Dystonia. Hypothermia-induced attacks are dystonic of nature.

<p>(A) Illustration showing the locations of the ECoG electrodes. ECoG was bilaterally recorded from the primary motor cortex with ground and reference electrodes placed above the superior colliculi. (B) Picture of the experimental setting showing a α₃^+/D801Y mouse freely moving in an empty cage while ECoG is recorded. (C) Representative example of ECoG (left) and corresponding power spectrum of a baseline measurement during which the mouse is exploring the cage. (D) As in C but the recording was made during an attack induced by cold water exposure in the same α₃^+/D801Y mouse. (E) As in C and D but recorded during a pilocarpine induced tonic-clonic seizure in the same mouse (note the difference in y-axis of both the ECoG and power spectrum). (F) Illustration indicating locations of EMG recordings from the tibialis and gastrocnemius in the hind limb. (G, H) Representative examples of EMG recorded from the same α₃^+/D801Y mouse from the anterior tibialis and gastrocnemius pre (B, blue) and post (C, green) a cold water induced attack. (I) Cross correlograms of the traces shown in G (blue) and H (green) showing a pronounced difference in correlation between activity of agonist and antagonist hind limb muscles indicative of dystonic postures during an attack.</p

Ataxia. α₃^+/D801Y mice display moderate motor deficits.

<p>(A) Gait analysis with fore and hind base width and stride length (n = 6 for both WT and α₃^+/D801Y). Front paws were colored blue, while hind paws were colored with red paint. (B) Hind limb clasping test (n = 10 for WT and n = 6 for α₃^+/D801Y). (C) Balance beam test over 3 consecutive days, with time to cross (left) and number of slips (right) (n = 24 for WT and n = 23 for α₃^+/D801Y). (D) Rope climb test with time to climb (n = 19 for WT and n = 23 α₃^+/D801Y). (E) Parallel rod floor test with distance traveled, number of slips and ataxia ratio defined by: number of slips/(distance*100) (n = 10 for WT and n = 12 for α₃^+/D801Y mice). (F) Grip strength (n = 12 for WT and n = 13 for α₃^+/D801Y). All data shown are means ± SEM. *p<0.05, **p<0.01, ***p<0.001.</p

Hypothermic attacks. Hypothermia causes dystonia-like attacks in α₃^+/D801Y mice.

<p>(A) Average occurrence (%) of an attack in α₃^+/D801Y mice, following restraining for 10 min (n = 5), tail suspension for 6 min (n = 6), randomly timed electric foot shocks (n = 5), exposure to fox urine (n = 5), warm incubator (43°C) (n = 5), temperate water swim (35°C) (n = 6), chronic variable stress protocol (n = 11), cold water swim (5–10°C) (n = 10), cold environment (-20°C) (n = 6) and Prazosin treatment before cold water swim (n = 5). Only hypothermia, caused by cold water swim or cold environment exposure, consistently induced attacks in the α₃^+/D801Y mice (n = 15 for cold water and n = 6 for cold environment). (B) Example of dystonic-like posture with hind limbs hyperextended caudally (left picture, arrow) and a period of convulsion with abnormal postures and twisting movements (right picture) in α₃^+/D801Y mice after cold water swim. WT mice never displayed similar abnormal symptoms (left picture). (C) Core body temperature measured by rectal probe at onset of attack induced by exposure to cold water or cold environment. Both methods induced a significant drop in body temperature just below about 20°C before symptoms occurred in α₃^+/D801Y mice. WT mice displayed identical drops in body temperature (n = 6 for both WT and α₃^+/D801Y). (D) Attack duration after induction by cold water when α₃^+/D801Y mice were left to recuperate at room temperature or on a 33.3°C heating pad (n = 6).</p

Cerebellar activity. <i>In vivo</i> recordings of awake α₃^+/D801Y mice revealed irregular firing of Purkinje cells and DCN neurons, which during dystonic spells was further exacerbated and turned into periods of abnormal high-frequency bursting.

