α3Na+/K+-ATPase deficiency causes brain ventricle dilation and abrupt embryonic motility in zebrafish

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.

FIGURE 1.

Expression of Atp1a3a and Atp1a3b mRNA in zebrafish embryos. A, Atp1a3a (black bars) and Atp1a3b (gray bars) mRNA expressions were quantified by qRT-PCR and normalized to Actb2 expression. Data are presented as mean ± S.E. of triplicate measurements. B, Atp1a3a mRNA expression analyzed by whole-mount in situ hybridization in 60 hpf zebrafish embryos; the inset shows sense probe hybridized control embryo. Atp1a3a is expressed in the brain and the spinal cord. The numbered vertical dashed lines, here and in C, show the positions of the transverse sections shown below in sections I–V. The abbreviations used are: C: cerebellum; Cg: cranial ganglia; D: diencephalon; E: epiphysis; H: hypothalamus; Hb: hindbrain; Mo: medulla oblongata; N: notochord; Oc: optic cup; T: tectum; Te: telencephalon; Tg: tegmentum; Sc: spinal cord. Scale bars represent 100 μm in whole-mount images and 50 μm in sections. C, Atp1a3b mRNA expression analyzed by whole-mount in situ hybridization in 60 hpf zebrafish embryos; the inset shows sense probe hybridized control embryo. Atp1a3b is expressed in specific brain regions.

FIGURE 2.

Knockdown of Atp1a3a causes brain ventricle dilation. A, significant brain ventricle dilation occurred in embryos upon Atp1a3a KD mediated by α_3a-MO or α_3a-SP-MO as compared with std-MO-injected control embryo. This phenotype was rescued by co-injection of Atp1a3a mRNA. p53-MO co-injections with any of the MOs did not rescue the brain ventricle dilation phenotype. B, brain ventricles of Tg(gfap:GFP) line, non-MO-injected and α_3a-MO-injected, were injected with rhodamine-conjugated dextran. Brain ventricles and the astrocytes are highlighted by red and green fluorescence, respectively. C, qRT-PCR tested efficiency of α_3a-SP-MO-mediated KD in terms of changes in the relative expression level of Atp1a3a. Data are presented as mean ± S.E. of triplicate measurements. Concentrations of MOs are indicated. D, mean percentages ± S.D. of the α_3a-SP-MO-injected embryos suffering from brain ventricle dilation of different severity, with (white columns, n = 117) and without (black columns, n = 96) Atp1a3a mRNA co-injection, are plotted. Embryos at the lower panel represent the extent of the brain ventricle dilation (VD) used as a criterion for grouping as severe (+++) (scale bar: ∼235 μm), moderate (++) (scale bar: ∼150 μm), and slight/no (+) (scale bar: ∼65 μm). *, p < 0.1; **, p < 0.01.

FIGURE 3.

Knockdown of Atp1a3b phenocopies Atp1a3a knockdown. A, brain ventricle dilation was observed in embryos upon Atp1a3b KD mediated by α_3b-MO- or α_3b-SP-MO-injected embryo as compared with the std-MO-injected control embryo. This phenotype was rescued by co-injection of Atp1a3b mRNA. p53-MO co-injections with any of the MOs did not rescue the brain ventricle dilation phenotype. B, brain ventricles of Tg(gfap:GFP) line, non-MO-injected and α_3b-MO-injected, were injected with rhodamine-conjugated dextran. Brain ventricles and the astrocytes are highlighted by red and green fluorescence, respectively. C, α_3b-SP-MO-mediated KD efficiency was tested by qRT-PCR in terms of changes in the relative expression level of Atp1a3b. Data are presented as mean ± S.E. of triplicate measurements. Concentrations of MOs are indicated. D, mean percentages ± S.D. of the α_3b-SP-MO-injected embryos suffering from brain ventricle dilation (VD) of different severity, with (white columns, n = 148) and without (black columns, n = 88) Atp1a3a mRNA co-injection, are plotted. The degree of brain ventricle dilation is as follows: +, slight/no; ++, moderate; +++, severe. **, p < 0.01; ***, p < 0.001. E, Atp1a3a mRNA did not rescue α_3b-SP-MO-injected embryos; similarly, Atp1a3b mRNA did not rescue α_3a-SP-MO-injected embryos from brain ventricle dilation.

FIGURE 4.

RB neurons are more depolarized in Atp1a3a KD zebrafish displaying severe brain ventricle dilation. A, RMP values of RB neurons from WT (n = 5), std-MO-injected (n = 6), and α_3a-MO-injected embryos (n = 10) are plotted. The α_3a-MO-injected embryos are divided into embryos displaying severe (+++) (n = 5) or slight/no (+) (n = 5) brain ventricle dilation (VD). The number of cells (n) recorded per group stems from at least three different animals. RMP data are presented as mean ± S.D. **, p < 0.01 between RMPs of α_3a-MO-injected embryos with severe ventricle dilation and control groups, WT, and std-MO-injected embryos. B, schematic representation summarizing the depolarization of the RMP in RB neurons in an Atp1a3a KD embryo. The scheme covers the time frame of 48–60 hpf. The dashed cross marks a malfunctioning α_3aNa⁺/K⁺-ATPase.

FIGURE 5.

Atp1a3a and Atp1a3b KD embryos respond to touch but have abnormal motility. Successive frame shots from touch response assay display three representative WT embryos with burst swimming response (top panel, supplemental Movie 1), a representative α_3a-MO-injected embryo that kept swirling around itself (middle panels, supplemental Movie 3), and a α_3b-MO-injected embryo that responded as a short distance recoil (bottom panel, supplemental Movie 4). Touch-stimulated embryos are marked with a color-coded asterisk, and the frame shot times are merged on images in seconds.

FIGURE 6.

Atp1a3a mRNA is present in DA neurons, but no loss of DA neuron was observed in Atp1a3a KD embryos. A, TH-positive DA neurons (green fluorescence) and Atp1a3a mRNA expression (red fluorescence) in the zebrafish brain are imaged along the dorsoventral axis (anterior to the left; posterior to the right). Scale bars represent 50 μm. Areas marked by squares are shown in the lower panel at higher magnification (scale bars represent 5 μm). B, Th mRNA expression analyzed by in situ hybridization in WT, α_3a-SP-MO-injected, and Atp1a3a mRNA-rescued embryos. C, total staining of Th mRNA in WT (n = 10) and α_3a-SP-MO-injected embryos (n = 10) was quantified using ImageJ, and mean values of integrated densities (IntDen) are plotted with standard deviations.

FIGURE 7.

Relative mRNA expressions of some of the regulated proteins detected in proteomics assay. A and B, mRNA expressions of selected proteins regulated by α_3a-MO-mediated Atp1a3a KD (A) or α_3b-MO-mediated Atp1a3b KD (B) were quantified by qRT-PCR and normalized to Actb2 expression in embryos at 60 hpf. Data are presented as mean ± S.E. of triplicate measurements.