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1.  Gene Profiling of Human Induced Pluripotent Stem Cell-Derived Astrocyte Progenitors Following Spinal Cord Engraftment 
This study investigated whether human induced pluripotent stem cell (hiPSC)-derived astrocyte progenitors could be engrafted to the rodent spinal cord and how the characteristics of these cells changed. 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.
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.
PMCID: PMC4006486  PMID: 24604284
Human induced pluripotent stem cells; Stem cell transplantation; Astrocytes; Gene profiling
2.  A Comprehensive Library of Familial Human Amyotrophic Lateral Sclerosis Induced Pluripotent Stem Cells 
PLoS ONE  2015;10(3):e0118266.
Amyotrophic lateral sclerosis is a progressive disease characterized by the loss of upper and lower motor neurons, leading to paralysis of voluntary muscles. About 10% of all ALS cases are familial (fALS), among which 15–20% are linked to Cu/Zn superoxide dismutase (SOD1) mutations, usually inherited in an autosomal dominant manner. To date only one FDA approved drug is available which increases survival moderately. Our understanding of ALS disease mechanisms is largely derived from rodent model studies, however due to the differences between rodents and humans, it is necessary to have humanized models for studies of disease pathogenesis as well as drug development. Therefore, we generated a comprehensive library of a total 22 of fALS patient-specific induced pluripotent stem cell (iPSC) lines. These cells were thoroughly characterized before being deposited into the library. The library of cells includes a variety of C9orf72 mutations, sod1 mutations, FUS, ANG and FIG4 mutations. Certain mutations are represented with more than one line, which allows for studies of variable genetic backgrounds. In addition, these iPSCs can be successfully differentiated to astroglia, a cell type known to play a critical role in ALS disease progression. This library represents a comprehensive resource that can be used for ALS disease modeling and the development of novel therapeutics.
PMCID: PMC4356618  PMID: 25760436
4.  Mechanisms, models and biomarkers in amyotrophic lateral sclerosis 
The last 30 years have seen a major advance in the understanding of the clinical and pathological heterogeneity of amyotrophic lateral sclerosis (ALS), and its overlap with frontotemporal dementia. Multiple, seemingly disparate biochemical pathways converge on a common clinical syndrome characterized by progressive loss of upper and lower motor neurons. Pathogenic themes in ALS include excitotoxicity, oxidative stress, mitochondrial dysfunction, neuroinflammation, altered energy metabolism, and most recently RNA mis-processing. The transgenic rodent, overexpressing mutant superoxide dismutase-1, is now only one of several models of ALS pathogenesis. The nematode, fruit fly and zebrafish all offer fresh insight, and the development of induced pluripotent stem cell-derived motor neurons holds promise for the screening of candidate therapeutics. The lack of useful biomarkers in ALS contributes to diagnostic delay, and the inability to stratify patients by prognosis may be an important factor in the failure of therapeutic trials. Biomarkers sensitive to disease activity might lessen reliance on clinical measures and survival as trial endpoints and reduce study length. Emerging proteomic markers of neuronal loss and glial activity in cerebrospinal fluid, a cortical signature derived from advanced structural and functional MRI, and the development of more sensitive measurements of lower motor neuron physiology are leading a new phase of biomarker-driven therapeutic discovery.
PMCID: PMC4284067  PMID: 23678877
ALS; biomarkers; pathogenesis; neuroimaging; neurophysiology
5.  C9orf72 Nucleotide Repeat Structures Initiate Molecular Cascades of Disease 
Nature  2014;507(7491):195-200.
A hexanucleotide repeat expansion (HRE), (GGGGCC)n, in C9orf72 is the most common genetic cause of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we identify a molecular mechanism by which structural polymorphism of the HRE leads to ALS/FTD pathology and defects. The HRE forms DNA and RNA G-quadruplexes with distinct structures and promotes RNA•DNA hybrids (R-loops). The structural polymorphism causes a repeat length-dependent accumulation of transcripts aborted in the HRE region. These transcribed repeats bind to ribonucleoproteins in a conformationdependent manner. Specifically, nucleolin (NCL), an essential nucleolar protein, preferentially binds the HRE G-quadruplex, and patient cells show evidence of nucleolar stress. Our results demonstrate that distinct C9orf72 HRE structural polymorphism at both DNA and RNA levels initiates molecular cascades leading to ALS/FTD pathologies, and provide the basis for a mechanistic model for repeat-associated neurodegenerative diseases.
