Skeletal condensation occurs when specified mesenchyme cells self-organize over several days to form a distinctive cartilage template. Here, we determine how and when specified mesenchyme cells integrate mechanical and molecular information from their environment, forming cartilage condensations in the pharyngeal arches of chick embryos. By disrupting cytoskeletal reorganization, we demonstrate that dynamic cell shape changes drive condensation and modulate the response of the condensing cells to Fibroblast Growth Factor (FGF), Bone Morphogenetic Protein (BMP) and Transforming Growth Factor beta (TGF-β) signaling pathways. Rho Kinase (ROCK)-driven actomyosin contractions and Myosin II-generated differential cell cortex tension regulate these cell shape changes. Disruption of the condensation process inhibits the differentiation of the mesenchyme cells into chondrocytes, demonstrating that condensation regulates the fate of the mesenchyme cells. We also find that dorsal and ventral condensations undergo distinct cell shape changes. BMP signaling is instructive for dorsal condensation-specific cell shape changes. Moreover, condensations exhibit ventral characteristics in the absence of BMP signaling, suggesting that in the pharyngeal arches ventral morphology is the ground pattern. Overall, this study characterizes the interplay between cytoskeletal dynamics and molecular signaling in a self-organizing system during tissue morphogenesis. A major objective in the skeletogenic field is to understand the sequential mechanisms that direct specification, condensation and overt differentiation during skeletal chondrogenesis. Local signaling from adjacent epithelia specify prechondrogenic fate in neural crest-derived mesenchyme, which then differentiate into chondrocytes several days later . The criThe classic model of condensation, based mainly on studies of limb and trunk mesenchymal stem cell populations in micromass culture, involves aggregation and rounding up of cells, cell migration towards the center, cell proliferation and an inability of the cells to move away from the center , 7. RemaWe investigated three questions related to the nature of the condensation process, (1) the timing and nature of dynamic cytoskeletal re-organization in specified prechondrogenic cells; (2) the identity of the principle molecular signaling pathways during cytoskeletal reorganization; and (3) and the effect of cytoskeletal reorganization on downstream gene expression required for chondrocyte differentiation. Our focus in this study was restricted to the nature of the condensation process with regard to timing and molecular signaling, and therefore, we did not investigate the magnitude of the physical forces involved.SOX9 expression and prevent chondrocyte differentiation. Thus, our findings indicate that cytoskeletal rearrangement is required for condensation and subsequence overt chondrocyte differentiation, and furthermore, that these cell shape changes regulate the downstream activity of several signaling pathways.Our results demonstrate that ROCK and Myosin II driven actomyosin contractions and differential cell cortex tension within the prechondrogenic mesenchyme drives cytoskeletal rearrangements, and the resultant cell shape changes are a prerequisite for mesenchymal condensation. Cytoskeletal reorganization is responsible for activating downstream BMP and FGF signaling, while negatively regulating TGF-β signaling. We have further determined that BMP signaling is instructive in dorsalizing the proximal pharyngeal condensations, but does not influence the ability of the mesenchyme to condense. Disrupting actomyosin contraction driven cytoskeletal rearrangements alone was sufficient to prevent condensation, inhibit Clemson University IACUC approved the study, protocol number 2011-041. Fertilized chicken eggs were obtained from the Clemson University Poultry Farm and incubated at 38.5°C in a humidified incubator to the desired stage. The embryos were harvested in normal saline solution and staged according to the Hamburger-Hamilton table of normal stages ..nd arch)To determine the timing and physical nature of mesenchyme condensation, morphological progression of the nascent columella was analyzed using fluorescent immunohistochemistry on transverse head sections . At low Progressive cellular and tissue morphogenesis between HH24-32 is revealed at higher magnification . CorrespAs the cytoskeletal components establish a new equilibrium, the previously irregular shaped cells and rounded nuclei adopt a stretched, rhomboid appearance , HH30. AAdditionally at HH28, otic capsule progenitors begin to differ visually, appearing lighter than the columella mesenchyme /(x+y), where x is the length of the major axis of a nucleus and y is the length of the minor axis of a nucleus. When the value of circularity is 1, it is a perfect circle, conversely, when NDI = 0, it is a perfect circle. Therefore, circularity and deformation index share an inverse relationship where the higher the deformation index, the lower the nuclear circularity.We observed significant reductions in circularity in the condensed columella cells at HH30, compared to specified mesenchyme (HH24) and overtly differentiated chondrocytes HH32, . CorrespIn summary, the formation of an organized condensation is observed at HH30, with the appearance of rhomboid-shaped cells, resulting from the synchronous transformation in cellular organization and cytoskeletal architecture. Thereafter, attaining a stable tissue conformation is a prerequisite to completion of chondrogenesis and the onset of overt chondrocyte differentiation at HH32.Extracolumella mesenchyme is located distal/ventral to the columella mesenchyme and is bound by the lateral surface ectoderm, medial pharyngeal endoderm and dorsally situated cardinal vein . The nasAt HH24, the extracolumella mesenchyme cells are densely packed with irregular bundles of cortical actin . By HH26The significant loss of nuclear circularity and increase in the deformation index leads us to conclude that extracolumella condensation occurs at HH26, in contrast to HH30 in the columella.In summary, these data indicate that the process of condensation occurs over a prolonged time period, relying on the combined influences of F-actin organization, cell-cell and cell-ECM contacts. The actual point of condensation coincides with cell nuclei being stretched and distorted from their original rounded shape, and is associated with the appearance of organized F-actin at HH26. This is followed by a period of tissue stabilization before the onset of overt differentiation of chondrocytes at HH34.Overall, although both columella and extracolumella condensation follow the same general pattern of cell shape changes, the timing of condensation and morphology of the condensed cells are unique for columella and extracolumella. We propose that this represents a general mechanism of condensation for the cartilage elements in the head, with the difference between columella and extracolumella being attributed to dorso-ventral positional identity. We will examine this idea in greater detail in the later part of this study.Condensations are defined as an aggregation of previously dispersed cells, resulting in an increase in cell density and upon reaching a critical density these cells will condense . In vivoCell numbers in the columella mesenchyme increased slightly, but not significantly, from HH24-27 during the period when no cell specific F-actin organization was observed . TUNEL labeling revealed that cell death levels were also non-detectable (data not shown). We propose that the cell shape changes occurring during condensation, together with ECM deposition and ECM realignment , 16, 18 In summary, cell shape changes and cytoskeletal re-organization are highly informative measures when defining the spatiotemporal aspects of condensation. These measures may prove more informative than the classical condensation definition , in the pharyngeal and other non-limb condensations. Next, we next tested the requirement for actin based cell shape changes and cytoskeletal reorganization.Middle ear mesenchyme specification in the columella occurs at HH18, condensation at HH30 and overt differentiation at HH32-34 . After HTo test the requirement for actin based cell shape changes and cytoskeletal reorganization we applied 50 μM of Cytochalasin D (an inhibitor of actin polymerization) in the growth medium at HH28, a day before the appearance of the long intercellular processes and rhomboid cell shapes that characterize condensation (HH30) , 21. AnaWe proposed that disrupting actin polymerization affects the pulling force generated by intercellular cell-cell adhesion and the traction forces of cell-ECM connections. The loss of opposing forces forestalled condensation, as indicated by the failure to achieve the rhomboid cell shapes and stretched nuclei observed in control tissue. Additionally, inhibiting polymerization disturbs the tension resistant forces produced by the interaction of cortical actin with the internal cytoskeleton interfering with tissue stabilization , 22.In summary, these data support a model of actin-based cytoskeletal rearrangements and associated cell shape changes that are required to establish the differential tensile forces  driving condensation. We next sought to determine if condensation and stabilization are required for chondrocyte cell fate.SOX9 and COL2A1 are markers of pre-chondrogenic and chondrogenic identity, respectively [SOX9 transcripts in specified chondrocytes in the columella and the otic capsule at HH30. In Cytochalasin D treated explants, however, reduced levels of SOX9 transcripts were detected . The extracolumella also normally exhibits negligible levels of Smad2 phosphorylation. Addition of recombinant BMP protein had no influence on the morphology of the dorsal or ventral condensation, indicating that the activation status downstream of the ligands is critical. Together these data indicate that the ventral condensation morphology is BMP-independent and indeed, is refractive to application of the BMP inhibitor.We also added SB43152, a selective inhibitor of TGF-β signaling along with LDN193189 (BMP inhibitor) to determine whether inhibiting TGF-β signaling rescues the effect of BMP inhibitor on the dorsal columella