Showing papers by "Herbert Budka published in 2012"

An antibody with high reactivity for disease-associated α-synuclein reveals extensive brain pathology

Abstract: α-Synuclein is the major protein associated with Lewy body dementia, Parkinson's disease and multiple system atrophy. Since α-synuclein is present in the brain in physiological conditions as a presynaptic protein, it is crucial to characterize disease-associated modifications to develop an in vivo biomarker. With the aim to develop antibodies showing high specificity and sensitivity for disease-associated α-synuclein, synthetic peptides containing different amino acid sequences were used for immunization of mice. After generation of α-synuclein aggregates, ELISA and immunoblotting were used to test the specificity of antibodies. Tissue microarray sections originating from different human α-synucleinopathies were used to compare immunostaining with other, commercially available antibodies. Immunization of mice with the peptide TKEGVVHGVATVAE (amino acid 44-57 of α-synuclein) resulted in the generation of a monoclonal antibody (5G4), which was able to bind aggregated α-synuclein preparation in sandwich ELISA or coated on magnetic beads. 5G4 proved to be superior to other antibodies in comparative immunohistochemical studies by revealing more widespread and distinct α-synuclein pathology. Immunoblotting of human brain tissue revealed an additional band seen in dementia with Lewy bodies, whereas the band representing monomeric α-synuclein was very weak or lacking. In summary, the 5G4 antibody is most promising for re-evaluation of archival material and may offer new perspective for the development of in vivo diagnostic assays for detecting disease-associated α-synuclein in body fluids.

A New Mechanism for Transmissible Prion Diseases

Abstract: The transmissible agent of prion disease consists of prion protein (PrP) in β-sheet-rich state (PrP(Sc)) that can replicate its conformation according to a template-assisted mechanism. This mechanism postulates that the folding pattern of a newly recruited polypeptide accurately reproduces that of the PrP(Sc) template. Here, three conformationally distinct amyloid states were prepared in vitro using Syrian hamster recombinant PrP (rPrP) in the absence of cellular cofactors. Surprisingly, no signs of prion infection were found in Syrian hamsters inoculated with rPrP fibrils that resembled PrP(Sc), whereas an alternative amyloid state, with a folding pattern different from that of PrP(Sc), induced a pathogenic process that led to transmissible prion disease. An atypical proteinase K-resistant, transmissible PrP form that resembled the structure of the amyloid seeds was observed during a clinically silent stage before authentic PrP(Sc) emerged. The dynamics between the two forms suggest that atypical proteinase K-resistant PrP (PrPres) gave rise to PrP(Sc). While no PrP(Sc) was found in preparations of fibrils using protein misfolding cyclic amplification with beads (PMCAb), rPrP fibrils gave rise to atypical PrPres in modified PMCAb, suggesting that atypical PrPres was the first product of PrP(C) misfolding triggered by fibrils. The current work demonstrates that a new mechanism responsible for prion diseases different from the PrP(Sc)-templated or spontaneous conversion of PrP(C) into PrP(Sc) exists. This study provides compelling evidence that noninfectious amyloids with a structure different from that of PrP(Sc) could lead to transmissible prion disease. This work has numerous implications for understanding the etiology of prion and other neurodegenerative diseases.