PMCID: PMC4046618  PMID: 24598541
ALS; FTD; C9orf72; G-quadruplex; R-loop; nucleolin; hnRNP; nucleolar stress; abortive transcription; tandem repeats; and repeat expansions
6.  RNA Toxicity from the ALS/FTD C9ORF72 Expansion Is Mitigated by Antisense Intervention 
Neuron  2013;80(2):415-428.
A hexanucleotide GGGGCC repeat expansion in the noncoding region of the C9ORF72 gene is the most common genetic abnormality in familial and sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The function of the C9ORF72 protein is unknown, as is the mechanism by which the repeat expansion could cause disease. Induced pluripotent stem cell (iPSC)-differentiated neurons from C9ORF72 ALS patients revealed disease-specific (1) intranuclear GGGGCCexp RNA foci, (2) dysregulated gene expression, (3) sequestration of GGGGCCexp RNA binding protein ADARB2, and (4) susceptibility to excitotoxicity. These pathological and pathogenic characteristics were confirmed in ALS brain and were mitigated with antisense oligonucleotide (ASO) therapeutics to the C9ORF72 transcript or repeat expansion despite the presence of repeat-associated non-ATG translation (RAN) products. These data indicate a toxic RNA gain-of-function mechanism as a cause of C9ORF72 ALS and provide candidate antisense therapeutics and candidate human pharmacodynamic markers for therapy.
PMCID: PMC4098943  PMID: 24139042
7.  Exome sequencing to identify de novo mutations in sporadic ALS trios 
Nature neuroscience  2013;16(7):10.1038/nn.3412.
ALS is a devastating neurodegenerative disease whose causes are still poorly understood. To identify additional genetic risk factors, here we assess the role of de novo mutations in ALS by sequencing the exomes of 47 ALS patients and both of their unaffected parents (n=141 exomes). We found that amino acid-altering de novo mutations are enriched in genes encoding chromatin regulators, including the neuronal chromatin remodeling complex component SS18L1/CREST. CREST mutations inhibit activity-dependent neurite outgrowth in primary neurons, and CREST associates with the ALS protein FUS. These findings expand our understanding of the ALS genetic landscape and provide a resource for future studies into the pathogenic mechanisms contributing to sporadic ALS.
PMCID: PMC3709464  PMID: 23708140
8.  Glia: an emerging target for neurological disease therapy 
Therapeutic strategies using stem cells for treating neurological diseases are receiving more attention as the scientific community appreciates cell-autonomous contributions to several diseases of the central nervous system. The transplantation of stem cells from various sources is now being employed for both neuronal and glial replacement. This review provides an assessment of glial contributions to some of the central nervous system diseases and the advancements in cellular replacement approaches. The rationale for glial replacement in individual diseases and the potential hurdles for cell-replacement strategies are also emphasized. The significant progress in the field of stem cell biology with the advent of tools such as induced pluripotent stem cells and imaging techniques holds promise for the clinical application of cell therapeutics.
PMCID: PMC3580428  PMID: 23021042
9.  Hemiparetic Primary Lateral Sclerosis: Revisiting Mills Syndrome 
Case Reports in Neurology  2013;7(3):191-195.
A slowly progressive hemiparesis beginning in a single limb with evolution to the ipsilateral limb was originally described in 8 patients in 1906 by Mills. We present 5 cases of progressive hemiparetic corticospinal tract degeneration, identified by the clinical presentation and the exclusion of other etiologies using serological, imaging, and electrodiagnostic studies.
PMCID: PMC4611067  PMID: 26500542
Prognosis; Primary lateral sclerosis; Mills syndrome; Asymmetry
10.  Electrical impedance myography as a biomarker to assess ALS progression 
Electrical impedance myography (EIM), a non-invasive, electrophysiological technique, has preliminarily shown value as an ALS biomarker. Here we perform a multicenter study to further assess EIM’s potential for tracking ALS. ALS patients were enrolled across eight sites. Each subject underwent EIM, handheld dynamometry (HHD), and the ALS Functional Rating Scale-revised (ALSFRS-R) regularly. Techniques were compared by assessing the coefficient of variation (CoV) in the rate of decline and each technique’s correlation to survival. Results showed that in the 60 patients followed for one year, EIM phase measured from the most rapidly progressing muscle in each patient had a CoV in the rate of decline of 0.62, compared to HHD (0.82) and the ALSFRS-R (0.74). Restricting the measurements to the first six months gave a CoV of 0.55 for EIM, 0.93 for HHD, and 0.84 for ALSFRS-R. For both time-periods, all three measures correlated with survival. Based on these data, a six-month clinical trial designed to detect a 20% treatment effect with 80% power using EIM would require only 95 patients/arm compared to the ALSFRS-R, which would require 220 subjects/arm. In conclusion, EIM can serve as a useful ALS biomarker that offers the prospect of greatly accelerating phase 2 clinical trials.
