The Ig-like domain of Punctin/MADD-4 is the primary determinant for interaction with the ectodomain of neuroligin NLG-1.

Punctin/MADD-4, a member of the ADAMTSL extracellular matrix protein family, was identified as an anterograde synaptic organizer in the nematode Caenorhabditis elegans. At GABAergic neuromuscular junctions, the short isoform MADD-4B binds the ectodomain of neuroligin NLG-1, itself a postsynaptic organizer of inhibitory synapses. To identify the molecular bases of their partnership, we generated recombinant forms of the two proteins and carried out a comprehensive biochemical and biophysical study of their interaction, complemented by an in vivo localization study. We show that spontaneous proteolysis of MADD-4B first generates a shorter N-MADD-4B form, which comprises four thrombospondin (TSP) domains and one Ig-like domain and binds NLG-1. A second processing event eliminates the C-terminal Ig-like domain along with the ability of N-MADD-4B to bind NLG-1. These data identify the Ig-like domain as the primary determinant for N-MADD-4B interaction with NLG-1 in vitro We further demonstrate in vivo that this Ig-like domain is essential, albeit not sufficient per se, for efficient recruitment of GABAA receptors at GABAergic synapses in C. elegans The interaction of N-MADD-4B with NLG-1 is also disrupted by heparin, used as a surrogate for the extracellular matrix component, heparan sulfate. High-affinity binding of heparin/heparan sulfate to the Ig-like domain may proceed from surface charge complementarity, as suggested by homology three-dimensional modeling. These data point to N-MADD-4B processing and cell-surface proteoglycan binding as two possible mechanisms to regulate the interaction between MADD-4B and NLG-1 at GABAergic synapses.

C. elegans Punctin specifies cholinergic versus GABAergic identity of postsynaptic domains.

Because most neurons receive thousands of synaptic inputs, the neuronal membrane is a mosaic of specialized microdomains where neurotransmitter receptors cluster in register with the corresponding presynaptic neurotransmitter release sites. In many cases the coordinated differentiation of presynaptic and postsynaptic domains implicates trans-synaptic interactions between membrane-associated proteins such as neurexins and neuroligins. The Caenorhabditis elegans neuromuscular junction (NMJ) provides a genetically tractable system in which to analyse the segregation of neurotransmitter receptors, because muscle cells receive excitatory innervation from cholinergic neurons and inhibitory innervation from GABAergic neurons. Here we show that Ce-Punctin/madd-4 (ref. 5), the C. elegans orthologue of mammalian punctin-1 and punctin-2, encodes neurally secreted isoforms that specify the excitatory or inhibitory identity of postsynaptic NMJ domains. These proteins belong to the ADAMTS (a disintegrin and metalloprotease with thrombospondin repeats)-like family, a class of extracellular matrix proteins related to the ADAM proteases but devoid of proteolytic activity. Ce-Punctin deletion causes the redistribution of synaptic acetylcholine and GABAA (γ-aminobutyric acid type A) receptors into extrasynaptic clusters, whereas neuronal presynaptic boutons remain unaltered. Alternative promoters generate different Ce-Punctin isoforms with distinct functions. A short isoform is expressed by cholinergic and GABAergic motoneurons and localizes to excitatory and inhibitory NMJs, whereas long isoforms are expressed exclusively by cholinergic motoneurons and are confined to cholinergic NMJs. The differential expression of these isoforms controls the congruence between presynaptic and postsynaptic domains: specific disruption of the short isoform relocalizes GABAA receptors from GABAergic to cholinergic synapses, whereas expression of a long isoform in GABAergic neurons recruits acetylcholine receptors to GABAergic NMJs. These results identify Ce-Punctin as a previously unknown synaptic organizer and show that presynaptic and postsynaptic domain identities can be genetically uncoupled in vivo. Because human punctin-2 was identified as a candidate gene for schizophrenia, ADAMTS-like proteins may also control synapse organization in the mammalian central nervous system.

Preventing Illegitimate Extrasynaptic Acetylcholine Receptor Clustering Requires the RSU-1 Protein.

