PRKCSH and SEC63. We previously found that, in patients with a PRKCSH mutation, over 76% of the cysts acquired a somatic ‘second-hit’ mutation in the wild type PRKCSH allele. We hypothesise that somatic second-hit mutations are a general mechanism of cyst formation in PCLD which also plays a role in PCLD patients carrying a SEC63 germline mutation. We collected cyst epithelial cells from 52 liver cysts from three different SEC63 patients using laser microdissection. DNA samples were sequenced to identify loss of heterozygosity (LOH) mutations and other somatic mutations in cyst epithelial DNA. We discovered somatic SEC63 mutations in patient 3 (1/14 cysts), but not in patient 1 and 2 (38 cysts). Upon review we found that the germline mutation of patient 1 and 2 (SEC63 c.1703_1705delAAG) was present in the same frequency in DNA samples from healthy controls, suggesting that this variant is not causative of PCLD. In conclusion, as somatic second-hit mutations also play a role in cyst formation in patients with a SEC63 germline mutation, this appears to be a general mechanism of cyst formation in PCLD.Polycystic liver disease (PCLD) is an autosomal dominant disorder characterised by multiple fluid filled cysts in the liver. This rare disease is caused by heterozygous germline mutations in  PRKCSH (MIM# 177060) SEC63 (MIM# 608648) PRKCSH mutations account for 15% of the PCLD cases, whereas SEC63 mutations can be found in 6% Polycystic liver disease  is a rare, dominantly inherited, disorder characterised by multiple fluid filled cysts in the liver. The cysts, which develop from bile duct epithelial cells (cholangiocytes), increase in size and number over time and can lead to a massive increase in liver volume In the case of a dominant disorder it is not always clear how the heterozygous mutations can lead to disease and which mechanisms are involved. The mutations can either lead to the production of a mutated protein which disrupts the cell function, or result in loss of functional gene product which can lead to misregulation of dosage dependent genes. Often, loss of a single allele will not have severe consequences for the function of the cell and only after the remaining allele is lost this will cause disease.PRKCSH mutation, over 76% of the cysts acquired a somatic ‘second-hit’ mutation in PRKCSHPRKCSH in the cyst epithelium suggesting that PCLD is recessive on a cellular level. This finding is consistent with reports on other cystic diseases, such as autosomal dominant polycystic kidney disease (ADPKD) PRKCSH gene product hepatocystin in these cells SEC63 lead to cyst formation. On an immunohistological level, cysts from PRKCSH and SEC63 patients show a different expression pattern for various proteins including MUC1 and C-erbB-2. Furthermore, no loss of SEC63 protein has been reported in cysts form patients with a germline mutation in this gene, which could reflect a different mechanism of cyst development in cysts from SEC63 patients Recently, we demonstrated that in PCLD patients harbouring a heterozygous PRKCSH, somatic second-hit mutations are also an important step in cyst formation in patients with a SEC63 germline mutation.Although there is genetic and immunohistological heterogeneity among PCLD patients, the clinical presentation, the presence of a normal bile duct system and the focal growth of the cysts on the other hand, are features shared by all PCLD patients SEC63 mutation. In one cyst sample we found loss of heterozygosity (LOH), whereas we did not find any somatic changes in 38 samples derived from the other two patients. After reviewing the different germline mutations we found that somatic inactivation only occurred against the background of the severe truncating germline mutation.To this end we analysed 52 cyst samples from 3 patients carrying a PRKCSH and SEC63 mutation carriers.These results show that somatic second-hit mutations play a role in cyst formation of both Liver tissue and blood samples of patients were obtained and stored in the course of treatment following the Dutch Code for the proper secondary use of human tissue. Use of this tissue for research was reviewed and approved by the regional ethics review board “Commissie Mensgebonden Onderzoek (CMO) regio Arnhem-Nijmegen”.SEC63 germline mutation carriers within the Netherlands. Patients were selected based on sequencing results from our molecular diagnostic laboratory, which routinely performs diagnostic tests for PCLD. Each PCLD patient is tested for PRKCSH as well as SEC63 and the database now holds 505 patients. Using this database we could readily identify 29 patients with a SEC63 mutation. We cross checked these patients against the Dutch National Pathology database to find those patients who had undergone laparoscopic cyst fenestration because of severe symptoms SEC63 germline mutation carriers for whom blood and tissue samples were available.We used the following strategy to obtain all known liver cyst samples from All patients were female, and age at the time of surgery varied between 33 and 41 years-of-age. We used fresh tissue samples that had been snap-frozen immediately after excision and stored at −80°C until analysis , or formalin-fixed paraffin-embedded liver tissue samples that had routinely been collected for pathological examination . All samples were collected with appropriate ethics approval: written informed consent for the use of secondary tissue was obtained from all patients.We collected whole blood from 1000 healthy controls (2000 chromosomes) which were recruited after advertisement in local papers from the same geographical region as our patients.SEC63 and PRKCSH using direct sequencing as described previously SEC63 mutations refer to the NM_007214.4 transcript according to the HGVS guidelines.We screened patient DNA from whole blood for germline mutations in SEC63 c.1703_1705delAAG mutation using PCR with the forward primer 5′-TAGTGAAATTGTCATCGAGTCAG-3′ and the reverse primer 5′-CGAGCAAGCAAACAAATGAA-3′ followed by high resolution melting (HRM). Melting curves were obtained from 65°C to 95°C with a ramp rate of 0.1°C/10 seconds in the CFX96TM Real-Time PCR Detection System  with EvaGreen  as fluorescent dye. Melting curves were analyzed using the Precision Melt software (Biorad). Abnormal curves were then sent for sequencing.We screened genomic DNA samples of 1000 healthy controls for the Tissue sections (10 µm), from frozen or formalin-fixed paraffin-embedded liver samples, were mounted on cross-linked PEN-membrane slides , stained with Mayers Hematoxylin (1 min) and rinsed in tap water. Paraffin embedded sections were deparaffinized using xylene and ethanol prior to hematoxylin nuclear stain. Specific isolation of the cyst epithelial cells (300 to 2000 cells/sample) was carried out using a Leica Laser Microdissection system (LMD 6000) equipped with an UV laser (Leica Microsystems GmbH) SEC63 and PRKCSH sequencing, cyst epithelial DNA was amplified using a commercially available whole genome amplification kit  and purified  prior to analysis. Sample amplification was performed in duplicate to control for any mutations resulting from the whole genome amplification procedure.DNA from the dissected cells was isolated using the QIAamp DNA Micro kit (Qiagen) according to instructions and with the use of carrier RNA. To increase the DNA yield and quality from formalin fixed tissue samples we made the following adjustments: samples were digested at 56°C for two days with occasional agitation; proteinase K solution (>600 mAU/ml) was added in two steps (5 µl on day 1 and 5 µl on day 2); after digestion and addition of buffer ATL, samples were incubated at 90°C for 1 hr to promote reverse cross-linking of the DNA. To obtain enough material for the We conducted the following analyses on DNA isolated from laser dissected samples:SEC63 LOH analysis. The region of the germline mutation was amplified using specific primers for the SEC63 c.958G>T mutation (forward 5′-TGAAAATTCCTGAGACCCTTG-3′ and reverse 5′-TGCTGCTTTCATCCCACTAA-3′) and the SEC63 c.1703_1705delAAG mutation (forward 5′-TAGTGAAATTGTCATCGAGTCAG-3′ and reverse 5′-CGAGCAAGCAAACAAATGAA-3′) followed by sequencing to determine the heterozygosity state of the germline mutation in both cyst epithelia and control liver cell samples.SEC63 and PRKCSH sequencing. All SEC63 and PRKCSH coding exons and flanking intronic sequences were sequenced (as described for genotyping) on amplified DNA to detect somatic mutations in the cysts. DNA isolated from whole blood of the patient was used as reference sample.5′-GGGCCAGCAGAAATAACTTG-3′, reverse 5′-GCTGTCGTTCTCATCATCCA-3′), rs13220047 , rs675117 , rs12210583  and rs10946279 , followed by sequencing.We used 5 heterozygous SNPs across chromosome 6 to analyse the heterozygosity state in cyst DNA using PCR with specific primers: rs2012025 , washed and bound to Horse-Radish-Peroxidase using the ABC method (Vector laboratories). Detection was carried out with the use of diaminobenzidine tetrahydrochloride (DAB) as substrate and enhanced with copper sulfate and nuclei were counterstained with Mayers Hematoxylin. After the staining samples were dehydrated with alcohol and xylol before mounting in Permount.Tissue sections (4 µm) from formalin-fixed paraffin-embedded PCLD liver tissue were obtained together with tissue sections for laser micro dissection. Sections were mounted on SuperFrost Plus glass slides  using a water bath and allowed to dry overnight at 37°C. Prior to the staining the tissue sections were deparaffinised with xylene and hydrated with alcohol and distilled water. Samples were microwave heated and boiled for 10 min in Na-Citrate buffer pH 6, cooled down to room temperature, washed in phosphate buffered saline (PBS) and incubated for 30 min with 1%BSA/PBS . Endogenous Avidin/Biotin was blocked  followed by overnight incubation with 1∶400 rabit anti-SEC63 antibody , 1∶200 mouse anti-hepatocystin antibody  or 1∶400 mouse anti-Cytokeratin 19  in 1%BSA/PBS. For each staining we included a negative control in which the primary antibody was omitted. Endogenous peroxidase was blocked in 0,3% HSEC63 c.1703_1705delAAG variant in DNA samples from PCLD patients with healthy controls, and to compare the presence of LOH and somatic point mutations in cyst between patients with a PRKCSH or a SEC63 germline mutation. The 95% confidence interval of the proportion of cysts with LOH was calculated with the Newcombe-Wilson method for proportions without continuity correction Fisher’s exact test was used to compare the frequency of the SEC63 germline mutation  to exon 6 in the same gene. The SNP (rs13220047) located at a distance of 82 kb remains heterozygous indicating that a genomic breakpoint occurred within this region. In tumour samples LOH regions are often telomeric We used 5 informative (heterozygous) single nucleotide