BRAF mutations in the papillary variant, offering promising options for targeted pharmacological treatment. The involvement of β-catenin and the Wnt pathway in the tumorigenesis of the adamantinomatous subtype has been previously established with the identification of stabilising mutations in exon 3 ofCTNNB1. Further understanding of the pathogenesis of this subtype has been facilitated with the use of mouse models and xenograft experiments. It has been proposed that the clusters of cells with upregulated Wnt/β-catenin signalling induce tumour formation in a paracrine manner; the complex interactions occurring between different cell populations need to be further clarified for further expansion of this hypothesis. This review outlines recent key advances in our understanding of the molecular pathology of craniopharyngiomas and discusses some of the challenges that need to be overcome for the development of targeted therapies that will hopefully improve the management and the outcomes of these patients.Craniopharyngiomas are rare epithelial tumours arising along the path of the craniopharyngeal duct. Two major histological subtypes have been recognised, the papillary and the adamantinomatous. Craniopharyngiomas remain challenging tumours to manage and are associated with significant morbidities and mortality. Recent advances in the molecular pathology of these neoplasms have identified They show a bimodal age distribution with peak incidence rates in children aged 5–14 and adults aged 50–745. No gender differences have been reported4.Craniopharyngiomas (CPs) are epithelial tumours (WHO grade I) arising along the path of the craniopharyngeal duct . Recent epidemiological data suggest incidence rates of 0.17–0.2 cases per 100,000 people6. The pCP has almost exclusively been described in adults (14–50% of adult cases and only up to 2% in children). Macroscopically, it tends to be solid or mixed with cystic and solid components, calcification is rare, and the cyst content is usually viscous and yellow. It is generally well circumscribed, and infiltration of adjacent brain tissue by neoplastic epithelium is less frequent than in aCP or even absent. Microscopically, it is composed of mature squamous epithelium forming pseudopapillae and of an anastomosing fibrovascular stroma without the presence of peripheral palisading of cells or stellate reticulum6.Histologically, two primary subtypes have been recognised, the adamantinomatous (aCP) and the papillary (pCP). The aCP is the most common and macroscopically shows cystic and/or solid components, necrotic debris, fibrous tissue, and calcification . The margins of aCP are sharp and irregular, often making the identification of the surgical planes difficult. The cytoarchitecture of aCP comprises a palisaded basal layer of small cells; above this, there is an intermediate layer of variable thickness composed of loose aggregates of stellate cells (termed “stellate reticulum”) and a top layer facing into the cyst lumen with abruptly enlarged, flattened, and keratinised flat plate-like squamous cells. The flat squames are desquamated singly or in distinctive stacked clusters, forming nodules of wet keratin, often heavily calcified and apparent grossly as white flecks7. Furthermore, these tumours are associated with significant long-term morbidity  and mortality, attributed to the damage of critical structures by the primary or recurrent tumour and/or to the adverse effects of therapeutic interventions11.The optimal management of CPs remains challenging, and the main options include surgery combined or not with radiotherapy; nonetheless, they can show aggressive and unpredictable behaviour with recurrence(s) difficult to treatBetter understanding of the molecular pathology of CPs is of major importance for the development of targeted therapies aiming to improve the outcomes of these patients. In recent years, there have been significant advances in this field, and we summarise them in this brief review.CTNNB1 that prevent phosphorylation and degradation of β-catenin have been identified in aCPs12. However, until recently, the genetic event underlying the development of pCP had remained unclear. Several studies employing whole exome sequencing, next-generation panel sequencing, pyrosequencing, and Sanger sequencing have demonstrated the presence of activating mutations inBRAF (V600E) in pCPs. Their reported prevalence varies according to the sequencing method used and ranges from 81 to 100%16. Extensive analysis of aCPs using the same methods has not identified recurrent mutations other than those in exon 3 ofCTNNB115. The lack of genetic complexity seen in these tumours is typical of their benign behaviour, and the almost-perfect segregation of mutation with tumour variant16 coupled with the existence of aBRAF V600E mutation-specific antibody (VE1) provides a useful diagnostic tool for specimens with scant epithelium or where diagnosis is challenging; it should be noted that there is some concern about the specificity of this antibody in pituitary tissue, necessitating cautious interpretation19. Much more promising, however, is the possibility for targeted pharmacological treatment with agents directed against theBRAF (V600E) mutation.The involvement of β-catenin and the Wnt pathway in the tumorigenesis of aCP has been established, since stabilising mutations in exon 3 ofBRAF (V600E) mutation has opened up the exciting possibility of repurposing existing drugs for the treatment of pCP cases refractory to surgery and radiotherapy. Pharmacological agents that specifically target and inhibit mutantBRAF (V600E) are very effective in malignant tumours positive for this mutation. Although the development of resistance to these agents is a potential drawback, the addition of other inhibitors of the MAPK pathway  can increase their efficacy20 and reduce the risk of cutaneous squamous-cell carcinoma, a common complication of BRAF inhibitor treatment.The finding that most pCPs harbour a21 described a patient with progressive visual deterioration due to a recurrent pCP previously managed with three surgeries and fractionated radiotherapy. The tumour harbouredBRAF (V600E) mutation and the patient was treated with vemurafenib. Stabilisation of vision was achieved within two weeks, along with a dramatic reduction in tumour size. Near-complete radiological remission occurred after three months, but the patient developed cerebrospinal fluid leak, pneumocephalus, and meningitis, necessitating antimicrobial therapy and surgical repair. Treatment was interrupted after three months, but the CP recurred within six weeks and vemurafenib was restarted. Tumour growth was stabilised until seven months after treatment initiation when progressive regrowth was detected. The second report from Brastianos and colleagues22 described a patient with multiply recurrent pCP withBRAF (V600E mutation). Four surgical decompressions were unsuccessful at controlling the growth of the tumour, which had a large cystic component. The patient suffered bilateral optic neuropathy and panhypopituitarism. Treatment was initially dabrafenib monotherapy, and after 17 days the solid and cystic components decreased by 50% and 70%, respectively. At day 21, the MEK inhibitor trametinib was added for a further 14 days to reduce the likelihood of resistance to BRAF inhibition. This regime led to solid and cystic components of the tumour decreasing in total by 85% and 81%, respectively. Subsequently, the patient underwent endoscopic transsphenoidal resection followed by radiotherapy and has remained symptom free 18 months after radiation.Two recent reports have shown significant reduction in tumour volume in cases of treatment-refractory pCP. Aylwin and colleaguesBoth cases illustrate the promising potential for the use of BRAF (V600E) inhibitors and remain to be validated with clinical trials.15. Additionally, unsupervised consensus clustering using the most variably expressed genes identified by microarray analysis resulted in clear separation of the two subtypes, indicating that they likely have distinct gene expression signatures23. Analysis of mRNA expression demonstrated up-regulation of Wnt/β-catenin pathway targets in aCP (LEF1 andAXIN2) and also components of the hedgehog signalling pathway . Additionally, the stem cell markerPROM1 (encoding CD133) was overexpressed. These findings are in keeping with previous published data from the same group that suggest the activation of Wnt and sonic hedgehog (SHH) pathways in the cell clusters of aCP (see below)28. For a detailed discussion of the opportunities for therapy targeting these upregulated pathways, seeAs well as their genetic differences, CP variants have been shown to have different epigenomic and transcriptomic signatures. Analysis of the most variably methylated CpG sites by both unsupervised hierarchical clustering and principal component analysis identified two distinct methylation clusters that separated aCP and pCP15.Interestingly, neither the methylation nor the mRNA expression signatures of paediatric aCP and adult aCP separated when analysed as described above, suggesting that they may not be distinct at the epigenomic or transcriptional level35. The function of these cluster cells remains enigmatic, but they represent a population strikingly different from the bulk of the tumour.The cells that accumulate β-catenin in aCP are small in number and often, but not always, accumulate to form small clusters with a whorl-like pattern near the infiltrating edges of the tumourCTNNB1 mutation in Rathke’s pouch progenitor cells is sufficient to drive aCP tumorigenesis. Expression ofCTNNB1 lacking exon 3 (degradation-resistant β-catenin) under the control of Hesx1 in the early stages of mouse pituitary development results in the formation of tumours that closely resemble human aCP, although they lack some of the regressive changes seen in the human tumour, e.g. wet keratin. Mutation of β-catenin in undifferentiated Rathke’s pouch progenitors is sufficient to drive tumorigenesis of aCP, but once lineage commitment has occurred, the mutation is no longer tumorigenic36. Interestingly, althoughCTNNB1 mutation was present in the whole population of progenitor cells, there was only cytosolic and/or nuclear accumulation of β-catenin in the characteristic cell clusters near the invading tumour edge36, indicating that an event other thanCTNNB1 mutation determined which cells formed clusters.A mouse model of aCP demonstrated thatet al. harvested cluster cells from human aCP using laser capture microdissection and sought to determine whether they harboured an activating mutation in exon 3 of the β-catenin gene and whether the Wnt pathway was activated by quantifyingAxin2 andBMP4. The cluster cells expressed mutatedCTNNB1, along with the surrounding tumour cells, but only the cluster cells expressed elevated levels of Axin2 and BMP4 RNA and protein. The authors concluded thatCTNNB1 mutation is not sufficient to drive nuclear β-catenin accumulation26. These cluster cells were also found to expressFascin, a Wnt target gene with a role in cytoskeletal organisation and cell migration26. Downregulation of both β-catenin and fascin expression in human primary aCP tumour cells using siRNA impaired their motility and migrational capacity24. The study did not separate cells derived from different features of the aCP prior to culture, so the culture was likely to contain non-cluster cells as well as the cluster cells the authors sought to characterise. Mutation inCTNNB1 was present in 66% of aCPs, but its effect on the behaviour of the