Genetic variants of human organic anion transporter 4 demonstrate altered transport of endogenous substrates.

Apical reabsorption from the urine has been shown to be important for such processes as the maintenance of critical metabolites in the blood and the excretion of nephrotoxic compounds. The solute carrier (SLC) transporter OAT4 (SLC22A11) is expressed on the apical membrane of renal proximal tubule cells and is known to mediate the transport of a variety of xenobiotic and endogenous organic anions. Functional characterization of genetic variants of apical transporters thought to mediate reabsorption, such as OAT4, may provide insight into the genetic factors influencing the complex pathways involved in drug elimination and metabolite reclamation occurring in the kidney. Naturally occurring genetic variants of OAT4 were identified in public databases and by resequencing DNA samples from 272 individuals comprising 4 distinct ethnic groups. Nine total nonsynonymous variants were identified and functionally assessed using uptake of three radiolabeled substrates. A nonsense variant, R48Stop, and three other variants (R121C, V155G, and V155M) were found at frequencies of at least 2% in an ethnic group specific fashion. The L29P, R48Stop, and H469R variants displayed a complete loss of function, and kinetic analysis identified a reduced V(max) in the common nonsynonymous variants. Plasma membrane levels of OAT4 protein were absent or reduced in the nonfunctional variants, providing a mechanistic reason for the observed loss of function. Characterization of the genetic variants of reabsorptive transporters such as OAT4 is an important step in understanding variability in tubular reabsorption with important implications in innate homeostatic processes and drug disposition.

Biological sulfur in the blood cells of Ascidia ceratodes: XAS spectroscopy and a cellular-enzymatic hypothesis for vanadium reduction in the ascidians.

Two samples of living blood cells and of cleared blood plasma from the Phlebobranch tunicate Ascidia ceratodes from Bodega Bay, California, and one of fresh Henze solution from A. ceratodes of Monterey Bay, California, have been examined using sulfur K-edge x-ray absorption spectroscopy (XAS). Biological sulfur included sulfate esters, sulfate and bisulfate ions, benzothiazole, thianthrene, epi-sulfide, thiol and disulfide. Glutathione dominated reduced sulfur, from which an average intracellular Voltage of -0.21 V was calculated. Sulfate-bisulfate ratios yielded blood cell pH values of 2.0 and 2.8. Total blood cell [sulfur] was 373±9 mM or 296±73 mM from BaSO4 gravimetry. Two plasma samples (pH 6.9 or 7.0; [S] = 33±6 mM or 26±4 mM) were dominated by sulfate and disulfide. Fresh Henze solution evidenced a sulfur inventory similar to blood cells, with calculated pH = 2.7. A V(III)-sulfonate fraction varied systematically with intracellular pH across six independent blood cell samples, implying a vanadium mobilization pathway. Bodega Bay and Monterey Bay A. ceratodes appear to maintain alternative suites of low-valent sulfur. The significance of the vanabins to vanadium metabolism is critically examined in terms of known protein - V(IV) biochemistry. Finally, a detailed hypothesis for the reduction of [VO4]3- to V(III) in ascidians is introduced. A vanadium oxido-reductase is proposed to span the signet ring membrane and to release V(III) into the inner acidic vacuole. The V(V) to V(III) reduction is predicted require an inner-sphere mechanism, a thiol reductant, 7-coordinate V(III), a biologically accessible Voltage, and proton-facilitated release of V(III).

Constructing and testing a disability index in a US sample of preschoolers with disabilities.

PURPOSE: To develop and test an index of disability severity in a heterogeneous population of preschoolers with disabilities. METHOD: Using a nationally representative sample of 3,104 children receiving special education services in the US, questions from a parent interview were used to develop an index of disability severity consisting of domains of functioning defined by gradients of severity. Regressions were used to examine the association between 15 functional domains and 8 cognitive, social/behavioural, and functional outcomes. Full and abbreviated versions of this disability index were compared. Correlations with proxy measures of disability in this population were used to establish concurrent validity, and the predictive ability of this measure was compared with that of the federally defined disability categories. RESULTS: Of the 15 domains examined, most were significant predictors of at least two outcomes. A shortened index of only six variables was found to be as effective as the longer version in characterising children's level of severity. The index was significantly correlated with intervention variables such as the age at which children began receiving special education or therapy service, r(2802) = -0.22, p

The uptake and fate of vanadyl ion in ascidian blood cells and a detailed hypothesis for the mechanism and location of biological vanadium reduction. A visible and X-ray absorption spectroscopic study.

