Category: aging

Melanocyte, the organelles that produce the skin and hair pigment melanin, require ATP generating mitochondria for their maturation. Mitofusin (Mfn) 2, the protein that allows mitochondria to fuse to the endoplasmic reticulum, is also found in the melanosome-mitochondrion contacts according to this 2014 electron microscope study. [1] The featured image of this post illustrates this fusion. These mitochondria contacts were found to be associated to the melanogenesis process. This study also demonstrated that pharmacological inhibition of mitochondrial ATP synthesis reduced not only the contact formation and impairs melanosome biogenesis. [1] The same year Wu and Hammer published a nice lay summary of this discovery. [2] The featured image is a truncated version of their Figure 1. [2]

Just as Wu and Hammer [2] published a short introduction to the Daniele 2014 TEM study [1] Philpott [3] gave a few interesting introductory insights into a new technique of watching hair turn gray [5].

Hair follicles are the “mini-organs” that produce hair shafts. The three stages are as follows:

1. Anagen, a period of active growth. This stage requires a lot of ATP and functioning mitochondria. It is during this phase that melanin production is turned on and off in a more plastic fashion than previously thought, [4,5]
2. Catagen, a period of regression
3. Telogen, a period of rest,

This image is a combination of a stock photo and the anatomy of a hair follicle from the Cruz review that we will com back to. [4] According to Philpott’s review of the literature, this cycle can last for as little as three months in eyebrow hair or as long as several years for scalp hair. [3]

Recent advances in hair follicle imaging and correlation of proteins therein have left us with the imagery that hair follicles are like tree rings with snap shots with what we might have been experiencing at the time.[4] Conventional wisdom states that stress can cause pigmented hair to turn white. White hairs can also regain their pigmentation. [4,3]

The proteomics of white and dark hair shafts

The Rosenberg study utilized two different protomics techniques in two different laboratories. Both techniques used 1 cm lengths of hair follicles. Both used the proteolytic enzyme trypsin to digest the hair proteins into peptides that were “sequenced” with two different mass spectrometry techniques. These sequences were used to search databases in order to identify the proteins within the hair samples.

Experiment 1: matched dark and white hairs collected at the same time from two closely age- and diet-matched individuals (one female and one male, both 35 years old, each dark and white HS measured twice, total n = 8)
Experiment 2 (validation): n = 17 hair segments from seven different individuals (four females and three males).

These figures are “Volcano Plots” that give us snap shots of significant changes in proteins. The higher the position of the dot, the more likely the protein is to be significantly more or less in white versus dark hairs. Note that we have a lot of mitochondria proteins (red dots) on the right hand “more in white hairs” side of the plots.

The next step is to look for proteins that are up and down regulated in both of these two rather diverse experiments.

1. CPT1A, Carnitine O-palmitoyltransferase catalyzes the transfer of the acyl group of long-chain fatty acid-CoA conjugates onto carnitine, an essential step for the mitochondrial uptake of long-chain fatty acids and their subsequent beta-oxidation in the mitochondrion
2. ACOT7 Cytosolic acyl coenzyme A thioester hydrolase catalyzes the hydrolysis of acyl-CoAs into free fatty acids and coenzyme A (CoASH), regulating their respective intracellular levels.
3. SOD1 Superoxide dismutase [Cu-Zn] Destroys (super oxide) radicals which are normally produced within the cells and which are toxic to biological systems.
4. CFL1 Cofilin binds to F-actin and exhibits pH-sensitive F-actin depolymerizing activity.
5. PGK1 phosphoglycerate kinase catalyzes one of the two ATP producing reactions in the glycolytic pathway via the reversible conversion of 1,3-diphosphoglycerate to 3-phosphoglycerate.
6. KRTAP3-1 Keratin associated protein 3-1…In the hair cortex, hair keratin intermediate filaments are embedded in an interfilamentous matrix, consisting of hair keratin-associated proteins (KRTAP), which are essential for the formation of a rigid and resistant hair shaft through their extensive disulfide bond cross-linking with abundant cysteine residues of hair keratins. The matrix proteins include the high-sulfur and high-glycine-tyrosine keratins.
7. KRTAP4-3 Keratin associated protein 4-3…In the hair cortex, hair keratin intermediate filaments are embedded in an interfilamentous matrix, consisting of hair keratin-associated proteins (KRTAP), which are essential for the formation of a rigid and resistant hair shaft through their extensive disulfide bond cross-linking with abundant cysteine residues of hair keratins. The matrix proteins include the high-sulfur and high-glycine-tyrosine keratins.

