The adhesion, mechanics, and motility of eukaryotic cells are highly sensitive to the ligand density and stiffness of the extracellular matrix (ECM). This relationship bears profound implications for stem cell engineering, tumor invasion and metastasis. Yet, our quantitative understanding of how ECM biophysical properties, mechanotransductive signals, and assembly of contractile and adhesive structures collude to control these cell behaviors remains extremely limited. Here we present a novel multiscale model of cell migration on ECMs of defined biophysical properties that integrates local activation of biochemical signals with adhesion and force generation at the cell-ECM interface. We capture the mechanosensitivity of individual cellular components by dynamically coupling ECM properties to the activation of Rho and Rac GTPases in specific portions of the cell with actomyosin contractility, cell-ECM adhesion bond formation and rupture, and process extension and retraction. We show that our framework is capable of recreating key experimentally-observed features of the relationship between cell migration and ECM biophysical properties. In particular, our model predicts for the first time recently reported transitions from filopodial to “stick-slip” to gliding motility on ECMs of increasing stiffness, previously observed dependences of migration speed on ECM stiffness and ligand density, and high-resolution measurements of mechanosensitive protrusion dynamics during cell motility we newly obtained for this study. It also relates the biphasic dependence of cell migration speed on ECM stiffness to the tendency of the cell to polarize. By enabling the investigation of experimentally-inaccessible microscale relationships between mechanotransductive signaling, adhesion, and motility, our model offers new insight into how these factors interact with one another to produce complex migration patterns across a variety of ECM conditions. The mechanical and geometric properties of the solid-state extracellular matrix (ECM) can profoundly influence cell motility, proliferation, death, and differentiation Cell motility is classically described as a stepwise process that involves protrusion of the leading edge of the cell, stabilization of nascent adhesions, contraction of the cell body, rupture of rear adhesions, and retraction of the trailing edge, which together lead to net translocation of the cell While it is widely acknowledged that these spatially- and temporally-coordinated signaling events are critical to motility, progress in this field is limited by a lack of computational models that couple these localized signals to cellular motility and force generation. The vast majority of existing models have either focused on isolated molecular-scale components or modeled the entire cell as a continuum structure without significant molecular detail To build upon these ongoing efforts and strengthen our understanding of the molecular basis of cell-ECM mechanosensing in migration, we developed a novel multiscale mathematical model of cell migration, which dynamically incorporates compartmentalized molecular signaling events with adhesion stabilization and rupture, stress fiber contractility, Rac GTPase-dependent protrusion and stabilization of adhesions, and Rho GTPase-dependent mechanical coordination between front and rear adhesions. This model enables us to probe a wide variety of complex cell-ECM adhesion and migration behaviors, ranging from localized spatio-temportal adhesion dynamics responsible for “stick-slip” migration patterns observed on intermediate-stiffness ECMs to the overall dependence of migration speed on ECM stiffness and ligand density. We also validate the model predictions with new, high-resolution phase contrast imaging of human glioma cells cultured on ECM substrates of defined stiffness.We conceptually reduce the cell to a one-dimensional geometry consisting of adhesion clusters with ell body . Our adhell body . Stress The front adhesion grows according to the protrusion/adhesion dynamics model, which includes Rac-dependent formation of lamellipodial protrusions, stabilization of nascent adhesions  exerted by the attached contractile stress fibers and the stored potential energy per bond where Rac-dependent actin polymerization at the leading edge of the cell regulates the formation of lamellipodial protrusions that adhere to the substrate and produce a net propulsive force N; (3) The energy associated with membrane ruffling Here, the first term represents the forward rate of formation of lamellipodial transient adhesions governed by the Rac activation level In addition, a positive feedback loop between Rac activation and rate of protrusion stabilization with a proportionality constant The forward and backward rates for the adhesion size due to receptor clustering, bond rupture, and protrusions, (Eqs. 1–3) are combined to yield a rate of adhesion growth of: F and R denote front and rear as before.where superscripts Contractile actin bundles (e.g. stress fibers) consist of antiparallel arrays of F-actin and a variety of accessory proteins interleaved with filaments of nonmuscle myosin II, whose motor function both directly underlies cell contractility and is regulated by Rho activated signaling through adhesions Here, the first term governs the growth of contractile force sustained by the stress fibers  are always balanced by the rearward forces (right side), which includes both contractile forces applied to rear adhesions and an effective “drag” force imposed by central adhesions, which must be disassembled in order for the cell to translocate. This cell-body drag force  are combined to yield a rate of adhesion growth of: F and R denote front and rear as before.where superscripts Thus, for a polarization factor Our simulations predict that the curtailment in cell polarization induces a biphasic relationship between migration speed and ECM stiffness . These red cells  where miTo gain further insight into the subcellular mechanisms that yield a biphasic relationship between migration speed and ECM stiffness for polarization factor tiffness . On softtiffness , whateve, , and  . The rea 30 min)  compared 30 min) . On inte 30 min) , the subThe overall finding is that cell types that do not have the inherent tendency to polarize  Migration speed is directly related to cycling times between protrusion, adhesion, and retraction; (2) Even on stiff ECMs, insufficient ligand density can reduce cell migration speed  and placsuperior job of simulating 3D migration than one that incorporated 2D details. In future models, it will be interesting to test this hypothesis more directly by explicitly incorporating these topological details. These next-generation models should also offer a good opportunity to include key structural details into the ECM, such as fiber diameter, mesh size, and nonlinear elastic properties, all of which are expected to play key roles in tumor cell invasion A potential limitation of this model is the use of a 1D geometry, which may fail to capture some of the complexities of migration in 2D and 3D topologies. Despite this important simplification, model predictions agree surprisingly well with our experimental observations of 2D cell migration, including migration speed and aspects of lamellipodial extension and force generation. Additionally, given the recent discovery that migration in tissue ECMs may more closely resemble 1D migration than 2D migration Our model also predicts several relationships which could be tested experimentally in the future, including the dependences of front and rear adhesion sizes, contractile forces, time between migration cycles, and migration speed on a wide range of ECM properties. We also provide a potentially testable mechanistic explanation for why some cell types exhibit a biphasic dependence of migration speed on ECM stiffness that is rooted in the tendency of that cell type to polarize. Experimental validation of these predictions should facilitate even more sophisticated and experimentally informed modeling efforts in the future, which may both lend greater insight into the underlying biological problem and permit simulations of cell migration in more complex microenvironments that better approximate the in vivo setting.Figure S1Model calculations of “stick-slip” migration phenotype in terms of normalized values of adhesion size ((EPS)Click here for additional data file.Video S1Phase contrast video of rear edge retraction of a U373-MG cell migrating on a 119 kPa fibronectin-conjugated polyacrylamide ECM.(AVI)Click here for additional data file.Video S2Phase contrast video of rear edge retraction of a U373-MG cell migrating on a 0.8 kPa fibronectin-conjugated polyacrylamide ECM.(AVI)Click here for additional data file.Text S1Glossary of time-dependent variables and model constants.(DOC)Click here for additional data file.


Up to 40% of Hodgkin lymphoma (HL) cases are associated with the Epstein-Barr virus (EBV). Clonal viral genomes can be found in the HL tumor cells, the Hodgkin Reed-Sternberg cells (HRS). The latent infection results in expression of the viral oncogenes LMP1 and LMP2A which contribute to generate the particular phenotype of the HRS cells. EBV does not only undergo epigenetic changes of its genome during latency, but also induces epigenetic changes in the host genome. The presence of EBV may alter the composition and activity of the immune cells surrounding the HRS cells. EBV favours a Th1 reaction, but this attempt at a cell mediated immune response appears to be ineffective. The presence of EBV in HL is associated with several clinicopathological characteristics: It is more frequent in cases with mixed cellular histology, in males, in children and older adults, and in developing countries, while the young-adult onset HL of nodular sclerosis type in industrialized countries is typically EBV-negative. Countries in the Mediterranean area often show an intermediate epidemiological pattern. Recent studies suggest a genetic predisposition to develop EBV-associated HL. Circulating EBV-DNA may serve as a biomarker to monitor response to therapy, and eventually, EBV will become a target for therapeutic intervention also in HL. Since its first description by Sir Thomas Hodgkin in 1832, the nature and cellular origin of Hodgkin’s disease has been an enigma. There was a long-lasting controversy as to whether Hodgkin’s disease was a malignant, inflammatory or infectious disease. Hodgkin’s disease is characterized by a rare population of Hodgkin Reed-Sternberg (HRS) cells which are surrounded by a massive inflammatory infiltrate. The paucity of the HRS cells hampered their biological characterizationIn the 1990s, finally, amplification of immunoglobulin genes by the polymerase chain reaction from isolated HRS cells helped to clarify the nature of the HRS cellsAn infectious aetiology has long been suspected in HL, and so far, Epstein-Barr virus (EBV) is the only candidate for the infectious agent causing HL. There are several lines of evidence linking EBV to the aetiology of some HL: the biological plausibility of EBV-mediated B cell transformation and presence of clonal EBV genomes within HL tumor cells, which implies that infection occurred before malignant transformation, epidemiologic associations with infectious mononucleosis (IM), representing symptomatic primary EBV infection, distinctive EBV antibody titer profiles and viral loads both pre- and post-HL diagnosis, and differing demographic, clinical, and epidemiologic characteristics of EBV+ and EBV− HL. Together this evidence strongly suggests that these virally defined variants of HL are distinct entities and that their pathogenesis should be considered separately. However, only a varying proportion of HL cases are EBV-associated. Despite extensive research, no other infectious agent has been so far identifiedThe large majority of Hodgkin lymphoma arising in the setting of HIV infection are pathogenetically linked to EBV, with rates of EBV positivity ranging from 80 to 100%This review will discuss the