Tyrosine Hydroxylase Deficiency: Studies in patient samples and in a cellular model

dc.contributor
Universitat de Barcelona. Facultat de Biologia
dc.contributor.author
Tristán Noguero, Alba
dc.date.accessioned
2020-01-21T08:41:26Z
dc.date.available
2020-11-21T01:00:13Z
dc.date.issued
2019-11-22
dc.identifier.uri
http://hdl.handle.net/10803/668313
dc.description
Programa de Doctorat en Biomedicina
dc.description.abstract
Monoamine Neurotransmitter diseases are a rare group of inherited disorders of metabolism that encompass 12 different genetic defects leading to abnormal dopamine and/or serotonin brain homeostasis. They correspond to enzymatic deficiencies involved in the biosynthesis, catabolism and transport of dopamine and serotonine. From a clinical point of view, they can appear at any age and manifest diverse clinical features. However, movement disorders ranging from dopa-responsive dystonia to severe parkinsonism, associated to variable degrees of cognitive impairment is the most common form of presentation. The pathophysiology underlying the wide spectrum of clinical phenotypes (from mild to severe) and response to neurotransmitter precursors (L-Dopa+carbidopa, BH4 and other dopaminergic enhancers) has not been studied in detail. Here we aim to address this important issue through different approaches: i) the study of a particular disease of neurotransmitters, Tyrosine Hydroxylase deficiency (THD), as a model of dopaminergic deficiency. Tyrosine hydroxylase (TH) enzyme catalyses the rate-limiting step in the biosynthesis of dopamine (DA). THD exhibits a wide spectrum of clinical manifestations that have been grouped according to the severity in two clinical phenotypes: “Type A” tends to present as L-Dopa responsive parkinsonism-dystonia whereas “Type B” produces a severe encephalopathy of early-onset with sub-optimal L-Dopa response. ii) The study of a large cohort of patients with neurotransmitter defects thanks to an international collaboration (I-NTD group). We have used both patients’ samples and an iPSc model to address these questions. A) Patients’ samples: A.1) We studied one THD B phenotype postmortem brain and we observed that the expression of key synaptic proteins and neurodevelopmental markers were altered: TH, VMAT 1 and 2 and dopamine receptors, especially D2DR were decreased. GABAergic and glutamatergic proteins such as GABAVT, NMDAR1 and calbindin were also altered. Finally, developmental markers for synapses, axons and dendrites were decreased, whereas markers of neuronal volume were preserved. A.2) 94 CSF samples of patients with neurotransmitter defects from 9 centres belonging to different countries/continents were collected in an international collaboration through the I-NTD working group. The proteomic study showed that the main category of overrepresented proteins was related to nervous system development. Moreover, different proteins were detected that could be useful biomarkers for severity prognosis and response to treatment that are specific of disorders. Four of them were correctly validated with an ELISA analysis: APOD, COL6A3, APOH and OMGP. These proteins are involved in diverse important biological functions such as myelination, phospholipid and other lipid related processes. B) iPSC model of THD: iPSC lines from Type A and B patients, controls and an isogenic corrected iPSC line were generated. Upon Dopaminergic differentiation, THD A and B neurons reproduced the disease-associated phenotype: decreased TH Protein, reduced enzyme activity and alteration on DA genes expression. A new neuronal phenotype was also described: less TH-immunoreactive cells and fiber density in both mutant TH+ neurons, Type A and B DAn presented altered morphology (reduced neuronal arborisation only in THD-Type B) and a reduced axonal TH localization was observed in THD-Type A. We were also able to test therapeutic approaches such as L-Dopa + carbidopa. To conclude, we have performed extensive and novel studies using different approaches and techniques to better characterize the pathophysiology underlying the spectrum of severity and response to the current pharmacological treatments of neurotransmitter defects. We have provided new information pointing towards a dysregulation of multiple neurodevelopmental functions in these diseases, and biomarkers of clinical severity that could be explored in the future as therapeutic targets. Additionally, an iPSC model for THD has been developed for the first time that introduces mechanistic and therapeutic insights in this early-parkinsonism model.
