Kinga Molnár

Intracellular processing of disease-associated α-synuclein in the human brain suggests prion-like cell-to-cell spread.

Dementia with Lewy bodies (DLB), Parkinsons disease (PD) and multiple system atrophy are characterized by the deposition of disease-associated α-synuclein. In the present study we 1) examined the molecular specificity of the novel anti-α-synuclein 5G4 antibody; 2) evaluated immunoreactivity patterns and their correlation in human brain tissue with micro- and astrogliosis in 57 cases with PD or DLB; and 3) performed a systematic immunoelectron microscopical mapping of subcellular localizations. 5G4 strongly binds to the high molecular weight fraction of β-sheet rich oligomers, while no binding to primarily disordered oligomers or monomers was observed. We show novel localizations of disease-associated α-synuclein including perivascular macrophages, ependyma and cranial nerves. α-Synuclein immunoreactive neuropil dots and thin threads associate more with glial reaction than Lewy bodies alone. Astrocytic α-synuclein is an important component of the pathology. Furthermore, we document ultrastructurally the pathway of processing of disease-associated α-synuclein within neurons and astroglial cells. Interaction of mitochondria and disease-associated α-synuclein plays a key role in the molecular-structural cytopathogenesis of disorders with Lewy bodies. We conclude that 1) the 5G4 antibody has strong selectivity for β-sheet rich α-synuclein oligomers; 2) Lewy bodies themselves are not the most relevant morphological substrate that evokes tissue lesioning; 3) both neurons and astrocytes internalize disease-associated α-synuclein in the human brain, suggesting prion-like cell-to-cell spread of α-synuclein by uptake from surrounding structures, as shown previously in experimental observations.

Intraneuronal Immunoreactivity for the Prion Protein Distinguishes a Subset of E200K Genetic From Sporadic Creutzfeldt-Jakob Disease

Abstract Recently, we reported widespread intraneuronal prion protein (PrP) immunoreactivity in genetic Creutzfeldt-Jakob disease (CJD) associated with the E200K mutation. Here, we evaluated 6 cases ofsporadic CJD MM type 1, 5 MV type 2, and 7 VV type 2 and compared their anatomical appearance with that of 29 E200K genetic CJD (gCJD) cases. We also performed double immunolabeling for ubiquitin, p62, early endosomal marker rab5, and immunogold electronmicroscopy in 3 cases. We identified 4 morphological types of intraneuronal PrP immunoreactivity: one type, defined as multiple globular structures, was significantly associated with a subset of E200K gCJD cases and was distinct from the intraneuronal small dotlike PrP immunoreactivity seen in sporadic CJD. Whereas the latter colocalized with rab5, there were single large (7.5 &mgr;m–15 &mgr;m) globular inclusion body–like structures detected predominantly but not exclusively in E200K gCJD; these were immunoreactive in part for ubiquitin and p62 and showed focal &ggr;-tubulin immunoreactivity, suggesting aggresome features. Ultrastructural examination using immunogold revealed PrP localization in aggresome-like structures and in autophagic vacuoles. These findings suggest that the permanent production of mutant PrP in the E200K gCJD cases overwhelms the ubiquitin-proteasome system and shifts the balance toward selectivemacroautophagy and/or to ubiquitinated inclusion body and aggresome formation as a cytoprotective effort to sequester the mutant protein.

Distribution of serpins in the tissues of the tobacco hornworm (Manduca sexta) larvae: Existence of new serpins possibly encoded by a gene distinct from the serpin-1 gene

Polyclonal antibodies were raised against the isolated hemolymph serine proteinase inhibitors (serpins) of Manduca sexta larvae. Two of these antibodies, MsH49a and MsH49b, displayed characteristic differences in labelling patterns of hemocytes, fat body, integumental epidermis and cuticle on immunoblots, and in light- and electronmicroscopic sections. The serpin composition of the latter three tissue homogenates was determined by native immunoblots and inhibitor binding assays. The results were compared to the hemolymph samples containing all the known inhibitors encoded by the well-characterized serpin-1 gene. The enzyme specificity of the MsH49b-labelled cuticular serpin was similar to serpin-1J, although its electrophoretic mobility on native PAGE was not identical with any of the known proteinase inhibitors encoded by the serpin-1 gene. Based on these data, we suggest that the cuticle and hemolymph may contain novel serpin(s) encoded by a gene other than the serpin-1 gene. Since the serpin-1J proved to be involved in the activation pathway of the prophenoloxidase system in the hemolymph, the in vivo function of cuticular MsH49b serpin was investigated by prophenoloxidase tests in native cuticular homogenates. Our results demonstrated that the cuticular serpin(s) that are labelled by the MsH49b antibody may play a determinant role in the regulation of the prophenoloxidase system of the integumental cuticle.

Isolation and characterization of an apically sorted 41-kDa protein from the midgut of tobacco hornworm (Manduca sexta).

