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Calcium, mitochondria and oxidative stress in neuronal pathology Novel aspects of an enduring theme Christos Chinopoulos and Vera Adam-Vizi Department of Medical Biochemistry, Semmelweis

Calcium, mitochondria and oxidative stress in neuronal pathology

Novel aspects of an enduring theme

Christos Chinopoulos and Vera Adam-Vizi

Department of Medical Biochemistry, Semmelweis University, Neurobiochemical Group, Hungarian Academy of Sciences, Szentagothai Knowledge Center, Budapest, Hungary

Background

A long-standing perception is that upon activation of

glutamate receptors followed by a robust Ca2+ influx,

in situ mitochondria generate reactive oxygen species

(ROS) [1–6] These studies inferred that mitochondrial

Ca2+ sequestration is a prerequisite for production of

ROS: abolition of mitochondrial membrane potential

(DYm) by mitochondrial poisons, and thus,

electro-phoretic calcium uptake or direct inhibition of the

uni-porter with ruthenium red prevented ROS generation

Parallel to these reports, the response of isolated

mito-chondria to calcium loading in terms of ROS

produc-tion has also been scrutinized; it was found that mitochondrial Ca2+uptake led to free radical produc-tion [7–12] On the other hand, it was shown that ROS formation depends steeply on DYm [13–15], and from

a thermodynamic point of view, Ca2+ uptake occur-ring at the expense of membrane potential should result in a decrease in ROS production (in the absence

of respiratory chain inhibitors), as it has also been demonstrated (reviewed in [16,17]) Nevertheless, brain mitochondria also generate ROS in a DYm-independ-ent manner [18–20] The reason behind the opposing observations that mitochondrial ROS production increases or decreases upon Ca2+ uptake is not

Keywords

alpha-ketoglutarate dehydrogenase;

oxidative stress; permeability transition

pore;store-operated Ca 2+ entry; transient

receptor potential; TRPM2; TRPM7

Correspondence

V Adam-Vizi, Semmelweis University,

Department of Medical Biochemistry,

Budapest H-1444, PO Box 262, Hungary

Fax: +36 1 2670031

Tel: +36 1 2662773

E-mail: av@puskin.sote.hu

(Received 18 October 2005, accepted

14 December 2005)

doi:10.1111/j.1742-4658.2005.05103.x

The interplay among reactive oxygen species (ROS) formation, elevated intracellular calcium concentration and mitochondrial demise is a recurring theme in research focusing on brain pathology, both for acute and chronic neurodegenerative states However, causality, extent of contribution or the sequence of these events prior to cell death is not yet firmly established Here we review the role of the alpha-ketoglutarate dehydrogenase complex

as a newly identified source of mitochondrial ROS production Further-more, based on contemporary reports we examine novel concepts as poten-tial mediators of neuronal injury connecting mitochondria, increased [Ca2+]c and ROS⁄ reactive nitrogen species (RNS) formation; specifically: (a) the possibility that plasmalemmal nonselective cationic channels con-tribute to the latent [Ca2+]c rise in the context of glutamate-induced delayed calcium deregulation; (b) the likelihood of the involvement of the channels in the phenomenon of ‘Ca2+paradox’ that might be implicated in ischemia⁄ reperfusion injury; and (c) how ROS ⁄ RNS and mitochondrial sta-tus could influence the activity of these channels leading to loss of ionic homeostasis and cell death

Abbreviations

2-APB, 2-aminoethoxydiphenyl borate; ADPR, ADP-ribose; DAG, diacylglycerols; DCD, delayed calcium deregulation; KGDHC, a-ketoglutarate dehydrogenase complex; NMDA, N-methyl- D -aspartate; PTP, permeability transition pore; RNS, reactive nitrogen species; ROS, reactive oxygen species; siRNA, short interfering RNA; SOC channel, store-operated Ca 2+ channel.

entirely clear; a plausible explanation lies in the

condi-tion in which mitochondria are probed for ROS,

specifically whether or not the organelles undergo

per-meability transition pore (PTP) formation Among the

many features accompanying mitochondrial

permeabil-ity transition (for a full list see [16] and references

therein) loss of glutathione, cytochrome c, substrates

and pyridine nucleotides are characteristic This leads

to an increase in ROS production from the impaired

mitochondria by multiple means: (a) loss of

glutathi-one from the matrix decreases the antioxidant capacity

resulting in a net ‘steady-state’ increase in the amount

of ROS [21]; (b) loss of cytochrome c impairs the flow

of electrons in the respiratory chain inducing

over-reduction of the complexes, favouring the generation

of ROS [16,17,22]; (c) reduction in the matrix

concen-tration of electron acceptors, i.e NAD+, results in

ROS emission from the a-ketoglutarate dehydrogenase

complex (KGDHC) [23,24]

Mitochondrial formation of ROS-the

role of KGDHC

The first observation of ROS production in

mitoch-ondrial fragments was reported in 1966 by Jensen [25]

