Journal: Cerebral Cortex (New York, NY)

Article Title: L1 and CHL1 Cooperate in Thalamocortical Axon Targeting

doi: 10.1093/cercor/bhq115

Figure Lengend Snippet: Analysis of CHL1−/−/L1−/y double mutant mice and expression of CHL1 and L1 in the thalamocortical pathway. (A) CHL1−/−/L1−/y double mutant mice are reduced in size compared with CHL1−/− mutant mice. (B) Nissl staining of primary somatosensory cortex in WT, L1−/y, CHL1−/−, and CHL1−/−/L1−/y mice (P21) in coronal sections. (C and D) Nissl staining of S1 cortex in WT (C) and CHL1−/−/L1−/y mice (D) (P7) showing normal barrels (circled in WT Cc and mutant Dd). (E–G) Normal boundary between V1 and V2 visualized by YFP-expressing layer V pyramidal neurons in WT (E), CHL1−/− (F), and CHL1−/−/L1−/y mice crossed to Thy1-YFP (line H) reporter mice at P21. (H–K) Normal size and location of DT nuclei visualized by Nissl staining of WT (H) and CHL1−/−/L1−/y double mutant mice (I) at P7. dLGN, dorsal LGN nuclei. (L–N) Immunofluorescence staining for L1 and CHL1 in the thalamocortical pathway of WT embryos (E14.5) in midcoronal sections. C1, L1−/y brain stained with L1 antibody. C2, CHL1−/− brain stained with CHL1 antibody. (O–P) Immunofluorescence staining for L1 and CHL1 in the VTe of WT embryos (E14.5) in coronal sections. (R–T) Higher magnification images of L–N. (U–W) Immunofluorescence staining for L1 and CHL1 in the DT of WT embryos (E14.5) in horizontal sections. R, rostral; C, caudal; m, medial, l, lateral. (X–Z) Immunofluorescence staining for L1 and CHL1 in the neocortex of WT embryos (E14.5). MZ, marginal zone; CP, cortical plate; VZ, ventricular zone. (Aa) In situ hybridization for L1 mRNA in serial horizontal sections of WT embryos (E14.5). L1 transcripts were present throughout the DT (dashed lines). (Bb) No expression was observed with the sense probe (control). Magnification bar = 100 μm in B; 200 μm in C and D; 500 μm in Aa, E–N, U–W; 300 μm in O–Q; 50 μm in X–Z.

Article Snippet: Immunostaining, Analysis of Cortical and Thalamic Areas, and Preparation of Neuronal Cultures Immunofluorescence staining was conducted using 10-μm frozen sections of mouse brain using antibodies directed against CHL1 (goat polyclonal anti-CHL1, R&D Systems; 1:200) and L1 (rat polyclonal anti-L1, Abcam; 1:50) as described ( Demyanenko et al. 1999 ), with FITC- or rhodamine-conjugated secondary antibodies.

Techniques: Mutagenesis, Expressing, Staining, Immunofluorescence, In Situ Hybridization

Journal: Cerebral Cortex (New York, NY)

Article Title: L1 and CHL1 Cooperate in Thalamocortical Axon Targeting

doi: 10.1093/cercor/bhq115

Figure Lengend Snippet: Retrograde tracing of thalamic axons from distinct cortical areas in WT, CHL1−/−, L1−/y, and CHL1−/−/L1−/y double mutant mice (P7). (A) Scheme showing results of retrograde tracing of thalamocortical axons with DiI or DiA from different neocortical areas to distinct DT nuclei in WT mice. (A1) Retrograde labeling of WT motor thalamic nuclei (VA/VL) but not VB nuclei (VPM, VPL) is shown following injection of DiI into M1. (A2) Retrograde labeling of WT dorsal LGN and VB is shown following dual injection of DiI into V1 and DiA into S1. Orientation of all sections in Figure 2 are indicated (R, rostral; L, lateral). Injection sites and position of the sections are diagrammatically illustrated in each panel. (B) Scheme showing results of retrograde tracing of thalamocortical axons with DiI or DiA from different neocortical areas to distinct DT nuclei in L1−/y mice. (B1) Retrograde labeling of VL but not VB nuclei (VPM, VPL) following injection of DiA into M1. (B2) Labeling of VB but not dorsal LGN following injection of DiI into S1. (B3) Retrograde labeling of dorsal LGN following injection of DiI into V1. (C) Scheme showing results of retrograde tracing of thalamocortical axons from different neocortical areas to distinct DT nuclei of CHL1−/− mice, in which axon contingents from the VB nucleus misproject caudally to V1. (C1) Retrograde labeling of VB and dorsal LGN following dual injections of DiA into S1 and DiI into V1. Neuronal soma in VB nuclei are inappropriately labeled with DiI. Injection sites are shown below the scheme. (D) Scheme showing results of retrograde tracing of thalamocortical axons from different neocortical areas to distinct DT nuclei of CHL1−/−/L1−/y mice, in which axon contingents from VA/VL and VB nuclei misprojected caudally to V1. (D1) Injection site of DiA into M1 of the neocortex (m, medial; l, lateral). (D2) Retrograde labeling of VA and VL but not VB nuclei (VPM, VPL) following DiA injection into M1. 4′,6-diamidino-2-phenylindole staining shows hippocampus (HC) and habenula (Hb). (D3) Injection site of DiI into S1 (m, medial; l, lateral). (D4) Labeling of VPM and VPL nuclei following DiI injection into S1. (D5) Labeling of dorsal LGN, VPM, and VPL nuclei following DiI injection into V1. (D6) Labeling of VA and VL nuclei following DiI injection into V1. (D7) Labeling of VPM and dorsal LGN following DiA injection into V1. (D8) Labeling of VA and VL nuclei following DiA injection into V1. M1, primary motor cortex; S1, primary somatosensory cortex; V1, primary visual cortex. Magnification bars equal 500 μm in all panels except D8 (300 μm).

