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.

Efficient conversion of normal prion protein (PrP) by abnormal hamster PrP is determined by homology at amino acid residue 155.

In the transmissible spongiform encephalopathies, disease is closely associated with the conversion of the normal proteinase K-sensitive host prion protein (PrP-sen) to the abnormal proteinase K-resistant form (PrP-res). Amino acid sequence homology between PrP-res and PrP-sen is important in the formation of new PrP-res and thus in the efficient transmission of infectivity across species barriers. It was previously shown that the generation of mouse PrP-res was strongly influenced by homology between PrP-sen and PrP-res at amino acid residue 138, a residue located in a region of loop structure common to PrP molecules from many different species. In order to determine if homology at residue 138 also affected the formation of PrP-res in a different animal species, we assayed the ability of hamster PrP-res to convert a panel of recombinant PrP-sen molecules to protease-resistant PrP in a cell-free conversion system. Homology at amino acid residue 138 was not critical for the formation of protease-resistant hamster PrP. Rather, homology between PrP-sen and hamster PrP-res at amino acid residue 155 determined the efficiency of formation of a protease-resistant product induced by hamster PrP-res. Structurally, residue 155 resides in a turn at the end of the first alpha helix in hamster PrP-sen; this feature is not present in mouse PrP-sen. Thus, our data suggest that PrP-res molecules isolated from scrapie-infected brains of different animal species have different PrP-sen structural requirements for the efficient formation of protease-resistant PrP.

In vivo cytotoxicity of the prion protein fragment 106-126.

Transmissible spongiform encephalopathies are fatal neurological diseases characterized by astroglyosis, neuronal loss, and by the accumulation of the abnormal isoform of the prion protein. The amyloid prion protein fragment 106-126 (P106-126) has been shown to be toxic in cultured hippocampal neurons (). Here, we show that P106-126 is also cytotoxic in vivo. Taking advantage of the fact that retina is an integral part of the central nervous system, the toxic effect of the peptide was investigated by direct intravitreous injection. Aged solutions of P106-126 induced apoptotic-mediated retinal cell death and irreversibly altered the electrical activity of the retina. Neither apoptosis nor electroretinogram damages were observed with freshly diluted P106-126, suggesting that the toxicity is linked to the aggregation state of the peptide. The retina provides a convenient in vivo system to look for potential inhibitors of cytotoxicity associated with spongiform encephalopathies.

Interactions between heterologous forms of prion protein: binding, inhibition of conversion, and species barriers.

The self-induced formation of the disease-associated, protease-resistant prion protein (PrP-res) from the normal protease-sensitive isoform (PrP-sen) appears to be a key event in the pathogenesis of transmissible spongiform encephalopathies. The amino acid sequence specificity of PrP-res formation correlates with, and may account for, the species specificity in transmission of transmissible spongiform encephalopathy agents in vivo. To analyze the mechanism controlling the sequence specificity of PrP-res formation, we compared the binding of PrP-sen to PrP-res with its subsequent acquisition of protease resistance by using cell-free systems consisting of heterologous versus homologous mouse and hamster PrP isoforms. Our studies showed that heterologous PrP-sen can bind to PrP-res with little conversion to the protease-resistant state and, in doing so, can interfere with the conversion of homologous PrP-sen. The interference occurred with molar ratios of homologous to heterologous PrP-sen molecules as low as 1:1. The interference was due primarily to the inhibition of conversion, but not the binding, of the homologous PrP-sen to PrP-res. The results provide evidence that the sequence specificity of PrP-res formation in this model is determined more by the conversion to protease resistance than by the initial binding step. These findings also imply that after the initial binding, further intermolecular interactions between PrP-sen and PrP-res are required to complete the process of conversion to the protease-resistant state.

Species-independent inhibition of abnormal prion protein (PrP) formation by a peptide containing a conserved PrP sequence.

Conversion of the normal protease-sensitive prion protein (PrP) to its abnormal protease-resistant isoform (PrP-res) is a major feature of the pathogenesis associated with transmissible spongiform encephalopathy (TSE) diseases. In previous experiments, PrP conversion was inhibited by a peptide composed of hamster PrP residues 109 to 141, suggesting that this region of the PrP molecule plays a crucial role in the conversion process. In this study, we used PrP-res derived from animals infected with two different mouse scrapie strains and one hamster scrapie strain to investigate the species specificity of these conversion reactions. Conversion of PrP was found to be completely species specific; however, despite having three amino acid differences, peptides corresponding to the hamster and mouse PrP sequences from residues 109 to 141 inhibited both the mouse and hamster PrP conversion systems equally. Furthermore, a peptide corresponding to hamster PrP residues 119 to 136, which was identical in both mouse and hamster PrP, was able to inhibit PrP-res formation in both the mouse and hamster cell-free systems as well as in scrapie-infected mouse neuroblastoma cell cultures. Because the PrP region from 119 to 136 is very conserved in most species, this peptide may have inhibitory effects on PrP conversion in a wide variety of TSE diseases.

