Mathematical kinetic modelling followed by in vitro and in vivo assays reveal the bifunctional rice GTPCHII/DHBPS enzymes and demonstrate the key roles of OsRibA proteins in the vitamin B2 pathway.

BACKGROUND: Riboflavin is the precursor of several cofactors essential for normal physical and cognitive development, but only plants and some microorganisms can produce it. Humans thus rely on their dietary intake, which at a global level is mainly constituted by cereals (> 50%). Understanding the riboflavin biosynthesis players is key for advancing our knowledge on this essential pathway and can hold promise for biofortification strategies in major crop species. In some bacteria and in Arabidopsis, it is known that RibA1 is a bifunctional protein with distinct GTP cyclohydrolase II (GTPCHII) and 3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS) domains. Arabidopsis harbors three RibA isoforms, but only one retained its bifunctionality. In rice, however, the identification and characterization of RibA has not yet been described. RESULTS: Through mathematical kinetic modeling, we identified RibA as the rate-limiting step of riboflavin pathway and by bioinformatic analysis we confirmed that rice RibA proteins carry both domains, DHBPS and GTPCHII. Phylogenetic analysis revealed that OsRibA isoforms 1 and 2 are similar to Arabidopsis bifunctional RibA1. Heterologous expression of OsRibA1 completely restored the growth of the rib3∆ yeast mutant, lacking DHBPS expression, while causing a 60% growth improvement of the rib1∆ mutant, lacking GTPCHII activity. Regarding OsRibA2, its heterologous expression fully complemented GTPCHII activity, and improved rib3∆ growth by 30%. In vitro activity assays confirmed that both OsRibA1 and OsRibA2 proteins carry GTPCHII/DHBPS activities, but that OsRibA1 has higher DHBPS activity. The overexpression of OsRibA1 in rice callus resulted in a 28% increase in riboflavin content. CONCLUSIONS: Our study elucidates the critical role of RibA in rice riboflavin biosynthesis pathway, establishing it as the rate-limiting step in the pathway. By identifying and characterizing OsRibA1 and OsRibA2, showcasing their GTPCHII and DHBPS activities, we have advanced the understanding of riboflavin biosynthesis in this staple crop. We further demonstrated that OsRibA1 overexpression in rice callus increases its riboflavin content, providing supporting information for bioengineering efforts.

The metabolite BH4 controls T cell proliferation in autoimmunity and cancer.

Genetic regulators and environmental stimuli modulate T cell activation in autoimmunity and cancer. The enzyme co-factor tetrahydrobiopterin (BH4) is involved in the production of monoamine neurotransmitters, the generation of nitric oxide, and pain1,2. Here we uncover a link between these processes, identifying a fundamental role for BH4 in T cell biology. We find that genetic inactivation of GTP cyclohydrolase 1 (GCH1, the rate-limiting enzyme in the synthesis of BH4) and inhibition of sepiapterin reductase (the terminal enzyme in the synthetic pathway for BH4) severely impair the proliferation of mature mouse and human T cells. BH4 production in activated T cells is linked to alterations in iron metabolism and mitochondrial bioenergetics. In vivo blockade of BH4 synthesis abrogates T-cell-mediated autoimmunity and allergic inflammation, and enhancing BH4 levels through GCH1 overexpression augments responses by CD4- and CD8-expressing T cells, increasing their antitumour activity in vivo. Administration of BH4 to mice markedly reduces tumour growth and expands the population of intratumoral effector T cells. Kynurenine-a tryptophan metabolite that blocks antitumour immunity-inhibits T cell proliferation in a manner that can be rescued by BH4. Finally, we report the development of a potent SPR antagonist for possible clinical use. Our data uncover GCH1, SPR and their downstream metabolite BH4 as critical regulators of T cell biology that can be readily manipulated to either block autoimmunity or enhance anticancer immunity.

GCH1 Deficiency Activates Brain Innate Immune Response and Impairs Tyrosine Hydroxylase Homeostasis.