Wait, this makes no sense!

You may be asking yourself why increased levels of Cu/Zn SOD1, generally considered a good thing, be associated with hair going white, generally considered a bad thing. The answer may be found in the Wiriyasermkul 2020 review on ion transporters in melanosomes. [6]

These are some images from the Wiriyasermkul 2020 review.

UniProt as some additional things to say about these copper and zinc enzymes.

• Tyrosinase: “In addition to hydroxylating tyrosine to DOPA (3,4-dihydroxyphenylalanine), also catalyzes the oxidation of DOPA to DOPA-quinone, and possibly the oxidation of DHI (5,6-dihydroxyindole) to indole-5,6 quinone (PubMed:28661582).
• L-dopachrome tautomerase TYRP2.:..”Plays a role in melanin biosynthesis (PubMed:33100333). Catalyzes the conversion of L-dopachrome into 5,6-dihydroxyindole-2-carboxylic acid (DHICA)”

The following are screen shots from the UniProt.org website illustrating the requirement of Cu and Zn for TYR and TYRP2, respectively. Both proteins contain two Cu/Zn per subunit. The Zn vs. Cu requirement of TYP1 is a little more ambiguous. The interesting thing is that mutations in both the TYR and TYRP2 genes are associated with albinism.

Hypothesis: how Cu cures white hair

Melanin production enzymes require Cu and Zn. SOD1 also requires Cu and Zn to function. Perhaps, in hair follicles, under conditions of stress, more SOD1 is produced that competes with TYR and DCT for Cu and Zn, respectively. That Cu supplementation cures white hair might simply be a matter of relative availability of Cu versus Zn.

We don’t know why stress that is associated with white hair [4] might increase certain mitochondria proteins. The Cruz review on hair have very little to say about the keratin associated proteins. [5] The Cruz review does mention growth factors, cytokines, hormones, and neurotransmitters as factors controlling hair cycling. [5] Surely these factors have the potential to also control the expression of the proteins in the Rosenberg study. [4] More mitochondria proteins and Cu/Zn SOD1 could be argued to be a good thing during times of stress that produce white hair. Do we also argue that the aging process is itself a source of stress? During these high stress times, could supplying Cu and/or Zn to Cu/Zn SOD1 reduce the Cu and/or Zn getting to the enzymes responsible for melanin synthesis?

References

1. Daniele T, Hurbain I, Vago R, Casari G, Raposo G, Tacchetti C, Schiaffino MV. Mitochondria and melanosomes establish physical contacts modulated by Mfn2 and involved in organelle biogenesis. Curr Biol. 2014 Feb 17;24(4):393-403. free article
2. Wu X, Hammer JA. Organelle interactions: melanosomes and mitochondria get cozy. Curr Biol. 2014 Mar 17;24(6):R240-2. free article
3. Philpott MP. Watching hair turn grey. Elife. 2021 Jun 30;10:e70584. PMC free article
4. Rosenberg AM, Rausser S, Ren J, Mosharov EV, Sturm G, Ogden RT, Patel P, Kumar Soni R, Lacefield C, Tobin DJ, Paus R, Picard M. Quantitative mapping of human hair greying and reversal in relation to life stress. Elife. 2021 Jun 22;10:e67437. PMC free article
5. Cruz CF, Costa C, Gomes AC, Matamá T, Cavaco-Paulo A. Human Hair and the Impact of Cosmetic Procedures: A Review on Cleansing and Shape-Modulating Cosmetics. Cosmetics. 2016; 3(3):26. free article
6. Wiriyasermkul P, Moriyama S, Nagamori S. Membrane transport proteins in melanosomes: Regulation of ions for pigmentation. Biochim Biophys Acta Biomembr. 2020 Dec 1;1862(12):183318. PMC free article

