Nicholas G. Irving

A mouse model for Down syndrome exhibits learning and behaviour deficits

Trisomy 21 or Down syndrome (DS) is the most frequent genetic cause of mental retardation, affecting one in 800 live born human beings. Mice with segmental trisomy 16 (Ts65Dn mice) are at dosage imbalance for genes corresponding to those on human chromosome 21q21–22.3—which includes the so–called DS ‘critical region’. They do not show early–onset of Alzheimer disease pathology; however, Ts65Dn mice do demonstrate impaired performance in a complex learning task requiring the integration of visual and spatial information. The reproducibility of this phenotype among Ts65Dn mice indicates that dosage imbalance for a gene or genes in this region contributes to this impairment. The corresponding dosage imbalance for the human homologues of these genes may contribute to cognitive deficits in DS.

Tau phosphorylation in transgenic mice expressing glycogen synthase kinase-3beta transgenes.

IN order to investigate the effect on tau of manipulating glycogen synthase kinase (GSK)-3β activity in the brain, we created transgenic mice harbouring wild-type GSK-3β genes or a mutant GSK-3β that is predicted to be more active. Transgene-derived mRNAs were detected in the brains of a number of the transgenic mouse lines and several of these transgenic lines displayed transgenic GSK-3β activity. Western blot analyses of the two lines with the highest levels of transgenic GSK-3β activity revealed that the phosphorylation status of tau was elevated at the AT8 epitope. These observations strongly suggest that GSK-3β is an in vivo tau kinase in the brain. Only low levels of expression of GSK-3β were obtained and it is possible that high levels of GSK-3β activity are lethal.

Mint2/X11-like colocalizes with the Alzheimer’s disease amyloid precursor protein and is associated with neuritic plaques in Alzheimer’s disease

Aberrant metabolism of the amyloid precursor protein (APP) is believed to be at least part of the pathogenic process in Alzheimers disease. The carboxy‐terminus of APP has been shown to interact with the Mint/X11 family of phosphotyrosine binding (PTB) domain‐bearing proteins. It is via their PTB domains that the Mints/X11s bind to APP. Here we report the cloning of full‐length mouse Mint2 and demonstrate that in primary cortical neurons, Mint2 and APP share highly similar distributions. Mint2 also colocalizes with APP in transfected CHO cells. In Mint2/APP‐cotransfected cells, Mint2 reorganizes the subcellular distribution of APP and also increases the steady‐state levels of APP. Finally, we demonstrate that Mint2 is associated with the neuritic plaques found in Alzheimers disease but not with neurofibrillary tangles. These results are consistent with a role for Mint2 in APP metabolism and trafficking, and suggest a possible role for the Mints/X11s in the pathogenesis of Alzheimers disease.

Overexpression of the mouse dishevelled-1 protein inhibits GSK-3β-mediated phosphorylation of tau in transfected mammalian cells

Tau is a neuronal microtubule‐associated protein whose function is modulated by phosphorylation. GSK‐3β is a tau kinase. GSK‐3β is part of the wingless signalling pathway and stimulation by wingless is predicted to down‐regulate GSK‐3β activity. In Drosophila imaginal disc cells, overexpression of dishevelled, a component of the wingless pathway, mimics the wingless signal. We have therefore studied the effect that overexpression of the murine dishevelled‐1 protein has on GSK‐3β‐mediated phosphorylation of tau in transfected CHO cells. We find that co‐transfection with dishevelled‐1 is inhibitory to GSK‐3β‐mediated tau phosphorylation. Tau is hyperphosphorylated in Alzheimers disease and the possible relevance of these findings to Alzheimers disease pathogenesis are discussed.

Fe65 and X11beta co-localize with and compete for binding to the amyloid precursor protein.

The Fe65s and X11s are two families of adaptor proteins that bind to the Alzheimers disease amyloid precursor protein (APP). Although both the X11s and Fe65s bind to similar regions of APP, they have opposing effects on Abeta production and hence may represent novel therapeutic targets. However, there is no evidence that the Fe65s and X11s are present within the same cell type or cell compartment and are thus capable of competing for binding to APP. Here we show that in neurones and transfected cells, APP, Fe65 and X11beta show overlapping subcellular distributions. Furthermore, we demonstrate that Fe65 and X11beta compete for binding to APP.

