Excitotoxicity Flashcards
What is the main excitatory nT in the brain?
Glutamate
What is glutamate key for?
Many physiological processes e.g. LTP and neurodevelopment
What is excitotoxicity?
Cell death resulting from the toxic actions of excitatory amino acids
What amino acids have been linked to excitatory toxic damage?
Cysteine Cysteine sulfonate Cysteic acid Homocysteine Glutamate Aspartate
Can oral glutamate be damaging?
Yes; can produce neurodegeneration in vivo
What diseases have been linked to excitotoxic damage?
Brain trauma Heavy metal toxicity Brain tumours Neurodegenerative disease CNS infections Autoimmune Stroke
What channel does an action potential at a synapse initially open?
Sodium channel - depolarization
Then K+ opens to result in repolarization
How is excess synaptic glutamate removed to avoid toxicity?
Reuptake into the presynaptic terminal
Uptake into glial cells (main route) - located close to excitatory synapse
Describe the presynaptic terminal action potential
Action potential fires in axon
Voltage gated Na+ open and Na+ floods in
DEPOLARIZATION
Delayed opening of K+ channels, K+ floods out
REPOLARIATION
Voltage dependent Ca2+ channels open, calcium influx
nT RELEASE
Describe the respective neuronal levels of glutamate
Vesicles; 100mM
Cytosol; 10mM
Extracellular 1 micromole
Synaptic space; 2mM
Are extracellular glutamate levels kept high or low?
Low; prevent endogenous toxicity
How is glutamate removed from the extracellular space to keep levels low?
ATP-dependent glutamate transporters (on plasma membrane)
THEREFORE; effective levels of ATP are required for glutamate transport and prevention of excitotoxicity
In what situations is glutamate uptake stopped or reversed?
Metabolic compromise; no ATP production
Mitochondria failure
E.g. ischaemic damage such as a stroke
What are the different types of glutamate receptors present on the postsynaptic neuron?
Metabotropic receptors (mGluR) - modulates intracellualr signal transduction Ionotropic receptors (GluR) - AMPA, kainate and NMDA Open cation channels
What cation channels do AMPA and NMDA open respectively?
AMPA = Na+ NMDA = Ca2+ and Na+ as well as K+ efflux
Which glutamate receptor plays a key role in excitoxicity?
NMDA
Describe the opening of NMDA receptors
NMDARs are highly permeable to Ca2+, therefore NMDAr activation allows Ca2+ entry
NMDARs are blocked by Mg2+ ions in a voltage dependent manner
NMDAr channel opening requires sufficient depolarization
Do NMDARs contribute to basal synaptic transmission?
No, Mg2+ block it
When will NMDARs contribute to synaptic transmission?
During intense synaptic activity, AMPARs are activated and if there is sufficient depolarization, then the Mg2+ is displaced and the NMDA opens
How do NMDARs mediate excitotoxicity?
High Ca2+ permeability and incomplete desensitisation
What does pathological activation of NMDARs contribute to?
Neuronal death after acute excitotoxic trauma
Chronic neurodegenerative disease
What is the dual component of a a glutamate mediated EPSC?
Fast AMPAR mediated
Slow NMDAR mediated
What are the 4 main types of glial cells in the CNS?
Astrocytes
Oligodendrocytes
Ependymal cells
Microglia
What are possible malfunctions are the glutamatergic synapse?
Excessive excitation; excitotoxicity
Deficient excitation
What is domoic acid?
Glutamate analog associated with certain harmful algal blooms
Are there any examples of human cases of excitotoxic damage due to oral ingestion?
Yes; domoic acid (glutamate analog) is assoc with harmful algal blooms and therefore can accumulate in high concentrations in tissues of plankton feeders (Shellfish)
DA is assoc with the phenomenon of amnesic shellfish poisoning (ASP)
In mammals, what is the action of DA?
Acts as a neurotoxin, causing a short-term memory loss, brain damage and in severe cases death
What do postmortem studies of people show of those who have been affected by DA?
Neuronal necrosis in hippocampus and amygdala
Survivors have irreversible loss of short term memory
What is the mechanism of action of domoic acid?
An agonist at non-NMDA receptors (AMPA/Kainate)
What action does DA have on AMPA/ Kainate receptors?
Increases intracellular Ca2+ promoting release of glutamate
This released glutamate activates NMDARs
What is the potency of DA at a neuronal level?