We tend to accept that our cancer risks increase as we age. In introductory remarks on an exciting new landmark paper Li and coauthors were commenting on the fate of poor CD8+ T cells in the center of tumor micro environment (TME). CD8+ cytotoxic T cells are antigen specific recognizing antigens from tumors, virus infected cells, and normal cell. CD8+ is a protein that binds to the constant chain of the MHC1 antigen presenting surface protein. Recognizing normal cells is considered an act of autoimmunity. When exposed to infected/dysfunctional somatic cells, CD8+ T cells release the cytotoxins perforin, granzymes, and granulysin, enzymes that activate the caspase protease cascade eventually leading to programmed cell death. Human Cell Bio. Not only are the T cells constantly exposed to antigen that sets them off, but they are also competing with the tumor for oxygen. [1] Naturally reactive oxygen species are part of the TME. [2] According to Healthline, age is not one of the risk factors for autoimmune diseases. In autoimmunity CD8+ T cells are also constantly exposed to self antigen. [3] It is desirable that they become exhausted and less self- reactive. Both of these featured studies used T cells from mice that are allergic to an egg protein ovalbumin. These dual roles of CD8+ T cells was addressed in a review by Collier and coauthors. [4]. This post will not get into the PD1/PDL1 checkpoint inhibitors that have been discussed elsewhere on this site. We have covered the inner-relationship between NADH/NADPH (derived from niacin) and reduced thiols. This post examines the possibility that cuprous niacin could find the happy middle ground between T cell exhaustion in cancer and viral infections and autoimmunity, both of these may be issues as we age. Perhaps age is not an autoimmune disease risk factor, but surely contributes to the symptoms.

1. Tales from the tumor micro environment [2]
   1. Figure 1, an increase in glycolysis
   2. Fig 2 less O2 reduction and ATP production with chronic stimulation
   3. Figure 3 mitochondria inhibition and T cell proliferation (not shown)
   4. reactive oxygen species and reduced thiol supplementation
2. Nicotinamide slowing down CD8+OT-I T cells? [3]
   1. Figures 1 and 2 Turning on cytokine production, or not…
   2. Figure 3 Some Sea Horse experiments
3. Concluding thoughts
4. References

Tales from the tumor micro environment [2]

Polyclonal CD8⁺ T cells, T cells were isolated from the spleen and inguinal lymph nodes of C57BL/6 mice. T cells were also isolated from OT-I mice. These mice contain transgenic inserts for mouse Tcra-V2 and Tcrb-V5 genes. The transgenic T cell receptor was designed to recognize ovalbumin peptide residues 257-264 (OVA_257-264) in the context of H2K^b (CD8 co-receptor interaction with MHC class I). The take home is that chronically stimulated T cells, by tumor antigens or ovalbumin, leads to impaired mitochondrial function and more reactive oxygen species generation. Both copper and niacin have the potential to improve this situation.

This results in MHC class I-restricted, ovalbumin specific, CD8+ T cells (OT-I cells). That is, the CD8 T cells of this mouse primarily recognize OVA_257-264 when presented by the MHC I molecule. Cells were cultured in the presence of 1 μM of SIINFEKL peptide, a peptide from ovalbumin. The melanoma cells supplied the MHC class I to present the transgenic T cells with their ovalbumin peptide. Vardhana used B16-F10, B16-OVA, EL4, and A20 meleanoma cell lines. An interesting nuance is that they added 50 μM of the reducing agent β-mercaptoethanol (β-ME).

Figure 1, an increase in glycolysis

In the melanoma cells, 1 ng/mL IFN-γ was to induce MHC-I expression. IL-2, 10 ng/mL, was also added with (chronic) or without (acute) 1 μM SIINFEKL. T cells were passaged into fresh co-cultures every 48 h until eight days following initial stimulation with the peptide.

