Additional file 1: Figure S1. of A novel multiplex assay for simultaneous quantification of total and S129 phosphorylated human alpha-synuclein

Principle and protocol of the AlphaLISA technique. In A Streptavidin-coated Donor-beads are bound to biotinylated antibodies. Acceptor-beads are directly coupled to antibodies. When both antibodies bind to an analyte, the beads are brought into close proximity. Donor-beads are excited at 680 nm and produce singlet oxygen molecules triggering a chemical reaction in the close-by Europium Acceptor-beads resulting in an emission of light at 615 nm. In B the protocol of a no-wash AlphaLISA is depicted. (PDF 315 kb

Additional file 2: Figure S2. of A novel multiplex assay for simultaneous quantification of total and S129 phosphorylated human alpha-synuclein

Quantification of S129 phosphorylation of human alpha-synuclein recombinant protein. An isoelectric focusing gel pH range 5–8 (A) was used to separate pS129 from non-phosphorylated h-asyn by loading recombinant h-asyn and pS129 h-asyn protein side by side. In B, blots were probed for total h-asyn (syn-1) or pS129 h-asyn (11A5) to determine pS129 h-asyn band. Quantification areas are indicated on total h-asyn blots by dashed boxes. This assay was run independently on three occasions, which yielded estimates of percent phosphorylation of h-asyn at S129 site to be 28.4, 29.5, and 26.0 %, respectively, with an average and standard deviation of 28.0 ± 1.8 %. (PDF 339 kb

Additional file 3: of Lewy body-like alpha-synuclein inclusions trigger reactive microgliosis prior to nigral degeneration

Figure S3. Antigen-presenting MHC-IIir microglia are not associated with peak of intraneuronal inclusions of pSyn in the striatum. Progression of pSyn pathology and MHC-IIir microglia in the striatum. (a) At 2 months p.i., pSyn inclusions are localized to neurites, presumably representing terminals from the SNc. (b–c) Over time pSyn inclusions become primarily localized to the soma of striatal neurons. (d) Abundant MHC-IIir microglia in the striatum primarily localized around the α-syn PFF injection site at 2 months. (e–f) MHC-IIir microglia in the striatum are largely absent during continuing accumulation of intraneuronal pSyn inclusions at 4 months (e) and 6 months (f) p.i. (g) Intrastriatal injection of PBS results abundant MHC-IIir microglia in the striatum localized near the site of injection at 2 months p.i., although appearing less abundant than MHC-IIir microglia in the striatum of α-syn PFF rats at the same time point (d). (h) MHC-IIir microglia are similarly absent from the parenchyma by 4 months (h) and 6 months p.i (i). Scale bars A–I = 50 μm. Abbreviations: p.i. = postinjection; PFFs = pre-formed alpha-synuclein fibrils; PBS = phosphate-buffered saline; pSyn = α-syn phosphorylated at serine 129, MHC-IIir = major-histocompatibility complex-II immunoreactive. (TIF 112368 kb

Additional file 1: of Lewy body-like alpha-synuclein inclusions trigger reactive microgliosis prior to nigral degeneration

Figure S1. Unilateral intrastriatal injection of α-syn PFFs, but not RSA or PBS, induces bilateral cortical and unilateral SNc Lewy-body like inclusions of phosphorylated α-syn (pSyn). (a) pSyn pathology is observed bilaterally in cortical areas after unilateral injection of α-syn PFFs, namely in layers 2/3 and orbital and agranular insular cortices. (b) Injection of PBS or (c) RSA did not induce pSyn accumulation. (d) pSyn accumulation in the ipsilateral substantia nigra pars compacta (SNc) at 2 months postinjection, with no evidence of pSyn inclusions in the contralateral SNc. Scale bars (A–D) = 50 μm. Abbreviations: α-syn = alpha-synuclein; PFFs = pre-formed alpha-synuclein fibrils; PBS = phosphate-buffered saline; RSA = rat serum albumin; pSyn = α-syn phosphorylated at serine 129. (TIF 117729 kb

Additional file 1: of Oligomeric and phosphorylated alpha-synuclein as potential CSF biomarkers for Parkinson’s disease

Supplementary Materials and Methods. (PDF 401 kb

Additional file 3: of Lewy body-like alpha-synuclein inclusions trigger reactive microgliosis prior to nigral degeneration

Figure S3. Antigen-presenting MHC-IIir microglia are not associated with peak of intraneuronal inclusions of pSyn in the striatum. Progression of pSyn pathology and MHC-IIir microglia in the striatum. (a) At 2 months p.i., pSyn inclusions are localized to neurites, presumably representing terminals from the SNc. (b–c) Over time pSyn inclusions become primarily localized to the soma of striatal neurons. (d) Abundant MHC-IIir microglia in the striatum primarily localized around the α-syn PFF injection site at 2 months. (e–f) MHC-IIir microglia in the striatum are largely absent during continuing accumulation of intraneuronal pSyn inclusions at 4 months (e) and 6 months (f) p.i. (g) Intrastriatal injection of PBS results abundant MHC-IIir microglia in the striatum localized near the site of injection at 2 months p.i., although appearing less abundant than MHC-IIir microglia in the striatum of α-syn PFF rats at the same time point (d). (h) MHC-IIir microglia are similarly absent from the parenchyma by 4 months (h) and 6 months p.i (i). Scale bars A–I = 50 μm. Abbreviations: p.i. = postinjection; PFFs = pre-formed alpha-synuclein fibrils; PBS = phosphate-buffered saline; pSyn = α-syn phosphorylated at serine 129, MHC-IIir = major-histocompatibility complex-II immunoreactive. (TIF 112368 kb

Additional file 2: of Lewy body-like alpha-synuclein inclusions trigger reactive microgliosis prior to nigral degeneration

Figure S2. Unilateral intrastriatal injection of α-syn PFFs induces widespread accumulation of Lewy-body like inclusions of phosphorylated α-syn (pSyn). Representative images illustrating the time course of pSyn pathology in regions innervating the striatum. (a–c) pSyn pathology in the ipsilateral agranular insular cortex localized to both the soma and neurites at 2 months p.i. (postinjection) that over time becomes primarily localized to the soma; scale bar = 50 μm, inset = 10 μm. (d–f) Ipsilateral accumulation of pSyn in the substantia nigra peaks at 2 months and becomes less abundant over time as neurons degenerate; scale bar = 200 μm, inset = 25 μm. (g–i) In contrast to other areas, pSyn in the striatum is primarily localized to neurites at 2 months and becomes more abundant and localized to the soma over time, scale bar = 50 μm, inset = 10 μm. Abbreviations: α-syn = alpha-synuclein; PFFs = pre-formed alpha-synuclein fibrils; pSyn = α-syn phosphorylated at serine 129; p.i. = postinjection. (TIF 33472 kb