EHP 120(5):623–626; Jackson et al].Organic brown rice syrup (OBRS) is a sweetener frequently used as an alternative to high-fructose corn syrup in organic and health food products. In a study of children’s foods, a team of researchers discovered high levels of arsenic in toddler formula products that contained OBRS. Given evidence that arsenic accumulates in rice at high levels, the researchers suspected OBRS was the source of the arsenic in the formulas—a suspicion corroborated by additional tests of several products both with and without the sweetener [The researchers tested 3 commercially available brown rice syrups, 29 cereal bars (18 with OBRS), 3 high-performance “energy shot blocks” with OBRS, 15 infant formulas without the sweetener, and 2 toddler formulas with it. The researchers measured inorganic arsenic as well as the organic chemical species dimethylarsenate (DMA) and monomethylarsenate (MMA), which are generally believed to be less toxic than inorganic arsenic.The OBRS-sweetened toddler formulas  had about 20 times the total arsenic concentrations of the non-OBRS infant formulas. Samples of prepared milk-based toddler formula had inorganic arsenic concentrations just below the current Environmental Protection Agency (EPA) drinking water standard of 10 µg/L, whereas inorganic arsenic in samples of soy-based toddler formula tested 1.5–2.5 times above the EPA standard.In the brown rice syrups tested, inorganic arsenic made up 80–90% of the arsenic content of two of the syrups and half the arsenic content of the third. All three also contained DMA, with lesser amounts of MMA. All 29 cereal bars tested contained some arsenic, but those without any rice-based ingredients had the lowest levels. Nearly 60% of the others contained inorganic arsenic. The three energy shot blocks contained enough inorganic arsenic that if an individual were to consume the manufacturer-recommended four servings during a two-hour workout, they would ingest the equivalent of drinking a liter of water containing 10 µg/L arsenic; total arsenic would be twice that.The two toddler formulas tested were the only ones the researchers could find that contained OBRS. However, they estimate that approximately half of all cereal and energy bars contain OBRS or other rice products, and they conclude that food containing OBRS may be a major pathway to arsenic exposure for some people. Although the health effects of low-level arsenic exposure over limited durations are unknown, childhood exposure may affect lifelong health and should be limited. The authors therefore urge U.S. regulators to establish limits for arsenic levels in food, particularly infant and toddler formulas.


Because cell signaling and cell metabolic pathways are executed through proteins, protein signatures in primary tumors are useful for identifying key nodes in signaling networks whose alteration is associated with malignancy and/or clinical outcomes. This study aimed to determine protein signatures in primary lung cancer tissues.p<0.05) by at least 30% between normal and tumor tissues. Most of those molecules play roles in cell proliferation, DNA repair, signal transduction and lipid metabolism, or function as cell surface/matrix proteins. We also validated RPPA results by Western blot and/or immunohistochemical analyses for some of those molecules. Statistical analyses showed that Ku80 levels were significantly higher in tumors of nonsmokers than in those of smokers. Cyclin B1 levels were significantly overexpressed in poorly differentiated tumors while Cox2 levels were significantly overexpressed in neuroendocrinal tumors. A high level of Stat5 is associated with favorable survival outcome for patients treated with surgery.We analyzed 126 proteins and/or protein phosphorylation sites in case-matched normal and tumor samples from 101 lung cancer patients with reverse-phase protein array (RPPA) assay. The results showed that 18 molecules were significantly different (Our results revealed that some molecules involved in DNA damage/repair, signal transductions, lipid metabolism, and cell proliferation were drastically aberrant in lung cancer tissues, and Stat5 may serve a molecular marker for prognosis of lung cancers. Molecular profiling of lung cancer through gene array assays for mRNA and microRNA has led to the identification of molecular signatures that are potentially useful for predicting patient survival and disease relapse and/or response to individual chemotherapeutic drugs based on hierarchical and probabilistic clustering of mRNA Efforts have been made to determine protein signatures in lung cancer by using two-dimensional gel electrophoresis and subsequent protein identification by mass spectrometry assay or by using direct mass spectrometry analyses The recent advent of protein microarray technology may allow us to identify critical nodes or interactions within the network of cellular signaling pathways. The advantage of the RPPA method is that a single test probe (antibody) is used for each array, so the testing condition is consistent for each antibody, thereby providing better reproducibility and sensitivity than other protein array techniques. With thoroughly assessed and validated antibodies, an RPPA can be used to detect signal differences in a few thousand molecules in testing samples We collected case-matched normal and malignant lung tissue samples from 101 patients. Characteristics of those patients and the tumors are summarized in p<0.05) in all the normal and tumor samples analyzed /]. Of 126 proteins or phosphorylation sites analyzed, 18 had signal differences that were greater than 30% and were statistically significant  on samples that showed difference between normal and tumor tissues. The results showed that the differences in the expression of all five molecules were derived from altered expression in cancer cells but not in stromal cells . Strikinet al. recently reported a RPPA profiling analysis on 46 lung cancer cases with 63 proteins or protein phosphorylation sites and identified several proteins were differentially expressed in primary lung cancer tissues Nanjundan Three  of four marker signature that differentiates NSCLC from normal lung in Nanjundan's study were also significantly different between normal and tumor tissues of the current studies. We therefore used Nanjundan's training set (25 cases) to test whether these three marker signature could be used to differentiate the whole data set (101 cases) of the current study. The result showed that these three markers, either alone or in combination, could distinguish tumor from the normal of the current study with various accuracies, sensitivities, and specificities . In genep = 0.004). Expression of cyclin B1 was significantly higher in poorly differentiated tumor tissues than in moderately or well-differentiated tumor tissues (p<0.025). On the other hand, expression of ATM, Ku80, and S6 was significantly higher in well-differentiated tumor tissues than in poorly or moderately differentiated tumor tissues (p<0.05). This result suggests that expression of certain molecules were dramatically different in neuroendocrinal tumors when compared with those in adenocarcinoma or squamous cell cancer, whereas the levels of the differentially expressed proteins listed in We analyzed whether expression of the 18 molecules listed in  tissues . When exTo determine whether levels of those proteins are associated with clinical outcomes, we performed survival analysis on the differentially expressed protein markers shown in p<0.05) different between normal and tumor samples. Western blot analysis and/or immunohistopathologic assays of several molecules validated the results obtained from RPPA arrays, demonstrating that the results from RPPA analysis are reliable. Moreover, a comparison with a RPPA study performed on another of patient samples showed that the results of RPPA profiling were highly repeatable and consistent in separate studies with separate set of patient samples. Our results also indicate that the expression of several