The GSK3β inhibitor, TDZD-8, rescues cognition in a zebrafish model of okadaic acid-induced Alzheimer's disease

Abstract

Currently, no treatments exist that are able to directly treat against Alzheimer's disease (AD), and we are facing an inevitable increase in the near future of the amount of patients who will suffer from AD. Most animal models of AD are limited by not being able to recapitulate the entire pathology of AD. Recently an AD model in zebrafish was established by using the protein phosphatase 2A inhibitor, okadaic acid (OKA). Administering OKA to zebrafish was able to recapitulate most of the neuropathology associated with AD. Therefore, providing a drug discovery model for AD that is also time and cost efficient. This study was designed to investigate the effects of GSK3β inhibition by 4-benzyl-2-methyl-1, 2, 4-thiadiazolidine-3, 5-dione (TDZD-8) on this newly developed AD model. Fish were divided into 4 groups and each group received a different treatment. The fish were divided into a control group, a group treated with 1 μM TDZD-8 only, a group treated with 1 μM TDZD-8 + 100 nM OKA, and a group treated with 100 nM OKA only. Administering the GSK3β inhibitor to zebrafish concomitantly with OKA proved to be protective. TDZD-8 treatment reduced the mortality rate, the ratio of active: inactive GSK3β, pTau (Ser199), and restored PP2A activity. This further corroborates the use of GSKβ inhibitors in the treatment against AD and bolsters the use of the OKA-induced AD-like zebrafish model for drug discovery.

Keywords: Alzheimer's disease; GSK3β; Okadaic acid; PP2A; TDZD-8; Tau; Zebrafish.

Fig. 1.

Mortality rate and necropsy observations of cause of death: A. No fish died during the control and TDZD-8 treatments. 1 fish died during TDZD-8 + OKA treatment. 3 fish died during OKA treatment. B. Hemorrhage is observed to because of death for all fish that died during the experiment. Ruptured vessels are marked by black boxes and arrows.

Fig. 2.

Learning (pre-treatment) and memory (post-treatment) data of control, TDZD-8, TDZD-8 + OKA, and OKA treated zebrafish. The dots on each graph represent the group’s running average at each trial point. The curved line represents a non-linear least-squares regression curve of the probability correct responses. A. Zebrafish were subject to the spatial alteration paradigm before being treated with their respective compounds. All 4 groups demonstrated the ability to learn by reaching 75% correct. B. After receiving their respective treatment, the zebrafish were again subject to the spatial alteration paradigm. The control group demonstrated the ability to remember by starting the behavioral task at 60% instead of the random chance probability of 50% and by reaching the 75% mark at an accelerated rate. The groups TDZD-8 and TDZD-8 + OKA demonstrated the ability to learn by reaching 75%. The OKA group did not demonstrate memory retention or the ability to learn by starting the post-treatment paradigm at random chance of 50% and never reaching a successful response of 75%. n = 12 (6 male and 6 female for pre-treatment control), n = 12 (6 male and 6 female for pre-treatment TDZD-8), n = 12 (6 male and 6 female for pre-treatment TDZD-8 + OKA), n = 12 (6 male and 6 female for pre-treatment OKA), n = 12 (6 male and 6 female for post-treatment control), n = 12 (6 male and 6 female for post-treatment TDZD-8), n = 11 (5 male and 6 female for post-treatment TDZD-8 + OKA), n = 9 (4 male and 5 female for post-treatment OKA).

Fig. 3.

Okadaic acid lowers PP2A activity in zebrafish forebrain. PP2A activity was analyzed using tissue taken from the telencephalon region of the zebrafish. PP2A activity was significantly reduced in the OKA treated zebrafish when compared to the control and TDZD-8 group. No significant difference of PP2A activity was determined between the control and TDZD-8 + OKA treated zebrafish. The bar graphs are presented as means ± SEM; *p < 0.05 n = 6 (3 male and 3 female for control), n = 6 (4 male and 2 female for TDZD-8), n = 5 (2 male and 3 female for TDZD-8 + OKA), n = 3 (1 male and 2 female for OKA).

Fig. 4.

TDZD-8 lowers Alzheimer’s related protein expression levels increased by okadaic acid. Western blotting on tissue taken from the telencephalon region of the zebrafish forebrain are shown. A. Immunoblotting for PP2A shows a decrease of PP2A expression in the OKA treated group when compared to the control and the TDZD-8 group. Reduction in PP2A expression appears in the TDZD-8 + OKA group, but no significant difference was found. B. Immunoblotting for active pGSK3β (Tyr216) and inactive pGSK3β (Ser9) shows an increase in the ratio of active to inactive pGSK3β in OKA treated zebrafish when compared to all other groups. No difference was in the ratio of active to inactive pGSK3β between the control, TDZD-8, and TDZD-8 + OKA treated zebrafish. C. Immunoblotting for pTau (Ser199) shows an increase in pTau expression of OKA treated zebrafish when compared to all other groups. No difference was found in pTau expression between the control, TDZD-8, and TDZD-8 + OKA treated zebrafish. The bar graphs are presented as means ± SEM; *p < 0.05 **p < 0.01, ***p < 0.001, n = 6 (3 male and 3 female for control), n = 6 (3 male and 3 female for TDZD-8), n = 6 (3 male and 3 female for TDZD-8 + OKA), n = 6 (3 male and 3 female for OKA).