NRP1 is expressed in smooth muscle cells (SMC) but the functional role of NRP1 in SMC has not been elucidated. We therefore investigated the biological relevance of NRP1 in SMC Nrp1 knockout mice (SMKONrp1) in which Cre recombinase is driven by the smooth muscle-specific myosin heavy chain (smMHC) promoter.Conditional gene targeting generated SMC-specific Nrp1 deficiency resulted in a significant reduction in intestinal length by 6 months, and, by 18 months, in severe constipation, and enlargement of the intestine consistent with chronic intestinal pseudo-obstruction. These effects were associated with significant thinning of the intestinal smooth muscle, and decreased intestinal contractility. Expression of contractile proteins was reduced in SMKONrp1 mice, including the smMHC isoform, SMB, whereas we observed a significant increase in the expression of the small-conductance calcium-activated potassium channel 3 (SK3/KCa2.3), implicated in negative regulation of smooth muscle contraction.SMC-specific Nrp1 deficiency in visceral SMC results in adult-onset defects in gastrointestinal contractility and motility and causes a shift to a less contractile SMC phenotype. These findings indicate a new role for Nrp1 in the maintenance of the visceral SMC contractile phenotype required for normal GI motility in aged mice. Nrp1-null mice die between embryonic day (E) 10.5 and E14.5, dependent on the genetic background, with a spectrum of cardiovascular and neuronal defects [Nrp1 in vascular endothelial cells are also embryonic lethal due to aberrant cardiovascular development [Nrp1 are viable and fertile with no discernible spontaneous phenotype, but exhibit increased mortality when challenged by cardiac pressure overload [Neuropilin 1 (NRP1) is a transmembrane glycoprotein receptor for two types of ligands, the class 3 semaphorins which regulate axonal guidance and neuronal patterning during development , and memelopment . Mice heoverload . The preoverload ,9,10, anIn vivo, apart from neurons, epicardium and endothelium, Nrp1 is reported to be most strongly expressed in late mouse embryos in the SMC of large vessels [Recent evidence indicates that NRP1 is expressed in other cell types , includi vessels . HoweverSmooth muscle is essential for the contractility of blood vessels, the uterus, and the organs of the GI tract, including the small and large intestines, the bladder and the urinary tract. Peristaltic gut motility, essential for the movement and digestion of food, and the excretion of solid waste, occurs as a result of the co-ordinated action of the circular and longitudinal smooth muscle layers in the GI wall. Genetically-targeted deficiency of components of the smooth muscle contractile apparatus, such as myosin light chain kinase (MLCK) , or smooSince NRP1 is expressed in SMC, we sought to investigate its role in SMC functions in vivo by generating mice with a SMC-specific loss of Nrp1 (Nrp1SMKO). These mice were viable, healthy, with no overt phenotype during early post-natal life. Unexpectedly, however, Nrp1SMKO mice developed a pronounced age-dependent defect in GI motility. Further studies of GI SMC function in aged Nrp1SMKO mice revealed marked defects in intestinal length and SMC architecture, combined with impaired GI motility and SMC contractility. These defects in GI tissue and function were associated with changes in expression of markers of SMC phenotypes indicating that loss of Nrp1 caused a switch to a less contractile SMC phenotype. Furthermore, examination of early post-natal mice revealed changes indicative of a similar early shift to a less contractile phenotype in Nrp1SMKO mice. These results show that NRP1 is essential for maintenance of the visceral SMC contractile phenotype and of the contractile functions of SMC required for normal GI motilityThis work was performed under UK Home Office Licence 70/7700, following full ethical approval by the University College London (UCL) Animal Welfare and Ethical Review Body, and review by the UK Home Office.Nrp1 knockout mice, two successive rounds of breeding were performed (+/−smMHC-Cre) [fl/fl)Nrp1  were healthy, viable and fertile. Male +/−; Nrp1fl/+smMHC-Cre mice, that were generated from the first cross, were then bred with female fl/flNrp1 mice to generate SMC-specific Nrp1 knockout mice . In smMHC-Cre/eGFP transgenic mice, transient expression of Cre recombinase occurs in the germ cells with expression ending prior to fertilization [Nrp1 (hereafter referred to as -) in all tissues and giving rise to mice heterozygous for global Nrp1 deficiency. Since Cre activity stops prior to fertilization, the resulting offspring will exhibit no universal deletion of any floxed allele of maternal origin. Consequently, in the second breeding round four different genotypes were generated: +/−;Nrp1fl/−smMHC-Cre , −/−;Nrp1fl/−smMHC-Cre  which we used as the controls for the SMC-specific Nrp1 knockout and globally Nrp1 heterozygous mice (SMKONrp1); +/−;Nrp1fl/+smMHC-Cre , and −/−;Nrp1fl/+smMHC-Cre (designated fl/+Nrp1 or WT). Genotyping of genomic DNA from ear biopsies was performed by polymerase chain reaction (PCR)  and the United Kingdom Home Office Animals (Scientific Procedures) Act 1986). In order to generate SMC-specific erformed B. First-Cre+/−) ,19,20 wi86. In oron (PCR) .Protein expression was determined in paraffin-embedded sections by immunostaining staining, and in tissue lysates by immunoblotting, as described ,22, usinProliferating cells in the colon were detected in P4, P7 and adult (6M) mice by immunostaining bromodeoxyuridine (BrdU) following intraperitoneal BrdU injection. To detect proliferating cells, each mouse was injected intraperitoneally with 100μg/g body weight bromodeoxyuridine (BrdU)  4 hours before harvesting the tissue. Tissue sections were treated with 2M HCl for 45 minutes followed by incubation with 0.1M Boric acid for 30 minutes prior to immunohistochemistry with anti-BrdU antibody (Dako # M0744). Proliferating cells were counted in the muscularis externa of the colon,Assessment of gastric emptying was carried out as described before , 25, excColonic contractility was measured in organ baths containing Krebs-Ringer buffer at 37°C. Colonic segments were stretched gradually to .5–1.0g tension and challenged with 80mM potassium chloride (KCl). Increasing doses of carbachol (CCh) or KCl were then administered. Changes in isometric tension were measured and recorded by LabChart software .t test. When the interaction was found significant in repeated measures two-way analysis of variance, one-tailed paired Student t test was performed. The significance of association between SMB isoform expression and the genotype was analysed in a 2×2 contingency table using a chi-squared test. P<.05 was considered statistically significant.Statistical significance was assessed either by repeated measures two-way analysis of variance followed by Bonferroni’s Multiple Comparison Test, or two-tailed Student in vivo we generated mice harbouring a deletion of Nrp1 in SMC by crossing Nrp1 floxed mice [The smMHC-Cre/eGFP mouse line, expressing Cre recombinase under the control of the smooth muscle-specific myosin heavy chain promoter ,20, has xed mice  with smMDetermination of eGFP expression in smMHC-Cre/eGFP mice confirmed that Cre recombinase activity was restricted to smooth muscle-rich tissues, including the large arteries, colon, bladder and uterus D. To fuNrp1 floxed alleles by Cre recombinase was verified by measurement of Nrp1 mRNA and protein expression. Nrp1 mRNA levels in the colon, measured using qPCR, were markedly reduced both in mice that had a SMC-specific Nrp1 knockout and were also globally heterozygous for Nrp1 (SMKONrp1) (32.87% ± 10.7%) and in globally heterozygous Nrp1 mice (+/−Nrp1) (38.93% ± 4.99%), as compared to wild-type fl/+Nrp1 (100%)  mice (+/−Nrp1 heterozygotes (p < 0.001) and in SMKONrp1 (p < 0.001) post-natal day (P) 7 pups as compared to wild-type (WT) fl/+Nrp1 pups. Immunohistochemical staining for NRP1 also revealed a marked reduction in NRP1 expression in the visceral SMC of colons from SMKONrp1 compared with WT fl/+Nrp1 mice and +/−Nrp1 heterozygotes  were compared with global Nrp1 heterozygotes , with additional comparisons with WT fl/+Nrp1 mice . See “The efficiency of recombination of the ozygotes B. Quanths) mice . Colonicozygotes . Double ozygotes . We alsoMKO mice . AdditioMKO mice . These rSMKONrp1 mutants were viable and fertile and appeared to develop normally and undergo normal post-natal growth. No overt vascular abnormalities were observed in adults either in large vessels, such as the aorta, or in the adult retinal vasculature (results not shown). However, by 18 months of age SMKONrp1 mice exhibited abdominal distension and a significantly reduced rate of gastric emptying, indicated by reduced overnight stool count (+/−Nrp1 controls and WT fl/+Nrp1 mice (SMKONrp1 mutants compared to the heterozygous controls (SMKONrp1 mutants (P<.0001) and width (P<.0009) mice were significantly greater in +/−;SMKONrp1 compared to +/−Nrp1 mice .To examine whether Nrp1 deficiency affected the GI smooth muscle architecture and organisation colonic tissue sections from ≥18 month-old  studies , reveale externa A. Immunrtrophic B. ExamiSMKONrp1 mutants by staining for Class III beta-tubulin, encoded by the Tubb3 gene, using an antibody that specifically recognises neurons in the adult mouse intestine [fl/+Nrp1 and +/−Nrp1 colon sections revealed clusters of neurons at the interface between circular and longitudinal smooth muscle layers, and neurons interspersed within the circular smooth muscle. TUBB3-positive neurons in SMKONrp1 mice had a flattened and elongated appearance  was observed at 80mM KCl  at which the maximal contraction was induced (data not shown), and a trend towards a decrease was observed at 48 mM KCl . Furthermore, the amplitude of spontaneous contractile activity from the SMKONrp1 mutants was reduced when compared to that of heterozygous +/−Nrp1 controls 7 pups and 6 month-old mice. No significant differences were observed in the length of the small intestine or colon in the SMKONrp1 mutants compared to their littermate +/−Nrp1 controls at P7 . Two widely studied markers of contractile SMC are smMHC and Sm22alpha [smMHC and Sm22alpha mRNA expression levels in colonic tissue extracts revealed a significant reduction of both contractile SMC markers in the +/−;SMKONrp1 mutants   A. A redstaining B. In co+/−Nrp1 samples analysed, in contrast, no expression of the SMB isoform could be detected in any of the SMKONrp1 samples   E. This +/− mice .in vivo. To investigate such a role we generated mutant mice with a conditional SMC-specific deficiency of SMKONrp1. Nrp1 mice were viable with no overt phenotype or significant mortality in development or in early post-natal life. However, aged mice with SMC-specific loss of Nrp1 unexpectedly exhibited signs of severely impaired GI motility characterised by increased size and reduced frequency of stool, reduced