DEFINITION
Excitotoxicity is a phenomenon that occurs in most Glu-synapse tetanicly stimulated in CNS, most evident in the regions of hippocampus and, obviously, the cortex. When a postsynaptic neuron is exposed to elevate doses of excitatory aminoacids, consequently to high-frequency stimulation of presynaptic neuron or elevate doses of Glu in neuropile, this cell undergoes to a process of apoptosis or in minor cases necrosis.
Excitotoxicity has been ipotized to be a dominant effector of neuropsychiatric and neuropathologic ethiology in Huntington, Alzheimer and Parkinson's diseases. In a novel interpretation, excitotoxicity is the key in competition among neurons.
The homoeostasis of glutamate and other aminoacids in neurons evict this cells from apoptosys and ischemic death, as explained above, primarily caused by an overload of Ca in the cytosol, associated to higher concentration of reactive specie of oxygen (ROS).
Glu RECEPTORS IN CNS
Glutammate plays a central role in neurophysiology, and is been identified like the first aminoacid able to induce an
EPSP in
CNS, and probably is the most important cause of excitotoxicity in
CNS, obviously acting on its postsynaptic receptors.
Glutammate receptors are distinguished in two big families: metabotropic and ionotropic receptors of glutammate, and both are usually expressed on the postsynaptic cell's membrane. On the presynatpic side, in glu-synapses, is expressed a glu-transporter for reuptake of the neurotrasmitter from the synaptic space.
The principal role in excitotoxicity may be attributed to ionotropic receptors: these are regrouped in three major-families:
AMPA, NMDA, and kainate. In the end we consider also metabotropic receptors of Glu.
- AMPA: AMPA receptors mediates rapid transmission of information from presynaptic to postsynaptic sides. AMPAs are tetramers, composed by subunities named GluR1, GluR2, GluR3 and GluR4. They are Na and Ca-permeable in order to induce an EPSP, and so they play an active role in excitotoxicity glu-induced, as explained belove. Studies on the stechiometry of the subunities, revealed two different classes of GluR2: GluR2Q and GluR2R. Post-trascritional mRNA reveals in the transmembrane dominion in position 586 a glutamine, a neutral aminoacid. During the post-transcriptional process, this residue could be replaced with arginine. Arg is a polar, basic aminoacids, and causes an huge impact on Ca-conductance of AMPA. There are evidences that the presence of many AMPA with GluR2R subunits is correlated to most evident excitotoxicity, consequently to higher influx of Ca during tetanic stimulation of the postsynaptic cell
- NMDA: NMDA receptors are strictly connected with synchronous AMPA activity. Without any stimulation, the pore of the receptors is occupied with one Mg ion, that blocks every flux of ions from extracellular space to cytosol. This ion can be evicted only with an elettrotonic depolarizing stimulation of the receptor, that occurs only when a near AMPA receptor is stimulated. NMDA receptors consequently acts like a logic gate (an AND gate), infact Na and mostly Ca-membrane conductance are present only with simultaneous direct stimulation of NMDAr and depolarization AMPA mediated. Molecularly, they are eterotetramers composed by two different couples of subunities: NR1 and NR2. Excitotoxicity is correlated to this receptors because of they elevate Ca conductance.
- KA: kainate receptors are tetramers similar to AMPA, and is possible to reveal the same mechanism of post-transcriptional editing of their subunits with exchange of glutamine with arginine (this phenomenon occurs in GluR5 and GluR6, not in GluR2), leading to a major Ca-permeability. The component of kainate receptors stimulation in an EPSP is very low (this caused the recent detection of this receptors), and is evident only in high-frequence stimulations. They are also sensible to the so-called spillover of glutammate: the diffusion from synaptic space of glu, thanks to the high affinity of KA receptors, can activate extrasynaptic KA receptors, that depolarizes extrasynaptic neurons, facilitating their stimulation.
- mGluR: these are metabotropic receptors, and so they are GPCR, linked to a G proteins. mGluR are divided in three great families: mGluR class I, II and III. Especially the ones in class I and III mediates, via activation of DAG-IP3 and PKC pathway, an huge influx of Ca in the postsynaptic cell, favouring the emerge of excitotoxicity.
Notably interesting, the correlation between
AMPA and
NMDA and consequent influx of Ca is not only fundamental in excitotoxicity but also in neuronal plasticity and so in establishing memory circuits.
