Quinolinic Acid Amyloid-like Fibrillar Assemblies Seed α-Synuclein Aggregation

Quinolinic acid (QA), a downstream neurometabolite in the kynurenine pathway, the biosynthetic pathway of tryptophan, is associated with neurodegenerative diseases pathology. Mutations in genes encoding kynurenine pathway enzymes, which control the level of QA production, are linked with elevated risk of developing Parkinson's disease. Recent findings have revealed the accumulation and deposition of QA in post-mortem samples, as well as in cellular models of Alzheimer's disease and related disorders. Furthermore, intrastriatal inoculation of mice with QA results in increased levels of phosphorylated α-synuclein and neurodegenerative pathological and behavioral characteristics. However, the cellular and molecular mechanisms underlying the involvement of QA accumulation in protein aggregation and neurodegeneration remain elusive. We recently established that self-assembled ordered structures are formed by various metabolites and hypothesized that these “metabolite amyloids” may seed amyloidogenic proteins. Here we demonstrate the formation of QA amyloid-like fibrillar assemblies and seeding of α-synuclein aggregation by these nanostructures both in vitro and in cell culture. Notably, α-synuclein aggregation kinetics was accelerated by an order of magnitude. Additional amyloid-like properties of QA assemblies were demonstrated using thioflavin T assay, powder X-ray diffraction and cell apoptosis analysis. Moreover, fluorescently labeled QA assemblies were internalized by neuronal cells and co-localized with α-synuclein aggregates. In addition, we observed cell-to-cell propagation of fluorescently labeled QA assemblies in a co-culture of treated and untreated cells. Our findings suggest that excess QA levels, due to mutations in the kynurenine pathway, for example, may lead to the formation of metabolite assemblies that seed α-synuclein aggregation, resulting in neuronal toxicity and induction of Parkinson's disease.     

Mammalian cell genetics. 3. Characterization of x-ray-induced forward mutations in Chinese hamster cell cultures

X-irradiation induces forward mutations from 8-azaguanine sensitvity to resistance in Chinese hamster cells in culture. At this locus the number of induced mutations increases non-linearly with X-ray exposure. The mutation rate increase from 4.2·10⁻⁷ per locus per R with 200 R to 1.8·10⁻⁶ per locus per R with 1200 R. Several factors including cell density markedly influence the mutational yield. Reversion tests using specific chemical mutagens on 72 randomly isolated, azaguanine-resistant mutants suggest that both point mutations and chromosome deletions might have occurred in the hamster cells after exposure to ionizing radiation.     

Influence of Age and Strain on Striatal Dopamine Loss in a Genetic Mouse Model of Lesch-Nyhan Disease

Abstract : Lesch-Nyhan disease is a neurogenetic disorder caused by deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT). Affected individuals exhibit a characteristic pattern of neurological and behavioral features attributable in part to dysfunction of basal ganglia dopamine systems. In the current studies, striatal dopamine loss was investigated in five different HPRT-deficient strains of mice carrying one of two different HPRT gene mutations. Caudoputamen dopamine concentrations were significantly reduced in all five of the strains, with deficits ranging from 50.7 to 61.1%. Mesolimbic dopamine was significantly reduced in only three of the five strains, with a range of 31.6-38.6%. The reduction of caudoputamen dopamine was age dependent, emerging between 4 and 12 weeks of age. Tyrosine hydroxylase and aromatic amino acid decarboxylase, two enzymes responsible for the synthesis of dopamine, were reduced by 22.4-37.3 and 22.2-43.1%, respectively. These results demonstrate that HPRT deficiency is strongly associated with a loss of basal ganglia dopamine. The magnitude of dopamine loss measurable is dependent on the genetic background of the mouse strain used, the basal ganglia sub-region examined, and the age of the animals at assessment.     

Nicotinamide phosphoribosyltransferase (NAMPT/PBEF/visfatin) is constitutively released from human hepatocytes

Circulating NAMPT (PBEF/visfatin) has pleiotropic functions and is secreted from adipocytes. Since it is doubtful whether serum levels can be explained by secretion from adipocytes alone, we asked whether hepatocytes are also able to liberate NAMPT. Using HepG2 cells and primary rat and human hepatocytes, release of NAMPT into the cell culture supernatant was found to occur constitutively in a time-dependent manner. In primary human hepatocytes, secretion within 24 h was far higher than the cellular content, but was neither influenced by inhibitors of secretion nor by glucose, insulin or TNFα. As determined by size exclusion chromatography, HepG2 lysates and supernatants primarily contained the dimeric form of NAMPT which exhibited similar in vitro specific enzymatic activity. In primary human hepatocytes, secreted NAMPT was less active. Our results demonstrate that human hepatocytes are a potential source of circulating NAMPT.     

