Saravanan Kolandaivelu

AIPL1, a protein associated with childhood blindness, interacts with α-subunit of rod phosphodiesterase (PDE6) and is essential for its proper assembly

Mutations in the gene coding for AIPL1 cause Leber congenital amaurosis (LCA), a severe form of childhood blindness. The severity in disease is reflected in the complete loss of vision and rapid photoreceptor degeneration in the retinas of mice deficient in AIPL1. Our previous observations suggest that rod photoreceptor degeneration in retinas lacking AIPL1 is due to the massive reduction in levels of rod cGMP phosphodiesterase (PDE6) subunits (α, β, and γ). To date, the crucial link between AIPL1 and the stability of PDE6 subunits is not known. In this study using ex vivo pulse label analysis, we demonstrate that AIPL1 is not involved in the synthesis of PDE6 subunits. However, ex vivo pulse-chase analysis clearly shows that in the absence of AIPL1, rod PDE6 subunits are rapidly degraded by proteasomes. We further demonstrate that this rapid degradation of PDE6 is due to the essential role of AIPL1 in the proper assembly of synthesized individual PDE6 subunits. In addition, using a novel monoclonal antibody generated against AIPL1, we show that the catalytic subunit (α) of PDE6 associates with AIPL1 in retinal extracts. Our studies establish that AIPL1 interacts with the catalytic subunit (α) of PDE6 and is needed for the proper assembly of functional rod PDE6 subunits.

RAS-converting enzyme 1-mediated endoproteolysis is required for trafficking of rod phosphodiesterase 6 to photoreceptor outer segments

Prenylation is the posttranslational modification of a carboxyl-terminal cysteine residue of proteins that terminate with a CAAX motif. Following prenylation, the last three amino acids are cleaved off by the endoprotease, RAS-converting enzyme 1 (RCE1), and the prenylcysteine residue is methylated. Although it is clear that prenylation increases membrane affinity of CAAX proteins, less is known about the importance of the postprenylation processing steps. RCE1 function has been studied in a variety of tissues but not in neuronal cells. To approach this issue, we generated mice lacking Rce1 in the retina. Retinal development proceeded normally in the absence of Rce1, but photoreceptor cells failed to respond to light and subsequently degenerated in a rapid fashion. In contrast, the inner nuclear and ganglion cell layers were unaffected. We found that the multimeric rod phosphodiesterase 6 (PDE6), a prenylated protein and RCE1 substrate, was unable to be transported to the outer segments in Rce1-deficient photoreceptor cells. PDE6 present in the inner segment of Rce1-deficient photoreceptor cells was assembled and functional. Synthesis and transport of transducin, and rhodopsin kinase 1 (GRK1), also prenylated substrates of RCE1, was unaffected by Rce1 deficiency. We conclude that RCE1 is essential for the intracellular trafficking of PDE6 and survival of photoreceptor cells.

Transducin translocation contributes to rod survival and enhances synaptic transmission from rods to rod bipolar cells

In rod photoreceptors, several phototransduction components display light-dependent translocation between cellular compartments. Notably, the G protein transducin translocates from rod outer segments to inner segments/spherules in bright light, but the functional consequences of translocation remain unclear. We generated transgenic mice where light-induced transducin translocation is impaired. These mice exhibited slow photoreceptor degeneration, which was prevented if they were dark-reared. Physiological recordings showed that control and transgenic rods and rod bipolar cells displayed similar sensitivity in darkness. After bright light exposure, control rods were more strongly desensitized than transgenic rods. However, in rod bipolar cells, this effect was reversed; transgenic rod bipolar cells were more strongly desensitized than control. This sensitivity reversal indicates that transducin translocation in rods enhances signaling to rod bipolar cells. The enhancement could not be explained by modulation of inner segment conductances or the voltage sensitivity of the synaptic Ca2+ current, suggesting interactions of transducin with the synaptic machinery.

Alternative Splicing Shapes the Phenotype of a Mutation in BBS8 To Cause Nonsyndromic Retinitis Pigmentosa

ABSTRACT Bardet-Biedl syndrome (BBS) is a genetic disorder affecting multiple systems and organs in the body. Several mutations in genes associated with BBS affect only photoreceptor cells and cause nonsyndromic retinitis pigmentosa (RP), raising the issue of why certain mutations manifest as a systemic disorder whereas other changes in the same gene affect only a specific cell type. Here, we show that cell-type-specific alternative splicing is responsible for confining the phenotype of the A-to-G substitution in the 3′ splice site of BBS8 exon 2A (IVS1-2A>G mutation) in the BBS8 gene to photoreceptor cells. The IVS1-2A>G mutation leads to missplicing of BBS8 exon 2A, producing a frameshift in the BBS8 reading frame and thus eliminating the protein specifically in photoreceptor cells. Cell types other than photoreceptors skip exon 2A from the mature BBS8 transcript, which renders them immune to the mutation. We also show that the splicing of Bbs8 exon 2A in photoreceptors is directed exclusively by redundant splicing enhancers located in the adjacent introns. These intronic sequences are sufficient for photoreceptor-cell-specific splicing of heterologous exons, including an exon with a randomized sequence.

