Journal of Neuroscience Research 89:808814 (2011)

Peripheral Reduction of b-Amyloid Is Sufcient To Reduce Brain b-Amyloid: Implications for Alzheimers Disease
J. Gregor Sutcliffe,1,2* Peter B. Hedlund,1 Elizabeth A. Thomas,1 Floyd E. Bloom,2 and Brian S. Hilbush2
Department of Molecular Biology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, California 2 ModGene LLC, Cardiff-by-the-Sea, California
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Three loci that modify b-amyloid (Ab) accumulation and deposition in the brains of a mouse model of Alzheimers disease have been previously described. One encompasses the Psen2 gene encoding presenilin 2, a component of the g-secretase activity responsible for generating Ab by proteolysis. We show that the activity of mouse Psen2, as measured by levels of mRNA accumulation, unexpectedly is heritable in the liver but not the brain, suggesting liver as the origin of brain Ab deposits. Administration of STI571, a cancer therapeutic that does not cross the bloodbrain barrier, reduced accumulation of Ab in both the blood and the brain, conrming brain Abs peripheral origin and suggesting that STI571 and related compounds might have therapeutic/prophylactic value in human Alzheimers disease. The genes Cib1 and Zfhx1b reside within the other modier loci and also exhibit heritable expression in the liver, suggesting that they too contribute to Ab C accumulation. V 2011 Wiley-Liss, Inc. Key words: Alzheimers disease; imatinib; Gleevec; amyloid; presenilin

Alzheimers disease (AD) is a neurodegenerative disorder characterized by the age-dependent deposition of b-amyloid (Ab) within vulnerable regions of the brain, particularly the frontal cortex and hippocampus (Terry, 2006). Ab has a pathogenic effect, leading to progressive neuronal loss that causes deterioration of the ability of those brain regions to orchestrate both higher order and basic neural processes. Some forms of human AD are highly heritable, caused by rare variations in genes that encode proteins that are associated with both familial and sporadic forms of this neurodegenerative disorder and play central roles in the initiation of the disease process. One of these encodes the amyloid precursor protein (APP; Tanzi, 1989), a membrane protein whose biochemical function is at present unknown. APP is a substrate for proteolysis by several endogenous proteases, liberating proteolytic fragments of various structures. Proteolysis of APP by b-secretase generates a fragment that can subsequently serve as a substrate for
' 2011 Wiley-Liss, Inc.

cleavage by g-secretase at multiple adjacent positions within the precursor to form Ab isoforms ranging from 37 to 43 amino acid residues. The 42-residue species is thought to be the most pathogenic (Wolfe, 2006) and forms oligomeric structures, which, in addition to depositing in plaques in the AD-affected brain, are thought to cause cognitive decits (Barten and Albright, 2008). AD-predisposing variations in APP cluster in the vicinity of the proteolytic cleavage sites, affecting the rate at which pathogenic Ab fragments are generated, their stability, and their ability to form oligomers (Selkoe, 2001). Individuals inheriting such APP variations usually show signs of AD in their 50s, whereas sporadic AD is not common until individuals reach their 70s (Waring and Rosenberg, 2008). Rare variations in two other genes, Presenilin 1 and Presenilin 2, also confer high risk for early-onset AD. These two genes encode independent proteins of similar structures that function as part of the g-secretase protein complex (Wolfe, 2006). As a consequence of these genetic observations and considerable experimentation, the etiologic model that has emerged holds that biochemical events that increase the production and accumulation of Ab, particularly Ab142, accelerate the onset and progression of AD. Transgenic mouse models have been developed that recapitulate critical features of human AD. In the R1.40 model, expression of a human APP transgene carrying the so-called Swedish mutations (K670N, M671L, variations that predispose those humans that inherit this mutant gene to develop early-onset AD) is driven from the natural human APP promoter (Kulnane and Lamb, 2001). Congenic lines were derived from the R1.40 model on the C57Bl/6 (B6) and DBA/2 (D2) backgrounds (Lehman et al., 2003). Although these two
*Correspondence to: J. Gregor Sutcliffe, Department of Molecular Biology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037. E-mail: gregorsutcliffe@aol.com Received 18 November 2010; Revised 31 December 2010; Accepted 31 December 2010 Published online 3 March 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jnr.22603

