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

MCNSCs

Polyornithine/laminin-coated plate

E15-E18 C57/BL6

Western Blotting

www.askabiologist.asu.edu

Figure 1(A): Results• MCNSCs capable of taking up extracellular α-

synuclein

?

Conclusions?

Confocal Microscopy

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

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 www.abcam.com

HRP

tyramide red

α-syn

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

MCNSCsα-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

mycmyc

mycmycmyc

SH-SY5Y

Q

QQ

QQ

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 myc-α-synuclein

Figure 4

SH-SY5YSH-SY5Y

Baf A1

myc-α-synuclein myc-α-synuclein

Figure 4

SH-SY5YSH-SY5Y

MG132

Figure 4(A-B): Results

Conclusions?

• Increased α-synuclein accumulation by lysosomal failure but no effect on proteosomal inhibition