Ca2+, mitochondria and selective motoneuron vulnerability: implications for ALS

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Ca2C, mitochondria and selective motoneuron vulnerability: implications for ALS Friederike von Lewinski1,2 and Bernhard U. Keller1 1

Zentrum Physiologie, Georg-August Universita¨t Go¨ttingen, Humboldtallee 23, 37073 Go¨ttingen, Germany Zentrum Neurologische Medizin, Abteilung fu¨r Klinische Neurophysiologie, Georg-August Universita¨t Go¨ttingen, Robert-Koch Strasse 40, 37075 Go¨ttingen, Germany

2

Motoneurons are selectively damaged in amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder. Although the underlying mechanisms are not completely understood, increasing evidence indicates that motoneurons are particularly sensitive to disruption of mitochondria and Ca2C-dependent signalling cascades. Comparison of ALS-vulnerable and ALS-resistant neurons identified low Ca2C-buffering capacity and a strong impact of mitochondrial signal cascades as important risk factors. Under physiological conditions, weak Ca2C buffers are valuable because they facilitate rapid relaxation times of Ca2C transients in motoneurons during high-frequency rhythmic activity. However, under pathological conditions, weak Ca2C buffers are potentially dangerous because they accelerate a vicious circle of mitochondrial disruption, Ca2C disregulation and excitotoxic cell damage. Introduction Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease clinically characterized by progressive loss of muscle force and breathing capacity, swallowing difficulties and limb spasticity [1]. ALS is sporadic in w90% of cases; the remaining 10% are of genetic origin, with a subset being induced by mutations in the enzyme superoxide dismutase 1 (SOD1). Clinically, there are no apparent differences in onset or progression of sporadic and familial ALS, which has led researchers to hypothesize that the two forms share at least some components in their pathogenesis and are likely to share a final pathway. Corresponding to the clinical picture, the characteristic hallmark of ALS pathology is the progressive and highly selective loss of cortical, spinal and brainstem motoneurons. As in most neurodegenerative diseases, the mechanisms leading to selective degeneration of motoneurons are far from being understood. However, several pathogenic factors have been proposed, including glutamate excitotoxicity [2], production of reactive oxygen species (ROS) [3], Ca2C-dependent formation of protein aggregates [4], axonal transport [5], mitochondrial dysfunction, deregulation of Ca2C homeostasis, and induction of pro-apoptotic pathways [6]. Although the involvement of each of these Corresponding author: Keller, B.U. ([email protected]). Available online 18 July 2005

factors has been well established, their temporal and spatial interplay remains elusive. Increasing evidence also suggests that ALS pathogenesis is not confined to motoneurons, but rather develops as a consequence of interplay between motoneurons and surrounding nonneuronal cells [7–11]. According to the selective pattern of motoneuron loss in ALS, it is generally believed that unique properties of affected motoneurons are responsible for their vulnerability during ALS-associated injury. These properties include weak buffering of cytosolic Ca2C [12,13], the presence of highly Ca2C-permeable AMPA receptors, which lack the GluR2 unit [4,14–16], a high neurofilament content [17], and an exceptional vulnerability to disruption of mitochondrial function [18]. This article will focus on what has been learned about Ca2C homeostasis and mitochondria in motoneurons under physiological conditions, and will also address their involvement during pathological states such as ALS. Finally we provide an integrative hypothesis for the role of Ca2C, mitochondria and ROS in selective motoneuron vulnerability. Cytosolic Ca2C homeostasis and selective motoneuron vulnerability Independent of the cellular and molecular event initiating motoneuron degeneration in ALS, disruption of intracellular Ca2C homeostasis is thought to have a key role in the disease process. Early evidence for involvement of Ca2C was provided by the observation that Ca2C-binding proteins such as calbindin-D28k and parvalbumin were absent in motoneuron populations lost early in ALS (cortical, spinal and lower cranial nerve motoneurons), whereas motoneurons damaged late or infrequently in the disease (those of Onuf ’s nucleus and the oculomotor, trochlear and abducens nerves) expressed markedly higher levels of calbindin-D28k and/or parvalbumin [13]. These findings are in good agreement with a quantitative comparison of Ca2C homeostasis in functionally intact vulnerable and resistant motoneurons in brain-slice preparations, which identified a low cytosolic Ca2C buffering capacity as an important risk factor for degeneration. Correspondingly, an increase in cytosolic Ca2C buffering capacity could protect vulnerable motoneurons from degeneration both in vitro and in vivo [19–21].