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transgenic strains produced the same amount of APP precursor (indicating that the transgene was expressed comparably in the two strain backgrounds), B6s accumulated more Ab than D2s, as measured by ELISA on brain homogenates and plasma at 21 and 60 days and developed amyloid deposits characteristic of human AD at 13.5 months, whereas the D2s were protected (no deposits at 2 years). This indicated that there were genetic differences that distinguish B6 and D2 mice and that modify the development of AD-like pathology, most likely by inuencing the accumulation of the pathogenic substance Ab (Lehman et al., 2003). The identities of the modier genes might suggest therapeutic or prophylactic modalities that would mimic the modier effect and delay or prevent the emergence of AD pathology. To assign the modifying genes to chromosomal intervals (quantitative trait loci; QTLs), Ryman and colleagues (2008) analyzed Ab accumulation in the brains of 516 F2 mice from a B6/D2 intercross population and mapped three modifying loci, assigned to broad regions centered on the following positions: chromosome 1, 182.049374 megabases (Mb); chromosome 2, 41.216315 Mb; and chromosome 7, 63.680922 Mb.
MATERIALS AND METHODS Wild-type B6 and D2 mice (age 812 weeks or 1518 months) were administered imatinib (methanesulfonate salt; LC Laboratories, Woburn, MA) in the doses indicated or vehicle twice daily for 7 days by intraperitoneal injection. Animals were sacriced 12 hr after the last injection. Individual mice were anesthetized with isourane and blood samples (100300 ll) taken by cardiac puncture with heparinized syringes. Samples were placed on ice for 30 min in the presence of EDTA and then centrifuged for 20 min at 16,000g at 48C. The plasma fraction was removed and stored at 808C. Brains were removed, frozen rapidly on dry ice, and stored at 808C. Oligomeric Ab was extracted in the SDS fraction essentially as described by Kawarabayashi et al. (2001). Samples were subjected to PAGE analysis and transferred to PVDF membranes, and the Ab hexamers were visualized using a monoclonal antibody, 4G8, directed against mouse Ab (1:1,000; Covance, Berkeley, CA), using the manufacturers recommended protocol. Blots were scanned by densitometry, and brain blots were then reprobed with an antibody to histone H3 (1:50,000; Abcam, Cambridge, MA) as a loading and transfer control. Uncleaved APP served as normalization control for blood samples. Data are depicted as normalized optical density. Animal care and handling were consistent with AVMA guidelines.

RESULTS Identication of a Modier Gene on Chromosome 1 The mouse gene encoding presenilin 2, Psen2, is located on chromosome 1 at 182.06371 Mb, the center of one of the trait locus intervals, suggesting it as a candidate for modifying Ab accumulation and deposit. This
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is consistent with its function as a component of g-secretase and with its demonstrated modier effect on early onset human AD. For Psen2 to represent the actual modier mapped to chromosome 1 by Ryman and colleagues (2008), its activity must vary heritably (in a Mendelian fashion) between B6 and D2 mouse strains, and the Psen2 activity should be greater in B6 mice than D2 mice because lower g-secretase activity would be expected to be protective in AD. One of the most profound mechanisms by which gene activity can vary is by the amount of mRNA that accumulates from its expression. We investigated this issue by determining the amount of Psen2 mRNA that accumulates in various tissues in B6 and D2 mouse strains and recombinant inbred lines produced by crossing B6 and D2 mice and subsequent inbreeding of the offspring. The concentrations of each of more than 20,000 mRNAs in 10 tissues (brain, cerebellum, liver, striatum, kidney, hippocampus, eye, prefrontal cortex, nucleus accumbens, and neocortex) of B6 and D2 mouse strains and as many as 89 recombinant inbred mouse lines are available in public databases compiled at http://www.GeneNetwork.org (Wang et al., 2003). For each of the recombinant inbred mouse lines, it has been determined by genotyping for each interval of its genome whether that interval was inherited from the B6 or D2 parent. We constructed a large database in which the genotype (B6 or D2) of each genetic interval that is dimorphic between the B6 and D2 parental strains was correlated by regression analysis with the amount of mRNA that accumulated from every gene in the various tissues of the RI lines. This gave us a map of so-called expression QTLs that dene the differences between the B6 and D2 genetic backgrounds that we could overlay on genetic maps of modier loci to locate coincident intervals containing both a modier gene and an expression QTL. Marker rs13476267 is located on chromosome 1 at 182.120454 Mb, within the modier locus that contains Psen2, and is genetically dimorphic between B6 and D2 strains. Using the WebQTL software (http://www.genenetwork.org/webqtl/WebQTL.py), we performed trait correlation regression analysis between the genotype of the rs13476267 interval and the amount of Psen2 mRNA that accumulates in each of the 10 tissues (Fig. 1 shows data from six of the tissues) in the recombinant inbred mice, calculating the Pearsons product-moment correlation values (Table I). None of the tissue samples derived from brain showed high heritability of Psen2 expression, as demonstrated by the overlapping distributions of expression levels between lines carrying the alternative genotypes at the Psen2 locus (Fig. 1). For the two brain regions that exhibit modest heritability of Psen2 mRNA expression, cerebellum and nucleus accumbens, more Psen2 mRNA was correlated with the D2 genotype than the B6 genotype: thus, Psen2 activity in the brain is not a modier of brain Ab accumulation. Unexpectedly, in the liver, the amount of Psen2 mRNA was highly correlated (P < 4.99 e41) with the genotype at the Psen2 locus (Fig. 1). Mice inheriting the B6

