Extracellular vesicle-mediated mitochondria delivery: Premise and promise

Abstract

Mitochondrial transfer is highly significant under physiological as well as pathological states given the emerging recognition of mitochondria as cellular "processors" akin to microchip processors that control the operation of a mobile device. Mitochondria play indispensable roles in healthy functioning of the brain, the organ with the highest energy demand in the human body and therefore, loss of mitochondrial function plays a causal role in multiple brain diseases. In this review, we will discuss various aspects of extracellular vesicle (EV)-mediated mitochondrial transfer and their effects in increasing recipient cell/tissue bioenergetics with a focus on these processes in brain cells. A subset of EVs with particle diameters >200 nm, referred to as medium-to-large EVs (m/lEVs), are known to entrap mitochondria during EV biogenesis. The entrapped mitochondria are likely a combination of either polarized, depolarized mitochondria or a mixture of both. We will also discuss engineering approaches to control the quality and quantity of mitochondria entrapped in the m/lEVs. Controlling mitochondrial quality can allow for optimizing/maximizing the therapeutic potential of m/lEV mitochondria-a novel drug with immense potential to treat a wide range of disorders associated with mitochondrial dysfunction.

Keywords: Extracellular vesicles (EVs); medium-to-large EVs; microvesicles; mitochondria; small EVs.

Figure 1.

Membrane contact sites formed between freshly-fissioned mitochondria and plasma membrane may result in the incorporation of those mitochondrial segments into the membrane buds that are then likely released as m/lEVs into the extracellular spaces via an ARRDC1-regulated mechanism described in the text.

Figure 2.

DLS Z-Average particle diameter, zeta potential and NTA particle concentration (a). Detection of EV marker proteins using western blotting. Each lane was loaded with 50 μg total protein, electrophoresed, immunoblotted and scanned on an Odyssey imager (b). EVs were cross sectioned as described by us previously [14–15]. m/lEVs contained one or more mitochondria noted as electron dense structures while sEV cross sections lacked electron dense structures. Images are representative sections obtained from a JEOL JEM 1400 Plus TEM (c). Oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were measured by treating cells with the indicated samples (EV particles) in OGD medium. We used a standard Mitochondrial Stress Test protocol on an Agilent Seahorse XFe24 Analyzer. All rates were normalized to cellular protein content measured using MicroBCA assay. Data are mean ± SEM, n = 3 (d). Quantification of total hemispheric infarct volume and neurological deficit scoring (NDS) at 24 h post-stroke (e). Data are mean ± SEM (n = 8). OGD primary HBMECs were incubated with the indicated samples for 24 h and were washed once in 1x PBS prior to measuring relative cellular ATP levels using a Cell Glo luminescence assay (f and g). Data represent average ± SD (n=3). Donor BECs were treated with 0.5 or 1 µM rotenone (RTN) to inhibit mitochondrial complex I prior to isolating EVs from mitochondria-impaired donor BECs (RTN-EVs). OGD HBMECs were then treated at the medium dose of 225,000 EV particles (g). Figures from our previous publications have been reproduced with permission from Elsevier (b to e) and Springer (g).

Figure 3.

Mitochondrial status in the donor cells (healthy (a) vs. damaged (b)/polarized vs. partially or fully depolarized) determines the quality of mitochondrial load that is packaged into the m/lEVs and ultimately, the resulting ATP levels in the recipient BECs.

Figure 4.

MitoTracker Red labeling of donor BECs allows tracking and intracellular uptake of mitoT-EVs into recipient BECs (a) using fluorescence microscopy (b) and flow cytometry (c). MitoT-EVs colocalized with recipient HBMEC mitochondria pre-transduced with CellLight Mitochondria-GFP BacMam to tag the alpha pyruvate mitochondrial matrix protein (d). MitoT-EVs were also internalized by cells in cortical and hippocampal mouse brain slices (e and f). Slice insets show MitoT signals without DAPI for clarity. Mean intensity values were normalized to control slices. Figures have been reproduced from our previous publications with permission from Elsevier (b to f).

Figure 5.

Activation of PGC-1α using resveratrol increased mitochondrial biogenesis in the donor BECs (a-b) followed by increased rates of m/lEV secretion into the extracellular spaces (c) compared to naïve m/lEVs collected from non-activated/basal cells. Mitochondria-enriched EVs (mito-m/lEVs) showed a greater magnitude of relative ATP increases compared to naïve m/lEVs in the recipient OGD BECs. Figures (c and d) have been reproduced from our previous publications with permission from Springer.