Selective mitochondrial Ca2+ uptake deficit in disease endstage vulnerable motoneurons of the SOD1G93A mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis is a progressive neurodegenerative disease that targets some somatic motoneuron populations, while others, e.g. those of the oculomotor system, are spared. The pathophysiological basis of this pattern of differential vulnerability, which is preserved in a transgenic mouse model of amyotrophic lateral sclerosis (SOD1(G93A)), and the mechanism of neurodegeneration in general are unknown. Hyperexcitability and calcium dysregulation have been proposed by others on the basis of data from juvenile mice that are, however, asymptomatic. No studies have been done with symptomatic mice following disease progression to the disease endstage. Here, we developed a new brainstem slice preparation for whole-cell patch-clamp recordings and single cell fura-2 calcium imaging to study motoneurons in adult wild-type and SOD1(G93A) mice up to disease endstage. We analysed disease-stage-dependent electrophysiological properties and intracellular Ca(2+) handling of vulnerable hypoglossal motoneurons in comparison to resistant oculomotor neurons. Thereby, we identified a transient hyperexcitability in presymptomatic but not in endstage vulnerable motoneurons. Additionally, we revealed a remodelling of intracellular Ca(2+) clearance within vulnerable but not resistant motoneurons at disease endstage characterised by a reduction of uniporter-dependent mitochondrial Ca(2+) uptake and enhanced Ca(2+) extrusion across the plasma membrane. Our study challenged the notion that hyperexcitability is a direct cause of neurodegeneration in SOD1(G93A) mice, but molecularly identified a Ca(2+) clearance deficit in motoneurons and an adaptive Ca(2+) handling strategy that might be targeted by future therapeutic strategies.

Fatigability of spinal reflex transmission in a mouse model (SOD1(G93A) ) of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive loss of motor neurons. To analyze the progressive motor deficits during the course of this disease, we investigated fatigability and ability of recovery of spinal motor neurons by testing monosynaptic reflex transmission with increasing stimulus frequencies in the lumbar spinal cord of the SOD1(G93A) mouse model for ALS in a comparison with wild-type (WT) mice. Monosynaptic reflexes in WT and SOD1(G93A) mice without behavioral deficits showed no difference with respect to their resistance to increasing stimulus frequencies. During the progression of motor deficits in SOD1(G93A) mice, the vulnerability of monosynaptic reflexes to higher frequencies increased, the required time for reflex recovery was extended, and recovery was often incomplete. Fatigability and demand for recovery of spinal motor neurons in SOD1(G93A) mice rose with increasing motor deficits. This supports the assumption that impairment of the energy supply may contribute to the pathogenesis of ALS.

Cu/Zn superoxide dismutase typical for familial amyotrophic lateral sclerosis increases the vulnerability of mitochondria and perturbs Ca2+ homeostasis in SOD1G93A mice.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the selective loss of defined motoneuron populations in the brainstem and spinal cord. Although low cytosolic calcium ([Ca(2+)](i)) buffering and a strong interaction between metabolic mechanisms and [Ca(2+)](i) have been associated with selective motoneuron vulnerability, the underlying cellular mechanisms are barely understood. To elucidate the underlying molecular events, we used rapid charge-cooled device imaging to evaluate Ca(2+) signaling and metabolic signatures in the brainstem slices of SOD1(G93A) mice, the mouse model of human ALS, at 8 to 9 and 14 to 15 weeks of age, corresponding to the presymptomatic and symptomatic stages of motor dysfunction, respectively, and compared the results with corresponding age-matched wild-type littermates. We also monitored the mitochondrial membrane potential (Delta(Psim)) of brainstem motoneurons, a valuable tool for characterizing the metabolic signature of intrinsic energy profiles and considered to be a good experimental measure for monitoring energy metabolism in cells. We found that different pharmacological interventions substantially disrupt Delta(Psim) in SOD1(G93A) motoneurons during the symptomatic stage. Furthermore, we investigated the impact of impaired mitochondrial mechanisms on [Ca(2+)](i) regulation by using the membrane-permeable indicator fura-acetoxy methyl ester. Taken together, the results indicate that mitochondrial disruptions are critical elements of SOD1(G93A)-mediated motoneuron degeneration in which selective motoneuron vulnerability results from a synergistic accumulation of risk factors, including the disruption of electrochemical potential, low Ca(2+) buffering, and strong mitochondrial control of [Ca(2+)](i). The stabilization of mitochondria-related signal cascades may represent a useful strategy for clinical neuroprotection in ALS.

