Rasal1: A candidate exercise mimetic to increase adult hippocampal neurogenesis in middle age

Rasal1: A candidate exercise mimetic to increase adult hippocampal neurogenesis in middle age | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Rasal1: A candidate exercise mimetic to increase adult hippocampal neurogenesis in middle age Yvonne Nolan, Maria Giovanna Caruso, Sebastian Dohm-Hansen, Sarah Nicolas, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8394751/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 12 You are reading this latest preprint version Abstract Physical exercise exerts several positive effects on the brain and is emerging as an approach for mitigating age-related functional decline. One benefit of exercise is the increase in adult hippocampal neurogenesis (AHN). However, physical exercise may not always be feasible in individuals due to physical or medical constraints which are more common in older age, highlighting the importance of understanding regulators of exercise-induced AHN. We demonstrate that Rasal1, a GTPase-activating protein, is downregulated in the dentate gyrus (DG) by voluntary running exercise in young adult rats. Middle age is recognised as a period of the lifespan prognostic of cognitive health in older age, and amenable to intervention. Lentiviral knockdown of Rasal1 in the DG promoted AHN and neurite growth in middle-aged rats that were otherwise reluctant to run. Treatment of hippocampal neuroprogenitor cells in vitro with serum from exercising animals mimicked the exercise-induced downregulation of Rasal1 and was associated with neuroprogenitor expansion. Lastly, multi-omic analysis of sera in conjunction with brain proteomics identified possible systemic mediators of Rasal1 downregulation. Taken together, our findings highlight Rasal1 as a novel candidate regulator of the pro-neurogenic effects of exercise. Identifying systemic factors underlying the Rasal1-driven effects of exercise could inform new therapies to enhance AHN and hippocampal function. Biological sciences/Neuroscience Biological sciences/Molecular biology Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Physical exercise has been shown to be neuroprotective by exerting beneficial effects at systemic, cellular, and molecular levels ( 1 , 2 ). One notable example is the stimulation of adult hippocampal neurogenesis (AHN) in the rodent dentate gyrus (DG) ( 3 , 4 ). As AHN is involved in regulating antidepressant action, anxiety, and certain forms of cognition, the ability to induce it via exercise could be useful in states associated with reduced AHN, such as aging or neurodegenerative disease ( 5 ). However, maintaining regular physical exercise may not always be feasible in such states due to physical or health constraints. Therefore, it is important to identify the mechanisms underlying exercise-induced AHN. AHN has been found to decline with advancing age in several species, including humans ( 6 – 8 ), mice ( 9 , 10 ), and rats ( 11 ). While a body of literature has found that exercise can reverse this age-related decline in rodents ( 12 ), it can also fail to induce AHN, especially at older ages ( 13 , 14 ), further highlighting the need to understand and identify novel regulators of AHN, such as proteins within the DG. Moreover, exercise influences AHN not just through effects in the brain but also via factors in the systemic circulation ( 2 , 15 ). For instance, the beneficial effects of voluntary wheel-running on AHN in both young and aged mice have been phenocopied by plasma transfer from exercising to sedentary animals, with circulating proteins as candidate mediators ( 16 ). With an increasingly older global population, there is significant interest in establishing tractable interventions that maintain cognitive and physical health, especially when exercise is not a viable intervention. Identifying AHN regulators in the systemic circulation would provide relevant targets to modify AHN for healthy brain aging. While studies on aging have historically focused on advanced age, emerging evidence suggests that middle age may be a period of accelerating change for the brain, as well as systemic circulation, that shape future cognitive trajectories ( 17 ). Thus, middle age presents a unique window of opportunity for such interventions. Herein, we used a proteomics approach to identify regulators of exercise-induced AHN in young adult rats that could potentially be harnessed to boost neurogenesis during middle age. We investigated whether genetic manipulation of an identified hippocampal protein candidate could mitigate the age-related decline in AHN in vivo in middle-aged rats. Using an in vitro approach, we subsequently investigated whether this AHN regulator was sensitive to regulation by exercise-induced changes in the blood circulation. Finally, we used a multi-omic approach to identify candidate blood-borne proteins and metabolites that may link exercise-induced alterations in the systemic circulation with proteins that regulate AHN in the brain. Materials and Methods Animals Adult (12 weeks old) and middle-aged (12 months old) male Sprague Dawley rats were obtained from Janvier Laboratories (France). All animal procedures were performed under authorizations (#AE19130/P149) issued by the Health Products Regulatory Authority (HPRA, Ireland), in accordance with the European Communities Council Directive (2010/63/EU) and approved by the Animal Experimentation Ethics Committee of University College Cork. Upon arrival, animals were pair-housed in either standard cages (Y-SED, MA-SED) or with free access to a running wheel (Activity wheel 33cm diameter, Techniplast, UK) (Y-EX, MA-EX). Running or sedentary conditions were maintained for the duration of the experiment, which lasted a total of 6 to 8 weeks ( Supplementary Dataset 5 ). Stereotactic brain surgery Rats were bilaterally, stereotactically infused with 3.0 µL of ultra-purified lentiviral-based short-hairpin RNA vectors (shRNA targeting rat Rasal1 > 1x10 8 IU mL − 1 or scrambled control > 1x10 8 IU mL − 1 ) into the dorsal hippocampus (from bregma: A.P. = -3.5, M.L. = +-2.4, D.V. = -3.6) at 1µl/min. qPCR analysis for the expression of the LV SCR and LV shRASAL1 viral vector transcripts was carried out in the dorsal hippocampus to further confirm the successful lentiviral transduction. See Supplementary Information for details. Proteomics and metabolomics Proteomics analysis of serum and hippocampal tissue, as well as metabolomics analysis of serum, are described in Supplementary Information. See Supplementary Table 1 for more details. IHC Ki67, DCX, Rasal1, NeuN, GFAP, Olig2, Iba1 immunohistochemistry was conducted in the hippocampus as described in Supplementary Information. All analyses were performed blind to treatment conditions. See Supplementary Table 2 for more details. Western blotting Western blotting Hippocampi tissue homogenates (20 µg of proteins per lane) were analysed by Western blot using a mouse anti-Rasal1 polyclonal antibody (Abnova #H00008437-B01P; Lot M4251). Protein band quantification was performed by densitometry analysis normalized against MemCode™ Reversible Total Protein Stain. Further details are provided in Supplementary Information. RNA extraction and quantitative real-time PCR Total RNA was extracted from hippocampi and from DIV5 neurospheres. Total RNA was converted to complementary DNA (cDNA), and measurement of relative gene expression of Rasal1 was performed by quantitative PCR (qPCR). Relative gene expression was normalized to β-actin and computed by the -Δ2CT method. Further details are provided in Supplementary Information. Ex-vivo Studies Primary embryonic day 18 (E18) hippocampal culture Bilateral hippocampi from E18 embryos were dissected out and collected in HBSS on ice. Hippocampal cells were isolated and seeded at a density of 2x10 5 cells/mL in 5 mL proliferation medium, supplemented with pooled, sterile-filtered sera (0.1%) from young adult sedentary or exercising rats. Cells were allowed to proliferate as neurospheres for 5 days in vitro (DIV). Neurospheres were imaged and sized at 4x magnification. See Supplementary Information for details. Results Voluntary running exercise and AHN were reduced in middle-aged rats Young adult rats ran on average 5.16 km/day, while middle-aged rats ran only 0.11 km/day, suggesting that middle-aged rats were less motivated to run (Fig. 1 A, B). Exercise reduced body weight gain ( p < 0.0001) (Fig. 1 C) and white adipose tissue ( p = 0.0001) (Fig. 1 D) in young adult rats, but not in middle-aged rats (weight gain: p = 0.71, white adipose tissue: p = 0.71). The density of doublecortin-positive (DCX) cells, a proxy for cellular plasticity changes linked to neurogenesis, was greater in the DG of young adult ( p < 0.05) but not middle-aged rats ( p = 0.13) after exercise compared to sedentary controls (Fig. 1 E). Given the functional segregation of AHN across the longitudinal axis of the hippocampus ( 18 ), we measured the effects of exercise on AHN in the dorsal and ventral regions separately. Exercise increased AHN in the ventral DG ( p < 0.05) and tended to increase AHN in the dorsal DG ( p = 0.06) of young adult rats, but exercise did not affect AHN in the dorsal ( p = 0.93) nor in the ventral ( p = 0.89) DG of the middle-aged rats (Fig. 1 E). Results suggest that exercise increased AHN in young, but not middle-aged rats with low intrinsic motivation to run voluntarily. Exercise in young adult rats downregulated hippocampal Rasal1 To identify possible regulators of the exercise-driven increase in AHN observed in young adult rats, discovery proteomics was performed on DG isolated from a second cohort of rats housed with or without free access to running wheels for 7 weeks (Fig. 2 A). As we observed region-specific effects of exercise on AHN along the dorso-ventral axis (Fig. 1 E), DG were segmented into dorsal and ventral regions. A total of 1,988 proteins across both regions met criteria for differential expression analysis, and among these, 8 and 13 proteins were differentially expressed ( Supplementary Dataset 1 ) in the dorsal (Fig. 2 B, left ) and ventral DG (Fig. 2 B, right ), respectively. This included known AHN regulators such as Vgf ( 19 ) and heat shock proteins ( 20 ), including Hspa1a and Hsph1. The top differentially expressed protein (DEP) according to FDR in the dorsal DG was the Ras GTPase-activating protein Rasal1, which was downregulated following exercise ( ꞵ = -0.58; FDR < 0.01) (Fig. 2 B, left ). The result was validated by proteomics analysis in an independent cohort ( Supplementary Fig. 1B ) as well as by Western blot (Y-SED vs. Y-EX: Welch’s t (10.63) = 1.15, p < 0.01, two-tailed) (Fig. 2 C). Downregulation of Rasal1 was also evident at the transcriptional level in response to exercise, as shown in the dorsal hippocampus from an independent cohort of rats ( p < 0.01) (Fig. 2 D). Rasal1 is a conserved member of the GAP family ( 21 ) and acts as a Ca 2+ spike frequency sensor via C2 domain-dependent plasma membrane association ( 22 ). Accordingly, when we examined enriched pathways among exercise DEPs where Rasal1 featured, terms relating to “Ras signaling”, “response to metal ion”, and “phospholipid