Dopamine and its metabolites in cathepsin D heterozygous mice before and after MPTP administration

Abstract

Cathepsin D (CD) is a lysosomal aspartyl protease which plays an important role in α-synuclein degradation, and neuronal survival. CD knockout mice die by post-natal day 25±1 due to intestinal necrosis. We analyzed the young adult male heterozygous mice, and found no behavior abnormalities in the heterozygous mice compared to wildtype littermates. LC3-II, p62, and α-synuclein levels are similar, while LAMP1 is higher in the striatum in CD heterozygous compared to wildtype mice. Interestingly, we found that dopamine and metabolites in the striatum and olfactory bulbs are at higher levels than wildtype littermates, while the DOPAC/DA and HVA/DA ratio remain similar between wildtype and CD heterozygous mice. In response to sub-chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration, dopamine, DOPAC, and HVA are depleted to similar levels in the striatum in both heterozygous and wildtype mice. Dopamine synthesizing enzyme tyrosine hydroxylase, metabolic enzyme monoamine oxidase, and catechol-O-methyltransferase (COMT) levels are similar in the striatum in wildtype and heterozygous mice. These studies provide valuable information regarding how lysosomal function may contribute to neurochemical homeostasis in animal models.

Fig. 1

Both CD+/+ and CD+/− mice exhibit MPTP-induced decrease in TH in the striatum. (A) Western blot analyses of TH protein levels in striatal protein extracts after sham or MPTP treatment. (B) Quantification of Western blot data from (A) showing that TH levels are significantly reduced in both CD+/+ and CD+/− mice in response to MPTP treatment. Data shown are mean ± SEM (*p < 0.01 sham > MPTP). There was no significant difference in TH levels between CD+/+ and CD+/− mice in the sham or MPTP-treated groups (p > 0.05). (C). Striatal DA levels are significantly higher in CD+/− mice than CD+/+ mice and are significantly reduced in both CD+/+ and CD+/− mice after MPTP. Data shown are mean ± SEM (#p < 0.05 sham CD+/− > sham CD+/+ and **p < 0.001 sham > MPTP). (D) Striatal DOPAC levels are significantly higher in CD+/− mice compared to CD+/+ mice and are significantly reduced in both CD+/+ and CD+/− mice after MPTP. Data shown are mean ± SEM (*p < 0.01 sham CD+/− > sham CD+/+ and **p < 0.001 sham > MPTP). (E) Striatal HVA levels are significantly higher in sham CD+/− mice compared to sham CD+/+ mice and are significantly reduced in both CD+/+ and CD+/− mice after MPTP. Data shown are mean ± SEM (*p < 0.05 sham CD+/− > sham CD+/+ and +p < 0.05 MPTP > SHAM in CD+/+). (F) Striatal DOPAC/DA ratio levels are similar between CD+/− and CD+/+ mice in sham mice, or mice after MPTP. Data shown are mean ± SEM (two way ANOVA was used to examine the effects of group (MPTP vs. sham) and genotype (+/+ vs.+/−). (G) Striatal HVA/DA ratio are similar between CD+/+ and CD+/− mice in sham and after MPTP, and are increased after MPTP in both genotypes. Data shown are mean ± SEM (+p < 0.05 sham < MPTP).

Fig. 2

CD+/− mice have significantly higher levels of DA and its metabolites than CD+/+ mice in the striatum and the olfactory bulbs. Striatal monoamines are higher in CD+/− mice than in CD+/+ controls (A and B). (A) DA levels are significantly higher in CD+/− mice than in CD+/+ controls. Data = mean ± SEM (*p < 0.01 CD+/+ < CD+/−). (B) DOPAC and HVA levels are significantly higher in CD+/− mice than in CD+/+ controls. Data = mean ± SEM (**p < 0.001 CD+/+ < CD+/−). Monoamine levels in the olfactory bulb are also higher in CD+/− mice than in CD+/+ controls (C and D). (C) Olfactory DA levels are significantly higher in CD+/− mice. Data = mean ± SEM (*p < 0.01 CD+/+ < CD+/−). (D) DOPAC levels in the olfactory bulb are significantly higher in CD+/− mice. Data = mean ± SEM (#p < 0.05 CD+/+ < CD+/−). HVA levels were consistently higher in the olfactory bulb of CD+/− mice than in CD+/+ mice but did not reach statistical significance. Data = mean ± SEM (p = 0.06 CD+/+ vs. CD+/−). (E) Striatal DOPAC/DA and HVA ratio are similar between CD+/+ and CD+/− mice. Data shown are mean ± SEM. (F) Olfactory DOPAC/DA and HVA/DA ratio are similar between CD+/+ and CD+/− mice. Data shown are mean ± SEM, p > 0.05 Student t-test.