The use of GAP-43 mRNA quantification in high throughput screening of putative neuroprotective agents in dorsal root ganglion cultures.
Large scale screening for neuroprotective drugs for peripheral neuropathies requires development of a high throughput system that is reliable and reproducible. Currently most accurate outcome measures of axonal degeneration are based on time-consuming, laborious measurement of morphological changes in neurites. In order to improve on the scalability of the screening procedure we developed a real-time RT-PCR based method of gene expression that correlates very well with morphological measures of neuritic degeneration. We examined the changes in GAP-43 expression in primary dorsal root ganglion (DRG) neurons in vitro with exposure to a zalcitabine (ddC), an antiretroviral drug that causes neuropathy in human immunodeficiency virus (HIV)-infected individuals, with and without FK506, an immunophilin ligand with neuroprotective and neuroregenerative properties. Similar to morphological measures of neuritic degeneration, in ddC-treated cultures there was a reduction in the expression of GAP-43 mRNA. This was prevented, in a dose-dependent manner, by co-administration of FK506. This assay, performed in a 96-well format, can easily be scaled for high throughput screening (HTS) using robotic systems.
Keswani
Sanjay C
SC
Department of Neurology, Johns Hopkins University, 600 N. Wolfe St., Path 509, Baltimore, MD 21287, USA.
Rosenberg
Brian
B
Hoke
Ahmet
A
D000818:Animals; D000831:Animals, Newborn; D018929:Cell Culture Techniques / Q000379:methods*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005333:Fetus; D019922:GAP-43 Protein / Q000235:genetics*; D005727:Ganglia, Spinal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects / Q000235:genetics; D009410:Nerve Degeneration / Q000188:drug therapy / Q000378:metabolism / Q000517:prevention & control; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D018696:Neuroprotective Agents / Q000494:pharmacology*; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D051381:Rats; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D016559:Tacrolimus / Q000494:pharmacology; D016047:Zalcitabine / Q000037:antagonists & inhibitors / Q000633:toxicity
D019922:GAP-43 Protein; D018696:Neuroprotective Agents; D012333:RNA, Messenger; D016047:Zalcitabine; D016559:Tacrolimus