RB1) tumor suppressor have been reported in lung cancer, mainly in small cell lung carcinoma, the tumor suppressive role of its relatives p107 and p130 is still a matter of debate. To begin to investigate the role of these two Rb family proteins in lung tumorigenesis, we have generated a conditional triple knockout mouse model (TKO) in which the three Rb family members can be inactivated in adult mice. We found that ablation of all three family members in the lung of mice induces tumorlets, benign neuroendocrine tumors that are remarkably similar to their human counterparts. Upon chemical carcinogenesis, DHPN and urethane accelerate tumor development; the TKO model displays increased sensitivity to DHPN, and urethane increases malignancy of tumors. All the tumors developing in TKO mice  have neuroendocrine features but do not progress to fully malignant tumors. Thus, loss of Rb and its family members confers partial tumor susceptibility in neuroendocrine lineages in the lungs of mice. Our data also imply the requirement of other oncogenic signaling pathways to achieve full transformation in neuroendocrine lung lesions mutant for the Rb family.Lung cancer is a deadly disease with increasing cases diagnosed worldwide and still a very poor prognosis. While mutations in the retinoblastoma ( Lung cancer is a major health problem worldwide, as it belongs to the most deadly cancer condition with overall poor prognosis and only a small percentage surviving more than 5 years. Much more effort is needed for understanding the molecular mechanisms underlying the disease and for the development of early detection techniques and novel effective therapies.Approximately 15–20% of lung tumors show characteristics of neuroendocrine cells. Pulmonary neuroendocrine (NE) tumors include, depending on the tumor size and the number of mitoses, a spectrum of tumors that range from the low-grade, typical carcinoid (TC), through intermediate-grade, atypical carcinoid (AC) to the high-grade, large-cell neuroendocrine carcinoma (LCNEC) and small-cell lung carcinoma (SCLC). The benign NE tumors that precede the carcinoid tumors are usually small nodular NE proliferations called tumorlets . TumorleRB1, RBL1 and RBL2 respectively), plays a crucial role in the control of cell cycle progression through its ability to bind the E2F transcription factors in a complex regulatory network. RB family also participates in multiple cellular processes beyond cell cycle progression. Observations in various human cancers in which genetic inactivation of RB1 has been reported, and the analysis of genetically engineered mice have identified pRb as a major tumor suppressor. The RB1 gene is inactivated in most cases of specific cancer types such as retinoblastoma, small cell lung cancer (SCLC) and osteosarcoma, whereas alterations of its family members p107 and/or p130 is still a matter of debate, being rarely mutated in human tumors . Overall, these data point to strict differences in tissue susceptibility and that further oncogenic signaling is required to account for malignant tumor development upon ablation of the three retinoblastoma family member genes in mouse lung.Another possibility would be related to potential oncogenic roles played by p130 in lung and thus, its elimination could preclude tumor progression. This alternative, besides being counterintuitive, is not supported by the actual data obtained from cancer mouse models. Indeed the loss of p130 in Rb-deficient cells promotes the development of neuroendocrine lesions in the lungs of mice , deletioRb1 and Trp53 is sufficient to cause SCLC development in mice [Regarding this last possibility, we observed moderate ERK and very mild Akt activation, probably due to the induction of PTEN in tumorlets. We also observed a weak induction of Stat3, which may play oncogenic and oncosuppressive roles in lung cancer . Further in mice , 42, 43.In an attempt to provide further clarification to these possible oncogenic inputs required, we performed chemical carcinogenesis using two well known lung carcinogens, DHPN and urethane, that cause Kras activation , 20, 22.On the other hand, upon urethane carcinogenesis, we observed that both control and TKO mice developed tumors with similar incidence and latency. However, TKO mice displayed a higher proportion of atypical carcinoids, which are considered a more malignant grade than the typical carcinoid. Collectively, the chemical carcinogenesis experiments reinforce the resistance to overt malignant progression observed upon ablation of the three retinoblastoma proteins in lung even upon activation of oncogenic signals such as Kras (denoted by ERK activation). Nonetheless, these experiments also indicate that the possible malignant evolution of the mouse lung tumors to typical and atypical carcinoids is facilitated by Rb-family loss.Rb1 mRNA in urethane induced tumors in different genetic backgrounds has also been reported [Of note, all the tumors that arose in our cohorts were of neuroendocrine type. In this case, given the absence of spontaneous tumorlets in the control untreated group, we could consider a potential role for p107 loss alone that could lead to the change to neuroendocrine cell phenotypes after urethane treatment. Urethane is known to induce lung adenomas/adenocarcinomas in A/J mice , 45 and reported  providinIn summary, our findings support that the inactivation of all the three members of the Rb family in the lungs renders neuroendocrine lung tumor formation but is not enough to allow spontaneous tumors to progress into a higher state of malignancy. These data provide evidence for the link between loss of Rb family members and development of specifically neuroendocrine tumors and points out to the requirement of other signaling pathways for the malignant progression.F/F; p130F/F;p107−/−Rb mice were previously described [Rb1 and Rbl2 in pulmonary cells was achieved by intratracheal or intranasal administration of adenovirus Ad5-CMVcre [8 pfu to 1010 pfu. Ad5-CMVcre was obtained from University of Iowa's Vector Core Facility (www.uiowa.edu). As control animals, F/F; p130F/F;p107−/−Rb littermates were used. Mice were sacrificed 9 to 24 months after the Ad5-CMVcre infection.All the animal experiments were approved by the Animal Ethical Committee and conducted in compliance with Centro de Investigaciones Energéticas, Medioambientales y Tecnologicas (CIEMAT) Guidelines. escribed . Rosa26Rescribed  were pur5-CMVcre  to 8–10 A group of animals were treated with chemical carcinogens (DHPN or urethane). Chemical carcinogen was administered both to adenovirus infected mice and to control group (non-infected littermates). Groups are then referred to as TKO+DHPN; control+DHPN; TKO+ urethane; control+urethane.n = 10) and infected mice (n = 12). DHPN administration began 4 or 22 weeks after Ad5-CMVcre delivery. Mice were sacrificed 35 weeks after starting DHPN treatment  was administered in drinking water in a 0.1% concentration for 8 weeks, both to uninfected (n = 11) or infected mice (n = 18). For the adenovirus infected mice urethane administration began 4 or 22 weeks after Ad5-CMVcre delivery. Mice were sacrificed 24 to 29 weeks after the beginning of the treatment.Urethane  was administered intraperitoneally (1 mg/g bodyweight) once a week for 10 weeks and additional 10 weeks after a month without treatment to uninfected control littermates  were stained with hematoxylin and eosin (H/E), for histological analysis, or processed for immunohistochemistry. Immunohistochemical and immunofluorescence analysis were performed essentially as previously described standard protocols , 50. AntFor quantification of the number of Ki-67and PH3 positive cells, analysis of tumor areas and images was done using ImageJ software. More than 1000 cell nuclei were counted.To detect β-galactosidase activity whole lungs were fixed in PBS containing 2% paraformaldehyde and 0.25% glutaraldehyde for 2 h at 4°C prior to incubation with X-gal solution at 37°C overnight. These tissues were then postfixed in 4% paraformaldehyde and embedded in paraffin. Sections were then processed for hematoxylin staining, immunohistochemistry or both, and examined by light microscopy.RNA was isolated from whole mouse lungs in control and TKO mice and from isolated tumors in urethane-treated mice using RNALater (Ambion) and miRNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. Genomic DNA was eliminated from the samples by a DNase treatment (Rnase-Free Dnase Set Qiagen). The Omniscript RT kit (Qiagen) and oligo dT primers were used to prepare cDNA from RNA of the mouse samples, using 2 μg of total RNA.RNA was obtained from isolated tumorlets embedded in paraffin using the RNeasy FFPE Kit (Qiagen) according to the manufacturer's instructions. Genomic DNA was eliminated from the samples by a DNase treatment (Rnase-Free Dnase Set Qiagen). The Omniscript RT kit (Qiagen), sequence-specific primers and random primers were used to prepare cDNA from RNA of the mouse samples, using 100 ng of total RNA.Real-time quantitative PCR was done on a 7500 Fast Real-Time PCR system (Applied Biosystems) with the GoTaq pPCR Master Mix (Promega), using 1 μl of cDNA (as a template). Melting curves were performed to verify specificity and absence of primer dimerization. Reaction efficiency was calculated for each primer combination and each sample was normalized using the values for the TATA binding protein gene (Tbp). The sequences of the specific oligonucleotides used are listed in t-test or Mann-Whitney test depending on the normal distribution of the data. The association between categorical variables was analyzed using Fisher's exact test. Statistical significance was accepted at p < 0.05. Graph prism 5.0 software was used.Comparisons between two groups were performed using Student's unpaired


MicroRNAs (miRNAs) are short, non-coding RNAs that regulate gene expression mainly through translational repression of target mRNA molecules. More than 2700 human miRNAs have been identified and some are known to be associated with disease phenotypes and to display tissue-specific patterns of expression.n = 28) or Parkinson’s disease (n = 29) and controls without neurological impairment (n = 36). A custom miRNA identification analysis pipeline was built, which utilizes miRDeep* miRNA identification and result filtering based on false positive rate estimates.We used high-throughput small RNA sequencing to discover novel miRNAs in 93 human post-mortem prefrontal cortex samples from individuals with Huntington’s disease  contains supplementary material, which is available to authorized users. MicroRNAs (miRNAs) are a class of small, noncoding regulatory RNAs. In their mature form, miRNAs are single-stranded, 19–23 nucleotides in length, most often generated from hairpin precursors transcribed from intergenic, intronic, or exonic regions of the genome. Mature miRNAs act as post-transcriptional regulators of gene expression through sequence-specific targeted binding of mRNA. The most common mechanism of regulation by miRNAs is translational repression of targeted mRNA. A single miRNA may target multiple mRNAs and a single mRNA may be targeted by multiple miRNAs [The importance of miRNA regulation of mRNAs and its effect on organism phenotype is not yet fully understood, but it is clear that it is a widespread phenomenon . miRNAs miRNAs can be species-specific and many display tissue-specific patterns of expression , 8. MoreThe goal of this study was to identify novel, potentially brain-specific miRNA species using miRNA sequencing from 93 human prefrontal cortex tissue samples, comprising persons with Huntington’s disease (HD) or Parkinson’s disease (PD) and neurologically healthy controls. Novel miRNAs were identified using the miRDeep* procedure  and finaSample Information is provided in Additional file Frozen brain tissue from the prefrontal cortex Brodmann Area 9 (BA9) was available from three brain banks: the National Brain and Tissue Resource for Parkinson’s Disease and Related Disorders at Banner Sun Health Research Institute, Sun City, Arizona, the Harvard Brain Tissue Resource Center McLean Hospital, Belmont, Massachusetts, and the Human Brain and Spinal Fluid Resource Center VA, West Los Angeles Healthcare Center, California.http://tucf-genomics.tufts.edu/) or Michigan State sequencing core facility (http://rtsf.natsci.msu.edu/genomics/). Batch 1 and batch 2 used eight samples per lane with equal numbers of HD and control samples per lane. In batch 3, all samples were given unique bar-codes