Atp1b2Atp1b1 Knock-In Mice Exhibit a Cone-Rod Dystrophy-Like Phenotype

Abstract

The Na,K-ATPase is a heterodimeric ion pump consisting of various combinations of a catalytic α-subunit (α1, α2, α3, or α4, encoded by ATP1A1-ATP1A4) and a β-subunit (β1, β2, or β3, encoded by ATP1B1-ATP1B3). We have previously shown that Atp1b2 knock-out (ko) mice exhibit rapid photoreceptor cell degeneration, whereas Atp1b2^Atp1b1 knock-in (ki) mice, which express the β1-subunit instead of the β2-subunit under regulatory elements of the Atp1b2 gene, exhibit slowly progressive retinal dystrophy. Here, we performed a detailed analysis of the retinal phenotype of the Atp1b2^Atp1b1 ki mouse. We found that the number of cone photoreceptor cells in the mutant retinas was significantly reduced by postnatal day 28. The retinas of 4-month-old mice were almost devoid of cones. The early onset and rapid loss of cones was followed by a slowly progressive degeneration of rods. Other retinal cell types were unaffected. Nonradioactive in situ hybridization and immunohistochemistry revealed that wild-type photoreceptors expressed β3 and high levels of β2, while Atp1b2^Atp1b1 ki photoreceptor cells expressed β3 and low levels of transgenic β1. Additionally, levels of retinoschisin, a secreted retina-specific protein that interacts directly with the β2-subunit, were greatly reduced in mutant retinas. The results demonstrate that the β1-subunit can functionally compensate, at least in part, for the absence of the β2-subunit. The results also show that cones are more susceptible to Na,K-ATPase dysfunction than rods. Taken together, the present study identifies the Atp1b2^Atp1b1 ki mutant as a novel animal model of an early-onset and rapidly progressive cone-rod dystrophy.

Keywords: ATP1A3; ATP1B2; Na,K-ATPase; bipolar cells; cones; cone–rod dystrophy; degeneration; photoreceptor cells; retina; retinoschisin; rods; voltage-gated potassium channel.

Figure 1

Neuroinflammation and retinal thinning in β2/β1 ki mice. (A) Expression of GFAP (Aa–Af), IBA1 (Ag–Al), and CD68 (Am–Ar) in β2/β1 ki and wild-type retinas at different ages. (B) Thickness of the total retina (Ba), the photoreceptor layer (Bb), and the inner nuclear layer (Bc) in β2/β1 ki (filled bars) and wild-type retinas (open bars) at different ages. Each bar represents the mean value (±SEM) of six animals. CD68: cluster of differentiation 68; gcl: ganglion cell layer; GFAP: glial fibrillary acidic protein; IBA1: ionized calcium-binding adapter molecule 1; inl: inner nuclear layer; ipl: inner plexiform layer; ki: knock-in; onl: outer nuclear layer; P: postnatal day; wt: wild-type; n.s.: not significant, **: p < 0.01, ***: p < 0.001 by two-way ANOVA followed by a Bonferroni post hoc test. Scale bar: 100 µm.

Figure 2

Expression of Na,K-ATPase subunits in wild-type and β2/β1 ki photoreceptors. In wild-type photoreceptors, α3 is strongly expressed in the inner segments (a–c) and co-localized with the β2-subunit (m–o), whereas in β2/β1 ki photoreceptors, α3 is weakly expressed in the inner segments (d–f) and co-localized with the β1-subunit (j–l). Inner segments of wild-type and β2/β1 ki photoreceptors are β1- and β2-negative, respectively (g–i and p–r, respectively). IS: inner segments; ki: knock-in; OS: outer segments; RHO: rhodopsin. Scale bar: 10 µm.

Figure 3

Expression of β-subunits in wild-type and β2/β1 ki retinas. Expression of Atp1b1, Atp1b2, and Atp1b3 transcripts in adult wild-type and β2/β1 ki retinas. Black arrows in (a,c,g,i,k) indicate the localization of the indicated transcripts in the photoreceptor inner segments. The retinal pigment epithelium and choroid are indicated by white arrowheads (a–k). To control for signal specificity, β2/β1 ki retinas were hybridized with an Atp1b2 antisense probe (e) or an Atp1b3 sense probe (Atp1b3s; (h)) and wild-type retinas were hybridized with an Atp1b1 sense probe (Atp1b1s; (d)). ki: knock-in; wt: wild-type. Scale bar in (h) for (a–h): 200 µm, in (k) for (i–k): 100 µm.

