The Role Of Autophagy During Neurite Degeneration And The Dynamics Of Autolysosomes In Neurites

Axon and dendrite (or neurite) degeneration plays a critical role during development of the nervous system, which is recognized as a self-destructive programme and distinct from somata apoptotic death. It occurs commonly in a wide range of neurodegenerative disorders. Neurite degeneration often precedes and sometimes leads to the death of cell soma, and may make a more important contribution to the patient's disability. Nevertheless, the mechanisms of neurite degeneration are poorly known.Autophagy is a well-characterized catabolic mechanism whereby cells degrade proteins and recycle cytoplasmic components and intracellular organelles in response to nutrient starvation. Accumulating evidence shows that autophagy has been linked to various human neurodegenerative diseases, while the existence of autophagy as a prodeath or prosurvival pathway is controversial and the mechanism by which autophagy programmes neurites to die is still unclear.We here investigated the involvement of the autophagic process in neurite degeneration induced by different experimental paradigms in mouse superior cervical ganglion (SCG) neurons. Our present study revealed the induction of autophagy in degenerating neurites of sympathetic neuron initiated by three different experimental paradigms, including in vitro Wallerian degeneration, nerve growth factor (NGF) deprivation and microtubule disruption. Autophagosomes/autolysosomes colocalized with collapsed cytoskeletal proteins in neuritic beadings during degeneration. Upregulation of microtubule-associated protein light chain 3-Ⅱ(LC3-Ⅱ), which is the most reliable marker for autophagy, was observed during the early stage of neurite degeneration. The autophagy inhibitor 3-Methyladenine (3-MA) efficiently suppressed neurite degeneration by protecting neurites from the loss of viability and mitochondrial function. Furthermore, knocking down the key autophagy-related gene atg7 or beclinl by RNA interference significantly delayed axonal and dendritic degeneration after NGF deprivation. Reduced expression of Atg7 also suppressed neurite fragmentation after transection.The accumulation of autolysosomes was also observed in neurites of PC12 cells after serum deprivation. In addition, fluorescence recovery after photobleaching (FRAP) technique showed the monomeric red fluorescence protein (mRFP)-LC3-labeled autolysosomes were motile in living cells. Real-time fluorescence imaging of serum-deprived PC 12 cells further demonstrated that autolysosomes moved along neurites in both anterograde and retrograde directions. They paused, re-started, and sometimes changed directions. By using image processing, quantitative analysis was made to show the dynamic biophysical characteristics of these vesicles. The speed of autolysosomes varied in size, as the movement of larger autolysosomes was relatively slow. Those small autolysosomes traveled along neurites in anterograde and retrograde directions rapidly, with an average velocity of approximately 0.33μm/s and 0.39μm/s respectively. The maximal speeds of anterograde and retrograde transport were 1.22μm/s and 1.51μm/s, and the maximal displacement of long-range moving autolysosomes we traced was longer than 50μm in 2 min. Disruption of microtubules by nocodazole completely abolished their movements, suggesting the neuritic transport of autolysosomes depends on microtubules. The directional transport of autolysosomes was also particularly affected by application of dynein or kinesin inhibitor.Collectively, our present study achieved the following novel findings:1) Induction of autophagy occurred during the early stage of neurite degeneration; 2) Application of autophagy inhibitor or knocking down the autophagy-related genes atg7 or beclinl significantly delayed neurite degeneration; 3) By using live-cell fluorescent imaging system, we found that autolysosomes moved along PC 12 neurites in both anterograde and retrograde directions. The highly dynamic movement of autolysosomes was traced and their biophysical characteristics were analyzed by image processing.4) Fluorescent double-label studies showed autolysosomes depended on microtubules for neuritic transport. Molecular motors kinesin and dynein regulated the anterograde and retrograde transport of autolysosomes respectively. Combined use of molecular and cellular biology, live cell imaging techniques and computer image processing allowed us to demonstrate the effect of autophagy during neurite degeneration and systematically describe the dynamics of autolysosomes in neurites for the first time. These results suggest the critical role of autophagy in neurite degeneration and may provide a valuable clue in understanding the mechanism of axonal and dendritic degeneration, which is pivotal for the development of new therapeutic approaches.