Journal: Neuron

Article Title: Kir4.1-Dependent Astrocyte-Fast Motor Neuron Interactions Are Required for Peak Strength

doi: 10.1016/j.neuron.2018.03.010

Figure Lengend Snippet: Kir4.1 Upregulation in Ventral Horn AS around Large FαMN MNs (A and B) Kir4.1 mRNA is enriched in spinal cord compared to other CNS regions in humans (A) and mice ( Kasukawa et al., 2011 ) (B). Data are represented as mean ± SEM. (C and D) Perineuronal enrichment of Kir4.1 expression in human and mouse ventral spinal cord. (C) KIR4.1 is expressed around SMI32 + neurons in adult human ventral spinal cord (scale bars, 1 mm, left; 50 μm, right). Representative image of n = 3 control human spinal cord. Patient data are provided in . (D) Kir4.1 is expressed around ChAT + MNs in mouse ventral lumbar spinal cord at P16 (scale bars, 200 μm, left; 50 μm, right). Dotted line denotes gray/white matter boundary. WM, white matter. Arrowheads denote Kir4.1 enrichment around ventral horn neurons. High-resolution images are single-plane confocal images. (E) Increased ventral (V) compared to dorsal (D) Kir4.1 protein by western blot from mouse lumbar spinal cord at P30 (n = 4 mice, mean ± SEM, Welch’s t test). (F) Left: fold change of Kir4.1 mRNA levels between ventral and dorsal samples from cultured (n = 6 mice, mean ± SEM, one-sample t test) neonatal mouse spinal cord AS. Right: FACS-purified AS from P5 Aldh1l1-GFP + (n = 3 mice, mean ± SEM, one-sample t test) mouse spinal cord. (G) Kir4.1 mRNA levels in P5 Aldh1l1-GFP + FACS-purified AS compared to Aldh1l1-GFP − non-AS cells from mouse ventral spinal cord (n = 3 mice, mean ± SEM, one-sample t test). (H) Kir4.1 protein is preferentially found around larger MMP-9 + FαMNs (white arrowheads) compared to smaller MMP-9 − SαMNs (yellow arrowheads) at P30 (scale bar, 50 μm). (I) Quantification of Kir4.1 signal intensity around individual MMP-9 + or MMP-9 − MN (n = 4 mice, >100 MN counts/animal, boxplot, Mann-Whitney test). (J) Kir4.1 loss in AS from VGLUT1 KO animals at P26. VGLUT1 KO mice were crossed with EAAT2-td-Tomato reporter for AS visualization. (K) Quantification of Kir4.1 immunofluorescence intensity per AS (EAAT2-td-tomato + ) (n = 2 mice, >50 AS counts/animal, boxplot, Mann-Whitney test; scale bar, 40 μm, insert: 20 μm). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Edges of boxplots denote interquartile range (25 th –75 th percentile) with whiskers denoting 1.5 times the interquartile range and the black line denoting the median value.

Article Snippet: Rabbit polyclonal anti-Kir4.1 intracellular , Alomone , Cat# APC-035, RRID: AB_2040120.

Techniques: Expressing, Western Blot, Cell Culture, Purification, MANN-WHITNEY, Immunofluorescence

Journal: Neuron

Article Title: Kir4.1-Dependent Astrocyte-Fast Motor Neuron Interactions Are Required for Peak Strength

doi: 10.1016/j.neuron.2018.03.010

Figure Lengend Snippet: AS Kir4.1 Is Required for Maintenance of FαMN Size (A) Left: breeding scheme. AS-Kir4.1cKO and cre-negative control animals were bred with ChAT-GFP mice for MN visualization. Right: total FαMN (ChAT + /MMP-9 + /NeuN + ), SαMN (ChAT + /MMP-9 − /NeuN + ), and γMN (ChAT + /MMP-9 − /NeuN − ) numbers are equivalent in AS-Kir4.1cKO and cre-negative control mice at the indicated ages (n = 3 mice/group, mean ± SEM, lumbar spinal cord, Welch’s t test). (B, D, and F) Representative images of lumbar MNs at P14 (B), P30 (D), and 6 months (F). Arrowheads denote example FαMNs (scale bar, 50 μm). (C, E, and G) Quantification of MN size at P14 (C), P30 (E), and 6 months (G) (n = 3 mice/group, >100 MN counts/animal, boxplot, Mann-Whitney test). (H) Schematic of retrograde labeling of MN pools using intramuscular injection of fluorescent cholera toxin subunit B (CTSB) in the tibialis anterior (TA) muscle. (I) Representative images of retrograde-labeled ventral horn MNs in AS-Kir4.1cKO and cre-negative control mice. Arrowheads denote example putative FαMNs (scale bar, 50 μm). (J) Quantification of ChAT-GFP + CTSB + MN soma area from (G) (n = 3 mice/group, >50 MN counts/animal, boxplot, Mann-Whitney test). ∗ p < 0.05, ∗∗∗ p < 0.001. Edges of boxplots denote interquartile range (25 th –75 th percentile) with whiskers denoting 1.5 times the interquartile range and black line denoting the median value.

