Transcript Figure 3(A) Hypothesis

Figure 3
• Purpose:
To further characterize cell-to-cell transmission of αsynuclein 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 αsynuclein will be detected in the donor cells via
immunofluorescence.
Figure 3
SH-SY5Y
myc
myc myc
myc
myc
SH-SY5Y + α-synuclein
Donor Cells
Q
Q
Q
Q Q
SH-SY5Y
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
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
*, p<0.05
**, p< 0.01
Conclusions?
Figure 3(B): Results
• Inclusion body formation occurs with prolonged
transmission of α-synuclein
Conclusions?
Figure 3(C)
• ~ ½ of the acceptor cells displayed ubiquitin
immunoreactivity
Figure 3(D)
• Cell-to-cell transmission occurs without cellular
membrane leakage
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.
Fig. S3A: Results
• Transmission of α-synuclein significantly reduced in
acceptor cells
**, p< 0.01
Conclusions?
Figure 4
• Increased α-synuclein accumulation by lysosomal
failure but no effect on proteosomal inhibition
Conclusions?
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.
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
Figure 5 (C): Results
• MCNSCs showed activated caspase 3 immunoreactivity
within 4 weeks
*, p<0.05
Conclusions?
Overall Conclusions
1. There is evidence for direct cell-to-cell transmission of αsynuclein.
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.
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
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?)