s2fieldsofstudy: Biology Psychology

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DNA Evidence for Strong Genome-Wide Pleiotropy of Cognitive and Learning Abilities

Very different neurocognitive processes appear to be involved in cognitive abilities such as verbal and non-verbal ability as compared to learning abilities taught in schools such as reading and mathematics. However, twin studies that compare similarity for monozygotic and dizygotic twins suggest that the same genes are largely responsible for genetic influence on these diverse aspects of cognitive function. It is now possible to test this evidence for strong pleiotropy using DNA alone from samples of unrelated individuals. Here we used this new method with 1.7 million DNA markers for a sample of 2,500 unrelated children at age 12 to investigate for the first time the extent of pleiotropy between general cognitive ability (aka intelligence) and learning abilities (reading, mathematics and language skills). We also compared these DNA results to results from twin analyses using the same sample and measures. The DNA-based method revealed strong genome-wide pleiotropy: Genetic correlations were greater than 0.70 between general cognitive ability and language, reading, and mathematics, results that were highly similar to twin study estimates of genetic correlations. These results indicate that genes related to diverse neurocognitive processes have general rather than specific effects.

Introduction
Very different neurocognitive processes appear to be involved in cognitive abilities such as reasoning and mathematics (Deary 2000) However, quantitative genetic research, largely based on twin studies, consistently indicates that genes that affect individual differences in performance in one domain are largely the same genes that affect performance in other domains, leading to the Generalist Genes Hypothesis (Plomin and Kovas 2005).
It is now possible to use DNA itself to estimate genetic influence in any sample of unrelated individuals rather than relying on comparisons between monozygotic and dizygotic twins. The method, implemented in a tool called Genome-wide Complex Trait Analysis (GCTA; Yang et al. 2011a) does not attempt to identify specific genes associated with traits. Instead, it correlates genomic similarity across hundreds of thousands of single nucleotide polymorphisms (SNPs) with phenotypic similarity in a large sample of unrelated individuals (Yang et al. 2010). This population-based approach does not rely on the strong assumptions made in classical twin studies.
Univariate Linear Mixed Model (LMM) implemented in the GCTA package has been used to estimate genetic influence for height and body mass index (Yang et al. 2010(Yang et al. , 2011b, psychiatric and medical disorders (Lee et al. 2011), personality (Vinkhuyzen et al. 2012), and cognitive abilities (Davies et al. 2011;Plomin et al. 2013b). In contrast to univariate genetic analysis, bivariate genetic analysis focuses on the genetic correlation, the correlation between genetic influences on different traits, called pleiotropy (Plomin et al. 2013a). High genetic correlations between phenotypes are often interpreted as an indication that the same genes affect the phenotypes. Genetic correlations between diverse cognitive abilities as estimated through twin studies are typically greater than 0.60, indicating that cognition-related genes largely have general pleiotropic effects (Calvin et al. 2012;Plomin and Kovas 2005). However, the genetic correlation estimated from twin studies could be biased due to misspecification of the model of twin similarity for genetic and non-genetic effects. In this study, we use the GCTA package to estimate the genetic correlation between traits in conventionally unrelated individuals based on DNA evidence alone; this estimate is free of bias if we assume that the sole reason for phenotypic similarity between conventionally unrelated individuals is shared additive genetic factors. For brevity, we refer to LMM used in the GCTA package simply as GCTA.
Here we use bivariate GCTA Yang et al. 2011a) to test the Generalist Genes Hypothesis by estimating genetic correlations between general cognitive ability ('g', aka intelligence) and language, reading, and mathematics. We compare these genetic correlation estimates from GCTA to those obtained from the twin design using the same sample assessed at the same age with the same measures. We also analyze the variables of height and weight for purposes of comparison.

Sample and genotyping
The sample was drawn from the Twins Early Development Study (TEDS), which is a multivariate longitudinal study that recruited over 11,000 twin pairs born in England and Wales in 1994, 1995and 1996(Haworth et al. 2012Oliver and Plomin 2007). TEDS has been shown to be representative of the UK population (Kovas et al. 2007). The project received approval from the Institute of Psychiatry ethics committee (05/Q0706/228) and parental consent was obtained prior to data collection.
Cognitive and DNA data were available for 3,747 11and 12-year-old children whose first language was English and had no major medical or psychiatric problems. From that sample, 3,665 DNA samples were successfully hybridized to Affymetrix GeneChip 6.0 SNP genotyping arrays using standard experimental protocols as part of the WTCCC2 project (for details see Trzaskowski et al. 2013). In addition to nearly 700,000 genotyped SNPs, more than one million other SNPs were imputed from HapMap 2, 3 and WTCCC controls using IMPUTE v.2 software (Howie et al. 2009). 3,152 DNA samples (1,446 males and 1,706 females) survived quality control criteria for ancestry, heterozygosity, relatedness, and hybridization intensity outliers. To control for ancestral stratification, we performed principal component analyses on a subset of 100,000 quality-controlled SNPs after removing SNPs in linkage disequilibrium (r 2 [ 0.2) (Fellay et al. 2007). Using the Tracy-Widom test (Patterson et al. 2006), we identified 8 axes with p \ 0.05, which were used as covariates in GCTA analyses.
The mean age of the sample was 11.5 years (SD = 0.66). The sample sizes for the GCTA results shown in Table 1 are 2,325 for 'g' and language, 2,238 for 'g' and mathematics, 2,250 for 'g' and reading, and 2,296 for height and weight. For the twin analyses, cognitive data were available for 5,434 twin pairs (Davis et al. 2009); however, the twin analyses presented here were based only on twins included in the GCTA analyses in order to provide a more precise comparison between GCTA and twin-study results. The numbers of twin pairs were 2,205, 2,095, 2,104 and 2,162, respectively.

Measures
Cognitive data were collected online via the Internet using, where possible, adaptive branching, which enabled measurement of the full range of ability using a relatively small number of items. Details about the following measures, including references, are available elsewhere (Kovas et al. 2007).

General cognitive ability (g)
'g' was assessed from two verbal tests and two non-verbal tests. The verbal tests included WISC-III-PI Multiple Choice Information (General Knowledge) and Vocabulary Composite measures for 'g', language, mathematics, and reading. For each cognitive measure, outliers above or below 3 SD from the mean were excluded. Scores were regressed on sex and age, and standardized residuals were derived and quantile normalized (Lehmann 1975;van der Waerden 1975). Composite measures for 'g', language, mathematics, and reading were created as unit-weighted means requiring complete data for at least 3 of the 4 tests for 'g' and reading and 2 of 3 tests for language and mathematics. All procedures were executed using R (www.r-project.org; R Development Core Team 2011). The phenotypic correlations among the composite measures were 0.63 for 'g' and language, 0.63 for 'g' and mathematics, and 0.57 for 'g' and reading.

Height and weight
Height and weight were assessed on the same sample (age 12) via self-report. Similar to the cognitive measures, outliers (± 3SD) were removed and scores were controlled for age and sex. The phenotypic correlation between height and weight was 0.63.

GCTA
Conceptually, the amount of phenotypic variance, or covariance, explained by genetic factors is estimated by a comparison of a matrix of pairwise genomic similarity to a matrix of pairwise phenotypic similarity (Yang et al. 2010). Before the variance or covariance can be decomposed into genetic and residual components, we need to calculate pairwise genomic similarity between all pairs of individuals in the sample using all genetic markers genotyped on the SNP array. Because the GCTA package uses a random effects model to estimate genetic effects from a sample of unrelated individuals in the population, any pair whose genetic similarity is equal to or greater than a fourth cousin is removed (estimate of pairwise relatedness [0.025). In univariate analysis, the variance of a trait can be partitioned using residual maximum likelihood into genetic and residual components. Detailed description of this method can be found in GCTA publications (Yang et al. 2010(Yang et al. , 2011a. The bivariate method extends the univariate model by relating the pairwise genetic similarity matrix to a phenotypic covariance matrix between traits 1 and 2 ). The eight principal components described earlier were used as covariates in our bivariate GCTA analyses; as mentioned in the previous section, all phenotypes were age-and sexregressed prior to analysis.
Twin modelling. The twin design and model-fitting is discussed elsewhere (Plomin et al. 2013a). We fit a bivariate Cholesky decomposition using OpenMx (Boker et al. 2011), which provided a direct comparison with the bivariate GCTA. The correlated factor solution is the least restricted model allowing variables to correlate with one another via genetic, shared environment, and non-shared environment. Because previous analyses of these data indicated nonsignificant differences in model-fitting results between males and females (Kovas et al. 2007), we combined same-sex and opposite-sex DZ twin pairs in order to increase the power of the analyses. Table 1 shows GCTA-estimated genetic correlations (and standard errors, SE) between 'g' and learning abilities for more than 2,238 12-year-old UK twins (randomly selecting only one member of each twin pair to control for potential confounds, such as birth order) based on 1.7 million SNPs measured from the Affymetrix 6.0 GeneChip or imputed from HapMap 2,3 and WTCCC controls (Trzaskowski et al. 2013). Genetic correlations are significant and Table 2 Bivariate Genome-wide Complex Trait Analysis (GCTA) results (with standard errors) for general cognitive ability ('g') versus language, mathematics, and reading, as well as comparison data for: g and height, g and weight, and height and weight Variables GCTA incorporates full-information maximum likelihood that uses the full sample of more than 2,900 individuals with data on trait 1 or trait 2. However, the variance estimates for each trait are based on individuals with data for that trait and the covariance estimates are based on individuals with data for both traits. The n reported in the last column is the most conservative, i.e., the n that was used for the estimation of the covariance V(G) variance explained by genetic factors for trait 1 and trait 2 (tr1, tr2), C(G) covariance between trait 1 and 2 explained by genetic factors;

Results
V(e) residual variance for trait 1 and trait 2, C(e) residual covariance between trait 1 and trait 2; Vp The current version of GCTA does not report the environmental correlation or its standard error. The environmental correlation was derived here from the GCTA estimates using the following algorithm: C(e)_tr12/(HV(e)_tr1 9 HV(e)_tr2), whereas the standard error was calculated using: Var(re) = re 9 re 9 (VarVe1/(4 9 Ve1 9 Ve1) ? VarVe2/(4 9 Ve2 9 EV) ? VarCe/ (Ce 9 Ce) ? CovVe1Ve2/(2 9 Ve1 9 Ve2 -CovVe1Ce/(Ve1 9 Ce) -CovVe2Ce/(Ve2 9 Ce)); SE(re) = sqrt[Var(re)], where re is the environmental correlation, Ve1 is the residual variance for trait 1, Ce is the residual covariance between two traits, VarVe1 is the sampling variance for Ve1 (residual variance for trait 1), VarCe is the sampling variance for Ce, CovVe1Ve2 is the sampling covariance between Ve1 and Ve2, and CovVe1Ce is the sampling covariance between Ve1 and Ce substantial for all three comparisons-between 'g' and language (0.81), mathematics (0.74), and reading (0.89). The GCTA-estimated genetic correlations between 'g' and learning abilities are similar in magnitude to the GCTAestimated genetic correlation between height and weight (0.76). In addition, Table 1 includes bivariate results for 'g' versus height and 'g' versus weight as 'negative controls'; their phenotypic correlations are both 0.07. As expected, these comparisons yielded negligible and nonsignificant genetic correlations (-0.03 and -0.06, respectively). Table 1 also includes analogous genetic correlations from twin model-fitting analyses, as estimated from the same twin sample but including the co-twins (more than 2,095 pairs of twins). The GCTA-estimated genetic correlations are highly similar to the twin study estimates and do not differ significantly, as indicated by their overlapping standard errors. The similarity of GCTA and twin estimates of genetic correlations extend to the comparison between height and weight as well as the negative control comparisons of 'g' and height and 'g' and weight. Tables 2 and 3 show full results from the bivariate GCTA and twin analyses, respectively.

