Hindlimb immobilization induces insulin resistance and elevates mitochondrial ROS production in the hippocampus of female rats.

Alzheimer's disease (AD) is the fifth leading cause of death in older adults, and treatment options are severely lacking. Recent findings demonstrate a strong relationship between skeletal muscle and cognitive function, with evidence supporting that muscle quality and cognitive function are positively correlated in older adults. Conversely, decreased muscle function is associated with a threefold increased risk of cognitive decline. Based on these observations, the purpose of this study was to investigate the negative effects of muscle disuse [via a model of hindlimb immobilization (HLI)] on hippocampal insulin sensitivity and mitochondrial function and identify the potential mechanisms involved. HLI for 10 days in 4-mo-old female Wistar rats resulted in the following novel findings: 1) hippocampal insulin resistance and deficits in whole body glucose homeostasis, 2) dramatically increased mitochondrial reactive oxygen species (ROS) production in the hippocampus, 3) elevated markers for amyloidogenic cleavage of amyloid precursor protein (APP) and tau protein in the hippocampus, 4) and reduced brain-derived neurotrophic factor (BDNF) expression. These findings were associated with global changes in iron homeostasis, with muscle disuse producing muscle iron accumulation in association with decreased serum and whole brain iron levels. We report the novel finding that muscle disuse alters brain iron homeostasis and reveal a strong negative correlation between muscle and brain iron content. Overall, HLI-induced muscle disuse has robust negative effects on hippocampal insulin sensitivity and ROS production in association with altered brain iron homeostasis. This work provides potential novel mechanisms that may help explain how loss of muscle function contributes to cognitive decline and AD risk.NEW & NOTEWORTHY Muscle disuse via hindlimb immobilization increased oxidative stress and insulin resistance in the hippocampus. These findings were in association with muscle iron overload in connection with iron dysregulation in the brain. Overall, our work identifies muscle disuse as a contributor to hippocampal dysfunction, potentially through an iron-based muscle-brain axis, highlighting iron dysregulation as a potential novel mechanism in the relationship between muscle health, cognitive function, and Alzheimer's disease risk.

Microglia-synapse engulfment via PtdSer-TREM2 ameliorates neuronal hyperactivity in Alzheimer's disease models.

Neuronal hyperactivity is a key feature of early stages of Alzheimer's disease (AD). Genetic studies in AD support that microglia act as potential cellular drivers of disease risk, but the molecular determinants of microglia-synapse engulfment associated with neuronal hyperactivity in AD are unclear. Here, using super-resolution microscopy, 3D-live imaging of co-cultures, and in vivo imaging of lipids in genetic models, we found that spines become hyperactive upon Aß oligomer stimulation and externalize phosphatidylserine (ePtdSer), a canonical "eat-me" signal. These apoptotic-like spines are targeted by microglia for engulfment via TREM2 leading to amelioration of Aß oligomer-induced synaptic hyperactivity. We also show the in vivo relevance of ePtdSer-TREM2 signaling in microglia-synapse engulfment in the hAPP NL-F knock-in mouse model of AD. Higher levels of apoptotic-like synapses in mice as well as humans that carry TREM2 loss-of-function variants were also observed. Our work supports that microglia remove hyperactive ePtdSer+ synapses in Aß-relevant context and suggest a potential beneficial role for microglia in the earliest stages of AD.

Selective breeding for physical inactivity produces cognitive deficits via altered hippocampal mitochondrial and synaptic function.

