5.2.3. Category 03: Metabolism

Overview. Inborn errors of metabolism constitute a heterogeneous group of disorders that affect metabolic pathways due to underlying genetic defects [201] and result in abnormalities in the synthesis or catabolism of biomolecules [201,202]. Many such inborn errors of metabolism are known to be associated with PR cell loss, manifested either as a primary ocular defect or as part of a systemic disease [201]. PR cells, with their high metabolic activity, are particularly vulnerable to defects in metabolism of biomolecules such as lipids, carbohydrates, nucleotides, and proteins, which provide energy and serve many other functions described below. Additionally, since organelles such as mitochondria and lysosomes are the major sites for cellular energy production and homeostasis, defects in organellar metabolism and function are also known to cause PR degeneration. The PR cell loss associated with different metabolic diseases varies in the age of onset, severity, and rate of progression (Figure 6, Table S1) and the underlying genetic defects can be categorized based on the type of biomolecular metabolism or the subcellular location of the pathways affected.

Biomolecular metabolism: lipids. PRs are extremely rich in lipids, which make up to 15% of their cellular wet weight as compared to 1% in most other cell types [203,204]. Phospholipids and cholesterol represent 90–95% and 4–6% (*w*/*w*) of total lipids, respectively [205]. The major phospholipids in rod outer segments include phosphatidylethanolamine, phosphatidylcholine, large amounts of phosphatidylserine, along with small amounts of sphingomyelin, phosphatidylinositol, and phosphatidic acid [205]. It has been suggested that the phospholipids in OS membranes are metabolically active and involved in generation of physiological mediators, and changes in metabolism of glycerolipids have been associated with transduction of visual stimuli [205]. Cholesterol has been reported to modulate the function of rhodopsin, a major protein of the OS membranes, by influencing membrane lipid properties [206]. Low-density lipoproteins (LDLs) are reported to be significant suppliers of PR lipids, especially cholesteryl esters [207,208]. The OSs of PRs are particularly rich in very-long-chain polyunsaturated fatty acids (PUFA), such as docasohexaenoic acid (DHA), which is considered to be essential for visual function [209], and phospholipid-containing DHA is suggested to help in isomerization of 11-*cis*-retinal to the all-*trans* form, which is further reduced for its entry into the visual cycle [210]. Recently, DHA has also been implicated in the maintenance of OS homeostasis [211] and mediating PR cell survival [212,213].

Thus, it is not surprising that disorders of lipid metabolism cause inherited PR degeneration. For example, mouse models for mutations in the elongation of very-long-chain fatty acids-like 4 (*Elovl4*) gene are reported to show features resembling Stargardt-like macular dystrophy in humans with cone degeneration preceding that of rods [214,215]. Mutations in genes involved in phospholipid metabolism such as *Lpcat1* cause rapid PR degeneration (90% and 75% degeneration in *Lpcat1rd11* and *Lpcat1rd11-2J* alleles, respectively by 47 days) [216]. Similarly, mutations in genes involved in cholesterol biosynthesis such as *Nsdhl* [217] or in the biosynthesis and regulation of DHA-containing phospholipids, such as *Agpat3* and *Adipor1,* respectively [210], also cause PR degeneration, confirming the importance of lipids in preserving PR integrity. Since membrane phospholipid asymmetry is critical to performing various biological functions, mutations in genes important for its generation and maintenance, also lead to PR degeneration. For example, mutations in *Atp8a2,* a type of P4-ATPase that translocates and maintains phospholipid asymmetry show a 30–40% PR degeneration by two months of age [218]. Similarly, conditional inactivation of *Tmem30a*, known to be required for folding and transport of several P4-ATPases to their plasma membrane destination [219,220], also results in severe PR degeneration [221]. *Tmem30a* knockout mice exhibit a more severe phenotype compared to *Atp8a2* knockout mice, possibly because *Tmem30a* binds multiple P4-ATPases [221].

