*3.3. The Role of D-Serine*

D-serine, an enantiomer of L-serine, is primarily released by glial cells, Müller cells and astrocytes [13]. Initially, it was discovered in the brain but has more recently been studied in the neural retina [13]. D-serine, formed by the racemization of L-serine by serine racemase (SRR) [13], functions as a neurotrophic factor and as a co-agonist for *N*-methyl-D-aspartate receptors (NMDARs) [37]. In the brain, NMDARs normally bind glutamate and glycine, but data suggests preferential or higher affinity binding to D-serine in the retina [13,38]. NMDARs locate at the synaptic terminals of RGCs and sparingly to photoreceptor terminals [39]. The role of NMDARs in the synaptic terminals has not been entirely substantiated, however, studies have indicated that they function in an excitatory role [13]. In the presence of D-serine, NMDA-mediated currents and light-evoked response showed increased amplitude over the D-serine absent control [13]. Irrespective of NMDAR functions, the importance of D-serine is evidenced by pathologies that are directly linked to D-serine irregularities. D-serine insufficiency has been associated with psychiatric and neurodevelopmental disorders [37]. Supplementation with D-serine has proven to mitigate some of the symptoms of psychosis [37]. Patients with DR, by contrast, suffer from increased SRR activity and over production of D-serine, which elicits an excitotoxic effect on RGCs and ultimately contributes to cell death [40]. This will be addressed in more detail in the following sections.

### *3.4. Serine and Epigenetic Regulation*

One carbon metabolism is a fundamental process for purine, thymidine, and amino acid biosynthesis that involves the transfer of a one carbon unit to generate critical metabolites [8]. Serine is essential in this group of metabolic reactions [41] and, in this context, is a precursor for the synthesis of methionine. Briefly, serine donates a methyl group which reacts with homocysteine, originally derived from aspartate [42,43]. This reaction is catalyzed by serine transhydroxymethylase to ultimately form methionine [42,43].

An adenosylation reaction of adenosine triphosphate (ATP) and methionine will yield *S*-adenosyl-L-methionine, which is the critical methyl donor for global methylation of DNA [41,44,45]. DNA methylation, an absolutely essential process to maintain cellular homeostasis [46], occurs on CpG islands of DNA and suppresses gene expression [47]. Therefore, irregularities in serine levels cause downstream hypo- or hyper-methylation of DNA [48], effecting stress responses, proliferation, metabolism, and even responses to extrinsic stimuli [46].

The link between epigenetic modulations and retinal disease is currently at a nascent phase [49], so correlations between aberrant serine metabolism, DNA methylation, and retinal disease remain unclear. However, recent studies performed on three pairs of monozygotic twins with different presentations of AMD indicated significant changes to DNA methylation patterns of genes that may be implicated in disease pathogenesis [50]. More critically, the diet of the studied twins also highlighted nutritional significance in epigenetic regulation. Subjects with reduced dietary methionine, vitamin D, and betaine had worse disease prognosis [50], implicating the importance of nutrient bioavailability and epigenetics. As serine is involved in the synthesis of methionine, further studies should explore serine levels in retinal disease in relation with epigenetic changes that may contribute to disease onset and/or progression.

### **4. Serine Is an Anti-Oxidant and Mediator of Inflammation**

The role that serine plays as a central junction for critical intermediates also extends to anti-oxidative and inflammatory mechanisms. Experimental evidence has indicated that the retina shares some metabolic patterns with neoplastic cells [16]. In these systems, the Warburg e ffect predominates [51], and energy is generated primarily through glycolysis to ensure accelerated ATP production [16]. As explained earlier, the retina is one of the highest energy consuming tissues and thus a source of excessive reactive oxygen species (ROS). Emerging data has shed light on extra-mitochondrial sources of ROS specifically in the outer retina and even in the outer segmen<sup>t</sup> of photoreceptors [52,53]. It was previously shown that oxygen is mainly absorbed at the level of the photoreceptors [54] and thus this site is most prone to oxidative stress, both in physiology and pathology [55]. To build on that, it seems that blue light exposure on outer segmen<sup>t</sup> discs as well as ectopic oxidative phosphorylation in the outer segmen<sup>t</sup> both pose a greater need for e ffective free radical scavengers in the outer segment–RPE interface [56–58]. Glycine and GSH and thus serine supply are essential for this ROS mitigation system of the rods and cones [59].

