Mitochondrial shape is determined by fission and fusion reactions catalyzed by large GTPases of the dynamin family, mutation of which can cause neurological dysfunction. While fission-inducing protein phosphatases have been identified, the identity of opposing kinase signaling complexes has remained elusive. We report here that in both neurons and non-neuronal cells, cAMP elevation and expression of an outer-mitochondrial membrane (OMM) targeted form of the protein kinase A (PKA) catalytic subunit reshapes mitochondria into an interconnected network. Conversely, OMM-targeting of the PKA inhibitor PKI promotes mitochondrial fragmentation upstream of neuronal death. RNAi and overexpression approaches identify mitochondria-localized A kinase anchoring protein 1 (AKAP1) as a neuroprotective and mitochondria-stabilizing factor in vitro and in vivo. According to epistasis studies with phosphorylation site-mutant dynamin-related protein 1 (Drp1), inhibition of the mitochondrial fission enzyme through a conserved PKA site is the principal mechanism by which cAMP and PKA/AKAP1 promote both mitochondrial elongation and neuronal survival. Phenocopied by a mutation that slows GTP hydrolysis, Drp1 phosphorylation inhibits the disassembly step of its catalytic cycle, accumulating large, slowly recycling Drp1 oligomers at the OMM. Unopposed fusion then promotes formation of a mitochondrial reticulum, which protects neurons from diverse insults. Mitochondria, the cellular powerhouse, are highly dynamic organelles shaped by opposing fission and fusion events. Research over the past decade has identified many components of the mitochondrial fission/fusion machinery and led to the discovery that mutations in genes coding for these proteins can cause human neurological diseases. While it is well established that mitochondrial shape changes are intimately involved in cellular responses to environmental stressors, we know very little about the mechanisms by which cells dynamically adjust mitochondrial form and function. In this report, we show that the scaffold protein AKAP1 brings the cAMP-dependent protein kinase PKA to the outer mitochondrial membrane to protect neurons from injury. The PKA/AKAP1 complex functions by inhibiting Drp1, an enzyme that mechanically constricts and eventually severs mitochondria. Whereas active, dephosphorylated Drp1 rapidly cycles between cytosol and mitochondria, phosphorylated Drp1 builds up in inactive mitochondrial complexes, allowing mitochondria to fuse into a neuroprotective reticulum. Our results suggest that altering the balance of kinase and phosphatase activities at the outer mitochondrial membrane may provide the basis for novel neuroprotective therapies.

Ca2+ is a key intermediary in a variety of signalling pathways and undergoes dynamic changes in its cytoplasmic concentration due to release from stores within the endoplasmic reticulum (ER) and influx from the extracellular environment. In addition to regulating cytoplasmic Ca2+ signals, these responses also affect the concentration of Ca2+ within the ER and mitochondria. Single fluorescent protein-based Ca2+ indicators, such as the GCaMP series based on GFP, are powerful tools for imaging changes in the concentration of Ca2+ associated with intracellular signalling pathways. Most GCaMP-type indicators have dissociation constants (Kd) for Ca2+ in the high nanomolar to low micromolar range and are therefore optimal for measuring cytoplasmic [Ca2+], but poorly suited for use in mitochondria and ER where [Ca2+] can reach concentrations of several hundred micromolar. We now report GCaMP-type low-affinity red fluorescent genetically encoded Ca2+ indicators for optical imaging (LAR-GECO), engineered to have Kd values of 24 μM (LAR-GECO1) and 12 μM (LAR-GECO1.2). We demonstrate that these indicators can be used to image mitochondrial and ER Ca2+ dynamics in several cell types. In addition, we perform two-colour imaging of intracellular Ca2+ dynamics in cells expressing both cytoplasmic GCaMP and ER-targeted LAR-GECO1. The development of these low-affinity intensiometric red fluorescent Ca2+ indicators enables monitoring of ER and mitochondrial Ca2+ in combination with GFP-based reporters.

