Intracellular signaling in neurons: unraveling specificity, compensatory mechanisms and essential gene function

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Intracellular signaling in neurons: unraveling specificity, compensatory mechanisms and essential gene function Gaiti Hasan Understanding how unique signaling outputs are generated in neurons using a limited set of intracellular signaling mechanisms has been a challenge. A combination of genetics and cell imaging, with tools developed to measure signaling outputs, has shown that the restricted presence of a signaling attenuator visibly alters the axonal range of the output and can be correlated with different behavioral outputs. Another question of interest is regarding the extent of genetic plasticity possible in the context of a single behavioral change. Recent molecular and genetic studies support the presence of parallel pathways that can compensate for the primary defect both at the level of physiology and behavior. Address National Centre for Biological Sciences, TIFR, Bellary Road, Bangalore 560065, India Corresponding author: Hasan, Gaiti ([email protected])

Current Opinion in Neurobiology 2013, 23:62–67 This review comes from a themed issue on Neurogenetics Edited by Ralph Greenspan and Christine Petit For a complete overview see the Issue and the Editorial Available online 8th August 2012 0959-4388/$ – see front matter, # 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.conb.2012.07.004

Introduction Molecular genetic studies in invertebrate model systems like Drosophila melanogaster and Caenorhabditis elegans have helped to identify genes for several behavioral paradigms including those required for responses to various sensory modalities, learning and memory, circadian rhythms, sleep and more recently behavioral links with metabolism. Not surprisingly neural circuits underlying these varied responses frequently utilize common neurotransmitters and receptors including metabotropic receptors whose intracellular signals fall under defined classes. Understanding how such ‘ubiquitous’ signaling mechanisms function in the context of a specific behavior has remained a challenge. Here I will discuss recent advances in the genetic dissection of two major intracellular signaling components, cyclic adenosine monophosphate (cAMP) and inositol 1,4,5-trisphosphate (IP3)/calcium (Ca2+), their cellular and systemic modes of function and how they affect neural circuit development, function and adult behavior. Current Opinion in Neurobiology 2013, 23:62–67

cAMP signaling The role of cAMP signaling in Drosophila neural function and behavior has been investigated primarily in the context of learning and memory. Two of the earliest learning and memory mutants, rutabaga (rut) and dunce (dnc), were discovered as encoding a calcium/calmodulin kinase dependant adenylate cyclase (AC) and a cAMP phosphodiesterase (PDE), respectively [1,2]. The presence of multiple genes in the Drosophila genome for these two key enzymes of cellular signaling very likely allowed for the isolation of viable null alleles of rut and dnc. Despite the fact that rut and dnc affect cAMP levels in opposing ways, their behavioral phenotypes which consist, among others, of 2 hour memory deficits for aversive conditioning are similar [3]. Recent work has analyzed the cellular basis of this apparent contradiction by measuring the amplitude and kinetics of Protein Kinase A (PKA) activation by a Fluorescent Resonance Energy Transfer (FRET) based sensor, in the mushroom body of rut and dnc mutants [4]. The mushroom body is a Drosophila brain region long associated with the behavioral tasks of learning and memory [5] and cAMP generation by AC activates PKA. Interestingly, the PKA response to dopamine (the neurotransmitter responsible for aversive olfactory conditioning) and octopamine (for appetitive olfactory learning) in the mushroom body is spatially different. While with dopamine it is restricted to the a lobe, with octopamine it is seen in all regions of the mushroom body. In dnc mutants the spatial restriction of PKA activity seen upon dopamine application is lost suggesting that dnc PDE acts to compartmentalize the response to aversive learning stimuli. The authors postulate that spatial restriction of the dopamine response maybe due to a scaffolding complex of the relevant dopamine receptor, Rut AC and PKA, associated with Dnc PDE (Figure 1). Thus the output behavior is dependent on the amplitude of the PKA response (reduced in rut mutants) and its spatial restriction to the a lobe of the mushroom body (altered in dnc mutants). The role of a scaffolding protein in maintaining signaling specificity has been directly demonstrated in Drosophila pacemaker neurons, where knockdown of the scaffolding protein nervy reduces cAMP signaling downstream of the PDF receptor. In the M pacemaker neurons the putative scaffolding complex functions to specifically link PDF receptor activation with adenylate cyclase 3 and not other ACs present in the same neurons [6]. Short-term memory, is thought to depend on the modification of existing proteins that alter synaptic strength www.sciencedirect.com

