Serpina1e mediates the exercise-induced enhancement of hippocampal memory in male mice

Domain: neuroscience
Published: 2026-04-09
Editorial outcome: major_revision

Abstract
Exercise enhances learning and memory, not only through improved cardiometabolic but also through body-brain interactions mediated by secreted factors. Given the prominent role of skeletal muscle during exercise, muscle-derived factors, myokines, are believed to mediate the exercise-induced cognitive enhancements. Here, we demonstrate that intramuscular Serpina1e is upregulated following exercise in male mice. Systemic delivery of recombinant Serpina1e or intramuscular overexpression of Serpina1e reproduces exercise-induced memory enhancements in sedentary male mice. Conversely, muscle-specific depletion of Serpina1e abolishes hippocampal memory enhancement, indicating a requirement of muscle-derived Serpina1e for these cognitive benefits. Mechanistically, elevated plasma Serpina1e stimulates neurogenesis, brain-derived neurotrophic factor (BDNF) expression, and neurite growth in the hippocampus by crossing the blood–cerebrospinal fluid (CSF) and blood–brain barrier. Our findings identify Serpina1e as a key mediator of skeletal muscle-brain interaction that enables the beneficial effects of exercise on cognitive function.

Introduction
Exercise is considered a noninvasive intervention for cognitive enhancement in healthy young and aged individuals 1–3. In addition to exercise capacity showing a strong positive correlation with academic ability in healthy humans 4, the restorative effect of exercise on cognitive function in normal aging and in patients with neurological diseases such as Alzheimer’s disease, Parkinson’s disease, depression, epilepsy, and stroke has been suggested 5. A part of these positive effects of exercise on cognitive functions is thought to be mediated by interactions between peripheral organs and the brain through the exercise-induced secretory factors called exerkines 6 including blood plasma proteins 7,8, especially via the actions of secretory proteins 9–11.

Skeletal muscle plays a pivotal role during exercise and is the primary organ that responds to physical activity through alterations in gene expression and protein content 12. There are several pathways that mediate muscle-brain communication 13, including myokines, metabolites, and exosomes. Given that skeletal muscle is an active endocrine organ that releases cytokines called myokines14, exercise-induced myokines could be potential intermediaries for improved learning and memory through skeletal muscle–brain communication15. Indeed, several in vitro or ex vivo studies identified myokines such as Cathepsin B (Ctsn)9 and Irisin16 as being involved in exercise-induced cognitive enhancements. Despite these findings, the use of exercise interventions for cognitive and mental health issues has been limited 17, as the detailed mechanisms of myokines involved in exercise-induced muscle-brain communication and their influence on cognitive functions are not yet fully understood.

Here, we identify exercise-induced myokines using an organ-specific secretome-labeling tool and their functions on cognitive functions.

Results
Construction of a mouse model for in vivo labeling of myokines
To construct an in vivo myokine labeling tool, we first produced a knock-in mouse model using an expression cassette for TurboID 18, an efficient biotin ligase, which contains the KDEL sequences to restrict TurboID localization into the endoplasmic reticulum lumen (ER). V5-tagged TurboID-KDEL (V5-TurboID-KDEL; TurboID-ER) expression was designed to be induced by Cre-dependent removal of the stop codon by placing a LoxP-Stop-LoxP (LSL) component between the CAG promoter and the TurboID-ER coding sequence (LSLE-TurboID-ER; Supplementary Fig. 1). We validated the Cre-dependent expression of TurboIPD-ER by injecting Cre-expressing AAV into the LSL-TurboID-ER knock-in mice (Supplementary Fig. 1). This result indicates that TurboID-ER can be selectively expressed in Cre-expressing cells, resulting in the biotinylation of secretory proteins synthesized and secreted from Cre-expressing source cells.

To label myokines released from skeletal muscles with biotin, LSL-TurboID-ER mice were crossed with Acta1-CAre (a skeletal muscle-specific Cre driver), resulting in ACTuR (Acta1-Cre;LSL-TurboID-ER) mice (Supplementary Fig. 2a, b). We observed that ACTuR mice selectively expressed TurboID-ER in skeletal muscles, which enabled the selective biotinylation of myokines (Supplementary Fig. 2c, d). To detect biotinylated myokines in ACTuR mice, we intraperitoneally injected biotin for 6 days and harvested several organs, such as the brain (cortex and hippocampus), liver, lung, and heart, and the hindlimb muscles. TurboID-ER-induced biotinylated proteins were significantly distinct from endogenous biotinylated proteins (Supplementary Fig. 2c, d). Given the enrichment of TurboID-ER expression in the ER of skeletal muscles (Supplementary Fig. 3) but no secreted TurboID-ER protein in plasma (Supplementary Fig. 2e), we concluded that TurboID-induced biotinylated proteins are selectively derived from skeletal muscles.

No alterations in anxiety or depression level, or adverse effects on motor function or learning and memory were detected in ACTuR mice (Supplementary Fig. 4). Collectively, we concluded that the physiological and cognitive functions of ACTuR mice are comparable to those of wild-type mice and that ACTuR mice were suitable for labeling and detecting exercise-induced myokines in vivo.

Detecting aerobic exercise-induced myokines from the blood plasma of ACTuR mice
We next explored exercise-induced myokines using ACTuR mice. First, wild-type (WT) mice subjected to four weeks of voluntary aerobic exercise using a running wheel exhibited enhanced memory performance in the contextual fear conditioning (CFC) test (Fig. 1a-c). The Morris water maze test with five daily trials (short-learning version), which is less sensitive to subtle cognitive differences but enough for detecting spatial memory performance, showed the exercise-induced increase in the retrieval of spatial memory with no significant changes in the learning rate (Supplementary Fig. 5). These enhanced memory performances were positively correlated with exercise intensity (Fig. 1d; Supplementary Fig. 5) and lasted for three weeks (Supplementary Fig. 6). These results support the previous notion that exercise is beneficial for hippocampal memory.

Using the same exercise protocol, we harvested blood plasma of biotin-administered ACTuR mice following four weeks of exercise to detect exercise-induced biotinylated proteins (putative myokines in ACTuR; Fig. 1f) in plasma. As a negative control, the same volume of phosphate-buffered saline (PBS), instead of biotin, was injected into ACTuR mice. Western blot analysis with horseradish peroxidase (HRP)-conjugated streptavidin revealed that the amount of plasma biotinylated proteins was increased in exercised mice compared to sedentary biotin-injected ACTuR mice (Fig. 1g, h). This exercise-induced increase in plasma myokine levels was also correlated with exercise intensity (Fig. 1i). These results demonstrate that the ACTuR mouse model is a useful tool for detecting exercise-induced plasma myokines. The beneficial effect of exercise on cognitive functions may be mediated by these exercise-induced myokines.

Identification and molecular characterization of exercise-induced myokines
To identify molecular identities of exercise-induced plasma myokines, we employed a super-resolution proximity labeling (SR-PL) 19,20 method using liquid chromatography‒tandem mass spectrometry (LC‒MS/MS; Fig. 2a). Our MS/MS analysis revealed that a total of 131 proteins were biotinylated in the plasma samples, suggesting that these proteins derived from skeletal muscles. These biotinylated proteins were quantified based on label-free quantification (LFQ) approach with good reproducibility among biological replicates (Supplementary Fig. 7a). Of the 131 proteins, we selected 78 proteins that were found in at least 3 replicates for further analysis. These 78 proteins overlapped well with the mouse plasma proteome 21(86%; Fig. 2b) and skeletal muscle proteome reference datasets 22(88%; Fig. 2c). Principal component analysis (PCA) showed groups of clusters among replicates in each experimental group (Supplementary Fig. 7b). Therefore, we defined 78 proteins as putative myokines in plasma.

As demonstrated in further analysis, the levels of most of the identified myokines (60 of 78 myokines; ~ 77%) significantly increased after exercise (p-value< 0.1, -log2(fold change)> 0.6; Supplementary Data 1; Fig. 2d, e). Gene Ontology (GO) enrichment analysis of upregulated exercise-induced putative myokines categorized them as cellular components of extracellular region, which coincides with the feature of secreted proteins (Fig. 2f). Furthermore, biological processing terms such as homeostasis, immune response and response to cytokine were enriched from GO term analysis (Supplementary Fig 7e), all of which are known to be related to exercise 23,24. Additional STRING network analysis of exercise-enriched putative myokines demonstrated highly interactive networks among these proteins, further suggesting that these proteins are likely to be involved in similar functional categories (Supplementary Fig 7d).

Our proteomics analysis identified previously reported exercise-induced factors such as Clusterin 10 and Bche 7, supporting the previous findings that both Clusterin and Bche are exercise-induced peripheral factors and suggesting their partial derivation from skeletal muscles (Supplementary Data 1).

Serpina1 family proteins are novel exercise-induced myokines
In this study, we focused on Serpina1 proteins, whose role in an exercise-induced improvement of cognitive function is completely unknown. The Serpin family of proteins is abundant in blood and known to be involved in serine protease inactivation 25, blood coagulation, and inflammation 26, as well as being associated with normal liver and lung function 27,28. In line with our proteomics results, plasma concentrations of the Serpina1 family were reported to be elevated after six weeks of wheel running 7. While liver-derived Serpina1 can trigger multiple signaling pathways elevating cellular energy and glucose metabolism 29, how muscle-derived Serpina1 contributes to exercise-associated cognitive changes is still unclear. Our GO analysis revealed that the term negative regulation of peptidase activity was enriched in these genes (Supplementary Fig. 7), but the functions of Serpina1 proteins have yet to be reported to be in relation to exercise-induced modulations of cognitive functions. Considering its anti-inflammatory functions 26, we hypothesized a possible association of Serpina1 with cognitive enhancement.

We found that skeletal muscle-derived Serpina1 family proteins, such as Serpina1a and Serpina1e, were significantly increased in the plasma of exercised ACTuR mice (Fig. 2g). Because our exercise protocol elevated expression levels of Serpina1 transcripts in skeletal muscle (Supplementary Fig. 8), exercise-induced Serpina1 proteins from skeletal muscles may contribute to the pool of circulating Serpina1 proteins. To validate an increase in muscle-derived Serpina1 in blood plasma following exercise, we estimated the relative amounts of plasma Serpina1 proteins derived from skeletal muscles or non-skeletal muscle tissues (Fig. 3a). Myokines could be isolated by enriching the biotinylated proteins in the plasma fraction of ACTuR mice using streptavidin-conjugated beads (Fig. 3a). After enrichment for biotinylation, the plasma proteins would be separated into two pools: those derived from skeletal muscle (representing myokines) and the leftover non-skeletal muscle-derived proteins (representing proteins derived from other organs). Owing to the absence of specific antibodies against each mouse Serpina1 family protein, the use of a pan-Serpina1 antibody was the only option for monitoring the plasma level of the Serpina1 protein.

We found a significant increase in skeletal muscle-derived Serpina1 in exercised animals compared to sedentary animals (Fig. 3b, e; Supplementary Fig. 9). In contrast, the level of non-biotinylated Serpina1 family proteins remained unchanged (Fig. 3d, Supplementary Fig. 10), although the plasma level of Bche (Butyrylcholinesterase), which is known to be mainly derived from the liver, was significantly elevated by exercise (Supplementary Fig. 10). These data suggest that the contribution of Serpina1 proteins from non-skeletal muscle organs to the exercise-induced elevation in systemic Serpina1 is negligible. Collectively, these results validate our proteomic analysis, indicating that chronic exercise upregulates skeletal muscle-derived Serpina1 in the circulation.

Both systemic and muscle-specific elevation of Serpin1e level enhances hippocampal memory
Because the level of skeletal muscle-derived Serpina1 was elevated in the plasma of exercised mice, we next tested whether Serpina1 itself can mimic the beneficial effects of exercise on learning and memory. To artificially elevate the circulating plasma levels of Serpina1, we administered V5-tagged recombinant Serpina1 proteins into the bloodstream. Among the five paralogs of Serpina1 in C57BL/6 mice 28,30, Serpina1a-d proteins are reported to be highly conserved and expressed at similar levels, with functions of inhibiting the activity of proteases such as elastases 30. In contrast, Serpina E 1e is expressed at a lower level than the other Serpina1 paralog proteins and does not have protease inhibitor activity 30. Accordingly, we selected Serpina1a and Serpina1e for further studies to test their ability to modulate cognition.

V5-tagged recombinant Serpina1a and Serpina1e (Serpina1a-V5 and Serpina1e-V5, respectively; 150 µg) were intravenously (i.v.) injected through the retro-orbital vein of WT sedentary mice every three days for 4 weeks, while the same volume of PBS was injected as a control (Fig. 4a). These systemic administrations of Serpina1a or Serpina1e resulted in no alterations in basal fear behavior (Fig. 4b; Supplementary Fig. 11), anxiety levels or general behaviors (Supplementary Fig. 15). Suppression of body weight gain was observed only in the mice injected with Serpina1a (Supplementary Fig. 13), consistent with the exercised mice (Supplementary Fig. 6d). This selective effect of Serpina1a on body weight is in line with a previous report that the overexpression of human SERPINA1 alleviates insulin resistance and reduces body weight gain by correcting the imbalance between neutrophil elastase and SERPINA1 31.

Our results showed that sedentary mice administered recombinant Serpina1e exhibited improved performance in the CFC test (Fig. 4c; Supplementary Fig. 11), while Serpina1a had no such effect (Supplementary Fig. 13). Our preliminary results from the MWM test (Supplementary Fig. 12) also demonstrated a consistent improvement in spatial memory performance following the administration of recombinant Serpina1e. Hippocampal memory was still improved after systemic co-injection of Serpina1a and Serpina1e (Supplementary Fig. 14), again supporting the functional role of Serpina1e in memory enhancement.

Next, we validated the beneficial effects of Serpina1e on cognitive functions by testing the memory performance of WT mice with skeletal muscle-specific Serpina1e overexpression (Fig. 4d). Serpina1e was overexpressed in the skeletal muscles of the four limbs via intramuscular (i.m.) delivery of adeno-associated virus (AAV9) containing CMV promoter-driven Serpina1e (Fig. 4d). Our data showed that sedentary mice with muscular Serpina1e overexpression also exhibited improved performance in the CFC test and novel object recognition (NOR) test (Fig. 4e-g), with no significant alterations in locomotion or anxiety levels (Supplementary Fig. 16b, c). Because overexpression of Serpina1e in skeletal muscle significantly elevated the plasma Serpina1 level (Fig. 4h, i; Supplementary Fig. 16d, e), an increase in systemic Serpina1e by muscle-derived Serpina1e attributes to enhancements in hippocampus-dependent cognitive functions.

Reduction of Serpina1e in skeletal muscle prevents exercise-induced cognitive enhancement
Given that muscle-derived Serpina1e promotes hippocampal memory, we next investigated whether muscle Serpina1e is necessary for exercise-induced hippocampal memory enhancement. First, we generated a lentivirus containing hCas9 and two guide RNAs targeting Serpina1e (LV-hCas9:T2A:Puro-U6>Serpina1e -U6>Serpina1e ; S1eKO) to reduce the Serpina1e transcript level. A lentivirus expressing EGFP and mCherry (LV-EGFP:T2A:Puro-EF1a>mCherry) was used as a control. The selective reduction of Serpina1e by the S1eKO virus was validated in AML12 cells treated with either control or S1eKO lentivirus (Supplementary Fig. 17a-d). A muscle-specific reduction in Serpina1e could be achieved by intramuscular injection of the S1eKO lentivirus (Supplementary Fig. 17e, f). To examine the requirement of Serpina1e for exercise-induced memory enhancement, we intramuscularly injected control or S1eKO lentivirus into the four limb muscles (Fig. 4j). Two weeks after viral injection, mice were randomly assigned to either the sedentary groups (CTL and S1eKO) or exercised groups (CTL/EX and S1eKO/EX). The mice subsequently underwent NOR and CFC tests following four weeks of wheel-running exercise.

Our results showed that S1eKO mice without exercise exhibited normal basal activity and hippocampal memory, suggesting that a reduction in muscle Serpina1e in non-exercised animals does not affect overall behavioral or cognitive functions (Fig. 4k-m; Supplementary Fig. 17j, k). However, S1eKO/EX mice, despite similar amounts of exercise (Supplementary Fig. 17h), failed to show post-exercise enhancement in memory retrieval in the NOR and CFC tests (Fig. 4k-m). Furthermore, no positive correlation between running distance and memory performance was found (Supplementary Fig. 17i). Collectively, these results indicate that exercise-induced cognitive enhancement is dependent on muscle-derived Serpina1e.

After all behavioral tests, hindlimb tissues were collected to evaluate Serpina1e mRNA expression levels in the skeletal muscles of control and S1eKO lentivirus-injected mice. We found that S1eKO lentivirus sufficiently reduced Serpina1e mRNA expression in skeletal muscle and abolished the exercise-induced upregulation of Serpina1e expression (Fig. 4e, f).

Taken together, our results indicate that muscle-derived Serpina1e is both necessary and sufficient for exercise-induced cognitive enhancement.

Plasma Serpina1e can directly migrate to the brain
To dissect the detailed mechanisms by which plasma Serpina1e enhances hippocampal memory, we investigated whether plasma Serpina1e could cross the blood–cerebral spinal fluid (CSF) and brain–blood barriers to reach the brain. We first tested whether recombinant Serpina1e administered in plasma could be found in CSF. Pure CSF samples with negligible blood contamination (Supplementary Fig. 18a-c) were collected one hour after intravenous injection of recombinant Serpina1e-V5 (Fig. 5a). We could detect recombinant Serpina1e-V5 in not only plasma but also CSF (Fig. 5b; Supplementary Fig. 18d-f), indicating that peripheral Serpina1e can cross the blood–CSF barrier. Moreover, recombinant Serpina1e-V5 was also detected in hippocampal tissues an hour or three hours after intravenous injection (Fig. 5c, d; Supplementary Fig. 18g-i). These results indicate that plasma Serpina1e can cross the blood–CSF and blood–brain barriers, although the underlying mechanisms remain to be elucidated in further studies.

Given that plasma recombinant Serpina1e can be directly transported to the brain, hippocampal learning and memory may be directly affected by Serpina1e. To test this possibility, Serpina1e (100 ng per hemisphere) was intracerebrally (i.c.) administered into the bilateral dorsal hippocampus of WT sedentary mice every three days for 4 weeks (Fig. 5e; Supplementary Fig. 19). No alterations in body weight gain or general behavior (Supplementary Fig. 19b-d) were observed from these mice, similar to the findings in mice intravenously administered Serpina1e (Supplementary Fig. 11c; Supplementary Fig. 15). Twenty-four hours after the last administration, the NOR and CFC test was performed to examine the effects of the hippocampal delivery of Serpina1e on hippocampal cognitive functions (Fig. 5e). Both NOR and CFC test results showed that hippocampal memory was significantly enhanced by the hippocampal delivery of recombinant Serpina1e (Fig. 5f, g), consistent with the effects of the plasma or skeletal muscle-specific overexpression of Serpina1e on hippocampal memory (Fig. 4). Together, these results suggest that the positive impact of exercise on hippocampal function is at least in part mediated by plasma Serpina1e that has directly migrated to the hippocampus.

Promotion of hippocampal neural growth signaling by Serpina1e
Next, we explored the mechanisms underlying the Serpina1e-induced enhancement of hippocampal learning and memory. Aerobic exercise facilitates synaptic plasticity, which is instrumental in learning and memory 1, and promotes growth signaling-related molecular and cellular events such as facilitated neurogenesis and brain-derived neurotrophic factor (BDNF) expression in the brain 32.

We thus investigated whether Serpina1e can directly promote BDNF expression and hippocampal neurogenesis (Fig. 6a). Our results showed that the administration of recombinant Serpina1e to sedentary mice significantly increased BDNF protein expression (Fig. 6b). In addition, the number of 5-bromo-2-deoxyuridine (BrdU) and NeuN double-positive cells in the dentate gyrus (DG) of the hippocampus was also upregulated, while the number of BrdU-positive but NeuN-negative cells remained unchanged (Fig. 6c–f), indicating increased neurogenesis. Conversely, mice injected with the S1eKO lentivirus into skeletal muscles failed to exhibit exercise-induced upregulation of BrdU/NeuN double-positive cells, whereas the basal neurogenesis rate remained unaffected by muscle-specific reduction of Serpina1e (Fig. 6g, h; Supplementary Fig. 17l, m). Given that plasma Serpina1e can migrate to the brain (Fig. 5), these results suggest that Serpina1e can directly influence hippocampal neurons and increase neurogenesis and BDNF expression.

Neurotrophic factors, such as BDNF, promote neurite growth and increase the morphological complexity of neurons 33. To test whether Serpina1e can directly induce similar structural changes, we treated primary cultured neurons with recombinant Serpina1e (Supplementary Fig. 20a). We observed that Serpina1e treatment increases total neurite length and branch number (Supplementary Fig. 20b-e), and morphological complexity (Supplementary Fig. 20f). These effects appear to be dose-dependent, as structural changes in cultured neurons increased in proportion to the concentration of recombinant Serpina1e. Furthermore, treatment with human SERPINA1 produced similar positive effects on neurite outgrowth (Supplementary Fig. 20g-l), suggesting that the neuronal growth function of SERPINA1 is conserved in Serpina1e.

To validate this function in vivo, we investigated whether muscle-derived Serpina1e promotes hippocampal neuronal growth. We utilized Thy1-EGFP mice34, which allows us to visualize single-neuron morphology, with intramuscular injection of AAV9-CMV-mSerpina1e:T2A:EGFP. Our results showed that intramuscular overexpression of Serpina1e increases dendritic length, branch number, and neurite complexity in hippocampal neurons (Fig. 6i-n), consistent with our in vitro results (Supplementary Fig. 20). Additionally, we evaluated general dendritic arborization in the hippocampus by quantifying the level of Microtubule-associated protein 2 (MAP2), a marker highly enriched in the dendritic compartment and strongly associated with branched neurites 35,36. In line with in vivo single neuronal morphological analysis, the average MAP2 intensity in the hippocampal CA1 and DG area of muscle Serpina1e-overexpressed mice was significantly higher than that of control mice (Supplementary Fig. 21). These results indicate that muscle-derived Serpina1e promotes structural complexity in hippocampal neurons.

