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Impaired PLP-Dependent Metabolism in Brain Samples from Huntington Disease Patients and Transgenic R6/1 Mice

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Impaired PLP-Dependent Metabolism in Brain Samples from Huntington Disease Patients and Transgenic R6/1 Mice View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Diposit Digital de la Universitat de Barcelona Metab Brain Dis DOI 10.1007/s11011-015-9777-7 ORIGINAL ARTICLE Impaired PLP-dependent metabolism in brain samples from Huntington disease patients and transgenic R6/1 mice M. Alba Sorolla1 & María José Rodríguez-Colman1 & Núria Vall-llaura1 & Celia Vived1 & Marta Fernández-Nogales2 & José J. Lucas2 & Isidre Ferrer3 & Elisa Cabiscol1 Received: 1 September 2015 /Accepted: 9 December 2015 # Springer Science+Business Media New York 2015 Abstract Oxidative stress has been described as important to Keywords Huntington disease . Oxidative stress . Pyridoxal Huntington disease (HD) progression. In a previous HD study, kinase . Pyridoxal 5-phosphate . Glutamate . Cystathionine we identified several carbonylated proteins, including pyri- doxal kinase and antiquitin, both of which are involved in the metabolism of pyridoxal 5´-phosphate (PLP), the active Abbreviations form of vitamin B6. In the present study, pyridoxal kinase ALDH7A1 α-aminoadipic semialdehyde dehydrogenase or levels were quantified and showed to be decreased both in antiquitin HD patients and a R6/1 mouse model, compared to control GSH Reduced glutathione samples. A metabolomic analysis was used to analyze metab- HD Huntington disease olites in brain samples of HD patients and R6/1 mice, com- Htt Huntingtin protein pared to control samples using mass spectrometry. This tech- mHtt Mutant huntingtin nique allowed detection of increased concentrations of pyri- LC Liquid chromatography doxal, the substrate of pyridoxal kinase. In addition, PLP, the MOPS 3-(N-Morpholino)propanesulfonic acid product of the reaction, was decreased in striatum from R6/1 MS Mass spectrometry mice. Furthermore, glutamate and cystathionine, both sub- PDXK Pyridoxal kinase strates of PLP-dependent enzymes were increased in HD. PLP Pyridoxal 5´-phosphate This reinforces the hypothesis that PLP synthesis is impaired, TOF Time of flight and could explain some alterations observed in the disease. Together, these results identify PLP as a potential therapeutic agent. Introduction Huntington disease (HD) is a dominantly inherited neurode- generative disease caused by the expansion of CAG trinucle- * M. Alba Sorolla otide repeats in exon 1 of the huntingtin gene that encodes a [email protected] stretch of polyglutamines (polyQ) in the huntingtin (Htt) pro- tein (Group 1993). The disease appears when the number of glutamines exceeds 40. Age of onset and disease severity are strongly correlated with the length of the CAG expansion 1 Departament de Ciències Mèdiques Bàsiques, Universitat de Lleida, IRBLleida, Av. Rovira Roure 80, 25198 Lleida, Catalonia, Spain (Benitez et al. 1994). Clinical symptoms include psychiatric and cognitive abnormalities, as well as involuntary move- 2 Centro de Biología Molecular BSevero Ochoa^, Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, ments (chorea). With HD progression, motor rigidity, depres- Madrid, Spain sion, and dementia predominate, and inevitably lead to death. 3 Institut de Neuropatologia, Servei Anatomia Patològica, Anatomically, HD is characterized by progressive brain atro- IDIBELL-Hospital Universitari de Bellvitge, Universitat de phy, affecting predominantly the striatum, and the cortex to a Barcelona, Barcelona, Spain lesser extent (Reiner et al. 1988). Htt is a widely expressed Metab Brain Dis 350-kDa protein that has been described to be related to sev- In the present study, we investigated the hypothesis that eral roles, however, its function is still unknown. One of the metabolic changes resulting from oxidation of PDXK and most significant findings in HD neuropathology was the pres- antiquitin are involved in HD pathogenesis due to decreased ence of mutant Htt (mHtt) deposits, mostly in striatum and PLP levels. We report decreased levels of PDXK, both in HD cortex (DiFiglia et al. 1997). Although the presence of aggre- patients and R6/1 transgenic mice. Using LC-MS metabolo- gates often correlates with toxicity (Perutz 1999), it has been mics several metabolites were quantified giving support to an described that the oligomeric species, formed before the con- impaired PLP synthesis and, as a consequence, an altered stitution of the mature mHtt deposits, are highly toxic and PLP-dependent metabolism in HD. The biological signifi- responsible for the cellular alterations observed in HD cance in HD pathology is discussed. (Arrasate et al. 2004). Increasing evidence supports the in- volvement of mitochondria in HD pathogenesis. Impaired en- ergy metabolism leading to