Differential expression of mRNA coding for the alpha-2-macroglobulin family and the LRP receptor system in C57BL/6J and C3H/HeJ male mice.

Expression of mouse A2M (MAM), murinoglobulin (MUG), the A2M receptor or LDL-Receptor related protein (A2MR/LRP) and the Receptor Associated Protein (RAP) were measured by northern blotting of mRNA isolated from liver, heart and peritoneal macrophages from C3H/HeJ and C57BL/6J (B6) mice. Marked differences between males of the two mouse strains were observed for MAM and MUG mRNA levels in liver, which were reflected in plasma levels of both proteinase inhibitors, as confirmed by immune-electrophoresis. C3H/HeJ mice had higher levels of the MAM and MUG mRNA and their corresponding plasma proteins than B6 mice. B6 mice expressed higher levels of LRP mRNA relative to C3H/HeJ mice but had lower levels of RAP mRNA. LRP receptor activity, assayed by fluoresceinated-A2M binding, was higher in B6 cells. The present data contribute to the knowledge of genetic background characteristics among male mouse of these two strains, which can take part in many biological events such as lipid metabolism, inflammation and immune response to different infectious agents.

The zebrafish space cadet gene controls axonal pathfinding of neurons that modulate fast turning movements.

All vertebrates depend on neural circuits to produce propulsive movements; however, the contribution of individual neural cell types to control such movements are not well understood. We report that zebrafish space cadet mutant larvae fail to initiate fast turning movements properly, and we show that this motor phenotype correlates with axonal defects in a small population of commissural hindbrain neurons, which we identify as spiral fiber neurons. Moreover, we demonstrate that severing spiral fiber axons produces space cadet-like locomotor defects, thereby providing compelling evidence that the space cadet gene plays an essential role in integrating these neurons into the circuitry that modulates fast turning movements. Finally, we show that axonal defects are restricted to a small set of commissural trajectories, including retinal ganglion cell axons and spiral fiber axons, and that the space cadet gene functions in axonal pathfinding. Together, our results provide a rare example in vertebrates of an individual neuronal cell type that contributes to the expression of a defined motor behavior. Movies available on-line

Trypanosoma cruzi: acute infection affects expression of alpha-2-macroglobulin and A2MR/LRP receptor differently in C3H and C57BL/6 mice.

Although a complete cellular and humoral immune response is elicited in Chagas' disease, recent data suggest that other natural elements of innate immunity may also contribute to the initial host primary defense. alpha-Macroglobulins are a family of plasma proteinase inhibitors that are acute-phase reactants in Trypanosoma cruzi-infected mice and humans. Mice contain a tetrameric alpha-2-macroglobulin (MAM) and a monomeric murinoglobulin (MUG). Heterogeneity in their reactions was observed in murine T. cruzi-infected plasma A2M levels despite an overall increase. In addition, up-regulation of the A2M receptor (A2MR/LRP) was observed in peritoneal macrophages during T. cruzi infection. Here, we show that during T. cruzi infection (Y strain), the MAM and MUG hepatic mRNA levels and the corresponding plasma protein levels were up-regulated in C3H and C57BL/6 (B6) mice, but with different kinetics. On the contrary, A2MR/LRP mRNA levels increased in acutely infected C3H mice, but decreased in B6 mice, in both liver and heart. Immunocytochemistry of infected B6 heart cryosections confirmed a less intense endothelium labeling by the fluoresceinated ligand for A2MR/LRP. On the other hand, infected B6 spleen cells displayed higher F-A2M-FITC binding and MAC1 expression, confirming higher A2MR/LRP expression in macrophages. In uninfected mice, as well as after T. cruzi infection, higher A2M plasma levels were measured in C3H mice than in B6 mice. The lower tissue T. cruzi parasitism found in C3H-infected mice could reflect an inhibitory effect of A2M on parasite invasion. Our present data further contribute to clarifying aspects of the role of A2MR/LRP in a model of acute Chagas' disease in different mouse strains.

