Banbury 2006 MEETING NOTES.doc
Fallon: Axonal FMRP
Translational regulation in axons involved in axonal guidance
(Holt, Yuang, Mary Bunge, 1973)
Translation in axons, campenot chamber work
Fallon: P15 brain; Olfactory bulb; reinnervation paradigm; FMRP granular staining in
glomeruli; in neuropil; not in other neuropil such as EPL; Fallon also sees FMRP
granules in CA3 hipp;
FMR2 and 2P ALSO co-localized in granules in OB and CA3 (SO; S. Lacunosum);
immuno-EM = FMRP in axons including presynaptic terminals (also post)
Axonal FMRP in CA3 mossy fibers (Ivanco paper); granules dense at P15; infrequent by
P60 in hipp; OB has later peak @ p30; --corr with synaptogenesis?
Regeneration of olfactory receptor neurons (methyl bromide cell death); in adult get
recovery and overshoot in axonal FMRP granules; recovery of area of granule precedes
reinnervation; FMRP granule expression correlates in time with synaptogenesis;
FXR2P: Co-localized for the most part with FMRP in granules; in FMR1 null there is a
compensatory up-regulation at a delay from the FMRP peak in the WT mouse
Proposes class II as mechanism of MPEP effect;
Koyoma et al 2002—mglur2 effect associ with Timm staining effect in CA3
Proposes presynaptic mGluR2 mechanism of action on FMRP expression in association
with synaptogenesis
Doing naris closure—odor exposure experiment now
Granules have S6 (ribo) immunoreactivity during synaptogenesis; not at other times
Bear; mfs have a lot of mGluR1;
Could these be “our” granules; very likely; but didn’t Soong Ho Not Find FXR2 in
granules?
FXR2 does not increase in dendrites during regeneration induced elevation in axons.
-----------------------------------------------
Jennifer Darnell, RNA targets of the KH2 and RGG domains
1304N – KH2 (change all to I304N)
Nova domain similar; these are SPLICING FACTORS!!!!!
DerI, look up Nova proteins
1304 is in the RNA binding pocket of the KH2 domain
1304 mutant protein does not associate with polysomes—stays at top of sucrose gradient
Is 1304 N responsible for the powerful syndrome?
Knock-in mouse with only 1304; cre- regulated
Expression level ok at mRNA level
Processed in 3 MW bands (lower expression than wt—20% in brain, 40% in testes)
Fallon Ab does not cross-react with FXRs – WHAT WAS TARGET EPITOPE?
mRNA encoding 1304N is largely not in polyribosome fraction
That which is associated may be dimerized with FXR1 or 2
I304N doesn’t seem to bind to mRNA per se but does so via heterodimerization
Patient may have the same low level of expression in the brain
LOF, RNA-binding
Motif (G-quartets, RGG binding)
G-quadriplex: combination of 3 stacked quartets plus linker region Gs between quartets
Can see the quadriplex in mass spectrometry
Quadriplex stabilized by Glycine-arginine-glycine triplet structure
Tertiary RNA structure important
Mutations in quadriplex interfere with binding
Compatible with Steph’s methylation data
KH2 double hairpin kissing complex
Kissing complex competes FMRP off mRNA; concentration dependent manner
Are the introns in the neighborhood of the binding region processed in the nucleus?
dFMR competes kissing complex
FXR c-termini do not bind to RGG/GQ motif
ONLY FMRP recognizes the G-quartet motif
Bardoni, different isoforms have different binding characteristics in RGG
(KH??)
Using U-V cross-linking to now identify targets
--not micro RNAs; appear to be mRNAs
no cross linking to any micro RNAs
No G-quartets found among cross-linked RNAs
Macro-orchid really appears late: 196 days?
Translation of targets would require helicase to unbind loop-loop structure
-------------------------------------------------------Steph
Arginine methyl transferase methlyates
Lysine, arginine and histidine can be methylated
No known arginine demethylase; lysine demethlyase has been identified
Is RGG box methylated; how does it affect binding?
FMRP is methylated post-translation
Deletion of RGG box reduces methylation by 88%; primary site of methylation
Multiple methylation sites in RGG box: 4 sites;
PRMT1 (Protein-arg-methyl-transferase)
Methylation affects mRNA binding to co-IP targets of Darnells
9 protein arginine methyl transferases are known
2 types
asymmetric; symmetric (methylation at symmetrical sites on second methylation of CH3
PRMT1 KO cell line; FMRP methylated just fine; not the one (at least the exclusive)
Knockdown PRMT3 and PRMT6; also not the ones
Now investigating 8 and 9
Question: if recycles through nucleus, must it be demethylayted?
