Targeting Talk: Retrograde Transport

I spoke with someone from your technical service over the phone and got the impression that your product dermorphin-SAP (Cat. #IT-12) is not a retrograde and will only affect the terminals or the cells that express mu opioid receptors in the injection site in the brain. I have three questions:

1) Do you have any written document on this issue?

That the peptide-toxins don’t undergo retrograde transport is an example of negative data, so people haven’t really been publishing too much on that. But two articles deal specifically with it: Lappi and Wiley[1] and Bugarith et al.[2] The latter, in particular, presents solid data on the inability of the peptide ligand toxin NPY-SAP (Cat. #IT-28) to undergo retrograde transport. I don’t think we have a single example of a peptide-ligand toxin that undergoes retrograde transport. In order for a peptide-toxin to kill cells, the cell body must have the receptor and the toxin must be injected within reach of the cell body. We’ve made a mistake in not putting that in the data sheets, and will begin to change that.

2) Will dermorphin-SAP also kill terminals in the injection site or just cell bodies?

Let me cite for you: Tokuno et al., Efferent projections from the striatal patch compartment: anterograde degeneration after selective ablation of neurons expressing mu-opioid receptor in rats.[3] As the title implies, they address the issue of elimination of processes following cell body destruction.

3) If it also kills terminals, will it affect their remote cell bodies?

I’m not sure I understand this question, but that won’t stop me from trying to answer it: The situation is the contrary, because the destruction of processes comes from the action taking place in the cell body. Our experience is that once the cell body is gone, it’s just a matter of time for the process to go away. This makes these toxins a little different than others. In fact, we recommend that you wait two weeks at least to see immunohistological evidence of a toxic effect after injection of a saporin toxin in vivo. That’s how long it takes the removal process to get rid of all the antigens that you might want to use for evidence of cell loss.

Can I inject NPY-SAP to destroy projections through retrograde transport?

Regarding NPY-SAP, a peptide-toxin, see previous response. The antibody-toxins such as 192-IgG-SAP (Cat. #IT-01) or anti-DBH-SAP (Cat. #IT-03) will undergo retrograde transport from terminals to cell bodies. Thus, you can put 192-IgG-SAP into the cortex and it will destroy neurons in the basal forebrain, because the saporin (probably the whole conjugate) is transported from the projection to the cell body. Likewise, anti-DBH-SAP in the spinal cord destroyed hindbrain catecholaminergic neurons by retrograde transport.[4] All the antibody-toxins appear to undergo retrograde transport. (See table on page 6.)

Finally, the lectin-toxins, CTB-SAP (Cat. #IT-14) and IB4-SAP (Cat. #IT-10) undergo retrograde transport, just like the native lectins do. CTB-SAP is well-described in Llewellyn-Smith et al.[5] and several others. Please see our website and the references on the CTB-SAP page. For IB4-SAP, Vulchanova et al.[6] describe use, along with several other articles on our reference page. In addition, detailed discussions are available in the book Molecular Neurosurgery with Targeted Toxins,[7] available from Humana Press.

References

  1. Lappi DA, Wiley RG (2000) Entering through the doors of perception: characterization of a highly selective Substance P receptor-targeted toxin. Neuropeptides 34:323-328.
  2. Bugarith K, Dinh TT, Li AJ, Speth RC, Ritter S. (2005) Endocrinology 146(3), 1179-1191.
  3. Tokuno H, Chiken S, Kametani K, Moriizumi T, Mounir S, Parent A. (2002) Efferent projections from the striatal patch compartment: anterograde degeneration after selective ablation of neurons expressing mu-opioid receptor in rats. Neurosci Lett 332:5-8.
  4. Ritter S, Bugarith K, Dinh TT. (2001) Immunotoxic destruction of distinct catecholamine subgroups produces selective impairment of glucoregulatory responses and neuronal activation. J Comp Neurol 432(2), 197-216.
  5. Llewellyn-Smith IJ, Martin CL, Arnolda LF, Minson JB. (1999) Retrogradely transported CTB-saporin kills sympathetic preganglionic neurons. NeuroReport 10, 307- 312.
  6. Vulchanova L, Olson TH, Stone LS, Riedl MS, Elde R, Honda CN. (2001) Cytotoxic targeting of isolectin IB4-binding sensory neurons. Neurosci 108(1):143-155.
  7. Molecular Neurosurgery With Targeted Toxins. Wiley RG, Lappi DA, eds. (2005) Humana Press, Totowa NJ.