PMCID: PMC3422377  PMID: 22670883
Clinical trials; amyotrophic lateral sclerosis; electrical impedance myography
11.  Selective Increase of Two ABC Drug Efflux Transporters at the Blood-Spinal Cord Barrier Suggests Induced Pharmacoresistance in ALS 
Neurobiology of Disease  2012;47(2):194-200.
ATP-binding cassette (ABC) drug efflux transporters in the CNS are predominantly localized to the luminal surface of endothelial cells in capillaries to impede CNS accumulation of xenobiotics. Inflammatory mediators and cellular stressors regulate their activity. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of upper and lower motor neurons characterized by extensive neuroinflammation. Here we tested the hypothesis that disease-driven changes in ABC transporter expression and function occur in ALS. Given the multitude of ABC transporters with their widespread substrate recognition, we began by examining expression levels of several ABC transporters. We found a selective increase in only two transporters; P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) both at mRNA and protein levels, in the SOD1-G93A mouse model of ALS, specifically in disease-affected CNS regions. Detailed analysis revealed a similar disease-driven increase in P-gp and BCRP levels in spinal cord microvessels, indicating that their altered expression occurs at the blood spinal cord barrier. Transport activity of P-gp and BCRP increased with disease progression in spinal cord and cerebral cortex capillaries. Finally, P-gp and BCRP protein expression also increased in spinal cords of ALS patients. Preclinical drug trials in the mouse model of ALS have failed to decisively slow or arrest disease progression; pharmacoresistance imparted by ABC transporters is one possible explanation for these failures. Our observations have large implications for ALS therapeutics in humans and suggest that the obstacle provided by these transporters to drug treatments must be overcome to develop effective ALS pharmacotherapies.
PMCID: PMC3367047  PMID: 22521463
Amyotrophic Lateral Sclerosis; ABC Transporters; P-glycoprotein; Breast Cancer Resistance Protein; Blood-Brain Barrier; Blood-Spinal Cord Barrier; Pharmacoresistance
12.  Reduction in expression of the astrocyte glutamate transporter, GLT1, worsens functional and histological outcomes following traumatic spinal cord injury 
Glia  2011;59(12):1996-2005.
The astrocyte glutamate transporter, GLT1, is responsible for the vast majority of glutamate uptake in the adult central nervous system (CNS), thereby regulating extracellular glutamate homeostasis and preventing excitotoxicity. Glutamate dysregulation plays a central role in outcome following traumatic spinal cord injury (SCI). To determine the role of GLT1 in secondary cell loss following SCI, mice heterozygous for the GLT1 astrocyte glutamate transporter (GLT1+/−) and wild-type mice received thoracic crush SCI. Compared to wild-type controls, GLT1+/− mice had an attenuated recovery in hindlimb motor function, increased lesion size, and decreased tissue sparing. GLT1+/− mice showed a decrease in intraspinal GLT1 protein and functional glutamate uptake compared to wild-type mice, accompanied by increased apoptosis and neuronal loss following crush injury. These results suggest that astrocyte GLT1 plays a role in limiting secondary cell death following SCI, and also show that compromise of key astrocyte functions has significant effects on outcome following traumatic CNS injury. These findings also suggest that increasing intraspinal GLT1 expression may represent a therapeutically relevant target for SCI treatment.
PMCID: PMC3269541  PMID: 21882244
secondary injury; GLT1+/− mice; crush injury; glutamate uptake; excitotoxicity
13.  Creation of an Open-Access, Mutation-Defined Fibroblast Resource for Neurological Disease Research 
PLoS ONE  2012;7(8):e43099.
Our understanding of the molecular mechanisms of many neurological disorders has been greatly enhanced by the discovery of mutations in genes linked to familial forms of these diseases. These have facilitated the generation of cell and animal models that can be used to understand the underlying molecular pathology. Recently, there has been a surge of interest in the use of patient-derived cells, due to the development of induced pluripotent stem cells and their subsequent differentiation into neurons and glia. Access to patient cell lines carrying the relevant mutations is a limiting factor for many centres wishing to pursue this research. We have therefore generated an open-access collection of fibroblast lines from patients carrying mutations linked to neurological disease. These cell lines have been deposited in the National Institute for Neurological Disorders and Stroke (NINDS) Repository at the Coriell Institute for Medical Research and can be requested by any research group for use in in vitro disease modelling. There are currently 71 mutation-defined cell lines available for request from a wide range of neurological disorders and this collection will be continually expanded. This represents a significant resource that will advance the use of patient cells as disease models by the scientific community.
