Alterations in markers of mitochondrial content with ketogenic diets (KD) have been reported in tissues of rodents, but morphological quantification of mitochondrial mass using transmission electron microscopy (TEM), the gold standard for mitochondrial quantification, is needed to further validate these findings and look at specific regions of interest within a tissue. In this study, red gastrocnemius muscle, the prefrontal cortex, the hippocampus, and the liver left lobe were used to investigate the impact of a 1-month KD on mitochondrial content in healthy middle-aged mice. The results showed that in red gastrocnemius muscle, the fractional area of both subsarcolemmal (SSM) and intermyofibrillar (IMM) mitochondria was increased, and this was driven by an increase in the number of mitochondria. Mitochondrial fractional area or number was not altered in the liver, prefrontal cortex, or hippocampus following 1 month of a KD. These results demonstrate tissue-specific changes in mitochondrial mass with a short-term KD and highlight the need to study different muscle groups or tissue regions with TEM to thoroughly determine the effects of a KD on mitochondrial mass.

The ε4 allele of apolipoprotein E (APOE4) is the strongest genetic risk factor for Alzheimer’s disease (AD), increasing AD risk about fourfold in ~ 34 million American and ~ 75 million European females. APOE4 carriers exhibit cerebral metabolic deficits decades before clinical onset. We previously demonstrated that ketogenic diet (KD), a low-carbohydrate, high-fat diet promoting ketone metabolism, confers cognitive benefits in aged C57BL/6 mice, and in the PS1/APP mouse model of early-onset AD. Here, we evaluated the effects of KD in a humanized APOE4 AD mouse model. KD significantly improved composite cognitive performance and spatial working memory, with pronounced effects in females. Synaptic plasticity, measured via long-term potentiation (LTP), was likewise enhanced exclusively in females. Transcriptomic and protein analyses revealed KD-induced activation of CREB pathway, marked by increased phosphorylation of ERK and CREB in female brains. Moreover, KD selectively reduced pro-inflammatory cytokine levels in females. These findings demonstrate sex-specific neuroprotective effects of KD in APOE4 mice and suggest its potential therapeutic role in mitigating AD risk in APOE4-positive women. Graphical Abstract