1. ENDOCANNABINOID REGULATION OF ALTERNATIVE MICROGLIA ACTIVATION IN MULTIPLE SCLEROSIS CAITLIN JAGLA

2. MULTIPLE SCLEROSIS: BASIC FACTS Chronic inflammatory and neurodegenerative disease Over 2.5 million people affected globally 2-3x more prevalent in women than men Symptoms, severity, & duration are highly variable between individuals Genetic & environmental factors seem to contribute Image: Initial MS Symptoms; multiplesclerosis.net

3. MS: IMMUNOLOGICAL PATHOGENESIS Reactive immune cells accumulate in the CNS Peripheral lymphocytes & macrophages (due to increased BBB permeability) Activated microglia Pathology: white matter demyelination leading to lesions in the brain Also grey matter demyelination, axon & neuron damage, loss of oligodendrocytes Multiple Sclerosis. NIH MedLine Plus (2012) 7:1 14-15.

4. ALTERNATIVE MICROGLIA ACTIVATION

5. DYSFUNCTIONAL SELF/NON-SELF SIGNALING BETWEEN NEURONS & MICROGLIA IN MS? NIRegs: neuroimmune regulatory proteins Expressed by neurons as ‘don’t eat me’ signals Modulate microglial function i.e. CD47, CD200 CD200 knock out / blockade aggravates murine models of MS

6. PAPER 1: CD200-CD200R1 INTERACTION CONTRIBUTES TO NEUROPROTECTIVE EFFECTS OF ANANDAMIDE ON EXPERIMENTALLY INDUCED INFLAMMATION HERNANGOMEZ ET AL. GLIA 2012, 60:1437-1450.

7. FIG. 1: AEA PROMOTES NEUROPROTECTION FROM ACTIVATED MICROGLIA-INDUCED CYTOTOXICITY CD200Fc or AEA treatment significantly reduced neuron death in WT but not CD200R1-/- cultures Demonstrates that AEA neuroprotection is mediated by CD200-CD200R1 interaction

8. FIG. 3: CD200R1 UPREGULATION BY AEA RELIES ON CB2 A) AEA does not effect neuron expression of CD200 regardless of LPS/IFNy B) LPS/IFNy stimulation reduces microglial expression of CD200R1; AEA rescues C) Microglial CD200R1 mRNA levels are decreased by LPS/IFNy treatment; AEA rescues D) AEA-induced recovery of CD200R1 expression by LPS/IFNy-treated microglia is dependent on CB2 SR1 (SR141716A): CB1 antagonist SR2 (SR144528): CB2 antagonist

9. FIG. 4: CD200R1 ACTIVATION BY CD200FC REGULATES MICROGLIAL CYTOKINE SECRETION IN RESPONSE TO LPS/IFNy A & B) LPS/IFN-y treatment induced secretion of IL-6 & IL-1B; addition of CD200Fc reduced C) LPS/IFN-y treatment induced secretion of IL-10; addition of CD200Fc enhanced

10. FIG. 5. IL-10 IS NEUROPROTECTIVE AND ENHANCES NEURON CD200 EXPRESSION. (A) IL-10 protects WT neurons from microglia-mediated inflammatory damage (B) CD200R1-/- reduces efficacy of IL-10 p <0.001) vs. p<0.0001 in WT (C) IL-10 increases neuronal CD200 expression (D) IL-10 knockout increases LPS/IFN-y induced neuronal death AEA requires IL-10 to exert neuroprotection (in comparison to Fig. 1c, 1d)

11. FIG. 6: TEMPORAL EXPRESSION OF CD200 AND CD200R1 mRNA IN THE SPINAL CORD OF TMEV-IDD MICE. A) CD200: Upregulated at 21 days post-infection (dpi) Downregulated at 35 (ns) & 60, 90 dpi B) CD200R1: Downregulated at 21 (ns) & 60, 90 dpi Upregulated at 35 (ns) dpi disparity between levels closer to infection, but similarity as dpi increases TMEV-IDD: virally-induced murine model of MS

12. FIG. 7. FAAH INHIBITION OR AEA TREATMENT INCREASES CD200 AND CD200R1 EXPRESSION IN TMEV-IDD MICE AA-5HT N-arachidonoyl-serotonin endogenous FAAH inhibitor FAAH fatty acid amide hydrolase Hydrolyzes AEA (C) & (D) – AEA was continuously delivered for 7 days using miniosmotic pumps implanted subcutaneously in TMEV-IDD mice

