shahana s. mahajan, ph.d research assistant professor nyu school of medicine. mechanisms of neuron...

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.