Download Banbury notes 05 - University of Illinois Archives

Photo of all people in the lab
Katie C
Head-banging
Mike T
Autism – almost all FX have elevated autism scores (CARS)
Anxiety – separation, panic (difficult situations—also can provoke seizures), social
phobia (gaze aversion), OCD, [PTSD?—emotional memories etched deeply]
Arousal—Hyperactivity, worse in early childhood
Attention—poor at all ages; significant capacity to attend to specific tasks, difficulty in
shifting set, aberrant motivational structure—need to understand this for experiment
design, typical reinforcement doesn’t work.
Affective lability—extremely unstable mood; unlike bipolar disorder, responds to
specific events around them; stimulants for arousal can induce early onset of mood
instability
Aggression—usually maladaptive responses to stimuli, events; reactions to stress
Randy, Reiss study, myelin abnormalities in FXS
Paylor
Hyperactive, LOW anxiety
Tube test, submissive
Direct interaction More interactive
Partition test, normal interactive
Partner, cage familiarity; initial social anxiety then over-social interaction when in a
familiar cage
Possible genetic background effects. Working on 57 X FVB
Genetic background effects on anxiety and social measures
Big background effects; some backgrounds normal behavior; some exacerbate pathology
D-2 mice; elevated marble burying (anxiety) and stereotypy
Backgrounds can affect direction of abnormality
 Overall KO effect across strains
Learning – conditioned fear;
Past work has emphasized hippo- dependent learning
Signaled leverpress avoidance (non-hippocampal):
KOs never learn to do this – none of KOs show any learning
In rats, Learning rate correlates with BDNF level in hippoc/ basal forebrain
MPEP – increases open field activity in a dose-dependent manner; decreases marble
burying
Flesinoxan
MPEP increases activity (ofa) on first exposure in both KO and WT; on second test
(familiar), effect on WT, not on KO
-----------------------------------------------------------------------------Porges; Nuc. V projects to n, ambig in moose, vagal loss in KO
-----------------------------------------------------------------------------Oostra:
Cerebellar deficits: Motor learning
Spine density, arborization normal in 8 week old mice
Spine length (head and neck) elongated
Cerebellar LTD induction is enhanced in the KO mouse
CPCCOet blocks LTD – mGluR1-specific antagonist
Eyeblink conditioning (delay—non-hippocampal): airpuff/tone: KOs basically fail
entirely to learn – ultimately some learning but far behind
Lesion dentate nucleus in controls reduces them to KO equivalent, implicates cerebellum
specifically
Conditional KO – CRE recombinase : Neo (hypomorph—low expression); CKO; KO
Can turn on full expression of protein late in life;
Want to study this cond KO;
Also want to study MPEP effect
Also, could we use ERPs to study trace vs delay and involvement of other
structures;
DCVs (from drosophila) elevated?
We have defined the phenotype well enough to use it in evaluating effects of various
kinds of drugs
Essential answers we still need to get; does phenotype reverse with onset of FMR1 in
adulthood, is phenotype established by withdrawal of FMR1 in adulthood?
Want to study axons, connectivity among systems
Rationale: why anatomy and molecular biology go hand in hand. Experiments to
separate developmental expression from later, etc.
Paylor, no one else gets delay effect in mice; others have published human effect
Selective KO in PCs
PC-specific KO is like full KO in eyeblink conditioning
Prepulse inhibition of startle: MPEP treatment reverses deficit to normal.
