Caloric restriction reinforces the stem cell pool in the aged brain without affecting overall proliferation status.

Overfeeding (OF) and obesity increase the risk for brain aging and neurodegenerative diseases due to increased oxidative stress and neuroinflammation, which likely contribute to cellular dysfunction. In contrast, caloric restriction (CR) is an intervention known for its effects on extending both life- and health-span. In the current study, the effects on the aging brain of two short-term feeding regimens, OF and CR, were investigated. We applied these diets for 12 weeks to both young and aged zebrafish. We performed protein and mRNA level analysis to examine diet-mediated effects on any potential age-related alterations in the brain. Markers implicated in the regulation of brain aging, cell cycle, proliferation, inflammation, and cytoskeleton were analyzed. The most prominent result observed was a downregulation in the expression levels of the stem cell marker, Sox2, in CR-fed animals as compared to OF-fed fish. Furthermore, our data highlighted significant age-related downregulations in Tp53, Myca, and L-plastin levels. The multivariate analyses of all datasets suggested that as opposed to OF, the adaptive mechanisms increasing lifespan via CR are likely exerting their effects by reinforcing the stem cell pool and downregulating inflammation. The data reveal important therapeutic targets with respect to the state of nutrient uptake for the slowing down of the detrimental effects of aging, resulting in a healthy and extended lifespan, as well as lowering the risk for neurodegenerative disease.

TERT distal promoter GC islands are critical for telomerase and together with DNMT3B silencing may serve as a senescence-inducing agent in gliomas.

Telomerase is reactivated in the majority of cancers. For instance, in gliomas, it is common that the TERT promoter is mutated. Research on telomere promoter GC islands have been focused primarily on proximal TERT promoter but little is known about the distal promoter. Therefore, in this study, we investigated the proximal and distal TERT promoter, in terms of DNA methylation. We did bisulfite sequencing in zebrafish tissue samples for the distal tert promoter. In the zebrafish brain tissues, we identified a hypomethylation site in the tert promoter, and found that this hypomethylation was associated with aging and shortened telomeres. Through site directed mutagenesis in glioma cell lines, we changed 10 GC spots individually, cloned into a reporter vector, and measured promoter activity. Finally, we silenced DNMT3B and measured telomerase activity along with vidaza and adriamycin treatments. Site directed mutagenesis of glioma cell lines revealed that each of the 10 GC spots are critical for telomerase activity. Changing GC to AT abolished promoter activity in all spots when transfected into glioma cell lines. Then, through silencing of DNMT3B, we observed a reduction in hTERT expression levels, while hTR remained the same, and a major increase in senescence-associated beta-galactosidase activity. Finally, we propose a model regarding the efficacy of two chemotherapeutic drugs, adriamycin and azacytidine, on gliomas. Here, we show that distal TERT promoter is critical; changing even one GC to AT abolishes TERT promoter activity. DNMT3B, a de novo methyltransferase, together with GC islands in distal TERT promoter plays an important role in regulation of telomerase expression and senescence.

Effects of caloric restriction on the antagonistic and integrative hallmarks of aging.

Aging is a significant risk factor for cognitive decline associated with neurodegenerative diseases, which makes understanding what promotes 'healthy brain aging' very important. Studies suggest that caloric restriction (CR) is a non-genetic intervention that reliably extends life- and healthspan. Here, we review the CR literature related to both the subject of aging and alterations in cell cycle machinery, especially surrounding the regulation of the E2F/DP1 complex, to elucidate the cellular protection mechanisms in the brain induced via dietary applications. The alterations extending lifespan via CR appear to exert their effects by promoting survival of individual cells, downregulating cell proliferation, and inducing stem cell quiescence, which results in keeping the stem cell reserve for extreme needs. This survival instinct of cells is believed to cause some molecular adaptations for their maintenance of the system. Avoiding energy waste of proliferation machinery promotes the long term survival of the individual cells and this is due to adaptations to the limited nutrient supply in the environment. Such a protective mechanism induced by diet could be promoted via the downregulation of crucial cell cycle-related transcription activators. This review article aims to bring attention to the importance of molecular adaptations induced by diet that promote healthy brain aging. It will provide insights into alternative targets for new treatments or neuroprotective approaches against neurodegenerative pathophysiologies.

