AGC2 (Citrin) Deficiency-From Recognition of the Disease till Construction of Therapeutic Procedures.

Can you imagine a disease in which intake of an excess amount of sugars or carbohydrates causes hyperammonemia? It is hard to imagine the intake causing hyperammonemia. AGC2 or citrin deficiency shows their symptoms following sugar/carbohydrates intake excess and this disease is now known as a pan-ethnic disease. AGC2 (aspartate glutamate carrier 2) or citrin is a mitochondrial transporter which transports aspartate (Asp) from mitochondria to cytosol in exchange with glutamate (Glu) and H+. Asp is originally supplied from mitochondria to cytosol where it is necessary for synthesis of proteins, nucleotides, and urea. In cytosol, Asp can be synthesized from oxaloacetate and Glu by cytosolic Asp aminotransferase, but oxaloacetate formation is limited by the amount of NAD+. This means an increase in NADH causes suppression of Asp formation in the cytosol. Metabolism of carbohydrates and other substances which produce cytosolic NADH such as alcohol and glycerol suppress oxaloacetate formation. It is forced under citrin deficiency since citrin is a member of malate/Asp shuttle. In this review, we will describe history of identification of the SLC25A13 gene as the causative gene for adult-onset type II citrullinemia (CTLN2), a type of citrin deficiency, pathophysiology of citrin deficiency together with animal models and possible treatments for citrin deficiency newly developing.

mRNA Therapy Improves Metabolic and Behavioral Abnormalities in a Murine Model of Citrin Deficiency.

Citrin deficiency is an autosomal recessive disorder caused by loss-of-function mutations in SLC25A13, encoding the liver-specific mitochondrial aspartate/glutamate transporter. It has a broad spectrum of clinical phenotypes, including life-threatening neurological complications. Conventional protein replacement therapy is not an option for these patients because of drug delivery hurdles, and current gene therapy approaches (e.g., AAV) have been hampered by immunogenicity and genotoxicity. Although dietary approaches have shown some benefits in managing citrin deficiency, the only curative treatment option for these patients is liver transplantation, which is high-risk and associated with long-term complications because of chronic immunosuppression. To develop a new class of therapy for citrin deficiency, codon-optimized mRNA encoding human citrin (hCitrin) was encapsulated in lipid nanoparticles (LNPs). We demonstrate the efficacy of hCitrin-mRNA-LNP therapy in cultured human cells and in a murine model of citrin deficiency that resembles the human condition. Of note, intravenous (i.v.) administration of the hCitrin-mRNA resulted in a significant reduction in (1) hepatic citrulline and blood ammonia levels following oral sucrose challenge and (2) sucrose aversion, hallmarks of hCitrin deficiency. In conclusion, mRNA-LNP therapy could have a significant therapeutic effect on the treatment of citrin deficiency and other mitochondrial enzymopathies with limited treatment options.

Pivotal role of inter-organ aspartate metabolism for treatment of mitochondrial aspartate-glutamate carrier 2 (citrin) deficiency, based on the mouse model.

Previous studies using citrin/mitochondrial glycerol-3-phosphate (G3P) dehydrogenase (mGPD) double-knockout mice have demonstrated that increased dietary protein reduces the extent of carbohydrate-induced hyperammonemia observed in these mice. This study aimed to further elucidate the mechanisms of this effect. Specific amino acids were initially found to decrease hepatic G3P, or increase aspartate or citrulline levels, in mGPD-knockout mice administered ethanol. Unexpectedly, oral glycine increased ammonia in addition to lowering G3P and increasing citrulline. Subsequently, simultaneous glycine-plus-sucrose (Gly + Suc) administration led to a more severe hyperammonemic state in double-KO mice compared to sucrose alone. Oral arginine, ornithine, aspartate, alanine, glutamate and medium-chain triglycerides all lowered blood ammonia following Gly + Suc administration, with combinations of ornithine-plus-aspartate (Orn + Asp) or ornithine-plus-alanine (Orn + Ala) suppressing levels similar to wild-type. Liver perfusion and portal vein-arterial amino acid differences suggest that oral aspartate, similar to alanine, likely activated ureagenesis from ammonia and lowered the cytosolic NADH/NAD+ ratio through conversion to alanine in the small intestine. In conclusion, Gly + Suc administration induces a more severe hyperammonemic state in double-KO mice that Orn + Asp or Orn + Ala both effectively suppress. Aspartate-to-alanine conversion in the small intestine allows for effective oral administration of either, demonstrating a pivotal role of inter-organ aspartate metabolism for the treatment of citrin deficiency.

Ubiquitous distribution of fluorescent protein in muscles of four species and two subspecies of eel (genus Anguilla).

In this study, the localization of fluorescent protein (FP) was characterized in the muscles of four species and two subspecies of eels Anguilla anguilla, A. australis, A. bicolor bicolor (b.), A. bicolor pacifica (p.) and A. mossambica in addition to the previously reported A. japonica. The open reading frame of each eel FP was 417 bp encoding 139 amino acid residues. The deduced amino acid sequences among the four species and two subspecies exhibited 91.4-100% identity, and belonged to the fatty-acid-binding protein (FABP) family. The gene structure of eel FPs in A. japonica, A. anguilla, A. australis, A. bicolor b., A. bicolor p. and A. mossambica have four exons and three introns, and were common to that of FABP family. The apo eel FPs expressed by Escherichia coli with recombinant eel FP genes were analysed for the fluorescent properties in the presence of bilirubin. The excitation and emission spectra of holo eel FPs had the maximum wavelengths of 490-496 and 527-530 nm, respectively. The holo eel FPs indicated that the fluorescent intensities were stronger in A. japonica and A. bicolor than in A. mossambica, A. australis and A. anguilla. The comparison of amino acid sequences revealed two common substitutions in A. mossambica, A. australis and A. anguilla with weak fluorescent intensity.

