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Cell Calcium 47 (2010) 165–174

Contents lists available at ScienceDirect

Cell Calcium

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alcium dysregulation in amyotrophic lateral sclerosis

ulian Grosskreutza,∗, Ludo Van Den Boschb,c, Bernhard U. Kellerd

Dept. of Neurology, Friedrich-Schiller University Hospital Jena, Erlanger Allee 101, 07747 Jena, GermanyDepartment of Neurobiology, K.U. Leuven, BelgiumVesalius Research Center, VIB, BelgiumCenter of Physiology, Georg August University Göttingen, Germany

r t i c l e i n f o

rticle history:vailable online 29 January 2010

eywords:myotrophic lateral sclerosisalcium deregulation

a b s t r a c t

In the fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS), motor neurons degeneratewith signs of organelle fragmentation, free radical damage, mitochondrial Ca2+ overload, impaired axonaltransport and accumulation of proteins in intracellular inclusion bodies. Subgroups of motor neurons ofthe brainstem and the spinal cord expressing low amounts of Ca2+ buffering proteins are particularlyvulnerable. In ALS, chronic excitotoxicity mediated by Ca2+-permeable AMPA type glutamate receptorsseems to initiate a self-perpetuating process of intracellular Ca2+ dysregulation with consecutive endo-

ndoplasmic reticulumitochondria

MPA receptorluR2eurodegenerationalcium bufferelective vulnerability

plasmic reticulum Ca2+ depletion and mitochondrial Ca2+ overload. The only known effective treatment,riluzole, seems to reduce glutamatergic input. This review introduces the hypothesis of a “toxic shiftof Ca2+” within the endoplasmic reticulum–mitochondria Ca2+ cycle (ERMCC) as a key mechanism inmotor neuron degeneration, and discusses molecular targets which may be of interest for future ERMCCmodulating neuroprotective therapies.

© 2009 Elsevier Ltd. All rights reserved.

otor neuron

. Introduction

Ca2+ dysregulation plays a central role in the pathophysiologyf amyotrophic lateral sclerosis (ALS). This incurable neurodegen-rative disorder leads to muscle atrophy, paralysis and death ofhe patient on average within 3–4 years after the start of therst symptoms. This relentlessly progressive disease is caused byelective degeneration of motor neurons in the spinal cord, brain-tem and motor cortex leading to loss of ambulation, speech andwallowing defects and finally in a compromised respiratory func-ion. Lifetime risk of ALS is 1 in 472 in women and 1 in 350 in

en [1] which is close to that of multiple sclerosis. In 10% of ALSatients, a family history of a mostly autosomal dominant disorderxists and in 20% of these familial cases the disease is caused byutations in the SOD1 gene encoding the Cu2+, Zn2+-dependent

uperoxide dismutase (SOD1) [2]. Transgenic mice overexpress-ng mutant SOD1 develop motor neuron abnormalities and have

reatly advanced the understanding of the mechanisms under-ying motor neuron degeneration [3]. Recently, mutations in theesicle-associated membrane protein-associated protein B (VAPB)4], TDP-43 [5,6] and FUS/TLS genes were identified as another

∗ Corresponding author. Tel.: +49 3641 932 3426; fax: +49 3641 932 3422.E-mail address: julian.grosskreutz@med.uni-jena.de (J. Grosskreutz).

143-4160/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.oi:10.1016/j.ceca.2009.12.002

cause of familial ALS [7,8]. The overall frequency of these mutationsis currently unknown.

One of the most striking characteristics of ALS is the selectivity ofthe disease process for motor neurons. This selectivity is also illus-trated by the fact that only motor neurons degenerate in mutantSOD1-associated familial ALS (FALS) despite the overexpression ofthe mutant protein in every cell [9]. An intriguing question is whichmechanism(s) is responsible for this selectivity. One possibilitycould be that excitatory neurotransmitters, in particular glutamate,can induce neuronal death because motor neurons are extremelysusceptible to this so-called excitotoxicity. Intrinsic properties ofmotor neurons could explain this high sensitivity to excessive stim-ulation of glutamate receptors. Spinal motor neurons receive verystrong glutamatergic input, express Ca2+-permeable �-amino-3-hydroxy-5-methylisoxazole proprionic acid (AMPA) receptors ontheir surface and have a low Ca2+ buffering capacity [10]. Thesecharacteristics are most likely crucial for motor neurons to be func-tionally normal, but under pathological conditions they could causeor speed up motor neuron death. It is indeed clear that additionalfactors and mechanisms are required to induce ALS. In the FALSpatients with mutations in the SOD1 gene, the presence of this

mutant protein could shift the balance from normal motor neu-ron excitation to excitotoxicity. This could be due to a decreasedglutamate uptake in the surrounding astrocytes and/or by interfer-ing with mitochondrial function. In general, the way motor neuronshandle Ca2+ could predispose them to the disease. In this review,

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Fig. 1. Schematic overview of excitotoxic motor neuron death and the role ofmutant SOD1. Glutamate released from the presynaptic neuron stimulates AMPAreceptors on the postsynaptic neuron. Motor neurons express a large number ofCa2+-permeable receptors lacking the GluR2 subunit. As a consequence, glutamater-gic stimulation increases the intracellular Ca2+ concentration and because of the lowCa2+-buffering capacity of motor neurons part of this Ca2+ will be taken up by themitochondria. The GLT1 transporter present in the astrocytes removes glutamatefrom the synaptic cleft and factors secreted by the astrocytes can increase the GluR2expression level in the surrounding motor neurons. As indicated on the figure, thepresence of mutant SOD1 in neurons as well as in astrocytes interferes at differ-

66 J. Grosskreutz et al. / Cel

e will give an overview of the evidence in favour of excitotoxicityn ALS and we will discuss the role of Ca2+ in motor neurons.

