Dysfunctional peroxisomal lipid metabolisms and their ocular manifestations

Retina is rich in lipids and dyslipidemia causes retinal dysfunction and eye diseases. In retina, lipids are not only important membrane component in cells and organelles but also fuel substrates for energy production. However, our current knowledge of lipid processing in the retina are very limited. Peroxisomes play a critical role in lipid homeostasis and genetic disorders with peroxisomal dysfunction have different types of ocular complications. In this review, we focus on the role of peroxisomes in lipid metabolism, including degradation and detoxification of very-long-chain fatty acids, branched-chain fatty acids, dicarboxylic acids, reactive oxygen/nitrogen species, glyoxylate, and amino acids, as well as biosynthesis of docosahexaenoic acid, plasmalogen and bile acids. We also discuss the potential contributions of peroxisomal pathways to eye health and summarize the reported cases of ocular symptoms in patients with peroxisomal disorders, corresponding to each disrupted peroxisomal pathway. We also review the cross-talk between peroxisomes and other organelles such as lysosomes, endoplasmic reticulum and mitochondria.

that peroxisomes are involved in common neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and multiple sclerosis (Lizard et al., 2012;Wojtowicz et al., 2020). Since neurodegeneration is associated with oxidative stress, lipid peroxidation, and cholesterol auto-oxidation, modifications of peroxisomal functions have been proposed as a potential mechanism for the altered metabolome. Retina is rich in lipids and highly metabolically demanding. Photoreceptors are very high in mitochondria, and nursed by retinal pigment epithelium (RPE) which uptakes nutrients from choroidal blood vessels and Müller glia which extends across the retinal layers and safeguards the blood-retinal barrier (Figure 1). This unique anatomical landscape and high metabolic demand made retina a vulnerable tissue prone to damage from metabolic insults such as peroxisomal disorders. In this review, we focus on the peroxisomal metabolism pathways that are involved in retinal health and the ocular dysfunction associated with peroxisomal disorders.

Catabolic function: α-oxidation of branched chain fatty acids
BCFAs are a specialized group of saturated fatty acids with mono-or poly-methyl branches on the carbon chain. Monomethyl BCFA are subdivided into iso and anteiso structure. Iso-BCFA have methyl branch on the penultimate carbon which is one carbon from the methyl end; meanwhile anteiso-BCFA have methyl branch on the antepenultimate carbon that is two carbons from the methyl end. Enriched sources of BCFA include ruminant-derived and fermented foods (Taormina et al., 2020). Phytanic acid and its metabolite pristanic acid are common 3-methyl BCFA synthesized in ruminant or derived from phytol that are bioaccumulated in the marine system from phytoplankton chlorophyll (Wanders et al., 2011a;Taormina et al., 2020). In humans, tissue phytanic acid levels are strictly derived from dietary sources as endogenous synthesis are limited (Steinberg et al., 1965).

Catabolic function: Glyoxylate detoxification and amino acid degradation
Glyoxylate is a highly reactive two carbon aldehyde form primarily from metabolisms of plant-source glycolate by peroxisomal glycolate oxidase (GO) and animal-source hydroxyproline by sequential breakdown via hydroxyproline oxidase (HPOX), Δ 1 -pyrroline-5carboxylate dehydrogenase (1P5CDH), aspartate aminotransferase (AspAT), and 4-hydroxy-2-oxoglutarate aldolase (HOGA) (Li et al., 2016;Wang et al., 2020). Glyoxylate is readily metabolized by dehydrogenase and oxidases to oxalate which then crystalizes with calcium to form calcium oxalate stone, a common source of kidney stones (Tsujihata, 2008). Since glyoxylate accumulation is correlated with dietary sources and intake, multiple detoxification mechanisms involving peroxisome and/or mitochondria are evolved depending on the food sources of the organisms (Danpure, 1997;Birdsey et al., 2005;Martin, 2010). Three enzymes are involved in the detoxification of glyoxylate: 1) peroxisomal and mitochondrial alanine:glyoxylate aminotransferase (AGT, encoded by the gene AGXT), 2) mitochondrial and cytosolic glyoxylate reductase/ hydroxypyruvate reductase (GRHPR), and 3) cytosolic lactate dehydrogenase (LDH) ( Figure 5). In contrast to rat where AGT is localized to both peroxisome and mitochondria, human AGT is exclusively expressed in peroxisome (Danpure, 1997). AGT catalyzes the transamination of glyoxylate and L-alanine to form pyruvate and glycine (Cellini et al., 2007). Cellular expressions of AGT in retina and RPE has not been elucidated.

