CrebB-17A

Mg²⁺ block of Drosophila NMDA receptors is required for long-term memory formation and CREB-dependent gene expression

NMDA receptor (NMDAR) channels allow Ca²⁺ influx only during correlated activation of both pre- and postsynaptic cells; a Mg²⁺ block mechanism suppresses NMDAR activity when the postsynaptic cell is inactive. Although the importance of NMDARs in associative learning and long-term memory (LTM) formation has been demonstrated, the role of Mg²⁺ block in these processes remains unclear. Using transgenic flies expressing NMDARs defective for Mg²⁺ block, it was found that Mg²⁺ block mutants are defective for LTM formation but not associative learning. It was demonstrated that LTM-dependent increases in expression of synaptic genes, including homer, staufen, and activin, are abolished in flies expressing Mg²⁺ block defective NMDARs. Furthermore, it was shown that genetic and pharmacological reduction of Mg²⁺ block significantly increases expression of a CREB repressor isoform. These results suggest that Mg²⁺ block of NMDARs functions to suppress basal expression of a CREB repressor, thus permitting CREB-dependent gene expression upon LTM induction (Miyashia, 2012).

Although the mechanism through which Mg²⁺ block restricts NMDAR activity is well known, the cellular and behavioral functions of Mg²⁺ block have not been extensively studied. In this study, transgenic flies expressing dNR1^N63IQ to show that Mg²⁺ block is important for formation of LTM. Previous studies of hypomorphic mutants have shown that NMDARs are required for both learning and LTM. In contrast, our Mg²⁺ block mutants do not have learning defects. This suggests that although Ca²⁺ influx through NMDARs is important for learning, inhibition of influx during uncorrelated activity is not. Notably, elav/dNR1^N63IQ flies have slightly enhanced learning. Consistent with this result, NMDAR-dependent induction of hippocampal LTP is enhanced in the absence of external Mg²⁺. In the current studies, Mg²⁺-block-defective dNR1 was overexpressed in an otherwise wildtype background, so it cannot be definitively concluded that Mg²⁺ block is dispensable for learning. However, electrophysiology experiments indicate that Mg²⁺ block is abolished in the flies at physiological potentials. Furthermore, it was demonstrated that expression of Mg²⁺-block-defective dNR1 rescues learning defects in dNR1 hypomorphs, consistent with a model in which Mg²⁺ block is not required for learning. Interestingly, the dNR1^N63IQ transgene does not rescue the semilethality of dNR1 hypomorphs, suggesting that Mg²⁺ block has an essential biological function unrelated to learning (Miyashia, 2012).

The results suggest that Mg²⁺-block-dependent suppression of NMDAR activity and Ca²⁺ influx at the resting state is critical for LTM formation. Supporting this idea, chronic reduction of NMDAR-mediated Ca²⁺ influx at the resting state has been shown to enhance long-term synaptic plasticity. Extending these results, it was found that Mg²⁺ block is required for CREB-dependent gene expression during LTM formation. A CREB-dependent increase in staufen expression upon spaced training is essential for LTM formation, and this study shows that Mg²⁺ block is required for this increase. Two other genes, activin and homer, were identified that are expressed upon LTM induction in a CREB-dependent manner. It is proposed that all three genes are maintained in an LTM-inducible state by Mg²⁺-block-dependent inhibition of CREB repressor, and it was shown that the amount of increase in expression of dCREB2-b in Mg²⁺ block mutants correlates with the ability of dCREB2-b to suppress LTM. The 4-fold increase in dCREB2-b protein in Mg²⁺ block mutant flies is comparable to the increase in dCREB2-b in heat-shocked hs-dCREB2-b flies showing equivalent defects in LTM (Miyashia, 2012).

Next the homer gene was further characterized and it was determined to be required specifically for LTM but not for learning or ARM. It was determined that spaced training increases HOMER expression in several brain regions, including the antennal lobes, lateral protocerebrum, protocerebral bridge, and calyx of the MBs. This increase does not occur in the absence of Mg²⁺ block. Significantly, when Mg²⁺ block is abolished by dNR1^N63IQ expression, specifically in the MBs, increased Homer expression is suppressed in the MBs but not in other regions, including the protocerebral bridge, indicating that Mg²⁺ block regulates CREB repressor and LTM-associated gene expressions in a cell autonomous manner (Miyashia, 2012).

Electrophyisiological experiments demonstrate that 20 mM Mg²⁺ is sufficient to block Drosophila NMDAR currents at the resting potential (-80 mV). Although this concentration is higher than the concentrations needed to block mammalian NMDARs, the Mg²⁺ concentration in Drosophila hemolymph has been shown by various groups to be between 20 and 33 mM, which is correspondingly higher than the Mg²⁺ concentration reported in mammalian plasma. In mammals, Mg²⁺ concentration is higher in cerebrospinal fluid than in plasma, further suggesting that the 20 mM Mg²⁺ concentration used in this study is likely to be within the physiologically relevant range (Miyashia, 2012).

An N/Q substitution at the Mg²⁺ block site of mammalian NR1 disrupts Mg²⁺ block and reduces Ca²⁺ permeability, while a W/L substitution in the TM2 domain of NR2B disrupts Mg²⁺ block and increases Mg²⁺ permeability. This raises the possibility that Mg²⁺-block-independent changes in channel kinetics and Mg²⁺ permeability may be responsible for the effects observed in the dNR1^N63IQ-expressing flies. While this possiblity cannot be completely ruled out, increases were observed in dCREB-2b protein in wild-type neurons in Mg²⁺-free conditions, indicating that disruption of Mg²⁺ block, rather than changes in other channel properties, causes increased CREB repressor expression and decreased expression of LTM-associated genes (Miyashia, 2012).

A chronic elevation in extracellular Mg²⁺ enhances Mg²⁺ block of NMDARs, leading to upregulation of NMDAR activity and potentiation of NMDA-induced responses at positive membrane potentials (during correlated activity) (Slutsky, 2010). This raised the possibility that the Mg²⁺ block mutations may cause a downregulation of NMDAR-dependent signaling and decreased NMDA-induced responses at positive membrane potentials. Since this study recorded NMDA-induced responses from various sizes of cells, it was not possible to directly compare amplitudes of NMDA-induced responses between elav/dNR1^wt cells and elav/dNR1^N63IQ cells. However, training-dependent increases in ERK activity, required for CREB activation, occurred normally in both elav/dNR1^wt cells and elav/dNR1^N63IQ cells, while it was significantly suppressed in dNR1 hypomorphs. These results suggest that the Mg²⁺ block mutations do not alter NMDA-induced responses at positive membrane potentials (Miyashia, 2012).