ISCHEMIC CASCADE IN EXCITOTOXICITY
As we have seen, AMPA, NMDA and KA mediates an huge influx of Ca in the neuron. Depending on the site of the synapsis, the ion can accumulates in body, axons or dendrites. In the last two cases, Ca is free to spread in the cytosol reaching neuron body's enzymes and mitochondria, unleashing toxic injuries to neuron.
The influx of Ca is the key and the first actor of excitotoxicity, correlated to its fundamental role of principal activator of a large number of enzymes.
So, the first step in the phenomenon of excitotoxicity is a elevate concentration of glutamate in the synaptic space, caused by a tetanic stimulation of presynaptic membrane or an high concentration of the aminoacid in the synaptic space. There is an important molecule used to evoke excitotoxicity in laboratory: THA .
Threohydroxyaspartate (THA) is an inhibitor of Glu-reuptake from synaptic space by presynaptic neuron; consequently, by subministrating THA and stimulating presynaptic neuron, we can observe an huge accumulation of neurotransmitter in synaptic space.
With high doses of glutamate, evoked by THA, lots of AMPA and NMDA channel open, thanks to the interaction ligand-receptor. As we have observed previously, NMDA and AMPA mediates Ca and Na-current on the cytoplasmatic membrane, following their elettro-chemical gradients.
Na mediates in some cases, with an influx of Cl via the Glu-receptors, to a sudden osmotic death, caused by an elettro-chemical disequilibrium occurring on the cytoplasmatic membrane: the massive influx of ion species mediates an huge influx of water, with disgregation of the membrane and necrosis.
Ca influx mediates the most common pathway of neuronal ischemic cascade.
This ion infact is one of the commonest biochemical transmitter in the cytosol, and a pathological increase of his concentration (usually is maintained in order of 10^-7 M) acts like a potent activator of a large number of enxymes, like endonucleases, APTases, calpaine, phospholipase; the activation of this enzymes and more other, causes an instability of the membrane of the neuron, and this may cause another release of Glu (that may lead to an increase of excitotoxicity) or another uptake of ions by the interstitium.
The key factor now is the instability of the membrane of the mitochondria: this organelle, above their role in maintaining low concentration of Ca and the oxidative phosphorylation, plays a major role in excitotoxicity determinating the fate of the cell. With the breakdown of mitocondria, there is a release obviously of Ca, but prevalently of toxins and procaspases, with activation of the casapases-mediated apoptosis. Now, I analyze more precisely the role of mitochondria in neuronal death.
Role of mitocondria
Mithocondria, as I have suggested, play a crucial role in excitotoxicity. Their disgregation usually lead to neuronal death. There are many factors that may lead to their distruction but they can be summed in a pathological rise of ROS and consequently cell death or cell survival.
ROS are free radicals: usually, they mediates a damage of the overall structures of the cell, but, in the last years, is emerged the notion of an redox-homeostasis, because several evidences revealed that this oxidant molecules have a physiological role in a large number of biochemical pathway linked with neuronal and cell survival.
Anyway, high concentration of ROS in excitotoxicity are mainly linked to neuronal death.
ROS mediates opening of MPTP and destabilization of mitochondria's membrane. Both of this process determine activation of apoptosis death mediated by caspases. Has been observed that low rate of ROS stress may cause neuronal survival: the trascription of NF-kB, mediated by caspase activation, could lead to trascription of crucial trascription factors that promote neuronal survival like c-Myc and p53 (p53 also is sort of receptor in physiological response to ROS). In most cases of high stress, NF-kB can't save the cell.
ROS may rise for lots causes: excessive amount of Ca in the cell, that lead to activation of several enzymes among which, as has been studied in last decade, nNOS with production of reactive nitrogen species (RNS), or uncoupling of the oxidative phosphorylation, or also by overload in the neuron of Fe that facilitates Fenton reaction.
Opening of MPTP may also be caused by direct action of Ca: this channel infact acts like a receptor for Ca. So, higher concentration of Ca caused by excessive NMDA and AMPA opening take to activation of MPTP.
Caspase mediated apoptosis
As we have seen, MPTP and consequently mitochondria are like a switch for the neuron between cell survival or apoptosis death.
When a mitochondrium is destructed, or MPTP are opened by ROS or Ca, in the most cases the fate of the neuron is determined, because there is the complete activation of apoptotic death.
