Targeting Trends Newsletter 01q3

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Newsletter Highlights

  • A Field Trip to Torrey Pines State Reserve (page 2)
  • Targeted Toxin Delivery (page 5)
  • Saporin, a Ribosome-Inactivating Protein (page 7)

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Dermorphin-SAP Kills MOR-Positive Cells

Product information related to cover article: Dermorphin-SAP (Cat. #IT-12)

A Tribute to Thomas J. Walsh, Ph.D.

Going to Work is Like a Walk in the Park

Featured Neuroscience Antibodies: Nerve Growth Factor (p75) Receptor

Recent Scientific References

Targeting Talk: In Vivo Delivery of Targeted Toxins

  • What are the options for delivery of targeted toxins?
  • When injecting directly into tissue, are there any special techniques that should be used?
  • What sort of special care should be given to the animal after administration of the targeted toxin?

Targeting Ticklers (Jokes)

Targeting Teaser Winners from last issue

Targeting Tools: Featured Products

  • Saporin (Cat. #PR-01): a ribosome-inactivating protein

Targeting Technology Tutorial

Targeting Teaser (Jumble)

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Targeting Tools: Saporin – a ribosome-inactivating protein

Saporin is a 30 kDa protein isolated from the seeds of the plant Saponaria officinalis. This pleasant resident of the banks of Southern European streams has long been known for medicinal properties, mainly due to its saponins, detergents that reside especially in the roots of the plant and which give the plant its name. It took the expertise of University of Bologna researcher Fiorenzo Stirpe to pull perhaps the most useful molecule from the plant, saporin.

Stirpe had screened several plants for ribosome-inactivating proteins. These proteins come in two different forms. One form is exemplified by the incredibly potent toxin ricin; it contains a cell-binding and internalization protein and an enzyme that upon entering the cell removes a single base from the ribosomal RNA of the large subunit of the ribosome. This enzyme action is a necessary characteristic of a ribosome- inactivating protein, cleverly termed a “RIP.” Other examples of toxins with cell-binding chains are abrin (Brooke Shields used it for her suicide in the film Blue Lagoon) and volkensin, which has been used in neuroscience research as a suicide transport agent.

[threecol_two][/threecol_two] [threecol_one_last]Figure 1. The white arrow indicates Saponaria officinalis (saporin) growing in Dr. Douglas Lappi’s garden. The yellow arrow points to Gangsta, Dr. Lappi’s cat, who is perfectly safe playing around this plant.  [/threecol_one_last]

Most RIPs found in plants are in the second form, without cell-binding
chains, and these plants are generally harmless (Fig. 1): RIPs from cucumber and asparagus are two examples. Stirpe could easily recognize the plants that had toxins, but had to screen the plants that weren’t known to be toxic. Since Saponaria had a medicinal reputation, he included it in his screening. In 1983, he published on a RIP from Saponaria, SO- 6, that was extremely active in cell-free protein synthesis inhibition, but also, in an observation that later would become very important, an unusual stability to denaturants, proteases and heat (1). This protein would become known as saporin. At that time, the RIP from ricin was widely used in targeted toxins. It had an unfortunate sensitivity to proteolytic attack when removed from its protective cell-binding chain, and this made it unable to function as a targeted toxin in cells that had high levels of proteases (2). This limited its use as a targeted toxin against, for instance, T lymphocytes, and caused considerable setbacks in the targeted toxin field. Stirpe demonstrated remarkable in vivo activities of the first targeted toxin made from saporin and, when targeted to T lymphocytes, it was devastating (3,4). Saporin was cloned by Doug Lappi and colleagues in Marco Soria’s group (5); this led to the use of recombinant saporin which Advanced Targeting Systems uses in the preclinical studies of SP-SAP. Presently, the highly active native saporin is sold by Advanced Targeting Systems in a sterile PBS solution ready for use in vitro and in vivo. Each lot is carefully assayed for full protein synthesis inhibition activity and not released unless it matches the highest levels (Fig. 2).

