CaM kinase II

Synaptic protein synthesis associated with memory is regulated by the RISC pathway in Drosophila

Long-lasting forms of memory require protein synthesis, but how the pattern of synthesis is related to the storage of a memory has not been determined. This study shows that neural activity directs the mRNA of the Drosophila Ca2+, Calcium/Calmodulin-dependent Kinase II (CaMKII), to postsynaptic sites, where it is rapidly translated. These features of CaMKII synthesis are recapitulated during the induction of a long-term memory and produce patterns of local protein synthesis specific to the memory. mRNA transport and synaptic protein synthesis are regulated by components of the RISC pathway, including the SDE3 helicase Armitage, which is specifically required for long-lasting memory. Armitage is localized to synapses and lost in a memory-specific pattern that is inversely related to the pattern of synaptic protein synthesis. Therefore, it is proposed that degradative control of the RISC pathway underlies the pattern of synaptic protein synthesis associated with a stable memory (Ashraf, 2006).

The CaMKII 3'UTR is necessary and sufficient for the robust localization of CaMKII to dendritic arbors: Since the mammalian aCaMKII is found at synapses, where its synthesis is regulated by neural activity, the attention of this study turned to the CaMKII gene of Drosophila. Drosophila CaMKII has a role in neuromuscular synaptic plasticity and memory in the courtship-conditioning paradigm. CaMKII is localized to both pre- and postsynaptic sites in the adult brain. Focus was placed on the olfactory system because of its well-described neural components, circuitry, and paradigms for the establishment of memory. This system consists of sensory neurons and interneurons that form an early receptive and processing circuit with synapses organized in bilaterally symmetric centers known as the antennal lobes. The first-order interneurons (Projection Neurons; PNs) collect sensory input in a stereotyped array of multisynaptic structures known as glomeruli, where the PN dendritic synapses collect cholinergic input via nicotinic acetylcholine receptors. The PNs direct-output to two brain centers via branching axons that project to the 'calyx' of the mushroom body and to the lateral horn. These terminals release acetylcholine from choline acyltransferase (ChAT)-positive boutons. The PN dendrites also form reciprocal synapses with local interneurons. On the PN dendrites, CaMKII was localized in postsynaptic puncta with the markers Discs Large (DLG) and ARD (a nAChR β-subunit. CaMKII was also concentrated at the PN presynaptic boutons in the calyx and lateral horn. Thus, within the same neuron, CaMKII is concentrated at both pre- and postsynaptic sites (Ashraf, 2006).

The mouse aCaMKII mRNA displays dendritic localization and activity-dependent synaptic translation, features conferred by sequences in its 3'UTR. To determine whether this is the case for Drosophila CaMKII, the 3'UTR was inserted downstream of the EYFP coding sequence in the reporter, UAS-EYFP^3'UTR. An additional pair of constructs was made bearing a translational fusion of EYFP to CaMKII, with the 3'UTR^CaMKII present (UAS-CaMKII::EYFP^3'UTR) or absent (UAS-CaMKII::EYFP^NUT) (Ashraf, 2006).

When expressed specifically in PNs using the GAL4, UAS binary system, EYFP^3'UTR fluorescence was strikingly localized to synapses on the PN dendritic and axonal termini and colocalized with ChAT at the presynaptic boutons and with the nAChR subunit ARD on dendritic branches in glomeruli. This distribution roughly matched that of CaMKII protein, though EYFP was somewhat more diffuse along the dendritic branches; presumably EYFP does not bind to the postsynaptic apparatus as CaMKII does. In contrast, a cytoplasmic EYFP reporter lacking CaMKII sequences was distributed poorly to the axons and dendrites (Ashraf, 2006).

The CaMKII::EYFP fusion protein synthesized from mRNA harboring the 3'UTR (CaMKII::EYFP^3'UTR) displayed synaptic localization in axons and dendrites like that of EYFP^3'UTR but was notably more concentrated in synaptic puncta. The same fusion protein made from mRNA lacking the 3'UTR (CaMKII::EYFP^NUT) was strongly localized to axonal presynaptic sites but found at a very low level on the antennal lobe dendrites, where it was localized to synaptic puncta. Thus the CaMKII 3'UTR is necessary and sufficient for the robust localization of CaMKII to dendritic arbors but not required for axonal localization (Ashraf, 2006).

Localization and rapid induction of CaMKII in dendrites is due to 3'UTR-dependent synaptic protein synthesis; To determine how neural activity might affect CaMKII expression, brains were explanted into bath culture with acetylcholine (ACh) or nicotine (an agonist of nAChRs). After 20 min, the tissue was examined by anti-CaMKII immunohistochemistry and quantitative confocal microscopy. On average, when cholinergic synapses were activated, CaMKII immunofluorescence in the antennal lobe increased by ~3- to 4-fold. In a time-course experiment, an increase in CaMKII level was detected within 5-10 min of nicotine exposure. The increase was widespread in the brain and reflected in a ~4-fold increase of CaMKII protein on Western blot analysis. The CaMKII increase was also specific, as the levels of synaptic proteins DLG and ARD were unchanged. Consistent with the notion that this regulation occurs via translational control, the effect of cholinergic stimulation was blocked by the ribosomal inhibitor Anisomycin but not by Actinomycin D, an inhibitor of transcription (Ashraf, 2006).

These results and the requirement of the CaMKII 3'UTR for dendritic localization suggest that cholinergic activity may induce the translation of CaMKII mRNA at postsynaptic sites. This was examined by monitoring EYFP^3'UTR reporter expression in explant culture. A 5 min nicotine incubation increased EYFP^3'UTR expression by 30% and, after 20 min, by 250%. The induced EYFP protein was found in large punctae. Cholinergic stimulation did not increase EYFP^3'UTR expression at the presynaptic terminals in the calyx. In contrast, CaMKII::EYFP^NUT expression was only slightly increased by nicotine or ACh exposure. Nicotine incubation did not alter the expression of cytoplasmic EYFP, CD8::GFP, or an EGFP construct harboring an a₁-tubulin 3'UTR (Hh::EGFP-3'UTR^tub). These observations indicate that the localization and rapid induction of CaMKII in dendrites is due to 3'UTR-dependent synaptic protein synthesis (Ashraf, 2006).

Odor-specific induction of synaptic protein synthesis occurs when conditioned and unconditioned stimuli are presented coincidentally and with temporal spacing, the experience that establishes an LTM: In Drosophila, an olfactory LTM is induced by 'spaced training,' a protocol where an odor (CS+) and electric shock (US) are presented coincidentally at temporally spaced intervals. A second odor (CS-) follows the CS+ odor in each interval without coincident shock. An LTM appears after several hours and lasts beyond 24 hr, as assayed by tactic behavior in a T-maze. This protocol was followed and EYFP^3'UTR was used to report synaptic protein synthesis in animals that developed an olfactory LTM. The analysis focused on the antennal lobe glomeruli because these structures can be reproducibly identified and display clustered synaptic activity. Furthermore, the first-order antennal lobe synapses might participate in an early stage of memory storage, including the storage of LTM. This analysis revealed an odorant-specific pattern of synaptic protein synthesis associated with the induction of a long-term memory (Ashraf, 2006).

Animals harboring the UAS-EYFP^3'UTR reporter driven by the PN-specific GH146-GAL4 were trained and analyzed at times from 4 to 24 hr posttraining. The brains of trained and untrained animals were processed for microscopy in parallel. For each glomerulus, a Z stack of 6-8 confocal microscopic images was recorded and analyzed via a thresholding protocol that isolated pixel groups corresponding to synaptic puncta. An average glomerulus intensity change (ΔF/F) was calculated for 5-8 brains in each experiment. Each experiment was repeated five times. LTM was in all cases verified by T-maze performance (Ashraf, 2006).

The analysis was restricted to a set of glomeruli that included those with a primary response to the odorants octanol (OCT) and methylcyclohexanol (MCH). Only particular glomeruli displayed a training-dependent increase in EYFP^3'UTR fluorescence, while others did not; their identities depended on the odorant (CS+) paired with shock. When OCT was the CS+, only glomeruli D and DL3 displayed increased fluorescence, by 115% and 108%, respectively. When MCH was the CS+, fluorescence increased significantly in glomeruli DA1 and VA1 by 95% and 70%, respectively. There were modest but possibly insignificant increases in glomeruli DM6 and VC2. The glomerulus-specific increases were noted as early as 4 hr posttraining and were not observed when odorant and/or electric shock was unpaired or left out or when temporal spacing was not employed ('massed training'). In animals that expressed a cytoplasmic EYFP reporter or CaMKII::EYFP^NUT, which lack the CaMKII 3'UTR, there were no significant fluorescence changes. This analysis revealed that an odor-specific induction of synaptic protein synthesis occurred when conditioned and unconditioned stimuli were presented coincidentally and with temporal spacing, the experience that establishes an LTM. This plasticity was evidently maintained for at least 24 hr (Ashraf, 2006).

A coordinated program for synaptic gene expression occurs during the storage of a memory: If CaMKII was synthesized at the synapse, its mRNA would be localized there. To address this question, an mRNA tracking system based on the bacteriophage coat protein MS2 and its RNA binding site were utilized. The fusion protein MS2::GFP::nls is concentrated in the nucleus by nuclear localization signals (nls) but can be diverted elsewhere by binding to an MS2 binding site (MS2-bs) tagged mRNA. Three mRNAs were tagged: the Drosophila CaMKII cDNA, its 3'UTR alone, and the mouse aCaMKII 3'UTR, which mediates dendritic localization and synaptic translation in the mouse (Ashraf, 2006).

GFP fluorescence was examined when MS2::GFP::nls was expressed in projection neurons (PNs) with or without an MS2-bs tagged mRNA. Punctate fluorescence was observed in the dendrites when an MS2-bs-tagged mRNA was coexpressed with MS2::GFP::nls but not with MS2::GFP::nls alone. For example, the tagged Drosophila CaMKII mRNA increased the intensity of glomerular fluorescence by 200%. In dendrites, particular mRNAs, including the mouse aCaMKII, are localized to particles containing the motor protein Kinesin. Consistent with this observation, the GFP-positive dendritic puncta were labeled with an antibody against the major kinesin heavy chain, KHC (Ashraf, 2006).

It was asked whether the synaptic CaMKII expression induced by cholinergic activity might be associated with enhanced dendritic localization of CaMKII mRNA, as has been found for the mouse aCaMKII and Arc mRNAs. When explanted into media with nicotine or ACh, brains harboring MS2::GFP::nls and the tagged Drosophila CaMKII mRNA displayed a striking increase in dendritic GFP fluorescence. The effect with cholinergic stimulation was similar with the tagged mouse aCaMKII 3'UTR: a 70%-73% increase relative to culture without nicotine. The activity-enhanced dendritic mRNA transport was blocked by Anisomycin but not by Actinomycin D. It is supposed that mainly existing mRNA can be translocated during the short period of culture. Thus, in Drosophila, like mammals, neural activity increases the rate of mRNA movement to the synapse by a protein synthesis-dependent mechanism (Ashraf, 2006).

It was then asked whether the induction of an LTM might affect mRNA transport to the synapse. When animals expressing MS2::GFP::nls and Drosophila ms2bs-CaMKII were subjected to the spaced training protocol, the number of GFP-labeled puncta in dendrites was substantially increased. The pattern of dendritic punctae did not display evident glomerular specificity, as observed for synaptic protein synthesis. However, the induced punctae were distributed along the dendritic branches, making a determination of glomerular specificity uncertain. These observations reveal that a coordinated program for synaptic gene expression occurs during the storage of a memory (Ashraf, 2006).

Regulation of mRNA transport and synaptic protein synthesis by the RISC pathway: The RNA interference (RISC) pathway silences gene expression by the targeted degradation of mRNAs or their nondestructive silencing. In Drosophila, RISC-mediated translational silencing controls oskar expression in the developing oocyte. An SDE3-class RNA helicase, Armitage (Armi) acts as part of RISC to control oskar translation and regulate cytoskeletal organization, possibly via control of Kinesin heavy chain (Khc) translation. Both the oskar and Khc 3'UTRs have putative binding sites for the microRNA (miRNA) miR-280. The CaMKII 3'UTR has a remarkably similar miR-280 binding site. This site and a nearby site for miR-289 satisfy the predictive rule that 7 of 8 nucleotides at the 5' end of an miRNA are cognate to a target mRNA. Kinesin is also a component of the RNA-containing dendritic particles that bring mRNA to the synapse. Staufen, likewise a mediator of RNA transport, has binding sites for miR-280 and miR-305 in its mRNA 3'UTR. Thus this study explored the role of RISC in CaMKII, KHC, and Staufen expression (Ashraf, 2006).

Dicer-2 is one of two Drosophila ribonucleases that produce short RNA components of RISC. CaMKII synaptic expression was dramatically increased in a dicer-2 mutant, particularly in the antennal lobe and mushroom body. In contrast, there was no difference in the expression of the cell adhesion protein Fasciclin II in the same animals. In Western analysis, there was a striking ~25-fold increase in CaMKII protein in dicer-2 mutant brains. Synaptic CaMKII expression was also elevated in aubergine and armitage mutant brains. The aubergine locus encodes an Argonaute protein involved in RISC assembly and function. The level of Staufen protein was also increased in the armitage mutant brain (Ashraf, 2006).

Whether the miRNA binding sites in the CaMKII 3'UTR might be involved in RISC-mediated regulation was examined with the EYFP^3'UTR transgene. When expressed in the PNs, EYFP fluorescence in glomeruli was 80% greater in armi than in the wild-type. The EYFP^3'UTR fluorescence was localized to large dendritic puncta like those found in brains explanted into nicotine-containing media. Indeed, EYFP^3'UTR expression in armi brains did not increase further upon explant with nicotine, consistent with the notion that cholinergic activation might act via antagonism of RISC. The expression of CaMKII::EYFP^NUT, which lacks the 3'UTR, increased slightly in the armi mutant background, while other control constructs, such as CD8::GFP, were essentially unchanged. In addition, RT-PCR analysis of wild-type and armi mutant brains did not reveal a difference in the levels of transgenic mRNAs. There was also a substantial increase in EYFP^3'UTR and CaMKII::EYFP^3'UTR synaptic fluorescence in dicer-2 and aubergine mutants. Therefore it is concluded that RISC regulates CaMKII expression by a posttranscriptional mechanism, utilizing sites in the CaMKII 3'UTR (Ashraf, 2006).

Armitage expression was found in multiple neuronal populations in the brain, including the PNs and mushroom-body Kenyon cells. It is distributed in puncta in cell bodies and dendrites and to axon termini. A GFP::Armi fusion protein, when expressed in the PNs, displayed a similar punctate distribution that overlaps synaptic puncta containing CaMKII. The GFP::Armi fusion protein retains armi⁺ activity such that neurons with high levels of GFP::Armi expression would have increased armi⁺ activity. Several observations indicate that a posttranscriptional autoregulatory circuit modulates Armi expression. Nonetheless, strong transgenic expression of GFP::Armi reduced the level of CaMKII expression, as revealed by Western blot analysis and immunohistochemistry. Neurons that expressed a high level of GFP::Armi displayed reduced expression of both CaMKII and KHC. A control UAS-CD8-GFP transgene was unaffected by GFP::Armi expression (Ashraf, 2006).

Since Armi regulates KHC and Staufen expression, the possibility was considered that it might also regulate the dendritic transport of CaMKII mRNA. When examined with the MS2::GFP system, armi^72.1 homozygotes indeed displayed a 78% increase in fluorescence by dendritic GFP-positive puncta, compared to an armi⁺ control. Therefore, Armi regulation of synaptic protein synthesis reflects a coordinated program with multiple miRNA targets, affecting both mRNA transport and translation at the synapse, where Armi protein is found (Ashraf, 2006).

Neural activity induces rapid proteasome-mediated degradation of Armitage: If mRNA silencing by RISC plays a role in LTM, this pathway would be expected to be somehow regulated by neural function. Given the inverse relationship between CaMKII expression and armi⁺ activity, whether Armi might be a regulatory target wad considered. The level of GFP::Armi fluorescence rapidly decreased (by 3.5-fold) in brains explanted into nicotine-containing medium. There was a correlated increase in CaMKII expression (by ~4.5-fold) in the PN dendritic arbors of the antennal lobe. A short incubation with nicotine (5 min) resulted in the complete disappearance of Armi protein in Western analysis. The GFP::Armi protein was also eliminated upon explant with nicotine. In contrast, the CaMKII protein level increased and a₁-tubulin was unchanged (Ashraf, 2006).

