Extracellular mitochondria

Mitochondria may be found outside the cell in four ways:

1. as cell free mitochondrial DNA in the plasma
2. encased in platelets
3. in exosome membranes
4. totally free

The authors of this review discuss the use of mitochondria isolated from the cerebral spinal fluid from sub arachnoid hemorrhage patients. [1] The mitochondria membrane potential indicator dye JCI was used to assess the health of released mitochondria. The origin of the mitochondria was determined by use of surface specific antigens in the flow cytometry system. [1] Mitochondria, in vesicles or not, may be taken up by new host cells. The Miliotis review discussed use of cell free mitochondria as a therapy. [1]

Exosomes and oxidative stress

Tefani and coworkers (2016) published an excellent review on oxidative stress that touched briefly on mitochondria debris being exported into exosomes. [2] In their review of the literature, these authors stated that healthy cells do not secrete many exosomes. Stressed cells do. They also discussed exosomes containing reactive oxygen species such the super oxide anion.

The other image just states that exosomes may contain messenger RNA (mRNA), micro RNA (miRNA), DNA, lipids, proteins, and presumably active protein enzymes. Exosomes contain protein antigens on their cell surface that my be used to identify their cell of origin. Exosomes are not only secreted by one cell type but may be absorbed by different cell types via antigen presentation and adhesion molecules. This is some background information for presentation of work done by the Goetzl Laboratory.

The Goetzl Lab general protocol

These protocol use only 0.25 mL of blood plasma that is then incubated with 0.1 mL of thromboplastin D for 30 min at room temperature for 30 minutes.  A balance salt solution with protease and phosphatase inhibitors is added.  The mixture is centrifuged at 3000xg at 4^oC for 30 minutes.  The supernatant was removed and exosomes precipitated with ExoQuik from System Biosciences.  The total population of exosomal vesicles was resuspended in the balanced salt solution.  Neuron or astrocyte specific antibodies were added.  The primary antibodies may be conjugated with biotin. Biotin binds to streptavidin with extremely high affinity. The antibody labeled exosomes can be removed from the mix with streptavidin agarose beads.  The eluted material was transferred to a tube containing 10% BSA and 1M Tris pH 8 to get the pH closer to physiological. 

ELISAs were used for quantitation.

1. neuron or astrocyte derived exosomes in psychosis [3]

Goetzl and coworkers were tracking mitochondrial proteins in exosomes in major depression disorder patients at the baseline and after treatment with SSRI. This study is of interest to Mitosynergy because some of the customers take cuprous nicotinic acid for depression. This particular study was using SSRIs to treat some sort of self diagnosed depression. This particular study compared baseline values of responders and non-responders (NR) with the mitochondrial markers after treatment. (TR)

• Mitofusin 2 (MFN2) Involved in the clearance of damaged mitochondria via selective autophagy (mitophagy) (PubMed:23620051). Is required for PRKN recruitment to dysfunctional mitochondria (PubMed:23620051).
• Cyclophilin D (CYPD)has multiple functions in ghe mitochondria. Perhaps most notable is regulation of the mitochondrial transition permeability pore with its binding partner VDAC, the voltage dependent anion channel.
• Humanin is a 24 amino acid mitochondrial genome coded peptide. It prevents the formation of Abeta 42 amyloid products. Human also protects neurons from diverse challenges, suppresses apoptosis, preserves synaptic proteins, reduces neuroinflammation and regulates aspects of glucose metabolism.
• MOTS-c In response to metabolic stress, translocates to the nucleus where it binds to antioxidant response elements (ARE) present in the promoter regions of a number of genes and plays a role in regulating nuclear gene expression in an NFE2L2-dependent manner and increasing cellular resistance to metabolic stress (PubMed:29983246). Increases mitochondrial respiration and levels of CPT1A and cytokines IL1B, IL6, IL8, IL10 and TNF in senescent cells

The nicotinic acid two thirds of Cu(I)NA₂

The Goetzl group may have chosen these proteins as specific electron transport chain proteins. They are of particular interest to mitosynergy because they involve the nicotinic acid derivative NADH.

• NMNAT2 ATP + β-nicotinamide D-ribonucleotide + H⁺ = diphosphate + NAD⁺
• NDUV1 6^th unit of complex I ubiquinone + 5 H⁺(in) + NADH = ubiquinol + 4 H⁺(out) + NAD⁺
• UQCR11 10^th sub unit of complex III
• NAD⁺ hydrolase SARM1 acts as a negative regulator of MYD88- and TRIF-dependent toll-like receptor signaling pathway by promoting Wallerian degeneration, an injury-induced form of programmed subcellular death which involves degeneration of an axon distal to the injury site.

2. In Alzheimer’s disease [4]

Complex IV was immunocaptured and assayed for its ability to reduce cytochrome C using a commercial kit.  What is reported in this publication as ATP synthase activity is really Complex V operating in reverse as an ATPase.

Figure 2 of the Yao (2021) is a bit perplexing.   A n approximately 4x reduction in subunit 1 of complex I was seen in the Alzheimer’s group compared to the controls.  There was an only 2x fold reduction of subunit 6 of complex I in the Alzheimer’s groups.  Complex IV and V were observed to be about 50% less in the Alzheimer’s group compared to the control group.  One possibility is that sub unit 1 of complex I is selectively damaged in AD.  The authors did not mention if they detected the mitochondrial super oxide dismutase Mn SOD2. 

