Disruption in copper homeostasis causes a number of cognitive and motor deficits. Wilson's disease and Menkes disease are neurodevelopmental disorders resulting from mutations in the copper transporters ATP7A and ATP7B, with ATP7A mutations also causing occipital horn syndrome, and distal motor neuropathy. A 65 year old male presenting with brachial amyotrophic diplegia and diagnosed with amyotrophic lateral sclerosis (ALS) was found to harbor a p.Met1311Val (M1311V) substitution variant in ATP7A. ALS is a fatal neurodegenerative disease associated with progressive muscle weakness, synaptic deficits and degeneration of upper and lower motor neurons. To investigate the potential contribution of the ATP7A(M1311V) variant to neurodegeneration, we obtained and characterized both patient-derived fibroblasts and patient-derived induced pluripotent stem cells differentiated into motor neurons (iPSC-MNs), and compared them to control cell lines. We found reduced localization of ATP7A(M1311V) to the trans-Golgi network (TGN) at basal copper levels in patient-derived fibroblasts and iPSC-MNs. In addition, redistribution of ATP7A(M1311V) out of the TGN in response to increased extracellular copper was defective in patient fibroblasts. This manifested in enhanced intracellular copper accumulation and reduced survival of ATP7A(M1311V) fibroblasts. iPSC-MNs harboring the ATP7A(M1311V) variant showed decreased dendritic complexity, aberrant spontaneous firing, and decreased survival. Finally, expression of the ATP7A(M1311V) variant in Drosophila motor neurons resulted in motor deficits. Apilimod, a drug that targets vesicular transport and recently shown to enhance survival of C9orf72-ALS/FTD iPSC-MNs, also increased survival of ATP7A(M1311V) iPSC-MNs and reduced motor deficits in Drosophila expressing ATP7A(M1311V). Taken together, these observations suggest that ATP7A(M1311V) negatively impacts its role as a copper transporter and impairs several aspects of motor neuron function and morphology.

Amyotrophic lateral sclerosis (ALS) is a fatal and progressive neurodegenerative disorder of undetermined etiology with no effective treatments. Motor weakness and bulbar dysfunction lead to premature death, usually resulting from respiratory failure. While much of the research has focused on the role of neuronal dysfunction in ALS etiology, evidence from human patients and animal models indicates that the neuromuscular junction (NMJ) shows significant functional and structural abnormalities prior to the onset of motor neuron degeneration and behavioral symptoms. The development of novel experimental approaches will allow the study and manipulation of human NMJs and significantly contribute to our understanding of ALS pathogenesis, leading to advances in pharmacological treatments for the disease. A novel approach that has been employed in recent years is the use of human induced pluripotent stem cells (iPSCs) to generate cell types contributing to synaptic connectivity at the NMJ. The generation and differentiation of cells derived from ALS patient iPSCs is a promising method for investigating disease mechanisms and drug screening at NMJs in vitro. In this review, we cover the theories underlying the mechanisms of ALS pathogenesis at the NMJ, an overview of the recent developments in the generation of functional human neuromuscular connectivity in vitro, and the advances in human iPSC programming and differentiation technology.

Human ALS/FTD patient iPSC-derived neurons are used to uncover mechanisms by which C9ORF72 mutations cause neurodegeneration. An intronic GGGGCC repeat expansion in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but the pathogenic mechanism of this repeat remains unclear. Using human induced motor neurons (iMNs), we found that repeat-expanded C9ORF72 was haploinsufficient in ALS. We found that C9ORF72 interacted with endosomes and was required for normal vesicle trafficking and lysosomal biogenesis in motor neurons. Repeat expansion reduced C9ORF72 expression, triggering neurodegeneration through two mechanisms: accumulation of glutamate receptors, leading to excitotoxicity, and impaired clearance of neurotoxic dipeptide repeat proteins derived from the repeat expansion. Thus, cooperativity between gain- and loss-of-function mechanisms led to neurodegeneration. Restoring C9ORF72 levels or augmenting its function with constitutively active RAB5 or chemical modulators of RAB5 effectors rescued patient neuron survival and ameliorated neurodegenerative processes in both gain- and loss-of-function C9ORF72 mouse models. Thus, modulating vesicle trafficking was able to rescue neurodegeneration caused by the C9ORF72 repeat expansion. Coupled with rare mutations in ALS2, FIG4, CHMP2B, OPTN and SQSTM1, our results reveal mechanistic convergence on vesicle trafficking in ALS and FTD.

