Convergence of adenosine and GABA signaling for synapse stabilization during development.

During development, neural circuit formation requires the stabilization of active γ-aminobutyric acid­mediated (GABAergic) synapses and the elimination of inactive ones. Here, we demonstrate that, although the activation of postsynaptic GABA type A receptors (GABAARs) and adenosine A2A receptors (A2ARs) stabilizes GABAergic synapses, only A2AR activation is sufficient. Both GABAAR- and A2AR-dependent signaling pathways act synergistically to produce adenosine 3',5'-monophosphate through the recruitment of the calcium­calmodulin­adenylyl cyclase pathway. Protein kinase A, thus activated, phosphorylates gephyrin on serine residue 303, which is required for GABAAR stabilization. Finally, the stabilization of pre- and postsynaptic GABAergic elements involves the interaction between gephyrin and the synaptogenic membrane protein Slitrk3. We propose that A2ARs act as detectors of active GABAergic synapses releasing GABA, adenosine triphosphate, and adenosine to regulate their fate toward stabilization or elimination.

Differential enhancement of ERK, PKA and Ca2+ signaling in direct and indirect striatal neurons of Parkinsonian mice.

Parkinson's disease (PD) is characterized by severe locomotor deficits due to the disappearance of dopamine (DA) from the dorsal striatum. The development of PD symptoms and treatment-related complications such as dyskinesia have been proposed to result from complex alterations in intracellular signaling in both direct and indirect pathway striatal projection neurons (dSPNs and iSPNs, respectively) following loss of DA afferents. To identify cell-specific and dynamical modifications of signaling pathways associated with PD, we used a hemiparkinsonian mouse model with 6-hydroxydopamine (6-OHDA) lesion combined with two-photon fluorescence biosensors imaging in adult corticostriatal slices. After DA lesion, extracellular signal-regulated kinase (ERK) activation was increased in response to DA D1 receptor (D1R) or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) stimulation. The cAMP-dependent protein kinase (PKA) pathway contributing to ERK activation displayed supersensitive responses to D1R stimulation after 6-OHDA lesion. This cAMP/PKA supersensitivity was specific of D1R-responding SPNs and resulted from Gαolf upregulation and deficient phosphodiesterase activity. In lesioned striatum, the number of D1R-SPNs with spontaneous Ca2+ transients augmented while Ca2+ response to AMPA receptor stimulation specifically increased in iSPNs. Our work reveals distinct cell type-specific signaling alterations in the striatum after DA denervation. It suggests that over-activation of ERK pathway, observed in PD striatum, known to contribute to dyskinesia, may be linked to the combined dysregulation of DA and glutamate signaling pathways in the two populations of SPNs. These findings bring new insights into the implication of these respective neuronal populations in PD motor symptoms and the occurrence of PD treatment complications.

ATP6AP2 variant impairs CNS development and neuronal survival to cause fulminant neurodegeneration.

Vacuolar H+-ATPase-dependent (V-ATPase-dependent) functions are critical for neural proteostasis and are involved in neurodegeneration and brain tumorigenesis. We identified a patient with fulminant neurodegeneration of the developing brain carrying a de novo splice site variant in ATP6AP2 encoding an accessory protein of the V-ATPase. Functional studies of induced pluripotent stem cell-derived (iPSC-derived) neurons from this patient revealed reduced spontaneous activity and severe deficiency in lysosomal acidification and protein degradation leading to neuronal cell death. These deficiencies could be rescued by expression of full-length ATP6AP2. Conditional deletion of Atp6ap2 in developing mouse brain impaired V-ATPase-dependent functions, causing impaired neural stem cell self-renewal, premature neuronal differentiation, and apoptosis resulting in degeneration of nearly the entire cortex. In vitro studies revealed that ATP6AP2 deficiency decreases V-ATPase membrane assembly and increases endosomal-lysosomal fusion. We conclude that ATP6AP2 is a key mediator of V-ATPase-dependent signaling and protein degradation in the developing human central nervous system.

Two-photon Imaging of Microglial Processes' Attraction Toward ATP or Serotonin in Acute Brain Slices.

Microglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of homeostasis, undergo rapid morphological changes. For example, a laser lesion induces in a few minutes an oriented growth of microglial processes, also called "directional motility", toward the site of injury. A similar effect can be obtained by delivering locally ATP or serotonin (5-hydroxytryptamine [5-HT]). In this article, we describe a protocol to induce a directional growth of microglial processes toward a local application of ATP or 5-HT in acute brain slices of young and adult mice and to image this attraction over time by multiphoton microscopy. A simple method of quantification with free and open-source image analysis software is proposed. A challenge that still characterizes acute brain slices is the limited time, decreasing with age, during which the cells remain in a physiological state. This protocol, thus, highlights some technical improvements (medium, air-liquid interface chamber, imaging chamber with a double perfusion) aimed at optimizing the viability of microglial cells over several hours, especially in slices from adult mice.

Molecular genetics of the transcription factor GLIS3 identifies its dual function in beta cells and neurons.

The GLIS family zinc finger 3 isoform (GLIS3) is a risk gene for Type 1 and Type 2 diabetes, glaucoma and Alzheimer's disease endophenotype. We identified GLIS3 binding sites in insulin secreting cells (INS1) (FDR q<0.05; enrichment range 1.40-9.11 fold) sharing the motif wrGTTCCCArTAGs, which were enriched in genes involved in neuronal function and autophagy and in risk genes for metabolic and neuro-behavioural diseases. We confirmed experimentally Glis3-mediated regulation of the expression of genes involved in autophagy and neuron function in INS1 and neuronal PC12 cells. Naturally-occurring coding polymorphisms in Glis3 in the Goto-Kakizaki rat model of type 2 diabetes were associated with increased insulin production in vitro and in vivo, suggestive alteration of autophagy in PC12 and INS1 and abnormal neurogenesis in hippocampus neurons. Our results support biological pleiotropy of GLIS3 in pathologies affecting ß-cells and neurons and underline the existence of trans­nosology pathways in diabetes and its co-morbidities.

Dendritic diameter influences the rate and magnitude of hippocampal cAMP and PKA transients during ß-adrenergic receptor activation.

In the hippocampus, cyclic-adenosine monophosphate (cAMP) and cAMP-dependent protein kinase (PKA) form a critical signaling cascade required for long-lasting synaptic plasticity, learning and memory. Plasticity and memory are known to occur following pathway-specific changes in synaptic strength that are thought to result from spatially and temporally coordinated intracellular signaling events. To better understand how cAMP and PKA dynamically operate within the structural complexity of hippocampal neurons, we used live two-photon imaging and genetically-encoded fluorescent biosensors to monitor cAMP levels or PKA activity in CA1 neurons of acute hippocampal slices. Stimulation of ß-adrenergic receptors (isoproterenol) or combined activation of adenylyl cyclase (forskolin) and inhibition of phosphodiesterase (IBMX) produced cAMP transients with greater amplitude and rapid on-rates in intermediate and distal dendrites compared to somata and proximal dendrites. In contrast, isoproterenol produced greater PKA activity in somata and proximal dendrites compared to intermediate and distal dendrites, and the on-rate of PKA activity did not differ between compartments. Computational models show that our observed compartmental difference in cAMP can be reproduced by a uniform distribution of PDE4 and a variable density of adenylyl cyclase that scales with compartment size to compensate for changes in surface to volume ratios. However, reproducing our observed compartmental difference in PKA activity required enrichment of protein phosphatase in small compartments; neither reduced PKA subunits nor increased PKA substrates were sufficient. Together, our imaging and computational results show that compartment diameter interacts with rate-limiting components like adenylyl cyclase, phosphodiesterase and protein phosphatase to shape the spatial and temporal components of cAMP and PKA signaling in CA1 neurons and suggests that small neuronal compartments are most sensitive to cAMP signals whereas large neuronal compartments accommodate a greater dynamic range in PKA activity.

DARPP-32 interaction with adducin may mediate rapid environmental effects on striatal neurons.