contractility of the colon, enlargement of the bladder and a distended and shortened large and small intestine, defects that were associated with a marked reduction in the thickness of the muscularis externa smooth muscle layer of the colon. Collectively, these defects indicate an essential role of NRP1 in the SMC of the gut in maintaining both healthy GI function and the normal architecture of visceral smooth muscle in later adult life.Recent findings indicate that NRP1 is expressed in VSMC, and mediates signaling and migratory responses in SMC to PDGF, raising the possibility that NRP1 plays an important role in physiological and pathophysiological roles of smooth muscle Nrp1 in SMC resulted in a shift of gut SMC to a less contractile and more synthetic state. In particular, the reduced expression in SMKONrp1 mice of smMHC and its SMB isoform is consistent with the reduced colonic contractility in these mutants. The SMB isoform is expressed mainly in bladder, intestine, stomach, and small muscular arteries, and is the predominant isoform in intestine [SMKONrp1 mutants of the small Ca2+-activated K+ channel, SK3, implicated in negative regulation of smooth muscle contraction, further indicates that mechanisms regulating smooth muscle contractility are disrupted in SMKONrp1 mice. This conclusion is also supported by immunofluorescent staining of colonic smooth muscle showing that SMC in SMKONrp1 mice had a less spindle-shaped appearance and were less closely arranged with larger intercellular spaces, characteristic of hypertrophic and more synthetic cells. Though mutant mice lacking Nrp1 in SMC appeared outwardly normal during development and early post-natal and adult life, we observed increased SMC proliferation and expression of SMemb, a marker of the synthetic phenotype, in early post-natal SMKONrp1 pups (P7). Furthermore, a significant reduction in the length of the small and large intestines was observed as early as 6 months in SMKONrp1 mice. Together, these findings indicate that loss of Nrp1 in gut SMC causes early post-natal changes in SMC resulting in a persistent loss of their contractile phenotype in turn contributing to progressively worsening defects in GI structure and late-onset impairment of GI contractility and motility. It is plausible that the increased proliferative capacity of SMC in SMKONrp1 mice and other changes associated with the synthetic phenotype make intestinal smooth muscle more susceptible to long-term degenerative processes resulting in thinning of the intestinal smooth muscle layer and loss of function. Since gastrointestinal SMC express PDGFRα and its major ligand PDGF-A, changes in SK3 expression might be due to expansion of SMC or non-SMC co-expressing PDGFR-alpha due to PDGF-A driven cell proliferation and/or migration. Though we found no significant changes Pdgfr-alpha mRNA expression in SMKONrp1 mutants as compared with WT fl/+Nrp1 or globally heterozygous +/−Nrp1 mice, it is plausible that PDGF-A signaling via PDGFRα may play a role in increasing SK3 expression, and in promoting a switch in gastrointestinal SMC to a more synthetic, less differentiated phenotype. This possibility warrants further study. Though we did not observe gross changes in the early post-natal GI tract, further investigation of the role of Nrp1 in the early stages of GI development may be warranted. It is of interest that mice null for Nrp2 exhibited enhanced bladder smooth muscle contractility [SMKONrp1 mice is consistent with the other pathological GI features described herein. It is possible therefore that NRP1 and NRP2 may play different, even opposing, roles in regulating visceral smooth muscle function.Analysis of markers of the contractile and synthetic/proliferative phenotypes indicated that loss of ntestine . Differentestine . This inntestine , and in ntestine . The incactility . We did SMKONrp1 mice indicates that loss of Nrp1 expression in SMC does not significantly impact upon cardiovascular development, angiogenesis, or arteriogenesis, though we do not preclude the possibility that targeted deletion of Nrp1 in SMC may have more subtle or organ-specific effects on cardiovascular integrity and development. Furthermore, it is plausible that residual SMC NRP1 expression perhaps due to incomplete Cre-mediated deletion of Nrp1 in our model is sufficient to perform some key functions of NRP1 during embryonic and post-natal development.The viability and apparently normal development of Ret or Gdnf, implicated in differentiation or patterning of the enteric nervous system, giving rise to an aganglionic megacolon [SMKONrp1 mice are similar, though less severe and later in onset, to the phenotype of mice with SMC-specific deletion of Serum Response Factor (SRF), a transcription factor for many contractile SMC genes [SMKOSRF mice exhibited chronic intestinal pseudo-obstruction (CIPO) resulting in cachexia and death between P13 to P22, and associated with decreased intestinal length and dilation of the large bowel, thinning of the intestinal SMC layer and reduced intestinal contractility. Several of these defects are similar to those observed in SMKONrp1 mice and in human CIPO. The loss of SMC-specific markers in intestinal SMC is associated with some forms of human GI motility dysfunction. For example, reduced jejunal α-SMA expression has been reported in idiopathic CIPO [Nrp1 in visceral smooth muscle could potentially be an underlying contributor to myopathy leading to impaired GI motility in human CIPO, a possibility that merits future investigation.Chronic obstruction of the large bowel and severe constipation in humans can be caused by defects either in the enteric nervous system or in the smooth muscle. Hirschsprung’s disease, the main genetic cause of chronic life-endangering constipation with a frequency of 1:5000 live births, results in many cases from mutations in specific genes, such as egacolon ,49. In cegacolon . The abeMC genes . SRFSMKOhic CIPO . The rolin vivo loss of Nrp1 in SMC, and the first to demonstrate a role of Nrp1 in visceral SMC function in vivo. The finding that Nrp1 is important for maintenance of adult mouse intestinal contractile smooth muscle function and for preservation of normal intestinal smooth muscle architecture identifies new roles for this molecule in vivo. Though we have not defined the precise mechanism involved, our results indicate a role for this molecule in promoting the sustained transition of visceral SMC to a more contractile phenotype from early post-natal life. This suggests that NRP1 mediates the actions of an extracellular factor or factors that promote differentiation of visceral SMC to the mature contractile state required for their functions in later adult life. Defining those differentiation factors that act via NRP1 in visceral SMC should be a focus of future research.This is the first study to report the effects of S1 FigSMKONrp1 adult mice (>18 months-old) compared to +/−Nrp1 controls (*P = .041). B. NRP1 protein is expressed in colonic smooth muscle cells as detected by immunofluorescence staining of colonic tissue sections from wild-type adult (5–6 months-old) mice. C. Western blotting also confirmed expression of NRP1 in extracts from purified murine bladder smooth muscle cells and whole colonic tissue from postnatal day 7 pups, this expression was markedly reduced in tissue extracts from +/−Nrp1 and SMKONrp1 littermates. D. NRP1 protein is also expressed in human colonic smooth muscle cells and is efficiently knocked down following treatment of the cells with siNRP1.A. Densitometry analysis shows a significant reduction in NRP1 protein expression in colonic tissue extracts from (TIFF)Click here for additional data file.S2 FigSMKONrp1 mice compared to +/−Nrp1 and fl/+Nrp1 controls  reflecting the larger stool size of these mice. Total weight of overnight stool  in +/−;SMKONrp1 mice was significantly reduced compared to fl/+Nrp1 and +/−Nrp1 controls , reflecting the reduction in overnight stool number which is likely due to slow gastric motility. No significant difference in stool length or width (C) could be detected in SMKONrp1 mice at 5–8 months of age (n = 4). No significant difference in body weight was detected in aged (>14 months-old) SMKONrp1 mice compared to +/−Nrp1 and fl/+Nrp1 controls .Mean stool weight was significantly larger in (TIFF)Click here for additional data file.S3 FigSMKONrp1 compared to controls (A), but Nrp1 deficiency in SMC did not significantly affect Tubb3 mRNA  and protein  expression. Expression of the pan-neuronal markers HuC (D) and HuD (E) were also not significantly different between fl/+Nrp1, +/−Nrp1 and SMKONrp1 mice (16–22 months-old), as determined by Q-PCR of mRNA prepared from whole colon extracts.Class III beta-tubulin/TUBB3 immunostaining of colon sections revealed an aberrant staining pattern in (TIFF)Click here for additional data file.S4 Figfl/+Nrp1. +/−Nrp1 and SMKONrp1 mice in organ bath experiments. The spontaneous contractile activity was similar between fl/+Nrp1 and +/−Nrp1 mice and markedly different from SMKONrp1 mice.The amplitude of spontaneous contractile activity was measured in colonic ring segments from (TIFF)Click here for additional data file.S5 FigP = .3344, n = 3) and colon  were detected in P7 SMKONrp1 compared to their littermate controls. B) At 6 months of age SMKONrp1 mutants began to display a significant shortening of the small intestine  and colon length  compared to the littermate controls, but no significant changes in the colon width were detected .A) No changes in the length of small intestine ((TIFF)Click here for additional data file.S6 FigSMKONrp1 neonates compared to their littermate controls (*P = .0255). B. A trend towards increased SMEMB expression was also detected in colonic tissue extracts from SMKONrp1 adult mice (>16 months-old) compared to controls, however, this did not reach statistical significance.A. A significant increase in SMEMB expression was detected in bladder tissue extracts from P7 (TIFF)Click here for additional data file.S7 FigSMKONrp1 and control littermates, +/−Nrp1.BrdU staining of adult colonic tissue sections showed no significant difference in SMC proliferation between (TIFF)Click here for additional data file.S8 Figfl/+Nrp1, +/−Nrp1 and SMKONrp1 16–22 months-old mice .No significant difference was seen in PDGFR-alpha mRNA levels by Q-PCR in colonic tissue extracts from (TIFF)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S1 Table(TIFF)Click here for additional data file.S2 Table(TIF)Click here for additional data file.S3 Table(TIF)Click here for additional data file.S4 TableAll primers work at 60°C annealing temperature.(TIFF)Click here for additional data file.S5 Tablefl/+Nrp1, SMHETNrp1, +/−Nrp1 and SMKONrp1 close to the expected Mendelian ratio of 1:1:1:1.Predicted and observed frequencies for each genotype, expressed as percentage of the total number of animals genotyped . Four different genotypes were generated: (TIFF)Click here for additional data file.