Two different cohorts were used for the ATPase assay.  The activity in the Alzheimer’s patients was less than half than the controls in the first cohort and 3-4x less In the second cohort.  Very little cytochrome C reductase activity was detected in the exosomes of Alzheimers’ patients in the second cohort.  The complex IV activity was undetectable in the Alzheimer’s patients even though protein was detected.  The Cu/Zn SOD1 activity was not assayed for in this study.  Both Cu/Zn SOD1 and complex IV use copper as a cofactor. It would be interesting to measure the copper content of these exosomes. Being from Alzheimer’s patients, we’d need to know if copper is complexed to proper enzymes or to amyloids.

3. In neuropsychiatric PASC [5]

The Goetzl group published a study comparing patients with PASC with and without neuro psychiatric (NP) symptoms.  Protein levels, but not enzyme activities, were published in this study. The truly incredible finding in this study was the presence of the Covid S1 spike protein receptor binding domain (RBD) and the nucleocapsid proteins.

The S1 spike protein immuno reactivity was about 3x greater in the asymptomatic former Covid patients compared to those that never had Covid.  Some background is to be expected in any immuno detection assay.  With the prospect of PASC having an autoimmune aspect needs to be considered in this system.  The S1 spike protein was increased in even asymptomatic former Covid patients in both the neuronal and the astrocyte derived exosomes.  The same spike protein was almost doubled in PASC patients with or without neuropsychiatric (NP) symptoms. 

The nucleocapsid protein (N) immunoreactivity was significantly greater in NDEV in asymptomatic former Covid patients compared to those that never had Covid, the difference was less than 2x.  In the astrocyte derived ADEV , nucleocapsid immunoreactivity became a predictor of neuropsychiatric symptoms. 

The significant changes in host mitochondrial proteins were seen in the astrocyte derived exosomes.

The mitochondrial Na⁺/Ca²⁺ exchanger was the only protein that was increased even 2x. 

Cool implications for Mitosynergy

The Goetzyl assay has a certain amount of simplistic beauty. Two unanswered questions are how these exosomes get past the blood brain barrier and whether FDA would accept this assay as a marker of efficacy. The latter may not be that important.

The MDD study that piggy backed on a clinical trial of effective and non effective SSRI was most interesting in that many of the enzymes analyzed had something to do with NAD⁺ pathways. Is some sort of Wallerian degeneration or other neurotoxicity associated with the etiology of major depression disease? Note: Mitosynergy and Charlie Barker’s patented improvement on cuprous nicotinic acid originated from his trip to Egypt for a treatment for his nephew’s spinal cord injury!

The Alzheimer’s Disease was truly incredible in that it examined the activity of complex IV and complex V going in reverse ATPase mode in exosomes. We’d like to see SOD1 activity measured. To the best of my knowledge, Alzheimer’s Disease is not an interest of Mitosynergy. Similar assays in spinal cord injury patients might be.

Mitosynergy is naturally interested in all aspect of Long Covid, now known as PASC. Astrosytes seem to be more effected. Is this due to their role as phagocytic role? Is seeing nucleo capsid protein in astrocyte exosomes a sign that they ore infected and/or that they are cleaning up debris from other infected cells? What markers to we want to see in healthy astrocytes that are doing their job versus reactive astrocytes?

References

1. Picca, A., Guerra, F., Calvani, R., Coelho-Junior, H. J., Bossola, M., Landi, F., Bernabei, R., Bucci, C., & Marzetti, E. (2020). Generation and Release of Mitochondrial-Derived Vesicles in Health, Aging and Disease. Journal of clinical medicine, 9(5), 1440. free paper PMC free article
2. Tafani, M., Sansone, L., Limana, F., Arcangeli, T., De Santis, E., Polese, M., Fini, M., & Russo, M. A. (2016). The Interplay of Reactive Oxygen Species, Hypoxia, Inflammation, and Sirtuins in Cancer Initiation and Progression. Oxidative medicine and cellular longevity, 2016, 3907147. PMC free article
3. Goetzl EJ, Wolkowitz OM, Srihari VH, Reus VI, Goetzl L, Kapogiannis D, Heninger GR, Mellon SH. Abnormal levels of mitochondrial proteins in plasma neuronal extracellular vesicles in major depressive disorder. Mol Psychiatry. 2021 Dec;26(12):7355-7362. PMC free article
4. Yao PJ, Eren E, Goetzl EJ, Kapogiannis D. (2021) Mitochondrial Electron Transport Chain Protein Abnormalities Detected in Plasma Extracellular Vesicles in Alzheimer’s Disease. Biomedicines. 2021 Oct 31;9(11):1587. doi: 10.3390/biomedicines9111587. PMC free article
5. Peluso MJ, Deeks SG, Mustapic M, Kapogiannis D, Henrich TJ, Lu S, Goldberg SA, Hoh R, Chen J, Martinez EO, Kelly JD, Martin JN, Goetzl EJ. (2022) SARS-CoV-2 and mitochondrial proteins in neural-derived exosomes of COVID-19. Ann Neurol. 2022 Mar 13 PMC free article