Stathmin is required for stability of the drosophila neuromuscular junction

Synaptic connections can be stably maintained for prolonged periods, yet can be rapidly disassembled during the developmental refinement of neural circuitry and following cytological insults that lead to neurodegeneration. To date, the molecular mechanisms that determine whether a synapse will persist versus being remodeled or eliminated remain poorly understood. Mutations in Drosophila stathmin were isolated in two independent genetic screens that sought mutations leading to impaired synapse stability at the Drosophila neuromuscular junction (NMJ). Here we demonstrate that Stathmin, a protein that associates with microtubules and can function as a point of signaling integration, is necessary to maintain the stability of the Drosophila NMJ. We show that Stathmin protein is widely distributed within motoneurons and that loss of Stathmin causes impaired NMJ growth and stability. In addition, we show that stathmin mutants display evidence of defective axonal transport, a common feature associated with neuronal degeneration and altered synapse stability. The disassembly of the NMJ in stathmin includes a predictable sequence of cytological events, suggesting that a common program of synapse disassembly is induced following the loss of Stathmin protein. These data define a required function for Stathmin during synapse maintenance in a model system in which there is only a single stathmin gene, enabling future genetic investigation of Stathmin function with potential relevance to the cause and progression of neuromuscular degenerative disease

Rab3-GEF controls active zone development at the Drosophila neuromuscular junction

Synaptic signaling involves the release of neurotransmitter from presynaptic active zones (AZs). Proteins that regulate vesicle exocytosis cluster at AZs, composing the cytomatrix at the active zone (CAZ). At the Drosophila neuromuscular junction (NMJ), the small GTPase Rab3 controls the distribution of CAZ proteins across release sites, thereby regulating the efficacy of individual AZs. Here we identify Rab3-GEF as a second protein that acts in conjunction with Rab3 to control AZ protein composition. At rab3-GEF mutant NMJs, Bruchpilot (Brp) and Ca(2+) channels are enriched at a subset of AZs, leaving the remaining sites devoid of key CAZ components in a manner that is indistinguishable from rab3 mutant NMJs. As the Drosophila homologue of mammalian DENN/MADD and Caenorhabditis elegans AEX-3, Rab3-GEF is a guanine nucleotide exchange factor (GEF) for Rab3 that stimulates GDP to GTP exchange. Mechanistic studies reveal that although Rab3 and Rab3-GEF act within the same mechanism to control AZ development, Rab3-GEF is involved in multiple roles. We show that Rab3-GEF is required for transport of Rab3. However, the synaptic phenotype in the rab3-GEF mutant cannot be fully explained by defective transport and loss of GEF activity. A transgenically expressed GTP-locked variant of Rab3 accumulates at the NMJ at wild-type levels and fully rescues the rab3 mutant but is unable to rescue the rab3-GEF mutant. Our results suggest that although Rab3-GEF acts upstream of Rab3 to control Rab3 localization and likely GTP-binding, it also acts downstream to regulate CAZ development, potentially as a Rab3 effector at the synapse

RIM promotes calcium channel accumulation at active zones of the Drosophila neuromuscular junction

Synaptic communication requires the controlled release of synaptic vesicles from presynaptic axon terminals. Release efficacy is regulated by the many proteins that comprise the presynaptic release apparatus, including Ca(2+) channels and proteins that influence Ca(2+) channel accumulation at release sites. Here we identify Drosophila RIM and demonstrate that it localizes to active zones at the larval neuromuscular junction. In Drosophila RIM mutants, there is a large decrease in evoked synaptic transmission, due to a significant reduction in both the clustering of Ca(2+) channels and the size of the readily releasable pool of synaptic vesicles at active zones. Hence, RIM plays an evolutionarily conserved role in regulating synaptic calcium channel localization and readily releasable pool size. Since RIM has traditionally been studied as an effector of Rab3 function, we investigate whether RIM is involved in the newly identified function of Rab3 in the distribution of presynaptic release machinery components across release sites. Bruchpilot (Brp), an essential component of the active zone cytomatrix T bar, is unaffected by RIM disruption, indicating that Brp localization and distribution across active zones does not require wild type RIM. In addition, larvae containing mutations in both RIM and rab3 have reduced Ca(2+) channel levels and a Brp distribution that is very similar to that of the rab3 single mutant, indicating that RIM functions to regulate Ca(2+) channel accumulation but is not a Rab3 effector for release machinery distribution across release sites