Patients—eyeblink conditioning
Listening to (watching) dvd – adolescent => adult
Headgear OK on patient – “no complaints”
FXS 25% CRs; Control >75%
Clear phenotype that can be measured on a single patient
Early efficacy detector for phase 1 trial (Will Spooren)
--------------------------Broadie
dFMRP
global regulator of neuronal growth
clones of FX- neurons at various devel periods
single mutant neurons in a normal brain
mushroom body neurons
Unipolar in WT; multipolar in mutants, axon like
Overexpression mutant, reduction in the small number of processes that do emerge
Negative regulation of AXON branching and growth
-Propensity in absence of dfmr to be more highly branched
-all classes of neurons: motor, sensory and central (overexpression simplifies;
underexpression promotes growth
presynaptic diff’n
photoconvertible marker for em localization of single cell processes
overproduction (or under release—increased density) of sv’s –twice as Nv
NMJ, same phenotype, elevated density of synaptic vesicles
cytoskeletal dynamics
dfmrp binds to futsch mRNA; futsch upregulated in null mutant (MAP1B)
futsch regulates cytoskeletal (microtubule) stability
stabilization is via acetylation of tubulin; reduced in null mutant
dfmr/futsch double mutant restores synaptic defects
microtubule stability in sperm tail is disrupted; immobile sperm; infertile
9+2 microtubule arrangement in WT; middle pair lost in dfmr null mutant; progressive
loss in development
monoamine synthesis, DA levels
2d westerns to examine proteins cy3 cy5
Differences very subtle
Merge: small number of changes
1500 prots; 33 change;
changes in both directions in mutant vs. wt
Punch –tryptophan hydroxylase (5HT) – rate limiting
Henna – phenylalanine and dopamine hydroxylase (DA)
Upregulation of enzymatic activity via posttranslational modification (P) in null,
upregulation of DA, 5HT levels
Dfmr is present in all kinds of neurons;
Increase in DCVs per bouton
Candidate Genes (DmGluR)
Mutate mGluR null
Expressed at synapse
Short term facilitation elevated in null mutant
Dfmr null is comparable in direction and size of effect
Looks like mGluR are in the same pathway, based on phenotype only
Novel Genes (modifier screens)
29 suppressor genes—suppress dfmr overexpression phenotype
e.g. increasing or decreasing dfmr expression
some are point mutations in dfmr; still early days
-------------------------------------------------------------------Porges
Polyvagal theory
Evolution
3 circuits
vagal myelinated system & Brainstem—striated muscles of face and head
Neuroception, danger threat engages these circuits
Detection of risk => mobilization of these adaptive neural system;
If vagal system fails, can pass out
Safety – spontaneously engages others, eye contact, ingestion -- myelinated vagus
activity, sucking, rocking
Life threat, death feightnhing death
1. Old Vagus, autonomic system of viscera, immobilization
2. Mobilization system, sympathetic nervous system, mobilize, engage movement of
large muscles
3. Corticobulbar, new vagus, myelinated at birth, safety, striated muscles of face & head
Social engagement system; cortex, brainstem, CNs V, VI x, x1
Heart rate rhythms, autonomic component of social engagement
Lifting eyelids (gaze) also promotes hearing of human voices; stapedius muscle in ear
Fight, flight, freeze systems – turn off to turn on social engagement
Central N. Amygdala -> PAG -> fight and flight patterns
Neuroception (safe), STS, shuts down central amygd, allowing motor cortex, medulla
system to take over engagement
FXS, features of are all examples of failed neuroceptive state (hyperarousal, hypoarousal,
difficulties in listening, sensory defensiveness, poor eye contact, anxiety, low cardiac
vagal tone)
Intervention to trigger soc eng syst enhances performance of autistics
Moves facial gaze into normal range (eye fixation to upper face)
HR patterns – intervention changed vagal input to heart
Pretty good fit for starting point hypothesis—social engagement system fits a lot of the
deficits of FXS
-----------------------------------------McCracken
Drug therapy for fragile X outline
Treatment targets & measures
Success
On to FXS
Diversity of treatments in use for autism; SSRI, Neuroleptics, stimulants, anti-epileptics,
alpha-agonists
consult psychiatrists?
Chose neuroleptics, need some evidence base for use of a treatment that powerful
Autism: 3 principal dimensions: communication, social interaction, repretitive behavior
as 3 core facets of disorder
Parental complaints – serious, often dangerous, always disruptive behaviors
Aggression, hyperactivity, self injury
Risperidone selected – 2nd generation antipsychotic (neuroleptic); reported beneficial in
case reports, reducing aggression, agitation
Dependent variables
Parent report has high expectation, can “erode” small drug effect – placebo response
Aberrant behavior checklist, Irritability scale
Scale improvement of much or very much improved required for criterion of efficacy
Double-blind placebo-controlled study
Crossover design
Dramatic effect (parental ratings of irritability). Clinically meaningful (physician ratings)
Genotyped polymorphisms in targets of Risperidone 5-HT2A receptor promoter SNP G
vs AA. AA showed much better effect.
Improved stereotypy, lethargy-social withdrawal (not significant w/ corr for mult tests),
hyperactivity
No effect inappropriate speech
Obsessive-compulsive scale, progressive change across study.
Cognition: Battery of tests, shot too high, only 40% of kids could complete the tests
Relapse with treatment withdrawal—low in risperidone group
Summary: keep endpoints focused, protect blind nature, assess cognition
Methylphenidate
Teachers and parents report
More modest effect sizes (relative to ADHD effect)
Social withdrawal increased by methylphenidate
“responders” had “a great deal” of improvement; individual variability profound
50% of sample!!!!!!