role of EBV in the pathogenesis of HL, the impact of EBV on immunological and clinical characteristics, the epidemiological evidences for a role of EBV in HL, and potential clinical applications. There have been excellent reviews on this topic, and therefore, we will focus on data from more recent publicationsEBV or human herpes virus 4 is closely associated with the GC B cell malignancies Burkitt lymphoma, post transplantation lymphomas and HL. HL is one of the most frequent lymphomas in the western world and can be divided into lymphocyte predominant (LP-HL) and classical (cHL) including mixed cellularity, nodular sclerosis and lymphocyte-rich subtypes6 B cells being latently-infectedEBV preferentially infects human B cells, in which it mainly persists as a harmless passenger. Over 90% of people worldwide are latently infected with the virusAfter infection of B cells the 172 kbp viral DNA genome circularizes to an episome in the nucleus and is subsequently amplified to 10–100 copies per cellIt is believed that the tumor cells in cHL originate from preapoptotic GC B cells. This is supported by the finding that in 25% of cHL cases the HRS cells carry B cell receptor (BCR)-destructive mutations in originally functional V-gene rearrangementsThe transcription factor NF-κB seems to play a major role in the rescue of HRS precursor cells. NF-κB activates the expression of the anti-apoptotic DISC-inhibitor c-FLIP and induces the expression of pro-inflammatory cytokinesRecent papers addressed the effect of EBV gene products on the expression of B cell-specific differentiation antigens. Vockerodt and colleagues used a non-viral vector-based method to express LMP1 in primary human GC B cellsA tissue microarray analysis on 288 HL biopsies showed a specific gene expression profile for EBV+ cases. The presence of EBV correlated strongly with the expression of STAT1 and STAT3, but inversely with the expression of cyclin E, CDK6, p27, p53, HDM2, and BCL-XLEBV may modulate cellular gene expression also by induction of epigenetic changes which result in inducing gene silencingModulation of the host methylation system may have implications for the pathogenesis of EBV-associated tumors. For gastric carcinomas, it has been reported that the methylation frequency of tumor suppressor genes is much higher in EBV+ than EBV− cases, indicating a possibility that EBV induces the hypermethylation of cellular genes critical to tumor pathogenesis. Interestingly, activation of DNMT1 appears not to be limited to EBV-induced tumorigenesis, but it was recently suggested to play an essential role in aberrant de novo methylation in various tumors which are associated with viral infection, such as tumors infected with the human papillomavirus-16 or the hepatitis B virusThe EBV genome undergoes changes in CpG methylation which appear to play an important role in regulating viral latency and limiting viral gene expression in normal lymphocytes and in tumorsThe clinical and pathologic features of cHL reflect an abnormal immune response that is thought to be due to the elaboration of a variety of cytokines by the malignant Reed-Sternberg (RS) cells or surrounding tissuesThe presence of EBV may alter the expression of cytokines and chemokines. In fact, EBV favors a Th1 reaction in the HL microenvironment. The expression of IL-12, which is responsible for Th1-cell differentiation, and chemokines that support a Th1 response  are expressed at higher levels in EBV+ HL cases than in EBV-casesIL-6 is another cytokine whose expression has been reported to be increased in EBV+ tumorsA recent study demonstrated an increased expression of CCL20, a chemokine capable of attracting Treg cells, in the microenvironment of EBV+ versus EBV− RS cellsThe development of molecular profiling techniques made it possible to establish more comprehensive gene expression patterns of EBV+ HL tissues. EBV+ tumors are characterized by a robust gene signature involving innate immunity and antiviral responsesWeiss et al were the first to demonstrate the presence of EBV DNA in HL tissue specimens using the cloned BamHI-W fragment of EBV, as a probe for in-situ hybridization (ISH)EBV genome products was shown to be clonal in the neoplastic Hodgkin-Reed-Sternberg (HRS) cellsThe complex epidemiology of HL suggests a multifactorial aetiology. Interactions between genetic and environmental factors are probable. The contribution of EBV to HL aetiology differs according to age group and geographic originThe individual’s age at the time of primary EBV-infection in combination with his genetic background appears to be important factors for the clinical manifestations of the infection. When primary infection with the virus occurs in early childhood, as is typical of developing countries, it is accompanied by, few if, any symptoms. If primary infection in contrast is delayed to adolescence, as often is seen in industrialized countries, it is associated with the clinical syndrome, infectious mononucleosis.A wealth of cohort and case–control studies has shown that mononucleosis is followed by an increased risk of HLAt variance from the typical bimodal age distribution of HL in most developed nations, with an initial peak occurring among young adults aged 15 to 39 years, in developing nations, the first peak occurs earlier, among children under age 15 years. . HL amonWith respect to geographic variation, EBV rates in HL tumors from North America and Europe have been reported to vary between 20–50%, while several studies from Central and South America showed an incidence rates varying from 50 to 95%A study on 277 biopsies from 10 countries revealed a particular high frequency of EBV-positivity (80–100%) in childhood HL from Kenya, Costa Rica, Iran, but also from Greece, while EBV was present in about 50% of pediatric HL cases from Egypt, Jordan, South Africa and the United KingdomThe pattern of HL in Israel is intermediate between the bimodal pattern of Western countries and the pediatric peak seen in developing countries. Patients born in Europe had a slightly lower rate of positivity to EBV (21.8%). This rate was higher in patients from Asia and Africa (27%). In contrast to these groups of Jews, the Bedouin patients, although representing a small group, showed a 66.7% rate of EBV infectionThree epidemiological patterns can be discerned. The type I pattern which is prevalent in developing countries, shows a relatively high incidence in male children, a low incidence in the third decade and a second peak of high incidence in older age groups. Type III, which is usually seen in developed countries, is characterised by a low rate in children and a pronounced initial peak in young adults. The third pattern (Type II), which is described in many Asian countries, is intermediate and reflects a transition between types I and III. In this pattern there is both a childhood and a third decade peak.An interesting question is whether the socioeconomic progress will change the incidence patterns of HL in developing countries. Hjalgrim and colleagues analysed HL incidence patterns in Singapore between 1968 and 2004, during which time period a socioeconomic transition towards Western World lifestyles took placeThe impact of socioeconomic and racial factors on the risk to develop EBV+ HL was studied by Glaser et al in a Californian populationGenetic predisposition to develop EBV+ HL is supported by the association of EBV+ HL with the highly polymorphic human leukocyte antigen (HLA) genes, which vary by racial/ethnic group. Genetic association of EBV+ HL was found with the HLA class I region, including the HLA-A geneThe distinct manifestations of EBV-infection are thought to be affected by the host’s different immune response to EBV, especially by cytokine production. The levels of interleukin (IL)-1a, IL-2, IL-6, and interferon (IFN)-γ have been reported to be elevated in the serum of patients with infectious mononucleosis. There is also increasing evidence indicating that cytokine gene polymorphisms, such as those of the IL-10 and IL-1a genes, have an impact on susceptibility to EBV infection. We studied several polymorphic allele variants of the cytokine genes IL-10  in HL patientsIn Japanese individuals, the polymorphism of TGF-β1 at codon 10 was associated with the development of EBV-related hematologic diseases, such as infectious mononucleosis, however associations with the development of EBV+ HL have not been reported so farThe impact of the tumor cell EBV status on the prognosis of patients with HL remains controversial. Some of the inter-study variation may be attributable to the different epidemiological features of the disease in different geographical settings and some may be related to case selection.Considering the age-stratified model discussed above seems to be important when analyzing the impact of EBV status on the outcome for HL patients. Thus, in young adults, there seems to be a marginal prognostic advantage when patients carry the EBV genome in their tumorDetection and quantitation of free plasma EBV viral DNA could potentially be used as a biomarker of disease activity in EBV+ HL. A number of groups have explored the value of cell-free EBV-DNA viral load quantification in EBV-associated malignancies including HLEBV genomes are detectable in the serum and plasma of EBV-associated HL cases. The origin of EBV genomes in serum/plasma varies in different disease states, in HL viral genomes are present as naked DNA and are probably shed from tumors. Consistent with the notion that cell-free viral DNA may be shed from circulating apoptotic malignant cells, it has been shown that cell-free DNA is present as “naked” DNA rather than as virionsThe variation in sensitivity and specificity of EBV-DNA as a biomarker between these studies and ours may in part be reflected by technical differences in the assays employed. In this regard, the use of new generation reagents are likely to improve results. The sensitivity of real-time PCR varies with the efficiency and purity of DNA extraction, the segment of DNA amplified, the fluorogenic probe used, and potentially, the source of DNA (serum versus plasma). Gandhi et al amplified a region of BALF5, a subunit of the EBV-DNA polymerase gene, from plasma samplesEBV genome copy number in serum/plasma may provide an indication of tumor burden and may prove to be a useful marker for monitoring HL patients. Additional prospective studies are required to further evaluate the use of free plasma EBV-DNA as a biomarker for monitoring response to treatment in patients with EBV+ HL.hi expression in HRS cells is an important negative regulator of HL tumour-associated EBV antigen-specific CD8+ T-cell immunity in cHL, and thus enables HRS cells to avoid T cell dependent immune attackAs the immunotherapeutic approach to EBV+ lymphoproliferative diseases including HL is the topic of another review in this journal, we will limit this subject to some few considerations. The presence of EBV latent antigens in EBV+ HL appear to be an excellent opportunity both for targeted cellular immunotherapy and antiviral strategies. These antigens could act as tumor-associated antigens for EBV-specific cytotoxic T cells (CTL). Antigen processing and presentation appear to remain intact in EBV+ HL, however an impaired CTL response is observed in cases of HL. The study by Gandhi et al. suggests that Gal-1Like other herpesviruses, EBV encodes a thymidine kinase (TK) enzyme which can be a target for purine nucleoside analogs, such as acyclovir (ACV) and ganciclovir (GCV). Latently infected B cells however do not express the EBV-TK transcript or protein, and are unaffected by these antiviral agents. However, exposure of these cells in vitro to arginine butyrate  results in modest induction of some lytic-phase genes and gene products, including TKIn conclusion, studies on the role of EBV in the immunopathogenesis of HL have delivered important insights which form the basis for therapeutic approaches targeting EBV. Recent progress may help to overcome obstacles encountered on the way to a targeted therapy in EBV+ HL.