Physical inactivity is the 4th leading cause of death globally and has been shown to significantly increase the risk for developing Alzheimer's Disease (AD). Recent work has demonstrated that exercise prior to breeding produces heritable benefits to the brains of offspring, suggesting that the physical activity status of previous generations could play an important role in one's brain health and their subsequent risk for neurodegenerative diseases. Thus, our study aimed to test the hypothesis that selective breeding for physical inactivity, or for high physical activity, preference produces heritable deficits and enhancements to brain health, respectively. To evaluate this hypothesis, male and female sedentary Low Voluntary Runners (LVR), wild type (WT), and High Voluntary Runner (HVR) rats underwent cognitive behavioral testing, analysis of hippocampal neurogenesis and mitochondrial respiration, and molecular analysis of the dentate gyrus. These analyses revealed that selecting for physical inactivity preference has produced major detriments to cognition, brain mitochondrial respiration, and neurogenesis in female LVR while female HVR display enhancements in brain glucose metabolism and hippocampal size. On the contrary, male LVR and HVR showed very few differences in these parameters relative to WT. Overall, we provide evidence that selective breeding for physical inactivity has a heritable and detrimental effect on brain health and that the female brain appears to be more susceptible to these effects. This emphasizes the importance of remaining physically active as chronic intergenerational physical inactivity likely increases susceptibility to neurodegenerative diseases for both the inactive individual and their offspring.

Perivascular cells induce microglial phagocytic states and synaptic engulfment via SPP1 in mouse models of Alzheimer's disease.

Alzheimer's disease (AD) is characterized by synaptic loss, which can result from dysfunctional microglial phagocytosis and complement activation. However, what signals drive aberrant microglia-mediated engulfment of synapses in AD is unclear. Here we report that secreted phosphoprotein 1 (SPP1/osteopontin) is upregulated predominantly by perivascular macrophages and, to a lesser extent, by perivascular fibroblasts. Perivascular SPP1 is required for microglia to engulf synapses and upregulate phagocytic markers including C1qa, Grn and Ctsb in presence of amyloid-ß oligomers. Absence of Spp1 expression in AD mouse models results in prevention of synaptic loss. Furthermore, single-cell RNA sequencing and putative cell-cell interaction analyses reveal that perivascular SPP1 induces microglial phagocytic states in the hippocampus of a mouse model of AD. Altogether, we suggest a functional role for SPP1 in perivascular cells-to-microglia crosstalk, whereby SPP1 modulates microglia-mediated synaptic engulfment in mouse models of AD.

Transcriptomic analysis reveals novel molecular signaling networks involved in low voluntary running behavior after AP-1 inhibition.

Understanding the neuro-molecular mechanisms that mediate the quantity of daily physical activity (PA) level is of medical significance, given the tremendous health benefits associated with greater physical activity. Here, we examined the effects of intra-nucleus accumbens (NAc) inhibition of activator protein-1 (AP-1), an important transcriptional factor downstream of cAMP response element binding protein (CREB; a reward-related transcriptional regulator), on voluntary wheel running behavior in wild-type (WT) and low voluntary running (LVR) female rats. Transcriptome analysis of the nucleus accumbens (NAc; a brain region critical for PA reward and motivation) was performed to further determine molecular responses to intra-NAc AP-1 inhibition in these rat lines. Within WT rats, intra-NAc AP-1 inhibition caused a significant decrease in overnight running distance in comparison to control rats (p = 0.009). Transcriptomic and bioinformatic analysis in WT rats identified involvement of gene products that regulate cellular proliferation and development, which were cellular processes regulated by AP-1. In contrast to above decreased WT distances, intra-NAc AP-1 inhibition in LVR rats increased nightly running distance in comparison to LVR control rats (p = 0.0008). Further analysis identified gene products that are associated with regulating intracellular Ca2+ homeostasis, calcium ion binding and neuronal excitability. In short, our study aims to gain a comprehensive understanding of transcriptional profile that was due to AP-1 inhibition in NAc, in which it could not only enhance the knowledge regarding molecular regulatory loops within NAc for modulating voluntary running behavior, but also provide further insights into molecular targets for future investigations.

Acute Wheel-Running Increases Markers of Stress and Aversion-Related Signaling in the Basolateral Amygdala of Male Rats.

