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1.  Small-molecule activator of glutamate transporter EAAT2 translation provides neuroprotection 
The Journal of Clinical Investigation  2014;124(3):1255-1267.
Glial glutamate transporter EAAT2 plays a major role in glutamate clearance in synaptic clefts. Several lines of evidence indicate that strategies designed to increase EAAT2 expression have potential for preventing excitotoxicity, which contributes to neuronal injury and death in neurodegenerative diseases. We previously discovered several classes of compounds that can increase EAAT2 expression through translational activation. Here, we present efficacy studies of the compound LDN/OSU-0212320, which is a pyridazine derivative from one of our lead series. In a murine model, LDN/OSU-0212320 had good potency, adequate pharmacokinetic properties, no observed toxicity at the doses examined, and low side effect/toxicity potential. Additionally, LDN/OSU-0212320 protected cultured neurons from glutamate-mediated excitotoxic injury and death via EAAT2 activation. Importantly, LDN/OSU-0212320 markedly delayed motor function decline and extended lifespan in an animal model of amyotrophic lateral sclerosis (ALS). We also found that LDN/OSU-0212320 substantially reduced mortality, neuronal death, and spontaneous recurrent seizures in a pilocarpine-induced temporal lobe epilepsy model. Moreover, our study demonstrated that LDN/OSU-0212320 treatment results in activation of PKC and subsequent Y-box–binding protein 1 (YB-1) activation, which regulates activation of EAAT2 translation. Our data indicate that the use of small molecules to enhance EAAT2 translation may be a therapeutic strategy for the treatment of neurodegenerative diseases.
doi:10.1172/JCI66163
PMCID: PMC3938250  PMID: 24569372
2.  Identification of Translational Activators of Glial Glutamate Transporter EAAT2 through Cell-Based High-Throughput Screening: An Approach to Prevent Excitotoxicity 
Journal of biomolecular screening  2010;15(6):653-662.
Excitotoxicity has been implicated as the mechanism of neuronal damage resulting from acute insults such as stroke, epilepsy, and trauma, as well as during the progression of adult-onset neurodegenerative disorders such as Alzheimer’s disease and amyotrophic lateral sclerosis (ALS). Excitotoxicity is defined as excessive exposure to the neurotransmitter glutamate or overstimulation of its membrane receptors, leading to neuronal injury or death. One potential approach to protect against excitotoxic neuronal damage is enhanced glutamate reuptake. The glial glutamate transporter EAAT2 is the quantitatively dominant glutamate transporter and plays a major role in clearance of glutamate. Expression of EAAT2 protein is highly regulated at the translational level. In an effort to identify compounds that can induce translation of EAAT2 transcripts, a cell-based enzyme-linked immunosorbent assay was developed using a primary astrocyte line stably transfected with a vector designed to identify modulators of EAAT2 translation. This assay was optimized for high-throughput screening, and a library of approximately 140,000 compounds was tested. In the initial screen, 293 compounds were identified as hits. These 293 hits were retested at 3 concentrations, and a total of 61 compounds showed a dose-dependent increase in EAAT2 protein levels. Selected compounds were tested in full 12-point dose-response experiments in the screening assay to assess potency as well as confirmed by Western blot, immunohistochemistry, and glutamate uptake assays to evaluate the localization and function of the elevated EAAT2 protein. These hits provide excellent starting points for developing therapeutic agents to prevent excitotoxicity.
doi:10.1177/1087057110370998
PMCID: PMC3016154  PMID: 20508255
excitotoxicity; glutamate transporter; EAAT2; high-throughput screen; neurodegeneration
3.  Increased glial glutamate transporter EAAT2 expression reduces epileptogenic processes following pilocarpine-induced status epilepticus 
Neurobiology of disease  2012;47(2):145-154.
