Journal: Frontiers in Cellular Neuroscience

Article Title: Validation of Hv 1 channel functions in BV2 microglial cells using small molecule modulators

doi: 10.3389/fncel.2025.1624224

Figure Lengend Snippet: Expression of Hv 1 channels in BV2 microglial cells. (A) Representative immunocytochemistry images of scramble shRNA transduced cells (control) and Hv 1 shRNA transduced cells stained with anti-Hv 1 antibody (red) and DAPI (blue). Scale bar = 40 μM (B) A comparison of Hv 1 channel protein expression by western blot in control cells and in Hv 1 shRNA transduced cells. (C) Hv 1 mRNA expression in control and Hv 1 shRNA cells was detected by using RT-PCR. (D) Representative whole-cell currents recorded in response to 3 s long pulses, stepping from a holding potential of −60 mV to levels ranging from −80 to +100 mV, with 20 mV increments in control (black traces) and Hv 1 shRNA cells (blue traces). (E) Mean current density versus membrane potential curves corresponding to the control condition and Hv 1 shRNA condition is plotted. Current density was measured by normalizing maximal peak currents with cell capacitance (pA/pF). *** p < 0.001, two-tailed unpaired t-test, N = 3.

Article Snippet: 10 μl of Lentiviral shRNA particles (mouse HVCN1; Origene-TL504897V and scramble; Origene–TR30021V) were added in culture medium with polybrene (final concentration 8 μg/ml) to the total volume of 1 ml DMEM/F-12 media and left for 72 h. Finally, the transduction efficiency was evaluated under the fluorescent microscope and gene knockdown was validated using immunocytochemistry, qPCR, western blot and patch clamp electrophysiology techniques.

Techniques: Expressing, Immunocytochemistry, shRNA, Control, Staining, Comparison, Western Blot, Reverse Transcription Polymerase Chain Reaction, Membrane, Two Tailed Test

Journal: Frontiers in Cellular Neuroscience

Article Title: Neutralization of Hv1/HVCN1 With Antibody Enhances Microglia/Macrophages Myelin Clearance by Promoting Their Migration in the Brain

doi: 10.3389/fncel.2021.768059

Figure Lengend Snippet: Expression of HVCN1 in microglia in vitro and in the normal adult cerebral cortex of mouse, marmoset, and human. (A) Hvcn1 mRNA is highly expressed in primary cultured mouse bone marrow derived macrophages (BMDM) and rat microglia, while exhibits much lower level in oligodendrocyte progenitor cells (OPCs) and astrocyte of the rat brain ( n = 3). (B) Specificity validation of the HVCN1 antibody in vitro by Western blot and Hvcn1 is overexpressed (OE) by transfecting with pLenti-CMV-HVCN1-HA-p2A-GFP plasmid whereas knocked down with siHVCN1 in COS-7 cells. (C) Representative images of Western blot of primary cultured cells: HVCN1 protein is expressed highly in rat microglia and mouse BMDM while exhibits low levels in rat OPCs, oligodendrocyte (OL), and astrocyte. (D,E) Specificity validation of HVCN1 antibody in vivo by immunofluorescence: targeted deleting Hvcn1 in CamKIIα + neurons of Hvcn1 fl/fl mice by focal injection of AAV9-CamKIIα-Cre-p2A-GFP virus into the cerebral cortex. In GFP + (green, CamKIIα + ) neurons in the cortex, immunofluorescence of HVCN1 (red) exhibits abundant signal in control (D) while eliminates labeling after Hvcn1 is targeted deleted (E) . As examples, arrows indicate GFP + (CamKIIα + ) neurons, while arrowheads indicate CamKIIα negative cells. (F,G) Representative immunofluorescence images: HVCN1 (red) is expressed in Iba1 + microglia (green) in the cerebral cortex of mouse (F) and marmoset (G) . (H) Immunofluorescence images: HVCN1 (red) is expressed in a few SOX10 + oligodendrocytes (green, upper) but not in GFAP + astrocyte (green, lower) in the cerebral cortex of the mouse. (I) Schematic drawing shows the site of human brain samples. (J) Representative Western blot images of HVCN1 in the postmortem human brain. (K) HVCN1 mRNA level in the cortex of the postmortem human brain ( n = 4) and the microglia isolated from the human brain ( n = 7) of patients with epilepsy . Scale bars (D–H) = 20 μm; the side length of the dashed-line square = 5 μm. Data are presented as mean ± SEM. Statistical tests: unpaired two-tailed t -test (K) ; * p < 0.05.

Article Snippet: Hvcn1 fl/fl mice (T019403) were obtained from GemPharmatech, Co., Ltd. (Nanjing, China).

