Skeletal Muscle MicroRNAs as Key Players in the Pathogenesis of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder, for which, to date, no effective treatment to ameliorate the clinical manifestations is available. The long-standing view of ALS as affecting only motor neurons has been challenged by the finding that the skeletal muscle plays an active role in the disease pathogenesis and can be a valuable target for therapeutic strategies. In recent years, non-coding RNAs, including microRNAs, have emerged as important molecules that play key roles in several cellular mechanisms involved in the pathogenic mechanisms underlying various human conditions. In this review, we summarize how the expression of some microRNAs is dysregulated in the skeletal muscle of ALS mouse models and patients. Shedding light on the mechanisms underlying microRNAs dysregulation in the skeletal muscle could clarify some of the processes involved in the pathogenesis of ALS and especially identify new promising therapeutic targets in patients.

Qualitative and quantitative differences of adipose-derived stromal cells from superficial and deep subcutaneous lipoaspirates: a matter of fat.

BACKGROUND AIMS: Subcutaneous fat represents a valuable reservoir of adipose-derived stem cells (ASCs) in the stromal vascular fraction (SVF), widely exploited in regenerative medicine applications, being easily harvested through lipoaspiration. The lack of standardized procedures for autologous fat grafting guided research efforts aimed at identifying possible differences related to the harvesting site, which may affect cell isolation yield, cell growth properties and clinical outcomes. Subcutaneous fat features a complex architecture: the superficial fascia separates superficial adipose tissue (SAT) from deep layer tissue (DAT). We aimed to unravel the differences between SAT and DAT, considering morphological structure, SVF composition, and ASC properties. METHODS: SAT and DAT were collected from female donors and comparatively analyzed to evaluate cellular yield and viability, morphology, immunophenotype and molecular profile. ASCs were isolated in primary culture and used for in vitro differentiation assays. SAT and DAT from cadaver donors were also analyzed through histology and immunohistochemistry to assess morphology and cell localization within the hypoderm. RESULTS: Liposuctioned SAT contained a higher stromal tissue compound, along with a higher proportion of CD105-positive cells, compared with DAT from the same harvesting site. Also, cells isolated from SAT displayed increased multipotency and stemness features. All differences were mainly evidenced in specimens harvested from the abdominal region. According to our results, SAT features overall increased stem properties. CONCLUSIONS: Given that subcutaneous adipose tissue is currently exploited as the gold standard source for high-yield isolation of adult stem cells, these results may provide precious hints toward the definition of standardized protocols for microharvesting.

Spinal fusion in the next generation: gene and cell therapy approaches.

Bone fusion represents a challenge in the orthopedics practice, being especially indicated for spine disorders. Spinal fusion can be defined as the bony union between two vertebral bodies obtained through the surgical introduction of an osteoconductive, osteoinductive, and osteogenic compound. Autogenous bone graft provides all these three qualities and is considered the gold standard. However, a high morbidity is associated with the harvest procedure. Intensive research efforts have been spent during the last decades to develop new approaches and technologies for successful spine fusion. In recent years, cell and gene therapies have attracted great interest from the scientific community. The improved knowledge of both mesenchymal stem cell biology and osteogenic molecules allowed their use in regenerative medicine, representing attractive approaches to achieve bone regeneration also in spinal surgery applications. In this review we aim to describe the developing gene- and cell-based bone regenerative approaches as promising future trends in spine fusion.

Characterization of SOD1-DT, a Divergent Long Non-Coding RNA in the Locus of the SOD1 Human Gene.

Researchers studying Amyotrophic Lateral Sclerosis (ALS) have made significant efforts to find a unique mechanism to explain the etiopathology of the different forms of the disease. However, despite several mutations associated with ALS having been discovered in recent years, the link between the mutated genes and its onset has not yet been fully elucidated. Among the genes associated with ALS, superoxide dismutase 1 (SOD1) was the first to be identified, but its role in the etiopathogenesis of the disease is still unclear. In recent years, research has been focused on the non-coding part of the genome to fully understand the mechanisms underlying gene regulation. Non-coding RNAs are conserved molecules and are not usually translated in protein. A total of 98% of the human genome is composed of non-protein coding sequences with roles in the transcriptional and post-transcriptional regulation of gene expression. In this study, we characterized a divergent nuclear lncRNA (SOD1-DT) transcribed in the antisense direction from the 5' region of the SOD1 coding gene in both the SH-SY5Y cell line and fibroblasts derived from ALS patients. Interestingly, this lncRNA seems to regulate gene expression, since its inhibition leads to the upregulation of surrounding genes including SOD1. SOD1-DT represents a very complex molecule, displaying allelic and transcriptional variability concerning transposable elements (TEs) included in its sequence, widening the scenario of gene expression regulation in ALS disease.

