Fig 1: Aß transcytosis across AD–derived endothelial monolayer and iPSC–derived endothelium carrying the rs3851179 PICALM variantsa, qRT–PCR and western blot analysis of PICALM in control and AD endothelial monolayers. b, Diminished Aß40 (1 nM) transcytosis across AD–derived endothelium and reversal by adenoviral–mediated Ad.PICALM re–expression. Ad.mLRP1, LRP1 minigene. Mean ± s.e.m., from 8 isolates in triplicate for control and AD monolayers. c, Diagram of CRISPR/Cas9–based generation of isogenic iPSC lines homozygous for the protective (A) or non–protective (G) allele of rs3851179. gRNA = guide RNA. d, SspI restriction digest of PCR products from iPSC genomic DNA at rs3851179 region. *denotes the CRISPR–Cas9 modified iPSC line. e, Sanger sequencing of iPSCs at rs3851179 confirming independent isogenic lines homozygous for either the G or A variant. f, FACS dot plot showing 15.7% of iPSC–derived endothelial cells via embryoid body (EB) formation are positive for endothelial markers CD31 and VE–Cadherin. g, iPSC–derived endothelial cells co–cultured with pericyte conditioned media form monolayer in vitro with ZO–1 positive tight junctions (green). Bar=100 µm. h–i, qRT–PCR and western blot analysis of PICALM (h) and Aß40 (1 nM) transcytosis (i) in human iPSC–derived endothelial monolayers carrying the protective rs3851179 (AA) variant and the non–protective rs3851179 (GG) variant. In h–i, means ± s.e.m., from 6 cultures for each rs3851179 variant in triplicates.
Fig 2: PICALM interacts with Rab5 and Rab11 during Aβ transcytosis across endothelial monolayera, Colocalization between PICALM (red) and Rab5 (green) in primary human brain endothelial cells (BEC) cultured with FAM–Aβ40 (250 nM) for 2 min. b, Lack of association between PICALM (red) and Rab7 (green) in BEC cultured with FAM–Aβ40 for 5 min. c, Colocalization between PICALM (red) and Rab11 (green) in BEC cultured with FAM–Aβ40 for 5 min. Dapi, nuclear staining (blue). Insets: high magnification depicting colocalization. Bar=10 µm. d, Quantification of colocalization between PICALM and Rab5, Rab7, or Rab11 puncta in a–c. e–f, Colocalization of FAM–Aβ40 (green) with Rab5 (magenta, upper) or RAB11b (magenta, bottom) 2 and 4 min after Aβ internalization at the basolateral side of endothelial monolayer, respectively. Arrows denote co–localized white puncta. g–h, PLA of PICALM–Rab11 association (g, bar=10 µm) and kinetics of PICALM association with Rab5, Rab7 and Rab11 in endothelium after addition of Aβ40 (1 nM) to the basolateral membrane (h). i, Coimmunoprecipitation of LRP1, Rab5, Rab7 and Rab11 by PICALM–specific antibody (IP: PICALM) 0, 2 and 4 min after addition of Aβ40 (1 nM) to the basloateral membrane. j–k, Basolateral–to–apical transcytosis of Aβ40 (1 nM) across monolayer expressing dominant negative Rab5–S34N, Rab7–T22N or Rab11b–S25N mutants compared to control EGFP (100%) over 60 min (j) and quantification of unidirectional Aβ40 transport within 30 min (k). Mean ± s.e.m. from 3 primary isolates in triplicates. p<0.05 by ANOVA followed by Tukey’s posthoc test. l–m, Inhibition of Rab5 (l) and Rab11 (m) GTPase activity by si.PICALM compared to si.Scramble control.
Fig 3: PICALM associates with LRP1 during Aß transcytosis across endothelial monolayera, PICALM (red), ZO–1 (green) and F–actin (magenta) in a monolayer. b, Colocalization of PICALM (red) with LRP1 (green) and FAM–Aß40 (magenta) 1 min after LRP1–Aß internalization at the basolateral membrane. c–d, Proximity ligation assay (PLA) depicting PICALM–LRP1 association (c, bar=10 µm) and kinetics of association between PICALM and LRP1 and clathrin and LRP1 in endothelium after Aß40 (1 nM) addition to the basolateral membrane studied by PLA (d). e, Internalization of Aß40 (1 nM) at the basolateral membrane of the monolayer transfected with si.LRP1, si.PICALM and si.CHC compared si.Scramble or untransfected control. f–g, Basolateral–to–apical transcytosis of Aß40 (1 nM) across endothelial monolayer transfected with si.PICALM and si.CHC compared to si.Scramble (100%) over 60 min (f) and quantification of unidirectional trans–endothelial Aß40 transport within 30 min (g). Mean ± s.e.m. from 3 primary isolates in triplicates. p<0.05 by ANOVA followed by Tukey’s posthoc test.
