LMO and ALK. The Unbelievable Likeness of Beings. Heberlein Lab UCSF and EGCRC

Transcription

1 LMO and ALK The Unbelievable Likeness of Beings Heberlein Lab UCSF and EGCRC

2 Because we believe: Why flies??? - similarity of drug-induced behaviors - conservation of synaptic machinery involved in drug responses - conservation of genetic bases of drug-related behaviors Because we can: - awesome classical and molecular genetics - economy of scale - robust behaviors - sophisticated tools to manipulate genome and neural circuits

3 The Approach Develop behavioral model Genetic screens Mutants Genes Candidate genes Mechanisms Targets for pharmacotherapy

4 100 µg cocaine Acute cocaine exposure McClung and Hirsh 1st min 2 ndmin 3 rd min 4 th min Linus Tsai

5 Acute cocaine exposure Mock exposure vehicle McClung and Hirsh 1st min 2 ndmin 3 rd min 4 th min Linus Tsai

6 A simple behavioral assay Drug Effect Score, % C o cain e, DES, % µg Bainton, Tsai et al., Current Biology, 2000

7 Cocaine Sensitivity Mutants (of ~ 400 lines screened) Line Phenotype Gene or homology EP1a EP1b RRR RRR RhoGAP18B (white rabbit) EP1c RR EP2 RRR Alpha-mannosidase EP3 RR MAPKKK (dtao) EP4a S Bx / LMO EP4b SS Bx / LMO EP5 EP6 EP7 EP8 EP9 EP10 SS SS SS S S S GPCR (moody) GSK3 (shaggy) mrna binding protein Neuropeptide (amnesiac) period Ethanolamine kinase (easily shocked) Linus Tsai, Roland Bainton

8 Effect of dlmo mutations on cocaine sensitivity Loss-of-function Gain-of-function Drug Effect Score, % C tl- 1 * * * EP1383 EP1306 C tl- 2 p drm C tl- 3 * hdp G enotype Genotype Drug Effect Score, % * * C tl B 1 x B J x Genotype dlmo expression in NS dlmo expression in NS Tsai et al. PLOS, 2004

9 Cocaine sensitivity is inversely proportional to [dlmo] 1 Drug sensitivity 0 lof wild type [dlmo] gof

10 How does dlmo work? LIM Domain Binding Protein (LDB) D D LMO LIM1 LIM2 D D LIM2 LIM1 LIM2 LIM1 HD HD LID LID LID LID LIM-HD Protein LIM2 LIM1 LIM2 LIM1 HD HD LIM1 LIM2 HD LIM1 LIM2 LIM1 LIM2 HD active inactive??? Milan and Cohen, 1999 Rincon-Limas et al., 2000

11 ALK: A Receptor Tyrosine Kinase ALK Pathway ALK - Oncogenic when fused to other proteins through chrom. translocations (Morris et al, 1994) - Activation of Alk promotes neurite outgrowth in culture (Souttou et al, 2001) - - Expressed primarily in brain, particularly during development (Iwahara et al, 1997; Vernersson et al, 2006) KO mice viable, exhibit antidepressant phenotype (Bilsland et al, 2008)

12 dlmo dalk 1 cocaine sensitivity dalk Drug sensitivity dlmo 0 lof wild type gof

13 dlmo dalk cocaine sensitivity

14

15 Mammalian LMOs interact with multiple partners bhlh GATA-1 (ZF) SMAD LMO4 Estrogen Receptor CREB HDAC BRCA1 LMOs can act as repressors or activators of transcription complexes

16 A null allele of Lmo4 generated from gene-trap ES cells insertion LMO4/ -galactosidase Heterozygous mice show enhanced cocaine sensitivity Lasek et al., 2010

17 LMO4 GAL fusion protein expression in adult Lmo4 gt /+ mouse brain PFC Hipp VTA Acb VTA Tu Lasek et al., 2010

18 Brain regions implicated in addiction-related behaviors Nestler, 2001

19 Where and when is Lmo4 function required to regulate cocaine-related behaviors? PFC CA3 Acb DA VTA Tu Virally-mediated RNA interference

20 Viral delivery to and infection of Nucleus Accumbens anti-gfp Lasek et al., 2010

21 shrna reduces Lmo4 mrna and protein in Acb LCM-QPCR IHH Relative Expression * SCR shrna Lmo4 shrna GFP Lmo4 Lasek et al., 2010

22 Sensitization: A long-lasting neuroadaptation induced by repeated drug exposure drug drug drug drug effect effect effect effect learning and memory liking wanting craving Robinson and Berridge, 1993

23 Cocaine sensitization enhanced by Lmo4 RNAi in Acb Distance traveled, cm x 10-3 n = * * * * Lmo4.3 cocaine Lmo4.3 saline SCR cocaine SCR saline Cocaine (15 mg/kg) or saline Lmo4 function in the adult Acb to regulate the acquisition and maintenance of cocaine sensitization Lasek et al., 2010

24 Does Lmo4 inhibit Alk expression in Acb? LCM-qPCR Lmo4 Alk Amy Lasek (unpublished)

25 Does LMO4 regulate Alk expression directly? ChIP anti-lmo4 Control IgG QPCR QPCR LMO4 is associated with Alk promoter in striatum, which is expected to inhibit Alk expression. Amy Lasek (unpublished)

26 Is Alk involved in cocaine sensitization? enhanced sensitization LMO4 Alk?? Prediction: Down-regulation of Alk should reduce/impair cocaine sensitization

27 Alk downregulation in Acb reduces rate of acquisition of cocaine sensitization Amy Lasek (unpublished)

28 LMO4-ALK does it matter? Cocaine sensitization Cocaine CPP drug saline Acute inhibition of ALK delays cocaine sensitization and reduces the rewarding properties of cocaine ALK inhibitors are in development for cancer, ALK may therefore be a novel therapeutic target for addiction. Amy Lasek (unpublished)

29 Not just ALK.. but its signaling pathways! ALK Pathway Pharmacotherapy available / in development

30 Not just ALK.. but its signaling pathways! ALK Pathway Pharmacotherapy available / in development

31 SUMMARY Validation of Lmo4 in cocaine sensitivity and sensitization Alk regulated by LMO4 dalk RTK regulated by dlmo Alk regulates cocaine sensitization dlmo mutants Cocaine sensitivity screen ALK small-molecule inhibitor reduces cocaine sensitization and reward

32

33 Linus Tsai Roland Bainton

34 The mouse team (EGCRC) Amy Lasek Francesco Giorgetti David Kapfhamer Julie Gesch

35 How does LMO4 regulate Alk expression?? Poster by Amy Lasek

36 Marco Milan, Steve Cohen, Nick Justice, Yuh-Nung Jan Steve Morris (St. Jude s) EGCRC Animal Core Facility EGCRC Histology Core Facility NIH/NIDA State of California Department of Defense

37 Thank you!