Similar to dNR1 Mg²⁺ block mutants, dNR1 hypomorphic mutants also have defects in CREB-dependent gene expression upon LTM formation. However, dNR1 hypomorphs and Mg²⁺ block mutants are likely to have opposing effects on Ca²⁺ influx. While hypomorphic dNR1 mutants should have decreased Ca²⁺ influx during spaced training because of a reduction in the number of dNMDARs, elav/dNR1^N63IQ flies are unlikely to have this effect. Conversely, while elav/ dNR1^N63IQ flies should have increased Ca²⁺ influx during the resting state when uncorrelated activity is likely to occur, dNR1 hypomorphs should not. Supporting a model in which dNR1 hypomorphs and Mg²⁺ block mutants inhibit LTM-dependent gene expression through different mechanisms, it was shown that Mg²⁺ block mutants increase basal expression of dCREB2-b repressor while NMDAR hypomorphs do not. Conversely, the data indicating that NMDAR hypomorphs are defective for training dependent increases in ERK activity, while elav/dNR1^N63IQ flies are not. These data fit a model in which there may be two equally important requirements for NMDARs in regulating LTM-dependent transcription. First, during correlated, LTM-inducing stimulation, a large Ca²⁺ influx through channels, including NMDARs, may be required to activate kinases, including ERK, necessary to activate CREB. dNR1 hypomorphs are defective for this process. However, a second and equally important requirement for NMDARs may be to inhibit low amounts of Ca²⁺ influx during uncorrelated activity to maintain the intracellular environment in a state conducive to CREB-dependent transcription. Mg²⁺ block is required for this process (Miyashia, 2012).

Although it is unclear what types of uncorrelated activity are suppressed by Mg²⁺ block, one type may be spontaneous, action potential (AP)-independent, single vesicle release events (referred to as 'minis'). Supporting this idea, an increase in dCREB2-b was observed in cultured wild-type brains in Mg²⁺-free medium in the presence of TTX, which suppresses AP-dependent vesicle releases but does not affect minis. In addition, a significant increase was observed in cytosolic Ca²⁺, [Ca²⁺]i, in response to 1 mM NMDA in the presence of extracellular Mg²⁺ in neurons from elav/dNR1^N63IQ pupae. In neurons from transgenic control and wild-type pupae, which have an intact Mg²⁺ block mechanism, 1 mM NMDA does not cause Ca²⁺ influx and membrane depolarization. The concentration of glutamate released by minis is on the order of 1 mM at the synaptic cleft, suggesting that an increase in frequency of mini-induced Ca²⁺ influx due to decreased Mg²⁺ block may contribute to the increase in dCREB2-b in elav/ dNR1^N63IQ flies (Miyashia, 2012).

Correlated, AP-mediated NMDAR activity has been proposed to facilitate dCREB2-dependent gene expression by increasing activity of a dCREB2 activator. The present study suggests that, conversely, Mg²⁺ block functions to inhibit uncorrelated activity, including mini-dependent Ca²⁺ influx through NMDARs, which would otherwise cause increased dCREB2-b expression and decreased LTM. Other studies have also suggested opposing roles of AP-mediated transmitter release and minis. For activity-dependent dendritic protein synthesis, local protein synthesis is stimulated by AP-mediated activity and inhibited by mini activity. In the case of NMDARs, the opposing role of low Ca²⁺ influx in inhibiting CREB activity must be suppressed by Mg²⁺ block for proper LTM formation (Miyashia, 2012).

A neuropeptide signaling pathway regulates synaptic growth in Drosophila

Neuropeptide signaling is integral to many aspects of neural communication, particularly modulation of membrane excitability and synaptic transmission. However, neuropeptides have not been clearly implicated in synaptic growth and development. This study demonstrates that cholecystokinin-like receptor (CCK-like receptor at 17D1; CCKLR), and Drosulfakinin (DSK), its predicted ligand, are strong positive growth regulators of the Drosophila melanogaster larval neuromuscular junction (NMJ). Mutations of CCKLR (CCKLR-17D1 but not CCKLR-17D3) or dsk produce severe NMJ undergrowth, whereas overexpression of CCKLR causes overgrowth. Presynaptic expression of CCKLR is necessary and sufficient for regulating NMJ growth. CCKLR and dsk mutants also reduce synaptic function in parallel with decreased NMJ size. Analysis of double mutants revealed that DSK/CCKLR regulation of NMJ growth occurs through the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA)-cAMP response element binding protein (CREB) pathway. These results demonstrate a novel role for neuropeptide signaling in synaptic development. Moreover, because the cAMP-PKA-CREB pathway is required for structural synaptic plasticity in learning and memory, DSK/CCKLR signaling may also contribute to these mechanisms (Chen, 2012).

Proper synaptic growth is essential for normal development of the nervous system and its function in mediating complex behaviors such as learning and memory. The Drosophila larval neuromuscular junction (NMJ) has become a powerful system for studying the molecular mechanisms underlying synaptic development and plasticity, and many of the key synaptic proteins are evolutionarily conserved (Chen, 2012).

Genetic and molecular analysis in Drosophila has uncovered numerous molecules and pathways that regulate NMJ growth, including proteins required for cell adhesion, endocytosis, cytoskeletal organization, and signal transduction via TGF-β, Wingless, JNK, cAMP, and other signaling molecules. For example, previous studies revealed that increased cAMP levels led to a down-regulation of the cell adhesion protein FasII at synapses, and the activation of the cAMP response element binding protein (CREB) transcription factor to achieve long-lasting changes in synaptic structure and function. Despite the identification and characterization of these various positive and negative regulators, understanding of the networks that govern synaptic growth is still incomplete, with many of the key components and mechanisms yet to be uncovered and analyzed (Chen, 2012).

To search for new regulators of synaptic growth, a forward genetic screen was conducted for mutants exhibiting altered NMJ morphology. In this screen, a new mutation was discovered that exhibits strikingly undergrown NMJs, which indicates disruption of a positive regulator of NMJ growth. The affected protein was identified as cholecystokinin (CCK)-like receptor (CCKLR), a putative neuropeptide receptor that belongs to the family of G-protein coupled receptors (GPCRs) sharing a uniform topology with seven transmembrane domains. When activated by their ligands, neuropeptide GPCRs affect levels of second messengers such as cAMP, diacylglycerol, inositol trisphosphate, and intracellular calcium. Through activation of their cognate receptors, secreted neuropeptides mediate communication among various sets of neurons as well as other cell types to regulate several physiological activities, including feeding and growth, molting, cuticle tanning, circadian rhythm, sleep, and learning and memory. In general, neuropeptides act by modulating neuronal activity through both short-term and long-term effects. Short-term effects include modifications of ion channel activity and alterations in release of or response to neurotransmitters. Long-term effects include changes in gene expression through activation of transcription factors and protein synthesis. In contrast with the well-known effects of neuropeptide signaling on neuronal activity and the strength of synaptic transmission, regulation of synaptic growth and development by neuropeptides has not previously been clearly established (Chen, 2012).