Techniques: Expressing, In Vitro, Cell Culture, Derivative Assay, Biomarker Discovery, Western Blot, Plasmid Preparation, In Vivo, Immunofluorescence, Injection, Virus, Control, Labeling, Isolation, Two Tailed Test

Journal: Frontiers in Cellular Neuroscience

Article Title: Neutralization of Hv1/HVCN1 With Antibody Enhances Microglia/Macrophages Myelin Clearance by Promoting Their Migration in the Brain

doi: 10.3389/fncel.2021.768059

Figure Lengend Snippet: Upregulation of HVCN1 in microglia/macrophage in multiple injuries. Representative immunofluorescence images (A) and quantification of the proportion (B) : HVCN1 (red) in Iba1 + (green) microglia/macrophages in focal demyelination lesion in corpus callosum of the mouse brain at 5 days after focal injection of lysolecithin (LPC). ( n = 4). Two HVCN1 + Iba1 + microglia are indicated as examples. (C) Immunofluorescence images show that HVCN1 (red) is expressed in both iNOS + (green, up) type and Arg1 + (green, down) type polarized IB4 + microglia/macrophages (gray). (D) The proportion of iNOS + and Arg1 + type microglia/macrophages in HVCN1 + cells ( n = 4/group). (E) The proportion of HVCN1 + cells in iNOS + and Arg1 + type microglia/macrophages ( n = 4/group). (F,G) Representative immunofluorescence images (E) and quantification of the proportion (F) : HVCN1 (red) in Iba1 + (green) microglia/macrophage in corpus callosum of mouse brain at 12 h after intraperitoneal (i.p.) injection of lipopolysaccharide (LPS). ( n = 3/group). Two HVCN1 + Iba1 + microglia are indicated as examples. Scale bars (A,C,E) = 20 μm; the side length of the dashed-line square = 5 μm. Data are presented as mean ± SEM. Statistical tests: unpaired two-tailed t -test (B,D,F) ; ** p < 0.01, *** p < 0.001.

Article Snippet: Hvcn1 fl/fl mice (T019403) were obtained from GemPharmatech, Co., Ltd. (Nanjing, China).

Techniques: Immunofluorescence, Injection, Two Tailed Test

Journal: Frontiers in Cellular Neuroscience

Article Title: Neutralization of Hv1/HVCN1 With Antibody Enhances Microglia/Macrophages Myelin Clearance by Promoting Their Migration in the Brain

doi: 10.3389/fncel.2021.768059

Figure Lengend Snippet: Upregulation of HVCN1 in microglia/macrophage in neurodegenerative diseases of the central nervous system. (A,B) Representative Western blot images (A) and quantification of densities (B) of HVCN1 in the whole brain of SOD1 G 93 A mice and their littermates B6SJL mice ( n = 5). (C–F) Representative immunofluorescence images (C,E) and quantification of the proportion (D,F) : HVCN1 (red) is expressed in Iba1 + microglia/macrophages (green) in the brain (C,D) and the spinal cord (E,F) of SOD1 G 93 A mice and their littermates B6SJL mice ( n = 3). (G,H) Representative immunofluorescence images (G) and quantification of the proportion (H): HVCN1 (red) is expressed in Iba1 + microglia/macrophages (green) in the brain of wild type (WT) and R6/2 mouse ( n = 3). (I) HVCN1 mRNA level in the peripheral blood of patients with Huntington’s Disease ( n = 14) in comparison with the healthy control subjects ( n = 12) . Scale bars (C,E,G) = 20 μm; the side length of the dashed-line square = 5 μm. Data are presented as mean ± SEM. Statistical tests: unpaired two-tailed t -test (B,D,F,H,I) ; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Hvcn1 fl/fl mice (T019403) were obtained from GemPharmatech, Co., Ltd. (Nanjing, China).

Techniques: Western Blot, Immunofluorescence, Comparison, Control, Two Tailed Test

Journal: Frontiers in Cellular Neuroscience

Article Title: Neutralization of Hv1/HVCN1 With Antibody Enhances Microglia/Macrophages Myelin Clearance by Promoting Their Migration in the Brain

doi: 10.3389/fncel.2021.768059

Figure Lengend Snippet: HVCN1 genetic deletion in vitro promotes cell migration. (A) HVCN1 knockout strategy by CRISPR/Cas9 in Hela cells. Black boxes, exons; line, introns; Arrows, primers for PCR detection; Underlined, sgRNA targeted sequences; Bold, protospacer adjacent motif (PAM). (B) Genomic PCR products of WT and HVCN1 –/– Hela cells. The position of primers is shown in (A) . (C) Western blot images of HVCN1 in WT and HVCN1 –/– Hela cells. (D) Representative images of transwell assay. Scale bars = 100 μm. (E) Quantification of transwell assay ( n = 5). (F) A heatmap of migration related gene expression changes between WT and HVCN1 –/– 293T cells by RNA-seq. Data are presented as mean ± SEM. Statistical tests: unpaired two-tailed t -test (E) ; ** p < 0.01.

