Transcript +ΔFosB
Neuron 2007 Speaker: Yu-han Lee Advisor: Yn-ho Huang Ph.D Dr. Chen-Jee Hong Escape deficit ( learned helplessness) repeated stress (eg. inescapable footshock) escape test No escape deficit (Non learned helplessness) ΔFosB ( brain: emotional regions) ﹖ role Introduction Learned Helplessness Animals exposed to inescapable shock present with a series of symptoms similar to those observed in depressed patients. -Willner et al.,1984 Seligman,1967 escapable shocks : ES Shuttle box Inescapable shocks : IS home-cage control : HC ΔFosB : a truncated splice variant of FosB, which lacks 101 amino acids from the C-terminal end of the full-length fosB AP-1 (activator protein - 1) transcription factor stimuli AP-1 : rapidly in response to a variety of stimuli AP-1 site Fos family protein : c-Fos, FosB, ΔFosB ,Fra-1, Fra-2 1. The nature of the biochemical modification that converts the unstable 33-kDa isoform into the stable 35- to 37-kDa isoforms has remained obscure.It has been speculated that phosphorylation may be responsible. 2. The 35- to 37-kDa isoforms of ΔFosB dimerize predominantly with JunD to form an active and long-lasting AP-1 complex within these brain regions. Eric J. Nestler et al, 2001 stimuli stimuli Colleen A. McClung et al, 2004 Escape deficit ( learned helplessness) repeated stress (inescapable footshock) escape test No escape deficit (Non learned helplessness) ΔFosB ( brain: emotional regions) ﹖ role Results and Methods C57BL/6 mice , 8 wks Shuttle box 120 inescapable footshock (IS) / day 2 days , 0.45mA No IS , 1hr / day , 2days Escape test (Shuttle box) : 15 trials , 25 sec / trial 24hr No IS IS - Escape deficit (LH : learned helplessness) IS – no escape deficit (NLH : non learned helplessness) 24hr Heterogeneous Escape Deficits Induced by IS in mice LH ＃ ★ NLH Define 2 subgroups C57BL/6 mice , 8 wks inescapable footshock (IS) 24hr escape test : No IS IS No IS - Yoked Immunohistochemistry ΔFosB (monoaminergic regions: dorsal raphe nucleus (DR) ventral tegmental area (VTA) locus coeruleus (LC) Full-length fosB Only ΔFosB only Middle region of both full-length fosB and ΔFosB ΔFosB Accumulates in the DR and LC, but Not in the VTA, after Repeated IS TPH : tryptophan hydroxylase vIPAG : ventrolateral periaqueductal gray (lateral margins of the rostral DR) ΔFosB Levels in the Ventrolateral Periaqueductal Gray Correlate Negatively with the Severity of the Behavioral Deficit ΔFosB NLH LH Rostral portion of DR vIPAG Repeated stress (IS) ΔFosB ΔFosB In “No escape deficit ” NLH In “escape deficit ” LH The level of ΔFosB induction by IS in this brain region predicts the individual degree of resilience to IS. (Also in repeated social defeat paradigm) - The vlPAG has been characterized previously as an important neural substrate for passive coping responses. - IS, which induces primarily passive behavioral responses, engages the vlPAG. Bandler and Shipley, 1994 Hypothesis : IS ΔFosB In vlPAG alterations in AP-1 transcription desensitize passive coping-related circuits in the vlPAG. PPT-A (preprotachykinin A) promoter : PPT-A enhancer or repressor PPT-A mRNA Alternative splicing substance P AP-1 Paterson et al,1995 substance P vlPAG ascending to nucleus accumbens(NAc), amygdala It has been shown that emotional stressors triggers the release of substance P in the NAc, Karl Ebner et al, 2004 IS ΔFosB alterations in AP-1 transcription desensitize passive coping-related circuits in the vlPAG. ？ AP-1(ΔFosB) substance P in vlPAG regulate ΔFosB Accumulates in Substance P-Positive Neurons after IS B6 after IS immunohistochemistry (ΔFosB) In situ hybridization ΔFosB Accumulates in Substance P-Positive Neurons after IS GABA 5-HT 63% 25% 10% ΔFosB Decreases Substance P Gene Expression In Vivo -ΔFosB +ΔFosB Substance P Hypothesis : IS ΔFosB alterations in AP-1 transcription desensitize passive coping-related circuits in the vlPAG. In vivo : AP-1(ΔFosB) substance P regulate In vitro ???? Luciferase assay PPT-A promoter -865 ~ +447 pGL3basic HSV-ΔFOSB or HSV-LacZ PC12 (substrate) ΔFosB Decreases Substance P Gene Expression In Vitro