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.

Specific inhibition of in vitro formation of protease-resistant prion protein by synthetic peptides.

The transmissible spongiform encephalopathies are characterized by the conversion of the protease-sensitive prion protein (PrPsen) into a protease-resistant isoform (PrPres) associated with the neuropathogenic process in vivo. Recently, PrPres has been shown to be capable of directly inducing the conversion of PrPsen to PrPres in a cell-free in vitro system. In the present experiments, various PrP peptides were studied for their ability to enhance or inhibit this cell-free conversion reaction. None of the synthetic peptides was able to confer protease-resistance to the labeled PrPsen molecules on their own. On the contrary, peptides from the central part of the hamster PrP sequence from 106 to 141 could completely inhibit the conversion induced by preformed PrPres. The presence of residues 119 and 120 from the highly hydrophobic sequence AGAAAAGA (position 113 to 120) was crucial for an efficient inhibitory effect. Fourier transform infrared spectroscopy analysis indicated that inhibitory peptides formed high beta-sheet aggregates under the conditions of the conversion reaction, but this was also true of certain peptides that were not inhibitory. Thus, the potential to form beta-sheeted aggregates may be necessary, but not sufficient, for peptides to act as inhibitors of PrPres formation. Clearly, the amino acid sequence of the peptide is also important for inhibition. The sequence specificity of the inhibition is consistent with the idea that residues in the vicinity of positions 106-141 of PrPres and/or PrPsen are critically involved in the intermolecular interactions that lead to PrPres formation.

Stable expression of the mouse levocabastine-sensitive neurotensin receptor in HEK 293 cell line: binding properties, photoaffinity labeling, and internalization mechanism.

The recently cloned new subtype of G protein-coupled neurotensin receptor (NTRL) was stably expressed in the HEK 293 cell line in order to investigate its binding and internalization properties. The expressed receptor exhibited the typical binding characteristics of the low affinity, levocabastine-sensitive binding site previously described in rat and mouse brain and was detected as a protein with an apparent MW of 45 kDa by photoaffinity labeling. Although intracellular modulation of adenylate cyclase, guanylate cyclase and phospholipase C was not detected after application of neurotensin or levocabastine on NTRL-transfected cells, this receptor was able to internalize iodinated neurotensin. The internalization process was followed by recycling of receptors to the cell membrane. By contrast, no recycling was observed with the high affinity neurotensin receptor (NTRH). The differential intracellular routing of NTRH and NTRL after internalization is most probably the consequence of their divergent carboxy-terminal sequences.

Identification in the rat neurotensin receptor of amino-acid residues critical for the binding of neurotensin.

In order to identify charged amino-acid residues of the cloned rat brain neurotensin (NT) receptor (NTR) that are critical for NT binding, we performed site-directed mutagenesis on the cDNA encoding this protein, followed by transient expression into mammalian COS-7 cells and in Xenopus laevis oocytes. Point substitutions of charged residues in the N-terminal part and in the 2nd and 3rd extracellular loop of the receptor either did not affect (125)I-Tyr3-NT binding or resulted in a decrease in binding affinity by a factor of 2-3. Mutations of amino acids Asp113 in the second transmembrane domain (TM) and of Arg149 or Asp150 in TM III yielded receptors that bound NT as efficiently as the native receptor. By contrast, replacement of the Asp139 residue in the 1st extracellular loop, or of Arg143 or Arg327-Arg328 residues at the top of TM III and in TM VI, respectively, completely abolished ligand binding. Confocal and EM immunocytochemical studies of the expression of these affected receptors, tagged with the C-terminal sequence of the vesicular stomatitis virus glycoprotein (VSV-G), indicated that this loss of binding was not due to altered receptor expression or to their improper insertion into the plasma membrane. When these mutated forms of neurotensin receptor were expressed into Xenopus oocytes, Asp139-Gly- and Arg143-Gly-modified receptors remained functional in spite of a lowered response to NT whereas the Arg327-Arg328 mutant form was totally insensitive to NT at concentrations up to 10 microM. In the case of the Arg327-Arg328 mutation, the observed insensibility to NT could be the result of a drastic conformational alteration of this mutant protein. By contrast, it would appear that Asp139 and Arg143 residues located in the first extracellular loop of the receptor may be directly involved in the interaction of the receptor with neurotensin.

[3H]SR 48692, the first nonpeptide neurotensin antagonist radioligand: characterization of binding properties and evidence for distinct agonist and antagonist binding domains on the rat neurotensin receptor.

