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1Confidential
Fragile X Syndrome
2Confidential
Background
• Caused by a CGG expansion in the FMR1 gene
• Males severely affected, females are mosaic
• Fragile X syndrome can be a cause of autism or related disorders, although not all children with fragile X syndrome have these conditions
• Symptoms include• Developmental delays: crawling, walking• Hand clapping or hand biting• Hyperactive or impulsive behavior• Mental retardation• Speech and language delay • Tendency to avoid eye contact• Physical signs: Flat feet, flexible joints and low muscle tone, large body size, large forehead or
ears with a prominent jaw, long face, large testicles, soft skin
3Confidential
Bakker et al., 2004; Han et al;. 2015; Huber et al. 2002; Krueger et al., 2011; Koga et al., 2015
FMR1 KO mice
• These mice have a knockout allele of the fragile X mental retardation syndrome 1 gene (Fmr1) on the X chromosome and exhibit many phenotypic characteristics of the Fragile X Syndrome in humans including hyperactivity, repetitive behavior and seizures.
• Absence of the Fragile X Mental Retardation protein (FMRP) in the mice causes activation of RAC1 protein resulting in abnormalities in dendritic spines in various regions of the brain. and altered synaptic function.
• The absence of FMRP also alters synaptic plasticity which results in an impairment of long-term potentiation in the cortex and hippocampus, as well as an augmentation of long-term depression in the hippocampus and cerebellum.
• Male FMR1 KO mice on FVB/n background bred at PsychoGenics are used in all studies
4Confidential
Behavioral Tests in Adult Mice
5Confidential
Male KO mice show similar BW compared to WT mice
Age (weeks)
8 9 10 11 12
Bo
dy W
eig
ht
(g)
15
20
25
30
35
40
FMR1_WTFMR1_KO
6Confidential
Male mice tested at 9 weeks of age
FMR1 KO mice are hyperactive compared to WT mice
Time (min)
5 10 15 20 25 30 35 40 45 50 55 60
Dis
tan
ce
Tra
ve
led
(c
m)
0
250
500
750
1000
1250
1500FMR1_WTFMR1_KO
Dis
tan
ce T
ravele
d in
Cen
ter
(cm
)
0
500
1000
1500
2000
2500FMR1_WTFMR1_KO
*
To
tal
Dis
tan
ce
Tra
ve
led
(c
m)
0
2000
4000
6000
8000
10000FMR1_WTFMR1_KO
*
Time (min)
5 10 15 20 25 30 35 40 45 50 55 60
Dis
tan
ce T
ravele
d i
n C
en
ter
(cm
)
0
100
200
300
400
500FMR1_WTFMR1_KO
7Confidential
Male mice tested at 9 weeks of age
Male FMR1 KO mice show increased rearing activity
Reari
ng
Fre
qu
en
cy (
co
un
t)0
250
500
750
1000
1250
1500
1750
2000FMR1_WTFMR1_KO
*
Time (min)
5 10 15 20 25 30 35 40 45 50 55 60
Re
ari
ng
Fre
qu
en
cy (
co
un
t)
0
50
100
150
200
250FMR1_WTFMR1_KO
8Confidential
KO mice show deficits in Contextual and Cued Fear Conditioning %
Fre
ezin
g
0
20
40
60
80
100FMR1_WTFMR1_KO
*
Time (min)
1 2 3 4 5
% F
reezin
g
0
20
40
60
80
100FMR1_WT FMR1_HMZ KO Context
Pre-Cue Cue Post-Cue
% F
ree
zin
g
0
10
20
30
40
50
60
70FMR1_WTFMR1_KO
**
Context
Male mice tested at 11 weeks of age
9Confidential
FMR1 KO mice show reduced startle response
Startle Intensity (dB)
70 75 80 85 90 95 100 105 110 115 120
Mean
Sta
rtle
Res
po
nse
0
200
400
600
800
1000
1200
FMR1_WTFMR1_KO
*
Male mice tested at 10 weeks of age
10Confidential
Audiogenic Seizures in 3 week old mice
11Confidential
Audiogenic seizures
• KO mice are tested at 3 weeks of age
