Abstract
Na(+)/K(+)-ATPases are transmembrane ion pumps that maintain ion gradients across the basolateral plasma membrane in all animal cells to facilitate essential biological functions. Mutations in the Na(+)/K(+)-ATPase α3 subunit gene (ATP1A3) cause rapid-onset dystonia-parkinsonism, a rare movement disorder characterized by sudden onset of dystonic spasms and slow movements. In the brain, ATP1A3 is principally expressed in neurons. In zebrafish, the transcripts of the two ATP1A3 orthologs, Atp1a3a and Atp1a3b, show distinct expression in the brain. Surprisingly, targeted knockdown of either Atp1a3a or Atp1a3b leads to brain ventricle dilation, a likely consequence of ion imbalances across the plasma membrane that cause accumulation of cerebrospinal fluid in the ventricle. The brain ventricle dilation is accompanied by a depolarization of spinal Rohon-Beard neurons in Atp1a3a knockdown embryos, suggesting impaired neuronal excitability. This is further supported by Atp1a3a or Atp1a3b knockdown results where altered responses to tactile stimuli as well as abnormal motility were observed. Finally, proteomic analysis identified several protein candidates highlighting proteome changes associated with the knockdown of Atp1a3a or Atp1a3b. Our data thus strongly support the role of α3Na(+)/K(+)-ATPase in zebrafish motility and brain development, associating for the first time the α3Na(+)/K(+)-ATPase deficiency with brain ventricle dilation.
Highlights
The Naϩ/Kϩ-ATPase maintains Naϩ/Kϩ gradients across the plasma membrane, essential for cellular functions
The highest expression of Atp1a3a occurred at the pectoral fin stage (60 hpf), which was almost 6-fold higher than the relative expression of the Atp1a3a transcript in adult zebrafish (Fig. 1A)
The extent of the brain ventricle dilation was reduced as compared with Atp1a3a KD embryos, and severe, moderate, and slight/no ventricle dilation was present in 21, 29, and 50%, respectively (Fig. 2D). These findings suggest that Atp1a3a KD had specific effects that led to the brain ventricle dilation in ␣3a- and ␣3a-SP MO-injected embryos
Summary
The Naϩ/Kϩ-ATPase maintains Naϩ/Kϩ gradients across the plasma membrane, essential for cellular functions. The brain ventricle dilation is accompanied by a depolarization of spinal Rohon-Beard neurons in Atp1a3a knockdown embryos, suggesting impaired neuronal excitability This is further supported by Atp1a3a or Atp1a3b knockdown results where altered responses to tactile stimuli as well as abnormal motility were observed. Two different genetically modified mouse models targeting the Atp1a3 gene exist: ␣3ϩ/ϪKII4 [14] and ␣3ϩ/ϪKII810N (Myshkin) [15] These mice display learning/memory deficits [14], epilepsy/seizures [15], stress-induced motor symptoms [16], anxious phenotype, and depression-like behavior [17]. To identify additional neuronal functions that may depend on correct ion homeostasis for normal performance, we used a proteomic approach This revealed several proteins, including cytoskeletal, ion-binding, muscle-associated proteins, etc., with expression levels that were affected by Atp1a3a or Atp1a3b KD. Zebrafish can serve as an advantageous model for analysis of brain ventricular volume maintenance and embryonic motility, both related to ion homeostasis
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