This study demonstrates that CCKLR is required presynaptically to promote NMJ growth. Moreover, mutations of drosulfakinin (dsk), which encode the predicted ligand of CCKLR, cause similar NMJ undergrowth and interact genetically with CCKLR mutations, indicating that DSK and CCKLR are components of a common signaling mechanism that regulates NMJ growth. In addition to the morphological phenotypes caused by mutations of CCKLR and dsk, mutant larvae also exhibit deficits in synaptic function. Through phenotypic analysis of double mutant combinations, it was shown that DSK/CCKLR signals through the cAMP-PKA-CREB pathway to regulate NMJ growth. The results suggest a novel role for neuropeptide signaling in regulation of synaptic development. Moreover, because the cAMP-PKA-CREB pathway is required for structural plasticity of synapses in learning and memory, DSK/CCKLR signaling may also contribute to these mechanisms (Chen, 2012).

Neuropeptides, whose effects have been extensively studied at NMJs, are usually described as neuromodulators because they modify the strength of synaptic transmission. For example, proctolin can potentiate the action of glutamate at certain NMJs in insects. However, involvement of neuropeptides in regulating neural development has not been well characterized. Recently, a C-type natriuretic peptide acting through a cGMP signaling cascade was found to be required for sensory axon bifurcation in mice, which suggests that neuropeptides may have a broader role in development than previously appreciated. The current studies demonstrate that DSK and its receptor, CCKLR, are strong positive regulators of NMJ growth in Drosophila (Chen, 2012).

DSK belongs to the family of FMRFamide-related peptides (FaRPs), which is very broadly distributed across invertebrate and vertebrate phyla. Originally identified in clams, FaRPs affect heart rate, blood pressure, gut motility, feeding behavior, and reproduction in invertebrates. They have been shown to enhance synaptic efficacy at NMJs in locust and to modulate presynaptic Ca²⁺ channel activity in crustaceans. In Drosophila, various neuropeptides derived from the FMRFa gene can modulate the strength of muscle contraction when perfused onto standard larval nerve-muscle preparations. To these previously described functions of FaRPs, this study adds a new role as a positive regulator of NMJ development (Chen, 2012).

Transgenic rescue experiments, RNAi expression, and overexpression of WT CCKLR demonstrate that CCKLR functions presynaptically in motor neurons to promote NMJ growth. Downstream components of this pathway were identified on the basis of known biochemistry of GPCRs and phenotypic interactions in double mutant combinations. GPCRs typically function by activating second messenger pathways via G proteins. Because loss-of-function mutations in dgs (which encodes the Gsα subunit in Drosophila) cause NMJ undergrowth, it is hypothesized that CCKLR signals through Gsα. Consistent with this idea, it was found that presynaptic constitutively active dgs overexpression rescues the NMJ undergrowth phenotype of CCKLR mutants. Conversely, dominant dose-dependent interactions were observed between CCKLR-null mutations and mutations of rut, which encodes an AC; or PKA-C1, which encodes a cAMP-dependent protein kinase, resulting in significant reductions in NMJ growth. These data place CCKLR together with the other genes in a common cAMP-dependent signaling pathway that regulates NMJ growth (Chen, 2012).

It is known that the AC encoded by rut is activated by Gsα, and on the basis of the results, it is proposed that Gsα is downstream of CCKLR signaling. However, the NMJ undergrowth in rut¹, which is a presumptive null mutant, is not as severe as that of a CCKLR-null mutant. This is likely due to the fact that the Drosophila genome contains up to seven different AC-encoding genes, all of which are stimulated by Gsα. Presumably, one or more additional AC-encoding genes share some functional overlap with rut in regulation of NMJ growth. This idea is in good agreement with the results of Wolfgang (2004), who found that the NMJ undergrowth phenotype of rut¹ is weaker than that of dgs mutants, which they also interpreted as an indication that multiple ACs are activated by the Gsα encoded by dgs (Chen, 2012).

The primary effector of this pathway is CREB2, a transcriptional regulatory protein that is activated upon phosphorylation by PKA. Consistent with the idea that CCKLR ultimately acts via activation of CREB, loss-of-function mutations of dCreb2 or neuronal overexpression of a dominant-negative dCreb2 transgene cause NMJ undergrowth similar to that of CCKLR-null mutants. Additionally, loss of one copy of dCreb2 in a CCKLR heterozygous background also causes NMJ undergrowth, and overexpression of WT dCreb2 fully rescues the NMJ undergrowth phenotype of CCKLR null, even leading to NMJ overgrowth. Thus, regulation of NMJ growth through the CCKLR signaling pathway is clearly mediated by dCreb2, whose activity is itself necessary and sufficient for regulating NMJ growth. This conclusion differs from another study that suggested that dCreb2 is required for NMJ function but not NMJ growth. One possible explanation for this discrepancy is that a weaker, inducible heat shock-driven transgene was used to express dCreb2 in the earlier work, whereas strong constitutive neuronal drivers were used this study. In any case, the current results demonstrate that in addition to its known role in NMJ function, CREB2 is also a strong positive regulator of NMJ growth and is likely to play a greater role in structural plasticity of synapses in learning and memory in Drosophila than previously suggested. This conclusion is consistent with a recent study indicating that sprouting of type II larval NMJs in response to starvation is stimulated by a cAMP/CREB-dependent pathway via activation of an octopamine GPCR (Chen, 2012).

In addition to being undergrown, NMJs in CCKLR mutant larvae also exhibit a functional deficit. This is perhaps less straightforward than it might seem. Previous analyses of mutations affecting growth of the larval NMJ in Drosophila have shown that there is no simple correlation between the size and complexity of the NMJ and the amplitude of EJPs or amount of neurotransmitter release. These discrepancies arise because of various homeostatic compensatory mechanisms and because some of the affected signaling pathways alter synaptic growth and synaptic function in different ways via distinct downstream targets. For example, a mutation in highwire, which has the most extreme NMJ overgrowth phenotype described, is associated with a decrease in synaptic transmission. Wallenda mutations have been shown to fully suppress the overgrowth phenotype, but have no effect on the deficit in synaptic transmission (Chen, 2012).

In the case of CCKLR mutants, however, there appears to be a very good correspondence between the morphological phenotype and the electrophysiological phenotype: the reduction in the total number of active zones in CCKLR larvae as measured morphologically correlates very well with the reduction in quantal content that was observe. In addition, no difference in CCKLR mutants was detected in calcium sensitivity of transmitter release or in the size or frequency of spontaneous release events. Thus, the synaptic growth phenotype of CCKLR mutant NMJs is sufficient to account for the functional phenotype. However, the possibility cannot be ruled out that DSK/CCKLR signaling also exerts some modulatory effect on NMJ function that is distinct from its effect on NMJ development (Chen, 2012).