A comparison of the same concentration of timcodar (1 μM) and rapamycin (1 μM) showed that both are inhibitors of lipid accumulation during adipogenesis. Importantly, timcodar potently (p < 0.01) suppressed transcriptional regulators of adipogenesis, PPARγ and C/EBPα, resulting in the inhibition of genes involved in lipid accumulation. These studies set the stage for timcodar as a possible antiobesity therapy, which is rapidly emerging as a pandemic.Nutrient overload and genetic factors have led to a worldwide epidemic of obesity that is the underlying cause of diabetes, atherosclerosis, and cardiovascular disease. In this study, we used macrolide drugs such as FK506, rapamycin, and macrolide derived, timcodar (VX-853), to determine their effects on lipid accumulation during adipogenesis. Rapamycin and FK506 bind to FK506-binding proteins (FKBPs), such as FKBP12, which causes suppression of the immune system and inhibition of mTOR. Rapamycin has been previously reported to inhibit the adipogenic process and lipid accumulation. However, rapamycin treatment in rodents caused immune suppression and glucose resistance, even though the mice lost weight. Here we show that timcodar (1  Drugs that are inhibitors of the adipogenic process are of much interest due to their capacity to reduce lipid accumulation in obese patients. Due to genetic factors and diets high in fat, obesity is on the rise and quickly emerging as an epidemic. The total excess cost related to obesity is estimated to be $254 billion/year in US health expenditures by 2030. We have recently shown that tetratricopeptide repeat (TPR) proteins, such as protein phosphatase 5 (PP5)  and FK50γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα)  containing 3% BSA. Subsequently, the membrane was incubated overnight at 4°C with antibodies to PPARt-tests. p values of 0.05 or smaller were considered statistically significant.Data were analyzed with Prism 5  using analysis of variance combined with Tukey's posttest to compare pairs of group means or unpaired In this investigation, we show for the first time that timcodar can inhibit lipid accumulation in a cellular model of adipogenesis. We have previously shown that FKBP proteins, FKBP52 and FKBP51, have a differential effect on nuclear receptor-regulated gene activity , 9, 35. μM of each of the compounds during an MTT assay (μM), however, had no effect on growth inhibition. Next, to determine the impact of timcodar on lipid accumulation, we treated the 3T3-L1 adipocytes with 0.1 and 1.0 μM (μM. A comparison of rapamycin (1 μM) and timcodar (1 μM) showed that they both significantly (p < 0.01) decreased lipid accumulation (p = 0.1318) change in lipid accumulation with FK506 (1 μM), even though the data were slightly elevated.Based on the above, we reasoned that timcodar, like rapamycin, may also regulate adipogenesis and lipid accumulation. As a first test, the effects of FK506, rapamycin, and timcodar on growth inhibitory properties and toxicity of 3T3-L1 cells were measured by treating with 0.1 and 1.0 TT assay . FK506 ad 1.0 μM , which dmulation . There wα, GRβ, GRγ, GR-A, and GR-P [α is the classic receptor type that is known to bind to GCs and activate or suppress genes at the GC response element (GRE) in their promoters [β does not bind GCs and inhibits GRα [α to GRβ (GRα/GRβ) can assist in determining GC sensitivity [β mRNA, but no change in the GRα/GRβ ratio was observed  increase GRα mRNA, but no change in GRβ expression, which did result in a significant increase in the ratio of GRα/GRβ (p = 0.0043). More importantly, GC-responsive genes pyruvate dehydrogenase kinase (PDK4) and glucocorticoid-induced leucine zipper (GILZ) were significantly (p < 0.05) increased with timcodar treatment (p < 0.0001) suppress growth in the MTT assay in γ transcription by binding directly to the promoter, which reduces adipocyte differentiation [We have previously shown that timcodar's most likely target is FKBP51 because it rescued glucocorticoid (GC) signaling in FKBP52 knockout MEF cells that retained normal amounts of FKBP51 . In compand GR-P . GRα is romoters . In contbits GRα , 39–41. sitivity , 39–41.  Figures . FK506 hreatment , which w p = 0.00. More imntiation .γ and C/EBPα. Drugs that inhibit the PPARγ-C/EBPα axis are considered to be antiadipogenic and reduce lipid accumulation. In Figures γ and C/EBPα protein and mRNA expression, demonstrating that they are antiadipogenic, at least in part by suppression of the PPARγ-C/EBPα axis. Interestingly, timcodar (1 μM) robustly inhibited expression of PPARγ protein and mRNA, and it was more significant than rapamycin . However, C/EBPα was suppressed at similar levels by rapamycin and timcodar. In contrast, FK506 (1 μM) treatment did not alter PPARγ and C/EBPα expression. Interestingly, Pref-1 was not affected by FK506 or timcodar but was induced by rapamycin (1 μM)  did significantly (p = 0.0415) enhance C/EBPβ expression in mature adipocytes. Possibly, long-term rapamycin treatment in animals may cause insulin resistance by chronically increasing C/EBPβ, or by GRβ [κB [The process of adipogenesis is mediated by two major regulators, PPARn (1 μM) . Pref-1 y C/EBPβ , 49. C/Etures [β . Howeverr by GRβ . A studyr by GRβ . The insr by GRβ , which sr by GRβ , 54, whi GRβ [κB . Further GRβ [κB . The alt GRβ [κB .μM) treatment suppressed genes involved in de novo lipid production and lipid storage, such as fatty acid synthase (FASN), sterol regulatory element binding protein 1 (SREBP1), stearoyl-CoA desaturase 1 (SCD1), and fatty acid binding protein 4 (FABP4)  treatment did suppress the expression of FASN and SCD1 but did not affect the expression of SREBP1 or FABP4. FK506 (1 μM) significantly (p = 0.0491) increased SCD1 expression , and de novo lipid production and storage. Inhibiting the expansion of adipocytes in the obese provides an avenue to regulate body mass size. Although its mechanism of action remains unresolved, these results suggest that timcodar may serve as a new therapeutic in the treatment of obesity, which is rapidly intensifying as an epidemic.Timcodar is a potent inhibitor of the adipogenic process that may prove useful in future therapies. Several studies have reported that rapamycin has antiobesity properties. However, long-term rapamycin administration may induce insulin resistance and possibly type II diabetes. Importantly, timcodar does not bind to the small FKBP proteins, such as FKBP12, which is necessary for inhibition of mTOR and the immune system. We did find that timcodar robustly inhibited lipid accumulation, by suppression of genes that regulate the adipogenic process (PPAR


Drosophila melanogaster,growth ceases in response to a peak of the molting hormone ecdysone that coincideswith a nutrition-dependent checkpoint, critical weight. Previous studies indicatethat insulin/insulin-like growth factor signaling (IIS)/Target of Rapamycin (TOR)signaling in the prothoracic glands (PGs) regulates ecdysone biosynthesis andcritical weight. Here we elucidate a mechanism through which this occurs. We showthat Forkhead Box class O (FoxO), a negative regulator of IIS/TOR, directly interactswith Ultraspiracle (Usp), part of the ecdysone receptor. While overexpressing FoxO inthe PGs delays ecdysone biosynthesis and critical weight, disrupting FoxO–Uspbinding reduces these delays. Further, feeding ecdysone to larvae eliminates theeffects of critical weight. Thus, nutrition controls ecdysone biosynthesis partiallyvia FoxO–Usp prior to critical weight, ensuring that growth only stops oncelarvae have achieved a target nutritional status.Despite their fundamental importance for body size regulation, the mechanisms thatstop growth are poorly understood. In DOI:http://dx.doi.org/10.7554/eLife.03091.001 The development of many of an animal's traits and characteristics are sensitive tothe conditions of its environment. The conditions experienced early on in life, inparticular, can alter how an animal grows and develops. The availability of foodduring development, for example, affects the final body size of manyanimals—including the larvae of fruit flies, which must reach a so-called‘critical weight’ before they can change into adults.A hormone called ecdysone controls when a larva turns into an adult insect. Thishormone's levels peak when a fruit fly larva has reached its critical weight, andthis peak triggers a cascade of events that ultimately will cause the larva to stopgrowing and to change into an adult fly. A signaling pathway involving insulin isalso known to help regulate body size in response to nutrition. But it was unclearhow the timing, and size, of the ecdysone peak is altered to match the larva'sdiet—and how the insulin-related pathway exerts its effect on body size.Now, Koyama et al. have found a molecular link between this signaling pathway and theecdysone hormone that can explain how nutrition can regulate the growth of the flies.First, Koyama et al. found that the ecdysone peak is delayed when young fruit flylarvae were starved before they had reached the critical weight. On the other hand,feeding these starved larvae the ecdysone hormone prevented this delay; and many ofthese larvae developed into underweight adults that were about three-quarters thesize of a typical fully-grown adult.The ecdysone hormone is made by cells within certain glands in the larvae. Koyama etal. also found that a protein called FoxO, which inhibits the insulin-relatedpathway, is transported out of the nuclei of these gland cells when growing larvaegain weight. But when larvae that had not reached their critical weight were starved,the FoxO protein was kept within the cell nuclei. Koyama et al. found that, in thenucleus, the FoxO protein blocks the production of ecdysone by interacting with aprotein that forms part of the ecdysone receptor. This protein complex delays theexpression of genes that are involved in making the hormone. When larvae were fed,the FoxO protein began to leave the nucleus, which allowed ecdysone production