Anabolic and catabolic function: ROS/ RNS metabolism
As its name defined, the core feature of peroxisome is the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) (De Duve and Baudhuin, 1966), which may cause biomolecular damage and cell death. As an organelle with high productions of ROS/RNS species, naturally, peroxisomes are equipped with an arsenal of antioxidant enzymes and non-enzymatic free radical scavengers ( Figure 5). To-date, there is no consensus regarding peroxisome's overall contribution as a net source of cellular ROS/RNS or sink for ROS/RNS detoxification (Fransen et al., 2013). Cellular reduction-oxidation (redox) state is dependent on several factors including the type, concentration, localization, synthetic rate, and degradation rate of oxidants formed (Trachootham et al., 2008;Forman et al., 2010). The magnitude of oxidative stress can have divergent effects on cellular fates where at high levels can induced irreversible damage to lipids, protein, and DNA resulting in apoptosis, but at low levels oxidants act as signaling messengers to promote cell survival and proliferation (Jones and Go, 2010;Holmstrom and Finkel, 2014).
The most abundant oxidants produced in peroxisomes is hydrogen peroxide (H 2 O 2 ) because of flavin-containing oxidases that reduce molecular oxygen (O 2 ) to H 2 O 2 (Antonenkov et al., 2010). Peroxisomes account up to 35% of total hydrogen peroxide generation in the mammalian tissues and 20% of total cellular oxygen consumption (Cipolla and Lodhi, 2017). In human peroxisomes, H 2 O 2 is degraded by catalase (CAT) and peroxiredoxin 5 (PRDX5).

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CAT is the most abundant peroxisomal antioxidant enzyme that is highly expressed in RPE as compared to neural retina. CAT activity exhibits a diurnal pattern where posttranslational modification of CAT increases antioxidant activity in the morning during peak VLCFA β-oxidation. While PRDX5 can assist in H 2 O 2 degradation, it is limited due to the small rate constant (Knoops et al., 2011). Superoxide anion radical (O 2
Redox imbalance is a significant contributor to multiple retinal metabolic diseases (Kowluru and Chan, 2007;Bellezza, 2018;Trachsel-Moncho et al., 2018;Graziosi et al., 2020). Restoring the redox homeostasis preserves retinal neuronal and vascular stability. However, the role of peroxisomal redox contribution to retinopathy is understudied. A recent report has shown that overactivation of peroxisomal oxidation with a gain function of ACOX1 (ACOX1 N237S ) causes elevated ROS and retinal glial loss (Chung et al., 2020). Further exploration of peroxisomal redox balance in retinal function is needed.

Anabolic function: Plasmalogen synthesis
Plasmalogen (Pls) are a subclass of glycerophospholipids defined by a vinyl-ether bond at sn-1 position of glycerol

Anabolic function: Docosahexaenoic acid (DHA) synthesis
DHA is the predominant omega-3 polyunsaturated fatty acids (n-3 PUFA) in retinal phospholipid membrane and its accretion may modulate membrane fluidity which affects assemblies of protein complexes and their retinal activity (Fliesler and Anderson, 1983;Treen et al., 1992;Litman and Mitchell, 1996). In human, DHA accounts for 15.3% of total fatty acids in photoreceptor rod cell outer segments as compared to 1-4% in other tissues (Fliesler and Anderson, 1983;Acar et al., 2012). In contrast to photoreceptor rod cells, the compositions of total DHA and diDHA (unique phospholipids with two esterlinked DHA) species in phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine were approximately 2-fold lower in cone-dominant retinas (Agbaga et al., 2018). The unique DHA accretion in the retina is maintained by three mechanisms: 1) dietary uptake of DHA (Young, 1976;Wang and Anderson, 1993b;Pifferi et al., 2012), 2) recycling of phospholipid DHA in rod outer segments via phagocytosis (Young, 1976;Stinson et al., 1991b;Gordon et al., 1992;Gordon and Bazan, 1993), and 3) synthesis of DHA from dietary precursors including α-linolenic acid (ALA, 18:3n-3), eicosapentaenoic acid (EPA, 20:5n-3), and DPAn-3 (22:5n-3) (Anderson et al., 1990;Wang and Anderson, 1993a;Alvarez et al., 1994;Delton-Vandenbroucke et al., 1997). Although biosynthesis of DHA from dietary n-3 PUFA precursor in retina and RPE is limited, impairment in peroxisomal β-oxidation of THA to DHA was observed in patients with X-linked retinitis pigmentosa (Wetzel et al., 1991;Hoffman et al., 2001). DHA synthesis was first described to be solely localized to the ER where a series of alternating elongation by ELOVL5/ 2 and desaturation by FADS1/2 to produce DPAn-3. DPAn-3 is subsequently desaturated to DHA by Δ4-desaturase. However, the presence of Δ4-desaturase in retina is still heavily debated (Metherel and Bazinet, 2019). Since the publication of Voss et al., in 1991, the dogma of DHA synthesis shifted from a localized ER pathway to concerted efforts between ER and peroxisome which is later termed the Sprecher Pathway (Voss et al., 1991;Sprecher et al., 1995) ( Figure 3). From fibroblasts of patients with Zellweger and Refsum disease, the final steps of DHA synthesis were characterized as such that DPAn-3 is elongated to tetrapentaenoic acid (TPA, 24:5n-3) by ELOVL2 and desaturated to THA by FADS2 in ER, then transported to peroxisome via ABCD2, where peroxisomal β-oxidation retroconvert THA to DHA for export (Moore et al., 1995;Morita and Imanaka, 2012). The other possibility is that peroxisomal dysfunction also affect the DHA recycling from photoreceptor outer segment shedding, which will be further discussed in Session 3.
Frontiers in Cell and Developmental Biology frontiersin.org Peroxisomal dysfunction in patients causes DHA deficiency in the brain and retina (Martinez, 1992). Lack of DHA contribute to the pathophysiology of multiple retinal disorders (Fu et al., 2019). In retinal diseases including diabetic retinopathy, retinopathy of prematurity (ROP), age-related macular degeneration (AMD) and inherited retinal degenerative diseases, DHA deficiency is observed. However, DHA supplementation shows positive impacts in some but not all studies (Fu et al., 2019).

