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.
Cerebellar Kv3.3 potassium channels activate TANK-binding kinase 1 to regulate trafficking of the cell survival protein Hax-1
Mutations in KCNC3, which encodes the Kv3.3 potassium channel, cause degeneration of the cerebellum, but exactly how the activity of an ion channel is linked to the survival of cerebellar neurons is not understood. Here, we report that Kv3.3 channels bind and stimulate Tank Binding Kinase 1 (TBK1), an enzyme that controls trafficking of membrane proteins into multivesicular bodies, and that this stimulation is greatly increased by a disease-causing Kv3.3 mutation. TBK1 activity is required for the binding of Kv3.3 to its auxiliary subunit Hax-1, which prevents channel inactivation with depolarization. Hax-1 is also an anti-apoptotic protein required for survival of cerebellar neurons. Overactivation of TBK1 by the mutant channel leads to the loss of Hax-1 by its accumulation in multivesicular bodies and lysosomes, and also stimulates exosome release from neurons. This process is coupled to activation of caspases and increased cell death. Our studies indicate that Kv3.3 channels are directly coupled to TBK1-dependent biochemical pathways that determine the trafficking of cellular constituents and neuronal survival.
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.
Cystatin C Plays a Sex-Dependent Detrimental Role in Experimental Autoimmune Encephalomyelitis
The cysteine protease inhibitor Cystatin C (CST3) is highly expressed in the brains of multiple sclerosis (MS) patients and C57BL/6J mice with experimental autoimmune encephalomyelitis (EAE; a model of MS), but its roles in the diseases are unknown. Here, we show that CST3 plays a detrimental function in myelin oligodendrocyte glycoprotein 35-55 (MOG35-55)-induced EAE but only in female animals. Female Cst3 null mice display significantly lower clinical signs of disease compared to wild-type (WT) littermates. This difference is associated with reduced interleukin-6 production and lower expression of key proteins (CD80, CD86, major histocompatibility complex [MHC] II, LC3A/B) involved in antigen processing, presentation, and co-stimulation in antigen-presenting cells (APCs). In contrast, male WT and Cst3-/- mice and cells show no differences in EAE signs or APC function. Further, the sex-dependent effect of CST3 in EAE is sensitive to gonadal hormones. Altogether, we have shown that CST3 has a sex-dependent role in MOG35-55-induced EAE.
Extracellular vesicles: where the amyloid precursor protein carboxyl-terminal fragments accumulate and amyloid-Î² oligomerizes
Pleiotropic roles are proposed for brain extracellular vesicles (EVs) in the development of Alzheimer's disease (AD). Our previous studies have suggested a beneficial role for EVs in AD, where the endosomal system in vulnerable neurons is compromised, contributing to the removal of accumulated material from neurons. However, the involvement of EVs in propagating AD amyloidosis throughout the brain has been considered because the amyloid-Î² precursor protein (APP), APP metabolites, and key APP cleaving enzymes were identified in association with EVs. Here, we undertook to determine whether the secretase machinery is actively processing APP in EVs isolated from the brains of wild-type and APP overexpressing Tg2576 mice. We found that full-length APP is cleaved in EVs incubated in the absence of cells. The resulting metabolites, both Î±- and Î²-APP carboxyl-terminal fragments and APP intracellular domain accumulate in EVs over time and amyloid-Î² dimerizes. Thus, EVs contribute to the removal from neurons and transport of APP-derived neurotoxic peptides. While this is potentially a venue for propagation of the pathology throughout the brain, it may contribute to efficient removal of neurotoxic peptides from the brain.
