Metabolic phenotyping of healthy and diseased human RPE cells.

Purpose: Metabolic defects in retinal pigment epithelium (RPE) are underlying many retinal degenerative diseases. This study aims to identify the nutrient requirements of healthy and diseased human RPE cells. Methods: We profiled the utilization of 183 nutrients in human RPE cells: 1) differentiated and dedifferentiated fetal RPE (fRPE), 2) induced pluripotent stem cell derived-RPE (iPSC RPE), 3) Sorsby fundus dystrophy (SFD) patient-derived iPSC RPE and its CRISPR-corrected isogenic SFD (cSFD) iPSC RPE, and 5) ARPE-19 cell lines cultured under different conditions. Results: Differentiated fRPE cells and healthy iPSC RPE cells can utilize 51 and 48 nutrients respectively, including sugars, intermediates from glycolysis and tricarboxylic acid (TCA) cycle, fatty acids, ketone bodies, amino acids, and dipeptides. However, when fRPE cells lose epithelial phenotype through dedifferentiated, they can only utilize 17 nutrients, primarily sugar and glutamine-related amino acids. SFD RPE cells can utilize 37 nutrients; however, Compared to cSFD RPE and healthy iPSC RPE, they are unable to utilize lactate, some TCA cycle intermediates, and short-chain fatty acids. Nonetheless, they show increased utilization of branch-chain amino acids (BCAAs) and BCAA-containing dipeptides. The dedifferentiated ARPE-19 cells in traditional culture media cannot utilize lactate and ketone bodies. In contrast, nicotinamide supplementation promotes differentiation into epithelial phenotype, restoring the ability to use these nutrients. Conclusions: Epithelial phenotype confers metabolic flexibility to the RPE for utilizing various nutrients. SFD RPE cells have reduced metabolic flexibility, relying on the oxidation of BCAAs. Our findings highlight the importance of nutrient availability and utilization in RPE differentiation and diseases.

CRISPR editing of anti-anemia drug target rescues independent preclinical models of retinitis pigmentosa.

Retinitis pigmentosa (RP) is one of the most common forms of hereditary neurodegeneration. It is caused by one or more of at least 3,100 mutations in over 80 genes that are primarily expressed in rod photoreceptors. In RP, the primary rod-death phase is followed by cone death, regardless of the underlying gene mutation that drove the initial rod degeneration. Dampening the oxidation of glycolytic end products in rod mitochondria enhances cone survival in divergent etiological disease models independent of the underlying rod-specific gene mutations. Therapeutic editing of the prolyl hydroxylase domain-containing protein gene (PHD2, also known as Egln1) in rod photoreceptors led to the sustained survival of both diseased rods and cones in both preclinical autosomal-recessive and dominant RP models. Adeno-associated virus-mediated CRISPR-based therapeutic reprogramming of the aerobic glycolysis node may serve as a gene-agnostic treatment for patients with various forms of RP.

Metabolic transcriptomics dictate responses of cone photoreceptors to retinitis pigmentosa.

Most mutations in retinitis pigmentosa (RP) arise in rod photoreceptors, but cone photoreceptors, responsible for high-resolution daylight and color vision, are subsequently affected, causing the most debilitating features of the disease. We used mass spectroscopy to follow 13C metabolites delivered to the outer retina and single-cell RNA sequencing to assess photoreceptor transcriptomes. The S cone metabolic transcriptome suggests engagement of the TCA cycle and ongoing response to ROS characteristic of oxidative phosphorylation, which we link to their histone modification transcriptome. Tumor necrosis factor (TNF) and its downstream effector RIP3, which drive ROS generation via mitochondrial dysfunction, are induced and activated as S cones undergo early apoptosis in RP. The long/medium-wavelength (L/M) cone transcriptome shows enhanced glycolytic capacity, which maintains their function as RP progresses. Then, as extracellular glucose eventually diminishes, L/M cones are sustained in long-term dormancy by lactate metabolism.