cells was not explored24. However, the observation that siRNA-mediated downregulation of β-catenin or fascin impairs motility in primary human aCP cells suggests a potential role for β-catenin, fascin, and (by inference) the cluster cells in the invasion into surrounding brain. Additionally, the same group demonstrated that claudin, a tight-junction component, was expressed in the finger-like protrusions of aCP but was absent from the cell clusters; this pattern of expression is consistent with a migratory phenotype for the cluster cells. Furthermore, inhibition of claudin expression increased significantly the motility of aCP tumour cells, and claudin expression was significantly lower in invasive than in non-invasive aCPs27.Hölskenet al., who used a xenotransplant model of human aCP in immunodeficient mice. Serial sectioning of a whole aCP xenograft provided a reconstruction of the formation of the finger-like protrusions that invade surrounding brain. From these serial sections, the authors inferred that the cluster cells themselves extrude through the palisading epithelial layer that encases the tumour to infiltrate the surrounding brain and then become enclosed again by a palisading epithelial layer to form the whorls characteristic of the finger-like protrusions of aCP. Consistent with this model for infiltration, the xenografts and human tumour specimens showed increased proliferation of the palisading cells and expression of p21WAF/Cip1 in the cluster cells, indicating cell cycle arrest. Although this model is a snapshot of the developing aCP and not a time course, the mechanism proposed is an attractive explanation for both the motile and the quiescent phenotype of cluster cells as well as their sustained presence in the infiltrating tumour projections.Further insight into the role of cluster cells in the pathogenesis of aCP was provided in a study by Stache36. Andoniadouet al. created a mouse model that expresses degradation-resistant β-catenin in Sox2+ cells upon tamoxifen induction to determine whether these cells could give rise to tumours at the embryonic stage and also in adult mice. The embryonic-induced mice developed tumours similar to those seen in the Hesx1 model36, with identifiable β-catenin-accumulating cluster cells reminiscent of those in human aCP that were quiescent or slow-dividing (did not express Ki-67) and were undifferentiated (did not express lineage commitment markers αGSU and PIT1)37. In the adult-induced model, the pattern of β-catenin expression was more complex, with some cells showing nucleocytoplasmic accumulation.The cluster cells in aCP are a clearly distinct subpopulation, but their function within the tumour is not yet well understood. There is growing evidence for a population of anterior pituitary stem cells expressing Sox2 that can give rise to the main anterior pituitary progenitor cell lineages and that, crucially, also have tumour-inducing potential+ cells simultaneously expressing both degradation-resistant β-catenin and yellow fluorescent protein (YFP) aiming to identify both these tumour progenitor cells and their descendants37. Interestingly, after induction of degradation-resistant β-catenin, the majority of tumour cells formed did not express YFP. However, there were populations of YFP-expressing cells adjacent to the tumour cells, some of which formed clusters. These findings indicate that the tumour cells were not derived from Sox2+ cells expressing degradation-resistant β-catenin. However, nucleocytoplasmic accumulation of β-catenin was observed in tumour cells that did not express YFP (and so were not derived from the Sox2+ progenitor cells), suggesting that this accumulation and activation of the Wnt pathway is non-cell-autonomous and may occur as a result of signalling from the adjacent Sox2+ cell population. Indeed, the β-catenin-accumulating, YFP– tumour cells were often found in close proximity to the YFP+ cells, suggesting a functional relationship37.Lineage tracing experiments used Sox2+ cells leads to the formation of β-catenin-accumulating cell clusters that resemble those in human aCP, implicating Sox2+ progenitor cells as the cells in which the tumour-initiating mutation in β-catenin occurs. However, these cells do not give rise in an autonomous manner to the tumour cells as would be expected if they were true mutation-sustaining cancer stem cells. Instead, they appear to drive tumour formation in a paracrine manner by inducing tumorigenic events in adjacent cells. The authors propose that induction of degradation-resistant β-catenin initiates a brief period of proliferation in a proportion of Sox2+ cells. The resulting daughter cells then form the β-catenin-accumulating clusters that are characteristic of aCP. These clusters become quiescent and secretory, signalling by means of secretory proteins including members of the fibroblast growth factor (FGF), transforming growth factor beta (TGFβ), epithelial growth factor, and SHH pathways, along with pro-inflammatory cytokines and chemokines,30 to induce transformation in surrounding cells and perhaps modify the tumour microenvironment. It is these non-Sox2+-derived cells that form the bulk of the tumour37.Expression of degradation-resistant β-catenin in Sox230, and they are also non-proliferative and undifferentiated41. A representation of the different cell populations at the invading edge of aCP and some of the differentially expressed proteins and factors is shown in40. Instead, even in highly invasive and destructive aCPs that destroy surrounding hypothalamic and pituitary tissue, clusters of β-catenin-accumulating, quiescent cells remain. Furthermore, these clusters are often located near to the invading tumour periphery, in regions where the proliferation of surrounding cells is high and selective pressure is likely to be significant. Reports differ as to the prevalence ofCTNNB1 mutation in aCPs and sequencing of a bulk aCP tumour is likely to result in false negative results if the mutation is present only in cluster cells, which may constitute a small proportion of the bulk tumour and so fall below the limit of detection of the method employed. However, immunohistochemical studies have suggested that even in aCP with no obvious cell clusters, there are β-catenin-accumulating cells found distributed throughout the tumour35. This is useful from a diagnostic perspective but may also be indicative of the importance of these cells for tumour pathogenesis. Further experiments will be required to determine whether cluster cells are essential paracrine signalling hubs for tumour pathogenesis after the tumour-initiating mutation inCTNNB1, but the current available evidence is consistent with this model.The mouse paracrine model of aCP pathogenesis includes a stem-cell-like progenitor that sustains the oncogenic mutation but is not the cell-of-origin of the tumour (comprehensively reviewed in37), while they allow the study and manipulation of tumorigenesis, lack some features of human aCP: for instance, they do not develop a defined palisading layer of epithelial cells at the invading edge of the tumour, nor do they show calcification or the anucleated ghost cells (wet keratin) that are pathognomonic for human aCP. It has been suggested that wet keratin is a feature of a more mature tumour and that the lack of this feature in the mouse models is likely a consequence of the relatively short period (weeks) over which the mouse tumour develops. A report by Scagliottiet al.43 of an intrauterine-diagnosed congenital aCP at 23 weeks of gestation described the presence of wet keratin and calcium deposits in the tumour. This is comparable to the 2–5-month period over which the Sox2+ aCP mouse model develops37, so perhaps the lack of wet keratin and calcification in the mouse tumour is due to species-specific differences in the pathogenesis of this tumour or perhaps the mouse model is lacking a facet of the human tumour that is not yet identified.Mouse models, primary cell cultures, and xenograft models are valuable tools for studying the pathogenesis of aCP, each of which has advantages for answering particular questions; however, none fully recapitulates the features of the human tumour. The mouse models  in which the aCP can grow and invade. However, opportunities for the manipulation of tumour pathogenesis are limited in this model, and its suitability for testing pharmacological agents may be compromised by the low availability of human tumour material and differences in responses to agents between species.Xenograft modelsAll the models discussed here have provided greater insight into the pathogenesis of aCP and represent useful tools for answering outstanding questions, but the particular advantages and disadvantages of each must be acknowledged and considered.BRAF (V600E) mutation in pCP has afforded us an opportunity for a promising pharmacological intervention, recent progress in understanding aCP reveals a morphologically complex tumour with distinct cell populations that have differing functions and cells of origin. If the paracrine model for aCP tumorigenesis withstands further investigation, it presents a potential target for therapeutic intervention, namely the cluster cells that initiate and support tumorigenesis in surrounding cells. However, the stem-like properties of these cells make them difficult targets for cytotoxic agents, which are often ineffective against quiescent cells. Any targeted therapy aimed at disrupting the paracrine signalling between clusters and surrounding cells will require deeper understanding of the signalling events and the effect on the surrounding tumour.This review has highlighted the very different architecture and pathogenesis of CP variants. While the discovery of theBRAF (V600E) mutations in pCP has led to the potential for new targeted pharmacological therapy. Our understanding of aCP has been greatly improved by mouse model and xenograft experiments revealing new and critical roles for β-catenin-accumulating cluster cells; however, there is still much to clarify about the complex interactions that occur between different cell populations within this neoplasm before we are in the position to develop effective targeted therapies.Understanding of CP pathogenesis has increased considerably in recent years. The identification of