The precise trafficking and spatial organization of signaling molecules within cells is critical for many fundamental cellular processes. Two interconnected microtubule-based organelles, the centrosome and primary cilium, have been making headlines recently due to their role as central “hubs” for coordinating such signaling events. The centrosome is the major microtubule-nucleating center in animal cells, which polarizes microtubule arrays and thereby directs microtubule-based trafficking toward itself and its associated structure, the primary cilium. Human diseases associated with centrosome-cilium aberrations generally result from defects in the proper assembly of the centrosome or cilium, or the trafficking of proteins to these structures. It is now well established that movement of proteins to and from the centrosome-cilium complex is facilitated by centriolar satellites, non-membranous 70–100 nm cytoplasmic granules that concentrate around the centrosome in animal cells  [6]. CenAzi1 was originally identified as a centrosomal protein Remarkably, Azi1 null mice are viable, and display none of the gross abnormalities commonly associated with either primary or motile cilia dysfunction. Examination of primary mouse embryonic fibroblasts (MEFs) isolated from Azi1 null mice showed that formation and compartmentalization of primary cilia appear normal, with normal distribution of ciliary membrane and transition zone proteins. Similarly, there were no gross defects with regard to centrosome duplication or centriolar satellite composition and distribution in cells lacking Azi1. Ultrastructure analysis of motile multiciliated tracheal epithelia also failed to detect any defects in centrioles/basal bodies and cilia. Collectively, and in stark contrast with the acute Azi1 loss in human and mouse cells, primary and motile cilia structure and function appear grossly normal in Azi1 null animals in vivo, as well as in Azi1 null MEFs grown in vitro.The difference in phenotypes observed following acute knockdown of Azi1 versus permanent deletion of the gene (referred to as “chronic” loss by the authors) is suggestive of a compensation mechanism that, in essence, rescues the defects caused by loss of Azi1. Intriguingly, while such compensation was seen in most tissues, Azi1 null male mice were infertile, exhibiting post-meiotic defects in spermatogenesis. Sperm cells lacked flagella and any remaining axonemes were truncated and immotile, likely due to mistrafficking of proteins required for flagellar assembly. This indicates that Azi1 (and by extension centriolar satellites) plays a critical role in regulating trafficking of proteins in the highly specialized sperm cells that, unlike other ciliated cell types, cannot compensate for loss of Azi1. This tissue-specific phenotype is reminiscent of human mutations in a number of genes required for centrosome assembly and duplication, where the predominant phenotype appears to be microcephaly The functional compensation reported by Hall et al. highlights the complexity and challenge in understanding cilia assembly and function in the context of a developing organism. The basis of the compensation, at this point, is unclear, as it cannot be simply attributed to genetic redundancy, but a mechanistic understanding of the process will be critical if more cases like Azi1 are discovered in the future. Interestingly, the Azi1 null MEFs isolated by Hall et al. are shown to be “active” in compensating for the loss of Azi1; it will be very useful to test whether the compensation can be reversed (or switched on/off) by inducibly expressing exogenous Azi1 in these cells. One interesting strategy would be to utilize the Azi1 null MEFs as an effective in vitro system to understand how functional compensation works in this case, or with centriolar satellites in general. Moreover, this study also highlights the importance of functional follow-up studies of siRNA-based data and cautions against direct extrapolation of transient loss-of-function phenotypes to the genetic in vivo phenotype. On the other hand, transient loss-of-function studies may expose roles of genes that would otherwise be overlooked due to compensation in long-term mutagenesis studies in vivo. As such, it will be important to combine both approaches in future studies.


Stem cells are the fundamental building blocks of life and contribute to the genesis and development of all higher organisms. The discovery of adult stem cells has led to an ongoing revolution of therapeutic and regenerative medicine and the proposal of novel therapies for previously terminal conditions. Hematopoietic stem cell transplantation was the first example of a successful stem cell therapy and is widely utilized for treating various diseases including adult T-cell leukemia-lymphoma and multiple myeloma. The autologous transplantation of mesenchymal stem cells is increasingly employed to catalyze the repair of mesenchymal tissue and others, including the lung and heart, and utilized in treating various conditions such as stroke, multiple sclerosis, and diabetes. There is also increasing interest in the therapeutic potential of other adult stem cells such as neural, mammary, intestinal, inner ear, and testicular stem cells. The discovery of induced pluripotent stem cells has led to an improved understanding of the underlying epigenetic keys of pluripotency and carcinogenesis. More in-depth studies of these epigenetic differences and the physiological changes that they effect will lead to the design of safer and more targeted therapies. Sox2, Oct4, Nanog, and Klf4 is most critical for the maintenance of ESCs [Sox2 is a member of the SRY-related HMB-box family and maintains ESC pluripotency by inducing Oct4 expression [Fgf4, Lefty1, Fbx15, Utf1, and Nanog via Oct-Sox enhancers [Nanog is a homeobox gene which is initially expressed monoallelically in 2–8 cell stage blastomeres and biallelically expressed only in the pluripotent inner cell mass as the embryo matures [Nanog expression seems to encourage differentiation while biallelic Nanog expression maintains pluripotency and is a key regulator in early embryonic development. Klf4 assists Oct4 and Sox2 in regulating various genes including Lefty1 expression, maintains stem cell pluripotency, and has been implicated in differentiation and proliferation [Oct4, and Klf4 can initiate the reprogramming of adult differentiated cells into Nanog expressing induced pluripotent stem cells (iPSCs) [Mammals are complex organisms populated by a cosmopolitan city of cells. Resembling the individual components of a metropolis, cells are the essential building blocks of all tissues and organs in an organism, ranging from the delicate construction of the inner ear to the sturdy femur. Like bricks in a skyscraper, the identity, role, and position of each cell must be carefully regulated to ensure the development of functional organs. However while buildings have to be designed and constructed, some bricks in each multicellular organism can mediate self-renewal and are commonly identified as stem cells (SCs). Embryonic SCs (ESCs) are pluripotent progenitors that retain the capacity to form cells from the three germ layers. These cells rely on a group of transcription factors that regulate a network of genes required for their maintenance and proliferation. Of these transcription factors, the activity of  of ESCs –3. Sox2 pression . Oct4 alnhancers –11. Nano matures . Hence mferation –16. Furt (iPSCs) .c-Myc, in transformed iPSCs which may increase the risk of tumorigenesis in iPSC-based therapies and the low transformation efficiency of adult cells to iPSCs (0.001–2%) [miR302/367 cluster which, when combined with lentiviral transduction, demonstrated 10% efficiency for reprogramming fibroblasts to iPSCs [Since the pioneering experiments that demonstrated the possibility of inducing iPSCs from mouse fibroblasts via retroviral transduction in 2006, increasing interest in iPSCs has led to the discovery of other alternative methods for producing iPSCs . Transdu.001–2%) . More re.001–2%) . More reto iPSCs . These iThe utilization of iPSCs in therapy design and research possesses several distinct advantages over ESCs. Firstly since iPSCs are derived from the patient, there will be a reduced risk of immune rejection compared to allografts or xenografts from ESCs. In addition, it is easier to obtain iPSC precursors from patients compared to the difficulty and ethical concerns associated with the derivation of ESCs. Finally iPSCs are epigenetically different from ESCs and retain predisposition to redifferentiate into their original cell type . This iPDppa5, Ddx43, Rcn3, Sp5) [Withania somnifera extract), and kinase inhibitors  which can inhibit phosphorylation of various genes [The reprogramming of iPSCs from normal somatic cells necessitates a complex epigenetic transformation. To appropriately design the perfect iPSC, it is essential to gain a better understanding of epigenetic differences between iPSCs and ESCs. Recent research has provided insight into the epigenetic memory of iPSCs, a signature artifact from parental cells and the reprogramming process which restricts the ability of iPSCs to differentiate and form cells from alternative lineages , 26. Then3, Sp5) , DNA metn3, Sp5) , 32, antus genes , 34. Staus genes , 36. Henus genes , 38. HowGenetic mutations and differences inherent in both iPSCs and other stem cells cultured in in vitro conditions also have to be evaluated before they can be safely utilized in medical therapies. While some studies have indicated that a subset of stem cells such as mesenchymal stem cells are able to maintain genetic stability for up to six months , 40, morSox2, Oct4, Nanog, and Klf4 have been linked to carcinogenesis, increased cancer malignancy and tumor drug resistance and are overexpressed in many cancers and cancer stem cells [p53 exhibits similar effects by encouraging iPSC formation at the expense of increased risk of tumorigenesis and genetic instability [Despite the limitations in understanding stem cell differentiation and iPSC reprogramming, there has been some progress in verifying the safety of adult stem-cell-based therapies for several diseases. This process is important because many genes activated in stem cells or considered useful in inducing iPSC formation are protooncogenes, and this raises the possibility that stem-cell-based therapies may increase the risk of cancer in patients. For example, the four transcription factors commonly utilized in iPSC reprogramming em cells –51. Additability .Multiple studies have been conducted in an attempt to verify the safety and effectiveness of various stem cell therapies. Some of these successful studies utilize adult stem cells such as bone-marrow-derived mesenchymal stem cells (MSCs) in trials of various regenerative therapies. These cells were first described as MSCs due to their ability to differentiate and form various mesenchymal cells such as bone and cartilage cells . MSCs foMSCs can also be extracted from adipose and synovial tissues, peripheral blood, skeleton muscles, and neonatal tissues like the umbilical cord . AdiposeWhile MSCs extracted from different regions exhibit similar differentiation potential and therapeutic effect, some distinct traits do exist. Human MSCs derived from marrow express different cellular markers from adipose MSCs. For example, adipose MSCs express higher levels of CD49d, CD34, and CD54 while marrow MSCs express higher levels of CD106 . These nHematopoietic stem cells (HSCs) have also been widely utilized in experimental therapies with pioneering studies of HSC transplantation (HSCT) dating back to the 1950s . HSCs coδ32 homozygous donor also resulted in the first successful cure of HIV and has renewed interest in the search of a remedy for this chronic epidemic which has contributed to over 30 million deaths worldwide [HSCT has also been tested in other experimental therapies including lysosomal storage diseases and other metabolic diseases such as Hurler syndrome and X-linked adrenoleukodystrophy leading to improved patient survival rates –98. Receorldwide . In summOct4 overexpression to generate other cell types [The therapeutic potential of neural stem cells (NSCs) has also received considerable interest. Studies have proven that NSCs can be isolated from the mouse bone marrow, striatum, and NSC lines with a stable chromosome number and have also been generated from human iPSCs –103. In ll types . Howeverll types , 106. Hell types –110. A cll types . Furtherll types .The study of how endogenous NSCs can be encouraged to mediate repair has also received considerable interest. NSCs exist in specific stem cell niches in adult mammals such as the ependymal layer and can migrate and differentiate to form functional neural cells in rat EAE models , 114. Th−, CD45−, Ter119−, Sca-1low, CD24+, and either CD49fhigh or CD29high [Many other different categories of naturally occurring stem cells have been identified and investigated for their therapeutic potentials including, mammary stem cells, intestinal stem cells, inner ear stem cells, and testicular stem cells. Mammary stem cell concentration can be enriched by fluorescence-activated cell sorting of mouse mammary glands for cells that are CD31CD29high , 122. ThCD29high , 123. InCD29high . More stCD29high , 126. ThCD29high –129. FurWhile the field of stem cell medicine is rapidly maturing, many