CaM kinase II: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - Calcium/calmodulin-dependent protein kinase II

Synonyms -

Cytological map position - 102E1--102F8

Function - Calcium/calmodulin-dependent kinase

Keywords - learning pathway, calcium dependent enzymes, endoderm, central nervous system, Visual signal transduction

Symbol - CaMKII

FlyBase ID:FBgn0264607

Genetic map position - 4-[3]

Classification - CaM Kinase, ATP binding motif

Cellular location - cytoplasmic and possibly nuclear



NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Nesler, K. R., Starke, E. L., Boin, N. G., Ritz, M. and Barbee, S. A. (2016). Presynaptic CamKII regulates activity-dependent axon terminal growth. Mol Cell Neurosci 76: 33-41. PubMed ID: 27567686
Summary:
Spaced synaptic depolarization induces rapid axon terminal growth and the formation of new synaptic boutons at the Drosophila larval neuromuscular junction (NMJ). This study identified a novel presynaptic function for the Calcium/Calmodulin-dependent Kinase II (CamKII) protein in the control of activity-dependent synaptic growth. Consistent with this function, both total and phosphorylated CamKII (p-CamKII) are were found to be enriched in axon terminals. Interestingly, p-CamKII appears to be enriched at the presynaptic axon terminal membrane. Moreover, levels of total CamKII protein within presynaptic boutons globally increase within one hour following stimulation. These effects correlate with the activity-dependent formation of new presynaptic boutons. The increase in presynaptic CamKII levels is inhibited by treatment with cyclohexamide suggesting a protein-synthesis dependent mechanism. Previous work has found that acute spaced stimulation rapidly downregulates levels of neuronal microRNAs (miRNAs) that are required for the control of activity-dependent axon terminal growth at this synapse. The rapid activity-dependent accumulation of CamKII protein within axon terminals is inhibited by overexpression of activity-regulated miR-289 in motor neurons. Experiments in vitro using a CamKII translational reporter show that miR-289 can directly repress the translation of CamKII via a sequence motif found within the CamKII 3' untranslated region (UTR). Collectively, these studies support the idea that presynaptic CamKII acts downstream of synaptic stimulation and the miRNA pathway to control rapid activity-dependent changes in synapse structure.
Castro-Rodrigues, A. F., Zhao, Y., Fonseca, F., Gabant, G., Cadene, M., Robertson, G. A. and Morais-Cabral, J. H. (2018). The interaction between the Drosophila EAG potassium channel and the protein kinase CaMKII involves an extensive interface at the active site of the kinase. J Mol Biol. PubMed ID: 30381148
Summary:
The Drosophila EAG (dEAG) potassium channel is the founding member of the superfamily of KNCH channels, which are involved in cardiac repolarization, neuronal excitability and cellular proliferation. In flies, dEAG is involved in regulation of neuron firing and assembles with CaMKII to form a complex implicated in memory formation. This study has characterized the interaction between the kinase domain of CaMKII and a 53-residue fragment of the dEAG channel that includes a canonical CaMKII recognition sequence. Crystal structures together with biochemical/biophysical analysis show a substrate-kinase complex with an unusually tight and extensive interface that appears to be strengthened by phosphorylation of the channel fragment. Electrophysiological recordings show that catalytically active CaMKII is required to observe active dEAG channels. A previously identified phosphorylation site in the recognition sequence is not the substrate for this crucial kinase activity, but rather contributes importantly to the tight interaction of the kinase with the channel. The available data suggest that the dEAG channel is a docking platform for the kinase and that phosphorylation of the channel's kinase recognition sequence modulates the strength of the interaction between the channel and the kinase.
BIOLOGICAL OVERVIEW

Multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase II) is one of the major protein kinases coordinating cellular responses to neurotransmitters and hormones. CaM kinase II is so named because of its requirement for calcium-bound calmodulin (CaM) for activation and its ability to phosphorylate and alter the function of a variety of substrates. This discussion will first focus on the effects of CaM kinase II on behavior and learning, and then describe the biochemical events involved. One begins by identifying the target(s) of CaM kinase II.

One target has been identified in Drosophila. Similar defects in both synaptic transmission and associative learning are produced in Drosophila by inhibition of calcium/calmodulin-dependent protein kinase II and mutations in the potassium channel subunit gene ether à go-go (eag), suggesting that EAG is targeted by CaM Kinase in learning. To test whether CaM Kinase influences learning, two behavior assays for learning have been employed: acoustic priming, a measure of nonassociative learning and sensitization, and courtship conditioning, a paradigm known to have characteristics of associative learning.

(1) acoustic priming

Courtship consists of a set of complex stereotyped behaviors, involving olfaction, hearing, vision and locomotion. This behavior was once thought to be completely "hard-wired", but is now known to exhibit significant experience-dependent features. Acoustic priming is an example of such plasticity. Exposure of virgin females to a correct species-specific male courtship song in the presence of wingless courting males (that is, males unable to sing) significantly enhances the speed of subsequently mating. A similar enhancement can be obtained by prestimulating the females with an artificial courtship song and subsequently adding normal winged males. The females appear to retain a memory of the prestimulation and consequently mate faster. This behavior is an example of sensitization: the enhancement of mating speed is the result of stimulation of the female with the male courtship song. The amount and duration of the priming effect appear to be dependent on the same cellular and biochemical mechanisms as other information storage and retrieval processes, since the classic learning mutants dunce, rutabaga and amnesiac are abnormal in this test of acoustic priming. These mutant flies seem to not retain memory of acoustic priming. To test whether learning is altered by an alteration in CaM Kinase activity, a peptide inhibitor of CaM Kinase was expressed from a heat shock promoter, allowing inhibition of CaM Kinase II in the adult female animals subjected to behavioral tests. Although the pathways mediating acoustic priming are intact in flies in which a CaM Kinase inhibitor is expressed, these lines are not normal with respect to their ability to retain the effects of sensitization if delays are introduced between the playing of the courtship song and the presentation of male flies. Such an abnormal loss of retention in flies carrying the CaM Kinase inhibitior takes place over the course of one to three minutes between prestimulation and test (Griffith, 1993b).

(2) courtship conditioning

The second behavioral assay employed was courtship conditioning, an associative learning assay. A normal male will court a virgin female vigorously, but his courtship activity becomes depressed if he is paired with a previously mated (and hence, unresponsive) female. This depression lasts for several hours; during this period, if the male is paired with a virgin female, he takes longer to initiate courtship and spends significantly less time courting. Production of this depression is associative, dependent on aversive chemical cues emitted by the mated female; courting behavior remains depressed in the simultaneous presence of virgin and previously mated females. It is not dependent on visual input, as both blind flies and flies kept in the dark can be conditioned.

In all previously characterized learning mutants of Drosophila, this inhibition of subsequent courtship of a virgin female is absent or decreased, (that is, the mutants show long term memory defects with regard to aversive conditioning), although being paired with a mated female initially leads to a decrease in courtship of that female. Introduction into mutant males of a heat shock activation of a CaM Kinase inhibitor, results in flies that are normal with respect to courtship of virgin females. Such inhibitor expressing lines can be trained by exposure to a mated female, indicating that these lines are responsive to the aversive signals given by a mated female. Nevertheless, the transgenic lines fail to retain the effects of training. When subsequently presented with a virgin female, instead of being inhibited with respect to time of initiation and percentage of time spent in courtship, they initiate rapidly and court vigorously. This failure to modify courtship behavior in an experience-dependent manner is manifested by an increase in courtship activity, again underscoring the fact that the transgenic lines are not simply sick but, rather, have a specific defect in plasticity (Griffith, 1993b).

Subsequent studies were carried out to unravel the relation between CaM kinase II and visual input in the neuronal circuit controlling courtship conditioning. Learning was measured by exposure of male flies to a mated female for 1 hour and then immediately placing the male with an anesthetized virgin female. Exposure to the mated female reduces the level of mating to the virgin female. Memory was tested by measuring a courtship index for the initial 10 minutes of exposure to an anesthetized virgin female after the previous day's training with a mated female. The role of visual input in producing this behavior and the effects of modifying visual input on CaM kinase-dependent memory formation were examined. Inhibition of CaM kinase blocks apparent learning regardless of visual input. Visual input selectively affects the memory phase of courtship conditioning: normal visual input masks (makes up for) the memory effects of inhibition of CaM kinase resulting in the generation of memory without apparent learning, whereas disruption of visual input reveals the CaM kinase-dependence of memory. Visual input is found to be important only during the training period, which implies that vision and CaM kinase are interacting in the formation rather than the retrieval of memory. These results suggest a model for courtship conditioning in which multiple sensory inputs are integrated at a CaM-kinase-dependent neuronal switch to modulate courtship behavior (Joiner, 1997).

What do these defects in learning have to do with potassium channels? At the molecular level, a portion of the putative cytoplasmic domain of Eag is a substrate of calcium/calmodulin-dependent protein kinase II. Flies expressing the inhibitor of CaM Kinase II, as well as flies mutant for eag show supernumary synaptic discharges. Spontaneous discharges occur at frequencies as high as 25 Hz, lasting up to ten seconds after electrical stimulation. These results are in contrast to controls, in which unevoked discharges are never seen. These results suggest a failure of the nerve terminal to repolarize properly after repetitive stimulation, and imply that both CaM kinase II activity and a normal Eag potassium channel subunit are required for repolarization. Thus, 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).

Morphological aberrations correlate with to the impaired associative conditioning observed in transformed strains expressing CaM kinase inhibitors. There are increased numbers of nerve terminal branches associated with large varicosities at the nerve terminals of motor neurons in transformed flies. Inhibitory peptide, under to control of a heat-shock promoter, results in morphological effects observed three days after induction of the inhibitor. Excess branching occurs in larger, type I boutons, but not at the smaller type II varicosities. Another striking feature is that inhibition of CaM kinase appears to cause branch misorientation. Frequently, aberrant extra nerve entry points are observed in heat shock flies (Wang, 1994).

Other targets of CaM Kinase II are know in vertebrates. One target is the enzyme tyrosine hydroxylase, the rate-limiting enzyme that catalyzes the hydroxylation of tyrosine to form dopa, a chemical precursor which is subsequently converted to neurotransmitters. Thus CaM kinase II regulates the formation of neurotransmitters. CaM kinase II regulates neural excitability, phosphorylating Synapsin I (see Drosophila Synapsin), a protein which functions in regulating vesicular movement. Vesicles release neurotransmitters at the synapse, and their dynamics is the focus of regulation of neural excitability. CaM kinase targets the AMPA receptor, known to be central to the process of long term potentiation, an experimental analog of learning (Braun, 1995 and references).

CaM kinase is also involved in the regulation of gene expression. The c-fos promoter is targeted by both CREB (Drosophila homolog: dCREB2) and C/EBP (Drosophila homolog: SLBO). Both CREB and C/EBP are targeted by CaM kinase II (Braun, 1995 and references).

