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, which renders serpin proteins very sensitive to point mutations that alter their folding. 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 created a 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 ameliorated by antioxidant molecules and is associated to a reduction in ER-mitochondria contact sites and alterations in neuronal morphology, suggesting a link between oxidative stress, mitochondrial dysfunction and cytoskeletal alterations in FENIB. This proposal is a continuation of our studies and focuses on the creation of a novel neuronal model with inducible expression of wild type or G392E NS, which will allow us to study the acute effects of NS polymer accumulation and to uncover new toxicity pathways by mRNA sequencing comparison of wild type and G392E NS neurons.
The molecular bases of the serpinopathies, particularly the dementia FENIB and alpha-1 antitrypsin (AAT) deficiency, which are caused by polymerisation of mutant NS and AAT respectively, are known since more than twenty years. Nevertheless, the exact nature of the cellular toxicity exerted by serpin polymers is still incompletely understood. As yet, there is no treatment for the dementia FENIB and only palliative treatments for the pathological manifestations of AAT deficiency. Recently, a small molecule specific for AAT able to prevent its polymerisation both in vitro and in cells expressing a polymerogenic variant has been described (Lomas et al., 2021), increasing the hopes of getting a pharmacological therapy useful for AAT deficiency patients, leading the way for all serpinopathies. We have in the past collaborated with groups at the CNR and University of Milan in the screening of small molecule inhibitors of NS polymerisation (Saga et al., 2016; Visentin et al., 2020), and future studies in this direction will highly benefit from our cell models of FENIB, in which the activity of potential drug candidates can be assessed at the cellular level.
In recent years, we have created a unique cell model overexpressing WT or the pathological G392E variant of NS in mouse neural progenitor cells, which recapitulates the main features of FENIB, and we are now using it to study the mechanisms behind the toxicity of NS polymers. In this model system, we have described for the first time a role for oxidative stress in the neuronal toxicity caused by polymerogenic NS (Guadagno et al., 2017), uncovering part of the mechanism that renders neurons more susceptible to apoptosis in FENIB brains. Our recent data has also revealed for the first time that neuronal expression of polymerogenic NS causes alterations in the mitochondrial network and in the cross talk between the ER and mitochondria (D¿Acunto et al., manuscript in preparation), providing the first evidence of mitochondrial dysfunction in this neurodegenerative condition, similarly to other forms of neurodegeneration. Our latest research is focused on yet another novel aspect of FENIB: the reduction in neurite extension observed in neurons overexpressing G392E NS. Physiologically, NS plays important roles in neuronal differentiation, neurite extension and synapse formation during the development of the nervous system, and in synapse remodelling during adulthood (Lee et al., 2017). Since our neuronal model is based on mouse neural progenitors that correctly express endogenous mouse NS, our results are the first demonstration of a dominant-negative effect for overexpressed polymerogenic NS during neuronal differentiation. All these results support our neuronal model system as highly useful to uncover the pathological events underlying FENIB, a field in which our group is leading the way in the international context.
The acute effects of polymerogenic neuroserpin expression and polymer accumulation within the ER of neurons are still unknown. In this proposal we intend to address this aspect by creating a novel inducible neuronal model system that, by allowing to switch neuroserpin expression on and off, can be used to study the initial alterations caused by polymerogenic NS expression before (neural progenitors), during and after neuronal differentiation. We will interrogate this system in two parallel ways: an informed approach, by verifying our previous data about oxidative stress and mitochondrial alterations obtained with the constitutive version of this cellular model, and an unbiased approach, based on mRNA sequencing comparison of WT and G392E NS cells after differentiation to neurons, using the isogenic lines in the `off¿ state as negative controls. This approach is completely novel in the field and addresses aspects of FENIB that have not been reported before, making our proposal highly innovative in the field. We expect that our research continues to expand the knowledge about 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 for other types of neurodegeneration.
Literature cited
Guadagno et al, 2017. Neurob Disease 103:32-44
Lee et al, 2017. Semin Cell Dev Biol 62:152-9
Lomas et al, 2021. EMBO Mol Med 13:e13167
Saga et al, 2016. Sci Rep 6:18769
Visentin et al, 2020. Life 10:111