Aging is characterized by functional decline of diverse organs and an increased risk for several diseases. Therefore, a high interest exists in understanding the molecular mechanisms that stimulate aging at all levels, from cells and tissues to organs and organisms, in order to develop ways to promote healthy aging. While many molecular and biochemical mechanisms are already understood in some detail, the role of changes in epigenetic regulation has only begun to be considered in recent years. The age-dependent global reduction in heterochromatin, along with site-specific changes in the patterns of DNA methylation and modification of histones, have been observed in several aging model systems. However, understanding of the precise role of such changes requires further research. In this review, we will discuss the role of epigenetic regulation in aging and indicate future research directions that will help elucidate the mechanistic details of it. Aging is an associated with an increased risk of several morbidities such cancer, cardiovascular disease, and autoimmune disease. It is associated with the body's altered capacity to cope with stress induced by metabolism, infection, and damage to cellular macromolecules. Understanding the molecular mechanisms that advance aging will help scientists to understand why aging individuals are more prone to those diseases and why they may be less stress-resistant.The cells in the aging body exhibit large differences to cells in the juvenile organism, ranging from changed gene expression and DNA methylation patterns , exonic (~10%), intronic (~45%), and intergenic (~35%) regions (Heyn et al., Analysis of DNA methylation in the genomes from CD4Age-dependent decrease in global genome methylation has also been observed in other aging model organisms such as mice (Wilson et al., While global DNA hypomethylation is observed in various tissues with age, the accumulation of domains of heterochromatin during the early steps leading to senescence has been described (Narita et al., When analyzing the observed changes in DNA methylomes of human blood cells in more detail, Heyn et al.  indicateThese findings are further backed up by a mathematical modeling study performed on 82 available Illumina DNA methylation array data sets to test for the presence of age predictor sites in the genome Horvath, . HorvathIt was observed that DNA hypermethylation is more site-specific as compared to hypomethylation. Importance of DNA hypomethylation for triggering senescence is demonstrated by two facts. First, hypomethylation occurs in senescing cells but not in immortalized cells (Wilson and Jones, As a result, the shift to hypomethylation of constitutive heterochromatin and hypermethylation of promoters in cell-cycle promoting genes, age-dependent changes in DNA methylation may trigger increased genomic instability and lead to a permanent cell-cycle arrest. Furthermore, the differential methylation of promoter CpGs may also contribute to the observed changes in senescence- and age-related gene expression profiles. The role of DNA hypomethylation and site-specific hypermethylation in aging and senescence would have to be clarified in the future research.Post-translational modifications of the histone amino tails affect their affinity to DNA and interacting proteins, and different combinations of such modification marks may have synergistic or antagonistic effects on those affinities. This indicates a role of histone modifications in formation of active or repressive chromatin states and led Jenuwein and Allis to the formulation of the “histone code” theory (Jenuwein and Allis, In addition to histone tail modifications, the nucleosome density affects the level of DNA packaging as well. Therefore, DNA regions with a high density of nucleosomes are likely transcriptionally inactive while transcriptionally active DNA regions are characterized by looser packaging and a low density of nucleosomes (Boeger et al., Both nucleosome occupancy and histone tail modifications experience changes as cells age Table . A decliIn addition to overall changes in nucleosome numbers, numerous changes in post-translational modifications of histone tails with age have been described Table . HoweverAforementioned histone marks are established by various histone-modifying enzymes including histone deacetylases and acetyltransferases (Kuo and Allis, Moreover, SUV39H1 seems to play an important role in controlling the proliferative status of a cell, through alterations of the chromatin structure at centromere region during the process of cell division (Aagaard et al., Furthermore, reduced SUV39H1 levels during cell senescence and aging (Sidler et al., Additionally, SUV39H1 is involved in regulating gene expression. For instance, SUV39H1 cooperates with E2F/RB to silence S-phase genes in terminally differentiating cells (Ait-Si-Ali et al., Studies of the effects of SUV39H1 mutations on lifespan are complicated as both the overexpression and the knockout lead to developmental defects (Czvitkovich et al., To sum up, the downregulation of SUV39H1 during the establishment of senescence may promote DNA repair in heterochromatin regions but eventually results in increased genomic instability promoting cell-cycle arrest. On the other hand, the altered expression and recruitment of SUV39H1 to promoter regions of genes may also shape the senescence-associated gene expression profile and further contribute to the establishment of senescence. Further research is needed to clearly define the regulation and role of SUV39H1 during the establishment of senescence.Both SUV39H1/2 knock-out mice and SUV39H1 transgenic mice showed severe developmental defects, while the knockout animals exhibited decreased viability, increased genomic instability, and susceptibility to tumor formation (Peters et al., Thus, it can be hypothesized that adequate SUV39H1 expression levels are required in order to balance genomic stability and proliferative potential. In such a model, increased SUV39H1 expression levels would protect the genome integrity and promote cell division, whereas decreased SUV39H1 expression levels would increase genomic instability and favor cell cycle arrest; however, if the SUV39H1 expression levels fall below a threshold level, the silencing of promoter regions of growth inhibitory genes may also be compromised and therefore result in tumor formation. In addition to mere differences in expression levels, the altered recruitment of SUV39H1 to specific DNA regions likely also contributes to senescence-associated changes in the distribution of H3K9me3 marks. In order to test this model for the role of SUV39H1 expression and recruitment in balancing genomic integrity and cell proliferation, it would be interesting to address the following questions.While the role of SUV39H1 in the formation of the heterochromatin in pericentric regions is well-accepted, the patterns of SUV39H1-mediated histone H3K9 trimethylation in other DNA sequence regions are less characterized. A better understanding of which transcription factors interact with SUV39H1 and may therefore recruit it to their target promoters could help researchers to understand the effects of SUV39H1-mediated H3K9 trimethylation on gene expression. One such example is the association of SUV39H1 with RB/HDAC1 resulting in the heterochromatin formation and suppression of genes involved in cell cycle progression (Bandyopadhyay et al., In addition to mediating the silencing of repetitive DNA regions, SUV39H1 also plays a significant role in the regulation of telomere length (Garcia-Cao et al., In order to address how gradual changes in SUV39H1 expression levels affect the cell-cycle dynamics, human diploid fibroblasts could be treated with increasing amounts of SUV39H1 targeting siRNA or transfected with increasing amounts of an SUV39H1 overexpression construct followed by analysis of cell proliferation by BrdU incorporation and senescence by the SA-beta-GAL assay. This would allow for testing of the hypotheses that have been formulated above and might help in determining the threshold of the SUV39H1 expression level that is necessary for the inhibition of cell division if such a threshold indeed exists.Another approach is to determine how the SUV39H1 expression levels correlate with the rate of proliferation in various cell types. For this, it would be interesting to compare various cancer cell lines, stem cells, and human diploid fibroblasts at different population doublings and primary cells derived from donors of different ages.A more detailed understanding of how SUV39H1 expression is controlled on the level of transcription and post-transcriptionally will help in studying what intra- and extra-cellular stimuli may trigger the downregulation of SUV39H1. Two studies have described an indirect role of p53 in regulating the expression of SUV39H1 by inducing the transcription of p21, which in turn represses E2F mediated transcription of SUV39H1 (Mungamuri et al., Several studies have described altered DNA methylomes that were characterized by extensive site-specific DNA hypomethylation alongside of some site-specific hypermethylation with increasing age (Heyn et al., de novo DNA methylation in a sequence-specific way (Schlesinger et al., The downregulation of DNMT1 during senescence could indicate that DNA methylation marks are passively lost through the deficiency in maintenance through cell divisions. However, 1,849 CpG sites were significantly differentially methylated when comparing three senescent to three dividing cell cultures in our study, pointing that the loss of DNA methylation during senescence may at least in part be non-random. Thus, it would be interesting to study whether the recruitment of DNMT1 to DNA is altered during senescence. This could be achieved through the immunoprecipitation of DNMT1 followed by mass-spectrometric analysis of its interacting proteins in order to determine whether DNMT1 is associated with different binding partners during senescence. Some studies suggest a role for the existing histone modification pattern in limiting the Changes in the DNA methylation patterns do not extensively correlate with altered gene expression, but the inhibition of DNA methylation was shown to trigger cell growth arrest (Vogt et al., In order to dissect the role of epigenetic changes in thymic involution further, it would be interesting to separate the different cell types that occur within the thymus, dendritic cells, epithelial cells, nurse cells, macrophages, and maturing T-cells. The measurement of DNMT1 and SUV39H1 protein levels and determination of global DNA methylation and H3K9 trimethylation in the different cell types could then indicate which cells are mainly affected by the loss of heterochromatin marks and corresponding increase in genomic instability.Furthermore, our study involved only three time points across the lifespan of the rats—1month-old animals, 4 month-old animals around the age of the onset of the thymic involution, and 18-month-old animals, which have a very small thymus (Sidler et al., If the downregulation of SUV39H1 and DNMT1 does occur relatively early during thymic involution, it would be interesting to further study their role in the onset of thymic involution by creating mouse-models that specifically overexpress or repress SUV39H1 or DNMT1 in the thymus or possibly even in a specific cell type that is affected by their age-dependent downregulation (Sidler et al., in vivo, through the repeated infection with a virus, such as CMV, followed by collection of blood samples in order to study the T-cell population that was clonally selected in response to each cycle of infection, or in vitro, by exposing virus-specific T-cells to their cognate antigen for several subcultures and studying changes in epigenetic regulation.On the other hand, since the proliferation of peripheral T-cells depends on antigen recognition, which is limited in the rats we studied as they were housed in a pathogen-free environment (Sidler et al., Considerable progress has been made in understanding regulatory pathways that are involved in aging over the recent years. Several studies have described age-dependent reduction in global and constitutive heterochromatin affecting both DNA methylation and histone modifications as well as site-specific changes in DNA methylation and histone modification patterns. A more detailed understanding of the regulation of the modifying enzymes and their recruitment during the establishment of senescence will likely pinpoint several molecular targets that are involved in the regulation of aging and may even serve as possible drug targets to promote a prolonged, healthy lifespan.CS: Wrote the manuscript; OK: Proposed the topic, revised the manuscript; IK: Proposed the topic, wrote the manuscript.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.


