The Ψ _m depolarization that accompanies mitochondrial Ca ²⁺ uptake is greater in mutant SOD1 than in wild-type mouse motor terminals

Abstract

The electrical gradient across the mitochondrial inner membrane (⌿ m) is established by electron transport chain (ETC) activity and permits mitochondrial Ca 2؉ sequestration. Using rhodamine-123, we determined how repetitive nerve stimulation (100 Hz) affects ⌿ m in motor terminals innervating mouse levator auris muscles. Stimulation-induced ⌿ m depolarizations in wild-type (WT) terminals were small (<5 mV at 30°C) and reversible. These depolarizations depended on Ca 2؉ influx into motor terminals, as they were inhibited when P/Q-type Ca 2ϩ channels were blocked with -agatoxin. Stimulation-induced ⌿m depolarization and elevation of cytosolic [Ca 2؉ ] both increased when complex I of the ETC was partially inhibited by low concentrations of rotenone (25-50 nmol/l). This finding is consistent with the hypothesis that acceleration of ETC proton extrusion normally limits the magnitude of ⌿ m depolarization during mitochondrial Ca 2؉ uptake, thereby permitting continued Ca 2؉ uptake. Compared with WT, stimulation-induced increases in rhodamine-123 fluorescence were Ϸ5 times larger in motor terminals from presymptomatic mice expressing mutations of human superoxide dismutase I (SOD1) that cause familial amyotrophic lateral sclerosis (SOD1-G85R, which lacks dismutase activity; SOD1-G93A, which retains dismutase activity). ⌿ m depolarizations were not significantly altered by expression of WT human SOD1 or knockout of SOD1 or by inhibiting opening of the mitochondrial permeability transition pore with cyclosporin A. We suggest that an early functional consequence of the association of SOD1-G85R or SOD1-G93A with motoneuronal mitochondria is reduced capacity of the ETC to limit Ca 2ϩ -induced ⌿m depolarization, and that this impairment contributes to disease progression in mutant SOD1 motor terminals.amyotrophic lateral sclerosis ͉ calcium ͉ mitochondria ͉ motor nerve terminals M itochondria sequester significant amounts of stimulationinduced Ca 2ϩ loads in many cell types (1-9). This mitochondrial Ca 2ϩ uptake occurs via a Ca 2ϩ uniporter/channel (10) down a potential gradient (⌿ m , 150-200 mV, matrix negative) established by ETC activity (11,12). Entry of Ca 2ϩ into mitochondria depolarizes ⌿ m , which would be expected to reduce the gradient driving Ca 2ϩ uptake. However, in motor nerve terminals, mitochondrial Ca 2ϩ uptake continues throughout prolonged trains of action potentials (13). One possible explanation for this apparent paradox is that the ⌿ m depolarization produced by Ca 2ϩ entry reduces the gradient against which ETC complexes I, III and IV extrude protons, thus accelerating ETC proton extrusion (14). This acceleration would limit the net ⌿ m depolarization, thereby allowing mitochondria to continue taking up Ca 2ϩ even during prolonged stimulation (15). We tested this hypothesis in mouse motor terminals, and found that the ⌿ m depolarizations produced by repetitive stimulation at 50-100 Hz were Ca 2ϩ dependent and reversible, and were small (or undetectable) at near-physiological temperatures (30°C). Par...

Mitochondrial dysfunction in familial amyotrophic lateral sclerosis

“…Consistent with this, mitochondrial depolarization has been observed in neuroblastoma cells expressing mutant SOD1 G93A (Carri et al 1997) or SOD1 G37R (Coussee et al 2011) and in motor neurons from mutant SOD1 G93A mice (Kruman et al 1999). Stimulationinduced mitochondrial membrane depolarizations in motor terminals of G93A and G85R transgenic mice were significantly enhanced and increased the likelihood of mitochondrial permeability transition pore opening during mitochondrial Ca 2+ uptake (Nguyen et al 2011;Nguyen et al 2009). …”

“…Impairment of intracellular Ca 2+ homeostasis has been reported in cells expressing mutant SOD1 G93A or SOD1 G37R (Carri et al 1997;Coussee et al 2011;Tradewell et al 2011) and in motor neurons from mutant SOD1 G93A transgenic mice Kruman et al 1999). Also isolated mitochondria from transgenic mutant SOD1 G93A and SOD1 G85R mice showed a significant decrease in Ca 2+ loading capacity (Damiano et al 2006;Nguyen et al 2009;Vila et al 2003). ATP deficit and Ca 2+ dysregulation may arise secondary to mutant SOD1-induced impairment of oxidative phosphorylation, or as a direct consequence of mutant SOD1 impeding on mitochondrial transport of ions and metabolites.…”

Molecular and Electrical Abnormalities in the Mouse Model of Amyotrophic Lateral Sclerosis

“…As these changes might suggest, there is a buildup of insoluble SOD1 proteins at this time (Johnston et al, 2000, Turner et al, 2003a, followed shortly by fragmentation of the Golgi (Mourelatos et al, 1996). The next signs of impairment appear in the mitochondria and in the cellular transport system (Zhang et al, 1997, Warita et al, 1999, Williamson and Cleveland, 1999, Mattiazzi et al, 2002, Kieran et al, 2005, Damiano et al, 2006, De Vos et al, 2007, Bilsland et al, 2008, Nguyen et al, 2009, Bilsland et al, 2010, Li et al, 2010. The immune response is initiated next (Alexianu et al, 2001, Chiu et al, 2008, Gowing et al, 2008, Chiu et al, 2009.…”

“…In motoneurons under normal conditions, the mitochondrial membrane potential powers both the Ca 2+ uniporter and ATP synthase, so in periods of heavy Ca 2+ influx, ATP production could be impaired (Mattson et al, 2008, Nguyen et al, 2009). The increased Ca 2+ influx in SOD1 motoneurons is likely to further impair the function of mitochondria under these conditions.…”

“…The increased Ca 2+ influx in SOD1 motoneurons is likely to further impair the function of mitochondria under these conditions. In addition, SOD1 mitochondria appear to be impaired in function under basal conditions (Mattiazzi et al, 2002, Nguyen et al, 2009, Li et al, 2010. Before the onset of symptoms, SOD1 mitochondria show decreased protein import, altered Ca 2+ sequestering, and an exaggerated response of the electrical gradient of the inner membrane to stimulation-induced Ca 2+ influx (Damiano et al, 2006, Bilsland et al, 2008, Nguyen et al, 2009, Li et al, 2010.…”