[threecol_two][/threecol_two] [threecol_one_last]Figure 2. Protein synthesis inhibition by three different lots of saporin. No statistical difference is seen in these extremely active RIPs. [/threecol_one_last]
References

  1. Stirpe F, Gasper-Campani A, Barbieri L, Falasca A, Abbondanza A, Stevens WA(1983) Ribosome-inactivating proteins from the seeds of Saponaria officinalis L. (soapwort) of Agrostemma githago L. (corn cockle) and of Asparagus officinalis (asparagus) and from the latex of Hura crepitans L. (sandbox tree). Biochem J 216:617-625.
  2. Bilge A, Howell-Clark J, Ramakrishnan S, Press OW (1994) Degradation of ricin A chain by endosomal and lysosomal enzymes – the protective role of ricin B chain. Therapeutic Immunol 1:197-204.
  3. Thorpe PE, Brown ANF, Bremner JAG, Foxwell BMJ, Stirpe F (1985) An immunotoxin composed of monoclonal anti-thy 1.1 antibody and a ribosome-inactivating protein from Saponaria officinalis: potent antitumor effects in vitro and in vivo. JNCI 75:151-159.
  4. Siena S, Lappi DA, Bregni M, Formosa A, Villa S, Soria M, Bonadonna G, Gianni AM (1988) Synthesis and characterization of an antihuman T-lymphocyte saporin immunotoxin (OKT1-SAP) with in vivo stability into nonhuman primates. Blood 72:756-765.
  5. Benatti L, Saccardo MB, Dani M, Nitti GP, Sassano M, Lorenzetti R, Lappi DA, Soria M (1989) Nucleotide sequence of cDNA coding for saporin-6, a type-1 ribosome-inactivating protein from Saponaria officinalis. Eur J Biochem 183:465-470.

Targeting Talk: In Vivo Delivery of Targeted Toxins

What are the options for delivery of targeted toxins?

The options for toxin delivery are varied and limited only by investigator ingenuity. Generally, injection has been the route of choice. Some toxins can be given intravenously, such as 192-Saporin (192-IgG-SAP, Cat. # IT-01) or anti-DBH-SAP (Cat. # IT-03), in which case all cells expressing p75 or dopamine beta-hydroxylase and exposed to the systemic circulation are potential targets. Intravenous injections will not deliver toxins to the CNS.

Subarachnoid injections have been used successfully for immunotoxins and peptide toxins such as SP-SAP (Cat. # IT-07).

Direct intraparenchymal injections have been used to restrict toxin application to just a few target cells. However, intraparenchymal injections require careful attention to injection technique and are impractical for large target structures.

When injecting directly into tissue, are there any special techniques that should be used?

Direct injections into brain or spinal cord have been used successfully by some investigators. Specifics of toxin dose, concentration, injection volume and speed of injection have varied considerably. If a high concentration of toxin is deposited locally, lesion specificity is often lost. Presumably, if toxin concentration is too high, cellular uptake by non-specific bulk fluid-phase endocytosis (pinocytosis) can internalize enough saporin to be lethal.

There is currently interest in "convective" delivery techniques developed in the laboratory of Dr. Edward Oldfield at the NIH. The basic principle is to deliver a relatively large concentration slowly over an extended period, often using a rather dilute solution. The parameters for any given species and injection site need to be determined by pilot experiments.

What sort of special care should be given to the animal after administration of the targeted toxin?

The toxins generally bind and internalize within minutes, although some immunotoxins circulate for longer periods if injected intravenously. However, no significant amount of active toxin is excreted. So, animals can be returned to group housing immediately after toxin injection. The only special requirements may derive from the specific target being studied. For example, rats given intraventricular 192-Saporin (192-IgG-SAP, Cat. # IT-01) develop decreased fluid and food intake for several days after injection. Since the adipsia is significant, providing the animals with fresh, juicy vegetables, such as cucumber or potatoes, can help.

Rats injected intraventricularly with anti-DBH-SAP (Cat. # IT-03) will lose considerable body weight and are slow to regain. They, too, may benefit from food supplements, including nuts and other high calorie appetizing treats. Otherwise, common sense care of any neurologic deficits is indicated depending on the target and toxin being used.