binding” were identified (Fig. 2 E). Rasal1 was also mapped to “positive regulation of nervous system development” and “cell projection organization” pathways related to neurogenesis and plasticity ( 23 ) (Fig. 2 E, Supplementary Fig. 1E ). These results suggest that Rasal1 downregulation in the dorsal DG could play a functional role in exercise-mediated plasticity and AHN regulation in young adulthood. The expression of Rasal1 was further confirmed in mature (NeuN-positive) and immature (DCX-positive) neurons, as well as glial cells (astrocytes: GFAP-positive) in the granular cell layer of the hippocampus in young adult male rats ( Supplementary Fig. 2 ). Microglial (Iba1-positive) and oligodendrocytic (Olig2-positive) cells did not express Rasal1 ( Supplementary Fig. 2 ). The neuronal expression of Rasal1 at the transcript and protein levels was independently confirmed using two recent publicly available proteomics and single-cell RNA-sequencing datasets ( 24 , 25 ) ( Supplementary Fig. 3 ). Rasal1 expression is associated with synaptic and neurogenic pathways that show divergent regulation in exercise and middle age To elucidate molecular processes in the DG that may be associated with Rasal1 expression independent of our study design, we regressed out the effect of experimental group and hippocampal region from the DG proteomics data and conducted Pearson correlations on the residuals. Following FDR correction, over 900 proteins in the dorsal DG were correlated with Rasal1 expression (Fig. 2 F). Pathway enrichment analysis stratified by correlation sign revealed that synapse and neurotransmission-related pathways featured heavily among the positively correlated proteins (Fig. 2 G, left ), whereas ribosomal, endocytic, phagosomal, and myelin pathway terms were most common among the negatively correlated proteins (Fig. 2 G, right ) ( Supplementary Dataset 4 ). These could relate to synaptic pruning processes, as the top negatively correlated protein was CD47 (r = − .74, FDR = 1.6E-05) (Fig. 2 F), which prevents microglia-mediated synaptic pruning through phagocytosis ( 26 ). We also found neurogenesis-related terms involving “neuron projection organization” and “nervous system development” ( Supplementary Dataset 4 ), further strengthening the possible link to AHN regulation. To investigate if Rasal1 is relevant to brain aging, differential expression analyses of dorsal and ventral DG proteomes of sedentary young and middle-aged rats were conducted, but Rasal1 was not identified among aging DEPs ( Supplementary Dataset 1 ), as we confirmed by western blot ( Supplementary Fig. 1 ). However, some Rasal1-associated pathways among exercise DEPs were also enriched among aging DEPs (Fig. 2 H). Furthermore, when we conducted pathway analyses with exercise DEPs and pathways from the Gene Expression Omnibus (GEO), we found that Rasal1 featured to pathways previously identified as upregulated in the CA1 and CA3 of 28- vs. 18-month-old rats (Fig. 2 I). This suggests that middle age could be a turning point in Rasal1 regulation. Knockdown of hippocampal Rasal1 increased AHN in middle-aged rats Since we identified that Rasal1 is downregulated by voluntary running exercise, we next tested whether reducing Rasal1 in middle age mimics the effects of exercise on AHN. We used a lentiviral approach to knock down Rasal1 in vivo in the dorsal hippocampus of middle-aged rats (Fig. 3 A; Supplementary Fig. 4A ). Lentiviral expression of a scrambled short hairpin (SCR) or a short hairpin directed at Rasal1 (shRASAL1) was confirmed by GFP autofluorescence in the granular cell layer of the dorsal hippocampus in LV SCR and LV shRASAL1 rats, respectively (Fig. 3 B). Reduced expression of Rasal1 transcript was also confirmed in the dorsal hippocampus of LV shRASAL1 compared to the LV SCR rats ( p = 0.06) (Fig. 3 C). To examine the effects of Rasal1 knockdown on AHN, the density of Ki67 + cells (cell proliferation) and DCX + cells (immature neurons) was measured along the longitudinal axis of the DG. The density of Ki67 + cells was greater in the dorsal ( p < 0.05), but not ventral ( p = 0.61) or whole DG ( p = 0.11) of LV shRASAL1 rats compared to LV SCR controls (Fig. 3 D). Similarly, the density of DCX + cells was greater in the dorsal ( p < 0.05) and whole DG ( p = 0.05), but not ventral ( p = 0.33) DG of LV shRASAL1 rats compared to LV SCR controls (Fig. 3 E), while there was no change in the area of the DG ( Supplementary Fig. 4B-C ). Previously published in vitro evidence suggests that Rasal1 regulates neuronal maturation ( 27 ). Thus, to explore the role of Rasal1 knockdown on the maturation of newly born neurons in vivo , morphometric analysis of individual DCX + cells in the dorsal hippocampus was carried out. While the length and the number of neurites per DCX + cell were not affected by Rasal1 downregulation ( p = 0.82 and p = 0.43, respectively), Rasal1 downregulation significantly increased the neurite length normalized by the number of neurites ( p < 0.05) (Fig. 3 F). Multi-omic analysis reveals serum factors associated with aging, exercise, and Rasal1 expression Since previous studies have reported that factors in the systemic circulation can mediate the beneficial effects of exercise on AHN ( 28 ) and that serum from exercising animals can increase hippocampal neurogenesis in vitro ( 29 ), we determined if serum factors could be driving the downregulation of Rasal1 using an in vitro approach. Embryonic day 18 (E18) rat hippocampal neuroprogenitor cells proliferating as neurospheres were treated with serum from exercising or sedentary young adult rats, and Rasal1 expression and neurosphere expansion were measured (Fig. 4 A). We found that relative to sedentary serum treatment Rasal1 was downregulated by treatment with serum from young adult exercising rats (one-sample t -test, H 0 : ratio = 1; t (6) = -2.7, p < 0.05, two-tailed) (Fig. 4 B), with a concomitant increase in neurosphere diameter (one-sample t -test, H 0 : ratio = 1; t (11) = 7.1, p < 0.0001, two-tailed) (Fig. 4 C, D, E). This suggests that serum factors are sufficient to phenocopy the effect of exercise on rasal1 gene expression in neuroprogenitor cells in vitro . Having established that Rasal1 expression is sensitive to serum factors, sera from animals with or without access to running wheels were screened for possible mediators using discovery proteomics and untargeted metabolomic analyses ( Supplementary Dataset 1 ). To fully leverage the multi-omic data from each sample, we performed multi-omic factor analysis using MOFA2 ( 30 ). This tool decomposes variance in multiple -omics modalities taken from the same samples, akin to a principal component analysis with multivariate data. The model returns latent factors that capture one or several -omics modalities, which can be used to cluster samples. We used this to identify factors that capture both proteomic and metabolomic variance (indicative of biologically plausible co-regulation) and then examined if they ordinated (“clustered”) samples by experimental group. To capture any possible opposing effects of exercise and age on serum -omes, we included samples from young adult sedentary, young adult exercising, and middle-aged sedentary animals. After filtering for high-quality features, 470 metabolites and 250 proteins were included. Proteins accounted for the largest amount of variance among serum samples compared to metabolites (28% vs. 12%) (Fig. 4 F left ). Factor 1 appeared the most biologically informative, as it displayed a similar degree of variance explained by proteins and metabolites (8.5% vs. 9%) (Fig. 4 F right ). Plotting the sample scores of each latent factor by experimental condition revealed that factor 1 captured an opposing relationship between age and exercise (Fig. 4 G). Indeed, unsupervised K-means clustering (with k = 3) of samples using factor 1 score captured the experimental groupings (Fig. 4 H). Next, we inspected the protein and metabolite feature loadings on factor 1 (Fig. 4 I). Among the top 10 absolute protein and metabolite feature loadings, biologically plausible features such as apolipoproteins and testosterone were identified, but also Platelet factor 4 (Pf4) (Fig. 4 J left ), which has been found to mediate effects of aging ( 31 ) and exercise ( 29 ) on AHN and cognition ( 31 ) in mice. The top-loading metabolite was 5-hydroxytryptophol (Fig. 4 J right ), a highly abundant peripheral serotonin metabolite in rats ( 32 ). To determine if individual variations in Rasal1 expression in the dorsal DG could be accounted for by variations in serum protein and metabolite abundances, the effects of experimental group and latent co-variance were regressed out in DG and serum -omics datasets, followed by Pearson correlation on the residuals. While 6 serum proteins (Fig. 4 K) and 19 metabolites (Fig. 4 L) were found to be nominally ( p < 0.05) correlated with Rasal1 expression in the dorsal DG, none remained statistically significant after applying FDR correction ( Supplementary Dataset 3 ). However, some of these nominally correlated features have plausible links to AHN, such as the (neuro)inflammatory regulator C-reactive protein (Crp) (r = .40, p < .05) (Fig. 4 K), the tryptophan metabolite Kynurenine (r = − .47, p < .05) (Fig. 4 L), and its metabolite Kyrurenic acid (r = − .41, p < .05) ( Supplementary Dataset 3 ). The positive correlations with (neuro)inflammatory regulators Crp (r = .40, p < .05) and Plg (r = .45, p < .05) ( 33 , 34 ) suggest that a heightened peripheral inflammatory state is associated with higher Rasal1 expression and possibly lower AHN. Taken together, these results point to a possible role of systemic factors in impacting Rasal1 expression through pro-inflammatory and tryptophan-related pathways in response to voluntary running exercise. Discussion Here, we have established that hippocampal Rasal1 is a candidate regulator of exercise-induced AHN that can be leveraged to mitigate the age-related AHN decline during middle age. Rasal1 is a GTPase-activating protein involved in different cellular functions in the brain, such as growth, differentiation, and survival ( 22 , 35 ). While there are no previous studies demonstrating that Rasal1 regulates AHN, induction of Ras signaling during exercise has been shown to recruit DG neural stem cells into the cell cycle ( 36 ), and RAS/ERK regulates neurogenesis through proneural activity programs ( 37 ), making Rasal1 an attractive candidate regulator of AHN. Our in vivo experiments show that Rasal1 is downregulated by exercise in young adult rats and demonstrate that its downregulation in middle-aged rats can mimic the neurogenic effects of exercise. We also show that Rasal1 expression in hippocampal neuroprogenitor cells is reduced by exposure to serum from exercising animals, with concomitant progenitor expansion. Lastly, we leveraged multi-omic analysis of sera in conjunction with brain proteomics and further identified possible regulators of Rasal1. The exercise-driven downregulation of Rasal1 and the related effects on AHN in young adult rats were mimicked by gene knockdown in middle-aged rats. This was prompted by the fact that the daily running distance by middle-aged rats was