Báo cáo khoa học: Calcium, mitochondria and oxidative stress in neuronal pathology Novel aspects of an enduring theme pdf

REVIEW ARTICLE
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 Ca
2+
influx,
in situ mitochondria generate reactive oxygen species
(ROS) [1–6]. These studies inferred that mitochondrial
Ca
2+
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 Ca
2+
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, Ca
2+

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 Ca
2+
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:
(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
[Ca
2+
]
c
and ROS ⁄ reactive nitrogen species (RNS) formation; specifically:
(a) the possibility that plasmalemmal nonselective cationic channels con-
tribute to the latent [Ca
2+
]
c
rise in the context of glutamate-induced
delayed calcium deregulation; (b) the likelihood of the involvement of the
channels in the phenomenon of ‘Ca
2+
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.
FEBS Journal 273 (2006) 433–450 ª 2006 The Authors Journal compilation ª 2006 FEBS 433
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
high DYm 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 H
2
O
2
. 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 H
2
O
2
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 regarding ROS production are at
present, unknown. The isolated Dld subunit is able to
form H
2
O
2

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 7.4 ¼ )283 mV) to allow
superoxide formation [50,51]. Moreover, H
2
O
2
produc-
Ca
2+
, mitochondria, ROS in neuronal disease C. Chinopoulos and V. Adam-Vizi
434 FEBS Journal 273 (2006) 433–450 ª 2006 The Authors Journal compilation ª 2006 FEBS
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 Ca
2+
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 Ca
2+
and ADP may perhaps
account for previous findings that mitochondrial ROS
production was increased by Ca
2+
[7–11,14] and ADP
[30]. Results obtained in our laboratory [23] demon-
strate that Ca
2+
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 phosphorylation or inhibition 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 H
2
O
2
[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 organello it 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 disease [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-
C. Chinopoulos and V. Adam-Vizi Ca
2+
, mitochondria, ROS in neuronal disease
FEBS Journal 273 (2006) 433–450 ª 2006 The Authors Journal compilation ª 2006 FEBS 435
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 Ca
2+
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 Ca
2+
-in-
duced mitochondrial pore formation [104,105]
however, in the presence of 3 mm ATP plus 1 mm free

Mg
2+
, cyclosporin A is only marginally effective, pro-
vided that mitochondria are challenged by boluses of
CaCl
2
[104]. In the case that Ca
2+
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 K
i
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 Ca
2+
[109]
during intense glutamate receptor stimulation for the
duration of seizure activity or reversal of glutamate
transporters throughout ischemia [110]. Ca
2+
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 situ mitochondrial 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 Ca
2+
-flux pathways and
relation to signal transduction
In general, the contribution of mitochondria to intra-
cellular Ca
2+
homeostasis is ascribed to uptake and
release through the uniporter, the mitochondrial
Na
+
⁄ Ca

2+
exchanger, the PTP (both high- and low-
Ca
2+
, mitochondria, ROS in neuronal disease C. Chinopoulos and V. Adam-Vizi
436 FEBS Journal 273 (2006) 433–450 ª 2006 The Authors Journal compilation ª 2006 FEBS
conductance mode) and other less well characterized
pathways, such as the ‘Na
+
-independent pathway for
Ca
2+
efflux’ and a H
+
⁄ Ca
2+
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 Ca
2+
and ROS [89].
Also the uniporter is supposed to be activated only if
extramitochondrial Ca
2+
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 Ca
2+
well below the
set-point, in permeabilized rat adrenal glomerulosal
cells [140]. Nonetheless, despite that mitochondria are
increasingly viewed as active mediators of [Ca
2+
]
c
regulation, the pathways that these organelles use to
achieve this task are rather passive.
To this repertoire of Ca
2+
influx and efflux mecha-
nisms across the mitochondrial membranes, a novel
Ca
2+
-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 Ca
2+
efflux in Ca
2+
-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 Ca
2+
efflux through DAG-sensitive
channels causes intense Ca
2+
cycling due to reuptake
by the uniporter, leading to PTP. However, cyclospo-
rin A fails to defend against the secondary Ca
2+
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 [Ca
2+
]
c
regulation can easily be envis-
aged: upon phosphatidylinositol (4,5) bisphosphate
(PIP
2
) hydrolysis, inositol-1,4,5-triphosphate (IP
3
) dif-
fuses in the cytosol to activate IP
3
receptors on the

endoplasmic reticulum releasing Ca
2+
to the cytoplasm,
followed by triggering of Ca
2+
influx from the extracel-
lular space [142]. The role of mitochondria in shaping
Ca
2+
transients during such events is recognized in lim-
iting Ca
2+
diffusion, and secondarily relieving Ca
2+
-
mediated negative feedback on the Ca
2+
flux pathways
themselves [143]. However, the other obligatory meta-
bolite of PIP
2
catabolism ) DAG ) may regulate the
role of mitochondria in shaping those [Ca
2+
]
c
tran-
sients: mitochondrial DAG-sensitive channels would
re-release sequestered matrix Ca
2+