Discussion
Using DNA evidence alone, these high genetic correlations estimated from GCTA support the Generalist Genes Hypothesis in showing strong pleiotropy between 'g' and learning abilities, especially because we show that these GCTA-estimated genetic correlations are as high as genetic correlations estimated from the twin design.
Although GCTA does not identify specific genes associated with these traits, it addresses a critical issue in genomewide association studies: the extent to which common SNPs used on commercially available SNP arrays can account for the heritability of quantitative traits (Yang et al. 2011b). We have shown in univariate GCTA analyses that, if samples were sufficiently large, common SNPs could account for more than two-thirds of the heritability of cognitive abilities estimated in twin studies (Yang et al. 2011b; see also Table 2). Why are univariate GCTA heritability estimates less than the twin study estimates of heritability? As discussed elsewhere (e.g. Yang et al. 2010), the main problem is imperfect tagging. The common SNPs used on all available commercial arrays only capture what is in LD with them. Rare variants, which have lower minor allele frequency, will thus not be 'tagged' and their influence will be missed. In addition, GCTA estimates additive genetic influence only, so that non-additive effects (gene-gene and gene-environment interaction) are not captured either.
A more novel question, and central to the present paper, is why, as we have shown here, bivariate genetic Table 3 Bivariate twin model-fitting results (with standard errors) for general cognitive ability ('g') versus language, mathematics, and reading, as well as comparison data for: g and height, g and weight, and height and weight Variables OpenMx twin model-fitting incorporates full-information maximum likelihood that uses the full sample of more than 2,000 pairs of twins with data on trait 1 or trait 2. However, the variance estimates for each trait are based on individuals with data for that trait. The covariance estimates are based on twin pairs with data for both traits, which is the conservative sample size shown in the last column V(G) proportion of the variance explained by genetic factors for trait 1 and trait 2 (tr1, tr2), C(G) proportion of the covariance between trait 1 and 2 explained by genetic factors; V(c) proportion of the variance explained by shared environment for trait 1 and trait 2 (tr1, tr2), C(c) proportion of the covariance between trait 1 and 2 explained by shared environment, V(e) proportion of the variance explained by non-shared environment for trait 1 and trait 2 (tr1, tr2), C(e) proportion of the covariance between trait 1 and 2 explained by non-shared environment, rG correlations estimated by GCTA are as great as twin study estimates. The likely reason is that attenuation of the estimated additive genetic variance due to imperfect linkage disequilibrium between causal variants and genotyped SNPs applies to both the additive genetic variance of the two traits and to their additive genetic covariance by the same proportion. Thus, the GCTA estimate of the genetic correlation is unbiased because it is derived from the ratio between genetic covariance and the genetic variances of the two traits.
Are generalist genes all in the mind (cognition) or are they in the brain as well? That is, genetic correlations between cognitive and learning abilities might be epiphenomenal in the sense that multiple genetically independent brain mechanisms could affect each ability, creating genetic correlations among abilities. However, the genetic principles of pleiotropy (each gene affects many traits) and polygenicity (many genes affect each trait) lead us to predict that generalist genes have their effects further upstream, creating genetic correlations among brain structures and functions, a prediction that supports a network view of brain structure and function.


extfieldsofstudy: Biology Medicine

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Internalization of B Cell Receptors in Human EU12 μHC+ Immature B Cells Specifically Alters Downstream Signaling Events

It has been recognized for a long time that engagement of B cell antigen receptors (BCRs) on immature B cells or mature B cells leads to completely opposite cell fate decisions. The underlying mechanism remains unclear. Here, we show that crosslinking of BCRs on human EU12 μHC+ immature B cells resulted in complete internalization of cell surface BCRs. After loss of cell surface BCRs, restimulation of EU12 μHC+ cells showed impaired Ca2+ flux, delayed SYK phosphorylation, and decreased CD19 and FOXO1 phosphorylation, which differ from those in mature Daudi or Ramos B cells with partial internalization of BCRs. In contrast, sustained phosphorylation and reactivation of ERK upon restimulation were observed in the EU12 μHC+ cells after BCR internalization. Taken together, these results show that complete internalization of cell surface BCRs in EU12 μHC+ cells specifically alters the downstream signaling events, which may favor receptor editing versus cell activation.

Introduction
The development of B lymphocytes from hematopoietic progenitor cells in the bone marrow (BM) into mature, circulating B cells can be divided into distinct stages according to the expression of specific cell surface markers as well as the rearrangement status of the IgH and L chain gene loci [1,2]. Immature B cells are the first to express the complete form of the B cell antigen receptors (BCRs), which can recognize specific antigens [3]. The BCRs expressed on immature and mature B cells are all composed of membrane bound IgH/IgL peptides with noncovalently linked signaling components, the Ig /Ig heterodimers [4,5]. Although the BCRs expressed on the immature and the mature B cells share the same components, stimulation through BCRs results in totally opposite outcomes. In mature B cells, BCR stimulation induces cell activation and proliferation, antibody affinity maturation, and class-switch recombination during T celldependent responses and ultimately drives B cells differentiating into memory B cells or antibody-secreting plasma cells [5][6][7]. In contrast, stimulation of immature B cells via BCRs leads to receptor editing or clonal deletion [8][9][10][11].
Most of our current understandings of BCR-mediated signaling events are derived from analyses of mature B cells. One of the early signal events following BCR crosslinking is protein tyrosine kinases (PTKs) Lyn and Syk-mediated phosphorylation of the tyrosine residues in the two immunoreceptor tyrosine-based activation motifs (ITAMs) in Ig and Ig [5,12,13]. Lyn predominantly phosphorylates the first ITAM tyrosine, whereas Syk phosphorylates and binds to both ITAM tyrosine residues [14]. The adaptor protein SH2 domain-containing leukocyte protein of 65 kDa (BLNK) is then recruited to the BCR complex, where it is phosphorylated by Syk [15]. Tyrosine phosphorylated BLNK serves as an adaptor to organize the signalosome by recruiting Btk, Vav, PLC 2, Grab2, and Rac1 [16]. Formation of this signalosome is important for full induction of Ca ++ influx and activation of the downstream PI3K, MAPK, and NF-B signaling pathways [17,18].
Human EU12 HC + cells have features of BM immature B cells [19][20][21]. Our previous studies have shown that replacement occurs spontaneously in the EU12 HC + cells [20]. Moreover, crosslinking of BCRs results in arrest of cell proliferation and induction of replacement [20,21]. Thus, the EU12 HC + cells were used as an experimental model to dissect the BCR-mediated signaling events in immature B cells versus those in mature B cells. We noticed that treatment of EU12 HC + cells with F(ab ) 2 anti-IgM antibodies resulted in complete internalization of cell surface BCRs, which is in sharp contrast to the partial internalization of BCRs observed in Daudi or Ramos mature B cells. Detailed analyses showed that complete BCR internalization specifically changed BCRmediated downstream signaling events in the EU12 HC + immature B cells with attenuated Ca ++ influx and CD19 and FOXO1 phosphorylation, but with sustained ERK activation. Such changes may favor the induction of receptor editing rather than cell proliferation in these cells.

Cell Lines and Treatments.
The EU12 HC + cells are purified by flow cytometric sorting from the parental EU12 cells, which were established from an acute lymphocytic childhood leukemia patient [20,21]. Ramos and Daudi are B cell lymphomas obtained by tissue culture adaptation of Burkitt's lymphomas [22][23][24]. In all experiments, cells were cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA), supplemented with 10% heat-inactivated FBS, 100 units/mL penicillin/streptomycin, 50 M -mercaptoethanol, and 2 mM Lglutamine. Cells were maintained at 0.1 to 1 × 10 6 cells per mL. For direct stimulation, cells were stimulated with goat F(ab ) 2 fragments of anti-human IgM antibodies (10 g/mL) (Southern Biotech, Birmingham, AL) at room temperature. For restimulation experiments, cells were pretreated with goat F(ab ) 2 anti-human IgM antibody fragments at 2 g/mL for 30 min; then, the cells are washed with fresh tissue culture medium to remove excessive anti-IgM antibodies, recovered in complete growth medium for 2 or 4 h, and then restimulated with 10 g/mL goat F(ab ) 2 anti-human IgM antibody or the monoclonal anti-human IgM antibody (BD Biosciences). Ca ++ influx, Western blot, and immunofluorescence analyses were all performed under these two conditions.

Cell Surface Staining and Flow Cytometric Analysis.
For cell surface staining, cells (1 × 10 6 cells) were blocked with PBS buffer with 2% FBS and stained with Cy5-labeled anti-human IgM F(ab ) 2 antibodies, FITC-labeled anti-HLA antibodies, or APC-labeled anti-CD86 antibodies on ice for 30 min [21]. After washing with PBS buffer with 2% FBS, samples were analyzed on an Accuri cytometric analyzer. Dead cells were excluded by PI staining. The results were then analyzed using the CFlow Plus software (Accuri Cytometers Inc., MI). 2+ Influx Assays. Intracellular Ca 2+ levels were monitored by flow cytometric analyses using the Fluo-3AM fluorescence dye (Molecular Probes, Eugene, OR) [21]. Briefly, EU12 HC + cells (10 6 cells/mL) were preloaded with 1 M Fluo-3AM with pluronic-F127 at 1 : 1 ratio for 30 min at 37 ∘ C in the dark. Cells were washed and resuspended in RPMI media containing 1% FBS. Cells were maintained in the dark at room temperature before use. Samples were analyzed on an Accuri cell analyzer. Unstimulated cells were used to establish the baseline of fluorescence. Different stimuli were added at 30 sec after running the samples on the Accuri cell analyzer and continued for 4 min. Flow cytometry data were analyzed using the CFlow Plus software (Accuri Cytometers Inc., MI).

