Journal: Cell Reports

Article Title: Oligodendrocyte-derived LGI3 and its receptor ADAM23 organize juxtaparanodal Kv1 channel clustering for short-term synaptic plasticity

doi: 10.1016/j.celrep.2023.113634

Figure Lengend Snippet: ID-related LGI3 is uniquely expressed in oligodendrocytes (A) Domain organization of human LGI3. SP, signal peptide; LRR, leucine-rich repeat domain; EPTP, epitempin domain; N- and C-cap, N- and C-terminal cysteine caps in LRR. (B) Mapping of the ID variant on the modeled LGI3 structure. Left, Asp331 is mapped as red spheres. Right, close-up view of the Ca 2+ -binding site in the LGI3 EPTP β-propeller. Ca 2+ and water molecules are shown as gray and red spheres, respectively. Sticks, Ca 2+ -interacting residues; dotted lines, hydrogen bonds. (C) Shown are western blots (WBs) of LGIs-V5 in the conditioned medium (secreted) and the cell lysates. Bottom, quantification of the amounts of secreted LGI proteins. n = 3 experiments. Mann-Whitney U test. ∗ p < 0.05. Mean ± SD. (D) Fluorescence in situ hybridization (FISH) analysis for Lgi3 , Lgi1 , and Lgi2 expression in the mouse brain (P125). Red, Lgi3 , Lgi1 , or Lgi2 ; green, Mbp . White arrows, Mbp + / Lgi3 + cells; yellow arrows, Mbp – / Lgi3 + cells; white arrowheads, Mbp + / Lgi1 – or Mbp + / Lgi2 – cells; yellow arrowheads, Mbp – / Lgi1 + or Mbp – / Lgi2 + cells. Nuclear DNA was stained by DAPI (blue). CC, corpus callosum; Cb, cerebellum; WM, white matter; L5/6, cortical layers V and VI. Scale bars, 10 μm. (E) WB analysis of LGI family proteins in the lysates of the mouse brains and primary cultured cortical neurons. N-cadherin, a loading control. P, postnatal days; DIV, days in vitro . (F) WB analysis of Lgi3 LoxP/ΔE1 and Lgi3 LoxP/ΔE1 ; Mbp -Cre mouse brain lysates (P35). See also Figure S1 and Table S1 .

Article Snippet: To generate the Lgi3 -FH knock-in mouse, the single guide RNA (sgRNA) target sequence of mouse Lgi3 (NM_145219), 5′-ACTGGTGTACCGACATGTTG-3’ (sgRNA#1), was subcloned into the pX330 (Addgene 42230) vector, which expresses Cas9 and sgRNA.

Techniques: Variant Assay, Binding Assay, Western Blot, MANN-WHITNEY, Fluorescence, In Situ Hybridization, Expressing, Staining, Cell Culture, Control, In Vitro

Journal: Cell Reports

Article Title: Oligodendrocyte-derived LGI3 and its receptor ADAM23 organize juxtaparanodal Kv1 channel clustering for short-term synaptic plasticity

doi: 10.1016/j.celrep.2023.113634

Figure Lengend Snippet: LGI3 is clustered at the JXP of myelinated axons (A) Schematic image of FLAG-Hisx6 (FH)-tagged LGI3 protein. (B) Distributions of LGI3-FH (red) and LGI1 (green) in the mouse brain. (C) LGI3-FH is co-localized with MBP in the subcortical white matter (P166). Insets, wild-type mouse brain. (D) Biochemical subcellular fractionation of LGI3 in the mouse brain (P34). (E) LGI3-FH is not detected in the soma of oligodendrocytes in Lgi3 FH/FH mice (P183). CC-1, cytoplasmic oligodendrocyte marker protein. White arrowheads, LGI3-FH clusters; yellow arrowheads, somata of oligodendrocytes. (F and G) LGI3 is clustered at the JXP in the mouse brain and sciatic nerves. Sections are triple labeled by antibodies to (F) FH tag (LGI3-FH, red), Caspr1 (green, a paranode marker), and ankyrinG (blue, a node marker); (G) Hisx6 tag (LGI3-FH, green), Caspr2 (red, a JXP marker), and ankyrinG (blue, in the merged image) in the internal capsule. Arrows (in F, bottom) indicate LGI3-FH signals at the JXP. CC, corpus callosum; Str, striatum; Cb, cerebellum; WM, white matter; SN, sciatic nerves. P100 mouse tissue sections were used (B, F, and G). Scale bars, 1 mm (B), 250 μm (C), 10 μm (E and F, top), and 5 μm (F, bottom, and G). See also Figure S2 .