• In Figure 1a we see the same story: The more PD-1 on the cell surface, the less TNF in vesicles. It is the chronically stimulated cells that have the most PD-1. Skipping some flow cytometry data….
• Panel d, The chronically stimulated T cells consume much more glucose than the T cells only stimulated once with the ovalbumin peptide. This glucose could be for glycolysis only or glycolysis plus OxPhos.
• Panel e shows an increase in lactate production indicating that glucose consumption is probably not going towards OxPhos.
• Panel f demonstrates that the extracellular acidification rate (ECAR) is increased when both the chronic and the acute stimulated T cells are stimulated with CD3 antibodies. The acute stimulation cells acidify more in the presence of electron transport chain blockers. And finally,
• panel g, the acute stimulation T cells divide almost 4x as fast as those chronically stimulated with the ovalbumin peptide .

Fig 2 less O₂ reduction and ATP production with chronic stimulation

Vardhala also used radioactive glucose to measure TCA cycle activity in these T cells (panels d-f not shown).. Some of the nucleotide ratios are not being shown in this post in effort to keep the narrative simple. It is just impossible to do this public access publication justice in a single post.

They saw a decrease in all intermediates in the TCA cycle. [2] Triphosphate and monophosphate nucleotide ratios were also reduced in the chronic stimulated T cells compared to the one time only acute stimulated cells. [2] Panel i from the chronic model documents that he problem gets worse with time.

Figure 3 mitochondria inhibition and T cell proliferation (not shown)

Cu(I)NA₂ is predicted to supply Cu/Zn SOD with the necessary cofactor to mitigate the ROS generation as well as keep the flow of electrons through through the electron transport chain. Both of these should increase the ATP/AMP ratio as well as decrease the NADH/NAD⁺ ratios.

• Panel a Mitotracker green is simply a fluorescent indicator there to measure mitochondria size irrespective of activity. These mitochondria might be undergoing a vain attempt to expand and keep up with demand.
• panel b This is a Western blot. The black bands are proteins. There does not appear to b a huge difference in protein amounts of various complex proteins.
• panel c This is something we might want to copy. A smaller NADH/NAD⁺ ratio in the acute T cells that were not over exposed to peptide suggest better functioning mitochondria.
• Panel d examines the total and mitochondrial reactive oxygen species upon stimulation. The gray shaded trace is the unstimulated reference.
• Panel e Various inhibitors of electron transport chain complexes were used to mimic he effect of chronic stimulation: complex I (rotenone), complex V (oligomycin),Fe-S cluster containing complexes, cobalt chloride (CoCl₂),

reactive oxygen species and reduced thiol supplementation

The rest of the Vardhana publication addressed the issue that the real root of reduced T cell proliferation was due to ROS generation rather than impaired mitochondria activity.

Nicotinamide slowing down CD8+OT-I T cells? [3]

The next paper tests the hypothesis that nicotinamide can shut down over responsive CD8+ T effector cells. [3] Just as a warning, this is the exact opposite of T cell exhaustion. The paper makes use of the OT-I mice genetically engineered to think that the ovalbumin egg protein is a virus. These authors introduced us to the mammalian target of Rapamycin, or mTOR. mTOR integrates all sorts of signals from everywhere and gives the cell permission to start translating mRNAs into proteins. Ribosomal S6 kinase is a target of mTOR. This kinase promotes translation of mRNAs. Note that increases in the ratios of ADP to ATP tend to turn mTOR off and sufficient nutrients tend to turn it on. These authors used the Sea Horse to measure extracellular acidification rate, i.e. glycolysis without the TCA cycle and electron transport chain. [3]

Figures 1 and 2 Turning on cytokine production, or not…

Naïve CD3⁺, CD4⁺, CD8⁺ T cells from spleen and lymph nodes from C57BL/6J mice were purified and differentiated using a commercial kit with added 30 mM or 10 mM NAM or the solution used to dissolve the NAM. Final activation was with CD3/CD28 or PMA + ionomicyn (PMAi), a combo used to stimulate T cell activation, proliferation, and cytokine production. The authors saw differences in cytokine production, but by their own admission, used an surrealistically high concentration of NAM in their cell culture experiments. [3] Not getting into the specifics, the reason has to do with mTOR.