Proline provides a nitrogen source in the retinal pigment epithelium to synthesize and export amino acids for the neural retina.

It is known that metabolic defects in the retinal pigment epithelium (RPE) can cause degeneration of its neighboring photoreceptors in the retina, leading to retinal degenerative diseases such as age-related macular degeneration. However, how RPE metabolism supports the health of the neural retina remains unclear. The retina requires exogenous nitrogen sources for protein synthesis, neurotransmission, and energy metabolism. Using 15N tracing coupled with mass spectrometry, we found human RPE can utilize the nitrogen in proline to produce and export 13 amino acids, including glutamate, aspartate, glutamine, alanine, and serine. Similarly, we found this proline nitrogen utilization in the mouse RPE/choroid but not in the neural retina of explant cultures. Coculture of human RPE with the retina showed that the retina can take up the amino acids, especially glutamate, aspartate, and glutamine, generated from proline nitrogen in the RPE. Intravenous delivery of 15N proline in vivo demonstrated 15N-derived amino acids appear earlier in the RPE before the retina. We also found proline dehydrogenase, the key enzyme in proline catabolism is highly enriched in the RPE but not the retina. The deletion of proline dehydrogenase blocks proline nitrogen utilization in RPE and the import of proline nitrogen-derived amino acids in the retina. Our findings highlight the importance of RPE metabolism in supporting nitrogen sources for the retina, providing insight into understanding the mechanisms of the retinal metabolic ecosystem and RPE-initiated retinal degenerative diseases.

Retinal Metabolic Profile on IMPG2 Deficiency Mice with Subretinal Lesions.

The interphotoreceptor matrix (IPM) is the extracellular matrix between the photoreceptors and the retinal pigment epithelium (RPE). The IPM has two proteoglycans: the IPM proteoglycans 1 and 2 (IMPG1 and IMPG2, respectively). Patients with mutations on IMPG2 develop subretinal vitelliform lesions that affect vision. We previously created an IMPG2 knockout (KO) mice model that generates subretinal lesions similar to those found in humans. These subretinal lesions in IMPG2 KO mice retinas are, in part, composed of mislocalized IMPG1. In addition, IMPG2 KO mice show microscopic IMPG1 material accumulation between the RPE and the photoreceptor outer segments. In this work we discuss the possibility that material accumulation on IMPG2 KO mice retinas affects photoreceptor metabolism. To further investigate this idea, we used targeted metabolomics to profile retinal metabolome on IMPG2 KO mice. The metabolite set enrichment analysis showed reduced glutamate metabolism, urea cycle, and galactose metabolism suggesting affected energy metabolism in mice retinas of IMPG2 KO mice with subretinal lesion.

A nitroalkene derivative of salicylate alleviates diet-induced obesity by activating creatine metabolism and non-shivering thermogenesis.

Obesity-related type II diabetes (diabesity) has increased global morbidity and mortality dramatically. Previously, the ancient drug salicylate demonstrated promise for the treatment of type II diabetes, but its clinical use was precluded due to high dose requirements. In this study, we present a nitroalkene derivative of salicylate, 5-(2-nitroethenyl)salicylic acid (SANA), a molecule with unprecedented beneficial effects in diet-induced obesity (DIO). SANA reduces DIO, liver steatosis and insulin resistance at doses up to 40 times lower than salicylate. Mechanistically, SANA stimulated mitochondrial respiration and increased creatine-dependent energy expenditure in adipose tissue. Indeed, depletion of creatine resulted in the loss of SANA action. Moreover, we found that SANA binds to creatine kinases CKMT1/2, and downregulation CKMT1 interferes with the effect of SANA in vivo. Together, these data demonstrate that SANA is a first-in-class activator of creatine-dependent energy expenditure and thermogenesis in adipose tissue and emerges as a candidate for the treatment of diabesity.

Proline provides a nitrogen source in the retinal pigment epithelium to synthesize and export amino acids for the neural retina.