ATP depletion, due to functional defects in mitochondrial complexes (Jenkins et al. 1993; Material and methods Shirendeb et al. 2011) suggest that defective mitochondria may be responsible for neuronal damage in HD. Such mito- Human samples chondrial damage might generate an oxidative stress situation welldescribedinHD(Duganetal.1995; Browne 2008; Human brain tissues (striatum and cortex) were obtained from Sorolla et al. 2008). Thus, cellular dysfunction in HD has been the Institute of Neuropathology and University of Barcelona related to loss of normal physiological function and especially Brain Banks (Barcelona, Spain), following the guidelines of to gain of toxic function of mHtt. the local ethics committees. Control cases had not suffered In previous publications using proteomic techniques neurologic or psychiatric disease and the neuropathology ex- (Sorolla et al. 2008; Sorolla et al. 2010), we demonstrated in amination was strictly normal, including lack of Alzheimer human HD samples and also in a mouse model that several disease-associated changes. Eight HD cases (range 41–62 enzymes were oxidized (carbonylated) in striatum. These re- CAG repeats) and eight controls were analyzed (Table 1). sults pointed to a possible disturbance in the metabolism of Control and HD groups were balanced for age, sex and post- pyridoxal 5´-phosphate (PLP), the active form of vitamin B6. mortem time, excluding these parameters as potential differ- Two enzymes, pyridoxal kinase (PDXK) and antiquitin 1, ences between these groups. After neurological examination, were identified as carbonylated proteins in human HD and all HD cases were classified as stage 4 according to Vonsattel the conditional HD mouse model Tet/HD94 (Sorolla et al. et al. (1985). All procedures were carried out according to the 2010). PDXK phosphorylates pyridoxal generating PLP. Helsinki declaration and conducted according to the guide- Antiquitin 1, encoded by the ALDH7A1 gene, acts as a δ-1- lines of the European Community Council. piperideine-6-carboxylate dehydrogenase or α-aminoadipic semialdehyde dehydrogenase. Mutations in the ALDH7A1 gene cause pyridoxine-dependent seizures, a disease affecting Table 1 Summary of controls (C) and HD (H) cases newborns and children. In these cases, mutations abolish Patient Sex Age Postmortem delay (h) antiquitin 1 activity and the accumulating substrate, piperideine-6-carboxylate, inactivates PLP by forming a C1 Female 73 7 Knoevenagel condensation product (Mills et al. 2006). The C2 Female 73 5.5 lack of PLP causes seizures and is associated with learning C3 Male 51 4 difficulties. These seizures are fully controlled by pyridoxine C4 Female 24 6 (Rankin et al. 2007). Thus, antiquitin 1 oxidation observed in C5 Male 70 13 HD could result in its inactivation, exacerbating the lack of C6 Female 47 9.6 PLP available as a cofactor. The active form of vitamin B6 is a C7 Male 53 3 necessary cofactor in more than 140 distinct enzymatic activ- C8 Male 46 15 ities catalogued by the Enzyme Commission (http://www. H1 Female 72 7 chem.qmul.ac.uk/iubmb/enzyme/). To study whether such H2 Female 72 5 enzymes might be affected in HD, the substrate of two key H3 Male 68 4 PLP-dependent enzymes, glutamate and cystathionine, were H4 Female 28 4.2 analyzed. They are involved in the synthesis of GABA, a H5 Male 71 10.2 neurotransmitter described to be decreased in striatal neurons H6 Female 65 15.2 of HD patients and, ii) synthesis of glutathione (GSH), a key H7 Male 59 5.5 molecule maintaining the cellular redox status and the GSH- H8 Male 60 13.1 dependent antioxidant defense system. Metab Brain Dis Murine samples min, 350 °C). The capillary voltage was 3500 V with a scan rate of 1 scan/s. Murine brain tissues (striatum, cortex and cerebellum) were Data Analysis and Quantitative Analysis of metabolites obtained from wild-type (WT) and R6/1 transgenic mice for were performed using Masshunter Software (Agilent the human exon-1-Htt gene (CAG repeats: 168–189), all of Technologies). The m/z values used for quantification were + them in B6CBAF1/J background (Mangiarini et al. 1996). m/z 150.056 [M + H] − [H2O] for pyridoxal, m/z 130.0503 + + Either 3-month-old (n =7,eachgroup)and7-month-old [M + H] − [H2O] for glutamate, and m/z 223.074 [M + H] (n = 4, each group) mice were bred at Severo Ochoa Center for cystathionine. Identities were confirmed using an orthog- (Madrid). Procedures using laboratory animals were in accor- onal characterization (based on exact mass <10 ppm and on dance with the European Union Directives and institutional retention time) and MS/MS spectrum, compared with authen- guidelines approved by the research ethics committee of tic standards (Jové et al. 2011). Data are presented as means ± Consejo Superior de Investigaciones Científicas (CSIC). All standard error from at least three independent experiments. efforts
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