Mitochondrial phosphagen kinases support the volatile power demands of motor nerve terminals.

Motor neurons are the longest neurons in the body, with axon terminals separated from the soma by as much as a meter. These terminals are largely autonomous with regard to their bioenergetic metabolism and must burn energy at a high rate to sustain muscle contraction. Here, through computer simulation and drawing on previously published empirical data, we determined that motor neuron terminals in Drosophila larvae experience highly volatile power demands. It might not be surprising then, that we discovered the mitochondria in the motor neuron terminals of both Drosophila and mice to be heavily decorated with phosphagen kinases - a key element in an energy storage and buffering system well-characterized in fast-twitch muscle fibres. Knockdown of arginine kinase 1 (ArgK1) in Drosophila larval motor neurons led to several bioenergetic deficits, including mitochondrial matrix acidification and a faster decline in the cytosol ATP to ADP ratio during axon burst firing. KEY POINTS: Neurons commonly fire in bursts imposing highly volatile demands on the bioenergetic machinery that generates ATP. Using a computational approach, we built profiles of presynaptic power demand at the level of single action potentials, as well as the transition from rest to sustained activity. Phosphagen systems are known to buffer ATP levels in muscles and we demonstrate that phosphagen kinases, which support such phosphagen systems, also localize to mitochondria in motor nerve terminals of fruit flies and mice. By knocking down phosphagen kinases in fruit fly motor nerve terminals, and using fluorescent reporters of the ATP:ADP ratio, lactate, pH and Ca2+ , we demonstrate a role for phosphagen kinases in stabilizing presynaptic ATP levels. These data indicate that the maintenance of phosphagen systems in motor neurons, and not just muscle, could be a beneficial initiative in sustaining musculoskeletal health and performance.

The Krebs Cycle Enzyme Isocitrate Dehydrogenase 3A Couples Mitochondrial Metabolism to Synaptic Transmission.

Neurotransmission is a tightly regulated Ca2+-dependent process. Upon Ca2+ influx, Synaptotagmin1 (Syt1) promotes fusion of synaptic vesicles (SVs) with the plasma membrane. This requires regulation at multiple levels, but the role of metabolites in SV release is unclear. Here, we uncover a role for isocitrate dehydrogenase 3a (idh3a), a Krebs cycle enzyme, in neurotransmission. Loss of idh3a leads to a reduction of the metabolite, alpha-ketoglutarate (αKG), causing defects in synaptic transmission similar to the loss of syt1. Supplementing idh3a flies with αKG suppresses these defects through an ATP or neurotransmitter-independent mechanism. Indeed, αKG, but not glutamate, enhances Syt1-dependent fusion in a reconstitution assay. αKG promotes interaction between the C2-domains of Syt1 and phospholipids. The data reveal conserved metabolic regulation of synaptic transmission via αKG. Our studies provide a synaptic role for αKG, a metabolite that has been proposed as a treatment for aging and neurodegenerative disorders.

Expression of multiple transgenes from a single construct using viral 2A peptides in Drosophila.

Expression of multiple reporter or effector transgenes in the same cell from a single construct is increasingly necessary in various experimental paradigms. The discovery of short, virus-derived peptide sequences that mediate a ribosome-skipping event enables generation of multiple separate peptide products from one mRNA. Here we describe methods and vectors to facilitate easy production of polycistronic-like sequences utilizing these 2A peptides tailored for expression in Drosophila both in vitro and in vivo. We tested the separation efficiency of different viral 2A peptides in cultured Drosophila cells and in vivo and found that the 2A peptides from porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A) worked best. To demonstrate the utility of this approach, we used the P2A peptide to co-express the red fluorescent protein tdTomato and the genetically-encoded calcium indicator GCaMP5G in larval motorneurons. This technique enabled ratiometric calcium imaging with motion correction allowing us to record synaptic activity at the neuromuscular junction in an intact larval preparation through the cuticle. The tools presented here should greatly facilitate the generation of 2A peptide-mediated expression of multiple transgenes in Drosophila.

Presynaptic mitochondria in functionally different motor neurons exhibit similar affinities for Ca2+ but exert little influence as Ca2+ buffers at nerve firing rates in situ.

Mitochondria accumulate within nerve terminals and support synaptic function, most notably through ATP production. They can also sequester Ca(2+) during nerve stimulation, but it is unknown whether this limits presynaptic Ca(2+) levels at physiological nerve firing rates. Similarly, it is unclear whether mitochondrial Ca(2+) sequestration differs between functionally different nerve terminals. We addressed these questions using a combination of synthetic and genetically encoded Ca(2+) indicators to examine cytosolic and mitochondrial Ca(2+) levels in presynaptic terminals of tonic (MN13-Ib) and phasic (MNSNb/d-Is) motor neurons in Drosophila, which, as we determined, fire during fictive locomotion at approximately 42 Hz and approximately 8 Hz, respectively. Mitochondrial Ca(2+) sequestration starts in both terminals at approximately 250 nM, exhibits a similar Ca(2+)-uptake affinity (approximately 410 nM), and does not require Ca(2+) release from the endoplasmic reticulum. Nonetheless, mitochondrial Ca(2+) uptake in type Is terminals is more responsive to low-frequency nerve stimulation and this is due to higher cytosolic Ca(2+) levels. Since type Ib terminals have a higher mitochondrial density than Is terminals, it seemed possible that greater mitochondrial Ca(2+) sequestration may be responsible for the lower cytosolic Ca(2+) levels in Ib terminals. However, genetic and pharmacological manipulations of mitochondrial Ca(2+) uptake did not significantly alter nerve-stimulated elevations in cytosolic Ca(2+) levels in either terminal type within physiologically relevant rates of stimulation. Our findings indicate that presynaptic mitochondria have a similar affinity for Ca(2+) in functionally different nerve terminals, but do not limit cytosolic Ca(2+) levels within the range of motor neuron firing rates in situ.

AP180 maintains the distribution of synaptic and vesicle proteins in the nerve terminal and indirectly regulates the efficacy of Ca2+-triggered exocytosis.

AP180 plays an important role in clathrin-mediated endocytosis of synaptic vesicles (SVs) and has also been implicated in retrieving SV proteins. In Drosophila, deletion of its homologue, Like-AP180 (LAP), has been shown to increase the size of SVs but decrease the number of SVs and transmitter release. However, it remains elusive whether a reduction in the total vesicle pool directly affects transmitter release. Further, it is unknown whether the lap mutation also affects vesicle protein retrieval and synaptic protein localization and, if so, how it might affect exocytosis. Using a combination of electrophysiology, optical imaging, electron microscopy, and immunocytochemistry, we have further characterized the lap mutant and hereby show that LAP plays additional roles in maintaining both normal synaptic transmission and protein distribution at synapses. While increasing the rate of spontaneous vesicle fusion, the lap mutation dramatically reduces impulse-evoked transmitter release at steps downstream of calcium entry and vesicle docking. Notably, lap mutations disrupt calcium coupling to exocytosis and reduce calcium cooperativity. These results suggest a primary defect in calcium sensors on the vesicles or on the release machinery. Consistent with this hypothesis, three vesicle proteins critical for calcium-mediated exocytosis, synaptotagmin I, cysteine-string protein, and neuronal synaptobrevin, are all mislocalized to the extrasynaptic axonal regions along with Dap160, an active zone marker (nc82), and glutamate receptors in the mutant. These results suggest that AP180 is required for either recycling vesicle proteins and/or maintaining the distribution of both vesicle and synaptic proteins in the nerve terminal.