F. Van Leuven

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.

Production of intracellular amyloid-containing fragments in hippocampal neurons expressing human amyloid precursor protein and protection against amyloidogenesis by subtle amino acid substitutions in the rodent sequence.

A distinguishing feature of Alzheimers disease (AD) is the deposition of amyloid plaques in brain parenchyma. These plaques arise by the abnormal accumulation of beta A4, a proteolytic fragment of amyloid precursor protein (APP). Despite the fact that neurons are dramatically affected in the course of the disease, little is known about the neuronal processing of APP. To address this question we have expressed in fully mature, synaptically active rat hippocampal neurons, the neuronal form of human APP (APP695), two mutant forms of human APP associated with AD, and the mouse form of APP (a species known not to develop amyloid plaques). Protein expression was achieved via the Semliki Forest Virus system. Expression of wild type human APP695 resulted in the secretion of beta A4‐amyloid peptide and the intracellular accumulation of potential amyloidogenic and non‐amyloidogenic fragments. The relative amount of amyloid‐containing fragments increased dramatically during expression of the clinical mutants, while it decreased strongly when the mouse form of APP was expressed. ‘Humanizing’ the rodent APP sequence by introducing three mutations in the beta A4‐region also led to increased production of amyloid peptide to levels similar to those obtained with human APP. The single Gly601 to Arg substitution alone was sufficient to triple the ratio of beta A4‐peptide to non‐amyloidogenic p3‐peptide. Due to the capacity of these cells to secrete and accumulate intracellular amyloid fragments, we hypothesize that in the pathogenesis of AD there is a positive feed‐back loop where neurons are both producers and victims of amyloid, leading to neuronal degeneration and dementia.(ABSTRACT TRUNCATED AT 250 WORDS)

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 Alzheimers disease.

Noninvasive in vivo MRI detection of neuritic plaques associated with iron in APP[V717I] transgenic mice, a model for Alzheimer's disease

Transgenic mice overexpressing the London mutant of human amyloid precursor protein (APP[V717I]) in neurons develop amyloid plaques in the brain, thus demonstrating the most prominent neuropathological hallmark of Alzheimers disease. In vivo 3D T2*‐weighted MRI on these mice (24 months of age) revealed hypointense brain inclusions that affected the thalamus almost exclusively. Upon correlating these MRI observations with a panel of different histologic staining techniques, it appeared that only plaques that were positive for both thioflavin‐S and iron were visible on the MR images. Numerous thioflavin‐S‐positive plaques in the cortex that did not display iron staining remained invisible to MRI. The in vivo detection of amyloid plaques in this mouse model, using the intrinsic MRI contrast arising from the iron associated with the plaques, creates an unexpected opportunity for the noninvasive investigation of the longitudinal development of the plaques in the same animal. Thus, this work provides further research opportunities for analyzing younger APP[V717I] mouse models with the knowledge of the final outcome at 24 months of age. Magn Reson Med 53:607–613, 2005.

The beta-amyloid domain is essential for axonal sorting of amyloid precursor protein.

We have analysed the axonal sorting signals of amyloid precursor protein (APP). Wild‐type and mutant versions of human APP were expressed in hippocampal neurons using the Semliki forest virus system. We show that wild‐type APP and mutations implicated in Alzheimers disease and another brain beta‐amyloidosis are sorted to the axon. By analysis of deletion mutants we found that the membrane‐inserted APP ectodomain but not the cytoplasmic tail is required for axonal sorting. Systematic deletions of the APP ectodomain identified two regions required for axonal delivery: one encoded by exons 11–15 in the carbohydrate domain, the other encoded by exons 16–17 in the juxtamembraneous beta‐amyloid domain. Treatment of the cells with the N‐glycosylation inhibitor tunicamycin induced missorting of wild‐type APP, supporting the importance of glycosylation in axonal sorting of APP. The data revealed a hierarchy of sorting signals on APP: the beta‐amyloid‐dependent membrane proximal signal was the major contributor to axonal sorting, while N‐glycosylation had a weaker effect. Furthermore, recessive somatodendritic signals, most likely in the cytoplasmic tail, directed the protein to the dendrites when the ectodomain was deleted. Analysis of detergent solubility of APP and another axonally delivered protein, hemagglutinin, demonstrated that only hemagglutinin formed CHAPS‐insoluble complexes, suggesting distinct mechanisms of axonal sorting for these two proteins. This study is the first delineation of sorting requirements of an axonally targeted protein in polarized neurons and indicates that the beta‐amyloid domain plays a major role in axonal delivery of APP.

Glycogen synthase kinase‐3β, or a link between amyloid and tau pathology?

Phosphorylation is the most common post‐translational modification of cellular proteins, essential for most physiological functions. Deregulation of phosphorylation has been invoked in disease mechanisms, and the case of Alzheimer’s disease (AD) is no exception: both in the amyloid pathology and in the tauopathy are kinases deeply implicated. The glycogen synthase kinase‐3 (GSK‐3) isozymes participate in diverse cellular processes and important signalling pathways and have been implicitly linked to diverse medical problems, i.e. from diabetes and cancer to mood disorders and schizophrenia, and in the neurodegeneration of AD. Here, we review specific aspects of GSK‐3 isozymes in the framework of recent data that we obtained in novel transgenic mouse models that robustly recapitulate the pathology and mechanistical problems of AD.