The Parkinson's disease (PD) risk gene GTP cyclohydrolase 1 (GCH1) catalyzes the rate-limiting step in tetrahydrobiopterin (BH4) synthesis, an essential cofactor in the synthesis of monoaminergic neurotransmitters. To investigate the mechanisms by which GCH1 deficiency may contribute to PD, we generated a loss of function zebrafish gch1 mutant (gch1-/-), using CRISPR/Cas technology. gch1-/- zebrafish develop marked monoaminergic neurotransmitter deficiencies by 5 d postfertilization (dpf), movement deficits by 8 dpf and lethality by 12 dpf. Tyrosine hydroxylase (Th) protein levels were markedly reduced without loss of ascending dopaminergic (DAergic) neurons. L-DOPA treatment of gch1-/- larvae improved survival without ameliorating the motor phenotype. RNAseq of gch1-/- larval brain tissue identified highly upregulated transcripts involved in innate immune response. Subsequent experiments provided morphologic and functional evidence of microglial activation in gch1-/- The results of our study suggest that GCH1 deficiency may unmask early, subclinical parkinsonism and only indirectly contribute to neuronal cell death via immune-mediated mechanisms. Our work highlights the importance of functional validation for genome-wide association studies (GWAS) risk factors and further emphasizes the important role of inflammation in the pathogenesis of PD.SIGNIFICANCE STATEMENT Genome-wide association studies have now identified at least 90 genetic risk factors for sporadic Parkinson's disease (PD). Zebrafish are an ideal tool to determine the mechanistic role of genome-wide association studies (GWAS) risk genes in a vertebrate animal model. The discovery of GTP cyclohydrolase 1 (GCH1) as a genetic risk factor for PD was counterintuitive, GCH1 is the rate-limiting enzyme in the synthesis of dopamine (DA), mutations had previously been described in the non-neurodegenerative movement disorder dopa-responsive dystonia (DRD). Rather than causing DAergic cell death (as previously hypothesized by others), we now demonstrate that GCH1 impairs tyrosine hydroxylase (Th) homeostasis and activates innate immune mechanisms in the brain and provide evidence of microglial activation and phagocytic activity.

Endothelial cell-specific roles for tetrahydrobiopterin in myocardial function, cardiac hypertrophy, and response to myocardial ischemia-reperfusion injury.

The cofactor tetrahydrobiopterin (BH4) is a critical regulator of nitric oxide synthase (NOS) function and redox signaling, with reduced BH4 implicated in multiple cardiovascular disease states. In the myocardium, augmentation of BH4 levels can impact on cardiomyocyte function, preventing hypertrophy and heart failure. However, the specific role of endothelial cell BH4 biosynthesis in the coronary circulation and its role in cardiac function and the response to ischemia has yet to be elucidated. Endothelial cell-specific Gch1 knockout mice were generated by crossing Gch1fl/fl with Tie2cre mice, generating Gch1fl/flTie2cre mice and littermate controls. GTP cyclohydrolase protein and BH4 levels were reduced in heart tissues from Gch1fl/flTie2cre mice, localized to endothelial cells, with normal cardiomyocyte BH4. Deficiency in coronary endothelial cell BH4 led to NOS uncoupling, decreased NO bioactivity, and increased superoxide and hydrogen peroxide productions in the hearts of Gch1fl/flTie2cre mice. Under physiological conditions, loss of endothelial cell-specific BH4 led to mild cardiac hypertrophy in Gch1fl/flTie2cre hearts. Endothelial cell BH4 loss was also associated with increased neuronal NOS protein, loss of endothelial NOS protein, and increased phospholamban phosphorylation at serine-17 in cardiomyocytes. Loss of cardiac endothelial cell BH4 led to coronary vascular dysfunction, reduced functional recovery, and increased myocardial infarct size following ischemia-reperfusion injury. Taken together, these studies reveal a specific role for endothelial cell Gch1/BH4 biosynthesis in cardiac function and the response to cardiac ischemia-reperfusion injury. Targeting endothelial cell Gch1 and BH4 biosynthesis may provide a novel therapeutic target for the prevention and treatment of cardiac dysfunction and ischemia-reperfusion injury.NEW & NOTEWORTHY We demonstrate a critical role for endothelial cell Gch1/BH4 biosynthesis in coronary vascular function and cardiac function. Loss of cardiac endothelial cell BH4 leads to coronary vascular dysfunction, reduced functional recovery, and increased myocardial infarct size following ischemia/reperfusion injury. Targeting endothelial cell Gch1 and BH4 biosynthesis may provide a novel therapeutic target for the prevention and treatment of cardiac dysfunction, ischemia injury, and heart failure.