Physical activity (PA) is a non-invasive, cost-effective means of reducing chronic disease. Most US citizens fail to meet PA guidelines, and individuals experiencing chronic stress are less likely to be physically active. To better understand the barriers to maintaining active lifestyles, we sought to determine the extent to which short- versus long-term PA increases stress- and aversion-related markers in wild-type (WT) and low voluntary running (LVR) rats, a unique genetic model of low physical activity motivation. Here, we tested the effects of 1 and 4 weeks of voluntary wheel-running on physiological, behavioral, and molecular measures of stress and Hypothalamic Pituitary Adrenal (HPA)-axis responsiveness (corticosterone levels, adrenal wet weights, and fecal boli counts). We further determined measures of aversion-related signaling (kappa opioid receptor, dynorphin, and corticotropin releasing hormone mRNA expression) in the basolateral amygdala (BLA), a brain region well characterized for its role in anxiety and aversion. Compared to sedentary values, 1, but not 4 weeks of voluntary wheel-running increased adrenal wet weights and plasma corticosterone levels, suggesting that HPA responsiveness normalizes following long-term PA. BLA mRNA expression of prodynorphin (Pdyn) was significantly elevated in WT and LVR rats following 1 week of wheel-running compared to sedentary levels, suggesting that aversion-related signaling is elevated following short- but not long-term wheel-running. In all, it appears that the stress effects of acute PA may increase molecular markers associated with aversion in the BLA, and that LVR rats may be more sensitive to these effects, providing a potential neural mechanism for their low PA motivation.

Resistance-exercise training attenuates LPS-induced astrocyte remodeling and neuroinflammatory cytokine expression in female Wistar rats.

Neuroinflammation is an early detectable marker of mild cognitive impairment, the transition state between normal cognition and dementia. Resistance-exercise training can attenuate the cognitive decline observed in patients with mild cognitive impairment. However, the underlying mechanisms of resistance training effects are largely unknown. To further elucidate mechanisms of the known cognitive health benefits from resistance-exercise training, we tested if resistance-exercise training could ameliorate lipopolysaccharide-induced neuroinflammation. Five-week-old female Wistar rats received intracerebroventricular injections of lipopolysaccharides to induce neuroinflammation and cognitive impairment. Rats then underwent 3 wk of progressive ladder climbing to recapitulate resistance-exercise training in humans. Cognition was assessed toward the end of the training period by novelty object recognition testing. Neuroinflammation was measured one and 24 h after the last resistance-exercise training workout. Resistance-exercise training ameliorated cognitive impairment, diminished lipopolysaccharide-induced neuroinflammatory cytokine expression, and attenuated astrocyte remodeling in the dentate gyrus 24 h post exercise. Here, we provide evidence that the ladder-climbing model of resistance-exercise training in rats can improve cognition as early as 3 wk. In addition, these data support the hypothesis that resistance exercise can reduce lipopolysaccharide-induced neuroinflammation in the dentate gyrus.NEW & NOTEWORTHY To further elucidate the known cognitive health benefits from resistance-exercise training, we tested if resistance-exercise training in rats would attenuate lipopolysaccharide-induced neuroinflammation. Our data demonstrated that resistance training had an anti-inflammatory effect in the brain as LPS-induced neuroinflammatory cytokine expression and reactive astrocytic remodeling were reduced in the dentate gyrus after 3 wk of progressive ladder climbing.

Creatine Supplementation Upregulates mTORC1 Signaling and Markers of Synaptic Plasticity in the Dentate Gyrus While Ameliorating LPS-Induced Cognitive Impairment in Female Rats.

