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Results: 5

1.
Figure 4

Figure 4. Effects of α-syn oligomers derived from symptomatic A53T α-syn transgenic mice on the kinetics of α-syn fibril formation. From: Distinct region-specific alpha-synuclein oligomers in A53T transgenic mice: implications for neurodegeneration.

The kinetics of forming fibrils from recombinant purified α-syn in vitro was assessed using the amyloid binding fluorescent dye Thioflavin T and expressed as relative fluorescence units (RFU). a) Oligomers isolated from SC significantly decreased the lag phase of fibril formation of recombinant α-syn whereas oligomers isolated from the OB b) prolong the lag phase of fibril formation. α-syn was depleted from oligomeric fractions by immunoprecipitation with MAB syn211. Values represent the mean ± SEM (n=4–5, two-way ANOVA *p=0.001, **p=0.05, ***p=0.01). c) Western blot analysis using syn211 of immunoprecipitated (I.P.) α-syn and the corresponding immunodepleted (I.D.) fractions from the OB used in the seeding assays (similar results were obtained with the SC fractions). Total protein was visualized by coomassie brilliant blue (CBB) staining showing equal loading of samples.

Elpida Tsika, et al. J Neurosci. ;30(9):3409-3418.
2.
Figure 3

Figure 3. Biochemical analysis of the 53Å α-syn oligomers isolated by SEC from A53T α-syn transgenic mice. From: Distinct region-specific alpha-synuclein oligomers in A53T transgenic mice: implications for neurodegeneration.

The fraction corresponding to the 53 Å-size oligomers is shown in lanes 2 and 4 while the monomer is shown in lanes 1 and 3 of each blot. a) 0.5 mg/ml of fractionated lysates were incubated with 8 M urea for 1 hour at 25 °C and b) 100 μg/ml proteinase K for 30minutes at 37 °C. The results of the treatments were the same for oligomers isolated either from the SC (shown here) or OB from both symptomatic and asymptomatic mice. The human specific α-syn MAB LB509 (shown here) recognizes an epitope at the C-terminus of the protein (residues 115–122). Analysis with MAB syn208 recognizing an epitope at the amyloidogenic region of the protein (residues 87–110) yielded the same results. c) Indication of biochemical diversity between oligomers isolated from regions bearing inclusions (SC) and pathology free (OB) is revealed by MAB syn303, which was generated against oxidized α-syn and preferentially recognizes oligomeric forms of α-syn in human brain tissue with α-syn aggregates (Duda et al., 2002).

Elpida Tsika, et al. J Neurosci. ;30(9):3409-3418.
3.
Figure 5

Figure 5. In vitro toxicity of α-syn oligomers derived from the spinal cord of symptomatic A53T α-syn transgenic mice. From: Distinct region-specific alpha-synuclein oligomers in A53T transgenic mice: implications for neurodegeneration.

a) Total cell number and volume of primary mouse cortical cultures was determined by a combination of Sapphire700 (non-specific cytosolic stain) and DRAQ5 (nuclear stain) after the addition of SEC fractions containing α-syn oligomers. b) neurite degeneration was assessed by quantifying neurofilament (NF) levels by immunostaining. 25 mM Hepes/150 mM NaCl pH 7.4 (Buffer) and equivalent SEC fractions from the hippocampus (Hipp) were utilized as controls. SEC fractions from the SC and OB were incubated with either IgG or anti-α-syn antibodies (syn211) to deplete α-syn oligomers and determine if neurodegeneration is mediated by the presence of α-syn. (n=5–10 for each time point, *p<0.05). Representative immunostaining of Sapphire700/DRAQ5 and NF from scans of the 96 well culture plate are shown under the graph. c) The amount of α-syn oligomers obtained from SC and OB SEC fractions added to the primary cultures were determined by I.P./western blot analysis. Samples were immunoprecipitated and blotted using MAB syn211 or mouse IgG as a control. The bands were analyzed by densitometry and found to be equal (integrated intensity of OB= 15.2; SC=15.3).

Elpida Tsika, et al. J Neurosci. ;30(9):3409-3418.
4.
Figure 2

Figure 2. Identification and regional distribution of α-syn oligomers in A53T α-syn transgenic mice. From: Distinct region-specific alpha-synuclein oligomers in A53T transgenic mice: implications for neurodegeneration.