Article Snippet: To generate the Lgi3 -FH knock-in mouse, the single guide RNA (sgRNA) target sequence of mouse Lgi3 (NM_145219), 5′-ACTGGTGTACCGACATGTTG-3’ (sgRNA#1), was subcloned into the pX330 (Addgene 42230) vector, which expresses Cas9 and sgRNA.

Techniques: Fractionation, Marker, Labeling

Journal: Cell Reports

Article Title: Oligodendrocyte-derived LGI3 and its receptor ADAM23 organize juxtaparanodal Kv1 channel clustering for short-term synaptic plasticity

doi: 10.1016/j.celrep.2023.113634

Figure Lengend Snippet: LGI3 interacts with ADAM23 family, LGI family, and Kv1 channel proteins and co-clusters at the JXP in the brain (A) In vivo LGI3-associated protein complex purified from the Lgi3 FH/FH mouse brain (∼P100). Asterisks, specific bands co-purified with LGI3-FH. (B) LGI3-interacting protein-protein networks in the mouse brain. Thresholds for enrichment are shown by gray dashed lines. Light gray dots, proteins with subthreshold values; gray dots above thresholds, proteins in other categories. All protein data (997 proteins) for the plot are shown in Table S2 . n = 2 independent TAP experiments (with two shotgun MS per sample). (C) Shown are WBs of input and TAP eluates with the indicated antibodies. (D and E) Co-localization of LGI3 with ADAM23 family proteins and Kv1.2 channels at the JXP. (F) The endogenous LGI1 protein (endoLGI1) ( Lgi1 +/+ , left) and neuronally expressed LGI1-FH driven by Thy1 promoter in an Lgi1 KO background ( Lgi1 −/− ; Thy1 - Lgi1 -FH mouse) were detected at the JXP. (G) Localization of LGI2 at the JXP in the cerebellar white matter (Cb). P100 (D, E, and G) and P36 (F) mouse brain sections were used. Scale bars, 10 μm (D and E, left), 5 μm (F and G), and 1 μm (D and E, right). See also Figures S3 and and Tables S2 and .

Article Snippet: To generate the Lgi3 -FH knock-in mouse, the single guide RNA (sgRNA) target sequence of mouse Lgi3 (NM_145219), 5′-ACTGGTGTACCGACATGTTG-3’ (sgRNA#1), was subcloned into the pX330 (Addgene 42230) vector, which expresses Cas9 and sgRNA.

Techniques: In Vivo, Purification

Journal: Cell Reports

Article Title: Oligodendrocyte-derived LGI3 and its receptor ADAM23 organize juxtaparanodal Kv1 channel clustering for short-term synaptic plasticity

doi: 10.1016/j.celrep.2023.113634

Figure Lengend Snippet: LGI3 and its associated proteins form nanoscale subclusters at the JXP (A) Two-color (2C) STED imaging of Caspr1 and ADAM23 at the perinodal axon in corpus callosum (CC). (B) STED imaging of the large axons in the medulla. Dashed boxes are magnified in the middle two images (proximal and distal). The size of subclusters at the distal JXP is measured (bottom graphs). Red lines represent the mean value. FWHM, full width at half maximum. (C) 2C STED imaging of the juxtaparanodal proteins. P100 Lgi3 FH/FH mouse brain sections were used. Scale bars, 1 μm (A, left, and B, top) and 100 nm (A and B, magnified, and C). See also Figure S5 .

Article Snippet: To generate the Lgi3 -FH knock-in mouse, the single guide RNA (sgRNA) target sequence of mouse Lgi3 (NM_145219), 5′-ACTGGTGTACCGACATGTTG-3’ (sgRNA#1), was subcloned into the pX330 (Addgene 42230) vector, which expresses Cas9 and sgRNA.

