A Nonsense Mutation in PDE6H Causes Autosomal-Recessive Incomplete Achromatopsia

Achromatopsia (ACHM) is an autosomal-recessive retinal dystrophy characterized by color blindness, photophobia, nystagmus, and severely reduced visual acuity. Its prevalence has been estimated to about 1 in 30,000 individuals. Four genes, GNAT2, PDE6C, CNGA3, and CNGB3, have been implicated in ACHM, and all encode functional components of the phototransduction cascade in cone photoreceptors. Applying a functional-candidate-gene approach that focused on screening additional genes involved in this process in a cohort of 611 index cases with ACHM or other cone photoreceptor disorders, we detected a homozygous single base change (c.35C>G) resulting in a nonsense mutation (p.Ser12^∗) in PDE6H, encoding the inhibitory γ subunit of the cone photoreceptor cyclic guanosine monophosphate phosphodiesterase. The c.35C>G mutation was present in three individuals from two independent families with a clinical diagnosis of incomplete ACHM and preserved short-wavelength-sensitive cone function. Moreover, we show through immunohistochemical colocalization studies in mouse retina that Pde6h is evenly present in all retinal cone photoreceptors, a fact that had been under debate in the past. These findings add PDE6H to the set of genes involved in autosomal-recessive cone disorders and demonstrate the importance of the inhibitory γ subunit in cone phototransduction.     

Biallelic Variants in TTLL5, Encoding a Tubulin Glutamylase,Cause Retinal Dystrophy

In a subset of inherited retinal degenerations (including cone, cone-rod, and macular dystrophies), cone photoreceptors are more severely affected than rods; ABCA4 mutations are the most common cause of this heterogeneous class of disorders. To identify retinal-disease-associated genes, we performed exome sequencing in 28 individuals with “cone-first” retinal disease and clinical features atypical for ABCA4 retinopathy. We then conducted a gene-based case-control association study with an internal exome data set as the control group. TTLL5, encoding a tubulin glutamylase, was highlighted as the most likely disease-associated gene; 2 of 28 affected subjects harbored presumed loss-of-function variants: c.[1586_1589delAGAG];[1586_1589delAGAG], p.[Glu529Valfs^∗2];[Glu529Valfs^∗2], and c.[401delT(;)3354G>A], p.[Leu134Argfs^∗45(;)Trp1118^∗]. We then inspected previously collected exome sequence data from individuals with related phenotypes and found two siblings with homozygous nonsense variant c.1627G>T (p.Glu543^∗) in TTLL5. Subsequently, we tested a panel of 55 probands with retinal dystrophy for TTLL5 mutations; one proband had a homozygous missense change (c.1627G>A [p.Glu543Lys]). The retinal phenotype was highly similar in three of four families; the sibling pair had a more severe, early-onset disease. In human and murine retinae, TTLL5 localized to the centrioles at the base of the connecting cilium. TTLL5 has been previously reported to be essential for the correct function of sperm flagella in mice and play a role in polyglutamylation of primary cilia in vitro. Notably, genes involved in the polyglutamylation and deglutamylation of tubulin have been associated with photoreceptor degeneration in mice. The electrophysiological and fundus autofluorescence imaging presented here should facilitate the molecular diagnosis in further families.     

Molecular determinants of cGMP binding to chicken cone photoreceptor phosphodiesterase

Structural studies on photoreceptor phosphodiesterases type 6 (PDE6s) have been hampered by an inability to express and purify substantial amounts of enzyme. Here we describe bacterial expression and characterization of the chicken cone PDE6 regulatory GAF-A and GAF-B domains. High affinity cGMP binding was found only for GAF-A as predicted from sequence alignments with the GAF domains of PDE2 and PDE5. A homology model of the GAF-A domain of chicken cone PDE6 based on the crystal structure of mouse PDE2A GAF-B was used to identify residues likely to make contact with cGMP. Alanine mutagenesis of 4 of these residues (F123A, D169A, T172A, and T176A) showed that each was absolutely required for cGMP binding. Three of these residues map to the H4 helical structure of the GAF-A domain indicating this region as a key structural component for cGMP binding. Mutagenesis of another residue, S97A, decreased cGMP binding affinity 5-fold. Finally mutagenesis of Glu-124 indicated that it is responsible for part but not all of the high specificity for cGMP binding to PDE6 GAF-A. Since little data is available on the properties of the chicken cone PDE6 holoenzyme, we also characterized the native PDEs of chicken retina. Two histone-activated PDE6 peaks were separated by ion exchange chromatography and identified by mass spectrometry as cone and rod photoreceptor PDE6s, respectively. Both of these PDEs had cGMP binding and kinetic properties similar to their corresponding bovine photoreceptor PDEs. Moreover the cGMP binding properties of chicken cone PDE6 holoenzyme were very similar to those of the bacterially expressed individual GAF-A or GAF-A/B domains.     

