ABSTRACT
Sensory neurons are afferent neurons in sensory systems that convert stimuli and transmit information to the central nervous system as electrical signals. Primary afferent neurons that are affected by non-noxious and noxious stimuli are present in the dorsal root ganglia (DRG), and the DRG sensory neurons are used as an in vitro model of the nociceptive response. However, DRG derived from mouse or rat give a low yield of neurons, and they are difficult to culture. To help alleviate this problem, we characterized human induced pluripotent stem cell (hiPSC) derived sensory neurons. They can solve the problems of interspecies differences and supply stability. We investigated expressions of sensory neuron related proteins and genes, and drug responses by Multi-Electrode Array (MEA) to analyze the properties and functions of sensory neurons. They expressed nociceptor, mechanoreceptor and proprioceptor related genes and proteins. They constitute a heterogeneous population of their subclasses. We confirmed that they could respond to both noxious and non-noxious stimuli. We showed that histamine inhibitors reduced histamine-induced neuronal excitability. Furthermore, incubation with a ProTx-II and Nav1.7 inhibitor reduced the spontaneous neural activity in hiPSC-derived sensory neurons. Their responsiveness was different from each drug. We have demonstrated that hiPSC-derived sensory neurons combined with MEA are good candidates for drug discovery studies where DRG in vitro modeling is necessary.
Subject(s)
Induced Pluripotent Stem Cells , Humans , Rats , Mice , Animals , Induced Pluripotent Stem Cells/physiology , Histamine/metabolism , Sensory Receptor Cells/metabolism , Ganglia, Spinal/metabolismABSTRACT
Purpose.We propose a linear network-based optimization model (LNBM) for high dose rate brachytherapy (HDR-BT) that uses a novel distance metric to measure the discrepancy between the dose delivered and the prescription. Unlike models in the literature, LNBM takes advantage of the adjacency structure of the patients' voxels by formalizing them into a network.Methods.We apply LNBM to a set of 7 cervical cancer cases treated with HDR-BT. State-of-the-art commercial optimization software solves LNBM to global optimality. The results of LNBM are compared with those of inverse planning by simulated annealing (IPSA) based on tumor coverage, dosimetric indices for the critical organs at risk (OARs), isodose contour plots, and two metrics of homogeneity new to this work (hot-spots volumes and diameters).Results.LNBM produces plans with improved tumor coverage and with improved isodose contour plots and dosimetric indices for OARs that receive highest dose (bladder and rectum in this study) when compared with IPSA. Using new metrics of homogeneity, we also demonstrate that LNBM produces more homogeneous plans on these cases. An analysis of the solutions of LNBM shows that they use a significant part of the voxel network structure, providing evidence that the plans produced are different from those created using traditional penalty approaches and are more directly guided by the geometry of the patients' anatomy.Conclusions.The proposed linear network-based optimization model efficiently generates more homogeneous high quality treatment plans for HDR-BT.
Subject(s)
Brachytherapy , Uterine Cervical Neoplasms , Female , Humans , Radiotherapy Dosage , Brachytherapy/methods , Radiotherapy Planning, Computer-Assisted/methods , Rectum/pathology , Uterine Cervical Neoplasms/radiotherapy , Uterine Cervical Neoplasms/pathologyABSTRACT
Emergency fire service (EFS) systems provide rescue operations for emergencies and accidents. If properly designed, they can decrease property loss and mortality. This paper proposes a distributionally robust model (DRM) for optimizing the location of fire stations, the number of fire trucks, and demand assignment for long term planning in an EFS system. This is achieved by minimizing the worst-case expected total cost, including fire station construction cost, purchase cost for fire trucks, transportation cost, and penalty cost for not providing adequate service. The ambiguity in demands and travel durations distributions are captured through moment information and mean absolute deviation. A cutting plane method is used to solve the problem. Due to fact that it is computationally intensive for larger problems, two approximate methods are introduced; one that uses linear decision rules (LDRs), and another that adopts three-point approximations of the distributions. The results show that the heuristic method is especially useful for solving large instances of DRM. Extensive numerical experiments are conducted to analyze the model's performance with respect to different parameters. Finally, data obtained from Hefei (China) demonstrates the practical applicability and value of the model in designing an EFS system in a large metropolitan setting.