A pleiotropic role for exosomes loaded with the amyloid Î² precursor protein carboxyl-terminal fragments in the brain of Down syndrome patients
Down syndrome (DS) is characterized by cognitive deficits throughout the life span and with the development of aging-dependent Alzheimer's type neuropathology, which is related to the triplication of the amyloid Î² precursor protein (APP) gene. A dysfunctional endosomal system in neurons is an early characteristic of DS and APP metabolites accumulate in endosomes in DS neurons. We have previously shown enhanced release of exosomes in the brain of DS patients and the mouse model of DS Ts[Rb(12.1716)]2Cje (Ts2), and by DS fibroblasts, as compared with diploid controls. Here, we demonstrate that exosome-enriched extracellular vesicles (hereafter called EVs) isolated from DS and Ts2 brains, and from the culture media of human DS fibroblasts are enriched in APP carboxyl-terminal fragments (APP-CTFs) as compared with diploid controls. Moreover, APP-CTFs levels increase in an age-dependent manner in EVs isolated from the brain of Ts2 mice. The release of APP-CTFs-enriched exosomes may have a pathogenic role by transporting APP-CTFs into naÃ¯ve neurons and propagating these neurotoxic metabolites, which are also a source of amyloid Î², throughout the brain, but also provides a benefit to DS neurons by shedding APP-CTFs accumulated intracellularly.
Neuroprotection mediated by cystatin C-loaded extracellular vesicles
Cystatin C (CysC) is implicated in neuroprotection and repair in the nervous system in response to diverse neurotoxic conditions. In addition to being secreted from cells in a soluble form, CysC is released by cells in association with extracellular vesicles (EVs), including exosomes. We demonstrate that EVs containing CysC protect cultured cells from starvation-induced death. Moreover, while EVs secreted by CysC-deficient cells were not protective, EVs secreted by CysC-deficient cells treated with exogenous human CysC significantly enhanced the survival of the cells. CysC also plays a role in modulating the secretion of EVs, enhancing secretion of EVs by primary cortical neurons and primary cortical smooth muscle cells. Confirming these in vitro findings, higher EV levels were observed in the brain extracellular space of transgenic mice expressing human CysC as compared to littermate controls. Regulation of cell-secreted EV levels and content in the brain is likely to be essential to maintaining normal brain function. We propose that enhanced EV release could rescue the deleterious effects of dysfunction of the endosomal-lysosomal system in neurodegenerative disorders. Moreover, a higher level of CysC-loaded EVs released from cells in the central nervous system has important protective functions, representing a potential therapeutic tool for disorders of the central nervous system.
Enhanced Generation of Intraluminal Vesicles in Neuronal Late Endosomes in the Brain of a Down Syndrome Mouse Model with Endosomal Dysfunction
Down syndrome (DS) is a human genetic disease caused by trisomy of chromosome 21 and characterized by early developmental brain abnormalities. Dysfunctional endosomal pathway in neurons is an early event of DS and Alzheimer's disease. Recently, we have demonstrated that exosome secretion is upregulated in human DS postmortem brains, in the brain of the trisomic mouse model Ts[Rb(12.1716)]2Cje (Ts2) and by DS fibroblasts as compared with disomic controls. High levels of the tetraspanin CD63, a regulator of exosome biogenesis, were observed in DS brains. Partially blocking exosome secretion by DS fibroblasts exacerbated a pre-existing early endosomal pathology. We thus hypothesized that enhanced CD63 expression induces generation of intraluminal vesicles (ILVs) in late endosomes/multivesicular bodies (MVBs), increasing exosome release as an endogenous mechanism to mitigate endosomal abnormalities in DS. Herein, we show a high-resolution electron microscopy analysis of MVBs in neurons of the frontal cortex of 12-month-old Ts2 mice and littermate diploid controls. Our quantitative analysis revealed that Ts2 MVBs are larger, more abundant, and contain a higher number of ILVs per neuron compared to controls. These findings were further corroborated biochemically by Western blot analysis of purified endosomal fractions showing higher levels of ILVs proteins in the same fractions containing endosomal markers in the brain of Ts2 mice compared to controls. These data suggest that upregulation of ILVs production may be a key homeostatic mechanism to alleviate endosomal dysregulation via the endosomal-exosomal pathway. This article is protected by copyright. All rights reserved.
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.