Development of a novel in vitro model of alzheimer's disease and its application to high-throughput screening assays
- Autores: Mateo Barro Fernández
- Directores de la Tesis: M. Isabel Loza García (dir. tes.), José Manuel Brea Floriani (dir. tes.)
- Lectura: En la Universidade de Santiago de Compostela ( España ) en 2025
- Idioma: inglés
- Tribunal Calificador de la Tesis: José Luis Labandeira-García (presid.), Luz Romero Alonso (secret.), Héctor Juan Caruncho Michinel (voc.)
- Programa de doctorado: Programa de Doctorado en Neurociencia y Psicología Clínica por la Universidad de A Coruña; la Universidad de Santiago de Compostela y la Universidad de Vigo
- Materias:
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- Resumen
Alzheimers disease (AD) is the leading neurodegenerative disorder worldwide and the most prevalent cause of dementia, accounting for 6080% of all cases. With an estimated 50 million individuals affected globally and the incidence projected to triple by 2050, AD represents a growing public health and socioeconomic burden. A small percentage of AD cases (15%) are familial in origin, caused by mutations in genes such as APP, PSEN1/2, and MAPT, which influence the amyloidogenic pathway or tau protein processing. Conversely, the sporadic form of AD, which constitutes the majority of cases, is linked to complex genetic and environmental interactions, including aging, previous pathologies, oxidative stress, and chronic inflammation. Alzheimers pathology is characterized by a progressive cognitive decline driven by the loss of synaptic integrity, neuronal death, and chronic neuroinflammation. These neurodegenerative changes and progression of the disease are underpinned by key hallmark features such as amyloid-ß and tau deposits. Despite extensive research over the past few decades, the pathogenesis of AD remains incompletely understood, primarily due to its multifactorial nature and the interplay of genetic, environmental, and biochemical risk factors.
Despite Alzheimers undeniable impact in current society, it remains as one of the diseases with the highest rate of failure in clinical trials and which therapeutic interventions remain palliative, addressing early-stage symptoms without halting disease progression. Taking in consideration these necessities, there is an unmet demand to develop translational models that can accelerate the pre-clinical drug discovery, enabling the detection of synergies and new mechanisms of action that are currently challenging to identify using traditional models. Therefore, the use of the differentiated SH-SY5Y neuroblastoma-derived cell line differentiated into a mature neuronal phenotype and transfected with fAD mutations could constitute an innovative biological platform for developing a scalable and automatable in vitro model for its application to high-throughput screening assays.
Therefore, in this doctoral thesis: i) A miniaturized and scalable neuronal phenotypic model was developed from the differentiation of the SH-SY5Y characterized by an increased number and density of neurites; ii) The transfection with APP V717I and MAPT P301L into induced significant morphological changes, including a reduction in the number of cells per well, a decrease in average neurite length, and reduced dendritic arborization; iii) It was demonstrated that treating the cell cultures with the reference neuroprotective compounds, melatonin and resveratrol, significantly reversed the morphological damage induced by the mutations; iv) A primary physiological assay based on intracellular calcium measurement was developed and optimized to study excitability changes in response to a depolarizing agent, registering a hypoexcitation in transfected cultures that melatonin and resveratrol were able to revert; v) It was confirmed that the model is suitable for high-throughput screening assays, by using the Prestwick® chemical library to evaluate the neuroprotective action of compounds against the hypo-excitability observed post-transfection (Z factor > 0.5). Eight compounds were identified for their ability to reverse the neuronal hypo-excitability with EC50 values ranging from 4 to 220 nm.
In conclusion, this doctoral thesis contributed to the development of a novel in vitro Alzheimers model, based on the use of the differentiated SH-SY5Y cell line to achieve a mature neuronal phenotype. This model proved to be applicable to scalable and automatable morphological and physiological assays for the characterization of neurodegenerative derived from cell transfection with APP V717I and MAPT P301L. Through these assays, it was possible to characterize the cytotoxic damage and hypo-excitability which were comparable to the neurodegenerative processes observed in AD. Furthermore, the compatibility of this model for application in high-throughput screening assays was validated using melatonin and resveratrol as reference compounds. Lastly, the model was applied to a screening of molecules from the Prestwick® chemical library, leading to the identification of eight hits with neuroprotective properties.