Over the past few decades, a variety of different reagents for stem cell maintenance and differentiation have been commercialized. These reagents share a common goal in facilitating the manufacture of products suitable for cell therapy while reducing the amount of non-defined components. Lessons from developmental biology have identified signalling molecules that can guide the differentiation process in vitro, but less attention has been paid to the extracellular matrix used. With the introduction of more biologically relevant and defined matrices, that better mimic specific cell niches, researchers now have powerful resources to fine-tune their in vitro differentiation systems, which may allow the manufacture of therapeutically relevant cell types. In this review article, we revisit the basics of the extra-cellular matrix, and explore the important role of the cell-matrix interaction. We focus on laminin proteins because they help to maintain pluripotency and drive cell fate specification. This article is part of the theme issue 'Designer human tissue: coming to a lab near you'.

Tongue weakness, like all weakness in Duchenne muscular dystrophy (DMD), occurs as a result of contraction-induced muscle damage and deficient muscular repair. Although membrane fragility is known to potentiate injury in DMD, whether muscle stem cells are implicated in deficient muscular repair remains unclear. We hypothesized that DMD myo-blasts are less sensitive to cues in the extracellular matrix designed to potentiate structure function relationships of healthy muscle. To test this hypothesis, we drew inspiration from the tongue and engineered contractile human muscle tissues on thin films. On this platform, DMD myoblasts formed fewer and smaller myotubes and exhibited impaired polarization of the cell nucleus and contractile cytoskeleton when compared with healthy cells. These structural aberrations were reflected in their functional behavior, as engineered tongues from DMD myoblasts failed to achieve the same contractile strength as healthy tongue structures. These data suggest that dystrophic muscle may fail to organize with respect to extracellular cues necessary to potentiate adaptive growth and remodeling.

Background: Large-scale expansion of myogenic progenitors is necessary to support the development of high-throughput cellular assays in vitro and to advance genetic engineering approaches necessary to develop cellular therapies for rare muscle diseases. However, optimization has not been performed in order to maintain the differentiation capacity of myogenic cells undergoing long-term cell culture. Multiple extracellular matrices have been utilized for myogenic cell studies, but it remains unclear how different matrices influence long-term myogenic activity in culture. To address this challenge, we have evaluated multiple extracellular matrices in myogenic studies over long-term expansion.Methods: We evaluated the consequence of propagating mouse and human myogenic stem cell progenitors on various extracellular matrices to determine if they could enhance long-term myogenic potential. For the first time reported, we comprehensively examine the effect of physiologically relevant laminins, laminin 211 and laminin 521, compared to traditionally utilized ECMs (e.g., laminin 111, gelatin, and Matrigel) to assess their capacity to preserve myogenic differentiation potential.Results: Laminin 521 supported increased proliferation in early phases of expansion and was the only substrate facilitating high-level fusion following eight passages in mouse myoblast cell cultures. In human myoblast cell cultures, laminin 521 supported increased proliferation during expansion and superior differentiation with myotube hypertrophy. Counterintuitively however, laminin 211, the native laminin isoform in resting skeletal muscle, resulted in low proliferation and poor differentiation in mouse and human cultures. Matrigel performed excellent in short-term mouse studies but showed high amounts of variability following long-term expansion.Conclusions: These results demonstrate laminin 521 is a superior substrate for both short-term and long-term myogenic cell culture applications compared to other commonly utilized substrates. Since Matrigel cannot be used for clinical applications, we propose that laminin 521 could possibly be employed in the future to provide myoblasts for cellular therapy directed clinical studies.

Multiple Sclerosis is a demyelinating disease of the CNS and the primary cause of neurological disability in young adults. Loss of myelinating oligodendrocytes leads to neuronal dysfunction and death and is an important contributing factor to this disease. Endogenous oligodendrocyte precursor cells (OPCs), which on differentiation are responsible for replacing myelin, are present in the adult CNS. As such, therapeutic agents that can stimulate OPCs to differentiate and remyelinate demyelinated axons under pathologic conditions may improve neuronal function and clinical outcome. We describe the details of an automated, cell-based, morphometric-based, high-content screen that is used to identify small molecules eliciting the differentiation of OPCs after 3 days. Primary screening was performed using rat CG-4 cells maintained in culture conditions that normally support a progenitor cell-like state. From a library of 73,000 diverse small molecules within the Sanofi collection, 342 compounds were identified that increased OPC morphological complexity as an indicator of oligodendrocyte maturation. Subsequent to the primary high-content screen, a suite of cellular assays was established that identified 22 nontoxic compounds that selectively stimulated primary rat OPCs but not C2C12 muscle cell differentiation. This rigorous triaging yielded several chemical series for further expansion and bio- or cheminformatics studies, and their compelling biological activity merits further investigation.

