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Because alpha-synuclein (Snca) has a role in brain lipid metabolism, we determined the impact that the loss of alpha-synuclein had on brain arachidonic acid (20:4n-6) metabolism in vivo using Snca-/- mice. We measured [1-(14)C]20:4n-6 incorporation and turnover kinetics in brain phospholipids using an established steady-state kinetic model. Liver was used as a negative control, and no changes were observed between groups. In Snca-/- brains, there was a marked reduction in 20:4n-6-CoA mass and in microsomal acyl-CoA synthetase (Acsl) activity toward 20:4n-6. Microsomal Acsl activity was completely restored after the addition of exogenous wild-type mouse or human alpha-synuclein, but not by A30P, E46K, and A53T forms of alpha-synuclein. Acsl and acyl-CoA hydrolase expression was not different between groups. The incorporation and turnover of 20:4n-6 into brain phospholipid pools were markedly reduced. The dilution coefficient lambda, which indicates 20:4n-6 recycling between the acyl-CoA pool and brain phospholipids, was increased 3.3-fold, indicating more 20:4n-6 was entering the 20:4n-6-CoA pool from the plasma relative to that being recycled from the phospholipids. This is consistent with the reduction in Acsl activity observed in the Snca-/- mice. Using titration microcalorimetry, we determined that alpha-synuclein bound free 20:4n-6 (Kd = 3.7 microM) but did not bind 20:4n-6-CoA. These data suggest alpha-synuclein is involved in substrate presentation to Acsl rather than product removal. In summary, our data demonstrate that alpha-synuclein has a major role in brain 20:4n-6 metabolism through its modulation of endoplasmic reticulum-localized acyl-CoA synthetase activity, although mutant forms of alpha-synuclein fail to restore this activity.