Structural targeting of a familial variant of neuroserpin (Ser52Arg) by epigallocatechin gallate binding that reduces polymerization and enhances the tissue plasminogen activator inhibition.

The serpinopathies result from conformational transitions in members of the serine proteinase inhibitor superfamily with aberrant tissue deposition or loss of function. They are typified by mutants of neuroserpin that are retained within the endoplasmic reticulum of neurons as ordered polymers in association with dementia. We show here that the S49P mutant of neuroserpin that causes the dementia familial encephalopathy with neuroserpin inclusion bodies (FENIB) forms a latent species in vitro and in vivo in addition to the formation of polymers. Latent neuroserpin is thermostable and inactive as a proteinase inhibitor, but activity can be restored by refolding. Strikingly, latent S49P neuroserpin is unlike any other latent serine proteinase inhibitor (serpin) in that it spontaneously forms polymers under physiological conditions. These data provide an alternative method for the inactivation of mutant neuroserpin as a proteinase inhibitor in FENIB and demonstrate a second pathway for the formation of intracellular polymers in association with disease.

Familial encephalopathy with neuroserpin inclusion bodies (FENIB) is a neurodegenerative pathology caused by accumulation of mutant neuroserpin (NS) polymers inside the endoplasmic reticulum (ER) of neurons, leading to cellular toxicity and neuronal death. To date, there is no cure for FENIB, and only palliative care is available for FENIB patients, underlining the urgency to develop therapeutic strategies. The purpose of this work was to create a cellular system designed for testing small molecules able to reduce the formation of NS polymers. Our results show the generation and characterisation of a novel cell culture model for FENIB based on neural stem progenitor cells (NPCs) with inducible expression of either wild type (WT) or G392E NS, a variant that causes severe FENIB. We also report the use of these novel cell lines to explore the effects of four different proteolysis targeting chimaera (PROTAC) compounds, small bivalent molecules engineered to bind to the E3 ubiquitin ligase cereblon, and to NS through a recruiting motif based on the small molecule embelin. This approach aims to enhance the degradation of mutant NS after retro-translocation to the cytosol by facilitating its targeting to the proteasome. Our results show little toxicity and no variation in NS levels with any of the compounds tested. In conclusion, this work sets the basis for future attempts to identify molecules able to prevent NS accumulation inside the ER of cultured cells.

Neuroserpin (NS) is a serpin inhibitor of tissue plasminogen activator (tPA) in the brain. The polymerisation of NS pathologic mutants is responsible for a genetic dementia known as familial encephalopathy with neuroserpin inclusion bodies (FENIB). So far, a pharmacological treatment of FENIB, i.e. an inhibitor of NS polymerisation, remains an unmet challenge. Here, we present a biophysical characterisation of the effects caused by embelin (EMB a small natural compound) on NS conformers and NS polymerisation. EMB destabilises all known NS conformers, specifically binding to NS molecules with a 1:1 NS:EMB molar ratio without unfolding the NS fold. In particular, NS polymers disaggregate in the presence of EMB, and their formation is prevented. The NS/EMB complex does not inhibit tPA proteolytic activity. Both effects are pharmacologically relevant: firstly by inhibiting the NS polymerisation associated to FENIB, and secondly by potentially antagonizing metastatic processes facilitated by NS activity in the brain.

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.

Contrasting conformational dynamics of β-sheet A and helix F with implications in neuroserpin inhibition and aggregation

Neuroserpin (NS) is a serine protease inhibitor (SERPIN) involved in different neurological pathologies, including the Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB), related to the aberrant polymerization of NS mutants. Here we present an in vitro and in silico characterization of native neuroserpin and its dysfunctional conformation isoforms: the proteolytically cleaved conformer, the inactive latent conformer, and the polymeric species. Based on circular dichroism and fluorescence spectroscopy, we present an experimental validation of the latent model and highlight the main structural features of the different conformers. In particular, emission spectra of aromatic residues yield distinct conformational fingerprints, that provide a novel and simple spectroscopic tool for selecting serpin conformers in vitro. Based on the structural relationship between cleaved and latent serpins, we propose a structural model for latent NS, for which an experimental crystallographic structure is lacking. Molecular Dynamics simulations suggest that NS conformational stability and flexibility arise from a spatial distribution of intramolecular salt-bridges and hydrogen bonds.

Neuroserpin (NS) is a member of the serine protease inhibitors superfamily. Specific point mutations are responsible for its accumulation in the endoplasmic reticulum of neurons that leads to a pathological condition named familial encephalopathy with neuroserpin inclusion bodies (FENIB). Wild-type NS presents two N-glycosylation chains and does not form polymers in vivo, while non-glycosylated NS causes aberrant polymer accumulation in cell models. To date, all in vitro studies have been conducted on bacterially expressed NS, de facto neglecting the role of glycosylation in the biochemical properties of NS. Here, we report the expression and purification of human glycosylated NS (gNS) using a novel eukaryotic expression system, LEXSY. Our results confirm the correct N-glycosylation of wild-type gNS. The fold and stability of gNS are not altered compared to bacterially expressed NS, as demonstrated by the circular dichroism and intrinsic tryptophan fluorescence assays. Intriguingly, gNS displays a remarkably reduced polymerisation propensity compared to non-glycosylated NS, in keeping with what was previously observed for wild-type NS in vivo and in cell models. Thus, our results support the relevance of gNS as a new in vitro tool to study the molecular bases of FENIB.