Genetic diversity of the Plasmodium falciparum GTP-cyclohydrolase 1, dihydrofolate reductase and dihydropteroate synthetase genes reveals new insights into sulfadoxine-pyrimethamine antimalarial drug resistance.

Plasmodium falciparum parasites resistant to antimalarial treatments have hindered malaria disease control. Sulfadoxine-pyrimethamine (SP) was used globally as a first-line treatment for malaria after wide-spread resistance to chloroquine emerged and, although replaced by artemisinin combinations, is currently used as intermittent preventive treatment of malaria in pregnancy and in young children as part of seasonal malaria chemoprophylaxis in sub-Saharan Africa. The emergence of SP-resistant parasites has been predominantly driven by cumulative build-up of mutations in the dihydrofolate reductase (pfdhfr) and dihydropteroate synthetase (pfdhps) genes, but additional amplifications in the folate pathway rate-limiting pfgch1 gene and promoter, have recently been described. However, the genetic make-up and prevalence of those amplifications is not fully understood. We analyse the whole genome sequence data of 4,134 P. falciparum isolates across 29 malaria endemic countries, and reveal that the pfgch1 gene and promoter amplifications have at least ten different forms, occurring collectively in 23% and 34% in Southeast Asian and African isolates, respectively. Amplifications are more likely to be present in isolates with a greater accumulation of pfdhfr and pfdhps substitutions (median of 1 additional mutations; P<0.00001), and there was evidence that the frequency of pfgch1 variants may be increasing in some African populations, presumably under the pressure of SP for chemoprophylaxis and anti-folate containing antibiotics used for the treatment of bacterial infections. The selection of P. falciparum with pfgch1 amplifications may enhance the fitness of parasites with pfdhfr and pfdhps substitutions, potentially threatening the efficacy of this regimen for prevention of malaria in vulnerable groups. Our work describes new pfgch1 amplifications that can be used to inform the surveillance of SP drug resistance, its prophylactic use, and future experimental work to understand functional mechanisms.

A hybrid approach reveals the allosteric regulation of GTP cyclohydrolase I.

Guanosine triphosphate (GTP) cyclohydrolase I (GCH1) catalyzes the conversion of GTP to dihydroneopterin triphosphate (H2NTP), the initiating step in the biosynthesis of tetrahydrobiopterin (BH4). Besides other roles, BH4 functions as cofactor in neurotransmitter biosynthesis. The BH4 biosynthetic pathway and GCH1 have been identified as promising targets to treat pain disorders in patients. The function of mammalian GCH1s is regulated by a metabolic sensing mechanism involving a regulator protein, GCH1 feedback regulatory protein (GFRP). GFRP binds to GCH1 to form inhibited or activated complexes dependent on availability of cofactor ligands, BH4 and phenylalanine, respectively. We determined high-resolution structures of human GCH1-GFRP complexes by cryoelectron microscopy (cryo-EM). Cryo-EM revealed structural flexibility of specific and relevant surface lining loops, which previously was not detected by X-ray crystallography due to crystal packing effects. Further, we studied allosteric regulation of isolated GCH1 by X-ray crystallography. Using the combined structural information, we are able to obtain a comprehensive picture of the mechanism of allosteric regulation. Local rearrangements in the allosteric pocket upon BH4 binding result in drastic changes in the quaternary structure of the enzyme, leading to a more compact, tense form of the inhibited protein, and translocate to the active site, leading to an open, more flexible structure of its surroundings. Inhibition of the enzymatic activity is not a result of hindrance of substrate binding, but rather a consequence of accelerated substrate binding kinetics as shown by saturation transfer difference NMR (STD-NMR) and site-directed mutagenesis. We propose a dissociation rate controlled mechanism of allosteric, noncompetitive inhibition.

In Silico Investigation of the Human GTP Cyclohydrolase 1 Enzyme Reveals the Potential of Drug Repurposing Approaches towards the Discovery of Effective BH4 Therapeutics.

