The adhesion G-protein-coupled receptor mayo/CG11318 controls midgut development in Drosophila.

Adhesion G-protein-coupled receptors (aGPCRs) form a large family of cell surface molecules with versatile tasks in organ development. Many aGPCRs still await their functional and pharmacological deorphanization. Here, we characterized the orphan aGPCR CG11318/mayo of Drosophila melanogaster and found it expressed in specific regions of the gastrointestinal canal and anal plates, epithelial specializations that control ion homeostasis. Genetic removal of mayo results in tachycardia, which is caused by hyperkalemia of the larval hemolymph. The hyperkalemic effect can be mimicked by a raise in ambient potassium concentration, while normal potassium levels in mayoKO mutants can be restored by pharmacological inhibition of potassium channels. Intriguingly, hyperkalemia and tachycardia are caused non-cell autonomously through mayo-dependent control of enterocyte proliferation in the larval midgut, which is the primary function of this aGPCR. These findings characterize the ancestral aGPCR Mayo as a homeostatic regulator of gut development.

Molecular sensing of mechano- and ligand-dependent adhesion GPCR dissociation.

Adhesion G-protein-coupled receptors (aGPCRs) bear notable similarity to Notch proteins1, a class of surface receptors poised for mechano-proteolytic activation2-4, including an evolutionarily conserved mechanism of cleavage5-8. However, so far there is no unifying explanation for why aGPCRs are autoproteolytically processed. Here we introduce a genetically encoded sensor system to detect the dissociation events of aGPCR heterodimers into their constituent N-terminal and C-terminal fragments (NTFs and CTFs, respectively). An NTF release sensor (NRS) of the neural latrophilin-type aGPCR Cirl (ADGRL)9-11, from Drosophila melanogaster, is stimulated by mechanical force. Cirl-NRS activation indicates that receptor dissociation occurs in neurons and cortex glial cells. The release of NTFs from cortex glial cells requires trans-interaction between Cirl and its ligand, the Toll-like receptor Tollo (Toll-8)12, on neural progenitor cells, whereas expressing Cirl and Tollo in cis suppresses dissociation of the aGPCR. This interaction is necessary to control the size of the neuroblast pool in the central nervous system. We conclude that receptor autoproteolysis enables non-cell-autonomous activities of aGPCRs, and that the dissociation of aGPCRs is controlled by their ligand expression profile and by mechanical force. The NRS system will be helpful in elucidating the physiological roles and signal modulators of aGPCRs, which constitute a large untapped reservoir of drug targets for cardiovascular, immune, neuropsychiatric and neoplastic diseases13.

p53-dependent c-Fos expression is a marker but not executor for motor neuron death in spinal muscular atrophy mouse models.

The activation of the p53 pathway has been associated with neuronal degeneration in different neurological disorders, including spinal muscular atrophy (SMA) where aberrant expression of p53 drives selective death of motor neurons destined to degenerate. Since direct p53 inhibition is an unsound therapeutic approach due carcinogenic effects, we investigated the expression of the cell death-associated p53 downstream targets c-fos, perp and fas in vulnerable motor neurons of SMA mice. Fluorescence in situ hybridization (FISH) of SMA motor neurons revealed c-fos RNA as a promising candidate. Accordingly, we identified p53-dependent nuclear upregulation of c-Fos protein in degenerating motor neurons from the severe SMNΔ7 and intermediate Smn2B/- SMA mouse models. Although motor neuron-specific c-fos genetic deletion in SMA mice did not improve motor neuron survival or motor behavior, p53-dependent c-Fos upregulation marks vulnerable motor neurons in different mouse models. Thus, nuclear c-Fos accumulation may serve as a readout for therapeutic approaches targeting neuronal death in SMA and possibly other p53-dependent neurodegenerative diseases.

Antisense oligonucleotide silencing of FUS expression as a therapeutic approach in amyotrophic lateral sclerosis.

Fused in sarcoma (FUS) is an RNA-binding protein that is genetically and pathologically associated with rare and aggressive forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). To explore the mechanisms by which mutant FUS causes neurodegeneration in ALS-FTD, we generated a series of FUS knock-in mouse lines that express the equivalent of ALS-associated mutant FUSP525L and FUSΔEX14 protein. In FUS mutant mice, we show progressive, age-dependent motor neuron loss as a consequence of a dose-dependent gain of toxic function, associated with the insolubility of FUS and related RNA-binding proteins. In this disease-relevant mouse model of ALS-FUS, we show that ION363, a non-allele-specific FUS antisense oligonucleotide, efficiently silences Fus and reduces postnatal levels of FUS protein in the brain and spinal cord, delaying motor neuron degeneration. In a patient with ALS with a FUSP525L mutation, we provide preliminary evidence that repeated intrathecal infusions of ION363 lower wild-type and mutant FUS levels in the central nervous system, resulting in a marked reduction in the burden of FUS aggregates that are a pathological hallmark of disease. In mouse genetic and human clinical studies, we provide evidence in support of FUS silencing as a therapeutic strategy in FUS-dependent ALS and FTD.

