InteractiveFly: GeneBrief

bruchpilot: Biological Overview | References

Gene name - bruchpilot

Synonyms -

Cytological map position- 2R

Function - cytoskeleton

Keywords - neuromuscular junctions, synaptogenesis

Symbol - brp

FlyBase ID: FBgn0259246

Genetic map position - 2R: 5,395,623..5,424,980 [+]

Classification - coiled-coil domain protein

Cellular location - cytoplasmic

NCBI links: Precomputed BLAST | EntrezGene
Recent literature
Sugie, A., Hakeda-Suzuki, S., Suzuki, E., Silies, M., Shimozono, M., Mohl, C., Suzuki, T. and Tavosanis, G. (2015). Molecular remodeling of the presynaptic active zone of Drosophila photoreceptors via activity-dependent feedback. Neuron [Epub ahead of print]. PubMed ID: 25892303
Neural activity contributes to the regulation of the properties of synapses in sensory systems, allowing for adjustment to a changing environment. Little is known about how synaptic molecular components are regulated to achieve activity-dependent plasticity at central synapses. This study found that after prolonged exposure to natural ambient light the presynaptic active zone in Drosophila photoreceptors undergoes reversible remodeling, including loss of Bruchpilot, DLiprin-alpha, and DRBP, but not of DSyd-1 or Cacophony. The level of depolarization of the postsynaptic neurons is critical for the light-induced changes in active zone composition in the photoreceptors, indicating the existence of a feedback signal. In search of this signal, this study has identified a crucial role of microtubule meshwork organization downstream of the divergent canonical Wnt pathway, potentially via Kinesin-3 Imac. These data reveal that active zone composition can be regulated in vivo and identify the underlying molecular machinery.

Woznicka, O., Gorlich, A., Sigrist, S. and Pyza, E. (2015). BRP-170 and BRP190 isoforms of Bruchpilot protein differentially contribute to the frequency of synapses and synaptic circadian plasticity in the visual system of Drosophila. Front Cell Neurosci 9: 238. PubMed ID: 26175667
In the first optic neuropil (lamina) of the optic lobe of Drosophila melanogaster, two classes of synapses, tetrad and feedback, show daily rhythms in the number and size of presynaptic profiles examined at the level of transmission electron microscopy (TEM). Number of tetrad presynaptic profiles increases twice a day, once in the morning and again in the evening, and their presynaptic ribbons are largest in the evening. In contrast, feedback synapses peak at night. The large scaffold protein Bruchpilot (BRP) is a major essential constituent of T-bars, with two major isoforms of 190 and 170 kD forming T-bars of the peripheral neuromuscular junctions (NMJ) synapses and in the brain. In the BRPDelta190 lacking BRP-190 there was almost 50% less tetrad synapses demonstrable than when both isoforms were present. The lack of BRP-170 and BRP-190 increased and decreased, respectively the number of feedback synapses, indicating that BRP-190 forms most of the feedback synapses. The oscillations in the number and size of presynaptic elements seem to depend on a different contribution of BRP isoforms in a presynaptic element at different time during the day and night and at various synapse types.
Sugie, A., Mohl, C., Hakeda-Suzuki, S., Matsui, H., Suzuki, T. and Tavosanis, G. (2017). Analyzing synaptic modulation of Drosophila melanogaster photoreceptors after exposure to prolonged light. J Vis Exp(120) [Epub ahead of print]. PubMed ID: 28287587
The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at the synaptic level. The Active Zone (AZ) is the region at the presynaptic membrane that mediates neurotransmitter release and is composed of a dense collection of scaffold proteins. AZs of Drosophila photoreceptors undergo molecular remodeling after prolonged exposure to natural ambient light. Thus the level of neuronal activity can rearrange the molecular composition of the AZ and contribute to the regulation of the functional output. Starting from the light exposure set-up preparation to the immunohistochemistry, this protocol details how to quantify the number, the spatial distribution, and the delocalization level of synaptic molecules at AZs in Drosophila photoreceptors. Using image analysis software, clusters of the GFP-fused AZ component Bruchpilot were identified for each R8 photoreceptor (R8) axon terminal. Detected Bruchpilot spots were automatically assigned to individual R8 axons. To calculate the distribution of spot frequency along the axon, a customized software plugin was used. Each axon's start-point and end-point were manually defined and the position of each Bruchpilot spot was projected onto the connecting line between start and end-point. Besides the number of Bruchpilot clusters, the delocalization level of Bruchpilot-GFP within the clusters was also quantified. These measurements reflect in detail the spatially resolved synaptic dynamics in a single neuron under different environmental conditions to stimuli.
Damulewicz, M., Mazzotta, G. M., Sartori, E., Rosato, E., Costa, R. and Pyza, E. M. (2017). Cryptochrome is a regulator of synaptic plasticity in the visual system of Drosophila melanogaster. Front Mol Neurosci 10: 165. PubMed ID: 28611590
Drosophila Cryptochrome (Cry) is a blue light sensitive protein with a key role in circadian photoreception. A main feature of Cry is that light promotes an interaction with the circadian protein Timeless (Tim) resulting in their ubiquitination and degradation, a mechanism that contributes to the synchronization of the circadian clock to the environment. Moreover, Cry participates in non-circadian functions such as magnetoreception, modulation of neuronal firing, phototransduction and regulation of synaptic plasticity. This study used co-immunoprecipitation, yeast 2 hybrid (Y2H) and in situ proximity ligation assay (PLA) to show that Cry can physically associate with the presynaptic protein Bruchpilot (Brp) and that Cry-Brp complexes are located mainly in the visual system. Additionally, evidence is presented that light-activated Cry may decrease Brp levels in photoreceptor termini in the distal lamina, probably targeting Brp for degradation.


Neurotransmitters are released at presynaptic active zones (AZs). In Drosophila, monoclonal antibody (MAB) nc82 specifically labels AZs. Nc82 was used to identify Bruchpilot protein (Brp), a previously unknown AZ component. BRP shows homology to human AZ protein ELKS/CAST/ERC (Note: CAST stands for cytomatrix at the active zone associated structural protein), which binds RIM1 in a complex with Bassoon and Munc13-1. The C terminus of Brp displays structural similarities to multifunctional cytoskeletal proteins. During development, transcription of bruchpilot coincides with neuronal differentiation. Panneural reduction of Brp expression by RNAi constructs permits a first functional characterization of this large AZ protein: larvae show reduced evoked but normal spontaneous transmission at neuromuscular junctions. In adults, loss of T bars at active zones, absence of synaptic components in electroretinogram, locomotor inactivity, and unstable flight (hence 'bruchpilot' - German for crash pilot), were observed. It is proposed that Brp is critical for intact AZ structure and normal-evoked neurotransmitter release at chemical synapses of Drosophila (Wagh, 2007). Further studies show that in brp mutants Ca2+ channels are reduced in density, evoked vesicle release is depressed, and short-term plasticity is altered. Brp-like proteins seem to establish proximity between Ca2+ channels and vesicles to allow efficient transmitter release and patterned synaptic plasticity (Kittel, 2006)

Neurotransmitter release during chemical communication between nerve cells takes place at synaptic active zones, specific sites characterized ultrastructurally by presynaptic membrane thickenings decorated with synaptic vesicles and surrounded by additional synaptic vesicle accumulations. Often, electron-dense projections of various shapes (plaques, pyramids, T-shaped structures, ribbons) extend from the active zone into the presynaptic cytoplasm. Considerable efforts have been undertaken in recent years to identify and functionally characterize the protein components of these projections and the cytoskeletal matrix associated with the active zone (CAZ). This complex meshwork of proteins most likely constitutes an essential part of the molecular machinery mediating neurotransmitter release. The fine regulation of this process is believed to be central to nervous system operation including higher functions such as learning, memory, and cognition (Wagh, 2007)

In vertebrates, several components associated with the presynaptic active zone have been characterized. In addition to the general cytoskeletal proteins actin and spectrin, the large protein Bassoon (420 kDa) (tom Dieck, 1998; Shapira, 2003) is specifically found at the CAZ. This protein has been shown to be required for structural active zone formation and/or maintenance. Piccolo (530 kDa) (Fenster, 2000) contains several putative protein-protein interaction domains and together with Bassoon is assumed to organize components of the active zone, including Rab3-interacting molecule (RIM1), Munc-13, and the CAZ-associated structural protein (ELKS/CAST/ERC) (Wagh, 2007)

Vertebrate ELKS/CAST/ERC proteins were identified as AZ components by purifying synaptic densities from rat brain followed by electrophoresis and mass spectrometry (Ohtsuka, 2002) and, independently, in a yeast-two-hybrid screen of a rat-brain cDNA library as proteins interacting with the RIM1α PDZ domain (Wang, 2002). Several isoforms have been reported to be transcribed from two genes (Wang, 2002; Deguchi-Tawarada, 2004). Two isoforms (CAST1/ERC2 and CAST2α/ERC1b) are brain-specific and contain several coiled-coil domains as well as a C-terminal IWA motif essential for binding the PDZ domain of RIM1 (Ohtsuka, 2002). ELKS/CAST/ERCs form large oligomeric protein complexes with the other known proteins of the CAZ (Munc-13, RIM1, Piccolo, Bassoon) and are believed to be involved in the molecular organization of presynaptic active zones (Ko, 2003) where they regulate the release of neurotransmitter (Takao-Rikitsu, 2004; Wagh, 2007 and references therein).

Although most proteins shown to be relevant for structure and/or function of the vertebrate nervous system are conserved in invertebrates, apparently no Bassoon or Piccolo homologs are encoded in the Drosophila genome. This study identified the Drosophila bruchpilot gene (brp) coding for a protein (Brp) that contains an N-terminal domain with significant sequence homology to vertebrate ELKS/CAST/ERC and a large C-terminal domain rich in coiled-coil structures similar to several cytoskeletal proteins. The structure of this gene was analyzed; the Drosophila Brp protein localizes at the presynaptic active zone. By analyzing various transgenic RNAi lines, the effects were characterized of reduction of Brp expression on synaptic ultrastructure, as well as neurotransmitter release and observe behavioral defects, including unstable flight (hence bruchpilot, crash pilot, named after an old German movie about a pilot who always crashes his planes but survives). It is speculated that Brp might combine functions of ELKS/CAST/ERC and a cytoskeletal structural protein in a single polypeptide that is highly conserved among insects (Wagh, 2007).

Over the last years, some insight into assembly and molecular composition of vertebrate presynaptic active zones has been gained. Cytoskeletal elements like actin and spectrin, as well as large active-zone-specific proteins like Piccolo and Bassoon, seem to form a structural meshwork organizing various components of the active zone. The third coiled-coil domain of Bassoon contains a motif that is highly homologous to the corresponding region of Piccolo and binds in competition to Piccolo the fifth predicted coiled-coil domain of ELKSα/CAST1. This binding of ELKSα/CAST1 to Bassoon seems to be involved in neurotransmitter release (Takao-Rikitsu, 2004). ELKSα/CAST1 itself has been shown to bind RIM1 via the C-terminal PDZ binding motif IWA (Ohtsuka, 2002). RIM1 is a target of the Rab3A small G protein (Wang, 1997) and interacts with Munc13-1. Together with vesicular proteins, this complex might control the recruitment of vesicles and regulate their subsequent fusion with the presynaptic membrane. Recently, ELKS has been shown to function in insulin exocytosis of pancreatic β cells (Ohara-Imaizumi, 2005). Deletion of the elks gene in C. elegans neither relocates RIM nor produces an obvious structural or functional phenotype, although C. elegans ELKS (Deken, 2005) like its mammalian homologs binds to the PDZ domain of RIM (Wagh, 2007)

Although there is a wealth of information on the ultrastructure of insect synapses, the molecular composition of their synaptic active zones is almost completely unknown. This work shows that in Drosophila, a protein with homology to the ELKS/CAST/ERC protein family of vertebrates localizes at the presynaptic active zones of neuronal terminals. The rather uniform distribution of tiny nc82 stained spots in all adult and larval neuropil regions suggests that Brp is present at active zones of most if not all synapses of Drosophila. Thus, the Brp protein provides an entry point to study general active-zone formation and function in this species (Wagh, 2007)

The open-reading frame of the cDNA identified by RT-PCR corresponds in size to the protein recognized by MAB nc82. The fact that the prominent Northern blot signal is about 5.5 kb larger than the known cDNA could indicate that the brp mRNA abundantly expressed in heads likely contains a long 3'UTR. Long 3'UTRs are found in several brain mRNAs (e.g., elav, fne). Possibly, the gene contains alternative polyadenylation sites giving rise to an abundant 11 kb mRNA and a less-abundant mRNA that contains the 3'end of cDNA AT09405 but is not detected in the Northern blot. This hypothesis is supported by two RT-PCR experiments with independent primer pairs downstream of the 3'end of cDNA AT09405. The signal at 2.0 kb cannot be interpreted with present cDNA information. Both signals were identically reproduced in two independent head mRNA blots hybridized with probes specific for either CG12933 and CG30336 or CG30337, or containing the entire cDNA sequence. No difference was observed with these three probes. Transcripts containing ORF CG12932, on the other hand, apparently are not abundantly expressed in adult heads and may or may not belong to the brp transcription unit (Wagh, 2007)

The combined evidence from MS analysis, bacterial cDNA expression, ectopic expression of GFP-labeled Brp, and the RNAi experiments proves that Brp represents the active-zone protein recognized by MAB nc82. Analysis of the amino acid sequence of Drosophila Brp predicts two leucine zipper domains and a glutamine-rich C terminus but no transmembrane domains. High homologies among human ELKSα, C. elegans CAST, and Brp are found in three regions of the proteins. However, no PDZ interaction motif (IWA) for RIM interaction as seen in several mammalian ELKS/CAST isoforms and, interestingly, in the C. elegans homolog seems to be present in insect Brp. No Drosophila protein is detected by BLAST analysis with conserved C-terminal sequences of worm or human. However, for the large C terminus of Brp (1260 aa) significant sequence similarities to cytoskeletal proteins such as plectin, myosin heavy chain, and restin are observed, suggesting a possible cytoskeletal role or interaction of the C terminus of Brp (Wagh, 2007)

The expression of Brp is not restricted to the glutamatergic type I boutons of the NMJ, but Brp is present in active zones of presumably all synapses. Consistently, those layers in the visual neuropil that contain high levels of either choline acetyltransferase or GABA/glutamic acid decarboxylase do not show reduced nc82 staining, and nc82 staining is found in both glutamatergic and nonglutamatergic synaptic boutons of larval NMJs. It was furthermore demonstrated that normal Brp levels are required for normal synaptic transmission at histaminergic synapses (Wagh, 2007)

This study directly address the structural, physiological, and systemic function of Brp, a member of the ELKS/CASTl/ERC family. The ultrastructure of synaptic active zones in terminals of larval motorneurons and adult photoreceptors is impaired when Brp protein levels are severely reduced. The loss of T bars in the lamina is compatible with the hypothesis that Brp may be required for anchoring the T bars to the presynaptic membrane. The fact that similar frequencies of T bars is seen in wild-type photoreceptor terminals (16.8%) and of conspicuous membrane densities in brp-RNAiC8-expressing terminals (19.5%) supports the proposition that these membrane thickenings may in fact represent presynaptic sites without typical T bars. However, it is unlikely that Brp is restricted to T bars because about 13%-37% of the active zones of type Ib boutons at NMJs of larval muscles 6/7 have no T bar, whereas in light microscopical preparations, only 3% of the active zones identified by their postsynaptic receptor fields contained no Brp label. Unfortunately, in these experiments the epitope recognized by MAB nc82 did not tolerate tissue fixation conditions required for immunoelectron microscopical analysis of T bars (Wagh, 2007)

Regarding a role of Brp in synaptic function, two RNAi lines with panneural expression were tested. Both showed a reduction in Brp protein levels and a decrease in evoked transmitter release as reflected by reduced EJC amplitude at larval NMJs, whereas miniature EJC amplitude and frequency were indistinguishable from wild-type controls (Wagh, 2007)

The genotype brp-RNAiB12/elav-Gal4 caused embryonic lethality. When Brp was suppressed only in the eye, adult brp-RNAiB12/gmr-Gal4 flies had a strong effect on eye development ('rough eye') and in some cases, in addition to the lack of ON/OFF transients, showed a much smaller ERG receptor potential than the other three lines. However, because this lethal and rough eye phenotype is observed only for a single RNAi line, it cannot be excluded that unspecific side effects might play a role. The description of the true loss-of-function phenotype will therefore have to await the generation of a genuine null mutant. A special advantage in this context is that Brp presumably is present at all synapses. Therefore, it is possible not only to asses the functionality of transgenic constructs at the electrophysiological level but also to score their effects at the behavioral level by genetically controlled, spatially, and/or temporally selective expression or suppression (Wagh, 2007)

In mammals ELKS/CAST/ERC isoforms apparently have both neuronal and nonneuronal roles. Drosophila Brp seems to correspond to the neuronal CAST isoforms, whereas the nonneuronal functions might be specific to vertebrates. Vesides the protein described here, the only published molecule localized specifically at Drosophila presynaptic active zones is the Ca2+ channel encoded by the cacophony gene. This channel seems to be responsible for providing the calcium trigger for evoked neurotransmitter release. The findings indicate, however, that the molecular structure of the presynaptic active zone might be more conserved between vertebrate and insect synapses than thought previously because of the lack of Piccolo and Bassoon homologs in insects. In the future, studying active-zone formation and function in Drosophila will be a valuable addition to similar studies in vertebrates, especially considering the powerful genetic tools available for Drosophila (Wagh, 2007)

Maturation of active zone assembly by Drosophila Bruchpilot

Synaptic vesicles fuse at active zone (AZ) membranes where Ca2+ channels are clustered and that are typically decorated by electron-dense projections. Recently, mutants of the Drosophila ERC/CAST family protein Bruchpilot (BRP) were shown to lack dense projections (T-bars) and to suffer from Ca2+ channel-clustering defects. This study used high resolution light microscopy, electron microscopy, and intravital imaging to analyze the function of BRP in AZ assembly. Consistent with truncated BRP variants forming shortened T-bars, BRP was identified as a direct T-bar component at the AZ center with its N terminus closer to the AZ membrane than its C terminus. In contrast, Drosophila Liprin-α, another AZ-organizing protein, precedes BRP during the assembly of newly forming AZs by several hours and surrounds the AZ center in few discrete punctae. BRP seems responsible for effectively clustering Ca2+ channels beneath the T-bar density late in a protracted AZ formation process, potentially through a direct molecular interaction with intracellular Ca2+ channel domains (Fouquet, 2009).

This study addressed whether BRP signals T-bar formation (without being a direct component of the T-bar) or whether the protein itself is an essential building block of this electron-dense structure. Evidence is provided that BRP is a direct T-bar component. Immuno-EM identifies the N terminus of BRP throughout the whole cross section of the T-bar, and genetic approaches show that a truncated BRP, lacking the C-terminal 30% of the protein's sequence, forms truncated T-bars. Immuno-EM and light microscopy consistently demonstrate that N- and C-terminal epitopes of BRP are segregated along an axis vertical to the AZ membrane and suggest that BRP is an elongated protein, which directly shapes the T-bar structure (Fouquet, 2009).

In brp5.45 (predicted as aa 1-866), T-bars were not detected, whereas brp1.3 (aa 1-1,389) formed T-bar-like structures, although fewer and smaller than normal. Moreover, the BRPD1-3GFP construct (1-1,226) did not rescue T-bar assembly. Thus, domains between aa 1,226 and 1,390 of BRP may also be important for the formation of T-bars. Clearly, however, the assembly scheme for T-bars is expected to be controlled at several levels (e.g., by phosphorylation) and might involve further protein components. Nonetheless, it is highly likely that the C-terminal half of BRP plays a crucial role (Fouquet, 2009).

Since BRP represents an essential component of the electron-dense T-bar subcompartment at the AZ center, it might link Ca2+ channel-dependent release sites to the synaptic vesicle cycle. Interestingly, light and electron microscopic analysis has located CAST at mammalian synapses both with and without ribbons. Overall, this study is one of the first to genetically identify a component of an electron-dense synaptic specialization and thus paves the way for further genetic analyses of this subcellular structure (Fouquet, 2009).

The N terminus of BRP is found significantly closer to the AZ membrane than the C terminus, where it covers a confined area very similar to the area defined by the CacGFP epitope. Electron tomography of frog NMJs suggested that the cytoplasmic domains of Ca2+ channels, reminiscent of pegs, are concentrated directly beneath a component of an electron-dense AZ matrix resembling ribs. In addition, freeze-fracture EM identified membrane-associated particles at flesh fly AZs, which, as judged by their dimensions, might well be Ca2+ channels. Peg-like structures were observed beneath the T-bar pedestal. Similar to fly T-bars, the frog AZ matrix extends up to 75 nm into the presynaptic cytoplasm. Based on the amount of cytoplasmic Ca2+ channel protein it has been concluded that Ca2+ channels are likely to extend into parts of the ribs. Thus, physical interactions between cytoplasmic domains of Ca2+ channels and components of ribs/T-bars might well control the formation of Ca2+ channel clusters at the AZ membrane. However, a short N-terminal fragment of BRP (aa 1-320) expressed in the brp-null background was unable to localize to AZs efficiently and consistently failed to restore Cac clustering (unpublished data) (Fouquet, 2009).

The mean Ca2+ channel density at AZs is reduced in brp-null alleles. In vitro assays indicate that the N-terminal 20% of BRP can physically interact with the intracellular C terminus of Cacaphony (Cac). Notably, it was found that the GFP epitope at the very C terminus of CacGFP was closer to the AZ membrane than the N-terminal epitope of BRP. It is conceivable that parts of the Cac C terminus extend into the pedestal region of the T-bar cytomatrix to locally interact with the BRP N terminus. This interaction might play a role in clustering Ca2+ channels beneath the T-bar pedestal (Fouquet, 2009).