Two experiments were performed to determine whether the activity-induced elimination of Armi required the proteasome. First, GFP::Armi was expressed along with a transgenic dominant-negative mutant of the proteasome β subunit. When the DTS5 transgene was present, the level of GFP::Armi fluorescence was elevated by 3.2-fold. In contrast, the DTS5 transgene did not alter the level of CD8::GFP. Second, incubation with the proteasome inhibitor lactacystin blocked the nicotine-induced loss of GFP::Armi and degradation of endogenous Armi protein. Preincubation with lactacystin also blocked nicotine-induced synaptic CaMKII synthesis, as determined by both Western analysis and by immunohistochemistry. Thus, cholinergic activity evidently acts via the proteasome to induce the degradation of Armitage and synaptic synthesis of CaMKII (Ashraf, 2006).

A degradative pathway for LTM: A key question is whether this degradative pathway has a role in synaptic protein synthesis associated with LTM. Animals expressing the GFP::Armi protein in projection neurons were subjected to olfactory spaced training and analyzed by the same microscopic methods used to assess LTM-associated EYFP^3'UTR expression. The GFP::Armi protein was found concentrated in synaptic puncta in the glomeruli. When examined at either 3 or 24 hr posttraining, GFP::Armi fluorescence was significantly reduced in many glomeruli and most strongly reduced in the glomeruli that had displayed the greatest increase in EYFP^3'UTR expression. Fluorescence in glomeruli DA1 and VA1 decreased by ~3.1- and 3.8-fold, respectively, when the odorant MCH was paired with shock. When the odorant OCT was paired with shock, the D and DL3 glomeruli displayed the most significant decreases (~2-fold). More modest losses of GFP fluorescence were observed in other glomeruli. These observations reveal an inverse relationship between synaptic Armi protein and CaMKII synthesis during the establishment of an LTM. Since these changes were still present at 24 hr posttraining, the change was evidently maintained long-term, perhaps for the term of the memory (Ashraf, 2006).

Given the role of Armi in the synaptic synthesis of CaMKII, it was wondered whether either of these genes might be required to form an olfactory LTM. Several armi hypomorphic alleles display normal adult viability and behavior, including normal odor and shock sensitivity. Given their normal performance in these tests, armi animals were examined for STM and LTM. The animals (armi^72.1/armi^72.1 or armi^72.1/Df(3L)E1) displayed normal memory in the short-term paradigm but were profoundly deficient in LTM. Expression of the GFP::Armi transgene rescued the armi^72.1/armi^72.1 LTM deficiency to a normal value. A nearly complete and tissue-specific loss of CaMKII was achieved by use of a construct that generates a CaMKII hairpin RNA. Animals expressing UAS-CaMKII^hpin in all CaMKII-positive neurons (with the CaMKII-GAL4 driver) retained normal short-term memory, but displayed a near-complete loss of LTM. Thus, both CaMKII and Armitage are required for LTM but not for STM (Ashraf, 2006).

It is concluded that memory-specific patterns of synaptic protein synthesis occur with the induction of a long-term memory in Drosophila. These patterns appear to be controlled by the proteasome-mediated degradation of a RISC pathway component, Armitage, to regulate the transport of mRNA to synapses and its translation once there (Ashraf, 2006).

To visualize synaptic protein synthesis, fluorescent reporters were used based on the Drosophila CaMKII gene, which has well-described roles in synaptic plasticity and memory. The 3'UTR of CaMKII shares regulatory motifs with the mammalian aCaMKII mRNA, which mediate dendritic mRNA localization and neural activity-dependent translation. The 3'UTR of Drosophila CaMKII was also necessary and sufficient for mRNA localization to dendrites and synaptic translation. This 3'UTR sufficed for the enhanced dendritic mRNA transport and translation induced by cholinergic stimulation. Hence a simple parallel was found between the synaptic regulation of CaMKII in Drosophila and mammals (Ashraf, 2006).

When these fluorescent reporters were utilized in vivo, the induction of synaptic protein synthesis was observed in several Drosophila brain centers following the spaced training paradigm of repetitive odor paired with electric shock that establishes a long-term memory. There were local patterns of memory specificity identifiable in glomeruli of the antennal lobe where synapses of similar function are clustered. When the odorant OCT was paired with electric shock, protein synthesis was induced selectively in the D and DL3 glomeruli. When the odorant MCH was paired with shock, the DA1 and VA1 glomeruli displayed the most robust enhancement of synaptic protein synthesis. Notably, the animals were exposed to both odorants during training; the pattern of synthesis depended on coincidence with shock. There was no significant induction of protein synthesis when exposure to odor and shock was nonoverlapping, with either stimulus presented alone, or in the absence of temporal spacing ('massed training'). Thus, an odor-specific pattern of synaptic protein synthesis was induced under conditions that produce an LTM (Ashraf, 2006).

Experiments in the honeybee suggest that the antennal lobe is a 'way station' for memory where stimuli are integrated to yield plasticity more labile than a short-term memory. A long-term memory can be formed in the honeybee antennal lobe in a spaced training paradigm. Experiments have revealed plasticity in the Drosophila antennal lobe, where particular glomeruli acquired enhanced synaptic activity after a single episode of paired odor and shock. Remarkably, the enhanced synaptic protein synthesis observed with spaced training occurred in essentially the same glomeruli that displayed enhanced synaptic activity in the STM protocol. These glomeruli are distinct from those that display the greatest odor or electric shock-evoked synaptic activity. Therefore, it is supposed that the mechanism that integrates a single paired odor and shock to produce new synaptic activity might also generate the trigger for synaptic protein synthesis when the paired stimuli are repeated with temporal spacing. It is believed this trigger includes the proteasome-mediated degradation of the RISC factor Armitage (Ashraf, 2006).

Though these 'memory traces' have been recorded in the antennal lobe, there is still no evidence for their role in memory. The mushroom body, in contrast, is required for LTM. The current methods cannot resolve patterns of synaptic protein synthesis in the mushroom body because it lacks the stereotyped synaptic architecture of the antennal lobe. When determined, a global brain map of synaptic protein synthesis will provide significant insights into the mechanisms of memory storage (Ashraf, 2006).

Synaptic protein synthesis and dendritic mRNA transport are well studied for the mammalian aCaMKII gene, which bears recognition motifs in its 3'UTR for CPEB and other proteins with transport and translation control functions. The presence of potential recognition motifs for the CPEB, Pumilio, and Staufen proteins in the Drosophila CaMKII 3'UTR suggests that these mechanisms are conserved in Drosophila. Indeed, Staufen, orb (a CPEB family member), and pumilio have been identified as LTM-deficient mutants. The roles of these genes remain to be fully explored (Ashraf, 2006).

Focus was placed instead on the RISC pathway because of apparent binding motifs for microRNAs miR-280 and miR-289 in the CaMKII 3'UTR. These sites are similar to those in the 3'UTRs of oskar and Kinesin heavy chain (Khc), which are targets for translational silencing by Armitage and other RISC components in the oocyte. Armitage was found in synaptic puncta on dendrites, colocalized with CaMKII. When the level of Armitage was decreased or increased by mutation or transgenic expression, CaMKII synaptic expression was modulated in a reciprocal and cell-autonomous fashion. This regulation could be recapitulated by an EYFP reporter bearing the CaMKII 3'UTR. Mutants for the RISC components Aubergine and Dicer-2 displayed similar phenotypes. It therefore seems likely that multiple tiers of control regulate CaMKII like oskar, where two systems (Bruno/Cup and RISC) act on distinct sites in its 3'UTR (Ashraf, 2006).

A second avenue for RISC control of CaMKII synthesis is via mRNA transport. By tagging CaMKII mRNA with a GFP reporter, dendritic punctae were observed whose frequency and intensity increased under the same conditions that induced synaptic protein synthesis: cholinergic activation and olfactory spaced training. The induction of mRNA transport required new protein synthesis but not transcription. Armitage was also found to regulate the frequency and intensity of the GFP-tagged dendritic puncta. Two proteins that play a role in mRNA transport, Kinesin heavy chain (KHC) and Staufen, recapitulate this pattern of regulation by cholinergic stimulation and Armitage. Both of their mRNAs bear targets for miRNA regulation in the 3'UTR. These studies leave open the possibility that the enhanced synaptic localization of CaMKII mRNA underlies the induction of its synaptic translation. However, the presence of miRNA binding sites in the CaMKII 3'UTR, the localization of Armitage with CaMKII in synaptic punctae, and the rapid induction of CaMKII synthesis by cholinergic activity all suggest that RISC acts at the synapse. Furthermore, local translational control may be required to impose the specificity that was not evident in the pattern of mRNA transport associated with the induction of an LTM (Ashraf, 2006).

A link between the induction of memory and synaptic protein synthesis is the proteasome-mediated degradation of Armitage. In explant culture, cholinergic induction of CaMKII synthesis was accompanied by the rapid degradation of Armi; both events were blocked by inhibition of the proteasome. The relationship between Armi degradation and CaMKII synaptic translation was recapitulated in the brain as animals formed and maintained an LTM. The same glomeruli that displayed the greatest increase in CaMKII synthesis displayed the largest decline in synaptic Armi. This reciprocal relationship between the Armi and CaMKII proteins was detected as early as 3 hr after training and maintained for at least 24 hr posttraining. The training-induced change of synaptic Armi was therefore 'locked in,' possibly for the term of the memory, consistent with a role in maintaining an alteration of synaptic function (Ashraf, 2006).

Therefore a new mechanism is proposed for stable memory in which an integrated sensory trigger induces the proteasome-mediated degradation of a RISC factor, releasing synaptic protein synthesis and mRNA transport from microRNA suppression. It is supposed that this mechanism is triggered with neuronal specificity in order to produce memory-specific patterns of protein synthesis. Whether this specificity is required for memory or extends to the level of a single synapse are questions that remain to be addressed (Ashraf, 2006).

The octopamine receptor OAMB mediates ovulation via Ca2+/calmodulin-dependent protein kinase II in the Drosophila oviduct epithelium

Ovulation is an essential physiological process in sexual reproduction; however, the underlying cellular mechanisms are poorly understood. OAMB, a Drosophila G-protein-coupled receptor for octopamine (the insect counterpart of mammalian norepinephrine), is required for ovulation induced upon mating. OAMB is expressed in the nervous and reproductive systems and has two isoforms (OAMB-AS and OAMB-K3) with distinct capacities to increase intracellular Ca2+ or intracellular Ca2+ and cAMP in vitro. This study investigated tissue specificity and intracellular signals required for OAMB's function in ovulation. Restricted OAMB expression in the adult oviduct epithelium, but not the nervous system, reinstated ovulation in oamb mutant females, in which either OAMB isoform was sufficient for the rescue. Consistently, strong immunoreactivities for both isoforms were observed in the wild-type oviduct epithelium. To delineate the cellular mechanism by which OAMB regulates ovulation, protein kinases functionally interacting with OAMB were explored by employing a new GAL4 driver with restricted expression in the oviduct epithelium. Conditional inhibition of Ca2+/Calmodulin-dependent protein kinase II (CaMKII), but not protein kinase A or C, in the oviduct epithelium inhibited ovulation. Moreover, constitutively active CaMKII, but not protein kinase A, expressed only in the adult oviduct epithelium fully rescued the oamb female's phenotype, demonstrating CaMKII as a major downstream molecule conveying the OAMB's ovulation signal. This is consistent with the ability of both OAMB isoforms, whose common intracellular signal in vitro is Ca2+, to reinstate ovulation in oamb females. These observations reveal the critical roles of the oviduct epithelium and its cellular components OAMB and CaMKII in ovulation. It is conceivable that the OAMB-mediated cellular activities stimulated upon mating are crucial for secretory activities suitable for egg transfer from the ovary to the uterus (Lee, 2009).

Mating activates highly coordinated physiological processes in the Drosophila female. During copulation, the female receives somatosensory stimulation, sperm and seminal proteins from the male. These mating signals act at multiple sites in the mated female to activate post-mating responses required for successful reproduction. For example, the seminal protein Ovulin stimulates egg-laying for 1 day after mating (Herndon, 1995). Ovulin is not only present in the base of the ovary right after copulation but it also enters the circulatory system, possibly acting at additional sites. Moreover, the seminal sex peptides Acp70A and DUP99B reduce sexual receptivity and stimulate egg-laying. While sex peptides have widespread binding sites in the central nervous system, endocrine glands, and reproductive tissues in the female, it is the sex peptide receptor SPR in the neurons expressing the sex determination factor Fruitless that is indispensable for reduced receptivity as well as increased egg-laying (Lee, 2009).

The downstream targets and mechanisms that Ovulin and SPR activate in the mated female are unknown. Several studies, nonetheless, indicate octopamine as a key neuromessenger for ovulation (Lee, 2003; Monastirioti, 2003; Cole, 2005; Rodriguez-Valentin, 2006; Middleton, 2006), suggesting it being a downstream signal of Ovulin or SPR for egg-laying. Octopamine is a major monoamine in insects and has similar functions to mammalian norepinephrine. Octopamine is synthesized from tyrosine by sequential actions of tyrosine decarboxylase (dTdc) and tyramine beta-hydroxylase (Tβh). The females defective in dTdc2 encoding neuronal dTdc or tβh are sterile due to defective egg-laying (Monastirioti, 2003; Cole, 2005). Octopaminergic neurons innervate numerous brain and thoracico-abdominal ganglion (TAG) areas. In addition, octopaminergic neurons in the TAG project to reproductive tissues such as the ovaries, oviducts, sperm storage organs and uterus. Indeed, the sterility of tβh females is rescued by restored Tβh expression in a subset of neurons including the TAG neurons that innervate the reproductive system. Consistently, octopamine, when applied to the dissected reproductive system, modulates muscle activities in a tissue-specific manner: it enhances muscle contraction in the ovary but inhibits it in the oviduct (Rodriguez-Valentin, 2006; Middleton, 2006). This suggests that distinct octopamine receptors present in the ovary and oviduct mediate the opposite actions of octopamine on muscle activity (Lee, 2009).

Drosophila has four known octopamine receptors: OAMB, Octβ1R, Octβ2R and Octβ3R. The oamb gene encodes two isoforms OAMB-K3 (K3) and OAMB-AS (AS), which are produced by alternative splicing of the last exon, and differ in the third cytoplasmic loop and downstream sequence. Both K3 and AS transcripts are found in the brain, TAG and reproductive system. When assayed in the heterologous cell lines, both isoforms activate an increase in intracellular Ca2+ while K3 also stimulates a cAMP increase. This implies that the two isoforms may activate distinct combinations of signal transduction pathways in vivo. To investigate OAMB's in vivo functions, several oamb mutants defective in both K3 and AS have been generated, and their prominent phenotype is female sterility (Lee, 2003). While oamb mutant females show normal mating, they are impaired in ovulation, causing abnormal retention of mature eggs in the ovary (Lee, 2003). This raises several important questions regarding mechanism of OAMB activity: where (brain, TAG or reproductive system) does OAMB regulate ovulation? Which isoform is critical for this process and what are the downstream signals? This study shows that the critical site for the OAMB's function in ovulation is the oviduct epithelium, in which transgenic expression of either K3 or AS isoform is sufficient to rescue the oamb female's ovulation defect. Moreover, OAMB recruits CaMKII as a key downstream effector for this function (Lee, 2009).

Octopamine, as a major neurotransmitter, neuromodulator and neurohormone, regulates diverse physiological processes in invertebrates that include sensory information processing, egg-laying, fight or flight responses, and complex neural functions such as learning and memory (Roeder, 2005). These astonishingly diverse effects of octopamine are initiated by the binding of octopamine to G-protein-coupled receptors expressed in distinct tissue or cell types; however, very little is known about relevant octopamine receptors and underlying cellular mechanisms that mediate octopamine's physiological functions. This work has shown that OAMB regulates ovulation in the oviduct epithelium and recruits CaMKII for this function. This role of OAMB is physiological, as opposed to developmental, since restored OAMB expression in the oviduct epithelium at the adult stage is sufficient for reinstating ovulation in oamb females. This is consistent with the findings observed in the octopamine-less dTdc2 and tβh females, in which feeding octopamine only at the adult stage rescues the sterility phenotype of both mutants (Cole, 2005; Lee, 2009 and references therein).