It is known that metabolic defects in the retinal pigment epithelium (RPE) can cause degeneration of its neighboring photoreceptors in the retina, leading to retinal degenerative diseases such as age-related macular degeneration. However, how RPE metabolism supports the health of the neural retina remains unclear. The retina requires exogenous nitrogen sources for protein synthesis, neurotransmission, and energy metabolism. Using 15N tracing coupled with mass spectrometry, we found human RPE can utilize the nitrogen in proline to produce and export 13 amino acids, including glutamate, aspartate, glutamine, alanine and serine. Similarly, we found this proline nitrogen utilization in the mouse RPE/choroid but not in the neural retina of explant cultures. Co-culture of human RPE with the retina showed that the retina can take up the amino acids, especially glutamate, aspartate and glutamine, generated from proline nitrogen in the RPE. Intravenous delivery of 15N proline in vivo demonstrated 15N-derived amino acids appear earlier in the RPE before the retina. We also found proline dehydrogenase (PRODH), the key enzyme in proline catabolism is highly enriched in the RPE but not the retina. The deletion of PRODH blocks proline nitrogen utilization in RPE and the import of proline nitrogen-derived amino acids in the retina. Our findings highlight the importance of RPE metabolism in supporting nitrogen sources for the retina, providing insight into understanding the mechanisms of the retinal metabolic ecosystem and RPE-initiated retinal degenerative diseases.

Tissue-Specific Sex Difference in Mouse Eye and Brain Metabolome Under Fed and Fasted States.

Purpose: Visual physiology and various ocular diseases demonstrate sexual dimorphisms; however, how sex influences metabolism in different eye tissues remains undetermined. This study aims to address common and tissue-specific sex differences in metabolism in the retina, RPE, lens, and brain under fed and fasted conditions. Methods: After ad libitum fed or being deprived of food for 18 hours, mouse eye tissues (retina, RPE/choroid, and lens), brain, and plasma were harvested for targeted metabolomics. The data were analyzed with both partial least squares-discriminant analysis and volcano plot analysis. Results: Among 133 metabolites that cover major metabolic pathways, we found 9 to 45 metabolites that are sex different in different tissues under the fed state and 6 to 18 metabolites under the fasted state. Among these sex-different metabolites, 33 were changed in 2 or more tissues, and 64 were tissue specific. Pantothenic acid, hypotaurine, and 4-hydroxyproline were the top commonly changed metabolites. The lens and the retina had the most tissue-specific, sex-different metabolites enriched in the metabolism of amino acid, nucleotide, lipids, and tricarboxylic acid cycle. The lens and the brain had more similar sex-different metabolites than other ocular tissues. The female RPE and female brain were more sensitive to fasting with more decreased metabolites in amino acid metabolism, tricarboxylic acid cycles, and glycolysis. The plasma had the fewest sex-different metabolites, with very few overlapping changes with tissues. Conclusions: Sex has a strong influence on eye and brain metabolism in tissue-specific and metabolic state-specific manners. Our findings may implicate the sexual dimorphisms in eye physiology and susceptibility to ocular diseases.

Metabolite Extraction from RPE Cells and Retinas Related to Retinitis Pigmentosa.

The application of metabolomics in ophthalmology helps to identify new biomarkers and elucidate disease mechanisms in different eye diseases, as well as aiding in the development of potential treatment options. Extracting metabolites successfully is essential for potential further analysis using mass spectrometry. In this chapter, we describe how to extract metabolites from a variety of sources including (1) cells on a dish, (2) cell culture medium, and (3) tissues in vivo with and without stable isotope tracers. Samples prepared using this protocol are suitable for a range of downstream mass spectrometry analyses and are stable in solvent for weeks at -80 °C.

Isocitrate dehydrogenase 3b is required for spermiogenesis but dispensable for retinal viability.