and distributed across all lanes, with the number of lanes and samples allocated to reach the target read depth of 16,000,000 reads per sample. Additional file Isolation and purification of total RNA was done using QIAzol Lysis Reagent and miRNeasy MinElute Cleanup columns, respectively. RNA quality was assessed either by RNA Quality number from Agilent’s BioAnalyzer 2100 and RNA 6000 Nano Kits, or by RNA Quality Number from Agilent 2200 TapeStation and Agilent DNA ScreenTape assay. Sequencing libraries were constructed using 1 μg of RNA per sample and Illumina’s TruSeq Small RNA Sample Prep Kit with the manufacturer’s protocol. Illumina’s HiSeq 2000 system was used for 1×51 nucleotides, single-end sequencing, conducted at Tufts University (v = 0) and up to 200 instances of multi-mapping (m = 200). More stringent read length filtering was done by miRDeep* before identification of novel miRNAs, discarding reads with length less than 18 nucleotides and greater than 23 nucleotides, miRDeep* default parameters. Differential expression analyses used the less stringent length filtered reads. Additional file Adaptor sequences were trimmed from the reads using cutadapt v. 1.2.1 , discardThe identification of miRNAs was performed using the tool miRDeep* v. 35 . miRDeepThe alignment files from all 93 samples were concatenated, divided by chromosome into 25 separate alignment files, and sorted. miRDeep* was run on each chromosome separately. The 99 final miRDeep* results were those that were both novel and passed the miRDeep* score threshold, which was determined using estimations of the false positive rate (FPR) and true positive rate (TPR). To make these estimations, the miRDeep* predictions for each chromosome were combined and divided into three categories based on hairpin mapping location: 1) known exons and small RNAs other than miRNAs, 2) known hairpin miRNAs, and 3) results that map to the remainder of the genome. This categorization was done using bedtools v2.22.1  with annFor every integer score value between the minimum and maximum scores of miRDeep* miRNA predictions, the FPR and TPR were estimated using the categorizations of feature overlap described above. The FPR was calculated as the number of miRDeep* predictions with score above the threshold overlapping exon or small RNA annotations, divided by the total number of miRDeep* predictions overlapping exon or small RNA annotations. Similarly, the TPR was calculated as the number of miRDeep* predictions overlapping known miRNAs with score above the threshold, divided by the total number of miRDeep* predictions overlapping known miRNAs. We chose the integer score threshold that yielded a FPR nearest 0.05. In order to get a more precise value than the integer score, we repeated this procedure with non-integer thresholds +/- 1 at 0.01 increments of the integer threshold.The miRDeep* novel mature sequences were aligned to mature miRNA sequences in miRBase v20 using the SSEARCH alignment algorithm from the FASTA program package , filterip-values. Bioconductor v. 3.0 and R v. 3.1.1 were used in these analyses. The HD/C (Huntington’s disease and control) analysis was conducted with 36 control and 28 HD brains, and the PD/C (Parkinson’s disease and control) analysis with the same 36 control brains and 29 PD brains. The differential expression analysis is limited by the data’s origination from separate studies, introducing potential biases on gene expression levels, though mitigated however possible.Differential expression analysis was performed with a set of miRNAs including the novel putative miRNAs identified in this study together with known human mature miRNAs from miRBase v20. The results of the novel miRNAs are presented here, and the differential expression of known miRNAs within these data has been previously characterized and published for both HD/control  and PD/cOur putative novel miRNAs were replicated using two publicly available small RNA sequencing data sets, obtained from the Gene Expression Omnibus. Santa-Maria et al. (GSE63501) contains 16 human post-mortem Brodmann Area 9 small RNA sequencing samples from individuals with Alzheimer’s disease, tangle-predominant dementia and normal neuropathology. Hebert et al. (GSE46131) contains 20 human post-mortem superior and mid-temporal neocortex grey matter small RNA sequencing samples from individuals with Alzheimer’s disease, dementia with Lewy bodies, hippocampal sclerosis of aging, frontotemporal lobar dementia (FTLD) and normal neuropathology. All 36 samples from these studies were concatenated for our replication analysis. miRNA results from the original and replication analyses were compared by genomic location using the bedtools v2.22.1 intersect function.Seven putative novel miRNAs were selected for orthogonal validation using the Exiqon miRCURY LNA Universal RT microRNA PCR system. RNA for these analyses was purified and extracted, as described above, from a set of twelve independent brains. The putative novel miRNAs selected for qRT-PCR analysis represent a wide range of mean expression values (5-5708 mean reads). We prioritized novel miRNAs with significant differential expression. Exiqon custom microRNA LNA PCR primers were designed using the miRNA primer design algorithm available online at exiqon.com. Total RNA was DNased using the TURBO DNA-free Kit following the manufacturer’s instructions (Ambion/Thermo Fisher Scientific). cDNA was generated from 100 ng of RNA using the Universal cDNA synthesis kit II and RNA Spike-in kit following the manufacturer’s instructions (Exiqon). For each sample, a cDNA reaction was performed omitting reverse transcriptase (-RT reaction). qPCR was performed using the ExiLENT SYBR Green Master mix kit following the manufacturer’s instructions (Exiqon) and using either a 1:80 or 1:20 dilution of the cDNA reaction as template. Two + RT reactions and one -RT reaction were performed for each sample and primer set. qPCR was done on a StepOnePlus Real-Time PCR System (ABI/Thermo Fisher Scientific) using the cycling and analysis parameters recommended by Exiqon .miRDeep* analysis of sequence alignment data from 93 pooled prefrontal cortex samples revealed 8891 miRNAs. Out of these, 3641 remained after filtering for known miRNAs from miRBase v20, other small RNAs and exons. The remaining miRNA results were filtered by the miRDeep* score, a measure of the likelihood that the miRDeep* result is a true miRNA, where higher scores indicate higher confidence of a miRNA result. In our miRDeep* results, score ranged from -9.98 to as high as 11 million, with a median of -0.85.The score value to be used as a threshold was determined by estimating false positive rate (FPR) and true positive rate (TPR) using the scores of the 4273 results overlapping with known exons and small RNAs  and of the 783 results overlapping with known miRNAs (true positives), excluding the 194 results overlapping both annotations. The performance of this method was evaluated with a receiver operating characteristic (ROC) curve, a plot of the FPR vs. TPR as the threshold is varied, shown in Additional file Figure N = 36), the replication analysis resulted in 30 putative miRNAs. Matching by overlap of genomic location, 19 of 30 were seen in our original 99 results. The remaining 11 were not found in our data. Thirty-three of the 99 putative novel mature miRNAs were reported in a recent novel miRNA study by Londin et al. [Nineteen of the 99 putative novel miRNAs were replicated using two independent human brain small RNA sequencing data sets obtained from the Gene Expression Omnibus (GEO access numbers GSE63501 and GSE46131) –26. UsinSeven of the 99 putative novel miRNAs plus two known miRNAs that represent the extremes of the expected range of expression were chosen for experimental validation using qRT-PCR  to at least one known mature miRNA from miRBase v20. Most of these 34 aligned to 1–4 distinct miRNAs, but 6 aligned to a large number 18–69) of sequences within the miRNA gene family hsa-miR-548. Pairwise alignments of the 99 putative novel mature miRNAs revealed that 34 align to at least one other result. Five of the six sequences that aligned to the hsa-miR-548 family also aligned to each other. Additional file –69 of sen = 36) and Huntington’s disease (n = 28) samples, and another 3 to be differentially expressed between control (n = 36) and Parkinson’s disease (n = 29) samples at False Discovery Rate of 5 % , and of the three PD-associated putative miRNAs, one has a negative log2 fold-change and two have positive log2 fold-changes.Differential expression analysis of known miRNAs miRBase v20) plus our 99 putative novel miRNAs was completed using linear models adjusting for age of death. This analysis showed 4 of the 99 putative miRNAs to be differentially expressed between control  similarity, as well as function similarity, conservation and common descent. While we have shown sequence similarity that may indicate potential miRNA familial membership, we cannot definitively determine membership with these data alone. The 65 novel results that did not align well to any currently known miRNA may be novel miRNAs that do not belong to any currently known family.miRNAs are known to exhibit tissue-specific patterns of expression , 8 and bWe performed differential expression analysis of the 99 putative novel miRNAs, of which, after adjusting for multiple comparisons with FDR q-value < 0.05, 3 are significantly differentially expressed for PD and 4 are significantly differentially expressed for HD. These seven are not only putative novel miRNAs and potentially specific to the brain, but are potentially associated with neurodegenerative disease. Five of the seven  have lower average expression within the disease samples relative to control samples. Currently, we cannot know whether these miRNAs are involved in the pathogenic mechanisms of disease or are only indirectly associated. Future studies would be required to understand the role of these seven miRNAs in PD or HD pathogenicity.It should be noted that the batching of samples was done originally for two separate studies, and that different brain banks specialize in different diseases (e.g. the HBTRC is the largest source of Huntington samples). Consequently, these data by necessity are limited by the association between brain bank and batch, and brain bank and disease status. Although we have attempted to mitigate possible biases by implementing identical sample preparation protocols for the studies and correction of batch effects with the Combat batch correction procedure, these differences may have introduced a bias on gene expression levels. This limitation should be appreciated for the differential expression analyses.We have developed a method utilizing miRDeep* to discover novel miRNAs from concatenated small RNA sequencing samples. Many existing miRNA discovery tools use samples individually, while others pool samples, similar to our approach . The maiWe have developed a pipeline that utilizes miRDeep*, pooled RNA sequencing samples and a score filtering method to discover novel miRNAs with high confidence. This pipeline was applied to 93 post-mortem human prefrontal cortex samples, yielding 99 putative novel miRNAs. Seven of these putative novel miRNAs were validated experimentally and 19 were replicated using an independent post-mortem human brain RNA sequencing data set. A subset of the 99 results may be specific to the brain or to the prefrontal cortex, and 7 show differential expression between HD and control or PD and control samples. More than a third of the putative miRNA sequences align well to known miRNAs and may be novel members of known miRNA families, while the remaining may be members of novel miRNA families.Our discovery and characterization of putative novel miRNAs may add to the rapidly-growing repository of known human miRNAs. These results reinforce the possibility that many miRNAs display tissue-specific expression and will only be discovered once many more tissue types are characterized for miRNA discovery. In addition, miRNAs have been implicated in neurodegenerative diseases, and seven of the miRNAs that we have discovered may not only be specific to the brain, but are associated with neurodegenerative disease in the samples available for the current study. We know very little about these miRNAs, and while they may be only peripherally or coincidentally associated with HD and PD, these results may eventually contribute to the understanding of the molecular mechanisms of these diseases.