Impairment of mitochondrial calcium handling in a mtSOD1 cell culture model of motoneuron disease.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the selective loss of motor neurons (MN) in the brain stem and spinal cord. Intracellular disruptions of cytosolic and mitochondrial calcium have been associated with selective MN degeneration, but the underlying mechanisms are not well understood. The present evidence supports a hypothesis that mitochondria are a target of mutant SOD1-mediated toxicity in familial amyotrophic lateral sclerosis (fALS) and intracellular alterations of cytosolic and mitochondrial calcium might aggravate the course of this neurodegenerative disease. In this study, we used a fluorescence charged cool device (CCD) imaging system to separate and simultaneously monitor cytosolic and mitochondrial calcium concentrations in individual cells in an established cellular model of ALS. RESULTS: To gain insights into the molecular mechanisms of SOD1(G93A) associated motor neuron disease, we simultaneously monitored cytosolic and mitochondrial calcium concentrations in individual cells. Voltage - dependent cytosolic Ca2+ elevations and mitochondria - controlled calcium release mechanisms were monitored after loading cells with fluorescent dyes fura-2 and rhod-2. Interestingly, comparable voltage-dependent cytosolic Ca2+ elevations in WT (SH-SY5Y(WT)) and G93A (SH-SY5Y(G93A)) expressing cells were observed. In contrast, mitochondrial intracellular Ca2+ release responses evoked by bath application of the mitochondrial toxin FCCP were significantly smaller in G93A expressing cells, suggesting impaired calcium stores. Pharmacological experiments further supported the concept that the presence of G93A severely disrupts mitochondrial Ca2+ regulation. CONCLUSION: In this study, by fluorescence measurement of cytosolic calcium and using simultaneous [Ca2+]i and [Ca2+]mito measurements, we are able to separate and simultaneously monitor cytosolic and mitochondrial calcium concentrations in individual cells an established cellular model of ALS. The primary goals of this paper are (1) method development, and (2) screening for deficits in mutant cells on the single cell level. On the technological level, our method promises to serve as a valuable tool to identify mitochondrial and Ca2+-related defects during G93A-mediated MN degeneration. In addition, our experiments support a model where a specialized interplay between cytosolic calcium profiles and mitochondrial mechanisms contribute to the selective degeneration of neurons in ALS.

Low Ca2+ buffering in hypoglossal motoneurons of mutant SOD1 (G93A) mice.

Mutations in the Cu/Zn superoxide dismutase (SOD1) gene are associated with amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder characterized by a selective degeneration of brainstem and spinal motoneurons. The pathomechanism of degeneration is still incompletely understood, but includes a disruption in cellular Ca2+ homeostasis. Here we report a quantitative microfluorometric analysis of the Ca2+ homeostasis in vulnerable hypoglossal motoneurons of neonatal mutant (G93A) SOD1 transgenic mice, a mouse model of human ALS. Ca2+ transient decay times (tau = 0.3 s), extrusion rates (gamma = 92 s(-1)) and exceptionally low intrinsic Ca2+ binding ratios (kappaS = 30) were found to be in the same range as compared to non-transgenic animals. Together with the previous observation of high Ca2+ binding ratios in ALS-resistant neurons (e.g. oculomotor), this supports the assumption that low Ca2+ buffering in vulnerable motoneurons represents a significant risk factor for degeneration. On the other hand, alterations in buffering properties by expression of mutant SOD1 are unlikely to be involved in disease initiation.

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

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 Ca(2+)-dependent signalling cascades. Comparison of ALS-vulnerable and ALS-resistant neurons identified low Ca(2+)-buffering capacity and a strong impact of mitochondrial signal cascades as important risk factors. Under physiological conditions, weak Ca(2+) buffers are valuable because they facilitate rapid relaxation times of Ca(2+) transients in motoneurons during high-frequency rhythmic activity. However, under pathological conditions, weak Ca(2+) buffers are potentially dangerous because they accelerate a vicious circle of mitochondrial disruption, Ca(2+) disregulation and excitotoxic cell damage.