polymorphisms (SNPs) across chromosome 6 to determine the extent of the region with LOH . This deImmunostaining of SEC63 and hepatocystin showed that the hepatocystin staining was similar between cysts, whereas for SEC63 the intensity of the staining was reduced in the cyst with LOH but appears normal in the cysts without loss of heterozygosity .SEC63 germline mutations and found that the germline mutation of patient #3 is potentially much more severe than those of the other 2 patients. Both patient #1 and #2 carry a heterozygous SEC63 c.1703_1705delAAG mutation which will lead to an in-frame deletion of a single amino acid: glutamate at position 568 in the protein. In contrast, the germline mutation of patient #3 (SEC63 c.958G>T) results in a premature stop codon after 319 amino acids (p.Glu320X), which deletes 441 out of 760 amino acids.We found LOH in one of the 14 cysts of patient #3, while somatic mutations were conspicuously absent from samples from the other 2 patients (38 cysts). We reviewed the different underlying SEC63 c.1703_1705delAAG mutation is a true pathogenic mutation or represents a rare (benign) polymorphism, we determined the frequency of this mutation in the normal population by high resolution melting analysis of this region in genomic DNA of 1000 healthy subjects. We found this specific amino acid deletion in 6 out of 2000 normal chromosomes , which is similar to the frequency present in a sample of 373 PCLD patients  as depicted in SEC63 c.1703_1705delAAG is not a pathogenic mutation causing PCLD which may explain the absence of any somatic mutations in this gene.To determine whether the SEC63 mutation carriers with PCLD, which is in line with the second-hit model for disease pathogenesis.Our data show that somatic second-hit mutations do occur in SEC63 c.1703_1705delAAG mutation did not differ between our PCLD patients and the healthy population. This indicates this variant is a (rare) polymorphism which is not associated with PCLD. The literature reports this mutation several times in relation to PCLD SEC63 mutation, but belong to the cohort of PCLD patients in which the genetic cause is still unknown. This is important to know as currently patients with PCLD are being screened, classified and advised based on wrong information.We did not detect somatic mutations in two out of three patients and we hypothesised this was related to the nature of the germline mutation. After reviewing the germline mutations we found that the allele frequency of the PRKCSH and SEC63 are ubiquitously expressed in the human body, it remains unclear why germline mutations would lead to a liver specific disorder. The gene product of SEC63, SEC63, is thought to play a role in protein transport across the endoplasmic reticulum (ER) membrane, whereas PRKCSH codes for hepatocystin and is part of a heterodimer complex involved in folding of glycoproteins in the ER PRKCSH and SEC63 in an ADPKD mouse model leads to a synergistic increase in disease severity, which suggests that these genes share a biological pathway As PRKCSH patients and this SEC63 patient is remarkable. We found LOH in only 7% (1/14) of SEC63 mutated cysts, whereas in PRKCSH germline carriers the majority of cysts (76%) acquired LOH SEC63 c.958G>T allele may no longer be translated into protein or is not recognised by the antibody.Although the frequency of somatic second-hit mutations varies between different genes and genetic disorders, the difference in somatic mutations between cysts from SEC63 germline mutation. Furthermore, the available tissue was formalin fixed which was not a problem for the laser microdissection procedure, but did reduce the yield of the DNA isolation. We were able to get a clear read on the LOH status of all samples, but the sequence efficiency was affected in material derived from formalin-fixed paraffin-embedded tissue. Therefore there may still be presence of, yet unidentified, somatic mutations in these samples.Our study was restricted by the limited amount of available patient tissue. However, we identified and collected all known PCLD liver tissue samples that had been stored within the Netherlands. We obtained samples from three patients, but only one patient carried a bonafide pathogenic AKT1SF3B1 gene can be demonstrated in subtypes of myelodysplastic syndromes in which ring sideroblasts are a prominent feature There is a body of evidence emanating from recent studies supporting the concept of somatic mutations as part of the genetic pathogenesis of benign and malignant disorders. It was recently shown that hamartomata that are part of the Proteus syndrome arise from somatic activating mutations in oncogene PRKCSH and SEC63 somatic second-hit mutations can occur which supports the notion that somatic second-hit mutations are part of the genetic mechanism in cyst formation in PCLD.In conclusion, we have now shown that in both


Absence of glial cell line-derived neurotrophic factor (GDNF) leads to intestinal aganglionosis. We recently demonstrated that patients with diverticular disease (DD) exhibit hypoganglionosis suggesting neurotrophic factor deprivation. Thus, we screened mRNA expression pattern of the GDNF system in DD and examined the effects of GDNF on cultured enteric neurons.Colonic specimens obtained from patients with DD (n = 21) and controls (n = 20) were assessed for mRNA expression levels of the GDNF system . To identify the tissue source of GDNF and its receptors, laser-microdissected (LMD) samples of human myenteric ganglia and intestinal muscle layers were analyzed separately by qPCR. Furthermore, the effects of GDNF treatment on cultured enteric neurons  were monitored.mRNA expression of GDNF and its receptors was significantly down-regulated in the muscularis propria of patients with DD. LMD samples revealed high expression of GDNF in circular and longitudinal muscle layers, whereas GDNF receptors were also expressed in myenteric ganglia. GDNF treatment of cultured enteric neurons increased mRNA expression of its receptors and promoted neuronal differentiation and plasticity revealed by synaptophysin mRNA and protein expression.Our results suggest that the GDNF system is compromised in DD. In vitro studies demonstrate that GDNF enhances expression of its receptors and promotes enteric neuronal differentiation and plasticity. Since patients with DD exhibit hypoganglionosis, we propose that the observed enteric neuronal loss in DD may be due to lacking neurotrophic support mediated by the GDNF system. Diverticular disease (DD) is a widespread disease in industrialized countries with increasing prevalence in the elderly. The initial pathology is characterized by multiple mucosal/submucosal outpouchings throughout the colonic muscle coat which may lead to a broad spectrum of symptoms with potentially lethal complications Traditionally, low fiber diet, elevated body mass index and connective tissue alterations are considered as risk factors for diverticulitis Survival, differentiation and maintenance of enteric neurons are strongly influenced by neurotrophic factors. Glial cell line-derived neurotrophic factor (GDNF) is a key neurotrophin for the ENS isolated originally from the supernatant of the glial cell line B49 and characterized by its ability to promote the survival of cultured dopaminergic neurons As GDNF-ablated mice exhibit total intestinal aganglionosis and DD is associated with intestinal hypoganglionosis, we raised the question whether the GDNF system might be compromised in DD thereby contributing to the partial loss of enteric neurons. Therefore, the tissue sources expressing GDNF and its receptors were identified in the human colon and gene expression profiles of the GDNF system were compared between patients with DD and controls. Moreover, the effects of GDNF on GDNF receptor expression as well as on the differentiation and plasticity of enteric neurons were monitored in rat postnatal enteric nerve cell cultures.Segments of sigmoid colon were obtained from patients  who underwent anterior rectal resection for non-obstructive colorectal carcinoma. Anorectal evacuation and colonic motility disorders as well as the presence of colonic diverticula were previously excluded. Full-thickness specimens were harvested at safe distance (>5 cm) from the tumor and immediately transferred from the operating room to the laboratory for tissue processing. The study of human tissue received approval from the Local Ethics Committee of the Faculty of Medicine, Christian-Albrechts-University of Kiel, Germany (B299/07).Segments of sigmoid colon were obtained from patients  who underwent sigmoid resection or left hemicolectomy for symptomatic DD. Patients were operated after two or more attacks of diverticulitis by elective surgery. Full-thickness specimens were harvested from sites adjacent to colonic diverticula. Diverticula-containing areas displaying an altered anatomy of the colonic wall due to transmural mucosal/submucosal outpouchings or signs of inflammation and fibrotic scaring were excluded from tissue sampling. The specimens were immediately transferred from the operating room to the laboratory for tissue processing. The study of human tissue received approval from the Local Ethics Committee of the Faculty of Medicine, Christian-Albrechts-University of Kiel, Germany (B299/07).The muscularis propria was isolated from full-thickness biopsies of the colonic wall, immediately frozen in isopentane and stored at −70°C until use. Prior to RNA isolation 20 orthogonal cryosections (10 µm) were cut on a cryostat and collected in RNA lysis buffer .Full-thickness biopsies (2 cm border length) were immediately frozen in isopentane and stored at −70°C until use. Orthogonal cryosections (10 µm) were placed on membrane-coated  slides. To visualize myenteric ganglia sections were ultra-rapidly (ca. 60 s) stained with toluidine blue and air-dried.2 were collected, immediately dissolved in 350 µl RNA lysis buffer  and stored at −70°C until further use.Laser-microdissection (LMD) was performed by a modified method described previously Preparation of myenteric ganglionic cells was performed according to a method described previously Extraction of total RNA from human tissue was performed using a Nucleospin II kit , RNA from enteric nerve cell cultures was isolated using a Nucleospin XS kit  according to the manufactureŕs guidelines. Prior to reverse transcription, contaminating genomic DNA was digested in a volume of 15 µl using 1.5 U of DNAse I . Reverse transcription was carried out in a total volume of 30 µl containing 200 ng RNA, 375 ng random hexamer primer , 0.5 mM dNTPs , 0.01 M DTT, 1×reaction buffer, and 150 U Superscript II Reverse Transcriptase . The annealing, elongation, and denaturation steps were carried out at 25°C for 10 min, at 42°C for 50 min, and at 70°C for 15 min, respectively.Quantitative PCR (qPCR) reactions were run on an ABI Prism 7700 Sequence Detection System . Amplification reactions were carried out in a 20 µl volume containing 1×qPCR Master Mix Plus , 900 nM primers, 225 nM hybridization probe, and 2 µl cDNA. Samples were run in duplicate and amplified over 50 cycles. Each cycle consisted of a denaturation phase of 15 s at 95°C and a hybridization/elongation phase of 1 min at 60°C. mRNA expression profiles were measured for GDNF, GFRα1, RET, synaptophysin, and the housekeeping gene HPRT in human samples, and for GFRα1, RET, synaptophysin and HPRT in rat enteric cell cultures. Forward and reverse primers and probes are listed as supporting document 2O2. Following blocking of unspecific signals with normal goat serum (1∶10) for 30 min, primary antibodies diluted in antibody diluent  were applied for 1 h: mouse anti-HuC/D  or mouse anti-tubulin ßIII, . HuC/D is a pan-neuronal marker staining specifically neuronal somata thereby allowing reliable identification and counting of nerve cells. Tubulin ßIII is a microtubular protein allowing reliable identification of neuronal processes and evaluation of the nervous network. After incubation with the secondary antibody, biotinylated goat anti-mouse IgG  for 30 min and ABC conjugated with horseradish peroxidase were applied for 30 min. DAB  was used as chromogen. Analysis was carried out with a Zeiss Axiophot microscope .Cells were fixed for 30 min with 4% paraformaldehyde, permeabilized for 10 min with methanol and treated for 10 min with 3% HAfter fixation and permeabilization as described above, samples were incubated with a rabbit anti-synaptophysin antibody  and a mouse anti-PGP 9.5 antibody  for 1 h. After 30 min incubation with the secondary antibodies, goat anti-rabbit-AlexaFlour488  and goat-anti-mouse-AlexaFluor546 , cells were counterstained with DAPI  to visualize cell nuclei. Analysis was carried out with a fluorescent microscope  coupled to a digital camera . Data acquisition was performed with Axiovision software .Morphometric analysis was carried out by using a light microscope  coupled to a digital camera . The software program Axiovision  was used for analysis. The data were transferred into Excel software (version 9.0) and further processed for statistical analysis and data plotting .The number of HuC/D-immunoreactive nerve cells was analyzed per optical field (objective with 20× magnification). Neurons were recorded and counted in three randomly assigned optical fields and the mean value was calculated. Experiments were carried out in quadruplicate (50 ng/ml GDNF vs. control).The area occupied by the tubulin ßIII-immunoreactive neuronal network was measured and related to the overall-area of the optical field (objective with 20× magnification). Five randomly chosen optical fields were analyzed using an area-analysis-tool  to allow reliable depiction of the entire nervous network. The mean value was calculated and expressed as relative area related to the optical field (percentage). Experiments were carried out in triplicate (50 ng/ml GDNF vs. control).Comparison of mRNA expression levels of the GDNF system between the control group and patients with DD as well as morphometric analysis of enteric nerve cell cultures comparing GDNF-treated cultures vs. controls were carried out by using non-parametric Mann-Whitney U-tests. The mRNA expression of the GDNF system in laser-microdissected myenteric ganglia and muscle layers and the effects of the GDNF treatment on rat enteric nerve cell cultures regarding gene expression studies were analyzed by one-way-ANOVA followed by Newman-Keuls post hoc test . Differences were considered significant if p<0.05.To determine the regulation of the GDNF system in patients with DD compared to controls mRNA expression levels of GDNF and its corresponding receptors GFRα1 und RET were monitored by qPCR. Analysis of the muscularis propria revealed that the GDNF mRNA expression was strikingly down-regulated in patients with DD (p<0.001), as mRNA levels dropped to 36% of control values . The GDNTo identify the tissue source of components of the GDNF system the intestinal compartments of interest were selectively isolated by LMD. Site-specific gene expression profiles of GDNF and its receptors GFRα1 and RET were monitored in myenteric ganglia as well as in the circular and longitudinal smooth muscle layers. GDNF mRNA levels were signifcantly higher in the circular (p<0.001) and longitudinal (p<0.05) muscle compared to the myenteric plexus. . HighestAs we aimed to investigate the putative effect of a reduced GDNF mRNA expression on enteric neurons, we implemented a cell culture model of rat postnatal dissociated myenteric plexus exposed to increasing concentrations of GDNF. First, the effect of GDNF on mRNA expression of its corresponding receptors GFRα1 and RET was assessed. After 1 week of GDNF treatment, both receptors showed up-regulation of their respective mRNA expression . A dose The neurotrophic capacity of GDNF was studied by assessing the neuronal number and differentation of the neuronal network in enteric nerve cell cultures exposed to GDNF. The rationale of these experiments was to conclude whether a lack of GDNF may contribute to the relative loss of enteric nerve cells observed in patients with DD. The numerical increase of cultured myenteric neurons was monitored by anti-HuC/D immunocytochemistry. After a culture time of 1 week, few dispersed HuC/D immunoreactive nerve cells were observed in control cultures . At the The positive effect of GDNF on neuronal differentiation of enteric nerve cell cultures was further addressed by assessing the influence of this neurotrophic factor on neuronal plasticity. Therefore, the effects of GDNF on the synaptic vesicle marker synaptophysin were studied in enteric nerve cell cultures. mRNA expression of synaptophysin was dose-dependently increased following GDNF treatment after one week of culture time . At a doTo our knowledge, this is the first study addressing neurotrophic factor deficits in patients with DD and investigating in vitro the underlying cellular and molecular mechanisms. The study reveals four important findings: (1) mRNA expression of GDNF and its receptors GFRα1 and RET is down-regulated in the muscularis propria of patients with DD; (2) the main source of GDNF is the intestinal smooth muscle, while RET is mainly expressed in enteric ganglia in which GFRα1 is also found; (3) GDNF up-regulates its corresponding receptors in enteric nerve cell cultures; (4) GDNF treatment promotes neuronal differentiation and plasticity of enteric nerve cell cultures.DD is a gastrointestinal disorder with a high prevalence but a still poorly understood pathogenesis. Traditional pathogenetical concepts have advocated over decades the impact of a low-fiber diet as mayor risk factor for DD. Whereas Painter and Burkitt Since GDNF- and GDNF receptor-knock-out mice exhibit total intestinal aganglionosis demonstrating the crucial role of this growth factor for the development and maintenance of the ENS Although all specimens studied from patients with DD were obtained from histologically un-inflamed areas showing no evidence for abscess formation, transmural inflammatory infiltrates nor fibrotic scarring, it cannot be excluded that recurrent inflammation might have contributed to alterations of the GDNF system in DD, as it has been previously shown that GDNF levels are modified in inflammatory bowel diseases  To investigate the cellular sources of the components of the GDNF system in the human colon, we applied LMD to those tissue compartments of interest followed by qPCR. The intestinal muscle layers could be identified as the main source of GDNF in the adult human colon, whereas RET was mainly expressed by enteric ganglia. GFRα1 mRNA expression exhibited highest levels in the longitudinal muscle but was also expressed in enteric ganglia. Thus, the so called “neurotrophic factor concept” originally postulated for the CNS might also apply for the ENS: The neurotrophic factor concept in its basic form envisages that innervated tissues produce a signal (neurotrophic factor) specifically directed towards the innervating neurons which express receptors for the target-derived neurotrophic factor for the selective limitation of neuronal death occurring during development To elucidate the role of GDNF on maintenance of enteric neurons, we addressed the effect of GDNF treatment on cultured myenteric neurons derived from postnatal rats. GDNF treatment increased mRNA levels of its corresponding receptors, i.e. at a dose of 50 ng/ml GDNF up-regulated mRNA levels of GFRα1 and RET in myenteric neurons. This observation is in accordance to the site-specific gene analysis of the GDNF system in the human colon by means of LMD showing that enteric neurons represent the main expression site of GDNF receptors. Thus, down-regulation of GDNF in the muscularis propria of patients with DD might result in lower receptor expression in the ENS thereby compromising the effects of the GDNF system. Similar findings have been obtained in animal studies and cell culture models of rodent gut components. In a culture model of rat intestinal muscle cells Rodrigues et al. GDNF treatment of enteric nerve cell cultures enhanced both neuronal numbers and the formation of an extensive nervous network after one week of culture demonstrating that GDNF effectively promotes the survival and differentiation of postnatal myenteric neurons. Consistent with these results, Rodrigues et al. could demonstrate after 72 h of incubation enhanced neuronal survival, axonal outgrowth and nerve fiber fasciculation induced by GDNF using co-cultures of neonatal rat myenteric neurons, smooth muscle and glial cells In addition to neuronal proliferation and differentiation GDNF also promoted the formation of synaptic vesicles in enteric nerve cell cultures as demonstrated by the upregulation of the synaptic vesicle marker synaptophysin. Outgrowing neuronal processes displayed multiple granular accumulations of synaptophysin resembling sites of synaptic communications also known as autonomic nerve fiber varicosities. These observations are consistent with a report of Zeng et al. Taken together, our results demonstrate for the first time that DD is associated with a decrease of the neurotrophic factor GDNF. The in vitro data illustrate that GDNF promotes differentiation and synaptic plasticity of postnatal enteric neurons arguing for a role in the maintenance of the ENS during postnatal life. The compromised GDNF system in patients with DD may help to explain the shortfall of enteric neurons in this condition and further strengthens the hypothesis that the pathogenesis of DD may be linked – amongst other etiological factors – to an underlying enteric neuropathy.Table S1Primer sequences.(DOCX)Click here for additional data file.