condensation. SB43152 inhibits TGF-β signaling mediated activation of SMAD proteins by blocking TGF-β superfamily type I activin receptor-like kinase  receptors . AdditioThe columella is formed from replacement cartilage, becoming ossified beginning at E13.5, whereas the extracolumella cartilage persists throughout life . Could tIn summary, these data indicate that dorsal skeletal mesenchyme, regardless of cellular origin, or fate (as persistent/replacement cartilage) requires instructive BMP signaling to adopt a dorsal morphology, whereas the more ventral mesenchyme situated proximally within the arches is BMP-independent. Thus, our results suggest that in the absence of an instructive BMP signal within dorsal condensations such as the columella condensation, dorsal condensations will adopt ventral morphology.Tissue organization during development exhibits coordinated cell behaviors involving tensile forces, differential cell adhesion and actomyosin based cell cortex tension, which regulate multiple morphogenetic processes : germ laOur results identify ROCK-mediated actomyosin contractions together with ROCK-dependent Myosin II generated differential cell cortex tension as essential generators of cytoskeletal reorganization. ROCK inhibition produces no obvious consequences on limb , 53, 54,The definition of condensation conjures images of cell compaction driven by compressive forces. Our results note two major deviations from this standard model; firstly, cell membranes that are stretched during condensation, producing rhomboid shaped cells and stretched nucleus, and secondly, the lowering of cell density in the pharyngeal dorsal and ventral condensations. Both effects are the results of ROCK/Myosin II induced cell shape changes, adaptive focal adhesions and ongoing production of ECM deposits, changes in ECM composition and realignment of ECM components , 54, 56.Remarkably, manipulating the polymerizing activity of cortical actin modulates the downstream molecular response of BMP, FGF and TGF-β signaling. In contrast, manipulating these signaling pathways had no impact on the ability of the mesenchyme cells to undergo condensation. Moreover, although we show that BMP signaling is irrelevant for the process of condensation, it is required for dorsal morphology, indicating that condensation and dorsal cell shape can be uncoupled. The loss of BMP signaling results in ventral condensation characteristics suggesting that the BMP-independent ventral pattern is the ground state for pharyngeal cartilage. FGF signaling from pharyngeal endoderm and the surface ectoderm of the arches during earlier specification  is a potIt appears that the level of Smad2 phosphorylation acts as a readout of condensation in the pharyngeal arches: 2–3% of pSmad2 positive cells occur in a normal dorsal condensation; 47% of pSmad2 positive cells in tissue with BMP inhibition that results in a dorsal to ventral transformation; and in cases of failure of condensation in tissue treated with cytoskeletal inhibitors there are 68–86% Smad2 positive cells. The percentage of pSmad2 expressing cells is significantly different  between the ventrally transformed condensation (BMP inhibitor—LDN193189) and in cases of failure of condensation . Thus, in the absence of negative regulation by BMP, the dorsal mesenchyme has an inherent predisposition to maintain TGF-β signaling, a property unique to the dorsal cells.These data settle an open question on the role of BMP signaling in condensation and overt differentiation. It was proposed that BMP signaling was required for condensation, however addition of BMP2 to a culture of chondrogenic cells prevented condensation, with overt differentiation still occurring in these experiments suggesting that condensation and chondrogenesis could be uncoupled . Our resWhat then, is the significance of BMP induced differential dorsoventral patterning in pharyngeal condensations? BMP acts in an instructive capacity by regulating differential cell cortex tension, which leads to the observed cell shape changes and most likely plays a crucial role in cell fate specification from a prechondrogenic identify toward overt differentiation. Moreover, the stretching of dorsal mesenchyme suggests that they are subject to greater differential cell cortex tension than the more compact, ventral, BMP-independent cells. Perhaps a threshold of differential cell cortex tension is required for cartilage condensation and transient downregulation of TGF-β signaling. Above a certain threshold, condensations adopt either dorsal or ventral morphology with the former having higher levels of differential cell cortex tension due to active BMP signaling. Others have demonstrated that these cytoskeletal dependent physical forces lead to deformation of the internal cytoskeleton and organelles, especially the nucleus . This coThis study together with our previous findings  suggests


Background and aims. Various morphologies are seen in different salivary gland tumorsor within an individual tumor, and the lesions show divers biological behaviors. Experimental results support the hypothesis that increased CrkII proto-oncogene is associated with cytokine-induced tumor initiation and progression by altering cell motility signaling pathway. The aim of this study was to assess the CrkII expression in common malignant salivary gland tumors and pleomorphic ade-noma.Materials and methods. Immunohistochemical analysis of CrkII expression was performed on paraffin blocks of 64 car-cinomas of salivary glands, 10 pleomorphic adenomas, and 10 normal salivary glands. Biopsies were subjected to immu-nostaining with EnVision detection system using monoclonal anti-CrkII. Evaluation of immunoreactivity of CrkII was based on the immunoreaction intensity and percentage of stained tumor cells which were scored semi-quantitatively on a scale with four grades 0 to 3. Kruskal-wallis test and additional Mann-Whitney statistical test were used for analysis of CrkII expression levels.Results. Increased expression of CrkII was seen (P=0.005) in malignant tumors including: mucoepidermoid carcinoma, adenoid cystic carcinoma, and carcinoma ex pleomorphic adenoma, but CrkII expression in acinic cell carcinoma was weak. CrkII expression in pleomorphic adenoma was weak or negative. A weak staining was sparsely seen in normal acinar serous cell.Conclusion. Increased expression of CrkII and its higher intensity of staining in tumors with more aggressive biologic behavior in carcinomas of salivary gland is consistent with a role for this proto-oncogene in salivary gland tumorigenesis and cancer progression. Evaluating the expression status of some tumor sup-pressor genes and proto-oncogenes as the markers of prognosis, differential diagnosis, and selecting therapies are currently being studied widely in sali-vary gland carcinomas. For example, increased Ki67 as a proliferative marker has shown an additional value in prediction of survival in various types of these tumors.7Salivary gland carcinomas constitute about 5% of head and neck cancers.8 It has been shown that Crk could transform mammalian cells in several characteristics like focal adhesion, lamelliopadia formation, cell motility, and epithelial-mesenchymal-like transition.10 It is involved in signaling cascades induced by various stimuli including integrin molecules and cytokines.11 Crk family can be subdivided into two classes, CrkI and CrkII, based on sequence homology.12CrkII is a member of the adapter proteins which contain Src homology-2 (SH2) and Src homology-3 (SH3) domains.13 CrkII plays a key role in intracytoplasmic signaling by tyrosine phosphorylation of cellular proteins.CT10 regulator kinase (Crk) was first discovered as V-Crk oncogene product in avian retroviruses.15 increased mRNA expression of CrkII genes was seen in more advanced stages of lung tumors with poor survival rates.16 CrkII has several important functions that involve regulation of the actin cytoskeleton, phagocytic entry of apoptotic cells, and pathogens into the host cells, as well as cell cycle, apoptosis, andmetabolism.12In addition to dysregulated Crk expression in some malignant neoplasms like glioblastoma and lung cancer,17 we decided to find the expression pattern of Crk II in different salivary gland tumors. Therefore, this study was designed to investigate the expression level of CrkII in common malignant salivary glands tumors with different biologic behaviors and comparison with the most common benign salivary gland tumor (pleomorphic adenoma) and normal salivary gland tissues.In the last decade, the role of CrkII and CrkI in human cancers has extended the reliability of this protein as a tumor marker; however, no studies have been carried out to elucidate the expression pattern of CrkII in salivary gland tumors. According to the well-recognized role of CrkII in breast cancer and its strong correlations with breast cancer staging and also the similarity of secretory carcinoma of breast and salivary gland acinic cell carcinoma,Tumor biopsies were obtained from 64 patients  who were operated between 2001 and 2006 at the University Hospital. The samples include 10 pleomorphic adenomas (PA), 11 acinic cell carcinomas (AcCC), 18 mucoepidermoid carcinomas (MEC), 12 carcinoma ex-pleomorphic adenomas (CaexPA) and 23adenoid cystic carcinomas (AdCC) paraffin blocks from patients with salivary glands neoplasm. Ten normal tissue samples of salivary gland from radical neck dissection surgery were also examined as normal controls.2O2 for 30 min. Immunohistochemical staining was performed using EnVision System . The slides were incubated overnight at 4°C with primary antibody directly against CrkII . The slides were immersed in diaminobenzidine (DAB) for signal visualization. Negative control was prepared by replacing the primary antibody with phosphate buffered saline (PBS).Four micrometers paraffin embedded tissue sections were mounted on glass slides. After the deparaffinization step in xylene, slides were immersed in target retrieval buffer solution . In the next step for antigen retrieval, slides were autoclaved to 121°C for 5 min. Endogenous peroxidase were blocked by incubation in methanol containing 0.3% H19Each slide was semi-quantitatively assigned a score of 0, 1, 2 or 3. Immunoreactivity of CrkII was based on intensity staining