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. 2021 Apr;41(4):761-770.
doi: 10.1177/0271678X20928147. Epub 2020 Jun 5.

Mitochondrial transfer from mesenchymal stem cells improves neuronal metabolism after oxidant injury in vitro: The role of Miro1

Nancy Tseng  1 , Scott C Lambie  1 , Christopher Q Huynh  2 , Bridget Sanford  3 , Manisha Patel  2 , Paco S Herson  4 , D Ryan Ormond  1
Affiliations

Mitochondrial transfer from mesenchymal stem cells improves neuronal metabolism after oxidant injury in vitro: The role of Miro1

Nancy Tseng et al. J Cereb Blood Flow Metab. 2021 Apr.

Abstract

Stroke-induced cerebral ischemia is a major cause of death and disability. The disruption of blood flow results in neuronal and glial cell death leading to brain injury. Reperfusion restores oxygen to the affected tissue, but can also cause damage through an enhanced oxidative stress and inflammatory response. This study examines mitochondrial transfer from MSC to neurons and the role it plays in neuronal preservation after oxidant injury. We observed the transfer of mitochondria from MSC to mouse neurons in vitro following hydrogen peroxide exposure. The observed transfer was dependent on cell-to-cell contact and led to increased neuronal survival and improved metabolism. A number of pro-inflammatory and mitochondrial motility genes were upregulated in neurons after hydrogen peroxide exposure. This included Miro1 and TNFAIP2, linking inflammation and mitochondrial transfer to oxidant injury. Increasing Miro1 expression in MSC improved the metabolic benefit of mitochondrial transfer after neuronal oxidant injury. Decreasing Miro1 expression had the opposite effect, decreasing the metabolic benefit of MSC co-culture. MSC transfer of mitochondria to oxidant-damaged neurons may help improve neuronal preservation and functional recovery after stroke.

Keywords: Brain ischemia; Miro1; mesenchymal stem cell transplant; mitochondrial transfer; neuronal injury.

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Conflict of interest statement

Declaration of conflicting interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1.
Figure 1.
(a) Oxidant-damaged neurons (white arrow) with mitochondria labeled with MitoTracker Green accepting mitochondria from a neighboring MSC (white arrowhead) with mitochondria labeled with Mitotracker Far Red after co-culture neuron at high power showing both red and green mitochondria. (b) Flow cytometry of primary cortical neurons labeled with MitoTracker Green. (c) Flow cytometry of MSC labeled with MitoTracker Far Red. (d) Flow cytometry of MitoTracker Green-labeled primary cortical neurons following hydrogen peroxide exposure and co-culture with MSC labeled with MitoTracker Far Red resulting in a double positive cell population. (e) Flow Cytometry of MitoTracker Green-labeled primary cortical neurons following oxidant injury and indirect co-culture with MSC labeled with MitoTracker Far Red. MSC and neurons were co-cultured with a 0.2 µm pore size membrane lying in between cells resulting in no double positive cell population of neurons.
Figure 2.
Figure 2.
Metabolic benefit of co-culture of MSC with oxidant-damaged neurons. Murine primary cortical neurons underwent in vitro oxidant injury with hydrogen peroxide followed by co-culture with MSC. (a) MTT assay. Neuronal cell viability declined after oxidant injury, improved after co-culture, but not if MSC were separated from neurons with a semi-porous membrane (CC membrane). (b–e) Seahorse XF Assay (pmol/min/60,000 cells). From left to right (see legend): MSC controls (blue); neuronal control (red); oxidant-damaged neurons in co-culture with MSC (green); neurons after hydrogen peroxide exposure (purple). (b) There is evidence of significant mitochondrial dysfunction in neurons after oxidant injury that recovers in co-culture with MSC when assessing mitochondrial respiration in neurons. If confidence intervals do not cross, differences are significant (see C for Figure key). (d) Basal mitochondrial respiration and spare respiratory capacity all worsened with oxidant injury and then improved with co-culture with MSC. (e) Proton leak and ATP production worsened in neurons after oxidant injury and recovered after co-culture. *Significant difference from control.
Figure 3.
Figure 3.
Clariom S Mouse array of murine neurons after hydrogen peroxide injury. (a) Rho Family GTPases and related pathways were upregulated. (b) Inflammatory pathways, cytoskeleton and microtubule pathways were also upregulated. (c) Gene expression microarray analysis comparing control neurons (1 A-C) with H2O2-oxidant damaged neurons (5 A-C): Clustering of the samples according to their expression profile. High expression is indicated in red. Low expression is indicated in blue. Hierarchical clustering was performed using the one minus Pearson correlation. (d and e) Graphical representation of two top scored networks identified by ingenuity pathway analysis (IPA). Canonical pathways from IPA for control neurons compared to oxidant damaged neurons. Molecular relationships between genes after treatment are shown demonstrating changes in genes related to mitochondrial motility (d) and upstream regulators of tumor necrosis factor (TNF) (e) with positive numbers representing upregulation with oxidant damage and negative numbers representing downregulation.
Figure 4.
Figure 4.
(a) Western Blot of Miro1 and TNFAIP2 expression before (Neuron (−)) or after (Neuron (+)) oxidant injury with H2O2 as well as after co-culture with MSC (CC). (b) Expression levels normalized to actin in Lanes 1–3 bar graphs, 25 ug protein per lane. Lane 1, untreated neurons (Neuron (−)). Lane 2, neurons after H2O2 treatment at 150 µM for 2 h (Neuron (+)). Lane 3, oxidant-damaged neurons co-cultured overnight with MSC (CC). Both Miro1 and TNFAIP2 expression were increased in primary cortical neurons after hydrogen peroxide exposure. This expression returned toward normal when oxidant-damaged neurons were co-cultured with MSC. (c) MTT assay. Some data repeated from Figure 2 for comparison purposes. Oxidant injury (neuron (+)) resulted in more neuronal cell death that recovered with co-culture with MSC (CC), but not if MSC were separated from neurons with a semi-porous membrane (CC (membrane)). This benefit is also decreased if Miro 1 was inhibited in MSC (CC (inhibited)) and enhanced if Miro 1 was overexpressed (CC (overexpressed)), implicating Miro 1 in the metabolic benefit of MSC co-culture with neurons. *Significant difference from control.
Figure 5.
Figure 5.
Proposed mechanism of intercellular transport of mitochondria from mesenchymal stem cells (MSC) to neurons via tunneling nanotubes (TNT). Ischemia damages neuronal mitochondria, but also induces an inflammatory response that increases TNFAIP2 and Miro 1 production. This, in turn, increases mitochondrial motility and the transfer of healthy mitochondria from MSC to neurons, potentially sparing neurons from apoptosis.
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