molecules in tumor tissues was associated with smoking history, differentiation, and histopathologic types of lung cancers.We analyzed molecular differences in protein or protein phosphorylation levels between normal and lung cancer tissues in 101 samples by RPPA assay. Of 126 molecules analyzed, we identified 18 molecules that were dramatically (>30%) and statistically significant  were increased in tumor tissues. Increased mRNA levels of ATM and DNA-PKcs, but not of Ku80, were detected in tumor tissues when compared with adjacent normal tissues Several deregulated proteins identified here have been investigated as therapeutic targets for cancer therapy. Small molecules or kinase inhibitors targeting growth factor receptors and the PI3K/AKT/mTOR, Src/Stat, p38, and ATM/CHK2 pathways were extensively investigated for cancer treatment, both preclinically and clinically, including treatment for lung cancers Normal and malignant lung tissue samples were collected between 2006 and 2009 from surgically removed specimens under a research protocol Lab-90-020 with informed consent from the patients. The study was approved by local ethics committee . The normal tissues were at least 5 cm away from the edge of corresponding tumors in the same specimens. Both normal and tumor tissues were collected from the operating room immediately after specimens were removed from patients. In all cases, histology quality control was performed by a thoracic pathologist on tissue sections. Tumor samples were included in the analysis if the percentage of malignant cells present in the sample were ≥70%. Normal lung samples from the same patients were reviewed to confirm that they contained no malignant cells. All samples were divided into two portions: one portion was instantly frozen and stored in liquid nitrogen for protein extraction; the other portion was fixed in formalin and embedded in paraffin for routine histological or immunohistochemical examinations. The pairs of matched samples were harvested, processed, and analyzed at the same time, under the same protocols.2, 1 mmol/L EGTA, 100 mmol/L NaF, 10 mmol/L NaPPi, 10% glycerol, 1 mmol/L Na3VO4, 1 mmol/L phenylmethylsulfonyl fluoride, and 10 µg/mL aprotinin]. After centrifugation, the supernatant was collected, and the protein concentration was determined by routine  assays and then adjusted to 1–1.5 mg/ml by addition lysis buffer. The tissue lysates were mixed with 1/4 volume of 4× SDS sample buffer containing 40% glycerol, 8% SDS, 0.25 M Tris-HCl (pH 6.8), and 10% (v/v) 2-mercaptoethanol (freshly added). Two-fold serially diluted tissue lysates (from undiluted to 1∶16 dilution) were printed on nitrocellulose-coated slides  by using a GeneTAC G3 arrayer (Genomic Solutions), along with corresponding positive and negative controls prepared from the dilution buffer. A total of 126 validated antibodies specific for proteins or their phosphorylated sites that are involved in various signaling pathways were available and used in the RPPA  were deparaffinized, hydrated, and heated in a steamer for antigen retrieval. The slides were then stained with various antibodies as described above. Tissues not incubated with a control antibody to mice IgG instead of a primary antibody were used as a negative control.t test was used for comparison between two groups. The diagonal linear discriminant analysis (DLDA) was used for classification and prediction of normal and tumor tissues. The survival data will be analyzed using the Kaplan-Meier method and Cox's proportional model. A p-value of <0.05 was considered statistically significant.Analysis of variance was performed by using STATISTICA software  for comparisons among groups. Student's Figure S1Protein levels detected by Western blot analysis in 6 additional cases for IGFBP2 and CHK2. IGFBP2 and CHK2 in normal (N) and primary lung tumor (T) tissues were analyzed by Western blot in additional 6 cases in which RPPA showed signal difference in normal and tumor tissues. β-actin was used as loading control.(TIF)Click here for additional data file.Table S1Expression difference in adenocarcinoma and squamous cancer.*(DOC)Click here for additional data file.Table S2Proteins and phosphorylation sites used in RPPA studies.(DOC)Click here for additional data file.


DNA methylation is an epigenetic modification involved in regulatory processes such as cell differentiation during development, X-chromosome inactivation, genomic imprinting and susceptibility to complex disease. However, the dynamics of DNA methylation changes between humans and their closest relatives are still poorly understood. We performed a comparative analysis of CpG methylation patterns between 9 humans and 23 primate samples including all species of great apes  using Illumina Methylation450 bead arrays. Our analysis identified ∼800 genes with significantly altered methylation patterns among the great apes, including ∼170 genes with a methylation pattern unique to human. Some of these are known to be involved in developmental and neurological features, suggesting that epigenetic changes have been frequent during recent human and primate evolution. We identified a significant positive relationship between the rate of coding variation and alterations of methylation at the promoter level, indicative of co-occurrence between evolution of protein sequence and gene regulation. In contrast, and supporting the idea that many phenotypic differences between humans and great apes are not due to amino acid differences, our analysis also identified 184 genes that are perfectly conserved at protein level between human and chimpanzee, yet show significant epigenetic differences between these two species. We conclude that epigenetic alterations are an important force during primate evolution and have been under-explored in evolutionary comparative genomics. Differences in protein coding sequences between humans and their closest relatives are too small to account for their phenotypic differences. It has been hypothesized that these differences may be explained by alterations of gene regulation rather than primary genome sequence. DNA methylation is an important epigenetic modification that is involved in many biological processes, but from an evolutionary point of view this modification is still poorly understood. To this end, we performed a comparative analysis of CpG methylation patterns between humans and great apes. Using this approach, we were able to study the dynamics of DNA methylation in recent primate evolution and to identify regions showing species-specific methylation pattern among humans and great apes. We find that genes with alterations of promoter methylation tend to show increased rates of divergence in their protein sequence, and in contrast we also identify many genes with regulatory changes between human and chimpanzee that have perfectly conserved protein sequence. Our study provides the first global view of evolutionary epigenetic changes that have occurred in the genomes of all species of great apes. The genomic era is characterized by different comparative approaches to understand the effect of genomic changes upon phenotypes. In the context of human evolution, the genomes of all species of great apes have now been sequenced DNA methylation is an important epigenetic modification found in many taxa. In mammals, it is involved in numerous biological processes such as cell differentiation, X-chromosome inactivation, genomic imprinting and susceptibility to complex diseases Most efforts to understand DNA methylation changes in primates have focused on the comparison of human with chimpanzee or macaque Here we present the first comparative analysis of DNA methylation patterns between humans and all great ape species, allowing us to recapitulate the evolution of CpG methylation over the last 15 million years in these species. We used Illumina Methylation450 BeadChips to profile DNA methylation genome-wide in blood-derived DNA from a total of 9 humans and 23 wild-born individuals of different species and sub-species