Excessive vigorous exercise impairs cognitive function through a muscle-derived mitochondrial pretender, 2025, Huang et al.

[SNT Gatchaman]

Excessive vigorous exercise impairs cognitive function through a muscle-derived mitochondrial pretender

Spoiler: Authors

Yan Huang; Biao Hu; Ya Liu; Ling-Qi Xie; Yu Dai; Yu-Ze An; Xin-Yi Peng; Ya-Lun Cheng; Yi-Fan Guo; Wei-Hong Kuang; Yao Xiao; Xin Chen; Yong-jun Zheng; Gen-Qing Xie; Jian-Ping Wang; Hui Peng; Xiang-Hang Luo

Excessive exercise impairs cognitive function, but the underlying mechanism remains unclear. Here, we show that excessive vigorous exercise-induced lactate accumulation stimulates muscles to secrete mitochondria-derived vesicles (MDVs), driving cognitive impairment.

These MDVs (named otMDVs) are characterized by high mtDNA levels and the surface marker PAF. They tend to migrate into hippocampal neurons, substituting endogenous mitochondria and triggering a synaptic energy crisis.

Mechanistically, otMDVs release mtDNA, which activates cGAS-STING-dependent inhibition of kinesin family member 5, preventing hippocampal mitochondria from transporting to synapses. Simultaneously, the otMDV marker PAF cooperates with syntaphilin to occupy mitochondrial anchoring sites, impairing synaptic energy supply.

Blocking otMDVs migration into the hippocampus with a PAF-neutralizing antibody alleviates excessive vigorous exercise-induced synapse loss and cognitive dysfunction. Notably, human studies link high circulating otMDV levels to cognitive impairment.

Together, our findings reveal that a unique muscle-derived MDV subpopulation, which displaces hippocampal mitochondria and disrupts their function, causes cognitive decline.

HIGHLIGHTS
• Excessive vigorous exercise causes cognitive impairment

• ATF5 lactylation promotes the secretion of otMDVs during excessive vigorous exercise

• otMDVs are characterized by enriched surface marker PAF and content mtDNA

• otMDVs induce synaptic energy deficits by occupying mitochondrial anchor sites

Web | DOI | PDF | Cell Metabolism | Paywall

 

[jnmaciuch]

Definitely fascinating if there’s a mechanism that shuttles mtDNA to the brain. This paper is on my list to read!

 

[DHagen]

This seems potentially huge - am very interested to hear from those better equipped to evaluate this paper than myself.

 

[Turtle]

Would 2 x 800 meters on an electric bike qualify as excessive vigorous exercise?

 

[SNT Gatchaman]

From the introduction —

Exercise is well-established as a strategy for disease prevention and health promotion, including benefits for brain structure and function that can mitigate age-related cognitive decline. However, the dose-response relationship between exercise and health follows an inverted J-shaped curve: excessive loads can cause adverse outcomes like muscular fatigue and cardiovascular dysfunction. Notably, several clinical investigations have indicated an association between excessive exercise and cognitive decline.

Synaptic loss represents an early pathological feature of cognitive decline. Given their substantial energy requirements, synapses depend on localized mitochondria for ATP production. The transport of mitochondria mediated by KIF5 and their synaptic anchoring by SNPH are each critical for sustaining this energy supply. Disruption of either process causes an energetic deficit, which impairs synaptic function and contributes to cognitive decline.

Mitochondria-derived vesicles (MDVs) are extracellular vesicles that selectively package mitochondrial fragments rather than intact mitochondria. They have garnered increasing attention across fields for their roles in cellular communication and mitochondrial quality control.

In the nervous system, research on MDVs is emerging, with growing evidence supporting their importance in neuronal health.

we identify a novel muscle-derived vesicle (otMDV), characterized by high mtDNA levels and surface expression of PCNA-associated factor (PAF) and regulated by activating transcription factor 5 (ATF5) lactylation, that causes excessive vigorous exercise-induced cognitive dysfunction. Upon migrating to hippocampal neurons, otMDVs trigger synaptic loss through activating the mtDNA-cGAS-STING pathway and occupying anchoring sites with PAF.