Central synaptopathy is the most conserved feature of motor circuit pathology across spinal muscular atrophy mouse models.

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by reduced survival motor neuron (SMN) protein. Recently, SMN dysfunction has been linked to individual aspects of motor circuit pathology in a severe SMA mouse model. To determine whether these disease mechanisms are conserved, we directly compared the motor circuit pathology of three SMA mouse models. The severe SMNΔ7 model exhibits vast motor circuit defects, including degeneration of motor neurons, spinal excitatory synapses, and neuromuscular junctions (NMJs). In contrast, the Taiwanese model shows very mild motor neuron pathology, but early central synaptic loss. In the intermediate Smn 2B/- model, strong pathology of central excitatory synapses and NMJs precedes the late onset of p53-dependent motor neuron death. These pathological events correlate with SMN-dependent splicing dysregulation of specific mRNAs. Our study provides a knowledge base for properly tailoring future studies and identifies central excitatory synaptopathy as a key feature of motor circuit pathology in SMA.

Chronic Pharmacological Increase of Neuronal Activity Improves Sensory-Motor Dysfunction in Spinal Muscular Atrophy Mice.

Dysfunction of neuronal circuits is an important determinant of neurodegenerative diseases. Synaptic dysfunction, death, and intrinsic activity of neurons are thought to contribute to the demise of normal behavior in the disease state. However, the interplay between these major pathogenic events during disease progression is poorly understood. Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by a deficiency in the ubiquitously expressed protein SMN and is characterized by motor neuron death, skeletal muscle atrophy, as well as dysfunction and loss of both central and peripheral excitatory synapses. These disease hallmarks result in an overall reduction of neuronal activity in the spinal sensory-motor circuit. Here, we show that increasing neuronal activity by chronic treatment with the FDA-approved potassium channel blocker 4-aminopyridine (4-AP) improves motor behavior in both sexes of a severe mouse model of SMA. 4-AP restores neurotransmission and number of proprioceptive synapses and neuromuscular junctions (NMJs), while having no effects on motor neuron death. In addition, 4-AP treatment with pharmacological inhibition of p53-dependent motor neuron death results in additive effects, leading to full correction of sensory-motor circuit pathology and enhanced phenotypic benefit in SMA mice. Our in vivo study reveals that 4-AP-induced increase of neuronal activity restores synaptic connectivity and function in the sensory-motor circuit to improve the SMA motor phenotype.SIGNIFICANCE STATEMENT Spinal muscular atrophy (SMA) is a neurodegenerative disease, characterized by synaptic loss, motor neuron death, and reduced neuronal activity in spinal sensory-motor circuits. However, whether these are parallel or dependent events is unclear. We show here that long-term increase of neuronal activity by the FDA-approved drug 4-aminopyridine (4-AP) rescues the number and function of central and peripheral synapses in a SMA mouse model, resulting in an improvement of the sensory-motor circuit and motor behavior. Combinatorial treatment of pharmacological inhibition of p53, which is responsible for motor neuron death and 4-AP, results in additive beneficial effects on the sensory-motor circuit in SMA. Thus, neuronal activity restores synaptic connections and improves significantly the severe SMA phenotype.

Implications of the Sap47 null mutation for synapsin phosphorylation, longevity, climbing proficiency and behavioural plasticity in adult Drosophila.

The Sap47 gene of Drosophila melanogaster encodes a highly abundant 47 kDa synaptic vesicle-associated protein. Sap47 null mutants show defects in synaptic plasticity and larval olfactory associative learning but the molecular function of Sap47 at the synapse is unknown. We demonstrate that Sap47 modulates the phosphorylation of another highly abundant conserved presynaptic protein, synapsin. Site-specific phosphorylation of Drosophila synapsin has repeatedly been shown to be important for behavioural plasticity but it was not known where these phospho-synapsin isoforms are localized in the brain. Here, we report the distribution of serine-6-phosphorylated synapsin in the adult brain and show that it is highly enriched in rings of synapses in the ellipsoid body and in large synapses near the lateral triangle. The effects of knockout of Sap47 or synapsin on olfactory associative learning/memory support the hypothesis that both proteins operate in the same molecular pathway. We therefore asked if this might also be true for other aspects of their function. We show that knockout of Sap47 but not synapsin reduces lifespan, whereas knockout of Sap47 and synapsin, either individually or together, affects climbing proficiency, as well as plasticity in circadian rhythms and sleep. Furthermore, electrophysiological assessment of synaptic properties at the larval neuromuscular junction (NMJ) reveals increased spontaneous synaptic vesicle fusion and reduced paired pulse facilitation in Sap47 and synapsin single and double mutants. Our results imply that Sap47 and synapsin cooperate non-uniformly in the control of synaptic properties in different behaviourally relevant neuronal networks of the fruitfly.