Lipoprotein receptor-related protein in brain and in cultured neurons of mice deficient in receptor-associated protein and transgenic for apolipoprotein E4 or amyloid precursor protein.

The role of the receptor-associated protein in controlling the expression of the low-density lipoprotein receptor-related protein was analysed in brain and in cultured neurons of receptor-associated protein - / - mice. In addition, the effect of two important ligands of lipoprotein receptor-related protein in brain, i.e. apolipoprotein E and amyloid precursor protein, was examined by crossing the receptor-associated protein - / - mice with transgenic mice overexpressing these proteins specifically in neurons. The immunohistochemical localization of lipoprotein receptor-related protein and receptor-associated protein in wild-type mouse brain was demonstrated to be congruent over all structures, including the cortex and hippocampus. In primary hippocampal neurons, lipoprotein receptor-related protein was distributed somatodendritically and receptor-associated protein was concentrated perinuclearly. In hippocampal neurons from receptor-associated protein - / - mice, lipoprotein receptor-related protein was redistributed over the cell body at the expense of the dendrites. In the absence of receptor-associated protein, maturation of lipoprotein receptor-related protein is slow, resulting in accumulation of the uncleaved 600,000 mol. wt precursor. Neither the added expression of apolipoprotein E4 nor that of amyloid precursor protein in cultured neurons influenced the maturation of lipoprotein receptor-related protein, in either the presence or absence of receptor-associated protein. This result shows that receptor-associated protein is not needed to allow co-expression of lipoprotein receptor-related protein with these ligands in neurons. Furthermore, the typical ramified neuronal morphology of cultured primary neurons and the histology and architecture of the brain were normal in receptor-associated protein - / - mice and in all of the double transgenic mice. Finally, we demonstrated that the survival of receptor-associated protein - /- hippocampal neurons was normal and unaffected by the genotype of the glial feeder cells, whether they were derived from wild-type mice or from mice deficient in receptor-associated protein or apolipoprotein E. These results show that, despite the dramatic effect on maturation and cellular localization of lipoprotein receptor-related protein, the absence of receptor-associated protein did not result in any notable physiological, functional or morphological effects.

Premature death in transgenic mice that overexpress a mutant amyloid precursor protein is preceded by severe neurodegeneration and apoptosis.

A mutant amyloid precursor protein (APP/RK) designed to interfere with processing by alpha-secretase caused a severe phenotype in transgenic mice, including behavioural abnormalities, i.e. neophobia, aggression, hypersensitivity to kainic acid, hyposensitivity to N-methyl-D-aspartate, and premature death [Moechars D. et al. (1996) Eur. molec. Biol. Org. J. 15, 1265-1274]. We now demonstrated that the APP/RK transgene did not disturb the expression of several other genes, i.e. endogenous amyloid precursor protein and amyloid precursor protein-like proteins, members of the low density lipoprotein receptor lipoprotein receptor family and several of their ligands, including apolipoprotein E, but expression of alpha-2-macroglobulin was never detected. Neither amyloid deposits nor neurofibrillary tangles were detected in the brain of APP/RK transgenic mice, even when 15-months-old. The tendency for seizures and hyposensitivity for N-methyl-D-aspartate was not due to or reflected in the distribution of the three major types of glutamate receptors. The major and consistent finding in transgenic APP/RK mice that died prematurely was extensive neurodegeneration and apoptosis, mainly in hippocampus and cortex, and accompanied by astrocytosis throughout the brain. Reduced synaptic density and dendritic damage was only observed in three transgenic mice that were killed shortly after positive observation of seizures. In addition, the distribution of cathepsin D and ubiquitin was abnormal in these mice.

Early phenotypic changes in transgenic mice that overexpress different mutants of amyloid precursor protein in brain.

