A PNAS Direct Submission (2009). Test if α-synuclein pathology involves direct neuron-to-...

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Transcript of A PNAS Direct Submission (2009). Test if α-synuclein pathology involves direct neuron-to-...

  • A PNAS Direct Submission (2009)

  • Test if -synuclein pathology involves direct neuron-to-neuron transmission of -synuclein aggregates via endocytosis

    Overall hypothesis:If -synuclein aggregates can be spread by direct neuron-to-neuron transmission, then we would expect accumulation of -synuclein aggregates in the uninfected neurons.

    Desplats Paper

  • Figure 1Figure 1A and 1B Hypothesis:

    If extracellular -synuclein can be taken up by the mouse cortical neuronal stem cells (MCNSCs), then -synuclein accumulation will be detected in the MCNSCs via Western blot and fluorescent microscopy.

  • MCNSCs E15-E18 C57/BL6

  • Western Blottingwww.askabiologist.asu.edu

  • Figure 1(A): ResultsMCNSCs capable of taking up extracellular -synucleinConclusions?

  • 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 -synuclein

    Conclusions?

  • Figure 1(C)Purpose: To determine whether -synuclein released from neuronal cells can be directly transferred to MCNSCs

    Hypothesis: If -synuclein is released by the neuronal cells, then we would expect to see uptake of -synuclein in the MCNSCs via immunofluoresence.

  • Figure 1(C)

  • Immunofluorescence -synuclein

  • Figure 1(C): Results47% of MCNSCs showed patterns of cytoplasmic accumulation of -synuclein MCNSCNeuronal Cells Neuronal Cells + MCNSC Conclusions?

  • Figure 2 Purpose: To analyze the propagation of -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 -synuclein will be detected in MCNSCs grafted into transgenic mice via immunofluoresence.

  • Immunostaining and TSA Adopted from www.abcam.comHRPtyramide red

  • Figure 2

    Injected GFP-labeled MCNSCs into the hippocampus of transgenic mice expressing human -synuclein. Transgenic (expresses human -synuclein via Thy-1 promoter)

  • Figure 2 (A-C): Results2.5% of MCNSCs showed human -synuclein immunoreactivity in transgenic mice after 1 week

  • Figure 2 (D-E): ResultsWhen MCNSCs not injected into -synuclein transgenic mice, no immunoreactivity (D) MCNSCs showed no human -synuclein immunoreactivity in non-transgenic mice (E)

    Controls

  • Figure 2 (F): Results 15% of -synuclein-positive MCNSCs developed LBs in cytoplasm after 4 weeks

  • Figure 2 (G): Results

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

  • Figure 3 Purpose: To further characterize cell-to-cell transmission of -synuclein using an in vitro coculture model

    Figure 3(A) Hypothesis: If myc-tagged -synuclein from donor cells can be released and transmitted to SH-SY5Y acceptor cells, then -synuclein will be detected in the donor cells via immunofluorescence.

  • Figure 3

  • Figure 3(A): Results After 24 hrs, myc-tagged -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 -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 -synuclein

    Conclusions?

  • Figure 3(C): Results

    ~ of the acceptor cells displayed ubiquitin immunoreactivity

    Conclusions?

  • Figure 3(D)

    Purpose: To examine the involvement of donor cell membrane leakage in transmission of -synuclein

    Lactate dehydrogenase release (LDH) assaySH-SY5Y cells overexpressing -galatosidase, -synuclein, and -synuclein-myc

  • Figure 3(D): Results

    Cell-to-cell transmission occurs without cellular membrane leakageConclusions?

  • Supplemental Fig. S2APurpose: To determine if cell-cell contact is required for inclusion body formationSH-SY5Y cells incubated in medium from SH-SY5Y cells expressing myc-tagged -synuclein.

    Hypothesis: If cell-cell contact is not required for inclusion body formation, then inclusion bodies will be detected in the cells in which -synuclein was taken up.

  • Results: Fig. S2A-synuclein inclusion bodies formed in the neuronal stem cellsConclusions?

  • Supplemental Fig. S3APurpose: To determine if transmission of -synuclein aggregates is dependent on endocytosisDynamin-1 K44A expressed in acceptor cells (blocks endocytic formation)Donor cells cocultured with acceptor cells

    Hypothesis: If transmission of -synuclein aggregates is dependent on endocytosis, then we would detect a reduction in the uptake of -synuclein in the cells expressing dynamin-1 K44A.

  • Results: Fig. S3ATransmission of -synuclein significantly reduced in acceptor cellsConclusions?

  • Figure 4Purpose: To determine the role of quality control failure in deposition of -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 -synuclein in the cells.

  • Figure 4Baf A1

  • Figure 4MG132

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

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