. Intrinsic properties of Ca2+ homeostasis in motoreurons

Several studies have investigated Ca2+ homeostasis in motoreurons, where a special focus has been the characterization ofarameters that distinguish ALS-vulnerable from resistant motoreuron types. For example, ALS-vulnerable spinal and brain stemotor neurons in mice have been shown to display low endogenous

a2+ buffering capacities [11,12] that are 5–6 times lower comparedo those found in ALS-resistant oculomotor neurons in the sameystem [13]. These findings have several interesting implicationsor the physiological function of motor neurons. For example, lowuffering capacities permit rapid recovery times of activity-relateda2+ transients at relatively low energy cost, which could be benefi-ial during ongoing motor activities like running or breathing [14].oreover, low buffering capacities account for large volume Ca2+

icrodomains around open channels [15], which might facilitateocal interactions of Ca2+ influx through voltage-dependent Ca2+

hannels with Ca2+-dependent mechanisms in nearby organellesike mitochondria or the endoplasmic reticulum (see below).

In an independent series of investigations, several groups havehown that both in mice and humans ALS-vulnerable motor neu-ons possess a high density of Ca2+-permeable glutamate receptorhannels of the AMPA receptor type [16,17]. Although the physio-ogical background of these receptors is not completely understood,igh densities of Ca2+-permeable AMPA receptor channels mightontribute to a high vulnerability of ALS-vulnerable motor neuronso excitotoxic conditions like elevated concentrations of glutamater excitotoxins in the extracellular space (see following chapter).

. Excitotoxicity and motor neuron death

.1. External excitotoxins induce motor neuron degeneration

Consumption of excitotoxins can give rise to selective motoreuron death [18]. Domoic acid concentrated in mussels wasesponsible for a food poisoning in Canada leading in some patientso a pure motor neuropathy [19]. Consumption of the chickling peaathyrus sativus caused lathyrism due to the abundant presencef the �-N-oxalyl-amino-l-alanine (BOAA) [20,21]. This potentMPA receptor agonist caused motor neuron degeneration [22,23].-methylamino-l-alanine (BMAA) is thought to be responsible

or Western Pacific amyotrophic lateral sclerosis-parkinsonismementia (Guam ALS) and BMAA is a potent glutamate receptorgonist causing a motor neuron syndrome [24,25]. All together,hese data indicate that (over)consumption of excitotoxins is neu-otoxic and that motor neurons seem to be most vulnerable to thisype of toxicity.

.2. Glutamate reuptake is disturbed in ALS

An important argument for the involvement of excitotoxicity inLS is that defective clearance of glutamate from the synaptic cleftas found in synaptosomes isolated from affected brain areas and

pinal cord of sporadic ALS patients [26,27]. This was due to theelective loss of the astroglial glutamate transporter, GLT1, in theotor cortex and spinal cord both in sporadic and in familial ALS

28–30]. A loss of GLT1 immunoreactivity was also found in the ven-ral horn of mutant SOD1 mice and rats [31–33]. It was also shownoth in vitro and in vivo that loss of GLT1 leads to selective motoreuron degeneration [34]. Also the opposite holds as stimulation ofLT1 expression using �-lactam antibiotics increased significantly

ent levels with this process. Consequently, the balance will be shifted from normalglutamatergic communication between neurons to excitotoxic motor neuron death.See text for further details.

the life span of the mutant SOD1 mice [35]. Although a lot of datafavour the hypothesis that glutamate transport deficiency plays acrucial role as a causal factor of motor neuron degeneration, it isworth mentioning that recent data challenge this concept [36]. Inthis recent in vivo study, prolonged elevation of extracellular glu-tamate due to its transport blockade did not induce the death ofspinal motor neurons in rats [36].

