Strain Differences in Responsiveness to Repeated Restraint Stress Affect Remote Contextual Fear Memory and Blood Transcriptomics.

The role of stress in altering fear memory is not well understood. Since individual variations in stress reactivity exist, and stress alters fear memory, exposing individuals with differing stress-reactivity to repeated stress would affect their fear memory to various degrees. We explored this question using the average stress-reactive Fisher 344 (F344) rat strain and the Wistar-Kyoto (WKY) strain with its heightened stress-reactivity. Male F344 and WKY rats were exposed to the contextual fear conditioning (CFC) paradigm and then chronic restraint stress (CRS) or no stress (NS) was administered for two weeks before a second CFC. Both recent and reinstated fear memory were greater in F344s than WKYs, regardless of the stress status. In contrast, remote memory was attenuated only in F344s after CRS. In determining whether this strain-specific response to CRS was mirrored by transcriptomic changes in the blood, RNA sequencing was carried out. Overlapping differentially expressed genes (DEGs) between NS and CRS in the blood of F344 and WKY suggest a convergence of stress-related molecular mechanisms, independent of stress-reactivity. In contrast, DEGs unique to the F344 and the WKY stress responses are divergent in their functionality and networks, beyond that of strain differences in their non-stressed state. These results suggest that in some individuals chronic or repeated stress, different from the original fear memory-provoking stress, can attenuate prior fear memory. Furthermore, the novel blood DEGs can report on the general state of stress of the individual, or can be associated with individual variation in stress-responsiveness.

Divergence in Morris Water Maze-Based Cognitive Performance under Chronic Stress Is Associated with the Hippocampal Whole Transcriptomic Modification in Mice.

Individual susceptibility determines the magnitude of stress effects on cognitive function. The hippocampus, a brain region of memory consolidation, is vulnerable to stressful environments, and the impact of stress on hippocampus may determine individual variability in cognitive performance. Therefore, the purpose of this study was to define the relationship between the divergence in spatial memory performance under chronically unpredictable stress and an associated transcriptomic alternation in hippocampus, the brain region of spatial memory consolidation. Multiple strains of BXD (B6 × D2) recombinant inbred mice went through a 4-week chronic variable stress (CVS) paradigm, and the Morris water maze (MWM) test was conducted during the last week of CVS to assess hippocampal-dependent spatial memory performance and grouped animals into low and high performing groups based on the cognitive performance. Using hippocampal whole transcriptome RNA-sequencing data, differential expression, PANTHER analysis, WGCNA, Ingenuity's upstream regulator analysis in the Ingenuity Pathway Analysis® and phenotype association analysis were conducted. Our data identified multiple genes and pathways that were significantly associated with chronic stress-associated cognitive modification and the divergence in hippocampal dependent memory performance under chronic stress. Biological pathways associated with memory performance following chronic stress included metabolism, neurotransmitter and receptor regulation, immune response and cellular process. The Ingenuity's upstream regulator analysis identified 247 upstream transcriptional regulators from 16 different molecule types. Transcripts predictive of cognitive performance under high stress included genes that are associated with a high occurrence of Alzheimer's and cognitive impairments (e.g., Ncl, Eno1, Scn9a, Slc19a3, Ncstn, Fos, Eif4h, Copa, etc.). Our results show that the variable effects of chronic stress on the hippocampal transcriptome are related to the ability to complete the MWM task and that the modulations of specific pathways are indicative of hippocampal dependent memory performance. Thus, the divergence in spatial memory performance following chronic stress is related to the unique pattern of gene expression within the hippocampus.

Nutritional Ketosis Affects Metabolism and Behavior in Sprague-Dawley Rats in Both Control and Chronic Stress Environments.

Nutritional ketosis may enhance cerebral energy metabolism and has received increased interest as a way to improve or preserve performance and resilience. Most studies to date have focused on metabolic or neurological disorders while anecdotal evidence suggests that ketosis may enhance performance in the absence of underlying dysfunction. Moreover, decreased availability of glucose in the brain following stressful events is associated with impaired cognition, suggesting the need for more efficient energy sources. We tested the hypotheses that ketosis induced by endogenous or exogenous ketones could: (a) augment cognitive outcomes in healthy subjects; and (b) prevent stress-induced detriments in cognitive parameters. Adult, male, Sprague Dawley rats were used to investigate metabolic and behavioral outcomes in 3 dietary conditions: ketogenic (KD), ketone supplemented (KS), or NIH-31 control diet in both control or chronic stress conditions. Acute administration of exogenous ketones resulted in reduction in blood glucose and sustained ketosis. Chronic experiments showed that in control conditions, only KD resulted in pronounced metabolic alterations and improved performance in the novel object recognition test. The hypothalamic-pituitary-adrenal (HPA) axis response revealed that KD-fed rats maintained peripheral ketosis despite increases in glucose whereas no diet effects were observed in ACTH or CORT levels. Both KD and KS-fed rats decreased escape latencies on the third day of water maze, whereas only KD prevented stress-induced deficits on the last testing day and improved probe test performance. Stress-induced decrease in hippocampal levels of ß-hydroxybutyrate was attenuated in KD group while both KD and KS prevented stress effects on BDNF levels. Mitochondrial enzymes associated with ketogenesis were increased in both KD and KS hippocampal samples and both endothelial and neuronal glucose transporters were affected by stress but only in the control diet group. Our results highlight the complex relationship between peripheral metabolism, behavioral performance and biochemical changes in the hippocampus. Endogenous ketosis improved behavioral and metabolic parameters associated with energy metabolism and cognition while ketone supplementation replicated the biochemical effects within the hippocampus but only showed modest effects on behavioral improvements.