Clearly, additional work will be needed to identify the contributions of discrete protein interactions in the potentially complex AZ protein interaction scheme. This study should pave the way for a genetic analysis of spatial relationships and structural linkages within the AZ organization. Moreover, the current findings should integrate in the framework of mechanisms for Ca2+ channel trafficking, clustering, and functional modulation (Fouquet, 2009).

The imaging assays allowed a temporally resolved analysis of AZ assembly in vivo. BRP is a late player in AZ assembly, arriving hours after DLiprin-α and also clearly after the postsynaptic accumulation of DGluRIIA. Accumulation of Cac was late as well, although it slightly preceded the arrival of BRP, and impaired Cac clustering at AZs lacking BRP became apparent only from a certain synapse size onwards, form at sites distant from preexisting ones and grow to reach a mature, fixed size. Thus, the late, BRP-dependent formation of the T-bar seems to be required for maintaining high Ca2+ channel levels at maturing AZs but not for initializing Ca2+ channel clustering at newly forming sites. As the dominant fraction of neuromuscular AZs is mature at a given time point, the overall impression is that of a general clustering defect in brp mutants. In reverse, it will be of interest to further differentiate the molecular mechanisms governing early Ca2+ channel clustering. Pre- to postsynaptic communication via neurexin-neuroligin interactions might well contribute to this process. A further candidate involved in early Ca2+ channel clustering is the Fuseless protein, which was recently shown to be crucial for proper Cac localization at AZs (Fouquet, 2009).

In summary, during the developmental formation of Drosophila NMJ synapses, the emergence of a presynaptic dense body, which is involved in accumulating Ca2+ channels, appears to be a central aspect of synapse maturation. This is likely to confer mature release probability to individual AZs and contribute to matching pre- and postsynaptic assembly by regulating glutamate receptor composition. Whether similar mechanisms operate during synapse formation and maturation in mammals remains an open question (Fouquet, 2009).

This study concentrated on developmental synapse formation and maturation. The question arises whether similar mechanisms to those relevant for AZ maturation might control activity-dependent plasticity as well and whether maturation-dependent changes might be reversible at the level of individual synapses. Notably, experience-dependent, bidirectional changes in the size and number of T-bars (occurring within minutes) were implied at Drosophila photoreceptor synapses by ultrastructural means. Moreover, at the crayfish NMJ, multiple complex AZs with double-dense body architecture were produced after stimulation and were associated with higher release probability. In fact, a recent study has correlated the ribbon size of inner hair cell synapses with Ca2+ microdomain amplitudes. Thus, a detailed understanding of the AZ architecture might permit a prediction of functional properties of individual AZs (Fouquet, 2009).

A high affinity RIM-binding protein/Aplip1 interaction prevents the formation of ectopic axonal active zones

Synaptic vesicles (SVs) fuse at active zones (AZs) covered by a protein scaffold, at Drosophila synapses comprised of ELKS family member Bruchpilot (BRP) and RIM-binding protein (RBP). This study demonstrates axonal co-transport of BRP and RBP using intravital live imaging, with both proteins co-accumulating in axonal aggregates of several transport mutants. RBP, via its C-terminal Src-homology 3 (SH3) domains, binds Aplip1/JIP1, a transport adaptor involved in kinesin-dependent SV transport. RBP C-terminal SH3 domains were shown in atomic detail to bind a proline-rich (PxxP) motif of Aplip1/JIP1 with submicromolar affinity. Point mutating this PxxP motif provoked formation of ectopic AZ-like structures at axonal membranes. Direct interactions between AZ proteins and transport adaptors seem to provide complex avidity and shield synaptic interaction surfaces of pre-assembled scaffold protein transport complexes, thus, favouring physiological synaptic AZ assembly over premature assembly at axonal membranes (Siebert, 2015).

Large multi-domain scaffold proteins such as BRP/RBP are ultimately destined to form stable scaffolds, characterized by remarkable tenacity and a low turnover, likely due to stabilization by multiple homo- and heterotypic interactions simultaneously. How these large and 'sticky'; AZ scaffold components engage into axonal transport processes to ensure their 'safe'; arrival at the synaptic terminal remains to be addressed. This study found that the AZ scaffold protein RBP binds the transport adaptor Aplip1 using a 'classic'; PxxP/SH3 interaction. Notably, the same RBP SH3 domain (II and III) interaction surfaces are used for binding the synaptic AZ ligands of RBP, that is, RIM and the voltage gated Ca2+ channel, though with clearly lower affinity than for Aplip1. A point mutation which disrupts the Aplip1-RBP interaction provoked a 'premature'; capture of RBP and the co-transported BRP at the axonal membrane, thus forming ectopic but, concerning T-bar shape and BRP/RBP arrangement, WT-like AZ scaffolds. The Aplip1 orthologue Jip1 has been shown to homo-dimerize via interaction of its SH3 domain. Thus, the multiplicity of interactions, with Aplip1 dimers binding to two SH3 domains of RBP as well as to KLC, might form transport complexes of sufficient avidity to ensure tight adaptor–cargo interaction and prevent premature capture of the scaffold components (Siebert, 2015).

Intravital imaging experiments showed that within axons RBP and BRP are co-transport in shared complexes together with Aplip1, whereas, despite efforts, no any co-transport of other AZ scaffold components, that is, Syd-1 or Liprin-α with BRP/RBP, were detected. In addition, STED analysis of axonal aggregates in srpk79D mutants showed BRP/RBP in stoichiometric amounts, but also failed to detect other AZ scaffold components. Moreover, BRP and RBP co-aggregated in the axoplasm of several other transport mutants tested (acsl, unc-51, appl, unc-76), consistent with both proteins entering synaptic AZ assembly from a common transport complex. Of note, during AZ assembly at the NMJ, BRP incorporation is invariably delayed compared to the 'early assembly'; phase which is driven by the accumulation of Syd-1/Liprin-α scaffolds. As the early assembly phase is, per se, still reversible, the transport of 'stoichiometric RBP/BRP complexes'; delivering building blocks for the 'mature scaffold'; might drive AZ assembly into a mature, irreversible state, and seems mechanistically distinct from early scaffold assembly mechanisms (Siebert, 2015).

Previous work suggested that AZ scaffold components (Piccolo, Bassoon, Munc-13 and ELKS) in rodent neurons are transported to assembling synapses as 'preformed complexes';, so-called Piccolo-Bassoon-Transport Vesicles (PTVs). The PTVs are thought to be co-transported with SV precursors anterogradely mediated via a KHC(KIF5B)/Syntabuli/Syntaxin-1 complex and retrogradely via a direct interaction between Dynein light chain and Bassoon. Since their initial description, however, further investigations of PTVs have been hampered by the apparent relative scarcity of PTVs, and by the lack of genetic or biochemical options for specifically interfering with their transport or final incorporation into AZs (Siebert, 2015).

A direct interaction of Aplip1 and BRP was not detected although their common transport can be uncoupled from the presence of RBP. One possible explanation could be a direct interaction of Aplip1 to other AZ proteins that are co-transported together with BRP and RBP. It is interesting that the very C-terminus of BRP is essential for SV clustering around the BRP-based AZ cytomatrix. Thus, it is tempting to speculate that adaptor/transport complex binding might block premature AZ protein/SV interactions before AZ assembly, but further analysis will have to await more atomic details than were obtained for the RBP::Aplip1 interaction (Siebert, 2015).

The down-regulation of the motor protein KHC also provoked severe axonal co-accumulations of BRP and RBP but per se should leave the adaptor protein-AZ cargo interaction intact. In contrast to aplip1, the axonal aggregations in khc mutants adapted irregular shapes most of the time, likely not representing T-bar-like structures. Thus, the data suggest a mechanistic difference when comparing the consequences between eliminating adaptor cargo interactions with a direct impairment of motor functions. Still, it cannot be excluded that trafficking of AZ complexes naturally antagonizes their ability to assemble into T-bars (Siebert, 2015).

The idea that proteins/molecules are held in an inactive state till they reach their final target has been observed in many other cell types. For example, in the context of local translation control, mRNAs are shielded or hidden in messenger ribonucleoprotein particles during transport so that they are withheld from cellular processing events such as translation and degradation. Shielding is thought to operate through proteins that bind to the mRNA and alter its conformation while at the correct time or place the masking protein is influenced by a signal that alleviates its shielding effect. As another example, hydrolytic enzymes, for example, lysosomes, are transported as proteolytically inactive precursors that become matured by proteolytic processing only within late endosomes or lysosomes. Particularly relevant in the context of AZ proteins involved in exocytosis, the Habc domain of Syntaxin-1 folds back on the central helix of the SNARE motif to generate a closed and inactive conformation which might prevent the interaction of Syntaxin-1 with other AZ proteins during diffusion (Siebert, 2015).

Previous genetic analysis of C. elegans axons forming en passant synapses suggested a tight balance between capture and dissociation of protein transport complexes to ensure proper positioning of presynaptic AZs. In this study, overexpression of the kinesin motor Unc-104/KIF1A reduced the capture rate and could suppress the premature axonal accumulations of AZ/SV proteins in mutants of the small, ARF-family G-protein Arl-8. Interestingly, large axonal accumulations in arl-8 mutants displayed a particularly high capture rate. Similarly, both aplip1 alleles exhibited enlarged axonal BRP/RBP accumulations. Thus, the capture/dissociation balance for AZ components might be shifted towards 'capture'; in these mutants, consistent with the ectopic axonal T-bar formation. It is tempting to speculate that loss of Aplip1-dependent scaffolding and/or kinesin binding provokes the exposure of critical 'sticky'; patches of scaffold components such as RBP and BRP. Such opening of interaction surfaces might increase 'premature'; interactions of cargo proteins actually destined for AZ assembly, thus increase overall size of the cargo complexes by oligomerization between AZ proteins and, finally, promote premature capture and ultimately ectopic AZ-like assembly. On the other hand, the need for the system to unload the AZ cargo at places of physiological assembly (i.e., presynaptic AZ) might pose a limit to the 'wrapping'; of AZ components and ask for a fine-tuned capture/dissociation balance (Siebert, 2015).

Several mechanisms for motor/cargo separation such as (1) conformational changes induced by guanosine-5′-triphosphate hydrolysis, (2) posttranslational modification as de/phosphorylation, or (3) acetylation affecting motor-tubulin affinity, have been suggested for cargo unloading. Notably, Aplip1 also functions as a scaffold for JNK pathway kinases, whose activity causes motor-cargo dissociation. JNK probably converges with a mitogen-activated protein kinase (MAPK) cascade (MAPK kinase kinase Wallenda phosphorylating MAPK kinase Hemipterous) in the phosphorylation of Aplip1, thereby dissociating Aplip1 from KLC. Thus, JNK signaling, co-ordinated by the Aplip1 scaffold, provides an attractive candidate mechanism for local unloading of SVs and, as shown in this study, AZ cargo at synaptic boutons. This study further emphasizes the role of the Aplip1 adaptor, whose direct scaffolding role through binding AZ proteins might well be integrated with upstream controls via JNK and MAP kinases. Intravital imaging in combination with genetics of newly assembling NMJ synapses should be ideally suited to further dissect the obviously delicate interplay between local cues mediating capturing and axonal transport with motor-cargo dissociation (Siebert, 2015).

Active zone scaffolds differentially accumulate Unc13 isoforms to tune Ca2+ channel-vesicle coupling

Brain function relies on fast and precisely timed synaptic vesicle (SV) release at active zones (AZs). Efficacy of SV release depends on distance from SV to Ca2+ channel, but molecular mechanisms controlling this are unknown. This study found that distances can be defined by targeting two unc-13 (Unc13) isoforms to presynaptic AZ subdomains. Super-resolution and intravital imaging of developing Drosophila melanogaster glutamatergic synapses revealed that the Unc13B isoform was recruited to nascent AZs by the scaffolding proteins RhoGAP100F/Syd-1 and Liprin-α, and Unc13A was positioned by Bruchpilot and Rim-binding protein complexes at maturing AZs. Unc13B localized 120 nm away from Ca2+ channels, whereas Unc13A localized only 70 nm away and was responsible for docking SVs at this distance. Unc13Anull mutants suffered from inefficient, delayed and EGTA-supersensitive release. Mathematical modeling suggested that synapses normally operate via two independent release pathways differentially positioned by either isoform. Isoform-specific Unc13-AZ scaffold interactions were identified, regulating SV-Ca2+-channel topology whose developmental tightening optimizes synaptic transmission (Bohme, 2016).

All presynaptic AZs accumulate scaffold proteins from a canonical set of few protein families, which are characterized by extended coiled-coil stretches, intrinsically unstructured regions and a few classical interaction domains, particularly PDZ and SH3 domains. These multidomain proteins collectively form a compact 'cytomatrix' often observable by electron-dense structures covering the AZ membrane, which have been found to physically contact SVs, and thus have been suggested to promote SV docking and priming as well as to recruit Ca2+ channels. Still, how the structural scaffold components (ELKS, RBP, RIM and Liprin-α) tune the functionality of the SV-release machinery has remained largely enigmatic. Liprin-α is crucial for the AZ assembly process and at Drosophila NMJ AZs, Liprin-α-Syd-1 cluster formation initializes the assembly of an 'early' scaffold complex, which subsequently guides the accumulation of a 'late' RBP-BRP scaffold complex. This study provides evidence that these scaffold complexes together operated as 'molecular rulers' that confer a remarkable degree of order, patterning AZ composition and function in space and time: the 'early' Liprin-α-Syd-1 clusters recruited Unc13B, and this scaffold served as a template to accumulate the 'late' BRP-RBP scaffold, which recruited Unc13A. Unc13 isoforms were precisely organized in the tens of nanometers range, which the data suggest to be instrumental to control SV release probability and SV-Ca2+ channel coupling. As a molecular basis of this patterning and recruitment, a multitude of molecular contacts was identified between the Unc13 N termini and the respective scaffold components using systematic Y2H analysis. As one out of several interactions, this study identified a cognate PxxP motif in the N terminus of Unc13A to interact with the second and third SH3 domains of RBP. Point mutants within the PxxP motif interfered with the binding of the RBP-SH3 domains II and III on the Y2H level but did not have a major impact on Unc13A localization and function when introduced into an Unc13 genomic transgene. Nonetheless, elimination of the scaffold components BRP and RBP on the one hand or Liprin-α on the other hand drastically impaired the accumulation of Unc13A or Unc13B. It is suggested that these results are explained by a multitude of parallel interactions that provide the avidity needed to enrich the respective Unc13 isoforms in their specific 'niches' and may cause a functional redundancy among interaction motifs, as was likely observed in the case of the Unc13A PxxP motif. Future analysis will be needed to investigate these interaction surfaces in greater detail, and address how exactly 'early' and 'late' scaffolds coordinate AZ assembly (Bohme, 2016).

Unc13 proteins have well-established functions in SV docking and priming. Accordingly, it was observed that loss of Unc13A resulted in overall reduced SV docking without affecting T-bar-tethered SVs, which is qualitatively opposite to a function of BRP in SV localization, whose C-terminal amino acids function in T-bar-tethering, but not docking. Variants lacking these residues suffer from increased synaptic depression, suggesting a role in SV replenishment. Therefore, in addition to its role in localizing Unc13A to the AZ reported here, BRP may also cooperate functionally with Unc13A by facilitating SV delivery to docking sites (Bohme, 2016).

Synapses are highly adapted to their specific features, varying widely concerning their release efficacy and short-term plasticity. These features impact information transfer and may provide neurons with the ability to detect input coherence, maintain stability and promote synchronization. Differences in the biochemical milieu of SVs can tune priming efficacy and release probability, which largely affects short-term plasticity. In the current experiments, it was found that loss of Unc13A resulted in dramatically (~90%) reduced synaptic transmission, which exceeded the (~50%) reduction in SV docking, pointing to an additional function in enhancing release efficacy. These changes were paralleled by drastically increased short-term facilitation as well as EGTA hypersensitivity and could be due to decreased Ca2+ sensitivity of the molecular release machinery, for example, mediated by different Synaptotagmin-type Ca2+ sensors, or different numbers of SNARE complexes. However, although a rightward shift was observed of the dependence of normalized release amplitudes on extracellular Ca2+ concentration at Unc13A-deficient synapses, its slope and thus Ca2+ cooperativity was unaltered, arguing against fundamentally different Ca2+-sensing mechanisms. Instead a scenario is favored in which SV Ca2+ sensing is conserved, but local Ca2+ signals at SV positions are attenuated because of their larger distances to Ca2+ channels upon loss of Unc13A. Both Unc13 isoforms were clearly segregated physically with different distances to the Ca2+ channel cluster, and loss of Unc13A selectively reduced the number of docked SVs in the AZ center. These findings are best explained by Unc13A promoting the docking and priming of SVs closer to Ca2+ channels than Unc13B. In fact, mathematical modeling reproduced the data by merely assuming release from two independent pathways with identical Ca2+ sensing and fusion mechanisms that only differed in their physical distance to the Ca2+ source in the AZ center. The distances estimated by the model were in very good agreement with the positions of the two Unc13 isoforms defined by STED microscopy. Thus, the data suggest that differences in the distance of SVs in the tens of nanometer range to the Ca2+ channels mediated by the two Unc13 isoforms likely contributed profoundly to the observed phenotypes. It is proposed that the role of the N terminus is to differentially target the isoforms into specific zones of the AZ, while the conserved C terminus confers identical docking and priming functions at both locations. Notably, recent work in Caenorhabditis elegans also characterized two Unc13 isoforms, with fast release being mediated by UNC-13L, whereas slow release required both UNC-13L and UNC-13S44. The proximity of the UNC-13L isoform to Ca2+ entry sites was mediated by the protein's N-terminal C2A-domain (not present in Drosophila) and was critical for accelerating neurotransmitter release, and for increasing/maintaining the probability of evoked release assayed by the fraction of AP- to sucrose-induced release. In contrast, the slow SV release form dominantly localized outside AZ regions. Thus it would be interesting to investigate the sub-AZ distribution of C. elegans Unc-13 isoforms and test whether the same scaffold complexes as in Drosophila mediate the localization of the different Unc-13 isoforms (Bohme, 2016).

Notable differences in short-term plasticity have been reported for mammalian Unc13 isoforms. The mammalian genome harbors five Munc13 genes. Of those, Munc13-1, -2 and -3 are expressed in the brain, and function in SV release; differential expression of Munc13 isoforms at individual synapses may represent a mechanism to control short-term plasticity. Thus, it might be warranted to analyze whether differences in the sub-active zone distribution of Munc13 isoforms contribute to these aspects of synapse diversity in the rodent brain (Bohme, 2016).

Fast and slow phases of release have recently been attributed to parallel release pathways operating in the calyx of Held of young rodents (56 nm and 135 nm) qualitatively matching the coexistence of two differentially positioned release pathways described in this study. The finding of discretely localized release pathways with distances larger than 60 nm is further in line with the recent suggestion that, at some synapses, SVs need to be positioned outside an 'exclusion zone' from the Ca2+ source (~50 nm distance to the center of the SV for the calyx of Held). At mammalian synapses, developmental changes in the coupling of SVs and Ca2+ channels have been described, which qualitatively matches the sequential arrival of loosely and tightly coupled Unc13B and Unc13A isoforms during synaptogenesis described here. Thus, this work suggests that differential positioning of Unc13 isoforms couples functional and structural maturation of AZs. To what degree modulation of this process contributes to the functional diversification of synapses is an interesting subject of future analysis (Bohme, 2016).

Nicotinamide mononucleotide adenylyltransferase maintains active zone structure by stabilizing Bruchpilot

Active zones are specialized presynaptic structures critical for neurotransmission. A neuronal maintenance factor, nicotinamide mononucleotide adenylyltransferase (NMNAT), is required for maintaining active zone structural integrity in Drosophila by interacting with the active zone protein, Bruchpilot (BRP), and shielding it from activity-induced ubiquitin-proteasome-mediated degradation. NMNAT localizes to the peri-active zone and interacts biochemically with BRP in an activity-dependent manner. Loss of NMNAT results in ubiquitination, mislocalization and aggregation of BRP, and subsequent active zone degeneration. It is proposed that, as a neuronal maintenance factor, NMNAT specifically maintains active zone structure by direct protein-protein interaction (Zang, 2013).

The findings of ubiquitinated, clustered and mislocalized BRP in loss-of-NMNAT neurons, and that increased activity leads to increased NMNAT-BRP interaction, together with the observation that active zone structure is maintained in nmnat-null neurons when neuronal activity is reduced, suggest the following model of the activity-dependent role of NMNAT in active zone maintenance. Under normal activity conditions, NMNAT is required to maintain active zone structure by interacting with BRP and to prevent the ubiquitination of BRP, inasmuch as loss of NMNAT results in BRP ubiquitination, mislocalization, aggregation and reduced active zone size. When neuronal activity is minimized (for example, by blocking light stimulation (dark rearing), or by blocking phototransduction (NorpA)), the demand on maintenance by NMNAT is reduced. These studies have revealed a specific role of NMNAT in regulating homeostasis of the active zone protein BRP. The role of NMNAT in protein-protein interaction is consistent with the chaperone function of NMNAT. Chaperones, such as CSP, have been implicated in maintaining synaptic integrity. Moreover, recent studies have shown that an elevated activity level poses stress to synaptic proteins by highlighting the effect of CSP in maintaining synaptic function. It is expected that increased neuronal and/or synaptic activity will lead to increased protein misfolding and turnover, and therefore to an increase in the load/demand of maintenance of synaptic protein homeostasis. This notion is supported by a study showing that the level of ubiquitin conjugation of synaptic proteins is altered by the level of synaptic activity. These studies describe NMNAT as a synapse maintenance factor under normal activity conditions post assembly, when most of the BRP protein is present at the active zone and NMNAT protein is localized to the active zone area to carry out its maintenance function. The interesting observation of clustered BRP protein in the cell body away from the synapse in loss-of-NMNAT neurons indicates a possible defect in the transport of BRP. Two possibilities might explain this phenotype. One, NMNAT facilitates the anterograde transport of BRP during activity. Reduced NMNAT level leads to inefficient transport and subsequent clustering of BRP in the cell body. Two, these BRP clusters are retrogradely transported from the active zone en route to degradation in the cell body. Further work will be required to determine the direction of transport. In summary, this work has identified NMNAT as a chaperone for maintaining active zones, and for facilitating their maintenance during neuronal activity by binding to active zone structural protein BRP, adding NMNAT to the list of synaptic chaperones that are required to maintain functional and structural integrity in neurons (Zang, 2013).

