Journal: bioRxiv

Article Title: ApoE4 disrupts interaction of sortilin with fatty acid-binding protein 7 essential to promote lipid signaling

doi: 10.1101/2021.05.20.444938

Figure Lengend Snippet: (A ) Chinese hamster ovary (CHO) cells stably overexpressing sortilin (CHO-S) or sortilin and FABP7 tagged with a myc epitope (CHO-S/F) were used for co-immunoprecipitation experiments. Expression of sortilin and FABP7-myc in total cell lysates of both cell lines is shown in the panel Input. In panel IP-FABP7, co-immunoprecipitation of sortilin with anti-myc affinity resin (+) is seen in lysates from CHO-S/F but not from CHO-S cells. No immunoprecipitation of sortilin with anti-myc affinity resin is seen in CHO-S cells, or in CHO-S/F in the absence of the affinity resin (-). ( B ) Co-immunoprecipitation of sortilin with FABP7-myc from CHO-S/F cells as described in panel A. Prior to immunoprecipitation with anti-myc affinity resin, cells were treated with conditioned medium from HEK293 cells containing 5 μg/ml of apoE3 (+E3) or apoE4 (+E4; see Suppl. methods for details) or blank medium (-) for 24 hours. (C) Proximity ligation assay (PLA) to assess close spatial proximity of sortilin and FABP7 in CHO cells. Primary antibodies were directed against sortilin or the myc epitope in FABP7-myc. Close proximity is detected in CHO cells expressing both sortilin and FABP7 (red signal). No PLA signal is seen in cells expressing sortilin only. Cell nuclei were counterstained with DAPI (blue). The inset shows a higher magnification image of cells positive for PLA signals for sortilin and FABP7-myc. Scale bar: 50 μm. ( D ) Subcellular fractionation of CHO-S/F cells using gradient ultracentrifugation. Fractions were identified based on markers for endoplasmic reticulum (ER; GRP78), plasma membrane (PM; β-integrin), trans -Golgi network (TGN; VTI1b), and early endosomes (Endo; EEA1). FABP7 co-localizes with sortilin to the PM (fractions 3-4), TGN (fractions 7-8), and endosomes (fraction 11).

Article Snippet: Parental Chinese hamster ovary (CHO-K1) cells or cell clones stably overexpressing mouse sortilin and/or myc-tagged mouse FABP7 (Origene, MR200772) were grown in DMEM (Gibco) supplemented with 10% FCS (Gibco) and 1% penicillin/streptomycin (Gibco).

Techniques: Stable Transfection, Immunoprecipitation, Expressing, Proximity Ligation Assay, Fractionation

Journal: bioRxiv

Article Title: ApoE4 disrupts interaction of sortilin with fatty acid-binding protein 7 essential to promote lipid signaling

doi: 10.1101/2021.05.20.444938

Figure Lengend Snippet: (A, B) Parental Chinese hamster ovary (CHO) cells or CHO cells stably expressing sortilin (CHO-S) were transiently transfected with expression constructs encoding for FABP7 and green fluorescent protein (GFP). An exemplary western blot documenting expression of sortilin and FABP7 in replicate lysates of CHO and CHO-S cells is shown in panel A. Detection of GFP and tubulin served as transfection and loading controls, respectively. Panel B shows FABP7 levels in CHO and CHO-S transfectants as determined by densitometric scanning of replicate western blots (n= 9 replicates from 3 independent experiments per cell line). Data are mean ± SEM given as percent of FABP7 levels in CHO (set to 100%). Levels of FABP7 (but not of GFP) are significantly increased by the presence of sortilin in CHO-S compared with CHO cells. **, p<0.01 (Student’s t test). (C, D) CHO and CHO-S cells were transiently transfected with a FABP7-myc expression construct. Forty-eight hours post transfection, replicate cultures of transfected cells were treated with 10 μg/ml of cycloheximide and collected at time points 0, 4, 8, and 12 hours later. Levels of FABP7 were determined by western blotting (C). Detection of GAPDH served as loading control. The decrease in FABP7 levels was significantly faster in CHO than in CHO-S cells as determined by densitometric scanning of replicate blots (D; n= 9 replicates per condition from 3 independent experiments). Data are mean ± SEM given as percent of FABP7 levels in CHO or CHO-S at 0 hour of treatment (set to 100%). The significance of data was determined by Two-way ANOVA, followed by Bonferroni post-hoc analysis (**, p<0.01; ****, p<0.0001).