potential stem cell therapies remain theoretical or restricted to successes in animal trials which may not translate directly to safe human stem cell therapies. In addition, a notable limitation of human clinical trials involving stem cell therapies is that they often only involve a small group of patients or that the studies were conducted over a short period of several years which may be insufficient to access the true risks of carcinogenesis. Hence the results may not be accurate when extrapolated to determine the long-term safety and effectiveness of these therapies in a larger population. Therefore stem cell therapies should continue to be utilized only as a last resort when conventional therapies have failed or are unavailable, and the condition and response of patients undergoing these therapies should be assessed at regular intervals to ensure their safety.It is also important to note that many currently utilized stem cell therapies and clinical trials have limited impact on alleviating disease symptoms. This is because most current therapies only attempt to treat one aspect of the disease. In addition a limited ability to diagnose the varied underlying causes of similar disease symptoms and the lack of tests to identify the complex differences between individual patients have led to the application of a generic therapy to a heterogeneous population of patients. Consequently better tools to determine the unique genetic and epigenetic factors in each patient that lead to each disease symptom are increasingly essential for maximizing the potential of customized stem cell therapies. Moreover combinational therapies capitalizing on the integration of various approaches such as stem cell transplantation, material science, gene therapy, developmental biology, and pharmacology for simultaneously targeting multiple aspects of each disease will progressively be required for developing next-generation therapies.An improved understanding of the intracellular molecular pathways regulating stem cell differentiation and new methods to manipulate these pathways to maintain genetic stability induce desired epigenetic modifications, and phenotypic changes will also be essential for guaranteeing the safety and potency of future stem cell therapies. To achieve this, the myriad genetic and epigenetic variations in seemingly homogeneous stem cell populations have to be studied in greater detail, and the impact of each variation on differentiation, proliferation, and pluripotency must be quantified.Finally a complete understanding of the mechanisms of stem cell signaling and intercellular communication, such as stromal cell-derived factor-1/CXC chemokine receptor-4 signaling for guiding hematopoietic stem cell mobilization, has to be elucidated . ElucidaIn conclusion it can be seen that although many pioneering discoveries have transformed the way we understand stem cell function, countless studies are still required on the expanding frontier of stem cell research before a complete mastery of stem cell manipulation for maximum therapeutic potential can be achieved.


Reticulons (RTNs) are a group of membrane proteins localized on the ER and known to regulate ER structure and functions. Several studies have suggested that RTNs are involved in different important cellular functions such as changes in calcium homeostasis, ER-stress-mediated cell death, and autophagy. RTNs have been demonstrated to exert a cancer specific proapoptotic function via the interaction or the modulation of specific proteins. Reticulons have also been implicated in different signaling pathways which are at the basis of the pathogenesis of several neurodegenerative diseases. In this paper we discuss the accumulating evidence identifying RTN-1C protein as a promising target in the treatment of different pathologies such as cancer or neurodegenerative disorders. Neuronal death occurs by necrosis or apoptosis, which differ morphologically and biochemically. Necrosis is the result of extreme perturbation of the cellular environment, as occurs in ischemic insults or trauma. In contrast apoptosis is dependent on intracellular pathways which lead to cellular commitment to a defined series of steps leading to cell suicide. Apoptosis is an important mechanism in normal cell turnover and in growth and development, as well as in aging. Alteration of this machinery results in the evolution of cancer and autoimmune or degenerative diseases. The process of neuronal apoptosis involves two principal pathways that converge on activation of caspases: the cell surface death receptor pathway and the mitochondrial pathway . However2+ stores, reduction of disulphide bonds, overexpression of certain proteins, and nutrient/glucose deprivation  (GAKRH)  Figure , the lysLysine acetylation is a reversible and highly regulated posttranslational modification, initially discovered on histones, and known to regulate diverse protein properties including DNA-protein interaction, subcellular localization, transcriptional activity and protein stability . AcetylaIn particular, inhibition of HDACs results in growth arrest and apoptosis of cancer cells while their deregulation has been linked to autophagy induction , suggestAs previously reported, an H4 histone consensus motif has been identified in the C-terminal domain of RTN-1C. All the three positively charged amino acids (KRH) in the H4 consensus are essential for RTN-1C-mediated apoptosis. The function of this reticulon protein has been shown to be modulated by posttranslational acetylation on lysine 204. Worth noting is a direct correlation between RTN-1C acetylation and HDACs activities; in fact the reticulon protein is able to negatively modulate their enzymatic activity . MoreoveConsidering the critical role of lysine acetylation in regulating diverse cellular functions, such as cancer development and human brain disorders , 51, HDAAnother interesting connection between RTN-1C and cancer development may be the reticulon possible involvement in autophagy induction. Autophagic cell death is another important physiological cell death process, involved in development and stress responses. Furthermore, like apoptosis, autophagic cell death is involved in tumorigenesis, even if currently there is no cancer therapeutic approaches that specifically target the autophagic cell death machine.It is now well accepted that there is a complex interplay between apoptosis and autophagy and that the molecular regulators are interconnected; numerous death stimuli are capable of activating either pathway and both pathways share several genes that are critical for their respective execution . In part2+ signalling and induces Ca2+-dependent autophagy. We can assume that autophagy may act as a prosurvival mechanism in response to ER stress and the accumulation of unfolded or misfolded proteins; thus autophagy machinery is perhaps activated to degrade the UPR proteins. Conversely, when the stress becomes severe and prolonged, these fine regulatory mechanisms are not sufficient and the cell undergoes apoptosis. According to this the upregulation of RTN1-C expression results in ER stress induction, finally leading to apoptotic cell death [It has been also suggested that inducing apoptosis via ER-stress and/or autophagy may represent a novel way to kill chemoresistant cells and a promising approach for enhancing efficiency of cancer chemotherapy , 63. Intll death . These dFuture studies should define the molecular details dictating the alternative behaviour of the different RTN family members in autophagy regulation.Another exciting frontier of reticulon research is the field of neurodegenerative diseases. It has been widely demonstrated that the ER stress pathway is implicated in various neurodegenerative pathologies . For exaStudies carried out on postmortem brain tissues have revealed changes in reticulon expression in the temporal and frontal cortex of patients with Alzheimer's disease . In partWe have recently shown, by the use of microarray analysis of the whole human genome, that RTN-1C is able to specifically regulate gene expression, modulating transcript clusters implicated in the onset of neurodegenerative disorders .In vivo studies have also established that enhanced expression of this reticulon family member in the cerebral cortex results in ER stress, leading to a neurodegenerative process characterized by an abnormal synaptic plasticity at corticostriatal synapses [synapses .Another important mechanism involved in neurodegeneration process is the regulation of calcium homeostasis. Dynamic changes in calcium concentration within the ER and alteration of ER calcium homeostasis may be important in regulation of cell function and survival and may trigger various forms of neurodegeneration and/or neuropathy , 79. In 2+ signalling [2+ from intracellular stores in response to receptor stimulation; this suggests that increased cytosolic calcium may be the primary molecular abnormality in this pathology [2+ levels by depleting the ER Ca2+ stores [2+homeostasis is critically involved in aberrant forms of synaptic plasticity in mouse models of AD and schizophrenia [Interestingly, RTN-1C modulates the expression of genes which has been demonstrated to be affected in schizophrenia with a common thread related to Cagnalling . It has athology . In keep+ stores . Matchin+ stores . Further+ stores . Conversophrenia , 83, 84.ophrenia . RTN-1C ophrenia , 87. Intophrenia  identifiReticulons have recently been implicated in other important neurodegenerative disorders; amyotrophic lateral sclerosis, which is a rapidly progressing fatal neurodegenerative disease, is characterized by the presence of protein inclusion in motor neurons. The induction of ER-stress-mediated apoptosis has proved to be an important event in the pathogenesis of ASL; moreover ER occurs early in the disease and involves the upregulation of protein disulphide isomerase (PDI), an important endoplasmic reticulum chaperone . EndoplaSeveral reports suggested that PDI function is particularly important for neuronal cell death because it is able to attenuate the neurotoxicity associated with the accumulation of aggregated proteins which is responsible for neurodegenerative processes . It has Interestingly, a very recent paper has shown that the reticulon family proteins, and in particular RTN-1C, represent novel regulators of PDI intracellular localization and that this phenomenon could be an important modulating factor in amyotrophic lateral sclerosis . In thisAnother interesting link between neurodegenerative disease and reticulons concerns the latter's previously mentioned involvement in autophagic signaling pathway. Autophagy dysfunction has been extensively described in neurodegenerative conditions linked to protein misfolding and aggregation . PharmacRecent studies have reported that autophagy is the major degradational pathway following UPR activation in neuronal cells and constitutes a connection between UPR activation and autophagic pathology in AD brain . In line2+-dependent autophagic pathway which is paralleled by changes in mitochondrial morphology and mitochondrial fission and fusion machinery. RTN-1C-mediated ER stress condition not only triggers alterations in Ca2+ homeostasis and mitochondrial dynamics, but is also capable of inducing an autophagic response. In the context of neurodegeneration, autophagy may constitute a prosurvival mechanism in response to ER stress and the accumulation of unfolded or misfolded proteins; reticulon-1C structural ER protein may modulate the neuronal stress at the basis of neurodegenerative pathologies.We have recently found that (manuscript submitted) RTN-1C induces the activation of a CaFinally very interesting recent work has reported that reticulons are implicated in axonopathy and, in particular, in hereditary spastic paraplegias (HSPs), a group of genetically heterogeneous neurodegenerative conditions. Specifically RTN-2 has been found to be mutated by a complete deletion or a frameshift mutation, producing a truncated protein which is the cause of HSP . These fReticulons are a family of integral membrane proteins implicated in a variety of important biological functions such as ER morphology and organization, nuclear envelope formation, calcium homeostasis, and cell death. In the last few years many experimental data have expanded the reticulons' functions to a wide array essential for neuronal cell homeostasis Studies from our group have focused on the reticulon-1C isoform of RTN1 gene which we originally identified as a GCS interacting protein. Considering the role of this enzyme in the mechanism of cancer development and MDR, we have been interested in characterizing the role of RTN-1C in tumour cells of neuroectodermal origin. During the last few years, we have obtained a series of very interesting results in the field of cancer development: these indicate that reticulon-1C is a promising molecular target for novel therapeutic approaches. We next expanded RTN-1C function to the modulation of neurodegeneration processes. We discovered that it participates in different signaling pathways, from ER-stress-induced cell death to autophagy, through the regulation of different enzymes such as HDACs or PDIs; future studies however would need to define the molecular details regulating the RTN-1C biological functions essential for clarifying its involvement in neural cell pathologies.


