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J Darnell, KH2 domain I304N RGG G-Q KH2 specific for I304N 2 stem loops in target GQ does not compete FMRP off brain polyribosomes Kissing complex competes FMRP off PRAs – specific to KH2 Also seen in FXRs—Kissing complex is not FMRP=specific FXRs don’t bind GQ, only FMRP Fly dfmr does not bind GQs RGG deletion does not disrupt FMRP association to PRAs RNAbob search program. Sean Eddy ? Wash U. Can’t search for tertiary structures MAP 1B (also Kendal Broadie, Yue Feng) = dendritic extension Nova CLIP RNA subset. Inhibitory synapse subset of mRNAs FMRP CLIP targets; not clear on this H Tiedge Small brain-specific RNAs in translational suppression Input specificity at synapse level Local protein synthesis control BC1 mRNA in dendrites (not in axons) Repressor Single nucleotide subst determines dendritic transport BC1 – eif3 – 40S –pretranslation complex, etc Binds AUG 60S, 80S -> elongation BC1 inhibition occurs before the AUG step EMCV-IRES translation does not require eif4E – cap recognition BC1 represses; not CAP mechanism CS-IRES is not suppressed—unwinding is critical step for BC1 BC1 binds PABP – 3’ Helicase, unwinds double stranded RNA (3’-5’ eif4A complex) BC1 inhibits unwinding BC1 has seemingly no specific/direct interaction with FMRP binding to translation complexes—interact only at different levels in the translation process (Denman) Why are there so many translational repressors at the synapse? Redundancy exists in the system Translation is by default “on” -- any mRNA free at synapse will be translated Repressors are functionally acting as activators Premature translation may be very detrimental to system -----------------------------------------------------------------------------------------------------------Sam Schacher – Columbia u Target and activity dependent regulation of mRNA distribution and local translation Trophin-like molecule in Aplysia Sensory to L7 motor neuron “plastic” synapse Neuron need not be present for synaptogenesis Synaptogenesis is anisomycin sensitive 80-100 mRNAs transported to neuritic region Sensorin—Orphan peptide Expressed in sensory neuron, not in L7 Translational regulation, not dependent on transcription Specifically activated by contact with L7 (vs. L11) Ab to sensorin blocks synapse formation in vitro (epsp size used to measure synapse formation; EM confirmation as well as visible varicosities; when a different target neuron is co-cultured these varicosities don’t form full synaptic specialization) Presynaptic growth is not affected by Ab; synapse-formation specific effect Sensorin increases MAPK translocation to sensory neuron nucleus Sensorin upregulates production of its own mRNA (model for FMR1?) mRNA accumulation is specific to branch contacting L7 (vs. L11) target neuron Long-term facilitation: assoc w increase in synaptic sites, new transcription and translation—5-HT induces facilitation Sensorin: increased only under conditions that elicit LTF Increase can occur without cell body of sensory neuron—local regulation of synthesis PI3K inhibitor blocks LTF and sensorin expression effect of 5-HT MapKinase activation involved in second wave of sensorin expression which is nucleusdependent (important for maintenance, stable phase, of LTF) 5-HT -> PKA receptor has not been identified ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Bear “exaggerated responses to group 1 mGluR1/5 activation” (mGluR theory) mGluR accelerator FMRP Brake Translation-dependent CA1 LTD controlled by mGluRs AMPAR cycled out of postsynaptic membrane Greater LTD in KO FMRP suppresses further translation—end product inhibition—after just enough protein synthesis to stabilize LTD Van Der Klish—elongation of dendritic spines in response to activation protein synthesis dependent MD-OD shift (17h) OD mouse model VEP amplitude; contra vs ipsi 7 days’ deprivation gives potentiation, 3 days gives depression KO, more potentiation (less depression) to 3 days’ deprivation ??? check mGluR5 cross restores plasticity in KO Randy Carpenter—company representative --------------------------------------------------------------------------------------------------Zukin AMPAR trafficking regulated by neuronal activity; Local synthesis of AMPAR subunits as possible mechanism of local synaptic plasticity AMPAR mRNA in dendrites; FISH/DiG; Banker Hipp Cultures GluR2 mRNA in clusters; granules; also GluR1 mRNA Localizes with MAP2 in dendrites—marker for dendrites; GluR mRNA is in spines; MAP 2 mRNA is not GluR not in axons (nice—axon wrapped around dendrite) Synapsin co-localization NMDAR activation reduces GluR2 mRNA level in dendrites with no change in Synapsin 1 mRNA Selective, not in somata Decrease in dendritic mRNA requires CA2 and ERK/MAPK signaling Bapta blocks decrease (ca2 absorbing) Calcium-dependent ERK/MAPK signaling PD9089 is a pathway blocker blocks nmda effect NMDA effect requires erk/mapk activation Reduces total cell GluR2 mRNA Transcription vs degradation mechanism? ??? no effect; not reduced