In hippocampal neurons, somato-dendritic CaV1.2 L-type voltage-gated Ca2+ channels (LTCC) function in excitation-transcription (E-T) coupling. Depolarizing stimuli that open LTCCs activate the transcription factors cAMP-response element binding protein (CREB) and nuclear factor of activated T-cells (NFAT) through Ca2+-regulated kinase and phosphatases. Importantly, LTCC transcriptional regulation is required for long-lasting forms of excitatory synaptic plasticity that underlie learning and memory, such as late-phase long-term potentiation (L-LTP). Thus, it is crucial to understand how LTCC activity and signaling are controlled to promote efficient, specific synapse to nucleus communication. The primary question in synapse-to-nucleus signaling is how local Ca2+ signals generated in dendrites are relayed remotely to the nucleus in the soma. In the last funding period, we established the postsynaptic scaffold protein A-kinase anchoring protein (AKAP) 79/150, which binds to CaV1.2 through a modified leucine zipper (LZ) motif and anchors the cAMP-dependent protein kinase (PKA) and Ca2+-calmodulin (CaM)-activated protein phosphatase-2B (calcineurin;CaN), as an essential regulator of neuronal LTCC currents and NFAT activation. We found that AKAP-anchored PKA promoted LTCC current enhancement that was strongly opposed by a Ca2+ negative feedback loop activating AKAP-anchored CaN to favor rapid, calcium-dependent inactivation (CDI). In addition, we found that local LTCC activation of AKAP-anchored CaN was required for NFAT translocation to the nucleus and transcription in response to depolarization. However, key questions remain regarding whether AKAP79/150 regulates LTCC Ca2+ influx specifically in dendritic spines in response to glutamate receptor activation and whether postsynaptic Ca2+ signals restricted in dendrites can locally activate CaN-NFAT signaling to the nucleus. We will explore these questions using a combination of whole-cell LTCC current recordings, local glutamate uncaging, Ca2+ imaging (Aim 1), NFAT imaging (Aim 2), transcriptional analyses, and extracellular recordings of L-LTP (Aim 3). In all three aims, AKAP79/150 regulation of LTCC activity and NFAT signaling will be investigated by expressing PKA anchoring deficient (delta-PKA), CaN anchoring deficient (delta-PIX), and LZ domain (delta-LZ) AKAP79 mutants in in rat neurons or using neurons from AKAP150 delta-PIX and delta-PKA knock-in mice. Overall, this project will test a central hypothesis in synapse-to-nucleus communication that postsynaptic Ca2+ signals are locally decoded in dendrites and then efficiently relayed to the nucleus to control gene expression linked to synaptic plasticity.
Excitation induced entry of calcium ions (Ca2+) into neurons through L-type voltage-gated Ca2+ channels (LTCC) influences synaptic plasticity by regulating gene expression. Elucidating LTCC mechanisms regulating neuronal plasticity is relevant for understanding normal learning and memory as well as learning deficits in intellectual disabilities, autism, schizophrenia, aging and neurodegenerative conditions such as Alzheimer's. Changes in LTCC channel function have been implicated in cognitive impairments associated with aging and a form of autism (Timothy syndrome), and alterations in Ca2+ and calcineurin (CaN) regulation of NFAT transcription factors involved in gene expression have been identified in Down syndrome and Alzheimer's. Previous studies have demonstrated that highly localized signaling processes occur near the LTCC that are important for both channel modulation and downstream signaling to transcription factors in the nucleus. However, the molecular mechanisms that coordinate phosphorylation regulation of neuronal LTCCs and coupling to transcription factors are not well understood. The scaffold protein AKAP79/150 could help provide these functions by promoting channel phosphorylation by PKA, by providing a novel Ca2+-activated CaN phosphatase negative feedback loop to oppose this regulation, and by positioning CaN and NFAT near the channel to regulate downstream transcriptional pathways linked to synaptic plasticity and learning and memory. Understanding how AKAP79/150 coordinates LTCC activity and CaN- NFAT transcriptional signaling may lead to development of novel therapeutics to treat cognitive impairments.
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