The DNA repair protein DNA-PKcs modulates synaptic plasticity via PSD-95 phosphorylation and stability
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ABSTRACT:
ORGANISM(S): Mus musculus (mouse)
SUBMITTER:
PROVIDER: S-BSST1420 | biostudies-other |
SECONDARY ACCESSION(S): 1
REPOSITORIES: biostudies-other
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Project description:The key DNA repair enzyme DNA-PKcs has several and important cellular functions. Loss of DNA-PKcs activity in mice has revealed essential roles in immune and nervous systems. In humans, DNA-PKcs is a critical factor for brain development and function since mutation of the prkdc gene causes severe neurological deficits such as microcephaly and seizures, predicting yet unknown roles of DNA-PKcs in neurons. Here we show that DNA-PKcs modulates synaptic plasticity. We demonstrate that DNA-PKcs localizes at synapses and phosphorylates PSD-95 at newly identified residues controlling PSD-95 protein stability. DNA-PKcs -/- mice are characterized by impaired Long-Term Potentiation (LTP), changes in neuronal morphology, and reduced levels of postsynaptic proteins. A PSD-95 mutant that is constitutively phosphorylated rescues LTP impairment when over-expressed in DNA-PKcs -/- mice. Our study identifies an emergent physiological function of DNA-PKcs in regulating neuronal plasticity, beyond genome stability.
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Project description:The key DNA repair enzyme DNA-PKcs has several and important cellular functions. Loss of DNA-PKcs activity in mice has revealed essential roles in immune and nervous systems. In humans, DNA-PKcs is a critical factor for brain development and function since mutation of the prkdc gene causes severe neurological deficits such as microcephaly and seizures, predicting yet unknown roles of DNA-PKcs in neurons. Here we show that DNA-PKcs modulates synaptic plasticity. We demonstrate that DNA-PKcs localizes at synapses and phosphorylates PSD-95 at newly identified residues controlling PSD-95 protein stability. DNA-PKcs -/- mice are characterized by impaired Long-Term Potentiation (LTP), changes in neuronal morphology, and reduced levels of postsynaptic proteins. A PSD-95 mutant that is constitutively phosphorylated rescues LTP impairment when over-expressed in DNA-PKcs -/- mice. Our study identifies an emergent physiological function of DNA-PKcs in regulating neuronal plasticity, beyond genome stability.
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Project description:Long-term potentiation (LTP) is accompanied by dendritic spine growth and changes in the composition of the postsynaptic density (PSD). We find that activity-dependent growth of apical spines of CA1 pyramidal neurons is accompanied by destabilization of the PSD that results in transient loss and rapid replacement of PSD-95 and SHANK2. Signaling through PSD-95 is required for activity-dependent spine growth and trafficking of SHANK2. N-terminal PDZ and C-terminal guanylate kinase domains of PSD-95 are required for both processes, indicating that PSD-95 coordinates multiple signals to regulate morphological plasticity. Activity-dependent trafficking of PSD-95 is triggered by phosphorylation at serine 73, a conserved calcium/calmodulin-dependent protein kinase II (CaMKII) consensus phosphorylation site, which negatively regulates spine growth and potentiation of synaptic currents. We propose that PSD-95 and CaMKII act at multiple steps during plasticity induction to initially trigger and later terminate spine growth by trafficking growth-promoting PSD proteins out of the active spine.
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Project description:Organization of signaling complexes at excitatory synapses by membrane-associated guanylate kinase (MAGUK) proteins regulates synapse development, plasticity, senescence and disease. Post-translational modification of MAGUK family proteins can drive their membrane localization, yet it is unclear how these intracellular proteins are targeted to sites of synaptic contact. Here we show using super-resolution imaging, biochemical approaches and in vivo models that the trans-synaptic organizing protein ephrin-B3 controls the synaptic localization and stability of PSD-95 and links these events to changes in neuronal activity via negative regulation of a newly identified mitogen-associated protein kinase (MAPK)-dependent phosphorylation site on ephrin-B3, Ser332. Unphosphorylated ephrin-B3 was enriched at synapses, and interacted directly with and stabilized PSD-95 at synapses. Activity-induced phosphorylation of Ser332 dispersed ephrin-B3 from synapses, prevented the interaction with PSD-95 and enhanced the turnover of PSD-95. Thus, ephrin-B3 specifies the synaptic localization of PSD-95 and likely links the synaptic stability of PSD-95 to changes in neuronal activity.
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Project description:The c-Abl tyrosine kinase is present in mouse brain synapses, but its precise synaptic function is unknown. We found that c-Abl levels in the rat hippocampus increase postnatally, with expression peaking at the first postnatal week. In 14 d in vitro hippocampal neuron cultures, c-Abl localizes primarily to the postsynaptic compartment, in which it colocalizes with the postsynaptic scaffold protein postsynaptic density protein-95 (PSD-95) in apposition to presynaptic markers. c-Abl associates with PSD-95, and chemical or genetic inhibition of c-Abl kinase activity reduces PSD-95 tyrosine phosphorylation, leading to reduced PSD-95 clustering and reduced synapses in treated neurons. c-Abl can phosphorylate PSD-95 on tyrosine 533, and mutation of this residue reduces the ability of PSD-95 to cluster at postsynaptic sites. Our results indicate that c-Abl regulates synapse formation by mediating tyrosine phosphorylation and clustering of PSD-95.