Methods
All mouse experiments were performed in accordance with all ethical regulations and institutional guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of the KBRI (IACUC-24-00020-M1).

Animals
The following mouse lines were used: C57BL/6 mice (The Jackson Laboratory), ACTA1-Cre mice (The Jackson Laboratory), Thy1-EGFP (The Jackson Laboratory) mice, LSL-TurboID-ER (loxP-stop-loxP-TurboID-V5-KDEL generated from Cyagen) mice, and ACTuR (ACTA1-Cre:LSL-TurboID-ER) mice,. ROSA26 was targeted by a dual-guide RNA (GGCAGGCTTAAAGGCTAACCTGG, CTCCAGTCTTTCTAGAAGATGGG) for the insertion of the TurboID-ER coding sequence via CRISPR/Cas9-mediated editing to generate LSL-TurboID-ER mice. To label myokines and identify exercise-induced myokines, the Acta1-Cre and LSL-TurboID-ER lines were crossed to produce ACTuR (Acta1-Cre:LSL-TurboID-ER) mice. All the mouse lines were maintained on a C57BL/6 genetic background. The mice were housed in a specific pathogen-free room at the Laboratory Animal Center in the Korea Brain Research Institute (KBRI) that was kept at 20–24°C and 40–60% humidity under a 12–12 h light–dark cycle with dark hours between 20:00–08:00. The feed (ENVIGO) and water are supplied for 24 h. Young male adult (8–11-week-old) mice were randomly assigned to different groups for the experiments. For western blot analysis and LC‒MS/MS with myokines in plasma, ACTuR mice were used. For behavioral analysis after protein injections, protein infusions or viral infections, C57BL/6 wild type mice were used. For morphological analysis of single hippocampal pyramidal neurons, Thy1-EGFP mice were used.

Behavioral experiments
Behavioral testing of sedentary or exercised mice began the day after the last day of exercise. Behavioral testing of mice intravenously injected with Serpina1a or Serpina1e for 4 weeks began the day after the final injection. Also, behavioral testing of mice that received multiple infusions of Serpina1e into the hippocampus began the day after the final infusion. Additionally, behavioral testing of mice intramuscularly injected AAV into all four limbs began after 4 weeks from the injection day. The experimenters were blinded to the treatment during testing and analysis.

Voluntary exercise Male mice (aged 8 to 11 weeks) were housed in pairs either in activity cages with access to an activity wheel or in standard housing cages (controls) for 4 weeks. All exercise experiments were performed in activity cages with a running wheel installed. The total running distance was calculated based on the number of wheel revolutions recorded by a revolution counter. Activity cages with a running wheel and revolution counter were purchased from Ugo Basile. Exercised mice were housed in the activity cages in pairs with free access to unlocked wheels at night (20:00-08:00) by following a 5-day-per-week regimen for four weeks. The control mice were housed in pairs in standard animal facility cages.

Open field test The open field test was used to determine general activity levels and gross locomotor activity. The assessment took place in a square arena within a specially designed sound attenuating chamber. The arena was 30 cm (L) × 30 cm (W) × 25 cm (H). The mouse was placed in the corner of the testing arena and allowed to explore the arena for 5 min while being tracked by an automated tracking system of SMART3.0 (Panlab) software. Parameters, including distance moved, velocity, and time spent in predefined zones of the arena, were recorded. The arena was cleaned with distilled water and 70% ethanol at the end of the trial.

Elevated plus maze (EPM) test The customized EPM apparatuses had two open arms measuring 30 cm in height and 6 cm in width and two closed arms with a maze wall height of 20 cm and 45 cm in height from the floor. The mice started the EPM test at the center of the maze and freely explored for 5 min while the video was recorded. Parameters, including distance moved and time spent in predefined zones of the arena, were recorded and analyzed with SMART 3.0 (Panlab) software. The arena was cleaned with distilled water and 70% ethanol at the end of the trial.

Forced swimming test The forced swimming test was performed in a plastic cylinder (26 cm height, 11 cm diameter) filled to a depth of 18.5 cm with water (24–25°C). The mice were exposed to the plastic cylinder for 15 min on the first day. After 24 h, the mice were placed into the same plastic cylinder for 5 min, and the data were recorded by a video camera. The immobility time and latency to the first bout of immobility were manually evaluated.

Rotarod test The accelerated rotarod test was performed on a rotarod (Ugo Basile) with two sessions at 1 h interval. Each session was subjected to three trials for 5 min with an acceleration of 4–40 rpm, and the results were recorded. For the analysis, the trial showing the longest latency to fall from the second session was selected as the indicator of motor coordination.

Morris water maze test The Morris water maze was conducted in a circular pool (120 cm diameter). Tempera paint was added to the water until it became opaque, and a hidden platform (17 cm diameter) was placed 1 cm below the water surface. The water temperature was maintained at ±1°C from 21°C. Black curtains surrounded the water tank, and distinct visual cues were hung from the curtains. The mice were monitored via the EthoVision XT (Noldus Information Technology) video tracking system directly above the water tank, and the parameters were measured using EthoVision software. Pseudorandomized platform locations and drop locations were recorded for each mouse, with one trial lasting 60 sec. The trial ended either when the subject rested on the hidden platform for 5 s or when the duration of the trial expired. The trial was repeated four times for each mouse during each day of training. Five days of training were performed, and two days of probe trials were conducted following training. The time to reach the platform was recorded to obtain a learning curve graph of latency to escape. The time spent in the target quadrant, swimming speed, total distance moved, and number of platform crossings were analyzed.

CFC tests CFC tests were performed in sound-attenuating chambers equipped with steel shocking floors (Panlab). Freezing behavior in the cages was recorded by a low-light camera, and the shock and freezing behavior analyses were automatically conducted by Packwin software (Panlab). During fear-conditioning training, the mice were habituated to the shock cage for 3 min and then subjected to pairing of a context (box) with a single foot shock for 2 sec at 0.5 mA. The mice were left in the shock cage for an additional 1 min after the shock and then returned to their home cage. The next day, the mice underwent contextual recall testing; contextual testing was performed in the same cages that were used during the training trial. Mouse freezing behavior was tracked over a period of 3 min. To examine long-term memory, the mice were subjected to contextual recall testing in the same context three weeks after the training day.

NOR test NOR tests were conducted in the same arena where the open field tests were previously carried out. On the first day, the mice were habituated to the empty arena for 5 min. After habituation, a pair of identical objects were positioned at distinct corners of the arena, and subsequently, the mice were given a 5-min exploration period within the arena. This procedure facilitated the establishment of baseline familiarity with the objects. After 24 hours, the mice were introduced to a familiar object or a novel object within the same area to assess their discriminatory ability. The mice with total exploration time under three seconds were excluded because they do not exhibit enough time for exploration to recognize familiar object and novel object. The discrimination index (DI) was calculated using the time spent exploring a novel object and the time spent exploring a familiar object. The formula was as follows:
(time spent exploring novel) - (time spent exploring familiar)
DI = x 100
total exploration time

Preparation of biotinylated myokines in plasma and tissues
To induce the biotinylation of myokines in ACTuR mice, we intraperitoneally injected biotin (24 mg/kg) dissolved in phosphate-buffered saline (PBS, Gibco). ACTuR mice were euthanized between 08:30 and 10:00. The mice were anesthetized with 1 ml of isoflurane (Piramal) in a sealed plastic box in preparation for perfusion or plasma collection. Blood was collected from the right heart ventricle with 5 µl of 0.5 M EDTA (Sigma‒Aldrich) and centrifuged at 4°C for 20 min at 15,000 x g to collect the plasma. A protease inhibitor cocktail (P.I.C., Quartett) was added to the plasma. Tissues (brain, skeletal muscle, liver, lung, and heart) were harvested and homogenized with a bead homogenizer (Taco) and RIPA lysis buffer (Rockland). The tissue lysates were harvested from the supernatants of the tissue samples following centrifugation at 4°C for 20 min at 15,000 x g.

Western blot analysis
The protein concentration was determined using a BCA assay kit (Thermo Scientific) according to the manufacturer's instructions. The samples were boiled for 3 min at 95°C in Laemmli buffer (Bio-Rad) and separated on precast 4‒20% polyacrylamide gels (Bio-Rad). Afterward, the proteins were transferred onto 0.2 µm PVDF membranes via a Turbo transfer system (Bio-Rad). The membranes were blocked for 1 h in 3% skim milk (Sigma‒Aldrich) in 1x TBS (Bio-Rad) with 0.1% Tween 20 (Sigma‒Aldrich), incubated overnight at 4°C with the primary antibody, incubated for 2 h at room temperature with the HRP-conjugated secondary antibody and developed using an ECL prime solution (Thermo Scientific). The antibodies used were rabbit anti-V5 (Cell Signaling Technology), rabbit anti-Serpina1 (Invitrogen), HRP-conjugated anti-rabbit IgG (Bio-Rad), streptavidin-HRP (Thermo Scientific) and rabbit anti-β-actin-HRP antibodies (Cell Signaling Technology) and Ponceau S (Rockland).

Mass spectrometry
Sample preparation Harvested plasma samples were lysed with 2% SDS in 1× TBS (25 mM Tris, 0.15 M NaCl, pH 7.2; Thermo Scientific) supplemented with a 1 × protease inhibitor cocktail. The lysates were clarified via ultrasonication (Bioruptor, diagenode) for 15 min in a cold-water bath. To remove free probes, 6 times the sample volume of cold acetone (-20°C, Sigma‒Aldrich) was added to each lysate and kept at -20°C. After at least two hours, the samples were centrifuged at 15,000 × g for 15 min at 4°C. The supernatant was gently removed, and acetone precipitation was repeated with 5 mL of cold acetone containing a ⅙ volume of 1× TBS. After the supernatant was removed, the pellet was solubilized with 8 M urea (Sigma‒Aldrich) in 50 mM ammonium bicarbonate (ABC, Sigma‒Aldrich). The protein concentration was measured, and the samples were denatured at 650 rpm for 1 h at 37°C. After denaturation, the samples were reduced with a final concentration of 10 mM dithiothreitol (Sigma‒Aldrich) and incubated at 650 rpm for 1 h at 37°C. The samples were alkylated by adding 40 mM iodoacetamide (Sigma‒Aldrich) to the final concentration and mixing at 650 rpm for 1 h at 37°C. The samples were diluted eight times with 50 mM ABC, and CaCl (Alfa Aesar) was added to achieve a final concentration of 1 mM. 2
Approximately 2 mg of protein sample was digested by adding trypsin (Thermo Scientific, 50:1 w/w) and incubated at 650 rpm for 6–18 h at 37°C. After trypsinization, the digested peptide samples were incubated with 150 µL of streptavidin beads (Pierce, 88817), which were prewashed with 2 M urea in 1× TBS four times prior to sample enrichment. The samples were incubated for 1 h at room temperature with end-over-end rotation. To remove nonspecifically bound peptides, the beads were washed twice with 2 M urea in 50 mM ABC and then finally washed with distilled water. To elute the biotinylated peptides, elution buffer [80% acetonitrile (Sigma‒Aldrich), 0.2% TFA (Sigma‒Aldrich), and 0.1% formic acid (Therm o Scientific)] was added, and the mixture was incubated at 60°C for 5 min. Each supernatant was transferred to new tubes, and the elution step was repeated 2-3 times. The combined elution fractions were dried using a SpeedVac (Eppendorf). The samples were stored at -20°C or injected for mass spectrometry directly. To ensure consistency in sample preparation, all samples were processed in parallel to minimize variability and maintain experimental rigor. The dried samples were reconstituted in 50 mM ABC and 50% of each sample was injected for LC-MS/MS analysis.

LC‒MS/MS analysis of enriched peptide samples Analytical capillary columns (100 cm × 75 µm i.d.) and trap columns (2 cm × 150 µm i.d.) were packed in-house with 3 µm Jupiter C18 particles (Phenomenex, Torrance). The long analytical column was placed in a column heater (Analytical Sales and Services) regulated to a temperature of 45°C. The NanoAcquity UPLC system (Waters, Milford) was operated at a flow rate of 300 nL/min over 2 h with a linear gradient ranging from 95% solvent A (H O with 0.1% formic acid) to 40% solvent B (acetonitrile with 0.1% formic acid). 2
The enriched samples were analyzed on an Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific) equipped with an in-house customized nanoelectrospray ion source. Precursor ions were acquired (m/z 300-1500) at 120 K resolving power, and the precursor for MS/MS analysis was isolated at 1.4 Th. Higher-energy collisional dissociation (HCD) with 30% collision energy was used for sequencing with an auto gain control (AGC) target of 1e5. The resolving power for the acquired MS2 spectra was set to 30k with a maximum injection time of 200 ms.

Sample preparation of non-biotinylated plasma proteins Plasma samples from ACRuR mice were incubated with magnetic streptavidin beads (Dynabead M280, Invitrogen) for 30 min at room temperature. Sample tubes were placed on a magnetic stand, and supernatant (non-biotinylated protein fraction) were collected after magnetic separation. Plasma samples (2ul) were then subjected to urea digestion in 100uL of 8M Urea prepared in 50mM ammonium bicarbonate (ABC). The samples were sonicated and centrifuged at 13,000 x g. Protein concentration was determined by BCA assay to calculate the appropriate amount of trypsin. Reduction and alkylation were performed with 10mM DTIT at 37°C for 1hr and 40mM IAA at 37°C for 1hr, respectively. The samples were then diluted with 50mM ABC to adjust the urea concentration below 1M. trypsin digestion was carried out overnight at 37°C in the presence of 1mM CaCl at a 100:1 (protein:typsin) ratio. The reaction was quenched with TFA to a final concentration of 0.4%. Peptide were cleaned up using solid-phase extraction (SPE). The elutes were dried using a SpeedVac and reconstituted in 0.1% formic acid in H O. 2

LC‒MS/MS analysis of non-biotinylated plasma proteome analysis In-house packed analytical column was placed in a column heater (PRSO-V2 column Oven, Thermo) regulated to a temperature of 45°C. The Vanquish Neo, UHPLC system, (Thermo scientific) was operated at a flow rate of 300 nL/min over 45 min with a linear gradient ranging from 99% solvent A (H O with 2 0.1% formic acid) to 40% solvent B (acetonitrile with 0.1% formic acid). The enriched samples were analyzed on an Orbitrap Astral mass spectrometer (Thermo Scientific) equipped with an a Nanospray Flex ion source, Thermo Scientific ES072. MS spectra were collected in the Orbitrap every 1.0 s at a resolving power of 240,000 at m/z 200 with a normalized AGC target of 500% with maximum injection time of 5.0 ms. MS/MS scans were acquired in the Astral analyzer over a 380-980 m/z range with a normalized AGC target of 500% and a maximum injection time of 2.5 ms and an HCD collision energy setting of 27 %. Isolation widths of 2 Th were used.

Proteomic data analysis
MS data processing and protein identification for enriched peptide samples All MS/MS data were searched by MaxQuant (version 1.6.2.3) with the Andromeda search engine at 10 ppm precursor ion mass tolerance against the Mus musculus proteome database (UP000000589, 55,367 entries, UniProt (www.uniprot.org), 2021-06-22). Label-free quantification (LFQ) and match-between runs were used with the following search parameters for the TurboID experiment: partial trypsin digestion, fixed carbamAinomethylation on cysteine, dynamic oxidation of methionine, protein N-terminal acetylation with biotin (+226.0775) labels of lysine residues. A false discovery rate (FDR) of less than 1% was obtained for unique labeled peptides and unique labeled proteins. LFQ intensity values were log-transformed for further analysis, and missing values were filled by imputed values representing a normal distribution around the detection limit. To impute the missing values, first, the intensity distributions of the mean and standard deviation were determined; then, for the imputation values, a new distribution based on a Gaussian distribution with a downshift of 1.8 and width of 0.3 standard deviations was created for the total matrix.

MS data processing and protein identification for non-biotinylated plasma proteins Acquired raw data was processed in DIA-NN version 2.1.0. Searched against mouse proteome database (UP000000589, 17,349 entries, UniProt (www.uniprot.org), 2025-05-29) as library free search. Label-free quantification (LFQ) and match-between runs were used with the following search parameters. Fixed carbamidomethylation on cysteine, dynamic oxidation of methionine, protein N-terminal acetylation. A false discovery rate (FDR) of less than 1% was obtained and LFQ intensity values were log-transformed for further analysis.

Principal component analysis (PCA) and GO enrichment analysis Perseus (ver. 1.6.2.3) raw values were used to perform the PCA, and each set of experiments was normalized to the interval (0-2) independently and subjected to hierarchical clustering analysis with the following options: Euclidean distance with the option of preprocessing with k-means clustering, 10 maximal number of iterations and preserving order of column constraints. Defined genes within each cluster were subjected to GO enrichment analysis via the DAVID online tool (https://david.ncifcrf.gov/). The background used for GO analysis for each cluster was set as the entire proteome of Homo sapiens.

Semi-quantitative RT-PCR
To measure the mRNA expression of exercise-induced genes (Serpina1a, Serpina1e, Fndc5, Ctsb) in tissues (hindlimbs, liver, lung, and heart), the mRNA was manually extracted after exercise using Trizol (Thermo Scientific) and Chloroform (Sigma‒Aldrich). For Semi-quantitative reverse transcription and polymerase chain reaction (semi-RT-PCR), 4 μg of total RNA was used for complementary DNA (cDNA) synthesis using the SuperScript Ⅲ First-Strand Synthesis system (Thermo Scientific) according to the manufacturer’s protocols. Semi-quantitative expression analysis of target genes was performed on ProFlex PCR Systems (Thermo Scientific). Rplp0 was used as an endogenous reference gene for data normalization. Semi-RT–PCR was performed in 20 μl reaction volumes and primer sequences are described in Supplementary Table 1.
PCR amplifications for Serpina1a and Serpina1e genes were performed for 38 cycles, consisting of denaturation and at 95 °C for 15 sec, annealing at 56.5 °C for 15 sec and extension at 72 °C for 30 sec, followed by an initial denaturation step at 95 °C for 1 min. For the Rplp0, Fndc5, and Ctsb genes, 28 cycles were performed using the same PCR protocol.

Immunohistochemistry
The mice were anesthetized as previously described and transcranially perfused with 4 % paraformaldehyde (PFA, Biosesang) following perfusion with 1x PBS. The muscle tissue was stored in 4% PFA for post-fixation overnight and then switched to 30% sucrose solution for an additional 48 h in preparation for sectioning.

TurboID-ER expression in the skeletal muscle To validate whether TurboID-ER is expressed in ER in skeletal muscle of ACTuR mice, limb muscle of ACTuR mice was collected for immunostaining. Serial horizontal sections of muscle tissues (thickness, 20 µm) were collected using a cryostat (Leica, CM3050S) and immediately mounted on the slide glass (Marienfeld). Attached muscle sections were washed with pre-warmed PBS and blocked with blocking solution (10% normal goat serum, 1% mouse on mouse blocking reagent, 0.2% Triton X-100 in PBS) for 1 h at room temperature and then incubated in primary antibody solution. The primary antibody used for immunostaining was a mouse anti-Calregulin antibody (1:20, Santa-Cruz) and a rabbit anti-V5 (1:400, Cell signaling), and the fluorescent secondary antibodies were diluted with blocking solution at a concentration of 1:250 and incubated with the sections for 2 h at room temperature. The fluorescent secondary antibody used was a goat anti-rabbit IgG Alexa Fluor 488-conjugated antibody (Thermo Scientific) and a goat anti-mouse IgG Alexa Fluor 568-conjugated antibody (Thermo Scientific). Nuclei were fluorescently labeled with DAPI containing mounting solution (Vectashield).

BrdU labeling To examine the effects of Serpina1e on hippocampal neurogenesis, 5-bromo-2′-deoxyuridine (BrdU, Sigma‒Aldrich) was injected intraperitoneally at 50 mg/kg into sedentary mice. The BrdU stock was resuspended in PBS at 10 mg/ml and stored at -20°C. The control mice were injected with an equivalent volume of PBS.
Serial coronal sections of the hippocampus (thickness, 40 µm) were collected using a cryostat (Leica, CM3050S) and washed with PBS. All brain sections were blocked with blocking solution (10% normal goat serum, 0.2% Triton X-100 in PBS) for 1 h at room temperature and then incubated in primary antibody solution. All brain sections stained for BrdU were pretreated with 2 M HCl for 30 min at 37°C before blocking. The primary antibody used for immunostaining was a rat anti-BrdU antibody (1:500, Abcam), and the fluorescent secondary antibodies were diluted with blocking solution at a concentration of 1:200 and incubated with the sections for 2 h at room temperature. For double staining with NeuN, rabbit anti-NeuN antibody (1:1000, Abcam) was treated with a rat anti-BrdU antibody. The fluorescent secondary antibody used was a goat anti-rat IgG Alexa Fluor 568-conjugated antibody (Thermo Scientific). For double staining with NeuN, a goat anti-rabbit IgG Alexa Fluor 488-conjugated antibody (Thermo Scientific) was used. Nuclei were fluorescently labeled with DAPI containing mounting solution (Vectashield). To count the number of BrdU-positive cells, six series of DG slices (at intervals of 120 µm) from the dorsal hippocampus of each hemisphere (12 sections per mouse) were selected. The BrdU(+)/NeuN(+) and BrdU(+)/NeuN(-) cell numbers from the six DG slices in each hemisphere were averaged to produce the mean cell numbers, which are shown as individual dots in the bar graph.

In vivo neurite arborization analysis To test whether Serpina1e can induce neuronal growth in vivo as shown in cultured cells (Fig. 6), neurite marker Microtubule-associated protein 2 (MAP2)-positive dendrites were measured. Serial coronal sections of the hippocampus (thickness, 20 µm) were collected using a cryostat (Leica, CM3050S) and washed with PBS. All brain sections were blocked with blocking solution (10% normal goat serum, 0.2% Triton X-100 in PBS) for 1 h at room temperature and then incubated in primary antibody solution. The primary antibody used for immunostaining was a rabbit anti-MAP2 antibody (1:1000, Sigma-Aldrich), and the fluorescent secondary antibodies were diluted with blocking solution at a concentration of 1:200 and incubated with the sections for 2 h at room temperature. The fluorescent secondary antibody used was a goat anti-rabbit IgG Alexa Fluor 488-conjugated antibody (Thermo Scientific) was used. Nuclei were fluorescently labeled with DAPI containing mounting solution (Vectashield).

