Extracellular Mitochondria for Therapy and Diagnosis in Acute Central Nervous System Injury

Abstract

OBJECTIVE

Acute central nervous system (CNS) injury after stroke and trauma remains a clinical challenge with limited diagnostic and therapeutic approaches. In this article, we review studies suggesting that after CNS injury, mitochondria can be released into extracellular space as a “help-me” signal to augment recovery. Results are taken from experimental studies in cell and animal models and an initial proof-of-concept analysis in humans suggesting the functional relevance of extracellular mitochondria after acute CNS injury.

OBSERVATIONS

After acute CNS injury, (1) mitochondria may be released into extracellular space, (2) mitochondria may be transferred between cells, and (3) levels of extracellular mitochondria may serve as potential biomarkers for recovery.

CONCLUSIONS AND RELEVANCE

Further translational and clinical studies are warranted to assess the overall hypothesis of using extracellular mitochondria as a therapy and biomarker in the CNS after stroke and trauma.

More than 2 billion years ago, atmospheric concentrations of oxygen dramatically increased, a shift commonly referred to in the geologic literature as the Great Oxidation Event.¹ Since then, oxidative phosphorylation has evolved into the primary biochemical basis of metabolism for mammalian life on earth.² For most eukaryotic cells, the mitochondrion represents the energetic core where the electron transport chain consumes oxygen and generates adenosine triphosphate (ATP).² Mitochondria play a vital homeostatic role in almost all aspects of cell physiology and pathophysiology. The reader is referred to excellent reviews conducted between 2014 and 2016 that cover the vast literature on mitochondrial regulation, dynamics, and its role in a wide spectrum of human conditions and disorders including aging, cancer, neurodegeneration, and cardiovascular disease.^3–9

As one of the most energy-intensive organs in the human body, the CNS is highly dependent on mitochondria to maintain homeostasis. Within neurons, mitochondria not only provide the basis for oxidative metabolism and ATP generation but also actively regulate the balance between prosurvival and prodeath signaling via antioxidant enzymes and apoptotic mediators. Emerging data suggest that mitochondria may also be released into extracellular space and potentially transferred between cells. In the context of a damaged or diseased brain, extracellular mitochondria might represent a novel class of help-me or danger signals, depending on context. In this review, we survey representative studies of extracellular mitochondria, discuss the overall hypothesis of mitochondrial transfer as a therapeutic approach for brain injury after stroke and trauma, and examine extracellular mitochondria as a potential biomarker for neuroprotection and neurorepair.

Mitochondria and Neuronal Survival

Because the mammalian CNS is extremely energy-intensive, any disruption of mitochondrial homeostasis should threaten neuronal survival. From a mechanistic perspective, the mitochondrion may link to all major pathways of neuronal cell death. Mitochondrial dysfunction leads to reductions in ATP levels that then impair sodium-potassium-ATP activity that is essential for sustaining membrane potentials and neuronal firing. Disruptions in neurotransmitter dynamics may perturb glutamate handling. Thus, mitochondrial dysfunction and energetic compromise can amplify excitotoxicity, a well-established mechanism for neuronal death.¹⁰

Beyond their involvement in energy loss per se, mitochondria may also help regulate neuronal responses to oxidative stress. In a damaged or diseased cell, disruptions in the electron transport chain can lead to electron leak and the generation of reactive oxygen species and the coordinated induction of reactive nitric oxide species.¹⁰ Conversely, the mitochondrion is enriched with a plethora of antioxidant systems including manganese superoxide dismutase, glutathione, thioredoxin, and thioredoxin reductase.¹⁰ Mitochondrial regulation of reactive oxygen species and reactive nitric oxide species represents a major mechanism for protecting against oxidative injury in the CNS.

Neuronal death via energetic compromise may be largely mediated via necrosis. However, in some conditions, programmed cell death mechanisms may also be induced.¹¹ In this context, mitochondrial response may again play a central role. Proapoptotic and antiapoptotic members of the Bcl family of proteins are known to converge on mitochondria, and the release of cytochrome c and the apoptosis-inducing factor from dysfunctional mitochondria underlie key steps in the control of apoptosis.¹¹ Taken together, mitochondria may represent a central point of convergence for regulating the balance between prolife and prodeath signaling in the CNS.