Environmental enrichment has multiple effects on behaviour, including modification of responses to psychostimulant drugs mediated by striatal neurons. However, the underlying molecular and cellular mechanisms are not known. Here we show that DARPP-32, a hub signalling protein in striatal neurons, interacts with adducins, which are cytoskeletal proteins that cap actin filaments' fast-growing ends and regulate synaptic stability. DARPP-32 binds to adducin MARCKS domain and this interaction is modulated by DARPP-32 Ser97 phosphorylation. Phospho-Thr75-DARPP-32 facilitates ß-adducin Ser713 phosphorylation through inhibition of a cAMP-dependent protein kinase/phosphatase-2A cascade. Caffeine or 24-h exposure to a novel enriched environment increases adducin phosphorylation in WT, but not T75A mutant mice. This cascade is implicated in the effects of brief exposure to novel enriched environment on dendritic spines in nucleus accumbens and cocaine locomotor response. Our results suggest a molecular pathway by which environmental changes may rapidly alter responsiveness of striatal neurons involved in the reward system.

Dendritic geometry shapes neuronal cAMP signalling to the nucleus.

Neurons have complex dendritic trees, receiving numerous inputs at various distances from the cell body. Yet the rules of molecular signal propagation from dendrites to nuclei are unknown. DARPP-32 is a phosphorylation-regulated signalling hub in striatal output neurons. We combine diffusion-reaction modelling and live imaging to investigate cAMP-activated DARPP-32 signalling to the nucleus. The model predicts maximal effects on the nucleus of cAMP production in secondary dendrites, due to segmental decrease of dendrite diameter. Variations in branching, perikaryon size or spines have less pronounced effects. Biosensor kinase activity measurement following cAMP or dopamine uncaging confirms these predictions. Histone 3 phosphorylation, regulated by this pathway, is best stimulated by cAMP released in secondary-like dendrites. Thus, unexpectedly, the efficacy of diffusion-based signalling from dendrites to nucleus is not inversely proportional to the distance. We suggest a general mechanism by which dendritic geometry counterbalances the effect of dendritic distance for signalling to the nucleus.

FAK dimerization controls its kinase-dependent functions at focal adhesions.

Focal adhesion kinase (FAK) controls adhesion-dependent cell motility, survival, and proliferation. FAK has kinase-dependent and kinase-independent functions, both of which play major roles in embryogenesis and tumor invasiveness. The precise mechanisms of FAK activation are not known. Using x-ray crystallography, small angle x-ray scattering, and biochemical and functional analyses, we show that the key step for activation of FAK's kinase-dependent functions--autophosphorylation of tyrosine-397--requires site-specific dimerization of FAK. The dimers form via the association of the N-terminal FERM domain of FAK and are stabilized by an interaction between FERM and the C-terminal FAT domain. FAT binds to a basic motif on FERM that regulates co-activation and nuclear localization. FAK dimerization requires local enrichment, which occurs specifically at focal adhesions. Paxillin plays a dual role, by recruiting FAK to focal adhesions and by reinforcing the FAT:FERM interaction. Our results provide a structural and mechanistic framework to explain how FAK combines multiple stimuli into a site-specific function. The dimer interfaces we describe are promising targets for blocking FAK activation.

Type 4 phosphodiesterase plays different integrating roles in different cellular domains in pyramidal cortical neurons.