FMR1) encoding the Fragile X Mental Retardation Protein (FMRP), an RNA-binding protein involved in RNA metabolism and modulating the expression level of many targets. Most cases of FXS are caused by silencing of FMR1 due to CGG expansions in the 5′-UTR of the gene. Humans also carry the FXR1 and FXR2 paralogs of FMR1 while flies have only one FMR1 gene, here called dFMR1, sharing the same level of sequence homology with all three human genes, but functionally most similar to FMR1. This enables a much easier approach for FMR1 genetic studies. Drosophila has been widely used to investigate FMR1 functions at genetic, cellular, and molecular levels since dFMR1 mutants have many phenotypes in common with the wide spectrum of FMR1 functions that underlay the disease. In this review, we present very recent Drosophila studies investigating FMRP functions at genetic, cellular, molecular, and electrophysiological levels in addition to research on pharmacological treatments in the fly model. These studies have the potential to aid the discovery of pharmacological therapies for FXS.Intellectual disability (ID) and autism are hallmarks of Fragile X Syndrome (FXS), a hereditary neurodevelopmental disorder. The gene responsible for FXS is Fragile X Mental Retardation gene 1 ( The Fragile X Syndrome (FXS), previously known as Martin-Bell syndrome or marker X syndrome or FRAXA, is the first X-linked intellectual disability (ID) syndrome described involving a DNA alteration and the most frequent heritable monogenic form of ID , the first gene associated with an X-linked ID . Through alternative splicing, at least 12 different isoforms of 67–80 kD are produced. The CGG repeats are polymorphic in the population ranging from 5 to 54 repeats in normal individuals to more than 200 (full mutation) in severely affected patients  in position Xq27.3  and Fragile X Related 2 (FXR2). While FXR2 has a distribution comparable to that of FMRP, some isoforms of FXR1 display a specific expression in brain while other isoforms are only present in muscle and heart  and in testes and absent from muscles and the heart  domains and one Arginine-Glycine-Glycine (RGG) box. The main function of FMRP is to regulate translation and indeed it has been found associated with polyribosomes in different cell lines and, importantly, in the brain . It is now named Fmr1 with a capital F, meaning that it has been identified through the human homolog FMR1. Here, we will call it dFMR1 to distinguish it from the mouse gene (Fmr1). FlyBase names the protein Fmr1, but here we will call it dFMRP with the “d” indicating Drosophila. The dFMR1 gene exhibits high sequence homology with all three human genes . All functional domains are highly conserved with the two KH domains being 75% identical and 85% similar between dFMR1 and hFMR1 , in the somatic musculature, in pole cells, in the gut and in the gonads   in 58 biological processes, which are summarized in Table In embryos, dFMRP has been localized in the brain, ventral nerve cord, and mesoderm  of Drosophila protein-coding genes, making it the largest collection of RNAi lines for all model systems. Currently, there are three different types of UAS-RNAi stocks available: GD and KK with long hairpins and shRNA with short hairpin micro RNAs. The existence of such a broad array of RNAi lines provides many experimental benefits for biological studies, including those focusing on FMRP. For example, tissue-specific RNAi studies uncovered the role of dFMRP in early developmental stages of fly larvae. It was shown that dFMRP regulates glial-dependent proper timing of neuroblast reactivation during brain development  enables the knockdown of a gene of interest at the post-transcriptional stage  that are oval structures hosting synapses differing in size, shape, physiology, and in the amount of sub-synaptic reticulum surrounding them. Type I boutons are glutamatergic and have been the focus of FXS studies. Immunohistological staining can be used to visualize these structures and observe different NMJ phenotypes. dFMR1 is expressed pre-synaptically in motor neurons  of dFMR1 mutants also exhibit fecundity and testes dysfunctions, which can be used to evaluate non-neuronal requirements  and dFMRP binds futsch mRNA, negatively regulating its translation  mRNA together with Ataxin-2  studies at NMJs in Drosophila showed that FMRP has clear pre-synaptic functions  and because it allows genetic experimentation. Also KO mutant rats present behavioral abnormalities related to ASD-like altered patterns of social interaction  Slack  medium has been exploited because it does not require heating and can simply be dissolved in room temperature water so that even heat-sensitive drugs can be tested pyridine (MPEP), three competitive mGluR antagonists or LiCl rescue naive courtship behavior, immediate recall memory and short-term memory of dFMR1 mutants . One reason could be the timing of the treatment or the need to combine different drugs since many other molecular mechanisms have been evoked as causative aspects of this disease. Importantly, in addition to FXS, many other neurodevelopmental disorders are correlated with defects in mGluR signaling.Glutamate is the main excitatory neurotransmitter in the CNS. Metabotropic glutamate receptors (mGluRs) at synapses control Long Term Depression (LTD), which mediates synaptic plasticity, thus weakening the synaptic response to stimuli. mGluR-dependent LTD was initially found to be enhanced in A and GABAB receptors (R) that are fast-acting inotropic and metabotropic receptors, respectively. A decrease in GABAA R expression and defects in GABAergic signaling were found in the FMR1 Knock Out (KO) mouse dFMR1 mutant, Chang et al.   restores the number of boutons in dFMR1 mutants. Using a newly developed larval crawling assay and a sophisticated algorithm (LarvaTrack) for drug screening, the locomotion defects (distance and velocity) of dFMR1 larvae were used as a readout of the larval bouton number phenotype  signal transduction pathway  2 are Ca2+-binding proteins that play a role in the control of the synapse number: loss-of-function mutations increase the synapse number and over-expression decreases it. Human NCS-1 is involved in schizophrenia  and its Drosophila models where it has been shown that FMRP binds the Dscam mRNA and down-regulates its translation , which mediates the exaggerated presynaptic terminal growth followed by Dscam over-expression probably because it is activated by DSCAM  is a conserved protein the levels of which have been found to be elevated in many brain disorders including FXS. This has been confirmed in the mouse and Drosophila, expression of dFMR1 specifically in the insulin-producing cells (IPSs) or in the whole nervous system is able to rescue the defective free-running locomotor rhythmicity of dFMR1 mutants, as well as short-term and long-term memory defects in olfactory learning tests , the authors were also able to obtain a rescue of the circadian rhythmicity of dFMR1 flies. These data are consistent with the fact that FMR1 is expressed in the IPSs of the mammalian pancreas and that insulin and IGFs have been involved in many neurological events, including synaptogenesis and progenitor cell growth  are evolutionarily highly conserved proteins that play an important role in growth and metabolism, but also in neurogenesis through their role in neuronal stem-cell homeostasis (reviewed in Lee et al., dFMR1 flies and mice brought significant advantages for understanding the functional roles of FMRP. These systems made it possible to decrypt several pathways responsible for phenotypes recalling human symptoms, such as cognitive and learning deficits. In comparison to the other animal models, Drosophila is less expensive and easier to maintain, lays many eggs that enable to perform genetic screens, has a shorter lifespan and generation time, a smaller entirely sequenced genome (Adams et al., in vivo functional studies (reviewed in Wangler et al., dFMR1 mutants exhibit defects that resemble those observed in human FXS patients, and flies and humans share many pathways that are altered in the disease and likely responsible for a specific set of phenotypes. Studies on FXS in Drosophila have set a paradigm to validate drug targets and gain a deeper insight into their molecular mechanisms for future research on FXS and other neurodevelopmental diseases. This is also because the dissection of the correlation between pathways and genotypes can be more easily realized in the fly than in any other model system.Individuals affected by FXS show a broad spectrum of clinical presentations, with a great variability of signs, symptoms and severity levels (Hagerman, All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


The present study evaluates the same parameters after response to highly active antiretroviral therapy (HAART). Blood samples were collected from patients prior to and after antiretroviral therapy, and from control subjects. Membrane fluidity and [Ca2+]int were assessed by fluorescence spectroscopy measurements, using three different probes: TMA-DPH and DPH for membrane fluidity, and fura-2 for Ca2+. When compared with the control group, both untreated and treated patients presented increased lymphocyte [Ca2+]int and decreased lymphocyte membrane fluidity, without significant differences between the two groups of patients. On the contrary, the therapy reversed the membrane fluidity variations observed in erythrocytes. The decreased erythrocyte [Ca2+]int of untreated patients was not reversed by HAART. AIDS patients present changes in lymphocyte (mostly noninfected) and erythrocyte properties, partially reversed by HAART, consistent with a process of facilitated propagation of the infection to new cells, stimulation of virion production, and maintenance of a reservoir of erythrocyte-bound infectious virus. These observations can be related with the action of the HIV Nef protein in the cell's proteins and lipid composition, as well as with the recently observed cell infection by HIV-1 via endocytosis.We previously showed that lymphocytes and erythrocytes of HIV-1–infected patients, prior to antiretroviral therapy, presented significant changes in intracellular calcium concentration ([Ca