toresume.Future studies could now test whether FoxO controls other traits that are affected bythe insect's diet (such as lifespan), and if growth-related hormones in other animalsare affected by a similar mechanism.DOI:http://dx.doi.org/10.7554/eLife.03091.002 Environmental conditions mould the developmental programs of many organisms to producedramatic differences in body size and shape, in developmental time and in pigmentationpatterns . In inseDrosophila melanogaster, provide a tractablemodel to address this question. Drosophila larvae regulate their bodysize and developmental timing in response to nutritional conditions, similar to manyother animals /Target of Rapamycin (TOR) signalingpathway . The IISUnder well-fed conditions, insulin-like peptides (ILPs) are secreted into the insectblood or hemolymph . By bindIn mammalian cells, FoxO binds to several nuclear hormone receptors (NHRs), such asconstitutive androstane receptor (CAR) and pregnane X receptor (PXR), to regulate CYP450expression . ThefunHere, we provide definitive evidence that critical weight results from the smallnutrition-sensitive ecdysone peak early in the L3. Further, we report that IIS/TORregulates the timing of ecdysone biosynthesis at critical weight via a novel mechanism,the direct association of FoxO and Usp. With these findings, we have constructed adetailed model of the molecular mechanisms underlying environmentally-sensitive ecdysonebiosynthesis during critical weight, an event that ultimately determines the duration ofthe growth period and accordingly final body size.Drosophila  . As the For FoxO to regulate ecdysone biosynthesis in a nutrition-dependent manner, we wouldexpect that it would remain in the nucleus in starved, pre-critical weight larvae. Inlarvae starved from 0–15 hr AL3E, FoxO remained in the nuclei of the PG cells. In contSince FoxO associates with NHRs to regulate CYP450 gene expression in mammaliancells, we hypothesized that FoxO could associate with either EcR or Usp to regulatethe nutrition-sensitive ecdysone peak by regulating the expression of CYP450 ecdysonebiosynthesis genes. Using GST-pulldown assays, we found that FoxO bound to Usp butnot to EcR in vitro .Co-immuBecause in vertebrates FoxO-NHR interactions sometimes change in the presence ofhormones , we testPhantom (Phm)-Gal4, a Gal4driver specific for the PG cells, to overexpress FoxO in the PGs. These larvaeattained critical weight at larger sizes and 10 hr later than in controls . Knocking down Usp resultedin a significant decrease in body size ,known to be sensitive to IIS/TOR signaling (e74B(eip74ef isoform B), which tracks the early effects of ecdysonesignaling  domain . This mosequence  and selesequence . We thensequence . Two of sequence , as detesequence . Becausesequence  and did- and MS1096-Gal4 drivers,and in the eyes, using the eyeless (Ey)- andGMR-Gal4 drivers. For all drivers, overexpression of FoxO andFoxO NK reduced organ size to a similar degree Exel8159) -Gal4  . When we the PGs . SinceP00)-Gal4 , and in g21-Gal4  all in tcontrols . Furtherody size . TakentFoxO wild type background,FoxO null larvae that overexpressed FoxO NK in their PGs reachedcritical weight earlier and at smaller sizes than larvae that overexpressed wild typeFoxO in their PGs  FoxO overexpression in the PGs has additional stage-specific effectson the time to pupariation after the critical weight ecdysone peak and this effect isnutrition-sensitive.We next tested whether exogenous ecdysone could rescue the delay in critical weightinduced by the FoxO/Usp complex. To do this, we assessed critical weight inenotypes . In the enotypes .When weof 57 hr at 12 hrP0206>FoxO, FoxO null and P0206,FoxO null parental control animals were significantly smaller when theywere continuously fed 20E-supplemented normal fly medium when compared to animalsreared on normal fly medium alone . Several extrinsic and intrinsicstimuli affect PTTH secretion, such as photoperiod, oxygen concentrations, signalsreleased from damaged imaginal discs, and the sesquiterpenoid ‘ hormone . Activat hormone  andabla hormone . Further hormone . Thus inFoxO also regulates the critical weight ecdysone peak independently of Usp;overexpressing FoxO NK in the PGs still induces delays in ecdysone biosynthesis andcritical weight, even if these delays are more moderate than those induced by wildtype FoxO. Thus, our data suggest that FoxO plays additional roles in regulatingecdysone biosynthesis, either on its own or through interaction with other bindingpartners.The effects of starvation on ecdysone biosynthesis do not appear to be the same forall stages of development. Even though starvation causes a delay in developmentbefore attaining critical weight, once they reach critical weight, starvation inducesmoderate acceleration in the time to metamorphosis . This suOur findings have broad implications for our understanding of the mechanisms of sizeregulation and the development of other environmentally-sensitive traits. In otherinsects, traits such as seasonal wing morphs in butterflies  or horn FoxO is also known to form complexes with many vertebrate NHRs, including thyroidhormone , androgeEcoRI andKpnI whose recognition sites are included on the primers, thenintegrated on the second chromosome by site-directed insertion using the phiC31integrase and an attP landing site carrying recipient line, y[1] w[1118];PBac{y[+]-attP-9A} VK00018 (Bloomington Drosophila Stock Center #9736) (FoxOnull mutant. We obtained thePromE(800)-Gal4  line from Dr Carlos Ribeiro. For Usp and FoxO knock down experiments, weused y[1] v[1]; P{y[+t7.7] v[+t1.8] = TRiP.JF02546}attP2  as UAS double-stranded (ds) Usp and Vienna Drosophila RNAi Center 107786as UAS dsFoxO.Wild type FoxO and FoxO NK were amplified by RT-PCR using cDNA made from whole bodyextract of post-feeding (wandering) w[1118] larvae. After sequencing, the constructswere inserted into pUAST attB vector using r #9736) . w; UAS Egg collections were performed on normal food plates and larvae were reared atcontrolled densities without additional yeast . Newly molted L3 larvae were collected every 2 hr. Collected larvaewere raised in a normal cornmeal/molasses medium without additional yeast until theappropriate time point. For starvation treatments, we used 1% non-nutritional agar.To determine the duration of the L3, pupariation time was observed every 2 hr untilall treated larvae pupariated or died. We defined pupariation as cessation ofmovement with evaginated spiracles. All treatments were performed at 25°C underconstant light to avoid the effect of circadian rhythm on PTTH secretion. To analyzecritical weight, we used a breakpoint analyses as previously described . We consFor ecdysone feeding experiments, we added 0.15 mg of 20E  to 1 g of normal fly medium or starvation medium(1% non-nutritive agar). After 20E was added, the media were well mixed and spun downa day before use. To measure time to pupariation from the onset of starvation, larvaewere collected as above in 2 hr intervals from the molt. They were then transferredto 20E-supplemented fly medium until they reached the desired age for transfer to20E-supplemented agar.As a proxy for adult body size, we individually weighed pharate adults. Pharateadults, which were about 6–14 hr before eclosion, were collected from vials,carefully cleaned off using distilled water and a paint brush, and then dried for 15min on paper towels. Once dry, pharate adults were individually weighed on anultra-microbalance . We observed the presence or absence of malespecific sex combs through pupal cases under a stereoscope to distinguish males fromfemales.Concentrations of 20E were quantified using a 20-Hydroxyecdysone EIA kit . Carefully staged larvae were washed in distilled water twice, brieflydried on paper towels, weighed and flash-frozen on dry ice. Larvae were preserved inthree-times their volume of ice cold methanol and kept at −80°C untiluse. Ecdysone extraction was performed as previously described . ConcentDrosophila foxo, ecr-A,ecr-B1 and usp cDNAs were isolated by RT-PCRusing cDNA made from w[1118] wandering larvae. For ecr-A,ecr-B1 and usp RT-PCR, forward primers weredesigned for gene specific sequences with a Flag-tag sequence on the 5′-endand reverse primers were designed for gene specific sequences including native stopcodons. To create point mutation constructs, we designed primers containing pointmutation(s) and performed standard site-directed mutagenesis methods with minormodifications. GST-tagged protein was purified by Glutathione Sepharose 4B . Flag-tagged protein was detected by the anti-Flag M2 monoclonal antibody. For co-immunoprecipitation assays, we used 500 µg of larvalprotein or cell extract, the AB11  [gifts from Drs ShoSakurai (Kanazawa University) and Lynn M Riddiford ] and DDA2.7 . For western blots, the antibodies we used were: anti-Usp ,anti-EcR , anti-FoxO (1:1000) (Entire coding regions of (1:1000)  and antiImmunocytochemistry was performed using standard methods as described previously. The antTotal RNA was extracted from entire larval bodies using TRIzol (Invitrogen). AfterDNase treatment, total RNA concentration was quantified and 1 µg total RNA wasconverted to cDNA using oligo dT primers and reverse transcriptase. qPCR wasperformed using SYBR Green PCR Master Mix (Applied Biosystems) and ABI 7900HT(Applied Biosystems). Primers are listed in ampr). InR- and4E-BP-luciferase constructs were made according to previous study(NotI and BamHI and inserted into modifiedpAc5-V5-His B vector (Invitrogen).The Dmel cell line was used for all cell culture experiments. Cells were cultured inthe Express Five SMF medium (Gibco) without any serum, insulin or additives, unlessmentioned. Transfection was performed using FuGENE HD Transfection Reagent (Roche),according to the manufacturer's instructions. For insulin treatment, transfectedcells were re-suspended 66 hr after transfection, and split into two groups. 