Mapping the anatomical circuit of CaM kinase-dependent courtship conditioning

Globally inhibiting CaM kinase activity in Drosophila, using a variety of genetic techniques, disrupts associative memory yet leaves visual and chemosensory perception intact. These studies implicate CaM kinase in the plastic processes underlying learning and memory but do not identify the neural circuitry that specifies the behavior. In this study, the GAL4/UAS binary expression system was used to define areas of the brain that require CaM kinase for modulation of courtship conditioning. The courtship-conditioning assay is divided into two distinct parts. The first is the training period during which the male is in contact with a mated female. The male normally modifies his behavior toward the trainer female during this period. This is the period in which the learning trait is measured. The second part is a test period in which the male is presented with a virgin female. His response to this female is a measure of his memory of associations made during the training period. The CaM kinase-dependent neurons that determine the response to the mated female during conditioning and those involved in formation and expression of memory are located in distinct areas of the brain. This supports the idea that courtship conditioning results in two independent behavioral modifications: a decrement in courtship during the conditioning period and an associative memory of conditioning. This study has allowed the circuit of information flow for a memory process in Drosophila to be genetically determined. The map generated dissects the behavior into multiple components and will provide tools that allow both molecular and electrophysiological access to this circuit (Joiner, 1999).

The anatomical location of CaM kinase-requiring neurons that are used for courtship conditioning was mapped using a total of 18 different P[GAL4] lines to express an inhibitor of CaM kinase in particular parts of the brain. In some of the lines, the expression pattern of GAL4 appears to be isolated within a single neural structure. In other lines, expression is predominantly localized to one or two structures with less intensive expression in other parts of the neuropil. By using, for example, five lines that predominantly express in the mushroom bodies, other areas of non-overlapping expression between the P[GAL4] lines can be ruled out as having a role in the observed phenotypes. Developmental effects can be controlled for using this approach, since there is a difference between the early temporal and spatial patterns of expression of independent lines. The use of multiple lines has also allowed for the determination that there are thresholds of inhibition below which few effects are seen (Joiner, 1999).

These experiments suggest that behavior during the conditioning period is determined by information processing at early synapses in the circuit in the antennal lobes. The main antennal lobes consist of primary sensory neurons that project from the olfactory and gustatory sense organs, as well as second-order intrinsic neurons. Cooling experiments in the honey bee have shown that initial processing of external stimuli occurs in the antennal glomeruli. Incoming information is processed in the first- and second-order neurons of the antennal nerve and lobes and neurons from the maxillary palps. This processing is required to modulate behavior toward the mated female during conditioning (Joiner, 1999).

Memory formation and modification of behavior toward subsequently presented females is determined by information processing deeper in the brain at higher-order synapses. Direct connections from the antennal nerve to the lateral protocerebrum have been described and P[GAL4] inserts (MJ146 and MJ286c) expressing ala (the CaM kinase inhibitor) in the lateral protocerebrum show that second- or third-order processing of this information occurs here. Memory is formed beyond the initial sensory processing centers in the brain at a number of different sites including the mushroom bodies, central complex, and lateral protocerebrum (Joiner, 1999).

The environmental inputs that drive this behavior in the absence of visual input are largely chemosensory. The neuronal circuit begins with chemosensory inputs, which send their processes to the antennal lobes where local inhibitory neurons and projection neurons interact. Of the two tracts from the antenna, mechanosensory and chemosensory, only a subset of the chemosensory input is used in this behavior. From select antennal glomeruli and directly from the antennal nerve, information is transferred to both the lateral protocerebrum and calyces of the mushroom bodies via unilateral connectives. Although the antennal nerve sends projections to both the lateral protocerebrum and indirectly to the central complex, the results presented here imply that processing of conditioning only occurs in the lateral protocerebrum (Joiner, 1999).

The mushroom bodies are used exclusively for memory in CaM kinase-dependent courtship conditioning; they send projections to the lateral protocerebrum. This is underscored by the results of the line 201Y, the only mushroom body line that shows a defect in response to the mated female. The most striking difference between the pattern of expression for 201Y and the other four mushroom-body expressing P[GAL4] lines are two pairs of cells, one in the lateral ventral protocerebrum and the other in the lateral dorsal region. The ventral pair resemble cellular expression of MJ146 and the dorsal pair resemble those of MJ162a. Both of these lines are defective for modulation of the response to the mated female (Joiner, 1999).

Unlike the lateral protocerebrum, the central complex (a neuropil involved in behavior output) is used solely for memory. Although no prominent neural tracts project directly to the central complex, it receives inputs from most areas of the brain, including the antennal nerve, the lateral protocerebrum, and the optic lobes, but not the mushroom bodies. The central complex is also known to be involved with motor output programs, and it is possible that inhibition of CaM kinase in this structure could impair output pathways. A strong argument against this is that global inhibition of CaM kinase has been shown to affect memory formation but not retrieval. In addition, there are no gross motor defects induced by CaM kinase inhibition; in fact failure to remember is associated with an increase in courtship behavior. These results would argue that the memory problems documented here are caused by impairment of formation, not retrieval (Joiner, 1999).

These experiments also demonstrate a difference between courtship conditioning and classical conditioning of odor avoidance in the circuitry and/or biochemistry underlying learning. Expression of activated Galphas in the mushroom bodies, but not in the central complex, using a subset of the lines used in this study, disrupts learning of odor avoidance (Connolly, 1996). Memory was not tested in this study. In particular, OK348, which expresses in the fan-shaped body, disrupts memory formation in the courtship-conditioning assay, but not learning in the odor avoidance assay. P-element insertion c232, which expresses in the ellipsoid body, does not affect either behavior. The relative magnitudes of disruption caused by mushroom body expression varies. 201Y, for example, is the least disruptive for classical conditioning, but the most disruptive for courtship conditioning. This biochemically defined circuit is not the equivalent of a connectivity circuit as defined electrophysiologically. The genetic manipulation of signal transduction pathways in discrete areas of the brain that are known to be connected directly or indirectly gives insight into the flow of information, but cannot rule out parallel pathways (Joiner, 1999).

In the courtship-conditioning assay, response to a virgin female is a measure of the male's memory of associations made during the training period. Previous studies have demonstrated that an association between attractive and aversive cues and courtship is required for the male to show intact memory in the test period. In flies in which CaM kinase activity is blocked in the mushroom bodies or parts of the central complex, a memory defect is observed with no change in the male's modification of his behavior during the training period. The decrement in courtship shown by wild-type males during the conditioning hour has been shown to be distinct from, and not necessary for, associative memory formation as assayed during the test period. One conclusion that can be drawn from these studies is that these two behaviors are not interdependent. This idea is supported strongly by the anatomical separation of these two responses, as demonstrated in this study. The decrement in courtship of the mated female is dependent upon intact CaM kinase in antennal lobes, whereas memory formation is dependent upon other brain structures. It is possible that this initial response to the mated female may even represent a form of nonassociative learning. A neural network model accounts for the differences between the phases of the assay in a more integrative way than by simply assigning nonassociative or associative labels to the behaviors (Joiner, 1999).

CaM Kinase II - biochemical activity

Ca2+ may enter the cell through extracellular voltage-senstive Ca2+ channels or through ligand-gated receptor channels. Ca2+ can also enter the cytoplasm from intracellular vesicular Ca2+ stores. Such intracellular stores are released through the action of inositol-3-phosphate, which is generated from lipids through the action of phospholipase-C (PLC). PLC is a target of G-protein coupled receptors. CaM kinase II transduces changes in intracellular free Ca2+ into changes in the phosphorylation state and activity of target proteins involved in neurotransmitter synthesis and release, neuronal plasticity and gene expression. Calmodulin, a ubiquitous enzyme, is the cell's calcium sensor, binding four calcium ions with high affinity. The Ca2+/calmodulin complex activates downstream targets, including CaM kinase II.

Structure/function analyses of the kinase reveal it is kept inactive in its basal state by a regulatory domain that is displaced by the binding of Ca2+/calmodulin. The crystal structures of calmodulin bound to the calmodulin-binding domain of CaM kinase II suggest that calmodulin wraps around the amphipathic calmodulin-binding domain, which then takes on an alpha-helical configuration. In essence, calmodulin may peel away a tight bound inhibitory segment from the active site. Calmodulin thereby activates the kinase by enabling the binding of both ATP and peptide substrates (Schulman, 1995 and references).

The CaM kinase II polypeptide consists of an N-terminal catalytic domain, a central regulatory domain, and a C-terminal association domain that is subject to alternative splicing in vertebrates, but not in Drosophila.

Whereas vertebrate CaM kinase II consists of four distinct classes of CaM kinase, each encoded by a separate gene, and each consisting of multiple isoforms generated by alternative splicing, Drosophila CaM kinase II is coded for by a single gene, whose transcript is subject to alternative splicing. Alternative splicing in the Drosophila enzyme takes place in the mRNA coding for a section near the C-terminus of the putative link segment, which is postulated to join the N-terminal to the C-terminal globules of the polypeptide. Alternative splicing in the Drosophila mRNA does not take place in mRNA coding for the C-terminal domain that is subject to alternative splicing in vertebrates (Griffith, 1993b).

Autophosphorylation provides a critical regulation of CaM kinase II. The autoinhibitory domain of the kinase is disrupted by binding of calmodulin at its C-terminal end, which leads to a de-inhibition of the kinase. The autoinhibitory domain can be further disrupted by phosphorylation of a key residue common to all isoforms. Phosphorylation of this site is not essential for kinase activity, but it does have two important consequences. First, autophosphorylation increases the affinity of the kinase for calmodulin several hundredfold by reducing the dissociation rate of the kinase-calmodulin complex. Second, the presence of a phosphate on this site is itself sufficient to disrupt the autoinhibitory domain, and the kinase retains partial activity even after calmodulin dissociates (Braun, 1995).

The frequency of Ca2+ oscillations or spikes, that is the extent of neural activity, may be decoded by CaM kinase via this autophosphorylation. Calmodulin is essentially trapped by autophosphorylation which converts CaM kinase into a high affinity calmodulin-binding protein. Repetitive stimulation of the kinase may promote recruitment of calmodulin to the kinase so that it becomes increasingly active with each stimulus in a frequency-dependent manner. The association domain at the C-terminal end of CaM kinase of vertebrates contains a variable region that targets isoforms of the kinase to the nucleus or cytoskeleton and assembles the kinase into a decameric structure. Alternative splicing introduces a short nuclear localization signal that targets transfected kinase to the nucleus where it may regulate nuclear functions. The regulatory properties of CaM kinase provide for molecular potentiation of Ca2+ signals and frequency detection whereas its association domain should enable it to decode such Ca2+ fluctuations in the nucleus (Schulman, 1995).

Thus the effects of CaM kinase are felt at different levels of nerve action, including production of neurotransmitters, vesicular release of neurotransmitters, activation of transcription factors known to be involved in the learning process, neural plasticity, as evidenced by axon sprouting, and various learning paradigms.

Presynaptic CamKII regulates activity-dependent axon terminal growth

Spaced synaptic depolarization induces rapid axon terminal growth and the formation of new synaptic boutons at the Drosophila larval neuromuscular junction (NMJ). This study identified a novel presynaptic function for the Calcium/Calmodulin-dependent Kinase II (CamKII) protein in the control of activity-dependent synaptic growth. Consistent with this function, both total and phosphorylated CamKII (p-CamKII) are were found to be enriched in axon terminals. Interestingly, p-CamKII appears to be enriched at the presynaptic axon terminal membrane. Moreover, levels of total CamKII protein within presynaptic boutons globally increase within one hour following stimulation. These effects correlate with the activity-dependent formation of new presynaptic boutons. The increase in presynaptic CamKII levels is inhibited by treatment with cyclohexamide suggesting a protein-synthesis dependent mechanism. Previous work has found that acute spaced stimulation rapidly downregulates levels of neuronal microRNAs (miRNAs) that are required for the control of activity-dependent axon terminal growth at this synapse. The rapid activity-dependent accumulation of CamKII protein within axon terminals is inhibited by overexpression of activity-regulated miR-289 in motor neurons. Experiments in vitro using a CamKII translational reporter show that miR-289 can directly repress the translation of CamKII via a sequence motif found within the CamKII 3' untranslated region (UTR). Collectively, these studies support the idea that presynaptic CamKII acts downstream of synaptic stimulation and the miRNA pathway to control rapid activity-dependent changes in synapse structure (Nesler, 2016).

