BIOLOGICAL OVERVIEW

Analysis of protein kinase A (PKA) has been initiated in Drosophila using transgenic techniques to modulate PKA activity in specific tissues during development. GAL4/UAS-regulated transgenes were constructed in active and mutant forms that encode PKAc, the catalytic subunit of PKA, and PKI(1-31), a competitive inhibitor of PKAc. Evidence is provided that the wild-type transgenes are active, and a summary is given of the phenotypes produced by a number of GAL4 enhancer-detector strains. The effects of transgenes encoding PKI(1-31) are compared with those encoding PKAr*, a mutant regulatory subunit that constitutively inhibits PKAc because of its inability to bind cyclic AMP. Both inhibitors block larval growth, but only PKAr* alters pattern formation by activating the Hedgehog signaling pathway. Therefore, transgenic PKI(1-31) should provide a tool to investigate the role of PKAc in larval growth regulation without concomitant changes in pattern formation. The different effects of PKI(1-31) and PKAr* suggest two distinct roles, cytoplasmic and nuclear, for PKAc in Hedgehog signal transduction. Alternatively, PKAr* may target proteins other than PKAc, suggesting a role for free PKAr in signal transduction, a role inhibited by PKAc in reversal of the classical relationship of these subunits (Kiger, 1999).

Phenotypes produced by PKI(1-31) and PKAr* are surprisingly different. The phenotypic effects of PKI(1-31) appear to represent a subset of those of PKAr*. Both retard or otherwise block larval growth. PKAr* alone affects patterning in embryos and imaginal discs by activating Hedgehog signaling, and it alone causes abnormal differentiation in imaginal discs (which may reflect minor aberrations in patterning). The origin of this difference might reside in some fundamental difference in the biological properties of PKI(1-31) and PKAr* or perhaps in their relative stabilities in different cell types. However, PKI(1-31) is demonstrably active in wing imaginal discs and in other tissues since it is capable of inhibiting ectopic PKAcF (epitope tagged PKA catalytic subunit). Regardless of the origin of the difference, it would appear that PKI(1-31) specifically targets larval growth (Kiger, 1999).

Newly hatched larvae consist of two cell types: (1) mitotic cells composing the imaginal discs, gonad, and some neuroblasts, and (2) endoreplicating cells making up the exclusively larval tissues. These latter cells do not divide after hatching, but they increase in size as the larva grows, being maintained by cycles of DNA replication without nuclear division. These two cell types are regulated in fundamentally different ways, as demonstrated by their responses to nutritional deprivation. It would appear that mitotic cells are not sensitive to expression of PKI(1-31), but only to expression of PKAr*, whereas endoreplicating cells are sensitive to both. Both proteins are effective inhibitors of the catalytic site of PKAc, possessing a pseudosubstrate binding site with a pair of adjacent Arg residues that interact with the catalytic site. PKAr, which is larger than PKI(1-31) or full-length PKI(1-77), makes additional contacts with PKAc that make the PKAr:PKAc complex more stable than the PKI(1-77):PKAc complex. PKI(5-24) and PKI(1-77) bind to PKAc with the same affinity, and PKI(1-31) is probably no different. The C terminus of PKI(5-24) is not involved in binding to PKAc, so the FLAG epitope at the C terminus of PKI(1-31) should not interfere with its inhibitory function. Free PKAc and PKI(1-77) are small enough to enter the nucleus by diffusion. PKI(1-77) possesses a nuclear export signal (residues 35-49) that is hidden until PKAc is bound, whereupon the PKI(1-77):PKAc complex is extruded from the nucleus. Moreover, the expression and intracellular distribution of PKI(1-77) is regulated during the cell cycle and is necessary for cell cycle progression. PKI(1-31) would be expected to inhibit both nuclear and cytoplasmic PKAc. In addition, PKI(1-31) could compete with a Drosophila homolog of PKI(1-77) for nuclear PKAc and block PKAc export. In contrast, PKAr is cytoplasmic whether or not it is complexed with PKAc because it is too large to enter the nucleus. In addition, anchoring proteins have been identified that bind PKAr to the membrane or cytoskeleton. PKAr* would be expected to inhibit cytoplasmic PKAc and to deplete nuclear PKAc by forming a cytoplasmic sink for PKAc that diffuses from the nucleus (Kiger, 1999 and references).

With regard to Hedgehog signaling, a possible target of PKAr* and PKI(1-31) in the cytoplasm would be the complex responsible for the proteolysis of the transcription factor Ci, where PKAr* and PKI(1-31) would inhibit phosphorylation of the PKA sites necessary for proteolysis of Ci155 to the repressor form Ci75. The ability of PKAr* to interact with anchoring or other proteins might give it greater access to this complex than PKI(1-31), accounting for the failure of the latter to activate Hedgehog target genes (Kiger, 1999).

Another possible explanation for the different actions of PKAr* and PKIF(1-31) is that free PKAr* (and by implication free PKAr) has a target other than PKAc through which it activates Hedgehog signaling. Precedent for such a role exists. In Dictyostelium, free PKAr binds and activates a cAMP-specific phosphodiesterase that is postulated to have functional homology to the cAMP-specific phosphodiesterase encoded by dunce. The Dictyostelium phosphodiesterase is also activated by bovine PKAr1a, and a synthetic monomeric form of this regulatory subunit is a more potent activator than the dimeric form. (The Dictyostelium PKAr protein lacks a dimerization domain, and its PKA exists as a heterodimer). In this scenario, in the absence of a cAMP signal, PKAc would bind to PKAr, inhibiting this novel activity. Reduction in the level of PKAc, e.g., in a mitotic clone of cells homozygous for a lethal allele of DC0, would lead to free PKAr that would activate Hedgehog signaling (Kiger, 1999 and references).

In an alternative scenario, the effect of PKAr* on Hedgehog target genes could be caused by its ability to deplete nuclear PKAc, a role that cannot be fulfilled by PKI(1-31). Since the normal role of PKI(1-77) is not only to inhibit, but to export, nuclear PKAc, it is possible that PKAc plays another critical role in the nucleus in addition to its catalytic role in phosphorylation. For example, PKAc might function as a corepressor with Ci75 to block transcription of Hedgehog target genes. Consistent with this hypothesis PKI(1-60) has been shown to activate Ci-mediated chloramphenicol acetyltransferase transcription from a model Gli enhancer in Drosophila Kc cells, a finding can be attribute solely to inhibition of proteolysis of cytoplasmic Ci155. It may be that PKAc can function as a corepressor even if its catalytic site is occupied by PKI(1-31). Corepression by PKI(1-31):PKAc and Ci75 might block transcription of target genes, even in the presence of Ci155 produced by concommitant inhibition of Ci155 proteolysis in the cytoplasm. Small changes in the ratio of Ci155 and Ci75 are believed to be critical for activation of Hedgehog target genes. In addition, PKI(1-77) may differ from PKI(1-31) because only the former reduces basal transcription from cAMP-stimulated promoters. If PKAc has such an additional role, then the R224 mutant must have lost this function, as well as its ability to bind PKAr* and PKIF, since PKAcR224F produces no abnormal phenotypes and has no effect on viability. On the other hand, the hypothesized nuclear role of PKAc might be catalytic if nuclear PKAc is in some way inaccessible to nuclear PKI(1-31) (Kiger, 1999 and references).

These considerations suggest that normal Hedgehog signal transduction may require both inhibition of cytoplasmic PKAc activity and export of nuclear PKAc. The Drosophila homolog of PKI(1-77) would be a good candidate for carrying out these functions. The fact that PKI(1-77) seems to play some role in regulating the cell cycle may help to explain why PKI(1-31) has different effects on endoreplicating cells and mitotic cells. Resolving the nature of the roles played by PKAc in the cytoplasm and in the nucleus may lead to simultaneous understanding of the effects seen here on pattern formation and on cell growth (Kiger, 1999 and references).

Direct comparisons of the effects of PKI(1-31) and of PKI(1-77) are needed to provide more insight into how different PKAc inhibitors are functioning. PKAc transgenes with specific catalytic site mutations should provide evidence for or against a noncatalytic nuclear role for PKAc. PKAr* transgenes with domain-specific mutations should provide insight into the role of PKAR* in Hedgehog signaling. Identification of a Drosophila homolog of PKI(1-77) and study of its regulation will be important to achieve a clear understanding of the roles of PKAc. From a practical standpoint, PKI(1-31) transgenes should provide a useful tool for investigating the role of PKA in larval growth regulation, independent of PKA's effects on pattern formation. Mutations that permit larvae to survive the effect of PKI(1-31) and develop to adults should help to identify elements controlling larval growth. Conversely, mutations that sensitize adults or embryos to PKI(1-31) may reveal elements important for pattern formation (Kiger, 1999).

Neural network computations are usually assumed to emerge from patterns of fast electrical activity. Challenging this view, this study shows that a male fly's decision to persist in mating hinges on a biochemical computation that enables processing over minutes to hours. Each neuron in a recurrent network contains slightly different internal molecular estimates of mating progress. Protein kinase A (PKA) activity contrasts this internal measurement with input from the other neurons to represent accumulated evidence that the goal of the network has been achieved. When consensus is reached, PKA pushes the network toward a large-scale and synchronized burst of calcium influx that is called an eruption. Eruptions transform continuous deliberation within the network into an all-or-nothing output, after which the male will no longer sacrifice his life to continue mating. Here, biochemical activity, invisible to most large-scale recording techniques, is the key computational currency directing behavior and motivational state (Thornquist, 2021).

The Corazonin-expressing (Crz) neurons of the male abdominal ganglion comprise an exceptionally tractable system for investigating neuronal networks and behavioral control. These four neurons drive two simultaneous and crucial events in the lives of the male and his mating partner: (1) the transfer of sperm from the male to the female and (2) a transition out of a period of insurmountably high motivation to continue mating. Both of these events occur 6 min after mating begins and are under the control of a molecular timer encoded by the slowly decaying autophosphorylation of the kinase CaMKII. This study shows that the Crz neurons use cyclic AMP (cAMP) signaling to average evidence about the passage of time across the network and generate an eruption that signals to downstream circuitry only when a consensus is reached. In addition to revealing this network phenomenon, these results explain the function of CaMKII in the only mechanism for neuronal interval timing yet to be described (Thornquist, 2021).

Decisions and behavioral control are thought to arise from long-lasting composite dynamics of neuronal networks, such as the seconds-long ramping of firing rates observed in premotor centers. This study provides mechanisms for the accumulation and storage of network information over much longer timescales, as well as insight into a thresholding mechanism for reporting the outcome. Here, the information is a spatially distributed estimate of elapsed time that emerges from interwoven biochemical and electrical processes. Cell-intrinsic evidence is read out within each neuron from the immediate activity of CaMKII, and network-level information is received from the electrical activity of other Crz neurons. The common currency is cAMP signaling, which accumulates intracellularly at the level of cAMP itself, PKA activity, and/or the accumulation of phosphate groups on the targets of PKA. A stark thresholding operation transforms the graded and distributed representation of evidence into a binary decision variable: the presence or absence of a network-wide eruption (Thornquist, 2021).