Sex peptides transferred to the female during copulation enhance egg-laying upon binding to the receptor SPR expressed in the Fruitless neurons. The mechanism by which the Fruitless neurons stimulate egg-laying is unknown; however, octopaminergic neurons in the TAG likely represent a downstream target since the egg-laying phenotype of tβh females is rescued by restored TβH expression in these neurons (Monastirioti, 2003). The TAG octopaminergic neurons project axons to various areas in the reproductive track including the ovary, lateral and common oviducts, sperm storage organs and the uterus (Rodriguez-Valentin, 2006; Middleton, 2003). Mating induces distinctive changes in vesicle release at the nerve terminals in different areas of the reproductive track, some of which may represent the TAG octopamine neuronal activities. In the dissected reproductive system, octopamine application augments the amplitude of myogenic contractions of the peritoneal sheath in the ovary while it inhibits stimulated muscle contractions of the oviduct (Rodriguez-Valentin, 2006; Middleton, 2003). These opposite effects of octopamine may be crucial for coordinated constriction and relaxation of the ovary and oviduct, respectively, in transferring a mature egg to the uterus. OAMB may serve as a receptor processing the octopamine's input in the oviduct while another octopamine receptor may mediate the constriction signal in the ovarian peritoneal sheath, which lacks OAMB expression (Lee, 2009).

Remarkably, OAMB's activity is required in the epithelium rather than the muscle for normal ovulation. Consistent with this, the histochemical analysis reported here reveals extensive innervation of the TAG octopamine neuronal processes into the oviduct epithelial layer where both OAMB isoforms are enriched, in addition to the muscle. This raises an important question regarding the nature of an OAMB's role in ovulation. While no information is available on the oviduct epithelium in Drosophila or other insects, studies of the mammalian oviduct indicate active roles of the epithelium in fluid secretion and ciliary activity for gamete and embryo transport. Similarly, it is possible that OAMB in the Drosophila oviduct epithelium is involved in regulating fluid secretion to establish proper luminal environment and possibly ciliary action for egg transport. The capacity of either OAMB-K3 or OAMB-AS to reinstate ovulation in oamb females strongly implicates intracellular Ca²⁺ rather than cAMP as a downstream effector. This is corroborated by findings demonstrating CaMKII as a key epithelial component downstream of OAMB. It is uncertain whether individual isoforms or two isoforms together have comparable efficacies in activating CaMKII and ovulation. Future studies employing quantitative manipulation of transgenic OAMB expression may clarify this issue. Taken together, the epithelial OAMB stimulated upon mating likely activates CaMKII via increased intracellular Ca²⁺, which may in turn trigger biochemical changes necessary for fluid secretion. Potential molecules involved in this process may include transporters, ion channels, Na⁺-K⁺-ATPase and the molecules involved in cilia movements. In the absence of OAMB, epithelial cell activities and fluid may be inadequate for egg movement, leading to ovulation failure. Since octopamine induces relaxation in the dissected oviduct, relaxation may involve another octopamine receptor in the muscle, and concerted activities of OAMB and a muscle receptor may be crucial for successful egg transport. This working model is currently under test (Lee, 2009).

Octopamine regulates oviduct activities in other insects as well. In the locust oviduct, octopamine inhibits the basal tonus and neurally evoked muscle contractions, which are mediated by cAMP-dependent mechanisms. These effects of octopamine may be mediated by an OAMB-like receptor with the different intracellular effector cAMP. Alternatively, they may involve another octopamine receptor(s) present in the muscle. Drosophila has three octopamine receptors (OctβR1, 2 and 3) that can also stimulate cAMP increases. Spatial expression patterns of three OctβRs are as yet unknown. It is conceivable that an OctβR or OctβR-like receptor, possibly present in the Drosophila or locust oviduct muscle, respectively, is additionally involved in ovulation by inducing muscle relaxation through a cAMP signaling pathway. At present, molecular components and cellular pathways controlling ovulation are largely unknown and likewise very little is known about the oviduct functions and mechanisms. The current findings uncover the critical roles of the oviduct epithelium and its cellular components OAMB and CaMKII in ovulation. Future studies to identify additional downstream effectors of OAMB and their functions should help further understanding of the important reproductive process ovulation and provide novel insights into the development of effective insecticides. Typically, intracellular signals activated by G-protein-coupled receptors are characterized in in vitro cell lines. This study has identified the intracellular signal activated by the G-protein-coupled receptor OAMB in vivo that has functional significance. Similar approaches could be applied to other receptors to investigate rather poorly defined cellular mechanisms that G-protein-coupled receptors activate for their in vivo functions (Lee, 2009 and references therein).

Norepinephrine, a mammalian counterpart of octopamine, also plays profound roles in female reproduction by acting on the reproductive and nervous systems. Sympathetic nerve terminals containing norepinephrine innervate the ovaries, oviducts, and uterus. Moreover, norepinephrine levels in the human fallopian tube vary in a region- and estrous cycle-dependent manner being the highest in the isthmus and the fimbriated end at the time of ovulation. When assayed in vitro, adrenergic receptor agonists not only modulate oviduct muscle activities but they also stimulate fluid secretion possibly via Ca²⁺-dependent mechanisms. Oviduct fluid in mammals is critical for egg transport, maturation and fertilization; however, the cellular process regulating its secretion is largely unknown. Damage in the oviduct epithelium is associated with pelvic inflammatory disorder, leading to hydrosalpinx formation and reduced fertility. Thus, enhanced understanding of physiological and cellular factors and processes controlling oviduct fluid will provide significant insights into healthy reproduction as well as impaired fertility associated with pelvic inflammatory disorder and other related disorders (Lee, 2009).

Presynaptic ryanodine receptor-CamKII signaling is required for activity-dependent capture of transiting vesicles

Activity elicits capture of dense-core vesicles (DCVs) that transit through resting Drosophila synaptic boutons to produce a rebound in presynaptic neuropeptide content following release. The onset of capture overlaps with an increase in the mobility of DCVs already present in synaptic boutons. Vesicle mobilization requires Ca²⁺-induced Ca²⁺ release by presynaptic endoplasmic reticulum (ER) ryanodine receptors (RyRs) that in turn stimulates Ca²⁺/calmodulin-dependent kinase II (CamKII). This study shows that the same signaling is required for activity-dependent capture of transiting DCVs. Specifically, the CamKII inhibitor KN-93, but not its inactive analog KN-92, eliminated the rebound replacement of neuropeptidergic DCVs in synaptic boutons. Furthermore, pharmacologically or genetically inhibiting neuronal sarco-endoplasmic reticulum calcium ATPase (SERCA) to deplete presynaptic ER Ca²⁺ stores or directly inhibiting RyRs prevented the capture response. These results show that the presynaptic RyR-CamKII pathway, which triggers mobilization of resident synaptic DCVs to facilitate exocytosis, also mediates activity-dependent capture of transiting DCVs to replenish neuropeptide stores (Wong, 2009).

The function of nerve terminals depends on vesicular delivery of proteins synthesized in the soma to synaptic boutons. Transport vesicles are known to contain channels, active zone constituents and neuropeptides. In contrast to synaptic membrane proteins and classical transmitters that are recycled following exocytosis, neuropeptide release is irreversible. Thus, peptidergic transmission depends on replacement of neuropeptide-containing dense core vesicles (DCVs). This is potentially a very slow process because delivery of vesicles synthesized in the soma to nerve terminals by fast axonal transport can take days. However, a cell biological strategy has been discovered that bypasses such delays. Activity-dependent capture of transiting vesicles utilizes a pool of DCVs that rapidly pass through the resting nerve terminal, but that are captured in response to a burst of activity (Shakiryanova, 2006). The onset of this capture, which is evident as decreased DCV efflux and increased neuropeptide content in synaptic boutons, occurs over a period of minutes instead of the hours that would be required for conventional steady state DCV replacement. Essentially, the nerve terminal can tap into the transiting DCV pool to rapidly replenish neuropeptide stores without any direct involvement of the soma. Hence, activity-dependent capture of transiting DCVs eliminates the delay in delivering nascent DCVs, apportions resources based on activity and places control of synaptic neuropeptide storage at sites of release instead of the site of synthesis (i.e., the soma) (Shakiryanova, 2006). A similar recruitment process also occurs with neurotrypsin-containing vesicles, which were concluded to rapidly undergo exocytosis following stimulated capture (Frischknecht, 2008). Likewise, vesicle capture appears to be involved in release of presynaptic Wnt/Wingless protein. Therefore, activity-dependent capture of transiting vesicles supports synaptic neuropeptide, enzyme and developmental peptide release (Wong, 2009).

The signaling required for activity-dependent capture of transiting DCVs is unknown. The long duration of this response in Drosophila motor neurons (i.e., for ~0.5 hour) coupled with the requirement for electrical activity suggests a potential involvement of Ca²⁺-induced phosphorylation. In fact, recent experiments have shown that such signaling increases the mobility of resident DCVs in synaptic boutons. Mobilization, which is triggered by Ca²⁺ influx and persists for ~10 minutes (Shakiryanova, 2005), requires Ca²⁺-induced Ca²⁺ release from presynaptic endoplasmic reticulum (ER) via ryanodine receptors (RyRs) (Shakiryanova, 2007). Subsequently, Ca²⁺/calmodulin-dependent protein kinase II (CamKII) is activated as a necessary step for DCV mobilization (Shakiryanova, 2007). The overlapping onset of the capture and mobilization responses in the first minutes following a brief tetanus stimulated an investigation of whether the RyR-CamKII pathway participates in activity-dependent capture of transiting vesicles (Wong, 2009).

In this study a GFP (Green Fluorescent Protein)-tagged neuropeptide was imaged at the intact Drosophila neuromuscular junction. The rebound in synaptic neuropeptide stores following activity-evoked release, which is caused by capture of transiting vesicles (Shakiryanova, 2006), requires RyR-mediated Ca²⁺ efflux from presynaptic ER and activation of CamKII. Therefore, RyR-CamKII signaling initiates both mobilization of resident DCVs within synaptic boutons and capture of DCVs from the rapidly transiting pool (Wong, 2009).

In vivo imaging has shown that a brief bout of activity elicits prolonged DCV mobilization and capture. These processes are independent because capture requires axonal transport while mobilization does not (Shakiryanova, 2005; Shakiryanova, 2006). Nevertheless, the onsets of mobilization of resident DCVs and capture of transiting DCVs overlap (i.e., both develop over minutes following seconds of activity). This observation stimulated a test of the hypothesis that these two mechanisms are initiated by the same signaling. Previous studies had established that Ca²⁺ influx triggers DCV mobilization by activating RyR-mediated Ca²⁺ release from presynaptic ER that in turn stimulates CamKII (Shakiryanova, 2007). The pharmacological and genetic experiments presented in this study establish that RyR-CamKII signaling is also required for activity-dependent capture of transiting DCVs (Wong, 2009).

This finding raises the issue of how a single signaling pathway produces responses with different durations: after seconds of activity, DCV mobilization lasts ~10 minutes, while the capture response lasts ~40 minutes (Shakiryanova, 2005; Shakiryanova, 2006). One possible consideration is that these kinetic differences could originate in the processes responsible for reversal of mobilization and capture. Specifically, RyR-CamKII signaling could initiate the two processes in parallel, but dephosphorylation of distinct CamKII substrates might occur at different rates, possibly because of the involvement of different phosphatases. This potential explanation suggests that identifying the CamKII substrates that mediate mobilization and capture will be important for understanding the diversity in long-lasting responses initiated by activity-triggered presynaptic RyR-CamKII signaling. Recently, CamKII-dependent phosphorylation of kinesin superfamily protein 17 (KIF17) was found to be essential for unloading NMDA receptor-carrying cargoes from microtubules near the postsynaptic density (Guillaud, 2008). Therefore, CamKII might induce capture by triggering dissociation of transiting DCVs from their molecular motor dynactin complex, which would contain both a kinesin-3 family member UNC-104/Kif1 and a dynein retrograde motor, while mobilization might depend on another CamKII substrate. Alternatively, some process downstream of dephosphorylation might be rate determining for reversal of capture. For example, once captured vesicles are committed to return to the transiting pool, they might need to recruit an unoccupied motor complex to support rapid transiting. If such complexes are rare, then recovery from capture would be very slow. In contrast, recovery from mobilization, which does not require exiting from the bouton, would not be limited in the same way. Regardless of the specific basis for the diverse time courses of mobilization and capture, the use of the same signaling pathway to induce both of these effects is an elegant means to ensure that facilitation of release is coupled to replacement of depleted synaptic neuropeptide stores (Wong, 2009).

Differential control of presynaptic CaMKII activation and translocation to active zones

The release of neurotransmitters, neurotrophins, and neuropeptides is modulated by Ca(2+) mobilization from the endoplasmic reticulum (ER) and activation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). Furthermore, when neuronal cultures are subjected to prolonged depolarization, presynaptic CaMKII redistributes from the cytoplasm to accumulate near active zones (AZs), a process that is reminiscent of CaMKII translocation to the postsynaptic side of the synapse. However, it is not known how presynaptic CaMKII activation and translocation depend on neuronal activity and ER Ca(2+) release. This study addresses these issues in Drosophila motoneuron terminals by imaging a fluorescent reporter of CaMKII activity and subcellular distribution. It is reported that neuronal excitation acts with ER Ca(2+) stores to induce CaMKII activation and translocation to a subset of AZs. Surprisingly, activation is slow, reflecting T286 autophosphorylation and the function of presynaptic ER ryanodine receptors (RyRs) and inositol trisphosphate receptors (IP3Rs). Furthermore, translocation is not simply proportional to CaMKII activity, as T286 autophosphorylation promotes activation, but does not affect translocation. In contrast, RNA interference-induced knockdown of the AZ scaffold protein Bruchpilot disrupts CaMKII translocation without affecting activation. Finally, RyRs comparably stimulate both activation and translocation, but IP3Rs preferentially promote translocation. Thus, Ca(2+) provided by different presynaptic ER Ca(2+) release channels is not equivalent. These results suggest that presynaptic CaMKII activation depends on autophosphorylation and global Ca(2+) in the terminal, while translocation to AZs requires Ca(2+) microdomains generated by IP3Rs (Shakiryanova, 2011).

Although CaMKII alters nerve terminal excitability, neurosecretion, and development, direct studies of CaMKII activation and translocation in living nerve terminals are lacking because of technical limitations. For example, the low level of CaMKII in the nerve terminal compared with the postsynaptic density, as well as the small size of most boutons, hinders light level in immunohistochemical assays of presynaptic CaMKII translocation. Electron microscopy coupled with immunodetection overcomes limitations in spatial resolution, but cannot follow CaMKII dynamics in a living preparation. Thus, the dependence of presynaptic CaMKII enzymatic activity and translocation on stimulation frequency, ER Ca2+ channels, and autophosphorylation was unknown. In this study a FRET indicator that detects both activation and translocation was shown to reproduce known features of presynaptic CaMKII behavior and was then used to determine how CaMKII dynamics is controlled in an intact living nerve terminal (Shakiryanova, 2011).

First, stimulation of presynaptic CaMKII by electrical stimulation, autophosphorylation, and ER Ca2+ channels was quantified in real time. These experiments established that CaMKII activation is surprisingly slow and frequency dependent. This property likely reflects the fact that Ca2+ entry via presynaptic Cav channels alone is not optimal for activating CaMKII. Rather, stimulation of CaMKII also is promoted by presynaptic ER Ca2+ release and autophosphorylation. The similarity of CaMKII kinetics induced by disrupting either of the latter processes suggests autophosphorylation is promoted by the increased activation of CaMKII by ER Ca2+. Furthermore, the participation of RyRs and IP3Rs, the requirement for Cav channels, and the insensitivity to knockdown of BRP, which enhances Cav channel clustering at AZs, imply that presynaptic CaMKII activation is driven by global presynaptic Ca2+ elevation induced by ER and surface Ca2+ channels (Shakiryanova, 2011).

