Journal: Molecular psychiatry

Article Title: Ankyrin-G isoform imbalance and interneuronopathy link epilepsy and bipolar disorder

doi: 10.1038/mp.2016.233

Figure Lengend Snippet: NT3-AnkG localizes Nav channels to the AIS of PV-interneurons. ( a ) ANK3 gene map labeled with BD-associated SNPs (*) and a BD-protective SNP(*). Alternative exons encoding NT1 (exon 1a and 1a′), NT2 (exon 1e) and NT3 (exon 1b) are labeled. ( b ) Brain AnkG protein isoforms generated by alternative first exon use (NT2 and NT3) and alternative splicing of the large exon (190, 270 and 480 kD). ( c–j ) NT2-AnkG and NT3-AnkG have distinct cell type specific expression in the hippocampus. ( c, e ) Low magnification: CA regions appear to contain only NT2-AnkG, while the dentate gyrus (DG) labels for both NT2- and NT3-AnkG. ( d, f ) Higher magnification (boxed regions from a, c): DG cells express NT2-AnkG always and NT3 variably. ( f, i ) High magnification: CA3 shows labeling of PV interneuron (PV; white) AISs by only NT3 (arrows). CA3 pyramidal cells (PC) express only NT2-AnkG. ( h, j ) High magnification (boxed regions from g , i ): CA3 PVs express only NT3 and PCs only NT2 ( k–l ) Heterozygous Ank3 exon 1b deletion proportionally reduces AnkG and Na V channel labeling at AISs of CA1 PV interneurons. ( k ) CA1 images from Ank3-1b KO/+ and WT mice, acquired under identical conditions. ( l ) Fluorescence intensity at PV AISs is significantly reduced for PanAnkG (p=0.0009), NT3-AnkG (p<0.0001) and PanNa V (p=0.0049) in Ank3-1b KO/+ compared WT. Scales: c, e : 100 μm; d, f, h, j : 10 μm; g, i : 50 μm; d, f subpanels: 5μm; h, j subpanels: 2 μm, k : 10 μm.

Article Snippet: Isoform-specific rabbit antibodies against portions of the NT1-AnkG, NT2-AnkG and NT3-AnkG peptides were raised (YenZym, Inc., S. San Francisco, CA) and affinity-purified using methods described previously( ).

Techniques: Labeling, Generated, Alternative Splicing, Expressing, Fluorescence

Journal: Molecular psychiatry

Article Title: Ankyrin-G isoform imbalance and interneuronopathy link epilepsy and bipolar disorder

doi: 10.1038/mp.2016.233

Figure Lengend Snippet: Cell type specific NT2-AnkG and NT3- AnkG expression is conserved in mouse and human cortex. ( a–f ) Mouse anterior cingulate cortex: ( a–b ) Widespread NT3 ( a : green) and NT2 ( b : green) labeling of neurons in area Cg2/24b. ( c, e ) High magnification: exclusive NT3 expression neocortical PV interneurons at AISs. ( d ) PCs show varied NT3 expression: weak NT3 labeling (◀), moderate NT3 labeling (◀◀)and strong NT3 labeling (◀◀◀). ( f ) PCs show varied NT2 expression: lack NT2 labeling (◀), moderate NT2 labeling ◀◀) and strong NT2 labeling (◀◀◀). ( g–l ) NT2 and NT3 expression pattern is conserved in human. ( g, j ) Human PV interneurons (frontal lateral neocortex) exclusively express NT3. Arrowheads in j mark a PV interneuron AIS lacking NT2 staining. ( h–i, k–l ) Pyramidal neurons variably express the two isoforms. Scales: a, b: 100 μm; c–f, g–l : 10 μm.

Article Snippet: Isoform-specific rabbit antibodies against portions of the NT1-AnkG, NT2-AnkG and NT3-AnkG peptides were raised (YenZym, Inc., S. San Francisco, CA) and affinity-purified using methods described previously( ).

Techniques: Expressing, Labeling, Staining

Journal: Molecular psychiatry

Article Title: Ankyrin-G isoform imbalance and interneuronopathy link epilepsy and bipolar disorder

doi: 10.1038/mp.2016.233

Figure Lengend Snippet: PV-interneurons in the basolateral amygdala (bla) express only NT3-AnkG. ( a ) BLA section labeled by the three indicated antibodies and DAPI. PV interneuron AISs (green arrows) appear yellow due to colabeling by NT3-AnkG (green) and all-AnkG (red) antibodies. Both strongly and weakly PV labeled interneurons (yellow boxes) express NT3. ( b–c ) Higher magnification views of two strongly NT3 labeled boxed cells from a exhibit strong ( b ) and light ( c ) PV labeling. ( d ) Two neighboring non-PV neurons: the upper AIS (single arrowhead) lacks detectable NT3 staining; the lower (double arrowheads) shows light NT3 labeling. Upper panel: merge; middle: NT2; lower: NT3. ( e ) AISs of PV interneurons (red arrows) are not labeled by NT2; both strongly and weakly PV labeled interneurons (yellow boxes) lack NT2. ( f–g ) Higher magnification views of two boxed cells from e, strongly ( f ) and lightly ( g ) PV labeled. Neither AIS stains for NT2. h, Two neighboring non-PV neurons: the upper AIS (single arrowhead) has light NT2 staining; the lower (double arrowheads) shows strong NT2 labeling. Upper panel: merge; middle: NT2; lower: NT3. Scales: a, e: 25 μm; b–d, g–h: 5 μm.

Article Snippet: Isoform-specific rabbit antibodies against portions of the NT1-AnkG, NT2-AnkG and NT3-AnkG peptides were raised (YenZym, Inc., S. San Francisco, CA) and affinity-purified using methods described previously( ).

Techniques: Labeling, Staining