HDAC6 is a Bruchpilot deacetylase that facilitates neurotransmitter release

Presynaptic densities are specialized structures involved in synaptic vesicle tethering and neurotransmission; however, the mechanisms regulating their function remain understudied. In Drosophila, Bruchpilot is a major constituent of the presynaptic density that tethers vesicles. This study shows that HDAC6 is necessary and sufficient for deacetylation of Bruchpilot. HDAC6 expression is also controlled by TDP-43, an RNA-binding protein deregulated in amyotrophic lateral sclerosis (see Drosophila as a Model for Human Diseases: Amyotrophic lateral sclerosis). Animals expressing TDP-43 harboring pathogenic mutations show increased HDAC6 expression, decreased Bruchpilot acetylation, larger vesicle-tethering sites, and increased neurotransmission, defects similar to those seen upon expression of HDAC6 and opposite to hdac6 null mutants. Consequently, reduced levels of HDAC6 or increased levels of ELP3, a Bruchpilot acetyltransferase, rescue the presynaptic density defects in TDP-43-expressing flies as well as the decreased adult locomotion. This work identifies HDAC6 as a Bruchpilot deacetylase and indicates that regulating acetylation of a presynaptic release-site protein is critical for maintaining normal neurotransmission (Miskiewicz, 2014).

This study finds that HDAC6 controls vesicle tethering and synaptic transmission by regulating BRP deacetylation, thereby antagonizing ELP3, a BRP acetyltransferase (Miśkiewicz, 2011). This work defines BRP as a deacetylation target of HDAC6. Acetylation of the C-terminal end of BRP results in more condensed T-bars, while deacetylation leads the protein to send excessive tentacles into the cytoplasm to contact more synaptic vesicles. Similar to chromatin structure being regulated by electrostatic mechanisms at the level of histone acetylation, it is proposed that electrostatic interactions between acetylated and deacetylated lysines in individual BRP strands regulate presynaptic density structure and function (Miskiewicz, 2014).

While many HDAC-like proteins are present in the nucleus to deacetylate histones, HDAC6 predominantly locates to the cytoplasm, where it has been implicated in the modification of different proteins, including α-tubulin, contractin, and HSP90. In neurons, HDAC6-dependent α-tubulin deacetylation may affect axonal transport by promoting kinesin-1 and dynein binding to microtubules. However, hdac6 null mutant flies did not show overt changes in synaptic features other than T-bar morphology as gauged by electron microscopy, suggesting that axonal transport as a consequence of tubulin defects was not massively affected, although more subtle transport defects cannot be excluded (Miskiewicz, 2014).

BRP is a presynaptic density structural component important to cluster calcium channels at release sites while tethering synaptic vesicles at its C-terminal end. The regulation of BRP by HDAC6-dependent deacetylation indicates the BRP C-terminal end is important to sustain neurotransmitter release during intense (60 Hz) stimulation by orchestrating vesicle tethering. Corroborating these results, mutations in the BRP C-terminal end (brpnude) cause defects in vesicle tethering and the maintenance of release during intense 60 Hz stimulation (Hallermann et al., 2010a). Similarly brp-isoform mutations that leave calcium channel clustering intact but result in a much more condensed T-bar top show a smaller readily releasable vesicle pool, very similar to the defects when BRP is excessively acetylated. The brpnude mutation shows somewhat less severe defects to maintain synaptic transmission, possibly because more vesicles still manage to tether in these mutants during stimulation compared to the conditions that result in strong shrinking of the T-bar top. Nonetheless, the data indicate that in flies, BRP orchestrates efficient synaptic transmission during intense activity (Miskiewicz, 2014).

In the model presented in this study, ELP3 and HDAC6 antagonistically control presynaptic function. TDP-43, a gene mutated in ALS, positively regulates HDAC6 expression, and in flies, increased HDAC6 activity or expression of pathogenic TDP-43 results in the deacetylation of active zone material and increased synaptic release. Remarkably, the presence of an ALS risk-associated ELP3 allele in humans correlates with reduced ELP3 expression in ALS patient spinal cords. In flies, elp3 mutants also cause active zone deacetylation and more synaptic release. Together with genetic interactions in fruit flies, the data suggest that decreased HDAC6 function and increased ELP3 function act antagonistically, both in flies and humans. However, the target(s) on which these enzymes converge in humans remains to be discovered. In flies, the data are consistent with ELP3-dependent acetylation to occur at the C-terminal tail of the BRP protein. However, the mammalian BRP counterpart, ELKS/CAST, that resides in the presynaptic density, does not contain a long C-terminal tail. ELKS/CAST in mammals has been found to be associated with filamentous structures, and the activity to concentrate synaptic vesicles near release sites may thus be executed by binding partners of ELKS/CAST such as Picollo or Bassoon. Hence, it will be interesting to test if ELP3 and HDAC6 regulate acetylation at the much shorter ELKS/CAST tail or whether ELKS/CAST binding partners are acetylated also in the context of ALS. It is in this perspective interesting to note that another active zone-associated protein, UNC13A, is implicated in ALS as well, but the pathomechanism of how UNC13A is implicated remains to be elucidated (Miskiewicz, 2014).

Quantitative super-resolution imaging of Bruchpilot distinguishes active zone states

The precise molecular architecture of synaptic active zones (AZs) gives rise to different structural and functional AZ states that fundamentally shape chemical neurotransmission. However, elucidating the nanoscopic protein arrangement at AZs is impeded by the diffraction-limited resolution of conventional light microscopy. This study introduces new approaches to quantify endogenous protein organization at single-molecule resolution in situ with super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM). Focusing on the Drosophila neuromuscular junction (NMJ), the AZ cytomatrix (CAZ) was found to be composed of units containing ~137 Bruchpilot (Brp) proteins, three quarters of which are organized into about 15 heptameric clusters. Tests were performed for a quantitative relationship between CAZ ultrastructure and neurotransmitter release properties by engaging Drosophila mutants and electrophysiology. The results indicate that the precise nanoscopic organization of Brp distinguishes different physiological AZ states and link functional diversification to a heretofore unrecognized neuronal gradient of the CAZ ultrastructure (Ehmann, 2014).

External and circadian inputs modulate synaptic protein expression in the visual system of Drosophila melanogaster

In the visual system of Drosophila the retina photoreceptors form tetrad synapses with the first order interneurons, amacrine cells and glial cells in the first optic neuropil (lamina), in order to transmit photic and visual information to the brain. Using the specific antibodies against synaptic proteins; Bruchpilot (BRP), Synapsin (SYN), and Disc Large (DLG), the synapses in the distal lamina were specifically labeled. Then their abundance was measured as immunofluorescence intensity in flies held in light/dark (LD 12:12), constant darkness (DD), and after locomotor and light stimulation. Moreover, the levels of proteins (SYN and DLG), and mRNAs of the brp, syn, and dlg genes, were measured in the fly's head and brain, respectively. In the head, SYN and DLG oscillations were not detected. It was found, however, that in the lamina, DLG oscillates in LD 12:12 and DD but SYN cycles only in DD. The abundance of all synaptic proteins was also changed in the lamina after locomotor and light stimulation. One hour locomotor stimulations at different time points in LD 12:12 affected the pattern of the daily rhythm of synaptic proteins. In turn, light stimulations in DD increased the level of all proteins studied. In the case of SYN, however, this effect was observed only after a short light pulse (15 min). In contrast to proteins studied in the lamina, the mRNA of brp, syn, and dlg genes in the brain was not cycling in LD 12:12 and DD, except the mRNA of dlg in LD 12:12. The abundance of BRP, SYN and DLG in the distal lamina, at the tetrad synapses, is regulated by light and a circadian clock while locomotor stimulation affects their daily pattern of expression. The observed changes in the level of synaptic markers reflect the circadian plasticity of tetrad synapses regulated by the circadian clock and external inputs, both specific and unspecific for the visual system.

CK2-alpha regulates the transcription of BRP in Drosophila

Development and plasticity of synapses are brought about by a complex interplay between various signaling pathways. Typically, either changing the number of synapses or strengthening an existing synapse can lead to changes during synaptic plasticity. Altering the machinery that governs the exocytosis of synaptic vesicles, which primarily fuse at specialized structures known as active zones on the presynaptic terminal, brings about these changes. Although signaling pathways that regulate the synaptic plasticity from the postsynaptic compartments are well defined, the pathways that control these changes presynaptically are poorly described. In a genetic screen for synapse development in Drosophila, this study found that mutations in CK2α lead to an increase in the levels of Bruchpilot (Brp), a scaffolding protein associated with the active zones. Using a combination of genetic and biochemical approaches, this study found that the increase in Brp in ck2α mutants is largely due to an increase in the transcription of brp. Interestingly, the transcripts of other active zone proteins that are important for function of active zones were also increased, while the transcripts from some other synaptic proteins were unchanged. Thus, these data suggest that CK2α might be important in regulating synaptic plasticity by modulating the transcription of Brp. Hence, it is proposed that CK2α is a novel regulator of the active zone protein, Brp, in Drosophila (Wairkar, 2013).

The Bruchpilot cytomatrix determines the size of the readily releasable pool of synaptic vesicles

Synaptic vesicles (SVs) fuse at a specialized membrane domain called the active zone (AZ), covered by a conserved cytomatrix. How exactly cytomatrix components intersect with SV release remains insufficiently understood. Previous studies have shown that loss of the Drosophila ELKS family protein Bruchpilot (BRP) eliminates the cytomatrix (T bar) and declusters Ca2+ channels. This paper explores additional functions of the cytomatrix, starting with the biochemical identification of two BRP isoforms. Both isoforms alternated in a circular array and are important for proper T-bar formation. Basal transmission is decreased in isoform-specific mutants, attributable to a reduction in the size of the readily releasable pool (RRP) of SVs. A corresponding reduction was found in the number of SVs docked close to the remaining cytomatrix. It is proposed that the macromolecular architecture created by the alternating pattern of the BRP isoforms determines the number of Ca2+ channel-coupled SV release slots available per AZ and thereby sets the size of the RRP (Matkovic, 2013).

An elaborate protein cytomatrix covering the AZ membrane is meant to facilitate and control the SV release process. Quantitative analysis of neurotransmitter release has provided evidence that the number of SV release sites per AZ might be fixed. Although these sites are thought to be located in close proximity to presynaptic Ca2+ channels, ultrastructural and molecular information is largely missing here. Potentially, specific interactions between SVs and certain cytomatrix components might be involved. This study provides evidence that the BRP-based cytomatrix plays a role in defining the number of readily releasable SVs, possibly by offering morphological and molecular-determined 'release slots' (Matkovic, 2013).

Previous studies have characterized the role of BRP based on null alleles, which result in a complete absence of AZ cytomatrix (T bar), partially declustered Ca2+ channels, and likely as a direct consequence, reduced vesicular release probability. In contrast, in the analysis of BRP isoform-specific mutants, the current study neither observed any Ca2+ channel clustering deficits nor changes in vesicular release probability (Matkovic, 2013).

Previous studies have found a binding site between the intracellular C terminus of the Cac Ca2+ channel and an N-terminal stretch of BRP, which is unique to BRP-190 (Fouquet, 2009). That solely losing BRP-190 is not sufficient to affect Ca2+ channel clustering could possibly be explained by the presence of redundant binding sites within BRP-170. Ca2+ channel clustering might well be a collective feature of the cytomatrix, and Ca2+ channels likely use multiple simultaneous interactions with several cytomatrix proteins to anchor within the AZ membrane (Matkovic, 2013).

In fact, RIM-binding protein family proteins at rodent and Drosophila AZs bind Ca2+ channels, and loss of the only RIM-binding protein in Drosophila results in partial loss of Ca2+ channels from AZs. RIM-binding protein levels at AZ were slightly but significantly reduced in the BRP isoform mutants. Clearly, it remains a possibility that RIM-binding protein is a major scaffold determinant of the release slots and that e.g., subtle mislocalizations of RIM-binding protein might in part contribute to the BRP isoform mutant phenotype. The brp-null phenotype can now be interpreted as a 'catastrophic event' in which a complete loss of this large scaffold protein leads to a severe decrease of cytomatrix avidity (potentially mediated via a loss of RIM-binding protein) below a critical level, resulting in a 'collapse' of the normal cytomatrix architecture. Thus, functionalities associated with discrete regions of BRP and RIM-binding protein can apparently be masked when the BRP-based AZ scaffold is completely eliminated (Matkovic, 2013).

The distal cytomatrix in brpnude is bare of SVs in EM, and SV replenishment is defective, resulting in short-term depression (and not facilitation as in brp nulls). However, no change of short-term plasticity could be detected in the brp isoform alleles with the same analyses, consistent with neither a change in Ca2+ channel clustering nor in SV clustering at the distal cytomatrix. Nevertheless, a basal release deficit was observed, which can be explained by a reduction in the size of the readily releasable vesicle pool, assigning an additional function to the BRP cytomatrix (Matkovic, 2013).

Release-ready SVs are meant to be molecularly and positionally primed for release. Important factors are the equipment with or the attachment to the proteins of the core release machinery and the localization of the SV in proximity to the Ca2+ source. At the Drosophila NMJ, SV release is insensitive to slow Ca2+ buffers such as EGT; therefore, SVs are thought to be spatially tightly coupled to Ca2+ channels (nanodomain coupling; Eggermann, 2012). Since Ca2+ channels are found localized directly underneath the T-bar pedestal composed of the N-terminal region of BRP (Fouquet, 2009), release-ready SVs might well correspond to the SVs that were found docked at the pedestal of the T bar and thus in very close proximity to the Ca2+ channels. This in turn is in agreement with BRP itself being important for defining the number of release-ready SVs determined by electrophysiology and EM (Matkovic, 2013).

Light microscopic inspection of an AB directed against the C terminus of BRP, common to both isoforms, with 50-nm STED resolution, typically revealed approximately five dots arranged as a circle or regular pentagon. Both isoforms were labelled individually, and it was found that (1) both isoforms seem to localize with their C termini similarly toward the distal edge of the cytomatrix and (2) both isoforms typically form an identical number of dots per AZ similar to the number of dots observed with the BRPC-Term AB recognizing both isoforms. Thus, the BRP isoforms seem to be arranged in neighboring but not overlapping clusters, forming a circular array. Consistent with both BRP isoforms not overlapping in space, there was neither efficient co-IP between them nor did elimination of one isoform substantially interfere with the AZ localization of the respective other isoform. Thus, BRP-190 and -170 seem to form discrete oligomers. The alternating pattern of BRP-190 and -170 appears to set a typical cytomatrix size, as both isoform mutants had a reduced T-bar width in EM and a reduced mean number of BRP dots per AZ. As this corresponded with a similar reduction in the number of SVs in the RRP, this AZ architecture could set a typical number of Ca2+ nanodomain-coupled RRP slots possibly located between BRP clusters. However, beyond providing a discrete morphological architecture, the two BRP isoforms described in this study might harbor additional functionalities. The brpΔ190 phenotype was more pronounced than the brpΔ170, leaving the possibility that the highly conserved N terminus of BRP-190 promotes release by further mechanisms going beyond the points analyzed in this study. Future analysis will also have to address whether localization and regulation of additional cytomatrix and release components, such as RIM-binding protein, Unc-13 family proteins, or RIM, contribute to the formation of release slots as well (Matkovic, 2013).

Ultimately, functional differences between individual synaptic sites must be defined by variances in their molecular organization. Functional features of a synapse can be extracted electrophysiologically. Thereby, the number of Ca2+ channels was recently identified as a major determinant of the release probability of single vesicles, Pvr, in rat calyces (Sheng, 2012). Furthermore, AZ size seems to scale with the overall likelihood of release from a given AZ (Holderith, 2012). The current results suggest that the BRP-based cytomatrix should be a general determinant of the release likelihood per AZ by establishing Pvr, through Ca2+ channel clustering, as shown previously, and, as shown in this study, by determining the size of the RRP. The genetic results show that the cytomatrix can, in principle, control the RRP size independent of Ca2+ channel clustering. A coupled increase in the size of the T-bar cytomatrix together with increasing SV release was previously observed at NMJs compensating for loss of the glutamate receptor subunit glurIIA. Moreover, an increase in the number of release-ready SVs together with an increase in the amount of BRP was recently described as part of a homeostatic presynaptic response after pharmacological block of postsynaptic GluRIIA (Weyhersmuller, 2011). In line with this scenario, it was recently shown that lack of acetylation of BRP in elp3 mutants led to an increase in the complexity of the AZ cytomatrix along with an increase in RRP size (Miskiewicz, 2011). Furthermore, in vivo imaging of synaptic transmission with single synapse resolution revealed that the likelihood of release correlates with the amount of BRP present at an individual AZ (Peled, 2011). This cytomatrix size-SV release scaling might be a general principle, as a correlation between the amount of SV exocytosis, measured by an optical assay, and the amount of the AZ protein Bassoon at individual synapses of cultured rat hippocampal neurons has also been observed (Matz, 2010). The current results suggest that not only the mere size, but also the distinct architecture of the cytomatrix influence release at individual synapses through determining RRP size (Matkovic, 2013).

Unc-51 controls active zone density and protein composition by downregulating ERK signaling

Efficient synaptic transmission requires the apposition of neurotransmitter release sites opposite clusters of postsynaptic neurotransmitter receptors. Transmitter is released at active zones, which are composed of a large complex of proteins necessary for synaptic development and function. Many active zone proteins have been identified, but little is known of the mechanisms that ensure that each active zone receives the proper complement of proteins. This study used a genetic analysis in Drosophila to demonstrate that the serine threonine kinase Unc-51 (see Atg1) acts in the presynaptic motoneuron to regulate the localization of the active zone protein Bruchpilot opposite to glutamate receptors at each synapse. In the absence of Unc-51, many glutamate receptor clusters are unapposed to Bruchpilot, and ultrastructural analysis demonstrates that fewer active zones contain dense body T-bars. In addition to the presence of these aberrant synapses, there is also a decrease in the density of all synapses. This decrease in synaptic density and abnormal active zone composition is associated with impaired evoked transmitter release. Mechanistically, Unc-51 inhibits the activity of the MAP kinase ERK to promote synaptic development. In the unc-51 mutant, increased ERK activity leads to the decrease in synaptic density and the absence of Bruchpilot from many synapses. Hence, activated ERK negatively regulates synapse formation, resulting in either the absence of active zones or the formation of active zones without their proper complement of proteins. The Unc-51-dependent inhibition of ERK activity provides a potential mechanism for synapse-specific control of active zone protein composition and release probability (Wairkar, 2009).

A large-scale anatomical screen was performed to identify mutants where not every glutamate receptor cluster is apposed to Bruchpilot. Mutants with a global decrease in Brp or DGluRIII across the NMJ were put aside, and instead focus was placed on mutants in which Brp was absent from a subset of synapses. Such mutants were identified by the presence of glutamate receptor clusters unapposed to Bruchpilot puncta. In this screen, mutants were identified in unc-51 (Wairkar, 2009).

In the unc-51 mutant many DGluRIII clusters are unappposed to Brp. Such misapposition could reflect either DGluRIII clusters unapposed to active zones, or receptor clusters apposed to abnormal active zones that do not contain Brp. The ideal experiment to distinguish between these possibilities would be to stain for other presynaptic active zone proteins. Unfortunately the only other such protein that can be visualized in Drosophila is the calcium channel Cacophony, and since its localization depends on Brp this experiment is not be informative. Nonetheless, two results strongly suggest that a subset of glutamate receptors is apposed to abnormal active zones. First, the decreased density of DGluRIII clusters observed via confocal microscopy approximates the decrease in active zone density observed via electron microscopy. If many DGluRIII clusters were unapposed to active zones, then a more dramatic decrease in active zone density would be expected. Second, ultrastructural analysis demonstrates a decrease in the proportion of active zones containing T-bars. Brp is not necessary for the formation of active zones, but is required for the localization of T-bars to active zones. If the absence of Brp were due to the absence of the entire active zone, then each active zone would contain Brp and a normal ratio of T-bars/active zones would be predicted. Instead, the decrease in T-bars/active zone is consistent with the presence of active zones missing Brp and, hence, lacking T-bars. Therefore, it is concluded that Unc-51 is required for the high fidelity of active zone assembly, ensuring that Brp is present at every active zone (Wairkar, 2009).

In addition to the presence of abnormal synapses in the unc-51 mutant, there is also a decrease in the number and density of synapses. It is speculated that the decrease in synaptic density and the presence of abnormal synapses may be related phenotypes that differ in severity. In this view, Unc-51 promotes synapse formation. In its absence, active zone assembly would be less efficient, resulting in either the formation of abnormal active zones missing crucial proteins such as Brp, or in more severe cases leading to complete failure of active zone assembly and, hence, the absence of a synapse. The complete suppression of both the synaptic density and apposition phenotypes by mutation of the downstream target ERK is consistent with these phenotypes sharing an underlying mechanism. As expected, this defect in the number and proper assembly of synapses leads to a dramatic decrease in synaptic efficacy (Wairkar, 2009).

