Investigació en biomarcadors de l’estat de la dopamina i la serotonina en pacients neuropediàtrics

[cat] Els errors congènits del metabolisme són malalties d’origen genètic amb una baixa prevalença dins de la població general. Aquestes malalties solen estar causades per mutacions en gens que provoquen un mal funcionament de diversos enzims o cofactors que controlen alguna ruta metabòlica de l’organisme. Moltes d’aquestes malalties poden presentar símptomes neurològics que poden comprometre el correcte desenvolupament del sistema nerviós central (SNC), particularment durant el període neonatal i la infància. Tot i la seva baixa prevalença, cada any es descriuen nous desordres i fenotips, motiu pel qual cal seguir investigant en nous biomarcadors. Les monoamines, com la dopamina i la serotonina, són neurotransmissors que intervenen en el correcte desenvolupament de les funcions motores, perceptives, cognitives i emocionals del SNC. Diverses condicions genètiques i ambientals han demostrat que afecten de manera primària el metabolisme d’ambdós neurotransmissors o bé d’aquells cofactors essencials per la correcta biosíntesis de la dopamina i serotonina. La detecció quantitativa de metabòlits de la dopamina i serotonina (àcid homovaníl·lic (HVA), i àcid 5-hidroxiindolacètic (5-HIAA), respectivament), així com dels seus cofactors (vitamina B6 i tetrahidrobiopterina), en líquid cefaloraquidi (LCR) es considera una important prova bioquímica pel diagnòstic i seguiment de les malalties mencionades anteriorment. El LCR és un fluid biològic que s’obté mitjançant una punció lumbar, intervenció invasiva que ha de realitzar personal especialitzat i seguint un estricte protocol estandarditzat. Els pacients amb dèficit de dopamina i serotonina d’origen genètic reben tractament amb precursors d’aquests neurotransmissors. El correcte seguiment del tractament es fa per puncions lumbars successives fent que els pacients s’hagin de sotmetre varies vegades a una punció lumbar. Seria de gran utilitat poder trobar un biomarcador relacionat amb la dopamina i/o serotonina, que no estigués influenciat per factors externs, que fos un reflex del que succeeix al SNC i que pogués determinar- se en d’altres fluids biològics menys invasius, com l’orina. Per això vam avaluar la utilitat de la melatonina en orina, una hormona sintetitzada per la glàndula pineal (localitzada al cervell) a partir de la serotonina cerebral. Una vegada és sintetitzada, és alliberada a la sang perquè pugui ser metabolitzada i posteriorment eliminada per orina com a 6-sulfatoximelatonina (aMT6s). D’altra banda, tot i que l’obtenció del LCR es realitza mitjançant un procediment protocol·litzat, pot està subjecte a alteracions preanalítiques, com ara la contaminació hemàtica per punció traumàtica. Està descrit que aquesta contaminació hemàtica té efectes sobre la concentració de glucosa i de proteïnes en el LCR, però no hi ha estudis on es descrigui l’efecte en d’altres biomarcadors que s’analitzen en LCR per l’estudi de patologies neurometabòliques. Tenint en compte aquests antecedents en aquesta tesi hem desenvolupat 3 objectius: 1. Avaluar l’efecte de la contaminació hemàtica en mostres de LCR a l’hora de realitzar i interpretar les anàlisis que realitzem al laboratori: aminoàcids, metabòlits dels neurotransmissors, 5-metiltetrahidrofolat, vitamina B6, pterines (neopterina i biopterina) i tiamina. Els resultats es van publicar a la revista “Fluids and Barriers of CNS” sota el títol Effect of blood contamination of cerebrospinal fluid on amino acids, biogenic amines, pterins and vitamins (2019). 2. Establir valors de referència de l’aMT6s (metabòlit de la melatonina) en orina en població sana, per tal d’avaluar si pot ser un bon biomarcador perifèric de l’estat de la serotonina cerebral en pacients amb mutacions patològiques de gens relacionats en la biosíntesis de la serotonina. Els resultats d’aquest estudi es van publicar a la revista “Scientific Reports” sota el títol Urinary sulphatoxymelatonin as a biomarker of serotonin status in biogenic amine-deficient patients (2017). 3. Avaluar retrospectivament el fenotip clínic i els perfils bioquímics d'amines biògenes en LCR de pacients diagnosticats genèticament d’una malaltia mitocondrial. Els resultats d’aquest estudi es van publicar a la revista “Journal of Inherited Metabolism Diseases” sota el títol Cerebrospinal fluid monoamines, pterins, and folate in patients with mitochondrial diseases: systematic review and hospital experience (2018). Paral·lelament a això vam poder publicar un protocol estandarditzat de l’anàlisi de les monoamines i els seus cofactors en LCR per HPLC a la revista “Nature Protocols” (Analysis of human cerebrospinal fluid monoamines and related cofactors by HPLC. 2017)