Several lines of evidence indicate that glutamate plays a crucial role in the initiation of seizures and their propagation; abnormal glutamate release causes synchronous firing of large populations of neurons, leading to seizures. In the present study, we investigated whether enhanced glutamate uptake by increased glial glutamate transporter EAAT2, the major glutamate transporter, could prevent seizure activity and reduce epileptogenic processes. EAAT2 transgenic mice, which have a 1.5-2 fold increase in EAAT2 protein levels as compared to their non-transgenic counterparts, were tested in a pilocarpine-induced status epilepticus (SE) model. Several striking phenomena were observed in EAAT2 transgenic mice compared with their non-transgenic littermates. First, the post-SE mortality rate and chronic seizure frequency were significantly decreased. Second, neuronal degeneration in hippocampal subfields after SE were significantly reduced. Third, the SE-induced neurogenesis and mossy fiber sprouting were significantly decreased. The severity of cell loss in epileptic mice was positively correlated with that of mossy fiber sprouting and chronic seizure frequency. Our results suggest that increased EAAT2 expression can protect mice against SE-induced death, neuropathological changes, and chronic seizure development. This study suggests that enhancing EAAT2 protein expression is a potential therapeutic approach.
doi:10.1016/j.nbd.2012.03.032
PMCID: PMC3572547  PMID: 22513140
Excitotoxicity; Glutamate transporter; EAAT2; Temporal lobe epilepsy; Pilocarpine; Epileptogenesis
4.  Glutamate transporter EAAT2: a new target for the treatment of neurodegenerative diseases 
Future medicinal chemistry  2012;4(13):1689-1700.
Glutamate is the primary excitatory amino acid neurotransmitter in the CNS. The concentration of glutamate in the synaptic cleft is tightly controlled by interplay between glutamate release and glutamate clearance. Abnormal glutamate release and/or dysfunction of glutamate clearance can cause overstimulation of glutamate receptors and result in neuronal injury known as excitotoxicity. The glial glutamate transporter EAAT2 plays a major role in glutamate clearance. Dysfunction or reduced expression of EAAT2 has been documented in many neurodegenerative diseases. In addition, many studies in animal models of disease indicate that increased EAAT2 expression provides neuronal protection. Here, we summarize these studies and suggest that EAAT2 is a potential target for the prevention of excitotoxicity. EAAT2 can be upregulated by transcriptional or translational activation. We discuss current progress in the search for EAAT2 activators, which is a promising direction for the treatment of neurodegenerative diseases.
doi:10.4155/fmc.12.122
PMCID: PMC3580837  PMID: 22924507
5.  Structure-activity relationship study of pyridazine derivatives as glutamate transporter EAAT2 activators 
Excitatory amino acid transporter 2 (EAAT2) is the major glutamate transporter and functions to remove glutamate from synapses. A thiopyridazine derivative has been found to increase EAAT2 protein levels in astrocytes. A structure-activity relationship study revealed that several components of the molecule were required for activity, such as the thioether and pyridazine. Modification of the benzylthioether resulted in several derivatives (7–13, 7–15 and 7–17) that enhanced EAAT2 levels by > 6 fold at concentrations < 5 μM after 24 h. In addition, one of the derivatives (7–22) enhanced EAAT2 levels 3.5 – 3.9 fold after 24 h with an EC50 of 0.5 μM.
doi:10.1016/j.bmcl.2011.08.009
PMCID: PMC3172717  PMID: 21875806
6.  Effects of axon degeneration on oligodendrocyte lineage cells: Dorsal rhizotomy evokes a repair response while axon degeneration rostral to spinal contusion induces both repair and apoptosis 
Glia  2010;58(11):1304-1319.