Article Snippet: Immunostaining, Analysis of Cortical and Thalamic Areas, and Preparation of Neuronal Cultures Immunofluorescence staining was conducted using 10-μm frozen sections of mouse brain using antibodies directed against CHL1 (goat polyclonal anti-CHL1, R&D Systems; 1:200) and L1 (rat polyclonal anti-L1, Abcam; 1:50) as described ( Demyanenko et al. 1999 ), with FITC- or rhodamine-conjugated secondary antibodies.

Techniques: Retrograde Tracing, Mutagenesis, Labeling, Injection, Staining

Journal: Cerebral Cortex (New York, NY)

Article Title: L1 and CHL1 Cooperate in Thalamocortical Axon Targeting

doi: 10.1093/cercor/bhq115

Figure Lengend Snippet: L1 and CHL1 mediate EphrinA5-induced growth cone collapse. (A) Dissociated cortical neurons from WT, L1−/y, and CHL1−/− embryos (E14.5) were cultured for 3 days, treated with 30 nM EphrinA5 or 30 nM IgG for 30 min, fixed, and stained with phalloidin for visualizing F-actin. Images are representative examples of noncollapsed and collapsed growth cones scored following IgG or EphrinA5-Fc treatment. Magnification bar = 5 μm. (B) Quantification of growth cone collapse in dissociated cortical neuron cultures shown in A, in response to control IgG or EphrinA5-Fc. Percent growth cone collapse is expressed as the mean ± standard error of the mean (SEM) (n = number of growth cones scored). Asterisk indicates significant differences (P < 0.05; 2-tailed t-test). (C) WT and CHL1−/− thalamic explants (E14.5) were cultured for 3 days, treated with 30 nM EphrinA5-AP or 30 nM Fc-AP, fixed, and stained with phalloidin. Representative examples of noncollapsed and collapsed growth cones following control Fc-AP or EphrinA5-AP treatment. Magnification bar in A = 5 μm. (D) Quantification of growth cone collapse from thalamic explants in C, in response to EphrinA5-AP. Percent growth cone collapse is mean ± SEM. n = number of growth cones scored. Asterisk indicates significant differences in means (P < 0.05; 2-tailed t-test). (E) Immunofluorescence staining showing colocalization of L1 and CHL1 in embryonic mouse cortical and thalamic neurons in dissociated cultures. Colocalization was evident in growth cones of cortical and thalamic neurons (lines). Some neurites of cortical neurons appeared enriched for L1 (arrows) and others for CHL1 (arrowheads). Control staining without primary antibodies is shown for cortical neurons (C1) and thalamic neurons (C2). Magnification bars = 20 μm.

Article Snippet: Immunostaining, Analysis of Cortical and Thalamic Areas, and Preparation of Neuronal Cultures Immunofluorescence staining was conducted using 10-μm frozen sections of mouse brain using antibodies directed against CHL1 (goat polyclonal anti-CHL1, R&D Systems; 1:200) and L1 (rat polyclonal anti-L1, Abcam; 1:50) as described ( Demyanenko et al. 1999 ), with FITC- or rhodamine-conjugated secondary antibodies.