Intravenous administration of Serpina1a and Serpina1e
Recombinant Serpina1a and Serpina1e with V5 and His tags were synthesized (GenScript). To conduct behavioral tests, neurogenesis analysis and BDNF analysis, we retro-orbitally injected Serpina1a or Serpina1e into sedentary C57BL/6 mice at 150 µg/100 µl for 4 weeks every three days, for a total of 9 times. For the Serpina1 transmission experiments, sedentary mice were injected retro-orbitally with Serpina1e at 300 µg/100 µl and anesthetized to collect CSF and hippocampal tissues.

Serpina1e-V5
MTPSISWCLLLLAGLCCLVPSFLAEDVQETDTSQKDQSPASHEIATNLGDFAISLYRELV
HQSNTSNIFFSPVSIATAFAMLSLGSKGDTHTQILEGLQFNLTQTSEADIHNSFQHLLQTL
NRPDSELQLSTGNGLFVNNDLKLVEKFLEEAKNHYQAEVFSVNFAESEEAKKVINDFVE
KGTQGKIVEAVKKLEQDTVFVLANYILFKGKWKKPFDPENTKQAEFHVDESTTVKVPM
MTLSGMLDVHHCSTLSSWVLLMDYAGNATAVFLLPDDGKMQHLEQTLNKELISKFLLN
RRRRLAQIHIPRLSISGNYNLETLMSPLGITRIFNSGADLSGITEENAPLKLSQAVHKAVLT
IDETGTEAAAATVLQGGFLSMPPILHFNRPFLFIIFEEHSQSPLFVGKVVDPTHKGKPIPN
PLLGLDSTHHHHHH

Serpina1a-V5
MTPSISWGLLLLAGLCCLVPSFLAEDVQETDTSQKDQSPASHEIATNLGDFAISLYRELV
HQSNTSNIFFSPVSIATAFAMLSLGSKGDTHTQILEGLQFNLTQTSEADIHKSFQHLLQTL
NRPDSELQLSTGNGLFVNNDLKLVEKFLEEAKNHYQAEVFSVNFAESEEAKKVINDFVE
KGTQGKIAEAVKKLDQDTVFALALNYILFKGKWKKPFDPENTEEAEFHVDESTTVKVPM
MTLSGMLHVHHCSTLSSWVILLMDYAGNATAVFLLPDDGKMQHLEQTLSKELISKFLLN
RRRRLAQIHFPRLSISGREYNLKTLMSPLGITRIFNNGADLSGITEENAPLKLSQAVHKAVL
TIDETGTEAAAVTVLQMVPMSMPPILRFDHPFLFIIFEEHTQSPIFLGKVVDPTHKGKPIPN
PLLGLDSTHHHHHH

Intramuscular injection of AAV9 and lentiviruses
To investigate whether muscular Serpina1e elicits cognitive enhancement, we delivered a single dose of AAV9-CMV-Serpina1e-T2A-EGFP (VectorBuilder; 1.06 × 10e11 GCs/mouse) into each of the four limbs of C57BL/6 mice. For the control mice, the same dose of AAV9-CMV-EGFP (VectorBuilder; 1.07 × 10e11 GC/mouse) was used instead. In addition, to evaluate whether muscular Serpina1e promotes neuronal outgrowth, the same strategies using AAVs were used to Thy1-EGFP mice. Behavioral assessments and morphological were performed four weeks after viral infection.
To examine whether the role of muscle-derived Serpina1e on exercise-induced beneficial effects, we intramuscularly single injected LV-hCas9:T2A:Puro-U6>Serpina1egRNA1-U6>Serpina1egRNA2 (VectorBuiler; 5 x 10e6 TU/mouse) to reduce the Serpina1e expression in skeletal muscle into four limbs. For the control mice, the same dose of LV-EGFP:T2A:Puro-EF1a>mCherry (VectorBuiler; 5 x 10e6 TU/mouse) was used. Behavioral assessments started two weeks after viral infection.

Infusion of Serpina1e into the hippocampus
To address the direct effects of Serpina1e treatment on the hippocampus, we infused Serpina1e into the molecular layer of the hippocampus (AP -1.95, ML ±1.20, and DV -1.70; Supplementary Fig. 19e) with the same timeline as systemic injections. A custom dual-guide cannula was designed with a 26-gauge, 7.8 mm pedestal height, 2.4 mm distance and 2 mm distance below the pedestal (RWD). The dual-guide cannula was implanted and covered with a dummy cannula with a 1 mm projection to fit the dual-guide cannula. A total of 100 ng/250 nl of Serpina1e per hemisphere was infused through the implanted cannula for 4 weeks every 3 days at 100 nl/min. The actual infused site in the hippocampus was determined by injecting DiI dye through the implanted cannula when all behavioral tests had concluded.

Enzyme-linked immunosorbent assays of BDNF
To measure BDNF levels in the hippocampus, C57BL/6 mice were intravenously injected with Serpina1e for 4 weeks every 3 days and then sacrificed 1 hour after the last injection. Hippocampal tissues were harvested following perfusion with PBS and lysed with lysis buffer (1% Triton X-100, 0.1% Tween 20, 100x P.I.C., and 1x PBS). The BDNF concentration was determined via enzyme-linked immunosorbent assays (Novusbio) according to the manufacturer’s instructions.

Cell culture
Cell line culture AML12 cell (ATCC) which is a hepatocyte cell line derived from a liver of mouse was cultured in normal medium (10% FBS (Thermo Scientific), 5,000 U/ml penicillin and 5,000 µg/ml streptomycin in DMEM (Gibco)) for Serpina1e knockdown efficiency tests in vitro. The cell line was authenticated by its supplier (ATCC) and not tested for mycoplasma contamination.
Primary cortical neuron culture Cortical neurons were prepared from the brains of C57BL/6 embryos at embryonic days 16-17. Cortices were dissected in dissection medium (10 mM HEPES in HBSS, Gibco), incubated in 0.25% trypsin-EDTA (Gibco) in a 37°C water bath for 20 min, and then triturated with 2 ml of plating medium (2 mM L-glutamine (Gibco), 0.45% glucose (Sigma‒Aldrich), 10% FBS (Thermo Scientific), 5,000 U/ml penicillin and 5,000 µg/ml streptomycin in MEM (Gibco)). Dissociated cells were filtered through a cell strainer and plated on poly-L-lysine-coated coverslips in 12-well plates (3 × 105 cells/well) in plating media. After one hour, the plates were replaced with maintenance medium (2% B27 supplement, Thermo Scientific), 2 mM L-glutamine, 5,000 U/ml penicillin and 5,000 µg/ml streptomycin in neurobasal medium (Gibco).

Acquisition and analysis of fluorescence images
Immunofluorescence images were obtained, quantified, and analyzed by an observer who was blinded to the treatment. To validate TurboID-ER expression in ER in skeletal muscle, fluorescence images were acquired by using a STELLARIS 8 microscope (Leica, 100x/1.40 NA) with the pinhole set to 1 Airy unit. For neurogenesis analysis via BrdU staining, fluorescence images were acquired by using a STELLARIS 8 microscope (Leica, 40x/1.10 NA) with the pinhole set to 1 Airy unit. Images were taken at the suggested optimal value interval based on magnification and analyzed using ImageJ (National Institutes of Health). Six series of DG slices (290.77 µm x 290.77 µm at intervals of 120 µm) from the dorsal hippocampus of each hemisphere (12 sections per mouse) were selected and the number of BrdU-labeled cells was manually counted.
For neurite analysis using MAP2 staining, Z-stack images of MAP2-positive dendrites (775.19 µm x 775.19 µm x 7 µm) were acquired by using a STELLARIS 8 microscope (Leica, 20x/0.75 NA) with the pinhole set to 1 Airy unit and zoom factor 0.75x. 2D-projected with maximum intensity was measured to compare the complexity of dendrites, represented by MAP2 intensity, between control and muscle Serpina1e-overexpressed mice. For the neurite morphological assessment of individual neurons, sparsely labeled GFP-positive neurons in the hippocampus were imaged using a Leica SP8 confocal microscope (20x/0.75 NA). Z-stacks were acquired with a 2 um z-step for a total of 40 steps, covering a total thickness of 80 um. The acquired z-stacks were processed into maximum intensity projections (MIP). Individual neurons were manually isolated from the MIP images via Region of Interest (ROI) extraction to ensure singular cell analysis. The isolated images were further processed for skeletonization using ImageJ/FIJI. Morphological parameters, including total neurite length and dendritic complexity via Sholl analysis (calculating the number of neurite crossings at intervals of 10 um from the soma), were quantified to evaluate the structural properties of the neurons. To analyze neurite morphology of primary neurons, cortical neurons at DIV7 were used. The neurons were imaged using a Leica SP8 confocal microscope (20x/0.75 NA). The images were processed for skeletonization via ImageJ/FIJI, and the total neurite length, branch number, and branch length were analyzed. The skeletonized images were further analyzed via the Sholl analysis plugin in ImageJ/FIJI, which calculates the number of neurite crossings at intervals of 10 µm from the cell body.

Statistics and Reproducibility
All the statistical analyses were performed via GraphPad Prism 10 with 95% confidence, and all the values are expressed as the mean ± SEM. One-way ANOVA with Dunnett’s post hoc tests was used to compare three or four groups, and unpaired two-tailed Student’s t tests were used to compare two groups. Because the data in Fig. 4k did not pass the normality test, we used a non-parametric Kruskal-Wallis test. The data in Fig. 6n and Supplementary Fig. 20f were analyzed via two-way ANOVA with Sidac’s and Dunnett’s post hoc test, respectively. The statistical tests for each experiment are provided in the legends of the respective figures. P values less than 0.05 were considered statistically significant. All experiments were repeated at least 3 times independently.

