targeted-toxins

Q: I have a question about Retrograde Transport. There is a comment in the FAQ “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.

A: 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. You can also view more References on our website.

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.

Q: 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.

A: 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: Targeted Toxins

References

1. Wiley RG et al. Suicide Transport and Immunolesioning. , 1994. R.G. Landes, Houston
2. Waite JJ et al. Time course of cholinergic and monoaminergic changes in rat brain after immunolesioning with 192 IgG-saporin. Neurosci Lett 169:154-158, 1994.
3. Mantyh PW et al. Inhibition of hyperalgesia by ablation of lamina I spinal neurons expressing the substance P receptor. Science 278:275-279, 1997.
4. Seeger G et al. Electron microscopic evidence for microglial phagocytotic activity and cholinergic cell death after administration of the immunotoxin 192IgG-saporin in rat. J Neurosci Res 48:465-476, 1997.

Allen JW (2006) Featured Article: Safety and efficacy of Substance P-SAP. Targeting Trends 7(3)

Related Products: SP-SAP (Cat. #IT-07), Saporin (Cat. #PR-01)

Read the featured article in Targeting Trends.

See Also:

• Allen JW et al. Safety evaluation of Intrathecal Substance P-Saporin, a targeted neurotoxin, in dogs. Toxicol Sci 91(1):286-298, 2006.

Fraley GS (2006) Immunolesions of glucoresponsive projections to the arcuate nucleus alter glucoprivic-induced alterations in food intake, luteinizing hormone secretion, and GALP mRNA, but not sex behavior in adult male rats. Neuroendocrinology 83(2):97-105. doi: 10.1159/000094375

Summary: In this work the author looked at the role hypothalamic glucose may play in reproductive function. 42 ng of anti-DBH-SAP (Cat. #IT-03) was injected dorsal of the arcuate nucleus of rats, which were then given glucoprivic challenges. Feeding and sex behavior were decreased during glucoprivation; sex behavior was also decreased in control animals. The data demonstrate the involvement of A1/C1 efferents to the ventromedial hypothalamus in glucostatic regulation of various processes.

Related Products: Anti-DBH-SAP (Cat. #IT-03)

Garrett JE, Kim I, Wilson RE, Wellman CL (2006) Effect of N-methyl-d-aspartate receptor blockade on plasticity of frontal cortex after cholinergic deafferentation in rat. Neuroscience 140(1):57-66. doi: 10.1016/j.neuroscience.2006.01.029

Summary: Acetylcholine from the nucleus basalis magnocellularis (NBM) plays roles in neocortical function and plasticity. Here the authors examined whether N-methyl-D-aspartate receptors mediate the increase in the GluR1 subunit of the a-amino-3-hydroxy-5-methylisoxazole-4-proprionate receptor in the frontal cortex following treatment of the NBM with 0.15 µg of 192-IgG-SAP (Cat. #IT-01). The data indicates that upregulation of GluR1 and spine density after cholinergic deafferentation is regulated by N-methyl-D-aspartate receptors.

Related Products: 192-IgG-SAP (Cat. #IT-01)

Kanai T, Uraushihara K, Totsuka T, Nemoto Y, Fujii R, Kawamura T, Makita S, Sawada D, Yagita H, Okumura K, Watanabe M (2006) Ameliorating effect of saporin-conjugated anti-CD11b monoclonal antibody in a murine T-cell-mediated chronic colitis. J Gastroenterol Hepatol 21(7):1136-1142. doi: 10.1111/j.1440-1746.2006.04391.x

Summary: Crohn’s disease is characterized by massive infiltration of macrophages and CD4(+) T-cells in the colon and small intestine. Using SCID mice, the authors evaluated the effects of Mac-1-SAP (Cat. #IT-06) on the development of chronic colitis. After transfer of T cells to the mice, 12.5 µg of Mac-1-SAP was injected into the intraperitoneal space. The reduction in CD4(+) T-cell infiltration of the colon, and suppression of IFNg and TNFa production indicate that macrophages play a significant role in the pathogenesis of Crohn’s disease.