The caspase's cascade originated by damage to mitochondria starts with the inactivation of antiapoptotic factors like Bcl2 and Bxl, that acts like “receptors” for pH and Ca concentration in the mitochondria, by Bad. Bcl2 and Bxl inhibited dishinibits proapoptotic factors like Bax, Bak, Noxa, Puma. Bad has a principal role: normally, is phosphorylated; Bax dephosphorylation forms an etherodimer with Bcl2 and Bxl, that inactivates this two antiapoptotic factor and favours Bax and Bak triggered apoptosis. Bax and Bak activated can polymerize on mitochondria's membrane forming pore (MAC), from which cytochrome c can exit and explicate its toxic function. Both Bax and Bak open also MPTP and both are phosphorylated and both are activated in presence of ROS . Bcl2 and Bxl physiologically prevent the flux of cytochrome c from mitochondria, consequent to a mitocondria disgregation, under survival signalation.
When cytochrome c spills over the mitochondria, it polymerize with two cytosolic proteins: APAF1 and procaspase 9, forming a complex that disrates procaspase 9 to caspase 9, the starting caspase of apoptosis. Caspase 9 activates consecutively procaspase 6, 7, 3, to active caspase 6, 7, 3, that are foundamentally proteases (Cysteine ASPartate Peptidases), that destruct all the components of the neuron, and also activate an Mg/Ca endonuclease that cuts DNA, breaking off all the genetic information of the neuron.
Obviously the apoptosis' process is largely more complex, with activation of a great number of other enzymes. It's interesting that also ER interferes in the process of excitotoxicity: infact, cytocrome c released from mithocondria can interact with I3PRs-linked ryanodynic receptors on ER, that lead to an huge release of Ca from ER. So, the toxic effect of Ca can be autosustained by this positive feedback cycle.
FLOGOSIS
The result of all this process is finally the apoptosis of the neuron, consequently to an excessive stimulation by Glu that leads to a loss of Ca homeostasis. The apoptosis, when doesn't lead to any release of cellular molecules in the interstitium, doesn't cause any inflammation process. But, more frequently, the apoptosis is mixed with process of necrosis, that logically take to a local inflammation near the overstimulated neuron.
This process usually causes, like every inflammation, to a recall of phagocyte mediated by PAMP receptor, dis-regulation of blood-brain barrier with cerebral edema and several other pathologies.
EXCITOTOXICITY LINKED PATHOLOGIES
Many pathologies has been linked to excitotoxicity-mediated cerebral damage, like Parkinson's disease and Huntington's corea.
- Parkinson's disease (PD) is described in literature like a chronic destruction mediated by apoptosis of the dopaminergic neurons of striatus' pars reticulata, consequently associated to hypocinetic movements and neurological symptoms. Early research demonstates that PARK2 (a PD's associated gene) downregulates postsynaptic expression of NMDA receptors, and so influencing and regulating eventual rise of excitotoxicity in striatum neurons. Mutations on this gene can cause an altered expression of Glu receptors, exposing the patient to a major vulnerability to excitotoxicity. Other studies proves that selective antagonists of NMDA have similar therapeutic effects in mice than give L-DOPA, the most known drug in PD's patients, proving that NMDA channel and excitotoxicity in PD plays an important role.
- Huntington's disease (HD) is an inherited neurodegenerative pathology that principally affects GABAergic spiny neurons of striatum. HD is strictly connected with mutations on the gene htt, and so this pathology has a notable genetic background. Is proven that htt has a strict interactions with PSD-95, a MAGUK associated with NMDA receptors on NR2 portion; some studies prove that PSD-95 acts like a bridge between htt and NMDA. Probably, but this hypothesis is in a phase of research, mutations of htt promote excitotoxicity obviously acting on NMDAr.
PROTECTIVE ISSUES
The emergent role of excitotoxicity as one of most important causes of neuronal death, especially in cerebral stroke, has determined the born of many studies on possible artificial or physiological molecules that can prevent neuronal ischemic death by excitotoxicity.
Here I quote only someone of the great number of articles about this interesting argument, that I think they are the most interesting:
HO-1 protects brain from acute excitotoxicity (2006): in this article is studied the strict relationship between expression of the anti-oxidant enzyme eme-oxygenase I and damages mediated by an induced excitotoxicity, probably caused not directly by the action on eme molecule but by its action against ROS' damages.
