A mitochondrial tipping point couples early hyperexcitability to late-stage failure in patient-derived ALS motor neurons

Amyotrophic lateral sclerosis (ALS) is a motor neuron (MN) disease characterized by profound alterations in energy metabolism and progressive degeneration of MNs. Evidence from patients and model systems point to MN hyperexcitability as an early hallmark of ALS. How altered electrical activity intersects with energy metabolism, however, remains largely unexplored. To directly examine this relationship, we performed long-term longitudinal recordings of neuronal firing and mitochondrial function in patient-derived MNs harboring the pathogenic TDP-43 mutation A382T, together with their isogenic controls. A382T MNs displayed an early, transient phase of hyperexcitability peaking around 35 days in culture, which was also observed in MNs carrying a different TDP-43 disease variant (M337V). This hyperexcitable phase coincided with elevated mitochondrial function (hyperpolarized membrane potential and accelerated electron flow across respiratory complexes) and was ultra-sensitive to mild Fo/F1 ATPase inhibition, revealing near maximal mitochondrial output to meet increased energy demands. This phase was followed by a sharp decline in A382T MN firing frequency, mitochondrial depolarization, and the loss of approximately 50% of firing-competent neurons. Acute bidirectional manipulations of MN firing rates elicited homeostatic mitochondrial responses, further demonstrating tight coupling between neuronal activity and mitochondrial metabolism. Together, these findings uncover a pathological trajectory in which early hyperexcitability drives mitochondrial hypermetabolism, with progressive erosion of mitochondrial capacity, ultimately leading to late-stage MN failure.