Anti-IgM Antibody-Mediated BCR Internalization.
Human EU12 HC + , Daudi, and Ramos cells or murine 70Z/3 cells were used in these experiments. Approximately 1 × 10 6 cells were incubated with goat anti-human IgM antibody F(ab ) 2 fragments (2 g/mL) for different human cell lines or goat anti-mouse IgM antibody F(ab ) 2 fragments (5 g/mL) (Southern Biotech, AL) at 37 ∘ C. Cells were collected at different time points and cell surface BCR levels were analyzed by flow cytometry.
2.6. Immunofluorescence Analysis. Different cells were directly stimulated or restimulated as described above. After treatment, cells were fixed with 3.7% formaldehyde at room temperature for 10 min in the dark, followed by incubation with 0.1% Triton X-100 for 5 min on ice. Untreated cells were used for both negative and secondary antibody staining controls. Cells were washed twice with 1 x PBS and blocked with PBS + 1% goat serum at room temperature for an hour. Anti-phospho-SYK or anti-phospho-ERK antibodies (1 : 200 dilution in PBS + 2% BSA) were added to the samples and incubated at room temperature for 1 h in the dark. After extensive washing, bound antibodies were detected with goat anti-rabbit IgG-Alxa488 secondary antibody (1 : 1000 dilution) (Invitrogen, Carlsbad, CA) and visualized under an Olympus X81 fluorescence microscope. Images were captured with 60X lens under oil and analyzed using the Slidebook 5 software.

Western Blot Analysis.
For analysis of global protein tyrosine phosphorylation, EU12 HC + , Daudi, and Ramos cells (10 × 10 6 /mL) were either directly stimulated with antihuman IgM F(ab ) 2 antibody fragments (10 g/mL) for 3 min or pretreated with goat anti-human IgM F(ab ) 2 antibody fragments (2 g/mL) for 30 min, washed, recovered in complete RPMI medium for 4 h, and then restimulated with goat anti-human IgM antibody F(ab ) 2 fragments (10 g/mL) for 3 min. Cells were washed once with 1 x PBS; whole cell lysates were prepared using 1% TNT buffer [21,25]. Samples were separated onto 10% SDS-PAGE. The phosphorylation status of the BCR signaling molecules CD19, LYN, SYK, BLNK, ERK, AKT, and FOXO1 was then analyzed by Western blot with phosphospecific antibodies (Cell Signaling Technology, Danvers, MA).

Crosslinking BCRs Induces Different Levels of BCR Internalization in Human Immature versus Mature B Cells.
Our previous studies showed that treatment of EU12 HC + cells with goat anti-human IgM antibody F(ab ) 2 fragments resulted in complete internalization of surface BCRs [21]. Here, we compared anti-IgM antibody-mediated BCR internalization in the EU12 HC + immature B cell, versus those in Daudi and Ramos mature B cells. In the EU12 HC + cells, crosslinking BCRs with anti-IgM antibodies resulted in quick BCR internalization, which can be seen as soon as 1 min (Figure 1 To determine if complete internalization of cell surface BCRs is a general mechanism for early B lineage cells, we analyzed a panel of murine early B lineage cells and found that crosslinking cell surface BCRs also induced complete internalization of cell surface BCRs in 70Z/3 cells (Figure 1(b)).
To further verify these findings, the three lines of cells were treated with or without Cy5 labeled F(ab ) 2 antihuman IgM antibody fragments (2 g/mL) at 37 ∘ C for 30 min, and cell surface BCR expression was visualized under immunofluorescence microscopy. In untreated cells, the labeled BCRs were distributed on the cell surface. After treatment, all the Cy5 labeled anti-IgM antibodies were internalized into the EU12 HC + cells. For Daudi and Ramos cells, a large fraction of Cy5 labeled anti-IgM antibodies still remained on the cell surface under the same treatment (Figure 1(c)). These results show that the EU12 HC + immature B cells are highly sensitive to BCR crosslinking and completely internalize their cell surface BCRs, which are different from that in the mature B cells.
Moreover, after treatment of EU12 HC + cells or Daudi cells with anti-IgM antibodies (2 g) for 24 hours, we analyzed cell surface HLA and CD86 expression as indicators for B cell activation. Upon anti-IgM antibody treatment, Daudi cells have elevated HLA and CD86 expression. In contrast, the expression levels of HLA and CD86 are not changed in EU12 HC + cells upon anti-IgM antibody treatment (Figure 1(d)). These results indicated that crosslinking cell surface BCRs activated different signaling in the EU12 HC + cells or in Daudi cells.

Complete Internalization of BCRs in the EU12 HC + Cells
Reduces Ca 2+ Influx upon Restimulation. To determine the effects of complete BCR internalization on subsequent BCRmediated signaling events, we compared EU12 HC + , Daudi, and Ramos cells for their capabilities to evoke calcium influx upon BCR stimulation before and after BCR internalization. Direct stimulation of the three lines of cells with goat anti-human IgM antibody F(ab ) 2 fragments (10 g/mL or 20 g/mL) induced Ca 2+ influx in a dose-dependent manner (Figure 2(b)). During the restimulation experiments, EU12 HC + cells showed dramatically reduced Ca 2+ influx compared to that during direct stimulation or those in restimulated Daudi and Ramos cells (Figures 2(a) and 2(b)). These results revealed that complete internalization of BCRs on the EU12 HC + cells reduced Ca 2+ influx during BCR restimulation.

Complete BCR Internalization in the EU12 HC + Cells Specifically Changes BCR-Mediated Downstream Signaling
Events. To further determine if BCR internalization affects BCR-mediated downstream signaling events, we performed Western blot analyses to determine the phosphorylation status of different BCR signaling molecules. As described before, the three lines of cells were either directly stimulated with goat anti-human IgM antibody F(ab ) 2 fragments (10 g/mL) or pretreated with goat anti-human IgM antibody F(ab ) 2 fragments (2 g/mL) at 37 ∘ C for 30 min to first internalize cell surface BCRs and then rechallenge them at 2 h or 4 h with goat anti-human IgM antibody F(ab ) 2 fragments (10 g/mL). With direct stimulation, EU12 HC + immature B cells exhibited similar activation of downstream signaling molecules, including CD19, SYK, ERK, AKT, and FOXO1, compared to those in mature Daudi and Ramos cells. However, BCR signaling induced BLNK phosphorylation was almost undetectable in the EU12 HC + cells (Figure 3(b)). Restimulation of EU12 HC + cells induced no apparent changes of global protein Tyrosine phosphorylation at 2 h or 4 h time points. Interestingly, restimulation of EU12 HC + cells did not induce SYK and FOXO1 phosphorylation at 3 min but still induced similar levels of ERK phosphorylation. Taken together, complete internalization of BCRs in the EU12 HC + cells not only attenuates BCRmediated signaling events, but also specifically changes the downstream signaling events with reduced SYK activation but with sustained ERK activation.  study, however, after complete internalization of BCRs in EU12 HC + cells, restimulation with anti-IgM antibody F(ab ) 2 fragments (10 g/mL) did not activate SYK at 3 min.

Delayed SYK Activation and
To further determine the activation status of SYK in the EU12 HC + cells during restimulation process, we analyzed SYK phosphorylation at different time points after either direct stimulation or restimulation. During direct stimulation, SYK phosphorylation can be detected as soon as 1 min after BCR crosslinking. It reached the peak level of phosphorylation at 3 min and then diminished after 10 min. In contrast, in restimulated EU12 HC + cells, SYK phosphorylation was barely detectable at 1 or 3 min, increased at 10 and 15 min, and lasted till 30 min (Figure 4). The level of CD19 phosphorylation was also reduced during restimulation. Surprisingly, ERK phosphorylation was sustained after 30 min even after all the cells lost their cell surface BCRs during the direct stimulation ( Figure 4, lane 6). The level of ERK phosphorylation was further enhanced during restimulation after 2 h (Figure 4,  lanes 8 and 9). Similar results were obtained using either goat F(ab ) 2 anti-IgM antibody fragments or monoclonal antihuman IgM antibodies. These results support that complete internalization of BCRs in the EU12 HC + immature B cells altered the downstream signaling pathways.

