Faculty Bibliography

The abnormal assembly of tau protein in neurons is a pathological hallmark of multiple neurodegenerative diseases, including Alzheimer's disease (AD). Assembled tau associates with extracellular vesicles (EVs) in the central nervous system of individuals with AD, which is linked to its clearance and prion-like propagation. However, the identities of the assembled tau species and EVs, as well as how they associate, are not known. Here, we combined quantitative mass spectrometry, cryo-electron tomography and single-particle cryo-electron microscopy to study brain EVs from individuals with AD. We found tau filaments composed mainly of truncated tau that were enclosed within EVs enriched in endo-lysosomal proteins. We observed multiple filament interactions, including with molecules that tethered filaments to the EV limiting membrane, suggesting selective packaging. Our findings will guide studies into the molecular mechanisms of EV-mediated secretion of assembled tau and inform the targeting of EV-associated tau as potential therapeutic and biomarker strategies for AD.

In the brain, mitochondrial components are released into the extracellular space via several mechanisms, including a recently identified type of extracellular vesicles called mitovesicles. While vesiculation of neuronal mitochondria yields various intracellular types of vesicles, with either a single or a double membrane, mitovesicles secreted into the extracellular space are a unique subtype of these mitochondria-derived vesicles, with a double membrane and a specific set of mitochondrial DNA, RNA, proteins, and lipids. Based on the most relevant literature describing mitochondrial vesiculation and mitochondrial exocytosis, we propose a model for their secretion when the amphisome, a hybrid endosome-autophagosome organelle, fuses with the plasma membrane, releasing mitovesicles and exosomes into the extracellular space. In aging and neurodegenerative disorders, mitochondrial dysfunction, in association with endolysosomal abnormalities, alter mitovesicle number and content, with downstream effect on brain health.

The polymorphic APOE gene is the greatest genetic determinant of sporadic Alzheimer's disease risk: the APOE4 allele increases risk, while the APOE2 allele is neuroprotective compared with the risk-neutral APOE3 allele. The neuronal endosomal system is inherently vulnerable during aging, and APOE4 exacerbates this vulnerability by driving an enlargement of early endosomes and reducing exosome release in the brain of humans and mice. We hypothesized that the protective effects of APOE2 are, in part, mediated through the endosomal pathway. Messenger RNA analyses showed that APOE2 leads to an enrichment of endosomal pathways in the brain when compared with both APOE3 and APOE4. Moreover, we show age-dependent alterations in the recruitment of key endosomal regulatory proteins to vesicle compartments when comparing APOE2 to APOE3. In contrast to the early endosome enlargement previously shown in Alzheimer's disease and APOE4 models, we detected similar morphology and abundance of early endosomes and retromer-associated vesicles within cortical neurons of aged APOE2 targeted-replacement mice compared with APOE3. Additionally, we observed increased brain extracellular levels of endosome-derived exosomes in APOE2 compared with APOE3 mice during aging, consistent with enhanced endosomal cargo clearance by exosomes to the extracellular space. Our findings thus demonstrate that APOE2 enhances an endosomal clearance pathway, which has been shown to be impaired by APOE4 and which may be protective due to APOE2 expression during brain aging.

BACKGROUND:Hypometabolism tied to mitochondrial dysfunction occurs in the aging brain and in neurodegenerative disorders, including in Alzheimer's disease, in Down syndrome, and in mouse models of these conditions. We have previously shown that mitovesicles, small extracellular vesicles (EVs) of mitochondrial origin, are altered in content and abundance in multiple brain conditions characterized by mitochondrial dysfunction. However, given their recent discovery, it is yet to be explored what mitovesicles regulate and modify, both under physiological conditions and in the diseased brain. In this study, we investigated the effects of mitovesicles on synaptic function, and the molecular players involved. METHODS:Hippocampal slices from wild-type mice were perfused with the three known types of EVs, mitovesicles, microvesicles, or exosomes, isolated from the brain of a mouse model of Down syndrome or of a diploid control and long-term potentiation (LTP) recorded. The role of the monoamine oxidases type B (MAO-B) and type A (MAO-A) in mitovesicle-driven LTP impairments was addressed by treatment of mitovesicles with the irreversible MAO inhibitors pargyline and clorgiline prior to perfusion of the hippocampal slices. RESULTS:Mitovesicles from the brain of the Down syndrome model reduced LTP within minutes of mitovesicle addition. Mitovesicles isolated from control brains did not trigger electrophysiological effects, nor did other types of brain EVs (microvesicles and exosomes) from any genotype tested. Depleting mitovesicles of their MAO-B, but not MAO-A, activity eliminated their ability to alter LTP. CONCLUSIONS:Mitovesicle impairment of LTP is a previously undescribed paracrine-like mechanism by which EVs modulate synaptic activity, demonstrating that mitovesicles are active participants in the propagation of cellular and functional homeostatic changes in the context of neurodegenerative disorders.