3.3. Motor neurons succumb to AMPA receptor-mediatedexcitotoxicity

Experimental evidence shows that motor neurons are extremelyvulnerable to AMPA receptor-mediated excitotoxicity. Intrathecalor intraspinal administration of AMPA receptor agonists inducedmotor neuron degeneration [37–39], motor neurons in organotypicspinal cord cultures were killed by overstimulation of AMPA recep-tors after inhibition of glutamate uptake [40,41] and AMPA receptoragonists killed selectively cultured motor neurons [16,17]. Appar-ently, motor neurons have intrinsic properties that make themextremely vulnerable to AMPA receptor-mediated excitotoxicity,while they are relatively resistant to overstimulation of NMDAreceptor agonists. In our opinion, these characteristics relate to theway motor neurons handle Ca2+. Two rather exceptional charac-teristics are combined: a high number of Ca2+-permeable AMPA

2+

receptors and a low Ca -buffering capacity. First, we will discussthe crucial role played by Ca2+-permeable AMPA receptors (Fig. 1)and subsequently we will focus on the cause and consequences ofthe absence of Ca2+-binding proteins. A direct consequence of thelower amount of Ca2+-buffering proteins in motor neurons is that

J. Grosskreutz et al. / Cell Calcium 47 (2010) 165–174 167

Fig. 2. Interaction of Ca2+ and mitochondria in ALS as a local disruptive feedback mechanism. Several research reports indicate an impairment of the mitochondrial respiratorychain in ALS (complex I – complex IV), which has multiple implications for Ca2+ regulation in motoneurons (see text for references). First, impairment of complex VI presumablyincreases ROS production, which leads to reduced glutamate uptake in neighbouring glia cells, elevated glutamate levels in the synaptic cleft and increased calcium influxt rbedC e innef

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hrough Ca-permeable glutamate receptor channels (see also Fig. 1). Second, distua2+ uptake / sequestration and an impaired function of the Na/Ca exchanger in theedback mechanism that contributes to motoneuron degeneration in ALS.

itochondria seem to play a more prominent role in Ca2+ buffer-ng (Fig. 2). Also, motor neurons as active excretory cells possess aarge and active endoplasmic reticulum (ER) which acts as a majorource of calcium and evidence of ER dysfunction in ALS is growings of late (Fig. 3).

. Ca2+ permeability of AMPA receptors in motor neurons

.1. Experimental evidence

Cultured motor neurons express a high proportion of Ca2+-ermeable AMPA receptors [17,42,43]. Ca2+ entry via thesea2+-permeable AMPA receptors is indeed responsible for selectiveotor neuron death [17]. This motor neuron death is completely

ependent on extracellular Ca2+ and is inhibited by selectivelockers of Ca2+-permeable AMPA receptors. Electrophysiologicalxperiments showed that AMPA receptors of motor neurons hadlower rectification index and a higher relative Ca2+ permeabil-

ty ratio than other neurons [44]. Also in vivo it was shown that

otor neurons express Ca2+-permeable AMPA receptors [45]. Per-

usion of AMPA in the rat spinal cord induced paralysis and aelective loss of spinal motor neurons. This AMPA-induced lossf motor neurons was reduced by a selective blocker of thea2+-permeable AMPA receptors and this selective motor neuron

mitochondria affect cytosolic calcium regulation by a reduction of mitochondrialr mitochondrial membrane. Taken together, these disturbances might form a local

death was triggered by an increase of intracellular Ca2+ via theseCa2+-permeable AMPA receptors [39]. In conclusion, motor neu-rons express Ca2+-permeable AMPA receptors which make themextremely vulnerable to excitotoxicity.

4.2. GluR2 regulates the Ca2+ permeability of the AMPA receptor

The Ca2+ permeability of AMPA receptors is determined by theabsence or presence of the GluR2 subunit in the receptor com-plex. In most conditions, AMPA receptor tetramers contain at leastone GluR2 subunit and have a very low Ca2+ permeability. How-ever, GluR2-lacking receptors are highly permeable to Ca2+. Therelative amount of GluR2 mRNA was significantly lower in motorneurons compared to other neurons, indicating that GluR2 is regu-lated at the transcriptional level [44]. Moreover, using laser capturemicroscopy and quantitative RT-PCR it was demonstrated in vivothat the expression level of the GluR2 subunit was lower in (human)spinal motor neurons compared to other neurons [46,47]. Studieswith mutant SOD1G93A mice confirmed the crucial role of GluR2 in

motor neuron viability. Lack of the GluR2 subunit accelerated dra-matically the motor neuron degeneration and shortened the lifespan of these mice [48]. Providing motor neurons with extra GluR2resulted in a significant increase of the life span of these doubletransgenic mice [49].

168 J. Grosskreutz et al. / Cell Calcium 47 (2010) 165–174

Fig. 3. Comprehensive model of the ER–mitochondria Ca2+ cycle (ERMCC) as the central regulatory mechanism in motor neuron (MN) Ca2+ homeostasis. Astrocytes (Ac) controlthe level of persisting glutamate (glu) at the glutamatergic synapse (S) through glutamate transporters (EAAT), but also exert life-supporting functions in motor neurons (i.e.BDNF, IGF, VEGF), some of which are not yet identified on a molecular level (X). Triggered by physiological activity of AMPA receptors with pathologically increased Ca2+-permeability, a chronic shift of Ca2+ from the ER compartment to the mitochondrial compartment (i.e. through Ca2+-induced Ca2+ release through ryanodine receptors RyRand mitochondrial uptake through the uniporter mUP) causes depletion of ER Ca2+ levels with protein folding dysfunction and chronic mitochondrial Ca2+ overload. Both cani the unE nd ther lemmN , ROS =