Arthropod diversity is unparalleled in the animal kingdom. The study of ontogeny is pivotal to understand which developmental processes underlie the incredible morphological disparity of arthropods and thus to eventually unravel evolutionary transformations leading to their success. Work on laboratory model organisms has yielded in-depth data on numerous developmental mechanisms in arthropods. Yet, although the range of studied taxa has increased noticeably since the advent of comparative evolutionary developmental biology (evo-devo), several smaller groups remain understudied. This includes the bizarre Pycnogonida (sea spiders) or “no-bodies”, a taxon occupying a crucial phylogenetic position for the interpretation of arthropod development and evolution.th century. We clarify and resolve terminological issues persisting in the pycnogonid literature and distinguish five developmental pathways, based on (1) type of the hatching stage, (2) developmental-morphological features during postembryonic development and (3) selected life history characteristics. Based on phylogenetic analyses and the fossil record, we discuss plausible plesiomorphic features of pycnogonid development that allow comparison to other arthropods. These features include (1) a holoblastic, irregular cleavage with equal-sized blastomeres, (2) initiation of gastrulation by a single bottle-shaped cell, (3) the lack of a morphologically distinct germ band during embryogenesis, (4) a parasitic free-living protonymphon larva as hatching stage and (5) a hemianamorphic development during the postlarval and juvenile phases. Further, we propose evolutionary developmental trajectories within crown-group Pycnogonida.Pycnogonid development is variable at familial and generic levels and sometimes even congeneric species exhibit different developmental modes. Here, we summarize the available data since the late 19A resurgence of studies on pycnogonid postembryonic development has provided various new insights in the last decades. However, the scarcity of modern-day embryonic data – including the virtual lack of gene expression and functional studies – needs to be addressed in future investigations to strengthen comparisons to other arthropods and arthropod outgroups in the framework of evo-devo. Our review may serve as a basis for an informed choice of target species for such studies, which will not only shed light on chelicerate development and evolution but furthermore hold the potential to contribute important insights into the anamorphic development of the arthropod ancestor. Arthropod evolution has led to an overwhelming species richness, which goes hand in hand with an extraordinary disparity of morphological forms . When aDrosophila genetics T.H. Morgan )Pycnogonum litorale ).Soon after walking limb bud outgrowth, the corresponding midgut diverticulum begins to extend into it Fig. . Data onReliable information on the location of the primordial germ cell(s) in the larval stages is missing, but the paired gonad anlagen become recognizable in the early postlarval phase in a dorsal position at the border of walking leg segments 1 and 2 . From thThe transition from postlarval to juvenile phase is here based on the molt that leads to a “miniature adult” with the full number of functional walking legs  Fig. . In mostPycnogonum litorale ).Nymphon hirtipes ),Large yolk-rich eggs and the hatching of an advanced postlarva (type 5) are characteristic of all Callipallenidae, but are also found in some nymphonids  may render the re-evaluation of this scheme necessary at some point in the future. In particular the recent success of the laboratory husbandry of a deep sea nymphonid  holds prWith no established laboratory model organism found among sea spiders, our understanding of many developmental processes at the cellular level and in terms of the underlying genetic mechanisms is still in its infancy. Clearly, additional studies are overdue and future investigations could address, among others, (1) the early embryonic development in “large egg species”, (2) the gastrulation in “large egg species” and the exact relationship of the mesodermal and entodermal cell lineages in pycnogonids in general, (3) the identification and localization of the germ line precursors during embryology, and the (4) understanding of axial growth and segmentation processes in the different developmental types. Ideally, such studies would include modern live imaging techniques, and their underpinning with gene expression and gene function data is needed. Although previous attempts to address the latter two issues have faced several challenges, first progress in the optimization of protocols has been made , bu, buPycnos e.g., , 11, 41), 41Pycnoades ago –44 – wouIt is noteworthy that Pycnogonida is the only extant chelicerate taxon that shares with many crustaceans a life cycle that includes a minute marine larva with only three limb-bearing segments [e.g., ). The co


We approached this issue by focusing on the small RNA transcriptome in grapevine berries of the two varieties Cabernet Sauvignon and Sangiovese, growing in adjacent vineyards in three different environments. Four different developmental stages were studied and a total of 48 libraries of small RNAs were produced and sequenced. Using a proximity-based pipeline, we determined the general landscape of small RNAs accumulation in grapevine berries. We also investigated the presence of known and novel miRNAs and analyzed their accumulation profile. The results showed that the distribution of small RNA-producing loci is variable between the two cultivars, and that the level of variation depends on the vineyard. Differently, the profile of miRNA accumulation mainly depends on the developmental stage. The vineyard in Riccione maximizes the differences between the varieties, promoting the production of more than 1000 specific small RNA loci and modulating their expression depending on the cultivar and the maturation stage. In total, 89 known vvi-miRNAs and 33 novel vvi-miRNA candidates were identified in our samples, many of them showing the accumulation profile modulated by at least one of the factors studied. The  The ability of a genotype to produce different phenotypes as a function of environmental cues is known as phenotypic plasticity  berries are characterized by high phenotypic plasticity  are ubiquitous and adjustable repressors of gene expression across a broad group of eukaryotic species and are directly involved in controlling, in a sequence specific manner, multiple epigenetic phenomena such as RNA-directed DNA methylation and chromatin remodeling , a red Italian grape variety, and Cabernet Sauvignon (CS), an international variety, were grown side by side in three different Italian locations, representing traditional areas of Sangiovese cultivation in Italy with a long-standing winemaking tradition.Two Vitis berlandieri × Vitis riparia), the cultivation techniques  and the health status were the same among all the locations.In order to reduce factors of variation, the age of the plants (between 10 and 12 years old), the clone type (Sangiovese clone R5 and Cabernet Sauvignon clone VCR23), the rootstock (The vineyards were located in Bolgheri (Bol), a coastal area of Tuscany, 50 m asl (above sea level) , in Montalcino (Mont) a mountain area of Tuscany, 195 m asl;  and in Riccione (Ric), a plain area of Emilia Romagna, 111 m asl; . Further details on the environmental conditions of the vineyards are provided in Supplementary Figure Berries from four developmental stages were collected in two biological replicates, during the 2011 growing season, for a total of 48 samples  and dissolved in DEPC water. All the steps suggested in the technical bulletin for small RNA isolation of plant tissues were followed except the “Filter Lysate” step, which was omitted. The quality and quantity of small RNAs were evaluated by a NanoDrop 1000 spectrometer (Thermo Fisher Scientific), and their integrity assessed by an Agilent 2100 Bioanalyzer using a small RNA chip (Agilent Technologies) according to the manufacturer's instructions.Small RNA libraries were prepared using the TruSeq Small RNA Sample Preparation Kit (Illumina®), following all manufacturers' instructions. Forty-eight bar-coded small RNA libraries were constructed starting from 50 ng of small RNAs. The quality of each library was assessed using an Agilent DNA 1000 chip for the Agilent 2100 Bioanalyzer. Libraries were grouped in pools with six libraries each (6-plex).The pools of libraries were sequenced on an Illumina Hiseq 2000 at IGA Technology Services .GSE85611.The sequencing data were submitted to GEO–NCBI under the accession number Vitis vinifera L. genomic sequence V1 , in order to reduce sequencing bias and to allow the comparison of small RNA accumulation from different libraries. The normalized abundance was calculated as:n base is 4,000,000.where To perform the clustering analysis, the “hits-normalized-abundance” (HNA) values were calculated as:TP4M is the normalized abundance of each small RNA sequence mapping in a giving cluster and a Hit is defined as the number of loci at which a given sequence perfectly matches the genome.where https://mpss.danforthcenter.org/dbs/index.php?SITE=grape_sRNA_GxE), in order to store and assist the visualization of all the sequenced libraries.One database was produced using the grapevine genome, and made available on the website , only those clusters where the HNA of each library in the comparison was greater than or equal to 5 (library A ≥ 5 HNA and library B ≥ 5 HNA) and the sum of the cluster abundance of these same libraries was higher than 30 (library A + library B > 30) were selected.All the clustering analyses were performed using only two developmental stages for each cultivar: bunch closure was used to represent “green tissues” (g) and 19 °Brix to represent “ripened tissues” (r).http://www.mirbase.org/). Subsequently, the whole set of small RNAs passed through the five filters designed according to the properties of validated plant miRNAs and their precursors  and novel (or specie-specific) miRNAs was carried out applying a conservative and robust pipeline as described by Jeong et al.  and ZhaiAll the known vvi-miRNAs identified by the pipeline were manually inspected, to ensure that the tags identified as known miRNAs were assigned correctly to their actual precursor, and to retrieve the most abundant isoform within the tags mapping in each precursor.Regarding the novel miRNA candidates identified using this pipeline, only those for which the most abundant tag was 20, 21, or 22 nt were retained. They were compared with all the known mature plant miRNAs in miRBase (version 20) to identify homologs. Finally, novel candidates passed through a manual evaluation of precursor secondary structures, using the plant version of the UEA sRNA hairpin folding and annotation tool  using multiple comparison analysis. We loaded the total raw redundant reads from our 48 libraries in the CLCbio package and trimmed the adaptors, considering only reads between 18 and 34 nt. We annotated miRNAs against the user defined database, comprehending our set of 122 MIRNA loci and their corresponding mature sequences. For each library, the total counts of read perfectly mapping to the miRNA precursors was considered as the input of the expression analysis.Differentially expressed miRNAs were identified using the CLCbio Genomics Workbench , or between the three vineyards in the same cultivar and in the same developmental stage . For this reason, we considered each developmental stage (12 libraries) and we performed the Empirical Analysis of digital gene expression (DGE), an implementation of the “Exact Test” present in the EdgeR Bioconductor package, as implemented in CLCbio software and estimating tagwise dispersion with pairwise comparisons and setting the significance threshold to FDR-adjusted ad-hoc normalization [log10(1+TP4M) or log10(1+HNA)] for correlation analysis. This normalization was chosen because of the enormous range of abundance values that produced a log-unimodal distribution and may cause significant biases in the correlation analysis when performed using TP4M or HNA values. A unity was then added to the abundance value due to the presence of zero entries. After this addition, a value of zero still corresponds to zero of the log10(1+TP4M) function, thus making consistent the comparisons among profiles.The normalized reads (TP4M) of all miRNAs identified in this study and also the cluster abundances obtained from the static clustering analysis were submitted to another 10(1+TP4M) or log10(1+HNA) values for miRNAs and sRNA-generating loci respectively. Pearson's correlation coefficients were converted into distance coefficients to define the height of the dendrogram.The