Fundamentally, intracellular redox homeostasis represents the equilibrium between oxidative and anti-oxidative species, creating a balance in which the environment is not cytotoxic and is sensitive enough to redox changes that may mandate an intracellular or extracellular response. Clearly, oxidative stress is caused by aberrations to this balance. Serine metabolism contributes to redox homeostasis through synthesis of glycine and its essential downstream products GSH and nicotinamide adenine dinucleotide phosphate (NADPH), as well as nicotinamide adenine dinucleotide (NADH) generation during serine biosynthesis [60]. In addition, GSH and NADPH deactivate ROS and other oxidative molecules [60]. GSH is the direct result of combining cysteine and glycine, both of which, as previously explored, are synthesized from serine [61]. NADPH is generated in many pathways, but recent studies sugges<sup>t</sup> that metabolism of serine is a significant contributor of NADPH to the mitochondria, especially during hypoxic conditions [11]. During serine synthesis from 3-phosphoglycerate, serine donates a single carbon to folate forming glycine as well as tetrahydrofolate (THF). THF reductase forms NADP<sup>+</sup>. Then, GSH mediated-reduction of electrophilic molecules helps to maintain the balance of NADPH to NADP<sup>+</sup>, which is principle to redox homeostasis.

GSH anti-oxidative activity is vital for proper retinal function [62]. So in malignancies where serine is deficient, there is an obvious reduction in GSH level and activity, and, as a consequence, the supply of the precursor molecules have been significantly a ffected. However, it is not as simple as reduced supply. 5' adenosine monophosphate-activated protein kinase (AMP-K) can also influence the availability of GSH. AMP-K, like nuclear factor erythroid-derived 2-like 2 (NRF-2), is a "cellular sensor", and responds to metabolic and redox irregularities [63]. AMP-K supports cell survival by upregulating anti-oxidant molecules such as GSH. Elevating serum serine levels in mice fed high fat diet showed increased levels of phosphorylated AMP-K, which resulted in reduced oxidative side-e ffects and increased GSH expression [63]. AMP-K and NRF-2 are, by no means, the only transcription factors involved in moderating the anti-oxidative response, but they represent the profound integration of serine metabolism and retinal oxidative homeostasis.

NRF-2 is an important redox-activated transcription factor under conditions of stress and imbalance of damaging oxidative species [64]. NRF-2 targets critical anti-oxidant genes such as superoxide dismutase, catalase, and GSH by upregulating transcription. In culture of non-small cell lung cancer cells (NSCLCs), gene enrichment analysis has demonstrated a correlation between PHGDH, the enzyme necessary for shunting 3-phosphoglycerate to serine synthesis, and genes that target the activation of NRF-2 [64]. The correlation suggests that biosynthesis of serine is involved

in the expression and regulation of essential anti-oxidant proteins [64]. In addition to the cohort of anti-oxidant proteins, NRF-2 also has some regulatory involvement in the bioavailability of nitric oxide (NO) [65], which has the ability to mitigate the e ffects of H2O2 and superoxide [66]. Treatment with serine increases NO levels in culture which directly links NO production to serine [65], whether endogenously or exogenously presented.

Oxidative stress can also elicit tremendous damage to membranes, DNA, and mitochondria. However, in many pathologies, oxidative stress and inflammation are intimately interlinked [67]. In particular, AMD and DR have been characterized by the slow infiltration of pro-inflammatory constituents of the innate immune system [68]. Serine is currently being explored as a possible therapy for addressing inflammation in these pathologies. However, the involvement of serine metabolism in inflammation and innate immunity remains unclear [69].

