Journal: Journal of Biological Chemistry

Article Title: Bone Overgrowth-associated Mutations in the LRP4 Gene Impair Sclerostin Facilitator Function

doi: 10.1074/jbc.m110.190330

Figure Lengend Snippet: FIGURE 3. LRP4 is expressed in human bone, and Lrp4 silencing blunts sclerostin-mediated inhibition of in vitro bone mineralization. A, LRP4 protein is expressed in human osteoblasts (arrowheads) and osteocytes (arrows). Immunohistochemistry of LRP4 in human femoral neck from a male subject aged 75 is shown. Scale bar, 50 m. B, LRP4 RNA is expressed in samples from human femoral neck. RNA was extracted from a female subject aged 65 and a male subject aged 80. PTH1R, LRP4, SOST, LRP5, and LRP6 RNA levels were assessed by real-time quantitative PCR and normalized with GAPDH, and relative expression compared with PTH1R is shown. C, knockdown of LRP4 in Kusa-A1 blocks the inhibitory effect of sclerostin on in vitro mineralization activity. Kusa-A1 cells were transducedwithlentiviralparticlesharboringshRNAagainstLrp4.ReductionofLrp4RNAlevelswasassessedbyreal-timequantitativePCR,priortotheaddition of an osteoblastic differentiation medium. Calcium content was assessed 4 days later. Error bars, S.E.

Article Snippet: Recombinant human DKK1 and LRP6 were purchased from R&D Systems.

Techniques: Inhibition, In Vitro, Immunohistochemistry, Real-time Polymerase Chain Reaction, Expressing, Knockdown, Activity Assay

Journal: Brain

Article Title: Inactivating Celsr2 promotes motor axon fasciculation and regeneration in mouse and human

doi: 10.1093/brain/awab317

Figure Lengend Snippet: Celsr2 expression is enriched in mouse and human spinal motor neurons. Using Celsr2 LacZ transgenic mice, Celsr2 expression is detected by anti-β-gal immunostaining. ( A – E ) E12.5 spinal sections were immunostained for Isl1 (red), β-gal (green) and counterstained for DAPI (blue). The merged image showed that all Isl1-positive cells in the ventral horn (VH) co-labelled by β-gal immunoreactivity ( E ). ( F – J ) In adult spinal sections, all ChAT-positive (red) spinal motor neurons co-expressed β-gal (green) indicated by arrows ( J ). ( K – O ) In WPC8 human spinal sections, ISL1 and CELSR2 double immunofluorescent staining showed that CELSR2 was expressed in ISL1-positive neurons in the ventral horn (VH) indicated by arrows ( O ). B – E , G – J and L – O correspond to boxed areas in A , F and K , respectively. Nuclei were counterstained by DAPI (blue).

Article Snippet: Primary antibodies were: goat anti-ChAT (1:500, ab144p, Millipore), chicken anti-β-gal (1:500, ab9361, Abcam), rabbit anti-Isl1 (1:1000, Abcam) and goat anti-human CELSR2 (1:100, AF6379, R&D systems).

Techniques: Expressing, Transgenic Assay, Immunostaining, Staining

Journal: Brain

Article Title: Inactivating Celsr2 promotes motor axon fasciculation and regeneration in mouse and human

doi: 10.1093/brain/awab317

Figure Lengend Snippet: Celsr2 knockout improves axon growth in mouse spinal motor explant culture . ( A ) Schema of the experimental procedure for explant culture and analysis. ( B – G ) Spinal motor neuron explants from E13.5 Celsr2 +/+ ( B – D ) and Celsr2 −/− mouse embryos ( E–G ) were cultured for 6 DIV and then immunostained for ChAT ( B and E ; red) and Tuj1 ( C and F ; green). Both signals co-localize ( D and G ; yellow). Representative axonal bundles are indicated in the insets of C and F . ( H – J ) Quantification of the maximal area covered by growing axons in 10 7 µm 2 ( H ; control: 0.86 ± 0.03, mutant: 1.02 ± 0.05; P < 0.05), maximal axon length in 10 3 µm ( I ; control: 1.68 ± 0.09, mutant: 2.04 ± 0.07; P < 0.001) and number of large axon bundles (>8 µm in diameter; J ; control: 3.11 ± 0.45, mutant: 6.77 ± 0.74; P < 0.001). These parameters are increased significantly in the mutant compared to the control. *** P < 0.001; ** P < 0.01; * P < 0.05; Student’s t -test; n = 19 in the control and n = 22 in the mutant.

