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Targeted Toxins

Ronald G. Wiley¹, Douglas A. Lappi²

¹Veterans Affairs Medical Center, Nashville, Tennessee
²Advanced Targeting Systems, San Diego, California

Publication Name:

Current Protocols in Neuroscience

Unit Number:

Unit 1.7

DOI:

10.1002/0471142301.ns0107s14

Online Posting Date:

May, 2001

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Abstract

Molecular neurosurgery can be used to make selective neural lesions by targeting cytotoxins to specific populations of neurons based on their common expression of a particular surface molecule. The targeted toxins employed in this unit consist of a targeting moiety (vector) and an effector moiety (cytotoxin). In all cases discussed in this unit, the cytotoxic moiety is an enzyme that catalytically inactivates the large ribosomal subunit, irreversibly inhibiting protein synthesis and resulting in cell death. These toxins appear to kill in an all-or-none fashion, indicating that one molecule of free cytotoxin in the cytoplasm of a cell is sufficient to kill the cell. Three general molecular neurosurgery protocols are presented in this unit. The first describes suicide transport, which refers to the use of targeted toxins to make anatomically restricted lesions based on retrograde axonal transport of the toxin. The second involves immunolesioning and uses anti-neuronal immunotoxins to make type-selective and anatomically restricted lesions. The final protocol uses neuropeptide-toxin conjugates to selectively destroy neurons expressing the receptor for the specific neuropeptide.

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Unit Introduction
Strategic Planning
Basic Protocol 1: Suicide Transport
Basic Protocol 2: Immunolesioning
Basic Protocol 3: Neuropeptide-Toxin Conjugates
Reagents and Solutions
Commentary
Bibliography
Figures
Tables

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Materials

Basic Protocol 1: Suicide Transport

Materials

Animal to be tested
Targeted toxin: immunotoxins, neuropeptide-saporin conjugates (Advanced Targeting Systems), or toxic lectins (e.g., ricin or abrin, Sigma)
Saline/BSA/fast green diluent (see recipe)
Hamilton syringes

Basic Protocol 2: Immunolesioning

Materials

Animal to be tested
Immunotoxin: e.g., 192-saporin, anti-DBH-saporin, or anti-DAT-saporin (Advanced Targeting Systems)
Saline/BSA/fast green diluent (see recipe)
Hamilton syringes

Basic Protocol 3: Neuropeptide-Toxin Conjugates

Materials

Animal to be tested
Neuropeptide-toxin conjugate: substance P–saporin or dermorphin-saporin (Advanced Targeting Systems)
Saline/BSA/fast green diluent (see recipe)
Hamilton syringe

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Figures

Figure 1.7.1

Schematic depiction of the molecular neurosurgery strategy. Neurons A to D are interconnected with the target cell, which is selectively destroyed (indicated by Xs inside cell) by a targeted toxin that recognizes a unique surface molecule on the target cell. The remaining four neurons lose inputs and efferent connections, but otherwise are unaffected. The toxin molecules are depicted as consisting of two parts; the Y shape is the targeting vector, which provides binding specificity, and the lightning bolt is the effector cytotoxin (usually the ribosome-inactivating protein, saporin).

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Figure 1.7.2

Low-power photomicrographs of rat cerebellar vermis stained for calbindin using indirect immunoperoxidase technique to demonstrate Purkinje neurons. (A,C) Normal, vehicle-injected rats. (B) A rat injected intracerebroventricularly (i.c.v.) 2 weeks previously with 4 µg of 192-saporin, showing spotty loss of Purkinje cells. (D) A rat injected 2 weeks previously with 1 µg of OX7-saporin i.c.v., showing a similar degree and distribution of Purkinje cell loss. In behavioral experiments, the OX7-saporin rats were compared to 192-saporin rats to control for the loss of Purkinje cells that accompanied destruction of the cholinergic basal forebrain by 192-saporin. Photomicrographs courtesy of C.C. Wrenn.

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Figure 1.7.3

Photomicrographs of lumbar dorsal horn of rat stained for the demonstration of dopamine -hydroxylase using indirect immunoperoxidase technique. (A) The typical normal appearance of beaded varicosities throughout the dorsal horn from a vehicle-injected rat. (B) The dorsal horn from an animal injected 3 months earlier with 250 ng anti-DBH-saporin into the lumbar subarachnoid space. Note the absence of noradrenergic fibers (a solitary fiber is visible at upper edge of field).