Expression of a Cu,Zn superoxide dismutase typical for familial amyotrophic lateral sclerosis increases the vulnerability of neuroblastoma cells to infectious injury.

BACKGROUND: Infections can aggravate the course of neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). Mutations in the anti-oxidant enzyme Cu,Zn superoxide dismutase (EC 1.15.1.1, SOD1) are associated with familial ALS. Streptococcus pneumoniae, the most frequent respiratory pathogen, causes damage by the action of the cholesterol-binding virulence factor pneumolysin and by stimulation of the innate immune system, particularly via Toll-like-receptor 2. METHODS: SH-SY5Y neuroblastoma cells transfected with the G93A mutant of SOD1 typical for familial ALS (G93A-SOD1) and SH-SY5Y neuroblastoma cells transfected with wildtype SOD1 were both exposed to pneumolysin and in co-cultures with cultured human macrophages treated with the Toll like receptor 2 agonist N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-cysteinyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]-lysine x 3 HCl (Pam3CSK4). Cell viability and apoptotic cell death were compared morphologically and by in-situ tailing. With the help of the WST-1 test, cell viability was quantified, and by measurement of neuron-specific enolase in the culture supernatant neuronal damage in co-cultures was investigated. Intracellular calcium levels were measured by fluorescence analysis using fura-2 AM. RESULTS: SH-SY5Y neuroblastoma cells transfected with the G93A mutant of SOD1 typical for familial ALS (G93A-SOD1) were more vulnerable to the neurotoxic action of pneumolysin and to the attack of monocytes stimulated by Pam3CSK4 than SH-SY5Y cells transfected with wild-type human SOD1. The enhanced pneumolysin toxicity in G93A-SOD1 neuronal cells depended on the inability of these cells to cope with an increased calcium influx caused by pores formed by pneumolysin. This inability was caused by an impaired capacity of the mitochondria to remove cytoplasmic calcium. Treatment of G93A-SOD1 SH-SY5Y neuroblastoma cells with the antioxidant N-acetylcysteine reduced the toxicity of pneumolysin. CONCLUSION: The particular vulnerability of G93A-SOD1 neuronal cells to hemolysins and inflammation may be partly responsible for the clinical deterioration of ALS patients during infections. These findings link infection and motor neuron disease and suggest early treatment of respiratory infections in ALS patients.

Mitochondrial Ca2+ buffering in hypoglossal motoneurons from mouse.

A variety of studies demonstrated a crucial role of mitochondria for clearance of Ca2+ loads in motoneurons. However, previous reports rarely addressed the potential influence of cell dialysis during patch-clamp recordings or temperature on mitochondrial processes. We therefore developed a protocol allowing investigation of Ca2+ dynamics in "undisturbed" AM-ester loaded hypoglossal motoneurons in a slice preparation. By comparing our findings to previous results, we argue against a significant disturbance of mitochondrial buffering by cell dialysis. By varying bath temperatures between 19 and 32 degrees C, we show that temperature alters the rate of mitochondrial uptake but not the relative contribution to maintenance of Ca2+ homeostasis. The results further indicate that mitochondria in hypoglossal motoneurons participate in intracellular Ca2+ regulation at concentrations much lower than has been generally observed for other neurons or neuroendocrine cells. Taken together, our findings further support the important role of mitochondria as regulators of Ca2+ homeostasis in motoneurons.

Functional analysis of glutamate transporters in excitatory synaptic transmission of GLAST1 and GLAST1/EAAC1 deficient mice.