Related Products: Mac-1-SAP mouse/human (Cat. #IT-06)

Traissard N, Herbeaux K, Cosquer B, Jeltsch H, Ferry B, Galani R, Pernon A, Majchrzak M, Cassel JC (2007) Combined damage to entorhinal cortex and cholinergic basal forebrain neurons, two early neurodegenerative features accompanying Alzheimer’s Disease: Effects on locomotor activity and memory functions in rats. Neuropsychopharmacology 32(4):851-871. doi: 10.1038/sj.npp.1301116

Summary: Two characteristics of Alzheimer’s disease (AD) are cholinergic dysfunction in the basal forebrain, and neuronal damage in the entorhinal cortex. Using 5 µg intracerebroventricular (icv) injections of 192-IgG-SAP (Cat. #IT-01), and 2.3 µg icv injections of OX7-SAP (Cat. #IT-02), locomotor activity, working, and reference memory of rats were examined. Although 192-IgG-SAP lesions caused limited deficits, rats receiving both lesions exhibited several behaviors associated with AD. The authors suggest that combining these lesions may be a more accurate model for AD than 192-IgG-SAP alone.

Related Products: 192-IgG-SAP (Cat. #IT-01), OX7-SAP (Cat. #IT-02)

Vera-Portocarrero LP, Yie JX, Kowal J, Ossipov MH, King T, Porreca F (2006) Descending facilitation from the rostral ventromedial medulla maintains visceral pain in rats with experimental pancreatitis. Gastroenterology 130(7):2155-2164. doi: 10.1053/j.gastro.2006.03.025

Summary: Here the authors investigated the role of ascending or descending pathways in the mediation of pain caused by pancreatitis. Rats received 1.5 pmol injections of dermorphin-SAP (Cat. #IT-12) into each side of the rostral ventromedial medulla. Abdominal hypersensitivity was tested using von Frey filaments. Although the ablation of mu-opioid receptor-expressing neurons by dermorphin-SAP did not prevent the initial expression of pancreatitis pain, maintenance of this pain was absent. The data link maintenance of pancreatitis pain to descending pathways.

Related Products: Dermorphin-SAP / MOR-SAP (Cat. #IT-12)

Basbaum AI, Julius D (2006) Toward better pain control. Sci Am 294(6):60-67. doi: 10.1038/scientificamerican0606-60

Summary: The authors discuss some of the advances in understanding and treating different types of pain, and specifically outline circuits, receptors, and ligands involved in pain pathways. Several treatments are described, one of which is the use of SP-SAP (Cat. #IT-07) to disrupt the chronic pain pathway in the spinal cord.

Related Products: SP-SAP (Cat. #IT-07)

Grimaldi P, Rossi F (2006) Lack of neurogenesis in the adult rat cerebellum after Purkinje cell degeneration and growth factor infusion. Eur J Neurosci 23(10):2657-2668. doi: 10.1111/j.1460-9568.2006.04803.x

Summary: Although neurogenesis occurs in very specific areas of the mammalian brain, neural progenitors can be found in many central nervous system sites. Here the authors examined neurogenesis in the rat cerebellum. 2.2 µg of 192-IgG-SAP (Cat. #IT-01) was injected into each lateral ventricle, and some animals were given exogenous EGF, bFGF, or FGF8. In this model, the local environment was not sufficient to direct neuronal differentiation, even with the addition of growth factors.

Related Products: 192-IgG-SAP (Cat. #IT-01)

Lu J, Sherman D, Devor M, Saper CB (2006) A putative flip-flop switch for control of REM sleep. Nature 441(1):589-594. doi: 10.1038/nature04767

Summary: The authors propose a REM sleep regulatory system that involves GABAergic and glutaminergic neurons in the mesopontine tegmentum. Among other work, 2 µl of 0.1% orexin-SAP (Cat. #IT-20) was injected into the medial medullary reticular formation of rats. This work suggests the sharp transitions into and out of REM sleep are controlled by reciprocal interactions between GABAergic REM-off and REM-on neuronal populations.

Related Products: Orexin-B-SAP (Cat. #IT-20)