significantly lower than that of young adult rats, in agreement with previously published data ( 38 ) and possibly due to the higher body weight of the middle-aged rats (final experimental week: Y-EX = 506.1 g, MA-EX = 715.5 g). Thus, we did not observe an exercise-driven increase in AHN in middle-aged rats, nor the Rasal1 downregulation that we observed in young adult rats. An age-related reduction in physical activity is also reported in older adult humans ( 39 ). As regular physical exercise may not always be feasible with aging, mechanisms such as the Rasal1-induced increase in AHN as we have identified may contribute to the development of strategies to mimic at least some of the benefits of physical activity. Given the observed downregulation of Rasal1 in young exercising rats, we examined whether lentiviral downregulation of Rasal1 could act as an exercise mimetic to increase AHN in sedentary middle-aged rats. We found that downregulation of Rasal1 was sufficient to promote AHN in middle-aged rats, thus recapitulating the neurogenic effects of voluntary running exercise seen in the young animals. Importantly, the downregulation of Rasal1 was sufficient to enhance the dendritic growth of new hippocampal neurons in middle-aged rats. Synaptic maturation and integration of adult-born neurons into existing neural circuits are stimulated by exercise and are important for memory processes ( 40 ). This is particularly relevant in the aging brain, where exercise has been shown to counteract the decline in cognitive functions by increasing the number and maturation of new hippocampal neurons ( 41 ). Beyond the established expression of Rasal1 by (im)mature neurons, we show that Rasal1 is expressed by hippocampal astrocytes in vivo and in vitro . Given the contribution of astrocytes to hippocampal functions ( 42 ) and the susceptibility of hippocampal astrocytes to exercise stimulation ( 43 ), our results support a possible role of astrocytes in the exercise-mediated regulation of Rasal1. Our multi-omic analysis of sera in conjunction with brain proteomics pinpointed candidate serum proteins and metabolites relevant to Rasal1 regulation, which map onto hallmark aging processes ( 44 ) and can be ameliorated by physical exercise ( 2 ). Those included inflammatory (Crp, Ahsg, Plg), metabolic (Ahsg), antioxidant (Carnosine), and tryptophan-related (Kynurenine, Kynurenic acid) factors. Notably, the metabolism of tryptophan into systemically circulating indole by gut microbes has been found to regulate AHN directly via the aryl hydrocarbon receptor( 45 ). Thus, it is plausible that the negative correlations between Kyurenine/Kynurenic acid and Rasal1 indicate that increased tryptophan metabolism is associated with lower Rasal1 and increased levels of AHN. Furthermore, given the detrimental role of peripheral inflammation to AHN ( 46 ), the positive correlation between Rasal1 and pro-inflammatory molecules could indicate a role of pro-inflammatory molecules in the stimulation of Rasal1 expression, resulting in impaired AHN. We also found a positive correlation between Rasal1 and Ahsg ( Supplementary Dataset 3 ), better known as Fetuin-A, which is lowered by exercise in humans ( 47 ) and rats ( 48 ). As a hepatokine, Fetuin-A is thought to exert its effects by modulating insulin signaling and sensitivity following exercise ( 49 ). Interestingly, we found a strong enrichment of SLC2A4 (GLUT4) as a common hub protein among exercise and age DEPs, as well as “central carbon metabolism” (i.e. glycolysis, Krebs cycle, pentose phosphate pathway) in our metabolomics data ( Supplementary Dataset 2 ). Thus, it is tempting to speculate that aging and exercise could exert opposing effects on Rasal1 (and consequently AHN and synaptic plasticity) through systemic regulation of pro-inflammatory signaling, tryptophan metabolism, or glucose homeostasis. Importantly, while Rasal1 was not differentially expressed in middle-aged rats, we found that it was upregulated in the rat hippocampus at older ages (Fig. 3 D). Additionally, in our independent analysis of Rasal1 transcript expression in the single-cell RNA-sequencing data from Wu et al. ( 25 ), we found qualitative evidence that it becomes more prominent in the mouse DG in old age compared to middle age ( Supplementary Fig. 2B ). This could imply a turning point in its regulation around middle age, albeit more research is needed to elucidate fluctuations in Rasal1 expression across the lifespan. In conclusion, we identified Rasal1 as a novel candidate regulator of exercise-induced AHN and demonstrated that it can be targeted to mitigate the age-related AHN decline during the critical time of middle age. Leveraging these findings to identify systemic factors responsible for the Rasal1-induced pro-neurogenic effects of exercise may support the development of novel therapeutic strategies aimed at enhancing AHN and improving hippocampal-related cognitive function. Such strategies are particularly important for individuals who are unable to exercise due to physical or pathological limitations, and who therefore may require pharmacological intervention to experience the brain health benefits of exercise otherwise inaccessible to them. Declarations Conflicts of Interest YMN is in receipt of research funding from Marigot Limited and has received honoraria from Yakult as an invited speaker. OFO has received research funding from Marigot Limited and Alkermes plc and honoraria as an invited speaker at a meeting organised by Janssen. All other authors declare that they have no conflict of interest. Author Contribution MGC: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Data Curation, Writing – original draft, review and editing, Visualisation; SD-H: Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Data Curation, Writing – original draft, review and editing, Visualisation; SN: Conceptualization, Methodology, Validation, Investigation, Data Curation, Writing – original draft, review and editing, Supervision, Project administration; JAE: Methodology, Software, Formal analysis, Resources, Data Curation, Writing – review and editing, Supervision; AL: Methodology, Software, Formal analysis, Data Curation, Writing – review and editing, Supervision; PJL: Conceptualization, Writing – review and editing, Supervision; JDM: Conceptualization, Writing – review and editing; OO’L: Conceptualization, Methodology, Writing – original draft, review and editing, Supervision, Project administration; YN: Conceptualization, Methodology, Resources, Writing – original draft, review and editing, Supervision, Project administration, Funding acquisition. 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Molecular and metabolomic effects of voluntary running wheel activity on skeletal muscle in late middle-aged rats. Physiol Rep. 2015;3(2):1–17. Sun F, Norman IJ, While AE. Physical activity in older people: a systematic review. BMC Public Health [Internet]. 2013 [cited 2025 Nov 20];13(1). Available from: https://pubmed.ncbi.nlm.nih.gov/23648225/ Vivar C, Peterson BD, van Praag H. Running rewires the neuronal network of adult-born dentate granule cells. Neuroimage [Internet]. 2016;131:29–41. Available from: http://dx.doi.org/10.1016/j.neuroimage.2015.11.031 Wu M V., Luna VM, Hen R. Running rescues a fear-based contextual discrimination deficit in aged mice. Front Syst Neurosci. 2015;9(AUGUST):1–10. Santello M, Toni N, Volterra A. Astrocyte function from information processing to cognition and cognitive impairment. Nat Neurosci [Internet]. 2019;22(2):154–66. Available from: http://dx.doi.org/10.1038/s41593-018-0325-8 Uda M, Ishido M, Kami K, Masuhara M. Effects of chronic treadmill running on neurogenesis in the dentate gyrus of the hippocampus of adult rat. Brain Res. 2006;1104(1):64–72. López-Otín C, Blasco MA, Partridge L, Serrano M, Kroemer G. Hallmarks of aging: An expanding universe. Cell. 2023;186(2):243–78. Wei GZ, Martin KA, Xing PY, Agrawal R, Whiley L, Wood TK, et al. Tryptophan-metabolizing gut microbes regulate adult neurogenesis via the aryl hydrocarbon receptor. Proc Natl Acad Sci U S A. 2021;118(27):1–10. Ryan SM, Kelly ÁM. Exercise as a pro-cognitive, pro-neurogenic and anti-inflammatory intervention in transgenic mouse models of Alzheimer’s disease. Ageing Res Rev [Internet]. 2016;27:77–92. Available from: http://dx.doi.org/10.1016/j.arr.2016.03.007 Ramírez-Amaya V, Vazdarjanova A, Mikhael D, Rosi S, Worley PF, Barnes CA. Spatial exploration-induced Arc mRNA and protein expression: Evidence for selective, network-specific reactivation. Journal of Neuroscience. 2005;25(7):1761–8. Saberi S, Askaripour M, Khaksari M, Amin Rajizadeh M, Abbas Bejeshk M, Akhbari M, et al. Exercise training improves diabetic renal injury by reducing fetuin-A, oxidative stress and inflammation in type 2 diabetic rats. Heliyon [Internet]. 2024 Mar 30 [cited 2025 Nov 20];10(6). Available from: https://pubmed.ncbi.nlm.nih.gov/38510054/ Lee S, Norheim F, Gulseth HL, Langleite TM, Kolnes KJ, Tangen DS, et al. Interaction between plasma fetuin-A and free fatty acids predicts changes in insulin sensitivity in response to long-term exercise. Physiol Rep [Internet]. 2017 Mar 1 [cited 2025 Nov 20];5(5). Available from: https://pubmed.ncbi.nlm.nih.gov/28270597/ Additional Declarations Yes YMN is in receipt of research funding from Marigot Limited and has received honoraria from Yakult as an invited speaker. OFO has received research funding from Marigot Limited and Alkermes plc and honoraria as an invited speaker at a meeting organised by Janssen. All other authors declare that they have no conflict of interest. Supplementary Files SupplementaryDataset5.xlsx Supplementary Dataset 5 SupplementaryDataset2.xlsx Supplementary Dataset 2 SupplementaryDataset3.xlsx Supplementary Dataset 3 SupplementaryFigure2.tif Supplementary Figure 2 SupplementaryFigure3.tif Supplementary Figure 3 SupplementaryDataset1.xlsx Supplementary Dataset 1 carusodohmhansenetalSupplementaryInformation.docx Supplementary Material SupplementaryFigure1.tif Supplementary Figure 1 SupplementaryDataset4.xlsx Supplementary Dataset 4 SupplementaryFigure4.tif Supplementary Figure 4 Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: revise 24 Mar, 2026 Review # 4 received at journal 04 Mar, 2026 Review # 2 received at journal 25 Feb, 2026 Review # 1 received at journal 21 Feb, 2026 Reviewer # 4 agreed at journal 17 Feb, 2026 Reviewer # 3 agreed at journal 17 Feb, 2026 Reviewer # 2 agreed at journal 10 Feb, 2026 Reviewer # 1 agreed at journal 09 Feb, 2026 Reviewers invited by journal 09 Feb, 2026 Editor assigned by journal 04 Jan, 2026 Submission checks completed at journal 04 Jan, 2026 First submitted to journal 18 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8394751","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":588597857,"identity":"463eec38-f2e4-4881-820d-52aef768b033","order_by":0,"name":"Yvonne Nolan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAApklEQVRIiWNgGAWjYLCCBwwMcmBGAtFagCqNSdeS2EC0aoMDvAcfJNTcSd9w/PADhgcVRGnhSzZIOPYsd8OZNAOGhDNEaeExk0hgO5y74QYP0HltRGv5dzjdAKzlH7FaEtsOJ0C0NBChRfIwj7FBYt9hw5lAvxxIOEaEFr7jPYYPPnw7LM93/PDDhz9qiNCicBiJc4AIDQwM8g1EKRsFo2AUjIIRDQDcUDhM6ZIHygAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-2426-234X","institution":"University