The presence of albuminuria has long been recognized as an adverse prognostic feature in patients with renal disease: the patients with appreciable albuminuria are much more likely to develop tubulointerstitial scarring and fibrosis and progress to end-stage renal failure. For many years, it was thought that excess albuminuria was simply a marker of a more severe renal disease, which was more likely to progress as a result of this severity rather than as a result of the albuminuria itself. This conviction was strengthened by the general assumption that albumin was a benign or inert molecule serving primarily to exert oncotic pressure and act as a carrier within the circulation. More recently, this view has been challenged with the accumulation of evidence suggesting that albumin is able to influence the function of cells with which it makes contact in the manner of a signalling molecule.AbbreviationsPTC =Proximal Tubular Epithelial Cells; PDZ acronym combining the first letters of three proteins =post synaptic density protein (PSD95), Drosophila disc large tumor suppressor (Dlg1), and zonula occludens-1 protein (zo-1); DNA=Dexoxyribonucleic Acid; PI 3-kinase= Phosphatidylinositol 3-kinase; mRNA= messenger Ribonucleic Acid. The majority of this reabsorbed albumin is subject to lysosomal degradation and the released amino acids reabsorbed into the circulation [2].A number of morphological studies have established that albumin is reabsorbed by proximal tubular epithelial cells (PTC) by receptor-mediated endocytosis [3], although binding studies suggest the presence of at Ieast two binding sides for albumin in these cells [5]. Ligand blotting experiments indicate that a number of lower-molecular weight proteins may also contribute to albumin binding activity in the proximal tubule [5]. The identity of the albumin receptors in PTC is not yet fully resolved. Compelling evidence indicates that megalin mediates at least a portion of albumin binding in PTC [6]. The accepted function of these receptors is intersperse of a variety of diverse ligands prior to lysosomal breakdown. Thus, megalin is a candidate for albumin binding in the proximal tubule. Interestingly, the cytoplasmic tail of megalin has no sequence homology with other members of the low-density lipoprotein receptor family except for short internalization sequences. This portion of the molecule, however, does contain Src-homology domains, PDZ domains, and a number of protein kinase phosphorylation sites [6]. Sequences such as these, strongly indicate a potentially novel role for megalin in signal transduction. Whether albumin binding to megalin transduces signals to the cell interior is unclear but is currently under investigation.Megalin, a single polypeptide of 4660 amino acids, is a member of the low-density lipoprotein receptor family, and as such, its extracellular domain possesses many similarities with the other members of this family [7]. If serum albumin, completed with bound fatty lipids, is applied to astrocytes, calcium spiking is observed together with an increase in DNA synthesis [8]. Rupture of the blood-brain barrier exposes cells of the nervous system to unusually high concentrations of albumin, and is thus analogous to the situation faced by PTC in nephrosis. Astrocytes exposed to albumin display a number of responses, and it has been postulated that these albumin-evoked responses may contribute to the development of cerebral scarring after brain haemorrhage. Exposure of astrocytes to lipid-free albumin precipitates a marked and sustained reduction of intracellular calcium. The mechanism appears to be via an as yet unidentified albumin receptor stimulating an intracellular messenger to cause calcium to enter intracellular stores [9]. Furthermore, binding of albumin to the endothelium activates an intracellular tyrosine kinase cascade [10]. This protein tyrosine kinase pathway probably regulates transcytosis of albumin across the endothelial monolayer but it is also likely to have other yet unidentified effects on cell function. One such effect may relate to cell survival. If cultured endothelial cells are starved of serum, they rapidly undergo apoptosis.In the circulation, interaction of albumin with endothelial cells critically regulates vascular permeability by modulating intracellular calcium concentrations [11]. These authors postulated that the albumin's ability to act as an endothelial survival factor was not due to its absorptive properties, but was rather a receptor-mediated event.Incubation with albumin abrogates this apoptotic response in a specific manner at physiologically relevant concentrations [12]. Clues to the mechanism of this mitogenic effect were obtained from studies into the regulation of albumin endocytosis by Opossum kidney PTC (OK cells). It was demonstrated that receptor-mediated endocytosis of albumin by these cells was not only regulated by the p85/p110 phosphatidyl-inositol 3-kinase (PI 3-kinase), but that incubation or OK cells with albumin also stimulated PI 3-kinase activity [13]. Subsequently, together with the use ultrapure yeast recombinant human albumin, it was demonstrated that albumin possessed the ability to stimulate a kinase cascade involving PI 3-kinase upstream of pp70s6 kinase [14]. Furthermore, incubation of OK cells with albumin results in activation of p44/p42 extracellular signal regulated mitogen activated protein kinase [15], the activity of all these enzymes being essential for albumin-evoked mitogenesis in OK cells.Several years ago, it was demonstrated that albumin was able to stimulate proliferation of cultured PTC [14]. In health, albumin is not totally excluded from glomerular filtrate, and accordingly micropuncture studies have revealed albumin concentrations in the range of 10-30 μg/ml in the proximal tubules of rodents. Taken together, these intriguing results suggest that low concentrations of albumin in tubular fluid may have an important role in the maintenance of PTC integrity and homeostasis in health. Conversely, chronically increased albumin stimulation in nephrosis may disturb this homeostatic balance, thus favouring the development of the typical renal histological changes of progressive tubulointerstitial disease. Appreciable stimulation of PI 3-kinase and pp70s6 kinase was observed with incubated albumin concentrations as low as 1-10 μg/ml [16]. Very recently, preliminary data has been presented indicating that albumin treatment of human immortalized PTC results in increased levels of hydrogen peroxide [17]. Clearly, some tension exists between the results of these various studies. Although differences in the models used may explain the discrepancies. More work is required to clarify this important area. Other studies have demonstrated a survival factor like the effect of albumin both in macrophages and mouse primary cultures of PTC. This was not associated with activation of PI 3-kinase, but appeared to be due to the ability of albumin to scavenge oxygen-free radicals accumulating in serum-free culture conditions [18]. Among the chemoattractants both monocyte chemoattractant protein-1 and RANTES are released by rat PTC in response to albumin exposure [20]. The production of these chemokines by PTC is dependent on albumin-induced activation of the transcription factor family nf-β [20]. Indeed, substantial evidence has established that the NF-β family of proteins controls the expression of many genes regulating immunological, inflammatory and growth related cellular responses [21]. It seems likely therefore, that albumin induction of NF-β in PTC may lead to the expression of a range of genes relevant to the development of tubulointerstitial inflammation and scarring. As well as the stimulation of intracellular kinase cascades, it is clear that exposure to albumin leads to phenotypic changes in PTC. In vitro studies have shown that a number of pro-inflammatory and vasoactive genes are upregulated in PTC exposed to albumin. Overload of cultured PTC with albumin provokes synthesis and release of endothelin-1 into the basolateral compartment [22]. Daily injection of 2 g of bovine serum albumin into the peritoneal cavity of rats renders the animals heavily proteinuric such that after 7 days they excrete up to 1 g urinary protein in 24 h. If sacrificed at this stage, considerable evidence of PTC proliferation is observed in proteinuric animals compared to controls, as assessed by in situ hybridization for histone mRNA [22]. This observation is in accordance with the effects of albumin on PTC proliferation seen in vitro. Predictably, the situation in the in vivo proximal tubule is more complex than that observed in cell culture. Marked apoptosis accompanies the proliferation seen in vivo in protein-overload nephrosis [22]. The stimulus for PTC-programmed cell death in proteinuria is unclear, but the contribution of albumin-bound fatty acids is likely to be important.The effect of proteinuria on PTC was studied in the non-immunologically mediated protein overload nephrosis model in rats [23]. By careful immunohistochemical analysis of kidneys from proteinuric rats, Abbate et al. [22] showed that, regardless the type of glomerular injury, interstitial macrophages are found preferentially in association with those proximal tubules displaying the most marked accumulation of intracellular protein. Furthermore, co-localization of the chemokine osteopontin with macrophage infiltrates and protein-overloaded tubules was observed [23]. Interstitial inflammation is observed in protein-overload nephrosis and is represented by a conspicuous macrophage infiltrate [ A considerable body of evidence now implicates albumin and other filtered proteins in the pathogenesis of tubulointerstitial disease in the glomerulonephrites. Manipulation of the cell signalling and proinflammatory effects of albumin on PTC may prove an effective approach to therapy of progressive renal disease.


