Targeting Topics 14q2

Habenular kisspeptin modulates fear in the zebrafish.

Ogawa S, Nathan FM, Parhar IS.

Proc Natl Acad Sci U S A 111(10):3841-3846, 2014.

The peptide kisspeptin can be found in several areas of the brain, but its role in regions other than the hypothalamus has not been studied. Zebrafish express kiss1 mRNA which is a conserved ortholog of the mammalian KISSI/KissI making zebrafish a viable model for investigating the role of kisspeptin in various brain systems. Animals received 1 μg of the custom conjugate kiss-SAP (see NK3-SAP, Cat. #IT-63) via an intracranial injection. Blank-SAP (Cat. #IT-21) was used as a control. Reducing Kiss1 immunoreactivity in the habenula and the raphe reduced an invoked fear response, indicating a role for kisspeptin in fear inhibition.

Selective actions of novel allosteric modulators reveal functional heteromers of metabotropic glutamate receptors in the CNS.

Yin S, Noetzel MJ, Johnson KA, Zamorano R, Jalan-Sakrikar N, Gregory KJ, Conn PJ, Niswender CM.

J Neurosci 34(1):79-94, 2014.

The eight mGlu receptor subtypes that have been discovered have been thought to function only as homodimers. Although recent studies have suggested that these receptors can form heterodimers in vitro, this has not been demonstrated in vivo. Using a variety of methods, including western blots, coimmunoprecipitation, thallium flux assays, transient transfections, and whole cell patch recordings the authors show that mGlu2 and mGlu4 form a hetero-complex in both rat and mouse tissues. An anti-mGluR2 (Cat. #AB-N32) was used in coimmunoprecipitation.

Medial septum-diagonal band of Broca (MSDB) GABAergic regulation of hippocampal acetylcholine efflux is dependent on cognitive demands.

Roland JJ, Stewart AL, Janke KL, Gielow MR, Kostek JA, Savage LM, Servatius RJ, Pang KC.

J Neurosci 34(2):506-514, 2014.

GABAergic and cholinergic neurons in the medial septum-diagonal band of Broca (MSDB) are both involved with spatial memory. In order to better understand the relationship between these two neuronal populations the authors administered 552.5 ng of GAT-1-SAP (Cat. #IT-32) to the MSDB of rats in several injections. Using a combination of behavioral assays and in vivo microdialysis it was shown that GAT-1-SAP lesions impaired hippocampal acetylcholine efflux as well as performance in the non-matching to position with delay test. The data indicate that GABAergic MSDB neurons are important during high memory load conditions.

Cholinergic contributions to supramodal attentional processes in rats.

Ljubojevic V, Luu P, De Rosa E.

J Neurosci 34(6):2264-2275, 2014.

Previous work has shown that cholinergic transmission is important for attentional processing of visual stimuli. It is not known whether the role of the cholinergic system is specifically in visual processing, or if it is involved in all types of attentional processing. The authors administrated bilateral 40 ng injections of 192-IgG-SAP (Cat. #IT-01) to the nucleus basalis magnocellularis of rats. Animals had been previously trained on both visual and olfactory 5-choice serial reaction time tasks. Lesioned animals displayed deficits on both the visual and olfactory tasks when the attentional demand of the task was increased. The data suggest a modular cortical network that services both visual and olfactory attentional processes.

Saporin Conjugated Monoclonal Antibody to the Transcobalamin Receptor TCblR/320 Is Effective in Targeting and Destroying Cancer Cells.

Quadros EV, Nakayama Y, Sequeira JM.

J Cancer Ther 4(6):1074-1081, 2013.

Although the transcobalamin receptor (TCb1R) is expressed in most cell types, the expression levels are increased in actively proliferating cells, and decreased in quiescent cells. This expression profile makes the TCb1R an attractive target for cancer therapy. The authors used a custom conjugate of antibodies generated against the TCb1R and saporin to eliminate cancer cell lines in culture. When applying the conjugate to cells in a dosing range of 0.156-5 nM, 2.5 nM was found to have the optimal effect. Given that the level of toxicity was determined by the level of TCb1R expression, targeting the TCb1R may have potential as part of a cancer treatment strategy.

Neutralization of Plasmodium falciparum Merozoites by Antibodies against PfRH5.

Douglas AD, Williams AR, Knuepfer E, Illingworth JJ, Furze JM, Crosnier C, Choudhary P, Bustamante LY, Zakutansky SE, Awuah DK, Alanine DG, Theron M, Worth A, Shimkets R, Rayner JC, Holder AA, Wright GJ, Draper SJ.