Figure 4

Photoreceptor cell degeneration in β2/β1 ki mice. (A) β2/β1 ki retinas show thinning of the outer nuclear layer (onl) and rapidly progressive loss of cone arrestin-positive (Aa–Af), m+s opsin-positive (Ag–Al), or PNA-labeled (Am–Ar) cone photoreceptor cells. Age-matched wild-type retinas are shown for comparison. (B) Number of rows of photoreceptor cell nuclei (Ba) and the density of cone arrestin- (Bb) or m+s opsin-positive (Bc) cones in β2/β1 ki (filled bars) and wild-type retinas (open bars) at different ages. Each bar represents the mean value (±SEM) of six animals. n.s.: not significant, *: p < 0.05, ***: p < 0.001 according to two-way ANOVA followed by a Bonferroni post hoc test. gcl: ganglion cell layer; inl: inner nuclear layer; ipl: inner plexiform layer; ki: knock-in; onl: outer nuclear layer; P: postnatal day; PNA: peanut agglutinin; wt: wild-type. Scale bar: 100 µm.

Figure 5

Expression of β1 and β2 in rod and cone bipolar cells. PKCα-positive rod bipolar cells and SCGN-positive cone bipolar cells in adult wild-type mice were β1-negative ((Aa–Ac) and (Ba–Bc), respectively) and β2-positive ((Ad–Af) and (Bd–Bf), respectively; some β2-positive cells are marked with white asterisks). In comparison, mutant rod and cone bipolar cells expressed β1 ((Ag–Ai) and (Bg–Bi), respectively; some β1-positive cells are marked with white asterisks) and were β2-negative ((Aj–Al) and (Bj–Bl), respectively). inl: inner nuclear layer; ki: knock-in; onl: outer nuclear layer; PKCα: protein kinase C alpha; SCGN: secretagogin; wt: wild-type. Scale bar: 20 µm.

Figure 6

Normal number of rod and cone bipolar cells and ganglion cells in β2/β1 ki retinas. Qualitative (A) and quantitative (B) analysis revealed similar densities of PKCα-positive rod bipolar cells (Aa,Ab,Ba), SCGN-positive cone bipolar cells (Ac,Ad,Bb), and BRN-3A-positive ganglion cells (Ae,Af,Bc) in wild-type and β2/β1 ki retinas. Each bar in (B) represents the mean value (±SEM) of six animals. BRN-3A: brain-specific homeobox/POU domain protein 3A; gcl: ganglion cell layer; inl: inner nuclear layer; ipl: inner plexiform layer; ki: knock-in; onl: outer nuclear layer; opl: outer plexiform layer; P: postnatal day; PKCα: protein kinase C alpha; SCGN: secretagogin; wt: wild-type; n.s.: not significant according to the Student’s t-test. Scale bar in (Ad) for (Aa–Ad) and (Af) for (Ae,Af): 50 µm.

Figure 7

Expression of retinoschisin in mutant and wild-type retinas. In wild-type retinas, retinoschisin (RS1; (Aa)) and β2 (Ab) were co-localized (Ac) in the photoreceptor inner segments, outer plexiform layer, and cell bodies in the inner nuclear layer. In β2/β1 ki retinas (Ae–Ag), retinoschisin was barely detectable and diffusely distributed (Ae). β2 was not detected in mutant retinas as expected (Af,Ag) is the overlay of (Ae,Af). As a negative control, sections were incubated with the secondary antibodies only (Ad,Ah). (B) Western blot analysis confirmed massively reduced levels of retinoschisin in mutant retinas when compared to wild-type retinas. Note that three-fold more mutant than wild-type samples were loaded. gcl: ganglion cell layer; inl: inner nuclear layer; ki: knock-in; onl: outer nuclear layer; RS1: retinoschisin; TPS: total protein stain; wt: wild-type. Scale bar: 50 µm.