Article Snippet: Parental Chinese hamster ovary (CHO-K1) cells or cell clones stably overexpressing mouse sortilin and/or myc-tagged mouse FABP7 (Origene, MR200772) were grown in DMEM (Gibco) supplemented with 10% FCS (Gibco) and 1% penicillin/streptomycin (Gibco).

Techniques: Stable Transfection, Expressing, Transfection, Construct, Western Blot

Journal: bioRxiv

Article Title: ApoE4 disrupts interaction of sortilin with fatty acid-binding protein 7 essential to promote lipid signaling

doi: 10.1101/2021.05.20.444938

Figure Lengend Snippet: (A, B) Western blot analysis of FABP5 and FABP7 levels in brain cortices of apoE3 (A) and apoE4 (B) targeted replacement mice, either wild-type (WT) or homozygous for the Sort1 null allele (KO) (at 3 months of age). Na/K ATPase and GAPDH served as loading controls for detection of FABP5 and FABP7, respectively. Detection of sortilin served as genotype control. ( C ) Quantitative analysis of FABP7 levels in brain cortices of apoE3- and apoE4-expressing mice of the indicated Sort1 genotype using densitometric scanning of replicate western blots. Values are mean ± SEM given as percent of the respective WT control (set to 100%). Student t test was used to determine significance of differences between genotypes (n=12-18 animals per group; **, p<0.01). ( D ) Levels of Fabp7 transcripts in brain extracts (cortex and hippocampus) of apoE3- and apoE4-expressing mice of the indicated Sort1 genotypes as determined by quantitative RT-PCR. Values are given as mean ± SEM. No statistically significant differences in transcript levels were observed using Student’s t test (n=5-8 animals per group). ( E, F ) Western blot analysis of FABP5 and FABP7 levels in prefrontal cortex specimens of AD patients homozygous for APOEe3 or APOEe4 (pathological characteristics given in ). An exemplary western blot is shown in panel E. Detection of GAPDH served as loading control. Panel F shows the result of densitometric scanning of replicate blots. Values are mean ± SEM given as percent of APOEe3/3 genotype (mean value set to 100%). Welch’s t -test was used to calculate the significance of data (n=12-34 individuals per group; *, p<0.05).

Article Snippet: Parental Chinese hamster ovary (CHO-K1) cells or cell clones stably overexpressing mouse sortilin and/or myc-tagged mouse FABP7 (Origene, MR200772) were grown in DMEM (Gibco) supplemented with 10% FCS (Gibco) and 1% penicillin/streptomycin (Gibco).

Techniques: Western Blot, Expressing, Quantitative RT-PCR

Journal: bioRxiv

Article Title: ApoE4 disrupts interaction of sortilin with fatty acid-binding protein 7 essential to promote lipid signaling

doi: 10.1101/2021.05.20.444938

Figure Lengend Snippet: ( A ) Immunohistological detection of FABP7 (red) in brain cortical sections from apoE3 mice, either wild-type (WT) or genetically deficient for Sort1 (KO). Additionally, the sections were stained for the neuronal marker NeuN (blue) and sortilin (green) as well as DAPI (white; in insets). Merged images show co-expression of FABP7 with NeuN. As a positive control, the insets document expression of FABP7 in glia in the cerebellum of E3/WT and E3/KO mice. Representative images from analysis of 3 mice per genotype are shown. Scale bar:100 μm. ( B ) Staining for FABP7 in hippocampal subfield CA1 and temporal cortex specimens of AD patients, showing immunoreactivity in glial cells (arrowheads) and light positivity in sparse neuronal cells (arrows, and insets). Representative images from one of three AD cases analyzed are shown (pathological characteristics given in ). Scale bars: 100 μm and 50 μm (inset).

Article Snippet: Parental Chinese hamster ovary (CHO-K1) cells or cell clones stably overexpressing mouse sortilin and/or myc-tagged mouse FABP7 (Origene, MR200772) were grown in DMEM (Gibco) supplemented with 10% FCS (Gibco) and 1% penicillin/streptomycin (Gibco).