Autosomal-Recessive Early-Onset Retinitis Pigmentosa Caused by a Mutation in PDE6G, the Gene Encoding the Gamma Subunit of Rod cGMP Phosphodiesterase

Retinitis pigmentosa (RP) is the most common form of hereditary retinal degeneration, with a worldwide prevalence of 1 in 4000. Over 30 genes and loci have been implicated in nonsyndromic autosomal-recessive (ar) RP. Genome-wide homozygosity mapping was conducted in two sibships from an extended consanguineous Muslim Arab Israeli family segregating ar severe early-onset RP. A shared homozygous region on chromosome 17q25.3 was identified in both sibships, with an overlap of 4.7 Mb. One of the genes located in this interval is PDE6G, encoding for the inhibitory γ subunit of rod photoreceptor cyclic GMP-phosphodiesterase. Mutations in the genes encoding for the catalytic subunits of this holoenzyme, PDE6A and PDE6B, cause arRP. Sequencing of all coding exons, including exon-intron boundaries, revealed a homozygous single base change (c.187+1G>T) located in the conserved intron 3 donor splice site of PDE6G. This mutation cosegregated with the disease in the extended family. We used an in vitro splicing assay to demonstrate that this mutation leads to incorrect splicing. Affected individuals had markedly constricted visual fields. Both scotopic and photopic electroretinograms were severely reduced or completely extinct. Funduscopy showed typical bone spicule-type pigment deposits spread mainly at the midperiphery, as well as pallor of the optic disk. Macular involvement was indicated by the lack of foveal reflex and typical cystoid macular edema, proved by optical coherence tomography. These findings demonstrate the positive role of the γ subunit in maintaining phosphodiesterase activity and confirm the contribution of PDE6G to the etiology of RP in humans.     

Genomic evidence for rod monochromacy in sloths and armadillos suggests early subterranean history for Xenarthra

Rod monochromacy is a rare condition in vertebrates characterized by the absence of cone photoreceptor cells. The resulting phenotype is colourblindness and low acuity vision in dim-light and blindness in bright-light conditions. Early reports of xenarthrans (armadillos, sloths and anteaters) suggest that they are rod monochromats, but this has not been tested with genomic data. We searched the genomes of Dasypus novemcinctus (nine-banded armadillo), Choloepus hoffmanni (Hoffmann''s two-toed sloth) and Mylodon darwinii (extinct ground sloth) for retinal photoreceptor genes and examined them for inactivating mutations. We performed PCR and Sanger sequencing on cone phototransduction genes of 10 additional xenarthrans to test for shared inactivating mutations and estimated the timing of inactivation for photoreceptor pseudogenes. We concluded that a stem xenarthran became an long-wavelength sensitive-cone monochromat following a missense mutation at a critical residue in SWS1, and a stem cingulate (armadillos, glyptodonts and pampatheres) and stem pilosan (sloths and anteaters) independently acquired rod monochromacy early in their evolutionary history following the inactivation of LWS and PDE6C, respectively. We hypothesize that rod monochromacy in armadillos and pilosans evolved as an adaptation to a subterranean habitat in the early history of Xenarthra. The presence of rod monochromacy has major implications for understanding xenarthran behavioural ecology and evolution.     

Targeted Ablation of the Pde6h Gene in Mice Reveals Cross-species Differences in Cone and Rod Phototransduction Protein Isoform Inventory