------------------------------------------------------------------------------------------------------Bardoni
FMRP-interacting proteins and affinity for RNA/RNA identification
FXRs, no evidence of RNA Targets
MSP58 protein binds G-quartets; co-localized in cells with FMRP
High affinity binding of FMR1-mRNA??? (G-quartet containing phantom target mRNA)
Competition; FXRs vs FMRP on phantom – FXR is first order competition; MSP58 is
higher order competition
Differential dissociation time; FMRP + FXR1P compete FMRP off phantom much faster
than either alone, suggesting they work as heterodimer that is more effective than
homodimer; same general rule for MSP58
Synergic effect; MSP58 acts by blocking the dissociation of FMRP from G-quartet RNA
Cyfips not RNA-binding proteins; in presence of activated RAC1, cy and FXR1p move
to microtubules or some aspect of cytoskeleton
Novel mRNA sequences/ structures; Miyashiro; Brown targets
Sod1 (reduced in dendrites in cerebellum and hipp
Corr with anxiety
Reduced binding target region to 64 NTs (no kissing complex structure)
Binding not Mg dependent
No GQ structure
We’re all in the same empirical hole
SOD1 expression is not reduced in presence of FMRP
--------------------------------------------------------------------------------------Yue Feng: translation regulation by FMRP during development
2 aspects proposed: network development and plasticity; presynaptic and postsynaptic;
Peak FMRP expression during second postnatal week
MAP1B; co-IPs with devel FMRP; regulated during same developmental window as
FMRP
Delayed decline of MAP1B expression relative to FMRP peak
Hypothesize FMRP is repressor of MAP1B expression
Map1b is expressed in astrocytes
Hilus: COLOcalization witth fmrp (in neurons)
Enhanced MAP1B mRNA in polyribosome fraction (or MRNPs) in KO neurons
GAP 43 not different in KO vs WT
Knockdown FMRP => increased MAP1B
She s “convinced” it is a regulatory target
MAP1B contains a GQ
Only present in some isoforms of MAP1B mRNA (predicted, I assume)
FMRP and miRNA
MAP1B has potential let7 binding sites; inhibits translation at the step of initiation;
Let-7 miRNA expression appears inversely co-regulated with FMRP level (not a great
match and only 3 time points)
Let-7 B suppresses MAP1B promoter activation of Luciferase
Confusing data from knockout on let-7 mechanism
RNAi FMRP facilitates neurite extension!!!!! (vs. control treatment)
Knockdown MAP1B: shorter neurites; more protrusions
FMR1 knockdown prevents neurite retraction caused by microtubule breakdown
DHPG stimulated MAP1B expression is abrogated in FMR1 KO neurons
PTZ induced seizure stimulates MAP1B expression in hippocampus (higher than wild
type in KO)
Does FMR1 reguate MAP1B expression in oligodendrocytes? How are glutamate effects
mediated; at what step of translation and via what mechanism
-----------------------------------------------------------------------------------------------question
Bob Wong; mGluR regulated plasticity (epileptogenesis) not involving NMDAR
-----------------------------------------------------------------------------------------------Gary Bassell
Rapid trafficking of FMRP granules; AMPA induced loss of FMRP from synapses
Statistical association of FMRP granules with postsynaptic markers
Role in trafficking, mRNA localization, regulation of synaptic synthesis;\\
Bidirectional movement at over 1 um/sec
Dominant negative Kinesin LC1 overexpression embargoes cargoes, reduces motility of
FMRP granules
Dhpg increases localization of poly A mRNA into dendrites
DHPG specifically induces localization of MAP1B mRNA into dendrites
Likewise CaMKIIalpha;
Lost in KO in both cases
Synaptoneurosomes: replicate rapid protein synthesis (S35 Methionine)
Cyclo, Rapamycin inhibitable
CamIIK, Psd95, robust synthesis; deficient in KO mice
Increased GluR1 synthesis in KO!!!??
FMRP ab pulls down camk2, glur1, psd95,nmdar1, fmr1, Beta Actin
Cultured Hippoc neuron spines (5DIC); labels actin for spines; synapsin for synapses
Fewer spines assoc with synapsin in KO—more spines that were not spine synapses
Protrusions in response to KCl depol; occurs it WT, not in KO (occluded by “more”)
Hyperabundance of filopodia in GCs from KO
Overexpression of FMRP downregulates filos in both KO and WT
Filopodia in KOs are more tight in pattern and less motile; “sluggish” movement; less
dynamic
Model includes retrograde return and recycling of FMRP through nucleus.
