Bin1 and CD 2 AP polarise the endocytic generation of beta‐amyloid
2016; Springer Nature; Volume: 18; Issue: 1 Linguagem: Inglês
10.15252/embr.201642738
ISSN1469-3178
AutoresFlorent Ubelmann, Tatiana Burrinha, Laura Salavessa, Ricardo A. Gomes, Cláudio Ferreira, Nuno Moreno, Cláudia G. Almeida,
Tópico(s)Cellular transport and secretion
ResumoArticle28 November 2016free access Source DataTransparent process Bin1 and CD2AP polarise the endocytic generation of beta-amyloid Florent Ubelmann Florent Ubelmann Neuronal Trafficking in Aging Lab, CEDOC, Chronic Diseases Research Centre, NOVA Medical School/Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, Lisboa, Portugal Search for more papers by this author Tatiana Burrinha Tatiana Burrinha Neuronal Trafficking in Aging Lab, CEDOC, Chronic Diseases Research Centre, NOVA Medical School/Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, Lisboa, Portugal Search for more papers by this author Laura Salavessa Laura Salavessa Neuronal Trafficking in Aging Lab, CEDOC, Chronic Diseases Research Centre, NOVA Medical School/Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, Lisboa, Portugal Search for more papers by this author Ricardo Gomes Ricardo Gomes Neuronal Trafficking in Aging Lab, CEDOC, Chronic Diseases Research Centre, NOVA Medical School/Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, Lisboa, Portugal Search for more papers by this author Cláudio Ferreira Cláudio Ferreira Neuronal Trafficking in Aging Lab, CEDOC, Chronic Diseases Research Centre, NOVA Medical School/Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, Lisboa, Portugal Search for more papers by this author Nuno Moreno Nuno Moreno Advance Imaging Lab, Instituto Gulbenkian de Ciência, Oeiras, Portugal Search for more papers by this author Cláudia Guimas Almeida Corresponding Author Cláudia Guimas Almeida [email protected] orcid.org/0000-0001-9384-2896 Neuronal Trafficking in Aging Lab, CEDOC, Chronic Diseases Research Centre, NOVA Medical School/Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, Lisboa, Portugal Search for more papers by this author Florent Ubelmann Florent Ubelmann Neuronal Trafficking in Aging Lab, CEDOC, Chronic Diseases Research Centre, NOVA Medical School/Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, Lisboa, Portugal Search for more papers by this author Tatiana Burrinha Tatiana Burrinha Neuronal Trafficking in Aging Lab, CEDOC, Chronic Diseases Research Centre, NOVA Medical School/Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, Lisboa, Portugal Search for more papers by this author Laura Salavessa Laura Salavessa Neuronal Trafficking in Aging Lab, CEDOC, Chronic Diseases Research Centre, NOVA Medical School/Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, Lisboa, Portugal Search for more papers by this author Ricardo Gomes Ricardo Gomes Neuronal Trafficking in Aging Lab, CEDOC, Chronic Diseases Research Centre, NOVA Medical School/Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, Lisboa, Portugal Search for more papers by this author Cláudio Ferreira Cláudio Ferreira Neuronal Trafficking in Aging Lab, CEDOC, Chronic Diseases Research Centre, NOVA Medical School/Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, Lisboa, Portugal Search for more papers by this author Nuno Moreno Nuno Moreno Advance Imaging Lab, Instituto Gulbenkian de Ciência, Oeiras, Portugal Search for more papers by this author Cláudia Guimas Almeida Corresponding Author Cláudia Guimas Almeida [email protected] orcid.org/0000-0001-9384-2896 Neuronal Trafficking in Aging Lab, CEDOC, Chronic Diseases Research Centre, NOVA Medical School/Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, Lisboa, Portugal