dendrogram was generated using the function hclust and the Pearson correlation was calculated using the function cor in R, based on the logHeat maps were produced using MeV  of the V2.1 annotation . This website provides a summary of the library information, including samples metadata, mapped reads, and GEO accession numbers. It also includes pages for data analysis, such as quick summary of the abundances of annotated microRNAs from grapevine or other species. Small RNA-related tools are available, for example target prediction for user-specified small RNA sequences and matching criteria. Finally, and perhaps most importantly, a customized browser allows users to examine specific loci (genes or intergenic regions) for the position, abundance, length, and genomic context of matched small RNAs; with this information, coupled with the target prediction output, users can develop and assess hypotheses about whether there is evidence for small RNA-mediated regulation of grapevine loci of interest.To facilitate access and utilization of these data, we have incorporated the small RNAs into a website (V. vinifera genotypes [Cabernet Sauvignon (CS) and Sangiovese (SG)] and environments , we investigated the regions in the grapevine genome from where a high number of small RNAs were being produced (sRNA-producing regions), by applying a proximity-based pipeline to group and quantify clusters of small RNAs as described by Lee et al.  windows or clusters. To determine the small RNA cluster abundance, we summed the hits-normalized-abundance (HNA) values of all the small RNAs mapping to each of the 500 bp clusters, for each library . To reduce the number of false positives, we considered a cluster as expressed when the cluster abundance was greater than the threshold (HNA = 30) for a given library, eliminating regions where few small RNAs were generated, possibly by chance. Libraries from bunch closure, representing green berries, and 19 °Brix representing ripened berries, where used in this analysis. From the 972,413 clusters covering the whole grapevine genome, 4408 (0.45%) were identified as expressed (sRNA-producing regions) in at least one sample. As showed in Figure Next, the small RNA-generating clusters were characterized on the basis of the genomic regions where they map, i.e., genic, intergenic and transposable elements. In general, when the berries were green, the numbers of sRNA-generating loci located in genic and intergenic regions were roughly equal in all environments and for both cultivars, except for Sangiovese berries collected in Riccione, which show a slight intergenic disposition of sRNA-producing regions Figure . DiffereWhen comparing the clusters abundance among libraries, we found that 462 clusters were expressed in all libraries. The remaining 3946 expressed clusters were either shared among groups of libraries or specific to unique libraries. Interestingly, 1335 (30.3%) of the 4408 expressed clusters were specific to Riccione-derived libraries Figure . The othWhen comparing the expressed clusters with the presence of transposable elements (TE) annotated in the grapevine genome (V1), we noticed that approximately 23% of the sRNA-generating regions were TE-associated. Sangiovese green berries from Riccione have the highest proportion (26%) of TE-associated expressed clusters, while Cabernet Sauvignon ripened berries also from Riccione show the lowest proportion (13%) of TE-associated expressed clusters. Sangiovese berries (both green and ripened) have the highest percentage of expressed clusters located in TE when cultivated in Riccione, compared to the other two vineyards. Interestingly, Cabernet Sauvignon berries show the lowest proportion of TE-associated clusters when growing in Riccione Figure , indepenIn all the libraries, Long Terminal Repeat (LTR) retrotransposons were the most represented TE. More specifically, the gypsy family was the LTR class associated with the highest number of sRNA hotspots. The other classes of TE associated with the sRNA-generating regions can be visualized in Figure To determine the global relationship of small RNA-producing loci in the different environments, cultivars and developmental stages, we performed a hierarchical clustering analysis. As showed in Figure Notwithstanding the evidence that developmental stage and variety have the strongest effect in terms of distinguishing samples clustering, we were interested to verify the environmental influence on small RNA loci expression in the two cultivars. Thus, for each sRNA-generating cluster we calculated the ratio between cluster abundance in Cabernet Sauvignon and Sangiovese (CS/SG) in each environment and developmental stage, thereby revealing the genomic regions with regulated clusters, considering a 2-fold change threshold, a minimum abundance of 5 HNA in each library and a minimum sum of abundance of 30 HNA . Figure Notably, we observed a small percentage of regulated clusters (from 0.5 to 5%) exhibiting at least a 10-fold higher abundance of small RNA in Cabernet Sauvignon or Sangiovese when compared to each other Figures . An examhttps://sourceforge.net/projects/mireap) as described by Jeong et al.  is known to be involved in the vacuolar accumulation of proanthocyanidins in grapevine  seem to be involved in the regulation of important secondary metabolites biosynthesis. Among the six targets predicted for grape-m1191, the Other novel vvi-miRNA candidates seem to be involved in cell proliferation  and in chloroplasts-related functions . Furthermore, for the new vvi-miRC482b candidate, besides the already known involvement of this miRNA family with disease resistance , i.e., their relative cloning frequency, we set an empirical cut off value equal to at least 10 TP4M in both biological replicates to consider a miRNA as expressed in a given library. Also, a miRNA was considered specific when it was expressed in one or more libraries of a unique cultivar, unique environment or unique developmental stage.According to our established cut off, 175 miRNAs were classified as expressed in at least one of our libraries  and represented in a heat map Figure . To examTo gain statistical evidence of miRNA differential expression driven by the environment and/or genotype, we made pairwise comparisons, keeping constant the developmental stage, and evaluating the miRNA modulation among vineyards  or between cultivars (Cabernet Sauvignon vs Sangiovese). The analyses (with an FDR ≤ 0.05) reveal that some miRNAs are differentially expressed between the two genotypes grown in the same environment, but also that a number of miRNAs are modulated by the environment. In particular the number of differentially expressed miRNAs is higher in ripened berries (19 °Brix and at harvest), while no miRNAs are differentially expressed at bunch closure stage  and known  are intriguingly connected to berry development and secondary metabolism, even though most of the modulated families are still uncharacterized, or with targets not clearly involved in berry ripening and development, and deserve further studies to fully understand their biological roles.Using high throughput sequencing coupled with robust bioinformatics pipelines we analyzed small RNAs derived from the berries of Cabernet Sauvignon and Sangiovese, grown side-by-side in three vineyards, representative of different grapevine cultivation areas in Italy . We obtained nearly 750 MB reads comprising a significant proportion of small (21–24 nt) RNAs. The size distribution profiles of our libraries were in general consistent with previous reports in berry grapevine, where the 21-nt class was more abundant than the 24-nt class . The gene in this locus, also known as VvRD22g, encodes a BURP domain-containing protein, involved in an ABA-mediated abiotic stress response, which persists still after long periods of stress  showed a 390-fold change in the small RNA abundance, when comparing Cabernet vs. Sangiovese  with fold change greater than 50 was found, where one of the libraries has 0 HNA and the other any number greater than 30 HNA. This fact suggests a very strong modulation of the expression of small RNAs between the two cultivars, which is more or less pronounced depending on the vineyard where the berries were cultivated. A similar situation was observed comparing the expression level of small RNAs between reciprocal hybrids of The ripening process of grapevine berries is highly affected by the environment  with sufficient warmth to fully ripen Poni, . ConsequApplying a conservative pipeline to the analysis of our 48 small RNA libraries, we recognized 89 known and annotated grapevine miRNAs. In addition, when compared to previous reports in grapevine  or nearly ubiquitous (18) to all the libraries, and very few miRNAs specific of a cultivar, vineyard or developmental stage. All these 39 miRNAs belong to known vvi-miRNA families. With few exceptions, the same set of miRNAs was also found expressed in all the small RNA libraries constructed with different tissues of the grapevine cv. Corvina  and was predicted to target a cinnamoyl reductase-like protein (CCR) (VIT_203s0110g00350), which is part of the of the polyphenol biosynthetic pathway (Leple et al., As for known miRNAs, several members of the miR395 family are differentially expressed at 19 °Brix and at harvest in Bolgheri and in both Bolgheri and Riccione, respectively, when comparing the two cultivars. Moreover, miR395f is differentially expressed also in CS at harvest between Montalcino and Bolgheri. This miRNA has been shown to target genes involved in Sulphate assimilation and metabolism (Liang and Yu, miR399 family members are also differentially expressed in several comparisons: at 19 °Brix between Riccione and Bolgheri in CS and between Riccione and Montalcino in SG, plus in Montalcino between CS and SG. At harvest, miR399 are differentially expressed in SG in all the three comparisons among vineyards and in Riccione between CS and SG. miR399 is implicated in Phosphate homeostasis being rapidly up-regulated upon Pi starvation (Fujii et al., miR396 family members are known to be regulated during organ development, targeting Growth Regulating Factors (Liu et al., Finally, the investigation of the global relationships of different small RNA classes and miRNAs expressed in different grapevine cultivars, collected in different vineyards and developmental stages, suggests that although the vineyard may influence their profile of abundance it probably does in less proportion than developmental stage and cultivar. Somehow, this behavior would be expected because although the epigenetic state is dynamic and responsive to both developmental and environmental signals, small RNAs in general and even more miRNAs are well known to play numerous crucial roles at each major stage of plants development (Jones-Rhoades et al., in silico analysis and wrote the paper. LB conceived the experimental plan and sampled biological material. SDS prepared plant material for RNA extraction, read critically the paper. GDL prepared plant material for RNA extraction, sampled the biological material, read critically the paper. MP conceived the work. MEP supported the lab work, contributed to data analysis and read critically the paper. BM gave a substantial contribution to in silico analysis. EM wrote the paper, prepared plant material for RNA extraction, supported small RNA libraries preparation and helped data analysis.DPP prepared small RNA libraries, performed the 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.