In addition to these synaptic defects, the unc-51 mutant also has a smaller NMJ and accumulations of synaptic material in the axons, suggesting defects in axonal transport. One mechanism that could link a small NMJ with defective transport is synaptic retraction, in which entire presynaptic boutons or branches retract leaving a footprint of postsynaptic proteins. However, no such footprints were observed in the unc-51 mutant, so this is not the cause of the small NMJ. Synaptic growth requires the retrograde transport of a BMP signal to the nucleus, however this study no change in the levels of phosphorylated MAD in motoneuron nuclei, suggesting that this is not a likely cause of the growth defect. Finally, in worms and mice Unc-51 is required for axon outgrowth, which may be somewhat analogous to defects in NMJ growth in Drosophila. However, to form an NMJ the axon must navigate out of the ventral nerve cord and cross a wide expanse of muscle before reaching its target and forming a junction. Since no defects were observed in the pattern of neuromuscular innervation, it is unlikely that a generic defect in axon outgrowth is responsible for the small NMJs. The apparent axonal transport defect is consistent with findings from mammals suggesting a function for Unc-51 in regulating axon transport. The role of Unc-51 for transport was not investigated, but note that it was possible to genetically separate the axonal transport and synapse development phenotypes, so the transport phenotypes may not be primary cause of the synaptic defects (Wairkar, 2009).

These data support the model that Unc-51 inhibits ERK activation to promote proper active zone development. In the unc-51 mutant a modest increase was observed in the levels of activated ERK, demonstrating that Unc-51 is a negative regulator of ERK activation in vivo. This increased ERK activity is responsible for the defects in active zone formation. Double mutants between unc-51 and the ERK hypomorph rl1 completely suppress the synapse density and apposition phenotypes of the unc-51 mutant, and restore synaptic strength to wild type levels. Hence, ERK is required for the synaptic phenotypes observed in the unc-51 mutant. The axonal transport defects were not suppressed in the double mutant, so Unc-51 must act through other pathways as well. In mammalian cells Unc-51 can downregulate ERK by inhibiting the binding of a scaffolding protein to the FGF receptor. To date, no receptor tyrosine kinase has been identified that regulates active zone formation in Drosophila. Future studies to characterize the mechanism by which Unc-51 inhibits ERK in Drosophila motoneurons may provide clues towards identification of such a pathway. In addition, it is unclear how ERK regulates active zone formation. A previous study demonstrated that phospho-ERK localizes to the active zone, which would suggest a direct mechanism. Unfortunately, these localization findings could not be replicated. The same study demonstrated that the transgenic expression of a constitutively active ras or a gain-of-function ERK allele both lead to an increase in the number of synaptic boutons, which is not consistent with the current finding of a smaller NMJ. Active zone structure and number were not assessed. It is speculated that the global activation of ERK may result in different phenotypes than relief of Unc-51 inhibition of ERK, which could show temporal and spatial specificity (Wairkar, 2009).

In mammalian and Drosophila neurons, release probability varies across release sites formed by a single neuron. One potential mechanism would be the differential localization or activity of core active zone proteins. In Drosophila, Bruchpilot is an excellent candidate for such a protein. It is required for the localization of calcium channels to the active zone, so changes in its localization or function would impact calcium influx and, hence, release probability at an active zone. The unc-51 mutant demonstrates that signaling pathways can differentially regulate the localization of Brp to individual release sites within a single neuron. As such, the Unc-51/Erk signaling pathway is a candidate mechanism to regulate active zone protein composition and release probability in a synapse-specific manner (Wairkar, 2009).

A Syd-1 homologue regulates pre- and postsynaptic maturation in Drosophila

Active zones (AZs) are presynaptic membrane domains mediating synaptic vesicle fusion opposite postsynaptic densities (PSDs). At the Drosophila neuromuscular junction, the ELKS family member Bruchpilot (BRP) is essential for dense body formation and functional maturation of AZs. Using a proteomics approach, Drosophila Syd-1 (DSyd-1: RhoGAP100F), homolog of Syd-1 (synapse defective 1), a multidomain RhoGAP-like protein, that is required for C. elegans HSNL synapse assembly (Dai, 2006; Patel, 2006). was identified as a BRP binding partner. In vivo imaging shows that DSyd-1 arrives early at nascent AZs together with DLiprin-alpha, and both proteins localize to the AZ edge as the AZ matures. Mutants in dsyd-1 form smaller terminals with fewer release sites, and release less neurotransmitter. The remaining AZs are often large and misshapen, and ectopic, electron-dense accumulations of BRP form in boutons and axons. Furthermore, glutamate receptor content at PSDs increases because of excessive DGluRIIA accumulation. The AZ protein DSyd-1 is needed to properly localize DLiprin-alpha at AZs, and seems to control effective nucleation of newly forming AZs together with DLiprin-alpha. DSyd-1 also organizes trans-synaptic signaling to control maturation of PSD composition independently of DLiprin-alpha (Owald, 2010).

Mechanisms which regulate assembly and maturation of presynaptic AZs are not well understood. This study identified the Drosophila Syd-1 homologue (DSyd-1) as a binding partner of BRP. DLiprin-α and DSyd-1 mark presynaptic sites where, subsequently, AZs (and adjunct PSDs) originate and mature, whereas BRP and Ca2+ channels accumulate at later time points than DLiprin-α and DSyd-1. DLiprin-α previously has been shown to be important for proper AZ formation. Thus, consistent with reduced numbers of AZs forming at NMJs of dsyd-1 and dliprin-α mutants and with both proteins being localized to AZs, the accumulation of DLiprin-α and DSyd-1 at nascent AZs may be instrumental for transforming selected sites into AZs, a process referred to as 'AZ nucleation activity.' However, as the morphological size of dsyd-1 NMJs is reduced, as is the AZ number, in principle, other growth processes might also become rate-limiting at dsyd-1 mutant NMJs. In other words, reduced AZ numbers could also be a consequence of a reduction in morphological NMJ growth. Studying the coupling between morphological growth and AZ formation will be important for determining the relevance of morphological size to total AZ number (Owald, 2010).

Work on en passant synapses of the C. elegans HSNL motor neuron implies that, in genetic terms, Syd-1 operates upstream of Syd-2/Liprin-α. This is based on the fact that a Syd-2/Liprin-α; dominant allele can bypass the requirement of syd-1, which indicates that the protein's essential role in AZ assembly at HSNL synapses is mediated via Syd-2/Liprin-α. This study provides evidence that DSyd-1 is required to properly target DLiprin-α to AZs. In the absence of DSyd-1, DLiprin-α distributes unevenly at NMJ terminals, sparing many AZs. Thus, direct evidence is provided that the RhoGAP DSyd-1 operates upstream in AZ assembly in vivo: DSyd-1 seemingly stalls DLiprin-α to developing AZs in order to allow for the AZ nucleation function of DLiprin-α to effectively operate (Owald, 2010).

DLiprin-α seems to be a direct substrate of DSyd-. The data imply that other presynaptic substrate proteins of DSyd-1 might exist at nascent synapses, a finding that is unexpected based on analysis of AZ formation in C. elegans. Therefore, it is deduced from these findings that presynaptic DSyd-1 (but apparently not DLiprin-α) plays an important role in shaping the PSD assembly. Embryos and larvae mutants for dsyd-1, and importantly, dliprin-α; dsyd-1 double mutant embryos (the double mutant is embryonic lethal), showed increased overall amounts of postsynaptic GluRs, whereas dliprin-α single mutant embryos and larvae did not. These increased amounts of GluRs in dsyd-1 mutants vanished after presynaptic reexpression of UAS–dsyd-1cDNA. It is tempting to speculate that the presynaptic DSyd-1 protein helps the AZ localization of an adhesion protein, which via trans-synaptic interaction might steer the incorporation of postsynaptic GluRs. A potential role of the Neurexin–Neuroligin axis should be evaluated in this context (Owald, 2010).

Drosophila NMJs express two functionally distinct GluR complexes, DGluRIIA and IIB, which influence the number of release sites formed. Individual PSDs form distinctly from preexisting ones, and mature over hours, switching from DGluRIIA to IIB incorporation throughout maturation in a manner dependant on presynaptic signaling. DSyd-1 might mediate such a maturation signal, as dsyd-1 mutants show excessive amounts of DGluRIIA incorporation at PSDs. This regulation is likely not (or only partially) due to compensation for reduced presynaptic glutamate release, as dliprin-α mutants (with similarly reduced transmission levels) do not show this dramatic increase in GluR levels (Owald, 2010).

Despite enlarged receptor fields and specifically elevated DGluRIIA levels, average miniature event amplitudes were comparable between dsyd-1 animals and controls, which currently cannot be accounted for. A possible explanation might comprise regulatory processes rendering populations of receptors non-/partially functional. Nonetheless, EJC decay time constants of dsyd-1 mutants resemble those found at dgluRIIB-deficient (and thus GluRIIA dominated) NMJs (Owald, 2010).

Which processes are downstream of the DSyd-1–mediated DLiprin-α activity at nascent AZs? Liprin family proteins steer transport in axons and dendrites (e.g., of AMPA receptors) to support synaptic specializations. Notably, in dsyd-1 mutants, although many AZs lacked proper amounts of DLiprin-α, large ectopic accumulations of DLiprin-α were observed. At the same time, ectopic accumulations of BRP/electron density were observed in the absence of DSyd-1. It is tempting to speculate that these ectopic pools of DLiprin-α provoke the aberrant accumulation of electron densities in dsyd-1 mutants, which is consistent with the transport function of DLiprin-α and the direct interaction of DLiprin-α/Syd-2 and ELKS/BRP. Consistently, large BRP accumulations observed in dsyd-1 embryos were no longer present in dsyd-1; dliprin-α double mutants, which indicates that the presence of DLiprin-α is needed to provoke these overaccumulations of BRP when DSyd-1 is missing (Owald, 2010).

In the absence of DSyd-1, BRP was inappropriately localized, even within the cytoplasm, forming ectopic electron-dense material (which is consistent with its role as building block for the electron-dense T bars). Such 'precipitates' also occurred at and close to non-AZ membranes. Moreover, at dsyd-1 AZs, large malformed T bars formed. Thus, it appears plausible that DSyd-1 keeps BRP 'in solution' to organize its proper consumption at AZs. An alternate and not mutually exclusive explanation may be that axonal BRP precipitates also reflect defects in axonal transport due to the absence of DSyd-1. The presence of several binding interfaces between BRP and DSyd-1 may be considered as a basis for regulating their interplay (Owald, 2010).

BRP accumulation in the center of the AZ is also in the center of the functional and structural AZ assembly process. It appears likely that BRP assembly is regulated on multiple levels. Notably, although BRP accumulation is severely compromised in mutants for the kinesin imac, it is not fully eliminated. Moreover, the serine/arginine protein kinase SRPK79D was recently shown to associate with BRP and to repress premature 'precipitation' of BRP in the axons. Furthermore, mutants for the serine/threonine kinase unc51 have recently been shown to suffer from BRP targeting defects. Phosphorylation of DSyd-1 (e.g., within serine-rich stretches toward the C terminus) might be involved in regulating proper longer-range transport ('blocking precipitation on the way') as well as proper delivery of BRP at nascent AZ sites (Owald, 2010).

Recently, the Rab3 GTPase has been shown to be crucial for effective nucleation of BRP at AZs (Graf, 2009). In an interesting parallel to dsyd-1 defects, rab3 mutant NMJs showed fewer BRP-positive AZs; however, if present, BRP levels were increased. Nonetheless, instead of overgrown T bars, as observed in dsyd-1 mutants, rab3 mutants rather showed multiple T bar AZs (Graf, 2009). It will be interesting to investigate whether these pathways act in parallel or converge, along with their relationships to other synaptogenic signals (Owald, 2010).

N-glycosylation requirements in neuromuscular synaptogenesis

Neural development requires N-glycosylation regulation of intercellular signaling, but the requirements in synaptogenesis have not been well tested. All complex and hybrid N-glycosylation requires MGAT1 (UDP-GlcNAc:alpha-3-D-mannoside-beta1,2-N-acetylglucosaminyl-transferase I) function, and Mgat1 nulls are the most compromised N-glycosylation condition that survive long enough to permit synaptogenesis studies. At the Drosophila neuromuscular junction (NMJ), Mgat1 mutants display selective loss of lectin-defined carbohydrates in the extracellular synaptomatrix, and an accompanying accumulation of the secreted endogenous Mind the gap (MTG) lectin, a key synaptogenesis regulator. Null Mgat1 mutants exhibit strongly overelaborated synaptic structural development, consistent with inhibitory roles for complex/hybrid N-glycans in morphological synaptogenesis, and strengthened functional synapse differentiation, consistent with synaptogenic MTG functions. Synapse molecular composition is surprisingly selectively altered, with decreases in presynaptic active zone Bruchpilot (BRP) and postsynaptic Glutamate receptor subtype B (GLURIIB), but no detectable change in a wide range of other synaptic components. Synaptogenesis is driven by bidirectional trans-synaptic signals that traverse the glycan-rich synaptomatrix, and Mgat1 mutation disrupts both anterograde and retrograde signals, consistent with MTG regulation of trans-synaptic signaling. Downstream of intercellular signaling, pre- and postsynaptic scaffolds are recruited to drive synaptogenesis, and Mgat1 mutants exhibit loss of both classic Discs large 1 (DLG1) and newly defined Lethal (2) giant larvae [L(2)gl] scaffolds. It is concluded that MGAT1-dependent N-glycosylation shapes the synaptomatrix carbohydrate environment and endogenous lectin localization within this domain, to modulate retention of trans-synaptic signaling ligands driving synaptic scaffold recruitment during synaptogenesis (Parkinson, 2013).

This study began with the hypothesis that disruption of synaptomatrix N-glycosylation would alter trans-synaptic signaling underlying NMJ synaptogenesis (Dani, 2012). MGAT1 loss transforms the synaptomatrix glycan environment. Complete absence of the HRP epitope, α1-3-fucosylated N-glycans, is expected to require MGAT1 activity: key HRP epitope synaptic proteins include fasciclins, Neurotactin and Neuroglian, among others. This study shows that HRP epitope modification of the key synaptogenic regulator Fasciclin 2 is not required for stabilization or localization, suggesting a role in protein function. However, complete loss of Vicia villosa (VVA) lectin reactivity synaptomatrix labeling is surprising because the epitope is a terminal β-GalNAc. This result suggests that the N-glycan LacdiNAc is enriched at the NMJ, and that the terminal GalNAc expected on O-glycans/glycosphingolipids may be present on N-glycans in this synaptic context. Importantly, VVA labels Dystroglycan and loss of Dystroglycan glycosylation blocks extracellular ligand binding and complex formation in Drosophila, and causes muscular dystrophies in humans. This study shows that VVA-recognized Dystroglycan glycosylation is not required for protein stabilization or synaptic localization, but did not test functionality or complex formation, which probably requires MGAT1-dependent modification. Conversely, the secreted endogenous lectin MTG is highly elevated in Mgat1 null synaptomatrix, probably owing to attempted compensation for complex and hybrid N-glycan losses that serve as MTG binding sites. MTG binds GlcNAc in a calcium-dependent manner and pulls down a number of HRP-epitope proteins by immunoprecipitation (Rushton, 2012), although the specific proteins have not been identified. It will be of interest to perform immunoprecipitation on Mgat1 samples to identify changes in HRP bands. Importantly, MTG is crucial for synaptomatrix glycan patterning and functional synaptic development. MTG regulates VVA synaptomatrix labeling, suggesting a mechanistic link between the VVA and MTG changes in Mgat1 mutants. The MTG elevation observed in Mgat1 nulls provides a plausible causative mechanism for strengthened functional differentiation (Parkinson, 2013).

Consistent with recent glycosylation gene screen findings (Dani, 2012), Mgat1 nulls exhibit increased synaptic growth and structural overelaboration. Therefore, complex and hybrid N-glycans overall provide a brake on synaptic morphogenesis, although individual N-glycans may provide positive regulation. Likely players include MGAT1-dependent HRP-epitope proteins (e.g., fasciclins, Neurotactin, Neuroglian), and position-specific (PS) integrin receptors and their ligands, all of which are heavily glycosylated and have well-characterized roles regulating synaptic architecture. An alternative hypothesis is that Mgat1 phenotypes may result from the presence of high-mannose glycans on sites normally carrying complex/hybrid structures, suggesting possible gain of function rather than loss of function of specific N-glycan classes. NMJ branch and bouton number play roles in determining functional strength, although active zones and GluRs are also regulated independently. Thus, the increased functional strength could be caused by increased structure at Mgat1 null NMJs. However, muscle-targeted UAS-Mgat1 rescues otherwise Mgat1 null function, but has no effect on structural defects, demonstrating that these two roles are separable. Presynaptic Mgat1 RNAi also causes strong functional defects, showing there is additionally a presynaptic requirement in functional differentiation. Neuron-targeted Mgat1 causes lethality, indicating that MGAT1 levels must be tightly regulated, but preventing independent assessment of Mgat1 presynaptic rescue of synaptogenesis defects (Parkinson, 2013).

Presynaptic glutamate release and postsynaptic glutamate receptor responses drive synapse function. Using lipophilic dye to visualize SV cycling, this study found Mgat1 null mutants endogenously cycle less than controls, but have greater cycling capacity upon depolarizing stimulation. The endogenous cycling defect is consistent with the sluggish locomotion of Mgat1 mutants, whereas the elevated stimulation-evoked cycling is consistent with electrophysiological measures of neurotransmission. Similarly, mutation of dPOMT1, which glycosylates VVA-labeled Dystroglycan, decreases SV release probability (Wairkar, 2008), although dPOMT1 adds mannose not GalNAc. Null Mgat1 mutants display no change in SV cycle components (e.g. Synaptobrevin, Synaptotagmin, Synaptogyrin, etc.), but exhibit reduced expression of the key active zone component Bruchpilot. Other examples of presynaptic glycosylation requirements include the Drosophila Fuseless (FUSL) glycan transporter, which is critical for Cacophony (CAC) voltage-gated calcium channel recruitment to active zones, and the mammalian GalNAc transferase (GALGT2), whose overexpression causes decreased active zone assembly. Postsynaptically, Mgat1 nulls show specific loss of GLURIIB-containing receptors. Similarly, dPOMT1 mutants exhibit specific GLURIIB loss (Wairkar, 2008), although dystroglycan nulls display GLURIIA loss. Selective GLURIIB loss in Mgat1 nulls may drive increased neurotransmission owing to channel kinetics differences in GLURIIA versus GLURIIB receptors (Parkinson, 2013).

Bidirectional trans-synaptic signaling regulates NMJ structure, function and pre/postsynaptic composition. This intercellular signaling requires ligand passage through, and containment within, the heavily glycosylated synaptomatrix, which is strongly compromised in Mgat1 mutants. In testing three well-characterized signaling pathways, this study found that Wingless (Wg) accumulates, whereas both GBB and JEB are reduced in the Mgat1 null synaptomatrix. WG has two N-glycosylation sites, but these do not regulate ligand expression, suggesting WG build-up occurs owing to lost synaptomatrix N-glycosylation. Importantly, WG overexpression increases NMJ bouton formation similarly to the phenotype of Mgat1 nulls, suggesting a possible causal mechanism. GBB is predicted to be N-glycosylated at four sites, but putative glycosylation roles have not yet been tested. Importantly, GBB loss impairs presynaptic active zone development similarly to Mgat1 nulls, suggesting a separable causal mechanism. JEB is not predicted to be N-glycosylated, indicating that JEB loss is caused by lost synaptomatrix N-glycosylation. Importantly, it has been shown that loss of JEB signaling increases functional synaptic differentiation similarly to Mgat1 nulls (Rohrbough, 2013). In addition, jeb mutants exhibit strongly suppressed NMJ endogenous activity, similarly to the reduced endogenous SV cycling in Mgat1 nulls. Moreover, the MTG lectin negatively regulates JEB accumulation in NMJ synaptomatrix, consistent with elevated MTG causing JEB downregulation in Mgat1 nulls (Parkinson, 2013).

Trans-synaptic signaling drives recruitment of scaffolds that, in turn, recruit pre- and postsynaptic molecular components. Specifically, DLG1 and L(2)GL scaffolds regulate the distribution and density of both active zone components (e.g. BRP) and postsynaptic GluRs, and both of these scaffolds are reduced at Mgat1 null NMJs. Importantly, dlg1 mutants display selective loss of GLURIIB, with GLURIIA unchanged, similar to Mgat1 nulls, suggesting a causal mechanism. Moreover, l(2)gl mutants display both a selective GLURIIB impairment as well as reduction of BRP aggregation in active zones, similarly to Mgat1 nulls, suggesting a separable involvement for this synaptic scaffold. DLG1 and L(2)GL are known to interact in other developmental contexts, indicating a likely interaction at the developing synapse. Although synaptic ultrastructure has not been examined in l(2)gl mutants, dlg1 mutants exhibit impaired NMJ development, including a deformed SSR. These synaptogenesis requirements predict similar ultrastructural defects in Mgat1 mutants, albeit presumably due to the combined loss of both DLG1 and L(2)GL scaffolds. Future work will focus on electron microscopy analyses to probe N-glycosylation mechanisms of synaptic development (Parkinson, 2013).