Phosphodiesterase-6 (PDE6) is a multisubunit enzyme that plays a key role in the visual transduction cascade in rod and cone photoreceptors. Each type of photoreceptor utilizes discrete catalytic and inhibitory PDE6 subunits to fulfill its physiological tasks, i.e. the degradation of cyclic guanosine-3′,5′-monophosphate at specifically tuned rates and kinetics. Recently, the human PDE6H gene was identified as a novel locus for autosomal recessive (incomplete) color blindness. However, the three different classes of cones were not affected to the same extent. Short wave cone function was more preserved than middle and long wave cone function indicating that some basic regulation of the PDE6 multisubunit enzyme was maintained albeit by a unknown mechanism. To study normal and disease-related functions of cone Pde6h in vivo, we generated Pde6h knock-out (Pde6h^−/−) mice. Expression of PDE6H in murine eyes was restricted to both outer segments and synaptic terminals of short and long/middle cone photoreceptors, whereas Pde6h^−/− retinae remained PDE6H-negative. Combined in vivo assessment of retinal morphology with histomorphological analyses revealed a normal overall integrity of the retinal organization and an unaltered distribution of the different cone photoreceptor subtypes upon Pde6h ablation. In contrast to human patients, our electroretinographic examinations of Pde6h^−/− mice suggest no defects in cone/rod-driven retinal signaling and therefore preserved visual functions. To this end, we were able to demonstrate the presence of rod PDE6G in cones indicating functional substitution of PDE6. The disparities between human and murine phenotypes caused by mutant Pde6h/PDE6H suggest species-to-species differences in the vulnerability of biochemical and neurosensory pathways of the visual signal transduction system.     

The structure of the GAF A domain from phosphodiesterase 6C reveals determinants of cGMP binding, a conserved binding surface, and a large cGMP-dependent conformational change

The photoreceptor phosphodiesterase (PDE6) regulates the intracellular levels of the second messenger cGMP in the outer segments of cone and rod photoreceptor cells. PDE6 contains two regulatory GAF domains, of which one (GAF A) binds cGMP and regulates the activity of the PDE6 holoenzyme. To increase our understanding of this allosteric regulation mechanism, we present the 2.6A crystal structure of the cGMP-bound GAF A domain of chicken cone PDE6. Nucleotide specificity appears to be provided in part by the orientation of Asn-116, which makes two hydrogen bonds to the guanine ring of cGMP but is not strictly conserved among PDE6 isoforms. The isolated PDE6C GAF A domain is monomeric and does not contain sufficient structural determinants to form a homodimer as found in full-length PDE6C. A highly conserved surface patch on GAF A indicates a potential binding site for the inhibitory subunit Pgamma. NMR studies reveal that the apo-PDE6C GAF A domain is structured but adopts a significantly altered structural state indicating a large conformational change with rearrangement of secondary structure elements upon cGMP binding. The presented crystal structure will help to define the cGMP-dependent regulation mechanism of the PDE6 holoenzyme and its inhibition through Pgamma binding.     

Rip3 knockdown rescues photoreceptor cell death in blind pde6c zebrafish

Achromatopsia is a progressive autosomal recessive retinal disease characterized by early loss of cone photoreceptors and later rod photoreceptor loss. In most cases, mutations have been identified in CNGA3, CNGB3, GNAT2, PDE6C or PDE6H genes. Owing to this genetic heterogeneity, mutation-independent therapeutic schemes aimed at preventing cone cell death are very attractive treatment strategies. In pde6c^w59 mutant zebrafish, cone photoreceptors expressed high levels of receptor-interacting protein kinase 1 (RIP1) and receptor-interacting protein kinase 3 (RIP3) kinases, key regulators of necroptotic cell death. In contrast, rod photoreceptor cells were alternatively immunopositive for caspase-3 indicating activation of caspase-dependent apoptosis in these cells. Morpholino gene knockdown of rip3 in pde6c^w59 embryos rescued the dying cone photoreceptors by inhibiting the formation of reactive oxygen species and by inhibiting second-order neuron remodelling in the inner retina. In rip3 morphant larvae, visual function was restored in the cones by upregulation of the rod phosphodiesterase genes (pde6a and pde6b), compensating for the lack of cone pde6c suggesting that cones are able to adapt to their local environment. Furthermore, we demonstrated through pharmacological inhibition of RIP1 and RIP3 activity that cone cell death was also delayed. Collectively, these results demonstrate that the underlying mechanism of cone cell death in the pde6c^w59 mutant retina is through necroptosis, whereas rod photoreceptor bystander death occurs through a caspase-dependent mechanism. This suggests that targeting the RIP kinase signalling pathway could be an effective therapeutic intervention in retinal degeneration patients. As bystander cell death is an important feature of many retinal diseases, combinatorial approaches targeting different cell death pathways may evolve as an important general principle in treatment.     

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.     