The S100B protein in biological fluids: more than a lifelong biomarker of brain distress.

S100B is a calcium-binding protein concentrated in glial cells, although it has also been detected in definite extra-neural cell types. Its biological role is still debated. When secreted, S100B is believed to have paracrine/autocrine trophic effects at physiological concentrations, but toxic effects at higher concentrations. Elevated S100B levels in biological fluids (CSF, blood, urine, saliva, amniotic fluid) are thus regarded as a biomarker of pathological conditions, including perinatal brain distress, acute brain injury, brain tumors, neuroinflammatory/neurodegenerative disorders, psychiatric disorders. In the majority of these conditions, high S100B levels offer an indicator of cell damage when standard diagnostic procedures are still silent. The key question remains as to whether S100B is merely leaked from injured cells or is released in concomitance with both physiological and pathological conditions, participating at high concentrations in the events leading to cell injury. In this respect, S100B levels in biological fluids have been shown to increase in physiological conditions characterized by stressful physical and mental activity, suggesting that it may be physiologically regulated and raised during conditions of stress, with a putatively active role. This possibility makes this protein a candidate not only for a biomarker but also for a potential therapeutic target.

Genome-wide gene expression profiling of human narcolepsy.

The objective of this study was to perform global gene expression profiling of patients affected by narcolepsy with cataplexy (NRLCP). This enabled identifying new potential biomarkers and relevant molecules possibly involved in the disease pathogenesis. In this study 10 NRLCP patients and 10 healthy controls were compared. Total RNA isolated from blood specimens was analyzed using microarray technology followed by statistical data analysis to detect genome-wide differential gene expression between patients and controls. Functional analysis of the gene list was performed in order to interpret the biological significance of the data. One hundred and seventy-three genes showed significant (p < 0.01) differential expression between the two tested conditions. The biological interpretation allowed categorizing differentially expressed genes involved in neurite outgrowth/extension and brain development, which could be possibly regarded as peripheral markers of the disease. Moreover, the NRLCP-related gene expression profiles indicated a dysregulation of metabolic and immune-related mechanisms. In conclusion, the gene expression profile associated to NRLCP suggested that molecular markers of neurological impairment, dysmetabolic and immune-related mechanisms, can be detected in blood of NRLCP patients.

Gene expression profiling in human craniosynostoses: a tool to investigate the molecular basis of suture ossification.

INTRODUCTION: Non-syndromic craniosynostoses (NSC) occur as isolated skull malformations due to the premature ossification of one (single-suture forms) or more (complex forms) calvarial sutures and represent the most frequent form of craniosynostosis worldwide. The etiology of NSC is still largely unknown as a genetic basis can be rarely demonstrated especially in single-suture forms. In these cases, during the prenatal/perinatal development of affected patients, only one suture undergoes a premature direct ossification within an otherwise physiologically grown skull. This could suggest that definite somatic alterations, possibly due to unclear environmental agents, occur locally at the site of premature suture fusion during skull development. A promising tool to investigate the molecular mechanisms that may orchestrate this event is the comparative analysis of suture- and synostosis-derived tissues and cells. PURPOSE: This review focuses on the different studies that attempted to clarify this issue using genome-wide microarray-based technologies for the comparative analysis of gene expression profiles. All relevant results have been comprehensively reviewed, possibly compared, and critically discussed. CONCLUSION: Due to the heterogeneity of the dataset available in the literature, a univocal CRS-associated molecular profile could not be deciphered. Most differentially expressed genes are found in different studies to be involved in extracellular matrix remodeling.

Genetic basis of single-suture synostoses: genes, chromosomes and clinical implications.

BACKGROUND: Non syndromic craniosynostoses are the most frequent craniofacial malformations worldwide. They represent a wide and heterogeneous group of entities, in which the dysmorphism may occur in a single (simple forms) or in multiple sutures (complex forms). Simple forms present a higher birth prevalence and are classified according to the involved suture and to the corresponding abnormal cranial shape: scaphocephaly (SC; sagittal suture), trigonocephaly (TC; metopic suture), anterior plagiocephaly (unilateral coronal suture), posterior plagiocephaly (unilateral lambdoid suture). They occur commonly as sporadic forms, although a familiar recurrence is sometimes observed, suggesting a mendelian inheritance. The genetic causes of simple craniosynostosis are still largely unknown, as mutations in common craniosynostosis-associated genes and structural chromosomal aberrations have been rarely found in these cases. AIMS: This review is intended to dissect comprehensively the state-of-the art on the genetic etiology of single suture craniosynostoses, in the attempt to categorize all known disease-associated genes and chromosomal aberrations. Possible genotype/phenotype correlations are discussed as useful clues towards the definition of optimized clinical management flowcharts.