<p>(A) Illustration of an <i>in vivo</i> recording of Purkinje cells in awake head-restrained mice. (B) Representative raw traces of Purkinje cells recorded from WT, α₃^+/D801Y at baseline, and α₃^+/D801Y mice during dystonic attack induced by cold water. Scale bars: 500 ms by 50 μV. (C) Average firing rate (upper), predominant firing rate (middle) and CV ISI (lower) of Purkinje cells from WT (N = 4 (animals), n = 19 (cells)), α₃^+/D801Y at baseline (N = 5, n = 23), control WT exposed to cold water (N = 3, n = 18) and α₃^+/D801Y mice during dystonic attacks induced by cold water (N = 4, n = 20). (D) Illustration of an <i>in vivo</i> recording of DCN neurons in awake head-restrained mice. (E) Representative raw traces of DCN neurons recorded from WT, α₃^+/D801Y at baseline, and α₃^+/D801Y mice during dystonic attack induced by cold water. (F) Average firing rate (upper), predominant firing rate (middle) and CV ISI (lower) of DCN neurons from WT (N = 4 (animals), n = 21 (cells)), α₃^+/D801Y at baseline (N = 5, n = 21), control WT mice exposed to cold water (N = 3, n = 18) and α₃^+/D801Y mice during dystonic attacks induced by cold water (N = 4, n = 20). All data shown are means ± SEM. *p<0.05, **p<0.01, ***p<0.001.</p

<i>In vitro</i> pump function. Functional assays of Na⁺/K⁺ ATPases with substitutions in the disease hotspot aspartate residue.

<p>(A, B, C) Currents recorded in Na⁺-loaded oocytes expressing exogenous ouabain-resistant Na⁺/K⁺-ATPases without (wild type, A), or with, a D-to-Y (B) or D-to-N (C) mutation at position 801 equivalent, held at -20 mV, exposed to 125 mM Na⁺ solution at pH 7.6 containing 1 μM ouabain (to silence endogenous Na⁺/K⁺-ATPases), with 15 mM K⁺ added as indicated by horizontal bars (Ko); the vertical lines are responses to 50-ms steps to other potentials. (D, E, F) Steady-state current levels plotted against voltage, from the recordings shown in (A, B, C) (filled symbols), in the presence (red) or absence (black) of K⁺, and from subsequent recordings in the same oocyte after inhibition of exogenously expressed pumps by 10 mM ouabain (empty symbols). (G, H, I) Average ± SEM 10 mM ouabain-sensitive steady currents (I ouab-sens) in 125 mM Na⁺, obtained by subtraction, at 0 mM K⁺ (black circle) or 15 mM K⁺ (red triangle), normalized to the maximum Na⁺ charge movement in each oocyte (J-O, below), a measure of the number of Na⁺/K⁺-ATPases; wild type (n = 4 oocytes), D-to-Y (n = 3 with K⁺, n = 6 without), D-to-N (n = 3). (J, K, L) 10 mM ouabain-sensitive pre-steady-state Na⁺ currents for wild type (J), D-to-Y (K), and D-to-N (L) Na⁺/K⁺-ATPases in 125 mM Na⁺ and 0 mM K⁺ solution obtained by subtraction of traces before and after pump inhibition; superimposed traces are from steps to voltages between -180 mV and +60 mV, and back to the holding potential, -20 mV. (M, N, O) Transient Na⁺ charge movements, ΔQ, obtained as the time integral of the transient currents at -20 mV after each voltage step, are plotted against potential during the step for wild type (M), D-to-Y (N), and D-to-N (O) Na⁺/K⁺-ATPases. Boltzmann relation fits to the ΔQ-V plots yielded maximum ΔQ values used for normalization (ΔQ norm), and mean fit values for effective valence, zq (wild type: 0.68±0.01, n = 9; D-to-Y: 0.38±0.02, n = 6; D-to-N: 0.48±0.02, n = 9), and for midpoint voltage (wild type: -24±1 mV, n = 9; D-to-Y: -51±3 mV, n = 6; D-to-N: -19±2 mV, n = 9); maximum ΔQ for D-to-Y pumps is likely underestimated due to the lower zq, so that D-to-Y currents normalized to maximum charge (H, above) may be overestimated; averaged ΔQ norm-V distributions are shown. See also Supplementary <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006763#pgen.1006763.s002" target="_blank">S2 Fig</a>.</p

Hypothermia-induced dystonia and abnormal cerebellar activity in a mouse model with a single disease-mutation in the sodium-potassium pump