Biomolecular metabolism: carbohydrate and nucleotide energy metabolism. The retina, and in particular PRs, have a high metabolic rate [222,223] to support functions that are energetically demanding, such as phototransduction during constant illumination, maintenance of ion gradients in darkness, and performing anabolic metabolism to replace the approximately 10% of OSs that are lost every day to phagocytosis by RPE cells [223]. RPE cells also perform many energy demanding functions, such as maintenance of appropriate ionic and fluid composition in the subretinal space, uptake and conversion of all-*trans*-retinol to 11-*cis*-retinal and its transport back to photoreceptor cells, and OS phagocytosis. This high energy requirement makes the retina and RPE particularly vulnerable to functional deficits induced by deficits in energy metabolism [222]. The retina relies on blood-derived glucose and oxygen for its energy requirements. Additionally, PR cells use excess lactate obtained from Müller glial cells and convert it to pyruvate to provide energy via oxidative

phosphorylation [222]. In addition to carbohydrates, the retina uses fatty acids [224] and nucleotides for its energy requirements [223].

Thus, neuronal activity and energy metabolism are tightly coupled and any mutations at the level of glucose, fatty acid or nucleotide biosynthesis can lead to PR degeneration. For example, mice lacking *Hkdc1*, which encodes a kinase found in the IS that phosphorylates glucose to glucose-6-phosphate, show 40% PR degeneration by 17 months [225]. Mice mutant for *Vldlr*, which encodes the receptor facilitating the uptake of triglyceride-derived fatty acids, show reduced cellular uptake and availability of fatty acids for energy production [224]. For some alleles of *Vldlr* (*Vldlrm1Btlr* and *Vldlrtm1Her*), more than 50% of PRs are lost by 12–14 months [226,227], with cones being affected more significantly than rods [228]. The decrease in net available energy may lead to greater cone loss, as cones have been reported to require three times more energy than rods [222]. Similarly, while in some cases, mutations in genes involved in nucleotide metabolism such as *Nampt*, show embryonic lethality [229], others such as mutation in *Nmnat1*, show severe PR degeneration by 4–6 months [230].

Biomolecular metabolism: hormones. The physiology of eye is also dependent on the action of several hormones [231]. Mouse models mutant for thyroid hormone metabolizing genes, such as *Dio3*, which is important for local amplification of triiodothyronine (T3), show selectively detrimental effects on cone cells [232]. This confirms the proposed role of thyroid hormone signaling in regulating cone viability and cone opsin expression [232,233]. Melatonin, a hormone that plays a role in sleep patterns, is known to have protective role against oxidative stress and apoptosis, and regulates retinal circadian rhythms [234]. A mouse model, mutant for the melatonin hormone receptor *Mtnr1a*, shows very slow PR degeneration (25% in 18 months) [235].

Biomolecular metabolism: oxidative stress. The eye is constantly subjected to oxidative stress due to daily exposure to light, atmospheric oxygen, and high metabolic activities [236]. Reactive oxygen species (ROS) are derived from diatomic oxygen and processes such as mitochondrial respiration that form superoxide anion radicals, toxic bis-retinoids that undergo photo-oxidation, and lipids, such as PUFAs, that undergo peroxidation [237]. Having unpaired electrons confers a grea<sup>t</sup> degree of ROS reactivity that can damage biomolecules such as DNA, lipids and proteins, and organelles including mitochondria and lysosomes [238,239], thereby impairing their biological functions [203,236]. Compared to other cells, non-proliferative postmitotic cells such as PRs and RPE cells are particularly sensitive to oxidative damage due to the apparent absence of a DNA damage detection system [240–242].