The GTP cyclohydrolase 1 enzyme (GTPCH1) is the rate-limiting enzyme of the tetrahydrobiopterin (BH4) biosynthetic pathway. Physiologically, BH4 plays a crucial role as an essential cofactor for the production of catecholamine neurotransmitters, including epinephrine, norepinephrine and dopamine, as well as the gaseous signaling molecule, nitric oxide. Pathological levels of the cofactor have been reported in a number of disease states, such as inflammatory conditions, neuropathic pain and cancer. Targeting the GTPCH1 enzyme has great potential in the management of a number of disease pathologies associated with dysregulated BH4 physiology. This study is an in silico investigation of the human GTPCH1 enzyme using virtual screening and molecular dynamic simulation to identify molecules that can be repurposed to therapeutically target the enzyme. A three-tier molecular docking protocol was employed in the virtual screening of a comprehensive library of over 7000 approved medications and nutraceuticals in order to identify hit compounds capable of binding to the GTPCH1 binding pocket with the highest affinity. Hit compounds were further verified by molecular dynamic simulation studies to provide a detailed insight regarding the stability and nature of the binding interaction. In this study, we identify a number of drugs and natural compounds with recognized anti-inflammatory, analgesic and cytotoxic effects, including the aminosalicylate olsalazine, the antiepileptic phenytoin catechol, and the phlorotannins phlorofucofuroeckol and eckol. Our results suggest that the therapeutic and clinical effects of hit compounds may be partially attributed to the inhibition of the GTPCH1 enzyme. Notably, this study offers an understanding of the off-target effects of a number of compounds and advocates the potential role of aminosalicylates in the regulation of BH4 production in inflammatory disease states. It highlights an in silico drug repurposing approach to identify a potential means of safely targeting the BH4 biosynthetic pathway using established therapeutic agents.

Relationship of Genotype, Phenotype, and Treatment in Dopa-Responsive Dystonia: MDSGene Review.

BACKGROUND: Pathogenic variants in 5 genes (GCH1, TH, PTS, SPR, and QDPR), involved in dopamine/tetrahydrobiopterin biosynthesis or recycling, have been linked to Dopa-responsive dystonia (DRD). Diagnosis and treatment are often delayed due to high between- and within-group variability. OBJECTIVES: Comprehensively analyzed individual genotype, phenotype, treatment response, and biochemistry information. METHODS: 734 DRD patients and 151 asymptomatic GCH1 mutation carriers were included using an MDSGene systematic literature review and an automated classification approach to distinguish between different forms of monogenic DRDs. RESULTS: Whereas dystonia, L-Dopa responsiveness, early age at onset, and diurnal fluctuations were identified as red flags, parkinsonism without dystonia was rarely reported (11%) and combined with dystonia in only 18% of patients. While sex was equally distributed in autosomal recessive DRD, there was female predominance in autosomal dominant DYT/PARK-GCH1 patients accompanied by a lower median age at onset and more dystonia in females compared to males. Accordingly, the majority of asymptomatic heterozygous GCH1 mutation carriers (>8 years of age) were males. Multiple other subgroup-specific characteristics were identified, showing high accuracy in the automated classification approach: Seizures and microcephaly were mostly seen in DYT/PARK-PTS, autonomic symptoms appeared commonly in DYT/PARK-TH and DYT/PARK-PTS, and sleep disorders and oculogyric crises in DYT/PARK-SPR. Biochemically, homovanillic acid and 5-hydroxyindoleacetic acid in CSF were reduced in most DRDs, but neopterin and biopterin were increased only in DYT/PARK-PTS and DYT/PARK-SPR. Hyperphenylalaninemia was seen in DYT/PARK-PTS, DYT/PARK-QDPR, and rarely reported in autosomal recessive DYT/PARK-GCH1. CONCLUSIONS: Our indicators will help to specify diagnosis and accelerate start of treatment. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

eIF4G-driven translation initiation of downstream ORFs in mammalian cells.