Article Snippet: Primary antibodies were: goat anti-ChAT (1:500, ab144p, Millipore), chicken anti-β-gal (1:500, ab9361, Abcam), rabbit anti-Isl1 (1:1000, Abcam) and goat anti-human CELSR2 (1:100, AF6379, R&D systems).

Techniques: Knock-Out, Cell Culture, Control, Mutagenesis

Journal: Brain

Article Title: Inactivating Celsr2 promotes motor axon fasciculation and regeneration in mouse and human

doi: 10.1093/brain/awab317

Figure Lengend Snippet: Celsr2 knockout contributes to neurite growth in primary spinal motor neuron culture. ( A and B ) E13.5 spinal motor neurons from Celsr2 +/+ ( A ) and Celsr2 –/– ( B ) mouse embryos were cultured for 6 DIV and immunostained for Tuj1 (red). DAPI counterstained nuclei (blue). ( C and D ) Double immunostaining of cultured neurons for F-actin (blue) and Tuj1 (red) disclosed the axon shafts and growth cones. ( E and F ) Statistical analysis of total neurite length ( E ; control: 165.25 ± 10.52 μm, mutant: 354.19 ± 24.89 μm; P < 0.0001, n = 41 in the control and n = 36 in the mutant) and growth cone areas ( F ; control: 17.48 ± 0.91 µm 2 , mutant: 84.89 ± 7.75 µm 2 ; P < 0.0001, n = 58 in the control and n = 40 in the mutant). ( G ) Protein extracts from E13.5 ventral horns of cervical spinal segments was subjected to western blots using anti-EB3 and β-III tubulin (tubulin). There was a dramatic increase of EB3 in the mutant compared to the control (control: 0.99 ± 0.09, mutant: 1.35 ± 0.01; P < 0.05, n = 3 animals in each group). * P < 0.05; **** P < 0.0001; Student’s t -test.

Article Snippet: Primary antibodies were: goat anti-ChAT (1:500, ab144p, Millipore), chicken anti-β-gal (1:500, ab9361, Abcam), rabbit anti-Isl1 (1:1000, Abcam) and goat anti-human CELSR2 (1:100, AF6379, R&D systems).

Techniques: Knock-Out, Cell Culture, Double Immunostaining, Control, Mutagenesis, Western Blot

Journal: Brain

Article Title: Inactivating Celsr2 promotes motor axon fasciculation and regeneration in mouse and human

doi: 10.1093/brain/awab317

Figure Lengend Snippet: Celsr2 cKO in spinal motor neurons improves functional recovery and NMJ formation after root avulsion/reimplantation. ( A and B ) After root avulsion/reimplantation, the function of the affected forelimb was assessed using the grooming ( A ) and climbing test ( B ). Scores were significantly higher in Celsr2 cKO ( Isl1-Cre; Celsr2 f/– ) compared to littermate controls ( Celsr2 f/– ) at Days 14, 21, 28, 35, 42, 49 and 56 post-injury. During the climbing test, usage of injured (R) and intact (L) forelimbs was compared; the R/L ratio was increased in the Celsr2 cKO. * P < 0.05; ** P < 0.01; Student’s t -test. ( C and D ) Biceps collected 56 days after injury were more atrophic on the injured than the intact side, in both mutant and control mice ( C ). Muscle wet weight on the intact side was comparable in both groups, whereas it was higher on the injured side in mutants versus controls, as reflected by the increased the R/L ratio (injured side to intact side) ( D ). Wet weight, control: 0.0339 ± 0.0008 g, mutant: 0.0339 ± 0.0011 g on the intact side, P < 0.05; control: 0.0237 ± 0.0024 g, mutant: 0.0300 ± 0.0010 g on the injured side, P < 0.05; the R/L ratio: 0.69 ± 0.06 and 0.89 ± 0.02 in the control and the mutant, respectively, P < 0.01. * P < 0.05; ** P < 0.01; Student’s t -test; n = 7 in the control and n = 8 in the mutant. ( E and F ) NMJs were examined using anti-NF200 and anti-ɑ-BT double staining 56 days post-surgery. On intact sides ( E ), several cholinergic receptor clusters showed axonal terminals overgrowth in Celsr2 cKO (17/32; indicated by arrows), but rarely in the control (4/52). On injury sides ( F ), there were more numerous growing axons and NMJs in the mutant than in the control (control: 56.33 ± 3.53, mutant: 213.00 ± 16.56 NMJs/muscle, P < 0.001, n = 3 animals in each group). ** P < 0.01; *** P < 0.001; Student’s t -test for NMJ number comparison and chi-square for receptor cluster comparison. ( G – I ) EMG of biceps was recorded 56 days post-surgery ( G ). The latencies were increased after injury, with no difference between both groups ( H ; control: 0.514 ± 0.014, mutant: 0.600 ± 0.033 ms on the intact side; 1.129 ± 0.170 ms for control and 1.632 ± 0.083 ms for mutant on the injured side; ratio of latency: 2.195 ± 0.335 and 1.632 ± 0.083 in the control and the mutant, respectively; P > 0.05 in all comparisons). Denervation resulted in a significant decrease of the peak–peak amplitude in both groups, but the amplitudes and the ratio of the injured to the intact muscle was significantly higher in the mutant compared to the control ( I ; control: 11.182 ± 0.923 mV, mutant: 11.269 ± 1.055 mV on the intact side, P > 0.05; control: 2.740 ± 0.523 mV, mutant: 5.572 ± 0.873 mV on the injured side, P < 0.05; ratio: 0.244 ± 0.044 and 0.492 ± 0.067 in the control and the mutant, respectively, P < 0.05). * P < 0.05; ** P < 0.01; *** P < 0.001; n.s, not significant; Student’s t -test; n = 7 in the control and n = 9 in the mutant.