Imaging experiments also revealed that CaMKII translocation toward AZs detected by immunoelectron microscopy in depolarized cultured neurons also occurs with native electrical activity in vivo. Experiments that used BRP immunolocalization or somewhat less efficient transgenic fluorescent protein labeling showed that presynaptic CaMKII clusters usually accumulate at AZs. This does not reflect a simple aggregation of CaMKII already localized near AZs because CaMKII initially localized away from AZs was depleted. Furthermore, once fully formed CaMKII clusters localized near AZs, their motion was limited. Therefore, it seems that initially diffuse presynaptic CaMKII forms clusters that are tethered near AZs (Shakiryanova, 2011).

Strikingly, disrupting autophosphorylation, which reduces activation, does not affect presynaptic CaMKII translocation. This might reflect that activating a subset of the subunits in the CaMKII dodecamer is sufficient for translocation. However, translocation is not a simple consequence of limited activation because, even though CaMKII activation tends to be greater after inhibiting IP3Rs than RyRs, translocation is more markedly disrupted by IP3R inhibition. Furthermore, CaMKII clustering at AZs can long outlast stimulated elevation of presynaptic Ca2+, implying that Ca2+ alone cannot account for persistent translocation. Finally, CaMKII translocates only to a subset of AZs, suggesting heterogeneity between AZs. Differential release between AZs in a single type Ib bouton has recently been demonstrated. Hence, it is possible that a single mechanism induces heterogeneity in AZ-mediated transmitter release and recruitment of CaMKII (Shakiryanova, 2011).

The results suggest that presynaptic CaMKII translocation is preferentially induced by IP3R-generated Ca2+ microdomains located at a subset of AZs. Consistent with this proposal, CaMKII translocation requires the AZ-associated scaffold protein BRP, which is long enough to separately integrate signals from Ca2+ microdomains generated by AZ Cav channel clusters at its C terminus and IP3Rs potentially located at its N terminus. BRP might affect CaMKII translocation also because it enhances Cav channel clustering and it is a potential CaMKII substrate. In contrast, RyRs may be positioned further away from BRP so that their local Ca2+ release cannot mimic the action of IP3Rs. Such a differential distribution of ER Ca2+ channels has not been studied in Drosophila nerve terminals, but is consistent with independent localization and function of RyRs and IP3Rs detected in other cell types and neuronal compartments. According to this view, diffuse CaMKII samples global Ca2+ changes induced by Cav and all ER Ca2+ channels to activate, but local Ca2+ changes generated by IP3Rs are needed for recruitment of CaMKII. With such a model, depletion of the IP3-sensitive Ca2+ pool would account for the observed dispersal of clusters that sometimes occurs during stimulation (Shakiryanova, 2011).

These experiments suggest that translocation is limited to a subset of AZs and is delayed compared with CaMKII activation and synaptic transmission. Thus, it is unlikely that translocation occurs for rapid acute regulation of transmitter release. Indeed, the protocols typically used for quantal analysis (e.g., low-frequency action potentials in the presence of low Ca2+) are unlikely to induce much CaMKII signaling. However, motor neurons fire in bursts that in vivo imaging experiments show are sufficient to translocate presynaptic CaMKII. Thus, future experiments will have to use more physiological stimuli to probe whether CaMKII is recruited to AZs to regulate synaptic transmission. Such effects could be selective, possibly because CaMKII is recruited to AZs that are most active and/or positioned near ER IP3Rs. Interestingly, another consequence of translocation to AZs is that CaMKII is depleted from the presynaptic cytoplasm. Hence, CaMKII activity can be redirected away from substrates that are excluded from AZs (e.g., neuropeptide vesicles and K+ channels) to substrates preferentially localized at AZs. In this way, the role of CaMKII in the nerve terminal changes dynamically depending both on its enzymatic activity and location (Shakiryanova, 2011).

Protein Interactions

The gene for Drosophila calcium/calmodulin-dependent protein kinase II is alternatively spliced to generate up to 18 different proteins that vary only in a region analogous to the point where mammalian alpha, beta, gamma, and delta isozymes show the greatest divergence from one another. By several criteria (domain organization, low affinity for calmodulin, holoenzyme structure, and ability to autophosphorylate and become independent of calcium), these proteins are functional homologs of the mammalian calcium/calmodulin-dependent protein kinase II. Two major isoform-specific catalytic differences are observed. First, the R3A isoform is found to have a significantly higher K of activation for calmodulin than the other isoforms. This indicates that the variable region, which is located distal to the calmodulin-binding domain, may play a role in activation of the enzyme by calmodulin. Decreased sensitivity to calmodulin may be biologically important if free calmodulin is limiting within the neuron. The second catalytic difference noted is that the R6 isoform has a significantly lower K(m) for the peptide substrate used in this study. Although the variable region is not in the catalytic part of the enzyme, it may have an indirect function in substrate selectivity (GuptaRoy, 1996a).

Isoforms of calcium/calmodulin-dependent protein kinase II from Drosophila (R1-R6 and R3A) showed differential activation by two series of mutant calmodulins: B1K-B4K and B1Q-B4Q (Maune, 1992). These mutant calmodulins were generated by changing a glutamic acid in each of the four calcium binding sites to either glutamine or lysine, altering their calcium binding properties. All mutations produce activation defects, the most severe being binding site 4 and B1Q mutants. Activation differs substantially between isoforms. R4, R5, and R6 are the least sensitive to mutations in calmodulin, while R1, R3, and R3A are the most sensitive. Activation of R1 and R2 by B4K and activation of R3 and R3A by B2K and B2Q produced significant (6-fold and almost 3-fold, respectively) differences in Kact between isoforms that differ structurally by a single amino acid. These differences could not be accounted for by differential binding, as all isoforms show almost identical binding patterns with the mutants. High binding affinity does not always correlate with ability to increase enzyme activity, implying that activation occurs in at least two steps. The isoform-specific differences seen in this study reflect a role for the COOH-terminal variable region in activation of CaM kinase II (GuptaRoy, 1996b).

Drosophila calcium/calmodulin-dependent protein kinase II is alternatively spliced to generate multiple isoforms that vary only in a region between the calmodulin-binding domain and the association domain. This variation has been shown to modulate activation of the enzyme by calmodulin. In this study the ability is examined of seven of the Drosophila isoforms to phosphorylate purified protein substrates and to be inhibited by a substrate analog, and the response of six of the isoforms to a mutant form of calmodulin (V91G) that was isolated in a genetic screen. Significant variation in Kms for Eag, a potassium channel, and Adf-1, a transcription factor, were found. In the case of a peptide inhibitor, AC3I, there are significant variations in Ki between isoforms. Kact for V91G calmodulin is increased for all of the isoforms. In addition, one isoform, RI, exhibits a lower Vmax when assayed with this mutant CaM. These results indicate that the variable domain of calcium/calmodulin-dependent protein kinase II is capable of altering the substrate specificity of the catalytic domain and the activation response to calmodulin (GuptaRoy, 2000).

Light activation of rhodopsin in the Drosophila photoreceptor induces a G protein-coupled signaling cascade that results in the influx of Ca2+ into the photoreceptor cells (the visual signal transduction pathway). Immediately following light activation, phosphorylation of a photoreceptor-specific protein, phosrestin I, is detected. Strong sequence similarity to mammalian arrestin and electroretinograms of phosrestin mutants suggest that phosrestin I is involved in light inactivation. The identity of the protein kinase responsible for the phosphorylation of phosrestin I was sought, to link the transmembrane signaling with the light-adaptive inactivation response. Type II Ca2+/calmodulin-dependent kinase is one of the major classes of protein kinases that regulate cellular responses to transmembrane signals. Partially purified phosrestin I kinase activity can be immunodepleted and immunodetected with antibodies to Ca2+/calmodulin-dependent kinase II; the kinase activity exhibits regulatory properties that are unique to Ca2+/calmodulin-dependent kinase II, such as Ca2+ independence after autophosphorylation and inhibition by synthetic peptides containing the Ca2+/calmodulin-dependent kinase II autoinhibitory domain. Ca2+/calmodulin-dependent kinase KII activity is present in Drosophila eye preparations. These results are consistent with the hypothesis that Ca2+/calmodulin-dependent kinase II phosphorylates phosrestin I. It is concluded that Ca2+/calmodulin-dependent kinase II plays a regulatory role in Drosophila photoreceptor light adaptation (Kahn, 1997).

Phototransduction in vertebrates and invertebrates is a complex signal transduction cascade, based on rhodopsin-G-protein coupling interactions. The phototransduction process has the capacity to amplify single photon events into large electrical signals and to regulate the photoresponse output in a broad dynamic range. Major advances have been made in characterizing the molecular components of phototransduction and the mechanisms of light adaptation and response termination. However, the modulation of these latter processes is not yet fully understood. The powerful combination of molecular genetics and electrophysiology makes Drosophila photoreceptors an exquisite preparation for studying these processes. In Drosophila, light activation of rhodopsin activates phospholipase C via G-proteins; phospholipase C hydrolyzes phosphatidylinositol-4,5-bisphosphate into inositol trisphosphate (IP3) and diacylglycerol (DAG). This process leads within a few tens of milliseconds to the opening of cation-selective channels encoded by the trp and trp-like genes. The feedback control of the activation process involves calcium-calmodulin (Ca2+/calmodulin), which tightly regulates the adaptation and termination of the light response (Peretz, 1998 and references).

The photoreceptor potential has a complex waveform that arises from the opening of light-activated channels as well as from voltage-dependent conductances. Interestingly, Drosophila photoreceptors are endowed with high densities of voltage-gated K+ channels. In neurons, K+ channels were recognized to regulate action potential duration, firing patterns, and resting membrane potential. A great diversity of K+ channel subtypes appears to underlie these pleiotropic functions. Analysis of Drosophila mutants enables the initial molecular characterization of several classes of K+ channels. Four different voltage-sensitive K+ channel genes are initially identified in Drosophila: Shaker and Shal, encoding A-type K+ currents (I_A), and Shab and Shaw, encoding delayed-rectifier K+ currents (I_K). Subsequently, other classes of K+ channels were characterized molecularly in Drosophila (Peretz, 1998 and references).

Drosophila photoreceptors express both I_A and I_K currents, with the former mediated by subunits encoded by the Shaker locus. However, the genes encoding the delayed-rectifier channel subunits have not yet been identified, and very little is known about I_K modulation. Although the functional significance of I_A and I_K remains to be clarified in Drosophila phototransduction, in Limulus and in the blowfly Calliphora vicina I_A and I_K may regulate the gain and frequency response during light and dark adaptation. In Drosophila photoreceptors, the sustained depolarization generated by light activation of transient receptor potential (TRP) and transient receptor potential-like (TRPL) cationic channels is expected to open voltage-gated K+ channels. One can predict that the subsequent hyperpolarizing K+ currents will oppose the light-induced depolarizing currents to shape the photoreceptor potential (Peretz, 1998).

Drosophila photoreceptors I_A andI_K are regulated by calcium-calmodulin (Ca2+/calmodulin). Photoreceptors I_K and I_A are specifically inhibited by different Ca2+/calmodulin antagonists such as W7 or TFP, with I_K being far more sensitive than I_A. This regulation occurs via a Ca2+/calmodulin-dependent protein kinase (CaM kinase), with I_K once again being far more sensitive than I_A. Inhibition of Ca2+/calmodulin by N-(6 aminohexyl)-5-chloro-1-naphthalenesulfonamide or trifluoperazine markedly reduces the K+ current amplitudes. Likewise, inhibition of CaM kinases by KN-93 potently depresses I_K and accelerates its C-type inactivation kinetics. Thus the modulation by the Ca2+/calmodulin antagonists is mimicked by two selective CaM kinase antagonists, KN-62 and KN-93. The effect of KN-93 is specific because its structurally related (but functionally inactive) analog KN-92 is totally ineffective. In Drosophila photoreceptor mutant ShKS133, which allows isolation of I_K, it is demonstrated by current-clamp recording that inhibition of I_K by quinidine or tetraethylammonium increases the amplitude of the photoreceptor potential, depresses light adaptation, and slows down the termination of the light response. Similar results are obtained when CaM kinases are blocked by KN-93. The mechanisms whereby I_K and I_A are depressed after exposure to CaM kinase inhibitors are not elucidated yet, but may involve either a direct channel phosphorylation or an indirect modulation. With respect to indirect regulation, it is possible that the CaM kinase mediates its effect via the Eag channel subunit. Shab 1 and Shab 2 isoforms as well as Shaw transcripts are expressed in Drosophila retina, indicating that Shab and Shaw gene products could possibly be direct or indirect substrates of CaM kinase regulation. In this regard, it is worth noting that the Shab channel subunit contains four consensus sites for phosphorylation by CaM kinase [XRXXS] at its intracellular amino and C termini. These findings place photoreceptor K+ channels as an additional target for Ca2+/calmodulin and suggest that I_K is well suited to act in concert with other components of the signaling machinery to sharpen light response termination and fine tune photoreceptor sensitivity during light adaptation (Peretz, 1998).

Slob has been identified as a novel protein that binds to the carboxy-terminal domain of Slowpoke. A yeast two-hybrid screen with Slob as bait identifies the zeta isoform of 14-3-3 as a Slob-binding protein. All three proteins are colocalized presynaptically at Drosophila neuromuscular junctions. 14-3-3 is known to be highly enriched in synaptic boutons at the neuromuscular junction and is present only at much lower levels in the motor axon and muscle (Broadie, 1997). Slob is also enriched in synaptic boutons, although its distribution appears to be less restricted than that of 14-3-3. Both 14-3-3 and dSlo are prominent in synaptic boutons, where they colocalize. Two serine residues in Slob are required for 14-3-3 binding, and the binding is dynamically regulated in Drosophila by calcium/calmodulin-dependent kinase II (CaMKII) phosphorylation of these residues. Slob itself increases the voltage sensitivity of dSlo, whereas 14-3-3 decreases the channel's voltage sensitivity (Zhou, 1999).

What are the molecular details of the profound downregulation of dSlo channel activity by 14-3-3? Members of the family of K_Ca channels are subject to modulation by a variety of molecular mechanisms, ranging from protein phosphorylation to oxidation/reduction reactions. It is conceivable that simply the binding of 14-3-3 to dSlo via Slob is sufficient to alter the gating of the channel, as appears to be the case for ß subunit interactions with Slowpoke and other potassium channels. Alternatively, 14-3-3 may act as another scaffolding component, to bring one of the protein kinases that it is known to bind into the proximity of the channel. Indeed, because 14-3-3 dimerizes, it might bridge the interactions of several different signaling proteins with the channel. It will be interesting to determine whether the Raf protein kinase, one of the kinases that binds 14-3-3, can phosphorylate and modulate dSlo, because Raf is a key player in the mitogen-activated protein (MAP) kinase pathway that conveys signals from the plasma membrane to the cell nucleus. Activation of this pathway can influence ion channel expression and activity, and potassium channel activity in turn can modulate tyrosine kinase signaling in cells. Thus, the present findings raise the intriguing possibility that a potassium channel regulatory complex is involved in MAP kinase signaling and the regulation of many fundamental cell processes (Zhou, 1999 and references).

The finding that the interaction of Slob with 14-3-3 requires Slob phosphorylation is consistent with studies of other 14-3-3 binding proteins. It is especially intriguing that the binding can be regulated in vivo by changes in the activity of CaMKII; these results suggest that there may be dynamic physiological regulation of dSlo channel activity by 14-3-3 that depends on the phosphorylation state of Slob. In view of the presynaptic colocalization of the three proteins described here, it is interesting that it is the CaMKII phosphorylation of Slob that regulates 14-3-3 binding. CaMKII is also present at a high concentration presynaptically in Drosophila, and thus the same calcium rise that evokes transmitter release might promote phosphorylation of Slob, binding of 14-3-3, and downregulation of dSlo (Zhou, 1999 and references).