dc.description.abstract
Los errores congénitos de las monoaminas (ECMM) son defectos en la síntesis, degradación o transporte de catecolaminas y serotonina. Las manifestaciones neurológicas incluyen trastornos del movimiento, retraso mental y encefalopatías. La Deficiencia de la TIrosina Hidroxilasa (THD) es un ECMM causado por un defecto en el enzima TH que cataliza el paso limitante en la biosíntesis de Dopamina (DA). Dos fenotipos clínicos se han descrito: el “A” que tiende a responder a la L-Dopa y el “B” que produce una encefalopatía grave de inicio temprano con mala respuesta a la L-Dopa. Para elucidar la gran variabilidad en cuanto a fenotipo y respuesta al tratamiento de estas enfermedades hemos realizado diferentes aproximaciones en muestras de pacientes y en un modelo celular. A) Pacientes: A.1) Estudiamos un cerebro postmortem THD de fenotipo B y observamos que la expresión de proteínas sinápticas clave y marcadores de neurodesarrollo estaban alteradas: reducción en la expresión de TH, VMAT 1 y 2 y los receptores de dopamina, especialmente D2DR. También se detectaron proteínas GABAérgicas y glutamatérgicas (GABAVT, NMDAR1 y calbindina) alteradas. Finalmente, los marcadores de desarrollo para sinapsis, axones y dendritas estaban disminuidos, mientras que los marcadores de volumen neuronal estaban preservados. A.2) 94 muestras de LCR de pacientes con ECMM fueron recogidas. El estudio de proteómica mostró que la categoría principal de proteínas sobrerrepresentada estaba relacionada con el desarrollo del sistema nervioso. Además, se detectaron diferentes proteínas que podrían ser biomarcadores útiles para el pronóstico de severidad y respuesta al tratamiento. Cuatro se validaron mediante un análisis ELISA: APOD, COL6A3, APOH y OMGP. B) Modelo celular Generamos líneas iPSC a partir de pacientes A y B, controles y una línea iPSC corregida. Tras la diferenciación DAn, las neuronas THD A y B reproducen el fenotipo de la enfermedad: proteína TH y actividad enzimática disminuidas y alteración en la expresión de genes DA. También se describió un nuevo fenotipo neuronal: menos células TH-inmunorreactivas, menor densidad fibrilar y alteraciones morfológicas en las nDA de ambos. Una reducción de la arborización neuronal en el B y la reducción de la localización axonal de TH en el A. Pudimos probar diferentes aproximaciones terapéuticas.
dc.format.extent
143 p.
dc.format.mimetype
application/pdf
dc.language.iso
eng
dc.publisher
Universitat de Barcelona
dc.rights.license
ADVERTIMENT. L'accés als continguts d'aquesta tesi doctoral i la seva utilització ha de respectar els drets de la persona autora. Pot ser utilitzada per a consulta o estudi personal, així com en activitats o materials d'investigació i docència en els termes establerts a l'art. 32 del Text Refós de la Llei de Propietat Intel·lectual (RDL 1/1996). Per altres utilitzacions es requereix l'autorització prèvia i expressa de la persona autora. En qualsevol cas, en la utilització dels seus continguts caldrà indicar de forma clara el nom i cognoms de la persona autora i el títol de la tesi doctoral. No s'autoritza la seva reproducció o altres formes d'explotació efectuades amb finalitats de lucre ni la seva comunicació pública des d'un lloc aliè al servei TDX. Tampoc s'autoritza la presentació del seu contingut en una finestra o marc aliè a TDX (framing). Aquesta reserva de drets afecta tant als continguts de la tesi com als seus resums i índexs.
dc.source
TDX (Tesis Doctorals en Xarxa)
dc.subject
Malalties de l'aparell locomotor
dc.subject
Enfermedades del aparato locomotor
dc.subject
Musculoskeletal system diseases
dc.subject
Cèl·lules mare
dc.subject
Células madre
dc.subject
Stem cells
dc.subject.other
Ciències Experimentals i Matemàtiques
dc.title
Tyrosine Hydroxylase Deficiency: Studies in patient samples and in a cellular model
dc.type
info:eu-repo/semantics/doctoralThesis
dc.type
info:eu-repo/semantics/publishedVersion
dc.subject.udc
577
dc.contributor.director
García Cazorla, Àngels
dc.contributor.director
Consiglio, Antonella
dc.contributor.tutor
Alcántara Horrillo, Soledad
dc.embargo.terms
12 mesos
dc.rights.accessLevel
info:eu-repo/semantics/openAccess


Documents

ATN_PhD_THESIS.pdf

11.69Mb PDF

Aquest element apareix en la col·lecció o col·leccions següent(s)