From what I read, A-beta plaques inhibit microtubule based transport of mitochondria when tau protein is present in the cell. How would I be able to do a test to see if one isoform of tau is more effective at conferring this A-beta sensitivity than another isofrom?


You can do a microscopy based assay to quantify the transport rate (both retrograde and anterograde). Mitochondria can be labeled with fluorescent proteins such as mito-dsRed, and its movement along the axon can be tracked by live cell imaging. You can check this paper; they have done this experiment. (Others have also done it but I remember this paper because someone told me about it recently).

How do you plan to see the effect of different isoforms? It may not be that easy. You will have to replace the common isoform with the others. It is not very easy to control splicing. So you may have to make lines that express only one variant (exon deletion). Though this study reports that alternative splicing can be controlled by using antisense oligonucleotides, the technology is not standardized yet; you can try it nonetheless, because making knockouts is difficult (you would have to make KO mice and then culture neurons from it).

  • $\begingroup$ Reference The transport is anterograde. I just need to think of an assay that will test which naturally occurring isoforms of tau will confer the greatest sensitivity to A-beta inhibition of MT based anterograde transport. Are you saying I would have to generate different lines, each expressing only one isoforms, and compare their vesicles transport rates along the MTs? $\endgroup$ – user6209 May 2 '14 at 4:49
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    $\begingroup$ does this answer your question ? The test that you intend to do is not that straightforward $\endgroup$ – WYSIWYG May 2 '14 at 4:52
  • $\begingroup$ Could I just take a tau KO line and introduce different tau isoforms to different neurons and then measure vesicles transport rate and compare them? $\endgroup$ – user6209 May 2 '14 at 4:57
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    $\begingroup$ You can do that too. But I am skeptical if tau KO mice will survive. Another trick is to intoduce loxP sites near exons/the gene and delete it by induction. You may also try working with neuroblastoma cell lines (e.g. Neuro2a) or PC12 cell line instead, for the ease of growing and manipulation. They can be differentiated to neuronal phenotype by retinoic acid/NGF treatment. $\endgroup$ – WYSIWYG May 2 '14 at 5:03
  • $\begingroup$ Ah I hadn't even thought of introducing loxP sites. That's a good idea. Thank you for all the help. $\endgroup$ – user6209 May 2 '14 at 5:15

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