Lanthionine ketimine-5-ethyl ester provides neuroprotection in a zebrafish model of okadaic acid-induced Alzheimer's disease

Abstract

Okadaic acid (OKA) is a protein phosphatase 2A inhibitor that is used to induce neurodegeneration and study disease states such as Alzheimer's disease (AD). Lanthionine ketimine-5-ethyl ester (LKE) is a bioavailable derivative of the naturally occurring brain sulfur metabolite, lanthionine ketimine (LK). In previously conducted studies, LKE exhibited neuroprotective and neurotrophic properties in murine models but its mechanism of action remains to be clarified. In this study, a recently established zebrafish OKA-induced AD model was utilized to further elucidate the neuroprotective and neurotrophic properties of LKE in the context of an AD-like condition. The fish were divided into 3 groups containing 8 fish per group. Group #1 = negative control, Group #2 = 100 nM OKA, Group #3 = 100 nM OKA +500 μM LKE. OKA caused severe cognitive impairments in the zebrafish, but concomitant treatment with LKE protected against cognitive impairments. Further, LKE significantly and substantially reduced the number of apoptotic brain cells, increased brain-derived neurotrophic factor (BDNF), and increased phospho-activation of the pro-survival factors pAkt (Ser 473) and pCREB (Ser133). These findings clarify the neuroprotective and neurotrophic effects of LKE by highlighting particular survival pathways that are bolstered by the experimental therapeutic LKE.

Keywords: BDNF; CREB; Lanthionine ketimine-5-ethyl-ester; Okadaic acid; PKB/Akt; Zebrafish.

Figure 1

Learning (pre-treatment) and memory (post-treatment) data of control, OKA treated, and LKE+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 3 groups demonstrated the ability to learn by reaching 70–75% correct. B. After receiving their respective treatment, the zebrafish were again subject to the spatial alteration paradigm. The control and LKE+OKA groups demonstrated the ability to remember by starting the behavioral task at 65% instead of the random chance probability of 50%. In addition to memory demonstration, the control and LKE+OKA zebrafish increased performance demonstrated by reaching 85%–90% correct. The OKA group did not demonstrate memory retention by starting the post-treatment paradigm at random chance of 50%. They also did not increase performance, demonstrated by their max correct response of 55–60%. n= 8 (4 male and 4 female for both pre and post-treatment control), n= 8 (4 male and 4 female for both pre and post-treatment LKE+OKA), n= 8 (4 male and 4 female for both pre and post-treatment OKA)

Figure 2

Pictures were taken of the dorsal lateral pallium located within the telencephalon of the zebrafish and analyzed by TUNEL assay. A. DAPI stain (blue) and TUNEL stain (green) and the overlay images taken using a 20× objective. B. DAPI stain (blue) and TUNEL stain (green) and the overlay images taken using a 100× objective. The images show an increase in apoptosis in the zebrafish dorsal lateral pallium treated with OKA which was analyzed and shown in C. Apoptosis was significantly increased in the dorsal lateral pallium of OKA treated zebrafish compared to the control and LKE+OKA treated zebrafish. No difference was found between the LKE+OKA and control group. The bar graphs are presented as means ± SEM; *p<0.05 **p<0.01, n= 4 (2 male and 2 female for control), n= 4 (2 male and 2 female for LKE+OKA), n= 4 (2 male and 2 female for OKA). D. Schematic overview of the whole adult zebrafish brain with sectioning scheme of the telencephalon. E. Schematic coronal section of the telencephalon indicating the specific area of interest, the dorsal lateral pallium. Zebrafish brain illustrations were adapted and modified from [48]. OB, Olfactory bulb; Ce, cerebellum; Dc, dorsal central pallium; Dl, dorsal lateral pallium; Dm, dorsal medial pallium; Dp, dorsal posterior pallium Tel, telencephalon; TO, tectum opticum.

Figure 3

Zebrafish forebrain showcases an increase in neurotrophic signaling expression by western blot analysis. A. Immunoblotting with an anti-BDNF antibody shows an increase in the BDNF expression of LKE+OKA zebrafish when compared to the control and the OKA zebrafish. OKA zebrafish show a reduction of BDNF but no significant difference was found. B. Immunoblotting for pAkt (Ser473) with an anti-pAkt(Ser473) shows an increase in pAkt expression of LKE+OKA zebrafish when compared to the control and the OKA zebrafish. No significant difference found between the control group and OKA group even though pAkt seems to be reduced in the OKA group. C. Immunoblotting with an anti-pCREB (Ser133) antibody shows an increase in pCREB of LKE+OKA zebrafish when compared to the control and OKA treated zebrafish. No significant difference found between the control group and OKA group even though it appears that pCREB is reduced in the OKA group. The bar graphs are presented as means ± SEM; *p<0.05 **p<0.01, n= 4 (2 male and 2 female for control), n= 4 (2 male and 2 female for LKE+OKA), n=4 (2 male and 2 female OKA)