Negative regulation of active zone assembly by a newly identified SR protein kinase

Presynaptic, electron-dense, cytoplasmic protrusions such as the T-bar (Drosophila) or ribbon (vertebrates) are believed to facilitate vesicle movement to the active zone (AZ) of synapses throughout the nervous system. The molecular composition of these structures including the T-bar and ribbon are largely unknown, as are the mechanisms that specify their synapse-specific assembly and distribution. In a large-scale, forward genetic screen, a mutation was identified termed air traffic controller (atc) that causes T-bar-like protein aggregates to form abnormally in motoneuron axons. This mutation disrupts a gene that encodes for a serine-arginine protein kinase (SRPK79D). This mutant phenotype is specific to SRPK79D and is not secondary to impaired kinesin-dependent axonal transport. The srpk79D gene is neuronally expressed, and transgenic rescue experiments are consistent with SRPK79D kinase activity being necessary in neurons. The SRPK79D protein colocalizes with the T-bar-associated protein Bruchpilot (Brp) in both the axon and synapse. It is proposed that SRPK79D is a novel T-bar-associated protein kinase that represses T-bar assembly in peripheral axons, and that SRPK79D-dependent repression must be relieved to facilitate site-specific AZ assembly. Consistent with this model, overexpression of SRPK79D disrupts AZ-specific Brp organization and significantly impairs presynaptic neurotransmitter release. These data identify a novel AZ-associated protein kinase and reveal a new mechanism of negative regulation involved in AZ assembly. This mechanism could contribute to the speed and specificity with which AZs are assembled throughout the nervous system (Johnson, 2009).

SRPK79D is one of very few proteins known to localize to T-bars or ribbon-like structures at the AZ and is the only known kinase to localize to this site. Genetic evidence is provided that SRPK79D functions to represses the premature assembly of T-bars in axons. In particular, it was shown that loss-of-function mutations in srpk79D cause the appearance of T-bar–like protein aggregates throughout peripheral axons, and the possibility was ruled out that this is an indirect consequence of impaired axonal transport. The appearance of ectopic T-bars is highly specific since numerous other synaptic proteins and mitochondria are normally distributed in the neuron and are normally trafficked to the presynaptic nerve terminal in the srpk79D mutant background. Thus, SRPK79D appears to have a specific function in repressing T-bar assembly prior to the AZ, consistent with the strong colocalization of SRPK79D protein with Brp and T-bar structures (Johnson, 2009).

Finally, a potential function was uncovered for SRPK79D at the active zone (AZ) where it is observed to colocalize with Brp. SRPK79D loss-of-function mutations do not alter the number, density, or organization of Brp puncta at the synapse and do not alter synaptic function. This is consistent with a negative regulatory role for SRPK79D during T-bar assembly and indicates that once SRPK79D-dependent repression of T-bar assembly is relieved, AZ assembly proceeds normally. Overexpression of SRPK79D, however, severely disrupts neurotransmission. The defect in presynaptic release is correlated with a disruption of Brp puncta organization and integrity. These phenotypes are consistent with a function for SRPK79D as a negative regulator of T-bar assembly and AZ maturation (Johnson, 2009).

SRPK79D is a member of the SRPK family of constitutively active cytoplasmic serine-threonine kinases that target serine-arginine–rich domains of SR proteins. Thus, it is interesting to postulate what the relevant kinase target might be. Given that SRPK79D and Brp colocalize, an obvious candidate is the Brp protein itself. However, the Brp protein does not have a consensus SR domain, and decreasing the genetic dosage of srpk79D does not potentiate axonal Brp accumulations that appear upon Brp overexpression. As such, Brp may not be the direct target of SRPK79D kinase activity. It is hypothesized, therefore, that SRPK79D colocalizes with Brp and another putative SR protein that is the direct target of SRPK79D kinase activity (Johnson, 2009).

The best-characterized role for SRPKs is in controlling the subcellular localization of SR proteins, thereby regulating their nuclear pre-mRNA splicing activity. SR protein involvement in several cytoplasmic mRNA regulatory roles has been reported. In particular, a phosphorylation-dependent role for SR proteins has been reported in both Drosophila and mammalian cell culture (Johnson, 2009).

It is interesting to speculate that the function of SRPK79D to prevent premature T-bar assembly might be related to the established function of SRPKs and SR-domain-containing proteins during RNA binding, processing, and translation. One interesting possibility is that RNA species are resident at the T-bar. In such a scenario, SRPK79D-dependent repression of RNA translation could prevent T-bar assembly in the axon, and relief of this repression would enable T-bar assembly at the AZ. The continued association of SRPK79D with the AZ could allow regulated control of further T-bar assembly during development, aging, and possibly as a mechanism of long-term synaptic plasticity. Several results provide evidence in support of such a possibility. First, local translation has been proposed to control local protein concentration within a navigating growth cone. There is also increasing evidence in support of local translation in dendrites and for the presence of Golgi outposts that could support local protein maturation. A specific role for RNA binding proteins at the presynaptic AZ is supported by the prior identification of the RIBEYE protein, which is a constituent of the vertebrate ribbon structure. RIBEYE contains a CtBP domain previously shown to bind RNA. The discovery of a different RNA binding protein (CtBP1) at the ribbon and this description of a putative RNA regulatory protein at the Drosophila T-bar further suggest that RNA processing might be involved in the formation or function of these presynaptic electron dense structures (Johnson, 2009).

In light of these data, the possibility was explored that SRPK79D might participate in translational control related to T-bar assembly. Therefore, mutations in genes that could represent SRPK79D-dependent negative regulators of translation, such as aret (bru), cup, pum, nos, and sqd were examined, reasoning that the loss of such a translational inhibitor might result in the ectopic synthesis of AZ proteins, ultimately leading to a phenotype similar to that observed in srpk79D mutants. Also, genomic deletions were generated for bru2 and bru3. However, no evidence was found of axonal Brp aggregation in any of these mutants. Next, mutations previously shown to be required for mRNA transport and local protein synthesis were assayed. If necessary for T-bar assembly, these mutations might disrupt synaptic Brp-dependent T-bar formation. These mutations, including orb, vas, and stau, have phenotypes at earlier stages of development, but show no defect in synaptic Brp staining. Thus, although these experiments do not rule out a function for SRPK79D in local translation, mutations were examined in several additional candidates and no evidence was uncovered in support of this model (Johnson, 2009).

Another possibility is that SRPK79D inhibits T-bar assembly through the constitutive phosphorylation-dependent control of a putative SR protein that colocalizes with SRPK79D and Brp within a nascent T-bar protein complex. Upon arrival of this nascent T-bar protein complex at the presynaptic nerve terminal, T-bar assembly could be initiated in a site-specific manner through the action of a phosphatase that is concentrated at a newly forming synapse. There are several examples of phosphatases that can be localized to sites of intercellular adhesion, some of which have been implicated in the mechanisms of synapse formation and remodeling. This model, therefore, proposes that negative regulation of T-bar assembly, via SRPK79D, is a critical process required for the rapid and site-specific assembly of the presynaptic AZ-associated T-bar structure. Finally, the possibility cannot be ruled out that SRPK79D normally functions to prevent T-bar superassembly as opposed to T-bar assembly per se. Consistent with this idea is the observation of T-bar aggregates in axons and prior observation that detached ribbon structures coalesce into large assemblies in vertebrate neurons (Johnson, 2009).

Synapse assembly is a remarkably rapid event. There is evidence that the initial stages of synapse assembly can occur in minutes to hours, followed by a more protracted period of synapse maturation. Synapses are also assembled at specific sites. In motoneurons and some central neurons, synapses are assembled when the growth cone reaches its muscle or neuron target. However, many central neurons form en passant synapses that are rapidly assembled at sites within the growing axon, behind the advancing growth cone. Current evidence supports the conclusion that intercellular signaling events mediated by cell adhesion and transmembrane signaling specify the position of the nascent synapse. The subsequent steps of presynaptic AZ assembly remain less clear. Calcium channels and other transmembrane and membrane-associated proteins appear to be delivered to the nascent synaptic site via transport vesicles that fuse at the site of synapse assembly. It has been proposed that cytoplasmic scaffolding molecules then gradually assemble at the nascent synapse by linking to the proteins that have been deposited previously. This model assumes, however, that the protein–protein interactions between the numerous scaffolding molecules that comprise the presynaptic particle web do not randomly or spontaneously occur in the cytoplasm prior to synapse assembly. What prevents these scaffolds from spontaneously assembling in the small volume of an axon, prior to synapse formation at the nerve terminal and between individual en passant synapses? Currently, nothing is known about how premature scaffold assembly is prevented. It is proposed that these studies of srpk79D identify one such mechanism of negative regulation that prevents premature, inappropriate assembly of a presynaptic protein complex. It is further proposed that such a mechanism of negative regulation, when relieved at a site of synapse assembly, could contribute to the speed with which presynaptic specializations are observed to assemble (Johnson, 2009).

Bruchpilot in ribbon-like axonal agglomerates, behavioral defects, and early death in SRPK79D kinase mutants of Drosophila

Defining the molecular structure and function of synapses is a central theme in brain research. In Drosophila the Bruchpilot (BRP) protein is associated with T-shaped ribbons ('T-bars') at presynaptic active zones (AZs). BRP is required for intact AZ structure and normal evoked neurotransmitter release. By screening for mutations that affect the tissue distribution of Bruchpilot, a P-transposon insertion was identified in gene CG11489 (location 79D) which shows high homology to mammalian genes for SR protein kinases (SRPKs). SRPKs phosphorylate serine-arginine rich splicing factors (SR proteins). Since proteins expressed from CG11489 cDNAs phosphorylate a peptide from a human SR protein in vitro, CG11489 was renamed the Drosophila Srpk79D (serine-arginine protein kinase at 79D) gene. Srpk79D transcripts and generated a null mutant. Mutation of the Srpk79D gene causes conspicuous accumulations of BRP in larval and adult nerves. At the ultrastructural level, these correspond to extensive axonal agglomerates of electron-dense ribbons surrounded by clear vesicles. Basic synaptic structure and function at larval neuromuscular junctions appears normal, whereas life expectancy and locomotor behavior of adult mutants are significantly impaired. All phenotypes of the mutant can be largely or completely rescued by panneural expression of SRPK79D isoforms. Isoform-specific antibodies recognize panneurally overexpressed GFP-tagged SRPK79D-PC isoform co-localized with BRP at presynaptic active zones while the tagged -PB isoform is found in spots within neuronal perikarya. SRPK79D concentrations in wild type apparently are too low to be revealed by these antisera. It is proposed that the Drosophila Srpk79D gene may be expressed at low levels throughout the nervous system to prevent the assembly of BRP containing agglomerates in axons and maintain intact brain function. The discovery of an SR protein kinase required for normal BRP distribution calls for the identification of its substrate and the detailed analysis of SRPK function for the maintenance of nervous system integrity (Nieratschker, 2009; full text of article).

These results demonstrate an important role of the kinase SRPK79D for the proper distribution of the active zone protein Bruchpilot. In larval and adult nerves the kinase is required for preventing the formation of conspicuous BRP-containing electron-dense ribbon-like agglomerates observed by electron microscopy in the Srpk79DVN mutant but not in wild-type controls. It is tempting to speculate that these ribbons may be molecularly related to T-bars beyond the association with BRP and that the kinase prevents the premature assembly of T-bars in peripheral axons. Whether BRP is also involved in generating the behavioral and survival defects observed when SRPK79D-PC/PF isoforms or all SRPK79D isoforms are missing is not known. Since BRP does not contain any serine-arginine rich domains it seems unlikely that BRP is a substrate for these kinases. in vitro phosphorylation data suggest that in Drosophila SRPK79D isoforms modify SR proteins and thus may be involved in splicing regulation. It will now be necessary to identify the endogenous substrates of the SRPK79D kinase and study the mechanisms by which the formation of the extensive BRP-containing electron-dense agglomerates in wild-type axons is prevented. The characterization of an SR protein kinase that appears to be localized at presynaptic active zones and has dramatic effects on the distribution of an active zone protein is likely to modify current views on vertebrate SRPK function and may initiate new approaches to the study of active zone assembly and function (Nieratschker, 2009).

Bruchpilot promotes active zone assembly, Ca2+ channel clustering, and vesicle release

The molecular organization of presynaptic active zones during calcium influx-triggered neurotransmitter release is the focus of intense investigation. The Drosophila coiled-coil domain protein Bruchpilot (BRP) was observed in donut-shaped structures centered at active zones of neuromuscular synapses by using subdiffraction resolution STED (stimulated emission depletion) fluorescence microscopy. At brp mutant active zones, electron-dense projections (T-bars) are entirely lost, Ca2+ channels are reduced in density, evoked vesicle release is depressed, and short-term plasticity is altered. BRP-like proteins seem to establish proximity between Ca2+ channels and vesicles to allow efficient transmitter release and patterned synaptic plasticity (Kittel, 2006).

Synaptic communication is mediated by the fusion of neurotransmitter-filled vesicles with the presynaptic membrane at the active zone, a process triggered by Ca2+ influx through clusters of voltage-gated channels. The spacing between Ca2+ channels and vesicles at active zones is particularly thought to influence the dynamic properties of synaptic transmission (Kittel, 2006).

The larval Drosophila neuromuscular junction (NMJ) is frequently used as a model of glutamatergic synapses. The monoclonal antibody Nc82 specifically stains individual active zones by recognizing a coiled-coil domain protein of roughly 200 kD named Bruchpilot (Brp). Brp shows homologies to the mammalian active zone components CAST [cytoskeletal matrix associated with the active zone (CAZ)-associated structural protein], also called ERC (ELKS, Rab6-interacting protein 2, and CAST). Whereas confocal microscopy recognized diffraction limited spots, the subdiffraction resolution of stimulated emission depletion (STED) fluorescence microscopy revealed donut-shaped Brp structures at active zones. Viewed perpendicular to the plane of synapses, both single and multiple 'rings' were uncovered, of similar size to freeze-fracture-derived estimates of fly active zones. The donuts were up to 0.16 µm high, as judged by images taken parallel to the synaptic plane (Kittel, 2006).

Brp seems to demark individual active zones associated with clusters of Ca2+ channels. Transposon-mediated mutagenesis allowed isolation of a mutant chromosome (brp69) in which nearly the entire open reading frame of Brp was deleted. brp mutants [brp69/df(2R)BSC29] develop into mature larvae but do not form pupae. The Nc82 label is completely lost from the active zones of brp mutant NMJs but can be restored by re-expressing the brp cDNA in the brp mutant background with use of the neuron-specific driver lines ok6-GAL4. This also rescued larval lethality. Mutants had slightly smaller NMJs and somewhat fewer individual synapses. However, individual receptor fields, identified by the glutamate receptor subunit GluRIID, were enlarged in brp mutants. Thus, principal synapse formation occurred in brp mutants, with individual postsynaptic receptor fields increased in size but moderately decreased in number (Kittel, 2006).

In electron micrographs of brp mutant NMJs, synapses with pre- and postsynaptic membranes in close apposition were present at regular density, and consistent with the enlarged glutamate receptor fields postsynaptic densities appeared larger while otherwise normal. However, intermittent rufflings of the presynaptic membrane were noted, and brp mutants completely lacked presynaptic dense projections (T-bars). Occasionally, very little residual electron-dense material attached to the presynaptic active zone membrane was identified. After re-expressing the Brp protein in the mutant background, T-bar formation could be partially restored, although these structures were occasionally somewhat aberrant in shape. Thus, Brp assists in the ultrastructural assembly of the active zone and is essential for T-bar formation (Kittel, 2006).

In brp mutant larvae a drastic decrease was noted in evoked excitatory junctional current (eEJC) amplitudes by using two-electrode voltage clamp recordings of postsynaptic currents at low stimulation frequencies. This drop in current amplitude could be partially rescued through brp re-expression within the presynaptic motoneurons by using either elav-GAL4 or ok6-GAL4. In contrast, the amplitude of miniature excitatory junctional currents (mEJCs) in response to single, spontaneous vesicle fusion events was increased over control levels. This is consistent with the enlarged individual glutamate receptor fields of brp mutants and excludes a lack of postsynaptic sensitivity as the cause of the reduced eEJC amplitudes (Kittel, 2006).

It follows that the number of vesicles released per presynaptic action potential (AP) (quantal content) was severely compromised at brp mutant NMJs and could not be attributed solely to the moderate decrease in synapse number. The ultrastructural defects of brp mutant synapses may interfere with the proper targeting of vesicles to the active zone membrane and thereby impair exocytosis. The number of vesicles directly docked to active zone membranes was slightly decreased in brp mutants. However, the amplitude distribution and sustained frequency of mEJCs showed that brp mutant synapses did not appear to suffer from extrasynaptic release, as would be caused by a misalignment of vesicle fusion sites with postsynaptic receptors. Consistent with the appropriate deposition of exo- and endocytotic proteins, an apparently normal distribution of Syntaxin, Dap160, and Dynamin was observed at brp mutant synapses (Kittel, 2006).

The exact amplitude and time course of AP-triggered Ca2+ influx in the nerve terminal governs the amplitude and time course of vesicle . Nerve-evoked responses of brp mutants were delayed when compared with controls, whereas in contrast mEJC rise times were unchanged. Thus, evoked vesicle fusion events were less synchronized with the invasion of the presynaptic terminal by an AP. Spatiotemporal changes in Ca2+ influx have a profound effect on short-term plasticity. Whereas at 10 Hz controls exhibited substantial short-term depression of eEJC amplitudes, brp mutants showed strong initial facilitation before stabilizing at a slightly lower but frequency-dependent steady-state current. As judged by the initial facilitation at 10 Hz, neither a reduction in the number of releasable vesicles nor available release sites could fully account for the low quantal content of brp mutants at moderate stimulation frequencies. Furthermore, the altered short-term plasticity of brp mutant synapses suggested a change in the highly Ca2+-dependent vesicle release probability. Paired-pulse protocols were applied to the NMJ. Closely spaced stimuli lead to a buildup of residual Ca2+ in the vicinity of presynaptic Ca2+ channels, enhancing the probability of a vesicle within this local Ca2+ domain to undergo fusion after the next pulse. The absence of marked facilitation at control synapses could be explained by a depletion of release-ready vesicles. At brp mutant NMJs, however, the prominent facilitation at short interpulse intervals showed that the enhancement of release probability strongly outweighed the depletion of releasable vesicles. Thus, initial vesicle release probability was low, and release at brp synapses particularly benefited from the accumulation of intracellular Ca2+ (Kittel, 2006).

Vesicle fusion is highly sensitive to the spacing between Ca2+ channels and vesicles at release sites. It has been calculated that doubling this distance from 25 to 50 nm decreases the release probability threefold, and the larger this distance, the more effective the slow synthetic Ca2+ buffer EGTA should become in suppressing release. Indeed, after bath application of membrane permeable EGTA-AM, the reduction of evoked vesicle release was more pronounced at brp mutant than at control NMJs (Kittel, 2006).

The Ca2+-channel subunit Cacophony governs release at Drosophila NMJs. By using a fully functional, GFP (green fluorescent protein)-labeled variant (CacGFP), Ca2+ channels were visualized in vivo. Consistently, Ca2+ channel expression was severely reduced over the entire NMJ and at synapses lacking Brp (Kittel, 2006).

Thus, it is concluded that brp mutants suffer from a diminished vesicle release probability due to a decrease in the density of presynaptic Ca2+ channel clusters. It is conceivable that Brp tightly surrounds but is not part of the T-bar structure, contained within the unlabeled center of donuts. Brp may establish a matrix, required for both T-bar assembly as well as the appropriate localization of active zone components including Ca2+ channels, possibly by mediating their integration into a restricted number of active zone slots. Related mechanisms might underlie functional impairments of mammalian central synapses lacking active zone components (Altrock, 2003) and natural physiological differences between synapse types. Electron microscopy has identified regular arrangements at active zones of mammalian CNS synapses ('particle web') and frog NMJs ('ribs'), where these structures have also been proposed to organize Ca2+ channel clustering. At calyx of Held synapses (an axosomatic synapse in the auditory brainstem), both a fast and a slow component of exocytosis have been described. The fast component recovers slowly and is believed to owe its properties to vesicles attached to a matrix tightly associated with Ca2+ channels, whereas the slow component recovers faster and is thought to be important for sustaining vesicle release during tetanic stimulation. In agreement with this concept, the absence or impairment of such a matrix at brp synapses has a profound effect on vesicle release at low stimulation frequencies, but this effect subsides as the frequency increases. The sustained frequency of mEJCs at brp synapses could be explained if spontaneous fusion events arise from the slow release component or a pathway independent of evoked vesicle fusion (Kittel, 2006).

Synapses lacking Brp and T-bars exhibited a defective coupling of Ca2+ influx with vesicle fusion, whereas the vesicle availability did not appear rate-limiting under low frequency stimulation. The activity-induced addition of presynaptic dense bodies has been proposed to elevate vesicle release probability. This work supports this hypothesis and suggests an involvement of Brp or related factors in synaptic plasticity by promoting Ca2+ channel clustering at the active zone membrane (Kittel, 2006).

Fos and Jun potentiate individual release sites and mobilize the reserve synaptic-vesicle pool at the Drosophila larval motor synapse

In all nervous systems, short-term enhancement of transmitter release is achieved by increasing the weights of unitary synapses; in contrast, long-term enhancement, which requires nuclear gene expression, is generally thought to be mediated by the addition of new synaptic vesicle release sites. In Drosophila motor neurons, induction of AP-1, a heterodimer of Fos and Jun, induces cAMP- and CREB-dependent forms of presynaptic enhancement. Light and electron microscopic studies indicate that this synaptic enhancement is caused by increasing the weight of unitary synapses and not through the insertion of additional release sites. Electrophysiological and optical measurements of vesicle dynamics demonstrate that enhanced neurotransmitter release is accompanied by an increase in the actively cycling synaptic vesicle pool at the expense of the reserve pool. Finally, the observation that AP-1 mediated enhancement eliminates tetanus-induced forms of presynaptic potentiation suggests: (1) that reserve-pool mobilization is required for tetanus-induced short-term synaptic plasticity; and (2) that long-term synaptic plasticity may, in some instances, be accomplished by stable recruitment of mechanisms that normally underlie short-term synaptic change (Kim, 2009).