DSK is identified as the Drosophila orthologue of CCK, the ligand of CCKLR in vertebrates, on the basis of sequence analysis. The genetic analysis strongly supports the conclusion that DSK is the ligand of CCKLR at the larval NMJ. First, mutations of dsk and expression of dsk RNAi result in NMJ undergrowth phenotypes similar to that of CCKLR mutants. Second, loss of one copy of both dsk and CCKLR in double heterozygotes results in NMJ undergrowth. Third, heterozygosity for dsk does not further enhance the phenotype of a CCKLR-null mutant as expected if DSK regulates NMJ growth through its action on CCKLR. Fourth, overexpression of UAS-dsk does not rescue the undergrowth phenotype of a CCKLR-null mutant, but CCKLR overexpression can rescue NMJ undergrowth of a dsk hypomorphic mutant (Chen, 2012).

The discovery of an entirely novel role for neuropeptide signaling in NMJ growth raises several questions about how this signaling is regulated and the biological significance of this mechanism. Although answers to these questions will require much additional work, an immediate question is whether a paracrine or autocrine mechanism is involved. In the case of octopamine-mediated synaptic sprouting in response to starvation, both autocrine and paracrine signaling are involved in the sprouting of type II and type I NMJs, respectively. In an early immunohistochemical investigation, it was reported that DSK was detected in medial neurosecretory cells in the larval CNS that extended projections anteriorly into the brain and posteriorly to the ventral ganglion. As it was not possible to obtain the original DSK antiserum and raising a new antiserum was not successful, the previous report has not been extended or confirmed. Instead, tissue-specific RNAi experiments were performed to examine the spatial requirement for DSK. Pan-neuronal dsk RNAi expression indicates that DSK expression in neurons is required to promote NMJ growth. In addition, C739-Gal4-driven dsk RNAi also causes NMJ undergrowth, whereas OK-Gal4-driven dsk RNAi in motor neurons does not. The expression pattern of C739-Gal4 overlaps with the DSK-positive cells previously identified by immunohistochemistry, which suggests that DSK produced by those neurosecretory cells is required for normal NMJ growth. Thus, from available data, it seems most likely that DSK is acting in paracrine fashion to regulate NMJ growth. However, further investigation will be necessary to determine the exact source of the DSK that promotes NMJ growth to fully understand how this neuropeptide regulates NMJ development (Chen, 2012).

Studies have demonstrated a role for CREB in long-term synaptic plasticity—structural changes in synaptic morphology that underlie the formation of long-term memories. This study shows that in addition to CREB’s role in structural modification of synapses in response to experience after development is complete, it is also a key regulator of growth and morphology during development of the larval NMJ. Moreover, although CREB is the transcriptional effector for many GPCRs, the fact that NMJs in CCKLR mutants are as undergrown as those of CREB mutants suggests that DSK/CCKLR signaling is a major input to CREB during NMJ growth. Many of the genes encoding intermediate components of the pathway such as dnc, rut, and PKA also have effects on NMJ growth and development as well as on synaptic plasticity and learning and memory, further emphasizing an overlap between the mechanisms that regulate synaptic growth during development and those that regulate postdevelopmental structural synaptic plasticity. These results raise the possibility that DSK/CCKLR signaling also plays a role in long-term synaptic plasticity and learning as well as in synaptic development (Chen, 2012).

Targets of Activity

Decapentaplegic (Dpp) is an extracellular signal of the transforming growth factor-beta family with multiple functions during Drosophila development. For example, it plays a key role in the embryo during endoderm induction. During this process, Dpp stimulates transcription of the homeotic genes Ultrabithorax in the visceral mesoderm and labial in the subjacent endoderm. A cAMP response element (CRE) from an Ultrabithorax enhancer mediates Dpp-responsive transcription in the embryonic midgut, and endoderm expression from a labial enhancer depends on multiple CREs. The enhancer, called Ubx B confers Wingless- and Decapentaplegic-dependent expression in the visceral mesoderm. Staining mediated by Ubx B is in two stripes of cells in the visceral mesoderm, a wide prominent one in parasegments 6-9 and a narrow weak one in parasegment 3. The Drosophila CRE-binding protein dCREB-2 binds to the Ultrabithorax CRE. Binding is at a palindromic sequence TGGCGTCA that resembles a typical cAMP response element (CRE) (TGACGTCA). Mutation of this site results in the elimination of response to Dpp, but a maintenance of response to Wg. This residual expression is in parasegment 8 and 9 coinciding with the main source of wg expression in the middle midgut. The Ubx CRE can also mediate response to Dpp signaling in the endoderm. Other transcription factors act through the Ubx B enhancer to confer its tissue-specific response to Dpp in the visceral mesoderm. CRE needs to cooperate with a LEF-1 binding site to respond to the Dpp signal in the visceral mesoderm. Schnurri, a transcription factor implicated in Dpp signaling, fails to interact with Ubx B. Adjacent to the CRE is another palindromic sequence that antagonizes the activating effects of Dpp and Wg signaling on the Ubx B enhancer. Ubiquitous expression of a dominant-negative form of dCREB-2 suppresses CRE-mediated reporter gene expression and reduces labial expression in the endoderm. Therefore, a dCREB-2 protein may act as a nuclear target, or as a partner of a nuclear target, for Dpp signaling in the embryonic midgut (Eresh, 1997).

Long term memory requires de novo gene expression mediated by CREB family genes. Using an inducible transgene that expresses CrebB-17A, the dominant negative member of the CREB family, long term memory has been specifically and completely blocked while short term memory (anasthesia-resistent memory) remains unaffected (Yin 1995b).

Induced expression of a CrebB-17A activator isoform enhances long term memory in Drosophila, so that maximum learning is achieved after only one training session. Memory requires phosphorylation of the activator isoform (Yin 1995b).

Fasciclin2 mutants lead to an increase in number of boutons at neuromuscular synapses without affecting quantal content. Increased cAMP in dunce mutants increases both synaptic structure and quantal content. Thus there must be other elements downstream of cAMP, but not downstream from Fas2, that are involved in increasing quantal content. CREB is a candidate for the cAMP target responsible for increasing quantal content. CREB acts in parallel with Fas2 to cause an increase in synaptic strength. Expression of an endogenous CREB repressor, CrebB-17A-a, in dunce mutants blocks functional but not structural plasticity. Expression of the activator isoform, CrebB-17A-a, increases synaptic strength, by increasing presynaptic transmitter release at single boutons, but only in Fas2 mutants that increase bouton number. Strong overexpression of CrebB-17A-a results in a significant increase in quantal content, independent of genetic background and with little effect on bouton number. Thus CREB-mediated increase in synaptic strength is due to increased presynaptic transmitter release and expression of CrebB-17A-a in a Fas2 mutant background genetically reconstitutes cAMP-dependent plasticity. It is concluded that cAMP initiates parallel changes in CREB and Fas2 to achieve long term synaptic enhancement (Davis, G. W 1996).