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How does altered motoneuron Ca2C homeostasis ultimately lead to degeneration of motoneurons in ALS? Several lines of evidence implicate disturbance of glutamate neurotransmission and subsequent glutamatetriggered Ca2C entry as important steps. For example, interfering with glutamate-mediated toxicity is so far the only neuroprotective therapeutic strategy that has proven beneficial in slowing disease progression in ALS patients. The presumed mechanisms of glutamate-mediated toxicity in ALS have been reviewed recently and are therefore only briefly outlined here [2,7]. Increased extracellular glutamate levels presumably result from reduced glial glutamate uptake, which can be caused by oxidative damage to the glutamate transporter EAAT2 or by aberrant RNA processing [22]. Damage to motoneurons by increased glutamate-mediated stimulation seems to involve mainly Ca2C-permeable AMPA receptors, which promote much larger elevations in intracellular Ca2C concentration ([Ca2C]i) in motoneurons than in ALS-resistant neurons [23]. The low Ca2C buffering properties, together with a high AMPA/kainate current density, could explain the particular vulnerability of motoneurons to increased stimulation by glutamate and concomitant Ca2C influx. For the well-studied familial form of ALS induced by mutant SOD1, the involvement of Ca 2C has been demonstrated in cell lines expressing the mutated protein and in the transgenic mouse model, where elevated Ca2C levels have been found [24–26]. A potential mechanism for [Ca2C]i disruption is given by the inhibition of glial glutamate transport by mutant SOD1 and the consecutive disturbance of Ca2C homeostasis by excitotoxic mechanisms similar to those proposed for sporadic ALS [27,28]. Indeed, various studies have pointed out the importance of Ca2C-permeable AMPA receptors in mutant SOD1-related ALS. In a cell-culture study, degeneration of motoneurons was prevented by joro spider toxin (a specific antagonist of Ca2C-permeable AMPA receptors), and survival times of transgenic SOD1 mice were significantly increased following chronic treatment with AMPA receptor antagonists [26,29]. In the cell-culture study, partial protection was also obtained by treatment with nifedipine, implicating Ca2C entry through voltage-gated Ca2C channels, in addition to glutamate receptors, in mediating the toxicity of mutant SOD1 in motoneurons. Recently, the crucial role of Ca2C-permeable AMPA receptors was further underlined by cross-breeding of transgenic SOD1 mice with mice that showed markedly reduced Ca2C permeability of AMPA/ kainate receptors, due to GluR2 overexpression. This resulted in a significant delay of disease onset, mortality and development of pathological hallmarks [4]. Mitochondrial disturbances in motoneuron degeneration In both sporadic and familial forms of ALS, there is increasing evidence for crucial involvement of mitochondria [30,31]. First clues were provided by histological observations of mitochondrial abnormalities, such as swelling, as some of the earliest signs of pathology in ALS mouse models and in human ALS [32–34]. Interestingly, the morphological alterations were not confined to the CNS, but were also detected in muscle and liver www.sciencedirect.com

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biopsies from ALS patients. More recently, research has focussed on mitochondrial dysfunction, in particular altered activity of the respiratory chain and increased production of ROS. In sporadic ALS, altered electron transport chain activity, most often a decrease in activity of complex IV has been linked to mutations in mitochondrial DNA (mtDNA) [35–37]. The question of whether alterations in the mitochondrial genome can lead to alterations in cell function has been addressed by transferring mtDNA from ALS subjects to mtDNA-depleted human neuroblastoma cells. This resulted in abnormal electron transport chain functioning, increases in activity of freeradical-scavenging enzymes, perturbed Ca2C homeostasis and altered mitochondrial ultrastructure, suggesting a pathological role for mtDNA mutations in some forms of ALS [38]. Other studies pointed out the increased production of ROS by mitochondria in motoneurons as a result of mitochondrial Ca2C overload following excitotoxic stimulation of AMPA/kainate receptors [39]. Evidence suggests that ROS generated in motoneurons can cross the plasma membrane and cause oxidative disruption of glutamate transporters in neighbouring astrocytes [7,40]. This in turn could enhance local excitotoxicity and initiate a vicious cycle of motoneuron damage. The model nicely integrates the hypotheses of excitotoxicity and oxidative damage, although exact mechanisms of how mitochondrial Ca2C uptake would increase ROS production are still unclear. In mutant-SOD1-related ALS, the importance of mitochondrial mechanisms was indicated not only by the striking morphological findings [34] but also by the observation that creatine treatment could prolong survival and reduce oxidative motoneuron damage in transgenic mice [41]. There is good reason to assume that mitochondria might be damaged by the mutated protein itself. Aggregates containing mutant SOD1 have been found within the mitochondrial matrix [42–44], and decreased enzyme activities of the electron transport chain at the levels of complexes I, II and IV have been demonstrated at early disease stages [45–47]. Most interestingly, recent work provided evidence that mutant SOD1 might disrupt association of complex IV (cytochrome c) with the inner mitochondrial membrane, and by this interfere with mitochondrial respiration [48]. An important consequence of such disturbed mitochondrial respiration seems to be increased production of ROS. This has been demonstrated for cultured motoneurons expressing mutant SOD1 [49], and is in line with a study of motoneurons in brain slices, where complex IV was inhibited by cyanide [18]. Independent of the cause of mitochondrial damage, various studies indicate that disturbance of mitochondrial respiration sensitizes motoneurons to stimulation by glutamate and to action of environmental toxins, and increases their vulnerability to degeneration [49,50]. For example, in vitro, chronic mitochondrial inhibition induced by the inhibitors malonate or sodium azide led to selective motoneuron death, which could be counteracted by free-radical scavengers and AMPA receptor blockers [51]. In vivo, there is evidence that chronic mitochondrial poisoning can induce selective motoneuron