Ectopic xenografting of testis is a feasible option for preservation of male fertility and angiogenesis plays a pivotal role in xenograft survival and functionality. When compared to immature testis, the adult testis is unable to establish functional xenografts due to potentially lower efficiency to induce angiogenesis. The precise molecular mechanism, however, remains elusive. In the present study, we compared adult and immature testis xenografts for survival, maturation and germ cell differentiation. Further, we evaluated differential expression of angiogenesis signalling-specific proteins in adult and immature testis and their xenografts. Results showed that adult testis xenografts degenerated whereas immature testis xenografts survived and established spermatogenesis with the production of haploid germ cells. Protein expression analysis demonstrated that immature testis xenografts were able to establish angiogenesis either through eNOS activation via VEGF and PI3K/AKT or through EGFR-mediated STAT3 pathway. The role of ERK/MAPK pathway in xenograft angiogenesis was ruled out. The absence or reduced expression of angiogenesis-specific proteins in adult testis and its xenografts possibly resulted in poor angiogenesis and in their subsequent degeneration. This study provides insight into angiogenesis mechanism that can be utilized to augment testis xenografting efficiency. Testis tissue xenografting allows for modulation/modification of spermatogenesis by manipulation of recipient mice environment. Several factors such as the size of the tissue, temperature at the grafting site and period of hypoxia play a vital role in the success of xenografting. The most important event that ensures survival of grafted testis tissue is the induction of angiogenesis. Interestingly, testis from differently aged donors has different potential for development when grafted onto recipient mice2. Till date, only testis from sexually immature hosts have resulted in successful progression of xenografts to complete spermatogenesis and emerged as successful models for studying testicular development in vivo. Fresh immature testis tissues from several species such as monkeys4, bulls6, buffalo7, goats1, pigs1, dogs8, cats9, hamsters10, rabbits11 and mice11 have shown complete spermatogenesis following xenografting onto mice recipients.Ectopic testis tissue xenografting is a feasible technique for studying spermatogenesis and testicular maturation. This technique has been used for the production of mature gametes by grafting small pieces of testis tissue under the dorsal skin of immunodeficient mice recipients12 or in species with seasonal reproductive patterns outside of mating season13. Xenografting of adult testis can also be used for studying the mechanism that causes impairment of spermatogenesis14. However it has been observed that in the xenografted human adult testis, germ cells fail to differentiate16 and spermatogenesis was arrested at meiosis in grafted horse17, dog8 and in the photo-regressed hamster18. Similarly, testes from mature adult donor goat, pig and cattle have not supported germ cell differentiation in xenograft18. Complete spermatogenesis leading to formation of elongated spermatids in xenografted testis from sexually mature species has not been reported so far. The failure of sexually mature testis to establish functional xenografts is attributed to their greater sensitivity to ischemia when compared with immature tissue. Testis from sexually mature animals are potentially less effective in inducing angiogenesis19. The angiogenesis in grafted tissues is regulated by the expression of angiogenesis signalling-specific proteins in the donor testis. Expression of several angiogenesis signalling-specific transcripts have been reported in immature bovine testis however, their role in angiogenesis needs to be determined2. There is no report so far which specifically identifies differentially expressed key angiogenesis signalling-specific proteins in immature and adult testis xenografts.Despite poor outcome of ectopic xenografting of adult testis tissue, it could be a feasible option to maintain spermatogenesis in testis that is rendered azoospermic by disease or cancer treatment20. The survival and function of testis xenografts majorly depend on the rapid formation of a stable microvasculature between the host and the grafted tissue21. The grafted tissue must establish an efficient blood vessel connection to the host’s circulatory system rapidly to facilitate the supply of oxygen and nutrients22. A successful xenograft should have adequate angiogenesis for functional Sertoli cells, which in turn are necessary for functional germ cells2. It has been reported that host contributes towards the formation of major connecting blood vessels associated with the graft and host22. However, cell signalling in testis tissue xenografts that induces angiogenesis is not yet known.Angiogenesis or the formation of new blood vessels involves a complex and dynamic interaction between endothelial cells and the corresponding extracellular environmentThe objective of the present study is to evaluate the differentially expressed key-angiogenesis signalling-specific proteins in adult and immature testis xenografts to understand their role in angiogenesis.25. The average weight of seminal vesicles recovered from recipients grafted with adult testis tissues was significantly lower than from those grafted with immature testis and non-grafted intact control mice . The inability of xenografts from adult testis to produce sufficient testosterone could be due to lack of mature Leydig cells. These results contradict previous findings where restoration of seminal vesicle weight in mice27 and, serum testosterone level in rats28 following adult testis autografting has been reported. This inconsistency could be due to failure or delay in the establishment of angiogenesis in xenografted adult testis leading to loss of Leydig cells29. The recovery of seminal vesicle weight in recipients grafted with immature testis suggests that not only the spermatogenic but also the steroidogenic function of the immature rat testis was restored in the xenografts. Induction and maintenance of spermatogenesis require a continuous and controlled interaction of several hormones in the hypothalamic–pituitary–testis axis30. This indicates that a synchronized hormonal interaction was established between the recipient’s hypothalamus and pituitary and the xenografted immature rat testis, which induced spermatogenesis in the xenografted tissue. Thus, suggesting that Sertoli cells and Leydig cells present in donor tissue were able to respond to mouse gonadotrophin stimulation.At 8 wk post grafting, xenografts and seminal vesicles were recovered from recipients grafted with adult testis tissues or immature rat testes and weighed Table . The weiAverage graft weight and total graft recovery were significantly lower from recipients grafted with adult rat testis tissue than in recipients grafted with immature rat testis (P < 0.05). The lower graft recovery from recipients grafted with adult rat testis tissue can be attributed to complete degeneration of xenografts and lower xenograft weight to progressive disintegration of the testicular architecture and extensive degeneration of seminiferous tubules.18. Moreover, the adult testis tissue xenografts are also highly sensitive to ischaemia10 and have a high demand for oxygen because of ongoing spermatogenesis22. The survival of sperm cells in otherwise degenerated tubules is interesting. It is likely that spermatozoa being terminally differentiated cells are rather resistant to degeneration and are able to survive in degenerated tubules. We had observed similar findings in autografted adult mice testis27 and another report also confirmed similar finding16. Testis from immature donors could establish spermatogenesis following ectopic xenografting and meiosis was induced as indicated by the presence of pachytene-stage spermatocytes as the most advanced germ cells , its xenograft was unable to do so. This could be due to initial ischemia before the blood supply to the grafts is established leading to a delay in initiation of spermatogenesis. Whether a longer grafting period is required for spermatogenesis to get completed in xenografted immature rat testis needs to be evaluated.Histological evaluation of 10-wk-old donor rat testis showed intact seminiferous tubules with normal spermatogenesis whereas 6-day-old rat donor testis showed seminiferous tubules with spermatogonia as the most advanced germ cell in 100% tubules Fig. . Xenogralls Fig. . These r31. Sertoli cell proliferation, maturation and establishment of spermatogenesis further confirmed that a synchronized hormonal interaction was indeed established between the recipient’s hypothalamus and pituitary and the xenografted immature rat testis. However, a significantly lower number of PCNA-positive Sertoli cells in grafts than that in the adult testis implied incomplete maturation. This delayed maturation of grafts could be a possible reason for incomplete spermatogenesis in the grafted testis. In grafts from adult donor, no PCNA-stained cells were evident in the tubules  in the seminiferous tubules Fig. . In 6-dales Fig. . These r32 hence, xenografts were analysed 1 and 2 wk post grafting. Growth factors and their receptors play an important role in the initiation of angiogenesis and the generation of new blood vessels critical for graft survival. VEGF is a key regulator of both vasculogenesis and angiogenesis33. In the present study, the expression of VEGF protein was present both in testis of immature and adult donors but its expression level was significantly lower in adult testis  identified only alpha subunit (85KDa) in immature fresh testis but both forms of PI3K were absent from adult testis. Interestingly, gamma (55KDa) subunit of activated PI3K was present in xenografts of immature testis at both collection time points and in xenografts of adult testis at 2 wk only. It is reported previously that activated PI3K subunits, p55γ and p85α act in concert to mediate microtubule stabilization and rearrangement68. In this study, simultaneous expression of both the subunits was absent in the immature and adult testis as well as their xenografts. Expression of AKT, another subsequent downstream protein in PI3K pathway, was absent in both immature and adult testes. Nevertheless, the expression of AKT protein was present in xenografts of immature and adult testes at both the collection time points  pups at age 6 days postpartum (refer to as immature) and at age 10 wk (refer to as adult) were used as testis donors. Immunodeficient male nude mice  were kept under specific pathogen-free conditions, and food, water, and bedding were autoclaved before use. All the animals used were housed in 12 hours light: 12 hours darkness cycle at constant temperature and provided food and water ad libitum.All animal procedures were performed in accordance with the relevant guidelines and regulations approved by the Institutional Animal Care and Use Committee (IACUC) of the Centre for Cellular and Molecular Biology (CCMB), Hyderabad, India (permit number 68/2015). Wistar rat . The testis pieces were placed on ice in Dulbecco’s modified Eagle’s medium/Ham’s F12 (DMEM/F12) HEPES  until grafting. Four rat testes (immature) or 4 pieces of testis (adult) were ectopically grafted onto the back of castrated nude mice . Briefly, mice were anesthetized with ketamine (0.1 mg/kg body weight (BW) and xylazine (0.5 mg/kg BW) in sterile physiological saline. For castration of recipient mice, a ventral medial incision