J Immunol 192(1):245-258, 2014.

Plasmodium falciparum is a protozoan parasite that can cause malaria in humans. The human malaria parasite reticulocyte –binding protein homolog 5 (PfRH5) is a potential target for neutralizing antibodies. PfRH5 binds basigin on erythrocytes, and through the use of anti-basigin (Cat. #AB-42), among other antibodies, the authors better characterized aspects of this binding that may be useful in preventing malaria.

The novel EpCAM-targeting monoclonal antibody 3-17I linked to saporin is highly cytotoxic after photochemical internalization in breast, pancreas and colon cancer cell lines.

Lund K, Bostad M, Skarpen E, Braunagel M, Krauss S, Duncan A, Hogset A, Selbo P.

MAbs 6(3)2014.

The epithelial cell adhesion molecule (EpCAM) is an attractive diagnostic and therapeutic target for a wide range of human carcinomas. It has also been found on cancer stem cells, increasing the interest in targeting and eliminating cells that express it. The authors have created a monoclonal antibody that binds EpCAM, and use several assays to demonstrate the antibody’s potential as an oncology tool. In one series of assays the biotinylated antibody was combined with streptavidin-ZAP (Cat. #IT-27), and in conjunction with photochemical internalization was shown to have specific cytotoxicity on several different cancer cell lines over a range of concentrations.

Depletion of alloreactive T cells by anti-CD137-saporin immunotoxin.

Lee SC, Seo KW, Kim HJ, Kang SW, Choi HJ, Kim A, Kwon BS, Cho HR, Kwon B.

Cell Transplant 24(6):1167-1181, 2015. PMID: 24594433

The ability to selectively remove T cells that mediate graft-versus-host disease (GVHD) would prevent graft rejection as well as preserve the T cells that mediate graft-versus-leukemia (GVL) effect – especially in cases of hematopoietic stem cell transplantation. In this work the authors used a custom conjugate of anti-mouse CD137 and saporin to eliminate alloreactive T cells during a T cell donor transfer in mice. Rat IgG-SAP (Cat. #IT-35) was used as a control. Transfer of T cells after the immunotoxin treatment did not cause GVHD, but the GVL effect was left intact, indicating the potential of selective T cell depletion for transplantation.

Neuroprotective effects of donepezil against cholinergic depletion.

Cutuli D, De Bartolo P, Caporali P, Tartaglione AM, Oddi D, D’Amato FR, Nobili A, D’Amelio M, Petrosini L.

Alzheimers Res Ther 5(5):50, 2013.

Acetylcholinesterase inhibitors appear to be one of the only pharmacological tools available to reduce cognitive deficits caused by the loss of cholinergic neurons in the basal forebrain. Here the authors pre-treated rats with the aceytlcholinesterase inhibitor donepezil before administering 0.5 μg of 192-IgG-SAP (Cat. #IT-01) into each side of the medial septum. Analysis of working memory, spatial discrimination, social novelty preference, and ultrasonic localizations, along with measuring hippocampal and neocortical caspase-3 activity indicates that donepezil pre-treatment ameliorates some effects of the cholinergic depletion.

Gabapentin increases extracellular glutamatergic level in the locus coeruleus via astroglial glutamate transporter-dependent mechanisms.

Suto T, Severino AL, Eisenach JC, Hayashida KI.

Neuropharmacology 81C:95-100, 2014.

Gabapentin is effective in reducing acute and chronic pain, but the mechanisms by which it works are not well understood. The authors assessed extracellular glutamate levels and glutamate interaction with several different cellular membrane proteins. Rats received a 0.25 μg injection of anti-DBH-SAP (Cat. #IT-03) into the locus coeruleus (LC) in order to deplete noradreline levels. Mouse IgG-SAP (Cat. #IT-18) was used as a control. The gabapentin-induced glutamate increase in the LC was not affected by the lesion, supporting data indicating that gabapentin induces glutamate release from astrocytes to stimulate descending inhibition.

Stimulation of feeding by three different glucose-sensing mechanisms requires hindbrain catecholamine neurons.

Li AJ, Wang Q, Dinh TT, Powers BR, Ritter S.

Am J Physiol Regul Integr Comp Physiol 306(4):R257-64, 2014.

The glucoregulatory system of the brain requires catecholamine neurons in the hindbrain. he sensory mechanisms and connected circuitry controlling the response to glucose deficit are not well understood. In order to investigate different drugs that stimulate food intake but interfere with cellular glucose metabolism and transport the authors administered 82 ng of anti-DBH-SAP (Cat. #IT-03) into the paraventricular nucleus as bilateral injections. Saporin (Cat. #PR-01) was used as a control. Lesioned animals did not increase food intake in response to any of the drugs, indicating that stimulation of food intake is activated through a catecholamine-dependent pathway.

