Fig. 1
Methods workflow. (A) After donor inclusion, autopsy was performed, and 8 cortical regions were dissected and grouped into regions affected at [10] Braak α-synuclein stage 4 (in blue), 5 (in yellow), and 6 (in red). (B) Brain tissue was processed for immunohistochemistry using antibodies targeting pSer129 α-synuclein, p-tau, Aβ, and NfL with bright-field, and synaptophysin and SV2A with fluorescence. Whole tissue slides were digitalized, and regions of interest (ROIs) were drawn in superficial (layer I-III) and deep (layer IV-VI) layers of the cortex on bright-field scans, and in the corresponding layer III and layer V-VI on fluorescent scans (detailed ROI placing within cortical minicolumns in C) and Fig. S1). D) Subsequently, pixel and object classifiers in QuPath [38] were used to quantify neuropathology load and NfL immunoreactivity on bright-field scans. The ROIs placed on fluorescent scans were E) pre-processed in Huygens Professional, and F) analysed with NIS elements to quantify synaptophysin+ and SV2A+ puncta per µm3 in the cortical neuropil volume. For example of artifact removal see Fig. S2. Aβ: amyloid-β; AF: autofluorescence; CTC: cross-talk correction; IHC: immunohistochemistry; LB: Lewy body; NfL: neurofilament light chain; pSer129-αSyn: α-synuclein phosphorylated at Ser129; p-tau: phosphorylated tau; ROI: region of interest; SV2A: synaptic vesicle glycoprotein 2 A