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Of Mice and MAGL (Monoacylglycerol Lipase) Bach, S; Bombinski, T; Daniels, M; Gross, D; Hogg, T; Martin, T; McMurray, D; Naber, E; Perez, N; Schulman, A; Tucker, S; Andera-Cato, S; Arnold, A; Blumberg, A; Bord, M; Feiertag, A; Greaves, M; Her, A; Kennedy, E; Orozco, C; Rice, C; Rodgers, A; Sauer, A; Schubert, J; Tubbs, C; Wray, T Teacher Advisor: G. Vogt - Brown Deer High School 8060 N. 60th, Brown Deer, WI 53223 Mentors: L. Shrestha, C. Hillard, Ph.D. Director of the Neuroscience Research Center Medical College of Wisconsin Abstract Figure 4: Inhibition of MAGL Hinders Hydrolysis of 2-AG Figure 2: Endocannabinoid Signaling System About 18.8 million American adults suffer depressive disorders that may occur with anxiety and substance abuse. Tetrahydrocannabinol (THC), a compound in marijuana, is a cannabinoid chemical that binds to and activates cannabinoid receptors (CB1) in the pre-synaptic cell membrane as part of neuron-to-neuron transmission in the endocannabinoid system (ECS). Glutamate in the pre-synaptic cell is released and binds to the post-synaptic cell triggering the synthesis and release of 2arachidonoylglycerol (2-AG). 2-AG returns to the pre-synaptic cell binding to and activating CB1 receptors. THC mimics 2-AG action, and is used to study the ECS retrograde signaling system and its effect on appetite and mood. A protein from the pre-synaptic cell, monoacylglycerol lipase (MAGL), hydrolyzes 2-AG into arachidonic acid (AA) and glycerol controlling 2-AG levels. When MAGL is hyperactive, too much 2-AG degrades, causing 2-AG shortage, prompting depression, anxiety, and other neurodegenerative diseases. Hypoactive MAGL activity creates an excess of 2-AG, causing obesity, diabetes, and addictive behaviors. The Brown Deer Students Modeling a Research Topic Team, in alliance with MSOE, built a MAGL model using a 3D printer. Study of MAGL hydrolysis sites may provide the key to regulating MAGL’s enzymatic activity leading to therapies that will prevent neurodegenerative disorders. Step 5: MAGL hydrolyzes 2-AG into arachidonic acid and glycerol. (See Figure 1) PRE-SYNAPTIC CELL Step 1: Glutamate is released from the pre-synaptic cell, and binds to the post-synaptic cell. MAGL Blockade of 2-Arachidonoylglycerol Hydrolysis by Selective Monoacylglycerol Lipase Inhibitor 4-Nitrophenyl 4-(Dibenzo[d[ [,13]dioxol-5yl(hydroxy)methyl)piperidine-1carboxylate (JXL184) Enhances Retrograde Endocannabinoid Signaling Bin Pan, Wei Wang, Jonathan Z. Long, Dalong Sun, Cecilia J Hillard, Benjamin F. Cravatt, Qing-sang Liu Electrodes placed in neurons from a slice of rat brain were used to detect the excitatory post-synaptic currents (EPSC) that run through the postsynaptic neurons when glutamate reacts with its receptors. These data were plotted on the y-axis of Figure 4. Small pulses were used to release glutamate from the pre-synaptic cell but not enough to trigger the synthesis of 2-AG. The baseline result between -50 and 0 seconds is at 100% of the EPSC amplitude because there is no 2-AG present. At time 0, the electrical pulses were vastly increased for a few milliseconds, triggering 2-AG synthesis. The presence of 2-AG decreased the release of glutamate reflected in a decrease in EPSC amplitude. The control line returned to 100% after a few seconds due to MAGL breaking down 2-AG. The other two curves demonstrate the inhibitory effect of JZL184 on MAGL activity; blocking MAGL delays the breakdown of 2-AG and delays the return time to control EPSC amplitude. Step 4: SYNAPTIC CLEFT 2-AG binds to CB1 receptor on the presynaptic cell inhibiting glutamate release. Introduction Approximately 80% of the 18.8 million American adults suffering from the serious neurological disorder known as depression are not receiving any treatment. Elucidation of neural signaling mechanisms in the brain may lead to more effective treatments. Specific brain cells called neurons transmit messages from pre-synaptic neurons to post-synaptic neurons across a gap called the synapse using neurotransmitter chemicals. One potential target of drug treatment is the regulation of the enzyme monoacylglycerol lipase (MAGL), found in the endocannabinoid system, shown in Figure 2. MAGL is an enzyme found in the pre-synaptic cell that hydrolyzes 2-AG: •Increase in MAGL activity decreases 2-AG concentrations resulting in depression and anxiety. •Decrease in MAGL activity increases 2-AG concentrations producing anti-depressant effects. Inhibition of MAGL will increase 2-AG, therefore making MAGL a good target for drug therapy of depression. Step 2: The binding of glutamate to the post-synaptic cellPOST-SYNAPTIC CELL Step 3: triggers the 2-AG travels back to synthesis and the pre-synaptic cell release of 2-AG. and binds to CB1 Figure 5: Inhibition of MAGL Calms Mouse Behavior 16 14 Marbles Buried receptors. POST-SYNAPTIC CELL (A Review on the Monoacylglycerol Lipase: At the Interface Between Fat and Endocannabinoid Signaling G. Labar, J. Wouters, D.M. Lambert “Current Medicinal Chemistry, 2010, 17, 2588-2607”) http://depressiontreatmenthelp.org/depression-statistics.php 12 10 8 6 4 2 0 Vehicle Figure 1: MAGL Hydrolyzes 2-AG to Glycerol and Arachidonic Acid 16 40 JZL-184 (mg/kg) MAGL Asp-239 Catalytic Triad His-269 3hju.pdb MAGL 2 Figure 3: Monoacylglycerol Lipase, in model form Ser-122 Ser-122 Research has been conducted using mice to study their marble burying behavior. Mice normally demonstrate anxious behavior when foreign objects are placed in their cage. When marbles were introduced the mice responded by frantically trying to bury them. The y-axis demonstrates the number of marbles buried with a vehicle and increasing concentrations of JZL184. The vehicle includes the experimental protocol used to inject the mice. The data shown in Figure 5 illustrates that injection of JZL184 reduces the anxious behavior of the mice causing them to bury fewer marbles. Kinsey SG, et al, Inhibition of endocannabinoid catabolic enzymes elicits anxiolytic-like effects in the marble burying assay, Pharmacol Biochem Behav (2010), doi:10.1016/j.pbb.2010.12.002 Biological Significance JZL184 has an inhibitory effect on MAGL activity. Regulation of MAGL activity could effectively treat anxiety and depression disorders in human. SMART Teams are supported by the National Institutes of Health (NIH)- National Center for Research Resources-Science Education Partnership Award (NCRR-SEPA), and an NIH CTSA Award to the Medical College of Wisconsin.