Eruption-like mechanisms seem well suited for diverse long-timescale computations. The linearity of evidence accumulation and dissipation allows undistorted evaluation, whether the system is near or far from its threshold. The magnitude of network activation distinguishes the information-gathering phase from the decisive output, allowing evidence to accumulate without triggering the downstream consequences. The accumulated signal persists even in the total absence of electrical input, enabling activity-silent memory that imparts the system with a history dependence that could not be discerned from purely electrophysiological measurements (Thornquist, 2021).

Although there has been no previous description of anything closely resembling an eruption in the nervous system, islets of pancreatic β cells synchronize their activity to coordinate insulin release using a similar positive feedback loop between electrical activity and cAMP signaling. In the brain, much of the cortex has been hypothesized to operate at near-criticality, giving rise to brief but expansive neural avalanches. Early in cortical development, nascent neuronal networks exhibit spontaneous, network-wide synchronous activation driven by positive feedback that is sustained for tens of seconds. Neurons in the primate lateral intraparietal cortex show trial-averaged ramping responses during decision-making tasks, but closer inspection shows that individual neurons jump to high activity rates as evidence accumulates. On much longer timescales, neurons in the mammalian suprachiasmatic nucleus (SCN) undergo 10-fold changes in activity depending on the time of day. In the SCN, each neuron expresses a cell-intrinsic representation of the time of day (encoded by levels of circadian clock proteins), but, as in the Crz neurons, the reliability of behavior is a network output that is much greater than would be expected from its individual oscillators (Thornquist, 2021).

This paper proposes to define an eruption as a thresholded jump in recurrent network activity, triggering downstream processes that had been blind to intranetwork computations. The eruption can be highly localized, as in the four-cell network examined in this study, and so it may have escaped detection by the population-level dimensionality reduction techniques used by most modern studies of neuronal decision making. Even in approaches that focus on individual cells, it is usually difficult or impossible to identify the network in which a given neuron functions. For example, the step changes in cortical spike rates observed during decision making in primates may indicate participation in an erupting network or reflect the enduring consequences of an upstream eruption (Thornquist, 2021).

Whether in existing datasets or from new experimental designs, it is believed eruptions are worth searching for. They may help explain the transformation of continuous brain activity into our discretized actions and experience and would therefore change our thinking about emergent brain properties. This study found that profound behavioral and motivational changes emerge from the interplay of population dynamics with molecular processing in a remarkably small group of cells. It is therefore argued that presuming neurons to be simple processing elements underestimates their computational power. It is believed that the computational capacity of individual neurons stems as much from their rich intracellular signaling pathways as from their interconnectedness, especially for computations in the regime of cellular supremacy, where biomolecular computations are proposed to be more efficient than implementations in the classic von Neumann and Turing frameworks. Studying the molecular-electrical interface in relatively simple systems such as the Crz network will provide still more guiding principles for linking the operations of neurons to our thoughts, emotions, precepts, and actions (Thornquist, 2021).

A major limitation of studying the Crz eruption has been an inability to monitor neuronal activity during mating. Nevertheless, the close fidelity between manipulations that alter the eruption in behaving animals and in ex vivo imaging preparation has allowed the authors to lay out the general structure of the Crz eruption. Still, many details remain unclear or are difficult to fully address with the data. Most prominent is the multifaceted role of calcium in this model. Calcium first activates the CaMKII timer, then drives the pre-eruption accumulation of cAMP, and finally a massive calcium influx essentially is the eruption. There are some clues as to how calcium could manage these diverse functions. Subcellular compartmentalization may explain some of this multifunctionality: the activation of CaMKII is voltage independent (Thornquist, 2020), whereas the eruption acts through VGCCs. Unlike the calcium-conducting ChR2-XXM, stimulation of the Crz neurons with the non-calcium conducting channel CsChrimson fails to activate CaMKII (Thornquist, 2020), further supporting the notion that VGCCs cannot activate CaMKII in this system. So, what does initiate the CaMKII timer? The most promising candidate may be the release of calcium from intracellular stores. The molecular screen presented here did not turn up hits in endoplasmic-reticular or mitochondrial calcium channels (or in the approximately two-thirds of the annotated GPCRs that were tested), but the screen will have missed manipulations that reduce CaMKII activation, as a premature eruption will not affect copulation duration. More directed explorations of the onset of the timer have so far failed to yield any clear leads, due either to limitations in tools or in the hypotheses (Thornquist, 2021).

Like action potentials in individual neurons, the system described in this study resets after an eruption. This is not fully understand either, although it may involve re-activation of CaMKII: elevation of intracellular calcium following strong bPAC stimulation activates CaMKII, while inhibiting CaMKII results in a high frequency of successive eruptions. A possible mechanism for CaMKII's ability to both delay and reset the eruption is indicated by work in mammalian cardiomyocytes, where CaMKII phosphorylates and activates phosphodiesterase 4D (PDE4), which degrades cAMP. In the Crz neurons, RNAi knockdown of the PDE4 homolog dunce shortens the duration of the voltage requirement, activation of CaMKII dramatically reduces baseline cAMP levels, and a potential CaMKII phosphorylation site on PDE4 seems to be conserved on dunce. However, it would not be surprising if CaMKII acts at multiple levels to inhibit cAMP signaling (although without strongly affecting baseline membrane voltage (Thornquist, 2020; Thornquist, 2021).

When CaMKII activity is low, cAMP activates PKA to drive the eruption, but what exactly does PKA do? In mammals, PKA is known to potentiate calcium influx through multiple calcium channels, suggesting a straightforward mechanism for driving the eruption that concludes the timer. However, other hits in the screen do not fit neatly into this pathway, suggesting further complexity and surprises upon deeper investigation (Thornquist, 2021).

Finally, what is the signal that is released by an eruption and how does it affect downstream neurons? The output signal is most likely different from the (also unknown) signal(s) and receptor(s) used for intranetwork recurrent excitation, and could consist of one or more neuropeptides, since they often require sustained excitation for release. The screens so far have only identified Unc13, a component of vesicle release machinery for both classical neurotransmitters and neuropeptides. The immediate downstream targets of the Crz neurons are also unknown, but ultimately the eruption drives the transfer of sperm and seminal fluid through the activation of a set of serotonergic neurons and adjusts the properties of drive-integrating neurons that control the immediate responsiveness of the male to challenges that arise during mating (Thornquist, 2021).

The cAMP effector PKA mediates Moody GPCR signaling in Drosophila blood-brain barrier formation and maturation

The blood-brain barrier (BBB) of Drosophila comprises a thin epithelial layer of subperineural glia (SPG), which ensheath the nerve cord and insulate it against the potassium-rich hemolymph by forming intercellular septate junctions (SJs). Previous work identified a novel Gi/Go protein-coupled receptor (GPCR), Moody, as a key factor in BBB formation at the embryonic stage. However, the molecular and cellular mechanisms of Moody signaling in BBB formation and maturation remain unclear. This study identified cAMP-dependent protein kinase A (PKA) as a crucial antagonistic Moody effector that is required for the formation, as well as for the continued SPG growth and BBB maintenance in the larva and adult stage. PKA is enriched at the basal side of the SPG cell, and this polarized activity of the Moody/PKA pathway finely tunes the enormous cell growth and BBB integrity. Moody/PKA signaling precisely regulates the actomyosin contractility, vesicle trafficking, and the proper SJ organization in a highly coordinated spatiotemporal manner. These effects are mediated in part by PKA's molecular targets MLCK and Rho1. Moreover, 3D reconstruction of SJ ultrastructure demonstrates that the continuity of individual SJ segments, and not their total length, is crucial for generating a proper paracellular seal. It is proposed that Moody/PKA signaling plays a central role in controlling the cell growth and maintaining BBB integrity (Li, 2021).

Previous studies implicated a novel GPCR signaling pathway in the formation of the Drosophila BBB in late embryos. This work also revealed that besides the GPCR Moody two heterotrimeric G proteins (Gαiβγ, Gαoβγ5) and the RGS Loco participate in this pathway. This study provides a comprehensive molecular and cellular analysis of the events downstream of G protein signaling using a candidate gene screening approach. New, more sensitive methods for phenotypic characterization are presented, and the analysis was extended to beyond the embryo into larval stages. This work identifies, together with some of its targets, as crucial antagonistic effectors in the continued cell growth of SPG and maintenance of the BBB sealing capacity. This role is critical to ensure proper neuronal function during BBB formation and maturation (Li, 2021).

Multiple lines of evidence demonstrate a role of PKA for proper sealing of the BBB: loss of PKA activity leads to BBB permeability defects, irregular growth of SPG during epithelium formation, reduced membrane overlap, and a narrower SJ belt at SPG cell-cell contacts. The role of PKA as an effector of the Moody signaling pathway is further supported by dominant genetic interaction experiments, which show that the dye penetration phenotype of PkaC1 heterozygous mutant embryos was partially rescued by removing one genomic copy of Gβ13F or loco. Moreover, the analysis of the larval phenotype with live SJ and cytoskeleton markers shows that PKA gain of function behaved similarly to Moody loss of function. Conversely, PKA loss of function resembled the overexpression of GαoGTP, which mimics Moody gain-of-function signaling (Li, 2021).

The results from modulating PKA activity suggest that the total cell contact and SJ areas are a major function of PKA activity: low levels of activity cause narrow contacts, and high levels give rise to broad contacts. Moreover, the analysis of various cellular markers (actin, microtubules, SJs, vesicles) indicates that the circumferential cytoskeleton and delivery of SJ components respond proportionately to PKA activity. This, in turn, promotes the changes in cell contact and junction areas coordinately at the lateral side of SPG. These experiments demonstrate that the modulation of the SPG membrane overlap by PKA proceeds, at least in part, through the regulation of actomyosin contractility, and that this involves the phosphorylation targets MLCK and Rho1. This suggests that crucial characteristics of PKA signaling are conserved across eukaryotic organisms (Li, 2021).

