hikaru genki


The matrix proteins Hasp and Hig exhibit segregated distribution within synaptic clefts and play distinct roles in synaptogenesis

The synaptic cleft is the space through which neurotransmitters convey neural information between two synaptic terminals. This space is presumably filled with extracellular matrix molecules involved in synaptic function or differentiation. However, little is known about the identities of the matrix components, and it remains unclear how these molecules organize the matrix in synaptic clefts. This study identified Hig-anchoring scaffold protein (Hasp), a Drosophila secretory protein containing CCP and WAP domains. Molecular genetic analysis revealed that Hasp diffuses extracellularly and is predominantly captured at synaptic clefts of cholinergic synapses. Furthermore, Hasp regulates levels of DLG and the nAChR subunits Dα6 and Dα7 at postsynaptic terminals. Hasp is required for trapping of another matrix protein, Hig, which is also secreted and diffused in the brain, at synaptic clefts of cholinergic synapses; however, Hig is dispensable for localization of Hasp at synaptic clefts. In addition, in the brains of triple mutants for the nAChR subunits Dα5, Dα6, and Dα7, the level of Hig, but not Hasp, was markedly reduced in synaptic regions, indicating that these nAChR subunits are required to anchor Hig to synaptic clefts. High-resolution microscopy revealed that Hasp and Hig exhibit segregated distribution within individual synaptic clefts, reflecting their differing roles in synaptogenesis. These data provide insight into how Hasp and Hig construct the synaptic cleft matrix and regulate the differentiation of cholinergic synapses, and also illuminate a previously unidentified architecture within synaptic clefts (Nakayama, 2016).

The synapse comprises presynaptic and postsynaptic terminals that are separated by a very narrow space, the synaptic cleft. Neurotransmitters traverse this extracellular space to convey neural information between the two terminals, a process that is essential for various neural functions. The synaptic cleft, which also serves as an interface that regulates the differentiation of synapses, is not simply an empty space; instead, it is filled with matrix proteins forming a scaffold that organizes membrane molecules on the synaptic terminals. To date, the matrix components in synaptic clefts have not been thoroughly identified, especially in the CNS (Nakayama, 2016).

Because synaptic function largely relies on neurotransmitter receptors localized at the postsynaptic membranes, the local density and efficiency of neurotransmitter receptors are critical for proper control of synaptic function. Previous studies showed that several proteins secreted into the extracellular space regulate clustering of neurotransmitter receptors. Agrin, found in vertebrates, is a proteoglycan that clusters AChR at neuromuscular junctions (NMJs). Multiple studies have investigated how Agrin released by motor neurons transmits the signal to various cytoplasmic proteins and eventually to AChR. In Caenorhabditis elegans, LEV-9 and OIG-4, which are released by muscles, promote clustering of AChR at NMJs. The long isoform of C. elegans Punctin/MADD-4, secreted by cholinergic motor neurons, clusters AChRs, whereas its short isoform, released by GABAergic motor neurons, clusters GABAA receptors at the NMJs. In Drosophila NMJs, which are mostly glutamatergic, clustering of glutamate receptors depends on the secreted protein Mind-the-Gap. In mice, Cbln1, which links Neurexin to the glutamate receptor GluD2 at cerebellar synapses, induces GluD2 clustering in culture cells. Thus, several secretory proteins involved in clustering receptors have been studied in cholinergic, GABAergic, and glutamatergic NMJs, as well as in glutamatergic synapses in the CNS. However, the molecular mechanisms underlying the differentiation of other types of synapses remain to be revealed. In addition, it remains unclear how the secreted proteins distribute and organize a matrix within an individual synaptic cleft (Nakayama, 2016).

Previous work identified the hikaru genki (hig) gene in a genetic screen for Drosophila mutants that exhibited reduced locomotor behavior. Hig, a secretory protein with one Ig domain and a maximum of five complement control protein (CCP) domains, localizes to the synaptic clefts of mature and nascent synapses in the brain. Hig localizes predominantly at synaptic clefts of cholinergic synapses in the CNS and regulates the levels of nAChR subunits and DLG, a Drosophila PSD-95 family member, in the postsynaptic terminals. Hig does not simply diffuse over the entire space of the synaptic cleft but, instead, is juxtaposed with the area of nAChR on the postsynaptic membrane. During synaptogenesis, Hig secreted from cholinergic or noncholinergic neurons or even from glia cells is captured in synaptic clefts of cholinergic synapses, suggesting that a specific mechanism is responsible for anchoring Hig to synaptic clefts (Nakayama, 2016).