Canine CNGA3 Gene Mutations Provide Novel Insights into Human Achromatopsia-Associated Channelopathies and Treatment

Cyclic nucleotide-gated (CNG) ion channels are key mediators underlying signal transduction in retinal and olfactory receptors. Genetic defects in CNGA3 and CNGB3, encoding two structurally related subunits of cone CNG channels, lead to achromatopsia (ACHM). ACHM is a congenital, autosomal recessive retinal disorder that manifests by cone photoreceptor dysfunction, severely reduced visual acuity, impaired or complete color blindness and photophobia. Here, we report the first canine models for CNGA3-associated channelopathy caused by R424W or V644del mutations in the canine CNGA3 ortholog that accurately mimic the clinical and molecular features of human CNGA3-associated ACHM. These two spontaneous mutations exposed CNGA3 residues essential for the preservation of channel function and biogenesis. The CNGA3-R424W results in complete loss of cone function in vivo and channel activity confirmed by in vitro electrophysiology. Structural modeling and molecular dynamics (MD) simulations revealed R424-E306 salt bridge formation and its disruption with the R424W mutant. Reversal of charges in a CNGA3-R424E-E306R double mutant channel rescued cGMP-activated currents uncovering new insights into channel gating. The CNGA3-V644del affects the C-terminal leucine zipper (CLZ) domain destabilizing intersubunit interactions of the coiled-coil complex in the MD simulations; the in vitro experiments showed incompetent trimeric CNGA3 subunit assembly consistent with abnormal biogenesis of in vivo channels. These newly characterized large animal models not only provide a valuable system for studying cone-specific CNG channel function in health and disease, but also represent prime candidates for proof-of-concept studies of CNGA3 gene replacement therapy for ACHM patients.     

Mutations in IMPG2, Encoding Interphotoreceptor Matrix Proteoglycan 2, Cause Autosomal-Recessive Retinitis Pigmentosa

Retinitis pigmentosa (RP) is a heterogeneous group of inherited retinal diseases caused by progressive degeneration of the photoreceptor cells. Using autozygosity mapping, we identified two families, each with three affected siblings sharing large overlapping homozygous regions that harbored the IMPG2 gene on chromosome 3. Sequence analysis of IMPG2 in the two index cases revealed homozygous mutations cosegregating with the disease in the respective families: three affected siblings of Iraqi Jewish ancestry displayed a nonsense mutation, and a Dutch family displayed a 1.8 kb genomic deletion that removes exon 9 and results in the absence of seven amino acids in a conserved SEA domain of the IMPG2 protein. Transient transfection of COS-1 cells showed that a construct expressing the wild-type SEA domain is properly targeted to the plasma membrane, whereas the mutant lacking the seven amino acids appears to be retained in the endoplasmic reticulum. Mutation analysis in ten additional index cases that were of Dutch, Israeli, Italian, and Pakistani origin and had homozygous regions encompassing IMPG2 revealed five additional mutations; four nonsense mutations and one missense mutation affecting a highly conserved phenylalanine residue. Most patients with IMPG2 mutations showed an early-onset form of RP with progressive visual-field loss and deterioration of visual acuity. The patient with the missense mutation, however, was diagnosed with maculopathy. The IMPG2 gene encodes the interphotoreceptor matrix proteoglycan IMPG2, which is a constituent of the interphotoreceptor matrix. Our data therefore show that mutations in a structural component of the interphotoreceptor matrix can cause arRP.     

Guanylate cyclases and associated activator proteins in retinal disease

Two isoforms of guanylate cyclase, GC1 and GC2 encoded by GUCY2D and GUCY2F, are responsible for the replenishment of cGMP in photoreceptors after exposure to light. Both are required for the normal kinetics of photoreceptor sensitivity and recovery, although disease mutations are restricted to GUCY2D. Recessive mutations in this gene cause the severe early-onset blinding disorder Leber congenital amaurosis whereas dominant mutations result in a later onset less severe cone–rod dystrophy. Cyclase activity is regulated by Ca²⁺ which binds to the GC-associated proteins, GCAP1 and GCAP2 encoded by GUCA1A and GUCA1B, respectively. No recessive mutations in either of these genes have been reported. Dominant missense mutations are largely confined to the Ca²⁺-binding EF hands of the proteins. In a similar fashion to the disease mechanism for the dominant GUCY2D mutations, these mutations generally alter the sensitivity of the cyclase to inhibition as Ca²⁺ levels rise following a light flash.     