See also: Targeted Toxins Catalog

Targeting Topics 01q3

Selective destruction of medial septal cholinergic neurons attenuates pyramidal cell suppression, but not excitation in dorsal hippocampus field CA1 induced by subcutaneous injection of formalin.

Zheng F, Khanna S.

Neuroscience 103(4):985-998, 2001. PMID: 11301206

Previously, the authors have shown that an injection of formalin in the hindpaw of rats will excite a select population of CA1 pyramidal cells within a larger suppressed population. This response is accompanied by increased theta activation. The authors selectively eliminated medial septal cholinergic neurons using 192-Saporin (0.4 μl; Cat. # IT-01) to investigate the role of these neurons in response to a persistent noxious stimulus such as a formalin injection. The data indicate a CA1 network modulated by cholinergic neurons in the medial septal region may influence pyramidal cell theta and pyramidal cell suppression.

Sequential upregulation of cell adhesion molecules in degenerating rat basal forebrain cholinergic neurons and in phagocytotic microglial cells.

Hartlage-Rubsamen M, Schliebs R.

Brain Res 897(1-2):20-26, 2001. PMID: 11282354

Neurodegeneration, found in brain disorders such as Alzheimer’s, Parkinson’s, and Huntington’s diseases, is marked by a significant microglial response. This microglial activation is characterized by increased migratory activity and potential cytotoxic action on injured neurons. The interaction of microglial cells with degenerating axons and neural somata is known to be mediated by expression of cell adhesion molecules. The authors use a single intracerebroventricular injection of 192- Saporin (4 μg; Cat. # IT-01) to initiate neurodegeneration of choline acetyltransferase-immunoreactive neurons and follow the expression of two cell adhesion molecules, ICAM-1 and LFA-1, using immunohisto- chemistry. The results indicate that these adhesion molecules may function as intercellular recognition signals through which degenerating cholinergic neurons actively participate in their own targeting and removal by microglia.

Immunotoxic destruction of distinct catecholamine subgroups produces selective impairment of glucoregulatory responses and neuronal activation.

Ritter S, Bugarith K, Dinh TT.

J Comp Neurol 432(2):197-216, 2001. PMID: 11241386

Control of regulatory responses to low glucose levels in the brain have been linked to catecholaminergic neurons. Studies of these neurons have been hindered by the lack of a selective and precise lesioning agent. Ritter et al. use anti-DBH-SAP (Cat. # IT-03) to create very precise lesions of catecholamine neurons in the paraventricular nucleus of the hypothalamus and spinal cord. Injection of anti-DBH-SAP into the spinal cord eliminates cells with caudal projections while injection into the paraventricular nucleus of the hypothalamus eliminated cells with rostral projections. This ability to selectively eliminate very specific subpopulations of cells is a valuable characteristic in dissecting neuronal function.

P75-expressing elements are necessary for anti-allodynic effects of spinal clonidine and neostigmine.

Paqueron X, Li X, Eisenach JC.

Neuroscience 102(3):681-686, 2001. PMID: 11226704

It has been suggested that α2-adrenergic agonists produce analgesia by activating spinal cholinergic neurons. The authors reason that since spinal cholinergic neurons in the ventral horn express p75 following peripheral nerve trauma, cholinergic dorsal horn neurons might also. Instead, they find that dorsal horn neurons express little or no p75 under normal conditions or following spinal nerve ligation. Since dorsal horn neurons do not express p75 they are not eliminated by 192-Saporin (0.1-0.6 μg; Cat. # IT-01), but the data indicate that p75-expressing elements do play a role in pain transmission in the dorsal horn. The authors note that when afferents that express p75 are eliminated, mechanical hypersensitivity is unaffected, but the reduction of hypersensitivity by α2- adrenergic agonists or cholinergic agents is blocked.

Neuropeptide-toxin conjugates in pain research and treatment.

Wiley RG.

