Further thoughts from the world of independent researchers

BSRA 2025 Poster - aka "still eating lots of citrate"

This contains the details of the poster being displayed at BSRA 2025 in Manchester. Unlike the poster it also includes links to papers which are implicitly referred to in the poster itself.



Abstract
Continuing experimentation in the world of independent research raises questions about Rapamycin, the role of menaquinones in mitochondrial function, whether Parkinson's Disease and ALS/MND should be considered diseases of aging, what the role of transitions, transversions and deletions are in terms of the effects of mtDNA damage on gene expression and whether follicular atresia should be considered as part of the aging process.

Introduction
There always have been independent researchers. One effect of the internet is to enable people with a rarer common interest to get in contact with each other and discuss things. This process has now been going on for some time such that the lifecycle of such systems has encountered its own challenges. For example more recently the Calorie Restriction Society Forum (http://crsociety.org/ - CR was being used for longevity reasons) encountered technical challenges because of the deaths of some of its original founders.

The author participates in a number of these including Rapamycin News and the Gerontology Research Group. At times original research is done jointly by participants such as some joint sponsoring of worm experiments by Rapamycin users and a member of the GRG Professor John Cramer has agreed to undergo the first human experiments by Mitrix Bio in mitochondrial transplantation.

Genomic Failure Hypothesis
The Genomic Failure Hypothesis is that the genome fails to produce the necessary proteins. This is because of a mixture of two factors driving citrate efflux from mitochondria:
  1. The mitochondrial membrane potential, which drives pyruvate intake and sits at the top of the TCA cycle - this is the primary issue
  2. The level of expression of the citrate carrier which is affected by Interleukin-10 in SASP through the Janus Kinase
Because the level of nuclear acetyl-CoA depends substantially on citrate efflux this depends on these factors. This drives the acetylation of various proteins most importantly the histones and splicing factors, but also other proteins.
Aging is an Extension of Development
Although one aspect of changed acetylation levels is the failure to express long genes, another aspect is a variation in the splices produced as part of the splicing process. Development may be a response to hormones, but the switches to turn on hormones arise from changed splicing.
Alternative Splicing Dynamics of the Hypothalamus Pituitary Ovary Axis During Pubertal Transition in Gilts
Two overall categories of diseases of aging
It is possible to divide diseases of aging into two categories. One is caused by stem cells failing to differentiate. Osteoporosis is a good example of this with the osteoblasts failing to be created in sufficient numbers whilst the osteoclasts continue to be created and remove bone. Similarly acinar cells are replaced in the pancreas by adipocytes. Another is as a result of splicing changes such as the failure to maintain elastin and keep the lens of the eye clear.
Infographic about the types of mtDNA mutations


