Ellen F. Barrett

Many studies of Ca effects on mitochondrial respiration in intact cells have used electrical and/or chemical stimulation to elevate intracellular [Ca ], and have reported increases in [NADH] and increased ADP/ATP ratios as dominant controllers of respiration. This study tested a different form of stimulation: brief temperature increases produced by pulses of infrared light (IR, 1863 nm, 8-10 C for ~5 s). Fluorescence imaging techniques applied to single PC-12 cells in low µM extracellular [Ca ] revealed IR stimulation-induced increases in both cytosolic (fluo5F) and mitochondrial (rhod2) [Ca [. IR stimulation increased O consumption (porphyrin fluorescence), and produced an alkaline shift in mitochondrial matrix pH (Snarf1), indicating activation of the electron transport chain (ETC). The increase in O consumption persisted in oligomycin, and began during a decrease in NADH, suggesting that the initial increase in ETC activity was not driven by increased ATP synthase activity or an increased fuel supply to ETC complex I. Imaging with two potentiometric dyes (TMRM and R123) indicated a depolarizing shift in ΔΨ that persisted in high [K ] medium. High-resolution fluorescence imaging disclosed large, reversible mitochondrial depolarizations that were inhibited by cyclosporin A (CSA), consistent with opening of transient mitochondrial permeability transition pores. IR stimulation also produced a Ca -dependent increase in superoxide production (Mitosox) that was not inhibited by CSA, indicating that the increase in superoxide did not require transition pore opening. Thus the intracellular Ca release that follows pulses of infrared light offers new insights into Ca -dependent processes controlling respiration and reactive oxygen species in intact cells.

Inner ear spiral ganglion neurons were cultured from day 4 postnatal mice and loaded with a fluorescent Ca 2+ indicator (fluo-4, -5F, or -5N). Pulses of infrared radiation (IR; 1,863 nm, 200 µs, 200–250 Hz for 2–5 s, delivered via an optical fiber) produced a rapid, transient temperature increase of 6–12°C (above a baseline of 24–30°C). These IR pulse trains evoked transient increases in both nuclear and cytosolic Ca 2+ concentration ([Ca 2+ ]) of 0.20–1.4 µM, with a simultaneous reduction of [Ca 2+ ] in regions containing endoplasmic reticulum (ER). IR-induced increases in cytosolic [Ca 2+ ] continued in medium containing no added Ca 2+ (±Ca 2+ buffers) and low [Na + ], indicating that the [Ca 2+ ] increase was mediated by release from intracellular stores. Consistent with this hypothesis, the IR-induced [Ca 2+ ] response was prolonged and eventually blocked by inhibition of ER Ca 2+ -ATPase with cyclopiazonic acid, and was also inhibited by a high concentration of ryanodine and by inhibitors of inositol (1,4,5)-trisphosphate (IP 3 )-mediated Ca 2+ release (xestospongin C and 2-aminoethoxydiphenyl borate). The thermal sensitivity of the response suggested involvement of warmth-sensitive transient receptor potential (TRP) channels. The IR-induced [Ca 2+ ] increase was inhibited by TRPV4 inhibitors (HC-067047 and GSK-2193874), and immunostaining of spiral ganglion cultures demonstrated the presence of TRPV4 and TRPM2 that colocalized with ER marker GRP78. These results suggest that the temperature sensitivity of IR-induced [Ca 2+ ] elevations is conferred by TRP channels on ER membranes, which facilitate Ca 2+ efflux into the cytosol and thereby contribute to Ca 2+ -induced Ca 2+ -release via IP 3 and ryanodine receptors. NEW & NOTEWORTHY Infrared radiation-induced photothermal effects release Ca 2+ from the endoplasmic reticulum of primary spiral ganglion neurons. This Ca 2+ release is mediated by activation of transient receptor potential (TRPV4) channels and involves amplification by Ca 2+ -induced Ca 2+ -release. The neurons immunostained for warmth-sensitive channels, TRPV4 and TRPM2, which colocalize with endoplasmic reticulum. Pulsed infrared radiation provides a novel experimental tool for releasing intracellular Ca 2+ , studying Ca 2+ regulatory mechanisms, and influencing neuronal excitability.