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 relation to the delayed, irreversible rise in
[Ca
2+
]
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 [Ca
2+
]
c
that 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 [Ca

2+
]
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 [Ca
2+
]
c
; this led to the proposal
of a ‘source specificity’ of Ca
2+
-induced neurotoxicity
[146]. However, this was subsequently challenged by
studies demonstrating that activation of NMDA recep-
tors produces much larger Ca
2+
entry than activation
of voltage-dependent Ca
2+
channels by high extracel-
lular K
+
[154].

This secondary [Ca
2+
]
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 Ca
2+
or Na
+
channels
[145,149,150,155]. Results supporting views that DCD
is comprised of an active Ca
2+
influx pathway
[93,146,149,150,155–159] as well as those indicating a
failure in Ca
2+
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
45
Ca
2+
within 30 s exposure to the isotope, without
any statistically significant difference seen in the pres-

ence or absence of N-methyl-d-aspartate receptors/non-
N-methyl-d-aspartate receptors/voltage dependent Ca
2+
C. Chinopoulos and V. Adam-Vizi Ca
2+
, mitochondria, ROS in neuronal disease
FEBS Journal 273 (2006) 433–450 ª 2006 The Authors Journal compilation ª 2006 FEBS 437
channels (NMDAR ⁄ non-NMDAR ⁄ VDCC blockers).
That attests to the presence of a discrete pathway for
Ca
2+
influx. Yet, it was recently demonstrated that in
an almost identical paradigm of excitotoxicity, the plas-
malemmal Na
+
⁄ Ca
2+
exchanger (in particular the
NCX3 isoform) is cleaved by calpain, severing the high
capacity Ca
2+
efflux pathway in neurons [161]. Provi-
ded that the Ca
2+
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 [Ca

2+
]
c
, probably coincides with the clea-
vage of the exchanger (but see [164]). Therefore, inhibi-
tion of the, as yet unidentified, Ca
2+
influx pathway or
prevention of NCX proteolysis should thwart DCD.
The question arises: what is the nature of the Ca
2+
influx pathway?
Non-selective cationic channel(s) and
the DCD
As mentioned above, inhibition of NMDAR⁄ non-
NMDAR ⁄ voltage-dependent Ca
2+
or Na
+
channels
after the initial Ca
2+
and Na
2+
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

Ca
2+
channels, such as glutamate receptor-operated or
voltage-gated Ca
2+
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 (I
pe
), is induced sub-
sequent to excitotoxic application of NMDA to
hippocampal neurons that probably contributes to the
delayed Ca
2+
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 Ca

2+
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 absence 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
La
3+
to cultured cortical neurons challenged by pro-
longed glutamatergic stimulation. We observed that
2-APB and La
3+
diminished the delayed Ca
2+
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
Ca
2+
(SOC) channels [171], the IP
3

receptor [172], the
sarco-endoplasmic reticulum Ca
2+
ATPase (SERCA)
pump [173], voltage-dependent K
+
channels [174], gap
junctions [175] and the cyclosporin A-insensitive PTP
[104], while La
3+
blocks SOC [176] and voltage-
dependent Ca
2+
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 Ca
2+
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) (PLA
2
) activation, and it does not
emanate from mitochondria. That may be in contrast
Ca
2+
, mitochondria, ROS in neuronal disease C. Chinopoulos and V. Adam-Vizi
438 FEBS Journal 273 (2006) 433–450 ª 2006 The Authors Journal compilation ª 2006 FEBS
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 Ca
2+
influx pathway of
DCD, as the increase in superoxide radical appears
after the secondary [Ca
2+
]
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 [Mg
2+
]
i
DCD is abolished in cul-
tured cortical neurons [93], and it is known that
TRPM7 receives strong negative feedback by intracel-
lular Mg
2+
[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 [Ca
2+
]
c
rise [181]. In our opinion, TRPM7
is one of the best possible candidates for the Ca
2+
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
’Ca
2+
paradox’
In spite of the widely accepted role of [Ca
2+
]
c
deregula-
tion in the manifestation of neurodegeneration, exactly
how Ca
2+
ions mediate neural cell death is less clear
[185]. One of the most important unresolved issues is the
mechanism by which [Ca
2+
]

