Truncation mutation in HRG4 (UNC119) leads to mitochondrial ANT-1-mediated photoreceptor synaptic and retinal degeneration by apoptosis.

PURPOSE: To characterize the time course of apoptosis and degeneration in a transgenic mouse model of retinal degeneration based on truncated mutant HRG4; to investigate the nature of binding of the mutant HRG4 to its target, ADP-ribosylation factor-like (ARL)2; to study its effects on the downstream molecules Binder-of-ARL2 (BART) and adenine nucleotide transporter (ANT)-1 and on the induction of apoptosis. METHODS: Saturation binding, microscopic morphometric, Western blot, immunofluorescence, and TUNEL analyses were used. RESULTS: Increased apoptosis did not occur until 20 months in the transgenic retina, consistent with the delayed-onset degeneration in this model. The truncated HRG4 protein exhibited approximately threefold greater affinity for ARL2 than the wild-type HRG4, likely resulting in nonfunctional sequestration of ARL2. A significant decrease in ARL2 was present by 20 months, accompanied by a 50% decrease in ANT-1 in the photoreceptor synaptic mitochondria, with evidence of mitochondrial dysfunction. Preapoptotic degeneration in the photoreceptor synapse was demonstrated with cytochrome c release and caspase 3 activation within the synapse-without evidence of TUNEL-positive apoptosis in the photoreceptor cell body-indicating an initial event in the synapse leading to apoptosis. Caspase 3 was activated in the accompanying secondary neuron, consistent with transsynaptic degeneration. CONCLUSIONS: The results support a novel mechanism of retinal degeneration in which preapoptotic degeneration starts in the photoreceptor synapse because of a deficiency in ANT-1 and spreads to the secondary neuron transsynaptically, followed by apoptosis and degeneration in the cell body of the photoreceptor.

Truncation and mutagenesis analysis of the human X-arrestin gene promoter.

X-arrestin (arrestin-3) is an arrestin present specifically in the outer segments of red-, green-, and blue-cone photoreceptors. The X-arrestin gene is on Xcen-q22, and consists of 17 exons with a promoter containing a TATA box and elements important for photoreceptor expression, including three CRX and one PCE-1-like element. In order to delineate the promoter structure necessary for the pan-cone-specific expression of X-arrestin, the expression of the gene in retinoblastoma cell lines was investigated, and a structure-function analysis of the promoter was conducted in the appropriate cellular substrate. Expression of X-arrestin was detected at a low level in the Y79 retinoblastoma cell line but not in the WERI retinoblastoma cell line. Truncation and expression analysis of the X-arrestin promoter in Y79 showed maximal activity in the proximal 378-bp region containing the CRX and PCE-1-like elements upstream of the TATA and CAAT boxes and a negative regulator in the distal 1-2-kbp region. Mutagenesis of the three CRX and PCE-1-like elements and expression analysis demonstrated complete elimination of the promoter activity. Mutagenesis of the TATA box and PCE-1-like element individually resulted in similar decrease in promoter activity, but the decrease in the promoter activity was greater when the CRX elements were mutagenized with a 5' to 3' spatial gradient in the negative effect, suggesting a cooperative effect of the three CRX elements. The regulation of expression from this promoter may involve the binding of a multi-protein enhanceosome complex at the CRX triplet and the PCE-1-like element, resulting in the recruitment and activation of the RNA polymerase II complex at the downstream TATA box.

Photoreceptor synaptic protein HRG4 (UNC119) interacts with ARL2 via a putative conserved domain.

Human retinal gene 4 (HRG4) (UNC119) is a photoreceptor synaptic protein of unknown function, shown when mutated to cause retinal degeneration in a patient and in a confirmatory transgenic model. ADP-ribosylation factor-like protein 2 (ARL2) was identified as an interactor of HRG4 by the yeast two-hybrid strategy. The presence of ARL2 in the retina and co-localization with HRG4 was confirmed by Western blot and double immunofluorescence analysis, respectively. The interaction of ARL2 with HRG4 was further confirmed by co-immunoprecipitation and direct binding analysis. Phosphodiesterase delta (PDEdelta) is an ARL2-binding protein homologous to HRG4. Amino acid residues of PDEdelta involved in binding ARL2 and forming a hydrophobic pocket were shown to be highly conserved in HRG4, suggesting similarity in binding mechanism and function.