Distribution of Phosphorylated SYK and ERK in
Stimulated EU12 HC + Cells. Next, we performed immunofluorescence staining to visualize phosphorylated SYK and ERK proteins in these three lines of cells upon direct stimulation or restimulation. In directly stimulated EU12 HC + , Daudi, or Ramos cells, crosslinking BCR with Cy5 labeled F(ab ) 2 anti-IgM antibodies (10 g/mL) induced SYK and ERK phosphorylation (Figures 5(a) and 5(b), left panels). In restimulated EU12 HC + cells, SYK phosphorylation was almost undetectable. Interestingly, phosphorylated ERK could be detected inside of the EU12 HC + cells even 4 h after the first stimulation and was further enhanced by restimulation with anti-IgM antibodies (Figures 5(a) and 5(b), right panels). These results are consistent with the Western blot results, which indicate that complete internalization of BCRs in the EU12 HC + cells specifically changes BCR-mediated downstream signaling with sustained ERK activation.
editing, clonal deletion, or anergy; for mature B cells, stimulation of BCR induces cell activation and proliferation. Understanding how the same BCRs can generate different signals is a long standing question in the immunology research field.
We are currently using the EU12 HC + cells as an experimental model system to study the molecular regulation of replacement. The EU12 HC + cells phenotypically resemble human bone marrow immature B cells [20]. We notice that  Figure 4: Internalization of BCR results in delayed activation of SYK and sustained activation of ERK1/2 in EU12 HC + cells upon restimulation. EU12 HC + cells were directly stimulated with goat anti-IgM antibody F(ab ) 2 fragments (10 g/mL) or pretreated with goat anti-IgM antibody F(ab ) 2 fragments (2 g/mL) for 30 min, washed, recovered, and restimulated upon goat anti-IgM antibody F(ab ) 2 fragments (10 g/mL). Cells were collected at 0, 1, 3, 10, 15, and 30 min and analyzed for CD19, SYK, and ERK phosphorylation. The membranes were stripped and blotted with anti-CD19, anti-SYK, anti-ERK, or anti--ACTIN antibodies as controls.
the EU12 HC + cells are highly sensitive to BCR crosslinking that internalizes their cell surface BCRs [21]. Treatment of EU12 HC + cells with low concentration of goat anti-human IgM antibody F(ab ) 2 fragments (2 g/mL) resulted in quick internalization of cell surface BCRs, which could be seen as soon as 1 min after adding the antibodies. After 30 min, almost all the EU12 HC + cells lost their cell surface BCRs. The complete BCR internalization observed with the EU12 HC + cells was different from the partial BCR internalization that occurred in mature B lineage Daudi or Ramos cells. Under the same stimulations, the majority of the Daudi and Ramos cells still have their cell surface BCRs, although the levels of cell surface BCRs are slightly reduced. Complete internalization of cell surface BCRs upon BCR crosslinking seems to be a general phenomenon for early B linage cells, because upon anti-IgM antibody treatment, murine 70Z/3 cells also completely internalize cell surface BCRs. Currently, it is not clear why EU12 HC + cells and 70Z/3 cells quickly and completely internalize their cell surface BCRs upon anti-IgM treatment. It has been shown that another murine B lineage cell line, WEHI231, which represents murine immature B cells, did not internalize cell surface BCRs upon anti-IgM antibody treatment [28]. It will be very interesting to further compare and contrast BCR signaling mediated events in these early B lineage cell lines to identify the responsive factors that control BCR internalization.
Treatment of EU12 HC + cells or Daudi cells with the same concentration of anti-IgM antibodies resulted in different activation of HLA and CD86. We speculate that internalization of cell surface BCRs will impact the downstream signaling events. Detailed analysis showed that restimulation of EU12 HC + cells after complete internalization of cell surface BCRs showed dramatic reduction of BCR induced Ca ++ influx. Furthermore, after BCR internalization, restimulated EU12 HC + cells failed to induce global protein tyrosine phosphorylation even with higher concentration of the stimulating anti-IgM antibodies at 2 h or 4 h. Under the same treatment, Ca ++ influx in Daudi or Ramos cells was also attenuated, but restimulation still induced strong protein tyrosine phosphorylation. Such results can be easily explained as that reducing the cell surface BCR expression levels attenuates BCR-mediated signaling events.
More specifically, after complete internalization of BCRs, restimulation of EU12 HC + cells resulted in a delayed SYK phosphorylation and reduced CD19 and FOXO1 phosphorylation. Such changes are consistent with the low level of BCR expression on the EU12 cell surface. However, anti-IgM antibody-mediated activation of ERK in the EU12 HC + cells was not affected by complete internalization of BCRs. on the contrary, sustained ERK phosphorylation was detected in the EU12 HC + cells even after they lost cell surface BCRs. Restimulation of EU12 HC + cells induced a similar wave of ERK activation. These results show that internalization of BCR in the EU12 HC + cells specifically changed BCRmediated downstream signaling events with sustained ERK activation but reduced SYK, CD19, and FOXO1 phosphorylation. Such changes may be due to the differential activation thresholds of individual signaling factors. For ERK, the activation threshold may be very low. With minimal levels of cell surface BCR stimulation, ERK can be phosphorylated to activate the downstream signaling events. On the other hand, the sustained ERK activation after BCR internalization suggested that the internalized BCRs within the endosomes might still be able to activate ERK. Such low threshold of ERK activation and sustained ERK activation might be critical for immature B cells for induction of receptor editing and negative selection with even low affinity selfantigens. However, after internalization of the majority of the cell surface BCRs, SYK can no longer be quickly and fully activated in the EU12 HC + immature B cells after restimulation. Given the important roles of SYK in BCRmediated early signaling events in mature B cells, delayed and attenuated SYK activation might affect the formation of the signalosome to fully activate Ca ++ influx and the downstream PI3K and NF-B pathways. Indeed, we observed reduced Ca ++ influx and reduced CD19 and FOXO1 phosphorylation upon restimulation of EU12 HC + cells. Recent studies have shown that ERK activation is involved in light chain receptor editing process [29]. Correlating with the sustained activation of ERK in the EU12 HC + cells, we suspect that ERK activation is important for induction of replacement in human immature B cells.
Among the different BCR-mediated downstream signaling pathways, induction of Ca ++ influx, activation of the PI3K, and NF-B pathways are always considered to be important for B cell activation and proliferation. Here, our results show that complete internalization of BCRs in the EU12 HC + cells specifically alters the BCR downstream signaling events with sustained activation of ERK but attenuated Ca ++ influx and CD19 and FOXO1 phosphorylation, which are different from that in mature B cells. We speculate that such changes in the EU12 HC + immature B cells may prevent B cell activation and proliferation from favoring the induction of replacement.

Conclusions
Crosslinking BCRs on EU12 HC + immature B cells completely internalizes cell surface BCRs and specifically alters the BCR downstream signaling events with sustained activation of ERK but reduced Ca ++ influx and SYK, CD19, and FOXO1 phosphorylation. Such changes may favor the induction of replacement rather than cell activation and proliferation in the EU12 HC + immature B cells.


extfieldsofstudy: Chemistry Medicine

text:
Increased DNA Damage in Progression Of COPD: A Response By Poly(ADP-Ribose) Polymerase-1

Chronic oxidative stress (OS), a major mechanism of chronic obstructive pulmonary disease (COPD), may cause significant damage to DNA. Poly(ADP-ribose) polymerase (PARP)-1 is rapidly activated by OS-induced DNA lesions. However, the degree of DNA damage along with the evolution of COPD is unclear. In peripheral blood mononuclear cells of non-smoking individuals, non-obstructive smokers, patients with COPD of all stages and those with COPD exacerbation, we evaluated DNA damage, PARP activity and PARP-1 mRNA expression using Comet Assay IV, biotinylated-NAD incorporation assay and qRT-PCR, respectively and subjected results to ordinal logistic regression modelling. Adjusted for demographics, smoking-related parameters and lung function, novel comet parameters, tail length/cell length ratio and tail migration/cell length ratio, showed the greatest increase along the study groups corresponding to the evolution of COPD [odds ratio (OR) 7.88, 95% CI 4.26–14.57; p<0.001 and OR 3.91, 95% CI 2.69–5.66; p<0.001, respectively]. Analogously, PARP activity increased significantly over the groups (OR = 1.01; 95%; p<0.001). An antioxidant tetrapeptide UPF17 significantly reduced the PARP-1 mRNA expression in COPD, compared to that in non-obstructive individuals (p = 0.040). Tail length/cell length and tail migration/cell length ratios provide novel progression-sensitive tools for assessment of DNA damage. However, it remains to be elucidated whether inhibition of an elevated PARP-1 activity has a safe enough potential to break the vicious cycle of the development and progression of COPD.

Introduction
Of the major mechanisms of chronic obstructive pulmonary disease (COPD), oxidative stress (OS) resulting from direct effects of smoking, but also from persistent inflammation, causes oxidative damage to important biomolecules, including DNA [1][2][3]. Spillover of the OS to the circulation is associated with a significant systemic disease in COPD [4,5]. The resulting fall in antioxidant capacity of blood cells could therefore be regarded as a measure of OS in COPD.
Systemic inammation and OS suggest a role for the nuclear enzyme poly(ADP-ribose) polymerase (PARP)-1 (E.C. 2.4.2.30) in the pathophysiology of COPD [6]. PARP-1, a member of the PARP superfamily of 18 enzymes, is responsible for more than 90% of the cellular poly(ADP-ribosyl)ation capacity [6]. PARP-1 is activated by reactive oxygen species (ROS)-induced DNA strand breaks, upon which it forms extensive poly(ADP-ribose) polymers from its substrate NAD + [7]. In extensive DNA damage, PARP-1 activation causes depletion of NAD + leading to cell death via reduced glycolysis and mitochondrial respiration [8]. Unlike that on the systemic effects of COPD in general, the data on DNA damage and expression of PARP-1 are inconsistent.
Animal models have shown that PARP-1 activation contributes significantly to the pathophysiology of chronic inflammatory diseases like asthma [9][10][11], diabetes [8], rheumatoid arthritis [12] and chronic colitis [13]. An elevation of PARP-1 in murine hippocampus could serve as proof of PARP-1 as a marker of systemic inflammation [14]. It has been shown, that PARP-1 activity is increased in peripheral blood lymphocytes in patients with COPD [7]. However, we currently tested the hypothesis that the expression of PARP-1 and PARP activity in human peripheral blood mononuclear cells (PBMC) are related to the progression of COPD. In parallel, we employed and developed further comet assay as a sensitive method for detection of DNA strand breaks, alkali-labile sites and delayed repair sites [15].
As far COPD is accompanied by systemic OS, the effect of novel tetrapeptide analogue of glutathione (GSH), UPF17 (4methoxy-L-tyrosinyl-a-L-glutamyl-L-cysteinyl-glycine) on the PARP-1 activity through changing mRNA expression of PARP-1 was measured.

Study Subjects
All participants, recruited from the Department of Pulmonary Medicine, Tartu University Hospital, Tartu, Estonia, were selected to create seven study groups: 1) non-obstructive neversmoking control individuals with normal lung function, 2) current smokers or ex-smokers with normal lung function matching the COPD patients for their demographics and smoking history and habits, 3) four patients groups formed by the four stages of stabile COPD (I-IV) according to the Global Initiative for COPD (GOLD) Guidelines, based on the post-bronchodilator forced expiratory volume in one second (FEV 1 ) and 4) patients with a COPD exacerbation according to the GOLD criteria requiring hospitalisation (table 1) [16]. All patients with COPD had their post-bronchodilator FEV 1 /forced vital capacity ratio ,0.7 and all showed ,12% improvement in FEV 1 compared with the prebronchodilator value. A current smoker was defined as a person, who currently smoked at least one cigarette per day, whereas an ex-smoker was defined as a person, who had quitted smoking for $6 month prior to the study. The exclusion criteria included pneumonia, bronchiectasis and any malignancy and all subjects except for those with COPD exacerbation had to be free of viral respiratory infections within the last 2 months. Patients were treated according to the severity of the disease and in accordance with the GOLD guidelines. The study was approved by the Ethics Committee on Human Research of the University of Tartu. Written informed consent was obtained from all participants.

Mononuclear Cell Extraction from Peripheral Blood
PBMC were separated in BD Vacutainer CPT tubes (Becton Dickinson, Franklin Lakes, NJ, USA), by centrifugation at 15006g for 21 min at 20uC. Isolated PBMC were washed twice with phosphate-buffered saline (PBS). 200 ml of PBMC suspension (6610 6 cells/ml) were frozen down in 10% dimethyl sulfoxide (DMSO) and RPMI-1640 [including 10% foetal calf serum (FCS) and 1% penicillin/streptomycin] for the comet assay and measurement of PARP activity. The remaining PBMC were cultured in RPMI-1640 medium for the PARP-1 mRNA expression studies.

