The conformational diseases are a group of pathologies characterised by the aggregation and tissue deposition of aberrant proteins. This is the molecular mechanism of a number of neurological conditions, including Alzheimer¿s and Huntington¿s diseases, the spongiform encephalopathies and the serpinopathies, human pathologies caused by mutations that promote polymerisation and intracellular deposition of proteins of the serpin superfamily, leading to a poorly understood cell toxicity and death. This mutations cause serpin polymerisation and retention within the endoplasmic reticulum of the cell of synthesis. Several such mutations have been found in neuroserpin. Human neuroserpin is a serpin mainly expressed by neurons and is responsible for a genetic disease known as Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB). Its main symptoms are early onset dementia, epilepsy and neurodegeneration. So far, the exact mechanism of polymer toxicity is not completely clear. In this proposal, we aim to characterise the cell toxicity of neuroserpin polymers in our neural model of FENIB, focusing in particular on mitochondria and oxidative stress: in fact, we have found that cells expressing polymerogenic NS overexpress several anti-oxidant genes and undergo apoptosis when the anti-oxidant defences are blocked. Our preliminary results also suggest an alteration of mitochondrial physiology. Here we propose to further characterise the involvement of oxidative stress and mitochondria in the neuronal degeneration underlying the dementia FENIB.
This project is part of a new and intriguing field of research, regarding the role of oxidative stress and mitochondrial dynamics in relation to neuroserpin accumulation in neural cells. Despite that the molecular bases of the serpinopathies, particularly AAT deficiency and the dementia FENIB, are known since more twenty years, the role of oxidative stress in FENIB had not been addressed until our recent publication (1). Furthermore, the exact nature of the toxicity exerted by serpin polymers within the ER is still incompletely understood, and currently there is no treatment for the dementia FENIB, while alpha-1 antitrypsin deficiency is treated by supplying exogenous AAT to the patients to slow down disease progression. In the last five years, we have created a novel cell model overexpressing wild type or the pathological variant G392E of NS in mouse neural progenitor cells, which recapitulates the main features of FENIB and is now being used to study the mechanism behind the toxicity of NS polymers. In this model system, we have recently described that expression of G392E NS in cells differentiated to neurons leads to upregulation of several anti-oxidant genes, and inhibition of these defences leads to apoptosis (1), thus uncovering part of the toxic effects of NS polymers in FENIB brains.
The accumulation of serpin polymers within the ER could upset the redox balance in this organelle. Protein folding and oxidation inside the ER leads to the generation of reactive oxygen species (ROS), while alterations in the redox status and generation of ROS lead to perturbation of ER homeostasis and protein folding (2). It has been shown that the polymerogenic Z variant of AAT leads to increased expression of redox-regulating genes in the liver of a mouse model of the disease, as well as higher levels of ROS and oxidative liver damage in aged mice (3). Moreover, increased oxidative stress markers and reduced antioxidant defences have been found in plasma samples from children suffering from this condition (4). In the case of FENIB, our recent publication has confirmed the activation of an anti-oxidative response in neural cells overexpressing the polymerogenic G392E NS variant that causes severe dementia FENIB (1). These findings are based in our RNA sequencing analysis of neural cells expressing polymerogenic G392E NS, which has uncovered the upregulation of several anti-oxidant genes but also shown alterations in the expression of other interesting genes, including several genes involved in actin cytoskeleton dynamics. We are currently analysing the distribution of mitochondria in our neural cell lines overexpressing NS, and our preliminary results show alterations related to cell toxicity in G392E NS cells. Based on these findings, we now propose to study mitochondrial alterations in the cellular response to NS polymer accumulation within the ER, and their relationship with the oxidative stress described in these cells and to the dynamics of the actin cytoskeleton, particularly looking at the genes hinted by our RNA sequencing analysis. These aspects are novel in the study of the neurodegeneration FENIB, so our research will generate new knowledge on the mechanisms that mediate the toxic effects of NS polymers in this fatal form of dementia.
(1) Guadagno et al, 2017. Neurob Disease, 103:32-44
(2) Malhotra and Kaufman, 2007. Antioxid Redox Signal, 9:2277¿2293
(3) Markus et al, 2012. Exp Biol Med, 237:1163-1172
(4) Escribano et al, 2015, Thorax, 70:82-83