A PNAS Direct Submission (2009)

Test if α-synuclein pathology involves direct neuron-to- neuron transmission of α-synuclein aggregates via endocytosis Overall hypothesis: If α-synuclein aggregates can be spread by direct neuron-to-neuron transmission, then we would expect accumulation of α-synuclein aggregates in the uninfected neurons. Desplats Paper

Figure 1 Figure 1A and 1B Hypothesis: If extracellular α-synuclein can be taken up by the mouse cortical neuronal stem cells (MCNSCs), then α-synuclein accumulation will be detected in the MCNSCs via Western blot and fluorescent microscopy.

Alexa-Fluor-488-α-synuclein MCNSCs Polyornithine/laminin-coated plate E15-E18 C57/BL6

Western Blotting

Figure 1(A): Results MCNSCs capable of taking up extracellular α-synuclein ? Conclusions?

Confocal Microscopy

Figure 1(B): Results MCNSCs took up extracellular Alexa-Fluor-488-labeled α-synuclein Conclusions?

Figure 1(C) Purpose: To determine whether α-synuclein released from neuronal cells can be directly transferred to MCNSCs Hypothesis: If α-synuclein is released by the neuronal cells, then we would expect to see uptake of α-synuclein in the MCNSCs via immunofluoresence.

Figure 1(C) Donor Cells Acceptor Cells MCNSCs-GFP Rat B103 + α-synuclein

Immunofluorescence α-synuclein

Figure 1(C): Results 47% of MCNSCs showed patterns of cytoplasmic accumulation of α-synuclein MCNSCNeuronal Cells Neuronal Cells + MCNSC Conclusions?

Figure 2 Purpose: To analyze the propagation of α-synuclein to transplanted stem cells in vivo. Figure 2 A-C Hypothesis: If a-syn can be transmitted directly from host to grafted neuronal stem cells, then α-synuclein will be detected in MCNSCs grafted into transgenic mice via immunofluoresence.

Immunostaining and TSA Adopted from HRP tyramide red α-syn HRP Secondary Antibody

Figure 2 Injected GFP-labeled MCNSCs into the hippocampus of transgenic mice expressing human α-synuclein. Transgenic (expresses human α-synuclein via Thy-1 promoter)

Figure 2 (A-C): Results ≈2.5% of MCNSCs showed human α-synuclein immunoreactivity in transgenic mice after 1 week 1 Week Later Hippocampus

Figure 2 (D-E): Results When MCNSCs not injected into α-synuclein transgenic mice, no immunoreactivity (D) MCNSCs showed no human α-synuclein immunoreactivity in non-transgenic mice (E) Controls

Figure 2 (F): Results 15% of α-synuclein-positive MCNSCs developed LBs in cytoplasm after 4 weeks 4 Weeks Later MCNSCs α-synuclein transgenic α-synuclein transgenic + MCSNCs

Figure 2 (G): Results Comments? 2.5% 15%

Figure 2 Suggests that α-synuclein pathology can be transmitted directly from host to grafted cells

Figure 3 Purpose: To further characterize cell-to-cell transmission of α- synuclein using an in vitro coculture model Figure 3(A) Hypothesis: If myc-tagged α-synuclein from donor cells can be released and transmitted to SH-SY5Y acceptor cells, then α- synuclein will be detected in the donor cells via immunofluorescence.

Figure 3 SH-SY5Y SH-SY5Y + α-synuclein myc SH-SY5Y Q Q Q Q Q Donor Cells Acceptor Cells

Figure 3(A): Results After 24 hrs, myc-tagged α-synuclein from donor cells was detected in acceptor cells Formation of inclusion bodies in some acceptors cells Conclusions?

Figure 3(A): Results After 24 hrs, myc-tagged α-synuclein from donor cells was detected in acceptor cells Formation of inclusion bodies in some acceptors cells Conclusions?

Figure 3(B): Results Inclusion body formation occurs with prolonged transmission of α-synuclein Conclusions?

Figure 3(C): Results ~ ½ of the acceptor cells displayed ubiquitin immunoreactivity Conclusions?

Figure 3(D) Purpose: To examine the involvement of donor cell membrane leakage in transmission of α-synuclein Lactate dehydrogenase release (LDH) assay SH-SY5Y cells overexpressing β-galatosidase, α- synuclein, and α-synuclein-myc

Figure 3(D): Results Cell-to-cell transmission occurs without cellular membrane leakage Conclusions?

Supplemental Fig. S2A Purpose: To determine if cell-cell contact is required for inclusion body formation – SH-SY5Y cells incubated in medium from SH-SY5Y cells expressing myc-tagged α-synuclein. Hypothesis: If cell-cell contact is not required for inclusion body formation, then inclusion bodies will be detected in the cells in which α-synuclein was taken up.

Results: Fig. S2A α-synuclein inclusion bodies formed in the neuronal stem cells Conclusions?

Supplemental Fig. S3A Purpose: To determine if transmission of α-synuclein aggregates is dependent on endocytosis – Dynamin-1 K44A expressed in acceptor cells (blocks endocytic formation) – Donor cells cocultured with acceptor cells Hypothesis: If transmission of α-synuclein aggregates is dependent on endocytosis, then we would detect a reduction in the uptake of α-synuclein in the cells expressing dynamin-1 K44A.

Results: Fig. S3A Transmission of α-synuclein significantly reduced in acceptor cells Conclusions?

Figure 4 Purpose: To determine the role of quality control failure in deposition of α-synuclein Hypothesis: If protein quality control systems are impaired due to being in the presence of MG132 proteosomal inhibitor or Baf A1 lysosomal inhibitor, then we would expect increased accumulation of transmitted α-synuclein in the cells.

myc-α-synuclein Figure 4 SH-SY5Y Baf A1

myc-α-synuclein Figure 4 SH-SY5Y MG132

Figure 4(A-B): Results Conclusions? Increased α-synuclein accumulation by lysosomal failure but no effect on proteosomal inhibition