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pathology [52,53]. In line with these observations, ALS-like symptoms and neuropathology can be produced in mice by a targeted deletion that eliminates the ability of vascular endothelial-cell growth factor (VEGF) to respond to tissue hypoxia [54]. Cross-breeding these mice with the SOD1 mutants severely enhanced motoneuron degeneration [55], whereas treatment of SOD-transgenic mice with VEGF delayed progression of symptoms and prolonged survival [56,57]. Although direct neurotrophic effects of VEGF might contribute to these phenomena, motoneurons seem selectively vulnerable to disturbances in mitochondrial respiration such as chronic hypoxia. Brainstem motoneurons as cellular model systems for studying selective vulnerability Various model systems have been used to investigate processes of mitochondrial disruption, disturbance of Ca2C homeostasis and selective vulnerability of motoneurons during ALS. Culture systems (i.e. slice cultures or motoneuron primary cultures) have proven valuable tools for physiological and biochemical characterization of ALS-related pathology over time, and for testing for protective strategies. Preparation of acute slices is particularly valuable because it preserves motoneurons in their physiological environment in a functionally intact state. This is highlighted by in vitro slice preparation of the mouse brainstem, where the hypoglossal motor nucleus can be preserved within a neuronal network receiving rhythmic excitatory input from the ‘PreBo¨tzinger complex’ as part of the respiratory system [58]. Combined patch-clamp and Ca2C-imaging experiments revealed that phases of activity of hypoglossal (a)

motoneurons are characterized by rapid transients in somatic and dendritic Ca2C levels, with amplitudes %200 nM [59,60] (Figure 1). As demonstrated by several groups, these Ca2C oscillations are closely related to the physiological regulation of motoneuron firing rates through Ca2C-dependent afterhyperpolarizations, primarily by coupling activity-evoked Ca2C influx to activation of Ca2C-dependent KC-channels [61–63]. An improvement in the understanding of motoneuron Ca2C homeostasis came with the application of the ‘added buffer’ approach, originally developed by Neher and Augustine [64] (Figure 2a). This strategy has enabled definition of different aspects of Ca2C homeostasis, such as endogenous buffering and clearance rates, in quantitative detail. For example, such experiments define the endogenous Ca2C-binding ratio Ks of a cell, which reflects the ability of the combined set of endogenous buffers to bind Ca2C. The Ks value of 40 defined for hypoglossal motoneurons indicates that for each Ca2C ion that appears as cytosolic free Ca2C, 40 ions are bound by endogenous buffers. Interestingly, in a linear model of cellular Ca2C homeostasis, this buffering capacity is directly proportional to the recovery time constant (t) of cytosolic Ca2C levels, indicating that low Ks is a useful adaptation for rapid relaxation times of cytosolic [Ca2C]i. Using this quantitative approach, it was possible to compare in detail specific parameters of the Ca2C homeostasis in different cell types and to correlate those parameters with the selective vulnerability of a given cell type in ALS (Figure 2b). For example, comparative studies demonstrated endogenous Ca2C-binding ratios of 264 in oculomotor neurons [65], which is six-times larger than values