Comet Assay
PBMC were embedded with 0.5% low melting point agarose (Gibco by Life Technologies, Carlsbad, CA, USA) in distilled water and the mixture was added to a microscope slide, pre-coated with 1% of normal melting point agarose in distilled water, and covered with a coverslip. The slide was placed briefly to 4uC for the agarose to achieve solidify. The slides were then immersed in lysis solution [2.5 M NaCl, 100 mM ethylenediaminetetraacetic acid (EDTA) and 10 mM Tris, pH 10.0] containing freshly added 1% triton X-100 and 10% DMSO for at least 1 h at 4uC. Subsequently, the slides were incubated in freshly prepared alkaline buffer (300 mM NaOH and 1 mM EDTA, pH.13) for 40 min for DNA unwinding and electrophoresed in the same buffer. The conditions for electrophoresis were 30 min at 300 mA and 25 V. Following electrophoresis, slides were neutralized with PBS, washed in distilled water and 70% ethanol and dried overnight. The gels were stained for DNA with 1 mg/ml 49,6diamidino-2-phenylindole dihydrochloride solution in distilled water and air-dried. Images of 100 randomly selected cells (50 cells from each of two replicate slides) were taken with Zeiss Axiolab microscope (Carl Zeiss, Oberkochen, Germany) and analysed using Comet Assay IV software (Perceptive Instruments Ltd., Suffolk, UK). All standard parameters originally provided by Table 1. Characteristics of the individuals involved in the measurement of DNA damage by Comet Assay IV and poly(ADP-ribose) polymerase (PARP) activity in peripheral blood mononuclear cells.  the Comet Assay IV were used. In addition, a set of novel parameters were tested for their ability to more precisely characterise the DNA damage by the progression of COPD: tail moment length, extent tail moment, cell length, tail length/cell length ratio and tail migration/cell length ratio. Tail moment length shows the distance from the centre of the head to the centre of the tail, extent tail moment was calculated by dividing DNA % in the tail by the tail length and cell length shows the distance from the beginning of the head until the end of the tail.

Detection of PARP Activity
PARP activity was measured using 6-biotin-17-nicotinamideadenine-dinucleotide (bio-NAD + ), as previously described [17]. PBMC were cultured on poly-L-lysine-coated coverslips in RPMI medium, supplemented with 10% FCS. After 20 min incubation allowing cells to attach to slides, the medium was removed and replaced with PARP reaction buffer [56 mM 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid, 28 mM KCl, 28 mM NaCl, 2 mM MgCl 2 , pH 8.0, complemented with 0.01% digitonin and 250 mM biotinylated NAD + immediately before use]. After 60-min incubation at 37uC, the cells were fixed at -20uC in 95% ethanol followed by 10% trichloroacetic acid. The coverslips were rinsed in PBS and endogenous peroxidase was blocked by 0.5% H 2 O 2 / methanol for 15 min. After rinses with PBS, the coverslips were blocked in 1% bovine serum albumin/PBS for 30 min, followed by rinses in PBS-Triton X-100 (0.1%). Incorporated biotin was detected by streptavidin-peroxidase (diluted 1:100 in PBS-Triton X-100) for 30 min. Coverslips were washed with PBS-Triton X-100 and colour was developed with cobalt-enhanced nickel-3,39diaminobenzidine substrate. The coverslips were mounted with glycerol on slides and viewed under a Zeiss Axiolab microscope (Carl Zeiss). The pictures were taken with a Zeiss Axiocam digital camera (Carl Zeiss) and analysed using AlphaView Software (ProteinSimple, Santa Clara, CA, USA).

Measurement of PARP-1 mRNA Expression
The PBMC suspension was divided into halves with one part cultured in the presence of 500 nM UPF17 and the other without UPF17 that served as a control in RPMI-1640 medium (includes 10% FCS and 1% penicillin/streptomycin). After 12 hours, RNA was extracted from PBMC with the Trizol method according to manufacturer's protocol (Invitrogen by Life Technologies) and stored at -80uC until cDNA synthesis. cDNA was synthesized with reverse transcriptase reaction, from total RNA (250 ng) using the SuperScript III enzyme (Invitrogen by Life Technologies), according to manufacturer's instructions.
The gene expression levels were detected using the TaqMan-qRT-PCR method (ABI Prism 7900HT Sequence Detection System, Applied Biosystems by Life Technologies) using Hs00242302_m1 for PARP-1 and hypoxanthine phosphoribosyltransferase-1 (HPRT-1) as house keeper gene (both from Applied Biosystems by Life Technologies). All reactions were carried out in quadruplicates. The comparative Ct method (DCt value) was used for quantification of mRNA, where the amount of target transcript was normalized to the level of endogenous HPRT-1.

Statistical Analysis
The equality of the baseline data across the study groups was evaluated with Kruskal-Wallis test in numeric variables and Pearson's chi-square test in nominal variables. Associations of the comet assay parameters, as well as the PARP-1 expression and PARP activity with the presumably increasing degree of impairment from healthy never-smokers through non-obstructive smokers, stable COPD patients of GOLD stages I-IV to patients with Table 2. Characteristics of the individuals involved in the measurement of poly(ADP-ribose) polymerase (PARP)-1 mRNA expression in peripheral blood mononuclear cells.  COPD exacerbation, were assessed with ordinal logistic regression analysis with calculating the odds ratios (OR) and their 95% confidence intervals (95% CI). Because of the very high collinearity between the comet assay indices due to their derivation, the comet parameters were not simultaneously included into the multivariate model. Instead, the ability of each of the comet assay parameters to indicate the increasing degree of DNA impairment throughout the study groups was assessed separately, though each of them was adjusted for age, gender, body mass index (BMI), lung function (FEV 1% predicted), packyears of smoking and status of a current smoker. The analyses of PARP-1 expression and PARP activity were adjusted similarly.-Mann-Whitney U-Test was performed to determine the difference between the COPD patients and non-COPD individuals for the effect of UPF17 on PARP-1 mRNA expression level. p-values below 0.05 were considered to designate statistical significance. All statistical analyses were performed with the Statistical Package for Social Sciences (SPSS, version 17.0, SPSS Inc., Chicago, IL, USA) software.

Study Subjects
Different cohorts were used to measure the DNA damage by comet assay and PARP activity (table 1) and the PARP-1 mRNA expression level (table 2). Amongst the patients, who were chosen for DNA damage and PARP activity analysis, there was a difference for smoking history because one patient with COPD exacerbation had a smoking history of 116 pack-years.

DNA Damage, PARP Activity and Expression of PARP-1 mRNA in PBMC
After adjustment for age, gender, BMI, smoking history (packyears), status of a current smoker and FEV 1% predicted, of the Comet IV standard parameters, olive tail moment, DNA % in the tail, head length, tail length and tail migration increased significantly (OR.1.0, p,0.05), whereas DNA % in the head, as well as mean grey level and total intensity decreased significantly (OR,1.0, p,0.05) along the order from healthy control individuals through non-obstructive smokers, patients with increasing stage of stable COPD to those with exacerbation of COPD (table 3, figure 1). Of the derived parameters, tail moment length, extent tail moment and cell length, as well as the novel parameters tail length to cell length ratio and tail migration to cell length ratio also increased significantly to designate escalation to the next group in our study (OR.1.0, p,0.05), (table 3, figure 1). In particular, the comet parameters from PBMC that had the highest OR to reflect the DNA changes along with the evolution of COPD turned out to be the novel parameters tail length/cell length and tail migration/cell length ratios. One unit increase in tail length/cell length ratio and tail migration/cell length ratio was associated with a 7.88-fold (95% CI 4.26-14.57, p,0.001) and a 3.91-fold (95% CI 2.69-5.66, p,0.001) higher probability, respectively, of progressing into the next group of individuals ( figure 1, table 3).
PARP activity was a significant factor associated with escalation into the next group in our study (OR 1.014, 95% CI 1.006-1.023, p,0.001) (table 4, figure 2). Unlike PARP activity, PARP-  Table 3. Changing DNA damage in peripheral blood mononuclear cells by Comet Assay IV parameters from never-smoking control individuals through current smokers or ex-smokers with normal lung function and patients with stabile chronic obstructive pulmonary disease (COPD) of the Global Initiative for Chronic Obstructive Lung Disease (GOLD) stages I-IV to patients with a COPD exacerbation, evaluated with ordinal logistic regression analysis.  Effect of UPF17 on the Expression of PARP-1 mRNA UPF17 had a stronger PARP-1 mRNA expression level lowering effect amongst patients with COPD, as compared to that amongst non-obstructive individuals (non-obstructive nonsmokers and non-obstructive smoking individuals) (p = 0.040) (figure 3).

