Neuroserpin is one of the serpins (serin protease inhibitors), a conserved superfamily of proteins that inhibit serin proteases by a mechanism that requires a high structural flexibility, and which renders serpin proteins very sensitive to point mutations that alter their folding and cellular handling. This molecular mechanism underlies a class of pathologies called the serpinopathies, where point mutations cause serpin polymerisation and retention within the endoplasmic reticulum of the cell of synthesis. To date, six different mutations have been found in neuroserpin, a secreted serpin mainly expressed by neurons, which cause polymer formation and a rare but deadly type of neurodegenerative dementia called FENIB. Although the pathological manifestations of serpin polymerisation depend on the inhibitory target and place of action of each specific serpin, the molecular mechanism is common and several aspects remain obscure for all serpinopathies. Particularly, little is known about the cellular toxicity effects of polymer accumulation inside the endoplasmic reticulum. We have recently published our novel neuronal cell model of FENIB, where we describe for the first time an oxidative stress response in cells expressing the highly polymerogenic G392E variant of NS. These cells undergo apoptosis when the anti-oxidant defences are inhibited pharmacologically, and our recent results show alterations in mitochondrial distribution in G392E NS neurons. This phenotype is worsened by a chelator of the antioxidant molecule glutathione and ameliorated by anti-oxidant molecules, and is associated to alterations in neuronal morphology, supporting a link between oxidative stress, mitochondrial dysfunction and cell toxicity in the neurodegeneration FENIB. This proposal aims to continue our studies on the alterations caused by polymers of NS to the endoplasmic reticulum and mitochondria, and how they lead to neuronal death in FENIB.
The molecular bases of the serpinopathies, particularly the dementia FENIB and alpha-1 antitrypsin deficiency, due to polymer formation by mutant NS and alpha-1 antitrypsin respectively, are known since more than twenty years. Nevertheless, the exact nature of the cellular toxicity exerted by serpin polymers is still incompletely understood, and as yet there is no treatment for the dementia FENIB and only palliative treatments for the pathological manifestations of alpha-1 antitrypsin deficiency. In recent 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 for the first time a role for oxidative stress in the neuronal toxicity caused by NS polymers (Guadagno et al., 2017), thus uncovering part of the mechanism that renders neurons more susceptible to apoptosis in FENIB brains. Moreover, our recent data demonstrates the presence of alterations in mitochondrial distribution in cells expressing mutant NS, in correlation with the observed oxidative stress, supporting a role for mitochondrial dysfunction in this neurodegenerative condition, as already described for other forms of neurodegeneration.
Accumulating evidence suggests that protein folding and generation of reactive oxygen species (ROS) as a by-product of protein oxidation in the ER are closely linked events. Conversely, alterations in the redox status and generation of ROS can alter ER homeostasis and protein folding (Malhotra et al., 2007). Both ER stress and oxidative stress, through ROS generation, may increase the leak of Ca2+ from the ER, as well as induce protein and lipid oxidation. Moreover, the very close proximity of ER and mitochondria in the mitochondria associated membrane (MAM) areas promotes the accumulation of cytosolic Ca2+ near mitochondria (Jacobson and Duchen, 2002), leading to ROS generation as a consequence of increased mitochondrial Ca2+ loading. High levels of mitochondrial ROS further increase Ca2+ release from the ER, generating a vicious cycle of ROS production that takes place during cellular oxidative stress.
The accumulation of serpin polymers within the ER may upset the redox balance in this organelle. It has been shown that the polymerogenic Z variant of alpha-1 antitrypsin, the archetypical serpin which undergoes polymerisation in the serpinopathy alpha-1 antitrypsin deficiency, 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 (Markus et al., 2012). The involvement of oxidative stress in alpha-1 antitrypsin deficiency in humans is supported by the presence of increased oxidative stress markers and reduced antioxidant defences in blood in a cohort of children suffering from this condition (Escribano et al., 2015). 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 (Guadagno et al., 2017), and more recently the alteration of the mitochondrial distribution in these cells, which is worsened by depletion of glutathione and rescued by anti-oxidant molecules (our unpublished results). In continuation with these findings, we now propose to study the neuronal morphology associated to each type of mitochondrial distribution, to investigate the functional properties of mitochondria and the distribution of ER-mitochondria contact sites in control and G392E NS cells, and to assess the activation of ER stress responses by NS polymers in our neuronal system. These studies are novel in the field of FENIB and the serpinopathies, so we expect that our research will shed new light into the intracellular pathways that mediate the toxic effects of NS polymers in the dementia FENIB, as well as be relevant for other diseases related to serpin polymerisation and other types of neurodegeneration.
Literature cited
Escribano et al, 2015, Thorax, 70:82-83
Guadagno et al, 2017. Neurob Disease, 103:32-44
Jacobson and Duchen, 2002. J Cell Science, 115:1175-1188
Malhotra and Kaufman, 2007. Antioxid Redox Signal, 9:2277¿2293
Markus et al, 2012. Exp Biol Med, 237:1163-1172