Changes in retinal synaptic proteins in the transgenic model expressing a mutant HRG4 (UNC119).

PURPOSE: HRG4 (UNC119) is a photoreceptor synaptic protein, a truncation mutant of which has been shown to cause late-onset cone-rod dystrophy in a patient and retinal degeneration with marked synaptic degeneration in a transgenic model. To investigate the mechanism of the retinal degeneration, the effect of the mutant protein expression on the other synaptic proteins was examined. METHODS: The status of 12 known synaptic proteins in the retinas of 5-month- and 13-month-old HRG4 transgenic and control mice was examined by Western blot analysis. Three selected proteins were analyzed by immunofluorescence in the 13-month-old retinas. The 12 proteins were tested for binding to HRG4 by a direct-binding assay and Western blot analysis. RESULTS: A decrease in three synaptic vesicle proteins and an increase in five cytoplasmic and plasma membrane proteins was detected by Western blot analysis in the older but not the younger transgenic retinas. These changes were demonstrated in both the outer and inner plexiform layers of the retina by immunofluorescence, along with a significant reduction in the thickness of the inner plexiform layer. A 23-kDa specie was found to bind to HRG4, but none of the 12 synaptic proteins matched it, according to immunoblot analysis. CONCLUSIONS: The expression of a mutant HRG4 protein in the photoreceptor synapses of the transgenic model had an intrasynaptic and transsynaptic effect, resulting in a decrease in three synaptic vesicle proteins, an increase in five cytoplasmic and plasma membrane proteins, and a significant reduction in the thickness of the inner plexiform layer. These changes were age dependent, similar to the pathologic phenotype of the transgenic model and the patient, and supported a close relationship of HRG4 with other participants in synaptic vesicle function. This interaction was not mediated by a direct coupling of HRG4 with any of the tested synaptic proteins but possibly through interaction with a 23-kDa protein.

Targeted inactivation of synaptic HRG4 (UNC119) causes dysfunction in the distal photoreceptor and slow retinal degeneration, revealing a new function.

HRG4 (UNC119) is a photoreceptor protein predominantly localized to the photoreceptor synapses and to the inner segments to a lesser degree. A heterozygous truncation mutation in HRG4 was found in a patient with late onset cone-rod dystrophy, and a transgenic (TG) mouse expressing the identical mutant protein developed late onset retinal degeneration, confirming the pathogenic potential of HRG4. Recently, the dominant negative pathogenic mechanism in the TG model was shown to involve increased affinity of the truncated mutant HRG4 for its target, ARL2, which leads to a delayed decrease in its downstream target, mitochondrial ANT1, mitochondrial stress, synaptic degeneration, trans-synaptic degeneration, and whole photoreceptor degeneration by apoptosis. In this study, the mouse HRG4 (MRG4) gene was cloned and targeted to construct a knock-out (KO) mouse model of HRG4 in order to study the effects of completely inactivating this protein. The KO model was examined by genomic Southern blotting, Western blotting, immunofluorescence, funduscopy, LM and EM histopathology, ERG, and TUNEL analyses. The KO model developed a slowly progressive retinal degeneration, characterized by mottling in the fundus, mild thinning of the photoreceptor layer, and increase in apoptosis as early as 6 months, dramatic acceleration at approximately 17 months, and virtual obliteration of the photoreceptors by 20 months. When compared to retinal degeneration in the TG model, significant differences existed in the KO consisting of more severe and early photoreceptor death without evidence of early synaptic and trans-synaptic degeneration as seen in the TG, confirmed by LM and EM histopathology, ERG, and Western blotting of synaptic proteins. The results indicated a dysfunction in the KO outside the synapses in the distal end of photoreceptors where MRG4 is also localized. Differences in the phenotypes of retinal degeneration in the KO and TG models reflect a dysfunction in the two opposite ends of photoreceptors, i.e., the distal inner/outer segments and proximal synapses, respectively, indicating a second function of MRG4 in the distal photoreceptor and dual functionality of MRG4. Thus, inactivation of MRG4 by gene targeting resulted in a retinal degeneration phenotype quite different from that previously seen in the TG, attesting to the multiplicity of MRG4 function, in addition to the importance of this protein for normal retinal function. These models will be useful in elucidating the functions of HRG4/MRG4 and the mechanism of slow retinal degeneration.