Factors affecting citrate efflux
The most important factor is the amount of citrate which is a result of pyruvate influx and the rate of conversion of acetyl-CoA to Citrate. Because the citrate carrier (SLC25A1) exchanges singly protonated citrate with a double negative charge with malate the balance of malate in the cytosol can affect transfer rates as well as the balance of citrate in the mitochondrial matrix. The next step in the TCA cycle after the production of citrate by Citrate synthase is the conversion of Citrate to Isocitrate by Aconitase. This uses the triply negatively charged ion of citrate. Hence the pH in the mitochondrial matrix is also a factor which drives the balance between citrate leaving via the citrate carrier and continuing round in the cycle. The balance between the different ionic forms of citrate can be calculated and at the normal pH of the matrix (7.6) the vast majority of citrate is in a form of the Citrate 3-. Just under 6% of citrate will be 2- and very small amounts in different ionic form. This does mean, however, that as the pH of the matrix varies the citrate efflux compared to the citrate continuing round the TCA cycle will also vary.
Palmieri EM, et al. Acetylation of human mitochondrial citrate carrier modulates mitochondrial citrate/malate exchange activity. Biochim Biophys Acta. 2015.. (Supports citrate–malate exchange and regulation.)
Chaouch A, et al. Mutations in the mitochondrial citrate carrier SLC25A1 Hum Mol Genet. 2014.. (Describes SLC25A1 function: citrate/isocitrate export in exchange for malate.)
Wiederkehr A, et al. A novel mitochondrial signal for sustained pancreatic beta-cell activity. J Biol Chem. 2009. . (Reports matrix pH values ~7.6, illustrating physiological range near 7.6.)
Poburko D, et al. Dynamic Regulation of the Mitochondrial Proton Gradient. J Biol Chem. 2011. . (Review on control of mitochondrial ΔpH and matrix pH.)
Palmieri F. The mitochondrial transporter family SLC25: identification, properties and physiopathology. Mol Aspects Med. 2013.
PreLights summary: SLC25A1 dosage regulates metabolic reprogramming and morphogenesis. 2023.
Chromatin remodeling due to degradation of citrate carrier impairs osteogenesis of aged mesenchymal stem cells. Nat Aging. 2021.
Mitochondrial Membrane Potential is the key variant
The mitochondrial membrane potential, however, is the key variant which limits the peak rate of citrate efflux. This is affected by the ATP/O efficiency of the mitochondria. When functioning normally this will depend on how well the electron transport chain is functioning. It appears that evolution has determined to expose only limited numbers of the subunits of the ETC to the risk of mitochondrial DNA damage. Complex II is entirely coded by the nucleus. Complex I has 7/45 subunits encoded in the mitochondrion, complex III 1/11 and complex IV 3/13. ATP synthase (Complex V) has 2/16.
Transitions, Transversions and Deletions have their role to play
Mitochondrial DNA (mtDNA) is particularly vulnerable to ROS from the Electron Transport Chain because it has no histone and is close to the ETC. There are three different general types of mutation that get through the relatively weak repair systems. Transitions are where one of the pyrimidines is swapped for the other. Interestingly it is thought that the majority of transitions occur because of replication errors by DNA Polymerase Gamma when the mtDNA is being copied. There is a minority view that a minority of transitions occur from Oxidative Stress, but there is a consensus that the majority of transitions are not caused by Oxidative Stress. Transversions (where a pyrimidine is swapped for a purine) and deletions are thought to arise from ROS.
What effect does this have on efficiency
There are a wide range of consequences that can arise from a mutation. Interestingly the most common deletion which takes out 4977 base pairs and removes the totality of six OxPhos genes and 5 tRNAs only has an effect on energy generation at a point when it has a high level of heteroplasmy. From a mechanistic perspective this is not surprising as deleting genes will not mean the wrong proteins are produced, but will mean that at a point a protein's homeostasis fails and the protein is not available. The author has not been able to find any research as to the impact of any specific individual transition or transversion on the electron transport chain, but there is plenty of research looking at well known mutations and it is known that they impact on mitochondrial efficiency. From a mechanistic perspective changing the amino acids in a location in the protein would be expected to have an effect. It might prevent the protein from folding properly. On the other hand the protein is likely to still be able to perform its function. It could be less efficient (which is more likely) or it even could create a more efficient ETC. Studies in the 2000s with mice with a mutation to DNA Polymerase Gamma which caused replication issues found that homozygous mice showed the signs of progeria, but the situation with heterozygous mice was not statistically clear. The vast majority of the mutations in this instance are transitions, there are small numbers of transversions, a few small deletions and no larger deletions. One can conclude from this that transitions don't have that much of an effect on mitochondrial function, but they do have some effect.
The exponential growth in deletions seen in human aging over around 65 is potentially caused by a mixture of the replication advantage of substantial deletions, failures of mitophagy and a reduction in pineal melatonin.

Infographic about the types of mtDNA mutations

Point mutations and the mutator mice
Vermulst et Al's paper from 2006 which studied mutation quantification in the heterozygous and homozygous mutator mice warrants further comment. Although they did not study lifespan analysis this paper reported median lifespan estimates from previous papers (Trifunovic et al., 2004; Kujoth et al., 2005) where wildtype mice survived 864 days, homozygous mice survived 423 days and heterozygous mice survived 758 days. So there was an estimated 41% reduction in lifespan for homozygous and an estimated 12% reduction in lifespan for heterozygous. Although accurately reported in the paper that no statistically significant difference was detected (p=0.875) between heterozygous and wild type mice, the paper has been considered elsewhere as it concluded there was no difference rather than it did not find a difference. What was also found by Vermulst was that young heterozygous mice had a higher level of mutations than old wild type mice. From this it could be hypothesised that transitions (the main replication error) appear to have much less effect, perhaps an order of magnitude less, on the efficiency of the mitochondria than deletions and transversions (the main errors from ROS). They recognise in their paper that their methods would not detect large mtDNA deletions although they would detect small mtDNA deletions. Hence their paper does not conflict with the thesis that the mutations caused by the replication errors in DNA Polymerase Gamma cause a lifespan reduction in both the heterozygous and homozygous mutator mice compared to wild type, but this has a significantly less effect than mutations caused by ROS rather than replication errors. Importantly their paper does not conflict with the thesis that the main cause of aging is mitochondrial mutations caused by ROS rather than replication errors, but that replication errors do have a minor effect on aging.

