Molecular and Functional Characterization of Neuromelanin and its Analogs in Neurodegeneration

• Autores: Alexandra Moreno García
• Directores de la Tesis: Miguel Calero Lara (dir. tes.), Olga Calero Rueda (codir. tes.)
• Lectura: En la Universidad Autónoma de Madrid ( España ) en 2022
• Idioma: inglés
• Número de páginas: 210
• Tribunal Calificador de la Tesis: Javier Saez Valero (presid.), Maria del Mar Pérez Martínez (secret.), Miguel Medina Padilla (voc.)
• Programa de doctorado: Programa de Doctorado en Biociencias Moleculares por la Universidad Autónoma de Madrid
• Materias:
  • Química
    • Bioquímica
      • Biología molecular
  • Ciencias médicas
    • Patología
      • Neuropatología
• Enlaces
  • Tesis en acceso abierto en: Biblos-e Archivo
• Resumen

  • Historically, neuromelanin (NM) has been considered a waste product of catecholaminergic metabolism. A key feature of NM, as well as all other melanins, is related to their heterocyclic ring-structure with conjugated double bonds that creates a π-bond system across multiple atoms that lowers the energy and stabilizes the molecule. Due to this conformation, melanins have been attributed with many functionalities from which stands out its ability to trap UV–light, free radicals, toxins, and metals.

    The presence in human brain of NM at localizations related to the main neurodegeneration foci, such as the sustantia nigra (SN) in Parkinson's disease (PD) and the locus coeruleous (LC) in Alzheimer's disease (AD), together with NM ability to absorb free radicals and chelate metals and drugs suggest a physiological role of NM in neurodegeneration. However, despite several reports suggesting the involvement of NM in neurodegenerative disorders, the molecular basis for such observations remains largely unknown.

    The focus of this doctoral thesis is the molecular and functional characterization of NM and three analogs based on three catecholamines potentially precursors of its synthesis in the brain. The results are divided into chapters composed of several experimental studies, focusing on the characterization at the molecular and functional level of NM and its analogs, while developing new approaches and methodologies for NM analysis.

    Since NM is a dynamic and complex substance with an undefined composition, as NM proxies, we selected three analogs produced by the oxidation of three catecholamines involved in NM synthesis: pLD, pDA and pNA from L-DOPA, DA or NA, respectively.

    In this work, by using a repertoire of techniques (thioflavin T aggregation kinetics, transmission electronic microscopy, transgenic C. elegans in vivo model), we demonstrated that NM analogs interact and modulate the aggregation kinetics of amyloid-prone proteins, pointing directly to one of the potential key roles of NM in neurodegeneration.

    We have also developed monoclonal antibodies against NM as a valuable biochemical tool for in vitro and ex-vivo NM characterization. Although the production of monoclonal antibodies is generally highly standardized for proteins, we were confronted with a polymeric substance with a nature very different from protein antigens that compelled us to develop alternative strategies for immunization by using NM coated to silica beads and an extended immunization protocol.

    Moreover, through plasma analysis by affinity chromatography and co-immunoprecipitation of NM, we have identified a subset of NM binding proteins and characterized their potential interaction mechanisms. Functional pathways analysis indicated that NM could interfere with inflammation and vascular processes, cytoplasm organization and the respiratory chain. All this events have been linked to AD and PD pathophysiology. Additionally, the use of cell cultures allowed us to study the cytotoxicity and proinflammatory effects triggered by NM.

    We have found that NM presents a characteristic fluorescence spectrum that can be exploited for the sensitive label-free detection of this substance in brain tissue. However, lipofuscin (LF) aggregates, another aging pigment, also presents an intense fluorescence throughout the brain tissue. By using confocal microscopy with specific settings for the collection of emission spectra in the near infrared region and the use of time-resolved fluorescence, we developed new methodologies to characterize the properties and distribution of NM, distinguishing structures composed of NM, LF or both.

    The data gathered in this thesis, together with previous findings, provide clues on the role of NM on neurodegenerative processes, changing the NM concept from metabolic “garbage” to an active substance, which is a key actor with a dual role in the physiopathology of neurodegeneration.