Drosophila CREB genes are implicated in regulation of the Drosophila homolog of mammalian JUN. A 43-bp 5' proximal promoter region is necessary for the transcription activity of DJUN (Perkins, 1988a). Deletion of this fragment decreases transcriptional activity 67-fold. This 43-bp sequence alone, containing a Drosophila transcription factor DTF-1 binding site and TATA box, however, is not sufficient for transcription activity. An 80-bp sequence including the start of transcription has considerable basal activity. This intragenic region containing an AP-1 site and a CRE site (presumably binding a Drosophila CREB) modulates or fine tunes activity of the promoter. An extragenic region containing two AP-1 sites similarly affects promoter activity (Wang, 1994).

The responsiveness of DJun to CREB suggests a role for DJun in the preservation of long term memory in the fly. To date, such a role has not been documented.

Time of day influences memory formation and dCREB2 proteins in Drosophila

Many biological phenomena oscillate under the control of the circadian system, exhibiting peaks and troughs of activity across the day/night cycle. In most animal models, memory formation also exhibits this property, but the underlying neuronal and molecular mechanisms remain unclear. The dCREB2 transcription factor shows circadian regulated oscillations in its activity, and has been shown to be important for both circadian biology and memory formation. This study shows that the time-of-day (TOD) of behavioral training affects Drosophila memory formation. dCREB2 exhibits complex changes in protein levels across the daytime and nighttime, and these changes in protein abundance are likely to contribute to oscillations in dCREB2 activity and TOD effects on memory formation. The results demonstrate notable correlations between the TOD behavioral effects and the circadian profile of dCREB2 proteins. At ZT = 20, there is a significant depression in memory formation, an event which coincides with apparent increases in blocker-related species clearly visible on the Western blots. At ZT = 16, a significant increase was measured in performance. This time point correlates with the end of a window (ZT = 13-15) when nuclear levels of the activator are elevated. Based on these relationships, it is hypothesized that the dynamics of dCREB2 protein levels contribute to the TOD effects on memory formation (Fropf, 2014).

Shifting transcriptional machinery is required for long-term memory maintenance and modification in Drosophila mushroom bodies

Accumulating evidence suggests that transcriptional regulation is required for maintenance of long-term memories (LTMs). This study characterized global transcriptional and epigenetic changes that occur during LTM storage in the Drosophila mushroom bodies (MBs), structures important for memory. Although LTM formation requires the CREB transcription factor and its coactivator, CBP, subsequent early maintenance requires CREB and a different coactivator, CRTC. Late maintenance becomes CREB independent and instead requires the transcription factor Beadex, also know as LIM-only. Bx expression initially depends on CREB/CRTC activity, but later becomes CREB/CRTC independent. The timing of the CREB/CRTC early maintenance phase correlates with the time window for LTM extinction and this study identified different subsets of CREB/CRTC target genes that are required for memory maintenance and extinction. Furthermore, it was found that prolonging CREB/CRTC-dependent transcription extends the time window for LTM extinction. These results demonstrate the dynamic nature of stored memory and its regulation by shifting transcription systems in the MBs (Hirano, 2016).

This study has identified Bx and Smr as LTM maintenance genes and has characterize a shift in transcription between CREB/CRTC-dependent maintenance (1-4 days) to Bx-dependent maintenance (4-7 days). In addition, a biological consequence of this shift was identified in defining a time window during which LTM can be modified, β-Spec was identified as being required for memory extinction (Hirano, 2016).

LTM maintenance mechanisms change dynamically during storage. In particular, CRTC, which is not required during memory formation, becomes necessary during 4-day LTM maintenance and then becomes dispensable again. Consistent with this, CRTC translocates from the cytoplasm to the nucleus of MB neurons during 4-day LTM maintenance and returns to the cytoplasm within 7 days. On the other hand, Bx expression is increased at both phases, suggesting that transcriptional regulation of memory maintenance genes may change between these two phases. Supporting this idea, it was found that Bx expression requires CRTC during 4-day LTM maintenance but becomes independent of CRTC 7 days after training. It is proposed that CREB/CRTC activity induces Bx expression, which subsequently activates a feedback loop where Bx maintains its own expression and that of other memory maintenance genes (Hirano, 2016).

Although it is proposed that the shifts in transcriptional regulation that were observed occur temporally in the same cells, the possibility cannot be discounted that LTM lasting 7 days is maintained in different cells from LTM lasting 4 days. MB Kenyon cells can be separated into different cell types, which exert differential effects on learning, short-term memory and LTM. Thus, it is possible that LTM itself consists of different types of memory that can be separated anatomically. In this case, CRTC in one cell type may exert non-direct effects on another cell type to activate downstream genes including Bx and Smr. However, as that CRTC binds to the Bx gene locus to promote Bx expression and both CRTC and Bx are required in the same α/β subtype of Kenyon cells, it is likely that the shift from CRTC-dependent to Bx-dependent transcription occurs within the α/β neurons (Hirano, 2016).

Currently, it is proposed that the alterations in histone acetylation and transcription that were uncovered are required for memory maintenance. However, it is noted that decreases in memory after formation could be caused by defects in retrieval and maintenance. Thus, it remains formally possible that the epigenetic and transcriptional changes reported in this study are required for recall, but not maintenance. However, this is unlikely, as inhibition of CRTC from 4 to 7 days after memory formation does not affect 7 day memory, whereas inhibition from 1 to 4 days does. This suggests that at least one function of CRTC is to maintain memory for later recall (Hirano, 2016).

Consistent with a previous study in mice, which suggests distinct transcriptional regulations in LTM formation and maintenance (Halder, 2016), the data indicate that memory formation and maintenance are distinct processes. Although the HAT, CBP, is required for formation but dispensable for maintenance, other HATs, GCN5 and Tip60, are required for maintenance but dispensable for formation. Through ChIP-seq analyses, those downstream genes, Smr and Bx, were identified as LTM maintenance genes and these are not required for LTM formation. Collectively, these results suggest differential requirements of histone modifications between LTM formation and maintenance. Although other histone modifiers besides GCN5 and Tip60 were identified in the screen, knockdown of these histone modifiers did not affect LTM maintenance. There are ~50 histone modifiers encoded in the fly genome, raising the possibility that the lack of phenotype in some knockdown lines is due to compensation by other modifiers (Hirano, 2016).

The results indicate some correlation of increase in CRTC binding with histone acetylation and gene expression. Interestingly, DNA methylation shows higher correlation to gene expression in comparison with histone acetylation in mice. Notably, flies lack several key DNA methylases and lack detectable DNA methylation patterns. Hence, histone acetylation rather than DNA methylation may have a higher correlation with transcription in flies. Reduction in histone acetylation was detected, overlapping with increase in CRTC binding. Those reductions could be due to CRTC interacting with a repressor isoform of CREB, CREB2b or other transcriptional repressor that binds near CREB/CRTC sites. These interactions would decrease histone acetylation and gene expression, and may be related to LTM maintenance. Although this study focused on the upregulation of gene expression through CREB/CRTC, downregulation of gene expression by transcriptional repressors may also be important in understanding the transcriptional regulation in LTM maintenance. The results demonstrate the importance of HATs for LTM maintenance; however, the data do not conclude that histone acetylation is a determinant for gene expression, but rather it might be a passive mark of gene expression. HATs also target non-histone proteins and also interact with various proteins, both of which could support gene expression in LTM maintenance (Hirano, 2016).

