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1.  Efficacy of local polymer-based and systemic delivery of the anti-glutamatergic agents riluzole and memantine in rat glioma models 
Journal of neurosurgery  2014;120(4):854-863.
The poor outcome of malignant gliomas is largely due to local invasiveness. Previous studies suggest that gliomas secrete excess glutamate and destroy surrounding normal peritumoral brain by means of excitotoxic mechanisms. In this study the authors assessed the effect on survival of 2 glutamate modulators (riluzole and memantine) in rodent glioma models.
In an in vitro growth inhibition assay, F98 and 9L cells were exposed to riluzole and memantine. Mouse cerebellar organotypic cultures were implanted with F98 glioma cells and treated with radiation, radiation + riluzole, or vehicle and assessed for tumor growth. Safety and tolerability of intracranially implanted riluzole and memantine CPP:SA polymers were tested in F344 rats. The efficacy of these drugs was tested against the 9L model and riluzole was further tested with and without radiation therapy (RT).
In vitro assays showed effective growth inhibition of both drugs on F98 and 9L cell lines. F98 organotypic cultures showed reduced growth of tumors treated with radiation and riluzole in comparison with untreated cultures or cultures treated with radiation or riluzole alone. Three separate efficacy experiments all showed that localized delivery of riluzole or memantine is efficacious against the 9L gliosarcoma tumor in vivo. Systemic riluzole monotherapy was ineffective; however, riluzole given with RT resulted in improved survival.
Riluzole and memantine can be safely and effectively delivered intracranially via polymer in rat glioma models. Both drugs demonstrate efficacy against the 9L gliosarcoma and F98 glioma in vitro and in vivo. Although systemic riluzole proved ineffective in increasing survival, riluzole acted synergistically with radiation and increased survival compared with RT or riluzole alone.
PMCID: PMC4322948  PMID: 24484234
riluzole; memantine; glioma; radiation; oncology
2.  Degeneration and impaired regeneration of gray matter oligodendrocytes in amyotrophic lateral sclerosis 
Nature neuroscience  2013;16(5):571-579.
Oligodendrocytes associate with axons to establish myelin and provide metabolic support to neurons. In the spinal cord of ALS mice, oligodendrocytes downregulate transporters that transfer glycolytic substrates to neurons and oligodendrocyte progenitors (NG2+ cells) exhibit enhanced proliferation and differentiation, although the cause of these changes in oligodendroglia is unknown. Here we report that there is extensive degeneration of gray matter oligodendrocytes in the spinal cord of ALS mice before disease onset. Although new oligodendrocytes were formed, they failed to mature, resulting in progressive demyelination. Oligodendrocyte dysfunction also is prevalent in human ALS, as gray matter demyelination and reactive changes in NG2+ cells were observed in motor cortex and spinal cord of ALS patients. Selective removal of mutant SOD1 from oligodendroglia substantially delayed disease onset and prolonged survival in ALS mice, suggesting that ALS-linked genes enhance the vulnerability of motor neurons and accelerate disease by directly impairing the function of oligodendrocytes.
PMCID: PMC3637847  PMID: 23542689
3.  Mitochondrial abnormalities and low grade inflammation are present in the skeletal muscle of a minority of patients with amyotrophic lateral sclerosis; an observational myopathology study 
Amyotrophic lateral sclerosis (ALS) is a primary progressive neurodegenerative disease characterised by neuronal loss of lower motor neurons (in the spinal cord and brainstem) and/or upper motor neurons (in the motor cortex) and subsequent denervation atrophy of skeletal muscle.
A comprehensive examination of muscle pathology from a cohort of clinically confirmed ALS patients, including an investigation of inflammation, complement activation, and deposition of abnormal proteins in order to compare them with findings from an age-matched, control group.
Material and methods
31 muscle biopsies from clinically confirmed ALS patients and 20 normal controls underwent a comprehensive protocol of histochemical and immunohistochemical stains, including HLA-ABC, C5b-9, p62, and TDP-43.