Gene therapy for achromatopsia

The present review summarizes the current status of achromatopsia (ACHM) gene therapy‐related research activities and provides an outlook for their clinical application. ACHM is an inherited eye disease characterized by a congenital absence of cone photoreceptor function. As a consequence, ACHM is associated with strongly impaired daylight vision, photophobia, nystagmus and a lack of color discrimination. Currently, six genes have been linked to ACHM. Up to 80% of the patients carry mutations in the genes CNGA3 and CNGB3 encoding the two subunits of the cone cyclic nucleotide‐gated channel. Various animal models of the disease have been established and their characterization has helped to increase our understanding of the pathophysiology associated with ACHM. With the advent of adeno‐associated virus vectors as valuable gene delivery tools for retinal photoreceptors, a number of promising gene supplementation therapy programs have been initiated. In recent years, huge progress has been made towards bringing a curative treatment for ACHM into clinics. The first clinical trials are ongoing or will be launched soon and are expected to contribute important data on the safety and efficacy of ACHM gene supplementation therapy.     

Retinitis pigmentosa: rapid neurodegeneration is governed by slow cell death mechanisms

For most neurodegenerative diseases the precise duration of an individual cell''s death is unknown, which is an obstacle when counteractive measures are being considered. To address this, we used the rd1 mouse model for retinal neurodegeneration, characterized by phosphodiesterase-6 (PDE6) dysfunction and photoreceptor death triggered by high cyclic guanosine-mono-phosphate (cGMP) levels. Using cellular data on cGMP accumulation, cell death, and survival, we created mathematical models to simulate the temporal development of the degeneration. We validated model predictions using organotypic retinal explant cultures derived from wild-type animals and exposed to the selective PDE6 inhibitor zaprinast. Together, photoreceptor data and modeling for the first time delineated three major cell death phases in a complex neuronal tissue: (1) initiation, taking up to 36 h, (2) execution, lasting another 40 h, and finally (3) clearance, lasting about 7 h. Surprisingly, photoreceptor neurodegeneration was noticeably slower than necrosis or apoptosis, suggesting a different mechanism of death for these neurons.     

AIPL1, a Protein Associated with Childhood Blindness, Interacts with ��-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.     

Characterization of a bovine cone photoreceptor phosphodiesterase purified by cyclic GMP-sepharose chromatography

The biochemical bases for the differences in cone and rod photoreceptor physiology have not been thoroughly examined because of the difficulty in obtaining cone photoreceptor components. We report here the purification and preliminary characterization of a bovine cyclic GMP phosphodiesterase (PDE) which is enriched in cone photoreceptors. The cone PDE was purified at least 15,000-fold to apparent homogeneity from bovine retinas by DEAE-cellulose and cGMP-Sepharose affinity chromatography. The trypsin-activated cone PDE hydrolyzed cGMP with efficiency similar to that of the rod PDE. However, a number of characteristics distinguished the cone PDE from the rod isozyme including the subunit structure. As previously reported, the apparent molecular weight of the cone PDE large subunit (alpha') was slightly larger than either of the large subunits of the rod PDE (93,500 versus 88,000 and 84,000). Three other smaller polypeptides were associated with the alpha' subunit (Mr = 11,000, 13,000, and 15,000), one of which (11,000) may be identical to the rod PDE gamma subunit. Cone phosphodiesterase binds at least 10-fold more cyclic GMP/mol of PDE than the rod photoreceptor isozyme. Cyclic GMP binds to this noncatalytic site with high affinity (Kd = 11 nM) and dissociates very slowly (t1/2 = 10-20 min at 37 degrees C). Purified rod transducin activated the cone PDE in solution to at least 90% of the trypsin-activated level. The concentration of rod transducin required for half-maximal activation of cone PDE (15 nM) was 50-fold lower than that necessary for half-maximal activation of rod PDE. Thus several properties of the cone phosphodiesterase clearly distinguish it from the rod isozyme and could account for some differences in cone and rod physiology.     