Peroxisomal disorders and ocular symptoms
Based on the results of enzyme activity studies and biochemical markers, the peroxisomal disorders was historically divided into three groups: 1) peroxisomal biogenesis disorders (PBD); 2) multiple enzyme deficiencies; and 3) single enzyme/transporter deficiency disorders. With recent development in the understanding of the biochemical and molecular genetic bases of these conditions, a new system has been proposed to divide the PBD into two subtypes: Zellweger Spectrum Disorder (PBD-ZSD) and the rhizomelic chondrodysplasia punctata (RCDP) spectrum (previously known as the multiple enzyme deficiencies). Other conditions such as Heimler Syndrome (HS), which was previously classified as a distinct syndrome consists of sensorineural hearing loss, amelogenesis inperfecta (AI) and nail anomalies with or without visual defect, is now considered as part of the PBD-ZSD (Ratbi et al., 2015). Single enzyme/ transporter deficiency disorders such as X-linked adrenoleukodystrophy (X-ALD) and peroxisomal acyl-CoA oxidase (ACOX) deficiency remains a separate entity.
Diagnosing patients with mild presentation of PBD-ZSD has been challenging due to the diverse and non-specific nature of the symptoms (Enns et al., 2021). Nonetheless, recent expansion in Frontiers in Cell and Developmental Biology frontiersin.org newborn screening programs have included screening of X-ALD using liquid chromatography tandem mass spectroscopy (LC-MS/MS) to detect VLCFA, C26:0-lysophosphatidyl choline (C26: 0-LPC), from dried blood spots (Turk et al., 2020). First introduced in 2013, newborn screening for X-ALD is now carried out in 20 States and the District of Columbia with 2 additional states currently running pilot programs (https:// adrenoleukodystrophy.info/clinical-diagnosis/ald-newbornscreening). Therefore, every newborn from these states will have C26:0-LPC testing at birth, which will identify all newborns with PBD-ZSD (Klemp et al., 2021). Concerns have been raised around the "incidental" identification of PBD-ZSD patients through the X-ALD screening due to lack of targeted treatment that modifies the course of PBD-ZSD. Therefore, development of targeted PBD-ZSD treatment is in dire need. The central nerves system (CNS) manifestations of PBD-ZSD involves structural brain malformations, predominantly involve the neuronal migration defects such as polymicrogyria, heterotopia, agenesis/hypoplasia of the corpus callosum, pachygyria, and hypoplastic olfactory bulb. Cerebral ventricular abnormalities such as colpocephaly and progressive dilatation of the ventricles were also reported (Nakai et al., 1995). Demyelination and global cerebral atrophy have also been reported in both early and late onset cases (Renaud et al., 2016). Ocular phenotypes related to PBD-ZSD includes anterior changes of cornea clouding, cataracts, and Brushfield spots (small, white or greyish/brown spots on the periphery of the iris). Posterior ocular findings include Leber congenital amaurosis (LCA), pigmentary retinopathy, and optic nerve dysplasia (Lambert et al., 1989;Mechaussier et al., 2019). LCA is a severe form of early onset retinal dystrophy. Patients with LCA typically have visual impairment since infancy. Symptoms of LCA include photophobia, nystagmus, keratoconus, and behavior of eye poking and picking (oculodigital signs). Retinal changes associated with PBD-ZSD involve all retinal layers from RPE to retinal ganglion cells (Haddad et al., 1976), with most severe loss in photoreceptors (Haddad et al., 1976;Kretzer et al., 1981;Garner et al., 1982). These changes are scattered from central to peripheral retina. The optic nerve of patients with PBD-ZSD can be profoundly demyelinated (Thomas et al., 1975). Fundoscopic changes in patients with PBD-ZSD can include retinal arteriolar attenuation, pale optic disc and loss of pigment epithelium most prevalent in the macular area (Folz and Trobe, 1991).
Heimler Syndrome is a condition found to be caused by pathogenic variants in PEX1 and PEX6 genes (Ratbi et al., 2015). It represents the mild end of PBD-ZSD. Long term follow-ups of index cases revealed "salt-and-pepper"-like mottling of RPE and abnormal electroretinogram (ERG) recording in young adulthood after complaints of decreased vision in a female patient. The patient also had cystoid macular edema in keeping with the diagnosis of macular dystrophy (Lima et al., 2011). After evaluating other patients with Heimler syndrome, three additional patients with PEX1 variants and one patient with PEX6 variants were also found to have retinal pigmentary changes (Heimler et al., 1991;Zaki et al., 2016). Given the rarity of these conditions, most literature is constituted with case studies describing the retinal phenotype of PBD-ZSD. Studies focusing on the natural history of retinal dystrophy in PBD-ZSD are required to understand its progressive impact on vision.