Subject(s)
Emergency Medical Services , Transportation , China , Motor Vehicles , UncertaintyABSTRACT
Connexin 43 (Cx43) gap junctions and hemichannels mediate astrocyte intercellular communication in the central nervous system under normal conditions and contribute to astrocyte-mediated neurotoxicity in amyotrophic lateral sclerosis (ALS). Here, we show that astrocyte-specific knockout of Cx43 in a mouse model of ALS slows disease progression both spatially and temporally, provides motor neuron (MN) protection, and improves survival. In addition, Cx43 expression is up-regulated in human postmortem tissue and cerebrospinal fluid from ALS patients. Using human induced pluripotent stem cellderived astrocytes (hiPSC-A) from both familial and sporadic ALS, we establish that Cx43 is up-regulated and that Cx43-hemichannels are enriched at the astrocyte membrane. We also demonstrate that the pharmacological blockade of Cx43-hemichannels in ALS astrocytes using GAP 19, a mimetic peptide blocker, and tonabersat, a clinically tested small molecule, provides neuroprotection of hiPSC-MN and reduces ALS astrocyte-mediated neuronal hyperexcitability. Extending the in vitro application of tonabersat with chronic administration to SOD1G93A mice results in MN protection with a reduction in reactive astrocytosis and microgliosis. Taking these data together, our studies identify Cx43 hemichannels as conduits of astrocyte-mediated disease progression and a pharmacological target for disease-modifying ALS therapies.
Subject(s)
Amyotrophic Lateral Sclerosis , Amyotrophic Lateral Sclerosis/genetics , Astrocytes , Connexin 43/genetics , Humans , Motor NeuronsABSTRACT
BACKGROUND: Spinal cord motor neurons (MNs) from human iPS cells (iPSCs) have wide applications in disease modeling and therapeutic development for amyotrophic lateral sclerosis (ALS) and other MN-associated neurodegenerative diseases. We need highly efficient MN differentiation strategies for generating iPSC-derived disease models that closely recapitulate the genetic and phenotypic complexity of ALS. An important application of these models is to understand molecular mechanisms of action of FDA-approved ALS drugs that only show modest clinical efficacy. Novel mechanistic insights will help us design optimal therapeutic strategies together with predictive biomarkers to achieve better efficacy. METHODS: We induce efficient MN differentiation from iPSCs in 4 days using synthetic mRNAs coding two transcription factors (Ngn2 and Olig2) with phosphosite modification. These MNs after extensive characterization were applied in electrophysiological and neurotoxicity assays as well as transcriptomic analysis, to study the neuroprotective effect and molecular mechanisms of edaravone, an FDA-approved drug for ALS, for improving its clinical efficacy. RESULTS: We generate highly pure and functional mRNA-induced MNs (miMNs) from control and ALS iPSCs, as well as embryonic stem cells. Edaravone alleviates H2O2-induced neurotoxicity and electrophysiological dysfunction in miMNs, demonstrating its neuroprotective effect that was also found in the glutamate-induced miMN neurotoxicity model. Guided by the transcriptomic analysis, we show a previously unrecognized effect of edaravone to induce the GDNF receptor RET and the GDNF/RET neurotrophic signaling in vitro and in vivo, suggesting a clinically translatable strategy to activate this key neuroprotective signaling. Notably, edaravone can replace required neurotrophic factors (BDNF and GDNF) to support long-term miMN survival and maturation, further supporting the neurotrophic function of edaravone-activated signaling. Furthermore, we show that edaravone and GDNF combined treatment more effectively protects miMNs from H2O2-induced neurotoxicity than single treatment, suggesting a potential combination strategy for ALS treatment. CONCLUSIONS: This study provides methodology to facilitate iPSC differentiation and disease modeling. Our discoveries will facilitate the development of optimal edaravone-based therapies for ALS and potentially other neurodegenerative diseases.
Subject(s)
Amyotrophic Lateral Sclerosis , Induced Pluripotent Stem Cells , Amyotrophic Lateral Sclerosis/drug therapy , Amyotrophic Lateral Sclerosis/metabolism , Edaravone/metabolism , Edaravone/pharmacology , Edaravone/therapeutic use , Glial Cell Line-Derived Neurotrophic Factor/metabolism , Glial Cell Line-Derived Neurotrophic Factor/pharmacology , Glial Cell Line-Derived Neurotrophic Factor/therapeutic use , Humans , Hydrogen Peroxide/metabolism , Hydrogen Peroxide/pharmacology , Hydrogen Peroxide/therapeutic use , Motor Neurons/metabolism , Proto-Oncogene Proteins c-ret/metabolism , Proto-Oncogene Proteins c-ret/therapeutic use , RNA, Messenger/metabolism , Signal TransductionABSTRACT
Since the opening of Earth Observation (EO) archives (USGS/NASA Landsat and EC/ESA Sentinels), large collections of EO data are freely available, offering scientists new possibilities to better understand and quantify environmental changes. Fully exploiting these satellite EO data will require new approaches for their acquisition, management, distribution, and analysis. Given rapid environmental changes and the emergence of big data, innovative solutions are needed to support policy frameworks and related actions toward sustainable development. Here we present the Swiss Data Cube (SDC), unleashing the information power of Big Earth Data for monitoring the environment, providing Analysis Ready Data over the geographic extent of Switzerland since 1984, which is updated on a daily basis. Based on a cloud-computing platform allowing to access, visualize and analyse optical (Sentinel-2; Landsat 5, 7, 8) and radar (Sentinel-1) imagery, the SDC minimizes the time and knowledge required for environmental analyses, by offering consistent calibrated and spatially co-registered satellite observations. SDC derived analysis ready data supports generation of environmental information, allowing to inform a variety of environmental policies with unprecedented timeliness and quality.