Article Snippet: Hvcn1 fl/fl mice (T019403) were obtained from GemPharmatech, Co., Ltd. (Nanjing, China).

Techniques: In Vitro, Migration, Knock-Out, CRISPR, Western Blot, Transwell Assay, Gene Expression, RNA Sequencing, Two Tailed Test

Journal: Frontiers in Cellular Neuroscience

Article Title: Neutralization of Hv1/HVCN1 With Antibody Enhances Microglia/Macrophages Myelin Clearance by Promoting Their Migration in the Brain

doi: 10.3389/fncel.2021.768059

Figure Lengend Snippet: Neutralization of HVCN1 with antibody promotes cell migration in vivo . (A) Schematic in vivo experiment design: CFSE-labeled myelin was co-injected with IgG on one side or with an HVCN1 antibody on the other side of the S1 cerebral cortex of the mouse. (B) Representative images show that neutralization with HVCN1 antibody increases CD68 + cell (red) density in both the core area (C) and the peripheral area (P) of the myelin (green) injection site. (C) Quantification of CD68 + microglia/macrophages density in the core area (left column) and the peripheral area (right column) of the myelin injection site ( n = 9). Scale bar (B) = 100 μm. Data are presented as mean ± SEM. Statistical tests: paired two-tailed t -test (C) ; ** p < 0.01, *** p < 0.001.

Article Snippet: Hvcn1 fl/fl mice (T019403) were obtained from GemPharmatech, Co., Ltd. (Nanjing, China).

Techniques: Neutralization, Migration, In Vivo, Labeling, Injection, Two Tailed Test

Journal: Frontiers in Cellular Neuroscience

Article Title: Neutralization of Hv1/HVCN1 With Antibody Enhances Microglia/Macrophages Myelin Clearance by Promoting Their Migration in the Brain

doi: 10.3389/fncel.2021.768059

Figure Lengend Snippet: Both genetic deletion and overexpression of HVCN1 do not affect phagocytosis of myelin in 293T cells. (A) HVCN1 gene knockout strategy by CRISPR/Cas9 in 293T cells. Black boxes, exons; line, introns; Arrows, primers for PCR detection; Underlined, sgRNA targeted sequences; Bold, PAM. (B) Genomic PCR products of WT and HVCN1 –/– 293T cells. The position of primers is shown in (A) . (C) Western blot images of HVCN1 in WT and HVCN1 –/– 293T cells. (D–F) Flow cytometry chart (D) and the quantification graphs (E,F) : HVCN1 knockout does not affect both the proportion of cells phagocytizing myelin (E) and the relative phagocytic index (F) in CFSE-labeled-myelin phagocytosis assay in 293T cells ( n = 3). (G–I) Flow cytometry chart (G) and the quantification graphs (H,I) show that overexpression of HVCN1 does not affect both the proportion of cells phagocytizing myelin (H) and the relative phagocytic index (I) in CFSE-labeled-myelin phagocytosis assay in 293T cells ( n = 3). Data are presented as mean ± SEM. Statistical tests: two-way ANOVA with Sidak’s multiple comparison posttest (E,F) or unpaired two-tailed t -test (H,I) .

Article Snippet: Hvcn1 fl/fl mice (T019403) were obtained from GemPharmatech, Co., Ltd. (Nanjing, China).

Techniques: Over Expression, Gene Knockout, CRISPR, Western Blot, Flow Cytometry, Knock-Out, Labeling, Phagocytosis Assay, Comparison, Two Tailed Test

Journal: Frontiers in Cellular Neuroscience

Article Title: Neutralization of Hv1/HVCN1 With Antibody Enhances Microglia/Macrophages Myelin Clearance by Promoting Their Migration in the Brain

doi: 10.3389/fncel.2021.768059

Figure Lengend Snippet: HVCN1 deletion impairs lysosome acidification during myelin phagocytosis in vitro . (A) Representative images show that HVCN1 (red) is colocalized with lysosome marker TMEM192 (green) in 293T cells overexpressed Plenti-HVCN1-mCherry and TMEM192-GFP plasmid. Scale bar = 50 μm (upper row) or 10 μm (lower row). Flow cytometry chart (B) and the quantification graphs (C,D) show that HVCN1 knockout decreases both the proportion of cells phagocytizing myelin (C) and the relative phagocytic index (D) in pHrodo-myelin phagocytosis assay in 293T cells ( n = 3). Flow cytometry chart (E) and the quantification graphs (F,G) show that overexpression of HVCN1 does not affect the proportion of phagocytic cells (F) and the relative phagocytic index (G) in pHrodo-myelin phagocytosis assay in 293T cell ( n = 3). Data are presented as mean ± SEM. Statistical tests: unpaired two-tailed t -test (C,D,F,G) ; * p < 0.05, ** p < 0.01.