Article Snippet: Rabbit polyclonal anti-Kir4.1 intracellular , Alomone , Cat# APC-035, RRID: AB_2040120.

Techniques: Negative Control, MANN-WHITNEY, Labeling, Injection

Journal: Neuron

Article Title: Kir4.1-Dependent Astrocyte-Fast Motor Neuron Interactions Are Required for Peak Strength

doi: 10.1016/j.neuron.2018.03.010

Figure Lengend Snippet: AS Kir4.1 Is Required for FαMN Function, Fast-Twitch Muscle Fiber Size, and Peak Force (A) Breeding scheme and electrophysiology recording schematic. (B) Two representative current steps at 3× rheobase (RB). (C–E) Rheobase (C), input resistance (D), and steady-state (SS) firing frequency (at 3× rheobase) (E) demonstrate altered intrinsic electrophysiological deficits in MNs from AS-Kir4.1cKO-ChAT-GFP animals at P12–P15 (n = 12 control MNs, n = 14 AS-Kir4.1cKO MNs from at least 3 animals per group, boxplot, Mann-Whitney test). (F) Input resistance versus rheobase scatterplot shows shift in electrophysiological properties from fast-like to slow-like in AS-Kir4.1cKO compared to control MNs. (G) Cross sections of TA muscle fibers immunolabeled for marker of fast-twitch muscle (myosin type 2) and laminin in P30 AS-Kir4.1cKO and cre-negative control animals (scale bar, 50 μm). (H) Quantification of TA muscle fiber cross-sectional area (n = 3 mice/group, >100 muscle fibers/animal, boxplot, Mann-Whitney test). (I–K) Abnormal muscle strength behavior in AS-Kir4.1cKO mice. Adult AS-Kir4.1cKO animals generate less peak force (>P50, n = 14–15 mice/group, boxplot, Welch’s t test) (I). AS-Kir4.1cKO animals have slower front and hindlimb movements as assessed by gait analysis with the catwalk behavioral test. Swing speed corresponds to the limb speed while in the air (same animals as in I, boxplot, Welch’s t test) (J). AS-Kir4.1cKO animals display a shorter latency to fall on the accelerating rotarod at P30–P35 (n ≥ 7 mice/group, mean ± SEM, two-way ANOVA, Bonferroni post hoc test) (K). Mice performed three trials (T, T2, and T3) per day on 3 consecutive days (D1, D2, and D3). ∗ p < 0.05, ∗∗ p < 0.01. Edges of boxplots denote interquartile range (25 th –75 th percentile) with whiskers denoting 1.5 times the interquartile range and black line denoting the median value.

Article Snippet: Rabbit polyclonal anti-Kir4.1 intracellular , Alomone , Cat# APC-035, RRID: AB_2040120.

Techniques: MANN-WHITNEY, Immunolabeling, Marker, Negative Control

Journal: Neuron

Article Title: Kir4.1-Dependent Astrocyte-Fast Motor Neuron Interactions Are Required for Peak Strength

doi: 10.1016/j.neuron.2018.03.010

Figure Lengend Snippet: Cell-Autonomous AS Kir4.1 Loss Does Not Alter MN Survival in ALS (A) Schematic of iPSC-derived AS from human SOD1D90A ALS patients and non-ALS controls (patient data are provided in ). (B) Representative images of iPSC-derived AS from human SOD1D90A ALS patients and non-ALS controls labeled with KIR4.1 and GFAP (scale bar, 40 μm). (C) KCNJ10 mRNA levels are downregulated in SOD1G90A iPSC-derived AS as compared to controls (n = 2–3 independent cultures, mean ± SEM, Mann-Whitney test). (D) Western blot of KIR4.1, ALDH1L1, and GFAP on iPSC-derived AS from human SOD1D90A ALS patients and non-ALS controls. (E) Quantification of KIR4.1, ALDH1L1, and GFAP protein levels from western blot in (D) (n = 3/group, mean ± SEM, Mann-Whitney test). (F) Breeding scheme used for the loss of function of AS Kir4.1 in SOD1G93A ( mSOD1 ) mutant background. (G) Representative images of ventral horn lumbar spinal cord at P80 from cre-negative control, mSOD1 , and mSOD1 ; AS-Kir4.1cKO ( mSOD1; cKO ) animals labeled with MN markers (scale bar, 50 μm). (H and I) Quantification of ChAT + NeuN + (H) and MMP-9 + NeuN + (I) MN numbers in cre-negative control, mSOD1 , and mSOD1; cKO animals (n = 4–7 mice/group, >100 MN counts/animal, mean ± SEM, Kruskal-Wallis test). ∗ p < 0.05, ∗∗ p < 0.01.

Article Snippet: Rabbit polyclonal anti-Kir4.1 intracellular , Alomone , Cat# APC-035, RRID: AB_2040120.