At the ultrastructural level, ssTEM analysis of the larval SPG epithelium clarifies the relationship between the inter-cell membrane overlaps and SJ organization and function. Across different PKA activity levels, the ratio of SJ areas to the total cell contact area remained constant at about 30%. This proportionality suggests a mechanism that couples cell contact with SJ formation. The primary role of Moody/PKA appears in this process to be the control of membrane contacting area between neighboring cells. This is consistent with the results of a temporal analysis of epithelium formation and SJ insertion in late embryos of WT and Moody pathway mutants, which shows that membrane contact precedes and is necessary for the appearance of SJs. The finding that the surface area that SJs occupy did not exceed a specific ratio, irrespective of the absolute area of cell contact, suggests an intrinsic, possibly steric limitation in how much junction can be fitted into a given cell contact space. While most phenotypic effects are indeed a major function of Moody and PKA activity, the discontinuity and shortening of individual SJ strands is not. It occurred with both increased and decreased signaling and appears to cause the leakiness of the BBB in both conditions. ssTEM-based 3D reconstruction thus demonstrates that the total area covered by SJs and the length of individual contiguous SJ segments are independent parameters. The latter appears to be critical for the paracellular seal, consistent with the idea that Moody plays a role in the formation of continuous SJ stands (Li, 2021).

The asymmetric localization of PKA that was observed sheds further light on the establishment and function of apical-basal polarity in the SPG epithelium. Prior to epithelium formation, contact with the basal lamina leads to the first sign of polarity. Moody becomes localized to the apical surface only after epithelial closure and SJ formation, suggesting that SJs are required as a diffusion barrier and that apical accumulation of Moody protein is the result of polarized exocytosis or endocytosis. This study now shows that the intracellular protein PKA catalytic subunit-PkaC1 accumulates on the basal side of SPG, and that this polarized accumulation requires (apical) Moody activity. Such an asymmetric, activity-dependent localization has not previously been described for PKA in endothelium, and while the underlying molecular mechanism is unknown, the finding underscores that generating polarized activity along the apical-basal axis of the SPG is a key element of Moody pathway function (Li, 2021).

An intriguing unresolved question is how increased SPG cell size and SJ length can keep up with the expanding brain without disrupting the BBB integrity during larva growth. The SJ was found to grow dramatically in length (0.57 ± 0.07 μm vs. 2.16 ± 0.14 μm, about 3.7-fold) from the late embryo to third instar larva, which matches the increased cell size of SPG (about fourfold). During the establishment of the SPG epithelium in the embryo, both increased and decreased Moody signaling resulted in asynchronous growth and cell contact formation along the circumference of SPG, which in turn led to irregular thickness of the SJ belt. Therefore, a similar relationship may exist during the continued growth of the SPG epithelium in larvae, with the loss of continuity of SJ segments in Moody/PKA mutants resulting from unsynchronized expansion of the cell contact area and an ensuing erratic insertion of SJ components. Since SJs form relatively static complexes, any irregularities in their delivery and insertion may linger for extended periods of time. The idea that shortened SJ segments are a secondary consequence of unsynchronized cell growth is strongly supported by the finding that disruption of actomyosin contractility in MLCK and Rho1 mutants compromises BBB permeability (Li, 2021).

Collectively, these data suggest the following model: polarized Moody/PKA signaling controls the cell growth and maintains BBB integrity during the continuous morphogenesis of the SPG secondary epithelium. On the apical side, Moody activity represses PKA activity (restricting local cAMP level within the apial-basal axis in SPG) and thereby promotes actomyosin contractility. On the basal side, which first adheres to the basal lamina and later to the PNG sheath, PKA activity suppresses actomyosin contractility via MLCK and Rho1 phosphorylation and repression. Throughout development, the SPG grow continuously while extending both their cell surface and expanding their cell contacts. The data suggest that the membrane extension occurs on the basolateral surface through insertion of plasma membrane and cell-adhesive proteins, with similar behavior in epithelial cell, but regulated by a distinct polarized Moody/PKA signaling in SPG. In analogy to motile cells, the basal side of the SPG would thus act as the 'leading edge' of the cell, while the apical side functions as the 'contractile rear'. According to this model, Moody/Rho1 regulate actomyosin to generate the contractile forces at the apical side to driving membrane contraction, which directs the basolateral insertion of new membrane material and SJs. In this way, differential contractility and membrane insertion act as a conveyor belt to move new formed membrane contacts and SJ from the basolateral to apical side. Loss of Moody signaling leads to symmetrical localization of PKA and to larger cell contact areas between SPG due to diminished apical constriction. Conversely, loss of PKA causes smaller cell contact areas due to increased basal constriction (Li, 2021).

These results may have important implications for the neuron-glia interaction in the nervous system and the development and maintenance of the BBB in vertebrates. SJs have several structural and functional components in common with paranodal junctions, which join myelinating glial cells to axons in the vertebrate nervous system, and they share similar regulation mechanisms. The vertebrate BBB consists of a secondary epithelium with interdigitations similar to the ones between the Drosophila SPG . While the sealing is performed by TJs, it will be interesting to investigate whether there are similarities in the underlying molecular and cellular mechanisms that mediate BBB function (Li, 2021).

Temporally and Spatially Localized PKA Activity within Learning and Memory Circuitry Regulated by Network Feedback

Dynamic functional connectivity within brain circuits requires coordination of intercellular signaling and intracellular signal transduction. Critical roles for cAMP-dependent protein kinase A (PKA) signaling are well established in the Drosophila mushroom body (MB) learning and memory circuitry, but local PKA activity within this well-mapped neuronal network is uncharacterized. This study use an in vivo PKA activity sensor (PKA-SPARK) to test spatiotemporal regulatory requirements in the MB axon lobes. Immature animals had little detectable PKA activity, whereas postcritical period adults showed high field-selective activation primarily in just 3/16 defined output regions. In addition to the age-dependent PKA activity in distinct α'/β' lobe nodes, females show sex-dependent elevation compared with males in these same restricted regions. Loss of neural cell body Fragile X mental retardation protein (FMRP) and Rugose [human Neurobeachin (NBEA)] suppresses localized PKA activity, whereas overexpression (OE) of MB lobe PKA-synergist Meng-Po (human SBK1) promotes PKA activity. Elevated Meng-Po subverts the PKA age-dependence, with elevated activity in immature animals, and spatial-restriction, with striking γ lobe activity. Testing circuit signaling requirements with temperature-sensitive shibire (human Dynamin) blockade, broadly expanded PKA activity was found within the MB lobes. Using transgenic tetanus toxin to block MB synaptic output, greatly heightened PKA activity was found in virtually all MB lobe fields, although the age-dependence is maintained. It is concluded spatiotemporally restricted PKA activity signaling within this well-mapped learning/memory circuit is age-dependent and sex-dependent, driven by FMRP-Rugose pathway activation, temporally promoted by Meng-Po kinase function, and restricted by output neurotransmission providing network feedback (Sears, 2022).

This study explored the spatiotemporal regulation of PKA activity within the MB lobes. PKA signaling initiates in early adulthood, with heightened activity in just 3/16 MB lobe output neuron fields (α'1, β'1, and β'2ap). In addition to age-dependence, this regional PKA signaling displays sex-dependence, with elevation in females over males. These findings were made possible with the PKA-SPARK biosensor; this fluorescent reporter uses motifs found in earlier PKA sensors, with pharmacological and genetic approaches promoting and preventing PKA activity verifying this new tool in cell culture and in vivo. While it is possible that the PKA-SPARK reporter is revealing only the strongest PKA activity, at the least α'1, β'1, and β'2ap connectivity regions have much higher PKA activity levels compared with the rest of the MB neuropil. In two disease models of intellectual disability and ASDs, from loss of either FMRP or Rugose/NBEA, PKA activity remains spatiotemporally restricted, but is dramatically reduced. There is surprisingly little change from overactivation of the FMRP→Rg control pathway, but PKA activity is profoundly altered by the PKA pathway Meng-Po kinase, with OE enhancing spatiotemporal PKA signaling and loss suppressing PKA activity. Network feedback downstream of KC neurotransmission strongly suppresses PKA activity, since blocking KC synaptic output with conditional shibire^ts or transgenic TNT induces widespread PKA activity signaling. Thus, localized PKA activity is highly regulated at the circuit level (Sears, 2022).

At a macro level, the α'1 and β'1 regions with the highest localized PKA activity levels have been linked to valence, i.e., whether local activation causes animals to approach or avoid a stimulus. The α'1 and β'2 fields exhibit opposing output valence (positive and negative, respectively), while the β'1 role appears less clear. However, higher PKA signaling can be activated in the γ3 region (via Meng-Po), which in combination with the β'1 region drives positive valence. Moreover, dopaminergic and serotonergic biosensor signals function through the β' region from a variety of external sense stimuli, to which the α'1/β'1 fields display high sensitivity, suggesting broad responsiveness. Very recent work shows high spontaneous activity in α'/β' restricted to young animals , suggesting α'/β' PKA signaling may be controlled by selective developmental activity. Moreover, inhibiting miR-92a in α/β and γ, but not α'/β', was shown to enhance memory, indicating another layer of lobe-selective circuit regulation. Note that the output neurons from these MB lobe regions are different (α'1 cholinergic, β'1 GABAergic, and β'2 glutamatergic), suggesting more complex integrative circuit functions. PKA signaling activation after the early-use critical period is consistent with sensory experience-dependent regulation. Importantly, MB sensory integration functions differ markedly between females and males, correlating with the report in this study of sex-dependent PKA signaling differences in females compared with males (Sears, 2022).

Two learning and memory proteins, FMRP and Rugose, are needed for full PKA activity in the α'1 and β'1 MB lobe output neuron fields. RNA-binding FMRP is a translational regulator known to facilitate PKA signaling, which is lost in the FXS, the commonest heritable cause of intellectual disability and ASD. Rugose/NBEA is a PKA-anchor that facilitates learning and memory, and is also associated with ASDs. Previous work has shown FMRP binds to rugose mRNA to drive KC expression. As predicted, disruption of this FMRP→Rg regulative pathway strongly impairs PKA activity in the MB lobes. In contrast, localized PKA signaling is dramatically strengthened by MB OE of the Meng-Po kinase, which induces early-onset PKA activity before adult sensory experience, spatially expands high PKA activity to the γ3 MBON field, and profoundly elevates PKA activity within all the normal MB lobe regions of heightened PKA signaling. Consistently, Meng-Po kinase OE also greatly improves learning and memory, via PKA phosphorylation, but additionally via signaling feedback synergy. Moreover, meng-po RNAi causes the opposite result of reducing localized PKA-SPARK puncta. Based on both loss and gain of function, it is suggested that Meng-Po enhances localized PKA activity, reflecting circuit level kinase regulation. Determining how Meng-Po-regulated PKA activity determines circuit excitability and regional balance will be a major subject of future research (Sears, 2022).