Techniques: Cell Culture, Staining, Immunofluorescence

Journal: Cerebral Cortex (New York, NY)

Article Title: L1 and CHL1 Cooperate in Thalamocortical Axon Targeting

doi: 10.1093/cercor/bhq115

Figure Lengend Snippet: L1 and CHL1 associate with EphA receptors. (A–D) CHL1 or L1 were coexpressed with EphA3, EphA4, EphA7, or EphB2 from pcDNA3 plasmids after transient transfection of HEK293T cells. Lysates (500-μg protein) were immunoprecipitated (IP) using antibodies against EphA3, EphA4, EphA7, L1, CHL1, or normal IgG and immunoblotted (IB) for the indicated protein. Blots were reprobed with antibodies used for IP. IB of cell lysates (25 μg) confirmed expression of each protein in transfected cells. Position of molecular weight markers are indicated by solid arrowheads (250 kDa) and open arrowheads (130 kDa). L1 protein was detected in (A) EphA3, (B) EphA4, and (C) EphA7 immunoprecipitates. CHL1 was not detected in the immunoprecipitates of (A) EphA3 or (B) EphA4 but was detected in (C) EphA7 immunoprecipitates. EphB2 was not detected in either (D) L1 or CHL1 immunoprecipitates. (E) EphA3, EphA4, EphA7, L1, or CHL1 were expressed alone from pcDNA3 plasmids by transient transfection of HEK293T cells. Lysates (25-μg protein) were immunoblotted with antibodies L1 or CHL1 to demonstrate their specificity and showed no cross-reactivity with EphA receptors (upper panels). In lower panels, blots were stripped and reprobed with EphA-specific antibodies to confirm expression of each protein in transfected cells.

Article Snippet: Immunostaining, Analysis of Cortical and Thalamic Areas, and Preparation of Neuronal Cultures Immunofluorescence staining was conducted using 10-μm frozen sections of mouse brain using antibodies directed against CHL1 (goat polyclonal anti-CHL1, R&D Systems; 1:200) and L1 (rat polyclonal anti-L1, Abcam; 1:50) as described ( Demyanenko et al. 1999 ), with FITC- or rhodamine-conjugated secondary antibodies.

Techniques: Transfection, Immunoprecipitation, Expressing, Molecular Weight

Journal: Cerebral Cortex (New York, NY)

Article Title: L1 and CHL1 Cooperate in Thalamocortical Axon Targeting

doi: 10.1093/cercor/bhq115

Figure Lengend Snippet: Schematic diagrams of thalamocortical trajectories based on axon tracing experiments in different mouse genotypes: (A) WT, (B) CHL1−/−, (C) CHL1−/− L1−/y, (D) L1−/y, (E) EphrinA5−/−/EphA4−/−, and (F) Neuropilin-1 mutant defective in Sema3A binding. Normal projections are depicted as solid lines and misprojections as dashed lines. In CHL1−/−/L1−/y and EphrinA5−/−/EphA4−/− mutants, VA or VL axon contingents caudally misproject to V1 and VB, respectively. Note similar misprojection of VB axon contingents to V1 in CHL1−/− and Neuropilin1Sema3A−/− mutants. L1−/y mutants show normal projections. The location of rostrocaudal gradients of ephrinA5, Sema3A, and EphA3, 4, 7 are indicated by shading.

Article Snippet: Immunostaining, Analysis of Cortical and Thalamic Areas, and Preparation of Neuronal Cultures Immunofluorescence staining was conducted using 10-μm frozen sections of mouse brain using antibodies directed against CHL1 (goat polyclonal anti-CHL1, R&D Systems; 1:200) and L1 (rat polyclonal anti-L1, Abcam; 1:50) as described ( Demyanenko et al. 1999 ), with FITC- or rhodamine-conjugated secondary antibodies.

Techniques: Mutagenesis, Binding Assay

Journal: Scientific Reports

Article Title: BACE1 partial deletion induces synaptic plasticity deficit in adult mice