Vanadium K-edge X-ray absorption spectroscopy (XAS) has been used to track the uptake and fate of VO(2+) ion in blood cells from Ascidia ceratodes, following exposure to dithiothreitol (DTT) or to DTT plus VO(2+). The full range of endogenous vanadium was queried by fitting the XAS of blood cells with the XAS spectra of model vanadium complexes. In cells exposed only to DTT, approximately 0.4% of a new V(III) species was found in a site similar to Na[V(edta)(H(2)O)]. With exposure to DTT and VO(2+), average intracellular [VO(aq)](2+) increased from 3% to 5%, and 6% of a new complexed form of vanadyl ion appeared evidencing a ligand array similar to [VO(edta)](2-). At the same time, the relative ratio of blood cell [V(H(2)O)(6)](3+) increased at the expense of [V(H(2)O)(5)(SO(4))](+) in a manner consistent with a significant increase in endogenous acidity. In new UV/Visible experiments, VO(2+) could be reduced to 7-coordinate [V(nta)(H(2)O)(3)] or [V(nta)(ida)](2-) with cysteine methyl ester in pH 6.5 solution. Ascorbate reduced [VO(edta)](2-) to 7-coordinate [V(edta)(H(2)O)](-), while [VO(trdta)](2-) was unreactive. These results corroborate the finding that the reductive EMF of VO(2+) is increased by the availability of a 7-coordinate V(III) product. Finally, a new and complete hypothesis is proposed for an ascidian vanadate reductase. The structure of the enzyme active site, the vanadate-vanadyl-vanadic reduction mechanism, the cellular locale, and elements of the regulatory machinery governing the biological reduction of vanadate and vanadyl ion by ascidians are all predicted. Together these constitute the new field of vanadium redox enzymology.

A national sample of preschoolers with autism spectrum disorders: special education services and parent satisfaction.

The Pre-Elementary Education Longitudinal Study (PEELS) examines the preschool and early elementary school experiences of a nationally representative sample of 3,104 children ages 3-5 with disabilities from 2004 through 2009. This paper describes the special education and related services received by a subsample of 186 preschoolers with autism spectrum disorders (ASD) in 2003-2004 and parental satisfaction with those services. Past research and patterns of litigation suggest that parents of children with ASD are not wholly satisfied with the special education and related services their children receive. In the current study, the authors found many similarities between children with ASD and children with other disabilities in the type of services received under IDEA and in parent satisfaction with these services. Still, some significant differences emerged in the number of services received, the amount of time children with ASD spent in special education settings, and parent satisfaction with the amount of time children spent with typically developing peers. Implications about the importance of parent satisfaction and social validity measures are discussed.

Validation of microsatellite markers for use in genotyping polyclonal Plasmodium falciparum infections.

Genotyping methods for Plasmodium falciparum drug efficacy trials have not been standardized and may fail to accurately distinguish recrudescence from new infection, especially in high transmission areas where polyclonal infections are common. We developed a simple method for genotyping using previously identified microsatellites and capillary electrophoresis, validated this method using mixtures of laboratory clones, and applied the method to field samples. Two microsatellite markers produced accurate results for single-clone but not polyclonal samples. Four other microsatellite markers were as sensitive as, and more specific than, commonly used genotyping techniques based on merozoite surface proteins 1 and 2. When applied to samples from 15 patients in Burkina Faso with recurrent parasitemia after treatment with sulphadoxine-pyrimethamine, the addition of these four microsatellite markers to msp1 and msp2 genotyping resulted in a reclassification of outcomes that strengthened the association between dhfr 59R, an anti-folate resistance mutation, and recrudescence (P = 0.31 versus P = 0.03). Four microsatellite markers performed well on polyclonal samples and may provide a valuable addition to genotyping for clinical drug efficacy studies in high transmission areas.

App gene dosage modulates endosomal abnormalities of Alzheimer's disease in a segmental trisomy 16 mouse model of down syndrome.