Data availability
All data are provided in the Supplementary Information and Source Data file. Source data are provided with this paper. The mass spectrometry proteomics data generated in this study have been deposited to the ProteomeXchange Consortium via the PRIDE [PMID:34723319] partner repository under the dataset identifier PXD055604. For further inquiries, please contact the Corresponding author.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This is a sophisticated study involving complex analyses that revealed a novel myokine (Serpina1e) that is influenced by exercise, can cross the blood brain barrier, and beneficially effect cognitive function, hippocampal neurogenesis and BDNF signaling. Strengths of the paper include the rigorous study design, application of advanced methods, and examination of experimental exercise identifying the Serpina1 family proteins along with both systemic and hippocampal delivery of Serpina1e to assess for BBB permeation and molecular mechanisms. The manuscript is novel, timely, and reveals a new and potentially potent mechanism underlying exercise effects on the brain that will be a significant addition to the field (of a similar magnitude as the breakthrough finding of irisin), with important translational and public health implications. However, there are several areas of the paper that require elaboration. Please consider the following comments and suggestions which may help to further strengthen the manuscript.\n1. Abstract, Lines 19-20. “The beneficial effects of exercise on cognitive functions appear to be mediated by muscle-derived secretory factors including myokines”. The authors may want to revise to ‘partially mediator’ or include another indicator to demonstrate that this is likely one of multiple pathways by which exercise beneficially effects cognition (e.g., improved cardiometabolic health, psychosocial factors (improved sleep) resulting from exercise that, in turn, improve cognition).\n2. Introduction. Lines 40-42. “…the restorative effect of exercise on cognitive function in patients with brain disorders has been suggested.” Consider being a bit more specific to better orient the readers ( e.g., the restorative effect of exercise on cognitive function in normal aging and in patients with neurologic conditions/neurodegenerative disorders has been suggested).\n3. Experimental methods appear adequate though reviewers with greater expertise in proteomic profiling and the ACTuR model may be able to comment further.\n4. Results. Authors report that the levels of most of the identified myokines (60 of 78 myokines; 123 ~ 77%) significantly increased after exercise. What led the authors to specifically focus on Serpina1 family proteins for the subsequent analysis? The authors nicely discuss Serpina1 proteins on pg. 8 lines 150-154, though further justification would be helpful in understanding the focus on Serpina1 vs. the number of other myokines increased after exercise. (e.g., are the functional roles of the other myokines relatively more well-established, compared to Serpina1 family proteins?)\n5. Were other myokines known to be influenced by exercise and related to exercise-induced cognitive enhancement (irisin, cathepsin B) impacted by the experimental exercise paradigm? Though these myokines are not the focus of the paper, given established evidence of their role, this would be helpful to mention in either the main text or supplement to better contextualize the findings. This data may be included in Fig. 2 panel D, but would be beneficial to explicitly state.\n6. Do the authors foresee any limitations that should be acknowledged in the discussion? For instance, does the age of the mice limit interpretation or generalizability in any way (e.g., should the study be replicated in aged mice to better ascertain whether these mechanisms vary across the lifespan)?\n7. Discussion. Overall, the conclusions are well supported by the evidence. It may be beneficial to include a few sentences relating the findings to recent work involving other core myokines (e.g., consider how the mechanisms of Serapina1 might interact with, amplify, or otherwise relate to other myokines thought to mediate exercise effects on the brain (e.g., irisin, cathepsin B))?\n8. There are a handful of very minor grammatical errors throughout (for example, page 8, lines 163-164 \"When assessed the relative amount of plasma Serpina1 protein...\"). These are easily addressable and do not detract from the quality of the work.", "reviewer_2": "Dear Editor,\nThe study addresses an important topic by investigating the role of the myokine Serpina1e in exercise-induced memory enhancement through advanced methods, including murine models, biotinylation, and proteomic analysis. I have outlined the primary points for improvement below to enhance the manuscript’s rigor and clarity.\nFirst, I recommend removing numeric citations in the abstract, as they detract from its clarity and independence. The abstract should be self-contained and easily understood without reference numbers.\nSecond, the discussion section closely aligns with an overly “perfect” narrative of experimental findings. To enhance scientific transparency, the authors should consider including study limitations, particularly concerning the applicability of murine models to human outcomes. Additionally, it would be beneficial for the authors to discuss any data that did not yield significant results, such as the Morris water maze data obtained using four daily trials, a setup that typically reduces sensitivity to subtle cognitive differences. Furthermore, it would be valuable to compare their findings on Serpina1e with other known myokines linked to exercise-induced cognitive benefits, highlighting any relevant similarities and differences.\nThese suggestions aim to enhance the study’s clarity and robustness, providing greater transparency and critical analysis of the results and methods.", "reviewer_3": "In this interesting manuscript, Kim et al. examine the role of the myokine Serpina 1e in mediating some of the beneficial effects of exercise on the brain. By using a novel mouse model, the authors identify myokines secreted by muscle in response to exercise, which include Serpina 1e. They further report that, like exercise, Serpina 1e improves cognitive functions, as determined by modulating Serpina 1e levels in muscle and by injecting it in the plasma and hippocampus. Overall, this is an interesting manuscript on an important topic. However, there are several major limitations of the current work that decrease the robustness of the study and confidence in the findings.\nMajor points\n1) The authors have generated an ER-targeted turboID mouse model to label myokines en route to be secreted. However, a better characterization of this mouse model is needed to determine whether the turboID indeed localizes to the ER of muscle cells. Moreover, the authors should determine by western blot whether the ER-targeted turboID can be secreted as well. This is an important control because if the ER-targeted turboID is secreted it can then label proteins in the plasma irrespective of their tissue origin.\n2) The authors report that several myokines, including Serpina 1a and 1e, are secreted into the plasm in response to exercise. However, it is unclear if these myokines are transcriptionally induced by exercise or, alternatively, whether there is a residing pool of Serpina 1a and 1e that is secreted only in response to exercise. Gene expression analysis, immunostaining, and western blot should be used to characterize the muscle pool of Serpina 1e and how it modulated by exercise.\n3) The analysis in Fig. 3 reports that the muscle-secreted pool of Serpina 1e increases in response to exercise whereas the liver pool does not. While this might be true, Serpina 1e and other serpina family members are highly expressed by the liver compared to skeletal muscle and therefore it remains undetermined whether the muscle-secreted Serpina 1e is indeed substantial enough to offset the circulating pool of Serpina 1e, which primarily derives from the liver given that this is the tissue that primarily expresses it. Therefore, the authors should compare on the same western blot membrane the levels of muscle-derived Serpina 1e versus the levels of Serpina 1e that are found in the leftover plasma from the same preparation (obtained as shown in Fig. 3a).\n4) On the same line as the point above, the authors should analyze by mass spectrometry the muscle-derived fraction vs. the leftover fraction (as shown in Fig. 3a) to determine whether the exercise-secreted myokines identified in Fig. 2 indeed represent a meaningful contribution to the systemic pool of these circulating factors. This is important because many of the exercise-induced myokines reported by the authors in Fig. 2 are known to be primarily expressed by non-muscle tissues such as the liver. Therefore, it remains unaddressed whether secretion of these factors by skeletal muscle is substantial enough to contribute to the systemic pool.\n5) In Fig. 4, the authors utilize AAVs to induce Serpina 1e expression in the skeletal muscle but they should better document whether there is indeed an upregulation of Serpina 1e in the producing skeletal muscle as expected.\n6) Throughout the manuscript, there is a wide range of variability in the number of mice utilized for the behavioral assays. Sometimes this number changes even within the same group of experimental animals that are tested in different assays. For example, Fig. 4d is done with n=5 (saline) and n=6 (Serpina 1e) mice but Fig. 4e (which analyzes the same groups of animals but with a different behavioral assay) is done with n=7/group. What is the reason for this change in the number of animals?\n7) The number of animals utilized in the behavioral assays seems rather small, also considering the inherent variability of these assays.\n8) The assessment of cognitive function is done with novel object recognition and with fear conditioning. However, there are other assays, such as the Morris water maze, listed in the methods but no data with these assays is reported in the figures for Serpina 1e (only a supplementary figure reports positive effects of exercise with this assay). The authors should perform this gold-standard assay for at least one of their models of Serpina 1e modulation.\n9) In Fig. 5a, the authors report that V5-tagged Serpina 1e that is retro-orbitally injected can reach the CSF and the hippocampus. However, the authors should also monitor the total levels of Serpina 1e (injected + endogenous) to determine whether the injection of Serpina 1e indeed increases the total levels of Serpina 1e in the CSF and hippocampus. For these studies, they can utilize the anti-Serpina 1 antibody used in Fig. 4f. On the same line, they should also monitor whether exercise modulates the CSF and hippocampal levels of Serpina 1e.\n10) In Figure 6, the authors report that Serpina 1e injection increases cell proliferation in the hippocampus and BDNF levels (by 10%). Are there corresponding changes in BDNF protein levels? This looks overall as a minor effect.\n11) It is unclear mechanistically how Serpina 1e would modulate BDNF levels, cell proliferation, and neurite outgrowth. What is the receptor mediating the action of Serpina 1e?\n12) The statistical analysis does not seem to be always appropriate. For example, the data in Fig. 4b was analyzed by Student’s t-test but there are 2 variables (before and retrieval) for 2 groups (saline and serpina) and therefore should be analyzed by 2-way ANOVA. This applies also to other graphs such as Fig. 4e and Fig. 5g.\nAdditional points:\n13) There is no loading control (such as Ponceau) for the western blot in Fig. 1e.\n14) In Fig. 2 the authors report a global increase in myokines in the plasma in response to exercise. However, how is this data normalized to ensure that this is indeed the case?\n15) It is unclear what is the control in Fig. 5e-g.\n16) The discussion of scientific background is a bit limited. The authors should better discuss studies that have examined communication between the skeletal muscle and the brain.", "reviewer_4": "In this manuscript Kim and colleagues identify Serpina1e as a myokine that is released by muscle into the plasma following 4 weeks of exercise. The also show that delivery of Serpina1e into the plasma, muscle or hippocampus can replicate the hippocampus-associated learning and memory enhancement observed following exercise. Overall, this is a very interesting study. However, several key points should be addressed before this manuscript would be suitable for publication in Nature Communications.\nMajor questions:\n1. The title of the paper states that Serpina1e “mediates” the exercise-induced enhancement of hippocampal memory. Likewise, in the abstract they state that Serpina1e…..”directly” supports long-term memory formation. I disagree that the data presented here shows this. The authors show that Serpina1e is released by muscle after exercise and that Serpina1e delivery can enhance cognition. However, whether Serpina1e is responsible/required for the exercise-induced enhancement in cognition has not been shown. To show this direct relationship, one would for example need to knock out Serpina1e in the muscle and show that exercise can no longer enhance cognition. These statements should all be toned down throughout the manuscript, including the title.\n2. The authors state that Serpina1e delivery increases neurogenesis in the hippocampal dentate gyrus. From the data presented in Figure 6 b one is not able to draw this conclusion. BrdU was injected for the last 5 days of prior to perfusion and BrdU+ cells quantified. These BrdU+ cells will be a mixture of proliferating neural precursor cells, immature neurons that may have already become post-mitotic since the first injection 5 days prior, proliferating microglia etc. To be able to quantify adult neurogenesis it would be best to inject BrdU at the start of the experiment and quantify BrdU+NeuN+ double-positive cells. Alternatively, on the available sections a proliferation marker such as Ki67 could be used to quantify the proliferating cells (ideally co-labelled with other markers such as Tbr2 or Sox2) and the immature newborn neurons could be quantified using DCX staining.\nAdditionally, it is unclear if the BrdU counts presented in Fig 6b are per section or per DG. There is no information in the methods about which sections were stained with BrdU (i.e., a 1 in 6 series through the whole brain or alike). It is stated that cells were counted from 12 sections – how were these sections chosen? The counts state they are # cells/DG and are around 20-30, which if it is BrdU cells per DG I would expect this count to be hundreds from a young mouse.\n3. Changes in dendritic arborization could also be assessed in the same mice in the newborn neurons by performing Sholl analysis on DCX+ cells in vivo. This would significantly strengthen the in vitro finding which was performed on embryonic cortical neurons rather than those from the adult hippocampus.\n4. Why were only male mice used in the study? This should be stated in the abstract and discussed in the discussion.\nMinor points:\n1. In the abstract, it is stated that “the beneficial effects of exercise on cognitive function appear to be mediated by muscle-derived secretory factors”. While muscle is certainly an important tissue that exerkines are secreted from, there are many others i.e. hepatokines, adipokines etc.\n2. The sentence “Each dot in the graphs represents a tested mouse” is repeated twice at the end of each supplemental figure legend.\n3. The discussion section is very brief. There are a lot of discussion points in the results section that could be moved/expanded on in the discussion."}, "author_response": "Responses to the Reviewer’s Comments\nWe would like to thank all reviewers for their productive comments on our\nmanuscript “Serpina1e mediates the exercise-induced enhancement of hippocampal\nmemory” (NCOMMS-24-60831-A) by Kim et al. Based on their excellent suggestions, we\nhave improved our manuscript, which is now considered suitable for publication in Nature\nCommunications.\nIn the revised manuscript, several substantial revisions were made as follows:\n1. ACTuR mice were further validated by providing additional results that (1) TurboID-ER\nis not secreted into blood vessels and (2) TurboID-ER is expressed in the ER.\n2. Additional information about the LC-MS/MS method was provided to clarify the\nunderstanding of our results.\n3. Additional semi-quantitative RT-PCR showed that our exercise protocol is sufficient for\ninducing not only the mRNA expression of Serpina1a and Serpina1e but also other\npreviously reported genes, such as Fndc5 and Ctsb, in skeletal muscle.\n4. By using a mouse model with muscle-specific reduction of Serpina1e, we provided\nadditional evidence for the requirement of muscle Serpina1e in exercise-induced\nhippocampal memory enhancement and neurogenesis.\n5. Immunohistochemical analysis of neurogenesis was supplemented by additional co-\nstaining of BrdU with NeuN in the hippocampal dentate gyrus (DG) of Serpina1e-\ninjected mice.\n6. Other additional immunohistology experiments showed that Serpina1e overexpression\nin skeletal muscle results in increased dendritic arborization of hippocampal DG and\nCA1 neurons. These results support our original in vitro findings on Serpina1e-induced\nneuronal growth in cultured neurons.\n7. We re-analyzed the original data according to the reviewers’ comments:\n• Data from the CFC test were re-analyzed using two-way ANOVA with Sidak’s post hoc tests.\n• The behavior dataset, which did not pass the normality test, was re-analyzed using a non-\nparametric Kruskal-Wallis test.\n• Additional details of the statistical analysis are included in the Methods section.\n• The number of tested mice used for behavior and western blot analysis was matched according\nto the criteria for behavior analysis.\n8. Additional discussions on our study have been included in the revised manuscript: (1)\nadvantages and disadvantages of the in vivo myokine labeling tool (ACTuR mouse)\nbased on TurboID-based labeling technique and LC-MS/MS analysis, (2) possible\nmechanisms of Serpina1e and their interactions with other myokines, and (3) the\nlimitations of our study and future directions study for application to human subjects.\n9. We corrected several errors in the text and figures in the revised manuscript.\nAll changes in the text of the revised manuscript have been highlighted in red.\nWe hope these revisions and additional results adequately address all reviewers’ points and\nstrengthen our findings on muscle-derived Serpina1e regulating exercise-induced cognitive\nenhancement.\nPlease find below our point-by-point response to the reviewers’ comments.\nReviewer #1 (Remarks to the Author):\nThis is a sophisticated study involving complex analyses that revealed a novel myokine\n(Serpina1e) that is influenced by exercise, can cross the blood brain barrier, and\nbeneficially effect cognitive function, hippocampal neurogenesis and BDNF signaling.\nStrengths of the paper include the rigorous study design, application of advanced\nmethods, and examination of experimental exercise identifying the Serpina1 family\nproteins along with both systemic and hippocampal delivery of Serpina1e to assess for\nBBB permeation and molecular mechanisms. The manuscript is novel, timely, and\nreveals a new and potentially potent mechanism underlying exercise effects on the\nbrain that will be a significant addition to the field (of a similar magnitude as the\nbreakthrough finding of irisin), with important translational and public health\nimplications. However, there are several areas of the paper that require elaboration.\nPlease consider the following comments and suggestions which may help to further\nstrengthen the manuscript.\n1. Abstract, Lines 19-20. “The beneficial effects of exercise on cognitive functions\nappear to be mediated by muscle-derived secretory factors including myokines”. The\nauthors may want to revise to ‘partially mediator’ or include another indicator to\ndemonstrate that this is likely one of multiple pathways by which exercise beneficially\neffects cognition (e.g., improved cardiometabolic health, psychosocial factors\n(improved sleep) resulting from exercise that, in turn, improve cognition).\n: We agree with the reviewer’s comment. The abstract is now revised.\nline 19: The exercise-induced enhancement of learning and memory is thought to be regulated not\nonly by improved cardiometabolic and psychosocial health but also by body‒brain\ninteractions mediated by metabolites and proteins derived from peripheral organs. Given\nthe prominent role of skeletal muscle during exercise, muscle-derived factors are believed\nto mediate the cognitive enhancements induced by exercise.\n2. Introduction. Lines 40-42. “…the restorative effect of exercise on cognitive function\nin patients with brain disorders has been suggested.” Consider being a bit more\nspecific to better orient the readers ( e.g., the restorative effect of exercise on cognitive\nfunction in normal aging and in patients with neurologic conditions/neurodegenerative\ndisorders has been suggested).\n: As suggested by the reviewer, we have modified the text in Introduction as below:\nline 47: … the restorative effect of exercise on cognitive function in normal aging and in patients\nwith neurological diseases such as Alzheimer’s disease, Parkinson’s disease, depression,\nepilepsy, and stroke has been suggested 5\n3. Experimental methods appear adequate though reviewers with greater expertise in\nproteomic profiling and the ACTuR model may be able to comment further.\n: Responding to the reviewer’s suggestion, we have included additional procedures of LC-\nMS/MS analysis in Methods in the revised manuscript.\nAdditionally, we have deposited our LC-MS dataset to ProteomeXchange Consortium\nrepository for reviewer and public access and describe this in Methods in the revised\nmanuscript.\nWe also included additional data showing TurboID-ER expression in the ER of skeletal\nmuscle (Supplementary Fig. 3 in the revised manuscript) as well as no detection of\nsecreted TurboID in blood plasma of ACTuR mice (Supplementary Fig. 2 in the revised\nmanuscript). These results support the idea that ACTuR model is a proper animal for\nlabeling skeletal muscle-derived secretory proteins.\n4. Results. Authors report that the levels of most of the identified myokines (60 of 78\nmyokines; 123 ~ 77%) significantly increased after exercise. What led the authors to\nspecifically focus on Serpina1 family proteins for the subsequent analysis? The\nauthors nicely discuss Serpina1 proteins on pg. 8 lines 150-154, though further\njustification would be helpful in understanding the focus on Serpina1 vs. the number\nof other myokines increased after exercise. (e.g., are the functional roles of the other\nmyokines relatively more well-established, compared to Serpina1 family proteins?)\n: Responding to the reviewer’s comment, we have included additional justification of why\nSerpina1 proteins are of interest in our study.\nOur proteomics analysis identified previously reported exercised-induced factors such as\nClusterin (De Miguel et al, 2021, Nature, 600:494-499) and Bche (Horowitz et al, 2020,\nScience 369(6500):167-173), supporting the previous findings that muscle-derived\nClusterin and Bche are exercise-induced myokines (Supplementary Table 1 in the\nrevised manuscript).\nBecause the main goal of this study is to explore novel exercise-induced myokines involved\nin cognitive enhancements, we have focused on Serpina1 proteins, whose role in an\nexercise-induced improvement of cognitive function is completely unknown. Liver-derived\nSerpina1 can activate Ephrin B2 receptor signaling, which can trigger multiple signaling\npathways elevating cellular energy and glucose metabolism (Okagawa et al, 2024, Nat\ncommun, 15:9585). However, whether muscle-derived Serpina1 has functional roles in\nexercise-associated changes in cognitive functions is completed unknown. Given its anti-\ninflammatory functions (Stockley et al, 2014, Trends. Mol. Med. 20:2), we hypothesized that\nSerpina1 is possibly associated with cognitive enhancement.\nThis additional text is now included in the Results of the revised manuscript.\nline 136: Our proteomics analysis identified previously reported exercise-induced factors such as\nClusterin 10 and Bche 7, supporting the previous findings that both Clusterin and Bche are\nexercise-induced peripheral factors and partially derived from skeletal muscles\n(Supplementary Table 1).\nline 146: Liver-derived Serpina1 can trigger multiple signaling pathways elevating cellular energy\nand glucose metabolism 29, but how muscle-derived Serpina1 contributes to exercise-\nassociated cognitive changes remains completely unknown. Given its anti-inflammatory\nfunctions 26, we hypothesized that Serpina1 may be possibly associated with cognitive\nenhancement.\n5. Were other myokines known to be influenced by exercise and related to exercise-\ninduced cognitive enhancement (irisin, cathepsin B) impacted by the experimental\nexercise paradigm? Though these myokines are not the focus of the paper, given\nestablished evidence of their role, this would be helpful to mention in either the main\ntext or supplement to better contextualize the findings. This data may be included in\nFig. 2 panel D, but would be beneficial to explicitly state.\n: To test whether our exercise paradigm is sufficient for inducing previously reported\nmyokines, such as FNDC5 and CTSB, from skeletal muscles, we extracted mRNA from the\nhindlimbs of exercised or control WT mice and performed semi-quantitative RT-PCR to\nmeasure Fndc5 and Ctsb transcripts.\nOur results showed that the mRNA expression of FNDC5 and CTSB was significantly\nincreased in the exercised animal group, with increases in Serpina1a and Serpina1e in\nparallel (Supplementary Fig. 8 in the revised manuscript). These data support the notion\nthat the experimental exercise paradigm in this study also impacts other exercise-induced\nmyokines.\nHowever, our proteomics analysis of blood plasma could not detect FNDC5/irisin and CTSB,\npossibly due to the following reasons:\nIn this study, we employed TurboID, a biotin ligase-based proximity labeling technique, to\ndirectly identify precise biotinylation sites on labeled proteins through LC-MS/MS analysis\n(Shin et al, 2024, Commun Biol, 7:554). This biotin ligase is known to selectively biotinylate\nlysine residues of target proteins, resulting in the limited identification of biotinylated\nproteins due to several factors: (1) The lysine residues are known to be extensively\nmodified by post-translational mechanisms. (2) The restricted surface accessibility of lysine\nresidues may limit the biotinylation of target proteins by TurboID, resulting in the inability to\nlabel peptides using LC-MS/MS. (3) Other factors, such as the expression level of proximity\nlabeling enzyme-tagged bait proteins, technical aspects of gathering sample amounts, and\nbiotin-labeling conditions (pH of subcellular organelles, biotin concentrations and etc.) may\nalso limit the depth of protein identification.