The binding of [3H]SR 48692, a new potent and specific nonpeptide neurotensin (NT) receptor antagonist, was characterized in membranes from mouse fibroblast LTK- cells stably transfected with the G protein-coupled rat NT receptor. The binding of [3H]SR 48692 was specific, time dependent, reversible, and saturable. Scatchard analysis of saturation experiments indicated that [3H]SR 48692 bound to a single population of sites, with a Kd of 3.4 nM and a Bmax value that was 30-40% greater than that observed in saturation experiments with [125I]NT. Two SR 48692-related enantiomers, SR 48527 and SR 49711, were 10 and 1000 times less potent, respectively, than unlabeled SR 48692 in inhibiting [3H]SR 48692. Unlabeled NT inhibited [3H]SR 48692 binding in a complex manner that was best analyzed with a three-site model, with high (Ki = 0.22 nM) and low (Ki = 57 nM) affinity NT binding sites and a site insensitive to unlabeled NT (up to 10 microM), which represented 60, 20, and 20%, respectively, of the total number of [3h]SR 48692 binding sites. Digitonin (10 micrograms/ml) markedly reduced the proportion of NT-insensitive sites without affecting [3H]SR 48692 binding. Na+ and guanosine-5'-(gamma-thio)triphosphate differentially modulated [3H]SR 48692 and [125I]NT binding and inverted the proportions of the high and low affinity NT binding sites. A mutant rat NT receptor that contained a deletion in a region (amino acids 45-60) of the amino-terminal extracellular domain near the first transmembrane helix and was expressed in COS M6 cells retained the same affinity for [3H]SR 48692 and the same stereoselectivity for SR 48527 and SR 49711 as the wild-type receptor. In contrast, it bound NT with 3000-fold lower potency. In conclusion, the data indicate that [3H]SR 48692 represents a new, potent, nonpeptide antagonist radioligand of the NT receptor that differentiates between agonist- and antagonist-receptor interactions. Furthermore, the data demonstrate that the peptide agonist and the nonpeptide antagonist bind to distinct regions of the NT receptor.

Thr-422 and Tyr-424 residues in the carboxyl terminus are critical for the internalization of the rat neurotensin receptor.

In order to identify the amino acid sequences responsible for the internalization of the cloned rat brain neurotensin receptor, we carried out site-directed mutagenesis of the cDNA encoding the receptor followed by expression of the receptor into mammalian COS 7 cells. In cells transfected with the full-length neurotensin receptor, 56% of iodinated neurotensin specifically bound to the cells after 60 min of incubation at 37 degrees C was internalized. Deletions made in the third intracellular loop did not affect receptor internalization. By contrast, internalization was reduced to 5% of total in cells in which almost all the carboxyl-terminal tail of the receptor had been deleted (R392stop). In order to determine which part of the tail was responsible for this effect, several Ser and Thr residues were deleted in the carboxyl cytoplasmic sequence of the receptor. Almost all of these receptors were internalized as efficiently as the wild type. Only the form of the neurotensin receptor truncated at Glu-421 (deletion of the last three residues, TLY) produced a significant decrease in the amount of ligand internalized. Finally, point mutations of Thr-422 and Tyr-424 residues to Gly led to an almost complete loss of ligand internalization demonstrating the involvement of these 2 residues in the internalization process. Replacement of the last three amino acids by the cytoplasmic endocytosis signal of the vesicular stomatitis virus did not restore the efficiency of neurotensin receptor internalization. These biochemical results were confirmed by confocal microscopic analysis. Cell transfected with the wild type receptor showed a temperature-dependent intracellular accumulation of a fluorescent analog of neurotensin, whereas cells transfected with a receptor truncated at the carboxyl terminus showed a clustering of the fluorescent peptide at the cell surface.

Stable expression of the cloned rat brain neurotensin receptor into fibroblasts: binding properties, photoaffinity labeling, transduction mechanisms, and internalization.

The study of the pharmacological, biochemical, and transduction properties of the cloned rat brain neurotensin receptor was carried out in thymidine kinase mutant fibroblasts stably transfected with the receptor cDNA. The interaction of neurotensin with transfected fibroblasts leads to a concentration-dependent stimulation of phosphatidylinositol hydrolysis and intracellular calcium. These effects are totally inhibited by the nonpeptide neurotensin antagonist SR48692. By contrast, this receptor remains unable to modulate intracellular levels of cyclic nucleotides. The transfected neurotensin receptor can be solubilized in an active form by digitonin with an identical pharmacological profile, whereas the detergent 3-[(3-cholamidopropyl) dimethylammonio]-1-propane-sulfonic acid is unable to solubilize the binding activity. The binding of iodinated neurotensin to transfected fibroblasts bearing the cloned receptor remains partly un-dissociated even after an acid washing step, indicating that the transfected neurotensin receptor retains the capacity to be internalized according to a temperature-dependent mechanism. Indeed, the sequestration of the neurotensin-receptor complex can be blocked by phenylarsine oxide. Finally, photoaffinity labeling experiments reveal that the cloned rat brain neurotensin receptor is expressed under two forms with molecular masses of 50 and 60 kDa. Labeling and internalization of these two proteins are totally blocked by the neurotensin antagonist SR48692.