Altered neuronal endocytosis is the earliest known pathology in sporadic Alzheimer's disease (AD) and Down syndrome (DS) brain and has been linked to increased Abeta production. Here, we show that a genetic model of DS (trisomy 21), the segmental trisomy 16 mouse Ts65Dn, develops enlarged neuronal early endosomes, increased immunoreactivity for markers of endosome fusion (rab5, early endosomal antigen 1, and rabaptin5), and endosome recycling (rab4) similar to those in AD and DS individuals. These abnormalities are most prominent in neurons of the basal forebrain, which later develop aging-related atrophy and degenerative changes, as in AD and DS. We also show that App, one of the triplicated genes in Ts65Dn mice and human DS, is critical to the development of these endocytic abnormalities. Selectively deleting one copy of App or a small portion of the chromosome 16 segment containing App from Ts65Dn mice eliminated the endosomal phenotype. Overexpressing App at high levels in mice did not alter early endosomes, implying that one or more additional genes on the triplicated segment of chromosome 16 are also required for the Ts65Dn endosomal phenotype. These results identify an essential role for App gene triplication in causing AD-related endosomal abnormalities and further establish the pathogenic significance of endosomal dysfunction in AD.

Analysis of four DLX homeobox genes in autistic probands.

BACKGROUND: Linkage studies in autism have identified susceptibility loci on chromosomes 2q and 7q, regions containing the DLX1/2 and DLX5/6 bigene clusters. The DLX genes encode homeodomain transcription factors that control craniofacial patterning and differentiation and survival of forebrain inhibitory neurons. We investigated the role that sequence variants in DLX genes play in autism by in-depth resequencing of these genes in 161 autism probands from the AGRE collection. RESULTS: Sequencing of exons, exon/intron boundaries and known enhancers of DLX1, 2, 5 and 6 identified several nonsynonymous variants in DLX2 and DLX5 and a variant in a DLX5/6 intragenic enhancer. The nonsynonymous variants were detected in 4 of 95 families from which samples were sequenced. Two of these four SNPs were not observed in 378 undiagnosed samples from North American populations, while the remaining 2 were seen in one sample each. CONCLUSION: Segregation of these variants in pedigrees did not generally support a contribution to autism susceptibility by these genes, although functional analyses may provide insight into the biological understanding of these important proteins.

Mortality of Rocky Mountain elk in Michigan due to meningeal worm.

Mortality from cerebrospinal parelaphostrongylosis caused by the meningeal worm (Parelaphostrongylus tenuis) has been hypothesized to limit elk (Cervus elaphus nelsoni) populations in areas where elk are conspecific with white-tailed deer (Odocoileus virginianus). Elk were reintroduced into Michigan (USA) in the early 1900s and subsequently greatly increased population size and distribution despite sympatric high-density (>or=12/km2) white-tailed deer populations. We monitored 100 radio-collared elk of all age and sex classes from 1981-94, during which time we documented 76 mortalities. Meningeal worm was a minor mortality factor for elk in Michigan and accounted for only 3% of mortalities, fewer than legal harvest (58%), illegal kills (22%), other diseases (7%), and malnutrition (4%). Across years, annual cause-specific mortality rates due to cerebrospinal parelaphostrongylosis were 0.033 (SE=0.006), 0.029 (SE=0.005), 0.000 (SE=0.000), and 0.000 (SE=0.000) for calves, 1-yr-old, 2-yr-old, and >or=3-yr-old, respectively. The overall population-level mortality rate due to cerebrospinal parelaphostrongylosis was 0.009 (SE=0.001). Thus, meningeal worm had little impact on elk in Michigan during our study despite greater than normal precipitation (favoring gastropods) and record (>or=14 km2) deer densities. Further, elk in Michigan have shown sustained population rates-of-increase of >or=18%/yr and among the highest levels of juvenile production and survival recorded for elk in North America, indicating that elk can persist in areas with meningeal worm at high levels of population productivity. It is likely that local ecologic characteristics among elk, white-tailed deer, and gastropods, and degree of exposure, age of elk, individual and population experience with meningeal worm, overall population vigor, and moisture determine the effects of meningeal worm on elk populations.

Sequence diversity and haplotype structure in the human ABCB1 (MDR1, multidrug resistance transporter) gene.

