Fig 1: Full-length and truncated DYRK1A levels in LCLs. (a) Antibodies used for full-length (D1694) and truncated forms of DYRK1A detection. (b) Western blotting showing full-length and truncated form levels of DYRK1A in LCLs from healthy individuals (2N) and DS patients with (dDS) or without (aDS) AD dementia. Proteins were performed by Western blotting and values were obtained by normalization of images to ßactin. (c,d) Data of full-length and truncated forms were normalized to the mean of healthy individuals (2N) and results are represented as mean ± SEM. n = number of LCLs. * p < 0.05; ** p < 0.005.; *** p < 0.001.
Fig 2: Age-dependent changes in plasma Dyrk1A levels. Blood was collected at the tail vein of Wistar and GK rats of 3 and 6 months of age, at 9:00 a.m. Analyses were performed in plasma. (A) Schematic representation of Dyrk1A with distinct epitopes recognized by different antibodies. Plasma levels of full-length Dyrk1A form (B) and full-length and truncated forms of Dyrk1A (C). The DYRK1A levels were assessed by a solid phase immobilized epitope- immunoassay set up for antibody 7D10 (Abnova; immunogen: 674 aa ~763 aa) and antibody D1694 (Sigma; immunogen: 32 aa ~51 aa) (73). After removal of unbound conjugates, bound enzyme activity was assessed by use of a chromogenic substrate for measurement at 450 nm by a microplate reader (Flex Station 3, Molecular Device, San Diego, CA, USA). All the assays were performed in duplicate. For multiple pairwise comparisons between genotypes and ages, statistical analysis was done with two-way ANOVA followed by Fisher's post-hoc test using Statview software. The results are expressed as means ± SEM (standard error of the mean). n = number of rats. Data were considered significant when p < 0.05.
Fig 3: L41 treatment prevents DYRK1A proteolysis in APP/PS1 mice hippocampus. a, b Western blot of hippocampus from APP/PS1 mice or littermates treated with vehicle or L41, showing lower levels of DYRK1A (90 kDa) immunoblotting with the a-DYRK1A-Cter antibody in vehicle-treated APP/PS1 mice (n = 6) compared to littermates (n = 6) (One-way ANOVA, p < 0.005). DYRK1A protein levels in L41-treated APP/PS1 mice (n = 7) were higher than in vehicle-treated APP/PS1 mice (One-way ANOVA, p < 0.005) and similar to what observed in littermates (One-way ANOVA, ns). c Calpain activity assessed by a fluorescent method, showing higher calpain activity in hippocampus from both vehicle-treated (n = 6) and L41-treated APP/PS1 mice (n = 7) compared to littermates (n = 6) (One-way ANOVA, p < 0.05 for both). There was no significant difference between L41-treated and vehicle-treated APP/PS1 mice (One-way ANOVA, ns). d DYRK1A protein levels did not correlate with calpain activity (r2 = 0.43; ns). e HPLC assay for total endogenous DYRK1A activity showing no differences between hippocampus from littermates (n = 9) and vehicle- or L41-treated APP/PS1 mice (n = 9 and 8, respectively) (One-way ANOVA, ns). f Representative images from immunohistochemical staining using the a-DYRK1A-Cter antibody, of hippocampal slices from littermates, vehicle- and L41-treated APP/PS1 mice, showing neuronal staining in the CA1 and Stratum Radiatum (StrR) regions (see enlargement at the bottom). g Representative images from immunohistochemical staining, using the a-DYRK1A-Nter antibody, of hippocampal slices, showing neuronal staining for L41-treated APP/PS1 mice and littermates in the CA1 and Stratum Radiatum (StrR) regions. Neuronal staining was observed in both the CA1 and StrR regions, whereas additional astrocyte staining was mostly observed in the Stratum Radiatum (StrR) region of vehicle-treated APP/PS1 mice. h Laser confocal microscopy showing double staining using a-DYRK1A-Cter (red) and anti-GFAP (green) antibodies. There were no differences in a-DYRK1A-Cter staining in GFAP positive cells between littermates (n = 6, astrocytes = 73), vehicle- (n = 6, astrocytes = 90), and L41-treated APP/PS1 mice (n = 6, astrocytes = 85) (One-way ANOVA, ns). i Laser confocal microscopy showing double staining using a-DYRK1A-Nter antibody (red) and anti-GFAP (green). a-DYRK1A-Nter staining was higher in the GFAP positive area in vehicle-treated APP/PS1 mice (n = 6, astrocytes = 105) compared to littermates (n = 6, astrocytes = 50) (One-way ANOVA, p < 0.0005). a-DYRK1A-Nter immunoreactivity was lower in the GFAP positive area of L41-treated (n = 6, astrocytes = 83) than vehicle-treated APP/PS1 mice (One-way ANOVA, p < 0.0005). Data represent the mean ± SEM and were analyzed by one-way ANOVA followed by Tukey’s post hoc test. Significant differences between littermates and vehicle-treated APP/PS1 mice are indicated by *p < 0.05, **p < 0.005 and ***p < 0.0005. Significant differences between vehicle- and L41-treated APP/PS1 mice are indicated by #p < 0.05, ##p < 0.005 and ###p < 0.0005
Fig 4: RNF169 mainly interacts with DYRK1A in the nucleus. (A) Relative enrichment of RNF169 in total cell extracts (light orange) and nuclear extracts (dark orange) of HeLa cells in the DYRK1A interactome analysis. The graph shows the enrichment score (ES) for each Ab and cellular compartment (mean ± SEM of two independent experiments). (B) DYRK1A and RNF169 co-immunoprecipitation experiments using HNEs and Abs against DYRK1A (Ab-C1) or RNF169 (a rabbit IgG was used as a control); *non-specific band. (C) HEK-293T soluble extracts transiently expressing the proteins indicated were immunoprecipitated with an anti-FLAG Ab (KR, DYRK1A K188R catalytically inactive mutant; CS, RNF169 C68S inactive mutant). The presence of the proteins was detected in WBs probed with anti-FLAG or anti-HA.
Fig 5: Identification of Leucettine L41 as a DYRK1A proteolysis inhibitor. a Control human hippocampus extract was incubated at 30 °C during 10 min with various concentrations of CaCl2 (0 to 4,0 mM) or with 2 mM of EGTA. Proteins were then analyzed by western blot using the a-DYRK1A-Nter antibody. b Control human hippocampus extract was incubated with 0 mM of CaCl2 or 2 mM of CaCl2 and various pharmacological compounds including Harmine (har), Leucettine LeuI (LeuI) or Leucettine L41 (L41). Proteins were analyzed by western blot using the a-DYRK1A-Nter antibody. c Control mouse (C57Bl6) hippocampus extract was incubated at 30 °C during 10 min with various concentrations of CaCl2 (0 to 4,0 mM) or/with 2 mM of EGTA or/with 2 mM of CaCl2 and various concentrations of Leucettine L41 (L41) (0,1 to 2 µM). Proteins were analyzed by western blot using the a-DYRK1A-Nter antibody. d Control mouse hippocampus extract was incubated at 30 °C during 10 min with 0 mM of CaCl2 or 2 mM of CaCl2 or 2 mM of CaCl2 with L41 at 1 µM. Protein extracts were then immunoprecipitated with the a-DYRK1A-Nter antibody overnight at 4 °C and immunoprecipitated protein extract were analyzed by western blot using a-DYRK1A-Nter, STAT3a and IkBa antibodies
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