Drosophila melanogaster to defend against systemic bacterial infection. The goal of the present study was to identify genes likely to inform the mechanism of this post-mating immunosuppression. We used microarrays to contrast genome-wide transcript levels in virgin vs. mated females before and after infection. Because the immunosuppressive effect of mating is contingent on the presence of a germline in females, we repeated the entire experiment by using female mutants that do not form a germline. We found that multiple genes involved in egg production show reduced expression in response to infection, and that this reduction is stronger in virgins than it is in mated females. In germline-less females, expression of egg-production genes was predictably low and not differentially affected by infection. We also identified several immune responsive genes that are differentially induced after infection in virgins vs. mated females. Immune genes affected by mating status and egg production genes altered by infection are candidates to inform the mechanism of the trade-off between mating and immune defense.Mating and consequent reproduction significantly reduce the ability of female  Although our knowledge of the invertebrate immune system is extensive and continues to expand . Females were randomly allocated to “virgin” or “mated” treatment groups and allowed to recover overnight. The following morning, within 2 hr of incubator “dawn,” a single virgin male was aspirated into each vial containing a female assigned to the “mated” treatment and individual copulations were observed. Males were removed from the presence of females shortly after copulation cessation to prevent additional courting or copulation attempts. Egg-producing females mated for an average of 23.8 min and eggless females mated for an average of 22.6 min. Females from copulations lasting fewer than 15 min were discarded and not used for infections to maximize the likelihood that all females used in the experiment received a full complement of sperm and seminal fluid from their mates.Eggless and egg-producing females were collected as virgins and aged for 3 d post-eclosion. The day before matings were to be set up, eggless and egg-producing females were lightly anesthetized with CO2 and infected; age- and rearing-matched virgin controls were infected in parallel. We have previously shown that females are already immunocompromised by 2.5 hr after mating  broth at 37°, then diluted in sterile LB to an optical density of A600 = 1.0 ± 0.05. In parallel, females to remain as uninjured controls were anesthetized on CO2 to control for effects of anesthesia. Infected mated and virgin females as well as uninjured virgin and mated controls were then put on fresh media in groups of approximately 10. We used uninjured controls in our experiment to detect the combined effects of both septic wounding and the presence of bacteria. A small number of flies were individually homogenized immediately after each round of infection, and an aliquot of undiluted homogenate was quantitatively plated on LB agar using a spiral plater . We found that our infection technique delivered an average dose of 1.4 × 103 (standard error = 4.7 × 102) bacteria to each female. We have found that the bacterial load of mated females begins to deviate from that of virgins at approximately 12 hr after infection  , and placed at -80°. The entire experimental set up was replicated on three independent days, resulting in three biological replicates for each of the eight experimental groups.At 2.5 hr (±15 min) after mating, mated eggless and egg-producing females were lightly anesthetized with COfree (Ambion), and the quality of the RNA from each sample was assessed using a BioAnalyzer 2100 (Agilent). The BioAnalyzer outputs for our samples showed strong, distinct peaks corresponding to 18S and 28S rRNA with little to no baseline signal between these peaks. This indicated that our samples were high quality with little degradation. Samples were labeled using Agilent’s Low Input Quick Amp Labeling kit and were hybridized to 4x44K Drosophila (V2) Gene Expression Microarrays (Agilent) according to the manufacturer’s instructions. RNA labeling, microarray hybridizations and feature extraction were performed by the Cornell University Life Sciences Core Laboratory Center.We extracted RNA from our samples using TRIZOL reagent according to the manufacturer’s protocol. Residual genomic DNA contamination was removed using TURBO DNA-Table S1 and Table S2). We also assayed for genes that showed a significant interaction between mating status and infection status (Table S3 and Table S4). These contrasts were initially performed within treatments of egg-producing females and then were separately repeated for arrays from eggless females. Many genes on the 4x44K Drosophila (V2) Gene Expression Microarrays (Agilent) were represented by multiple probesets with distinct probe sequences. We performed our analyses at the probeset level and report all difference values for all probesets in Table S1 and Table S2. For simplicity, we present results at the level of gene rather than probeset in the text. In figures, when more than one probeset showed significantly altered expression for a particular gene, we report the probeset with the largest fold change.The microarray data were analyzed using the Bioconductor package limma . Data wewww.flybase.org). Hypergeometric tests for enrichment of genes with shared Biological Process Gene Ontology (GO) terms were performed using the online tool GOrilla  . Of these 124 genes whose expression changed in response to infection, 103 were up-regulated. Most of these genes are known immunity genes (Table S1).By examining gene expression changes that occur in response to infection in both virgin , as was that of many peptidoglycan recognition proteins . We also found infection-induced increases in transcript abundance in multiple genes in the Turandot gene family . At least one gene in the Tot family (TotA) is regulated by the JAK/STAT signaling pathway . We note that some of the genes we detected as being up-regulated have negative regulatory roles in the humoral immune response , illustrating mechanisms by which the host modulates immune system activity . Notably, this set was enriched for genes involved in egg formation, specifically vitelline membrane and chorion formation . Given that female D. melanogaster suffer a germline-dependent reduction in immune defense after mating . As was the case for females with intact germlines, the genes whose expression increased in response to infection included many known immunity genes, such as those encoding antimicrobial peptides , peptidoglycan recognition proteins , and other known infection responsive genes . Thus, the general response to infection is not germline dependent. Notably missing from this list, however, are the Tot genes. More detailed inspection revealed that expression of TotA, TotC, and TotM increases significantly after infection in virgin but not mated eggless females , Vm34Ca (down a maximum of 7.89-fold), Vml (down 7.06-fold), and Vm26Ab (down 4.47-fold) (Table S1). These data suggest that nonreproductive  females preferentially suppress expression of genes in egg formation when faced with systemic bacterial infection. These genes are not significantly affected by infection in mated females , likely because mated females continue to produce mature eggs even while combating infection ; therefore, we think it is unlikely that this higher immune gene transcript abundance reflects increased positive stimulation of the immune system through higher pathogen load.We performed a reciprocal analysis to identify changes in gene expression in response to infection that were significant only in mated females but not in virgins . This is not unexpected because the germline-less females do not produce eggs, but it does provide a clear example of a germline-dependent difference in the transcriptional response to infection of virgin and mated females. This finding is consistent with our model that post-mating suppression of immune defense is related to energetic expenditure on the production of fertile eggs . There were 335 genes that met this criterion. We found that for 68 of these genes, the virgin response to infection was significantly different from the mated response to infection at a nominal (uncorrected) p-value of 0.05 (Table S3). GO analysis of the 335 genes showed significant enrichment for four Biological Process terms: “defense response to gram-positive bacterium,” “defense response,” “ATP biosynthetic process,” and “vitelline membrane formation involved in chorion-containing eggshell formation” . All of the Attacin genes and TotM are also more strongly induced in virgin females relative to mated females, although not significantly so (Table S3). PGRP-SD and IM4 show significantly greater expression in mated females than in virgins , whereas sphinx2, r2d2, and Gr28b are increased in response to infection in mated females but decreased in virgins . These data reveal that virgins respond differently to infection than do mated females, although the differences are complex. The Attacin genes and TepII, which are induced to a greater degree in virgins, are directly involved in bacterial elimination. PGRP-SD, which is induced to a greater degree in mated females, is best characterized as encoding a protein that recognizes Gram-positive bacterial infection  and ion transport  (Table S3), suggesting that basic metabolic functions may be differentially affected by infection depending on mating status.The GO category “ATP biosynthetic process” contained genes encoding proteins with multiple roles in basic metabolic processes, such as ATP synthesis . Of these, Vml, Vm26Ab, and Vm34Ca all exhibited nominally significant expression changes . For all six vitelline membrane genes (the five above plus Vm26Ac), transcript abundance was greater in mated females compared to virgins, which is expected given that mated females actively produce higher numbers of eggs  being greater than 1.0. We found only 32 genes that met this basic criterion (Table S4). Of these 32, only six genes showed a nominally significant difference in expression between virgin and mated eggless females . Notably, three genes from the list of 32 were also significant in this same comparison in egg-producing females . Takeout (to) shows sequence similarity with Manduca sexta juvenile hormone binding protein and has been hypothesized to act as a carrier of juvenile hormone  may in some way be contingent on the presence of a germline.We also identified genes that exhibited different magnitudes of expression change in response to infection in virgins  defense , but eggTable S1) and/or infected females (comparison D in Table S1). In several microarray studies, authors have investigated the differences in transcript abundance due to mating in females outside the context of infection . There were 489 genes whose expression was altered by mating in both uninfected (comparison C in Table S1). Of these, 286 genes were significantly up-regulated in both uninfected and infected females and 203 genes were significantly down-regulated in both treatments (Table S1). A large number of genes were specifically altered in either uninfected or infected females. There were 101 genes significantly up-regulated and 101 genes significantly down-regulated after mating in uninfected females, but mating did not significantly alter the expression of these 202 genes in infected females (Table S1). Reciprocally, there were 225 genes that were up-regulated and 288 genes down-regulated in response to mating in infected females only (Table S1).In our study, females were assayed at 12.5 hr after mating cessation for expression of genes significantly altered by mating in one or both infection states (comparison C and/or D in Table S1), Vm26Aa, Vm26Ab, and Vm34Ca). They also reported increased expression in yolk protein genes (Yp1 and Yp2) and an odorant binding protein (Obp99a), which we found to be significantly affected by mating