Journal: PLoS ONE

Article Title: The MMP-9/TIMP-1 Axis Controls the Status of Differentiation and Function of Myelin-Forming Schwann Cells in Nerve Regeneration

doi: 10.1371/journal.pone.0033664

Figure Lengend Snippet: A, A schematic of BrdU administration (100 mg/kg, i.p.) at days 2–4, followed by BrdU detection during remyelination (day 27 post-crush). B, Methylene blue Azure II staining of transverse araldite-embedded nerves at day 27 post-crush, the crush site (top panel). Effective remyelination in MMP-9 +/+ and MMP-9 −/− mice (representative of n = 3/group). Immunostaining for BrdU (red) and laminin (green) after A. (bottom panel); longitudinal nerve section, the crush site, day 27 post-crush. Increased number of cells that incorporate BrdU at days 2–4 post-crush remain in remyelinated nerves in MMP-9 −/− mice (representative of n = 3/group). Scale bars, 50 µm. C, The mean BrdU+ cell counts and pan-laminin staining from B. ± SEM in n = 4/group (*, p<0.05). D, Teased fibers in normal and remyelinated (day 27 post-crush) nerves in MMP-9 +/+ and MMP-9 −/− mice: immunofluorescent staining for laminin a2 (green), Caspr (red) and DAPI (blue). The distance between two arrows represents a myelin internode. The graph represents the mean internodal length (µm) ± SEM presented in A (*, p<0.05). N.S., not significant. Scale bars: A, 30 µm (insets, 1200× magnification). E, Electron microscopy of the nodes of Ranvier and the adjacent paranodes in normal and remyelinated (day 27 post-crush) nerves in MMP-9 +/+ and MMP −/− mice. (a), Paranodal loops in contact with the axolemma of a small myelinated fiber in MMP-9 +/+ mouse. (b), Desmosomal junctions between adjacent paranodal loops (arrow) and nodal microvilli (arrowhead) in MMP-9 −/− mouse. Inset shows a heminode with an axon-SC network (asterisk) in the juxtaparanodal region. (c), The lipid-laden macrophages (M, upper left) in the crushed nerves of MMP-9 +/+ mouse. (d) The heminode (left) is myelinated in the crushed nerve of MMP-9 −/− mouse. The lipid-laden macrophages in close contact with the axolemma in the non-myelinated region (M, right). Scale bar 1.33 µm in “a”; 0.87 µm in “b” (2.0 µm in inset); and 1.67 µm in c–d. F, Immunostaining for Na v 1.6 (red) and MAG (green) in teased nerve fibers. The graph represents the mean Na v 1.6 reactive clusters (red) signal area or the intensity ± SEM (*, p<0.05). Scale bars, 3 µm.

Article Snippet: Rabbit anti-Na v 1.6 (cat. #S0438, 1∶350), mouse anti-β-actin (cat. #A53166, 1∶10,000), rabbit anti-laminin (cat. #L9393, 1∶300), rat anti-laminin a2 (cat. #L0663, 1∶100) were from Sigma.

Techniques: Staining, Immunostaining, Electron Microscopy

Journal: Frontiers in Pharmacology

Article Title: The Na + /Ca 2+ Exchanger 3 Is Functionally Coupled With the Na V 1.6 Voltage-Gated Channel and Promotes an Endoplasmic Reticulum Ca 2+ Refilling in a Transgenic Model of Alzheimer’s Disease

doi: 10.3389/fphar.2021.775271

Figure Lengend Snippet: Distribution of Na V 1.6 channels and NCX3 in Tg2576 primary hippocampal neurons. (A) Confocal microscopic images displaying the distribution of Na V 1.6 (red) and NCX3 (green) immunoreactivities in hippocampal neurons isolated from WT and Tg2576 mouse embryos and cultured for 12 DIV (scale bars: in a–f: 20 µm). (B) Confocal microscopic images displaying the distribution of Na V 1.6 (red) and NCX3 (green) immunoreactivities in Tg2576 primary hippocampal neurons at 12 DIV. Arrows in a-c point to the intense co-localization of Na V 1.6 and NCX3 immunostaining along neurites (scale bars: 20 µm). (C) Densitometric analysis of Na V 1.6 (left) and NCX3 (right) fluorescence intensities in WT and Tg2576 neurons at 12 DIV. The data are expressed in arbitrary units (* p < 0.05 vs. WT). (D) Confocal microscopic images displaying the distribution of Na V 1.6 (red) and NCX3 (green) immunoreactivities in WT and Tg2576 primary hippocampal neurons at 12 DIV. Arrows in a–f point to Na V 1.6 and NCX3 immunoreactivities along the somatic plasma membrane of both WT and Tg2576 neurons. Panels g and h show higher magnification images of the frame depicted in c and f, respectively. Nuclei were counterstained with DAPI (blue) (scale bars: in a–f: 10 μm; in g and h: 2 µm). Panels i and j show the line profiling of Na V 1.6 (red) and NCX3 (green) fluorescence intensities along the line selected on the somatic plasma membrane of both WT (c) and Tg2576 (f) hippocampal neurons.