Krabbe disease (KD) is a neurodegenerative disorder caused by the lack of β- galactosylceramidase enzymatic activity and by widespread accumulation of the cytotoxic galactosyl-sphingosine in neuronal, myelinating and endothelial cells. Despite the wide use of Twitcher mice as experimental model for KD, the ultrastructure of this model is partial and mainly addressing peripheral nerves. More details are requested to elucidate the basis of the motor defects, which are the first to appear during KD onset. Here we use transmission electron microscopy (TEM) to focus on the alterations produced by KD in the lower motor system at postnatal day 15 (P15), a nearly asymptomatic stage, and in the juvenile P30 mouse. We find mild effects on motorneuron soma, severe ones on sciatic nerves and very severe effects on nerve terminals and neuromuscular junctions at P30, with peripheral damage being already detectable at P15. Finally, we find that the gastrocnemius muscle undergoes atrophy and structural changes that are independent of denervation at P15. Our data further characterize the ultrastructural analysis of the KD mouse model, and support recent theories of a dying-back mechanism for neuronal degeneration, which is independent of demyelination. It is caused by the autosomal recessive mutation of lysosomal galactosylceramidase (GALC) enzyme, that leads to accumulation of galactosyl-sphingosine (psychosine), a potent lipid raft-associated neurotoxin6. Degeneration and death of myelinating cells are the main pathological hallmark of KD. More recently, psychosine accumulation was shown to influence neuron survival independently of the demyelination process7. Overall, both CNS and PNS are affected in KD patients8.Krabbe disease (KD)11, homozygous for the inactive form of GALC. Combination of cell therapy with gene therapy14, substrate reduction therapy15, pharmacological chaperone therapy16 and/or anti-oxidant therapy17, has resulted in a synergic therapeutic effect. Nonetheless, these strategies just afforded a slowdown of KD progression and a limited lifespan extension, suggesting that other targets should be identified and treated. Investigation of the cause-effect relationship between different overlapping mechanisms of the pathology is crucial to address this issue.The only accepted treatment for KD in humans is hematopoietic stem cells transplantation, with scarce clinical outcome. Several other strategies have been proposed in a murine model for KD, the Twitcher mice (TWI)18: indeed, Caspase3  is increased in KD sciatic nerves compared with spinal cords8. Furthermore, axonal loss in sciatic nerves occurs before apoptosis of cell bodies, even before clinical onset of KD symptoms19. These findings represent an alternative to older theories of the dying-forward and demyelination-dependent progression for KD7, based on the analysis of peripheral nerves ultrastructure20. Additionally, the affection of endothelial cells-especially blood vessel walls architecture-in KD mice22 further suggests that therapeutic approaches based only on the rescue of GALC activity in the brain may not be sufficient to cure KD disease.Mechanism of KD neural progression is currently debated. Recent studies proposed a dying-back mechanism for neuronal degeneration, independent of demyelination23 and highlight here unprecedented details. Furthermore, we also analyzed the upstream spinal cord regions where somata of sciatic-nerves-forming motor neurons are located, and the downstream muscle innervated by sciatic nerve synapses. Our data unveil possible novel mechanisms taking part into KD progression that should be taken into account in the search for new therapeutic approaches.In this study we exploited TEM to analyze the lower motor system of TWI mice in comparison with their wild-type (WT) littermates before onset and upon complete development of KD . We have chosen the lower motor system owing to the presence of both CNS  and PNS (sciatic nerves), and also skeletal muscle. We started our analysis from sciatic nerves for consistency with previous findings2, n = 42; TWI: 0.007 ± 0.0004 axons/μm2, n = 47; ****P < 0.0001). We performed a morphometric analysis on myelinated axons . Additionally, perimeter and diameter evaluation indicates that TWI axons are swollen compared with WT ones . Furthermore, the MST of TWI sciatic nerves is larger than that of WT for every couple , in agreement with the average value . Due to this increased cytoskeletal density, the number of trapped mitochondria is larger in TWI mice than in WT littermates .The organization and density of cytoskeletal components is altered in TWI sciatic nerves Fig. . Axonal 2, to avoid differences in myelin sheaths thickness related to axonal size. A gray-level profile of the image along a line crossing the myelin sheath shows disorganized structure of the MCLs through the entire TWI myelin sheath section  are visible on the external side of the sheath . Instead, the TWI SCs appear enlarged  exhibiting degenerated organelles, mainly damaged mitochondria and swollen cisternae of endoplasmic reticulum . Finally, some organelles -probably Golgi’s apparatus cisternae- are so degenerated that even at higher magnification it is possible to observe only a few discontinuous membranes (arrowheads).Surprisingly, severe morphological alterations were also observed in non-myelinating SCs, which in sciatic nerves are those surrounding Remak fibers Fig. . Remak bWe also observed, in TWI sciatic nerves, a peculiar sub-population of myelinating cells close to Remak bundles that enclose more than one axon (panels 3 G-I). Since this multiple myelination cannot be due to preparation artifacts (see Methods) we can conclude that a variable architecture of myelination is a specific feature of the TWI sciatic nerve. We classified three different levels of multiple myelination processes : (1) one regular-sized axon close to one small axon Fig. ; (2) oneWe analyzed 10 to 20 motorneurons (MNs) bodies from TWI versus WT spinal cords. The ultrastructural features  of the MNs soma were similar in the two experimental groups Fig. . We onlyNucleus, nucleolus and nuclear envelope -and all other cytoplasmic organelles of TWI MNs are not different from those of WT MNs Fig. . In geneBoth axonal and myelin sheath architectures are altered in the white matter of spinal cord of TWI mice compared with WT ones. We found that in several cases a clear separation is present between axons and their surrounding myelin sheaths. Cytoskeletal density follows the same trend observed in peripheral nerves indicating that neurofilaments and tubules are densely packed .A morphometric analysis of the same parameters evaluated in sciatic nerves on those axons corresponding to the ventral root region indicates again that in TWI mice the value of axonal area and the MST are significantly larger than in WT ones .26. These have been well characterized to be more prone than other skeletal muscles bearing slower fibers (e.g. diaphragm), to degenerative processes in pathological conditions29.We focused our examination on motor units  of the Gastrocnemius muscle because this is a Fast twitch muscle predominantly formed by fast fatigable MU31. We quantified the area of single myofibers ; (ii) Remak bundles structures smaller than WT ones, both in terms of size and number of sensorial axons per bundle . Moreover, RB envelopes are more electron-dense and wrap each axon two or three times ; (iii) reduced diameter of myofibers  and higher percentage of altered/innervated NMJ  in the gastrocnemius muscle.Similarly to what found at P30, P15 TWI samples displayed: (i) enlargement of sciatic nerve diameter, increased dispersion of myelinated axons, and change of morphometric parameters describing axonal features of the proximal  and distal  sciatic nerve. However, differences between the two experimental groups are usually milder than at latter pathological stages  no significant alteration in the MCL structure, as we did not find unmyelinated axons . Furthermore, although some swelling of SC cytosolic compartment was observed, this is likely a very early phenomenon as it is not associated with subcellular compartment degeneration; (ii) lack of cytoskeletal alteration in the perinuclear region of the MN body ; (iii) unaltered state of ependymocytes as well as endothelial cells of capillary walls .Thus the principal ultrastructural differences between P15 and P30 are summarized in Table The therapies proposed so far to cure KD are only symptomatic and supportive. Increasing evidence points to KD onset and progression as a multifactorial pathology, in which several different cell types undergo degeneration. Understanding the causal and temporal links between these pathological phenotypes is crucially important to achieve an adequate understanding of this pathology.The work presented here provides an ultrastructural characterization of the whole lower motor system of the TWI mouse model of KD disease. Neurons, myelinating and non-myelinating Schwann cells, ependymocytes, endothelial cells and muscular fibers were analyzed at a late pathological stage of disease progression (P30) and compared with those at an early stage (P15). We chose this experimental plan because it allowed us to study the progression of KD on two different scales. First, we gained details of the spatial progression of neural degeneration. Additionally, comparison between early and late disease stages allowed us to monitor the temporal progression of the pathology in the central and peripheral districts of the lower motor system, where the clinical symptoms first appear.11. Recent theories point to a dying-back mechanism for neurodegeneration which is independent of demyelination in KD19; however, this view is in marked contrast with the older dying-forward view of KD progression20. To reconcile these two hypotheses we investigated all-at-once three different districts: (i) the lumbar region of the spinal cord, focusing on the ventral horns of the gray matter where motor neuron soma are located; (ii) the sciatic nerve, made by axons of the same motoneurons, and (iii) the gastrocnemius muscle, which is innervated by the distal portions of the same motoneuronal axons. This allowed us to map the whole route of motor neurons from the CNS to the peripheral skeletal muscle, thus monitoring how the neural damage is related to the specific district investigated. Our results indicate that the bodies of TWI motor neurons are only slightly altered in the cytoskeletal organization . It is tempting to surmise that mitochondria are stuck as a consequence of axonal transport defects due to the dense network of both neurofilaments and microtubules . Indeed, impaired axonal transport has been related to several neuro-muscular disorders33. Increased cytoskeletal density is also represented in the most distal portion of sciatic nerves: terminal branches and NMJ  drives the conversion of non-myelinating in myelinating SCs to afford an endogenous remyelination mechanism with a compensatory effect, as already described in tumorigenesis34. Furthermore, it was demonstrated that the distinction between myelinating and non-myelinating SCs is not absolute because a reversion between them could manifest in demyelinating pathology or upon proper signaling coming from neurons37. Further experimental work will be required to provide a conclusive answer to this hypothesis. However, a proper understanding of the molecular basis of MMP would be an invaluable contribution: indeed, this could eventually lead to the development of myelin restoring approaches in demyelinating diseases.Despite the lack of damage in the MCLs at P15, we observed that TWI SCs display an enlarged cytosolic portion  compared with WT littermates and that this presumably anticipates the degeneration of cytoplasmic organelles at P30 Fig.  and in t38. Indeed, we found marked rearrangements in the spinal cord both in endothelial and epithelial cells at P30 .Finally, we provide here the first example of ultrastructural characterization of skeletal muscle in KD. Muscular damage in KD has been documented in TWI mice as an early symptom of disease progression with a general muscular weakness and tremors; however, analysis was limited to hind limb muscles and muscle innervated by cranial nervesles Fig. , in agre40. In this case, the skeletal muscle might represent a primary target of psychosine toxicity: psychosine could cause SR degeneration, and this in turn could lead to loss of calcium storage districts, finally resulting in reduced muscular strength. However, a high concentration of free oxygen radicals, possibly mediated by imbalance of calcium homeostasis and inflammation, should also be taken into account as a cause of the observed phenotype, as demonstrated in cellular and animal models of KD15. Indeed, we believe that therapeutic actions aimed at reducing oxidative stress and increasing muscular strength should be encouraged in KD treatment, as this approach has already proved to be useful to rescue muscular damage in a model of ALS disease27.Subsequent ultrastructural