Acute spaced synaptic depolarization rapidly induces the formation of new synaptic boutons at the larval NMJ. These immature presynaptic outgrowths, also known as "ghost boutons", are characterized by the presence of synaptic vesicles but by a lack of active zones and postsynaptic specializations. IA wild-type third instar larval NMJ will typically have about 2 ghost boutons. Using an established synaptic growth protocol, a robust increase in the number of ghost boutons was observed following 5 x K+ spaced stimulation. It has been shown that activity-dependent ghost bouton formation involves both new gene transcription and protein synthesis. Furthermore, new presynaptic expansions can form within 30 min of stimulation even after the axon innervating the NMJ has been severed. These findings suggest that a local mechanism (i.e. local signaling and/or translation) is required for the budding and outgrowth of new axon terminals. As expected, application of the translational inhibitor cyclohexamide during the recovery phase prevented the formation of new ghost boutons (Nesler, 2016).

It has been shown previously that the outgrowth of new synaptic boutons in response to spaced depolarization requires the function of activity-regulated neuronal miRNAs including miR-8, miR-289, and miR-958 (Nesler, 2013). This implies that mRNAs encoding for synaptic proteins might be targets for regulation by these miRNAs. Focused was placed on CamKII for three reasons. (1) CamKII has been shown to have a conserved role in the control of long-term synaptic plasticity and its expression at synapses requires components of the miRNA pathway. Furthermore, the fly CamKII mRNA contains two predicted binding sites for activity-regulated miR-289. (2) CamKII and PKA both phosphorylate and actives synapsin. At the fly NMJ, a synapsin-dependent mechanism is required for a transient increase in neurotransmitter release in response to tetanic stimulation. Synapsin also redistributes to sites of activity-dependent axon terminal growth and regulates outgrowth via a PKA-dependent pathway. (3) Presynaptic CamKII has been shown to function in axon pathfinding in cultured Xenopus neurons. It seemed likely that activity-dependent ghost bouton formation and axon guidance might share similar molecular machinery (Nesler, 2016).

It was postulated that presynaptic CamKII was required to control activity-dependent axon terminal growth at the larval NMJ. To address this question, CamKII expression was disrupted in motor neurons using two transgenic RNAi constructs. Depletion of presynaptic CamKII with both transgenes prevented the formation of new ghost boutons in response to spaced stimulation. Thus, presynaptic CamKII is necessary to control the formation of new synaptic boutons (Nesler, 2016).

To further confirm that presynaptic CamKII function was required for activity-dependent growth, a transgenic line was used that inducibly expressed an inhibitory peptide (UAS-CamKIIAla). As in mammals, the activation of Drosophila CamKII by exposure to calcium leads to the autophosphorylation of a conserved threonine residue within the autoinhibitory domain (T287 in Drosophila). Activation of CamKII then confers an independence to calcium levels that persists until threonine-287 is dephosphorylated. The synthetic Ala peptide mimics the autoinhibitory domain and its transgenic expression is sufficient to substantially inhibit endogenous CamKII activity. Expression of the Ala inhibitory peptide in larval motor neurons disrupted the formation of new ghost boutons following spaced synaptic depolarization. These observations are consistent with results from CamKII RNAi (Nesler, 2016).

Together, these data suggest that presynaptic CamKII function is required to control new ghost bouton formation in response to acute synaptic activity. Similarly, presynaptic CamKII has been implicated in controlling both bouton number and morphology during development of the larval NMJ. Reducing neuronal CamKII levels by RNAi has recently been shown to significantly reduce the number of type 1b boutons at the larval NMJ suggesting that presynaptic CamKII is required to control normal synapse development (Gillespie, 2013). In contrast, presynaptic expression of the Ala inhibitory peptide has no effect on the total number of type 1 synaptic boutons. Given that the Ala peptide does not completely inhibit CamKII autophosphorylation, it is suggested that the activation of CamKII in response to acute spaced synaptic depolarization is likely to be more sensitive to disruption then during NMJ development (Nesler, 2016).

It was next asked if presynaptic CamKII could induce activity-dependent axon terminal growth at the NMJ. The overexpression of genes that are necessary for the control of ghost bouton formation generally does not cause an increase in the overall number of new synaptic boutons following 5 x high K+ spaced. Instead, overexpression often leads to an increased sensitization of the synapse to subsequent stimuli (for example, significant growth is observed after 3 x instead of 5 x high K+). The overexpression of a wild-type CamKII transgene in motor neurons caused an increase of 71% in ghost bouton numbers in 3 x K+ spaced stimulation larvae compared to 0 x K+ controls. While this is trending towards an increase, it did not reach statistical significance, even though expression levels were substantially higher than endogenous CamKII. Thus, increased CamKII is not sufficient to stimulate activity-dependent axon terminal growth (Nesler, 2016).

To further investigate the role of presynaptic CamKII in activity-dependent axon terminal growth, the effect of transgenic neuronal overexpression of either an overactive form of CamKII (CamKIIT287D) or a form that is incapable of remaining active in the absence of elevated calcium (CamKIIT287A). Much like C380-Gal4/+ controls, presynaptic expression of either transgene had no significant effect on the number of ghost boutons in 3 x high K+ stimulation. Again, levels of CamKII protein in axon terminals in both transgenic lines were elevated relative to controls. Collectively, these data suggest that constitutive activation of CamKII is not sufficient to sensitize the NMJ to stimulation (Nesler, 2016).

The results suggest that the temporal and/or spatial regulation of CamKII expression or activation is likely required to control activity-dependent growth. In support, the Drosophila CamKII protein has been shown to phosphorylate and regulate the activity of the Ether-a-go-go (Eag) potassium channel in motor neuron axon terminals. In turn, CamKII is bound and locally activated by phosphorylated Eag. This local activation can persist even after calcium levels have been reduced. CamKII autophosphorylation and Eag localization to synapses requires the activity of the membrane-associated Calcium/Calmodulin-associated Serine Kinase, CASK. The presynaptic coexpression of CASK with CamKIIT287D reverses (to wild-type levels) the increase in type 1b boutons observed when CamKIIT287D is overexpressed alone. Thus, a mechanism exists at the larval NMJ that allows for the persistence of local CamKII activation in the absence of additional stimul (Nesler, 2016).

After establishing that CamKII has a novel presynaptic function in activity-dependent ghost bouton formation, the distribution of CamKII protein at the larval NMJ was examined closely. It has previously been reported that CamKII strongly colocalizes with postsynaptic Discs large (DLG), the Drosophila ortholog of mammalian PSD-95, around the borders of type 1 synaptic boutons. In support, an anti-CamKII antibody coimmunoprecipitates DLG from larval body wall extracts. Interestingly, while DLG is pre-dominantly postsynaptic at the developing NMJ it is also initially expressed in the presynaptic cell and at least partially overlaps with presynaptic membrane markers in axon terminals. It has been demonstrated that while fly CamKII colocalizes with DLG within dendrites of adult olfactory projection neurons (PNs), it also localizes to presynaptic boutons within those same neurons. Consistent with the latter observations (using a different CamKII antibody), it has been shown that CamKII is substantially enriched in presynaptic terminals of type 1b boutons. To resolve these inconsistent results, both antibodies against CamKII were used to more closely analyze the localization of CamKII at the third instar larval NMJ. First, double labeling of wild-type NMJs with a monoclonal CamKII antibody and anti-horseradish peroxidase (HRP), a marker for Drosophila neurons, confirmed that CamKII was enriched in presynaptic boutons in a pattern very similar to that of HRP. A closer examination of confocal optical sections revealed that almost all CamKII localized to the presynaptic terminal and was not significantly enriched either (1) at sites surrounding presynaptic boutons, or (2) in the axons innervating synaptic arbors (Nesler, 2016).

Within boutons, CamKII appeared to be predominantly cytoplasmic but was sometimes localized to discrete puncta that were reminiscent of antibody staining for active zones. Prolonged depolarization of hippocampal neurons with K+ leads to mobilization of CamKII from the cytoplasm to sites near active zones. Moreover, using a fluorescent reporter for CamKII activity, high frequency stimulation causes the very rapid (on the order of minutes) activation of presynaptic CamKII and promotes its translocation from the cytoplasm to sites near active zones. To address this possibility, larval NMJs were double labeled with antibodies targeting both CamKII and DVGLUT, the Drosophila vesicular glutamate transporter, in order to visualize active zones. As predicted, it was found that some presynaptic CamKII colocalized with DVGLUT in type 1b and 1s boutons. Thus, in some type 1 synaptic boutons, CamKII protein is enriched in or near active zones (Nesler, 2016).

To confirm that CamKII was enriched in presynaptic boutons, wild-type NMJs were double labelled with a polyclonal CamKII antibody and anti-DLG. CamKII did partially colocalize with DLG at the border of type 1 synaptic boutons. However, in this study, CamKII was primarily localized to the presynaptic side of the synapse. Collectively, this study provides strong evidence that CamKII is expressed on both the pre- and postsynaptic side of the synapse but that it is clearly enriched within presynaptic boutons at the larval NMJ. This localization is analogous to CamKII distribution in mammalian axons (Nesler, 2016).

After demonstrating that total CamKII was enriched in presynaptic axon terminals, it was next asked if any of this protein was active by assessing phosphorylation of threonine-287 using a phospho-specific polyclonal antibody. It was found that pT287 CamKII staining intensity was strong and fairly uniform in presynaptic boutons and weakly stained axons innervating synaptic arbor. Closer examination of confocal optical sections revealed that almost all p-CamKII colocalized with HRP in the presynaptic terminal and only sparsely stained the body wall muscle (Fig. 3A′). Presynaptic CamKII RNAi almost completely disrupted p-CamKII in axon terminals leaving some residual staining in the presynaptic bouton and surrounding muscle suggesting that the antibody is specific. To further demonstrate this presynaptic localization, it was found that p-CamKII staining clearly does not overlap with postsynaptic DLG but does colocalize strongly with immunostaining using the monoclonal total CamKII antibody (Nesler, 2016).

Collectively, three different antibodies were used to show that CamKII enriched in presynaptic axon terminals. Next, it was asked as to how this enrichment was occurring. In Drosophila and mammalian neurons, the CamKII mRNA is transported to dendritic compartments and locally translated in response to synaptic stimulation. This spatial and temporal regulation requires sequence motifs found within the 5' and 3' UTRs of the CamKII transcript. In contrast, the localization of CamKII to axon terminals of Drosophila PNs does not strictly require the CamKII 3'UTR suggesting that enrichment in presynaptic boutons occurs through a mechanism that does not strictly require local translation. In mammalian neurons, CamKII is enriched in axon terminals where it can associate with synaptic vesicles and synapsin I. Recently, it has been shown that mammalian CamKII and the synapsin proteins are both conveyed to distal axons at rates consistent with slow axonal transport, with a small fraction of synapsin cotransported with vesicles via fast transport (Nesler, 2016).