Two different KC synaptic output blocking methods dramatically expand PKA activity signaling in the MB. Both conditional shibire^ts and transgenic tetanus toxin tools block KC neurotransmission, but through quite different mechanisms. At 33°C, KC-targeted shibire^ts drives PKA activity expansion in the MB γ lobe. This change could indicate cross-compartment network interactions between the γ lobe and other MB regions. Increasing γ1 PKA activity is especially interesting, as γ1 toggles inhibition of other MB regions. The tetanus toxin protease blocks neurotransmission through eliminating SV exocytosis, and therefore provides a stronger and more selective means to silence KC synaptic output. Consistently, TNT animals show a more profound expansion of PKA activity throughout the MB lobes, albeit again affecting only spatial and not temporal patterning. Neither shibire^ts shibire^ts nor TNT blockade alters early PKA activity, suggesting induction of PKA signaling is determined primarily by later experience-driven activity. Localized PKA activity changes with KC output block implies active circuit balance; for example, weighing aversive versus attractive responses to sensory stimuli. The widespread PKA activity upregulation with KC output block leads to a hypothesis that enhancing KC neuron activity should result in elevated PKA signaling (Sears, 2022).

At the MB circuit level, multiple candidate synaptic pathways need to be explored for roles in local PKA activity regulation in different MB lobe output neuron fields. GABAergic inputs to dopaminergic neurons are one likely candidate, since GABA treatment has been shown to correct dfmr1 mutant circuit defects exacerbated by glutamate exposure. Moreover, GABAergic anterior paired lateral (APL) neurons broadly control MB activity through feedback to the KCs, and are most strongly activated by the α'/β' lobes. Recent work shows that treatment with a dopamine transport inhibitor also ameliorates rugose mutant social interaction and memory deficits. Another candidate is the Amnesiac neuropeptide from the serotonergic dorsal paired medial neurons, required for their normal development in the broad innervation of the MB lobes. In the context of these studies, de facto depression may feedback onto KCs to promote PKA activity signaling. The impact of upstream input onto the MB lobes is an important consideration, including how this circuitry combines with spontaneous MB activity and internal lobe circuitry to determine PKA signaling. Future research directions should attempt to dissect how these different layers of neuromodulation control localized PKA activity signaling within the MB lobe circuit, and between females and males, by manipulating input-specific neuronal activity in targeted transgenic studies (Sears, 2022).

In conclusion, this study reports that PKA activity signaling in the Drosophila brain MB learning and memory center is highly induced during early experiential adulthood, with selective upregulation in the α'1, β'1, and β'2ap MB lobe output neuron regions. Age-specific and sex-specific PKA signaling controlled within KCs and downstream of KC output shows that spatiotemporally restricted MB lobe PKA activity is regulated through a combination of both intracellular control and intercellular network-level mechanisms. Importantly, PKA signaling can be precociously promoted and spatial expanded though the activity of the PKA-synergist Meng-Po kinase. Moreover, KC neurotransmission inhibits localized PKA signaling within the MB circuit. Future studies will be aimed toward generating new genetic responder tools to test KC signaling with both neurotransmission output blockade and activity promotion of upstream and downstream MB circuit components, simultaneously and independently of KCs. PKA activity signaling will be tested with the manipulation of specific KC partners, by altering neurotransmission signaling in combination with postsynaptic neurotransmitter receptor mutants to determine network communication cues. Taken together with the current work, these ongoing studies will continue to expand understanding of circuit-level PKA signaling regulation in normal function and in neurologic disease model contexts (Sears, 2022).

Dual PDF Signaling Pathways Reset Clocks Via TIMELESS and Acutely Excite Target Neurons to Control Circadian Behavior

Molecular circadian clocks are interconnected via neural networks. In Drosophila, Pigment-Dispersing Factor (PDF) acts as a master network regulator with dual functions in synchronizing molecular oscillations between disparate PDF⁺ and PDF^- circadian pacemaker neurons and controlling pacemaker neuron output. Yet the mechanisms by which PDF functions are not clear. This study has demonstrated that genetic inhibition of protein kinase A (PKA) in PDF^- clock neurons can phenocopy PDF mutants, while activated PKA can partially rescue PDF receptor mutants. PKA subunit transcripts are also under clock control in non-PDF DN1p neurons. To address the core clock target of PDF, per was rescued in PDF neurons of arrhythmic per⁰¹ mutants. PDF neuron rescue induced high amplitude rhythms in the clock component Timeless (Tim) in per-less DN1p neurons. Complete loss of PDF or PKA inhibition also results in reduced Tim levels in non-PDF neurons of per⁰¹ flies. To address how PDF impacts pacemaker neuron output, PDF was focally applied to DN1p neurons and was found to acutely depolarize and increase firing rates of DN1p neurons. Surprisingly, these effects are reduced in the presence of an adenylate cyclase inhibitor, yet persist in the presence of PKA inhibition. Evidence is provided for a signaling mechanism (PKA) and a molecular target (Tim) by which PDF resets and synchronizes clocks; PDF exhibits an acute direct excitatory effect on target neurons to control neuronal output. The identification of Tim as a target of PDF signaling suggests it is a multimodal integrator of cell autonomous clock, environmental light, and neural network signaling. Moreover, these data reveal a bifurcation of PKA-dependent clock effects and PKA-independent output effects. Taken together, these results provide a molecular and cellular basis for the dual functions of PDF in clock resetting and pacemaker output (Seluzicki, 2014).

Suppression of inhibitory GABAergic transmission by cAMP signaling pathway: alterations in learning and memory mutants

The cAMP signaling pathway mediates synaptic plasticity and is essential for memory formation in both vertebrates and invertebrates. In the fruit fly Drosophila melanogaster, mutations in the cAMP pathway lead to impaired olfactory learning. These mutant genes are preferentially expressed in the mushroom body (MB), an anatomical structure essential for learning. While cAMP-mediated synaptic plasticity is known to be involved in facilitation at the excitatory synapses, little is known about its function in GABAergic synaptic plasticity and learning. Using whole-cell patch-clamp techniques on Drosophila primary neuronal cultures, this study demonstrates that focal application of an adenylate cyclase activator forskolin (FSK) suppressed inhibitory GABAergic postsynaptic currents (IPSCs). A dual regulatory role of FSK on GABAergic transmission was observed, where it increases overall excitability at GABAergic synapses, while simultaneously acting on postsynaptic GABA receptors to suppress GABAergic IPSCs. Further, it was shown that cAMP decreased GABAergic IPSCs in a PKA-dependent manner through a postsynaptic mechanism. PKA acts through the modulation of ionotropic GABA receptor sensitivity to the neurotransmitter GABA. This regulation of GABAergic IPSCs is altered in the cAMP pathway and short-term memory mutants dunce and rutabaga, with both showing altered GABA receptor sensitivity. Interestingly, this effect is also conserved in the MB neurons of both these mutants. Thus, this study suggests that alterations in cAMP-mediated GABAergic plasticity, particularly in the MB neurons of cAMP mutants, account for their defects in olfactory learning (Ganguly, 2013).

Ca2+/CaM dependent adenylate cyclase (AC) produces cAMP and is also known to function as a co-incidence detector during learning in both Drosophila and Aplysia. In addition, AC-dependent cAMP activation changes the strength of Drosophila excitatory synapses which may be the cellular mechanism underlying learning and memory. Although inhibitory synaptic transmission is equally important for proper neuronal communication, the effects of cAMP at the inhibitory GABAergic synapses have remained unexplored. This study shows that forskolin (FSK), an activator of cAMP, suppresses the frequency of inhibitory GABAergic IPSCs in Drosophila primary neuronal cultures. A concentration dependent effect of FSK on GABAergic IPSCs was observed in the same physiological range as described in recent imaging studies in intact fly brains. Further cAMP was shown to decrease GABAergic IPSCs in a PKA-dependent manner through a postsynaptic mechanism (Ganguly, 2013).

Sparsening of odor representation through GABAergic inhibition in the mushroom body (MB) neurons is thought to be a possible mechanism for information storage in locusts (Perez-Orive, 2002). GABAergic local neurons are known to be involved in olfactory information processing in Drosophila (Wilson, 2005; Olsen, 2008) indicating that GABAergic transmission plays a crucial role in shaping odor response. The MB shows extensive GABAergic innervation in both locusts (Perez-Orive., 2002) and Drosophila (Yasuyama, 2002). This, along with the observation that cAMP pathway genes like dunce and rutabaga are preferentially expressed in the MB (Davis, 2011), indicates that cAMP mediated GABAergic plasticity may be important for learning in Drosophila. Consistent with this hypothesis, this study observed altered cAMP mediated GABAergic IPSCs in the cAMP mutants dnc1 and rut1. The effect of cAMP on suppression of GABAergic currents was less pronounced in the mutants. This suggests that the altered inhibition contributes to their observed learning defects. In fact, recent studies have shown that GABAA RDL receptors expressed in the MB and GABAergic neurons projecting to the MB are essential for olfactory learning (Liu, 2007; Liu, 2009). It is thus possible that altered cAMP mediated GABAergic plasticity at the MB neurons may account for some forms of the learning defects in Drosophila (Ganguly, 2013).

GABAergic IPSCs are known to act through picrotoxin-sensitive postsynaptic GABA receptors in both Drosophila embryonic and pupal neuronal cultures. This study observed that the suppression of GABAergic IPSCs by FSK is completely abolished in the presence of a membrane impermeable PKA inhibitor restricted to the postsynaptic neuron. This indicates that PKA may modulate GABAergic IPSCs by regulating GABA receptor sensitivity by phosphorylation, similar to what has been suggested in the mammalian hippocampus (Ganguly, 2013).

There are three known ionotrophic GABA receptor gene homologs in Drosophila - RDL, LCCH3 and GRD. Amongst them, the GABA RDL subunit is widely expressed in several regions of the Drosophila brain and its expression in the MB is inversely correlated to olfactory learning. Therefore, RDL-containing GABA receptors may play an important role in cAMP-dependent synaptic plasticity and thus be involved in learning and memory. The data suggests that the majority of synaptic GABA receptors contain the RDL subunit while a small fraction of synaptic GABA receptors lack RDL, providing evidence of heterogeneous synaptic GABA receptors in Drosophila for the first time. However, it is still not known what particular subunit of GABA receptors is involved in regulation of cAMP-dependent GABAergic plasticity. Based on the observation that RDL containing GABA receptors mediate the majority of GABAergic IPSCs in Drosophila primary neuronal cultures, the action of FSK on GABAergic IPSCs is probably through the GABA RDL subunit. While the detailed molecular mechanism remains to be explored, it is proposed that PKA-mediated phosphorylation of RDL subunits and subsequent GABA receptor internalization may occur in the postsynaptic region. In this scenario, the only functional synaptic GABA receptors will be those lacking the RDL subunit at the postsynaptic regions. This will account for a decrease in mIPSC frequency with response to FSK, while leaving mIPSC amplitude almost unchanged (Ganguly, 2013).

Although GABA receptor subunits would be the target of cAMP-PKA signaling the possibility that other molecules can be phosphorylated and then indirectly regulate GABA receptor subunits can still not be rule out. Future work using heterologous expression systems for GABA subunits will help to determine whether GABA receptors are directly phosphorylated (Ganguly, 2013).