Mild cognitive impairment (MCI) designates the boundary area between cognitive function in natural aging and dementia, and this is viewed as a therapeutic window to prevent the occurrence of dementia. The current study investigated the neurocognitive effects of oral creatine (Cr) supplementation in young female Wistar rats that received intracerebroventricular injections of lipopolysaccharide (LPS) to mimic MCI. Neuromolecular changes within the dentate gyrus were analyzed following behavioral testing. We also investigated both neurocognitive and neuromolecular changes following Cr supplementation in the absence of LPS in young female Wistar rats to further investigate mechanisms. Interestingly, based on trial 2 of Barnes maze test, Cr supplementation ameliorated spatial learning and memory deficit induced by LPS, shown by decreased latency time and errors to reach the escape box (p < 0.0001, n = 12). Cr supplementation also attenuated recognition memory deficit induced by LPS, shown by increased amount of time taken to explore the new object (p = 0.002, n = 12) during novel object recognition testing. Within the dentate gyrus, Cr supplementation in LPS injected rats upregulated mTORC1 signaling (p = 0.026 for mTOR phosphorylation, p = 0.002 for p70S6K phosphorylation, n = 8) as well as the synapsin (p = 0.008) and PSD-95 synaptic proteins (p = 0.015), in comparisons to LPS injected rats. However, Cr supplementation failed to further enhance spatial memory and recognition memory in the absence of LPS. In conclusion, Cr ameliorates LPS-induced cognitive impairment in a rodent MCI model. Mechanistically, these phenotypic effects may, in part, be mitigated via an upregulation of mTORC1 signaling, and an enhancement in synaptogenesis in the dentate gyrus. While preliminary, these findings may inform future research investigating neurocognitive effects of Cr for MCI patients.

Insight into the role of phosphatidylserine in complement-mediated synapse loss in Alzheimer's disease.

The innate immune system plays an integral role in the brain. Synaptic pruning, a fundamental process in developmental circuit refinement, is partially mediated by neuroimmune signalling at the synapse. In particular, microglia, the major tissue-resident macrophages of the brain, and the classical complement cascade, an innate immune pathway that aids in the clearance of unwanted material, have been implicated in mediating synapse elimination. Emerging data suggest that improper signalling of the innate immune pathway at the synapse leads to pathological synapse loss in age-related neurodegenerative diseases, including Alzheimer's disease. Now the key questions are whether synapses are targeted by complement and, if so, which synapses are vulnerable to elimination. Here, we review recent work implicating C1q, the initiator of the classical complement cascade, and surrounding glia as mediators of synapse loss. We examine how synapses could undergo apoptosis-like pathways in the Alzheimer brain, which may lead to the externalisation of phosphatidylserine on synapses. Finally, we discuss potential roles for microglia and astrocytes in this 'synaptic apoptosis'. Critical insight into neuroimmune regulatory pathways on synapses will be key to developing effective targets against pathological synapse loss in dementia.

The role of nucleus accumbens CREB attenuation in rescuing low voluntary running behavior in female rats.

Given the integral role of nucleus accumbens (NAc) cAMP response element binding protein (CREB) activity in motivational processes, the goal of the current study was to determine whether blunting chronic NAc CREB activity could rescue the low physical activity motivation of female, low voluntary running (LVR) rats. NAc CREB phosphorylation is elevated in these rats, a state previously attributed to deficits in reward valuation. It was recently shown that overexpression of the upstream CREB inhibitor, protein kinase inhibitor alpha (PKIα), increased LVR nightly running by ~threefold. Therefore, the current study addresses the extent to which NAc CREB attenuation influences female LVR and wild-type (WT) wheel-running behavior. Inducible reductions in NAc neuronal activity using Gi-coupled hM4Di DREADDs increased running behavior in LVR, but not in WT, rats. Similarly, site-directed pharmacological inhibition of NAc CREB activity significantly increased LVR nightly running distance and time by ~twofold, with no effect in WT rats. Finally, environmentally enriched LVR rats exhibit higher levels of running compared to socially isolated rats in what appeared to be a CREB-related manner. Considering the positive outcomes of upstream CREB modulation and environmental enrichment on LVR behavior, we believe that blunting NAc CREB activity has the neuromolecular potential to partially reverse low physical activity motivation, as exemplified by the LVR model. The positive physical activity outcome of early life enrichment adds translatable value to human childhood enrichment and highlights its importance on motivational processes later in life.

Five months of voluntary wheel running downregulates skeletal muscle LINE-1 gene expression in rats.