• Mice are are individually placed in a Plexiglas chamber and allowed to explore for 15 sec. They are then exposed to a 125 dB tone for 2 minutes, followed by 1 minute of no sound, and then a repeat 2 minute tone. The mice are scored based on their response, latency, and seizure intensity:
0: no response 1: wild running and jumping2: clonic seizures3: clonic/tonic seizures4: tonic seizures5: respiratory arrest
12Confidential
Late
nc
y t
o s
eiz
e (
sec
)
0
50
100
150
200
250
300
350
Salineseizure: 66.6 seizure: 256 seizure: 300 seizure: 270
* **
SalineMPEP 3 mg/kg MPEP 10 mg/kg MPEP 30 mg/kg MTEP 30 mg/kg
Effects of MPEP and MTEP on audiogenic seizures
Late
ncy t
o r
esp
irato
ry a
rrest
(sec)
0
50
100
150
200
250
300
350
arrest arrest: 99.875 arrest: 101.3 arrest: 275.4 arrest: 300 arrest: 300
* * *SalineMPEP 3 mg/kg MPEP 10 mg/kg MPEP 30 mg/kg MTEP 30 mg/kg
13Confidential
Effects of MPEP and MTEP on survival
Vehicle
MTEP (30 mg/kg)
MPEP 1 mg/kg 3 mg/kg 30 mg/kg
died
survived
14Confidential
Effects of diazepam on audiogenic seizures
Vehicle Diazepam 0.03 mg/kgDiazepam 0.1 mg/kgDiazepam 0.3 mg/kgDiazepam 1 mg/kg
Late
ncy t
o s
eiz
e (
sec)
0
50
100
150
200
250
300
350
seizure: 151
seizure: 168.75
seizure: 246.375
seizure: 300
seizure: 300
* * *
Late
ncy t
o r
esp
irato
ry a
rrest
(sec)
0
50
100
150
200
250
300
350
Col 2: 162.6667 Col 2 Col 2: 162.6667 Col 2: 180.625 Col 2: 277.5 Col 2: 300 Col 2: 300
* * *
died
survived
Diazepam 0 0.03 0.1 0.3 1 mg/kg
15Confidential
Effects of dicyclomine on audiogenic seizures
Late
ncy t
o r
esp
irato
ry a
rrest
(sec)
0
50
100
150
200
250
300
350
*
* *
Dicyclomine 0 3 10 30 mg/kg
Late
nc
y t
o s
eiz
e (
se
c)
0
50
100
150
200
250
300
350
*
**
Dicyclomine 0 3 10 30 mg/kg
Dicyclomine 0 3 10 30 mg/kg
died
survived
16Confidential
Electrophysiology
17Confidential
Fmr1 mice exhibit enhanced hippocampal mGluR-dependent long-term potentiation (LTD), which is reversed by mGluR antagonist 8-OH-DPAT
Time course of the changes in responses recorded inCA1 area of hippocampus induced with an applicationof an mGluR agonist (S)-DHPG (100μM). DHPG wasapplied from 0 to 5 min and the subsequent mGluRantagonist 8-OH-DPAT (100nM) was applied from 10to 15 minutes
fEP
SP
Slo
pe (
% B
aselin
e)
0
20
40
60
80
100
120FMR1_WT FMR1_KOFMR1_KO - 8OH DPAT(100uM)
*
#
TIme (min)
-10 0 10 20 30 40 50 60
fEP
SP
Slo
pe
(%
Ba
se
lin
e)
20
40
60
80
100
FMR1_WT FMR1_ KO FMR_KO - 8OH DPAT
Summary of the data for the last 5 min ofexperiment. *p
18Confidential
A significant reduction in tonic inhibitory currents, a critical factor modulating neuronal excitability, was observed in dentate granule cells of 2 month-old male FMR1-KO mice in agreement with prior observations (Zhang, N. et al (2017). Exp. Neurol., PMID: 28822839).
Tonic inhibitory currents are significantly reduced in dentate granule cells of FMR1-KO mice.Whole-cell patch clamp recordings were made in dentate granule cells of hippocampal slices from 2-monthold male wild-type (WT) (n=22 cells, 6 mice) and FMR1-KO mice (n=24 cells, 5 mice), Vhold=-70 mV.Left Following a stable baseline (-THIP) and subsequent enhancement of tonic GABA currents by the dsubunit-selective agonist THIP (gaboxadol, 1 mM; +THIP), tonic currents were unmasked by blockingGABAA receptors with 100 mM picrotoxin. *p