10µg/ml bovine insulin (Sigma) was added into the medium of one of these groups.Cells were kept for additional 6 hr at 25°C. Luciferase assays were performedusing the Luciferase Assay System (Promega), according to the manufacturer'sinstructions. To transfect equal amount of plasmid between all treatments, we usedbacterial ampicillin resistance gene(us study. Brieflyus study and ampldoi:10.5061/dryad.75940)  . In addi reviewprocess). Similarly, the author response typically shows only responsesto the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of thepublished articles . An edited version of theletter sent to the authors after peer review is shown, indicating the substantiveconcerns or comments; minor concerns are not usually shown. Reviewers have theopportunity to discuss the decision before the letter is sent , a Reviewing editor, and 3 reviewers.Thank you for sending your work entitled “Nutritional Control of Body SizePlasticity through FoxO-Ultraspiracle Mediated Ecdysone Biosynthesis” forconsideration at The Reviewing editor and the other reviewers discussed their comments before we reachedthis decision, and the Reviewing editor has assembled the following comments to help youprepare a revised submission.Overall, all of the reviewers felt that the manuscript was interesting, largely welldone, and addressed the important issue of the achievement of “criticalweight”. The proposal that FoxO binds to Usp to form a novel complex to inhibit apeak of ecdysone, thus regulating the critical weight transition is intriguing, and ingeneral the experiments demonstrating FoxO's involvement in CW are convincing.However the reviewers agreed that additional experiments were needed to support the mainconclusions of the manuscript.1) Show that FOXO and Usp interact in Co-IPs in fed wildtype L3 larvae. In 2) Demonstrate the effects of feeding ecdysone at precritical weight times. If the modelis correct then one would expect that feeding 20E just after ecdysis to L3 will lead toa smaller critical weight and precocious metamorphosis.3) Concerns were raised about the specificity of the Gal4 drivers used. The P0206 driverused as a driver to express UAS-FoxO in the PG is also expressed in the CA and inoenocytes, which are known to regulate larval growth. The reviewers felt that it wasimportant to use other drivers (Aug21 for the CA and the oenocyte 'specific”driver PromE-GAL4 from Joel Levine) as additional controls. Although the P0206-GAL4driver has been used in similar studies of critical weight, the authors note thatphm-GAL4 driven expression of UAS-FoxO is lethal in FoxO mutants but not in controlanimals. This observation may indicate that the presence of FoxO in the peripheraltissues of control animals is modifying the phenotypic effects of PG-specific FoxOover-expression. Although the authors suggest that animals expressing P0206-FoxO areviable due to the moderate levels of FoxO expression, these animals could be survivingdue to the presence of FoxO in oenocytes. Therefore, the observations described in 4) The authors' interpretations of the in vivo effects of UAS-FoxO NK  onwards Additional comments:Reviewer #1:1) The presence of ecdysone receptor (EcR) is shown to reduce the Usp-FoxO interactionin vitro  but ther2) Is there any functional relevance for a well-fed larva of dFoxO/Usp repression ofecdysone biosynthesis and its release near critical age? Loss of dFoxO activity is knownto have no obvious effect on the final size/viability of adult flies from well-fedlarvae MA . Moreove3) The present manuscript will be almost impenetrable for readers outside the immediatefield and, even for insiders, it will be challenging in places. For example, manyResults sections finish with a raw result and a figure citation but they would benefitfrom one sentence of interpretation/conclusion. Also, the figures and legends needclarifying in several places such as, for 4) It will be confusing to readers that phm and dib expression often peak only aftercritical weight is attained in controls and in some genetic manipulations e.g. . MoreoveReviewer #2:I have a minor comment regarding Reviewer #3:It is unclear how the authors determine CW. The plots they provide in The authors play up the fact that they have discovered a key regulator of the smallecdysone peak that is associated with CW. In this regard, I was surprised that theauthors never mention a recent paper from the King-Jones lab :e1001160.) which showed that PTTH also regulates the smallecdysone peak through effects on the nuclear localization of the NHR HR4. Since HR4 isproposed to negatively regulate biosynthetic enzymes prior to CW just like the FoxO/Uspcomplex proposed here, it would seem to me that this warrants significant mention. Arethese two processes related or coordinated? Does HNR4 associate with FoxO? It would seemthat at some level they have to be and this should be discussed.Lastly, In Manduca and to some extent in Drosophila, there is data suggesting thatlimiting oxygen is the key to determination of critical weight. Once again there is nodiscussion of this point. 1) Show that FOXO and Usp interact in Co-IPs in fed wildtype L3 larvae.In, it is shownthat FoxO binds Usp in Co-IPs of extracts from starved but not fed larvae. For thisinteraction to be relevant to the regulation of the critical weight transition, theauthors need to show that the dFoxO-Usp interaction also occurs in fed early L3larvae . While the GST pulldowns with recombinant proteinsinprovide convincing evidence that FoxO and Usp have the potential tobind in vitro, the westerns are less convincing. The westerns show aweak Co-IPed band above the background smear that we are told corresponds to FoxO in. Given that demonstrating this point is so essential for the main conclusion of thepaper about a molecular FoxO-Usp interaction, further controls need to be shown. Onesuggestion is to show that the FoxO band present in wild type animals is missing in FoxO null mutants.We have performed a new Co-IP experiment examining FoxO-Usp interactions in: 1) fedpre-critical weight wild-type larvae (0-5 h AL3E), 2) fed pre-critical weight FoxO nulllarvae, 2) starved pre-critical weight wild-type larvae and 4) fed, post-criticalweight, wild-type larvae. This experiment is now in 2) Demonstrate the effects of feeding ecdysone at precritical weight times. Ifthe model is correct then one would expect that feeding 20E just after ecdysis to L3will lead to a smaller critical weight and precocious metamorphosis.We have created a new Figure now showing 3) Concerns were raised about the specificity of the Gal4 drivers used. TheP0206 driver used as a driver to express UAS-FoxO in the PG is also expressed in theCA and in oenocytes, which are known to regulate larval growth. The reviewers feltthat it was important to use other drivers (Aug21 for the CA and the oenocyte'specific” driver PromE-GAL4 from Joel Levine) as additional controls.Although the P0206-GAL4 driver has been used in similar studies of critical weight,the authors note that phm-GAL4 driven expression of UAS-FoxO is lethal in FoxOmutants but not in control animals. This observation may indicate that the presenceof FoxO in the peripheral tissues of control animals is modifying the phenotypiceffects of PG-specific FoxO over-expression. Although the authors suggest thatanimals expressing P0206-FoxO are viable due to the moderate levels of FoxOexpression, these animals could be surviving due to the presence of FoxO inoenocytes. Therefore, the observations described inandcould result from FoxO activity in either the oenocytes and not the PG or asynergistic interaction between these two tissues. The authors may address theseproblems with two simple experiments. Does oenocyte-specific expression of UAS-FoxOin a FoxO mutant delay the onset of pupariation? And, if so, is this delay 20Edependent? Finally, both of these Figures lack the necessary P0206-GAL4 negativecontrol.We have used both Aug21- and PromE(800)-Gal4 to drive FoxO expression in the CA andoenocytes of FoxO mutant animals . We founWe’ve added the P0206>+ control to 4) The authors' interpretations of the in vivo effects ofUAS-FoxO NK (onwards) as a way of specifically impeding the FoxO-Usp interaction are notnecessarily correct. The experiments have been done very thoroughly (and oftensensibly using a FoxO null background) but the main result is that the effects ofUAS-FoxO NK upon critical age/weight, pupal weight, gene expression and ecdysone tend to be INTERMEDIATE between controls and UAS-FoxO animals. Thisindicates that, at best, the FoxO-Usp interaction only makes a partial contributiontowards the overall FoxO activity relevant to ecdysone gene repression. At worst, ifgeneral FoxO functions are compromised in the NK protein then the authors would notbe demonstrating any specific in vivo role for Usp-FoxOinteractions, simply that partially reducing the general activity of FoxO gives ahypomorphic phenotype. Therefore, the authors should examine the effects of FoxONKoverexpression while simultaneously over expressing Usp. If the complex is trulyimportant then coexpression of foxONK together with Usp should look just like foxONK.If however, Usp coexpression still augments the delay to critical weight, the resultis more consistent for a role of Usp in CW determination that is independent ofcomplex formation.We agree with the reviewers that if FoxO NK is a weak hypomorph of FoxO, this wouldexplain the intermediate phenotype. In the first version of this manuscript, weaddressed whether FoxO NK may simply be a hypomorph of FoxO through several means.First, we found that FoxO NK induced the expression of known FoxO target, InR and 4E-BP,to the same levels as FoxO. Secondly, we explored whether FoxO NK overexpression reducedthe size of tissues to the same extent as FoxO. FoxO NK significantly reduced the sizeof wings, and eyes, although not as much as FoxO. Importantly, FoxO NK and FoxO bothreduced the size of the prothoracic gland to the same extent.Further, because overexpressing Usp in the prothoracic gland on its own did notsignificantly change either CW