Parallel Genomic Engineering of Two Drosophila Genes Using Orthogonal attB/attP Sites.

Precise modification of sequences in the Drosophila melanogaster genome underlies the powerful capacity to study molecular structure-function relationships in this model species. The emergence of CRISPR/Cas9 tools in combination with recombinase systems such as the bacteriophage serine integrase ΦC31 has rendered Drosophila mutagenesis a straightforward enterprise for deleting, inserting and modifying genetic elements to study their functional relevance. However, while combined modifications of non-linked genetic elements can be easily constructed with these tools and classical genetics, the independent manipulation of linked genes through the established ΦC31-mediated transgenesis pipeline has not been feasible due to the limitation to one attB/attP site pair. Here we extend the repertoire of ΦC31 transgenesis by introducing a second pair of attB/attP targeting and transgenesis vectors that operate in parallel and independently of existing tools. We show that two syntenic orthologous genes, CG11318 and CG15556, located within a 25 kb region can be genomically engineered to harbor attPTT and attPCC sites. These landing pads can then independently receive transgenes through ΦC31-assisted integration and facilitate the manipulation and analysis of either gene in the same animal. These results expand the repertoire of site-specific genomic engineering in Drosophila while retaining the well established advantages and utility of the ΦC31 transgenesis system.

Synapsin function in GABA-ergic interneurons is required for short-term olfactory habituation.

In Drosophila, short-term (STH) and long-term habituation (LTH) of olfactory avoidance behavior are believed to arise from the selective potentiation of GABAergic synapses between multiglomerular local circuit interneurons (LNs) and projection neurons in the antennal lobe. However, the underlying mechanisms remain poorly understood. Here, we show that synapsin (syn) function is necessary for STH and that syn(97)-null mutant defects in STH can be rescued by syn(+) cDNA expression solely in the LN1 subset of GABAergic local interneurons. As synapsin is a synaptic vesicle-clustering phosphoprotein, these observations identify a presynaptic mechanism for STH as well as the inhibitory interneurons in which this mechanism is deployed. Serine residues 6 and/or 533, potential kinase target sites of synapsin, are necessary for synapsin function suggesting that synapsin phosphorylation is essential for STH. Consistently, biochemical analyses using a phospho-synapsin-specific antiserum show that synapsin is a target of Ca(2+) calmodulin-dependent kinase II (CaMKII) phosphorylation in vivo. Additional behavioral and genetic observations demonstrate that CaMKII function is necessary in LNs for STH. Together, these data support a model in which CaMKII-mediated synapsin phosphorylation in LNs induces synaptic vesicle mobilization and thereby presynaptic facilitation of GABA release that underlies olfactory STH. Finally, the striking observation that LTH occurs normally in syn(97) mutants indicates that signaling pathways for STH and LTH diverge upstream of synapsin function in GABAergic interneurons.

Identification and structural characterization of interneurons of the Drosophila brain by monoclonal antibodies of the würzburg hybridoma library.

Several novel synaptic proteins have been identified by monoclonal antibodies (mAbs) of the Würzburg hybridoma library generated against homogenized Drosophila brains, e.g. cysteine string protein, synapse-associated protein of 47 kDa, and Bruchpilot. However, at present no routine technique exists to identify the antigens of mAbs of our library that label only a small number of cells in the brain. Yet these antibodies can be used to reproducibly label and thereby identify these cells by immunohistochemical staining. Here we describe the staining patterns in the Drosophila brain for ten mAbs of the Würzburg hybridoma library. Besides revealing the neuroanatomical structure and distribution of ten different sets of cells we compare the staining patterns with those of antibodies against known antigens and GFP expression patterns driven by selected Gal4 lines employing regulatory sequences of neuronal genes. We present examples where our antibodies apparently stain the same cells in different Gal4 lines suggesting that the corresponding regulatory sequences can be exploited by the split-Gal4 technique for transgene expression exclusively in these cells. The detection of Gal4 expression in cells labeled by mAbs may also help in the identification of the antigens recognized by the antibodies which then in addition to their value for neuroanatomy will represent important tools for the characterization of the antigens. Implications and future strategies for the identification of the antigens are discussed.