Immunohistochemistry analysis of rat brain injected with active human alpha synuclein PFFs (GTX17669-pro). Species: Female Sprague-Dawley Rat. Rat was injected with 2μL active human alpha synuclein PFFs (GTX17669-pro) in each of 2 injection sites: AP+1.6, ML+2.4, DV-4.2 from skull; and AP-1.4, ML+0.2, DV-2.8 from skull. 30-days post-injection. Fixation: Saline perfusion followed by 4% PFA fixation for 48 hrs. Secondary Antibody: Biotin-SP Donkey Anti-Rabbit IgG (H+L) at 1:500 for 2 hours in cold room with shaking. ABC signal amplification, DAB staining. Magnification: 20X. Alpha synuclein pathology is seen in the striatum close to an injection site.

Primary rat hippocampal neurons show lewy body inclusion formation when treated with active Alpha Synuclein Protein Preformed Fibrils (GTX17669-pro) at 4 μg/ml (D-F), but not when treated with control Alpha Synuclein Protein Preformed Fibrils (GTX17667-pro) at 4 μg/ml (A-C).
Tissue: Primary hippocampal neurons. Species: Sprague-Dawley rat. Fixation: 4% formaldehyde from PFA. Primary Antibody: Mouse anti-pSer129 Antibody at 1:1000 24 hours at 4ºC. Secondary Antibody: FITC Goat Anti-Mouse (green) at 1:700 for 1 hours at RT. Counterstain: Hoechst (blue) nuclear stain at 1:4000 for 1 hour at RT. Localization: Lewy body inclusions. Magnification: 20x.

Fluorescently-labelled Alpha Synuclein Protein Preformed Fibrils (GTX17669-pro) were taken up, transported into the soma, and induced alpha synuclein aggregation in mouse neurocortical primary cells.
Left : Neurites filled with fluorescently-labelled alpha synuclein seeds in a microfluidic co-culture system after 24 hours.
Right : Alpha synuclein seeds within the soma and neurites of mouse neurocortical primary cells after 24 hours.

Confocal imaging shows NeuN+ (mature) primary cortical neurons filled with Fluorescently-labelled Alpha Synuclein Protein Preformed Fibrils (GTX17669-pro). GTX17669-pro seed endogenous alpha synculein phosphorylation after 7 days.

ICC/IF analysis of human iPSC-derived neurons treated with Alpha Synuclein Protein Preformed Fibrils (GTX17669-pro).
Figure A : Negative control; no fibrils added to well.
Figure B : 7 days after addition of active recombinant human pre-formed fibrils (Type 1). Fibrils were sonicated before use and applied 2.5 ug per well
Green : Primary Antibody : Mouse Anti-Alpha Synuclein (phospho Ser129) Monoclonal Antibody at 1:1000
Blue : Hoechst
Magenta : beta Actin

Transmission electron microscopy (TEM) image of GTX17669-pro Human alpha Synuclein protein (active, Pre-Formed Fibrils).

Transmission electron microscopy (TEM) image of GTX17669-pro Human alpha Synuclein protein (active, Pre-Formed Fibrils).

Toxicity results comparing active Human alpha Synuclein protein (Pre-Formed Fibrils) (GTX17667-pro) and active Alpha Synuclein Protein Preformed Fibrils (GTX17669-pro). Data was graphed after live cell imaging results were obtained using the following procedure: After 8 days in vitro, primary rat mixed cortical neuron cells were treated with 500 μg/ml of Type 1 and Type 2 Alpha Synuclein Proteins for 20 hours at 37˚C.

Alpha Synuclein Protein Preformed Fibrils (GTX17669-pro) was shown to be taken up by SH-SY5Y cells and transmitted to neuronal iPSCs within 14 days.
Blue : Hoechst
Green : SH-SY5Y-GFP
Red : Alpha Synuclein Protein Preformed Fibrils (GTX17669-pro)
Purple : Tubulin

SDS-PAGE of ~14 kDa active Human Recombinant Alpha Synuclein Protein Preformed Fibrils (GTX17669-pro). Lane 1: Molecular Weight Ladder (MW). Lane 2: active Alpha Synuclein Protein Preformed Fibrils (GTX17669-pro).

Active alpha synuclein preformed fibrils (GTX17669-pro) seed the formation of new alpha synuclein fibrils from the pool of alpha synuclein monomers (GTX17668-pro). Thioflavin T is a fluorescent dye that binds to beta sheet-rich structures, such as those in alpha synuclein fibrils. Upon binding, the emission spectrum of the dye experiences a red-shift, and increased fluorescence intensity. Thioflavin T emission curves show increased fluorescence (correlated to alpha synuclein protein aggregation) over time when 10 nM of active alpha synuclein preformed fibrils (GTX17669-pro) is combined with 100 μM of alpha synuclein monomer (GTX17668-pro), as compared to active alpha synuclein preformed fibrils (GTX17669-pro) alone and alpha synuclein monomer (GTX17668-pro) alone. Thioflavin T ex = 450 nm, em = 485 nm.