PCNA is Proliferating cell nuclear antigen (Wikipedia)

PAF is aka PCNA Clamp Associated Factor (GeneCards)

 

[SNT Gatchaman]

From results —

we analyzed UK Biobank cross-sectional data using metabolic equivalents (METs) to quantify physical activity volume. […] 316,678 participants with complete data were included, then were stratified into activity quintiles and assessed with multiple cognitive metrics. After adjusting for age, sex, educational background, BMI, etc., we identified a paradoxical association: higher physical activity levels correlated with poorer cognitive performance. Notably, the highest activity quintile (Q5) demonstrated significantly lower standardized scores across cognitive domains versus the population mean.

We further assessed long-term cognitive outcomes across MET levels over a median 13.7-year follow-up (5,338 cognitive impairment cases and 21,575 deaths). Notably, total physical activity exhibited a J-shaped association with cognitive impairment risk, with the optimal dose at 3,972 MET-min/week (hazard ratio = 0.73; 95% confidence interval [CI]: 0.68–0.78; 21.76% participants above nadir).

(Then investigated with mice doing moderate or vigorous exercise, assessed via novel object recognition test and the Morris water maze.)

The moderate-intensity group showed cognitive benefits when MET exceeded 281 MET-min/week. Conversely, the vigorous-intensity group initially showed cognitive improvements, but these turned to adverse effects when MET was surpassed, with detrimental effects persisting.

In the MWM, excessively exercised mice exhibited longer escape latencies […] along with fewer platform crossings during the subsequent probe trials and less time spent in the target quadrant […] indicating that excessive vigorous exercise impaired both learning and memory abilities. Additionally, excessively exercised mice spent less time exploring the new object […] indicating reduced nonspatial learning abilities.

Golgi staining revealed that excessively exercised mice exhibited significantly reduced dendritic spine density in hippocampal neurons […] Additionally, we quantified synaptic density by detecting the expression of the presynaptic marker synapsin and the postsynaptic marker PSD95 in the hippocampus and found decreased levels of both […] transmission electron microscopy (TEM) also demonstrated that excessive vigorous exercise reduced the length of the PSD in neuronal synapses.

 

[SNT Gatchaman]

Results (cont'd) —

TEM revealed that in mice subjected to excessive vigorous exercise, muscular mitochondria developed vacuolar-like changes and swelling […] isolated mitochondria from the muscles of these excessively exercised mice showed increased budding structures.

Emerging studies have demonstrated that under various stimuli, mitochondria can generate and release MDVs, which subsequently regulate the function of other organs. […] observed a significant increase in total MDV levels in excessively exercised mice compared with controls.

TEM further revealed that the isolated MDVs had a diameter of approximately 100 nm and a cup-shaped morphology. […] Distinct from typical EVs, MDVs were equipped with mitochondrial protein (including HSP60 and VDAC), higher mtDNA content, and increased ATP production capacity.

We isolated plasma MDVs from control mice and excessively exercised mice, which are referred to as conMDVs and otMDVs

I didn't see it defined but I think otMDV means "over-trained mitochondrial derived vesicles" (and con for control.)

otMDVs were intravenously administered to recipient wild-type (WT) mice to observe the distribution of MDVs through in vivo imaging systems (IVISs). We found that DiR probe-labeled MDVs migrated to the brain. […] PKH67-labeled MDVs were found to distribute to the hippocampal region, which were mainly localized in neurons (marker: NeuN) rather than in astrocytes (marker: Gfap) and microglia (marker: Iba1)

findings indicated that MDVs cross the BBB through both transcytosis and paracellular mechanisms […] fluorescence intensity ratio of intracerebral to peripheral was approximately 12% in control mice versus 25% in the excessive-exercise group, indicating that MDVs can cross the BBB while excessive exercise enhances the permeability.

we administered MDVs via injection three times per week for 8 weeks. Following this […] behavioral tests […] revealed that otMDVs treatment induced cognitive decline

otMDVs disrupted synaptic density, morphology, and the expression of synaptic markers[…]. We also found the decreased PSD length in neuronal synapses of the hippocampal region […]. These findings suggested that otMDVs disrupted synaptic structure and contributed to cognitive decline.

PINK1 plays a crucial role in MDV release by promoting the packaging of damaged mitochondria, a process important for cellular responses to mitochondrial stress. […] suppression of muscular MDV secretion restored the impaired cognition and synaptic function

Among all upregulated proteins in otMDVs, PAF was the most highly expressed […] PAF, a PCNA-associated factor, has been previously reported to be involved in cell proliferation, apoptosis, and cell cycle regulation.