(b) Dendritic distance (µm) [Ca]i Brainstem motoneuron

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Figure 1. Spontaneous Ca2C oscillations in rhythmically active motoneurons. (a) Micrograph of soma and proximal dendrites of a patch-clamped hypoglossal motoneuron in a 700-mm-thick mouse brainstem slice. The Ca2C indicator dye fura-2 was introduced via a patch-pipette at 400 mM. (b) Whole-cell patch-clamp recording (current-clamp mode, bottom) and ratiometric Ca2C imaging (above) of rhythmic spontaneous discharges and concurrent Ca2C transients in the soma and in six dendritic compartments at distances 24–76 mm from the soma (compartment positions are represented by boxes in (a)). Spontaneous burst of action potentials (shown in the bottom trace as changes in membrane voltage, Vm) are accompanied by [Ca2C]i rises %200 nM. Modified from [59]. www.sciencedirect.com

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Figure 2. Ca2C-buffering capacities correlate with the vulnerability of neurons in ALS. (a) The Ca2C buffering capacity (KS) of a cell, which reflects the relative fraction of bound versus free Ca2C in the cell, can be calculated by using the ‘added buffer’ approach [64]. In this linear one-compartment model, the recovery time of cytosolic [Ca2C]i elevations – represented by the recovery time constant (t) – depends on the amount of endogenous buffer (S; corresponding to Ca2C-binding proteins), the amount of exogenous buffer (B; i.e. fura-2) and the transport rate (g) of Ca2C across cellular membranes. Gradual introduction of exogenous buffer via the patch pipette enables the cytosolic KS to be determined. KB indicates the buffer capacity of the exogenous buffer (i.e. fura-2). (b) Cytosolic Ca2C-buffering capacities have been calculated for various cell types [12,64–66,70–72]. Note that KS varies substantially between cells and that neurons that are typically damaged in ALS – hypoglossal, spinal and facial motoneurons (MN) – display KS values that are several times lower than cells usually resistant in ALS [i.e. oculomotor neurons and cerebellar (Cb) Purkinje cells].

importantly, such studies therefore identified an exceptionally low endogenous buffering capacity as a cellular risk factor for selective motoneuron vulnerability. It is interesting to note that experimentally measured buffering capacities correlated well with the expression profiles

of 41 and 50 found in hypoglossal and spinal motoneurons, respectively. Interestingly, values in resistant oculomotor neurons are comparable to those found in cortical and hippocampal cells [66], which are also hardly affected in ALS patients and corresponding mouse models. Most

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Figure 3. Specialized Ca2C homeostasis in weakly buffered motoneurons. Low cytosolic Ca2C-buffering capacity has several implications for spatial and temporal Ca2C signalling. For a given amount of influx, the amplitude of Ca2C transients is several times larger in weakly buffered cells (e.g. hypoglossal and spinal motoneurons) than in strongly buffered ones (e.g. oculomotor neurons), and the recovery time is significantly accelerated. As illustrated, low cytosolic Ca2C buffering capacity promotes Ca2C accumulation and formation of subcellular domains around influx sites (red), and by this facilitates the interaction with intracellular organelles such as mitochondria. Indeed, various studies have demonstrated substantial mitochondrial Ca2C uptake following influx through the plasma membrane (e.g. through voltage-dependent Ca2C channels, VDCC) in weakly buffered vulnerable motoneurons [18,39,69]. Modified from [65]. www.sciencedirect.com

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of Ca2C-binding proteins such as parvalbumin and calbindin-D28k, supporting the notion that they might represent the major structural components responsible for endogenous Ca2C buffering. Regarding the increased risk of neuronal degeneration imposed by low Ca2C-buffering ability, it is important to note that low buffering also provides an important functional advantage in rhythmically active cells such as hypoglossal motoneurons. As pointed out in Figure 2, low buffering Ks directly accounts for rapid relaxation times of cytosolic Ca2C transients if all other parameters are held constant. In rhythmically active hypoglossal motoneurons, activity-related Ca2C oscillations occur at frequencies up to 10 Hz [60]. In this case, an extremely rapid recovery of Ca2C transients within hundreds of milliseconds is essential for physiological cell function. Although fast recovery times of [Ca2C]i can be achieved for high buffering capacities by more effective extrusion, this strategy is associated with higher energy consumption in a permanently oscillating system [60]. Taken together, these results therefore indicate that low buffering capacities account for rapid Ca2C signalling at relatively low energy cost, but also enhance the selective vulnerability of hypoglossal motoneurons during ALS-related motoneuron degeneration.

