Huntingtin Associated Protein 1 Is Required For The Endocytosis And Retrograde Trafficking Of BDNF And P75NTR

Objective This project aims to elucidate whether Huntingtin associated protein 1(HAP1) plays a role in the endocytosis and retrograde transport of BDNF and its receptors,which may offer a way to investigate the molecular biology mechanism of Huntington disease.Methods 1.Plasmids were constructed firstly.PC12 cells were transfected with plasmids,or followed by triple labeling with an immunofluorescence method.Co-localization was analyzed and quantified with confocal imaging software:1) Plasmids of HAP1A-CPF,BDNF-DsRed,pro-BDNF-DsRed and pre-BDNF-DsRed were constructed.The biotin labeled BDNF, pro-BDNF,pre-BDNF,and medium containing DsRed labeled mature BDNF,were prepared.2) We have co-transfected PC12 cells with HAP1A-CPF and BDNF-DsRed,and incubated cells with the conditioning medium containing exogenous DsRed labeled mature BDNF,in the absence,or presence,of the antibodies to mature BDNF or to p75NTR extracellular domain.Confocal images were taken and merged.The same methods were used with pro-BDNF and pre-BDNF.3) PC12 cells were used to co-transfected with the plasmid HAP1-A-CFP and p75NTR-YFP.After co-transfection,differentiated PC12 cells were incubated in the presence or absence of DsRed-labeled BDNF conditioning medium to allow internalization,followed by immunofluorescence confocal imaging.4) We compared the intracellular distribution of p75NTR and HAP1 in differentiated PC12 cells which were incubated with biotin labeled BDNF,followed by triple labeling with an immunofluorescence method. Co-localization was analyzed and quantified with confocal imaging software.2.Cortical neurons from transgenic mice were incubated with biotin-labeled BDNF,followed by triple labeling with an immunofluorescence confocal imaging,or analyzed by Western blot,in order to elucidate whether HAP1 plays a role in the endocytosis and retrograde transport of BDNF:1) Cortical neurons from HAP1^+/+,HAP1^+/-,and HAP1^-/- from mice at postnatal day 1 were cultured.Then applied biotin labeled BDNF to trigger endocytosis,followed by immunostaining experiments and confocal imaging.2) We transfected the HAP1^-/- neurons with HAP1-CFP plasmids and incubated with incubated with biotin-labeled BDNF,followed by immunostaining experiments and confocal imaging.3) Cortical neurons were incubated with biotin labeled BDNF for 1 hour,lysed and analyzed by Western blot. 3.The p75NTRs on the surface of cultured cortical neurons from transgenic mice were biotin labeled,followed by incubation,precipitation and Western-Blot;the apFRET technology was used to see the fluorescence resonance energy transfer between HAP1 A-CFP and p75NTR-YFP;finally,a Co-immunoprecipitation assay was done to elucidate whether and how HAP1 plays a role in the endocytosis and retrograde transport of BDNF receptors-p75NTR:1) The p75NTRs on the surface of cultured cortical neurons from HAP1^+/+ and HAP1^-/- neonatal mice were biotin labeled using a reversible, membrane-impermeable cross-linker(sulfo-NHS-S-S-biotin).Then we incubated neurons with BDNF(25nM) or not for 1 hour.The cell surface biotin was then cleaved from the remaining surface- exposed receptors as described,and the neurons were lysed in RIPA buffer.Equal amounts of protein lysates containing internalized biotinylated receptors were subjected to precipitation with streptavidin- agarose.Immunoblotting was performed with p75 antibodies.2) The apFRET technology was used to see the fluorescence resonance energy transfer between HAP1 A-CFP and p75NTR-YFP, which were expressed by co- transfected PC12 cells with plasmids of HAP1A-CFP and p75NTR-YFP,by BDNF stimulation.3) A Co-immunoprecipitation assay was done using HEK293 cell lysate which co-expressed HAP1A-CFP and p75NTR-YFP by co-transfection,followed by precipitation with p75NTR antibody and protein A Sepharose and immunoblotting.Results 1.Internalized BDNF in PC12 cells is highly co-localized with HAP1 but the co-localization was completely abolished by the antibodies to BDNF or to p75NTR.2.Internalized pro-BDNF in PC12 cells is highly co-localized with HAP1 but the co-localization was completely abolished by the antibodies to BDNF,and partly to p75NTR or sortilin.3.Internalized pre-BDNF in PC12 cells is highly co-localized with HAP1 but the co-localization was completely abolished by the antibodies to BDNF or to sortilin,not to p75NTR.4.The co-localization of the HAP1A-CFP with p75NTR-YFP,which were expressed by co-transfected PC12 cells with plasmids of HAP1A-CFP and p75NTR- YFP,was found and the co-localization was increased by BDNF stimulation.5.Substantial co-localization of the intracellular distribution of HAP1 with p75NTR was found in differentiated PC12 cells,and the co-localization was increased by BDNF stimulation.6.Upon incubation of cortical neurons with exogenous labeled BDNF,the labeled factor was detected on the cell surface,and within neurites and cell bodies,in most HAP1^+/+ and HAP1^+/-,but not in HAP1^-/- neurons,suggesting HAP1 is required for BDNF endocytosis and intracellular trafficking.7.Moreover,the intensity of internalized BDNF was attenuated in HAP1^+/- neurons compared with WT neurons.8.The defect in BDNF endocytosis in HAP1^-/- neurons could be rescued by HAP1 plasmid transfection.9.A Western blot assay showed endocytosed BDNF at 14 kDa was detected in HAP1^+/+,but not in HAP1^-/- neurons.10.Incubation of cortical neurons with sulfo-NHS-S-S-biotin,a reversible membrane-impermeable cross-linker of biotin,followed by precipitation and immunoblotting,showed that the endocytosed p75NTR were abolished in HAP1^-/- mice,suggesting HAP1 is significant for the signaling of p75NTR in the endocytosis of BDNF in cortical neurons.11.The FRET technology suggested that there were fluorescence resonance energy transfer between HAP1 A-CFP and p75NTR-YFP, which were expressed by co-transfected PC12 cells with plasmids of HAP1A-CFP and p75NTR-YFP,by BDNF stimulation,suggesting that HAP1 may interact with p75NTR.12.A Co-immunoprecipitation assay showed that HAP1 A-CFP at about 100Kd was detected by precipitation with p75NTR antibody and protein A Sepharose using HEK293 cell lysate which co-expressed HAP1A-CFP and p75NTR-YFP by co-transfection,and immunoblotting. Results verify that HAP1 could binds with p75NTR in the endocytosis and retrograde transport of BDNF.Conclusions We found that HAP1 is an essential molecule required for the endocytosis and trafficking of BDNF and its associated receptor p75NTR.The mechanism underlying the effect is that HAP1 could binds with p75NTR in the endocytosis and retrograde transport of BDNF.