Dystroglycan Mediates Clustering of Essential GABAergic Components in Cerebellar Purkinje Cells.

Muscle dystrophin-glycoprotein complex (DGC) links the intracellular cytoskeleton to the extracellular matrix. In neurons, dystroglycan and dystrophin, two major components of the DGC, localize in a subset of GABAergic synapses, where their function is unclear. Here we used mouse models to analyze the specific role of the DGC in the organization and function of inhibitory synapses. Loss of full-length dystrophin in mdx mice resulted in a selective depletion of the transmembrane ß-dystroglycan isoform from inhibitory post-synaptic sites in cerebellar Purkinje cells. Remarkably, there were no differences in the synaptic distribution of the extracellular α-dystroglycan subunit, of GABAA receptors and neuroligin 2. In contrast, conditional deletion of the dystroglycan gene from Purkinje cells caused a disruption of the DGC and severely impaired post-synaptic clustering of neuroligin 2, GABAA receptors and scaffolding proteins. Accordingly, whole-cell patch-clamp analysis revealed a significant reduction in the frequency and amplitude of spontaneous IPSCs recorded from Purkinje cells. In the long-term, deletion of dystroglycan resulted in a significant decrease of GABAergic innervation of Purkinje cells and caused an impairment of motor learning functions. These results show that dystroglycan is an essential synaptic organizer at GABAergic synapses in Purkinje cells.

Mapping and phasing of structural variation in patient genomes using nanopore sequencing.

Despite improvements in genomics technology, the detection of structural variants (SVs) from short-read sequencing still poses challenges, particularly for complex variation. Here we analyse the genomes of two patients with congenital abnormalities using the MinION nanopore sequencer and a novel computational pipeline-NanoSV. We demonstrate that nanopore long reads are superior to short reads with regard to detection of de novo chromothripsis rearrangements. The long reads also enable efficient phasing of genetic variations, which we leveraged to determine the parental origin of all de novo chromothripsis breakpoints and to resolve the structure of these complex rearrangements. Additionally, genome-wide surveillance of inherited SVs reveals novel variants, missed in short-read data sets, a large proportion of which are retrotransposon insertions. We provide a first exploration of patient genome sequencing with a nanopore sequencer and demonstrate the value of long-read sequencing in mapping and phasing of SVs for both clinical and research applications.

Defining the diverse spectrum of inversions, complex structural variation, and chromothripsis in the morbid human genome.

BACKGROUND: Structural variation (SV) influences genome organization and contributes to human disease. However, the complete mutational spectrum of SV has not been routinely captured in disease association studies. RESULTS: We sequenced 689 participants with autism spectrum disorder (ASD) and other developmental abnormalities to construct a genome-wide map of large SV. Using long-insert jumping libraries at 105X mean physical coverage and linked-read whole-genome sequencing from 10X Genomics, we document seven major SV classes at ~5 kb SV resolution. Our results encompass 11,735 distinct large SV sites, 38.1% of which are novel and 16.8% of which are balanced or complex. We characterize 16 recurrent subclasses of complex SV (cxSV), revealing that: (1) cxSV are larger and rarer than canonical SV; (2) each genome harbors 14 large cxSV on average; (3) 84.4% of large cxSVs involve inversion; and (4) most large cxSV (93.8%) have not been delineated in previous studies. Rare SVs are more likely to disrupt coding and regulatory non-coding loci, particularly when truncating constrained and disease-associated genes. We also identify multiple cases of catastrophic chromosomal rearrangements known as chromoanagenesis, including somatic chromoanasynthesis, and extreme balanced germline chromothripsis events involving up to 65 breakpoints and 60.6 Mb across four chromosomes, further defining rare categories of extreme cxSV. CONCLUSIONS: These data provide a foundational map of large SV in the morbid human genome and demonstrate a previously underappreciated abundance and diversity of cxSV that should be considered in genomic studies of human disease.

