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Project Plan

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Rhalena Thomas

on 29 March 2010

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Transcript of Project Plan

Important information
From Papers
Conclusions form Liturature
Hypothesis and Questions
Experiments
LGI1 binds to the surface of hippocampal neurons
Molecular and Cellular Neuroscience (2009)
Owuor, Strittmatter et al.
LGI1 binds to the surface of chick DRGs and E18 rat hippocampal neurons
The Kd of Binding of LGI1 for Adam23 is 10nM
Addition of 50nm and 100nM LGI1 increases neurite outgrowth of hippocampal neurons
There is no effect of 20nM LGI1 on outgrowth
They test if Adam23-/+ are more likely to have seizures by treating 6 month the injected PTZ every 30minutes most Adam23-/+ reached a seizure grade of 4 by 30mg some reached level 4 by 10-20mg
They say the dose at which mice were considerd kindled was lower in Adam23-/+ than wt mice
Adam23-/- mice have lower density of dendritic arbors determined by Scholl analysis of golgi staining counting for 6 mice about 7 cells per mouse (adult)
drawings were made from z-stacks of 40uM sections
Also found the same decrease in P2 mice but only have n=2
LGI1 mRNA is expressed highly in the hippocampal formation and entorhinal cortex
LGI1 mRNA was not found in anywhere outside of the telenchephalon
Brain Research 2010
Herranz-Perez, Perez-Tur
Project 1
Project 2
Other LGI proteins - do they bind NgR1-2, Adam22/23/11
Binding domain of LGI1 for NgR1
Binding domain of NgR1 for LGI1
Competition with P4 and smaller peptides
Bioimformatics study shows LGI2 is in a locus with chromosomal deletion of 24 genes in PEPS
Partial Epilepsy with pericentral Spikes
Neuron 2006
Schulte et al
Perform mass spect to find interactors of Kv1
They find LGI1 as well as Adam22, GIT1 and other pre-post synaptic proteins
They look for LGI1 interacators and find Kv channel but don't list the rest
Subcellular fractionation of Adult rat brain showed LGI1 was in the soluable membrane fraction and not in the cytoplasm or insoluable membrane fraction
We co-expressed with subunits of Kv1 channels in xenopus oocytes LGI1 antagonize the ability of KvB1 to increase inactivation time of Kv1 channels
Mutants of LGI1 do not exhibit this effect
PSD95, SAP97,
Caspr-2, contactin,
Na/K ATPase, Ca ATPase,
synataxin, synaptotagmin
Genomics 2010
Kunapuli, Cowell et al
They preform a gene expression array comparing T98G cells with T98G cells overexpressing LGI1
They find several proteins involved in axon guidance are altered
EGFR is down regulated
Stress fibres and cell motility are decreased
MAPK/ erk activation is decreased
Science 2006
Fukata et al
MS screen for proteins interacting with PSD95
Determine Adam22 interacts with PSD95 and LGI1 interacts with Adam22
LGI1 increases amplitude of mEPSP in hippocampal slices
LGI1 does not effect LTP
Nature Medicine 2009
Zhou, Anderson et al.
Analyze two transgenic mice LGI1 OE and mLGI1
The mLGI1 mice do not have spontaneous seizures
LGI1 OE reduces PPF in P23/adult mice and mLGI1 increase PPF
When synapses mature the ratio of NR2B/NR2A decreases at P21 measured in hippocampal slices
adult mLGI1 mice have higher NR2B/NR2A ratios than adult wt mice and OE mice have lower ratios
This switch seems to be mediated by src as inhibitor of src PP2 reduces NR2B/NR2A ratio in imature wt mice and in adult mLGI1
They do not show ratios of immature mLGI and OELGI1 mice it would be interesting know if OELGI1 immature mice already have reduced ratios
At P21 dentric arbors of hippocampal granual neurons change from widely branched to refined they measure arbors by scholl analsysis out to 300uM from cell soma
Analysis of Dendritic arbor morphology shows that mLGI1 have arbors resembling the immature wt mice, OE-LGI1 look like wt
