InteractiveFly: GeneBrief

Hig-anchoring scaffold protein: Biological Overview | References

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 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 (Briseno-Roa, 2014). 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 (Rapti, 2011). 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 (Briseno-Roa, 2014). 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).

Search PubMed for articles about Drosophila Hasp

Briseno-Roa, L. and Bessereau, J. L. (2014). Proteolytic processing of the extracellular scaffolding protein LEV-9 is required for clustering acetylcholine receptors. J Biol Chem 289: 10967-10974. PubMed ID: 24619422

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

Rapti, G., Richmond, J. and Bessereau, J. L. (2011). A single immunoglobulin-domain protein required for clustering acetylcholine receptors in C. elegans. EMBO J 30: 706-718. PubMed ID: 21252855

date revised: 12 February 2016

Home page: The Interactive Fly © 2011 Thomas Brody, Ph.D.