Reg Anesth Pain Med 25(5):546-548, 2000. PMID: 11009244

Several lines of evidence indicate dorsal horn neurons that respond to substance P (SP) play a role in nociception. Wiley discusses the attributes of SP-SAP (Cat. # IT-07), a targeted toxin that eliminates cells expressing the neurokinin-1 receptor. Animals treated with this material using a lumbar intrathecal injection show a decrease in both hyperalgesia and allodynia in several pain models. The success of SP-SAP indicates that other neuropeptides, hormones, and growth factors would be useful as targeted toxins.

Rat basal forebrain cholinergic lesion affects neuronal nitric oxide synthase activity in hippocampal and neocortical target regions.

Hartlage-Rubsamen M, Schliebs R.

Brain Res 889(1-2):155-164, 2001. PMID: 11166699

Nitric oxide (NO) mediates a variety of mechanisms in the brain including cortical perfusion, learning and memory, and neuronal plasticity. Cholinergic dysfunction has been associated with some of these same processes, notably reduced cortical cerebral blood flow and impaired performance in learning and memory tasks. The authors use a single intracerebroventricular injection of 192- Saporin (2.8 μg; Cat. # IT-01) to deplete the cholinergic neurons of the basal forebrain. Although total cortical neuronal NO synthase levels are not affected, the activity levels in select neocortical hippocampal neurons are reduced. The data suggest the ratio of catalytically active and inactive cortical NO synthase may be driven in part by basal cholinergic forebrain input.

Behavioural, histological and immunocytochemical consequences following 192 IgG-saporin immunolesions of the basal forebrain cholinergic system.

Perry T, Hodges H, Gray JA.

Brain Res Bull 54(1):29-48, 2001. PMID: 11226712

192-Saporin (Cat. #IT-01) has been used extensively as a model for Alzheimer’s Disease. The neuronal deficits caused by intraparenchymal forebrain injections (0.3-0.51 μg/μl) are apparent during tasks demanding attentional processing, but not standard tasks of learning and memory. Perry et al. compare the testing strategies for each deficit. They find that the water maze may not demand enough attentional processing to demonstrate deficits caused by this lesion. The authors also study long-term effects of 192-Saporin in rats. Although the authors produced very useful data at five to six months, they found evidence of an inflammatory response and non-specific cell death eleven months post treatment, indicating 192-Saporin may be problematic for very long-term experiments.

Septal cholinergic neurons suppress seizure development in hippocampal kindling in rats: comparison with noradrenergic neurons.

Ferencz I, Leanza G, Nanobashvili A, Kokaia Z, Kokaia M, Lindvall O.

Neuroscience 102(4):819-832, 2001. PMID: 11182245

Kindling can be caused in rats by lesioning forebrain cholinergic or noradrenergic projections. Ferencz et al. utilize 192-Saporin (2.5 μg; Cat. # IT- 01) to lesion forebrain cholinergic neurons and 6-hydroxydopamine to lesion noradrenergic neurons, administering both compounds by intraventricular injection. Upon comparing various aspects of hippocampal kindling, the authors determine that while both noradrenergic and cholinergic projections to the forebrain exert inhibitory effects, the cholinergic effect is less pronounced and occurs prior to seizure generalization.

Toxin-induced death of neurotrophin-sensitive neurons.

Wiley RG.

Methods Mol Biol 169:217-222, 2001. PMID: 11142013

Wiley discusses some of the specifics of using 192-Saporin (Cat. #IT-01) to eliminate cells expressing the rat p75 low-affinity nerve growth factor receptor. Wiley also describes the sequence of events following treatment with 192-Saporin from binding of the immunotoxin through ribosomal inactivation and cell death. Methods of handling the immunotoxin and injection are also addressed.

Model for aging in the basal forebrain cholinergic system.

Gu Z, Wortwein G, Yu J, Perez-Polo JR.

Antioxid Redox Signal 2(3):437-447, 2000. PMID: 11229357

A wide range of evidence indicates that cholinergic neurons play a role in memory and learning. Loss of these neurons is seen both in aged subjects and Alzheimer’s Disease patients. The authors discuss the use of 192-Saporin (Cat. #IT-01) to model this phenomenon. Many lesioning methods have been developed, including fimbria-fornix transections, mechanical lesions with radiofrequency or electrolysis, and intracerebral injections of excitotoxins. Information obtained through these methods suffers because non-cholinergic neurons are depleted as well as the desired cholinergic neurons. 192-Saporin provides a solution by specifically targeting and eliminating cholinergic neurons expressing p75 in the basal forebrain, closely mimicking a key component of aging.