Mapping of the human and murine X11-like genes (APBA2 and Apba2), the murine Fe65 gene (Apbb1), and the human Fe65-like gene (APBB2): genes encoding phosphotyrosine-binding domain proteins that interact with the Alzheimer's disease amyloid precursor protein

Abnormal processing of the membrane-spanning amyloid precursor protein (APP), resulting in the production of increased amounts of fibrillogenic b-amyloid peptide (Ab), is considered to be one of the key metabolic events underlying Alzheimer’s disease (AD; Selkoe 1994). The function of APP is not fully understood, and the precise cellular mechanisms that lead to A b production are not clearly defined. However, one pathway for A b production involves the re-internalization of membrane-bound APP into lysosomes where fragments of APP containing intact A b are generated (Selkoe 1994). In common with a number of cell surface receptors, the carboxy terminal cytoplasmic domain of APP contains an AsnPro-Thr-Tyr (NPTY) motif which mediates re-internalization via clathrin-coated pits (Chen et al. 1990). This motif has also been demonstrated to be a consensus sequence for binding to phosphotyrosine binding/interacting domain (PTB)-bearing proteins (van der Geer and Pawson 1995). We and others have recently reported that the cytoplasmic domain of APP binds to four human PTB proteins: X11, X11-like, Fe65, and Fe65-like (Borg et al. 1996; Bressler et al. 1996; Fiore et al. 1995; Gue ́nette et al. 1996; McLoughlin and Miller 1996). It has been confirmed that the YENPTY sequence in the cytoplasmic domain of APP is responsible for mediating the interactions between the PTB domain in X11 and the second of two PTB domains in Fe65 (Borg et al. 1996; Fiore et al. 1995). PTB domain proteins are believed to be involved in signal transduction processes (van der Geer and Pawson 1995), and the interaction of APP with X11, X11-like, Fe65, and Fe65-like suggests a role for APP in such signal transduction mechanisms. Furthermore, as they interact with the YENPTY motif in APP, these PTB proteins may modulate processing of APP and hence formation of A b. Therefore, mapping of the genes coding for these proteins is important as they represent new candidate susceptibility genes for AD. The approved gene symbols for the members of these APP binding protein (APB) families are presented in Table 1. The gene for human X11 (APBA1) is already known to be on Chromosome (Chr) 9 close to marker D9S411E (Duclos et al. 1993), and the gene for human Fe65 (APBB1) has been localized to Chr 11 at 11p15 (Bressler et al. 1996). The existence of murine X11 and murine Fe65-like has not yet been reported. Here we report the chromosomal assignment of human APBA2 and APBB2 plus the chromosomal mapping of the murine homologs of X11-like (Apba2) and Fe65 ( Apbb1). In order to map the human APBA2 and APBB2 genes, we selected PCR primers from the previously identified cDNA clones (McLoughlin and Miller 1996) and overlapping sequences deposited in the databases (accession numbers R89683, R13010, R18654, and T16098 for APBA2 and accession number HSU62325 for APBB2). For APBA2 the following primer pair: forward, 58-TTACAAGTCGTGTCCTGGGAG-38, and reverse, 58-GACGTCTGGGGTCCTGTG-3 8, generated a small PCR product of 103 bp. For APBB2 the following primer pair: forward, 58-CACAGAGAAGAGTCTGGCCC-38 and reverse, 5 8-AGGTTGCTTGTGACAGGTCC-38, generated a PCR product of 114 bp. These PCR products were sequenced to confirm they originated from the correct genes. Both human APBA2 and APBB2 genes were mapped using the Genebridge 4 radiation hybrid panel (HGMP Resource Centre, Cambridge, UK) consisting of 94 hamster-derived cell lines. PCR amplification of human DNA with PCR primers designed for these genes resulted in products of the expected size, while no amplification products were obtained from the hamster DNA control sample. Scores for individual cell lines were submitted at the WICGR mapping service at http:// www.genome.wi.mit.edu. APBA2 was assigned to human Chr 15 between the markers WI-5590 (10.31 cR) and D15S144 (21.7 cR). APBB2 was assigned to human Chr 4 between the markers D4S405 (4.6 cR) and D4S496 (10.1 cR). To map theApba2andApbb1loci in the mouse, we used the EUCIB resource which comprises 982 interspecific backcross progeny for high-resolution genetic mapping across the mouse genome (Breen et al. 1994). It is clear from sequence alignments that the mouse sequence L34676 available in the Genbank database corresponds to the mouse homolog of APBA2 ( pba2) rather than to the mouse homolog of APBA1 (McLoughlin and Miller 1996). The following primer pair was selected for mouse Apba2 PCR amplifications: forward, 5 8-GCGCTCTGATCTCAATGG38; reverse, 58-GGAAATGATGCCACCTTC-38. This generated an approximately 1000-bp PCR product. Primers for mouse Apbb1 were designed from the published rat sequence (accession number X60468). The following primer pair was designed for mouse Apbb1 PCR amplifications: forward, 5 8-CTGGCACATCCCAACAGG-38; reverse, 58-AGCAAAGCCAGTCCAGGT-38. The PCR product was 202 bp. Both of these murine PCR products were sequenced to confirmed their origin. The mouseApba2andApbb1PCR products did not show any allelic size difference between C57BL/6 and Mus spretus, the two parental strains of the EUCIB interspecific backcross. However, in both cases, SSCP analysis (Chang et al. 1993) did show a clear polymorphism between C57BL/6 and Mus spretus.In the case of Apba2the large 1-kb PCR product was Sau3AI digested prior to loading on the SSCP gel. 92 random samples from the EUCIB backcross were analyzed for the segregation of C57BL/6 and Mus Correspondence to: D.M. McLoughlin at Dept. of Neuroscience Mammalian Genome 9, 473–475 (1998).

