Targeting Topics 16q4

Neural activity promotes long-distance, target-specific regeneration of adult retinal axons.

Lim JH, Stafford BK, Nguyen PL, Lien BV, Wang C, Zukor K, He Z, Huberman AD.

Nat Neurosci 19(8):1073-1084, 2016. PMID: 27399843

Axons in the CNS fail to regenerate after injury. Scientists sought to identify strategies that would allow retinal ganglion cell (RGC) axons to regenerate in the eye-to-brain pathway, and if that was possible, whether the axons could reconnect with their correct targets and restore visual function. It was previously shown that increasing mTOR signaling could trigger RGC axon regeneration. Several conditions were tested, but combining increased mTOR signaling and then exposing mice to high-contrast visual stimulation daily for 3 weeks scientists after optic nerve crush resulted in long distance RGC axon regeneration, re-innervation of the brain and partial recovery of a subset of visual behaviors. A 1:1000 dilution of Anti-Melanopsin (Cat. #AB-N38) was used for the immunohistochemical analysis of retinas, optic nerves and brain tissue.

Effective antitumor therapy based on a novel antibody-drug conjugate targeting the Tn carbohydrate antigen.

Sedlik C, Heitzmann A, Viel S, Ait Sarkouh R, Batisse C, Schmidt F, De La Rochere P, Amzallag N, Osinaga E, Oppezzo P, Pritsch O, Sastre-Garau X, Hubert P, Amigorena S, Piaggio E.

Oncoimmunology 5(7):e1171434, 2016. PMID: 27622021

Scientists wanted to study the potential of Chi-Tn, a monoclonal antibody against a glycol-peptidic tumor-associated antigen, as an anticancer antibody-drug conjugate. They demonstrated that Chi-Tn specifically targeted tumor cells in vivo, using flow cytometry and deconvolution microscopy to show that Chi-Tn is rapidly internalized. Chi-Tn-SAP (ATS Custom Services) effectively killed Tn-positive cells, but had no effect on Tn-negative cells. Saporin (Cat. #PR-01) was used as control. The cytotoxicity of the Chi-Tn-SAP correlated with the level of tumoral Tn expression.

Limiting glucocorticoid secretion increases the anorexigenic property of Exendin-4.

Lee SJ, Diener K, Kaufman S, Krieger JP, Pettersen KG, Jejelava N, Arnold M, Watts AG, Langhans W.

Mol Metab 5(7):552-565, 2016. PMID: 27408779

Glucagon-like peptide-1 (GLP-1) analogs lower blood surgar levels and cause a loss of appetite. Exendin-4 (Ex-4) is a GLP-1 receptor agonist, and also increases glucocorticoid secretion. Several tests were conducted to determine if the released glucocorticoids interact with Ex-4’s anorexigneic effect. One method involved ablating hindbrain catecholaminergic neurons by stereotaxically injecting 42 ng of Anti-DBH-SAP (Cat. #IT-03) bilaterally into the paraventricular nucleus of the hypothalamus in rats. Animals were injected with equimolar concentrations of unconjugated Saporin (Cat. #PR-01) as a control. Anti-DBH-SAP lesions reduced the efficacy of Ex-4 to increase corticosterone secretion but increased the anorexigenic effect, indicating that Ex-4-dependent corticosterone secretion opposes Ex-4’s actions. Anti-DBH-SAP lesions increased Ex-4’s ability to reduce food intake and body weight.

Brain penetration, target engagement, and disposition of the blood-brain barrier-crossing bispecific antibody antagonist of metabotropic glutamate receptor type 1.

Webster CI, Caram-Salas N, Haqqani AS, Thom G, Brown L, Rennie K, Yogi A, Costain W, Brunette E, Stanimirovic DB.

FASEB J 30(5):1927-1940, 2016. PMID: 26839377

To generate a BBB-transmigrating antibody that could be reformatted to full IgG, scientists started with the BBB-crossing llama?? single domain antibody FC5. Standard phage display protocols were used to isolate single-chain variable fragments (scFv) from the FC5-scFv library. 6His Mouse Monoclonal antibody (Cat. #AB-213) was used to assess cell binding of scFvs of FC5 using fluorescence microvolume assay technology. An scFv that competed with FC5 binding was selected for further testing. An antibody antagonist of the metabotropic glutamate receptor-1 was fused with this scFv antibody fragment (BBB-mGluR1) and tested in an in vitro BBB model. The resulting bispecific antibody retained selective mGluR1 binding and saw a 20-fold enhanced rate of transcytosis across the BBB compared to fusion with control antibody fragment. Intravenous injection of BBB-mGluR1 had analgesic properties in a rat model of persistent inflammatory pain.