\nWe also included the extended discussion about the advantages and disadvantages of the\nTurboID-based protein labeling and detection technique in Discussion in the revised\nmanuscript (line 303~314).\n6. Do the authors foresee any limitations that should be acknowledged in the\ndiscussion? For instance, does the age of the mice limit interpretation or\ngeneralizability in any way (e.g., should the study be replicated in aged mice to better\nascertain whether these mechanisms vary across the lifespan)?\n: We thank the reviewer for the valuable comment helpful for improving our manuscript and\ndesigning future studies.\nWe have not yet tested the age-dependent effects of Serpina1e on cellular and cognitive\nchanges. By using young adult mice, our study revealed that exercise-induced Serpina1e\nis effective for elevating neurogenesis and the expression of BDNF protein, all of which are\nhallmarks of exercise-induced effects in the hippocampus. Because both neuronal loss and\ndecreased BDNF levels in the hippocampus are prominent in the aged brain (Erickson et\nal., 2010, J Neurosci, 30(15):5368-75; Fan et al., 2017, Ann Rev Neurosci 40: 251-272),\nexercise has been suggested to improve cognitive functions in middle-aged or old mice\n(Marlatt et al., 2012, Developmental Neurobiology 72: 943-952; van Praag et al., 2005, J\nNeurosci 25: 8680-8685). It is thus possible that Serpina1e-dependent mechanisms, along\nwith other exercise-induced myokines, play significant roles in the exercise-induced\nrestoration of age-associated cognitive decline.\nThese future directions of our study are now included in Discussion in the revised\nmanuscript.\nline 361: By using young adult mice, our study revealed that exercise-induced Serpina1e is effective\nin elevating neurogenesis and the expression of BDNF protein, both of which are hallmarks\nof exercise-induced effects in the hippocampus. By restoring both neuronal loss and\ndecreased BDNF level in the hippocampus prominent in the aged brain 41,42, exercise has\nbeen suggested to ameliorate cognitive decline in middle-aged or old mice 2,43. It is thus\npossible that Serpina1e-dependent mechanisms, along with other exercise-induced\nmyokines, play significant roles in the exercise-induced restoration of age-associated\ncognitive decline. Further studies are required to test whether Serpina1e can restore this\ncognitive decline.\n7. Discussion. Overall, the conclusions are well supported by the evidence. It may\nbe beneficial to include a few sentences relating the findings to recent work involving\nother core myokines (e.g., consider how the mechanisms of Serapina1 might interact\nwith, amplify, or otherwise relate to other myokines thought to mediate exercise\neffects on the brain (e.g., irisin, cathepsin B)?\n: As the reviewer commented, it is possible that Serpina1 may interact with or modify other\nexercise-induced myokines, because neurogenesis and BDNF expression are common\nconsequences of exercise-induced changes in the brain by Serpina1e and/or irisin and\ncathepsin B.\nHowever, the differential mechanisms of exercise-induced factors on cognitive functions\ncannot be excluded. Exercise is known to increase the plasma levels of anti-inflammatory\nperipheral proteins such as Clusterin (Fig. 2d; Supplementary Table 1 in the revised\nmanuscript), and the migration of such proteins across several physiological barriers to\nthe brain could reduce neuroinflammatory gene expression (De Miguel, Z. et al. 2021,\nNature 600:494–499). Similarly, human SERPINA1 could possess anti-inflammatory\nproperties independent of its anti-protease activity (Jonigk et al., 2013, PNAS, 110: 15007-\n15012). Because Serpina1e was reported to have no anti-protease activity, which is not\ntypical of other paralogs (Serpina1a-d), possibly due to a variance in the P1 amino acid in\nthe reactive center loop (RCL; Forsyth et al., 2003, Genomics 81: 336–345), Serpina1e\nmay be implicated in exercise-induced anti-inflammatory functions in the brain, resulting in\nupregulation of neuronal growth factors, such as BDNF.\nOn the other hand, the main effector of Irisin is cAMP/PKA-mediated CREB transcription\n(Lourenco et al, 2019, Nat. Med. 25:165-175), and CTSB not only act as a lysosomal\nprotease but also contributes to the cell growth signaling by activating the Wnt and Notch\nreceptor signaling (Yu et al., 2025, Trends in Molecular Medicine, S1471-4914(24)00343-\n5).\nGiven such differences of myokine-associated signaling, exercise-triggered secretion and\ndelivery of multiple myokines to the brain may evoke distinct downstream mechanisms, all\nof which may concurrently facilitate neurotrophic events in the hippocampus. Whether\nthese signaling pathways can elicit cognitive enhancement in a cooperative or differential\nmanner remains to be addressed.\nThis additional discussion is now included in Discussion in the revised manuscript (line\n338~360).\n8. There are a handful of very minor grammatical errors throughout (for example, page\n8, lines 163-164 \"When assessed the relative amount of plasma Serpina1 protein...\").\nThese are easily addressable and do not detract from the quality of the work.\n: As the reviewer recommended, we reviewed the manuscript and corrected errors.\nReviewer #2 (Remarks to the Author):\nThe study addresses an important topic by investigating the role of the myokine\nSerpina1e in exercise-induced memory enhancement through advanced methods,\nincluding murine models, biotinylation, and proteomic analysis. I have outlined the\nprimary points for improvement below to enhance the manuscript’s rigor and clarity.\n1. I recommend removing numeric citations in the abstract, as they detract from its\nclarity and independence. The abstract should be self-contained and easily understood\nwithout reference numbers.\n: As the reviewer commented, we deleted all numeric citations in Abstract in the revised\nmanuscript.\n2. The discussion section closely aligns with an overly “perfect” narrative of\nexperimental findings. To enhance scientific transparency, the authors should consider\nincluding study limitations, particularly concerning the applicability of murine models\nto human outcomes.\n: As the reviewer commented, we have included the limitations of our study in the\nDiscussion, particularly concerning the applicability of findings from murine models to\nhuman outcomes.\nOur study reveals the novel function of Serpina1e in enhancing hippocampal memory.\nUnlike the human SERPINA1 gene, the mouse Serpina1 has five different paralogs. One\nof the main differences between Serpina1e and SERPINA1 is the reactive center loop (RCL)\ndomain, which is responsible for the specificity of anti-protease activity (Sanrattana et al.,\n2021, Proc. Natl. Acad. Sci. U. S. A. 118(45):e2108458118). In fact, mouse Serpina1e is\nreported to have no anti-protease activity (Paterson et al., 1995, Biochem Biophys Res\nCommun, 219(1):64-9), which is atypical of the other paralogs (Serpina1a-d) and human\nSERPINA1. This structural difference between the human and mouse Serpina1 genes may\nlead to selective and differential effects of human and mouse Serpina1 on neuronal growth\nsignaling and cognitive enhancements.\nHowever, it is also possible that common structural and functional domains between human\nSERPINA1 and mouse Serpina1 are responsible for modulating brain functions, as our\nresults showed that both human SERPINA1 and mouse Serpina1e can induce neurite\noutgrowth in cultured neurons (Fig. 6). Furthermore, human SERPINA1 could possess anti-\ninflammatory properties independent of its anti-protease activity (Jonigk et al., 2013, PNAS,\n110: 15007-15012), raising the possibility that mouse Serpina1e is also implicated in anti-\ninflammatory signaling.\nFurther studies are thus required to compare the exact molecular features of SERPINA1\nand Serpina1e involved in neuronal growth signaling and to dissect their differential effects\non cognitive enhancements.\nReflecting our responses to the reviewer’s comment, the additional statement is now\nincluded in Discussion in the revised manuscript (line 348~353; 369~372).\n3. Additionally, it would be beneficial for the authors to discuss any data that did not\nyield significant results, such as the Morris water maze data obtained using four daily\ntrials, a setup that typically reduces sensitivity to subtle cognitive differences.\n: As recommended by the reviewer, we have included the limitation of our Morris water\nmaze test in Result in the revised manuscript.\nline 97: … The Morris water maze test with five daily trials (short-learning version), which is less\nsensitive to subtle cognitive differences but enough for detecting spatial memory\nperformance, showed the exercise-induced increase in the retrieval of spatial memory with\nno significant changes in the learning rate (Supplementary Fig. 5).\n4. Furthermore, it would be valuable to compare their findings on Serpina1e with other\nknown myokines linked to exercise-induced cognitive benefits, highlighting any\nrelevant similarities and differences.\n: As recommended by the reviewer, we have included additional discussion highlighting the\nsimilarities and differences between Serpina1e and other myokines that have been\nreported to be involved in exercise-induced cognitive enhancements.\nBoth an increase in the proliferation of neural progenitor cells and BDNF induction are\ncommon consequences of exercise-induced Serpina1e and other myokines. Therefore, it\nis possible that Serpina1e and other myokines may share downstream signaling pathways.\nHowever, it is also possible that Serpina1e and other myokines independently and\ncooperatively affect neuronal growth signaling. Serpina1e has been reported to\ndownregulate inflammatory signaling molecules such as TNF-alpha, CXCL1, MHCII, and\nToll-like receptors (Jonigk et al., 2013, PNAS, 110: 15007-15012). In contrast, Irisin can\nstimulate cAMP/PKA-mediated CREB-dependent transcription in the brain (Lourenco et al.,\n2019, Nat. Med. 25:165-175). Additionally, by reducing unnecessary proteins as a\nlysosomal protease, Ctsb can contribute to the cell growth signaling of neural progenitor\ncells by activating the Wnt and Notch receptor pathways (Yu et al., 2025, Trends in\nMolecular Medicine, S1471-4914(24)00343-5).\nTherefore, it is possible that exercise influences cognitive function through multiple\nsignaling pathways mediated by different myokines. However, whether these myokine-\ninduced signaling pathways are interconnected or differentially influence cognitive functions\nremains to be addressed.\nThis additional statement is now included in the Discussion in the revised manuscript\n(line 338~360).\nReviewer #3 (Remarks to the Author):\nIn this interesting manuscript, Kim et al. examine the role of the myokine Serpina 1e in\nmediating some of the beneficial effects of exercise on the brain. By using a novel\nmouse model, the authors identify myokines secreted by muscle in response to\nexercise, which include Serpina 1e. They further report that, like exercise, Serpina 1e\nimproves cognitive functions, as determined by modulating Serpina 1e levels in muscle\nand by injecting it in the plasma and hippocampus. Overall, this is an interesting\nmanuscript on an important topic. However, there are several major limitations of the\ncurrent work that decrease the robustness of the study and confidence in the findings.\nMajor comments\n1. The authors have generated an ER-targeted TurboID mouse model to label myokines\nen route to be secreted. However, a better characterization of this mouse model is\nneeded to determine whether the TurboID indeed localizes to the ER of muscle cells.\n: As suggested by the reviewer, we additionally tested whether ACTuR mice express the\nV5-tagged TurboID-ER construct in the ER of muscle cells, by performing\nimmunohistochemistry using anti-V5. Our immunohistochemistry results showed that\nTurboID-ER is well colocalized with the ER marker (Calregulin) as shown below. This data\nis now included in Supplementary Fig. 3 in the revised manuscript.\nMoreover, the authors should determine by western blot whether the ER-targeted\nTurboID can be secreted as well. This is an important control because if the ER-targeted\nTurboID is secreted it can then label proteins in the plasma irrespective of their tissue\norigin.\n: To address the reviewer’s point about whether TurboID-ER is secreted into the blood, we\ndetected TurboID-ER protein in the blood plasma fraction by western blot using anti-\nV5 (Supplementary Fig. 2e in the revised manuscript). TurboID-ER was not detected in\neither the input or streptavidin enrichment fraction.\nSince TurboID frequently undergoes self-biotinylation (Shin et al., 2024, Commun. Biol.\n7(1):554), which can be detected through LC-MS analysis, we attempted to identify self-\nlabeled TurboID peptides. Consistent with the western blot result, we did not detect any\nbiotinylated peptides corresponding to TurboID in the plasma sample through enriched\nsample analysis via LC-MS (Supplementary Table 1 in the revised manuscript).\n2. The authors report that several myokines, including Serpina 1a and 1e, are secreted\ninto the plasma in response to exercise. However, it is unclear if these myokines are\ntranscriptionally induced by exercise or, alternatively, whether there is a residing pool\nof Serpina 1a and 1e that is secreted only in response to exercise. Gene expression\nanalysis, immunostaining, and western blot should be used to characterize the muscle\npool of Serpina 1e and how it modulated by exercise.\n: Previous studies have shown that the plasma levels of myokines such as FNDC5 and\nCTSB can be elevated by exercise-induced upregulation of their mRNA transcription\n(Pedersen et al, 2019, Nat. Rev. Endocrinol. 15:383-392; Moon et al, 2016, Cell Metab,\n24:332-340). Therefore, it is also possible that the upregulation of plasma Serpina1 results\nfrom increased mRNA expression in skeletal muscles.\nTo test this possibility, we measured transcript levels of Serpina1a and Serpina1e, as well\nas other known exercise-induced myokines such as Fndc5 and Ctsb from skeletal muscle\ntissues, using semi-quantitative RT-PCR. Our results showed that four weeks of running\nwheel exercise is sufficient for inducing not only Fndc5 and Ctsb but also Serpina1a and\nSerpina1e in skeletal muscles (Supplementary Fig. 8 in the revised manuscript). Given\nthe finding of the increased plasma level of Serpina1 by AAV-mediated overexpression of\nSerpina1e mRNA in skeletal muscles (Fig. 4f, g in the revised manuscript), these results\nsuggest that the exercise modulates the synthesis of Serpina1 proteins, which later\ncontribute to the circulating pool of Serpina1.\nHowever, the possibility of exercise-modulation of the secretion of myokines cannot be\nexcluded and is needed to be tested in future studies.\n3. The analysis in Fig. 3 reports that the muscle-secreted pool of Serpina 1e increases\nin response to exercise whereas the liver pool does not. While this might be true,\nSerpina1e and other serpina family members are highly expressed by the liver\ncompared to skeletal muscle and therefore it remains undetermined whether the\nmuscle-secreted Serpina 1e is indeed substantial enough to offset the circulating pool\nof Serpina 1e, which primarily derives from the liver given that this is the tissue that\nprimarily expresses it. Therefore, the authors should compare on the same western blot\nmembrane the levels of muscle-derived Serpina 1e versus the levels of Serpina 1e that\nare found in the leftover plasma from the same preparation (obtained as shown in Fig.\n3a).\n: We would like to thank the reviewer for the opportunity to improve our study by providing\nthe valuable comment.\nAs described in the manuscript already, there is only pan-Serpina1 antibody available, and\nit is thus impossible to compare the amount of muscle-derived Serpina1e with non-muscle-\nderived one by the western blot experiment.\nInstead, to directly test a requirement of skeletal muscle Serpia1e for exercise-induced\nmemory enhancement, we achieved a muscle-specific reduction of Serpina1e by using a\nCRISPR-Cas9 technique.\nFirst, we generated the lentivirus containing hCas9 and two gRNAs targeting Serpina1e\n(LV-hCas9:T2A:Puro-U6>Serpina1e -U6> Serpina1e ; S1eKO). Lentivirus\ngRNA1 gRNA2\nexpressing EGFP and mCherry (LV-EGFP:T2A:Puro-EF1a>mCherry; CTL) was used as a\ncontrol. The selective reduction in Serpina1e by S1eKO virus was validated in AML12 cells\ntreated with control or S1eKO lentiviruses (Supplementary Fig. 15a-d in the revised\nmanuscript).\nNext, we injected control or S1eKO virus into four limbs of WT mice to test whether both\nexercise-induced neuronal growth and memory enhancement are affected by the muscle-\nspecific reduction of Serpina1e. Two weeks after virus injection, mice were subjected to 4\nweek of wheel running exercise (Fig. 4h in the revised manuscript). Then, novel object\nrecognition and contextual fear conditioning tests were carried out using mice injected with\nCTL or S1eKO virus.\nOur data showed that sedentary mice with S1eKO exhibit comparable basal activity and\nhippocampal memory with sedentary CTL mice (Fig. 4k, l; Supplementary Fig. 15 in the\nrevised manuscript). However, exercised S1eKO-injected mice failed to show enhanced\nhippocampal memory (Fig. 4k, l in the revised manuscript).\nWhen Serpina1e expression in each group was tested after behavior experiments, a\nsignificant reduction in Serpina1e mRNA in skeletal muscles by in vivo delivery of S1eKO\nwas still observed (Fig. 4i, j in the revised manuscript).\nTaken together, these additional findings support our conclusion that muscle-derived\nSerpina1e, despite its lower amount than liver-derived one, is essential for exercise-\ninduced cognitive enhancement. Whether the muscle-secreted Serpina1e is indeed\nsubstantial enough to offset the circulating pool of Serpina1e remains elusive, but our data\nstill emphasize the necessary role of muscle-derived Serpina1e in the modulation of\ncognitive functions.\n4. On the same line as the point above, the authors should analyze by mass\nspectrometry the muscle-derived fraction vs. the leftover fraction (as shown in Fig. 3a)\nto determine whether the exercise-secreted myokines identified in Fig. 2 indeed\nrepresent a meaningful contribution to the systemic pool of these circulating factors.\nThis is important because many of the exercise-induced myokines reported by the\nauthors in Fig. 2 are known to be primarily expressed by non-muscle tissues such as\nthe liver. Therefore, it remains unaddressed whether secretion of these factors by\nskeletal muscle is substantial enough to contribute to the systemic pool.\n: Our LC-MS/MS method for detecting muscle-derived secretory proteins from ACTuR mice\ndirectly analyzed the enriched fraction of biotinylated proteins (muscle-derived fraction)\nfrom blood plasma (Fig. 2a in the revised manuscript). Therefore, we believe that\nadditional mass spectrometry of the muscle-derived fraction would be redundant and\nunnecessary.\nRegarding LC-MS analysis of the leftover plasma fraction, it is well established that plasma\nprotein concentrations span up to 12 orders of magnitude, with a few abundant proteins\naccounting for ~95% of the total plasma proteome, including albumin, which constitutes\nmore than half of the total protein content (Anderson et al., 2002, Mol. Cell\nProteomics1(11):845-867; Hortin et al., 2006, Clin. Chem. 52(7):1218-1222). Given the\ninherent limitations of mass spectrometry and proteomic analyses, these high-abundance\nproteins tend to dominate the results, hindering the detection of low-abundance\ncomponents (Anderson et al., 2002; Hortin et al., 2006; Hortin et al., 2008, Clin.\nChem. 54(10):1608-1616; Hortin, 2006, Clin. Chem. 52(7):1223-1237). Therefore, we\nbelieve that additional LC-MS analysis of the non-biotinylated plasma fraction would yield\nno meaningful insights relevant to our study.\nOur results indicate that while many plasma proteins originate from the liver, they are not\nexclusively expressed there. It is possible that a smaller fraction of non-liver-derived\nproteins contributes to the systemic pool.\nOur mass spectrometry method, based on a biotin ligase-mediated protein labeling\ntechnique, has been demonstrated to efficiently identify precise biotinylation sites on\nlabeled proteins (Shin et al., 2024, Commun Biol. 7:554). Consistent with this, our\nproteomics analysis of blood plasma from ACTuR mice detected biotinylated peptides that\nare likely derived from skeletal muscle, given the selective expression of TurboID-ER in\nmuscle (Fig. 1, Supplementary Figs. 2 and 3 in the revised manuscript). By analyzing\nthe fraction enriched using streptavidin beads, our LC-MS/MS analysis identified 78\nputative myokines in plasma, which overlapped significantly with both the mouse plasma\nproteome (86%; Fig. 2b) and the skeletal muscle proteome reference datasets (88%; Fig.\n2c). Notably, 60 of these 78 putative myokines were significantly increased following\nexercise.\nAmong these 60 exercise-induced myokines, we identified Clusterin (De Miguel et al.,\n2021, Nature 600:494-499) and BChe (Horowitz et al., 2020, Science 369(6500):167-173),\nboth previously reported as exercise-induced plasma proteins. While Clusterin is expressed\nubiquitously with higher levels in the brain, endocrine tissues, liver, and reproductive organs,\nBChe is primarily produced in the liver. Our findings indicate that a portion of systemic\nClusterin and BChe induced by exercise originates from muscle tissue.\nA similar case applies to Serpina1. While Serpina1 is predominantly expressed in the liver\nand lungs, it is also present at lower levels in skeletal muscle. Plasma concentrations\nof Serpina1 family proteins were previously reported to increase after six weeks of wheel\nrunning (Horowitz et al., 2020, Science 369:167-173), but the tissue origin of this exercise-\ninduced increase had not been directly examined.\nIn our study, Serpina1 family proteins were classified as putative myokines based on our\nproteomic analysis. We further detected a significant increase in\nbiotinylated Serpina1 (muscle-derived due to the muscle-specific expression of ER-\ntargeted TurboID) in blood plasma from ACTuR mice after four weeks of exercise (Fig. 3\nin the revised manuscript). Importantly, this occurred without any exercise-induced\nchange in total or non-biotinylated (i.e., non-muscle-derived, primarily liver-\nderived) Serpina1 levels. Furthermore, our data show that selectively reducing muscle-\nderived Serpina1e prevents exercise-induced memory enhancement and neurogenesis\n(Fig. 4h~l in the revised manuscript), supporting our conclusion that muscle-\nderived Serpina1e plays a key role in exercise-induced brain adaptations.\nIn summary, given the sensitivity and selectivity of the biotin ligase-based protein labeling\nmethod and the limitations of LC-MS/MS analysis of unlabeled plasma samples, our\napproach—leveraging the ACTuR mouse model and LC-MS/MS analysis—effectively\ndetermines the precise tissue origin of circulating secretory proteins in plasma, where\nproteins from multiple tissues coexist.\n5. In Fig. 4, the authors utilize AAVs to induce Serpina 1e expression in the skeletal\nmuscle, but they should better document whether there is indeed an upregulation of\nSerpina 1e in the producing skeletal muscle as expected.\n: As suggested by the reviewer, we provide additional evidence for the AAV-induced\nupregulation of Serpina1e mRNA in skeletal muscle. Semi-quantitative RT-PCR analysis of\nskeletal muscle tissues (limb muscles) from mice injected with AAVs containing Serpina1e-\nEGFP (AAV9-CMV-mSerpina1e:T2A:EGFP; S1eOE) demonstrated that Serpina1e is\nsignificantly overexpressed (~ 350 %), while the expression level of Serpina1a remains\nunaffected by AAV injection.\nThese results are now included in Supplementary Figure 14 of the revised manuscript.\n6. Throughout the manuscript, there is a wide range of variability in the number of mice\nutilized for the behavioral assays. Sometimes this number changes even within the\nsame group of experimental animals that are tested in different assays. For example,\nFig. 4d is done with n=5 (saline) and n=6 (Serpina 1e) mice but Fig. 4e (which analyzes\nthe same groups of animals but with a different behavioral assay) is done with\nn=7/group. What is the reason for this change in the number of animals?\n: In response to the reviewer’s comment, we re-examined all behavioral data and the\nnumber of mice used in our analyses.\nTo assess the effect of Serpina1e overexpression (S1eOE) in skeletal muscle on behavior\n(Fig. 4c-e), control and S1eOE mice were sequentially subjected to the open field test\n(OFT), novel object recognition test (NORT), and contextual fear conditioning test (CFC).\nFollowing the behavioral tests, tissues and plasma fractions from these mice were collected\n(Fig. 4g in the revised manuscript).\nInitially, each group consisted of seven mice. However, during NORT analysis, two control\nmice and one S1eOE mouse were excluded because they did not meet the exploration\ntime criteria (this was now included in Methods in the revised manuscript, line 601~603),\nresulting in a final sample size of control = 5 and S1eOE = 6 for NORT. Since these three\nmice exhibited normal behavior in the other tests, their data were retained for OFT and\nCFC (control = 7, S1eOE = 7) as well as western blot analysis in the original study.\nTo prevent any confusion, we have now excluded all behavioral and western blot results\nobtained from the mice that did not meet the behavioral analysis criteria. The updated\nsample sizes (control = 5, S1eOE = 6) have been corrected in the corresponding figure\nlegends (figure legend for Fig. 4c-g in the revised manuscript).\n7. The number of animals utilized in the behavioral assays seems rather small, also\nconsidering the inherent variability of these assays.\n: To minimize unnecessary sacrifice of animals, we aimed to use the minimal number of\nmice required for our study. To assess whether our dataset deviated significantly from\nnormality, we performed a normality test.\nMost behavioral datasets followed a normal distribution (see below). However, the dataset\nfrom exercised WT mice expressing lentivirus S1eKO (LV-hCas9:T2A:Puro-U6>gRNA-\nU6>gRNA) did not pass the normality test.\nTherefore, we used a non-parametric Kruskal-Wallis test to analyze whether the reduced\nexpression of muscle-derived Serpina1e affects exercise-induced memory enhancement\n(Fig. 4l in the revised manuscript).\nDespite the limited sample size in the behavioral assays, we observed significant changes\nin memory performance following muscle Serpina1e manipulation.\nAdditionally, we have revised and expanded the details of statistical tests in the Methods\nsection of the revised manuscript.\n8. The assessment of cognitive function is done with novel object recognition and with\nfear conditioning. However, there are other assays, such as the Morris water maze,\nlisted in the methods but no data with these assays is reported in the figures for Serpina\n1e (only a supplementary figure reports positive effects of exercise with this assay).