Cover Article: Dermorphin-SAP Kills MOR-Positive Cells

Advanced Targeting Systems announces the release of its new, very exciting targeted toxin, dermorphin-SAP. It is a conjugate of the mu opioid receptor (MOR) agonist dermorphin and the ribosome-inactivating protein, saporin. Its cytotoxicity to cells that express the MOR promise to make it an important tool in the discovery and definition of the role of these cells in many biological processes.

In the latest issue of the Journal of Neuroscience, Porreca et al. (1) use this molecule for an important characterization of the descending pain pathways and the possible role of “ON” cells, the MOR-expressing cells of the rostroventromedial medulla (RVM), in the processes of chronic pain models. They injected dermorphin-SAP into the RVM and demonstrated loss of MOR-expressing cells near the injection site (Fig. 1). These neurons project to the spinal cord and it has been suggested by Howard Fields that they are responsible for a tonic discharge that mediates descending facilitation of nerve injury-induced pain. In fact, Porreca et al. demonstrate that with the loss of these cells, the expression of experimental neuropathic pain is ablated. This striking demonstration of supraspinal neurons having such a powerful effect on spinal cord properties is, well, sensational.

[threecol_two][/threecol_two] [threecol_one_last]Figure 1. MOR staining in the dorsal horn of rats treated as indicated. Loss of staining in dermorphin-SAP-treated animals is evident.
Figures supplied by Drs. Frank Porreca and Josephine Lai [/threecol_one_last]

The conjugate is made with dermorphin, first characterized from the skin of Phyllomedusa sauvagei by Montecucchi et al. (2). This agonist has one of the best profiles of specificity for the MOR of any known molecule, with exquisite affinity for the MOR (Fig. 2), while much lower affinity for the delta receptor (3). It has been documented to be internalized upon receptor binding, and with saporin attached takes in the ribosome-inactivating agent, causing protein synthesis inhibition and subsequent cell death. This specific lesioning tool is exemplary of many of Advanced Targeting Systems’ products.

Dermorphin-SAP was developed from a collaboration with Ron Wiley, and a glimpse of the activity of this cytotoxin was published in the journal Neuropeptides (4). MOR-expressing neurons have long been considered some of the most important cells in the nervous systems because of their participation in pain, pain control, addiction, gastrointestinal motility, and mast cell function, among others. This specific cytotoxin provides new methods for understanding these neurons and how they work.

[threecol_two][/threecol_two] [threecol_one_last]Figure 2. Inhibition of binding of DAMGO to MOR by dermorphin and dermorphin-SAP. Data demonstrate retention of binding after conjugation of dermorphin to saporin. [/threecol_one_last]

References

  1. Porreca F, Burgess SE, Gardell LR, Vanderah TW, Malan TP, Jr, Ossipov MH, Lappi DA, Lai J (2001) Inhibition of neuropathic pain by selective ablation of brainstem medullary cells expressing the μ-opioid receptor. J Neurosci 21(14):5281- 5288.
  2. Montecucchi PC, de Castiglione R, Piani S, Gozzini L, Erspamer V (1981) Amino acid composition and sequence of dermorphin, a novel opiate-like peptide from the skin of Phyllomedusa sauvagei. Int J Pept Prot Res 17(3):275-283.
  3. Attila M, Salvadori S, Balboni G, Bryant SD, Lazarus LH (1993) Synthesis and receptor binding analysis of dermorphin hepta-, hexa- and pentapeptides. Int J Pept Prot Res 42:550-559.
  4. Lappi DA, Wiley RG (2000) Entering through the doors of perception: characterization of a highly selective Substance P receptor-targeted toxin. Neuropeptides 34(5):323-328.

Related product information: Dermorphin-SAP (Cat. #IT-12)