In hippocampal neurons, somato-dendritic CaV1.2 L-type voltage-gated Ca2+ channels (LTCC) function in excitation-transcription (E-T) coupling. Depolarizations that open LTCCs in postsynaptic neurons activate the transcription factors cAMP-response element binding protein (CREB) and nuclear factor of activated T-cells (NFAT) through Ca2+-regulated kinases and phosphatases. Because LTCC transcriptional regulation is required for long-lasting forms of excitatory synaptic plasticity that underlie learning and memory, it is crucial to understand how LTCC signaling leads to efficient, spatiotemporally specific synapse-to-nucleus communication. A question of fundamental importance in synapse-to-nucleus signaling is: how are early signals in E-T coupling Ca2+ signals in dendritic postsynaptic nanodomains transduced into signals that are reliably relayed over long distances to the nucleus? The postsynaptic scaffold protein A-kinase anchoring protein (AKAP) 79/150 binds to CaV1.2 through a modified leucine zipper (LZ) motif. This AKAP anchors both the cAMP- dependent protein kinase (PKA), via an amphipathic alpha-helical motif, and the Ca2+-calmodulin (CaM)-activated protein phosphatase-2B (calcineurin; CaN), via an atypical PxIxIT docking motif. Anchoring of PKA to AKAP79/150 supports enhancement of neuronal LTCC current amplitude that is potently opposed by Ca2+-dependent feedback through AKAP-anchored CaN. LTCC activation of AKAP-localized CaN is also required for K+ depolarization-triggered NFAT translocation to the nucleus and activation of transcription. However, key synapse-to-nucleus signaling questions remain for the LTCC-AKAP-CaN-NFAT pathway: (1) does the AKAP79/150 signaling complex regulate LTCC Ca2+ influx specifically in dendrites excited by postsynaptic glutamate receptor activation; (2) do these Ca2+ signals in dendrites locally activate CaN-NFAT signaling that ultimately acts in the nucleus; (3) what are the neuronal target genes regulated by this signaling pathway; and (4) is this process engaged during synaptic plasticity? We will explore these crucial questions in three aims that rely upon a combination of Ca2+ imaging (Aim 1), CaN and NFAT imaging (Aim 2), and gene transcription analyses (Aim 3). AKAP79/150 regulation of LTCC Ca2+ influx, CaN-NFAT signaling dynamics, and activity-dependent gene transcription will be investigated in neurons or brain slices expressing AKAP mutants that alter PKA anchoring, CaN anchoring, or LZ domain binding. The overall goal of this project is to test a central hypothesis in synapse-to-nucleus communication that postsynaptic Ca2+ signals are locally re-coded in dendrites as protein-based signals (e.g., NFAT), and relayed to the nucleus to control plasticity-associated gene expression.
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, neuropsychiatric disorders, aging and neurodegenerative diseases. Changes in LTCC channel function have been implicated in cognitive impairments associated with aging and an inherited 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. Recent genome-wide association studies have also linked single nucleotide polymorphisms in the genes encoding LTCC subunits to increased risks of autism spectrum disorder, attention deficit-hyperactivity disorder, bipolar disorder, major depressive disorder, an schizophrenia. Linkage of LTCC's to multiple neuropsychiatric and neurological diseases further supports a key role for these channels in regulating central gene expression programs that control neuronal plasticity throughout the brain. 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 coupling of LTCCs at synapses to transcription factors that ultimately act in the nucleus are not well understood. The scaffold protein AKAP79/150 could help provide these functions by promoting channel 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 neuropsychiatric and neurological disease.