Eel green fluorescent protein is associated with resistance to oxidative stress.

Green fluorescent protein (GFP) from eel (Anguilla japonica) muscle (eelGFP) is unique in the vertebrates and requires bilirubin as a ligand to emit fluorescence. This study was performed to clarify the physiological function of the unique GFP. Investigation of susceptibility to oxidative stress was carried out using three types of cell lines including jellyfish (Aequorea coerulescens) GFP (jfGFP)-, or eel GFP (eelGFP)-expressing HEK293 cells, and control vector-transfected HEK293 cells. Binding of eelGFP to bilirubin was confirmed by the observation of green fluorescence in HEK293-eelGFP cells. The growth rate was compared with the three types of cells in the presence or absence of phenol red which possessed antioxidant activity. The growth rates of HEK293-CV and HEK293-jfGFP under phenol red-free conditions were reduced to 52 and 31% of those under phenol red. Under the phenol red-free condition, HEK293-eelGFP had a growth rate of approximately 70% of the phenol red-containing condition. The eelGFP-expressing cells were approximately 2-fold resistant to oxidative stress such as H2O2 exposure. The fluorescence intensity partially decreased or disappeared after exposure to H2O2, and heterogeneous intensity of fluorescence was also observed in isolated eel skeletal muscle cells. These results suggested eelGFP, but not jfGFP, coupled with bilirubin provided the antioxidant activity to the cells as compared to non-bound free bilirubin.

Lysosomal localization of Japanese medaka (Oryzias latipes) Neu1 sialidase and its highly conserved enzymatic profiles with human.

Desialylation in the lysosome is a crucial step for glycoprotein degradation. The abnormality of lysosomal desialylation by NEU1 sialidase is involved in diseases of mammals such as sialidosis and galactosialidosis. Mammalian Neu1 sialidase is also localized at plasma membrane where it regulates several signaling pathways through glycoprotein desialylation. In fish, on the other hand, the mechanism of desialylation in the lysosome and functions of Neu1 sialidase are still unclear. Here, to understand the significance of fish Neu1 sialidase, neu1 gene was cloned from medaka brain and the profiles of its polypeptides were analyzed. Open reading frame of medaka neu1 consisted 1,182 bp and the similarity of its deduced amino acids with human NEU1 was 57%. As this recombinant polypeptide did not show significant sialidase activity, medaka cathepsin A, known in mammals as protective protein activating Neu1, was cloned and then co-expressed with medaka Neu1 to examine whether medaka cathepsin A activates Neu1 activity. As a result, Neu1/cathepsin A showed a drastic increase of sialidase activity toward MU-NANA. Major substrate of medaka Neu1 was 3-sialyllactose and its optimal pH was 4.0. With immunofluorescence analysis, signal of overexpressed medaka Neu1 was found to coincide with Lysotracker signals (organelle marker of lysosome) and co-localized with medaka cathepsin A in fish hepatic Hepa-T1 cells. Furthermore, part of medaka Neu1 was also detected at plasma membrane. Medaka Neu1 possessed signal peptide sequence at N-terminal and incomplete lysosomal targeting sequence at C-terminus. Medaka neu1 gene was ubiquitously expressed in various medaka tissues, and its expression level was significantly higher than other sialidase genes such as neu3a, neu3b and neu4. The present study revealed the profiles of fish Neu1 sialidase and indicated its high conservation with human NEU1 for the first time, suggesting the presence of similar desialylation system in the medaka lysosome to human. Moreover, the present study showed the possibility of medaka as a model animal of human NEU1 sialidase.

Cytochrome P450 1C1 complementary DNA cloning, sequence analysis and constitutive expression induced by benzo-a-pyrene in Nile tilapia (Oreochromis niloticus).

CYP1C is the newest member of the CYP1 family of P450s; however, its physiological significance, inducers, and metabolic functions are unknown. In this study, a new complementary DNA of the CYP1C subfamily encoding CYP1C1 was isolated from Nile tilapia (Oreochromis niloticus) liver after intracoelomic injection with benzo-a-pyrene (BaP). The full-length cDNA was 2223 base pair (bp) long and contained an open reading frame of 1581 bp encoding a protein of 526 amino acids and a stop codon. The sequence exhibited 3' non-coding region of 642 bp. The deduced amino acid sequence of O. niloticus CYP1C1 shows similarities of 86, 82.5, 79.7, 78.7, 77.8, 75.5, 69.6 and 61.3% with scup CYP1C1, killifish CYP1C1,1C2, Japanese eel CYP1C1, zebra fish CYP1C1, common carp CYP1C1, scup CYP1C2, common carp CYP1C2 and zebra fish CYP1C2, respectively. Phylogenetic tree based on the amino acids sequences clearly shows tilapia CYP1C1 and scup CYP1C1 to be more closely related to each other than to CYP1C genes from other species. Furthermore, for measuring BaP induction of CYP1C1 mRNA in different organs of tilapia (O. niloticus), ß-actin gene as internal control was selected based on previous studies to assess their expression variability. Real time RCR results revealed that there was a large increase in CYP1C1 mRNA in liver (43.1), intestine (5.1) and muscle (2.4).