Dose-dependent HSV-LacZ(control) Hypothesis : IS ΔFosB alterations in AP-1 transcription desensitize passive coping-related circuits in the vlPAG. AP-1(ΔFosB) substance P regulate In vitro ???? YES PPT-A repressor ΔFosB PPT-A mRNA Alternative splicing substance P AP-1 Hypothesis : IS ΔFosB alterations in AP-1 transcription desensitize passive coping-related circuits in the vlPAG. AP-1(ΔFosB) substance P regulate Question: Whether this regulation represents a direct effect of ΔFosB on the PPT-A gene??? Mice - IS: 2hr , 24hr ChIP(chromatin immunoprecipitation) assay control : 2hr , 24hr vlPAG of 3~4 mice Crosslink with 1% formaldehyde sonication Anti-fosB Ab (c-terminal Ab) (full-length fosB only) Anti-fosB Ab (middle region of both fulllength fosB and ΔFosB) Reverse crosslink Real-time PCR PCR analysis(164bp) ΔFosB accumulating in substance P-positive neurons in the vlPAG after IS binds directly to the promoter region of the PPT-A gene in vivo. IS ΔFosB alterations in AP-1 transcription desensitize passive coping-related circuits in the vlPAG. AP-1(ΔFosB) substance P regulate Question: Whether the regulation of substance P gene expression in vlPAG neurons by ΔFosB may contribute to functional alterations of forebrain substance P neurotransmission?? Microdialysis with simultaneous behavioral analysis to mimic the endogenous pattern of ΔFosB induction after IS baseline Rats: HSV- LacZ HSV- ΔFosB In NAc 4 samples for baseline (every 30min,homecage ) In vlPAG Swim tank 10 min 3 samples (every 30 min, homecage) IS, 20min 5 samples, (every 30 min, homecage) After viral Overexpression of ΔFosB in the vlPAG After IS Fig.1B Hypothesis : IS ΔFosB alterations in AP-1 transcription desensitize passive coping-related circuits in the vlPAG. AP-1(ΔFosB) In vlPAG substance P regulate In NAc Hypothesis : IS ΔFosB alterations in AP-1 transcription desensitize passive coping-related circuits in the vlPAG. desensitize substance P input to the NAc Substance P vIPAG NAc NK1(substance P receptor) drug Function ? D1 D2 D3 D4 IS IS rest test Drug drug drug drug 30min before test NK1 antagonist antidepressant Decreased substance P signaling in the NAc exerts an antidepressant-like effect and promotes active escape responses in the learned helplessness paradigm. ΔFosB Hypothesis : IS Antidepressant-like effect ??? alterations in AP-1 transcription desensitize passive coping-related circuits in the vlPAG. desensitize substance P input to the NAc Substance P vIPAG NAc NK1(substance P receptor) drug antidepressant-like effect Overexpression of ΔFosB in the vlPAG Opposes Behavioral Despair ΔJunD, a truncated isoform lacking 48 amino acids at the N terminus , is a truncated variant of JunD, the major in vivo partner for ΔFosB (Chen et al., 1995; Hiroi et al., 1998) Forced swim test Antidepressant-like effect No change sensitivity to foot shocks Rotarod test of motor coordination Locomotor activity conclusion ΔFosB Repeated stress stress substance P accumulate in vlPAG release in NAc In resilient animals Repeated stress ΔFosB accumulate in vlPAG repress substance P gene in vlPAG substance P release in NAc Promoting active coping responses ascending to In resilient mice ΔFosB substance P accumulate sufficiently to repress substance P signaling In helpless mice substance P Not accumulate sufficiently to repress substance P signaling ΔFosB Rotarod • A reliable test to study motor function and learning • Bar is rotated at constant or increasing speed • Time to fall is recorded manually or automatically RT Series滾筒式跑步機主要是應用於研究藥物對動作協調性和抗疲勞特性的實驗 △Ct = (Nacute,chronic - Navecontrol) x Ctavecontrol, (= CtPPT / Ct whole lysate - Ctavesynaptophysin / Ct whole lysate ) x Ctavesynaptophysin - N is the normalized Ct value of PPT [Ct(PPT)/Ct(Input)] - Nave is the mean N value for the control - Ctave is the mean Ct value for the control. - Fold differences (stress ChIP relative to control ChIP) were then determined by raising 2 to the Ct power. Input : One hundred microliters of the pre-immunoprecipitated lysate Control ChIP : samples were immunoprecipitated with 5 µg nonimmune rabbit IgG