of chimpanzee, bonobo, gorilla and orangutan. We observed that the methylation values recapitulate the known phylogenetic relationships of the species, and we were able to characterize methylation differences that have occurred exclusively in the human lineage and among different great apes species. We also identified a significant positive relationship between the rate of coding variation and alterations of methylation at the promoter level, indicative of co-occurrence between evolution of protein sequence and gene regulationWe obtained cytosine methylation profiles of peripheral blood DNA isolated from a set of males and females of nine humans, five chimpanzees, six bonobos, six gorillas and six orangutans  using thCell heterogeneity may also act as a confounder when measuring DNA methylation, particularly from whole blood In this work we used two different datasets: i) we confined our analysis to 114,739 autosomal probes and 3,680 probes on the X chromosome that were directly comparable across all the species to facilitate an unbiased comparison of human and all great apes , and ii) we used 291,553 shared autosomal probes between humans and chimpanzees to compare these two species. We performed separate analyses of autosomal and sex-linked probes to prevent confounding effects of X chromosome inactivation on DNA methylation between males and females 2 = 0.98, p = 0.0003)  to each of the primate reference genome and contain no common polymorphisms suggesting that that methylation levels are associated with the evolutionary history of these species  whole-genome multiple alignments of human, chimpanzee, gorilla, and orangutan [Ensemble Compara.6_primates_EPO]  0.0003) . We then 0.0003) . This tr species .Pan sp., 5,289 in Gorilla sp. and 5,740 in Pongo sp., with the resulting final set being 99,919 CpGs shared across all five species, covering 12,593 genes  to 3′UTR). The proportion of sites removed in this step are consistent with the relative population diversity within each of these species Due to the relatively recent origin of all partitions within genera of great apes Pan species (1.2%), 1,374 specific to Gorilla species (1.4%). and 5,501 changes specific to Pongo species (5.5%)  showed no significant changes among any of the species . Comparison of genes linked with these sites showed an enrichment of Gene Ontology (GO) categories for fundamental cellular processes. In contrast, we identified 2,284 human-specific (2.3%) differentially methylated CpGs, 1,245 specific to  species . We obse species  and a si −5, see .Pan genus (chimpanzee and bonobo) we observed significant enrichments among genes involved in epithelial development and the respiratory system, while in Pongo species (orangutan) enriched categories included a variety of basic metabolic and reproductive processes (Using the Genomic Regions Enrichment of Annotation Tool (GREAT) rocesses .ARTN, COL2A1 and PGAM2  and CpG islands showed relative conservation of methylation.We observed a highly non-random distribution of the differentially methylated CpGs  in relat−16 Kolmogorov-Smirnov test), showing intermediate methylation levels, which has been shown to be a hallmark of distal regulatory elements. We also observed a significant difference in the distribution of methylation levels at differentially methylated sites compared to the rest of the genome . While tMID1 gene which has been previously reported as a gene subject to X-inactivation in humans but not in mouse Pan lineage, but not in all other great apes. Similarly the HTR2C gene shows multiple probes upstream of the TSS with similar patterns of methylation in both male and female humans, potentially suggesting that this gene escapes XCI in the human lineage. In contrast, the same sites show significantly higher methylation levels in females compared to males in all other primate species, suggesting that in these species HTR2C may be subject to XCI  is maintained via a number of epigenetic marks, including altered DNA methylation t to XCI . Using pTo maximize the identification of altered methylation patterns between human and our closest living relative, the chimpanzee, we performed a pairwise comparison of these two species using a larger dataset of 289,007 filtered probes common to human and chimpanzee. We used the chimpanzee species and not the whole genera to make use of the better annotation in the genome reference assembly for this species compared to the rest of non-human primate genome reference assemblies st exon, see A/KI ratio  of these differentially methylated genes against the remainder BRCA1 gene, which contains 32 amino acid changes between human and chimpanzee and has a KA/KI ratio of 0.69 . This gene shows large differences in methylation ∼1–1.5 kb upstream of the TSS . We estimate that over 2,500 genes present at least some methylation changes between human and chimpanzees , a higher number than that known to be affected by copy number variation or under positive selection in the same species We observed that the great ape phylogeny can be recapitulated from methylation data alone. Potential explanations for this are that methylation values could be driven by proximal DNA changes that were not controlled in this study, or that epigenetic changes have occurred independently of DNA sequence but are subject to similar rates of change either through selective pressures or neutral drift.Interestingly, we also identified a significant positive relationship between the rate of coding variation within genes and alterations of promoter methylation, suggesting a co-occurrence between changes in protein sequence and gene regulation that may be related to expression changes in fast evolving genes Overall our analysis identified >800 genes with significantly altered methylation patterns specifically within each species of human and great apes, including 171 with a methylation pattern unique to humans. Analysis of these 171 genes identified interesting enrichments for a number of functional categories that could suggest a relationship to human-specific traits. For example, we observed that genes involved in the regulation of blood pressure and development of the semicircular canal of the inner ear among others, were all highly enriched for DNA methylation changes specifically in the human lineage. While major changes in circulatory physiology are required for upright locomotion, the inner ear provides sensory input crucial for maintaining balance. Furthermore, a previous study of primates and other mammals has shown that the size of the semicircular canals is correlated with locomotion and with relatively larger canals found in species that utilize fast or agile movement Although sequencing studies have undoubtedly provided major advances in our understanding of primate evolution, our analysis of primate epigenomes unveils many novel differences among the great apes that are not apparent from purely sequence-based approaches. Of particular note is the fact that we identify enrichments in multiple independent functional gene categories which suggests that regulatory changes may have played a key role in the acquisition of human-specific trait. Therefore, epigenetic alterations likely represent an important facet of evolutionary change in primate genomes. Future studies that integrate epigenetic data with recent detailed maps of functional elements, selective constraint and chromatin interactions in the human genome The non-human research has been approved by the ethical committee of the European Research Union. No living animal has been used and DNA has been obtained during standard veterinary checks. Methylation profiling of human subjects was approved by the Institutional Review Board of the Icahn School of Medicine at Mount Sinai (HS#: 12-00567 HG).We obtained methylation data from peripheral blood DNA extracted from 9 adult humans, 5 chimpanzees, 6 bonobos, 6 gorillas and 6 orangutans. All individuals were unrelated adults and the non-human primates were all wild born. DNA samples were bisulfite converted, whole-genome