UNLABELLED: Diffuse extrasynaptic neurotransmitter receptors constitute an abundant pool of receptors that can be recruited to modulate synaptic strength. Whether the diffuse distribution of receptors in extrasynaptic membranes is a default state or is actively controlled remains essentially unknown. Here we show that RSU-1 (Ras Suppressor-1) is required for the proper distribution of extrasynaptic acetylcholine receptors (AChRs) in Caenorhabditis elegans muscle cells. RSU-1 is an evolutionary conserved cytoplasmic protein that contains multiple leucine-rich repeats (LRRs) and interacts with integrin-dependent adhesion complexes. In rsu-1 mutants, neuromuscular junctions differentiate as in the wild type, but AChRs assemble into ectopic clusters that progressively enlarge during development. As a consequence, the synaptic content of AChRs is reduced. Our study provides the first evidence that an RSU-1-dependent active mechanism maintains extrasynaptic receptors dispersed and indirectly regulates synapse maturation. SIGNIFICANCE STATEMENT: Using Caenorhabditis elegans neuromuscular junction as a model synapse, we uncovered a novel mechanism that regulates the distribution of acetylcholine receptors (AChRs). In an unbiased visual screen for mutants with abnormal AChR distribution, we isolated the ras suppressor 1 (rsu-1) mutant based on the presence of large extrasynaptic clusters. We show that disrupting rsu-1 causes spontaneous clustering of extrasynaptic receptors that are normally dispersed, independently of synaptic cues. These clusters outcompete synaptic domains and cause a decrease of synaptic receptor content. These results indicate that the diffuse state of extrasynaptic receptors is not a default state that is simply explained by the lack of synaptic cues but necessitates additional proteins to prevent spontaneous clustering, a concept that is relevant for developmental and pathological situations.

The core apoptotic executioner proteins CED-3 and CED-4 promote initiation of neuronal regeneration in Caenorhabditis elegans.

A critical accomplishment in the rapidly developing field of regenerative medicine will be the ability to foster repair of neurons severed by injury, disease, or microsurgery. In C. elegans, individual visualized axons can be laser-cut in vivo and neuronal responses to damage can be monitored to decipher genetic requirements for regeneration. With an initial interest in how local environments manage cellular debris, we performed femtosecond laser axotomies in genetic backgrounds lacking cell death gene activities. Unexpectedly, we found that the CED-3 caspase, well known as the core apoptotic cell death executioner, acts in early responses to neuronal injury to promote rapid regeneration of dissociated axons. In ced-3 mutants, initial regenerative outgrowth dynamics are impaired and axon repair through reconnection of the two dissociated ends is delayed. The CED-3 activator, CED-4/Apaf-1, similarly promotes regeneration, but the upstream regulators of apoptosis CED-9/Bcl2 and BH3-domain proteins EGL-1 and CED-13 are not essential. Thus, a novel regulatory mechanism must be utilized to activate core apoptotic proteins for neuronal repair. Since calcium plays a conserved modulatory role in regeneration, we hypothesized calcium might play a critical regulatory role in the CED-3/CED-4 repair pathway. We used the calcium reporter cameleon to track in vivo calcium fluxes in the axotomized neuron. We show that when the endoplasmic reticulum calcium-storing chaperone calreticulin, CRT-1, is deleted, both calcium dynamics and initial regenerative outgrowth are impaired. Genetic data suggest that CED-3, CED-4, and CRT-1 act in the same pathway to promote early events in regeneration and that CED-3 might act downstream of CRT-1, but upstream of the conserved DLK-1 kinase implicated in regeneration across species. This study documents reconstructive roles for proteins known to orchestrate apoptotic death and links previously unconnected observations in the vertebrate literature to suggest a similar pathway may be conserved in higher organisms.

UNC-30/PITX coordinates neurotransmitter identity with postsynaptic GABA receptor clustering.

Terminal selectors are transcription factors that control neuronal identity by regulating the expression of key effector molecules, such as neurotransmitter (NT) biosynthesis proteins, ion channels and neuropeptides. Whether and how terminal selectors control neuronal connectivity is poorly understood. Here, we report that UNC-30 (PITX2/3), the terminal selector of GABA motor neuron identity in C. elegans , is required for NT receptor clustering, a hallmark of postsynaptic differentiation. Animals lacking unc-30 or madd-4B, the short isoform of the MN-secreted synapse organizer madd-4 ( Punctin/ADAMTSL ), display severe GABA receptor type A (GABA A R) clustering defects in postsynaptic muscle cells. Mechanistically, UNC-30 acts directly to induce and maintain transcription of madd-4B and GABA biosynthesis genes (e.g., unc-25/GAD , unc-47/VGAT ). Hence, UNC-30 controls GABA A R clustering on postsynaptic muscle cells and GABA biosynthesis in presynaptic cells, transcriptionally coordinating two critical processes for GABA neurotransmission. Further, we uncover multiple target genes and a dual role for UNC-30 both as an activator and repressor of gene transcription. Our findings on UNC-30 function may contribute to our molecular understanding of human conditions, such as Axenfeld-Rieger syndrome, caused by PITX2 and PITX3 gene mutations.