Neurogenic changes were confirmed in 30/31 ALS cases. In one case, no neurogenic changes could be detected. Muscle fibre necrosis was seen in 5/31 cases and chronic mononuclear inflammatory cell infiltration in 5/31 (2 of them overlapped with those showing muscle necrosis). In four biopsies there was an increase in the proportion of cytochrome oxidase (COX) negative fibres (2-3%). p62 faintly stained cytoplasmic bodies in eight cases and none were immunoreactive to TDP-43.
This large series of muscle biopsies from patients with ALS demonstrates neurogenic atrophy is a nearly uniform finding and that mild mitochondrial abnormalities and low-grade inflammation can be seen and do not rule out the diagnosis of ALS. These findings could lend support to the notion that ALS is a complex and heterogeneous disorder.
PMCID: PMC4297389  PMID: 25510661
Amyotrophic lateral Sclerosis; Mitochondria; Inflammation; Pathology and Muscle
4.  Oligodendroglia metabolically support axons and maintain structural integrity 
Trends in cell biology  2013;23(12):10.1016/j.tcb.2013.07.007.
Axons are specialized extensions of neurons that are critical for the organization of the nervous system. In order to maintain function in axons that often extend some distance from the cell body, specialized mechanisms of energy delivery are likely necessary. Over the last decade, greater understanding of human demyelinating diseases and the development of animal models have suggested that oligodendroglia are critical for maintaining the function of axons. In this review, we will discuss evidence for the vulnerability of neurons to energy deprivation, the importance of oligodendrocytes for axon function and survival, and very recent data suggesting that transfer of energy metabolites from oligodendroglia to axons through monocarboxylate transporter 1 may be critical for the survival of axons. This pathway has important implications both for the basic biology of the nervous system as well as for human neurologic disease. New insights into the role of oligodendroglial biology provide an exciting opportunity for revisions in nervous system biology, understanding myelin-based disorders and in therapeutics development.
PMCID: PMC3842360  PMID: 23988427
MCT1; oligodendroglia; neurodegeneration; myelin; ALS; lactate
5.  Human stem cell-derived spinal cord astrocytes with defined mature or reactive phenotypes 
Cell reports  2013;4(5):1035-1048.
Differentiation of astrocytes from human stem cells has significant potential for analyzing their role in normal brain function and disease, but existing protocols generate only immature astrocytes. Using early neuralization, we generated spinal cord astrocytes from mouse or human embryonic (ESCs) or induced pluripotent (hiPSCs) stem cells with high efficiency. Remarkably, short exposure to FGF1 or FGF2 was sufficient to direct these astrocytes selectively toward a mature quiescent phenotype, as judged both by marker expression and functional analysis. In contrast, TNFα and IL-1β but not FGFs, induced multiple elements of a reactive phenotype but did not affect maturation. These phenotypically defined, scalable populations of spinal cord astrocytes will be important both for studying normal astrocyte function and for modeling human pathological processes in vitro.
PMCID: PMC4229657  PMID: 23994478
Embryonic stem cells; human induced pluripotent stem cells; astrocytes; glutamate transporter; fibroblast growth factor; tumor necrosis factor-alpha; maturation; reactivity
6.  Mutations in the Matrin 3 gene cause familial amyotrophic lateral sclerosis 
Nature neuroscience  2014;17(5):664-666.
MATR3 is an RNA/DNA binding protein that interacts with TDP-43, a major disease protein linked to amyotrophic lateral sclerosis (ALS) and fronto-temporal dementia. Using exome sequencing, we identified mutations in MATR3 in ALS kindreds. We also observed MATR3 pathology in the spinal cords of ALS cases with and without MATR3 mutations. Our data provide additional evidence supporting the role of aberrant RNA processing in motor neuron degeneration.
PMCID: PMC4000579  PMID: 24686783
7.  Oligodendroglia metabolically support axons and contribute to neurodegeneration 
Nature  2012;487(7408):443-448.