Comprehensive genome-wide analysis has revealed the presence of translational elements in the 3' untranslated regions (UTRs) of human transcripts. However, the mechanisms by which translation is initiated in 3' UTRs and the physiological function of their products remain unclear. This study showed that eIF4G drives the translation of various downstream open reading frames (dORFs) in 3' UTRs. The 3' UTR of GCH1, which encodes GTP cyclohydrolase 1, contains an internal ribosome entry site (IRES) that initiates the translation of dORFs. An in vitro reconstituted translation system showed that the IRES in the 3' UTR of GCH1 required eIF4G and conventional translation initiation factors, except eIF4E, for AUG-initiated translation of dORFs. The 3' UTR of GCH1-mediated translation was resistant to the mTOR inhibitor Torin 1, which inhibits cap-dependent initiation by increasing eIF4E-unbound eIF4G. eIF4G was also required for the activity of various elements, including polyU and poliovirus type 2, a short element thought to recruit ribosomes by base-pairing with 18S rRNA. These findings indicate that eIF4G mediates translation initiation of various ORFs in mammalian cells, suggesting that the 3' UTRs of mRNAs may encode various products.

Molecular and metabolic bases of tetrahydrobiopterin (BH4) deficiencies.

Tetrahydrobiopterin (BH4) deficiency is caused by genetic variants in the three genes involved in de novo cofactor biosynthesis, GTP cyclohydrolase I (GTPCH/GCH1), 6-pyruvoyl-tetrahydropterin synthase (PTPS/PTS), sepiapterin reductase (SR/SPR), and the two genes involved in cofactor recycling, carbinolamine-4α-dehydratase (PCD/PCBD1) and dihydropteridine reductase (DHPR/QDPR). Dysfunction in BH4 metabolism leads to reduced cofactor levels and may result in systemic hyperphenylalaninemia and/or neurological sequelae due to secondary deficiency in monoamine neurotransmitters in the central nervous system. More than 1100 patients with BH4 deficiency and 800 different allelic variants distributed throughout the individual genes are tabulated in database of pediatric neurotransmitter disorders PNDdb. Here we provide an update on the molecular-genetic analysis and structural considerations of these variants, including the clinical courses of the genotypes. From a total of 324 alleles, 11 are associated with the autosomal recessive form of GTPCH deficiency presenting with hyperphenylalaninemia (HPA) and neurotransmitter deficiency, 295 GCH1 variant alleles are detected in the dominant form of L-dopa-responsive dystonia (DRD or Segawa disease) while phenotypes of 18 alleles remained undefined. Autosomal recessive variants observed in the PTS (199 variants), PCBD1 (32 variants), and QDPR (141 variants) genes lead to HPA concomitant with central monoamine neurotransmitter deficiency, while SPR deficiency (104 variants) presents without hyperphenylalaninemia. The clinical impact of reported variants is essential for genetic counseling and important for development of precision medicine.

GCH1 mutations in hereditary spastic paraplegia.

GCH1 mutations have been associated with dopa-responsive dystonia (DRD), Parkinson's disease (PD) and tetrahydrobiopterin (BH4 )-deficient hyperphenylalaninemia B. Recently, GCH1 mutations have been reported in five patients with hereditary spastic paraplegia (HSP). Here, we analyzed a total of 400 HSP patients (291 families) from different centers across Canada by whole exome sequencing (WES). Three patients with heterozygous GCH1 variants were identified: monozygotic twins with a p.(Ser77_Leu82del) variant, and a patient with a p.(Val205Glu) variant. The former variant is predicted to be likely pathogenic and the latter is pathogenic. The three patients presented with childhood-onset lower limb spasticity, hyperreflexia and abnormal plantar responses. One of the patients had diurnal fluctuations, and none had parkinsonism or dystonia. Phenotypic differences between the monozygotic twins were observed, who responded well to levodopa treatment. Pathway enrichment analysis suggested that GCH1 shares processes and pathways with other HSP-associated genes, and structural analysis of the variants indicated a disruptive effect. In conclusion, GCH1 mutations may cause HSP; therefore, we suggest a levodopa trial in HSP patients and including GCH1 in the screening panels of HSP genes. Clinical differences between monozygotic twins suggest that environmental factors, epigenetics, and stochasticity could play a role in the clinical presentation.

GTP-cyclohydrolase deficiency induced peripheral and deep microcirculation dysfunction with age.

