A <i>KCNC3</i> mutation causes a neurodevelopmental, non-progressive SCA13 subtype associated with dominant negative effects and aberrant EGFR trafficking

<div><p>The autosomal dominant spinocerebellar ataxias (SCAs) are a diverse group of neurological disorders anchored by the phenotypes of motor incoordination and cerebellar atrophy. Disease heterogeneity is appreciated through varying comorbidities: dysarthria, dysphagia, oculomotor and/or retinal abnormalities, motor neuron pathology, epilepsy, cognitive impairment, autonomic dysfunction, and psychiatric manifestations. Our study focuses on SCA13, which is caused by several allelic variants in the voltage-gated potassium channel KCNC3 (Kv3.3). We detail the clinical phenotype of four SCA13 kindreds that confirm causation of the <i>KCNC3</i>^<i>R423H</i> allele. The heralding features demonstrate congenital onset with non-progressive, neurodevelopmental cerebellar hypoplasia and lifetime improvement in motor and cognitive function that implicate compensatory neural mechanisms. Targeted expression of human KCNC3^R423H in <i>Drosophila</i> triggers aberrant wing veins, maldeveloped eyes, and fused ommatidia consistent with the neurodevelopmental presentation of patients. Furthermore, human KCNC3^R423H expression in mammalian cells results in altered glycosylation and aberrant retention of the channel in anterograde and/or endosomal vesicles. Confirmation of the absence of plasma membrane targeting was based on the loss of current conductance in cells expressing the mutant channel. Mechanistically, genetic studies in <i>Drosophila</i>, along with cellular and biophysical studies in mammalian systems, demonstrate the dominant negative effect exerted by the mutant on the wild-type (WT) protein, which explains dominant inheritance. We demonstrate that ocular co-expression of KCNC3^R423H with <i>Drosophila</i> epidermal growth factor receptor (dEgfr) results in striking rescue of the eye phenotype, whereas KCNC3^R423H expression in mammalian cells results in aberrant intracellular retention of human epidermal growth factor receptor (EGFR). Together, these results indicate that the neurodevelopmental consequences of KCNC3^R423H may be mediated through indirect effects on EGFR signaling in the developing cerebellum. Our results therefore confirm the <i>KCNC3</i>^<i>R423H</i> allele as causative for SCA13, through a dominant negative effect on KCNC3^WT and links with EGFR that account for dominant inheritance, congenital onset, and disease pathology.</p></div

KCNC3^R423H displays aberrant intracellular trafficking, glycosylation, and failure to express current in cell culture.

<p>(A-C) Immunofluorescence of CHO cells expressing human KCNC3^WT, KCNC3^R420H, or KCNC3^R423H using a KCNC3 antibody. Insets illustrate other representative cells from each transfection. (D-G) Confocal fluorescence images of CHO cells transiently expressing Clover-tagged human KCNC3^WT, KCNC3^R420H, KCNC3^R423H, or KCNC3^F448L. Fluorescence images of CHO cells transiently expressing (H-J) an ER marker, SIGMAR1^CFP with KCNC3^R423H-mRuby2; (K-M) an anterograde vesicle marker, JMY^GFP with KCNC3^R423H-mRuby2; and (N-P) a Golgi–endosome vesicle marker, PI4K2A^GFP with KCNC3^R423H-mRuby2. (N) Boxed area is magnified in (Q-S), with inset in S clearly demonstrating intravesicular retention of KCNC3^R423H-mRuby2. Cropped immunoblot of human KCNC3^WT, KCNC3^R423H, or KCNC3^R420H expressed in (T) CHO cells or (U) human U87 glioblastoma cells illustrating aberrant glycosylation for both causative mutant alleles (full-length blot shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173565#pone.0173565.s006" target="_blank">S1 Fig</a>). Representative currents evoked by commands to potentials between −80 mV and +70 mV, recorded in CHO cells expressing (V) KCNC3^WT-Clover or (W) KCNC3^R423H-Clover.</p

KCNC3^R423H causes dominant electrophysiological and trafficking effects on KCNC3^WT.

<p>(A) Representative currents evoked by a step from −70 mV to +70 mV in CHO cells expressing KCNC3^WT-Clover or KCNC3^R423H-Clover, and in those transfected with both constructs KCNC3^WT-Clover:KCNC3^R423H-mRuby2 in a 1:1 ratio. (B) Mean current densities recorded in CHO cells expressing either wild-type KCNC3^WT-Clover (n = 7) or KCNC3^R423H-Clover (n = 5) and in those expressing both KCNC3^WT-Clover: KCNC3^R423H-mRuby2 (1:1) constructs (n = 6). Current density was calculated by dividing the peak current evoked by a step from −70 to +70 mV by cell capacitance. Values are shown as mean±SEM, and significance was tested using a one-way ANOVA. (C) Current-voltage relations for cells in the three conditions shown in (A) and (B). Confocal fluorescence microscopy of cells expressing KCNC3^WT-Clover (D) or KCNC3^R423H-mRuby2 (G) individually, with no channel bleed-through (E,F). (H-S) Confocal fluorescence microscopy of cells co-expressing KCNC3^WT-Clover and KCNC3^R423H-mRuby2 at ratios of 1:1 to 6:1 (<i>KCNC3</i>^<i>WT</i>:<i>KCNC3</i>^<i>R423H</i>) showing co-localization and intracellular retention of both proteins, even at the highest concentration of <i>KCNC3</i>^<i>WT</i>. The total amount of DNA used in the co-transfection experiments was kept constant across ratios by adding control plasmid pcDNA 3.1.</p

Effects of Egfr on the <i>KCNC3</i>^<i>R423H</i> <i>Drosophila</i> eye and wing phenotypes.