Adamtsl3 mediates DCC signaling to selectively promote GABAergic synapse function.

The molecular code that controls synapse formation and maintenance in vivo has remained quite sparse. Here, we identify that the secreted protein Adamtsl3 functions as critical hippocampal synapse organizer acting through the transmembrane receptor DCC (deleted in colorectal cancer). Traditionally, DCC function has been associated with glutamatergic synaptogenesis and plasticity in response to Netrin-1 signaling. We demonstrate that early post-natal deletion of Adamtsl3 in neurons impairs DCC protein expression, causing reduced density of both glutamatergic and GABAergic synapses. Adult deletion of Adamtsl3 in either GABAergic or glutamatergic neurons does not interfere with DCC-Netrin-1 function at glutamatergic synapses but controls DCC signaling at GABAergic synapses. The Adamtsl3-DCC signaling unit is further essential for activity-dependent adaptations at GABAergic synapses, involving DCC phosphorylation and Src kinase activation. These findings might be particularly relevant for schizophrenia because genetic variants in Adamtsl3 and DCC have been independently linked with schizophrenia in patients.

The netrin receptor UNC-40/DCC assembles a postsynaptic scaffold and sets the synaptic content of GABAA receptors.

Increasing evidence indicates that guidance molecules used during development for cellular and axonal navigation also play roles in synapse maturation and homeostasis. In C. elegans the netrin receptor UNC-40/DCC controls the growth of dendritic-like muscle cell extensions towards motoneurons and is required to recruit type A GABA receptors (GABAARs) at inhibitory neuromuscular junctions. Here we show that activation of UNC-40 assembles an intracellular synaptic scaffold by physically interacting with FRM-3, a FERM protein orthologous to FARP1/2. FRM-3 then recruits LIN-2, the ortholog of CASK, that binds the synaptic adhesion molecule NLG-1/Neuroligin and physically connects GABAARs to prepositioned NLG-1 clusters. These processes are orchestrated by the synaptic organizer CePunctin/MADD-4, which controls the localization of GABAARs by positioning NLG-1/neuroligin at synapses and regulates the synaptic content of GABAARs through the UNC-40-dependent intracellular scaffold. Since DCC is detected at GABA synapses in mammals, DCC might also tune inhibitory neurotransmission in the mammalian brain.

Transcriptional coordination of synaptogenesis and neurotransmitter signaling.

During nervous system development, postmitotic neurons face the challenge of generating and structurally organizing specific synapses with appropriate synaptic partners. An important unexplored question is whether the process of synaptogenesis is coordinated with the adoption of specific signaling properties of a neuron. Such signaling properties are defined by the neurotransmitter system that a neuron uses to communicate with postsynaptic partners, the neurotransmitter receptor type used to receive input from presynaptic neurons, and, potentially, other sensory receptors that activate a neuron. Elucidating the mechanisms that coordinate synaptogenesis, neuronal activation, and neurotransmitter signaling in a postmitotic neuron represents one key approach to understanding how neurons develop as functional units. Using the SAB class of Caenorhabditis elegans motor neurons as a model system, we show here that the phylogenetically conserved COE-type transcription factor UNC-3 is required for synaptogenesis. UNC-3 directly controls the expression of the ADAMTS-like protein MADD-4/Punctin, a presynaptically secreted synapse-organizing molecule that clusters postsynaptic receptors. UNC-3 also controls the assembly of presynaptic specializations and ensures the coordinated expression of enzymes and transporters that define the cholinergic neurotransmitter identity of the SAB neurons. Furthermore, synaptic output properties of the SAB neurons are coordinated with neuronal activation and synaptic input, as evidenced by UNC-3 also regulating the expression of ionotropic neurotransmitter receptors and putative stretch receptors. Our study shows how synaptogenesis and distinct, function-defining signaling features of a postmitotic neuron are hardwired together through coordinated transcriptional control.