pattern of nuclei and cytoplasm with anti CrkII and percentage of stained tumor cells which were analyzed by consensus of two pathologists, as follows: 0, no staining; 1, weak to moderate in <25% of tumor cells; 2, moderate to severe in 25-50% of tumor cells; and 3, strong staining in >50% tumor cells. Scores ≥2 was defined as over expression.Kruskal-Wallis test was used for analysis of CrkII expression levels. To compare CrkII expression levels among different tumor groups, additional Mann-Whitney U statistical test was used. A Pvalue of less than 0.05 was considered to be statistically significant.The frequency distribution of cancer types showed no statistically significant difference in the studied population according to the gender or age group .Staining was not seen in normal salivary glands . A weak Only 5 of the 10 cases of PA cases showed a weak staining and CrkII expression was totally absent in other PA specimens Figure. Expression of CrkII Significantly increased in MEC, CaexPA and AdCC in comparison with normal salivary gland tissue and PA .Additionally, the intensity of staining was pronounced in epithelial cells with more pleomorphic and atypical changes, and in invasive areas (front).In all cases, cytoplasmic staining was seen; however, nuclear staining was not seen. In AdCC, no differences were seen in cribriform, tubular, or solid histologic types.Staining was more in ExPA cells with malignant changes and in areas that were more associated with the invasion to surrounding tissue.Given the potential role of CrkII in tumor initiation and progression, we tested the CrkII expression in malignant salivary gland tumors and PA. The results of our study showed significantly increased expression of CrkII in MEC, CaexPA and AdCC in comparison with normal salivary gland tissue, PA and AcCC. These findings support the hypothesis that this proto-oncogene may alter the signaling pathway of tyrosine phosphorylation and perhaps also some cellular processes like cell growth and cell motility which play a key role in cancer metastasis and migration.20have shown that CrkI/II knock down in breast cancer, a condition relatively similar to salivary glands carcinoma,17 could decrease cell spreading on extracellular matrix. The mechanism mainly related to the ability of CrkII in cell spreading is reducing actin fiber stress and formation of focal adhesion by the ability of Crk II SH2 domain to bind specific related tyrosine kinases. The study by Pasquale et al21shows that suppression of migration of the human malignant breast cancer by EphrinB2 requires phosphorylation of CrkII on Tyr221 by Abl which could possibly disassemble Crk II mediated p130cas/ Crk/DOCK180 complex.To the best of our knowledge, no research has addressed the role and mechanism of CrkII in salivary gland tumours to date; however, Rodrigues et al13 These domains are responsible for assembly of protein-protein complexes.22 Interactions of the SH2 domain of CrkII with specific phosphorylated tyrosine residue motif like YXXXP play a major role in some cellular processes like cell growth and development, cell motility, focal adhesion and migration.24 Studies have shown increased mRNA expression and protein level of CrkII in various types of cancers like glyioblastoma and lung cancer.15 Analysis of different specimens of lung adenocarcinomas and breast tumors have also shown a significant increase of CrkII in stage III compared to stage I.19 We also noted that CrkII expression levels in low grade carcinoma AcCC as well as in benign neoplasm PA of salivary gland was less than those in invasive and metastatic salivary gland tumors. These findings are mostly consistent with the results of previous studies by Yamada et al19 which show overexpression of CrkII is strongly correlated with higher grades in T classification of cancers.CrkII does not have any intrinsic tyrosine kinase activity. However, it has been shown that increased expression level of adaptor protein CrkII could alter the cell signaling pathway by the two Src homology domains SH2 and SH3.26 Thus, increased expression of CrkII in CaexPA, MEC and AdCC are directly associated with enhanced invasive and metastatic properties of these tumors.Cell movement is a complex phenomenon which is dependent on cytoskeleton actin remodeling. It has been shown that recognizing specific phosphorylated tyrosine residues on p130Cas and paxillin by SH2 domain of Crk and DOCK180 by SH3 domain of Crk will activate cell signaling pathway, which triggers cytoskeketal movement and cancer cell invasion and migration.28 These data are in agreement with our observation of more CrkII expression in cells with higher malignant changes and in invasive areas inMEC and ExPA groups.Expression analysis in kidney has shown that CrkII plays a key role in the acquisition of a mesenchymal phenotype by epithelial cells. Down regulation or mutation of E-cadherin or β-catenin will lead to acquisition of a motile mesenchymal phenotype in the MDCK kidney epithelial cells.29Regarding to a great similarity between cell of salivary and mammary gland tumors and the crucial role of CRKII in morphogenesis, migration, invasion and motility of cells in many of malignancy like breast cancer.30 First, CrkII may be used as a potential tool for prognosis of cancers. Second, it may serve as a therapeutic target in some cancers like glioblastoma and breast cancer using RNAitechnology.16 It has been found that siRNA-mediated gene silencing of Crk significantly reduced migration and invasive behavior of tumor cells in both glioblastoma30 and breast cancer.31 However, there seems to be no study on the status of CrkII expression in malignant and benign tumors of salivary glands.We investigated the expression status of CrkII, and its possible relationship with invasion and metastasis in salivary gland tumors. This study reports CrkII protein expression in carcinomas of salivary glands and provides evidence that the CrkII proto-oncogene may plays an important function in aggressiveness of salivary gland cancers. Recent studies described two roles for CrkII, as a marker in tumor cells.In conclusion, increased expression of CrkII and its higher expression in the cells with higher malignant changes and in invasive areas (front) at carcinomas of salivary gland with more aggressive biologic behavior may be consistent with a role for this proto-oncogene in salivary gland tumorigenesis and cancer progression. Further studies are required to establish the role of CrkII suppression as a possible novel therapeutic approach for salivary gland tumor metastasis. Also, investigating its relation with clinicohistopathologic factors influencing prognosis and treatment is recommended.This study was supported by a grant (388149) from Tehran University of Medical Sciences. A part of the study was performed as a DMD thesis. The authors acknowledge the Dental and Periodontal Research Center at Tabriz University of Medical Sciences.


Recent progresses in stem cell field, in particular in the generation of induced Pluripotent Stem Cells (iPSCs) has made possible for the first time obtaining substantial amounts of human cells to recapitulate in vitro some of the key pathogenetic processes underlying MNDs. In the present review, recently published studies involving the use of iPSCs to unravel aspects of ALS and SMA pathogenesis are discussed with an overview of their implications in the process of finding a cure for these still orphan disorders.Motor neuron diseases (MNDs) are neuromuscular disorders affecting rather exclusively upper motor neurons (UMNs) and/or lower motor neurons (LMNs). The clinical phenotype is characterized by muscular weakness and atrophy leading to paralysis and almost invariably death due to respiratory failure. Adult MNDs include sporadic and familial amyotrophic lateral sclerosis (sALS-fALS), while the most common infantile MND is represented by spinal muscular atrophy (SMA). No effective treatment is ccurrently available for MNDs, as for the vast majority of neurodegenerative disorders, and cures are limited to supportive care and symptom relief. The lack of a deep understanding of MND pathogenesis accounts for the difficulties in finding a cure, together with the scarcity of reliable  Concerning their epidemiology, the prevalence of MNDs accounts for about 5–7 cases in every 100,000 people, with an annual incidence of approximately two new cases per 100,000 people [ type 1) .The biological substrate of MNDs is responsible for the extremely disabling clinical phenotype, characterized by progressive weakness with muscle wasting, eventually leading to paralysis and death, mostly secondary to respiratory insufficiency . No effeMNDs are usually differentiated relating to the subset of motor neurons that are mainly involved in the disease course. Upper motor neurons are especially affected in primary lateral sclerosis (PLS) and hereditary spastic paraplegias (HSP). On the other hand, SMA, progressive muscular atrophy (PMA), SBMA and hereditary motor neuropathies (HMNs) involve mainly lower motor neurons.C9ORF72 are found in 40% of cases, followed by mutations of superoxide dismutase-1 (SOD1) (20% of cases), TARDBP (4% of cases), FUS (4% of cases) and other genes [ALS is the most common adult form of MND, characterized by the simultaneous degeneration of UMNs and LMNs. ALS can be divided into a sporadic form (sALS), representing 90%–95% of all cases, and a familial form (fALS), accounting for the remaining 5%–10% of cases . The spoThe group of SMAs comprises a series of LMN disorders characterized by extreme heterogeneity in both clinical presentation and genetic condition.SMN) gene mapped in 5q11.2–q13.3 [SMN are localized on chromosome 5q13: the telomeric SMN1 gene and its inverted centromeric homologue SMN2. SMN2 only differs from SMN1 for five base pair changes, of which a C to T substitution at +6 of exon 7 (c.840C > T) is the only nucleotide change in the coding region [pre-mRNA of SMN2 that excludes exon 7 from the majority of SMN2 transcripts. The result is the production of 10%–50% of the full-length functional protein and 50%–90% of a truncated, non-functional and unstable transcript (SMNΔ7) [SMN2, which correlates to the severity of the disease. The exact functions of the SMN protein, as much as the reasons accounting for the