Discs large (DLG) mediates the clustering of synaptic molecules. Synaptic localization of Dlg itself is regulated by CaMKII. Dlg and CaMKII colocalize at synapses and exist in the same protein complex. Constitutively activated CaMKII phenocopies structural abnormalities of dlg mutant synapses and dramatically increases extrajunctional Dlg. Decreased CaMKII activity causes opposite alterations. This was most clearly demonstrated by examining the size of the postsynaptic junctional membrane -- the SSR. Constitutive CaMKII activation results in poorly developed SSR, while CaMKII inhibition results in an overdeveloped SSR. These alterations phenocopy previously described effects of changing Dlg levels at the synaptic membrane. Mutations in dlg result in a poorly developed SSR, while overexpressing wild-type Dlg results in an overdeveloped SSR. The similarity between the phenotypes elicited by constitutive activation of CaMKII and those observed in dlg mutants is not solely restricted to the morphology of the SSR. An increase in the number of active zones, an enlargement of the boutons, and an abnormal Fas II clustering around these boutons is observed. Thus, these results strongly suggest that the changes in synaptic structure and composition elicited by changing CaMKII activity are the result of changes in DLG distribution. In vitro, CaMKII phosphorylates a Dlg fragment with a stoichiometry close to one. Moreover, expression of site-directed dlg mutants that block or mimic phosphorylation has effects similar to those observed upon inhibiting or constitutively activating CaMKII. It is proposed that CaMKII-dependent Dlg phosphorylation regulates the association of Dlg with the synaptic complex during development and plasticity, thus providing a link between synaptic activity and structure (Koh, 1999).

Antibodies against Drosophila CaMKII were used to determine its distribution at the larval NMJ. CaMKII is localized at wild-type NMJs around type I synaptic boutons in a pattern similar to Dlg. Double labeling with anti-Dlg reveals that Dlg and CaMKII are colocalized at bouton borders. In addition, transgenic CaMKII is targeted to synaptic boutons, as visualized by increased immunoreactivity levels at NMJs, upon expression of a CaMKII transgene in both motor neurons and muscles, by using muscle specific and motor neuron specific GAL4 activators. CaMKII and DLG colocalization is supported by immunoprecipitation experiments. Both proteins exist in the same complex in body wall muscles. When anti-CaMKII is used for immunoprecipitations of body wall muscle extracts, Dlg coimmunoprecipitates from wild-type, CaMKII+, or CaMKII-T287D overexpressing extracts. Moreover, transgenic Dlg expressed in dlg^X1-2 mutants is also coimmunoprecipitated by CaMKII. Thus, Dlg and CaMKII are closely associated at type I NMJs (Koh, 1999).

It is proposed that the anchoring of Dlg at the synapse is optimal when Dlg is in the dephosphorylated state. Upon phosphorylation, Dlg is less restricted to the synaptic complex. Because Dlg is essential for proper synaptic localization of Fas II and Shaker, these binding partners are similarly free to move away from the synaptic complex upon CaMKII-dependent phosphorylation. An alternative possibility however, and one that is not mutually exclusive, is that CaMKII-dependent phosphorylation of Dlg affects its targeting to the synapse during development. However, CaMKII and DLG colocalize at synapses. Therefore, it is likely that CaMKII-dependent phosphorylation of Dlg occurs at the synapse. It is known that synaptic Fas II is downregulated by increased cAMP levels, suggesting that PKA is also involved in coupling synaptic activity to structural plasticity at NMJs. Both PKA and CaMKII can be activated by various synaptic stimuli. Whether both signal transduction pathways act together or in parallel to regulate Dlg-dependent localization of Fas II remains to be determined (Koh, 1999 and references).

Drosophila Uba2 and Ubc9 (Lesswright) SUMO-1 conjugation enzyme homologs (DmUba2 and DmUbc9) were isolated as calcium/calmodulin-dependent kinase II (CaMKII) interacting proteins by yeast two-hybrid screening of an adult head cDNA library. At least one isoform of Drosophila neuronal CaMKII is conjugated to DmSUMO-1 (see SUMO) in vivo. The interactions observed in the two-hybrid screen may therefore reflect catalytic events. To understand the role of SUMO conjugation in the brain, a characterization of the system was undertaken. The other required components of the system, Drosophila Aos1 and SUMO-1 (DmAos1 and DmSUMO-1), were identified in expressed sequence tag data base searches. Purified recombinant DmUba2/DmAos1 dimer can activate DmSUMO-1 in vitro and transfer DmSUMO-1 to recombinant DmUbc9. DmSUMO-1 conjugation occurs in all developmental stages of Drosophila and in the adult central nervous system. Overexpression of a putative dominant negative DmUba2(C175S) mutant protein in the Drosophila central nervous system resulted in an increase in overall DmSUMO-1 conjugates and a base-sensitive p120 species, which is likely to be DmUba2(C175S) linked to endogenous DmSUMO-1 through an oxygen ester bond. Overexpression of DmUba2(wt) protein in vivo also led to increased levels of DmSUMO-1 conjugates. High level overexpression of either DmUba2(wt) or DmUba2(C175S) in the Drosophila central nervous system caused pupal and earlier stage lethality. Expression in the developing eye led to a rough eye phenotype with retinal degeneration. These results suggest that normal SUMO conjugation is essential in the differentiated nervous system and reveal a potential novel mechanism that regulates neuronal calcium/calmodulin-dependent protein kinase II function (Long, 2000).

Calcium/calmodulin dependent kinase II (CaMKII), PDZ-domain scaffolding protein Discs-large (Dlg), immunoglobin superfamily cell adhesion molecule Fasciclin 2 (Fas2) and the position specific (PS) integrin receptors, including ßPS (Myospheroid) and its alpha partners (alphaPS1, alphaPS2, PS3/alpha Volado), are all known to regulate the postembryonic development of synaptic terminal arborization at the Drosophila neuromuscular junction (NMJ). Recent work has shown that Dlg and Fas2 function together to modulate activity-dependent synaptic development and that this role is regulated by activation of CaMKII. PS integrins function upstream of CaMKII in the development of synaptic architecture at the NMJ. ßPS integrin physically associates with the synaptic complex anchored by the Dlg scaffolding protein, which contains CaMKII and Fas2. This study demonstrates an alteration of the Fas2 molecular cascade in integrin regulatory mutants, as a result of CaMKII/integrin interactions. Regulatory ßPS integrin mutations increase the expression and synaptic localization of Fas2. Synaptic structural defects in ßPS integrin mutants are rescued by transgenic overexpression of CaMKII (proximal in pathway) or genetic reduction of Fas2 (distal in pathway). These studies demonstrate that ßPS integrins act through CaMKII activation to control the localization of synaptic proteins involved in the development of NMJ synaptic morphology (Beumer, 2002).

ßPS integrin is a transmembrane receptor present in both pre- and post-synaptic membranes at the larval NMJ. Dlg is a synaptic scaffolding protein associated with both pre-and post-synaptic membranes and is also involved in the regulation of synaptic morphology, through the localization of diverse transmembrane proteins (including Fas2). Studies were undertaken to determine whether ßPS integrin associates with the DLG complex to tie together these disparate receptor components in a common molecular machine. In confocal analyses, ßPS integrin and Dlg co-localize at the larval NMJ. Dlg clearly has less extensive expression, more tightly localized at NMJ boutons, whereas ßPS is more extensive through the subsynaptic reticulum (SSR) and also localized at extrasynaptic sites in the muscle, including the muscle sarcomere and attachment sites. However, both proteins are most intensively expressed at the NMJ presynaptic/postsynaptic interface, where they co-localize. Therefore, tests were performed to determine if ßPS integrins form part of the synaptic complex linked by Dlg. Protein immunoprecipitation assays were performed using rabbit anti-DLG to probe Oregon R head extracts. Dlg antibodies clearly co-immunoprecipitate Dlg and ßPS integrin protein, consistent with co-localization observed in confocal analysis. Inspection of the ratio of both bound proteins and proteins not bound to beads (immunoprecipitation versus flow through lanes) indicates that a large portion of the ßPS integrin protein associates with a complex containing Dlg. The fact that ßPS was found to co-immunoprecipitate with the complex mediated by Dlg provides support for integrins existing in a synaptic complex with Fas2 and CaMKII at the synapse (Beumer, 2002).

To assay the mys requirement in synaptic development comprehensively, two regulatory alleles of mys with opposing structural phenotypes were compared and contrasted: mys^b9, which causes NMJ undergrowth, and mys^ts1, which causes NMJ overgrowth. In both mutants, a correlation was observed between the morphological alterations and synaptic function, in contrast to the homeostasis seen in Fas2 mutant synapses but consistent with the parallel alterations seen in CaMKII-inhibited synapses. However, the correlation between synaptic structural and functional alterations in mys mutants is not striking, and it is clear that integrins primarily mediate architectural regulation. Therefore, the focus in this study was exclusively on the mechanism(s) by which integrins modulate NMJ structural development. This article presents molecular and genetic evidence that strongly support the hypothesis that synaptic integrin receptors containing ßPS modulate CaMKII activation on both sides of the synaptic cleft and, through CaMKII, control both the expression and the synaptic localization of Fas2 at the synapse. The primary experimental results supporting these conclusions are detailed below (Beumer, 2002).

(1) Transgenic expression of CaMKII is sufficient to completely rescue all synaptic structure defects in mys mutants. Genetically increasing CaMKII in postsynaptic muscle, but not presynaptic neuron, completely rescues the structural undergrowth of the mys^b9 integrin mutant, whereas it is necessary to elevate CaMKII both pre- and post-synaptically to rescue structural overgrowth in the mys^ts1 mutant. These results are consistent with the postsynaptic mislocalization of ßPS integrin in the mys^b9 mutant, as opposed to the global loss of synaptic integrin in the mys^ts1 mutant. These data indicate that coordinate regulation of CaMKII in the muscle and motoneuron is necessary for proper development of synaptic architecture (Beumer, 2002).

(2) At the distal end of the cascade, both the expression and synaptic localization of Fas2 are increased in mys mutants, although the extent of Fas2 misregulation is significantly different between the two regulatory mutants. Importantly, both the NMJ overgrowth (mys^ts1) and undergrowth (mys^b9) phenotypes are rescued towards wild-type structure by genetically reducing the amount of Fas2 available at the synapse to near normal levels. In mys^ts1 mutants, correcting for Fas2 level suppresses the synaptic overgrowth, while in mys^b9 mutants, correcting the Fas2 level suppresses the synaptic undergrowth. These results support the conclusion that Fas2 is centrally involved in mediating synaptic growth, but suggest that the Fas2-mediated mechanism is more complex than previously thought (Beumer, 2002).

The results demonstrate that integrins regulate morphological growth at the postembryonic NMJ through activation of CaMKII in both pre- and post-synaptic compartments. One important target of CaMKII is Fas2 and it is clear that regulation of Fas2 expression and localization is an important component of integrin regulation. However, the modulation of Fas2 levels alone is unlikely to account fully for the alterations in synaptic architecture in integrin mutants. In particular, both mys^ts1 and mys^b9 upregulate synaptic Fas2 levels, albeit to different degrees, yet show opposite alterations in synaptic growth. Moreover, reducing Fas2 levels rescues both under- and overgrowth defects, but the rescue is not perfect (Beumer, 2002).

Precise control of Fas2 levels finely tunes morphological development at the NMJ. Different degrees of reduced Fas2 expression can either facilitate or inhibit the growth/maintenance of the NMJ, and reduced Fas2 expression has been demonstrated in other overgrowth mutants. However, overexpression of Fas2 in specific muscles drives increased NMJ elaboration/bouton differentiation and selective preference for a high-expressing muscle over a low-expressing muscle. How can this complexity be interpreted? One likely explanation is interaction between Fas2 and other developmental pathways regulated by CaMKII. To date, the only known Fas2-independent regulation of synaptic morphology involves a deubiquitinating protease encoded by fat facets (faf), and a putative ubiquitin ligase encoded by highwire (hiw), which have been shown to work together to modulate the degree of NMJ growth. Loss-of-function hiw mutants display NMJ structural overgrowth, importantly without a concomitant decrease in Fas2. Indeed, overexpression of Fas2 cannot suppress the overgrowth seen in hiw mutants. These observations are consistent with the overgrowth combined with overexpression of Fas2 observed in mys^ts1 mutants in this study. However, any putative interaction between Fas2-dependent and -independent mechanisms of morphological growth regulation at the Drosophila NMJ synapse remain to be elucidated. It is expected that further study will reveal an additional structural control mechanism regulated by CaMKII, acting in parallel to and interacting with Fas2. The ubiquination mechanism discussed here is one candidate mechanism, but as CaMKII is known to have many synaptic targets, it is clearly not the only candidate (Beumer, 2002).

In summary, it is concluded that architectural developmental defects observed in NMJ synapses mutant for ßPS integrin are due to the loss of the ability to regulate synaptic CaMKII properly. One function of CaMKII is to phosphorylate the scaffolding protein Dlg, and thus regulate the proteins synaptically localized by this scaffold. Fas2 is the central known output of this regulatory cascade. Loss of this regulation is central to the mys mutant defects in the postembryonic elaboration of NMJ structure. It is clear from this study, however, that regulation of Fas2 localization via CaMKII is only one facet of how integrins function at the synapse (Beumer, 2002).

Regulation of the Ca2+/CaM-responsive pool of CaMKII by scaffold-dependent autophosphorylation

CaMKII is critical for structural and functional plasticity. Camguk (Cmg, also known as Caki), the Drosophila homolog of CASK/Lin-2, associates in an ATP-regulated manner with CaMKII to catalyze formation of a pool of calcium-insensitive CaMKII. In the presence of Ca2+/CaM, CaMKII complexed to Cmg can autophosphorylate at T287 and become constitutively active. In the absence of Ca2+/CaM, ATP hydrolysis results in phosphorylation of T306 and inactivation of CaMKII. Cmg coexpression suppresses CaMKII activity in transfected cells, and the level of Cmg expression in Drosophila modulates postsynaptic T306 phosphorylation. These results suggest that Cmg, in the presence of Ca2+/CaM, can provide a localized source of active kinase. When Ca2+/CaM or synaptic activity is low, Cmg promotes inactivating autophosphorylation, producing CaMKII that requires phosphatase to reactivate. This interaction provides a mechanism by which the active postsynaptic pool of CaMKII can be controlled locally to differentiate active and inactive synapses (Lu, 2003).

Cmg, mammalian CASK, and C. elegans Lin-2 form a MAGUK subfamily that is defined by an N-terminal CaMKII-like domain in addition to the SH3, PDZ, and guanylate kinase domains typical of the MAGUK family. In mammalian neurons, CASK has been shown to be localized to synapses both pre- and postsynaptically where it associates with cell surface molecules like neurexins, calcium channels, and syndecans, the cytoskeletal protein 4.1, PSD95, and other adaptor molecules. CASK may also participate in signaling to the nucleus by directly interacting with a transcription factor (Lu, 2003 and references therein).

Cmg null animals have impaired locomotion, but their ability to learn had not been tested. Modification of courtship behavior in Drosophila has been used to test both associative and nonassociative memory formation; CaMKII is required for both. Exposure of a male to a mated female leads to decreased courtship of a subsequently presented virgin. This suppression reflects formation of an associative memory that requires mushroom body and central complex circuits. Courtship suppression also occurs during the training period via a non-mushroom body central circuit. Habituation to a courtship stimulating cue can be demonstrated after training with immature males. In these behavioral assays, the locomotor defects of the Cmg null flies could only cause an underestimate of behavioral defects since 'failure' in the assay is signified by an increase in courtship activity. Wild-type, Cmg null, and cmg deficiency heterozygotes were tested for mated female conditioning. All genotypes were normal for associative memory formation when compared to males that had been sham conditioned in an empty chamber. Behavior during the training period, however, was abnormal; Cmg null males fail to decrease courtship during training. Deficiency heterozygotes are intermediate. To determine if the Cmg null has a habituation defect, the response to immature males, who emit courtship promoting pheromones distinct from the female type, was examined. Cmg null animals fail to habituate. Courting Cmg null males have normal initial courtship levels and the ability to discriminate between virgin and mated females, indicating that peripheral sensory pathways are intact (Lu, 2003).