in our study as well (Table S1).We tested for enrichment of GO terms among the genes whose expression was significantly altered by mating in the uninfected and/or infected females . Among tThe post-mating increases in vitelline membrane gene expression we found are expected given that vitelline membrane genes are highly expressed during the vitellogenic stages of oogenesis . Jonah genes have previously been reported to be expressed only in the midgut . The induction of Jonah genes by mating and their repression by infection may indicate one potential antagonistic pleiotropy between immunity and reproduction, perhaps mediated by differences in feeding behavior or nutritional uptake.Genes encoding proteolysis regulators could be involved in many possible post-mating functions, including the processing of seminal fluid proteins e.g., . Many ofe midgut . Jonah gGenes with reduced transcript abundance after mating in both uninfected and infected females were enriched for many GO terms involved in cellular replication, including chromosome segregation, regulation of cell cycle, DNA replication, and spindle organization . These aP = 6.36 × 10−5, corrected P = 0.102, data not shown). Because our multiple testing correction was rather stringent, we thought that this result warranted further investigation. This GO term included two immune-induced molecules (IM4 and IM10) and five genes with lysozyme activity  whose expression was significantly greater after mating in infected females but not in uninfected females (Table S1). The lysozyme genes up-regulated in response to mating comprise the LysD-like gene family, which is thought to be expressed only in the gut of adult flies  was differentially regulated after infection in virgins compared to mated females. We also found that a number of gut-specific genes were affected by mating status but in an infection-status specific manner . Although the full implications of these results remain to be explored, the initial observations suggest that the differences we see in immune defense between virgins and mated females may stem partially from differential changes in feeding behavior after mating and infection.In this work, we identified changes in gene expression that occur in response to mating and infection with the goal of gaining a better understanding of the molecular mechanisms that contribute to post-mating immunosuppression in female


Mitochondrial DNA (mtDNA) mutations lead to decrements in mitochondrial function and accelerated rates of these mutations has been linked to skeletal muscle loss (sarcopenia). The purpose of this study was to investigate the effect of mtDNA mutations on mitochondrial quality control processes in skeletal muscle from animals  expressing a proofreading-deficient version of mtDNA polymerase gamma (PolG). This progeroid aging model exhibits elevated mtDNA mutation rates, mitochondrial dysfunction, and a premature aging phenotype that includes sarcopenia. We found increased expression of the mitochondrial biogenesis regulator peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) and its target proteins, nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A (Tfam) in PolG animals compared to wild-type (WT) (P<0.05). Muscle from older PolG animals displayed higher mitochondrial fission protein 1 (Fis1) concurrent with greater induction of autophagy, as indicated by changes in Atg5 and p62 protein content (P<0.05). Additionally, levels of the Tom22 import protein were higher in PolG animals when compared to WT (P<0.05). In contrast, muscle from normally-aged animals exhibited a distinctly different expression profile compared to PolG animals. Older WT animals appeared to have higher fusion  and lower autophagy (Beclin-1 and p62) compared to young WT suggesting that autophagy is impaired in aging muscle. In conclusion, muscle from mtDNA mutator mice display higher mitochondrial fission and autophagy levels that likely contribute to the sarcopenic phenotype observed in premature aging and this differs from the response observed in normally-aged muscle. Mitochondria play a fundamental role in several key cellular processes which include but are not limited to energy production, calcium signaling, reactive oxygen species (ROS) generation, and cell death (apoptosis) Increased levels of mtDNA point mutations and deletions are documented in a wide range of aged tissues in both humans and animals, and have been linked to a number of pathological conditions Mitochondria have many defense pathways to combat excessive damage and maintain quality control, and this is particularly important for post-mitotic tissue such as skeletal muscle. Mitochondria are not static organelles but instead quite dynamic, continuously being reorganized and/or recycled via fusion and fission processes. Biogenesis of new organelles is regulated by the peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), as well as a number of fusion and fission proteins. The latter proteins not only dictate the morphology of this complex interconnected network but are also important for energy metabolism, redox signaling, and cell death. Moreover, mitochondrial fusion facilitates the mixing of mtDNA from one organelle to another and prevents a high concentration of mutant mtDNA from accumulating The purpose of this study was to investigate whether mitochondrial quality control processes are altered in an animal model of progeroid aging that exhibits high mtDNA mutations, mitochondrial dysfunction, and most importantly a sarcopenic phenotype that is commonly observed in normally-aging mice. These animals were not used as a model of chronological aging but as a tool to study and understand the specific effects of mtDNA mutations/mitochondrial dysfunction on muscle wasting. MtDNA mutator mice (PolG) begin to display muscle wasting as early as 8 months of age 2 and stored for further analysis.All animal research was conducted in accordance with the regulatory policies of the Institutional Animal Care and Use Committees of the University of Florida (CL approved protocol #D420) and all efforts were made to minimize suffering to the animals. Transgenic mice expressing a proofreading-deficient version of the mitochondrial DNA polymerase gamma (D257A) gene referred to as PolG were generated as previously described 2PO4+2 mM EDTA, pH 7.2) and sonicated (3×5 s) on ice. Following a brief spin, the supernatant was removed and enzyme activity determined by the maximal oxidation rate of completely reduced cytochrome c, evaluated as a change in absorbance at 550 nm using a multi-detection microplate reader .Cytochrome c oxidase (COX) activity was measured as previously described Total DNA was prepared from 2 mg of muscle using NucleoSpin Tissue Kit (Macherey Nagel) and stored at −20°C. Real Time PCR (qRT-PCR) was used to measure mtDNA content as previously described Frozen quadriceps muscles were pulverized and protein extracts prepared as previously described P<0.05. Data are presented as mean ± standard error (SE).All statistical analyses were carried out by two-way analysis of variance (ANOVA) to compare differences among multiple groups, followed by Bonferonni post-hoc analysis to specifically test individual differences between groups. Differences were considered statistically significant if We measured the relative levels of mtDNA, as well as cytochrome c oxidase (COX) activity as indicators of mitochondrial content and function Mitochondrial biogenesis is required for the synthesis of new mitochondria and is also important for maintaining mitochondrial turnover. We found higher levels (P<0.05) of peroxisome proliferator-activated receptor gamma coactivator-1 alpha PGC-1α; , as wellTo determine whether mitochondrial dynamics was altered in muscle from mtDNA mutator mice, we measured key fusion proteins mitofusin 1 and 2 (Mfn1 and Mfn2) and optic atrophy 1 (Opa1), and fission proteins, fission 1 protein (Fis1) and dynamin-related protein (Drp1). Both Mfn1 and Mfn2 levels were significantly higher with age P<0.05; , while FThe regulation of mitochondrial turnover by autophagy occurs through lysosomal-mediated clearance of damaged organelles Mitochondrial protein import machinery plays a crucial role in mitochondrial biogenesis and turnover by regulating the import and translocation of hundreds of nuclear-encoded mitochondrial proteins into subcellular organelle compartments. Levels of the cytosolic and mitochondrial heat shock proteins  were reduced (P<0.05) in aging muscle  while noMutations and deletions in mtDNA have been reported in aging skeletal muscle The ability of skeletal muscle to adapt to cellular perturbations is highly dependent on mitochondrial regulation/biogenesis. Mitochondrial biogenesis involves the synthesis of new organelles from existing mitochondria and this process is predominantly regulated by PGC-1α It is now well established that mitochondria are not static but rather dynamic organelles capable of altering their shape and size in response to ever changing cellular demands. Mitochondrial fusion and fission events not only dictate the morphology of the organelle but are also responsible for maintaining mitochondrial turnover through their contributions to the synthesis (biogenesis) and degradation of organelles Excessive amounts of fission and autophagy have been proposed to precede muscle atrophy in experimental models of muscle disuse (i.e. denervation and fasting) et al. recently reported greater levels of autophagy-related machinery Atg7 and Parkin in muscle from 35-month old Fischer 344×Brown Norway rats In normal-aging muscle, autophagy proteins were downregulated and this is consistent with previous observations by our group and others showing that autophagy is impaired in aging muscle Finally, we investigated the effect of mtDNA mutations on the mitochondrial protein import machinery (PIM). Since the majority of mitochondrial proteins required for biogenesis are encoded in the nucleus, a specialized group of machinery composed of chaperones and translocases of the outer and inner membrane  exists to facilitate their import into the organelle In summary, data from this study provides further evidence for the importance of mitochondrial control processes in muscle atrophy. Moreover, although higher mtDNA mutations are central to both the etiology of muscle loss in mtDNA mutator mice and normal-aging mice, the protein expression pattern in these conditions appears to be quite distinct . The div