Gene mutations in peroxisomal β-oxidation
Peroxisomal acyl-CoA oxidase deficiency disorder caused by biallelic pathogenic variants in ACOX1 gene and D-bifunctional protein (DBP) deficiency disorder caused by either or both MFE 2-enoyl-CoA hydratase and 3hydroxyacyl-CoA dehydrogenase deficiency, are disorders of peroxisomal fatty acid β oxidation (Figure 3). They can present with neonatal onset severe neurological sequela with progressive white-matter demyelination, seizures, and cortical malformations. Both conditions were associated with visual impairment, abolished electroretinogram possibly related to retinal degeneration or subretinal pigmentary retinopathy (Suzuki et al., 2002;Kurian et al., 2004;Bae et al., 2020). ACOX1 is abundantly expressed in the RPE as compared to neural retina, whereas the distribution of ACOX2 and ACOX3 in RPE and retinal layers remains unclear (Das et al., 2019). ACOX1 deficiency results in premature death in adolescence and are associated with abnormal retinal pigmentation, retinal degeneration, and optic atrophy (Das et al., 2021). Patients with ACOX1 deficiency have elevated plasma and fibroblast saturated VLCFA and comparable saturated LCFA, BCFA (phytanic acid), and DHCA/THCA to control (Ferdinandusse et al., 2007). MFE-2 is equally or more expressed in the neural retina than RPE, whereas expressions of MFE-1 has yet to be elucidated in the eyes (Das et al., 2019). Severe MFE-2 deficiency may manifest symptoms comparable to that of PBD-ZSD; however milder juvenile onset deficiency may present with abnormal retinal pigmentation with normal visual acuity (Das et al., 2021). Patients with MFE-2 deficiency have elevated levels of saturated VLCFA, BCFA (pristanic acid), and DHCA/ THCA and reduced levels of plasma and retinal DHA (Martinez, 1992;Ferdinandusse et al., 2006a). Patients with peroxisomal ACOX deficiency and MFE-2 deficiency show no accumulation of pipecolic acid, with no reduction in erythrocyte plasmalogens. MFE-2 deficiency is associated with increased plasma levels of bile acid intermediates, but ACOX deficiency is not (Ferdinandusse et al., 2007).
SCPx deficiency was first reported in 2006 in a patient with normal level of VLCFA but elevated level of pristanic acid (Ferdinandusse et al., 2006b). The only ocular finding Frontiers in Cell and Developmental Biology frontiersin.org reported was pathological saccadic eye movements indicating potential pathology in the brain stem. Another patient with SCPx deficiency had mild symptoms of nyctalopia. This individual had normal visual acuity. Detailed ocular investigation revealed peripapillary atrophy, irregular autofluorescence associated with the major vascular arcades, with no peripheral pigmentary retinal changes (Horvath et al., 2015;Morarji et al., 2017). ACAA1 deficiency has yet to be associated with human disease.

Gene mutations in peroxisomal αoxidation
FALDH deficiency leads to Sjögren-Larsson Syndrome, a condition associated with accumulation of phytol. Sjögren-Larsson Syndrome is associated with reduced visual acuity, degeneration of Müller cells in the inner retina, retinal thinning, RPE atrophy, and photoreceptor dysfunction (van den Brink et al., 2004;Nanda and Kovach, 2019). PHYH deficiency is one of the primary causes of Refsum disease (Jansen et al., 1997) (Figure 4). Patient with Refsum disease have elevated levels of phytanic acid in blood and tissues. Clinical symptoms of Refsum disease includes retinitis pigmentosa, miosis with attenuated pupillary light response, iris atrophy, and cataract (Ruether et al., 2010). Phytanic acid exposure cause morphological changes in both fetal bovine and human RPE in vitro, mimicking the RPE pathology observed in patients with Refsum's disease (Bernstein et al., 1992). HACL1 deficiency has yet to be reported in humans; however, animal model suggests that dietary intake of phytol in Hacl1 −/+ mice exhibited elevated levels of phytanic acid in blood and tissues similar to the biochemical profile of patients with Refsum disease (Mezzar et al., 2017). Interestingly, the human HACL1 is only three amino acids different from its mouse analog. Clinical phenotypes of AMACR deficiency include retinitis pigmentosa and decreased visual acuity (Thompson et al., 2009;Stewart et al., 2011).

Gene mutations in glyoxylate detoxification
Defects in AGXT gene results in primary hyperoxaluria type 1, which is an inborn error of metabolism characterized by accumulation of calcium oxalate ( Figure 5). This process leads to calcinosis cutis metastatica of the skin, tooth mobility, vascular spasm/occlusion, and nephrocalcinosis. Retinal complications include optic atrophy, retinopathy and choroidal neovascularization (Leumann and Hoppe, 2001;Cochat et al., 2006;Lorenzo et al., 2006). Patients with primary hyperoxaluria type 1 also exhibit crystalline deposit in RPE leading to bilateral vision loss (Roth et al., 2012;Kourti et al., 2020).