The abnormal assembly of tau protein in neurons is the pathological hallmark of multiple neurodegenerative diseases, including Alzheimer's disease (AD). In addition, assembled tau associates with extracellular vesicles (EVs) in the central nervous system of patients with AD, which is linked to its clearance and prion-like propagation between neurons. However, the identities of the assembled tau species and the EVs, as well as how they associate, are not known. Here, we combined quantitative mass spectrometry, cryo-electron tomography and single-particle cryo-electron microscopy to study brain EVs from AD patients. We found filaments of truncated tau enclosed within EVs enriched in endo-lysosomal proteins. We observed multiple filament interactions, including with molecules that tethered filaments to the EV limiting membrane, suggesting selective packaging. Our findings will guide studies into the molecular mechanisms of EV-mediated secretion of assembled tau and inform the targeting of EV-associated tau as potential therapeutic and biomarker strategies for AD.

Cocaine, an addictive psychostimulant, has a broad mechanism of action, including the induction of a wide range of alterations in brain metabolism and mitochondrial homeostasis. Our group recently identified a subpopulation of non-microvesicular, non-exosomal extracellular vesicles of mitochondrial origin (mitovesicles) and developed a method to isolate mitovesicles from brain parenchyma. We hypothesised that the generation and secretion of mitovesicles is affected by mitochondrial abnormalities induced by chronic cocaine exposure. Mitovesicles from the brain extracellular space of cocaine-administered mice were enlarged and more numerous when compared to controls, supporting a model in which mitovesicle biogenesis is enhanced in the presence of mitochondrial alterations. This interrelationship was confirmed in vitro. Moreover, cocaine affected mitovesicle protein composition, causing a functional alteration in mitovesicle ATP production capacity. These data suggest that mitovesicles are previously unidentified players in the biology of cocaine addiction and that target therapies to fine-tune brain mitovesicle functionality may be beneficial to mitigate the effects of chronic cocaine exposure.

Extracellular vesicles (EVs) in the brain play a role in neuronal homeostasis by removing intracellular material and regulating cell-to-cell communication. Given that sex and aging differentially modulate brain networks, we investigated sex-dependent differences in EV levels and content in the brain during aging. EVs were isolated from the brains of 3, 6, 12, 18, and 24 month-old female and male C57BL/6 J mice, and the levels of different EV species determined. While the number of plasma membrane-derived microvesicles and a subset of late endosomes-derived exosomes increased with age in the brain of female mice, no significant changes were seen in males. Mitochondria-derived mitovesicles in the brain increased during aging in both sexes, a change that may reflect aging-dependent alterations in mitochondrial function. These findings reveal enhanced turnover during aging in female brains, suggesting a mechanism for advantageous successful female brain aging and sex-depending different susceptibility to age-related neurodegenerative diseases.

Extracellular vesicles (EVs) are nanoscale vesicles secreted into the extracellular space by all cell types, including neurons and astrocytes in the brain. EVs play pivotal roles in physiological and pathophysiological processes such as waste removal, cell-to-cell communication and transport of either protective or pathogenic material into the extracellular space. Here we describe a detailed protocol for the reliable and consistent isolation of EVs from both murine and human brains, intended for anyone with basic laboratory experience and performed in a total time of 27 h. The method includes a mild extracellular matrix digestion of the brain tissue, a series of filtration and centrifugation steps to purify EVs and an iodixanol-based high-resolution density step gradient that fractionates different EV populations, including mitovesicles, a newly identified type of EV of mitochondrial origin. We also report detailed downstream protocols for the characterization and analysis of brain EV preparations using nanotrack analysis, electron microscopy and western blotting, as well as for measuring mitovesicular ATP kinetics. Furthermore, we compared this novel iodixanol-based high-resolution density step gradient to the previously described sucrose-based gradient. Although the yield of total EVs recovered was similar, the iodixanol-based gradient better separated distinct EV species as compared with the sucrose-based gradient, including subpopulations of microvesicles, exosomes and mitovesicles. This technique allows quantitative, highly reproducible analyses of brain EV subtypes under normal physiological processes and pathological brain conditions, including neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease.

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 2.5 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 α-synuclein per exosome in the brain of females but did not affect exosomal α-synuclein content in the brain of males, a sex-difference eliminated by ovariectomy. Enhanced packaging of α-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.

The role of mitophagy, a process that allows the removal of damaged mitochondria from cells, remains unknown in multiple sclerosis (MS), a disease that is found associated with dysfunctional mitochondria. Here we have qualitatively and quantitatively studied the main players in PINK1-mediated mitophagy in peripheral blood mononuclear cells (PBMCs) of patients with relapsing-remitting MS. We found the variant c.491G>A (rs550510, p.G140E) of NDP52, one of the major mitophagy receptor genes, associated with a MS cohort. Through the characterization of this variant, we discovered that the residue 140 of human NDP52 is a crucial modulator of NDP52/LC3C binding, promoting the formation of autophagosomes in order to drive efficient mitophagy. In addition, we found that in the PBMC population, NDP52 is mainly expressed in B cells and by ensuring efficient mitophagy, it is able to limit the production of the proinflammatory cytokine TNF-α following cell stimulation. In sum, our results contribute to a better understanding of the role of NDP52 in mitophagy and underline, for the first time, a possible role of NDP52 in MS.