Mechanisms that regulate cellular metabolism are a fundamental requirement of all cells. Most eukaryotic cells rely on aerobic mitochondrial metabolism to generate ATP. Nevertheless, regulation of mitochondrial activity is incompletely understood. Here we identified an unexpected and essential role for constitutive InsP3R-mediated Ca2+ release in maintaining cellular bioenergetics. Macroautophagy provides eukaryotes with an adaptive response to nutrient deprivation that prolongs survival. Constitutive InsP3R Ca2+ signaling is required for macroautophagy suppression in cells in nutrient-replete media. In its absence, cells become metabolically compromised due to diminished mitochondrial Ca2+ uptake. Mitochondrial uptake of InsP3R released Ca2+ is fundamentally required to provide optimal bioenergetics by providing sufficient reducing equivalents to support oxidative phosphorylation. Absence of this Ca2+ transfer results in enhanced phosphorylation of pyruvate dehydrogenase and activation of AMPK, which activates pro-survival macroautophagy. Thus, constitutive InsP3R Ca2+ release to mitochondria is an essential cellular process that is required for efficient mitochondrial respiration and maintenance of normal cell bioenergetics.

SUMMARY Mitochondrial Ca2+ (Ca2+m) uptake is mediated by an inner membrane Ca2+ channel called the uniporter. Ca2+ uptake is driven by the considerable voltage present across the inner membrane (ΔΨm) generated by proton pumping by the respiratory chain. Mitochondrial matrix Ca2+ concentration is maintained 5–6 orders of magnitude lower than its equilibrium level, but the molecular mechanisms for how this is achieved are not clear. Here we demonstrate that the mitochondrial protein MICU1 is required to preserve normal [Ca2+]m under basal conditions. In its absence, mitochondria become constitutively loaded with Ca2+, triggering excessive reactive oxygen species generation and sensitivity to apoptotic stress. MICU1 interacts with the uniporter pore-forming subunit MCU and sets a Ca2+ threshold for Ca2+m uptake without affecting the kinetic properties of MCU-mediated Ca2+ uptake. Thus, MICU1 is a gatekeeper of MCU-mediated Ca2+m uptake that is essential to prevent [Ca2+]m overload and associated stress.

Blockade of acetylcholine release by botulinum neurotoxin type A at the neuromuscular junction induces the formation of an extensive network of nerve-terminal sprouts. By repeated in vivo imaging of N-(3-triethyl ammonium propyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide uptake into identified nerve endings of the mouse sternomastoid muscle after a single intramuscular injection of the toxin, inhibition of stimulated uptake of the dye at the terminals was detected within a few days, together with an increase in staining of the newly formed sprouts. After 28 days, when nerve stimulation again elicited muscle contraction, regulated vesicle recycling occurred only in the sprouts [shown to contain certain soluble N-ethylmaleimide-sensitive factor attachment proteins (SNAREs) and to abut acetylcholine receptors] and not at the parent terminals. Therefore, only these sprouts could be responsible for nerve-muscle transmission at this time. However, a second, distinct phase of the rehabilitation process followed with a return of vesicle turnover to the original terminals, accompanied by an elimination of the by then superf luous sprouts. This extension and later removal of ''functional'' sprouts indicate their fundamental importance in the repair of paralyzed endplates, a finding with ramifications for the vital process of nerve regeneration.The ability of nerve endings at the neuromuscular junction to sprout after the potent blockade of neurotransmission by botulinum neurotoxin type A (BoTx͞A) is a striking example of synaptic plasticity (1-4). Although significant progress already has been made in identifying the factors triggering such outgrowth (see ref. 5), as well as that induced on, for example, partial denervation by nerve crush (1, 6), the significance of endplate remodeling in the intricate series of interactions between presynaptic and postsynaptic cells during recovery of transmission at paralyzed synapses has yet to be shown. The selective blockade of the regulated exocytosis of acetylcholine (ACh) by BoTx͞A after its intracellular proteolytic cleavage of synaptosomal-associated protein with a molecular mass of 25 kDa ref. 7 and 8) causes an unique and long-term eradication of synaptic activity, while, advantageously, avoiding removal of the nerve endings (9, 10). Despite the extent of the paralysis induced by this toxin, recovery of neurotransmission does occur eventually, as manifested in animal experiments (2, 11-13) and in the clinical treatment of dystonias involving involuntary movement of certain skeletal muscles (see ref. 14). Although BoTx͞A is known to promote nerve sprouting, it has remained unclear what the precise contribution, if any, these newly formed outgrowths make to the recovery from this initially dramatic, but ultimately temporary, paralysis.Limitations encountered in earlier studies on this fundamentally important question originated from the need to employ conventional histological techniques in vitro to excised tissues. These limitations have been overcome in our stu...