Techniques: Staining, Marker, Expressing, Positive Control

Journal: bioRxiv

Article Title: ApoE4 disrupts interaction of sortilin with fatty acid-binding protein 7 essential to promote lipid signaling

doi: 10.1101/2021.05.20.444938

Figure Lengend Snippet: (A) Immunodetection of FABP7 (red) and sortilin (green) in primary neuronal cultures from apoE3 mice either (WT) or genetically deficient for Sort1 (KO). FAPB7-stained cells are identified as neurons by expression of MAP2 (blue). Merged images show co-expression of FABP7 and sortilin. Scale bar: 20 μm. ( B ) Detection of apoE3 and apoE4 in medium and lysate of neuronal cultures of the indicated genotypes. Detection of recombinant apoE4 served as control (ctr). (C-D) Western blot analysis of sortilin, FABP7, and FABP5 in primary neurons from apoE3 or apoE4-expressing mice, either wild-type (WT) or genetically deficient for Sort1 (KO). Detection of sortilin and GAPDH served as controls. Levels of FABP7 are reduced in primary cortical neurons from apoE3 mice lacking sortilin (E3/KO) compared with neurons from (E3/WT) animals as determined by densitometric scanning of replicate blots (exemplified in panel C). No statistically significant difference in FABP7 levels is seen comparing neurons from apoE4 mice with (E4/WT) and without (E4/KO) sortilin (panel D). Data are given as mean ± SEM with the respective mean WT levels set to 100% (n=4-8 biological replicates per genotype; Student’s t test). *, p<0.05. ( E ) Quantitative RT-PCR analysis of Gfap transcripts in primary neuronal cultures from E3/WT and E3/KO mice. Values are given as log2 fold changes relative to E3/WT set to value 0 (n=4 independent cultures per genotype). ( F ) Levels of Fabp7 transcript were determined in mouse primary neuronal cultures of the indicated genotypes by quantitative RT-PCR (n=7-10 biological replicates per group for apoE3; n=5-6 biological replicates per group for apoE4). Values are given as log2 fold change relative to transcript levels in the respective WT (set to value 0).

Article Snippet: Parental Chinese hamster ovary (CHO-K1) cells or cell clones stably overexpressing mouse sortilin and/or myc-tagged mouse FABP7 (Origene, MR200772) were grown in DMEM (Gibco) supplemented with 10% FCS (Gibco) and 1% penicillin/streptomycin (Gibco).

Techniques: Immunodetection, Staining, Expressing, Recombinant, Western Blot, Quantitative RT-PCR

Journal: bioRxiv

Article Title: ApoE4 disrupts interaction of sortilin with fatty acid-binding protein 7 essential to promote lipid signaling

doi: 10.1101/2021.05.20.444938

Figure Lengend Snippet: ( A ) Immunodetection of FABP7 (red) and sortilin (green) in primary astrocytic cultures from apoE3 mice either (WT) or genetically deficient for Sort1 (KO). FAPB7-stained cells are identified as astrocytes by expression of GFAP (blue). Merged images show co-expression of FABP7 and sortilin. Images represent single z-planes. Scale bar: 20 μm. ( B-C ) Exemplary western blot analyses of sortilin and FABP7 in primary astrocytes from apoE3 or apoE4 targeted replacement mice, either wild-type (WT) or homozygous for the Sort1 null allele (KO). Detection of sortilin and tubulin served as controls. ( D ) Quantitative analysis of FABP7 levels in primary astrocyte cultures using densitometric scanning of replicate blots (exemplified in panels b and c). No genotype-dependent alterations in FABP7 levels were seen comparing cells of the indicated APOE and Sort1 genotypes. Data are given as mean ± SEM with the respective WT level set to 100% (n=5 independent cultures per genotype). ( E ) Levels of Fabp7 transcript were identical in primary astrocytes from apoE3- and apoE4-expressing mice, either WT or KO for Sort1 , as determined by quantitative RT-PCR (n=5 independent cultures per genotype). Values are given as log2 fold change relative to the respective WT (set to value 0).

Article Snippet: Parental Chinese hamster ovary (CHO-K1) cells or cell clones stably overexpressing mouse sortilin and/or myc-tagged mouse FABP7 (Origene, MR200772) were grown in DMEM (Gibco) supplemented with 10% FCS (Gibco) and 1% penicillin/streptomycin (Gibco).

Techniques: Immunodetection, Staining, Expressing, Western Blot, Quantitative RT-PCR

Journal: bioRxiv

Article Title: ApoE4 disrupts interaction of sortilin with fatty acid-binding protein 7 essential to promote lipid signaling