Techniques: Imaging

Journal: Cell Reports

Article Title: Oligodendrocyte-derived LGI3 and its receptor ADAM23 organize juxtaparanodal Kv1 channel clustering for short-term synaptic plasticity

doi: 10.1016/j.celrep.2023.113634

Figure Lengend Snippet: Loss of LGI3 specifically reduces ADAM23, ADAM22, and Kv1 channels in the myelin fraction without affecting myelination (A) Shown are representative WBs of the whole-brain lysates and subcellular fractions of wild-type ( +/+ ) and Lgi3 −/− mice (∼P100) (left). The graph shows protein levels of the Lgi3 −/− mouse brains relative to those of wild type (right). n = 5 mice per genotype. Mann-Whitney U test. ∗∗ p < 0.01. Mean ± SD. (B) Immunostaining for MBP in the neocortex and subcortical regions of the young (P10) and adult (P100) mouse brains. CC, corpus callosum; Str, striatum; Ctx, cortex. (C) Immunohistochemistry for CC-1 (mature oligodendrocyte marker, green) and Olig2 (expressed throughout the oligodendrocyte lineage, red) in the CC (P50). Nuclear DNA was stained by TO-PRO3 (blue). Bottom, quantification of the number of CC-1 + cells and Olig2 + cells. n = 3 mice per genotype. Two-tailed Student’s t test. NS, not significant. Mean ± SD. Scale bars, 100 μm (B) and 50 μm (C). See also Figure S6 .

Article Snippet: To generate the Lgi3 -FH knock-in mouse, the single guide RNA (sgRNA) target sequence of mouse Lgi3 (NM_145219), 5′-ACTGGTGTACCGACATGTTG-3’ (sgRNA#1), was subcloned into the pX330 (Addgene 42230) vector, which expresses Cas9 and sgRNA.

Techniques: MANN-WHITNEY, Immunostaining, Immunohistochemistry, Marker, Staining, Two Tailed Test

Journal: Cell Reports

Article Title: Oligodendrocyte-derived LGI3 and its receptor ADAM23 organize juxtaparanodal Kv1 channel clustering for short-term synaptic plasticity

doi: 10.1016/j.celrep.2023.113634

Figure Lengend Snippet: Loss of LGI3 disturbs ADAM23 and Kv1 channel clustering at the JXP (A and B) Immunohistochemistry for Kv1.1 and axonal domain marker proteins in the L5/6 (A) and CC (B) of the wild-type ( +/+ ) and Lgi3 −/− mice (left). Magnified images are shown at the bottom. The number and size of Caspr1 clusters and the number and length of juxtaparanodal Kv1.1 clusters are quantified (right graphs). (C and D) The number of ADAM23 clusters at the JXP is significantly reduced in the L5/6 and CC of Lgi3 −/− mice (C), while that of ADAM11 clusters is not affected (D). (E) Representative STED images of juxtaparanodal Kv1.1 clustering in the L5/6 of wild-type ( +/+ ) and Lgi3 −/− mice. (F) Schematic illustration of stereotaxic injection of AAV for Adam23 family gene KO by the CRISPR-Cas9 system. AAV-DJ-expressing sgRNA for Adam23 or Adam22 and a reporter mCherry were injected into the primary motor cortex of Rosa26 Cas9/Cas9 knockin mice (∼P40). Juxtaparanodal cluster formation on the mCherry + axons in the contralateral cortex (blue square) was evaluated at 8 weeks after injection. (G) Representative images of the juxtaparanodal proteins (grayscale or blue) on the mCherry + (red) axons are shown. The number of juxtaparanodal protein clusters (ratio to Caspr1 clusters, green) was significantly reduced in the Adam23 KO axons. (A–E) Wild-type mice, P100–P119; Lgi3 −/− mice, P100. (A–D) n = 3 mice per genotype. Mann-Whitney U test. ∗∗ p < 0.01 and ∗∗∗ p < 0.001. Mean ± SD. (G) n = 4 experiments, one-way ANOVA post hoc Tukey’s test. NS, not significant. ∗∗ p < 0.01. mean ± SD. Scale bars, 10 μm (A and B, top), 5 μm (A and B, bottom, C, and D), 1 μm (E), and 10 μm (G). See also Figure S7 .

Article Snippet: To generate the Lgi3 -FH knock-in mouse, the single guide RNA (sgRNA) target sequence of mouse Lgi3 (NM_145219), 5′-ACTGGTGTACCGACATGTTG-3’ (sgRNA#1), was subcloned into the pX330 (Addgene 42230) vector, which expresses Cas9 and sgRNA.