C. elegans Punctin Clusters GABA(A) Receptors via Neuroligin Binding and UNC-40/DCC Recruitment.

Positioning type A GABA receptors (GABA(A)Rs) in front of GABA release sites sets the strength of inhibitory synapses. The evolutionarily conserved Ce-Punctin/MADD-4 is an anterograde synaptic organizer that specifies GABAergic versus cholinergic identity of postsynaptic domains at the C. elegans neuromuscular junctions (NMJs). Here we show that the Ce-Punctin secreted by GABAergic motor neurons controls the clustering of GABA(A)Rs through the synaptic adhesion molecule neuroligin (NLG-1) and the netrin receptor UNC-40/DCC. The short isoform of Ce-Punctin binds and clusters NLG-1 postsynaptically at GABAergic NMJs. NLG-1 disruption causes a strong reduction of GABA(A)R content at GABAergic synapses. Ce-Punctin also binds and localizes UNC-40 receptors in the postsynaptic membrane of NMJs, which promotes the recruitment of GABA(A)Rs by NLG-1. Since the mammalian orthologs of these genes are expressed in the central nervous system and their mutations are implicated in neuropsychiatric diseases, this molecular pathway might have been evolutionarily conserved.

Monitoring autophagy in the filamentous fungus Podospora anserina.

Autophagy has been monitored in the filamentous fungus Podospora anserina using electron, light, and fluorescence microscopy. In this organism autophagy can be induced either by starvation or rapamycin treatment or by het gene incompatibility. Incompatible HET products signal a cell death reaction referred to as cell death by incompatibility. In het-R het-V strain bearing the two incompatible het-R and het-V genes, cell death is induced by a simple shift in growth temperature, as incompatibility is thermosensitive. In this strain large autophagosomes are formed as revealed by electron microscopy or using the GFP-PaATG8 marker. This strain constitutes an alternative model to study autophagy. Analysis of the three autophagy mutants, DeltaPaATG1, DeltaPaATG8, and DeltapspA, reveals that autophagy is essential for aerial hyphae and female organ differentiation and involved in spore germination. During the incompatibility reaction, autophagy might protect cells from cell death as suggested by accelerated cell death observed in autophagy mutants.

The genome sequence of the model ascomycete fungus Podospora anserina.

BACKGROUND: The dung-inhabiting ascomycete fungus Podospora anserina is a model used to study various aspects of eukaryotic and fungal biology, such as ageing, prions and sexual development. RESULTS: We present a 10X draft sequence of P. anserina genome, linked to the sequences of a large expressed sequence tag collection. Similar to higher eukaryotes, the P. anserina transcription/splicing machinery generates numerous non-conventional transcripts. Comparison of the P. anserina genome and orthologous gene set with the one of its close relatives, Neurospora crassa, shows that synteny is poorly conserved, the main result of evolution being gene shuffling in the same chromosome. The P. anserina genome contains fewer repeated sequences and has evolved new genes by duplication since its separation from N. crassa, despite the presence of the repeat induced point mutation mechanism that mutates duplicated sequences. We also provide evidence that frequent gene loss took place in the lineages leading to P. anserina and N. crassa. P. anserina contains a large and highly specialized set of genes involved in utilization of natural carbon sources commonly found in its natural biotope. It includes genes potentially involved in lignin degradation and efficient cellulose breakdown. CONCLUSION: The features of the P. anserina genome indicate a highly dynamic evolution since the divergence of P. anserina and N. crassa, leading to the ability of the former to use specific complex carbon sources that match its needs in its natural biotope.

Cell death by incompatibility in the fungus Podospora.

Filamentous fungi are naturally able of somatic fusions. When cells of unlike genotype at specific het loci fuse, non-self recognition operates in the fusion cell and a cell death reaction termed cell death by incompatibility is triggered. In Podospora anserina cell death by incompatibility is characterized by a dramatic vacuolar enlargement, induction of autophagy and cell lysis. Autophagy contributes neither to vacuolar morphological changes nor to cell death but rather protects cells against death. Autophagy could be involved in selective elimination of pro-death signals. Vacuole collapse and cytoplasm acidification might be the cause of cell death by incompatibility.

Podospora anserina target of rapamycin.

We have isolated the Podospora anserina TOR gene. The PaTOR protein displayed strong identities with TOR proteins from other eukaryotes especially in the FRB domain and the kinase domain. Genome analysis suggests that a single TOR gene exists in Podospora. The serine residue known to be one site of missense mutations conferring rapamycin resistance in other organisms is conserved in the PaTOR protein (S1895). A PaTOR-S1895R mutated allele has been constructed and introduced in the wild-type strain, as expected strains expressing the PaTOR-S1895R gene become resistant to rapamycin. The dominance of the PaTOR-S1895R allele indicates that apparently the mutation does not impair the kinase activity. We confirm that all cytological modifications associated with rapamycin treatment in Podospora are indeed mediated by PaTOR. We conclude that the PaTOR gene is likely to be essential and that rapamycin treatment might be useful to further investigate rapamycin-sensitive TOR functions in Podospora and especially newly identified rapamycin-sensitive functions such as the autophagy-independent control of vacuole remodeling and septation.