The present study assessed the impact of impaired tetrahydrobiopterin (BH4) production on vasoreactivity from conduit and small arteries along the vascular tree as seen during aging. For this purpose, the mutant hyperphenylalaninemic mouse (hph-1) was used. This model is reported to be deficient in GTP cyclohydrolase I, a rate limiting enzyme in BH4 biosynthesis. BH4 is a key regulator of vascular homeostasis by regulating the nitric oxide synthase 3 (NOS3) activity. In GTP-CH deficient mice, the aortic BH4 levels were decreased, by -77% in 12 week-middle-aged mice (young) and by -83% in 35-45 week-middle-aged mice (middle-aged). In young hph-1, the mesenteric artery ability to respond to flow was slightly reduced by 9%. Aging induced huge modification in many vascular functions. In middle-aged hph-1, we observed a decrease in aortic cGMP levels, biomarker of NO availability (-46%), in flow-mediated vasodilation of mesenteric artery (-31%), in coronary hyperemia response measured in isolated heart following transient ischemia (-27%) and in cutaneous microcirculation dilation in response to acetylcholine assessed in vivo by laser-doppler technic (-69%). In parallel, the endothelium-dependent relaxation in response to acetylcholine in conduit blood vessel, measured on isolated aorta rings, was unchanged in hph-1 mice whatever the age. Our findings demonstrate that in middle-aged GTP-CH depleted mice, the reduction of BH4 was characterized by an alteration of microcirculation dilatory properties observed in various parts of the vascular tree. Large conduit blood vessels vasoreactivity, ie aorta, was unaltered even in middle-aged mice emphasizing the main BH4-deletion impact on the microcirculation.

GTP Cyclohydrolase I as a Potential Drug Target: New Insights into Its Allosteric Modulation via Normal Mode Analysis.

Guanosine triphosphate (GTP) cyclohydrolase I (GCH1) catalyzes the conversion of GTP into dihydroneopterin triphosphate (DHNP). DHNP is the first intermediate of the folate de novo biosynthesis pathway in prokaryotic and lower eukaryotic microorganisms and the tetrahydrobiopterin (BH4) biosynthesis pathway in higher eukaryotes. The de novo folate biosynthesis provides essential cofactors for DNA replication, cell division, and synthesis of key amino acids in rapidly replicating pathogen cells, such as Plasmodium falciparum (P. falciparum), a causative agent of malaria. In eukaryotes, the product of the BH4 biosynthesis pathway is essential for the production of nitric oxide and several neurotransmitter precursors. An increased copy number of the malaria parasite P. falciparum GCH1 gene has been reported to influence antimalarial antifolate drug resistance evolution, whereas mutations in the human GCH1 are associated with neuropathic and inflammatory pain disorders. Thus, GCH1 stands as an important and attractive drug target for developing therapeutics. The GCH1 intrinsic dynamics that modulate its activity remains unclear, and key sites that exert allosteric effects across the structure are yet to be elucidated. This study employed the anisotropic network model to analyze the intrinsic motions of the GCH1 structure alone and in complex with its regulatory partner protein. We showed that the GCH1 tunnel-gating mechanism is regulated by a global shear motion and an outward expansion of the central five-helix bundle. We further identified hotspot residues within sites of structural significance for the GCH1 intrinsic allosteric modulation. The obtained results can provide a solid starting point to design novel antineuropathic treatments for humans and novel antimalarial drugs against the malaria parasite P. falciparum GCH1 enzyme.

Comprehensive analysis of GTP cyclohydrolase I activity in Mycobacterium tuberculosis H37 Rv via in silico studies.

GTP cyclohydrolase I enzyme (GTPCH-I) is a rate limiting enzyme in the biosynthesis pathway of tetrahydrobiopterin (BH4) and tetrahydrofolate (THF) compounds; latter being are an essential compounds involved in many biological functions. This enzyme has been evaluated structurally and functionally in many organisms to understand its putative role in cell processes, kinetics, regulations, drug targeting in infectious diseases, pain sensitivity in humans, and so on. In Mycobacterium tuberculosis (a human pathogen causing tuberculosis), this GTPCH-I activity has been predicted to be present in Rv3609c gene (folE) of H37 Rv strain, which till date has not been studied in detail. In order to understand in depth, the structure and function of folE protein in M. tuberculosis H37 Rv, in silico study was designed by using many different bioinformatics tools. Comparative and structural analysis predicts that Rv3609c gene is similar to folE protein ortholog of Listeria monocytogenes (cause food born disease), and uses zinc ion as a cofactor for its catalysis. Result shows that mutation of folE protein at 52th residue from tyrosine to glycine or variation in pH and temperature can lead to high destability in protein structure. Studies here have also predicted about the functional regions and interacting partners involved with folE protein. This study has provided clues to carry out experimentally the analysis of folE protein in mycobacteria and if found suitable will be used for drug targeting.

