Chronically shortened rod outer segments accompany photoreceptor cell death in Choroideremia

X-linked choroideremia (CHM) is a disease characterized by gradual retinal degeneration caused by loss of the Rab Escort Protein, REP1. Despite partial compensation by REP2 the disease is characterized by prenylation defects in multiple members of the Rab protein family that are master regulators of membrane traffic. Remarkably, the eye is the only organ affected in CHM patients, possibly because of the huge membrane traffic burden of the post mitotic photoreceptors, which synthesise outer segments, and the adjacent retinal pigment epithelium that degrades the spent portions each day. In this study, we aimed to identify defects in membrane traffic that might lead to photoreceptor cell death in CHM. In a heterozygous null female mouse model of CHM (Chmnull/WT), degeneration of the photoreceptor layer was clearly evident from increased numbers of TUNEL positive cells compared to age matched controls, small numbers of cells exhibiting signs of mitochondrial stress and greatly increased microglial infiltration. However, most rod photoreceptors exhibited remarkably normal morphology with well-formed outer segments and no discernible accumulation of transport vesicles in the inner segment. The major evidence of membrane trafficking defects was a shortening of rod outer segments that was evident at 2 months of age but remained constant over the period during which the cells die. A decrease in rhodopsin density found in the outer segment may underlie the outer segment shortening but does not lead to rhodopsin accumulation in the inner segment. Our data argue against defects in rhodopsin transport or outer segment renewal as triggers of cell death in CHM.

Commonalities and differences in the Oxidative Phosphorylation of Mitochondria and Neuronal Membranes

ABSTRACTMitochondria are considered the exclusive site of aerobic metabolism. However, in recent years, the functional expression of the oxidative phosphorylation (OxPhos) machinery has been reported in several other membranous structures, including the plasma membrane, endoplasmic reticulum, nucleus, myelin sheath and disks of rod outer segments. Thus, to underline commonalities and differences between extra-mitochondrial and mitochondrial aerobic metabolism, we characterized the aerobic ATP synthesis in isolated myelin sheath (IM) and rod outer segment (OS) disks, using mitochondria-enriched fractions, as a positive control. Oxygen consumption and ATP synthesis were evaluated in the presence of conventional (pyruvate + malate or succinate) and unconventional (NADH) substrates. ATP synthesis was also assayed in the presence of 10-100 µM ATP in the assay medium. Data show that IM and OS disks consumed oxygen and synthesized ATP both in the presence of conventional and unconventional respiratory substrates, while the mitochondria-enriched fraction did not utilize NADH. Only in mitochondria, ATP synthesis was progressively lost in the presence of increasing ATP concentrations. Conversely, only myelin sheath and rod OS disks produced ATP at a later time or after the removal of respiratory substrates, reflecting their ability to accumulate energy and this opens up exciting perspectives in the study of sleep. Thus, these data suggest that the extramitochondrial OxPhos in IM and rod OS displays a different behavior concerning the classic mitochondrial aerobic metabolism, representing a possible basic molecular process involved in the physiology of the nervous system.Significance StatementMitochondria are considered the cell powerhouse, being the site of the oxidative phosphorylation (OxPhos), which produces the major part of cellular chemical energy by oxygen consumption. However, proteomics, microscopy, and biochemical analyses have described the ectopic functional expression of the OxPhos machinery also in other membranous structures, such as isolated myelin (IM) and rod outer segments (OS). The results reported in this work shows that, although the proteins involved in IM and rod OS OxPhos appear the same expressed in mitochondria, the comparison of mitochondrial and extramitochondrial OxPhos display some differences, opening a new scenario about the energy metabolism modulation.Graphical Abstract