Intracellular signaling in neurons Hasan 63

Figure 1

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Scaffolding Protein2 (SP2) Effector: Adenylyl cyclase/ PLCβ Second Messenger (SM): cAMP/IP3 Signal Amplifier (SA): PKA/IP3R Active Target protein Signal Restrictor (SR): PDE/BP

Effector: Adenylyl cyclase/ PLCβ Signal Amplifier (SA): PKA/ IP3R Inactive Target protein Signal Restrictor (SR): PDE/BP

Current Opinion in Neurobiology

Spatial localization of signaling in neurons. The presence of a signal restricting (SR) protein within the signaling complex in an axonal branch can serve to localize the signaling range (green in b) of either the effector or the signal amplifier (SA). In the absence of the signal restrictor the signal is more spread out (blue in b). Examples of each class of proteins from either the cAMP or the IP3 pathway are mentioned in the figure. G-protein coupled receptor, GPCR; Phospholipase Cb, PLCb; Protein Kinase A, PKA; Phosphodiesterase, PDE; Binding protein, BP.

over 2–3 hours. Direct measurements of altered synaptic strength post short-term learning have not been technically possible so far. However, the effects of rut and dnc mutants on synaptic function have been measured at the more accessible larval neuromuscular junction [7,8]. These studies show that in rut mutants synaptic arbors are reduced and this is concurrent with reduced and more variable synaptic transmission as measured by the amplitude of excitatory junctional currents (ejc). dnc mutants exhibit extended arbors but do not have a strong effect on synaptic properties on their own. In rut dnc double mutants, ejc amplitudes are restored to an extent, but they still remain variable. If both genes affected the same pathway for synaptic function in opposing ways one would expect that double mutants would be compensated and relatively ‘normal’. Sequestration of rut and dnc in different compartments at the larval nmj similar to what has been proposed in the mushroom body (above) could explain these results. Thus, both in a short-term memory paradigm that functions acutely and in synaptic function at the nmj, where rut and dnc have developmental effects; the two mutants do not have directly opposing phenotypes as might be predicted from their enzymatic activity. The Rut AC is also required for the formation of longterm memory (LTM) that lasts for 24 hours in Drosophila and is generally considered to arise by translocation of activated PKA to the nucleus followed by CREB-dependent transcription and translation of new proteins [9]. Changes in the transcriptional profile of rut and dnc brains found very few common genes, suggesting that at www.sciencedirect.com

steady state, which is in the absence of specific memory training, the two genes affect different aspects of cell function [8]. When transcriptional changes in wild-type Drosophila during LTM consolidation were measured genes affecting mRNA translocation and protein synthesis were found to be upregulated [10]. Interestingly, transcripts encoding AGO2, an integral component of the RNA silencing complex, were significantly downregulated in the rut microarray [8]. A recent study has shown that CREB-dependent transcription is responsible for the reduced synaptic arbors in the larval nmj of rut mutants (see above). CCKR, a neuropeptide receptor, signals through the heterotrimeric G-protein subunit Gas to the Rut-PKA-CREB pathway [11]. Since the phenotypes of CCKR mutants are much stronger than rut mutants the authors propose parallel activation of other ACs by CCKR.