Transgenic mice overexpressing different forms of amyloid precursor protein (APP), i.e. wild type or clinical mutants, displayed an essentially comparable early phenotype in terms of behavior, differential glutamatergic responses, deficits in maintenance of long term potentiation, and premature death. The cognitive impairment, demonstrated in F1 hybrids of the different APP transgenic lines, was significantly different from nontransgenic littermates as early as 3 months of age. Biochemical analysis of secreted and membrane-bound APP, C-terminal "stubs," and Abeta(40) and Abeta(42) peptides in brain indicated that no single intermediate can be responsible for the complex of phenotypic dysfunctions. As expected, the Abeta(42) levels were most prominent in APP/London transgenic mice and correlated directly with the formation of amyloid plaques in older mice of this line. Plaques were associated with immunoreactivity for hyperphosphorylated tau, eventually signaling some form of tau pathology. In conclusion, the different APP transgenic mouse lines studied display cognitive deficits and phenotypic traits early in life that dissociated in time from the formation of amyloid plaques and will be good models for both early and late neuropathological and clinical aspects of Alzheimer's disease.

Transgenic mice expressing an alpha-secretion mutant of the amyloid precursor protein in the brain develop a progressive CNS disorder.

Expression of alpha-secretion mutant APP/RK in mouse brain results in a progressive disorganization of the central nervous system, exemplified by behavioral deficits, premature death and neuropathology. Here we report on the progressive nature of this CNS disorder as indicated by the age dependency of the neophobic reaction in the open-field test. The earlier reported NMDA hypo-sensitivity in the transgenic APP/RK mice is likely to represent a subtle functional disturbance, since no changes in NMDA receptor density or distribution could be detected. None of the typical neuropathological hallmarks of Alzheimer's Disease, i.e. amyloid deposits and neurofibrillary tangles are detected in the brain of these transgenic mice. Nevertheless, the progressive CNS disorder elicited in the transgenic APP/RK mice recapitulates certain features and symptoms of patients with Alzheimer's disease as discussed.

Expression in brain of amyloid precursor protein mutated in the alpha-secretase site causes disturbed behavior, neuronal degeneration and premature death in transgenic mice.

A double mutation in the alpha-secretase site in the betaA4 region of mouse amyloid precursor protein (APP) reduced its secretion from COS cells, polarized MDCK cells and rat primary neurons. Expression of this mutant in the brain of mice, using the neuron-specific elements of the mouse Thy-1 gene promoter, resulted in transgenic mice that became progressively hyperactive, displayed seizures and died prematurely. In three different transgenic lines the severity of the phenotype was related directly to the expression levels of the transgene, estimated by both mRNA and protein levels. In addition, homozygous mice derived from each transgenic strain showed more severe symptoms which also occurred earlier in life than in heterozygotes. The observed symptoms were, however, not essentially different in the different lines. Increased aggressiveness, disturbed responses to kainic acid and N-methyl-D-aspartate, neophobia and deficiency in exploratory behavior were demonstrated in these mice. In the brain, the observed neuropathological changes included necrosis, apoptosis and astrogliosis in the hippocampus, cortex and other areas. The data demonstrate that incomplete or incorrect alpha-secretase processing of APP results in severe neurotoxicity and that this effect is expressed in a dominant manner.

Expression of mouse alpha-macroglobulins, lipoprotein receptor-related protein, LDL receptor, apolipoprotein E, and lipoprotein lipase in pregnancy.

The expression of the proteinase inhibitors of the alpha-macroglobulin family and of their clearance receptor was analyzed in the mouse during pregnancy, embryonal development, and adolescence. In total we studied seven partners of a complicated network of interactions in proteolysis and lipid metabolism:alpha-2-macroglobulin, murinoglobulin, the alpha-2-macroglobulin receptor/lipoprotein receptor related protein, the murine equivalent of the receptor associated protein or the 44 kDa heparin binding protein, the low density lipoprotein receptor, apolipoprotein E, and lipoprotein lipase. The data demonstrate that: i) the regulation of expression of mouse tetrameric alpha-2-macroglobulin results in very constant levels, similar to alpha-2-macroglobulin in humans; ii) single chain murinoglobulin, not alpha-2-macroglobulin, is subject to regulation of expression during pregnancy, around birth, and in adolescence; iii) an important role seems implicated for the alpha-2-macroglobulin receptor in placental lipid metabolism, probably making it the most important lipoprotein receptor to supply the fetus; iv) the massive increase in apolipoprotein E synthesis in uterus and placenta accentuate the changed lipid metabolism during pregnancy to an apolipoprotein E-based uptake by the alpha-2-macroglobulin receptor/lipoprotein receptor related protein; v) the increased expression of lipoprotein lipase underlines its role in the generation of free fatty acids in uterus and placenta as another mechanism of supply, next to receptor mediated endocytosis of lipoproteins.