Oligodendroglia support axon survival and function through mechanisms independent of myelination and their dysfunction leads to axon degeneration in several diseases. The cause of this degeneration has not been determined, but lack of energy metabolites such as glucose or lactate has been hypothesized. Lactate is transported exclusively by monocarboxylate transporters, and changes to these transporters alter lactate production and utilization. We show the most abundant lactate transporter in the CNS, monocarboxylate transporter 1 (MCT1), is highly enriched within oligodendroglia and that disruption of this transporter produces axon damage and neuron loss in animal and cell culture models. In addition, this same transporter is reduced in patients with, and mouse models of, amyotrophic lateral sclerosis (ALS), suggesting a role for oligodendroglial MCT1 in pathogenesis. The role of oligodendroglia in axon function and neuron survival has been elusive; this study defines a new fundamental mechanism by which oligodendroglia support neurons and axons.
PMCID: PMC3408792  PMID: 22801498
8.  Human Nasal Olfactory Epithelium as a Dynamic Marker for CNS Therapy Development 
Experimental neurology  2011;232(2):203-211.
Discovery of new central nervous system (CNS) acting therapeutics has been slowed down by the lack of useful applicable biomarkers of disease or drug action often due to inaccessibility of relevant human CNS tissue and cell types. In recent years, non-neuronal cells, such as astrocytes, have been reported to play a highly significant role in neurodegenerative diseases, CNS trauma, as well as psychiatric disease and have become a target for small molecule and biologic therapies. We report the development of a method for measuring pharmacodynamic changes induced by potential CNS therapeutics using nasal olfactory neural tissue biopsy. We validated this approach using a potential astrocyte-targeted therapeutic, thiamphenicol, in a pre-clinical rodent study as well as a phase 1 human trial. In both settings, analysis of the olfactory epithelial tissue revealed biological activity of thiamphenicol at the drug target, the excitatory amino acid transporter 2 (EAAT2). Therefore, this biomarker approach may provide a reliable evaluation of CNS glial-directed therapies and hopefully improve throughput for nervous system drug discovery.
PMCID: PMC3220936  PMID: 21945230
Nasal biopsy; olfactory epithelial tissue; glutamate transporter; ALS; astroglia; astrocyte; surrogate marker
9.  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
10.  β-III spectrin is critical for development of Purkinje cell dendritic tree and spine morphogenesis 
The Journal of Neuroscience  2011;31(46):16581-16590.
Mutations in the gene encoding β-III spectrin give rise to spinocerebellar ataxia type 5 (SCA5), a neurodegenerative disease characterized by progressive thinning of the molecular layer, loss of Purkinje cells and increasing motor deficits. A mouse lacking full-length β-III spectrin (β-III−/−) displays a similar phenotype. In vitro and in vivo analyses of Purkinje cells lacking β-III spectrin, reveal a critical role for β-III spectrin in Purkinje cell morphological development. Disruption of the normally well-ordered dendritic arborization occurs in Purkinje cells from β-III−/− mice, specifically showing a loss of monoplanar organization, smaller average dendritic diameter and reduced densities of Purkinje cell spines and synapses. Early morphological defects appear to affect distribution of dendritic, but not axonal, proteins. This study confirms that thinning of the molecular layer associated with disease pathogenesis is a consequence of Purkinje cell dendritic degeneration, as Purkinje cells from 8-month old β-III−/− mice have drastically reduced dendritic volumes, surface areas and total dendritic lengths compared to 5–6 week old β-III−/− mice. These findings highlight a critical role of β-III spectrin in dendritic biology and are consistent with an early developmental defect in β-III−/− mice, with abnormal Purkinje cell dendritic morphology potentially underlying disease pathogenesis.
PMCID: PMC3374928  PMID: 22090485
11.  Harmine, A Natural Beta-Carboline Alkaloid, Upregulates Astroglial Glutamate Transporter Expression 
Neuropharmacology  2010;60(7-8):1168-1175.