College Cork","correspondingAuthor":true,"prefix":"","firstName":"Yvonne","middleName":"","lastName":"Nolan","suffix":""},{"id":588597858,"identity":"eaf76fd2-7021-4155-b2e0-37ab636cad60","order_by":1,"name":"Maria Giovanna Caruso","email":"","orcid":"https://orcid.org/0009-0008-2011-7130","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"Giovanna","lastName":"Caruso","suffix":""},{"id":588597859,"identity":"68a9309e-9966-4fdb-8bf8-9c64d2461a31","order_by":2,"name":"Sebastian Dohm-Hansen","email":"","orcid":"https://orcid.org/0000-0002-3309-9380","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Sebastian","middleName":"","lastName":"Dohm-Hansen","suffix":""},{"id":588597860,"identity":"e3871d67-602d-43cf-820a-a9f07ec43b6d","order_by":3,"name":"Sarah Nicolas","email":"","orcid":"https://orcid.org/0000-0002-2581-3484","institution":"University College Cork","correspondingAuthor":false,"prefix":"","firstName":"Sarah","middleName":"","lastName":"Nicolas","suffix":""},{"id":588597861,"identity":"c7d000c8-d2a5-446c-a979-6bc1a498f4dc","order_by":4,"name":"Jane English","email":"","orcid":"https://orcid.org/0000-0001-9930-7050","institution":"University College Cork","correspondingAuthor":false,"prefix":"","firstName":"Jane","middleName":"","lastName":"English","suffix":""},{"id":588597862,"identity":"4e534fa2-70d0-45f7-a98f-9c7982148306","order_by":5,"name":"Aonghus Lavelle","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Aonghus","middleName":"","lastName":"Lavelle","suffix":""},{"id":588597863,"identity":"d9fc78bd-ce9c-4d22-a45b-1df8ba6933c9","order_by":6,"name":"Paul Lucassen","email":"","orcid":"https://orcid.org/0000-0001-9708-9133","institution":"Swammerdam Institute for Life Sciences, University of Amsterdam,","correspondingAuthor":false,"prefix":"","firstName":"Paul","middleName":"","lastName":"Lucassen","suffix":""},{"id":588597864,"identity":"7275b4be-6d11-4867-9663-78ccdcc78926","order_by":7,"name":"Joram Mul","email":"","orcid":"https://orcid.org/0000-0003-0453-2568","institution":"University of Amsterdam","correspondingAuthor":false,"prefix":"","firstName":"Joram","middleName":"","lastName":"Mul","suffix":""},{"id":588597865,"identity":"fb3e092c-4483-47dc-8625-de805a41c9ca","order_by":8,"name":"Olivia O'Leary","email":"","orcid":"https://orcid.org/0000-0002-9171-2032","institution":"University College Cork","correspondingAuthor":false,"prefix":"","firstName":"Olivia","middleName":"","lastName":"O'Leary","suffix":""}],"badges":[],"createdAt":"2025-12-18 11:20:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8394751/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8394751/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102747234,"identity":"64cc5354-fd92-4fc7-a65d-d328bf8cbe8f","added_by":"auto","created_at":"2026-02-16 09:04:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":771310,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eReduced voluntary running exercise and AHN in middle aged rats\u0026nbsp; \u003c/strong\u003eA) Study design. B) Average running distance (km/day). C) Body weight gain. D) White adipose tissue normalized to body weight and expressed as percentage. E) \u003cem\u003eTop\u003c/em\u003e: Number of DCX+ cells / mm\u003csup\u003e2\u003c/sup\u003e in the whole, dorsal and ventral dentate gyrus (DG) of young adult (Y) and middle aged (MA) rats in sedentary (SED) and exercise (EX) conditions. \u003cem\u003eBottom\u003c/em\u003e: Representative images of DCX+ cells in the DG of Y and MA rats in SED and EX conditions. Scale bar 200 µm. Data are analysed with two-tailed Welch’s t-test and presented as mean +SEM. *\u003cem\u003ep\u003c/em\u003e\u0026lt;.05, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;.001, ****\u003cem\u003ep\u003c/em\u003e\u0026lt;.0001.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8394751/v1/3059af211adc7f763641887a.png"},{"id":102577533,"identity":"9b37f796-af15-45a9-9bbe-d1b362e7b1d9","added_by":"auto","created_at":"2026-02-13 08:35:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":656968,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExercise in young adult rats downregulated hippocampal Rasal1.\u003c/strong\u003e A) Study design. B) Volcano plots of proteomic analysis of isolated dorsal (\u003cem\u003eleft\u003c/em\u003e) and ventral (\u003cem\u003eright\u003c/em\u003e) dentate gyri (DG) from young adult (Y) rats in sedentary (SED) and exercise (EX) conditions. Top 3 up- and down-regulated proteins highlighted. C) Western blot (WB) validation of Rasal1 expression in dorsal DG samples (Welch's \u003cem\u003et\u003c/em\u003e-test, two-tailed). D) qPCR analysis of dorsal hippocampal Rasal1 expression (fold change) in an independent cohort of Y-SED and Y-EX rats (Welch's \u003cem\u003et\u003c/em\u003e-test, two-tailed). \u0026nbsp;E) Enriched pathways featuring Rasal1 among exercise differentially expressed proteins (DEP). F) Pearson correlation of residualized dorsal dentate gyrus (DG) protein abundance with Rasal1 expression in the discovery cohort. Top 3 significant positive and negative correlations highlighted. G) Top 5 enriched pathways among dorsal DG proteins that correlate positively (\u003cem\u003eleft\u003c/em\u003e) or negatively (\u003cem\u003eright\u003c/em\u003e) with dorsal Rasal1 expression. H) Common Gene Ontology (GO) pathways that feature Rasal1 enriched among age and exercise differentially expressed proteins (DEP) in discovery cohort DG proteomics. Fold changes of enriched DEP in each pathway are plotted. The red vertical lines indicate average fold change of DEP. I) Enriched ageing-related Gene Expression Omnibus (GEO) pathways among exercise DEPs that feature Rasal1. Data in D are expressed as mean + SEM \u0026nbsp;**\u003cem\u003ep\u003c/em\u003e\u0026lt;.01.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8394751/v1/60d5dfd0103c27c30e5dd602.png"},{"id":102747429,"identity":"18d5f356-da49-47ff-bbc0-fade744c3b26","added_by":"auto","created_at":"2026-02-16 09:04:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":715692,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKnockdown of Rasal1 increased AHN in middle aged rats \u003c/strong\u003eA) Study design. B) Representative images of GFP+ cells in the dorsal and ventral dentate gyrus (DG). Scale bar 200 µm. C) Rasal1 transcript fold change normalized to β-actin in dorsal hippocampus. D)\u003cem\u003e Left:\u003c/em\u003e Representative images of Ki67+ cells in the DG of LV\u003csup\u003eSCR\u003c/sup\u003e and LV\u003csup\u003eshRASAL1\u003c/sup\u003e middle aged rats. Scale bar 200µm.\u003cem\u003e Right:\u003c/em\u003e Number of Ki67+ cells / mm\u003csup\u003e2\u003c/sup\u003e in the whole, dorsal and ventral DG of LV\u003csup\u003eSCR\u003c/sup\u003e and LV\u003csup\u003eshRASAL1\u003c/sup\u003e middle aged rats. E) \u003cem\u003eLeft:\u003c/em\u003e Representative images of DCX+ cells in the DG of LV\u003csup\u003eSCR\u003c/sup\u003e and LV\u003csup\u003eshRASAL1\u003c/sup\u003e middle aged rats. Scale bar 200 µm. \u003cem\u003eRight:\u003c/em\u003e Number of DCX+ cells / mm\u003csup\u003e2\u003c/sup\u003e in the whole, dorsal and ventral DG of LV\u003csup\u003eSCR\u003c/sup\u003e and LV\u003csup\u003eshRASAL1\u003c/sup\u003e middle aged rats. F) \u003cem\u003eLeft:\u003c/em\u003e Morphometric reconstruction of DCX+ cells in the dorsal DG of LV\u003csup\u003eSCR\u003c/sup\u003e and LV\u003csup\u003eshRASAL1\u003c/sup\u003e middle aged rats. Scale bar 50 µm. \u003cem\u003eRight:\u003c/em\u003e Total neurite length, number of neurites and neurite length normalized by number of neurites per DCX+ cell in the dorsal DG of LV\u003csup\u003eSCR\u003c/sup\u003e and LV\u003csup\u003eshRASAL1\u003c/sup\u003e middle aged rats. Data are analysed with two-tailed Welch’s t-test and presented as mean +SEM. *\u003cem\u003ep\u003c/em\u003e\u0026lt;.05.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8394751/v1/f9ee73a2a574f80daf715f03.png"},{"id":102577538,"identity":"01f0a3a7-aa45-4270-b0ef-84397d4245ef","added_by":"auto","created_at":"2026-02-13 08:35:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":603136,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRasal1 is downregulated by serum from exercising animals. \u003c/strong\u003eA) \u003cem\u003eIn vitro\u003c/em\u003e experimental overview. B) Rasal1 transcript fold change in neurosphere cultures after 5 \u003cem\u003edays in vitro\u003c/em\u003e (DIV) of serum treatment (one-sample \u003cem\u003et\u003c/em\u003e-test, two-tailed). C) Median neurosphere diameter ratio after 5 DIV of serum treatment (one-sample \u003cem\u003et\u003c/em\u003e-test, two-tailed). D) Representative images of primary hippocampal neurospheres at 5 DIV following treatment with 0.1% serum from sedentary (SED; \u003cem\u003eleft\u003c/em\u003e) or exercising (EX; \u003cem\u003eright\u003c/em\u003e) young adult (Y) rats from the discovery cohort\u003cem\u003e.\u003c/em\u003eScale bar 200 µm. E) Relationship between Rasal1 transcript fold change and neurosphere diameter ratio (normalized to SED serum treatment). F-J) Multi-omic factor analysis (MOFA) of discovery cohort serum proteomics and metabolomics. \u0026nbsp;F) Variance explained by each -ome in the MOFA model \u003cem\u003e(left\u003c/em\u003e)\u003cem\u003e,\u003c/em\u003e and variance explained by each -ome within each MOFA latent factor \u003cem\u003e(right\u003c/em\u003e)\u003cem\u003e.\u003c/em\u003eG) Sample scores on each latent factor. H) K-means clustering (\u003cem\u003ek\u003c/em\u003e=3) of samples using factor 1 scores. I) Protein (\u003cem\u003etop\u003c/em\u003e) and metabolite (\u003cem\u003ebottom\u003c/em\u003e) feature weights on factor 1. J) Top 10 protein (\u003cem\u003eleft\u003c/em\u003e) and metabolite (\u003cem\u003eright\u003c/em\u003e) weights on factor 1. K-L) Pearson correlation of residualized serum protein (K) and metabolite (L) abundance with dorsal dentate Rasal1 expression. The top 3 nominally (i.e. unadjusted) significant correlations are highlighted. Data in B and C are expressed as mean + SEM \u0026nbsp;*\u003cem\u003ep\u003c/em\u003e\u0026lt;.05, ****\u003cem\u003ep\u003c/em\u003e\u0026lt;.0001.