Yusoff AAM, et al. A comprehensive overview of mitochondrial DNA 4977-bp deletion. Mitochondrial DNA A. 2019. . (Details the "common deletion" removing OxPhos genes and tRNAs.)
Nanopore sequencing identifies a higher frequency and expanded spectrum of mitochondrial DNA deletion mutations in human aging
Species Variations
An important point, however, is that although different species have different lifespans the rules of chemistry and physics are the same. Hence it is entirely possible that the human metabolism is better at clearing deletions than mice, but as humans live for a lot longer the effect of transitions and transversions becomes important. It is clear from the cycle that germline mtDNA follows that the body makes a lot of efforts to protect mtDNA and the quality control system rejected more than 99.99% of Oocytes as having insufficiently good nucDNA and mtDNA. Hence we should not ignore completely transitions and transversions. (Transversions are likely (on average) to be more damaging than transitions)
Mitochondrial point mutations do not limit the natural lifespan of mice
Mitochondrial DNA Damage Patterns and Aging: Revising the Evidences for Humans and Mice
Summary of hypotheses to look at next points
Aging and Development occurs through a change to gene expression caused by changes in acetylation levels, primarily as a result of a reduction in mitochondrial efficiency, but also affected by senescent cells producing SASP which acts on the same acetyl-CoA pathway. An important point, however, is that some changes to mtDNA occur merely from replication and do not require ROS.
How does ROS get to the mtDNA
ROS is primarily created by Complexes I and III. Hence ordinarily ROS will come from the ETC. With higher partial pressures of O2 and higher glucose levels the levels of ROS can be increased and hence more rapid damage be caused to mtDNA. This accelerates aging/development. For Stem Cells and oocytes, Complexes I and III are reduced in activity and the cells are generally kept in a hypoxic environment. The cells are provided with pyruvate so that the redox balance can be maintained whilst generating energy substantially from glycolysis without OxPhos.
What about the dynamic equilibrium of the mtDNA
There are a number of factors that affect the dynamic equilibrium of mtDNA status. There are endogenous anti-oxidants. The role of melatonin is particularly interesting. Although it is primarily generated within the mitochondria, an amount of melatonin is generated by the pineal gland and injected directly into the cerebrospinal fluid via the third ventricle. This has the effect of providing a higher concentration of melatonin to cells in the CNS and thereby holding back the aging/development process. In children higher levels of melatonin hold back puberty. Precocious puberty can occur when pineal melatonin production is disrupted. On the other side of the dynamic equilibrium is selective mitophagy where the PINK1/Parkin demarcation process is used to identify mitochondria for autophagy. Fission is particularly useful when a mitochondrion has a number of copies of mtDNA some of which are in a good state and some of which are badly damaged. By dividing, the mitochondrial fragments with lower membrane potential can be selected for mitophagy.
Tricoire H, et al. Melatonin Enters the Cerebrospinal Fluid through the Pineal Recess. Endocrinology. 2002. . (Direct secretion into third-ventricle CSF.)
Reiter RJ, et al. Role of age, sleep, and the cerebrospinal fluid melatonin rhythm. 2023.. (Contemporary overview of high third-ventricle CSF melatonin vs plasma.)
Should follicular atresia be considered as part of the aging process?
The handling of the germline should be considered part of the aging process as it needs to act to maintain mitochondrial quality. Oocytes are created within the female embryo and a process that limits the variation of mtDNA to a maximum of 3 different copies of mtDNA occurs as part of this. However, this does not select on mitochondrial quality. In humans 6-7 million oocytes are initially created peaking in number when the female embryo is about 20 weeks old. This then reduces to around 1-2 million by birth. Multiple lines of evidence (TUNEL staining, caspase activation, gene knockouts) indicate that pre- and perinatal germ-cell loss is largely mediated by the intrinsic (mitochondrial) apoptotic pathway. This process depends on mitochondrial outer membrane permeabilization (MOMP) regulated by BCL-2 family proteins, rather than being directly controlled by mitochondrial membrane potential. DNA-damage checkpoints, such as p63-dependent responses to unrepaired meiotic double-strand breaks, are a major trigger of oocyte elimination. In addition, retrotransposon activation (e.g., LINE-1) can promote prenatal oocyte loss through DNA damage and checkpoint activation. Following apoptosis, clearance mechanisms are engaged: transcriptomic and imaging data suggest that complement activation and macrophage recruitment facilitate the removal of dying germ cells, particularly during postnatal follicular atresia. Together, these pathways act as quality-control systems, preferentially eliminating oocytes with unrepaired DNA lesions, excessive retrotransposon activity, or defective mitochondria. Hence the quality control system operating here will clear a proportion of oocytes that have inefficient mitochondria.