Recordings from embryonic and pupal MB neurons of both dunce and rutabaga mutants show a defect in ionotropic GABA receptor response in the presence of FSK. Interestingly, this response to FSK is similar in both the mutants despite their contrasting levels of cellular cAMP. Recent imaging studies in the rutabaga mutant have shown that AC is required for co-incidence detection in the MB neurons. FSK application also fails to increase PKA to wild-type levels in the MB neurons of rutabaga. Thus in the current experiments, the changes in receptor response in rutabaga can be explained by a lack of increase in cAMP/PKA levels due to defects in FSK-mediated AC activation (Ganguly, 2013).

The dunce mutants with high levels of cAMP also show defects in short-term memory due to alterations in the spatiotemporal restriction of dunce phosphodiesterase to the Drosophila MB. Further, the dunce MB neurons show an increase in PKA levels on FSK application similar to the wild-type strains. These findings suggest that FSK-mediated inhibition of GABA receptor should be greater in dunce neurons. However, in the current results the dunce and rutabaga mutants, despite having opposing effects on cellular cAMP levels, showed very similar FSK mediated effects on GABAergic IPSCs. Several other studies have also shown that dunce and rutabaga have similar defects in growth cone motility, excitatory synaptic plasticity and more importantly, short-term memory. Even though the effect of FSK on GABAergic IPSCs in dunce and rutabaga mutants is similar, it is very likely that the molecular mechanisms underlying these responses differ in the two mutants. It has been shown that elevated cAMP signaling reduces phosphorylation in rat kidney cells through activation of protein phosphatase 2A. In addition, increased PKA activity in mouse hippocampus hyper-phosphorylates several downstream molecular targets including a tyrosine phosphatase (STEP), correlates with decreased phosphodiesterase protein (PDE4) levels and results in memory defects. Therefore, it is tempting to speculate that high levels of cAMP due to the dunce mutation leads to the activation of phosphatase(s) and thus reduces the effects of FSK as seen in the current study. Taken together, all these findings strongly suggest that the disruption of cellular cAMP homeostasis can alter inhibitory GABAergic synaptic plasticity and hence cause defects in olfactory learning, although the underlying mechanisms leading to this effect can be different (e.g. reduced PKA activity in rut1 versus increased phosphatase activity in dnc1) (Ganguly, 2013).

Strengthening in the efficacy of excitatory transmission underlies enhanced synaptic plasticity such as hippocampal long-term potentiation (LTP) and facilitation (LTF) in Aplysia. It is thus possible that the suppression of inhibitory transmission by a common second messenger like cAMP, which can enhance excitatory synaptic transmission, may lead to synaptic strengthening. Previous work has shown that the cAMP activator FSK increases excitability at the cholinergic synapses in Drosophila primary neuronal cultures. However the effect of FSK on other synapses like the GABAergic synapses has not been explored. This study shows that FSK elevates overall cellular excitability at GABAergic synapses as demonstrated by the increase in spontaneous AP frequency. Moreover, when PKA in the postsynaptic neuron is completely blocked by an inhibitor, an increase is seen in the frequency of GABAergic IPSCs. Together with previous studies on cholinergic synapses (Yuan, 2007), the current results indicate that FSK/cAMP act as common molecules regulating globally presynaptic excitability at both the cholinergic as well as GABAergic synapses. It is also noted that FSK inhibits the response of postsynaptic GABA receptors in a specific manner leading to a decrease in GABAergic synaptic strength. These studies demonstrate a novel dual regulatory role of cAMP by showing that it increases overall presynaptic function on one hand; and, acts specifically on postsynaptic GABA receptors to decrease GABAergic plasticity on the other. This action of cAMP could result in global increases in excitability and learning (Ganguly, 2013).

Null EPAC mutants reveal a sequential order of versatile cAMP effects during Drosophila aversive odor learning

This study defines a role of the cAMP intermediate EPAC in Drosophila aversive odor learning by means of null epac mutants. Complementation analysis revealed that EPAC acts downstream from the rutabaga adenylyl cyclase and in parallel to protein kinase A. By means of targeted knockdown and genetic rescue, mushroom body Kenyon cells (KCs) were identified as a necessary and sufficient site of EPAC action. Mechanistic insights were provided by analyzing acquisition dynamics and using the 'performance increment' as a means to access the trial-based sequential organization of odor learning. Thereby it was shown that versatile cAMP-dependent mechanisms are engaged within a sequential order that correlate to individual trials of the training session (Richlitzki, 2017).

The cAMP signaling pathway is central to the regulation of plasticity and can mediate cellular responses via different intermediaries, i.e., PKA (protein kinase A), EPACs (exchange proteins directly activated by cAMP), and CNGs (cyclic nucleotide gated channels). While the numerous contributions of PKA to the regulation of plasticity have been described in great detail, the role of EPAC was not recognized until 1998. Since then, its operation as a noncanonical cAMP sensor has been proven in numerous studies, aided by the development of selective cAMP analogs and/or genetic models that allow discrimination between PKA and EPAC functions. Epac has been shown to enhance neurotransmitter release, activate neuronal excitability via Ca2+-dependent K+-channels, and enhance hippocampal long-term potentiation and memory consolidation. This study investigated a potential role of Epac in the Drosophila aversive odor-learning paradigm (Richlitzki, 2017).

This study has distinguish epac-dependent from epac-independent learning by means of a Drosophila null-epac mutant and used the performance increment as a means to address the functional disparity of individual training trials. Disparity impacts on the learning rate suggesting an evolutionary benefit of alternative cAMP mediators as this provides a mechanism for transforming rut-derived cAMP signals into behavioral output following different strategies (Richlitzki, 2017).

Rutabaga adenylyl cyclase (rut-AC1) is supposed to act as coincidence detector between US- and CS-derived impulses that converge at the level of KC synapses and consequently induce cAMP-dependent plasticity within an odor specific matrix of KCs. This KC synapse matrix is widely accepted as a neuronal representation of learning and thought of as an engram of an odor memory. Dopaminergic neurons (DANs) provide the major share of KCs' dopamine-induced cAMP signals, however KC synapses are not uniform but exhibit different sensitivities for cAMP. Likewise, learning is not an instantaneous process but develops over the multiple trials of a training session. As a consequence, cAMP gain over the time course of training would not be uniform but effectively determined by the sensitivity of a particular KC synapse within the odor specific matrix. This disparity would proceed to the next levels, i.e., activation gain of alternative downstream cAMP intermediaries that, in turn, are unequally sensitive as reflected by characteristic half-maximal cAMP concentrations (IC50), i.e., IC50 values for PKA ~80 nm; for HCN channels ~ 100 nm; and for EPAC ~ 800 nm. By this design, KCs diversify into high and low cAMP gain fractions that activate mediators responsive to either high and/or low cAMP amplitudes at quite different rates. Thereby, a particular odor-specific KC matrix can interconnect to multiple outputs via different mechanisms of cAMP-dependent plasticity as its synaptic elements exhibit disparate hysteresis, i.e., different time-dependent changes in the cellular cAMP levels (Richlitzki, 2017).

The surprising result that insensitive EPAC recruits early, i.e., during the second trial of the training session, suggests existence of a fast, high-amplitude cAMP signal that meets EPAC's thresholds early during training. Similarly, one has to assume that the efficacy of later training trials is mediated by a perpetuating low-amplitude signal that develops with low gain over multiple trials. Given the fact that PKA acts isogenic to EPAC and null epac mutants show regular learning during late training trials-that is from the third trial onward-neither of these intermediates is likely to account for late trial learning. One plausible mechanism would be HCN channels, i.e., subthreshold, voltage-gated ion channels that reduce membrane resistance and promote neuronal firing probability. Within sparsely firing KCs such channels might stabilize an odor-specific synaptic matrix, i.e., the CS-representation, over the 1-min time course of the training cycle and promote its transfer to mushroom body output neurons (MBONs), the recognized convergence site for KCs. In contrast, epac has been shown to enhance neurotransmitter release and/or activated neuronal excitability via different mechanisms (Richlitzki, 2017).

While epac-dependent and independent learning mechanisms clearly dissociate at the molecular level, their learning rates appear counter-intuitive as a high-amplitude signal precedes a low-amplitude one, but both originate from rut-AC1. If one considers the animal's need to trade off certainty of a prediction against its computation time, this seemingly counterintuitive design appears deliberate and beneficial as it holds the possibility of combining both contrarian needs (DasGupta, 2014): First, epac-dependent learning requires a short computation time, i.e., after two trials, compared with the slow and cumbersome integration over multiple trials needed for epac-independent learning. Second, epac-dependent learning is restricted to salient conditions, i.e., high voltages that represent a serious threat to the animal's health, while this part of the animal's memory is spared with the 15 V DC US. In fact, wild-type strains trained with 15 V exhibited similar learning to epac-null mutants trained with 120 V suggesting that epac amplifies conditioned avoidance under trusted environmental circumstances (Richlitzki, 2017).

Do training trials clock recurrent computation within the learning network? How individual training trials are represented within the fly brain is unclear. However, functional studies have identified DANs as critical substrates of the US that tightly innervate KCs at the level of the MBs. In general, DANs and KCs work together with MBONs, the recognized readout routes of aversive odor memory. Moreover, their anatomy suggests that MBONs serve as critical inter-loops that reiterate the MBs' computational output to DANs, i.e., its major modulatory input: This recurrent connectivity exhibits a remarkable zonal architecture as dendrites of MBONs tile the length of KC axons in a nonoverlapping manner, where they meet with dopaminergic neurons (DANs), the other main innervation of the MB lobes. The DANs tile the MB lobes in a corresponding manner so that the dendrites of a particular MBON meet axonal projections of cognate DANs. Moreover, dendrites of DANs overlap with MBON axons within the MBON projection zones outside the MBs suggesting that MBONs modulate the activity of DANs and thereby generate recurrent loops. By this design MBON activity is dually modulated by DANs, first via a direct connection, and second via a KC detour that undergoes cAMP-dependent plasticity. However, further research will be required to understand the rules by which repetitive trials clock the computation within the DAN/KC/MBON network (Richlitzki, 2017).

A kinase-dependent feedforward loop affects CREBB stability and long term memory formation

In Drosophila, long-term memory (LTM) requires the cAMP-dependent transcription factor CREBB, expressed in the mushroom bodies (MB) and phosphorylated by PKA. To identify other kinases required for memory formation, Trojan exons encoding T2A-GAL4 were integrated into genes encoding putative kinases and genes expressed in MB were selected for. These lines were screened for learning/memory deficits using UAS-RNAi knockdown based on an olfactory aversive conditioning assay. A novel, conserved kinase, Meng-Po (MP, CG11221, SBK1 in human) was identified; loss severely affects 3 hr memory and 24 hr LTM, but not learning. Remarkably, memory is lost upon removal of the MP protein in adult MB but restored upon its reintroduction. Overexpression of MP in MB significantly increases LTM in wild-type flies showing that MP is a limiting factor for LTM. PKA phosphorylates MP and both proteins synergize in a feedforward loop to control CREBB levels and LTM (Lee, 2018).