Novel mutations in SAR1B and MTTP genes in Tunisian children with chylomicron retention disease and abetalipoproteinemia

Monogenic hypobetalipoproteinemias include three disorders: abetalipoproteinemia (ABL) and chylomicron retention disease (CMRD) with recessive transmission and familial hypobetalipoproteinemia (FHBL) with dominant transmission. We investigated three unrelated Tunisian children born from consanguineous marriages, presenting hypobetalipoproteinemia associated with chronic diarrhea and retarded growth. Proband HBL-108 had a moderate hypobetalipoproteinemia, apparently transmitted as dominant trait, suggesting the diagnosis of FHBL. However, she had no mutations in FHBL candidate genes (APOB, PCSK9 and ANGPTL3). The analysis of MTTP gene was also negative, whereas SAR1B gene resequencing showed that the patient was homozygous for a novel mutation (c.184G>A), resulting in an amino acid substitution (p.Glu62Lys), located in a conserved region of Sar1b protein. In the HBL-103 and HBL-148 probands, the severity of hypobetalipoproteinemia and its recessive transmission suggested the diagnosis of ABL. The MTTP gene resequencing showed that probands HBL-103 and HBL-148 were homozygous for a nucleotide substitution in the donor splice site of intron 9 (c.1236+2T>G) and intron 16 (c.2342+1G>A) respectively. Both mutations were predicted in silico to abolish the function of the splice site. In vitro functional assay with splicing mutation reporter MTTP minigenes showed that the intron 9 mutation caused the skipping of exon 9, while the intron 16 mutation caused a partial retention of this intron in the mature mRNA. The predicted translation products of these mRNAs are non-functional truncated proteins.     

Allosteric Regulation of Rod Photoreceptor Phosphodiesterase 6 (PDE6) Elucidated by Chemical Cross-Linking and Quantitative Mass Spectrometry

Photoreceptor phosphodiesterase (PDE6) is the central effector enzyme in the visual excitation pathway in rod and cone photoreceptors. Its tight regulation is essential for the speed, sensitivity, recovery, and adaptation of visual signaling. The rod PDE6 holoenzyme (Pαβγ₂) is composed of a catalytic heterodimer (Pαβ) that binds two inhibitory γ subunits. Each of the two catalytic subunits (Pα and Pβ) contains a catalytic domain responsible for cGMP hydrolysis and two tandem GAF domains, one of which binds cGMP noncatalytically. Unlike related GAF-containing PDEs where cGMP binding allosterically activates catalysis, the physiological significance of cGMP binding to the GAF domains of PDE6 is unknown. To elucidate the structural determinants of PDE6 allosteric regulators, we biochemically characterized PDE6 complexes in various allosteric states (Pαβ, Pαβ–cGMP, Pαβγ₂, and Pαβγ₂–cGMP) with a quantitative cross-linking/mass spectrometry approach. We employed a normalization strategy to dissect the cross-linking reactivity of individual residues in order to assess the spatial cross-linking propensity of detected pairs. In addition to identifying cross-linked pairs that undergo conformational changes upon ligand binding, we observed an asymmetric binding of the inhibitory γ-subunit and the noncatalytic cGMP to the GAFa domains of rod PDE6, as well as a stable open conformation of Pαβ catalytic dimer in different allosteric states. These results advance our understanding of the exquisite regulatory control of the lifetime of rod PDE6 activation/deactivation during visual signaling, as well as providing a structural basis for interpreting how mutations in rod PDE6 subunits can lead to retinal diseases.     

Involvement of Endoplasmic Reticulum Stress in TULP1 Induced Retinal Degeneration

Inherited retinal disorders (IRDs) result in severe visual impairments in children and adults. A challenge in the field of retinal degenerations is identifying mechanisms of photoreceptor cell death related to specific genetic mutations. Mutations in the gene TULP1 have been associated with two forms of IRDs, early-onset retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA). TULP1 is a cytoplasmic, membrane-associated protein shown to be involved in transportation of newly synthesized proteins destined for the outer segment compartment of photoreceptor cells; however, how mutant TULP1 causes cell death is not understood. In this study, we provide evidence that common missense mutations in TULP1 express as misfolded protein products that accumulate within the endoplasmic reticulum (ER) causing prolonged ER stress. In an effort to maintain protein homeostasis, photoreceptor cells then activate the unfolded protein response (UPR) complex. Our results indicate that the two major apoptotic arms of the UPR pathway, PERK and IRE1, are activated. Additionally, we show that retinas expressing mutant TULP1 significantly upregulate the expression of CHOP, a UPR signaling protein promoting apoptosis, and undergo photoreceptor cell death. Our study demonstrates that the ER-UPR, a known mechanism of apoptosis secondary to an overwhelming accumulation of misfolded protein, is involved in photoreceptor degeneration caused by missense mutations in TULP1. These observations suggest that modulating the UPR pathways might be a strategy for therapeutic intervention.