Primordial follicles form around week 20 and a process of follicular atresia starts. By puberty, however, only around 300,000 remain. During the menstrual cycle, however, a process of selection of a follicle (an oocyte and surrounding diploid granulosa cells) is used to select one egg as part of this. The author hypothesises that the effect of splicing on the oocyte's gene expression is amplified by the granulosa cells so that atresia can occur for the eggs with the lower quality mtDNA. Additionally zygotes with insufficiently efficient mitochondria are more likely to fail to go to term. In the child and adult female the oocytes are kept in a dormant state with hypoxia until the selection process. During the selection process hypoxia is reduced slightly for the follicles in the selection process through vascularisation. The unselected follicles then return to hypoxia whilst going through atresia and apoptosis

Infographic about the maintenance of germline mtDNA quality

Female Age-Related Fertility Decline
Role of maternal age and pregnancy history in risk of miscarriage: prospective register based study
Integrative proteome analysis implicates aberrant RNA splicing in impaired developmental potential of aged mouse oocytes
The rate and nature of mitochondrial DNA mutations in human pedigrees("Finally, we show that the fate of new mutations is determined by a drastic germline bottleneck, amounting to an average of 3 mtDNA units effectively transmitted from mother to child.")
The proteostatic landscape of healthy human oocytes
The developmental potential of the human oocyte is related to the dissolved oxygen content of follicular fluid: association with vascular endothelial growth factor levels and perifollicular blood flow characteristics
SRSF3 maintains transcriptome integrity in oocytes by regulation of alternative splicing and transposable elements
SRSF1 regulates primordial follicle formation and number determination during meiotic prophase I
Loss of ESRP1 blocks mouse oocyte development and leads to female infertility
BCAS2 regulates oocyte meiotic prophase I by participating in mRNA alternative splicing
Nuclear m6A reader YTHDC1 regulates alternative polyadenylation and splicing during mouse oocyte development
Integrative proteome analysis implicates aberrant RNA splicing in impaired developmental potential of aged mouse oocytes
Mechanisms of ovarian aging

Variations in mitochondrial quality
Unsurprisingly there is a variation in mitochondrial quality that occurs in young mammals at birth notwithstanding the selection of few eggs for ovulation. This has been seen in the children born to older mothers. This will, of course, be a multigenerational issue as it feeds back into the germline and raises complex issues that need some detailed thought. To what extent it is possible to intervene to maintain or improve mitochondrial quality in the germline is unclear, but it will have substantial long term health benefits.
Maternal age effect and severe germ-line bottleneck in the inheritance of human mitochondrial DNA
Modelling variations in mitochondrial quality
Given the importance to human society of maintaining the quality of the mitochondrial germline a considerable amount of research needs to be done to model the state of mtDNA. In principle certain conclusions are likely. Firstly, the subset of oocytes selected for ovulation vary in their mitochondrial quality. Secondly, it appears that the process of quality control has the ability to improve mitochondrial quality on average such that some are more efficient than the original fertilised egg of the mother. Thirdly, although the cells are in a quiescent state they are metabolising although probably at a slower rate prior to puberty (because of higher melatonin levels) than after puberty. Hence the average movement can be approximated by a simplistic formula for each generation:

M2=M1+I-Ytp*D1-Yap*D2.

M1 is the initial average membrane potential and can probably be measured in millivolts. (Mitochondria tend to have a membrane potential around 150 mV) M2 is the average membrane potential of the oocyte on fertilisation. I is an assessment as to the ability of follicular atresia to improve average membrane potential - this may vary over time, however, Ytp is the years to puberty, D1 is the rate of deterioration through metabolism prior to puberty. Yap is the years from puberty to conception, D2 is the rate of deterioration after puberty. Such a formula is worth the research effort to guide people into what decisions to take such as what interventions to take to reduce D2 (potentially even D1, but that is likely to be lower anyway) or when to freeze eggs or indeed when to target conception. The option of a mitochondrial transplant is also known to be a possibility, that may be an option to consider in situations which do not have formally confirmed mitochondrial disease. It is unlikely that simple linear relationships will hold, but a start needs to be made. Additionally there may be a variation in mitochondrial delta pH that needs to be taken into account, but similar principles will apply.

"for each generation" above is emphasised because this is potentially a cumulative process. Although there is an element of randomness in each generation and a potential improvement and further there will be people whose genes involve a lower rate of ROS generation, it should be assumed that this is cumulative.