Transposable elements (TEs) are mobile DNA and constitute approximately half of the human genome. LINE-1 (L1) is the only active autonomous TE in the mammalian genome and has been implicated in a number of diseases as well as aging. We have previously reported that skeletal muscle L1 expression is lower following acute and chronic exercise training in humans. Herein, we used a rodent model of voluntary wheel running to determine whether long-term exercise training affects markers of skeletal muscle L1 regulation. Selectively bred high-running female Wistar rats (n = 11 per group) were either given access to a running wheel (EX) or not (SED) at 5 wk of age, and these conditions were maintained until 27 wk of age. Thereafter, mixed gastrocnemius tissue was harvested and analyzed for L1 mRNA expression and DNA content along with other L1 regulation markers. We observed significantly (P < 0.05) lower L1 mRNA expression, higher L1 DNA methylation, and less L1 DNA in accessible chromatin regions in EX versus SED rats. We followed these experiments with 3-h in vitro drug treatments in L6 myotubes to mimic transient exercise-specific signaling events. The AMP-activated protein kinase (AMPK) agonist 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR; 4 mM) significantly decreased L1 mRNA expression in L6 myotubes. However, this effect was not facilitated through increased L1 DNA methylation. Collectively, these data suggest that long-term voluntary wheel running downregulates skeletal muscle L1 mRNA, and this may occur through chromatin modifications. Enhanced AMPK signaling with repetitive exercise bouts may also decrease L1 mRNA expression, although the mechanism of action remains unknown.

Resistance-exercise training ameliorates LPS-induced cognitive impairment concurrent with molecular signaling changes in the rat dentate gyrus.

Effective treatments preventing brain neuroinflammatory diseases are lacking. Resistance-exercise training (RT) ameliorates mild cognitive impairment (MCI), a forerunner to neuroinflammatory diseases. However, few studies have addressed the molecular basis by which RT abates MCI. Thus experiments were performed to identify some molecular changes occurring in response to RT in young, female Wistar rats. To induce MCI, intraventricular lipopolysaccharide (LPS) injections were used to increase dentate gyrus inflammation, reflected by significantly increased TNF-α (~400%) and IL-1ß (~1,500%) mRNA (P < 0.0001) after 6 wk. Five days after LPS injections, half of LPS-injected rats performed RT by ladder climbing for 6 wk, 3 days/wk, whereas half remained without ladders. RT for 6 wk increased lean body mass percentage (P < 0.05), individual muscle masses (gastrocnemius and tibialis anterior) (P < 0.05), and maximum lifting capacity (P < 0.001). The RT group, compared with sedentary controls, had 1) ameliorated spatial learning deficits (P < 0.05), 2) increased dentate gyrus phosphorylation of IGF-1R, protein kinase B, and GSK-3ß proteins (P < 0.05), components of downstream IGF-1 signaling, and 3) increased dentate gyrus synaptic plasticity marker synapsin protein (P < 0.05). Two follow-up experiments (without LPS) characterized dentate gyrus signaling during short-term RT. Twenty-four hours following the third workout in a 1-wk training duration, phosphorylation of ERK1/2 and GSK-3ß proteins, as well as proliferation marker protein, PCNA, were significantly increased (P < 0.05). Similar changes did not occur in a separate group of rats following a single RT workout. Taken together, these data indicate that RT ameliorates LPS-induced MCI after RT, possibly mediated by increased IGF-1 signaling pathway components within the dentate gyrus. NEW & NOTEWORTHY The data suggest that resistance-exercise training restores cognitive deficits induced by lipopolysaccharides and can activate associated IGF-1 signaling in the dentate gyrus. Our data show, for the first time, that as few as three resistance-exercise workouts (spread over 1 wk) can activate IGF-1 downstream signaling and increase proliferation marker PCNA in the dentate gyrus.

Overexpression of Protein Kinase Inhibitor Alpha Reverses Rat Low Voluntary Running Behavior.