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8394751/v1/481518ffc444f32d214d50b4.png"},{"id":103056277,"identity":"6fc7b562-41f1-4b72-9bb3-a85455bc269c","added_by":"auto","created_at":"2026-02-20 09:02:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3661322,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8394751/v1/e7242861-d6b8-4a45-89dd-4cbc24944212.pdf"},{"id":102577532,"identity":"3c402dcb-3832-4a36-a841-24b91001f7ff","added_by":"auto","created_at":"2026-02-13 08:35:46","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":52861,"visible":true,"origin":"","legend":"Supplementary Dataset 5","description":"","filename":"SupplementaryDataset5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8394751/v1/cb9c8edc30b84abbfd94ae57.xlsx"},{"id":102577537,"identity":"be33d675-a309-482c-a086-d8d8f8f57ea8","added_by":"auto","created_at":"2026-02-13 08:35:46","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":313023,"visible":true,"origin":"","legend":"Supplementary Dataset 2","description":"","filename":"SupplementaryDataset2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8394751/v1/b83f8a5814a2e79ead54a9e8.xlsx"},{"id":102746619,"identity":"48307ca2-276c-4242-9cda-5d0fcf1ae7aa","added_by":"auto","created_at":"2026-02-16 08:58:47","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":378132,"visible":true,"origin":"","legend":"Supplementary Dataset 3","description":"","filename":"SupplementaryDataset3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8394751/v1/1e72981fbb41b0b570adb6af.xlsx"},{"id":102746542,"identity":"a8fbcb7f-99b3-4801-b9d8-45f95a6e6c93","added_by":"auto","created_at":"2026-02-16 08:58:07","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1412507,"visible":true,"origin":"","legend":"Supplementary Figure 2","description":"","filename":"SupplementaryFigure2.tif","url":"https://assets-eu.researchsquare.com/files/rs-8394751/v1/c6d2c1a7a6bb8e5aabbbdfef.tif"},{"id":102577546,"identity":"98f724d4-5881-46f6-99c2-9c2a843af130","added_by":"auto","created_at":"2026-02-13 08:35:47","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":897742,"visible":true,"origin":"","legend":"Supplementary Figure 3","description":"","filename":"SupplementaryFigure3.tif","url":"https://assets-eu.researchsquare.com/files/rs-8394751/v1/9b6a10972863c9efaaa6b953.tif"},{"id":102577544,"identity":"5fcba7c2-c672-4178-9ba5-70f40000369d","added_by":"auto","created_at":"2026-02-13 08:35:46","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":3022666,"visible":true,"origin":"","legend":"Supplementary Dataset 1","description":"","filename":"SupplementaryDataset1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8394751/v1/410a9a500322ad42bfdc4832.xlsx"},{"id":102747098,"identity":"9c3a6d06-0dd8-41a8-9990-4d39becacb9d","added_by":"auto","created_at":"2026-02-16 09:03:50","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":139488,"visible":true,"origin":"","legend":"Supplementary Material","description":"","filename":"carusodohmhansenetalSupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-8394751/v1/4b34dc40b387d40e27501843.docx"},{"id":102577540,"identity":"9666c213-13a5-4f25-b89a-4779c8ce56d2","added_by":"auto","created_at":"2026-02-13 08:35:46","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":1421862,"visible":true,"origin":"","legend":"Supplementary Figure 1","description":"","filename":"SupplementaryFigure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-8394751/v1/938cde6a7c4b076e184dfb1e.tif"},{"id":102577545,"identity":"bb7bf2ad-ce73-4a35-bbbe-c60c2cf6949e","added_by":"auto","created_at":"2026-02-13 08:35:47","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":137781,"visible":true,"origin":"","legend":"Supplementary Dataset 4","description":"","filename":"SupplementaryDataset4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8394751/v1/01a9106c732e8b09065b661b.xlsx"},{"id":102577542,"identity":"23aadd7b-502d-4e3d-8cbf-0dac7ccd4078","added_by":"auto","created_at":"2026-02-13 08:35:46","extension":"tif","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":376211,"visible":true,"origin":"","legend":"Supplementary Figure 4","description":"","filename":"SupplementaryFigure4.tif","url":"https://assets-eu.researchsquare.com/files/rs-8394751/v1/2af50bc7271ae2897cc86c46.tif"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e\nYMN is in receipt of research funding from Marigot Limited and has received honoraria from Yakult as an invited speaker. OFO has received research funding from Marigot Limited and Alkermes plc and honoraria as an invited speaker at a meeting organised by Janssen. All other authors declare that they have no conflict of interest.","formattedTitle":"Rasal1: A candidate exercise mimetic to increase adult hippocampal neurogenesis in middle age","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePhysical exercise has been shown to be neuroprotective by exerting beneficial effects at systemic, cellular, and molecular levels (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). One notable example is the stimulation of adult hippocampal neurogenesis (AHN) in the rodent dentate gyrus (DG) (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). As AHN is involved in regulating antidepressant action, anxiety, and certain forms of cognition, the ability to induce it via exercise could be useful in states associated with reduced AHN, such as aging or neurodegenerative disease (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). However, maintaining regular physical exercise may not always be feasible in such states due to physical or health constraints. Therefore, it is important to identify the mechanisms underlying exercise-induced AHN.\u003c/p\u003e \u003cp\u003eAHN has been found to decline with advancing age in several species, including humans (\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e), mice (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e), and rats (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). While a body of literature has found that exercise can reverse this age-related decline in rodents (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e), it can also fail to induce AHN, especially at older ages (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e), further highlighting the need to understand and identify novel regulators of AHN, such as proteins within the DG. Moreover, exercise influences AHN not just through effects in the brain but also via factors in the systemic circulation (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). For instance, the beneficial effects of voluntary wheel-running on AHN in both young and aged mice have been phenocopied by plasma transfer from exercising to sedentary animals, with circulating proteins as candidate mediators (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWith an increasingly older global population, there is significant interest in establishing tractable interventions that maintain cognitive and physical health, especially when exercise is not a viable intervention. Identifying AHN regulators in the systemic circulation would provide relevant targets to modify AHN for healthy brain aging. While studies on aging have historically focused on advanced age, emerging evidence suggests that middle age may be a period of accelerating change for the brain, as well as systemic circulation, that shape future cognitive trajectories (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Thus, middle age presents a unique window of opportunity for such interventions.\u003c/p\u003e \u003cp\u003eHerein, we used a proteomics approach to identify regulators of exercise-induced AHN in young adult rats that could potentially be harnessed to boost neurogenesis during middle age. We investigated whether genetic manipulation of an identified hippocampal protein candidate could mitigate the age-related decline in AHN \u003cem\u003ein vivo\u003c/em\u003e in middle-aged rats. Using an \u003cem\u003ein vitro\u003c/em\u003e approach, we subsequently investigated whether this AHN regulator was sensitive to regulation by exercise-induced changes in the blood circulation. Finally, we used a multi-omic approach to identify candidate blood-borne proteins and metabolites that may link exercise-induced alterations in the systemic circulation with proteins that regulate AHN in the brain.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eAdult (12 weeks old) and middle-aged (12 months old) male Sprague Dawley rats were obtained from Janvier Laboratories (France). All animal procedures were performed under authorizations (#AE19130/P149) issued by the Health Products Regulatory Authority (HPRA, Ireland), in accordance with the European Communities Council Directive (2010/63/EU) and approved by the Animal Experimentation Ethics Committee of University College Cork. Upon arrival, animals were pair-housed in either standard cages (Y-SED, MA-SED) or with free access to a running wheel (Activity wheel 33cm diameter, Techniplast, UK) (Y-EX, MA-EX). Running or sedentary conditions were maintained for the duration of the experiment, which lasted a total of 6 to 8 weeks (\u003cb\u003eSupplementary Dataset 5\u003c/b\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eStereotactic brain surgery\u003c/h3\u003e\n\u003cp\u003eRats were bilaterally, stereotactically infused with 3.0 \u0026micro;L of ultra-purified lentiviral-based short-hairpin RNA vectors (shRNA targeting rat Rasal1\u0026thinsp;\u0026gt;\u0026thinsp;1x10\u003csup\u003e8\u003c/sup\u003e IU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e or scrambled control\u0026thinsp;\u0026gt;\u0026thinsp;1x10\u003csup\u003e8\u003c/sup\u003e IU mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e ) into the dorsal hippocampus (from bregma: A.P. = -3.5, M.L. = +-2.4, D.V. = -3.6) at 1\u0026micro;l/min. qPCR analysis for the expression of the LV\u003csup\u003eSCR\u003c/sup\u003e and LV\u003csup\u003eshRASAL1\u003c/sup\u003e viral vector transcripts was carried out in the dorsal hippocampus to further confirm the successful lentiviral transduction. See Supplementary Information for details.\u003c/p\u003e\n\u003ch3\u003eProteomics and metabolomics\u003c/h3\u003e\n\u003cp\u003eProteomics analysis of serum and hippocampal tissue, as well as metabolomics analysis of serum, are described in Supplementary Information. See Supplementary Table\u0026nbsp;1 for more details.\u003c/p\u003e\n\u003ch3\u003eIHC\u003c/h3\u003e\n\u003cp\u003eKi67, DCX, Rasal1, NeuN, GFAP, Olig2, Iba1 immunohistochemistry was conducted in the hippocampus as described in Supplementary Information. All analyses were performed blind to treatment conditions. See Supplementary Table\u0026nbsp;2 for more details.\u003c/p\u003e\n\u003ch3\u003eWestern blotting\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eWestern blotting\u003c/div\u003e \u003cp\u003eHippocampi tissue homogenates (20 \u0026micro;g of proteins per lane) were analysed by Western blot using a mouse anti-Rasal1 polyclonal antibody (Abnova #H00008437-B01P; Lot M4251). Protein band quantification was performed by densitometry analysis normalized against MemCode\u0026trade; Reversible Total Protein Stain. Further details are provided in Supplementary Information.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction and quantitative real-time PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from hippocampi and from DIV5 neurospheres. Total RNA was converted to complementary DNA (cDNA), and measurement of relative gene expression of Rasal1 was performed by quantitative PCR (qPCR). Relative gene expression was normalized to β-actin and computed by the -Δ2CT method. Further details are provided in Supplementary Information.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEx-vivo Studies\u003c/h3\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003ePrimary embryonic day 18 (E18) hippocampal culture\u003c/h2\u003e \u003cp\u003eBilateral hippocampi from E18 embryos were dissected out and collected in HBSS on ice. Hippocampal cells were isolated and seeded at a density of 2x10\u003csup\u003e5\u003c/sup\u003e cells/mL in 5 mL proliferation medium, supplemented with pooled, sterile-filtered sera (0.1%) from young adult sedentary or exercising rats. Cells were allowed to proliferate as neurospheres for 5 days \u003cem\u003ein vitro\u003c/em\u003e (DIV). Neurospheres were imaged and sized at 4x magnification. See Supplementary Information for details.