Extracellular Mitochondria as a Help-Me Signal

Emerging evidence now suggest that in some conditions, mitochondria can surprisingly be released and transferred between cells. Intercellular mitochondrial transfer has been observed to take place between cancer cells and mesenchymal stem cells or endothelial cells.¹² The ability of stem cells to release extracellular particles containing mitochondria may also allow an amplification of potentially beneficial bystander effects.¹³ In damaged lung, bone marrow– derived stromal cells release mitochondria that may be transferred into pulmonary alveoli to suppress injury.¹⁴ Similar pathways may also exist within the CNS. For example, retinal neurons may transfer mitochondria to astrocytes for disposal and recycling.¹⁵ If mitochondria or mitochondria-containing vesicles can indeed be exchanged between different cell types, is it possible that these extracellular particles may represent an important mode of cell-cell signaling within the CNS?

In the context of tissue damage or disease, mitochondria play a regulatory role for coordinating immune and inflammatory responses.¹⁶ It is well known that some mitochondrial components may contain damage-associated molecular pattern motifs and thus act as “danger signals.”¹⁶ However, the good vs bad of extracellular mitochondria may depend on context. A 2016 study proposed that within some experimental conditions, astrocytes may transfer mitochondria into neurons as a help-me signal after stroke.¹⁷ In primary cortical astrocyte cultures, electron microscopy confirmed the presence of extracellular mitochondria-containing particles in conditioned media. Measurements of ATP, JC-1 fluorescence ratio, and oxygen consumption suggested that extracellular mitochondria may retain some of their functionality even outside the cell. When astrocyte-conditioned media were then added to primary neurons, these extracellular mitochondria appeared to enter into neurons and protect them against oxygen-glucose deprivation as well as promote dendritic markers of neuroplasticity. Similar results were obtained in vivo. In mouse models of focal cerebral ischemia, injections of labeled extracellular mitochondria also appeared to enter into neurons and upregulate prosurvival antiapoptotic signals. Conversely, blocking the transfer of mitochondria from astrocytes into adjacent neurons worsened stroke recovery outcomes in these in vivo mouse models of focal ischemia. Taken together, these findings suggest that astrocytes may somehow sense stress and transfer mitochondria into adjacent neurons at risk as a help-me signal. Nevertheless, the underlying mechanisms of intercellular release and entry remain to be fully defined. Initial data suggested that extracellular mitochondria may not merely comprise nonspecific cellular debris. Active mechanisms may be involved because calcium-dependent CD38 signaling can upregulate mitochondrial release from astrocytes. Similarly, active mechanisms may be involved for cellular entry because blockade of integrin and src/syk signaling decreased mitochondrial transfer into neurons. Ultimately, it is likely that multifactorial pathways contribute to this phenomenon. Indeed, actual release of mitochondrial particles may not even be required because in other model systems, tunneling nanotubes can also allow mitochondria to move between cells.¹⁸

Translation Implications of Mitochondrial Transfer

In a multicellular organ, such as the brain, communication between neuronal, glial, and vascular cells is required for normal function, a concept formalized as the “neurovascular unit.”¹⁹ The mechanisms of CNS disorders can then be interpreted as a disruption of homeostatic signals within the neurovascular unit. Similarly, any endogenous response to CNS injury and disease will surely recruit noncell autonomous mechanisms as neuronal, glial, and vascular cells exchange a plethora of signals to repair and rebuild their internal networks.²⁰ If viable mitochondria can indeed be released into extracellular space and transferred between cells, this phenomenon may offer novel opportunities to protect, repair, and report on the CNS (Figure).^21–23

Figure. Extracellular Mitochondria for Therapy and Diagnosis.

Mitochondrial release or transfer has been reported in mesenchymal stem cells (MSC),¹⁴ neurons,^15,17 astrocytes,^15,17 microglia,²¹ and endothelial cells.^12,21 Whether other central nervous system (CNS) cells can also participate in this phenomenon should be investigated in future studies. Transplant of exogenous mitochondria may offer novel ways to augment neurorepair after CNS injury.^22,23 To complement transplant approaches, extracellular mitochondria may also be preconditioned to further amplify therapeutic benefit.¹⁰ Finally, insofar as extracellular mitochondria may reflect intracellular metabolism, they may also represent a novel class of biomarkers in the CNS. EPC indicates endothelial progenitor cells; NSC, neuronal stem cells; OPC, oligodendrocyte precursor cells.