ABSTRACT
More than 40% of non-small cell lung cancer (NSCLC) patients lack actionable targets and require non-targeted chemotherapeutics. Many become refractory to drugs due to underlying resistance-associated mutations. KEAP1 mutant NSCLCs further activate NRF2 and upregulate its client PTGR1. LP-184, a novel alkylating agent belonging to the acylfulvene class is a prodrug dependent upon PTGR1. We hypothesized that NSCLC with KEAP1 mutations would continue to remain sensitive to LP-184. LP-184 demonstrated highly potent anticancer activity both in primary NSCLC cell lines and in those originating from brain metastases of primary lung cancers. LP-184 activity correlated with PTGR1 transcript levels but was independent of mutations in key oncogenes (KRAS and KEAP1) and tumor suppressors (TP53 and STK11). LP-184 was orders of magnitude more potent in vitro than cisplatin and pemetrexed. Correlative analyses of sensitivity with cell line gene expression patterns indicated that alterations in NRF2, MET, EGFR and BRAF consistently modulated LP-184 sensitivity. These correlations were then extended to TCGA analysis of 517 lung adenocarcinoma patients, out of which 35% showed elevated PTGR1, and 40% of those further displayed statistically significant co-occurrence of KEAP1 mutations. The gene correlates of LP-184 sensitivity allow additional personalization of therapeutic options for future treatment of NSCLC.
ABSTRACT
BACKGROUND: Non-targeted cytotoxics with anticancer activity are often developed through preclinical stages using response criteria observed in cell lines and xenografts. A panel of the NCI-60 cell lines is frequently the first line to define tumor types that are optimally responsive. Open data on the gene expression of the NCI-60 cell lines, provides a unique opportunity to add another dimension to the preclinical development of such drugs by interrogating correlations with gene expression patterns. Machine learning can be used to reduce the complexity of whole genome gene expression patterns to derive manageable signatures of response. Application of machine learning in early phases of preclinical development is likely to allow a better positioning and ultimate clinical success of molecules. LP-184 is a highly potent novel alkylating agent where the preclinical development is being guided by a dedicated machine learning-derived response signature. We show the feasibility and the accuracy of such a signature of response by accurately predicting the response to LP-184 validated using wet lab derived IC50s on a panel of cell lines. RESULTS: We applied our proprietary RADR® platform to an NCI-60 discovery dataset encompassing LP-184 IC50s and publicly available gene expression data. We used multiple feature selection layers followed by the XGBoost regression model and reduced the complexity of 20,000 gene expression values to generate a 16-gene signature leading to the identification of a set of predictive candidate biomarkers which form an LP-184 response gene signature. We further validated this signature and predicted response to an additional panel of cell lines. Considering fold change differences and correlation between actual and predicted LP-184 IC50 values as validation performance measures, we obtained 86% accuracy at four-fold cut-off, and a strong (r = 0.70) and significant (p value 1.36e-06) correlation between actual and predicted LP-184 sensitivity. In agreement with the perceived mechanism of action of LP-184, PTGR1 emerged as the top weighted gene. CONCLUSION: Integration of a machine learning-derived signature of response with in vitro assessment of LP-184 efficacy facilitated the derivation of manageable yet robust biomarkers which can be used to predict drug sensitivity with high accuracy and clinical value.