Linking tumor-associated macrophages, inflammation, and intestinal tumorigenesis: role of MCP-1.

Tumor-associated macrophages are associated with poor prognosis in certain cancers. Monocyte chemoattractant protein 1 (MCP-1) is thought to be the most important chemokine for recruitment of macrophages to the tumor microenvironment. However, its role on tumorigenesis in a genetic mouse model of colon cancer has not been explored. We examined the role of MCP-1 on tumor-associated macrophages, inflammation, and intestinal tumorigenesis. Male Apc(Min/+), Apc(Min/+)/MCP-1(-/-) or wild-type mice were euthanized at 18 wk of age and intestines were analyzed for polyp burden, apoptosis, proliferation, ß-catenin, macrophage number and phenotype, markers for cytotoxic T lymphocytes and regulatory T cells, and inflammatory mediators. MCP-1 deficiency decreased overall polyp number by 20% and specifically large polyp number by 45% (P < 0.05). This was consistent with an increase in apoptotic cells (P < 0.05), but there was no change detected in proliferation or ß-catenin. MCP-1 deficiency decreased F4/80-positive cells in both the polyp tissue and surrounding intestinal tissue (P < 0.05) as well as expression of markers associated with M1 (IL-12 and IL-23) and M2 macrophages (IL-13, CD206, TGF-ß, and CCL17) (P < 0.05). MCP-1 knockout was also associated with increased cytotoxic T lymphocytes and decreased regulatory T cells (P < 0.05). In addition, MCP-1(-/-) offset the increased mRNA expression of IL-1ß and IL-6 in intestinal tissue and IL-1ß and TNF-α in polyp tissue (P < 0.05), and prevented the decrease in SOCS1 expression (P < 0.05). We demonstrate that MCP-1 is an important mediator of tumor growth and immune regulation that may serve as an important biomarker and/or therapeutic target in colon cancer.

Plantaris muscle of aged rats demonstrates iron accumulation and altered expression of iron regulation proteins.

Increased free radical production and oxidative damage in ageing muscle may be a contributing factor to the development of sarcopenia. It has been suggested that the accumulation of iron may be an underlying factor in the development of oxidative stress in ageing tissues, including skeletal muscle. At present, however, the mechanisms responsible for ageing-associated muscle iron accumulation are unknown. These experiments tested the hypothesis that ageing-associated elevations in skeletal muscle iron are accompanied by altered expression of key regulators of intracellular iron status. We determined non-haem iron, oxidative injury, and expression levels of iron regulation proteins in plantaris muscles harvested from 6- and 24- to 26-month-old Fisher 344 rats (n = 10 per group). Ageing resulted in a 62% elevation in skeletal muscle non-haem iron (P < 0.05) and higher protein oxidative damage (P < 0.05). Notably, ageing was associated with elevated expression of ferritin (heavy chain, +56.2-fold; light chain, +7.3-fold), an important iron storage protein. Conversely, the iron transport protein transferrin receptor-1 demonstrated dramatic downregulation (-10.8-fold; P < 0.05) in old muscle, whereas the level of divalent metal transporter-1 protein expression was unaltered. No change in protein level of iron regulatory protein-1 was observed. In summary, these results demonstrate the occurrence of altered iron regulation concomitant with iron accumulation and oxidative stress in aged skeletal muscle. Importantly, the maintenance of divalent metal transporter-1 protein expression into old age could play a role in the accumulation of skeletal muscle iron.

Insulin-mimetic and insulin-sensitizing activities of a pentacyclic triterpenoid insulin receptor activator.

Five pentacyclic triterpenoids isolated from Campsis grandiflora were tested for insulin-mimetic and insulin-sensitizing activity. The compounds enhanced the activity of insulin on tyrosine phosphorylation of the IR (insulin receptor) beta-subunit in CHO/IR (Chinese-hamster ovary cells expressing human IR). Among the compounds tested, CG7 (ursolic acid) showed the greatest enhancement and CG11 (myrianthic acid) the least. We characterized the effect of CG7 further, and showed that it acted as an effective insulin-mimetic agent at doses above 50 mug/ml and as an insulin-sensitizer at doses as low as 1 mug/ml. Additional experiments showed that CG7 increased the number of IRs that were activated by insulin. This indicates that a major mechanism by which CG7 enhances total IR auto-phosphorylation is by promoting the tyrosine phosphorylation of additional IRs. CG7 not only potentiated insulin-mediated signalling (tyrosine phosphorylation of the IR beta-subunit, phosphorylation of Akt and glycogen synthase kinase-3beta), but also enhanced the effect of insulin on translocation of glucose transporter 4 in a classical insulin-sensitive cell line, 3T3-L1 adipocytes. The results of the present study demonstrate that a specific pentacyclic triterpenoid, CG7, exerts an insulin-sensitizing effect as an IR activator in CHO/IR cells and adipocytes. The enhancement of insulin activity by CG7 may be useful for developing a new class of specific IR activators for treatment of Type 1 and Type 2 diabetes.