Again they don't show an anlysis of the immature OE-mice which should have pre-mature refinement
Excitatory synaptic transmisssion is altered : OE LGI1 mice have decreases AMPAR and NMDA currents compared to wt mice and mLGI1 have increased currents - they should have looked at the AMPAR/NMDAR ratio as well
mLGI1 have increases in mEPSPs and OE-LGI1 have decreases mEPSPs frequency and not apllitude indicating a presynaptic effect
To determine if EPSP changes were Kv dependent they added DTX and found that the mLGI1 increase was still present but the OE-LGI1 decrease was absent
They test inhibitory transmission and find no effect. However it appears that mLGI1 slightly decreases IPSPs and OE-LGI slightly increases IPSPs but the variation is very high
They tested seizure suspectabliity in hippocampal slices stimulating MPP and measuring field potentials in the granual layer there was no difference in evoked field potentials
When a low dose 50uM was added evoke potential increased the same in wt and OE-LGI1 in the mLGI1 mice the increase in both spike number and amplitude was about 40% higher
Induced epliptic activity with 30mg PTZ injections and measured EEG of hippocampus and cortex mLGI1 have higger EEG scores after the first injection and reach SE with less injections than wt and OE-LGI1
I would exspect if LGI1 acted at the synaptic level that OE-LGI1 would be less susceptable to seizures
LGI1 alters short term plasticity
In immature mice OELGI1 decreases src levels and mLGI1 increases src levels in adults compared to wt
Human Molecular Genetics 2010
Yu, Cowell et al
PNAS 2010
Fukata et al
LGI1 null mouse made using Cre-Flox didn't make conditional
LGI1-/- mice have spontaneous seizures
LGI1-/+ do not have sponateous seizures however seizures can be more readily induced in these animals
One month old mice were given one injection of 35mg/kg PTZ and observed more LGI1-/+ had level 4 or 5 responses than in wt
LGI1 is required for Co-IP of PSD95 and Kv1, Adam22/23 with Adam22/23
LGI1 effect the distribution of Adam22, Adam23 and Kv1 just that the levels are reduced in synaptic fractions in the absense of LGI1
The effect is rescued by transgenic with wtLGI1 but not mutant
LGI1-/- have lower AMPAR/NMDAR ratios than wt mice but no change in PPF
LGI1-/- have decreases mEPSP amplitute but no change in mEPSP frequency
Molecular and Cellular Neuroscience 2010
Owuor, Strittmatter et al
Find the Kd of bind of LGI1 for Adam23 is 10 looks the same for Adam22 and Adam11
50 and 100nM LGI1 increase neurite outgrowth from hippocampal neurons
Create LGI1-/- mice using Cre/Flox system
They deleted from exon 3 to exon 8
mRNA levels of LGI1 in the hippocampus are constant from P4-P23
LGI1-/- mice have a slight reduction in body size (mentioned not measured)
At 12-20 days LGI1-/- have generalized myoclonic seizures that lasted about 20min
LGI1-/+ did not have seizures
Measured sponanteous discharge in field recordings in CA1 and found LGI1-/- mice exhibit more sponaneous discharge
Whole cell current clamp recordings of pyramidal neurons showed no difference in spikes or firing frequency or resting membrane potential in LGI1-/- compared to wt
They remark that the lack of changes indicates there is not intrinsic change in cell properties AND there is not morphological change in the pyramidal neurons and circuit (I'm not sure how they can make that conclusion)
They measure mEPSPs in CA1 pyramidal neurons they find no change in ampliture but and increase in frequency in the LGI1-/- *** indicates presynaptic changes
They find both AMPAR and NMDAR mediated EPSP are larger in LGI1-/- mice
They test the mIPSPs and eIPSPs for changes in inhibitory transmission and find no differences it looks to me like they might be slightly decreased
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