A new physiological medium uncovers biochemical and cellular alterations in Lesch-Nyhan disease fibroblasts.

BACKGROUND: Lesch-Nyhan disease (LND) is a severe neurological disorder caused by the genetic deficiency of hypoxanthine-guanine phosphoribosyltransferase (HGprt), an enzyme involved in the salvage synthesis of purines. To compensate this deficiency, there is an acceleration of the de novo purine biosynthetic pathway. Most studies have failed to find any consistent abnormalities of purine nucleotides in cultured cells obtained from the patients. Recently, it has been shown that 5-aminoimidazole-4-carboxamide riboside 5'-monophosphate (ZMP), an intermediate of the de novo pathway, accumulates in LND fibroblasts maintained with RPMI containing physiological levels (25 nM) of folic acid (FA), which strongly differs from FA levels of regular cell culture media (2200 nM). However, RPMI and other standard media contain non-physiological levels of many nutrients, having a great impact in cell metabolism that does not precisely recapitulate the in vivo behavior of cells. METHODS: We prepared a new culture medium containing physiological levels of all nutrients, including vitamins (Plasmax-PV), to study the potential alterations of LND fibroblasts that may have been masked by the usage of non-physiological media. We quantified ZMP accumulation under different culture conditions and evaluated the activity of two known ZMP-target proteins (AMPK and ADSL), the mRNA expression of the folate carrier SLC19A1, possible mitochondrial alterations and functional consequences in LND fibroblasts. RESULTS: LND fibroblasts maintained with Plasmax-PV show metabolic adaptations such a higher glycolytic capacity, increased expression of the folate carrier SCL19A1, and functional alterations such a decreased mitochondrial potential and reduced cell migration compared to controls. These alterations can be reverted with high levels of folic acid, suggesting that folic acid supplements might be a potential treatment for LND. CONCLUSIONS: A complete physiological cell culture medium reveals new alterations in Lesch-Nyhan disease. This work emphasizes the importance of using physiological cell culture conditions when studying a metabolic disorder.

Purine Nucleotide Alterations in Tumoral Cell Lines Maintained with Physiological Levels of Folic Acid.

Most cancer cells have an increased synthesis of purine nucleotides to fulfil their enhanced division rate. The de novo synthesis of purines requires folic acid in the form of N10-formyltetrahydrofolate (10-formyl-THF). However, regular cell culture media contain very high, non-physiological concentrations of folic acid, which may have an impact on cell metabolism. Using cell culture media with physiological levels of folic acid (25 nM), we uncover purine alterations in several human cell lines. HEK293T, Jurkat, and A549 cells accumulate 5'-aminoimidazole-4-carboxamide ribonucleotide (ZMP), an intermediary of the de novo biosynthetic pathway, at physiological levels of folic acid, but not with the artificially high levels (2200 nM) present in regular media. Interestingly, HEK293T and Jurkat cells do not accumulate high levels of ZMP when AICAr, the precursor of ZMP, is added to medium containing 2200 nM folate; instead, ATP levels are increased, suggesting an enhanced de novo synthesis. On the other hand, HeLa and EHEB cells do not accumulate ZMP at physiological levels of folic acid, but they do accumulate in medium containing AICAr plus 2200 nM folate. Expression of SLC19A1, which encodes the reduced folate carrier (RFC), is increased in HEK293T and Jurkat cells compared with HeLa and EHEB, and it is correlated with the total purine nucleotide content at high levels of folic acid or with ZMP accumulation at physiological levels of folic acid. In conclusion, tumoral cell lines show a heterogenous response to folate changes in the media, some of them accumulating ZMP at physiological levels of folic acid. Further research is needed to clarify the ZMP downstream targets and their impact on cell function.

A fraction of barrier-to-autointegration factor (BAF) associates with centromeres and controls mitosis progression.

Barrier-to-Autointegration Factor (BAF) is a conserved nuclear envelope (NE) component that binds chromatin and helps its anchoring to the NE. Cycles of phosphorylation and dephosphorylation control BAF function. Entering mitosis, phosphorylation releases BAF from chromatin and facilitates NE-disassembly. At mitotic exit, PP2A-mediated dephosphorylation restores chromatin binding and nucleates NE-reassembly. Here, we show that in Drosophila a small fraction of BAF (cenBAF) associates with centromeres. We also find that PP4 phosphatase, which is recruited to centromeres by CENP-C, prevents phosphorylation and release of cenBAF during mitosis. cenBAF is necessary for proper centromere assembly and accurate chromosome segregation, being critical for mitosis progression. Disrupting cenBAF localization prevents PP2A inactivation in mitosis compromising global BAF phosphorylation, which in turn leads to its persistent association with chromatin, delays anaphase onset and causes NE defects. These results suggest that, together with PP4 and CENP-C, cenBAF forms a centromere-based mechanism that controls chromosome segregation and mitosis progression.