Isabelle Boutet
Tests: sensitivity to improvements and side effects; test retest reliability; probe a targeted
function; allow inferences to underlying brain regions (and tests of)
And PRACTICALITIES: duration, mobility, motivation expertise cost
cognitive level—few tests aimed at low cognitive range
Spatial frequency grating contrast sensitivity
Very interesting, develops then falls with age (optical and cortical effects combine)
Magnocellular pathway (high speed flicker), parvo—nonflickering
Parvo: no effect of FXS
Low spatial frequency (magno) deficient in FXS 0.3 cy/deg
Why specific to Magno? Could this reflect
Magno, visual control of action dorsal stream
Vs. ventral, parvo, object identification path
Non-verbal associative learning
Adapted from monkey testing (Mishkin)
WGTA object discrimination task
ODL, one object always rewarded (nickel)
OD reversal (inhib prev rewarded response)
DNMT – recognize new object
Visual spatial
Egocentric Disc Lng (leftmost or rightmost identical object)
Delayed non-match to position identical object
ODL, impaired
ODR, very impaired
DNMTS, no deficit at 5 s delay
EDL good
DNMTP – extremely poor
----------------------------------------------------Berry-Kravis
CX516 Trial
CARS, GARS,CGI etc; Memory for words; Peabody PVT attn; impulsivity; vis spat
memory at outset and repeated at week 5
4 weeks on drug
No serious adverse events
Tolerate repeat visits and testing
Liittle statistical evidence for improvement on any of the cognitive tests; often tests gave
good, reliability, reproducibility,
Incomplete analysis at this point.
Fairly non-potent ampakine. Treatment period may not have been long enough and dose
should be 3X higher.
Some hopeful signs in individual protocols
Some tests are simply too difficult for many of the subjects.
---------------------------------------------------------Hagerman
Lithium discovery in spas good for mania
Lithium decreased in use with atypicals avail;
Lithium and neuroprotection; increases bcl2 levels neuroprotective protein; decreases
pro-apoptotic proteins
Good list of lithium effects
Li downregulates mGluR5
Lithium affects PIP pathway
“enhanced mGluR5 activity that occurs in FXS…”
May decrease seizures
Cog activity, etc,
Inhibitory control, arousal regulation (PPI startle; incr vagal tone)
Phase 1: 2 mo trial; ages 6-25; overlap with pruning process
Phase 2; year long trial – experimentals are those who respond to li in phase 1;
controls are those who do not respond or tolerate lithium during phase 1!!!!!!!!!!!
Behavioral Outcome measures: various scales of aberrant behavior; clinical impression;
Cog outcome measures: Range modified from L B-K results
Vagal measures; eeg; PPI; social challenge; parental diaries (seizure)
Not started.
Li inhibits phosphorylation of MAP1B
TUESDAY
J Darnell
KH2 domain
I304N protein does not associate with polyribosomes- substitution is in RNA-binding
pocket
Majority of FMRP is PRA assoc – heavy polyribosomes
(nova 1 has KH1 and 2 domains)
in vitro RNA selection
SELEX
Random 52 mer
Enriched for those that bind protein
Intramolecular g-quartet
Targets RGG box, not KH domains
Re-do selex, got ligand specific to KH2 domain
Single point mutation abolishes binding
GaBA-R is an example
Molec details, tandem pair of loops critical to binding, mg-dependent binding
The loops form “kissing complex” with each other by base pairing
Stefani et al binding to polysomes
G-quartet ligand does not compete FMRP off
Kisssing complex (kc) ligand competes FMRP off of polyribosomes
Hu protein not competed off PRAs
RNAs with kc could be important in FXS
Large loop in fmrp not present in dfmr, fxr1, 2, chicken FMRP
Iso 7 has not exon 12
Iso 1 has loop
Exon 12 is critical
FXR1 and 2 are competed off PRAs by kcRNA
G-quartet binding by fmr1 mRNA, not by FXRs
Only FMRP recognizes G-quartet, not other FXRs
They have a knock in of I304N Mouse, interested in collaborative phenotyping
What is the implication of a largely procedural learning deficit?????
Polysome profile is not FMRP-dependent (we knew this—KO still has polysomes
Experiment???? What mRNAs are not in PRA fraction in KO’s
---------------------------------------------------------------Fallon
Exp dep transcription
Devel reg “
Repeat expansion methylation regulation
Experince transl regulation
Transl reg of cargoes
Regulated degredation
DR mice/ light exposure at p60—transient synaptoneurosome expression of FMRP at 30
min, gone by 60 min (become sighted)
P45 rats response is faster
Increase in neuropil expression, also increase in soma expression
NO CHANGE OCCURS IN FMR1 mRNA; this is translational regulation
NMDA-R – dependent
Disappearance rapid, suggests degredation regulation
15 minutes of fame – epoch of synaptic plasticity
New, Olfactory system
Highest level of fmr1-FMRp expression
Naris closure
Experience- amyl acetate (banana)
Neurogenesis
FMRP expressed in perigranular cells, most cells in OB
Developmental expression peaks at P20
Particularly in granule cells
Experience, odor exposure increases fmrp/fmr1 in olf bulb, biggest effect at p4, gone by
p30
Odor deprivation (occlusion) reduces fmr1 expression in bulb, p 5 and 20
Mech of transcription?