Because activity-dependent growth at the larval NMJ requires the miRNA pathway and new protein synthesis, it was asked if the localization of CamKII protein to axon terminals might require the CamKII 3'UTR. As expected, when expression was specifically driven in larval motor neurons, a transgenic CamKII:EYFP fusion protein regulated by the CamKII 3'UTR localized strongly to presynaptic boutons at the larval NMJ. However, very similar results were observed using the same CamKII:EYFP fusion protein regulated by a heterologous 3'UTR. Taken together, these data suggest that localization of CamKII protein to presynaptic boutons at the NMJ does not require mRNA transport and local translation. Thus, it is concluded that most of the Drosophila CamKII protein found in motoneuron axon terminals is likely there due to the transport of cytosolic CamKII from the cell body to synapses via a mechanism involving axonal transport. (Nesler, 2016).

It was of interest to determining how CamKII might be regulating activity-dependent axon terminal growth, and it was speculated that either the levels or distribution of CamKII protein might be altered in response to spaced depolarization. It first asked if high K+ stimulation resulted in an increase in CamKII protein within motoneuron axon terminals. Larval preparations were stimulated, and changes in the levels of CamKII protein in presynaptic boutons was examined by immunohistochemistry and quantitative confocal microscopy. Following spaced stimulation, CamKII staining within boutons rapidly increased (in ~ 1 h) by an average of 26%. This increase in immunofluorescence was global and did not appear to be localized to particular regions of the NMJ (i.e., near obvious presynaptic outgrowths). CamKII has been reported to very rapidly translocate to regions near active zones in response to high frequency stimulation. However, when compared to DVGLUT levels in unstimulated and stimulated larvae, no significant increase in CamKII immunofluorescence was observed indicating that translocation does not occur or does not persist in the current assay. To determine if this increase in CamKII enrichment required new protein synthesis, larval preparations were incubated with the translational inhibitor cyclohexamide during the recovery phase. Surprisingly, this treatment completely blocked the activity-dependent affects on presynaptic CamKII enrichment within axon terminals. Thus, spaced high K+ stimulation results in a rapid increase in CamKII levels in presynaptic boutons via some mechanism that requires activity-dependent protein synthesis (Nesler, 2016).

Next, it was asked if the levels or distribution of p-CamKII changed in response to spaced stimulation. Larval preparations were stimulated exactly as described above and analyzed by confocal microscopy. Interestingly, p-CamKII staining was enriched at the presynaptic membrane of many axon terminals following spaced depolarization (Nesler, 2016).

Given the requirement for new protein synthesis, it was speculated that the additional CamKII protein in axon terminals could be derived from a pool of CamKII mRNA that is rapidly transcribed and translated in the soma in response to spaced depolarization. This newly translated CamKII would then be actively transported out to axon terminals via standard mechanisms. If this were true, it would be expected that elevated CamKII levels could be detected in the larval ventral ganglion. To examine this process more closely, global total CamKII expression levels within the larval ventral ganglion were assayed by Western blot analysis. It was found that two distinct isoforms of CamKII are expressed in explanted larval ventral ganglia, Surprisingly, no increase was observed in CamKII protein levels in the ventral ganglion (Nesler, 2016).

What is the source of this new presynaptic CamKII protein? Three possible explanations are proposed. First, new CamKII protein might be transcribed and translated in the motor neuron cell body. However, this new protein would be rapidly transported away to axon terminals in response to spaced depolarization. Second, some CamKII protein is found in the axons innervating the NMJ (seen using the p-CamKII antibody). It is possible that activity stimulates the rapid transport of an existing pool of CamKII protein from distal axons into axon terminals. This process would be sensitive to translational inhibitors. Finally, a pool of CamKII mRNA might be actively transported into axon terminals and then locally translated in response to spaced depolarization. This would account for the both the dependence on translation and for increased CamKII enrichment in presynaptic boutons (Nesler, 2016).

Thus far, this study has shown that activity-dependent ghost bouton formation correlates with a protein synthesis-dependent increase in CamKII levels within presynaptic boutons at the larval NMJ. The activity-dependent translation of CamKII in olfactory neuron dendrites in the adult Drosophila brain requires components of the miRNA pathway. Within the CamKII 3'UTR, there are two putative binding sites for activity-regulated miR-289. These two binding sites were of particular interest. It was previously shown that levels of mature miR-289 are rapidly downregulated in the larval brain in response to 5 x high K+ spaced training (Nesler, 2013). Moreover, presynaptic overexpression of miR-289 significantly inhibits activity-dependent ghost bouton formation at the larval NMJ (Nesler, 2013). Based on these data, it was speculated that CamKII might be a target for regulation by miR-289 (Nesler, 2016).

To determine if CamKII is a target for repression by miR-289 in vivo, a transgenic construct containing the primary miR-289 transcript was overexpressed in motor neurons and CamKII enrichment was examined by anti-CamKII immunostaining and quantitative confocal microscopy. Relative to controls, the presynaptic overexpression of miR-289 completely abolished the observed activity-dependent increase in CamKII immunofluorescence. When analyzing global CamKII levels within axon terminals during NMJ development, presynaptic miR-289 expression led to a slight decrease in CamKII immunofluorescence. This trend is similar to results observed following treatment with cyclohexamide during the recover period. The lack of full repression by miR-289 is not surprising given that one miRNA alone is often not sufficient to completely repress target gene expression (Nesler, 2016).

To directly test the ability of miR-289 to repress translation of CamKII, a reporter was developed where the coding sequence for firefly luciferase (FLuc) was fused to the regulatory CamKII 3'UTR (FLuc-CamKII 3'UTR). When this wild-type reporter was coexpressed with miR-289 in Drosophila S2 cells, expression of FLuc was significantly reduced. In contrast, when this reporter was coexpressed with miR-279a, a miRNA not predicted to bind to the CamKII 3'UTR, no repression was observed. To confirm that repression of the FLuc-CamKII reporter by miR-289 was via a specific interaction, the second of two predicted miR-289 binding sites was mutagenized. Binding site 2 (BS2) was a stronger candidate for regulation because it is flanked by AU-rich elements (AREs) and miR-289 has been shown to promote ARE-mediated mRNA instability through these sequences. Moreover, it is well established that the stabilization and destabilization of neuronal mRNAs via interactions between AREs and ARE-binding factors plays a significant role in the establishment and maintenance of long-term synaptic plasticity in both vertebrates and invertebrates. Altering three nucleotides within BS2 in the required seed region binding site was sufficient to significantly disrupt repression of the reporter by miR-289. The minimal predicted BS2 sequence was cloned into an unrelated 3'UTR and it was asked if miR-289 could repress translation. Coexpression of the FLuc-SV-mBS2 reporter with miR-289 led to significant repression. Taken together, these results indicate that the BS2 sequence is both necessary and sufficient for miR-289 regulation via the CamKII 3′UTR (Nesler, 2016).

The most important conclusion of this study is that presynaptic CamKII is required to control activity-dependent axon terminal growth at the Drosophila larval NMJ. First, it was shown that CamKII is necessary to control ghost bouton formation in response to spaced synaptic depolarization. Next, it was demonstrated that spaced stimulation correlates with a rapid protein synthesis dependent increase in CamKII immunofluorescence in presynaptic boutons. This increase is suppressed by presynaptic overexpression of activity-regulated miR-289. Previous work has shown that overexpression of miR-289 in larval motor neurons can suppress activity-dependent axon terminal growth (Nesler, 2013). This study demonstrated that miR-289 can repress the translation of a FLuc-CamKII 3'UTR reporter via a specific interaction with a binding site within the CamKII 3'UTR ( Fig. 6C-E). Collectively, this experimental evidence suggests that CamKII functions downstream of the miRNA pathway to control activity-dependent changes in synapse structure. (Nesler, 2016).

Thus, CamKII protein is expressed in the right place to regulate rapid events that are occurring within presynaptic boutons. Several questions remain regarding CamKII function in the control of activity-dependent axon terminal growth. First, it is unclear what the significance might be of a rapid increase of total CamKII in presynaptic terminals. Why is the pool of CamKII protein that is already present not sufficient to control these processes? Similar questions have been asked regarding activity-dependent processes occurring within dendrites. It is postulated that the CamKII mRNA might be locally translated in axon terminals. It has been proposed that local mRNA translation might be (1) required for efficient targeting of some synaptic proteins to specific sites, or (2) local translation may in and of itself be required to control activity-dependent processes at the synapse. Second, the impact of spaced depolarization on CamKII function needs to be assessed and downstream targets of CamKII phosphorylation involved in these processes need to be identified. One very strong candidate is synapsin which, at the Drosophila NMJ, has been shown to rapidly redistribute to sites of new ghost bouton outgrowth in response to spaced stimulation. Finally, the idea that CamKII might work through a Eag/CASK-dependent mechanism to control activity-dependent axon terminal growth needs to be examined (Nesler, 2016).

The long 3'UTR mRNA of CaMKII is essential for translation-dependent plasticity of spontaneous release in Drosophila melanogaster

A null mutation of the Drosophila calcium/calmodulin-dependent protein kinase II gene (CaMKII) was generated using homologous recombination. Null animals survive to larval and pupal stages due to a large maternal contribution of CaMKII mRNA, which consists of a short 3'-UTR form lacking regulatory elements that guide local translation. The selective loss of the long 3'UTR mRNA in CaMKII null larvae allows testing its role in plasticity. Development and evoked function of the larval neuromuscular junction are surprisingly normal, but the resting rate of miniature excitatory junctional potentials (mEJPs) is significantly lower in CaMKII mutants. Mutants also lack the ability to increase mEJP rate in response to spaced depolarization, a type of activity-dependent plasticity shown to require both transcription and translation. Consistent with this, overexpression of miR-289 in wild-type animals blocks plasticity of spontaneous release. In addition to the defects in regulation of mEJP rate, CaMKII protein is largely lost from synapses in the mutant. All phenotypes are non-sex-specific and rescued by a fosmid containing the entire wild-type CaMKII locus, but only viability and CaMKII localization are rescued by genomic fosmids lacking the long 3'UTR. This suggests that synaptic CaMKII accumulates by two distinct mechanisms: local synthesis requiring the long 3'UTR form of CaMKII mRNA and a process which requires zygotic transcription of CaMKII mRNA. The origin of synaptic CaMKII also dictates its functionality. Locally translated CaMKII has a privileged role in regulation of spontaneous release which cannot be fulfilled by synaptic CaMKII from the other pool (Kuklin, 2017).

CaMKII is both ubiquitous and abundant. In mammals, CaMKII constitutes around 1% of 96 total brain protein and it is also highly expressed in fly heads. Unsurprisingly, CaMKII has been shown to have a plethora of important functions in the nervous system including roles in multiple stages and forms of learning and memory. At the Drosophila neuromuscular junction (NMJ) the roles of CaMKII encompass both development of the synapse and its activity-dependent plasticity. To date, these functions have been revealed using transgenes encoding CaMK II inhibitors or RNAi to decrease kinase activity or activated forms of the kinase to increase activity (Kuklin, 2017).

Early studies at the larval NMJ showed that global inhibition of CaMKII with a heat shock-inducible inhibitor peptide transgene (hs-ala lines) increased branching and bouton number. This same manipulation also increased the amplitude of evoked currents and blocked paired pulse facilitation. Global inhibition of CaMKII was also associatedwith increased presynaptic excitability while expression of constitutively active CaMKII in motor neurons suppressed excitability. Subsequent studies looking at postsynaptic inhibition of CaMKII with both ala peptide and another inhibitor (CaMKIINtide) showed that muscle CaMKII activity could stimulate a retrograde signaling pathway which increased quantal content without changes in mEJP amplitude. The larger quantal content correlated with an increase in morphologically identified release sites and, for high levels of CaMKIINtide expression, an increase in mini frequency (Kuklin, 2017).