Myelination in the central nervous system is the process by which oligodendrocytes form myelin sheaths around the axons of neurons. Myelination enables neurons to transmit information more quickly and more efficiently and allows for more complex brain functions; yet, remarkably, the underlying mechanism by which myelination occurs is still not fully understood. A reliable in vitro assay is essential to dissect oligodendrocyte and myelin biology. Hence, we developed a protocol to generate myelinating oligodendrocytes from mouse embryonic stem cells and established a myelin formation assay with embryonic stem cell-derived neurons in microfluidic devices. Myelin formation was quantified using a custom semi-automated method that is suitable for larger scale analysis. Finally, early myelination was followed in real time over several days and the results have led us to propose a new model for myelin formation.

Glioblastoma multiform (GBM) remains clinical indication with significant "unmet medical need". Innovative new therapy to eliminate residual tumor cells and prevent tumor recurrences is critically needed for this deadly disease. A major challenge of GBM research has been the identification of novel molecular therapeutic targets and accurate diagnostic/prognostic biomarkers. Many of the current clinical therapeutic targets of immunotoxins and ligand-directed toxins for high-grade glioma (HGG) cells are surface sialylated glycoproteins. Therefore, methods that systematically and quantitatively analyze cell surface sialoglycoproteins in human clinical tumor samples would be useful for the identification of potential diagnostic markers and therapeutic targets for malignant gliomas. In this study, we used the bioorthogonal chemical reporter strategy (BOCR) in combination with label-free quantitative mass spectrometry (LFQ-MS) to characterize and accurately quantify the individual cell surface sialoproteome in human GBM tissues, in fetal, adult human astrocytes, and in human neural progenitor cells (NPCs). We identified and quantified a total of 843 proteins, including 801 glycoproteins. Among the 843 proteins, 606 (72%) are known cell surface or secreted glycoproteins, including 156 CD-antigens, all major classes of cell surface receptor proteins, transporters, and adhesion proteins. Our findings identified several known as well as new cell surface antigens whose expression is predominantly restricted to human GBM tumors as confirmed by microarray transcription profiling, quantitative RT-PCR and immunohistochemical staining. This report presents the comprehensive identification of new biomarkers and therapeutic targets for the treatment of malignant gliomas using quantitative sialoglycoproteomics with clinically relevant, patient derived primary glioma cells.

Abstract The coenzyme, NAD, is as an essential redox factor in cellular biochemical processes such as respiration and metabolism. NAD is also a substrate for cellular enzymes including sirtuins and poly (ADP ribose) polymerases. NAMPT, which catalyzes the rate limiting step of NAD biosynthesis, has been considered as promising cancer target for more than a decade. NAMPT inhibitors profoundly inhibit tumor cell growth; however, the mechanism that leads to the tumor cell growth inhibition is not understood. In this work, we report the results of our exploration of the metabolic and transcriptional alterations post-NAMPT inhibition in HCT116 cell line. Our results show that NAMPTi induces a time-dependent modulation of tumor metabolism which in turn induces the reprogramming of tumor cell transcriptome concomitant with cell growth inhibition. The transcriptional profile revealed different metabolic and oncogenic signaling pathways are modulated by NAMPT inhibition. The wnt/b-catenin pathway was highlighted as one of the most modulated pathways by NAMPTi. We indeed confirmed that the NAMPTi induces the inhibition of b-catenin/TCF reporter system transfected in HCT116 cells or in HEK293 cells constitutively expressing Wnt3A. NAMPT inhibition induces a strong decrease of cyclinD1 and c-myc expression in agreement with the modulation of Wnt/b-catenin pathways. The NAMPTi impact on Wnt/b-catenin pathways can be seen in CRC cell lines bearing APC wt or APC mut and in cell lines with heterozygote but not homozygote b-catenin mutation suggesting that the effect of NAMPTi effect is upstream b-catenin. Our studies highlighted two potential mechanisms that contribute to wnt/b-catenin pathway modulation by NAMPTi. First, we observed an inhibition of disheveled activation and secondly, a decrease in the activity of tankyrase. The results obtained from these studies highlight the key role of NAMPT in tumor metabolism and the wnt/b-catenin oncogenic pathway. This suggests that modulation of wnt /b-catenin contributes to the antitumor activity of NAMPTi. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B158. Citation Format: Maysoun Shomali, Patrick McGlynn, Dmitri Wiederchain, Paul August, Francisco Adrian, Carlos Garcia-Echeverria, Joachim Theilhaber, Monsif Bouaboula. Transcriptional profile of NAMPTi unveils modulation of Tankyrase and Wnt/B-catenin pathway. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B158.

