Preliminary Study On The Role Of RAB39B In Regulating Autophagy

Parkinson's disease(PD),the second largest progressive neurodegenerative disease after Alzheimer's disease,is characterized by behavioral and cognitive disorders.At present,the recognized pathological features are the degeneration and death of dopaminergic neurons and the occurrence of Lewy body pathology in the brain.Through the application of genome-wide association study(GWA S),second-generation sequencing and other technologies,novel PD risk gene mutations have been constantly identified,including mutations in the RAB39B gene.In addition to PD,RAB39B has also been linked to neurologic diseases such as autism,epilepsy,macrocephaly and X-linked mental disorders.RAB39B,like other members of the RABs,is a kind of small GTPase that may be involved in vesicular traffiking and regulation of autophagy.Nevertheless,the physiological and pathological functions of RAB39B are poorly understood.In this study,we found that both 7-month and 10-month old Rab39b knockout(KO)mice showed deficient motor balance skills during rota-rod tests when compared to wild type(WT)mice.In addition,10-month old Rab39b KO mice showed worsen motor balance skills than 7-month old Rab39b KO mice.By western blotting analysis,we found that the LC3B-? level that is a maj or autophagy marker,was markedly increased in the cortex and hippocampus of 2-month old Rab39b KO mice when compared to those of WT mice.Consistently,LC3B-II levels were increased in cultured primary neurons from Rab39b KO mice.When treated with the autophagy stimulator rapamycin,we found that LC3B-? levels were dramatically increased in WT primary neurons but not in Rab39b KO neurons.We also transfect the mRFP-GFP-LC3B plasmid into cultured neurons,and found that the number of yellow dots and red dots was markedly increased in Rab39b KO neurons when compared to those in WT neurons.Moreover,transmission electron microscope studies revealed trends of increases in autophagolysosome numbers in both the cortex and hippocampus of Rab39b KO mice when compared to those of WT mice.Together,these results indicate that RAB39B may be involved in rapamycin-induced autophagy and that loss of RAB39B may promote the formation of autophagolysosome.Finally,we found that both the length and width of post-synaptic density(PSD)in the cortex of Rab39b KO mice were decreased when compared to those of WT mice.This result implies that loss of RAB39B may alter synapse structures and compromise synaptic functions.In conclusion,our findings elucidate the participation of RAB39B in autophagy and synapse structures,suggesting that RAB39B loss-of function mutations may lead to PD pathogenesis through disturbing autophagy and synaptic functions.This study not only expands our understanding on the physiological function of RAB39B,but also provides important clues for the pathological mechanism underlying PD and its drug development strategies.