Gene mutations in plasmalogen synthesis
GNPAT deficiency results in RCDP type 2   (Figure 3). Patients with GNPAT deficiency have low plasmalogen levels in erythrocytes. They typically have a shortened life expectancy not exceeding a decade (Itzkovitz et al., 2012). Congenital cataracts have been reported . AGPS deficiency results in RCDP type 3 and patients exhibited lower levels of plasmalogen in erythrocytes (Wanders et al., 1994). FAR1 deficiency results in autosomal recessive RCDP type 4 (Buchert et al., 2014). FAR1deficient patients have markedly lower levels of plasmalogens, and congenital cataracts is a cardinal symptom (Buchert et al., 2014). Interestingly, patients with autosomal dominant FAR1 de novo variants also shares similar clinical phenotypes including congenital and juvenile cataracts, but a divergent biochemical phenotype of elevated plasmalogens in fibroblasts (Ferdinandusse et al., 2021). FAR1 de novo variant disrupted negative feedback regulation leading to un-inhibited biosynthesis of plasmalogen (Ferdinandusse et al., 2021). Deficiency in FAR2 has yet to be associated with human diseases.

Gene mutations in peroxisomal membrane transporter
Although, ABCD1 was shown to be expressed ubiquitously between RPE and neural retina in rodent model (Das et al., 2019), patients with X-ALD do not exhibit direct retinal pathology. The visual impairment in X-ALD patients are caused by two mechanisms: 1) the loss of color vision in a subgroup of X-ALD patients are caused by continuous gene deletions in X chromosome where ABCD1 gene and red pigment gene are in close proximity (Sack et al., 1989) or by chromosome rearrangements (Alpern et al., 1993), 2) the cortical blindness associated with X-ALD is caused by visual track demyelination, which can subsequently lead to retrograde degeneration of ganglion cells (Kaplan et al., 1995;Ohkuma et al., 2014).
Patients with ABCD3 deficiency have elevated levels of DHCA and THCA, but ocular abnormalities have not yet been identified . Patients with BAAT deficiency exhibited elevated levels of unconjugated bile acids (Carlton et al., 2003). BAAT expression in retinal cells are unknown and patients with BAAT deficiency has no reported ocular abnormalities.
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Current therapy
Currently, there is no curative or targeted disease modifying treatment for PBD-ZSD. Cholic acid (CA) therapy was approved by Food and Drug Administration (FDA) in March 2015 to treat liver disease in patients with PBD-ZSD. CA has been shown to decrease toxic bile acid intermediates, improve transaminase levels, and reduce liver inflammation, with improvement in growth parameters in pediatric populations (Anderson et al., 2021). Since PBD-ZSD patients have markedly low level of DHA (Janssen et al., 2000), trials of DHA supplementation were made as an attempt to restore the neurological, ocular and hepatic function in PBD-ZSD patients. M. Martinez conducted a cohort study in the 1990s treating 20 PBD-ZSD patients with DHA supplementation. Patients were treated with variable doses of DHA ethyl ether (depends on age and blood DHA level) between 9 months and 9 years and more than half of the patients not only had improved biochemical markers such as reduced VLCFA and increased erythrocyte plasmalogen level, but also improved brain and retinal function compared to pre-treatment (Martinez, 2001). Nonetheless, the clinical improvement was not subjectively quantified. A follow up study focusing on the visual function of this cohort demonstrated resolution of nystagmus, improvement of ERG recording, and amelioration of visual behaviors in some cases and stabilization in the rest. The improvement was better appreciated in patients who had baseline ophthalmological assessment (Noguer and Martinez, 2010). Based on this preliminary work, a randomized double-blind case control trial was conducted supplementing a fixed dose (100 mg/kg/d) of DHA triglyceride (47% DHA) and arachidonic acid (ARA; 20:4n-6) triglyceride (46% ARA) (Paker et al., 2010). This is a short-term study in 50 patients (25 in treatment group and 25 in control) with follow ups in 1 year. The study also focused exclusively on quantitative biometrics of patients such as ERG changes, weight, height and biochemical markers such as VLCFA, plasmalogen and liver enzymes. Given the short-term intervention and the broad spectrum of clinical variability of PBD-ZSD patients, it was not surprising that no statistically significant changes were obtained between the two groups. At this time, there is very limited evidence supporting the efficacy of clinical use of DHA in patients with PBD-ZSD.

