Shahana S. Mahajan, Ph.DResearch Assistant ProfessorNYU School of Medicine.
Mechanisms of Neuron Death in Neurodegenerative Diseases
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Central nervous system has specialized cells
A typical neuron
Neurons signal through synapses
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GluR1-4
AMPA receptors are glutamate gated cationic channels
AMPA receptors are homo or heterotertamers of four subunitsGluR1, 2, 3 and 4
Properties of AMPA heteromer depend on subunit composition
R1 R1
Ca2+
Ca2+
R3 R3
Ca2+
R2R2GluR2 lacking GluR2 containing
GluR2 structure
N
C
TM1 TM2 TM3TM4
Ligand binding domain
Flip/Flop region
NSF binding domain
RNA editingR607Q
Unedited GluR2 (mutated)
N
C
TM1 TM2 TM3 TM4Q
GluR2Q
Unedited Q
GluR2(Q)-containing AMPA receptors are Ca 2+ permeable
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Motor system comprised on motor neurons controls musclesin our body
Amyotrophic lateral sclerosis (ALS) /Lou Gehrig disease(motor neuron disease)
Neurodegenerative disease with progressive paralysis leading to death in 3-5 years.
Selective loss of motor neurons in the ventral horn and motor cortex.
Motor neurons from ALS patients have reduced GluR2 editing
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Kawahara and Kwak, 2004
Rat embryonic (E18) hippocampal neurons 14 DIV on coverslips
Infect with sindbis virus expressing GluR subunits
Treat with glutamate/AMPA/other 17 hours after infection.
Incubate for 8 hrs.
Fix, TUNEL assay and immuncytochemistry
Image on confocal
Quantitate
TUNEL assay to measure toxicity of AMPA receptor subunits
Excitotoxicity in GluR overexpressing neurons
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GluR2Q GluR2Q+AMPA
GluR2- GreenTUNEL- Red
0
10
20
30
40
50
60
R2 R2+AMPA R2Q R2Q+AMPA
% TUNEL positive neurons
0
5
10
15
20
25
30
35
40
45
R1R1+AR1(R)
R1(R)+AR2(R)R2(R)+A
R2(Q)R2(Q)+A
R3R3+A
% TUNEL positive neurons
A
B
0
10
20
30
40
50
60
70
R1 R1(R) R2 R2(Q) R3 R3(R)
Surface expression
GluR2(Q) is the most toxic of all AMPA receptor subunits
GluR1 and GluR2/3 follow different trafficking pathways to reach neuron surface.
Only GluR2 binds NSF, a trafficking protein.
0
10
20
30
40
50
60
R2R2+A
R2NONSFR2NONSF+A
R2QR2Q+A
R2QNONSFR2QNONSF+A
% TUNEL positive
0
10
20
30
40
50
60
R2R2+A
R2NONSFR2NONSF+A
R2QR2Q+A
R2QNONSFR2QNONSF+A
Normalized TUNEL positive
GluR2(Q) mutant that does not bind NSF does not reach surface efficiently and shows reduced toxicity
N
C
TM1 TM2 TM3TM4
Ligand binding domain
Flip/Flop region
NSF binding domain
RNA editingR607Q
Inhibitory peptidepep2m
0
20
40
60
80
100
120
140
R2Q
R2Q+PEP2MR2Q+SCR R2QNONSF
R2QNONSF+PEP2MR2QNONSF+SCR
Surface expression
0
5
10
15
20
25
30
35
40
45
50
R2QR2Q+A
R2Q+PEP2MR2Q+PEP2M+A
R2Q+SCRR2Q+SCR+A
R2QNONSFR2QNONSF+A
R2QNONSF+PEP2MR2QNONSF+P+A
R2QNONSF+SR2QNONSF+S+A
% TUNEL positive
A B
Conclusions:
Failure of GluR2 editing at the Q/R site greatly enhances the excitotoxic potential of GluR2.
NSF helps maintain surface GluR2 levels hence contributes to unedited GluR2 toxicity.
Blocking the interaction of GluR2 and NSF with pep2m reducesthe toxicity.
Pep2m may be employed as a potential therapeutic reagent.
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