Journal: Experimental eye research

Article Title: Elevated ocular pressure reduces voltage-gated sodium channel NaV1.2 protein expression in retinal ganglion cell axons

doi: 10.1016/j.exer.2019.107873

Figure Lengend Snippet: Assessment of NaV1.2 channel protein level and physiology in retinal ganglion cells. (A) NaV1.2 (red) is expressed along retinal ganglion cell (RGC) axon proximal sections (dashed line segment) as identified by SMI32 (green) and z-axis depth within whole mount retinas. NaV1.2 is also localized within distal axons (arrowheads) marked by SMI32. (B) Intensity profiles of NaV1.2 and SMI32 along the axon proximal segment indicated in A. The heavy dashed horizontal line indicates mean intensity for each signal. (C) NaV1.2 is localized within varicosities of distal axons (arrowheads). (D) Overlap of NaV1.2 and SMI32 signals within distal axon example shown in C. The heavy dashed horizontal line marks the mean intensity for each signal. (E) NaV1.2 outlines Brn3a-positive (blue) somas. (F) NaV1.2 intensity profile of example RGC soma (dashed square) shown in E. Scale bar = 10μm. (G) Example light-evoked trace from a typical αON-sustained RGC prior to blockade of NaV1.2. (H) The NaV1.2-specific blocker, Phrixotoxin-3 (Phx3), reduces light-evoked spiking in a dose-dependent manner in an example ON-sustained RGC (histogram bin width = 300 ms). (I) Light-evoked spike rate was normalized to pre-drug light response for each cell (n=10). Spike rate was significantly (*, p≤0.04) reduced by application of 25nM Phx3, and spiking was completely abolished by 400nM for all cells analyzed. Solid line is the best-fit exponential decay (R2=0.88). Based on the exponential decay function 40nM Phx3 would reduce the light-evoked spike rate by 50% (Ic50, dashed gray line). The heavy solid gray horizontal lines in G and H indicates light duration (3 s). Data represented as mean ± sem. Statistics: One-Way ANOVA, Tukey Post-hoc comparisons.

Article Snippet: NaV1.2 channels were modulated by bath application of 25-400nM Phrixotoxin-3 (Alomone labs ( Bosmans et al., 2006 )).

Techniques:

Journal: Experimental eye research

Article Title: Elevated ocular pressure reduces voltage-gated sodium channel NaV1.2 protein expression in retinal ganglion cell axons

doi: 10.1016/j.exer.2019.107873

Figure Lengend Snippet: Retinal NaV1.2 channel protein expression decreases following increased IOP. (A) Relative NaV1.2 mRNA expression (microbead:saline) following two (n=4), three (n=4), and four (n=4) weeks of raised intraocular pressure (*, p=0.039). (B) Retinal vertical sections labeled for NaV1.2 (red), ChAT (green), and DAPI (blue) in saline-injected control, 2-week microbead, and 4-week microbead retinas. OPL, outer plexiform layer, IPL, inner plexiform layer. Scale bar = 20 μm. (C) Quantification of NaV1.2 intensity within the IPL relative to control retinas revealed no significant difference in protein level after 2 weeks (p=0.31) but reduced expression after 4 weeks (*, p=0.02) of raised IOP (30% above baseline). (D) Maximum projection micrographs of whole-mounted retinas show 4 weeks of elevated IOP decreases NaV1.2 (red) staining within RGCs co-labeled by SMI32 (cell bodies and axon bundles, green) and the transcription factor Brn3a (cell bodies, blue). Scale bar = 50 μm. (E) NaV1.2 expression was analyzed in RGC axons within the plane of most intense SMI32 labelling and somas within the plane of densest Brn3a staining. (F) Colocalization of SMI32 to Nav1.2 (tM1) and NaV1.2 to SMI32 (tM2) is not influenced by increased IOP (p≥0.11). (G) Quantification of NaV1.2 within SMI32-positive axon layer and Brn3a-positive soma layer show expression significantly diminishes after 4 weeks of elevated IOP (*, p≤0.003). (H) NaV1.2 (red) localizes in SMI32-labeled (green) RGC somas and axons (arrowheads). Scale bar = 20 μm. (I) Following 4 weeks of raised IOP, NaV1.2 intensity significantly decreases within RGC axon varicosities and somas (*, p<0.001). (J) NaV1.2 intensity distribution normalized by length (bin width = 0.02μm). NaV1.2 significantly decreases within distal axon sections (*, p≤0.01). Data represented as mean ± sem. Statistics: A: One-way ANOVA, Tukey Post hoc; C-J: Independent samples t-tests.

Article Snippet: NaV1.2 channels were modulated by bath application of 25-400nM Phrixotoxin-3 (Alomone labs ( Bosmans et al., 2006 )).

Techniques: Expressing, Labeling, Injection, Staining

Journal: bioRxiv

Article Title: Distinctive in vitro phenotypes in iPSC-derived neurons from patients with gain- and loss-of-function SCN2A developmental and epileptic encephalopathy

doi: 10.1101/2023.02.14.528217

Figure Lengend Snippet: (A) Schematic of Na v 1.2 protein showing the position of R853Q and R1882Q mutations (D, Domain; N, N-Terminal; C, C-Terminal). (B) Sanger sequencing of R1882Q and R853Q iPS cell lines and corresponding CRISPR-mediated corrected cell lines (top) maintained normal morphology (bottom). (C) Schematic overview of the NGN2 differentiation protocol with details of crucial molecules exposure and analysis timeline. RT-qPCR analysis of neuronal maturation marker MAP2 and cell identity markers GAD1 and VGLUT at 22 DIV in R1882Q (D) and R853Q (E) iPSC-derived neurons and corresponding control. RT-qPCR analysis of sodium channel genes SCN1A, SCN2A, SCN3A, SCN8A in R1882Q (F) and R853Q (G) iPSC-derived neurons and corresponding control. Data presented as mean ± SD (n=3 experimental biological replicates with n=3 samples per experiment). Unpaired t-test was used to assess statistical significance. Immunofluorescence analyses of MAP2 and SCN2A expression in R1882Q (H) and R853Q (I) iPSC-derived neurons and corresponding control at 22 DIV. Scale bars, (B) 50 μm, (F, G) 20 μm. * p <0.05.

Article Snippet: Immunoreactivity was tested against SCN2A (Na v 1.2) (#AGP-026, Alomone; 1:250), and MAP2 (#ab5622, Abcam; 1:500).

Techniques: Sequencing, CRISPR, Quantitative RT-PCR, Marker, Derivative Assay, Control, Immunofluorescence, Expressing