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nduce apoptosis through Bcl-2 dependent mechanisms, the ER in particular whenRMCC may reduce ER or mitochondrial Ca2+ release to reduce total Ca2+ turnover aeceptors, VGCC = voltage gated Ca2+ channels, Na/K = Na+/K+ pump, pNCE = plasmaa+/Ca2+ exchanger, Cyt C = cytochrome c, SERCA = sarco-endoplasmic Ca2+ ATPase

.3. Regulation of the GluR2 expression

In view of the crucial role of GluR2 in the determination of bothhe Ca2+ permeability of the AMPA receptor and the sensitivity of

otor neurons to excitotoxicity, it is highly relevant to understandhe regulation of this subunit. Surrounding astrocytes can influencehe expression level of the GluR2 subunit in motor neurons [50].oluble factor(s) released from these astrocytes influence the tran-cription of the GluR2 gene resulting in motor neurons with a lowermount of Ca2+-permeable AMPA receptors and a lower suscepti-ility of motor neurons to excitotoxicity both in vitro and in vivo.nterestingly, the presence of mutant SOD1 interferes with the pro-uction and/or secretion of this factor(s). The identity of all theseactors is not yet known although it was recently discovered thatascular endothelial growth factor (VEGF) secreted by the astro-ytes can upregulate GluR2 expression at the transcriptional level51]. Other known factors that upregulate GluR2 expression arehe neurotrophic factors: brain-derived neurotrophic factor (BDNF)nd glial cell-derived neurotrophic factor (GDNF) [52].

.4. Disturbance of GluR2 editing

The crucial importance of GluR2 in neuronal survival is fur-her illustrated by the phenotype of transgenic mice in whichhe RNA editing at the Q/R site is reduced. This editing in theluR2 pre-mRNA introduces a positively charged arginine at the/R site of the GluR2 peptide instead of the genetically encodedeutral glutamine and is responsible for the impermeability toa2+ of GluR2-containing AMPA receptors [53]. Transgenic miceith a reduced editing have a lethal phenotype with seizures

nd acute neurodegeneration [54,55]. Transgenic mice carrying ainigene with the GluR2 gene encoding an asparagine (GluR2-) at the Q/R site, which makes editing impossible, are viablend fertile [56]. AMPA receptor channels incorporating GluR2-Nre permeable to Ca2+ [53] and the combined expression of the

folded protein reaction (UPR) is exhausted. Pharmacological routes to stabilize theresulting toxic shift of Ca2+ within the ERMCC in motor neurons. (NMDAR = NMDA

al Na+/Ca2+ exchanger, PMCA = plasmalemmal Ca2+ ATPase, mNCE = mitochondrialreactive oxygen species, SOD = superoxide dismutase).

GluR2-N transgene and endogenous GluR2 alleles results in a two-fold increase in permeability for Ca2+ [56]. These transgenic micedeveloped motor neuron degeneration late in their life [56] andGluR2-N overexpression induced a progressive decline in function,as well as a degeneration of spinal motor neurons [57]. Inter-estingly, crossbreeding these GluR2-N transgenics with mutantSOD1 mice aggravated the course of the motor neuron diseaseand reduced survival confirming the role for edited GluR2 in ALS[57].

Under normal conditions GluR2 pre-mRNA editing is virtuallycomplete, but under pathological conditions it could become lessefficient resulting in more Ca2+-permeable AMPA receptors. Indeed,evidence exists that the editing of the pre-mRNA of GluR2 is defec-tive in the spinal motor neurons of individuals affected by sporadicALS [58]. This was only checked in a limited number of ALS patientsand this very interesting observation awaits independent confirma-tion.

5. Ca2+ and mitochondria in motor neurons

5.1. Mitochondria are heavily affected in ALS

In the last years, an increasing number of studies has providedsubstantial evidence for a crucial involvement of mitochondriaboth in sporadic and familial forms of ALS (e.g. [59–63]). Some ofthe first observations were provided by histopathological inves-tigations of mitochondrial abnormalities such as swelling as anearly sign of pathology in ALS mouse models and human ALS[64,65]. More recent studies have focussed on ALS-related func-tional disruptions of mitochondria, in particular on impairments

of the mitochondrial respiratory chain. For example, in spo-radic forms of ALS, a decrease in complex IV of the electrontransport chain activity has been linked to mutations in mito-chondrial DNA [66,67]. The question of whether alterations inthe mitochondrial genome can lead to alterations in cell function

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as been addressed by transferring mitochondrial DNA (mtDNA)rom ALS subjects to mtDNA-depleted human neuroblastomaells. This resulted in abnormal electron transport chain func-ioning, increases in free radical scavenging enzyme activities,erturbed Ca2+ homeostasis and altered mitochondrial ultrastruc-ure [68].