Discussion
After adjustment for demographic variables, BMI, smokingrelated indices and lung function, in the PBMC, the common Comet Assay IV parameters indicative of DNA damage, such as tail length, tail migration, olive tail moment and DNA % in the tail, showed a significant positive dependence on progression throughout the scale from healthy non-smoking controls to severe exacerbations of COPD. Formerly, the issue of stage-or progression-dependence of the DNA damage in COPD has remained controversial. Ceylan et al. showed an increased DNA damage in peripheral blood mononuclear leukocytes in patients with COPD [1], but using the same technique, Maluf et al. did not detect a correlation between the increased DNA damage in PBMC and COPD severity [18]. The major difference between the current study and those conducted by Ceylan et al. and Maluf et al. was that in the current one, a special software (Comet Assay IV) was used to analyse at least 10 different variables of the comet assay instead of visual scoring of the tail intensity only. Furthermore, in our study 7 groups were analysed using ordinal logistic regression to test for the progressing DNA injury from nonsmoking control and non-obstructive smokers through the four COPD stages to severe COPD exacerbation. The DNA damage along with PARP activity and PARP-1 mRNA expression has not been investigated formerly in this way. In-vitro studies have shown relations of PARP activity and cigarette smoke [19,20]. Also, differential activation of PARP in PBMC from controls and COPD patients has been shown previously [7], but in-vivo studies on the relationships between PARP activity and COPD progression have not been performed before. Not less importantly, all variables indicative of DNA damage were adjusted for parameters like age, gender, BMI, smoking habits and lung function. In the context that COPD is a disease of systemic senescence [21], the results adjusted for the demographic and clinical parameters point out that the increase in DNA damage is likely to increase accordingly to the advancement of the OS and inflammation from non-smoking controls up to COPD exacerbation, rather than caused by age, by fall in FEV 1 % or by cigarette smoke (CM) alone. Adjustment to gender might be of particular importance, as the PBMC PARP activity has been shown to be significantly lower amongst female subjects compared with male subjects [22]. Moreover, according to rodent [23] and porcine [24] models, the regulation of PARP has been reported to be more profound in males than in females. In addition to the variables provided by the Comet Assay IV, 5 new parameters were introduced and found to reflect the relationship between DNA damage and the progression of the disease even better. Out of the novel or newly introduced parameters, the tail length/cell length ratio and tail migration/cell length ratio were found to show the highest ORs with the development and progression of COPD up to COPD exacerbation. A one unit increase in tail length/cell length ratio and tail migration/cell length ratio was associated with a 7.88-fold or 3.91fold higher probability, respectively, for the process progressing to the next stage and therefore can appear most suitable to describe the DNA damage in PBMC over the evolution of COPD. This is the first report to show that these new parameters are most sensitive for the advancement of COPD and to suggest the tail length/cell length ratio and the tail migration/cell length ratio as new precision tools for independent assessment of the DNA damage in smokers and in patients with COPD.
Along with the comet assay indices, the current study revealed a strong relationship between the evolution of COPD and PARP activity in PBMC. These data suggest an intensified DNA repair occurring in PBMC of patients with higher COPD stages and adds a valuable adjunct to understanding of the systemic component of COPD. PARP activity was somehow heightened amongst the nonobstructive smokers, as compared to both the non-smoking Table 4. Changing poly(ADP-ribose) polymerase (PARP) activity and PARP-1 mRNA expression in peripheral blood mononuclear cells from never-smoking control individuals through current smokers or ex-smokers with normal lung function and patients with stabile chronic obstructive pulmonary disease (COPD) of the Global Initiative for Chronic Obstructive Lung Disease (GOLD) stages I-IV to patients with a COPD exacerbation, evaluated with ordinal logistic regression analysis.  controls on one hand and to patients with mild COPD on the other (table 4, figure 2). This may indicate that those smokers, who do not develop COPD, are still sensitive to various antioxidant influences than do COPD patients, at least those with a milder disease. This suggests that although PARP is a very important player in the DNA repair, it does not represent the only pathway to prevent smokers from developing COPD. In mammalian cells, the basal enzymatic activity of PARP is very low, but oxidative and nitrosative stress can trigger DNA strand breakage, which in turn abruptly stimulates the PARP-1 activity [20,[25][26][27]. Moderate activation of PARP is of physiological importance and could be beneficial via facilitating the DNA repair. However, overactivation of PARP is connected to numerous pathological conditions associated with oxidative and nitrosative stress, including COPD [28]. In particular, in parallel with energy depletion by the cells caused by PARP over-activation, PARP contributes to the synthesis of inflammatory mediators including cytokines, chemokines and enzymes like inducible nitric oxide synthase, but also up-regulates expression of pro-inflammatory genes, which leads to augmented inflammation [6]. Thus, the DNA damage caused by chronic OS as one of the main mechanisms of COPD can activate PARP. As a consequence, systemic PARP over-activation may play an important role by upregulating the expression of pro-inflammatory genes in COPD [29,30] leading to a vicious circle of consecutively increased OS and inflammation. In this light, one could speculate that the overactivation of PARP can be one of the intermediate mechanisms for the abnormally augmented inflammation in COPD [31]. If true, inhibition of PARP activity could provide protection against OSrelated inflammation and thereby afford therapeutic benefits in COPD [32,33].
Our study showed that UPF17, a non-toxic and ,3000-fold better hydroxyl radical scavenger than GSH [34,35], has a significant inhibitory effect on PARP-1 mRNA expression in patients with COPD, compared to that in non-obstructive individuals. Our latest experiments on HepG2 cells suggested that the activity of UPF17 is mediated via the nuclear factor (erythroidderived 2)-like 2 (Nrf2) (unpublished data). Although, inhibiting PARP can have anti-inflammatory effects, inhibiting PARP in a greater degree reduces DNA repair in cells [36], which can be considered as a side effect in the context of COPD. Collectively, the clinical significance of PARP-1 down-regulation remains elusive.
Prospective nature, inclusion of people from healthy controls through non-obstructive smokers to severe exacerbation of COPD into ordinary logistic regression models adjusted for major demographic, clinical and smoking-related parameters can be considered as strengths of the study. Small and unequal study groups and different populations for comet assay and assessment of PARP-1 mRNA expression are clear limitations.
Taken together, the present results show that COPD is a systemic disease and that DNA damage and PARP activation in PBMC are related both to the progression of COPD, but also to its exacerbation. The newly derived comet assay parameters (tail length/cell length ratio and tail migration/cell length ratio) can better reflect the changes occurring along with the evolution of COPD than the standard parameters that were used up to now. Furthermore, UPF17 was effective in modulating the expression of PARP-1 mRNA. However, it remains to be elucidated whether a modest inhibition of PARP-1 may have a safe enough potential to break the vicious cycle of the development and progression of COPD.


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Novel Neuroprotective Function of Apical-Basal Polarity Gene Crumbs in Amyloid Beta 42 (Aβ42) Mediated Neurodegeneration

Alzheimer's disease (AD, OMIM: 104300), a progressive neurodegenerative disorder with no cure to date, is caused by the generation of amyloid-beta-42 (Aβ42) aggregates that trigger neuronal cell death by unknown mechanism(s). We have developed a transgenic Drosophila eye model where misexpression of human Aβ42 results in AD-like neuropathology in the neural retina. We have identified an apical-basal polarity gene crumbs (crb) as a genetic modifier of Aβ42-mediated-neuropathology. Misexpression of Aβ42 caused upregulation of Crb expression, whereas downregulation of Crb either by RNAi or null allele approach rescued the Aβ42-mediated-neurodegeneration. Co-expression of full length Crb with Aβ42 increased severity of Aβ42-mediated-neurodegeneration, due to three fold induction of cell death in comparison to the wild type. Higher Crb levels affect axonal targeting from the retina to the brain. The structure function analysis identified intracellular domain of Crb to be required for Aβ42-mediated-neurodegeneration. We demonstrate a novel neuroprotective role of Crb in Aβ42-mediated-neurodegeneration.

Background
Alzheimer's disease (AD) is a progressive neurodegenerative disorder with no effective cure to date. AD is characterized by the progressive loss of neurons in the hippocampus and cortex causing decline in cognitive and behavioral functions eventually leading to the death of the patient [1,2]. AD neuropathology is associated with two types of abnormal protein deposition in the human brain viz.: (1) neurofibrillary tangles (NFTs) containing hyperphosphorylated forms of a microtubule associated protein Tau, and (2) the accumulation of the amyloid-beta (Ab42) peptide [1][2][3][4][5][6][7][8]. Ab42 is generated by improper (b-and c-) cleavage of the transmembrane receptor amyloid precursor protein (APP), as well as by mutations linked to familial AD that affect APP processing [9]. The abnormal cleavage of APP causes the protein to be 42 amino acids long (Ab42), whereas, the normal length of the protein is 40 amino acids long (Ab40) [1,2,7,8]. The amyloid hypothesis suggests that Ab42 forms protofibrils and fibrils. Accumulation of Ab42 impairs basic cellular processes due to oxidative stress, misregulation of intracellular calcium, ER stress [10], and aberrant signaling through interaction with several receptors [3,5,6,8], which results in the death of neurons [7]. Therefore, it is important to understand the mechanism underlying Ab42 mediated cell death and neurotoxicity.
Since the genetic machinery and basic cell biological pathways are conserved from insects to humans, several animal models have been employed to model AD. Despite the immense amount of information available from modeling AD in animal models such as the mouse [2,7] and the fruit fly [7,[11][12][13][14][15], the exact mechanism(s) mediating Ab42-dependent cell death are yet to be determined. The fruit fly has been a model organism for human diseases for many years since nearly 70% of human disease genes are conserved in flies [16]. We have used a Drosophila melanogaster eye model to express the human Ab42 peptide [3].
The Drosophila eye model has been extensively employed to investigate patterning, growth, and cell biological processes [7,[14][15][16]. The adult Drosophila compound eye develops from an epithelial bi-layer structure housed inside the larva called the eye-antennal imaginal disc, which gives rise to an eye, antenna and head cuticle of the adult fly [17]. A synchronous differentiation event in the developing third instar larval eye imaginal disc differentiates retinal precursor cells to photoreceptor neurons [18]. The eye imaginal disc metamorphose to a pupal retina which develops into the adult eye comprising of about 800 units called ommatidia [18]. Each ommatidium contains eight photoreceptors, pigment cells and several support cells. In the pupal retina, the extra undifferentiated cells are eliminated by programmed cell death (PCD) [19]. PCD is not observed during earlier stages of larval eye development, however, abnormal extracellular signaling due to inappropriate levels of morphogens may trigger cell death in the developing larval eye imaginal disc [20]. We have found that Ab42 dependent cell death is mediated, in part, through activation of the JNK signaling pathway [3]. However, blocking the JNK signaling pathway does not completely rescue the Ab42dependent cell death [3]. Therefore, there may be other genetic components that remain to be identified.
Using the Gal4/UAS system [21], we have developed an AD model with transgenic flies [3] where high levels of Ab42 are misexpressed in the differentiating photoreceptor neurons of the fly retina using a Glass Multiple Repeat driver [22] (GMR-Gal4.UAS-Ab42, hereafter GMR.Ab42). These GMR.Ab42 transgenic flies exhibit progressive neurodegenerative pathology in the developing retina, which is similar to that observed in AD [3]. Moreover, the misexpression of Ab42 in the differentiating retina (GMR.Ab42) exhibits a stronger neurodegenerative phenotype at 29uC [3]. The expression of the cell fate marker like disc large (dlg, a membrane specific marker) in the developing eye imaginal disc was studied. In comparison to the wild type adult eye ( Figure 1A) and the larval eye imaginal disc ( Figure 1B), misexpression of Ab42 (GMR.Ab42) in the Drosophila eye imaginal disc resulted in a reduced eye size with disorganized photoreceptors on the posterior margin as evident from the expression of pan neural marker, Elav (DSHB), in the photoreceptor neurons ( Figure 1G), and a highly reduced adult eye which did not show any wild type ommatidium within the compound eye ( Figure 1F) [3].
Our earlier studies showed that in the GMR.Ab42 retina, the ommatidia delaminated from the retinal layers possibly due to loss of polarity and/or cell adhesion [3]. We tested various components of the apical-basal polarity gene pathway in a forward gain of function genetic screen by individually co-expressing the apical basal polarity genes with Ab42 (GMR.Ab42+apical basal polarity genes) in the differentiating photoreceptor neurons. From this screen, we identified a transmembrane protein Crumbs (Crb), as a strong genetic modifier of the Ab42 mediated neurodegenerative phenotype. Crb is highly conserved and has three homologs CRB1, CRB2 and CRB3 in humans. An apical basal polarity gene crb encodes Crb protein, which is localized to the apical domain of the epithelial cells, where it is involved in setting up the apico-basal axis of the cell [23]. Furthermore, Crb is required for organizing apico-basal polarity specification, adherens junctions (AJ) and remodeling in epithelial cells [23,24]. Crb works by forming a complex with Stardust (Sdt/Pals1) [25]. Sdt, in turn, binds to the intracellular domain of Crb and recruits Pals associated tight junction protein (Patj) [26] and Lin7 [27]. To date, Crb has not been reported to play any role in Ab42 mediated neurodegeneration.
We have employed Gal4/UAS system for targeted misexpression studies [21]. All Gal4/UAS crosses were maintained at 18uC, 25uC and 29uC, unless specified, to sample different induction levels. The adult fly cultures were maintained at 25uC, while the egg laying (progeny) were transferred to 29uC. Misexpression of Ab42 in the differentiating retina (GMRGal4.UAS-Ab42, GMR.Ab42) exhibits a stronger neurodegenerative phenotype at 29uC [3]. All the targeted misexpression experiments were conducted using the Glass Multiple Repeat driver line (GMR-Gal4), which directs expression of transgenes in the differentiating retinal precursor cells of the developing eye imaginal disc and pupal retina [22].

