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cbd oil for mitochondrial disease

Cbd oil for mitochondrial disease

For those that aren’t, below are the chemical structures of both the THC-A and THC molecules.

With that being the case, a question to ask is this: What impact do cannabis molecules have on mitochondria?

Although THC is the most psychoactive molecule found in Whole Plant Cannabis, it has many medical benefits, including treatment for pain, cancer, nausea, neuropathy and much more. By contrast, until a few years ago, very little was done with THC-A.

However, since THC-A is obviously “made” quite easily in nature, more recently we have been able to witness just how patients benefit that have a variety of disorders, including those with cancer, seizures, Myasthenia Gravis, and other neurological disorders and myopathies.

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Cbd oil for mitochondrial disease

A direct role for mitochondria in CBD signaling was confirmed here by the use of dual-loaded hippocampal neurons with Ca 2+ -sensitive probes for mitochondrial and cytosolic compartments, with changes in mitochondrial Ca 2+ levels preceding the rise of cytosolic Ca 2+ . Moreover, the depletion of Ca 2+ from mitochondria (using FCCP) resulted in the inability of CBD to yield any subsequent response. CBD’s point of action upon mitochondria was identified to be the NCX (and not the mPTP), as CBD responses were no longer present when this exchanger was blocked by CGP, yet unaffected by CsA. The NCX functions normally to remove Ca 2+ from the mitochondria but can reverse when the ionic gradients are sufficiently altered, especially in disease states (Jung et al., 1995; Griffiths, 1999; Poburko et al., 2006). A fundamental role for the mitochondrial NCX in Ca 2+ signaling associated with ischemia and excitotoxic events, where the influx of Na + into the cell causes release of Ca 2+ from mitochondria, has previously been identified in hippocampal tissue (Zhang and Lipton, 1999).

The plant Cannabis sativa has for many centuries been reputed to possess therapeutically relevant properties. Its most widely studied and characterized component, Δ 9 -tetrahydrocannabinol (THC), is one of 60+ compounds from Cannabis sativa, collectively known as phytocannabinoids. However, THC may have a limited usefulness due to psychoactivity, dependence, and tolerance (Sim-Selley and Martin, 2002); therefore, attention has turned to some of the nonpsychoactive phytocannabinoids, most notably cannabidiol (CBD). CBD has little agonistic activity at the known cannabinoid receptors (CB1 and CB2) (Pertwee, 2004), and may possess therapeutic potential, e.g., anti-epileptic (Cunha et al., 1980), anxiolytic (Guimarães et al., 1994), anti-inflammatory (Carrier et al., 2006), and even anti-psychotic properties (Leweke et al., 2000) [for review, see Pertwee (2004) and Drysdale and Platt (2003)]. In addition, CBD has shown neuroprotection in a range of in vivo (Lastres-Becker et al., 2005) and in vitro models (Esposito et al., 2006), some in association with a reduction in [Ca 2+ ]i (Iuvone et al., 2004).

SH-SY5Y cells were grown on 96-well plates and treated with FCCP overnight as described above. MitoCapture reagent (Calbiochem) was diluted 1:1000 in PBS (at room temperature) before use. The medium was removed from the wells of the plate and replaced with 50 μl of reagent solution and placed in an incubator (37°C at 5% CO2) for 15–20 min. The cells were then washed twice with PBS and run through the plate reader (Synergy HT, Bio-Tek) with two fluorescence channels measured (green monomers: excitation 488 nm and emission 530 nm; red aggregates: excitation 488 nm and emission 590 nm). Control and toxin groups were run in 6 samples (wells) per experiment and performed three times.

