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Authors
Advisor(s)
Abstract(s)
Age-related synaptic dysfunctions comprise a range of pathophysiological mechanisms that are
correlated with cognitive decline. It has been demonstrated, both in animal models and human samples,
that there is an upsurge of A2A receptors within aging. In animals, this aberrant expression may be the
explanation for the mGluR-5 dependent overactivation of NMDA receptors, enhanced calcium influx,
dysfunctionalities in synaptic plasticity and increased glutamate release. Modeling aging in
differentiated human neurons has been limited, and alternative models are needed to better understand
the underlying mechanisms.
In this study, we followed a direct conversion protocol that, according to the literature, retain
important aging signatures, unlike iPSC-based reprogramming. Using a protocol from Dr. Jerome
Mertens and the Gage lab., we generated induced neurons (iNs) from three skin fibroblast samples,
including two from adult (< 65 years) and one from an old (> 65 years) individual. We have optimized
and adapted this protocol that combines the delivery of two transcription factors (TFs) and a cocktail of
small molecules, by identifying the appropriate cell density and plating method for further experiments
such as RT-qPCR, immunocytochemistry, and electrophysiology. We also characterized iNs according
to specific cell markers and electrophysiologic properties.
Our results demonstrated a decrease in fibroblast marker vimentin and increased expression of the
neuronal marker βIII-tubulin at 21 days, as well as VGLUT1 expression in both adult- and aged-derived
iNs, confirmed by RT-qPCR and immunocytochemistry. However, we did notice that iNs at 21 days
were still immature, as they lacked expression of some synaptic markers and the ability to fire action
potentials.
Overall, direct conversion represents a promising model for studying age-related synaptic
dysfunctions in humans. Optimal protocol adaptation and characterization of iNs made in this study can
facilitate further research to better assess the underlying mechanisms of age-related synaptic
dysfunctions. Futures studies will focus on maturation prospects so that iNs acquire functional synapses,
including co-culturing with astrocytes,
Description
Tese de mestrado, Engenharia Biomédica e Biofísica, 2023, Universidade de Lisboa, Faculdade de Ciências
Keywords
Envelhecimento Humano Fibroblastos Neurónios Conversão Direta Teses de mestrado - 2023