------------------------------------------------------------------------------------------------Pete Van der Klish
Non-synaptic excitability plasticity—intrinsic property
L V, somatosensory ctx
Most properties not different in KO, WT
LTP-IE; sustained increase in number of spikes to a specific depol. current triggered by
mGluR activation; not different in KO, WT—not dependent on translation
Synaptic Plasticity
Deficit in potentiation; normal depression (presynaptic) (spike timing dependent
plasticity); Bear LTD is postsynaptic
High throughput proteomics on KO mouse:
Mass Spectrometry:
Synaptosomes as “compromise” between PSD and synaptoneurosomes
Heavy vs. light isotope labeling; mix KO and WT cultures
BDNF induced changes in the synaptic proteome: 363 proteins upregulated by 15’ to 2 h
BDNF exposure
Translation components
Many others
FMR1 KO mouse: small percentage of proteins change (baseline between genotypes)
Adenomatous polyposis coli prtien – part of NMDAR complex
About 20-25 that go up or down in KO
----------------------------------------------------------------------Mansuo Hayashi (Tonegawa)
Spine development and abnormal spine morphol; need to credit Purpura
PAK – LMK – Cofilin (spines)
Opposite spine phenotype to FMR1KO—more short g type spines, fewer number of
spines
dnPAK TG (dom neg) has enhanced LTP ? in cortex—proposes opposite signaling
pathways at synapse
FMRP and PAK1 are in same complex based on IP
(blocking peptide control)
FMRP pulls down GST-PAK, not GST alone (specificity)
Crossed double mutant:
II=IIIpyr spines
Double mutant is normalized to WT level in density – rescue
EM, spine size analysis:
PSD length: PAK elevated, FMR1 KO not statistically reduced; tendency towards mutant
rescue
Perfs elevated in dnPAK, double mutant rescued (KO didn’t look like a phenotype but
says lower perfs than WT)
Behavioral phenotype: (prelim) (n=8), age 4 months
Hyperactivity: high in KO, partial rescue by dnPAK; ditto center squares
Fear conditioning: coterminating CS/footshock: context vs. tone
dnPAK, contextual memory deficit; no tone deficit
FMR1; just not clear but argues that dnPAK rescues deficit in performance???
Hypothesis; PAK (kinase) activates synthesis of proteins repressed by FMRP???
------------Catania:
mGluR5 receptor expression and interaction with homer proteins
can RE expression using Yac construct rescue phenotype
Audiogenic seizure phenotype is rescued in FMR1 KO mice
This in older animals;
Looking at receptor expression:
Nodifference in mRNA for ampa, mglur
No difference in protein levels wt KO
Immunohisotchemistry: GluR1; mGluR5 no dif expression
How about expression differences reported by others?
Artifact of Triton solubility?
mGluR5 is more soluble, more Triton Extractable
does not account
Homer, homer 1a, no difference KO - WT
Lost in a morass of binding and membrane proteins
--------------------------------------------------------------------------------------------IJ
--------------------------------------------------------------------------------------------Holly Cline
Retinotectal synaptic transmission regulates dendritic arbor development in optic tectum
Single cell electroporation of plasmid DNA into a single neuron in vivo – dye GFP YFP
Visual stimulation enhances dendritic arbor growth. Flashing LED array – Tadpoles.
Dark – limited process outgrowth – 4H of visual stimulation enhances rate of arbor
proliferation;
GluR?/NMDA blockers prevent this
CPEB [ poly adenyl binding protein] – Phos required for local translation
Also regulates transport out into the dendrite; mutant deficient in microtubule assoc.
protein binding domain
FMR1 contains 3’UTR PEBs
Measuring cpeb puncta and dendritic growth
CPEB promotes growth compared to “phospho dead” construct mutant
Arbor size and complexity is greatly increased; distance apart branch tips about the same
(no stimulation)
Phospho-dead mutant renders neurons completely incapable of responding to visual
stimulation which enhances development of normal CPEB-containing cells
Sites of structural plasticity are enriched in CPEB-containing puncta—probably moving
on microtubules.
Puncta dynamics not affected by visual stimulation
Branch dynamics: stable, maintained; transient; added and persisted; retracted:
No difference between CPEB and Phosphodead
CPEB and phosphodead BOTH correlated with more stable branches
Both CPEB and phosphodead accumulate in stable branches. (Can’t dump cargoes?)
