Inflammation and endocannabinoid system: the role of epigenetic modifications in BV2 cells treated with amyloid beta peptide

Numerous studies indicate that inflammatory processes and disturbed neuron-microglia interactions may mediate the pathogenesis of Alzheimer disease (AD), which is characterized by progressive memory impairment and cognitive deficits. The disease is associated with the presence of extracellular senile plaques mainly composed of amyloid-beta peptide (Abeta) and intraneuronal neurofibrillary tangles containing hyperphosphorylated tau protein (1). To date, the pathophysiology of AD is not completely unveiled, but increasing evidences suggest that epigenetic mechanisms may play a role in tracking the course and development of AD. Recent studies have indicated that Endocannabinoid (eCB) levels and metabolic enzymes change during the progression of AD and the inhibition of the major N-Arachidonoylethanolamine (AEA)-hydrolyzing enzyme, fatty acid amide hydrolase (FAAH), has beneficial effects in AD mice (2, 3). Our study is aimed at revealing the immune-modulatory effects induced by targeting FAAH with URB597, a well known FAAH inhibitor. This compound, an aryl ester of carbamic acid, inhibits the degradative metabolism of AEA as well as other eCBs. We have investigated whether epigenetic actors as the histone post-translational modifications, may affect inflammatory response in BV2 murine microglia cells challenged with Abeta in presence or absence of URB597. In order to evaluate the possible URB597 cytotoxicity on BV2 cells and to define the optimal concentration for the purpose analyzes, cell viability experiments by MTT assay were performed. The results show that URB597, used at concentrations up to 10 uM, does not exert negative effects on cell viability, even when treatment is continued for 72 h, indicating that the inhibitor does not interfere with the survival capability of the cells. According to the literature data, we used a concentration of URB597 of 5 uM. Therefore, BV2 cells were treated with 30 uM Abeta25-35 in presence or absence of 5 uM URB597. To evaluate the protein level of pan-acetylated histone H3 and methylated histone H3 on lysine 27 (H3K27me3) in our experimental model, we performed Western blot analyses. We observed an increase of pan-acetylated histone H3 protein level in all treated samples with respect to control. Conversely a diminution in of H3K27me3 level were found in URB597 treated sample compared to those treated only with Abeta and to control. To determine the level of inflammatory state in our BV2 cellular models, we analyzed, by qRT-PCR, the expression of pro-inflammatory interleukins as IL-1beta, TNFalfa and IL-6 as well as anti-inflammatory interleukins as TGFbeta, L-10, IL-4. The results demonstrate that pro-inflammatory interleukins expression as IL-1beta, TNFalfa increases within 1-3 hours (short time) in BV2 cells treated with Abeta compared to untreated control, whereas IL-6 expression increases after 6 hours of treatment. URB597 did not affect the expression of the same pro-inflammatory genes, whereas FAAH inhibitor is able to modulate the TGFbeta, IL-10 interleukins gene expression within 6-24 h. Chromatin immune-precipitation analyses (ChIp) using anti-H3 pan-acetyl and anti-H3K27me3 antibodies were performed to study promoter chromatin modifications that may accompany changes of pro and anti inflammatory ILs expression. Although not conclusive, our data show that TGFbeta and IL-10 chromatin promoters acquire a chromatin enriched of H3 pan acetyl modification and reduce levels of H3K27me3 gene expression when contemporary treated with Abeta and URB597. The obtained data suggest that an increase of AEA, due to FAAH inhibition, may contribute to decrease the inflammation cellular response modulating pro-inflammatory interleukins at chromatin level. References: 1) Selkoe DJ (2001) Alzheimer's disease: genes, proteins, and therapy. Physiol Rev 81: 741-766. 2) Bisogno T., Di Marzo V. (2008) The role of the endocannabinoid system in Alzheimer's disease: facts and hypotheses. Curr Pharm Des. 2008;14(23):2299-3305 3)Basavarajappa B, Shivakumar M, Joshi V, Subbanna S. (2017) Endocannabinoid system in neurodegenerative disorders. Neurochem. 142(5):624-648.