Mutations in human neuroserpin gene cause an autosomal dementia, familial encephalopathy with neuroserpin inclusion bodies (FENIB). We generated and analyzed transgenic mice expressing high levels of either FENIB-type (G392E) or wild-type human neuroserpin in neurons of the central nervous system. G392E neuroserpin accumulated age-dependently in neurons of the neocortex, thalamus, amygdala, pons, and spinal cord of homozygous transgenic mice. Such accumulations were not observed in hemizygous transgenic mice nor in transgenic mice for wild-type neuroserpin. In differential centrifugation of brain homogenates, G392E neuroserpin recovered in the nucleus-rich fraction dramatically increased along with aging, suggesting that the aggregations gradually increase their densities presumably by their conversion into heavier and more compact configurations. In immunoelectron microscopical analyses, immunopositivities for G392E neuroserpin were found not only in endoplasmic reticulum but also in lysosomes. G392E neuroserpin transgenic mice were much more susceptible to seizures induced by kainate administration than nontransgenic mice. Overall, G392E neuroserpin accumulated in the central nervous system neurons of transgenic mice in mutation-, aging-, and gene dosage-dependent manners. The established transgenic mice will be valuable to elucidate not only mechanisms for the formation of G392E neuroserpin aggregations but also pathways for the degradation and/or clearance of the already formed aggregations in neurons.

Neuroserpin (NS) is predominantly expressed in the brain and is the primary inhibitor of tissue plasminogen activator (tPA). NS variants are associated with the neurogenerative disease termed familial encephalopathy with neuroserpin inclusion bodies (FENIB). The disease is characterized by variable age of onset and severity. The reactive center loop (RCL) insertion-based inhibitory mechanism of NS requires a coordinated conformational change leading to a shift in the strands of the β-sheet A and movement of helix F. Strand 1A is connected to the helix F at its C terminal end and with the strand 2A at its N terminal, both these domain move for accommodating the inserting loop; therefore, a variant that influences their movement may alter the inhibition rates. A molecular dynamic simulation analysis of a H138C NS variant from strand 1A showed a large decrease in conformational fluctuations as compared with wild-type NS. H138 was mutated, expressed, purified and a native-PAGE and transmission electron microscopy (TEM) analysis showed that this variant forms large molecular weight aggregates on a slight increase in temperature. However, a circular dichroism analysis showed its secondary structure to be largely conserved. Surprisingly, its tPA inhibition activity and complex formation remain unhindered even after the site-specific labeling of H138C with Alexa fluor C5 maleimide. Further, a helix F-strand 1A (W154C-H138C) double variant still shows appreciable inhibitory activity. Increasingly, it appears that aggregation and not loss of inhibition is the more likely cause of shutter region-based variants phenotypes, indicating that hindering polymer formation using small molecules may retain inhibitory activity in pathological variants of NS.

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.

Neuroserpin (NS) is an inhibitory protein belonging to the serpin family and involved in several pathologies, including the dementia Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB), a genetic neurodegenerative disease caused by accumulation of NS polymers. Our Molecular Dynamics simulations revealed the formation of a persistent salt bridge between Glu289 on strand s2C and Arg362 on the Reactive Centre Loop (RCL), a region important for the inhibitory activity of NS. Here, we validated this structural feature by simulating the Glu289Ala mutant, where the salt bridge is not present. Further, MD predictions were tested in vitro by purifying recombinant Glu289Ala NS from E. coli. The thermal and chemical stability along with the polymerisation propensity of both Wild Type and Glu289Ala NS were characterised by circular dichroism, emission spectroscopy and non-denaturant gel electrophoresis, respectively. The activity of both variants against the main target protease, tissue-type plasminogen activator (tPA), was assessed by SDS-PAGE and chromogenic kinetic assay. Our results showed that deletion of the salt bridge leads to a moderate but clear reduction of the overall protein stability and activity.