Search for more papers by this author Author Information Florent Ubelmann1, Tatiana Burrinha1, Laura Salavessa1, Ricardo Gomes1, Cláudio Ferreira1, Nuno Moreno2 and Cláudia Guimas Almeida *,1 1Neuronal Trafficking in Aging Lab, CEDOC, Chronic Diseases Research Centre, NOVA Medical School/Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, Lisboa, Portugal 2Advance Imaging Lab, Instituto Gulbenkian de Ciência, Oeiras, Portugal *Corresponding author. Tel: +35 1969 941245; E-mail: [email protected] EMBO Reports (2017)18:102-122https://doi.org/10.15252/embr.201642738 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The mechanisms driving pathological beta-amyloid (Aβ) generation in late-onset Alzheimer's disease (AD) are unclear. Two late-onset AD risk factors, Bin1 and CD2AP, are regulators of endocytic trafficking, but it is unclear how their endocytic function regulates Aβ generation in neurons. We identify a novel neuron-specific polarisation of Aβ generation controlled by Bin1 and CD2AP. We discover that Bin1 and CD2AP control Aβ generation in axonal and dendritic early endosomes, respectively. Both Bin1 loss of function and CD2AP loss of function raise Aβ generation by increasing APP and BACE1 convergence in early endosomes, however via distinct sorting events. When Bin1 levels are reduced, BACE1 is trapped in tubules of early endosomes and fails to recycle in axons. When CD2AP levels are reduced, APP is trapped at the limiting membrane of early endosomes and fails to be sorted for degradation in dendrites. Hence, Bin1 and CD2AP keep APP and BACE1 apart in early endosomes by distinct mechanisms in axon and dendrites. Individuals carrying variants of either factor would slowly accumulate Aβ in neurons increasing the risk for late-onset AD. Synopsis This study shows that Bin1 and CD2AP, genetic risk factors for Alzheimer's disease, control Aβ generation by keeping APP and BACE1 apart in early endosomes via distinct sorting mechanisms in axon and dendrites. Loss of function of Bin1 and CD2AP increases endogenous Aβ generation. Loss of function of Bin1 and CD2AP increases APP and BACE1 convergence in early endosomes via distinct sorting events. Bin1 is required for BACE1 exit from early endosomes by cutting off BACE1 tubules in axons. CD2AP is required for an efficient sorting of APP, away from processing at the early endosomal membrane, for degradation in dendrites. Introduction Beta-amyloid (Aβ), the Alzheimer's disease's (AD) established primary trigger 1, is produced normally by neurons. The amyloid precursor protein (APP) undergoes sequential cleavages by the rate limiting β-secretase (BACE1) and γ-secretase originating mainly Aβ40 and Aβ42 23. Aβ42 is more hydrophobic, aggregates faster and the most synaptotoxic 4. In familial forms of AD, Aβ excessive generation and/or higher ratio of Aβ42 are sufficient for neurodegeneration. However, in the most frequent late-onset forms of AD, the transition to pathological generation of Aβ remains poorly understood at a cellular and molecular level. Once generated, Aβ is secreted to the extracellular space or accumulates within endosomes 356. Endocytosis of APP and BACE1 to early endosomes, also named sorting endosomes, is required for the processing of APP generating Aβ 7891011. The neuronal trafficking of APP and BACE1 is largely segregated keeping to a minimum normal Aβ generation 10121314. During early endosome maturation, APP is sorted for degradation in the lysosome 1516, while BACE1 recycles back to the plasma membrane 121617. Consequently, the deregulation of APP and BACE1 segregation at early endosomes may boost Aβ generation 3. Yet the mechanisms underlying APP and