Under physiological conditions, cellular redox homeostasis is maintained by a balance between ROS generation and antioxidant systems [236]. Antioxidant enzymes such as *Sod1, Sod2,* and *Gpx4* are known to play a major role in ROS scavenging and changes in their expression or activity or both are reported to cause increased oxidative stress and are associated with diseases such as age-related macular degeneration (AMD) [243]. For example, mutations in the *Sod1* gene, encoding a cytosolic Cu-Zn superoxide dismutase that catalyzes the conversion of superoxide to hydrogen peroxide, are known to cause PR degeneration [244]. *Sod2,* which encodes a mitochondrial Mn superoxide dismutase, is required for survival and mutations in this gene lead to embryonic lethality [244,245]. Genes such as *Nxnl1* and *Nxnl2*, known as rod-derived cone viability factors are also suggested to have antioxidant function and show cone degeneration when mutated [246,247], with *Nxnl1* also showing a progressive rod cell loss [246]. Similarly, a mouse model for loss of *Ttpa*, coding for a protein that transports vitamin E, which is known to have antioxidant function, also shows 40% PR degeneration by 20 months [248].

Organellar metabolism: lysosomes. The lysosome, a subcellular organelle is critical for performing several vital functions such as degradation of extracellular and intracellular material, nutrient sensing, energy metabolism, and maintaining cellular homeostasis [249]. Lysosomes contain a wide variety of hydrolytic enzymes that enzymatically degrade biomolecules such as polysaccharides, lipids, etc. [250]. Defects in lysosomal function results in lysosomal storage disorders, a group of inherited metabolic disorders sharing a common biochemical feature of accumulating incompletely degraded metabolites within the lysosomes. Lysosomal storage disorders are generally classified by the composition of the

material accumulated within them and often di ffer depending on the lysosomal proteins a ffected, which reflect di fferent cell biological processes that are a ffected but terminating in a similar pathology of reduced clearance of metabolic aggregates.

RD is an early consequence of lysosomal storage diseases, especially in neuronal ceroid lipofuscinoses (NCL) [251], also called Batten disease, an early-onset neurodegenerative disease with other systemic features such as dementia and epilepsy [252]. NCL may be caused by disruption of genes encoding lysosomal enzymes (*Ppt1* and *Cln5*) and membrane proteins (*Mfsd8*) as well as ER membrane (*Cln6* and *Cln8*) and secretory pathway (*Grn*) proteins, and is characterized by a common lysosomal accumulation of ceroid. Similar to the early retinal phenotype reported for most human NCLs, most mouse models for NCL disease show an early onset of PR degeneration, beginning at 1 month of age and showing greater than 60% degeneration by 6–9 months [253–256]. Additionally, similar to the adult-onset reported for mutations in human GRN, the mouse model for loss of *Grn* also shows a late onset PR degeneration by 12 months [257].

Mouse models for other lysosomal disorders, namely, mucopolysaccharidosis and mucolipidosis due to mutations in lysosomal proteins required for the breakdown of glycosaminoglycans and enzymes required for phosphorylation of glycoproteins, respectively, also develop PR degeneration. For example, mouse models for mucopolysaccharidosis with a mutation in *Naglu* present with a slowly progressive rod-cone degeneration [258], and for mucolipidosis with a mutation in *Gnptab* develop a severe PR degeneration with complete PR loss by 10 months [259].

The lysosome receives materials for degradation via two major pathways, autophagy and phagocytosis. Phagocytosis has an important function in maintaining retinal health since 10% of the OSs are phagocytosed daily by the RPE cells to dispose of waste such as photo-oxidative products while retaining and recycling useful contents back to the PR cells [260]. Phagocytosis by RPE requires its own machinery for processes such as recognition (e.g., *Cd36*), engulfment (e.g., *Mertk*), and degradation (lysosomal enzymes) of the extracellular material. Disruption of the phagocytic machinery due to absence/mutations in proteins involved in the phagocytic pathway, therefore, have severe consequences for PRs and can lead to PR cell death. Mouse models for mutations in genes involved in phagocytosis such as *Mertk*, *Cd36,* and *Rab28* show PR degeneration with the loss of *Mertk* showing a more severe phenotype (>80% degeneration by 60 days for *Mertktm1Grl* and *Mertktm1Gkm*) [261,262] than loss of *Cd36* (17% degeneration at 12 month) [263], and the model for *Rab28* loss showing a more cone-specific response [264].