doi: 10.1101/2021.05.20.444938

Figure Lengend Snippet: ( A-B ) CHO cells stably co-expressing sortilin and FABP7 (CHO-S/F) were treated for 24 hours with conditioned medium containing 5 μg/ml of apoE3 (+E3) or apoE4 (+E4; see methods for details). Western blot analysis (panel A) and densitometric scanning of replicate blots (panel B) document reduced levels of FABP7 in CHO-S/F cells in the presence of apoE4 as compared with apoE3 (n=10 replicate cultures from 4 independent experiments). Data are mean ± SEM given as percent of FABP7 levels in apoE3-treated cells (set to 100%). *, p<0.05 (Student’s t test). ( C-D ) Experiment as in panels A and B but using conditioned medium from primary astrocytes secreting apoE3 or apoE4 (n=3 replicates from one culture). Data are mean ± SEM given as percent of FABP7 levels in apoE3-treated cells (set to 100%). *, p<0.05 (Student’s t test). ( E ) Proximity ligation assay (PLA) to visualize the intracellular localization of sortilin-FABP7 complexes (red signal) in CHO-S/F cells treated with apoE3- or apoE4-conditioned HEK293 medium for 24 hours (left panel). For comparison, immunostaining of total sortilin (middle panels) and FABP7 (right panels) in treated cells are shown as well. Cell nuclei were counterstained with DAPI (blue). PLA signals for sortilin-FABP7 complexes change from a dispersed vesicular pattern with apoE3 to a perinuclear pattern with apoE4 (arrowheads). A similar change is seen for total sortilin, whereas the pattern for total FABP7 remains unaffected by apoE4. ( F-G ) Colocalization studies in CHO-S/F cells documenting the presence of sortilin-FABP7 complexes (as deduced by PLA; red signal) in early endosomes, marked by antibodies against Rab5 (green signal; panel F) or recycling endosomes marked by Rab11 (green signal, panel G). ( H-I ) Extent of colocalization of PLA for sortilin and FABP7 with Rab5 (panel H) and Rab11 (panel I) as documented by thresholded Mander’s coefficient tM1. This experiment was replicated 3 times (n=33-38 cells per condition). *, p <0.05; **, p <0.01 (Student’s t test). Scale bars: 20 μm

Article Snippet: Parental Chinese hamster ovary (CHO-K1) cells or cell clones stably overexpressing mouse sortilin and/or myc-tagged mouse FABP7 (Origene, MR200772) were grown in DMEM (Gibco) supplemented with 10% FCS (Gibco) and 1% penicillin/streptomycin (Gibco).

Techniques: Stable Transfection, Expressing, Western Blot, Proximity Ligation Assay, Immunostaining

Journal: bioRxiv

Article Title: ApoE4 disrupts interaction of sortilin with fatty acid-binding protein 7 essential to promote lipid signaling

doi: 10.1101/2021.05.20.444938

Figure Lengend Snippet: ( A ) CHO cells stably expressing sortilin (CHO-S) were transfected with reporter gene constructs encoding a PPAR-responsive firefly luciferase gene (Luc) and a constitutively expressed renilla luciferase gene (Ren). Where indicated, transfectants were also treated with rosiglitazone (+ RGS) for 24 hours. Forty-eight hours after transfection, the activities of firefly and renilla luciferases were determined in cell lysates using a luminometer (n= 3-8 replicates per cell line). Values are given as ratio of firefly to renilla luciferase activity (mean ± SEM, condition without RGS as 100%). ***, p<0.001; ****, p<0.0001 (Student’s t test). ( B ) Replicate layers (n=4) of CHO cells stably expressing sortilin (CHO-S), FABP7 (CHO-F), or both proteins (CHO-S/F) were transfected with constructs encoding a PPAR-responsive firefly luciferase and a constitutively expressed renilla luciferase and analyzed for luciferase activity as described above. Values are given as ratio of firefly to renilla luciferase (mean ± SEM, CHO-S set to 100%). **, p<0.01; ***, p<0.001 (Student’s t test). ( C ) CHO-S/F were transfected with reporter gene constructs encoding a PPAR-responsive firefly luciferase reporter gene and a constitutively expressed renilla luciferase. Then, transfectants were treated with conditioned medium containing 5 μg/ml of human apoE3 or apoE4 for 24 hours. Forty-eight hours after transfection, the activities of firefly and renilla luciferases were determined in cell lysates using a luminometer (n= 9 replicates from 3 independent experiments per condition. Values are given as ratio of firefly to renilla luciferase (mean ± SEM, + apoE3 set to 100%). *, p<0.05 (Student’s t test). ( D-E ) Proposed model of the interaction of sortilin with FABP7 and apoE3 (D) or apoE4 (E) in cellular lipid metabolism (see discussion section for details).

Article Snippet: Parental Chinese hamster ovary (CHO-K1) cells or cell clones stably overexpressing mouse sortilin and/or myc-tagged mouse FABP7 (Origene, MR200772) were grown in DMEM (Gibco) supplemented with 10% FCS (Gibco) and 1% penicillin/streptomycin (Gibco).

Techniques: Stable Transfection, Expressing, Transfection, Construct, Luciferase, Activity Assay