They are likely to do the FMR1 experiment in some way, by introducing a construct into
some part of this pathway.
---------------------------------------------------------------------------------------------------Eric Klann,
mGluR-dependent LTD – Bear hypothesis
FMRP levels increase transiently (5-10 min) after mGluR LTD induction (mGluR5 ; little
contribution from mGluR1)
FMRP winds up back in soma or else is synthesized there; is it degraded rapidly?
Proteasome inhibitors block decline
Ubiquitination occurs that could lead to proteasome degredation
FMRP over-expression necessary for LTD – as well as degredation of FMRP
KO reduced levels of Map1B and CamKII
LTD in KO is not protein synthesis dependent (but not in the WT mouse)
Stim => degredation of fmrp => translation of fmrp binding mRNAs => ampa receptor
internalization
FMRP increased synth with DHPG is increased by PI3K, mTor blockers
4E-BP KO mice (repressor of translation, blocks translation initiation scaffold)
mGluR phenotype identical to fmr1 ko mice
should decrease cap dependent translation
P10 model for autism, upregulating signaling pathways for cap-dep translation
--------------------------------------------------------------------------------------------Story Landis
General words about NIH funding
Big chunk of doubling went into bioterrorism
NINDS, as many grants in 06 as in 05
Pay line down from 26%ile; Now 12%ile
Number of grant proposals doubled
Continuation levels cut approx 2%
Senate $7B; House critical on this
Budget won’t be settled till after midterm elections – could be a major shift
Tension—basic (curiousity-driven), translational, clinical (trials)
RO1 vs. “big” science/roadmap
New/experienced investigators
FXS: NICHD is lead institute; MH; NiNDS.
-------------------------------------------------------------------------------------------------------Os Steward
Focus on synaptic signaling mechs; brain in a “developed” state; much of deficits hard to
reverse
Polyribosomes rare; limited numbers of mRNAs but a lot compared to synthetic
machinery.
Size of mRNAs, stretched out really quite large compared to the size of a synapse.
Polysome just sort of fits in a small synapse
Not a lot of room for a lot of mRNA trafficking at a synapse
[spine neck diameter could really limit mRNA access to translation machinery]
Could the kinetics be different in a closed space like a synapse?
Could knowing cargoes/targets enable different therapeutic approaches?
[betsy quinlan; reversibility of early visual deficits after MD]
translation at induction stage [ltd] MUST involve RNA already present at the synapse
phosph. Of transl factors etc.,--changes that ramp up synthetic capacity locally at synapse
General, non-specific increase vs. targeted translation of specific mRNAs
Selection mechanism for mRNAs – signal transduction pathways
Map K pathway in DG in response to activation of perforant path; strong phosphorylation
of ERK; (unilateral in dentate)
PP inputs, middle of granule cell dendrites
p-ERK migrates to all layers of dendrite and to cell soma very rapidly; within minutes;
CaMKII increases follow ERK phosph
Arc mRNA is transcribed in nucleus and translated in dendrites (30-40 minutes after HF
stim of pp)
EF1 Alpha, localized in dendrites (elongation factor; promotes GTP dependent binding of
amino acyl tRNA to the ribosome during peptide elongation) – accumulated in inner
molecular layer; translation turned on during induction of LTD
DHPG causes local synthesis of EF1alpha—seen from leakage around pipette tip.
(no increases in Arc or CaMKII in response to leaky pipette)
Selective Activation of translation of different mRNAs depending on differences in the
signal transduction cascade.
Don’t know who does what to whom
Rats give better separation of molecular correlates than mice
Mice don’t show ERK Phos when LTP induced electrically but do show LTP
Signaling altered in FMR1 KO mice – reduced ERK phosphorylation in response to
shock learning task; time course of activation is different; WT returns to control level
after 3h while in KO ERK phosph remains elevated.
-------------------------------------------------------------------------------------------------Kim Huber
Synapse development
Model, endocytosis of AMPA-Rs => LTD
“LTD proteins” => persistence
FMRP as neg feedback to ihibit further translation
But mGluR stimulation of prot synth is not present in fmr1 ko mouse
Elevated “ltd proteins” in Basal state?
DHPG LTD is insensitive to Prot synth inhib in Fmr1 KO mice (also Klann)
WT mice have prot synth dependent LTD
Young animals, mGluR-dependent LTD, immature, not prot. Synth. Dependent
Surface/total ratio of AMPAR surface GluR2/3 unaffected;
Missed something here
KOs seem to have elevated “ltd protein” levels
Would they be cargo proteins?