OBJECTIVES: There is increasing evidence that polymorphism of the ABCB1 (MDR1) gene contributes to interindividual variability in bioavailability and tissue distribution of P-glycoprotein substrates. The aim of the present study was to (1) identify and describe novel variants in the ABCB1 gene, (2) understand the extent of variation in ABCB1 at the population level, (3) analyze how variation in ABCB1 is structured in haplotypes, and (4) functionally characterize the effect of the most common amino acid change in P-glycoprotein. METHODS AND RESULTS: Forty-eight variant sites, including 30 novel variants and 13 coding for amino acid changes, were identified in a collection of 247 ethnically diverse DNA samples. These variants comprised 64 statistically inferred haplotypes, 33 of which accounted for 92% of chromosomes analyzed. The two most common haplotypes, ABCB1*1 and ABCB1*13, differed at six sites (three intronic, two synonymous, and one non-synonymous) and were present in 36% of all chromosomes. Significant population substructure was detected at both the nucleotide and haplotype level. Linkage disequilibrium was significant across the entire ABCB1 gene, especially between the variant sites found in ABCB1*13, and recombination was inferred. The Ala893Ser change found in the common ABCB1*13 haplotype did not affect P-glycoprotein function. CONCLUSION: This study represents a comprehensive analysis of ABCB1 nucleotide diversity and haplotype structure in different populations and illustrates the importance of haplotype considerations in characterizing the functional consequences of ABCB1 polymorphisms.

Increased sensitivity of homozygous Sod2 mutant mice to oxygen toxicity.

Induction or overexpression of pulmonary manganese superoxide dismutase (MnSOD) has been shown to protect against oxygen (O2) toxicity. Genetic inactivation of MnSOD (Sod2) results in multiple organ failure and early neonatal death. However, lungs or O2-tolerance of Sod2 knockout mice have not been investigated. We evaluated survival, lung histopathology, and other pulmonary antioxidants (glutathione cycle) of homozygous (-/-) and heterozygous (+/-) Sod2 mutant mice compared with wild-type controls (Sod2+/+) following 48 h exposure to either room air or to O2. The ability of antioxidant N-acetylcysteine to compensate for the loss of MnSOD was explored. Mortality of Sod2-/- mice increased from 0% in room air to 18 and 83% in 50 and 80% O2, respectively. N-acetylcysteine did not alter mortality of Sod2-/- mice. Histopathological analysis revealed abnormalities in saccules of Sod2-/- mice exposed either to room air or to 50% O2 suggestive of delayed postnatal lung development. In 50% O2, activities of glutamate-cysteine ligase (GCL) (previously known as gamma-glutamylcysteine synthetase, gamma-GCS) and glutathione peroxidase increased in Sod2-/- (35 and 70%, respectively) and Sod2+/- (12 and 70%, respectively) mice, but glutathione levels remained unaltered. We conclude that MnSOD is required for normal O2 tolerance and that in the absence of MnSOD there is a compensatory increase in pulmonary glutathione-dependent antioxidant defense in hyperoxia.

Craniofacial phenotypes in segmentally trisomic mouse models for Down syndrome.

Trisomy for chromosome 21 (Chr 21) has profound effects on development that result in a constellation of phenotypes known as Down syndrome (DS). Distinctive craniofacial manifestations are among the few features common to all individuals with DS. The characteristic face of a person with DS results primarily from maldevelopment of the underlying craniofacial skeleton. The Ts65Dn mouse, which has segmental trisomy 16, producing dosage imbalance for about half the genes found on human Chr 21, exhibits specific skeletal malformations corresponding directly to the craniofacial dysmorphogenesis in DS. Here we demonstrate that Ts1Cje mice, which are at dosage imbalance for about 3/4 of the genes triplicated in Ts65Dn, demonstrate a very similar pattern of anomalies in the craniofacial skeleton. However, one characteristic of Ts65Dn mice, a broadening of the cranial vault contributing to brachycephaly, is not seen in Ts1Cje mice. These observations independently confirm that a dosage imbalance for mouse genes orthologous to those on human Chr 21 has corresponding effects in both species. The subtle differences in the craniofacial phenotypes of Ts1Cje and Ts65Dn mice have implications for elucidation of the mechanisms by which this aneuploidy disrupts development.

The vanadium environment in blood cells of Ascidia ceratodes is divergent at all organismal levels: an XAS and EPR spectroscopic study.

K-edge X-ray absorption and EPR spectroscopies were used to test the variation in blood cell vanadium between and within specimens of the tunicate Ascidia ceratodes from Bodega Bay, California. Intracellular vanadium was speciated by fitting the XAS spectra of whole blood cells with linear combinations of the XAS spectra of models. Blood cell samples representing one specimen each, respectively, revealed 92.5 and 38.7% of endogenous vanadium as [V(H(2)O)(6)](3+), indicating dissimilar distributions. Conversely, vanadium distributions within blood cell samples respectively representing one and six specimens proved very similar. The derived array of V(III) complexes was consistent with multiple intracellular regions that differ both in pH and c(sulfate), both within and between specimens. No systematic effect on vanadium distribution was apparent on mixing blood cells. EPR and XAS results indicated at least three forms of endogenous vanadyl ion, two of which may be dimeric. An inverse linear correlation was found between soluble and complexed forms of vanadyl ion, implying co-regulation. The EPR A value of endogenous vanadyl ion [A(0)=(1.062+/-0.008)x10(-2) cm(-1)] was marginally different from that representing Monterey Bay A. ceratodes [A(0)=(1.092+/-0.006) x10(-2) cm(-1)]. Comparisons indicate that Bodega Bay A. ceratodes maintain V(III) in a more acidic intracellular environment on average than do those from Monterey Bay, showing variation across populations. Blood cell vanadium thus noticeably diverges at all organismal levels among A. ceratodes.

