Aging Rewires Neuronal Metabolism, Exacerbating Cell Death After Ischemic Stroke: A Hidden Reason for the Failure of Neuroprotection

Aging profoundly modifies neuronal responses to ischemia. We aimed to define age-dependent features of neuronal metabolism and cell death after ischemic stroke by assessing NeuN, NSE, and Caspase-3 in human cortical neurons and by comparing transcriptional activity within PI3K/Akt/mTOR and PI3K/Akt/FOXO3a pathways across age groups. The aim of this study was to determine age-dependent features of neuronal metabolism and cellular degradation in ischemic stroke based on immunohistochemical assessment of NeuN, NSE, and Caspase-3 markers in human cerebral cortex neurons, as well as to conduct a comparative analysis of gene expression in the PI3K/Akt/mTOR and PI3K/Akt/FOXO3a signaling pathways involved in the regulation of neuronal survival and apoptosis. For the investigation, frontal cortex autopsies from patients with ischemic stroke (n = 154; “young”, “middle” and “elderly”; death ≤7 days post-onset) were examined. Histology (hematoxylin–eosin) and Nissl staining were used for morphology and neuron counts. Multiplex immunofluorescence (NeuN, NSE, Caspase-3) quantified metabolically active and apoptotic neurons, and the percentage of Caspase-3+ among NeuN+ cells was calculated. qRT-PCR measured PIK3CA, AKT2, MTOR, and FOXO3A expression in the infarct border zone. Based on our results, neuronal density and NeuN/NSE expression declined with aging, and the fraction of Caspase-3+ among NeuN+ neurons in the penumbra rose (young 42%, middle 82%, elderly 89%). Morphologically “intact” penumbral neurons frequently lacked NeuN/NSE, revealing covert dysfunction. Young brains showed balanced activation of PI3K/Akt/mTOR and PI3K/Akt/FOXO3a, whereas elderly brains exhibited reduced Akt/mTOR activity with FOXO3A predominance, consistent with pro-apoptotic, inflammatory, and dysregulated autophagic signaling. Thus, aging markedly reduces neuronal metabolic activity and increases apoptotic death in the infarct border zone after ischemic stroke. In older patients, there is an almost complete loss of NeuN and NSE expression in penumbral neurons with robust activation of the caspase cascade, whereas younger patients retain a pool of metabolically active neurons. Age-dependent dysregulation of PI3K/Akt signaling—characterized by FOXO3a hyperactivation and mTOR suppression—further promotes apoptosis and dysregulated autophagy. These changes likely underlie the limited efficacy of standard neuroprotection in ischemic stroke and support the need for age-tailored neurotropic therapy aimed at enhancing pro-survival pathways within the infarct border zone. © 2025 by the authors.