stability Actinomycin to block transcription—transcriptional arrest implicated Must be blocking transcription DHPG—mGluR local protein synthesis Promotes local synthesis of AMPARs ROBUST increase in GluR1,2 mRNA!!!!!! Visible in tissue culture ICC Nmdar > ca > ERK/MAPK +RISC > nucleus > receptor for transcription increases mRNA NOW we need to re-report our receptor bypass experiments again????? Does it matter that the effect of FMRP is post mGluR? --------------------------------------------------------------------------------------------------------- Sean McBride (Jongens) p-element modification of gene; block coding regions Rescue wild-type dfmr rescues all phenotypes so far (frame shift control) Phenotype: Circadian deficit in darkness Mushroom Body—critical for STM and LTM in courtship conditioning paradigm courtship conditioning paradigm: courtship conditioning with previously mated female decreases courtship; learn not to court????? Virgin female courtship suppressed— suppression is index of memory dfmr—reduced courtship in males No detectable immediate recall in mutants—cognitive deficit mGluR – one actually confirmed in Dm MPEP has been shown to antagonize DmGluRA (mGluRB not shown to bind glutamate) MPEP treatment during development rescues courtship deficit Adult MPEP also rescues courtship WE SHOULD SEE IF MPEP RESCUES KO SYNAPTIC PROTEIN SYNTHESIS MPEP adult or devel rescues stm deficit in courtship LY mGlu1 antag also rescues courtship LiCl is an angagonist?, also rescues; also MPPG, MTPG rescue Mushroom body phenotype goes away with mpep, ly, mtpg during development Adult, no brain rescue Brain phenotype does not consistently associate with behavioral measures ------------------------------------------------------------------------------------------Toth Behavior Missed part FX mice don’t show normal diurnal variation of activity—is similar to humans Sensory system—auditory—hyperreactive; could contribute to speech anomalies Easy to overdrive brainstem with auditory stim in mouse, not so in human Audiogenic seizures in mice; elevated c-fos activity to sound stimuli in KO; Seizures: Coch N > IC > PAG > Sp cord Startle reaction should go down in KO due to inhibitory input from PPTg Prepulse would further inhibit; more suppression in KO via this pathway Get both effects: no habituation to startle stimuli; elevated prepulse inhibition Human data, Frankland et al, opposite; but, higher IQ individuals show increased ppi May reflect greater cortical input GABA (B): How to reduce excitability – metabotropic receptor, 2 subtypes, 1 and 2 At GABA synapses, presynaptic decreases activity; sometimes postsynaptic (hyperpolarization, decreases activity) Baclofen (reduces audiogenic seizure in IC; Feingold, 1994) Should have a positive effect on seizure activity Rescues startle in KO mouse (chronic treatment) Hyperactivity, make mouse anxious; reduce activity More sensitive in open field activity to low doses of baclofen No effect on rotarod performance except at high doses that affect muscle tone Very short half-life; too short even for minipump; put in drinking water At proper dose, hyperactivity is reduced by baclofen? Thinks not direct effect of fmrp on GABA-B receptor—rather, some kind of compensatory reaction to FMRP effects -----------------------------------------------------------------------------------------------------------Bureau Barrel ctx Caged Glutamate Patch recording—synaptic currents Laser beam probe (UV) measures excitatory inputs—apparently voltage-sensitive dye local activation; matrix approach Can assess level of presynaptic inputs (No cells, No inputs; drive of inputs) Study neocortical circuits Columnar organization less ‘tight’ in ko mouse; strength of connections weaker (lay 4 to 2/3—not statistically significant, however (2nd and 3rd postnatal weeks) Fewer axons or weaker axons? Not said, but have capacity to determine this Plasticity: whisker trimming; deprived whisker lay 4 cells are reduced in input; layer 5 deprived cells have strengthened input in deprived; In FMR1KO deprivation effects are blocked; suggests it is because weaker input is blocked Are KOs like normal mice that have been deprived—no farther to go? “Loss of experience dependent plasticity” “Weaker connections” (despite lack of significance) No details on underlying changes; hypothesis synapse strength or number ----------------------------------------------------------------------------------------------Dobkin Seizure phenotype in mouse GABA (A), Cl- entry Reduced GAR in corte brainstem diencephalon, hippocampus, not cerebellum 30-40% reduction GAD? (makes GABA): expression is increased; except cerebellum GFP-FMRP transgene; mosaic (one X chr) Label cells having fmrp Absence of fmrp does not predict GAD+, not directly related to absence of FMRP but to a general increase in activity N=1 – “just a couple of sections” GABA-A receptor also not closely coupled to presence-absence of FMRP! => Compensatory response?? What a disappointment; two in a