Asymmetry of neurodegenerative disease-related pathologies: a cautionary note

Abstract: Alzheimer’s disease (AD) is characterized neuropathologically by the presence of amyloid plaques (Ab) and neurofibrillary tangles (tau). In addition, molecular pathologic examinations revealed concomitant protein depositions (in particular TDP-43 or a-synuclein), in various combinations with AD associated pathology [8]. Earlier papers evaluating senile changes in the brain noted variable asymmetric pathology without predominance of either hemisphere [6]. Two further papers, one on the evaluation of 5 patients [14] and another on 56 [7] indicated differences between the left and right hippocampus; differences were highly significant for plaques, tangles and also for granulovacuolar degeneration. Diagnostic and staging criteria developed after these studies did not provide recommendations for the side to be evaluated [2–4, 10]. Furthermore, asymmetric distribution of neuronal loss and gliosis correlating with clinical symptoms (e.g., progressive aphasia or apraxia) is a feature of various molecular forms of frontotemporal lobar degeneration. Unilateral Creutzfeldt–Jakob disease has been also reported [15]. A recent comprehensive study indicated asymmetric involvement of the medial temporal lobe in patients with advanced argyrophilic grain disease [1]. Accordingly, we aimed to evaluate whether sidedness of the examined block has any influence on the diagnosis or staging of disease process, or on the interpretation of concomitant deposition of proteins in AD. We performed our study on cases from our Archives in the period of 1994–2011 (over 3,500 brain autopsies with various diagnoses). The minimum selection criteria were the presence of some degree of neurofibrillary degeneration according to the original neuropathology report and the availability of left and right hippocampus, irrespective of the clinical data. Formalin-fixed, paraffin-embedded tissue blocks (2.5 9 2.0 cm) were evaluated. Our cohort included 19 men and 20 women; the mean age was 78.9 (ranging from 63 to 91 years). Neuropathological diagnoses included pure AD-related pathology in 20; with brain infarction in 4; and with Lewy body disease in 15. The following monoclonal antibodies were used for immunohistochemistry: anti-tau AT8 (pS202, 1:200, Pierce Biotechnology, Rockford, IL, USA), anti-phospho-TDP-43 (pTDP-43, pS409/ 410, 1:2,000, Cosmo Bio, Tokyo, Japan), anti-a-synuclein (1:2,000, clone 5G4, Roboscreen, Leipzig, Germany), and anti-Ab (1:50, clone 6F/3D, Dako, Glostrup, Denmark). The Dako EnVision detection kit was used for visualization of antibody reactions. Braak and Braak staging was performed in tissue sections stained for AT8, according to published criteria [2, 3]. In addition, neuropil threads and neuronal tau immunoreactivity was semiquantitatively scored (none, some, moderate, and many) in the dentate gyrus, CA4, and CA1 subregion of the hippocampus. AntipTDP43 staining was evaluated similarly in the dentate gyrus, CA1 subregion, and entorhinal cortex. a-Synuclein immunoreactivity and the presence of Ab deposits was evaluated in the same way in CA2/3 subregions and in the entorhinal cortex (for a-synuclein) and in the entorhinal cortex, CA1 and CA4 subregions (for Ab). Neurofibrillary degeneration was staged as V–VI in 17 cases, III–IV in 19 cases, and I–II in 3 cases according to the original diagnostic procedure using blocks from one side only. In 6 cases we observed significant difference between the two sides (Fig. 1a, b), resulting in a staging switch from transentorhinal to limbic in five (in two cases left was higher), or from limbic to isocortical stage in a single case (right higher) (Fig. 2). Evaluation of tau H. Stefanits H. Budka G. G. Kovacs (&) Institute of Neurology, Medical University of Vienna, AKH 4J, Währinger Gürtel 18-20, 1097 Vienna, Austria e-mail: gabor.kovacs@meduniwien.ac.at

Antibody 9D5 recognizes oligomeric pyroglutamate amyloid-β in a fraction of amyloid-β deposits in Alzheimer's disease without cross-reactivity with other protein aggregates