Drosophila larval motor synapses show increased synaptic strength when AP-1 is overexpressed in motor neurons (Sanyal, 2002). This synaptic enhancement is accompanied by increases in the quantal content of neurotransmitter release, and increases in the number of presynaptic varicosities (Sanyal, 2002). This study asked whether AP-1 mediated synapse enhancement can be explained by increases in synapse number, Ca2+ influx, Ca2+ sensitivity of vesicle fusion or synaptic vesicle number. The observations support a model in which: (1) AP-1 induced synaptic enhancement occurs without an accompanying increase in synapse number; (2) AP-1 increases the size of the cycling synaptic vesicle pool through mobilization of the reserve pool; (3) that AP-1 causes persistent synaptic change by stably recruiting a cellular mechanism transiently used for posttetanic potentiation, a ubiquitous but poorly understood form of short-term synaptic facilitation (Kim, 2009).

Previous studies have shown AP-1 overexpression in Drosophila motor neurons enhances glutamate release from motor terminals in a manner that is accompanied by an increase in bouton number (Sanyal, 2002). These conclusions were confirmed using failure frequency analysis, which, under conditions of very low Ca2+, measures frequency of 'failure' to release even a single quantum of neurotransmitter. At 0.3 mM Ca2+, frequencies of failure events are reduced in C155/+;UAS Fos/+;UAS Jun/+ (hereafter referred to as 'AP-1') compared with control C155/+ hereafter 'control') synapses. Therefore, this analysis confirmed quantal content (m = ln [number of events/number failures]) is significantly increased in motor synapses from AP-1 animals. Similar results were obtained under nonfailure conditions where quantal content is calculated by m = EJP/mEJP. Because quantal amplitude is not increased by AP-1 (SF1Fig. S1), presynaptic mechanisms completely account for the measured synaptic strengthening. These observations, taken together with previous work (Sanyal, 2002), show that AP-1 increases quantal content of transmitter release at both low and physiological Ca2+ concentrations (Kim, 2009).

Although AP-1 overexpression increases the number of presynaptic boutons (Sanyal, 2002), the average bouton size is significantly reduced. For this reason, and because individual boutons contain multiple release sites, bouton number is not necessarily a reliable measure of synapse number. The following strategy was used to assess whether AP-1-terminals have more functional synapses, which is defined as presynaptic release sites apposed to postsynaptic receptor clusters. In wild-type neuromuscular junctions (NMJ), ~95% of GluR clusters are coupled to Bruchpilot (brp/CAST) immunopositive presynaptic puncta (Rasse, 2005). This fraction is not altered by AP-1 expression. Thus, the number of Brp-positive puncta provides a measure of synapse number in AP-1 synapses (Kim, 2009).

Since individual Brp spots are clearly resolved, they could be counted and analyzed with a spot-detection/analysis program. This method yielded values that were in good agreement with those derived from previous serial EM studies of wild-type NMJs. Surprisingly, total Brp positive puncta (per NMJ) decreased by 21% in AP-1 synapses. AP-1 induction did not detectably alter the distribution of T-Bar or synapse size assessed by quantitative fluoresence and electron microscopy respectively. Thus, it is concluded that although AP-1 increases total bouton number, the number of functional synapses is significantly reduced. Because the quantal content of neurotransmitter release is N × p (where N is synapse number and p is the average probability of vesicle release per synapse), these observations point to an increase in p at AP-1 terminals (Kim, 2009).

If AP-1 overexpression leads to changes in the probability of release, it was reasoned that forms of short-term plasticity, which also alter p, might be altered at these motor terminals. To test this idea, two separable forms of short-term plasticity observed at the Drosophila larval NMJ at low Ca2+ concentrations were measured. The first form, paired-pulse facilitation (PPF) is short-lived and decays within milliseconds. This is easily distinguished from longer-lived presynaptic plasticity, observed during and after tetanic stimulation, which decays more slowly (10s of seconds to minutes). Although multiple processes (e.g., augmentation and posttetanic potentiation) could contribute to this longer-lived form of plasticity, this phenomenon is referred by a single term, tetanus-induced potentiation (TIP) (Kim, 2009).

At interstimulus intervals (ISI) of 25 ms, 50 ms, 100 ms, and 1,000 ms, the paired pulse ratios exhibited by control and AP-1 motor terminals did not differ significantly. The site of action for residual Ca2+ during paired pulse facilitation (PPF) has been demonstrated in previous studies to be located in the Ca2+ microdomain immediately surrounding clustered Ca2+ channels and vesicle release sites (Blatow, 2003; Zucker, 2002). The observation that PPF is normal in AP-1 synapses suggests that Ca2+ dynamics in this microdomain are not significantly altered by AP-1 (Kim, 2009).

In contrast, TIP was strikingly altered by AP-1 expression. In control synapses, transmitter release increases during a 2-min train of 10-Hz stimulation, eventually reaching a plateau. Contributions from both facilitation and TIP processes underlie the potentiated response during delivery of the tetanic stimulus train. Facilitation, however, decays within a few hundred milliseconds. Thus, longer-lived components (TIP), which decay on the order of seconds to minutes, can be isolated in the potentiated response after the tetanic train ends. TIP is greatly reduced in AP-1 terminals compared with the control. The potentiation factor immediately after the tetanus is 2.53 ± 0.13 for control and 1.15 ± 0.10 for AP-1. This early potentiation decays with time but lasts for several minutes as evidenced by the values for PF2.75 measured 2.75 min after stimulation cessation, which are 1.54 ± 0.14 for control and 0.93 ± 0.11 for AP-1. Thus, in AP-1 appears to affect both PF0 (Kim, 2009).

The absence of TIP components in AP-1 synapses is consistent with a model where individual release sites are 'prepotentiated' in AP-1 motor terminals. Loss of TIP cannot be explained by postsynaptic receptor saturation, because EJPs of twice this magnitude can easily be detected at this motor synapse. The observation that one form of short-term plasticity (PPF) remains unaltered, whereas longer lived forms (TIP) are dramatically diminished argues that AP-1 acts through a selective and relatively specific mechanism normally used for tetanus-induced presynaptic plasticity (Kim, 2009).

To determine the underlying mechanism of synaptic enhancement by AP-1, three key parameters that influence the efficiency of neurotransmitter release were measured: (1) presynaptic Ca2+ entry; (2) sensitivity of the exocytotic machinery to Ca2+; and (3) the available pool of synaptic vesicles (Kim, 2009).

A simple mechanism for increasing the probability of exocytosis from an active zone is enhanced Ca2+ entry, e.g., because of a decreased presynaptic potassium conductance and/or an increased Ca2+ current. The highly comparable paired-pulse ratios in AP-1 and control terminals suggest presynaptic Ca2+ entry and, particularly, the molecular target of residual Ca2+ during PPF, is unchanged in AP-1 expressing motor neurons (Kim, 2009).

Direct Ca2+ imaging to support the above argument is difficult, because small changes in single-action potential induced Ca2+ entry potentially can account for the observed increase in quantal content. Using an indirect approach, it was instead asked whether summed Ca2+ entry during 40-Hz nerve stimulation was increased in AP-1 expressing animals (Kim, 2009).

In motor terminals expressing the genetically encoded Ca2+ indicator, GCaMP 1.6, fluorescence was imaged during sustained 40-Hz stimulation. Values for DF/F at a plateau reached in ~2 seconds were similar in AP-1 and control synapses. Unexpectedly, Ca2+ rise times in AP-1 terminals were slightly slower than in the control. This cannot be ascribed to faster Ca2+ extrusion as GCaMP signal does not decay any faster in AP-1 synapses after stimulation cessation. Instead, these data indicate that less Ca2+ enters AP-1 presynaptic terminals per action potential, at least during high-frequency stimulation. Although GCaMP imaging does not provide absolute measurement of presynaptic Ca2+ before and after stimulation, these data argue against increased evoked Ca2+ entry as being the primary mechanism for AP-1's effect on transmitter release (Kim, 2009).

Another mechanism to enhance transmitter release is to increase sensitivity of the exocytotic machinery to free Ca2+. Measurements, however, show Ca2+ cooperativity of transmitter release was not significantly altered by AP-1 expression (Kim, 2009).

The last major parameter that influences and often correlates with quantal content is the size of the active cycling vesicle pool (also referred to as exo-endo cycling pool, ECP) available for release (Murthy, 1999). At Drosophila motor synapses, the ECP contributes to transmitter release at low to moderate rates of nerve stimulation, e.g., 3 Hz. A second 'reserve' pool of vesicles (RP) poorly accessed at 3-Hz stimulation, is efficiently mobilized during high frequency stimulation >10 Hz. Two independent approaches, one electrophysiological and the other, optical allow the sizes of the cycling and total synaptic vesicle pools to be compared at the Drosophila NMJ (Kim, 2009).

ECP sizes were compared as follows. First, AP-1 and control synapses were stimulated continuously at 3 Hz in the presence of 1 μM bafilomycin A1, a drug that pharmacologically blocks the refilling of vesicles with neurotransmitter. Initial rates of synaptic depression under these experimental conditions largely reflect depletion of the cycling pool of vesicles. The later phase in the decay plot, after significant ECP depletion, represents vescles that arise from slow mixing between RP and ECP. The initial phase is extended in AP-1 compared with control, consistent with a larger ECP. To quantitatively estimate ECP size, Y-intercept values were determined by linear regression of the points from the later slow phase of depression in a cumulative plot. These ECP estimates were consistent with substantial enlargement of the ECP in AP-1 motor terminals. Because these estimates derive from fitting the observed curves to a specific (previously suggested) model (Delgado, 2000), a second and completely independent technique was used to estimate the ECP. In this technique, optical measurements of styryl dye uptake into individual varicosities were used. Consistent with predictions from electrophysiological measurements, varicosities at AP-1 synapses were more brightly labeled than control synapses when the ECP was loaded with FM1-43 dye by 3-Hz stimulation for 7 min, indicating a larger ECP (Kim, 2009).

To test whether this increased ECP in AP-1 synapses occurs at the expense of the reserve poolwe measured the total vesicle content in AP-1 and control terminals was measured by stimulating them to depletion at 10-Hz frequency in the presence of Bafilomycin. Total vesicle pool size was estimated by integrating the complete depression curve of quantal content versus stimulus number. This direct electrophysiological estimate showed a slightly smaller total pool size in AP-1 terminals. To independently assess the sizes of the total vesicle pool FM1-43 uptake into presynaptic boutons was measured after 7 min of 30-Hz stimulation, conditions that should label both ECP and RP. Remarkably, both control and AP-1 terminals were labeled to very similar levels under these conditions, with AP-1 showing slightly lower labeling. This indicates that the total number of synaptic vesicles is similar in control and AP-1 synapses. Thus, 2 independent approaches-electrophysiological and optical establish that AP-1 increases the actively cycling vesicle pool by partially mobilizing the reserve pool of synaptic vesicles. EM analyses of synaptic-vesicle density in AP-1 and control nerve terminals are also conistent with this conclusion (Kim, 2009).

Based on these observations, AP-1 synapses show 2 major differences from the wild-type. First, they have a larger fraction of actively cycling vesicles. Second, they exhibit highly reduced TIP. These 2 phenotypes can be linked if one proposes that mobilization from the reserve vesicle pool is required for TIP. In such a model, AP-1 synapses cannot be further potentiated because the RP has already been mobilized. It was therefore asked whether tetanus-induced potentiation requires RP mobilization (Kim, 2009).

Previous work has established that RP mobilization depends on activity of the myosin light chain kinase (MLCK) in Drosophila motor terminals (Verstreken, 2005). Blocking the activity of this enzyme results in failure to recruit vesicles from the inactive pool under high frequency stimulation (Verstreken, 2005). Strikingly, the MLCK inhibitor ML-7 also inhibited tetanus-induced potentiation; PF was not examined at later time points because in relevant control preparations, the small amount of DMSO required to dissolve MLCK increased the rate of decay of TIP]. Taken together, the above experiments indicate that (1) TIP requires synaptic-vesicle mobilization from the reserve pool; and, by inference, (2) AP-1 driven prepotentiation of transmitter release is accompanied by a stable expansion of the cycling pool of vesicles through reserve pool mobilization (Kim, 2009).

One important conclusion from this work is that Fos and Jun enhance synaptic strength, not by increasing synapse number, but rather by increasing the average probability of release from individual active zones. This conclusion is based on the following: (1) increases in synaptic strength in AP-1 motor terminals can be completely accounted for by increased transmitter release; and (2) light microscopic studies show no increases in the number of release sites. Thus, there is an increase in the average probability of synaptic vesicle fusion at release sites of AP-1 motor neurons. There are some caveats to this argument. First, the definition of functional synapses as Brp-positive puncta is based on the assumptions that Brp puncta: (1) mark the large majority of release sites; and (2) are mostly capable of transmitter release postsynaptic stimulation. These assumptions are supported by the tight colocalization of presynaptic Ca2+ channels and postsynaptic receptors with Brp puncta (Kim, 2009).

Increased vesicle-release probability from active zones could conceivably be explained by several different mechanisms. In AP-1 synapses, a large increase in the size of the actively cycling synaptic vesicle pool (ECP), which arises at the expense of the reserve pool (RP), was demonstrated. An increased ECP can account for the observed synaptic enhancement in AP-1 motor terminals if it increases the number of synaptic vesicles immediately available for fusion. RP mobilization has been associated with specific instances of short-term plasticity: e.g., cocaine-induced increases in dopamine release from rat striatal neurons. This study shows that this process can be initiated by nuclear gene expression (Kim, 2009).

How widely might RP mobilization be deployed for synaptic change in vivo? Studies of the reserve pool in hippocampal synapses do not easily support the idea that vesicle trafficking from this source controls synaptic vesicle availability within these small axonal terminals. However, RP mobilization can regulate the output from larger synapses such as neuromuscular junctions or the calyx of Held, where inhibition of the myosin light chain kinase required for vesicle mobilization has been shown to reduce the stability of the synaptic firing during repetitive stimulation (Verstreken, 2005). Because the actual mechanism of RP mobilization is poorly understood, more experiments will be required to understand exactly how Fos and Jun regulate this process. In one model, phosphorylation of synapsin, which tethers synaptic vesicles to an actin-based cytoskeleton in the central domain of synaptic boutons, may mobilize the reserve pool by triggering the dissociation of vesicles from the cytoskeleton, and their transport/diffusion to peripheral release sites. In Drosophila NMJs, mitochondria within the presynaptic terminal are required for sustained release of neurotransmitter under high frequency stimulation. One study suggests ATP production from mitochondria is required to fuel MLCK-activated, myosin-propelled transport of reserve vesicles from central to peripheral sites (Verstreken, 2005). Although such processes might be involved, it is also possible that different pathways are used for Fos and Jun mediated RP mobilization. For instance, smaller sized boutons, such as those observed in AP-1 animals, may be less efficient at holding a central pool of reserve vesicles. In such a scenario, bouton geometry, rather than a specific regulatory protein, may prove to be the relevant target of AP-1 activity (Kim, 2009).

The simplest interpretation of these observations is that stable reserve pool mobilization underlies the observed loss of tetanus-induced potentiation in AP-1 synapses. Other work at the Drosophila neuromuscular junction has associated mobilization of the reserve pool with the expression of TIP (Kim, 2009).

Cytosolic Ca2+ accumulation and signaling is required for the induction of TIP. However, links between Ca2+ signaling and the expression of TIP are poorly defined; indeed, tetanus induced potentiation could include multiple Ca2+-dependent processes including augmentation and posttetanic potentiation/PTP. If TIP expression requires RP mobilization, then it would be occluded in 1 of 2 ways. Either: (1) by preexisting mobilization of the reserve pool; or (2) by inhibition of reserve pool mobilization. Drosophila dnc mutants with enhanced cAMP signaling, and rut mutants with reduced cAMP signaling respectively illustrate these 2 different mechanisms of inhibition. Both mutants do not show tetanus-induced potentiation. Although dnc mutants show a greatly increased ECP and already enhanced transmitter release, rut mutants show a large, stable reserve pool that cannot be mobilized by tetanic stimulation. These data indicate that AP-1 synapses behave like dnc mutants in which reserve vesicles have already been mobilized (Kim, 2009).

If reserve pool mobilization is required for TIP, then mutations or drugs that inhibit reserve pool mobilization would also be expected to block TIP. Consistent with this prediction, application of an MLCK inhibitor, which blocks reserve pool mobilization, was shown to dramatically inhibit TIP induction in wild-type motor synapses. Thus, although alternative contributing mechanisms cannot be ruled out, this study shows that tetanus induced presynaptic potentiation is tightly linked to reserve pool mobilization (Kim, 2009).

It is possible that many different direct and indirect targets of AP-1 contribute to various observed AP-1 dependent neuronal phenomena: e.g., increased bouton number, reduced bouton size, increased dendritic growth, elevated evoked transmitter release and increased ECP size. In addition, AP-1 may have effects on some phenomena that are not yet measured, e.g., kinetic and spatial features of synaptic Ca2+ dynamics. Nonetheless, this work shows functions of Fos and Jun in neurons, and provides substantial evidence for a model in which transcription-dependent changes in synaptic function occur through stable recruitment of mechanisms used in short-term plasticity. Recent observations that short-term forms of presynaptic plasticity are altered following synaptic enhancements induced by either BDNF or postsynaptic PSD-95 overexpression suggest that this could be a viable strategy for long-term information storage in central synapses (Zakharenko, 2003). If long-term plasticity requires stable recruitment of short-term plasticity mechanisms, then the lability of long-term memory traces, as observed in studies of reconsolidation, may not require the elimination of stable synaptic connections representing the stored memory (Kim, 2009).

Rab3 dynamically controls protein composition at active zones

Synaptic transmission requires the localization of presynaptic release machinery to active zones. Mechanisms regulating the abundance of such synaptic proteins at individual release sites are likely determinants of site-specific synaptic efficacy. A role for the small GTPase Rab3 has been identified in regulating the distribution of presynaptic components to active zones. At Drosophila rab3 mutant NMJs, the presynaptic protein Bruchpilot, calcium channels, and electron-dense T bars are concentrated at a fraction of available active zones, leaving the majority of sites devoid of these key presynaptic release components. Late addition of Rab3 to mutant NMJs rapidly reverses this phenotype by recruiting Brp to sites previously lacking the protein, demonstrating that Rab3 can dynamically control the composition of the presynaptic release machinery. While previous studies of Rab3 have focused on its role in the synaptic vesicle cycle, these findings demonstrate an additional and unexpected function for Rab3 in the localization of presynaptic proteins to active zones (Graf, 2009).

Individual neurons can form thousands of discrete synaptic connections with their postsynaptic partners. Each synapse comprises tightly apposed pre- and post-synaptic membranes, a postsynaptic cluster of neurotransmitter receptors, and a presynaptic complex of proteins that promotes neurotransmitter release. For a synapse to function, the proper complement of proteins must localize to the presynaptic release machinery, and the protein composition at the release site is a likely determinant of its synaptic efficacy. While the general properties of synapses formed by a single axon are similar, the release probability of such synapses can vary dramatically. This presynaptic heterogeneity is likely due to mechanisms that control synapse specific plasticity and may represent one aspect of the molecular basis of learning and memory. Thus, identifying mechanisms that control the protein composition and presynaptic release properties of individual synapses will provide insights into plasticity mechanisms in the brain (Graf, 2009).

The Drosophila neuromuscular junction (NMJ) is an excellent system for identifying mechanisms that regulate the protein composition of individual active zones. A single Drosophila motoneuron and single muscle cell form an NMJ comprising hundreds of individual release sites, or presynaptic active zones, each apposed to a postsynaptic glutamate receptor (GluR) cluster. Each release site is akin to a single mammalian central nervous system synapse, and like CNS synapses, there is heterogeneity in their release properties. Drosophila contains orthologs of all of the major vertebrate presynaptic proteins with the exception of Bassoon and Piccolo. Among these, Bruchpilot (Brp), the Drosophila ortholog of CAST, plays an essential role in organizing the presynaptic release machinery. This role is similar in mammals where CAST acts as a molecular scaffold within the cytomatrix at the active zone, interacting with Piccolo, Bassoon, Rim1α, and α-liprins/SYD-2 and in C. elegans where the Brp homolog ELKS-1 acts with SYD-2/α-liprin to promote the assembly of presynaptic active zone components. In Drosophila, Bruchpilot localizes to every active zone, but its distribution is heterogeneous, and the abundance of Brp at an active zone appears to correlate with the release probability of that site. Brp is not required for active zone formation per se, but is an integral component of T bars, electron-dense active zone specializations that likely promote transmitter release, and is required for the continuous accumulation of Ca2+-channels at active zones during synapse maturation. These findings with Brp imply that mechanisms exist to (1) ensure that Brp is present at each release site and (2) regulate the level of Brp at each site. Such mechanisms would likely impact site-specific release probability by controlling the protein composition of the release machinery at each site. To identify such mechanisms, a large-scale genetic screen was performed to identify genes required for the proper localization of Brp to active zones (Graf, 2009).

This study found that the small GTPase Rab3 functions to influence the distribution of Brp and other crucial presynaptic active zone components to release sites. In the absence of Rab3, key constituents of the presynaptic release machinery are concentrated at a fraction of available sites, resulting in the formation of a small number of super sites with enhanced release probability and a larger number of sites devoid of key presynaptic release proteins. Rab3 can rapidly recruit Brp to active zones, demonstrating that the protein composition of the release machinery is under dynamic control and that Rab3 is well positioned to participate in synapse-specific plasticity mechanisms. Whereas previous studies have implicated Rab3 in the cycling and docking of synaptic vesicles, this study reports a role for Rab3 in influencing the protein composition of the presynaptic release apparatus at individual active zones (Graf, 2009).