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 Ca
2+
influx that may per-
tain during ischemia ⁄ reperfusion or other type of
pathology. Large [Ca
2+
]
c
increases are known to be trig-
gered by reintroduction of ‘normal’ Ca
2+
concentra-
tions to the extracellular milieu after the tissue has
experienced a [Ca
2+
]
e
-free challenge, or at least a severe
reduction in extracellular calcium concentration, termed
‘Ca
2+
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 Ca
2+
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 Ca
2+
paradox
Paradoxical Ca
2+
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 Ca
2+
influx contributes
to neuronal degeneration was put forward almost
20 years ago [199], but the vast majority of subsequent
work on [Ca
2+
]
c
elevation during excitotoxicity has
since concentrated on other Ca
2+

entry routes, inclu-
ding glutamate receptors and voltage-gated Ca
2+
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 Ca
2+
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 Ca
2+
rise
per se remains a poorly understood phenomenon. What
is known though, is that abolition of in situ mitochond-
rial respiration and oxidative phosphorylation protects
against the Ca
2+
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 Ca
2+
that
can only happen in respiring mitochondria.
Possible mechanisms underlying neuronal

paradoxical Ca
2+
-increases
While multiple mechanisms could contribute to para-
doxical Ca
2+
increases, the most current interest is the
activation of novel nonselective cation channels. It is
known that reduction of [Ca
2+
]
e
activates 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 Ca
2+
increases
C. Chinopoulos and V. Adam-Vizi Ca
2+
, mitochondria, ROS in neuronal disease
FEBS Journal 273 (2006) 433–450 ª 2006 The Authors Journal compilation ª 2006 FEBS 439
activated by transient [Ca
2+
]
e
removal. We have also
observed the appearance of a nonselective, noninacti-

vating cation conductance upon reducing extracellular
Ca
2+
and Mg
2+
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 Ca
2+
entry [93] and it is conceivable that the
[Ca
2+
]
c
rise is a result of the ‘tails’ of these currents.
Alternative mechanisms for paradoxical Ca
2+
rise lie
in a diversity of molecular complexities: lowering
[Ca
2+
]
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 I
crac
-conducting channel [209,210],
voltage-gated Ca
2+
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 [Ca
2+
]
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 [Ca
2+
]
e
: (a) the Ca
2+

-sensing receptor is activated by
millimolar changes in [Ca
2+
]
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 [Ca
2+
]
e
decreases [221] and it is likely that
gap junctional regulation could be strongly modified by
[Ca
2+
]
e
in the central nervous system [222]; (c) metabo-
tropic glutamate receptors 1, 3 and 5 [223] are activated
by physiological [Ca
2+
]
e
fluctuations in the synaptic
cleft [224]; and (d) the Gamma-aminobutyric acid (B)
GABA
B
receptor also possesses Ca
2+

sensing proper-
ties, potentiating GABA responses upon increase of
[Ca
2+
]
e
[225]. It is not yet known whether these addi-
tional Ca
2+
-sensing mechanisms may act alone or in
concert with nonselective Ca
2+
channels in producing
significant excitotoxic Ca
2+
increases following ischemic
insults.
TRP channels as candidates for paradoxical
Ca
2+
-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 [Ca
2+
]
c
decay during the
paradoxical Ca
2+
rise and converts it to a progressive
[Ca
2+
]
c
rise (C. Chinopoulos, unpublished data).
Of particular interest however, are the observations
that a number of TRP channels are activated by a
decrease in [Ca
2+
]
e
, raising the possibility that they
could contribute to paradoxical Ca
2+

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 Ca
2+
and Na
+

load to
enter the cytosol. Since both high [Ca
2+
]
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
Ca
2+
, mitochondria, ROS in neuronal disease C. Chinopoulos and V. Adam-Vizi
440 FEBS Journal 273 (2006) 433–450 ª 2006 The Authors Journal compilation ª 2006 FEBS
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 Ca
2+

ions to enter the
cytosol they also permit Na
+
influx and K
+
efflux
[226,253,254]. The ominous effects of an elevated
[Na
+
]
i
are mostly associated with cell swelling and acti-
vation of the Na
+
⁄ Ca
2+
exchanger causing Ca
2+
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 Ca
2+
[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

Ca
2+
, 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
+
]
i
acts deleteriously on in
situ mitochondria [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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