Peroxisomes and other cell organelles
Peroxisomes are highly dependent on the interaction with other organelles (ER, mitochondria and lysosomes) in order to perform proper metabolism. For example, some of the peroxisomal substrates (e.g., VLCFAs) are synthesized and elongated from shorter chain fatty acids (SCFAs) in ER. In addition, the end products of peroxisomal β-oxidation subsequently enter mitochondria for metabolism and energy production.
Frontiers in Cell and Developmental Biology frontiersin.org primary ER-peroxisome tether, and loss of VAP or ACBD5 increases peroxisome mobility. VAP-ACBD5mediated contact between the ER and peroxisomes is also important for peroxisome growth and lipid homeostasis (Costello et al., 2017;Hua et al., 2017). In addition, plasmalogen synthesis starts in peroxisomes and completes in ER (Nagan and Zoeller, 2001;Braverman and Moser, 2012). The initial two steps are catalyzed by peroxisomal matrix enzymes dihydroxyacetone phosphate acyltransferase (DHAPAT/GNPAT) for transfer of acyl-DHAP across the enzyme active sites, and AGPS for the exchange of the acyl group (fatty acid) for an alkyl group (fatty alcohol, supplied by FAR1). Alkyl-DHAP is further reduced and plasmalogen synthesis proceeds in the ER (Figure 2).
Moreover, ER is involved in peroxisome biogenesis. Peroxisomal membrane proteins (PMP) are thought to be first inserted in ER and subsequently exit from ER to peroxisomes (Dimitrov et al., 2013). It has also been proposed that peroxisomes originate from ER and the avoidance of toxic lipid by-products is the driving force for the separation of the peroxisomes (Gabaldon, 2014). In mammalian cells, PEX16 with an appended NH 2 -terminal type I signal anchor sequence moves to peroxisomes after being cotranslationally synthesized in the ER (Kim et al., 2006). In mouse dendritic cells, PEX13 and PMP70 are present in the ER domain with a peroxisomal reticulum from which mature peroxisomes are derived (Geuze et al., 2003). Meanwhile, the peroxisome biogenesis factors PEX19 and PEX3 cooperate and mediates the posttranslational targeting of proteins such as UBXD8, a lipid droplet-destined membrane protein and reticulon homology domain-containing protein to ER (Schrul and Kopito, 2016;Yamamoto and Sakisaka, 2018). Therefore, the close connection between peroxisome and ER is evident.

Peroxisomes and lysosomes
Peroxisomal substrates such as VLCFAs can also be generated from lysosomal degradation of different lipid species. Rod photoreceptor outer segment is rich in VLCPUFA and LCPUFA (DHA and ARA), which comprisẽ 60% of total phospholipids (Stinson et al., 1991a). To maintain normal retinal function, there is a continuous shedding of photoreceptor outer segment on daily basis (Fliesler & Anderson, 1983). The sheded outer segments are ingested by RPE via phagocytosis and packaged into membrane-bound phagosomes, which are then fused with lysosomes for subsequent degradation (Bosch et al., 1993;Ferrington et al., 2016). It has been reported that there is a dynamic membrane contact between peroxisomes and lysosomes mediated by lysosomal Synaptotagmin VII (Syt7) binding to the lipid PI(4,5)P 2 on peroxisomal membrane, and the organelle contact facilitates the transport of cholesterol from lysosomes to peroxisomes (Chu et al., 2015). Cholesterol is accumulated in cells and animal models of peroxisomal disorders (Chu et al., 2015). These findings suggest the potential involvement of peroxisomes in VLCFAs recycling during photoreceptor outer segment shedding. Although RPE immediately recycles DHA released from the disc membrane during degradation back to photoreceptors for disc membrane synthesis , the involvement of peroxisome in this process is yet to be characterized. Moreover, compromised lysosomes with impaired degradative enzyme activity leads to lipofuscin-like autofluorescence in RPE (Guha et al., 2014). Understanding of the interactions between peroxisome and lysosome will uncover the basic lipid biology and balance in retina.