To identify mechanisms that control the molecular composition of individual release sites, a collection of Drosophila mutants was screened for those with defects that differentially affect presynaptic active zones within an NMJ. An anatomical genetic screen was performed on a collection of ∼1500 lines that carry unique insertions of transposable elements in or near genes on the second chromosome. Third-instar homozygous mutant larvae were dissected from each line and stained for the presynaptic active zone protein Bruchpilot (Brp) and the essential glutamate receptor subunit DGluRIII. The immunostained NMJs were stained with fluorescence microscopy and mutants were identified with altered active zones, including changes in Brp puncta size, number, or intensity, as well as those with defects in the apposition of presynaptic Brp and postsynaptic DGluRIII puncta. Within this group, one line, P{SUPor-P}KG07292, was identified that has an active zone phenotype. In this mutant, there is a dramatic loss of Brp-positive active zones, yet the morphology and number of DGluRIII clusters appears grossly normal. As such, most GluR clusters are unapposed to a Brp-positive active zone. The remaining Brp puncta are apposed to GluR clusters, and these Brp puncta are significantly larger than in wild-type. Due to the large number of unapposed GluR clusters, this mutant was named running-unapposed (rup) (Graf, 2009).

While Brp morphology is altered in rup, the gross morphology of the mutant NMJ is normal, and the synaptic terminal area is not significantly different than in wild-type. Staining with an antibody against the vesicular glutamate transporter (DVGLUT) demonstrates that synaptic vesicles are distributed throughout the NMJ, and co-staining for the postsynaptic scaffolding protein Discs-large (Dlg) reveals that the presynaptic terminal is apposed to the postsynaptic specialization across its length. Hence, the presence of unapposed GluR clusters is not due to synaptic retraction of synaptic boutons or branches; instead, affected synapses distribute in a salt-and-pepper pattern throughout the synaptic terminal, suggesting that the defect occurs at the level of individual synapses. Glutamate receptors colocalize with the serine-threonine kinase Pak at the Drosophila NMJ. Pak distribution appears normal in the rup mutant, and like GluR clusters, most Pak clusters are unapposed to Brp-positive active zones. Hence, in rup postsynaptic morphology is relatively normal and the primary morphological defect is likely presynaptic. Despite their abnormal active zones, rup mutant animals are viable and fertile (Graf, 2009).

To investigate the mechanism underlying the defective active zones in rup, it was necessary to identify the responsible gene. Although rup was found in a collection of insertional mutants, the phenotype does not map to the P{SUPor-P}KG07292 transposable element. Instead, rup is a second-site mutation fortuitously present on the chromosome. rup was roughly mapped by meiotic recombination to position 43-48 on the right arm of the second chromosome and a deficiency chromosome (Df(2R)ED2076) was identified that fails to complement the mutation. This deficiency deletes 26 predicted genes in the region between 47A10 and 47C1. The list of candidates was narrowed to 21 by complementation testing with known null mutants in the region. The coding regions of candidate genes was sequenced, ultimately identifying a five base pair deletion near the 3' end of the rab3 gene. This deletion throws rab3 out of frame and would lead to a deletion of the last 35 amino acids of the protein, including the final CXC motif that in other systems is required for lipid modification, the binding of Rab3 to synaptic vesicles, and proper Rab3 localization (Graf, 2009 and references therein).

A single ortholog of Drosophila rab3 was previously cloned and demonstrated to be highly conserved. It was further shown to be expressed throughout the fly nervous system; however, no functional studies were performed. To investigate the localization of Rab3 protein and the nature of the mutant allele, we generated a polyclonal antibody to a peptide epitope in the unique C-terminal region of Drosophila Rab3. This antibody stains synaptic terminals of wild-type NMJs in a pattern similar to synaptic vesicle markers such as synapsin and DVGLUT. However, unlike synapsin and DVGLUT, Rab3 staining is further concentrated in a punctate pattern at active zones, as visualized by costaining with Brp. This punctate localization of Rab3 at active zones is not observed in brp mutant NMJs (brp69/Df(2R)BSC29) even though the synaptic vesicle-like distribution of Rab3 staining remains. In addition, the antibody recognizes a single band of the predicted size on immunoblots from wild-type larvae. Both the synaptic staining at the NMJ and the band on the immunoblot are absent in the rup mutant, demonstrating that the antibody is specific for Rab3 and that the rup mutant does not express wild-type Rab3 protein. Since the mutation in rup is located in the C-terminal region of rab3 just upstream of the epitope, it is possible that a truncated protein could be expressed. While such a mutant protein could have residual function, the active zone phenotype of rup homozygotes and transheterozygotes of rup and Df(2R)ED2076 are similar in terms of the percentage of GluR clusters apposed to Brp and the average area of individual Brp punctum. Therefore, rup behaves as a genetic null or a very strong hypomorph (Graf, 2009).

This study shows that the small GTPase Rab3 controls the protein composition and release probability of individual active zones at the Drosophila neuromuscular junction. In a rab3 mutant, key constituents of the presynaptic release machinery are enriched at a subset of active zones while the remaining release sites are apparently devoid of these proteins. Expression of Rab3 rapidly and reversibly rescues this altered protein distribution. Physiological studies are consistent with these morphological findings, demonstrating an increase in release probability from an apparently decreased number of release sites. Mechanistic studies indicate that Rab3 functions to increase the probability that the essential synaptic organizing molecule Bruchpilot will cluster at an active zone. This Rab3-dependent regulation of active zone protein composition and release probability provides a potential mechanism for the synapse-specific control of synaptic efficacy (Graf, 2009).

The Drosophila NMJ consists of a motoneuron axon terminal arranged as a chain of synaptic boutons closely associated with the postsynaptic muscle membrane. Within each string of boutons are hundreds of individual synapses, discrete sites of neurotransmitter release where a presynaptic active zone is directly apposed to a postsynaptic glutamate receptor cluster. Such a synapse comprises (1) the site where the axon and muscle membranes are in closest proximity, likely tethered by trans-synaptic cell adhesion molecules; (2) the presynaptic release apparatus that influences the Ca2+-mediated release of the neurotransmitter-filled vesicles; and (3) the neurotransmitter receptors and scaffolding and signaling proteins of the postsynaptic density. This study demonstrates that disrupting rab3 alters the distribution of proteins that make up the presynaptic release machinery without grossly disturbing the other two components of the synapse (Graf, 2009).

In the absence of Rab3, a subset of synapses contain increased amounts of the active zone protein Bruchpilot, higher levels of the calcium channel Cacophony, and more electron dense T bars at the active zone. Since Brp is a component of T bars and influences Ca2+-channel accumulation, the altered distribution of these components is likely a direct consequence of changes in Brp distribution. The creation of additional active zone markers will be necessary to determine the full extent of this altered distribution. However, since all three components examined influence the probability of evoked vesicle release, the active zones where they accumulate likely are sites of enhanced vesicle release. Conversely, the remaining sites that are devoid of these components likely exhibit impaired evoked release. Two lines of evidence support this conclusion. First, glutamate receptors preferentially cluster opposite sites with the highest release probability. In the rab3rup mutant, GluR clusters are larger at Brp-positive than Brp-negative sites, suggesting that those active zones containing Brp have a higher release probability. Second, facilitation resulting from short stimulus trains is reduced in the mutant, consistent with an increased release probability (p). However, since quantal content and quantal size are unchanged, the increase in p must be balanced by a decrease in the number of sites that are firing. Hence, both the morphological and electrophysiological data are consistent with the model that Rab3 controls the distribution of active zone proteins to influence the efficacy of individual release sites (Graf, 2009).

Other Drosophila mutants have active zone phenotypes, but none have the combination of phenotypes described in this study. Mutations in synaptojanin, neurexin, and spectrin affect the size and spacing of the entire array of active zones. Mutations in the Unc-51 kinase and the protein phosphatase PP2A have differential affects on active zones, resulting in a subset of glutamate receptors unapposed to Bruchpilot puncta as in the rab3rup mutant. However, in the unc-51 and PP2A mutants, the remaining Brp puncta are not enlarged and there is no increase in the proportion of active zones with multiple T bars. Such phenotypes are consistent with defects in active zone formation, rather than in the distribution of proteins across active zones. Finally, GluR clusters unapposed to Brp puncta occurs following synaptic retraction, but in such mutants the active zone defects are secondary to the loss of the entire presynaptic terminal. Hence, Rab3 participates in a previously undescribed mechanism that differentially regulates active zones within an NMJ (Graf, 2009).

These findings demonstrate that Rab3 plays a central role in the localization of Bruchpilot to individual active zones. In the absence of Rab3, approximately 70% of active zones are devoid of Brp while the other 30% contain an excess of Brp. What is the function of Rab3 such that its loss leads to this altered Brp distribution? It is suggested that Brp is present in two pools: one fraction bound in complexes at active zones and a second mobile fraction in the cytosol. It is further suggested that Brp is dynamic and may alternate between these two pools by associating with or dissociating from the active zone complex. As such, unbound Brp in the cytosol may either nucleate a cluster at an active zone, creating a new Brp punctum, or add to a pre-existing Brp punctum making it larger. Given this scenario, the rab3 phenotype may be explained by two alternative models of Rab3 function: (1) Rab3 limits Brp puncta size, or (2) Rab3 increases the ability of Brp to nucleate new Brp clusters at active zones. If Rab3 functions to limit the addition of Brp to already existing sites, disruption of Rab3 would allow Brp clusters to grow to a maximal size, reducing the availability of cytosolic Brp to create new puncta and consequently constraining the number of puncta formed. In such a model, it would be predicted that Brp puncta size would be large at rab3rup mutant NMJs regardless of Brp expression levels. Instead, decreasing Brp levels in the rab3rup mutant decreases the size of Brp puncta. Even more telling, increasing Brp levels in the rab3rup mutant also reduces the size of Brp puncta. These results are inconsistent with the model that primary function of Rab3 is to limit the size of Brp puncta (Graf, 2009).

Instead, it is suggested that Rab3 functions to increase the probability that Brp will nucleate a new cluster at an active zone. The presence of Brp at some active zones demonstrates that Rab3 is not absolutely required for Brp localization. Why then is Rab3 required for Brp localization to the 70% of active zones that are bereft of Brp? Rather than posit that these two classes of active zones are fundamentally different in the rab3rup mutant, it is suggested that in the absence of Rab3, Brp is much less likely to nucleate a cluster at an active zone (Graf, 2009).

The data presented in this study are consistent with this model. First, late rescue with rab3 leads to the rapid addition of new, small Brp puncta and, on a slower time scale, a decrease in the size of the large Brp puncta. This demonstrates that Brp is dynamic and can move into and out of active zones. Second, reducing the levels of Brp at wild-type synapses leads to a decrease in both the number of Brp puncta formed as well as their size. An increase in Brp expression at a wild-type synapse cannot increase the number of puncta since essentially 100% of active zones already contain a Brp puncta, but it does lead to an increase in the size of the puncta. Hence, Brp levels affect both the likelihood of forming a Brp puncta at an active zone as well as the ultimate size of the Brp puncta. Third, increased Brp expression enhances the ability of Brp to cluster at active zones, overcoming the absence of Rab3 and leading to the formation of more Brp puncta in the rab3rup mutant. This demonstrates that these mutant active zones do have the capacity to cluster Brp, but that it requires the stronger driving force provided by the additional Brp to overcome the absence of Rab3. Finally, when Brp is overexpressed in the rab3rup mutant the Brp puncta are smaller than when Brp is expressed at wild-type levels. This apparent paradox suggests that Brp puncta compete for unbound Brp and that the large increase in the number of Brp puncta provides more sites for unbound Brp and so ultimately results in smaller puncta. Hence, this model explains the variation in the number and size of Brp puncta present in the various genetic backgrounds tested above, and highlights a novel role for Rab3 in controlling the protein composition of active zones (Graf, 2009).

Brp appears to play a prominent role in the mechanism by which Rab3 regulates the distribution of active zone components to release sites. However, there is no evidence that Rab3 interacts directly with Brp, and such a direct interaction between Rab3 and members of the CAST/ERC family, of which Brp is an ortholog, has not previously been reported. Other proteins could mediate the interaction between Rab3 and Brp. In other species, Rab3 is known to interact with proteins involved in the Rab3 GTPase cycle such as Rab3-GEF, Rab3-GAP, and GDI, as well as the putative Rab3 effectors Sec15, Rabphilin, and Rim. Among the Rab3 effectors, Rabphilin is an unlikely candidate because Rabphilin knockout mice and worms have no observable morphological or physiological synaptic defects. Rim is a more plausible candidate because it is a constituent of the presynaptic release apparatus and binds to many other presynaptic active zone proteins including orthologs of Brp. Alternatively, Rab3 may act on a yet unidentified target to regulate the molecular properties of Brp. Understanding Rab3 function at the fly NMJ will require the identification of the protein(s) Rab3 interacts with to distribute active zone components among release sites (Graf, 2009).

Previous studies of rab3 knockouts in other organisms suggest that Rab3 is involved in regulating vesicle cycling, docking, and exocytosis. While Rab3 may play a direct role in vesicle dynamics and release at the Drosophila NMJ, this study also suggesta that Rab3 plays a second, separate role in influencing the distribution of the presynaptic release apparatus. Defects at the active zone in the rab3rup mutant are unlikely to be secondary to altered synaptic vesicle release because (1) other mutants affecting release do not disrupt the composition of the active zone and (2) neither increasing nor decreasing activity in the rab3rup mutant exacerbates or ameliorates the active zone phenotype (Graf, 2009).

If Rab3 controls the protein composition of the active zone, then why have genetic analyses of rab3 in mice and C. elegans not identified structural abnormalities at the synapse? In Drosophila, loss of rab3 results in a very specific ultrastructural phenotype. The active zone, visualized as an electron dense thickening of tightly apposed pre- and postsynaptic membranes, is normal in Drosophila rab3rup mutants as it is in worms and mice. However, some synapses, including Drosophila NMJ synapses, contain prominent electron-dense specializations such as T bars that are thought to promote transmitter release. It is the distribution of these T bars that is altered in the Drosophila rab3rup mutant, which would not be apparent at, for example, hippocampal synapses, where such dense bodies are not readily observed. While structural defects have not been detected in other organisms, the electrophysiological findings in mice show interesting parallels to the fly phenotype. The quadruple knockout of Rab3A, Rab3B, Rab3C, and Rab3D in mice demonstrates that Rab3 increases the release probability of a subset of vesicles in the readily releasable pool. Two hypotheses were proposed to explain these findings. The first stays within the traditional vesicle-centric framework for Rab3, suggesting that Rab3 docks specific vesicles to sites of high release probability. The second hypothesis posits that Rab3 recruits additional proteins to the release machinery at certain synapses, thereby making Ca2+-mediated release more efficient. This second possibility is consistent with the findings in Drosophila that Rab3 regulates the distribution of release apparatus proteins to control the efficacy of individual sites (Graf, 2009).

Many neurons differentially regulate the release properties of individual release sites along their axonal lengths through presynaptic, synapse-specific mechanisms. These include the regulation of Ca2+-channel localization and function and the selective accumulation of group III metabotropic glutamate receptors to specific presynaptic active zones. It is suggested that Rab3 is well positioned to participate in such synapse-specific plasticity mechanisms. The finding that late expression of Rab3 can rapidly reverse the apposition phenotype of the mutant and redistribute Brp to active zones that previously lacked the protein indicates that (1) Brp is highly mobile and (2) Rab3 can rapidly modulate its distribution among individual sites. Multiple proteins control Rab3 function via its GTPase cycle, so mechanisms that locally activate or inhibit Rab3 could lead to rapid and local changes in active zone structure and function. Thus, Rab3 is a candidate to participate in plasticity mechanisms that regulate the protein composition and efficacy of individual release sites (Graf, 2009).

ELP3 controls active zone morphology by acetylating the ELKS family member Bruchpilot

Elongator protein 3 (ELP3) acetylates histones in the nucleus but also plays a role in the cytoplasm. This study reports that in Drosophila neurons, ELP3 is necessary and sufficient to acetylate the ELKS family member Bruchpilot, an integral component of the presynaptic density where neurotransmitters are released. In elp3 mutants, presynaptic densities assemble normally, but they show morphological defects such that their cytoplasmic extensions cover a larger area, resulting in increased vesicle tethering as well as a more proficient neurotransmitter release. A model is proposed where ELP3-dependent acetylation of Bruchpilot at synapses regulates the structure of individual presynaptic densities and neurotransmitter release efficiency (Miskiewicz, 2011).

Naked dense bodies provoke depression

At presynaptic active zones (AZs), the frequently observed tethering of synaptic vesicles to an electron-dense cytomatrix represents a process of largely unknown functional significance. This study identified a hypomorphic allele, brpnude, lacking merely the last 1% of the C-terminal amino acids (17 of 1740) of the active zone protein Bruchpilot. In brpnude, electron-dense bodies were properly shaped, though entirely bare of synaptic vesicles. While basal glutamate release was unchanged, paired-pulse and sustained stimulation provoked depression. Furthermore, rapid recovery following sustained release was slowed. These results causally link, with intramolecular precision, the tethering of vesicles at the AZ cytomatrix to synaptic depression (Hallermann, 2010).

The specific impairment of vesicle tethering reported in this study delivers the first direct demonstration that efficient sustained release relies on the ability of the AZ to tether vesicles. While the overall AZ structure, including the distribution of Ca2+ channels, was unaffected, the impairment of vesicle tethering provoked pronounced synaptic depression and a slowed first component of recovery (Hallermann, 2010).

The C-terminal half of BRP consists of ~1000 aa of essentially contiguous coiled-coil sequence, reminiscent of Golgi/ER-resident tethering factors such as, e.g., GM130. These coiled-coils typically form rod-like structures, where 100 aa residues extend ~15 nm when dimerized, and proteins such as Uso1p extend over 150 nm. These rod-like proteins are believed to act before SNARE protein assembly by forming contacts between membranes at a distance, thereby increasing the specificity or efficiency of the initial attachment of vesicles (tethering). This study has provided morphological and functional evidence that BRP filaments tether vesicles, and thus further mechanistic comparisons between AZ and Golgi/ER trafficking, e.g., concerning the role of small GTPases, might well be informative (Hallermann, 2010).

The C-terminal half of BRP is very highly conserved in insects but not elsewhere. Interestingly, the Drosophila genome does not appear to encode homologs of the vertebrate AZ components Piccolo and Bassoon, which are key regulators of the vertebrate cytomatrix. At central vertebrate synapses, CAST and Bassoon immunoreactivities (closer and further from the AZ membrane, respectively) were recently found to be associated with filaments that may connect vesicles to the AZ. It is tempting to speculate that at AZs of central vertebrate synapses, CAST associates with coiled-coil domain proteins, such as bassoon, to perform the dual functions of Ca2+ channel clustering and vesicle tethering executed by the N-terminal and the C-terminal domains of BRP, respectively (Hallermann, 2010).

How synapses manage to repetitively release transmitter with high precision is intensely investigated. Vesicles tethered to electron-dense bodies may represent a reservoir of vesicles required for sustained release. Consistent with this hypothesis, synaptic stimulation provokes depletion of vesicles tethered at dense bodies. While the supply of vesicles appears rate limiting during the train and the first component of recovery, the maturation of vesicles closer to Ca2+ channels appears rate limiting during the second component of recovery. One may argue that the rapid component of depression observed at brpnude synapses could be partially attributed to fewer docked vesicles (though not significantl) with a higher initial release probability. However, a functional estimation of the number of readily releasable vesicles using back-extrapolation from the cumulative EPSC amplitudes in the trains revealed similar numbers of readily releasable vesicles in brpnude and controls. Finally, it is pointed out that the C-term of BRP could be involved in endocytotic mechanisms, which have been shown to be crucial for sustained release. Novel techniques have begun to address the spatial organization of local vesicle reuse within active zones. It will have to be clarified via which routes vesicles move within active zones and in which direction Bruchpilot steers their translocation (Hallermann, 2010).

Genetic dissection of aversive associative olfactory learning and memory in Drosophila larvae

Memory formation is a highly complex and dynamic process. It consists of different phases, which depend on various neuronal and molecular mechanisms. In adult Drosophila it was shown that memory formation after aversive Pavlovian conditioning includes-besides other forms-a labile short-term component that consolidates within hours to a longer-lasting memory. Accordingly, memory formation requires the timely controlled action of different neuronal circuits, neurotransmitters, neuromodulators and molecules that were initially identified by classical forward genetic approaches. Compared to adult Drosophila, memory formation was only sporadically analyzed at its larval stage. This study deconstructed the larval mnemonic organization after aversive olfactory conditioning. After odor-high salt conditioning (establishing an aversive olfactory memory) larvae form two parallel memory phases; a short lasting component that depends on cyclic adenosine 3'5'-monophosphate (cAMP) signaling and synapsin gene function. In addition, this study shows for the first time for Drosophila larvae an anesthesia resistant component, which relies on radish and bruchpilot gene function, protein kinase C (PKC) activity, requires presynaptic output of mushroom body Kenyon cells and dopamine function. Given the numerical simplicity of the larval nervous system this work offers a unique prospect for studying memory formation of defined specifications, at full-brain scope with single-cell, and single-synapse resolution (Widmann, 2016).

Memory formation and consolidation usually describes a chronological order, parallel existence or completion of distinct short-, intermediate- and/or long-lasting memory phases. For example, in honeybees, in Aplysia, and also in mammals two longer-lasting memory phases can be distinguished based on their dependence on de novo protein synthesis. In adult Drosophila classical odor-electric shock conditioning establishes two co-existing and interacting forms of memory--ARM and LTM--that are encoded by separate molecular pathways (Widmann, 2016).