doi: 10.1038/s41598-019-56329-7

Figure Lengend Snippet: BACE1-mediated processing of APP and CHL1 is reduced in cortex of young BACE1 cKO mice following tamoxifen treatment. Cortex homogenates from TAM- or VEH-treated mice were resolved by SDS-PAGE for Western blot analysis of APP and CHL1 processing. Homogenates from aged-matched BACE +/− and BACE1 −/− were also loaded as control samples. Representative blots of ( a ) APP-full length (APP-FL) (C1/6.1), ( b ) APP-Carboxy Terminal Fragments (CTFs) (C1/6.1) and ( c ) CHL1. ( d ) Densitometry analysis of protein expression. Protein amount was normalized to protein levels in control mice (set at 1). APP-FL, pC99 and pC89 were normalized to GAPDH (MAB374) while CHL1-FL and CHL1-NTF were normalized to β-tubulin (JDR.3B8). APP processing was reduced in TAM-treated mice as demonstrated by the accumulation of APP-FL (C1/6.1), and reduced levels of the βCTFs pC99 and pC89. βCTFs were clearly identified because missing in the BACE1 −/− sample. CHL1-FL (AF2147) levels were increased while CHL1-N Terminal Fragment (CHL1-NTF) levels were not affected in cortex of TAM-treated mice. However, the CHL1-NTF/CHL1-FL ratio was significantly decreased in TAM-treated mice demonstrating reduced BACE1 processing (VEH n = 8; TAM n = 8). ( e ) Aβx-40 was quantified from brain homogenates by ELISA (VEH n = 8; TAM n = 8). Levels of Aβx-40 expressed as pMol/g of cortex were significantly reduced in TAM-treated mice (~50% decrease). Results were plotted as Mean ± SEM, ***p < 0.001; ****p < 0.0001; n.s. = not significant, Student’s t test.

Article Snippet: Immunoblot and serial fractionation were performed as previously described with the following antibodies: rabbit monoclonal anti-BACE1 (1:1000; D10E5; Cell signaling technology); mouse monoclonal anti-APP (and APP CTFs) antibody (1:5000; C1/6.1; BioLegend); goat polyclonal anti-N-terminal CHL1 antibody (for CHL1-FL and CHL1-NTF) (1:1000; AF2147; R&D Systems); mouse monoclonal anti-GAPDH (1:10,000; MAP374; Millipore); mouse monoclonal anti-β-tubulin (1:10,000; JDR.3BR; Sigma); mouse monoclonal anti-calnexin (1:2000; 610523; BD biosciences); rabbit polyclonal anti-ADAM10 (1:1000;AB19026; Millipore); rabbit polyclonal anti-PS1 AB14 (1:1000) and rat monoclonal anti-SEZ6 (1:250) , rat monoclonal anti-APPsβ (1:40) and HRP-conjugated secondary antibodies visualized by ECL (GE Healthcare).

Techniques: SDS Page, Western Blot, Control, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: BACE1 partial deletion induces synaptic plasticity deficit in adult mice

doi: 10.1038/s41598-019-56329-7

Figure Lengend Snippet: BACE1-mediated processing of APP and CHL1 is reduced in cortex of aged BACE1 cKO mice following tamoxifen treatment. Cortex homogenates from TAM- or VEH-treated mice were resolved by SDS-PAGE for Western blot analysis of APP and CHL1 processing. Homogenates from aged-matched BACE +/− and BACE1 −/− were also loaded as control samples. APP-FL, pC99 and pC89 were normalized to GAPDH (MAB374) while CHL1-FL and CHL1-NTF were normalized to β-tubulin (JDR.3B8). Protein amount was normalized to protein levels in control mice injected with vehicle (set at 1). Representative blots of ( a ) APP-FL (C1/6.1), ( b ) APP-CTFs (C1/6.1) and (c ) CHL1. ( d ) Densitometry analysis of protein expression. APP processing was reduced in TAM-treated mice as demonstrated by the accumulation of APP-FL (C1/6.1), and reduced levels of the βCTFs pC99 and pC89. βCTFs were clearly identified because missing in the BACE1 −/− sample. CHL1-FL (AF2147) levels were increased and CHL1-NTF levels were significantly reduced. Furthermore, the CHL1-NTF/CHL1-FL ratio was significantly decreased in TAM-treated mice demonstrating reduced BACE1 processing (VEH n = 7; TAM n = 7). ( e ) Quantification of Aβx-40 was performed by MSD immunoassay on cortex homogenates and expressed as pMol/g of cortex. The decrease of levels of Aβx-40 in TAM-treated mice was comparable to the one observed in samples collected from young TAM-treated mice (~50% decrease) (VEH n = 7; TAM n = 7). Results were plotted as Mean ± SEM, *p < 0.05; **p < 0.005; ***p < 0.001; ****p < 0.0001; n.s. = not significant, Student’s t test.