<p>(A-C) gmr-Gal4–driven expression of controls LacZ, Egfr, and KCNC3^R423H individually. (D) Co-expression of Egfr and KCNC3^R423H shows rescue of the mutant phenotype. (E-G) dpp-Gal4–driven expression of KCNC3^R423H and Egfr-RNAi, individually and co-expressed. Red arrows and boxes (dashed lines) indicate areas of aberrations.</p

KCNC3^R423H causes aberrant EGFR trafficking resulting in intracellular retention.

<p>(A) Immunoblot with α-EGFR illustrating positive immunoprecipitation (IP) of EGFR in the eluent from beads bound to anti-EGFR antibody (left panel), with absence of KCNC3 in the same complex (α-KCNC3, right panel). (B) Immunoblot (α-KCNC3) showing presence of KCNC3 in the lysate. (C-E) Confocal fluorescence images of cells co-expressing KCNC3^{WT-mCerulean3}:human EGFR^Citrine (5:1 ratio), showing membrane localization for both proteins. (F-H) Representative cell co-expressing KCNC3^{R423H-mCerulean3}: human EGFR^Citrine (5:1 ratio) showing that both proteins do not reach the plasma membrane and are retained in intracellular vesicles. Insets provide additional examples. (I-K) Representative cell co-expressing KCNC3^{R423H-mCerulean3}:human EGFR^Citrine (4:1 ratio) also showing aberrant trafficking for both proteins. Insets magnify the co-localization of the two proteins in vesicles. (L-N) Representative cell co-expressing KCNC3^{R423H-mCerulean3}:human EGFR^Citrine (2:1 ratio) showing both plasma membrane trafficking and intracellular retention for EGFR. (O-Q) Confocal fluorescence images of cells co-expressing KCNC3^{R423H-mCerulean3}:human EGFR^Citrine (1:1 ratio) also demonstrating membrane and intracellular trafficking for EGFR with continued intracellular retention for KCNC3^R423H. The total amount of DNA used in the co-transfection experiments was kept constant across ratios by adding control plasmid pcDNA 3.1.</p

KCNC3^R423H causes dominant effects on KCNC3^WT in <i>Drosophila</i>.

<p>Eye images from gmr-Gal4 flies expressing (A) KCNC3^R423H; (B) two copies of <i>KCNC3</i>^<i>R423H</i>; and (C) <i>KCNC3</i>^<i>WT</i> with <i>KCNC3</i>^<i>R423H</i>. Wing images from dpp-Gal4 flies expressing (D) KCNC3^R423H; (E) two copies of <i>KCNC3</i>^<i>R423H</i>; and (F) <i>KCNC3</i>^<i>WT</i> with <i>KCNC3</i>^<i>R423H</i>. Red arrows and boxes (dashed lines) indicate areas of aberrations.</p

Expression of human <i>KCNC3</i>^WT and <i>KCNC3</i>^R423H in the <i>Drosophila</i> wing and eye.

<p>(A) Cropped immunoblot using antibodies directed against mammalian <i>KCNC3</i> and <i>Drosophila</i> tubulin illustrates da-Gal4 expression of the indicated transgenes (human <i>KCNC3</i>^<i>WT</i>, <i>KCNC3</i>^<i>R423H</i>, or β-galactosidase (<i>LacZ</i>)) under control of the ubiquitous da-Gal4 driver (full-length blot shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173565#pone.0173565.s006" target="_blank">S1 Fig</a>). (B-D) Wing images from adult flies maintained at 29°C, where the dpp-Gal4 drives expression of LacZ, KCNC3^WT, or KCNC3^R423H in the anterioposterior border of the wing. (E-G) Magnified images showing loss of the anterior crossvein (ACV [E]; red arrow [G]) and disruption of the longitudinal vein L3 as a consequence of KCNC3^R423H expression (dotted brackets [G]). (H-J) Wing images from adult flies where A9-Gal4 drives expression in the wing compartment. (K-M) Eye images from adult flies where gmr-Gal4 drives expression of LacZ, KCNC3^WT, or KCNC3^R423H, demonstrating small, maldeveloped eyes in the mutant. (N-P) Scanning electron microscopy images of whole eyes from <i>LacZ</i>, <i>KCNC3</i>^<i>WT</i>, or <i>KCNC3</i>^<i>R423H</i> flies. Yellow square highlights region of eye shown at high resolution (Q-S).</p

Pedigrees of the probands’ families and MRI.

<p>Four pedigrees (A) 423–1, (B) 423–2, (C) 423–3, and (D) 423–4, illustrating the inheritance pattern of KCNC3^R423H. De novo inheritance in patient II-2 is illustrated in 423–2. Midline T1-weighted sagittal magnetic resonance images (MRIs) of (E) a 35-year-old control; (F) patient 423–1, II-3 at age 42 years; (G) patient 423–1, III-1 at age 10 months (inset shows age-matched control); (H) de novo patient 423–2, II-2 at age 21 months. Midline T1-weighted sagittal MRIs of (I,J) patient 423–3, III-2 at age 7 and 17 years, respectively; and (K,L) patient 423–4, III-4 at age 16 and 26 years, respectively, demonstrating the lack of progressive cerebellar hypoplasia and/or atrophy.</p