AIPL1, A protein linked to blindness, is essential for the stability of enzymes mediating cGMP metabolism in cone photoreceptor cells

Defects in the photoreceptor-specific gene encoding aryl hydrocarbon receptor interacting protein like-1 (AIPL1) are linked to blinding diseases, including Leber congenital amaurosis (LCA) and cone dystrophy. While it is apparent that AIPL1 is needed for rod and cone function, the role of AIPL1 in cones is not clear. In this study, using an all-cone animal model lacking Aipl1, we show a light-independent degeneration of M- and S-opsin containing cones that proceeds in a ventral-to-dorsal gradient. Aipl1 is needed for stability, assembly and membrane association of cone PDE6, an enzyme crucial for photoreceptor function and survival. Furthermore, RetGC1, a protein linked to LCA that is needed for cGMP synthesis, was dramatically reduced in cones lacking Aipl1. A defect in RetGC1 is supported by our finding that cones lacking Aipl1 exhibited reduced levels of cGMP. These findings are in contrast to the role of Aipl1 in rods, where destabilization of rod PDE6 results in an increase in cGMP levels, which drives rapid rod degeneration. Our results illustrate mechanistic differences behind the death of rods and cones in retinal degenerative disease caused by deficiencies in AIPL1.

Rod Phosphodiesterase-6 (PDE6) Catalytic Subunits Restore Cone Function in a Mouse Model Lacking Cone PDE6 Catalytic Subunit

Rod and cone photoreceptor neurons utilize discrete PDE6 enzymes that are crucial for phototransduction. Rod PDE6 is composed of heterodimeric catalytic subunits (αβ), while the catalytic core of cone PDE6 (α′) is a homodimer. It is not known if variations between PDE6 subunits preclude rod PDE6 catalytic subunits from coupling to the cone phototransduction pathway. To study this issue, we generated a cone-dominated mouse model lacking cone PDE6 (Nrl−/− cpfl1). In this animal model, using several independent experimental approaches, we demonstrated the expression of rod PDE6 (αβ) and the absence of cone PDE6 (α′) catalytic subunits. The rod PDE6 enzyme expressed in cone cells is active and contributes to the hydrolysis of cGMP in response to light. In addition, rod PDE6 expressed in cone cells couples to the light signaling pathway to produce S-cone responses. However, S-cone responses and light-dependent cGMP hydrolysis were eliminated when the β-subunit of rod PDE6 was removed (Nrl−/− cpfl1 rd). We conclude that either rod or cone PDE6 can effectively couple to the cone phototransduction pathway to mediate visual signaling. Interestingly, we also found that functional PDE6 is required for trafficking of M-opsin to cone outer segments.

Cone Phosphodiesterase-6α′ Restores Rod Function and Confers Distinct Physiological Properties in the Rod Phosphodiesterase-6β-Deficient rd10 Mouse

Phosphodiesterase-6 (PDE6) is the key effector enzyme of the vertebrate phototransduction pathway in rods and cones. Rod PDE6 catalytic core is composed of two distinct subunits, PDE6α and PDE6β, whereas two identical PDE6α′ subunits form the cone PDE6 catalytic core. It is not known whether this difference in PDE6 catalytic subunit identity contributes to the functional differences between rods and cones. To address this question, we expressed cone PDE6α′ in the photoreceptor cells of the retinal degeneration 10 (rd10) mouse that carries a mutation in rod PDEβ subunit. We show that adeno-associated virus-mediated subretinal delivery of PDE6α′ rescues rod electroretinogram responses and preserves retinal structure, indicating that cone PDE6α′ can couple effectively to the rod phototransduction pathway. We also show that restoration of light sensitivity in rd10 rods is attributable to assembly of PDE6α′ with rod PDE6γ. Single-cell recordings revealed that, surprisingly, rods expressing cone PDE6α′ are twofold more sensitive to light than wild-type rods, most likely because of the slower shutoff of their light responses. Unlike in wild-type rods, the response kinetics in PDE6α′-treated rd10 rods accelerated with increasing flash intensity, indicating a possible direct feedback modulation of cone PDE6α′ activity. Together, these results demonstrate that cone PDE6α′ can functionally substitute for rod PDEαβ in vivo, conferring treated rods with distinct physiological properties.

