HAP1 Is An In Vivo UBE3A Target That Augments Autophagy In A Mouse Model Of Angelman Syndrome

Angelman syndrome(AS)is a severe neurodevelopmental disorder caused by maternal mutation and paternal imprinting of the gene encoding UBE3 A,an E3 ubiquitin ligase.Although several potential target proteins of UBE3 A have been reported,how these proteins regulate neuronal development remains unclear.We performed a large-scale quantitative proteomic analysis using stable-isotope labeling of amino acids in mammals(SILAM)in mice with maternal Ube3 a mutation.Among the up-reg?lated potential substrates,we identified huntingtin(Htt)-associated protein(HAP1),a protein that is involved in Huntington's disease(HD),as a new in vivo target of UBE3 A.Stability and ubiquitination experiments for HAP1 were performed for substrate confirmation.Mass spectrometry analysis of HAP1 modifications further identified the ubiquitination sites regulated by UBE3 A.Remarkably,we observed the accelerated autophagy existed both in AS mouse and cortical neuron,while m TOR1 activity was not inhibited.Further,we demonstrate that HAP1 acts as a critical reg?lator of the Ptd Ins3 K complex to promote autophagy by interacting with ATG14,and reveal that the coiled coil domain of HAP1 is sufficient for the interaction.What's more,HAP1 participates in the formation of ATG14-VPS34-Beclin1 complex to a larger one in HEK293 T cells and mouse brain lysate.The increased activity of Ptd Ins3 K was mitigated by knocking down the expression of HAP1 in AS primary cortical neuron.HAP1 is co-localized with DFCP1,and this distribution pattern coincides with Ptd Ins3 P targeting to isolation membranes and promotion the nucleation of autophagosomes.MAP1LC3(LC3),as the mature autophagosome marker,is also co-expressed with HAP1 in the form of puncta,suggesting a role of HAP1 in mediating the autophagosome formation.In the final step,HAP1 targets Stx17 to enhance the fusion between autophagosome and lysosome.As a consequence,the autophagic flux is accelerated upon overexpressed HAP1 by monitoring the tandem RFP-GFP-LC3 expression signal,and glucose starvation treatment further enhances the flux progress.Strikingly,knocking down of HAP1 alleviated aberrant autophagy in primary neurons from AS mice.Concordantly,treatment of AS neurons with an autophagy inhibitor alleviated the reduction in density of dendritic spines.Together,our results identify HAP1 as an in vivo UBE3 A target that contributes to deregulated autophagy and synaptic dysfunction in the central nervous system of AS mouse.