This study identified Hasp (Hig-anchoring scaffold protein), a CCP domain-containing synaptic matrix protein predominantly localized at synaptic clefts of cholinergic synapses in the Drosophila brain. Hasp has a domain organization resembling that of LEV-9 of Caenorhabditis elegans. The data show that Hasp is required for the synaptic localization of Hig and nAChR subunits; however, Hig and nAChR subunits are not reciprocally required for Hasp localization. High-resolution microscopy revealed that Hig and Hasp are nonuniformly distributed in individual synaptic clefts, suggesting the presence of functionally distinct matrix compartments (Nakayama, 2016).

This study has revealed that Hasp, an matrix component, occupies cholinergic synaptic clefts. Both Hig and Hasp proteins contain multiple CCP domains, and the loss of either protein causes similar behavioral and molecular phenotypes, suggesting that both proteins are involved in the same process of synaptic development or function. Consistent with this, Hasp and Hig localize close to each other at cholinergic synapses. However, high-resolution imaging revealed that these proteins occupy distinct areas within synaptic clefts. These results provide novel insight into the molecular architecture of the synaptic cleft matrix in the CNS and suggest that each of the areas containing Hig or Hasp plays a distinct role in synaptogenesis (Nakayama, 2016).

Genetic analysis revealed that the roles of Hasp and Hig proteins in synaptic differentiation are not identical: although both proteins similarly affect the levels of nAChR subunits and DLG, Hasp is required for Hig to localize at the synaptic cleft, whereas Hig is dispensable for the synaptic localization of Hasp. These functional relationships raise the possibility that Hasp directly regulates the levels of nAChR subunits, as well as those of DLG, and simultaneously mediates anchoring of Hig at synapses. Alternatively, Hasp may only be involved in capture of Hig and regulates the distribution of the synaptic proteins as a secondary consequence of its main function. The data indicate that the altered levels of AChR subunits Dα6, Dα7, and DLG in hasp and hig single mutants and hasp hig double mutants are quantitatively similar, strongly suggesting that the primary role of Hasp is localizing Hig to the synaptic clefts. The close interaction between Hig and nAChR subunits was corroborated by genetic data showing that Dα5, Dα6, and Dα7 are redundantly required for localization of Hig, but not Hasp at synaptic clefts, and also by coimmunoprecipitation of Hig with Dα6 and Dα7. Thus, Hig and the nAChR subunits mutually interact for their synaptic distribution, and the physiologically important role of Hasp is localizing Hig at synaptic clefts (Nakayama, 2016).

In C. elegans, LEV-9, a Hasp homolog, LEV-10, a transmembrane protein containing CUB domains, and Oig-4, a secretory protein containing an Ig domain, are required for LAChR clustering; the absence of any of these proteins, including LAChR, causes the loss of all the other proteins on NMJs. In Drosophila, however, Hasp is localized normally at the synaptic cleft in the CNS when Hig or a subset of nAChR subunits is missing. This difference between the mechanisms underlying synaptic localization of LEV-9 and Hasp could be explained simply by evolutionary diversification among species, or alternatively by differences in synaptic architecture between NMJ and CNS synapses (Nakayama, 2016).