as seen in Targeting Trends Newsletter, Jul-Aug-Sep 2006

I have a question about the issues raised in the last edition of Targeting Trends (Volume 7, Issue 2, see above). There is a comment “In fact, we recommend that you wait two weeks at least to see immunohistological evidence of a toxic effect after injection of a saporin toxin in vivo.” Are there data to support this recommendation? As a researcher who utilizes your toxin products I often get asked about the time course of toxin action. It’s difficult to answer because the literature is currently limited with regard to in vivo toxin application. Any citations, advice, or comments would be greatly appreciated.

Actually there’s quite a bit of in vivo use. Or is it just that I think the glass is half full? If you search PubMed for the use of the immunotoxin 192-IgG-SAP using the terms ‘192’ and ‘saporin,’ you’ll get 223 hits, and all of these describe in vivo use.

As far as the two-week idea, you’re right, it’s a bit more challenging to pin that down in the literature. In our book, Suicide Transport and Immunolesioning [1], Ron Wiley discusses at several points the microglial infiltration that occurs and subsides by 14 days. That is what cleans out the antigens that you probably would use to demonstrate cell death — that is, they aren’t there any more. You might want to see if it’s in your library; it’s a good basic source of info. To get a full list of articles, select the References button on the home page of the ATS website and click on some of the toxins.

As far as the process, Waite et al. [2] show the appearance of behavioral effects associated with neuronal loss at day four and plateauing at day 7. This coincides with the time course seen in vitro [3]. At this point, microglia will infiltrate; this is nicely described in Seeger et al. [4]. However, they stop at 7 days, which is probably the peak day for infiltration. Once there is complete removal of the detritus, microglia down-regulate and at 14 days, you don’t see them, or the antigens that belonged to the cells that were eliminated. So that’s the idea behind waiting.

Do you have a product which can be used to produce retrograde lesions WITHOUT killing cells at the site of injection? What I’d like to do is to kill neurons that project to an efferent nucleus without damaging neurons in the efferent nucleus itself.

Making a selective retrograde neural lesion based only on the criterion that the cells to be lesioned are afferent to a particular nucleus or population of neurons, is a formidable challenge at present. Conceptually, this would seem to require a targeted toxin that was taken up only by afferent terminals and not by dendrites, cell bodies and/or axonal membranes of the neurons that are to be de-afferented.

There are some instances in which you can avoid local killing, but only in the case in which there are no receptors in that area, except from projections. So for instance, cholinergic neurons will project to the cortex. You can inject 192-IgG-SAP (Cat. #IT-01) there; it will be taken up and eliminate basal forebrain neurons with little harm to other cortical neurons. Or you can inject anti-DBH-SAP (Cat. #IT-03) into the spinal cord; it will eliminate brainstem neurons that project to there. Currently, this task is best suited to immunotoxins since there is little data on using neuropeptide toxin conjugates to produce retrograde lesions. If armed to kill, a growth factor such as NGF might also work, if toxin conjugation did not damage binding and intracellular trafficking of the NGF. But these are special cases (which you can find in our reference lists for these products).

Targeting presynaptic antigens that are common to all types of axon terminals would seem a dubious undertaking since success with an immunotoxin requires the target molecule be displayed on the external surface of the terminal and not be present at all on cell bodies or dendrites. I do not know of a suitable target molecule for this purpose.

 

See also: Targeted Toxins Catalog