Using MiMIC technology, 27 genes encoding putative protein kinases were converted with the Trojan T2A-GAL4 exon, and an image screen was performed for genes expressed in MBs. This tagging approach is especially useful for genes that are expressed at low levels in the CNS. By tagging the proteins with GFP, a conditional and reversible knockdown can be achieved in almost any tissue or cell. This allowed identification of a novel serine/threonine protein kinase, Meng-Po (MP), that is a critical player in LTM formation in Drosophila. MP is a homologue of SBK1 in mammals, a gene that is expressed in the hippocampus and the cortex. Loss of this gene in mice is associated with embryonic lethality, whereas in flies, loss of MP leads to a reduction in viability as well as sterility (Lee, 2018).

The data show that CREBB stability is highly susceptible to loss of MP. CREBB activity is modulated by phosphorylation via PKA and CamKII in Drosophila. Although the findings indicate that MP kinase activity is critical for maintaining CREBB levels and that MP kinase activity acts in synergy with PKA, it has not been possible to demonstrate that CREBB is a direct target of MP. However, some kinases require a previously phosphorylated residue as part of their recognition sequence, and various kinases were not mixed with MP in in vitro assays. Hence, it remains to be established how CREBB is degraded in the absence of MP (Lee, 2018).

A reduction in CREB levels has been shown to be associated with an age-dependent memory loss in rodents. Interestingly, delivery of CREB protein in the hippocampus using somatic cell transfer attenuated LTM impairement. However, no gene has so far been shown to affect CREBB stability in vivo and the current findings that MP, together with PKA, synergize to dramatically affect CREBB levels via a feedforward loop, reveal another mechanism to control CREBB levels during memory formation. This model is supported by the observation that overexpression of MP increases CREBB activity and promotes memory formation, suggesting that it is a central player in LTM (Lee, 2018).

Separate roles of PKA and EPAC in renal function unraveled by the optogenetic control of cAMP levels in vivo

Cyclic AMP (cAMP) is a ubiquitous second messenger that regulates a variety of essential processes in diverse cell types, functioning via cAMP-dependent effectors such as protein kinase A (PKA) and/or exchange proteins directly activated by cAMP (EPAC). In an intact tissue it is difficult to separate the contribution of each cAMP effector in a particular cell type using genetic or pharmacological approaches alone. This study, therefore, utilized optogenetics to overcome the difficulties associated with examining a multicellular tissue. The transgenic photoactive adenylyl cyclase bPAC can be activated to rapidly and reversibly generate cAMP pulses in a cell-type-specific manner. This optogenetic approach to cAMP manipulation was validated in vivo using GAL4-driven UAS-bPAC in a simple epithelium, the Drosophila renal (Malpighian) tubules. As bPAC was expressed under the control of cell-type-specific promoters, each cAMP signal could be directed to either the stellate or principal cells, the two major cell types of the Drosophila renal tubule. By combining the bPAC transgene with genetic and pharmacological manipulation of either PKA or EPAC it was possible to investigate the functional impact of PKA and EPAC independently of each other. The results of this investigation suggest that both PKA and EPAC are involved in cAMP sensing, but are engaged in very different downstream physiological functions in each cell type: PKA is necessary for basal secretion in principal cells only, and for stimulated fluid secretion in stellate cells only. By contrast, EPAC is important in stimulated fluid secretion in both cell types. It is proposed that such optogenetic control of cellular cAMP levels can be applied to other systems, for example the heart or the central nervous system, to investigate the physiological impact of cAMP-dependent signaling pathways with unprecedented precision (Efetova, 2013).

This study has pioneered the use of bPAC, a photoactive adenylyl cyclase, as an optogenetic tool to distinguish between the functions of alternative cAMP effectors in the regulation of a physiological process in vivo. To validate bPAC as an in vivo tool Drosophila renal tubules were used to confirm the bPAC transgene could be stimulated with blue light to generate cAMP signals in a cell-type-specific manner. This optogenetic approach was combined with standard techniques that targeted PKA or EPAC to resolve the complex regulatory network of discrete cAMP pathways involved in the control of fluid secretion (Efetova, 2013).

Primary urine is generated within the main segment of the Malpighian tubules, where the principal cells establish an electrochemical gradient that provides the driving force for fluid secretion, by actively transporting potassium from the basolateral to the apical surface via a defined array of ion transporters. In parallel, the stellate cells control the anion shunt conductance and water flux of the tubules, via the action of tightly regulated aquaporins and chloride channels (Efetova, 2013).

As revealed by the current analysis, two distinct cAMP pathways are deployed within the principal cells to sustain fluid secretion: firstly, the basal principal cell PKA pathway, which regulates the rate of basal fluid secretion; and secondly the stimulatory principal cell EPAC pathway, which stimulates fluid secretion above basal levels in a cAMP-dependent manner. Manipulation of EPAC activity altered stimulated secretion but not basal secretion, and manipulation of PKA altered basal secretion but not stimulated secretion. In this respect, the two principal cell secretory control pathways appear to be independent of one another (Efetova, 2013).

Could these downstream pathways be controlled independently in vivo, through a single second messenger? While imposed cAMP signals feeding into each pathway could be generated by activation of the bPAC transgene with a defined light intensity, in vivo the neuropeptides DH44, related to corticotropin releasing factor (CRF), and DH31, related to calcitonin/calcitonin gene-related peptide (CGRP), both increase fluid secretion by raising cAMP in the principal cells. However, there is evidence in other insects that these two neuropeptides might have distinct downstream effects; in the related malarial mosquito Anopheles gambiae, DH31, but not DH44, acts as a natriuretic peptide by increasing basolateral Na+ conductance. Moreover, DH31 and DH44 have an additive stimulatory effect on fluid secretion, suggesting that they target different transport processes. Cellular association of specific GPCRs with either PKA or EPAC might well account for the different outputs observed from each GPCR. Another tempting possibility involves a class of soluble adenylyl cyclases (sACs) that are localized near the apical membrane and activated by cellular ionic concentrations rather than GPCRs, as seen in the mammalian kidney (Efetova, 2013).

The apical plasma membrane H+ V-ATPase is the driving force for ion transport in the principal cells, and is therefore an obvious downstream target for stimulatory or inhibitory cAMP signals. Formation of a functional V-ATPase complex requires PKA-dependent phosphorylation, which prevents the complex from disassembly. In blowfly salivary gland (another insect epithelium energized by a V-ATPase), cAMP has been shown to promote assembly of the V-ATPase complex. However, V-ATPase assembly - and thus activation - has also been reported via EPAC signaling within the rat renal collecting duct. By contrast, intracellular calcium has been shown to activate tubule H+ V-ATPase by directly activating mitochondria, and so increasing the ATP supply. In this complex field, optogenetic control of cellular cAMP levels in the principal cells will provide a valuable analytical tool to investigate such issues (Efetova, 2013).

A surprising feature of cAMP-dependent fluid secretion is the complete inhibition (below basal) observed with millimolar levels of cell-permeable 8-Br-cAMP, or at very high illumination levels in bPAC-transgenic tubules. Through targeted use of bPAC this effect was localized to the principal cells, and formally established an inhibitory principal cell cAMP signal. It is likely that these manipulations bring intracellular cAMP levels to abnormally high levels that are unlikely to be reached in vivo, where the resting intracellular cAMP concentration is typically in the range 0.1-1.5 μM; nonetheless, there is a real effect to be explained. At present, it is only possible to speculate on the underlying mechanisms, but it is likely that saturation or desensitization of some component of the signaling pathway is occurring; or that there is cross-talk to, for example calcium signaling via cyclic nucleotide gated calcium channels, which are known to play a role in tubule (Efetova, 2013).

In the stellate cells this study identified a stimulatory stellate cAMP signal that stimulates fluid secretion via PKA, with moderate illuminations of bPAC. In contrast, high illuminations return fluid secretion to the baseline level, suggesting that dual modulation, i.e., augmentation with low levels and inhibition with high levels of cAMP, is a common theme within the stellate and principal cells. However, further experiments will be required to substantiate this speculation (Efetova, 2013).

Interestingly, the stellate cells are known to be controlled by leucokinin, which acts though calcium, rather than cAMP, so no extracellular ligand for the stellate cAMP pathway is presently known. Tyramine has also been shown to act on stellate cells, but its second messenger is yet to be established (Efetova, 2013).

Selective elevation of cAMP in stellate cells shows that both PKA and EPAC can stimulate fluid secretion. However, these pathways do not act in parallel in the stellate cells; PKA must be upstream of EPAC, because RNAi knockdown of DC0 in stellate cells abolishes the ability of bPAC to stimulate fluid secretion. In contrast, EPAC is sufficient for secretion when activated in a cAMP-independent manner via the EPAC-specific agonist 8-pCPT-2'-O-Me-cAMP. Therefore, cAMP is likely to signal through PKA to EPAC. In turn, EPAC levels are likely to be rate limiting, as stellate-specific overexpression of epac enormously enhanced secretion (Efetova, 2013).

This study has established the use of photoactive adenylyl cyclases (PACs) as a potent tool for investigating organotypic physiological processes in vivo. A unique advantage of this optogenetic transgene is that it acts as a 'Trojan horse', allowing cell-type-specific control of cellular cAMP levels with temporal and spatial precision, through simple blue light illumination. It is this feature that has allowed deconstruction of the complex regulatory network of cAMP pathways involved in fluid secretion control, and to assign function within the Drosophila renal (Malpighian) tubule. This experimental approach can easily be adapted to other physiological preparations, for example the central nervous system or the cardiac system, to address similar physiological questions (Efetova, 2013).

Further improvements to bPAC could be achieved; for example it would be beneficial to further reduce the residual dark activity, which must be considered during experimental analysis. Although functional imaging of cAMP has been achieved, further development of this complementary technology would be advantageous for studying complex cellular signaling networks. Another feature of light-induced cAMP signals is that, as bPAC is cytoplasmic, the elevation of cAMP is uniform across the cell. In contrast, naturally occurring cAMP is often unevenly distributed on a sub-cellular level, and concentrated in local microdomains. In addition to the compartmentalization of cAMP, the cAMP sensors PKA and EPAC are also spatially regulated by binding to scaffolding proteins, such as A-kinase anchoring proteins (AKAPs). In future, it should be possible to localize genetically encoded PACs to specific subcellular domains, and embark on a new era of precision optogenetics (Efetova, 2013).