Techniques: Immunohistochemistry, Marker, Injection, CRISPR, Expressing, Knock-In, MANN-WHITNEY

Journal: Cell Reports

Article Title: Oligodendrocyte-derived LGI3 and its receptor ADAM23 organize juxtaparanodal Kv1 channel clustering for short-term synaptic plasticity

doi: 10.1016/j.celrep.2023.113634

Figure Lengend Snippet: LGI3 regulates short-term synaptic plasticity through juxtaparanodal Kv1 channels (A) Schematic electrophysiological experiments. Blue and green circles represent light illumination to the white matter and to the center of recording L5a pyramidal cell (PC) soma, respectively. (B) Representative averaged current trace of L5a PC in response to repetitive stimulations by light illumination (1 ms duration, 20 Hz, light blue bars) to the white matter axons of ChR2 + L2/3 callosal neurons. (C and D) EPSC amplitude ratio (second to first and third to first responses, respectively) of L5a PCs in the wild-type ( +/+ ) or Lgi3 −/− mouse brain slices evoked by white matter (C) or soma stimulation (D). Wild-type, n = 13 and 12; Lgi3 −/− , n = 16 and 15 (white matter and soma stimulation, respectively). Welch’s t test. ∗ p < 0.01, ∗∗ p < 0.001, ∗∗∗ p < 0.0001, and ∗∗∗∗ p < 0.00001. Mean ± SD. (E and F) Blockade of Kv1 channels suppresses STD inductions in wild-type but not in Lgi3 −/− mice. Ratio of EPSC evoked by white matter stimulation before and during bath application of (E) 100 μM 4-AP and (F) 100 nM DTX-I. Wilcoxon signed-rank test. NS, not significant. ∗ p < 0.05. (G and H) Presynaptic terminals on recorded L5a PCs were directly stimulated by soma stimulation in the presence of 1 μM TTX before and during bath application of 100 μM 4-AP (G) or 100 nM DTX-I (H). Wilcoxon signed-rank test. NS, not significant. (I) LGI3 is exclusively localized in non-presynaptic regions of the mouse cortex in contrast to LGI1. Dashed white square area is magnified (bottom). Manders co-localization coefficient of vGlut1/2 with LGI3 or LGI1 is shown in the graph. Scale bars, 10 μm (top) and 5 μm (magnified, bottom). n = 3 experiments. Mean ± SD. (J) Presynaptic Kv1 channels are not altered in Lgi3 −/− mice. The intensity of the Kv1.2 is quantified (graph). n = 6 experiments. Two-tailed Mann-Whitney U test. NS, not significant. Mean ± SD. Scale bars, 10 μm. (K and L) Loose-patch single-axon recordings in the CC. (K) Inset shows the schematic loose-patch single-axon recording. S, stimulating electrode. (L) Wilcoxon signed-rank test. NS, not significant. ∗ p < 0.05. Red lines represent the mean value (E–H and L). See also Tables S4 and .

Article Snippet: To generate the Lgi3 -FH knock-in mouse, the single guide RNA (sgRNA) target sequence of mouse Lgi3 (NM_145219), 5′-ACTGGTGTACCGACATGTTG-3’ (sgRNA#1), was subcloned into the pX330 (Addgene 42230) vector, which expresses Cas9 and sgRNA.

Techniques: Two Tailed Test, MANN-WHITNEY

Journal: Cell Reports

Article Title: Oligodendrocyte-derived LGI3 and its receptor ADAM23 organize juxtaparanodal Kv1 channel clustering for short-term synaptic plasticity

doi: 10.1016/j.celrep.2023.113634

Figure Lengend Snippet:

Article Snippet: To generate the Lgi3 -FH knock-in mouse, the single guide RNA (sgRNA) target sequence of mouse Lgi3 (NM_145219), 5′-ACTGGTGTACCGACATGTTG-3’ (sgRNA#1), was subcloned into the pX330 (Addgene 42230) vector, which expresses Cas9 and sgRNA.

Techniques: Recombinant, Virus, Sequencing, Modification, RNAscope, Multiplex Assay, Positive Control, Negative Control, Mass Spectrometry, Knock-In, Knock-Out, Transgenic Assay, Control, Software