Similar to traumatic fear memory in rodents, this study found that aversive LTM in flies can be extinguished by exposing them to an extinction protocol specifically during 4-day LTM maintenance. These observations suggest the time-limited activation of molecules that allows LTM extinction only during the early storage. Supporting this concept, it was found that CRTC is activated during the extinguishable phase of LTM maintenance and prolonging CRTC activity extends the time window for extinction. Thus, CRTC is the time-limited activated factor determining the time window for LTM extinction in flies. In cultured rodent hippocampal neurons, CRTC nuclear translocation is not sustained, suggesting that other transcription factors may function in mammals to restrict LTM extinction (Hirano, 2016).

This work demonstrates that LTM formation and maintenance are distinct, and involve a shifting array of transcription factors, coactivators and HATs. A key factor in this shift is CRTC, which shows a sustained but time-limited translocation to the nucleus after spaced training. Thus, MB neurons recruit different transcriptional programmes that enable LTM to be formed, maintained and extinguished (Hirano, 2016).

Protein Interactions

In the cyclic AMP signal transduction pathway, protein kinase A (PKA) activates CREB by phosphorylation (Drain, 1991). The isoform CrebB-17A-a is a PKA dependent activator of transcription (Yin 1994a). The isoform CrebB-17A-b does not function as a PKA dependent activator, but works as a direct antagonist of PKA-dependent activation by CrebB-17A-a (Yin 1995a).

Interactions of Creb-binding Protein

Drosophila CBP (Nejire) is a co-activator of cubitus interruptus in hedgehog signaling. Drosophila CBP predicts a protein of relative molecular mass 332,000; the gene maps to position 8F/9A on the X chromosome. Mutants for dCBP gene, nejire, die at stages 9 or 10 during embryogenesis, although some embryos survive to hatching. The most severe phenotype of the nej hemizygotes is the twisting of the embryo at germband elongation. The expression of wingless is strikingly reduced at the posterior margin of each parasegment in mutants. In addition, engrailed expression, which is maintained by WG protein, is significantly lower than in wild type. These observations suggest the Drosophila CBP might contribute to the functioning of some transcription factors involved in the wingless-engrailed signaling pathway. Cubitus interruptus protein physically interacts with Drosophila CBP (dCBP). A series of deletion mutants of ci indicate that a region of CI between amino acids 1020 and 1160 is required for phosphorylation independent interaction with dCBP. This region is part of the CI transactivation domain, C-terminal to five putative PKA sites. dCBP expression augments transactivation by CI up to a maximum of 62 fold. The dominant gain-of-function ciD mutant phenotype in which the longitudinal vein 4 of the adult wing is shortened, some posterior row hairs are missing, and the posterior wing margin is flattened, can be explained by the inappropriate expression of ci in the posterior compartment of the wing imaginal disc, where it is usually repressed by Engrailed. A subset of the ciD wing defects is suppressed by haploinsufficiency of dCBP. Thus dCBP is required for the activation of Cubitus interruptus target genes such as patched, and CBP is required for the activator function of CI but not for the repressor function. dCBP binds to dCREB2, the Drosophila homolog of CREB, in a phosphorylation-dependent manner, whereas the dCBP-CI interaction is phosphorylation-independent. These findings raise the possiblilty that a limited amount of dCBP might be recruited to PKA-phosphorylated dCREB2, resulting in a decrease in CI activity, explaining the antagonistic actions of PKA and Hedgehog (Akimaru, 1997a).

Attempts to demonstrate trans-activation activity by the Drosophila Myb gene product have been unsuccessful so far. Co-transfection of Schneider cells with a plasmid expressing the Drosophila homolog of transcriptional co-activator CBP (dCBP) results in transactivation by Myb. Using this assay system, the functional domains of Myb have been analyzed. Two domains located in the N-proximal region, one of which is required for DNA binding and the other for dCBP binding, are both necessary and sufficient for trans-activation. In this respect, D-Myb is similar to c-Myb and A-Myb, but different from mammalian B-Myb. These results shed light on how the myb gene diverged during the course of evolution (Hou, 1997).

Although CREB-binding protein (CBP) functions as a co-activator of many transcription factors, relatively little is known about the physiological role of CBP. Mutations in the human CBP gene are associated with Rubinstein-Taybi syndrome, a haplo-insufficiency disorder characterized by abnormal pattern formation. Drosophila CBP is maternally expressed, suggesting that it plays a role in early embryogenesis. Mesoderm formation is one of the most important events during early embryogenesis. To initiate the differentiation of the mesoderm in Drosophila, multiple zygotic genes such as twist (twi) and snail (sna), which encode a basic-helix-loop-helix and a zinc finger transcription factor, respectively, are required. The transcription of these genes is induced by maternal Dorsal protein, a transcription factor that is homologous to the NF-kappa B family of proteins. Drosophila CBP mutants fail to express twi and generate twisted embryos. This is explained by results showing that dCBP is necessary for Dorsal-mediated activation of the twi promoter (Akimaru, 1997b).

T-cell factor (TCF), a high-mobility-group domain protein, is the transcription factor activated by Wnt/Wingless signaling. When signaling occurs, TCF binds to its coactivator, beta-catenin/Armadillo, and stimulates the transcription of the target genes of Wnt/Wingless by binding to TCF-responsive enhancers. Inappropriate activation of TCF in the colon epithelium and other cells leads to cancer. It is therefore desirable for unstimulated cells to have a negative control mechanism to keep TCF inactive. Drosophila CREB-binding protein (dCBP) binds to Drosophila TCF (Pangolin). dCBP mutants show mild Wingless overactivation phenotypes in various tissues. Consistent with this, dCBP loss-of-function suppresses the effects of armadillo mutation. Moreover, dCBP is shown to acetylate a conserved lysine in the Armadillo-binding domain of dTCF, and this acetylation lowers the affinity of Armadillo binding to dTCF. Although CBP is a coactivator of other transcription factors, these data show that CBP represses TCF (Waltzer, 1998).

The insulin-regulated CREB coactivator TORC promotes stress resistance in Drosophila

In fasted mammals, glucose homeostasis is maintained through induction of the cAMP response element-binding protein (CREB; see Drosophila CrebB-17A coactivator transducer of regulated CREB activity 2 (TORC2), which stimulates the gluconeogenic program in concert with the forkhead factor FOXO1. Starvation also triggers TORC activation in Drosophila, where it maintains energy balance through induction of CREB target genes in the brain. TORC mutant flies have reduced glycogen and lipid stores and are sensitive to starvation and oxidative stress. Neuronal TORC expression rescued stress sensitivity as well as CREB target gene expression in TORC mutants. During refeeding, increases in insulin signaling inhibited TORC activity through the salt-inducible kinase 2 (SIK2)-mediated phosphorylation and subsequent degradation of TORC. Depletion of neuronal SIK2 increased TORC activity and enhanced stress resistance. As disruption of insulin signaling also augments TORC activity in adult flies, these results illustrate the importance of an insulin-regulated pathway that functions in the brain to maintain energy balance (Wang, 2008).