Subsequent studies by Britton Chance’s group,

estab-lished that mitochondria generate ROS [26,27] The

sites of ROS formation within the organelle have been

extensively reviewed elsewhere [17,20,28] Among

them, complex I [29–31] and III [32–35] of the

respirat-ory chain have attracted most attention However, in

light of recent results on the substantial contribution

of matrix enzymes (especially KGDHC) on ROS

gen-eration, we believe that in addition to the respiratory

chain, the components of the Krebs cycle should also

be considered as a possible important source of ROS

in mitochondria

Almost all studies have used respiratory chain

inhib-itors as tools to maximize and to identify potential

sites of ROS production in isolated mitochondria

They revealed that inhibition of complexes I and III,

respectively, with specific mitochondrial toxins such as

rotenone and antimycin A, results in high rates of

ROS production [29,36,37] For complex I in

partic-ular, the ‘reverse electron transport’ mode of ROS

pro-duction has gained momentum throughout the past

four decades [38]; reverse electron transport requires

highDYm and is abolished by the complex I inhibitor,

rotenone [18], but the pathophysiological relevance of

this mode of ROS generation is questionable Similar

approaches have been used successfully to study ROS

production in in situ brain mitochondria present in

isolated nerve terminals (synaptosomes) [39], but no

information is yet available regarding the specific sites

or mechanisms of ROS generation in the absence of respiratory chain inhibitors

Numerous reports in isolated or in situ mitochondria support complex I being regarded as a major site of ROS production, however, a lingering assumption remains that all ROS production caused by complex I inhibitors occurs at the complex I site There are other sources of ROS within the mitochondrial matrix that are in equilibrium with the ratio NAD(P)H⁄ NAD(P)+, such as the dihydrolipoyl dehydrogenase (Dld) compo-nent of KGDHC [40] In intact mitochondria, complex

I inhibition by any means, inevitably results in over-reduction of most if not all NAD+-linked matrix enzymes

Among the NAD+-linked dehydrogenases that gen-erate ROS, KGDHC deserves special attention KGDHC is a mitochondrial enzyme tightly bound to the inner mitochondrial membrane on the matrix side [41] It (as well as other but not all dehydrogenases) binds to complex I of the mitochondrial respiratory chain [42] and may form a part of the TCA cycle enzyme supercomplex [43] Mammalian KGDHC is composed of multiple copies of three enzymes: a-keto-glutarate dehydrogenase (E1; EC 1.2.4.2), dihydrolipo-amide succinyltransferase (E2; EC 2.3.1.61), and dihydrolipoamide dehydrogenase (E3 or Dld; EC 1.8.1.4) Dld is also a part of other multienzyme com-plexes such as the pyruvate dehydrogenase complex (PDHC), the branched chain ketoacid dehydrogenase complex, and the glycine cleavage system [44–47] The catalytic mechanism of the a-ketoacid dehydrogenase complex was reviewed by Bunik [40]

Isolated KGDHC [23] as well as PDHC [24] in isola-ted and in in situ mitochondria respectively produce superoxide and H2O2 Quantitatively, it seems likely that KGDHC generates the majority of ROS among dehydrogenases: under conditions of maximum respir-ation induced with either ADP or an uncoupler, a-ketoglutarate supports the highest rate of H2O2 pro-duction [24] The Dld component of KGDHC, and to

a lesser degree of PDHC, generate ROS in isolated mouse brain mitochondria [24] The reasons behind this quantitative discrepancy among the Dld-contain-ing dehydrogenases regardDld-contain-ing ROS production are at present, unknown The isolated Dld subunit is able to form H2O2 and superoxide radical, accompanying NADH oxidation [40,48,49] This observation is important as to the mechanisms and sites of ROS pro-duction in mitochondria because the flavin of the Dld subunit is abundant and possesses a sufficiently negat-ive redox potential (Em 7.4¼)283 mV) to allow superoxide formation [50,51] Moreover, H2O2

produc-tion by brain mitochondria isolated from heterozygous

knockout mice deficient in Dld is significantly

dimin-ished, as compared to wild-type littermates [24]

Within KGDHC, it is the flavin or the neighbouring

disulfide bridge in the catalytic centre of the Dld

com-ponent that could act as an electron donor for

superox-ide formation [52] KGDHC is activated by low

concentrations of Ca2+and matrix ADP [53–56]

Con-sidering that KGDHC-mediated ROS production

requires a fully active complex with all the cofactors

and substrates (except NAD+), the fact that the enzyme

activity is stimulated by Ca2+ and ADP may perhaps

account for previous findings that mitochondrial ROS

production was increased by Ca2+[7–11,14] and ADP

[30] Results obtained in our laboratory [23]

demon-strate that Ca2+activates ROS production by isolated

KGDHC both in the presence and in the absence of

pyridine nucleotides Still, the reduced Dld subunit is

the most likely source of ROS under conditions of an

elevated NADPH⁄ NADP+ ratio in the mitochondrial

matrix [23,24] The conditions promoting

KGDHC-mediated ROS production may be any that increase the

intramitochondrial NADH⁄ NAD+ ratio (e.g

inhibi-tion of oxidative phosphorylainhibi-tion or inhibiinhibi-tion of any

segment of the mitochondrial electron transport chain)

This hypothesis is favoured by our results showing

that ROS production by isolated KGDHC is strongly

dependent on the NADH⁄ NAD+ratio [23]