SMAD7 gene. Our objective was to confirm the association between SMAD7 SNPs and colorectal cancer risk in the multi-center Colon Cancer Family Registry.Recent genome-wide studies identified a risk locus for colorectal cancer at 18q21, which maps to the SMAD7 gene were genotyped among 1,592 population-based and 253 clinic-based families. The SNP-colorectal cancer associations were assessed in multivariable conditional logistic regression.23 tagging SNPs in the Among the population-based families, both SNPs rs12953717 , and rs11874392  were associated with risk of colorectal cancer. These associations were similar among the population- and the clinic-based families, though they were significant only among the former. Marginally significant differences in the SNP-colorectal cancer associations were observed by use of nonsteroidal anti-inflammatory drugs, cigarette smoking, body mass index, and history of polyps.SMAD7 SNPs were associated with colorectal cancer risk in the Colon Cancer Family Registry. There was evidence suggesting that the association between rs12953717 and colorectal cancer risk may be modified by factors such as smoking and use of nonsteroidal anti-inflammatory drugs. SMAD7, a functional candidate gene for CRC. Smad7 plays inhibitory roles in the transforming growth factor beta (TGF-β) signaling pathway SMAD7 associated with CRC and the SNP rs4939827 was later replicated as the top-ranking SNP at 18q21 by Tenesa et al. SMAD7 expression was found to be lower in colorectal cancers than in adenomas regardless of 18q copy number status SMAD7 expression in lymphoblastoid cell lines SMAD7 is likely to be the biological mechanism underlying the association between variations in 18q21 and CRC risk.It is estimated that inherited susceptibility contributes to ∼35% of all colorectal cancer (CRC) cases SMAD7 SNPs and CRC risk in a large family-based case-control study based on the multi-center Colon Cancer Family Registry (Colon CFR), and to examine gene X environmental interactions to identify risk/protective factors that may affect the association between SMAD7 SNPs and CRC risk. Our case-unaffected sibling study design has been shown to be more powerful for controlling potential confounding from population stratification and detecting gene-environment interactions Given the SMAD7's role in the TGF-β signaling pathway Data for this study were obtained through the Colon CFR, a National Cancer Institute (NCI)–funded registry of CRC cases, unaffected family members, and population-based controls. The registry is described in detail in Newcomb et al. n = 429) from one of the Colon CFR sites (Fred Hutchinson Cancer Research Center). A total of 1,923 cases  and their 3,115 unaffected sibling controls  were included in the analyses.In the Colon CFR, population-based controls were only obtained from one of the Colon CFR sites (Fred Hutchinson Cancer Research Center), and the total sample size (N = 429) is much smaller than that of the sibling controls . To make the most use of the available genetic data, in this investigation we used a case/unaffected sibling control designAll subjects signed an informed consent before providing data to the Colon CFR. Ethics approval for the study was obtained from the Institutional Review Boards at each CCFR site: University of Southern California Health Sciences Institutional Review Board, Mount Sinai Hospital Ethics Research Board, University of Hawaii Institutional Review Board, The University of Melbourne Central Human Research Ethics Committee, Fred Hutchinson Cancer Research Center Institutional Review Board, and Mayo Clinic Institutional Review Board.SMAD7 was genotyped as part of an ongoing study of genes relevant to lipid peroxidation and apoptosis (5R01CA114472-02). Tagging single nucleotide polymorphisms (SNPs) were selected using the program Snagger20 2 of ≥0.80 in the region covering each gene of interest as well as 20 kb upstream and 10 kb downstream of the gene. The linkage disequilibrium blocks were determined using data from the International HapMap Project White CEPH (Utah residents with ancestry from northern and western Europe) population . Finally, three GWAS-identified SNPs, rs4939827, rs12953717, rs4464148, were also included. SNPs were genotyped on the Illumina GoldenGate platform  SMAD7. A total of 133 blinded duplicate pairs were included for genotyping. Concordance for the duplicate samples was >99%.All available tumors from the Colon CFR's Jeremy Jass Memorial Pathology Bank were assayed for instability at the following 10 microsatellites: BAT25, BAT26, BAT40, BAT34C4, D5S346, D17S250, D18S55, D10S197, ACTC, and MYCL as described previously Tumor location was obtained from the pathology report and was available for 1,778 (92.1%) of cases . Right colon was defined as the cecum through the splenic flexure; left colon included the descending colon through the sigmoid colon; rectal tumors included the rectosigmoid junction and the rectum.All statistical analyses were conducted using the R programming language and SAS v9.1 .R2) and D-prime (D′) between markers. We also evaluated Hardy-Weinberg equilibrium for each SNP. No deviations were observed for all SNPs except rs1873190. Among the unrelated population-based controls, statistically significant reduced numbers of heterozygous genotypes of rs1873190  were observed. The genotyping data of this SNP showed clear genotype separation with a call rate of 98% and concordance rates of 100% among replicates.MAF was estimated from the genotype data from unrelated population-based controls. Pairwise linkage disequilibrium between SNPs was estimated using the square of the correlation coefficient  and sex. We assessed the SNP-CRC associations assuming a log-additive model. In all analyses, the lower frequency allele was coded as the “risk” allele and individuals were assigned a 0, 1, or 2 representing the number of risk alleles they possessed for that SNP. Test for haplotypic association was performed using SAS/Genetics and haplotype frequencies were estimated by the expectation–maximization algorithm.Among the population-based families, we also examined the possibility that the SNP-CRC association was modified by other factors including gender, age, use of nonsteroidal anti-inflammatory drugs (NSAIDs), cigarette smoking, alcohol drinking, body mass index (BMI), physical activity  hours of physical activity throughout adulthood), history of diabetes, polyps, and ulcerative colitis, and family history of CRC in a first-degree relative as reported by the proband. All analyses within the exposure strata were specified in advance based on indications for potential effect modification in the literature. To test this hypothesis, dummy variables representing stratum-specific exposure were created for estimating stratum-specific results in one single conditional logistic regression model. P-values for interactions were estimated from likelihood ratio tests. We also evaluated differences in the association by tumor location  and MSI status  by stratifying the matched sets on the tumor characteristics of the case. We assigned the sets the MSI or tumor-site category of the case and included interaction terms in the conditional logistic regression models to estimate these stratum-specific odds ratios. Finally, we considered whether inclusion of cases recruited >2 years after diagnosis resulted in biased estimates and results were fundamentally unchanged after exclusion of these cases (data not shown).SMAD7 gene, only two SNPs, rs11874392 and rs12953717, remained significantly associated with CRC risk among the population-based families. Each minor allele (T) of rs12953717 SNP was associated with a significantly increased risk of CRC , whereas each minor allele (T) of rs11874392 was associated with a statistically significantly reduced risk of CRC . These associations were similar among the population-based families and the clinic-based families, though they were only significant among the population-based families with its larger sample size. The two SNPs were highly correlated with each other . In a logistic regression analysis, the inclusion of rs11874392 did not significantly improve the fit of the model over that with rs12953717 alone (P = 0.88). Analysis of rs12953717 and rs11874392 revealed that CT and TA were the most common haplotypes (−5). Haplotype CA was not associated with CRC risk (P = 0.53). No significant associations were observed for the rs4464148 SNP or other SNPs evaluated in SMAD7 after adjustment for multiple testing.Results of the single SNP analysis are shown in plotypes  and onlyAmong the population-based families, we assessed the association between rs12953717 and CRC risk after stratification by related risk/protective factors . We obseWe also examined the possible heterogeneity of the SNP effect by tumor location and MSI status . The strTo address potential survival bias due to the inclusion of cases interviewed up to 5 years after diagnosis, we repeated the analyses of main effects including only probands interviewed within 2 years of diagnosis and their unaffected siblings and results were fundamentally unchanged (data not shown).SMAD7 polymorphisms, rs12953717 and rs11874392, and CRC in this large family-based case-control study. We observed suggestions that the association of rs12953717 and CRC risk may be modified by use of NSAIDs, cigarette smoking, BMI and history of polyps.Consistent with the two GWAS SMAD7 expression and subsequent TGF-β signaling. Compared to the C allele, the risk allele G forms weaker protein–DNA complexes with nuclear extracts and was associated with a reduced expression of SMAD7 in the colorectum. However, it remains unclear how such alterations may promote carcinogenesis. It has been suggested that SMAD7 may induce tumorigenicity by blocking TGF-β-induced growth inhibition and apoptosis SMAD7 is very low in epithelial tissues, but is up-regulated in several cancers. Over-expression of SMAD7 has been shown to inhibit TGF-β-mediated induction of endogenous heme oxygenase-1 (HO-1) gene expression SMAD7 blocks the TGF-β-mediated activation of NFκBSMAD7 were found to have a favorable clinical outcome SMAD7 gene copy number The underlying causal variant for the association between 18q21 variation and CRC risk remains unknown. Pittman et al. SMAD7 is involved in intestinal inflammation through its regulation of TGF-β signaling SMAD7 SNPs and CRC risk may be modified by factors affecting inflammation. In human inflammatory bowel tissues, TGF-β1 signaling is disrupted by the up-regulation of SMAD7, leading to an enhanced production of inflammatory cytokines. Inhibition of SMAD7 with a specific antisense oligonucleotide can restore the immunosuppressive TGF-β1 signaling and suppress the inflammatory cytokine production SMAD7 genotype (rs4939827 and rs4464148) among patients who were regularly using NSAIDs before diagnosis, but not among never-users and former users of NSAIDs SMAD7 is a feature of colitis and blocking SMAD7 restores TGF- β1 signaling in colitis SMAD7 SNP association among non-/former smokers would lend some support to the inflammation hypothesis. We consistently observed a higher CRC OR for rs12953717 among individuals with ulcerative colitis than those without, although this difference did not reach statistical significance, possibly due to the small number of individuals with ulcerative colitis. On the other hand, smoking increases the risk of the other form of IBD, Crohn's disease, but we were unable to examine the SNP-CRC association stratified by history of Crohn's disease due to the small number of individuals with this disease (N = 32). In addition, the observation of a BMI interaction could also suggest the involvement of inflammation pathways, since obesity is associated with a state of chronic inflammation Given that SMAD7 may also function through insulin-related pathways. SMAD7 protein expression in the renal cortex is decreased in diabetes SMAD7 in pancreatic β cells disrupts TGF-β signaling and induces reversible diabetes mellitus SMAD7 SNP rs3764482 (IVS2 –21C>T) has been associated with a reduced risk of type 2 diabetes in mice SMAD7 SNPs and CRC risk, we further defined these associations according to other known risk/protective factors for CRC and tumor characteristics. We found a stronger SNP association among individuals with a prior history of polyps. Further exploration by the type, number and size of polyps was not performed because detailed information on these characteristics is lacking. The exact biological mechanism for the interaction between polyps and SMAD7 is unknown; however, a recent study in prostate cancer cells found that SMAD7 can interact with adenomatous polyposis coli (APC) protein in linking the TGF-β type I receptors to the microtubule system to promote cell migration SMAD7 in the progression from adenomatous polyps to CRCs. Interpretation of this result needs to be cautious, because the proportion of subjects who had been examined for polyps prior to cancer diagnosis is unknown. There was some indication of differences in the effect of SMAD7 SNPs by tumor location. We observed a significant association of rs12953717 for distal colon tumors, but not for proximal colon tumors and rectal tumors. Such finding is consistent with Curtin et al. SMAD7 SNPs but not for proximal colon and rectal tumors. Similarly, Slattery et al. In addition to confirming the associations between 2 = 1.0 in HapMap CEU data Release 28.) between rs4939827 and rs11874392, there is unlikely to have been any loss of information due to this exclusion. Second, the Bonferroni method to adjust for multiple comparisons may be so conservative that some potentially important polymorphisms, such as the variant identified in a previous GWAS of CRC rs4464148 SMAD7 SNPs and CRC risk in the clinic-based families, possibly due to the limited sample size in the clinic-based dataset. Alternatively, the genetic predisposition in the multiple-case, clinic-based families may be largely due to other yet-to-be-identified genetic variations with stronger effects.Our study has a number of limitations. First, SNP rs4939827 was excluded from our analysis due to genotyping failure; however, given the perfect linkage disequilibrium (RSMAD7 and CRC found in GWAS. Further studies are needed to confirm our results stratified by demographic factors and tumor characteristics and to elucidate the relevant biological mechanisms. With the growing epidemiological evidence linking SMAD7 to CRC susceptibility, studies are needed to investigate potential biological mechanisms by which SMAD7 contributes to the development of CRC.In summary, using data from the Colorectal Cancer Family Registry, we confirmed the association between