was given in the abdomen and the testes were removed following which the peritoneum and skin sutured closed using absorbable suture . During the same surgery, each mouse received two incisions (~5 mm) on each side of the back . One piece of testis was inserted through each incision. The incisions were sutured closed and the mice were allowed to recover and returned to their cages.Testes were collected from immature (n = 44) and adult (n = 6) rats following euthanasia by CO2 inhalation. The fresh testis from immature and adult rats served as starting material . Xenografts were collected from mice at 1 and 2 wk post grafting to observe the expression of angiogenesis signalling-specific proteins by western blot analysis (n = 6 for each time point). At 8-wk-post grafting, the xenografts were recovered, weighed and taken for histological analysis. The weight of the seminal vesicle from recipient mice was measured as an indicator of bioactive testosterone production by the grafted tissue7. Average weight of seminal vesicle from non-grafted intact adult males  was taken as control. The percentage of recovered xenografts was calculated for mice in which xenografts were found. It was also recorded when no xenografts were recovered from a mouse. For histological analysis, xenografts and control testis tissues were fixed in Bouin’s solution followed by tissue dehydration with increased concentrations of alcohol in a series. After processing, the tissues were embedded in paraffin, sectioned (7 µm thick), stained with haematoxylin and eosin (H&E), dehydrated, mounted in Vectamount  and observed under a light microscope  for morphological evaluation. Establishment of spermatogenesis was assessed by morphological evaluation as previously described72. In xenografts where seminiferous tubules were observed, all tubules were analysed and classified either as degenerated or non-degenerated. A graft was classified as degenerated which did not contain distinct cell types and no tubules were found and, non-degenerated if it contained even a single seminiferous tubule with spermatogenesis. The percentage of seminiferous tubules and degenerated tubules within a graft was also calculated. Spermatogenesis was assessed by examining the most advanced germ cell type in the recovered xenografts.For the collection of xenografts and seminal vesicles, recipient mice were euthanized by CO2O2 for 10 min, washed three times with PBS, incubated with rabbit anti-mouse horseradish peroxidise conjugated secondary antibody  for 30 min at 37 °C, rinsed three times with PBS, incubated for 3 to 5 min using a DAB Substrate Kit (Vector Laboratories) according to the manufacturer’s instruction, rinsed thoroughly in distilled water, and mounted. Negative control sections were incubated in mouse isotype control antibodies  instead of primary antibodies at the same concentration, but all other procedures remained the same. PCNA-stained tubule cross sections were scored for the presence of PCNA-positive germ cells and Sertoli cells  in each section. An average of 50–100 tubules/cross-section and 15–30 random fields at 400x magnification per recipient mouse were examined.The tissue sections from xenografts collected at 8 wk post grafting were stained with proliferating cell nuclear antigen (PCNA) antibody to evaluate cell proliferation in seminiferous tubules. The fresh testis from immature and adult rats served as control (T0). Dilutions of primary and secondary antibodies were done in PBS with 1% BSA . Briefly, after deparaffinization and rehydration, sections were blocked with 10% FBS and 3% BSA in PBS for 30 minutes, incubated with mouse anti-PCNA antibody  overnight at 4 °C, washed several times with PBS, incubated with 3% HTotal protein from xenografts collected 1 and 2 wk post grafting was extracted upon homogenization by sonication in a dissolving buffer . Single-strength ProteCEASE-50, EDTA-free protease inhibitor  was added to the dissolving buffer before protein extraction. Protein from testis of immature and adult rats served as a representation of starting material (T0). The lysed samples (40 µg) were subjected to electrophoresis on 12% SDS–polyacrylamide gel. The gels were transferred onto PVDF membranes . The membranes were blocked with Starting Block TBS blocking buffer  for 1 h at room temperature. The blocked membranes were incubated with one of the following primary antibodies for overnight at 4 °C: hypoxia inducible factor1 alpha , epidermal growth factor receptor , angiopoietin receptor TIE2 , mitogen-activated protein kinase , phospho-p44/42 MAPK , serine-threonine protein kinase AKT , endothelial NOS , glyceraldehyde-3-phosphate dehydrogenase   vascular endothelial growth factor receptor 1 ; Angiopoietin 1  ; vascular endothelial growth factor , vascular endothelial growth factor receptor 2 , platelet-derived growth factor , platelet-derived growth factor receptor beta , AKT-pThr308 (1:100), AKT-pSer473 (1:500), PI3K-p85 alpha/p55 gamma(pY467/199) 1:500, mammalian target of rapamycin , mammalian target of rapamycin pSer2448 , phosphatase and tensin homolog , serine/threonine-protein kinase c-RAF , mitogen-activated protein kinase kinase 1/2 , signal transducer and activator of transcription 3 , STAT3-pY705 (1:100), janus kinase 1 . The membranes were then washed with TBS-T and incubated with goat anti-rabbit, goat anti-mouse or rabbit anti-goat HRP-conjugated secondary antibody  in TBS-T for 1 h at room temperature. After washing with TBS-T, immunoreactivity was detected by chemoluminescence using a C-DiGit Blot Scanner  against SuperSignal West Femto chemiluminescent substrate (Thermo Scientific), and the generated signal was analysed using a densitometer. To control protein loading on the gels, the membranes were further probed with GAPDH antibody.The results were presented as mean ± S.E.M. The statistical analyses were performed by ANOVA. Significant differences between the means were determined by analysing the data using the Tukey honest significance difference (Tukey HSD) test. The level of significance was set at P < 0.05.


In vitro experiments were conducted with preadipocytes isolated from VAT and SAT biopsies. The expression of 23 out of 33 PCP genes was enriched in VAT compared to SAT. Strong positive expression correlations of individual PCP genes were observed in VAT. WNT5A expression in VAT, but not in SAT, correlated with indexes of JNK signaling activity, IL6, waist-to-hip ratio and hsCRP. In vitro, WNT5A promoted the expression of IL6 in human preadipocytes. In conclusion, elevated non-canonical WNT5A signaling in VAT contributes to the exacerbated IL-6 production in this depot and the low-grade systemic inflammation typically associated with visceral adiposity.The accumulation of visceral adiposity is strongly associated with systemic inflammation and increased cardiometabolic risk. WNT5A, a non-canonical WNT ligand, has been shown to promote adipose tissue inflammation and insulin resistance in animal studies. Among other non-canonical pathways, WNT5A activates planar cell polarity (PCP) signaling. The current study investigated the potential contribution of non-canonical WNT5A/PCP signaling to visceral adipose tissue (VAT) inflammation and associated metabolic dysfunction in individuals with obesity. VAT and subcutaneous adipose tissue (SAT) samples obtained from subjects undergoing bariatric surgery were analyzed by qRT-PCR for expression of WNT/PCP genes.  However, a wealth of evidence has now confirmed that the regional distribution of adipose tissue is as important, if not more important, than overall adiposity in determining cardiometabolic risk1. It is widely accepted that accumulation of intra-abdominal visceral fat is a major contributor to systemic metabolic dysfunction and cardiovascular risk. In this regard, an increasing body of evidence supports the notion that visceral (VAT) and subcutaneous (SAT) adipose tissue exhibit different intrinsic properties, which make VAT a more pathogenic depot2. More specifically, the increased cardiometabolic risk associated with visceral adiposity has been linked with an overproduction of pro-inflammatory and pro-atherogenic cytokines, which contribute to establish a low-grade systemic inflammatory state that drives cardiometabolic disease8. However, the specific signaling pathways in VAT that account for this exacerbated inflammatory response remain largely unexplored.Obesity, defined as a body mass index (BMI) ≥30 kg/m11. Wingless-type MMTV integration site family (WNT) proteins are secreted signaling molecules that play fundamental roles during embryonic development and have been implicated in numerous critical aspects of physiology and disease in the adult. There are 19 WNT family members in mammals, which typically act in an autocrine/paracrine fashion and activate a number of different signaling pathways, typically classified as either canonical (β-catenin-dependent) or non-canonical (β-catenin-independent). While the role of canonical WNT signaling in the regulation of adipose tissue expansion is generally accepted12, the potential effects of non-canonical WNT signaling activation in this tissue and its contribution to fat depot heterogeneity in humans remain poorly defined. We recently reported that WNT5A, a WNT ligand that predominantly activates non-canonical WNT signaling contributes to obesity-induced adipose tissue inflammation and systemic insulin resistance in obese mice14. Although WNT5A is able to activate various non-canonical signaling pathways, its roles in embryo development are mostly mediated by activation of the WNT/planar cell polarity (PCP) pathway, which frequently leads to activation of JUN N-terminal kinase (JNK) signaling16. In adults, WNT/PCP signaling has been shown to contribute to cancer development in different settings, but its specific contribution to other disease processes has just recently begun to be explored22. The present study provides evidence supporting that WNT5A/PCP signaling is overactivated in VAT compared to SAT and contributes to the increased local and systemic inflammation associated with visceral adiposity.Emerging evidence suggests that developmental genes play central roles in fat distribution and the different properties of specific adipose tissue depots23, there was a marked upregulation (14.72-fold) of WNT5A expression in VAT compared with SAT , but it was greater in men  and in subjects with diabetes .To investigate the role of WNT5A and downstream signaling in obesity-associated adipose tissue dysfunction in humans, we used quantitative real-time PCR (qRT-PCR) to evaluate transcript expression of 33 genes involved in WNT/PCP signaling in visceral  and subcutaneous fat obtained from 44 individuals at the time of bariatric surgery. The clinical characteristics of this human population are summarized in Supplementary Table SAT Fig. . The difWNT5A in the PCP pathway were markedly and uniformly overrepresented in VAT. FRIZZLED (FZD) proteins are the main receptors for WNT ligands, and many FZD genes, including FZD1, 2, 3 and 7, were overrepresented 2- or more-fold in VAT compared with SAT  , Dishevelled (DSH) 1, 2 and 3, and PRICKLE1  Fig. , one of VAT Fig. . SimilarWNT5A, the increased expression of PCP genes in VAT was not affected by gender or diabetes status (data not shown). However, there were strong positive correlations between the expressions of individual WNT/PCP receptor components in VAT. Expression levels of ROR2, FZD7, VANGL2 and PRICKLE1 correlated with each other . Consistent with a previous report30, we found increased levels of both phosphorylated and total JNK1/2 in VAT  expression in