Isocitrate dehydrogenase 3 (IDH3) is a key enzyme in the mitochondrial tricarboxylic acid (TCA) cycle, which catalyzes the decarboxylation of isocitrate into α-ketoglutarate and concurrently converts NAD+ into NADH. Dysfunction of IDH3B, the ß subunit of IDH3, has been previously correlated with retinal degeneration and male infertility in humans, but tissue-specific effects of IDH3 dysfunction are unclear. Here, we generated Idh3b-KO mice and found that IDH3B is essential for IDH3 activity in multiple tissues. We determined that loss of Idh3b in mice causes substantial accumulation of isocitrate and its precursors in the TCA cycle, particularly in the testes, whereas the levels of the downstream metabolites remain unchanged or slightly increased. However, the Idh3b-KO mice did not fully recapitulate the defects observed in humans. Global deletion of Idh3b only causes male infertility but not retinal degeneration in mice. Our investigation showed that loss of Idh3b causes an energetic deficit and disrupts the biogenesis of acrosome and flagellum, resulting in spermiogenesis arrestment in sperm cells. Together, we demonstrate that IDH3B controls its substrate levels in the TCA cycle, and it is required for sperm mitochondrial metabolism and spermiogenesis, highlighting the importance of the tissue-specific function of the ubiquitous TCA cycle.

Transketolase in human Müller cells is critical to resist light stress through the pentose phosphate and NRF2 pathways.

The Pentose Phosphate Pathway (PPP), a metabolic offshoot of the glycolytic pathway, provides protective metabolites and molecules essential for cell redox balance and survival. Transketolase (TKT) is the critical enzyme that controls the extent of "traffic flow" through the PPP. Here, we explored the role of TKT in maintaining the health of the human retina. We found that Müller cells were the primary retinal cell type expressing TKT in the human retina. We further explored the role of TKT in human Müller cells by knocking down its expression in primary cultured Müller cells (huPMCs), isolated from the human retina (11 human donors in total), under light-induced oxidative stress. TKT knockdown and light stress reduced TKT enzymatic activities and the overall metabolic activities of huPMCs with no detectable cell death. TKT knockdown restrained the PPP traffic flow, reduced the expression of NAD(P)H Quinone Dehydrogenase 1 (NQO1), impaired the antioxidative response of NRF2 to light stress and aggravated the endoplasmic reticulum (ER) stress. TKT knockdown also inhibited overall glucose intake, reduced expression of Dihydrolipoamide dehydrogenase (DLD) and impaired the energy supply of the huPMCs. In summary, Müller cell-mediated TKT activity plays a critical protective role in the stressed retina. Knockdown of TKT disrupted the PPP and impaired overall glucose utilisation by huPMCs and rendered huPMCs more vulnerable to light stress by impairing energy supply and antioxidative NRF2 responses.

Glucose uptake by GLUT1 in photoreceptors is essential for outer segment renewal and rod photoreceptor survival.

Photoreceptors consume glucose supplied by the choriocapillaris to support phototransduction and outer segment (OS) renewal. Reduced glucose supply underlies photoreceptor cell death in inherited retinal degeneration and age-related retinal disease. We have previously shown that restricting glucose transport into the outer retina by conditional deletion of Slc2a1 encoding GLUT1 resulted in photoreceptor loss and impaired OS renewal. However, retinal neurons, glia, and the retinal pigment epithelium play specialized, synergistic roles in metabolite supply and exchange, and the cell-specific map of glucose uptake and utilization in the retina is incomplete. In these studies, we conditionally deleted Slc2a1 in a pan-retinal or rod-specific manner to better understand how glucose is utilized in the retina. Using non-invasive ocular imaging, electroretinography, and histochemical and biochemical analyses we show that genetic deletion of Slc2a1 from retinal neurons and Müller glia results in reduced OS growth and progressive rod but not cone photoreceptor cell death. Rhodopsin levels were severely decreased even at postnatal day 20 when OS length was relatively normal. Arrestin levels were not changed suggesting that glucose uptake is required to synthesize membrane glycoproteins. Rod-specific deletion of Slc2a1 resulted in similar changes in OS length and rod photoreceptor cell death. These studies demonstrate that glucose is an essential carbon source for rod photoreceptor cell OS maintenance and viability.