\nThe authors should perform this gold-standard assay for at least one of their models of\nSerpina 1e modulation.\n: We acknowledge that the Morris water maze is a well-established test for assessing\nspatial learning and memory. However, other behavioral tests, such as contextual fear\nconditioning (CFC) and novel object recognition (NOR), are also widely recognized\nmethods for evaluating hippocampus-dependent cognitive functions.\nWe also observed that skeletal muscle-specific reduction of Serpina1e expression prevents\nthe exercise-induced improvement in memory retrieval in both the NOR and CFC tests (Fig.\n4k, l in the revised manuscript). Furthermore, the same manipulation abolishes the\nexercise-induced increase in the number of NeuN/BrdU double-positive cells (Fig. 6g, h in\nthe revised manuscript).\nTherefore, these findings not only highlight the beneficial and essential roles of muscle-\nderived Serpina1e in hippocampal memory through the promotion of neurogenesis but also\nsupport the notion that NOR and CFC tests are equally effective as the Morris water maze\nin evaluating exercise-modulated hippocampal cognitive functions.\n9. In Fig. 5a, the authors report that V5-tagged Serpina 1e that is retro-orbitally injected\ncan reach the CSF and the hippocampus. However, the authors should also monitor the\ntotal levels of Serpina 1e (injected + endogenous) to determine whether the injection of\nSerpina 1e indeed increases the total levels of Serpina 1e in the CSF and hippocampus.\nFor these studies, they can utilize the anti-Serpina 1 antibody used in Fig. 4f.\n: As the reviewer suggested, we attempted to detect changes in total Serpina1e protein\nlevels (injected + endogenous) in the hippocampus following administration of\nrecombinant Serpina1e-V5 protein by using anti-Serpina1 antibody targeting.\nConsistent with our findings in original study, we detected a significant increase in total\nplasma Serpina1 level from mice administered with Serpina1e-V5 (Supplementary Fig.\n16d, e in the revised manuscript). Further analysis with CSF collected from mice with\nSerpina1e-V5 administration revealed that total Serpina1 is slightly increased\n(Supplementary Fig. 16f in the revised manuscript), although western blot analysis with\nCSF was not fully replicated due to the limited number of available mice.\nWe next attempted to detect total Serpina1 level in hippocampal extract from mice with\nSerpina1e-V5 administration. Despite some non-specific signals, our western blot results\nshowed a weak but significant anti-Serpina1 signal in hippocampal extracts from mice\nadministered with Serpina1e-V5. The detected band matched the expected size of\nthe Serpina1e-V5 protein and the detected Serpina1 band in plasma (Supplementary Fig.\n16d, g-i in the revised manuscript).\nImportantly, this corresponding signal was absent in hippocampal extracts from control\n(PBS-injected) mice, suggesting that endogenous Serpina1 protein levels in the\nhippocampus are negligible or too low to be detected by western blot. This finding aligns\nwith publicly available data, such as the Human Protein Atlas\n(https://www.proteinatlas.org/ENSG00000197249-SERPINA1), which reports that both\nmRNA and protein levels of endogenous SERPINA1 are very low in the hippocampus\ncompared to other tissues.\nOn the same line, they should also monitor whether exercise modulates the CSF and\nhippocampal levels of Serpina 1e.\n: As suggested by the reviewer, we attempted several times to detect changes in\ntotal Serpina1 protein levels by performing western blot analysis on hippocampal extracts\nusing a pan-Serpina1 antibody. However, we were unable to detect any significant signal\ncorresponding to the expected size of Serpina1 proteins in all groups of mice (please see\nthe image below).\nWe suspect that this result is due to the concentration of hippocampus-transmitted\nSerpina1(e) being too low to be detected using conventional antibody-based methods such\nas western blot. Despite this negative finding, we have already demonstrated that\nsystemically administered recombinant Serpina1e protein can be detected in the\nhippocampus (Fig. 5c,d; Supplementary Fig. 16 in the revised manuscript). This\nsuggests that Serpina1e is indeed capable of crossing from the bloodstream into the\nhippocampus.\n10. In Figure 6, the authors report that Serpina 1e injection increases cell proliferation\nin the hippocampus and BDNF levels (by 10%). Are there corresponding changes in\nBDNF protein levels? This looks overall as a minor effect.\n: The BDNF levels shown in Fig. 6c of the original manuscript were measured using an\nELISA assay and directly correspond to BDNF protein levels in the hippocampus. To avoid\nany confusion, we will clearly indicate this in the revised manuscript (line 269).\nBDNF protein exists at relatively low concentrations, however, even subtle changes in its\nlevels are known to effectively influence downstream signaling via its high-affinity receptor,\nTrkB (Kd ~ 10⁻¹¹ M; Rodrigues-Tebar et al., 1990, Neuron, 4:487-92). Previous studies\nhave reported that BDNF protein is detectable at 108–256 ng/g of tissue in the\nhippocampus, frontal cortex, and neostriatum of the rodent brain (Radka et al., 1996, Brain\nResearch, 709:122-301). Furthermore, treating cultured hippocampal neurons with as little\nas 20 ng/ml of recombinant BDNF is sufficient to elicit BDNF-TrkB-dependent neurite\ngrowth (Righi et al., 2000, J. Neurosci., 20:3165-3174). Additionally, a recent study\ndemonstrated that even a ~10% change in BDNF levels can alter dendritic growth and\nrescue memory impairment induced by hypoxic stress (Li et al., 2025, Sci. Rep., 15:1135).\nOn the other hand, BDNF secretion and clearance have been reported to occur on the\nscale of seconds to minutes (Bergami et al., 2008, JCB, 183:213-221; Han et al., 2021, Sci.\nRep., 11:21203; Park and Poo, 2013, Nat. Rev. Neurosci., 14:7-23), indicating the rapid\nand transient kinetics of BDNF concentration in the brain. These unique properties of BDNF\nmay lead to an underestimation of the extent to which Serpina1 upregulates BDNF when\nmeasured using hippocampal extracts and antibody-based detection methods such as\nELISA.\n11) It is unclear mechanistically how Serpina 1e would modulate BDNF levels, cell\nproliferation, and neurite outgrowth. What is the receptor mediating the action of\nSerpina 1e?\n: The receptor for Serpina1e in the brain is totally unknown and remains to be identified. In\nperipheral tissues, such as adipocytes, mouse Serpina1 proteins have been reported to\ninteract with Ephrin type-B receptor 2 (EphB2). This interaction could elicit cell proliferation\nand lead to UCP1-induced improvements in thermogenesis and glucose metabolism\n(Okagawa et al., 2024, Nature Communications, 15:9585). However, whether EphB2 is\nassociated with the beneficial effects of Serpina1e on growth signaling has not yet been\nexamined. Further investigation is required to elucidate the molecular mechanisms\nunderlying Serpina1e-induced neuronal growth signaling and cognitive enhancement.\nWe have included additional discussion about the expected downstream signaling of\nSerpina1e in the brain in Discussion of the revised manuscript (line 338 ~ 360).\n12) The statistical analysis does not seem to be always appropriate. For example,\nthe data in Fig. 4b was analyzed by Student’s t-test but there are 2 variables (before\nand retrieval) for 2 groups (saline and serpina) and therefore should be analyzed by\n2-way ANOVA. This applies also to other graphs such as Fig. 4e and Fig. 5g.\n: As the reviewer suggested, a two-way ANOVA with Sidak’s post hoc test was used for\ncontextual conditioning tests in Fig. 4b, 4e, 4l, and 5g in the revised manuscript. Other\ndata in Supplementary Fig. 10b, 11b, and 12b in the revised manuscript were tested\nwith a two-way ANOVA with Sidak’s post hoc test.\nThe change from one-way ANOVA to two-way ANOVA did not result in any changes in the\nconclusion of our findings. We have also included detailed information of the statistical\nanalysis in the Methods of the revised manuscript.\nAdditional points:\n13) There is no loading control (such as Ponceau) for the western blot in Fig. 1e.\n: We added ponceau stained membrane image in Fig. 1e in the revised manuscript.\n14) In Fig. 2 the authors report a global increase in myokines in the plasma in\nresponse to exercise. However, how is this data normalized to ensure that this is\nindeed the case?\n: To guide the normalization strategy for LC-MS experiments, we were hinted from Western\nblot analysis results.\nDuring Western blot analysis, equal amounts of the plasma fraction acquired from AcTuR\nmice were loaded to estimate the total amount of input plasma proteins and the abundance\nof biotinylated proteins in plasma. Because the overall intensities of Ponceau staining were\ncomparable, we assumed that the total input proteins are comparable across all\nexperimental groups (Fig. 1e, right, in the revised manuscript). However, the levels of\nbiotinylated proteins were increased in plasma samples from mice provided with biotin, and\nthe overall amounts of biotinylated proteins were further elevated when exercise was\nintroduced (Fig. 1e-f in the revised manuscript).\nBased on this result, sample preparation for LC-MS/MS analysis was performed with an\nequal amount of plasma from each experimental group/mouse as in the Western blot\nanalysis had been done, to ensure the consistency of the amounts of total plasma proteins.\nAll samples were processed in parallel to minimize variability and maintain experimental\nrigor. When the biotinylated proteome samples were ready, we consistently used the\nidentical fraction of samples (50% of each sample) for LC-MS/MS analysis, and the\nquantification of biotinylated proteins was performed based on label-free quantification\n(LFQ) (Cox et al., 2014, Mol. Cell. Proteomics, 13: 2513-2526). Once we obtained the\nquantification result of biotinylated proteins, we did not perform any further post-\nnormalization of the quantified dataset.\nConsistent with the Western blot analysis, LC-MS/MS data demonstrated a reproducible\nincrease in overall biotinylated protein levels upon biotin supplementation, with a further\nenhancement observed following exercise combined with biotin supplementation\n(Supplementary Fig. 7c in the revised manuscript, ****p<0.0001, one-way ANOVA with\nDunnett’s post hoc test), as shown in Fig. 1e-f in the revised manuscript.\nAs these two independent experiments showed consistent results, we confirmed that our\nobservations accurately reflect the actual phenomenon of increased secretion of\nbiotinylated myokines from muscle upon exercise.\nThis additional result and description of protein quantification are now updated in\nSupplementary Fig. 7 and Methods in the revised manuscript.\n15) It is unclear what is the control in Fig. 5e-g.\n: Control mouse was injected with the same volume of PBS instead of Serpina1e-V5.\nThis information is now included in the legend of Figure 5 and Methods in the revised\nmanuscript.\n16) The discussion of scientific background is a bit limited. The authors should better\ndiscuss studies that have examined communication between the skeletal muscle and\nthe brain.\n: We thank the reviewer for helpful comment to improve our manuscript.\nNow the revised manuscript includes the summary of previous studies showing functional\nroles and related mechanisms of exercise-induced myokines such as FNDC5/irisin and\nCTSB in brain functions in the Discussion of the revised manuscript.\nReviewer #4 (Remarks to the Author):\nIn this manuscript Kim and colleagues identify Serpina1e as a myokine that is released\nby muscle into the plasma following 4 weeks of exercise. The also show that delivery\nof Serpina1e into the plasma, muscle or hippocampus can replicate the hippocampus-\nassociated learning and memory enhancement observed following exercise. Overall,\nthis is a very interesting study. However, several key points should be addressed before\nthis manuscript would be suitable for publication in Nature Communications.\nMajor questions:\n1. The title of the paper states that Serpina1e “mediates” the exercise-induced\nenhancement of hippocampal memory. Likewise, in the abstract they state that\nSerpina1e…..”directly” supports long-term memory formation. I disagree that the data\npresented here shows this. The authors show that Serpina1e is released by muscle\na muscle after exercise and that Serpina1e\ndelivery can enhance cognition. However, whether Serpina1e is responsible/required for the exercise-induced\nenhancement in cognition has not been shown. To show this direct relationship, one would for example need to knock out\nSerpina1e in the muscle and show that exercise can no longer enhance cognition. These statements should all be toned down throughout the manuscript, including the title.\n: We thank the reviewer for the valuable comment.\nTo support our conclusion that Serpina1e mediates exercise-induced enhancement of\nhippocampal memory, we tested whether exercise-induced cognitive improvements are\nprevented by the reduced Serpina1e expression in skeletal muscles.\nTo achieve this, we produced a lentivirus containing hCas9 and two gRNAs targeting\nSerpina1e (LV-hCas9:T2A:Puro-U6>gRNA-U6>gRNA; S1eKO). This lentivirus was\ninjected into the limb muscles of wild-type mice, while the lentivirus expressing EGFP and\nmCherry (LV-EGFP:T2A:Puro-EF1a>mCherry) was used as a control. Semi-quantitative\nRT-PCR analysis of limb muscles injected with the S1eKO lentivirus confirmed that\nSerpina1e transcript levels were significantly reduced (Supplementary Fig. 15a-d; Fig.\n4h-j in the revised manuscript).\nNext, wild-type mice injected with either the S1eKO or control lentivirus were subjected to\nnovel object recognition (NOR) and contextual fear conditioning (CFC) tests after four\nweeks of wheel-running exercise. Our results showed that S1eKO-injected animals without\nexercise exhibited normal basal activity and hippocampal memory, indicating that muscle-\nderived Serpina1e in non-exercised animals does not affect overall behavioral or cognitive\nfunctions (Fig. 4k, l; Supplementary Fig. 15e-i in the revised manuscript). However,\nS1eKO-injected animals failed to show enhanced memory retrieval in NOR and CFC tests,\nindicating that exercise-induced cognitive enhancement requires muscle-derived\nSerpina1e (Fig. 4k, l in the revised manuscript). Taken together with the results from the\noriginal manuscript, this additional data supports our conclusion that muscle-derived\nSerpina1e mediates exercise-induced cognitive enhancement.\n2. The authors state that Serpina1e delivery increases neurogenesis in the hippocampal\ndentate gyrus. From the data presented in Figure 6 b one is not able to draw this\nconclusion. BrdU was injected for the last 5 days of prior to perfusion and BrdU+ cells\nquantified. These BrdU+ cells will be a mixture of proliferating neural precursor cells,\nimmature neurons that may have already become post-mitotic since the first injection\n5 days prior, proliferating microglia etc. To be able to quantify adult neurogenesis it\nwould be best to inject BrdU at the start of the experiment and quantify BrdU+NeuN+\ndouble-positive cells. Alternatively, on the available sections a proliferation marker\nsuch as Ki67 could be used to quantify the proliferating cells (ideally co-labelled with\nother markers such as Tbr2 or Sox2) and the immature newborn neurons could be\nquantified using DCX staining.\n: As the reviewer suggested, we re-assessed the amount of neurogenesis stimulated by\nSerpina1e delivery by measuring BrdU/NeuN-double positive cells in the mouse\nhippocampus following Serpina1e administration. Our new immunohistochemistry results\nshowed an increased number of BrdU/NeuN-double positive cells after Serpina1e\nadministration, while the number of BrdU+/NeuN- cells was unaffected (Fig. 6c-f in the\nrevised manuscript). These results indicate that Serpina1e-evoked proliferating cells are\nneuronally differentiated.\nIn our experiment, BrdU injection was started 5 days before brain harvest, as done\npreviously, to examine the effect of chronic Serpina1e administration on neurogenesis. As\nthe reviewer pointed out, exercise is effective for both the survival and proliferation of\nhippocampal neurons (van Praag, 2008, NeuroMolecular Medicine, 10-128-140). Many\nstudies have shown that chronic exercise (at least three weeks or longer) is most efficient\nfor the proliferation and survival of hippocampal neurons, as demonstrated by BrdU labeling\nat the start of the exercise training. On the other hand, our results, with BrdU injection\nstarting 5 days prior to the end of Serpina1e administration, reveal only the effect of\nSerpina1e on the proliferation of hippocampal cells, not the survival of newborn neurons.\nWhether Serpina1e is also effective in upregulating the survival of newborn neurons\nremains unclear. Given the crucial role of BDNF signaling in cell survival and the increased\nBDNF level after 4 weeks of Serpina1e administration (Fig. 6b in the revised manuscript),\nit is possible that Serpina1e administration may also be beneficial for the survival of\nnewborn neurons.\nAdditionally, it is unclear if the BrdU counts presented in Fig 6b are per section or per\nDG. There is no information in the methods about which sections were stained with\nBrdU (i.e., a 1 in 6 series through the whole brain or alike). It is stated that cells were\ncounted from 12 sections – how were these sections chosen?\n: To count the number of BrdU-positive cells, six series of DG slices (at intervals of 120 µm)\nfrom the dorsal hippocampus of each hemisphere (12 sections per mouse) were selected.\nThe BrdU(+)/NeuN(+) and BrdU(+)/NeuN(-) cell numbers from the six DG slices in each\nhemisphere were averaged to produce the mean cell numbers, which are shown as\nindividual dots in the bar graph (Fig. 6d-f in the revised manuscript).\nThis detailed information on image analysis has now been included in the Methods section\nof the revised manuscript (line 743~747).\nThe counts state they are # cells/DG and are around 20-30, which if it is BrdU cells per\nDG I would expect this count to be hundreds from a young mouse.\n: There was our mistake in Fig. 6b in the original manuscript.\nThe range of Y-axis should be 0-20, and it is now corrected in Fig. 6d in revised\nmanuscript. This result is consistent with previous reports that the mean number of BrdU-\npositive cells in the hippocampus of adult mice is around 15 cells (Zhang et al, 2016, Mol\nMed Rep, 14:2231-2237; Kang et al, 2018, Exp Neurobiol, 27(5):419-436).\n3. Changes in dendritic arborization could also be assessed in the same mice in the\nnewborn neurons by performing Sholl analysis on DCX+ cells in vivo. This would\nsignificantly strengthen the in vitro finding which was performed on embryonic cortical\nneurons rather than those from the adult hippocampus.\n: Instead of DCX staining, we evaluated dendritic arborization in the hippocampal CA1 and\nDG areas by measuring Microtubule-associated protein 2 (MAP2)-positive dendrites, as\nMAP2 is known to be highly enriched in the dendritic compartment and strongly associated\nwith branched neurites (Chamak et al., 1987, J Neurosci, 7(10):3163-3170; Chou et al.,\n2024, eLife13:RP103127).\nTo increase the plasma level of Serpina1e, we overexpressed Serpina1e in skeletal\nmuscles by delivering an AAV virus expressing Serpina1e-EGFP (AAV9-CMV-\nmSerpina1e:T2A:EGFP) into the four limbs of WT mice. Control mice were injected with\nAAV9-CMV-EGFP. This intramuscular overexpression of Serpina1e was sufficient to\nelevate Serpina1e mRNA expression in skeletal muscles and plasma Serpina1e protein\nlevels (Fig. 4f-g; Supplementary Fig. 14 in the revised manuscript), as well as\nhippocampal memory performance (Fig. 4c-e in the revised manuscript). Given our\nfindings that plasma Serpina1e can be delivered to the hippocampus by crossing the blood-\nCSF and blood-brain barriers (Fig. 5a-d; Supplementary Fig. 16 g-i in the revised\nmanuscript), this intramuscular overexpression of the Serpina1e gene is expected to\nupregulate hippocampal Serpina1e protein levels and serve as a useful model for testing\nthe in vivo effects of Serpina1e on neurite growth.\nZ-stack images of MAP2-positive dendrites (775.19 µm x 775.19 µm x 7 µm) were 2D-\nprojected with maximum intensity to compare the complexity of CA1 dendrites, represented\nby MAP2 intensity, between control and muscle Serpina1e-overexpressed mice. Our\nresults show that the average MAP2 intensity in the hippocampal CA1 and DG area of\nmuscle Serpina1e-overexpressed mice is significantly higher than that of control mice\n(Supplementary Fig. 18 in the revised manuscript). These results indicate that\nSerpina1e promotes neuronal dendritic growth in vivo and is consistent with in vitro finding\nof elevated neurite growth following treatment with recombinant Serpina1e protein in\ncultured neurons.\n4. Why were only male mice used in the study? This should be stated in the abstract\nand discussed in the discussion.\n: To avoid any unexpected effects of sex-dependent physiological and behavioral\nphenotypes, we only used male mice in this study.\nAs human SERPINA1 gene is reported to be a direct target of estrogen receptor and a\npredictor of survival rate in patients with breast cancer (Chan et al., 2016, Oncotarget. 6:\n25815-25817), it is possible that Serpina1e-induced effects can be influenced by sex-\nspecific biological mechanisms. Future studies will need to consider comparative\ninvestigations of Serpina1e-mediated cognitive modulation in both sexes.\nAs the reviewer suggested, we include the statement of the usage of male mice and the\nfuture need of studies addressing the sex-specific role of Serpina1e in cognitive modulation,\nin the Abstract (line 27) and Discussion (line 375~376) in the revised manuscript.\nMinor points:\n1. In the abstract, it is stated that “the beneficial effects of exercise on cognitive\nfunction appear to be mediated by muscle-derived secretory factors”. While muscle is\ncertainly an important tissue that exerkines are secreted from, there are many others\ni.e. hepatokines, adipokines etc.\n: Agreeing with the reviewer’s point. We have revised the abstract as following:\nline 19: The exercise-induced enhancement of learning and memory is thought to be\nregulated not only by improved cardiometabolic and psychosocial health but also\nby body‒brain interactions mediated by metabolites and proteins derived from\nperipheral organs. Given the prominent role of skeletal muscle during exercise,\nmuscle-derived factors are believed to mediate the cognitive enhancements induced\nby exercise.\n2. The sentence “Each dot in the graphs represents a tested mouse” is repeated twice\nat the end of each supplemental figure legend.\n: We re-examine our manuscript and corrects all errors in the revised manuscript.\n3. The discussion section is very brief. There are a lot of discussion points in the results\nsection that could be moved/expanded on in the discussion.\n: We thank the reviewer for valuable feedback to improve our manuscript.\nReflecting all reviewer’s comments, we have expanded the Discussion section by\nincorporating the relevant points and limitations of our results as well as future directions\nto understand detailed molecular mechanisms underlying Serpina1e-mediated cognitive\nenhancements. Please find revised Discussion in the revised manuscript for changes\n(marked in red texts)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have thoughtfully responded to the critiques. The revisions have significantly strengthened the manuscript, which will no doubt be an impactful addition to the field.", "reviewer_2": "The manuscript titled \"Serpina1e mediates the exercise-induced enhancement of hippocampal memory\" presents robust and novel findings identifying Serpina1e as a key myokine mediating the cognitive benefits of exercise. The study is methodologically sound, with comprehensive in vivo and in vitro validation, and the conclusions are well supported by the data.\nThe work is highly significant for the field, offering insights comparable to previous discoveries such as Irisin, and expanding our understanding of muscle-brain communication. The authors satisfactorily addressed reviewer comments, improving the clarity, methodological transparency, and discussion of their findings. The experimental design, data analysis, and reporting meet the expected standards, with sufficient detail provided to ensure reproducibility. In my evaluation, the manuscript is ready for publication without further revisions.", "reviewer_3": "The authors have made improvements to this interesting manuscript; however, I remain skeptical about several key findings due to significant limitations in both the previous and newly added data.\n1) The authors did not conduct the straightforward analysis I requested (previous point 3) to assess whether muscle-contributed Serpina1 is a significant source of the systemic Serpina1 pool, which is primarily expressed in the liver. The authors have access to pan-Serpina1 antibodies and mass spectrometry that could be used for these analyses. At present it remains uncertain whether Serpina 1e secreted by muscle significantly contributes to the circulating pool of Serpina 1e and total Serpina1.\n2) The authors now suggest that the muscle pool of Serpina1e plays a key role because they injected lentiviral vectors to knock out Serpina1 in 4 limb muscles, which they claim prevents exercise-induced changes in behavior (discrimination index and freezing). However, these experiments (Fig. 4h-l, as well as others such as those in Fig. 7a-h) are conducted suboptimally, as a control exercise group is not included, leaving it unclear whether the exercise training was performed at optimal levels to induce a response. Additionally, there is no evidence that the lentiviral vectors used are muscle-specific, as they do not appear to carry a muscle-specific promoter and could potentially target Serpina1e in the liver or other tissues. Finally, even if the authors' model is correct and Serpina1e is indeed specifically knocked out in 4 limb muscles, it is still uncertain how this could impair the beneficial effects of exercise on the brain, given that many other muscles, both in the limbs and throughout the body, remain unaffected by the lentiviral vectors (Serpina1e should still increase following exercise in the vast majority of these muscles which are not targeted by the lentiviral vectors and hence promote exercise-induced improvements in behavior).\n3) As mentioned in my previous comments, the cognitive assays that are currently utilized in the manuscript (novel object recognition and fear conditioning) are inherently variable. Robust gold-standard assays such as the Morris Water Maze should be performed for at least some of the key experiments."}, "author_response": "Responses to the Reviewer’s Comments\nWe would like to thank the Reviewers #1 and #2 for their favorable evaluation, indicating that\nour manuscript, “Serpina1e mediates the exercise-induced enhancement of\nhippocampal memory” (NCOMMS-24-60831-A) by Kim et al.” is now suitable for\npublication in Nature Communications.