More recently, presynaptic expression of either ala peptide or CaMKII RNAi was shown to block activity-dependent bouton sprouting. The abundance of roles is consistent with the presence of the kinase at high levels on both sides of the NMJ, and in multiple cellular compartments (Kuklin, 2017).

The use of genetics to investigate the role of signal transduction molecules in neuronal function has been standard practice for many years in Drosophila. Although Drosophila CaMKII was cloned over 20 years ago, the location of the gene on heterochromatin-rich chromosome complicated standard mutational approaches. To begin genetic analysis of CaMKII, a null mutation was generated in the CaMKII gene by homologous recombination, inserting two stop codons into the N-terminal coding sequence. This study shows that this mutation is comp letely lethal before adulthood in the homozygous state (Kuklin, 2017).

Homozygous mutant animals survive into late larval and pupal stages, due to a large amount of maternally contributed CaMKII mRNA which has a short 3'UTR lacking regulatory information, including binding site for miR-289 which are present in the long form. The fact that null animals survive to pupate, and show essentially normal morphological development of the neuromuscular junction (NMJ), implies that maternally-derived short 3'UTR CaMKII is able to support the majority of basic processes. Indeed transgenic expression of the short 3'UTR form can partially rescue viability indicating that lack of CaMKII protein is the cause of lethality rather than some critical role of the long 3'UTR form of the mRNA. Third instar null animals, however, lack the synaptic enrichment of CaMKII seen in wild-type animals. They also show very specific defects in miniature excitatory junctional potentials (mEJPs) and do not exhibit transcription/translation-dependent plasticity of mEJP frequency. CaMKII derived from newly transcribed mRNA can rescue synaptic localization and viability independent of the 3'UTR, but plasticity of mEJPs requires the long 3'UTR mRNA. Consistent with this, suppression of CaMKII translation in wild-type animals by overexpression of miR-289 also blocks mEJP plasticity. These results argue that synaptic localization of CaMKII can occur via multiple mechanisms, and that locally translated kinase has a special role in plasticity (Kuklin, 2017).

Maternal CaMKII mRNA allows initiation of normal development in null larvae but cannot support metamorphosis. Given the numerous and important functions of CaMKII, it is not unexpected that loss of CaMKII is lethal by adulthood. Surprisingly, however, these mutants appear to be fairly normal with respect to the structure and function of the nervous system in larval stages. This is likely because Drosophila embryos receive large amounts of mRNA from maternally-derived support cells in the ovary. Because the maternal genotype is CaMKII w+/+, this means that even genetically null oocytes will contain mRNA encoding CaMKII. This mRNA is able to provide normal initial levels of the protein, and accordingly there is no lethality during embryonic development. By late larval stages the amount of CaMKII falls, reflecting either degradation or dilution of maternally encoded kinase. By the time animals reach third instar, functional problems can beseen at the NMJ and there is significant lethality. The severely reduced CaMKII level in pupal stages blocks the ability to complete metamorphosis. Production of new CaMKII mRNA is clearly necessary for continued viability as the animal enters this stage (Kuklin, 2017).

One major difference between the maternal and zygotic mRNAs is that the maternal message has a truncated 3'UTR and lacks sequences known to confer post-transcriptional regulation. Interestingly, viability does not seem to require the long 3'UTR . Animals containing either a rescue fosmid lacking long UTR sequences or expressing a neuronal transgene with a truncated UTR are able to reach adulthood and reproduce. This suggests that producing new protein is sufficient for survival through metamorphosis and that the long UTR form has a specialized role. This underscores the need for future studies to utilize cell-specific and temporally-controlled genetic manipulations of kinase protein and mRNA structure (Kuklin, 2017).

The CaMKII null NMJ phenotype differs from that of animals expressing CaMKII inhibitors. The ability to obtain CaMKII null third instar larvae allowed characterization both the structure of the NMJ and its function in the absence of zygotic transcription. What was immediately obvious was that the phenotypes of the null animals did not resemble the phenotypes reported for animals expressing CaMK II inhibitors or RNAi, manipulations that should affect CaMKII activity levels regardless of the mRNA template. Null animals had no obvious morphological defects or changes in excitability or evoked release, only a decrease in the rate of spontaneous release. In contrast, in animals with strong postsynaptic inhibition of CaMKII an increase in mini rate was reported, likely due to an increase in the number of presynaptic release sites. In the CaMKII null, the number of release sites, as assessed by staining for Brp, is unchanged indicating that the decrease in minis is due to an alteration in release probability (Kuklin, 2017).

These qualitatively distinct phenotypes suggest that inhibition of CaMKII enzymatic activity is not the same as loss of zygotic transcription on a background of maternally-provided kinase. On the face of it, these differences are surprising since both types of manipulation, transgenic inhibition of CaMKII and loss of new transcription of the gene, should produce animals with reduced CaMKII enzyme activity. What might account for these differences? One possibility is that the absolute levels of CaMKII activity might be different. This would imply that different magnitudes of activity loss have qualitatively distinct effects. This possibility seems unlikely, however, given previous findings with the heat shock driven ala lines where different levels of peptide inhibitor were tested: high and low levels of ala expression did not differ qualitatively, only in severity. A second possibility is that the time window in which CaMKII activity is lost is the key difference between the two manipulations. Expression of inhibitors using GAL4 lines that turn on early in development could reduce activity earlier than slow depletion of maternal mRNA does. This would imply that early loss of CaMKII activity has qualitatively different effects than later loss. A third possibility is that RNAi and inhibitor peptides have off-target effects, perhaps on CaMKI, a poorly studied enzyme in the fly. A fourth possibility, and one that is favored, is that inhibition and mutation might be different because they disrupt distinct pools of CaMKII which are specified by both transcriptional and translational mechanisms (Kuklin, 2017).

How is CaMKII from newly transcribed mRNA distinct from that encoded by maternal mRNA? Maternal CaMKII mRNA differs in two ways from zygotic mRNA. First, it differs in structure. Drosophila CaMKII has multiple polyadenylation sites and can have either a short or long 3'UTR. Based on publicly available RNA seq data sets and 3' RACE PCR, mRNA from 0-2 h embryos (which reflects maternal contribution) contains exclusively the short 3'UTR. The RNA seq data also suggest it originates from a distinct transcription start site and differs in its 3'UTR. The second difference is that zygotic mRNA has a different history. Maternal mRNA is synthesized in the nuclei of ovarian nurse cells and never sees the inside of a neuronal nucleus. For CaMK II and other mRNAs the association with mRNA transport machinery occurs in the nucleus. Newly transcribed nuclear mRNAs therefore have preferential access to the machinery that mediates RNA localization. This machinery can be cell type-specific and change over development, meaning that maternal mRNA, even if it has the correct regulatory sequences, may not be competent to localize correctly. Thus while maternal and zygotic CaMKII mRNAs encode the same protein, they do not contain the same regulatory information and may not have the same access to localization or processing factors (Kuklin, 2017).

How do these two differences influence neuronal structure and function? CaMKII null mutants have two obvious defects: a decrease in basal and stimulated mEJP rate and a lack of synaptically localized CaMKII. These two deficits appear to be mechanistically distinct. Rescue of synaptic localization was seen with both the WT gene and a fosmid lacking long UTR sequences. Localization is therefore 3'UTR -independent but appears to require newly transcribed mRNA. Whether this is due to an mRNA-based mechanism (transport of mRNA to synaptic sites and local translation), or whether it is due to preferential transport or diffusion of protein synthesized in the soma from new mRNA templates, will require further investigation (Kuklin, 2017).

In contrast to synaptic CaMKII localization, the presence of the long 3'UTR is absolutely required for establishing a normal basal level of spontaneous release, and for activity-dependent increases in mEJP rate. This plasticity is translation-dependent and is suppressed by miR-289 which has been previously shown to regulate activity-dependent presynaptic synthesis of CaMKII at the NMJr. The partial suppression seen with pre synaptic miR-289 could be due to relative expression levels of the mRNA and miR or to a requirement for other regulators. The postsynaptic suppression of plasticity points to involvement of CaMKII- dependent retrograde signaling in spontaneous release. Taken together, however, these data imply that there is a population of synaptic long 3'UTR CaMKII mRNA that is locally translated and acts to increase the probability of release. Why newly translated CaMKII is required is unknown, but in rodent neurons, synaptically synthesized CaMKII has preferential access to certain binding partners. These results also revealed differences be tween the NMJ and adult olfactory system synapses. In adult projection neurons, the 3'UTR was required for both localization and activity- dependent regulation of CaMKII translation in dendrites. Further investigation of the mechanisms of RNA and protein localization will be required to resolve these differences, but it is likely that there will be multiple mechanisms for regulation of synaptic CaMKII levels (Kuklin, 2017).

Activity-dependent synthesis of CaMKII is clearly a critical feature of the enzyme and is conserved across species and developmental stages. Previous work in the adult fly brain has shown that CaMKII mRNA contains sequences that regulate activity-dependent translation. Importantly, this conserved in mammals where 3'UTR sequences in the CAMK2A gene have been shown to drive localization and activity-dependent translation, though it has been suggested that there may also be a role for 3'UTR sequences. The fly will provide a powerful model system for understanding how and why CaMKII is targeted to multiple subcellular compartments. The discovery that local translation of CaMKII is a key driver of plasticity of mini rate also provides a foothold for obtaining an understanding of this process. Spontaneous release is increasingly being recognized as mechanistically and functionally distinct from evoked release (Kuklin, 2017).

The regulation of spontaneous release, and even the sites at which it occurs, are separate from action potential evoked activity. These miniature events can regulate nuclear gene expression, local translation and participate in developmental processes such as circuit wiring. The dependence of activity-dependent plasticity of spontaneous release on local translation of CaMKII on both sides of the synapse suggests that there are complex mechanisms for fine tuning this important type of synaptic activity (Kuklin, 2017).

The conserved, disease-associated RNA binding protein dNab2 interacts with the Fragile X Protein ortholog in Drosophila neurons

The Drosophila dNab2 protein is an ortholog of human ZC3H14, a poly(A) RNA binding protein required for intellectual function. dNab2 supports memory and axon projection, but its molecular role in neurons is undefined. This study presents a network of interactions that links dNab2 to cytoplasmic control of neuronal mRNAs in conjunction with the fragile X protein ortholog dFMRP. dNab2 and dfmr1 interact genetically in control of neurodevelopment and olfactory memory, and their encoded proteins co-localize in puncta within neuronal processes. dNab2 regulates CaMKII, but not futsch, implying a selective role in control of dFMRP-bound transcripts. Reciprocally, dFMRP and vertebrate FMRP restrict mRNA poly(A) tail length, similar to dNab2/ZC3H14. Parallel studies of murine hippocampal neurons indicate that ZC3H14 is also a cytoplasmic regulator of neuronal mRNAs. Altogether, these findings suggest that dNab2 represses expression of a subset of dFMRP-target mRNAs, which could underlie brain-specific defects in patients lacking ZC3H14 (Bienkowski, 2017).