Data-driven evolution of neurosurgical gene therapy delivery in Parkinson's disease.

Loss of nigrostriatal dopaminergic projection neurons is a key pathology in Parkinson's disease, leading to abnormal function of basal ganglia motor circuits and the accompanying characteristic motor features. A number of intraparenchymally delivered gene therapies designed to modify underlying disease and/or improve clinical symptoms have shown promise in preclinical studies and subsequently were evaluated in clinical trials. Here we review the challenges with surgical delivery of gene therapy vectors that limited therapeutic outcomes in these trials, particularly the lack of real-time monitoring of vector administration. These challenges have recently been addressed during the evolution of novel techniques for vector delivery that include the use of intraoperative MRI. The preclinical development of these techniques are described in relation to recent clinical translation in an adeno-associated virus serotype 2-mediated human aromatic L-amino acid decarboxylase gene therapy development programme. This new paradigm allows visualisation of the accuracy and adequacy of viral vector delivery within target structures, enabling intertrial modifications in surgical approaches, cannula design, vector volumes and dosing. The rapid, data-driven evolution of these procedures is unique and has led to improved vector delivery.

Vector-mediated l-3,4-dihydroxyphenylalanine delivery reverses motor impairments in a primate model of Parkinson's disease.

Ever since its introduction 40 years ago l-3,4-dihydroxyphenylalanine (l-DOPA) therapy has retained its role as the leading standard medication for patients with Parkinson's disease. With time, however, the shortcomings of oral l-DOPA treatment have become apparent, particularly the motor fluctuations and troublesome dyskinetic side effects. These side effects, which are caused by the excessive swings in striatal dopamine caused by intermittent oral delivery, can be avoided by delivering l-DOPA in a more continuous manner. Local gene delivery of the l-DOPA synthesizing enzymes, tyrosine hydroxylase and guanosine-tri-phosphate-cyclohydrolase-1, offers a new approach to a more refined dopaminergic therapy where l-DOPA is delivered continuously at the site where it is needed i.e. the striatum. In this study we have explored the therapeutic efficacy of adeno-associated viral vector-mediated l-DOPA delivery to the putamen in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated rhesus monkeys, the standard non-human primate model of Parkinson's disease. Viral vector delivery of the two enzymes, tyrosine hydroxylase and guanosine-5'-tri-phosphate-cyclohydrolase-1, bilaterally into the dopamine-depleted putamen, induced a significant, dose-dependent improvement of motor behaviour up to a level identical to that obtained with the optimal dose of peripheral l-DOPA. Importantly, this improvement in motor function was obtained without any adverse dyskinetic effects. These results provide proof-of-principle for continuous vector-mediated l-DOPA synthesis as a novel therapeutic strategy for Parkinson's disease. The constant, local supply of l-DOPA obtained with this approach holds promise as an efficient one-time treatment that can provide long-lasting clinical improvement and at the same time prevent the appearance of motor fluctuations and dyskinetic side effects associated with standard oral dopaminergic medication.

SV-Pop: population-based structural variant analysis and visualization.

BACKGROUND: Genetic structural variation underpins a multitude of phenotypes, with significant implications for a range of biological outcomes. Despite their crucial role, structural variants (SVs) are often neglected and overshadowed by single nucleotide polymorphisms (SNPs), which are used in large-scale analysis such as genome-wide association and population genetic studies. RESULTS: To facilitate the high-throughput analysis of structural variation we have developed an analytical pipeline and visualisation tool, called SV-Pop. The utility of this pipeline was then demonstrated through application with a large, multi-population P. falciparum dataset. CONCLUSIONS: Designed to facilitate downstream analysis and visualisation post-discovery, SV-Pop allows for straightforward integration of multi-population analysis, method and sample-based concordance metrics, and signals of selection.

Mast cell tetrahydrobiopterin contributes to itch in mice.