Glutamate is the predominant excitatory amino acid neurotransmitter in the mammalian central nervous system (CNS). Glutamate transporter EAAT2 /GLT-1 is the physiologically dominant astroglial protein that inactivates synaptic glutamate. Previous studies have shown that EAAT2 dysfunction leads to excessive extracellular glutamate and may contribute to various neurological disorders including amyotrophic lateral sclerosis (ALS). The recent discovery of the neuroprotective properties of ceftriaxone, a beta lactam antibiotic, suggested that increasing EAAT2 /GLT-1 gene expression might be beneficial in ALS and other neurological/psychiatric disorders by augmenting astrocytic glutamate uptake. Here we report our efforts to develop a new screening assay for identifying compounds that activate EAAT2 gene expression. We generated fetal derived-human immortalized astroglial cells that are stably expressing a firefly luciferase reporter under the control of the human EAAT2 promoter. When screening a library of 1040 FDA approved compounds and natural products, we identified harmine, a naturally occurring beta-carboline alkaloid, as one of the top hits for activating the EAAT2 promoter. We further tested harmine in our in vitro cell culture systems and confirmed its ability to increase EAAT2/GLT1 gene expression and functional glutamate uptake activity. We next tested its efficacy in both wild type animals and in an ALS animal model of disease and demonstrated that harmine effectively increased GLT-1 protein and glutamate transporter activity in vivo. Our studies provide potential novel neurotherapeutics by modulating the activity of glutamate transporters via gene activation.
PMCID: PMC3220934  PMID: 21034752
harmine; GLT-1; EAAT2; glutamate transporter; astroglia; ALS
12.  Molecular Comparison of GLT1+ and ALDH1L1+ Astrocytes In Vivo In Astroglial Reporter Mice 
Glia  2011;59(2):200-207.
Astrocyte heterogeneity remains largely unknown in the CNS due to lack of specific astroglial markers. In this study, molecular identity of in vivo astrocytes was characterized in BAC ALDH1L1 and BAC GLT1 eGFP promoter reporter transgenic mice. ALDH1L1 promoter is selectively activated in adult cortical and spinal cord astrocytes, indicated by the overlap of eGFP expression with ALDH1L1 and GFAP, but not with NeuN, APC, Olig2, IbaI, PDGFRα immunoreactivity in BAC ALDH1L1 eGFP reporter mice. Interestingly, ALDH1L1 expression levels (protein, mRNA, and promoter activity) in spinal cord were selectively decreased during postnatal maturation. In contrast, its expression was up-regulated in reactive astrocytes in both acute neural injury and chronic neurodegenerative (G93A mutant SOD1) conditions, similar to GFAP, but opposite of GLT1. ALDH1L1+ and GLT1+ cells isolated through fluorescence activated cell sorting (FACS) from BAC ALDH1L1 and BAC GLT1 eGFP mice share a highly similar gene expression profile, suggesting ALDH1L1 and GLT1 are co-expressed in the same population of astrocytes. This observation was further supported by overlap of the eGFP driven by the ALDH1L1 genomic promoter and the tdTomato driven by a 8.3kb EAAT2 promoter fragment in astrocytes of BAC ALDH1L1 eGFP X EAAT2-tdTomato mice. These studies support ALDH1L1 as a general CNS astroglial marker and investigated astrocyte heterogeneity in the CNS by comparing the molecular identity of the ALDH1L1+ and GLT1+ astrocytes from astroglial reporter mice. These astroglial reporter mice provide useful in vivo tools for the molecular analysis of astrocytes in physiological and pathological conditions.
PMCID: PMC3199134  PMID: 21046559
astroglia; BAC; ALDH1L1; GLT1; GFAP; oligodendroglia; ALS
13.  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
14.  Increased expression of glutamate transporter GLT-1 in peritumoral tissue associated with prolonged survival and decreases in tumor growth in a rat model of experimental malignant glioma 
Journal of neurosurgery  2013;119(4):878-886.
Gliomas are known to release excessive amounts of glutamate, inducing glutamate excitotoxic cell death in the peritumoral region and allowing the tumor to grow and to expand. Glutamate transporter upregulation has been shown to be neuroprotective by removing extracellular glutamate in a number of preclinical animal models of neurodegenerative diseases, including amyotrophic lateral sclerosis and Parkinson disease as well as psychiatric disorders such as depression. The authors therefore hypothesized that the protective mechanism of glutamate transporter upregulation would be useful for the treatment of gliomas as well.
In this study 9L gliosarcoma cells were treated with a glutamate transporter upregulating agent, thiamphenicol, an antibiotic approved in Europe, which has been shown previously to increase glutamate transporter expression and has recently been validated in a human Phase I biomarker trial for glutamate transporter upregulation. Cells were monitored in vitro for glutamate transporter levels and cell proliferation. In vivo, rats were injected intracranially with 9L cells and were treated with increasing doses of thiamphenicol. Animals were monitored for survival. In addition, postmortem brain tissue was analyzed for tumor size, glutamate transporter levels, and neuron count.
