Heterozygous mice deficient in Atp1a3 exhibit motor deficits by chronic restraint stress

doi:10.1016/j.bbr.2014.06.048

Behavioural Brain Research

Volume 272, 1 October 2014, Pages 100-110

https://doi.org/10.1016/j.bbr.2014.06.048 Get rights and content

Highlights

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Atp1a3^+/− exhibited shorter stride length at 4 weeks of age.
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Shorter stride length was persistently seen in chronically-stressed Atp1a3^+/− mice.
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Shorter hanging time was observed after chronic restraint stress in Atp1a3^+/−.
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Atp1a3^+/− is a useful animal model of RDP.

Abstract

Dystonia is a neurological disorder with involuntary and simultaneous contractions of agonist and antagonist muscles. Rapid-onset dystonia parkinsonism (RDP), one of the heredity forms of dystonia, is caused by mutations of Na,K-ATPase α3 subunit gene (ATP1A3). The abrupt onset of bulbar and limb symptoms of RDP are often triggered by physical and/or emotional stress. We reported previously that Atp1a3-deficient heterozygous mice showed higher locomotor activity and developed enhanced dystonia symptoms after kainate injection into the cerebellum, but not spontaneous movement disorder like RDP patients. Here we show that Atp1a3-deficient heterozygous mice exhibited shorter stride length at 4 weeks of age without stress and at later stages under chronic restraint stress loading. Shorter hanging time in the hanging box test was also observed after stress loading. Shorter stride length and hanging time may be relevant to certain phenotypes, such as gait abnormality, observed in RDP patients. Atp1a3 was widely expressed in the brain, including basal ganglia and cerebellum, and spinal cord of young mice, and the expression pattern was compatible with movement abnormalities under lack of one of alleles. Our results demonstrated the usefulness of Atp1a3-deficient heterozygous mice as an animal model of RDP and its potential use to explore the pathophysiology of movement abnormality in this disorder.

Introduction

Dystonia is a neurological movement disorder characterized by involuntary movement and simultaneous contractions of agonist and antagonist muscles [8], [4]. DYT12, known as rapid-onset dystonia-parkinsonism (RDP), is one of the heredity dystonia forms and often triggered by physical and emotional stress. Abrupt onset of bulbar symptoms and limb dystonia is observed between ages of 4 and 55 years, and symptoms stay for life [6]. The causative gene of DYT12 is ATP1A3, which encodes the α3 subunit of the Na,K-ATPase [14]. In an autosomal dominant manner, missense mutations, a deletion, or an insertion in ATP1A3 are linked to DYT12 [12], [7], [26], [3]. Recently, ATP1A3 was also identified as the affected gene in alternating hemiplegia of childhood (AHC) [21], [42], [25]. AHC is a neurological disorder characterized by transient hemiplegia with other paroxysmal symptoms, and sometimes presents with symptoms common with RDP, i.e., dystonia [42]. Manifestation of symptoms begins before 18 months of age in AHC patients [42]. Both diseases show a phenotypical continuum caused by mutation of ATP1A3 [42], and substitutions of amino acids at different positions or substitution of different amino acids at the same positions in ATP1A3 is thought to affect the manifestations of RDP and AHC [25]. It is possible that AHC is the severe phenotype of RDP [42].

The Na,K-ATPase consists of α (α1–α4) and β subunits (β1–β3), and maintains the electrochemical gradient of Na⁺ and K⁺ across the cell membrane using the energy of ATP hydrolysis [31], [2]. The α3 subunit is predominantly expressed in neurons, including the basal ganglia and cerebellar cortex, which play important roles in motor function in both adult and juvenile mice [11], [24]. So far, two lines of Atp1a3-deficient mice, Atp1a3^tm1Ling/+ [36] and our Atp1a3^+/− [24], and a line of mice with point mutation in Atp1a3 (Myk/⁺) [13] have been reported. Atp1a3^tm1Ling/+ mice exhibit lower memory function [36] and motor deficits in rotarod and balanced beam tests after stress loading [15]. Atp1a3^+/− mice show higher locomotor activity and enhanced dystonia symptoms following intracerebellar administration of kainate [24]. Myk/⁺ mice have low body weight, motor deficits including gait abnormality, and cognitive impairment under non-stress conditions. Such phenotypes are also observed in AHC patients [13], [28]. However, none of the above mice are reported to show spontaneous dystonia movement. Because stress is known to trigger the expression of dystonia in RDP and the stress loading such as application of chronic stress to mice is a useful method for induction of neurobehavioral signs [33], [20], [49], [27], [15], [32], [51], we performed in the present study various behavioral tests under specific stress loading (restraint) and examined whether Atp1a3^+/− of both sexes show phenotypes relevant to the symptoms of RDP, such as gait abnormality, slowness of movement, postural instability, and psychiatric depression-like feature [41]. We also completed comprehensive examination of Atp1a3 mRNA expression in young wild-type mice.