TOR1A; DYT6/THAP1; DYT4/TUBB4a; DYT7; DYT13; DYT21; DYT23/CIZ1; DYT24/ANO3; DYT25/GNAL) and recessive  forms. However most sporadic cases, especially those with late-onset, are likely multifactorial, with genetic and environmental factors interplaying to reach a threshold of disease. At present, genetic counseling of dystonia patients remains a difficult task. Recently non-motor clinical findings in dystonias, new highlights in the pathophysiology of the disease, and the availability of high-throughput genome-wide techniques are proving useful tools to better understand the complexity of PTD genetics. We briefly review the genetic basis of the most common forms of hereditary PTDs, and discuss relevant issues related to molecular diagnosis and genetic counseling.Dystonias are heterogeneous hyperkinetic movement disorders characterized by involuntary muscle contractions which result in twisting and repetitive movements and abnormal postures. Several causative genes have been identified, but their genetic bases still remain elusive. Primary Torsion Dystonias (PTDs), in which dystonia is the only clinical sign, can be inherited in a monogenic fashion, and many genes and loci have been identified for autosomal dominant (DYT1/ Dystonias are a heterogeneous group of hyperkinetic movement disorders characterized by involuntary muscle contractions, resulting in twisting and repetitive movements and abnormal postures Fahn, . AlthougIn PTDs, dystonia is the only clinical sign (apart of tremor), without evidence of identifiable exogenous causes or neurodegeneration. PTDs represent the third most common movement disorder after Parkinson’s disease (PD) and essential tremor, and include about 75% of dystonia cases  gene on chromosome 9q34.11 is found in nearly all mutated cases  gene on chromosome 8p21–p22 were initially identified in Amish–Mennonite families  and variable expressivity  has been identified by exome sequencing , ANO3 , and GNAL , have been identified in families with juvenile-adult-onset cervical or cranial-cervical dystonia and in a few sporadic cases with similar phenotypes , suggesting they could represent a major cause of adult-onset cranial-cervical dystonia PTD.Recently, thanks to the advent of Whole Exome Sequencing (WES) technology, four new genes have been implicated as causative of AD focal/segmental dystonia. In one large Australian family with fully penetrant juvenile-adult-onset “whispering dysphonia,” occasional generalization and alcohol benefit  Collaborative Group, n polymorphism in the dopamine D5 receptor gene has been associated with adult-onset focal dystonia in patients of European ancestry, but not in other cohorts. Similarly, a functional polymorphism  in the brain-derived neurotrophic factor (BDNF) gene and variants in the THAP1 gene have been associated to PTD risk, but with inconsistent results. More extensive work has been performed on TOR1A variants. The p.D216H SNP was reported as a risk factor in patients with familial focal/segmental dystonia, but these results were not replicated in other studies. Several SNPs from the 3′ untranslated region of TOR1A have also been implicated in focal dystonia as predisposing factors in some studies but protective in others. These contradictory data are likely due to confounding factors including the small number of SNPs tested, limited samples size, clinical heterogeneity of PTDs and different ethnicity Although 15 years have passed from the discovery of the first PTD gene  have been found altered not only in focal dystonia patients  and has a protective role by maintaining a homeostatic threshold against the endothelial reticulum (ER) stress. When mutated, its buffering capacity is greatly diminished, its ATPase activity reduced and its ER translocation prevented  have boosted these fields , a rare AD episodic dystonic syndrome characterized by recurrent and brief attacks of involuntary movements triggered by sudden voluntary movements. Overlapping phenotypes include Infantile Convulsions and Paroxysmal Choreoathetosis (ICCA) and Benign Familial Infantile Seizures syndrome (BFIS). PKD, ICCA, and other related phenotypes had been mapped to distinct loci at the pericentromeric region of chromosome 16  can be simultaneously genotyped in large cohorts of patients and controls, followed by a stringent statistical analysis to discriminate real hits from false positive results. In movement disorders, this approach has been largely adapted to PD, although the number of significant hits has been unexpectedly low and only few genes, such as TOR1A mutations are extremely rare and whole gene sequencing is not recommended in clinical practice  should be considered in patients with predominant cranial-cervical and laryngeal involvement, especially if family history is positive, with prioritization according to the age at onset and distribution of dystonia.Diagnosis of dystonia is mainly clinical, yet genetic analysis and counseling is being requested more and more often. In primary dystonias, genetic testing is only available for those forms of early-onset PTD for which the gene is known Figure . TestingThe identification of mutations in a proband requires a detailed assessment of family history, given their dominant inheritance and variable expression. Once a mutation is identified in a proband, carrier testing is available for at-risk asymptomatic relatives who may request it. This type of testing should never be performed on minors and should always be accompanied by pre-test genetic counseling. Since most PTD mutations show reduced penetrance, it is impossible to predict whether a mutation carrier will develop the disease, although such probability will progressively decrease with age, especially for early-onset conditions such as DYT1 and DYT6. Yet, in case of positive testing, they need to understand the 50% risk to transmit the mutation to their offspring who could eventually develop the disease, based on estimated penetrance.Genetic diagnosis in sporadic late-onset PTD is much more complex as several genetic and environmental factors are likely to be involved. GWAS are significantly improving our understanding of the complexity of sporadic PTDs, allowing the identification of genetic susceptibility or protective factors for the development of the disease. Yet, to avoid false expectation from commercially available genetic profiles, it should be remembered that such genetic factors are relevant only in large population studies, while they are of limited significance in personalized medicine (Kumar et al., Prof. Valente serves on the editorial boards of BMC Neurology, Movement Disorders, and Pediatric Research. She is the recipient of a European Research Council Starting Grant, and of research grants from the Italian Ministry of Health, the Italian Ministry of University and Research, the Telethon Foundation Italy and the European Community FP7 Program.


Degeneration of the axon is an important step in the pathomechanism of traumatic, inflammatory and degenerative neurological diseases. Increasing evidence suggests that axonal degeneration occurs early in the course of these diseases and therefore represents a promising target for future therapeutic strategies. We review the evidence for axonal destruction from pathological findings and animal models with particular emphasis on neurodegenerative and neurotraumatic disorders. We discuss the basic morphological and temporal modalities of axonal degeneration . Based on the mechanistic concepts, we then delineate in detail the major molecular mechanisms that underlie the degenerative cascade, such as calcium influx, axonal transport, protein aggregation and autophagy. We finally concentrate on putative therapeutic targets based on the mechanistic prerequisites. The axon represents the largest functional entity in many neuronal populations, e.g. spanning up to more than one meter in human motoneurons. Whereas the dysfunction of the cell soma and its consecutive degeneration in the course of neurological disorders dominated the perception in neuropathology for many years, an increasing amount of data supports the concept of axonal degeneration as the initial pathological mechanism. Thus, it comes to no surprise that many approaches aiming at neuroprotection and mainly somatic mechanisms  have failed in functional long-term trials and in humans. The consideration of axonal degeneration as a major mechanistic step is therefore mandatory for the design of successful therapeutic strategies in disorders of the central nervous system (CNS).The discovery of mutant mice that show a delay in Wallerian degeneration after injury through expression of the WldS  mutant protein , and degenerative diseases, such as Alzheimer’s disease (AD) and Parkinson’s disease (PD), also show pronounced axonal pathology. We have to bear in mind that, although some parts of the degenerative cascade might indeed be similar in different pathologies, their origins may vary fundamentally. Membrane disruption in traumatic brain or spinal cord injury, for example, might eventually result in calcium influx, calpain-mediated cleavage and axonal transport breakdown. Axonal transport impairment, however, may also be caused by aggregation of proteins. Both membrane disruption and protein aggregation can thus converge on the very same common final path having a provenance in completely different pathologies. This illustrates that axonal degeneration cannot be regarded as a uniform sequence of events, but rather should be seen as a process with numerous variants, some of which share common key features.In the following section, we discuss the neuropathological evidence with respect to axonal degeneration from traumatic, inflammatory and degenerative CNS disorders and their corresponding animal models.Traumatic spinal cord injury is a prime example of axonal damage that affects the longest projecting CNS neurons and peripheral afferents. Most cases of traumatic spinal cord injury result in a partial transection of ascending and/or descending tracts and incomplete impairment of sensory or motor functions, whereas only a small fraction represents complete transections  to primary dopaminergic neurons also leads to significant alterations of axonal transport mechanisms in vitro. Interestingly, MPP+ shows effects on mitochondrial movement, but not on synaptophysin-tagged vesicles or other moving particles, demonstrating that substrate specificity contributes to the particular toxicity of MPP+ in axons. In this model, neurite degeneration and an induction of autophagy -mediated expression of mutant human alpha-synuclein (aSyn.A53T) in the substantia nigra, neuronal loss occurs in a significant manner after 17 weeks. However, well before that, as early as 4 weeks after AAV-injection, dystrophic dopaminergic neurons in the striatum are found and a marked change occurs in proteins with functions in synaptic transmission and axonal transport  mutant results in mislocalization of tau protein in dendrites and causes dendrite degeneration. The mechanism is suggested to involve an increased recruitment of the Drosophila glycogen synthase kinase 3 (GSK-3β) homolog (shaggy) by LRRK2 (G2019S), which in turn induces hyperphosphorylation and mislocalization of tau  belong to the most frequent causes of familial PD. Large genome-wide association studies have identified LRRK2 in addition to alpha-synuclein as an important risk locus for the development of PD /MPPAxonal degeneration in amyotrophic lateral sclerosis (ALS) has long been considered to occur only as a secondary process, the result of motoneuron apoptosis comparable to Wallerian degeneration  mouse model of ALS, which is based on a mutation found in familial ALS cases and faithfully reproduces the clinical course of disease in humans  model induced by immunization with myelin oligodendrocyte glycoprotein, show axonal degeneration as an important feature and this can even precede the demyelinating component  salvage pathway in mammals. UBE4b is an E4-type ubiquitin ligase that can add multiubiquitin chains to substrates of the ubiquitin/proteasome degradation pathway -binding site of UBE4b, as has been shown by knock-out experiments. Both a functional NMNAT1 and a functional UBE4b fragment seem to be required for the neuroprotective action of WldS. This is suggested by the observation that, although disruption of the enzymatic activity of NMNAT1 in transgenic WldS mice results in a strongly reduced neuroprotective phenotype  calcium influx from the extracellular space through disrupted membranes, (2) calcium-channel-mediated calcium influx from the extracellular space and (3) release of calcium from intracellular calcium stores  and spinal cord injury  possess alpha-synuclein aggregates in the axons of the sciatic nerve. These animals display increased Wallerian degeneration after axotomy of the sciatic nerve, whereas in mice without alpha-synuclein expression (C57BL/6-Ola-hsd strain by Harlan B6), axonal degeneration occurs at a significantly slower rate . Ubiquitin-positive aggregates have been observed in axonal projections that are known to degenerate in SCA3 patients. It is likely that these aggregates are detrimental to axonal transport mechanisms; this however remains to be established  in axonal lesion models in Kinases involved in inhibitory myelin signalling are also involved in the regulation of axonal stability. Myelin-derived inhibitory molecules, such as Nogo (the neurite outgrowth