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Literature Cited

Literature Cited
	Cusick, C.G., Wall, J.T., Whiting, J.H. Jr., and Wiley, R.G. 1990. Temporal progression of cortical reorganization following nerve injury. Brain Res. 537:355-358.
	de la Cruz, R.R., Baker, R., and Delgado-Garcia, J.M. 1991. Response of adult cat abducens internuclear interneurons to selective removal of their target motoneurons. Exp. Brain Res. 84:167-172.
	de la Cruz, R.R., Pastor, A.M., and Delgado-Garcia, J.M. 1994a. Neurotoxic lesion of oculomotor neurons: Evidence for rearrangement of axon terminals of surviving afferent neurons. Neurotoxicology 15:633-636.
	de la Cruz, R.R., Pastor, A.M., and Delgado-Garcia, J.M. 1994b. Effects of target depletion on adult mammalian central neurons: Functional correlates. Neuroscience 58:81-97.
	de la Cruz, R.R., Pastor, A.M., and Delgado-Garcia, J.M. 1994c. Effects of target depletion on adult mammalian central neurons: Morphological correlates. Neuroscience 58:59-79.
	Harrison, M.B., Roberts, R.C., and Wiley, R.G. 1993. A selective lesion of striatonigral neurons decreases presynaptic binding of [3H]hemicholinium-3 to striatal interneurons. Brain Res. 630:169-177.
	Helke, C.J., Charlton, C.G., and Wiley, R.G. 1985. Suicide transport of ricin demonstrates the presence of substance P receptors on medullary somatic and autonomic motor neurons. Brain Res. 328:190-195.
	Helke, C.J., Charlton, C.G., and Wiley, R.G. 1986. Studies on the cellular localization of spinal cord substance P receptors. Neuroscience 19:523-533.
	Lee, M.G., Chrobak, J.J., Sik, A., Wiley, R.G., and Buzsaki, G. 1994. Hippocampal theta activity following selective lesion of the septal cholinergic system. Neuroscience 62:1033-1047.
	Mantyh, P.W., Rogers, S.D., Honore, P., Allen, B.J., Ghilardi, J.R., Li, J., Daughters, R.S., Lappi, D.A., Wiley, R.G., and Simone, D.A. 1997. Inhibition of hyperalgesia by ablation of lamina I neurons expressing substance P receptor. Science 278:275-279.
	Roberts, R.C., Harrison, M.B., Francis, S.M., and Wiley, R.G. 1993. Differential effects of suicide transport lesions of the striatonigral or striatopallidal pathways on subsets of striatal neurons. Exp. Neurol. 124:242-252.
	Stirpe, F., Barbieri, L., Battelli, M.G., Soria, M., and Lappi, D.A. 1992. Ribosome-inactivating proteins from plants: Present status and future prospects. Bio/Technology 10:405-412.
	Wall, J.T., Cusick, C.G., Migani-Wall, S.A., and Wiley, R.G. 1988. Cortical organization after treatment of a peripheral nerve with ricin: An evaluation of the relationship between sensory neuron death and cortical adjustments after nerve injury. J. Comp. Neurol. 277:578-592.
	Wiley, R.G. 1992. Neural lesioning with ribosome-inactivating proteins: Suicide transport and immunolesioning. Trends Neurosci. 15:285-290.
	Wiley, R.G. 1997. Findings about the cholinergic basal forebrain using immunotoxin to the nerve growth factor receptor. Ann. N.Y. Acad. Sci. 835:20-29.
	Wiley, R.G. and Lappi, D.A. 1994. Suicide Transport and Immunolesioning. pp. 1-120. R.G. Landes, Austin, Tex.
	Wiley, R.G. and Lappi, D.A. 1997. Destruction of neurokinin-1 receptor expressing cells in vitro and in vivo using substance P-saporin in rats. Neurosci. Lett. 230:97-100.
	Wiley, R.G. and Stirpe, F. 1988. Modeccin and volkensin but not abrin are effective suicide transport agents in rat CNS. Brain Res. 438:145-154.
	Wiley, R.G., Blessing, W.W., and Reis, D.J. 1982. Suicide transport: Destruction of neurons by retrograde transport of ricin, abrin, and modeccin. Science 216:889-890.
	Wiley, R.G., Oeltmann, T.N., and Lappi, D.A. 1991. Immunolesioning: Selective destruction of neurons using immunotoxin to rat NGF receptor. Brain Res. 562:149-153.
Key References
	Mantyh et al., 1997. See above.
Example of use of substance P–saporin to selectively lesion the dorsal horn of the spinal cord and initial observations on changes in pain behavior.
	Salo, P.T., Theriault, E., and Wiley, R.G. 1997. Selective ablation of rat knee joint innervation with injected immunotoxin: A potential new model for the study of neuropathic arthritis. J.Orthopaed. Res. 15:622-628.
Good example of using OX7-saporin to denervate a target tissue by suicide transport destruction of primary afferent neurons innervating the joint capsule of the knee.
	Wiley, R.G. and Lappi, D.A. 1999. Targeting NK-1R expressing neurons with SSP-saporin. Neurosci. Lett. 277:1-4.
Examples of modifying vector to enhance selective targeting and assessment of collateral damage to nontargeted neurons.
	Wrenn, C.C. and Wiley, R.G. 1998. The behavioral functions of the cholinergic basal forebrain: Lessons from 192-saporin. Int. J. Dev. Neurosci. 16:595-602.
Comprehensive review of behavioral studies using 192 IgG–saporin to produce selective destruction of the cholinergic neurons of the basal forebrain.
	Wrenn, C.C., Picklo, M.J., Lappi, D.A., Robertson, D.R., and Wiley, R.G. 1996. Intraventricular injection of anti-DH-saporin: Anatomical findings. Brain Res. 740:175-186.
Initial description of use of anti-DBH-saporin to lesion CNS noradrenergic neurons. Good example of anatomical verification of the lesion.
Internet Resources
	http://www.ATSbio.com
Advanced Targeting Systems' online catalog of available immunotoxins and neuropeptide toxins. Offers custom synthesis of new agents.