CAMP at Perinuclear mAKAPα Signalosomes Is Regulated by Local Ca2+ Signaling in Primary Hippocampal Neurons.

Tomasz Boczek,Ying Zhu,Kimberly L Dodge-Kafka,Jeffrey L Goldberg,Michael S Kapiloff,Qian Yu

doi:10.1523/eneuro.0298-20.2021

Tomasz Boczek, Ying Zhu + Show 4 more

Open Access

https://doi.org/10.1523/eneuro.0298-20.2021

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Abstract

The second messenger cyclic adenosine monophosphate (cAMP) is important for the regulation of neuronal structure and function, including neurite extension. A perinuclear cAMP compartment organized by the scaffold protein muscle A-kinase anchoring protein α (mAKAPα/AKAP6α) is sufficient and necessary for axon growth by rat hippocampal neurons in vitro. Here, we report that cAMP at mAKAPα signalosomes is regulated by local Ca2+ signaling that mediates activity-dependent cAMP elevation within that compartment. Simultaneous Forster resonance energy transfer (FRET) imaging using the protein kinase A (PKA) activity reporter AKAR4 and intensiometric imaging using the RCaMP1h fluorescent Ca2+ sensor revealed that membrane depolarization by KCl selectively induced activation of perinuclear PKA activity. Activity-dependent perinuclear PKA activity was dependent on expression of the mAKAPα scaffold, while both perinuclear Ca2+ elevation and PKA activation were dependent on voltage-dependent L-type Ca2+ channel activity. Importantly, chelation of Ca2+ by a nuclear envelope-localized parvalbumin fusion protein inhibited both activity-induced perinuclear PKA activity and axon elongation. Together, this study provides evidence for a model in which a neuronal perinuclear cAMP compartment is locally regulated by activity-dependent Ca2+ influx, providing local control for the enhancement of neurite extension.

Highlights

Central nervous system (CNS) neurons responsible for higher order functions fail to survive or regenerate their axons after injury, resulting in permanent disability in common diseases such as stroke, Alzheimer’s disease, Parkinson's disease and glaucoma
Given that perinuclear cyclic adenosine monophosphate (cAMP) signaling has been shown to be required for activity-dependent neurite extension (Boczek et al, 2019), we considered whether KCl-mediated depolarization can regulate cAMP at mAKAPα signalosomes
Cultured primary e18 rat hippocampal neurons were transfected on day 4-5 in culture with expression plasmids for perinuclear-localized PN-AKAR4 or diffusely localized parent AKAR4 protein kinase A (PKA) activity Forster resonance energy transfer (FRET)

Summary

Introduction

Central nervous system (CNS) neurons responsible for higher order functions fail to survive or regenerate their axons after injury, resulting in permanent disability in common diseases such as stroke, Alzheimer’s disease, Parkinson's disease and glaucoma. To combat this disability, strategies are being sought to promote CNS neuron survival and axon regeneration after injury, including the identification of intracellular signaling pathways whose activation might be beneficial in disease. Enhanced cyclic adenosine monophosphate (cAMP) signaling has been shown to potentiate neurotrophic signaling and to promote neuron survival and axon regeneration (Wang et al, 2015; Wild and Dell'Acqua, 2018).

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