experimental modelsipt Fig.  and protipt Fig.  level. NVAT Fig. , but notVAT Fig. . FurtherVAT Fig. . IL6 traWNT5A knockdown affect IL6 transcript levels in human primary preadipocytes. Supporting the validity of this cell culture model, we observed that transcript levels of both WNT5A and IL6 are higher in omental versus subcutaneous preadipocytes, consistent with their higher expression in VAT  and this did not have a statistically significant effect on IL6 transcript levels  levels was observed after exclusion of patients on anti-inflammatory drugs (NSAIDs and immunosuppressive drugs)  Fig. . This cogs) Fig. . In contgs) Fig. . Similar11. In this study we analyzed gene expression patterns in visceral and subcutaneous adipose tissue to gain insight into the potential role of non-canonical WNT5A/PCP signaling in adipose tissue inflammation in the setting of obesity. Our data show an over-activation of this signaling pathway in VAT under obesity conditions, which may contribute to the exacerbated local and systemic inflammation associated with visceral adiposity and its metabolic and cardiovascular complications.The differential expression of developmental genes has been suggested to contribute to the qualitative differences between visceral and subcutaneous adipose tissue and to the different cardiometabolic risk associated with the expansion of these depots in the context of obesity23, a finding that we corroborated in the current study. However, the expression of the many signaling genes that modulate WNT5A-mediated PCP signaling had remained largely unexplored. In this regard, our study adds insight into the role of non-canonical WNT signaling in adipose tissue heterogeneity by demonstrating that most of the downstream components of the non-canonical WNT/PCP signaling pathway  are markedly overrepresented in VAT compared to SAT. These data suggest that the over-activation of this signaling pathway extends beyond the increased expression of the WNT5A ligand in this depot. It is noteworthy that many of the WNT/PCP receptor components that we found most highly upregulated in VAT  have been shown to be essential for WNT5A signaling in different experimental settings33. We are unaware of any other signaling pathway that has been reported to be so uniformly and robustly upregulated in visceral fat. These findings suggest that PCP signaling modulators represent unique nodal drivers of VAT dysfunction in the setting of obesity.We and others have previously reported that WNT5A is expressed at higher levels in VAT than SAT in the setting of obesity34, although not to the extent that is observed for some WNT/PCP genes. On the other hand, we have previously reported that myeloid cells are a significant source of Wnt5a in mouse adipose tissue14, and pro-inflammatory TLR signaling has been reported to increase WNT5A expression in human monocyte/macrophages35. Thus, the differential expression of WNT5A in VAT and SAT could also reflect differences in macrophage infiltration and/or activation. Regardless of the underlying mechanism, these data suggest that not only VAT does produce more WNT5A, but also that this fat depot is likely to be more responsive to its actions, given its higher expression of WNT/PCP receptors and other WNT signaling intermediates. Consistent with this notion, we find that WNT5A is associated with increased JNK signaling in VAT, but not in SAT. Although WNT5A is able to activate a plethora of signaling pathways in a context-dependent manner, JNK is the main signaling kinase activated by the WNT/PCP pathway in mammals16, and it frequently mediates the effects of WNT5A. Notably, experimental studies suggest that JNK signaling is particularly relevant in the setting of obesity-induced inflammation and associated metabolic dysfunction29.Our data also reveals a remarkable correlation among the expression of different PCP genes in VAT, particularly in subjects with diabetes, suggesting the existence of common regulatory mechanisms that coordinate their expression. The regulators of PCP gene expression in omental fat are probably multiple. Omental and subcutaneous fat have different embryological origins, and this could contribute to the differential expression of WNT/PCP genes. In this regard, the greater expression of WNT5A observed in omental versus subcutaneous preadipocytes is consistent with this possibility. Several developmental genes have been reported to be differentially expressed in the different adipose tissue depots36. The pro-inflammatory cytokine IL-6 is generally believed to be a particularly relevant contributor to the high cardiometabolic risk associated with intra-abdominal fat accumulation. It is produced at higher levels in VAT compared to SAT7 and circulating IL-6 levels exhibit a stronger association with visceral adiposity than other pro-inflammatory cytokines, such as TNF31. Despite of these findings, the specific mechanisms that account for the increased production of IL-6 in VAT remain poorly defined. In this regard, we recently reported that Wnt5a promotes adipose tissue inflammation and systemic insulin resistance in mouse models of obesity, at least in part through an increased production of IL-6 in adipose tissue14. However, the extrapolation of these mouse results to human obesity remained challenging due to the many differences between adipose tissue depots in mice and humans37. While omental fat is the prototypical visceral fat depot in humans, this depot is essentially absent in mice. Furthermore, inflammatory responses in mice and humans are known to differ significantly38, and the biology of IL-6 is likely to exhibit many differences between species, as suggested by the low amino acid sequence identity between human and mouse IL-6 proteins (~41%)39. The results of the current studies with subcutaneous and omental preadipocytes in culture and the in vivo association between the expression of WNT5A and IL6 in VAT presented herein supports our previous mouse studies14, and provides evidence that WNT5A/PCP signaling is a clinically-relevant contributor to the elevated IL-6 production in VAT. The correlation observed in VAT between WNT5A and the pJNK/JNK ratio, an index of JNK signaling, also supports this possibility. JNK signaling is known to promote IL-6 expression in adipose tissue29, and our previous mouse studies suggest that JNK signaling is a main mechanism linking WNT5A to IL-6 production14. Further supporting this WNT5A/IL-6 connection, we also observed a significant association between WNT5A expression in VAT and circulating levels of CRP, a marker of systemic inflammation. The connection between WNT5A expression in VAT and IL-6/CRP levels is of particular relevance, given the large body of clinical evidence that links increased IL-6 signaling and/or increased circulating CRP levels to insulin resistance, diabetes and atherosclerotic CVD43. Some of our data suggest that the contribution of non-canonical WNT5A signaling to IL-6 production and inflammation may be particularly relevant in subjects with diabetes, which typically exhibit high levels of systemic inflammation. However, the differences between subjects with and without diabetes observed in our study need to be interpreted with caution, given our limited sample size and the substantial differences in clinical characteristics between subjects with and without diabetes. Future studies with greater sample size will be needed to assess whether a direct connection between diabetes and WNT5A expression in VAT exists in humans, as suggested by our previous mouse studies14.Systemic low-grade inflammation is one of the main mechanisms that link obesity with insulin resistance, diabetes and cardiovascular disease. While conflicting data have been reported on the contribution of subcutaneous fat, a consistent body of evidence suggests a strong association between the accumulation of intra-abdominal visceral fat and systemic inflammation2. The analysis of such population may preclude the detection of pathophysiologically relevant associations that might be clearer in more diverse populations. Another limitation of our study is that most patients were on treatment with a number of drugs, including thiazolidinediones, anti-inflammatory drugs and statins, which may have confounded our results. Indeed, the association between WNT5A expression in VAT and circulating CRP levels only reached statistical significance after exclusion of subjects on anti-inflammatory drugs. Similarly, we cannot discard that the low calorie diet and associated body weight loss that is required prior to bariatric surgery may have affected gene expression or systemic inflammation in the subjects included in our study. Overall, these limitations highlight the need for more extensive studies that include the analysis of PCP gene expression in VAT and/or SAT of individuals across different BMI groups and before and after bariatric surgery. Future studies are also warranted to evaluate the potential association between WNT5A/PCP signaling in adipose tissue and atherosclerotic CVD, given the major role that IL-6 signaling plays in this condition as revealed by large Mendelian randomization studies45.Our study is limited by its cross-sectional nature and the specific characteristics of the studied patient population, which included mostly patients with BMI ≥ 35 kg/mIn summary, the present study reveals a remarkable overactivation of non-canonical WNT5A/PCP signaling in visceral fat under obesity conditions, which contributes to the higher IL-6 production and systemic inflammation typically associated with visceral adiposity, in particular in individuals with diabetes. Future studies are warranted to evaluate the mechanisms underlying PCP signaling activation in visceral adipose tissue and the therapeutic or prognostic potential of this pathway in the setting of obesity and associated cardiometabolic disease.2, age >18 years) who were referred to undergo bariatric weight loss surgery at Boston Medical Center were recruited for this study. Subjects with unstable medical conditions such as active coronary syndromes, congestive heart failure, systemic infection, acute illness, malignancy or pregnancy were excluded from participation. Anthropometric measures and blood draws were obtained during a single baseline pre-surgical visit, which occurred within 1–2 weeks before their planned surgery and adipose tissue collection. Clinical characteristics including blood pressure, heart rate, height, weight, BMI, and waist circumference were recorded by established methods. Subjects were in a fasting state and their medications held in the morning when blood samples were drawn for biochemical analyses including glucose and insulin levels at the time of the baseline pre-surgical visit. Subjects were diagnosed with diabetes if they had fasting plasma glucose ≥126 mg/dl and/or HbA1C ≥6.5%, were taking glucose lowering medications or had history of diabetes. Study population characteristics are summarized in Supplementary Table Consecutive men and women with obesity  and stored at −80 °C. mRNA of whole adipose tissue samples was isolated using RNeasy Lipid Tissue Mini Kit (Qiagen) and RNA quality was confirmed by gel electrophoresis. Reverse transcription to cDNA was performed with High Capacity cDNA Reverse Transcription Kit (Applied Biosystems), and a pre-amplification step of selected target genes was conducted using TaqMan PreAmp Master Mix and specific TaqMan gene expression assays (Applied Biosystems). Real-time PCR was performed in a Viia7 thermal cycler (Applied Biosystems) using TaqMan gene expression assays and TaqMan Gene Expression Master Mix. Expression data for all target genes was normalized to Protein extracts from human fat samples were obtained by tissue homogenization in a TissueLyser II disruptor (Qiagen) in the presence of ice-cold lysis buffer  supplemented with protease/phosphatase inhibitors (Roche Applied Science). Commercially available ELISA kits were used to quantify relative levels of phosphorylated and total JNK1/2  and IL-6 (R&D Systems) following manufacturer’s instructions.in vitro experiments. Cells on passage 4 were treated with recombinant human recombinant WNT5A protein (R&D) or vehicle as control, and siRNA against WNT5A or scrambled siRNA as control . RNA from cells was isolated using RNeasy Micro Kit (Qiagen). cDNA synthesis and RT-PCR to asses WNT5A and IL6 mRNA levels were performed as described above (without pre-amplification).Preadipocytes isolated from subcutaneous and omental adipose tissue biopsies obtained from individuals with obesity during bariatric surgery were purchased from the Boston Nutrition and Obesity Research Center (BNORC). Cells isolated from 5 patients were used for the t tests. Pearson’s correlation coefficients (r) were used to analyze the association between variables. A two-way ANOVA was used for the rWNT5A in vitro study. All statistical tests were performed using GraphPad Prism software (GraphPad Software Inc.).Data are shown as mean ± SEM unless otherwise stated. Statistical significance of differences between gene expression in omental and subcutaneous fat were assessed by paired Student’s The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.Supplementary Information