RNA binding proteins (RBPs) play important roles in the biogenesis and expression of virtually all types of eukaryotic RNAs, including protein-coding mRNAs. Despite these broad roles, mutations in genes that encode RBPs often lead to tissue-specific disease pathology, particularly within the brain and nervous system. Examples of this link include the fragile X mental retardation protein (FMRP) and the spinal muscular atrophy protein SMN. The prevalence of neurological disorders caused by defects in RBPs likely reflects the enhanced role post-transcriptional mechanisms play in translational control within distal neuronal processes (Bienkowski, 2017).

The ZC3H14 (zinc-finger CysCysCysHis [CCCH]-type 14) gene encodes a ubiquitously expressed RBP that is lost in an inherited form of autosomal, recessive, non-syndromic intellectual disability (Pak, 2011). Patients homozygous for nonsense mutations in ZC3H14 have reduced IQ but lack associated dysmorphic features. Loss of the ubiquitously expressed Drosophila ZC3H14 homolog, dNab2, produces defects in adult viability, motor function, and brain morphology that are fully rescued by neuronal dNab2 re-expression and partially rescued by human ZC3H14 expression. These data reveal an important, and evidently conserved, role for human ZC3H14 and fly dNab2 in neurons (Bienkowski, 2017).

ZC3H14 and dNab2 are predominantly localized to the nucleus but are members of a conserved protein family whose founding member, S. cerevisiae Nab2, shuttles between the nucleus and the cytoplasm. ZC3H14 and dNab2 share a domain structure of an N-terminal PWI (proline/tryptophan/isoleucine)-like domain, a nuclear localization sequence, and five well-conserved C-terminal CCCH-type zinc fingers (ZnFs). These ZnF domains bind synthetic polyadenosine RNA probes in vitro, implying that dNab2 and ZC3H14 interact with adenosine-rich tracts in vivo. In support of this hypothesis, ZC3H14 co-localizes with poly(A) mRNA speckles in rodent hippocampal neurons, and its loss increases bulk poly(A) tail (PAT) length among RNAs in cultured N2a cells. dNab2 also restricts PAT length in vivo and genetic interactions between dNab2, and components of the polyadenylation machinery (e.g., the PABP poly(A) binding protein and the hiiragi poly(A) polymerase) indicate that altered PAT length may underlie dNab2 mutant phenotypes (Pak, 2011). Altered PAT length can affect multiple steps in RNA metabolism, including turnover and translational efficiency (Bienkowski, 2017 and references therein).

dNab2 plays important roles within the central nervous system (CNS). Pan-neuron dNab2 depletion within the peripheral nervous system (PNS) and CNS replicates almost all phenotypes resulting from zygotic loss of dNab2, while dNab2 depletion from motor neurons does not. Moreover, pan-neuron dNab2 depletion impairs short-term memory and disrupts axon projection into the α/β lobes of the mushroom bodies (MBs), twin neuropil structures in the brain required for associative olfactory learning and memory. In dNab2 mutants, β axons misproject across the brain midline and α axons show a high frequency of branching defects. Selective depletion of dNab2 in Kenyon cells, which give rise to MB α/β axons, is sufficient to phenocopy these dNab2 zygotic defects, and dNab2 re-expression in these cells is sufficient to rescue them. However, there is little evidence of how dNab2 regulates bound RNAs and whether this regulation occurs exclusively in the nucleus, as suggested by the nuclear steady-state localization of dNab2, Nab2, and ZC3H14, or involves a role for dNab2 in cytoplasm (Bienkowski, 2017).

This study describes a genetic screen for dNab2-interacting factors in the Drosophila eye that uncovers physical and functional interactions between dNab2 and the Drosophila ortholog of the FMRP. The FMRP RBP is lost in fragile X syndrome (FXS), the most common genetic cause of intellectual disability. FMRP undergoes nucleocytoplasmic shuttling but is enriched in the cytoplasm at steady state. Cytoplasmic FMRP regulates ~800 polyadenylated neuronal mRNAs, allowing for finely tuned pre- and post-synaptic translation of their encoded proteins. Genetic interactions between dNab2 and the Drosophila FMRP gene (dfmr1) correspond at a molecular level to an RNase-resistant physical association of dNab2 and Drosophila FMRP (dFMRP) proteins in neurons. Within brain neurons, dNab2 and dFMRP co-localize in the soma but are also detected within discrete messenger ribonucleoprotein (mRNP)-like foci distributed along neuronal processes. A corresponding memory defect in dNab2/+,dfmr1/+ trans-heterozygotes indicates that dNab2 may co-regulate a subset of mRNAs bound by dFMRP. dNab2 associates with the dFMRP-regulated mRNA encoding CaMKII (calmodulin-dependent protein kinase-II) and is required for repression of a CaMKII translational reporter in neurons. By contrast, dNab2 does not appear to regulate a second dFMRP-target mRNA encoding Futsch/Map1β, implying that the spectrum of dNab2-regulated mRNAs only partially overlaps with dFMRP. Moreover, this study has found evidence that dFMRP and FMRP restrict PAT length of neuronal mRNAs in a manner similar to dNab2 and ZC3H14. Finally, ZC3H14 was shown to be present in hippocampal axons and dendrites, where it is enriched in ribonucleoprotein (RNP) and 80S ribosomal fractions. Altogether, these data represent a significant advance in understanding dNab2/ZC3H14 by defining a role for these disease-associated RBPs in translational control of neuronal mRNAs that, in Drosophila, occurs in conjunction with the dFMRP protein (Bienkowski, 2017).

This study reports the results of a candidate-based screen for factors that interact genetically with the Drosophila dNab2 gene, which encodes an RBP whose human ortholog is lost in an inherited intellectual disability. Identified interacting genes include components of the translation machinery (PABC1, EF-1α, and eIF-4e) and elements of a pathway centered on the Drosophila ortholog of the FMRP translational repressor (dfmr1 itself, Argonaute-1, Gw182, Rm62, staufen, and Ataxin-2), suggesting that dNab2 functions within the dFMRP pathway. Additional genetic tests support this hypothesis. dfmr1 alleles suppress a rough-eye phenotype caused by transgenic expression of dNab2 in retinal neurons, while dfmr1 alleles enhance a locomotor defect caused by neuronal RNAi of dNab2. Genetic interactions also occur in the CNS, where dfmr1 heterozygosity enhances the frequency of MB α lobe defects in dNab2 mutants. dNab2 heterozygosity suppresses MB α lobe defects in dfmr1 mutants, implying a functional hierarchy in which dNab2 effects are dependent on dFMRP status. The inability of either RBP to rescue phenotypes caused by loss of the other argues for a model in which dNab2 and dFMRP participate in a common mechanism or mechanisms but are not functionally redundant (Bienkowski, 2017).

Genetic interactions between the dNab2 and the dfmr1 genes are paralleled by a dNab2:dFMRP protein complex detected in neurons. This dNab2:dFMRP interaction, which could involve other factors, includes a cytoplasmic pool of dNab2 that partially co-localizes with dFMRP in mRNP-like granules in neuronal processes, suggesting that the two RBPs may associate with some of the same RNAs. dNab2 can interact with and regulate the CaMKII mRNA, a dFMRP target, but is not required to regulate futsch, a second dFMRP target. The finding that trans-heterozygosity for dNab2 and dfmr1 impairs olfactory memory provides additional evidence that dNab2:dFMRP co-regulate some neuronal mRNAs. Finally, this study found that murine ZC3H14 is present in axons and dendrites of murine hippocampal neurons and associates with mRNPs and elements of the translational machinery. FMRP also localizes to dendrites and axons and regulates filopodial dynamics and motility of axonal growth cones. In aggregate, these data significantly advance understanding of the role of dNab2/ZC3H14 proteins in neurons by defining a cytoplasmic pool of these proteins associated with translational control of mRNAs that, in Drosophila, occurs in conjunction with dFMRP (Bienkowski, 2017).

This study highlights the dNab2:dFMRP association but also suggests that dNab2 can function independently of dFMRP. For example, dNab2 and dFMRP are each required for MB αβ lobe structure, yet dosage-sensitive interactions between dNab2 and dfmr1 alleles are only evident in α lobes, suggesting that dNab2 and dFMRP may co-regulate RNAs within specific axon branches. In addition, dNab2 selectively regulates CaMKII, but not futsch, and that asymmetry is reflected at the level of the futsch PAT, which is unchanged in dNab2 mutant brains but extended in dfmr1 mutant brains. The failure of dNab2 alleles to alter Futsch protein levels is consistent with their lack of effect on the Futsch-dependent process of NMJ development. Altogether, these data suggest that the futsch mRNA is not a physiological target of dNab2 and that dNab2 only regulates a subset of dFMRP-bound transcripts (Bienkowski, 2017).

dFMRP protein is a well-established translational repressor, but the data reveal a previously unappreciated requirement for dFMRP/FMRP to inhibit mRNA poly(A) tail (PAT) length, which in the case of futsch, is likely to stem from direct binding by dFMRP. This effect on PAT length could simply be a secondary consequence of enhanced futsch translation in dfmr1/Fmr1 mutant cells. However, loss of the cytoplasmic polyadenylation element binding protein (CPEB), which promotes cytoplasmic PAT extension in mammals and flies, rescues FXS phenotypes in Fmr1 knockout mice. One interpretation of this result is that inappropriate PAT elongation contributes to excess translation in FXS, similar to the positive correlation between PAT length and translation observed among germline and embryonic mRNAs. These data thus raise the possibility that altered mRNA polyadenylation may be an unappreciated feature of translational dysregulation in neurons lacking dfmr1/Fmr1 (Bienkowski, 2017).

The dNab2:dFMRP complex suggests that dNab2 may regulate gene expression through its interaction with dFMRP. FMRP inhibits translational initiation, blocks ribosome movement along polyribosome-associated mRNAs, and interacts with elements of the miRNA machinery. The dNab2-sensitive CaMKII-3'UTR GFP sensor is also regulated by the miRNA pathway, and multiple factors involved in miRNA-induced silencing interact genetically with dNab2. The precise role dNab2 plays on bound mRNAs is not clear. PAT elongation induced by dNab2 loss could enhance recruitment of cytoplasmic PABPs that promote translation-coupled circularization of mRNAs. dNab2 and its ortholog ZC3H14 both repress PAT length and may thus indirectly limit the binding of cytoplasmic PABPs to key transcripts. Alternatively, they may directly compete with these PABPs for binding to polyadenosine tails and thus occlude access of other factors involved in translation (Bienkowski, 2017).

Consistent with the role of dNab2 in translational regulation, its ortholog ZC3H14 localizes to axons, dendrites, and dendritic spines in hippocampal neurons and co-sediments with 80S ribosomes. FMRP is primarily associated with polysomes and can inhibit translation by ribosome stalling. The FMRP-target CamKIIα mRNA is enriched in anti-ZC3H14 precipitates, and CaMKIIα levels increase in the hippocampus of Zc3h14Δ13/Δ13 knockout mice compared to control mice, raising the possibility that Drosophila and vertebrate CaMKII mRNAs are conserved targets of dNab2/ZC3H14. The FMRP-related protein Fxr1 co-precipitates with the zinc-finger domain of ZC3H14, suggesting that ZC3H14 may interact with FMRP family members in a manner analogous to dNab2 and dFMRP (Bienkowski, 2017).