Article Snippet: The cell cultures were fixed in 4% wt/vol paraformaldehyde in phosphate buffer for 30 min. After blocking with 3% BSA, the cells were incubated with monoclonal anti-NCX3 (1:1,000, Trans Genic Inc., Japan) and rabbit policlonal anti-Na V 1.6 (1:1,000, Alomone Labs, Israel).

Techniques: Isolation, Cell Culture, Immunostaining, Fluorescence

Journal: Scientific Reports

Article Title: Amyloid β-Induced Upregulation of Na v 1.6 Underlies Neuronal Hyperactivity in Tg2576 Alzheimer’s Disease Mouse Model

doi: 10.1038/s41598-019-50018-1

Figure Lengend Snippet: Effect of Aβ 1–42 exposure on Na V 1.6 protein expression and activity in primary hippocampal neurons at 10–12 DIV. ( A ) Representative western blot (top) and densitometric quantification (bottom) of Na V 1.1 protein expression in primary hippocampal neurons under control conditions and after 5 μM Aβ 1–42 (24 h). Values are expressed as mean ± SEM of 3 independent experimental sessions. ( B ) Representative western blot (top) and densitometric quantification (bottom) of Na V 1.2 protein expression in primary hippocampal neurons under control conditions and after 5 μM Aβ 1–42 (24 h). Values are expressed as mean ± SEM of 3 independent experimental sessions. ( C ) Representative western blot (top) and densitometric quantification (bottom) of Na V 1.6 protein expression in primary hippocampal neurons under control conditions and after 5 μM Aβ 1–42 (24 h). Values are expressed as mean ± SEM of 3 independent experimental sessions. ** p < 0.01 versus control. ( D ) Representative traces of Na + currents recorded under control conditions, in the presence of siNa V 1.6 (50 nM; 48 h) and in the presence of anisomycin (10 μM; 30 min) in primary hippocampal neurons. (E) Representative traces of Na + currents recorded after 5 μM Aβ 1–42 (24 h) alone, in the presence of siNa V 1.6 (50 nM; 48 h) and in the presence of anisomycin (10 μM; 30 min) in primary hippocampal neurons. ( F ) Normalization of Na + current densities, at −20 mV, represented in panel D and E. The number of cells used for each experimental condition is noted on the bars, values are expressed as percentage mean ± SEM of 3 independent experimental sessions. *** p < 0.001 versus control, # p < 0.001 versus control Aβ 1–42 . (G) Representative western blot of Na V 1.6 protein expression (top) in the presence of siNa V 1.6 (50 nM; 48 h) in primary hippocampal neurons at 12 DIV. Quantification of siNav1.6 inhibition in primary hippocampal neurons (bottom). Values are expressed as percentage mean ± SEM of 3 independent experimental sessions. ** p < 0.01 versus control neurons.

Article Snippet: In brief, cell cultures were fixed in 4% paraformaldehyde in PBS for 30 min. After blockage with Rodent M Block (Biocare Medical, Concord, CA, USA) for 1 hour, cells were incubated with rabbit polyclonal anti-Na V 1.6 antibody (1:2000 Alomone Labs, Israel) and mouse monoclonal anti-MAP2 antibody (1:2000, Sigma Aldrich, Milan, Italy) at 4 °C overnight for 24 hours.