Subject(s)
Alkylating Agents , Antineoplastic Agents , Machine Learning , Biomarkers , Cell Line, Tumor , Humans , Neoplasms/drug therapyABSTRACT
The ability to generate human-induced pluripotent stem cell (hiPSC)-derived neural cells displaying region-specific phenotypes is of particular interest for modeling central nervous system biology in vitro. We describe a unique method by which spinal cord hiPSC-derived astrocytes (hiPSC-A) are cultured with spinal cord hiPSC-derived motor neurons (hiPSC-MN) in a multielectrode array (MEA) system to record electrophysiological activity over time. We show that hiPSC-A enhance hiPSC-MN electrophysiological maturation in a time-dependent fashion. The sequence of plating, density, and age in which hiPSC-A are cocultured with MN, but not their respective hiPSC line origin, are factors that influence neuronal electrophysiology. When compared to coculture with mouse primary spinal cord astrocytes, we observe an earlier and more robust electrophysiological maturation in the fully human cultures, suggesting that the human origin is relevant to the recapitulation of astrocyte/motor neuron crosstalk. Finally, we test pharmacological compounds on our MEA platform and observe changes in electrophysiological activity, which confirm hiPSC-MN maturation. These findings are supported by immunocytochemistry and real-time PCR studies in parallel cultures demonstrating human astrocyte mediated changes in the structural maturation and protein expression profiles of the neurons. Interestingly, this relationship is reciprocal and coculture with neurons influences astrocyte maturation as well. Taken together, these data indicate that in a human in vitro spinal cord culture system, astrocytes support hiPSC-MN maturation in a time-dependent and species-specific manner and suggest a closer approximation of in vivo conditions. Stem Cells Translational Medicine 2019;8:1272&1285.
Subject(s)
Action Potentials , Astrocytes/cytology , Electrodes , Induced Pluripotent Stem Cells/cytology , Motor Neurons/physiology , Spinal Cord/cytology , Animals , Cell Differentiation , Cells, Cultured , Coculture Techniques , Electrophysiological Phenomena , Humans , Mice , Motor Neurons/cytology , NeurogenesisABSTRACT
One of the fundamental limitations in assessing potential efficacy in Central Nervous System (CNS) transplantation of stem cells is the capacity for monitoring cell survival and migration noninvasively and longitudinally. Human glial-restricted progenitor (hGRP) cells (Q-Cells) have been investigated for their utility in providing neuroprotection following transplantation into models of amyotrophic lateral sclerosis (ALS) and have been granted a Food and Drug Administration (FDA) Investigational New Drug (IND) for intraspinal transplantation in ALS patients. Furthermore, clinical development of these cells for therapeutic use will rely on the ability to track the cells using noninvasive imaging methodologies as well as the verification that the transplanted GRPs have disease-relevant activity. As a first step in development, we investigated the use of a perfluorocarbon (PFC) dual-modal (19 F magnetic resonance imaging [MRI] and fluorescence) tracer agent to label Q-Cells in culture and following spinal cord transplantation. PFCs have a number of potential benefits that make them appealing for clinical use. They are quantitative, noninvasive, biologically inert, and highly specific. In this study, we developed optimized PFC labeling protocols for Q-Cells and demonstrate that PFCs do not significantly alter the glial identity of Q-Cells. We also show that PFCs do not interfere with the capacity for differentiation into astrocytes either in vitro or following transplantation into the ventral horn of the mouse spinal cord, and can be visualized in vivo by hot spot 19 F MRI. These studies provide a foundation for further preclinical development of PFCs within the context of evaluating Q-Cell transplantation in the brain and spinal cord of future ALS patients using 19 F MRI. Stem Cells Translational Medicine 2019;8:355-365.
Subject(s)
Fluorocarbons/administration & dosage , Neuroglia/cytology , Stem Cells/cytology , Amyotrophic Lateral Sclerosis/diagnosis , Amyotrophic Lateral Sclerosis/therapy , Animals , Astrocytes/cytology , Cell Differentiation/physiology , Cell Survival/physiology , Cells, Cultured , Fluorine-19 Magnetic Resonance Imaging/methods , Humans , Male , Mice , Spinal Cord/cytology , Spinal Cord/diagnostic imaging , Stem Cell Transplantation/methodsABSTRACT
We recently demonstrated that microRNA-218 (miR-218) is greatly enriched in motor neurons and is released extracellularly in amyotrophic lateral sclerosis model rats. To determine if the released, motor neuron-derived miR-218 may have a functional role in amyotrophic lateral sclerosis, we examined the effect of miR-218 on neighbouring astrocytes. Surprisingly, we found that extracellular, motor neuron-derived miR-218 can be taken up by astrocytes and is sufficient to downregulate an important glutamate transporter in astrocytes [excitatory amino acid transporter 2 (EAAT2)]. The effect of miR-218 on astrocytes extends beyond EAAT2 since miR-218 binding sites are enriched in mRNAs translationally downregulated in amyotrophic lateral sclerosis astrocytes. Inhibiting miR-218 with antisense oligonucleotides in amyotrophic lateral sclerosis model mice mitigates the loss of EAAT2 and other miR-218-mediated changes, providing an important in vivo demonstration of the relevance of microRNA-mediated communication between neurons and astrocytes. These data define a novel mechanism in neurodegeneration whereby microRNAs derived from dying neurons can directly modify the glial phenotype and cause astrocyte dysfunction.