The relationship of ROS to KGDHC is extended in

an ‘ouroboros’ fashion to the self-inactivation of the

enzyme by ROS We demonstrated previously, that

KGDHC is sensitive to inhibition by H2O2 [57] That

inevitably leads to a decrease in complex I function, as

repeatedly demonstrated [57–61], since KGDHC which

is the rate-limiting step of the TCA cycle provides

NADH as a substrate for the respiratory chain complex

It is difficult to establish the extent of contribution

of KGDHC and other enzymes to overall ROS

pro-duction in mitochondria, as this is prone to be

condi-tion-dependent (e.g choice of substrate), in addition to

heavily reliant on non-Krebs cycle enzyme mediated

ROS formation through the respiratory chain; i.e both

complex I and KGDHC are in equilibrium with the

NAD(P)H⁄ NAD(P)+ratio, and therefore

interdepend-ent on each other concerning ROS formation Thus,

in organelloit might not be possible to accurately

esti-mate the degree of contribution of each ROS-forming

site, because inhibition of ROS production in the one

may aggravate ROS formation in the other, and vice

versa

The observation that KGDHC generates and is also

self-inactivated by ROS, is of paramount importance in

neuronal pathology A compelling body of evidence

indicates that mitochondria are the major source of ROS in several neurodegenerative conditions [37,62] Also, KGDHC activity is severely reduced in a variety

of neurodegenerative diseases associated with impaired mitochondrial functions, specifically, Alzheimer’s dis-ease [63–67], Parkinson’s disdis-ease [68–71], progressive su-pranuclear palsy [72,73] and Wernicke–Korsakoff syndrome [74] It is not known if the physical associ-ation of KGDHC with complex I (see above) plays a role in the dual deficiency of these protein complexes in Parkinson’s disease It appears that neuronal pathology

is preferentially associated with KGDHC deficiency: in

an animal model of diminished KGDHC activity caused

by thiamine deprivation in the diet, neurons are dying, while endothelial cells, astrocytes and microglia are not affected In fact, KGDHC activity is increased in these non-neuronal cell types [63], which might indicate that KGDHC deficiency has an etiologic role in the manifes-tation of some neurodegenerative diseases [75,76] It must be emphasized that this multienzyme is the rate-limiting step of the Krebs cycle, and if altered that would inpact on the overall energy production in the affected tissue Moreover, in vivo studies suggested that reduced activity of KGDHC predisposes to damage by toxins, such as 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine (MPTP) or malonate, reducing the capacity of neurons to respond to stress [77,78] In addition, it was shown recently that reduction in the E2 subunit of KGDHC is associated with diminished growth of cells and impaired antioxidant defence systems, without a reduction in the overall activity of the complex [79] This finding should come at no surprise: several enzymes of the TCA cycle (and at least one glycolytic enzyme [80]) have roles beyond those of just being cycle participants for the provision of reducing equivalents: aconitase, isocitrate dehydrogenase and kgd2p (a sub-unit of KGDHC in yeast equivalent to E2 in mammals), have two or more different functions, in addition to having supporting functions for oxidative defences [79], involving the thioredoxin system [40] Aconitase acts also as an iron-responsive element binding protein, iso-citrate dehydrogenase is an RNA-binding protein, while kgd2p is a mitochondrial DNA binding protein [81–84] Mitochondria from different brain regions contain different amounts of KGDHC [85,86], which may account for regional vulnerability For instance, the cholinergic neurons of the nucleus basalis of Meynert have high levels of KGDHC, and these neurons are particularly vulnerable in Alzheimer disease [64] Nevertheless, the relationship between KGDHC activity and mitochondrial damage per se is much less clear One can speculate that KGDHC-mediated oxidative stress predisposes the cell to succumb to

con-comitant adverse conditions; in addition, a diminished

KGDHC activity will lead to insufficient provision of

reducing equivalents, lowering the energetic capacity of

the mitochondria of the affected cell However, studies

with the KGDHC inhibitor KMV

(alpha-keto-beta-methyl-n-valeric acid) suggest that inhibition of the

enzyme might contribute to cell death by induction of

permeability transition [87]

Permeability transition pore in situ

Permeability transition pore is considered to be a

chan-nel with a large conductance provided by proteins

resi-ding in both the inner and outer mitochondrial

membrane, that is activated by mitochondrial Ca2+

overloading and other factors including oxidative stress

[88,89] In neurons the presence of PTP in situ has not

gained wide acceptance among investigators and

results published in the literature support views of both

its presence and absence in several in vitro models of

neurodegeneration [90–98] One of the possible reasons

for this discrepancy is that sensitivity to cyclosporin A

is considered pathognomonic for mitochondrial PTP

(see also [90]) Cyclosporin A is a potent inhibitor of

PTP in isolated liver mitochondria [99] that has been

demonstrated to be effective also in situ in this and

other organs [100–103] The sensitivity of isolated

brain mitochondria to cyclosporin A depends highly

on the conditions: in the absence of adenine

nucleo-tides and magnesium, cyclosporin A mitigates Ca2+

-in-duced mitochondrial pore formation [104,105]