Mitochondrial Transplant

Proof of principle has been obtained in the heart. Masuzawa et al²⁴ demonstrated that direct injections of extracellular mitochondrial particles into ischemic myocardium rescued cell viability and function in a rabbit model of cardiac ischemia and reperfusion. Promising findings are beginning to emerge in humans as well. Direct transplant of autologous mitochondria isolated from the patient’s own body into the heart was feasible without inducing immune or autoimmune responses.²⁵ In this study, mitochondria were injected into the ischemic myocardium at 8 to 10 sites (0.1 mL per site) using a tuberculin syringe with a 28-Gneedle.To allow for clinical use, a rapid method for the isolation and purification of mitochondria was developed. Using a filtration method instead of centrifugation, the isolation of intact viable mitochondria was performed in less than 30 minutes. Intriguingly, these studies also suggested that relatively small numbers of mitochondria (approximately 2 × 10⁵/mL) may still have a cardioprotective effect against ischemia. Similar mitochondrial transplant approaches might also work in the CNS. Pep-1-conjugated mitochondrial transplant protected dopaminergic neurons in hydroxydopamine rat model of Parkinson disease.²⁶ In a rat model of focal cerebral ischemia, intracerebral or intravenous injections of exogenous mitochondria appeared to reduce infarction.²⁷ For the purpose of treating traumatic CNS injury, injections of transgenically labeled cell culture-derived mitochondria were effectively transferred into microglia and endothelial cells in rodent spinal cords in vivo.²¹

Modulating Mitochondria

Beyond direct transplant of exogenous mitochondria per se, modulating endogenous mitochondrial response may offer complementary ways to augment this class of intercellular help-me signals. Mitochondria may be evolutionarily derived from photosynthetic organisms, so it is possible that some mitochondrial elements might retain the ability to respond to light.²⁸ Indeed, nearinfrared photons (approximately 800–900–nm wavelengths) may stimulate mitochondrial function and protect neurons against oxygen-glucose deprivation.²⁹ Additionally, the induction of hypoxiarelated adaptive gene pathways has also been shown to be protect mitochondria against stress and dysfunction.³⁰ Looking ahead, it is possible to hypothesize that preconditioning extracellular mitochondrial particles before transplant might offer novel ways to enhance therapeutic effects.

Extracellular Mitochondria as Biomarkers

Finally, extracellular mitochondria may also make up a novel class of biomarkers. The basic premise would be that the functional status of extracellular mitochondria reflects intracellular metabolism, so regardless of release or reuptake mechanisms, they can be used to indirectly assess the underlying CNS metabolic integrity. A proof-of-principle study was performed in a small cohort of patients with subarachnoid hemorrhage (SAH).³¹ After SAH, extracellular particles containing mitochondria were detected in cerebrospinal fluid. Measurements of JC1 fluorescence ratios suggested that membrane potentials in CSF extracellular mitochondria were reduced after SAH, consistent with the presence of brain injury. Recovery of JC1 mitochondrial membrane potentials at day 3 after SAH was correlated with improved clinical outcomes at 3 months. If mitochondria are indeed released and exchanged between cells, they may allow one to eavesdrop on cellular metabolism.

Conclusions

Although it may still be too early for large clinical trials, follow-up translational studies may be envisioned. After cerebral ischemia, mitochondrial transfer appeared to take place from astrocytes into vulnerable neurons. Whether extracellular mitochondria can be released from and exchanged between other cell types should be examined. Initial studies suggest that mitochondrial transplant may protect and boost recovery in animal models of acute CNS injury. But whether these potential benefits are sustained for long-term neurological outcomes should be investigated. An initial proof-of-concept human study suggested that extracellular mitochondria are indeed detected in CSF from patients with SAH. Larger replication studies are warranted not only in SAH but also in other types of strokes and CNS trauma. Finally, perturbations in mitochondrial function and dynamics are also implicated in neurodegeneration. Therefore, it may be interesting ask whether extracellular mitochondria may also be detected in CSF in the context of Alzheimer disease, Parkinson disease, and other CNS disorders. Taken together, emerging findings in cellular, animal, and preliminary human studies suggest that mitochondria can be released and potentially transferred between cells. Further studies in both experimental and clinical settings are warranted to investigate and translate potential therapeutic and biomarker applications for these extracellular help-me signals in the CNS.