Article Snippet: Hvcn1 fl/fl mice (T019403) were obtained from GemPharmatech, Co., Ltd. (Nanjing, China).

Techniques: In Vitro, Marker, Plasmid Preparation, Flow Cytometry, Knock-Out, Phagocytosis Assay, Over Expression, Two Tailed Test

Journal: Frontiers in Cellular Neuroscience

Article Title: Neutralization of Hv1/HVCN1 With Antibody Enhances Microglia/Macrophages Myelin Clearance by Promoting Their Migration in the Brain

doi: 10.3389/fncel.2021.768059

Figure Lengend Snippet: Knockdown of HVCN1 does not affect myelin phagocytosis and lysosome acidification in vitro . (A) Representative immunofluorescence images show that Hvcn1 knockdown by Cy3 labeled siRNA (red) does not affect myelin phagocytosis (green) in CD11b + BMDM (blue). Scale bar = 100 μm. Arrows indicate two example BMDM cells that phagocytized myelin. Flow cytometry chart (B) and the quantification graphs (C,D) show that Hvcn1 knockdown does not affect both the proportion of cells phagocytizing myelin (C) and the relative phagocytic index (D) in CFSE-labeled myelin phagocytosis assay in BMDM cells ( n = 3). Flow cytometry chart (E) and the quantification graphs (F,G) show that Hvcn1 knockout does not affect both the proportion of cells phagocytizing myelin (F) and the relative phagocytic index (G) in CFSE-labeled myelin phagocytosis assay in primary cultured microglia cells ( n = 5). Flow cytometry chart (H) and the quantification graphs (I,J) show that Hvcn1 knockdown does not affect the proportion of cells phagocytizing myelin (I) and the relative phagocytic index (J) in pHrodo-myelin phagocytosis assay in BMDM cells ( n = 7). Data are presented as mean ± SEM. Statistical tests: unpaired two-tailed t -test (C,D,F,G,I,J) .

Article Snippet: Hvcn1 fl/fl mice (T019403) were obtained from GemPharmatech, Co., Ltd. (Nanjing, China).

Techniques: Knockdown, In Vitro, Immunofluorescence, Labeling, Flow Cytometry, Phagocytosis Assay, Knock-Out, Cell Culture, Two Tailed Test

Journal: Frontiers in Cellular Neuroscience

Article Title: Neutralization of Hv1/HVCN1 With Antibody Enhances Microglia/Macrophages Myelin Clearance by Promoting Their Migration in the Brain

doi: 10.3389/fncel.2021.768059

Figure Lengend Snippet: Neutralizing HVCN1 with antibody enhances myelin debris clearance in the brain. (A,B) The binding patterns of the HVCN1 antibody to cells without (upper row) or with Triton X-100 permeabilization (lower row). Confocal images of GFP (green), HVCN1 (red) in 293T cells overexpressed with Plenti-HVCN1-GFP (A) . Confocal images of CD11b (green), HVCN1 (red) in BMDM cells (B) . (C) Schematic in vivo experiment design: CFSE labeled myelin was co-injected with IgG on one side or with an HVCN1 antibody on the other side of the S1 cerebral cortex of the mouse. (D–F) Representative images of the residual myelin (green) taken in every 36 or 48 μm thickness of brain section (D) and quantification of its volume (E) and its fluorescence intensity (F) ( n = 9). (G) Confocal images of the Iba1 + microglia (red), HVCN1 (gray), and phagocytized myelin (green). Scale bars = 100 μm (D) or 20 μm (A,B,G) , and the side length of the dashed-line square = 5 μm (A,B,G) . Data are presented as mean ± SEM. Statistical tests: paired two-tailed t -test (E,F) ; * p < 0.05, ** p < 0.01.

Article Snippet: Hvcn1 fl/fl mice (T019403) were obtained from GemPharmatech, Co., Ltd. (Nanjing, China).

Techniques: Binding Assay, In Vivo, Labeling, Injection, Fluorescence, Two Tailed Test