Reduced nuclear NAD+ drives DNA damage and subsequent immune activation in the retina.

Mutations in NMNAT1, a key enzyme involved in the synthesis of NAD+ in the nucleus, lead to an early onset severe inherited retinal degeneration (IRD). We aimed to understand the role of nuclear NAD+ in the retina and to identify the molecular mechanisms underlying NMNAT1-associated disease, using a mouse model that harbors the p.V9M mutation in Nmnat1 (Nmnat1V9M/V9M). We identified temporal transcriptional reprogramming in the retinas of Nmnat1V9M/V9M mice prior to retinal degeneration, which begins at 4 weeks of age, with no significant alterations in gene expression at 2 weeks of age and over 2600 differentially expressed genes by 3 weeks of age. Expression of the primary consumer of NAD+ in the nucleus, PARP1, an enzyme involved in DNA damage repair and transcriptional regulation, as well as 7 other PARP family enzymes, was elevated in the retinas of Nmnat1V9M/V9M. This was associated with elevated levels of DNA damage, PARP-mediated NAD+ consumption and migration of Iba1+/CD45+ microglia/macrophages to the subretinal space in the retinas of Nmnat1V9M/V9M mice. These findings suggest that photoreceptor cells are especially sensitive to perturbation of genome homeostasis, and that PARP-mediated cell death may play a role in other genetic forms of IRDs, and potentially other forms of neurodegeneration.

Extracellular matrix dysfunction in Sorsby patient-derived retinal pigment epithelium.

Sorsby Fundus Dystrophy (SFD) is a rare form of macular degeneration that is clinically similar to age-related macular degeneration (AMD), and a histologic hallmark of SFD is a thick layer of extracellular deposits beneath the retinal pigment epithelium (RPE). Previous studies of SFD patient-induced pluripotent stem cell (iPSC) derived RPE differ as to whether these cultures recapitulate this key clinical feature by forming increased drusenoid deposits. The primary purpose of this study is to examine whether SFD patient-derived iPSC-RPE form basal deposits similar to what is found in affected family member SFD globes and to determine whether SFD iPSC RPE may be more oxidatively stressed. We performed a careful comparison of iPSC RPE from three control individuals, multiple iPSC clones from two SFD patients' iPSC RPE, and post-mortem eyes of affected SFD family members. We also examined the effect of CRISPR-Cas9 gene correction of the S204C TIMP3 mutation on RPE phenotype. Finally, targeted metabolomics with liquid chromatography and mass spectrometry analysis and stable isotope-labeled metabolite analysis were performed to determine whether SFD RPE are more oxidatively stressed. We found that SFD iPSC-RPE formed significantly more sub-RPE deposits (∼6-90 µm in height) compared to control RPE at 8 weeks. These deposits were similar in composition to the thick layer of sub-RPE deposits found in SFD family member globes by immunofluorescence staining and TEM imaging. S204C TIMP3 correction by CRISPR-Cas9 gene editing in SFD iPSC RPE cells resulted in significantly reduced basal laminar and sub-RPE calcium deposits. We detected a ∼18-fold increase in TIMP3 accumulation in the extracellular matrix (ECM) of SFD RPE, and targeted metabolomics showed that intracellular 4-hydroxyproline, a major breakdown product of collagen, is significantly elevated in SFD RPE, suggesting increased ECM turnover. Finally, SFD RPE cells have decreased intracellular reduced glutathione and were found to be more vulnerable to oxidative stress. Our findings suggest that elements of SFD pathology can be demonstrated in culture which may lead to insights into disease mechanisms.

A highly conserved zebrafish IMPDH retinal isoform produces the majority of guanine and forms dynamic protein filaments in photoreceptor cells.

