Identification of translational activators of glial glutamate transporter EAAT2 through a cell-based high-throughput screening: An approach for preventing excitotoxicity

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

Characterization of Novel Src Family Kinase Inhibitors to Attenuate Microgliosis

Microgliosis is a major hallmark of Alzheimer’s disease (AD) brain pathology. Aβ peptide is hypothesized to act as a stimulus for microglia leading to activation of non-receptor tyrosine kinases and subsequent secretion of pro-inflammatory cytokines. Therefore, the signaling pathways mediating microglial activation may be important therapeutic targets of anti-inflammatory therapy for AD. Four novel compounds were chosen after high throughput screening kinase activity assays determined them as potential Lyn kinase inhibitors. Their kinase inhibitory and anti-inflammatory effect on Aβ-stimulated activation was assessed using the murine microglial cell line, BV2. Cells were treated with the compounds to determine effects on active, phosphorylated levels of Src family kinases, Src and Lyn, as well as MAP kinases ERK, JNK and p38. Only one compound, LDDN-0003499, produced a dose dependent decrease in basal levels of active, phosphorylated Src and Lyn in the BV2 cells. LDDN-0003499 treatment also attenuated the Aβ-stimulated increase in active, phosphorylated levels of Lyn/Src and TNFα and IL-6 secretion. This study identifies a novel small molecule Src family tyrosine kinase inhibitor with anti-inflammatory effects in response to Aβ stimulation of microglia. Further in vitro/in vivo characterization of LDDN-0003499 as well as structural modification may provide a new tool for attenuating microglial-mediated brain inflammatory conditions such as that occurring in AD

An anti-large T-antigen strategy to develop anti-JCV drugs

There are currently no JCV-specific therapies available for clinical use. This study evaluates viral large T antigen (LTA) as a potential target for drug development. LTA is a hexameric protein with a helicase activity that is powered by ATP binding and hydrolysis. The helicase and ATPase function is critical for viral replication and inhibition by small molecules would disrupt the viral life cycle. LTA is a valid target for discovery of anti-JCV drugs. The hits identified are reasonable starting points for medicinal chemistry to improve potency and selectivity. Screening of additional chemical libraries could also be considered to identify chemical structures that may be more potent with acceptable cytotoxicity

Application of Particle Image Velocimetry - Theory and Practice

For investigations of flow fields with pronounced spatial structures and/or rapid temporal or spatical chances new experimental techniques, such as Particle Image Velocimetry (PIV) are required which allow to capture large floe velocity fields instantaneoulsy. A number ofdifferent approaches for the recording and evaluation of PIV images has been described in literature. This course will mainly concetrate on those aspects of the theorey of PIV relevant to applications. Besides giving lectureson the fundamental aspects, special emphasis is placed on the presentation of practical and reliable solutions of problems which are faced during the implementation of this technique in wind tunnelsand other test facilities. During practice the participants will have the opportunity to carry out the recording and the evaluation of PIV photographs for themselves in small groups

LDDN-0125694 treatment did not attenuate active, phosphorylated Src, Lyn, ERK, JNK, or p38 protein levels in BV2 cells.

<p>Microglial BV2 cells were treated with vehicle (DMSO), 0.5nM, 5nM, 50nM, 0.5 μM, 5μM, and 50 μM LDDN-0075935 for 1h. Cells lysates were used for western-blot analyses with (B) anti-pLyn (Tyr 396), (C) anti-pSrc (Tyr 416), (D) anti-pERK, (E) anti-pJNK, and (F) anti-p-p38 antibodies with Lyn, Src, ERK2, JNK, and p38 antibodies as their respective loading controls. Optical densities from three independent experiments were graphed and averaged ± SD (*p<0.05 vs. vehicle). (A) A representative western blot is shown.</p

LDDN-0003499 treatment attenuated an Aβ-dependent increase in active phosphorylated Src and Lyn but not pERK protein levels in BV2 cells.

<p>(A) Human AD temporal lobe sections along with age-matched controls were immunostained using anti-pLyn antibody and anti-Aβ (4G8) antibody. A double label with anti-pLyn (grey) and anti-Aβ (red) antibodies is shown. (B) BV2 cells were treated with vehicle DMSO (v) or 1 μM Aβ in presence and absence of increasing concentrations of LDDN-0003499 (0.0005, 0.005, 0.05, 0.5, 5, 50 μM). Cell lysates were resolved by SDS-PAGE and western blotted using anti-pSrc, anti-pLyn and p-ERK antibodies with Src, Lyn, and ERK as loading controls. (C) Optical densities from three independent experiments were averaged and graphed ± SD (*p<0.05 respective control vs Aβ, $p<0.05 vs. Aβ only, #p<0.05 vs. vehicle).</p