Journal: bioRxiv

Article Title: Müller glial Kir4.1 channel Dysfunction in APOE4 -KI model of Alzheimer’s disease

doi: 10.1101/2025.02.26.640427

Figure Lengend Snippet: (a) Representative images of retinal slices showing Glutamine synthase (GS) and Kir4.1 staining pattern in APOE3 and APOE4 mice, scale 20µm (n: APOE3 = 3, APOE4 = 3). (b) Bar graph showing quantification of immunofluorescence for Kir4.1 and GS (n: 11-12 images/ group). (c) Current densities of Kir4.1 from freshly isolated Müller cells from APOE3 and APOE4 mice. Currents were elicited by a 50-ms hyperpolarization of −140 mV from a holding potential of −60 mV. (n: APOE3 = 26 cells/ 9 mice, APOE4 = 33 cells/ 8 mice). Values are expressed as mean ± SEM. Unpaired t-test was used for statistical analysis. **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Sections were then incubated overnight at 4 0 C with primary antibodies, including Kir4.1 (Cat. #APC-035-GP, Alomone Labs, 1:200), glutamine synthetase (GS, Cat. #MAB302, Millipore, 1:200) and TOMM20 (Cat. #MA5-32148, Invitrogen, 1:100).

Techniques: Staining, Immunofluorescence, Isolation

Journal: bioRxiv

Article Title: Müller glial Kir4.1 channel Dysfunction in APOE4 -KI model of Alzheimer’s disease

doi: 10.1101/2025.02.26.640427

Figure Lengend Snippet: (a) An illustration suggests a potential mechanism by which APOE4 disrupts Kir4.1 expression and causes Müller cell dysfunction. APOE4 may disrupt nuclear-encoded mitochondrial gene expression, reducing membrane potential (ΔΨm) and increasing reactive oxygen species (ROS). These changes can impair mitochondrial function, resulting in the downregulation of Kir4.1 channels in Müller cells and driving inflammation, aging, and cell death, contributing to retinal dysfunction in Alzheimer’s disease. (b) Representative images of retinal slices showing Glutamine synthase (GS) and TOMM20 staining pattern in APOE3 and APOE4 mice, scale 20µm (n: APOE3 = 3, APOE4 = 3). (c) Bar graph showing quantification of immunofluorescence for TOMM20 and GS (n: 10-11 images/ group). Values are expressed as mean ± SEM. Unpaired t-test was used for statistical analysis. *p<0.05, ***p<0.001.

Article Snippet: Sections were then incubated overnight at 4 0 C with primary antibodies, including Kir4.1 (Cat. #APC-035-GP, Alomone Labs, 1:200), glutamine synthetase (GS, Cat. #MAB302, Millipore, 1:200) and TOMM20 (Cat. #MA5-32148, Invitrogen, 1:100).

Techniques: Expressing, Gene Expression, Membrane, Staining, Immunofluorescence

Journal: bioRxiv

Article Title: Müller glial Kir4.1 channel Dysfunction in APOE4 -KI model of Alzheimer’s disease

doi: 10.1101/2025.02.26.640427

Figure Lengend Snippet: (a) Schematic showing generation of rMC-1 expressing human APOE isoforms. rMC-1 were transiently transfected with human APOE2 / APOE3 / APOE4 and EV was used as a control. (b) mRNA expression of Kcnj10 gene for Kir4.1 normalized to housekeeping gene β-actin. (c) Representative western blots of Kir4.1 expression and quantification of integrated optical density (IOD) ratio of Kir4.1 and α-tubulin showing decreased protein expression of Kir4.1 in APOE4 -transfected rMC-1. Values are expressed as mean ± SEM. One-way ANOVA followed by Tukey’s multiple comparison test was used for statistical analysis. *p<0.05, **p<0.01. (n: 4 independent experiments)

Article Snippet: Sections were then incubated overnight at 4 0 C with primary antibodies, including Kir4.1 (Cat. #APC-035-GP, Alomone Labs, 1:200), glutamine synthetase (GS, Cat. #MAB302, Millipore, 1:200) and TOMM20 (Cat. #MA5-32148, Invitrogen, 1:100).

Techniques: Expressing, Transfection, Control, Western Blot, Comparison

Journal: bioRxiv

Article Title: Müller glial Kir4.1 channel Dysfunction in APOE4 -KI model of Alzheimer’s disease

doi: 10.1101/2025.02.26.640427

Figure Lengend Snippet: (a) mRNA expression of KCNJ10 gene for Kir4.1 normalized to housekeeping gene for β-actin after treating rMC-1 with 1µM MitoQ and vehicle. mRNA expression of Kir4.1 was significantly increased in APOE4 -transfected rMC-1 upon treatment with 1µM MitoQ compared to the vehicle. (b) Representative western blots of Kir4.1 expression and quantification of IOD ratio of Kir4.1 and α-tubulin showing comparable protein expression of Kir4.1 in APOE4 -transfected rMC-1 as compared to EV/ APOE2 / APOE3 transfected rMC-1 after treating with 1µM MitoQ. Values are expressed as mean ± SEM. Two-way ANOVA followed by Tukey’s multiple comparison test was used for statistical analysis. *p<0.05, **p<0.01. (n: 3-4 independent experiments)

Article Snippet: Sections were then incubated overnight at 4 0 C with primary antibodies, including Kir4.1 (Cat. #APC-035-GP, Alomone Labs, 1:200), glutamine synthetase (GS, Cat. #MAB302, Millipore, 1:200) and TOMM20 (Cat. #MA5-32148, Invitrogen, 1:100).