however, in the presence of 3 mm ATP plus 1 mm free

Mg2+, cyclosporin A is only marginally effective,

pro-vided that mitochondria are challenged by boluses of

CaCl2 [104] In the case that Ca2+ loading occurs

slowly, cyclosporin A delays onset of PTP in brain

mitochondria extensively, even in the presence of

aden-ine nucleotides and magnesium [106] The caveat here

is that despite the decreased ATP levels to less than

the millimolar range during ischemic deenergizing,

ADP levels approximate 400 lm [107], and the Ki for

inhibition of the PTP by ADP is in the low

micromo-lar range [108] Moreover, in situ neuronal

mitochon-dria are exposed to bolus-like additions of Ca2+ [109]

during intense glutamate receptor stimulation for the

duration of seizure activity or reversal of glutamate

transporters throughout ischemia [110] Ca2+ cycling

across the mitochondrial inner membrane ensues

sub-sequently [111] On the other hand, intense stimulation

of N-methyl-d-aspartate (NMDA) receptors on

cul-tured cerebellar granule and hippocampal neurons

cau-ses major ultrastructural alterations of mitochondria,

implying the activation of some form of PTP [112,113]

Mitochondrial alterations suggestive of pore opening is also demonstrated in vivo, during the postischemic per-iod in the gerbil brain [114] Yet, to identify these

in situmitochondrial alterations as the PTP on the basis

of the functional⁄ morphological ⁄ pharmacological cri-teria applied for isolated mitochondria is rather hasty Collectively, the sensitivity of glutamate-induced neuronal damage to cyclosporin A as diagnostic for PTP occurrence is unreliable This ambiguity is also nur-tured by the complex pharmacology of cyclosporin A and its affinity to non-PTP targets [90,115] that could be involved in the manifestation of neuronal injury [116], in addition to the fact that PTP may not have a causal role

in excitotoxic cell death It is to be noted that the magni-tude of the literature involving cyclosporin A unrelated

to mitochondria is 12 times larger than that implicating PTP! The nonimmunosuppressant analogue, N-methyl-valine-4-cyclosporin also gave contrasting results, con-ferring neuronal protection against excitotoxicity in some studies [92,117,118], but not in others [94]

What could be important though, is the role of the

in situ mitochondrial pore formation in dictating the type of death that the ill-fated neuron will follow A most simplistic view is that this pore will promote apoptosis due to release of cytochrome c followed by activation of caspases [119,120], provided that pertain-ing conditions divert the type of cell death from the necrotic to the apoptotic pathway [121,122] The role

of mitochondria in apoptosis and necrosis has been extensively reviewed elsewhere [121,123–131] Recently however, a blow was delivered to the conception that PTP contributes to apoptotic cell death by three almost simultaneous and independent reports using cyclophilin D knockout mice [132–134] Cyclophilin D

is a component of the PTP complex [135,136] and it is the target for cyclosporin A As expected, mitochon-dria isolated from the cyclophilin D knockout mice were much less susceptible to various PTP-inducing regimes, that are otherwise sensitive to cyclosporin A treatment (see also [137]) Unexpectedly though, tissues obtained from mutant mice were not more resistant to several apoptotic stimuli than those from their wild-type littermates; however, the resistance of the mutant mice to treatments known to result in necrotic cell death was much higher than in control mice

Mitochondrial Ca2+-flux pathways and relation to signal transduction

In general, the contribution of mitochondria to intra-cellular Ca2+ homeostasis is ascribed to uptake and release through the uniporter, the mitochondrial

Na+⁄ Ca2+ exchanger, the PTP (both high- and

low-conductance mode) and other less well characterized

pathways, such as the ‘Na+-independent pathway for

Ca2+efflux’ and a H+⁄ Ca2+antiporter [89,138] With

the exception of the high-conductance mode of PTP

and the uniporter, none of these molecular

complexit-ies have been described to be modulated by any signal

transduction mediators High-conductance PTP is

known to be affected by matrix Ca2+ and ROS [89]

Also the uniporter is supposed to be activated only if

extramitochondrial Ca2+levels exceed a certain

thresh-old concentration, termed the ‘set-point’ [139];

how-ever, this has been challenged recently, showing that

in situ mitochondria accumulate Ca2+ well below the

set-point, in permeabilized rat adrenal glomerulosal

cells [140] Nonetheless, despite that mitochondria are

increasingly viewed as active mediators of [Ca2+]c

regulation, the pathways that these organelles use to

achieve this task are rather passive

To this repertoire of Ca2+ influx and efflux

mecha-nisms across the mitochondrial membranes, a novel

Ca2+-efflux-only machinery has been recently added: a

channel located in the inner membrane activated by

dia-cylglycerols (DAGs) [141] This is either a single channel

with numerous substates (mean conductance
200 pS),

or multiple channels with unequal conductance DAGs

cause a biphasic form of Ca2+ efflux in Ca2+-loaded

mitochondria: the first wave of efflux is attributed to the

activation of the DAG-sensitive nonselective cationic

channels; the second wave is due to opening of the PTP

It is not yet known how activation of the former leads

to induction of the latter One is tempted to hypothesize

that the initial Ca2+ efflux through DAG-sensitive

channels causes intense Ca2+ cycling due to reuptake

by the uniporter, leading to PTP However,

cyclospo-rin A fails to defend against the secondary Ca2+efflux

in liver mitochondria in the presence of DAGs, in which

the immunosuppressant otherwise confers significant

protection against PTP induction

The role of DAG-sensitive mitochondrial channels in

physiological [Ca2+]c regulation can easily be

envis-aged: upon phosphatidylinositol (4,5) bisphosphate

(PIP2) hydrolysis, inositol-1,4,5-triphosphate (IP3)