Fasting triggers concerted changes in behavior, physical activity, and metabolism that are remarkably well conserved through evolution. In mammals, such responses are often coordinated by transcriptional coactivators that are themselves targets for regulation by environmental cues, but the extent to which these coactivators function in model organisms such as Drosophila is less clear (Wang, 2008).

In the basal state, mammalian TORCs are phosphorylated by salt-inducible kinases (SIKs) and sequestered in the cytoplasm via phosphorylation-dependent association with 14-3-3 proteins (Koo, 2005; Screaton, 2004). During fasting, elevations in circulating pancreatic glucagon promote TORC dephosphorylation via the protein kinase A (PKA)-mediated phosphorylation and inhibition of SIK2 (Wang, 2008).

Increases in intracellular calcium have also been found to stimulate cAMP response element-binding protein (CREB) target gene expression through the activation of calcineurin/PP2B, a calcium/calmodulin-dependent serine/threonine (Ser/Thr) phosphatase that binds directly to and dephosphorylates mammalian TORCs (Koo, 2005; Screaton, 2004). Indeed, cAMP and calcium signals stimulate TORC dephosphorylation cooperatively through their effects on SIKs and PP2B, respectively. Following their liberation from 14-3-3 proteins, dephosphorylated TORCs shuttle to the nucleus, where they mediate cellular gene expression by associating with CREB over relevant promoters (Wang, 2008).

TORC2 is thought to function in parallel with FOXO1 to maintain energy balance during fasting. Knockdown and knockout studies support a critical role for both proteins in regulating catabolic programs in the liver (Dentin, 2007, Koo, 2005; Matsumoto, 2007). In Drosophila, starvation promotes the mobilization of glycogen and lipid stores in response to increases in circulating adipokinetic hormone (AKH), the fly homolog of mammalian glucagon (Kim, 2004; Lee, 2004). In parallel, decreases in insulin/IGF signaling (IIS) also stimulate the dephosphorylation and nuclear translocation of Drosophila FOXO, which in turn stimulates a wide array of nutrient-regulated genes (Wang, 2008).

The accumulation of lipid and glycogen stores in adult flies is highly correlated with resistance to starvation in Drosophila (Djawdan, 1998). Indeed, disruption of the IIS pathway promotes lipid accumulation and correspondingly increases resistance to starvation and oxidative stress. Although FOXO does not appear to be required for starvation resistance in adult flies, overexpression of FOXO has been found to mimic the starvation phenotype in larvae (Wang, 2008).

This study addresses the importance of Drosophila TORC, the single Drosophila homolog of mammalian TORCs, in metabolic regulation. It was found that increases in TORC activity during starvation enhance survival through the activation of CREB target genes in the brain. During feeding, increases in insulin signaling inhibit TORC activity through phosphorylation by a Drosophila homolog of mammalian SIK2. These studies indicate that TORC is part of an insulin-regulated pathway that functions in concert with FOXO to promote energy balance and stress resistance (Wang, 2008).

Drosophila TORC shares considerable sequence homology with mammalian TORCs in its CREB binding domain (CBD), transactivation domain (TAD), calcineurin (Cn) recognition motif, and regulatory site (Ser157), which is phosphorylated by members of the AMPK family of stress- and energy-sensing Ser/Thr kinases in mammals. Drosophila TORC protein is expressed at low levels during larval and pupal stages, with the highest amounts detected in adults. TORC mRNA levels are also increased in adults relative to larvae, although to a lesser extent (Wang, 2008).

In the basal state, Drosophila TORC is highly phosphorylated at Ser157 and localized to the cytoplasm in Drosophila S2 and KC-167 cells. Demonstrating the importance of Ser157 phosphorylation in sequestering TORC, Ser157Ala mutant TORC shows only low-level binding to 14-3-3 proteins relative to wild-type TORC in HEK293T cells. Exposure to the adenylyl cyclase activator forskolin (FSK) or to staurosporine (STS), an inhibitor of SIKs and other protein kinases, promotes TORC dephosphorylation, liberation from 14-3-3 proteins, and nuclear translocation (Wang, 2008).

Consistent with these changes, overexpression of wild-type Drosophila TORC potentiated CRE-luciferase (CRE-luc) reporter activity following exposure of HEK293T cells to FSK, whereas phosphorylation-defective Ser157Ala TORC stimulated CRE-luc activity under basal as well as FSK-induced conditions. CRE-luc activity is blocked by coexpression of the dominant-negative CREB inhibitor ACREB. Taken together, these results indicate that Drosophila TORC modulates CREB target gene expression following its dephosphorylation at Ser157 and nuclear entry in response to cAMP (Wang, 2008).

Based on the ability of mammalian TORCs to promote fasting metabolism (Koo, 2005), this study examined whether Drosophila TORC performs a similar function in adult flies. Amounts of dephosphorylated, active TORC increased progressively during water-only starvation. Feeding adult flies paraquat, a respiratory chain inhibitor that stimulates the production of reactive oxygen species, also promoted the accumulation of dephosphorylated TORC, suggesting a broader role for this coactivator in stress resistance. Similar to mammalian TORCs, the upregulation of TORC in Drosophila appears to reflect an increase in TORC protein stability, as amounts of TORC mRNA did not change significantly in response to fasting or paraquat treatment (Wang, 2008).

Insulin signaling regulates lipid and glucose metabolism in both C. elegans and Drosophila in part by inhibiting FOXO-dependent transcription. Lipid stores are increased in flies with mutations in the IIS pathway; these animals are resistant to starvation as well as oxidative stress. This study found that TORC enhances survival during starvation in part by stimulating target gene expression in neurons. Although TORC appears to act in parallel with FOXO, the increase in FOXO activity observed in TORC mutant flies indicates that TORC may also impact on this pathway (Wang, 2008).

TORC appears to be required for the expression of genes that promote lipid and glucose metabolism, amino acid transport, and proteolysis. Consistent with this idea, paralogs for a number of TORC-regulated genes (TrxT, CAT, and UCP4c) appear to be required for starvation and oxidative stress resistance. Superimposed on these effects, neuronal TORC may also promote systemic resistance to starvation and oxidative stress by modulating the expression of neuropeptide hormones and other circulating factors that regulate peripheral glucose and lipid metabolism (Wang, 2008).

In mammals, refeeding has been found to increase SIK2 kinase activity during refeeding through the AKT-mediated phosphorylation of SIK2. Phosphorylated TORC2 is subsequently ubiquitinated and degraded through the E3 ligase COP1. Supporting a similar mechanism in Drosophila, RNAi-mediated knockdown of AKT in Drosophila is sufficient to increase TORC activity. Conversely, depletion of neuronal SIK2 enhances TORC activity and increases resistance to both starvation and paraquat feeding. Although a Drosophila homolog for COP1 has not been identified, it is imagined that the ubiquitin-dependent degradation of Drosophila TORC is also critical in modulating its activity in brain as well as other tissues (Wang, 2008).