Peroxisomes and mitochondria
Peroxisomes and mitochondria tightly interact with each other in fatty acid metabolism and undergo close communication through three major pathways: 1) there is a physical association between these two organelles, and the degree of contact changes dynamically with the cell types and microenvironment (Fransen et al., 2017). For example, the peroxisomal membrane protein PEX34 (conserved to humans) and the outer mitochondrial membrane protein FZO1 (mammalian homologue Mitofusin 2) tether the peroxisome and mitochondria in the yeast (Shai et al., 2018). 2) mitochondria-derived vesicles containing mitochondriaanchored protein ligase that can fuse with a subset of peroxisomes (Neuspiel et al., 2008); 3) peroxisomes and mitochondria communicates through releasing of biological messengers such as ROS, lipids and NAD + etc.
VLCFAs, which cannot be degraded in mitochondria, need to be shortened by peroxisome β-oxidation. Shortened VLCFAs are further oxidized in mitochondria for energy production. Peroxisomal β-oxidation can also compensate defective mitochondrial fatty oxidation by breaking down medium-chain and long-chain fatty acids (Violante et al., 2019;Ranea-Robles et al., 2021). Studies of RPE from AMD vs control patients indicated that proliferation of peroxisomes is accompanied with mitochondrial abnormalities (Feher et al., 2006). Abnormal peroxisomal metabolism and redox status can rapidly disturb mitochondrial redox state. The antioxidant enzyme CAT is mainly localized in peroxisomes. Deficiency of CAT increases redox state of mitochondria and peroxisome-derived oxidative stress causes mitochondrial redox imbalance (Ivashchenko et al., 2011). Retinal transfection of AAV-expressing CAT and superoxide dismutases 2 (SOD2) prolonged cone photoreceptor survival in mice during retinal degeneration (Xiong et al., 2015), and AAV-CAT also exerts Frontiers in Cell and Developmental Biology frontiersin.org neuroprotective effects on retinal ganglion cells in ischemiainduced retinal injury in rats (Chen and Tang, 2011). In addition, peroxisomal oxidation also regulates lipid composition in mitochondria membrane, which in turn regulates mitochondrial dynamics and ATP production (Petrushka et al., 1959;Hatch, 2004). Oxidative stress induces release of cytochrome c from mitochondria, and cytochrome c cleaves plasmenylcholine (plasmalogens of the phosphocholine head group class) in membrane bilayers (Jenkins et al., 2018). Loss of plasmalogens in mitochondrial membrane causes inefficient respiration (Kimura et al., 2019). As VLCPUFAs are recycled back to the retina during photoreceptor outer segment renewal Gordon et al., 1992), mitochondrial DHA and ARA content could also be potentially modulated by peroxisomal oxidation. Both dietary DHA (no ARA) and ARA (no DHA) in rats profoundly alters cardiac mitochondrial phospholipid fatty acid compositions, and suppresses Ca 2+ -induced opening of the mitochondrial permeability transition pore (MPTP), which causes cell death (Khairallah et al., 2010;Khairallah et al., 2012); dietary DHA (no ARA) also depletes cardiac mitochondrial ARA content (Khairallah et al., 2012). These findings suggest that peroxisomal oxidation may control mitochondrial membrane composition and activity via VLCPUFA recycling. More recently, it has been reported that adipose specific knockout of the peroxisomal biogenesis factor PEX16 or the plasmalogen synthetic enzyme GNPAT blocks cold-induced mitochondrial fission, decreases mitochondrial copy number and causes abnormal mitochondrial function. Dietary plasmalogen increases mitochondrial copy number, improves mitochondria function in mice with Pex16 deficiency (Park et al., 2019). This work provides direct evidence of peroxisome-derived ether lipid regulating mitochondrial activity.
Moreover, mitochondrial respiration is also critical to reoxidize NADH formed in peroxisomes and maintain peroxisomal β-oxidation (Wanders et al., 2015). Altered NAD (+)/NADH redox state with age-dependent increases in intracellular NADH and decreases in NAD (+) content is shown in human brain (Zhu et al., 2015), reflecting declined mitochondrial function. NAD (+) levels also decrease in mouse RPE cells with aging (Jadeja et al., 2018). Mutations of gene encoding nicotinamide mononucleotide adenylyltransferase 1 involved in NAD (+) biosynthesis reduce the enzymatic activity and causes blindness within the first year after birth (Koenekoop et al., 2012). Taken together, peroxisomal metabolic and redox homeostasis is important to maintain mitochondrial function. Further understanding of peroxisomal and mitochondrial interaction will provide more clues to understand cellular activity in healthy and diseased retinas.

Knowledge from the brain
Although there are limited studies regarding the disease pathogenesis of peroxisome-related retinal dysfunction, we could gain some knowledge from the brain investigations as retina is part of the CNS (London et al., 2013). Peroxisomal dysfunction leads to compromised antioxidant defense with decreased CAT expression, attenuated D-serine degradation, disrupted lipid composition in myelin sheath and cellular membranes in the CNS (Uzor et al., 2020), which in turn cause abnormalities in the myelinated axons and synaptic transmission (Berger et al., 2016). Accumulated VLCFAs and decreased plasmalogen ethanolamine levels are observed in the brain of patients with Alzheimer's disease (Ginsberg et al., 1995;Kou et al., 2011). Reduced DHA, ARA and plasmalogen ethanolamine levels are found in the brain of patients with Parkinson's disease, and supplementation of DHA-containing plasmalogen precursor protects against striatal dopamine loss in mice modeling Parkinson's disease (Fabelo et al., 2011;Miville-Godbout et al., 2017). These findings suggest an essential role of peroxisomes and the related lipid metabolism in maintaining brain health.
The contribution of peroxisomes to brain function could be cell-dependent. In mice, dysfunction of peroxisome in neural cells leads to abnormal cortical neuronal migration and maturation of the cerebellum (Janssen et al., 2003;Krysko et al., 2007;Muller et al., 2011). Peroxisome deficiency in astrocytes disturbs brain-derived neurotrophic factor levels and dysregulates axogenesis of the hippocampus neurons (Abe et al., 2020), suggesting that astrocytic peroxisomes modulate neuronal integrity. Moreover, microglial peroxisomal dysfunction induces an inflammatory activated and proliferative state in the mouse brain, but does not affect neuronal health up to one-year old and the long-term impacts is yet to be evaluated (Beckers et al., 2019). Therefore, it would be important to uncover the contribution of neuronal and glial peroxisomal activity to neuronal function in the CNS.