BACE1 segregation at endosomes are unknown. Genetic studies of late-onset AD found variants in sortilin-related receptor 1 (SORL1), phosphatidylinositol binding clathrin assembly protein (PICALM), bridging integrator 1 (BIN1), and CD2-associated protein (CD2AP), known as endocytic regulators 1819202122232425, associated with increased risk for AD 2627282930. Sorl1 and PICALM have been shown to be reduced in the AD brain 313233. Loss of function of Sorl1 and PICALM potentiates Aβ production and decreases Aβ clearance, respectively 3234. Bin1 and CD2AP expression in AD patients has begun to be analysed 353637383940. Bin1 has mainly two isoforms in the brain, a longer and neuronal specific and a shorter and ubiquitous. The expression of the neuronal isoform has been found decreased in AD 3641. However, Bin1 and CD2AP expression in patients carrying BIN1 and CD2AP variants is unknown. Importantly, Bin1 variants were associated with poorer memory performance 42. Bin1 lower expression correlates with earlier AD onset 38. Bin1 knockdown impact on Aβ accumulation is not clear since increased Aβ secretion was observed in HeLa cells overexpressing APP with a familial AD mutation 43 but not in wild-type neuronal-like cells 36. CD2AP susceptibility loci were described to correlate with neuritic plaque burden in AD patients 44. Reduced CD2AP levels increased Aβ intracellularly but not extracellularly in neuronal-like cells overexpressing APP without detectable changes on amyloid load in a familial AD mouse model 45. Yet how Bin1 and CD2AP modulate Aβ generation is unknown. To investigate how Bin1 and CD2AP impact Aβ generation in late-onset AD, we used a knockdown approach in otherwise normal neurons. By analysing endogenous intracellular Aβ accumulation with a sensitive assay, by following the kinetics of APP and BACE1 endocytic trafficking, and by directly visualising early endosomal sorting events important for Aβ generation using fast live imaging or super-resolution techniques, we made the following mechanistic discoveries relevant for the AD field and more broadly to cell biology: Bin1 and CD2AP polarise the normal endogenous Aβ generation by specifically controlling BACE1 sorting for recycling in axons, and by specifically controlling APP sorting for degradation in dendrites, respectively. Mechanistically, we found Bin1 to be required for BACE1 exit from early endosomes by cutting off BACE1 tubules; and CD2AP to be required for an efficient sorting of APP away from processing at the early endosomal membrane by translocating APP to the lumen during multivesicular body biogenesis. Their loss of function potentiates Aβ accumulation and may thus contribute to the development of late-onset AD. We identify the mechanisms implicated in a novel neuron-specific polarisation of the amyloidogenic pathway regulated by two AD risk factors. Results Downregulation of Bin1 and CD2AP increases polarised endogenous Aβ generation To determine whether Bin1 and CD2AP impact endogenous Aβ, we first established a semi-quantitative assay for intracellular endogenous Aβ based on Aβ42 immunofluorescence 4647 (Fig EV1). Next, we efficiently downregulated Bin1 and CD2AP in wild-type primary cortical neurons (neurons) using established siRNAs (Fig EV2A) 20484950. Bin1 and CD2AP depletion increased intracellular endogenous Aβ42 in neuronal cell bodies (Fig 1A and D). However, when we analysed the levels of Aβ42 in neuronal dendrites and axons, morphologically identified based on soluble GFP expression or AnkG (axon marker), we unexpectedly found that downregulation of Bin1 resulted in a more pronounced increase in Aβ42 in axons than in dendrites and cell bodies (Fig 1A, C and