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 is at the base of a class of pathologies called the serpinopathies, which are due to mutations that cause serpin polymerisation and retention within the endoplasmic reticulum of the cell of synthesis. Six different polymerisation-causing mutations have been found in neuroserpin, a secreted serpin mainly expressed by neurons, as the cause of a rare but deadly type of 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 cell toxicity effects of polymer accumulation inside the endoplasmic reticulum. We have recently created a neural expression system and performed a RNA sequencing comparison of control cells and cells expressing a severe pathological FENIB variant of NS. 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 mitochondrial and cytoskeleton dynamics, focusing on several genes identified in our RNA sequencing of G392E NS cells, in the neuronal degeneration underlying the dementia FENIB.
The molecular bases of the serpinopathies, particularly alpha-1 antitrypsin deficiency and the dementia FENIB due to polymer formation by mutant alpha-1 antitrypsin and NS respectively, are known since more twenty years. Nevertheless, 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 and only palliative treatment for alpha-1 antitrypsin deficiency. 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 for the first time a role for oxidative stress in the neuronal toxicity caused by the FENIB-causing variant G392E NS (Guadagno et al, 2017), thus uncovering part of the mechanism that renders neurons more susceptible to apoptosis in FENIB brains. These findings have been possible thanks to our RNA sequencing analysis of neural cells expressing polymerogenic G392E NS, which has produced other interesting hits that are the subject of our ongoing studies.
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 alter ER homeostasis and protein folding (reviewed in Malhotra et al., 2007). Both ER stress and oxidative stress, through ROS generation, may increase the leak of Ca2+ from the ER lumen as well as induce protein and lipid oxidation. Moreover, the very close proximity of ER and mitochondria easily leads to 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: cellular oxidative stress.
In this context, the accumulation of serpin polymers within the ER could upset the redox balance in this organelle. It has been shown that the polymerogenic Z variant of alpha-1 antitrypsin, the canonical serpin and causative of 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 is 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). In line with these findings, we now propose to look at the mitochondrial state and the role of Ca2+ in the cellular response to NS polymer accumulation within the ER, and their relationship with the oxidative stress described in these cells. This is supported by previous findings showing a role for Ca2+ in the ER stress response to NS polymers (Davies et al, 2009), by our preliminary findings of an altered distribution of mitochondria in G392E NS cells differentiated to neurons, and by the identification by RNA sequencing of G392E NS cells of several genes involved with cytoskeleton behaviour that show altered expression. These studies are novel in the field of the serpinopathies, so our research will shed new light into the intracellular pathways that mediate the toxic effects of NS polymers in the dementia FENIB, and will also be relevant for other diseases related to serpin polymerisation and/or leading to neurodegeneration.
Specific literature
Davies MJ et al, 2009. J. Biol. Chem 284:18202-18209
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