Exposure-dependent alterations in hippocampal cell-death-associated markers following repeated methamphetamine...

Methamphetamine (METH) abuse can cause persistent cognitive impairment and neuronal injury, but exposure-dependent alterations in cell-death-associated markers during repeated exposure remain incompletely characterized. We combined behavioral testing, exposure-dependent molecular profiling, transmission electron microscopy, and terminal hippocampal RNA sequencing to characterize METH-induced hippocampal injury. Repeated METH exposure impaired spatial learning and memory in mice. qPCR and Western blot analyses from animals sampled across five days of repeated METH exposure revealed that changes in autophagy-associated markers were most prominent following a single day of exposure, whereas changes in apoptosis- and pyroptosis-associated markers became more pronounced after three or more days of cumulative exposure. GPX4, FDX1, ATP7A/B, and lipoylated DLAT also showed exposure-dependent alterations, although these marker alterations did not establish the execution of ferroptosis or cuproptosis. Transmission electron microscopy revealed chromatin condensation and margination, double-membrane vacuolar structures, and mitochondrial swelling, cristae disruption, or increased electron density. These features were compatible with apoptotic stress, autophagic processing, and mitochondrial injury but were not specific to individual cell-death modalities. Terminal RNA sequencing identified 517 candidate differentially expressed genes, including 289 upregulated and 228 downregulated genes. Functional enrichment analysis primarily highlighted antigen processing and presentation, endoplasmic-reticulum-associated compartments, extracellular matrix organization, and transmembrane transport. Sample-level single-sample gene set enrichment analysis (ssGSEA) identified no significant differences among five cell-death programs after multiple-testing correction. While sample-level ssGSEA did not indicate activation of any specific cell-death program, a global enrichment analysis (GSEA) revealed a coordinated transcriptional shift in genes associated with autophagy and apoptosis. This suggests that the transcriptomic landscape is permissive to or co-regulated with these processes, but not that the programs themselves are functionally active. Pyroptosis, ferroptosis, and necrosis gene sets showed no significant enrichment. Together, these findings document parallel exposure-dependent marker alterations, mitochondrial ultrastructural injury, and immune- and stress-related transcriptional remodeling following repeated METH exposure across the experimental cohorts. The results provide an observational correlative framework for future causal studies aiming to dissect METH-induced hippocampal neurotoxicity.