1. Role of lipid rafts in TrkB-BDNF signaling Natália Assaife-Lopes Institute of Pharmacology and Neurosciences, Faculty of Medicine and Unit of Neurosciences, IMM, University of Lisbon Unit of Neurosciences Neurosciences

2. Lipid rafts Membrane microdomains enriched in glycosphingolipids, cholesterol and certain proteins Contains the major part of tyrosine kinase activity of the plasma membrane Required for proper signaling of different receptors and neurotransmitter release “Membrane rafts are small (10-200nm), heterogeneous, highly dynamic, sterol- and sphingolipid-enriched domains that compartmentalize cellular processes...” Pike, 2006.

3. Physiological role of lipid rafts InternalizationCompartmentalization Allen et al, Nat Rev Neurosci, 2007Simons and Vaz, Annual Review Biophy Biomol Struct, 2004

4. Lipid rafts, cholesterol and the brain Cholesterol is essential to lipid raft formation The brain contains 25% of total body cholesterol and cholesterol derivatives Brain cholesterol (CLT) is synthesized locally. Neurons can either produce CLT or uptake glial-derived CLT Aging alters the transbilayer distribution of cholesterol → Lipid raft abundance and composition may be altered

5. Lipid rafts and diseases

6. Lipid rafts and Alzheimer’s disease Wolozin, PNAS 2001 ApoE ε4 allele is a risk factor for AD High cholesterol levels in mid-life correlate with Aβ deposition and risk of developing AD. AD has a lower incidence in individuals taking statins (Wolozin 2004) Direct correlation between ↑ cholesterol and size/number of lipid rafts is still to be established Corder et al, Science, 1993.

7. Lipid rafts and Aβ aggregation GM1 ganglioside-bound Aβ (GM1/Aβ) possesses an extremely high aggregation potential and an altered pattern of immunoreactivity (Yanagisawa et al, Nat Med 1995) GM1/Aβ seems to be directly injurious to cellular membranes (McLaurin and Chakrabartty, J Biol Chem 1996) Cholesterol and sphingomyelin metabolism are regulated by Aβ and presenilin (Grimm et al, Nat Cell Biol 2005) DIG: detergent-insoluble glycosphingolipid-rich domainKatsuhiko Yanagisawa

8. BDNF low affinity high affinity Chao, Nat Rev Neurosci (4):299, 2003 BDNF and TrkB receptors Depression Alzheimer’s disease Huntington’s disease Epilepsy Schizophrenia Development Neuronal survival Synaptic plasticity

9. BDNF and cholesterol metabolism Neuron-specific

10. BDNF and lipid rafts

11. 1.BDNF induces TrkB translocation to lipid rafts 2.It is necessary for some BDNF effects Suzuki et al., J Cell Biol(167):1205, 2004 1.Lipid raft disruption impairs TrkB phosphorylation and PLCγ activation 2.BDNF-induced TrkB translocation occurs intracellularly Pereira and Chao, J Neurosci (27):4859-69, 2007 BDNF (TrkB) CLT synthesis + Lipid rafts + + Adenosine A 2A receptors?

12. Cross-talk between TrkB and A 2A receptors Synaptic transmission at CNS (Diógenes et al, 2004) GABA uptake (Vaz et al, 2008) Synaptic plasticity (Fontinha et al, 2008) Most trophic actions (Neves-Tomé, unpublished) A 2A required A 2A dispensable What are the differences? What makes it specific? BDNF effect Transmission at the neuromuscular junction (Pousinha et al, 2006) Diógenes et al., J Neurosci(24):2905, 2004

13. Objectives 1.To study the role of A 2A receptors in TrkB sublocalization in the lipid rafts 2.What are the molecular mechanisms involved? 3.What are the functional consequences of A 2A -induced TrkB translocation to lipid rafts? A 2A receptors ?

14. Isolation of lipid rafts Corticohippocampal neurons (8-10 days in vitro) Pregnant rat E18 embryos 20 nM CGS 21680 (30’), before 20 ng/ml BDNF Cell lysis in an 0.5% triton X- 100 containing buffer @ 4ºC (lipid rafts remain insoluble) 8 x 1.5 mL fractions Optiprep gradient 0% 30% 35% Lipid rafts Western blotting Fyn transferrin receptor 36400rpm 6h @ 4 o C

15. Results: 20ng/ml BDNF for 5 minutes! Activation of adenosine receptors induces TrkB translocation to lipid rafts TrkB Fyn control 20nM CGS21680 + BDNF 20ng/ml BDNF 20 ng/ml 20nM CGS21680 50nM ZM241385 + BDNF 20ng/ml CGS 21680: adenosine A 2A agonist ZM 241385: adenosine A 2A antagonist Fyn: lipid raft marker

16. Activation of adenosine receptors increases pTrkB staining in the lipid rafts + BDNF 20ng/ml 20nM CGS 21680 ++ ++ 50nM ZM 241385 ++ -- - - - - -- -- - Fraction #2 analysis Fyn Flotillin-1 pTrkB TrkB CGS 21680: adenosine A 2A agonist ZM 241385: adenosine A 2A antagonist Fyn: lipid raft marker Flotillin-1: protein enriched in lipid rafts