disruption of MNs in SMA, are yet to be fully disclosed.The most prevalent forms of SMAs go under the name of “Proximal Spinal Muscular Atrophy”, often referred to simply as “SMA”, and are caused by genetic mutations on chromosome 5q (hence the term “5q-SMA”). SMA is the most common MND during childhood: with an incidence of 1:6000–1:10,000 live births and a carrier frequency of 1:40–1:60, it is the leading genetic cause of infantile mortality ,12. SMA .2–q13.3 . In the g region . This is (SMNΔ7) . All indSMN1 and present with early denervation weakness, but different clinical symptoms than those stated above, including joint contractures (infantile SMA with arthrogryposis—XL-SMA), distal rather than proximal weakness , diaphragmatic paralysis (SMA with respiratory distress 1—SMARD1), and pontocerebellar degeneration (SMA with pontocerebellar hypoplasia—SMA-PCH) [Further, less common forms of SMAs recognize defects in genes other than SMA-PCH) ,18.The inaccessibility to the cell type of interest majorly involved in MNDs and the lack of established models for the elucidation of pathogenetic mechanisms underlying such disorders represent a fundamental obstacle for progresses to be made in the field of therapies development. In order to resolve these issues and make durable discoveries, new strategies should be taken into account. In this respect, stem cell technology may represent a valuable solution.in vitro and give rise to teratomas in vivo. They can be identified with stem cell markers and resemble embryonic stem cells (ESCs) both in morphology and behaviour, but they are not burdened by ethical concerns [in vitro models that recapitulate mechanisms responsible for the establishment of pathologies [in vitro models obtained from iPSC-derived differentiated cells, like iPSC-MNs, exhibiting the affected genotype peculiar to the disease. So far, the great potential of iPSCs led to the generation of patient-specific cells for several neurodegenerative disorders, including Alzheimer’s Disease [Induced pluripotent stem cells (iPSCs) are originated from patients’ differentiated cells (oftentimes fibroblasts) through the use of reprogramming factors, first identified by Yamanaka in 2006 . IPSCs aconcerns . Indeed,hologies . Obtainehologies . Further Disease , Hunting Disease , Parkins Disease  and Amyo Disease . These cSOD1) [i.e., the role of the oxidative damage). The identification of C9ORF72 repeat expansion as the major factor responsible for ALS onset in the familial forms has focused attention on the causative role of alterations in RNA metabolism [TARDBP and FUS genes (encoding DNA/RNA binding proteins) in ALS development [Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with adult onset. Symptoms reflect the dysfunction and death of motor neurons (MNs) and are characterized by muscular weakness and atrophy progressively leading to paralysis . Upper aSOD1) , researctabolism ,34,35, aelopment .in vitro some of these pathogenetic hypotheses and to model and investigate early disease mechanisms. Eggan’s group pioneered the field in 2008, when they investigated the potential of human MNs derived from embryonic stem cells (ESCs) to provide key data on ALS pathogenesis [SOD1 mutated mice. After 10 days in culture, a significant decrease in MN number could be observed in a time-dependent manner, thus suggesting a strong implication of non-cell autonomous mechanisms in ALS onset. Moreover, they tested if the same toxic glial effect could be detected with human interneurons and they found that the glia-induced toxicity is rather specific for MNs: interneurons treated for 20 days with SOD1G93A glia-conditioned medium appeared fully preserved. Complementary, MNs co-cultured with SOD1G93A mouse embryonic fibroblasts remained unaffected, thus indicating that the toxic effect is specifically related to the presence of astrocytes. Oligonucleotide arrays were exploited to identify genes differentially expressed in mutant glia. After these analyses, Di Giorgio and colleagues focused their investigation on the role of prostaglandin D2, which resulted responsible for a significant decrease of MN survival in culture. This work proved the advantages to use human cells for in vitro disease studies and provided the basis for further investigations on ALS pathogenesis.The establishment of human cell platforms has allowed for the first time to test ogenesis . ESC-derSOD1 in human MNs [SOD1 iPSC lines presented a further reduced survival when co-cultured with SOD1 glia, but this effect was much more amplified in the latter case implying the presence of a strong cell-autonomous component in ALS pathology. SOD1 MNs presented also an unhealthy morphology with shorter processes and reduced soma, thus summarizing the changes observed in the human pathology. Importantly, ZFN-mediated gene correction of SOD1 mutation resulted in the rescue of both altered morphology and reduced lifespan. Data from RNA-seq of SOD1 MNs highlighted a strong down-regulation of genes related to mitochondria functions and protein translation. Further analyses showed impairment in mitochondria motility in addition to an enrichment of mitochondria number located in neuronal processes. A substrate of ER stress has been found in healthy human MNs, which could be related to the cell size; this result is in line with the well-known early degeneration of largest alpha MNs in ALS. A combination of oxidative and ER stress and the up-regulation of the unfolded protein response (UPR) have been shown and could be additive causative mechanisms of neuronal toxicity. Moreover, the electrical activity of MNs could be involved in the ER stress, a result which nicely correlates with the report of the intrinsic hyperexcitability displayed by iPSCs-derived MNs from ALS patients [C9ORF72 and SOD1 lines led to the discovery of common pathways downstream of these mutations related to enhanced oxidative stress response and decreased mitochondria activity.The same research group proceeded beyond these results with a recent study aiming to elucidate pathways which are impaired by the expression of mutated uman MNs . Kiskinipatients . FinallyC9ORF72 iPSC lines were obtained by Sareen et al. to investigate the pathological processes underlying the most common genetic form of ALS [C9ORF72 was due to either gain of function or loss of function or both mechanisms. To address this question, iPSCs lines were developed from different C9ORF72 hexanucleotide expansion carriers affected by ALS and/or frontotemporal lobar degeneration (FTLD). Southern blot analyses were performed on derived MNs assessing the expansion and also highlighting differences among patients, which in some cases could be reflected in more severe forms of the disease. FISH analyses detected the presence of RNA foci within C9ORF72-ALS MNs , in line with previous data from patients’ tissues [C9ORF72 and TDP43 ALS forms. This work provided data in the direction of a “gain of function” mechanism linked to C9ORF72 hexanucleotide expansion. It has been shown that the mutated allele is usually transcribed and its downregulation using antisense oligonucleotides did not affect cell viability, but resulted in the correction of cell transcriptional profile. The specific pathogenetic role of the involvement of different RNA binding proteins interacting with RNA foci needs further investigations and could represent a common causative mechanism shared by different forms of ALS. Indeed, the aberrant cytoplasmatic aggregation of TDP43 represents a rather common pathological hallmark both in familial and sporadic ALS. Processes leading from the cytoplasmic aggregation to the selective MN loss appear to be exquisitely human and not easily detectable in animal models, where overexpression of TDP43 is ubiquitously provoked. Indeed, the majority of cell platforms and animal models considered in ALS pathogenesis studies relied on the overexpression of TDP43 in nonhuman or nonneuronal cells. Reason underlying the selective vulnerability of MNs to the disease process could be misled as well as the investigation of key molecular events that cause the human disease [et al. generated iPSCs from a patient affected by ALS carrying the TDP43 M337V mutation and used them as a tool to investigate TDP43 pathology in human neuronal cells [m of ALS . They in tissues . Observe tissues  and its  disease . The estal cells . No diffet al. proceeded beyond these data to elucidate the role of TDP43 in physiological conditions and its impairment in ALS related disease [Alami et al. could investigate the causative role of mutant SOD1 in impairing neurofilament (NF) turnover within MNs [D90ASOD1 mutation, thus obtaining isogenic controls. A modified differentiation protocol was applied to originate synchronized mature cells avoiding the generation of later-born MNs or glial cells.Neurofilament aggregation is a well-known pathological hallmark of ALS. Thanks to iPSC-based technology, Chen thin MNs . To bypaIndeed, challenges that need to be overcome when producing iPSC-derived neurons involve both the generation of immature cells and significant differences in the time rate of differentiation among iPSC colonies. These issues could lead to the production of heterogonous populations in terms of neural differentiated phenotype. The optimization of differentiation protocols has allowed generating neural cell populations that are synchronized regarding their growth in culture, in order to properly observe and investigate all the phases of their development .in vivo when patients present phenotypically asymptomatic or with very mild symptoms. This effect was due to the presence of mutated SOD1, as demonstrated by gain of function and loss of function experiments. Further analyses revealed that binding of SOD1 to 3′ UTR of NF-L mRNA could cause the alteration of NF structure, making them prone to aggregate. These events were selectively present in ALS-MNs and not in control cells or non-MNs. Moreover, human ALS MNs presented in vitro lower/normal levels of SOD1 (and increased amount of NF) compared to control MNs, strongly contrasting data from animal models where mutant SOD1 levels are much higher.NF subunits in ALS-MNs appeared to be unbalanced and developed a tendency to aggregate leading to neurite degeneration, an effect that probably interests the early