Techniques: Expressing, Transfection, Western Blot, Comparison

Journal: Neuron

Article Title: TREK-1 and TRAAK are principal K + channels at the nodes of Ranvier for rapid action potential conduction on mammalian myelinated afferent nerves

doi: 10.1016/j.neuron.2019.08.042

Figure Lengend Snippet: A) Left, TREK-1 immunoreactivity (TREK-1-ir) at a NR and MBP immunoreactivity (MBP-ir) on myelin sheath. Right, TREK-1-ir at a NR and CASPR-ir in paranodal regions. B) Left, TRAAK-ir at a NR and MBP-ir on myelin sheath. Right, TRAAK-ir at a NR and CASPR-ir in paranodal regions. C) Similar to A&B except TREK-2-ir was examined and was negative at NRs. In A-C, NRs are indicated by arrows. MBP, myelin basic protein. CASPR, contactin associated protein. D) Summary of immunoreactive nodes for experiments represented in A-C: 112/129 nodes were TREK-1-ir positive, 118/129 nodes were TRAAK-ir positive. 0/129 nodes were TREK-2-ir positive. E) HEK293 cells transfected with TREK-1/eGFP (left), TRAAK/mCherry (middle), and both TREK-1/EGFP and TRAAK/mCherry (right). F) Traces illustrate single channel currents recorded at −80 mV from an HEK293 cell transfected with TREK-1/eGFP (upper, homomeric TREK-1) or an HEK293 cell transfected with TRAAK/mCherry (lower, homomeric TRAAK). Bottom, I-V curves of single channel currents recorded at different transmembrane voltages for homomeric TREK-1 channels (open circles, n = 7) or homomeric TRAAK (solid circles, n = 6). G) Sample traces show two types of single channels recorded at −80 mV from a HEK293 cell co-transfected with TREK-1/eGFP and TRAAK/mCherry plasmids, one type (upper, TREK-1/TRAAK) has unitary currents apparently larger than homomeric channels and another type (lower, TREK-1-like) has unitary currents similar to homomeric TREK-1 channels shown in F. Bottom panel, I-V curves of the currents of TREK-1/TRAAK single channels (n = 12, solid triangles) and TREK-1-like single channels (n = 13, open triangles). H) Summary of single channel conductance at −80 mV (open bars) and 80 mV (closed bars) for homomeric TREK-1 (n = 5 at −80 mV, n = 6 at 80 mV), homomeric TRAAK (n = 6 at both voltages), TREK-1-like (n = 12 at −80 mV, n = 5 at 80 mV), and TREK-1/TRAAK channels (n = 8 at −80 mV, n = 5 at 80 mV) expressed in HEK293 cells. The single channel conductance of nodal K2P channels (n = 13 at both voltages) is also included in the graph for a comparison. The single channel conductance at −80 mV was used for comparison. All recordings were performed under the cell-attached mode. Data represent Mean ± SEM, ns, no significant difference, *p < 0.05, ***p < 0.001, one-way ANOVA with the Tukey post hoc test. See also Fig. S5–9

Article Snippet: Following primary antibodies were used: rabbit anti-K2P 2.1 ( TREK-1, 1:1000 or 1:500, Alomone labs, Jerusalem, Israel), rabbit anti-K2P 10.1 ( TREK-2, 1:1000, Alomone labs, Jerusalem, Israel), rabbit anti-K2P 4.1 ( TRAAK, 1:1000 or 1:500, Alomone labs, Jerusalem, Israel), chicken anti-Myelin basic protein (1:1000, Abcam, Cambridge, MA, USA), mouse anti-contactin associated protein 1 (CASPR, 1:1000 or 1:500, MilliporeSigma, Burlington, MA, USA), chicken anti-GFP (1:2000, Aves Labs Inc., Tigard, OR, USA) and mouse anti-mCherry (1:2000, Abcam, Cambridge, MA, USA).

Techniques: Transfection

Journal: Neuron

Article Title: TREK-1 and TRAAK are principal K + channels at the nodes of Ranvier for rapid action potential conduction on mammalian myelinated afferent nerves

doi: 10.1016/j.neuron.2019.08.042

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Following primary antibodies were used: rabbit anti-K2P 2.1 ( TREK-1, 1:1000 or 1:500, Alomone labs, Jerusalem, Israel), rabbit anti-K2P 10.1 ( TREK-2, 1:1000, Alomone labs, Jerusalem, Israel), rabbit anti-K2P 4.1 ( TRAAK, 1:1000 or 1:500, Alomone labs, Jerusalem, Israel), chicken anti-Myelin basic protein (1:1000, Abcam, Cambridge, MA, USA), mouse anti-contactin associated protein 1 (CASPR, 1:1000 or 1:500, MilliporeSigma, Burlington, MA, USA), chicken anti-GFP (1:2000, Aves Labs Inc., Tigard, OR, USA) and mouse anti-mCherry (1:2000, Abcam, Cambridge, MA, USA).

Techniques: Recombinant, shRNA, Software