Mitochondrial and cytosolic CBD (1 μ m ) responses in naive cultures loaded with Rhod-FF, AM and fura-2 AM. A, B, Sample traces from neurons showing delayed cytosolic (black trace) and early biphasic mitochondrial (gray trace) Ca 2+ responses. NMDA application was used as an indicator of neuronal viability and to make a clear distinction between neurons and glia. C, Application of FCCP (1 μ m ) led to a drop in mitochondrial Ca 2+ levels and prevented a further Ca 2+ rise by CBD. Vertical lines have been added to visualize the order of responses. All Rhod-FF data are raw fluorescence values (OD, optic density), and fura-2 AM responses are presented as ratio values.

Enhanced excitability and epileptiform activity

CGP may act not only upon mitochondrial NCX, but also as an inhibitor of VGCCs in dorsal root ganglion neurons (Baron and Thayer, 1997). However, our previous data with VGCC blockers (Drysdale et al., 2006) are not consistent with an effect of CBD on this target. Others have found CGP to inhibit the NCX in the plasma membrane of cerebellar granule cells (Czyz and Kiedrowski, 2003), although with an IC50 of 13 μ m , a concentration higher than that used here, and higher than CGP’s IC50 (4 μ m ) for the mitochondrial NCX in cultured rat DRGs (Baron and Thayer, 1997). Indeed, the concentration of CGP used here is in keeping with recent work by others in cultured neurons (Medvedeva et al., 2008).

For calcium imaging experiments (see also Ryan et al., 2006), hippocampal cultures were washed with HBS (as above) at room temperature and loaded with the cell-permeable fluorescent calcium indicator fura-2 AM (10 μ m , Invitrogen) for 1 h in the dark. To allow the monitoring of postsynaptic events uncontaminated by spontaneous activity and transmitter release, the sodium channel blocker tetrodotoxin (TTX, 0.5 μ m , Alomone Labs) was added to all perfusion media (except in experiments with 4AP). Cultures were perfused with HBS or low-Mg 2+ (0.1 m m ) HBS, using a gravity perfusion system at a flow rate of 1–2 ml/min.

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 Ca 2+ homeostasis and mediates neuroprotection in neuronal preparations. Imaging studies in hippocampal cultures using fura-2 AM suggested that CBD-mediated Ca 2+ regulation is bidirectional, depending on the excitability of cells. Under physiological K + /Ca 2+ levels, CBD caused a subtle rise in [Ca 2+ ]i, whereas CBD reduced [Ca 2+ ]i and prevented Ca 2+ oscillations under high-excitability conditions (high K + or exposure to the K + channel antagonist 4AP). Regulation of [Ca 2+ ]i was not primarily mediated by interactions with ryanodine or IP3 receptors 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 [Ca 2+ ]i regulation. Application of carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP) and the mitochondrial Na + /Ca 2+ exchange inhibitor, CGP 37157, but not the mitochondrial permeability transition pore inhibitor cyclosporin A, prevented subsequent CBD-induced Ca 2+ responses. In established human neuroblastoma cell lines (SH-SY5Y) treated with mitochondrial toxins, CBD (0.1 and 1 μ m ) 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 Ca 2+ dysregulation, CBD may prove beneficial in preventing apoptotic signaling via a restoration of Ca 2+ homeostasis.


Cannabinoids as potential neuroprotectants against mitochondrial stressors in SH-SY5Y cells. Data are expressed as percentage protection (+SEM) relative to within-experiment controls and shown for peroxide (0.1 m m ) (A) and oligomycin (20 μ m ) (B) toxicity. CBD conferred protection against oligomycin toxicity in both SH-SY5Y cells and hippocampal cultures (HIPP.). Pre-Inc., Following 1 h preincubation; *p < 0.05, ***p < 0.001.

The apparent mitochondrial site of action of CBD led to the hypothesis that CBD may act as a neuroprotectant against mitochondrially acting toxins, acting either directly on mitochondrial sites or downstream thereof ( Fig. 1 ). Initial tests used the mitochondria-reliant viability assay Alamar Blue in SH-SY5Y cells, with protective actions of CBD confirmed in hippocampal cultures using a live–dead stain ( Fig. 7 ).