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Project description:Postsynaptic density protein 95 (PSD-95), a specialized scaffold protein with multiple protein interaction domains, forms the backbone of an extensive postsynaptic protein complex that organizes receptors and signal transduction molecules at the synaptic contact zone. Large, detergent-insoluble PSD-95-based postsynaptic complexes can be affinity-purified from conventional PSD fractions using magnetic beads coated with a PSD-95 antibody. In the present study purified PSD-95 complexes were analyzed by LC/MS/MS. A semiquantitative measure of the relative abundances of proteins in the purified PSD-95 complexes and the parent PSD fraction was estimated based on the cumulative ion current intensities of corresponding peptides. The affinity-purified preparation was largely depleted of presynaptic proteins, spectrin, intermediate filaments, and other contaminants prominent in the parent PSD fraction. We identified 525 of the proteins previously reported in parent PSD fractions, but only 288 of these were detected after affinity purification. We discuss 26 proteins that are major components in the PSD-95 complex based upon abundance ranking and affinity co-purification with PSD-95. This subset represents a minimal list of constituent proteins of the PSD-95 complex and includes, in addition to the specialized scaffolds and N-methyl-d-aspartate (NMDA) receptors, an abundance of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, small G-protein regulators, cell adhesion molecules, and hypothetical proteins. The identification of two Arf regulators, BRAG1 and BRAG2b, as co-purifying components of the complex implies pivotal functions in spine plasticity such as the reorganization of the actin cytoskeleton and insertion and retrieval of proteins to and from the plasma membrane. Another co-purifying protein (Q8BZM2) with two sterile alpha motif domains may represent a novel structural core element of the PSD.
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Project description:Postsynaptic density 95 (PSD-95) is a major synaptic scaffolding protein that plays a key role in bidirectional synaptic plasticity, which is a process important for learning and memory. It is known that PSD-95 shows increased dynamics upon induction of plasticity. However, the underlying structural and functional changes in PSD-95 that mediate its role in plasticity remain unclear. Here we show that phosphorylation of PSD-95 at Ser-561 in its guanylate kinase (GK) domain, which is mediated by the partitioning-defective 1 (Par1) kinases, regulates a conformational switch and is important for bidirectional plasticity. Using a fluorescence resonance energy transfer (FRET) biosensor, we show that a phosphomimetic mutation of Ser-561 promotes an intramolecular interaction between GK and the nearby Src homology 3 (SH3) domain, leading to a closed conformation, whereas a non-phosphorylatable S561A mutation or inhibition of Par1 kinase activity decreases SH3-GK interaction, causing PSD-95 to adopt an open conformation. In addition, S561A mutation facilitates the interaction between PSD-95 and its binding partners. Fluorescence recovery after photobleaching imaging reveals that the S561A mutant shows increased stability, whereas the phosphomimetic S561D mutation increases PSD-95 dynamics at the synapse. Moreover, molecular replacement of endogenous PSD-95 with the S561A mutant blocks dendritic spine structural plasticity during chemical long-term potentiation and long-term depression. Endogenous Ser-561 phosphorylation is induced by synaptic NMDA receptor activation, and the SH3-GK domains exhibit a Ser-561 phosphorylation-dependent switch to a closed conformation during synaptic plasticity. Our results provide novel mechanistic insight into the regulation of PSD-95 in dendritic spine structural plasticity through phosphorylation-mediated regulation of protein dynamics and conformation.
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Project description:Recent studies highlighted the importance of astrocyte-secreted molecules, such as ATP, for the slow modulation of synaptic transmission in central neurones. Biophysical mechanisms underlying the impact of gliotransmitters on the strength of individual synapse remain, however, unclear. Here we show that purinergic P2X receptors can bring significant contribution to the signalling in the individual synaptic boutons. ATP released from astrocytes facilitates a recruitment of P2X receptors into excitatory synapses by Ca(2+)-dependent mechanism. P2X receptors, co-localized with NMDA receptors in the excitatory synapses, can be activated by ATP co-released with glutamate from pre-synaptic terminals and by glia-derived ATP. An activation of P2X receptors in turn leads to down-regulation of postsynaptic NMDA receptors via Ca(2+)-dependent de-phosphorylation and interaction with PSD-95 multi-protein complex. Genetic deletion of the PSD-95 or P2X4 receptors obliterated ATP-mediated down-regulation of NMDA receptors. Impairment of purinergic modulation of NMDA receptors in the PSD-95 mutants dramatically decreased the threshold of LTP induction and increased the net magnitude of LTP. Our findings show that synergistic action of glia- and neurone-derived ATP can pre-modulate efficacy of excitatory synapses and thereby can have an important role in the glia-neuron communications and brain meta-plasticity.
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Project description:The persistent activity of protein kinase Mzeta (PKM?), a brain-specific, constitutively active protein kinase C isoform, maintains synaptic long-term potentiation (LTP). Structural remodeling of the postsynaptic density is believed to contribute to the expression of LTP. We therefore examined the role of PKM? in reconfiguring PSD-95, the major postsynaptic scaffolding protein at excitatory synapses. In primary cultures of hippocampal neurons, PKM? activity was critical for increasing the size of PSD-95 clusters during chemical LTP (cLTP). Increasing PKM? activity by overexpressing the kinase in hippocampal neurons was sufficient to increase PSD-95 cluster size, spine size, and postsynaptic AMPAR subunit GluA2. Overexpression of an inactive mutant of PKM? did not increase PSD-95 clustering, and applications of the ?-pseudosubstrate inhibitor ZIP reversed the PKM?-mediated increases in PSD-95 clustering, indicating that the activity of PKM? is necessary to induce and maintain the increased size of PSD-95 clusters. Thus the persistent activity of PKM? is both necessary and sufficient for maintaining increases of PSD-95 clusters, providing a unified mechanism for long-term functional and structural modifications of synapses.
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