Previous research on germline mtDNA - added after the BSRA conference
At the BSRA Conference a slide was shown which demonstrated quite a variation in the lifespan of C. Elegans controls. It seemed clear that the controls were not controlled for the initial value of mtDNA even if they were genetically the same from a nuclear DNA perspective. An obvious experiment would be to see the effect of varying the worm parental age in order to vary the starting mtDNA. However, this is experiment was reported in 2023 and I link to it below this paragraph. What I find interesting is that the paper makes reference to the effect of deterioration of the mtDNA germline without identifying that in fact it is the mtDNA germline that is causing these effects. Obviously there are individuals in a range of species that age at slower rates. For those people the effect is not as great, but for most people the issue of maintaining mtDNA germline quality is a material issue. The paper itself is worth reading and there is no sense me rephrasing a lot of it for this web page.
Parental age effect on the longevity and healthspan in Drosophila melanogaster and Caenorhabditis elegans
Interventions to improve germline mtDNA
Germline mtDNA operates differently to somatic mtDNA. That is because the sharing of mitochondria that occurs between cells does not happen with the germline. That is a necessary limit because the processes of oocyte selection and the mtDNA bottleneck enable some of the germline oocytes to have mitochondria that are more efficient than those which were in the mother's oocyte when she was conceived (remember the oocytes are created in the embryo). Also necessarily the germline cells are in a dormant state. Hence although the granulosa cells provide nutrients (importantly pyruvate) to the oocytes, they do not provide mitochondria. However, systems such as selective mitophagy still work although more so following fertilisation. Further research is needed with interventions such as mTOR inhibitors to identify the extent to which PINK1/Parkin selective mitophagy can be used to both maintain and also improve the germline.

COVID, mtDNA and Fertility
Obviously anything that damages mtDNA is likely to reduce fertility and COVID damages mtDNA. Hence actions to respond to the reduction of global fertility which is probably now in part caused by covid infection (but not by vaccination) as well as additional metabolism are similar in nature to any other interventions to improve the quality of mtDNA.

This table looks at links between COVID and mitochondrial damage. I have not checked it yet and obtained it from chatGPT. Obviously oocyte mtDNA is more protected, but not necessarily immune from Covid and any damage from Covid is cumulative with that of metabolism.

Mechanism / finding Tissue / model Key result Evidence type Year Link
ORF9b binds TOM70, innate immune evasion Structural biology Crystal structure of SARS-CoV-2 ORF9b bound to human TOM70; supports mitochondrial targeting and IFN suppression. Primary (structure) 2021 Gao et al., Nat Commun
ORF9b modulates mitochondrial function via TOM70 Human cells ORF9b localizes to mitochondria in a TOM70-dependent manner and alters host mitochondrial responses. Primary (cell biology) 2023 J Cell Biol
OXPHOS gene suppression; glycolytic shift Human nasopharyngeal & autopsy tissues; animals Down-regulation of nuclear-encoded OXPHOS genes; HIF-1α/glycolysis program activation with infection. Primary (transcriptomics + validation) 2023 Guarnieri et al., 2023 (PMC)
Mitochondrial metabolic & epigenomic remodeling Cells / patient data (reanalysis) SARS-CoV-2 inhibits OXPHOS and increases mitochondrial ROS; broad mito-metabolic reprogramming. Primary/Analysis 2024 Guarnieri et al., Exp Cell Res
cGAS–STING activation via (host) DNA leakage Human tissues; models Type-I IFN immunopathology in COVID-19 driven by cGAS–STING; endothelial contribution implicated. Primary 2022 Domizio et al., Nature
Platelet mitochondrial dysfunction & oxidative stress Patient platelets Mitochondrial depolarization, ROS, hyporeactivity; patient plasma can induce depolarization in control platelets. Clinical observational 2023 Léopold et al., Res Pract Thromb Haemost
Lymphocyte immunometabolic dysfunction Patient T cells Dysregulated immunometabolism associates with exhaustion and impaired effector/memory responses. Primary 2023 Gurshaney et al., Commun Biol
Circulating mitochondrial cfDNA predicts severity COVID-19 patients (plasma) Higher plasma mtDNA levels associate with worse oxygenation and outcomes. Clinical cohort 2021 Valdés-Aguayo et al., Front Cell Infect Microbiol
Long-COVID skeletal muscle: post-exertional worsening Biopsies; exercise challenge Severe exercise-induced myopathy; structural and metabolic disturbances that worsen after PEM. Primary (clinical biopsy study) 2024 Appelman et al., Nat Commun
Long-COVID skeletal muscle mitochondrial dysfunction Clinical cohort Intrinsic muscle mitochondrial dysfunction, endothelial abnormalities, fiber-type shift in long COVID. Primary 2024 Charlton et al., 2024
Cardiomyocytes: mitochondrial damage with SARS-CoV-2 Human cardiomyocytes Infection damages mitochondrial integrity and impairs respiration in cardiomyocytes. Primary 2025 Che et al., 2025
Overview: mitochondrial dysfunction in acute & post-acute COVID Review Synthesizes mitochondrial impacts across organs; links to long-term sequelae. Review 2024 Molnar et al., 2024 (Review)
Stem Cells, pyruvate metabolism and hypoxia
A similar pattern of quiescence to protect mtDNA from metabolism is seen with stem cells. These are also kept in a low oxygen environment and require exogenous pyruvate. There appears, however, to be a conflict in the literature as to exactly what happens in asymmetric division. The relevance to this page is for now just that they are kept in a quiescent state which protects mtDNA and that they divide their mitochondria asymmetrically. This is of course, quite significant from the perspective of mtDNA quality, but the literature seems contradictory on exactly what happens.

