Steroid-triggered programmed cell death of a motoneuron is autophagic and involves structural changes in mitochondria.

Neuronal death occurs during normal development and disease and can be regulated by steroid hormones. In the hawkmoth, Manduca sexta, individual accessory planta retractor (APR) motoneurons undergo a segment-specific pattern of programmed cell death (PCD) at pupation that is triggered directly and cell autonomously by the steroid hormone 20-hydroxyecdysone (20E). APRs from abdominal segment six [APR(6)s] die by 48 hours after pupal ecdysis (PE; entry into the pupal stage), whereas APR(4)s survive until adulthood. Cell culture experiments showed previously that 20E acts directly on APRs to trigger PCD, with intrinsic segmental identity determining which APRs die. The APR(6) death pathway includes caspase activation and loss of mitochondrial function. We used transmission electron microscopy to investigate the ultrastructure of APR somata before and during PCD. APR(4)s showed normal ultrastructure at all stages examined, as did APR(6)s until approximately stage PE. During APR(6) death, there was massive accumulation of autophagic bodies and vacuoles, mitochondria became ultracondensed and aggregated into compact clusters, and ribosomes aggregated in large blocks. Nuclear ultrastructure remained normal, without chromatin condensation, until the nuclear envelope fragmented late in the death process. Light microscopic immunocytochemistry showed that dying APR(6)s were TUNEL-positive, which is diagnostic of fragmented DNA. These observations indicate that the steroid-induced, caspase-dependent, cell-autonomous PCD of APR(6)s is autophagic, not apoptotic, and support an early role for mitochondrial alterations during PCD. This system permits the study of neuronal death in response to its bona fide developmental signal, the rise in a steroid hormone.