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Extracellular vesicles: where the amyloid precursor protein carboxyl-terminal fragments accumulate and amyloid-β oligomerizes

Pérez-González, Rocío; Kim, Yohan; Miller, Chelsea; Pacheco-Quinto, Javier; Eckman, Elizabeth A; Levy, Efrat
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-β 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 α- and β-APP carboxyl-terminal fragments and APP intracellular domain accumulate in EVs over time and amyloid-β 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.
PMID: 32772431
ISSN: 1530-6860
CID: 4560212

A pleiotropic role for exosomes loaded with the amyloid β precursor protein carboxyl-terminal fragments in the brain of Down syndrome patients

Pérez-González, Rocío; Gauthier, Sébastien A; Sharma, Ajay; Miller, Chelsea; Pawlik, Monika; Kaur, Gurjinder; Kim, Yohan; Levy, Efrat
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 β 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 naïve neurons and propagating these neurotoxic metabolites, which are also a source of amyloid β, throughout the brain, but also provides a benefit to DS neurons by shedding APP-CTFs accumulated intracellularly.
PMID: 31479861
ISSN: 1558-1497
CID: 4069032

Neuroprotection mediated by cystatin C-loaded extracellular vesicles

Pérez-González, Rocío; Sahoo, Susmita; Gauthier, Sebastien A; Kim, Yohan; Li, Meihua; Kumar, Asok; Pawlik, Monika; Benussi, Luisa; Ghidoni, Roberta; Levy, Efrat
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.
PMID: 31367000
ISSN: 2045-2322
CID: 4015362

Enhanced Generation of Intraluminal Vesicles in Neuronal Late Endosomes in the Brain of a Down Syndrome Mouse Model with Endosomal Dysfunction

D'Acunzo, Pasquale; Hargash, Tal; Pawlik, Monika; Goulbourne, Chris N; Pérez-González, Rocío; Levy, Efrat
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.
PMID: 31278881
ISSN: 1932-846x
CID: 3968442

Apolipoprotein E4 genotype compromises brain exosome production

Peng, Katherine Y; Pérez-González, Rocío; Alldred, Melissa J; Goulbourne, Chris N; Morales-Corraliza, Jose; Saito, Mariko; Saito, Mitsuo; Ginsberg, Stephen D; Mathews, Paul M; Levy, Efrat
In addition to being the greatest genetic risk factor for Alzheimer's disease, expression of the ɛ4 allele of apolipoprotein E can lead to cognitive decline during ageing that is independent of Alzheimer's amyloid-β 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 ɛ3 allele we show that the ɛ4 allele, whether homozygous or heterozygous with an ɛ3 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-β 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.
PMID: 30496349
ISSN: 1460-2156
CID: 3500172

Exosome Production Is Key to Neuronal Endosomal Pathway Integrity in Neurodegenerative Diseases

Mathews, Paul M; Levy, Efrat
Dysfunction of the endosomal-lysosomal system is a prominent pathogenic factor in Alzheimer's disease (AD) and other neurodevelopmental and neurodegenerative disorders. We and others have extensively characterized the neuronal endosomal pathway pathology that results from either triplication of the amyloid-β precursor protein (APP) gene in Down syndrome (DS) or from expression of the apolipoprotein E ε4 allele (APOE4), the greatest genetic risk factor for late-onset AD. More recently brain exosomes, extracellular vesicles that are generated within and released from endosomal compartments, have been shown to be altered in DS and by APOE4 expression. In this review, we discuss the emerging data arguing for an interdependence between exosome production and endosomal pathway integrity in the brain. In vitro and in vivo studies indicate that altered trafficking through the endosomal pathway or compromised cargo turnover within lysosomes can affect the production, secretion, and content of exosomes. Conversely, exosome biogenesis can affect the endosomal-lysosomal system. Indeed, we propose that efficient exosome release helps to modulate flux through the neuronal endosomal pathway by decompressing potential "traffic jams." Exosome secretion may have the added benefit of unburdening the neuron's lysosomal system by delivering endosomal-lysosomal material into the extracellular space, where other cell types may contribute to the degradation of neuronal debris. Thus, maintaining robust neuronal exosome production may prevent or mitigate endosomal and lysosomal abnormalities linked to aging and neurodegenerative diseases. While the current evidence suggests that the exosomal system in the brain can be modulated both by membrane lipid composition and the expression of key proteins that contribute to the formation and secretion of exosomes, how exosomal pathway-regulatory elements sense and respond to perturbations in the endosomal pathway is not well understood. Based upon findings from the extensively studied DS and APOE4 models, we propose that enhanced neuronal exosome secretion can be a protective response, reducing pathological disruption of the endosomal-lysosomal system in disease-vulnerable neurons. Developing therapeutic approaches that help to maintain or enhance neuronal exosome biogenesis and release may be beneficial in a range of disorders of the central nervous system.
PMCID:6920185
PMID: 31911768
ISSN: 1662-4548
CID: 4257312

