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Epilepsies are characterized by abnormal electrophysiological activity of the brain. Among various types of inherited epilepsies different epilepsy syndromes, among them progressive myoclonus epilepsies with features of ataxia and neurodegeneration, are counted. The progressive myoclonus epilepsy of type 1 (EPM1), also known as Unverricht-Lundborg disease presents with features of cerebellar atrophy and increased oxidative stress. It has been found that EPM1 is caused by mutations in human cystatin B gene (human stefin B). We first describe the role of protein aggregation in other neurodegenerative conditions. Protein aggregates appear intraneurally but are also excreted, such as is the case with senile plaques of amyloid-β (Aβ) that accumulate in the brain parenchyma and vessel walls. A common characteristic of such diseases is the change of the protein conformation toward β secondary structure that accounts for the strong tendency of such proteins to aggregate and form amyloid fibrils. Second, we describe the patho-physiology of EPM1 and the normal and aberrant roles of stefin B in a mouse model of the disease. Furthermore, we discuss how the increased protein aggregation observed with some of the mutants of human stefin B may relate to the neurodegeneration that occurs in rare EPM1 patients. Our hypothesis (Ceru et al., 2005) states that some of the EPM1 mutants of human stefin B may undergo aggregation in neural cells, thus gaining additional toxic function (apart from loss of normal function). Our in vitro experiments thus far have confirmed that four mutants undergo increased aggregation relative to the wild-type protein. It has been shown that the R68X mutant forms amyloid-fibrils very rapidly, even at neutral pH and forms perinuclear inclusions, whereas the G4R mutant exhibits a prolonged lag phase, during which the toxic prefibrillar aggregates accumulate and are scattered more diffusely over the cytoplasm. Initial experiments on the G50E and Q71P missense EPM1 mutants are described.

Background: Protein aggregation is a major contributor to the pathogenic mechanisms of human neurodegenerative diseases. Mutations in the CSTB (cystatin B) gene [StB (stefin B)] cause EPM1 (progressive myoclonus epilepsy of type 1), an epilepsy syndrome with features of neurodegeneration and increased oxidative stress. Oligomerization and aggregation of StB in mammalian cells have recently been reported. It has also been observed that StB is overexpressed after seizures and in certain neurodegenerative conditions, which could potentially lead to its aggregation. Human StB proved to be a good model system to study amyloid fibril formation in vitro and, as we show here, to study protein aggregation in cells., Results: Endogenous human StB formed smaller, occasional cytoplasmic aggregates and chemical inhibition of the UPS (ubiquitin-proteasome system) led to an increase in the amount of the endogenous protein and also increased its aggregation. Further, we characterized both the untagged and T-Sapphire-tagged StB on overexpression in mammalian cells. Compared with wild-type StB, the EPM1 missense mutant (G4R), the aggregate-prone EPM1 mutant (R68X) and the Y31 StB variant (both tagged and untagged) formed larger cytosolic and often perinuclear aggregates accompanied by cytoskeletal reorganization. Non-homogeneous morphology of these large aggregates was revealed using TEM (transmission electron microscopy) with StB detected by immunogold labelling. StB-positive cytoplasmic aggregates were partially co-localized with ubiquitin, proteasome subunits S20 and S26 and components of microfilament and microtubular cytoskeleton using confocal microscopy. StB aggregates also co-localized with LC3 and the protein adaptor p62, markers of autophagy. Flow cytometry showed that protein aggregation was associated with reduced cell viability., Conclusions: We have shown that endogenous StB aggregates within cells, and that aggregation is increased upon protein overexpression or proteasome inhibition. From confocal and TEM analyses, we conclude that aggregates of StB show some of the molecular characteristics of aggresomes and may be eliminated from the cell by autophagy. Intracellular StB aggregation shows a negative correlation with cell survival.

To contribute to the question of the putative role of cystatins in Alzheimer disease and in neuroprotection in general, we studied the interaction between human stefin B (cystatin B) and amyloid-beta-(1-40) peptide (Abeta). Using surface plasmon resonance and electrospray mass spectrometry we were able to show a direct interaction between the two proteins. As an interesting new fact, we show that stefin B binding to Abeta is oligomer specific. The dimers and tetramers of stefin B, which bind Abeta, are domain-swapped as judged from structural studies. Consistent with the binding results, the same oligomers of stefin B inhibit Abeta fibril formation. When expressed in cultured cells, stefin B co-localizes with Abeta intracellular inclusions. It also co-immunoprecipitates with the APP fragment containing the Abeta epitope. Thus, stefin B is another APP/Abeta-binding protein in vitro and likely in cells.

Amyloid-induced toxicity is a well-known phenomenon but the molecular background remains unclear. One hypothesis relates toxicity to amyloid-membrane interactions, predicting that amyloid oligomers make pores into membranes. Therefore, the toxicity and membrane interaction of prefibrillar aggregates and individual oligomers of a non-pathological yet highly amyloidogenic protein human stefin B (cystatin B) was examined. By monitoring caspase-3 activity and by testing cell viability, we showed that the lag phase aggregates obtained at pH 5 and 3 were toxic to neuroblastoma cells. Of equal toxicity were the higher-order oligomers prepared at pH 7 by freeze-thaw cycles. The higher-order oligomers eluted on size-exclusion chromatography (SEC) as a broad peak comprising hexamers, octamers, 12- and 16-mers, well separated from monomers, dimers and tetramers. Only oligomers higher than the tetramers (Rh >3.5 nm) proved toxic, in contrast to dimers and tetramers. In accordance with data from SEC, dynamic light scattering and atomic force microscopy data indicate that the toxic oligomers have diameters larger than 4 nm. Critical pressure measurements showed that the toxic higher-order oligomers inserted more effectively into model lipid monolayers than dimers and tetramers. They also bound, similarly to prefibrillar aggregates, to the plasma membrane and became internalized. Taken together, our results confirm the importance of membrane interaction and perforation in the phenomenon of cytotoxicity.