Techniques: Expressing, Activity Assay, Western Blot, Inhibition

Journal: Scientific Reports

Article Title: Amyloid β-Induced Upregulation of Na v 1.6 Underlies Neuronal Hyperactivity in Tg2576 Alzheimer’s Disease Mouse Model

doi: 10.1038/s41598-019-50018-1

Figure Lengend Snippet: Expression and activity of Na V 1.6 channels in Tg2576 primary hippocampal neurons. ( A ) Representative traces of Na + currents recorded in WT and Tg2576 primary hippocampal neurons after 8 and 12 DIV. ( B ) Normalization of Na + current densities at −20 mV represented in panel A. Values are expressed as mean ± SEM of current densities of 3 independent experimental sessions. The number of cells used for each experimental condition is noted on the bars, values are expressed as percentage mean ± SEM of 3 independent experimental sessions. *** p < 0.001 versus WT. ( C ) Representative western blot (top) and densitometric quantification (bottom) of Na V 1.1 protein expression in WT and Tg2576 primary hippocampal neurons after 12 DIV. Values are expressed as mean ± SEM of 3 independent experimental sessions. ( D ) Representative western blot (top) and densitometric quantification (bottom) of Na V 1.2 protein expression in WT and Tg2576 primary hippocampal neurons after 12 DIV. Values are expressed as mean ± SEM of 3 independent experimental sessions. ( E ) Representative western blot (top) and densitometric quantification (bottom) of Na V 1.6 protein expression in WT and Tg2576 primary hippocampal neurons after 12 DIV. Values are expressed as mean ± SEM of 3 independent experimental sessions. ** p < 0.01 versus WT. (F) Representative confocal images displaying Na V 1.6 distribution in WT (left) and Tg2576 (right) primary hippocampal neurons after 12 DIV. Scale bars: 20 μm.

Article Snippet: In brief, cell cultures were fixed in 4% paraformaldehyde in PBS for 30 min. After blockage with Rodent M Block (Biocare Medical, Concord, CA, USA) for 1 hour, cells were incubated with rabbit polyclonal anti-Na V 1.6 antibody (1:2000 Alomone Labs, Israel) and mouse monoclonal anti-MAP2 antibody (1:2000, Sigma Aldrich, Milan, Italy) at 4 °C overnight for 24 hours.

Techniques: Expressing, Activity Assay, Western Blot

Journal: Scientific Reports

Article Title: Amyloid β-Induced Upregulation of Na v 1.6 Underlies Neuronal Hyperactivity in Tg2576 Alzheimer’s Disease Mouse Model

doi: 10.1038/s41598-019-50018-1

Figure Lengend Snippet: Effect of siNa V 1.6 and anisomycin on Na V 1.6 protein expression and activity in Tg2576 primary hippocampal neurons. ( A ) Representative traces of Na + currents recorded in WT primary hippocampal neurons after 12 DIV under control conditions, in the presence of siNa V 1.6 (50 nM; 48 h) and in the presence of anisomycin (10 μM; 30 min). ( B ) Representative traces of Na + currents recorded in Tg2576 primary hippocampal neurons after 12 DIV under control conditions, in the presence of siNa V 1.6 (50 nM; 48 h) and in the presence of anisomycin (10 μM; 30 min). ( C ) Normalization of Na + current densities at −20 mV represented in panel A and B. The number of cells used for each experimental condition is noted on the bars, values are expressed as percentage mean ± SEM of 3 independent experimental sessions. ** p < 0.01 versus control WT, *** p < 0.001 versus control WT, # p < 0.001 versus control Tg2576. ( D ) Representative current tracings recorded in the gap-free mode in WT and Tg2576 hippocampal neurons after 12 DIV under control conditions, in the presence of siNa V 1.6 (50 nM; 48 h) and in the presence of anisomycin (10 μM; 30 min). ( E ) Quantification of spike frequency recorded in WT and Tg2576 hippocampal neurons after 12 DIV under control conditions, in the presence of siNa V 1.6 (50 nM; 48 h) and in the presence of anisomycin (10 μM; 30 min). The number of cells used for each experimental condition is noted on the bars, values are expressed as percentage mean ± SEM of 3 independent experimental sessions. *** p < 0.001 versus WT. # p < 0.001 versus control Tg2576. ( F ) Quantification of membrane depolarization recorded in WT and Tg2576 primary hippocampal neurons after 12 DIV under control conditions, in the presence of siNa V 1.6 (50 nM; 48 h) and in the presence of anisomycin (10 μM; 30 min). The number of cells used for each experimental condition is noted on the bars, values are expressed as percentage mean ± SEM of 3 independent experimental sessions. ** p < 0.01 versus WT. # p < 0.001 versus control Tg2576

Article Snippet: In brief, cell cultures were fixed in 4% paraformaldehyde in PBS for 30 min. After blockage with Rodent M Block (Biocare Medical, Concord, CA, USA) for 1 hour, cells were incubated with rabbit polyclonal anti-Na V 1.6 antibody (1:2000 Alomone Labs, Israel) and mouse monoclonal anti-MAP2 antibody (1:2000, Sigma Aldrich, Milan, Italy) at 4 °C overnight for 24 hours.