Subject(s)
Amyotrophic Lateral Sclerosis/genetics , Astrocytes/physiology , MicroRNAs/metabolism , Amino Acid Transport System X-AG/genetics , Amino Acid Transport System X-AG/physiology , Animals , Astrocytes/metabolism , Cells, Cultured , Disease Models, Animal , Down-Regulation , Excitatory Amino Acid Transporter 2/genetics , Excitatory Amino Acid Transporter 2/physiology , Glutamic Acid/metabolism , HEK293 Cells , Humans , Mice , Mice, Inbred C57BL , MicroRNAs/genetics , MicroRNAs/physiology , Motor Neurons/metabolism , Motor Neurons/physiology , Neuroglia/metabolismABSTRACT
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder marked by the loss of motor neurons (MNs) in the brain and spinal cord, leading to fatally debilitating weakness. Because this disease predominantly affects MNs, we aimed to characterize the distinct expression profile of that cell type to elucidate underlying disease mechanisms and to identify novel targets that inform on MN health during ALS disease time course. microRNAs (miRNAs) are short, noncoding RNAs that can shape the expression profile of a cell and thus often exhibit cell-type-enriched expression. To determine MN-enriched miRNA expression, we used Cre recombinase-dependent miRNA tagging and affinity purification in mice. By defining the in vivo miRNA expression of MNs, all neurons, astrocytes, and microglia, we then focused on MN-enriched miRNAs via a comparative analysis and found that they may functionally distinguish MNs postnatally from other spinal neurons. Characterizing the levels of the MN-enriched miRNAs in CSF harvested from ALS models of MN disease demonstrated that one miRNA (miR-218) tracked with MN loss and was responsive to an ALS therapy in rodent models. Therefore, we have used cellular expression profiling tools to define the distinct miRNA expression of MNs, which is likely to enrich future studies of MN disease. This approach enabled the development of a novel, drug-responsive marker of MN disease in ALS rodents.SIGNIFICANCE STATEMENT Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease in which motor neurons (MNs) in the brain and spinal cord are selectively lost. To develop tools to aid in our understanding of the distinct expression profiles of MNs and, ultimately, to monitor MN disease progression, we identified small regulatory microRNAs (miRNAs) that were highly enriched or exclusive in MNs. The signal for one of these MN-enriched miRNAs is detectable in spinal tap biofluid from an ALS rat model, where its levels change as disease progresses, suggesting that it may be a clinically useful marker of disease status. Furthermore, rats treated with ALS therapy have restored expression of this MN RNA marker, making it an MN-specific and drug-responsive marker for ALS rodents.
Subject(s)
Amyotrophic Lateral Sclerosis/metabolism , Gene Expression Profiling/methods , MicroRNAs/metabolism , Motor Neurons/metabolism , Animals , Biomarkers/metabolism , Female , Male , Mice , Mice, Inbred C57BL , Reproducibility of Results , Sensitivity and Specificity , Transcriptome/geneticsABSTRACT
The efficacy of drugs targeting the CNS is influenced by their limited brain access, which can lead to complete pharmacoresistance. Recently a tissue-specific and selective upregulation of the multidrug efflux transporter ABCB1 or P-glycoprotein (P-gp) in the spinal cord of both patients and the mutant SOD1-G93A mouse model of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease that prevalently kills motor neurons has been reported. Here, we extended the analysis of P-gp expression in the SOD1-G93A ALS mouse model and found that P-gp upregulation was restricted to endothelial cells of the capillaries, while P-gp expression was not detected in other cells of the spinal cord parenchyma such as astrocytes, oligodendrocytes, and neurons. Using both in vitro human and mouse models of the blood-brain barrier (BBB), we found that mutant SOD1 astrocytes were driving P-gp upregulation in endothelial cells. In addition, a significant increase in reactive oxygen species production, Nrf2 and NFκB activation in endothelial cells exposed to mutant SOD1 astrocytes in both human and murine BBB models were observed. Most interestingly, astrocytes expressing FUS-H517Q, a different familial ALS-linked mutated gene, also drove NFκB-dependent upregulation of P-gp. However, the pathway was not dependent on oxidative stress but rather involved TNF-α release. Overall, these findings indicated that nuclear translocation of NFκB was a converging mechanism used by endothelial cells of the BBB to upregulate P-gp expression in mutant SOD1-linked ALS and possibly other forms of familial ALS. GLIA 2016 GLIA 2016;64:1298-1313.