amplified, enzymatically fragmented, and hybridized to the Infinium HumanMethylation450 BeadChip which provides quantitative estimates of methylation levels at 482,421 CpG sites distributed genome-wide. The assay was performed according to the manufacturer's instructions. The BeadChip array data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE41782. Due to the low density of probes targeting non-CpG dinucleotides (<0.7%) on the array, we focused our study on CpG sites.Since the 50 bp probes on the array were designed against the human reference genome but we performed hybridizations utilizing DNA from different great ape species, we first mapped the probe sequences to the chimpanzee (panTro3), bonobo (panPan1), gorilla (gorGor3) and orangutan (ponAbe2) reference genomes using BWA Methylation values for CpG sites in each sample were obtained as β-values, calculated as the ratio of the methylated signal intensity to the sum of both methylated and unmethylated signals after background subtraction . Within each individual, probes with a detection p>0.01 were excluded. We performed a two color channel signal adjustment and quantile normalization on the pooled signals from both channels and recalculation of average β-values as implemented in “lumi” package of R Using a published human data set To investigate the global correspondence of DNA sequence differences between species and the degree of methylation changes, we examined the Enredo-Pecan-Orthus (EPO) whole-genome multiple alignments of human, chimpanzee, gorilla, and orangutan [Ensemble Compara.6_primates_EPO] We built phylogenetic trees based on the methylation states of 114,739 filtered probes (perfect match probes and probes containing 1 or 2 mismatches in the first 45 bp) . We usedPan genus, 5,289 in Gorilla and 5,740 in Pongo, with the resulting final set being 99,919 CpGs shared across all five species.To identify only those methylation differences that represent fixed changes between genera, we retained only those CpGs with low methylation variance within each genus (intragenus standard deviation <0.1). This filtering step resulted in the removal of 1,377 CpGs in human, 5,224 in the Pan species/Human-Gorilla species/Human-Pongo species/Pan species-Gorilla species/Pan species-Pongo species/Gorilla species-Pongo species). We defined a site to be genus-specific differentially methylated if all three comparisons with other groups were significant  and mean β-value difference in each case ≥0.1. We also tried other statistical approaches . We defined a gene to be differentially methylated if there were at least two differentially methylated CpG sites separated by ≤1 kb. To assess significance of these observations we performed a permutation test, as follows. Based on the number of differentially methylated sites detected in each species  we randomly sampled from the 99,919 CpGs and then determined the number of clusters , repeating this process 10,000 times to create the null distribution. The p-value corresponded to the number of times that differentially methylated clusters appeared within the null distribution divided by the number of permutations .The Genomic Regions Enrichment of Annotations Tool (GREAT version 2.0.1) We performed two color channel signal adjustment and quantile normalization on males and females separately. Due to the different methylation pattern in females no BMIQ normalization was done in this data set. For studies of DNA methylation on the X-chromosome that might be linked with XCI between species, we searched for CpG sites presenting no significant changes between males and females in a specific lineage  but showing significant changes in all the other species .The number of probes shared between human and chimpanzee after applying our mapping and SNP filters was 291,554. Based on this set of probes, we performed a separate two color channel signal adjustment and quantile normalization of the raw data using only human and chimpanzee samples. We performed a BMIQ normalization to correct the probe design bias. After excluding probes with a standard deviation within either species >0.1 we retained a total of 289,007 probes. Differentially methylated sites were those with p<0.05  and a mean β-value difference ≥0.1.A/KI ratio of genes with robust alterations of promoter methylation  versus those without methylation changes . The Gene Ontology enRIchment anaLysis and visuaLizAtion tool (GOrilla) From the total set of 13,454 human:chimpanzee orthologous genes Figure S1Effect of sequence mismatches on probe performance. Difference in mean β-values between human and chimpanzee (A), gorilla (B) and orangutan (C) for each category of probes. Probes with mismatches located in the last 5 bp at the 3′ end, C>T transitions of the CpG site being measured, or ≥3 bp of mismatch showed an excess of variation compared to probes with ≤2 mismatches, and were removed from further analysis.(PDF)Click here for additional data file.Figure S2Effect of sequence mismatches on probe performance. Distribution of differences in mean β-value between human and (A) chimpanzee, (B) gorilla and (C) orangutan. Dotted lines represent the probes used in this study (perfect match and 1–2 mismatches).(PDF)Click here for additional data file.Figure S3Neighbor-joining tree based on 114,739 autosomal CpGs measured in all individuals and species. Bootstrap values  are shown for each node.(PDF)Click here for additional data file.Figure S4HTR2C gene on the X chromosome shows a relative reduction in promoter methylation specifically in human females compared to other great ape species. Probes upstream of the TSS of HTR2C show similar patterns of methylation in both male and female humans. The same sites show significantly increased methylation in females versus males in all other primate species tested. These observations suggest evolutionary changes in the X chromosome inactivation status of HTR2C specifically in humans compared to other primates.(A) Density plot showing the distribution of methylation levels of CpG sites in males and females. (B) The (PDF)Click here for additional data file.Figure S5A/KI) between human and chimpanzee in two data sets. (A) Probes with perfect match to the chimpanzee genome. Differentially methylated genes: n = 334, genes without significant changes in methylation: n = 5,655. (B) Probes with 1 or 2 mismatches in the first 45 bp in the chimpanzee genome. Differentially methylated genes: n = 247, genes without significant changes in methylation: n = 4,840.Comparison of alterations in promoter methylation with frequency of amino-acid changes and the relative rate of coding to non-coding variation within genes (K(PDF)Click here for additional data file.Figure S6(A) β-value distribution of the 114,739 sites shared among the five species before and after BMIQ. . (B) β-value distribution of the 291,553 sites shared among human and chimpanzee before and after BMIQ, .(PDF)Click here for additional data file.Table S1Sample information. NA: Information not available for these samples.(XLS)Click here for additional data file.Table S210,404 CpG sites showing species-specific differential methylation.(XLS)Click here for additional data file.Table S3815 genes associated with species-specific methylation changes.(XLS)Click here for additional data file.Table S4Pan species, and Pongo species. All GO terms shown have p<0.05 (5% FDR).(XLS)Click here for additional data file.Table S5CpG sites on the X chromosome showing gender-specific methylation changes.(XLS)Click here for additional data file.Table S6184 genes that show perfect conservation of amino acid sequence between human and chimpanzee, but which have significant epigenetic changes within their promoter regions.(XLS)Click here for additional data file.Table S7184 genes that show perfect conservation of amino acid sequence between human and chimpanzee, but which have significant epigenetic changes within their promoter regions.(XLSX)Click here for additional data file.Text S1Supplementary methods.(DOCX)Click here for additional data file.