D). In contrast, downregulation of CD2AP led to an increase in Aβ42 in dendrites, but not in axons (Fig 1A–D). We rescued the increase in Aβ42 by re-expressing CD2AP and neuronal Bin1 but not ubiquitous Bin1 confirming the specificity of the siRNA (Fig EV2B and C). Click here to expand this figure. Figure EV1. Validation of intracellular endogenous Aβ42 semi-quantitative assay Intracellular endogenous Aβ42 changes positively induced by overexpressing APP-RFP and BACE1-GFP or BACE1-GFP alone; or negatively induced by overexpressing Rab11SN, a dominant-negative mutant of Rab11 89 in N2a cells immunolabelled with anti-Aβ42 (clone H31L21). Single-cell semi-quantitative analysis of Aβ42 immunofluorescence normalised to Aβ42 fluorescence intensity in GFP-transfected cells (n = 3–5, NGFP = 133, NAPP + BACE1 = 98, NBACE1 = 65, NRab11SN = 101; ****P < 0.0001 vs. GFP, Mann–Whitney test, mean ± SEM). Intracellular endogenous Aβ42 changes upon the indicated treatment with inhibitors of Aβ generation, DAPT that blocks γ-secretase 90, or compound IV that blocks BACE1 91, or their vehicle DMSO in N2a cells immunolabelled with anti-Aβ42 (clones H31L21 or 12F4). Single-cell semi-quantitative analysis of Aβ42 immunofluorescence normalised to Aβ42 fluorescence intensity in cells treated with DMSO (n = 2–3, H31L21: NDMSO 24 h = 195, NDMSO 48 h = 145, NDAPT 24 h = 178, NDAPT 48 h = 128, NComp. IV = 141, 12F4: NDMSO 24 h = 243, NDMSO 48 h = 138, NDAPT 24 h = 139, NDAPT 48 h = 128, NComp. IV = 97; ****P < 0.0001 vs. DMSO, t-test, mean ± SEM). Intracellular endogenous Aβ42 (green) and C99 (magenta) in N2a cells expressing C99 treated with DAPT or its vehicle DMSO, immunolabelled with anti-Aβ42 (clones H31L21 or 12F4 as indicated) and with anti-C-terminal APP (Y188 or 4G8), analysed by epifluorescence microscopy. Scale bar, 10 μm. Intracellular endogenous Aβ42 changes upon expression of C99 and treatment with DAPT or its vehicle DMSO in N2a cells immunolabelled with anti-Aβ42 (clones H31L21 or 12F4). Single-cell semi-quantitative analysis of Aβ42 immunofluorescence normalised to Aβ42 fluorescence intensity in cells treated with DMSO (n = 3, H31L21: NC99 + DMSO = 70, NC99 + DAPT = 116, 12F4: NC99 + DMSO = 84, NC99 + DAPT = 80; ****P < 0.0001 vs. C99 + DMSO, t-test, mean ± SEM). Download figure Download PowerPoint Click here to expand this figure. Figure EV2. CD2AP and Bin1 knockdown efficiency, intracellular Aβ and APP-CTFs levels upon DAPT treatment Bin1 and CD2AP knockdown efficiency in siBin1-, siCD2AP- and siControl-treated neurons by Western blot analysis with anti-Bin1, anti-CD2AP and anti-tubulin as a loading control. The quantification of Bin1 and CD2AP levels normalised to tubulin levels is shown on the right (nsiBin1 = 5, ****P < 0.0001, t-test, mean ± SEM; nsiCD2AP = 3, **P = 0.075, t-test, mean ± SEM). Bin1 and CD2AP knockdown efficiency in siBin1-, siCD2AP- and siControl-treated N2a cells by Western blot analysis with anti-Bin1, anti-CD2AP and anti-tubulin as a loading control. Quantification of Bin1 and CD2AP levels normalised to tubulin levels is shown on the right (nsiBin1 = 3; *P = 0.0137, t-test, mean ± SEM; nsiCD2AP = 3; **P = 0.0028, t-test, mean ± SEM). Intracellular endogenous Aβ42 immunolabelled with anti-Aβ42 (clone H31L21) in N2a cells treated with siBin1, siCD2AP or siControl alone or upon expression of the siRNA-resistant plasmids: CD2AP-GFP, neuronal Bin1 or ubiquitous Bin1 (right insets), analysed by epifluorescence microscopy. Scale bars, 10 μm. The quantification of Aβ42 intensity per cell is shown on the right (n = 3, NsiControl = 197, NsiCD2AP = 208, NsiCD2AP + CD2AP-GFP = 170, NsiBin1 = 186, NsiBin1 + neuronal Bin1 = 117, NsiBin1 + ubiquitous Bin1 = 203; ****P < 0.0001 siCD2AP vs. siControl, siBin1 vs. siControl and siBin1 + ubiquitous Bin1 vs. siControl, ####P < 0.0001 siCD2AP + CD2AP-GFP vs. siCD2AP and siBin1 vs. siBin1 + neuronal Bin1 and siBin1 + ubiquitous Bin1 vs. siBin1 + neuronal Bin1, one-way ANOVA with Tukey's test; mean ± SEM). Intracellular endogenous Aβ40 in N2a cells treated with siBin1, siCD2AP or siControl, immunolabelled with anti-Aβ40, analysed by epifluorescence microscopy. Scale bar, 10 μm. The quantification of Aβ40 intensity per cell is shown on the right (n = 3, NsiControl = 268, NsiBin1 = 234, NsiCD2AP = 257; ****P < 0.0001 siBin1 vs. siControl, t-test, mean ± SEM). Endogenous APP and APP C-terminal fragments (APP-CTFs) levels by Western blot analysis with anti-APP antibody (Y188) of siBin1-, siCD2AP- or siControl-treated neurons upon DAPT treatment. The graph on the right shows the quantification of APP-CTFs normalised to APP (n = 4; **P = 0.0057 siBin1 vs. siControl, t-test, mean ± SEM). Endogenous nicastrin levels by Western blot analysis with anti-nicastrin antibody (PA1-758) and tubulin as a loading control in siBin1-, siCD2AP- or siControl-treated neurons. The quantification of nicastrin levels normalised to tubulin levels is shown on the right (n = 3, mean ± SEM). Source data are available online for this figure. Download figure Download PowerPoint Figure 1. Downregulation of Bin1 and CD2AP increases polarised endogenous Aβ generation A. Intracellular endogenous Aβ42 (green), Ankyrin-G (AnkG; magenta) and GFP (blue) in siBin1-, siCD2AP- and siControl-treated primary cortical neurons (neurons) expressing GFP immunolabelled at 9 DIV with anti-Aβ42 (clone 12F4) and anti-AnkG, analysed by spinning-disc confocal microscopy. The white rectangles indicate the dendrites (Dd) and axons (Ax) magnified below showing Aβ42 in axons and dendrites outlined based on AnkG (magenta) and GFP (blue), respectively. Scale bars, 10 μm. B, C. Aβ42 line profiles in dendrites (Dd; B) and axons (Ax; C) of siControl (grey line), siBin1 (blue line) and siCD2AP (red line) neurons shown in (A). D. Quantification of Aβ42 (12F4) intensity in cell body (CB), dendrite (Dd) and axon (Ax) (n = 5, NCB = 44–53, NDd = 90–120, NAx = 60–74; ****PCB < 0.0001 siBin1 vs. siControl, ***PCB < 0.001 siCD2AP vs. siControl, ****PDd < 0.0001 siBin1 vs. siControl, ****PDd < 0.0001 siCD2AP vs. siControl, ****PAx < 0.0001 siBin1 vs. siControl, t-test, mean ± SEM). E. Quantification of extracellular endogenous Aβ40, Aβ42 and of Aβ42/Aβ40 ratio by ELISA analysis of conditioned media of 9 DIV siBin1, siCD2AP or siControl neurons (n = 6; *PAβ40 = 0.0270 siBin1 vs. siControl, *PAβ42/40 = 0.0378 siBin1 vs. siControl, *PAβ42/40 = 0.0463 siCD2AP vs. siControl, t-test, mean ± SEM). F. Endogenous APP and APP-CTFs levels by Western blot with anti-APP antibody (Y188) of siBin1-, siCD2AP- or siControl-treated neurons at 9 DIV. G. Quantification of APP and APP-CTFs levels normalised to APP (n = 4; *PAPP = 0.0355 siBin1 vs. siControl, ***PAPP-CTFs/APP < 0.001 siBin1 vs. siControl, ****PAPP-CTFs/APP < 0.0001 siCD2AP vs. siControl, t-test, mean ± SEM). Source data are available online for this figure. Source Data for Figure 1 [embr201642738-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint Analysis of extracellular endogenous Aβ evidenced a small increase in the ratio of Aβ42/Aβ40 both in Bin1- and CD2AP-depleted neurons likely due to the significant decrease in Aβ40 secretion (Fig 1E). This decrease in extracellular Aβ40 was paralleled by a significant increase in intracellular Aβ40 when Bin1 but not CD2AP was depleted (Fig EV2D). Could the rise in Aβ42 by downregulation of Bin1 and CD2AP be due to