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Fig. 1. Plot of Psen2 locus genotype (B6/B6 or D2/D2) vs. Psen2 mRNA concentration in six tissues (arbitrary units) from the up to 89 recombinant inbred (RI) lines. The parental C57 and DBA values are plotted next to those from the RI lines. Some tissues have data from single RI lines that are heterozygous at the Psen2 locus; these are represented on the plots as B6/D2. Data were obtained from GeneNetwork.org (Wang et al., 2003). For liver, expression data were initially expressed as the ratio of the liver uorescence signal to that generated by the reference mRNA sample for each probe. Data were normalized using a robust LOWESS smoothing method that

adjusts for nonlinearity of signal in the two channels. We then computed the log base 2 of these ratios (median). A value of 1 indicates that expression in liver is roughly half that in the control; a value of 2 indicates that expression in the liver is roughly one-fourth that in the control, etc. Conversely, a value of 1 2 indicates that the expression in liver is fourfold greater in liver. Liver data set from 40 recombinant inbred lines described by Gatti et al. (2007). For other tissues, expression values and alternative normalization methods are as indicated (Wang et al., 2003).

genotype express two- to eightfold more Psen2 mRNA in the liver than do mice inheriting the D2 genotype, an observation consistent with the notion that higher g-secretase activity might contribute to greater generation of Ab peptides. These data demonstrate that Psen2 expression in the liver or in one or more peripheral tissues modies Ab accumulation and suggest the hypothesis that reduction of Ab in the periphery would be sufcient to modify its deposition in the mouse brain. That is, despite the tacit, natural assumption that this brain disease would be caused by events that occur within the brain (and the valid observation that APP itself is expressed within the brain), an effective therapeutic or prophylactic treatment for AD that reduces Ab accumulation might not have to cross the bloodbrain barrier and enter the brain. Inhibition of Psen2 or g-secretase activity or reduction of Ab production or accumulation by other means, outside of

the central nervous system, should be sufcient to protect the brain from Ab deposition. Peripheral Administration of STI571 Reduces Ab in Brain The tyrosine kinase inhibitor imatinib mesylate (STI571, trade name Gleevec) is presently approved for treatment of chronic myelogenous leukemia and gastrointestinal stromal tumors. STI571 potently reduces the production of Ab, both in APP-transfected neuroblastoma cells and in cell-free extracts prepared from the transfected cells, via a mechanism that does not require the Abl tyrosine kinase, one of the important targets of this drug in leukemia cells (Netzer et al., 2003). Recent work suggests that STI571 acts as an inhibitor of the interaction between g-secretase and the g-secretase activating protein (GSAP; He et al., 2010) by a mechanism
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Pathogenic Ab Has a Peripheral Origin

TABLE 1. Heritability of Psen2 mRNA Accumulation in Various Tissues* r Brain Cerebellum Eye Hippocampus Kidney Liver Nucleus accumbens Neocortex Prefrontal cortex Striatum |r| < 0.05 r 5 0.6344 |r| < 0.35 |r| < 0.36 r 5 0.4733 r 5 0.9402 r 5 0.7260 r 5 0.5500 |r| < 0.51 r 5 0.5329 r2 <0.0025 0.4025 <0.1225 <0.1296 0.2240 0.8840 0.5271 0.3025 <0.2601 0.2840

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*Pearsons product-moment correlation value (r), negative correlation values indicate that expression of Psen2 mRNA in B6 genotypes is higher than in D2 genotypes. |r| < 0.8 represents incomplete heritabilities unlikely to account for mapped QTLs because of overlapping distributions (see Fig. 1). r Values are from GeneNetwork.org (Wang et al., 2003).