Based on its ability to potentiate CREB target gene expression in neurons, TORC may function in other biological settings. Indeed, Drosophila CREB appears to have an important role in learning and memory, circadian rhythmicity, rest homeostasis, and addictive behavior. Future studies should reveal the extent to which TORC participates in these contexts as well (Wang, 2008).

Kinetics of doubletime kinase-dependent degradation of the Drosophila period protein

Robust circadian oscillations of the proteins Period (Per) and Timeless (Tim) are hallmarks of a functional clock in the fruit fly Drosophila melanogaster. Early morning phosphorylation of Per by the kinase Doubletime (Dbt) and subsequent Per turnover is an essential step in the functioning of the Drosophila circadian clock. Using time-lapse fluorescence microscopy, Per stability in the presence of Dbt and its short, long, arrhythmic, and inactive mutants were studied in S2 cells. Robust Per degradation was observed in a Dbt allele-specific manner. With the exception of doubletime-short (DBT^S), all mutants produce differential Per degradation profiles that show direct correspondence with their respective Drosophila behavioral phenotypes. The kinetics of Per degradation with DBT^S in cell culture resembles that with wild-type Dbt and posits that, in flies DBT^S likely does not modulate the clock by simply affecting Per degradation kinetics. For all the other tested Dbt alleles, the study provides a simple model in which the changes in Drosophila behavioral rhythms can be explained solely by changes in the rate of Per degradation (Syed, 2011).

These studies following the temporal pattern of fluorescently labeled Per and Dbt indicate that the onset of Per degradation is very tightly regulated in S2 cells and Per abundance starts to diminish typically within 3-5 h after protein induction with only ~10% of the substrate remaining at the end of the degradation process. In contrast, when TIM is coexpressed with Per, only a small fraction of the substrate is degraded because of Dbt activity while most of the Per remains bound to Tim and protected from degradation. The data show that the undegraded Per, Tim, and Dbt translocate to the nucleus ~5.5 h postinduction, suggesting that Dbt does not substantially modify timing of Per/Tim entry into the nucleus. Although the average timing of nuclear entry is unaffected by Dbt, presence of the kinase appears to reduce the translocation stochasticity observed in the cell population. It has been reported that Per/Tim nuclear entry events uniformly distribute over an interval of ~5 h. In the present studies, over 70% of cells coexpressing Dbt show Per/Tim nuclear entry within a narrower temporal window of ~3 h. This reduction in the variation of nuclear translocation is consistent with the reconstitution of an interval timer that more closely resembles the one found in vivo. Indeed, immunostaining in pacemaker neurons shows an ~2 h variation in the appearance of nuclear Per, when compared among multiple wild-type fly brains. Additionally, the Per/Tim/Dbt nuclear entry data reveal that Dbt can translocate to the nucleus 1-2 h prior to Per. These data further refine a model that was based on in vivo results with lower temporal resolution indicating that Per/Dbt nuclear accumulation occurs concurrently (Syed, 2011).

The behavioral phenotypes of a number of dbt mutants have been described previously. However, a detailed molecular description of how these mutations on dbt ultimately give rise to behavioral changes in Drosophila has been missing. This study was a thorough cell-based study aimed at elucidating the effects of Dbt mutations on Per turnover kinetics. To quantify the data, a multiple parameter-based hypothesis was formulated and the onset of Per turnover, the degradation half-life, and the fraction of the substrate that is degraded in individual cells were quantitated. Analysis of the data shows that Per half-life ranges between ~1.2 and 6.5 h, depending on the allele of the kinase that is coexpressed with the substrate. In the presence of wild-type, short-period, and long-period alleles of Dbt, Per stability appears to be independent of the substrate concentration. However, in the cells expressing only endogenous Dbt or overexpressing the catalytically compromised kinases Dbt^ar and Dbt^K38R, Per stability varies strongly with its abundance. Crucially, the substrate abundance remains steady for hours in the cells expressing high levels of Per, perhaps due to multimerization of the protein with other cellular components. The data do not reveal identity of these components but they are speculated to be Per itself or other endogenous proteins that titrate the substrate and hinder its interaction with low concentration of endogenous kinase molecules. These findings also provide a possible explanation for why several others may have concluded that singly overexpressed Per is stable in S2 cells. Because these studies sample bulk population of cells, it is likely that their measurement signal is dominated by cells expressing maximal levels of Per (equivalent of Per > 500 A.U.), the regime where single-cell measurements show that the protein is indeed in a more stable form (Syed, 2011).

Most importantly, the data permit direct comparison between kinetic parameters determined in cell culture to behavioral measurements in vivo. In particular, a plot of Per half-life in the presence of the five different alleles of Dbt and the period of daily activity from animals carrying those forms of Dbt shows a remarkable correspondence between the two quantities. A simple linear relationship emerges from this comparison with one noticeable outlier. The emergent model from that comparison suggests that alterations in the period of circadian rhythms that are observed in animals carrying the variants of Dbt can be explained mostly by the resultant changes in the rate of Per degradation (Syed, 2011).

Two previous studies addressing Dbt^S activity in Drosophila concluded that the mutation causes increased kinase activity. However, the first study did not reveal a discernible difference in Per phosphorylation or degradation when dbt was replaced by dbt^S, and data from the second study were derived from the human short-period mutant and substrate to argue about the Drosophila system. Since the latter work, it has been demonstrated that expression of mammalian casein kinase I in the fly does not rescue Drosophila dbt mutants, indicating that comparisons of mutant forms of the two proteins across these systems would be difficult. The current detailed measurements on Per degradation with Dbt^S show negligible changes on substrate stability due to the Pro-47 -> Ser mutation on the kinase (Syed, 2011).

In summary, these data indicate that several mutations of the kinase Dbt affect a common feature in the circadian clock in altering length of the period. Most of the mutations appear to modulate activity rhythm simply by changing Per stability. Deviation of the Dbt^S mutation from the proposed model raises the possibility that it likely affects period length through a mechanism other than changing the rate of Per turnover in vivo. It has been conjectured that Dbt^S might modulate Per activity as a repressor by producing a qualitatively different phosphorylation pattern of the substrate. Additionally, data has suggested that dbt^S causes early termination of per transcription, consistent with the previous finding that Per starts to decrease ~6 h earlier in dbt^S mutants compared with wild-type animals. Another possibility is that Dbt^S activity is modified by co-factors in vivo. These cofactors are missing in S2 cells but are presumably present in the nuclei of clock neurons. Regardless of the actual mechanism through which Dbt^S shortens period length, the current results suggest it is different from that of all the other variants of Dbt examined in this study (Syed, 2011).

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