Mushtaq, Z.; Lasser, D. A.; Lion, L. M.; Ebding, J.; Escribano, B.; Burkhalter, D.; Moreno, E.; Maritzen, T.; Stephan, R.; Pielage, J.

Progressive, axon length-dependent degeneration of nerve terminals is a defining feature of dying-back neuropathies; yet, whether defects in axonal transport initiate or are a consequence of this process remains unresolved. Here, we show that NudE, a scaffold for dynein motor activation, is required for the initiation of retrograde axonal transport at Drosophila motoneuron synapses in vivo. Loss of NudE impairs dynein activation at the synaptic terminal, severely reducing the proportion of cargo entering retrograde transport and decreasing retrograde motor velocity. Live imaging and temporal analysis establish that this transport initiation defect is the earliest event in a degenerative cascade, followed by progressive microtubule destabilization, impaired synaptic transmission, and structural degeneration in a distal-to-proximal gradient that recapitulates dying-back neuropathies. Structure-function analysis validates the biochemically defined NudE-dynein binding domains in vivo, with graded disruption producing correspondingly graded phenotypes. Genetic two-hit experiments uncover a reciprocal dependence between transport initiation and microtubule maintenance: perturbations in either process that are individually tolerated synergistically trigger degeneration when combined, while increasing microtubule levels alone cannot compensate for failed dynein activation. These findings reveal retrograde transport initiation as a critical vulnerability point in motoneurons and provide a mechanistic basis for how defects in dynein activation produce progressive neurodegeneration.