1. 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.

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

3. 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

4. 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? *, p<0.05 **, p< 0.01

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

6. Figure 3(C) ~ ½ of the acceptor cells displayed ubiquitin immunoreactivity Conclusions? Figure 3(D) Cell-to-cell transmission occurs without cellular membrane leakage

7. 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.

8. Fig. S3A: Results Transmission of α-synuclein significantly reduced in acceptor cells **, p< 0.01 Conclusions?

9. Figure 4 Conclusions? Increased α-synuclein accumulation by lysosomal failure but no effect on proteosomal inhibition

10. Figure 5 Purpose: To examine the toxicity of endocytosed neuron-derived extracellular α-synuclein to the neurons Hypothesis: If secreted/endocytosed α-synuclein is toxic to the cells, then we would expect to detect neuronal degeneration and caspase 3 activation.

11. Figure 5 (A & B): Results Neurons showed nuclear fragmentation and increased immunoreactivity of activated caspase 3 over 3 days *, p<0.05 **, p< 0.01

12. Figure 5 (C): Results MCNSCs showed activated caspase 3 immunoreactivity within 4 weeks *, p<0.05 Conclusions?

13. Overall Conclusions 1. There is evidence for direct cell-to-cell transmission of α- synuclein. 2. Nerve cells that overexpress tagged α-synuclein can transmit the protein to neural stem cells both in vivo and in vitro. 3. Inclusion body formation occurs with prolonged transmission of α-synuclein. 4. α-synuclein accumulation is achieved when protein quality control systems of the acceptor cells are inhibited. 5. Acceptor neuronal stem cells exhibit cell death and activated caspase 3 due to α-synuclein propagation.

14. So, why do we care? Is Parkinson’s Disease really a problem of protein aggregation? PD is characterized by neuron death and the surviving neurons show aggregations of α-synuclein If α-synuclein aggregates into an insoluble Lewy Body, PD develops

15. So, why do we care? Deposition of Lewy-like inclusions from host to grafted neurons in human PD patients is age-dependent Understanding how to control α-synuclein aggregation in & around neurons could lead to revolutionary treatment of Parkinson’s Disease How can we treat PD (based on Desplats et al.’s paper?)