Inosine monophosphate dehydrogenase (IMPDH) is a key regulatory enzyme in the de novo synthesis of the purine base guanine. Dominant mutations in human IMPDH1 cause photoreceptor degeneration for reasons that are unknown. Here, we sought to provide some foundational information on Impdh1a in the zebrafish retina. We found that in zebrafish, gene subfunctionalization due to ancestral duplication resulted in a predominant retinal variant expressed exclusively in rod and cone photoreceptors. This variant is structurally and functionally similar to the human IMPDH1 retinal variant and shares a reduced sensitivity to GTP-mediated inhibition. We also demonstrated that Impdh1a forms prominent protein filaments in vitro and in vivo in both rod and cone photoreceptor cell bodies, synapses, and to a lesser degree, in outer segments. These filaments changed length and cellular distribution throughout the day consistent with diurnal changes in both mRNA and protein levels. The loss of Impdh1a resulted in a substantial reduction of guanine levels, although cellular morphology and cGMP levels remained normal. Our findings demonstrate a significant role for IMPDH1 in photoreceptor guanine production and provide fundamental new information on the details of this protein in the zebrafish retina.

Nuclear NAD+-biosynthetic enzyme NMNAT1 facilitates development and early survival of retinal neurons.

Despite mounting evidence that the mammalian retina is exceptionally reliant on proper NAD+ homeostasis for health and function, the specific roles of subcellular NAD+ pools in retinal development, maintenance, and disease remain obscure. Here, we show that deletion of the nuclear-localized NAD+ synthase nicotinamide mononucleotide adenylyltransferase-1 (NMNAT1) in the developing murine retina causes early and severe degeneration of photoreceptors and select inner retinal neurons via multiple distinct cell death pathways. This severe phenotype is associated with disruptions to retinal central carbon metabolism, purine nucleotide synthesis, and amino acid pathways. Furthermore, transcriptomic and immunostaining approaches reveal dysregulation of a collection of photoreceptor and synapse-specific genes in NMNAT1 knockout retinas prior to detectable morphological or metabolic alterations. Collectively, our study reveals previously unrecognized complexity in NMNAT1-associated retinal degeneration and suggests a yet-undescribed role for NMNAT1 in gene regulation during photoreceptor terminal differentiation.

Proline metabolism and transport in retinal health and disease.

The retina is one of the most energy-demanding tissues in the human body. Photoreceptors in the outer retina rely on nutrient support from the neighboring retinal pigment epithelium (RPE), a monolayer of epithelial cells that separate the retina and choroidal blood supply. RPE dysfunction or cell death can result in photoreceptor degeneration, leading to blindness in retinal degenerative diseases including some inherited retinal degenerations and age-related macular degeneration (AMD). In addition to having ready access to rich nutrients from blood, the RPE is also supplied with lactate from adjacent photoreceptors. Moreover, RPE can phagocytose lipid-rich outer segments for degradation and recycling on a daily basis. Recent studies show RPE cells prefer proline as a major metabolic substrate, and they are highly enriched for the proline transporter, SLC6A20. In contrast, dysfunctional or poorly differentiated RPE fails to utilize proline. RPE uses proline to fuel mitochondrial metabolism, synthesize amino acids, build the extracellular matrix, fight against oxidative stress, and sustain differentiation. Remarkably, the neural retina rarely imports proline directly, but it uptakes and utilizes intermediates and amino acids derived from proline catabolism in the RPE. Mutations of genes in proline metabolism are associated with retinal degenerative diseases, and proline supplementation is reported to improve RPE-initiated vision loss. This review will cover proline metabolism in RPE and highlight the importance of proline transport and utilization in maintaining retinal metabolism and health.

Effect of selectively knocking down key metabolic genes in Müller glia on photoreceptor health.