13. FIG. 7. FAAH INHIBITION OR AEA TREATMENT IMPROVES MOTOR BEHAVIOR IN TMEV-IDD MICE HACTV: Horizontal activity VACTV: Vertical activity Assessed using electronic activity monitoring system

14. FIG. 8: FAAH INHIBITION OR AEA TREATMENT INCREASES IL-10 AND DECREASES IL-1B AND IL-6 mRNA LEVELS IN THE SPINAL CORD OF TMEV-IDD MICE

15. ANANDAMIDE PROMOTES CD200-CD200R INTERACTION IN TMEV-IDD MODEL OF MS: CONCLUSIONS AEA is neuroprotective and provides symptom relief in MS models CD200-CD200R1 interaction is necessary CB2 activation is involved, at least in LPS/IFN-y stimulated microglia AEA treatment induces IL-10 upregulation & IL-6, IL-1B downregulation in TMEV-IDD and LPS/IFN-y stimulated microglia IL-10 is necessary for AEA neuroprotection (in LPS/IFN-y model)

16. PAPER 2: ANANDAMIDE, ACTING VIA CB2 RECEPTORS, ALLEVIATES LPS-INDUCED NEUROINFLAMMATION IN RAT PRIMARY MICROGLIAL CULTURES MALEK ET AL. NEURAL PLASTICITY 2015, 130639.

17. FIG. 2: NITRIC OXIDE RELEASE AFTER LPS STIMULATION OF PRIMARY RAT MICROGLIA CULTURES

18. FIG. 3: qPCR, RECEPTOR LEVELS FOLLOWING LPS-ONLY OR LPS + AEA/ANTAGONIST TREATMENT CB1 (a) LPS (+ all) Decrease vs. null (*) CB2 (b) LPS + AM-630 Increase vs. null (*) & LPS-only (#) GPR18 (c) LPS (+ all) Increase vs. null (*) LPS + AM-630 Increase vs. null (*) & LPS-only (#) GPR55 (d) LPS + AM-630 LPS + AM-251 LPS + CID-1602 Increase vs. null (*) & LPS-only

19. FIG. 4: qPCR, M1 MICROGLIA MARKERS: IL-18, TNF- α, NOS2 LPS treatment tended to upregulate all 6 markers of M1 microglia phenotype LPS-stimulated upregulation of NOS2 was significantly decreased by: AEA treatment GPR18/55 antagonism CB1 antagonism CB2 antagonism significantly upregulated LPS-induced IL-18 and TNF- α levels (and NOS2 vs. null only)

20. FIG. 4: qPCR, M1 MICROGLIA MARKERS: IL-1 β, IL-6, COX2 LPS treatment tended to upregulate all 6 markers of M1 microglia phenotype LPS-stimulated upregulation of COX2 and IL-6 was significantly decreased by: AEA treatment GPR18/55 antagonism CB1 antagonism (IL-6 only) CB2 antagonism significantly upregulated LPS-induced COX2 levels (and IL-1 β & IL-6 vs. null only)

21. FIG. 5: qPCR, M2 MICROGLIA MARKERS: IL-10, NGF IL-10: low levels in non-stimulated microglia LPS stimulation significantly upregulated IL-10 only in the presence of AEA or AM-251 (CB1 antagonist) IL-10 levels increased non-significantly following LPS stimulation in other conditions NGF: high levels in non-stimulated microglia LPS stimulation significantly decreased NGF levels on its own and in the presence of AM-630 or CID-1602 AEA or CB1 antagonism with AM-251 essentially blocked LPS-induced decrease in NGF levels

22. ANANDAMIDE ACTS PARTIALLY THROUGH CB2 ACTIVATION TO ALLEVIATE LPS-INDUCED NEUROINFLAMMATION: CONCLUSIONS AEA treatment of LPS-stimulated microglia reduces NO release, dependent on functional CB2 AEA treatment tends to downregulate M1-associated cytokines and enzymes, while promoting M2-related Involvement of IL-1B, IL-6, and COX2 is interesting due to previous work showing relationship to endocannabinoid system

23. QUESTIONS/HYPOTHESIS Does AEA act through any other putative CB receptors to effect microglial alternative activation? (PPARs, TRPV?) Previous work shows that PPARy ligands can regulate CD200-CD200R1 expression/interaction AEA neuroprotection in murine models of MS is mediated by multiple pathways CB2-dependent upregulation of CD200R1 in microglia (as previously suggested) PPARy binding to induce IL-10 expression, which in turn promotes CD200 expression by neurons