Mohyeldin A, et al. Oxygen in Stem Cell Biology: A Critical Component of the Stem Cell Niche. Cell Stem Cell. 2010. . (Stem cells reside in hypoxic niches and rely more on glycolysis.)
Old mitochondria regulate niche renewal via α-ketoglutarate metabolism in stem cells
Asymmetrically Segregated Mitochondria Provide Cellular Memory of Hematopoietic Stem Cell Replicative History and Drive HSC Attrition - where it goes wrong
Asymmetric division of stem cells and its cancer relevance
Metabolic determination of cell fate through selective inheritance of mitochondria




Mechanistic hypothesis drives synergistic interventions
The above hypothesis assists determining synergistic interventions as it is possible to look at how they intervene in the pathway through mtDNA damage and senescence via citrate metabolism into acetylation as a PTM. Preventing mtDNA damage, destroying damaged mtDNA, making mitochondria more efficient, directly increasing cytosolic acetyl-CoA and inhibiting PTM deacetylation can all have a synergistic effect.
Infographic about acetylation interventions

What about Rapamycin?
Rapamycin is an inhibitor of the eponymously named mTOR complex. Because the cell uses mTOR as a signal of the availability of nutrition, inhibiting mTOR makes autophagy more likely. The autophagy stimulated by Rapamycin includes selective mitophagy prioritising the mitochondria with lower membrane potential. Rapamycin, therefore causes cells to improve the average quality of mitochondria and mtDNA. Doing lifespan experiments in Homo sapiens with Rapamycin is not practical because of the time requirements. It has, however, done well in other species. The Rapamycin News forum of self experimenters unsurprisingly includes many users of Rapamycin and is the normal source of anecdotal information about its effects. The question, that is difficult to answer, is what dosing to take and what frequency of dosing. The evidence points to Rapamycin running a cyclical spring clean of mitochondria and dosing at different stages of life in model organisms has resulted in extensions in median lifespan. The author takes the view that a high level of mTOR inhibition is likely to result in a greater removal of a cohort of mitochondria with a lower membrane potential and that a higher dosage at a lower frequency than weekly is advised. The author, therefore, takes a high dose of rapamycin combined with a CYP3A4 inhibitor every few months. In late 2024 he took 22mg of Rapamycin combined with a grapefruit (CYP3A4 inhibitor) which is estimated to be an equivalent of 77mg on a one off basis. (Given Rapamycin's estimated 60 hour half life this was well within the estimated safe peak serum levels). He wore a CGM (apart from when it fell off) for about 4 weeks and did a full panel blood test twice a week. The detailed results of this are published on Rapamycin News (see this poster on the internet for the URL). The results were not particularly surprising. Because of the short half life of neutrophils compared to lymphocytes the numbers of neutrophils dropped rapidly and then picked up as the Rapamycin was metabolised. In a sense this is more interesting as it appears to be the results of a threshold rather than purely being dose dependent. Similarly as a form of hepatic insulin resistance was created average glucose levels crept up and then there was an overswing as the body generated higher levels of insulin and the levels of glucose were driven down to a daily average of 4.8 mmol/l.