row. ------------------------------------------------------------------------------------------------------Lauder Common genes Smith-Lemli-Opitz syndrome and fxs Features of the two syndromes overlap Differential strain effect: c57 (social—goes to target compartment with mouse), fvb – no difference in response to mouse or empty compartment SLOS mice, may also have deficit in social approach Really nothing there yet—SLOS data didn’t replicate in new building; background strains different, etc. Microarray data: s-score program for statistical analysis of microarray data; free (zhang) Huge genetic background effect on expression; fvb and bl6 often have opposite wt/ko results Cholesterol biosynthesis genes; cytoskeleton; neurodegeneration; synaptic function SLOS gene pattern more or less obverse of fragile x Common genes affected; direction often opposite More serotonin neurons in SLOS Most neurotransmitters receptors require cholesterol for proper insertion into membrane [a little knowledge is a dangerous thing?] --------------------------------------------------------------------------------------------------Dan Kelly (Richard Davidson) Blood cell fmrp defect, consistent across samples No-data of my own Does berry kravis think that FMRP directly controls Adenylate cyclase Question Warren identified Adenylate Cyclase as a FMRP cargo ---------------------------------------------------------------------------------------------Fallon Transcriptional regulation NOTE: starting with Simple, Consensus diagrams Dark rearing 45 or 60 days; light exposure 0-60 min “maintain the critical period” Transient expression of FMRP Synaptoneurosomes 15 min light exposure increases fmrp in somata; increases in neuropil expression; more still at 30 min. Post-transcriptional; NMDAR-dependent New model: Olf bulb (lifelong neurogenesis) p20; fmrp in all layers—peak of expression at p20 (GC; MC) Isoamyl acetate odor exposure increases fmrp expression at p10, p15; not (or less) @ p30 Naris closure: decreases developmental fmrp expression in early postnatal period (unilat) Parallel—fmrp expression and critical period FMR1 promotor: AP2 alpha; Ap2 alpha response element in promoter; multiple devel roles; parallel fxs (craniofacial; cns devel; epidermis; neural crest) Ap2 alpha KO has reduced FMR1 expression (die at e-16) Cre-Lox; delete Ap2alpha in eye; dramatic reduction in fmr1 In adulthood; fmr1 comes back; Xenopus oocytes; dominant negative ap2alpha reduces fmr1 expression Transcription/DNA methylation: FMR1 promoter during olf bulb development: Bisulfide sequencing: recognize methlyation status base by base (Ann Booker) Selective demethlyation (WT) during olf bulb critical period; regulating accessibility of promoter to regulators; Ap2 site initially unmethlylated, becomes methlyated at p1, stays methylated; shutting down a site that was active embryonically; interaction of methylation status with activators Finds a promoter domain that is unmethylated at p20—period of peak developmental fmrp expression; having been methylated up to p20 and becoming methylated again after p20; this corresponds to the end of granule cell proliferation This is important; opening up of fmr1 expression at specific times during development; correlate with timing of mitral dendrite loss. He is collaborating with Peter Brunjes Responses to questions: Rapid fmrp in response to light exposure: alpha CAM2 kinase stable; Thinks rapid elevation of fmrp is in part reduced degredation ------------------------------------------------------------------------------------------------Klann Klann & Dever – NRN 2004 – excellent summary diagram of mglurs and pathways LTD and translation Model Learn Mem 11 365 2004 FMRP increased during LTD [K I S S] DHPG increases fmrp levels in ca1; protein synthesis dependent Principally in CB layer; some in dendrites; increase largely in soma Not compatible with a synaptic synthesis model Cargoes: PSD 95; rapid increase with DHPG in WT; KO no further response with DHPG—looks like KO is already saturated; identical result for MAP1B—already elevated in KO Looks like KO is already turned on; in this model FMRP is a repressor (compatible with our selective regulation model)’ ERK: no total effect; DHPG increases phospho-ERK – KO is already elevated in phospho-ERK mGluR LTD induced by dhpg is anisomycin-sensitive in WT; independent in KO ERK paper has got to go out; Mgur-LTD is not ERK dependent in KO mouse DHPG-induced FMRP increase in CA1 Proteosome inhiitors block DHPG increase in FMRP (MG132 increases FMRP) Is FMRP ubiquinated during mGluR LTD? Steph’s ubiquinated antibody; FMRP becomes poly-ubiquinated; increased western blot molecular weight MG132 blocks DHPG-induced LTD mGluR LTD enhanced in KO mouse; yac transgenic rescue inhibits LTD wild type mouse; mg132 inhibits DHPG induced LTD two-dimensional gels; look at KO, WT with LTD 20 proteins increase; 8 proteins decrease Posttranslational modifications – phosphorylation CaMKIIa; Peroxiredoxin II increased in KO; Neuron specific anti-oxidant protein; increased under oxidative