The zinc-finger proteins WOC and ROW play distinct functions within the HP1c transcription complex.

In Drosophila, the Heterochromatin Protein 1c (HP1c) forms a transcriptional complex with the zinc-finger proteins WOC and ROW, and the extraproteasomal ubiquitin receptor Dsk2. This complex localizes at promoters of active genes and it is required for transcription. The functions played by the different components of the HP1c complex are not fully understood. In this study we show that WOC and ROW are required for chromatin binding of both Dsk2 and HP1c. However, while impairing chromatin binding strongly destabilizes HP1c, it does not affect Dsk2 stability. We also show that WOC, but not ROW, is required for nuclear localization of Dsk2. Moreover, WOC and Dsk2 co-immunoprecitate upon ROW depletion. These results suggest that WOC and Dsk2 interact to form a subcomplex that mediates nuclear translocation of Dsk2. We also show that ROW mediates chromatin binding of the WOC/Dsk2 subcomplex, as well as of HP1c. Altogether these observations favor a model by which the interaction with WOC recruits Dsk2 to the HP1c complex that, in its turn, binds chromatin in a ROW-dependent manner.

Fluorescently-labelled CPD and 6-4PP photolyases: new tools for live-cell DNA damage quantification and laser-assisted repair.

UV light induces cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts (6-4PPs), which can result in carcinogenesis and aging, if not properly repaired by nucleotide excision repair (NER). Assays to determine DNA damage load and repair rates are invaluable tools for fundamental and clinical NER research. However, most current assays to quantify DNA damage and repair cannot be performed in real time. To overcome this limitation, we made use of the damage recognition characteristics of CPD and 6-4PP photolyases (PLs). Fluorescently-tagged PLs efficiently recognize UV-induced DNA damage without blocking NER activity, and therefore can be used as sensitive live-cell damage sensors. Importantly, FRAP-based assays showed that PLs bind to damaged DNA in a highly sensitive and dose-dependent manner, and can be used to quantify DNA damage load and to determine repair kinetics in real time. Additionally, PLs can instantly reverse DNA damage by 405 nm laser-assisted photo-reactivation during live-cell imaging, opening new possibilities to study lesion-specific NER dynamics and cellular responses to damage removal. Our results show that fluorescently-tagged PLs can be used as a versatile tool to sense, quantify and repair DNA damage, and to study NER kinetics and UV-induced DNA damage response in living cells.

Novel Candidate Genes and a Wide Spectrum of Structural and Point Mutations Responsible for Inherited Retinal Dystrophies Revealed by Exome Sequencing.

BACKGROUND: NGS-based genetic diagnosis has completely revolutionized the human genetics field. In this study, we have aimed to identify new genes and mutations by Whole Exome Sequencing (WES) responsible for inherited retinal dystrophies (IRD). METHODS: A cohort of 33 pedigrees affected with a variety of retinal disorders was analysed by WES. Initial prioritization analysis included around 300 IRD-associated genes. In non-diagnosed families a search for pathogenic mutations in novel genes was undertaken. RESULTS: Genetic diagnosis was attained in 18 families. Moreover, a plausible candidate is proposed for 10 more cases. Two thirds of the mutations were novel, including 4 chromosomal rearrangements, which expand the IRD allelic heterogeneity and highlight the contribution of private mutations. Our results prompted clinical re-evaluation of some patients resulting in assignment to a syndromic instead of non-syndromic IRD. Notably, WES unveiled four new candidates for non-syndromic IRD: SEMA6B, CEP78, CEP250, SCLT1, the two latter previously associated to syndromic disorders. We provide functional data supporting that missense mutations in CEP250 alter cilia formation. CONCLUSION: The diagnostic efficiency of WES, and strictly following the ACMG/AMP criteria is 55% in reported causative genes or functionally supported new candidates, plus 30% families in which likely pathogenic or VGUS/VUS variants were identified in plausible candidates. Our results highlight the clinical utility of WES for molecular diagnosis of IRD, provide a wider spectrum of mutations and concomitant genetic variants, and challenge our view on syndromic vs non-syndromic, and causative vs modifier genes.