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Amyotrophic Lateral Sclerosis/metabolism , Astrocytes/metabolism , Blood-Brain Barrier/metabolism , Endothelial Cells/metabolism , ATP Binding Cassette Transporter, Subfamily B/metabolism , Amyotrophic Lateral Sclerosis/pathology , Animals , Astrocytes/pathology , Blood-Brain Barrier/pathology , Capillaries/metabolism , Capillaries/pathology , Cell Line , Coculture Techniques , Disease Models, Animal , Humans , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/pathology , Mice, Transgenic , NF-kappa B/metabolism , RNA-Binding Protein FUS/metabolism , Spinal Cord/metabolism , Spinal Cord/pathology , Superoxide Dismutase-1/genetics , Superoxide Dismutase-1/metabolism , Up-Regulation , ATP-Binding Cassette Sub-Family B Member 4ABSTRACT
Duchenne muscular dystrophy (DMD) remains an intractable genetic disease. Althogh there are several animal models of DMD, there is no human cell model that carries patient-specific DYSTROPHIN mutations. Here, we present a human DMD model using human induced pluripotent stem cells (hiPSCs). Our model reveals concordant disease-related phenotypes with patient-dependent variation, which are partially reversed by genetic and pharmacological approaches. Our "chemical-compound-based" strategy successfully directs hiPSCs into expandable myoblasts, which exhibit a myogenic transcriptional program, forming striated contractile myofibers and participating in muscle regeneration in vivo. DMD-hiPSC-derived myoblasts show disease-related phenotypes with patient-to-patient variability, including aberrant expression of inflammation or immune-response genes and collagens, increased BMP/TGFß signaling, and reduced fusion competence. Furthermore, by genetic correction and pharmacological "dual-SMAD" inhibition, the DMD-hiPSC-derived myoblasts and genetically corrected isogenic myoblasts form "rescued" multi-nucleated myotubes. In conclusion, our findings demonstrate the feasibility of establishing a human "DMD-in-a-dish" model using hiPSC-based disease modeling.
Subject(s)
Induced Pluripotent Stem Cells/pathology , Models, Biological , Muscular Dystrophy, Duchenne/pathology , Myoblasts/pathology , Animals , Cell Line , Flow Cytometry , Humans , Mice , Muscle Development , Muscle Fibers, Skeletal/metabolism , Muscular Dystrophy, Duchenne/genetics , Myoblasts/metabolism , Phenotype , Signal Transduction , Smad Proteins/metabolismABSTRACT
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive loss of motor neurons in the CNS. Astrocytes play a critical role in disease progression of ALS. Astrocytes are interconnected through a family of gap junction proteins known as connexins (Cx). Cx43 is a major astrocyte connexin conducting crucial homeostatic functions in the CNS. Under pathological conditions, connexin expression and functions are altered. Here we report that an abnormal increase in Cx43 expression serves as one of the mechanisms for astrocyte-mediated toxicity in ALS. We observed a progressive increase in Cx43 expression in the SOD1(G93A) mouse model of ALS during the disease course. Notably, this increase in Cx43 was also detected in the motor cortex and spinal cord of ALS patients. Astrocytes isolated from SOD1(G93A) mice as well as human induced pluripotent stem cell (iPSC)-derived astrocytes showed an increase in Cx43 protein, which was found to be an endogenous phenomenon independent of neuronal co-culture. Increased Cx43 expression led to important functional consequences when tested in SOD1(G93A) astrocytes when compared to control astrocytes over-expressing wild-type SOD1 (SOD1(WT) ). We observed SOD1(G93A) astrocytes exhibited enhanced gap junction coupling, increased hemichannel-mediated activity, and elevated intracellular calcium levels. Finally, we tested the impact of increased expression of Cx43 on MN survival and observed that use of both a pan Cx43 blocker and Cx43 hemichannel blocker conferred neuroprotection to MNs cultured with SOD1(G93A) astrocytes. These novel findings show a previously unrecognized role of Cx43 in ALS-related motor neuron loss. GLIA 2016;64:1154-1169.
