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

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

Glomerular membrane attack complex is not a reliable marker of ongoing C5 activation in lupus nephritis

Complement plays an important role in the pathogenesis of lupus nephritis (LN). With the emergence of therapeutic complement inhibition, there is a need to identify patients in whom complement-driven inflammation is a major cause of kidney injury in LN. Clinical and histopathological data from 57 biopsies with class III, IV and V LN (both active and chronic) were obtained retrospectively and biopsies stained for complement components (C9, C5b-9, C3c, C3d) and CD68. C9 staining was equivalent to C5b-9 (capillary wall r=0.92, p<0.0001). C5b-9 was detected in the mesangium and/or capillary wall of both active and chronic proliferative (class III and IV) LN in all but one biopsy and in the capillary wall of class V LN in all biopsies. C5b-9 staining intensity in the tubular basement membrane correlated with markers of tubulointerstitial damage (r=0.50, p=0.0001). Glomerular C5b-9 staining intensity did not differ between active and chronic disease, however C3c and CD68 were associated with active disease. More intense capillary wall C5b-9 staining was significantly associated with non-response to treatment (p=0.01). Study of serial biopsies and comparison of active and chronic biopsies indicated that C5b-9 staining persisted for months to years. In summary, C5b-9 staining is almost always present in LN, resolves slowly and is not a reliable marker of ongoing glomerular C5 activation. This limits its use in identifying patients most likely to benefit from C5 inhibition