inhibitor), myelin-associated glycoprotein or oligodendrocyte myelin glycoprotein confer their signal through the trimeric NgR/p75/LINGO-1 or NgR/TROY/LINGO-1 receptor complexes  in axonal stability. Knockout mice for Pi4k2a develop a progressive neurological disease with tremor, limb weakness, urinary incontinence and premature mortality. Axonal degeneration in the spinal cord has been found to be a major histological correlate of the symptoms, whereas peripheral nerves appear essentially normal. Thus, Pi4k2a signalling appears to be crucial for axonal integrity and therefore Degradation of proteins or even organelles occurs in a homeostatic manner via several degradative routes, one of them being autophagy. Autophagy is a collective term and, in this text, we use it to refer to macroautophagy. Because autophagy is a highly conserved and tightly regulated mechanism any disturbance (be it an increase or a decrease) can result in cellular dysfunction.Following mechanical axonal damage in a crush model of the optic nerve, an increased number of autophagosomes has been observed in the vicinity of the lesion site and this is dependent on calcium influx. Inhibition of autophagy attenuates axonal degeneration in this model  has also been shown to be relevant for axonal degeneration. This has been demonstrated by the inhibition of UPS in an optic nerve crush lesion model in which Wallerian degeneration is attenuated by proteasome inhibition. Fragmentation of microtubuli as an early event following crush lesion can be diminished by the application of the proteasome inhibitor MG132 , for which particular axonoprotective effects have been described.Interference with ROCK function has recently been shown to be beneficial in various disease models as it is able to confer neuroprotection and promote axonal regeneration -17 and a marked decrease of the interferon-gamma/IL-4 ratio, CNS demyelination and acute axonal transections are robustly attenuated  cell culture models of PD  inhibitor rapamycin attenuates the accumulation of mutant huntingtin and is neuroprotective in a The regulation of autophagy in a therapeutic manner will largely depend on increasing knowledge of the regulation of this pathway in specific pathologies. Part of this improved understanding must be a more detailed temporal resolution. Autophagic flux might be increased at one point after lesion but may return to normal levels or be inhibited at another. A unidirectional approach, which does not take into account the kinetics of autophagic activity, might therefore result in unwanted detrimental effects by interference with this conserved physiological mechanism.The search for effective compounds or strategies to prevent protein aggregation is one of the hottest topics in current neurodegeneration research. Because of primary axonal damage in the course of aggregopathies, this approach holds the promise of achieving true neuroprotective and possibly neurorestorative effects. One therapeutic approach that has been tested extensively in AD mouse models is the immunization of animals with antibodies against Aβ. In an APP mutant mouse model (APPswe/PS1dE9), the application of an anti-Aβ antibody has not only resulted in a modest reduction of Aβ in the brain, but can also significantly attenuate axonal degeneration resulting in higher axon densities in the cortex and hippocampus of the treated mutant mice  in the substantia nigra is able to improve axonal stability and somatic survival of dopaminergic neurons in the 6-OHDA model of PD. Thus, 6-OHDA-mediated axonal dying-back pathology can be rescued, at least partially, by GDNF Mandel . Kirik eIn an axotomy model, local application of ciliary neurotrophic factor is able to prevent the degeneration of rat facial motoneurons  is a multifunctional cytokine that not only controls erythropoiesis, but has also been attributed neuroprotective properties (e.g. Grunfeld et al. The molecular machinery of axonal degeneration still requires a much better understanding and this can be partially achieved through in vivo visualization of the implicated processes. Here, life-imaging techniques (Misgeld and Kerschensteiner


Protein misfolding cyclic amplification showed that conformation of PrPd partly determines host susceptibility.To assess interspecies barriers to transmission of transmissible spongiform encephalopathies, we investigated the ability of disease-associated prion proteins (PrP C) to the pathognomonic disease-associated isoform (PrPd) (d (d isoforms uses limited proteolysis and classification of the protease-resistant prion protein (PrPres) in terms of the sizes of the nonglycosylated fragment(s) produced and the ratio of the 3 possible glycoforms  and onto PrPC of a different primary sequence (when transmitted between species). In support of this theory, the agent responsible for the TSE of cattle, called bovine spongiform encephalopathy (BSE), the accepted cause of variant Creutzfeldt-Jakob disease (vCJD) in humans  are called prions. Although their precise biochemical composition is a matter of debate, they are known to occur in a series of strains, each with a characteristic disease phenotype and host range  into substrate containing excess PrPC and other essential conversion cofactors can be amplified to readily detectable levels by sequential cycles of sonication and incubation. We have previously reported that the molecular characteristics, electrophoretic mobility, and glycoform ratio of the PrPres associated with the vCJD PrPd conformer were faithfully reproduced by PMCA (PRNP-129) genotype. The most efficient amplification was achieved in a methionine homozygous (PRNP-129MM) substrate; the least efficient, in a valine homozygous (PRNP-129VV) substrate. To estimate the molecular component of transmission barriers for particular TSE agents between species, we used PMCA reactions to amplify PrPd associated with vCJD   6H4 after limited proteinase K digestion. The reaction mixes were split into 2 aliquots; 1 aliquot was stored immediately at –80°C (−PMCA), and the other was subjected to 48 cycles of PMCA (+PMCA)  and MAb 3F4 .We prepared seed and substrate homogenates as 10% (wt/vol) homogenates in PMCA conversion buffer . In contrast, the PrPd conformer associated with the ovine scrapie strain, although sharing the same PrP amino acid sequence as the PrPd in ovine BSE, could not be amplified in any of the PRNP humanized mouse substrates but could be amplified in a sheep brain substrate. These observations are consistent with conformation of a TSE agent’s PrPd (rather than solely its amino acid sequence) having a role in determining the susceptibility of a host’s PrPC to conversion. They similarly suggest that these molecular factors could in turn have a powerful influence on disease susceptibility and incubation time.Our results are best appreciated in terms of the molecular interaction between seed PrPinvolved . RegardlPRNP-129MM genotype, as might be predicted from the efficiency of amplification of BSE-related PrPd shown here. Extrapolating from these results, one would predict that the next genotypic group most likely to show susceptibility to the BSE agent would be heterozygous (MV) at codon 129 of the PRNP gene, as previously suggested from the corresponding in vivo transmission studies (To date, all clinical cases of vCJD have occurred in persons with the In the wake of BSE epidemics in the United Kingdom and elsewhere, enhanced surveillance has identified apparently new TSEs (


Epigenetic-based mechanisms are reversible and the possibility of “resetting” the abnormal cancer epigenome by applying pharmacological or genetic strategies is an attractive, novel approach. Gliomas are incurable with all current therapeutic approaches and new strategies are urgently needed. Increasing evidence suggests the role of epigenetic events in development and/or progression of gliomas. In this review, we summarize current data on the occurrence and significance of mutations in the epigenetic and metabolic enzymes in pathobiology of gliomas. We discuss emerging therapies targeting specific epigenetic modifications or chromatin modifying enzymes either alone or in combination with other treatment regimens.Epigenetic modifications control gene expression by regulating the access of nuclear proteins to their target DNA and have been implicated in both normal cell differentiation and oncogenic transformation. Epigenetic abnormalities can occur both as a cause and as a consequence of cancer. Oncogenic transformation can deeply alter the epigenetic information enclosed in the pattern of DNA methylation or histone modifications. In addition, in some cancers epigenetic dysfunctions can drive oncogenic transformation. Growing evidence emphasizes the interplay between metabolic disturbances, epigenomic changes and cancer,  The extent of chromatin condensation at each locus plays an important role in regulating the access of nuclear proteins to their target sites in DNA and hence directly influences cell functions . The staN-terminal tails, have been described. For example, acetylation of N-terminal lysine residues of histones H3 and H4 is associated with active chromatin, while methylation of lysines 9 and 27 of histone H3 appears to be the hallmark of condensed chromatin at silent loci  Binding Protein 1) binds to single stranded DNA, in particular the FUSE of MYC, and in a complex with PUF60 negatively regulates MYC expression. FUBP1 mutations may lead to activation of a MYC oncogene [ATRX, CIC (homolog of the Drosophila capicua gene), and FUBP1 mutations in gliomas [The protein encoded by oncogene . The HMGoncogene . The mut gliomas . ATRX wa gliomas .RRP22 (Ras-related protein on chromosome 22) has been suggested as a candidate tumor suppressor in human cancers. Its expression was decreased in high-grade gliomas (WHO grades III and IV) compared with low-grade gliomas (WHO grade II) and was associated with shorter overall survival in 180 glioblastoma patients included in the NIH REMBRANDT database. Decreased RRP22 expression was in part explained by 5'-CpG island hypermethylation and acetylated histone H3 and H4. In primary human glioblastomas, the increased levels of H3K9me3-bound and the decreased levels of pan-Ac-H3-bound RRP22 were observed as compared to non-neoplastic brain tissue. It shows that 5'-CpG island hypermethylation and histone modifications may contribute to the frequent and prognostically unfavorable transcriptional down-regulation of RRP22 in malignant gliomas .i.e., inhibitors of DNA methyltransferase (5-azacytidin and decitabin) were approved by the Food and Drug Administration (FDA) in the treatment of myelodysplastic syndrome and a few inhibitors of histone acetylases  are approved in the treatment of hematological malignancies, particularly in refractory or relapsed cutaneous T-cell lymphoma. Other compounds are presently in phase II and III clinical trials [As epigenetic modifications by its nature are reversible, therefore changes in the epigenome associated with cancer are potentially reversible. It opens up the possibility of using “epigenetic drugs” which may have a powerful impact on various cancers, including glioblastomas. A number of small-molecule inhibitors against chromatin regulators have been developed and some of them are already used in clinical cancer treatment or are currently in preclinical and clinical trials . Indeed,l trials ,103. HypTo date, there are a few studies testing the impact of the histone modifying enzymes inhibitors on glioblastomas, some of them show promising results. For example the histone deacetylation inhibitor, suberoylanilide hydroxamic acid crossed the blood-brain barrier, inhibited the proliferation of intracranial gliomas and increased mice survival . The AGIIDH1/2 mutations in secondary glioblastomas which have progressed from low-grade gliomas, suggests that these tumors share a common progenitor cell population which through sequential molecular evolution gives rise to the IDH1 mutant glioblastoma. The observed patterns of DNA changes, DNA methylation and copy number alterations suggest molecular evolution in which the appearance of mutant IDH1 protein in spatially and temporally restricted neural precursors is an initial event, followed by production of TP53 mutant protein, and finally by copy number alterations of PTEN and EGFR [Occurrence of somatic mutations in genes encoding the replication-independent histone H3 variant H3.3 and the canonical histone H3.1, the presence of mutant IDH1 protein in spatially and temporally restricted neural precursors, deregulated expression/activity of epigenetic enzymes may lead to aberrant histone modification and DNA methylation profiles which are intimately linked to glioma pathology. Growing evidence demonstrates a variety of mechanisms, all of which lead to global deregulation in the epigenetic landscape in glioblastomas and support an idea of glioblastoma being an epigenetic malignancy. The high frequency >80%) of % of IDH1Unlike DNA mutations, changes in the epigenome associated with cancer are potentially reversible, which opens up the possibility that “epigenetic drugs” may have a powerful impact within the treatment regiments of various cancers. Though, there are a few studies testing the impact of epigenetic inhibitors on glioblastomas, some of them show promising results and undoubtedly will lead to further preclinical and clinical studies of those compounds in glioblastoma therapy.