It has not yet been determined how Hasp localizes Hig at synaptic clefts. Hasp may either trap extracellularly diffusing Hig or prevent degradation of Hig localized at synaptic clefts. Hasp contains a WAP domain, which has been implicated in protease inhibition, implying that Hasp stabilizes Hig by preventing its degradation. However, immunoblot analysis indicated that the amounts of full-length and short form Hig polypeptides were unchanged in extracts from hasp mutants, suggesting instead that Hasp recruits Hig at synaptic clefts. Hasp and Hig occupy their respective areas, which may be completely separate or partly overlap with each other. This regional distribution suggests that a single Hasp molecule may not be sufficient to trap Hig. Rather, a number of Hasp molecules may construct a Hasp compartment, which could serve as a scaffold for Hig or a Hig-based compartment maintained within synaptic clefts. A previous study showed that C. elegans LEV-9 must be processed into fragments to cluster AChR at NMJs. Consistent with this, Hasp and Hig are processed to produce truncated polypeptides. Therefore, the patterns of Hig and Hasp staining observed in this study may represent the distribution of a mixture of Hig and Hasp fragments containing their respective N-terminal amino acid-sequences (the antigens used to raise the antibodies) and may not reflect the entire fragment distribution. Further studies are required to reveal the details of Hig and Hasp cleavage, as well as the distribution of the processed fragments in synaptic clefts (Nakayama, 2016).

Hig could regulate the accumulation of nAChR on postsynaptic membranes via either of two mechanisms. Hig has an Ig domain and a maximum of five CCP domains in its C-terminal half and the residual N-terminal half contains an RGD sequence, a putative integrin binding site. This domain organization can be used to form a scaffold complex that may physically interact with nAChR subunits and thereby either maintain clustering of the receptors on postsynaptic membranes or prevent their degradation. Alternatively, Hig may transduce signals through transmembrane proteins into the cytoplasm of postsynaptic terminals and induce clustering of nAChRs that move laterally on the membrane, as reported for Agrin-mediated AChR clustering (Nakayama, 2016).

Mutant analysis revealed that loss of Hig or Hasp resulted in an increase in the level of DLG, as well as a reduction in the levels of Dα6 and Dα7, indicating that Hig also affects the accumulation of cytoplasmic proteins in postsynaptic terminals. It is notable that PSD-95 family members in vertebrates are present at cholinergic synapses, where they function as scaffolds for AChR, as they do for glutamate receptors at glutamatergic synapses. Moreover, synaptic PSD-95 accumulation is increased by reduced synaptic activity and decreased by elevated activity via regulation of phosphorylation or palmitoylation in glutamatergic synapses. The increase of DLG in hasp mutant brains may reflect similar homeostatic regulation in the Drosophila cholinergic synapses: the reduced synaptic activity caused by the decrease in Dα6 and Dα7 levels may activate a compensatory mechanism by which DLG accumulates to a greater extent on postsynaptic membranes (Nakayama, 2016).

On the basis of the current data, a model is proposed that illustrates how the synaptic cleft matrix is constructed during synaptogenesis. During the early stages of synaptogenesis, when synaptic structures are immature, Hasp is secreted extracellularly, diffused, and trapped by an unknown molecule, occupying a particular space in the synaptic clefts of cholinergic synapses. The molecule involved in trapping Hasp may be a secretory or membrane protein localized specifically to the cholinergic synapses. During this and later stages, the Hasp-containing scaffold increases its volume by incorporating new Hasp molecules, and nAChR subunits start to accumulate on postsynaptic membranes. Following Hasp localization, secreted Hig molecules are continuously captured in the differentiating matrix architecture containing the Hasp scaffold, as well as maintained by nAChR subunits, thereby increasing the volume of the Hig-containing scaffold. Reciprocally, the Hig scaffold stabilizes nAChR subunits on the postsynaptic membranes by a physical interaction in synaptic clefts or signaling into the cytoplasm of postsynaptic terminals. In mature cholinergic synapses, the two scaffolding complexes divide synaptic clefts into compartments, reflecting their distinct roles in synaptic differentiation. To further understand the entire process of matrix construction, it will be important to identify other matrix components in the Hasp and Hig scaffold complexes, and especially the Hasp-anchoring molecules (Nakayama, 2016).