The nuclear factor-kappa B (NF-κB) family of transcription factors has recently emerged as a major regulator of the growth and elaboration of neural processes. NF-κB signaling has been implicated in controlling axon initiation, elongation, guidance and branching and in regulating dendrite arbor size and complexity during development and dendritic spine density in the adult. NF-κB is activated by a variety of extracellular signals, and either promotes or inhibits growth depending on the phosphorylation status of the p65 NF-κB subunit. These novel roles for NF-κB, together with recent evidence implicating NF-κB in the regulation of neurogenesis in the embryo and adult, have important implications for neural development and for learning and memory in the mature nervous system. After introducing the NF-κB family and the basics of NF-κB signaling, we provide an in-depth analysis of the ways in which NF-κB affects the elaboration and structural modification of neural processes. Furthermore, we discuss the broader significance of these findings and other recent advances with respect to the neurodevelopmental roles of NF-κB and its importance in learning and memory.In mammals, the NF-κB family of transcription factors consists of five structurally related proteins, p65, RelB, c-Rel, p50 and p52, that form homodimers and heterodimers that induce or repress gene expression by binding to DNA sequences (κB elements) within the promoters and enhancers of target genes A great variety of stimuli activate NF-κB in different cell types via several distinct pathways . In the The function of nuclear NF-κB is controlled by numerous post-translational changes to the NF-κB subunits, including phosphorylation and acetylation on specific residues that can affect transcriptional activity, target gene specificity or termination of the NF-κB response The growth, branching and spatial arrangement of axons and dendrites in the developing nervous system is regulated by a multitude of extracellular proteins and by changing patterns of neural activity as neurons establish and modify synaptic connections Dendritic spines, the small protrusions that are the site of most of the excitatory synapses on principal neurons of the central nervous system (CNS), rapidly accumulate with the growth and elaboration of dendritic arbors In the following sections, we describe recent work that implicates NF-κB as a major player in the intracellular signaling and transcriptional networks that regulate the growth and elaboration of axons and dendrites during development . This woEvidence for the regulation of axonal growth by NF-κB has come from studying the effects of manipulating NF-κB signaling pathways in primary cultures of nodose ganglion sensory neurons established from developing mice a. These Although NF-κB signaling makes a major contribution to BDNF-promoted neurite growth throughout late fetal and early postnatal development, shortly before birth there is an abrupt switch in the NF-κB signaling network required for BDNF-promoted neurite growth. During a narrow developmental window between embryonic day 15 (E15) and E17, BDNF strongly activates the TK-dependent NF-κB signaling pathway through activation of its receptor TrkB and subsequent activation of two members of the Src family of tyrosine kinases, Src and Lck. These kinases in turn phosphorylate IκBα on Tyr 42 in vivo levels of phospho-Tyr42-IκBα in late fetal nodose ganglia and the subsequent marked decrease in the level of IκBα phosphorylated at Tyr42 residue during the early postnatal period is consistent with the in vivo operation of the TK-dependent NF-κB activation mechanism prenatally and its subsequent loss postnatally Subsequently, during a narrow developmental window between E18 and postnatal day 1 (P1), blocking the canonical signaling pathway, but not the TK-dependent NF-κB signaling pathway, significantly reduces BDNF-promoted axon growth and branching Like BDNF, CNTF also activates the TK-dependent NF-κB signaling pathway in developing nodose neurons In addition to enhancing growth, NF-κB signaling can also exert a potent inhibitory effect on axon growth. Whether NF-κB promotes or inhibits growth depends on the mechanism of NF-κB activation and the resulting phosphorylation status of the p65 NF-κB subunit. Neonatal sympathetic neurons, which survive and extend axons in response to the neurotrophin nerve growth factor (NGF), contain p65 that is constitutively phosphorylated on Ser 536 (phospho-S536–p65). Inhibiting NF-κB transcriptional activity in these neurons does not affect NGF-promoted neurite growth, and enhancing NF-κB transcriptional activity by tumor necrosis factor α (TNFα) treatment or by overexpressing p65, p65/p50, or IKKβ strongly inhibits NGF-promoted neurite growth in vivode novo neurite growth in primary cultures of sympathetic neuroblasts and early post-mitotic sympathetic neurons.Although inhibiting NF-κB activity in neonatal sympathetic neurons does not affect NGF-promoted neurite growth Drosophila, causes photoreceptor axons to reach incorrect positions within the optic lobe In addition to regulating the growth of sensory and sympathetic axons during the stage of development when they are growing and branching extensively under the influence of target-derived neurotrophins, NF-κB signaling has recently been implicated in the earlier stages of axon initiation and guidance. In cultures of E17 mouse hippocampal neurons, phospho-S32/36–IκBα is selectively distributed along the length of early axons and becomes progressively concentrated in the axon initial segment (AIS) with axon elongation NTR) Several studies have demonstrated a role for NF-κB signaling in regulating the growth and morphology of dendritic arbors in several regions of the developing and mature CNS. In the intact neuropil of somatosensory cortical slice cultures prepared from P3/P4 mice, inhibiting either NF-κB canonical signaling or NF-κB-mediated transcriptional activation causes highly significant reductions in dendritic length and branching of layer 2/3 pyramidal neurons NTR and Hes1/5 in CA3 pyramidal neurons of the hippocampus NF-κB-dependent enhanced expression of the helix-loop-helix transcription factors, Hes1 and Hes5, plays a key role in bringing about the effects of NGF on dendrite number and growth Purkinje cell degeneration mutant mouse, psdSid, which has an exon 7 deletion in Nna1 and is characterized by underdevelopment of Purkinje cell dendrites in vivo and in vitro. Knockdown of p65 by short hairpin RNA (shRNA) in developing wild type Purkinje cells in culture mimics the detrimental effect of Nna1 knockdown on dendritic arbor size and complexity Evidence for the participation of NF-κB in dendritogenesis in the developing cerebellum has come from analysis of the It has recently been reported that manipulating the function of IKKβ in medium spiny neurons (MSNs) of the nucleus accumbens (NAc) of adult mice in the context of reward and stress behavioral paradigms influences dendritic spine density and morphology psdSid mice exhibit reduced expression of MAP2 and MAP1B, and shRNA knockdown of these proteins in wild type cerebellar neurons also suppresses dendrite growth in culture Although the available evidence suggests that the effects of NF-κB signaling on the growth of neural processes are brought about by NF-κB-dependent changes in gene transcription, the relevant transcriptional targets are largely obscure. Only in the case of Purkinje dendritic growth have two of the required NF-κB transcriptional targets been identified, namely those encoding the microtubule-associated proteins, MAP2 and MAP1B Of the multitude of genes regulated by NF-κB in various cell types, several encode proteins that have been implicated in cell adhesion and neurite growth, such as β1 integrin p50−/− mice have a selective deficit in short-term spatial memory performance c-rel−/− mice have impaired hippocampus-dependent memory formation p65–/–/Tnfr1−/− mice c-rel−/− mice Numerous pharmacological and genetic studies utilizing a variety of experimental paradigms have implicated NF-κB signaling in learning and memory in diverse species. For example, pharmacological inhibition of NF-κB interferes with memory formation in the crab in vivo and in synaptosomes isolated from the cerebral cortex and hippocampus 2+ channels suppresses basal NF-κB activity in vivoLinks between synaptic function and NF-κB signaling that are potentially relevant for learning and memory have been increasingly recognized. The p65 and p50 NF-κB subunits are detectable at pre- and postsynaptic sites in vivoAlternative ways in which NF-κB signaling