Regulation of tyrosine phosphatase STEP61 by protein kinase A during motor skill learning in mice

Abstract

Recently, striatal-enriched protein tyrosine phosphatase (STEP) and its upstream regulator protein kinase A (PKA) have been suspected to play a role in the intracellular mechanisms of fear conditioning and spatial memory. However, whether they contribute to the learning and memory of motor skills is totally unknown. In this study, we have investigated the role of STEP and PKA activities during motor skill learning associated with the accelerating rotarod task. We observed that learning the rotarod task differentially modulated the levels of phosphorylated STEP61 at serine 221, a site directly regulated by PKA, in the hippocampus, motor cortex and striatum. In a second set of experiments, we have pharmacologically inhibited PKA by the injection of Rp-cAMPS directly into the dorsal striatum of mice before rotarod trainings. PKA phosphorylation of STEP prevents the dephosphorylation of STEP substrates, whereas inhibition of PKA promotes STEP activity. Striatal PKA inhibitions dose-dependently impaired mice performances on the accelerating rotarod task. General motor abilities testing revealed an intact motor control in mice treated with 5 and 20 µg of Rp-cAMPS, but not at the highest dose of 40 µg. This suggested that motor learning was selectively affected by PKA inhibition at lower doses. Most notably, striatal inhibition of PKA reduced the levels of phosphorylated STEP61 at serine 221. Our data support that inactivation of STEP61 by the PKA activity is part of the molecular process associated with motor skill learning.

Figure 1. Experimental design.

(A) Analysis of STEP expression during motor skill learning. Drug naïve mice were trained on the accelerating rotarod and sacrificed immediately after the end of the last trial at each day of training (day 1, 2, 3 and 4). Levels of total and phosphorylated STEP proteins in selected brain regions were analyzed by western blot techniques. (B) Role of PKA in motor learning. A guide cannula was implanted into the dorsal striatum of mice 7 days before rotarod training. Rp-cAMPS or vehicle were injected directly into the dorsal striatum of mice before the first trial at each rotarod training day (day 1, 2, 3 and 4). At the fourth day of training, mice were also tested for their motor abilities. Another cohort of mice received Rp-cAMPS or vehicle before the first trial at each rotarod training day. Mice were sacrificed at the second training day to performed western blot experiments. (C) Role of PKA activity in the motor coordination requested during rotarod testing. A guide cannula was implanted into the dorsal striatum of mice 7 days before rotarod training. Mice were trained during four consecutive days and Rp-cAMPS or vehicle was injected before the first trial at the fifth training day.

Figure 2. Levels of phosphorylated STEP61 in the brain of mice during motor learning.

(A) Drug-naïve mice were trained on the accelerating rotarod during 4 consecutive days and sacrificed at the end of each training day. Protein levels were evaluated by western blot. Proteins were extracted from selected brain regions of untrained or trained mice. Protein levels of phosphorylated STEP61 at Ser221, total STEP61 as well as GAPDH were measured in (B) hippocampus, (C) anterior cortex and (D) striatum. Data represent the mean of p-STEP61 relative optical density (expressed as a percentage of control values) ± S.E.M. Values are expressed relative to total STEP and are from triplicate experiments/animal, n = 4 mice/group. *p<0.05, **p<0.01 vs. untrained mice; ^§ p<0.05 vs day 1; ^# p<0.05, ^### p<0.001 vs. day 3; ^± p<0.05, ^±± p<0.01 vs. day 4.

Figure 3. Effect of intrastriatal inhibition of PKA in mice during rotarod training.

(A) Time spent on the rod of the accelerating rotarod for every trial completed at days 1, 2, 3 and 4. Mice were treated directly into the dorsal striatum, 15 min before each training day, with vehicle (saline) or Rp-cAMPS at doses: 5, 20 or 40 µg/side. Data represent the mean of latency to fall per trial expressed for every training day ± S.E.M. Lower panels represent the average scores of (B) the two first trials and (C) the two last trials of each training day. Values represent the average mean latency to fall expressed in seconds ± S.E.M. n = 4 to 11 mice/group. *p<0.05, **p<0.01 ***p<0.001 vs. respective vehicle-treated mice; ^# p<0.05 vs. mice treated with 5 µg/side.

Figure 4. Motor abilities in mice with intrastriatal inhibition of PKA.

(A) Pole, (B) wire suspension and (C) stepping tests were performed to assess motor abilities in saline (vehicle)-treated mice as well as mice treated with 5, 20, or 40 µg/side of Rp-cAMPS into the dorsal striatum. Data represents the mean of time require to perform the test (pole and wire suspension test) and the mean of numbers of adjusting steps (stepping test) ± S.E.M. n = 4 to 7 mice/group. *p<0.05, **p<0.01 vs. respective vehicle-treated mice; ^# p<0.05 vs. mice treated with 5 µg/side; ^± p<0.05 vs. mice treated with 20 µg/side. (D) Evaluation of motor coordination on the accelerating rotarod. Drug-naïve mice were trained on the accelerating rotarod during 4 consecutive days and treated into the dorsal striatum at the fifth day with vehicle (saline), 20 or 40 µg/side Rp-cAMPS. The total average latency to fall is shown at every training day in mice treated with vehicle or Rp-cAMPS. Data represent the means of all trials per training day ± S.E.M. n = 4 mice/group.

Figure 5. Levels of phosphorylated STEP61 and CREB in mice with intrastriatal inhibition of PKA.

(A) Levels of phosphorylated STEP61 at Ser221 and (B) phosphorylated CREB at Ser133 were evaluated in the dorsal striatum of trained mice injected with vehicle (saline) or Rp-cAMPS at the second day of training. Data represent the mean of relative optical density of phosphorylated STEP61 and phosphorylated CREB (expressed as a percentage of control values) ± S.E.M. Values are respectively expressed relative to total STEP and total CREB. Values are from triplicate experiments/animal, n = 4 mice/group. ***p<0.001 vs. vehicle-treated mice.