DA is up to 20 fold more potent than kainate at KARs
Is DA readily removed from the synapse?
No; not readily removed by glutamate transporters and is therefore very toxic
What is BMAA?
A neurotoxin that is very similar to the amino alanina
What produces BMAA?
Cyanobacteria
What are the sequelae of BMAA ingestion?
ALS/ PD-LB dementia complex
Where can BMAA be found?
Cyad seeds and fruit bats
How does BMA cause ALS like symptoms?
Kills motor neurones by activated AMPA and kainate glutamate receptors and by the formation of oxygen free radicals
What is the evidence that excitotoxicity is implicated in ischaemic brain damage?
After a stroke, extracellular levels of glutamate increase by up to 100 micromoles
In stroke models, NMDAR antagonists can reduce ischaemic neuronal damage in vivo
Which glutamate receptors are most sensitive to glutamate?
NMDARs; EC50 around 2micromoles
AMPA/KAR EC50 around 300 micromoles
NMDARs are the most sensitive to glutamate, and this is why they are most implicated in excitotoxic damage
What changes occur at the presynaptic membrane that are linked to excitotoxic damage?
Increased firing rate of action potential; enhanced nT release
Change in calcium influx mediated by voltage dependent calcium channels (threshold lowered)
Altered glutamate release; altered requirement for calcium influx, altered vesicle loading
Is there anything that can enhance glutamate toxicity?
Reverse uptake; outpouring of glutamate
Depletion of cytosolic ATP
What changes can occur at the postsynaptic membrane that can lead to excitotoxic damage?
Increased affinity of glutamate binding site
Increased density of glutamate receptors
Deficient cation selectivity of the ionophore (R/Q editing) - particularly AMPA receptors
How can the ion selectivity at glutamate (AMPA) gated channels change?
Under certain conditions, AMPARs can become permeable to Ca2+
If the RNA editing fails, the GluA2 subunit changes from an R to a Q
The Q form of GluA2 is calcium permeable
Why are most AMPA receptors impermeable to calcium at rest?
Contain GluA2 subunits (R) that are calcium impermeable - this is RNA edited
What are the 2 main ingestible excitotoxins?
Domoic acid
BMAA
Summarise the abnormalities that can lead to excessive synaptic excitation?
Ingestion of excitotoxins (DA, BMAA)
Presynaptic anomalies; excessive triggering via AP, excessive release
Post synaptic anomalies; loss of cation selectivity (altered R/Q editing), changes in receptor, modulation of receptor number
Deficiencies in glutamate uptake
What are the cellular mechanisms that can cause neuronal death?
Apoptosis
Necrosis
Describe neuronal apoptosis?
Destruction of individual cells induced by a lack of physiological stimuli (lack of growth factors, hormonal changes)
NO INFLAMMATIO N
Shrinking of cytoplasm, condensation of nucleus
Blebbing of plasma membrane with no loss of integrity
Energy (ATP) - dependent active process
Cell death pathways
Describe neuronal necrosis
Affects GROUPS of cells Evoked by non-physiological events (virus, ischaemia, metabolic poisons) INFLAMMATION Swelling of cytoplasm and mitochondria Loss of plasma membrane integrity No energy required; passive process Calcium overload
Chain of events in the cell death pathway - mild excitotoxic damage
Mild excitotoxicity allows NMDAR activation by ambient conc of glutamate
Increased mitochondrial calcium and free radical production
HOWEVER< there is preserved ATP generation (apoptosis is ATPdependent process)
Cytochrome C (Cytc), caspase 9, apoptosis inducing factor (AIF) released
Chain of events in necrosis - severe excitotoxicity
Severe insults e.g. ischaemia, NMDAR activation is enhanced resulting in a large rise in intracellular calcium
This activated NOS (nitric oxide synthase) which increases mitochondrial calcium and superoxide generation
This allows the formation of peroxynitrite (ONOO-)
All of these chemicals result in cellular damage including DNA damage, and the activation of poly-ADP ribose polymerase (PARS)
Mitochondrial calcium accumulation and oxidative damage leads to the activation of permeability transition pore (PTP) which is linked to excitotoxic cell death
What are the actions of peroxynitrite?
Protein oxidation
Lipid peroxidation
DNA/RNA oxidation
What are the actions of PARS?
