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. 2017 Dec;174(23):4263-4276.
doi: 10.1111/bph.14019. Epub 2017 Nov 2.

Tetrahydrocannabinolic Acid Is a Potent PPARγ Agonist With Neuroprotective Activity

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Free PMC article

Tetrahydrocannabinolic Acid Is a Potent PPARγ Agonist With Neuroprotective Activity

Xavier Nadal et al. Br J Pharmacol. .
Free PMC article

Abstract

Background and purpose: Phytocannabinoids are produced in Cannabis sativa L. in acidic form and are decarboxylated upon heating, processing and storage. While the biological effects of decarboxylated cannabinoids such as Δ9 -tetrahydrocannabinol have been extensively investigated, the bioactivity of Δ9 -tetahydrocannabinol acid (Δ9 -THCA) is largely unknown, despite its occurrence in different Cannabis preparations. Here we have assessed possible neuroprotective actions of Δ9 -THCA through modulation of PPARγ pathways.

Experimental approach: The effects of six phytocannabinoids on PPARγ binding and transcriptional activity were investigated. The effect of Δ9 -THCA on mitochondrial biogenesis and PPARγ coactivator 1-α expression was investigated in Neuro-2a (N2a) cells. The neuroprotective effect was analysed in STHdhQ111/Q111 cells expressing a mutated form of the huntingtin protein and in N2a cells infected with an adenovirus carrying human huntingtin containing 94 polyQ repeats (mHtt-q94). The in vivo neuroprotective activity of Δ9 -THCA was investigated in mice intoxicated with the mitochondrial toxin 3-nitropropionic acid (3-NPA).

Key results: Cannabinoid acids bind and activate PPARγ with higher potency than their decarboxylated products. Δ9 -THCA increased mitochondrial mass in neuroblastoma N2a cells and prevented cytotoxicity induced by serum deprivation in STHdhQ111/Q111 cells and by mutHtt-q94 in N2a cells. Δ9 -THCA, through a PPARγ-dependent pathway, was neuroprotective in mice treated with 3-NPA, improving motor deficits and preventing striatal degeneration. In addition, Δ9 -THCA attenuated microgliosis, astrogliosis and up-regulation of proinflammatory markers induced by 3-NPA.

Conclusions and implications: Δ9 -THCA shows potent neuroprotective activity, which is worth considering for the treatment of Huntington's disease and possibly other neurodegenerative and neuroinflammatory diseases.

Figures

Figure 1
Figure 1
The carboxylic acid group of phytocannabinoids is critical for enhanced PPARγ binding. Cannabinoid binding affinities were tested at the indicated concentrations and compared with the binding affinity of rosiglitazone (RGZ). Data were transformed to a logarithmic function, and the K i values were calculated and are shown in the Figure (n = 5).
Figure 2
Figure 2
Cannabinoid acids induce PPARγ transcriptional activity and PPARγ degradation. (A–C) HEK‐293T cells were transiently transfected with PPARγ‐GAL4 plus GAL4‐luc and incubated with increasing concentrations of the indicated neutral and cannabinoid acids for 6 h (n = 5). (D) Transfected HEK‐293T cells were stimulated with two phytoextracts derived from the Cannabis variety MONIEK before and after decarboxylation (n = 5). (E) STHdh Q7/Q7 cells were treated with Δ9‐THCA, Δ9‐THC and rosiglitazone (RGZ) for 6 h, and the steady‐state levels of endogenous PPARγ and β‐actin detected by Western blots (n = 5). (F) STHdh Q7/Q7 cells were transiently transfected with PPARγ‐GAL4 plus GAL4‐luc and incubated with increasing concentrations of Δ9‐THCA or Δ9‐THC for 6 h (n = 5). * P < 0.05, significantly different from untreated cells.
Figure 3
Figure 3
Δ9‐THCA competes with rosiglitazone and activates PPARγ in a reversible manner. HEK‐293T cells were transiently transfected with PPARγ‐GAL4 plus GAL4‐luc. (A) Cells were pretreated with Δ9‐THCA for 1 h and then incubated for 6 h in the presence or absence of rosiglitazone (RGZ) (n = 5). (B) Cells were pretreated with Δ9‐THCA for 1 h and then washed or not with PBS and incubated in complete medium for 6 h (n = 5). Cells were lysed and tested for luciferase activity. * P < 0.05, significantly different from untreated cells # P < 0.05, significantly different from rosiglitazone‐treated cells.
Figure 4
Figure 4
Δ9‐THCA increases mitochondrial biogenesis in N2a cells. (A) The cells were treated with Δ9‐THCA, Δ9‐THC and rosiglitazone (RGZ) for 72 h, and mitochondria stained with the Mitotracker Green dye (n = 5). (B) Quantification of fluorescence intensity (100% = control untreated cells). (C) Δ9‐THCA up‐regulated the expression of PGC‐1α. N2a cells were stimulated with rosiglitazone, Δ9‐THCA or Δ9‐THC, and the levels of PGC‐1α mRNA were analysed by qPCR (n = 5). * P < 0.05, significantly different from control.
Figure 5
Figure 5
Δ9‐THCA prevents mutated huntingtin‐induced cytotoxicity via PPARγ. (A) Serum deprivation induces neuronal death in STHdh Q111/Q111 but not in STHdh Q7/Q7 cells. Cellular viability was measured by the MTT method (n = 5). (B) Δ9‐THCA and rosiglitazone (RGZ) prevent cell death induced by serum deprivation. STHdh Q111/Q111 cells were cultured under serum deprivation conditions and treated with Δ9‐THCA in the absence or the presence of the PPARγ antagonist GW9662 (5 μM). Cell viability was calculated using the MTT method and referred to control cells (n = 5). * P < 0.05, significantly different from untreated cells; # P < 0.05, significantly different from serum‐starved cells; P < 0.05, significantly different from serum‐starved cells treated with Δ9‐THCA.
Figure 6
Figure 6
Δ9‐THCA is neuroprotective in 3‐NPA‐treated mice. (A) Behavioural score was determined 12 h after 3‐NPA injections. Mice were treated with Δ9‐THCA (20 mg·kg−1). Hindlimb clasping, general locomotor activity, hindlimb dystonia and kyphosis were measured, and values are expressed as means ± SEM (n = 9). (B) Δ9‐THCA down‐regulates the expression of inflammatory genes in mice brain. RNA was isolated from the striatum, retrotranscribed and analysed by real‐time PCR. Tnf‐α, Inos, Il‐6 and Cox‐2 gene were studied. * P < 0.05, significantly different from control group; # P < 0.05, significantly different from 3‐NPA only group; P < 0.05, significantly different from 3‐NPA plus Δ9‐THCA group (n = 9 animals per group).
Figure 7
Figure 7
Δ9‐THCA prevents neuronal loss, microgliosis and astrogliosis induced by 3‐NPA administration. (A) Representative images of Nissl, Iba‐1 and GAFP staining performed on coronal striatal brain sections (original magnification 20×). (B) Quantification of the different markers was performed with ImageJ software. Total average number of neurons (Nissl), microglia (Iba‐1+) and astrocytes (GFAP+) is shown. Values are expressed as means ± SEM (n = 6). * P < 0.05, significantly different from control group; # P < 0.05, significantly different from 3‐NPA group (n = 9 animals per group).

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