Proteins in exosomes

Ten years ago miRNA was the hot cargo of exosomes. This image came from the review of Tefani and coauthors (2016) [1] The main point is that exosomes contain bits distressed mitochondria, cell membranes and membrane proteins of the cell of origin, DNA, mRNA, and miRNA. The contents of phagosomes is another interesting possibility.

2. The Goetzl Lab protocol, easy and inexpensive [2]

1. 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.  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 is removed and exosomes precipitated with ExoQuik from System Biosciences.  The total population of exosomal vesicles is resuspended in the balanced salt solution. 
2. Neuron or astrocyte specific antibodies can be added to the mixture.  The primary antibodies are labeled with biotin. Biotin binds to streptavidin with extremely high affinity. The antibody labeled exosomes are removed from other exosomes by use of streptavidin agarose beads.  
3. The exosomes are eluted from the antibodies by use of a pH 3 glycine solution. The eluted material can be transferred to a tube containing 10% BSA and 1M Tris pH 8 to get the pH closer to physiological. 
4. The eluted material is transferred to the bottom of a microtiter plate. An enzyme linked immuno sorption is used to detect proteins of interest. Conversely, a tryptic digest could be performed to detect proteins using mass spectrometry.

3. Brain exosomes in major depressive disorder (MDD) [3]

Selective serotonin reuptake inhibitors (SSRI) are used to treat depression. Goetzl and coworkers piggy backed on a clinical trial of multiple SSRI. Some worked. Some did not. [3] 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 the 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 neuro inflammation and regulates aspects of glucose metabolism.
• MOTS-c In response to metabolic stress, MOTs 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

Brain exosomes: Covid-19 is a chronic infection

Post Actute Sequelae of Covid-19 (PASC) is the new term for “Long Covid.” Some think that PASC is a chronic infection. 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 in brain specific exosomes.[3]

All data in the above table were divided by the average signal from the matched control group.

Source of EVs	SARS-CoV2	Control	Covid no PASC	PASC w/out NP	PASC w/NP	PASC w/severe NP
NDEVs	S1	1	3	4.3	6	5
	N	1	12	16	29	25
ADEVs	S1	1	4.4	8.7	9	8
	N	1	23	33	48	46

There are a few things that become more evident when looking at approximate ratio changes

1. The change in nucleocapsid (N) is greater than that of the spike protein (S1) in going from “Covid no PASC” to “PASC w/NP”.
2. Compared to controls, astrocyte EV contained more Covid protein than did the neuron EV.
3. The nucleocapsid, on a very qualitative level, seems to predict neuro-PASC better than the spike protein.

Are astrocytes more likely to become infected with Covid-19 or simply have more of the proteins because they have phagocytic potential? If this is the case, are we also looking at proteolytic fragments of these vesicles?

Exosomes and the intestinal microflora

In 2016 Liu and coworkers isolated exosomes from mice and human feces, and intestinal contents from various regions of the mouse gut. [4] miRNA binds to the 3′ untranslated portion of messenger RNA (mRNA) in such a way as to prevent translation of mRNAQ into protein. Liu et al found miRNA that could inhibit the growth of some bacteria. Total RNA from feces was isolated using the MirVana isolation kit. [4] Liu used a strain of mutant mice that could not produce miRNA. These animals had altered GI microflora. Intestinal exosomes released into the lumen were collected from germ free animals.

Note the size of the gut exosomes. This might have implications for health care providers considering the use of fecal filtrate transplantation to treat gastroinestinal conditions and even autism. The major goal of this procedure is to isolate bacteriophage from healthy donors. The donors are hypothesized to have a beneficial flrora because bacteriophage are killing off the “bad” bacteria. These protocols usually call for relatively low speed centrifugation to pellet most of the bacteria. The bacteriophage are separated by remaining bacteria and other debris by filtration through a 0.2 or 0.45μm filter. (There are 1000nm in 1 μm) Perhaps the slightly larger 0.45μm would be better because it would spare a larger portion of the exosomes released into the GI tract. Chiaporri and coworkers (2022) took this concept to compare small, non cellular RNA in feces of neurotyipical and autistic children. [5] These authors merely isolated total RNA and focused on characterizing mRNA translation inhibiting miRNA and gene silencing piRNA. These data were selected from the text comparing two matched siblings: one with autism (ASV) and a control neurotypical (CSV)

This post is not showing piRNA and changes in the mycobiome (fungi) in the Chiappori report [5]. The fecal filtrate might contain more than just bacteriophage. These exosomes might contain protein in addition to the miRNA. Right now, this might be a distraction from making autistic kids better.

References

1. 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
2. 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
3. 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
4. Liu, S., da Cunha, A. P., Rezende, R. M., Cialic, R., Wei, Z., Bry, L., Comstock, L. E., Gandhi, R., & Weiner, H. L. (2016). The Host Shapes the Gut Microbiota via Fecal MicroRNA. Cell host & microbe, 19(1), 32–43. PMC free article
5. Chiappori, F., Cupaioli, F. A., Consiglio, A., Di Nanni, N., Mosca, E., Licciulli, V. F., & Mezzelani, A. (2022). Analysis of Faecal Microbiota and Small ncRNAs in Autism: Detection of miRNAs and piRNAs with Possible Implications in Host-Gut Microbiota Cross-Talk. Nutrients, 14(7), 1340. PMC free paper
7. Gonzales, Patricia A et al. “Large-scale proteomics and phosphoproteomics of urinary exosomes.” Journal of the American Society of Nephrology : JASN vol. 20,2 (2009): 363-79. PMC free article
8. Erdbrügger, U., Blijdorp, C. J., Bijnsdorp, I. V., Borràs, F. E., Burger, D., Bussolati, B., Byrd, J. B., Clayton, A., Dear, J. W., Falcón-Pérez, J. M., Grange, C., Hill, A. F., Holthöfer, H., Hoorn, E. J., Jenster, G., Jimenez, C. R., Junker, K., Klein, J., Knepper, M. A., Koritzinsky, E. H., … Martens-Uzunova, E. S. (2021). Urinary extracellular vesicles: A position paper by the Urine Task Force of the International Society for Extracellular Vesicles. Journal of extracellular vesicles, 10(7), e12093. PMC free article