Subject(s)
Amyotrophic Lateral Sclerosis/pathology , Astrocytes/physiology , Cerebral Cortex/pathology , Connexin 43/metabolism , Gene Expression Regulation/genetics , Motor Neurons/physiology , Spinal Cord/pathology , Adenosine Triphosphate/pharmacology , Aged , Aged, 80 and over , Amyotrophic Lateral Sclerosis/genetics , Animals , Astrocytes/drug effects , Cells, Cultured , Connexin 43/genetics , Female , Gene Expression Regulation, Developmental/genetics , Glial Fibrillary Acidic Protein/genetics , Glial Fibrillary Acidic Protein/metabolism , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/physiology , Interleukin-1beta/pharmacology , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Middle Aged , Motor Neurons/drug effects , Peptides/pharmacology , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
Transplantation-based replacement of lost and/or dysfunctional astrocytes is a promising therapy for spinal cord injury (SCI) that has not been extensively explored, despite the integral roles played by astrocytes in the central nervous system (CNS). Induced pluripotent stem (iPS) cells are a clinically-relevant source of pluripotent cells that both avoid ethical issues of embryonic stem cells and allow for homogeneous derivation of mature cell types in large quantities, potentially in an autologous fashion. Despite their promise, the iPS cell field is in its infancy with respect to evaluating in vivo graft integration and therapeutic efficacy in SCI models. Astrocytes express the major glutamate transporter, GLT1, which is responsible for the vast majority of glutamate uptake in spinal cord. Following SCI, compromised GLT1 expression/function can increase susceptibility to excitotoxicity. We therefore evaluated intraspinal transplantation of human iPS cell-derived astrocytes (hIPSAs) following cervical contusion SCI as a novel strategy for reconstituting GLT1 expression and for protecting diaphragmatic respiratory neural circuitry. Transplant-derived cells showed robust long-term survival post-injection and efficiently differentiated into astrocytes in injured spinal cord of both immunesuppressed mice and rats. However, the majority of transplant-derived astrocytes did not express high levels of GLT1, particularly at early times post-injection. To enhance their ability to modulate extracellular glutamate levels, we engineered hIPSAs with lentivirus to constitutively express GLT1. Overexpression significantly increased GLT1 protein and functional GLT1-mediated glutamate uptake levels in hIPSAs both in vitro and in vivo post-transplantation. Compared to human fibroblast control and unmodified hIPSA transplantation, GLT1-overexpressing hIPSAs reduced (1) lesion size within the injured cervical spinal cord, (2) morphological denervation by respiratory phrenic motor neurons at the diaphragm neuromuscular junction, and (3) functional diaphragm denervation as measured by recording of spontaneous EMGs and evoked compound muscle action potentials. Our findings demonstrate that hiPSA transplantation is a therapeutically-powerful approach for SCI.
Subject(s)
Astrocytes/physiology , Induced Pluripotent Stem Cells/physiology , Induced Pluripotent Stem Cells/transplantation , Spinal Cord Injuries/surgery , Action Potentials/physiology , Animals , Astrocytes/transplantation , Cell Differentiation , Cell Proliferation , Cells, Cultured , Diaphragm/physiopathology , Disease Models, Animal , Excitatory Amino Acid Transporter 2 , Female , Gene Expression Regulation , Glutamate Plasma Membrane Transport Proteins/genetics , Glutamate Plasma Membrane Transport Proteins/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Mice , Mice, Inbred C57BL , Neuromuscular Junction/pathology , Neuromuscular Junction/physiopathology , Rats , Rats, Sprague-Dawley , Spinal Cord Injuries/pathologyABSTRACT
The ability to reprogram adult somatic cells into pluripotent stem cells that can differentiate into all three germ layers of the developing human has fundamentally changed the landscape of biomedical research. For a neurodegenerative disease like Amyotrophic Lateral Sclerosis (ALS), which does not manifest itself until adulthood and is a heterogeneous disease with few animal models, this technology may be particularly important. Induced pluripotent stem cells (iPSC) have been created from patients with several familial forms of ALS as well as some sporadic forms of ALS. These cells have been differentiated into ALS-relevant cell subtypes including motor neurons and astrocytes, among others. ALS-relevant pathologies have also been identified in motor neurons from these cells and may provide a window into understanding disease mechanisms in vitro. Given that this is a relatively new field of research, numerous challenges remain before iPSC methodologies can fulfill their potential as tools for modeling ALS as well as providing a platform for the investigation of ALS therapeutics. This article is part of a Special Issue entitled ALS complex pathogenesis.
Subject(s)
Amyotrophic Lateral Sclerosis/physiopathology , Induced Pluripotent Stem Cells/physiology , Amyotrophic Lateral Sclerosis/drug therapy , Animals , Drug Evaluation, Preclinical/economics , Drug Evaluation, Preclinical/methods , HumansABSTRACT
The generation of human induced pluripotent stem cells (hiPSCs) represents an exciting advancement with promise for stem cell transplantation therapies as well as for neurological disease modeling. Based on the emerging roles for astrocytes in neurological disorders, we investigated whether hiPSC-derived astrocyte progenitors could be engrafted to the rodent spinal cord and how the characteristics of these cells changed between in vitro culture and after transplantation to the in vivo spinal cord environment. Our results show that human embryonic stem cell- and hiPSC-derived astrocyte progenitors survive long-term after spinal cord engraftment and differentiate to astrocytes in vivo with few cells from other lineages present. Gene profiling of the transplanted cells demonstrates the astrocyte progenitors continue to mature in vivo and upregulate a variety of astrocyte-specific genes. Given this mature astrocyte gene profile, this work highlights hiPSCs as a tool to investigate disease-related astrocyte biology using in vivo disease modeling with significant implications for human neurological diseases currently lacking animal models.
