In mammalian CNS neurons, the majority of axonal mitochondria lack mitochondrial DNA and associated transcripts and proteins, originating from the soma in this state, and function metabolically in reverse mode by hydrolyzing ATP through Complex V to maintain membrane potential rather than producing ATP through oxidative phosphorylation, while dendritic mitochondria in the same neurons contain abundant mtDNA and actively generate ATP.

In neurons of the mammalian central nervous system (CNS), axonal mitochondria are thought to be indispensable for supplying ATP during energy-consuming processes such as neurotransmitter release. We have previously shown that glutamatergic excitatory cortical pyramidal neurons (CPNs) are characterized by a striking compartmentalization of mitochondrial structure. Axonal mitochondria are maintained at a small uniform size (∼1 microns length) by high levels of MFF-dependent fission, whereas dendritic mitochondria form a highly fused network of mitochondria throughout the entire dendritic arbor. Here, we tested whether these striking structural differences reflect functional and molecular compartmentalization. Using four independent techniques, we reveal that in CPNs as well as in two subtypes of GABAergic inhibitory cortical interneurons and neuromodulatory dopaminergic neurons of the substantia nigra, the majority of axonal, but not dendritic, mitochondria lack mitochondrial DNA (mtDNA) in vivo. We also show that axonal mitochondria are depleted in both mtDNA-encoded mRNAs and proteins compared to dendritic mitochondria. Timelapse imaging establishes that most mitochondria entering nascent dendritic and axonal processes from the soma are small and lack mtDNA. Using dynamic, optical imaging of genetically encoded sensors for ATP and pH targeted to the mitochondrial matrix, we demonstrate that in axons of CPNs, but not in their dendrites, mitochondrial F1F0-ATP synthase (Complex V) functions in a reverse way, hydrolyzing ATP and extruding H+ out of the matrix to maintain mitochondrial membrane potential. Our results indicate that in neurons of the mammalian CNS, the majority of axonal mitochondria lack mtDNA and likely do not play a major role in ATP generation, despite playing other functions such as regulation of neurotransmission via presynaptic Ca2+ buffering.