Detection of cell death
Cell death was detected using TUNEL assays from Roche Diagnostics [3,31]. TUNEL assays were used to identify the cells undergoing cell death where the cleavage of double and single stranded DNA is labeled by a Fluorescein. The fluorescently labeled nucleotides are added to 39 OH ends in a templateindependent manner by Terminal Deoxynucleotidyl transferase (TdT). The fluorescent label tagged fragmented DNA within a dying cell can be detected by fluorescence microscopy. Eye antennal discs after secondary antibody staining [32] were blocked in 10% normal donkey serum in phosphate buffered saline with 0.2% Triton X-100 (PBT) and labeled for TUNEL assays using a cell death detection kit from Roche Diagnostics.
The TUNEL positive cells were counted from five sets of imaginal discs and were used for statistical analysis using Microsoft Excel 2010. The P-values were calculated using one-tailed t-test and the error bars represent Standard Deviation from Mean [3].

Adult eye imaging
Adult eye images were taken on the Axioimager.Z1 Zeiss Apotome. Adult flies were mounted onto a needle and the image was completed by using extended depth of focus function of the Axiovision software version 4.6.3 by compiling the individual stacks from the Z-sectioning approach. The final images and figures were prepared using Adobe Photoshop CS4 software.

Results
We tested Crb protein levels using Crb antibody (Cq4, DSHB) [23] in the GMR.Ab42 eye imaginal disc using a modified protocol [30]. The Crb protein is localized to the apical domain of the epithelial cells. We observed higher levels of Crb protein in the GMR.Ab42 background ( Figure 1F) as compared to the wild type eye imaginal disc ( Figure 1C). Misexpression of Ab42 peptide with full length Crb [28] using GMR-Gal4 driver (GMR.Ab42+Crb (FL), as evident from Crb antibody staining ( Figure 1I), resulted in increased neurodegeneration as shown by highly disorganized morphology due to clumping of photoreceptor neurons (Red channel, marked by Elav) of neighboring ommatidia of the eye imaginal disc ( Figure 1H). Large gaps were observed among the photoreceptors of the ommatidia where the cells begin to die or clump together. The adults failed to form due to early pupal lethality ( Figure 1G). These animals died in the early pupal stages; as a result we could not observe any pupal retina like structures (data not shown). Downregulating Crb levels by using a heterozygous combination of FRT82B crb 11A22 allele [33] ( Figure 1L) or crb RNAi (Figure 1 O) resulted in the rescue of the GMR.Ab42 mediated neurodegeneration as seen in the eye imaginal disc ( Figure 1K, N) as well as in the adult eye ( Figure 1J, M). We found significant rescue although complete restoration to the wild type eye was not seen. These results suggested that higher levels of crbs are associated with the retina undergoing neurodegeneration due to misexpression of Ab42. Furthermore, Ab42 mediated neurodegeneration can be rescued by downregulating crb function.
We employed TUNEL staining to discern the mechanism of neurodegeneration due to misexpression of Crb in the developing retina. The TUNEL staining marks the nuclei of the dying cells, where the cleavage of double and single stranded DNA is labeled by Fluorescein [31]. Here we utilized TUNEL staining to quantitate the effects of Crb protein levels on neurodegeneration in the GMR.Ab42 background (Figure 2A-F). The TUNEL positive cells were counted from five sets of imaginal discs and were used for statistical analysis using Microsoft Excel 2010. The P-values were calculated using one-tailed t-test and the error bars represent Standard Deviation from the Mean [3]. It is known that a few cells undergo cell death in the wild-type eye imaginal disc ( Figure 2A) which does not affect the final morphology of the adult compound eye ( Figure 1A). The number of TUNEL positive nuclei of the dying cells in the GMR.Ab42 flies ( Figure 2B) was almost three times as high when compared to the wild-type eye imaginal disc (p = 1.943610 26 ; Figure 2F). We investigated the levels of Crb with reference to the induction of cell death and found that when Crb levels were increased in a GMR.Ab42 background (GMR.Ab42+Crb FL), the TUNEL positive cell number increased ( Figure 2C) and was almost seven times higher than the wild type eye imaginal disc (p = 9.536610 28 ; Figure 2F) and nearly two times higher than the GMR.Ab42 eye imaginal disc ( Figure 2F). Reducing levels of crb by using crb 11A22 allele [33] ( Figure 2D) or crb RNAi ( Figure 2E) reduced cell death as evident from reduction in the number of TUNEL positive cells to almost two fold with respect to the GMR.Ab42 eye imaginal disc (for crb 11A22 p = 8.386610 25 , for crb RNAi p = 8.030610 25 ; Figure 2F).
Next, we investigated the effects of modulating levels of Crb on retinal axon targeting from the retina to the brain using chaoptin (24B10, a marker for photoreceptor cells and their axons [34], DSHB) staining. Disruption of axonal transport mechanisms that leads to axonal vesicle stalling has been shown to contribute to the neurodegenerative phenotypes in the AD fly model [35]. During Drosophila visual system development, stereotypical targeting of the axons from the retinal neurons to the special layers of the optic ganglion, medulla and lamina of the brain occurs. The axons of the eight photoreceptor neurons from each ommatidium [36] fasciculate together and project as a single bundle towards the optic lobes of the brain. The Drosophila photoreceptors (R cells) seek specific targets to connect in distinct layers of the optic lobes of the brain, viz., R1-R6 axons project to the lamina; R7 and R8 axons project to the separate layers of the medulla [37]. In comparison to the wild-type eye disc where retinal neurons innervate different layers (medulla and lamina) in the brain ( Figure 3A), the GMR.Ab42 eye disc shows complete loss of axonal targeting ( Figure 3B). Additional upregulation of full length Crb levels in GMR.Ab42 (GMR.Ab42+Crb FL) strongly affected the retinal axon targeting from the retina to the brain ( Figure 3C) as compared to the wild type ( Figure 3A) and the GMR.Ab42 alone ( Figure 3B). The axonal targeting was restored when crb levels were reduced in the GMR.Ab42 background by using either FRT82B crb 11A22 allele ( Figure 3D) or crb RNAi ( Figure 3E). These results further validated our hypothesis that higher levels of Crb enhanced the neurodegenerative phenotype of Ab42 aggregate accumulation.
In order to discern how different domains of Crb protein ( Figure 4A) are involved in preventing GMR.Ab42 mediated neurodegeneration, we used the structure function analysis approach. The full length Crb, a type I transmembrane protein, has 28 EGF domains and four Laminin-AG like repeats in its large extracellular domain (ECD), a transmembrane domain (TM), and a short intracellular domain (ICD) ( Figure 4A). The Crb protein's TM domain consists of 37 amino acids spanning the region of the membrane [38]. The ICD contains two motifs, juxtamembrane FERM-binding motif (FBM or JM) domain and C-terminal PDZ (Postsynaptic density/Discs large/ZO-1) binding motif (PBM) domain ( Figure 4A). Through its PBM domain, Crb forms a complex with PDZ domain proteins, Stardust and PatJ [25]. It is important to note that the ICD of Crb protein interacts with a variety of conserved proteins including apical basal polarity genes Par6 and aPKC [39,40]. Prior structure-function studies using the different Crb domains, for example, in the gastrulating embryo, showed that the ubiquitous expression of a membranebound cytoplasmic ICD, suppressed the crb mutant phenotype to the same extent as full length crb [23,28]. Thus, the different domains of Crb carry out different downstream signaling interactions of the protein, so it is important to investigate which domains are involved in the rescue or enhancement of the neurodegeneration caused by Ab42.
We employed targeted misexpression [21] of Ab42 and various domains of Crb protein using the GMR-Gal4 driver [22] for a structure function analysis. The rationale of these studies was to determine which domain of Crb protein is required for its function in Ab42 mediated neurodegeneration ( Figure 1D, E). As discussed previously, in comparison to the wild type eye ( Figure 4B), GMR.Ab42 exhibited strong reduction in size due to neurodegeneration as seen in the adult eye ( Figure 4C), whereas GMR.Crb [41] resulted in an increase of the adult eye size with minimal necrosis on the margin ( Figure 4D) [42]. Targeted misexpression of Crb ICD (the Crb ICD construct used has been Figure 1. Levels of apical basal polarity gene crb modulates Ab42 mediated neurodegeneration. Wild type (A) adult compound eye, a highly organized structure comprising of 750-800 ommatidia [18], which develops from (B, C) eye-imaginal disc. Third-instar eye imaginal disc stained with membrane specific marker, Disc large (Dlg; white), a pan neural marker Elav (red, marks photoreceptors), and (C) Crb protein expression. The Crb expression is localized on the apical surface of epithelial cells and accumulates at the apical membrane's outer margin [51]. (D-F) Misexpression of Ab42 using GMR-Gal4 driver [22] in the differentiating photoreceptor neurons results in the induction of neurodegeneration as seen in (D) the highly reduced adult eye with a glazed surface and (E, F) eye imaginal disc. Note that in GMR.Ab42 eye imaginal discs (E) pan neural marker, Elav, exhibits clumping of the photoreceptor neurons and holes in the developing retina, and (F) strong enrichment of Crb expression in the GMR domain. (G-I) Misexpression of Crb full length [41] in GMR.Ab42 background (GMR.Ab42+Crb FL) strongly enhances the neurodegeneration phenotype which results in (G) pupal lethality (adults failed to form due to early pupal lethality and as a result lacked the adult eye structure) and (H, I) severe neurodegeneration observed in the eye imaginal disc as evident from (H)  Role of Crumbs in Ab42 Mediated Neurodegeneration PLOS ONE | www.plosone.org referred to as Crb intra [28]; Figure 4A) in a GMR.Ab42 background (GMR.Ab42+Crb intra ) resulted in strong enhancement of the neurodegenerative phenotype of GMR.Ab42 alone as seen in the eye imaginal disc ( Figure 4G) as well as in the adult eye ( Figure 4F). The GMR.Ab42+Crb intra adult eye showed strong neurodegeneration as evident from the dark necrotic patch in place of the adult eye ( Figure 4F). However, the control GMR.Crb intra also showed some neurodegeneration ( Figure 4E), which was not as strong as GMR.Ab42+Crb intra ( Figure 4F). Since both the control ( Figure 4E) as well as GMR.Ab42+  Crb intra (Figure 4F, G) showed a neurodegenerative phenotype, it raised the possibility of an additive effect. Further experimentation using the truncated constructs of Crb intra domains disproved this additive effect hypothesis. Targeted misexpression of GMR.Ab42 with Crb intra DPBM [28] or Crb intra DJM [28] in developing retina resulted in the rescue of the GMR.Ab42 neurodegenerative phenotype as seen in the eye imaginal disc ( Figure 4J, M) as well as the adult eye ( Figure 4I, L). The controls GMR.Crb intra DPBM ( Figure 4H) and GMR.Crb intra DJM ( Figure 4K) exhibit a slightly reduced adult eye. The Crb intra construct lacking both the JM and PBM domains (GMR. Crb intra DJM DPBM ( Figure 4A)) resulted in a near normal adult eye ( Figure 4N). Targeted misexpression of GMR.Ab42 with Crb intra DJM DPBM resulted in the rescue of the GMR.Ab42 neurodegenerative phenotype as seen in the eye imaginal disc (Fig. 4P), and the adult eye (Fig. 4O). All these results clearly demonstrated that like the full length Crb (Crb FL), the entire ICD (Crb intra ) is also responsible for the enhancement of the neurodegenerative phenotype of GMR.Ab42. It suggests that Crb ICD is sufficient enough to carry out the Crb FL function in Ab42 mediated neurodegeneration. When we remove either one or both of the JM and PBM domains from the ICD of Crb, the GMR.Ab42 phenotype is rescued and the ommatidia are restored to near wild-type. This data strongly indicates that both the JM and PBM domains in Crb are essential to suppress the Ab42 effects. There might be a correlative interaction between the JM and PBM domains of Crb in the Ab42 mediated neurodegeneration. However, when we have an intact ICD or full length Crb, there is a severe enhancement of the GMR.Ab42 phenotype. Also, in the loss-of-function crb flies (GMR.Ab42+crb 11A22 and GMR.Ab42+crb RNAi) where we see reduced Crb level expression ( Figure 1L, O) as compared to the wild-type ( Figure 1C), there is a rescue of Ab42 mediated neurodegeneration further validating our hypothesis that Crb levels can modify the neurodegenerative phenotype of Ab42 accumulation. Thus, Crb levels can serve as an excellent biomarker for AD.
To further verify the structure function analysis results, TUNEL assays were performed on all of the constructs. The rationale was to examine if the reduced eye phenotype seen in GMR.Ab42+Crb intra was due to cell death or, on the other hand, if the restored eye as shown by removing either or both of the JM and PBM domains of ICD motifs ( Figure 4A) is due to reduced number of TUNEL cells. As mentioned earlier, TUNEL marks the nuclei of dying cells, therefore a reduced number of TUNEL positive cells nuclei corresponds to less cells dying, which will lead to a rescue of GMR.Ab42 neurodegenerative phenotype in the adult eye. We found that the severely reduced adult eye phenotype of GMR.Ab42+Crb intra is in fact due to an increase in the number of TUNEL positive cells as compared to the wild-type and the GMR.Ab42 eye disc ( Figure 5A, B, I). The GMR.Ab42+Crb intra exhibits strong neurodegenerative phenotype as evident from disorganized photoreceptor neurons (marked by Elav, green) in the ommatidia. Furthermore, the number of TUNEL positive cells nuclei are increased ( Figure 5A, B; red). The TUNEL staining explains the reason for a highly reduced adult eye in GMR.Ab42+Crb intra ( Figure 2F). Additionally, when any either JM or PBM or both JM and PBM domains of the ICD motifs were removed in the GMR.Ab42 background, the severity of neurodegenerative phenotypes was significantly reduced. In GMR.Ab42+Crb intra DJM ( Figure 5C, D), GMR.Ab42+ Crb intra DPBM ( Figure 5E, F), or GMR.Ab42+Crb intra DJM DPBM ( Figure 5G, H), the number of TUNEL positive dying cells nuclei were significantly less than GMR.Ab42 and GMR.Ab42+ Crb intra ( Figure 5I). All of these results further validate the data shown in Figure 4 and conforms to the adult eye phenotypes of each of these structures.
For all the ICD motifs of Crb, the TUNEL positive cells were counted from five sets of imaginal discs and were used for statistical analysis using Microsoft Excel 2010. The P-values were calculated using one-tailed t-test and the error bars represent Standard Deviation from the Mean [3]. All the p-values showed the TUNEL count to be significantly different from GMR.Ab42 and the wild-type ( Figure 5I). By studying the domains of Crb with reference to the cell death, we found that misexpression of intact Crb ICD domain in GMR.Ab42 background (GMR.Ab42 +Crb intra ), resulted in the increased number of TUNEL positive cell ( Figure 5I) and was almost six times higher than the wild type eye imaginal disc (p = 1.5559610 27 ) and nearly two times higher than the GMR.Ab42 eye imaginal disc (p = 8.  Crb intra DJM DPBM p = 8.9278610 26 ; Figure 5I). This TUNEL assay further validated our hypothesis that the reduced eye phenotype seen in GMR.Ab42+Crb intra (with its fully intact ICD) is primarily due to induction of cell death and the restored eye phenotypes seen when any one or both of the ICD motifs of Crb is/are removed, does indeed have reduced number of dying cells as evident from TUNEL staining.
To further test our hypothesis, we looked at the axonal targeting from the retina to the brain using 24B10 (Chaoptin) in these constructs ( Figure 4A). As mentioned earlier, 24B10 shows an organized and orderly axon branching from the retina to the brain in the wild-type background ( Figure 3A). However, when we observed the 24B10 staining in the GMR.Ab42+Crb intra eye there is extreme disorganization marked by the clumping of axons, as well as Elav (red) positive photoreceptors which results in impairing of axonal targeting from retina to the brain ( Figure 6A, B). This data further confirms our TUNEL data using GMR.Ab42+Crb intra . Additionally, when we analyzed other constructs of Crb by removing either or both of the JM or PBM domains from the ICD motif, there is a rescue of the adult eye ( Figure 4A, H-P), a reduction in the number of TUNEL positive ( Figure 5C-I), and restoration of the organization of axons from the retina to the brain (Fig. 6 C-H) in all three constructs (GMR.Ab42+Crb intra DJM ( Figure 6C, D), GMR.Ab42+Crb intra DPBM ( Figure 6E, F), GMR.Ab42+Crb intra DJM DPBM (Figure 6G, H). When the JM motif (GMR.Ab42+Crb intra DJM ( Figure 6C, D) or the PBM motif (GMR.Ab42+Crb intra DPBM ( Figure 6E, F) was removed, there is restoration of the axonal targeting as evident from the 24B10 staining and marking the axonal projections innervate the two layers of the brain. Furthermore, when we remove both of the ICD motifs (GMR.Ab42+Crb intra DJMDPBM ( Figure 6G, H), the axonal connection to the brain is restored to near wild type axonal targeting. These data further validates that the ICD domain of Crb is sufficient enough for Crb function in Ab42 mediated neurodegeneration.