phases of the disease in vitro has provided crucial data for this purpose  were able to reduce MN degeneration and increase lifespan in a murine model of mutant SOD1 ALS [Overall, these results highlight the importance of validating data obtained from animal models in human cells and using them as a precious tool to elucidate mechanisms which are peculiar of human physiology and pathology. The identification of the mechanisms underlying the development of ALS is crucial for the discovery of new therapeutic compounds. Indeed, the elucidation of the role of misregulated neurofilament turnover , granula purpose . The optentified . FinallySOD1 ALS . ALS astSMN1 gene. RT-PCR analysis also detected a few truncated transcripts lacking exon 7, along with the full-length SMN transcripts, witnessing the maintenance of functional SMN2 gene and its alternative splicing. Since lower alpha MNs are the main target of the pathological processes of SMA, iPSCs from SMA and WT fibroblasts were directed towards a motor neuronal and glial fate, in order to disclose in which phase of maturation the pathogenetic events occur. If in the early stages of the differentiation protocol a robust production of MNs was described and no significant difference was documented between the SMA- and WT-derived MNs, interestingly, around week 10 of the protocol, the SMA-derived MNs appeared to be decreased both from a quantitative and a qualitative point of view, as they were fewer and smaller compared to the WT-derived MNs. It was then speculated that the SMA phenotype hampers MN ontogenesis at later developmental time periods, either by inhibiting the generation of new cells or increasing the degeneration of those already matured. This was the first study to report that human iPSCs can be adopted as a reliable model of a genetically inherited disease, such as SMA, and serve as a powerful tool for a better understanding of its etiopathogenesis.Ebert and Svendsen were the first authors to demonstrate that human iPSCs can be employed to recapitulate the specific pathology that underlies SMA . Until tin vitro [Sareen and Svendsen’s group carried on the experiment for a more precise definition of the mechanisms that are responsible for the post-developmental damage that leads to cell death, as observed in vitro . Sareen in vitro . This wain vitro . Taken tSMN2 sequence-specific oligodeoxynucleotides. The correction of SMA iPSCs consisted in the exchange of a T to C at position +6 of exon 7, thus converting SMN2 into SMN1 through the inclusion of exon 7 in SMN2 transcripts. Subsequently, the model required the differentiation of SMA fibroblasts-derived iPSCs, both treated and untreated, into MNs, the cell-type majorly involved in SMA, in order to evaluate the effects of SMN1 deficiency on these cells. At week 8 of the multistage differentiation protocol, based on the administration of Retinoic Acid and Sonic Hedgehog, they observed specific disease effects of the SMN1 defect on untreated SMA iPSCs-derived MNs compared to the cultures treated with oligodeoxynucleotides. The statistically significant alterations included an overall reduction in cell soma size, axonal elongation and in the ability to form neuromuscular junctions, resulting in a decreased survival of MNs derived from untreated iPSCs. This experiment well demonstrates that a SMA model reproducing at least some aspects of the disease can be generated, and that MN dysfunction in SMA is the result of multiple neuropathological events likely occurring at a late differentiative state. Genetic correction of SMN2 via oligodeoxynucleotides proved to rescue the cellular damage to the defective SMN1 gene and to significantly increase the number of detectable gems, nuclear aggregates of SMN protein which correlate with the rate of translation of the full-length protein.Another contribution to the knowledge on the pathogenic mechanisms of SMA was given by Corti and colleagues . Using aSMN1 deficiency.Given the recent hypothesis on the role of RNA and splicing abnormalities as a primary determinant of selective MN death in SMA ,53, the SOD1 mutated models of ALS [Following the hypothesis that a cell population other than MNs may be implied in the activation of the apoptotic cascade, the attention was then focused on glial cells. Indeed, astrocytes had already proved to contribute to MN dysfunction in s of ALS ,55 and ts of ALS . In ordes of ALS , the moss of ALS . They deOverall, the possibility to isolate cell types that are specifically damaged in the disease process, like MNs and glia cells in SMA, makes iPSCs an unprecedented tool to disclose pathogenetic mechanisms that have been inaccessible so far . MoreoveSOD1 which characterizes mutant SOD1 mice may influence observed molecular phenotypes [SMN2 gene, which plays a crucial role in determining clinical phenotypes [in vitro studies. On the other hand, the development of iPSC-based technology has allowed to generate human pluripotent cells in abundance to perform any in vitro study bypassing ethical constraints [i.e., fibroblasts or astrocytes) into relevant cell types  [The pathogenesis of several MNDs is still obscure under multiple aspects, thus hampering the development of potential therapies . Experimenotypes . With reenotypes . As a coenotypes ,61, but straints . Furtherstraints . Anotherstraints . It is cstraints . Recentlneurons) . These rThe development and optimization of iPSC-based platforms has allowed elucidating disease-specific mechanisms, which are exquisitely human . Further


Deductions about the ecology of high taxonomic bacterial ranks  are often based on their abundance patterns, yet few studies have quantified how accurately variations in abundance of these bacterial groups represent the dynamics of the taxa within them. Using 454-pyrosequencing of the 16S rRNA gene, we investigated whether the changes in abundance of six dominant bacterial classes  along a large dam-regulated river are reflected by those of their constituent Operational Taxonomic Units . The environmental impact generated by the reservoirs promoted clear compositional shifts in all bacterial classes that resulted from changes in the abundance of individual OTUs rather than from the appearance of new taxa along the river. Abundance patterns at the class level represented the dynamics of only a small but variable proportion of their constituting OTUs, which were not necessarily the most abundant ones. Within most classes, we detected sub-groups of OTUs showing contrasting responses to reservoir-induced environmental changes. Overall, we show that the patterns observed at the class level fail to capture the dynamics of a significant fraction of their constituent members, calling for caution when the ecological attributes of high-ranks are to be interpreted. Despite recent advances in our knowledge of bacterial communities associated to the development of high throughput sequencing technologies, it is still under debate whether bacterial high taxonomic ranks  form ecologically coherent units and to which extent their constituent taxa share functional traits and environmental preferences  would behave as an ecologically coherent clade if most taxa within it respond similarly to changes in environmental conditions. More specifically, if a given high-rank group shows a decrease in abundance following a change in the environment, then such group could be regarded as an ecologically coherent entity if most of its constituting taxa also showed proportional reductions in their abundances without major changes in community composition. However, cases where changes in high-rank abundance are due to a complete substitution of the existing taxa by other better adapted to the new conditions, or due to a large increase in the relative abundances of previously rare taxa, would imply the presence of ecologically different sub-clusters within that high-rank group. The latter scenarios contradict the idea of functionally coherent units, and could lead to misleading interpretations of the ecological attributes of a particular lineage depending on the fraction of the constituent taxa that actually holds such attributes.Considerable experimental and empirical evidence has led to the idea that high taxonomic ranks can show a certain degree of ecological coherence, despite the enormous phylogenetic, and physiological diversity that they harbor. For example, studies have reported recurrent seasonal or spatial patterns in the abundances of deeply branching bacterial lineages, such as phyla or classes  provides an ideal environment to examine the compositional succession within bacterial high-rank groups, since the presence of large reservoirs in the mid-lower reach creates pronounced changes in environmental conditions, which cause large and contrasting shifts in the relative abundance of dominant bacterial classes and phyla along the river  and the sampling design have been previously described in Ruiz-González et al. . Briefly3 s−1) than in upstream (34–64 m3 s−1) or reservoir (51–78 m3 s−1) sites. At each station, water temperature, conductivity, pH, and dissolved oxygen were determined in situ with probes, and triplicate samples were filtered and stored until analysis for soluble reactive phosphorus, dissolved organic carbon, dissolved inorganic nitrogen, suspended solids, and chlorophyll-a concentrations. These values and methodological details are presented in Table Three samplings were carried out during mid and late summer  and in winter (December) 2011, each conducted over a 3-day period. Surface water samples were taken at six sites upstream and five downstream of the reservoir system, as well as one site located at the reservoir, covering a 330 km transect  and 1–2.5 L were filtered onto 0.22 μm pore size filters  to obtain the free-living bacterial assemblage. Genomic DNA was extracted from the 0.22 μm filters with the Power Soil DNA extraction kit  following the manufacturer's protocol. The bacterial 16S rRNA gene V3/V4 region was amplified with the primers 341F (5′-CCTACGGGAGGCAGCAG-3′) and 907R  were checked for quality [sliding window Phred average (50 bp) > 25] and denoised with DeNoiser . Otherwise the non-rarefied OTU table was used . Although this number of sequences likely comprises the most abundant bacteria, we defined two abundance categories for