We investigated the role of phosphodiesterases (PDEs) in the integration of cAMP signals and protein kinase A (PKA) activity following beta-adrenergic stimulation, by carrying out real-time imaging of male mouse pyramidal cortical neurons expressing biosensors to monitor cAMP levels (Epac1-camps and Epac2-camps300) or PKA activity (AKAR2). In the soma, isoproterenol (ISO) increased the PKA signal to approximately half the maximal response obtained with forskolin, with a characteristic beta(1) pharmacology and an EC(50) of 4.5 nm. This response was related to free cAMP levels in the submicromolar range. The specific type 4 PDE (PDE4) inhibitor rolipram had a very small effect alone, but strongly potentiated the PKA response to ISO. Blockers of other PDEs had no effect. PDE4 thus acts as a brake in the propagation of the beta(1)-adrenergic signal from the membrane to the bulk somatic cytosol. The results for a submembrane domain were markedly different, whether recorded with a PKA-sensitive potassium current related to the slow AHP or by two-photon imaging of small distal dendrites. The responses to ISO were stronger than in the bulk cytosol. This is consistent with the cAMP/PKA signal being strong at the membrane, as shown by electrophysiology, and favored in cellular domains with a high surface area to volume ratio, in which this signal was detected by imaging. Rolipram alone also produced a strong cAMP/PKA signal, revealing tonic cAMP production. PDE4 thus appears as a crucial integrator with different physiological implications in different subcellular domains.

PKA dynamics in a Drosophila learning center: coincidence detection by rutabaga adenylyl cyclase and spatial regulation by dunce phosphodiesterase.

The dynamics of PKA activity in the olfactory learning and memory center, the mushroom bodies (MBs), are still poorly understood. We addressed this issue in vivo using a PKA FRET probe. Application of dopamine, the main neuromodulator involved in aversive learning, resulted in PKA activation specifically in the vertical lobe, whereas octopamine, involved in appetitive learning, stimulated PKA in all MB lobes. Strikingly, MB lobes were homogeneously activated by dopamine in the learning mutant dunce, showing that Dunce phosphodiesterase plays a major role in the spatial regulation of cAMP dynamics. Furthermore, costimulation with acetylcholine and either dopamine or octopamine led to a synergistic activation of PKA in the MBs that depends on Rutabaga adenylyl cyclase. Our results suggest that Rutabaga acts as a coincidence detector and demonstrate the existence of subcellular domains of PKA activity that could underlie the functional specialization of MB lobes in aversive and appetitive learning.

Real-time monitoring of cyclic nucleotide signaling in neurons using genetically encoded FRET probes.

Signaling cascades involving cyclic nucleotides play key roles in signal transduction in virtually all cell types. Elucidation of the spatiotemporal regulation of cyclic nucleotide signaling requires methods for tracking the dynamics of cyclic nucleotides and the activities of their regulators and effectors in the native biological context. Here we review a series of genetically encoded FRET-based probes for real-time monitoring of cyclic nucleotide signaling with a particular focus on their implementation in neurons. Current data indicate that neurons have a very active metabolism in cyclic nucleotide signaling, which is tightly regulated through a variety of homeostatic regulations.

Dynamics of protein kinase A signaling at the membrane, in the cytosol, and in the nucleus of neurons in mouse brain slices.

The cAMP-dependent protein kinase A (PKA) plays a ubiquitous role in the regulation of neuronal activity, but the dynamics of its activation have been difficult to investigate. We used the genetically encoded fluorescent probe AKAR2 to record PKA activation in the cytosol and the nucleus of neurons in mouse brain slice preparations, whereas the potassium current underlying the slow afterhyperpolarization potential (sAHP) in thalamic intralaminar neurons was used to monitor PKA activation at the membrane. Adenylyl cyclase was stimulated either directly using forskolin or via activation of 5-HT7 receptors. Both stimulations produced a maximal effect on sAHP, whereas in the cytosol, the amplitude of the 5-HT7 receptor-mediated response was half of that after direct adenylyl cyclase stimulation with forskolin. 5-HT7-mediated PKA responses were obtained in 30 s at the membrane, in 2.5 min in the cytosol, and in 13 min in the nucleus. Our results show in morphologically intact mammalian neurons the potential physiological relevance of PKA signal integration at the subcellular level: neuromodulators produce fast and powerful effects on membrane excitability, consistent with a highly efficient functional coupling between adenylyl cyclases, PKA, and target channels. Phosphorylation in the cytosol is slower and of graded amplitude, showing a differential integration of the PKA signal between the membrane and the cytosol. The nucleus integrates these cytosolic signals over periods of tens of minutes, consistent with passive diffusion of the free catalytic subunit of PKA into the nucleus, eventually resulting in a graded modulation of gene expression.