dif-fuses in the cytosol to activate IP3 receptors on the

endoplasmic reticulum releasing Ca2+to the cytoplasm,

followed by triggering of Ca2+influx from the

extracel-lular space [142] The role of mitochondria in shaping

Ca2+transients during such events is recognized in

lim-iting Ca2+ diffusion, and secondarily relieving Ca2+

-mediated negative feedback on the Ca2+flux pathways

themselves [143] However, the other obligatory

meta-bolite of PIP2 catabolism) DAG ) may regulate the

role of mitochondria in shaping those [Ca2+]c

tran-sients: mitochondrial DAG-sensitive channels would

re-release sequestered matrix Ca2+ only in the vicinity where DAGs are formed most likely in microdomains, since this second messenger is extremely lipophilic and does not diffuse into the aqueous cytosol

Mitochondrial permeabilization and the delayed calcium deregulation

The association of ROS to a possible PTP induction prior to neuronal cell death has received much atten-tion in relaatten-tion to the delayed, irreversible rise in [Ca2+]c following a prolonged glutamate stimulus, coined by Nicholls’ group as ‘delayed calcium deregu-lation, DCD’ [144] that commits a neuron to die [145–148] DCD was originally described by Manev and colleagues [149], further characterized by the groups of Thayer [150] and Tymianski [146] However, credit should also be given to an earlier work by Con-nor and colleagues, showing that a short exposure (1–3 s) of CA1 hippocampal neurons to NMDA causes

an abrupt elevation in [Ca2+]cthat returns to baseline;

a subsequent exposure to NMDA of the same duration

a few minutes later leads to an irreversible and sus-tained increase in intracellular [Ca2+]c in apical dend-rites [151] DCD is invariably demonstrated in every neuronal cell type studied, i.e spinal [146], hippocam-pal [150], cerebellar granule [152], striatal [117] and cortical neurons [93,153] The phenomenon is not observed if high extracellular K+ is alternatively employed to elevate [Ca2+]c; this led to the proposal

of a ‘source specificity’ of Ca2+-induced neurotoxicity [146] However, this was subsequently challenged by studies demonstrating that activation of NMDA recep-tors produces much larger Ca2+ entry than activation

of voltage-dependent Ca2+ channels by high extracel-lular K+[154]

This secondary [Ca2+]c rise is not inhibitable by postglutamate addition of antagonists of NMDA or non-NMDA receptors [94,145,149,150], nor by block-ing voltage-dependent Ca2+ or Na+ channels [145,149,150,155] Results supporting views that DCD

is comprised of an active Ca2+ influx pathway [93,146,149,150,155–159] as well as those indicating a failure in Ca2+efflux mechanisms [160–162], are avail-able in the literature It is anticipated that these seem-ingly opposing observations represent two-facets of the same problem: even in the earliest report on DCD by Manev and colleagues [149] it was shown that during the postglutamate period neurons still accumulate

45Ca2+ within 30 s exposure to the isotope, without any statistically significant difference seen in the pres-ence or abspres-ence of N-methyl-d-aspartate receptors/non-N-methyl-d-aspartate receptors/voltage dependent Ca2+

channels (NMDAR⁄ non-NMDAR ⁄ VDCC blockers).

That attests to the presence of a discrete pathway for

Ca2+ influx Yet, it was recently demonstrated that in

an almost identical paradigm of excitotoxicity, the

plas-malemmal Na+⁄ Ca2+ exchanger (in particular the

NCX3 isoform) is cleaved by calpain, severing the high

capacity Ca2+ efflux pathway in neurons [161]

Provi-ded that the Ca2+influx pathway is most likely a

chan-nel, it must saturate [163] imposing a continuous load of

calcium to the neuron The turning point upon which

the cell looses the ability to buffer the incoming calcium

resulting in an abrupt, sustained and irreversible

increase in [Ca2+]c, probably coincides with the

clea-vage of the exchanger (but see [164]) Therefore,

inhibi-tion of the, as yet unidentified, Ca2+influx pathway or

prevention of NCX proteolysis should thwart DCD

The question arises: what is the nature of the Ca2+

influx pathway?

