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
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
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