Autophagy is another lysosome-mediated degradation process essential for maintaining cellular homeostasis [265]. Autophagic flux, the complete dynamic process of autophagy, includes multiple steps involving the formation of phagosomes and autophagosomes, autophagosome fusion with lysosomes, the degradation of the intra-autophagosomal contents, and recycling [266]. Thus, both lysosomal function and autophagy are interconnected wherein disruption of the hydrolytic functions of lysosomes impairs autophagic flux and, conversely, lysosomal function requires normal flux through autophagy [267,268]. In the retina, autophagy plays a dual role: promoting cell survival against harmful stress, and cell death. High basal autophagic levels are maintained in RPE and PR cells. RPE cells being post-mitotic phagocytes are not self-renewing; the autophagy of intracellular components is therefore essential for a normal cellular function of the RPE [265]. In PR cells, autophagy occurs during various cellular activities such as OS degeneration [269], rhodopsin protein expression [270], visual cycle function, and PR apoptosis [271]. Mouse models of conditional inactivation of autophagy genes such as *Atg5*, *Atg7*, and *Rb1cc1* in RPE cells show that these genes are indeed important for survival of the animal and show PR degeneration.

Organellar metabolism: mitochondria. Mitochondria, often referred to as "the powerhouse of the cell", are the major site for cellular energy production in the form of ATP via oxidative phosphorylation. They also perform other important functions such as ROS generation and scavenging, calcium regulation, steroid, and nucleotide metabolism, regulation of intermediary metabolism, and initiation of apoptosis [272]. Oxidative phosphorylation is carried out by the mitochondrial respiratory chain, which consists of five complexes located along the inner mitochondrial membrane. These complexes, in an intricately organized series of biochemical events, synthesize ATP from ADP in response to cellular energy demands. A large number of mitochondria are present in the rod and cone IS and in RPE cells. The total surface area of the inner mitochondrial membrane in cones is 3-fold greater than in rods, presumably accommodating more respiratory chain enzymes to generate more ATP. Cones require more ATP than rods as they do not saturate in bright light and use more ATP/sec for light transduction and phosphorylation [222].

Defective cellular energy production due to abnormal oxidative phosphorylation in mitochondria can therefore lead to PR degeneration. A mouse model for the Leu122Pro mutation of OPA3, a protein hypothesized to be important for maintaining the inner mitochondrial membrane, is reported to cause a multisystemic disease characterized by severely reduced vision, loss of ganglion cells and PR degeneration (by 50%) at 3–4 months of age, a much more severe progression than observed in humans [273]. Similarly, a mouse model for a mutation in the gene for NAD-specific mitochondrial enzyme isocitrate dehydrogenase 3 (*Idh3a*), catalyzing the rate limiting step of TCA cycle, also causes an early and severe PR degeneration (more than 90%) by 90 days [274].

Extra-mitochondrial components of the tricarboxylic acid cycle and oxidative phosphorylation machinery have been localized to the rod OS [275]. It has been hypothesized that perturbation of this machinery results in excess ROS production, leading to PR cell death due to oxidative stress [275–277]. Mutations in a subset of mouse RD models in Table S1 alter genes (*Mpc1, Opa3, Idh3a, Impdh1,* and *Oat*) that encode mouse homologs of mitochondria-associated proteins identified in bovine rod OS [275]. Of these, only IDH3A is directly involved in cellular energy production [274]; the others may influence oxidative phosphorylation or the TCA cycle indirectly, possibly altering the generation of ROS. It may be of interest to determine whether PR cell loss in these mouse models correlates with an altered distribution of extra-mitochondrial oxidative phosphorylation proteins in the rod OS [278], or an increased ROS production, which can be measured in retinal explants [279].