Section snippets

Animals

Atp1a3-deficient mice were established as described previously [24]. The heterozygous mice of Atp1a3 (Atp1a3^+/−) were backcrossed to C57BL6/J for 22–24 generations. Atp1a3^+/− and wild-type littermates were used in this study. Mice were housed under a 12 h light/dark cycle (light from 7:00 to 19:00) in a temperature-controlled room (22 ± 2 °C). Food and water were provided ad libitum.

Experimental protocol

For each sex and genotype, 4-week mice were divided into two groups (stressed and non-stressed). Footprint analysis,

Restraint-stress loading does not affect body weight of Atp1a3^+/−

Body weight is an index of general health. We measured body weight of mice during stress loading and evaluated whether motor deficits result from differences in body weight. Restraint stress was applied to the stressed group from 4 to 12 weeks of age, and body weight was measured every two weeks during this period. In female and male mice, body weight of Atp1a3^+/− was similar to that of the wild type at 4 weeks of age before the restraint stress loading (Fig. 1A and B). During the restraint

Chronic restraint stress induces motor deficits in Atp1a3^+/−

To investigate whether stress-load Atp1a3^+/− mice show motor deficits and other abnormal behaviors that are observed in RDP patients, comprehensive behavioral analyses were performed. Footprint analysis, which evaluates motor coordination and synchrony [10], is a behavioral test suitable for validation of the usefulness of Atp1a3^+/− as an animal model of RDP, because patients with RDP often show gait abnormalities [5], [30], [45]. Patients with RDP and other parkinsonism or Parkinson's disease

Acknowledgments

We thank all members of the Division of Biology for the helpful discussion and technical assistance. This work was supported by The Science Research Promotion Fund of the Promotion and Mutual Aid Corporation for Private Schools of Japan (to H.S.), and Jichi Medical University Young Investigator Award (to H.S.).

