Behavioral and anatomical abnormalities in Mecp2 mutant mice: a model for Rett syndrome.

Over 90% of Rett syndrome (RTT) cases have a mutation in the X-linked gene encoding methyl CpG binding-protein 2 (MeCP2). A mouse model that reprises clinical manifestations of the disease would be valuable for examining disease mechanisms. Here, we characterize physical and behavioral measures, as well as brain region volumes in young adult mice that have mutations in mouse methyl CpG binding-protein 2 gene (Mecp2) to serve as a baseline for other studies. Hemizygous males, which produce no functional protein, exhibit hypoactivity and abnormalities in locomotion, stereotypies, and anxiety reminiscent of the clinical condition. The mutant males also exhibit cognitive deficits in fear conditioning and object recognition relative to wildtypes. Volumetric analyses of male brains revealed a 25% reduction in whole brain volume in mutants relative to wildtypes; regional differences were also apparent. Mutants had decreased volumes in three specific brain regions: the amygdala (39%), hippocampus (21%), and striatum (29%). Heterozygous females, which produce varying amounts of functional protein, displayed a less severe behavioral phenotype. The mutant females exhibit abnormalities in locomotion, anxiety measures, and cognitive deficits in object recognition in an open field. This study provides the first evidence that the abnormal motor and cognitive behavioral phenotype in Mecp2 mice is consistent with specific volume reductions in brain regions associated with these behaviors, and shows how these data parallel the human condition. The Mecp2 mutant mice provide a very good model in which to examine molecular and behavioral mechanisms, as well as potential therapeutic interventions in RTT.

Selective immunolesions of cholinergic neurons in mice: effects on neuroanatomy, neurochemistry, and behavior.

The ability to selectively lesion mouse basal forebrain cholinergic neurons would permit experimental examination of interactions between cholinergic functional loss and genetic factors associated with neurodegenerative disease. We developed a selective toxin for mouse basal forebrain cholinergic neurons by conjugating saporin (SAP), a ribosome-inactivating protein, to a rat monoclonal antibody against the mouse p75 nerve growth factor (NGF) receptor (anti-murine-p75). The toxin proved effective and selective in vitro and in vivo. Intracerebroventricular injections of anti-murine-p75-SAP produced a dose-dependent loss of choline acetyltransferase (ChAT) activity in the hippocampus and neocortex without affecting glutamic acid decarboxylase (GAD) activity. Hippocampal ChAT depletions induced by the immunotoxin were consistently greater than neocortical depletions. Immunohistochemical analysis revealed a dose-dependent loss of cholinergic neurons in the medial septum (MS) but no marked loss of cholinergic neurons in the nucleus basalis magnocellularis after intracerebroventricular injection of the toxin. No loss of noncholinergic neurons in the MS was apparent, nor could we detect loss of noncholinergic cerebellar Purkinje cells, which also express p75. Behavioral analysis suggested a spatial learning deficit in anti-murine-p75-SAP-lesioned mice, based on a correlation between a loss of hippocampal ChAT activity and impairment in Morris water maze performance. Our results indicate that we have developed a specific cholinergic immunotoxin for mice. They also suggest possible functional differences in the mouse and rat cholinergic systems, which may be of particular significance in attempts to develop animal models of human diseases, such as Alzheimer's disease, which are associated with impaired cholinergic function.

COP-1, a member of the CCN family, is a heparin-induced growth arrest specific gene in vascular smooth muscle cells.