Implication of various forms of neurotensin receptors in the mechanism of internalization of neurotensin in cerebral neurons.

This report describes the kinetics and molecular aspects of neurotensin internalization in neurons. Incubation of alpha-125I-Bolton-Hunter neurotensin-(2-13) with cortical neurons at 37 degrees C was followed by a rapid internalization of the peptide bound to its receptors. This process was completed within 20 min and was inhibited either irreversibly by the general endocytosis inhibitor phenylarsine oxide or reversibly by incubation at low temperature (0-4 degrees C). The discrepancy of maximal binding capacities observed in the presence (150 fmol/mg of protein) or in the absence (250 fmol/mg of protein) of internalization inhibitors could be attributed to the appearance of a new pool of neurotensin binding sites on the cell surface rather than a recycling of internalized receptors. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis in denaturing conditions revealed that three different protein subunits of 50, 60, and 100 kDa were covalently labeled at 37 degrees C with a radioactive photoreactive analogue of neurotensin. The 50- and 60-kDa subunits remained labeled when internalization was blocked, whereas the specific labeling of the 100-kDa protein was abolished. These results suggest that neurotensin-induced internalization of the 50- and 60-kDa subunits initially present on the cell surface triggers insertion of the 100-kDa subunit into the membrane from an intracellular compartment. Subcellular fractionation experiments have shown that, in the absence of neurotensin, the 100-kDa protein is located in an intracellular vesicular fraction of neurons.

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.

Binding and internalization of iodinated neurotensin in neuronal cultures from embryonic mouse brain.

The binding and internalization of labeled neurotensin were studied by means of biochemical and light microscopic radioautography techniques in primary cultures of neurons from whole cerebral hemispheres of mouse embryos. Saturable, high affinity neurotensin binding was detected 5-7 days postplating in cells incubated with 0.1 nM 125I-Tyr3-neurotensin at 37 degrees C or 10 degrees C. The binding capacity at equilibrium was 3 times higher at 37 degrees C than at 10 degrees C. Moreover, whereas virtually all the radioactivity bound at 10 degrees C was membrane-bound (i.e. was readily washable by a hypertonic, high pH, NaCl solution), more than 70% of the radioactivity bound at 37 degrees C was intracellular (i.e. resisted the same treatment). Light microscopic radioautograms of whole cells revealed that approximately 16% of neurons were labeled with 125I-Tyr3-neurotensin at either 37 degrees C or 10 degrees C. The labeling was observed over cell bodies and processes, and the density of silver grains associated with perikarya, as compared to processes, was proportionally higher at 37 degrees C than at 10 degrees C. Semi-thin (1 micron thick) sections through cells incubated at 37 degrees C confirmed that a major fraction of the radioactivity was intracellular and showed that it was mainly confined to the cytoplasm. These results indicate that 125I-Tyr3-neurotensin binds to a distinct subset of primary cultured neurons and that a large proportion of the bound radioactivity undergoes rapid internalization in a temperature-dependent manner. It is proposed that this internalization is ligand-induced and that it may play a role in the modulation of central neurotensin receptor levels.

Colocalization of neurotensin receptors and of the neurotensin-degrading enzyme endopeptidase 24-16 in primary cultures of neurons.

This paper compares the localization of neurotensin receptors and of endopeptidase 24-16, a peptidase likely involved in the inactivation of neurotensin in primary cultures of neurons. Neurotensin binding sites were radiolabeled with 125I-Tyr3-neurotensin, whereas endopeptidase 24-16 was stained by immunohistochemical techniques using a monospecific polyclonal antibody. Endopeptidase 24-16 is present in 80-85% of the nondifferentiated neurons. The proportion of immunoreactive neurons decreased during maturation to reach 35-40% after 4-8 d of culture. By contrast, neurotensin receptors were not detectable in nondifferentiated cells and appear during maturation. Specific 125I-Tyr3-neurotensin labeling is maximal after 4 d of culture and is located on about 10% of differentiated neurons. Double-labeling experiments show that about 90% of cortical, hypothalamic, and mesencephalic neurons bearing the neurotensin receptor also contained endopeptidase 24-16, supporting the hypothesis that one of the functions of endopeptidase 24-16 is the physiological inactivation of neurotensin. However, the presence of endopeptidase 24-16 on numerous neurons that do not contain neurotensin receptors also suggests that the enzyme could be involved in the degradation and/or maturation of other neuropeptides.

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.