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eVoluntary running exercise and AHN were reduced in middle-aged rats\u003c/h2\u003e \u003cp\u003eYoung adult rats ran on average 5.16 km/day, while middle-aged rats ran only 0.11 km/day, suggesting that middle-aged rats were less motivated to run (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). Exercise reduced body weight gain (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) and white adipose tissue (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) in young adult rats, but not in middle-aged rats (weight gain: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.71, white adipose tissue: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.71).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe density of doublecortin-positive (DCX) cells, a proxy for cellular plasticity changes linked to neurogenesis, was greater in the DG of young adult (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) but not middle-aged rats (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.13) after exercise compared to sedentary controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Given the functional segregation of AHN across the longitudinal axis of the hippocampus (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e), we measured the effects of exercise on AHN in the dorsal and ventral regions separately. Exercise increased AHN in the ventral DG (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and tended to increase AHN in the dorsal DG (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.06) of young adult rats, but exercise did not affect AHN in the dorsal (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.93) nor in the ventral (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.89) DG of the middle-aged rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Results suggest that exercise increased AHN in young, but not middle-aged rats with low intrinsic motivation to run voluntarily.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eExercise in young adult rats downregulated hippocampal Rasal1\u003c/h2\u003e \u003cp\u003eTo identify possible regulators of the exercise-driven increase in AHN observed in young adult rats, discovery proteomics was performed on DG isolated from a second cohort of rats housed with or without free access to running wheels for 7 weeks (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). As we observed region-specific effects of exercise on AHN along the dorso-ventral axis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), DG were segmented into dorsal and ventral regions. A total of 1,988 proteins across both regions met criteria for differential expression analysis, and among these, 8 and 13 proteins were differentially expressed (\u003cb\u003eSupplementary Dataset 1\u003c/b\u003e) in the dorsal (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, \u003cb\u003eleft\u003c/b\u003e) and ventral DG (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, \u003cb\u003eright\u003c/b\u003e), respectively. This included known AHN regulators such as Vgf (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e) and heat shock proteins (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e), including Hspa1a and Hsph1. The top differentially expressed protein (DEP) according to FDR in the dorsal DG was the Ras GTPase-activating protein Rasal1, which was downregulated following exercise (\u003cem\u003eꞵ\u003c/em\u003e = -0.58; FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, \u003cb\u003eleft\u003c/b\u003e). The result was validated by proteomics analysis in an independent cohort (\u003cb\u003eSupplementary Fig.\u0026nbsp;1B\u003c/b\u003e) as well as by Western blot (Y-SED vs. Y-EX: Welch\u0026rsquo;s \u003cem\u003et\u003c/em\u003e\u003csub\u003e(10.63)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.15, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, two-tailed) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Downregulation of Rasal1 was also evident at the transcriptional level in response to exercise, as shown in the dorsal hippocampus from an independent cohort of rats (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Rasal1 is a conserved member of the GAP family (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e) and acts as a Ca\u003csup\u003e2+\u003c/sup\u003e spike frequency sensor via C2 domain-dependent plasma membrane association (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Accordingly, when we examined enriched pathways among exercise DEPs where Rasal1 featured, terms relating to \u0026ldquo;Ras signaling\u0026rdquo;, \u0026ldquo;response to metal ion\u0026rdquo;, and \u0026ldquo;phospholipid binding\u0026rdquo; were identified (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Rasal1 was also mapped to \u0026ldquo;positive regulation of nervous system development\u0026rdquo; and \u0026ldquo;cell projection organization\u0026rdquo; pathways related to neurogenesis and plasticity (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, \u003cb\u003eSupplementary Fig.\u0026nbsp;1E\u003c/b\u003e). These results suggest that Rasal1 downregulation in the dorsal DG could play a functional role in exercise-mediated plasticity and AHN regulation in young adulthood. The expression of Rasal1 was further confirmed in mature (NeuN-positive) and immature (DCX-positive) neurons, as well as glial cells (astrocytes: GFAP-positive) in the granular cell layer of the hippocampus in young adult male rats (\u003cb\u003eSupplementary Fig.\u0026nbsp;2\u003c/b\u003e). Microglial (Iba1-positive) and oligodendrocytic (Olig2-positive) cells did not express Rasal1 (\u003cb\u003eSupplementary Fig.\u0026nbsp;2\u003c/b\u003e). The neuronal expression of Rasal1 at the transcript and protein levels was independently confirmed using two recent publicly available proteomics and single-cell RNA-sequencing datasets (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e) (\u003cb\u003eSupplementary Fig.\u0026nbsp;3\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eRasal1 expression is associated with synaptic and neurogenic pathways that show divergent regulation in exercise and middle age\u003c/em\u003e \u003c/p\u003e \u003cp\u003eTo elucidate molecular processes in the DG that may be associated with Rasal1 expression independent of our study design, we regressed out the effect of experimental group and hippocampal region from the DG proteomics data and conducted Pearson correlations on the residuals. Following FDR correction, over 900 proteins in the dorsal DG were correlated with Rasal1 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Pathway enrichment analysis stratified by correlation sign revealed that synapse and neurotransmission-related pathways featured heavily among the positively correlated proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, \u003cb\u003eleft\u003c/b\u003e), whereas ribosomal, endocytic, phagosomal, and myelin pathway terms were most common among the negatively correlated proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, \u003cb\u003eright\u003c/b\u003e) (\u003cb\u003eSupplementary Dataset 4\u003c/b\u003e). These could relate to synaptic pruning processes, as the top negatively correlated protein was CD47 (r\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;.74, FDR\u0026thinsp;=\u0026thinsp;1.6E-05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF), which prevents microglia-mediated synaptic pruning through phagocytosis (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). We also found neurogenesis-related terms involving \u0026ldquo;neuron projection organization\u0026rdquo; and \u0026ldquo;nervous system development\u0026rdquo; (\u003cb\u003eSupplementary Dataset 4\u003c/b\u003e), further strengthening the possible link to AHN regulation.\u003c/p\u003e \u003cp\u003eTo investigate if Rasal1 is relevant to brain aging, differential expression analyses of dorsal and ventral DG proteomes of sedentary young and middle-aged rats were conducted, but Rasal1 was not identified among aging DEPs (\u003cb\u003eSupplementary Dataset 1\u003c/b\u003e), as we confirmed by western blot (\u003cb\u003eSupplementary Fig.\u0026nbsp;1\u003c/b\u003e). However, some Rasal1-associated pathways among exercise DEPs were also enriched among aging DEPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). Furthermore, when we conducted pathway analyses with exercise DEPs and pathways from the Gene Expression Omnibus (GEO), we found that Rasal1 featured to pathways previously identified as upregulated in the CA1 and CA3 of 28- vs. 18-month-old rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI). This suggests that middle age could be a turning point in Rasal1 regulation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eKnockdown of hippocampal Rasal1 increased AHN in middle-aged rats\u003c/h2\u003e \u003cp\u003eSince we identified that Rasal1 is downregulated by voluntary running exercise, we next tested whether reducing Rasal1 in middle age mimics the effects of exercise on AHN. We used a lentiviral approach to knock down Rasal1 \u003cem\u003ein vivo\u003c/em\u003e in the dorsal hippocampus of middle-aged rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA; \u003cb\u003eSupplementary Fig.\u0026nbsp;4A\u003c/b\u003e). Lentiviral expression of a scrambled short hairpin (SCR) or a short hairpin directed at Rasal1 (shRASAL1) was confirmed by GFP autofluorescence in the granular cell layer of the dorsal hippocampus in LV\u003csup\u003eSCR\u003c/sup\u003e and LV\u003csup\u003eshRASAL1\u003c/sup\u003e rats, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Reduced expression of Rasal1 transcript was also confirmed in the dorsal hippocampus of LV\u003csup\u003eshRASAL1\u003c/sup\u003e compared to the LV\u003csup\u003eSCR\u003c/sup\u003e rats (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.06) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo examine the effects of Rasal1 knockdown on AHN, the density of Ki67\u0026thinsp;+\u0026thinsp;cells (cell proliferation) and DCX\u0026thinsp;+\u0026thinsp;cells (immature neurons) was measured along the longitudinal axis of the DG. The density of Ki67\u0026thinsp;+\u0026thinsp;cells was greater in the dorsal (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), but not ventral (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.61) or whole DG (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.11) of LV\u003csup\u003eshRASAL1\u003c/sup\u003e rats compared to LV\u003csup\u003eSCR\u003c/sup\u003e controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Similarly, the density of DCX\u0026thinsp;+\u0026thinsp;cells was greater in the dorsal (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and whole DG (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.05), but not ventral (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.33) DG of LV\u003csup\u003eshRASAL1\u003c/sup\u003e rats compared to LV\u003csup\u003eSCR\u003c/sup\u003e controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), while there was no change in the area of the DG (\u003cb\u003eSupplementary Fig.