Cannabinoids and the endocannabinoid system have attracted considerable interest for therapeutic applications. Nevertheless, the mechanism of action of one of the main nonpsychoactive phytocannabinoids, cannabidiol (CBD), remains elusive despite potentially beneficial properties as an anti-convulsant and neuroprotectant. Here, we characterize the mechanisms by which CBD regulates Ca²⁺homeostasis and mediates neuroprotection in neuronal preparations. Imaging studies in hippocampal cultures using fura-2 AM suggested that CBD-mediated Ca²⁺regulation is bidirectional, depending on the excitability of cells. Under physiological K⁺/Ca²⁺levels, CBD caused a subtle rise in [Ca²⁺]_i, whereas CBD reduced [Ca²⁺]_iand prevented Ca²⁺oscillations under high-excitability conditions (high K⁺or exposure to the K⁺channel antagonist 4AP). Regulation of [Ca²⁺]_iwas not primarily mediated by interactions with ryanodine or IP₃receptors of the endoplasmic reticulum. Instead, dual-calcium imaging experiments with a cytosolic (fura-2 AM) and a mitochondrial (Rhod-FF, AM) fluorophore implied that mitochondria act as sinks and sources for CBD's [Ca²⁺]_iregulation. Application of carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP) and the mitochondrial Na⁺/Ca²⁺exchange inhibitor, CGP 37157, but not the mitochondrial permeability transition pore inhibitor cyclosporin A, prevented subsequent CBD-induced Ca²⁺responses. In established human neuroblastoma cell lines (SH-SY5Y) treated with mitochondrial toxins, CBD (0.1 and 1 μm) was neuroprotective against the uncoupler FCCP (53% protection), and modestly protective against hydrogen peroxide- (16%) and oligomycin- (15%) mediated cell death, a pattern also confirmed in cultured hippocampal neurons. Thus, under pathological conditions involving mitochondrial dysfunction and Ca²⁺dysregulation, CBD may prove beneficial in preventing apoptotic signaling via a restoration of Ca²⁺homeostasis.

Post-mortem investigations of human Alzheimer’s disease (AD) have largely failed to provide unequivocal evidence in support of the original amyloid cascade hypothesis, which postulated deposition of β-amyloid (Aβ) aggregates to be the cause of a demented state as well as inductive to tau neurofibrillary tangles (NFTs). Conflicting evidence suggests, however, that Aβ plaques and NFTs, albeit to a lesser extent, are present in a substantial subset of non-demented individuals. Hence, a range of soluble tau and Aβ species has more recently been implicated as the disease-relevant toxic entities. Despite the incorporation of soluble proteins into a revised amyloid cascade hypothesis, a detailed characterization of these species in the context of human AD onset, progression and cognitive decline has been lacking. Here, lateral temporal lobe samples (Brodmann area 21) of 46 human cases were profiled via tau and Aβ Western blot and native state dot blot protocols. Elevations in phospho-tau (antibodies: CP13, AT8 and PHF-1), pathological tau conformations (MC-1) and oligomeric tau (TOC1) agreed with medical diagnosis (non-AD cf. AD) and Braak stage classification (low, intermediate and high), alongside elevations in soluble Aβ species (MOAB-2 and pyro-glu Aβ) and a decline in levels of the amyloid precursor protein. Strong correlations were observed between individual Braak stages and multiple cognitive measures with all tau markers as well as total soluble Aβ. In contrast to previous reports, SDS-stable Aβ oligomers (*56) were not found to be reliable for all classifications and appeared likely to be a technical artefact. Critically, the robust predictive value of total soluble Aβ was dependent on native state quantification. Elevations in tau and Aβ within soluble fractions (Braak stage 2–3 cf. 0) were evident earlier than previously established in fibril-focused disease progression scales. Together, these data provide strong evidence that soluble forms of tau and Aβ co-localise early in AD and are closely linked to disease progression and cognitive decline.