Neuroserpin (NS) 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 (ER). To date, six different mutations have been described in NS, a secreted serpin expressed mainly 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 ER. We have recently published our neuronal cell model of FENIB and described for the first time an oxidative stress response in cells expressing the highly polymerogenic G392E variant of NS. These cells undergo apoptosis when the antioxidant 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 antioxidant molecules, and is associated to a reduction in ER-mitochondria contact sites and alterations in neuronal morphology, supporting a link between oxidative stress, mitochondrial dysfunction and cytoskeletal alterations in the neurodegeneration FENIB. This proposal is thus a continuation of our studies on the cellular alterations, with a novel focus on cytoskeletal effects, caused by polymerogenic mutant NS and how they lead to neuronal death in FENIB.
The molecular bases of the serpinopathies, particularly the dementia FENIB and alpha-1 antitrypsin deficiency, which are caused by polymerisation of 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.
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). 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 polymerogenic NS (Guadagno et al., 2017), thus uncovering part of the mechanism that renders neurons more susceptible to apoptosis in FENIB brains. Our recent data has also revealed alterations in mitochondrial distribution, mitochondrial inner membrane potential and ER-mitochondria cross-talk in cells expressing G392E NS (D¿Acunto et al., manuscript in preparation), supporting for the first time a role for mitochondrial dysfunction in this neurodegenerative condition, similar to other forms of neurodegeneration.
We now propose to address the role of the cytoskeleton in FENIB, based on our preliminary results showing morphological alterations in G392E NS neurons, which display shorter neurites than WT NS controls (Fig. 1), and the changes in expression levels for genes involved in actin cytoskeleton dynamics that we observed in our mRNA sequencing analysis (Guadagno et al., 2017). The actin cytoskeleton is a highly dynamic cellular structure active in the intracellular transport of vesicles and organelles, the control of cellular shape and in cell movement. Neurons are particularly dependent on the microtubule and actin cytoskeletons for organising their highly branched morphology, both in shape and function, including mitochondrial distribution. Our findings of defective mitochondrial distribution and reduced neurite extension in neurons overexpressing polymerogenic NS strongly suggests the involvement of the cytoskeleton in the cellular responses associated to FENIB, and our mRNA analysis has provided several candidates to investigate, reinforcing the rational for this project. The role of the cytoskeleton is the object of active research for other neurodegenerative conditions (Lai and Wonk, 2020), and has not been addressed before for FENIB or for other serpinopathies that present upon polymer formation by a mutant serpin in other cell types, making our proposal highly innovative in the field. We expect that our research will expand the knowledge of the intracellular pathways that mediate the neurotoxic effects of polymerogenic mutant NS 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
Lai and Wonk, 2020. Ageing Res Rev, 58:101021
Markus et al, 2012. Exp Biol Med, 237:1163-1172