Neuroserpin: structure, function, physiology and pathology

Neuroserpin is a serine protease inhibitor identified in a search for proteins implicated in neuronal axon growth and synapse formation. Since its discovery over 30 years ago, it has been the focus of active research. Many efforts have concentrated in elucidating its neuroprotective role in brain ischemic lesions, the structural bases of neuroserpin conformational change and the effects of neuroserpin polymers that underlie the neurodegenerative disease FENIB (familial encephalopathy with neuroserpin inclusion bodies), but the investigation of the physiological roles of neuroserpin has increased over the last years. In this review, we present an updated and critical revision of the current literature dealing with neuroserpin, covering all aspects of research including the expression and physiological roles of neuroserpin, both inside and outside the nervous system; its inhibitory and non-inhibitory mechanisms of action; the molecular structure of the monomeric and polymeric conformations of neuroserpin, including a detailed description of the polymerisation mechanism; and the involvement of neuroserpin in human disease, with particular emphasis on FENIB. Finally, we briefly discuss the identification by genome-wide screening of novel neuroserpin variants and their possible pathogenicity.

Neuroserpin Inclusion Bodies in a FENIB Yeast Model

FENIB (familial encephalopathy with neuroserpin inclusion bodies) is a human monogenic disease caused by point mutations in the SERPINI1 gene, characterized by the intracellular deposition of polymers of neuroserpin (NS), which leads to proteotoxicity and cell death. Despite the different cell and animal models developed thus far, the exact mechanism of cell toxicity elicited by NS polymers remains unclear. Here, we report that human wild-type NS and the polymerogenic variant G392E NS form protein aggregates mainly localized within the endoplasmic reticulum (ER) when expressed in the yeast S. cerevisiae. The expression of NS in yeast delayed the exit from the lag phase, suggesting that NS inclusions cause cellular stress. The cells also showed a higher resistance following mild oxidative stress treatments when compared to control cells. Furthermore, the expression of NS in a pro-apoptotic mutant strain-induced cell death during aging. Overall, these data recapitulate phenotypes observed in mammalian cells, thereby validating S. cerevisiae as a model for FENIB.

G392E neuroserpin causing the dementia FENIB is secreted from cells but is not synaptotoxic

Familial encephalopathy with neuroserpin inclusion bodies (FENIB) is a progressive neurodegenerative disease caused by point mutations in the gene for neuroserpin, a serine protease inhibitor of the nervous system. Different mutations are known that are responsible for mutant neuroserpin polymerization and accumulation as inclusion bodies in many cortical and subcortical neurons, thereby leading to cell death, dementia and epilepsy. Many efforts have been undertaken to elucidate the molecular pathways responsible for neuronal death. Most investigations have concentrated on analysis of intracellular mechanisms such as endoplasmic reticulum (ER) stress, ER-associated protein degradation (ERAD) and oxidative stress. We have generated a HEK-293 cell model of FENIB by overexpressing G392E-mutant neuroserpin and in this study we examine trafficking and toxicity of this polymerogenic variant. We observed that a small fraction of mutant neuroserpin is secreted via the ER-to-Golgi pathway, and that this release can be pharmacologically regulated. Overexpression of the mutant form of neuroserpin did not stimulate cell death in the HEK-293 cell model. Finally, when treating primary hippocampal neurons with G392E neuroserpin polymers, we did not detect cytotoxicity or synaptotoxicity. Altogether, we report here that a polymerogenic mutant form of neuroserpin is secreted from cells but is not toxic in the extracellular milieu.

Embelin as Lead Compound for New Neuroserpin Polymerization Inhibitors

Familial encephalopathy with neuroserpin inclusion bodies (FENIB) is a severe and lethal neurodegenerative disease. Upon specific point mutations in the SERPINI1gene-coding for the human protein neuroserpin (NS) the resulting pathologic NS variants polymerize and accumulate within the endoplasmic reticulum of neurons in the central nervous system. To date, embelin (EMB) is the only known inhibitor of NS polymerization in vitro. This molecule is capable of preventing NS polymerization and dissolving preformed polymers. Here, we show that lowering EMB concentration results in increasing size of NS oligomers in vitro. Moreover, we observe that in cells expressing NS, the polymerization of G392E NS is reduced, but this effect is mediated by an increased proteasomal degradation rather than polymerization impairment. For these reasons we designed a systematic chemical evolution of the EMB scaffold aimed to improve its anti-polymerization properties. The effect of EMB analogs against NS polymerization was assessed in vitro. None of the EMB analogs displayed an anti-polymerization activity better than the one reported for EMB, indicating that the EMB–NS interaction surface is very specific and highly optimized. Thus, our results indicate that EMB is, to date, still the best candidate for developing a treatment against NS polymerization.

Calcium signalling in mammalian cell lines expressing wild type and mutant human α1-Antitrypsin

A possible role for calcium signalling in the autosomal dominant form of dementia, familial encephalopathy with neuroserpin inclusion bodies (FENIB), has been proposed, which may point towards a mechanism by which cells could sense and respond to the accumulation of mutant serpin polymers in the endoplasmic reticulum (ER). We therefore explored possible defects in Ca2+-signalling, which may contribute to the pathology associated with another serpinopathy, α1-antitrypsin (AAT) deficiency. Using CHO K1 cell lines stably expressing a wild type human AAT (MAAT) and a disease-causing polymer-forming variant (ZAAT) and the truncated variant (NHK AAT), we measured basal intracellular free Ca2+, its responses to thapsigargin (TG), an ER Ca2+-ATPase blocker, and store-operated Ca2+-entry (SOCE). Our fura2 based Ca2+ measurements detected no differences between these 3 parameters in cell lines expressing MAAT and cell lines expressing ZAAT and NHK AAT mutants. Thus, in our cell-based models of α1-antitrypsin (AAT) deficiency, unlike the case for FENIB, we were unable to detect defects in calcium signalling.