Lysophosphatidylcholine acyltransferase 1 protects against cytotoxicity induced by polyunsaturated fatty acids.

Akagi S, Kono N, Ariyama H, Shindou H, Shimizu T, Arai H.

FASEB J 30(5):2027-2039, 2016. PMID: 26887439

Dietary consumption of polyunsaturated fatty acids can influence the degree of fatty acid unsaturation in membrane phospholipids, and consequently membrane-associated functions. Scientists set out to investigate how mammalian cells change their membrane lipid composition in response to loading with excess polyunsaturated fatty acids (PUFAs). Lipidomic analysis showed that PUFA treatment induces production of dipalmitoylphosphatidylcholine (DPPC). By suppressing phospholipid metabolism-related genes by RNA interference, they found that Lysophosphatidylcholine acyltransferase 1 (LPCAT1) was involved in DPPC production. To reveal the role of DPPC produced by PUFA treatment, HeLa cells were transfected with a siRNA against LPCAT1 to reduce its protein expression. The cells were lysed after treatment with a PUFA and subjected to western blot analysis using a 1:1000 dilution of Anti-SCD-1 (Cat. #AB-259) as the primary. SCD-1 desaturates the substrate of LPCAT1 for producing DPPC. PUFAs significantly reduced both the protein and mRNA expression of SCD-1. They showed that inhibiting DPPC production by LPCAT1 knockdown enhanced apoptosis, suggesting that DPPC produced via LPCAT1 protects against PUFA-induced cytotoxicity.

Selective noradrenaline depletion impairs working memory and hippocampal neurogenesis.

Coradazzi M, Gulino R, Fieramosca F, Falzacappa LV, Riggi M, Leanza G.

Neurobiol Aging 48:93-102, 2016. PMID: 27644078

Neuronal loss in the locus coeruleus (LC) of Alzheimer’s patients is well known, but the contribution of LC-derived noradrenergic afferents to learning and memory function is unknown. To model noradrenergic neuron degeneration in the LC, rats were bilaterally injected directly into the LC with 0.2 ug of Anti-DBH-SAP (Cat. #IT-03). Lesioned and sham-lesioned animals were tested behaviorally and exhibited robust working memory deficits but lesioning did not affect reference memory. They concluded that ascending noradrenergic afferents might be involved in more complex aspects of working memory, possibly via newly generated progenitors in the hippocampus.

Nucleus incertus contributes to an anxiogenic effect of buspirone in rats: Involvement of 5-HT1A receptors.

Kumar JR, Rajkumar R, Lee LC, Dawe GS.

Neuropharmacology 110(Pt A):1-14, 2016. PMID: 27436722

The nucleus incertus (NI) is involved in stress and anxiety responses. The NI is a cluster of GABAergic neurons in the brainstem, and coexpresses CRF, 5-HT1A and D2 receptors. Buspirone is a partial agonist of 5-HT1A receptors, an antagonist of D2 receptors, and activates the NI. Buspirone is an anti-anxiety drug, but preclinical studies showed that it induces anxiety at high doses. To see if the NI is necessary for the anxiogenic effects of high doses of buspirone, rats were bilaterally injected with 86 ng of CRF-SAP (Cat. #IT-13) into the NI. Blank-SAP (Cat. #IT-21) was used as a control. NI lesioning alone had an anxiogenic effect in several anxiety screening tests compared to sham-lesioned rats, which suggests that the NI reduces anxiety physiologically. Lesioning with CRF-SAP reduced the anxiogenic effects of intra-NI injections of buspirone. No significant difference in the anxiety screening tests resulted from injecting quinpiole, a D2 agonist, which suggests that the 5HT1A receptors in the NI are involved in the anxiogenic effects of buspirone.

Substituting mouse transcription factor Pou4f2 with a sea urchin orthologue restores retinal ganglion cell development.

Mao CA, Agca C, Mocko-Strand JA, Wang J, Ullrich-Luter E, Pan P, Wang SW, Arnone MI, Frishman LJ, Klein WH.