The high affinity, Na(+)-dependent, electrogenic glial L-glutamate transporters GLAST1 and GLT1, and two neuronal EAAC1 and EAAT4, regulate the neurotransmitter concentration in excitatory synapses of the central nervous system. We dissected the function of the individual transporters in the monogenic null allelic mouse lines, glast1(-/-) and eaac1(-/-), and the derived double mutant glast(-/-)eaac1(-/-). Unexpectedly, the biochemical analysis and the behavioral phenotypes of these null allelic mouse lines were inconspicuous. Inhibition studies of the Na(+)-dependent glutamate transport by plasma membrane vesicles and by isolated astrocytes of wt and glast1(-/-) mouse brains indicated the pivotal compensatory role of GLT1 in the absence particularly of GLAST1 and GLAST1 and EAAC1 mutant mice. In electrophysiological studies, the decay rate of excitatory postsynaptic currents (EPSCs) of Purkinje cells (PC) after selective activation of parallel and climbing fibers proved to be similar in wt and eaac1(-/-), but was significantly prolonged in glast1(-/-) PCs. Bath application of the glutamate uptake blocker SYM2081 prolonged EPSC decay profiles in both wt and double mutant glast1(-/-)eaac1(-/-) PCs by 286% and 229%, respectively, indicating a prominent role of compensatory glutamate transport in shaping glast1(-/-)eaac1(-/-) EPSCs.

Serotonergic modulation of intracellular calcium dynamics in neonatal hypoglossal motoneurons from mouse.

(1) Serotonin (5HT)-mediated calcium signaling was investigated in hypoglossal motoneurons (HGMs) in brain stem slices of neonatal mice. Electrical activity and associated calcium signaling were studied by simultaneous patch clamp recordings and high resolution calcium imaging. (2) Bath application of 5HT (5-50 microM) depolarized membrane potential of HGMs and generated action potential discharges that were accompanied by elevations in intracellular calcium concentrations ([Ca2+]i) in the soma and dendrites. Current-evoked bursts of action potentials were more intense in the presence of 5HT; however, the corresponding calcium signals were reduced. (3) The 5HT2 receptor agonist alpha-Methyl-5HT (25, 50 microM) had effects on membrane potential, discharge properties and [Ca]i that were identical to those observed for 5HT, whereas the 5HT3 receptor agonist 1-(m-chlorophenyl) biguanide (50 microM) had no effect on membrane properties or intracellular calcium levels. (4) 8-OHDPAT (25, 50 microM), a 5HT1A receptor agonist, was without effect on steady-state membrane potential or basal [Ca]i. Similar to 5HT and alpha-Methyl-5HT, 8-OHDPAT depressed stimulus-evoked calcium transients in current and voltage clamp mode. (5) Our results suggest that calcium profiles in hypoglossal motoneurons are differentially regulated by 5HT1A and 5HT2 receptors. Activation of 5HT1A receptors primarily reduced voltage-activated Ca2+ signals without a significant impact on basal [Ca]i. In contrast, activation of 5HT2 receptors initiated a net inward current followed by membrane depolarization, where the resulting pattern of action potential discharges represents the essential determinant of global elevations in [Ca2+]i. Taken together, our results therefore identify 5HT-dependent signal pathways as a versatile tool to modulate hypoglossal motoneuron excitability under various physiological and pathophysiological conditions.

Calcium dysregulation in amyotrophic lateral sclerosis.

In the fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS), motor neurons degenerate with signs of organelle fragmentation, free radical damage, mitochondrial Ca2+ overload, impaired axonal transport and accumulation of proteins in intracellular inclusion bodies. Subgroups of motor neurons of the brainstem and the spinal cord expressing low amounts of Ca2+ buffering proteins are particularly vulnerable. In ALS, chronic excitotoxicity mediated by Ca2+-permeable AMPA type glutamate receptors seems to initiate a self-perpetuating process of intracellular Ca2+ dysregulation with consecutive endoplasmic 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 shift of Ca2+" within the endoplasmic reticulum-mitochondria Ca2+ cycle (ERMCC) as a key mechanism in motor neuron degeneration, and discusses molecular targets which may be of interest for future ERMCC modulating neuroprotective therapies.

Impact of mitochondrial inhibition on excitability and cytosolic Ca2+ levels in brainstem motoneurones from mouse.