increased Aβ generation by APP processing? To answer this question, we analysed APP processing into its C-terminal fragments (APP-CTFs). APP-CTFs increased when Bin1 was depleted and decreased when CD2AP was depleted without major change in APP (Fig 1F and G), indicating altered APP processing. Bin1 depletion likely raises APP-CTFs by increasing BACE1 processing of APP, the rate limiting step to generate Aβ. CD2AP depletion likely decreases APP-CTFs by increasing APP processing since inhibiting γ-secretase with DAPT treatment of CD2AP-depleted neurons restored the levels of APP-CTFs to that of control neurons (Fig EV2E). We ruled out that these results could be explained by an increase in γ-secretase levels, since levels of nicastrin, a subunit of γ-secretase, were unaltered (Fig EV2F). Together, our results indicate that loss of function of Bin1 and CD2AP increases polarised Aβ generation in axons and dendrites, respectively. Downregulation of Bin1 and CD2AP impacts on APP and BACE1 endocytic trafficking Bin1 and CD2AP can associate with the endocytic machinery 51 and localise to early endosomes in non-neuronal cells 2023. Bin1 has been described to control endocytosis, recycling and more recently the degradative pathway 21222352. CD2AP functions in the degradative pathway 20. Thus, we investigated if the endocytic trafficking of APP was altered upon Bin1 and CD2AP knockdown by pulse/chase assays using an antibody against N-terminal APP (22C11) 5354 (Fig 2A) in N2a cells transiently expressing human wild-type APP C-terminally tagged with RFP. 22C11 surface labelling (Fig EV3A) and endocytosis (10-min pulse; Fig 2B and C) in Bin1- and CD2AP-depleted cells were similar to control cells. This indicates that neither Bin1 nor CD2AP knockdown significantly alter cell surface APP or its endocytosis. After a 60-min chase, however, we observed a 40% loss in 22C11 labelling in control and Bin1-depleted cells, consistent with APP degradation in the lysosome (Figs 2D and E, and EV3C and D) 5556. In contrast, in CD2AP-depleted cells, we did not observe a similar loss in 22C11 after the same chase period (Fig 2D and E), indicating that CD2AP knockdown delays APP degradation. Importantly, we rescued the defect in APP degradation by re-expressing siRNA-resistant CD2AP in CD2AP-depleted cells (Fig EV3E). These results indicate that CD2AP is specifically required for efficient APP degradation. Overall, these results suggest that CD2AP, but not Bin1, controls APP endocytic trafficking. Figure 2. Downregulation of Bin1 and CD2AP impacts on APP and BACE1 endocytic traffickingN2a cells treated with siBin1, siCD2AP or siControl. Scheme illustrating APP endocytosis trafficking assayed in N2a cells transiently expressing APP-RFP using a pulse/chase assay with anti-N-terminal APP (22C11). A 10-min pulse to assay APP endocytosis (B) followed by a 60-min chase to assay the degradation of endocytosed APP (D). Endocytosed APP detected after a 10-min pulse with 22C11 and APP-RFP (insets), analysed by epifluorescence microscopy. Scale bars, 10 μm. The amount of endocytosed APP fluorescence at 10 min was quantified and normalised to APP-RFP fluorescence (n = 3, NsiControl = 56, NsiBin1 = 70, NsiCD2AP = 42; mean ± SEM). Non-degraded APP detected after 10-min pulse and 60-min chase with 22C11 and APP-RFP (insets), analysed by epifluorescence microscopy. Cells are outlined in white. Scale bars, 10 μm. APP degradation was assessed by the decrease in the amount of endocytosed APP fluorescence at 60 min relative to time 0 (10 min pulse) in siControl cells normalised to APP-RFP fluorescence (n60 min = 3, NsiControl = 68, NsiBin1 = 43, NsiCD2AP = 64; ****PAPP60 min < 0.0001, t-test, mean ± SEM). Scheme illustrating BACE1 endocytic trafficking assayed in N2a cells transiently expressing BACE1-GFP N-terminally tagged with FLAG using a pulse–chase assay with anti-FLAG antibody (M1). A 5-min pulse to assay BACE1 endocytosis (G), a 10-min pulse and 20-min chase to assay BACE1 recycling to the plasma membrane (I) and the pool of endocytosed BACE1 that did not recycle (K). Endocytosed BACE1 detected upon 5-min pulse with M1 and BACE1-GFP (insets), analysed by epifluorescence microscopy. Scale bars, 10 μm. The amount of endocytosed BACE1 per cell was quantified as percentage of siControl normalised to BACE1-GFP (n = 3, NsiControl = 86, NsiBin1 = 72, NsiCD2AP = 113; mean ± SEM). Recycled BACE1 detected at the plasma membrane of non-permeabilised cells with M1, upon a 10-min pulse, acid stripping and 20-min chase, and BACE1-GFP (insets), analysed by epifluorescence microscopy. Scale bars, 10 μm. The amount of recycled BACE1 was quantified as in (H) (n = 3, NsiControl = 94, NsiBin1 = 58, NsiCD2AP = 109; ****P < 0.0001 siBin1 vs. siControl, t-test, mean ± SEM). Non-recycled BACE1 detected in acid-stripped permeabilised cells pulse-chased as in (I) with M1 and BACE1-GFP (insets), analysed by epifluorescence microscopy. Scale bars, 10 μm. The amount of non-recycled BACE1 was quantified as in (H) (n = 3, NsiControl = 61, NsiBin1 = 51; ****P < 0.0001 siBin1 vs. siControl, t-test; n = 2, NsiCD2AP = 37; mean ± SEM). Download figure Download PowerPoint Click here to expand this figure. Figure EV3. APP trafficking assays upon CD2AP and Bin1 depletionN2a cells treated with siBin1, siCD2AP or siControl Surface APP detected after a 4-min pulse with anti-N-terminal APP (22C11) and immunofluorescence of non-permeabilised N2a cells expressing APP-RFP (insets), analysed by epifluorescence microscopy. Scale bars, 10 μm. The graph on the right shows the amount of cell surface APP fluorescence per cell quantified and normalised to APP-RFP fluorescence (n = 4, NsiControl = 91, NsiBin1 = 49, NsiCD2AP = 85; mean ± SEM). Recycled APP detected with 22C11 at the plasma membrane of non-permeabilised cells expressing APP-RFP (insets), upon a 10-min pulse with 22C11, membrane acid stripping and 20-min chase, analysed by epifluorescence microscopy. Scale bars, 10 μm. The graph on the right shows the amount of recycled APP fluorescence per cell quantified and normalised to APP-RFP fluorescence (n = 4, NsiControl = 75, NsiBin1 = 61, NsiCD2AP = 63; mean ± SEM). Endocytosed APP detected with 22C11 upon 10-min pulse (left panels) and a 60-min chase (right panels) in DMSO- or leupeptin-treated N2a cells expressing APP-RFP (insets), analysed by epifluorescence microscopy. Scale bars, 10 μm. The graph on the right shows APP degradation assessed by the decrease in the amount of endocytosed APP fluorescence at 60 min relative to time 0 (10-min pulse) in DMSO-treated cells normalised to APP-RFP fluorescence (n = 3, NDMSO 10 min = 111, NDMSO 60 min = 109, NLeu 10 min = 118, NLeu 60 min = 99, ****P60 min < 0.0001, t-test, mean ± SEM). APP levels by Western blot with anti-APP (Y188) of DMSO- or leupeptin-treated neurons at 11–12DIV. Quantification of APP levels normalised to tubulin levels is shown on the right (n = 3, *P = 0.0296 leupeptin vs. DMSO, t-test, mean ± SEM). Non-degraded APP detected with 22C11 (10-min pulse and 60-min chase) in N2a cells expressing APP-RFP (left insets) treated with siCD2AP, siControl alone or upon expression of siRNA-resistant CD2AP-GFP (right insets), analysed by epifluorescence microscopy. Scale bars, 10 μm. The amount of endocytosed APP fluorescence per cell quantified and normalised to APP-RFP fluorescence is shown on the right (n = 3, NsiControl = 109, NsiCD2AP = 82, NsiCD2AP + CD2AP-GFP = 83; ****P < 0.0001 siCD2AP vs. siControl, ####P < 0.0001 siCD2AP+CD2AP-GFP vs. siCD2AP, one-way ANOVA with Tukey's test, mean ± SEM). Source data are available online for this figure. Download figure Download PowerPoint We next tested if Bin1 could instead impact BACE1 endocytic trafficking. To analyse BACE1 endocytosis, we introduced a FLAG tag at the N-terminus of BACE1-GFP that has a localisation similar to endogenous BACE1 57. Upon transient expression of FLAG-BACE1-GFP in N2a cells, we performed pulse/chase assays using an antibody against FLAG (M1) (Fig 2F). M1 surface labelling (Fig EV4A) and endocytosis (5-min pulse; Fig 2G and H) were unaltered in cells depleted for Bin1 or CD2AP, indicating that neither Bin1 nor CD2AP knockdown alters cell surface BACE1 or its endocytosis. To measure BACE1 recycling back to the plasma membrane, we acid-stripped non-endocytosed M1 (Fig EV4B) and further chased endocytosed M1 for 20 min. Recycled M1 was then detected at the surface of non-permeabilised cells (Fig 2I). We observed a reduction in the amount of recycled M1 at the surface of Bin1-depleted cells as compared to CD2AP-depleted and control cells (Fig 2I and J). These results indicate that Bin1 knockdown affects recycling of BACE1 but importantly not of APP (Fig EV3B). Click here to expand this figure. Figure EV4. BACE1 trafficking assays upon CD2AP and Bin1 depletionN2a cells treated with siBin1, siCD2AP or siControl Surface BACE1 detected after a 4-min pulse with anti-FLAG (M1) and immunofluorescence of non-permeabilised cells expressing BACE1-GFP (insets), analysed by epifluorescence microscopy. Scale bars, 10 μm. The graph on the right shows the amount of cell surface BACE1 fluorescence per cell quantified and normalised to BACE1-GFP fluorescence (n = 4, NsiControl = 70, NsiBin1 = 43, NsiCD2AP = 68; mean ± SEM). Surface BACE1 detected as in (A) before and after acid stripping, analysed by epifluorescence microscopy. Scale bars, 10 μm. Endocytosed BACE1 detected with M1 (10-min pulse and 60-min chase) and BACE1-GFP (insets), analysed by epifluorescence microscopy. Scale bars, 10 μm. The graph on the right shows the amount of endocytosed BACE1 fluorescence per cell quantified and normalised to BACE1-GFP fluorescence (n = 4, NsiControl = 82, NsiBin1 = 72, NsiCD2AP = 76; mean ± SEM). Non-recycled BACE1 detected with M1 (10-min pulse, acid stripping and 20-min chase) in acid-stripped permeabilised N2a cells expressing BACE1-GFP (left insets) treated with siBin1 or siControl alone or upon expression of neuronal Bin1 or ubiquitous Bin1 (right insets), analysed by epifluorescence microscopy. Scale bars, 10 μm. The graph on the right shows the amount of recycled BACE1 fluorescence per cell quantified and normalised to BACE1-GFP fluorescence (n = 3, NsiControl = 95, NsiBin1 = 120, NsiBin1 + neuronal Bin1 = 99, NsiBin1 + ubiquitous Bin1 = 89; ****P < 0.0001 siBin1 vs. siControl and siBin1 + ubiquitous Bin1 vs. siControl, ####P < 0.0001 siBin1 vs. siBin1 + neuronal Bin1 and siBin1 + ubiquitous Bin1 vs. siBin1 + neuronal Bin1, one-way ANOVA with Tukey's test; mean ± SEM). Endogenous BACE1 levels by Western blot analysis with anti-BACE1 antibody and tubulin as a loading control in siBin1-, siCD2AP- or siControl-treated neurons. Quantification of BACE1 levels normalised to tubulin levels is shown on the right (n = 5, mean ± SEM). Source data are available online for this figure. Download figure Download PowerPoint The defective recycling of BACE1 observed in Bin1-depleted cells could be explained by intracellular retention and/or increased degradation
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