Abstract: Recent evidence suggests that soluble oligomeric amyloid-β (Aβ) assemblies are critically involved in the pathogenesis of Alzheimer's disease (AD). We have generated a conformation-dependent monoclonal antibody (9D5) that selectively recognizes low-molecular weight AβpE3 oligomers, and demonstrated its diagnostic and therapeutic potential. Here, we further characterize the specificity of this antibody by evaluating a spectrum of neurodegeneration-related protein deposits for cross-reactivity, and by comparing the staining pattern of 9D5 with a generic Aβ antibody that targets a linear epitope (mAb NT244), and with another conformation-dependent Aβ antibody that selectively labels amyloid fibrils of various molecular weights (pAb OC). The 9D5 antibody does not cross-react with other aggregated protein deposits in brains of progressive supranuclear palsy, corticobasal degeneration, argyrophilic grain disease, Pick's disease, Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, frontotemporal lobar degeneration or amyotrophic lateral sclerosis with TDP-43 inclusions, Creutzfeldt-Jakob disease, and vessel changes in Binswanger encephalopathy, demonstrating the specificity of 9D5 for Aβ deposits. While NT244 and OC showed a comparable plaque load, 9D5 detected only approximately 15% of the total Aβ plaque load in the entorhinal cortex, the CA1 region, and the temporal neocortex. Our study further supports a possible therapeutic advantage of 9D5 by the highly specific recognition of an epitope found only in oligomeric assemblies of AβpE3 of AD patients. Moreover, selective binding to only a pathogenetically relevant fraction of Aβ deposits serves as rationale for passive immunization with 9D5-derivatives by limiting potential side effects of vaccination due to dissolvement of existing amyloid deposits.

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 μm-15 μm) 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 γ-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.

Prion protein (PrP) deposits in the tectum of experimental Gerstmann-Sträussler-Scheinker disease following intraocular inoculation

Abstract: The abnormal misfolded isoform of prion protein (PrPd; "d" for disease) is considered as a surrogate marker for infectivity in the transmissible spongiform encephalopathies (TSEs) or prion diseases, including Creutzfeldt-Jakob disease (CJD). In this experiment, we used intraocular inoculation to study PrPd deposition in the visual system of the brain of mice infected with the Fujisaki (K.Fu) strain of Gerstmann-Straussler-Scheinker (GSS) disease. We report here that PrPd is deposited in the superior colliculus following contralateral intraocular inoculation and thus follows neuronal connections when it spreads into the brain. Until 26 weeks postinoculation, no PrPd-specific immunostaining was observed in the brain. At 27 weeks postinoculation, PrPd targeted to the contralateral superior colliculus as delicate granular synaptic deposits located in the superficial part of this structure. As already reported, a few spongiform vacuoles were visible in the same area by conventional H and E staining. In several other sections, vacuoles were visible but no PrPd staining could be detected.

Novel crystalloid oligodendrogliopathy in hereditary spastic paraplegia

Abstract: Hereditary spastic paraplegia (HSP) comprises a group of clinically and genetically heterogeneous disorders associated with spastic paraparesis (pure HSP) with or without additional neurological symptoms (complicated HSP). Here we present a case of an adult-onset, apparently autosomal-dominant, complicated form of HSP. Onset of clinical symptoms was at the age 40 years and characterised by slowly progressive corticospinal tract dysfunction, dysarthria, disorientation, extrapyramidal symptoms, and bilateral ptosis. Cranial MRI revealed hyperintensities on T2-weighted sequences mostly in the posterior limb of the internal capsule. The proband deceased at the age of 64 years. As morphological substrate for the slowly progressive clinical symptoms, comprehensive neuropathological and ultrastructural evaluation revealed a novel oligodendrogliopathy with distinctive, partly ubiquitinated and p62 positive fibrillar inclusions evolving into crystalloid deposits, containing elements of the oligodendroglial cytoskeleton (α- and β-tubulin, TPPP/p25). In the central nervous system, accumulation of crystalloid structures has been related to histiocytes but not to glial cells. This study has implications for the understanding on how the human central nervous system reacts to protracted dysfunction and disruption of the oligodendroglial cytoskeleton, including development of crystalloid structures, which have not yet been reported in neurodegenerative diseases including HSP.