Thiamphenicol showed little effects on proliferation of 9L gliosarcoma cells in vitro and did not change glutamate transporter levels in these cells. However, when delivered locally in an experimental glioma model in rats, thiamphenicol dose dependently (10–5000 μM) significantly increased survival up to 7 days and concomitantly decreased tumor size from 46.2 mm2 to 10.2 mm2 when compared with lesions in nontreated controls. Furthermore, immunohistochemical and biochemical analysis of peritumoral tissue confirmed an 84% increase in levels of glutamate transporter protein and a 72% increase in the number of neuronal cells in the tissue adjacent to the tumor.
These results show that increasing glutamate transporter expression in peritumoral tissue is neuroprotective. It suggests that glutamate transporter upregulation for the treatment of gliomas should be further investigated and potentially be part of a combination therapy with standard chemotherapeutic agents.
PMCID: PMC4086647  PMID: 23909244
glioblastoma; EAAT2; thiamphenicol; glutamate; glutamate transporter; GLT-1; oncology; rat
15.  Identification of a Unique TGF-β Dependent Molecular and Functional Signature in Microglia 
Nature neuroscience  2013;17(1):131-143.
Microglia are myeloid cells of the central nervous system (CNS) that participate both in normal CNS function and disease. We investigated the molecular signature of microglia and identified 239 genes and 8 microRNAs that were uniquely or highly expressed in microglia vs. myeloid and other immune cells. Out of 239 genes, 106 were enriched in microglia as compared to astrocytes, oligodendrocytes and neurons. This microglia signature was not observed in microglial lines or in monocytes recruited to the CNS and was also observed in human microglia. Based on this signature, we found a crucial role for TGF-β in microglial biology that included: 1) the requirement of TGF-β for the in vitro development of microglia that express the microglial molecular signature characteristic of adult microglia; and 2) the absence of microglia in CNS TGF-β1 deficient mice. Our results identify a unique microglial signature that is dependent on TGF-β signaling which provides insights into microglial biology and the possibility of targeting microglia for the treatment of CNS disease.
PMCID: PMC4066672  PMID: 24316888
16.  Safety, Pharmacokinetic, and Functional Effects of the Nogo-A Monoclonal Antibody in Amyotrophic Lateral Sclerosis: A Randomized, First-In-Human Clinical Trial 
PLoS ONE  2014;9(5):e97803.
The neurite outgrowth inhibitor, Nogo-A, has been shown to be overexpressed in skeletal muscle in amyotrophic lateral sclerosis (ALS); it is both a potential biomarker and therapeutic target. We performed a double-blind, two-part, dose-escalation study, in subjects with ALS, assessing safety, pharmacokinetics (PK) and functional effects of ozanezumab, a humanized monoclonal antibody against Nogo-A. In Part 1, 40 subjects were randomized (3∶1) to receive single dose intravenous ozanezumab (0.01, 0.1, 1, 5, or 15 mg/kg) or placebo. In Part 2, 36 subjects were randomized (3∶1) to receive two repeat doses of intravenous ozanezumab (0.5, 2.5, or 15 mg/kg) or placebo, approximately 4 weeks apart. The primary endpoints were safety and tolerability (adverse events [AEs], vital signs, electrocardiogram (ECG), and clinical laboratory tests). Secondary endpoints included PK, immunogenicity, functional endpoints (clinical and electrophysiological), and biomarker parameters. Overall, ozanezumab treatment (0.01–15 mg/kg) was well tolerated. The overall incidence of AEs in the repeat dose 2.5 mg/kg and 15 mg/kg ozanezumab groups was higher than in the repeat dose placebo group and repeat dose 0.5 mg/kg ozanezumab group. The majority were considered not related to study drug by the investigators. Six serious AEs were reported in three subjects receiving ozanezumab; none were considered related to study drug. No study drug-related patterns were identified for ECG, laboratory, or vital signs parameters. One subject (repeat dose 15 mg/kg ozanezumab) showed a weak, positive anti-ozanezumab-antibody result. PK results were generally consistent with monoclonal antibody treatments. No apparent treatment effects were observed for functional endpoints or muscle biomarkers. Immunohistochemical staining showed dose-dependent co-localization of ozanezumab with Nogo-A in skeletal muscle. In conclusion, single and repeat dose ozanezumab treatment was well tolerated and demonstrated co-localization at the site of action. These findings support future studies with ozanezumab in ALS.
