Glu and Stroke

Question 1

Briefly describe ionotropic glutamate receptors

Answer 1

Receptor-ionophore complex; ligand gated non-selective cation channel

Question 2

Briefly describe metabotropic glutamate receptors

Answer 2

G protein coupled receptor

Question 3

iGlu-R families and homology

Answer 3

18-40% homology between families
AMPA: GluR1-4 (56-73%) [GluA1-4]
Kainate: GluR5-7 (75-80%) [GluK1-3]; KA1-2 (68%) [GluK4-5] } 45%
NMDA: NR1; NR2A-2D (38-53%) [GluN1-3]; NR3A-3B (50)%

Question 4

mGlu-R families and homology

Answer 4

Group I (ACPD): mGlu1&5 (62%)
Group II (ACPD): mGlu2&3 (68%)
Group III (L-AP4): mGlu4,6,7&8 (69-74%) (mGlu6 retina only)

Question 5

NMDA receptors

Answer 5

N-methyl-D-aspartate, is a specific agonist; D-2-amino-5-phosphponopentanoate (D-AP5) is a specific antagonist (competitive)

Question 6

AMPA receptors:

Answer 6

alpha-amino-3-hydroxy-5-ethyl-4-isoxazolepropionic acid, prefer AMPA to kainate; NB QX is a specific antagonist for non NMDA-Rs (competitive)

Question 7

Kainate receptors

Answer 7

Like AMPA but kainate > AMPA

Question 8

IGlu-R stoichiometry and signalling

Answer 8

GluA1-4(AMPA) & GluK1-3 (kainate): homomeric or heteromeric
GluK4/5 (kainate) heteromeric only, must be with GluK1-3; have dual mechanism of action (diagram)
GluN1-2: heteromeric only- dual agonism

Question 9

Outline mGlu-R structure

Answer 9

Cys rich domain
7 transmembrane domains
Intracellular carboxy terminal
G-protein binding to intracellular loops 2 and 3
Functional receptors are homodimers linked by S-S bridge between VFT domains

Question 10

First toxic effect of L-glu

Answer 10

(Lucas & Newhouse 1957)- systemic IV MSG in young mice P2-16 led to inner retina degeneration → complete cell loss in 2 weeks

Question 11

MSG as flavouring raised concerns when…

Answer 11

Olney (1969) demonstrated brain damage in primates and mice following systemic subcutaneous IV administration

Question 12

Olney-1971

Answer 12

described the neurotoxicity of a number of amino acids that caused necrotic cell death (L-Glu, L-Asp, NMDA); in 1980’s terminology updated to excitotoxicity as understanding of L-Glu and iGlu-Rs in excitatory neurotransmission increased
Glu has been previously used to treat epilepsy (TCA link)

Question 13

Which disease has Glu been previously used to treat?

Answer 13

Glu has been previously used to treat epilepsy (TCA link)

Question 14

Definition of excitotoxicity (Olney)

Answer 14

Over-activation iGlu-Rs triggering cell death via necrosis +/apoptosis (NB high L-Glu not toxic per se); possibly involves permissive or facilitatory role of L-Glu

Question 15

How many types of excitotoxicity have been described in vitro?

Answer 15

3
Acute (1-3 hours)
Delayed (2-12 hours)
Slow (24-72 hours)

Question 16

Acute Glu excitotoxicity

Answer 16

Induced by high levels of EC Glu applied up to 30 mins, mainly NMDA-R mediated
Non-NMDA-R not involved as non-NMDA-R desensitised (due to duration)
Not Ca2+ dependent
Depol- Na+ and Ca 2+ influx and passive Cl- influx
Osmotic concentration of cytosol increase
Oedema
Mitochondrial collapse and cell membrane lysis

Question 17

Delayed Glu excitotoxicity (Ca2+ dependent)

Answer 17

Induced by high levels of EC Glu applied up to 5 mins, neurones recover from acute swelling
All iGlu-Rs involved
Ca2+ dependent, with prolonged rise in IC Ca2+ from influx through NMDA and some CA2+ permeable AMPA/KA-Rs
Probably self propagating (1st wave of cell death initiates further Glu release
Similarities with progressive neurodegeneration seen in disease states in vivo

Question 18

Slow excitotoxicity

Answer 18

Induce by prolonged exposure to AMPA or KA agonists for 24-72 hours, non NMDA-R mediated
Probably Ca2+ dependent with a prolonged rise in IC Ca2+ from influx through AMPA/KA-Rs or VGCCs

Question 19

Clinical causes of excitotoxicity

Answer 19

Epilepsy
Ischaemia
Hypoxia
Neurodegeneration

Question 20

Types of stroke

Answer 20

Ischaemic- 85%, blockage of blood vessel from thrombotic or embolic small artery occlusion
Haemorrhagic- 15%, rupture of blood vessels from intracerebral (hypertensive) haemorrhage or subarachnoid haemorrhage (ruptured aneurysm)

Question 21

Impact of ischaemia

Answer 21

Hypoxia: reduced blood supply, reduced O2, decreased ATP:ADP; Na+/Ka+ ATPase less effective thus Na+i increase and K+o increase; ion gradients reduced so ErevS tends to 0
Depolarisation of presynaptic terminals increases vesicular Glu release; Glu transporter mechanisms reversed

Question 22

MK-801 in animal models of ischemia (middle cerebral artery occlusion)

Answer 22

Very effective
BUT
Narrow therapeutic window
Low therapeutic index
Schizophrenic like psychosis, memory impairment and neurotoxic syndrome

Question 23

NBQX is a poor clinical agent because

Answer 23

it has poor water solubility

Question 24

Zonampanel

Answer 24

Reduces MCAO infarct volume in rats by 50-60% if given up 3 hours after ischaemic episode, abandoned at phase II trials

Question 25

NMDA-Rs in cell death

Answer 25

come back to