- There are two articles of 1996 and '97 that studies the relationship between actin depolymerization and excitotoxicity:
Evidence that actin depolymerization protects hippocampal neurons against excitotoxicity by stabilizing [Ca2+]i and
The actin-severing protein gelsolin modulates calcium channel and NMDA receptor activities and vulnerability to excitotoxicity in hippocampal neurons.; particularly interesting is the first one, in which, with cytochalasin D, was artificially depolymerized the actin tubules. With an induced excitotoxicity was oserved that probably actin monomers mediate a negative feedback regulating directly (they haven't a scavenger effect) influx of Ca in neurons when this becomes toxic. Instead, no effect is evidentiable whit antitumoral like colchicine. In the second, there is a deeper analysis of the negative feedback regulation: in this article is studied the relationship between gelsolin and the feedback cycle of Ca and actin, and emerges that gelsolin plays obviously a strict role in the regulation of neuron cytoskeleton, and according to this regulates the feedback and indirectly the excitotoxicity. The relevance of the extreme complexity of Ca and actin regulation in excitotoxicity is revealed by the interaction between Ca and gelsolin: indeed, Ca is a potent activator of gelsolin.
-
Role of actin in anchoring postsynaptic receptors in cultured hippocampal neurons: differential attachment of NMDA versus AMPA receptors. (1997) As this article explains, actin and gelsolin as a so important role in excitotoxicity probably because they regulates cytoskeleton and so the quantity of AMPA and NMDA receptors on the postsynaptic cell.
-
The protective effects of plasma gelsolin on stroke outcome in rats (2011) this recent study analyse the effect of plasma gelsolin amid excitotoxicity: gelsolin is a Ca binding protein, directly regulating Ca influx, and a depolimerazing factor of actin, another crucial actor on excitotoxicity regulation.This article maybe is the most important, infact we can see the analysis of a potential therapeutic method against excitotoxicity-linked pathologies.
- Neuroprotective effect of CPDT on THA-induced cortical motor neuron death in an organotypic culture model.
illustrates the role of 5,6-Dihydrocyclopenta-1,2-dithiole-3-thione (CPDT) in preventing brain damages caused by THA-induced excitotoxicity going to activate Nrf2 that establishes transcription of anti-oxidant enzymes, that directly prevent damages induced by artificial excitotoxicity; one of this is eme-oxygenase I (see above)
Conclusions
The phenomenon of excitotoxicity could advise us of many neurodegenerative disorders strictly linked with disequilibrium on the Ca balance but mostly with mithocondrium and energetic disorders, that allows the apoptotic death of the neurons. Studies in a not-so-distant future could
give us new drugs or new therapeutic and diagnostic methods that have to do with this phenomenon.
In a more speculative way, excitotoxicity could be interpreted with a competitive vision of biological and molecular pathways, in wich “strongest” neurons led to apoptosis and death other postsynaptic neurons, and consequently manipulating and finely regulating cortical and subcortical circuitry; excitotoxicity so could run parallel to LTP and LTD, also mediated by AMPA and NMDA receptors.
SOURCES AND QUOTES:
http://it.wikipedia.org/wiki/Eccitotossicit%C3%A0
http://en.wikipedia.org/wiki/Excitotoxicity
http://en.wikipedia.org/wiki/Ischemic_cascade
http://en.wikipedia.org/wiki/Mitochondrial_permeability_transition
http://en.wikipedia.org/wiki/Glutamate_transporter
http://www.ncbi.nlm.nih.gov/pubmed/21998617
http://www.ncbi.nlm.nih.gov/pubmed/19135430
http://www.ncbi.nlm.nih.gov//19096131
http://www.ncbi.nlm.nih.gov/pubmed/16828975
http://it.wikipedia.org/wiki/Eccitotossicit%C3%A0
http://en.wikipedia.org/wiki/Excitotoxicity
http://en.wikipedia.org/wiki/Ischemic_cascade
http://en.wikipedia.org/wiki/Mitochondrial_permeability_transition
http://en.wikipedia.org/wiki/Glutamate_transporter
Fisiologia Medica, F. Conti, Vol. 1 e 2
http://www.ncbi.nlm.nih.gov/pubmed/12909079
http://www.nature.com/aps/journal/v30/n4/full/aps200924a.html
http://www.nature.com/nature/journal/v341/n6242/abs/341533a0.html
http://www.ncbi.nlm.nih.gov/pubmed/16828975
http://www.ncbi.nlm.nih.gov/pubmed/7649222
http://www.ncbi.nlm.nih.gov/pubmed/9334393
http://www.ncbi.nlm.nih.gov/pubmed/22047744
http://www.ncbi.nlm.nih.gov/pubmed/9502803
http://www.ncbi.nlm.nih.gov/pubmed/10627577
http://www.ncbi.nlm.nih.gov/pubmed/20851746
http://www.jneurosci.org/content/31/39/13710.long
http://www.ncbi.nlm.nih.gov//19096131
http://www.ncbi.nlm.nih.gov/pubmed/19135430