A gene was sought that could reverse low voluntary running distances in a model of low voluntary wheel-running behavior. In order to confirm the low motivation to wheel-run in our model does not result from defects in reward valuation, we employed sucrose preference and conditioned place preference for voluntary wheel-access. We observed no differences between our model and wild-type rats regarding the aforementioned behavioral testing. Instead, low voluntary runners seemed to require less running to obtain similar rewards for low voluntary running levels compared to wild-type rats. Previous work in our lab identified protein kinase inhibitor alpha as being lower in low voluntary running than wild-type rats. Next, nucleus accumbens injections of an adenoviral-associated virus that overexpressed the protein kinase inhibitor alpha gene increased running distance in low voluntary running, but not wild-type rats. Endogenous mRNA levels for protein kinase inhibitor alpha, dopamine receptor D1, dopamine receptor D2, and Fos were all only lower in wild-type rats following overexpression compared to low voluntary runners, suggesting a potential molecular and behavioral resistance in wild-type rats. Utilizing a nucleus accumbens preparation, three intermediate early gene mRNAs increased in low voluntary running slices after dopamine receptor agonist SKF-38393 exposure, while wild-type had no response. In summary, the results suggest that protein kinase inhibitor alpha is a promising gene candidate to partially rescue physical activity in the polygenic model of low voluntary running. Importantly, there were divergent molecular responses to protein kinase inhibitor alpha overexpression in low voluntary runners compared to wild-type rats.

Correction to: Overexpression of Protein Kinase Inhibitor Alpha Reverses Rat Low Voluntary Running Behavior.

The original version of this article unfortunately contained mistake in Table 2 to where two directionality arrows were inverted.

5-Aminoimidazole-4-carboxamide ribonucleotide prevents fat gain following the cessation of voluntary physical activity.

NEW FINDINGS: What is the central question of this study? We investigated whether 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) could prevent acute increases in body fat and changes in omental and subcutaneous adipose tissue following the sudden transition from physical activity to physical inactivity. What is the main finding and its importance? AICAR prevented fat gains following the transition from physical activity to inactivity to levels comparable to rats that remained physically active. AICAR and continuous physical activity produced depot-specific changes in cyclin A1 mRNA and protein that were associated with the prevention of fat gain. These findings suggest that targeting AMP-activated protein kinase signalling could oppose rapid adipose mass growth. The transition from physical activity to inactivity is associated with drastic increases in 'catch-up' fat that in turn foster the development of many obesity-associated maladies. We tested whether 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) treatment would prevent gains in body fat following the sudden transition from a physically active state to an inactive state by locking a voluntary running wheel. Male Wistar rats were either sedentary (SED) or given wheel access for 4 weeks, at which time rats with wheels continued running (RUN), had their wheel locked (WL) or had WL with daily AICAR injection (WL + AICAR) for 1 week. RUN and WL + AICAR prevented gains in body fat compared with SED and WL (P < 0.001). Cyclin A1 mRNA, a marker of cell proliferation, was decreased in omental, but not subcutaneous adipose tissue, in RUN and WL + AICAR compared with SED and WL groups (P < 0.05). Both cyclin A1 mRNA and protein were positively associated with gains in fat mass (P < 0.05). Cyclin A1 mRNA in omental, but not subcutaneous, adipose tissue was negatively correlated with p-AMPK levels (P < 0.05). Differences in fat gain and omental mRNA and protein levels were independent of changes in food intake and in differences in select hypothalamic mRNAs. These findings suggest that AICAR treatment prevents acute gains in adipose tissue following physical inactivity to levels of rats that continuously run, and that together, continuous physical activity and AICAR could, at least initially in these conditions, exert similar inhibitory effects on adipogenesis in a depot-specific manner.

Maternal Western diet age-specifically alters female offspring voluntary physical activity and dopamine- and leptin-related gene expression.