GlyT2GFP and GAD67GFP mice showed the majority of inhibitory LDPNs project either ipsilaterally or adjacent to the midline. Additionally, we used several transgenic mouse lines to define the developmental provenance of LDPNs and found that V2b positive neurons form a subset of ipsilaterally projecting LDPNs. Finally, a population of Neurobiotin (NB) labelled LDPNs were assessed in detail to examine morphology and plot the spatial distribution of contacts from a variety of neurochemically distinct axon terminals. These results provide important baseline data in mice for future work on their role in locomotion and recovery from SCI.Long descending propriospinal neurons (LDPNs) are interneurons that form direct connections between cervical and lumbar spinal circuits. LDPNs are involved in interlimb coordination and are important mediators of functional recovery after spinal cord injury (SCI). Much of what we know about LDPNs comes from a range of species, however, the increased use of transgenic mouse lines to better define neuronal populations calls for a more complete characterisation of LDPNs in mice. In this study, we examined the cell body location, inhibitory neurotransmitter phenotype, developmental provenance, morphology and synaptic inputs of mouse LDPNs throughout the cervical and upper thoracic spinal cord. LDPNs were retrogradely labelled from the lumbar spinal cord to map cell body locations throughout the cervical and upper thoracic segments. Ipsilateral LDPNs were distributed throughout the dorsal, intermediate and ventral grey matter as well as the lateral spinal nucleus and lateral cervical nucleus. In contrast, contralateral LDPNs were more densely concentrated in the ventromedial grey matter. Retrograde labelling in  The propriospinal system is comprised of spinal interneurons with longitudinal axonal projections that extend outside their segment of origin, forming a network that connects motor and sensory circuits throughout the length of the spinal cord . This intraspinal network is important for the propagation of supraspinal signals , is currently the subject of a similar anatomical and molecular investigation  and transgenic mice were anaesthetised with isoflurane  and a T12 laminectomy was performed to expose the L2 spinal segment. The overlying dura mater was cleared and an injection pipette filled with 4% (w/v) Fluorogold dissolved in normal saline  was advanced to a depth of 0.75 mm from the surface of the spinal cord, 0.4 mm lateral to the midline Figure . FluorogCervical and upper thoracic segments were sectioned at 40 μm in the transverse plane on a freezing sledge-microtome. Every fifth section was processed with anti-Fluorogold antibody to reveal retrograde-labelled LDPNs, and tissues expressing transgenic GFP/YFP were additionally treated with anti-GFP antibody (see Table To map the location of Fluorogold labelled LDPNs in wild-type (ICR) and transgenic mice, photomicrographs of immunostained sections between C2 to T3 were imported into Adobe Illustrator CS4  and superimposed on transverse spinal cord slice templates , were matched to each section containing LDPN cell bodies. Each square within the grid was scored based on the number of LDPN cell bodies it contained. The individual squares within each spinal segment grid were then coloured based on their score using a conditional formatting function . The colour-coded grids were overlaid onto the original spinal cord templates in Adobe Illustrator.First, LDPNs were retrogradely labelled in wild-type (C57BL6) mice with 2.5% (w/v) DiI dissolved in DMSO using the same surgical procedure described above for Fluorogold injections. DiI was used instead of Fluorogold to enable easier identification of LDPNs as it has brighter fluorescence in thick spinal cord slices. Targeted whole-cell patch-clamp NB fills were carried out in fresh transverse spinal cord slices (250 μm thick) that were maintained in oxygenated artificial cerebrospinal fluid  to label NB-filled neurons. The entire somatodendritic arbour of recovered neurons was then scanned on a Bio-Rad Radiance 2100 confocal microscope  with a krypton–argon laser at 20× magnification. This laser exposure during this initial scanning step potentially quenched some of the fluorescence. Therefore, maximal labelling for subsequent high-power scanning was achieved by re-sectioning slices at 60 μm and re-incubating these sections in Avidin-rhodamine to increase label penetration and replenish the potentially quenched flurophore in LDPN processes. To label putative synaptic inputs onto the filled neurons, selected sections containing the rhodamine-labelled neurons were incubated in a cocktail of primary antibodies against vesicular GABA transporter (VGAT), vesicular glutamate transporter type 1 (VGLUT1) and parvalbumin (PV) for 48 h at 4°C . The precise location of direct appositions from four types of axon terminals were then plotted on the reconstructed neurons, based on their relative expression patterns for VGAT, VGLUT1 and PV, and assigned to one of four potential synaptic input populations. Terminals expressing VGAT were considered segmental inhibitory interneurons . A Sholl analysis, also carried out using Neurolucida Explorer 10 software, was performed to determine the number of terminals expressing VGAT, VGLUT1 and PV that apposed the NB labelled neurons on different segments of their somatodendritic arbour. The first shell was set at 10 μm and any contacts found within this were deemed to be on the cell body. All subsequent shells were spaced at 20 μm intervals.t-test when data sets were normally distributed (assessed by the Kolmogorov-Smirnov test) and had equal variance (SD of both groups differed by less than a factor of 2). In cases where data sets were normally distributed but had unequal variances, Welch’s t-test was applied. Non-normally distributed data sets were compared with the Mann-Whitney U test. All statistical analysis was performed with GraphPad Prism 6 software . Statistical significance was set at p < 0.05. Values are presented as mean ± SEM unless otherwise noted.Group comparisons were made with an unpaired Student’s GlyT2GFP (glycinergic) and GAD67GFP (GABAergic) mice. LDPNs that express markers of developmentally defined interneurons were quantified in En1Cre;Thy1floxstop−YFP (V1), Gata3Cre;Thy1floxstop−YFP (V2b) and Sim1Cre;Thy1floxstop−YFP (V3).The cell body locations of lumbar projecting LDPNs were mapped throughout the cervical and upper thoracic spinal cord in wild-type (ICR) mice. Inhibitory LDPNs were mapped in Injection sites were located within the L2 spinal segment (right side of cord) and confined within the borders of the grey matter Figure . An injeThe cell bodies of lumbar projecting LDPNs were mapped between C2 and T3 spinal segments. Retrogradely-labelled LDPNs were found both ipsilateral and contralateral to the lumbar injection site Figures . A similIpsilateral LDPNs were distributed widely throughout the grey matter, including the deep dorsal horn , lamina VII (11.4 ± 0.9%) and lamina VIII (7.0 ± 1.5%). Retrogradely-labelled LDPNs were also found in the ipsilateral lateral spinal nucleus and lateral cervical nucleus . LDPNs were compared with control neurons  from the same region (n = 4). All NB-filled neurons were from wild-type mice (C57BL6) and located within spinal segments C5 to T1. LDPN and control neurons had relatively simple, radiating dendritic arbours that extended throughout the intermediate zone and ventral horn . The number of dendritic branch points in LDPNs and control neurons ranged from 7 to 25 and 12–42, respectively, and these values were also similar for each population . Immunohistochemical labelling of excitatory and inhibitory terminals in close apposition to this population of LDPNs showed a diverse range of convergent inputs including putative premotor inhibitory interneurons, corticospinal fibres and proprioceptive afferents.This study characterised the cell body location, inhibitory neurotransmitter profile, developmental genetics, morphology and likely synaptic inputs of LDPNs in mice. LDPNs were found both ipsilateral and contralateral to the lumbar injection site in approximately even numbers but were localised to different laminae on each side. Ipsilaterally projecting LDPNs were spread diffusely throughout the deep dorsal horn, intermediate zone and ventral horn, with a dense population in the LSN/LCN. In contrast, contralaterally projecting LDPNs are concentrated within the ventromedial quadrant of the ventral horn. Another population was also identified in the midline, located in lamina IV and the dorsal part of lamina X. Inhibitory LDPNs, identified by co-localisation of Fluorogold and GFP in ascending propriospinal neurons (LAPNs) inhibits conventional interlimb coordination in mice , which is further compounded by the well-established co-expression of glycine and GABA  and V2c (Sox1 positive) interneurons , inhibitory premotor interneurons such as Renshaw cells and group Ia inhibitory interneurons (VGAT/PV), excitatory corticospinal or myelinated afferent (VGLUT1) and proprioceptive afferent input from the central terminals of group Ia muscle afferents (VGLUT1/PV). While we are unable to confirm the origin of the various axon terminals apposed to the LDPN or unlabelled comparison group in our study, recent work has confirmed the presence of corticospinal, descending 5-HT and intraspinal inputs onto LDPNs  spinal neurons located in lamina V , the National Institutes of Health (NS090919 to MDG), the Hunter Medical Research Institute  and the Biotechnology and Biological Sciences Research Council .