that does not involve kinase inhibition. Importantly, STI571 does not interfere with Notch cleavage, a shortcoming that reduces the utility of typical g-secretase inhibitors. STI571 has poor penetration of the blood brain barrier in both humans and mice, as shown in an STI571-treated leukemia patient whose cerebral spinal uid (CSF) level of the drug was 92-fold lower than in the blood (Takayama et al., 2002) and STI571-treated Balb/c mice whose CSF drug levels were 155-fold lower than in plasma (Wolff et al., 2003). The drug also has poor brain penetrance in B6 mice (Williams et al, 2007). To test the hypothesis that an effective therapeutic compound for AD need not cross the bloodbrain barrier, we administered STI571 peripherally to wild-type mice twice daily for 1 week and collected plasma and brain tissue. We studied wild-type mice in order to replicate most closely the natural, endogenous Ab-producing environment. In an initial study using D2 mice, vehicle groups were injected intraperitoneally with 100 ll

Fig. 2. STI571 reduces Ab in plasma and whole brain. Wild-type B6 and D2 mice [age 812 weeks (A,B) or 1518 months (C,D)] were administered drug or saline vehicle twice daily for 7 days by intraperitoneal injection and tissue extracts, prepared as described in Materials and Methods, and were analyzed by Western blotting using Ab oligomers as molecular size markers. A: Bar graph quantication of Western blot images of the Ab-immunoreactive band comigrating
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with Ab hexamers in plasma from young D2 mice treated with vehicle or STI571 at three doses (n 5 4 per group), *P < 0.02, **P < 0.001. B: Brain extracts from young B6 mice treated with vehicle or STI571 at 20 mg/kg (n 5 10 per group); plasma (C) or brain extracts (D) of old B6 mice treated with vehicle or STI571 at 20 mg/kg (n 5 4 per group), *P < 0.05, **P < 0.01.

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saline, and drug treatment groups received 1, 10, or 100 mg/kg STI571. We analyzed plasma by Western blotting using the 4G8 antibody directed against Ab. A dose-dependent reduction in a 4G8-immunoreactive plasma protein that comigrated with marker Ab hexamers was observed (Fig. 2A), and the highest dose reduced circulating immunoreactive target by approximately 75%. We selected an intermediate dose, 20 mg/kg, to study brain effects. This dose reduced brain and plasma levels of the 4G8-immunoreactive target by approximately 50% in young and old B6 mice (Fig. 2BD), and proportional levels of reduction would be protective in the R1.40 mouse model (Lehman et al., 2003). Identication of Candidate Chromosome 2 and 7 Modier Genes Because the studies described above demonstrated that pathogenic Ab in the B6 mouse likely derives from the liver and/or other peripheral tissues, we searched for genes that mapped to the chromosome 2 and 7 intervals and whose activities in the liver varied heritably between B6 and D2 mouse strains (expression QTLs), using the same database and methodology as described above. The dimorphic genetic marker rs4226715 is located on chromosome 7 at 80.138616 Mb, within the modier locus for that chromosome. Two genes from this interval showed extremely high heritability of expression within the liver but not within the brain; the Ngrn gene (80.138736 Mb), which encodes neugrin, a widely expressed protein of unknown function whose expression increases in some cancers and has been associated with neuroblastoma differentiation (Ishigaki et al., 2002; Hustinx et al., 2004), and the Cib1 gene (80.101507 Mb), which encodes calmyrin, a myristoylated calcium- and integrin-binding membrane-associated protein originally discovered because of its preferential interaction with presenilin 2 in two-hybrid screens (Stabler et al., 1999), showed the highest correlations: Pearsons values r 5 0.945, and r 5 0.913, respectively, both P < 4.9939. However, the demonstrated interaction of calmyrin with presenilin 2 makes it the preferred candidate AD modier. Because the calmyrin distribution in the brain does not correlate well with either presenilin distribution or regions most susceptible to AD pathology, its potential role in contributing to Ab production in the forebrain has been considered but judged unlikely (Blazejczyk et al., 2006). However, calmyrin is highly expressed by the liver (Stabler et al., 1999). The heritability of liver calmyrin mRNA expression was extremely high. In every line that inherited its Cib1 genes from the B6 parents, the amount of calmyrin mRNA was considerably higher than in strains that inherited their Cib1 genes from the D2 parents (Fig. 3A). One line (line 73) appears to be heterozygous at the marker but expressed D2-like amounts of calmyrin mRNA. This suggests that low calmyrin expression in liver decreases the accumulation of Ab in the brain and protects mice from its adverse effects. One suggested calmyrin activity is as a protein ligand for the inositol

Fig. 3. Plot of Cib1 (A) or Zfhx1b (B) genotype (B6/B6, B6/D2, or D2/D2) vs. calmyrin (A) or Zfhx1b (B) mRNA concentration in liver (arbitrary units) for 40 recombinant inbred lines, as in Figure 1. Data were obtained from GeneNetwork.org (Wang et al., 2003); the liver data set was described by Gatti et al. (2007).

