Optimal neuromuscular performance requires motor neuron phosphagen kinases

Phosphagen systems are crucial for muscle bioenergetics by rapidly regenerating ATP to support the high metabolic demands of intense musculoskeletal activity. However their role in motor neurons (MNs) that drive muscle contraction has received little attention. Here we knocked down expression of the primary phosphagen kinase (arginine kinase 1 (ArgK1)) in Drosophila larval MNs and assessed the impact on presynaptic energy metabolism and neurotransmission in situ. Fluorescent metabolic probes showed a deficit in presynaptic energy metabolism and some glycolytic compensation. Glycolytic compensation was revealed through a faster elevation in lactate at high firing frequencies and the accumulation of pyruvate subsequent to firing. Our performance assays included two tests of endurance: enforced cycles of presynaptic calcium pumping and, separately, enforced body-wall contractions for extended periods. Neither test of endurance revealed deficits when ArgK1 was knocked down. The only performance deficits were detected at firing frequencies that approached, or exceeded, twice the firing frequencies recorded during fictive locomotion, where both electrophysiology and SynaptopHluorin imaging showed an inability to sustain neurotransmitter release. Our computational modelling of presynaptic bioenergetics indicates that the phosphagen system's contribution to MN performance is likely through the removal of ADP in microdomains close to sites of ATP hydrolysis rather than the provision of a deeper reservoir of ATP. Taken together these data demonstrate that, as in muscle fibres, MNs rely on phosphagen systems during activity that imposes intense energetic demands. KEY POINTS: Phosphagen systems are well characterized in muscle, but their role in neurons remains largely unexplored, despite evidence linking phosphagen deficits to behavioural and learning impairments. Knockdown of the primary phosphagen kinase in Drosophila larval motor neurons (MNs) caused deficits in exocytosis at the neuromuscular junction within 50 ms of burst firing. Endurance activities such as prolonged cycles of presynaptic Ca²⁺ pumping or body-wall contractions were unaffected by phosphagen kinase knockdown. Deficits in neurotransmission were consistent with our computational model, which predicts deficits in ATP levels and the ATP-to-ADP ratio only at high firing frequencies. The performance deficits we observe in phosphagen kinase-deficient Drosophila MNs phenocopy deficits in phosphagen kinase-deficient mouse muscle fibres, where tetanus contraction force is reduced but endurance remains intact.