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A Receptors (GABAARs) at the nanometric scale during the induction of inhibitory long-term potentiation (iLTP). Additional electron tomography (ET) experiments on immunolabelled hippocampal neurons allowed the visualization of synaptic contacts and confirmed the reorganization of post-synaptic GABAAR clusters in response to chemical iLTP inducing protocol. Altogether, these approaches revealed that, following the induction of inhibitory synaptic potentiation, GABAAR clusters increase in size and number at the post-synaptic membrane with no other major structural changes of the pre- and post-synaptic elements.Both excitatory and inhibitory synaptic contacts display activity dependent dynamic changes in their efficacy that are globally termed synaptic plasticity. Although the molecular mechanisms underlying glutamatergic synaptic plasticity have been extensively investigated and described, those responsible for inhibitory synaptic plasticity are only beginning to be unveiled. In this framework, the ultrastructural changes of the inhibitory synapses during plasticity have been poorly investigated. Here we combined confocal fluorescence microscopy (CFM) with high resolution scanning electron microscopy (HRSEM) to characterize the fine structural rearrangements of post-synaptic GABA While most of the works of the last three decades have mainly focused on excitatory synaptic plasticity, the plasticity of inhibitory connections is far less understood. More recently, it has been demonstrated that inhibitory synapses express several forms of plasticity, indicating that plastic changes of both excitatory and inhibitory synapses play a crucial role in setting the Excitation/Inhibition balance (E/I), a major determinant of network functioning. At inhibitory synapses, several forms of synaptic plasticity have been comprehensively characterized at the pre-synaptic level. Indeed, retrograde messengers released from post-synaptic side  and nitric oxide (NO)) diffuse back to the presynaptic element, modulating the release machinery and regulating the amount of GABA released in the synaptic cleft3. In contrast, at the post-synaptic level, the molecular mechanisms responsible for the activity-dependent tuning of inhibitory synaptic responses are only beginning to be revealed7. An increasing body of evidence has demonstrated that phosphorylation of the GABAARs by the calcium dependent kinase CaMKII is a key determinant for the changes in GABAARs functioning8 and localization at synapses9. Along the same lines, diverse post-translational modifications of the inhibitory scaffold protein gephyrin modulate the aggregation state of synaptic gephyrin, thus regulating the GABAAR anchoring/clustering at synaptic sites12. Previous work by our group showed that, during potentiation of inhibitory synapses, gephyrin is redistributed from the extrasynaptic to the synaptic area upon CaMKII phosphorylation of GABAAR with consequent immobilization and accumulation of GABAAR at synaptic sites14.One of the most distinctive features of neuronal networks is their ability to undergo activity dependent plastic changes that modify synaptic strengthmilieu, dendritic spines play a pivotal role in the synapse functioning both in basal conditions and during synaptic plasticity15. Indeed, activity-dependent structural remodelling of dendritic spines has been observed during synaptic plasticity by super-resolution fluorescence microscopy and electron microscopy19. While a large number of studies addressed the structural plasticity of glutamatergic spines, the fine changes in the morphology of GABAergic synapses during synaptic plasticity have been far less characterized. Inhibitory synapses do not have a clear post-synaptic density and their structural remodelling has not been studied as thoroughly as that of excitatory post-synaptic sites. Nevertheless a proteomic study has recently identified a far more complex protein network at inhibitory postsynaptic sites than previously thought20. The aim of the present work is to reveal at nanometric resolution the modifications of inhibitory GABAergic synapses following the expression of a post-synaptic form of inhibitory long-term potentiation (iLTP). In particular, by correlative light-high resolution scanning electron microscopy (CL-HRSEM) and electron tomography (ET) we examined the degree of synaptic clustering of surface GABAARs and the properties of the pre- and post-synaptic membranes before and after the delivery of the plasticity-inducing protocol. In detail we combined confocal fluorescence microscopy (CFM) with high resolution scanning electron microscopy (HRSEM) on the same region of interest33 to immuno-localize GABAARs epitopes at the surface of the neuronal membrane, and we performed electron tomography (ET) to explore the intracellular ultrastructure in 3D at nanometric scale38.Differently from inhibitory synapses, which are defined symmetric, asymmetric excitatory synapses are located on dendritic varicosities called “spines”. By partially segregating the post-synaptic ARs at nanometre scale, neurons grown on glass coverslips with a photo-etched finder grid were labelled with primary antibodies directed against the GABAAR α1 subunit (GABAARα1) and with secondary antibodies conjugated with FluoroNanogoldTM  after iLTP induction or a control treatment. The fluorescence of the FluoroNanogoldTM was first imaged by confocal microscopy (CFM) in order to get a first map of GABAAR clusters and localize neurons of interest on the gridded coverslips, with the typical CFM lateral resolution. Samples were then processed for HRSEM after a gold enhancement reaction to increase the size of gold nanoseeds . The images of the detected backscattered electron (BSE) and the secondary electron (SE) signals were then simultaneously acquired in order to localize gold nanoparticles,  while imaging the fine surface morphology by SE signal. In another set of experiments we performed ET on hippocampal neurons immuno-labelled for GABAARα1. This allowed us to precisely localize gold particle clusters on post-synaptic membranes facing GABAergic presynaptic boutons in control conditions and during iLTP. The aforementioned approaches revealed an increased clustering of GABAARα1 receptors at GABAergic synapses upon the induction of inhibitory synaptic potentiation. Importantly, the expression of such inhibitory plasticity occurred in the absence of major structural rearrangements of the pre- and post-synaptic membranes. Those results indicate that, upon the conditions examined here, the expression of inhibitory synaptic potentiation does not require structural plasticity, leaving the modulation of GABAARα1 receptor trafficking and lateral diffusion as the main determinants of iLTP, differently from what happens in excitatory synapses.To acquire information on the distribution of GABAAR α1 subunit (GABAARα1) primary antibody followed FluoroNanogoldTM Fab secondary antibody revealed the presence of GABAARα1 gold clusters of variable size (due to gold-enhancement reaction) on both soma and neurites  was revealed with a 10 nm colloidal gold particle-conjugated secondary antibody, instead of the FluoroNanogoldTM , their size (average number of gold particles/cluster) and the number of clusters formed respectively by n ≤ 5 and n > 5 gold particles. In agreement with the CFM results, following plasticity induction with NMDA treatment, the average density of gold clusters significantly increased as compared to controls . In the NMDA treated neurons we measured a general increase in the number of gold clusters belonging to each class with respect to controls, especially the 2nd class (>5 gold particles) that nearly tripled  imaging was conducted to obtain a map of GABAons Fig. . In lineane Fig. . As a sisee Fig.  for NMDAsee Fig. . Along tsee Fig. . In othesee Fig. . Only insee Fig. . Next, tARα1 signal specifically at inhibitory synapses we performed ET on primary cultured hippocampal neurons immunolabelled for GABAARα1 using FluoroNanogoldTM Fab secondary antibody followed by gold enhancement. In both controls and NMDA-treated neurons we observed the presence of gold clusters along the post-synaptic membrane facing the pre-synaptic terminal of symmetrical synapses , several recent papers have shown the feasibility of CL-HRSEM by proposing new probes32, new substrates for growing cells40 and new coating strategies to provide electrical conductivity42. Through the years, immuno-SEM has been used to localize at the nanometre scale the spatial distribution of a variety of surface molecular targets, ranging from phosphorylated histone H330, cell adhesion molecules47, bacterial and cancer surface50, collagen fibrils51, and cell focal adhesion sites21 in a variety of organelles, cell types and tissues. In particular HRSEM in combination with fluorescence microscopy has been used to study a variety of molecular targets on the surface of a multiplicity of cells and tissue types. Two main approaches have been applied: i) the use of exclusively fluorescent probes to mark molecules of interest, and then the superimposition of the fluorescence images with the corresponding high resolution SE image54 or ii) the use of dual tagged probes56 or of a mixture of fluorescent and electron-dense probes28 to visualize the molecular tags at the HRSEM using BSE, after the overlay of the fluorescent images with the corresponding SE images.By coupling fluorescence microscopy with the high-resolution power of HRSEM, correlative light - high resolution scanning electron microscopy (CL-HRSEM) is an excellent method to study the spatial distribution of target molecules at the cellular surface at nanometric levelARα1 fluorescent labelling we observed on neuron cell bodies and neurites showed a punctate pattern57. The same distribution was observed at the HRSEM as intense BSE bright spots. GABAARα1 clusters were mainly located at GABAergic symmetrical synapses, although a sizable amount of receptor immunolabelling was detected at extrasynaptic sites, i.e. dendritic regions that were not coupled to axonal counterparts58. At synapses, GABAARα1 immunoreactivity was exclusively post-synaptic59. The aggregation of GABAARα1 receptors at symmetric synapses is consistent with their role in fast inhibitory synaptic transmission. In contrast, the GABAARα1 receptors observed at extrasynaptic areas are those that dynamically exchange between synapses and exocytosis/endocytosis sites7, and may contribute to tonic inhibitory current58. The evidence that during iLTP we observed increased GABAA R clustering without major remodelling of the pre- and post-synaptic membranes indicates that inhibitory synaptic potentiation does not require structural plasticity. Hence, the mechanisms underlying iLTP fundamentally differ from those of excitatory LTP, which imply profound modifications of the post-synaptic membrane at spines60. These data are in line with recent studies reporting that the synaptic cluster of gephyrin, the main GABAAR anchoring protein at inhibitory synapses, showed increased size and complexity following iLTP14. Thus, we emphasize here that during inhibitory synaptic plasticity, the redistribution of postsynaptic proteins at post-synaptic sites is the main molecular event in the potentiation of GABAergic synapses. The correlative labelling protocol described here could be also applied for wet environmental scanning electron microscopy (wet-ESEM) imaging61. This might reduce the spatial resolution as compared to high-vacuum techniques, but would allow investigating the dynamics of receptor redistribution.As expected for proteins enriched at synaptic sites, the GABAAll experiments were carried out in accordance with the guidelines established by the European Communities Council (Directive 2010/63/EU of 22 September 2010) were permitted by the Italian Ministry of Health and followed the rules approved by the Italian Institute of Technology. All animal surgeries were done in agreement with the Italian Ministry of Health Regulation and Authorization