Deregulation of NMDA-receptor function and down-stream signaling in APP(V717I) transgenic mice

Evidence is accumulating for a role for amyloid peptides in impaired synaptic plasticity and cognition, while the underlying mechanisms remain unclear. We here analyzed the effects of amyloid peptides on NMDA-receptor function in vitro and in vivo. A synthetic amyloid peptide preparation containing monomeric and oligomeric A beta (1-42) peptides was used and demonstrated to bind to synapses expressing NMDA-receptors in cultured hippocampal and cortical neurons. Pre-incubation of primary neuronal cultures with A beta peptides significantly inhibited NMDA-receptor function, albeit not by a direct pharmacological inhibition of NMDA-receptors, since acute application of A beta peptides did not change NMDA-receptor currents in autaptic hippocampal cultures nor in xenopus oocytes expressing recombinant NMDA-receptors. Pre-incubation of primary neuronal cultures with A beta peptides however decreased NR2B-immunoreactive synaptic spines and surface expression of NR2B containing NMDA-receptors. Furthermore, we extended these findings for the first time in vivo, demonstrating decreased concentrations of NMDA-receptor subunit NR2B and PSD-95 as well as activated alpha-CaMKII in postsynaptic density preparations of APP[V717I] transgenic mice. This was associated with impaired NMDA-dependent LTP and decreased NMDA- and AMPA-receptor currents in hippocampal CA1 region in APP[V717I] transgenic mice. In addition, induction of c-Fos following cued and contextual fear conditioning was significantly impaired in the basolateral amygdala and hippocampus of APP[V717I] transgenic mice. Our data demonstrate defects in NMDA-receptor function and learning dependent signaling cascades in vivo in APP[V717I] transgenic mice and point to decreased surface expression of NMDA-receptors as a mechanism involved in early synaptic defects in APP[V717I] transgenic mice in vivo.

Amyloidogenic Processing of Human Amyloid Precursor Protein in Hippocampal Neurons Devoid of Cathepsin D

βA4-Amyloid peptide, the main component of the amyloid plaques in the brain of Alzheimers disease patients is produced from amyloid precursor protein (APP) by proteolytical processing. Several lines of evidence suggest a direct role for cathepsin D, the major endosomal/lysosomal aspartic endopeptidase, in βA4-amyloid peptide generation. Here we tested this hypothesis using primary cultures of hippocampal neurons derived from cathepsin D-deficient (knock out) mice and expressing wild-type human APP and two clinical APP variants via recombinant Semliki Forest virus. We demonstrate APP secretory processing, production of carboxyl-terminal amyloid fragments, and secretion of the βA4-amyloid peptide in the complete absence of cathepsin D. The results rule out cathepsin D as a critical component of α-, β-, or γ-secretase and therefore as a primary target for drugs aimed at decreasing the βA4-amyloid peptide burden in Alzheimers disease.

The major tuber storage protein of araceae species is a lectin. Characterization and molecular cloning of the lectin from Arum maculatum L.

A new lectin was purified from tubers of Arum maculatum L. by affinity chromatography on immobilized asialofetuin. Although this lectin is also retained on mannose-Sepharose 4B, under the appropriate conditions free mannose is a poor inhibitor of its agglutination activity. Pure preparations of the Arum lectin apparently yielded a single polypeptide band of approximately 12 kD upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, N-terminal sequencing of the purified protein combined with molecular cloning of the lectin have shown that the lectin is composed of two different 12-kD lectin subunits that are synthesized on a single large precursor translated from and mRNA of approximately 1400 nucleotides. Lectins with similar properties were also isolated from the Araceae species Colocasia esculenta (L.) Schott, Xanthosoma sagittifolium (L.) Schott, and Dieffenbachia sequina Schott. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration of the different Araceae lectins have shown that they are tetrameric proteins composed of lectin subunits of 12 to 14 kD. Interestingly, these lectins are the most prominent proteins in the tuber tissue. Evidence is presented that a previously described major storage protein of Colocasia tubers corresponds to the lectin.

Neonatal neuronal overexpression of glycogen synthase kinase-3β reduces brain size in transgenic mice

Glycogen synthase kinase-3beta (GSK-3beta) is important in neurogenesis. Here we demonstrate that the kinase influenced post-natal maturation and differentiation of neurons in vivo in transgenic mice that overexpress a constitutively active GSK-3beta[S9A]. Magnetic resonance imaging revealed a reduced volume of the entire brain, concordant with a nearly 20% reduction in wet brain weight. The reduced volume was most prominent for the cerebral cortex, without however, disturbing the normal cortical layering. The resulting compacted architecture was further demonstrated by an increased neuronal density, by reduced size of neuronal cell bodies and of the somatodendritic compartment of pyramidal neurons in the cortex. No evidence for apoptosis was obtained. The marked overall reduction in the level of the microtubule-associated protein 2 in brain and in spinal cord, did not affect the ultrastructure of the microtubular cytoskeleton in the proximal apical dendrites. The overall reduction in size of the entire CNS induced by constitutive active GSK-3beta caused only very subtle changes in the psychomotoric ability of adult and ageing GSK-3beta transgenic mice.