exploratory purposes, “moderately abundant” (<0.1% of the pooled sequences) and “dominant” (>0.1% of the pooled sequences). A total of 32 samples were analyzed, since four winter samples provided too few sequences.p < 0.05) differences in taxonomic diversity or phylogenetic relatedness among categories were analyzed through One-way analysis of variance (ANOVA).The Shannon index was calculated as an estimate of bacterial taxonomic diversity, and the mean phylogenetic distance (MPD) was used to analyze changes in phylogenetic relatedness . The two distances were visualized by nonmetric multidimensional scaling (NMDS) analyses done in R. The differences in beta diversity between upstream or downstream communities or among sampling campaigns were tested using ANOSIM . After the rarefaction, 31,930 quality-checked sequences remained that clustered into 832 OTUs. Altogether, 15 bacterial phyla were detected, yet bacterial assemblages were mainly dominated by the phyla Actinobacteria , followed by Proteobacteria (22%), and Bacteroidetes (7%). The former included exclusively the class Actinobacteria, while among Proteobacteria, the class Betaproteobacteria comprised 15% of the total sequences, Alphaproteobacteria 6%, and Gammaproteobacteria 0.6%. Finally, sequences belonging to the classes Flavobacteria and Sphingobacteria comprised most of Bacteroidetes sequences, and accounted for 4 and 3% of total sequences, respectively. Twelve other rarer phyla  were also identified (details not shown). The OTU accumulation curve showed a clear plateau, suggesting that we had good coverage of the bacterial richness of the system Figure .R = 0.93, p < 0.0001) regardless of the season, whereas the temporal variability was remarkably smaller . These clear spatial patterns were most likely caused by the large changes in environmental conditions associated to the presence of reservoirs, which resulted in abrupt decreases in conductivity, DIN and chlorophyll a concentrations and suspended particles from upstream to downstream reaches  than the non-correlating taxa Table . AccordiWe investigated the responses of the OTUs within each class to the environmental gradients generated by the reservoirs, and compared them to those observed for their harboring class. We run a PCA based on the environmental parameters Figure , and expDespite some recent efforts to use trait-based approaches for understanding the ecology of bacterial groups  were associated to different taxonomic orders, this was not the case for the rest of the classes Figure . This suIn general, the fraction of taxa within the subgroups of OTUs covarying with their corresponding class was less taxonomically and phylogenetically diverse than the uncorrelated taxa Figure , Table 2Had the OTUs not represented by the class's dynamics comprised only rare and non-responsive taxa, the abundance patterns at the class level would provide good insight into the ecological preferences of its dominant members. However, the fact that the non-correlated OTUs actually harbored a larger proportion of the “dominant” taxa within each class Table , and thaIn summary, we show that, even in a single hydrologically connected system, a significant proportion of OTUs within dominant bacterial classes did not share the ecological trends observed for the class, including some of the dominant taxa. The presence of different subgroups of taxa within each class showing contrasting responses to the riverine environmental gradient calls for caution when the ecological attributes of high bacterial ranks are to be interpreted, and further highlights the need to elucidate ecologically coherent lower rank sub-clusters within bacteria for a more accurate understanding of the functioning of bacterial assemblages and their potential responses to changing environmental conditions.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pioneering genetic studies in Drosophila first revealed a key function of Miro in regulating the axonal transport of mitochondria, during which Miro forms a multi-protein transport complex with Milton and Kinesin heavy chain (KHC) to link trafficking mitochondria with the microtubule (MT) cytoskeleton. Recent studies showed that through binding to the EF hands of Miro and causing conformational changes of Miro and alteration of protein-protein interactions within the transport complex, Ca2+ can alter the engagement of mitochondria with the MT/kinesin network, offering one mechanism to match mitochondrial distribution with neuronal activity. Despite the importance of the Miro/Milton/Kinesin complex in regulating mitochondrial transport in metazoans, not all components of the transport complex are conserved in lower organisms, and transport-independent functions of Miro are emerging. Here we review the diverse functions of the evolutionarily conserved Miro proteins that are relevant to the development, maintenance, and functioning of the nervous system and discuss the potential contribution of Miro dysfunction to the pathogenesis of diseases of the nervous system.Mitochondrial rho GTPase (Miro) is a mitochondrial outer membrane protein containing two GTPase domains and two helix-loop-helix Ca These proteins have tandem GTP-binding domains separated by a linker region containing putative calcium-binding EF hand motifs  content were observed . The authors observed that mitochondria in dmiro mutant muscles and neurons are abnormally distributed. They accumulate in neuronal soma instead of being transported into axons and dendrites. As a result, mutant neuromuscular junctions (NMJs) lack presynaptic mitochondria, and neurotransmitter release and acute Ca2+ buffering is impaired during prolonged high frequency stimulation but not under basal conditions. Conversely, gain of dMiro function causes an abnormal accumulation of mitochondria in distal synaptic boutons of NMJs, supporting that dMiro promotes anterograde transport of mitochondria and their proper distribution within nerve terminals. A similar phenotype was observed in the Drosophila milton mutant, in which mitochondria are missing from synaptic terminals and axons, but abundant in cell bodies . Despite the agreement on the essential role of Miro in sensing Ca2+ and confer Ca2+-dependent arrest of mitochondrial motility, there is less consensus on the detailed molecular mechanisms by which Ca2+ regulates mitochondrial motility. Several models have been proposed. In one model, it is proposed that increased Ca2+ causes the KIF5 to dissociate from microtubules and to interact with Miro on mitochondria, dissociating the motors from microtubules  contact sites P3R on the ER, the voltage-dependent anion-selective channel protein 1 (VDAC1) on the mitochondrial outer membrane, and the mitochondrial Ca2+ uniporter (MCU) on the mitochondrial inner membrane , leading to cytochrome c release, propagation of the caspase cascade and ultimately apoptosis, the function of Miro in regulating intramitochondrial Ca2+ homeostasis may be highly relevant to the pro-apoptotic effects of Miro discussed later.One possible function of Miro proteins localized at the mitochondria-ER contact sites is to regulate mitochondria-ER Cag Figure . At thosDrosophila, it was found that dmiro mutants exhibited altered structures of synaptic boutons at the NMJ , reduction of function of Miro as well as components of the transport machinery effectively rescued the degenerative phenotype in dopaminergic neurons seen in PINK1 mutant, suggesting that at least in this setting altered mitochondrial transport contributes to PINK1 pathogenesis (Liu et al., Drosophila larval motor neurons or mammalian hippocampal neurons. Biochemically, it was found that PINK1 and another PD associated gene product, Parkin, acted together to promote the degradation of Miro in vivo in Drosophila tissues, or in cultured mammalian cells treated with the mitochondrial toxin CCCP (Wang et al., In the first functional characterization of human Miro proteins, it was found that overexpression of the constitutively active form of Miro1 led to increased rate of apoptosis in cultured non-neuronal cells (Fransson et al., Drosophila showed that inhibition of Miro activated the PAR-1/MARK family kinases and promoted the pathological phosphorylation of tau (Iijima-Ando et al., 2+ level, thereby disrupting anterograde mitochondrial transport in neuronal axons, and a Ca2+-insensitive form of Miro1 rescued the effects of VAPB-P56S on axonal mitochondria transport (Mórotz et al., The PINK1/Parkin pathway has been implicated in mitochondrial quality control, during which PINK1 becomes stabilized on damaged mitochondria, recruits Parkin from the cytosol, and targets damaged mitochondria for clearance by autophagy (mitophagy; Narendra and Youle, 2+ buffering to synaptic differentiation and plasticity. The intracellular distribution of mitochondria needs to be precisely matched to the demand for these organelles, a task particularly difficult for neurons due to their highly polarized morphology and their dynamic patterns of neuronal activity and synaptic plasticity in vivo. Because abnormal mitochondrial distribution and function has been consistently observed at early stages of neurodegenerative disorders such as AD and PD as well as neuropsychiatric disorders, understanding the genetic control of mitochondrial distribution in neurons has assumed a high priority in neuroscience research. The identification of the Miro/Milton/Khc complex as a conserved mitochondrial transport machinery has offered tremendous insights into the role of the evolutionarily conserved Miro proteins in mitochondrial trafficking in neurons. Studies of Miro proteins in a number of organisms have suggested that the function of Miro may go beyond transport of mitochondria in neurons. Newly revealed functions of Miro proteins include mitochondrial morphogenesis, mitochondria-ER communication, MT dynamics, and apoptosis. Futures studies of Miro function in diverse experimental systems, from model organisms such as yeast and Drosophila to patient-derived neurons should further our understanding of the myriad function of Miro in the nervous system and its relevance to human health and disease.Mitochondria play essential roles in normal neuronal physiology, from energy production to CaThe 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.