AIPL1 Protein and its Indispensable Role in Cone Photoreceptor Function and Survival

Mutations in Aryl hydrocarbon receptor interacting protein like-1 (AIPL1) are linked to Leber congenital amaurosis (LCA), a severe blinding disease that occurs in early childhood. The severity of disease is due to requirement for AIPL1 in both rod and cone photoreceptor cell survival and function. Aipl1 is expressed very early during retinal development in both rods and cones. In adult primates, robust expression of Aipl1 is found in rods but not in cones. Mouse models revealed the importance of AIPL1 in stability and function of heteromeric phosphodiesterase 6 (PDE6), an enzyme needed for visual response. However, the need for AIPL1 in cone cell survival and function is not clearly understood. In this chapter, using results obtained from multiple lines of animal models, we discuss the role for AIPL1 in photoreceptors.

Early Alteration of Retinal Neurons in Aipl1−/− Animals

PURPOSE Mutations in the photoreceptor cell-specific gene encoding aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) lead to Leber congenital amaurosis (LCA4), retinitis pigmentosa, and cone-rod dystrophy. Gene therapy appears to be promising in the treatment for AIPL1-mediated vision loss in humans. Prior to initiating these treatments, however, it is crucial to understand how the retinal neurons remodel themselves in response to photoreceptor cell degeneration. In this study, using an animal model for AIPL1-LCA, Aipl1(-/-) mice, we investigate the changes in postreceptoral retinal neurons during the course of photoreceptor cell loss. METHODS Morphology of the Aipl1(-/-) retina from postnatal day 8 to 150 was compared to that of age-matched, wild-type C57Bl6/J retina (WT) by immunocytochemistry using cell-specific markers. RESULTS Expression of postsynaptic proteins in bipolar cells is reduced prior to photoreceptor cell degeneration at postnatal day 8. Bipolar and horizontal cells retract their dendrites. Cell bodies and axons of bipolar and horizontal cells are disorganized during the course of degeneration. Müller cell processes become hypertrophic and form a dense fibrotic layer outside the inner nuclear layer. CONCLUSIONS An early defect in photoreceptor cells in the AIPL1-LCA mouse model affects the expression of postsynaptic markers, suggesting abnormal development of bipolar synapses. Once degeneration of photoreceptor cells is initiated, remodeling of retinal neurons in the Aipl1(-/-) animal is rapid.

Deficiency of Isoprenylcysteine Carboxyl Methyltransferase (ICMT) Leads to Progressive Loss of Photoreceptor Function

Retinal neurons use multiple strategies to fine-tune visual signal transduction, including post-translational modifications of proteins, such as addition of an isoprenyl lipid to a carboxyl-terminal cysteine in proteins that terminate with a “CAAX motif.” We previously showed that RAS converting enzyme 1 (RCE1)-mediated processing of isoprenylated proteins is required for photoreceptor maintenance and function. However, it is not yet known whether the requirement for the RCE1-mediated protein processing is related to the absence of the endoproteolytic processing step, the absence of the subsequent methylation step by isoprenylcysteine methyltransferase (ICMT), or both. To approach this issue and to understand the significance of protein methylation, we generated mice lacking Icmt expression in the retina. In the absence of Icmt expression, rod and cone light-mediated responses diminished progressively. Lack of ICMT-mediated methylation led to defective association of isoprenylated transducin and cone phosphodiesterase 6 (PDE6α′) with photoreceptor membranes and resulted in decreased levels of transducin, PDE6α′, and cone G-protein coupled receptor kinase-1 (GRK1). In contrast to our earlier findings with retina-specific Rce1 knock-out mice, rod PDE6 in Icmt-deficient mice trafficked normally to the photoreceptor outer segment, suggesting that the failure to remove the −AAX is responsible for blocking the movement of PDE6 to the outer segment. Our findings demonstrate that carboxyl methylation of isoprenylated proteins is crucial for maintenance of photoreceptor function. SIGNIFICANCE STATEMENT In this report, we show that an absence of isoprenylcysteine methyltransferase-mediated protein methylation leads to progressive loss of vision. Photoreceptors also degenerate, although at a slower pace than the rate of visual loss. The reduction in photoresponses is due to defective association of crucial players in phototransduction cascade. Unlike the situation with RCE1 deficiency, where both methylation and removal of −AAX were affected, the transport of isoprenylated proteins in isoprenylcysteine methyltransferase-deficient retinas was not dependent on methylation. This finding implies that the retention of the −AAX in PDE6 catalytic subunits in Rce1−/− mice is responsible for impeding their transport to the rod photoreceptor outer segment. In conclusion, lack of methylation of isoprenylcysteines leads to age-dependent photoreceptor dysfunction.