Techniques: Expressing, Activity Assay

Journal: Scientific Reports

Article Title: Amyloid β-Induced Upregulation of Na v 1.6 Underlies Neuronal Hyperactivity in Tg2576 Alzheimer’s Disease Mouse Model

doi: 10.1038/s41598-019-50018-1

Figure Lengend Snippet: Immunocytochemical analysis of Na V 1.6 protein expression after anisomycin treatment in Tg2576 primary hippocampal neurons at 12 DIV. ( A ) Confocal double immunofluorescence images displaying Na V 1.6 (green) and MAP2 (red) distribution in WT (a-c) and Tg2576 primary hippocampal neurons in the absence (d-f) or in the presence (g-i) of anisomycin. Scale bars in a-i: 20 μm. ( B ) Quantitative analyses of Na V 1.6-positive puncta within the soma of WT and Tg2576 primary hippocampal neurons in the absence or in the presence of anisomycin. Scale bars: 5 μm. Data are expressed as mean ± SEM of values obtained from 20 cells per group in 3 independent experimental sessions. ** p < 0.01 versus WT; # p < 0.001 versus Tg2576.

Article Snippet: In brief, cell cultures were fixed in 4% paraformaldehyde in PBS for 30 min. After blockage with Rodent M Block (Biocare Medical, Concord, CA, USA) for 1 hour, cells were incubated with rabbit polyclonal anti-Na V 1.6 antibody (1:2000 Alomone Labs, Israel) and mouse monoclonal anti-MAP2 antibody (1:2000, Sigma Aldrich, Milan, Italy) at 4 °C overnight for 24 hours.

Techniques: Expressing, Immunofluorescence

Journal: Scientific Reports

Article Title: Amyloid β-Induced Upregulation of Na v 1.6 Underlies Neuronal Hyperactivity in Tg2576 Alzheimer’s Disease Mouse Model

doi: 10.1038/s41598-019-50018-1

Figure Lengend Snippet: Evaluation of Na V 1.6 protein expression in the hippocampus of 3-month-old WT and Tg2576 mice. ( A ) Representative western blot (top) and densitometric quantification (bottom) of Na V 1.1 protein expression in the hippocampus of WT and Tg2576 mice. Values are expressed as mean ± SEM of 3 independent experimental sessions. ( B ) Representative western blot (top) and densitometric quantification (bottom) of Na V 1.2 protein expression in the hippocampus of WT and Tg2576 mice. Values are expressed as mean ± SEM of 3 independent experimental sessions. ( C ) Representative western blot (top) and densitometric quantification (bottom) of Na V 1.6 protein expression in the hippocampus of WT and Tg2576 mice. Values are expressed as mean ± SEM of 3 independent experimental sessions. ** p < 0.01 versus WT. ( D ) Confocal double immunofluorescence images displaying Na V 1.6 (green) and MAP2 (red) distribution in the hippocampus of 3-month-old WT (a-c) and Tg2576 mice (d-f). Scale bars in a-f: 20 μm. (E) Quantitative analyses of Na V 1.6-positive puncta within the soma of WT and Tg2576 neurons in the hippocampus of 3-month-old mice. Values are expressed as mean ± SEM of 3 independent experimental sessions. **p < 0.01 versus WT.

Article Snippet: In brief, cell cultures were fixed in 4% paraformaldehyde in PBS for 30 min. After blockage with Rodent M Block (Biocare Medical, Concord, CA, USA) for 1 hour, cells were incubated with rabbit polyclonal anti-Na V 1.6 antibody (1:2000 Alomone Labs, Israel) and mouse monoclonal anti-MAP2 antibody (1:2000, Sigma Aldrich, Milan, Italy) at 4 °C overnight for 24 hours.

Techniques: Expressing, Western Blot, Immunofluorescence