IP3 and calcium signaling Scaffolding of signaling complexes in ‘signalosomes’ as a means for restricting signaling in cellular compartments and maintaining specificity is likely to be a general concept encompassing many modes of signaling [12]. In the worm C. elegans compartmentalized Ca2+ signals in two axonal regions of a single interneuron, the RIA, were correlated directly with the direction of head bends. These Ca2+ dynamics in the RIA axon are due to stimulation of the muscarinic acetylcholine receptor encoded by gar3, which acts through its effector PLCb, encoded by egl8. Apparently, the Ca2+ dynamics arise in the axonal Current Opinion in Neurobiology 2013, 23:62–67

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compartments of the RIA, in response to head movements and very likely modulate the turning behavior of C. elegans [13]. In this case the proteins responsible for restricting the signal are yet to be identified, though a likely possibility could be differential localization of Ca2+ binding proteins through a scaffolding complex (Figure 1). Unlike cAMP signaling components, genes that encode key elements of IP3 and intracellular Ca2+ signaling are often found as single copies in invertebrate genomes including Drosophila melanogaster and C. elegans. Thus in Drosophila, dgq encodes the Gaq subunit of the heterotrimeric G protein, Gq [14,15]; itpr is the gene for the inositol 1,4,5-trisphosphate receptor or IP3R [16,17]; dstim codes for the Drosophila stromal interacting molecule [18], that senses the fall of calcium in endoplasmic reticular stores (ER) after Ca2+-release by the

IP3R and dorai or Drosophila orai, codes for the surface membrane channel that replenishes ER Ca2+ stores after depletion [19,20]. Together Stim and Orai constitute the key elements of store-operated Ca2+ entry (SOCE) in metazoans and have been extensively reviewed recently [21,22]. In Drosophila the behavioral consequences of disrupted intracellular Ca2+ signaling (through the IP3R and SOCE) have been investigated primarily in the context of air-puff induced flight. These studies have shown that neuronal IP3/Ca2+ signals and SOCE are both required for Drosophila flight circuit development [23,24,25]. Even though connections and neurons that make up the air-puff induced flight circuit in Drosophila remain to be mapped precisely, it is known that the circuit forms in the first 48 hours of pupal development [26]. By controlling knockdown of the IP3R with a temperature sensitive repressor of the inducible expression system used [27,28], expression of the RNAi transgene for the

Figure 2

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Parallel pathways can rescue behavioral and physiological outputs. (a) In normal Drosophila adults an air-puff stimulus triggers flight by activating the flight central pattern generator (CPG) in the ventral ganglion. The flight CPG in turn drives the rhythmic firing pattern (shown below each figure) of the flight motor neurons and the post-synaptic indirect flight muscles (DLM). (b) Flight physiology and behavior are compromised in Drosophila IP3R mutants [30]. (c) Flight physiology and behavior can be rescued by expression of a wild-type IP3R cDNA in two non-overlapping neuronal domains [24]. The systemic rescue suggests that parallel modes of signaling are capable of rescuing flight. Current Opinion in Neurobiology 2013, 23:62–67