or critical age, we concluded that Usp did not play anindependent role in CW. However, overexpressing both FoxO NK and Usp would providebetter evidence that Usp is not acting on its own to regulate CW. We found thatco-overexpressing Usp and FoxO NK did not affect the size and the age at critical weight.FurtherAdditional comments: Reviewer #1: 1) The presence of ecdysone receptor(EcR) is shown to reduce the Usp-FoxO interaction in vitro but there is no follow up of the in vivofunctional significance of EcR in this context. This issue is left hangingand so adds little to the paper. At present, the reader is left wondering whether ornot EcR directly regulates ecdysone biosynthetic gene expression in the prothoracicgland and whether or not this regulation is antagonized by FoxO. Addressing this mayadd significant extra mechanism to the model.In the in vitro method (GST-pulldown), we used standardized quantities of FoxO, Usp andEcR protein that are not adjusted to the physiological levels. Our in vitro results showthat FoxO and EcR can compete for Usp binding in principle. However in vivo, our co-IPresults show that this is unlikely to be the case. We see high levels of Usp in theco-IP overall, and in starved larvae FoxO-Usp binding does not qualitatively diminishthe amount of EcR-Usp binding .2) Is there any functional relevance for a well-fed larva of dFoxO/Usprepression of ecdysone biosynthesis and its release near critical age? Loss of dFoxOactivity is known to have no obvious effect on the final size/viability of adultflies from well-fed larvae (MA).Moreover, FoxO overexpression only delays the small L3 ecdysone peak rather thanabolishing it.Our new co-IP experiment shows that FoxO and Usp make complexes in fed, pre-criticalweight larvae , suggest3) The present manuscript will be almost impenetrable for readers outside theimmediate field and, even for insiders, it will be challenging in places. Forexample, many Results sections finish with a raw result and a figure citation butthey would benefit from one sentence of interpretation/conclusion. Also, the figuresand legends need clarifying in several places such as, for, when (and whennot) FoxO null mutant backgrounds are being used in an experiment (better indicatedon the figure itself).Thank you for your feedback, we’ve worked hard to clarify the text and make theresults and figure legends more accessible to a broader audience.4) It will be confusing to readers that phm and dib expression often peak only aftercritical weight is attained in controls and in some genetic manipulations  larvae  and use linear regression tocalculate the size at CW from the age at which larvae reach CW. We have included a newfigure supplement for ) larvae , and theThe authors play up the fact that they have discovered a key regulator of thesmall ecdysone peak that is associated with CW. In this regard, I was surprised thatthe authors never mention a recent paper from the King-Jones lab :e1001160.) which showed that PTTH alsoregulates the small ecdysone peak through effects on the nuclear localization of theNHR HR4. Since HR4 is proposed to negatively regulate biosynthetic enzymes prior toCW just like the FoxO/Usp complex proposed here, it would seem to me that thiswarrants significant mention. Are these two processes related or coordinated? DoesHNR4 associate with FoxO? It would seem that at some level they have to be and thisshould be discussed.Yes, you are right, this was an oversight. We are focusing on how environmental cuesregulate the CW pulse, but should not neglect the lovely work from the O’Connorand King-Jones labs on this subject.We have added these references in the introduction, and expanded our discussion todiscuss the relative roles of insulin/TOR versus PTTH (via DHR4).Lastly, In Manduca and to some extent in Drosophila, there is data suggestingthat limiting oxygen is the key to determination of critical weight. Once again thereis no discussion of this point.We have added this to our text.


CyPGs are produced in ischemic brain and disrupt the ubiquitin-proteasome system (UPS). Ubiquitin-C-terminal hydrolase L1 (UCH-L1) is a brain-specific deubiquitinating enzyme that has been linked to neurodegenerative diseases. Using tandem mass spectrometry (MS) analyses, we found that the C152 site of UCH-L1 is adducted by CyPGs. Mutation of C152 to alanine (C152A) inhibited CyPG modification and conserved recombinant UCH-L1 protein hydrolase activity after 15dPGJ2 treatment. A knock-in (KI) mouse expressing the UCH-L1 C152A mutation was constructed with the bacterial artificial chromosome (BAC) technique. Brain expression and distribution of UCH-L1 in the KI mouse was similar to that of wild type (WT) as determined by western blotting. Primary cortical neurons derived from KI mice were resistant to 15dPGJ2 cytotoxicity compared with neurons from WT mice as detected by the WST-1 cell viability assay and caspase-3 and poly ADP ribose polymerase (PARP) cleavage. This protective effect was accompanied with significantly less ubiquitinated protein accumulation and aggregation as well as less UCH-L1 aggregation in C152A KI primary neurons after 15dPGJ2 treatment. Additionally, 15dPGJ2-induced axonal injury was also significantly attenuated in KI neurons as compared with WT. Taken together, these studies indicate that UCH-L1 function is important in hypoxic neuronal death, and the C152 site of UCH-L1 has a significant role in neuronal survival after hypoxic/ischemic injury.Cyclopentenone prostaglandins (CyPGs), such as 15-deoxy-Δ We then constructed a knock-in (KI) mouse using the bacterial artificial chromosome (BAC) technique with a cysteine to alanine mutation at this 15dPGJ2 binding site on UCH-L1. Primary neurons derived from KI and wild-type (WT) mice were used to determine the effect of CyPG binding to UCH-L1 on cell death and disruption of the UPS. These studies address a potential role for modification of UCH-L1 by CyPGs and other reactive lipid species in stroke and neurodegenerative diseases.UCH-L1 is a target of CyPG modification.15 UCH-L1 has six cysteine residues with C90 considered as the core for protein hydrolase activity. To explore which cysteine residues in UCH-L1 are the primary targets for 15dPGJ2 adduction, Flag-tagged UCH-L1 was overexpressed in the rat primary neurons by lentivirus (LV)-Flag UCHL-WT infection and cells were then incubated with 15dPGJ2 (20 μM) for 24 h before harvest. Flag-tagged UCH-L1 was immunoprecipitated with an anti-Flag antibody and the resulting precipitants were separated by PAGE, digested with trypsin, and subjected to nanoLC-MS and -MS/MS analyses.Our and others' previous work has demonstrated that 15dPGJ2 can modify UCH-L1 on cysteine residues through Michael addition and therefore profoundly alter the protein's folding and functions.m/z=748.36. This ion yielded the MS/MS spectrum (from an average of six tandem mass spectra) shown in One of the peptides from the digest with a retention time range of 56.87–57.07 min was observed in the MS experiment as a doubly charged ion at 14 To examine whether the C152A mutation preserves hydrolase activity after incubation with 15dPGJ2, an in vitro hydrolase activity assay was performed using recombinant UCH-L1 WT and mutant C152A proteins with ubiquitin-AMC, a substrate that fluoresces when hydrolyzed by UCH-L1. Recombinant proteins were incubated with 12.5 μM 15dPGJ2 and hydrolase activity was measured at 0–12 min after the addition of ubiquitin-AMC. As shown in As a deubiquitinating enzyme, UCH-L1 hydrolase activity cleaves ubiquitin from a poly-ubiquitinated (Ub) protein. In our previous report, 15dPGJ2 modification of UCH-L1 abolished the protein's hydrolase activity.days in vitro (DIV2), and then treated with 5 μM 15dPGJ2 or vehicle for 48 h. Cell death was measured by lactate dehydrogenase (LDH) assay. As shown in μM2 and small particles measuring less than 0.5 μM2. The numbers of large and small particles per cell were counted in both UCH-L1 WT and C152A overexpressing primary neurons after 15dPGJ2 or vehicle treatment. 15dPGJ2-induced UCH-L1 protein aggregation was observed in primary neurons overexpressing WT UCH-L1; this effect was not observed in cells overexpressing the UCH-L1 C152A mutation  at 2 mutation .in vitro model in which to investigate the functions of UCH-L1. However, because the protein level of endogenous UCH-L1 is very high in primary neurons and because the lentiviral infection rate was limited to 40–50% in primary neurons, a KI mouse expressing the UCH-L1 C152A mutation was developed to overcome these obstacles. In collaboration with the University of Michigan Transgenic Core, a targeting vector was constructed and UCH-L1 C152A heterozygous KI male mice were produced on a C57Bl6 background  were observed, suggesting the activation of the apoptosis signal transduction pathway. The activation of caspase 3 and PARP was attenuated in cells produced from UCH-L1 C152A KI mice as compared with WT, indicating that 15dPGJ2-induced apoptosis was significantly inhibited in KI cells -soluble and -insoluble fractions. PolyUb proteins were detected in each fraction using anti-polyubiquitin antibody. As shown in in vivo and act as a universal signal for proteins targeted for proteasomal degradation.16 K48-polyUb is the most abundant ubiquitin linkage type in cells and has an important role in the UPS. Specific K48-polyubiquitin proteins were analyzed by immunoblotting in both WT and UCH-L1 C152A KI cells. Comparable to the results for polyUb protein, K48-polyUb was significantly induced by 15dPGJ2 treatment in both RIPA-soluble and -insoluble fractions and this induction was significantly reduced in UCH-L1 C152A KI cells as compared with WT cells. Taken together, the UCH-L1 C152A mutation attenuated the disruption of the UPS induced by 15dPGJ2 treatment in primary neurons. In contrast to the K48-polyUb linkage, the K63-polyUb linkage is not associated with proteasome degradation and may have a role in endosome transport and other intracellular functions. Unlike K48-polyUb, 15dPGJ2 treatment significantly decreased K63-polyUb detected by immunoblots in WT primary neurons, and this effect was significantly attenuated in UCH-L1 C152A KI cells  for 24 h. The neurites were visualized by immunostaining with anti-neurofilament L antibody. As shown in μM 15dPGJ2 in WT neuronal cells compared with 7.5 μM 15dPGJ2 required to induce cell death. 