Targeting of prion-infected lymphoid cells to the central nervous system accelerates prion infection

Abstract: Prions, composed of a misfolded protein designated PrPSc, are infectious agents causing fatal neurodegenerative diseases. We have shown previously that, following induction of experimental autoimmune encephalomyelitis, prion-infected mice succumb to disease significantly earlier than controls, concomitant with the deposition of PrPSc aggregates in inflamed white matter areas. In the present work, we asked whether prion disease acceleration by experimental autoimmune encephalomyelitis results from infiltration of viable prion-infected immune cells into the central nervous system. C57Bl/6 J mice underwent intraperitoneal inoculation with scrapie brain homogenates and were later induced with experimental autoimmune encephalomyelitis by inoculation of MOG35-55 in complete Freund's adjuvant supplemented with pertussis toxin. Spleen and lymph node cells from the co-induced animals were reactivated and subsequently injected into naive mice as viable cells or as cell homogenates. Control groups were infected with viable and homogenized scrapie immune cells only with complete Freund's adjuvant. Prion disease incubation times as well as levels and sites of PrPSc deposition were next evaluated. We first show that acceleration of prion disease by experimental autoimmune encephalomyelitis requires the presence of high levels of spleen PrPSc. Next, we present evidence that mice infected with activated prion-experimental autoimmune encephalomyelitis viable cells succumb to prion disease considerably faster than do mice infected with equivalent cell extracts or other controls, concomitant with the deposition of PrPSc aggregates in white matter areas in brains and spinal cords. Our results indicate that inflammatory targeting of viable prion-infected immune cells to the central nervous system accelerates prion disease propagation. We also show that in the absence of such targeting it is the load of PrPSc in the inoculum that determines the infectivity titers for subsequent transmissions. Both of these conclusions have important clinical implications as related to the risk of prion disease contamination of blood products.

Creutzfeldt-Jakob disease with unusually extensive neuropathology in a child treated with native human growth hormone

Abstract: We report a case of iatrogenic Creutzfeldt-Jakob disease(iCJD) in a child with a neonatal growth hormone (GH) deficiency that was treated with native human growth hormone (hGH) between the ages of 9 months and 7 years. Three years after the end of treatment a progressive neurological syndrome consistent with Creutzfeldt-Jakob disease (CJD) developed, leading to death within a year, at age 11. Neuropathological examination showed an unusual widespread form of CJD, notably characterized by (i) involvement of the cerebellar white matter, (ii) cortico-spinal degeneration and (iii) ballooned neurons. A transitional form of the disease between common iatrogenic and panencephalopathic CJD is suggested.

Statement on a request from the European Commission for the assessment of the scientific elements supporting the prohibition for the placing on the market of GM potato EH92-527-1 for cultivation purposes in Austria

Abstract: Austria notified to the European Commission its ordinance implementing a national safeguard measure prohibiting the placing on the market of GM potato EH92-527-1 for cultivation purposes in Austria, after which the European Commission asked the European Food Safety Authority to assess the scientific elements supporting the prohibition. Having considered the information package provided by Austria and all relevant scientific publications, the GMO and BIOHAZ Panels concluded that: i) no new data specific to the safety of the nptII gene have been provided; ii) the risk posed by the formation of mosaic structures of aminoglycoside phosphotransferase genes could not be assessed without data documenting the existence of such structures among the existing gene variants and such data were not provided; iii) the therapeutic relevance of kanamycin and neomycin was already addressed in the EFSA’s opinion on ARM genes and as of yet there is no evidence to indicate that resistance to these antibiotics in clinically-relevant bacteria has developed as a result of acquisition of the nptII gene; iv) the knowledge gaps and uncertainties highlighted in the Austrian document have already been considered in the EFSA’s opinion on ARM genes. Austria did not provide any new or additional information on the molecular characterisation or PMEM of potato EH92-527-1 after the date of consent for this GM event that would require the reassessment of existing information. The EFSA GMO Panel reiterates its scientific opinion on the 2010 monitoring report of GM potato EH92-527-1 in which it provides specific recommendations to improve the methodology of the PMEM of the GM potato. Further, the EFSA GMO Panel concludes that no grounds exist to date that would lead to reconsideration of its opinion on GM potato EH92-527-1.