(FWIW, pathways I recall we see repeatedly highlighted in ME transcriptomics studies though that may be pretty generic.)

Using the Seahorse assay, we measured the oxygen consumption of MDVs, and results showed that otMDVs had significantly lower basal respiration, ATP production, and maximal respiration than conMDVs. […] ATP assays further confirmed an ATP deficit in otMDVs, suggesting that these otMDVs have a lower capacity to generate ATP compared with conMDVs.

 

[SNT Gatchaman]

Results (cont'd) —

Our results indicated that there were no obvious changes in circulating cortisol, but BDNF levels were increased in the excessive-exercise group. […] BDNF is beneficial for cognition

Notably, lactate, but not other factors, exclusively promoted PAF+ MDV levels […] we demonstrated that lactate-stimulated C2C12 cells elevated PAF+ MDV release and decreased neuronal synaptic density

short vigorous exercise (15–45 min) causes transient lactate accumulation, with no significant PAF+ MDV release. By contrast, prolonged vigorous exercise (over 60 min) leads to higher, sustained lactate, along with significant PAF+MDV release

we intramuscularly injected graded lactate injections […] a higher level of mtDNA was detected in MDVs from mice receiving high-dose lactate treatment, compared with those with vehicle or low-dose lactate treatment. […] mice with high-load lactate injection in muscle exhibited cognitive decline, as reflected by longer escape latency and less exploration time for the novel object.

we mimicked intramuscular injection effects by intravenously administering lactate to achieve comparable circulating levels. Notably, this elevation in blood lactate did not induce cognitive impairment […] Lactate treatment significantly increased plasma lactate levels but slightly increased muscle lactate levels, while PAF+MDV levels remained unchanged.

Lactylation, a newly identified post-translational modification, has been shown to regulate various biological processes. Here, we found that lactyl modification was significantly enhanced in the muscle tissue of excessively exercised mice

See our tag for lactylation.

Among all lactyl-antibody-immunoprecipitated proteins identified via lactylation mass spectrometry of lactate-stimulated C2C12 cells, ATF5 has been reported to be involved in regulating mitochondrial homeostasis under various stimuli. We thus focused on ATF5 for further investigation.

Increased ATF5 lactylation was confirmed in the muscle of excessively exercised mice […] We observed that mutations on the lactylation sites reduced PAF+ MDV secretion along with decreased mtDNA content within MDVs […] exhibited significantly alleviated excessive exercise-related cognitive decline

Considering that PAF functions as the surface marker of otMDVs, we generated a neutralizing antibody specifically targeting PAF […] Significantly, PAF-NAb treatment effectively mitigated these functional and structural impairments caused by PAF+ MDVs.

These findings confirm the crucial role of PAF+ MDVs in regulating cognitive impairment and demonstrate the therapeutic potential of PAF-NAb for excessive exercise-related cognitive dysregulation.

 

[SNT Gatchaman]

Results cont'd —

To investigate the necessity of the PAF marker on otMDVs, we treated hippocampal primary neurons with conMDVs, otMDVs, and otMDV PAF−, respectively.

Notably, compared with conMDVs, otMDV treatment led to decreased dendritic density, while otMDV PAF−partially reversed the synaptic loss.

Live imaging experiments were performed […] we inferred that the surface marker PAF is indispensable for otMDVs to occupy synapses.

SNPH [Syntaphilin, see GeneCards] has been identified as a ‘‘static anchor’’ that anchors axonal mitochondria onto microtubules close to presynaptic terminals.

Immunofluorescence revealed co-localization of otMDVs and SNPH, which was dramatically reduced in the otMDV PAF− group

Then, we treated hippocampal neurons with conMDVs, otMDVs, and otMDVs with PAF-NAb simultaneously. Notably, PAF-NAb reduced the synaptic occupation of otMDVs and reversed the aberrant hippocampal mitochondrial distribution induced by otMDVs, further ameliorating the synaptic ATP deficit in synapses. Furthermore, PAF-NAb restored synaptic deficits and reversed otMDV-impaired dendritic spine morphology and density.

using Snph small interfering RNA […] significantly decreased otMDV anchoring at synaptic terminals. […] findings support our assumption that otMDV anchoring in synapses depends on the binding of the surface marker PAF with SNPH.