Association of Cmg with CaMKII is stimulated by ATP bound to the CaMKII catalytic domain. At physiological levels of ATP, Cmg and CaMKII are likely to be associated. ATP hydrolysis in the presence of Ca2+/CaM leads to autophosphorylation of CaMKII on T287 and production of a Cmg-localized constitutively active kinase. Cmg can also promote ATP hydrolysis by CaMKII in the absence of Ca2+/CaM. Under these conditions and without prior T287 autophosphorylation, CaMKII preferentially autophosphorylates its own CaM binding domain. This reaction leads to the fast release of the CaMKII in a form that cannot be activated by Ca2+/CaM. The ability of genetic manipulation of Cmg, PP2A, and synaptic activity levels in vivo to alter the amount of postsynaptic CaMKII that is in the inactivated state argues strongly that these reactions are physiologically relevant and define a new mechanism for the regulation of CaMKII activity (Lu, 2003).

What is the function of this interaction in vivo? One possible role is to provide a basis for maintaining differences between active and inactive synapses. The effect of cmg on courtship conditioning, a behavior known to require CaMKII, is suggestive of a role for this interaction in behavioral plasticity. Differences in synaptic strength can fine tune networks and are crucial to most memory models. Local CaMKII inactivation by Cmg could serve to decrease the gain on CaMKII-mediated calcium signaling at synapses that have been inactive. At low Ca2+, T306 autophosphorylation would occur, and inactive kinase would be released from Cmg. This provides a 'use it or lose it' mechanism for preserving synapse-specific strength differentials, since CaMKII tethered to Cmg at synapses that see higher Ca2+ levels would be protected from inactivating autophosphorylation by bound CaM. This complexed kinase can undergo T287 autophosphorylation, resulting in constitutive kinase activity and a slowed CaM dissociation rate that would further prolong the lifetime of the complex. Whether the Cmg-mediated inactivation of CaMKII operates locally or globally remains to be determined and likely depends on the ability of the inactivated kinase to escape from complexes with its other binding partners in the postsynaptic density (Lu, 2003).

Establishment of a pool of autophosphorylated CaMKII that cannot be directly stimulated by Ca2+/CaM also provides a mechanism for phosphatase activity to regulate CaMKII. In vitro, both PP1 and PP2A are able to dephosphorylate the CaM binding domain of CaMKII. In Drosophila, PP2A is the relevant in vivo phosphatase for postsynaptic pT306. The opposing actions of Cmg and PP2A provide machinery for dynamic regulation of the maximum level of calcium-activatable CaMKII at synapses (Lu, 2003).

In mouse, knockin of a CaMKII T305D mutation blocks LTP and learning and decreases postsynaptic localization of the kinase, suggesting that this mutant is unable to interact with one or more of its normal synaptic binding partners. Inability to undergo inactivating autophosphorylation (T305VT306A knockin) is associated with a lower threshold for LTP and loss of behavioral fine tuning. These data argue that, in mammals, autophosphorylation of the CaM binding domain of CaMKII is also an important regulatory mechanism for both function and localization of CaMKII. Whether in mammals CASK (the Cmg homolog) is the critical synaptic binding partner and regulator of synaptic T305 autophosphorylation is unknown (Lu, 2003).

This study also demonstrates a novel binding mechanism for CaMKII on a MAGUK scaffolding protein. It is proposed that this interaction consists of two discrete steps. First, binding of ATP to the catalytic domain of CaMKII reveals an interaction surface on the kinase that forms an initial bond to the Cmg protein. This initial interaction can occur either in the presence or absence of Ca2+/CaM, implying that it does not involve the C-terminal CaM binding sequences of the autoinhibitory domain. The ability of a peptide corresponding to the N terminus of the autoinhibitory domain to partially block complex formation suggests that this region, which is known to be involved in regulation of ATP binding or the catalytic domain surface that the N terminus of the autoinhibitory domain binds to, is the site of this initial interaction (Lu, 2003).

Once an initial interaction is established, bound ATP is no longer required. This could be due to a stabilization of the ATP-bound conformation by Cmg binding. Stabilization of the open form of the kinase autoregulatory domain is postulated to occur after Ca2+/CaM-dependent NR2B binding, since this interaction is insensitive to CaM stripping. Another possibility for the loss of ATP requirement is that a secondary interaction occurs that abrogates the need for the initial interaction. Such a secondary interaction does occur, but this does not rule out a concurrent stabilization of the ATP-bound conformation (Lu, 2003).

The secondary interaction that forms after ATP-dependent binding of Cmg involves the C-terminal region of the autoinhibitory domain, including the CaM binding sequences. Three lines of evidence suggest that this secondary interaction provides the most stable form of the complex. (1) N-terminal peptides only partially block complex formation, while peptides that contain the CaM binding domain completely block complex formation. (2) CaMKIIs with mutations of T306/7 do not bind to Cmg at all. These mutants are fully capable of binding ATP, but the exposure of the initial binding domain is insufficient to maintain a salt-stable interaction in the absence of a normal CaM binding domain. The failure of the relatively subtle threonine to serine mutant to bind suggests a direct interaction of Cmg with T306/7. (3) The natural release mechanism of this complex is via autophosphorylation of the CaM binding domain. Protection of this region by Ca2+/CaM can prevent both the autophosphorylation and dissociation of CaMKII (Lu, 2003).

The two-step association mechanism and the autophosphorylation-regulated dissociation of CaMKII from Cmg provide a new way in which CaMKII can be localized and regulated using its autoinhibitory domain. As defined for catalytic activation, the regulatory region of CaMKII is a small but complicated protein interaction domain. It is becoming clear that this region can support multiple intra- and intermolecular interactions that have profound effects on local CaMKII activity and synaptic plasticity (Lu, 2003).

Activity-dependent gating of CaMKII autonomous activity by Drosophila CASK

The ability of CaMKII to act as a molecular switch, becoming Ca²⁺ independent after activation and autophosphorylation at T287, is critical for experience-dependent synaptic plasticity. This study shows that Caki, the Drosophila homolog of CASK, also known as Camguk, can act as a gain controller on the transition to calcium-independence in vivo. Genetic loss of dCASK significantly increases synapse-specific, activity-dependent autophosphorylation of CaMKII T287. In wild-type adult animals, simple and complex sensory stimuli cause region-specific increases in pT287. dCASK-deficient adults have a reduced dynamic range for activity-dependent T287 phosphorylation and have circuit-level defects that result in inappropriate activation of the kinase. dCASK control of the CaMKII switch occurs via its ability to induce autophosphorylation of T306 in the kinase's Calmodulin (CaM) binding domain. Phosphorylation of T306 blocks Ca²⁺/CaM binding, lowering the probability of intersubunit T287 phosphorylation, which requires CaM binding to both the substrate and catalytic subunits. dCASK is the first CaMKII-interacting protein other than CaM found to regulate this plasticity-controlling molecular switch (Hodge, 2006).

Autophosphorylation of CaMKII at a site in the N terminus of its autoregulatory domain (T287 in Drosophila and T286 in mammalian αCaMKII) confers Ca²⁺-independent activity on the enzyme. This switch-like property of the kinase is crucial to its role in long-term potentiation and memory formation in mammals and flies, and generation of the autonomous form of the kinase can be stimulated by a number of different behaviors and activity paradigms. Interestingly, in mice, both mutations that prevent T286 phosphorylation, and transgenes that increase constitutive activity have been shown to block plasticity. This implies that the level of constitutive activity needs to be tightly controlled to remain in a range optimal for learning. This balance has been believed to be exerted by the positive effects of Ca²⁺/CaM and the negative effects of phosphatases. To date, no proteins other than calmodulin have been shown to influence the development of autonomous activity via T287 autophosphorylation (Hodge, 2006).

The function of autophosphorylation in the C terminus of the regulatory domain within the CaM binding region (T306 in Drosophila and T305 in mammalian αCaMKII) has been more mysterious, but also has consequences for plasticity. In the test tube, with purified kinase, this phosphorylation occurs only after T287 phosphorylation and removal of CaM with EGTA because the site is protected from phosphorylation by bound CaM. With purified kinase, this means that pT306 is only found in doubly phosphorylated pT287, pT306 enzyme. Recently, a novel mechanism for phosphorylation of T306 in the absence of pT287 has been described (Lu, 2003). dCASK interacts with the regulatory domain of the kinase and, when the CaM binding site of the kinase is unoccupied (e.g., when synaptic calcium is low), stimulates the kinase to autophosphorylate at T306. This reaction releases CaMKII from the dCASK complex in a form that is incapable of binding Ca²⁺/CaM and has been suggested to provide a mechanism for downregulation of the activatable kinase pool at quiescent synapses. The effects of dCASK on T287 phosphorylation have not been addressed, and modulation of this site would have important consequences for plasticity (Hodge, 2006).

This study presents evidence that dCASK can also act as an activity-dependent modulator of the levels of constitutively active CaMKII in vivo. CaMKII pT287 autophosphorylation occurs within the dodecameric holoenzyme and requires Ca²⁺/CaM to be bound to both the subunit doing the catalysis and the subunit that contains the substrate threonine. This cooperativity implies that modifications that alter the ability of Ca²⁺/CaM to bind would affect the ability of the kinase to become Ca²⁺ independent. Because it promotes phosphorylation of T306, interaction with dCASK regulates T287 phosphorylation by altering the occupancy of CaM binding sites in the holoenzyme. In vivo this postsynaptic interaction provides a synapse-specific mechanism to alter the probability of generating autonomous activity that is controlled by the activity history of the synapse. This makes dCASK an important gain controller for the CaMKII molecular switch (Hodge, 2006).

Drosophila adducin regulates Dlg phosphorylation and targeting of Dlg to the synapse and epithelial membrane

Adducin is a cytoskeletal protein having regulatory roles that involve actin filaments, functions that are inhibited by phosphorylation of adducin by protein kinase C. Adducin is hyperphosphorylated in nervous system tissue in patients with the neurodegenerative disease amyotrophic lateral sclerosis, and mice lacking β-adducin have impaired synaptic plasticity and learning. This study found that Drosophila adducin, encoded by hu-li tai shao (hts), is localized to the post-synaptic larval neuromuscular junction (NMJ) in a complex with the scaffolding protein Discs large (Dlg), a regulator of synaptic plasticity during growth of the NMJ. hts mutant NMJs are underdeveloped, whereas over-expression of Hts promotes Dlg phosphorylation, delocalizes Dlg away from the NMJ, and causes NMJ overgrowth. Dlg is a component of septate junctions at the lateral membrane of epithelial cells, and this study show that Hts regulates Dlg localization in the amnioserosa, an embryonic epithelium, and that embryos doubly mutant for hts and dlg exhibit defects in epithelial morphogenesis. The phosphorylation of Dlg by the kinases PAR-1 and CaMKII has been shown to disrupt Dlg targeting to the NMJ and evidence is presented that Hts regulates Dlg targeting to the NMJ in muscle and the lateral membrane of epithelial cells by controlling the protein levels of PAR-1 and CaMKII, and consequently the extent of Dlg phosphorylation (Wang, 2011).

Dlg is a Drosophila member of a family of MAGUK scaffolding proteins that has four mammalian members, SAP97/hDlg, PSD-93/Chapsyn-110, PSD-95/SAP90 and SAP102/NE-Dlg, and which has important developmental and regulatory roles in the nervous system and in epithelia. Phosphorylation is emerging as a mechanism for the regulation of the localization and function of the Dlg family. The post-synaptic targeting of Dlg to the Drosophila NMJ is inhibited by phosphorylation of the first PDZ domain by CaMKII and of the GUK domain by PAR-1. In mammalian neurons, CaMKII phosphorylation of PDZ1 of PSD-95 terminates long term potentiation-induced spine growth by inducing translocation of PSD-95 out of the active spine. Furthermore, phosphorylation of PDZ1 of either PSD-95 or SAP97/hDlg by CaMKII regulates their interaction with NMDA subunits, and additional cell culture studies in epithelial cells demonstrate effects of SAP97/hDlg phosphorylation by various kinases (Wang, 2011).

An important function of Dlg at the NMJ is involvement in synaptic plasticity during muscle growth, at least in part by controlling the localization at the pre-synaptic and post-synaptic membranes of Fas2, a homophilic cell adhesion molecule of the immunoglobulin superfamily that binds Dlg. The breaking and restoration of Dlg/Fas2-mediated adhesion between the pre- and post-synaptic membranes is likely critical to synaptic growth during development. This study has shown that Hts exists in a complex with Dlg, probably at both the post-synaptic membrane of the NMJ and at the lateral membrane in epithelia, where the two proteins are likely brought into close proximity by a shared association with the spectrin-actin junction. Hts is therefore positioned to locally regulate Dlg and participate in synaptic plasticity at sites of association with the membrane cytoskeleton, and it positively contributes to phosphorylation of Dlg on its GUK domain. This site of phosphorylation is a target for PAR-1, and consistent with this it was found that Hts can regulate the levels of PAR-1. Phosphorylation of Dlg impedes its targeting to the post-synaptic membrane and it is proposed that Hts regulates this targeting at the synapse by controlling the levels of PAR-1 at the post-synaptic membrane. This could occur through Hts acting as a scaffold participating in localized stabilization or translation of PAR-1 at the post-synaptic membrane and, consistent with the latter mechanism, localized post-synaptic translation of NMJ proteins has been reported in Drosophila. Over-expression of Hts permits synaptic overgrowth, which may in part be due to effects on Dlg targeting to the post-synaptic membrane, but unpublished results indicating additional routes of action. Furthermore, if Hts were acting mainly by elevating PAR-1 levels, synaptic undergrowth would be expected rather than overgrowth with Hts over-expression, as the post-synaptic over-expression of PAR-1 leads to decreased bouton number and an oversimplified synapse. CaMKII also regulates synaptic growth and Dlg targeting at the NMJ through phosphorylation. There is currently no antibody available to examine phosphorylation of Dlg by CaMKII in vivo, but it was determined that Hts controls the levels of this kinase similarly to PAR-1, suggesting that Hts may also be regulating Dlg targeting via CaMKII-dependent phosphorylation (Wang, 2011).

Two other studies address Hts function in neurons, one describing a role in photoreceptor axon guidance and the other characterizing pre-synaptic Hts function at the NMJ (Ohler, 2011; Pielage, 2011). The latter study reported a phenotype of synaptic retraction at the hts mutant NMJ, revealed by the absence of the pre-synaptic marker Bruchpilot from extensive stretches of the NMJ, which is consistent with the observation of small synapses in hts mutants stained for the synaptic vesicle marker CSP-2 (Pielage, 2011). However, in addition to retraction, this group observed overgrowth of the hts mutant NMJ, visible with anti-HRP staining. Although no quantification was performed using anti-HRP, qualitative examination of hts mutant muscle 6/7 NMJs in the current study did not indicate an overgrowth phenotype. A possible explanation for this discrepancy is the choice of the muscle 6/7 NMJ for analysis in contrast to Pielage, who focused on muscle 4. Unlike most other larval muscles, muscles 6 and 7 are not innervated by type II boutons. A component of the synaptic overgrowth reported by Pielage is the extension of thin, actin-rich extensions somewhat similar in appearance to type II and type III processes and, accordingly, growth of these processes is impaired with pre-synaptic over-expression of Hts (Pielage, 2011). Pielage proposes that pre-synaptic Hts restricts synaptic growth through its function as an actin-capping protein. At the muscle 6/7 NMJ this role may not be so prevalent and the post-synaptic growth-promoting function of Hts prevails. By examining NMJs other than muscle 6/7 in hts mutant larvae it was possible to confirm the overgrowth phenotype observed by Pielage (Wang, 2011).

Synaptic plasticity at the Drosophila NMJ has parallels with the structural changes seen at synapses in cellular models of learning such as long-term facilitation (LTF) in Aplysia and long-term potentiation in the mammalian hippocampus, and several studies indicate that the current results with hts at the NMJ may be relevant to these other systems. Phosphorylation of the conserved serine residue in the MARCKS domain of adducin is increased during LTF in Aplysia, and mice lacking β-adducin exhibit impaired synaptic plasticity and learning (Bednarek, 2011; Gruenbaum, 2003; Porro, 2010; Rabenstein, 2005; Ruediger, 2011; Wang, 2011 and references therein).