Association of PKA with the AMPA receptor GluR1 subunit via the A kinase anchor protein AKAP150 is crucial for GluR1 phosphorylation. Mutating the AKAP150 gene to specifically prevent PKA binding reduced PKA within postsynaptic densities (>70%). It abolished hippocampal LTP in 7–12 but not 4‐week‐old mice. Inhibitors of PKA and of GluR2‐lacking AMPA receptors blocked single tetanus LTP in hippocampal slices of 8 but not 4‐week‐old WT mice. Inhibitors of GluR2‐lacking AMPA receptors also prevented LTP in 2 but not 3‐week‐old mice. Other studies demonstrate that GluR1 homomeric AMPA receptors are the main GluR2‐lacking AMPA receptors in adult hippocampus and require PKA for their functional postsynaptic expression during potentiation. AKAP150‐anchored PKA might thus critically contribute to LTP in adult hippocampus in part by phosphorylating GluR1 to foster postsynaptic accumulation of homomeric GluR1 AMPA receptors during initial LTP in 8‐week‐old mice.

Phosphorylation-dependent modulation of the vanilloid receptor TRPV1 is one of the key mechanisms mediating the hyperalgesic effects of inflammatory mediators, such as prostaglandin E₂(PGE₂). However, little is known about the molecular organization of the TRPV1 phosphorylation complex and specifically about scaffolding proteins that position the protein kinase A (PKA) holoenzyme proximal to TRPV1 for effective and selective regulation of the receptor. Here, we demonstrate the critical role of the A-kinase anchoring protein AKAP150 in PKA-dependent modulation of TRPV1 function in adult mouse dorsal root ganglion (DRG) neurons. We found that AKAP150 is expressed in ∼80% of TRPV1-positive DRG neurons and is coimmunoprecipitated with the capsaicin receptor. In functional studies, PKA stimulation with forskolin markedly reduced desensitization of TRPV1. This effect was blocked by the PKA selective inhibitors KT5720 [(9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylicacid hexyl ester] and H89 (N-[2-(p-bromo-cinnamylamino)-ethyl]-5-isoquinoline-sulfon-amide 2HCl), as well as by the AKAP inhibitory peptide Ht31. Similarly, PGE₂decreased TRPV1 desensitization in a manner sensitive to the PKA inhibitor KT5720. Both the forskolin and PGE₂effects were strongly impaired in DRG neurons from knock-in mice that express a mutant AKAP150 lacking the PKA-binding domain (Δ36 mice). Protein kinase C-dependent sensitization of TRPV1 remained intact in Δ36 mice. The PGE₂/PKA signaling defect in DRG neurons from Δ36 mice was rescued by overexpressing the full-length human ortholog of AKAP150 in these cells. In behavioral testing, PGE₂-induced thermal hyperalgesia was significantly diminished in Δ36 mice. Together, these data suggest that PKA anchoring by AKAP150 is essential for the enhancement of TRPV1 function by activation of the PGE₂/PKA signaling pathway.

Mitochondria are essential for proper cellular function through their critical roles in ATP synthesis, reactive oxygen species production, calcium (Ca²⁺) buffering, and apoptotic signaling. In neurons, Ca²⁺ buffering is particularly important as it helps to shape Ca²⁺ signals and to regulate numerous Ca²⁺-dependent functions including neuronal excitability, synaptic transmission, gene expression, and neuronal toxicity. Over the past decade, identification of the mitochondrial Ca²⁺ uniporter (MCU) and other molecular components of mitochondrial Ca²⁺ transport has provided insight into the roles that mitochondrial Ca²⁺ regulation plays in neuronal function in health and disease. In this review, we discuss the many roles of mitochondrial Ca²⁺ uptake and release mechanisms in normal neuronal function and highlight new insights into the Ca²⁺-dependent mechanisms that drive mitochondrial dysfunction in neurologic diseases including epilepsy, Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. We also consider how targeting Ca²⁺ uptake and release mechanisms could facilitate the development of novel therapeutic strategies for neurological diseases.