and have been approved by the Italian Institute of Technology.14. Neurons were plated on coverslips with a photo-etched counting grid (BELLCO GLASS INC.) and kept in Neurobasal medium in incubator at 37 °C with 5% CO2. To induce inhibitory synaptic potentiation, at 13–16 days in vitro (DIV) neurons were treated for 2 minutes with NMDA 20 μM  and CNQX 10 μM  dissolved in an extracellular recording solution  and then washed and incubated in the extracellular solution to allow recovery and expression of iLTP14. During the recovery period, 12 minutes after the end of the NMDA treatment, live immunolabelling of GABAARα1 subunits was performed, namely cells were incubated 13 minutes in a solution containing primary antibody against GABAARα1  diluted 1:30 in 0.5% bovine serum albumin (BSA), 350 mM sucrose in PBS, followed by 13 minutes incubation with gold-conjugated secondary antibody. A first set of experiments was performed using secondary antibody conjugated to 10 nm gold particles . To reduce the size of our probe and to directly correlate CFM and electron microscopy we then used FluoroNanogoldTM  since it exhibits both an Alexa 488 fluorochrome and a colloidal nanogold particle (1.4 nm) conjugated to an IgG Fab fragment. In control samples, NMDA and CNQX were omitted. To check for non-specific binding of the secondary antibodies, in control experiments the primary antibody was omitted.Primary cultures of hippocampal neurons were prepared from C57BL/6 J mice at postnatal day 0 (P0) as previously describedARα1 clusters. Subsequently, the same samples were prepared for SEM analysis. Cells were fixed for 1 hour in 1.2% glutaraldehyde in PBS followed by enhancement of the FluoroNanogoldTM particles size with a gold enhancement kit  until reaching a final size of about 10 nm to allow their detection by backscattered electrons (BSE) signal in HRSEM. Neurons were then post-fixed in 1% osmium tetroxide in 0.1 M sodium cacodylate (pH 7.4), dehydrated in a graded series of cold ethanol followed by complete dehydration in hexamethyldesilanzane (HMDS). Complete evaporation of HMDS was obtained overnight and the following days samples were sputter-coated with a thin (5 nm) layer of chromium to allow surface conductivity. Samples were then transferred in the SEM right after the sputter coating to limit the fast chromium oxidation. HRSEM imaging was performed by a JEOL JSM 7500 F microscope equipped with a cold field emission gun and working at an acceleration voltage of 15 kV. In-lens detector and in-chamber multi-quadrant annular retractable solid state detector collected the secondary electrons and backscattered electrons signals, respectively. Both SE and BSE signals were simultaneously recorded, keeping the samples at a fixed working distance of 8 mm.Live confocal and transmitted fluorescence images where acquired with a Nikon Live scan SFC inverted microscope at various magnifications . Lower magnifications were used to localize neurons on the reference photo-etched grid, while the 60x objective was used to acquire fluorescence images of GABAARα1 (see above) were then fixed for 1 hr at room temperature in 1.2% glutaraldehyde in 66 mM sodium cacodylate buffer (pH 7.4) and processed for conventional resin embedding as described by Giacomini and colleagues36. Semi-thin sections of about 350 nm of the embedded cell monolayer were cut with an ultramicrotome  equipped with a diamond knife  and collected on Formvar/carbon coated copper slot grids. Electron tomography was performed in scanning transmission electron microscopy (STEM) working in high angular annular dark field (HAADF) geometry, using a FEI Tecnai F20 transmission electron microscope operating at 200 kV and equipped with a Schottky field-emission gun. Such geometry was chosen in order to rule out any diffraction contrast contribution to the collected images. The tomographic series where collected from 350 nm thick sections tilted over ± 60 degrees with the following tilt scheme: 1 degree angular step for tilt angle higher/lower than ± 30 degrees, and 2 degrees angular step between ± 30 degrees. The images were acquired with magnification ranging from 40.000 and 56.000, corresponding to a pixel size comprised between 2.59 and 1.85 nm respectively. Computation of tomograms was done by weighted back projection (WBP)62 with IMOD 4.8 software package63. Both segmentation and 3D visualization were performed using the Amira package .For ET 3D imaging neurons that were live-labelled for GABA64. The gold clusters density has been calculated using the “analyse particles” function of ImageJ on a total of 54 HRSEM BSE and SE images of iLTP and control samples . The clusters size (number of gold particles/cluster) has been calculated on a total of 2244 randomly picked gold clusters from HRSEM images coming from different experiments . Moreover more than 4800 randomly picked gold clusters  were arbitrary subdivided in classes formed by n ≤ 5 and n > 5 gold particles. The analysis on the tomographic 3D models of inhibitory synapses in both control and iLTP hippocampal neurons have been performed using the Amira package . The post-synaptic terminal surface and the number and volume of gold clusters at the inhibitory synapse have been extracted using the MaterialStatistics module as implemented in Amira from a total of 26 tomographic reconstructions from four different immuno-EM experiments . To analyse CLM puncta which did not show nanogold clusters at the SEM level, we correlated CLM and HRSEM areas with the help of the finder photo-etched grid and clusters were manually quantified using ImageJ 1.50i64. Statistical significance of normally distributed datasets was tested using a two tailed Student’s t-test. A p < 0.05 level was considered as statistically significant; to test for normality the Kolmogorov–Smirnov was performed prior to statistical analysis.Image analysis on HRSEM data has been performed using ImageJ 1.50iSupplementary InformationMovie S1Movie S2


Secretagogin was not released in parallel with glucose stimulated insulin release, but was markedly elevated in response to endoplasmic reticulum stressors and cytokines. These findings indicate that secretagogin is a potential novel biomarker, reflecting stress and islet cell dysfunction in T2D patients.Beta cell dysfunction accompanies and drives the progression of type 2 diabetes mellitus (T2D), but there are few clinical biomarkers available to assess islet cell stress in humans. Secretagogin, a protein enriched in pancreatic islets, demonstrates protective effects on beta cell function in animals. However, its potential as a circulating biomarker released from human beta cells and islets has not been studied. In this study primary human islets, beta cells and plasma samples were used to explore secretion and expression of secretagogin in relation to the T2D pathology. Secretagogin was abundantly and specifically expressed and secreted from human islets. Furthermore, T2D patients had an elevated plasma level of secretagogin compared with matched healthy controls, which was confirmed in plasma of diabetic mice transplanted with human islets. Additionally, the plasma secretagogin level of the human cohort had an inverse correlation to clinical assessments of beta cell function. To explore the mechanism of secretagogin release  Type 2 diabetes mellitus (T2D) is the most common form of diabetes ,2. One o1c, and biomarkers of beta cell capacity for insulin release, such as insulin and C-peptide. However, these biomarkers do not provide a comprehensive picture of the status of islet cells, for instance overall mass, viability or ability to cope with micro-environmental stressors at the islet level ) were used. The pro-inflammatory stress mechanism was mimicked by treatment using the combination of IFN-γ, IL1-β and TNF-α as a cytokine cocktail. These in vitro studies showed that:To investigate whether hyperglycemia and/or induced islet stress resulted in increased secretagogin release the pattern of its release was assessed Secretagogin was released in response to both ER stress and cytokine stimuli in human beta cells. The siRNA knockdown of intracellular secretagogin increased apoptosis of EndoC cells only in response to ER stress inducers.Secretagogin was not released in parallel with insulin and not in response to hyperglycemic conditions. The intracellular level of secretagogin was slightly upregulated by high glucose level in EndoC cells.2+ sensor and not a buffering protein [2+ and redox state secretagogin may have different characteristics [2+ sensor, while in an oxidizing milieu (such as in ER and under oxidative stress conditions) secretagogin may act as a Ca2+ buffer [2+ in the ER can severely impact protein folding capacity, causing UPR and triggering cell death [2+ fluctuations and promote survival. These results are also in line with a recent finding that intracellular secretagogin promotes pancreatic beta cell survival and decreases ER stress by stabilizing deubiquitinating proteins [Our findings that intracellular secretagogin protected beta cells from apoptosis is supported by recent findings in cancer cells by Bai and colleagues  which sh protein . Howevereristics . In the + buffer . Fluctuall death . Thus, iproteins .Secretagogin has previously been suggested to be involved in beta cell homeostasis by protecting against IL-1β-mediated beta cell destruction in diabetes-prone biobreeding rat islets . These rPrevious studies based on rodent models, or rodent beta cell lines ,24,27 haThe fact that secretagogin release was not fluctuating in relation to insulin release is an important feature of a novel biomarker. It could enable secretagogin to reflect a certain disease condition that cannot be captured by insulin, C-peptide or proinsulin levels. A suggestion may be that secretagogin may function as a stress responsive endocrine or paracrine factor, released in response to increased cell stress. But further studies on effects of released secretagogin on surrounding cells are needed to test this hypothesis.In conclusion, our findings suggest that released secretagogin is a candidate plasma biomarker of islet cell stress with independent value compared to insulin. To further validate secretagogin as a biomarker for islet stress, studies in larger cohorts are warranted.S1 FigHigh resolution immunohistofluorescence staining for secretagogin (showed in red), insulin (showed in green) and glucagon (showed in turquoise), and nucleus counterstained with DAPI (pseudo-colored in white). In the left image secretagogin is visualized together with the nuclear counterstaining. In the middle image, the insulin staining is added to the secretagogin and nuclear staining. In the right-hand image, secretagogin, insulin and nuclear staining is visualized together with the staining for glucagon.(TIF)Click here for additional data file.S2 FigFour pre-designed  silencing RNA oligos of secretagogin (siSCGN) as well as a pool of #4-#6 were assessed for their effect on mRNA silencing of secretagogin expression in EndoC cells as compared with the expression of a scrambled siRNA (Scr siRNA) as negative control.(TIF)Click here for additional data file.S3 FigIntracellular CCAAT-enhancer-binding protein homologous protein (CHOP) expression was assessed using western blotting, analyzing 10μg total protein per well of EndoC cells treated with stress induction by either tunicamycin, thapsigargin or cytokine cocktail  for 24h. All substances were dissolved in DMSO (1:1000) and control cells were incubated in DMSO (1:1000).(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S1 Material and Methods(DOCX)Click here for additional data file.