Article Snippet: Primary antibodies were: goat anti-ChAT (1:500, ab144p, Millipore), chicken anti-β-gal (1:500, ab9361, Abcam), rabbit anti-Isl1 (1:1000, Abcam) and goat anti-human CELSR2 (1:100, AF6379, R&D systems).

Techniques: Functional Assay, Mutagenesis, Control, Double Staining, Comparison

Journal: Brain

Article Title: Inactivating Celsr2 promotes motor axon fasciculation and regeneration in mouse and human

doi: 10.1093/brain/awab317

Figure Lengend Snippet: Improved axon regeneration in Celsr2 cKO mice after root avulsion/reimplantation. ( A ) Toluidine Blue staining of musculocutaneous nerves from intact and injured sides 56 days after root avulsion/reimplantation. ( B ) Electron microscope images of musculocutaneous nerves from intact and injured sides. On the intact side, axons were more closely packed in the mutant than in the control. ( C and D ) Statistical analysis showed that axon number was comparable on the intact side in the two genotypes but higher in the mutant on the injured side ( C ; control: 734.0 ± 24.8, mutant: 696.3 ± 20.2 axons/section on the intact side, P > 0.05; control: 201.0 ± 21.5, mutant: 300.3 ± 25.8 axons/section on the injured side, P < 0.05; the ratio: 0.244 ± 0.044 and 0.492 ± 0.067, P < 0.05; n = 7 in the control and n = 9 in the mutant). The ratio of the injured to the intact side (R/L) was higher in the mutant ( C ; control: 0.27 ± 0.02, mutant: 0.43 ± 0.03, P < 0.05; n = 3 in each group). The distribution of axons according to their diameter showed no differences in the two genotypes on the intact side and an increased number of axons with 2–6 µm diameter on the operated side in the mutant relative to the control ( D ). * P < 0.05; ** P < 0.01; *** P < 0.001; n.s, not significant; Student’s t -test.

Article Snippet: Primary antibodies were: goat anti-ChAT (1:500, ab144p, Millipore), chicken anti-β-gal (1:500, ab9361, Abcam), rabbit anti-Isl1 (1:1000, Abcam) and goat anti-human CELSR2 (1:100, AF6379, R&D systems).