Tau phosphorylation in cells transfected with wild-type or an Alzheimer's disease mutant Presenilin 1.

We have studied the effect of overexpressing either wild-type or an Alzheimers disease mutant Presenilin 1 (PS1) on tau phosphorylation in transfected Chinese hamster ovary (CHO) and COS cells. Tau transfected into these cells is predominantly non-phosphorylated at many PHF-tau sites but co-transfection with the tau kinase glycogen synthase kinase-3 beta (GSK-3 beta) induces phosphorylation that generates epitopes for several phosphorylation-dependent antibodies. Co-transfection of tau with either wild-type or mutant PS1 did not alter tau phosphorylation as detected by five different antibodies. Likewise, co-transfection of the PS1s did not influence GSK-3 beta-mediated tau phosphorylation. The implications of these results for the pathogenesis of Alzheimers disease are discussed.

Alzheimer’s disease, amyotrophic lateral sclerosis, and transgenic mice

Transgenic mouse technology has contributed much to our understanding of the function and dysfunction of the nervous system. It has helped us to model and test hypotheses relating to neurodegenerative diseases, such as Alzheimer’s disease and motor neuron disease, and has also provided insight into the molecular basis of higher brain functions such as learning and memory. Alteration of the mouse genome is carried out by one of two methods: the first involves the addition of new genes (known as transgenes) and the second involves the modification of endogenous mouse genes. The first method usually involves microinjection of the recombinant DNA into the pronucleus of a one cell mouse embryo. The resulting manipulated embryos are reimplanted into the oviducts of a pseudopregnant female mouse and the offspring are then screened for the presence of the transgene so as to identify transgenic progeny. The frequency of production of successful transgenic progeny by this method is surprisingly good with a rate of about one in five to six offspring being transgenic. Manipulation of endogenous mouse genes is a more complex process and involves the use of embryonic stem cells. Embryonic stem cells are derived from the inner cell mass of blastocysts, and retain the ability to differentiate into any cell type of the original mouse. Embryonic stem cells can be expanded in vitro and transfected so as to introduce new DNA. To generate transgenic mice, the modified embryonic stem cells cells are injected into host blastocysts and the resulting embryos are then reintroduced into the reproductive tracts of pseudopregnant females. Animals derived from these manipulated blastocysts are chimeric—that is, some of their cells are derived from the inner cell mass of the host blastocyst and some from the injected embryonic stem cells. Coat colour is often used to identify such chimeric animals. …

The alpha 2 chain of type 1 collagen does not map to mouse chromosome 16 but maps close to the Met proto-oncogene on mouse chromosome 6.

The gene locus for the alpha 2 chain of type 1 collagen (Cola-2) was previously assigned to chromosome 16. Here we demonstrate, utilising both somatic cell hybrid analysis and genetic linkage analysis, in an interspecific Mus domesticus x Mus spretus cross that Cola-2 fails to cosegregate with mouse chromosome 16, but is linked to the Met proto-oncogene on chromosome 6.

The multipoint genetic mapping of mouse chromosome 16

Utilizing a Mus spretus/Mus domesticus (C57BL/10) interspecific backcross, we have constructed a multipoint genetic map of mouse chromosome 16 that extends 43.2 cM from the proximal Prm-1 locus to the distal Ets-2 locus. The genetic map incorporates three new markers: D16Smh6, a random genomic clone; Pgk-1ps1, a phosphoglycerate kinase pseudogene; and the growth-associated protein Gap43. The map position of Gap43 indicates the presence, on mouse chromosome 16, of a significant-size conserved linkage group with human chromosome 3.