A flexible estimating equations approach for mapping function-valued traits.

In genetic studies, many interesting traits, including growth curves and skeletal shape, have temporal or spatial structure. They are better treated as curves or function-valued traits. Identification of genetic loci contributing to such traits is facilitated by specialized methods that explicitly address the function-valued nature of the data. Current methods for mapping function-valued traits are mostly likelihood-based, requiring specification of the distribution and error structure. However, such specification is difficult or impractical in many scenarios. We propose a general functional regression approach based on estimating equations that is robust to misspecification of the covariance structure. Estimation is based on a two-step least-squares algorithm, which is fast and applicable even when the number of time points exceeds the number of samples. It is also flexible due to a general linear functional model; changing the number of covariates does not necessitate a new set of formulas and programs. In addition, many meaningful extensions are straightforward. For example, we can accommodate incomplete genotype data, and the algorithm can be trivially parallelized. The framework is an attractive alternative to likelihood-based methods when the covariance structure of the data is not known. It provides a good compromise between model simplicity, statistical efficiency, and computational speed. We illustrate our method and its advantages using circadian mouse behavioral data.

Large-scale SNP genotyping with canine buccal swab DNA.

The dog is an attractive model for genetic studies of complex disease. With drafts of the canine genome complete, a large number of single-nucleotide polymorphisms (SNPs) that are potentially useful for gene-mapping studies and empirical estimations of canine diversity and linkage disequilibrium (LD) are now available. Unfortunately, most canine SNPs remain uncharacterized, and the amount and quality of DNA available from population-based samples are limited. We assessed how these real-world challenges influence automated SNP genotyping methods such as Illumina's GoldenGate assay. We examined 384 SNPs on canine chromosome 9 and successfully genotyped a minimum of 217 and a maximum of 275 SNPs using buccal swab samples for 181 dogs (86 beagles, 76 border collies, and 15 Australian shepherds). Call rates per SNP and sample averaged 97%, with reproducibility within and between analyses averaging 98%. The majority of these SNPs were polymorphic across all 3 breeds. We observed extensive LD, albeit less than reported for surveys using fewer dogs, consistent between breeds. Analyses of population substructure indicated that beagles are distinct from border collies and Australian shepherds. These results demonstrate the suitability of amplified canine buccal samples for high-throughput multiplex genotyping and confirm extensive LD in the dog.

Functional effects of protein sequence polymorphisms in the organic cation/ergothioneine transporter OCTN1 (SLC22A4).

BACKGROUND: OCTN1 is a multispecific transporter of organic cations and zwitterions, including several clinically important drugs as well as the antioxidant ergothioneine. OCTN1 is highly expressed in the kidney, where it is thought to aid in active secretion of organic cations, and may facilitate the active reabsorption of ergothioneine. Genetic variation in OCTN1 may help to explain interindividual variability in the pharmacokinetics of many cationic or zwitterionic drugs. METHODS: We screened for human genetic variants in the OCTN1 coding region by direct sequencing in a large sample (n=270) of ethnically diverse healthy volunteers. RESULTS: Six protein sequence-altering variants were identified, including five-amino-acid substitutions and one nonsense mutation. Two of the variants, T306I and L503F, were polymorphic, occurring at frequencies of 37 and 19%, respectively, in the total sample. Allele frequencies are varied by ethnicity. In biochemical assays, two of the variants (D165G and R282X) resulted in complete loss of transport function, and one variant (M205I) caused a reduction in activity to approximately 50% of the reference sequence protein. One variant, L503F, showed altered substrate specificity; this variant occurred at particularly high allele frequency (42%) in the European-American participants in our sample. Subcellular localization and ergothioneine inhibition kinetics were similar among the common amino-acid sequence variants of OCTN1. CONCLUSIONS: The common OCTN1-L503F variant may explain a significant amount of population variation in the pharmacokinetics of OCTN1 substrate drugs. The rare loss-of-function variants provide a rational tool for studying the importance of ergothioneine in humans in vivo.

