Age and Ketogenic Diet Have Dissociable Effects on Synapse-Related Gene Expression Between Hippocampal Subregions

Abstract

As the number of individuals living beyond the age of 65 is rapidly increasing, so is the need to develop strategies to combat the age-related cognitive decline that may threaten independent living. Although the link between altered neuronal signaling and age-related cognitive impairments is not completely understood, it is evident that declining cognitive abilities are at least partially due to synaptic dysfunction. Aging is accompanied by well-documented changes in both excitatory and inhibitory synaptic signaling across species. Age-related synaptic alterations are not uniform across the brain, however, with different regions showing unique patterns of vulnerability in advanced age. In the hippocampus, increased activity within the CA3 subregion has been observed across species, and this can be reversed with anti-epileptic medication. In contrast to CA3, the dentate gyrus shows reduced activity with age and declining metabolic activity. Ketogenic diets have been shown to decrease seizure incidence and severity in epilepsy, improve metabolic function in diabetes type II, and improve cognitive function in aged rats. This link between neuronal activity and metabolism suggests that metabolic interventions may be able to ameliorate synaptic signaling deficits accompanying advanced age. We therefore investigated the ability of a dietary regimen capable of inducing nutritional ketosis and improving cognition to alter synapse-related gene expression across the dentate gyrus, CA3 and CA1 subregions of the hippocampus. Following 12 weeks of a ketogenic or calorie-matched standard diet, RTq-PCR was used to quantify expression levels of excitatory and inhibitory synaptic signaling genes within CA1, CA3 and dentate gyrus. While there were no age or diet-related changes in CA1 gene expression, expression levels were significantly altered within CA3 by age and within the dentate gyrus by diet for several genes involved in presynaptic glutamate regulation and postsynaptic excitation and plasticity. These data demonstrate subregion-specific alterations in synaptic signaling with age and the potential for a ketogenic diet to alter these processes in dissociable ways across different brain structures that are uniquely vulnerable in older animals.

Keywords: CA1; CA3; GABA; ROCK; dentate gyrus; glutamate; metabolism.

FIGURE 1

The ketogenic diet-fed rats had significantly higher (A) BHB, lower (B) glucose and lower (C) GKI relative to standard diet-fed rats, regardless of age group. Boxes represent the upper and lower quartiles around the median, the whiskers are the minimum and maximum observed values and + indicates the group mean. ^∗ = main effect of diet.

FIGURE 2

Cycle threshold (C_t) for housekeeping gene RPLP1 across age and diet groups in all hippocampal subregions examined. In the (A) dentate gyrus (DG), (B) CA3, and (C) CA1 subregions, the C_t did not vary across age and diet groups, nor was there a significant interaction. Thus, RPLP1 was utilized as a housekeeping gene to normalize the quantification of RNA levels for all other genes of interest. Boxes represent the upper and lower quartiles around the median, the whiskers are the minimum and maximum observed values and + indicates the group mean.

FIGURE 3

Genes affected by diet in the dentate gyrus. The heat map represents mean z-scores of all genes that were significantly reduced (FDR q = 0.005 threshold) as a function of diet. Genes were categorized into biologically relevant processes, and experimental groups were ordered to reflect the main effect of diet. Yellow represents greater gene expression, whereas blue represents lower gene expression. Black represents no change.

FIGURE 4

Genes within the ionotropic glutamate receptor (iGluR) pathway in the DG reliably changed with diet. (A) 8 genes associated with presynaptic glutamate regulation were reduced as a function of diet. (B) 6 genes associated with postsynaptic excitation were reduced as a function of diet. (C) 7 genes associated with postsynaptic plasticity were reduced as a function of diet. Black bars represent young; gray bars represent aged; open bars represent standard diet; and filled bars represent ketogenic diet. Boxes represent the upper and lower quartiles around the median, the whiskers are the minimum and maximum observed values and +indicates the group mean.

FIGURE 5

Genes affected by age in CA3. The heat map represents mean z-scores of all genes that were significantly reduced (FDR q = 0.005 threshold) as a function of age. Genes were categorized into biologically relevant processes, and experimental groups were ordered to reflect the main effect of age. Yellow represents greater gene expression, whereas blue represents lower gene expression. Black represents no change.

FIGURE 6

Genes within the ionotropic glutamate receptor (iGluR) pathway reliably changed with age within CA3. (A) 4 genes associated with presynaptic glutamate regulation were reduced as a function of age. (B) 4 genes associated with postsynaptic excitation were reduced as a function of age. (C) 4 genes associated with postsynaptic plasticity were reduced as a function of age. Black bars represent young; gray bars represent aged; open bars represent standard diet; and filled bars represent ketogenic diet. Boxes represent the upper and lower quartiles around the median, the whiskers are the minimum and maximum observed values and +indicates the group mean.

FIGURE 7

Hippocampal ROCK2 protein expression. (A) Aged rats demonstrated significantly higher levels of ROCK2 expression in the hippocampus relative to young standard-fed (SD) rats, but levels were restored to that of the young SD-fed rats in ketogenic-fed (KD) aged subjects. (B) Representative ROCK2 bands (top), which were then normalized to total protein stains (bottom) to account for variability in total protein loading. Note total protein was less in aged compared to young animals. Boxes represent the upper and lower quartiles around the median, the whiskers are the minimum and maximum observed values and + indicates the group mean. All values are expressed as percent of young SD-fed controls (dotted line). ^∗ indicates a significant effect of age across standard-fed subjects and ^∗∗ indicates a significant effect of diet across aged subjects.

FIGURE 8

Model of gene contributions to significant ionotropic glutamate receptor (glutamatergic synapse; KEGG) pathway enrichment as determined by DAVID for (A) inputs to the DG, (B) projections from DG to CA3, and (C) projections from CA3 to CA1. Significant reductions in receptors/genes by diet (red) and age (blue) are indicated by color.