It is hard to quantify whether this approach of a high intermittent dose is more effective than less frequent lower doses. The author has not had the usual side effects from this and believes that the canker sores caused by longevity dosing may be more likely where a body tolerates a higher level of infection and rapamycin is used such that the immune system remains inhibited over a long period of time.
Link to rapamycin news forum for detailed results
Li Q, et al. Rapamycin Enhances Mitophagy and Attenuates Apoptosis in Models of Injury. Mol Neurobiol. 2018. . (Evidence that rapamycin enhances mitophagy.)
mTOR modulator project
This is a project run by the Rapamycin Longevity Lab. They describe this as:
Searching for a better Rapamycin
Rapamycin is the gold standard for lifespan extension, but better options may exist. Rapamycin Longevity Lab has already screened 300 mTOR-modulating compounds. Five of these outperformed Rapamycin’s 27% lifespan effect, with Omipalisib achieving a remarkable 63% increase.

Additional 300 compounds are soon to be screened once we obtain the remaining $23,400 funds needed. The goal with this project is to provide the field with important unique data to improve human longevity.

They are looking for donations at the link https://masteronething.com/mtor


Hair regrowth


Hair regrowth is often an issue which is studied as part of the aging pathway. With male pattern baldness there is also a factor relating to DHT. Hence reversing hair growth is a challenge because cells which have not functioned for decades need be made to function. What tends to happen is that vellus hairs start up and then become terminal. After that there can be an increase in the width of the hair. The areas compared above are from August 2023 to August 2025.

Phenotypic changes

A number of subjective phenotypic changes have occurred. Here are some photos.
Pictures of the author Pictures of the author at various dates in the last 7 years


the role of menaquinones in mitochondrial function?
Vitamin K2 is available in supplement form as MK4, MK7 and MK9. The number indicates the number of isoprene residues. There is a material difference between MK4 and MK7 because the additional three isoprene residues make MK7 hydrophobic and capable of sitting in the mitochondrial membrane wall accepting electrons from the ETC. Research in Drosophila has pointed to how K2 (in the MK4 form) can rescue failing mitochondria. However, this conflicts with other research. Research in human cells with MK4 has found it was not able to substitute CoQ10 as an electron carrier. Various self-experimenters including some suffering from Parkinson's Disease have experimented with higher levels of MK7. (1mg per day plus) The results are consistent with an improvement in mitochondrial efficiency. However, the conclusions are undermined by polypharmacy. Nonetheless MK7 is a good candidate when looking for a synergistic manner in which to improve mitochondrial function. It has a longer half life (3 days) than Methylene Blue which is thought to have some similar benefits. Caution should be taken with moving to a high systemic concentration of MK7 rapidly as it can shift some of the body's homeostases in a way that the body does not handle that well.

Vitamin K2 is a mitochondrial electron carrier that rescues pink1 deficiency
Vitamin K2 cannot substitute Coenzyme Q10 as electron carrier in the mitochondrial respiratory chain of mammalian cells
Chemistry of Lipoquinones: Properties, Synthesis, and Membrane Location of Ubiquinones, Plastoquinones, and Menaquinones
Sato T, et al. Comparison of menaquinone-4 and menaquinone-7 bioavailability. Nutr J. 2012. . (MK-7 exhibits substantially longer half-life than MK-4.)
Maresz K. Growing Evidence of a Proven Mechanism Shows Vitamin K2 is Important. 2021. . (Review noting MK-7's long half-life and extrahepatic activity.)
Infographic about neurodegeneration

whether Parkinson's Disease and ALS/MND should be considered diseases of aging,
Neurodegenerative diseases in general and Parkinson's and ALS/MND specifically have suffered from a difficulty in identifying causes. A July 2025 paper found that movements in the Basal Forebrain Volume were predictive of the phenoconversion of isolated rapid eye movement sleep behaviour disorder to PD. Arguably this could be because of a factor affecting both the Basal Forebrain and then later the substantia nigra. Both diseases have links to splicing problems and are associated with mitochondrial fission. There is limited research studying cerebrospinal fluid in people with Parkinson's Disease. However, there is evidence both of a reduction of melatonin levels in the CSF and of a phase shift in serum levels of melatonin. This would be consistent with melatonin failing to inject normally from the pineal into the third ventricle of the CSF and as a consequence the reduction in exogenous melatonin for the CNS. Similarly some ALS/MND is associated with difficulties in the flow of CSF. Both diseases clearly have mitochondrial aspects and because dopaminergic and motor neurons have high energy usage and, unlike cones and rods, a high dependency on OxPhos they are more vulnerable to high levels of ROS from the ETC. Furthermore cones and rods may produce their own melatonin which compensates for CSF melatonin not being available to the retina (this is still a matter of debate). Some individual researchers with PD are experimenting with interventions to improve mitochondrial function. Hence it is possible to see the role of melatonin in the CSF as something that decelerates the aging of cells in the CNS. At the same time a reduction in melatonin supply will increase the rate of aging of high energy using cells. This approach will be promoted by a team in the ALS Longitude prize.

