Isolation of mitochondria-derived mitovesicles and subpopulations of microvesicles and exosomes from brain tissues

Cocaine perturbs mitovesicle biology in the brain

“…As assessed by Western blot analysis (Figure 1a), with our fractionation system microvesicle proteins, such as Annexin A2, were enriched in low‐density fractions (with the highest signal in fractions 1–3) while exosome proteins, such as ALIX, were mainly found in the intermediate‐density fractions 4–6 (with a minor presence in fractions 3 and 7), consistent with previous reports (Barreto et al., 2022; Jeppesen et al., 2019; D'Acunzo et al, 2021, 2022). The brain EVs in the highest density fractions, particularly in Fr8, were largely negative for microvesicle and exosome proteins and contained mitovesicles, as assessed by the levels of mitochondrial proteins such as PDHE1α, found in the mitovesicle matrix (MMx), COX‐IV, found in the inner mitovesicle membrane (IMM), and VDAC, found in the outer mitovesicle membrane (OMM).…”

“…The resulting supernatant was ultra‐centrifuged at 100,000 g ( k ‐factor: 207.5, 45Ti rotor type, Beckman Coulter, Brea, CA, US) for 70 min at 4°C to obtain a crude brain EV pellet. Mitovesicles were separated from other small EVs and contaminant using a high‐resolution iodixanol step‐gradient approach (D'Acunzo et al., 2021; D'Acunzo et al., 2022; Y. Zhang et al., 2021) through which mitovesicles were enriched in the fraction number 8 (Fr8) of the gradient (Figure 1a). Although the secretion of both nude mitochondria and mitochondria‐encapsulated microvesicles into the extracellular space was reported before, at least in vitro (Puhm et al., 2019), the strategy employed in this study to isolate brain mitovesicles was designed to separate and pellet these particles out from the other types of EVs.…”

“…Upon treatments, cells were washed once in PBS and lysed in RIPA buffer (formulation as described; D'Acunzo et al., 2022) supplemented with protease inhibitors (5 μg/ml leupeptin, 5 μg/ml antipain dihydrochloride, 5 μg/ml pepstatin A, 1 mM phenylmethanesulfonyl fluoride, 1 μM E64; all reagents from Sigma‐Aldrich), while the medium was collected and serially centrifuged at 300 g for 10 min at 4°C, at 2,000 g for 10 min at 4°C, and at 10,000 g for 30 min at 4°C. The resulting supernatant was ultracentrifuged at 100,000 g (36,000 rpm) for 70 min at 4°C in a 45Ti rotor type (Beckman Coulter) to obtain a crude EV pellet, which was later washed in phosphate‐buffered saline (PBS, Sigma‐Aldrich) and centrifuged again at 100,000 g (50,000 rpm) for 70 min at 4°C in a TLA‐55 rotor type ( k ‐factor: 79.9, Beckman Coulter).…”

“…Murine brain mitovesicles and EVs from conditioned cell media were analyzed by cryo‐EM, TEM, NTA, and ATP determination assays as previously described in detail (D'Acunzo et al., 2021; D'Acunzo et al., 2022). Per MISEV2018 guidelines, the level of EVs, including mitovesicles, should be determined using at least two unbiased, independent methods (Thery et al., 2018).…”

“…Unlike exosomes and microvesicles, mitovesicles are characterised by: (I) specific biophysical and biochemical properties, including a higher density and a higher protein/lipid ratio; (II) the presence of a double membrane and an electron‐dense core when visualised by cryogenic electron microscopy (cryo‐EM); (III) the lack of microvesicle‐ and exosome‐specific proteins; (IV) a specific lipidome, including the enrichment of the mitochondrial lipid cardiolipin; (V) the incorporation of mitochondria‐specific dyes (e.g., MitoTracker); and (VI) the ability to produce ATP via ETC in vitro (D'Acunzo et al., 2021; D'Acunzo et al., 2022). Mitovesicles are also distinguishable from microvesicles by their smaller size (D'Acunzo et al., 2021; D'Acunzo et al., 2022), while the hydrodynamic diameters of mitovesicles and exosomes largely overlap when studied by nanoparticle tracking analysis (NTA) (D'Acunzo et al., 2021; D'Acunzo et al., 2022). However, mitovesicles are distinctively characterised by a double membrane and an electron‐dense core under cryo‐EM, while exosomes and microvesicles are surrounded by a single membrane and display a more electron‐lucent lumen (D'Acunzo et al., 2021).…”