I304N mutation [should we borrow some mice from J Darnell?]
[Spine anatomy?]
mGluR LTD is protein synth insensitive in I304N knock in mice
Theory: LTD proteins are “kissing complex” cargoes
Dendritic translation can regulate dendritic growth (e.g., Holly Cline)
Transfected KO slice DG cells (culture???) with FMRP-GFP; dual patch clamp recording
in vivo apparently
Punctate expression pattern in dendrites (Bassell like)
Reduces AMPAR synaptic transmission
*Are these LTD’d???
Synapse number? Change in mEPSC number (axon still a KO)
Reduced NMDAR conductance
Synaptic failure rate estimates release probability; decreased amplitude of successes –
indicative of changes in synapse number rather than changes in receptors on surface of
postsynaptic cell
EPhys says decrease in failure rate indicates decrease in synapse number
(is receptor hypothesis dead?)
RGG box mutant see decrease in receptor, protein synths dependent?????
I304N, receptors are spread out all over, not punctate expression;
Dissociated neuron culture – surface GluR1 – PSD95 (I think not punctate)
Suggests FMRP regulates synapse number via formation or pruning
Regulation of translation can regulate synapse number
Alternatively, FMRP may stim inhibitor of LTD
Target issue is rearing its lovely head.
---------------------------------------------------------------------------------------------Mark Bear – tests of the mGluR theory
“By mGluR I mean group 1”
NOW there is lots of mGluR1 in the hippocampal formation
Smith data; elevated basal prot synth in KO
Could we correct FXS by reducing prot synth with a mgluR blocker?
Cross mglur5 Hets with fmr1KO
Mglur5 expression rescued to lower level in het
Seizure (audiogenic); wild running, seizure, status epilepticus (c57)
mGluR5 hets; reduction of seizure
Weight gain: FXS kids tend to be obese; accelerated early growth; adolescent cross-over;
See same crossover in mice
Het cross (FMRk0 with 1 bad mglur5 gene) rescues
OD plasticity
Hubel & Wiesel
Doing a Quinlan expt
Visual Evoked Potential
Contra > Ipsi
MD, p28
3d Dep > closed eye depression
shrinkresp
Longer dep > open eye potentiation
Fragile x?
More rapid potentiation of non-deprived eye;
Het; impairment in dep eye depression
Cross – rescue – no difference in depression or potentiation in deprivation paradigm
Methionine incorporation in slices: Basal rate restored to wt levels in het??
Testis size phenotype – needs older animal; no rescue;
-----------------------------------------------------------------------------------------------Sumantra Chattarji –Bangalore India
Amygdala, fear memories; hipp= factual memories
Stress enhances fear memories; stress response neg fdbk hipp; amygd pos fdbk!!
Dendritic atrophy in hipp following chronic stress
Dendritic/spine hypertrophy after chronic stress in amygdala
Stress that did not trigger anxiety did not affect dendritic measures (amyG)
10 days of chronic stress increases spine density in amygdala
proximal spine effect; not in distal dendrites;
Plus maze measure of anxiety
Stress may enhance anxiety by altering spine synapses in basolateral amygdala
This might get to the heart of the stress matter; much more relevant to syndrome
Silent synapse formation following chronic stress?
Coefficient of Variation of epscs reveals silent synapses.
AMPA synapses get NMDA receptors; or NMDA comes with new synapses?
Record epscs that have both components, without Mg++ and with glycine
NMDA is added to new spines—giving rise to larger evoked response with stress in
amygdala
Fear conditioning in stressed vs unstressed rats:
Stressed learn a bit faster
Weakened shock level – weakened memory
Different context, fear memory is lost in unstressed rats at 24 h delay; stressed rats have
much higher level of fear memory in different context; loss of contextual memory so they
give a fear response; (is it stronger fear memory or weaker context memory?)
Link to spines in rat analysis; mgluR5 antagonists are potent anxiolytics;
DHPG LTD paradigm in amygdala; induces potentiation of epsps; mpep blockable;
anisomycin blocks late phase LTP
No effect on baseline synaptic transmission???
FMR-KO mice (with Mark’s help—my error not to work with him at SfN)
Spine density higher in basolateral amygd of ko vs WT mice. Both basal and apical
dendrite.
Basically Amygd mirrors Hipp but wrt fear
KO – anxiety lower in plus maze and open field?
