Journal: Acta Neuropathologica

Article Title: Distinct tau and alpha-synuclein molecular signatures in Alzheimer’s disease with and without Lewy bodies and Parkinson’s disease with dementia

doi: 10.1007/s00401-023-02657-y

Figure Lengend Snippet: Heatmaps depicting epitope mapping of aSyn in the human brain. MJFR14 (a.a. 133–138) showed best overall binding to both the MTG and AMY brain tissues and showed increased detection of aSyn in the AMY of AD-LB and PDD cases when compared to controls and AD cases. C-terminal antibodies LB509, MJFR1, Syn211 and Syn202 detected also more aSyn in the AD-LB cases when compared to controls and/or AD cases in the AMY. EP1536Y detected more pS129 aSyn in the MTG and AMY of AD-LB cases compared to controls and AD cases, while only detecting more pS129 aSyn in the AMY when comparing PDD to controls cases. Each tile displays the averaged normalized value per group, as is depicted by the scale bar. PTM-specific antibodies are highlighted in red. NAC non-amyloid-β component region, CTT C-terminal truncation site, n nitration site, p phosphorylation site, a.a. amino acid

Article Snippet: As a positive control for aSyn gels, 20 ng of recombinant aSyn (rPeptide, cat# S1-1001) and 20 ng of recombinant pS129 aSyn (Proteos, cat# RP-004) were loaded per gel, and for tau gels, 2 µL of tau protein ladder (rPeptide, cat# T-1007, containing 5 ng of each tau isoform) was loaded per gel.

Techniques: Binding Assay, Nitration

Journal: Acta Neuropathologica

Article Title: Distinct tau and alpha-synuclein molecular signatures in Alzheimer’s disease with and without Lewy bodies and Parkinson’s disease with dementia

doi: 10.1007/s00401-023-02657-y

Figure Lengend Snippet: Multiplexed western blots for aSyn in soluble and insoluble fractions of the MTG. All antibodies, except EP1536Y targeting pS129 aSyn, showed strong detection of aSyn in the soluble fractions of all cases. Interestingly, Syn-1 showed stronger detection of soluble aSyn in the soluble fraction of PDD cases compared to all other groups. EP1536 signal was only detected in the insoluble fractions of 2/3 AD-LB cases and very minimally in 2/3 PDD cases, while no signal was detected in any of the other groups. All other antibodies were also able to detected aSyn mainly in the insoluble fraction of the AD-LB cases and to a very limited extent in all other groups

Article Snippet: As a positive control for aSyn gels, 20 ng of recombinant aSyn (rPeptide, cat# S1-1001) and 20 ng of recombinant pS129 aSyn (Proteos, cat# RP-004) were loaded per gel, and for tau gels, 2 µL of tau protein ladder (rPeptide, cat# T-1007, containing 5 ng of each tau isoform) was loaded per gel.

Techniques: Western Blot

Journal: Acta Neuropathologica

Article Title: Distinct tau and alpha-synuclein molecular signatures in Alzheimer’s disease with and without Lewy bodies and Parkinson’s disease with dementia

doi: 10.1007/s00401-023-02657-y

Figure Lengend Snippet: Multiplexed western blots for aSyn in soluble and insoluble fractions of the amygdala. All antibodies, except EP1536Y targeting pS129 aSyn, showed strong detection of aSyn in the soluble fractions of all cases. Syn-1, showed stronger detection of soluble aSyn in the soluble fraction of PDD cases compared to the AD group. Meanwhile, A15115A, 4B12 and MJFR1 detected less soluble aSyn in AD and/or AD-LB cases compared to the other groups. pS129 aSyn (EP1536Y) was significantly upregulated in the insoluble fractions of AD-LB cases compared to all other groups. MJFR14 specifically detected insoluble aSyn in AD-LB and PDD cases, while MJFR1 showed additionally also slight detection of insoluble aSyn in controls and PDD cases

Article Snippet: As a positive control for aSyn gels, 20 ng of recombinant aSyn (rPeptide, cat# S1-1001) and 20 ng of recombinant pS129 aSyn (Proteos, cat# RP-004) were loaded per gel, and for tau gels, 2 µL of tau protein ladder (rPeptide, cat# T-1007, containing 5 ng of each tau isoform) was loaded per gel.