15dPGJ2 treatment significantly decreased the number of intact neurites and increased broken fragments in WT cells in a dose-dependent manner. Neurite damage was significantly reduced in UCH-L1 C152A mutant cells, suggesting that the UCH-L1 C152A mutation preserves axonal integrity against 15dPGJ2-induced toxicity.To determine the role of the UCH-L1 C152A mutation in 15dPGJ2-induced neuronal cell axonal injury, primary neurons from UCH-L1-C152A KI or WT mice were seeded on coverslips and incubated with 15dPGJ2 (1.25–5 The major findings of this study are (1) CyPGs such as 15dPGJ2 adduct the C152 of UCH-L1 and mutation of the C152 of UCH-L1 attenuates the loss of hydrolase activity after 15dPGJ2 treatment; (2) the UCH-L1 C152A mutation decreases 15dPGJ2-induced accumulation and aggregation of UCH-L1 and ubiquitinated proteins in primary neurons; and (3) primary neurons derived from UCH-L1-C152A mutant mice are resistant to cell death and neurite injury induced by treatment with 15dPGJ2.1H-15N HSQC spectrum of recombinant UCH-L1 protein consistent with unfolding of the enzyme.15 The effect of CyPG binding upon protein structure was abolished by mutation of C152, but not by mutation of other UCH-L1 cysteines.15 UCH-L1 hydrolyzes ubiquitin from poly-ubiquitinated proteins and thus may be a component of the neuronal UPS.19 The UCH-L1 C152A mutation tempered but did not abolish the decrease in hydrolase activity after incubation with 12.5 μM 15dPGJ2. Mutation of C152A prevents the unfolding of UCH-L1 induced by CyPGs, thus preserving protein function.15 But the UCH-L1 hydrolase active site contains a cysteine (C90) that also maybe adducted by 15dPGJ2. Accordingly, mutation of C152 only partially prevents the loss of hydrolase activity produced by CyPGs. The C152A mutation may exert its protective effects through mechanisms in addition to hydrolase activity. UCH-L1 may have other neuron-specific functions including regulation of axon transport and synaptic function.21 Unfolding of UCH-L1 induced by CyPGs may have an important role in disrupting these functions, and these functions may be dependent upon protein–protein interactions with other components of the axon and synaptosome.15MS/MS analysis  indicate24 Their reactive carbonyl group can adduct cysteine in many proteins and have been proposed to have significant biological effects in many signal transduction pathways.28 CyPGs have been shown to induce cell death in neurons characterized by apoptosis and accumulation of Ub proteins.31 The results in 32 The concentration of 15dPGJ2 has been measured to increase to an average concentration of 100 nM in ischemic brain,34 but this concentration is less than what is reported to produce toxicity in culture. However, whole brain measures of CyPG concentrations may not be reflective of localized intracellular concentrations. Additionally, there are a variety of other reactive lipid species produced under pathological conditions, which also contain reactive carbonyl groups that may interact with the C152 site. Thus, binding of reactive lipids to the C152 site may be important to the pathogenesis of ischemic injury and related disorders.CyPGs are reactive lipid species that have been shown to have a wide variety of potential protein targets.19 Similarly, UCH-L1 has been proposed to ligate ubiquitin to proteins;2 thus, UCH-L1 may have a major role in the neuronal UPS. The UCH-L1 C152A mutation also attenuated the accumulation of insoluble Ub proteins and specifically K48 polyUb proteins, and decreased the number of intraneuronal particles containing polyUb proteins and UCH-L1. Accumulation of insoluble Ub proteins disrupts multiple protein degradation pathways including both the UPS and autophagy, and results in formation of large protein aggregates.35 Binding of CyPGs to UCH-L1 produces dramatic structural changes in the molecule and produces aggregation of the protein in vitro.15 Accumulation of insoluble protein aggregates occurs in neurodegenerative disease, such as PD, AD, and ischemic brain injury.37 Modification of UCH-L1 by CyPGs and other reactive lipids may result in aggregation of UCH-L1 and insoluble protein aggregates, and thus could be important in the pathogenesis of these and related disorders.38Mutation of the C152 CyPG adduction site of UCH-L1, the site identified with conformational change of the protein with CyPG adduction, afforded protection from 15dPGJ2-induced cell death in primary cortical neurons. The UCH-L1 C152A mutation also attenuated the accumulation of both soluble and insoluble Ub proteins within the neurons. There are increased numbers of aggregates containing Ub proteins and UCH-L1 within the neuron after 15dPGJ2 treatment that are also attenuated by the C152A mutation. An increase in soluble Ub proteins may occur when there is either an increase in the number of misfolded proteins to be labeled for proteasomal degradation, or there is dysfunction in the UPS resulting in impaired de-ubiquitination or processing of Ub-tagged proteins by the proteasome. These data suggest that a significant portion of the effect of CyPGs upon accumulation of RIPA-soluble Ub proteins is due to its binding to UCH-L1. UCH-L1 hydrolyzes ubiquitin from polyUb-tagged proteins allowing the tagged protein to enter the proteasome and recycle free ubiquitin.40 A recent report has demonstrated that impairment of K63-polyUb response during oxidative stress affects polysome stability and protein expression, rendering cells more sensitive to stress.41 Twenty-four hour treatment with 15dPGJ2 significantly decreased the K63-polyUb level in WT primary neurons, and this effect was significantly attenuated in UCH-L1 C152A KI cells. Therefore, besides the effects on the proteasome system, the UCH-L1 C152A mutation may protect primary neurons against CyPG insults through other mechanisms such as protection against oxidative stress.K63-linked Ub proteins were also detected in WT and KI cells by immunoblotting . Unlike 42 UCH-L1 is selectively expressed in neurons, suggesting that it has a neuron-specific function. UCH-L1 interacts with cytoskeletal and synaptic proteins, and thus may have an important role in axonal transport and synaptic function.46 Humans with mutations in UCH-L1 have been reported to have extensive white-matter abnormalities.5 Furthermore, UCH-L1 activity modulates LTP in hippocampus and is deficient in a mouse model of AD.47 These data indicate that neuronal processes are more sensitive to 15dPGJ2-induced injury than the neuronal cell body and that the UCH-L1 C152A mutation protects neuronal processes from the toxic effects of CyPGs. Thus, UCH-L1 may have an important role in maintaining the structure and function of axons and dendrites.While UCH-L1 may have a role in the neuronal UPS, there are other enzymes expressed in brain such as UCH-L3 with much higher hydrolase activity that are known to be major constituents of the UPS in other cell types.et al.4 have shown that it is feasible to construct a HIV-1-trans-activating (TAT) domain fused UCH-L1 protein (TAT-UCH-L1). TAT-UCH-L1 proteins cross the blood brain barrier, transduce neurons, and improve memory function in AD mouse models. Additional modification of the C152 by the TAT-UCH-L1 protein may confer additional potency and stability to these proteins enhancing their therapeutic potential. Furthermore, conventional drugs that compete for the binding of the reactive lipids to the C152 site could be designed.These results demonstrate that the binding of CyPGs and other reactive lipid species may have a role in the pathogenesis of ischemia and neurodegenerative disease. Gong In summary, CyPGs and other reactive lipids produced in cerebral ischemia may bind to UCH-L1 and disrupt its structure and function, and the metabolism of Ub proteins in the neuron. The resultant accumulation of insoluble protein aggregates may produce pathogenic effects similar to those seen in stroke and neurodegenerative diseases. Neuronal processes are particularly sensitive to CyPG toxicity mediated through CyPG binding to UCH-L1. Binding of CyPGs and other reactive lipid species to UCH-L1 may be important in the pathogenesis of stroke and neurodegenerative disease, and is a potential target for future therapeutic interventions.Animal studies were performed with the approval of the University of Pittsburgh Institutional Animal Care and Use Committee and conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Animals were housed in a temperature- and humidity-controlled environment with 12 h light cycles and free access to food and water.