17. Results: BDNF for 40 minutes! Fyn Flotillin-1 pTrkB TrkB + BDNF 20ng/ml 20nM CGS 21680 ++ ++ 50nM ZM 241385 ++ -- - - - - -- -- - CGS 21680: adenosine A 2A agonist ZM 241385: adenosine A 2A antagonist Fyn: lipid raft marker Flotillin-1: protein enriched in lipid rafts

18. Activation of A 2A receptors potentiates BDNF-induced pTrkB localization in the lipid rafts p≥0.05, n=4 Fyn Flotillin-1 pTrkB TrkB + BDNF 20ng/ml 20nM CGS 21680 ++ ++ 50nM ZM 241385 ++ -- - - - - -- -- -

19. Conclusions (I) Activation of A 2A receptors induces TrkB translocation to lipid rafts (independent of pTrkB formation!) Pre-activation of adenosine A 2A receptors potentiates BDNF-induced pTrkB staining in lipid rafts The effects of CGS 21680 and BDNF are additive on TrkB sublocalization

20. Objectives 1.To study the effect of an A 2A agonist in TrkB sublocalization in the lipid rafts 2.What are the molecular mechanisms involved in CGS 21680 effects? 3.What are the functional consequences of A 2A -induced TrkB translocation to lipid rafts? adenosine

21. Mechanisms involved in the CGS 21680-induced TrkB translocation to lipid rafts FSK: Adenylate cyclase activator H-89: PKA inhibitor U73122: PLC inhibitor PP2: Src family kinase inhibitor H-89 H-89 + CGS 21680 Fyn TrkB control CGS 21680 FSK FSK + CGS 21680 control CGS 21680 control CGS 21680 PP2 PP2 + CGS 21680 U73122 U73122 + CGS 21680 Rp-cAMPs Rp-cAMPs + CGS 21680

22. Comparison between CGS 21680-induced and BDNF-induced TrkB receptors on lipid rafts MDC: Dansyl cadaverine: Inhibits clathrin-dependent endocytosis Fyn pTrkB TrkB control CGS MDC BDNF 40’ MDC + CGS MDC + BDNF 40’

23. Effects of lipid raft disrupting drugs on A 2A -induced effects on TrkB sublocalization Fyn Flotillin-1 TrkB control CGS MβCD wsCLT wsCLT + CGS MβCD + CGS Methyl-β-cyclodextrin: Binds to cholesterol (removing it from membranes) wsCLT: water-soluble cholesterol (MβCD:chol complexes)

24. FSK mimics CGS 21680 effects on TrkB translocation to lipid rafts The PLC pathway is not involved in CGS 21680-induced TrkB staining in the lipid rafts Inhibition of clathrin-dependent endocytosis affects BDNF-induced but not CGS 21680-induced TrkB translocation to lipid rafts Disruption of lipid rafts with 3mM MβCD greatly inhibits TrkB staining in fraction #2 (lipid rafts) and cholesterol addition promotes TrkB translocation 3 [H] ZM 241385 binding to cortical membranes is not affected by lipid rafts disruption or cholesterol addition Conclusions (II)

25. Objectives 1.To study the effect of an A 2A agonist in TrkB sublocalization in the lipid rafts 2.What are the molecular mechanisms involved? 3.What are the functional consequences of A 2A -induced TrkB translocation to lipid rafts? 3 [H] glutamate release (in cortical synaptosomes) TrkB sublocalization after HFS Role of lipid rafts in BDNF-mediated enhancement of LTP

26. 3 [H] glutamate release from cortical synaptosomes Is BDNF effect on glutamate release dependent on A 2A receptors and lipid raft integrity? *

27. Role of A 2A receptors on BDNF-induced 3 [H] glutamate release from cortical synaptosomes CGS 21680: adenosine A 2A receptor agonist ADA: adenosine deaminase: converts adenosine into inosine (inactive on A 2A receptors)

28. Role of lipid rafts on BDNF-induced 3 [H] glutamate release from cortical synaptosomes MCD: methyl-β-cyclodextrin: Binds to (and removes) cholesterol C.O.: cholesterol oxidase: Oxidizes cholesterol to cholest-4-en-3-one

29. Nagappan and Lu, TINS, 2005 Proposed mechanisms for synapse-specific effect of BDNF on LTP: Effects of synaptic activity on TrkB receptors

30. High frequency stimulation of hippocampal slices increases TrkB staining in lipid rafts TrkB Fyn Lipid rafts (fraction #2)

31. The effects of BDNF upon LTP are dependent on lipid raft integrity

32. Conclusions (III) BDNF-induced glutamate release from cortical synaptosomes is totally dependent on endogenous adenosine and at least partially dependent on lipid raft integrity High frequency stimulation of hippocampal slices induces translocation of TrkB receptors to lipid rafts in an adenosine-dependent manner The effect of BDNF on LTP is dependent on lipid raft integrity

33. ANY QUESTIONS??