Anti-obesity sodium tungstate treatment triggers axonal and glial plasticity in hypothalamic feeding centers

Objective: This study aims at exploring the effects of sodium tungstate treatment on hypothalamic plasticity, which is known to have an important role in the control of energy metabolism. Methods: Adult lean and high-fat diet-induced obese mice were orally treated with sodium tungstate. Arcuate and paraventricular nuclei and lateral hypothalamus were separated and subjected to proteomic analysis by DIGE and mass spectrometry. Immunohistochemistry and in vivo magnetic resonance imaging were also performed. Results: Sodium tungstate treatment reduced body weight gain, food intake, and blood glucose and triglyceride levels. These effects were associated with transcriptional and functional changes in the hypothalamus. Proteomic analysis revealed that sodium tungstate modified the expression levels of proteins involved in cell morphology, axonal growth, and tissue remodeling, such as actin, CRMP2 and neurofilaments, and of proteins related to energy metabolism. Moreover, immunohistochemistry studies confirmed results for some targets and further revealed tungstate-dependent regulation of SNAP25 and HPC-1 proteins, suggesting an effect on synaptogenesis as well. Functional test for cell activity based on c-fos- positive cell counting also suggested that sodium tungstate modified hypothalamic basal activity. Finally, in vivo magnetic resonance imaging showed that tungstate treatment can affect neuronal organization in the hypothalamus. Conclusions: Altogether, these results suggest that sodium tungstate regulates proteins involved in axonal and glial plasticity. The fact that sodium tungstate could modulate hypothalamic plasticity and networks in adulthood makes it a possible and interesting therapeutic strategy not only for obesity management, but also for other neurodegenerative illnesses like Alzheimer's disease

Dual and Opposite Costimulatory Targeting with a Novel Human Fusion Recombinant Protein Effectively Prevents Renal Warm Ischemia Reperfusion Injury and Allograft Rejection in Murine Models

Many studies have shown both the CD28-D80/86 costimulatory pathway and the PD-1-PD-L1/L2 coinhibitory pathway to be important signals in modulating or decreasing the inflammatory profile in ischemia-reperfusion injury (IRI) or in a solid organ transplant setting. The importance of these two opposing pathways and their potential synergistic effect led our group to design a human fusion recombinant protein with CTLA4 and PD-L2 domains named HYBRI. The objective of our study was to determine the HYBRI binding to the postulated ligands of CTLA4 (CD80) and PD-L2 (PD-1) using the Surface Plasmon Resonance technique and to evaluate the in vivo HYBRI effects on two representative kidney inflammatory models-rat renal IRI and allogeneic kidney transplant. The Surface Plasmon Resonance assay demonstrated the avidity and binding of HYBRI to its targets. HYBRI treatment in the models exerted a high functional and morphological improvement. HYBRI produced a significant amelioration of renal function on day one and two after bilateral warm ischemia and on days seven and nine after transplant, clearly prolonging the animal survival in a life-sustaining renal allograft model. In both models, a significant reduction in histological damage and CD3 and CD68 infiltrating cells was observed. HYBRI decreased the circulating inflammatory cytokines and enriched the FoxP3 peripheral circulating, apart from reducing renal inflammation. In conclusion, the dual and opposite costimulatory targeting with that novel protein offers a good microenvironment profile to protect the ischemic process in the kidney and to prevent the kidney rejection, increasing the animal's chances of survival. HYBRI largely prevents the progression of inflammation in these rat models

Discovery of biomarker panels for neural dysfunction in inborn errors of amino acid metabolism.

Patients with inborn errors of amino acid metabolism frequently show neuropsychiatric symptoms despite accurate metabolic control. This study aimed to gain insight into the underlying mechanisms of neural dysfunction. Here we analyzed the expression of brain-derived neurotrophic factor (BDNF) and 10 genes required for correct brain functioning in plasma and blood of patients with Urea Cycle Disorders (UCD), Maple Syrup Urine Disease (MSUD) and controls. Receiver-operating characteristic (ROC) analysis was used to evaluate sensitivity and specificity of potential biomarkers. CACNA2D2 (α2δ2 subunit of voltage-gated calcium channels) and MECP2 (methyl-CpG binding protein 2) mRNA and protein showed an excellent neural function biomarker signature (AUC ≥ 0,925) for recognition of MSUD. THBS3 (thrombospondin 3) mRNA and AABA gave a very good biomarker signature (AUC 0,911) for executive-attention deficits. THBS3, LIN28A mRNA, and alanine showed a perfect biomarker signature (AUC 1) for behavioral and mood disorders. Finally, a panel of BDNF protein and at least two large neural AAs showed a perfect biomarker signature (AUC 1) for recognition of psychomotor delay, pointing to excessive protein restriction as central causative of psychomotor delay. To conclude, our study has identified promising biomarker panels for neural function evaluation, providing a base for future studies with larger samples

Tobacco cessation among smokers under substance use treatment for alcohol and/or cannabis: study protocol and pilot study