Interesting observations have demonstrated a contradictory picture. Exogenously administered serine has been shown to reduce levels of inflammatory cytokines such as interleukin-1β and interleukin-6 [63]. Serine has also been shown to weaken the pro-inflammatory response necessary for macrophage recruitment after bacterial infections in mice [70]. The conflict emanates from the upregulation of inflammatory elements by way of increased glycolytic flux. In DR and AMD, dietary mismanagement and genetic mutations lead to metabolic dysregulation [71–74], which may increase glycolysis in cells. This may lead to increased synthesis of serine since it is produced from the PHGDH shunt. The increase in serine glycolytic synthesis is linked to the amplified activation of toll-like receptor 4 (TLR-4) after activation by lipopolysaccharide (LPS) [69] or hydrogen peroxide [75]. TLR-4 then induces a cytokine response, namely interleukin-1β [69]. Furthermore, GSH, whose activity and expression is upregulated by stress and higher levels of serine, further contributes to cytokine production and maintenance of redox balance in the cellular environment [69].

Serine deprivation was shown to be e ffective in reducing the activation of TLR-4, and reducing cytokine levels [69]. Activation of TLR-4 has also been implicated in the etiology of AMD [76]. Contrarily, serine supplementation is being explored as a potential therapy. Exogenous supplementation has proven to be e ffective in many cases reducing oxidative stress and reducing cytokine levels, but endogenous synthesis (as discussed above) augments the inflammatory response [77]. Therefore, balancing the anti-oxidant e ffects of serine with the pro-inflammatory nature of its endogenous synthesis is something to consider when developing a serine-based therapy for retinopathies in which inflammation contributes to the pathogenesis.

### **5. Consequences of Aberrations in Serine Metabolism**

Our exploration has thus far presented the extensive involvement of serine metabolism in retinal homeostasis. Therefore, aberrations to this important keg in the metabolic machinery can negatively influence retinal pathologies. Glycolytic serine synthesis provides the largest contribution to intracellular serine stores, so deficiencies generally result from abnormalities in that synthetic process. For instance, deleterious mutations in PHGDH have been associated with microcephaly, reduced cognition, and psychomotor abnormalities [78]. Neu-Laxova is a fatal congenital disease marked by serious systemic abnormalities and is attributed to homozygous mutations in enzymes involved in serine biosynthesis [1]. This is recapitulated in the PHGDH−/− mouse model, where mice su ffer from embryonic lethality [1]. These irregularities can a ffect every part of the cell, from mitochondrial biosynthesis to oxidative imbalance.

An extensive comparative metabolomics study was performed by Gao et al., analyzing the metabolic and mitochondrial changes of colon cancer cell lines that occur as a result of serine deprivation [79]. Primarily, glycine, serine, threonine, pyrimidine, and sphingolipid pathways were the most significantly a ffected. Cells exhibited reduced fatty acid synthesis, and reduced TCA intermediates, consequently cells had a 45% reduction in ATP. Serine deprivation also caused major changes in mitochondrial membrane potential and increased fragmentation. However, phospholipid and phosphatidylserine levels remained comparably similar to standard culture conditions; sphingosine and

ceramide levels on the other hand were significantly lowered [79]. It was concluded that mitochondrial fragmentation was exacerbated by reduced production of ceramides that serve as major constituents of the mitochondrial membrane. Ultimately, Gao et al. was able to isolate e ffected pathways that attenuated proper mitochondrial functions critical for cellular viability [79]. Considering the metabolic demands in the retina, such deficits would be extremely detrimental and could play a significant role in the etiology of many retinal diseases associated with metabolic dysregulation.

Cytotoxic aggregation of deoxysphingolipids is another important feature of serine deficiency [78]. As plasma serine levels decline, alanine flux increases and promotes the production of deoxysphingolipids [78]. As previously indicated, serine sphingolipids are synthesized through the condensation of serine and palmitoyl-CoA mediated by SPT. In cases of serine deficiency, SPT may incorporate alanine or glycine into the formation of the lipid, forming a cytotoxic analogue [78]. These lipids have been shown to aggregate and induce apoptosis in in vitro and in vivo models [78]. Retinal organoids treated with deoxysphingolipids exhibited dose-dependent apoptosis [9].

### **6. Retinal Degeneration and Dysregulated Serine Metabolism**
