Fig 2: Impact of Dyrk1a inactivation in glutamatergic neurons on general behavior, locomotor activity and cognition.(A) The exploratory behavior of a new environment was analyzed by the percentage of time spent in the center of an open field over 30 min of test. Dyrk1aC/C mice spent more time in the center of the arena, suggesting that they are less anxious. (B) Confirmation of the phenotype in a new group of mice using the Elevated Plus Maze test (EPM) with Dyrk1aC/C mice showing a higher percentage of time spent in the open arms of the maze. (C) Mouse activity, measured by the number of entries in both open and closed arms, was increased in Dyrk1aC/C animals. (D) Evaluation of the locomotor performance on the rotarod during consecutive trials with increased rotational speeds. The latency is the mean of 3 independent trials. Dyrk1aC/C mice showed increased performance. (E) In the NOR test, the percentage of time spent exploring the familiar (FO) and the new (NO) objects show that control mice spend significantly more time on the NO while Dyrk1aC/C mice do not make any difference between the two objects. (F) Exploration times of the two identical objects during the presentation phase of the NOR show that Dyrk1aC/C mice tend to have an increased exploration time. (G) Total exploratory time of the two objects during the test indicates that the absence of object discrimination of the Dyrk1aC/C mice is not due to a lack of interest of the objects. (H) Percentage of freezing time during the habituation phase (before the foot shock; basal level of activity) (Habituation; Mann-Whitney rank sum test, p = 0.71) and during the 6 min of contextual exposure 24 hours later indicate a deficit of contextual learning in Dyrk1aC/C mice. (I-J) Pain sensitivity was evaluated by measuring the mouse latency to elicit a response to pain when put on a plate (52°C). In this test, Dyrk1aC/C and Dyrk1a+/- mice had a lower threshold than their control littermates. (K) Interest in social interaction was measured by the time spent sniffing the cage containing a congener (C1) during the Crawley test. This time was reduced for Dyrk1aC/C mice compared to control mice (unpaired t-test p = 0.01) while the time spent exploring the empty cage (EC) was the same between the two groups (unpaired t-test p = 0.23). Data are presented as point plots with mean ± SD. Statistical analyses were done with unpaired Student’s t-test or Mann-Whitney rank sum test if normality test failed, except E: paired T-test FO vs NO and one sample T-test vs 50% mean (in red: ***p<0.001); A, D, H, I and K: tests were done on young males (1.5–3.5 months old depending on the test with animals aged ± 3 weeks), n = 8–10 per genotype,. B-C (EPM) and E-G (NOR) were done with another batch of males a 4 months of age, n = 18 and 16 controls for each test respectively and 13 Dyrk1aC/C(2 animals were removed from the NOR test because they did not reach the minimum exploration time during the presentation session); *p<0.05, **p<0.01, ***p<0.001.

Fig 3: PPM1B reduces the oligomerization and aggregation of pTau.A, HEK293 cells were transfected for 24 h with plasmid encoding GFP-Tau, V5-Tau, HA-DYRK1A, or Myc-PPM1B alone or in combination. Cell lysates were immunoprecipitated with anti-V5 antibody, followed by immunoblotting with anti-GFP antibody. The proper expression of transiently transfected proteins in cell lysates was identified with immunoblot analysis using anti-GFP, anti-V5, anti-HA or anti-Myc antibodies. Hsp90 served as a loading control. B, all data represent the mean ± SD of three independent experiments (***p < 0.001). C, HEK293 cells were transfected for 48 h with plasmid encoding V5-Tau, HA-DYRK1A, or Myc-PPM1B alone or in combination. Cell lysates were analyzed by western blotting (Inputs), and the extent of tau oligomerization was analyzed by filter trap assay. The blots of the filter trap assay were subjected to either immunoblotting with anti-V5 antibody or Ponceau S staining. The proper expression of transiently transfected proteins in cell lysates was identified with immunoblot analysis using anti-V5, anti-HA or anti-Myc antibodies. Asterisk indicates a nonspecific band. D, all data represent the mean ± SD of three independent experiments (***p < 0.001). E, HEK293 cells were transfected for 48 h with plasmid encoding V5-Tau, HA-DYRK1A, or Myc-PPM1B alone or in combination. Cells were lysed in either oligomerization buffer containing Triton X-100 or lysis buffer containing 0.5% Nonidet P-40. The proper expression of transiently transfected proteins in cell lysates was identified with immunoblotting with anti-V5 (Insoluble or Inputs), anti-HA, or anti-Myc antibodies (Inputs). F, all data represent the mean ± SD of three independent experiments (**p < 0.01, ***p < 0.001).

Fig 4: Dual specificity tyrosine-phosphorylation-regulated kinase 1A mediated phosphorylation of GluN2A at S1048 increases its surface expression in primary cortical neurons. Primary cultures of mouse embryo cortices were transiently transfected with GFP-GluN2A (wt, wild-type; S1048A, phospho-deficient mutant; S1048E, phospho-mimetic mutant) on day in vitro 8 (DIV8), in the presence or absence of heterologous HA-DYRK1A. The effect of DYRK1A on the surface:intracellular ratio of GluN1/GluN2A in primary mouse cortical neurons was evaluated by immunofluorescence. Prior to permeabilization, anti-GFP/Alexa488 was used to detect the surface chimeric receptors (represented in green), whereas intracellular GFP-GluN2A receptors were visualized after permeabilizing the cells, using an anti-GFP/Alexa555 antibody. Scale bar = 5 µm. The histogram represents the mean ± SEM GluN2A surface expression normalized to the intracellular GFP-GluN2A signal (n = 31–52 dendrites from, at least, three independent experiments per condition, *p < 0.05, **p < 0.01, ***p < 0.001, ANOVA).

Fig 5: Knockdown of DYRK1A disproportionately increases HIV replication in nondividing cells. (A) Western blot showing the knockdown of DYRK1A in HeLa, THP-1, and differentiated THP-1 (dTHP-1) cells. Cell lysates taken at 48 h prior to viral infections are shown for each cell type. (B) HeLa cells were transfected with control or DYRK1A siRNA for 48 h and infected with VSV-G-pseudotyped HIV-1Luc for 48 h. (C and D) Stable THP-1 cells expressing control or DYRK1A shRNA were either directly infected with HIVLuc or differentiated into macrophages and then infected with HIVLuc for 48 h. HIV infection was measured by luciferase luminescence in cell lysates normalized to total protein concentration. (E) Knockdown DYRK1A in monocyte-derived macrophages (MDMs) isolated from HIV-negative donors after 48 h of siRNA transfection. (F to H) MDMs from three different donors were infected with VSV-G-pseudotyped HIV-1Luc for 48 h and HIV infection was measured as described above. (I and J) Differentiated THP-1 cells with control of DYRK1A shRNA were infected with HIV-1 for 48 h and the MTT assay was performed as described in Materials and Methods. (K) MDMs with control or DYRK1A siRNA were infected with HIV-1 for 48 h and the MTT assay was performed. Data are means, and error bars indicate SEM (n = 3). **, P < 0.001; *, P < 0.05, Student’s t test.