AP2 site in promoter
Developmental functions of AP2 sites
AP2 alpha binds to FMR1 promoter
AP2alpha KO reduces FMR1 expression; severe craniofacial abnormalities
(six3cre mouse)
embryonic reduction of fmr1
(no major birth defects in fmr1 KO of course)
later fmr1 transcription must use different promoter sites
dominant negative ap2 alpha suppresses fmr1 expression
(Not fxr1)
does methlyation regulate fmr1 normally?
Dynamic regulation of FMR1 p0 methylation in olf bulb; demethylated at p20. Meth
again at p30—not static, dynamic across development
Differential time course of methylation of different cytosines at different locations in
promoters including ap2 site – transient de-methylation
Epigenetic regulation of methylation in development
-------------------------------------------------------------Klann
Huber enhanced mGluR LTD
FMRP as feedback inhibitor; see fig in his Learn Mem 2004
paper
LTD induction; western blots CA1
Phosphorylation
DHPG-induced increases in FMRP levels
LTD induction
Declines to baseline over 20 minutes (like fallon’s vis cx)
Anisomycin blocks, prot-synth dependent
FMRP in proximal dendrites
Not clear if synth in soma and shipped, or synth in dendrites
PDK/mtor pathway require both mGluR1 and MGluR5 – both appear to be involed in
LTD
DHPG effect is enhanced (on FMRP levels) by mGluR5 antagonists (inhibit
degredation?)
Very fast rise and fall of DHPG, decline blocked by MG132 (proteosome ihibitor)
Very rapid synthesis and degredation
Makes IJ’s finding even more interesting; only the fast effect occurs in WT; slow effect
in KO could be completely different phenomenon
Increased ubiquitination with LTD – degredation
MG132 allows LTD to return to baseline; LTD is dependent on degredation of FMRP
The retention of plasticity requires that FMRP go away!!!!!!!!!!!!!
FMR1 KO has enhanced ltd, Bear
KO overexpression transgenic has inhibition of LTD
Predicts synapse structure changes assoc with LTD will be inhibited as well
PSD 95 increase produced by DHPG (Todd) G quartet protein
Not seen in KO
We need to be on top of this
DHPG > CAM2 levels, also not present in KO – also looks like a rapidly degraded
protein
Phospho-ERK increased by LTD/DHPG; no further increase in KO
Inappropriately regulated translation in KO.
“not in agreement with mGluR theory”
Not the area in which to compete
Doing 2D gels like Broadie
Some proteins increase, some decrease
Need immediately to do a Don Hunt experiment????????
Sees differences among phosphorylation of proteins in 2d westerns
Several proteins are chronically elevated in KO vs WT
Van der Klish in vivo mGluRs “prime,” NMDA-Rs induce
IJ, could be translocation of FMRP to granules like stress granules (Khandjian)-------------------------------------------------------IJ fxr1 on slide
Receptor for drug target must also be on lymphocyte (Liz). Imperfect screen, potentially.
Mike, compare pathways in autistics, etc.
Arrange through CAN to get autistic lymphocytes or blood samples!!!!!!!!
--------------------------------------------------------------------------------------------------Discussion: Rapid degredation of FMRP in brain (if it is ubiquitin, sumo, etc. degredation
rather than sequestering of protein in granules.
Is it a particular isoform? Justin says the 3 isoforms all go together in his sensory
experiements
Kissing complex as blocker of function of FMRP??? Using electroporation in cultured
cells (slices?)
Fallon. Degredation is also fmrp-dependent.
Methylation defects; Ben has methylation mosaics and unmethylyated people who have
children that are fully methylated
Demethylation of repeat without replication – Oostra
MicroRNAs assoc w FMRP complex; dependent on state of phosphorylation – something
I need to know more about
----------------------------------------------------------------Hayashi
Altering actin remodeling
P21 –activated kinase (PAK)
Pak3 mutation linked to mental Xlinked retardation
FXS like dendritic morphology
Dominant negative PAK; forebrain-specific expression postnatal
Downregulates PAK# 50%
Golgi: Fewer spines 22%; proximal to distal dendrites—whole extent
Spine length shorter, head larger – opposite of FMR1 KO
No branch number differences
EM
Enhanced ltp, reduced LTD (matches shape, sort of) in cortex
No phenotype in spine morphology in hippoc
Morris task: no retention deficit at 1 day; major deficit in retention at 3 weeks
No deficit in theta burst LTP in hippocampus. Suggest cortical LTM deficit
Cortex enhanced LTP
Rac1 target of dfmr Gao
Crossed PAK FMR1 rescues longer psd length in PAK3 mutant
More Larger perforated synapses in PAK mutant, also reversed in FMR1 KO
Genetic interaction between these 2 molecules?
