RIM1alpha phosphorylation at serine-413 by protein kinase A is not required for presynaptic long-term plasticity or learning

Abstract

Activation of presynaptic cAMP-dependent protein kinase A (PKA) triggers presynaptic long-term plasticity in synapses such as cerebellar parallel fiber and hippocampal mossy fiber synapses. RIM1alpha, a large multidomain protein that forms a scaffold at the presynaptic active zone, is essential for presynaptic long-term plasticity in these synapses and is phosphorylated by PKA at serine-413. Previous studies suggested that phosphorylation of RIM1alpha at serine-413 is required for presynaptic long-term potentiation in parallel fiber synapses formed in vitro by cultured cerebellar neurons and that this type of presynaptic long-term potentiation is mediated by binding of 14-3-3 proteins to phosphorylated serine-413. To test the role of serine-413 phosphorylation in vivo, we have now produced knockin mice in which serine-413 is mutated to alanine. Surprisingly, we find that in these mutant mice, three different forms of presynaptic PKA-dependent long-term plasticity are normal. Furthermore, we observed that in contrast to RIM1alpha KO mice, RIM1 knockin mice containing the serine-413 substitution exhibit normal learning capabilities. The lack of an effect of the serine-413 mutation of RIM1alpha is not due to compensation by RIM2alpha because mice carrying both the serine-413 substitution and a RIM2alpha deletion still exhibited normal long-term presynaptic plasticity. Thus, phosphorylation of serine-413 of RIM1alpha is not essential for PKA-dependent long-term presynaptic plasticity in vivo, suggesting that PKA operates by a different mechanism despite the dependence of long-term presynaptic plasticity on RIM1alpha.

Fig. 1.

RIM1α contains a single tight 14-3-3 binding site. (A) Schematic overview of RIM1α and its protein domains. GST-fusion proteins that were used for the biochemical characterization are indicated. SSA, SSB, SSC, splice site A, B, and C; PxxP, proline-rich sequence; S, serine-413 phosphorylation site. (B) Coomassie blue staining of the eluates of the affinity column that contained phosphorylated GST-RIM1 351–596 and nonphosphorylated control RIM1α. The arrow indicates the phospho-specific band that was submitted to mass spectrometry. (C) In vitro binding of wild-type (wt) and mutant GST-RIM1 351–429 to 14-3-3ε in GST-pulldown experiments from rat brain homogenates. (D) In vitro binding of PKA phosphorylated and nonphosphorylated GST-RIM1 fusion proteins to 14-3-3ε in GST pulldown experiments from brain homogenates. S/A, S413A-point mutant RIM1α fragment; wt, wild-type RIM1 fragment; *, cross-reactive band that sometimes appears in fresh brain homogenates with the 14-3-3ε antibody.

Fig. 2.

Generation of RIM1 S413A-KI mice. (A) KI strategy for the RIM1 S413A-KI mice showing (from top to bottom) the wild-type (WT) RIM1 allele, the S413A KI targeting construct, the RIM1 mutant allele after homologous recombination, and the flp-excised KI allele. 5, 6, and 7, exons 5, 6 and 7; DT, diphtherotoxin-expressing cassette; NR, neomycin resistance cassette; *, S413A point mutation in exon 6; N, NcoI restriction sites that were used for Southern blotting. (B) Southern blot from heterozygous embryonic stem cells after homologous recombination and wild-type control cells, NcoI digest, and a 5′ outside probe were used. The wild-type band is 8.0 kilobases (kb), the KI band 3.8 kb. (C) PCR genotyping of the RIM1 S413A-KI mice including heterozygous and wild-type controls for (from top to bottom) the 5′ loxP site, the 3′ loxP/frt site after flp-recombination, and the S413A point mutation/BglI restriction site after BglI digest of the amplified fragments. (D) Sequencing result of the homozygous KI allele in RIM1 S413A-KI mice. Wild-type (WT) DNA and protein sequences are indicated for comparison.

Fig. 3.

Basic characterization of the RIM1 S413A-KI mice. (A) Survival ratio of offsprings from heterozygous matings of RIM1 S413A-KI mice; the gray shaded area indicates the expected Mendelian ratio. (B) Body weight of RIM1 S413A-KI and control littermate mice from postnatal day 6 to 60. (C) Western blotting for RIM1α with antibodies against the N terminus (Q703), the central part including the PDZ domain (R809), the phosphoserine-413 residue (T2798; arrow, absence of serine-413-phosphorylated RIM1α; star, cross-reactive band at a slightly lower molecular weight), and loading controls. GDI, GDP dissociation inhibitor; VCP, valosin-containing protein). (D) Western blotting of brain homogenates for several presynaptic and other proteins appears normal. Rph, rabphilin; RIM-BP2, RIM-binding protein 2; NSF, N-ethylmaleimide-sensitive factor; Syn, synapsin; Syt 1, synaptotagmin-1.

Fig. 4.

Cerebellar parallel fiber LTP, mossy fiber LTP, and I-LTD in the hippocampus are normal in RIM1 S413A-KI mice. (A) LTP at parallel fiber to Purkinje cell synapses in response to a single tetanus (200 stimuli at 10 Hz, vertical arrow). EPSC, excitatory postsynaptic current. (B) LTP at mossy fiber to CA3 pyramidal cell synapses evoked by a tetanus (125 pulses at 25 Hz) delivered to mossy fibers (vertical arrow). A few data points right after tetanization were removed for clarity. fEPSP, field excitatory postsynaptic potential. (C) Endocannabinoid-mediated long-term depression at hippocampal inhibitory synapses (I-LTD) in wild-type and mutant mice was induced by theta-burst stimulation (vertical arrow) consisting of a series of 10 bursts of 5 stimuli (100-Hz burst, 200-ms interburst interval), which was repeated 4 times (5 s apart). IPSC, inhibitory postsynaptic current. (D) Mossy fiber LTP in RIM1 S413A-KI/RIM2α KO double-mutant and RIM1 S413A-KI control mice. In A–D, averaged sample traces taken at times indicated by numbers are shown above summary graphs.

Fig. 5.

Short-term synaptic plasticity in RIM1 S413A-KI mice in excitatory Schaffer collateral to CA1 pyramidal neuron synapses. (A) Summary graph (left) and averaged sample traces (right) of paired pulse facilitation (PPF) measured at 40-ms interstimulus interval in RIM1 S413A-KI and control mice. (B) Synaptic responses [field excitatory postsynaptic potentials (fEPSPs)] elicited by a 14-Hz train of 25 stimuli in RIM1 S413A-KI and control mice. Values are normalized to the first synaptic response of the stimulation train.

Fig. 6.

RIM1 S413A-KI mice displayed normal locomotor activity, anxiety-like behavior, motor coordination, and spatial and emotional learning and memory. (A) Measurements of the spontaneous motor activity of RIM1 S413A-KI and control mice were determined as the number of beam breaks per minute in a fresh home cage equipped with photodetector beams. (B) Anxiety-related behaviors probed as latency to enter the light side of a dark/light box, as the time spent in the open arms of an elevated plus maze, and as the time spent in the center of an open field. (C) Measurements of motor coordination monitored as the time mice stay on an accelerating rotarod as a function of trial number. (D) Freezing behavior to both context- and cue-dependent fear conditioning 24 h after training. (E) Morris water maze analysis of spatial learning during the initial 11 days of training as measured by latency to reach the submerged platform. (F) Spatial memory test on day 12 of the Morris water maze analysis after removal of the platform from the target quadrant. Percent time spent in each quadrant is indicated.