INSIGHTS INTO THE PATHOLOGY OF THE α2-Na+/K+-ATPase IN NEUROLOGICAL DISORDERS; LESSONS FROM ANIMAL MODELS

A functional Na+/K+-ATPase consists of a catalytic α subunit and a regulatory β subunit. Four α isoforms of the Na+/K+-ATPase are found in mammals, each with a unique expression pattern and catalytic activity. The α2 isoform, encoded by the ATP1A2 gene, is primarily found in the central nervous system (CNS) and in heart-, skeletal- and smooth muscle tissues. In the CNS, the α2 isoform is mainly expressed in neuroglial cells. In particular, the α2 isoform is found in astrocytes, and is important for astrocytic K+ clearance and, consequently, the indirect uptake of neurotransmitters. Both processes are essential for proper brain activity, and autosomal dominantly mutations in the ATP1A2 gene cause the neurological disorder Familial hemiplegic migraine type 2 (FHM2). FHM2 is a severe subtype of migraine with aura that involving temporary numbness or weakness, and affecting only one side of the body. FHM2 patients often suffer from neurological comorbidities such as seizures, sensory disturbances, cognitive impairment and psychiatric manifestations. The functional consequences of FHM2 disease mutations leads to a partial or complete loss of function of pump activity; however a clear phenotype-genotype correlation has yet to be elucidated. Gene-modified mouse models targeting the Atp1a2 gene have proved instrumental in the understanding of the pathology of FHM2. Several Atp1a2 knockout (KO) mice targeting different exons have been reported. Homozygous Atp1a2 KO mice die shortly after birth due to respiratory malfunction resulting from abnormal Cl- homeostasis in brainstem neurons. Heterozygous KO mice are viable, but display altered behavior and neurological deficits such as altered spatial learning, decreased motor activity and enhanced fear/anxiety compared to wild type mice. FHM2 knock-in (KI) mouse models carrying the human in vivo disease mutations W887R and G301R have also been reported. Both models display altered cortical spreading depression (CSD) and points to deficits in the glutamatergic system as the main underlying mechanism of FHM2

The loss-of-function disease-mutation G301R in the Na+/K+-ATPase α2 isoform decreases lesion volume and improves functional outcome after acute spinal cord injury in mice

Abstract Background The Na+/K+-ATPases are transmembrane ion pumps important for maintenance of ion gradients across the plasma membrane that serve to support multiple cellular functions, such as membrane potentials, regulation of cellular volume and pH, and co-transport of signaling transmitters in all animal cells. The α2Na+/K+-ATPase subunit isoform is predominantly expressed in astrocytes, which us the sharp Na+-gradient maintained by the sodium pump necessary for astroglial metabolism. Prolonged ischemia induces an elevation of [Na+]i, decreased ATP levels and intracellular pH owing to anaerobic metabolism and lactate accumulation. During ischemia, Na+/K+-ATPase-related functions will naturally increase the energy demand of the Na+/K+-ATPase ion pump. However, the role of the α2Na+/K+-ATPase in contusion injury to the spinal cord remains unknown. We used mice heterozygous mice for the loss-of-function disease-mutation G301R in the Atp1a2 gene (α 2 +/G301R ) to study the effect of reduced α2Na+/K+-ATPase expression in a moderate contusion spinal cord injury (SCI) model. Results We found that α 2 +/G301R mice display significantly improved functional recovery and decreased lesion volume compared to littermate controls (α 2 +/+ ) 7 days after SCI. The protein level of the α1 isoform was significantly increased, in contrast to the α3 isoform that significantly decreased 3 days after SCI in both α 2 +/G301R and α 2 +/+ mice. The level of the α2 isoform was significantly decreased in α 2 +/G301R mice both under naïve conditions and 3 days after SCI compared to α 2 +/+ mice. We found no differences in astroglial aquaporin 4 levels and no changes in the expression of chemokines (CCL2, CCL5 and CXCL1) and cytokines (TNF, IL-6, IL-1β, IL-10 and IL-5) between genotypes, just as no apparent differences were observed in location and activation of CD45 and F4/80 positive microglia and infiltrating leukocytes. Conclusion Our proof of concept study demonstrates that reduced expression of the α2 isoform in the spinal cord is protective following SCI. Importantly, the BMS and lesion volume were assessed at 7 days after SCI, and longer time points after SCI were not evaluated. However, the α2 isoform is a potential possible target of therapeutic strategies for the treatment of SCI

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

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

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

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

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

<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

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