\nHowever, Reviewer #3 still raised several concerns regarding our findings and requested\nadditional evidence to support our conclusion. Specifically, Reviewer #3 asked us to provide\nevidence for: (1) whether Serpina1e derived from non-muscle sources contributes to the\nexercise-induced plasma pool of Serpina1e, (2) whether the experimental design for reducing\nSerpina1e in skeletal muscle was appropriately optimized, and (3) whether Serpina1e\nimproves hippocampal memory in the Morris water maze test.\nIn response to these points, we have improved our manuscript with additional results and\nsubstantial revisions as follows:\n1. To detect exercise-induced Serpina1 family proteins derived from non-muscle tissues,\nwe performed additional LC-MS/MS analysis on plasma proteins remaining after the\ndepletion of biotinylated proteins. Our results showed that exercise does not\nincrease the amount of non-muscle-derived Serpina1 family proteins.\n2. To address the concern that the exercise training of the S1eKO/EX group was\ninsufficient for memory enhancement, we compared the exercise intensities of\nS1eKO/EX mice to those of exercised control mice (normal Serpina1e in muscles).\nOur results showed that the S1eKO/EX mice performed similar amounts of\nexercise compared to the control mice.\n3. In response to the concern that our lentiviral vector-mediated reduction of muscular\nSerpina1e may be non-specific, we measured Serpina1e mRNA levels in lentiviral\nvector-injected muscles and other non-injected tissues. Our semi-quantitative RT-\nPCR results showed that intramuscular delivery of lentiviral vectors does not\naffect Serpina1e expression in other tissues.\n4. Because Reviewer #3 suggested that “robust gold-standard assays” such as the\nMorris Water Maze (MWM) should be performed for at least some of the key\nexperiments in our study, we additionally conducted the MWM test after systemic\nadministration of recombinant Serpina1e. Our results showed that Serpina1e-\nadministered mice exhibited improved memory performance in the MWM test,\nconsistent with our findings from the contextual fear conditioning test.\nAll changes in the text of the revised manuscript have been highlighted in red.\nWe hope these revisions and additional results adequately address all reviewers’ points and\nstrengthen our findings on muscle-derived Serpina1e mediating exercise-induced cognitive\nenhancement. We believe that this revised manuscript is suitable for publication in Nature\nCommunications.\nPlease find below our point-by-point response to the reviewers’ comments.\nReviewer #1 (Remarks to the Author):\nThe authors have thoughtfully responded to the critiques. The revisions have\nsignificantly strengthened the manuscript, which will no doubt be an impactful addition\nto the field.\n: We thank the reviewer for the thoughtful and encouraging comments, which were helpful\nin improving our manuscript.\nReviewer #2 (Remarks to the Author):\nThe manuscript titled \"Serpina1e mediates the exercise-induced enhancement of\nhippocampal memory\" presents robust and novel findings identifying Serpina1e as a\nkey myokine mediating the cognitive benefits of exercise. The study is\nmethodologically sound, with comprehensive in vivo and in vitro validation, and the\nconclusions are well supported by the data.\nThe work is highly significant for the field, offering insights comparable to previous\ndiscoveries such as Irisin, and expanding our understanding of muscle-brain\ncommunication. The authors satisfactorily addressed reviewer comments, improving\nthe clarity, methodological transparency, and discussion of their findings. The\nexperimental design, data analysis, and reporting meet the expected standards, with\nsufficient detail provided to ensure reproducibility. In my evaluation, the manuscript is\nready for publication without further revisions.\n: We greatly appreciate the reviewer’s comments and are truly honored by the recognition\nand appreciation of our work.\nReviewer #3 (Remarks to the Author):\nThe authors have made improvements to this interesting manuscript; however, I remain\nskeptical about several key findings due to significant limitations in both the previous\nand newly added data.\n1) The authors did not conduct the straightforward analysis I requested (previous point\n3) to assess whether muscle-contributed Serpina1 is a significant source of the\nsystemic Serpina1 pool, which is primarily expressed in the liver. The authors have\naccess to pan-Serpina1 antibodies and mass spectrometry that could be used for these\nanalyses. At present it remains uncertain whether Serpina 1e secreted by muscle\nsignificantly contributes to the circulating pool of Serpina 1e and total Serpina1.\n: To directly address whether Serpina1e derived from non-muscle tissues is also a\nsignificant source of exercise-induced systemic Serpina1e, we conducted additional LC-\nMS/MS analysis of plasma proteins in the non-biotinylated fraction (the fraction remaining\nafter the depletion of biotinylated proteins). This fraction primarily contains proteins\noriginating from non-muscle tissues.\nOur additional proteomic analysis revealed that the levels of Serpina1 family proteins\n(Serpina1a, Serpina1b, and Serpina1e) in the non-biotinylated fraction remained\nunchanged following exercise (Supplementary Fig. 10 in the revised manuscript). This\ncontrasts with the significant exercise-induced increase observed for biotinylated (muscle-\nderived) Serpina1 family members (Fig. 3 in the revised manuscript). It is important to\nnote that Serpina1c was detected but could not be reliably quantified due to its low\nabundance, precluding accurate mapping and measurement of Serpina1c-derived peptides\nunder our experimental conditions. Serpina1d was not included in this analysis, as this\nprotein was not detected in the biotinylated plasma fraction (Fig. 3 in the revised\nmanuscript).\nBy contrast, we found that the level of Bche (Butyrylcholinesterase), an exercise-induced\nfactor predominantly expressed in the liver (Horowitz et al., 2020, Science 369(6500):167-\n173) was significantly increased in the non-biotinylated fraction of the plasma of exercised\nmice (p = 0.0146, unpaired t-test; Supplementary Table 2 and Supplementary Fig. 10b\nin the revised manuscript). This indicates that our exercise training conditions were\nsufficient for eliciting systematic elevations of exercise-induced factors in the plasma.\nTherefore, these data support our findings in Fig. 3 that the exercise-upregulated plasma\nSerpina1e proteins are predominantly derived from muscle tissues.\nAdditional text explaining these results is now included in the Results of the revised\nmanuscript:\nline 168: There was no exercise-induced change in the plasma levels of non-biotinylated\nSerpina1 family proteins (derived from non-skeletal muscle tissues; Fig. 3d,\nSupplementary Fig. 10), while the plasma level of Bche (Butyrylcholinesterase),\nwhich is known to be mainly derived from the liver, was significantly elevated by\nexercise (Supplementary Fig. 10). These results indicate that the contribution of\nSerpina1 proteins from non-skeletal muscle tissues or organs to the systemic\nSerpina1 levels post-exercise is negligible.\n2) The authors now suggest that the muscle pool of Serpina1e plays a key role because\nthey injected lentiviral vectors to knock out Serpina1 in 4 limb muscles, which they\nclaim prevents exercise-induced changes in behavior (discrimination index and\nfreezing). However, these experiments (Fig. 4h-l, as well as others such as those in Fig.\n7a-h) are conducted suboptimally, as a control exercise group is not included, leaving\nit unclear whether the exercise training was performed at optimal levels to induce a\nresponse.\n: To address the reviewer’s concern, we compared the exercise intensity of mice in the\nS1eKO/EX group (S1eKO/EX) to that of the control group. We also examined whether\nS1eKO mice exhibit a positive relationship between exercise intensity (running distance in\nour study) and the memory performance, because exercise intensity is strongly correlated\nwith hippocampal memory performance (Fig. 1C).\nOur results demonstrated that the running distance of S1eKO/EX is comparable to that of\ncontrol mice (Fig.4 and 6; Supplementary Fig. 17f in the revised manuscript). However,\ndespite similar levels of exercise, S1eKO/EX failed to show a positive correlation between\nrunning distance and memory performance (Supplementary Fig. 17g in the revised\nmanuscript). These findings indicate that the lack of exercise-induced cognitive\nimprovement in S1eKO/EX is caused by minimal contribution of exercise-induced\nSerpina1e from skeletal muscles rather than an insufficient amount of exercise\nThe additional text is now included in the Results of the revised manuscript.\nline 229: We found that S1eKO/EX mice showed no enhancement in memory retrieval in\nthe NOR and CFC tests (Fig. 4k, l) and no positive correlation between running\ndistance and memory performance (Supplementary Fig. 17i), despite similar\namounts of exercise (Supplementary Fig. 17h). These results indicate that\nexercise-induced cognitive enhancement requires muscle-derived Serpina1e.\nAdditionally, there is no evidence that the lentiviral vectors used are muscle-specific,\nas they do not appear to carry a muscle-specific promoter and could potentially target\nSerpina1e in the liver or other tissues.\n: As concerned by the reviewer, it is possible that some of the lentiviral vector injected into\nlimb muscles may be transmitted to other non-muscle tissues, such as the liver. To\ninvestigate this possibility, we measured changes in Serpina1e mRNA levels in the liver,\nlung, and heart in S1eKO mice, by performing semi-quantitative RT-PCR on the Serpina1e\ntranscript.\nOur results replicate the finding of the significant downregulation of Serpina1e expression\nin limb muscles (where the lentiviral vector was injected) following the injection. However,\nno differences in Serpina1e mRNA levels were observed in other tissues (Supplementary\nFig. 17l, m in the revised manuscript). These results indicate that our method of reducing\nmuscle-derived Serpina1e through intramuscular injection of the lentiviral vector even\nwithout a muscle-specific promoter is effective for achieving limb muscle-selective\nSerpina1e knockdown.\nThis additional text is now included in the Results of the revised manuscript.\nline 219: The selective reduction of Serpina1e by the S1eKO virus was validated in AML12\ncells with either control or S1eKO lentiviruses (Supplementary Fig. 17a-d). A\nmuscle-specific reduction in Serpina1e was achieved by intramuscular injection of\nthe lentiviral vector (Supplementary Fig. 17e, f).\nFinally, even if the authors' model is correct and Serpina1e is indeed specifically\nknocked out in 4 limb muscles, it is still uncertain how this could impair the beneficial\neffects of exercise on the brain, given that many other muscles, both in the limbs and\nthroughout the body, remain unaffected by the lentiviral vectors (Serpina1e should still\nincrease following exercise in the vast majority of these muscles which are not targeted\nby the lentiviral vectors and hence promote exercise-induced improvements in\nbehavior).\n: As the reviewer pointed out, skeletal muscles that are unaffected by the Serpina1e-\ntargeting lentiviral vectors may still respond to physical activity during exercise.\nHuman limb skeletal muscles account for approximately 73–75% of total skeletal muscle\nmass (Sergi et al., 2015, Clinical Nutrition, 34(4): 667-673). Similarly, hindlimb skeletal\nmuscles in rodents constitute over 70% of total hindlimb mass (Armstrong, R. B. & Phelps,\nR. O., 1984, Am. J. Anat., 171(3): 259-272). Therefore, it is plausible that limb skeletal\nmuscles in mice also represent a significant portion of the total skeletal muscle mass in the\nbody. Thus, we suggest that Serpina1e knockdown in four limb muscles can effectively\ntarget most of the skeletal muscles involved in exercise.\n3) As mentioned in my previous comments, the cognitive assays that are currently\nutilized in the manuscript (novel object recognition and fear conditioning) are\ninherently variable. Robust gold-standard assays such as the Morris Water Maze\nshould be performed for at least some of the key experiments.\n: As suggested by the reviewer, we conducted the Morris water maze (MWM) test to\ndetermine whether the systematic delivery of recombinant Serpina1e can replicate the\nexercise-induced enhancement of hippocampal memory as shown in Fig. 4a and b in the\nrevised manuscript.\nOur results from the MWM test, which included five daily trials (short-learning version;\nSupplementary Fig. 5 in the revised manuscript), demonstrated that Serpina1e\nadministration increases the retrieval of spatial memory (Supplementary Fig. 12 in\nrevised manuscript). These improved memory performances in the MWM test align with\nour findings from the contextual fear conditioning tests (Fig. 4a, b in the revised\nmanuscript), indicating that systemic Serpina1e administration is beneficial for\nhippocampal memory.\nThis additional text is now included in the Results of the revised manuscript.\nline 197: Sedentary mice administered with recombinant Serpina1e exhibited improved\nperformance in the CFC test (Fig. 4b; Supplementary Fig. 11) and the MWM test\n(Supplementary Fig. 12), whereas Serpina1a had no such effect (Supplementary\nFig. 13)."}, {"round": 3, "reviewer_comments": {"reviewer_3": "I thank the authors for considering my points and for revising the text accordingly. However, it seems that no new experiments were done to address the outstanding issues. Therefore, I remain skeptical of the overall conclusions of this manuscript as they are not fully supported by the analyses. In particular, I remain not convinced that the skeletal muscle is a significant source of circulating Serpina1e even after exercise because this circulating factor is more highly (>100x) expressed in other organs (such as the liver) and therefore it seems unlikely that the muscle pool would significantly impact the circulating levels of Serpina 1e.\nWhile I understand there are limitations of the TurboID approach, the authors have previously utilized mass spectrometry to measure Serpina 1e levels in the circulation. Therefore, the authors could utilize this approach to define how much Serpina 1e is secreted by muscle vs. other organs by measuring Serpina 1e levels in the mice with muscle-targeted knockdown of Serpina 1e compared to controls. This approach is independent of TurboID and, therefore, I do not see any technical limitations in applying it to determine what proportion of circulating Serpina 1e is secreted by muscle in control conditions and following exercise.\nAltogether, without addressing experimentally this issue, it remains unclear whether muscle-secreted Serpina 1e is a meaningful contributor to the circulating pool of Serpina 1e.\nLikewise, I remain unconvinced that Serpina 1e knockdown in muscle abrogates the protective effects of exercise on the brain as claimed by the authors because there are many circulating factors (secreted by muscle and other tissues) such as irisin and cathepsin B that have been reported to exert this function. Therefore, I find it odd that Serpina 1e knockdown would completely abrogate the positive effects of exercise on memory, as found by the authors.\n“We found that S1eKO/EX mice showed no enhancement in memory retrieval in the NOR and CFC tests (Fig. 4k, l) and no positive correlation between running distance and memory performance (Supplementary Fig. 17i)”.\nAltogether, I find this manuscript interesting but several conclusions are based on suboptimal or incomplete experiments. While I suggested a few key analyses that would have mitigated these outstanding issues, the authors have not provided any new data to address these points despite standard experimental approaches (e.g. mass spec) available for these analyses. Therefore, I remain unconvinced about the overall robustness and validity of this study."}, "author_response": "Responses to the REVIEWER COMMENTS\nReviewer #3 (Remarks to the Author):\nI thank the authors for their work in addressing my queries. While I appreciate their efforts,\nthe pressing questions I previously asked have been answered only in part. Therefore, I\nwould please request the authors to address these remaining points:\n(1) Please clearly define what % of the circulating Serpina 1e derives from the skeletal\nmuscle versus other tissues in control conditions and in response to exercise. To this\npurpose, you can utilize the LC-MS/MS mass spec approach you previously used for the\nanalysis of immunoprecipitated biotinylated proteins, which are muscle-derived, vs.\nleftover fractions, which consist of proteins secreted from other tissues. Please display\nthe resulting data on the same graph so that it is possible to appreciate whether the\nskeletal muscle contributes a meaningful amount of Serpina 1e in control conditions\nand/or following exercise compared to other tissues. Without this analysis, it remains\nunclear whether muscle-secreted Serpina 1e is a significant contributor to the circulating\npool of Serpina 1e. Please clearly define what % of the circulating Serpina 1e derives from\nthe skeletal muscle compared to other tissues.\n: We thank the reviewer for this excellent question. Defining the relative contribution of different\ntissues to the circulating Serpina1e pool is a critical step in understanding its systemic function.\nWe fully agree that this information would further clarify the tissue origin of circulating Serpina1e.\nHowever, accurately quantifying the relative contribution of skeletal muscle versus other tissues\nto the circulating Serpina1e pool is currently not feasible due to inherent limitations of in vivo\nproximity labeling systems, including the TurboID system. As the reviewer requested, this would\nrequire comparing the biotinylated (muscle-derived) protein to the non-biotinylated (non-muscle-\nderived) fraction. However, this comparison would not produce biologically meaningful results for\ntwo main reasons:\n1. Low and unknown labeling efficiency: The biotinylation efficiency of TurboID and the\ndetection of biotinylated proteins are known to be very low and inconsistent even under\noptimal conditions due to the several factors, such as expression level of proximity labeling\nenzyme, efficiencies of protein biotinylation and enrichment of biotinylated proteins,\nand dynamic range of LC-MS (Qian et al., 2006, Molecular & Cellular Proteomics 5: 1727–\n1744; Kim et al., 2014, Proc Natl Acad Sci U S A, 111(24):E2453-61; Branon et al., 2018,\nNat Biotechnol., 36, 880–898; Cho et al., 2020, Nat Protoc., 15, 3971–3999; Jiang et al.,\n2024, ACS Meas Sci Au., 4: 338-417; Yang et al., 2024, bioRxiv\n12.17.628942; doi: https://doi.org/10.1101/2024.12.17.628942).\nIn addition, it has been reported that biotinylation efficiency of TurboID is relatively low in\nspecific cellular compartments like the ER, where Serpina1e is processed for secretion\n(Branon et al., 2018, Nat Biotechnol., 36, 880–898; Cho et al., 2020, Nat Protoc., 15,\n3971–3999). Taken together, these suggest that our system cannot exactly determine\nwhat fraction of muscle-derived Serpina1e is actually labeled in vivo.\n2. Confounding analytic variables: Quantitative comparison would be confounded by\ninconsistent variables, such as in vivo biotin delivery kinetics and the limited labeling\nradius of the TurboID enzyme (Kim et al., 2014, Proc Natl Acad Sci U S A, 111(24): E2453-\n61; Qian et al., 2006, Molecular & Cellular Proteomics 5: 1727–1744). Therefore, these\ninconsistent and uncontrollable variables would render any ratiometric comparison\nbetween the labeled and unlabeled fractions unreliable.\nWhile these technical issues prevent us from calculating an exact percentage of muscle-derived\nSerpina1e in the systemic pool, our data already provide indirect yet compelling evidence\naddressing the reviewer's core question, whether skeletal muscle represents a significant source\nof circulating Serpina1e. We demonstrated that:\n- A muscle-derived pool of biotinylated Serpina1e becomes detectable in the circulation\nfollowing exercise (Fig. 2 and 3).\n- Limb muscles exhibited elevated Serpina1e mRNA following exercise (Supplementary Fig.\n17f), despite lower baseline expression than in the liver.\n- Muscle-specific Serpina1e knockdown (Supplementary Fig. 17f) abolishes both the exercise-\ninduced increase in the intramuscular Serpina1e transcript and associated enhancements in\nhippocampal memory (Fig. 4k, l) and neurogenesis (Fig. 6g, h).\nTaken together, these findings clearly indicate that skeletal muscle is a physiologically relevant\nand functionally essential source of circulating Serpina1e in response to exercise.\nIn summary, although our proximity labeling approach is powerful for specifically identifying\nproteins originating from muscle, it is not a quantitative tool for determining their absolute\nproportion in circulation. Nonetheless, our data demonstrate that skeletal muscle is a significant\nsource of a circulating, exercise-responsive pool of Serpina1e, which is the central conclusion\nrelevant to our study.\nWe believe that accurately quantifying the contributions of muscle-derived Serpina1e to the\nsystemic pool may be possible if Serpina1e-specific antibodies or other precise quantitative\nprotein labeling tools become available in future studies.\nIn response to the reviewer’s point, we have included the limitations of the TurboID-based\nproximity labeling technique as below:\nline 322-330: Additionally, the in vivo labeling efficiency of TurboID is relatively low 36 and is inherently\ninfluenced by the subcellular localization of both the enzyme and its target proteins 18. In\nour model, TurboID was targeted to the endoplasmic reticulum (ER) of skeletal muscle cells,\nthereby preferentially labeling proteins that transit through or reside within the ER lumen.\nConsequently, cytosolic or non-classically secreted myokines, such as Irisin and Cathepsin\nB—which may bypass the conventional ER-Golgi secretion pathway—are unlikely to be\nbiotinylated and thus could not be detected in our system. Furthermore, even for proteins\nprocessed through the classical secretory pathway like Serpina1e, the inherent limitations\nof TurboID can lead to incomplete labeling. This reduces detection sensitivity and precludes\nreliable quantitative measurements in vivo.\nWe also emphasize that the skeletal muscle is the essential source of exercise-induced Serpina1e\nin the Discussion part of the revised manuscript as below:\nline 336-338: Despite the dominant expression in the liver 29,36, our data demonstrate a muscle-specific\nupregulation following exercise, both at the mRNA and protein level (Fig. 2 and 3;\nSupplementary Fig. 8 and 9) after 4 weeks of wheel running exercise.\n(2) Please utilize the same mass spec approach as above to better characterize the\nlentiviral model of Serpina1e knockdown. Specifically, the authors should measure the\ncirculating levels of Serpina1e in mice with or without Serpina1e knockdown in control\nconditions and following exercise. Also in this case, the data for all conditions should be\ndisplayed on the same graph.\n: We appreciate the reviewer’s suggestion to further characterize our knockdown model at the\nprotein level. While we agree that measuring circulating Serpina1e following knockdown would\nbe of interest, we respectfully argue that our existing data provide a definitive validation of the\nfunctional relevance of our knockdown model for two key reasons:\n1. Technical limitations of detecting a partial reduction in circulating Serpina1: As discussed\nin our previous response, accurately quantifying total circulating Serpina1e via LC-MS is\ntechnically challenging. Detecting a modest reduction in circulating Serpina1e, resulting\nfrom a partial knockdown in muscles, would likely fall below the sensitivity and reliability\nthreshold of this method. These technical limitations would make the measurement\npotentially inconclusive and difficult to interpret.\n2. Conclusive validation of knockdown via functional outcomes: The validity of our\nknockdown model is best demonstrated by its clear biological consequences. Our data\nshow that a significant reduction of Serpina1e mRNA at the source (Fig. 4i; Supplementary\nFig. 17e, f), a well-established indicator of knockdown efficacy, is accompanied by a\nmarked loss of the cognitive benefits of exercise and its associated neurogenesis (Fig. 4k,\nl; Fig. 6g, h). This direct link between local transcript loss and the impairment in systemic\nfunctions provides compelling evidence for the critical role of muscle-derived Serpina1e.\nIn summary, we acknowledge the importance of protein-level quantification for addressing the\nexact contribution of muscle-derived Serpina1e to the systemic pool. However, we believe the\nfunctional consequences observed following Serpina1e knockdown provide more robust and\nbiologically meaningful validation of the role of Serpina1e, rather than a technically challenging\nmeasurement that may not be reliable.\n(3) I am not convinced that lentiviral injection in the limbs will target all limb muscles and\nrepresent a knockdown of Serpina 1e in 73-75% of all body muscles as claimed by the\nauthors. This is unlikely because major muscles such as the diaphragm are not targeted\nand because the limb injection that the authors do is most likely an intramuscular injection\nthat delivers the virus primarily to only one limb muscle, whereas the other limb muscles\nremain most likely untargeted. The authors should test whether indeed all limb muscles\nare targeted by doing a qRT-PCR for Serpina1e from each of the individually dissected\nlimb muscles, western blots for Serpina1 from dissected limb muscles, and/or mass spec.\n: We thank the reviewer for insightful concerns regarding the extent and validation of our lentiviral\nknockdown model.\nFirst, to clarify a key point, our manuscript does not claim a knockdown in '73-75% of all body\nmuscles.' Our goal was to achieve a functionally significant knockdown in the large limb muscles,\nwhich are the primary effectors in our exercise model.\nSecond, while we understand the request for analyzing individual muscle with qRT-PCR,\nwestern blot, or mass spec to validate comprehensive knockdown efficacy across all limb muscles,\nwe respectfully disagree that this level of detail is essential for validating the core conclusions of our\nstudy.\n1. Contribution of untargeted muscles is negligible:\nAs the reviewer pointed out, the diaphragm or other muscle was not targeted by the lentiviral\nvector in our study. However, its contribution to total body muscle is negligible.