\u0026nbsp;4B-C\u003c/b\u003e). Previously published \u003cem\u003ein vitro\u003c/em\u003e evidence suggests that Rasal1 regulates neuronal maturation (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Thus, to explore the role of Rasal1 knockdown on the maturation of newly born neurons \u003cem\u003ein vivo\u003c/em\u003e, morphometric analysis of individual DCX\u0026thinsp;+\u0026thinsp;cells in the dorsal hippocampus was carried out. While the length and the number of neurites per DCX\u0026thinsp;+\u0026thinsp;cell were not affected by Rasal1 downregulation (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.82 and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.43, respectively), Rasal1 downregulation significantly increased the neurite length normalized by the number of neurites (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eMulti-omic analysis reveals serum factors associated with aging, exercise, and Rasal1 expression\u003c/h2\u003e \u003cp\u003eSince previous studies have reported that factors in the systemic circulation can mediate the beneficial effects of exercise on AHN (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e) and that serum from exercising animals can increase hippocampal neurogenesis \u003cem\u003ein vitro\u003c/em\u003e (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e), we determined if serum factors could be driving the downregulation of Rasal1 using an \u003cem\u003ein vitro\u003c/em\u003e approach. Embryonic day 18 (E18) rat hippocampal neuroprogenitor cells proliferating as neurospheres were treated with serum from exercising or sedentary young adult rats, and Rasal1 expression and neurosphere expansion were measured (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). We found that relative to sedentary serum treatment Rasal1 was downregulated by treatment with serum from young adult exercising rats (one-sample \u003cem\u003et\u003c/em\u003e-test, H\u003csub\u003e0\u003c/sub\u003e: ratio\u0026thinsp;=\u0026thinsp;1; \u003cem\u003et\u003c/em\u003e\u003csub\u003e(6)\u003c/sub\u003e = -2.7, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, two-tailed) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), with a concomitant increase in neurosphere diameter (one-sample \u003cem\u003et\u003c/em\u003e-test, H\u003csub\u003e0\u003c/sub\u003e: ratio\u0026thinsp;=\u0026thinsp;1; \u003cem\u003et\u003c/em\u003e\u003csub\u003e(11)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;7.1, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, two-tailed) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D, E). This suggests that serum factors are sufficient to phenocopy the effect of exercise on rasal1 gene expression in neuroprogenitor cells \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHaving established that Rasal1 expression is sensitive to serum factors, sera from animals with or without access to running wheels were screened for possible mediators using discovery proteomics and untargeted metabolomic analyses (\u003cb\u003eSupplementary Dataset 1\u003c/b\u003e). To fully leverage the multi-omic data from each sample, we performed multi-omic factor analysis using MOFA2 (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). This tool decomposes variance in multiple -omics modalities taken from the same samples, akin to a principal component analysis with multivariate data. The model returns latent factors that capture one or several -omics modalities, which can be used to cluster samples. We used this to identify factors that capture both proteomic and metabolomic variance (indicative of biologically plausible co-regulation) and then examined if they ordinated (\u0026ldquo;clustered\u0026rdquo;) samples by experimental group. To capture any possible opposing effects of exercise and age on serum -omes, we included samples from young adult sedentary, young adult exercising, and middle-aged sedentary animals. After filtering for high-quality features, 470 metabolites and 250 proteins were included. Proteins accounted for the largest amount of variance among serum samples compared to metabolites (28% vs. 12%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF \u003cb\u003eleft\u003c/b\u003e). Factor 1 appeared the most biologically informative, as it displayed a similar degree of variance explained by proteins and metabolites (8.5% vs. 9%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF \u003cb\u003eright\u003c/b\u003e). Plotting the sample scores of each latent factor by experimental condition revealed that factor 1 captured an opposing relationship between age and exercise (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). Indeed, unsupervised K-means clustering (with \u003cem\u003ek\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3) of samples using factor 1 score captured the experimental groupings (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). Next, we inspected the protein and metabolite feature loadings on factor 1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). Among the top 10 absolute protein and metabolite feature loadings, biologically plausible features such as apolipoproteins and testosterone were identified, but also Platelet factor 4 (Pf4) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ \u003cb\u003eleft\u003c/b\u003e), which has been found to mediate effects of aging (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e) and exercise (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e) on AHN and cognition (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e) in mice. The top-loading metabolite was 5-hydroxytryptophol (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ \u003cb\u003eright\u003c/b\u003e), a highly abundant peripheral serotonin metabolite in rats (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo determine if individual variations in Rasal1 expression in the dorsal DG could be accounted for by variations in serum protein and metabolite abundances, the effects of experimental group and latent co-variance were regressed out in DG and serum -omics datasets, followed by Pearson correlation on the residuals. While 6 serum proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK) and 19 metabolites (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eL) were found to be nominally (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) correlated with Rasal1 expression in the dorsal DG, none remained statistically significant after applying FDR correction (\u003cb\u003eSupplementary Dataset 3\u003c/b\u003e). However, some of these nominally correlated features have plausible links to AHN, such as the (neuro)inflammatory regulator C-reactive protein (Crp) (r\u0026thinsp;=\u0026thinsp;.40, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK), the tryptophan metabolite Kynurenine (r\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;.47, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eL), and its metabolite Kyrurenic acid (r\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;.41, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;.05) (\u003cb\u003eSupplementary Dataset 3\u003c/b\u003e). The positive correlations with (neuro)inflammatory regulators Crp (r\u0026thinsp;=\u0026thinsp;.40, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;.05) and Plg (r\u0026thinsp;=\u0026thinsp;.45, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;.05) (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e) suggest that a heightened peripheral inflammatory state is associated with higher Rasal1 expression and possibly lower AHN. Taken together, these results point to a possible role of systemic factors in impacting Rasal1 expression through pro-inflammatory and tryptophan-related pathways in response to voluntary running exercise.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eHere, we have established that hippocampal Rasal1 is a candidate regulator of exercise-induced AHN that can be leveraged to mitigate the age-related AHN decline during middle age. Rasal1 is a GTPase-activating protein involved in different cellular functions in the brain, such as growth, differentiation, and survival (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). While there are no previous studies demonstrating that Rasal1 regulates AHN, induction of Ras signaling during exercise has been shown to recruit DG neural stem cells into the cell cycle (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e), and RAS/ERK regulates neurogenesis through proneural activity programs (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e), making Rasal1 an attractive candidate regulator of AHN. Our \u003cem\u003ein vivo\u003c/em\u003e experiments show that Rasal1 is downregulated by exercise in young adult rats and demonstrate that its downregulation in middle-aged rats can mimic the neurogenic effects of exercise. We also show that Rasal1 expression in hippocampal neuroprogenitor cells is reduced by exposure to serum from exercising animals, with concomitant progenitor expansion. Lastly, we leveraged multi-omic analysis of sera in conjunction with brain proteomics and further identified possible regulators of Rasal1.\u003c/p\u003e \u003cp\u003eThe exercise-driven downregulation of Rasal1 and the related effects on AHN in young adult rats were mimicked by gene knockdown in middle-aged rats. This was prompted by the fact that the daily running distance by middle-aged rats was significantly lower than that of young adult rats, in agreement with previously published data (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e) and possibly due to the higher body weight of the middle-aged rats (final experimental week: Y-EX\u0026thinsp;=\u0026thinsp;506.1 g, MA-EX\u0026thinsp;=\u0026thinsp;715.5 g). Thus, we did not observe an exercise-driven increase in AHN in middle-aged rats, nor the Rasal1 downregulation that we observed in young adult rats. An age-related reduction in physical activity is also reported in older adult humans (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). As regular physical exercise may not always be feasible with aging, mechanisms such as the Rasal1-induced increase in AHN as we have identified may contribute to the development of strategies to mimic at least some of the benefits of physical activity. Given the observed downregulation of Rasal1 in young exercising rats, we examined whether lentiviral downregulation of Rasal1 could act as an exercise mimetic to increase AHN in sedentary middle-aged rats. We found that downregulation of Rasal1 was sufficient to promote AHN in middle-aged rats, thus recapitulating the neurogenic effects of voluntary running exercise seen in the young animals. Importantly, the downregulation of Rasal1 was sufficient to enhance the dendritic growth of new hippocampal neurons in middle-aged rats. Synaptic maturation and integration of adult-born neurons into existing neural circuits are stimulated by exercise and are important for memory processes (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). This is particularly relevant in the aging brain, where exercise has been shown to counteract the decline in cognitive functions by increasing the number and maturation of new hippocampal neurons (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Beyond the established expression of Rasal1 by (im)mature neurons, we show that Rasal1 is expressed by hippocampal astrocytes \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e. Given the contribution of astrocytes to hippocampal functions (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e) and the susceptibility of hippocampal astrocytes to exercise stimulation (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e), our results support a possible role of astrocytes in the exercise-mediated regulation of Rasal1.