Mutation in HRG4 (UNC119) Leads To Mitochondrial ANT1–Mediated Apoptosis and Retinal Degeneration

Abstract

Abstract: : Purpose: HRG4 (UNC119) is a novel photoreceptor synaptic protein we have isolated by a differential cloning approach. A truncation mutation of HRG4 has been shown to cause late–onset cone–rod dystrophy in a patient, and a transgenic model expressing the identical mutation has been shown to develop late–onset retinal degeneration with ERG abnormality just as in the patient. HRG4 was shown to bind ARL2 and the mutant truncated HRG4 was shown to bind ARL2 3–fold as much as the wild–type HRG4, most likely sequestering it in a non–functional manner. This was shown to result in eventual decrease of ARL2, increase in the ARL2–binding protein, BART, and decrease in mitochondrial ANT1, the target of the ARL2–BART complex. We have investigated the role of ANT1 decrease and mitochondrial abnormality in the mechanism of retinal degeneration in this model. Methods: Single and double immunofluorescence was used to analyze the status of HRG4, ANT1, cytochrome C and caspase 3 proteins in the normal and transgenic retinas. Mitochondria was analyzed by Mitotracker staining. TUNEL method was used to demonstrate apoptosis of retinal cells. Retinal sections of different ages were analyzed microscopically. Results: The colocalization of HRG4 and ANT1 in the photoreceptor synapses was confirmed. In transgenic retinas 20 months or older, decrease of ANT1 and stress–induced mitochondrial proliferation were confirmed. Synapses filled with cytochrome C consistent with increased mitochondria and possible leakage into the synaptoplasm were observed. Apoptosis of retinal cells was confirmed by TUNEL assay and caspase 3 activation. Histologic changes consisting of wandering photoreceptor nuclei, abnormal shape of the outer nuclear layer (ONL), eventual thinning of the ONL, and transsynaptic degeneration were seen. These changes occurred slowly with age and accelerated after 20 months. Conclusions: In this model of truncated mutation of HRG4, a mechanism of retinal degeneration was shown to involve an eventual decrease of mitochondrial ANT1 caused by the sequestration of ARL2, leading to mitochondrial stress and apoptosis, confirmed by cytochrome C increase, caspase 3 activation, and TUNEL positivity. These changes occur slowly with age and accelerate after 20 months, making this model an excellent model for study of slow retinal degeneration.

Keywords: retinal degenerations: hereditary • proteins encoded by disease genes • apoptosis/cell death