Non-selective cationic channel(s) and

the DCD

As mentioned above, inhibition of NMDAR⁄

non-NMDAR⁄ voltage-dependent Ca2+ or Na+ channels

after the initial Ca2+and Na2+influx through the

glu-tamate receptors, failed to prevent DCD Yet, DCD

demands the existence of a discrete pathway as it

pre-cedes, and eventually leads to, plasma membrane

leaki-ness and cell death [145,146,148] The notion that

DCD is not attributed to the ‘traditionally’ recognized

Ca2+channels, such as glutamate receptor-operated or

voltage-gated Ca2+ channels has been proposed

previ-ously [157,158] Along this line, it was shown that a

secondary activation of a nonselective cation

conduct-ance, termed postexposure current (Ipe), is induced

sub-sequent to excitotoxic application of NMDA to

hippocampal neurons that probably contributes to the

delayed Ca2+rise [156]

Relevant to the inability of the glutamate receptor

blockers to prevent DCD, antiexcitotoxic therapy

util-izing these compounds failed to produce a better

out-come in clinical trials concerning stroke treatment

[165–167] To address this setback, Aarts and

collea-gues [159] examined the possibility that an overlooked

neurotoxic process was occurring in a well-established

in vitro model of excitotoxicity, by subjecting cultured

neurons to oxygen–glucose deprivation This treatment

results in neuronal demise through NMDAR

activa-tion [168,169] It was found that a member of the

melastatin branch of the transient receptor potential

channel (TRP) family, TRPM7 [170], mediates a lethal

cation current loading the neurons with Ca2+ and

Na+ This nonselective current was activated by ROS

and reactive nitrogen species (RNS), and its abolition permitted the survival of neurons previously destined

to die from prolonged anoxia, regardless of the pres-ence or abspres-ence of NMDAR blockers

In a subsequent study, we explored the hypothesis that a TRP channel contributes to the manifestation of DCD [93] A pharmacological approach was used, applying 2-aminoethoxydiphenyl borate (2-APB) or

La3+ to cultured cortical neurons challenged by pro-longed glutamatergic stimulation We observed that 2-APB and La3+ diminished the delayed Ca2+ rise with a 50% inhibitory concentration of 62 ± 9 lm and 7.2 ± 3 lm, respectively Both substances are known to inhibit TRP channels in addition to acting

on many other targets; 2-APB blocks store-operated

Ca2+(SOC) channels [171], the IP3 receptor [172], the sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) pump [173], voltage-dependent K+channels [174], gap junctions [175] and the cyclosporin A-insensitive PTP [104], while La3+ blocks SOC [176] and voltage-dependent Ca2+ channels [177] Almost all non-TRP targets are irrelevant or have been previously excluded concerning the origin of DCD, except for the cyclospo-rin A-insensitive PTP that is abolished by 2-APB in isolated brain mitochondria [104] However, in our hands, bongkrekic acid ameliorated the cyclosporin A-insensitive PTP but not the DCD [93,104] From this study we concluded that a TRP channel could be responsible for the Ca2+influx part of DCD In gen-eral, the two inhibitors that we used do not distinguish among individual members of the TRP family, but for reasons explained below, it is tempting to speculate that it is the TRPM7 Unfortunately, we could not achieve silencing of TRPM7 expression in our cultures with short interfering RNA (siRNA); primary neurons are notoriously vulnerable to transfection techniques,

as opposed to the ease and the high efficiency of the procedure in cell lines Hopefully, the development of novel approaches such as the conjugation of siRNA to penetratins [178,179] will assist transfection protocols and allow research on primary neuronal cultures to benefit from the tremendous potential of siRNA The connection of TRPM7 to DCD may lie in the observation that this channel is activated by ROS and RNS [159] For a long time, ROS were considered to

be responsible for DCD [180]; however, in a recent study it was deduced that the increased ROS produc-tion is a consequence, rather than a cause of DCD [181] In the latter study the authors also demonstrated that the increase in superoxide radical formation is predominantly associated with extramitochondrial phospholipase A(2) (PLA2) activation, and it does not emanate from mitochondria That may be in contrast

with previous reports claiming that ROS are the

induc-ers of DCD However over the years concerns have

arisen as for the reliability of ROS-detecting dyes,

given that some are affected by confounding

parame-ters such as mitochondrial membrane potential (see

discussion in [181]) The development of new dyes

des-cribed recently will no doubt contribute to the

clarifi-cation of these matters [182]

In light of the recent observations though, one could

argue that TRPM7 is not the Ca2+ influx pathway of

DCD, as the increase in superoxide radical appears

after the secondary [Ca2+]c rise However, the exact

species activating TRPM7 is not known, and the extent

of ROS production necessary to activate the channel

maybe less than the detection level of the probes used

In addition, ROS⁄ RNS could be just one of the many

activators of the channel [183], while others that might

play a significant role could be also mobilized upon

prolonged glutamate exposure We have found that by

elevating intracellular [Mg2+]iDCD is abolished in

cul-tured cortical neurons [93], and it is known that

TRPM7 receives strong negative feedback by

intracel-lular Mg2+ [170] In addition, TRPM7 currents

induced by oxygen–glucose deprivation promote

fur-ther ROS production [159], and this could partially

explain the results of Vesce and colleagues, detecting

an increase in superoxide formation after the delayed

secondary [Ca2+]c rise [181] In our opinion, TRPM7

is one of the best possible candidates for the Ca2+

influx part of DCD; other good candidates are TRPM2

(see below) and the calcium-permeable acid-sensing ion

channel [184] (not reviewed here)