Zebrafish brain RNA sequencing reveals that cell adhesion molecules are critical in brain aging.

Brain aging is a complex process, which involves multiple pathways including various components from cellular to molecular. This study aimed to investigate the gene expression changes in zebrafish brains through young-adult to adult, and adult to old age. RNA sequencing was performed on isolated neuronal cells from zebrafish brains. The cells were enriched in progenitor cell markers, which are known to diminish throughout the aging process. We found 176 statistically significant, differentially expressed genes among the groups, and identified a group of genes based on gene ontology descriptions, which were classified as cell adhesion molecules. The relevance of these genes was further tested in another set of zebrafish brains, human healthy, and Alzheimer's disease brain samples, as well as in Allen Brain Atlas data. We observed that the expression change of 2 genes, GJC2 and ALCAM, during the aging process was consistent in all experimental sets. Our findings provide a new set of markers for healthy brain aging and suggest new targets for therapeutic approaches to neurodegenerative diseases.

Short-term dietary restriction in old zebrafish changes cell senescence mechanisms.

Brain aging is marked by a decline in cognitive abilities and associated with neurodegenerative disorders. Recent studies have shown, neurogenesis continues into adulthood but is known to be decreasing during advancing age and these changes may contribute to cognitive alterations. Advances, which aim to promote better aging are of paramount importance. Dietary restriction (DR) is the only non-genetic intervention that reliably extends life- and health-span. Mechanisms of how and why DR and age affect neurogenesis are not well-understood, and have not been utilized much in the zebrafish, which has become a popular model to study brain aging and neurodegenerative disease due to widely available genetic tools. In this study we used young (8-8.5months) and old (26-32.5months) zebrafish as the model to investigate the effects of a short-term DR on actively proliferating cells. We successfully applied a 10-week DR to young and old fish, which resulted in a significant loss of body weight in both groups with no effect on normal age-related changes in body growth. We found that age decreased cell proliferation and increased senescence associated ß-galactosidase, as well as shortened telomere lengths. In contrast, DR shortened telomere lengths only in young animals. Neither age nor DR changed the differentiation patterns of glial cells. Our results suggest that the potential effects of DR could be mediated by telomere regulation and whether these are beneficial or negative remains to be determined.

Mild expression differences of MECP2 influencing aggressive social behavior.

The X-chromosomal MECP2/Mecp2 gene encodes methyl-CpG-binding protein 2, a transcriptional activator and repressor regulating many other genes. We discovered in male FVB/N mice that mild (~50%) transgenic overexpression of Mecp2 enhances aggression. Surprisingly, when the same transgene was expressed in C57BL/6N mice, transgenics showed reduced aggression and social interaction. This suggests that Mecp2 modulates aggressive social behavior. To test this hypothesis in humans, we performed a phenotype-based genetic association study (PGAS) in >1000 schizophrenic individuals. We found MECP2 SNPs rs2239464 (G/A) and rs2734647 (C/T; 3'UTR) associated with aggression, with the G and C carriers, respectively, being more aggressive. This finding was replicated in an independent schizophrenia cohort. Allele-specific MECP2 mRNA expression differs in peripheral blood mononuclear cells by ~50% (rs2734647: C > T). Notably, the brain-expressed, species-conserved miR-511 binds to MECP2 3'UTR only in T carriers, thereby suppressing gene expression. To conclude, subtle MECP2/Mecp2 expression alterations impact aggression. While the mouse data provides evidence of an interaction between genetic background and mild Mecp2 overexpression, the human data convey means by which genetic variation affects MECP2 expression and behavior.