Disrupted Plasma Membrane Protein Homeostasis in a Xenopus Laevis Model of Retinitis Pigmentosa

Abstract

Rhodopsin mislocalization is frequently observed in retinitis pigmentosa (RP) patients. For example, class I mutant rhodopsin is deficient in the VxPx trafficking signal, mislocalizes to the plasma membrane (PM) of rod photoreceptor inner segments (ISs), and causes autosomal dominant RP. Mislocalized rhodopsin causes photoreceptor degeneration in a manner independent of light-activation. In this manuscript, we took advantage of Xenopus laevis models of both sexes expressing wild-type human rhodopsin or its class I Q344ter mutant fused to Dendra2 fluorescent protein to characterize a novel light-independent mechanism of photoreceptor degeneration caused by mislocalized rhodopsin. We found that rhodopsin mislocalized to the PM is actively internalized and transported to lysosomes where it is degraded. This degradation process results in the downregulation of a crucial component of the photoreceptor IS PM: the sodiumpotassium ATPase ␣-subunit (NKA␣). The downregulation of NKA␣ is not because of decreased NKA␣ mRNA, but due to cotransport of mislocalized rhodopsin and NKA␣ to lysosomes or autophagolysosomes. In a separate set of experiments, we found that class I mutant rhodopsin, which causes NKA␣ downregulation, also causes shortening and loss of rod outer segments (OSs); the symptoms frequently observed in the early stages of human RP. Likewise, pharmacological inhibition of NKA␣ led to shortening and loss of rod OSs. These combined studies suggest that mislocalized rhodopsin leads to photoreceptor dysfunction through disruption of the PM protein homeostasis and compromised NKA␣ function. This study unveiled a novel role of lysosome-mediated degradation in causing inherited disorders manifested by mislocalization of ciliary receptors. Significance StatementRetinal ciliopathy is the most common form of inherited blinding disorder frequently manifesting rhodopsin mislocalization. Our understanding of the relationships between rhodopsin mislocalization and photoreceptor dysfunction/degeneration has been far from complete. This study uncovers a hitherto uncharacterized consequence of rhodopsin mislocalization: the activation of the lysosomal pathway, which negatively regulates the amount of the sodium-potassium ATPase (NKA␣) on the inner segment plasma membrane. On the plasma membrane, mislocalized rhodopsin extracts NKA␣ and sends it to lysosomes where they are co-degraded. Compromised NKA␣ function leads to shortening and loss of the photoreceptor outer segments as observed for various inherited blinding disorders. In summary, this study revealed a novel pathogenic mechanism applicable to various forms of blinding disorders caused by rhodopsin mislocalization.

RPE Cells Engulf Microvesicles Secreted by Degenerating Rod Photoreceptors

“…Immunohistochemistry. X. laevis retinal sections (12 mm) were prepared and stored at À80°C, as described previously (Lodowski et al, 2013;Ropelewski and Imanishi, 2019). Slides were removed from À80°C storage conditions and warmed using a 37°C incubator (CCC 0.5d, Boekel Scientific).…”

“…Preparation of retina explant for confocal imaging. Tadpoles were killed, decapitated, and their eyes were removed as described previously (Ropelewski and Imanishi, 2019). The RPE layers were separated from the retinas, which were immediately placed into a glass-bottom dish (catalog #P35G-1.5-14-C, MatTek) containing modified Wolf medium (D-glucose, 700 mg/L, 30 mM NaHCO 3 , 55% MEM, 31% sodium-free BBS, 10% FBS; Lodowski et al, 2013).…”

“…Confocal microscopy, image analysis, quantification, and statistical analysis. All images were acquired using a Leica TCS SP2 laser-scanning confocal/multiphoton microscope system equipped with the following four lasers for excitation: 488 nm argon ion, 543 nm HeNe, 633 nm HeNe, and tunable Chameleon XR Ti:Sapphire laser (Leica Microsystems), as described previously (Ropelewski and Imanishi, 2019). An HCX PL APO CS 40.0 Â 1.25 oil UV objective lens was used for imaging.…”

“…Thus, we have initiated an effort to understand the renewal of IS PM proteins, especially focusing on class I mutant rhodopsin. In addition to the vesicle-mediated removal described above, mislocalized class I mutant rhodopsin is degraded intracellularly: once reaching the IS PM, mislocalized rhodopsin becomes internalized and subsequently degraded by lysosomes (Ropelewski and Imanishi, 2019). The IS PM component Na 1 /K 1 -ATPase (NKA) plays a critical role in maintaining both the dark current of photoreceptor cells (Yau and Baylor, 1989) and interactions between bipolar and photoreceptor cells (Molday et al, 2007;Friedrich et al, 2011).…”

“…The IS PM component Na 1 /K 1 -ATPase (NKA) plays a critical role in maintaining both the dark current of photoreceptor cells (Yau and Baylor, 1989) and interactions between bipolar and photoreceptor cells (Molday et al, 2007;Friedrich et al, 2011). The lysosome-mediated removal of class I mutant rhodopsin induces co-internalization and codegradation of native NKA, compromising the structure and function of rod photoreceptors (Ropelewski and Imanishi, 2019). Another IS PM protein, HCN1 channel, plays a role in the normal electrophysiological response of photoreceptor cells, and its deficiency worsens the symptoms of retinitis pigmentosa (Schön et al, 2016).…”