Abstract Immunocytochemical localization and sorting properties of a newly purified 41-kDa protein (MsM41) were investigated in an insect, the tobacco hornworm Manduca sexta. The protein purified from midgut homogenates of feeding fifth-stadium larvae was found exclusively in this tissue on Western blots. Presence of MsM41 protein was indicated in both anterior and posterior regions of the midgut during the whole fifth stadium. However, in the posterior region an additional 39-kDa protein was also detected during the feeding period of the last larval stage. Upon light-microscopic examination immunoreactivity was localized in the columnar cells, while the goblet, endocrine and regenerative cells remained unlabeled. Distribution of the label during the feeding period was different in the anterior and posterior regions. In the anterior region immunoreactivity was localized only to the brush border membrane of columnar cells, while in the posterior region some cytoplasmic structures identified as large trans-Golgi vesicles, endoplasmic reticulum and small secretory vesicles were also labeled. Large, apical extrusions remained immunonegative. In vitro translation confirmed that our protein was expressed only in the posterior region of the midgut. The primary translation product was a 39-kDa protein. Putative post-translational modifications yielded the 41-kDa form, which was then secreted apically. Its presence in the region of the anterior part microvilli was probably due to the countercurrent flux of the ectoperitrophic fluid.

Lysosomal response in relation to α-synuclein pathology differs between Parkinson's disease and multiple system atrophy

Intracellular deposition of pathologically altered α-synuclein mostly in neurons characterises Parkinsons disease (PD), while its accumulation predominantly in oligodendrocytes is a feature of multiple system atrophy (MSA). Recently a prion-like spreading of pathologic α-synuclein has been suggested to play a role in the pathogenesis of PD and MSA. This implicates a role of protein processing systems, including lysosomes, supported also by genetic studies in PD. However, particularly for MSA, the mechanism of cell-to-cell propagation of α-synuclein is yet not fully understood. To evaluate the significance of lysosomal response, we systematically compared differently affected neuronal populations in PD, MSA, and non-diseased brains using morphometric immunohistochemistry (cathepsin D), double immunolabelling (cathepsin D/α-synuclein) laser confocal microscopy, and immunogold electron microscopy for the disease associated α-synuclein. We found that i) irrespective of the presence of neuronal inclusions, the volume density of cathepsin D immunoreactivity significantly increases in affected neurons of the pontine base in MSA brains; ii) volume density of cathepsin D immunoreactivity increases in nigral neurons in PD without inclusions and with non-ubiquitinated pre-aggregates of α-synuclein, but not in neurons with Lewy bodies; iii) cathepsin D immunoreactivity frequently colocalises with α-synuclein pre-aggregates in nigral neurons in PD; iv) ultrastructural observations confirm disease-associated α-synuclein in neuronal and astrocytic lysosomes in PD; v) lysosome-associated α-synuclein is observed in astroglia and rarely in oligodendroglia and in neurons in MSA. Our observations support a crucial role for the neuronal endosomal-lysosomal system in the processing of α-synuclein in PD. We suggest a distinct contribution of lysosomes to the pathogenesis of MSA, including the possibility of oligodendroglial and eventually neuronal uptake of exogenous α-synuclein in MSA.

Baculovirus-mediated trans-epithelial transport of proteins in infected caterpillars

Baculovirus-mediated abnormal protein trafficking was studied in infected caterpillars by using heterologous proteins. The gene for human complement C1r was expressed in larvae of Mamestra brassicae by a recombinant Autographa californica nuclear polyhedrosis virus (AcMNPV) vector. By following the time-course of recombinant C1r distribution among various tissues, cell types and cell organelles, we concluded that the dominant site of recombinant protein synthesis was the fat body, although some production in the haemocytes and midgut was also observed. Only about 4% of the cells of the infected organs expressed recombinant C1r, which was then secreted into the haemolymph. The tracheal and integumental cuticle was rich in recombinant protein from the fourth day after infection although epidermal cells did not synthesize recombinant C1r. The morphological picture suggested that the accumulation was a consequence of a trans-epithelial transport. This transport process was checked by following the fate of the 49 kDa haemolymph protein and injected ovalbumin in AcMNPV-infected Mamestra brassicae and in Lymantria dispar nuclear polyhedrosis virus-infected Lymantria dispar larvae. Both proteins were able to pass the basal membrane of the epidermis and accumulated in the cuticle, while in control larvae neither was transported. The observed trans-epithelial transport points to the role of baculoviruses in directing recombinant, endo- and exo-genous proteins to cuticulated tissues. Based on these results we conclude that the permeability of basal membranes undergoes a characteristic change during the course of baculovirus infection.