Abnormal purine and pyrimidine nucleotide content in primary astroglia cultures from hypoxanthine-guanine phosphoribosyltransferase-deficient transgenic mice

Lesch-Nyhan syndrome is a pediatric metabolic-neurological syndrome caused by the X-linked deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT). The cause of the metabolic consequences of HGPRT deficiency has been clarified, but the connection between the enzyme deficiency and the neurological manifestations is still unknown. In search for this connection, in the present study, we characterized purine nucleotide metabolism in primary astroglia cultures from HGPRT-deficient transgenic mice. The HGPRT-deficient astroglia exhibited the basic abnormalities in purine metabolism reported before in neurons and various other HGPRT-deficient cells. The following abnormalities were found: absence of detectable uptake of guanine and of hypoxanthine into intact cell nucleotides; 27.8% increase in the availability of 5-phosphoribosyl-1-pyrophosphate; 9.4-fold acceleration of the rate of de novo nucleotide synthesis; manyfold increase in the excretion into the culture media of hypoxanthine (but normal excretion of xanthine); enhanced loss of label from prelabeled adenine nucleotides (loss of 71% in 24 h, in comparison with 52.7% in the normal cells), due to 4.2-fold greater excretion into the media of labeled hypoxanthine. In addition, the HGPRT-deficient astroglia were shown to contain lower cellular levels of ADP, ATP, and GTP, indicating that the accelerated de novo purine synthesis does not compensate adequately for the deficiency of salvage nucleotide synthesis, and higher level of UTP, probably due to enhanced de novo synthesis of pyrimidine nucleotides. Altered nucleotide content in the brain may have a role in the pathogenesis of the neurological deficit in Lesch-Nyhan syndrome.     

Changes in quinolinic acid production and its related enzymes following D-galactosamine and lipopolysaccharide-induced hepatic injury

Increases in quinolinic acid (QUIN), a neurotoxic L-tryptophan metabolite, have been observed in human serum and cerebrospinal fluid and in animal models of severe hepatic injury. The aim of this study was to evaluate the changes in QUIN accumulation and its related enzymes after acute hepatic injury induced by D-galactosamine and endotoxin. Gerbils were given an intraperitoneal injection of pyrogen-free saline alone as control, lipopolysaccharide (LPS) alone (150 ng/kg), D-galactosamine alone (500 mg/kg) or a combination of D-galactosamine with LPS. Concentrations of QUIN, its related metabolites, and related enzyme activities were determined. D-Galactosamine treatment significantly decreased activities of hepatic aminocarboxymuconate-semialdehyde decarboxylase (ACMSDase) resulting in increased QUIN concentrations in serum and tissues. The magnitude of QUIN responses was markedly increased by endotoxin due to the increased availability of L-kynurenine, a rate-limiting substrate for QUIN synthesis. Further, infiltration of monocytes/macrophages, which is a possible major source of QUIN production in the liver, was shown by immunohistochemistry after hepatic injury induced by D-galactosamine and endotoxin. Increased serum QUIN concentrations are probably due to the increased substrate availability and the decreased activity of aminocarboxymuconate-semialdehyde decarboxylase in the liver, accompanying the increased monocyte/macrophage infiltration into the liver after hepatic injury.     

Suppression of intestinal polyposis in Apcmin/+ mice by targeting the nitric oxide or poly(ADP-ribose) pathways

Mutant frequency at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene in the peripheral blood lymphocytes obtained from 44 healthy individuals (23 non-smokers and 21 smokers) of an Indian male population was studied using T-lymphocyte cloning assay. It was found that ln MF increased with age at a rate of 2.5% per year (P < 0.001). Blood samples from smokers showed a significant (P < 0.037) increase in HPRT mutant frequency (MF) (10.43 ± 4.74 × 10⁻⁶) as compared to that obtained from non-smokers (7.69 ± 3.69 × 10⁻⁶). This study also showed a significant (P < 0.027) inverse correlation between ln MF and non-selected cloning efficiency (CE). However, with respect to age no variation was observed in cloning efficiency. The results obtained in this study showed a good comparison with those reported in different populations of the world.     