Figure 3 Some Sea Horse experiments

Let us continue with the Agliano [3] publication, which also used T cells from OT-I mice. Recall that these cells were engineered to react with the ovalbumin peptide provided that it is presented via MHC1 surface proteins.

• Panel A the general protocol. They are activating the T cells with the peptide, phorbol ester, or a bunch of cytokines.
• Panels Band C Nicotinamide only slows down production of INFγ in response to the peptide, but not the phorbol ester plus ionomycin Ca²⁺ ionophore. PMA activates protein kinase C. ionomycin pokes Ca²⁺ permeable holes in cell membranes. This is sort of a double whammy way of activating T cells. Panel D examined response to interleukins.
• Panel E. Here Angliano and coauthors are comparing 10 mM nicotinamide (NAM) and the solution (vehicle) used to dissolve NAM. When glucose (gluc) is added the cell culture medium starts acidifying. Adding the ATP synthase inhibitor oligomysin (Olig.) does nothing. At 122 g per mole, an average human male with 5 liters of blood would have to consume about 6.1 g of nicotinamide to theoretically achieve this concentration.
• Panel F The large extracellular concentrations of nicotinamide are increasing the intracellular concentrations of NAD⁺.
• Panel G used only 1 mM NAD⁺. Consuming 0.6 g of NAD⁺ probably won’t happen either.
• Panel H, FK866 is a highly specific noncompetitive inhibitor of nicotinamide phospho ribosyltransferase . The authors presented another narrative suggesting that the large doses of niotininamide were slowing the translation of cytokine transcripts via phospho-AMP kinase pathways involving mTOR. [3]

Figure 4 demonstrates that NAM down regulates mTORC1 independently of NAD⁺. [3] Figures 5 and 6 looked at the effect of NAM on human T effector cells. This post is not going to get into this part of the study.

Concluding thoughts

The Vardhana paper made the cause for CD8+ T cell exhaustion being tied to reactive oxygen species and proposed use of reduced thiol supplements like N-acetyl cysteine. [2] We’d like to point out that (1) copper is a cofactor for Cu/Zn superoxide dismutase and (2) niacin is a precursor for NADH/NADPH. NADPH, in particular, plays a role in keeping thiols reduced. The Agliano paper made the case niacin’s close relative nicotinamide, being a good treatment autoimmune diseases when CD8+ T cells become cytotoxic against self, i.e. autoimmunity. [3] This site is in not way making any claims to treat disease. That cuprous niacin could treat two opposite ends of immune dysregulation would be an audacious claim and perhaps a worthy hypothesis to test!

References

1. Li W, Cheng H, Li G, Zhang L. Mitochondrial Damage and the Road to Exhaustion. Cell Metab. 2020 Dec 1;32(6):905-907. free article
2. Vardhana, S. A., Hwee, M. A., Berisa, M., Wells, D. K., Yost, K. E., King, B., Smith, M., Herrera, P. S., Chang, H. Y., Satpathy, A. T., van den Brink, M., Cross, J. R., & Thompson, C. B. (2020). Impaired mitochondrial oxidative phosphorylation limits the self-renewal of T cells exposed to persistent antigen. Nature immunology, 21(9), 1022–1033. PMC free article
3. Agliano, Federica et al. “Nicotinamide breaks effector CD8 T cell responses by targeting mTOR signaling.” iScience vol. 25,3 103932. 15 Feb. 2022, free article
4. Collier JL, Weiss SA, Pauken KE, Sen DR, Sharpe AH. Not-so-opposite ends of the spectrum: CD8⁺ T cell dysfunction across chronic infection, cancer and autoimmunity. Nat Immunol. 2021 Jul;22(7):809-819. PMC free article