Techniques: Derivative Assay, Labeling, MANN-WHITNEY, Western Blot, Mutagenesis, Negative Control

Journal: Neuron

Article Title: Kir4.1-Dependent Astrocyte-Fast Motor Neuron Interactions Are Required for Peak Strength

doi: 10.1016/j.neuron.2018.03.010

Figure Lengend Snippet: Kir4.1 Viral-Mediated Overexpression Is Sufficient to Increase MN Size (A and B) Left: schematic of intracerebroventricular injections in neonatal mice (P2–P3) of AAV-encoding Kir4.1-eGFP or control td-Tomato vectors for gain-of-function (GOF) experiments (A). Right: viral transduction of ventral spinal cord with AAV-td-Tomato or AAV-Kir4.1-eGFP quantified in (B) (n = 3–7 mice/group, mean ± SEM, Mann-Whitney test). (C) ChAT (right) and MMP-9 (left) immunofluorescent staining in the ventral spinal cord of AAV-td-Tomato and AAV-Kir4.1-eGFP-injected mice. (D and E) Quantifications of ChAT + (D) and MMP-9 + (E) MN soma area in P60 mice (2 months post injection) (n = 9–13 mice/group, 70–100 MNs counts/animal, boxplot, Mann-Whitney test, scale bar, 40 μm). (F) Example of MNs in contact (right) or not (left) with Kir4.1-overexpressing AS. (G) Ratio of soma area of MN contacting/non-contacting transduced AS (n = 9–13 mice/group, mean ± SEM, Mann-Whitney test). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Edges of boxplots denote interquartile range (25 th –75 th percentile) with whiskers denoting 1.5 times the interquartile range and black line denoting the median value.

Article Snippet: Rabbit polyclonal anti-Kir4.1 intracellular , Alomone , Cat# APC-035, RRID: AB_2040120.

Techniques: Over Expression, Transduction, MANN-WHITNEY, Staining, Injection

Journal: Neuron

Article Title: Kir4.1-Dependent Astrocyte-Fast Motor Neuron Interactions Are Required for Peak Strength

doi: 10.1016/j.neuron.2018.03.010

Figure Lengend Snippet: AS Kir4.1 Regulates MN Size through PI3K/mTOR/pS6 Pathway (A and C) Immunofluorescence co-staining of mTOR downstream effector pS6 and MMP-9 + MNs in P30 AS-Kir4.1cKO LOF mice (A) and in P60 AAV-Kir4.1 GOF mice (C). (B and D) Quantification of pS6 fluorescence intensity per MMP-9 + MN (B, LOF: n = 3 mice/group, 80 MNs counts/animal; D, GOF: n = 5 mice/group, 200 MN counts/animal, boxplot, Mann-Whitney test, scale bar, 25 μm). (E) Rapamycin treatment in AAV-Kir4.1 GOF mice. (F) Immunofluorescent co-staining of ChAT (left) and MMP-9 (right) in vehicle-treated AAV-td-Tomato and AAV-Kir4.1-eGFP mice (top two panels) or rapamycin-treated AAV-Kir4.1-eGFP mice (lower panel). (G and H) Quantification of ChAT + (G) and MMP-9 + (H) MN size in vehicle-treated AAV-td-Tomato and AAV-Kir4.1-eGFP and rapamycin-treated AAV-Kir4.1-eGFP mice (n = 3–4 mice/group, 100 MN counts/animal, boxplot, Kruskal-Wallis test, scale bar, 30 μm). (I) Incubation of acute spinal cord slices in solutions with high K + concentration. (J) Detection of ChAT-GFP + MNs in the ventral horn of P14 mice after 2 hr incubation in 3 mM KCl, 12 mM KCl, or 5 mM mannitol ACSF. (K) Quantification of MN soma area in the corresponding conditions (n = 4 mice/group, average 250 MN counts/animal, mean ± SEM, Kruskal-Wallis test, scale bar, 25 μm).

Article Snippet: Rabbit polyclonal anti-Kir4.1 intracellular , Alomone , Cat# APC-035, RRID: AB_2040120.

Techniques: Immunofluorescence, Staining, Fluorescence, MANN-WHITNEY, Incubation, Concentration Assay

Journal: Neuron

Article Title: Kir4.1-Dependent Astrocyte-Fast Motor Neuron Interactions Are Required for Peak Strength

doi: 10.1016/j.neuron.2018.03.010

Figure Lengend Snippet: AS-Encoded Kir4.1 Is Required for FαMN Function and Peak Force Generation AS Kir4.1 deletion leads to decreased FαMN size with mTOR downregulation, loss of fast-firing MN frequency, and decrease in fast-twitch muscle fiber size and peak strength without affecting MN survival.

Article Snippet: Rabbit polyclonal anti-Kir4.1 intracellular , Alomone , Cat# APC-035, RRID: AB_2040120.

Techniques:

Journal: Neuron

Article Title: Kir4.1-Dependent Astrocyte-Fast Motor Neuron Interactions Are Required for Peak Strength

doi: 10.1016/j.neuron.2018.03.010

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-Kir4.1 intracellular , Alomone , Cat# APC-035, RRID: AB_2040120.

Techniques: Plasmid Preparation, Recombinant, SYBR Green Assay, Mutagenesis, Software