Motoneurones (MNs) are particularly affected by the inhibition of mitochondrial metabolism, which has been linked to their selective vulnerability during pathophysiological states like hypoxia and amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder. To elucidate underlying events, we used sodium cyanide (CN) as a pharmacological inhibitor of complex IV of the mitochondrial respiratory chain ('chemical hypoxia') and investigated the cellular response in vulnerable and resistant neurone types. Bath application of 2 mm CN activated TTX-insensitive Na+ conductances in vulnerable hypoglossal MNs, which depolarized these MNs by 10.2 +/- 1.1 mV and increased their action potential activity. This response was mimicked by sodium azide (2 mm) and largely prevented by preincubation with the antioxidants ascorbic acid (1 mm) and Trolox (750 microm), indicating an involvement of reactive oxygen species (ROS) in the activation mechanism. CN also elevated cytosolic [Ca2+] levels through (i) Ca2+ release from mitochondria-controlled stores, (ii) significant retardation of cytosolic Ca2+ clearance rates, even when cytosolic ATP levels were held constant during whole-cell recording, and (iii) secondary Ca2+ influx during elevated firing rates. Blocking mitochondrial ATP production additionally raised cytosolic Ca2+ levels and prolonged recovery of Ca2+ transients with a delay of 5-6 min. Comparative studies on hypoglossal MNs, facial MNs and dorsal vagal neurones suggested that CN responses were dominated by the activation of K+ conductances in resistant neurones, thus reducing excitability during mitochondrial inhibition. In summary, our observations therefore support a model where selective MN vulnerability results from a synergistic accumulation of risk factors, including low cytosolic Ca2+ buffering, strong mitochondrial impact on [Ca2+]i, and a mitochondria-controlled increase in electrical excitability during metabolic disturbances.

Spatial profiles of store-dependent calcium release in motoneurones of the nucleus hypoglossus from newborn mouse.

Hypoglossal motoneurones (HMN) are selectively damaged in both human amyotrophic lateral sclerosis (ALS) and corresponding mouse models of this neurodegenerative disease, a process which has been linked to their low endogenous Ca2+ buffering capacity and an exceptional vulnerability to Ca2+-mediated excitotoxic events. In this report, we investigated local Ca2+ profiles in low buffered HMNs by utilizing multiphoton microscopy, CCD imaging and patch clamp recordings in slice preparations. Bath application of caffeine induced highly localized Ca2+ release events, which displayed an initial peak followed by a slow 'shoulder' lasting several seconds. Peak amplitudes were paralleled by Ca2+-activated, apamin-sensitive K+ currents (IKCa), demonstrating a functional link between Ca2+ stores and HMN excitability. The potential involvement of mitochondria was investigated by bath application of CCCP, which collapses the electrochemical potential across the inner mitochondrial membrane. CCCP reduced peak amplitudes of caffeine responses and consequently IKCa, indicating that functionally intact mitochondria were critical for store-dependent modulation of HMN excitability. Taken together, our results indicate localized Ca2+ release profiles in HMNs, where low buffering capacities enhance the role of Ca2+-regulating organelles as local determinants of [Ca2+]i. This might expose HMN to exceptional risks during pathophysiological organelle disruptions and other ALS-related, cellular disturbances.

Neurotoxicity of pneumolysin, a major pneumococcal virulence factor, involves calcium influx and depends on activation of p38 mitogen-activated protein kinase.

Neuronal injury in bacterial meningitis is caused by the interplay of host inflammatory responses and direct bacterial toxicity. We investigated the mechanisms by which pneumolysin, a cytosolic pneumococcal protein, induces damage to neurons. The toxicity after exposure of human SH-SY5Y neuroblastoma cells and hippocampal organotypic cultures to pneumolysin was time- and dose-dependent. Pneumolysin led to a strong calcium influx apparently mediated by pores on the cell membrane formed by the toxin itself and not by voltage-gated calcium channels. Buffering of intracellular calcium with BAPTA-AM [1, 2-bis (o-aminophenoxy) ethane N, N, N', N'-tetraacetic acid tetra(acetomethoxyl) ester] improved survival of neuronal cells following challenge with pneumolysin. Western blotting revealed increased phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) as early as 30 min after challenge with pneumolysin. SB 203580, a potent and selective inhibitor of p38 MAPK, rescued human neuronal cells from pneumolysin-induced death. Inhibition of the mitochondrial permeability transition pore using bongkrekate and caspase inhibition also improved survival following challenge with the toxin. Modulation of cell death pathways activated by pneumolysin may influence the outcome of pneumococcal meningitis.