The importance of Müller glia for retinal homeostasis suggests that they may have vulnerabilities that lead to retinal disease. Here, we studied the effect of selectively knocking down key metabolic genes in Müller glia on photoreceptor health. Immunostaining indicated that murine Müller glia expressed insulin receptor (IR), hexokinase 2 (HK2) and phosphoglycerate dehydrogenase (PHGDH) but very little pyruvate dehydrogenase E1 alpha 1 (PDH-E1α) and lactate dehydrogenase A (LDH-A). We crossed Müller glial cell-CreER (MC-CreER) mice with transgenic mice carrying a floxed IR, HK2, PDH-E1α, LDH-A, or PHGDH gene to study the effect of selectively knocking down key metabolic genes in Müller glia cells on retinal health. Selectively knocking down IR, HK2, or PHGDH led to photoreceptor degeneration and reduced electroretinographic responses. Supplementing exogenous l-serine prevented photoreceptor degeneration and improved retinal function in MC-PHGDH knockdown mice. We unexpectedly found that the levels of retinal serine and glycine were not reduced but, on the contrary, highly increased in MC-PHGDH knockdown mice. Moreover, dietary serine supplementation, while rescuing the retinal phenotypes caused by genetic deletion of PHGDH in Müller glial cells, restored retinal serine and glycine homeostasis probably through regulation of serine transport. No retinal abnormalities were observed in MC-CreER mice crossed with PDH-E1α- or LDH-A-floxed mice despite Cre expression. Our findings suggest that Müller glia do not complete glycolysis but use glucose to produce serine to support photoreceptors. Supplementation with exogenous serine is effective in preventing photoreceptor degeneration caused by PHGDH deficiency in Müller glia.

Tracing Nitrogen Metabolism in Mouse Tissues with Gas Chromatography-Mass Spectrometry.

Nitrogen-containing metabolites including ammonia, amino acids, and nucleotides, are essential for cell metabolism, growth, and neural transmission. Nitrogen metabolism is tightly coordinated with carbon metabolism in the breakdown and biosynthesis of amino acids and nucleotides. Both nuclear magnetic resonance spectroscopy and mass spectrometry including gas chromatography-mass spectrometry (GC MS) and liquid chromatography (LC MS) have been used to measure nitrogen metabolism. Here we describe a protocol to trace nitrogen metabolism in multiple mouse tissues using 15N-ammonia coupled with GC MS. This protocol includes detailed procedures in tracer injection, tissue preparation, metabolite extraction, GC MS analysis and natural abundance corrections. This protocol will provide a useful tool to study tissue-specific nitrogen in metabolically active tissues such as the retina, brain, liver, and tumor.

Mutant Nmnat1 leads to a retina-specific decrease of NAD+ accompanied by increased poly(ADP-ribose) in a mouse model of NMNAT1-associated retinal degeneration.

Nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1) is required for nuclear nicotinamide adenine mononucleotide (NAD+) biosynthesis in all nucleated cells, and despite its functional ubiquity, mutations in this gene lead to an isolated retinal degeneration. The mechanisms underlying how mutant NMNAT1 causes disease are not well understood, nor is the reason why the pathology is confined to the retina. Using a mouse model of NMNAT1-associated retinal degeneration that harbors the p.Val9Met mutation, we tested the hypothesis that decreased function of mutant NMNAT1 has a greater effect on the levels of NAD+ in the retina than elsewhere in the body. Measurements by liquid chromatography with tandem mass spectrometry showed an early and sustained decrease of NAD+ in mutant retinas that was not observed in other tissues. To understand how consumers of nuclear NAD+ are affected by the reduced availability of NAD+ in mutant retinas, poly(ADP-ribose) polymerase (PARP) and nuclear sirtuin activity were evaluated. PARP activity was elevated during disease progression, as evidenced by overproduction of poly(ADP-ribose) (PAR) in photoreceptors, whereas histone deacetylation activity of nuclear sirtuins was not altered. We hypothesized that PARP could be activated because of elevated levels of oxidative stress; however, we did not observe oxidative DNA damage, lipid peroxidation, or a low glutathione to oxidized glutathione ratio. Terminal deoxynucleotidyl transferase dUTP nick end labeling staining revealed that photoreceptors appear to ultimately die by apoptosis, although the low NAD+ levels and overproduction of PAR suggest that cell death may include aspects of the parthanatos cell death pathway.