Magnetic resonance imaging is a noninvasive method of evaluating embryonic development. Diffusion tensor imaging (DTI), based on the directional diffusivity of water molecules, is an established method of evaluating tissue structure. Yet embryonic motion degrades the in vivo acquisition of long-duration DTI. We used a dual-cooling technique to avoid motion artifact and aimed to investigate whether DTI can be used to monitor chick embryonic skeletal muscle development in ovo, and to investigate the correlation between quantitative DTI parameters fractional anisotropy (FA) and fiber length and quantitative histologic parameters fiber area percentage (FiberArea%) and limb length.From 84 normally developing chick embryos, 5 were randomly chosen each day from incubation days 5 to 18 and scanned using 3.0 Tesla magnetic resonance imaging. A dual-cooling technique is used before and during imaging. Eggs were cracked for making histological specimen after imaging. 3 eggs were serially imaged from days 5 to 18. We show that skeletal muscle fibers can be tracked in hind limb in DTI beginning with incubation day 8. Our data shows a good positive correlation between quantitative DTI and histologic parameters . The result of tracked fibers in DTI during incubation corresponds to the development of chick embryonic skeletal muscle as reported in the literature.Diffusion tensor imaging can provide a noninvasive means of evaluating skeletal muscle development in ovo. Animal models play a central role and are essential to basic medical research. Having an established pedigree of use in developmental biology, transplantation research, and cancer research, the chick embryo provides an in vivo model which is both accessible and economical, as it contains all components necessary for development, excepting heat and oxygen . PluripoDiffusion tensor imaging (DTI) is a method of observing the displacement distribution of water molecules present within an imaging voxel, allowing evaluation of tissue structure at a scale below the spatial resolution of MRI . FractioWe were able to discriminate and draw a region of interest (ROI) on the hind limb or hind limb bud on T2 weighted images from day 5. Beginning with day 8, skeletal muscle fibers of the hind limb could be tracked with DTI and the fiber length derived. As shown in Table The result of correlation analysis between FA and FiberArea%, and between length of tracked fibers (Fiber_length) and length of hind limb (Limb_length) is shown in Figure A single typical example of serially DTI-derived skeletal muscle fiber tracking is shown in Figure Our data show that DTI reflects skeletal muscle fiber formation and growth in hind limb during the incubation period. In contrast to invasive methods of analysis, repeated observations on a single embryo throughout incubation can be implemented using DTI and subsequent phenotypic characteristics would be evident at hatching.Chick embryo skeletal muscle development has been investigated in detail by developmental biologists -15. MultThe tracked fiber length of the hind limb in DTI was not entirely consistent with the length of hind limb. For this study, we used a simple measurement method, the length of tracked fibers, which is an average value and it is affected by the tractography stopping threshold parameters . Therefore, while the DTI measured tracking lengths are not necessarily representative of a single myofiber, it can be considered to be a useful relative surrogate given the correlation obtained between Fiber_length and Limb_length.This study has several limitations. The spatial resolution of 3.0 T MRI limits evaluation of small embryonic structures, particularly during the early incubation period (before 5 days); spatial resolution would almost certainly be improved with a higher field strength magnet. Matching of MRI imaging planes and histologic specimens was performed with careful attention to detail but acquiring exact matches is technically challenging and exact matching was not feasible in all cases. The reverse method, used by some researches, would be to use reconstructions of the isotropic or nearly-isotropic 3D MRI dataset to match the histological sections, possibly obtaining more accurate imaging-histologic correlation. Our dual-cooling method, which reduced the temperature, may influence diffusion measurements (such as apparent diffusion coefficient and mean diffusion) compared to room temperature; we intend to explore this possibility in our next work. Furthermore, there may be developmental differences among embryos of the same incubation day, though this could be accounted for by increasing statistical power.Given that DTI has the ability to evaluate skeletal muscle differentiation and development, it may be possible to monitor the effect of mutations which affect skeletal muscle migration and development ,19. DTI In conclusion, this study shows that a widely-available (3.0 T) MRI system can be used as a powerful investigative tool to evaluate embryonic development with diffusion tensor imaging in ovo from 5 days incubation to hatching. We show that acquired skeletal muscle diffusion tensor imaging parameters correlate with histologic specimens from sacrificed embryos. Noninvasive evaluation of developmental processes in embryonic models has traditionally been technically difficult and largely restricted to anatomic evaluation. Functional imaging with DTI MRI and other advanced MRI techniques permit the evaluation of tissues and cellular processes below the conventional spatial resolution of MRI, and is a potentially promising method of improving the understanding of early developmental processes.The experimental protocol and procedures were approved by the Institutional Ethics Committee of the Shanghai Jiaotong University School of Medicine. Ninety (90) fertile Hy-Line White eggs each weighing 50–55 g were obtained from a commercial hatchery and placed in a digital tabletop incubator with temperature (37.8°C) and humidity (60%) controlled automatically. After four days of incubation, eggs were “candled”  to determine if they were fertile and developing normally. Six eggs were removed from the incubator for underdevelopment. From the 84 remaining eggs, 5 eggs were chosen at random each day, from incubation day 5 to 18 . Eggs were first removed from the incubator and air-cooled for one hour at 3.5-4°C prior to imaging. The surface temperature of the egg before air-cooling was 24-26°C. During imaging, the egg was wrapped in a single piece of Techni-Ice . Techni Ice egg-contact surface temperature was 0-2°C, as measured immediately prior to and after imaging. Temperature monitoring during imaging was not performed. The dual-cooling method was used to suppress embryonic motion throughout the imaging session (about 32 minutes). In order to show the in ovo serial skeletal muscle diffusion tractography of the developing chick embryo using DTI, an additional 3 eggs were imaged serially without making histological specimen from day 5 to 18.Eggs wrapped in Techni-Ice were imaged in a 3 T Philips Achieva  using a four-channel dedicated animal coil with a 5 cm inner diameter. Image acquisition consisted of high-resolution T2 weighted SENSE TSE (turbo spin echo) images , slice thickness 1.2 mm, no gap, 12 min 23 s duration), and SENSE DTI using SE (spin echo)-DWI , b=500 s/mm2, NEX=2, slice thickness 1.2 mm, no gap, 20 minute duration). A 16th unweighted (b=0) DTI image was also acquired. All imaging planes were sagittal.Skeletal muscle fibers in the hind limb were tracked using FiberTrak software . Three parameters, minimum fractional anisotropy (FA), maximum angle change, and minimum fiber length needed to be set to determine the stopping threshold of tractography. We chose a small minimum FA (0.15) and small maximum angle change (27°) to balance the sensitivity of myofiber tracking during early development stage and the precision during later development; these values were held constant throughout the incubation period. Small values for minimum fiber length (5 mm) were used during early incubation (days 5–11), and minimum fiber length was increased to 10 mm during later incubation (days 12–18).T2-weighted images were used to guide placement of the rectangular region of interest (ROI) for fractional anisotropy (FA) measurement. The sagittal image containing the largest section of a single hind limb was selected. An ROI was then placed on the hind limb. ROI size changed with embryonic development. As shown in Figure After MR imaging, the egg was cracked and the corresponding embryonic hind limb bud or hind limb was resected as a specimen and fixed in a 10% formaldehyde solution for one week. The length of hind limb bud or hind limb was simply measured by a ruler before resection. As shown in Figure Fractional anisotropy (FA) value and tracked fiber length of tissue within ROI was automatically computed by the FiberTrak software. The percentage of skeletal muscle fiber area (termed FiberArea%) was determined using custom software based on the Insight Segmentation and Registration Toolkit . The algorithm used for calculation of FiberArea% is based on grey-level differences. Color specimen micrographs were converted to an 8-bit grey-scale pictures, with each pixel measuring from 0 (black) to 255 (white). Pixels were classified via thresholding and binning; pixel values below 90 were associated with nucleus or myoblast structures, and values above 200 were associated with background mesenchymal tissue. Pixel values between 90 and 200 were considered to represent skeletal muscle fiber. FiberArea% represents the ratio of binned pixels representing skeletal muscle fiber relative to the total number of pixels within the micrograph. An example is shown in Figure Data management and statistical analysis was performed using MedCalc . A linear model was used to evaluate the relationship between day of incubation and ROI-derived fractional anisotropy (FA) values, length of tracked fibers, FiberArea% and length of hind limb. To evaluate the relationship between imaging and histology during embryonic development, two correlation analyses were performed with p values < 0.05 considered to be significant. One was between FA and FiberArea%, the other was between length of tracked fibers (Fiber_length) and length of hind limb (Limb_length).MRI: Magnetic resonance imaging; DTI: Diffusion tensor imaging; FA: Factional anisotropy; ROI: Region of interest; FiberArea%: The percentage of skeletal muscle Fiber within a given histologic specimen.The authors declare that they have no competing interests.Research idea and study design, JRX JNH EMH, Chick embryo hatching, ZEZ, MRI acquisition and measurement of diffusion parameters, ZEZ JH YQY ZSZ, Histological specimen making and analysis, LMW, Statistical analysis, ZEZ LMW JRX JNH ZD YZ, Manuscript drafting, ZEZ JH ZD YZ, Manuscript revision for important intellectual content, all authors. Approval of final version of submitted manuscript, all authors. All authors read and approved the final manuscript.