(no evidence except at level of phenotype)
IP, Pak pulls down FMRP – colocalized in cytoplasm
Model: PAK > actin polymerization > spine formation and stabilization
FMRP promotes synthesis of other targets at spine: Map1b, RAC, PAK, Arc, alphaCAMKII
Maybe PAK regulates FMRP?
No Evidence for regulation
PAK phosphorylates Erk (Weiler talk referred to)
-----------------------------------------------------------------Bear
Regulation of prot. Synthesis near synapse is an uncited “assumption”
Get slide, distribution of mGluR1 vs 5 (forebrain)
Relates symptoms of fxs incliding hyperalgesia and gut motility to mGlurs
GluR internalization (ampa)
Triggered by mGluR, DHPG; protein synthesis dependent
Shows puncta constituting individual synapses internalized in cultured neurons
KO much greater effect of applying DHPG on receptor internalization as a mechanism
for LTD; blocked by anisomycin; in WT mouse response is minimal (despite LTD???)
Lateral amygdala synaptic plasticity in thalamic afferent inputs (done with Chattarji)
DHPG increases epsp; synaptic potentiation at this synapse
Anisomycin blocks; ActinoD does not block > translational from pre-formed mRNA
Assumes reversal of internalization of ampa r’s underlies this; the type of synapse
determines LTD vs. LTP
OD plasticity in visual system of mice
Monoc occlusion reduces contralateral VEP
Deprivation at 4 weeks reduces contra VEP amplitude
ODom shift
Dep eye rapidly reduces response
Non-dep eye potentiates (over days) NMDA-R dependent
Think changed subunit composition NR2A/2B ratio increase
KO mice have smaller VEP effect (but clearly present)
Non-deprived eye response goes up much faster in KO than in WT
Changes in NR2B in cortex.
PSD 95 higher in KO—
------------------------------------------------------------Bauchwitz
MPEP
Blocks pain
Phenotypes
AGS
OFA central square activity
Inferior colliculus nec to seizures
AGS much more easy to evoke in FVB than C57; harder yet in hybrid
By 30 days all but FVB mice “grow out of” seizure susceptibility
More MPEP required for more seizure sensitive strains
OFA CSA
KO more willing to go out into field; MPEP blocks this; less of an effect in WT
Odor sequential order learning task – 2 sequence plus distractor odor – new odor set on
each trial
No actual data presented
Using hybrids for most experiments
Tolerance to MPEP; pre-administration rapidly increases tolerance (reduced efficacy) in
seizure model (5 days); wild-type animals do not show tolerance; KO specific tolerance
MPEP reduces mGluR5 level in inferior colliculus of KO more than in WT
Fetal human stem cells
14 week human brain tissue (neurospheres) contain stem cells
Map2 neuron marker – all cells express early, ditto GFAP
Astrocytes (and neurons) express fmrp and mglur5
Fragile X culture neurons have reduced level of neurite present; WT larger neurons
No effect on astrocyte numbers; neuron number effect: FX lower, WT humans have
higher neuron to astrocyte ratio; 50% reduction in FX neuron number
MPEP reduces Neurite length in Both genotypes
MPEP reduces normal but not fx neuron size;
All MPEP responses are u-shaped dose-response curves???
FMRP negative regulator of map 1B
Li reduces inositol levels, downregulates PLC, etc.;
In FX mice, Li is an anticonvulsant – FX-specific
In culture from human es cells, Li decreases normal neurite length; increases fx neurite
length.
“We haven’t found a strain difference yet but we’ve only done the FVB.”
Katie: WAISMAN CTR has fragile X neurospheres available (see FRAXA website)
---------------------------------------------------------------------------------------------------Denman
Does mGluR5 mRNA bind to FMRP?
Binds in vitro
Biotinylated FMRP
Luciferase reporter
Atlas human neurobiology array (lots of mGluRs)
Lights up to mGluR 3, 5 and 7; all but mGluR2 on array
Motif
3’ utr of FMR1 binds specifically with FMRP in vitro
localizes
mGluR5 RNA is enriched in synaptoneurosomes; other dendrite RNAs are often not;
suggest it is at synapses and translated there
HeLa cells transfected with mGluR
KO mGluR5 levels:
Significant decrease in mGluR5 protein levels (65% reduction in cerebral cortex at 8
weeks); total cortex homogenate
FX transgenics have even more reduction of mGluR5 protein
Protein but not RNA levels altered by FX KO
Is it possible that mouse mglur5 is not regulated by fmrp but human is????