could affect learning and memory relate to its roles in regulating the growth and morphology of axons and dendrites, as discussed above. In particular, the possible influence of NF-κB signaling on dendritic spine number and morphology in the adult NAc An additional way in which NF-κB signaling might influence learning and memory relates to its role in neurogenesis . Severalp50–/– mice, there is a marked deficiency in neurogenesis in the hippocampus of these mice as a result of a defect in the late maturation of newly born neurons in vitro proliferative response of adult SVZ NSCs to TNFα In the adult CNS, neurogenesis occurs in the subventricular zone (SVZ) of the lateral wall of the lateral ventricle and in the subgranular zone (SGZ) of the dentate gyrus. In adult rats, stress activates interleukin-1β (IL-1β)/IL-1RI signaling, followed by the activation of NF-κB in NSCs in the SGV p50–/– mice Numerous studies demonstrating increased hippocampal neurogenesis following hippocampus-dependent, but not hippocampus-independent, learning tasks together with the detrimental effects of neurogenesis suppression on performance of certain hippocampus-dependent learning tasks, has established a potential link between neurogenesis in the adult hippocampus and learning and memory in vivo using viral vectors to manipulate NF-κB signaling and by using conditional gain-of-function or loss-of-function approaches in transgenic mice. Such studies will likely reveal important insights into the transcriptional networks that regulate the establishment and refinement of neuronal morphology and neural circuitry during development and structural plasticity in the mature nervous system.NF-κB signaling is becoming increasingly recognized as a major regulator of the growth and morphology of neural processes in the developing and mature nervous system. From the small number of peripheral and central neurons that have so far been investigated, NF-κB has been implicated in axonal and dendritic growth regulation from the earliest stages of process initiation to stages when neurons are establishing functional connections during development to the regulation of dendrite spine number in adult neurons. These observations raise many outstanding questions relating to the regulation and modulation of NF-κB signaling and the identification of relevant NF-κB target genes that influence the development and structural plasticity of neural processes . To date


In silico analyses highlighted significant clustering of proteins contributing to functional pathways regulating synaptic transmission and neurite development. Molecular markers of degeneration were conserved in injury and disease, with comparable responses observed in synapse-enriched fractions isolated from mouse models of Huntington's disease (HD) and spinocerebellar ataxia type 5. An initial screen targeting thirteen degeneration-associated proteins using mutant Drosophila lines revealed six potential regulators of synaptic and axonal degeneration in vivo. Mutations in CALB2, ROCK2, DNAJC5/CSP, and HIBCH partially delayed injury-induced neurodegeneration. Conversely, mutations in DNAJC6 and ALDHA1 led to spontaneous degeneration of distal axons and synapses. A more detailed genetic analysis of DNAJC5/CSP mutants confirmed that loss of DNAJC5/CSP was neuroprotective, robustly delaying degeneration in axonal and synaptic compartments. Our study has identified conserved molecular responses occurring within synapse-enriched fractions of the mouse brain during the early stages of neurodegeneration, focused on functional networks modulating synaptic transmission and incorporating molecular chaperones, cytoskeletal modifiers, and calcium-binding proteins. We propose that the proteins and functional pathways identified in the current study represent attractive targets for developing therapeutics aimed at modulating synaptic and axonal stability and neurodegeneration in vivo.Degeneration of synaptic and axonal compartments of neurons is an early event contributing to the pathogenesis of many neurodegenerative diseases, but the underlying molecular mechanisms remain unclear. Here, we demonstrate the effectiveness of a novel “top-down” approach for identifying proteins and functional pathways regulating neurodegeneration in distal compartments of neurons. A series of comparative quantitative proteomic screens on synapse-enriched fractions isolated from the mouse brain following injury identified dynamic perturbations occurring within the proteome during both initiation and onset phases of degeneration.  Drosophila, in which we found that at least six of the individual proteins modified in degenerating synapses from mice were capable of independently regulating neuronal stability and degeneration in vivo. Designing novel therapeutics to target these proteins and pathways may help to delay or prevent neurodegeneration across a range of diseases.In diseases affecting the nervous system, such as Alzheimer's disease and motor neuron disease, the breakdown of synaptic connections between neurons is a critical early event, contributing to disease onset and progression. However, we still know very little about the molecular machinery present in synaptic and axonal compartments of neurons that regulate their stability and cause breakdown during neurodegeneration. In this study we examined the protein composition of healthy and degenerating synapse-enriched fractions isolated from the brains of mice in order to identify early molecular changes occurring during neurodegeneration. We identified a range of proteins and cellular pathways that were modulated in synapse-enriched fractions during the early phases of degeneration, many of which were already known to regulate synaptic function. Similar molecular alterations were found in synapse-enriched fractions prepared from mouse models of Huntington's disease (HD) and spinocerebellar ataxia type 5. Data from these proteomic studies were then used to design experiments in  Synaptic and axonal compartments of neurons are exceptionally vulnerable to a wide array of neurodegenerative stimuli, ranging from physical trauma through to genetic disease SWld) protein in vivo are relatively rare, including cysteine string protein alpha  in vivo. However, this is likely to require the development of integrated experimental approaches capable of identifying and characterizing molecular responses to degeneration in distal compartments of neurons.Despite an increasing awareness of the scientific and clinical importance of synaptic and axonal degeneration, little is known about why distal compartments of neurons are particularly vulnerable. Furthermore, our understanding of molecular and genetic mechanisms regulating neurodegeneration remains in its infancy. Of the many proteins present in synapses and distal axons, only a few have been shown to be capable of directly modulating neurodegeneration. One of the most extensively characterised, the chimeric Wallerian degeneration slow  showed that molecular pathways underlying distal neuron degeneration were conserved from injury to disease. Genetic manipulation of 13 synaptic proteins using mutant Drosophila lines led to the identification of 6 potential regulators of axonal and synaptic degeneration in vivo: ALDHA1 (Aldehyde dehydrogenase), CALB2 , DNAJC5/CSP (DnaJ (Hsp40) homolog, subfamily C, member 5), DNAJC6 (DnaJ (Hsp40) homolog, subfamily B, member 6), HIBCH (3-hydroxyisobutyryl-CoA hydrolase) and ROCK2 . A more robust genetic analysis of DNAJC5/CSP confirmed that loss of this synaptic protein was neuroprotective, robustly delaying degeneration in axonal and synaptic compartments of neurons in vivo. We conclude that conserved molecular responses are instigated locally within distal compartments of neurons during the early stages of neurodegeneration. Such responses are focused around networks of proteins modulating synaptic transmission, incorporating molecular chaperones, cytoskeletal modifiers, and calcium binding proteins.Here, we report on the development of a novel ‘top-down’ approach for identifying proteins and functional pathways regulating neurodegeneration in distal compartments of neurons To uncover molecular pathways activated in synapses and axons during the early stages of neurodegeneration we initially wanted to obtain a global overview of protein expression changes occurring locally within distal neuronal compartments undergoing degeneration in response to a defined stimulus. We therefore performed a series of comparative, unbiased proteomic screens on synapse-enriched fractions biochemically isolated from the mouse