Cystatin C prevents neuronal loss and behavioral deficits via the endosomal pathway in a mouse model of down syndrome

Kaur, Gurjinder; Gauthier, Sebastien A; Perez-Gonzalez, Rocio; Pawlik, Monika; Singh, Amol Bikram; Cosby, Benjamin; Mohan, Panaiyur V; Smiley, John F; Levy, Efrat
Cystatin C (CysC) plays diverse protective roles under conditions of neuronal challenge. We investigated whether CysC protects from trisomy-induced pathologies in a mouse model of Down syndrome (DS), the most common cause of developmental cognitive and behavioral impairments in humans. We have previously shown that the segmental trisomy mouse model, Ts[Rb(12.1716)]2Cje (Ts2) has DS-like neuronal and behavioral deficiencies. The current study reveals that transgene-mediated low levels of human CysC overexpression has a preventive effect on numerous neuropathologies in the brains of Ts2 mice, including reducing early and late endosome enlargement in cortical neurons and decreasing loss of basal forebrain cholinergic neurons (BFCNs). Consistent with these cellular benefits, behavioral dysfunctions were also prevented, including deficits in nesting behavior and spatial memory. We determined that the CysC-induced neuroprotective mechanism involves activation of the phosphotidylinositol kinase (PI3K)/AKT pathway. Activating this pathway leads to enhanced clearance of accumulated endosomal substrates, protecting cells from DS-mediated dysfunctions in the endosomal system and, for BFCNs, from neurodegeneration. Our findings suggest that modulation of the PI3/AKT pathway offers novel therapeutic interventions for patients with DS.
PMID: 30176349
ISSN: 1095-953x
CID: 3271052

Intracellular metalloprotease activity controls intraneuronal Aβ aggregation and limits secretion of Aβ via exosomes

Pacheco-Quinto, Javier; Clausen, Dana; Pérez-González, Rocío; Peng, Hui; Meszaros, Austin; Eckman, Christopher B; Levy, Efrat; Eckman, Elizabeth A
Accumulating evidence suggests that the abnormal aggregation of amyloid-β (Αβ) peptide in Alzheimer's disease (AD) begins intraneuronally, within vesicles of the endosomal-lysosomal pathway where Aβ is both generated and degraded. Metalloproteases, including endothelin-converting enzyme (ECE)-1 and -2, reside within these vesicles and normally limit the accumulation of intraneuronally produced Aβ. In this study, we determined whether disruption of Aβ catabolism could trigger Aβ aggregation within neurons and increase the amount of Aβ associated with exosomes, small extracellular vesicles derived from endosomal multivesicular bodies. Using cultured cell lines, primary neurons, and organotypic brain slices from an AD mouse model, we found that pharmacological inhibition of the ECE family of metalloproteases increased intracellular and extracellular Aβ levels and promoted the intracellular formation of Aβ oligomers, a process that did not require internalization of secreted Aβ. In vivo, the accumulation of intraneuronal Aβ aggregates was accompanied by increased levels of both extracellular and exosome-associated Aβ, including oligomeric species. Neuronal exosomes were found to contain both ECE-1 and -2 activities, suggesting that multivesicular bodies are intracellular sites of Aβ degradation by these enzymes. ECE dysfunction could lead to the accumulation of intraneuronal Aβ aggregates and their subsequent release into the extracellular space via exosomes.-Pacheco-Quinto, J., Clausen, D., Pérez-González, R., Peng, H., Meszaros, A., Eckman, C. B., Levy, E., Eckman, E. A. Intracellular metalloprotease activity controls intraneuronal Aβ aggregation and limits secretion of Aβ via exosomes.
PMID: 30481490
ISSN: 1530-6860
CID: 3629702