Fig 4: PICALM/clathrin–dependent endocytosis of Aß–LRP1 complex by brain endothelial cellsa–b, Colocalization of LRP1–Aß40 complex with PICALM (a) and clathrin heavy chain (CHC) (b) in human brain endothelial cells (BEC) within 30 s of FAM–Aß40 (250 nM) treatment. c, Immunostaining for LRP1, PICALM and CHC without Aß (– Aß). Dapi, nuclear staining (blue). Insets: higher magnification. Bar=10 µm. d, Quantification of LRP1 puncta colocalized with PICALM in a, c and with CHC in b, c, and FAM–Aß40 puncta colocalized with LRP1 and PICALM in a, b. Means ± s.d. from 3 primary isolates in triplicate. p<0.05 by Student’s t–test. e, Coimmunoprecipitation of PICALM, CHC and clathrin adaptor protein a–adaptin (AP–2) by LRP1–specific antibody (IP: LRP1) in BEC 30 s or 5 min after stimulation with Aß40 (1 nM); IgG, non–immune IgG. f, LRP1 internalization in control BEC (vehicle) and after transfection with si.Scramble RNA and/or si.RNAs targeting PICALM or CHC. Aß40 (1 nM) was applied for 15 min at 4°C followed by 1 min at 37°C to initiate LRP1 internalization. Values at 4°C were taken as 100%. Means ± s.d. from 3 primary isolates in triplicate. p<0.05 by ANOVA followed by Tukey’s posthoc tests. g, In vitro binding of human recombinant PICALM to GST–tagged LRP1 C–terminus fusion protein (GST–LRP1C). h, C–terminal mutants of the human LRP1 minigene (LRP4T100). i, Coimmunoprecipitation of HA–tagged C–terminal LRP1 mutants (LRP4T100) by anti–Flag antibody (IP: Flag) in HEK293T cells after transfection with Flag–PICALM and HA–LRP4T100 mutants. HA–LRP4 and Flag–PICALM were used as loading controls.
Fig 5: PICALM reductions in brain capillary endothelium in Alzheimer’s diseasea, PICALM and Aß immunostaining in the prefrontal cortex of an age–matched control (Braak I, left) and AD case (Braak V–VI, right). Bar=20 µm. b, Immunoblotting for PICALM, von Willebrand Factor (vWF), ß3–tubulin, glial fibrillar acidic protein (GFAP), and GAPDH (loading control) in isolated microvessels and microvessel–depleted brains from controls (Braak 0–I) and AD cases (Braak V–VI). c, Relative abundance of PICALM in microvessels and microvessel–depleted brains from control and AD cases determined by densitometry analysis relative to GAPDH. Mean ± s.e.m., n=6/group; p<0.05 by ANOVA followed by Tukey’s posthoc tests. d, PICALM (green), lectin–positive endothelial capillary profiles (magenta) and microtubule–associated protein 2 (MAP2)–positive neurons (red) in the hippocampus (CA1) of an age–matched control (Braak I) and AD cases (Braak III and V–VI). Bar=20 µm. e, Quantification of PICALM–positive area (percentage) occupying lectin–positive endothelial capillary profiles in the prefrontal cortex and the CA1 hippocampal subfield. Mean ± s.d., n=9 controls (Braak 0–I) and 7 AD cases (Braak V–VI); p<0.01 by Student’s t–test. f–i, Correlations between PICALM–positive area occupying lectin–positive endothelial capillary profiles (percentage) in the prefrontal cortex and Aß load (f), Braak stages (g), Clinical Dementia Rating (CDR) (h), and Mini–Mental State Examination (MMSE) (i). Each point in f–i is an individual value from 50 (f–g), 28 (h) and 37 (i) controls and AD cases. CDR and MMSE were not available for all cases. Significance by Pearson and Spearman rank correlation analysis.
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