In mutant superoxide dismutase 1 (SOD1) mouse models of familial amyotrophic lateral sclerosis (fALS) some of the earliest signs of morphological and functional damage occur in the motor nerve terminals that innervate fast limb muscles. This study tested whether localized peripheral application of a protective drug could effectively preserve neuromuscular junctions in late-stage disease. Methylene blue (MB), which has mitochondria-protective properties, was infused via an osmotic pump into the anterior muscle compartment of one hind limb of late pre- symptomatic SOD1-G93A mice for ≥3weeks. When mice reached end-stage disease, peak twitch and tetanic contractions evoked by stimulation of the muscle nerve were measured in two anterior compartment muscles (tibialis anterior [TA] and extensor digitorum longus [EDL], both predominantly fast muscles). With 400μM MB in the infusion reservoir, muscles on the MB-infused side exhibited on average a ~100% increase in nerve-evoked contractile force compared to muscles on the contralateral non-infused side (p<0.01 for both twitch and tetanus in EDL and TA). Pairwise comparisons of endplate innervation also revealed a beneficial effect of MB infusion, with an average of 65% of endplates innervated in infused EDL, compared to only 35% on the non-infused side (p<0.01). Results suggested that MB's protective effects required an extracellular [MB] of ~1μM, were initiated peripherally (no evidence of retrograde transport into the spinal cord), and involved MB's reduced form. Thus peripherally-initiated actions of MB can help preserve neuromuscular structure and function in SOD1-G93A mice, even at late stages of disease. [Display omitted] •In SOD1-G93A mice, motor nerve terminals die before spinal motor neurons.•Methylene blue (MB) was infused for ≥3weeks into a hind limb muscle compartment.•On the MB-infused side more endplates remained innervated at end-stage disease.•Nerve-evoked muscle contractions were 2× greater on the MB-infused side.•Thus treatments aimed at the periphery can preserve neuromuscular transmission.

Abundant evidence indicates that mitochondrial dysfunction and Ca 2+ dysregulation contribute to the muscle denervation and motor neuron death that occur in mouse models of familial amyotrophic lateral sclerosis (fALS). This perspective considers measurements of mitochondrial function and Ca 2+ handling made in both motor neuron somata and motor nerve terminals of SOD1-G93A mice at different disease stages. These complementary studies are integrated into a model of how mitochondrial dysfunction disrupts handling of stimulation-induced Ca 2+ loads in presymptomatic and end-stages of this disease. Also considered are possible mechanisms underlying the findings that some treatments that preserve motor neuron somata fail to postpone degeneration of motor axons and terminals.

Muscle endplates become denervated in mice that express mutations of human superoxide dismutase 1 (hSOD1), models of familial amyotrophic lateral sclerosis. This denervation is especially marked in fast limb muscles, and precedes death of motor neuron somata. This study used mice that expressed yellow fluorescent protein (YFP) in neurons to investigate changes in the morphology and function of axons and motor terminals innervating a fast forelimb muscle (epitrochleoanconeus, ETA) in presymptomatic and symptomatic hSOD1-G85R mice, compared to those in mice that express wild-type (wt) hSOD1. The percentage of endplates (identified using fluorescently-labeled α-bungarotoxin) innervated by motor terminals remained high in presymptomatic SOD1-G85R mice, but fell to ~50% in symptomatic mice. The number of large diameter (≥4μm) axons in the ETA nerve also decreased as mice became symptomatic, and endplate innervation correlated best with the number of large diameter axons. Motor terminal function was assessed using changes in terminal YFP fluorescence evoked by trains of action potentials; different components of the pH-dependent YFP signals reflect stimulation-induced Ca2+ entry and vesicular exo/endocytosis. Most visible motor terminals (>90%) remained capable of responding to nerve stimulation in both pre- and symptomatic hSOD1-G85R mice, but with functional alterations. Responses in presymptomatic terminals suggested reduced acidification and increased vesicular release, whereas symptomatic terminals exhibited increased acidification and reduced vesicular release. The fact that most remaining terminals were able to respond to nerve stimulation suggests that motor terminal-protective therapies might contribute to preserving neuromuscular function in fALS mice. ► Innervation of mouse forelimb muscles was assayed by imaging neuronally-expressed YFP. ► Symptomatic SOD1-G85R mice lost motor terminals and large-diameter motor axons. ► Activation by action potentials was detected as YFP florescence changes in terminals. ► >90% of surviving SOD1-G85R motor terminals remained responsive to stimulation.

Previous work demonstrated that hyperthermia (43°C for 2 h) results in delayed, apoptotic-like death in striatal neuronal cultures. We investigated early changes in mitochondrial function induced by this heat stress. Partial depolarization of the mitochondrial membrane potential (ΔΨ(m)) began about 1 h after the onset of hyperthermia and increased as the stress continued. When the heat stress ended, there was a partial recovery of ΔΨ(m), followed hours later by a progressive, irreversible depolarization of ΔΨ(m). During the heat stress, O(2) consumption initially increased but after 20-30 min began a progressive, irreversible decline to about one-half the initial rate by the end of the stress. The percentage of oligomycin-insensitive respiration increased during the heat stress, suggesting an increased mitochondrial leak conductance. Analysis using inhibitors and substrates for specific respiratory chain complexes indicated hyperthermia-induced dysfunction at or upstream of complex I. ATP levels remained near normal for ∼4 h after the heat stress. Mitochondrial movement along neurites was markedly slowed during and just after the heat stress. The early, persisting mitochondrial dysfunction described here likely contributes to the later (>10 h) caspase activation and neuronal death produced by this heat stress. Consistent with this idea, proton carrier-induced ΔΨ(m) depolarizations comparable in duration to those produced by the heat stress also reduced neuronal viability. Post-stress ΔΨ(m) depolarization and/or delayed neuronal death were modestly reduced/postponed by nicotinamide adenine dinucleotide, a calpain inhibitor, and increased expression of Bcl-xL.