A complementary line of research has pointed out the impor-ance of ALS-related disruptions of mitochondrial transport63,69,70]. In this case, mutant SOD1 has been shown to slow downoth fast axonal transport of mitochondria and other membraneound organelles [71,72]. Interestingly, mitochondrial transportas primarily inhibited in the anterograde direction, suggesting

hat energy supply in presynaptic terminals of motor endplates isarticularly compromised. Other studies pointed out the increasedroduction of reactive oxygen species (ROS) by mitochondria inotor neurons as a result of mitochondrial Ca2+ overload following

xcitotoxic stimulation of AMPA/kainate receptors [16]. Comple-entary evidence suggests that ROS generated in motor neurons

an even cross the plasma membrane and cause oxidative dis-uption of glutamate transporters in neighbouring astrocytes [73].his in turn may enhance local excitotoxicity and initiate a viciousycle of motor neuron damage. The model nicely integrates theypothesis of excitotoxicity and oxidative damage, although exactechanisms, how mitochondrial Ca2+ uptake would increase ROS

roduction, are still unclear.Although the detailed molecular mechanisms are still unre-

olved, several pieces of evidence suggest that mutant SOD1 proteinirectly disrupts mitochondrial function. Aggregates containingutant SOD1 have been found within the mitochondrial matrix

74–76] and a decrease in enzyme activities of the electron trans-ort chain at the level of complex I, II and IV has been demonstratedt early disease stages [77–79]. Most interestingly, recent workrovided evidence that mutant SOD1 might disrupt association ofomplex IV (cytochrome c) with the inner mitochondrial mem-rane and by this interfere with mitochondrial respiration [80]. An

mportant consequence of such a disturbed mitochondrial respi-ation seems to be an increased production of ROS. This has beenemonstrated for cultured motor neurons expressing mutant SOD181] and is in line with a study on motor neurons in brain slices,here complex IV was inhibited by cyanide [82].

.2. Interaction of Ca2+ and mitochondria in ALS

To study in more detail the interaction between Ca2+ anditochondria, motor nerve terminals have served as a valuableodel system (e.g. [83–85]). In nerve terminals of lizard, David

t al. studied mitochondrial Ca2+ regulation during brief, repeti-ive trains of action potentials [83]. For each train, they observed

biphasic increase in the cytosolic [Ca2+]i followed by a risen mitochondrial Ca2+ levels during ongoing stimulation. Whileytosolic Ca2+ elevations returned to basal Ca2+ levels relativelyast, mitochondrial Ca2+ levels showed much slower recoveryimes. Application of the mitochondrial uncoupler carbonyl cyanide

-chlorophenylhydrazone (CCCP) not only multiplied stimulus-voked Ca2+ responses, but also slowed the rise of cytosolic Ca2+

ransients after stimulation. Taken together, these measurementstrongly suggest that mitochondrial uptake is not only a criti-al determinant of cytosolic Ca2+ profiles, but also represents theayor clearance mechanism for cytosolic Ca2+ loads under physi-

logical conditions.In a subsequent series of experiments, David [86] investigated

itochondrial Ca2+ responses during prolonged trains of actionotentials. Interestingly, Ca2+ concentrations in the mitochondrialatrix saturated at plateau levels of 1 �M, although pharmacolog-

cal experiments clearly showed a sustained uptake of Ca2+ fromhe cytosol during ongoing stimulation protocols. This apparent

um 47 (2010) 165–174 169

discrepancy might be explained by the reversible formation of aninsoluble Ca2+ salt in the mitochondrial lumen. This mechanismprovides one explanation how mitochondria might take up largeamounts of Ca2+ while maintaining free Ca2+ levels in the mito-chondrial lumen at constant levels [86,87].

To relate these observations to motor neuron degeneration,Nguyen et al. [85] investigated the role of mitochondrial Ca2+

regulation in motor nerve terminals of several mutant SOD1mouse models of ALS-related motor neuron disease. In this system,stimulus-evoked Ca2+ elevations are paralleled by mitochondrialCa2+ uptake as monitored by a corresponding depolarization inthe mitochondrial electrochemical potential. Interestingly, Nguyenet al. found that Ca2+-induced depolarization is greater in mutantSOD1 animals compared to wild type controls [85]. These datasupport a model where mitochondrial Ca2+ uptake effectivelyaccelerates the mitochondrial electron transport chain with a cor-responding increase in mitochondrial proton extrusion. Underphysiological conditions, this mechanism provides an efficientfeedback loop to speed up mitochondrial Ca2+ regulation duringperiods of enhanced electrical activity. Under pathophysiologi-cal conditions, this feedback mechanism appears to be disruptedand mitochondria suffer from an impaired ability to handle large,activity-induced Ca2+ loads (Fig. 2).

