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1.  Mutant Disrupted-In-Schizophrenia 1 in astrocytes: focus on glutamate metabolism 
Journal of neuroscience research  2014;92(12):1659-1668.
Disrupted-In-Schizophrenia 1 (DISC1) is a genetic risk factor that has been implicated in major mental disorders. DISC1 binds to and stabilizes serine racemase (SR) to regulate production of D-serine by astrocytes, contributing to glutamate (GLU) neurotransmission. However, the possible involvement of astrocytic DISC1 in synthesis, metabolism, re-uptake or secretion of GLU remains unexplored. Thus, we studied the effects of dominant-negative mutant DISC1 on various aspects of GLU metabolism using primary astrocyte cultures and the hippocampal tissue from transgenic mice with astrocyte-restricted expression of mutant DISC1. While mutant DISC1 had no significant effects on astrocyte proliferation, GLU re-uptake, Glutaminase or Glutamate carboxypeptidase II activity, expression of mutant DISC1 was associated with increased levels of alanine-serine-cysteine transporter 2, vesicular glutamate transporters 1 and 3 in primary astrocytes and in the hippocampus as well as elevated expression of the NR1 subunit and diminished expression of the NR2A subunit of NMDA receptors in the hippocampus at postnatal day 21. Our findings indicate that decreased D-serine production by astrocytic mutant DISC1 may lead to compensatory changes in levels of the amino acid transporters and NMDA receptors in the context of tripartite synapse.
PMCID: PMC4247786  PMID: 25131692
glutamate uptake; VGLUT; ASCT2; D-serine; NMDA; psychiatric disease
2.  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
3.  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
4.  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
5.  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
6.  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
7.  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
8.  GluR1 Controls Dendrite Growth through Its Binding Partner, SAP97 
Activity-dependent dendrite elaboration influences the pattern of interneuronal connectivity and network function. In the present study, we examined the mechanism by which the GluR1 subunit of AMPA receptors controls dendrite morphogenesis. GluR1 binds to SAP97, a scaffolding protein that is a component of the postsynaptic density, via its C-terminal 7 aa. We find that elimination of this interaction in vitro or in vivo (by deleting the C-terminal 7 aa of GluR1, GluR1Δ7) does not influence trafficking, processing, or cell surface GluR1 expression but does prevent translocation of SAP97 from the cytosol to membranes. GluR1 and SAP97 together at the plasma membrane promotes dendrite branching in an activity-dependent manner, although this does not require physical association. Our findings suggest that the C-terminal 7 aa of GluR1 are essential for bringing SAP97 to the plasma membrane, where it acts to translate the activity of AMPA receptors into dendrite growth.
PMCID: PMC2699678  PMID: 18842882
motor neurons; spinal cord; synaptic activity; postsynaptic density; trafficking; scaffold protein

Results 1-8 (8)