Interaction of Positively Charged Gold Nanoparticles with Cancer Cells Monitored by an in Situ Label-Free Optical Biosensor and Transmission Electron Microscopy

Functionalized nanoparticles (NPs) can penetrate into living cells and vesicles, opening up an extensive range of novel directions. For example, NPs are intensively employed in targeted drug delivery and biomedical imaging. However, the real-time kinetics and dynamics of NP-living cell interactions remained uncovered. In this study, we in situ monitored the cellular uptake of gold NPs-functionalized with positively charged alkaline thiol-into surface-adhered cancer cells, by using a high-throughput label-free optical biosensor employing resonant waveguide gratings. The characteristic kinetic curves upon NP exposure of cell-coated biosensor surfaces were recorded and compared to the kinetics of NP adsorption onto bare sensor surfaces. We demonstrated that from the above kinetic information, one can conclude about the interactions between the living cells and the NPs. Real-time biosensor data suggested the cellular uptake of the functionalized NPs by an active process. It was found that positively charged particles penetrate into the cells more effectively than negatively charged control particles, and the optimal size for the cellular uptake of the positively charged particles is around 5 nm. These conclusions were obtained in a cost-effective, fast, and high-throughput manner. The fate of the NPs was further revealed by electron microscopy on NP-exposed and subsequently fixed cells, well confirming the results obtained by the biosensor. Moreover, an ultrastructural study demonstrated the involvement of the endosomal-lysosomal system in the uptake of functionalized NPs and suggested the type of the internalization pathway.

The inhibitory effect of Panax ginseng extract on amyloid-like fibril formation of trypsin in aqueous ethanol

BACKGROUND In case of several chronic diseases, prevention is could be more effective than treatment. Functional foods that contain significant amounts of bioactive components gained considerable attention not only in traditional but in modern medicine as well. We have investigated how P. ginseng extract inhibits the in vitro formation of amyloid-like fibrils of phenylmethylsulfonyl- trypsin (PMS-trypsin) in 60% ethanol at pH 7.0. The model system used is non-physiological, but it is capable of detecting the anti-amyloidogenic effect of the various agents. OBJECTIVE The main objective of this study was to examine the possible inhibitory effect of ginseng extract on amyloid-like fibril formation of trypsin in aqueous ethanol. METHODS The amyloid formation and aggregation kinetics of PMS-trypsin was studied by turbidity measurements, Congo Red (CR) binding assays, size exclusion chromatography and Electronic Circular Dichroism (ECD) measurements and the shapes of amyloid fibrils became visible by Transmission Electron Microscopy (TEM). RESULTS In the presence of 500-fold diluted P. ginseng extract in the incubation mixture, the absorption at 350 nm decreased to 47.1% after incubation for 24 h, compared relative to the sample which contained no additives. CR binding experiments suggested that the aggregates in our samples have amyloid-like properties, and P. ginseng extract inhibits the amyloid-like fibril formation of PMS-trypsin depending on concentration. Our results show that the ginseng extract does not bind to the fibrils. In the absence of P. ginseng extracts large sized colloid aggregates were abundant. Adding P. ginseng extracts to our samples decreased the light dispersion of the solution. This is due to the decrease of the rate of the aggregation or to the smaller size of the aggregates evolved. Our results show that the presence of ginseng extract helps to maintain the native structure of the protein. In the presence of 500-fold diluted P. ginseng extract, TEM images demonstrated, that P. ginseng extract has inhibitory effect on the formation of amyloid-like fibrils of PMS-trypsin. CONCLUSION The results indicated that P. ginseng extract significantly inhibits the formation of amyloid-like fibrils of PMS-trypsin in aqueous ethanol, and helps to maintain the native structure of the protein. The rate of inhibition depends on concentration. P. ginseng extract is an efficient antiamyloidogenic agent.

Examination of a Planarian

The simplest animals that are bilaterally symmetrical and triploblastic (having three germ layers) are the flatworms (Platyhelminthes). Flatworms have no body cavity (acoelomate) and lack an anus. One of their groups is the freshwater triclads (Tricladida), or planarians. They are large free-living flatworms which are commonly found on the underside of stones or submerged leaves or sticks in freshwater springs, ponds, and streams. Planarians are mobile and use cilia on their ventral surface to glide over surfaces (Fig. 2.1).

Examination of a Hydra

The cnidarian body consists of a central blind sac, the coelenteron (gastrovascular cavity), enclosed by a body wall comprising two epithelia, the outer epidermis and the inner gastrodermis (Fig. 1.1). A gelatinous connective tissue layer, the mesolamella (mesogloea), lies between the two epithelia. The mouth opens at one end of the coelenteron and marks the oral end. The mouth is at the tip of a process, the hypostome that elevates it above the oral surface. The opposite pole is the aboral end forming the pedal disc. The imaginary line connecting the oral and aboral poles is the axis of symmetry around which the radial symmetry of the body is organised. The mouth is usually surrounded by a ring of hollow tentacles, which are well endowed with cnidocyte batteries (white spots in Fig. 1.1).