Pyridoxal 5'-phosphate (PLP), the active form of vitamin B, functions as a cofactor in humans for more than 140 enzymes, many of which are involved in neurotransmitter synthesis and degradation. A deficiency of PLP can present, therefore, as seizures and other symptoms that are treatable with PLP and/or pyridoxine. Deficiency of PLP in the brain can be caused by inborn errors affecting B vitamer metabolism or by inactivation of PLP, which can occur when compounds accumulate as a result of inborn errors of other pathways or when small molecules are ingested. Whole-exome sequencing of two children from a consanguineous family with pyridoxine-dependent epilepsy revealed a homozygous nonsense mutation in proline synthetase co-transcribed homolog (bacterial), PROSC, which encodes a PLP-binding protein of hitherto unknown function. Subsequent sequencing of 29 unrelated indivduals with pyridoxine-responsive epilepsy identified four additional children with biallelic PROSC mutations. Pre-treatment cerebrospinal fluid samples showed low PLP concentrations and evidence of reduced activity of PLP-dependent enzymes. However, cultured fibroblasts showed excessive PLP accumulation. An E.coli mutant lacking the PROSC homolog (ΔYggS) is pyridoxine sensitive; complementation with human PROSC restored growth whereas hPROSC encoding p.Leu175Pro, p.Arg241Gln, and p.Ser78Ter did not. PLP, a highly reactive aldehyde, poses a problem for cells, which is how to supply enough PLP for apoenzymes while maintaining free PLP concentrations low enough to avoid unwanted reactions with other important cellular nucleophiles. Although the mechanism involved is not fully understood, our studies suggest that PROSC is involved in intracellular homeostatic regulation of PLP, supplying this cofactor to apoenzymes while minimizing any toxic side reactions.

BackgroundCurrently there are no effective treatments for many neurodegenerative diseases. Reliable biomarkers for identifying and stratifying these diseases will be important in the development of future novel therapies. Lewy Body Dementia (LBD) is considered an under diagnosed form of dementia for which markers are needed to discriminate LBD from other forms of dementia such as Alzheimer’s Disease (AD). This work describes a Label-Free proteomic profiling analysis of cerebral spinal fluid (CSF) from non-neurodegenerative controls and patients with LBD. Using this technology we identified several potential novel markers for LBD. These were then combined with other biomarkers from previously published studies, to create a 10 min multiplexed targeted and translational MRM-LC-MS/MS assay. This test was used to validate our new assay in a larger cohort of samples including controls and the other neurodegenerative conditions of Alzheimer’s and Parkinson’s disease (PD).ResultsThirty eight proteins showed significantly (p < 0.05) altered expression in LBD CSF by proteomic profiling. The targeted MRM-LC-MS/MS assay revealed 4 proteins that were specific for the identification of AD from LBD: ectonucleotide pyrophosphatase/phosphodiesterase 2 (p < 0.0001), lysosome-associated membrane protein 1 (p < 0.0001), pro-orexin (p < 0.0017) and transthyretin (p < 0.0001). Nineteen proteins were elevated significantly in both AD and LBD versus the control group of which 4 proteins are novel (malate dehydrogenase 1, serum amyloid A4, GM2−activator protein, and prosaposin). Protein-DJ1 was only elevated significantly in the PD group and not in either LBD or AD samples. Correlations with Alzheimer-associated amyloid β-42 levels, determined by ELISA, were observed for transthyretin, GM2 activator protein and IGF2 in the AD disease group (r2 ≥ 0.39, p ≤ 0.012). Cystatin C, ubiquitin and osteopontin showed a strong significant linear relationship (r2 ≥ 0.4, p ≤ 0.03) with phosphorylated–tau levels in all groups, whilst malate dehydrogenase and apolipoprotein E demonstrated a linear relationship with phosphorylated-tau and total-tau levels in only AD and LBD disease groups.ConclusionsUsing proteomics we have identified several potential and novel markers of neurodegeneration and subsequently validated them using a rapid, multiplexed mass spectral test. This targeted proteomic platform can measure common markers of neurodegeneration that correlate with existing diagnostic makers as well as some that have potential to show changes between AD from LBD.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-015-0059-y) contains supplementary material, which is available to authorized users.