---------------------------------------------------------Huber
KO LTD+
mGluR > endocytosis of Ampars > LTD proteins > persistent LTD
FMRP as negative regulator of mRNA translation
Predictions
More mGluR stimulated protein synthesis (Not confirmed)
Revised model
Mglur-LTD should be independent of protein synthesis in KO
(confirmed, LTD not blocked by ani in KO; is in WT)
Elevated LTD proteins or persistence of an immature form of LTD?
Immature LTD independent of prot synth, presynaptic mech;
Mature ltd postsynaptic, prot synth dependent
Revised model
LTD not saturated in KO? Maybe must have some internalization????
Hipp slice culture 6do ko or wt mice Darnells have GFP-frmp promoter; gene gun
transfection; light up transfected ko neurons; separate receptor currents with clamp;
Overexpression of FMRP in cultured KO decreases AMPAR mediated synaptic
transmission—no change in NMDA epsps—decrease in mEPSCs frequency but not size
suggesting removal of AMPA receptors—decrease in ampar mediated synaptic
transmission; same effect in wt mouse or rat cultures
We may want to get the constructs that Darnell and others have. Want a list.
I304N abolishes in vitro translation inhibition
RGG box not required for FMRP effect on LTD/synapses
I304N, no effect;
Clumps, punctate distribution of FMRP in neurons; also clumped in RGG-delete
Not in I304N – not in granules!!!!!!! Ben Oostra: smaller granules
Young synapses, NMDA-only transmission; FMRP might negatively regulate synapse
maturation; prevent expression of AMPA-Rs which is characteristic of mature synapses;
psd95, camKII as possible mediators; KO has more ampars
Can’t do slices after about p14; can’t do devel studies
-----------------------------------------------------------------------BASSELL
Shuttling, granule assembly, transport, anchoring, translation, retrograde transport
Granules contain FMRP in dendrites
FMRP in 74% of spines
Co-loc with synapsin
Dhpg induced FMRP in dendrites; blocked by mGlur pathway antagonists
Mpep decreased;
Rapid loss of fmrp from the spine with activation, (maybe rapid degredation), then
replacing fmrp with pre-existing pool in soma
KCl enhances movement of fmrp granules into dendrites from soma
Both trajectories, just like ours. Over 1 micron per second; kinesin-like rate
DHPG speeds FMRp movement into dendrites
Kinesin 1 motor; dominant negative KLC construct impairs DHPG enhanced transport of
FMRP granules into dendrites
DHPG induces assn of FXR1p with Kif5B (Kinesin heavy chain) – nice
Can see co-localization and do pull-down with antibody to KHC
Target mRNAs (FISH):
Map1B – clear association with fmrp
Staufen not interacting with Map1B (control)
Spine development; filopodia can transform to spines
Harris filo > shaft > spine
Cultured hipp KO neurons, hyperabundance of filopodial structures absent presynaptic
boutons (Antar)
KCl 30’ increases number of filopodia in WT; in KO no further increase produced
KCl increased number of innervated spines—in KO KCL drives synapses to filopodia
I304N parallels KO excess filopodia, loss of dhpg ncreases in filopodia
FMRP overexpression decreases filopodia
Dhpg synaptosomes increase CAMK2, psd95 in wt, not KO mouse
Fallon, why so hard to see FMRP at synapses in vivo?
Does fixation hurt immunoreactivity?
------------------------------------------------------------------------------Vanderklish
3 plasticities req local prot synth; mglur LTDmglur; LTPnmdar, BDNF induction TrkB
eif4g???
LTP prot synth “almost immediately”
LTP far outlasts half life of new proteins
Me: Are there some very long term stable proteins????
Differential translation; unique cocktails of proteins arranged by different initiation
mechanisms
Differential initiation – IRESs in several dendritic mRNAs: CAMD RC3 Dendrin, Arc,
FMRP
Cap-dependent, Ires balance regulated; aplysia elh involves switch from cap to Ires
translation
mGluRs strongly activate mTor & erk pathways
Granules (heterogeneous mRNAs, motors, silent state, can go into spines, translation
factors, actin binding proteins, etc)
May be perturbed in fr x synd
Sucrose gradient polysomes and other fractions; fmrp is in polysome fraction;
Granules are brain-specific
We should check our lymphocytes
Granules reorganized with stimulated translation; em studies
Kanai, 45 proteins in granules identified
Granules reduced in KO and restored by MPEP as seen on sucrose density gradient
Granules regulated by endogenous mGluR5 activity.