Genetic modifiers of the phenotype of mice deficient in mitochondrial superoxide dismutase.

Sod2-/- mice, which are deficient in the mitochondrial form of superoxide dismutase (MnSOD), have a short survival time that is strongly affected by genetic background. This suggests the existence of genetic modifiers that are capable of modulating the degree of mitochondrial oxidative damage caused by the MnSOD deficiency, thereby altering longevity. To identify these modifier(s), we generated recombinant congenic mice with quantitative trait loci (QTL) containing the putative genetic modifiers on the short-lived C57BL/6J genetic background. MnSOD deficient C57BL/6J mice with a QTL from the distal region of chromosome 13 from DBA/2J were able to survive for as long as those generated on the long-lived DBA/2J background. Within this region, the gene encoding nicotinamide nucleotide transhydrogenase (Nnt) was found to be defective in C57BL/6J mice, and no mature NNT protein could be detected. The forward reaction of NNT, a nuclear-encoded mitochondrial inner membrane protein, couples the generation of NADPH to proton transport and provides NADPH for the regeneration of two important antioxidant compounds, glutathione and thioredoxin, in the mitochondria. This action of NNT could explain its putative protective role in MnSOD-deficient mice.

Functional genetic diversity in the high-affinity carnitine transporter OCTN2 (SLC22A5).

Systemic carnitine deficiency (SCD) is a rare autosomal recessive disease resulting from defects in the OCTN2 (SLC22A5) gene, which encodes the high-affinity plasma membrane carnitine transporter. Although OCTN2 is fairly well studied in its relationship with SCD, little is known about the carrier frequency of disease-causing alleles of OCTN2, or of more common functional polymorphisms in this gene. To address these issues, we screened for genetic variants in the OCTN2 coding region by direct sequencing of the exons and flanking intronic region of OCTN2 in a large sample (n = 276) of ethnically diverse subjects. In addition, we established lymphoblastoid cell lines from subjects homozygous for either allele of the previously identified promoter region variant, -207G>C. We found eight amino acid sequence variants of OCTN2, of which three (Phe17Leu, Leu144Phe, and Pro549Ser) were polymorphic in at least one ethnic group. When assayed for functional activity by expression in human embryonic kidney 293 cells, using as probes both the endogenous substrate (l-carnitine) and the organic cation tetraethylammonium, three variants showed functional differences from the reference OCTN2 (Phe17Leu, Tyr449Asp, Val481Phe; p < 0.05). Further studies of the Phe17Leu polymorphism showed a reduced V(max) for l-carnitine transport to approximately 50% of the reference OCTN2. Confocal microscopy studies using an OCTN2-GFP fusion protein showed that Phe17Leu had distinct subcellular localization from the reference OCTN2, with diffuse cytoplasmic retention of Phe17Leu, in contrast to reference OCTN2, which localized specifically to the plasma membrane. Lymphoblasts from subjects homozygous for the -207G allele showed increased l-carnitine transport compared with the -207C/C homozygotes (p < 0.05). This study suggests that although loss-of-function mutations in OCTN2 are likely to be rare, common variants of OCTN2 found in healthy populations may contribute to variation in the disposition of carnitine and some clinically used drugs.

Reversal of Alzheimer's-like pathology and behavior in human APP transgenic mice by mutation of Asp664.

The deficits characteristic of Alzheimer's disease (AD) are believed to result, at least in part, from the neurotoxic effects of beta-amyloid peptides, a set of 39-43 amino acid fragments derived proteolytically from beta-amyloid precursor protein (APP). APP also is cleaved intracytoplasmically at Asp-664 to generate a second cytotoxic peptide, APP-C31, but whether this C-terminal processing of APP plays a role in the pathogenesis of AD is unknown. Therefore, we compared elements of the Alzheimer's phenotype in transgenic mice modeling AD with vs. without a functional Asp-664 caspase cleavage site. Surprisingly, whereas beta-amyloid production and plaque formation were unaltered, synaptic loss, astrogliosis, dentate gyral atrophy, increased neuronal precursor proliferation, and behavioral abnormalities were completely prevented by a mutation at Asp-664. These results suggest that Asp-664 plays a critical role in the generation of Alzheimer-related pathophysiological and behavioral changes in human APP transgenic mice, possibly as a cleavage site or via protein-protein interactions.