1,4,5-trisphosphate receptor Ca21 release channel (White et al., 2006), whose gating activity is aberrant in chicken cells transfected with mutant presenilin genes (Cheung et al., 2008). This argument cannot rule out an additional role for neugrin, whose abundance in liver is inversely correlated with Ab accumulation. The dimorphic genetic marker rs3669981 is located on chromosome 2 at 44.943029 Mb, within the fairly broad modier locus for that chromosome. The Zfhx1b gene (44.810557 Mb), which encodes zinc nger homeobox 1b protein, showed the highest expression heritability correlation in the liver (r 5 0.919, P < 4.9939), but none in brain. The Zfhxb1 protein is a Smad-interacting transcriptional corepressor involved in Wnt and hedgehog signaling (Bassez et al., 2004; Verstappen et al., 2008; Isohata et al., 2009). Detrimental variants of the gene cause the developmental disorder Mowat-Wilson syndrome, which presents with multiple congenital decits, including mental retardation (Zweier et al., 2002). Although the Zfhx1b mRNA is widely expressed during development, especially within the nervous system, in the adult mouse it is most highly expressed in the liver (Bassez ey al., 2004). In nearly every line that inherited its Zfhx1b genes from the B6 parents, the amount of Zfhx1b mRNA in liver was greater than in lines that inherited their Zfhx1b genes from the D2 parents (Fig. 3B). Strains 12 and 36 differed in genotype at the probe but had similar mRNA levels. These data suggest that low Zfhx1b expression in liver lowers the accumulation of Ab in the brain and protects mice from its adverse effects. DISCUSSION Although Alzheimers disease is a devastating neurological disorder whose pathology is believed to be
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caused by the toxicity and deposition of Ab within the brain, the present study suggests that, at least in the mouse, the origin of a substantial portion of the pathogenic Ab is the periphery, probably the liver. Lamb and his collaborators (Ryman et al., 2008) mapped three genes whose activities modify Ab accumulation in the mouse brain to broad QTLs. By examining the correlation of mRNA expression levels with genotypes in recombinant inbred mice, we demonstrate here that the modier activity of the chromosome 1 gene, Psen2, is not heritable in the brain but is heritable in the liver. We mimicked the liver modier effect by short-term (1 week) administration of STI571, a drug approved for human cancer treatment that does not readily penetrate the bloodbrain barrier. STI571 signicantly lowers immunoreactive Ab levels not only in the plasma, but also in the brain. The magnitude of the drug effect on the mouse brain compartment suggests that it would be sufcient to protect nonaffected humans from developing AD pathology and might be benecial to those whom have already presented with symptoms, although human clinical testing will be required to establish appropriate doses and treatment regimens and the extent, if any, to which disease progression can be reversed. The identication of the liver as the probable origin of the pathogenic levels of brain Ab allowed us to identify candidates for the other two genes mapped by Ryman and colleagues (2008), namely, Cib1 and Zfhx1b, both of which exhibit liver expression heritability. Proof that these are indeed the modiers of Ab will require additional genetic and/or drug activity-modication studies. However, these observations suggest the model that the activities of the Psen2, Cib1, and Zfhx1b gene products cooperate in the liver to produce Ab, which enters the circulation, forms oligomers, penetrates the brain (fairly rapidly judging by the time course of the STI571 effect), and eventually accumulates to a pathogenic level. Each of these targets presents an orthogonal opportunity for designing therapeutic antagonists. The human AD modiers encoding APP, Psen1, and apolipoprotein E (and the more recently implicated clusterin and PICALM) might also exert their effects in liver but would not have been picked up in the R1.40 mouse screen because their activities do not vary signicantly between B6 and D2 mice. Netzer and colleagues (2003) demonstrated that STI571 inhibits Ab production by cultured cells. To test the activity of STI571 on brain Ab accumulation in vivo, they implanted osmotic minipumps to deliver STI571 intrathecally to the brains of guinea pigs and observed a decrease in Ab accumulation. They concluded that . . . the ability to achieve a high degree of penetration of the bloodbrain barrier would be necessary to improve the likelihood of therapeutic benet. Our study suggests not only that penetration of the bloodbrain barrier by STI571 is unnecessary but also that avoiding brain penetration might avert some possible adverse side effects. Discovery of the liver origin of brain Ab might explain the successes of some Ab immunotherapeutic approaches (Schenk et al., 1999).
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ACKNOWLEDGMENTS We thank Rob Williams for helpful and enthusiastic discussions about the GeneNetwork databases, Eddie Koo for advice on Ab detection, and Bin Tang and Patria Danielson for assistance with Western blotting. REFERENCES
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