Altered protein homeostasis underlies degenerative diseases triggered by misfolded proteins, including spinal and bulbar muscular atrophy (SBMA), a neuromuscular disorder caused by a CAG/glutamine expansion in the androgen receptor. Here we show that the unfolded protein response (UPR), an ER protein quality control pathway, is induced in skeletal muscle from SBMA patients, AR113Q knock-in male mice, and surgically denervated wild-type mice. To probe the consequence of UPR induction, we deleted CHOP (C/EBP homologous protein), a transcription factor induced following ER stress. CHOP deficiency accentuated atrophy in both AR113Q and surgically denervated muscle through activation of macroautophagy, a lysosomal protein quality control pathway. Conversely, impaired autophagy due to Beclin-1 haploinsufficiency decreased muscle wasting and extended lifespan of AR113Q males, producing a significant and unexpected amelioration of the disease phenotype. Our findings highlight critical cross-talk between the UPR and macroautophagy, and they indicate that autophagy activation accentuates aspects of the SBMA phenotype. In many age-dependent neurodegenerative diseases, the accumulation of misfolded or mutant proteins drives pathogenesis. Several protein quality control pathways have emerged as central regulators of the turnover of these toxic proteins and therefore impact phenotypic severity. In spinal and bulbar muscular atrophy (SBMA), the mutant androgen receptor with an expanded glutamine tract undergoes hormone-dependent nuclear translocation, unfolding, and oligomerization—steps that are critical to the development of progressive proximal limb and bulbar muscle weakness in men. Here we show that the unfolded protein response (UPR), an endoplasmic reticulum stress response, is triggered in skeletal muscle from SBMA patients and knock-in mice. We find that disruption of the UPR exacerbates skeletal muscle atrophy through the induction of macroautophagy, a lysosomal protein quality pathway. In contrast, impaired autophagy diminishes muscle wasting and prolongs lifespan of SBMA mice. Our findings highlight cross-talk between the UPR and autophagy, and they suggest that limited activation of the autophagic pathway may be beneficial in certain neuromuscular diseases such as SBMA where the nucleus is the essential site of toxicity. Many adult onset neurodegenerative disorders are characterized by the accumulation of abnormally folded proteins that self-associate into soluble oligomeric species or coalesce into insoluble protein aggregates. Among these disorders are ones caused by expansions of CAG/glutamine tracts Ar exon 1 with human sequence containing 21 or 113 CAG repeats Studies in mouse models have defined several general principles that guide our understanding of SBMA pathogenesis. Transgenic over-expression of the expanded glutamine AR leads to disease, consistent with the notion that toxicity is predominantly mediated through a gain-of-function mechanism The cellular pathways by which the expanded glutamine AR mediates toxicity are complex and incompletely understood, with evidence in several model systems showing disruption of gene expression in vitroIn addition to the chaperone machinery, other pathways regulating protein quality control have been implicated in SBMA pathogenesis. Here we explored the role of the unfolded protein response (UPR), an integrated signal transduction pathway that transmits information about protein folding within the ER lumen to the nucleus and cytosol to regulate protein synthesis and folding and to influence cell survival To determine whether the ER stress response is activated in SBMA we obtained skeletal muscle from patients and male controls. Gene expression analysis by qPCR demonstrated that SBMA muscle contained significantly higher levels of several mRNAs that are induced in response to ER stress  [34], [3Ar gene Analysis of proximal hind limb muscle from adult AR113Q male mice similarly demonstrated the induction of mRNAs encoding BiP, ATF4, CHOP and PDI . This wain vivo. To accomplish this, we generated AR113Q males deficient in CHOP, a regulator of cell survival during ER stress that we found to be up-regulated in SBMA muscle. CHOP null mice exhibit impaired programmed cell death following pharmacological induction of ER stress As the UPR plays a central role in protein homeostasis in the ER and influences survival in a cellular model of SBMA CHOP deficiency markedly affected AR113Q muscle, the site of UPR activation, by accentuating skeletal muscle atrophy . This unTo determine the mechanism by which CHOP deficiency increased skeletal muscle atrophy, we initially considered the possibility that motor neuron degeneration was more severe in AR113Q mice deficient in CHOP, resulting in enhanced neurogenic atrophy. However, we found no evidence of increased motor neuron loss in the spinal cords of these double mutants (not shown). Furthermore, skeletal muscle expression of mRNAs induced following denervation These findings prompted us to consider the possibility that another protein degradation pathway underlies the increased atrophy triggered by CHOP deficiency. As recent studies demonstrate that autophagy contributes to skeletal muscle wasting Our observation of robust UPR activation in AR113Q skeletal muscle raised the possibility that muscle denervation induces ER stress, and that disruption of the UPR by CHOP deficiency enhances wasting by altering the cellular response to ER stress. To first test whether denervation is sufficient to activate the UPR in skeletal muscle, wild type male mice underwent unilateral sciatic nerve transection, and denervated and intact gastrocnemius muscles were harvested at 3 or 7 days post surgery. Denervation significantly increased phosphorylation of eIF2 alpha and splicing of XBP1 mRNA  indicatiThese results encouraged us to use this system to further explore the relationship between the UPR and autophagy, and to test the notion that CHOP deficiency enhances muscle wasting through the induction of autophagy. Surgical denervation of male mice expressing the wild type AR demonstrated that CHOP deficiency significantly increased activity of the autophagic pathway, similar to our findings in AR113Q muscle. Denervated CHOP null muscle harvested 7 days post surgery contained ∼2.5 fold more LC3-II than did wild type muscle . p62 didBecn1) is a critical regulator of autophagy that binds class III phosphoinositide 3-kinase and is both required for the initiation of autophagosome formation and contributes to autophagosome maturation To confirm that autophagy contributes to muscle atrophy following surgical denervation, we transected the sciatic nerve of Beclin-1 haploinsufficient male mice Our finding that enhanced autophagy triggered by CHOP deficiency promoted muscle wasting in AR113Q mice prompted us to determine the consequences of limiting autophagy on the SBMA phenotype. To accomplish this, we generated AR113Q males haploinsufficient for Beclin-1. Similar to effects following surgical denervation, Beclin-1 haploinsufficiency significantly increased AR113Q muscle fiber size, although in this case the effect was less robust . LimitinDespite the limited changes in AR113Q muscle, Beclin-1 haploinsufficiency had a striking effect on survival. The lifespan of AR113Q males haploinsufficient for Beclin-1 was extended on average by ∼10 wks compared to AR113Q, Beclin-1 wild type littermates . AR113Q The accumulation of misfolded, mutant proteins is a common basis for adult onset neurodegenerative diseases including those caused by CAG/glutamine tract expansions Activation of the UPR has been reported previously in yeast and mammalian cell culture models of polyglutamine disease The functional consequences of altered autophagy in SBMA mice were unexpected and suggest that limiting activity of this pathway is beneficial for certain aspects of the disease phenotype. As the polyglutamine AR resides in the nucleus in the presence of ligand and largely escapes degradation through this pathway Of the clinical symptoms experienced by SBMA patients, muscle wasting is a substantial contributor to morbidity. Here we show that activation of autophagy significantly enhances atrophy of surgically denervated and AR113Q muscle. In contrast, limiting autophagy prolongs lifespan and maintains motor function in SBMA mice. While the effects of Beclin-1 haploinsufficiency are relatively mild in AR113Q muscle, lifespan extension is striking, and likely reflects benefits of limited autophagy in cell types other than muscle fibers, perhaps including effects on metabolism. Defining the targets affected by Beclin-1 haploinsufficiency that mediate lifespan extension remains an important goal for future work. Notably, strategies to modulate the activity of the autophagic pathway have attracted considerable attention as studies in several polyglutamine disease models have documented degradation of cytoplasmic protein aggregates through autophagy Ar alleles containing 21 or 113 CAG repeats in exon 1 was described previously Ar gene in CJ7 embryonic stem cells. Male chimeras were mated with C57BL/6J females, and females heterozygous for the targeted Ar allele were backcrossed to C57BL/6J to generate mice used in this study. Surgical castration of 5–6 wk old males was as previously described tm1Dron/J) Becn1 null allele were previously reported Derivation of mice with targeted Becn1 haploinsufficient male mice congenic to C57BL6/J were used for studies of denervated muscle. Under deep inhaled anesthesia with 2% isoflurane, the right sciatic nerve was exposed at the thigh just below the sciatic notch. Both the proximal and distal sides were ligated with monocryl 4-0 suture, and about 2 mm of sciatic nerve was cut between the ligations to prevent axonal regeneration. At 3 and 7 days after surgery, the right gastrocnemius and tibialis anterior muscle were dissected and frozen for histology or RNA and protein analysis. The contralateral side was used as control.7 wk old C57BL/6J, CHOP deficient or Anonymized SBMA muscle and control biopsy samples were obtained from the University of Michigan Medical School in accordance with IRB procedures and in a manner that assured patient privacy. Additionally, anonymized skeletal muscle was harvested from SBMA patients at autopsy, as approved by the ethics committee of the Nagoya University Graduate School of Medicine and in accordance with the Declaration of Helsinki (Hong Kong Amendment).2 according to scale. 100 adjacent fibers from each section were measured.Muscle was frozen in isopentane chilled by liquid nitrogen, cut in cross section at a thickness of 5 µm and stained by H&E. Digital images were captured using a Zeiss Axioplan 2 imaging system. The area of each muscle fiber was defined using Adobe Photoshop CS4 or ImageJ, and the pixel number was converted to µm5′-GAACCAGGAGTTAAGAAC-3′ and 5′-AGGCAACAGTGTCAGAGT-3′; human: 5′-GAATGAGTGAGCTGGAACAG-3′ and 5′-GAGTCAATACCGCCAGAATC-3′) to amplify 10 ng of cDNA through 22 cycles. One tenth of the total PCR products were resolved on 15% nondenaturing polyacrylamide gels and stained with SYBR Green 1  after electrophoresis. Bands were visualized on a Typhoon Trio+ scanner  and analyzed with AlphaImager 2200 software .Total RNA isolated from tissues with Trizol  served as a template for cDNA synthesis using the high capacity cDNA archive kit from Applied Biosystems . Gene-specific primers and FAM labeled probes  were purchased from Applied Biosystems. TaqMan assays were performed in duplicate using 5 ng aliquots of cDNA on an ABI 7500 Real Time PCR system. Relative expression levels were calculated comparing with the expression of 18S rRNA. Semi-quantitative RT-PCR analysis of Xbp1 RNA splicing was performed using primers . Blots were probed with primary antibodies and proteins were visualized by chemiluminescence . The AR (N-20), HSP90 and eIF2α antibodies were from Santa Cruz Biotechnology , phospho-eIF2α (Ser51) and phospho-JNK antibodies were from Cell Signaling Technology , LC3B antibody was from Novus Biologicals , GAPDH, BiP and PDI antibodies were from AbCam , 20S proteasome antibody was from Calbiochem  and p62 antibody was from American Research Products . Western blot quantification was performed using ImageJ.Muscle tissue was homogenized in RIPA buffer containing complete protease inhibitor cocktail  and phosphatase inhibitor  using a motor homogenizer . Sample lysates were incubated on a rotator at 4°C for 1 hour and the pre-cleared by centrifugation at 15,000 Frozen muscle tissue was sectioned at 5 µm with a cryostat and stained with H&E or NADH. For immunofluorescence, 5 µM frozen sections were stained with an antibody against AR and an Alexa Fluor 594 conjugated secondary antibody (Invitrogen). Confocal images were captured with a Zeiss LSM 510 microscope and a water immersion lens (×63).The grip strength meter (Columbus Instruments) was positioned horizontally and mice were lowered toward the apparatus. Mice were allowed to grasp the smooth metal triangular pull bar with their fore limbs only, and then were pulled backward in the horizontal plane. The force applied to the bar at the moment the grasp was released was recorded as the peak tension (kg). The test was repeated 5 consecutive times within the same session, and the highest value from the 5 trials was recorded as the grip strength for that animal.t-test or by ANOVA with the Newman-Keuls multiple comparison test. The distribution of muscle fiber size was analyzed by Mann-Whitney test. All statistics was performed by the Prism 5 . P values less than 0.05 were considered significant.Statistical significance was assessed by two-tailed Student's Figure S1n = 5–6/genotype) by qPCR. ***p<0.001 by ANOVA, n. s. = not significant.MyoD and myogenin mRNA expression. Relative expression of MyoD and myogenin mRNAs was determined in proximal hind limb muscle of 12 wk mice ((TIF)Click here for additional data file.Figure S220S proteasome expression. Western blot shows expression of 20S proteasome subunit in proximal hind limb muscle. Lower panel shows relative quantification of signal intensity. n. s. = not significant.(TIF)Click here for additional data file.