MLC1 gene (>75% of patients) or the GlialCAM gene (<20% of patients) are responsible for the disease. Recently, the GlialCAM adhesion protein was found essential for the membrane expression and function of the chloride channel ClC-2 indicating MLC disease caused by mutation in GlialCAM as the first channelopathy among leukodystrophies. On the contrary, the function of MLC1 protein, which binds GlialCAM, its functional relationship with ClC-2 and the molecular mechanisms underlying MLC1 mutation-induced functional defects are not fully understood yet. The human MLC1 gene encodes a 377-amino acid membrane protein with eight predicted transmembrane domains which shows very low homology with voltage-dependent potassium (K+) channel subunits. The high expression of MLC1 in brain astrocytes contacting blood vessels and meninges and brain alterations observed in MLC patients have led to hypothesize a role for MLC1 in the regulation of ion and water homeostasis. Recent studies have shown that MLC1 establishes structural and/or functional interactions with several ion/water channels and transporters and ion channel accessory proteins, and that these interactions are affected by MLC1 mutations causing MLC. Here, we review data on MLC1 functional properties obtained in in vitro and in vivo models and discuss evidence linking the effects of MLC1 mutations to brain channelopathies.Megalencephalic leukoencephalopathy with subcortical cysts (MLCs) disease is a rare inherited, autosomal recessive form of childhood-onset spongiform leukodystrophy characterized by macrocephaly, deterioration of motor functions, epileptic seizures and mental decline. Brain edema, subcortical fluid cysts, myelin and astrocyte vacuolation are the histopathological hallmarks of MLC. Mutations in either the  Channelopathies are a heterogeneous group of disorders of genetic or acquired origin caused by altered function of ion channel subunits or the proteins that regulate them. These include diseases of the cardiovascular, muscular, respiratory, urinary, endocrine, and immune systems. Due to the fundamental role played by ion channels in neuronal activity, channelopathies are involved in a growing number of nervous system disorders . The rarhttp://ela-asso.com).Leukodystrophies comprise a large number of rare genetic disorders that affect mainly the CNS with different etiology and a common target. The European Leukodystrophies Association (ELA) offers a classification including the following types: peroxisomal, lysosomal, vacuolating, hypomyelinating, atypical, undetermined. Against such a heterogeneous etiopathogenesis, the common target is myelin: “either the myelin does not form, degrades, or is too abundant”  and brain alterations observed in MLC patients  suggest that MLC1 can play a role in the regulation of ion and water fluxes and cell volume. However, although several recent studies support this hypothesis  channel Kv1.1 α subunit . However, the results of endoglycosydase treatment and site-directed mutagenesis of the putative glycosylation site  ruled out that MLC1 is glycosylated when expressed in heterologous cells . Furthermore, it was found that treatment with substances activating PKC or PKA and phosphatase inhibitors  can modify MLC1 plasma membrane expression and formation of multimeric structures . Changes in the extent of phosphorylation at sites adjacent to the RXR-type ER retention motif may enable a given protein to interact with the forward secretory machinery by hiding or hampering the retention signals, therefore allowing channel/receptor trafficking to the plasma membrane  suggests that the same regulation takes place for MLC1, similarly to what observed for some ion channels and transporters , and the identification by LC-MS analysis of several kinases and phosphatases among MLC1 interactors in primary rat astrocytes  suggest a more complex phosphorylation-mediated regulation of MLC1 localization, and probably function, in astrocytes. Experiments are in progress in our laboratory to better understand the role of kinases in MLC1 protein stability, molecular interactions, and function. This knowledge could be relevant for the therapy of MLC since several kinases are currently under investigation as drug targets to modulate the activity of ion channels involved in tumoral, inflammatory, and neurologic diseases  were recorded in monocyte-derived macrophages from MLC patients, though the impact of such alterations is unknown since MLC patients do not show overt deficits in blood or immune system functions.One of the few certainties on MLC1 protein is its predominant expression in brain astrocytes, particularly in astrocytic end-feet contacting cerebral blood vessels. Due to this finding, MLC1 is emerging as a promising marker for perivascular astrocytes . The firlt brain . These rlt brain . Using slt brain . Outsidein vitro . AccordiFigure 2). Presence of MLC1 protein in caveolar rafts is supported by binding of cav-1 to the NH2 intracellular terminal of the MLC1 protein containing the putative caveolin/cholesterol binding site .Biochemical studies in cultured astrocytes and rat brain tissue showed that MLC1 is present in specialized areas of the plasma membrane called lipid rafts . Lipid rFigure 3). By cell fractionation, immunofluorescence and electron microscopy analysis of rat primary astrocytes and human astrocytoma cells, MLC1 protein was detected in early endosomes . By using this approach, GlialCAM has been identified as the second MLC disease-associated gene and the molecular chaperon that targets MLC1 to astrocyte cell–cell junctions . As this article focuses on the role of MLC1 protein, we refer the reader to specific publications for detailed studies on GlialCAM involvement in MLC disease  animal models have allowed to better understand the relationship between GlialCAM and MLC1  disease .Kir4.1 gene in mice causes myelin vacuolation and cyst formation that are reminiscent of the MLC phenotype . The same study reports that MLC1 does not co-immunoprecipitate with DGC components in cultured rat astrocytes acquiring a differentiated morphology upon prolonged treatment with cytosine arabinoside  associated protein complex dystrophin–dystroglycan complex DGC;  promptedhenotype . The MLChenotype  and thathenotype . In contbinoside . Differebinoside . FurtherTable 2). Using complementary biochemical, proteomic and in vivo protein interaction assays  in cultured rat astrocytes and human astrocytoma cells, we found that MLC1 binds the β1 subunit of the Na, K-ATPase enzyme (Table 1). The functional relationship of these molecules with MLC1 is under investigation. Cell fractionation and immunolocalization experiments revealed that in astrocytes MLC1 interaction with Na, K-ATPase and V-ATPase occurs mainly in the membrane of intracellular organelles, like early and recycling endosomes, in basal culture conditions and also in the plasma membrane after exposure to osmotic stress  expressing mutated MLC1 protein showed that many missense pathological mutations impaired MLC1 translocation to the plasma membrane. MLC1 mutations also affected the protein half-life by causing an increase in protein degradation  we found that not all the mutated proteins were retained in the ER. For example, MLC1 protein carrying the S426R mutation was able to reach the plasma membrane, similarly to WT MLC1 . This re channel , support channel  and in mull mice , but didull mice . CollectMLC1 ortholog gene  results. Some discrepancies may reflect the inability of the in vitro systems to reproduce the complexity of astrocyte functional and structural connections in vivo. It is generally accepted that gene inactivation in vivo represents an essential approach to study the physiological roles of several proteins. However, KO mice, while useful for a rough characterization of gene function, have some disadvantages for deciphering precisely pathogenetic mechanisms, including gene redundancy, and developmental issues. Furthermore, not all the MLC1 mutations analyzed in vitro lead to complete protein degradation, a phenotype recapitulated by null-mouse models. Noteworthy, astrocytes display heterogeneous characteristics depending on species of origin, brain region, developmental stage, environmental factors, and disease states, all of which may render experimental results highly variable. The complexity of the glia themselves as well as the marked increase in the ratio of glia to neurons in higher mammals  that occurs after cell swelling in astrocytes. It has been shown that the cation channel TRPV4 co-localizes with AQP4 in the astrocyte end-feet and is responsible for an outward-rectifying Ca2+ conductance and Ca2+ influx following cell swelling induced by mechanical and osmotic stress . The TRPV4-mediated Ca2+ influx is an essential step in the activation of RVD in astrocytes which is needed to rescue the temporary cell swelling induced by hyposmosis. Using the U251 astrocytoma cell model, we found that MLC1 mutations causing retention of the protein in the cytoplasmic perinuclear area affect TRPV4-mediated Ca2+ influx induced by hyposmotic stress  and maximal amplitude of the Ca2+ changes . Further studies are needed to understand if MLC1 affects TRPV4 function by acting as a docking site for mechanical sensors. We recently demonstrated that MLC1, by modulating endosome acidity (see below), can influence TRPV4 trafficking favoring channel recycling versus degradation. We cannot exclude that this effect might mechanistically explain the functional cooperation between MLC1 and TRPV4. Accordingly, defects in calcium influx during hyposmosis were also observed in monocyte-derived macrophages from MLC patients . VRAC is not only involved in the RVD, but also in pathological events like apoptotic volume decrease, necrotic volume increase following lactacidosis and excitotoxicity . Moreover, ablation of GlialCAM did not result in the expected changes  and caused a mild decrease of the current, compatible with the decrease in ClC-2 expression observed in MLC1-/- and GlialCAM-/- mice by immunohistochemistry. So far, the most likely interpretation of these results is that MLC1 plays a role as a chaperone contributing to the correct localization of ClC-2 proteins, but is not involved in the modulation of the biophysical features of ClC-2 channel.ClC-2 channels are almost ubiquitous and their whole-cell current is characterized by hyperpolarization-induced slow activation, slow deactivating tail currents at more positive potentials, strong inward rectification, and blockade by iodide but not by classical Clamoxifen . In the cipitate . In the cipitate . Overexpcipitate . Howevercipitate . A recenarations . As in parations , also inin vitro experiments  showed a higher pH compared to early endosomes from the parental U251 cells .Does MLC1 directly function as an ion/water permeation site or as a transport protein? Ion channels herein referred to are crucial for the transepithelial transport of salt and water, the regulation of cellular volume and pH, the acidification of intracellular organelles, and chemical signaling. They may be regulated by calcium, pH, phosphorylation, and lipids. Many channels are oligomers of identical or homologous pore-forming α subunits and assemble in complexes with β/γ subunits which may be essential for their function or modulate their properties. Most of these features have been described for MLC1 (see above). Experimental studies revealed that several different  mechanisms control MLC1 membrane localization  suggesting that a strict temporal and spatial regulation of MLC1 is necessary in astrocytes. Although at present conclusive results ruling out MLC1 direct involvement in ion/water permeation/transport in astrocytes are missing, the ensemble of the data collected so far converge to delineate a dual role for MLC1 ranging from a chaperoning to a regulatory role of functional events.The observation that several of the pathological MLC1 mutations cause a mislocalization of the protein together with that of some of its functional interactors suggests a chaperoning-like role of MLC1 in the correct assembly of those specific proteins. On the other hand, the variety of events affected by MLC1 ablation or mutations, together with the recognition of several putative binding sites that could act as docking sites for regulatory enzymes, prompt to consider its regulatory role in the complex scenario of events taking place in specific intracellular domains and involving water/ion movements and pH control.Besides the exact definition of the multifaceted role played by MLC1, the advent of MLC1 in animals developing myelin prompts to consider in a phylogenetic perspective the importance of this protein for the new needs of such a complex functional structure comprising astrocytes, oligodendrocytes, and neurons. In MLC disease myelin degeneration is not attributable to directmyelin or oligodendrocyte protein alterations but to an indirect effect mediated by astrocytic dysfunction. Although at present the molecular mechanisms causing myelin degeneration in MLC disease are not known it is established that defects in astrocyte maturation, astrocyte functional impairment causing accumulation of toxic substrates, and/or failure of specific astrocyte-mediated homeostatic pathways can affect myelin formation and maintenance leading to cystic or vacuolating myelin degeneration (reviewed in Channelopathies are a diverse set of disorders associated with defects in ion channel (and transporter) function that are mostly linked with inherited mutations impairing the biophysical properties of the channels. The rapid progress made in molecular genetics and electrophysiology has allowed to expand the field of channelopathies revealing that many disorders reflect dysfunctions in regulatory proteins that alter ion channel synthesis, membrane trafficking and/or posttranslational modifications.+ buffering (Moreover, associated proteins can define tissue/organ functional specificity, making ion channels at a specific location more susceptible to a pathological condition even if their expression is not tissue-specific. This could be the case of MLC1 which is highly and exclusively expressed by astrocytes in the CNS. The results obtained so far demonstrate MLC1 involvement in astrocyte volume control. Astrocytes not only undergo dramatic volume changes as a result of the ionic dysregulation that occurs in pathological conditions such as ischemia, trauma, and epilepsy but also during neuronal activity in conjunction with extracellular KThe 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.