Vascular smooth muscle cell (VSMC) hyperplasia is responsible for the failure of 15-30% of vascular surgical procedures such as coronary artery bypass grafts and angioplasties. We and others have shown that heparin suppresses VSMC proliferation in vivo and in cell culture. We hypothesize that heparin inhibits VSMC proliferation by binding to cell surface receptors, resulting in selective modulation of mitogenic signal transduction pathways and altered transcription of a specific subset of growth regulatory genes. To test this idea, we used subtractive hybridization to identify differentially expressed mRNAs in heparin-treated and untreated VSMC. We identified a heparin induced mRNA identical to Cop-1, a member of the CCN family of proteins which are secreted, cysteine-rich modular proteins involved in growth regulation and migration. Cop-1 from smooth muscle cells appears to have a different expression pattern and possibly different functions than Cop-1 from other cells. Cop-1 mRNA is expressed at high levels in quiescent VSMC and at low levels in proliferating VSMC, an expression pattern highly characteristic of growth arrest specific genes. Cop-1 mRNA is expressed at high levels in heparin treated VSMC and COP-1 protein is secreted into culture medium. In tissues, Cop-1 expression is observed in the uninjured rat aorta suggesting a possible role for Cop-1 in vivo. We found PDGF, but not EGF, inhibits the expression of Cop-1 in VSMC. Neither TGF-beta nor interferon-beta, two inhibitors of VSMC proliferation, were able to induce Cop-1 expression. In addition, heparin does not induce Cop-1 mRNA in endothelial cells and VSMC resistant to the antiproliferative effect of heparin. Conditioned medium from cells over-expressing COP-1 protein inhibits VSMC proliferation in culture. Together, our data indicate that COP-1 may play a role in the antiproliferative mechanism of action of heparin.

Cholinergic basal forebrain is critical for social transmission of food preferences.

Studies using selective lesions of basal forebrain cholinergic neurons suggest that these neurons play a role in attentional processing, but not learning and memory. However, the tests of learning and memory used thus far have been restricted largely to spatial tasks. In the present study, we examined whether the cholinergic basal forebrain plays a role in a form of nonspatial associative memory, the social transmission of food preferences. Sham-operated control rats were compared to rats with 192 IgG-saporin lesions of the medial septum/diagonal band cholinergic projections to hippocampus or nucleus basalis magnocellularis/substantia innominata cholinergic projections to neocortex. Both lesions impaired 24-h retention of a learned social food preference relative to controls, despite performance on an immediate retention trial that was indistinguishable from controls. Moreover, 24-h retention of the socially learned food preference correlated strongly with cholinergic enzymatic activity in the neocortex, but not in the hippocampus. Immunohistochemical data confirmed significant and selective lesion-induced cholinergic depletions in the intended brain regions. These data provide evidence that the cholinergic basal forebrain, particularly the cholinergic projection to neocortex, is involved in the formation and/or retrieval of social memories related to food preference, and suggest a role for cortical acetylcholine in consolidation of associative memory processes.

Synthesis and characterization of highly sensitive heparin probes for detection of heparin-binding proteins.

Three labeled heparin species were synthesized as probes for heparin-binding protein detection. Heparin conjugated with 5([4,6-dichlorotriazin-2-yl]amino)fluorescein can be iodinated to a high specific activity. This probe specifically detected 40 pg histone on a dot blot without affinity purification. Heparin biotinylated on its naturally occurring primary amino groups also detected known heparin-binding proteins in a specific manner. This probe detected lower amounts of collagen I and basic fibroblast growth factor on nitrocellulose membranes than did the iodinated probe, with comparable detection times. To create more attachment sites for biotin, we covalently attached amino groups to the hydroxyl groups of heparin using 3-bromopropylamine hydrobromide. After biotinylation, the amino-rich probe detected heparin-binding proteins at the same or higher sensitivity as the biotinylated native heparin probe, using 100-fold less probe and much shorter detection times. This method of labeling is generally applicable to other polysaccharides, and would be useful when the amount of ligand is limited. We show that these three probes detect essentially the same spectrum of proteins in detergent extract of smooth muscle cell plasma membrane, and expect them to be useful probes for detection of cell-surface heparin receptors.

The proregion of cathepsin L is required for proper folding, stability, and ER exit.