brain . In order to improve our dataset, leaving only molecular responses directly associated with active processes of degeneration, we refined our analysis by undertaking a subsequent comparison with proteomic data obtained from synapse-enriched fractions in mice genetically protected from neurodegeneration by the g injury , we refig injury . This apDrosophila (see below), we wanted to exclude any of the 93 putative proteins identified for which we could not be certain of their identity based on the peptide sequences reported from the proteomics analysis. We therefore excluded all putative proteins identified only by 1 unique peptide that we could not subsequently validate by western blotting. Examples of validation western blots for one up-regulated (ABLIM1) and one down-regulated (UBR4) protein are shown in in vivo.Given that we planned to use data generated by our proteomics analysis to directly guide subsequent molecular genetic experiments in Expression mapping of all 47 proteins identified as having modified expression levels in degenerating synapse-enriched fractions allowed them to be grouped according to the temporal dynamics of their responses, as well as magnitude of expression change . Given tDiverse temporal patterns of expression changes were observed across the 47 proteins examined, suggesting that our analyses had detected proteins contributing both to early initiating phases and onset phases of degeneration . Of the in silico systems level analysis on the proteomics data using Ingenuity Pathway Analysis (IPA) software. This analysis identifies statistically significant functional clustering of proteins, based on known protein interactions and biological functions reported in the published literature P value of <0.05 were considered as being significant. These experiments revealed that the 47 identified proteins were functionally clustered into a relatively small group of networks . These included candidates from the top five cellular process categories identified in our functional clustering analysis see . We quanThe pathophysiology of HD involves aggregation of mutated huntingtin (Htt) protein, transcriptional dysregulation, altered energy metabolism, excitotoxicity, impaired axonal transport and synaptic pathology The βIII-spectrin knockout mouse models many of the human aspects of spinocerebellar ataxia type 5, including; synaptic dysfunction, postural abnormalities, progressive loss of motor coordination, and cerebellar degeneration Comparisons of protein expression data obtained from the cortical lesion model, R6/2 model and βIII-spectrin knockout revealed that 9 of the examined proteins showed expression changes occurring in the same direction across all three models . Althougin vivo. We therefore used a molecular genetic approach in Drosophila to screen individual proteins for a direct role in neurodegeneration.Although we had obtained a clear understanding of conserved molecular alterations occurring in synapse-enriched fractions undergoing neurodegeneration, it remained unclear whether or not any of the proteins and pathways identified were capable of actively modulating synaptic and axonal stability and degeneration Drosophila olfactory system to screen a collection of existing mutants, or lines with transposon insertions in a subset of these genes. Briefly, mutants and insertion lines were crossed in to a background that allowed visualization of a subset of olfactory receptor neurons . Distal axons and their synaptic fields in the antennal lobe were examined in uninjured controls as well as 7 days after surgical ablation of antennae. Examining uninjured controls allowed us to screen individual mutant lines and test whether they modified basal synaptic and axonal stability (e.g. do synapses and axons degenerate spontaneously in the mutant line?). Spontaneous degeneration was identified by the presence of fragmented axons and absence/decrease of GFP signal in the glomeruli housing synaptic terminals of ORNs We examined the role of individual proteins in regulating synaptic stability and degeneration using the Drosophila lines for 21 different genes that harbored either defined mutations known to affect that gene, or P element insertions within the locus identified by the Drosophila Genome Project (see methods). Of the 34 mutant lines obtained, 14 produced viable flies suitable for analyses of axonal and synaptic stability and degeneration Exel6138, a deletion which completely removes the csp locus. Both X1csp and Df(3R)Exel6138 failed to complement the delay in axonal degeneration observed with our original allele (DG29203csp), thereby mapping this phenotype to the csp locus  in axonal and synaptic degeneration, we obtained two additional alleles: sp locus . We furtDrosophila. In addition, our work rigorously defines the role of DNAJC5/CSP as an in vivo regulator of synaptic and axonal degeneration.Thus, several individual proteins initially identified as a result of having modified expression levels in synapse-enriched fractions undergoing neurodegeneration appear capable of directly influencing synaptic and axonal stability and degeneration in Drosophila that revealed roles for 6 proteins in regulating synaptic and axonal degeneration in vivo. These findings further our understanding of mechanisms regulating the active degeneration of synapses and axons, providing a basis from which to develop novel neuroprotective strategies for a range of neurodegenerative conditions.In this study we report on the effective use of a novel ‘top-down’ approach for identifying individual proteins and functional pathways responsible for regulating neurodegeneration in synaptic and axonal compartments of neurons. By undertaking a series of comparative quantitative proteomic screens on degenerating synapse-enriched fractions isolated from the mouse brain we identified 47 proteins with robustly modified expression levels during the early stages of neurodegeneration. We showed that molecular responses to degeneration occurring in synapse-enriched fractions following injury were recapitulated in synapse-enriched fractions undergoing pathological changes as a result of disease-causing genetic mutations. We also used our proteomic data to design molecular genetic screens in Drosophila screen reveals a diverse range of biological functions. For example, both DNAJC5/CSP and DNAJC6 belong to the evolutionarily conserved DNAJ/HSP40 family of proteins that regulate molecular chaperone activity by stimulating ATPase activity in silico analysis of data generated by our proteomics experiments highlighted significant clustering of proteins within functional networks that regulate synaptic transmission. This finding is further reinforced by comparisons of the biological roles of the 6 proteins found to independently regulate degeneration in our Drosophila screen, 5 of which have been implicated in the control of synaptic function: both CALB2/Calretinin and ALDHA1 modulate synaptic long-term potentiation (LTP) An initial comparison of the 6 individual proteins found to directly mediate synaptic and axonal stability and degeneration in our Drosophila screen  is unlikely to impact directly on synaptic transmission pathways. HIBCH plays an important role in valine catabolism, disruption of which is sufficient to induce progressive infantile neurodegeneration in humans in silico analysis revealing that several of the proteins identified in our screen also contribute to pathways regulating neurite development. This supports previous observations from Drosophila models linking ubiquitin-mediated developmental processes with neurodegenerative processes occurring in axonal compartments of neurons in vivo.It is worth noting, however, that at least one of the other proteins found to influence degeneration in our in vivo. DNAJC5/CSP has been implicated in synaptic degeneration contributing to the pathogenesis of neurodegenerative diseases increased in degenerating synapse-enriched fractions following injury and in synapse-enriched fractions from mouse models of neurodegenerative disease. Moreover, a thorough genetic analysis in Drosophila using well-defined mutants in DNAJC5/CSP revealed that loss of CSP is neuroprotective, delaying degeneration in axonal and synaptic compartments. Thus, whilst it is clear that DNAJC5/CSP needs to be regarded as a critical regulator of-neuronal stability and degeneration in vivo, precise details correlating expression levels with its role in stabilizing distal axons and synapses during disease-induced degeneration remain to be determined.Only one of proteins we identified as a direct mediator of