Proc Biol Sci 283(1826):20152978, 2016. PMID: 26962139

Pou4f2 is Pou domain transcription factor that is essential for the development of retinal ganglion cells (RGCs) in the vertebrate retina. The sea urchin genome contains SpPou4f1/2, a distant orthologue of Pou4f2, but they have no obvious eyes and their photoreceptors are located around their tube feet disc. Scientists replaced genomic Pou4f2 with an SpPou4f1/2 cDNA to see if SpPou4f1/2 could support RGC development in mice. Mice expressing SpPou4f1/2 developed retinas that looked like wild-type mice. Immunolabeling of retinas with a 1:1000 dilution of Anti-Melanopsin (Cat. #AB-N39) showed the presence of many well-bundled axons emanating from SpPou4f1/2-expressing RGCs. Electroretinogram recordings from these mice indicate that their RGCs are functionally active. These results suggest that there is a high degree of functional conservation between the two genes despite more than 540 million years of divergence from the common ancestor of mice and sea urchins.

GABA-A receptor activity in the noradrenergic locus coeruleus drives trigeminal neuropathic pain in the rat; contribution of NAalpha1 receptors in the medial prefrontal cortex.

Kaushal R, Taylor BK, Jamal AB, Zhang L, Ma F, Donahue R, Westlund KN.

Neuroscience 334:148-159, 2016. PMID: 27520081

The goal of this study was to investigate the role of the locus coeruleus (LC) in a rat orofacial pain model of trigeminal neuropathy induced by chronic constrictive injury of the infraorbital nerve (CCI-ION). Mechanical thresholds to von Frey filaments were tested on whisker pads to evaluate neuropathic pain behavior; pain was indicated by development of mechanical hypersensitivity. Noradrenergic (NA) neurons were lesioned with 5-mcg injections of Anti-DBH-SAP (Cat. #IT-03) into the left lateral ventricle. Mouse-IgG-SAP (Cat. #IT-18) was used as a control. After ablation of NA neurons there was a notable increase in the mechanical threshold compared to control animals. They also targeted coeruleotrigeminal NA neurons by injecting Anti-DBH-SAP into the trigeminal brainstem nuclei bilaterally in one animal and saw similar results. Injecting a GABAA receptor antagonist into the LC after injury had an inhibitory effect on nerve injury induced hypersensitivity. Injection of a NAα1 receptor antagonist, but not a NAα2 receptor antagonist, into the medial prefrontal cortex (mPFC) alleviates mechanical hypersensitivity. They conclude that GABAA-mediated activation of NA neurons during CCI-ION can facilitate hypersensitivity through NAα1 receptors in the mPFC, and that the LC is a chronic pain generator.

A2 noradrenergic neurons regulate forced swim test immobility.

Nam H, Kerman IA.

Physiol Behav 165:339-349, 2016. PMID: 27553574

Wistar-Kyoto rats are often used as a model of depression, and exhibit high levels of immobility when subjected to a forced swim test (FST). Researchers discovered relative hyperactivation in the locus coeruleus of WKY rats compared to the genetically related Wistar rats when exposed to one- and two-day FSTs. Lesser activation of A2 noradrenergic cell group was seen by diminished levels of FOS after both days of the FST. A2 noradrenergic neurons of Winstar rats were lesioned by injecting 2.2 ug of Anti-DBH-SAP (Cat. #IT-03) into the nucleus tractus solitaris (NTS). Lesioned rats exhibited increased FST immobility on both days of the test, similar to natural WKY behavior in the same test. These data indicate that the A2 noradrenergic cell group regulates FST behavior and that its hypoactivation may contribute to the unique behavioral phenotype of WKY rats.

Saponins from Saponaria officinalis L. Augment the Efficacy of a Rituximab-Immunotoxin.

Gilabert-Oriol R, Thakur M, Haussmann K, Niesler N, Bhargava C, Gorick C, Fuchs H, Weng A.

Planta Med 2016. PMID: 27392242

It is known that triterpenoidal saponins that come from Saponaria officinalis, the plant that saporin comes from, increases the cytotoxicity of saporin by modulating its intracellular trafficking. Investigators wanted to know if this could increase the therapeutic affect of Rituximab-Saporin. In the presence of saponins, Rituximab-Saporin had a 700-fold increase in efficacy. Concentrations of 0.0001-1nM of Anti-CD22-SAP (Cat. #IT-37) and 0.001-10nM of Anti-CD25-SAP (Cat. #IT-24) were also tested in vitro with saponins for comparison. They saw a 170-fold and 25-fold increase in cytotoxicity, respectively. All conjugates were tested on Ramos cells, and differing levels of receptor expression could explain the drastic differences in cytotoxicity enhancement.