We report that a mutant of human stefin B is in a molten globule conformation. It has all the spectroscopic characteristics for such a state. We also demonstrate that the molten globule is oligomeric, eluting on SEC within a similar MW range than the higher order oligomers of the wild type protein, which is confirmed by DLS and AFM. Both, the higher oligomers and the molten globule state bind ANS, implying a high degree of hydrophobic patches exposure and partial opening of the structure. Finally, we demonstrate that the oligomeric molten globule is as toxic as the prefibrillar aggregates obtained at acid pH or the higher order oligomers prepared at neutral pH.

Protein aggregation is central to most neurodegenerative diseases, as shown by familial case studies and by animal models. A modified 'amyloid cascade' hypothesis for Alzheimer's disease states that prefibrillar oligomers, also called amyloid-beta-derived diffusible ligands or globular oligomers, are the responsible toxic agent. It has been proposed that these oligomeric species, as shown for amyloid-beta, beta2-microglobulin or prion fragments, exert toxicity by forming pores in membranes, initiating a cascade of detrimental events for the cell. Interaction of granular aggregates and globular oligomers of an amyloidogenic protein, human stefin B, with model lipid membranes and monolayers was studied. Prefibrillar oligomers/aggregates of stefin B are shown to cause concentration-dependent membrane leaking, in contrast to the homologous stefin A. Prefibrillar oligomers/aggregates of stefin B also increase the surface pressure at an air-water interface, i.e. they have amphipathic character and are surface seeking. In addition, they show stronger interaction with 1,2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] monolayers than native stefin A or nonaggregated stefin B. Prefibrillar aggregates interact predominantly with acidic phospholipids, such as dioleoylphosphatidylglycerol or dipalmitoylphosphatidylserine, as shown by calcein release experiments and surface plasmon resonance. The same preparations are toxic to neuroblastoma cells, as determined by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay, again in contrast to the homologue stefin A, which does not aggregate under any of the conditions studied. This study is aimed to contribute to the general model of cellular toxicity induced by prefibrillar oligomers of amyloidogenic proteins, not necessarily involved in pathology.

Loss of function mutations in the gene (CSTB) encoding human cystatin B, a widely expressed cysteine protease inhibitor, are responsible for a severe neurological disorder known as an Unverricht-Lundborg disease (EPM1). EPM1 had been linked to chromosome 21q22.3 in Finnish families and it is an autosomal recessive inherited disorder with a homozygous minisatellite expansion in the cystatin B gene (stefin B gene). This disease is difficult to treat because it is refractory to most antiepileptic drugs and using multiple medications had been unsuccessful so far. To come a step closer to understanding of the nature of this disease, especially about the events on the molecular level, in vitro properties of missense EPM1 mutant G4R were determined. It was observed that the mutant has a prolonged lag phase of fibrillation at the same protein stability, which could indicate it were more toxic to the cells. Similar experiments with the N-terminal fragment of 67 aminoacid residues are ongoing, showing higher propensity to aggregate. Therefore, a hypothesis is launched that at least in a subset of Unverricht-Lundborg disease patients, cystatin B protein may aggregate in the cell. Protein aggregation can be secondary to external insults or aging, however, inherited forms of neurodegenerative diseases, such as familial Parkinson's, Huntington's or familial Alzheimer's disease, are directly linked to the mutant proteins aggregation. Protein aggregates in the form of amyloid plaques, neurofibrilary tangles, intra-cytoplasmic or intra-nuclear inclusions lead to increased production of the reactive oxygen species and dysfunction of the ubiquitine/proteasome system. Finally, mitochondrial dysfunction and cell death are observed. Certainly, it remains to be checked by experiments whether overexpression in cell culture of the missense mutants G4R and N-terminal fragment to residue 68 lead to cellular inclusions and the accompanying changes characteristic for the conformational disorders.

Objective: To evaluate the effects of iloprost infusion on the microcirculation in patients suffering from severe Raynaud's phenomenon secondary to systemic sclerosis., Methods: Eight patients received a 7-hour infusion of iloprost for five consecutive days and then for one day 3 months later. The effects on vascular distensibility were evaluated by piezoelectric plethysmography before and after the treatment and at 2, 4 and 6 weeks., Results: The beneficial effects on the peripheral microcirculation were statistically significant after five days of infusion (distensibility index: 0.18 +/- 0.01 vs 0.23 +/- 0.01, p < 0.002) and lasted for less than four weeks, whereas no difference (0.22 +/- 0.04 vs 0.24 +/- 0.02, p: ns) was seen after one day of treatment. One patient suffered from typical angina pectoris with electrocardiographic changes of the ST wave detected during the infusion., Conclusion: Our results show that a five-day infusion of iloprost has an effect which lasts from two to four weeks; after four weeks the distensibility index returned to the baseline value. The one-day infusion had no effect on the vascular bed, studied by the piezoelectric pletysmographic method. Treatment with five consecutive days of infusion every four weeks is an impracticable scheme to adopt, however. We have therefore instituted a treatment schedule of a single daily infusion every four weeks with the aim of maintaining the effects induced by the initial five-day infusion. The preliminary results obtained with this schedule are reported.