In a complementary series of experiments, Ladewig et al. [88]directly investigated mitochondrial Ca2+ regulation in the soma anddendrites of hypoglossal motor neurons in acutely isolated slicepreparations of the mouse brain stem. Also under these condi-tions, brief trains of action potentials evoked substantial elevationsof cytosolic Ca2+ levels both in the soma and dendritic compart-ments. Interestingly, peak amplitudes and recovery time constantsof Ca2+ transients increased with increasing distance from thesoma. Application of the mitochondrial uncoupler CCCP prolongeddecay times in the soma 2-fold and in dendrites 3-fold respectively.Significant uptake of activity-related Ca2+ loads into the lumenof the mitochondria could be demonstrated directly by utilizingthe fluorescent dye Rhod-2. Mitochondrial Ca2+ profiles were sig-nificantly retarded compared to those in the cytosol, which wasin agreement with previous observations in motor nerve termi-nals. In this system, complementary pharmacological experimentswere performed to investigate the interaction between caffeine-operated Ca2+ stores, mitochondrial Ca2+ regulation and electricalexcitability of motor neurons. These observations demonstrated aclose functional link between caffeine-operated Ca2+ stores, mito-chondrial Ca2+ regulation and the activation of Ca2+-dependentpotassium conductances. Under pathological conditions, this closefunctional link might expose vulnerable hypoglossal motor neu-rons to exceptional risks for organelle and/or mitochondria-relateddisturbances.

Using a different approach, Bergmann and Keller [82] investi-gated the role of mitochondria in motor neurons by using specificinhibitors of the complex IV of the respiratory chain (“chemicalhypoxia”). Inhibition of complex IV by cyanide depolarised theplasma membrane potential by 10 mV and increased the spon-taneous activity of hypoglossal motor neurons. All these effectswere reversible and values returned to basal levels after removalof cyanide. Interestingly, cyanide application was paralleled by anincreased level of basal cytosolic Ca2+ concentrations, presumablyreflecting mitochondria-controlled Ca2+ release. Inhibition of com-plex IV also retarded recovery rates of cytosolic Ca2+ transientstwo-fold, which confirms the model that mitochondrial Ca2+ uptakerepresents the dominant process.

In summary, studies on hypoglossal motor neurons support amodel where ALS-related motor neuron degeneration results froma synergistic combination of mitochondria-related risk factors,including low endogenous calcium buffering capacities resultingfrom low concentrations of cytosolic Ca2+ binding proteins, strong

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itochondrial impact on [Ca2+]i and a mitochondria-dependentontrol of electrical activity during metabolic disturbances.

In an independent series of experiments, Grosskreutz et al.89] investigated the role of mitochondria during agonist-induceda2+ elevations in cultured motor neurons. Using a fast applicationystem for AMPA receptor agonists, Grosskreutz et al. demon-trated substantial kainate-induced Ca2+ responses in somaticnd dendritic compartments. Amplitudes and recovery times ofgonist-induced responses were significantly increased after appli-ation of the mitochondrial uptake blocker RU360, demonstratinghe central role of mitochondrial Ca2+ uptake also in this system.

. Endoplasmic reticulum dysfunction in ALS

The endoplasmic reticulum (ER) is the main site of protein pro-essing and folding, reacts to a number of Ca2+ dependent stimulind constitutes a large Ca2+ store. It interacts closely with theucleus, mitochondria, and the Golgi apparatus to pass on pro-eins to axonal transport and exocytosis, and protein degradationathways. ER and mitochondria interact by exchanging Ca2+ in ayclic manner, which is thought to synchronize energy demandnd rates of protein folding, and may initiate apoptosis (for reviewee [90–92]). ER Ca2+ uptake and release are determined by molec-lar events which tend to equilibrate a balance between the ERs a Ca2+ store and its surroundings, in particular mitochondria inhe vicinity [93]. A derangement of this interplay can induce Ca2+

epletion of the ER, accumulation of misfolded proteins and con-ecutive activation of the unfolded protein response (UPR) [94]. ThePR shuts down most protein translation and promotes expressionf ER bound chaperones via the XBP-1 gene and helps to restoreR protein folding ability. Failure of the UPR is thought to play anmportant role in neurodegenerative diseases (for further reviewee [95]). In ALS, no direct evidence has as yet demonstrated ERalcium signalling pathology, but a number of indirect findingsmplicate disease-associated changes in the ER.

.1. ALS-related ER pathology

In ALS patients, a number of early reports have described ERnd Golgi apparatus fragmentation with unusual “virus-like” inclu-ions and mitochondrial crystalline particles [96–98]. A single postortem observation revealed a proliferation of the ER and focaleurofibrillar accumulations in axons [99]. In another case, the

nclusions were found to contain RNA-protein compounds whichonsisted of randomly interwoven tubules with granular ER andree ribosomes [100]. A particular 14-year prolongation of life byrtificial ventilation in an ALS patient revealed cytoplasmic virus-ike inclusions in neurons and glial cells as well as changes in theranular ER in astrocytes consisting of large accumulations of ribo-omes. The discovered inclusions were similar to particles found inuscle fibres in ALS patients [101]. Adding to the aspect of a sys-

emic disease of neuronal phenotype, in 21 ALS patients the liverhowed bizarre giant mitochondria; intramitochondrial paracrys-alline inclusions; disorganization of the lamellar structure of roughR; increased numbers of smooth ER; and parasinusoidal fibrosis.bove all, paracrystalline inclusions appeared to be an ALS specificnding [102].