Early transcriptional events during osteogenic differentiation of human bone marrow stromal cells induced by Lim mineralization protein 3.

Lim mineralization protein-3 (LMP3) induces osteoblast differentiation by regulating the expression and activity of certain molecules involved in the osteogenic cascade, including those belonging to the bone morphogenetic protein (BMP) family. The complete network of molecular events involved in LMP3-mediated osteogenesis is still unknown. The aim of this study was to analyze the genome-wide gene expression profiles in human mesenchymal stem cells (hMSC) induced by exogenous LMP3 to mediate osteogenesis. For this purpose hMSC were transduced with a defective adenoviral vector expressing the human LMP3 gene and microarray analysis was performed 1 day post-adenoviral transduction. Cells transduced with the vector backbone and untransduced cells were used as independent controls in the experiments. Microarray data were independently validated by means of real-time PCR on selected transcripts. The statistical analysis of microarray data produced a list of 263 significantly (p < 0.01) differentially expressed transcripts. The biological interpretation of the results indicated, among the most noteworthy effects, the modulation of genes involved in the TGF-beta1 pathway: 88 genes coding for key regulators of the cell cycle regulatory machinery and 28 genes implicated in the regulation of cell proliferation along with the development of connective, muscular, and skeletal tissues. These results suggested that LMP3 could affect the fine balance between cell proliferation/differentiation of mesenchymal cells mostly by modulating the TGF-beta1 signaling pathway.

A pRb-independent mechanism preserves the postmitotic state in terminally differentiated skeletal muscle cells.

In skeletal muscle differentiation, the retinoblastoma protein (pRb) is absolutely necessary to establish definitive mitotic arrest. It is widely assumed that pRb is equally essential to sustain the postmitotic state, but this contention has never been tested. Here, we show that terminal proliferation arrest is maintained in skeletal muscle cells by a pRb-independent mechanism. Acute Rb excision from conditional knockout myotubes caused reexpression of E2F transcriptional activity, cyclin-E and -A kinase activities, PCNA, DNA ligase I, RPA, and MCM2, but did not induce DNA synthesis, showing that pRb is not indispensable to preserve the postmitotic state of these cells. Muscle-specific gene expression was significantly down-regulated, showing that pRb is constantly required for optimal implementation of the muscle differentiation program. Rb-deleted myotubes were efficiently reactivated by forced expression of cyclin D1 and Cdk4, indicating a functionally significant target other than pRb for these molecules. Finally, Rb removal induced no DNA synthesis even in pocket-protein null cells. Thus, the postmitotic state of myotubes is maintained by at least two mechanisms, one of which is pocket-protein independent.

Gene expression profiling in the early phases of DMD: a constant molecular signature characterizes DMD muscle from early postnatal life throughout disease progression.

Genome-wide gene expression profiling of skeletal muscle from Duchenne muscular dystrophy (DMD) patients has been used to describe muscle tissue alterations in DMD children older than 5 years. By studying the expression profile of 19 patients younger than 2 years, we describe with high resolution the gene expression signature that characterizes DMD muscle during the initial or "presymptomatic" phase of the disease. We show that in the first 2 years of the disease, DMD muscle is already set to express a distinctive gene expression pattern considerably different from the one expressed by normal, age-matched muscle. This "dystrophic" molecular signature is characterized by a coordinate induction of genes involved in the inflammatory response, extracellular matrix (ECM) remodeling and muscle regeneration, and the reduced transcription of those involved in energy metabolism. Despite the lower degree of muscle dysfunction experienced, our younger patients showed abnormal expression of most of the genes reported as differentially expressed in more advanced stages of the disease. By analyzing our patients as a time series, we provide evidence that some genes, including members of three pathways involved in morphogenetic signaling-Wnt, Notch, and BMP-are progressively induced or repressed in the natural history of DMD.

Effects of Class II-Selective Histone Deacetylase Inhibitor on Neuromuscular Function and Disease Progression in SOD1-ALS Mice.