degeneration, DNAJC5/CSP, belongs to the small group of endogenous genes and proteins previously reported to directly affect synaptic stability and degeneration Drosophila provides a powerful approach for elucidating mechanisms of neurodegeneration in vivo.Given that only partial coverage of the entire synaptic proteome is possible through the coupling of subcellular fractionation with current proteomics technologies, alongside the stringent 20% cut off threshold employed, the refinement methodologies applied in the current study and the limited number of viable fly lines that we screened, it is highly likely that additional genes and proteins capable of regulating neurodegeneration remain to be discovered. Our uncovering of molecular responses underlying neurodegeneration in distal compartments of neurons, alongside the identification of 5 novel mediators of degeneration and new experimental insights into the role of DNAJC5/CSP, suggests that combining proteomic screens on synapse-enriched fractions with axonal/synaptic degeneration assays in All animal experiments were approved by a University of Edinburgh internal ethics committee and were performed under license by the UK Home Office (project license number 60/3891).sWld mice were obtained from Harlan Olac Laboratories  and housed within the animal care facilities in Edinburgh. Care was taken to ensure that the wild-type mice did not contain the alpha-synuclein gene deletion that was present in a sub-strain of Harlan Olac Bl6 mice Two month old, female C57Bl/6 (wild-type) and R62 mice with a CAG repeat number of 259–266 from breeding colonies at the University of Cambridge were sacrificed at 9–10 weeks old. Mice were genotyped as previously described sWld mice subjected to a cortical lesion and R6/2 mice). Synapse-enriched fractions were prepared as previously described g for 10 min and the supernatant (S1) was collected. The pellet (P1) was resuspended in sucrose solution and centrifuged again at 900 g for 10 min. The resulting supernatant (S1′) was combined with S1 and centrifuged in a fixed angle rotor at 20,000 g for 15 min. The supernatant (S2) was discarded and the pellet (P2) containing crude synapse-enriched fractions was washed in a Krebs-like buffer  then centrifuged at 14,000 g for 10 min.Brains were rapidly removed following sacrifice and required brain regions microdissected out  before acetone precipitation and labeling for iTRAQ analysis as previously described Aliquots of each sample equivalent to 100 µg were made up to 28 µl using the iTRAQ sample buffer minus denaturant. To each sample 1 µl of 84 mM IAA was added, the samples mixed and spun prior to incubation at room temperature in the dark for 30 minutes. To each sample 10 µl of a 1 µl/µl solution of trypsin  in water was added and the samples incubated overnight on a shaking platform at 30°C. To each vial of iTRAQ reagent  70 µl of ethanol was added, mixed and spun prior to transfer to each sample vial . The pH was checked for each sample to ensure pH was greater than 8.0 prior to incubation for 1 hour at room temperature. 100 µl of water was added to each sample to quench the reaction prior to pooling of the six iTRAQ labelled samples and subsequent drying by vacuum centrifugation as previously described The pooled iTRAQ sample was resuspended in 50 µl of 25% acetonitrile in 0.1% formic acid prior to loading through a home made ziptip using 10 µl of 10% slurry of Poros HS in 50∶50 methanol∶water. The ziptip was then washed with 3×25 µl of 25% acetonitrile in 0.1% formic acid prior to loading of the pooled iTRAQ sample. The ziptip was then washed with 3×25 µl of 25% acetonitrile in 0.1% formic acid prior to elution with a stepped NaCl gradient in 25% acetonitrile in 0.1% formic acid. A fraction of iTRAQ labelled peptides were then eluted with 2×25 µl of 5–800 mM NaCl  in 25% acetonitrile in 0.1% formic acid. A final elution of the ziptip with 200 mM NH4OH and 50% propanol was used to remove the most hydrophobic peptides. Each fraction was then dried by vacuum centrifugation and stored until mass spectrometry analysis of pooled iTRAQ samples by nano liquid chromatography-mass spectrometry/mass spectrometry (nLC-MS/MS). Prior to the analysis, each dried SCX fraction was re-suspended in 35 ml of 1% formic acid and 10 ml aliquots were injected onto an Agilent 6520 Q-TOF using an Agilent 1200 series nanoLC system with microfluidic interface as previously described Raw data files were converted to mascot generic file (mgf) by MassHunter workstation software prior to merging of the files with Mascot Daemon and subsequent database (IPI Mouse) searching with the Mascot search engine . To be considered as a protein with modified relative expression, the peptide abundance  had to be modified by greater than 20% (up or down) Ingenuity Pathways Analysis (IPA) statistical network analyses were performed as previously described Drosophila orthologs of the designated mouse proteins were identified, when available, using a reciprocal BLASTing approach and the Ensembl website (ensemble.org). Available mutations were identified in Flybase the following Drosophila mutant stocks were obtained from the Bloomington Stock Center (Bloomington ID #): 12900, 26277, 18382, 7762, 20497, 12301, 30075, 24665, 18046, 5595, 23173, 6671, 13491, 20672, 25213, 18502, 13446, 9109, 11876, 7084, 25107, 7938, 5708, 11734, 29228, 2247, 13491, 15889, 8479, 18884, 23097, 15642, 382, 32035, and 7617. Mutant stocks on the X chromosome were crossed to OR22a-Gal4, UAS-mCD8::GFP flies and males were used for the degeneration screen. Stocks for mutants on the second chromosome were crossed to Sp/Cyo; OR22a-Gal4, UAS-mCD8::GFP, and the progeny were self-crossed to obtain homozygous mutant flies on the second and OR22a-Gal4, UAS-mCD8::GFP on the third chromosome. Similarly, stocks for mutants on the third chromosome were crossed to OR22a-Gal4, UAS-mCD8::GFP; Dr/TM3 and the progeny self-crossed to obtain homozygous mutant flies on the third chromosome and OR22a-Gal4, UAS-mCD8::GFP on the second chromosome.rd antennal segment was then removed, and the flies aged for another 7 days. The right antennal ORNs served as unlesioned controls. The fly heads were then fixed in 4% formaldehyde/PBS/0.1% Tween, and dissected. Fly brains were mounted in Vectashield and examined by confocal microscopy where they were phenotypically scored by an investigator who was unaware of the genotype. A minimum of 10 animals were examined and assessed per mutant strain.The Wallerian degeneration assay was performed as described Spontaneous degeneration in uninjured axons and synapses was scored by eye using the following criteria: 0 = no evidence for fragmentation of axons or loss of GFP fluorescence in the glomerulus; 1 = <10% of axons showing fragmentation and/or mild loss of GFP fluorescence in the glomerulus; 2 = <25% of axons showing fragmentation and/or mild to moderate loss of GFP fluorescence in the glomerulus; 3 = >50% of axons showing fragmentation and/or moderate to severe loss of GFP fluorescence in the glomerulus; 4 = only fragmented axons remaining and/or severe loss of GFP fluorescence in the glomerulus; 5 = no GFP signal remaining in axons or the glomerulus. Delayed degeneration in injured axons and synapses was scored by eye using the following criteria: 0 = no intact axons and no fragments remaining; 1 = no intact axons but fragmented debris remaining; 2 = ∼25% of axons intact but with extensive fragmentation of surrounding axons; 3 = ∼50% of axons intact with evidence for fragmentation in surrounding axons; 4 = >75% of axons intact with only modest amounts of fragmentation in surrounding axons; 5 = intact axons and synapses with no evidence for fragmentation.P<0.05 was considered to be statistically significant.Statistical analyses were performed using either Ingenuity Pathways Analysis (IPA) software  or GraphPad Prism software (for data from quantitative western blots). Table S1All proteins with altered expression levels >20% in wild-type striatal synaptosome preparations at 24 and/or 48 hrs after cortical lesion.(PDF)Click here for additional data file.Table S2S striatal synaptosome preparations at 24 and/or 48 hrs after cortical lesion.All proteins with altered expression levels >20% in Wld(PDF)Click here for additional data file.Table S3Proteins with altered expression levels >20% in striatal synapse-enriched preparations from wild-type mice 48 hrs after cortical lesion .(PDF)Click here for additional data file.Table S4Systems level analysis of functional clustering of proteins modified in degenerating synapses .(PDF)Click here for additional data file.