Vitamin B6 in the form of pyridoxine (PN) is one of the most widespread pharmacological therapies for inherited diseases involving pyridoxal phosphate (PLP)-dependent enzymes, including primary hyperoxaluria type I (PH1). PH1 is caused by a deficiency of liver-peroxisomal alanine: glyoxylate aminotransferase (AGT), which allows glyoxylate oxidation to oxalate leading to the deposition of insoluble calcium oxalate in the kidney. Only a minority of PH1 patients, mostly bearing the F152I and G170R mutations, respond to PN, the only pharmacological treatment currently available. Moreover, excessive doses of PN reduce the specific activity of AGT in a PH1 cellular model. Nevertheless, the possible effect(s) of other B6 vitamers has not been investigated previously. Here, we compared the ability of PN in rescuing the effects of the F152I and G170R mutations with that of pyridoxamine (PM) and PL. We found that supplementation with PN raises the intracellular concentration of PN phosphate (PNP), which competes with PLP for apoenzyme binding leading to the formation of an inactive AGT-PNP complex. In contrast, PNP does not accumulate in the cell upon PM or PL supplementation, but higher levels of PLP and PM phosphate (PMP), the two active forms of the AGT coenzyme, are found. This leads to an increased ability of PM and PL to rescue the effects of the F152I and G170R mutations compared with PN. A similar effect was also observed for other folding-defective AGT variants. Thus, PM and PL should be investigated as matter of importance as therapeutics for PH1 patients bearing folding mutations.

There is increasing evidence that vitamin B 6 , given either as pyridoxine or pyridoxal 5 0 -phosphate, can sometimes result in improved seizure control in idiopathic epilepsy. Whole-exome sequencing was used to identify a de novo mutation (c.629G>A; p.Arg210His) in KCNQ2 in a 7-year-old patient whose neonatal seizures showed a response to pyridoxine and who had a high plasma to CSF pyridoxal 5 0 -phosphate ratio, usually indicative of an inborn error of vitamin B 6 metabolism. This mutation has been described in three other patients with neonatal epileptic encephalopathy. A review of the literature was performed to assess the effectiveness of vitamin B 6 treatment in patients with a KCNQ2 channelopathy. Twenty-three patients have been reported to have been trialled with B 6

Targeted gene panels can be used to establish molecular diagnoses in paediatric cohorts. Reid et al. report that this approach is accurate, efficient and can be preferred to whole-exome or genome sequencing for patients with neurological symptomatology and clues suggestive of an inherited metabolic disorder.

Mutations in SLC25A22 are known to cause neonatal epileptic encephalopathy and migrating partial seizures in infancy. Using whole exome sequencing we identified four novel SLC25A22 mutations in six children from three families. Five patients presented clinical features similar to those in the literature including hypotonia, refractory neonatal-onset seizures and developmental delay. However, the sixth patients presented atypically with isolated developmental delay, developing late-onset (absence) seizures only at 7 years of age. Abnormal metabolite levels have not been documented in the nine patients described previously. One patient in our series was referred to the metabolic clinic because of persistent hyperprolinaemia and another three had raised plasma proline when tested. Analysis of the post-prandial plasma amino acid response in one patient showed abnormally high concentrations of several amino acids. This suggested that, in the fed state, when amino acids are the preferred fuel for the liver, trans-deamination of amino acids requires transportation of glutamate into liver mitochondria by SLC25A22 for deamination by glutamate dehydrogenase; SLC25A22 is an important mitochondrial glutamate transporter in liver as well as in brain. Electron microscopy of patient fibroblasts demonstrated widespread vacuolation containing neutral and phospho-lipids as demonstrated by Oil Red O and Sudan Black tinctorial staining; this might be explained by impaired activity of the proline/pyrroline-5-carboxylate (P5C) shuttle if SLC25A22 transports pyrroline-5-carboxylate/glutamate-γ-semialdehyde as well as glutamate.Electronic supplementary materialThe online version of this article (doi:10.1007/s10545-017-0025-7) contains supplementary material, which is available to authorized users.

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