In this study, we sought to shed light on the molecular mechanisms underlying the beneficial effects of exercise in AD by examining a priori hypothesized molecular effectors in plasma NDEVs from AD patients participating in a RCT of exercise. We found that in patients with mild to moderate AD, 16 weeks of aerobic exercise increased NDEV levels of BDNF, proBDNF and humanin (Fig. 1). These findings strengthen the notion that NDEV cargo reflects the molecular state of brain neurons and any dynamic changes to it, a thesis supported by previous evidence showing that NDEV biomarkers can track AD progression (34) and demonstrate target engagement in clinical trials (20).
BDNF is a neurotrophin produced upon proteolytic cleavage of its precursor, proBDNF, which is also bioactive. Physical activity increases BDNF concentrations in brain (35) and plasma (36). Previous studies have shown that proBDNF and mature BDNF mRNA and protein, are decreased in early and end-stage AD brain in correlation with cognitive measures (14). The upregulation of BDNF by exercise has been observed in both animal (37) and human studies (38) supporting its development as a non-pharmacological intervention for AD. Proposed mechanisms on how exercise may increase BDNF levels in neurons include: (i) effects through the PGC1α-dependent myokine, irisin (33), (ii) the myokine Cathepsin B, which when increased peripherally by exercise can cross the BBB and enhance BDNF production and hence neurogenesis (39) and (iii) β-hydroxybutyrate, which also increases during aerobic exercise and increases BDNF expression in the brain (37). As previously shown, both proBDNF and BDNF are present in NDEVs, at higher levels compared to plasma (40). Moreover, NDEV proBDNF, but not BDNF, was associated with physical activity in a large longitudinal cohort of aging (41). Future studies, unrestrained by the amount of plasma available, may use NDEVs as a tool to further dissect the downstream effects of proBDNF and BDNF, such as by measuring their receptor levels (p75 NTR and TrkB), downstream effectors and functional outcomes, such as levels of synaptic proteins (34).
Humanin is a mitochondria-derived peptide that suppresses neuronal apoptosis, preserves synapses, reduces inflammation and supports glucose and oxidative metabolism (42). Plasma humanin decreases with age in humans and mice (43), and upon replenishment, cognition in aged mice is improved (44). Humanin mRNA in plasma EVs has been found decreased in AD compared to control individuals (45), whereas protein levels of humanin in NDEVs have been found decreased in multiple neuropsychiatric disorders (46, 47). Interestingly, physical activity increases humanin in plasma (48). Humanin can be destabilized via ubiquitination assisted by TRIM11 (49), a process that could be interrupted by aerobic exercise, which inhibits the ubiquitin-proteosome pathway (50). In turn, increased humanin levels could inhibit the pro-apoptotic factors BAX and BID, thus preventing mitochondrial-outer membrane permeabilization and enhancing neuronal survival and cognitive performance in AD (51). These observations suggesting that humanin depletion is associated with mitochondrial function deficits in AD further support humanin augmentation via physical activity as a non-pharmacological intervention for AD.
Exercise-induced factors released from muscle and other organs, collectively coined exerkines, can mediate systemic responses to physical activity (13). The extracellular milieu is a harsh environment for soluble exerkines, which may have driven the evolution of a parallel lipid-enclosed delivery mode via EVs, which are known to exert autocrine, paracrine and endocrine signaling functions (13). Previous studies have shown that endurance exercise increases circulating EVs, including a muscle-derived subpopulation capable of being biodistributed to the brain after peripheral injection (13, 52). Thus, it has been widely hypothesized that physical activity modulates neuronal function indirectly, through the activity of soluble or EV-associated peripheral exerkines capable of crossing the BBB. Although many of these peripheral exerkines are also expressed in neurons, they remained unchanged in NDEVs after exercise (Table S2), suggesting that physical activity engages different pathways in neurons and peripheral organs.
Our study also indicates that neuronal exercise effects are coordinated across multiple inter-connected pathways; this is suggested by the correlation between changes in NDEV BDNF and irisin (Fig. S2), proteins whose expression is known to be co-regulated (53), as well as correlations between humanin with irisin and proBDNF, both at baseline and after exercise, (Fig. S2) which have not been previously reported.
We explored whether exercise effects may differ by APOE genotype, as previous studies have shown that, in both healthy elderly and AD individuals, beneficial effects of physical activity on cognition and hippocampal volume are stronger in ε4 carriers (54). These studies suggest that that the presence of ε4 increases the responsiveness of neurons to exercise; in favor of such a hypothesis, we found more prominent increases in NDEV proBDNF and humanin with exercise in ε4 carriers (Fig. 2).