Similar to the plasticity exhibited by the NMJ during development, the morphogenesis of epithelia involves what has been referred to as 'epithelial plasticity' in which cell-cell adhesions are disassembled and cells become motile, for example in epithelial-mesenchymal transitions. The acquisition of motility by epithelial cells is also involved in tumor cell invasion and metastasis. The Drosophila follicular epithelium is a model for studying both developmental and pathological epithelial plasticity, and recent observations indicate that Dlg and Fas2 collaborate to prevent inappropriate invasion of follicle cells between neighboring germ cells. Furthermore, border cell migration, an invasion of a subset of follicle cells between germ cells occurring during normal oogenesis, requires downregulation of Fas2 expression in the border cells. PAR-1 is required for detachment of border cells from the follicular epithelium and it is interesting to speculate that this might involve regulation of Dlg localization. These various results indicate that regulation of the Dlg/Fas2 complex is important for the epithelial plasticity exhibited by follicle cells and this mechanism likely applies to epithelia in the developing embryo. As in muscle, Hts over-expression causes elevated levels of PAR-1 and CaMKII in epithelial cells. While Hts over-expression does not cause discernible Dlg delocalization in the epidermis, it disrupts the membrane localization of Dlg in amnioserosa cells. Dlg in the amnioserosa might be particularly susceptible to Hts function, as it not incorporated into septate junctions in this tissue, unlike in the epidermis. Furthermore, if the delocalization of Dlg by Hts is CaMKII-dependent, it would be more pronounced in the amnioserosa than the epidermis as Hts can only weakly promote CaMKII accumulation in the epidermis. In addition to showing that Hts can regulate Dlg localization in the amnioserosa, this study has determined that proper cortical localization of Hts in amnioserosa cells in the early stages of dorsal closure is dependent on Dlg. This result suggests that where Dlg and Hts are together in a complex, Dlg stabilizes the membrane localization of Hts. This stabilization may occur in cells other than the amnioserosa but might not be readily detectable. Hts localizes all around the epithelial membrane, with much of it not co-localizing with Dlg. In the very flat, squamous cells of the amnioserosa early in dorsal closure, the proportion of lateral membrane Hts dependent on Dlg for stabilization may be greater than in more columnar epithelial cells such as those in the epidermis (Wang, 2011).

Consistent with the effects that they have on each other's localization, the frequency of cuticle defects seen in dlg hts zygotic double mutant embryos suggests that Hts and Dlg co-operate in epithelial development, and it is of interest that mammalian adducin has recently been implicated in the stabilization and remodeling of epithelial junctions (Naydenov, 2010). Embryos deficient in Hts likely have a diminished ability to delocalize Dlg, effectively reducing the pool of Dlg available for de novo junction formation, and this situation would be worsened by reducing Dlg levels with a dlg allele. Conversely, reducing Dlg in an embryo deficient in Hts may further compromise Hts function through effects on Hts membrane localization. One of the phenotypes seen in hts mutants, the frequency of which is increased by additionally reducing Dlg, is that of embryos which secrete only small pieces of cuticle. This phenotype is characteristic of maternal zygotic dlg mutant embryos (Wang, 2011).

The interaction of Hts with Dlg suggests that adducin could be a regulator of the septate junction and it will be of interest to examine adducin effects on septate junction morphology in the nervous system. In the Drosophila nervous system septate junctions are formed between glial cells, whereas in mammals they are found between glial and neuronal membranes at the paranodal junction. Interestingly, β-adducin was recently identified as a paranodal junction protein localized to the neuronal membrane where it co-localizes with NCP1, the vertebrate homolog of the Drosophila septate junction protein Neurexin IV (Ogawa and Rasband, 2009) (Wang, 2011).

DEVELOPMENTAL BIOLOGY

Embryonic

A trace amount of transcript is detected in cleavage stage embryos. After blastoderm formation, basal levels of zygotic expression are observed throughout the embryo. During germ band elongation high levels of expression become apparent in the anterior and posterior midgut. Expression in the midgut becomes more prominent during germ band shortening. At the same time, expression is observed in the neuroblasts of the central nervous system. Transcripts of CaM kinase II are expressed in great quantities in the central and peripheral nervous systems in the late embryonic stage of development (Ohsako, 1993).

In situ hybridization shows CaM kinase mRNA is present in both neuronal and nonneuronal tissues in adult Drosophila. No differential tissue distribution of isoforms was observed (Griffith, 1993b).

Adult

Transcripts are more abundant in the head than in the body of the adult fly (Ohsako, 1993).

Expression is seen in ovaries in the somatic follicle cells and germ line oocytes. Adult expression is also observed in gut epithelium (Griffith, 1993b).

Courtship and courtship conditioning are behaviors that are regulated by multiple sensory inputs, including chemosensation and vision. Globally inhibiting CaMKII activity in Drosophila disrupts courtship plasticity while leaving visual and chemosensory perception intact. Light has been shown to modulate CaMKII-dependent memory formation in this paradigm and the circuitry for the nonvisual version of this behavior has been investigated. In this paradigm, volatile and tactile pheromones provide the primary driving force for courtship, and memory formation is dependent upon intact mushroom bodies and parts of the central complex. In the present study, the GAL4/UAS binary expression system has been used to define areas of the brain that require CaMKII for modulation of courtship conditioning in the presence of visual, as well as chemosensory, information. Visual input suppresses the ability of mushroom body- and central complex-specific CaMKII inhibition to disrupt memory formation, indicating that the cellular circuitry underlying this behavior can be remodeled by changing the driving sensory modality. These findings suggest that the potential for plasticity in courtship behavior is distributed among multiple biochemically and anatomically distinct cellular circuits (Joiner, 2000).

To analyze the distribution of Drosophila calcium/calmodulin-dependent protein kinase II in the adult brain, monoclonal antibodies were generated against the bacterially expressed 490-amino acid form of CaMKII. One of those, named #18 antibody, was used for this study. Western blot analysis of the adult head extracts showed that the antibody specifically detects multiple bands between 55 and 60 kDa corresponding to the molecular weights of the splicing isoforms of CaMKII. Epitope mapping revealed that it was in the region between amino acids 199 and 283 of CaMKII. Preferential CaMKII immunoreactivity in the embryonic nervous system, adult thoracic ganglion and gut, and larval neuro-muscular junction (NMJ) was consistent with previous observations by in situ hybridization and immunostaining with a polyclonal antibody at the NMJ, indicating that the antibody is applicable to immunohistochemistry. Although CaMKII immunoreactive signal was low in the retina, it was found at regular intervals in the outer margin of the compound eye. These signals were most likely to be interommatidial bristle mechanosensory neurons. CaMKII immunoreactivity in the brain was observed in almost all regions and relatively higher staining was found in the neuropilar region than in the cortex. Higher CaMKII immunoreactivity in the mushroom body (MB) was found in the entire gamma lobe including the heel, and dorsal tips of the alpha and alpha' lobes, while cores of alpha and beta lobes were stained lightly. Finding abundant CaMKII accumulation in the gamma lobe suggests that this lobe might especially require high levels of CaMKII expression to function properly among MB lobes (Takamatsu, 2003).

A model for the role of sarah in egg activation

The Drosophila modulatory calcineurin-interacting protein (MCIP) sarah (sra) is essential for meiotic progression in oocytes. Activation of mature oocytes initiates development by releasing the prior arrest of female meiosis, degrading certain maternal mRNAs while initiating the translation of others, and modifying egg coverings. In vertebrates and marine invertebrates, the fertilizing sperm triggers activation events through a rise in free calcium within the egg. In insects, egg activation occurs independently of sperm and is instead triggered by passage of the egg through the female reproductive tract; it is unknown whether calcium signaling is involved. MCIPs [also termed regulators of calcineurin (RCNs), calcipressins, or DSCR1 (Down's syndrome critical region 1)] are highly conserved regulators of calcineurin, a Ca2+/calmodulin-dependent protein phosphatase 1 and 2. Although overexpression experiments in several organisms have revealed that MCIPs inhibit calcineurin activity, their in vivo functions remain unclear. Eggs from sra null mothers are arrested at anaphase of meiosis I. This phenotype was due to loss of function of sra specifically in the female germline. Sra is physically associated with the catalytic subunit of calcineurin, and its overexpression suppresses the phenotypes caused by constitutively activated calcineurin, such as rough eye or loss of wing veins. Hyperactivation of calcineurin signaling in the germline cells resulted in a meiotic-arrestphenotype, which can also be suppressed by overexpression of Sra. All these results support the hypothesis that Sra regulates female meiosis by controlling calcineurin activity in the germline. This is the first unambiguous demonstration that the regulation of calcineurin signaling by MCIPs plays a critical role in a defined biological process (Takeo, 2006; Horner, 2006).

In vertebrates and marine invertebrates, fertilization triggers a transient rise in free Ca²⁺, and this rise is responsible for subsequent activation events such as modification of the eggshell, prevention of polyspermy, and cell-cycle resumption. This study shows that a protein involved in a calcium signal-transduction pathway is necessary for several egg-activation events in Drosophila. The sarah gene product is a calcipressin; the human calcipressin DSCR1 can directly bind to and inhibit calcineurin, the only known phosphatase that is dependent on both calcium and calmodulin. The accompanying paper by Takeo (2006) verifies that the fly Sarah protein shares these biochemical properties (Horner, 2006).

The present understanding of egg activation in Xenopus and the activities of calcipressin make strong predictions for the role of sra in meiotic reactivation. Upon fertilization of frog eggs, Ca²⁺ activates calmodulin-dependent protein kinase II (CaMKII), which in turn directs the inactivation of Anaphase Promoting Complex (APC) inhibitors such as Erp1/Emi2. The APC in turn inactivates MPF through the destruction of cyclin, relieving the meiotic block and initiating other egg-activation events. The current results suggest that essentially the same pathway operates during the activation of Drosophila eggs. It is hypothesized that Sarah acts early in the pathway by mediating the antagonistic relationship between calcineurin and CaMKII; that is, CaMKII activity upon egg activation requires the inhibition of calcineurin by Sarah (Horner, 2006).

Consistent with this model is the similarity of the sarah phenotype to that associated with mutations in another Drosophila gene called cortex. The Cortex protein is a member of the Cdc20 protein family, whose members serve as specificity factors and activators for the APC. Meiosis arrests normally at metaphase I in cortex oocytes and resumes when the eggs are laid but then soon arrests again at metaphase II. The variation in the phase of the meiotic arrest (metaphase II versus anaphase I) being an exception, other aspects of egg activation are similarly affected by mutations in cortex and sra (Horner, 2006).

The meiotic arrest in both sra and cortex eggs is most simply explained by the failure of the APC to target certain molecules for degradation upon egg laying. In cortex eggs, cyclin A fails to be degraded. The cyclin B component of MPF remains undegraded in sra eggs. These results are consistent with the mitotic-arrest phenotypes seen in early Drosophila embryos expressing nondegradable cyclin A (metaphase arrest) or nondegradable cyclin B (early anaphase arrest). Different APC targets could remain undegraded in sra and cortex eggs because there are at least two Cdc20-like proteins (Cortex and Fzy) in Drosophila eggs. APC^Cortex and APC^Fzy may have different substrate specificities and may be differently regulated by APC inhibitors downstream of CaMKII (Horner, 2006).

The failure of the eggs laid by sra mutant mothers to translate the maternal bcd mRNA can also be understood in the same theoretical framework. The translation of bcd requires the polyadenylation of its mRNA; the enzyme poly(A) polymerase that catalyzes polyadenylation is phosphorylated and thus negatively regulated by MPF. Failure of APC activation in sra mutant eggs would therefore prevent both MPF inactivation and bcd mRNA translation (Horner, 2006).

Although meiosis and bcd translation are both disrupted in sra eggs, vitelline membrane (VM) cross-linking is apparently normal. VM cross-linking also occurs in other mutants that block female meiotic progression; such mutants include cortex, grauzone, prage, and wispy. In fact, VM reorganization can occur independently of every other known egg-activation event, including bcd mRNA translation, the degradation of other maternal mRNAs, and microtubule depolymerization. If there is only a single trigger for egg activation, it must therefore be able to activate at least two autonomous downstream pathways (one for VM cross-linking and a second for other events). Because in vitro activated eggs are defective in several aspects of embryonic development, it is difficult to interpret the finding of delayed VM modification in sra eggs upon in vitro activation. Although sra function is not formally required for VM organization, sra-dependent processes might nonetheless impinge on its efficiency (Horner, 2006).

In summary, results indicate that despite its independence from a sperm trigger, egg activation in Drosophila involves calcium-mediated pathways that are likely to be analogous to those in other animals. It is intriguing that among these downstream events is the acquisition of the egg's competence to remodel the sperm nucleus into the male pronucleus (Horner, 2006).

Effects of Mutation or Deletion

CaM kinase II is capable of directly regulating its own activity by autophosphorylation. To assess the involvement of CaM kinase in experience-dependent behavior in an intact animal, a specific peptide inhibitor of CaM kinase was expressed in transgenic Drosophila under control of an inducible promoter. These flies fail to learn normally in two behavioral plasticity paradigms: acoustic priming, a nonassociative measure of sensitization, and courtship conditioning, a measure of associative learning. The magnitude of the learning defect in the associative paradigm appears to be proportional to the level of expression of the peptide gene in the two transgenic lines and can be increased by heat shock induction of gene expression. These results suggest that CaM kinase activity is required for plastic behaviors in an intact animal (Griffith, 1993a).

CaM kinase II has been implicated in neural plasticity that underlies learning and memory processes. Transformant strains of Drosophila, ala1 and ala2, expressing a specific inhibitor of CaM kinase have altered short-term plasticity in synaptic transmission along with abnormal nerve terminal sprouting and directionality of outgrowth. These results represent an interesting parallel with the activity-dependent regulation of synaptic physiology and morphology by the cAMP cascade in Aplysia and Drosophila (See cAMP dependent protein kinase 1). In contrast to the learning mutants dunce and rutabaga, which are defective in the cAMP cascade, inhibition of CaM kinase in ala transformants causes increased sprouting at larval neuromuscular junctions near the nerve entry point, rather than altering the higher order branch segments. In addition, synaptic facilitation and potentiation are altered in a manner different from that observed in the cAMP mutants. Furthermore, synaptic currents in ala transformants are characterized by greater variability, suggesting an important role of CaM kinase in the stability of transmission (Wang, 1994).

Similar defects in both synaptic transmission and associative learning are produced in Drosophila melanogaster by inhibition of calcium/calmodulin-dependent protein kinase II and mutations in the potassium channel subunit gene ether à go-go (eag). These behavioral and synaptic defects are not simply additive in animals carrying both an eag mutation and a transgene for a protein kinase inhibitor. This raises the possibility that the phenotypes share a common pathway. At the molecular level, a portion of the putative cytoplasmic domain of Eag is a substrate of calcium/calmodulin-dependent protein kinase II. These similarities in behavior and synaptic physiology, the genetic interaction, and the in vitro biochemical interaction of the two molecules suggest that an important component of neural and behavioral plasticity may be mediated by modulation of Eag function by calcium/calmodulin-dependent protein kinase II (Griffith, 1994).

Transgenic Drosophila strains expressing an inhibitory peptide of CaM Kinase II, or a constitutive activated CaM kinase show altered neuronal process morphology compared to wild type in scanning electron microscopy of cultured mature neurons from embryonic neuroblasts. Significantly enhanced neural process growth is observed in cells with inhibited enzyme, and reduced process growth is observed with activated enzyme, suggesting that active CaM kinase is involved in the inhibition of neurite growth during development. The subcellular distribution of CaM kinase was examined in wild type neuronal cultures. Prior to neuronal contact there is little CaM kinase in neuronal processes, but after connections have been made the processes exhibit a high level of CaM kinase. These results suggest that the transport of CaM kinase to terminals does not occur until after or during the formation of neuronal connections when a functional synapse might be formed. These results also suggest a target-dependent transport of the enzyme along processes and an inhibitory role for CaM kinase on neurite branching. One of the roles of CaM kinase in the mature synapse is the phosphorylation of synapsin I, which is involve in the Ca2+ dependent release of neurotransmitter from neurosecretory vesicles. Synapsin, like CaM kinase translocates to the terminus following contact with the postsynaptic target. The down-regulatory role of CaM kinase contrasts with the apparent up-regulatory role of protein kinase C in neurite elongation (Broughton, 1996).

