Download J Darnell, KH2 domain I304N RGG G

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