Basal Forebrain Volume Predicts Disease Conversion in Prodromal Synucleinopathy
The Energy balance between Glycolysis and OxPhos in Neurons
What causes Parkinson's Disease - is it actually an accelerated form of brain aging?
Melatonin in Retinal Physiology and Pathology: The Case of Age-Related Macular Degeneration - discusses the debate about human retinal melatonin
Monepantel and the Blood Brain Barrier
A lot of mTOR inhibitors have problems getting to neurons probably because there are transport proteins actively pumping them out. However, there is some experimentation going on with Monepantel.
Trial / Study Condition / Focus Link
PharmAust - Phase I trial Refractory metastatic cancer View
PharmAust - MND/ALS trial details Motor Neurone Disease (ALS) View
ClinicalTrials.gov - NCT04894240 MND/ALS (Phase I safety/tolerability) View
Biospace / PR Newswire MEND trial results (ALS) View
PharmAust - MND/ALS summary Study results & development plans View
ClinicalTrials.gov - NCT06177431 MND/ALS (Open-label extension) View
PubMed - Phase I trial Solid tumours, dose-escalation View
Longevity Escape Velocity
The hypothesis accepts that DNA damage occurs including situations such as males losing Y chromosomes. It is hard to improve mitochondria generally and some replication damage to mtDNA may be really challenging. If people are looking to hit LEV then issues other than the mitochondria may need to be a target. However, the diseases known as diseases of aging are substantially linked to mitochondrial inefficiency through mtDNA damage.

Aging factors found in Blood
IL-10 in SASP - inhibits NF kappa B, reduces SLC25A1 via Janus Kinase
ACBP (Acyl-CoA-binding protein) in SASP - reduces autophagy activates mTOR
Acyl-CoA-binding protein as a driver of pathological aging


When it comes to longevity indoles are not all indolent
Psilocybin, which is an illegal substance in the UK, has been more recently found to have life extending properties in mice. It is an indole. Melatonin is also an indole. Perhaps the indole with the most effective lifespan results was Indolepropionamide (IPAM) which was found to give a 268% lifespan increase in Rotifers. This attracted the interest of the Rapamycin fan club a group of whom clubbed together to get some lifespan experiments done with C. elegans. Sadly it did not seem to make that much difference, but the results are published below. Further experimentation is being done with higher doses. The highest dose (60uM) resulted in an increase in the size of the worms similar to the size increase in rotifers. This, in combination with no evidence of toxicity at these doses, indicates that similar lifespan effects may be possible at higher doses, and this is why higher doses are being explored. Some individual self experimenters (not the author on this occasion) are experimenting with IPAM. There are not really any anecdotal conclusions, but it is worth reporting the interim negative results.




See forum entry on Rapamycin News
268% increase in lifespan of rotifers
Psilocybin treatment extends cellular lifespan and improves survival of aged mice
Effects of exogenous melatonin--a review
Monitoring functional changes
There are continual debates about how to measure the effects of the aging pathway with a wide range of biomarkers being offered. However, the end objective is functional changes. Physical changes like osteoporosis and muscle strength are often measured. Gingivitis is also an area that requires measurement. However, subclinical cataracts are often ignored. Subclinical cataracts where there is no impact on vision, but a slight clouding of the lens are not necessarily noted during a vision test. However, the author has persuaded his optician (Pabari of Moseley) to monitor the lens of the eye at a higher frequency than normal (every 3-4 weeks). Interestingly a clearing of the lens in the left eye has been seen whilst the right eye maintains some subclinical clouding. This is consistent with anecdotal reports from members of Rapamycin News. It is well known that people who have diabetes, but bring this under control can end up with a reduction in cataracts. However, for people without diabetes an improvement in the lens does not appear in the literature. This monitoring process continues.

Conflicts of interest
The author has made patent applications in connection with his discoveries and has a company selling interventions based upon his interventions.

Use of AI
LLMs have been used for search, proofreading and review, but not for generation of hypotheses.

About the author
Born 1960
Educated: King Edwards School, Birmingham.
Scholarship to Magdalen College (Oxford), MA (Oxon) in Physics.
Formed his first business in 1983 which was sold in 2019
Councillor on Birmingham City Council 1990-2008
Deputy Leader of Birmingham City Council 2004-2005
Member of Parliament Birmingham, Yardley 2005-2015
Has been drummer in a heavy metal and punk band in the 1970s then moved into prog rock as a drummer, but more recently is the keyboard player in "John Hemming and the Jazz Lobbyists" a Birmingham based jazz band.
Contact Email: john@hemming.email