Alteration of the endocannabinoid system in mouse brain during prion disease.

Prion diseases are neurodegenerative disorders characterized by deposition of the pathological prion protein (PrPsc) within the brain of affected humans and animals. Microglial cell activation is a common feature of prion diseases; alterations of various neurotransmitter systems and neurotransmission have been also reported. Owing to its ability to modulate both neuroimmune responses and neurotransmission, it was of interest to study the brain endocannabinoid system in a prion-infected mouse model. The production of the endocannabinoid, 2-arachidonoyglycerol (2-AG), was enhanced 10 weeks post-infection, without alteration of the other endocannabinoid, anandamide. The CB2 receptor expression was up-regulated in brains of prion-infected mice as early as 10 weeks and up to 32 weeks post-infection whereas the mRNAs of other cannabinoid receptors (CBRs) remain unchanged. The observed alterations of the endocannabinoid system were specific for prion infection since no significant changes were observed in the brain of prion-resistant mice, that is, mice devoid of the Prnp gene. Our study highlights important alterations of the endocannabinoid system during early stages of the disease long before the clinical signs of the disease.

Binding of a pure 125I-monoiodoleptin analog to mouse tissues: a developmental study.

The preparation of a pure 125I-labeled monoiododerivative of mouse leptin is described. This radiolabeled analog has been used to characterize and localize central and peripheral leptin binding sites (Ob-R) of the mouse at different stages of its development. The affinity values found in membrane homogenates of various mouse tissues are similar and range between 0.1 and 0.3 nM, indicating that all the Ob-R isoforms have a similar affinity. Leptin binding sites are highly expressed at the membrane level in lung, intestine, kidney, liver, and skin and to a lesser degree in stomach, heart, and spleen. Brain, thymus, and pancreas homogenates are devoid of any specific binding. The distribution of mouse Ob-R has also been explored by autoradiography and dipping techniques on whole mouse sections. In lung, leptin binding sites are located at the pulmonary parenchyma and at the bronchiolar epithelial level. Binding sites are expressed all along the digestive tract from the tongue to the rectum (esophagus, stomach, intestine, colon, and rectum). In muscular visceral structures (stomach, intestine, and bladder) the binding is mainly present in the lamina propria. During development, leptin receptors are early expressed in the liver, kidney, and bone. In the lung, the Ob-R level increased gradually from birth to adulthood where the expression is maximal. By contrast, leptin receptors located in the medulla of the kidney remain remarkably constant all along the development. A broad signal is present in cartilage and bone particularly in vertebrae, limb, and ribs. Interestingly, leptin receptors are barely detectable in the mouse brain except in the choroid plexus and leptomeninges, whereas in the rat brain leptin binding sites are located in the thalamus, the piriform cortex, the cerebellum (at the granular and molecular cell layer), and the pineal gland.

The 100-kDa neurotensin receptor is gp95/sortilin, a non-G-protein-coupled receptor.

In this work, the 100-kDa neurotensin (NT) receptor previously purified from human brain by affinity chromatography (Zsürger, N., Mazella, J., and Vincent, J. P. (1994) Brain Res. 639, 245-252) was cloned from a human brain cDNA library. This cDNA encodes a 833-amino acid protein 100% identical to the recently cloned gp95/sortilin and was then designated NT3 receptor-gp95/sortilin. The N terminus of the purified protein is identical to the sequence of the purified gp95/sortilin located immediately after the furin cleavage site. The binding of iodinated NT to 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid-solubilized extracts of COS-7 cells transfected with the cloned cDNA was saturable and reversible with an affinity of 10-15 nM. The localization of the NT3 receptor-gp95/sortilin into intracellular vesicles was in agreement with previous results obtained with the purified receptor and with gp95/sortilin. Affinity labeling and binding experiments showed that the 110-kDa NT3 receptor can be partly transformed into a higher affinity (Kd = 0.3 nM) 100-kDa protein receptor by cotransfection with furin. This 100-kDa NT receptor corresponded to the mature form of the receptor. The NT3/gp95/sortilin protein is the first transmembrane neuropeptide receptor that does not belong to the superfamily of G-protein-coupled receptors.

Preparation and binding properties of radioiodinated analogues of dermorphin and deltorphin with high specificity for the mu- and delta-opioid receptors.

The synthesis, purification, chemical characterization, and binding properties of two 125I-labeled analogues of dermorphin and deltorphin-I are described. Native deltorphin-I and [Lys7] dermorphin sequences were elongated by an aminopentyl chain on their C-terminal amide function and alkylated with the 125I-labeled monoiodinated derivative of Bolton-Hunter reagent (BH*). The resulting radiolabeled peptides, epsilon-BH* [Lys7] dermorphin 5-aminopentylamide and omega-BH* deltorphin-I 5-aminopentylamide, have kept most of the original properties of the parent peptides. They bind with high selectivity and specificity to the mu- (dermorphin analogue) or delta- (deltorphin-I analogue) opioid receptors from rat brain or from cells transfected with cDNAs encoding the mu and delta receptors. The autoradiographic distribution of specific binding sites for the 125I-labeled dermorphin and deltorphin-I analogues in rat brain is in complete agreement with previously reported localizations of mu- and delta-opioid receptors. The two radiolabeled peptides are the best ligands of mu- and delta-opioid receptors currently available in terms of sensitivity, specificity, and selectivity.

Radiolabeled ligands specific for the G protein-coupled state of neurotensin receptors.