Nonselective cationic channels and the

’Ca2+paradox’

In spite of the widely accepted role of [Ca2+]c

deregula-tion in the manifestaderegula-tion of neurodegeneraderegula-tion, exactly

how Ca2+ ions mediate neural cell death is less clear

[185] One of the most important unresolved issues is the

mechanism by which [Ca2+]c increases to excessively

high levels in neurons following periods of intense

neur-onal activation Reaching further from the possibility of

the involvement of TRP channels in the delayed calcium

deregulation, these proteins could participate in an

addi-tional overlooked pathway of Ca2+influx that may

per-tain during ischemia⁄ reperfusion or other type of

pathology Large [Ca2+]cincreases are known to be

trig-gered by reintroduction of ‘normal’ Ca2+

concentra-tions to the extracellular milieu after the tissue has

experienced a [Ca2+]e-free challenge, or at least a severe

reduction in extracellular calcium concentration, termed

‘Ca2+paradox’ The free extracellular calcium

concen-tration falls dramatically in several brain disease states: (a) during or after ischemia (0.1–0.28 mm [186–189]); (b) traumatic brain injury (0.1 mm [190]); (c) severe hypo-glycemia (0.12 mm [191]); and (d) spreading depression (0.06–0.08 mm [192]) Reduction of extracellular Ca2+

is mostly due to robust influx of the cation to the intra-cellular milieu, although the appearance of lactate in the interstitium during ischemia, with the ability to chelate divalent ions significantly, also plays a role [193,194]

The Ca2+paradox Paradoxical Ca2+increases were originally described in isolated heart preparations [195] and subsequently shown to be associated with tissue damage in this and other organs, including the kidney and skeletal muscle [196,197], but not in others, i.e liver [198] Interestingly, the possibility that paradoxical Ca2+influx contributes

to neuronal degeneration was put forward almost

20 years ago [199], but the vast majority of subsequent work on [Ca2+]c elevation during excitotoxicity has since concentrated on other Ca2+ entry routes, inclu-ding glutamate receptors and voltage-gated Ca2+ chan-nels Unfortunately, this emphasis has not resulted in any clinically useful intervention to limit the neuronal damage following ischemia⁄ reperfusion or other brain injury Inescapably, within a context of ischemia⁄ reper-fusion in which a Ca2+ paradox is encompassed [200], concomitant adverse conditions, e.g oxygen–glucose deprivation, associated ROS production and many more) reviewed in [201] ) contribute to irreversible tissue damage Nevertheless, the paradoxical Ca2+rise per seremains a poorly understood phenomenon What

is known though, is that abolition of in situ mitochond-rial respiration and oxidative phosphorylation protects against the Ca2+ paradox [202] The reasons behind this unexpected finding are not yet understood A num-ber of theories were put forward, including the deleteri-ous effect of overloading mitochondria with Ca2+ that can only happen in respiring mitochondria

Possible mechanisms underlying neuronal paradoxical Ca2+-increases

While multiple mechanisms could contribute to para-doxical Ca2+increases, the most current interest is the activation of novel nonselective cation channels It is known that reduction of [Ca2+]eactivates nonselective cation currents in hippocampal neurons [203] and neo-cortical nerve terminals [204] termed csNSC and NSC, respectively, as well as in thalamic neurons [205], vagal afferent nerves [206] and ventricular myocytes [207] Such currents may underlie paradoxical Ca2+increases

activated by transient [Ca2+]e removal We have also

observed the appearance of a nonselective,

noninacti-vating cation conductance upon reducing extracellular

Ca2+and Mg2+in cultures of cortical neurons, as well

as in cortical and hippocampal neurons in brain slices

from adult mice, raising the possibility that such

cur-rents are readily available in these cells (C

Chinopou-los, unpublished data) Furthermore, we have recently

reported that cultured cortical neurons exhibit

para-doxical Ca2+ entry [93] and it is conceivable that the

[Ca2+]c rise is a result of the ‘tails’ of these currents

Alternative mechanisms for paradoxical Ca2+ rise lie

in a diversity of molecular complexities: lowering

[Ca2+]e reduces the shielding of negatively charged

groups located at the membrane surface affecting the

voltage-dependent activation of various ion channels

[163,208] In addition, it is the biophysical property of

many types of channels to conduct monovalents in a

less controlled manner in the absence of divalent

cati-ons, such as the Icrac-conducting channel [209,210],

voltage-gated Ca2+ channels [211–215], Na+channels

[216,217], K+ channels [218], other unidentified

chan-nels [203–207] and many members of the TRP family

of channels (see below) In extreme cases, channel

selectivity is lost when [Ca2+]e is reduced to ultra-low

(<1 lm) concentrations [219]

Apart from this biophysical property of channels, a

number of receptor-based mechanisms are modulated

by [Ca2+]e: (a) the Ca2+-sensing receptor is activated by

millimolar changes in [Ca2+]e, and is widely distributed

in mammalian tissues including brain [220]; (b)

hemi-gap channels in horizontal cells of the catfish retina are

activated by [Ca2+]edecreases [221] and it is likely that

gap junctional regulation could be strongly modified by

[Ca2+]ein the central nervous system [222]; (c)

metabo-tropic glutamate receptors 1, 3 and 5 [223] are activated

by physiological [Ca2+]e fluctuations in the synaptic

cleft [224]; and (d) the Gamma-aminobutyric acid (B)