Targeted inactivation of the mouse alpha 2-macroglobulin gene.

The mouse alpha 2-macroglobulin gene was inactivated in embryonic stem cells by homologous recombination. Liver alpha 2-macroglobulin mRNA and plasma protein was absent in homozygotes and reduced to 50% in heterozygotes. alpha 2-Macroglobulin-deficient mice were viable and produced normally sized litters with normal sex ratio over 3 generations. Characterization of adult homozygotes included diets with different fat content, treatments with endotoxin, bleomycin, carbon tetrachloride, and ethionine to test for immune system, lung, liver, and pancreas toxicity, respectively. Knock-out mice were more resistant to endotoxin but more sensitive to a choline-free diet supplemented with ethionine. Regulation of murinoglobulin mRNA expression during pregnancy was analyzed as a possible back-up mechanism for the deficiency in alpha 2-macroglobulin. In addition, expression of mRNA was studied, coding for alpha 2-macroglobulin receptor/lipoprotein receptor-related protein, low density lipoprotein receptor, and very low density lipoprotein receptor and for some common ligands, i.e. apolipoprotein E, lipoprotein lipase, and the 44-kDa heparin binding protein. Their differential regulation in the knock-out mice relative to C57B1 mice was evident and is discussed. The impressive 15-fold increase in maternal liver murinoglobulin mRNA at partum in the knock-out mice indicated increased consumption, compared to only 4-fold in normal mice. Thus, murinoglobulin appears as the major proteinase inhibitor around partum, obviously solicited to a much greater extend in alpha 2-macroglobulin-deficient mice.

Expression in mouse embryos and in adult mouse brain of three members of the amyloid precursor protein family, of the alpha-2-macroglobulin receptor/low density lipoprotein receptor-related protein and of its ligands apolipoprotein E, lipoprotein lipase, alpha-2-macroglobulin and the 40,000 molecular weight receptor-associated protein.

We have analysed by northern blotting and by in situ hybridization the expression patterns of eight different genes during the second half of mouse embryonic development and in adult mouse brain: we compared the messenger RNA levels of amyloid precursor protein and of the two amyloid precursor protein-like proteins 1 and 2 and we have analysed expression of apolipoprotein E and of its main receptor in brain, the alpha-2-macroglobulin/low density lipoprotein receptor-related protein and three other ligands: the proteinase inhibitor alpha-2-macroglobulin, the modifying enzyme lipoprotein lipase and the 44,000 molecular weight heparin binding protein, a ligand of unknown function. During embryogenesis the temporal expression pattern differs considerably for the three members of the amyloid precursor proteins. Total embryo messenger RNA levels of amyloid precursor protein and amyloid precursor protein-like protein 2 increased progressively, while amyloid precursor protein-like protein 1 messenger RNA showed a burst of synthesis between days 10 and 13 post-coitum. Significantly, expression of the alpha-2-macroglobulin/low density lipoprotein receptor-related protein and of its associated protein, the 44,000 molecular weight heparin binding protein, exhibited their most important increase very similar to that of amyloid precursor protein-like protein 1, between days 10 and 13 post-coitum. Apolipoprotein E, lipoprotein lipase and alpha-2-macroglobulin messenger RNA levels in total embryos increased progressively, beginning most pronounced at days 13, 15 and 17, respectively. In mouse embryos, in situ hybridization established amyloid precursor protein, amyloid precursor protein-like protein 2 and alpha-2-macroglobulin/low density lipoprotein receptor-related protein messenger RNA to be expressed in most organs, with the notable exception of the liver, while expression of the other studied proteins was much more restricted. Among adult mouse tissues, the genes investigated were expressed very prominently in brain, except for lipoprotein lipase and for the complete absence of alpha-2-macroglobulin messenger RNA. In adult mouse brain, the cortex and hippocampus exhibited strong signals for most genes analysed. Exceptions are lipoprotein lipase and apolipoprotein E messenger RNAs, and the absent alpha-2-macroglobulin messenger RNA. Several interesting features, similarities as well as differences, between brain tissue sections hybridized with probes for amyloid precursor protein, amyloid precursor protein-like proteins 1 and 2 and between alpha-2-macroglobulin/low density lipoprotein receptor-related protein and heparin binding protein-44 were observed and are described. The results are further discussed in view of the known or anticipated physiological functions of the proteins examined and of their possible role in the etiology of Alzheimer's disease.