ATP depletion
NAD depletion
What is the evidence to show than NMDAR is neuroprotective?
Blockade of NMDARs in vivo REDUCES neuronal viability postnatally
Elimination of NMDARs in vivo causes widespread apoptosis in developing neurons
In adult CNS, NMDAR blockage enhances neuronal loss
What is the role of physiological NMDAR activity?
Neuronal survival
Synaptic plasticity - LTP
Neuronal development
What curve do NMDAR show to glutamate?
Classical hormetic dose-response curve
Too much - pro death (excitotoxic)
Too little - neuronal cell death and lack of development
Where are the NMDA receptors located that represent survival promotion?
Synaptic NMDARs
Where are the NMDAARs located that promote death by chronically elevated glutamate levels?
Extra synaptic - linked to signalling pathways such as CREB function and mitochondrial depolarization
PRO-DEATH PATHWAYS
What are the synaptic factors that result in good neuronal health e.g. neuroprotective factors?
Increased local mitochondrial function
Enhanced antioxidant defences
Enhanced trophic support
Suppressed apoptotic pathway
How do NMDARs confer neuroprotective activity in connection with suppressed apoptotic activity?
Activate key signalling pathways such as P13 kinase which is a cell survival pathway
What is PI3-kinase?
Pathway that phosphorylates PIP2 into PIP3 which is part of the phospholipase C signalling pathway
Can activate AkT
This is a cell surviving pathway
How does Akt promote cell survival?
Inhibits activity GSK3-beta (inhibition of this kinase promotes neuronal survival)
Reduce level of BAD which has been linked to cell death. BAD can also reduce levels of Bcl-2 (Bcl-2 is a cell surviving protein)
Reduction in p53, which reduces levels of Bax. Bax is a cell death molecule and so the reduction enhances neuronal survival
In summary, how does Akt promote cell survival?
Reduced GSK30beta Reduced levels of BAD Increased Bcl-2 Reduced p53 Reduced Bax
In terms of gene expression, what is the role of NMDARs?
NMDAR synaptic activity can induce the expression of pro-survival genes but supresses pro-death genes
How does NMDAR induce the expression of pro-survival genes?
Calcium influx in the cell allows nuclear calcium signalling which can target CREB genes
NMDAR receptors allows communication between the synapse and the nucleus via calcium transport
Which CREB target genes are implicated in NMDAR dependent neuroprotection?
AID; Activity-dependent Inhibitors of Death
NFAT; Nuclear Factor of Activated T cells - a calcium responsive transcription factor
What is the role of AID and NFAT genes?
Activation of these genes has been visible to show neuroprotective effects in vivo and in vitro
Why does oxidative damage occur in cells?
Imbalance in ROS (reactive oxygen species) production and the cells capacity to neutralize ROS
Correct redox regulation is essential in all cells
What is the role of NMDAR in the oxidative stress of neurones?
The vulnerability of neurones to oxidative death is regulated by synaptic NMDAR activity
Neurones with a high NMDAR activity withstand oxidative damage more readily that electrically quiet neurones
Neurones are highly vulnerable to oxidative damage after NMDAR blockade in vivo and in vitro
How does NMDAR activity boost antioxidant mechanisms of cells?
Synaptic activity exerts changes in the thioredoxin-peroxiredoxin system
Synaptic activity also promotes a series of gene expression changes that boost anti-oxidant defences
What is the role of the thioredoxin-peroxiredoxin system?
Enhanced thioredoxin activity will facilitate the reduction of hyperoxidized peroxiredoxins (antioxidant enzyme)
What are the changes that can occur to gene transcription that confer an antioxidant boost in NMDAR synapses?
Triggers transcriptional suppression of an inhibitor TXNIP (FOXO target gene)
This ultimately boosts anti-oxidant defences
What is the main role of mitochondria in neuronal health?
Energy demands (ATP) Calcium homeostasis
What is the role of neuronal activity on mitochondrial function?
Regulated mitochondrial fission/fusion and intracellular trafficking
This balances energy demands with localised availability
In summary, what is the role of synaptic activity on mitochondrial action?
Enhances mitochondrial fission
Reduced mitochondrial mobility
Localises mitochondria to dendritic spines
How does synaptic activity regulate neurotrophic factors?