Seen in this light, memory formation in Drosophila larvae established via classical odor-high salt conditioning seems to follow a similar logic. It consist of LSTM (larval short lasting component) and LARM (anesthesia resistant memory). Aversive olfactory LSTM was already described in two larval studies using different negative reinforcers (electric shock and quinine) and different training protocols (differential and absolute conditioning). The current results introduce for the first time LARM that was also evident directly after conditioning but lasts longer than LSTM. LARM was established following different training protocols that varied in the number of applied training cycles and the type of negative or appetitive reinforcer. Thus, LSTM and LARM likely constitute general aspects of memory formation in Drosophila larvae that are separated on the molecular level (Widmann, 2016).

Memory formation depends on the action of distinct molecular pathways that strengthen or weaken synaptic contacts of defined sets of neurons. The cAMP/PKA pathway is conserved throughout the animal kingdom and plays a key role in regulating synaptic plasticity. Amongst other examples it was shown to be crucial for sensitization and synaptic facilitation in Aplysia, associative olfactory learning in adult Drosophila and honeybees, long-term associative memory and long-term potentiation in mammals (Widmann, 2016).

For Drosophila larvae two studies by Honjo (2005) and Khurana (2009) suggest that aversive LSTM depends on intact cAMP signaling. In detail, they showed an impaired memory for rut and dnc mutants following absolute odor-bitter quinine conditioning and following differential odor-electric shock conditioning. Thus, both studies support the interpretation of the current results. It is argued that odor-high salt training established a cAMP dependent LSTM due to the observed phenotypes of rut, dnc and syn mutant larvae. The current molecular model is summarized in A molecular working hypothesis for LARM formation. Yet, it has to be mentioned that all studies on aversive LSTM in Drosophila larvae did not clearly distinguish between the acquisition, consolidation and retrieval of memory. Thus, future work has to relate the observed genetic functions to these specific processes (Widmann, 2016).

In contrast, LARM formation utilizes a different molecular pathway. Based on different experiments, it was ascertained, that LARM formation, consolidation and retrieval is independent of cAMP signaling itself, PKA function, upstream and downstream targets of PKA, and de-novo protein synthesis. Instead it was found that LARM formation, consolidation and/or retrieval depends on radish (rsh) gene function, brp gene function, dopaminergic signaling and requires presynaptic signaling of MB KCs (Widmann, 2016).

Interestingly, studies on adult Drosophila show that rsh and brp gene function, as well as dopaminergic signaling and presynaptic MB KC output are also necessary for adult ARM formation. Thus, although a direct comparison of larval and adult ARM is somehow limited due to several variables (differences in CS, US, training protocols, test intervals, developmental stages, and coexisting memories), both forms share some genetic aspects. This is remarkable as adult ARM and LARM use different neuronal substrates. The larval MB is completely reconstructed during metamorphosis and the initial formation of adult ARM requires a set of MB α/β KCs that is born after larval life during puparium formation (Widmann, 2016).

In addition, this study has demonstrated the necessity of PKC signaling for LARM formation in MB KCs. The involvement of the PKC pathway for memory formation is also conserved throughout the animal kingdom. For example, it has been shown that PKC signaling is an integral component in memory formation in Aplysia, long-term potentiation and contextual fear conditioning in mammals and associative learning in honeybees. In Drosophila it was shown that PKC induced phosphorylation cascade is involved in LTM as well as in ARM formation. Although the exact signaling cascade involved in ARM formation in Drosophila still remains unclear, this study has established a working hypothesis for the underlying genetic pathway forming LARM based on the current findings and on prior studies in different model organisms. Thereby this study does not take into account findings in adult Drosophila. These studies showed that PKA mutants have increased ARM and that dnc sensitive cAMP signaling supports ARM. Thus both studies directly link PKA signaling with ARM formation. (Widmann, 2016).

KCs have been shown to act on MB output neurons to trigger a conditioned response after training. Work from different insects suggests that the presynaptic output of an odor activated KCs is strengthened if it receives at the same time a dopaminergic, punishment representing signal. The current results support these models as they show that LARM formation requires accurate dopaminergic signaling and presynaptic output of MB KCs. Yet, for LARM formation dopamine receptor function seems to be linked with PKC pathway activation. Indeed, in honeybees, adult Drosophila and vertebrates it was shown that dopamine receptors can be coupled to Gαq proteins and activate the PKC pathway via PLC and IP3/DAG signaling. As potential downstream targets of PKC radish and bruchpilot are suggested. Interference with the function of both genes impairs LARM. The radish gene encodes a functionally unknown protein that has many potential phosphorylation sites for PKA and PKC. Thus considerable intersection between the proteins Rsh and PKC signaling pathway can be forecasted. Whether this is also the case for the bruchpilot gene that encodes for a member of the active zone complex remains unknown. The detailed analysis of the molecular interactions has to be a focus of future approaches. Therefore, the current working hypothesis can be used to define educated guesses. For instance, it is not clear how the coincidence of the odor stimulus and the punishing stimulus are encoded molecularly. The same is true for ARM formation in adult Drosophila. Based on the working hypothesis it can be speculated that PKC may directly serve as a coincidence detector via a US dependent DAG signal and CS dependent Ca2+ activation (Widmann, 2016).

Do the current findings in general apply to learning and memory in Drosophila larvae? To this the most comprehensive set of data can be found on sugar reward learning. Drosophila larva are able to form positive associations between an odor and a number of sugars that differ in their nutritional value. Using high concentrations of fructose as a reinforcer in a three cycle differential training paradigm (comparable to the one used in this study for high salt learning and fructose learning) other studies found that learning and/or memory in syn97 mutant larvae is reduced to ~50% of wild type levels. Thus, half of the memory seen directly after conditioning seems to depend on the cAMP-PKA-synapsin pathway. The current results in turn suggest that the residual memory seen in syn97 mutant larvae is likely LARM. Thus, aversive and appetitive olfactory learning and memory share general molecular aspects. Yet, the precise ratio of the cAMP-dependent and independent components rely on the specificities of the used odor-reinforcer pairings. Two additional findings support this conclusion. First, a recent study has shown that memory scores in syn97 mutant larvae are only lower than in wild type animals when more salient, higher concentrations of odor or fructose reward are used. Usage of low odor or sugar concentrations does not give rise to a cAMP-PKA-synapsin dependent learning and memory phenotype. Second, another study showed that learning and/or memory following absolute one cycle conditioning using sucrose sugar reward is completely impaired in rut1, rut2080 and dnc1 mutants. Thus, for this particular odor-reinforcer pairing only the cAMP pathway seems to be important. Therefore, a basic understanding of the molecular pathways involved in larval memory formation is emerging. Further studies, however, will be necessary in order to understand how Drosophila larvae make use of the different molecular pathways with respect to a specific CS/US pairing (Widmann, 2016).

Bruchpilot, a synaptic active zone protein for anesthesia-resistant memory

In Drosophila, aversive associative memory of an odor consists of heterogeneous components with different stabilities. This study report that Bruchpilot (Brp), a ubiquitous presynaptic active zone protein, is required for olfactory memory. Brp was shown before to facilitate efficient vesicle release, particularly at low stimulation frequencies. Transgenic knockdown in the Kenyon cells of the mushroom body, the second-order olfactory interneurons, revealed that Brp is required for olfactory memory. It was further demonstrated that Brp in the Kenyon cells preferentially functions for anesthesia-resistant memory. Another presynaptic protein, Synapsin, was shown previously to be required selectively for the labile anesthesia-sensitive memory, which is less affected in brp knockdown. Thus, consolidated and labile components of aversive olfactory memory can be dissociated by the function of different presynaptic proteins (Knapek, 2011).

In Drosophila, middle-term olfactory memory after a single training cycle comprises functionally dissociable forms of memory: the labile ASM and the stable ARM. In contrast to ASM, the molecular basis of ARM formation is poorly understood. Only a few molecules have been shown to be important for ARM so far. This study demonstrates that Brp in the Kenyon cells of the mushroom body is preferentially required for ARM. Although there is no apparent developmental defect in the downregulation of Brp, the requirement for ARM in the adult was not specified in previous studies (Knapek, 2011).

The Brp protein is specifically localized to the active zone at the presynaptic terminals, in which it forms electron dense projections. Interestingly, the Radish protein that is also required for ARM is highly enriched in the lobes of the mushroom body. Thus, Brp and Radish might interact at the active zones to regulate neurotransmission underlying ARM (Knapek, 2011).

Several memory mutants have been shown to have a selective phenotype in ASM. Consistent with the parallel memory formation of ASM and ARM, the brp knockdown and a rutabaga mutation caused an additive memory deficit. Interestingly, Synapsin is required for ASM preferentially, and the null mutation caused no augmentation of the memory phenotype in the rutabaga single mutant (Knapek, 2010). Thus, the complementary forms of memory might recruit differential signaling mechanisms that rely on distinct presynaptic machineries (Knapek, 2011).

In a current model of memory dynamics, ARM gradually develops after training, whereas ASM occupies the largest part of early memory and decays more quickly. Although Radish and Brp are selectively required for ARM measured at 3 h after training, flies lacking either of the proteins are impaired also in immediate memory. By applying cold anesthesia for STM, it was found that STM does contain a significant ARM component. The consistent requirement of Brp for short-term and 3 h ARM may contribute to a synaptic mechanism of memory that is stable against amnesic treatment (Knapek, 2011).

If ARM and ASM were formed at the same synapses of Kenyon cells, how could the two synaptic proteins Brp and Synapsin dissociate these different forms of memory? Notably, Brp and Synapsin are meant to be required for distinct components of action potential-evoked vesicle release. In vertebrates, Synapsin has been shown to be particularly important for recruitment of synaptic vesicles from reserve pools at high stimulation frequencies. Consistently, synapsin mutants show normal quantal content (number of synaptic vesicles released per action potential) at moderate action potential frequencies. Similarly, Drosophila Synapsin maintains the reserve pool of vesicles and mediates mobilization of the reserve pool during intense stimulation. The brp null mutant in contrast shows decreased quantal contents particularly in response to the first arrival of an action potential. Vesicle release after subsequently following high-frequency spikes however is less affected, suggesting the importance in vesicle release at low-frequency stimulation. The two different modes of neurotransmission (e.g., different release probabilities during high- vs low-frequency stimulation) could therefore differentiate ASM and ARM, even if the traces of these different forms of memory resided in the same synapses of the Kenyon cells (Knapek, 2011).

Alternatively, the memory traces of ASM and ARM could be spatially separated within the same neurons, i.e., localized at different synapse populations. In the lobes, Kenyon cell axons have multiple compartments that are intersected by transverse extrinsic neurons. This study found that brp knockdown in the α/β neurons affected ARM. This is consistent with a previous report, in which inhibition of the output of the α/β neurons impaired ARM. Interestingly, inhibition of a specific type of dopaminergic neurons that synapse onto another restricted compartment of the β lobe selectively affected ASM (Aso, 2010). Thus, associative plasticity underlying ASM and ARM could be formed by stimulating different synapses of the same neurons. This hypothesis may be tested in future by the identification of extrinsic neurons that are specifically required for ARM and corresponding functional imaging of memory traces (Knapek, 2011).

Defects in Synapse Structure and Function Precede Motor Neuron Degeneration in Drosophila Models of FUS-Related ALS

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease that leads invariably to fatal paralysis associated with motor neuron degeneration and muscular atrophy. One gene associated with ALS encodes the DNA/RNA-binding protein Fused in Sarcoma (FUS). There now exist two Drosophila models of ALS. In one, human FUS with ALS-causing mutations is expressed in fly motor neurons; in the other, the gene cabeza (caz), the fly homolog of FUS, is ablated. These FUS-ALS flies exhibit larval locomotor defects indicative of neuromuscular dysfunction and early death. The locus and site of initiation of this neuromuscular dysfunction remain unclear. This study shows that in FUS-ALS flies, motor neuron cell bodies fire action potentials that propagate along the axon and voltage-dependent inward and outward currents in the cell bodies are indistinguishable in wild-type and FUS-ALS motor neurons. In marked contrast, the amplitude of synaptic currents evoked in the postsynaptic muscle cell is decreased by >80% in FUS-ALS larvae. Furthermore, the frequency but not unitary amplitude of spontaneous miniature synaptic currents is decreased dramatically in FUS-ALS flies, consistent with a change in quantal content but not quantal size. Although standard confocal microscopic analysis of the larval neuromuscular junction reveals no gross abnormalities, superresolution stimulated emission depletion (STED) microscopy demonstrates that the presynaptic active zone protein Bruchpilot is aberrantly organized in FUS-ALS larvae. The results are consistent with the idea that defects in presynaptic terminal structure and function precede, and may contribute to, the later motor neuron degeneration that is characteristic of ALS (Shahidullah, 2013).

Age-associated increase of the active zone protein Bruchpilot within the honeybee mushroom body

In honeybees, age-associated structural modifications can be observed in the mushroom bodies. Prominent examples are the synaptic complexes (microglomeruli, MG) in the mushroom body calyces, which were shown to alter their size and density with age. It is not known whether the amount of intracellular synaptic proteins in the MG is altered as well. The presynaptic protein Bruchpilot (BRP) is localized at active zones and is involved in regulating the probability of neurotransmitter release in the fruit fly, Drosophila melanogaster. This study explored the localization of the honeybee BRP (Apis mellifera BRP, AmBRP) in the bee brain and examined age-related changes in the AmBRP abundance in the central bee brain and in microglomeruli of the mushroom body calyces. Predominant AmBRP localization is reported near the membrane of presynaptic boutons within the mushroom body MG. The relative amount of AmBRP was increased in the central brain of two-week old bees whereas the amount of Synapsin, another presynaptic protein involved in the regulation of neurotransmitter release, shows an increase during the first two weeks followed by a decrease. In addition, an age-associated modulation was demonstrated of AmBRP located near the membrane of presynaptic boutons within MG located in mushroom body calyces where sensory input is conveyed to mushroom body intrinsic neurons. The observed age-associated AmBRP modulation might be related to maturation processes or to homeostatic mechanisms that might help to maintain synaptic functionality in old animals (Gehring, 2017).

In Drosophila, it has been demonstrated that Bruchpilot (DmBRP) in Kenyon cells plays a critical role in the formation of an anesthesia-resistant memory (Knapek, 2011): a 70% reduction of DmBRP in the Kenyon cells reduces this type of memory significantly. Accordingly, an age-associated increase of BRP, as observed in this experiment, might facilitate memory formation in fruit fly and possibly also in honeybees. However, Gupta (2016) demonstrated that an age-induced increase of DmBRP, which could be mimicked by an increase of the BRP copy number, did not facilitate anesthesia-resistant memory but instead blocked a cold-sensitive, anesthesia-sensitive memory. Based on these results, it was proposed that, in the Drosophila nervous system, aging synapses might steer towards the upper limit of their operational range by increasing BRP levels. This age-dependent process might limit synaptic plasticity and contribute to impairment of memory formation with age (Gehring, 2017).

Previous studies demonstrated that the packing density of boutons in lip and dense collar decreases with age resulting in fewer boutons in a defined area, i.e. a region of interest (ROI), of these neuropils. Thus, one would predict that presynaptic proteins in lip and dense collar are decreasing with age due to the decreased packing density of boutons resulting in fewer boutons per ROI that were analyzed. Indeed, this prediction proves true for Synapsin in the dense collar in this study, since an age-associated reduction was observed of the number of anti-SYNORF1-positive pixels. However, this is not the case for Synapsin in the lip where the number of anti-SYNORF1-positive pixels does not change with age. What might be the reason for this finding? It was shown that, in addition to the decrease in density, the mean volume of individual boutons increases with age in the lip and the dense collar. This increase is stronger in the lip than in the collar. Thus, the decrease in bouton density and the increase in bouton volume most likely counteract each other in the lip and this might be the reason why no change is seen in the amount of Synapsin in the lip (Gehring, 2017).

As it is the case with Synapsin, age-associated alterations in the structural organization of lip and collar boutons might influence the detection of anti-BRPlast200-positive pixels. Thus, the ratio between the median number of anti-BRPlast200-positive pixels to the median number of anti-SYNORF1-positive pixels per ROI was calculated, thereby factoring out the influence of morphological changes in the density and volume of the boutons on the detection of anti-BRPlast200-positive pixels. The ratios, i.e. the relative area, and thus probably the amount, of AmBRP increased in an age-associated manner in both, lip and collar: In the dense collar and the lip, the relative amount of AmBRP is significantly increased in 43-day-old bees. In addition, an increase was observed in the relative amount of AmBRP in the first week after emergence in the lip (Gehring, 2017).

AmBRP is a protein predominately located at presynapses. Due to the age-associated increase in bouton volume, boutons with a larger surface might also have more active zones. Increased numbers of active zones per bouton would lead to increased AmBRP levels which would provide an explanation for the observed age-associated increase in the relative amount of AmBRP. Indeed, this hypothesis could hold true for the collar as it was shown that the number of active zones per bouton is increased in 35-day-old bees compared with 1-day-old bees and that the proportion of ribbon vs. non-ribbon type active zones is increased in 35-day-old bees compared to 1-day-old bees. The latter is interesting, because ribbon-synapses in bees resemble T-bar-shaped synapses in fruit flies, that contain BRP, whereas non-ribbon synapses do not resemble this synapse type. Thus, these data are in line with findings of an increase of AmBRP from day 1, day 8 and day 15 to day 43 (Gehring, 2017).

In contrast to the collar, the number of active zones per bouton remains unchanged between 1- and 35-day-old bees in the lip. However, the same study showed that also in lip boutons the proportion of ribbon vs. non-ribbon type active zones increases. Thus, the AmBRP increase in the lip might not be indicative for the formation of new active zones and thus new synapses. Rather, it is suggested that, in the lip, it is the amount of AmBRP at existing active zones that is altered in an age-associated manner. As mentioned above, this alteration seems to take place twice: Early after emergence and late in the bees' lifetime between day 29 and 43. It might well be that an alteration of the amount of AmBRP at existing synapses shifts the proportion of ribbon vs non-ribbon active zones such that ribbon-active zones are increasing in an age-dependent manner (Gehring, 2017).

What might be the cause of the observed age-associated alterations of AmBRP in the lip and collar? Based on the existing literature, the first increase of AmBRP in the lip could be due to maturation processes in the olfactory system. The lip can be regarded as part of this olfactory system as projection neurons from the antennal lobes convey odor information onto MB Kenyon cells in this region. Neuropils belonging to the olfactory system such as the antennal lobes are not yet fully developed in newly emerged bees and mature during the first days after emergence. These maturation processes occurring in the antennal lobe might also influence synaptic connections, and thereby probably the amount of AmBRP, in upstream odor processing centers such as the lip (Gehring, 2017).

In addition to an AmBRP increase during the first week of a bee's life, increased AmBRP levels were found in very old bees (43-day-old) in the lip, but also in the collar. Similar results were observed at neuromuscular junctions of aged fruit flies (Gupta, 2016). The authors found that, with progression of age, the number of BRP-labeled spots, which indicate active zones, per bouton increased up to an age of 42 days and that this increase is accompanied by an increase in bouton volume. It is known from studies on endocytosis mutants, that an increase in number of boutons and active zones compensates a decrease of synaptic vesicle exocytosis. Thus, increased AmBRP levels at boutons in older insects might represent compensatory mechanisms for age-associated lower synaptic transmission. This hypothesis is in line with the view that age-associated synaptic alterations might be the consequence of adaptive processes due to neuronal plasticity that compensate for age-dependent cognitive impairments. Indeed, it has been demonstrated that a drop in postsynaptic excitability drives an increase of presynaptic scaffolds. According to the authors, this increase of presynaptic scaffolds might lead to an increase of synaptic vesicle release, which has been shown to be age-dependent (Gupta, 2016). In line, in a fruit fly model of Alzheimer's disease, an age-dependent reduction of the amount of BRP and the synaptic vesicle release probability has been observed suggesting that presynaptic β-amyloid plaques in the fruit fly brain might hinder a compensation of age-dependent processes that could be related to the amount of BRP (Gehring, 2017).

A striking feature of honeybee workers is their age-related division of labor. Individual workers perform different tasks within and outside the hive in an age-dependent manner: For the first 2-3 weeks after adult emergence, workers perform in-hive duties such as brood care and food processing, and start to forage for nectar and pollen outside the hive thereafter. This behavioral plasticity has been suggested to have both age- and experience-related determinants. Therefore, it should be taken into account that age-associated processes observed in honeybees are not only due to their chronological age but also due to the task they fulfill because of their age and because of the state of the colony. Thus, the age-associated effects observed in this study could be due to the (unknown) age-dependent signal that triggers the switch between the two tasks, due to experiences made when fulfilling the age-associated task, or due to the internal state of the colony. In the latter case, the observed effects would not be due to the bees' age but to the state of the colony. Since bees of defined ages were observed in a colony that was not manipulated, it is proposed that this study observed 'normally' aging bees and that the effects that were observed are directly or indirectly associated with the bees' age (Gehring, 2017).

This study reports that the level of the presynaptic proteins, Synapsin and AmBRP, are modified in an age-associated manner in the honeybee brain. An early increase was found in the relative amount of AmBRP during the first week after emergence in the MB lip, which was hypothesized to be due to maturation processes in the olfactory system. This study has shown that both MB regions, lip and collar, have increased amounts of AmBRP in 43-day-old bees. Given that BRP is homologous to the vertebrate ELKS/CAST/ERC protein, which is part of the presynaptic active zone, it will be interesting if these proteins are altered in an age-associated manner in vertebrates as well and if an AmBRP increase compensates for age-dependent cognitive impairments (Gehring, 2017).


Search PubMed for articles about Drosophila Bruchpilot

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Biological Overview

date revised: 20 August 2017

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