Trial Registration NCT00875446 GSK ID 111330
PMCID: PMC4026380  PMID: 24841795
17.  Pre-synaptic regulation of astroglial excitatory neurotransmitter transporter GLT1 
Neuron  2009;61(6):880-894.
The neuron-astrocyte synaptic complex is a fundamental operational unit of the nervous system. Astroglia play a central role in the regulation of synaptic glutamate, via neurotransmitter transport by GLT1/EAAT2. The astroglial mechanisms underlying this essential neuron-glial communication are not known. Here we show that presynaptic terminals are sufficient and necessary for GLT1/EAAT2 transcriptional activation and have identified the molecular pathway that regulates astroglial responses to presynaptic input. Presynaptic terminals regulate astroglial GLT1/EAAT2 via kappa B-motif binding phosphoprotein (KBBP), the mouse homologue of human heterogeneous nuclear ribonucleoprotein K (hnRNP K), which binds to an essential element of GLT1/EAAT2 promoter. This neuron-stimulated factor is required for GLT1/EATT2 transcriptional activation and is responsible for astroglial alterations in neural injury. Denervation of neuron-astrocyte signaling in vivo, by acute corticospinal tract transection, ricin-induced motor neuron death, or chronic neurodegeneration in amyotrophic lateral sclerosis (ALS) all result in reduced astroglial KBBP expression and transcriptional dysfunction of astroglial transporter expression. Our studies indicate that presynaptic elements dynamically coordinate normal astroglial function and also provide a fundamental signaling mechanism by which altered neuronal function and injury leads to dysregulated astroglia in CNS disease.
PMCID: PMC2743171  PMID: 19323997
18.  Epigenetic Regulation of Neuron-Dependent Induction of Astroglial Synaptic Protein GLT1 
Glia  2010;58(3):277-286.
Astroglial glutamate transporter EAAT2/GLT1 prevents glutamate-induced excitotoxicity in the central nervous system. Expression of EAAT2/GLT1 is dynamically regulated by neurons. The pathogenesis of amyotrophic lateral sclerosis (ALS) involves astroglial dysfunction, including dramatic loss of EAAT2/GLT1. DNA methylation of gene promoters represents one of the most important epigenetic mechanisms in regulating gene expression. The involvement of DNA methylation in the regulation of astroglial EAAT2/GLT1 expression in different conditions, especially in ALS has not been explored. In this study, we established a procedure to selectively isolate a pure astrocyte population in vitro and in vivo from BAC GLT1 eGFP mice using an eGFP-based fluorescence-activated cell sorting approach. Astrocytes isolated from this procedure are GFAP+ and GLT1+ and respond to neuronal stimulation, enabling direct methylation analysis of GLT1 promoter in these astrocytes. To investigate the role of DNA methylation in physiological and pathological EAAT2/GLT1 expression, methylation status of the EAAT2/GLT1 promoter was analyzed in astrocytes from in vitro and in vivo paradigms or postmortem ALS motor cortex by bisulfite sequencing method. DNA demethylation on selective CpG sites of the GLT1 promoter was highly correlated to increased GLT1 mRNA levels in astrocytes in response to neuronal stimulation; however, low level of methylation was found on CpG sites of EAAT2 promoter from postmortem motor cortex of human amyotrophic lateral sclerosis patients. In summary, hypermethylation on selective CpG sites of the GLT1 promoter is involved in repression of GLT1 promoter activation, but this regulation does not play a role in astroglial dysfunction of EAAT2 expression in patients with ALS.