Structure of the gene (LRP1) coding for the human alpha 2-macroglobulin receptor lipoprotein receptor-related protein.

The alpha 2-macroglobulin receptor or lipoprotein receptor-related protein (A2MR/LRP) is an amazingly large and multifunctional receptor. The active receptor protein is derived from a 600-kDa precursor, encoded by a 15-kb mRNA, cloned and sequenced in human, mouse, and chicken. We report here the cloning of the entire human gene (LRP1) coding for A2MR/LRP. The gene covered about 92 kb and a total of 89 exons were identified, varying in size from 65 bases (exon 86) to 925 bases (exon 89). The introns varied from 82 bases (intron 53) to about 8 kb (intron 6). In the introns, 3 complete and 4 partial Alu sequences were identified. In intron 44 a complex repetitive sequence posed a cloning problem since it was not retrieved from any genomic library screened. Interexon PCR from exon 43 to 45 yielded a fragment of 2.5 kb. Attempts to subclone this fragment yielded inserts ranging between 0.8 and 1.6 kb. Sequencing of 3 subclones with different-size inserts revealed a complex repetitive element with a different size in each subclone. In the mouse LRP gene this intron was much smaller, and no repetitive sequence was observed. In 18 unrelated individuals no difference in size was observed when analyzed by interexon PCR.

Identification of four genes coding for isoforms of murinoglobulin, the monomeric mouse alpha 2-macroglobulin: characterization of the exons coding for the bait region.

Murinoglobulins are the single chain members of the alpha 2-macroglobulin family of proteinase inhibitors in the mouse. DNA clones representing the genes coding for four different murinoglobulins were isolated from three independent mouse genomic DNA libraries. Sequence analysis demonstrated that in each gene two exons are coding for the bait region. This is the specific protein sequence in each alpha-macroglobulin, which is functionally important since it is extremely sensitive to cleavage by different proteinases. The molecular data established the existence of at least four different murinoglobulin genes. Three of these corresponded to the three cDNA clones previously identified. Sequencing of intron-exon boundaries and intron sizing allowed us to construct physical maps of the region from exon 15 to exon 25 (numbered in comparison to mouse alpha 2-macroglobulin) in each murinoglobulin gene. Southern blotting of genomic DNA from five different mouse strains confirmed this analysis and even suggested the possible existence of a fifth murinoglobulin gene. These data indicate that the mouse presents a genetic repertoire of the alpha 2-macroglobulin family much more complex than originally anticipated. The bait region exon sequences showed a considerably higher degree of divergence (72 to 88% sequence identity) than that of the flanking exon sequences coding for adjacent, structural domains of the murinoglobulin proteinase inhibitors (91 to 96%). Even more surprising was that adjacent intron sequences are conserved as faithfully as the nonbait region coding exons (90 to 96%). These data demonstrate a unique property of the bait region coding sequences, as they apparently are allowed to mutate considerably. This divergency must then confer divergent proteinase inhibitory properties to the resulting proteins.

Distribution of mRNA coding for alpha-2-macroglobulin, the murinoglobulins, the alpha-2-macroglobulin receptor and the alpha-2-macroglobulin receptor associated protein during mouse embryogenesis and in adult tissues.