Dear Editor,With interest, we read the article by Sidy Secket et al. on the mineral and bone disease in black African hemodialysis patients: A report from Senegal. The authors evaluated 118 end-stage renal disease (ESRD) patients under hemodialysis and showed that chronic kidney disease related mineral and bone disease (CKD-MBD) are frequent in Senegalese hemodialysis patients with prevalence of 66.9 % and hyperparathyroidism is the most commonplace disorder followed by adynamic bone disease and osteomalacia  osteitis fibrosa cystic, in which bone turnover is increased due to secondary hyperparathyroidism, 2) adynamic or aplastic bone disease, in which bone turnover is low due primarily to excessive suppression of the parathyroid glands and in some cases due to aluminum deposition, 3) osteomalacia, in which bone turnover is also low in combination with a marked increase in the volume of unmineralized bone, most current cases result from aluminum deposition in bone among patients who used aluminum-containing antacids as phosphate binders, 4) mixed renal osteodystrophy, a mixture of the first and three type, in which elements of both high and low bone turnover may be observed and presented as marrow fibrosis and increased unmineralized osteoid, and 5- beta 2-microglobulin-associated amyloid deposit’s bone disease in which occur in patients on long-term dialysis and characterized by bone cysts .It appears that the prevalence of the different types of bone disease is considerably different between developed and developing countries , 9. For However in developed countries, the prevalence of secondary hyperparathyroidism among dialysis patients has markedly decreased compared to last few decades and at present, the most common disorder is non-aluminum-induced adynamic bone disease, with osteitis fibrosa due to secondary hyperparathyroidism, osteomalacia, and mixed disease less frequently observed -13.High frequency of non-aluminum-induced adynamic bone disease in developed countries can be explained by multiple factors, including changes in therapeutic strategies such as the increased and earlier use of vitamin D analogs and calcium-containing phosphate binders , 15.Patients' demographics (older and increased number of diabetic patients) may also be implicated in the increasing number of patients with adynamic bone disease among these countries. For example in a bone biopsy study in 84 patients with stage 5 CKD not yet on maintenance dialysis, adynamic bone disease was the most common type of renal bone disease among patients with diabetes mellitus .We have a methodological comment on the study by Sidy Secket et al. The authors did not perform bone biopsy for diagnosing the type of renal bone disease and it is a limited factor for the study . Bone biThe discrepancy between biochemical parameters and the type of renal bone disease was shown in retrospective study of Gal-Moscovici A et al. They compared serum intact PTH levels with results from bone biopsy and showed no correlation between bone biopsy results and PTH levels in 150 to 500 pg/mL and 500 to 1,200 pg/mL. However a significant correlation is observed in this study between adynamic bone disease and PTH levels less than 150 pg/mL .


Historically, the molecular and cellular mechanisms of cerebellar development were investigated through structural descriptions and studying spontaneous mutations in animal models and humans. Advances in experimental embryology, genetic engineering, and neuroimaging techniques render today the possibility to approach the analysis of molecular mechanisms underlying histogenesis and morphogenesis of the cerebellum by experimental designs. Several genes and molecules were identified to be involved in the cerebellar plate regionalization, specification, and differentiation of cerebellar neurons, as well as the establishment of cellular migratory routes and the subsequent neuronal connectivity. Indeed, pattern formation of the cerebellum requires the adequate orchestration of both key morphogenetic signals, arising from distinct brain regions, and local expression of specific transcription factors. Thus, the present review wants to revisit and discuss these morphogenetic and molecular mechanisms taking place during cerebellar development in order to understand causal processes regulating cerebellar cytoarchitecture, its highly topographically ordered circuitry and its role in brain function. The vertebrate brain is a remarkably complex anatomical structure that contains diverse subdivisions and neuronal subtypes with specific, sometimes prodigal, synaptic connections that contribute to the complexity of its function. During development the primordial brain  has to be progressively regionalized. A precise spatial and temporal arrangement of gene expression regulates intercellular and intracellular signals driving a proper molecular patterning that is required for this regionalization. Pioneering genoarchitectural studies and fate mapping experiments established correlations on how morphogens, transcription factors, and other signaling molecules modulate the specification of neuroepithelial territories, to generate the structural complexity and cellular diversity that characterizes the brain  and moves caudally after stage HH15, to coincide with the pre-existent midbrain hindbrain constriction at around HH20–21  and of the medio-lateral cerebellum into four regions that have distinct molecular coding and represent functional domains.The homeodomain transcription factors of Engrailed family  Figures ,E′ are e Figures . Thus, mDistinct neural and glial identities are acquired by neuroepithelial progenitor cells through progressive restriction of histogenetic potential under the influence of local environmental signals. Evidence for morphogenetic regulatory processes at specific locations of the developing neural primordium has led to the concept of secondary organizers, which regulate the identity and regional polarity of neighboring neuroepithelial regions  at the anterior end of the neural plate, the zona limitans intrathalamica (ZLI) in the diencephalon, and the isthmic organizer (IsO) at the mid-hindbrain boundary  seem required for the expression of Otx2, Gbx2, and Fgf8 and the proper formation of the mesencephalic and rhombencephalic vesicles  and transforming growth factor (TGF)-β family members  and lateral (hemisphere) foliation patterns in mammalian cerebellum. Thus, these genes are proposed as a new class of genes that are fundamental for patterning cerebellum foliation throughout the medio-lateral axis acting late in development . However, and despite the well-known participation in coordinating proprioceptive-motor processing, the cerebellum is found to be involved in other very important higher functions such as cognition, emotion, and language processing , rich in neuropil consisting mainly of parallel fibers, purkinje dendrites, and glial cell processes as well as neurons allocated at superficial and deep zones, such stellate and basket cells Figure . The PC , rich inMath1  as early as embryonic stage E9.5 in mice. Math1 is induced by bone morphogenetic protein (BMP) from the roof plate, which itself is differentiating into the Chp  and five GABAergic subtypes . The deep cerebellar nuclei , which encodes a bHLH transcription factor, is expressed at the VZ  and the upper RL (Math1 positive), which generate GABAergic and glutamatergic neurons, respectively  overlap with that of Ptf1a in the VZ  cells were not affected. On the other hand, the lack of Ascl1 led to a dramatic reduction of Pax2 and Olig2 precursors. Interestingly, no change was found in PC development in any of the experiments mentioned above. Thus, the latter evidence suggests that Ascl1 contributes to the generation of GABAergic interneurons and DCN as well as to PC development but not to their specification.The second germinal center, the VZ, has been thought to give rise to cerebellar GABAergic neurons . In addition radial Bergmann fibers act as essential guide rails for the migration of granule cells Rakic,  and contGdf10; Figures Gdf10 was identified as a marker expressed in the PCL of the cerebellum (Zhao et al., Gdf10 is expressed in PCL but not in GCL. Finally, it was further demonstrated that Gdf10 is expressed in BG cells (Koirala and Corfas, Gdf10 localizes to the cerebellar VZ as early as E13.5 and by E15.5, Gdf10-positive cells actively migrate toward the pial surface, as part of a migration process that these cells undergo until postnatal day P7 (Yamada and Watanabe, Gdf10 expression in the PCL becomes progressively more restricted to fine band of BG cells located between the soma of Purkinje neurons (Figures Gdf10 is very limited and Gdf10 null mutant mice develop normally (Zhao et al., Gdf10 is reduced to 15% of the signal obtained from wild type littermates of the same age (4 postnatal months; Rong et al., BG cells, like interneurons and PCs, are born in the VZ of the fourth ventricle, where they express among other lineage-restricted markers growth and differentiation factor 10 ( Figures ,V. Our kAs we mentioned above, the cerebellum and its stereotyped circuitry, contributes not only to motor learning and correction of motor acts, but also to cognitive and emotional functions. Clumsiness and abnormal motor behavior have been well-documented in disorders such as autism and Asperger's syndrome (see Frith, GBX2 predominance over OTX2 and a consequent rostral shift of the IsO (Chizhikov and Millen, MID1 gene. Neuroimaging of human brain patients lacking this gene showed hypoplasia of the anterior cerebellar vermis (Pinson et al., Mid1-null mice show also vermis hypoplasia among other motor coordination defects (Lancioni et al., The combination of a shortened midbrain and/or elongated pons is associated with an enlarged anterior vermis in humans presumably due to a rostral displacement of the IsO, with loss of mesencephalic tissue and gain of cerebellar tissue Figure . In factThe 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.