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.

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.

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.

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.

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.

Medial preoptic estrogen receptor-beta blunts the estrogen receptor-alpha mediated increases in wheel-running behavior of female rats.

Estrogens are believed to enhance rodent voluntary wheel-running through medial preoptic (mPOA) estrogen receptor α (ERα) signaling, with little role attributed to estrogen receptor ß (ERß). Systemic ERß activation has been shown to mitigate ERα driven increases in wheel-running. Therefore, the present goal was to determine whether ERß signaling in the mPOA plays a similar modulatory role over ERα. We utilized outbred wild-type (WT) and rats selectively bred for low voluntary running (LVR) behavior to address whether mPOA ERß signaling blunts ERα driven wheel-running behavior and immediate-early gene (Fos, Zif268, and Homer1) mRNA induction. Further, we addressed baseline mPOA mRNA expressions and circulating 17ß-estradiol levels between female WT and LVR rats. Following ovariectomy, WT rats reduced running behavior ∼40 %, with no effect in LVR rats. Intra-medial preoptic injection of the ERα-agonist propylpyrazoletriol (PPT) increased wheel-running ∼3.5-fold in WT rats, while injections of the ERß-agonist diarylpropionitrile (DPN) or a combination of the two agonists had no effect. Similarly, ERα-agonism (PPT) increased Fos and Homer1 induction ∼3-fold in WT and LVR isolated mPOA neurons, with no effect of the ERß-agonist DPN alone or in combination with PPT, suggesting medial-preoptic ERß activity may blunt ERα signaling. LVR rats exhibited higher mPOA mRNA expressions of Esr1, Esr2 and Cyp19a1, lower normalized uterine wet weights and lower 17ß-estradiol plasma levels compared to WT, suggesting their low running may be due to low circulating estrogen levels. Collectively, these findings highlight mPOA ERß as a potential neuro-molecular modulator of the estrogenic control of wheel-running behavior.

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.

Cross talk between androgen and Wnt signaling potentially contributes to age-related skeletal muscle atrophy in rats.

We sought to determine whether age-related gastrocnemius muscle mass loss was associated with parallel decrements in androgen receptor (AR) or select Wnt signaling markers. To test this hypothesis, serum-free and total testosterone (TEST) and gastrocnemius AR and Wnt signaling markers were analyzed in male Fischer 344 rats that were 3, 6, 12, 18, and 24 mo (mo) old ( n = 9 per group). Free and total TEST was greatest in 6 mo rats, and AR protein and Wnt5 protein levels linearly declined with aging. There were associations between Wnt5 protein levels and relative gastrocnemius mass ( r = 0.395, P = 0.007) as well as AR and Wnt5 protein levels (r = 0.670, P < 0.001). We next tested the hypothesis that Wnt5 affects muscle fiber size by treating C2C12-derived myotubes with lower (75 ng/ml) and higher (150 ng/ml) concentrations of recombinant Wnt5a protein. Both treatments increased myotube size ( P < 0.05) suggesting this ligand may affect muscle fiber size in vivo. We next tested if Wnt5a protein levels were androgen-modulated by examining 10-mo-old male Fischer 344 rats ( n = 10-11 per group) that were orchiectomized and treated with testosterone-enanthate (TEST-E); trenbolone enanthate (TREN), a nonaromatizable synthetic testosterone analogue; or a vehicle (ORX only) for 4 wk. Interestingly, TEST-E and TREN treatments increased Wnt5a protein in the androgen-sensitive levator ani/bulbocavernosus muscle compared with ORX only ( P < 0.05). To summarize, aromatizable and nonaromatizable androgens increase Wnt5a protein expression in skeletal muscle, age-related decrements in muscle AR may contribute Wnt5a protein decrements, and our in vitro data imply this mechanism may contribute to age-related muscle loss. NEW & NOTEWORTHY Results from this study demonstrate androgen and Wnt5 protein expression decrease with aging, and this may be a mechanism involved with age-related muscle loss.

The 1-Week and 8-Month Effects of a Ketogenic Diet or Ketone Salt Supplementation on Multi-Organ Markers of Oxidative Stress and Mitochondrial Function in Rats.

We determined the short- and long-term effects of a ketogenic diet (KD) or ketone salt (KS) supplementation on multi-organ oxidative stress and mitochondrial markers. For short-term feedings, 4 month-old male rats were provided isocaloric amounts of KD (n = 10), standard chow (SC) (n = 10) or SC + KS (~1.2 g/day, n = 10). For long-term feedings, 4 month-old male rats were provided KD (n = 8), SC (n = 7) or SC + KS (n = 7) for 8 months and rotarod tested every 2 months. Blood, brain (whole cortex), liver and gastrocnemius muscle were harvested from all rats for biochemical analyses. Additionally, mitochondria from the brain, muscle and liver tissue of long-term-fed rats were analyzed for mitochondrial quantity (maximal citrate synthase activity), quality (state 3 and 4 respiration) and reactive oxygen species (ROS) assays. Liver antioxidant capacity trended higher in short-term KD- and SC + KS-fed versus SC-fed rats, and short-term KD-fed rats exhibited significantly greater serum ketones compared to SC + KS-fed rats indicating that the diet (not KS supplementation) induced ketonemia. In long term-fed rats: (a) serum ketones were significantly greater in KD- versus SC- and SC + KS-fed rats; (b) liver antioxidant capacity and glutathione peroxidase protein was significantly greater in KD- versus SC-fed rats, respectively, while liver protein carbonyls were lowest in KD-fed rats; and (c) gastrocnemius mitochondrial ROS production was significantly greater in KD-fed rats versus other groups, and this paralleled lower mitochondrial glutathione levels. Additionally, the gastrocnemius pyruvate-malate mitochondrial respiratory control ratio was significantly impaired in long-term KD-fed rats, and gastrocnemius mitochondrial quantity was lowest in these animals. Rotarod performance was greatest in KD-fed rats versus all other groups at 2, 4 and 8 months, although there was a significant age-related decline in performance existed in KD-fed rats which was not evident in the other two groups. In conclusion, short- and long-term KD improves select markers of liver oxidative stress compared to SC feeding, although long-term KD feeding may negatively affect skeletal muscle mitochondrial physiology.