Discussion
Our studies strongly suggest that transmembrane protein Crb is involved in Ab42 mediated neurodegeneration. During wing development, N upregulates crb transcription at the dorso-ventral (DV) boundary, and the ability of Crb to inhibit the activity of the c-secretase complex has been proposed to help refine the N activity domain [43]. Crb functions as a negative regulator of the N signaling pathway [42]. Notch is involved in the development and organization of the dorso-ventral boundary through cell proliferation of the developing eye. Because N and Amyloid Precurssor Protein (APP) are cleaved by similar secretases [44] and Crb regulates N, the Crb effects on Ab42 could be caused through N regulation. However, in the GMR.Ab42 model used in our studies, the Ab42 protein is already cleaved from of APP and does not require cleavage by band c-secretase. Therefore, our data using the transgenic model suggests that Crb also acts downstream of c-secretase mediated cleavage of APP. Furthermore, higher levels of Crb can enhance human Ab42 mediated neurodegeneration [3]. Thus, Crb role in modulating Ab42 mediated neurodegeneration is downstream of N signaling pathway.
In addition, Crb is an upstream regulator of the organ size growth control pathway viz., Hippo signaling pathway. Recently, it was shown that Crb interacts with its juxtamembrane FERMbinding motif (JM) with the FERM domain of Expanded (Ex) to regulate growth by affecting the Hippo pathway activity [45][46][47]. Our structure function analysis studies exhibited that ICD of Crb is sufficient for its role in Ab42 mediated neurodegeneration suggesting that Crb may act independent of its interaction with Hippo pathway member Ex in Ab42 mediated neurodegeneration.
Since Crb ICD is involved in its interaction with apical basal polarity gene localization, there is a strong possibility that higher level of Crb in a GMR.Ab42 background might affect the apical basal polarity of the retinal photoreceptor neurons which result in neurodegeneration. Mutations in Crb homolog 1 (CRB1) has been shown to cause autosomal recessive retinitis pigmentosa (arRP) and autosomal Leber congenital amaurosis (arLCA) [36]. During Drosophila eye development, Crb is required in photoreceptors for stalk elongation [42,48], and in preventing light-dependent retinal degeneration [49]. Mutations in the human Crb homolog (CRB1) result in abnormalities like thick retina and lamination problems [50]. Furthermore, mutant Crb protein is thought to be responsible for retinal degenerations [50]. However, in GMR.Ab42 background higher levels of Crb protein were responsible for neurodegeneration. Therefore, it is a strong possibility that higher Crb levels may impair apical basal polarity leading to the Ab42 neurodegeneration. Thus, regulating Crb levels can help prevent the onset of neurodegeneration and Crb may serve as one of the biomarker as well as the key therapeutic targets for the AD. Figure 6. Misexpression of Crb intracellular domain (ICD) can impair axonal targeting. The eye-antennal disc is stained with Elav (red), which marks the photoreceptors, and 24B10 (chaoptin; green), which stains the axons from the retina to the brain [34]. (A, B) Misexpression of intact instar cellular domain ICD GMR.Ab42+Crb intra results in clumping of photoreceptors (Elav; A), disorganization of axonal targeting from the retina to the brain as evident from 24B10 staining. (C, D)When the JM motif using GMR.Ab42+Crb intra DJM , photoreceptor organization as well as the axonal targeting is restored to the wild type. Similarly, removing the (E, F) PBM motif of the ICD using GMR.Ab42+Crb intra DPBM , or both PBM and JM domain in GMR.Ab42+Crb intra DJMDPBM , result in restoration of axonal targeting and photoreceptors. Thus, ICD domain of Crb is required for its role in neurodegeneration. doi:10.1371/journal.pone.0078717.g006