Techniques: Western Blot

Journal: Acta Neuropathologica

Article Title: Distinct tau and alpha-synuclein molecular signatures in Alzheimer’s disease with and without Lewy bodies and Parkinson’s disease with dementia

doi: 10.1007/s00401-023-02657-y

Figure Lengend Snippet: Schematic overview highlighting available epitopes on tau and alpha-synuclein for antibody binding in the human brain under non-denaturing conditions, distinct tau and aSyn profiles in pure versus mixed cases and tau/aSyn co-aggregates in astrocytes in AD-LB cases. This study highlights the proline-rich regions and C-terminus of tau and the C-terminus of aSyn to be the most available for antibody binding in detergent-free crude human brain homogenates. These regions were also characteristic for pathological variants of both proteins, as preferential binding was seen in AD/AD-LB cases for tau and AD-LB/PDD cases for aSyn. Differences were observed in the molecular profile between AD-LB and AD cases for tau, as tau load was similar (AT8) while more insoluble pTau (pS396) was detected in AD versus AD-LB cases. Differences were also observed in the molecular aSyn profile between AD-LB and PDD cases, as both aSyn load (MJFR1) was higher and an increased detection of insoluble pS129 aSyn was seen in AD-LB versus PDD cases. Co-localization between pTau (pS422) and aSyn (NAC-region) could be detected in astrocytes (GFAP) in the MTG of AD-LB cases. A theoretical model highlights the uptake of tau and aSyn seeds from the tripartite synapse, which leads to translocation of these seeds to the cell body for targeted degradation, after which impartial degradation results in the formation of co-aggregates. Created with BioRender.com

Article Snippet: As a positive control for aSyn gels, 20 ng of recombinant aSyn (rPeptide, cat# S1-1001) and 20 ng of recombinant pS129 aSyn (Proteos, cat# RP-004) were loaded per gel, and for tau gels, 2 µL of tau protein ladder (rPeptide, cat# T-1007, containing 5 ng of each tau isoform) was loaded per gel.

Techniques: Binding Assay, Translocation Assay

Journal: Acta Neuropathologica

Article Title: Molecular profiling of alpha-synuclein pathology and seeding activity in Parkinson’s disease

doi: 10.1007/s00401-026-03026-1

Figure Lengend Snippet: Measurement of total and pSer129-aSyn levels in detergent-insoluble brain fractions by AlphaLISA. ( a and b ) total and pSer129-aSyn levels in PD and control (C) groups. ( c and d ) total and pSer129-aSyn levels across controls, EOPD and LOPD. ( e and f ) Comparison of total and pSer129-aSyn levels in FP-LO and SP-LO. Data represent mean ± SD. Each dot represents the average value for an individual case. Violin plots show the distribution of the data, with the median indicated by the central line and quartiles by the dashed lines. Statistical significance was determined using Kruskal–Wallis test followed by Dunn’s multiple comparison test for group comparisons (c and d) and the Mann–Whitney U test for pairwise comparisons (a, b, c and e), and Student’s t-test (f)

Article Snippet: The reaction mixture contained 1X phosphate-buffered saline (KD Medical, PBS 10X, pH 8.0), 0.0006% sodium dodecyl sulfate (SDS), 10 μM Thioflavin T (ThT), and 0.1 mg/mL recombinant human aSyn monomer (Proteos, RP-010).

Techniques: Control, Comparison, MANN-WHITNEY

Journal: Acta Neuropathologica

Article Title: Molecular profiling of alpha-synuclein pathology and seeding activity in Parkinson’s disease

doi: 10.1007/s00401-026-03026-1

Figure Lengend Snippet: aSyn seeding bioactivity measured by FRET FC assay. ( a ) Comparison of aSyn seeding activity in PD and control (C) groups. ( b ) Comparison of aSyn seeding activity in EOPD and LOPD disease subgroups. ( c ) Comparison of aSyn seeding activity in FP-LO and SP-LO subgroups. ( d ) Correlation between pSer129-aSyn levels and FRET seeding bioactivity in PD cases. ( e ) Cell viability analysis following treatment with detergent-insoluble brain fractions. Data represent mean ± SD. Each dot represents the average value for an individual case. Violin plots show the distribution of the data, with the median indicated by the central line and quartiles by the dashed lines. Statistical significance was determined using Kruskal–Wallis test followed by Dunn’s multiple comparison test for group comparisons and the Mann–Whitney U test for pairwise comparisons. For the correlation analysis, a two-tailed Spearman’s rank correlation test was used, and r and p values are indicated on the plot

Article Snippet: The reaction mixture contained 1X phosphate-buffered saline (KD Medical, PBS 10X, pH 8.0), 0.0006% sodium dodecyl sulfate (SDS), 10 μM Thioflavin T (ThT), and 0.1 mg/mL recombinant human aSyn monomer (Proteos, RP-010).

