Proyecto FONDECY Adjudicado por el Dr. Jorge Patricio Fuentealba en el Concurso Regular FONDECYT 2013

Se transcribe el Resumen del Proyecto FONDECYT obtenido por el Dr. Jorge Patricio Fuentealba Arcos, luego que 9 grupos de investigación de la Facultad de Ciencias Biológicas se adjudicaron proyectos presentados al Concurso de Proyectos Regular Fondecyt  2013.

Título  del  Proyecto

Cellular and Molecular Determinants of Purinérgic Contribution to Neuronal Dishomeostasis on Alzheimer Disease

Abstract

Alzheimer´s disease (AD) is probably the most common senile dementia, accompanied by symptoms like cognitive dysfunction, memory loss and behavioural changes as consequences of synaptic damage and neuronal death. The beta-amyloid peptide (βA) has been described as a key element in the onset and progression of AD, however the specific cellular alterations at the synapse are not fully understood. Among the spectrum of aggregate forms from monomers to plaques, soluble oligomers of beta amyloid peptide (SO-βA, 50-70 kD) have been widely described as the main neurotoxic species. The principal mechanism proposed has been the property of forming a non selective pore in the plasma membrane, inducing membrane damage that results in cytosolic Ca2+ overload, leakage of key intracellular molecules and cell death.

In our laboratory, acute exposure of neurons to SO-βA induced several effects in agreement with the above hypothesis of membrane damage (i.e. fast Ca2+ increase, strong increase in synaptic activity (80%) and miniature currents (two fold) in neurons), suggesting early excitotoxicity. Chronic treatment (24-48 h) of the neurons with SO-βA, on the other hand, showed a potent synaptic silencing (≈85%), reduction in spontaneous Ca2+ activity (≈75%) in the neuronal network and a sustained increase in cell death. More recently, using an in vitro AD model induced by βA, we have found that the neurons undergo cytosolic ATP leakage (Fuentealba et al., 2011) and mitochondrial Ca2+ oscillations (unpublished data) through the amyloid pore (Sepulveda et al., 2010). It is possible that this ATP leakage might induce an increment in auto/paracrine activation of purinergic receptors, mainly P2X subtypes present in the neuronal membrane that could enhance Ca2+ overload, mitochondrial dysfunction, and contribute to the synaptic failure and cellular toxicity present in AD.

This suggests a potential new target in the cellular physiopathology of AD that could contribute to toxicity. Observations made in our laboratory show an overexpression of P2X2 receptors in hippocampal neurons, but not in PC12 cells treated with SO-βA (24 h), supporting the existence of a new element in the AD neurotoxicity induced by SO-βA that needs to be characterized and studied in detail.

A pore formation in neurons induce a membranebTaken together, we postulate that

dyshomeostasis, which includes a sustained leak of intracellular ATP that activates neuronal

P2X purinergic receptors (auto- and/or paracrinally). Subsequently, this stimulation produces

A onbchanges in the expression and function of these receptors in neurons, enhancing the toxicity of  neuronal Alzheimer Disease models. Classically, alterations in purinergic transmission have been described as a glial response to CNS injury (inflammation, stroke, etc), but information about the consequences that this could have on the expression of specific P2X2 subunits (up/down regulation) in the neuronal membrane has not been proposed, nor a direct correlation with aging as a potentiating factor of cellular βA toxicity. In this proposal, using two cellular neurodegenerative models of AD A, we will evaluate the neuronal purinergic P2X receptor contribution on the toxicbconditioned by SO-A, characterizing the changes in their expression levels (Western blot, RT-PCR and QPCR),bmechanism of  functional and pharmacological properties (electrophysiology and purinergic modulators), spatial distribution, changes in Ca2+ signalling and interaction with membrane proteins (confocal techniques,

ratiometric and non-ratiometric fluorescent proteins). Our preliminary results demonstrate that the use of suramine or PPADs prevent the SO-βA toxicity in neurons, suggesting a role of P2X2 receptors (see above). On the other hand, the direct interaction between SO-βA and isolated mitochondria abolished the Ca2+ oscillations observed in intact cells, suggesting that the plasma membrane is an important element for the onset of toxic effects.

The results obtained from this proposal, will be of high relevance to define a new target on the physiopathology of AD, contributing to establish new possible therapeutics targets and treatments.