Sara Mertel

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 melanogaster ERC/CAST family protein Bruchpilot (BRP) were shown to lack dense projections (T-bars) and to suffer from Ca2+ channel–clustering defects. In this study, we 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, we identify BRP 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.

Glutamate receptor dynamics organizing synapse formation in vivo

Insight into how glutamatergic synapses form in vivo is important for understanding developmental and experience-triggered changes of excitatory circuits. Here, we imaged postsynaptic densities (PSDs) expressing a functional, GFP-tagged glutamate receptor subunit (GluR-IIAGFP) at neuromuscular junctions of Drosophila melanogaster larvae for several days in vivo. New PSDs, associated with functional and structural presynaptic markers, formed independently of existing synapses and grew continuously until reaching a stable size within hours. Both in vivo photoactivation and photobleaching experiments showed that extrasynaptic receptors derived from diffuse, cell-wide pools preferentially entered growing PSDs. After entering PSDs, receptors were largely immobilized. In comparison, other postsynaptic proteins tested (PSD-95, NCAM and PAK homologs) exchanged faster and with no apparent preference for growing synapses. We show here that new glutamatergic synapses form de novo and not by partitioning processes from existing synapses, suggesting that the site-specific entry of particular glutamate receptor complexes directly controls the assembly of individual PSDs.

RIM-binding protein, a central part of the active zone, is essential for neurotransmitter release

Transmitter release at the fly neuromuscular junction is abolished in the absence of a scaffold protein. The molecular machinery mediating the fusion of synaptic vesicles (SVs) at presynaptic active zone (AZ) membranes has been studied in detail, and several essential components have been identified. AZ-associated protein scaffolds are viewed as only modulatory for transmission. We discovered that Drosophila Rab3-interacting molecule (RIM)–binding protein (DRBP) is essential not only for the integrity of the AZ scaffold but also for exocytotic neurotransmitter release. Two-color stimulated emission depletion microscopy showed that DRBP surrounds the central Ca2+ channel field. In drbp mutants, Ca2+ channel clustering and Ca2+ influx were impaired, and synaptic release probability was drastically reduced. Our data identify RBP family proteins as prime effectors of the AZ scaffold that are essential for the coupling of SVs, Ca2+ channels, and the SV fusion machinery.

Activity-dependent site-specific changes of glutamate receptor composition in vivo

The subunit composition of postsynaptic non–NMDA-type glutamate receptors (GluRs) determines the function and trafficking of the receptor. Changes in GluR composition have been implicated in the homeostasis of neuronal excitability and synaptic plasticity underlying learning. Here, we imaged GluRs in vivo during the formation of new postsynaptic densities (PSDs) at Drosophila neuromuscular junctions coexpressing GluRIIA and GluRIIB subunits. GluR composition was independently regulated at directly neighboring PSDs on a submicron scale. Immature PSDs typically had large amounts of GluRIIA and small amounts of GluRIIB. During subsequent PSD maturation, however, the GluRIIA/GluRIIB composition changed and became more balanced. Reducing presynaptic glutamate release increased GluRIIA, but decreased GluRIIB incorporation. Moreover, the maturation of GluR composition correlated in a site-specific manner with the level of Bruchpilot, an active zone protein that is essential for mature glutamate release. Thus, we show that an activity-dependent, site-specific control of GluR composition can contribute to match pre- and postsynaptic assembly.

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

A proteomics approach identifies Drosophila Syd-1 as a Bruchpilot binding partner that controls maturation on both sides of the neuromuscular junction.