Techniques: Comparison, Activity Assay, Control, MANN-WHITNEY, Two Tailed Test

Journal: Acta Neuropathologica

Article Title: Molecular profiling of alpha-synuclein pathology and seeding activity in Parkinson’s disease

doi: 10.1007/s00401-026-03026-1

Figure Lengend Snippet: Intracellular aSyn aggregates in biosensor cells. ( a ) HEK.A53T-C/Y cells were treated with detergent-insoluble brain fractions and imaged under conditions in which excitation was applied at CFP and emission was detected at YFP wavelengths, enabling FRET-based visualization of intracellular aggregate formation. The scale bar represents 50 µm. ( b ) Quantification of intracellular aggregates in PD and control (C) samples. ( c ) Quantification of intracellular aggregates across controls, EOPD and LOPD. ( d ) Within LOPD subgroups, comparison of aggregate burden in FP-LO and SP-LO patients. Data represent mean ± SD. Each dot represents the average value for an individual case. Violin plots show the distribution of the data, with the median indicated by the central line and quartiles by the dashed lines. Data are shown as violin plots normalized to the negative control condition (lipofectamine-treated cells set to 1). Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test for group comparisons and unpaired two-tailed Student’s t-tests for pairwise comparisons

Article Snippet: The reaction mixture contained 1X phosphate-buffered saline (KD Medical, PBS 10X, pH 8.0), 0.0006% sodium dodecyl sulfate (SDS), 10 μM Thioflavin T (ThT), and 0.1 mg/mL recombinant human aSyn monomer (Proteos, RP-010).

Techniques: Control, Comparison, Negative Control, Two Tailed Test

Journal: Acta Neuropathologica

Article Title: Molecular profiling of alpha-synuclein pathology and seeding activity in Parkinson’s disease

doi: 10.1007/s00401-026-03026-1

Figure Lengend Snippet: Proteinase K (PK) digestion of seed amplification assay products identified differential proteolytic resistance profiles between controls (C) and PD patients. ( a ) Undigested product is shown on a Silver Stain at the expected band size for aSyn monomer (~ 15 kDa). PK digestion broke this product down over time. ( b ) PD patient products were more resistant than controls at all time points. ( c ) Although patient groups remained more resistant than controls, no differences within patient sub-groups were found in relation to disease onset. ( d ) Between late-onset patients, a trend toward higher resistance was observed in FP-LO patients. Data represent mean ± SD. Statistical significance was determined using Kruskal–Wallis test followed by Dunn’s multiple comparison test for group comparisons and the Mann–Whitney U test for pairwise comparisons

Article Snippet: The reaction mixture contained 1X phosphate-buffered saline (KD Medical, PBS 10X, pH 8.0), 0.0006% sodium dodecyl sulfate (SDS), 10 μM Thioflavin T (ThT), and 0.1 mg/mL recombinant human aSyn monomer (Proteos, RP-010).

Techniques: Amplification, Silver Staining, Comparison, MANN-WHITNEY

Journal: Acta Neuropathologica

Article Title: Molecular profiling of alpha-synuclein pathology and seeding activity in Parkinson’s disease

doi: 10.1007/s00401-026-03026-1

Figure Lengend Snippet: Gross pathology of the cingulate gyrus and correlations with Lewy body (LB) burden and biochemical measures. Representative coronal brain sections of the cingulate gyrus showing the cingulate cortex (white arrowheads) in an exceptional case ( a ), a typical case ( b ), and a non-LB control ( c ). Gross examination shows cortical atrophy and pallor of the cingulate region in PD cases compared to controls. ( d and e ) Immunohistochemistry with anti-NACP antibody in the cingulate cortex; (a) and (d) are from the same patient, and (b) and (e) are from the same patient. Inset in (d) shows an LB at higher magnification. Scale bars: 5 mm (a–c); 100 μm (d and e); 20 μm (inset). ( f ) Quantitative analysis of LB counts across PD patients did not reveal significant differences between clinical subgroups, although substantial variability was observed within each group. ( g ) Spearman correlation analyses show a positive association between LB counts and integrated FRET density, pSer129-aSyn, and total aSyn levels in PD cases

Article Snippet: The reaction mixture contained 1X phosphate-buffered saline (KD Medical, PBS 10X, pH 8.0), 0.0006% sodium dodecyl sulfate (SDS), 10 μM Thioflavin T (ThT), and 0.1 mg/mL recombinant human aSyn monomer (Proteos, RP-010).

Techniques: Control, Immunohistochemistry