β-actin antibody and anti-UCH-L1 antibody were from Sigma-Aldrich ; anti-NeuN antibody was from Millipore; and anti-GAPDH antibody was from Ambion . Cy3- and Alexafluor 488-conjugated secondary antibodies were from Jackson Immunoresearch Lab . Ultra performance liquid chromatography organic solvents and water were from VWR . The lentiviral expression vector, pLVX-IRES-ZsGreen1 vector, and Lenti-X HTX concentrator and packaging system were purchased from Clontech Laboratories . WST-1 cell proliferation assay kit and Lenti-X 293 T cell line were purchased from Clontech.15dPGJ2 was purchased from Cayman Chemical . Antibody sources were as follows: mouse monoclonal anti-mono- and poly-ubiquitinated proteins antibody (clone FK2) and anti-poly-ubiquitinated proteins antibody (clone FK1) were from Enzo Life Sciences . Monoclonal anti-ubiquitin antibody was from Covance  and anti-ubiquitin Lys48-specific antibody was from Millipore , anti-ubiquitin Lys63-specific antibody was from Abcam ; anti-PARP, anti-caspase-3, and anti-Neurofilament-L antibodies were from Cell Signaling ; anti-XhoI restriction sites. A UCH-L1 point mutation substituting alanine for cysteine (UCH-L1 C152A) was introduced by PCR. Constructs were confirmed by sequencing. For lentiviral expression vector construction, UCH-L1 WT or UCH-L1 C152A cDNA was inserted into pLVX-IRES-ZsGreen1 vector with XhoI and BamHI restrictive sites to generate lenti-UCH-L1 WT or lenti-UCH-L1 C152A.24 This vector expresses UCH-L1 WT or its mutant C152A and the green fluorescent protein ZsGreen from a bicistronic mRNA transcript, allowing ZsGreen to be used as an indicator of transduction. The resulting constructs were confirmed by DNA sequencing. To generate infectious lentiviral particles, Lenti-X 293 T cells were transfected with the lenti-UCH-L1 WT and KI vectors, or empty lentiviral vector together with Lenti-X packaging mix using XFect (Clontech) following the manufacturer's instructions. Lentiviral particles were then collected, concentrated, and purified. UCH-L1 C152A and WT lentiviral titrations were determined with a Lenti-X qRT-PCR titration kit (Clontech).The DNA sequence encoding full-length rat UCH-L1 WT was amplified by PCR, and cloned into pET22b vector , using NdeI and Escherichia coli Rosetta (DE3) strains (Novagen) producing 6 × His-tagged full-length UCH-L1 WT or the mutant protein UCH-L1 C152A. Protein production was induced by shaking the E. coli Rosetta cells in ‘overnight express autoinduction system' (Novagen) at 300 r.p.m. for 16 h, then purified with a His-select column (Sigma-Aldrich) according to the manufacturer's protocol. Protein purity was assessed by SDS-PAGE and Coomassie blue staining. Recombinant UCH-L1 WT and UCH-L1 C152A proteins were further confirmed by immunoblotting.Plasmids were introduced into μM) for 24 h at DIV11 before harvest. The cell lysates were immunoprecipitated with an anti-Flag antibody, and the resulting precipitants were subjected to polyacrylamide gel electrophoretic separation, in-gel digestion, and nanoLC-MS and -MS/MS analyses.Rat primary neurons were infected with LV-Flag UCHL-WT at DIV2 to overexpress Flag-tagged UCH-L1-WT. Cells were then incubated with 15dPGJ2  connected to a Waters Acquity Ultra Performance Liquid Chromatography (UPLC) system  consisting of sample trap  and a C18 column  interfaced with a nanospray ionization source. Solvent A was 0.1% formic acid in water. Solvent B was 0.1% formic acid in acetonitrile. Samples were loaded on the sample trap with 1% Solvent B at a flow rate of 15 μl/min for 1 min. Peptides were then eluted off the column with a 90-min gradient running at 300 nl/min: 5% B for 3 min, 5–55% B in 60 min, 30–95% B in 1 min, 95% B for 5 min, 95–5% B in 1 min, 5% B for 20 min. All MS spectra were acquired in the Orbitrap detector at a resolution of 60 000 in profile mode, while top nine data-dependent MS/MS spectra were yielded by fragmentation of precursor peptide ions using collision-induced dissociation (CID) normalized collision energy setting of 35% in the ion trap of the LTQ mass spectrometer, then acquired in the Orbitrap at a resolution of 7500.The gel was then stained with Coomassie blue, protein bands were excised, minced, and digested with porcine Trypsin Gold , as previously describedDatabase searches were done with Proteome Discoverer 1.4 against the complete Uniprot human database appended to a contaminant database  with the Sequest search engine for a trypsin digest with two missed cleavages, three dynamic modifications . The mass tolerance was set at 10 p.p.m. for precursor mass and 0.8 Da for CID fragment ion masses. Peptide identifications were validated using the Percolator algorithm with a 5% global false discovery rate (FDR). The spectra of 15dPGJ2-adducted peptides were manually inspected.14 Briefly, 2 μM UCH-L1 protein was incubated with 12.5 μM 15dPGJ2 or vehicle for 2 h before dilution with UCH-L1 hydrolase buffer. Samples were then mixed with 500 nM ubiquitin-AMC . The final concentration for UCH-L1 was 100 nM. Free AMC fluorophore was read using a fluorescent plate reader .The UCH-L1 hydrolase activity assay was performed as previously described with slight modifications.50and used for experiments after 9 DIV. Cells were grown in serum-free Neurobasal medium  supplemented with B27 and GlutaMAX (Invitrogen). To overexpress UCH-L1 or mutant UCH-L1 C152A in primary neurons, cells were seeded in 96-well plates and lenti-UCH-L1, lenti-UCH-L1 C152A, or empty lentiviral particles were added to the culture medium at 1.2 × 108 particles/well for 18 h. Viral infection was performed at DIV 2, and cells underwent 15dPGJ2 treatment at DIV10. Cell viability and cell death were quantitatively assessed by LDH assay and WST-1 cell viability assay according to the manufacturer's protocols (n=6–12 wells per group).Rat cortical primary neuronal cultures were prepared from E17 fetal rats  as previously described54 Homologous recombination of DNA fragments was used to modify a BAC containing UCH-L1 to produce a point mutation in UCH-L1 that converts the 152 cysteine to alanine (TGT>GCT) within the normal chromosome of the mouse  cells. The vector was then linearized and electroporated into mouse C57BL/6J ES cells. After neomycin selection, Southern blots were performed to identify ES cells having homologous recombination. These ES cells were shipped to Taconic Artemis  and injected into blastocysts to generate heterozygous UCH-L1 C152A KI mice on a C57Bl6 background. Male heterozygous UCH-L1 C152A mice were then bred with female C57Bl6 mice. Heterozygous offspring were crossed to produce homozygous UCH-L1 C152A and WT UCH-L1 lines. Mouse genotype was identified by genomic DNA extraction and PCR amplification with primers 5′-CGAGGTAATAGAAAATGCTTCTGG-3′ and 5′-GAATATGCTCCAAACAGCTTGC-3′. The details of targeting vector, Southern blotting, and genotyping protocols are available upon request. Mouse cortical primary neuronal cultures were prepared from E17 WT and KI fetal mice as described above and used for experiments after 9 DIV.In collaboration with the University of Michigan Transgenic Core, the BAC technique was used to construct a UCH-L1 C152A KI mouse.he mouse . In brieμM2 and ≥0.5 μM2 were counted in 23–25 individual cells per group using NIH ImageJ software . Data were analyzed by a blinded investigator using one-way ANOVA with Bonferroni post hoc testing.Primary neurons were seeded on glass coverslips and incubated with 15dPGJ2 or methyl acetate (veh) for 24 h before fixation with 4% PFA in phosphate-buffered saline (PBS). Immunocytochemistry using anti-Ub-proteins and anti-UCH-L1 antibodies followed by Alexafluor 488 and DyLight 549-conjugated secondary antibodies was performed, and cells were photographed using an Olympus confocal microscope with FluoView FV1000 software . For particle size counting, cells were photographed at × 240. UCH-L1 particles <0.5 34 In brief, primary neurons were treated with vehicle (methyl acetate) or 15dPGJ2 (2.5 or 5 μM) for 24 h. Cells were lysed in RIPA lysis and extraction buffer  containing 25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, and protease inhibitor cocktail (Pierce). Cells were incubated with RIPA buffer for 15 min on ice then centrifuged at 16,800 × g at 4 °C for 15 min. The supernatants were collected and the insoluble pellets were washed twice with cold PBS, and then re-suspended in RIPA buffer containing 0.5% SDS for 1 h on ice and sonicated three times for protein solubilization. Protein concentrations were determined by the bicinchoninic acid (BCA) method (Pierce).The cell lysate detergent-insoluble protein aggregate fraction was prepared as previously described.14 For caspase-3, PARP, and UCH-L1 detection, cell lysates were resolved on 10 or 12% SDS-PAGE. After blocking with 5% non-fat milk in TBS/Tween-20, membranes were incubated with anti-caspase-3, anti-PARP, or anti-UCH-L1 antibodies at 4 °C overnight. For Ub-protein detection, cell lysates were resolved on a 4–20% linear gradient polyacrylamide gel  before incubation with anti-poly-ubiquitinated conjugates, anti-ubiquitin Lys48-specific or anti-ubiquitin Lys63-specific antibodies . Blots were washed and the appropriate secondary antibodies applied. Protein signal was visualized with ECL reagents (Pierce). Blots were subsequently stripped and re-probed using anti-GAPDH or β-actin antibodies for verification of equal protein loading. Brain cortex, hippocampus, and striatum were dissected from male 12-week-old WT and KI mice (n=4 per group) and rapidly frozen on dry ice until homogenization with T-PER tissue protein extraction reagent (Pierce) and protein measurement. Equal amounts of protein were loaded for SDS-PAGE and immunoblotted using anti-UCH-L1 antibody as described above.Western blotting was performed as previously described.l-poly d-lysine-coated glass coverslips. 15dPGJ2 treatment was performed on DIV11, and cells were fixed with 4% PFA in PBS at different time points . After permeabilization with blocking buffer  for 1 h at room temperature, cells were immunostained with anti-Neurofilament-L antibody  and Alexafluor 488-conjugated secondary antibody sequentially and photographed using a confocal microscope . Image acquisition and analysis was performed on × 60 images randomly taken from two coverslips for each treatment condition per genotype (n=8 fields per group). Quantification of axon degeneration was performed by a blinded investigator as previously described.56 The axon/neurite outgrowth was evaluated based on morphological characteristics: (1) intact: length of neurite outgrowth >2 × the length of the soma and (2) fragmented: isolated neurite swellings. The numbers of intact neurites and neurites fragments were normalized to the number of neurons examined.Primary neurons from WT or UCH-L1 C152A KI mice were seeded onto post hoc testing where appropriate using SPSS software . Results were considered to be significant when P<0.05.Data are expressed as means±S.E. and were analyzed using one-way ANOVA with Bonferroni or Dunnett's