It is interesting to note that several other studies have provided evidence that low cytosolic Ca2C buffering capacity might correlate with a particularly close interaction of Ca2C influx pathways and intracellular organelles such as mitochondria and the endoplasmic reticulum [67,68]. In hypoglossal motoneurons, Ca2C influx through the plasma membrane is directly associated with massive mitochondrial Ca2C uptake [69]. Interestingly, such mitochondrial Ca2C uptake is comparably small in highly buffered ALS-resistant cells [39,70], suggesting that mitochondria partially compensate for weak cytosolic Ca2C buffering in vulnerable cells. Theoretical considerations indicate that mitochondrial Ca2C uptake in weakly buffered cells is facilitated by formation of large Ca2C domains around influx sites, and by a larger rise in [Ca2C]i for a given amount of influx than in strongly buffered cells (Figure 3). There is experimental evidence that mitochondria control Ca2C homeostasis in motoneurons not only by buffering but also by ROS-dependent regulation of excitability [18]. When respiration is inhibited by cyanide, subsequent ROS formation induces opening of NaC conductances, increases action potential discharge and enhances voltage-dependent Ca2C influx. This mechanism is potentially relevant for ALS because various studies have demonstrated mitochondrial inhibition and increased ROS production.

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Figure 4. Ca2C, mitochondria and motoneuron degeneration in ALS – an integrative model that combines different aspects of ALS-related motoneuron degeneration. ALS-related disturbance of mitochondrial respiration, by mutant superoxide dismutase 1 (SOD1) or hypoxia, results in increased formation of reactive oxygen species (ROS) [48,49]. Evidence suggests that ROS formation enhances motoneuron excitability via induction of a NaC current, leading to increased discharge of action potentials and Ca2C influx through voltage-dependent Ca2C channels (VDCC) [18]. ROS are also suspected to leave the neuron and damage glial glutamate transporters [40]. Inflammatory processes and microglial activation contribute to propagation of oxidative damage [73]. Given the increased motoneuron excitability, impaired glutamate transport substantially increases the risk for glutamate-mediated toxicity involving Ca2C influx through Ca2C-permeable AMPA receptors [7]. Ca2C influx is buffered in large part by mitochondria [39,69]. However, once mitochondrial disturbance results in loss of the potential gradient, mitochondrial buffering becomes inefficient and stored Ca2C can be released. A final step in the cascade could be the inability of mitochondria to serve the increased metabolic demand following disturbed ion homeostasis. The circle of motoneuron damage could alternatively be initiated at different sites – that is, start with glutamate toxicity or be triggered by oxidative stress. The figure excludes phenomena such as aggregate formation, neurofilament disruption or induction of pro-apoptotic pathways, which could be integrated into the model as consequences of mitochondrial disturbance, ROS formation or increased [Ca2C]i. www.sciencedirect.com

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Ca2C, mitochondria and selective motoneuron vulnerability: an integrative view In conclusion, it seems that ALS is a multifactorial disease, where motoneuron degeneration can be initiated in different ways – by disruption of mitochondrial processes or of excitatory synaptic transmission. Figure 4 illustrates how the different aspects can be combined to fit a unifying hypothesis of motoneuron damage. Briefly, mitochondrial respiration can be disturbed by mutations in SOD1, hypoxia, Ca2C overload or alterations in the mitochondrial genome. Although exact molecular mechanisms still have to be worked-out, these alterations seem to increase formation of ROS. It is hypothesized that ROS have a central role in propagating damage by targeting surrounding glia and by increasing the excitability of motoneurons. Perhaps as a consequence, excitotoxicity develops with increased activity-dependent Ca2C influx and concomitant mitochondrial Ca2C cycling. Given the mitochondrial disturbance, Ca2C buffering becomes inefficient and cytosolic Ca2C levels rise. Other observed phenomena, such as aggregate formation, neurofilament disruption and induction of apoptotic pathways, could well be consequences of either ROS generation or a rise in [Ca2C]i. Forthcoming studies could add to the understanding of why these processes preferentially damage motoneurons. We hypothesize that vulnerability is a consequence of specific physiological features, particularly highly specialized Ca2C homeostasis. This includes not only highly Ca2C-permeable AMPA/kainate receptor channels and low cytosolic buffering capacity, but also continuous activity-dependent Ca2C cycling and a predominant role of mitochondria in buffering of Ca2C transients. Additionally, motoneurons seem particularly sensitive to metabolic disturbances, owing both to their high metabolic demand and to mitochondrial control of electrical excitability. Accordingly, therapeutic measures aimed at protecting mitochondrial function could be useful in various forms of ALS.

Acknowledgements We would like to thank Drs Saju Balakrishnan, Michael Mu¨ller, Erwin Neher, Diethelm Richter and Eike Schomburg for valuable discussions. This research was supported by grants from the DFG, Sonderforschungsbereich SFB 406 and the Center of Molecular Physiology of the Brain (CMPB).

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