PMCID: PMC3428297  PMID: 22952635
14.  Selected statins produce rapid spinal motor neuron loss in vitro 
Hmg-CoA reductase inhibitors (statins) are widely used to prevent disease associated with vascular disease and hyperlipidemia. Although side effects are uncommon, clinical observations suggest statin exposure may exacerbate neuromuscular diseases, including peripheral neuropathy and amyotrophic lateral sclerosis. Although some have postulated class-effects, prior studies of hepatocytes and myocytes indicate that the statins may exhibit differential effects. Studies of neuronal cells have been limited.
We examined the effects of statins on cultured neurons and Schwann cells. Cultured spinal motor neurons were grown on transwell inserts and assessed for viability using immunochemical staining for SMI-32. Cultured cortical neurons and Schwann cells were assessed using dynamic viability markers.
7 days of exposure to fluvastatin depleted spinal motor neurons in a dose-dependent manner with a KD of < 2 μM. Profound neurite loss was observed after 4 days exposure in culture. Other statins were found to produce toxic effects at much higher concentrations. In contrast, no such toxicity was observed for cultured Schwann cells or cortical neurons.
It is known from pharmacokinetic studies that daily treatment of young adults with fluvastatin can produce serum levels in the single micromolar range. We conclude that specific mechanisms may explain neuromuscular disease worsening with statins and further study is needed.
PMCID: PMC3487793  PMID: 22703530
ALS; Peripheral neuropathy; Statins; Toxicity; Motorneuronopathy
15.  New Treatments in Amyotrophic Lateral Sclerosis 
Neuropsychopharmacology  2010;36(1):370-372.
PMCID: PMC3055527  PMID: 21116264
16.  Human Glial-Restricted Progenitor Transplantation into Cervical Spinal Cord of the SOD1G93A Mouse Model of ALS 
PLoS ONE  2011;6(10):e25968.
Cellular abnormalities are not limited to motor neurons in amyotrophic lateral sclerosis (ALS). There are numerous observations of astrocyte dysfunction in both humans with ALS and in SOD1G93A rodents, a widely studied ALS model. The present study therapeutically targeted astrocyte replacement in this model via transplantation of human Glial-Restricted Progenitors (hGRPs), lineage-restricted progenitors derived from human fetal neural tissue. Our previous findings demonstrated that transplantation of rodent-derived GRPs into cervical spinal cord ventral gray matter (in order to target therapy to diaphragmatic function) resulted in therapeutic efficacy in the SOD1G93A rat. Those findings demonstrated the feasibility and efficacy of transplantation-based astrocyte replacement for ALS, and also show that targeted multi-segmental cell delivery to cervical spinal cord is a promising therapeutic strategy, particularly because of its relevance to addressing respiratory compromise associated with ALS. The present study investigated the safety and in vivo survival, distribution, differentiation, and potential efficacy of hGRPs in the SOD1G93A mouse. hGRP transplants robustly survived and migrated in both gray and white matter and differentiated into astrocytes in SOD1G93A mice spinal cord, despite ongoing disease progression. However, cervical spinal cord transplants did not result in motor neuron protection or any therapeutic benefits on functional outcome measures. This study provides an in vivo characterization of this glial progenitor cell and provides a foundation for understanding their capacity for survival, integration within host tissues, differentiation into glial subtypes, migration, and lack of toxicity or tumor formation.
PMCID: PMC3187829  PMID: 21998733
17.  Peripheral Hyperstimulation Alters Site of Disease Onset and Course in SOD1 Rats 
Neurobiology of disease  2010;39(3):252-264.
In amyotrophic lateral sclerosis (ALS), the exogenous temporal triggers that result in initial motor neuron death are not understood. Overactivation and consequent accelerated loss of vulnerable motor neurons is one theory of disease initiation. The vulnerability of motor neurons in response to chronic peripheral nerve hyperstimulation was tested in the SOD1G93A rat model of ALS. A novel in vivo technique for peripheral phrenic nerve stimulation was developed via intra-diaphragm muscle electrode implantation at the phrenic motor endpoint. Chronic bilateral phrenic nerve hyperstimulation in SOD1G93A rats accelerated disease progression, including shortened lifespan, hastened motor neuron loss and increased denervation at diaphragm neuromuscular junctions. Hyperstimulation also resulted in focal decline in adjacent forelimb function. These results show that peripheral phrenic nerve hyperstimulation accelerates cell death of vulnerable spinal motor neurons, modifies both temporal and anatomical onset of disease, and leads to involvement of disease in adjacent anatomical regions in this ALS model.