Flexing the femorotibial joint elicits a resistance response from the tibial extensor motor neurons, and this reflex response decays on repetition. The frequency of excitatory junction potentials (EJPs) was monitored throughout a repetitive series of sinusoidal flexion-extension movements, and the data were collected in 50 msec bins. The peak response corresponds to the flexion phase of the movement. With 2 Hz of stimulation the peak response decays smoothly, with a time constant of one second in these examples to a plateau of ~40% of the initial response. This response was readily fit with a single exponential decay function, as is the case for many other examples of habituation. This response decrement conforms to other features of habituation as well. When the 2 Hz stimulus is terminated, the initial response recovers spontaneously to ~75% of the initial amplitude in 2 sec and is fully recovered within 60 sec. As is the case for most other examples of habituation, the rate of decrement in this reflex is dependent on the frequency and amplitude of the stimulus. For example, decreasing the rate of flexion from 2 to 0.5 Hz increases the time constant of decay from 1 to 22 sec. The time constant also is correlated with stimulus strength. Finally, the response can be dishabituated by tactile stimuli to the abdomen. When an animal was stimulated with 15 successive flexion movements and a tactile stimulus is provided during the ninth and tenth stimulus, the response recovers to ~60% of the initial response (Jin, 1998).

To study the presynaptic function of CaMKII in this nonassociative form of learning, a P[Gal4] insertion line was used to target the expression of mutant forms of CaMKII to the sensory neurons controlling the reflex. Targeted expression of a calcium-independent CaMKII construct (T287D) in the sensory neurons eliminates habituation. Targeted expression of a mutant CaMKII incapable of achieving calcium independence (T287A) reduces the initial reflex response, but a strong facilitation then occurs, and this eliminates most of the habituation. Finally, when a CaMKII inhibitory peptide (ala) is expressed in sensory neurons, the initial response is reduced, followed by facilitation. Thus, increasing the amount of calcium-independent kinase activity with the T287D construct eliminates habituation, and the response level remains at its maximum throughout the stimulus trial. This implies that the additional kinase activity is able to mobilize transmitter release and relieve the presynaptic depression that is normally responsible for habituation. Even with reduced stimulus strength that enhances depression in most habituation paradigms, only a weak depression is seen. An increased amount of calcium-dependent activity with the T287A construct has a similar effect, slowing the rate of depression and supporting the idea that additional CaMKII activity can relieve habituation. These results suggest that basal CaMKII levels in the presynaptic neurons set the response level and dynamics of the entire neural circuit (Jin, 1998).

Retrograde control of synaptic transmission by postsynaptic CaMKII at the Drosophila neuromuscular junction

Retrograde signaling plays an important role in synaptic homeostasis, growth, and plasticity. A retrograde signal at the neuromuscular junction (NMJ) of Drosophila controls the homeostasis of neurotransmitter release. This retrograde signal is regulated by the postsynaptic activity of Ca2+/calmodulin-dependent protein kinase II (CaMKII). Reducing CaMKII activity in muscles enhances the signal and increases neurotransmitter release, while constitutive activation of CaMKII in muscles inhibits the signal and decreases neurotransmitter release. Postsynaptic inhibition of CaMKII increases the number of presynaptic, vesicle-associated T bars at the active zones. Consistently, it is shown that glutamate receptor mutants also have a higher number of T bars; this increase is suppressed by postsynaptic activation of CaMKII. Furthermore, presynaptic BMP receptor Wishful thinking is required for the retrograde signal to function. These results indicate that CaMKII plays a key role in the retrograde control of homeostasis of synaptic transmission at the NMJ of Drosophila (Haghighi, 2003).

Reducing the function of postsynaptic glutamate receptors at the neuromuscular junction (NMJ) of Drosophila triggers a retrograde signal from the postsynaptic muscle to the presynaptic motor neuron, leading to an increase in the amount of neurotransmitter release. This retrograde signal is regulated by the postsynaptic activity of CaMKII. Reducing postsynaptic CaMKII activity by expressing a CaMKII inhibitory peptide in somatic muscles increases quantal content, mimicking the effect of reducing postsynaptic glutamate receptor activity. Furthermore, in glutamate receptor GluRIIA^-/- mutants, constitutive activation of CaMKII in muscles inhibits the retrograde signal and decreases quantal content. These changes in retrograde signaling and neurotransmitter release are not accompanied by any significant changes in the number of synaptic boutons per muscle surface area or any gross structural or ultrastructural alterations. However, upon inhibition of CaMKII postsynaptically the number of T bars per active zone in presynaptic boutons is significantly increased. Similarly, the number of T bars per active zone is doubled in GluRIIA^-/- mutant larvae. This increase is suppressed by constitutive activation of CaMKII in postsynaptic muscles in GluRIIA^-/- mutants. These results point to CaMKII as a key regulator of the retrograde signal controlling homeostasis of synaptic transmission at the NMJ of Drosophila (Haghighi, 2003).

Postsynaptic inhibition of CaMKII activity is sufficient to increase presynaptic neurotransmitter release in a retrograde fashion. This increase in quantal content can be potentiated by expressing additional doses of the inhibitory transgene and suppressed by expressing a constitutively active CaMKII transgene simultaneously. These results suggest a direct involvement of CaMKII in controlling the retrograde signal that maintains the homeostasis of neurotransmitter release at the Drosophila NMJ (Haghighi, 2003).

While increasing the postsynaptic activity of CaMKII in wild-type larvae has no effect on neurotransmitter release, once the retrograde signal is induced (i.e., in GluRIIA^-/- mutants), activation of CaMKII can inhibit the signal. This is consistent with the observation that while removal of GluRIIA causes a decrease in quantal size and an increase in quantal content, overexpression of GluRIIA, which leads to an increase in quantal size, does not change quantal content. These results suggest that quantal content may be increased only when CaMKII activity is reduced to a critical threshold. As long as CaMKII activity remains above this critical threshold, quantal content is unchanged. This could be a mechanism through which the synapse can compensate for any reduction in muscle activity and ultimately maintain homeostasis (Haghighi, 2003).

In a recent study, Kazama (2003) has provided evidence for the involvement of postsynaptic CaMKII in the retrograde control of neurotransmission at the Drosophila NMJ. Changes in the activity of postsynaptic CaMKII have been shown to affect both neurotransmitter release and synaptic structure in early first instar larvae. Kazama also reports an apparent change in localization of postsynaptic glutamate receptors in response to postsynaptic activation of CaMKII. This is in contrast to the current observations; no changes were found in the Highaghi (2003) study in overall synaptic structure or localization of glutamate receptors in response to either inhibition or activation of postsynaptic CaMKII. The differences in these findings could be partially due to differences in the level and pattern of expression of transgenes (using different Gal4 lines) or due to the fact that the NMJ was examined at very different developmental stages. Haghighi (2003) examined late third instar larvae, while Kazama (2003) examined early first instar larvae. Interestingly, the results are in agreement in that both studies observed no changes in the amplitude or kinetics of spontaneous potentials, indicating that CaMKII does not directly modulate glutamate receptors at the Drosophila NMJ; this is not the case with vertebrates (Haghighi, 2003).

When the activity of postsynaptic glutamate receptors at the Drosophila NMJ is reduced, a retrograde signal from the muscle to the motor neuron is triggered that causes an increase in quantal content. It has been suggested that this retrograde signal could be triggered in response to changes in muscle depolarization or in response to Ca²⁺ conducted by glutamate receptors. The data indicate that postsynaptic activity of CaMKII plays an important role in controlling the signal. Both muscle depolarization and Ca²⁺ flux through glutamate receptors could be involved in changing the levels of intracellular Ca²⁺ and thus that of CaMKII. The idea is favored that calcium influx through glutamate receptors is at least in part responsible for activating CaMKII and triggering the retrograde signal. There are several lines of evidence that support this hypothesis (Haghighi, 2003).

One line of evidence is based on the glutamate receptor ion channel properties and how they are changed in GluRIIA^-/- mutants. Compared to wild-type receptors, glutamate receptors in these mutants have a greatly reduced single-channel mean open time; this also affects the kinetics of EPSPs. Therefore, evoked currents that give rise to similar EPSP peak amplitudes in wild-type and GluRIIA^-/- mutants will lead to less ion influx in the mutants (ion flux is a product of time and current). Considering the high Ca²⁺ permeability of glutamate receptors (P_Ca/P_Na = 9.55), due to this reduced ion influx, Ca²⁺ influx will also be reduced in GluRIIA^-/- mutants both during spontaneous and evoked activities. Therefore, it is conceivable that this change in Ca²⁺ influx, monitored by CaMKII, could act as a trigger for the retrograde signal (Haghighi, 2003).

This hypothesis is further supported by data demonstrating that the retrograde increase in quantal content in GluRIIA^-/- mutants can be counteracted by overexpressing multiple copies of GluRIIA, independent of the size of EPSPs. The conclusion from these results is that the retrograde control of presynaptic release for these genotypes is not directly related to muscle depolarization. In addition, it is argued that if muscle depolarization were the sole trigger for the homeostatic retrograde signaling, then quantal content in highwire (hiw) mutants should have been compensated for. hiw mutants have 60%-70% less quantal release, while retrograde control of neurotransmission is still intact in these mutants. Therefore, it is proposed that postsynaptic CaMKII regulates presynaptic release by responding to calcium influx through glutamate receptors during evoked and spontaneous neurotransmitter release (Haghighi, 2003).

The role of postsynaptic membrane depolarization in homeostatic control of presynaptic release at the Drosophila NMJ has been investigated by Paradis (2001). This study demonstrates that the expression of an inward-rectifying potassium channel, Kir2.1, in postsynaptic muscles leads to an increase in quantal content. Kir2.1-expressing muscles show severe defects in muscle properties, including input resistance and membrane potential. More importantly, muscle excitability is affected to the point that mEPSP amplitude is reduced to less than half of wild-type levels (Paradis, 2001). The authors further show that mEPSCs are still wild-type under voltage-clamp conditions, suggesting no change in glutamate receptor function. However, considering the kinetics of membrane depolarization, it is conceivable that, under physiological conditions, GluRIIA function could be compromised during an evoked response that is severely reduced in duration. In other words, while glutamate receptor function is not affected directly, these results suggest that ion influx through glutamate receptors could be affected due to membrane defects. Therefore, the moderate increase in quantal content could be partially due to this apparent reduction in glutamate receptor activity (Haghighi, 2003).

How does the motor neuron respond to the retrograde signal? Based on the results, inhibition of postsynaptic CaMKII mimics the reduction in postsynaptic activity in glutamate receptor mutants and triggers the retrograde signal, leading to an increase in neurotransmitter release at the NMJ. This increase in neurotransmitter release does not appear to induce the NMJ to grow more synaptic boutons, since the numbers of synaptic boutons remained unchanged. Similarly, the overall ultrastructure of boutons remained indistinguishable from wild-type. In contrast, Koh (1999) has reported an overdevelopment of the subsynaptic reticulum in larvae expressing a CaMKII inhibitory peptide (Ala) (Haghighi, 2003).

In that study, Ala or CaMKII^T287D were expressed in both muscles and the nervous system simultaneously, whereas Haghighi (2003) manipulated CaMKII only in neurons or muscles exclusively. It is conceivable that the level and the pattern of expression of these transgenes could have led to differences between experiments. Furthermore, Koh analyzed boutons at the midline section only, while the Haghighi study looked at complete serial sections of boutons. These differences in the levels or pattern of Ala expression as well as differences in analyses could underlie this discrepancy (Haghighi, 2003).

The Haghighi study found a 60% increase in the number of T bars per active zone in response to inhibition of CaMKII in muscles. Often present at active zones at the Drosophila NMJ, T bars are electron-dense structures associated with clusters of synaptic vesicles. Higher numbers of active zones and T bars appear to correlate with an increase in the strength of synaptic transmission. For example, hyperexcitable eag shaker mutants contain a higher number of T bars than wild-type at NMJ synapses. The Haghighi study further demonstrates that induction of retrograde signaling in GluRIIA^-/- mutants leads to a doubling of the number of T bars per active zone, similar to the effect of postsynaptic inhibition of CaMKII. Reiff (2002) has recently reported an increase in T bars in another allelic combination of glutamate receptor mutants. Finally, the Haghighi study shows that the increase in T bars could be surpressed by postsynaptic activation of CaMKII. Since postsynaptic activation of CaMKII in glutamate receptor mutants also suppresses quantal content, these results further support a direct correlation between presynaptic T bars and neurotransmitter release at the Drosophila NMJ. These findings suggest that postsynaptic reduction of CaMKII activity may boost presynaptic neurotransmitter release by upregulating T bars at active zones in presynaptic boutons, a potential mechanism for the control of synaptic transmission induced by the retrograde signal. The number of T bars per active zone could therefore be used as an index for the presence of the homeostatic retrograde signal, independent of quantal content measurements (Haghighi, 2003).

It has been demonstrated that a BMP type II receptor, wishful thinking (wit), is required for both growth and function of the NMJ in Drosophila. To further explore the mechanism by which motor neurons respond to the retrograde signal, whether the retrograde enhancement of quantal content can occur in wit mutants was examined. The results indicate that the retrograde signal cannot increase neurotransmitter release in the absence of Wit. Activation of the retrograde signal by either postsynaptic expression of GluRIIA^M/R or postsynaptic inhibition of CaMKII did not lead to any increase in quantal content. These results indicate a requirement for wit presynaptically for the functioning of the retrograde mechanism that controls the homeostasis of neurotransmitter release at the NMJ of Drosophila and that postsynaptic inhibition of CaMKII requires the function of presynaptic BMP signaling to enhance quantal release (Haghighi, 2003).

Glass bottom boat (Gbb), a BMP ortholog, functions as a retrograde ligand for Wit at the Drosophila NMJ. Mutations in gbb lead to NMJ defects similar to those observed in wit mutants, and postsynaptic transgenic expression of Gbb can rescue many of these defects. In light of these findings, it is possible that there is a link between postsynaptic activity of CaMKII and the level and function of Gbb at the NMJ of Drosophila (Haghighi, 2003).

Another candidate protein for interacting with CaMKII in controlling retrograde signaling is Discs large (DLG). DLG has been shown to be phosphorylated by CaMKII (Koh, 1999) and to be involved in synaptic transmission at the NMJ of Drosophila. However, the defects in synaptic transmission in dlg mutants are rescued by presynaptic rather than postsynaptic expression of DLG. This suggests that the role of DLG in neurotransmission is primarily presynaptic. Furthermore, in the rho-type guanine nucleotide exchange factor dpix mutants quantal release is not greatly affected, while DLG levels are reduced by 80%. Therefore, it seems unlikely that the effects observed are due to changes in DLG phosphorylation levels. Additional experiments are needed to further elucidate the mechanism through which CaMKII activity controls the homeostasis of neurotransmitter release and to identify target proteins that CaMKII may interact with in the postsynaptic cell (Haghighi, 2003).

Calcium-independent calcium/calmodulin-dependent protein kinase II in the adult Drosophila CNS enhances the training of pheromonal cues

Calcium/calmodulin-dependent protein kinase II (CaMKII) is abundant in the CNS and is crucial for cellular and behavioral plasticity. It is thought that the ability of CaMKII to autophosphorylate and become Ca2+ independent allows it to act as a molecular memory switch. Inhibition of Drosophila CaMKII leads to impaired performance in the courtship conditioning associative memory assay, but it was unknown whether the constitutive form of the kinase had a special role in learning. In this study, a tripartite transgenic system combining GAL4/UAS with the tetracycline-off system was used to spatially and temporally manipulate levels of Ca2+-independent CaMKII activity in Drosophila. An enhancement of information processing during the training period was found with Ca2+-independent, but not Ca2+-dependent, CaMKII. During training, control animals have a lag before active suppression of courtship begins. Animals expressing Ca2+-independent CaMKII have no lag, implying that there is a threshold level of Ca2+-independent activity that must be present to suppress courtship. This is the first demonstration, in any organism, of enhanced behavioral plasticity with overexpression of constitutively active CaMKII. Anatomical studies indicate that transgene expression in antennal lobes and extrinsic mushroom body neurons drives this behavioral enhancement. Interestingly, immediate memory was unaffected by expression of T287D CaMKII in mushroom bodies, although previous studies have shown that CaMKII activity is required in this brain region for memory formation. These results suggest that the biochemical mechanisms of CaMKII-dependent memory formation are threshold based in only a subset of neurons (Mehren, 2004; full text or article).

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