The specific localization of both Hig and Hasp at cholinergic synapses suggests that the molecular composition of synaptic matrix may be related to the type of synapse and the distinct complement of neurotransmitters and receptors. In mice, >30 genes encoding predicted CCP proteins are expressed in the CNS. One of these proteins, SRPX2, regulates the formation of glutamatergic synapses in the brain. Further work should attempt to elucidate how these CCP proteins participate in synaptogenesis and how their combinatorial repertoire is involved in the diversification of synaptic properties. Because synaptic clefts are the space through which neurotransmitters disperse, the molecular composition of the matrix may also affect the behavior of neurotransmitters, thereby influencing synaptic plasticity and the efficiency of neurotransmission. Further studies focusing on the matrix architecture of synaptic clefts may reveal novel aspects of synaptic differentiation and function (Nakayama, 2016).



Messenger RNA is detected in 8-16 hour embryos and is abundant at 16-24 hours. Expression is first detected in the head region of stage 10 embryos. Expression is observed in several cylindrical cells, some segregating from the surface in the procephalic neurogenic region. In stage 14 embryos, transcripts accumulate in several clusters of cells in each of the hemispheres of the brain and persist until later stages. In the ventral cord, expression starts at stage 11. In stage 13 embryos, a pair of cells on both sides of the midline in each segment and an additional pair on the lateral-most edge contain HIG mRNA. Slightly older embryos begin expressing mRNA in another pair of cells in each segment, located posterior to the early expressing cells. Anterior expressing cells along the midline are located ventrally and between the anterior and posterior commissures; posterior cells are behind the posterior fascicles. Based on its position relative to the fascicles, the anterior cell along the midline is one of the RP cells and the posterior cell is pCC. (See Chris Doe's Hyper-Neuroblast map site for information on identity of specific neuroblasts.) Expression does not take place in aCCs that are siblings of pCCs produced from the same precursors. At a later stage, positive cells increase in number, and by stages 16 and 17 more than 40 cells express hig in each neuromere (Hoshino, 1993).

Hikaru genki (Hig) is a putative secreted protein of Drosophila that belongs to immunoglobulin and complement-binding protein superfamilies. Previous studies have reported that, during pupal and adult stages, Hig protein is synthesized in subsets of neurons and appears to be secreted to the synaptic clefts of neuron-neuron synapses in the central nervous system (CNS). Reported here are the analyses of distribution patterns of Hig protein at embryonic and larval stages. In embryos, Hig is mainly observed in subsets of neurons of the CNS that include pCC interneurons and RP5 motorneurons. Hig is possibly localized in cholinergic neurons (e.g. many neurons in the optic lobes of the adult brain) and in histaminergic neurons (e.g. pCC neuron). Thus Hig expression is detected in various types of neurons and does not correlate with any neuronal markers examined to date (Hoshinom, 1999).

Larval and Pupal stages

mRNA levels decrease in first instar larvae and are low throughout the early pupal stage. In late pupae, it increases again (Hoshino, 1993).

In the peripheral nervous system of the third instar larva, axons containing Hig run around a body wall muscle, muscle 8, in each hemisegment. Axons innervating muscle 8 route via the transverse nerve (TN) that runs along the posterior edge of the muscle and via the SNa branch of the segmental nerve. A few axons in SNa appear to cross muscle 8 at the ventrolateral side and merge with TN vertically from the anterior side. Hig protein is found along both nerve tracts; for TN, the immunoreactivity can be traced along the nerve from muscle 7 toward muscle 8, but a strong staining associated with many varicosities is confined in a portion abutting the posterior edge of muscle 8. For SNa, staining is only found around muscle 8 but not in the region more proximal to the CNS. In addition, a lateral bipolar dendrite cell (LBD), which is located on the posterior edge of muscle 8 and sends two projections in opposite directions along TN, exhibits Hig staining in the cell body and processes. The LBD processes could not be distinguised from other axonal processes in most regions of the transvese nerve, but it is possible that LBD partly contributes to the staining of TN (Hoshino, 1999).

In the CNS of the third instar larva, cells expressing Hig were found both in the brain and ventral cord. In the brain hemispheres, a subset of cells stain for Hig. In the ventral cord, cells expressing Hig are arranged metamerically along the midline and in the lateral cortex (Hoshino, 1999).

