the anatomical and physiological impairments observed in adults. Here we explored cognitive function, diurnal rhythms (activity, temperature), SWS, and parvalbumin (PV) and perineuronal net (PNN)-positive cell densities in adult mice that had received a single day of EtOH exposure on P7 and saline-treated littermate controls. Half of the animals also received a LiCl injection on P7. The results suggest that developmental EtOH resulted in adult behavioral hyperactivity, cognitive impairment, and reduced SWS compared to saline controls. Both of these effects were reduced by LiCl treatment on the day of EtOH exposure. Finally, developmental EtOH resulted in decreased PV/PNN-expressing cells in retrosplenial (RS) cortex and dorsal CA3 hippocampus at P90. As with sleep and behavioral activity, LiCl treatment reduced this decrease in PV expression. Together, these results further clarify the long-lasting effects of developmental EtOH on adult behavior, physiology, and anatomy. Furthermore, they demonstrate the neuroprotective effects of LiCl co-treatment on this wide range of developmental EtOH's long-lasting consequences.