Altogether, the data presented in this study provide evidence that dNab2 localizes to both the nucleus and the cytoplasm of Drosophila neuronal processes and that it interacts physically and functionally with the dFMRP protein. Additional data provide evidence of an equivalent pool of cytoplasmic ZC3H14 that interacts with RNP complexes found in the axons and dendrites in the mouse brain. Given the link between FMRP and intellectual disability in humans, these interactions raise the possibility that defects in translational silencing of mRNAs transported to distal sites within neuronal processes contribute to neurodevelopmental and cognitive defects in Drosophila lacking dNab2 or in humans lacking ZC3H14 (Bienkowski, 2017).

Synapse-specific and compartmentalized expression of presynaptic homeostatic potentiation

Postsynaptic compartments can be specifically modulated during various forms of synaptic plasticity, but it is unclear whether this precision is shared at presynaptic terminals. Presynaptic Homeostatic Plasticity (PHP) stabilizes neurotransmission at the Drosophila neuromuscular junction, where a retrograde enhancement of presynaptic neurotransmitter release compensates for diminished postsynaptic receptor functionality. To test the specificity of PHP induction and expression, this study has developed a genetic manipulation to reduce postsynaptic receptor expression at one of the two muscles innervated by a single motor neuron. PHP can be induced and expressed at a subset of synapses, over both acute and chronic time scales, without influencing transmission at adjacent release sites. Further, homeostatic modulations to CaMKII, vesicle pools, and functional release sites are compartmentalized and do not spread to neighboring pre- or post-synaptic structures. Thus, both PHP induction and expression mechanisms are locally transmitted and restricted to specific synaptic compartments (Li, 2018a).

Although the genes and mechanisms that mediate retrograde homeostatic potentiation have been intensively investigated, whether this process can be expressed and restricted to a subset of synapses within a single neuron has not been determined. This study has developed a manipulation that enables the loss of GluRs on only one of the two postsynaptic targets innervated by a Type Ib motor neuron at the Drosophila NMJ. The analysis of synaptic structure and function in this condition has revealed the spectacular degree of compartmentalization in postsynaptic signaling and presynaptic expression that ultimately orchestrate the synapse- specific modulation of presynaptic efficacy (Li, 2018a).

Compartmentalization of postsynaptic PHP signaling GluRs are dynamically trafficked in postsynaptic compartments where they mediate the synapse-specific expression of Hebbian plasticity such as LTP and homeostatic plasticity, including receptor scaling. In contrast, homeostatic plasticity at the human, mouse, and fly NMJ is expressed through a presynaptic enhancement in neurotransmitter release, but is induced through a diminishment of postsynaptic neurotransmitter receptor functionality. Using biased expression of Gal4 to reduce GluR levels on only one of the two muscle targets innervated by a single motor neuron, this study demonstrates that the inductive signaling underlying PHP is compartmentalized at the postsynaptic density, and does not influence activity at synapses innervating the adjacent muscle (Li, 2018a).

Postsynaptic changes in CaMKII function and activity have been associated with PHP retrograde signaling. Consistent with this compartmentalized inductive signaling, this study observed pCaMKII levels to be specifically reduced at postsynaptic densities of Ib boutons in which GluR expression is perturbed, while pCaMKII was unchanged at postsynaptic compartments opposite to Is boutons and at NMJs in the adjacent muscle with normal GluR expression. Further, postsynaptic overexpression of the constitutively active CaMKII occludes the expression of PHP. Similar synapse-specific control of postsynaptic CaMKII phosphorylation, modulated by activity, has been previously observed. As noted in other studies, this localized reduction in pCaMKII provides a plausible mechanism for the inductive PHP signaling restricted to and compartmentalized at Ib synapses (Li, 2018a).

How does a perturbation to GluR function lead to a reduction in CaMKII activity that is restricted to postsynaptic densities opposing Type Ib boutons? Recent evidence suggests that distinct mechanisms regulate pCaMKII levels during retrograde PHP signaling depending on pharmacologic or genetic perturbation to glutamate receptors and the role of protein synthesis. Scaffolds at postsynaptic densities are associated in complexes with GluRs and CaMKII. Intriguingly, the scaffold dCASK is capable of modulating CaMKII activity at specific densities in an activity-dependent fashion. Further, CaMKII activity can regulate plasticity with specificity at subsets of synapses in Drosophila and other systems. Although intra-cellular 'cross talk' between Is and Ib boutons cannot be ruled out, as GluRIIA is reduced at postsynaptic sites of both neuronal subtypes, it is striking that reductions in pCaMKII are restricted to Ib postsynaptic compartments. An attractive model, therefore, is that the postsynaptic density isolates calcium signaling over chronic time scales to compartmentalize PHP induction. The membranous complexity and geometry of the SSR at the Drosophila NMJ may be the key to restricting calcium signaling at these sites, as this structure can have major impacts on ionic signaling during synaptic transmission. These properties, in turn, may lead to local modulation of CaMKII function. Interestingly, Drosophila mutants with defective SSR elaboration and complexity have been associated with defects in PHP expression. In the mammalian central nervous system, it is well established that dendritic spines function as biochemical compartments that isolate calcium signaling while enabling propagation of voltage changes, and it is tempting to speculate that the SSR may subserve similar functions at the Drosophila NMJ to enable synapse-specific retrograde signaling (Li, 2018a).

The homeostatic modulation of presynaptic neurotransmitter release is compartmentalized at the terminals of Type Ib motor neurons. It was previously known that PHP can be acutely induced and expressed without any information from the cell body of motor neurons. The current data suggests that the signaling necessary for PHP expression is even further restricted to specific postsynaptic densities and presynaptic boutons, demonstrated through several lines of evidence. First, quantal content is specifically enhanced at boutons innervating muscle 6 in M6>GluRIIARNAi without measurably impacting transmission on the neighboring boutons innervating muscle 7. In addition, PHP can be acutely induced at synapses innervating muscle 7 despite PHP having been chronically expressed at muscle 6. Finally, the homeostatic modulation of the RRP and enhancement of the functional number of release sites is fully expressed regardless of whether PHP is induced at all Type Ib boutons or only a subset. Thus, PHP signaling is orchestrated at specific boutons according to the state of GluR functionality of their synaptic partners and does not influence neighboring boutons within the same motor neuron. Although the compartmentalized expression of PHP was not unexpected, there was precedent to suspect inter-bouton crosstalk during homeostatic signaling. In the dynamic propagation of action potentials along the axon, the waveform could, in principle, change following PHP expression to globally modulate neurotransmission at all release sites in the same neuron. However, voltage imaging did not identify any change in the action potential waveform at individual boutons following PHP signaling, and this study did not observe any impact on neighboring boutons despite PHP being induced at a subset of synapses in the same motor neuron. Further, mobilization of an enhanced readily releasable synaptic vesicle pool is necessary for the expression of PHP, and synaptic vesicles and pools are highly mobile within and between presynaptic compartments. Hence, it was conceivable that a mobilized RRP, induced at some presynaptic compartments, may be promiscuously shared between other boutons. However, while a large enhancement was observed in the RRP at synapses innervating muscle 6 in M6>GluRIIARNAi, this adaptation had no impact on the RRP at adjacent presynaptic compartments innervating muscle 7. Thus, PHP signaling is constrained to boutons innervating one of two postsynaptic targets and does not 'spread' to synapses innervating the adjacent target despite sharing common cytosol, voltage, and synaptic vesicles (Li, 2018a).

What molecular mechanisms mediate the remarkable specificity of PHP expression at presynaptic compartments? One attractive possibility is that active zones themselves are fundamental units and act as substrates for the homeostatic modulation of presynaptic function. The active zone scaffold BRP remodels during both acute and chronic PHP expression (Weyhersmuller, 2011), and other active zone proteins are likely to participate in this remodeling. Indeed, many genes encoding active zone components are required for PHP expression, including the calcium channel cac and auxiliary subunit α2-δ, the piccolo homolog fife, the scaffolds RIM (Rab3-interacting Molecule) and RIM-binding protein (RBP), and the kainite receptor DKaiR1D. If individual active zones can undergo the adaptations necessary and sufficient for PHP expression, this would imply that PHP can be induced and expressed with specificity at individual active zones. Indeed, the BRP cytomatrix stabilizes calcium channel levels at the active zone, and also controls the size of the RRP, two key presynaptic expression mechanisms that drive PHP. Further, the recruitment of new functional release sites have been observed following both chronic and acute PHP expression, suggesting that previously silent active zones become 'awakened' and utilized to potentiate presynaptic neurotransmitter release (Li, 2018a).

Interestingly, presynaptic GluRs, localized near active zones, are necessary for PHP expression and have the capacity to modulate release with specificity at individual active zones. Thus, active zones have the capacity to remodel with both the specificity and precision necessary and sufficient for compartmentalized PHP expression. If each active zone operates as an independent homeostat to adjust release efficacy in response to target-specific changes, how is information transfer at individual sites integrated to ensure stable and stereotypic 'global' levels of neurotransmission? One speculative possibility is that active zones at terminals of each neuron are endowed with a total abundance of material that is tightly controlled and sets stable global levels of presynaptic neurotransmitter release. Such active zone material may be 'sculpted' with considerable heterogeneity within presynaptic terminals, varying in number, size, and density. Consistent with such a possibility, mutations in the synaptic vesicle component Rab3 exhibit extreme changes in active zone size, number, and density, but stable global levels of neurotransmission. Within this global context, plasticity mechanisms may operate at individual active zones, superimposed as independent homeostats to adaptively modulate synaptic strength. In addition, there is intriguing evidence for the existence of 'nanocolumns' between presynaptic active zones and postsynaptic GluRs that form structural and functional signaling complexes (Biederer, 2017; Tang, 2016). One particularly appealing possibility, therefore, is that a dialogue traversing synaptic nanocolumns functions to convey the retrograde signaling and active zone remodeling necessary for PHP expression at individual release sites. Studies in mammalian neurons have revealed parallel links between the functional plasticity of active zones, including their structure and size, and the homeostatic modulation of neurotransmitter release. Such intercellular signaling systems are likely to modify synaptic structure and function to not only establish precise pre- and post-synaptic apposition during development, but also to maintain the plasticity necessary for synapses to persist with the flexibility and stability to last a lifetime (Li, 2018a).

A glutamate homeostat controls the presynaptic inhibition of neurotransmitter release

This study has interrogated the synaptic dialog that enables the bi-directional, homeostatic control of presynaptic efficacy at the glutamatergic Drosophila neuromuscular junction (NMJ). Homeostatic depression and potentiation use disparate genetic, induction, and expression mechanisms. Specifically, homeostatic potentiation is achieved through reduced CaMKII activity postsynaptically and increased abundance of active zone material presynaptically at one of the two neuronal subtypes innervating the NMJ, while homeostatic depression occurs without alterations in CaMKII activity and is expressed at both neuronal subtypes. Furthermore, homeostatic depression is only induced through excess presynaptic glutamate release and operates with disregard to the postsynaptic response. It is proposed that two independent homeostats modulate presynaptic efficacy at the Drosophila NMJ: one is an intercellular signaling system that potentiates synaptic strength following diminished postsynaptic excitability, while the other adaptively modulates presynaptic glutamate release through an autocrine mechanism without feedback from the postsynaptic compartment (Li, 2018b).