Background: Approximately 80% of people with a substance use disorder (SUD) are smokers. Starting SUD treatment offers the opportunity to also quit smoking. The ACT-ATAC project aims to identify the predictors associated with smoking cessation among persons treated for alcohol and/or cannabis use disorder in Barcelona. This manuscript reports its methodology and the experience of carrying it out during the COVID-19 pandemic. Methods: Mixed methods project with three substudies. Substudy 1 (S1) comprises heterogeneous discussion groups among clinicians. S2 has two prospective cohorts composed of smokers under treatment for alcohol and/or cannabis use disorder and the clinicians in charge of these patients. Participating smokers will be followed for 12 months and interviewed about their substance use and the tobacco cessation services received using the Spanish version of the users' Knowledge, Attitudes, and Services (S-KAS) scale. The clinicians will be asked about their self-reported practices in smoking cessation using the Knowledge, Attitudes, and Practices (S-KAP) scale. S3 comprises heterogeneous discussion groups with smokers. Data will be triangulated using qualitative and quantitative analyses. To facilitate the recruitment process, the researchers have introduced several strategies (design clear protocols, set monthly online meetings, extend the project, provide gift cards, etc.). Discussion: The results of S1 were used to develop the questionnaires. S2 required some adjustments due to the COVID-19 pandemic, particularly the follow-up interviews being conducted by phone instead of face-to-face, and the recruitment rhythm was lower than expected. Recruitment will last until reaching at least 200-250 users. The fieldwork could not have been possible without the collaboration of the ACT-ATAC team and the introduction of several strategies

Anti-obesity sodium tungstate treatment triggers axonal and glial plasticity in hypothalamic feeding centers.

International audienceOBJECTIVE: This study aims at exploring the effects of sodium tungstate treatment on hypothalamic plasticity, which is known to have an important role in the control of energy metabolism. METHODS: Adult lean and high-fat diet-induced obese mice were orally treated with sodium tungstate. Arcuate and paraventricular nuclei and lateral hypothalamus were separated and subjected to proteomic analysis by DIGE and mass spectrometry. Immunohistochemistry and in vivo magnetic resonance imaging were also performed. RESULTS: Sodium tungstate treatment reduced body weight gain, food intake, and blood glucose and triglyceride levels. These effects were associated with transcriptional and functional changes in the hypothalamus. Proteomic analysis revealed that sodium tungstate modified the expression levels of proteins involved in cell morphology, axonal growth, and tissue remodeling, such as actin, CRMP2 and neurofilaments, and of proteins related to energy metabolism. Moreover, immunohistochemistry studies confirmed results for some targets and further revealed tungstate-dependent regulation of SNAP25 and HPC-1 proteins, suggesting an effect on synaptogenesis as well. Functional test for cell activity based on c-fos-positive cell counting also suggested that sodium tungstate modified hypothalamic basal activity. Finally, in vivo magnetic resonance imaging showed that tungstate treatment can affect neuronal organization in the hypothalamus. CONCLUSIONS: Altogether, these results suggest that sodium tungstate regulates proteins involved in axonal and glial plasticity. The fact that sodium tungstate could modulate hypothalamic plasticity and networks in adulthood makes it a possible and interesting therapeutic strategy not only for obesity management, but also for other neurodegenerative illnesses like Alzheimer's disease

Urinary sulphatoxymelatonin as a biomarker of serotonin status in biogenic amine-deficient patients

Abstract Melatonin is synthesized from serotonin and it is excreted as sulphatoxymelatonin in urine. We aim to evaluate urinary sulphatoxymelatonin as a biomarker of brain serotonin status in a cohort of patients with mutations in genes related to serotonin biosynthesis. We analized urinary sulphatoxymelatonin from 65 healthy subjects and from 28 patients with genetic defects. A total of 18 patients were studied: 14 with autosomal dominant and recessive guanosine triphosphate cyclohydrolase-I deficiency; 3 with sepiapterin reductase deficiency; and 1 with aromatic L-amino acid decarboxylase deficiency. Further 11 patients were studied after receiving serotoninergic treatment (serotonin precursors, monoamine oxidase inhibitors, selective serotonin re-uptake inhibitors): 5 with aromatic L-amino acid decarboxylase deficiency; 1 with sepiapterin reductase deficiency; 3 with dihydropteridine reductase deficiency; and 2 with 6-pyruvoyltetrahydropterin synthase deficiency. Among the patients without therapy, 6 presented low urinary sulphatoxymelatonin values, while most of the patients with guanosine triphosphate cyclohydrolase-I deficiency showed normal values. 5 of 11 patients under treatment presented low urine sulphatoxymelatonin values. Thus, decreased excretion of sulphatoxymelatonin is frequently observed in cases with severe genetic disorders affecting serotonin biosynthesis. In conclusion, sulphatoxymelatonin can be a good biomarker to estimate serotonin status in the brain, especially for treatment monitoring purposes