A PNAS Direct Submission (2009)

1. A PNAS Direct Submission (2009)

2. Desplats Paper • 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.

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

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

5. Western Blotting www.askabiologist.asu.edu

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

7. Confocal Microscopy http://www.lucid-tech.com/client_images/catalog19974/pages/images/vivascopy/graphic_scopy.gif

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

9. 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.

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

11. Immunofluorescence α-synuclein

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

13. 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, thenα-synuclein will be detected in MCNSCs grafted into transgenic mice via immunofluoresence.

14. Immunostaining and TSA HRP HRP Secondary Antibody α-syn tyramide red α-syn Adopted from www.abcam.com

15. 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)

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

17. 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

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

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

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

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

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

23. 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?

24. 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?

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

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

27. 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

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

29. 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.

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

31. Supplemental Fig. S3A • Purpose: To determine if transmission of α-synucleinaggregates 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-synucleinaggregates is dependent on endocytosis, then we would detect a reduction in the uptake of α-synucleinin the cells expressing dynamin-1 K44A.

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

33. 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.

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

35. Figure 4 SH-SY5Y SH-SY5Y MG132 myc-α-synuclein myc-α-synuclein

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