Immunoelectron microscopic analyses have revealed the sub-cellular localization of Hig in the peripheral nervous system. In the third instar larvae, a couple of processes in or associated with TN contain numerous Hig-positive vesicles and frequently bulge with the vesicles to make varicosities along the posterior ridge of muscle 8. A few axons appear to make many NMJs on muscle 8, but only a subset of presynaptic terminals are found to be filled with vesicles heavily labelled with Hig staining. These terminals do not have widely spread subsynaptic reticulum characteristic for the type I NMJ and their morphology is rather similar to type III terminals. It should be noted that the immunoreactivity is not clearly detected in the synaptic cleft even in the NMJ stained for Hig. This staining pattern shows a striking contrast to the pattern found in the adult brain, where Hig is clearly localized in the synaptic clefts of neuron-neuron synapses but is not detected inside the synaptic terminals (Hoshino, 1999).

The localization of Hig protein was also observed in the specialized cells under the electron microscope. For instance, the LBD cell in the third instar larva, which may be a neurosecretory cell, exhibits Hig staining in a number of small dots that represent vesicles. In the ventral ganglion of adult flies, characteristic cells are occasionally observed that have a large number of dense core vesicles as well as clear vesicles in their cell bodies: subsets of the vesicles are immunoreactive to Hig antibody. The cells are morphologically different from the surrounding neurons, where a much smaller number of vesicles are present and Hig is predominantly associated with the nuclear membrane and endoplasmic reticulum/Golgi apparatus. Therefore, the vesicle-rich cells in the adult ventral ganglion seem to be specialized for neurosecretion. Hig protein thus appears to be produced by neurosecretory cells as well as neurons (Hoshino, 1999).

Loss of yata, a novel gene regulating the subcellular localization of APPL, induces deterioration of neural tissues and lifespan shortening

The subcellular localization of membrane and secreted proteins is finely and dynamically regulated through intracellular vesicular trafficking for permitting various biological processes. Drosophila Amyloid precursor protein like (APPL) and Hikaru genki (HIG) are examples of proteins that show differential subcellular localization among several developmental stages. During the study of the localization mechanisms of APPL and HIG, a novel mutant was isolated of the gene, CG1973, which was named yata. This molecule interacted genetically with Appl and is structurally similar to mouse NTKL/SCYL1, whose mutation was reported to cause neurodegeneration. yata null mutants showed phenotypes that included developmental abnormalities, progressive eye vacuolization, brain volume reduction, and lifespan shortening. Exogenous expression of Appl or hig in neurons partially rescued the mutant phenotypes of yata. Conversely, the phenotypes were exacerbated in double null mutants for yata and Appl. The subcellular localization of endogenous APPL and exogenously pulse-induced APPL tagged with FLAG was examined by immunostaining the pupal brain and larval motor neurons in yata mutants. These data revealed that yata mutants showed impaired subcellular localization of APPL. Finally, yata mutant pupal brains occasionally showed aberrant accumulation of Sec23p, a component of the COPII coat of secretory vesicles traveling from the endoplasmic reticulum (ER) to the Golgi. Thus this study identified a novel gene, yata, which is essential for the normal development and survival of tissues. Loss of yata resulted in the progressive deterioration of the nervous system and premature lethality. The genetic data showed a functional relationship between yata and Appl. As a candidate mechanism of the abnormalities, it was found that yata regulates the subcellular localization of APPL and possibly other proteins (Sone, 2009).


In adults, transcripts are most abundant in the head and absent in bodies. In the adult CNS, the optic lobes have very high levels, and weaker activity is found in cells of the central brain and ventral ganglion (Hoshino, 1993). In the brain, the laminar cells in the optic lobe and some cells in the central brain show strong protein staining. HIG protein is found both in neuropils in the internal region of the brain (in which a multitude of synapses are formed), and in the cellular cortical regions. In the ventral ganglion, stain is observed in a dotted pattern along nerves that run on the surface of the ganglion and extend to the peripheral tissues. Staining is detected along motor nerves and boutons of specific central muscles (Hoshino, 1996).