Techniques: Staining, Microscopy, Mutagenesis, Control

Journal: Brain

Article Title: Inactivating Celsr2 promotes motor axon fasciculation and regeneration in mouse and human

doi: 10.1093/brain/awab317

Figure Lengend Snippet: CELSR2 knockdown increases axonal regeneration in human embryonic spinal motor explant culture . ( A – D ) Cultured spinal motor neuron explants from WPC7 human embryos were transfected with a CELSR2 scrambled shRNA as control ( A and B ) and with CELSR2-shRNA ( C and D ). After 5 DIV, cultured explants were immunostained for Tuj1. A′ , B′ , C′ and D′ are enlarged areas from A , B , C and D , respectively. Upon CELSR2-shRNA knockdown, growing axons grew in circles (one example indicated in C ) and formed large axonal bundles (arrows in C′ and D′ ). ( E – H ) The maximal area in 10 7 µm 2 ( E ; control: 0.82 ± 0.12, CELSR2-shRNA: 3.93 ± 0.40; P < 0.0001, n = 23 in each group), maximal axon length in 10 3 µm ( F ; control: 1.20 ± 0.05, and CELSR2-shRNA: 3.30 ± 0.25; P < 0.0001, n = 23 in each group), explants with axons growing into circles ( G ; 1/26 in the control and 11/27 in the CELSR2-shRNA) and the number of large axon bundles ( H ; control : 7.83 ± 1.37, mutant: 53.96 ± 3.98, P < 0.0001, n = 23 in each group) were significantly increased in the CELSR2-shRNA knockdown explants compared to control. **** P < 0.0001; Student’s t -test ( E , F and H ). ** P < 0.01; chi-square test ( G ).

Article Snippet: Primary antibodies were: goat anti-ChAT (1:500, ab144p, Millipore), chicken anti-β-gal (1:500, ab9361, Abcam), rabbit anti-Isl1 (1:1000, Abcam) and goat anti-human CELSR2 (1:100, AF6379, R&D systems).

Techniques: Knockdown, Cell Culture, Transfection, shRNA, Control, Mutagenesis

Journal: Brain

Article Title: Inactivating Celsr2 promotes motor axon fasciculation and regeneration in mouse and human

doi: 10.1093/brain/awab317

Figure Lengend Snippet: CELSR2 knockdown promotes axonal growth in primary human spinal motor neuron culture. ( A – C ) CELSR2 scrambled shRNA ( A , control) and CELSR2-shRNA ( B ) were used to transfect cultured primary spinal motor neurons from WPC7 and WPC8 human embryos. Transfected neurons were visualized by virus-encoded GFP (green). After 5 DIV, neurons were immunostained for Tuj1 (red). GFP- and Tuj1-immunoreactivity overlapped in the somas and neurites as shown in the merged images. A significant increase of total neurite length was observed in CELSR2-shRNA transfected neurons ( C ; in µm, control: 118.70 ± 5.66, CELSR2-shRNA: 321.28 ± 21.90, P < 0.0001, n = 43 in the control and n = 36 in the CELSR2-shRNA). ( D – F ) Double immunostaining for F-actin (blue) and Tuj1 (red) reveal axon shafts and growth cones in control ( D ) and CELSR2-shRNA ( E ) transfected neurons (GFP labelling, green). The growth cone area was significantly increased in CELSR2-shRNA versus control transfected neurons ( F ; control: 16.92 ± 1.40 µm 2 , and CELSR2-shRNA: 109.00 ± 9.12, P < 0.0001, n = 52 in the control and 55 in the CELSR2-shRNA). ( G ) Western blot analysis of 5-DIV cultured neurons with antibodies to EB3 and β-III tubulin (tubulin, reference) showed an increase of EB3 levels in CELSR2-shRNA transfected neurons (control: 0.99 ± 0.09, CELSR2-shRNA: 1.34 ± 0.02, P < 0.05, n = 3 independent experiments). ( H ) Intracellular calcium influx was evaluated in 19 DIV-cultured neurons by measuring Fluoro-4 AM fluorescence intensity. The curves were drawn from captured images before and after potassium application. There was an increase of the fluorescent peaks in CELSR2-shRNA transfected neurons (control: 0.84 ± 0.13, and CELSR2-shRNA: 2.22 ± 0.09, P < 0.05, n = 3 independent experiments). * P < 0.05; **** P < 0.0001; Student’s t -test.

Article Snippet: Primary antibodies were: goat anti-ChAT (1:500, ab144p, Millipore), chicken anti-β-gal (1:500, ab9361, Abcam), rabbit anti-Isl1 (1:1000, Abcam) and goat anti-human CELSR2 (1:100, AF6379, R&D systems).

