The PKC-β selective inhibitor, Enzastaurin, impairs memory in middle-aged rats

Enzastaurin is a Protein Kinase C-β selective inhibitor that was developed to treat cancers. Protein Kinase C-β is an important enzyme for a variety of neuronal functions; in particular, previous rodent studies have reported deficits in spatial and fear-conditioned learning and memory with lower levels of Protein Kinase C-β. Due to Enzastaurin’s mechanism of action, the present study investigated the consequences of Enzastaurin exposure on learning and memory in 12-month-old Fischer-344 male rats. Rats were treated daily with subcutaneous injections of either vehicle or Enzastaurin, and behaviorally tested using the spatial reference memory Morris Water Maze. Rats treated with Enzastaurin exhibited decreased overnight retention and poorer performance on the latter testing day, indicating a mild, but significant, memory impairment. There were no differences during the probe trial indicating that all animals were able to spatially localize the platform to the proper quadrant by the end of testing. RNA isolated from the hippocampus was analyzed using Next Generation Sequencing (Illumina). No statistically significant transcriptional differences were noted. Our findings suggest that acute Enzastaurin treatment can impair hippocampal-based learning and memory performance, with no effects on transcription in the hippocampus. We propose that care should be taken in future clinical trials that utilize Protein Kinase C-ß inhibitors, to monitor for possible cognitive effects, future research should examine if these effects are fully reversible. Middle-aged (12 months) virgin male Fischer-344 rats born and raised at the National Institute on Aging colony at Harlan Laboratories (Indianapolis, IN) were used. The rats were pair housed at Arizona State University, had exposure to food and water ad-lib, and were maintained on a 12-h light/dark cycle at 23°C. Rats were randomly divided into two treatment groups, receiving either vehicle (n=10) or ENZ (n=8). Rats received either daily subcutaneous injections of ENZ (25mg/kg; 0.4mL) (Selleck Chemicals, Houston, TX) suspended in polyethylene glycol (PEG, Sigma-Aldrich, St. Louis, MO) or vehicle (100% PEG) for three days concurrent to behavioral testing. Injections started the first day of behavioral testing. Maze testing commenced 30-45 minutes after injection. Animals were sacrificed immediately after the probe trial on day 3 of testing. Rats received six trials/day for three days, with a 15 min delay given between trials three and four (Markham et al. 2002). A video camera recorded each rat and a tracking system (Ethovision XT 5.1, Noldus) analyzed each rat’s path. Immediately after the probe trial on day 3 of testing, animals were anesthetized with isofluorane gas and decapitated using a guillotine, and brains were rapidly dissected and frozen. Dissected tissues were stored in pre-weighed microcentrifuge tubes at -70°C until analysis. Dissection of the CA1/CA2 region of the hippocampus (plates 39-42) was according to plate designations in Paxinos and Watson (1998). The brain was cut in the coronal plane to obtain access to the ventral CA1/CA2 region of the hippocampus. The dentate gyrus and the alveus were excluded.