Motor nerve terminals are especially sensitive to an ischemia/reperfusion stress. We applied an in vitro model of this stress, oxygen/glucose deprivation (OGD), to mouse neuromuscular preparations to investigate how Ca2+ contributes to stress-induced motor terminal damage. Measurements using an ionophoretically-injected fluorescent [Ca2+] indicator demonstrated an increase in intra-terminal [Ca2+] following OGD onset. When OGD was terminated within 20–30min of the increase in resting [Ca2+], these changes were sometimes reversible; in other cases [Ca2+] remained high and the terminal degenerated. Endplate innervation was assessed morphometrically following 22min OGD and 120min reoxygenation (32.5°C). Stress-induced motor terminal degeneration was Ca2+-dependent. Median post-stress endplate occupancy was only 26% when the bath contained the normal 1.8mM Ca2+, but increased to 81% when Ca2+ was absent. Removal of Ca2+ only during OGD was more protective than removal of Ca2+ only during reoxygenation. Post-stress endplate occupancy was partially preserved by pharmacological inhibition of various routes of Ca2+ entry into motor terminals, including voltage-dependent Ca2+ channels (ω-agatoxin-IVA, nimodipine) and the plasma membrane Na+/Ca2+ exchanger (KB-R7943). Inhibition of a Ca2+-dependent protease with calpain inhibitor VI was also protective. These results suggest that most of the OGD-induced motor terminal damage is Ca2+-dependent, and that inhibition of Ca2+ entry or Ca2+-dependent proteolysis can reduce this damage. There was no significant difference between the response of wild-type and presymptomatic superoxide dismutase 1 G93A mutant terminals to OGD, or in their response to the protective effect of the tested drugs. ► An oxygen/glucose deprivation stress was applied to mouse neuromuscular preparations. ► This stress elevated [Ca2+] and depolarized mitochondria within motor terminals. ► Removal of bath Ca2+ during the stress preserved motor terminals and axons. ► Inhibitors of Ca2+ influx and of calpain activation preserved endplate innervation. ► Motor terminal degeneration following oxygen/glucose deprivation is Ca2+-dependent.

Mitochondria contribute to neuronal function not only via their ability to generate ATP, but also via their ability to buffer large Ca2+ loads. This review summarizes evidence that mitochondrial Ca2+ sequestration is especially important for sustaining the function of vertebrate motor nerve terminals during repetitive stimulation. Motor terminal mitochondria can sequester large amounts of Ca2+ because they have mechanisms for limiting both the mitochondrial depolarization and the increase in matrix free [Ca2+] associated with Ca2+ influx. In mice expressing mutations of human superoxide dismutase −1 (SOD1) that cause some cases of familial amyotrophic lateral sclerosis (fALS), motor terminals degenerate well before the death of motor neuron cell bodies. This review presents evidence for early and progressive mitochondrial dysfunction in motor terminals of mutant SOD1 mice (G93A, G85R). This dysfunction would impair mitochondrial ability to sequester stimulation-associated Ca2+ loads, and thus likely contributes to the early degeneration of motor terminals.

Mitochondria in motor nerve terminals temporarily sequester large Ca2+ loads during repetitive stimulation. In wild-type mice this Ca2+ uptake produces a small (<5mV), transient depolarization of the mitochondrial membrane potential (Ψm, motor nerve stimulated at 100Hz for 5s). We demonstrate that this stimulation-induced Ψm depolarization attains much higher amplitudes in motor terminals of symptomatic mice expressing the G93A or G85R mutation of human superoxide dismutase 1 (SOD1), models of familial amyotrophic lateral sclerosis (fALS). These large Ψm depolarizations decayed slowly and incremented with successive stimulus trains. Additional Ψm depolarizations occurred that were not synchronized with stimulation. These large Ψm depolarizations were reduced (a) by cyclosporin A (CsA, 1–2μM), which inhibits opening of the mitochondrial permeability transition pore (mPTP), or (b) by replacing bath Ca2+ with Sr2+, which enters motor terminals and mitochondria but does not support mPTP opening. These results are consistent with the hypothesis that the large Ψm depolarizations evoked by repetitive stimulation in motor terminals of symptomatic fALS mice result from mitochondrial dysfunction that increases the likelihood of transient mPTP opening during Ca2+ influx. Such mPTP openings, a sign of mitochondrial stress, would disrupt motor terminal handling of Ca2+ loads and might thereby contribute to motor terminal degeneration in fALS mice. Ψm depolarizations resembling those in symptomatic fALS mice could be elicited in wild-type mice following a 0.5–1h exposure to diamide (200μM), which produces an oxidative stress, but these depolarizations were not reduced by CsA. ► Repetitive nerve stimulation transiently depolarizes mitochondria in motor terminals. ► This Ψm depolarization increases in symptomatic fALS mice (SOD1-G93A, -G85R). ► Cyclosporin A or substitution of Sr2+ for Ca2+ reduces these large Ψm depolarizations. ► Diamide increases stimulation-evoked Ψm depolarizations in wild-type mice. ► Mitochondrial transition pore openings may contribute to motor terminal degeneration.