Subject(s)
Astrocytes , Cell Differentiation , Gene Expression Profiling , Induced Pluripotent Stem Cells , Neural Stem Cells , Spinal Cord , Animals , Astrocytes/cytology , Astrocytes/metabolism , Gene Expression Regulation , Heterografts , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/transplantation , Mice , Neural Stem Cells/cytology , Neural Stem Cells/metabolism , Rats , Rats, Sprague-Dawley , Spinal Cord/cytology , Spinal Cord/metabolismABSTRACT
Tumor-initiating stem cells (also referred to as cancer stem cells, CSCs) are a subpopulation of cancer cells that play unique roles in tumor propagation, therapeutic resistance and tumor recurrence. It is increasingly important to understand how molecular signaling regulates the self-renewal and differentiation of CSCs. Basic helix-loop-helix (bHLH) transcription factors are critical for the differentiation of normal stem cells, yet their roles in neoplastic stem cells are not well understood. In glioblastoma neurosphere cultures that contain cancer stem cells (GBM-CSCs), the bHLH family member inhibitors of DNA binding protein 2 and 4 (Id2 and Id4) were found to be upregulated during the differentiation of GBM-CSCs in response to histone deacetylase inhibitors. In this study, we examined the functions of Id2 and Id4 in GBM neurosphere cells and identified Id proteins as efficient differentiation regulators of GBM-CSCs. Overexpression of Id2 and Id4 promoted the lineage-specific differentiation of GBM neurosphere cells as evidenced by the induction of neuronal/astroglial differentiation markers Tuj1 and GFAP and the inhibition of the oligodendroglial marker GalC. Id protein overexpression also reduced both stem cell marker expression and neurosphere formation potential, a biological marker of cancer cell "stemness." We further showed that Id2 and Id4 regulated GBM neurosphere differentiation through downregulating of another bHLH family member, the oligodendroglial lineage-associated transcription factors (Olig) 1 and 2. Our results provide evidence for distinct functions of Id proteins in neoplastic stem cells, which supports Id proteins and their downstream targets as potential candidates for differentiation therapy in CSCs.
Subject(s)
Glioblastoma/metabolism , Glioblastoma/pathology , Inhibitor of Differentiation Protein 2/metabolism , Inhibitor of Differentiation Proteins/metabolism , Neoplastic Stem Cells/metabolism , Basic Helix-Loop-Helix Transcription Factors/biosynthesis , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Differentiation , Cell Line, Tumor , DNA-Binding Proteins/antagonists & inhibitors , Galactosylceramidase/antagonists & inhibitors , Galactosylceramidase/biosynthesis , Humans , Inhibitor of Differentiation Protein 2/biosynthesis , Inhibitor of Differentiation Proteins/biosynthesis , Neoplastic Stem Cells/pathology , Nerve Tissue Proteins/biosynthesis , Nerve Tissue Proteins/genetics , Oligodendrocyte Transcription Factor 2 , Oligodendroglia/metabolism , RNA Interference , RNA, Small Interfering , Tubulin/biosynthesisABSTRACT
Cell-to-cell fusion plays an important role in normal physiology and in different pathological conditions. Early fusion stages mediated by specialized proteins and yielding fusion pores are followed by a pore expansion stage that is dependent on cell metabolism and yet unidentified machinery. Because of a similarity of membrane bending in the fusion pore rim and in highly curved intracellular membrane compartments, in the present study we explored whether changes in the activity of the proteins that generate these compartments affect cell fusion initiated by protein fusogens of influenza virus and baculovirus. We raised the intracellular concentration of curvature-generating proteins in cells by either expressing or microinjecting the ENTH (epsin N-terminal homology) domain of epsin or by expressing the GRAF1 (GTPase regulator associated with focal adhesion kinase 1) BAR (Bin/amphiphysin/Rvs) domain or the FCHo2 (FCH domain-only protein 2) F-BAR domain. Each of these treatments promoted syncytium formation. Cell fusion extents were also influenced by treatments targeting the function of another curvature-generating protein, dynamin. Cell-membrane-permeant inhibitors of dynamin GTPase blocked expansion of fusion pores and dominant-negative mutants of dynamin influenced the syncytium formation extents. We also report that syncytium formation is inhibited by reagents lowering the content and accessibility of PtdIns(4,5)P(2), an important regulator of intracellular membrane remodelling. Our findings indicate that fusion pore expansion at late stages of cell-to-cell fusion is mediated, directly or indirectly, by intracellular membrane-shaping proteins.