Drosophila middle-term memory: Amnesiac is required for PKA activation in the mushroom bodies, a function modulated by Neprilysin 1

In Drosophila, the mushroom bodies (MB) constitute the central brain structure for olfactory associative memory. As in mammals, the cAMP/PKA pathway plays a key role in memory formation. In the MB, Rutabaga adenylate cyclase acts as a coincidence detector during associative conditioning to integrate calcium influx resulting from acetylcholine stimulation and G protein activation resulting from dopaminergic stimulation. Amnesiac encodes a secreted neuropeptide required in the MB for two phases of aversive olfactory memory. Previous sequence analysis has revealed strong homology with the mammalian pituitary adenylate cyclase-activating peptide (PACAP). This study examined whether amnesiac is involved in cAMP/PKA dynamics in response to dopamine and acetylcholine co-stimulation in living flies. Experiments were carried out with both sexes, or with either sex. The data show that amnesiac is necessary for the PKA activation process that results from coincidence detection in the MB. Since PACAP peptide is cleaved by the human membrane neprilysin hNEP, an interaction was sought between Amnesiac and Neprilysin 1 (Nep1), a fly neprilysin involved in memory. When Nep1 expression is acutely knocked down in adult MB, memory deficits displayed by amn hypomorphic mutants are rescued. Consistently, Nep1 inhibition also restores normal PKA activation in amn mutant flies. Taken together the results suggest that Nep1 targets Amnesiac degradation in order to terminate its signaling function. This work thus highlights a key role for Amnesiac in establishing within the MB the PKA dynamics that sustain middle-term memory formation, a function modulated by Nep1 (Turrel, 2020).

Associative learning, which temporally pairs a conditioned stimulus (CS) to an unconditioned stimulus (US), is a powerful way of acquiring adaptive behavior. At the molecular and cellular levels, the association between CS and US is mediated by coincidence detection mechanisms that reflect the superadditive activation of a molecular pathway in the presence of both stimuli. One of the major coincidence detectors is the cAMP/PKA pathway, which depends on Type-I adenylate cyclases stimulated by both calcium/calmodulin, via acetylcholine signaling representing the CS, and G-protein coupled to dopamine metabotropic receptors activated by dopaminergic neurons encoding the US (Turrel, 2020).

In Drosophila, the mushroom bodies (MB) constitute the central integrative brain structure for olfactory memory. The MB are composed of 4000 intrinsic neurons called Kenyon cells (KC), and classed into three subtypes whose axons form two vertical (a and a9) and three medial (b, b9, and g) lobes. Using a classical conditioning paradigm in which an odorant (CS) was paired to electric shocks (US), Bouzaiane (2015) revealed that flies are capable of forming six discrete memory phases reflected at the neural network level. Among these phases are middle-term memory (MTM) and long-term memory (LTM), which are both encoded in a/b KC. As in mammals, the fly cAMP/PKA pathway plays a key role in associative memory, wherein the adenylate cyclase Rutabaga (Rut) acts as a coincidence detector in the MB to associate the CS and US pathways (Turrel, 2020).

The amnesiac Drosophila mutant (amn) was isolated in a memory defect behavioral screen. As with other components of the cAMP/PKA pathway involved in Drosophila memory, amn is expressed in the MB. It was recently showen that amn expression in the MB is specifically required for MTM and LTM (Turrel, 2018). amn encodes a neuropeptide precursor with a signal sequence. Sequence analyses suggest the existence of three peptides, with one of them homologous to mammalian pituitary adenylate cyclase-activating peptide (PACAP). PACAP is widely expressed throughout the brain, acting as a neuromodulator or neurotrophic factor through activation of G-protein-linked receptors to regulate a variety of physiological processes through stimulation of cAMP production. Furthermore, PACAP may exert a role in learning and memory (Turrel, 2020).

After its release, a neurotransmitter's action is terminated either by diffusion, re-uptake by the presynaptic neuron, or enzymatic degradation. In contrast, neuropeptide signaling is exclusively terminated by enzymatic degradation. Possible enzyme candidates include neprilysins, type 1 metalloproteinases whose main function is the degradation of signaling peptides at the cell surface (Turner, 2001). Indeed, the human neprilysin hNEP is capable of cleaving a PACAP neuropeptide. Drosophila express four neprilysins that are all required for MTM and LTM, establishing that neuropeptide degradation is a central process for memory formation. Among the four neprilysins, Neprilysin 1 (Nep1) is the only one whose expression is required for MTM in the MBx (Turrel, 2020).

This study aimed to confirm whether AMN intervenes in memory by modulating cAMP concentration, as suggested by its sequence homology. For this, PKA dynamics were analyzed in the MB vertical lobes. The results show that amn mutant brains fail to display PKA activation in the a lobe in response to co-application of dopamine and acetylcholine. Whether Nep is involved in terminating AMN action was examined. Using RNAi, it was shown that Nep1 knock-down restores normal MTM and normal PKA dynamics in amn mutants, establishing a functional interaction between Nep1 and AMN in the MB (Turrel, 2020).

Previous work has shown that AMN expression is required in the MB for Drosophila memory. This study established that AMN expression in the MB is necessary for the synergistic activation of PKA observed on co-stimulation by dopamine and acetylcholine in the a lobe, a process that is thought to mimic the coincidence detection event underlying memory formation. Furthermore, the data demonstrate a functional interaction between AMN and Nep1, suggesting that Nep1 targets AMN degradation, thereby terminating AMN signaling (Turrel, 2020).

Six different aversive memory phases that are spatially segregated have been described in Drosophila (Bouzaiane, 2015). Their formation relies on distinct neuronal circuits, as well as distinct molecular and cellular mechanisms. rut mutants are impaired in specific memory phases, including short-term memory (STM), encoded in g KC, and MTM encoded in a/b KC. It was previously shown that Rut expression restricted to g KC is sufficient to restore STM, but not MTM, in rut mutant flies. It is thus likely that distinct Rut-mediated coincidence detection events occur in parallel in g and a/b KC, resulting in STM and MTM formation, respectively. Interestingly, mutants expressing a reduced amn level display normal STM. Thus, AMN is most likely not required for the coincidence detection process that leads to STM, a process that remains to be identified. Using in vivo imaging, previous work showed that coapplication of dopamine and acetylcholine induces a strong synergistic PKA response, which is Rut dependent and occurs specifically in MB vertical lobes. This study shows that this coincident PKA activation in the a lobe is abolished in amn mutants, while neither calcium signaling nor cAMP signaling following dopaminergic stimulation alone are altered. It is proposed that PKA activation mimics the coincidence detection event that occurs in a/b KC during MTM formation, and that AMN intervenes in this process by enabling a sustained Rut-mediated PKA activation in the MB a lobe (Turrel, 2020).

AMN might thus act at a step that ranges from the initial coincidence detection event that provokes Rut activation, to the final level of PKA activation. This is consistent with previous reports that AMN and DC0, the fly PKA catalytic subunit, act in a common pathway, and that AMN function is upstream of DC0 function. If AMN plays a role posterior to the coincidence detection event, it could be involved in an increase in cAMP concentration through the inhibition of phosphodiesterases that degrade cAMP. Indeed, dopamine receptors positively coupled to adenylate cyclases are equally distributed in all MB lobes as are DC0 and Rut, whereas 100 mM dopamine only induces a PKA response in the a lobe. This spatial control is achieved by the cAMP-specific phosphodiesterase Dunce (Dnc) which preferentially degrades cAMP in the b and g lobes, thus restricting high dopamine-induced PKA activation to the a lobe. AMN could thus be involved in the specific inhibition of Dnc in the a lobe (Turrel, 2020).

One attractive alternative hypothesis is that AMN action could take place at the level of Rut activation itself. Indeed, the fact that one of the AMN peptides is homologous to PACAP suggests that AMN might play a role in activating the adenylate cyclase Rut through G-protein-coupled receptors. This hypothesis fits with sequence prediction , and is supported by studies showing that AMN is functionally related to human PACAP. It was initially reported that Rut is activated by the application of human PACAP-38 (Zhong, 1995), and later shown that bath application of PACAP-38 rescues L-type current deficiency in amnX8 larval muscle fibers . Such rescue is abolished by application of an antagonist to Type-I PACAP-receptor as well as by application of an inhibitor of AC (Turrel, 2020).

Although STM and MTM both rely on the cAMP/PKA pathway, not only these processes occur in separate KC, but while STM is instantaneously acquired, MTM is acquired in a dynamic fashion following a two-step mechanism. It is proposed that AMN function is specifically required in the incremental build-up of MTM by boosting Rut activation following the initial event of coincidence detection, namely the first CS/US association of the training protocol. In this model, this first association results in an initial moderate level of Rut activation, followed by a moderate level of PKA activation (Turrel, 2020).

This moderate level of PKA activation does not mediate MTM formation and is below detection threshold. It is hypothesized that this initial increase in PKA activity, directly or indirectly, triggers the second step of the process, namely AMN secretion, and thus generate an activation loop whereby AMN activates Rut, hence creating a much higher level of Rut activation and subsequent high levels of PKA activation that is observable with the AKAR2 probe. MTM formation would rely on an AMN-dependent PKA-activation loop terminated on AMN degradation by Nep1 (Turrel, 2020).

One previous study has indicated that human PACAP is a substrate for hNEP (Gourlet, 1997), and this present work in Drosophila describes a functional interaction between AMN and Nep1. Importantly, whereas Nep1 knock-down rescues the amn mutant memory phenotype in a genetic context where the AMN level is reduced to ~50% versus wild-type flies (heterozygous for the amn null allele), it fails to do so in a genetic context where AMN is absent (i.e., in flies hemizygous for the amn null allele). Namely, the memory rescue observed on Nep1 inhibition is dependent on the presence of AMN, suggesting that this latter is targeted by Nep1. While a biochemical confirmation of this hypothesis would be welcome, it is technically difficult to achieve. Specifically, not only are AMN antibodies not available, but amn mRNA is expressed at very low levels, indicating that AMN peptide may be very scarce (Turrel, 2020).

The observation that the AMN peptide may be targeted by Nep1 is in agreement with a neuromodulatory function. Once released, a signaling molecule must be removed from its site of action to prevent continued stimulation, and to allow new signals to propagate. If neurotransmitter's action is terminated either by diffusion, re-uptake by the presynaptic neuron, or enzymatic degradation, signaling neuropeptides are specifically removed by degradation. The intensity and duration of neuropeptide-mediated signals are thus controlled via the cleavage of these neuropeptides by peptidases like neprilysins. Despite a few exceptions, neprilysins occur as integral membrane endopeptidases whose catalytic site faces the extracellular compartment. It is hypothesized that on conditioning, AMN is secreted by the KC to participate in Rut activation via G-protein-coupled receptors, and is ultimately removed from the extracellular compartment by Nep1 anchored at the KC membrane. Importantly, AMN expression in the MB restores normal PKA dynamics in amn null mutant flies, suggesting that the AMN peptide secreted by the MB on conditioning should act in an autocrine-like way to sustain Rut activity in the a/b neurons. Interestingly, the effects of neuropeptide transmitters are very diverse and often long-lived, which fits well with the specific involvement of AMN peptide in non-immediate memory phases via sustained PKA activation (Turrel, 2020).