PMCID: PMC2794958  PMID: 19672971
epigenetic; astrocyte; GLT1
19.  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
20.  A Neuronal Glutamate Transporter Contributes to Neurotransmitter GABA Synthesis and Epilepsy 
The predominant neuronal glutamate transporter, EAAC1 (for excitatory amino acid carrier-1), is localized to the dendrites and somata of many neurons. Rare presynaptic localization is restricted to GABA terminals. Because glutamate is a precursor for GABA synthesis, we hypothesized that EAAC1 may play a role in regulating GABA synthesis and, thus, could cause epilepsy in rats when inactivated. Reduced expression of EAAC1 by antisense treatment led to behavioral abnormalities, including staring–freezing episodes and electrographic (EEG) seizures. Extracellular hippocampal and thalamocortical slice recordings showed excessive excitability in antisense-treated rats. Patch-clamp recordings of miniature IPSCs (mIPSCs) conducted in CA1 pyramidal neurons in slices from EAAC1 antisense-treated animals demonstrated a significant decrease in mIPSC amplitude, indicating decreased tonic inhibition. There was a 50% loss of hippocampal GABA levels associated with knockdown of EAAC1, and newly synthesized GABA from extracellular glutamate was significantly impaired by reduction of EAAC1 expression. EAAC1 may participate in normal GABA neurosynthesis and limbic hyperexcitability, whereas epilepsy can result from a disruption of the interaction between EAAC1 and GABA metabolism.
PMCID: PMC2483507  PMID: 12151515
EAAC1; transport; antisense; GABA; metabolism; epilepsy
21.  Design and Initial Results of a Multi-Phase Randomized Trial of Ceftriaxone in Amyotrophic Lateral Sclerosis 
PLoS ONE  2013;8(4):e61177.
Ceftriaxone increases expression of the astrocytic glutamate transporter, EAAT2, which might protect from glutamate-mediated excitotoxicity. A trial using a novel three stage nonstop design, incorporating Phases I-III, tested ceftriaxone in ALS. Stage 1 determined the cerebrospinal fluid pharmacokinetics of ceftriaxone in subjects with ALS. Stage 2 evaluated safety and tolerability for 20-weeks. Analysis of the pharmacokinetics, tolerability, and safety was used to determine the ceftriaxone dosage for Stage 3 efficacy testing.
In Stage 1, 66 subjects at ten clinical sites were enrolled and randomized equally into three study groups receiving intravenous placebo, ceftriaxone 2 grams daily or ceftriaxone 4 grams daily divided BID. Participants provided serum and cerebrospinal fluid for pharmacokinetic analysis on study day 7. Participants continued their assigned treatment in Stage 2. The Data and Safety Monitoring Board (DSMB) reviewed the data after the last participants completed 20 weeks on study drug.
Stage 1 analysis revealed linear pharmacokinetics, and CSF trough levels for both dosage levels exceeding the pre-specified target trough level of 1 µM (0.55 µg/mL). Tolerability (Stages 1 and 2) results showed that ceftriaxone at dosages up to 4 grams/day was well tolerated at 20 weeks. Biliary adverse events were more common with ceftriaxone but not dose-dependent and improved with ursodeoxycholic (ursodiol) therapy.
The goals of Stages 1 and 2 of the ceftriaxone trial were successfully achieved. Based on the pre-specified decision rules, the DSMB recommended the use of ceftriaxone 4 g/d (divided BID) for Stage 3, which recently closed.
Trial Registration NCT00349622.
PMCID: PMC3629222  PMID: 23613806
22.  The blood-brain barrier: an engineering perspective 
It has been more than 100 years since Paul Ehrlich reported that various water-soluble dyes injected into the circulation did not enter the brain. Since Ehrlich's first experiments, only a small number of molecules, such as alcohol and caffeine have been found to cross the blood-brain barrier, and this selective permeability remains the major roadblock to treatment of many central nervous system diseases. At the same time, many central nervous system diseases are associated with disruption of the blood-brain barrier that can lead to changes in permeability, modulation of immune cell transport, and trafficking of pathogens into the brain. Therefore, advances in our understanding of the structure and function of the blood-brain barrier are key to developing effective treatments for a wide range of central nervous system diseases. Over the past 10 years it has become recognized that the blood-brain barrier is a complex, dynamic system that involves biomechanical and biochemical signaling between the vascular system and the brain. Here we reconstruct the structure, function, and transport properties of the blood-brain barrier from an engineering perspective. New insight into the physics of the blood-brain barrier could ultimately lead to clinical advances in the treatment of central nervous system diseases.