Chemical diagnosis of Lesch-Nyhan syndrome using gas chromatography-mass spectrometry detection

Lesch-Nyhan syndrome (LNS) is caused by a severe deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT) and clinically characterized by self-injurious behavior and nephrolithiasis; the latter is treatable with allopurinol, an inhibitor of xanthine oxidase which converts xanthine and hypoxanthine into uric acid. In the HPRT gene, more than 200 different mutations are known, and de novo mutation occurs at a high rate. Thus, there is a great need to develop a highly specific method to detect patients with HPRT dysfunction by quantifying the metabolites related to this enzyme. A simplified urease pretreatment of urine, gas chromatography-mass spectrometry, and stable isotope dilution method, developed for cutting-edge metabonomics, was further applied to quantify hypoxanthine, xanthine, urate, guanine and adenine in 100 microl or less urine or eluate from filter-paper-urine strips by additional use of stable isotope labeled guanine and adenine as the internal standards. In this procedure, the recoveries were above 93% and linearities (r(2)=0.9947-1.000) and CV values (below 7%) of the indicators were satisfactory. In four patients with proven LNS, hypoxanthine was elevated to 8.4-9.0 SD above the normal mean, xanthine to 4-6 SD above the normal mean, guanine to 1.9-3.7 SD, and adenine was decreased. Because of the allopurinol treatment for all the four patients, their level of urate was not elevated, orotate increased, and uracil was unchanged as compared with the control value. It was concluded that even in the presence of treatment with allopurinol, patients with LNS can be chemically diagnosed by this procedure. Abnormality in the levels of hypoxanthine and xanthine was quite prominent and n, the number of standard deviations above the normal mean, combined for the two, was above 12.9.     

Crystal structures of free,IMP-, and GMP-bound Escherichia coli hypoxanthine phosphoribosyltransferase

Crystal structures have been determined for free Escherichia coli hypoxanthine phosphoribosyltransferase (HPRT) (2.9 A resolution) and for the enzyme in complex with the reaction products, inosine 5'-monophosphate (IMP) and guanosine 5'-monophosphate (GMP) (2.8 A resolution). Of the known 6-oxopurine phosphoribosyltransferase (PRTase) structures, E. coli HPRT is most similar in structure to that of Tritrichomonas foetus HGXPRT, with a rmsd for 150 Calpha atoms of 1.0 A. Comparison of the free and product bound structures shows that the side chain of Phe156 and the polypeptide backbone in this vicinity move to bind IMP or GMP. A nonproline cis peptide bond, also found in some other 6-oxopurine PRTases, is observed between Leu46 and Arg47 in both the free and complexed structures. For catalysis to occur, the 6-oxopurine PRTases have a requirement for divalent metal ion, usually Mg(2+) in vivo. In the free structure, a Mg(2+) is coordinated to the side chains of Glu103 and Asp104. This interaction may be important for stabilization of the enzyme before catalysis. E. coli HPRT is unique among the known 6-oxopurine PRTases in that it exhibits a marked preference for hypoxanthine as substrate over both xanthine and guanine. The structures suggest that its substrate specificity is due to the modes of binding of the bases. In E. coli HPRT, the carbonyl oxygen of Asp163 would likely form a hydrogen bond with the 2-exocyclic nitrogen of guanine (in the HPRT-guanine-PRib-PP-Mg(2+) complex). However, hypoxanthine does not have a 2-exocyclic atom and the HPRT-IMP structure suggests that hypoxanthine is likely to occupy a different position in the purine-binding pocket.     

Failure of caffeine to influence induced mutation frequencies and the independence of cell killing and mutation induction in V79 Chinese hamster cells

Using V79 Chinese hamster cells and replating assay, no effect of caffeine post-treatment on spontaneous or UV- or EMS-induced mutation frequencies to 8-azaguanine resistance was demonstrable. However, considerable potentiation of cell killing was observed. Previous reports that caffeine enhances induced mutation frequencies are explained by an artefact in the in situ method used; a similar artefact may also explain the cumulative in situ mutation dose-responde curves. Furthermore, the relationship between mutation induction and dose has been shown to be qualitatively distinct from that between cell killing an dose. These differences suggest that cell killing and mutation induction are mediated via independent mechanisms and that pre-mutational lesions may be qualitatively distinct from pre-lethal lesions.