Chromatin conformation shapes the environment in which our genome is transcribed into RNA. Transcription is a source of DNA damage, thus it often occurs concomitantly to DNA damage signaling. Growing amounts of evidence suggest that different types of RNAs can, independently from their protein-coding properties, directly affect chromatin conformation, transcription and splicing, as well as promote the activation of the DNA damage response (DDR) and DNA repair. Therefore, transcription paradoxically functions to both threaten and safeguard genome integrity. On the other hand, DNA damage signaling is known to modulate chromatin to suppress transcription of the surrounding genetic unit. It is thus intriguing to understand how transcription can modulate DDR signaling while, in turn, DDR signaling represses transcription of chromatin around the DNA lesion. An unexpected player in this field is the RNA interference (RNAi) machinery, which play roles in transcription, splicing and chromatin modulation in several organisms. Non-coding RNAs (ncRNAs) and several protein factors involved in the RNAi pathway are well known master regulators of chromatin while only recent reports show their involvement in DDR. Here, we discuss the experimental evidence supporting the idea that ncRNAs act at the genomic loci from which they are transcribed to modulate chromatin, DDR signaling and DNA repair. Genetic information is transmitted as DNA, yet is functional as RNA in cellular organisms. Genome integrity and as a consequence transcription fidelity is continuously harmed by DNA lesions. The cascade of events that starts with the detection of DNA lesions proceeds through signaling pathways and eventually triggers repair is known as the DNA damage response DDR; . Among tThe generation of a DSB causes the appearance of transcripts mapping to sequences flanking the site of damage  referredThe use of deep sequencing technology lead to a revolution in our understanding of epigenetic mechanisms behind eukaryotic genome functions by uncovering the existence of ncRNAs which act as master chromatin regulators . DDRNAs Xist lncRNA is active at a very low copy number per cells  which has also been proposed to participate in XCI  are considered all non-coding transcript longer then 200 base-pairs up to several kilobases; while below this arbitrary threshold ncRNAs are generally referred to as small ncRNAs (sncRNAs). However, this gross classification only defines two overarching categories, each of which includes several different families based on ncRNA biogenesis, localization and function . Apart fer cells . Intrigue in XCI , 2004. Ne in XCI . Similar locally . A numbeed genes  while PAA damage .in cis. Intriguing examples are the p15AS (p15 antisense transcript), which is expressed in antisense orientation to the cyclin-dependent kinase inhibitor p15 and controls its silencing by heterochromatin formation in a DICER-dependent manner (KCNQ1ot1) which represses KCNQ1 by recruiting the H3K9 histone methyltransferase G9a and the PRC2 complex increasing the level of H3K9me3 and H3K27me3 in cis to its locus , which originates from the HOXC locus, represses transcription of the HOXD locus in trans by recruiting PRC2  and the activating chromatin effector Pygopus homolog 2 (Pygo2), thereby enhancing selective looping of AR-bound enhancers to target gene promoters  are also divided in several different classes. Among a number of sncRNAs families, the more characterized are microRNAs (miRNAs or miR): DICER- and DROSHA-dependent sncRNAs which control the expression of more than 30% of human coding transcripts both at post-transcriptional and transcriptional level . Certainl miRNAs .DNA damage and DDR activation can also regulate miRNA expression by modulating the miRNA processing at maturation steps. Following DNA damage, the main DDR kinase Ataxia telangiectasia mutated ATM phosphorylates the KH-type splicing regulatory protein KSRP , a multiTranscripts coding for key factors involved in DDR and chromatin modulation are also targeted by specific miRNAs at the post-transcriptional level following DNA damage. For example, one of the earliest events in the DDR is the ATM-dependent phosphorylation of the histone variant H2AX , the codHowever, a different group of miRNA has a positive impact on DDR activation. The recently described miR339-5p, and miR542-3p, indirectly stabilize TP53 by repressing MDM2 . OverallTranscripts originating from DNA breaks are processed by two ribonucleases of the RNAi machinery DROSHA and DICER, into sncRNAs  and haveAlthough in all of these contexts sncRNAs recruit chromatin repressive complexes that induce silencing of target loci, DICER-dependent sncRNAs have also been proposed to play the opposite role of restraining the spreading of constitutive heterochromatin at the boundaries of centromeres  suggestiDrosophila melanogaster Dicer 2 and Ago 2 interact with RNAPII and are required for positioning the transcription machinery on gene promoters in euchromatin  molecules, however, DICER is able to cleave only double stranded RNA (dsRNA) precursors . How sucocessing . In suppocessing . It has ocessing . Howeverocessing . An examocessing . These aocessing .Figure 1 for a schematic representation).Another interesting source of dsRNA has been proposed at human promoters where an engaged RNAPII is loaded in an opposite orientation respect to the gene and synthesizes complementary RNAs of few hundred bases but does not elongate beyond the promoter . In suppIn plants and yeasts an additional source of dsRNA precursors is provided by RNA dependent RNA polymerases (RdRP), able to synthesize the complementary strand of a single stranded RNA template . Most hiBoth condensation and relaxation have been show to occur at damaged chromatin. Recently, it has been shown that chromatin compaction after DNA damage is a secondary event that follows an initial chromatin relaxation . AdjacenA number of other processes that promote chromatin relaxation and are required for proper DDR activation have been reported, such as the mobilization of heterochromatin-associated factors HP1 and KAP-1  away froFigure 1), , .A genomic locus is repaired more efficiently when actively transcribed than when kept silent , an obsein cis to the DSB cluster  and Short Interspersed Nuclear Elements (SINE), which derive from retrotransposons and Alu repeats. LINE and SINE are present in millions of copies in our genome occupying respectively 17% and 11% of it. Indeed, by inducing transcriptional gene silencing of repetitive sequences, RNAi represents one of the main forms of innate immunity against viruses and invading genetic elements in all organisms . From arIn plants, RNAi is a major source of immunity functioning through induction of exogenous DNA silencing via methylation , while iRNA-mediated control of transposon activation is particularly active in the mammalian germ line where a devoted class of small-interfering RNA, PIWI-interacting RNA (piRNA), has been linked to both epigenetic and post-transcriptional gene silencing of retrotransposons . piRNAs Neurospora crassa (Caenorhabditis elegans (N. crassa. Similarly to what occur during quelling, DNA breaks in endogenous epetitive sequences might induce the expression of complementary transcript processed into sncRNAs, which in turn stimulate repair and silencing of the repetitive DNA (Another interesting phenomenon linking RNAi-guided gene silencing and control of genome stability is quelling in a crassa  a post t elegans . During  elegans ,b. DsRNA elegans . Both sn elegans , a fact tive DNA .It seems that sncRNAs play an evolutionarily conserved role in both chromatin dynamics and genome stability. Thus, the discoveries of DDRNAs and diRNAs highlight the existence of a consistent, yet previously unknown, sncRNA-mediated layer in the regulation of DDR signaling and DNA repair. This discovery raises the possibility of novel approaches in designing chemical or other pharmacological molecules that might act on, or through, the DDR pathway in hopes of identifying novel strategies for disease treatment . The ideGiven the involvement of DDR in a number of physiological and pathological processes such as immunodeficiency, neurodegeneration, sterility and development , combineThe author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.