mGluR5 may regulate translation in behaving rodent
DHPG eradicates granule peak
All these things are done with immature neurons
15-25 ribosomes; 200-300nm in diameter
translationally silent granules > mglur5 > active polysomes
doing mass spec on proteins in granules – we need to hurry if we’re going to do this with
Hunt
DHPG causes some spines to elongate
Long spines have Fewer ampa rs – inducing ltd elongates spines
Synaptic uncoupling of FMRP granules triggered by mGluR initiates local synthesis
------------------------------------------------------------------------------Wong
Interictal bursts not seizure associated
Ictal discharges associated with seizures
1.How neurons become synchromized (Ionotropic GluR)
recurrent collaterals synchronize cells and spread excitation rapidly; glutamatergic
synapses; normally regulated by inhibitory neurons
increased ion flow increases synchronization
2.Evolution of short interictal to ictal
gluRs
DHPG opens, via mGluR pathway, a new nonsynaptic channel that allows neurons to fire
long duration bursts.
mGluR agonists > ERK pathway > protein synthesis > I mGluRV (newly appearing
channel, different from extant calcium current channels)
Once activated, this current will hang around for a while in absence of DHPG, resulting
in longer bursts of action potentials
Burst firing produces/is transition from ictal to interictal state
ERK activation is necessary for the current change (tyrosine kinase dependent); does so
via protein translation
Believes FMRP is involved; KO is more likely to convert to ictal discharges
3.Epileptogenesis?
One type prot synth dependent and regulated by FMRP
------------------------------------------------------------------------------------Jongens
KO fly
Arrhythmic in total darkness
Beta lobe midline crossover connection in brain mushroom body
Courtship/mating
Visual, pheromonal, behavioral cues
Deficit in naïve courtship in males; short intervals of courtship activity
Courtship with a mated female—female rejects—male learns to reduce activity; male
normally goes from high to no courtship; mutant flies show normal learning;
“memory” of this lasts about 3 hours; mutants show deficit in memory;
“cognitive” deficit in mutant fly
Neuronal
Over elaboration of synaptic terminals; excessive dendritic branching; synaptic
transmission defects.
Fly has 1 mGluR (vertebrates have 8)
MPEP and competitive antagonists; LiCl (blocks Plc-Ip3 pathway)
All mGluR antagonists rescue naïve courtship as does Li
Negative effect on rescue flies
Learning not affected by antagonists
Memory rescued by mpep
Holds true for all mGluR antagonists
Mushroom body defect is rescued if given during development!!!!!!
Does mpep during development reverse/mitigate neuronal and myelin
deficits in the mouse? This combines well with Ben’s/Dave’s switchable
onset construct
Aging memory deficits show up earlier in mutant; mpep rescues this
Is the mpep brain structure work published?????
Think about 3 parts—myelin; synapse morphology; devel process?
Are the apra targets that phosphorylate erk differently expressed in KO vs WT?
------------------------------------------------------------------------Spooren
Psychopharmacology, in press
Mpep vs diazepam?
mGlu5 – anxiolytic?
[The brain expn mgluR slide is Shigemoto, okazaki, japan]
Broad anxiolytic effects in rodent models of anxiety; compare with benzodiazepines;
Toughest tests: Conflict:
Geller Seifter
Cond emot resp
Vogel Conflict test
Mpep reported to disrupt spatial and working memory tasks
At conflict-effective doses, does mpep disrupt cognition (Morris;
DMTPosition)
Geller Seifter Conflict: punished responding; non rewarded responding; unpunished
responding; mpep overcomes tendency not to respond during punished period; [not very
functional???]
CER: Cue light signals rewarded lever press will be punished; test with no shock; mpep
increases responding; dzp similar to mpep in both the Geller Seifter and CER;
Punished drinking similarly increased by dzp and mpep
IJ what does mpep do to phospho-ERK?