Mutations in NSUN2 cause autosomal-recessive intellectual disability

With a prevalence between 1 and 3%, hereditary forms of intellectual disability (ID) are among the most important problems in health care. Particularly, autosomal-recessive forms of the disorder have a very heterogeneous molecular basis, and genes with an increased number of disease-causing mutations are not common. Here, we report on three different mutations (two nonsense mutations, c.679C>T [p.Gln227(∗)] and c.1114C>T [p.Gln372(∗)], as well as one splicing mutation, g.6622224A>C [p.Ile179Argfs(∗)192]) that cause a loss of the tRNA-methyltransferase-encoding NSUN2 main transcript in homozygotes. We identified the mutations by sequencing exons and exon-intron boundaries within the genomic region where the linkage intervals of three independent consanguineous families of Iranian and Kurdish origin overlapped with the previously described MRT5 locus. In order to gain further evidence concerning the effect of a loss of NSUN2 on memory and learning, we constructed a Drosophila model by deleting the NSUN2 ortholog, CG6133, and investigated the mutants by using molecular and behavioral approaches. When the Drosophila melanogaster NSUN2 ortholog was deleted, severe short-term-memory (STM) deficits were observed; STM could be rescued by re-expression of the wild-type protein in the nervous system. The humans homozygous for NSUN2 mutations showed an overlapping phenotype consisting of moderate to severe ID and facial dysmorphism (which includes a long face, characteristic eyebrows, a long nose, and a small chin), suggesting that mutations in this gene might even induce a syndromic form of ID. Moreover, our observations from the Drosophila model point toward an evolutionarily conserved role of RNA methylation in normal cognitive development.

Restoring polyamines protects from age-induced memory impairment in an autophagy-dependent manner

Age-dependent memory impairment is known to occur in several organisms, including Drosophila, mouse and human. However, the fundamental cellular mechanisms that underlie these impairments are still poorly understood, effectively hampering the development of pharmacological strategies to treat the condition. Polyamines are among the substances found to decrease with age in the human brain. We found that levels of polyamines (spermidine, putrescine) decreased in aging fruit flies, concomitant with declining memory abilities. Simple spermidine feeding not only restored juvenile polyamine levels, but also suppressed age-induced memory impairment. Ornithine decarboxylase-1, the rate-limiting enzyme for de novo polyamine synthesis, also protected olfactory memories in aged flies when expressed specifically in Kenyon cells, which are crucial for olfactory memory formation. Spermidine-fed flies showed enhanced autophagy (a form of cellular self-digestion), and genetic deficits in the autophagic machinery prevented spermidine-mediated rescue of memory impairments. Our findings indicate that autophagy is critical for suppression of memory impairments by spermidine and that polyamines, which are endogenously present, are candidates for pharmacological intervention.

Cooperation of Syd-1 with Neurexin synchronizes pre- with postsynaptic assembly

Synapse formation and maturation requires bidirectional communication across the synaptic cleft. The trans-synaptic Neurexin-Neuroligin complex can bridge this cleft, and severe synapse assembly deficits are found in Drosophila melanogaster neuroligin (Nlg1, dnlg1) and neurexin (Nrx-1, dnrx) mutants. We show that the presynaptic active zone protein Syd-1 interacts with Nrx-1 to control synapse formation at the Drosophila neuromuscular junction. Mutants in Syd-1 (RhoGAP100F, dsyd-1), Nrx-1 and Nlg1 shared active zone cytomatrix defects, which were nonadditive. Syd-1 and Nrx-1 formed a complex in vivo, and Syd-1 was important for synaptic clustering and immobilization of Nrx-1. Consequently, postsynaptic clustering of Nlg1 was affected in Syd-1 mutants, and in vivo glutamate receptor incorporation was changed in Syd-1, Nrx-1 and Nlg1 mutants. Stabilization of nascent Syd-1–Liprin-α (DLiprin-α) clusters, important to initialize active zone formation, was Nlg1 dependent. Thus, cooperation between Syd-1 and Nrx-1–Nlg1 seems to orchestrate early assembly processes between pre- and postsynaptic membranes, promoting avidity of newly forming synaptic scaffolds.

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. Here, we 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. Our results causally link, with intramolecular precision, the tethering of vesicles at the AZ cytomatrix to synaptic depression.

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

Two Bruchpilot isoforms create a stereotypic arrangement of the cytomatrix that defines the size of the readily releasable pool of synaptic vesicles.