Differential regulation of neurexin at glutamatergic and GABAergic synapses.

Neurexins (Nrxs) have emerged as potential determinants of synaptic specificity, but little is known about their localization at central synapses. Here we show that Nrxs have a remarkably selective localization at distinct types of glutamatergic synapses and we reveal an unexpected ontogenetic regulation of Nrx expression at GABAergic synapses. Our data indicate that synapses are specified by molecular interactions that involve both Nrx-dependent and Nrx-independent mechanisms. We propose that differences in the spatio-temporal profile of Nrx expression may contribute to specify the molecular identity of synapses.

Neuregulin1/ErbB4-induced migration in ST14A striatal progenitors: calcium-dependent mechanisms and modulation by NMDA receptor activation.

BACKGROUND: A number of studies have separately shown that the neuregulin1 (NRG1)/ErbB4 system and NMDA-type glutamate receptors (NMDARs) are involved in several aspects of neuronal migration. In addition, intracellular calcium fluctuations play central roles in neuronal motility. Stable expression of the tyrosine kinase receptor ErbB4 promotes migratory activity in the neural progenitor cell line ST14A upon NRG1 stimulation. In this work we analyzed the potential interactions between the NRG1/ErbB4 system and NMDARs in the ST14A migratory process as well as its calcium dependence. RESULTS: RT-PCR studies have shown that both native ST14A cells (non-expressing ErbB4), as well as ErbB4-transfected cells express low levels of a restricted number of NMDAR subunits: NR1, NR2C, NR2D and NR3B. The resulting NMDAR would form Ca(2+) channels characterized by low Mg(2+)-sensitivity and low Ca(2+)-permeability, generating small, long-lasting currents. Ca(2+)-imaging experiments showed slow [Ca(2+)](i) increases in 45% of the cells following 8 µM NMDA stimulation. Basal migration of ErbB4-transfected ST14A cells was unaffected by 18 hrs NMDA incubation. However, over the same incubation time, NMDA was able to significantly enhance NRG1-induced migration. Pre-incubation with the intracellular calcium chelator BAPTA-AM reduced both NRG1- and NRG1/NMDA-stimulated migration, suggesting the involvement of Ca(2+) in these processes. NRG1 stimulation of ErbB4-transfected ST14A cells induced a sustained, long-lasting increase in [Ca(2+)](i), in 99% of the cells. These intracellular Ca(2+) signals could be ascribed to both release from intracellular stores and influx from the extracellular medium trough a mechanism of store-operated calcium entry (SOCE). Short-time co-incubation of NMDA and NRG1 did not substantially modify the NRG1-induced intracellular calcium signals. CONCLUSIONS: In summary, NRG1 stimulation of the ErbB4 receptor exerts a sustained [Ca(2+)](i) increase in ST14A neural progenitors; NRG1-induced migration is Ca(2+)-dependent and can be positively modulated by activation of the NMDA receptor.

Understanding the molecular diversity of GABAergic synapses.

GABAergic synapses exhibit a high degree of subcellular and molecular specialization, which contrasts with their apparent simplicity in ultrastructural appearance. Indeed, when observed in the electron microscope, GABAergic synapses fit in the symmetric, or Gray's type II category, being characterized by a relatively simple postsynaptic specialization. The inhibitory postsynaptic density cannot be readily isolated, and progress in understanding its molecular composition has lagged behind that of excitatory synapses. However, recent studies have brought significant progress in the identification of new synaptic proteins, revealing an unexpected complexity in the molecular machinery that regulates GABAergic synaptogenesis. In this article, we provide an overview of the molecular diversity of GABAergic synapses, and we consider how synapse specificity may be encoded by selective trans-synaptic interactions between pre- and postsynaptic adhesion molecules and secreted factors that reside in the synaptic cleft. We also discuss the importance of developing cataloguing tools that could be used to decipher the molecular diversity of synapses and to predict alterations of inhibitory transmission in the course of neurological diseases.