In mutant SOD1 mice lesions of the Golgi apparatus were founddentical to human sporadic ALS. The stacks of the cisternae ofhe fragmented Golgi apparatus were shorter, and Golgi-associated

esicles and adjacent cisternae of the rough ER were reduced.he fragmentation of the Golgi apparatus occurred in an early,resymptomatic stage and preceded the development of the vacuo-

ar changes. In familial ALS, an early lesion of the Golgi apparatus ofotor neurons may have adverse functional effects, because newly

um 47 (2010) 165–174

synthesized proteins destined for fast axoplasmic transport passthrough the Golgi apparatus [103].

In contrast, the only specific hallmark of human ALS inlower motor neurons were Bunina bodies (BBs), which aresmall eosinophilic intraneuronal inclusions. Small deposits ofimmunoperoxidase products were seen in the cytoplasms and den-drites, some forming a tubular or vesicular pattern, but no roughER, mitochondria, lipofuscin granules or nuclei were detected. BBswere thought to represent an abnormal accumulation of unknownproteinous material associated with the Golgi apparatus [104].

6.2. The ER is stressed in ALS

Recent studies on autopsied ALS patients and studies usingmutant SOD1 transgenic mice suggested that ER stress-relatedtoxicity might be a relevant ALS pathomechanism. Levels of ERstress-related proteins were upregulated in motor neurons in thespinal cords of ALS patients. It was also shown that mutant SOD1,translocated to the ER, caused ER stress in neurons in the spinalcord of mutant SOD1 transgenic mice [105]. A newly identified ALS-causative gene, vesicle-associated membrane protein-associatedprotein B (VAPB), plays a pivotal role in the UPR [106]. The ALS-linked P56S mutation in VAPB nullifies the function of VAPB,resulting in increased motor neuronal vulnerability to ER stress[107]. The full UPR with induction of stress sensor kinases, chaper-ones and apoptotic mediators, was present in spinal cords of humanpatients with sporadic ALS [108]. Against expectations, XBP-1 defi-cient mice crossbred with mutant SOD1 mice were more resistantto the progressive motor neuron degeneration. While the clear-ance of mutant SOD1 aggregates by macroautophagy was genderindependent, only females were resistant to mutant SOD1 toxicity[109].

6.3. ER signalling in motor neurons and ALS

The ER interacts with adjacent structures through a numberof mechanism. One of the fastest and most immediate links isprovided by Ca2+-induced Ca2+ release (CICR) from the ER, i.e.through ryanodine receptors (RyR). Furthermore, inositol triphos-phate receptors (IPTR) release Ca2+ from the ER. Mitochondria takeup Ca2+ through the uniporter (mUP), and release, i.e. through theNa+/Ca2+ exchanger (mNSE). Reuptake into the ER is mediated bythe sarco-endoplasmic reticulum calcium ATPases (SERCA) (Fig. 3,for review see [92]). As one mechanism to release Ca2+ from intra-cellular stores, caffeine sensitivity seems to increase over time withsome evidence for a role of voltage-gated calcium channels (VGCC)in this process [110,111].

While only some subtypes of RyR seem to be expressed inmotoneurons, IP3 receptors are present in all isoforms [111,112].ITPR2 encodes the type 2 receptor for IP3, and is the most abundantisoform in motor neurons [112]. A strong association was foundbetween a single nucleotide polymorphism (SNP) in intron 42 ofthe ITPR2 gene on chromosome 12p11 and the occurrence of ALSin a Dutch, Swedish and Belgian population [113], although thisassociation could not be confirmed in a German cohort [114]. Thenature of the association between the SNP and ALS is unknown,but mRNA levels of IP3 receptor 2 were significantly higher in ALSpatients than in controls.

Ca2+ entry through AMPA receptors caused RyR mediatedcalcium-induced calcium release from the ER in embryonic motorneurons in co-culture with neonatal Schwann cells [115]. RyR are

opened by calcium itself which may induce propagated calciumrelease on the ER surface provided that RyR density is sufficient.The release patterns suggest different levels of sensitivity to cal-cium, modulate exocytosis in motor terminals and can be detectedfrom the inside of the ER using, i.e. aequorines [116–118]. Although

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igh affinity calcium dyes may interfere with CICR [119], in embry-nic motor neurons CICR was shown to contribute greatly to AMPAeceptor stimulation induced CICR through RyR and Ca2+ dysregu-ation with very brief (1–2 s) kainate application [120] and calciumransients in spontaneously active motor neuron networks [115]sing low concentrations of FURA-2.

Repetitive tetanic stimulation in frog motor nerve terminalst 50 Hz can trigger CICR until ER Ca2+ stores become depleted121,122]. Dantrolene as a blocker of RyRs greatly reduces Ca2+

ransients both in kainate-induced transients in motor neurons andn spontaneous network activity in motor neuron cultures [115].

According to one study on motor neurons, all three SERCAsoforms are expressed [111]. Others detected a SERCA2b spliceariant only [112]. In days 7–14 in vitro motor neuron culturesn Schwann-cell feeder layers CPA, a reversible blocker of SERCAumps, greatly reduced large synaptically evoked cytosolic Ca2+

ransients while surrounding Schwann cells responded with anncrease of [Ca2+]c [115].

