Thesis Defense Q's

  1. What does an upstream regulator mean?
    Usually a transcription factor. APP has intracellular domains that gets cleaved, and then act as co-transcription activators. For example, Fe65
  2. Explain canonical pathways
    A natural established pathway inside a cell
  3. How do you purify the ribosome?
    Using a GFP antibody, you can pull it down. We use a chemical to freeze the ribosomes in place
  4. APP Western
    Most of APP protein remains uncleaved on the membrane. We see a strong band migrating between 75-100kDa using both 22C11 and Y188. This is consistent with what Eddie K observed. We thought using the Y188 antibody will help identify which secretase is active in learning, that may show us which pathway is involved
  5. Which secretase is cleaving APP (Y188 antibody)?
    The AICD fragments for APPalpha is 9kDa, and APPbeta is 12kDa. However, the Y188 fragment we saw is around 20kDa. This doesn't correspond to any of the classical C terminal fragments. However, there is a new more recent secretase that cleaves APP called meprin beta. This secretase competes with beta secretase binding, thereby inhibiting beta secretase cleavage of APP. Right now, we are not sure, but we're investigating this.
  6. What is APP doing?
    APP has been shown to play a role in synapse formation, synapse strengthening, and synapse maturation. It also plays a role in long-term memory which suggests it is involved in memory consolidation.
  7. What evidence supports APP's role in memory?
    We see APP protein identified as a central hub in the learning transcriptome. We also saw APP protein upregulated in response to learning, and proteolytically cleaved in response to learning. APP protein was also localized to the corticospinal neurons that project to C8, and this was upregulated in learning.
  8. How are the secretases changed during learning?
    We haven't looked at protein levels using western blot analysis. But from RNA sequencing, the gamma and beta secretase mRNA are up only at 14 days, but the alpha secretase mRNA are up at 4, 7 and 14 days of learning.
  9. Why does histology of APP show significant increase of APP but western blot doesn't?
    Because with Western blot, our signal is diluted because our protein extraction included layers 1-6 of the cortex. With histology, we were able to focus on layer 5 projecting to the C8 spinal cord only.
  10. How was the tissue prepared?
    First you perfuse the animal with paraformaldehyde, then you cryoprotect in sucrose. Brain tissue is then isolated and you cut on a microtome in thin small slices to get tissue sections. We then stain those tissue for APP antibody.
  11. How did you quantify the histology images?
    • You outline neuron cell bodies, then you measure the integrated density of APP inside the neuron using image J. You also measure the background signal which is noise and subtract the noise from the APP signal and take into account the area selected. 
    • APP histology = APP signal - (area of cell - background)
  12. Why is HOLO APP not upregulated that much, but Y188 is?
    Because we don't fully understand the proteolytic processing of APP in response to learning. There might be an alternative secretase pathway that's activated in response to learning. But we don't know. Also, western blot is the total motor cortex with all layers.
  13. What is western blot analysis?
    It's a way to separate proteins by molecular weight. Small proteins will be at the bottom, and heavy proteins will be at the top. The proteins are then transferred to a membrane, that will fix the proteins in place. You can then use a specific antibody like APP to see protein size.
  14. Why do we use GAPDH?
    It's a house keeping gene.
  15. Where does Y188 bind?
    It binds at the C terminus. The fragments we see most likely correspond to the AICD fragments. But we need to do more experiments to verify this. (mass spec - sequencing for proteins).
  16. Explain to me densitometry for western blots?
    You measure the area under the curve for each band. And then you divide the APP curve by the GAPDH curve.
  17. What is the protein size of secreted APP alpha and secreted APP beta?
    • APP full length = 100kDa
    • sAPPa = 83kDa
    • sAPPb = 81kDa

    You cannot distinguish sAPPa and sAPPb. Further analysis needs to be done to determine if this increase in protein is full length APP or one of the secreted fragments. Like mass spec. 22C11 is used to detect holo APP.
  18. What about total APP mRNA? What did that show?
    I don't know the total levels. But we are isolating the active transcriptome, which is translated into protein.
  19. What is a hub gene?
    A hub is a gene with high correlation and connectivity in a network. So it would be centrally connected to all the proteins in the network.
  20. Presinilin 1?
    Part of the gamma secretase complex
  21. What is a principal component analysis?
    It's a dimensional way of looking how principal components drive data separation
  22. Why is it called the corticospinal tract?
    Because the neuron cell bodies are in the cortex, and the axons extend into the spinal cord.
  23. What about mice age?
    This skill was used in mice aged 4 weeks and 12 weeks of age. Previous studies have shown that young and aged mice did not significantly differ in their behavioral performance in forelimb skilled grasp. However, experience driven topographical reorganization of the motor cortex varies with age and time, as younger mice display more forms of plasticity in comparison to aged rats.
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Thesis Defense Q's
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