Wallerian degeneration in the dorsal columns (DC) after spinal cord injury (SCI) is associated with microglial activation and prolonged oligodendrocyte (OL) apoptosis that may contribute to demyelination and dysfunction after SCI. But, there is an increase in OL lineage cells after SCI that may represent a reparative response, and there is evidence for remyelination after SCI. To assess the role of axonal degeneration per se in OL apoptosis and proliferation, we cut the L2-S2 dorsal roots producing massive axonal degeneration and microglial activation in the DC, and found no evidence of OL loss or apoptosis. Rather, the numbers of OL-lineage cells positive for NG2 and APC (CC1) increased, and BrdU studies suggested new OL formation. We then tested contusion SCI (cSCI) that results in comparable degeneration in the DC rostral to the injury, microglial activation, and apoptosis of DC OLs by 8 days. NG2+ cell proliferation and oligodendrogenesis was seen as after rhizotomy. The net result of this combination of proliferation and apoptosis was a reduction in DC OLs, confirming earlier studies. Using an antibody to oxidized nucleic acids, we found rapid and prolonged RNA oxidation in OLs rostral to cSCI, but no evidence of oxidative stress in DC OLs after rhizotomy. These results suggest that signals associated with axonal degeneration are sufficient to induce OL proliferation, and that secondary injury processes associated with the central SCI, including oxidative stress, rather than axonal degeneration per se, are responsible for OL apoptosis.
doi:10.1002/glia.21009
PMCID: PMC3045846  PMID: 20607865
cell death; cell proliferation; progenitor cells; Wallerian degeneration; spinal cord injury; dorsal root; dorsal columns; oxidative stress
7.  Oxidative damage to RNA: mechanisms, consequences, and diseases 
Overproduction of free radicals can damage cellular components resulting in progressive physiological dysfunction, which has been implicated in many human diseases. Oxidative damage to RNA received little attention until the past decade. Recent studies indicate that RNA, such as messenger RNA and ribosomal RNA, is very vulnerable to oxidative damage. RNA oxidation is not a consequence of dying cells but an early event involved in pathogenesis. Oxidative modification to RNA results in disturbance of the translational process and impairment of protein synthesis, which can cause cell deterioration or even cell death. In this review, we discuss the mechanisms of oxidative damage to RNA and the possible biological consequences of damaged RNA. Furthermore, we review recent evidence suggesting that oxidative damage to RNA may contribute to progression of many human diseases.
doi:10.1007/s00018-010-0277-y
PMCID: PMC3010397  PMID: 20148281
RNA oxidation; Oxidative damage; Oxidative stress; Free radical; Neurodegeneration; Alzheimer’s disease; ALS
8.  Messenger RNA Oxidation Occurs Early in Disease Pathogenesis and Promotes Motor Neuron Degeneration in ALS 
PLoS ONE  2008;3(8):e2849.
Background
Accumulating evidence indicates that RNA oxidation is involved in a wide variety of neurological diseases and may be associated with neuronal deterioration during the process of neurodegeneration. However, previous studies were done in postmortem tissues or cultured neurons. Here, we used transgenic mice to demonstrate the role of RNA oxidation in the process of neurodegeneration.
Methodology/Principal Findings
We demonstrated that messenger RNA (mRNA) oxidation is a common feature in amyotrophic lateral sclerosis (ALS) patients as well as in many different transgenic mice expressing familial ALS-linked mutant copper-zinc superoxide dismutase (SOD1). In mutant SOD1 mice, increased mRNA oxidation primarily occurs in the motor neurons and oligodendrocytes of the spinal cord at an early, pre-symptomatic stage. Identification of oxidized mRNA species revealed that some species are more vulnerable to oxidative damage, and importantly, many oxidized mRNA species have been implicated in the pathogenesis of ALS. Oxidative modification of mRNA causes reduced protein expression. Reduced mRNA oxidation by vitamin E restores protein expression and partially protects motor neurons.
Conclusion/Significance
These findings suggest that mRNA oxidation is an early event associated with motor neuron deterioration in ALS, and may be also a common early event preceding neuron degeneration in other neurological diseases.
doi:10.1371/journal.pone.0002849
PMCID: PMC2481395  PMID: 18682740

Results 1-8 (8)