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IP3R was limited to neurons during specific developmental stages in pupae and adults. These studies established that the major requirement for IP3R/SOCE was during the first 48 hours of pupal development, and only a minor deficit was observed if the RNAi was induced in adults (S Sadaf, G Hasan, unpublished data). The behavioral studies were accompanied by cellular calcium measurements in cultured itpr mutant neurons, which showed compromised Ca2+ release through the IP3R, and in addition reduced SOCE. Importantly, restoration of SOCE in itpr mutant neurons by overexpression of either dSTIM or dOrai correlated with the ability to maintain flight [24,25]. A limitation of the studies described above was their inability to identify the precise neurons that require itpr function and SOCE in the context of flight, though RNAi knockdowns demonstrate that SOCE is required in glutamatergic neurons, which in Drosophila include all motor neurons and a subset of interneurons. Attempts at identifying neuronal classes that require IP3R function for flight were made by expressing an inducible itpr+ cDNA transgene in defined neuronal subsets of itpr mutants. These experiments implicated two non-overlapping neuronal expression domains. Of these one domain is of dopaminergic and serotonergic neurons (Dopa decarboxylase GAL4, DdcGAL4; [29,30]) and the other is the insulin-like peptide (ILP) producing cells of the brain (Dilp2GAL4; [24,31]). Surprisingly the same RNAi knockdown constructs that work in the glutamatergic domain had no effect on flight when expressed in the rescue domains of either Ddc or ILP expressing neurons [24]. Taken together these findings support the idea that restoring intracellular Ca2+ signaling in Ddc/ILP neurons of itpr mutants leads to rescue of flight circuit development through parallel pathways (Figure 2). Absence of phenotypes with RNAi knockdowns could thus be in part due to compensation by parallel pathways and in part due to differential strengths of the GAL4 strains used. Microarray experiments that compared differentially regulated genes between itpr mutants with either a rescue transgene in the ILP neurons or a suppressor mutant that raises basal cytosolic Ca2+ provide a further insight. Only a small subset of transcriptionally regulated genes in the itpr mutant strain, revert back towards wild type levels in rescue and suppressor conditions. Moreover, there is minimal overlap between genes whose expression levels revert in the two conditions [32] suggesting that loss or reduction of a key intracellular signaling component can be compensated for in multiple ways by an organism. Finally, while cAMP and IP3/Ca2+ signaling are generally considered as mutually exclusive there are indications that both pathways could be activated by a single receptor (also see discussion below). Drosophila Insulin Producing Cells (IPCs) express the OAMB form of the octopamine www.sciencedirect.com

receptor, stimulation of which can lead to elevation of both cAMP and Ca2+ signals. cAMP signaling downstream of OAMB inactivates Slowpoke (SLO), a Ca2+ gated K channel. Biochemical evidence from mammalian arterial smooth muscle cells has shown that function of the slo homolog in vertebrates, the BKCa channel, is regulated by Ca2+ release through IP3R1 [33]. Reduced function of SLO in the IPCs is thought to be responsible for the wake-promoting affects of octopamine on flies [34]. In an independent set of experiments, SLO activity in the IPCs has been shown to influence downstream insulin signaling and metabolism [35]. Thus, as suggested previously, Drosophila brain IPCs could function as integrators of neuronal activity and metabolic status of the animal in a manner similar to the mammalian hypothalamus [36].

Conclusions A cellular understanding of how ubiquitous signaling mechanisms within neurons lead to specific behavior, is beginning to emerge from the recent genetic studies. Compartmentalization of signaling complexes is clearly one mechanism employed across signaling pathways and species to spatially restrict signaling in neuronal subcompartments or subdomains [37,38] (Figure 1). Mechanisms that are responsible for the development of such spatial restriction and the extent of its plasticity will be of interest in future studies. At the systemic level, the combination of genetics and microarray studies has produced some unexpected results which suggest that multiple parallel pathways can lead to the same end result as measured by behavior (Figure 2). Moreover, parallel signaling mechanisms can operate not only at the systemic level but also at tissue-specific and cellular levels. In this context the following recent studies are informative. For example, in Drosophila larvae two Gprotein coupled receptors, both of which respond to the neuropeptide FMRF, activate IP3/Ca2+ signals in neurons and knockdowns of both attenuate larval repulsion to bright light [39]. In adult Drosophila, when activity levels of two G-proteins Gas and Gao were manipulated, circadian rhythms were affected in similar ways. While Gas acts through cAMP, Gao was shown to act through PLCb. This study elegantly elucidates the existence of parallel signaling mechanisms in the same set of circadian neurons [40]. Even in the well-studied context of learning and memory, it has long been discussed that parallel pathways must contribute to the memory that is retained in rut mutants. Understanding the genetics of signaling mechanisms and whole organism behavior thus needs further thoughtful analysis.

Acknowledgments The author acknowledges gratefully Sufia Sadaf’s help with drawing the figures in this article. Work in the author’s laboratory is funded by NCBS (TIFR), Dept of Science and Technology and the Dept. of Biotechnology, Govt. of India. Current Opinion in Neurobiology 2013, 23:62–67

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