GTP cyclohydrolase (GCH1) governs de novo synthesis of the enzyme cofactor, tetrahydrobiopterin (BH4), which is essential for biogenic amine production, bioactive lipid metabolism and redox coupling of nitric oxide synthases. Overproduction of BH4 via upregulation of GCH1 in sensory neurons is associated with nociceptive hypersensitivity in rodents, and neuron-specific GCH1 deletion normalizes nociception. The translational relevance is revealed by protective polymorphisms of GCH1 in humans, which are associated with a reduced chronic pain. Because myeloid cells constitute a major non-neuronal source of BH4 that may contribute to BH4-dependent phenotypes, we studied here the contribution of myeloid-derived BH4 to pain and itch in lysozyme M Cre-mediated GCH1 knockout (LysM-GCH1-/- ) and overexpressing mice (LysM-GCH1-HA). Unexpectedly, knockout or overexpression in myeloid cells had no effect on nociceptive behaviour, but LysM-driven GCH1 knockout reduced, and its overexpression increased the scratching response in Compound 48/80 and hydroxychloroquine-evoked itch models, which involve histamine and non-histamine dependent signalling pathways. Mechanistically, GCH1 overexpression increased BH4, nitric oxide and hydrogen peroxide, and these changes were associated with increased release of histamine and serotonin and degranulation of mast cells. LysM-driven GCH1 knockout had opposite effects, and pharmacologic inhibition of GCH1 provided even stronger itch suppression. Inversely, intradermal BH4 provoked scratching behaviour in vivo and BH4 evoked an influx of calcium in sensory neurons. Together, these loss- and gain-of-function experiments suggest that itch in mice is contributed by BH4 release plus BH4-driven mediator release from myeloid immune cells, which leads to activation of itch-responsive sensory neurons.

Dystonia and Parkinson's disease: What is the relationship?

Dystonia and Parkinson's disease are closely linked disorders sharing many pathophysiological overlaps. Dystonia can be seen in 30% or more of the patients suffering with PD and sometimes can precede the overt parkinsonism. The response of early dystonia to the introduction of dopamine replacement therapy (levodopa, dopamine agonists) is variable; dystonia commonly occurs in PD patients following levodopa initiation. Similarly, parkinsonism is commonly seen in patients with mutations in various DYT genes including those involved in the dopamine synthesis pathway. Pharmacological blockade of dopamine receptors can cause both tardive dystonia and parkinsonism and these movement disorders syndromes can occur in many other neurodegenerative, genetic, toxic and metabolic diseases. Pallidotomy in the past and currently deep brain stimulation largely involving the GPi are effective treatment options for both dystonia and parkinsonism. However, the physiological mechanisms underlying the response of these two different movement disorder syndromes are poorly understood. Interestingly, DBS for PD can cause dystonia such as blepharospasm and bilateral pallidal DBS for dystonia can result in features of parkinsonism. Advances in our understanding of these responses may provide better explanations for the relationship between dystonia and Parkinson's disease.

Improved folate accumulation in genetically modified maize and wheat.

Folates are indispensable co-factors for one-carbon metabolism in all organisms. In humans, suboptimal folate intake results in serious disorders. One promising strategy for improving human folate status is to enhance folate levels in food crops by metabolic engineering. In this study, we cloned two GmGCHI (GTP cyclohydrolase I) genes (Gm8gGCHI and Gm3gGCHI) and one GmADCS (aminodeoxychorismate synthase) gene from soybean, which are responsible for synthesizing the folate precursors pterin and p-aminobenzoate, respectively. We initially confirmed their functions in transgenic Arabidopsis plants and found that Gm8gGCHI increased pterin and folate production more than Gm3gGCHI did. We then co-expressed Gm8gGCHI and GmADCS driven by endosperm-specific promoters in maize and wheat, two major staple crops, to boost their folate metabolic flux. A 4.2-fold and 2.3-fold increase in folate levels were observed in transgenic maize and wheat grains, respectively. To optimize wheat folate enhancement, codon-optimized Gm8gGCHI and tomato LeADCS genes under the control of a wheat endosperm-specific glutenin promoter (1Dx5) were co-transformed. This yielded a 5.6-fold increase in folate in transgenic wheat grains (Gm8gGCHI+/LeADCS+). This two-gene co-expression strategy therefore has the potential to greatly enhance folate levels in maize and wheat, thus improving their nutritional value.