PMCID: PMC2910141  PMID: 20381620
motor neuron; neurodegeneration; ALS; amyotrophic lateral sclerosis; SOD1; phrenic nerve; diaphragm; diaphragm pacing; diaphragm stimulation; respiratory; disease onset; environment
18.  A targeted neuroglial reporter line generated by homologous recombination in human embryonic stem cells 
Stem cells (Dayton, Ohio)  2009;27(8):1836-1846.
In this study we targeted Olig2, a basic helix-loop-helix transcription factor that plays an important role in motoneuron and oligodendrocyte development, in human embryonic stem cell (hESC) line BG01 by homologous recombination. One allele of Olig2 locus was replaced by a GFP cassette with a targeting efficiency of 5.7%. Targeted clone R-Olig2 (like the other clones) retained pluripotency, a typical hESC morphology and a normal parental karyotype 46, XY. Most importantly, GFP expression recapitulated endogenous Olig2 expression when R-Olig2 was induced by sonic hedgehog and retinoic acid, and GFP+ cells could be purified by fluorescence-activated cell sorting (FACS). Consistent with previous reports on rodents, early GFP-expressing cells appeared biased to a neuronal fate whereas late GFP-expressing cells appeared biased to an oligodendrocytic fate. This was corroborated by myoblast coculture, transplantation into the rat spinal cords and whole genome expression profiling. The present work reports an hESC reporter line generated by homologous recombination targeting a neural lineage specific gene, which can be differentiated and sorted to obtain pure neural progenitor populations.
PMCID: PMC2741170  PMID: 19544414
Gene targeting; neurogenesis; gliogenesis; bHLH transcription factor; Olig2; ESC
19.  Focal Transplantation-based Astrocyte Replacement is Neuroprotective in a Model of Motor Neuron Disease 
Nature neuroscience  2008;11(11):1294-1301.
Cellular abnormalities in amyotrophic lateral sclerosis (ALS) are not limited to motor neurons. Astrocyte dysfunction occurs in human ALS and SOD1G93A animal models. Therefore, the value of focal enrichment of normal astrocytes was investigated using transplantation of lineage-restricted astrocyte precursors, Glial-Restricted Precursors (GRPs). GRPs were transplanted around cervical spinal cord respiratory motor neuron pools, the principal cells responsible for death in this neurodegenerative disease. GRPs survived in diseased tissue, differentiated efficiently into astrocytes, and reduced microgliosis in SOD1G93A rat cervical spinal cord. GRPs extended survival and disease duration, attenuated motor neuron loss, and slowed declines in fore-limb motor and respiratory physiological function. Neuroprotection was mediated in part by the primary astrocyte glutamate transporter, GLT1. These findings demonstrate the feasibility and efficacy of transplantation-based astrocyte replacement, and show that targeted multi-segmental cell delivery to cervical spinal cord is a promising therapeutic strategy for slowing focal motor neuron loss associated with ALS.
PMCID: PMC2656686  PMID: 18931666
stem cell; grafting; transplantation; motor neuron; neurodegeneration; replacement; neuroprotection; non-cell autonomous; astroglia; astrocyte; neural precursor cell; progenitor; lineage-restricted precursor; glial precursor; ALS; amyotrophic lateral sclerosis; SOD1
20.  Intraparenchymal spinal cord delivery of adeno-associated virus IGF-1 is protective in the SOD1G93A model of ALS 
Brain research  2007;1185:256-265.
The potent neuroprotective activities of neurotrophic factors, including insulin-like growth factor 1 (IGF-1), make them promising candidates for treatment of amyotrophic lateral sclerosis (ALS). In an effort to maximize rate of motor neuron transduction, achieve high levels of spinal IGF-1, and thus enhance therapeutic benefit, we injected an adeno-associated virus 2 (AAV2)-based vector encoding human IGF-1 (CERE-130) into lumbar spinal cord parenchyma of SOD1G93A mice. We observed robust and long-term intraspinal IGF-1 expression and partial rescue of lumbar spinal cord motor neurons, as well as sex-specific delayed disease onset, weight loss, decline in hindlimb grip strength and increased animal survival.
PMCID: PMC2265207  PMID: 17963733
Adeno; associated virus; insulin; like growth factor 1; gene therapy; neurodegeneration; amyotrophic lateral sclerosis; neuroprotection

Results 1-20 (20)