Future perspectives
Defective peroxisomal function causes disturbed retinal homeostasis and retinal degeneration. However, the mechanisms behind are largely unknown. We speculate that the disrupted lipid metabolism and redox balance typically regulated through the interaction between peroxisomes, lysosomes, mitochondria and ER may contribute to the disease development and progression. Further investigations in the pathophysiology and natural history of peroxisomeal disorder associated retinal dystrophy will greatly advance the potential for therapeutic development.
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Retinal lipid homeostasis and alterations
The retinal lipid homeostasis is essential in maintaining normal retinal neuronal and vascular function (Fu et al., 2019;Fliesler, 2021). As there are extensive lipid alterations accompanied with peroxisomal dysfunction, it is important to know that whether all or certain altered lipids are the significant contributors to the development of retinal diseases. Lipids are important membrane components of cells and organelles. Change in lipid composition alters organelle function and cell status. In addition, lipids and their downstream lipid metabolites can function as signaling molecules to induce various cellular responses in metabolism, inflammation, cell survival and apoptosis. Moreover, fatty acids such as palmitate are also possible fuel substrates for photoreceptor, but other type and source of fatty acids are understudied (Joyal et al., 2016). Supplementation of specific deficient lipids and modulating the downstream targeting pathways may be protective against retinal degeneration.
Furthermore, the interaction between RPE, Müller glia, and neuronal cells in retina is also important for the modulation of lipid availability and clearance of neighboring photoreceptors (Fu et al., 2021). For example, disrupted lipid homeostasis in RPE causes lipid droplet formation and RPE dysfunction with aging (Yako et al., 2022). Interestingly, peroxisomes and lipid droplets have physical interaction within cells. Peroxisomal β-oxidationderived ROS regulate the levels of adipose triglyceride lipase, which control lipolysis of intracellular lipids (Ding et al., 2021). Therefore, increasing peroxisomal activity may decrease lipid droplets in RPE, preserve RPE function and in turn photoreceptor health. Fatty acid profiling focuses on the entire tissue and radiolabeled lipid tracing aids the understanding of the kinetic incorporation, turnover and loss of fatty acids in the retina (Rodriguez de Turco et al., 1990;Gordon and Bazan, 1993;Santos et al., 1995). However, the knowledge of cell-specific uses of lipids and the interactions among different lipid metabolic pathways are limited. The feasibility of single-cell transcriptomics and cell-specific lipidomics in retinal research may provide insight to the cell specific lipid and fatty acid metabolism. Furthermore, progress in characterizing dynamic processing of fatty acids in retina is often limited by the high cost of radiotracer and retinal tissue size. Recently, a cost-effective, tracer-free novel methods using compound-specific isotope analysis (CSIA) has been established to trace fatty acid metabolism by utilizing food sources differing in natural carbon isotope abundance. Briefly, because the carbon isotopic composition of a molecule is conserved following dietary intake, highly precise measurement of carbon 13 ( 13 C) abundance can provide insights to the dietary source that contributed to the incorporation of 13 C to an endogenous molecule. Therefore, by designing well-controlled dietary switch experiments that switches diets made from foods less enriched in 13 C foods (C3 plants) to foods more enriched in 13 C (C4 plants), fatty acid incorporation, turnover, and synthesis can be measured as effectively as radiotracer experiments (Lacombe et al., 2020;Lacombe and Bazinet, 2021). Moreover, due to the high precision of 13 C measurements by gas chromatography-isotope ratio mass spectrometry (GC-IRMS), cell-specific retinal extract can be quantified.

Implications of peroxisomes on other retinal disorders
Altered peroxisomal function has been implicated in agerelated neurodegenerative diseases and diabetes-related complications (Cipolla and Lodhi, 2017). In RPE from AMD vs control patients, mitochondrial changes are associated with proliferation of peroxisomes (Feher et al., 2006). However, it is unclear that whether this mitochondrial response is protective or detrimental. Further investigation is required.
Furthermore, the knowledge of DHA metabolism and the application of dietary DHA for prevention of retinal dysfunction is translatable to a number of retinal diseases. Some (but not all) clinical trials showed that fish oil (rich in DHA and EPA) supplementation reduces risk for severe ROP (Pawlik et al., 2011;Beken et al., 2014;Pawlik et al., 2014;Molloy et al., 2016;Najm et al., 2017). Fish oil supplementation or dietary intake of food rich in DHA decreased the risk for AMD in a number of trials (Sangiovanni et al., 2009;Tan et al., 2009;Christen et al., 2011;Ho et al., 2011). However, dietary supplementation of DHA (350 mg/day) and EPA (650 mg/ day) did not reduce the risk of advanced AMD in Age-Related Eye Disease Studies (AREDS/AREDS2) (Age-Related Eye Disease Study 2 Research Group, 2013). The reasons for the inconsistent observations are unclear. It has been noted that DHA and EPA supplements may lower circulating ARA levels (D'ascenzo et al., 2014;Zhao et al., 2015) and decrease the ARA/ DHA ratio in preterm infants (Najm et al., 2017). Low serum ARA level in the postnatal period is strongly associated with development of clinically significant ROP (Lofqvist et al., 2018) and sufficient ARA is required for DHA to protect against severe ROP . In addition, DHA-and ARAoxylipins generated via cytochrome P450 oxidases exaggerates while DHA-and ARA-derived oxylipins via lipoxygenases inhibit pathological retinal neovascularization (Sapieha et al., 2011;Gong et al., 2016;Gong et al., 2017). Therefore, metabolic imbalance among these pathways may also contribute to the inconsistent observations among different trials.
In conclusion, understanding of retinal cell-specific peroxisomal function, VLCFA metabolism and natural history of retinal dysfunction in peroxisomal disorders is a key to development of disease modifying therapeutics. Given the involvement of peroxisomal dysfunction in various retinal pathophysiology, this therapeutic intervention is likely to Frontiers in Cell and Developmental Biology frontiersin.org benefit common multifactorial retinal degenerative disorders as well.

Author contributions
All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.

Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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