GABAB receptor also possesses Ca2+ sensing

proper-ties, potentiating GABA responses upon increase of

[Ca2+]e[225] It is not yet known whether these

addi-tional Ca2+-sensing mechanisms may act alone or in

concert with nonselective Ca2+ channels in producing

significant excitotoxic Ca2+increases following ischemic

insults

TRP channels as candidates for paradoxical

Ca2+-increases

TRP channels are widely expressed in mammalian

tis-sues, especially in neurons of the central nervous

system [226] With a few notable exceptions, the

phy-siological roles of TRP channels in neurons remain

largely unknown [226–231] Diverse neuropathological conditions were also found to implicate TRP family members: (a) mucolipidosis type IV [232] involving a channel from the distant polycystin branch (TRPP); (b) TRPV4 in neuropathic pain [233], and – as dis-cussed above) (c) TRPM7 in neuronal death caused

by oxygen–glucose deprivation [159]; the latter study also proposed the possibility of TRPM2 involvement,

a view supported by more recent observations on oxi-dative stress-induced cell death [234] Furthermore, ROS were specifically shown to trigger the opening of TRPC3 [235], TRPM2 [236–238] and TRPM7 [159] In preliminary experiments, we have observed that the presence of ROS abolishes [Ca2+]c decay during the paradoxical Ca2+rise and converts it to a progressive [Ca2+]crise (C Chinopoulos, unpublished data)

Of particular interest however, are the observations that a number of TRP channels are activated by a decrease in [Ca2+]e, raising the possibility that they could contribute to paradoxical Ca2+increases Recent descriptions have included the Drosophila TRP channel [239], TRPC1 and TRPC3 [240], TRPC6 [241], TRPC7 [242,243], and TRPM7 [159]

Mitochondrial permeabilization and a possible link to TRP channel activation

Among the known activators of some members of the TRP family, NAD+ and its catabolite ADP-ribose (ADPR) were described to activate TRPM2 [244–247],

in addition to the fact that the channel is stimulated

by ROS⁄ RNS [236,238,246] Furthermore, it was dem-onstrated that the major source of free ADPR medi-ating the activation of TRPM2 in cultured cells were the mitochondria [248] One could link these observa-tions to the fact that opening of the PTP causes the release of mitochondrial NAD+followed by its hydro-lysis by an extramitochondrial NAD+ glycohydrolase

to ADPR [103,249] It is tempting to speculate that this ADPR in conjunction with ROS produced upon loss of mitochondrial integrity, activates the nonselec-tive TRPM2 allowing a large Ca2+ and Na+ load to enter the cytosol Since both high [Ca2+]c and ROS promote mitochondrial pore formation, it seems that the order of appearance of a pore or TRPM2 activa-tion is trivial; what is probably more important is that activation of the one can lead to activation of the other, completing a vicious cycle Intriguingly, silen-cing the expression of TRPM7 with siRNA, led to an accompanying decrease in TRPM2 expression This suggests that the two transcripts might be coordinately regulated, raising the possibility that a fraction of the oxygen–glucose deprivation-induced current recorded

earlier [159] is mediated by TRPM2 or TRPM7

hetero-multimers, a structural arrangement commonly

occur-ring among TRP channels [250,251] Further

implications of TRP channels in relation to the overall

metabolic state of the cell in hypoxia have been

reviewed elsewhere [252]

Trp channels and ionic homeostasis

In view of the fact that most TRP channels are

nonse-lective, in addition to allowing Ca2+ions to enter the

cytosol they also permit Na+ influx and K+ efflux

[226,253,254] The ominous effects of an elevated

[Na+]iare mostly associated with cell swelling and

acti-vation of the Na+⁄ Ca2+ exchanger causing Ca2+

influx However, it is possible that the effect of an

increased [Na+]i may be directly on mitochondria as

recently demonstrated, diminishing the half-life of

mit-ochondrially encoded mRNA, without involving Ca2+

[255,256] In addition it was recently shown that in

mature hippocampal slices, NAD(P)H transients during

postsynaptic neuronal activation are not mediated by

Ca2+, but rather reflect alterations in [Na+]i That may

explain our previous results in isolated nerve terminals

showing that in the presence of an oxidative stress a

concomitant elevation in [Na+]iacts deleteriously on in

situmitochondria [257] The effect of K+loss from the

cytoplasm is commonly ignored; however, it was shown

that it can promote neuronal apoptosis [258–260] To

what extent) if any ) the activation of TRP channels

is associated with alterations of Na+and K+

homeos-tasis in neurodegeneration, is currently unknown

Nev-ertheless, the fact that these proteins are intensely

expressed in the central nervous system [251,254,261]

and their ever-increasing roles in physiology and

pathology being discovered [253,262], identify them as

excellent novel targets amenable to pharmacological

manipulation [254,263,264]

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