Techniques: Knockdown, shRNA, Control, Cell Culture, Transfection, Virus, Double Immunostaining, Western Blot, Fluorescence

Journal: Brain

Article Title: Inactivating Celsr2 promotes motor axon fasciculation and regeneration in mouse and human

doi: 10.1093/brain/awab317

Figure Lengend Snippet: Celsr2 knockout increases Cdc42/Rac1 and JNK/c-Jun signalling in injured ventral horns. ( A – D ) Western blot analysis of GST-pulldown proteins and samples from spinal ventral columns 3 days after root avulsion, using anti-Cdc42 ( A ), anti-Rac1 ( B ), anti-JNK ( C ), anti-c-Jun ( D ) antibodies. Anti-GADPH antibody was used as reference. ( E – H ) GTP-bound Cdc42 ( E ; control: 1 ± 0, mutant: 2.22 ± 0.17, P < 0.05) and Rac1 ( F ; control: 1 ± 0, mutant: 1.90 ± 0.13, P < 0.05) proteins, as well as concentrations of JNK ( G ; control: 2.01 ± 0.12, mutant: 3.65 ± 0.44, P < 0.05) and c-Jun ( H ; control: 0.48 ± 0.02, mutant: 0.84 ± 0.07, P < 0.05) were increased in mutant compared to control samples. * P < 0.05; Student’s t -test; n = 3 animals in each group.

Article Snippet: Primary antibodies were: goat anti-ChAT (1:500, ab144p, Millipore), chicken anti-β-gal (1:500, ab9361, Abcam), rabbit anti-Isl1 (1:1000, Abcam) and goat anti-human CELSR2 (1:100, AF6379, R&D systems).

Techniques: Knock-Out, Western Blot, Control, Mutagenesis

Journal: Nature Communications

Article Title: Brain-targeted drug delivery by manipulating protein corona functions

doi: 10.1038/s41467-019-11593-z

Figure Lengend Snippet: Characterization of ApoE functions in protein corona. a sLip or SP-sLip (100 μL, 10 mg mL −1 lipid) were incubated with BSA (10 μg mL −1 ) with or without rhApoE (10 μg mL −1 ) for 1 h at 37 °C, then recombinant human LRP1 (rhLRP1) was added and incubated for another 1 h at 37 °C. Protein coronas were isolated by centrifugation at 14k RCF, separated by SDS-PAGE and stained with silver. b SP-PEG3400-DSPE dissolved in methanol (0.1 μg per well) was coated in 96-well ELISA plate. After dry in room temperature, it was rinsed by cold PBS for three times. BSA (3%) was used to block for 2 h at room temperature. After thrice PBST rinses, anti-SP antibody (rabbit IgG, containing 0.1% BSA) was added and incubated for 1 h at 37 °C. Different concentrations of free SP (from 0 to 100 μg mL −1 ) or liposomes (sLip or SP-sLip, 0–5 mg mL −1 lipid) with or without plasma were incubated for another 1 h at 37 °C. HRP-anti-rabbit IgG was applied to detect the primary antibody using TMB kit. c sLip or SP-sLip were incubated with plasma for 1 h at 37 °C, then I 125 radiolabeled rhLRP1 was added in the mixture for another 1 h incubation. The radioactivity of liposomes after three cycles centrifugation and rinses was counted for detecting the absorbed rhLRP1. d Microscopic observation of cellular uptake of DiI-loaded sLip (sLip/DiI) or SP-sLip (SP-sLip/DiI) by bEnd.3 cells. sLip/DiI or SP-sLip/DiI was preincubated with PBS, rhApoE (10 μg mL −1 ) or mouse plasma for 1 h at 37 °C, then incubated with bEnd.3 cells for 4 h at 37 °C. Cellular uptake was detected by confocal laser scanning microscopy. Scale bar = 20 μm. e Quantitative analysis of DiI positive bEnd.3 cells by flow cytometry. Data are means ± SD ( n = 3), and analyzed with GraphPad Prism 6.0. *** p < 0.001 by student’s t -test

Article Snippet: Recombinant human LRP1 Cluster II Fc Chinera, Human IgG1 was from R&D system (Minneapolis, MN).

Techniques: Incubation, Recombinant, Isolation, Centrifugation, SDS Page, Staining, Enzyme-linked Immunosorbent Assay, Blocking Assay, Liposomes, Clinical Proteomics, Radioactivity, Confocal Laser Scanning Microscopy, Flow Cytometry