Ethanol-withdrawal Seizures Are Controlled by Tissue Plasminogen Activator via Modulation of NR2B-containing NMDA Receptors Pawlak R, Melchor JP, Matys T, Skrzypiec AE, Strickland S Proc Natl Acad Sci USA 2005;102:443–448 Chronic ethanol abuse causes upregulation of N-methyl-D-aspartate (NMDA) receptors, which underlies seizures and brain damage on ethanol withdrawal (EW). Here we show that tissue plasminogen activator (tPA), a protease implicated in neuronal plasticity and seizures, is induced in the limbic system by chronic ethanol consumption, temporally coinciding with upregulation of NMDA receptors. tPA interacts with NR2B-containing NMDA receptors and is required for upregulation of the NR2B subunit in response to ethanol. As a consequence, tPA-deficient mice have reduced NR2B, extracellular signal-regulated kinase 1/2 phosphorylation, and seizures after EW. tPA-mediated facilitation of EW seizures is abolished by NR2B-specific NMDA antagonist ifenprodil. These results indicate that tPA mediates the development of physical dependence on ethanol by regulating NR2B-containing NMDA receptors. Neuroserpin Portland (Ser52Arg) Is Trapped as an Inactive Intermediate That Rapidly Forms Polymers: Implications for the Epilepsy Seen in the Dementia FENIB Belorgey D, Sharp LK, Crowther DC, Onda M, Johansson J, Lomas DA Eur J Biochem 2004;271:3360–3367 The dementia, familial encephalopathy with neuroserpin inclusion bodies (FENIB), is caused by point mutations in the neuroserpin gene. We have shown a correlation between the predicted effect of the mutation and the number of intracerebral inclusions, and an inverse relation with the age at onset of disease. Our previous work has shown that the intraneuronal inclusions in FENIB result from the sequential interaction between the reactive center loop of one neuroserpin molecule with β-sheet A of the next. We show here that neuroserpin Portland (Ser52Arg), which causes a severe form of FENIB, also forms loop-sheet polymers but at a faster rate, in keeping with the more severe clinical phenotype. The Portland mutant has a normal unfolding transition in urea and a normal melting temperature but is inactive as a proteinase inhibitor. This results in part from the reactive loop being in a less accessible conformation to bind to the target enzyme, tissue plasminogen activator. These results, with those of the CD analysis, are in keeping with the reactive center loop of neuroserpin Portland being partially inserted into β-sheet A to adopt a conformation similar to an intermediate on the polymerization pathway. Our data provide an explanation for the number of inclusions and the severity of dementia in FENIB associated with neuroserpin Portland. Moreover, the inactivity of the mutant may result in uncontrolled activity of tissue plasminogen activator, and so explain the epileptic seizures seen in individuals with more severe forms of the disease.

The dementia familial encephalopathy with neuroserpin inclusion bodies (FENIB) is caused by point mutations in the neuroserpin gene. We have shown a correlation between the predicted effect of the mutation and the number of intracerebral inclusions, and an inverse relationship with the age of onset of disease. Our previous work has shown that the intraneuronal inclusions in FENIB result from the sequential interaction between the reactive centre loop of one neuroserpin molecule with beta-sheet A of the next. We show here that neuroserpin Portland (Ser52Arg), which causes a severe form of FENIB, also forms loop-sheet polymers but at a faster rate, in keeping with the more severe clinical phenotype. The Portland mutant has a normal unfolding transition in urea and a normal melting temperature but is inactive as a proteinase inhibitor. This results in part from the reactive loop being in a less accessible conformation to bind to the target enzyme, tissue plasminogen activator. These results, with those of the CD analysis, are in keeping with the reactive centre loop of neuroserpin Portland being partially inserted into beta-sheet A to adopt a conformation similar to an intermediate on the polymerization pathway. Our data provide an explanation for the number of inclusions and the severity of dementia in FENIB associated with neuroserpin Portland. Moreover the inactivity of the mutant may result in uncontrolled activity of tissue plasminogen activator, and so explain the epileptic seizures seen in individuals with more severe forms of the disease.

Familial encephalopathy with neuroserpin inclusion bodies (FENIB) is a conformational proteinopathy characterised by neuronal inclusion bodies composed of the serine protease inhibitor (SERPIN), neuroserpin. Presenting clinically as a familial dementia-epilepsy syndrome, the molecular mechanism of the pathogenic abnormalities in neuroserpin has been characterised at atomic resolution. There is a remarkable genotype-phenotype correlation between the degree of molecular destabilisation of the several variants of the neuroserpin protein, their propensity to self-associate and the age of onset of the dementia-epilepsy complex. As with other serpinopathies there appears to be a mix of cell-autonomous toxicity, due to neuronal accumulation of neuroserpin, and non-cell autonomous toxicity, caused by loss of protease inhibition, in this case the dysregulated protease is likely to be tissue plasminogen activator (tPA). FENIB should be considered in cases of progressive myoclonic epilepsy and dementia particularly where there is family history of neuropsychiatric disease.

Familial encephalopathy with neuroserpin inclusion bodies is a neurodegenerative disorder characterized by the accumulation of neuroserpin polymers in the endoplasmic reticulum (ER) of cortical and subcortical neurons in the CNS because of neuroserpin point mutations. ER-associated degradation (ERAD) is involved in mutant neuroserpin degradation. In this study, we demonstrate that two ER-associated E3 ligases, Hrd1 and gp78, are involved in the ubiquitination and degradation of mutant neuroserpin. Overexpression of Hrd1 and gp78 decreases the mutant neuroserpin protein level, whereas Hrd1 and gp78 knockdown increases mutant neuroserpin stability. Moreover, ERAD impairment by mutant valosin-containing protein increases the mutant neuroserpin protein level and aggregate formation. Thus, these findings identify mutant neuroserpin as an ERAD target and show that Hrd1 and gp78 mediate mutant neuroserpin turnover through the ERAD pathway.