Synaptosomes isolated from cryopreserved MND motor cortex reveal altered calcium handling and reduced complex IV–linked respiration

Motor neuron disease (MND) is marked by progressive neurodegeneration in which presynaptic Ca²⁺-handling and mitochondrial metabolism are thought to be vulnerable, but direct functional studies in human brain are scarce because most material is frozen long-term. Here, we show that synaptosomes isolated from paired fresh and experimentally frozen mouse cortex, and from cryopreserved human motor cortex, retain recognisable synaptosome ultrastructural features, synaptic proteome enrichment, and depolarisation-evoked Ca²⁺-mobilisation. K⁺ and veratridine elicited robust, pharmacologically suppressible Ca²⁺ influx across preparations, and response amplitudes in human samples varied by region but did not correlate with donor age, post-mortem interval (PMI), or years in storage. Synaptosomes from neuropathologically confirmed MND motor cortex and hSOD1^G93A mouse cortex showed significantly greater depolarisation-evoked Ca²⁺ entry than their respective controls, suggesting that increased presynaptic Ca²⁺ influx is shared across our human MND cohort and the hSOD1^G93A mouse model. Using synaptosome preparations from MND and control motor cortices in Seahorse respiratory assays, we found that Complex IV-driven oxygen consumption (TMPD/ascorbate–evoked and azide-sensitive) was reduced in MND synaptosomes, whereas donor-matched free-mitochondrial fractions showed no group difference, supporting a Complex IV defect detectable in the synaptosome-enriched fraction within this cohort. By defining protein-to-OCR relationships for both fractions, we provide practical parameters for applying these assays to archived human cohorts. Together, these data suggest that archived cryopreserved human brain tissues can support informative synaptosome Ca²⁺ and bioenergetic readouts, and that synaptosome-enriched preparations may reveal disease-relevant presynaptic phenotypes in MND that are not evident in donor-matched bulk mitochondrial isolates.