Effects of mutation or deletion

The hikaru genki locus was identified as a P element insertion, isolated using an enhancer trap screen. Homozygous mutant adults have reduced activity levels for locomotion as well as lower fertility and reduced longevity. Mutant flies remain motionless most of the time, moving only occasionally and then only slowly. Although adults die within 1-4 days after eclosion, more than half of the mutant embryos develop and survive to the adult stage. Females lay no eggs. Mutants rarely fly and never jump, but they do show grooming behavior. When placed under a strong light or in the presence of a chemical odorant, they quickly respond and move vigorously. This phenotype gives its name to the gene: in Japanese, hikaru genki means "light activated." Upon hatching, wild-type larvae elongate a significant amount during the process of escaping from the egg shell, but mutant larvae do not elongate. Mutant larvae have uncoordinated locomotion. Mutant larvae rarely move in a straight line and frequently move backward in response to stimuli that normally causes forward locomotion. Their movements are characterized by muscle contractions that are both laterally and vertically abnormal (Hoshino, 1993).

On occasion, mutant flies exhibit body and wing tremors while either standing or walking. Neural circuits of mutants show unusually frequent bursting activity, rarely observed in wild-type flies. The bursting is observed simultaneously in muscles located at both sides (Hoshino, 1996).

The matrix protein Hikaru genki localizes to cholinergic synaptic clefts and regulates postsynaptic organization in the Drosophila brain

The synaptic cleft, a crucial space involved in neurotransmission, is filled with extracellular matrix that serves as a scaffold for synaptic differentiation. However, little is known about the proteins present in the matrix and their functions in synaptogenesis, especially in the CNS. This study reports that Hikaru genki (Hig), a secreted protein with an Ig motif and complement control protein domains, localizes specifically to the synaptic clefts of cholinergic synapses in the Drosophila CNS. The data indicate that this specific localization is achieved by capture of secreted Hig in synaptic clefts, even when it is ectopically expressed in glia. In the absence of Hig, the cytoskeletal scaffold protein DLG accumulates abnormally in cholinergic postsynapses, and the synaptic distribution of acetylcholine receptor (AchR) subunits Dalpha6 and Dalpha7 significantly decreased. hig mutant flies consistently exhibited resistance to the AchR agonist spinosad, which causes lethality by specifically activating the Dalpha6 subunit, suggesting that loss of Hig compromises the cholinergic synaptic activity mediated by Dalpha6. These results indicate that Hig is a specific component of the synaptic cleft matrix of cholinergic synapses and regulates their postsynaptic organization in the CNS (Nakayama, 2014).


Hoshino, M., Matsuzaki, F., Nabeshima, Y. and Hama, C. (1993). hikaru genki, a CNS-specific gene identified by abnormal locomotion in Drosophila, encodes a novel type of protein. Neuron 10: 395-407. 8461133

Hoshino, M., et al. (1996). Hikaru genki protein is secreted into synaptic clefts from an early stage of synapse formation in Drosophila. Development 122: 589-597. PubMed Citation: 8625810

Hoshinom, M., et al. (1999). Neural expression of hikaru genki protein during embryonic and larval development of Drosophila melanogaster. Dev. Genes Evol. 209(1): 1-9. 9914413

Nakayama, M., Matsushita, F. and Hama, C. (2014). The matrix protein Hikaru genki localizes to cholinergic synaptic clefts and regulates postsynaptic organization in the Drosophila brain. J Neurosci 34: 13872-13877. PubMed ID: 25319684

Nakayama, M., Suzuki, E., Tsunoda, S. and Hama, C. (2016). The matrix proteins Hasp and Hig exhibit segregated distribution within synaptic clefts and play distinct roles in synaptogenesis. J Neurosci 36: 590-606. PubMed ID: 26758847

Sia, G. M., Clem, R. L. and Huganir, R. L. (2013). The human language-associated gene SRPX2 regulates synapse formation and vocalization in mice. Science 342(6161): 987-91. PubMed ID: 24179158

Sone, M., Uchida, A., Komatsu, A., Suzuki, E., Ibuki, I., Asada, M., Shiwaku, H., Tamura, T., Hoshino, M., Okazawa, H. and Nabeshima, Y. (2009). Loss of yata, a novel gene regulating the subcellular localization of APPL, induces deterioration of neural tissues and lifespan shortening. PLoS One 4: e4466. PubMed ID: 19209226

hikaru genki: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 10 March 2016 

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