Mitochondrial motility is essential for sustaining synaptic energy. KIF5 acts as the principal anterograde motor for axonal mitochondrial transport and governs mitochondrial distribution at synaptic sites. [some observations] we hypothesized that mtDNA contributed to the reduction of synaptic mitochondria and ATP supply.

Many studies have revealed that mtDNA robustly activates the cGAS-STING signaling pathway. […] findings revealed that otMDV PAF− dampened synaptic function through mtDNA release and subsequently cGAS-STING activation.

Given that stress can affect the free circulating mtDNA level and BBB permeability, we also verified the elevation of free circulating mtDNA levels alongside BBB permeability. […] findings indicate that free mtDNA can cross the BBB, either through active transport or via barrier leakage. […] permeabilized otMDVs failed to induce cognitive impairment, underscoring the critical role of vesicular packaging.

We found that Kif5 overexpression reversed the impaired mitochondrial transportation, along with improved ATP levels and dendritic spine and synaptic deficits. […] mice with Kif5 overexpression showed enhanced cognitive function.

In summary, synaptic dysfunction and cognitive impairment are mediated by local energy deficits in synapses through dual mechanisms. Firstly, the PAF marker on otMDVs occupies synaptic anchoring sites through the PAF-SNPH complex. Secondly, mtDNA released from otMDVs decreases mitochondrial transport to synapses through cGAS-STING-mediated KIF5 downregulation.

 

[SNT Gatchaman]

we further investigated the implications of otMDVs in humans. We recruited participants engaged in different types of exercise, including badminton (n = 15), running (n = 15), and weightlifting (n = 15).

A significant positive correlation was observed between circulating lactate levels and PAF + MDVs across all exercise modalities.

we conducted [an RCT] recruited 40 participants (with ∼1,000 MET-min/week in the past 3 months) […] Participants were assigned to either the moderate-duration vigorous exercise group or the excessive vigorous exercise group. […] circulating lactate levels were elevated in both groups […] with significantly greater increases in the excessive group.

both PAF+ MDV levels and mtDNA levels within MDVs were significantly elevated in the excessive group after intervention, while remaining unchanged in the control group

We further conducted proton magnetic resonance spectroscopy (MRS) in the hippocampus and analyzed the ratio of N-acetyl aspartate to creatine (NAA/Cr). Here, NAA/Cr was reduced in the excessive group but not in the control group. Moreover, participants in the excessive group exhibited lower fluid intelligence scores and numeric memory capacity.

Importantly, we observed a significant negative correlation between the changes in PAF+ MDV levels and both the changes in fluid intelligence and numeric memory after adjusting for confounding factors, including cortisol, BDNF, and interleukin (IL)-6, establishing PAF+ MDVs as an independent risk factor for excessive exercise-induced cognitive dysfunction.

 

[SNT Gatchaman]

Over to @jnmaciuch and others for expert assessment / evaluation of methods.

But a couple of initial thoughts after reading this in the context of findings related to synaptic genes, AMPA receptors etc.

1. What's our muscle lactate doing? Following CPET. Do we know?

Ie if there is muscle capillary basement membrane thickening, it's possibly impeding lactate clearance to blood when formed with exertion. We already know it's normal at rest.

Eg Brain and muscle chemistry in myalgic encephalitis/chronic fatigue syndrome ME/CFS and long COVID: a 7T magnetic resonance spectroscopy study (2025) —

underwent brain […] and calf muscle MRS scanning at 7 Tesla […] Compared to HC, ME/CFS patients had elevated levels of lactate in both pgACC and dACC […] By contrast, skeletal muscle metabolites at rest did not significantly differ between the groups.

But remembering Excessive Intracellular Acidosis Of Skeletal Muscle On Exercise In A Patient With A Post-Viral Exhaustion/Fatigue Syndrome: A 31P Nuclear Magnetic Resonance Study (1984)

2. We should probably measure our MDV and PAF+ MDV levels. At rest and over 6, 24, 48 hrs following CPET (as per Hanson/Grimson).

If the above concept of capillary basement membrane thickening impeding O2 flux and increasing muscle lactate were valid, then likely the extracellular vesicles would be similarly impeded in getting to the bloodstream.

However, if muscle cells packaged up these vesicles regardless and they elevated concentration in plasma and transported to the brain over say, the next 6 - 48 hours, then that would comport with PEM timeframes.