Emerging evidence indicates that transcriptome alterations due to epigenetic deregulation concur to ALS pathogenesis. Accordingly, pan-histone deacetylase (HDAC) inhibitors delay ALS development in mice, but these compounds failed when tested in ALS patients. Possibly, lack of selectivity toward specific classes of HDACs weakens the therapeutic effects of pan-HDAC inhibitors. Here, we tested the effects of the HDAC Class II selective inhibitor MC1568 on disease evolution, motor neuron survival as well as skeletal muscle function in SOD1G93A mice. We report that HDACs did not undergo expression changes during disease evolution in isolated motor neurons of adult mice. Conversely, increase in specific Class II HDACs (-4, -5 and -6) occurs in skeletal muscle of mice with severe neuromuscular impairment. Importantly, treatment with MC1568 causes early improvement of motor performances that vanishes at later stages of disease. Notably, motor improvement is not paralleled by reduced motor neuron degeneration but by increased skeletal muscle electrical potentials, reduced activation of mir206/FGFBP1-dependent muscle reinnervation signaling, and increased muscle expression of myogenic genes.

BBS9 gene in nonsyndromic craniosynostosis: Role of the primary cilium in the aberrant ossification of the suture osteogenic niche.

Nonsyndromic craniosynostosis (NCS) is the premature ossification of skull sutures, without associated clinical features. Mutations in several genes account for a small number of NCS patients; thus, the molecular etiopathogenesis of NCS remains largely unclear. Our study aimed at characterizing the molecular signaling implicated in the aberrant ossification of sutures in NCS patients. Comparative gene expression profiling of NCS patient sutures identified a fused suture-specific signature, including 17 genes involved in primary cilium signaling and assembly. Cells from fused sutures displayed a reduced potential to form primary cilia compared to cells from control patent sutures of the same patient. We identified specific upregulated splice variants of the Bardet Biedl syndrome-associated gene 9 (BBS9), which encodes a structural component of the ciliary BBSome complex. BBS9 expression increased during in vitro osteogenic differentiation of suture-derived mesenchymal cells of NCS patients. Also, Bbs9 expression increased during in vivo ossification of rat sutures. BBS9 functional knockdown affected the expression of primary cilia on patient suture cells and their osteogenic potential. Computational modeling of the upregulated protein isoforms (observed in patients) predicted that their binding affinity within the BBSome may be affected, providing a possible explanation for the aberrant suture ossification in NCS.

Potential therapeutic targets for ALS: MIR206, MIR208b and MIR499 are modulated during disease progression in the skeletal muscle of patients.

Amyotrophic lateral sclerosis (ALS) is characterized by the progressive loss of motor neurons followed by muscle weakness, paralysis and death. The disease progression is extremely variable among patients, and reliable prognostic markers have not been identified. The aim of the study was to functionally characterize selected genes and microRNAs acting in the skeletal muscle of ALS patients, taking into account the duration and evolution of the disease, in order to obtain information regarding the muscle response to ALS progression. This prospective, longitudinal study enrolled 14 ALS patients and 24 age- and sex-matched healthy controls. Gene expression and histological analysis indicated an increase of MIR208B and MIR499 levels and the predominance of slow fibres, respectively, in the muscles of patients with a slower disease progression. A decreased expression of MIR206 and increased levels of HDAC4, during the progression of the disease were also observed. Taken together, our data suggest that the molecular signalling that regulates re-innervation and muscle regeneration is hampered during the progression of skeletal muscle impairment in ALS. This could provide precious hints towards defining prognostic protocols, and designing novel tailored therapeutic approaches, to improve ALS patients' care and delay disease progression.

Muscle Expression of SOD1(G93A) Modulates microRNA and mRNA Transcription Pattern Associated with the Myelination Process in the Spinal Cord of Transgenic Mice.

A crucial system severely affected in several neuromuscular diseases is the loss of effective connection between muscle and nerve, leading to a pathological non-communication between the two tissues. One of the best examples of impaired interplay between muscle and nerve is Amyotrophic Lateral Sclerosis, a neurodegenerative disease characterized by degeneration of motor neurons and muscle atrophy. Increasing evidences suggest that damage to motor neurons is enhanced by alterations in the neighboring non-neuronal cells and indicate that altered skeletal muscle might be the source of signals that impinge motor neuron activity and survival. Here we investigated whether muscle selective expression of SOD1(G93A) mutant gene modulates mRNAs and miRNAs expression at the level of spinal cord of MLC/SOD1(G93A) mice. Using a Taqman array, the Affymetrix Mouse Gene 2.0 ST approach and the MiRwalk 2.0 database, which provides information on miRNA and their predicted target genes, we revealed that muscle specific expression of SOD1(G93A) modulates relevant molecules of the genetic and epigenetic circuitry of myelin homeostasis in spinal cord of transgenic mice. Our study provides insights into the pathophysiological interplay between muscle and nerve and supports the hypothesis that muscle is a source of signals that can either positively or negatively affect the nervous system.