Prenatal overnutrition affects development into adulthood and influences risk of obesity. We assessed the transgenerational effect of maternal Western diet (WD) consumption on offspring physical activity. Voluntary wheel running was increased in juvenile (4-7 wk of age), but decreased in adult (16-19 wk of age), F1 female WD offspring In contrast, no wheel-running differences in F1 male offspring were observed. Increased wheel running in juvenile female WD offspring was associated with up-regulated dopamine receptor (DRD)-1 and -2 in the nucleus accumbens (NAc) and with down-regulated Lepr in the ventral tegmental area (VTA). Conversely, decreased wheel running by adult female WD offspring was associated with down-regulated DRD1 in the NAc and with up-regulated Lepr in the VTA. Body fat, leptin, and insulin were increased in male, but not in female, F1 WD offspring. Recombinant virus (rAAV) leptin antagonism in the VTA decreased wheel running in standard diet but not in WD F1 female offspring. Analysis of F2 offspring found no differences in wheel running or adiposity in male or female offspring, suggesting that changes in the F1 generation were related to in utero somatic reprogramming. Our findings indicate prenatal WD exposure leads to age-specific changes in voluntary physical activity in female offspring that are differentially influenced by VTA leptin antagonism.-Ruegsegger, G. N., Grigsby, K. B., Kelty, T. J., Zidon, T. M., Childs, T. E., Vieira-Potter, V. J., Klinkebiel, D. L., Matheny, M., Scarpace, P. J., Booth, F. W. Maternal Western diet age-specifically alters female offspring voluntary physical activity and dopamine- and leptin-related gene expression.

Loss of Cdk5 function in the nucleus accumbens decreases wheel running and may mediate age-related declines in voluntary physical activity.

KEY POINTS: Physical inactivity, which drastically increases with advancing age, is associated with numerous chronic diseases. The nucleus accumbens (the pleasure and reward 'hub' in the brain) influences wheel running behaviour in rodents. RNA-sequencing and subsequent bioinformatics analysis led us to hypothesize a potential relationship between the regulation of dendritic spine density, the molecules involved in synaptic transmission, and age-related reductions in wheel running. Upon completion of follow-up studies, we developed the working model that synaptic plasticity in the nucleus accumbens is central to age-related changes in voluntary running. Testing this hypothesis, inhibition of Cdk5 (comprising a molecule central to the processes described above) in the nucleus accumbens reduced wheel running. The results of the present study show that reductions in synaptic transmission and Cdk5 function are related to decreases in voluntary running behaviour and provide guidance for understanding the neural mechanisms that underlie age-dependent reductions in the motivation to be physically active. ABSTRACT: Increases in age are often associated with reduced levels of physical activity, which, in turn, associates with the development of numerous chronic diseases. We aimed to assess molecular differences in the nucleus accumbens (NAc) (a specific brain nucleus postulated to influence rewarding behaviour) with respect to wheel running and sedentary female Wistar rats at 8 and 14 weeks of age. RNA-sequencing was used to interrogate transcriptomic changes between 8- and 14-week-old wheel running rats, and select transcripts were later analysed by quantitative RT-PCR in age-matched sedentary rats. Voluntary wheel running was greatest at 8 weeks and had significantly decreased by 12 weeks. From 619 differentially expressed mRNAs, bioinformatics suggested that cAMP-mediated signalling, dopamine- and cAMP-regulated neuronal phosphoprotein of 32 kDa feedback, and synaptic plasticity were greater in 8- vs. 14-week-old rats. In depth analysis of these networks showed significant (∼20-30%; P < 0.05) decreases in cell adhesion molecule (Cadm)4 and p39 mRNAs, as well as their proteins from 8 to 14 weeks of age in running and sedentary rats. Furthermore, Cadm4, cyclin-dependent kinase 5 (Cdk5) and p39 mRNAs were significantly correlated with voluntary running distance. Analysis of dendritic spine density in the NAc showed that wheel access increased spine density (P < 0.001), whereas spine density was lower in 14- vs. 8-week-old sedentary rats (P = 0.03). Intriguingly, intra-NAc injection of the Cdk5 inhibitor roscovitine, dose-dependently decreased wheel running. Collectively, these experiments suggest that an age-dependent loss in synaptic function and Cdk5/p39 activity in the NAc may be partially responsible for age-related declines in voluntary running behaviour.