\u003c/p\u003e \u003cp\u003eOur multi-omic analysis of sera in conjunction with brain proteomics pinpointed candidate serum proteins and metabolites relevant to Rasal1 regulation, which map onto hallmark aging processes (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e) and can be ameliorated by physical exercise (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Those included inflammatory (Crp, Ahsg, Plg), metabolic (Ahsg), antioxidant (Carnosine), and tryptophan-related (Kynurenine, Kynurenic acid) factors. Notably, the metabolism of tryptophan into systemically circulating indole by gut microbes has been found to regulate AHN directly via the aryl hydrocarbon receptor(\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). Thus, it is plausible that the negative correlations between Kyurenine/Kynurenic acid and Rasal1 indicate that increased tryptophan metabolism is associated with lower Rasal1 and increased levels of AHN. Furthermore, given the detrimental role of peripheral inflammation to AHN (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e), the positive correlation between Rasal1 and pro-inflammatory molecules could indicate a role of pro-inflammatory molecules in the stimulation of Rasal1 expression, resulting in impaired AHN. We also found a positive correlation between Rasal1 and Ahsg (\u003cb\u003eSupplementary Dataset 3\u003c/b\u003e), better known as Fetuin-A, which is lowered by exercise in humans (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e) and rats (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). As a hepatokine, Fetuin-A is thought to exert its effects by modulating insulin signaling and sensitivity following exercise (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). Interestingly, we found a strong enrichment of SLC2A4 (GLUT4) as a common hub protein among exercise and age DEPs, as well as \u0026ldquo;central carbon metabolism\u0026rdquo; (i.e. glycolysis, Krebs cycle, pentose phosphate pathway) in our metabolomics data (\u003cb\u003eSupplementary Dataset 2\u003c/b\u003e). Thus, it is tempting to speculate that aging and exercise could exert opposing effects on Rasal1 (and consequently AHN and synaptic plasticity) through systemic regulation of pro-inflammatory signaling, tryptophan metabolism, or glucose homeostasis. Importantly, while Rasal1 was not differentially expressed in middle-aged rats, we found that it was upregulated in the rat hippocampus at older ages (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Additionally, in our independent analysis of Rasal1 transcript expression in the single-cell RNA-sequencing data from Wu et al. (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e), we found qualitative evidence that it becomes more prominent in the mouse DG in old age compared to middle age (\u003cb\u003eSupplementary Fig.\u0026nbsp;2B\u003c/b\u003e). This could imply a turning point in its regulation around middle age, albeit more research is needed to elucidate fluctuations in Rasal1 expression across the lifespan.\u003c/p\u003e \u003cp\u003eIn conclusion, we identified Rasal1 as a novel candidate regulator of exercise-induced AHN and demonstrated that it can be targeted to mitigate the age-related AHN decline during the critical time of middle age. Leveraging these findings to identify systemic factors responsible for the Rasal1-induced pro-neurogenic effects of exercise may support the development of novel therapeutic strategies aimed at enhancing AHN and improving hippocampal-related cognitive function. Such strategies are particularly important for individuals who are unable to exercise due to physical or pathological limitations, and who therefore may require pharmacological intervention to experience the brain health benefits of exercise otherwise inaccessible to them.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflicts of Interest\u003c/h2\u003e \u003cp\u003eYMN is in receipt of research funding from Marigot Limited and has received honoraria from Yakult as an invited speaker. OFO has received research funding from Marigot Limited and Alkermes plc and honoraria as an invited speaker at a meeting organised by Janssen. All other authors declare that they have no conflict of interest.\u003c/p\u003e \u003ch2\u003eAuthor Contribution\u003c/h2\u003e \u003cp\u003eMGC: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Data Curation, Writing \u0026ndash; original draft, review and editing, Visualisation; SD-H: Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Data Curation, Writing \u0026ndash; original draft, review and editing, Visualisation; SN: Conceptualization, Methodology, Validation, Investigation, Data Curation, Writing \u0026ndash; original draft, review and editing, Supervision, Project administration; JAE: Methodology, Software, Formal analysis, Resources, Data Curation, Writing \u0026ndash; review and editing, Supervision; AL: Methodology, Software, Formal analysis, Data Curation, Writing \u0026ndash; review and editing, Supervision; PJL: Conceptualization, Writing \u0026ndash; review and editing, Supervision; JDM: Conceptualization, Writing \u0026ndash; review and editing; OO\u0026rsquo;L: Conceptualization, Methodology, Writing \u0026ndash; original draft, review and editing, Supervision, Project administration; YN: Conceptualization, Methodology, Resources, Writing \u0026ndash; original draft, review and editing, Supervision, Project administration, Funding acquisition.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis work was supported by Research Ireland (formerly Science Foundation Ireland (SFI)) under Grant Number SFI/FFP/6820. We thank Dr. Thieza Melo, Dr. Andrew Sasmita, Tara Foley, Anna Golubeva, and Patrick Fitzgerald for technical assistance.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKandola A, Hendrikse J, Lucassen PJ, Y\u0026uuml;cel M. Aerobic Exercise as a Tool to Improve Hippocampal Plasticity and Function in Humans: Practical Implications for Mental Health Treatment. Front Hum Neurosci. 2016;10(July):1\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTari AR, Walker TL, Huuha AM, Sando SB, Wisloff U. Neuroprotective mechanisms of exercise and the importance of fitness for healthy brain ageing. Lancet [Internet]. 2025 Mar 29 [cited 2025 Nov 20];405(10484):1093\u0026ndash;118. 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Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubmed.ncbi.nlm.nih.gov/28270597/\u003c/span\u003e\u003cspan address=\"https://pubmed.ncbi.nlm.nih.gov/28270597/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-psychiatry","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"mp","sideBox":"Learn more about [Molecular Psychiatry](http://www.nature.com/mp/)","snPcode":"41380","submissionUrl":"https://mts-mp.nature.com/cgi-bin/main.plex","title":"Molecular Psychiatry","twitterHandle":"@molpsychiatry","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8394751/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8394751/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePhysical exercise exerts several positive effects on the brain and is emerging as an approach for mitigating age-related functional decline. One benefit of exercise is the increase in adult hippocampal neurogenesis (AHN). However, physical exercise may not always be feasible in individuals due to physical or medical constraints which are more common in older age, highlighting the importance of understanding regulators of exercise-induced AHN. We demonstrate that Rasal1, a GTPase-activating protein, is downregulated in the dentate gyrus (DG) by voluntary running exercise in young adult rats. Middle age is recognised as a period of the lifespan prognostic of cognitive health in older age, and amenable to intervention. Lentiviral knockdown of Rasal1 in the DG promoted AHN and neurite growth in middle-aged rats that were otherwise reluctant to run. Treatment of hippocampal neuroprogenitor cells \u003cem\u003ein vitro\u003c/em\u003e with serum from exercising animals mimicked the exercise-induced downregulation of Rasal1 and was associated with neuroprogenitor expansion. Lastly, multi-omic analysis of sera in conjunction with brain proteomics identified possible systemic mediators of Rasal1 downregulation. Taken together, our findings highlight Rasal1 as a novel candidate regulator of the pro-neurogenic effects of exercise. Identifying systemic factors underlying the Rasal1-driven effects of exercise could inform new therapies to enhance AHN and hippocampal function.\u003c/p\u003e","manuscriptTitle":"Rasal1: A candidate exercise mimetic to increase adult hippocampal neurogenesis in middle age","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-13 08:35:41","doi":"10.21203/rs.3.rs-8394751/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2026-03-24T09:59:17+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-03-04T17:47:42+00:00","index":4,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-02-25T11:48:20+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-02-21T16:08:24+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-02-17T15:47:10+00:00","index":4,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-02-17T14:14:14+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-02-10T17:37:07+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-02-10T01:54:08+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2026-02-09T23:47:13+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-04T15:27:09+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-04T15:23:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Psychiatry","date":"2025-12-18T11:15:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-psychiatry","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"mp","sideBox":"Learn more about [Molecular Psychiatry](http://www.nature.com/mp/)","snPcode":"41380","submissionUrl":"https://mts-mp.nature.com/cgi-bin/main.plex","title":"Molecular Psychiatry","twitterHandle":"@molpsychiatry","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ee964087-9daa-4261-95ef-06063dfd7954","owner":[],"postedDate":"February 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":62621180,"name":"Biological sciences/Neuroscience"},{"id":62621181,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2026-03-24T10:02:21+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-13 08:35:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8394751","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8394751","identity":"rs-8394751","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}