characterization of the gastrocnemius muscle shows that several structures of TWI muscles are severely damaged at P30, in particular some organelles as the sarcoplasmic reticulum and mitochondria  was generously donated by Dr. A. Biffi . Animals were maintained and used according to the protocols and ethical guidelines approved by the Ministry of Health, as per Italian law (Permit Number: 0004419).The Twitcher mouse colony , and all efforts were made to minimize mice suffering.Genomic DNA was extracted from the clipped tails of mice by Proteinase K lysis buffer as previously describedPostnatal day 30 (P30) TWI mice and their WT littermates  and one P15 TWI mouse versus its WT littermate were perfused with a fixative solution . Sciatic nerves, spinal cords and gastrocnemius muscles were dissected and post-fixed for 4 hours at room temperature in the same fixative solution.3 in volume. Sciatic nerves were processed without further sectioning.Spinal cords were dissected in the lumbar region, isolating four 1-mm-thick sections in the lumbar enlargement region and the gastrocnemius muscles were cut in small portions, approximately 1 mm43. Briefly, the samples were deeper fixed in 2–2.5% glutaraldehyde in cacodylate buffer . After rinsing, specimens were post-fixed with osmium tetroxide (1%)-potassium ferricyanide (1%) in cacodylate buffer, rinsed again, en bloc stained with 3% uranyl acetate in ethanol, dehydrated and embedded in epoxy resin, that was baked for 48 h at 60 °C. Thin sections were obtained with an ultramicrotome  and collected on G300Cu grids (EMS). Finally, sections were examined with a Zeiss LIBRA 120 plus transmission electron microscope equipped with an in-column omega filter.The selected tissues were further treated for epoxy resin embedding as previously describedImages of all the grid holes containing samples were collected at low magnifications (between 250X to 400X) and post-processed using Photoshop CS6 software (Adobe) to reconstruct the whole nerve section, as shown in Fig. Electron digital micrographs were used for the evaluation of nerve architecture and for the distribution of neurofilaments within axons.The morphology of the myelin sheaths was fully evaluated for integrity, thickness, size and shape. Data were analyzed using the software Fiji.The evaluation of cytoskeleton density was performed on 10 images of correctly-myelinated axons for each experimental point among three different experiments. Furthermore, we chose those axons that showed at least one region of myelin concentric layers stacking in the myelin sheaths to check the orthogonal sectioning. After an automatic adjustment of brightness and contrast, axons were isolated from the surrounding myelin sheath and the axonal area evaluated. Cytoplasmic organelles were deleted from the micrograph and, following an automatic thresholding of the images the generation of bit-maps, the number of identified particle was counted and normalized for axonal area.We first evaluated the normality of each data distribution with Shapiro-Wilk normality test and then we used Mann-Whitney non-parametric test to check the difference between the experimental groups. Values are reported as mean, standard deviation, number of values of experimental group and value of statistical analysis.In supplementary materials we reported the evaluation of single experiments repeated two or three times for a more relevant analysis.Supplementary Information


A wide spectrum of comorbidities has been associated with Parkinson’s disease (PD), a progressive neurodegenerative disease that affects more than seven million people worldwide. Emerging evidence indicates that chronic diseases including diabetes, depression, anemia and cancer may be implicated in the pathogenesis and progression of PD. Recent epidemiological studies suggest that some of these comorbidities may increase the risk of PD and precede the onset of motor symptoms. Further, drugs to treat diabetes and cancer have elicited neuroprotective effects in PD models. Nonetheless, the mechanisms underlying the occurrence of these comorbidities remain elusive. Herein, we discuss the biological and clinical implications of comorbidities in the pathogenesis, progression, and clinical management, with an emphasis on personalized medicine applications for PD. Parkinson’s disease (PD) is an incurable neurodegenerative disease affecting 7–10 million people worldwideAlthough PD is primarily categorized as a movement disorder, a wide range of non-motor conditions are increasingly being recognized as early features of the disease  motor scores and more severe Hoehn and Yahr staging  receptor. GLP-1 mimetics including, exenatide, liraglutide and lixisenatide have shown efficacy in alleviating some of the motor symptoms in PD patients. Among these drugs, PD patients treated with exenatide showed improvement in motor symptoms and cognitive domains after 1 year of treatment  mRNA, the most highly ranked gene in the meta-analysis, was upregulated in drug-naïve PD patients compared to healthy controls , alpha hemoglobin stabilizing protein (ASHP) and solute carrier family 11 member 2 (SLC11A2) in blood of PD, thus reinforcing the relevance of these pathways in the disease pathogenesis , a key gene involved in human pigmentation, have been associated with risk of PD in several populations. A large case-control study including more than 120,000 US men and women reported that red hair and the associated MC1R p.R151C polymorphism, both of which confer high melanoma risk, were associated with greater risk of PD  and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxins  was capable to distinguish untreated PD patients from healthy controls with an overall diagnostic accuracy of 86%. Interestingly, pharmacological intervention with a highly specific NAMPT inhibitor conferred neuroprotection in a 6-hydroxydopamine (OHDA) in vitro model of PD  induced-toxicity (Keane et al., Network analyses have been key to identify potential therapeutic targets for PD. For example, network analysis combining genetic and toxicogenomic information identified alvespimycin as a potential neuroprotective agent in PD (Gao et al., Past clinical trials investigating drugs and neuroprotective agents for PD have largely failed to achieve disease modification in part due to the inadequate definition of PD (Espay et al., SNCA (Mittal et al., Given the influence of comorbidities in the disease pathogenesis and progression, we propose that chronic comorbidities such as diabetes and depression should also be taken into consideration when defining PD subtypes Figure . For insCollectively, comorbidities have important implications in the health outcomes and clinical management of PD patients. Increasing evidence from epidemiological studies suggest that diabetes, depression and anemia may appear before the onset of PD thus highlighting the importance of recognizing these comorbidities as potential risk factors for PD. Notably, understanding the molecular and clinical evidence of comorbidities in PD has paved the way for the discovery of novel therapeutic strategies for PD. In this context, common drugs to treat diabetes and cancer are now being tested in clinical trials for PD. In parallel, emerging “big-data” and system-biology approaches are helping in the discovery of biomarkers, therapeutic targets and understanding the molecular mechanisms underlying the occurrence of these comorbidities in PD. We expect that a multidimensional approach to PD incorporating comorbidities will provide new venues for the advancement of individualized treatment in PD patients.JAS and VB wrote the first draft of the manuscript. JAS, VB and JAP edited and reviewed the final draft of the manuscript.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.


As per the requirement of metabolism and fitness, normal cellular functions are controlled by several proteins, and their interactive molecular and signaling events at multiple levels. Protein quality control (PQC) mechanisms ensure the correct folding and proper utilization of these proteins to avoid their misfolding and aggregation. To maintain the optimum environment of complex proteome PQC system employs various E3 ubiquitin ligases for the selective degradation of aberrant proteins. Glycoprotein 78 (Gp78) is an E3 ubiquitin ligase that prevents multifactorial deleterious accumulation of different misfolded proteins via endoplasmic reticulum-associated degradation (ERAD). However, the precise role of Gp78 under stress conditions to avoid bulk misfolded aggregation is unclear, which can act as a crucial resource to establish the dynamic nature of the proteome. Present article systematically explains the detailed molecular characterization of Gp78 and also addresses its various cellular physiological functions, which could be crucial to achieving protein homeostasis. Here, we comprehensively represent the current findings of Gp78, which shows its PQC roles in different physiological functions and diseases; and thereby propose novel opportunities to better understand the unsolved questions for therapeutic interventions linked with different protein misfolding disorders. Glycoproteins are a special class of macromolecules composed of carbohydrate moieties attached covalently to proteins, thus achieving specialized structures to perform a variety of functions Spiro, . The N-gmilieu  cells , which comes into play for binding of AMFR with Ube2G2, an E2-conjugating enzyme  for their maturation and post-translational modifications , where it recognizes already conjugated ubiquitin molecules to substrate protein mediated by another upstream E3 ubiquitin ligase Ram 1 homolog , an enzyme involved in the synthesis of triacylglycerol, interacts directly with Gp78 for polyubiquitination and proteasomal degradation  and ataxin-3, which are targeted by Gp78 for ubiquitination and proteasomal degradation , E6AP and Parkin etc. have thoroughly been investigated, and found to be implicated in multiple cellular pathways, and become the cause of incurable diseases when present in non-functional state  isoforms and GTPases Rac1 and RhoA, while no significant change in Cdc42 was observed -hydrooxyeicosatetraenoic acid (12-(S)-HETE), which causes alterations in cellular architecture and helps in cell motility , were also developed against AMFR to suppress invasion and metastasis. miR-139-5p in colorectal cancer cells and edited miR-376a* in glioblastoma target AMFR showing different effects; while the edited form of miR-376a* leads to increase in AMFR expression, miR-139-5p suppresses metastasis by downregulating AMFR  infected HeLa cells reported increased mRNA levels of AMFR, Insig and BiP, along with upregulated autophagy, whereas several ERAD-associated components were significantly downregulated causing ER stress and unfolded protein response (UPR) inside the cells. Despite the increase in expressions of AMFR and BiP, upregulated UPR and autophagy, and an increase in ER size draw an elusive line between all these components and pathways, which further need to be explored (Carpenter and Grose, Like molecular chaperones, QC E3 ubiquitin ligases also have the roles of surveilling the misfolded or accumulated forms of proteins and degrade them through UPS or autophagy (Chhangani et al., Thirty years since the identification of AMFR as a signaling protein implicated in mediating the metastasis of transformed cells inducing tumor progression, this membrane receptor has been thoroughly investigated later for its many other functions (Nabi and Raz, The importance of AMFR has also increased with establishing knowledge and understanding of its beneficial roles in protection against proteotoxic stresses. Still, much more work is required to elucidate how exactly AMFR E3 ubiquitin ligase bears the burden of maintaining the cellular PQC. It has also been shown that AMFR has a potential to work as an E4 enzyme (Morito et al., Although the indispensable association of AMFR with ERAD pathway has been thoroughly reported, where, in association with other ER membrane-bound E3 ubiquitin ligase complexes and ER resident chaperones, AMFR clears proteotoxic load of unfolded or misfolded protein species generated inside this cellular compartment due to several types of extra-and intracellular stresses. Multiple studies have also provided crucial insights into neuroprotective roles of AMFR; still, more work is needed to understand the applicability of this protein for consideration as a possible future therapeutic target against multiple neurodegenerative diseases. Roles of this E3 ubiquitin ligase in the maintenance of healthy cellular proteome by selectively targeting its substrate proteins for proteasomal degradation, is an area to explore further for developing a better understanding of the therapeutic potential of AMFR gene.VJ and AU executed complete drawing of figures. AK provided critical inputs. AM formulated the entire concept of the manuscript and designed the initial draft of figures. All authors reviewed the manuscript.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.