\nIn case of normal human adults, the average weight of the diaphragm is approximately 200 g,\naccounting for about 3 to 4% of body weight (e.g., Thurlbeck, 1978, Thorax, 33: 483-487). Given\nthat skeletal muscles comprise approximately 40% of total body weight (e.g., Frontera and Ochala,\n2015, Calcif Tissue Int. 96: 183-195), the diaphragm's mass constitutes only about 1% of total\nmuscle mass. It appears that rodents have a similar or even smaller proportion of diaphragm\nmass. Therefore, any contribution from the diaphragm or similar size of the muscle to the exercise-\ninduced pool of Serpina1e is expected to be insignificant compared to the large limb muscles we\ntargeted.\n2. Widespread transduction of lentiviral vectors in limb muscles is well-established:\nRegarding the concern about localized delivery, we respectfully disagree that injection targets\nonly a single muscle.\nThe intramuscular injection of viral vectors is a well-established technique for achieving\ntransduction of foreign genes in multiple muscle fibers. Given our conditions for injecting the\nlentiviral vector (5 x 10^8 TU / 25 µL per limb), it is highly likely that the injected lentiviral vectors\nspread and transduced a wider region of the limb musculature around the injected sites. Previous\nstudies have demonstrated that lentiviral particles can transduce multiple adjacent muscles after\na single intramuscular injection (e.g., O'Rourke et al., 2003, Mol. Ther., 7: 632-639; Talbot et al.,\n2009, Mol. Ther., 18: 601-608; Kimura et al., 2009, Mol. Ther., 18(1): 206-213). We used similar\nranges of viral titer and volume for our intramuscular injection of the lentiviral vector.\n3. Validation of our lentiviral knockdown model with functional outcome:\nOur primary objective was to investigate the impact of lowering Serpina1e expression on exercise-\ninduced cognitive enhancement. The validity of our knockdown model is best demonstrated by its\nbiological impact: the lentiviral vector-mediated knockdown resulted in a significant reduction\nof Serpina1e transcripts in limb muscle, leading to the complete abolishment of cognitive and\nneurogenic benefits of exercise (Fig. 4h-l, Fig. 6g, h, Supplementary Fig. 17).\nIn summary, the definitive functional outcome of our knockdown experiments provides the most\ncritical validation of our lentiviral Serpina1e knockdown in skeletal muscles. Our functional\nevidence, combined with the quantitative insignificance of untargeted muscles, provides a robust\nand reliable basis for our conclusions. While individual muscle analysis would provide a more\ndetailed and through assessment, it is unlikely to alter the overall conclusion that reducing\nSerpina1e levels in skeletal muscle diminishes the effect of exercise on cognitive functions.\n(4) The Morris Water Maze results are interesting but preliminary because they are done\nwith a low number of animals (n=5-7). Normally, a higher number of animals is used in\nbehavioral tests like this to ensure the robustness and reproducibility of the findings.\n: We acknowledge the reviewer’s concern regarding the limited sample size used in the Morris\nwater maze (MWM) test. To minimize animal sacrifice while maintaining statistical rigor, we\nemployed a relatively small cohort of mice for the behavioral assessments. In the revised\nmanuscript, we have designated the MWM results as ‘preliminary’ to reflect this limitation.\nHowever, we would like to emphasize that these findings are in line with the significant\nimprovements in memory performance in other behavioral tests. Our MWM results demonstrate\na trend consistent with findings from contextual fear conditioning (Fig. 4a, b). Furthermore, an\nalternative approach to increase the systemic Serpina1e level – intramuscular overexpression of\nSerpina1e – also yielded significant improvements in memory performance in the novel object\nrecognition and contextual fear conditioning tests (Fig. 4c-e). Therefore, we believe that\nincreasing the number of animals in the MWM would not substantially alter these consistent\nfindings.\nThe description of our findings were revised as below:\nline 197~201: Sedentary mice administered recombinant Serpina1e exhibited improved performance in the\nCFC test (Fig. 4b; Supplementary Fig. 11), while Serpina1a had no such effect\n(Supplementary Fig. 13). Our preliminary results from the MWM test (Supplementary Fig.\n12) also demonstrated a consistent improvement in spatial memory performance following\nthe administration of recombinant Serpina1e.\n(5) There are many factors secreted by skeletal muscle following exercise that contribute\nto increasing cognitive capacity (e.g. irisin and cathepsin B), in addition to exercise-\ninduced signals secreted by other tissues (such as the liver) that similarly improve\ncognition. Therefore, I find it odd that Serpina 1e knockdown would completely abrogate\nthe positive effects of exercise on memory, as found by the authors: “We found that\nS1eKO/EX mice showed no enhancement in memory retrieval in the NOR and CFC tests\n(Fig. 4k, l) and no positive correlation between running distance and memory performance\n(Supplementary Fig. 17i)”.\n: As the reviewer pointed out, there are known myokines, such as FNDC5/irisin and Cathepsin B,\nas well as other exercise-induced factors (“exerkines”) that have been shown to improve cognitive\nfunction.\nWhile our data demonstrated the essential role of muscle-derived Serpina1e in exercise-induced\ncognitive functions, we did not exclude the possibility that other exerkines play a role in cognitive\nenhancement. In the Discussion of the previous version of the manuscript (line 343~349), we\nalready suggest that Serpina1e may share downstream signaling with other myokine such as\nIrisin. We now extend this point by providing additional explanations as below:\nThe beneficial effects of exercise on cognitive functions appear to be mediated by the complex\nand cooperative actions of multiple exerkines rather than by the individual effect of a single\nexerkine. This is because intense and chronic physical activities trigger systemic interactions\namong various organs, including muscles, liver, bone, and adipose tissue (Severinsen et al.,\n2020, Endocrine Reviews, 41(4): 594-609).\nIn fact, elevations in BDNF levels, neurogenesis, and neurite growth by Serpina1e are common\nphysiological consequences of exerkines in the hippocampus. These functional similarities\nbetween Serpina1e and other exerkines raise the possibility that exercise-induced changes in\nbrain function are mediated by the cooperative functions of multiple exerkines.\nFor instance, lactate produced during intense exercise can be transported to the hippocampus,\nresulting in the upregulation of BDNF expression through SIRT1-PGC1a signaling (Hayek et al.,\n2019, J. Neurosci., 39(13): 2369–2382). This lactate-induced BDNF was mediated\nby PGC1a/FNDC5 pathway, suggesting that the beneficial effects of exercise on cognitive\nfunctions are dependent on complex interactions among multiple exerkine-related signaling\npathways. Additionally, the anti-inflammatory functions of Serpina1e may correlate with those of\nother myokines, such as irisin, because irisin can suppress glial activation and inflammatory\nsignaling in the diseased brain (Islam et al., 2021, Nat. Metab., 3: 1058-1070; Peng et al.,\n2017, Mol. Immunol., 91: 185-194). While the molecular identities of exerkines that functionally\ninteract with Serpina1e remain unclear, it is possible that Serpina1e plays a crucial role as either\nan upstream or downstream signaling component essential for exerkine-induced cognitive\nenhancement.\nTherefore, our data showing no exercise-induced cognitive enhancement or related changes in\nthe hippocampus following Serpina1e knockdown suggest that Serpina1e is an essential\ncomponent for the exercise-induced modulation of cognitive functions. Moreover, the beneficial\neffects of other exerkines on cognitive functions appear to depend on Serpina1e-related signaling.\nIn our future studies, we will explore whether Serpina1e signaling interacts with other exercise-\ninduced factors and how Serpina1e interacts with other exerkines during exercise-induced\ncognitive enhancements.\nIn response to the reviewer’s feedback, we strengthened our discussion on the potential\ninteraction between Serpina1e and other exercise-induced factors in the Discussion section of\nthe revised manuscript, as below:\nline 354~378: The beneficial effects of exercise on cognitive functions may be mediated by the complex\nand cooperative actions of multiple exerkines, rather than by the individual effect of a single\nexerkine. Exercise triggers systemic interactions among various organs, including muscles,\nliver, bone, and adipose tissue. Indeed, the elevations in BDNF levels, neurogenesis, and\nneurite growth induced by Serpina1e (Fig. 6) are common physiological consequences\nshared among various exerkines, raising the possibility that exercise-induced changes in\nbrain function are mediated by a cooperative functional network. This potential cooperation\nis exemplified by the functional overlap between Serpina1e and Irisin, both of which\nincrease the number of newborn neurons and the expression of BDNF. Moreover, a recent\nstudy indicated a possible overlap in the downstream signaling pathways of Serpina1 and\nIrisin. Serpina1 proteins interacts with Ephrin type-B receptor 2 (EphB2) to promote cell\nproliferation and improve thermogenesis and glucose metabolism by the uncoupling protein\n1 (UCP1) 29. Meanwhile, Irisin also upregulates browning-associated genes and UCP1\nprotein in cultured adipocytes and adipose tissues 38. Additionally, the anti-inflammatory\nfunctions of Serpina1e may correlate with those of other myokines, such as irisin, because\nirisin can suppress glial activation and inflammatory signaling in the diseased brain 39,40.\nCrosstalk among multiple exerkine pathways is further highlighted by findings that lactate,\nproduced during intense exercise, can upregulate BDNF expression through\nPGC1a/FNDC5 signaling 41. Therefore, the abolishment of exercise-induced cognitive\nenhancement by muscle Serpina1e knockdown (Fig. 4k, l; Supplementary Fig. 17i) and\nrelated hippocampal changes (Fig. 6g, h) not only indicates that Serpina1e is essential for\nthe exercise-induced upregulation of hippocampal functions but also suggests that the\nbeneficial effects of other exerkines depend on Serpina1e-related signaling. Although the\nmolecular identities of exerkines that functionally interact with Serpina1e remain unclear,\nit is plausible that Serpina1e plays a crucial role as either an upstream or downstream\nsignaling component within this complex exerkine crosstalk.\nResponses to the reviewer’s points:\nReviewer #3 (Remarks to the Author):\no I thank the authors for considering my points and for revising the text accordingly. However,\nit seems that no new experiments were done to address the outstanding issues. Therefore, I\nremain skeptical of the overall conclusions of this manuscript as they are not fully\nsupported by the analyses. In particular, I remain not convinced that the skeletal muscle is\na significant source of circulating Serpina1e even after exercise because this circulating\nfactor is more highly (>100x) expressed in other organs (such as the liver) and therefore it\nseems unlikely that the muscle pool would significantly impact the circulating levels of\nSerpina 1e.\nWhile I understand there are limitations of the TurboID approach, the authors have\npreviously utilized mass spectrometry to measure Serpina 1e levels in the circulation.\nTherefore, the authors could utilize this approach to define how much Serpina 1e is secreted\nby muscle vs. other organs by measuring Serpina 1e levels in the mice with muscle-targeted\nknockdown of Serpina 1e compared to controls. This approach is independent of TurboID\nand, therefore, I do not see any technical limitations in applying it to determine what\nproportion of circulating Serpina 1e is secreted by muscle in control conditions and\nfollowing exercise.\nAltogether, without addressing experimentally this issue, it remains unclear whether\nmuscle-secreted Serpina 1e is a meaningful contributor to the circulating pool of Serpina 1e.\n- We thank the reviewer for the constructive feedback. We agree that the question of\nwhether skeletal muscle contributes meaningfully to circulating Serpina1e is\nimportant.\nBelow we provide three lines of evidence that clarify this point:\n(1) High transcript abundance of Serpina1e does not equate to high Serpina1e\nprotein secretion from the liver\nWhile Serpina1e transcripts are indeed more abundant in the liver than in skeletal\nmuscle (for the very recent example, Okagawa et al., 2024, Nat. Commun., 15:9585),\nproteomic studies show that transcript abundance does not necessarily equate to\namount of systemic secretion.\nFor example, studies employing the in situ secretory protein labeling using the ER-\nanchored TurboID approach (Kim et al., 2021, Nature Comm., 12: 5204 and Wei et al.,\n2021, Nat Chem Biol., 17: 326-334) demonstrated liver-secreted proteins but failed\ndetect Serpina1e among circulating liver-derived proteins, although other\nSerpina1 proteins were detected.\nThis result indicates that liver-derived Serpina1e protein in the plasma is not well\ncorrelated with its high transcript abundance. The similar poor correlation between\nmRNA and protein expression has widely been observed in diverse cell types (for\nexample, Nusinow et al., 2020, Cell, 180: 387-402).\nFurthermore, a recent organism-wide secretome atlas (Wei et al., 2023, Cell Metab.,\n35: 1261-1279) also support the notion that the liver is not a dominant source of\ncirculating Serpina1e. In this dataset, Serpina1e was identified as a secretory protein\nfrom multiple tissues, including both liver and skeletal muscle. Our quantitative re-\nanalysis of these data shows that relative contributions of liver and muscle to the\ncirculating Serpina1e are comparable under sedentary conditions (below Figure 1).\ndEPeV+G\"E\n-S+ELMS)\"G\"2MPE4\naVbEPL>)c2MPE4\nB<KD)ELBMC2MPE4\n@G+DPEGKEKL@IA\"2MPE4\n>IPE+G)CTVKK<EL?P:G\"2MPE4\n;<TL=V)\"2MPE4\n5+IST8E)VG)CDE))KLMI8:2MPE4\nBCDE))CG+IC-CDE))KLMN#2MPE4\n! \"! #! A!\naSI#Cg+TE+KVTh\nFigure 1. Serpina1e level secreted by corresponding tissue types (left). Serpina1e proportion\nsecreted by corresponding tissue types (right), based on re-analysis of the mass spectrometry\nresults from Wei et al., 2023, Cell Metab., 35: 1261-1279.\n(2) Exercise induces muscle-specific upregulation of Serpina1e transcript\nGiven the limited contribution of the liver Serpina1e under the sedentary condition,\nwe reason that the exercise-induced increase in circulating Serpina1e primarily originates\nfrom skeletal muscle.\nConsistent with this idea, we observed a significant exercise-induced increase in\nSerpina1e transcripts in muscle, whereas liver or other tissue-derived Serpina1e\nexpression remained unchanged (Supplementary Fig. 17 in the revised\nmanuscript).\nThese data strongly suggest that skeletal muscle is the main source of the elevated\nplasma Serpina1e following exercise.\n!\"\nBone(Col1a1 TcellandBc\n-Cre) ells(CD2-Cre)\n13% 9%\nEndothelialc\nells(Cdh5-\nCre)\n10%\nLiver(Alb-\nCre)\n18% Gut(Vil1-Cre)\n16%\nMuscle(MCK-\nCre)\n17%\nAdrenaltissu\nPancreases( e(Nr5a1-Cre)\nPdx1-Cre) 10%\n7%\n(3) No increase in the non-muscle-derived Serpina1e protein following exercise\nOur streptavidin-based fractionation of biotinylated- and non-biotinylated proteins in\nplasma of ACTuR mice showed that only the biotinylated (muscle-derived) Serpina1\nprotein (Serpina1a-e) fraction increased in plasma after exercise, while the non-\nbiotinylated fraction (leftover) remained unchanged (Fig. 3d, e in the revised\nmanuscript).\nIn direct response to the reviewer’s suggestion, we further conducted additional LC-\nMS/MS analysis on the non-biotinylated fraction (the biotinylated fraction was\nalready analyzed in Fig. 2 of the original manuscript) during the previous round of\nrevision.\nThese additional results confirmed that Serpina1e level in the non-biotinylated fraction\ndid not change following exercise (Supplementary Fig. 10 in the revised\nmanuscript).\nPlease see Fig. 2 below for direct comparison of Serpina1e levels in the biotinylated\nand non-biotinylated fractions.\nFigure 2. Serpina1e level in plasma of ACTuR mouse analyzed by LC-MS/MS\na. Serpina1e level in the biotinylated fraction of the plasma (secreted from skeletal muscles,\nadapted from Fig. 2 in the original manuscript).\nb. Serpina1e level in the non-biotinylated fractioin of the plasma (secreted from non-skeletal\nmuscles, adapted from Supplementary Fig. 10 in the revised manuscript).\nTaken together, these independent in vivo secretome-mapping studies as well as our\nfindings provide strong orthogonal support for our conclusion that skeletal muscle is\nthe physiologically relevant source of circulating Serpina1e.\no Likewise, I remain unconvinced that Serpina1e knockdown in muscle abrogates the\nprotective effects of exercise on the brain as claimed by the authors because there are many\ncirculating factors (secreted by muscle and other tissues) such as irisin and cathepsin B that\nhave been reported to exert this function. Therefore, I find it odd that Serpina 1e knockdown\nwould completely abrogate the positive effects of exercise on memory, as found by the\nauthors. Furthermore, it seems unlikely the authors’ claim that the activity of cathepsin B\nand irisin (and of any other exercise-induced protective factor, secreted by muscle and other\ntissues) would strictly depend on optimal Serpina 1e levels.\n- We thank the reviewer for raising this important point regarding the dominant role of\nSerpina1e in mediating cognitive benefits of exercise.\nAs pointed by the reviewer, other exercise-induced myokines, including FNDC5/Irisin\nand Cathepsin B, have been shown to be required for promoting hippocampal memory.\nHowever, given evidence from our work and other studies, these results are thought\nto be due to the mechanisms that exercise-induced myokines may act cooperatively\nrather than independently.\n(1) Interdependent actions of myokines in exercise-induced cognitive benefits\nPrevious studies have demonstrated that the genetic deletion of Fndc5 (Islam et al.,\n2023, Nat. Metab., 5:1058–1070) or Ctsb (Moon et al., 2018, Cell Metab., 24:332–340)\nleads to an impairment in exercise-induced memory enhancement, even though other\nmyokines presumably remain intact.\nThese findings, along with our results, raise the possibility that multiple exercise-\ninduced myokines functionally interact to support cognitive benefits, and that disruption\nof any key component can attenuate the overall effect of myokines.\n(2) New data suggesting a functional Serpina1e-Fndc5 interaction\nTo test whether Serpina1e influences other myokines associated with cognitive\nenhancement, we conducted additional experiments.\nOur new results showed that Serpina1e regulates the Fndc5 expression in\nskeletal muscle (Fig. 3 below):\n(a) AAV-mediated overexpression of Serpina1e in skeletal muscle (S1eOE)\nmodestly increased Fndc5 levels\n(b) Conversely, lentiviral knockdown of Serpina1e in skeletal muscle (S1eKO)\nsignificantly reduced Fndc5 levels\n(c) These effects were selective, because Ctsb expression remained\nunchanged under both conditions.\nTogether, these findings suggest that Serpina1e acts as a functional upstream\nregulator of Fndc5 in skeletal muscle, providing a plausible mechanistic explanation\nfor why Serpina1e knockdown abolishes the cognitive benefits of exercise.\nIn conclusion, these findings collectively support the notion that exercise-induced\ncognitive enhancement depends on a coordinated functional network of multiple\nmyokines—as exemplified by the Serpina1e–FNDC5 axis in this study—rather than a\nsingle independent signaling pathway.\nFigure 3. Selective association between Serpina1e and Fndc5 expression in skeletal\nmuscles\na. Representative gel images showing mRNA expression of Fndc5, Cathepsin B,\nSerpina1e, and Rplp0 in skeletal muscle derived from control or S1eOE mice.\nb. Bar graphs depicting the average mRNA expression levels of Fndc5, Cathepsin B, and\nSerpina1e normalized to Rplp0. ****p< 0.0001 (Serpina1e); p= 0.0775 (Fndc5) and p=\n0.9835 (Cathepsin B); unpaired two-tailed Student’s t-test.\nc. Representative gel images showing mRNA expression of Fndc5, Cathepsin B,\nSerpina1e, and Rplp0 in skeletal muscle derived from control, S1eKO, or S1eKO+EX mice.\nd. Bar graphs depicting the average mRNA expression levels of Fndc5, Cathepsin B, and\nSerpina1e normalized to Rplp0. ***p= 0.0007 and **p= 0.0076 (Serpina1e); *p= 0.0173 and\np= 0.0802 (Fndc5); p= 0.4305 and p= 0.1678 (Cathepsin B), one-way ANOVA with\nDunnett’s post hoc test.\nAll the data in the bar graphs are presented as the mean ± SEM. Each dot in the graphs\nrepresents an individual mouse.\no Altogether, I find this manuscript interesting but several conclusions are based on\nsuboptimal or incomplete experiments. While I suggested a few key analyses that would\nhave mitigated these outstanding issues, the authors have not provided any new data to\naddress these points despite standard experimental approaches (e.g. mass spec) available\nfor these analyses. Therefore, I remain unconvinced about the overall robustness and\nvalidity of this study."}, {"round": 4, "reviewer_comments": {"reviewer_4": "The authors have performed an additional experiment (Figure 4) to address my original query whether Serpina1e is responsible/required for the exercise-induced enhancement in cognition. To address this they have reduced S1e expression in muscle tissue using a lentiviral strategy. Here they show that there is no significant difference in cognitive function between the control, S1eKO and the S1e+ exercice mice and they conclude that this supports the conclusion that muscle-derived Serpina1e mediates exercise-induced cognitive enhancement. The data however, showing that this exercise paradigm leads to enhancement in in NORT and CFCT in these mice is absent. It is hard to determine whether the effect of exercise on cognition is blocked by the knockdown or is absent.\nThe authors have also added in additional neurogenesis quantification in Figure 6. I query why only a n=3 mice were phenotyped for BrdU/NeuN (Fig 6e,f), when there were BrdU counts for 6-7 mice per group in panel D. This is a very small N.\nTo assess dendritic arborization, the authors have measured MAP2 intensity as a proxy of dendritic complexity. It would have been much more informative to make measures from individual neurons from each group of mice to assess parameters such as dendritic length, number of branches etc."}, "author_response": "Responses to the reviewer’s points:\nReviewer (Remarks to the Author):\n● The authors have performed an additional experiment (Figure 4) to address my\noriginal query whether Serpina1e is responsible/required for the exercise-induced\nenhancement in cognition. To address this, they have reduced S1e expression in\nmuscle tissue using a lentiviral strategy. Here they show that there is no significant\ndifference in cognitive function between the control, S1eKO and the S1e+ exercise\nmice and they conclude that this supports the conclusion that muscle-derived\nSerpina1e mediates exercise-induced cognitive enhancement. The data however,\nshowing that this exercise paradigm leads to enhancement in NORT and CFCT in\nthese mice is absent. It is hard to determine whether the effect of exercise on\ncognition is blocked by the knockdown or is absent.\n: We thank the reviewer for raising this important point.\nSince our exercise paradigm was proven to induce the hippocampal memory\nenhancement, which is highly correlated with running distance (Fig. 1a-c in the revised\nmanuscript), we expected that exercise intervention would successfully induce the\nmemory performance of control (CTL) mice in this experimental setup.\nHowever, as the reviewer pointed out, all NORT and CFCT performances results from\nexercised CTL mice and mice with Serpina1e knockdown in skeletal muscles (S1eKO) are\nessential to determine the precise role of muscle-derived Serpina1e in exercise-induced\ncognitive enhancement.\nIn this revision, we conducted additional behavioral tests to include all results from CTL\nand S1eKO mice in either sedentary or exercised condition (CTL, CTL/EX, S1eKO,\nS1eKO/EX). Our new results still confirmed the positive effect of exercise on the memory\nperformance of CTL mice. However, this beneficial effect was abolished in S1eKO/EX\nmice, while exercise significantly enhances memory performance (Fig. 4k-m in the\nrevised manuscript).\nThese results support our conclusion that muscle-derived Serpina1e is necessary for\nexercise-induced memory enhancement.\n● The authors have also added in additional neurogenesis quantification in Figure 6.\nI query why only a n=3 mice were phenotyped for BrdU/NeuN (Fig 6e, f), when there\nwere BrdU counts for 6-7 mice per group in panel D. This is a very small N.\n: We agree with the reviewer’s concern regarding the small sample size.\nTo address this, we have included more BrdU/NeuN co-staining results acquired from\nadditional preparation of mice. Now, our revised data contains at least five mice (Fig. 6d-\nf in the revised manuscript). Please note that the data presented in Fig. 6d-f of the\nrevised manuscript has been replaced with results obtained from the same preparation,\nwhich consequently led to changes in the reported N values.\nConsistent with our previous data, our new results showed that an increase in plasma\nSerpina1e level leads to upregulated neurogenesis.\nTo address the same concern regarding the sample size, we performed additional analysis\nof newborn neurons from the hippocampus of S1eKO mice. These results are now\nincluded in Fig. 6h and Supplementary Fig. 17l, m in the revised manuscript.\n● To assess dendritic arborization, the authors have measured MAP2 intensity as a\nproxy of dendritic complexity. It would have been much more informative to make\nmeasures from individual neurons from each group of mice to assess parameters\nsuch as dendritic length, number of branches etc.\n: We thank the reviewer for the helpful comment to improve our manuscript.\nTo measure changes in neurite lengths and branch numbers of individual hippocampal\nneurons in vivo, we utilized the Thy1-EGFP mouse model to visualize and analyze the\nsingle neuronal morphology. After 4 weeks of intramuscular overexpression in Serpina1e\n(S1eOE) in Thy1-EGFP mice, morphological parameters of 3D-reconstructed\nhippocampal neurons with isolated EGFP expression were assessed to test whether\nmuscle-derived Serpina1e promotes the hippocampal neuronal growth.\nIndeed, our new results demonstrate that muscle-derived Serpina1e produces a significant\nincrease in neuronal outgrowth in vivo, as evidenced by a longer dendritic length,\nincreased number of branches and neurite complexity (Fig. 6i-n in the revised\nmanuscript). These results are consistent with our previous data showing the neurite\noutgrowths of in vitro primary neurons by recombinant Serpina1e protein (Supplementary\nFig. 20 in the revised manuscript) and elevated MAP2 intensity by muscle-derived\nSerpina1e (Supplementary Fig. 21 in the revised manuscript)."}, {"round": 5, "reviewer_comments": {"reviewer_4": "The authors have now addressed all of my previous queries."}, "author_response": ""}]

Decision letter