The Drosophila neuromuscular junction (NMJ) is a powerful model system to study the bi-directional, homeostatic control of synaptic strength. At this glutamatergic synapse, acute pharmacological and chronic genetic manipulations that reduce postsynaptic glutamate receptor (GluR) function activate a retrograde, trans-synaptic signaling system that triggers a compensatory increase in presynaptic glutamate release, restoring baseline levels of synaptic strength. Because the expression of this form of plasticity requires a presynaptic increase in neurotransmitter release, this process is referred to as presynaptic homeostatic potentiation (PHP). Multiple lines of evidence have established that the homeostat that governs PHP is exquisitely sensitive to diminished postsynaptic excitability and operates through a retrograde enhancement of presynaptic efficacy, stabilizing overall synaptic strength. Parallel phenomena have been observed at cholinergic NMJs in rodents and humans, suggesting this is a fundamental and conserved form of synaptic plasticity that does not depend on the neurotransmitter system (Li, 2018b).

In contrast to PHP, far less is known about the homeostat that governs an inverse process at the Drosophila NMJ, referred to as presynaptic homeostatic depression (PHD). The first evidence for PHD, although not appreciated as such, was discovered while characterizing mutations in synaptic vesicle endocytosis genes, in which increased synaptic vesicle size was found to result from defects in vesicle re-formation mechanisms. Independently, evidence for PHD was found using a separate manipulation that also increased synaptic vesicle size through overexpression of the vesicular glutamate transporter (vGlut; vGlut-OE). Both defective endocytosis and vGlut-OE result in enlargement of individual synaptic vesicles, leading to excess glutamate emitted from each synaptic vesicle and enhanced postsynaptic responsiveness (quantal size). However, normal levels of synaptic strength (excitatory postsynaptic potential [EPSP] amplitude) were observed due to a homeostatic reduction in the number of synaptic vesicles released (quantal content). When the phenomenon of PHD was initially defined, one hypothesis put forward was that PHD may be induced as an adaptive response to excess glutamate. More recently, PHD has been considered a mechanism that stabilizes neurotransmission in the same way that PHP operates, implying that PHD is calibrated as a homeostat that responds to overall synaptic strength. Despite these studies, the nature of the homeostat that controls PHD, as well as the genes and mechanisms involved, remains much less understood relative to PHP. It is not even clear whether trans-synaptic communication is required to induce, express, or modulate PHD (Li, 2018b).

This study has characterized the adaptations to synaptic physiology, growth, structure, and plasticity when PHP and PHD are induced and expressed alone and in conjunction at an individual synapse. Several lines of evidence demonstrate that PHP and PHD are independent processes that use distinct mechanisms to modulate presynaptic efficacy in opposing directions and operate at separate neuronal subtypes. However, PHP and PHD are not simply independent signaling systems that each tune presynaptic efficacy to maintain stable levels of synaptic strength. Rather, the data indicate that PHP is indeed a homeostat dedicated to maintaining synaptic strength, induced through retrograde signaling in the postsynaptic compartment. In contrast, PHD operates with indifference to the state of the postsynaptic cell and is oblivious to overall synaptic strength, instead functioning cell autonomously in the presynaptic neuron as a negative feedback system to homeostatically modulate glutamate release (Li, 2018b).

Clearly, distinct genetic mechanisms underlie PHP and PHD signaling, because genes necessary for PHP have no role in PHD. This is illustrated by loss of the gene dysbindin, which is required for PHP expression but has no impact on PHD, consistent with findings that other genes necessary for PHP do not impact PHD. Thus, while PHP and PHD appear to be parallel processes that modulate presynaptic neurotransmitter release in inverse directions, they do not employ overlapping genetic machinery (Li, 2018b).

PHP and PHD also use distinct physiological expression mechanisms. PHD reduces probability of release at both type Ib and Is motor neurons through an apparent reduction in Ca2+ influx yet without a change in BRP or the size of the RRP (Gavino, 2015). In contrast, the current study and others have found PHP adaptations involve an increase in presynaptic Pr mediated through increased Ca2+ influx, active zone scaffolding, Ca2+ channel abundance, and enhancement of the RRP. Indeed, remodeling of BRP at active zones appears to be unique to PHP and to terminals of type Ib boutons. The BRP scaffold controls the size of the RRP and stabilizes Cac channels at the active zone. Therefore, an attractive hypothesis is that PHP requires enhancements in Cac and BRP abundance to promote both Ca2+ influx and increase RRP size. Ca2+ channels and active zone scaffolds are also homeostatically regulated to control presynaptic neurotransmitter release in mammalian neurons, suggesting that such plasticity mechanisms may be evolutionarily conserved (Li, 2018b).

The postsynaptic induction mechanisms that orchestrate PHP signaling are enigmatic. However, it is clear that PHP signaling is extremely sensitive to reductions in postsynaptic excitability, which triggers a compartmentalized intercellular signaling system that originates in the postsynaptic muscle and requires a reduction in CaMKII activity to potentiate neurotransmitter release in the presynaptic neuron (Haghighi, 2003, Li, 2018b, Newman, 2017). If PHD were a homeostat designed to stabilize synaptic strength in a way that parallels PHP, then enhanced muscle excitability should induce a retrograde signaling system to depress presynaptic glutamate release. However, the data and previous work demonstrate that homeostatic depression is not induced when quantal size is increased. Rather, excess glutamate release from the motor neuron appears to be necessary and sufficient to induce and express PHD. This suggests that an autocrine mechanism triggers PHD signaling, in which excess glutamate is sensed and transduced into a reduction in presynaptic efficacy. Such an autocrine mechanism was astutely proposed as a possibility in the original vGlut-OE study (Li, 2018b).

If an autocrine mechanism mediates PHD induction, this would imply the existence of presynaptic GluRs that can sense excess glutamate and initiate presynaptic inhibition in response. Presynaptic autoreceptors are present and modulate presynaptic function at glutamatergic NMJs of invertebrates and vertebrates. One attractive candidate is the lone metabotropic GluR encoded in the Drosophila genome, mGluRA. mGluRA is present at presynaptic terminals of motor neurons at the larval NMJ and promotes presynaptic inhibition following excess glutamate released during high-frequency stimulation (Bogdanik, 2004). Other possibilities include presynaptic NMDA receptors, which mediate presynaptic inhibition in response to excess glutamate release in the mammalian hippocampus. Notably, NMDA receptors have been reported to be present at the Drosophila NMJ. The nature of the glutamate sensor and autocrine signaling system that govern the induction and expression of PHD remains to be defined (Li, 2018b).

Why doesn't a homeostat governing synaptic strength exist at the NMJ that is responsive to enhanced postsynaptic excitability? Proper control of muscle contraction is essential to life, and NMJs in many systems use a safety factor that ensures neurotransmitter is released in excess to stably promote muscle contraction. Hence, given this safety factor, it is not clear that a postsynaptic signaling system at the NMJ is necessary to detect and respond to heightened neurotransmitter release or sensitivity. Pharmacological perturbations to cholinergic NMJs that inhibit the enzymatic breakdown of neurotransmitter in worms and mammals lead to rapid paralysis and death, and there is no evidence that homeostatic retrograde signaling systems are initiated to inhibit presynaptic neurotransmitter release during these challenges. Thus, a retrograde homeostatic signaling system to depress presynaptic efficacy in response to increased postsynaptic excitability may not have developed due to a lack of evolutionary pressure (Li, 2018b).

Why, then, does a process like PHD exist at the Drosophila NMJ, designed to inhibit glutamate release through an autocrine presynaptic signaling system? One attractive possibility is that PHD may be a process that maintains stable glutamate levels at the larval NMJ of Drosophila in lieu of classical glutamate reuptake mechanisms. In the CNS, various clearance mechanisms homeostatically maintain ambient glutamate levels to prevent excitotoxicity, and excitatory GluRs are present at presynaptic terminals of the larval Drosophila NMJ. The major mechanism for glutamate clearance in the mammalian brain requires glutamate transporter proteins in the plasma membrane of both glial cells and neurons. In Drosophila, there a single excitatory amino acid transporter specific for glutamate reuptake encoded in the genome, dEAAT1. dEAAT1 is expressed in the central nervous system and in peripheral glia at the adult NMJ, where it is involved in glutamate clearance. However, dEAAT1 is not expressed at the embryonic or larval NMJ, and it is unclear how glutamate is controlled in this system. Accordingly, PHD may serve as an adaptive cell autonomous mechanism that responds to excess glutamate and inhibits release to maintain glutamate homeostasis, a process that may have parallels in the mammalian CNS (Li, 2018b).


GENE STRUCTURE

The Drosophila CaM kinase II gene consists of at least 16 exons spanning approximately 20 kilobase pairs. Alternative splicing generates four forms of the enzyme from a single gene. The four forms differ only by amino acid insertions or deletions near the C-terminus of the putative link segment, which is postulated to join the N-terminal to the C-terminal globules of the polypeptide, forming a dumbbell-like shape (Ohsako, 1993).

cDNA clone length - 2722 (Cho, 1991) and 2734 + (Ohsako, 1993)

Bases in 5' UTR - 250

Exons - at least 16

Bases in 3' UTR - 1230 (Ohsako, 1993)


PROTEIN STRUCTURE

A monoclonal antibody against rat brain type II Ca2+/calmodulin-dependent protein kinase (CaM kinase) precipitates three proteins from Drosophila heads with apparent molecular weights similar to those of the subunits of the rat brain kinase. Fly heads also contain a CaM kinase activity that becomes partially independent of Ca2+ after autophosphorylation, as does the rat brain kinase. A Drosophila cDNA encodes an amino acid sequence that is 77% identical to the sequence of the rat alpha subunit. All known autophosphorylation sites are conserved, including the site that controls Ca(2+)-independent activity (Cho, 1991).

The four cDNA sequences encoding Ca2+/calmodulin-dependent protein kinase II encode polypeptides of 490, 509, 516, and 530 amino acids. They are identical to one another except for amino acid insertions or deletions near the carboxyl-terminal of the putative "link" segment. These polypeptides show considerable similarity to rat brain CaM kinase II with more than 70% of the amino acids being identical. The Drosophila adult head contains three major species of CaM kinase II, with molecular masses of 55, 58, and 60 kDa. These cross-react with anti-rat CaM kinase II antibody. An expression study of the four Drosophila cDNA sequences in mammalian cells reveals that the polypeptides of 490, 509, and 530 amino acids that had been predicted from the cDNA sequences correspond to the 55-, 58-, and 60-kDa polypeptides found in the Drosophila head, respectively, and all exhibit enzymatic properties similar to those of rat brain CaM kinase II, including self-regulation (Ohsako, 1993).

Eight different CaM kinase II cDNA sequences, varying only at the junction of the regulatory and association domains of the kinase have been isolated. The diversity of CaM kinase in Drosophila is greater than previously appreciated and is generated by alternative splicing of a single gene (Griffith, 1993b).

Four forms of the Drosophila Ca2+/calmodulin-dependent protein kinase II are generated from a single gene by alternative splicing. A fifth form of the cDNA is maternally derived, and encodes the enzyme expressed in the ovary, unfertilized egg and early embryos. The fifth form is also generated from the gene by alternative splicing and is identical to the cDNA encoding the 530-amino-acid polypeptide, the longest of the four forms previously identified, except that it lacks exon 11. Three splicing derivatives which have lost one amino acid from the 509- and 530-amino-acid polypeptides are also found in 4 to 10 h embryos (Takamatsu, 1994).

Amino Acids - 490, 509, 516 and 530 (Cho, 1991 and Ohsako, 1993)

Structural Domains

See above: biological overview

The database Online Mendelian Inheritance in Man (OMIM) is an excellent source of information about mammalian CaMK2A.


CaM kinase II: | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References
date revised:  22 December 2018
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