---------------------------------------------------------------------------------------------------Oostra
Cerebellar deficits, eyeblink experiments
Big deficit in FXS - % response much lower (males)
Females, smaller deficit but impaired
LTM, 6 month delay: saving in controls and patients but still a large deficit in males and
asymptoting lower
One Pt did exceptionally well, better than mean of controls; “exceptional case”; why?
90 IQ; mother ambitious to stimulate learning starting at a young age.
FMRP expression in hair roots (same lineage as neurons): (affected – below 30 %
expression) 6%! FMRP in blood – 18%
=> Deletion, taking out repeat plus a few bases on each end; still able to make protein
(start site present)
Pt has 3 genotypes; mosaic?; full mutation; premutation and deletion
=> No correlation between FMRP expression and phenotype
Rescue of phenotype; YAC transgenic into KO – overexpression, Nelson paper
Does it rescue eyeblink? Yes, improved acquisition to control levels;
Prepulse inhibition of startle diminished in traditional KO; MPEP, LY367385 (mGluR1
antagonist): Mpep partially rescues phenotype. Not a learning phenomenon.
MPEP Impairs eyeblink conditioning below KO level; LY data not in yet
MPEP blocks induction of LTP in WT mouse neocortex (Vignes et al., 2005), may
impair other learning tests (not to mention early development)
Bear: could mpep have an analgesic effect?
Spooren: MPEP blocks associative conditioning
***Propose to Katie; mpep and barrel development/spine development; Aaron to
supervise for 2 years; basic control, then enrichment (frostig?)***
-----------------------------------------------------------------------------------------------Cox
Explain components, e.g. fiber volley
M/cPG all mglurs; blocks cortical ltp in wt
MPEP also blocks ltp in WT
All data in 14 – 20 day old animals
Neuronal excitability studies:
ACPD general agonist bath applied; depol and hyperpol responses across subjects
(neurons)
KO – see only depolarization, no hyperpolarization (yet)
Increase overall in net excitation, consistent with seizure phenotype
---------------John Larson
(slow speaking style; understandable but paced slower than preferable)
Theta burst potentiation
“associational connections” in layer Ib of olfactory cortex
I/O curves – no KO effect on baseline
No paired pulse differences
Significant reduction in LTP in KO – 6 mo old
Younger animals no effect; older animals dramatic effect
(GABA-A receptors blocked; same effect without blockage)
Effect appears monosynaptic; no indication of interneuron effects
Effect specific to anterior pyriform cortex; not in hippocampus
Olfactory learning set; smell paired with reward; FMR1KO mice show impairment
(haven’t studied mice at the age that LTP deficits occur)
---------------------------------------------------------------------------------------------------Bureau
L 4 cells, whisker barrel cortex; anatomical columns
Layer 2/3
Laser scaning photostimulation. Allows mapping of synaptic functional connectivity of
all cells in focal plane in a slice of tissue. Excitatory circuits only.
Average connectivity measure
WT vs KO; ko weaker projection from layer 4
Between barrel cells (septae) – no difference in
Axons more spread out in ko than in wt—mistargeting of axons
Don’t see any difference in dendrites – this is at day 13-15
Differences disappear in later development
Abnormal targeting of L4 cells to septal regions in KO—they infer a weaker synapse
strength because there are more axons and not more functional connectivity
Clip whiskers pnd9 – strengthening of projections (Layer5a=>Layer 2/3) – weakening of
others (L4>L2/3); KO mouse only shows the strengthening; block of plasticity
Gary B: local effects: Netrin?
Mark B: deprivation induced depression in layer 4
-------------------------------------------------------------------------------------------------------
Bob Wong
mGluR induced epileptogenesis
1. Hypersynchronized, 2. inter ictal bursts, 3. ictal discharges (spread in space and time
with development of epilept.
3HpG (group 1 agonist) induces bursts propogated via glutamatergic interconnections
Receptors: NMDA, AMPA, Kainate, mGluRs
Interictal > ictal
Bicuculline increases interictal discharges; lasts 4 hours
DHPG prolongs inter ictal – permanent transformation
KO – DHPG transforms to Ictal Discharges
FMRP stops the signal transduction, blocks system from inctal discharge spread
Induction requires protein synthesis;
WT: Bic + DHPG induction is blocked by Anisomycin
Significant rise in ERK phosphorylation – is it nec. For induction?
DHPG > perk
Can block ionic GluRs and occlude seizures but still get induction
Blocking MKK blocking ERK phosphorylation blocks induction
KO: without anisomycin, Bic + DHPG > epileptogenesis
mGluR5 alone induces epileptogenesis; mGluR1 alone won’t generate epileptogenesis
Assumes ERK drives translation
DHPG alone can transform interictal to ictal discharges (???)