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DYT-TOR1A or DYT1 early-onset generalized dystonia is an inherited movement disorder characterized by sustained muscle contractions causing twisting, repetitive movements, or abnormal postures. The majority of the DYT1 dystonia patients have a trinucleotide GAG deletion in DYT1/TOR1A. Trihexyphenidyl (THP), an antagonist for excitatory muscarinic acetylcholine receptor M1, is commonly used to treat dystonia. Dyt1 heterozygous ΔGAG knock-in (KI) mice, which have the corresponding mutation, exhibit impaired motor-skill transfer. Here, the effect of THP injection during the treadmill training period on the motor-skill transfer to the accelerated rotarod performance was examined. THP treatment reversed the motor-skill transfer impairment in Dyt1 KI mice. Immunohistochemistry showed that Dyt1 KI mice had a significant reduction of the dorsolateral striatal cholinergic interneurons. In contrast, Western blot analysis showed no significant alteration in the expression levels of the striatal enzymes and transporters involved in the acetylcholine metabolism. The results suggest a functional alteration of the cholinergic system underlying the impairment of motor-skill transfer and the pathogenesis of DYT1 dystonia. Training with THP in a motor task may improve another motor skill performance in DYT1 dystonia.
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Mouse models for DYT/PARK-ATP1A3 dystonia, which is well-known to be stress-induced, are either asymptomatic or present symptoms not recapitulating the human disease such as seizures. Interestingly, most attempts to induce dystonia-like movements in transgenic DYT/PARK-ATP1A3 mouse models have failed so far (DeAndrade et al., 2011; Sugimoto et al., 2014). An argument could be made that the use of chronic restraint stress was insufficient and too mild for the induction of dystonia-like movements in these rodent models.
One of the great mysteries in dystonia pathophysiology is the role of environmental factors in disease onset and development. Progress has been made in defining the genetic components of dystonic syndromes, still the mechanisms behind the discrepant relationship between dystonic genotype and phenotype remain largely unclear. Within this review, the preclinical and clinical evidence for environmental stressors as disease modifiers in dystonia pathogenesis are summarized and critically evaluated. The potential role of extragenetic factors is discussed in monogenic as well as adult-onset isolated dystonia. The available clinical evidence for a “second hit” is analyzed in light of the reduced penetrance of monogenic dystonic syndromes and put into context with evidence from animal and cellular models. The contradictory studies on adult-onset dystonia are discussed in detail and backed up by evidence from animal models. Taken together, there is clear evidence of a gene-environment interaction in dystonia, which should be considered in the continued quest to unravel dystonia pathophysiology.
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In the current study, we demonstrated the occurrence of central endocrinopathies including precocious puberty and growth hormone deficiency. The pathophysiology of precocious puberty may originate from the arcuate nucleus (ARH) and the preoptic/anterior nucleus (POA) areas both of which demonstrate robust Atp1a3 expression in the mouse [10,16–18]. Kisspeptin-expressing neurons reside in the anteroventral periventricular nucleus and the ARH, among other hypothalamic nuclei, and send projections into the medial POA, where there is an abundance of gonadotropin-releasing hormone cell bodies that play a role in GnRH secretion [10,19].
Many central nervous system disorders result in hypothalamic-pituitary (HP) axis dysfunction. Alternating Hemiplegia of Childhood (AHC) is usually caused by mutations in the ATP1A3 subunit of the Na⁺/K⁺ ATPase, predominantly affecting GABAergic interneurons. GABAergic interneurons and the ATP1A3 subunit are both important for function of the hypothalamus. However, whether HP dysfunction occurs in AHC and, if so, how such dysfunction manifests remains to be investigated.
We conducted a retrospective review of a cohort of 50 consecutive AHC patients for occurrence of HP related manifestations and analyzed the findings of the 6 patients, from that cohort, with such manifestations.
Six out of 50 AHC patients manifested HP dysfunction. Three of these patients were mutation positive and 3 were mutation negative. Of the 6 patients with HP dysfunction, 3 had central precocious puberty. A fourth had short stature due to growth hormone deficiency. Two other patients had recurrent episodes of fever of unknown origin (FUO) diagnosed, after workups, as being secondary to central fever. All patients were evaluated and co-managed by pediatric neurology and endocrinology or rheumatology.
AHC was associated with HP dysfunction in about 12% of patients. Awareness of such dysfunction is important for anticipatory guidance and management particularly in the case of FUO which often presents a diagnostic dilemma. Our findings are also consistent with current understandings of the underlying pathophysiology of AHC and of the HP axis.
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Furthermore, the correlation of this phenotype with epilepsy or non-epileptic symptoms of AHC was not determined. The phenomenon of stress-induced triggers is a shared phenotype of AHC with other ATP1A3 mutant diseases, and has been recapitulated in other disease models including heat stress induced paralysis in a Drosophila model (Helseth et al., 2018; Holm and Lykke-Hartmann, 2016; Isaksen et al., 2017; Palladino et al., 2003; Sugimoto et al., 2014). Intriguingly, another study has identified a temperature-sensitive ion leakage phenomenon in a closely related Type II P-Type ATPase family member, after identifying an uncoordination phenotype in Drosophila harboring mutations in SERCA (Kaneko et al., 2014).
Alternating hemiplegia of childhood (AHC) is a rare neurodevelopmental disease caused by heterozygous de novo missense mutations in the ATP1A3 gene that encodes the neuronal specific α3 subunit of the Na,K-ATPase (NKA) pump. Mechanisms underlying patient episodes including environmental triggers remain poorly understood, and there are no empirically proven treatments for AHC. In this study, we generated patient-specific induced pluripotent stem cells (iPSCs) and isogenic controls for the E815K ATP1A3 mutation that causes the most phenotypically severe form of AHC. Using an in vitro iPSC-derived cortical neuron disease model, we found elevated levels of ATP1A3 mRNA in AHC lines compared to controls, without significant perturbations in protein expression. Microelectrode array analyses demonstrated that in cortical neuronal cultures, ATP1A3^+/E815K iPSC-derived neurons displayed less overall activity than neurons differentiated from isogenic mutation-corrected and unrelated control cell lines. However, induction of cellular stress by elevated temperature revealed a hyperactivity phenotype following heat stress in ATP1A3^+/E815K neurons compared to control lines. Treatment with flunarizine, a drug commonly used to prevent AHC episodes, did not impact this stress-triggered phenotype. These findings support the use of iPSC-derived neuronal cultures for studying complex neurodevelopmental conditions such as AHC and provide a platform for mechanistic discovery in a human disease model.
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The prevalence of delivery through cesarean-section (C-section) has been increasing worldwide. Although different modes of delivery, such as vaginal birth and C-section, are associated with incidence of some diseases in humans, little is known about how delivery stimuli affect short- and long-term brain function. Phenotypic analyses of Atp1a2 homozygous knockout (Atp1a2^−/−) neonates showed that the mode of delivery affected neural phenotypes; Atp1a2^−/− mice born by vaginal delivery started spontaneous breathing, while Atp1a2^−/− mice born by C-section showed a complete absence of breathing followed by their death. This life or death phenotype prompted us to examine several aspects of the neonatal brain following C-section or vaginal delivery. We found significantly different levels of several monoamines and transporters/channel proteins and a different c-Fos expression pattern. Furthermore, these mice showed different behaviors in adulthood. Our results suggest that birth mode impacts neurotransmission and functional network formation in the neonatal brain.
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Mutations in ATP1A3 lead to different phenotypes having in common acute neurological decompensation episodes triggered by a specific circumstance and followed by sequelae. Alongside Alternating Hemiplegia of Childhood (AHC), Rapid-onset Dystonia Parkinsonism (RDP) and Cerebellar ataxia, Areflexia, Pes cavus, Optic atrophy, Sensorineural hearing loss syndrome (CAPOS), a new Relapsing Encephalopathy with Cerebellar Ataxia (RECA) phenotype was published in 2015. We describe herein eight new pediatric cases. Most of them had no specific history when the first neurological decompensation episode occurred, before the age of 5 years, triggered by fever with severe paralytic hypotonia followed by ataxia with or without abnormal movements. Neurological sequelae with ataxia as the predominant symptom were present after the first episode in three cases and after at least one subsequent relapse in five cases. Five of the eight cases had a familial involvement with one of the two parents affected. The phenotype–genotype correlation is unequivocal with the causal substitution always located at position 756. The pathophysiology of the dysfunctions of the mutated ATPase pump, triggered by fever is unknown. Severe recurrent neurological decompensation episodes triggered by fever, without any metabolic cause, should lead to the sequencing of ATP1A3.