Elevates expression of BDNF which contributes to neuroprotection
Releases and matures pro-NGF (nerve growth factor) activity
Upregulates FGF2 which has shown to delay photoreceptor death
In terms of NMDAR mediated cell death, describe the role of mitochondrial dysfunction
Mitochondrial dysfunction due to excessive calcium uptake is key for severe excitotoxicity
Describe how mitochondria uptake excessive calcium which ultimately leads to dysfunction in NMDAR mediated excitotoxicity
Mitochondrial membrane is depolarized by calcium uptake which inhibits ATP production - this can deplete cytosolic ATP due to reversal of mitochondrial ATPase
Loss of ATP decreases the ability of neurons to regulate calcium
Mitochondrial calcium uptake also promotes production of ROS which further damages the mitochondria
What mediates calcium efflux in neurons?
PMCA and NCX
Plasma membrane calcium ATPase
Na/Ca exchanger
What are calpains?
Calcium dependent proteases
What is the role of calpain in NMDAR mediated cell death?
Cleaves the major NCX3 isoform which impairs its function
Inactivates PMCA following excitotoxic insults
Impairs the efflux of calcium
THEREFORE
Excessive calcium influx IMPAIRS calcium efflux mechanisms
What are SAPKs?
Stress activated protein kinases
Which SAPKs are implicated in NMDAR driven cell death?
P38 MAPK and JNK
In cerebellar and cortical neurons, NMDAR dependent p38 MAPK activation involves nNOS (neuronal nitric oxide synthase)
What determines if NMDAR activity is neuroprotective or excitotoxic?
Stimulus intensity
Distribution (locus) of NMDAR - synaptic or extra synaptic
Molecular density/ subunit composition of NMDAR
What is the evidence that the intensity of the stimulus to NMDAR is key in whether they act in a neuroprotective manner or excitotoxic?
Modest/ physiological NMDAR activity promotes neuroprotection
Too much or too little = cell death = hormetic dose response curve
Pro survival requires lower calcium levels than pro death
What is the significance of NMDAR locus (synaptic or extrasynaptic) in whether they play a role as neuroprotective or excitotoxic?
Synaptic locus; calcium influx well tolerated. Activated ERK pathway. Activated CREB gene expression (AIB, NFAT). Activated pro-survival P13-kinase/ Akt pathway
Extrasynaptic; calcium influx triggers cell death/ Induces ERK inactivation. CREB dephosphorylation. No activation of PI3-kinase/ Akt
What is the significance of the NMDAR subunit composition that differentiates their functional ability (pro survival or pro death)?
Evidence isn’t clear
One study: NR2B promotes neuronal death but NR2A promotes survival
BUT NR2A can promote excitotoxicity and NR2B can mediate pro-survival signalling
What is the composition of NMDARs?
NR1, NR2 (NR2A/B/C/D) +/- NR3
What do the NR2 subunits of NMDARs determine?
Biophysics and pharmacology
In the hippocampus, which NMDAR subunits predomimnate?
NR2A and NR2B
What is the difference between NR2A and NR2B?
NR2A: synaptic
NR2B: extra-synaptic
Can NMDARs be targeted therapeutically?
Animal studies implicate NMDARs in ischaemic damage but NMDAR antagonists for stroke failed due to poor tolerance and efficacy
Describe memantine
NMDAR antagonist that blocks chronic NMDAR activity
Describe the role of NMDA in AD?
Synaptic NMDA activation:
Reduces A-beta production and release
Reduces intraneuronal Ab
Increases components of non-amyloidogenic pathway (soluble APP alpha)
Recruits putative a-secretase ADAM-10
THEREFORE; synaptic NMDA receptors have protective factors against amyloid factors in AD
What is the role of NMDA in SMA and ALS?
Degeneration of motor neurones
SMA-like mice; daily NMDA in vivo can reduce motor neuron death and improve motor behaviour and lifespan
ALS mouse model; significant decrease in NR2A subunit identified with coincident alterations in synaptic plasticity and dendritic outgrowth
What is the role of NMDA in HD?
Synaptic activity increases the non-toxic mutant huntingtin inclusions via NMDAR
YAC 128 HD mouse model; increased extrasynaptic NMDAR expression (increase in pro-death)
Is there a role for memantine in HD?
Memantine which preferentially targets extrasynaptic NMDAR improves neuropathological behaviour in HD mouse models