To investigate the role of the proregion in the biosynthesis and trafficking of mouse cathepsin L, cathepsin L cDNAs encoding proteins with altered proregions were constructed and their expression in COS cells was examined. As in transformed cells, normal mouse cathepsin L was secreted by COS cells. In contrast, two altered proregion cathepsin L proteins, one in which the proregion was deleted and a second in which the proregion was replaced with that of a homologous protein (aleurain), were retained within the cell and degraded over a period of 2-6 h. Immunofluorescence localization and the lack of effect of NH4Cl and brefeldin A on the turnover of the altered cathepsin L proteins indicated that their degradation occurred in the endoplasmic reticulum (ER). By using brefeldin A to induce colocalization of the UDPGlcNAc: lysosomal enzyme N-acetylglucosamine-1-phosphotransferase with the cathepsin L proteins in the ER, it was shown that the altered proteins were not susceptible to mannose phosphorylation as they exist in the ER. Trypsin sensitivity assays indicated that altered proregion proteins synthesized in COS cells or in vitro are misfolded. Taken together, these results indicate that the proregion plays an essential role in proper folding of cathepsin L. ER retention, decreased stability, and lack of mannose phosphorylation of the altered proteins are most likely secondary effects resulting from improper folding.

Comparison of cathepsin L synthesized by normal and transformed cells at the gene, message, protein, and oligosaccharide levels.

The major excreted protein of transformed mouse fibroblasts (MEP) has recently been identified as the lysosomal cysteine protease, cathepsin L. The synthesis and intracellular trafficking of this protein in mouse fibroblasts are regulated by growth factors and malignant transformation. To further define the basis for this regulation, a cDNA encoding MEP/cathepsin L was isolated from a mouse liver cDNA library and used to compare cathepsin L of normal and Kirsten sarcoma virus-transformed NIH 3T3 fibroblasts. Although cathepsin L message levels were elevated 20-fold in the transformed fibroblasts, normal and transformed cells displayed similar cathepsin L genomic DNA digest patterns and gene copy numbers, and cathepsin L mRNA sequences appeared identical by RNase protection analysis. These findings indicate that (i) cathepsin L is synthesized from the same gene in normal and transformed cells and (ii) cathepsin L polypeptides made by these cells are translated with the same primary sequence. Cathepsin L polypeptides synthesized by quiescent, growing, and transformed cells displayed similar isoelectric focusing patterns, suggesting similar post-translational modification. Site-directed mutagenesis of the mouse liver cDNA and expression in COS monkey cells was used to examine the glycosylation of mouse cathepsin L. The results indicated that only one of the two potential N-linked glycosylation sites (the one at Asn221) is glycosylated. Analysis by ion exchange chromatography on QAE-Sephadex, and affinity chromatography on mannose 6-phosphate receptor-Affi-Gel 10, indicated that the cathepsin L oligosaccharide was phosphorylated similarly in normal and transformed cells. Although several phosphorylated oligosaccharide species were observed, the major species contained two phosphomonoester moieties and bound efficiently to the receptor. These findings suggest that cathepsin L made by normal and transformed mouse fibroblasts are identical and substantiate the hypothesis that trafficking of cathepsin L in these cells is regulated by growth-induced changes in the lysosomal protein transport system.

Immunological distinction of mycobacterial beta-lactamases.

Beta-lactamase-containing fractions have been isolated from Myobacterium tuberculosis strain R(1)R(v) and M. smegmatis strains NCTC 8158 and T-64. Antisera raised in rabbits to these fractions demonstrated no cross-reactivity with a commercial Bacillus cereus beta-lactamase. Antiserum to either strain of M. smegmatis revealed no cross-reactivity with M. tuberculosis R(1)R(v), one specificity in common with the other M. smegmatis strain, and two specificities unique to the immunizing fractions. Further characterization of the common specificity was not possible, but beta-lactamase activity was related to the strain-specific precipitin lines in each case. These studies demonstrate the feasibility of isolating beta-lactamase enzyme fractions that may prove useful in the classification and diagnosis of mycobacteria.