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Research reportHeterozygous mice deficient in Atp1a3 exhibit motor deficits by chronic restraint stress

Highlights

Abstract

Introduction

Section snippets

Animals

Experimental protocol

Restraint-stress loading does not affect body weight of Atp1a3+/−

Chronic restraint stress induces motor deficits in Atp1a3+/−

Acknowledgments

Neurobiol Dis

Neurosci Biobehav Rev

Neuron

Behav Brain Res

J Neurosci Methods

Physiol Behav

Jpn J Pharmacol

Brain Res

Lancet Neurol

Behav Process

Neurosci Lett

NeuroScience

Late-onset Parkinsonism in NFκB/c-Rel-deficient mice

Brain

Na+, K+-ATPase: functions in the nervous system and involvement in neurologic disease

Neurology

A C-terminal mutation of ATP1A3 underscores the crucial role of sodium affinity in the pathophysiology of rapid-onset dystonia-parkinsonism

Hum Mol Genet

Rapid-onset dystonia-parkinsonism in a second family

Neurology

The phenotypic spectrum of rapid-onset dystonia-parkinsonism (RDP) and mutations in the ATP1A3 gene

Brain

ATP1A3 mutations in infants: a new rapid-onset dystonia-Parkinsonism phenotype characterized by motor delay and ataxia

Dev Med Child Neurol

The pathophysiological basis of dystonias

Nat Rev Neurosci

Behavioural profiles of inbred mouse strains used as transgenic backgrounds. I: Motor tests

Genes Brain Behav

Tests to assess motor phenotype in mice: a user's guide

Nat Rev Neurosci

Distribution of Na/K-ATPase alpha 3 isoform, a sodium–potassium P-type pump associated with rapid-onset of dystonia parkinsonism (RDP) in the adult mouse brain

J Comp Neurol

Mutation I810N in the α3 isoform of Na+,K+-ATPase causes impairments in the sodium pump and hyperexcitability in the CNS

Proc Nat Acad Sci USA

The clinical spectrum of freezing of gait in atypical parkinsonism

Mov Disord

Depressive state with anxiety in repeated cold-stressed mice in forced swimming tests

Jpn J Pharmacol

Research report
Heterozygous mice deficient in Atp1a3 exhibit motor deficits by chronic restraint stress

Restraint-stress loading does not affect body weight of Atp1a3^+/−

Chronic restraint stress induces motor deficits in Atp1a3^+/−

Na⁺, K⁺-ATPase: functions in the nervous system and involvement in neurologic disease

Mutation I810N in the α3 isoform of Na⁺,K⁺-ATPase causes impairments in the sodium pump and hyperexcitability in the CNS