Ablating Spinal NK1-Bearing Neurons Eliminates the Development of Pain and Reduces Spinal Neuronal Hyperexcitability and Inflammation From Mechanical Joint Injury in the Rat.

Weisshaar CL, Winkelstein BA.

J Pain 15(4):378-386, 2014.

A high percentage of chronic neck pain involves the facet joint. Although the facet joint is innvervated by peptide-responsive nociceptive afferents, the role of these cells in the development and modulation of nociceptive signaling remains unclear. Using a previously developed rat model of facet joint injury, the authors examined the role of neurokinin-1 receptor-expressing spinal cells in this pathway. Rats received 100 ng SSP-SAP (Cat. #IT-11) via lumbar puncture. Blank-SAP (Cat. #IT-21) was used as a control. The results demonstrate that spinal NK1r-expressing cells are essential for nociception and inflammation due to a mechanical joint injury.

The p75 receptor is associated with inflammatory thermal hypersensitivity.

Watanabe T, Ito T, Inoue G, Ohtori S, Kitajo K, Doya H, Takahashi K, Yamashita T.

J Neurosci Res 86(16):3566-3574, 2008.

Inflammation, which can lead to pain hypersensitivity, not only elevates NGF levels, but also can increase the sensitivity of high-threshold receptors. NGF is thought to be a major contributor to heightened pain states due to inflammation, and in this work the authors investigated the role that the p75 neurotrophin receptor might play in NGF-induced hyperalgesia. 30 minutes prior to injection with Freund’s adjuvant, mice received 10 μl of polyclonal anti-p75 (Cat. #AB-N01) into the plantar surface of the paw. The antibody administration blocked hyperalgesia, indicating that p75 plays an important role in inflammation-induced hyperalgesia.

Phox2b-expressing retrotrapezoid neurons and the integration of central and peripheral chemosensory control of breathing in conscious rats.

Takakura AC, Barna BF, Cruz JC, Colombari E, Moreira TS.

Exp Physiol 99(3):571-585, 2014.

Previous work has shown that lesions to the retrotrapezoid nucleus (RTN) have at least a modest effect on breathing, but it is unclear whether those lesions affected the entire nucleus or were incomplete. The authors used bilateral lesions of the RTN with 0.3 to 1.2 ng total of SSP-SAP (Cat. #IT-11) to eliminate neurokinin-1 receptor-expressing neurons; these are also Phox2b+TH- neurons. The results indicate that loss of Phox2b(+)TH(-) neurons may cause deficits seen after RTN lesion, and help define the ways in which these cells are involved in controlling central and peripheral chemoreflexes.

IB4(+) and TRPV1(+) sensory neurons mediate pain but not proliferation in a mouse model of squamous cell carcinoma.

Ye Y, Bae S, Viet CT, Troob S, Bernabe D, Schmidt BL.

Behav Brain Funct 10(1):5, 2014.

The peptidergic and nonpeptidergic neurons in the spinal cord are thought to have different responsibilities in the transmission of nociceptive information. In this work the authors more specifically defined the roles of each neuronal type in cancer-induced mechanical allodynia and thermal hyperalgesia. Mice received 2.5 μg intrathecal injections of IB4-SAP (Cat. #IT-10) followed by injection of carcinoma cells into the hindpaw. Saporin (Cat. #PR-01) was used as a control. The data show that peptidergic neurons mediate thermal hyperalgesia, and nonpeptidergic neurons are involved with mechanical allodynia.

Expression of Different Neurokinin-1 Receptor (NK1R) Isoforms in Glioblastoma Multiforme: Potential Implications for Targeted Therapy.

Cordier D, Sailer M, Gerber A, Kluba C, Bauman A, Hutter G, Mindt TL, Mariani L.

Cancer Biother Radiopharm Epub2014.

The neurokinin-1 receptor (NK1r) has been found to be consistently over-expressed in gliomas, making it a potential target for therapeutic strategies. However, treatments with therapies utilizing substance P (SP), the ligand for the NK1r, have at best yielded uneven results. In this work the authors investigated factors that may predict the response to therapies directed at NK1r gliomas. SSP-SAP (Cat. #IT-11) was used at a concentration of 1 nM in cytotoxicity assays on several different glioma cell lines. Using this and other data it was shown that only the cell line with the most full-length NK1r RNA transcripts displayed high levels of binding, internalization, and cell killing necessary for NK1r to be a therapeutic target using SP.