Human apolipoprotein E genotype differentially affects olfactory behavior and sensory physiology in mice [Meeting Abstract]

East, B S; Fleming, G; Peng, K; Olofsson, J K; Levy, E; Mathews, P M; Wilson, D A
Apolipoprotein E (ApoE) is an important lipid carrier in both the periphery and the brain. The ApoE e4 allele (ApoE4) is the single most important genetic risk-factor for Alzheimer's disease (AD) while the e2 allele (ApoE2) is associated with a lower risk of AD-related neurodegeneration compared to the most common variant, e3 (ApoE3). ApoE genotype affects a variety of neural circuits; however, the olfactory system appears to provide early biomarkers of ApoE genotype effects. Here, we directly compared olfactory behavior and olfactory system physiology across all three ApoE genotypes in 6-month- and 12-month-old mice with targeted replacement for the human ApoE2, ApoE3, or ApoE4 genes. Odor investigation and habituation were assessed, along with, olfactory bulb and piriform cortical local field potential activity. The results demonstrate that while initial odor investigation was unaffected by ApoE genotype, odor habituation was impaired in E4 relative to E2 mice, with E3 mice intermediate in function. There was also significant deterioration of odor habituation from 6 to 12 months of age regardless of the ApoE genotype. Olfactory system excitability and odor responsiveness were similarly determined by ApoE genotype, with an ApoE4 > ApoE3 > ApoE2 excitability ranking. The hyper-excitability of ApoE4 mice may contribute to the impairment of odor habituation memory, while the hypo-excitability of ApoE2 mice may contribute to its protective effects. Given that these ApoE mice do not have AD pathology, our results demonstrate the potential process by which ApoE affects the olfactory system at early stages, prior to the development of AD
EMBASE:626768858
ISSN: 1464-3553
CID: 3757222

Human apolipoprotein E genotype differentially affects olfactory behavior and sensory physiology in mice

East, Brett S; Fleming, Gloria; Peng, Kathy; Olofsson, Jonas K; Levy, Efrat; Mathews, Paul M; Wilson, Donald A
Apolipoprotein E (ApoE) is an important lipid carrier in both the periphery and the brain. The ApoE ε4 allele (ApoE4) is the single most important genetic risk-factor for Alzheimer's disease (AD) while the ε 2 allele (ApoE2) is associated with a lower risk of AD-related neurodegeneration compared to the most common variant, ε 3 (ApoE3). ApoE genotype affects a variety of neural circuits; however, the olfactory system appears to provide early biomarkers of ApoE genotype effects. Here, we directly compared olfactory behavior and olfactory system physiology across all three ApoE genotypes in 6-month- and 12-month-old mice with targeted replacement for the human ApoE2, ApoE3, or ApoE4 genes. Odor investigation and habituation were assessed, along with, olfactory bulb and piriform cortical local field potential activity. The results demonstrate that while initial odor investigation was unaffected by ApoE genotype, odor habituation was impaired in E4 relative to E2 mice, with E3 mice intermediate in function. There was also significant deterioration of odor habituation from 6 to 12 months of age regardless of the ApoE genotype. Olfactory system excitability and odor responsiveness were similarly determined by ApoE genotype, with an ApoE4 > ApoE3 > ApoE2 excitability ranking. Although motivated behavior is influenced by many processes, hyper-excitability of ApoE4 mice may contribute to impaired odor habituation, while hypo-excitability of ApoE2 mice may contribute to its protective effects. Given that these ApoE mice do not have AD pathology, our results demonstrate how ApoE affects the olfactory system at early stages, prior to the development of AD.
PMCID:5959295
PMID: 29678753
ISSN: 1873-7544
CID: 3043232