Accelerated cell death in Podospora autophagy mutants.

Although autophagy is characteristic of type II programmed cell death (PCD), its role in cell death is currently debated. Both cell death-promoting and prosurvival roles of autophagy have been reported depending on the organism and the cell type. In filamentous fungi, a cell death reaction known as an incompatibility reaction occurs when cells of unlike genotype fuse. Cell death by incompatibility is characterized by a dramatic vacuolar enlargement and cell lysis. In Podospora anserina, autophagy is induced early during this cell death reaction. Cell death by incompatibility in Podospora is a model of type II PCD used here to assess the role of autophagy in this type of cell death. We have inactivated PaATG1, the Podospora ortholog of the Saccharomyces cerevisiae ATG1 gene involved in the early steps of autophagy in yeast. The DeltaPaATG1 mutant displays developmental defects characteristic of abrogated autophagy in Podospora. Using the green fluorescent protein-PaATG8 autophagosome marker, we show that autophagy is abolished in this mutant. Neither cell death by incompatibility nor vacuolization are suppressed in DeltaPaATG1 and DeltaPaATG8 autophagy mutants, indicating that a vacuolar cell death reaction without autophagy occurs in Podospora. Our results thus provide a novel example of a type II PCD reaction in which autophagy is not the cause of cell death. In addition, we found that cell death is accelerated in DeltaPaATG null mutants, suggesting that autophagy has a protective role in this type II PCD reaction.

Autophagy is induced during cell death by incompatibility and is essential for differentiation in the filamentous fungus Podospora anserina.

In filamentous fungi, a cell death reaction occurs when cells of unlike genotype fuse. This cell death reaction, known as incompatibility reaction, is genetically controlled by a set of loci termed het loci (for heterokaryon incompatibility loci). In Podospora anserina, genes induced during this cell death reaction (idi genes) have been identified. The idi-6/pspA gene encodes a serine protease that is the orthologue of the vacuolar protease B of Saccharomyces cerevisiae involved in autophagy. We report here that the PSPA protease participates in the degradative autophagic pathway in Podospora. We have identified the Podospora orthologue of the AUT7 gene of S. cerevisiae involved in the early steps of autophagy in yeast. This gene is induced during the development of the incompatibility reaction and was designated idi-7. We have used a GFP-IDI7 fusion protein as a cytological marker of the induction of autophagy. Relocalization of this fusion protein and detection of autophagic bodies inside the vacuoles during the development of the incompatibility reaction provide cytological evidence of induction of autophagy during this cell death reaction. Therefore, cell death by incompatibility in fungi appears to be related to type II programmed cell death in metazoans. In addition, we found that pspA and idi-7 null mutations confer differentiation defects such as the absence of female reproductive structures, indicating that autophagy is required for differentiation in Podospora.

Rapamycin mimics the incompatibility reaction in the fungus Podospora anserina.

In filamentous fungi, a programmed cell death (PCD) reaction occurs when cells of unlike genotype fuse. This reaction is caused by genetic differences at specific loci termed het loci (for heterokaryon incompatibility). Although several het genes have been characterized, the mechanism of this cell death reaction and its relation to PCD in higher eukaryotes remains largely unknown. In Podospora anserina, genes induced during the cell death reaction triggered by the het-R het-V interaction have been identified and termed idi genes. Herein, we describe the functional characterization of one idi gene (idi-1) and explore the connection between incompatibility and the response to nutrient starvation. We show that IDI-1 is a cell wall protein which localizes at the septum during normal growth. We found that induction of idi-1 and of the other known idi genes is not specific of the incompatibility reaction. The idi genes are induced upon nitrogen and carbon starvation and by rapamycin, a specific inhibitor of the TOR kinase pathway. The cytological hallmarks of het-R het-V incompatibility (increased septation, vacuolization, coalescence of lipid droplets, induction of autophagy, and cell death) are also observed during rapamycin treatment. Globally the cytological alterations and modifications in gene expression occurring during the incompatibility reaction are similar to those observed during starvation or rapamycin treatment.