Up to date, fly neprilysins have been involved in several behaviors: in the control of circadian rhythms, via hydrolysis of the pigment dispersing factor neurotransmitter, and in the control of food intake via cleavage of insulin-like regulatory peptides. In the latter study, it was shown that both Neprilysin 4 knock-down and overexpression in the larval CNS cause reduced food intake (Hallier, 2016). In a similar way, this study shows that both Nep1 knock-down and overexpression in a/b KC impairs MTM, consistent with the need for a proper control of AMN levels. It is suggested that Nep1 overexpression results in amn loss of function, whereas Nep1 knock-down causes the prolongation of AMN action, thus generating a prolonged activation of the cAMP/PKA pathway, a process deleterious for memory. This is in agreement with a previous study demonstrating that overexpressing DC0 in the MB impairs MTM (Turrel, 2020).

In conclusion, this study reports an acute role for AMN in memory formation via the PKA pathway in the a/b MB neurons, a function modulated by Nep1. These results thus support a role for AMN as an activating adenylate cyclase peptide, much like the role of PACAP, bringing clarity to the role PACAP may play in memory consolidation in mammals (Turrel, 2020).

Earlier PKA literature

Drosophila research is currently uncovering the role G-protein coupled receptors play in development. G-protein coupled receptors have been extensively characterized for their involvement in adult physiology (see The Learning Pathway). In mammals for example, G-protein coupled receptors are commonly found for hormones, neuropeptides and neurotransmitters. However, the involvement of G-protein coupled receptors in developmental pathways is a fairly new area of inquiry.

Cyclic AMP is the classic second messenger (see Cyclic AMP Second Messenger System for more information). It is used in Drosophila to transduce external signals sensed by G protein coupled receptors (e.g., Smoothened) or as a signal transducer of cytoplasmic events such as a change in Ca++ levels, for example, sensed by Calmodulin, which then interacts with Rutabaga).

Mammalian cyclic AMP-dependent protein kinase A (PKA) may serve here as a model for its Drosophila protein counterpart. The mammalian protein is a tetramer composed of two regulatory subunits and two catalytic subunits. In the holoenzyme, the enzymatic activity of the catalytic subunit, coded for by the gene described here, PKA-C1) is latent because of the inhibition conferred by the inhibitory domain of the regulatory subunit. When two mammalian cAMP molecules bind per regulatory subunit, the affinity of the regulatory subunit for the catalytic subunit decreases 10,000-100,000 fold, the tetramer dissociates into dimeric regulatory subunits, two monomers of the catalytic subunit are released, and inhibition is relieved. The catalytic subunit is subject to autophosphosphorylation that may stabilize the catalytic site and foster optimal alignment of peptide substrate within the site for phosphotransfer. Likewise, the catalytic subunit phosphorylates the regulatory subunit in an autoinhibitory domain (Francis, 1994).

Mutation in the catalytic subunit of PKA mimics the phenotype generated by ectopic expression of hedgehog in the anterior compartment of wing and leg discs and ahead of the morphogenetic furrow in the developing eye. That is, either PKA mutation or hedgehog ectopic expression induce ectopic expression of decapentaplegic, wingless and patched. Based on this evidence, it has been suggested that PKA is a component of a signaling pathway that represses dpp, wg and ptc expression and that hh antagonizes this pathway to maintain target gene expression at the anterior-posterior compartment border of appendages and in the morphogenetic furrow (Pan, 1995 and Li, 1995).

With the recent purification of Smoothened, the HH receptor, these findings can now be placed in a broader context. Smoothened is a 7 pass transmembrane receptor, characteristic of G-protein coupled receptors. As mentioned above, these receptors have well characterized roles as both hormone and neurotransmitter signal transducers, but Smoothened has a developmental role as HH receptor (Alcedo, 1996). Like other members of the serpentine receptor family, Smoothened is also coupled to G proteins. The presumed target of SMO is Adenylate cyclase (See Rutabaga), an enzyme that converts ATP to cyclic AMP. In turn, cAMP regulates Protein kinase A.

The target of PKA in segmentation is not yet known, although recent work suggests that this target is likely to be Cubitus interruptus (see below). In the learning pathway the target is CREB, the cyclic AMP response element binding protein, a transcription factor activated by PKA phosphorylation. Likewise, PKA targeted CREB is implicated in the regulation of proliferation of anterior pituitary somatotropic cells. Blocking CREB function in transgenic mice prevents proliferation of somatotropic cells, resulting in pituitary atrophy and dwarfism (Struthers, 1991).

PKA has a another documented role in Drosophila, in addition to signal transduction in segmentation and learning. PKA is associated with germ cell membranes and involved in establishment of oocyte polarity, acting to transduce a signal for microtubule reorganization involved in oocyte mRNA localization (Lane, 1994 and 1995).

Genetic analysis of glutamate receptors in Drosophila reveals a retrograde signal regulating presynaptic transmitter release

PKA controls quantal size in the Drosophila neuromuscular junction (NMJ). The NMJ is a glutamatergic synapse (the neurotransmitter is glutamate) with ultrastructural similarities to glutamatergic central synapses of vertebrates. Postsynaptic sensitivity to glutamate was genetically manipulated at the Drosophila neuromuscular junction (NMJ) to test whether postsynaptic activity can regulate presynaptic function during development. The gene encoding a second muscle-specific glutamate receptor, DGluRIIB, has been cloned and is closely related to the previously identified DGluRIIA, which is located adjacent to it in the genome. Both are non-NMDA type but cannot be classified as either AMPA or kainate type receptors. Both genes are expressed in muscle from stage 12, and neither gene is expressed in the nervous system. Mutations that eliminate DGluRIIA (but not DGluRIIB) or transgenic constructs that increase DGluRIIA expression were generated. When DGluRIIA is missing, the response of the muscle to a single vesicle of transmitter is substantially decreased. However, the response of the muscle to nerve stimulation is normal, because quantal content is significantly increased. Thus, a decrease in postsynaptic receptors leads to an increase in presynaptic transmitter release, indicating that postsynaptic activity controls a retrograde signal that regulates presynaptic function (Petersen, 1997).

The cyclic AMP system and Drosophila learning

Two distinct mechanisms regulate synaptic efficacy at the Drosophila neuromuscular junction: a PKA-dependent modulation of quantal size and a retrograde regulation of presynaptic release. Postsynaptic expression of a constitutively active PKA catalytic subunit decreases quantal size, whereas overexpression of a mutant PKA regulatory subunit (inhibiting PKA activity) increases quantal size. Increased PKA activity also decreases the response to direct iontophoresis of glutamate onto postsynaptic receptors. The PKA-dependent modulation of quantal size requires the presence of the muscle-specific glutamate receptor DGluRIIA, since PKA-dependent modulation of quantal size is lost in viable homozygous DGluRIIA- mutants. The DGluRIIA sequence contains an optimal PKA consensus phosphorylation site on the C-terminal tail (RRXS), believed to be located in the cytoplasmic portion of the protein. Elevated postsynaptic PKA reduces the quantal amplitude and the time constant of miniature excitatory junctional potential (mEJP) decay to values that are nearly identical to those observed in DGluRIIA mutants. PKA modulation of quantal size is sensitive to the copy number of DGluRIIA. Larvae heterozygous for a deletion of DGluRIIA show significantly less modulation by PKA than wild-type controls. This suggests that PKA-dependent modulation of receptor function may be influenced by the subunit composition of postsynaptic receptors. PKA activity appears to constitutively regulate synaptic function at the wild-type synapse. The demonstration that inhibition of PKA leads to a large increase in quantal size suggests that there is a high basal phosphorylation of DGluRIIA at the wild-type synapse. The PKA-dependent reduction in quantal size is accompanied developmentally by an increase in presynaptic quantal content, indicating the presence of a retrograde signal that regulates presynaptic release. A retrograde regulation of presynaptic transmitter release may serve to maintain postsynaptic excitation during the developmental growth of this synapse (Davis, 1998).

GENE STRUCTURE

At least four RNA species with differing sizes derive from the catalytic subunit gene. The relative abundance of these RNA forms varies at different times during development; the shortest species is particularly prominent in embryos and the longest in adults. The size differences among these RNA species occur principally at the 3' ends, perhaps by the use of different sites of polyadenylation (Kalderon, 1988).

Exons - 2

PROTEIN STRUCTURE

Amino Acids - 353

Structural Domains

Pka-C1 is 82% identical to the mammalian catalytic subunit of the cAMP-dependent protein kinase (Kalderon, 1988).

Since a basic surface on the catalytic (C) subunit of mammalian cAMP-dependent protein kinase is important for binding to the regulatory (R) subunit, acidic residues in R were sought that might contribute to R-C interaction. Seven specific carboxylates in RIalpha have been identified that are protected from chemical modification in the holoenzyme; each was then replaced with alanine. Of these, mutated rRI amino acids E15, E106, D107, E105, D140, E143, and D258 all are defective in holoenzyme formation and define negative electrostatic surfaces on RIalpha. An additional conserved carboxylate, Glu101 in RIalpha and the equivalent, Glu99 in RIIalpha, were mutated to Ala. Replacement of Glu101 has no effect while rRII E99 is very defective. RIalpha and RIIalpha thus differ in the molecular details of how they recognize C. Unlike wild-type RI, two additional mutants, D170A and K242A, inhibited C-subunit stoichiometrically in the presence of cAMP and show increases in both on- and off-rates. Asp170, which contributes directly to the hydrogen bonding network in cAMP-binding site A, thus contributes also to holoenzyme stability (Gibson, 1997).

Protein kinases constitute a large family of regulatory enzymes, each with a distinct specificity to restrict its action to its physiological target(s) only. The catalytic (C) subunit of protein kinase A, regarded as a structural prototype for this family, is composed of a conserved core flanked by two nonconserved segments at the amino and carboxyl termini. The active site consists of an extended network of interactions that weave together both domains of the core as well as both segments that flank the core. Also, the opening and closing of the active site cleft, including the dynamic and coordinated movement of the carboxyl terminal tail, contributes directly to substrate recognition and catalysis. In addition to peptide and ATP, the active site contains six structured water molecules that constitute a conserved structural element of the active site. One of these active-site conserved water molecules is locked into place by its interactions with the nucleotide, the peptide substrate/inhibitor, the small and large domains of the conserved core, and Tyr-330 from the carboxyl-terminal "tail" (Shaltiel, 1998).

date revised: 18 February 2024 

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