Radiolabeled analogues of neuromedin N have been prepared by acylation of the alpha, epsilon 1, and epsilon 2 amino groups of [Lys2]neuromedin N (Lys-Lys-Pro-Tyr-Ile-Leu) either with the 125l-labeled Bolton-Hunter reagent or with N-succinimidyl[2,3-3H]propionate. The binding properties of the purified analogues toward newborn mouse brain homogenate or toward membranes of cells transitorily (COS) or permanently (AA1) transfected with the cloned rat brain neurotensin receptor cDNA were evaluated and compared with those of radiolabeled neurotensin. The alpha-modified analogue of [Lys2]neuromedin N behaves exactly like neurotensin in these binding experiments, whereas the epsilon 1- and epsilon 2-modified analogues selectively recognize the fraction of neurotensin binding sites that is sensitive to GTP gamma S. The proportion of neurotensin receptors coupled to GTP binding proteins is approximately 50% in membranes of newborn mouse brain or of AA1 cells that respond to neurotensin by an increase of the intracellular inositol trisphosphate concentration. By contrast, membranes of transitorily transfected COS cells that do not respond to neurotensin exhibit very low levels of GTP-sensitive receptors labeled with the epsilon 1- or epsilon 2-modified analogues. These radiolabeled peptides offer new tools to selectively detect active neurotensin receptors.

Solubilization and purification of a high affinity neurotensin receptor from newborn human brain.

High affinity neurotensin receptors were solubilized in an active form from newborn human brain using the non-denaturing detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS). The solubilized receptor was purified in a single step by affinity chromatography. The binding properties of the purified receptor towards [125I-Tyr3]neurotensin are very similar to those of the membrane bound and of the crude CHAPS-solubilized receptor in terms of affinity and specificity. The purified receptor is a single protein chain of molecular weight 100 kDa as shown by gel filtration and by affinity labelling with [125I-Tyr3]neurotensin in the presence of the cross-linking agent disuccinimidyl suberate.

Compared binding properties of 125I-labeled analogues of neurotensin and neuromedin N in rat and mouse brain.

Neurotensin and neuromedin N are two structurally related peptides that are synthesized by a common precursor. The purpose of the present work was to characterize neuromedin N receptors in rat and mouse brain and to compare these receptors with those of neurotensin. A radiolabeled analogue of neuromedin N has been prepared by acylation of the N-terminal amino group of the peptide with the 125I-labeled Bolton-Hunter reagent. This 125I-labeled derivative of neuromedin N bound to newborn mouse brain homogenate with high affinity (KD = 0.5 nM). Cross-competition experiments between radiolabeled and unlabeled neurotensin and neuromedin N indicated that each peptide was able to displace completely and specifically the other peptide from its interaction with its receptor. Independently of the radioligand used, the affinity of neurotensin was always better than that of neuromedin N. Quantitative radioautographic studies demonstrated that the ratio of labeling intensities obtained with 125I-labeled analogues of neurotensin and neuromedin N remained constant in all the brain areas. Our results do not support the existence of a specific neuromedin N receptor in rat and mouse brain and can be explained by the presence of a common receptor for both peptides.

Ontogenesis and binding properties of high-affinity neurotensin receptors in human brain.

The ontogenesis of neurotensin binding sites was studied in human brain of subjects deceased from Sudden Infant Death Syndrome. Monoiodo-Tyr3 neurotensin specifically recognized 2 distinct classes of binding sites in human brain homogenate. The high affinity sites were already present at birth and increased to a maximal level of 240 fmol/mg protein 1 month after birth. Thereafter, the density of these sites decreased to reach a value of 8 fmol/mg protein in 15-month-old brain, a value similar to that found in adult brain. The dissociation constant of the high-affinity sites (about 0.3 nM) did not vary from birth to adulthood. The high-affinity binding sites were sensitive to GTP which decreased their affinity for neurotensin by a factor of 3, indicating that these sites are functional receptors coupled to GTP-binding proteins. By contrast, the low-affinity sites were insensitive to GTP and could be partly blocked by the antihistaminic drug levocabastine. These sites were absent in human brain during the first post-natal year and could be detected only in brain homogenate of 15-month-old infants. The transient increase in high-affinity neurotensin binding sites after birth suggests that neurotensin could act as a regulatory peptide during brain development.

Purification of the neurotensin receptor from mouse brain by affinity chromatography.

The neurotensin receptor was purified from newborn mouse brain by affinity chromatography. Active neurotensin binding sites were solubilized from brain homogenates using the nondenaturing detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS) in the presence of cholesteryl hemisuccinate. Chromatography of the soluble extract on SP-Sephadex C-25 and hydroxylapatite eliminated 50% of proteins without loss of neurotensin binding activity. This prepurified material was loaded into an affinity column prepared by coupling neurotensin (2-13) to glutaraldehyde-activated Ultrogel AcA22. Nonspecifically adsorbed proteins were eliminated by extensive washing, and the receptor was eluted with a buffer containing 1 M NaCl, 0.1% CHAPS, and 0.02% cholesteryl hemisuccinate. After desalting, the purified receptor bound 125I-labeled neurotensin with a dissociation constant of 0.26 nM and retained its specificity towards a series of neurotensin analogues. The desalted NaCl eluate appeared on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a major band of molecular weight 100,000 which was identified as the receptor by affinity labeling with 125I-labeled neurotensin in the presence of disuccinimidyl suberate. The purity of the mouse brain receptor eluted from the affinity column was estimated to be 78%. Electroelution of the 100-kDa protein band gave an homogenous preparation of receptor. Very similar results were obtained with CHAPS-solubilized neurotensin receptors from rat and rabbit brain.