Fmr1KO – completely missed this
Maintenance
DHPG prolongs induced discharges; MPEP blocks maintenance (also mGluR1 blocker
blocks maintenance)
WT: There is a current turned on by DHPG (ImGluR); DHPG turns on an associated
channel; stays persistently opened, even after DHPG washout
Induction: mGluR5 agonist; NMDA-R unimportant; ERK phos is necessary;
In KO, ERK-p is necessary; mechanism of induction appears to be different from WT
Maintenance: WT: mGlur1 > mGluR5; persistent ImGluR is the basis.
-------------------------------------------------------------------------------------------------------Min Zhuo (Toronto)
Pain pathway – projections to anterior cingulated cortex
LTP, pairing pain pathway stim with cortical stim
NR2A, B both required for LTP (Roche compounds); also NR1
Postsynaptic expression; NMDAR current
Cond fear paradigm; elect stim of cingulate ctx or L 4 Ant cing Ctx (ACC) gives cond
fear memory to tone or context; hippoc. Or somatosensory cortex serves as negative
control.
Receptor medicine cabinet; NR2B
Trace fear memory; 30 sec between tone and shock; KO learn more slowly
ACC LTP is abolished in FMR1KO mice
----------------------------------------------------------------------------------------------------Richard Paylor
Assays for anxiety:
Marble burying
Genetic background influences on FMR1
2 show reduced anxiety; 1 strain shows it increased
1 shows increased social interaction, 1 shows it decreased
etc.
Genetic modifiers:
(potential therapeutic targets)
mGluR5 Het cross
MAP1B
This is actually a modified cargo hypothesis project. What a nightmare; few rescues.
MAP1b
Paired pulse inhib; map1b rescues over-inhibition ko phenotype; however the ko mice
have better ppi whereas humans have worse ppi
mGluR5
OFA; KO high; het does not rescue
Anxiety OFA; exacerbates phenotype
Marble no; light dark anx no
Acustric startle, no rescue
Ppi Fmr1 enhanced; reduced a little in het – only sign of a rescue
***if not rescuing but rescuing seizure, neural phenotypes, what does that mean?**
at threshold; not enough dosage change
compensation for het effect?
Phenotypes to come will show effects
Wrong genetic background
***Me: is behavior a more sensitive test of rescued brain function?****
FXR2 has several rescue effects ppi, cond fear
----------------------------------------------------------------------------------------------------Carolyn Smith
Regional glucose metab increased 26% in FMR1KO
Hypotheses: hyperexcitability; unstabilized synapses?; long, thin dendritic spines?
Metabolic abnormality.
**how long were animals adapted to the conditions during the experiment?
Protein synthesis, regional: elevated in some limbic regions in KO (CA1, 2, 3); 2 of 10
neocortical areas showed sig. elevations in KO
Hypothalamus: PVN, SON, LH, SCN increased in KO
Various thalamic nuclei
**Any areas where WT > KO?
Spine length increased in CA1, SCN [PVN?]
MPEP reduced prot synth in both WT and KO; not statistically significant;
I hold to my original evaluation. Marian D lives.
--------------------------------------------------------------------------------------------------------David Nelson
FXR influences on FMRKO mouse
Neuronal role for FXR1 (vs complete null)
FMR1 influences per gene
FXR1 KO lethal, muscle abnormalities;
FXR2 KO
**Could I get your slide of the conditional ko’s—about 3rd or 4th slide of talk**
cerebellar conditional Fxr1 ko shows impaired rotarod
Jongens circadian deficit in flies.