<div><p>Mutations in the neuron-specific α₃ isoform of the Na⁺/K⁺-ATPase are found in patients suffering from Rapid onset Dystonia Parkinsonism and Alternating Hemiplegia of Childhood, two closely related movement disorders. We show that mice harboring a heterozygous hot spot disease mutation, D801Y (α₃^+/D801Y), suffer abrupt hypothermia-induced dystonia identified by electromyographic recordings. Single-neuron <i>in vivo</i> recordings in awake α₃^+/D801Y mice revealed irregular firing of Purkinje cells and their synaptic targets, the deep cerebellar nuclei neurons, which was further exacerbated during dystonia and evolved into abnormal high-frequency burst-like firing. Biophysically, we show that the D-to-Y mutation abolished pump-mediated Na⁺/K⁺ exchange, but allowed the pumps to bind Na⁺ and become phosphorylated. These findings implicate aberrant cerebellar activity in α₃ isoform-related dystonia and add to the functional understanding of the scarce and severe mutations in the α₃ isoform Na⁺/K⁺-ATPase.</p></div

α₃ in cerebellum. Na⁺/K⁺-ATPase expression and gross cerebellar morphology in α₃^+/D801Y mice.

<p>(A) Western blot of cerebellar lysates from p0 and p70 α₃^+/D801Y mice and WT littermates with antibodies against α₁, α₂ and α₃ Na⁺/K⁺-ATPase isoform and actin as loading control. Quantification of blots is presented below as expression relative to WT (n = 6 for each group). Full-length Western blots are shown in Supplementary <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006763#pgen.1006763.s001" target="_blank">S1 Fig</a>. (B) Immunofluorescence staining of cerebellum from WT and α₃^+/D801Y mice using antibodies against the α₁ (magenta) and α₃ (green) isoform, with Hoechst (blue) for nuclear stain. Scale bars: 20 μm; gcl: granular cell layer; pc: purkinje cell layer; ml: molecular layer. (C) Picture of brains from a WT and a α₃^+/D801Y mouse, no gross mass change of cerebellum was observed. Scale bar represent 1 mm per tick. (D) Hematoxylin and eosin staining of cerebellar slices from WT and α₃^+/D801Y mice. (E) Immunofluorescent calbindin staining of Purkinje cells in cerebellar slices from WT and α₃^+/D801Y mice. Number of Purkinje cells was quantified as mean number of Purkinje cells per 100 μm (N = 3 (animals), n = 6 (slices) for both WT and α₃^+/D801Y). Scale bar 100 μm. All data shown are means ± SEM. *p<0.05, **P<0.01.</p

Twenty-seven genes relevant to lens placode formation and lens morphogenesis show differential expression in Pax6^−/− E9.5 mutated lens placodes.

<p>(A) A list of 27 genes includes a combination of well-characterized genes in lens biology and selected differentially expressed genes in Pax6 null (<i>Sey</i>) cortex <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054507#pone.0054507-Holm1" target="_blank">[45]</a>. The differentially expressed genes in Pax6^−/− E9.5 wild type and mutated lens placodes were identified using the Illumina Mouse6 bead microarrays as described elsewhere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054507#pone.0054507-Huang1" target="_blank">[96]</a>. Twenty-four of the 27 genes were differentially expressed in at least 50% of experiments. (B) Relative expression levels of <i>Fat4</i>, <i>Trpm3</i>, <i>Pax6</i>, <i>Has2</i>, <i>Efnb2</i>, and <i>Nav1</i> in wild type (WT, black bars) and Pax6^−/− (open bars) lens placode and mutated ectoderm were determined using qRT-PCR as described in Methods.</p

Pax6 regulates expression of Snca.

<p>(A) Identification of Pax6-binding region by ChIP-Chip in lens chromatin and corresponding luciferase reporter constructs for transfection assays. (B) Pax6 regulates <i>Snca</i> promoter/distal region in cultured cells. Transient transfections were performed in P19 embryonic carcinoma and in αTN4-1 lens cell as described in Methods. (C) Prediction of Pax6 binding sites with novel Pax6 DNA binding motifs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054507#pone.0054507-Xie1" target="_blank">[48]</a>. (D) EMSA validation of Pax6 binding to the probes identified by motif 1-1, 1-2 and 3-3. PD/HD, recombinant Pax6 protein containing both Pax6 paired domain (PD) and homeodomain (HD). P6CON, DNA-binding concensus for Pax6 paired domain.</p

Down-regulation of Kif1b and Snca in Sye/Sey forebrains.

<p>In situ hybridization analysis of E14 wild type (WT) and Pax6^Sey/Sey mouse embryos. Ganglionic eminence, GE.</p