The multikinase inhibitor Sorafenib enhances glycolysis and synergizes with glycolysis blockade for cancer cell killing.

Although the only effective drug against primary hepatocarcinoma, the multikinase inhibitor Sorafenib (SFB) usually fails to eradicate liver cancer. Since SFB targets mitochondria, cell metabolic reprogramming may underlie intrinsic tumor resistance. To characterize cancer cell metabolic response to SFB, we measured oxygen consumption, generation of reactive oxygen species (ROS) and ATP content in rat LCSC (Liver Cancer Stem Cells) -2 cells exposed to the drug. Genome wide analysis of gene expression was performed by Affymetrix technology. SFB cytotoxicity was evaluated by multiple assays in the presence or absence of metabolic inhibitors, or in cells genetically depleted of mitochondria. We found that low concentrations (2.5-5 µM) of SFB had a relatively modest effect on LCSC-2 or 293 T cell growth, but damaged mitochondria and increased intracellular ROS. Gene expression profiling of SFB-treated cells was consistent with a shift toward aerobic glycolysis and, accordingly, SFB cytotoxicity was dramatically increased by glucose withdrawal or the glycolytic inhibitor 2-DG. Under metabolic stress, activation of the AMP dependent Protein Kinase (AMPK), but not ROS blockade, protected cells from death. We conclude that mitochondrial damage and ROS drive cell killing by SFB, while glycolytic cell reprogramming may represent a resistance strategy potentially targetable by combination therapies.

Gene regulation networks in early phase of Duchenne muscular dystrophy.

The aim of this study was to analyze previously published gene expression data of skeletal muscle biopsies of Duchenne muscular dystrophy (DMD) patients and controls (gene expression omnibus database, accession #GSE6011) using systems biology approaches. We applied an unsupervised method to discriminate patient and control populations, based on principal component analysis, using the gene expressions as units and patients as variables. The genes having the highest absolute scores in the discrimination between the groups, were then analyzed in terms of gene expression networks, on the basis of their mutual correlation in the two groups. The correlation network structures suggest two different modes of gene regulation in the two groups, reminiscent of important aspects of DMD pathogenesis.

Adipose-derived mesenchymal cells for bone regereneration: state of the art.

Adipose tissue represents a hot topic in regenerative medicine because of the tissue source abundance, the relatively easy retrieval, and the inherent biological properties of mesenchymal stem cells residing in its stroma. Adipose-derived mesenchymal stem cells (ASCs) are indeed multipotent somatic stem cells exhibiting growth kinetics and plasticity, proved to induce efficient tissue regeneration in several biomedical applications. A defined consensus for their isolation, classification, and characterization has been very recently achieved. In particular, bone tissue reconstruction and regeneration based on ASCs has emerged as a promising approach to restore structure and function of bone compromised by injury or disease. ASCs have been used in combination with osteoinductive biomaterial and/or osteogenic molecules, in either static or dynamic culture systems, to improve bone regeneration in several animal models. To date, few clinical trials on ASC-based bone reconstruction have been concluded and proved effective. The aim of this review is to dissect the state of the art on ASC use in bone regenerative applications in the attempt to provide a comprehensive coverage of the topics, from the basic laboratory to recent clinical applications.

Mitochondrial network genes in the skeletal muscle of amyotrophic lateral sclerosis patients.

Recent evidence suggested that muscle degeneration might lead and/or contribute to neurodegeneration, thus it possibly play a key role in the etiopathogenesis and progression of amyotrophic lateral sclerosis (ALS). To test this hypothesis, this study attempted to categorize functionally relevant genes within the genome-wide expression profile of human ALS skeletal muscle, using microarray technology and gene regulatory network analysis. The correlation network structures significantly change between patients and controls, indicating an increased inter-gene connection in patients compared to controls. The gene network observed in the ALS group seems to reflect the perturbation of muscle homeostasis and metabolic balance occurring in affected individuals. In particular, the network observed in the ALS muscles includes genes (PRKR1A, FOXO1, TRIM32, ACTN3, among others), whose functions connect the sarcomere integrity to mitochondrial oxidative metabolism. Overall, the analytical approach used in this study offer the possibility to observe higher levels of correlation (i.e. common expression trends) among genes, whose function seems to be aberrantly activated during the progression of muscle atrophy.