Double KO FXR2 shows rhythm disappearance
FMR1 O is rhythmic, FXR2 or double KO blows out the entrainment
Much worse than Per 1/ per 2 ko – don’t entrain
Double KO or FXR2 alters per1 circadian expression pattern
Also alters pattern of other clock/rhythm genes—different patterns of expression for
different genes
Double KO shows profound LTD (hippoc)
---------------------------------------------------------------------------------------------Warren 2000 compound screen
Rescue on lethal food
*GABA, MPEP, *Nipecotic acid, *creatinine, clomihene citrate; *isopilocarpine nitrate,
ergonovine maleate;
Mushroom body defect rescued by* G, C, I, N (at optimum dose)
Futsch overexpression defect rescued by CGPN
Newborn screen, hypermethylated CGG region, blood spots
Bisulfite conversion of cytosine to thymidine; methylated cytosine doesn’t convert
Probe with fluor and quencher; pcr probes
Also multiplex klinefelter and turner syndrome; 5Cents/screened individual
Said MPEP was not one of the drugs screened? In answer to question
----------------------------------------------------------------------------------------------------Bilbe
Monogenetic drive; environmental overlay
[not really monogenetic]
Gene therapy making a comeback; went out of fashion due to regulatory issues
Gene locus modulation; histone deacetylase modulators (HDACS); histone methyl
transferase inhibitors
Intervention in the FMRP pathway (mGluRs)
Hyperglutamterticity => anxiety (MPEP)
Lilly, mGluR2 – anxiety; was way ahead of mGluR5; found to be convulsive in phase 3
trials
Novartis has a mGluR5 antagonist in Phase 2; good indications
PSD95-NMDAR complex, NMDAR antagonists (Memantine)
AMPA agonists (Cortex withdrew CX516)
Glucocorticoid antagonist??? Write Graham***
ERK—lots of compounds available, animal screened
mTOR (Klann)
PAK inhibitors exist—should we take a look at this in mice? Also p-ERK blockers.
Talk to Graham****
----Basic approaches:
Understanding role of the protein
Need animal models
Need translational models for drug action: KO mouse is a blunt tool
Diagnostic tools/Biomarkers; IJ is hot
Clinical markers; new endophenotypes (sleep? Learning and cognition?)
Interplay between clinical and preclinical scientists
Drug discovery > proof of concept > translational medicine
**IJ go ahead on patent**
KO rats are coming; less straightforward than mice
Ben; a KO rat could be made for around $10K in Holland
---------------------------------------------------------------------------------------------------Gasparini
PET imaging agents for the mGlu5 receptor
Ligand displacement assays—show selective receptor effects; bypass nonspecific binding
Mpep analogs with high affinity (Merck)
Fluorinated derivitave for imaging (Hamill, Synapse, 2005)
Monkey has mGluR5 in cerebellum by displacement criterion; rat & mouse don’t
ABP688 – excellent mGlu5 antagonist
Great explanation of how displacement assays at high brain resolution can assess quality
of a drug and its use to determine receptor occupancy of other antagonists
The drug guys NEVER put their slides on someone else’s computer
-----------------------------------------------------------------------------------------------------Spooren
Fenobam, non benzo anxiolytic
(drug spy stories)
elevated plus, stress induced hyperthermia, marble burying, social exploration;
conditioned emotional response, fear potentiated startle
public information approach; mimic that avoids a patent
random screen of chemical library; binding assays, functional assays.
Hits – high affinity for receptor\
Fenobam was a hit
Atypical anxiolytic; no clue as to mechanism of action
Clinical development stopped for unknown reasons; no sedation, no interaction with etoh,
benzodiaz receptor-independent
Kd Bmax, slightly less potent than mpep
Clean; selective mGluR5 affinity
Non-competitive receptor antagonist; inverse agonist activity at mGlur5
Same receptor pocket as mpep
Stress-induced hyperthermia blocked
Vogel conflict; punished drinking
CER
Geller Seifter fear conditioning test
Looks inferior to mpep in general
Poor & erratic PK (Pharmacokinetic) profile
Varation in exposure
Excessive, extensive metabolism;
Poor correlation of plasma levels with therapeutic effect
Side effects; nausea, insomnia, dizziness, agitation
PoC for mGlu5 antagonists as anti anxiety agents
--------------------------------------------------------------------------------------------------Berry-Kravis
CX516 – no efficacy on memory measure or any assessment
Li inhibits pi turnover – parallel to mpep
4/5 showed improvement; 4/4 showed p-ERK effect
-------------------------------------------------------------------------------------------------Randy Carpenter – seaside therapeutics – sention – mGluR5 antag
Small financial returns
Regulatory hassles
Goal: P o C on drug
Funded by Wealthy donor with nephew having FXS
Collaborate openly with anyone
Patents for mGluR1 as treatment for FX disease
Merck had stopped mGluR5 program
Licensed to Seaside
In vitro: characteristics of ligands look good
In vivo: criteria behavioral assays; side effects; tox;
Production scale-up
Efficacy in audiogenic seizure model (Bauchwitz)
Other monogenic disorders
Autism (5%)
Prkcb1 > PP2A >V Mek < Patented
Graeme – will run screens on ideas – tools for biochemical or other assays related to
fragile X (ERK compounds?????)