stress; Ox stress implicated in phenotypes of autism Contact Nelson to get transgenic for SOD1 immunocytochemistry --------------------------------------------------------------------------------------------------Yue Feng – MAP 1B translation control by FMRP Devel upregulation of fmrp in hippocampus at 7 d postnatal; down by p14 (synaptogenesis?) Timm staining axonal proliferation (dentate; CA3) GET COPY of summary fig from Weiler 2004 Make master set of slides FX Map1b is earliest microtubule protein expressed in devel; drops after birth Day 10 drop in Map 1b not seen in KO; just WT; translational not transcriptional Modulating microtubule dynamics; pathfinding IP demonstration of fmrp binding to MAP 1B (not 1A) mRNA Aberrant expression of Maplb in Hilus in KO; in pyramidal and GC layers; 300% differential overxpresssion in KO; ICC not very good quality Sucrose gradients; map1b mRNA associated with large polyribosomes (whole gradient, really) GAP only on small ribosomes In KO more association of map1b with large pras Should be more translation in KO; faster run off Control – miRNA: Jin/Warren paper MAP 1B mRNA has several let-7 binding sites; conserved in mouse and human Let-7 co-IP’s with MAP1B I should have gone to my strength; the fx phenotype, period FMR1 KO: microtubule polymers are more resistant to disruption – more stable; consistent with function of MAP1B posttranslational modification of microtubules—MT stability Nocodazole disruption: KO more stable Basis for lack of dendritic withdrawal? Is there a barrel experiment here? Actin dynamics in GC; Gorden-Weeks 2004 GC theory; pathfinding; mtubule stabilization role This should be testable Olfactory bulb; both strains MAP1B upregulated during process extension Would it make sense to compare c57 with FVB – map levels in dendrites RNAi FMR Kd – increases MAP1B translation (Pulse label reveals increase) RNAi FMRP Kd facilitates neurite extension (Not clear what cells) Also (siRNA) prevents neurite retraction caused by nicodozole disruption of microtubules This is very convincing as a mechanism ***Send a copy of Robert’s olfact bulb ms -----------------------------------------------------------------------------------------------------------Bob Wong mGluR-induced protein synthesis underlies epilepsy FMRP suppresses Short duration (interictal) discharge – ionotropic receptors Ictal discharges, strong, diverging connections > synchronization One neuron can potentially ignite the whole network Strategically-situated GABA-ergic neurons can stop this spread GABA-ergic system is compromised in FXS mGluR agonists translate interictal to ictal discharges induce prolongation of interictal discharge prolongation extends beyond period of DHPG activation (“forever”) ERK activation required Anisomycin suppresses FMRP as brake; prevents runaway seizure Only KO gives seizure, not WT Bicucculine mimics KO Protein synthesis is enhanced (?) in KO Target for anti-epileptic drug Autosensitive process: mGluR is involved in maintenance of ictal discharges Could there be covalent receptor modification occurring? Or is it a downstream effect? ------------------------------------------------------------------------------------------------------CB Smith – regional protein synthesis in KO vs wt KO has higher glucose utilization than WT in adult Could it be that our sns are misleading because of the degree of fmrp expression in early postnatal animals? 14C Leucine intravenous; ages 4 & 6 Mo KO has higher prot synth in some basal forebrain regions RCPS (cerebral protein synthesis) declines across this age range Attributes to possible synapse pruning in WT Brain as a whole – genotype not significant (but small possible effect; small n) Age significant Looked at 75 regions of brain; age effects in all but 2; genotype effects in 26 regions Frontal assoc cortex & post parietal cortex were the only significant cortical regions (KO > WT) – although small effects favoring KO in all cases 5 of 16 thalamic areas show genotype effect Hippocampus: CA3 highest synthesis; biggest KO effect, but also in CA1 and dentate – presubiculum Largest changes in hypothalamus: PVN, SON 34%, SCN big difference favoring KO High fmrp expression in SON Pons, brainstem: DMNVagus; N. Raphe favor KO Aangenehpour sfn monkey fmrp distribution We could do this – Jenny; fraxa proposal? This is just autoradiography I think ------------------------------------------------------------------------------------------------------Kaufmann – involved in initial ARC work; naming, with Worley Bagni paper (BC1) proposed Arc as regulated expression cargo Was ARC on our microarray? Early gene Activity-regulated – NMDAR, AMPAR, BDNF Steward—loc at activated synapses Cytoskeletal protein homology (spectrin) CREB-initiated effector protein—possible dendritic structural modifications Arc is DHPG-induced—mGlur PSD as a postsynaptic compartment regulating interactions of second msgr systems Kim & Sheng, NRN 5 2004 Homer involvement in ARC regulation FMRP as a modulator of the postsynaptic compartment