As we age, tiny vesicles released by brain cells called astrocytes begin carrying a sticky protein, transglutaminase 2, that gums up the connections between neurons. This project tests the idea that this molecular gunk, not neuron death, is what causes the memory lapses and slower thinking that come with normal ageing—even in people without dementia. The problem is that scientists understand how synapses fail in Alzheimer’s disease, but not in healthy ageing. This research fills that gap by using human brain tissue and lab-grown mini-brains on a chip, rather than relying on animal models that don’t fully capture human biology. If the hypothesis holds, it could point to a drug target—perhaps a transglutaminase 2 inhibitor—that preserves cognitive function in older adults. That would shift the focus from treating dementia to preventing the subtle, universal decline that affects nearly everyone over 60. For now, the work is fundamental science: it asks *how* the ageing brain loses its edge, not yet *how to fix it*. But understanding that mechanism is the first step toward any future intervention.
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Ageing is inevitable and brings with it a general and progressive decline even in the absence of pathology. The brain is particularly affected by ageing. In fact, cognitive defects involving different brain processes, including attention, learning, and memory, are common even in individuals who are ageing physiologically. Several lines of evidence indicate that age-related cognitive impairment that occurs in the absence of neurodegenerative diseases are not associated with death of neurons, but with subtle alterations of the synapses, functional connections between neurons, mainly related to dysregulation of Ca2+ homeostasis in these cells. Still, the molecular processes underlying these alterations are largely obscure. In this project I aim to explore the molecular mechanisms at the basis of synaptic dysfunction typical of physiological brain ageing. I will build on our recent findings demonstrating that astrocytes, brain cells active at the synapse, can increase neuronal Ca2+ and alter synapse functionality through secretion of small membrane vesicles called extracellular vesicles (EVs) displaying on their surface transglutaminase 2 (TG2), a multifunctional protein whose levels and activity are increased in the ageing brain. TG2 may also affect synaptic function indirectly by influencing extracellular matrix (ECM), a network of extracellular molecules that stabilize synapses but should be remodelled to allow plasticity. Interestingly, synaptic alterations induced by astrocyte-derived EVs (AEVs) via TG2 resemble the ones found in aged brains. Still, the contribution of TG2 to synapse dysfunction in normal ageing is largely unexplored. In this work I will test the hypothesis that augmented TG2 expression and secretion from astrocytes via EVs, and/or TG2 altered activity, may cause synaptic dysfunction, affecting cognition during physiological ageing in humans. To this aim, I will employ state-of-the art human models that allow a better understanding of human biology compared to animals. In particular, I will: Aim 1) Analyse the changes in TG2 expression/activity and in the proteome of AEV during physiological ageing, by isolating AEV from the brain of aged (>60 years old) and adult (20-30 years old) healthy human subjects. AEVs from aged and adult brains will be measured by TRPS-qNano, the levels and activity of TG2 associated with AEVs will be evaluated by high-sensitivity ExoView technology and a specific TG2 activity assay; their proteome will be analysed by high-resolution mass spectrometry. Aim 2) Investigate how age-related alteration of TG2 content/activity in AEVs impacts Ca2+ and synaptic function in human iPSC-derived 2D neuronal cultures. Human induced pluripotent stem cell (iPSC)-derived neurons will be exposed to AEVs from aged/adult brains and EV effects on synaptic activity and plasticity will be evaluated by Ca2+-imaging, electrophysiology, immunocytochemistry for synaptic markers and dendritic spine analysis. TG2 involvement in aged AEV effects will be evaluated using TG2 inhibitors. Aim 3) Investigate whether age-related alteration of TG2 content/activity in AEVs affects ECM deposition, neuronal Ca2+, and synaptic transmission in a human 3D brain-on-a-chip. I will employ a 3D co-culture of human iPSC-derived brain cells in a microfluidic device, to test the effects of AEV from aged/adult brain and vesicular TG2 on the synapse in a system that recapitulates some aspects of cortical brain tissue and allows ECM studies. Filling a substantial gap of knowledge, this study will bring us to a deeper understanding of the processes underlying age-related physiological decline, and may drive the development of possible interventions aimed at preserving cognitive functions in elders.
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