Left ventricle transcriptomic analysis reveals connective tissue accumulation associates with initial age-dependent decline in V̇o2peak from its lifetime apex.

Peak oxygen consumption (V̇o2peak) strongly predicts morbidity and mortality better than other established risk factors, yet mechanisms associated with its age-associated decline are unknown. Our laboratory has shown that V̇o2peak first begins to decrease at the same age of 19-20 wk in both sedentary and wheel-running, female Wistar rats (Toedebusch et al., Physiol Genomics 48: 101-115, 2016). Here, we employed a total systemic approach using unsupervised interrogation of mRNA with RNA sequencing. The purpose of our study was to analyze transcriptomic profiles from both sedentary (SED) and wheel-running (RUN) conditions as a strategy to identify pathways in the left ventricle that may contribute to the initial reductions in V̇o2peak occurring between 19 and 27 wk of age. Transcriptomic comparisons were made within both SED and RUN rats between 19 and 27 wk (n = 5-8). Analysis of mRNAs shared in SED and RUN between 19 and 27 wk found 17 upregulated (e.g., Adra1d, Rpl17, Xpo7) and 8 downregulated (e.g., Cdo1, Ctfg, Sfrp1) mRNAs, at 19 wk, respectively. Furthermore, bioinformatics analysis of mRNAs common to SED and RUN produced networks suggestive of increased connective tissue development at 27 vs. 19 wk. Additionally, Ctfg mRNA was negatively associated with V̇o2peak in both SED and RUN (P < 0.05). In summary, transcriptomic analysis revealed mRNAs and networks associated with increased connective tissue development, decreased α-adrenergic activity, and decreased protein translation in the left ventricle that could, in part, potentially influence the initiation of the lifelong reduction in V̇o2peak, independent of physical activity levels.

Hypothalamic Npy mRNA is correlated with increased wheel running and decreased body fat in calorie-restricted rats.

The neuro-molecular mechanisms that regulate the relationship between physical activity level, energy homeostasis regulation, and body fat are unclear. Thus, we aimed to investigate the relationship between mRNAs in the hypothalamic arcuate nucleus (ARC) related to energy homeostasis, wheel running distance, and body fat in ad lib (AL) and calorie-restricted (CR) growing rats. We hypothesized that changes in select mRNAs (Pomc, Cart, Agrp, Npy, Lepr, Insr, Mc4r, Ampk, Sirt1, Sirt3) in CR would be associated with decreases in body fat percentage and increased wheel running behavior. Male Wistar rats were given access to voluntary running wheels at 4 weeks of age and randomized into AL (n=8) and CR (70% of AL; n=7) groups at 5 weeks of age until study termination at 12 weeks of age. Body composition, serum leptin, insulin, and adiponectin, and ARC mRNA expression in AL and CR rats were assessed and correlated with week-12 running distance to examine potential relationships that may exist. By 12 weeks of age, wheel running was increased ∼3.3-fold (p=0.03) while body fat percentage was ∼2-fold lower in CR compared to AL (p=0.001). Compared to AL, ARC Npy mRNA expression was ∼2-fold greater in CR (p=0.02), while Lepr, Insr, Ampk, and Sirt1 mRNA were additionally increased in CR (p<0.05). Significant correlations existed between ARC Npy mRNA levels versus week-12 wheel running distance (r=0.81, p=0.03), body fat (r=-0.93, p<0.01), and between body fat and wheel running (r=-0.83, p=0.02) in CR, but not in AL. These results reveal possible mechanisms by which fat-brain crosstalk may influence physical activity during energy deficit. These data suggest that below a 'threshold' fat content, body fat may drive activity levels, potentially through hypothalamic Npy action.