PMCID: PMC3757302  PMID: 24009582
blood-brain barrier; neurovascular unit; capillary; microvasculature; transport
23.  Deletion of galectin-3 exacerbates microglial activation and accelerates disease progression and demise in a SOD1G93A mouse model of amyotrophic lateral sclerosis 
Brain and Behavior  2012;2(5):563-575.
Galectins are pleiotropic carbohydrate-binding lectins involved in inflammation, growth/differentiation, and tissue remodeling. The functional role of galectins in amyotrophic lateral sclerosis (ALS) is unknown. Expression studies revealed increases in galectin-1 mRNA and protein in spinal cords from SOD1G93A mice, and in galectin-3 and -9 mRNAs and proteins in spinal cords of both SOD1G93A mice and sporadic ALS patients. As the increase in galectin-3 appeared in early presymptomatic stages and increased progressively through to end stage of disease in the mouse, it was selected for additional study, where it was found to be mainly expressed by microglia. Galectin-3 antagonists are not selective and do not readily cross the blood–brain barrier; therefore, we generated SOD1G93A/Gal-3−/− transgenic mice to evaluate galectin-3 deletion in a widely used mouse model of ALS. Disease progression, neurological symptoms, survival, and inflammation were assessed to determine the effect of galectin-3 deletion on the SOD1G93A disease phenotype. Galectin-3 deletion did not change disease onset, but resulted in more rapid progression through functionally defined disease stages, more severely impaired neurological symptoms at all stages of disease, and expiration, on average, 25 days earlier than SOD1G93A/Gal-3+/+ cohorts. In addition, microglial staining, as well as TNF-α, and oxidative injury were increased in SOD1G93A/Gal-3−/− mice compared with SOD1G93A/Gal-3+/+ cohorts. These data support an important functional role for microglial galectin-3 in neuroinflammation during chronic neurodegenerative disease. We suggest that elevations in galectin-3 by microglia as disease progresses may represent a protective, anti-inflammatory innate immune response to chronic motor neuron degeneration.
PMCID: PMC3489809  PMID: 23139902
Alternative activation; amyotrophic lateral sclerosis; microglia; motor neuron disease; SOD1
24.  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
25.  Derivation of Glial Restricted Precursors from E13 mice 
This is a protocol for derivation of glial restricted precursor (GRP) cells from the spinal cord of E13 mouse fetuses. These cells are early precursors within the oligodendrocytic cell lineage. Recently, these cells have been studied as potential source for restorative therapies in white matter diseases. Periventricular leukomalacia (PVL) is the leading cause of non-genetic white matter disease in childhood and affects up to 50% of extremely premature infants. The data suggest a heightened susceptibility of the developing brain to hypoxia-ischemia, oxidative stress and excitotoxicity that selectively targets nascent white matter. Glial restricted precursors (GRP), oligodendrocyte progenitor cells (OPC) and immature oligodendrocytes (preOL) seem to be key players in the development of PVL and are the subject of continuing studies. Furthermore, previous studies have identified a subset of CNS tissue that has increased susceptibility to glutamate excitotoxicity as well as a developmental pattern to this susceptibility. Our laboratory is currently investigating the role of oligodendrocyte progenitors in PVL and use cells at the GRP stage of development. We utilize these derived GRP cells in several experimental paradigms to test their response to select stresses consistent with PVL. GRP cells can be manipulated in vitro into OPCs and preOL for transplantation experiments with mouse PVL models and in vitro models of PVL-like insults including hypoxia-ischemia. By using cultured cells and in vitro studies there would be reduced variability between experiments which facilitates interpretation of the data. Cultured cells also allows for enrichment of the GRP population while minimizing the impact of contaminating cells of non-GRP phenotype.
PMCID: PMC3399460  PMID: 22760029
Neuroscience;  Issue 64;  Physiology;  Medicine;  periventricular leukomalacia;  oligodendrocytes;  glial restricted precursors;  spinal cord;  cell culture

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