Organellar metabolism: peroxisomes. Peroxisomes are subcellular organelles with various catabolic and anabolic functions such as catabolism of long chain fatty acids and biosynthesis of DHA and bile acids [280]. Several childhood multisystem disorders with prominent ophthalmological manifestations have been ascribed to the malfunction of the peroxisomes, either at the level of peroxisomal biogenesis (PBD) or single enzyme deficiencies [281]. While little is known about the metabolic role of these organelles in retina, studies have shown the presence of peroxisomes in nearly all layers of retina and RPE, albeit with differential expression of lipid metabolizing enzymes, suggesting different functions in different cell types [282]. For example, Zellweger spectrum disorder (ZSD) is a disease continuum known to result from inherited defects in *Pex* genes essential for normal peroxisome assembly. Mice homozygous for the G844D point mutation in *Pex1* show a decreased ERG response and loss of cone PRs (up to 80%) by 22 weeks, recapitulating the abnormal retinal function phenotype in ZSD patients with mild disease [283]. The retinal pathology in such disorders suggests the importance of peroxisomes in maintaining retinal homeostasis and function.

### 5.2.4. Category 04: Visual Cycle and Retinoids

The visual cycle reisomerizes vitamin A retinal that has been released from visual pigments in PR cells, allowing regeneration of the bleached pigments and the subsequent detection of additional light stimuli. The process is catalyzed by enzymes located in PR and RPE cells, so the retinoid intermediates in the process must be transported between them. Mutation of genes involved in the visual cycle pathway cause PR degeneration, in most instances with a moderate to slow progression depending on the allele and the genetic background. Most *Rpe65* mutant alleles show moderately slow PR cell loss (D50 = 7–11 months) [284–288]. Allelic effects are observed in models bearing missense mutations, *Rpe65tm1Lrcb* [289] or *Rpe65tm1.1Kpal* [290], which cause slower progression than observed in *Rpe65tm1Tmr* knockout mice [285–288]. *Abca4tm1Ght* on the BALB/c strain, which also carries a homozygous *Rpe65* Leu450Met mutation, show a late-onset PR degeneration with 40% loss by 11 months of age [291].

By contrast, the same *Abca4tm1Ght* mutation on a 129S4/SvJae background results in abnormal thickening of Bruch's membrane but normal ONL nuclei count and thickness [292]. Several visual cycle mutant alleles have other retinal abnormalities but normal ONL nuclei/thickness. For example, *Abca4tm1.1Rsmy* causes only autofluorescence and A2E accumulation [293] and *Abca4tm2.1Kpal* on C57BL/6\*129Sv leads to a RPE defect but normal ONL nuclei count and thickness [294]. In addition, PR degeneration in *Abca4* mutants can be induced by light exposure [295] or through interaction with other genes such as *Rdh8* [296–298]. The *Lrattm1Kpal* mutation on a 129S6/SvEvTac\*C57BL/6J background results in mild PR degeneration, with <10% loss at 4–5 months [299]. However, a 35% decrease in rod OS length was also reported in this model, indicating the importance of the visual cycle for OS maintenance. Another allele, *Lrattm1.1Bok*, showed a similar loss of rod OS length and 18% PR degeneration at 6 months of age [300]. The *Rbp3tmGil* mutation results in the most rapid PR cell loss in this category (D50 = 0.79 months), possibly attributable to an early developmental role of the protein [301]. The *Rbp4tm2Zhel* congenic mutation on C57BL/6J showed 20% PR cell loss in some peripheral areas and 10% in the central retina an age of 40 weeks [302]. Mutations in two genes that play a role in retinoid uptake in the eye also result in PR cell loss. The *Rtbdntm1.1Itl* allele causes a slow degeneration with a 20% and 37% loss of PR nuclei at 240 days of age in heterozygotes and homozygotes, respectively. *Stra6tm1Nbg* mice exhibit a normal number of rod PR nuclei but significant cone PR cell loss as detected by the cone-specific marker peanut agglutinin [303]. PR cell loss in *Stra6tm1.1Jvil* mice was more pronounced with vitamin A restriction [304].