Cognition:
Delayed match to position; responses increasingly incorrect with delays
Essentially no cognitive effect at doses with anxiolytic activity (does at 10 times the
effective anxiolytic dose)
Diazepam disrupts working memory at effective anxiolytic dose
Morris: Likewise mpep has almost no effect on cognition
Diazepam – severe cognitive disruption at doses clinically anxiolytic
Benzos also have severe interaction with alcohol
----------------------------------------------------------------------Toth
Psy traits: harm avoidance, reward dependence, novelty seeking, persistence determine
risk-taking > anxiety dimension
Genetic basis of pathological behavior
How animal tests relate: emotionality, avoidance, activity, arousal
Anxiety pathways
FXS hyperactivity, sensory hyperreactivity
Activity box 2 hours, KO >> WT “genuine hyperactivity”
Caudate DA reduced; methylphenidate reduces hyperactivity by blocking DA uptake
activating Caudate driving of motor function
{light blue-green slide background – virtually all colors show up well}
Central audiogenic pathway: PeriAqueductal Gray—pure brainstem seizure; not like
human seizure
Humans are very resistant to brainstem seizures and don’t exhibit audiogenic seizures
Auditory deprivation in rodent during critical period susceptibilizes them to acoustic
seizures
Pathway overlaps with startle pathway in brainstem
Startle deficit in KO; don’t habituate
Auditory is a more dominant modality in rodents
Prepulse – inhibition of startle elevated in KO mouse; reduced in human
GABA-B on Glu and GABA-A terminals; both pre and postsynaptic
Inhibitory both presynaptic and postsynaptic
Baclofen (agonist); KO mice respond more sensitively to Baclofen, normalizes KO
activity to WT levels
Rotarod not affected at equivalent dose levels
Baclofen short half-life, need minipump administration for multi-hour tests
Startle deficit in KO; minipump??? Baclofen
Reduced startle in KO reversed to wild type level by Baclofen chronic treatment from 2-4
weeks; a single injection at 2-4 weeks apparently does this as well, but only if tested
under drug?????
NOT a clear speaker;
Baclofen affects both motor and auditory systems, normalizing function
Good plug for a non-glutamatergic drug
Baclofen has anxiolytic activity as well
--------------------------------------------------------------------------------------Lauterborn
AMPa trafficking Less insertion into membrane in KO
>reduced glutamatergic/AMPAR function in KO
Ampakines
Trophic, neuroprotection, learning/memory, LTP, schizophrenia (?)
Screening for gene regulation effects in hippoc slice cultures
BDNF expression as dependent variable
(blocks LTD; enhances LTP)
Memory effects; spine density effects;
CX 614 induces BDNF briefly in both KO & WT
Greater induction in FMR1 KO
Also in cortex; lasts less than 48 h
Different effects of different ampakines in vivo and in vitro
Effect may be potentiated in animals in complex environment
Continued ampakine treatment: BDnf effect goes away as does increase seen in GluR1 in
response to ampakines; chronic treatment does not give stable responses; similar declines
in response to glutamate, carbachol; property of both WT and KO
This does not seem good for a therapeutic drug
How to prevent
Repeated dosing, on and off, maintains response
Recovery period and drug on period interact complexly
------------------------------------------------------------------Steve Johnson (Cortex)
Julie’s doses very high; well above therapeutic level 50 micromolar plasma would be
lethal, 50X typical ED50
New Ampakine CX717; binds a different site on AMPAR
50 X more potent; safety margin 50; half life 9h
Does a powerful ampakine enhance the effect of a complex environment on brain
anatomy rescue?
DMTS (Deadwyler, monkey), new objects on each trial; varying complexity (number of
incorrect unmatching distractors)
CX717 enhances performance
VERY IMPRESSIVE!!!!
------------------------------------------------------------Gasparini
PET imaging agents for mGluR5
Screening 960 compounds (now hundreds of thousands for such a search)
Identified SIB1757, refined to MPEP
Short-lived radioisotopes
Receptor occupancy => dose finding; treatment regimen
Determination of receptor distribution/expression
Patient selection, diagnostic, treatment regimen
Can look at competitive interactions—displacement from receptor, for example, to assess
specificity of binding
Can’t determine dose regimen from competition
Labeled derivatives: figure out where does not interfere with binding
ABP688 – highly evolved receptor ligand for use with PET mGluR5 detection—not
mPep
mGluR5 KO mouse exists (does it look like FMR1 KO?)
No specific uptake in brain
-----------------------------------------------------------------------------Shiosaki
Devel of mGluR5 antagonist SN107
Specificity, safe, access to brain, convenient for use, etc
MTEP better than MPEP: longer half life, better solubility
ESTATE of mGlur antagonists
SN107 lead compound
In vitro—potent and selective antagonist of mgr5
Vivo—active behaviorally
Oral bioavailability; half life 6-8h
BBB penetration
Safety
Chemistry
Dose response receptor occupancy (displacement) fairly linear plasma to occupancy ratio
MPEP MTEP both decrease locomotor activity; SN107 does not
Cog. Deficits? Impairs cognition at 75% R.O. – learning of a sequence of stimuli to space
relationship;; no effect at 30 mg/kg
Object recognition: no effect at 30 mg/kg
Repeated administration; tolerance to cog impairment—disappears
Needs Safety; CMC?; clinical application pathway
How about an anatomy study—does it reverse anatomy in development? Does it
potentiate environmental effects?
Phase 1 late 2006-2007
Mark Bear owns Sention
The original rationale for mpep was elevated mGluR5 due to lack of FMRP inhibition of
synthesis. This does not appear to be the case. Why do you think mglluR antag is
effective?