Biology subjects

Gallardo, M. D.

Publications and source records attributed to Gallardo, M. D..

3 recordsLinked to original sources

bioRxiv2023

COGNITIVE REJUVENATION IN OLD RATS BY HIPPOCAMPAL OSKM GENE THERAPY

Impaired performance in spatial learning and memory during aging in rats is associated with morphological and molecular changes in the brain, particularly in the hippocampus. Here, we assessed the cognitive performance of young (3.5 mo.) untreated rats and old (25.3 mo.) treated and control rats. Treatment was carried out by intrahippocampal injection of an adenovector that carries the GFP reporter gene as well as the 4 Yamanaka genes. Learning and spatial memory performance were assessed by means of the Barnes maze test. The learning performance of the OSKM-treated old rats was significantly improved compared to that of the control old counterparts. A marginal (P=0.06) improvement in the spatial memory was recorded in the treated versus control old rats. OSKM gene expression induced no pathological changes in the brain. The morphology and number of hippocampal cell populations like astrocytes and mature neurons did not show any changes with the treatment in the old rats as compared with the control old counterparts. The rat pan tissue DNAm age marker revealed that old OSKM gene-treated rats show a trend towards a decrease in epigenetic age. The Limma package was used to assess differential methylation by fitting linear models to the methylation data for specific group comparisons. Comparison of differential methylation between old treated and old control hippocampal DNA samples identified 671 differentially methylated CpGs probes (DPMs) in the DNA of OSKM-treated hippocampi (p<0.05). Assessment of the DPMs in old versus young controls revealed the presence of 1,279 hypomethylated CpGs near the promoter regions in young hippocampi (versus old controls) and 914 hypermethylated CpGs near the promoter in young hippocampi compared to old control hippocampi. We found a subset of 174 hypomethylated CpGs in the hippocampal DNA from old OSKM rats and young controls both compared with old control hippocampi. This means that in the hippocampal DNA there is a common set of CpGs which are hypermethylated during aging and are demethylated by the OSKM genes. This observation suggested that in these 174 CpGs the hypermethylation induced by aging is reversed by the demethylation effect of the OSKM genes on the same 174 CpGs. This observation can be interpreted as a rejuvenation effect of the OSKM genes of the old hippocampal methylome. Our results extend to the rat the evidence that viral vector-mediated delivery of the Yamanaka genes in the brain has strong regenerative effects without adverse side effects.

neuroscience↗

bioRxiv2023

REGENERATIVE GENE THERAPY IN THE HYPOTHALAMUS PROLONGS FERTILITY IN FEMALE RATS

There is substantial evidence that age-related ovarian failure in rats is preceded by abnormal responsiveness of the neuroendocrine axis to estrogen positive feedback. In middle-aged (M-A) female rats, we have demonstrated that intrahypothalamic gene therapy for insulin-like growth factor-I (IGF-I) started at 6 months of age extends the regular cyclicity of the animals beyond 10 month (the age at which MA rats stop ovulating) and preserves the integrity of the ovarian structure. Here, we implemented long-term regenerative gene therapy in the hypothalamus of young females. The goal was to extend fertility in the treated animals. We constructed a helper-dependent adenovector that harbors the green fluorescent protein (GFP) reporter gene as well as a gene tandem, termed STEMCCA, which harbors the 4 Yamanaka genes (oct4, sox2, klf4, and c-myc, OSKM), both under the control of a Tet-Off bidirectional promoter. An adenovector that only carries the gene for GFP was used as control. At 4 months of age 12 female rats received an intrahypothalamic injection of our OSKM-GFP vector (treated rats); 12 control rats a vector expressing GFP only (control rats). At 9.3 months of age control and treated rats were mated with young males. A group of 12 young intact female rats was also mated. The rate of pregnancy recorded was 83%, 8.3% and 25% for young, M-A control and M-A treated animals, respectively. Average litter size was 9, 3 and 3 for the corresponding groups. Mean pup BW was slightly higher in the M-A rats. In a preliminary study we confirmed that ovulation in our M-A females ceases at 10 months of age. Our results are in line with the evidence that viral vector-mediated delivery of the Yamanaka genes in the brain has strong regenerative effects without adverse side effects. The particular significance of the present results is that, for the first time, they show that long-term OSKM gene therapy in the hypothalamus is able to extend the functionality of such a complex system as the hypothalamo-pituitary-ovarian axis.

neuroscience↗

bioRxiv2022

YOUNG PLASMA REJUVENATES BLOOD DNA METHYLATION PROFILE, PROLONGS MEAN LIFESPAN AND IMPROVES HEALTH IN OLD RATS

There is converging evidence that young blood conveys cells, vesicles and molecules able to revitalize function and restore organ integrity in old individuals. Here, we assessed the effects of young rat plasma on the lifespan, epigenetic age and healthspan of old female rats. Beginning at 25.3 months of age, a group of 9 rats (group T) was intraperitoneally injected with plasma from young rats (2 months) until their natural death. A group of control rats of the same age, received no treatment. Blood samples were collected every other week. Survival curves showed that from age 26 to 30 months, none of the T animals died, whereas the survival curve of C rats began to decline at age 26 months. The external appearance of the T rats was healthier than that of the C counterparts. Blood DNA methylation (DNAm) was assessed using the HorvathMammalMethylChip320. Blood DNAm age versus chronological age showed that DNAm age in young animals increased faster than chronological age then slowed down progressively, entering a plateau after 27 months. Immediately after the start of the treatment, the DNAm age (i.e., epigenetic age) of the treated rats fell below the DNAm age of controls and remained consistently lower until the end of their lives. Assessment of each experimental group showed that the blood DNA methylation levels of 1638 CpGs were different between treated and control blood samples (false discovery rate q-value<0.05). Of these, 1007 CpGs exhibited increased methylation, with age while 631 CpGs showed decreased methylation levels. When rats were grouped according to the similarities in their differential blood DNA methylation profile, samples from the treated and control rats clustered in separate groups. Analysis of promoter differential methylation in genes involved in systemic regulatory activities revealed specific GO term enrichment related to the insulin-like factors (IGFs) pathways as well as to cytokines and chemokines associated with immune and homeostatic functions. We conclude that young plasma therapy may constitute a natural noninvasive intervention for epigenetic rejuvenation and health enhancement, readily translatable to the clinic.

bioinformatics↗