Article Snippet: Immunoblot and serial fractionation were performed as previously described with the following antibodies: rabbit monoclonal anti-BACE1 (1:1000; D10E5; Cell signaling technology); mouse monoclonal anti-APP (and APP CTFs) antibody (1:5000; C1/6.1; BioLegend); goat polyclonal anti-N-terminal CHL1 antibody (for CHL1-FL and CHL1-NTF) (1:1000; AF2147; R&D Systems); mouse monoclonal anti-GAPDH (1:10,000; MAP374; Millipore); mouse monoclonal anti-β-tubulin (1:10,000; JDR.3BR; Sigma); mouse monoclonal anti-calnexin (1:2000; 610523; BD biosciences); rabbit polyclonal anti-ADAM10 (1:1000;AB19026; Millipore); rabbit polyclonal anti-PS1 AB14 (1:1000) and rat monoclonal anti-SEZ6 (1:250) , rat monoclonal anti-APPsβ (1:40) and HRP-conjugated secondary antibodies visualized by ECL (GE Healthcare).

Techniques: SDS Page, Western Blot, Control, Injection, Expressing

Journal: Scientific Reports

Article Title: BACE1 partial deletion induces synaptic plasticity deficit in adult mice

doi: 10.1038/s41598-019-56329-7

Figure Lengend Snippet: Axon guidance defects were absent in hippocampus mossy fibers of aged BACE1 cKO mice following partial BACE1 deletion. ( a ) Coronal sections collected from aged mice were stained with anti-synaptoporin (SPO) antibody (green) and DAPI (blue). Scale bar 50 μm. ( b) Quantification of IPB length showed no alteration in TAM-treated mice compared to controls. IPB length was normalized on the length of the CA3 stratum lucidum (VEH n = 8; TAM n = 7, 3 to 4 sections per mouse). ( c ) Representative microscopy images showing reduced BACE1 (D10E5) expression in the hippocampus of TAM-treated mice. BACE1 signal was totally absent in BACE −/− mice, used as control to evaluate the amount of background in the staining. Scale bar 200 μm. Hippocampus full homogenates from TAM- or VEH-treated mice were resolved by SDS-PAGE for analysis of APP processing and fractionated (soluble and membrane fractions) for the analysis of SEZ6 and CHL1 processing. Homogenates from aged-matched BACE +/− and BACE1 −/− were loaded as control samples. Representative blots of ( d ) APP-FL (C1/6.1) and APP- CTFs (C1/6.1), ( e ) fractionation blots of sAPPβ (BAWT), SEZ6 (14E5) and CHL1 (AF2147). ( f ) Densitometry analysis of protein expression. APP processing was reduced in TAM-treated mice as demonstrated by the accumulation of APP-FL (C1/6.1), and reduced levels of the βCTFs pC99 and pC89, and sAPPβ. βCTFs and sAPPβ were identified because missing in the BACE1 −/− sample. SEZ6 processing was decreased in TAM-treated mice with accumulation of the full length and decreased levels of the ectodomain (SEZ6-NTF) as well as decreased SEZ6-NTF/SEZ6FL ratio. Processing of CHL1 was also impaired as showed by increased of CHL1-FL levels, while CHL1-NTF was not altered. CHL1-NTF/CHL1-FL ratio was significantly decreased. APP-FL, CTFs, SEZ6-NTF and CHL1-NTF were normalized to GAPDH (MAB374), SEZ6-FL and CHL1-FL were normalized to Calnexin (610523) (VEH n = 5; TAM n = 5). ( g ) Aβx-40 was quantified from hippocampus homogenates by MSD immunoassay. TAM-treated group displayed a significant reduction of Aβx-40 levels (~50% decrease) compared to control (VEH n = 7; TAM n = 7). Results were plotted as Mean ± SEM, **p < 0.005; ***p < 0.001; n.s. = not significant, Student’s t test. DG: dentate gyrus, IPB: infrapyramidal bundle, slu: stratum lucidum, MB: main bundle.

Article Snippet: Immunoblot and serial fractionation were performed as previously described with the following antibodies: rabbit monoclonal anti-BACE1 (1:1000; D10E5; Cell signaling technology); mouse monoclonal anti-APP (and APP CTFs) antibody (1:5000; C1/6.1; BioLegend); goat polyclonal anti-N-terminal CHL1 antibody (for CHL1-FL and CHL1-NTF) (1:1000; AF2147; R&D Systems); mouse monoclonal anti-GAPDH (1:10,000; MAP374; Millipore); mouse monoclonal anti-β-tubulin (1:10,000; JDR.3BR; Sigma); mouse monoclonal anti-calnexin (1:2000; 610523; BD biosciences); rabbit polyclonal anti-ADAM10 (1:1000;AB19026; Millipore); rabbit polyclonal anti-PS1 AB14 (1:1000) and rat monoclonal anti-SEZ6 (1:250) , rat monoclonal anti-APPsβ (1:40) and HRP-conjugated secondary antibodies visualized by ECL (GE Healthcare).

Techniques: Staining, Microscopy, Expressing, Control, SDS Page, Membrane, Fractionation