The distribution of mRNA coding for the members of the wide-spectrum proteinase scavenging system of the alpha-2-macroglobulin family was examined in the mouse: Alpha-2-macroglobulin (MAM), the murinoglobulins (MUG), the alpha-2-macroglobulin receptor (alpha 2MR) and the receptor associated protein, the heparin binding protein-44 (alpha 2MRAP/HBP-44), a component of unknown function. The results demonstrate that MAM is expressed in the mouse embryo exclusively in the liver and not before day 13 of gestation. MUG mRNA was never detected during embryogenesis. On the other hand, both the alpha 2MR and the alpha 2MRAP/HBP-44 messages were present throughout all embryonal stages examined. The distribution of the alpha 2MR mRNA was widespread in most tissues, with stronger signals observed in developing mouse brain, in whisker follicles and in the perifollicular mesenchyme, in lung, liver, kidney, intestine and placenta. The alpha 2MRAP/HBP-44 mRNA was detected predominantly in brain, lung, liver, kidney and placenta. Interestingly, within each tissue the cellular distribution of the alpha 2MR and alpha 2MRAP/HBP-44 mRNA was quite different with the most remarkable extremes observed in kidney and in placenta. The implication of these observations for receptor expression and function are discussed. Northern analysis of adult tissues extended these observations: major signals for MAM and MUG were seen only in liver, while the expression of the alpha 2MR and the alpha 2MRAP/HBP-44 was widespread with highest levels of the 15-kb alpha 2MR mRNA in liver. Kidney was the most abundant source of alpha 2MRAP/HBP-44 mRNA with the 1.8- and 3.6-kb mRNAs, derived from the same gene by alternative mRNA splicing, present in nearly constant ratios in most tissues, except in testis. The notable absence of expression of MAM in the first half of gestation indicates that during this period the receptor is scavenging for proteinases complexed to MAM derived from the maternal circulation or is being used for endocytosis of the other documented ligands, such as plasminogen activator complexes or apolipoprotein E-containing lipoprotein particles.

Molecular cloning and sequencing of the murine alpha-2-macroglobulin receptor cDNA.

We have molecularly cloned and sequenced the mouse alpha-2-macroglobulin receptor cDNA. The cDNA contained 14849 bases with one large open reading frame of 4545 codons which is one more than in the corresponding human cDNA. Comparison of the predicted mouse and human receptor proteins revealed the very conserved nature of this receptor with an overall amino acid identity of more than 97%. A dramatic example of this is the presence of 331 cysteine residues predicted in the mouse protein, of which 327 are positionally conserved relative to human.

The primary sequence and the subunit structure of mouse alpha-2-macroglobulin, deduced from protein sequencing of the isolated subunits and from molecular cloning of the cDNA.

Mouse plasma alpha-2-macroglobulin (m alpha 2M) was isolated and the N-terminal amino-acid sequences determined after separation of the 165-kDa and 35-kDa subunits. These sequences were compared to the protein sequence predicted by the cDNA, which was cloned from a mouse liver library and sequenced. From these data it is evident that both subunits are encoded by one mRNA of approximately 5 kb expressed predominantly in liver. The smaller subunit, with the N-terminal sequence DLSSSDLT, comprises the C-terminal 257 residues of m alpha 2M and is derived from a single-chain precursor probably by proteolytic processing at an arginine residue in the sequence PTRDLSS. Analysis of the predicted protein further showed all the salient features of a proteinase inhibitor of the macroglobulin family: a bait region that deviates from all known sequences in this family, a very conserved internal thiolester site and conserved cysteine residues and putative N-glycosylation sites. The synthesis of m alpha 2M in adult liver was demonstrated by Northern blotting and in fetal liver by in-situ hybridization. Transient transfection of COS cells with the cDNA under control of a viral promoter demonstrated the secretion and partial processing of m alpha 2M in the culture medium. In plasma the level of m alpha 2M was found to be stable as expected for the murine counterpart of human plasma alpha-2-macroglobulin. The possibilities of using the mouse as a genetic model to study this proteinase inhibitor in vivo are discussed.