Abstract SAR154782 is a tight binder, low reversible NAMPT inhibitor that has been identified by a phenotypic screen and chemogenomic approaches. This class of NAMPT inhibitor induces a profound and time dependent modulation of tumor metabolism which in turn induces the reprogrammation of tumor cell transcriptome. The transcriptional profile revealed that different metabolic and oncogenic signaling pathways are modulated by SAR154782. In order to define the context of tumor cell sensitivity to NAMPT inhibition, the effects of this compound on tumor cell growth and viability have been assessed against a panel of about 240 cell lines from different tissue origin. Based on the difference of tumor cell lines sensitivity to NAMPT inhibitor, a panel of 60 genes that are differentially expressed in the sensitive versus non-sensitive cell lines has been identified. Interestingly, the low expression of NAMPT concomitant with the high expression of Sirtuin1 and PARP1 are the main outstanding features in the panel of genes identified for the most sensitive cell lines. This finding is in agreement with high NAD turnover. The relevance of the preceding biomarkers to determine the potential cancer target population that could benefit upon treatment with a NAMPT inhibitor has been investigated in in vivo settings by evaluating the antitumor activity of an advanced new class of NAMPT inhibitors in several mouse tumor xenografts and human primary tumor models. The results obtained from these studies highlighted the modulation upon NAMPT inhibition of important tumor growth pathways and revealed for the first time a potential molecular signature of tumor sensitivity and support further investigation into use of the signature for patient stratification in the clinical setting Citation Format: Sandrine Hilairet, Jerome Arigon, Frederico Nardi, Samir Jegham, Annick Peleraux, Sylvie Cosnier, Regine Floutard, Omar Jbilo, Paul August, Maysoun Shomali, Francois Autelitano, Rosalia Arrebola, Stephan Reiling, Jack Pollard, Francisco Adrian, Jason Sager, Christoph Lengauer, Marion Dorsh, Carlos Garcia-Echeverria, Monsif Bouaboula. The molecular signature of tumor cells that are sensitives to a new tight binder NAMPT inhibitor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2201. doi:10.1158/1538-7445.AM2013-2201

Articular cartilage, which is mainly composed of collagen II, enables smooth skeletal movement. Degeneration of collagen II can be caused by various events, such as injury, but degeneration especially increases over the course of normal aging. Unfortunately, the body does not fully repair itself from this type of degeneration, resulting in impaired movement. Microfracture, an articular cartilage repair surgical technique, has been commonly used in the clinic to induce the repair of tissue at damage sites. Mesenchymal stem cells (MSC) have also been used as cell therapy to repair degenerated cartilage. However, the therapeutic outcomes of all these techniques vary in different patients depending on their age, health, lesion size and the extent of damage to the cartilage. The repairing tissues either form fibrocartilage or go into a hypertrophic stage, both of which do not reproduce the equivalent functionality of endogenous hyaline cartilage. One of the reasons for this is inefficient chondrogenesis by endogenous and exogenous MSC. Drugs that promote chondrogenesis could be used to induce self-repair of damaged cartilage as a non-invasive approach alone, or combined with other techniques to greatly assist the therapeutic outcomes. The recent development of human induced pluripotent stem cell (iPSCs), which are able to self-renew and differentiate into multiple cell types, provides a potentially valuable cell resource for drug screening in a "more relevant" cell type. Here we report a screening platform using human iPSCs in a multi-well plate format to identify compounds that could promote chondrogenesis.