. Excitotoxicity and the ER–mitochondria Ca2+ cycleERMCC) in ALS: implications for therapy

The most important argument that excitotoxicity plays a rolen ALS is that riluzole, the only drug that slows the disease inumans, has anti-excitotoxic properties [123,124]. Riluzole slowshe disease progression and significantly increases survival by aew months. This drug inhibits the release of glutamate due to thenactivation of voltage-dependent Na+ channels on glutamatergicerve terminals, as well as to activation of a G-protein dependentignal transduction process [125]. Moreover, riluzole can also blockMDA and AMPA receptors [124,126,127] and has a protectiveffect in the mutant SOD1 mouse model [128] and a number ofharmacological interventions that all block AMPA receptors havesignificant effect on survival. RPR119990, 1,2,3,4-tetrahydro-

-nitro-2,3-dioxobenzo[f]quinoxaline-7-sulfonamide (NBQX) andK187638 all increased significantly the life span of the mutantOD1 mouse model [129–131]. Moreover, intrathecal infusion of apecific blocker of Ca2+-permeable AMPA receptors slowed the lossf motor neurons in mutant SOD1 rats [132]. The use of AMPARntagonists is restricted due to severe side effects.

Based on the multitude of pathways, particles and organelleseported to be involved in ALS pathology an extended model of exci-otoxicity has been proposed [89] to explain motor neuron deathnd identify putative therapeutic targets. Based on models delin-ated by Berridge [90], it postulates a chronic toxic shift of Ca2+

rom the ER compartment to mitochondria, which induces longerm accumulation of misfolded proteins in the ER due to a chronicow intraluminal calcium concentration; as of today this has noteen shown in motor neurons or ALS neurons. Chronic mitochon-rial Ca2+ overload on the other hand has indeed been reported inerve terminals of ALS patients [133]. Ca2+ seems to be shuttledack and forth between the ER and mitochondria; the process wasalled ER–mitochondria Ca2+ cycle (ERMCC) accordingly which ineurons is driven by synaptic excitatory input and electrical activ-

ty rather than occurring spontaneously as in non excitable cells. Inig. 3, the hypothesis is delineated in more detail.

As ALS motor neuron degeneration occurs mostly in the centralervous system, it is crucial for neuroprotective drugs to overcomehe liver first pass effect, and cross the blood-brain barrier. Previ-us anti-excitotoxic strategies focused on blocking receptors on the

urface of motor neurons. Intrathecal application of VEGF provedeneficial in mutant SOD1 mice [134] and one of the possible mech-nism(s) of action could be the increase in expression of GluR2hich renders AMPARs less Ca2+ permeable [51]. Given that ER

alcium release acts as a potent amplifier of minute calcium influx

um 47 (2010) 165–174 171

through AMPAR [115] even small changes in AMPAR calcium per-meability may exert major changes in intracellular calcium shifts.

The ERMCC model may help to circumvent AMPA receptorantagonist related central nervous system (CNS) side effects byproviding a number of intracellular targets for pharmacologicalsubstances which may stabilize intracellular Ca2+ signalling with-out interference with the electrical integration tasks of neurons.Given the hypothetical nature of the model, in particular the asyet unproven toxic shift of Ca2+ from the ER to mitochondria, onecan speculate that reducing ER Ca2+ release may indeed return bal-ance to the ERMCC at the cost of reducing ER protein throughputbut increasing protein folding reliability. This may be achieved bydrugs like dantrolene or other partial RyR blockers as well as IPTRblockers. Other strategies may include blocking the mitochondrialuniporter to prevent mitochondrial calcium overload and consec-utive release of cytochrome c which triggers apoptosis (Fig. 3).Preventing reuptake of calcium into the ER by SERCA blockingagents, on the other hand, may seem counterproductive but mayindeed provide protection by slowing hyperactive calcium cyclingwhich has been found in localized areas of impaired mitochondrialfunction in skeletal muscles of SOD1 mice [135].

The therapeutic window of such drugs may be narrow, com-plicated by the fact that it may primarily reach the ER in nerveterminals rather than cell bodies and axons, and have quite differ-ent effective doses in different regions of the target motor neurons.However, intrathecal application may largely circumvent theseproblems and open a new perspective of intracellular modulation ofthe ERMCC which when effective in ALS may provide new therapeu-tic strategies in other neurodegenerative diseases like Parkinson’sor Alzheimer’s disease.

Acknowledgements

Research of LVDB is supported by grants from the Fund forScientific Research Flanders (F.W.O. Vlaanderen), the Universityof Leuven and the Interuniversity Attraction Poles Program P6/43of the Belgian Federal Science Policy Office (Molecular Geneticsand Cell Biology). BK is supported by the Bundesministerium fürBildung und Forschung (BMBF grant no. 0313610), the BernsteinCenter for Computational Neuroscience (BCCN) Göttingen and bya BMBF grant from the EU-ERA-Net program. JG is supported bygrants from the Deutsche Forschungsgemeinschaft (GR 1578/2-1)and the EU-ERA-Net program.

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