N-Acylethanolamine-hydrolizing Acid Amidase (NAAA) in the EAE model of multiple sclerosis

V Capurro1, S Pontis1, M Dionisi1, P Busquet1, R Bertorelli1, G Tarozzo1, A Reggiani1, D Piomelli1,2. 1Drug Discovery and Development, Fondazione Istituto Italiano di Tecnologia, Genova, 16163, Italy, 2Departments of Pharmacology and Biological Chemistry, University of California, Irvine, 92697-4621, USA

Aberrant inflammatory response involving T cells, macrophages and microglia leads to the gradual loss of myelin sheath that protects nerve fibers in multiple sclerosis (MS) (1). Clinical studies have shown that both plasma and cerebrospinal fluid levels of N-Acylethanolamines are altered in MS patients suggesting that the endocannabinoid system could play a role in the disease. N-Acylethanolamine-hydrolizing Acid Amidase (NAAA) has been identified as the enzyme responsible for the degradation of palmitoylethanolamide (PEA) (2). Previous studies revealed that PEA shows anti-inflammatory effects acting mainly through PPAR-α receptors in several models of inflammatory diseases (2). Moreover, oral administration of PPAR-α agonists gemfibrozil and fenofibrate inhibits clinical signs of experimental autoimmune encephalomyelitis (EAE), an experimental model of MS in mice (3). In order to further investigate the role of NAAA and PPAR-α in the disease, we tested the susceptibility of PPAR-α KO mice in the EAE relapsing-remitting model of MS and examined the expression of NAAA in the microglia of C57BL6/J EAE mice. EAE was induced in PPAR-α KO and C57BL/6J mice (n=5 and 10, respectively) by immunization with myelin oligodendrocyte peptide (MOG) 35-55 peptide, 200 μg/mouse. Mice also received intraperitoneal injections of 200ng pertussis toxin (PTX) in 200μL PBS after MOG injection and 48 h later. Control-immunized mice (injected with both MOG vehicle and PTX in the absence of MOG) in addition to unimmunized mice served as negative controls. Mice were weighted and scored daily for clinical signs as described by Stromnes and Goverman (4). Mice have been killed by anaesthetic overdose (chloral hydrate, 450 mg/kg, IP) at different time points (7-10-21 days post-immunization) and spinal cord tissue processed for qPCR and immunohistochemistry. Gene expression of NAAA and of inflammation marker iNOS was evaluated. Immunohistochemistry studies were conducted in order to identify the phenotype of NAAA expressing cells: Iba1 as a marker of microglia, GFAP of astrocytes, CNPase of oligodendrocytes.

PPAR-α KO animals showed more severe symptoms and higher clinical score compared to WTs (p<0.001, 2way ANOVA, Bonferroni’s test) confirming previous studies in which they exhibited a greater vulnerability to inflammation (5). In C57BL/6J mice, analysis of qPCR data showed that, at day 7 and 10 post immunization, NAAA and iNOS mRNA levels were significantly upregulated in mice showing symptoms at the moment of sacrifice (for NAAA: non-symptomatic 0.85 ± 0.03 at day 7 and 0.74 ± 0.02 at day 10 vs symptomatic 1.57 ± 0.01 and 2.31 ± 0.07; for iNOS: non-symptomatic 0.86 ± 0.04 at day 7 and 0.73 ± 0.05 at day 10 vs symptomatic 13.62 ± 0.07 at day 7 and 26.74 ± 0.08 at day 10; p<0.0001, Student’s t test). Immunohistochemistry confirmed that levels of NAAA were increased in immunized mice compared to controls, at day 7 already. In particular, analysis of fluorescence in spinal cord sections showed significant induction of NAAA in EAE mice vs control immunized mice (integrated density average in EAE mice 136 ±16 vs control immunized 0.29 ± 0.11; p<0,001 Student’s t test). Overall, our preliminary results suggest that NAAA and the endocannabinoid system are altered during the development of the disease. Further studies are needed to clarify whether their modulation could be beneficial for the treatment of MS.

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