Triton-soluble extracts from the indicated regions of the nervous system of symptomatic mice were subjected to native SEC. The SEC fractions were then analyzed by SDS-PAGE/western blot for α-syn using the human specific anti-α-syn MAB, syn211. NSE was used as loading control. α-Syn monomer eluted at a volume corresponding to a 34 Å-size particle, migrating as a 19 kDa species by SDS-PAGE. High molecular weight oligomers eluted between 36 and 113 Å. The horizontal marker indicates the apparent molecular radius in angstroms (Å) that corresponds to the elution volume of globular protein standards analyzed by SEC, while the vertical marker indicates the mobility of protein standards with known molecular mass on SDS-PAGE, in kDa. Representative blots of each region analyzed are shown. The graph on the lower right panel shows a quantitative densitometry analysis of the SEC/SDS-PAGE blots from symptomatic and asymptomatic mice, revealing equal relative levels of soluble oligomeric α-syn from tissues of asymptomatic and symptomatic mice. A quantitative analysis of each oligomeric form is shown in supplemental Fig. 2. Total α-syn oligomers levels are expressed as percentage of total soluble α-syn. Mean values ± SEM, n=3–5 for all tissues were plotted. The asterisk denotes statistical significance (p=0.02, SC symptomatic vs Hipp symptomatic, using a one-way ANOVA with Tukey’s post-hoc test). Combination of oligomer levels from all inclusion-bearing regions were also compared to those found in all non-inclusion-bearing regions and also did not reveal a statistically significant difference (% total oligomers: regions with inclusions 44.1±9.4, n=9; regions without inclusions 41.5±4.6, n=28; mean values ± SEM, p=0.63, student’s t-test analysis).

Elpida Tsika, et al. J Neurosci. ;30(9):3409-3418.
5.
Figure 1

Figure 1. Regional specific accumulation of Triton-insoluble α-syn in symptomatic A53T α-syn transgenic mice. From: Distinct region-specific alpha-synuclein oligomers in A53T transgenic mice: implications for neurodegeneration.

a) Tissues from asymptomatic (4–6 month old) and symptomatic (11–12 month old) A53T α-syn transgenic mice, as well as 11 month old transgenic mice expressing human wild-type α-syn, were subjected to extraction with 1 % Triton X-100 buffer (T-soluble) followed by western blot analysis using the human specific anti-α-syn MAB, LB509. Neural specific enolase (NSE) was used as a loading control. Tyrosine hydroxylase (TH) served as a marker of catecholaminergic regions. Regions containing α-syn lesions [Spinal cord (SC) and cerebellum (Cr)] as well histologically unaffected regions [substantia nigra (SN), olfactory bulbs (OB), and hippocampus (H)] were analyzed. b) The insoluble pellets obtained from Triton fractions were extracted with buffer containing 2 % SDS (T-insoluble). Vimentin (Vim) was used as a loading control. The markers on the left indicate the mobility of standards with known molecular masses on SDS-PAGE in kDa. c) Immunohistochemical analysis demonstrating gliosis in areas of the nervous system associated with α-syn pathological inclusions. Tissue sections from olfactory bulb (A,B,E,F) and spinal cord (C,D,G,H) from 12 month old non-transgenic (nTg) (A,C,E,G) and symptomatic A53T α-syn transgenic mice (B,D,F,H) were stained with an antibody to GFAP (A,B,C,D) or α-syn (syn505) (E,F,G,H). Staining with syn505 reveals abundant α-syn pathological inclusions in spinal cord of the A53T α-syn transgenic mice (H), and the lack thereof in the olfactory bulb (F) and in the tissues from the nTg mice (E,G). Staining with an anti-GFAP antibody demonstrates significant gliosis in the spinal cord of the symptomatic A53T α-syn transgenic mice (D), while the olfactory bulbs of A53T α-syn transgenic mice (B) as well as tissues from nTg mice (A,C) demonstrate less gliosis. (Bar scale = 100 μm)

Elpida Tsika, et al. J Neurosci. ;30(9):3409-3418.

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