Significant differences were observed between the intensity of the staining of the transgenic mice and the diseased human brains by the 96C105, 116C125, and 131C140 antibodies

Significant differences were observed between the intensity of the staining of the transgenic mice and the diseased human brains by the 96C105, 116C125, and 131C140 antibodies. Open in a separate window Fig.?6 Semi-quantitative assessment (0C3) of the intensity of the immunostaining of four (Thy-1)-h[A30P] alpha-synuclein transgenic mouse brains and four alpha-synucleinopathy patients. human and transgenic mice was compared. Taken together, the study shows that although comparable epitopes are uncovered in both in vitro and in vivo formed alpha-synuclein inclusions, structural heterogeneity Roflumilast N-oxide can be observed between different molecular species. Keywords: Parkinsons disease, Dementia with Lewy bodies, Alpha-synuclein, Epitope mapping Introduction The alpha-synucleinopathies are a subset of neurodegenerative diseases that include Parkinsons disease (PD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA), and the Lewy body variant of Alzheimers disease (Goedert 2001). The common pathological feature of the alpha-synucleinopathies is the presence of insoluble cytoplasmic aggregates of alpha-synuclein, termed Lewy bodies and Lewy neurites (Spillantini et al. 1997). In PD, the inclusions are mainly found in dopaminergic neurons in the substantia nigra and other subcortical regions, while in DLB, they are also located in the cerebral cortex (Spillantini et al. 1997, 1998). In MSA, the aggregates are predominantly found in oligodendrocytes and are then referred to as glial cytoplasmic inclusions (Papp et al. 1989). Alpha-synuclein consists of three distinct structural domains: a lipid-interacting amino-terminus (1C60), a hydrophobic mid-region (61C95), and an acidic carboxyl-terminus with a random Roflumilast N-oxide coil structure (96C140) (George et al. 1995). It has been believed that alpha-synuclein mainly exists as an intrinsically disordered protein, which adopts an alpha-helical structure upon binding to lipids via its N-terminus and central region (Davidson et al. 1998). The physiological function of alpha-synuclein remains largely unknown, but increasing evidence suggest that it is involved in neurotransmitter release (Abeliovich et al. 2000; Nemani et Roflumilast N-oxide al. 2010). For example, alpha-synuclein can promote the formation of the soluble diagnosis, Parkinsons disease, Parkinsons disease dementia, dementia with Lewy bodies, neurologically normal, postmortem delay Immunohistochemistry of Mouse and Human Brain Tissue Paraffin sections were deparaffinized through ethanol baths of decreasing concentration (99.9C70%) and then washed with distilled water. The sections were pretreated with proteinase K (Life Technologies, Carlsbad, CA) at 50?g/ml in a buffer containing 10?mM TrisCHCl, 100?mM NaCl, 0.1% Nonidet-P40 (United States Biochemical Corporation, Cleveland, OH) at pH 7.8 and 37?C for 5?min. Additionally, the human tissue was microwaved in preheated 25?mM citrate buffer for 15?s and allowed to cool down at room temperature for 40?min. All sections were then permeabilized with 0.4% Triton X-100 (Sigma-Aldrich) in TBS for 10?min and treated with 0.3% H2O2 for 5?min to block endogenous peroxidase reactivity. The sections were blocked with Background Sniper (Biocare Medical, Concord, CA) and incubated at 4?C overnight with the IgY antibodies at a concentration of 0.5?g/ml. Detection was performed using a biotinylated secondary anti-chicken (1.5?g/ml, Jackson ImmunoResearch Laboratories Inc.), followed by incubation with Streptavidin-HRP (1:30, 3310-9, Mabtech AB, Nacka, Sweden). The signal was visualized using the NovaRed substrate kit (Vector Laboratories). The sections were counterstained Rabbit Polyclonal to OR2T2 with hematoxylin (Histolab, Gothenburg, Sweden), dehydrated and mounted with DPX (VWR, Stockholm, Sweden). As a negative control, sections were incubated with secondary antibody alone. Neuropathological Analysis of Mouse and Human Brain Tissue Two independent assessors analyzed immunohistochemically stained sections semi-quantitatively in a blinded manner. The intensity of the staining was scored on a predetermined scale of 0C3 (0?=?no signal, 1?=?overall faint signal, orange/yellow-colored LB in human tissue, 2?=?overall positive signal, red-colored LB Roflumilast N-oxide in human tissue, 3?=?very strong signal, dark red/brown-colored LB Roflumilast N-oxide in human tissue), and scores were averaged. The differences between mouse and human brain were assessed by a two-way ANOVA and Bonferroni post hoc tests. Results Characterization of Immunoglobulin Y Antibodies In order to determine the surface-exposed epitopes of alpha-synuclein, we generated 18 polyclonal IgY antibodies against short linear peptides spanning most of the alpha-synuclein molecule (Fig.?1). We chose to generate chicken IgY antibodies because of the increased phylogenetic distance between chicken and humans compared to other mammalian species typically used for antibody production (e.g., rabbit) (Hadge and Ambrosius 1984). The sequence identity of chicken alpha-synuclein and human alpha-synuclein is 86.7% compared to 95% between rabbits and humans, which could potentially increase the immunogenicity of the alpha-synuclein peptides. The antibodies were affinity purified against their respective immunizing peptide and showed similar reactivity against them as observed by indirect ELISA (Fig.?2a). Open in a separate window Fig.?1 Sequence of immunizing peptides and human alpha-synuclein with its three structural regions: the N-terminus (1C60 showed no cross-reactivity and were selected for the following experiments (b). represent the SEM. Each experiment was performed three times (except in the phospholipid ELISA; 20?m In the human PD, DLB, and PDD brain tissue, 1C10 and the C-terminal antibodies 111C120 and 116C125 showed the.