Genomic Biomarkers and Genome-Wide Loss-of-Heterozygosity Scores in Metastatic Prostate Cancer Following Progression on Androgen-Targeting Therapies

Genomic biomarkers; Prostate cancer; Targeting therapiesBiomarcadores genómicos; Cáncer de próstata; Terapias dirigidasBiomarcadors genòmics; Càncer de pròstata; Teràpies dirigidesPURPOSE To study the impact of standard-of-care hormonal therapies on metastatic prostate cancer (mPC) clinical genomic profiles in real-world practice, with a focus on homologous recombination-repair (HRR) genes. PATIENTS AND METHODS Targeted next-generation sequencing of 1,302 patients with mPC was pursued using the FoundationOne or FoundationOne CDx assays. Longitudinal clinical data for correlative analysis were curated via technology-enabled abstraction of electronic health records. Genomic biomarkers, including individual gene aberrations and genome-wide loss-of-heterozygosity (gLOH) scores, were compared according to biopsy location and time of sample acquisition (androgen deprivation therapy [ADT]-naïve, ADT-progression and post-ADT, and novel hormonal therapies [NHT]-progression), using chi-square and Wilcoxon rank-sum tests. Multivariable analysis used linear regression. False-discovery rate of 0.05 was applied to account for multiple comparisons. RESULTS Eight hundred forty (65%), 132 (10%), and 330 (25%) biopsies were ADT-naïve, ADT-progression, and NHT-progression, respectively. Later-stage samples were enriched for AR, MYC, TP53, PTEN, and RB1 aberrations (all adjusted P values < .05), but prevalence of HRR-related BRCA2, ATM, and CDK12 aberrations remained stable. Primary and metastatic ADT-naïve biopsies presented similar prevalence of TP53 (36% v 31%) and BRCA2 (8% v 7%) aberrations; 81% of ADT-naïve BRCA2-mutated samples presented BRCA2 biallelic loss. Higher gLOH scores were independently associated with HRR genes (BRCA2, PALB2, and FANCA), TP53, and RB1 aberrations, and with prior exposure to hormonal therapies in multivariable analysis. CONCLUSION Prevalence of HRR-gene aberrations remains stable along mPC progression, supporting the use of diagnostic biopsies to guide poly (ADP-ribose) polymerase inhibitor treatment in metastatic castration-resistant prostate cancer. gLOH scores increase with emerging resistance to hormonal therapies, independently of individual HRR gene mutations

Network Structure Implied by Initial Axon Outgrowth in Rodent Cortex: Empirical Measurement and Models

The developmental mechanisms by which the network organization of the adult cortex is established are incompletely understood. Here we report on empirical data on the development of connections in hamster isocortex and use these data to parameterize a network model of early cortical connectivity. Using anterograde tracers at a series of postnatal ages, we investigate the growth of connections in the early cortical sheet and systematically map initial axon extension from sites in anterior (motor), middle (somatosensory) and posterior (visual) cortex. As a general rule, developing axons extend from all sites to cover relatively large portions of the cortical field that include multiple cortical areas. From all sites, outgrowth is anisotropic, covering a greater distance along the medial/lateral axis than along the anterior/posterior axis. These observations are summarized as 2-dimensional probability distributions of axon terminal sites over the cortical sheet. Our network model consists of nodes, representing parcels of cortex, embedded in 2-dimensional space. Network nodes are connected via directed edges, representing axons, drawn according to the empirically derived anisotropic probability distribution. The networks generated are described by a number of graph theoretic measurements including graph efficiency, node betweenness centrality and average shortest path length. To determine if connectional anisotropy helps reduce the total volume occupied by axons, we define and measure a simple metric for the extra volume required by axons crossing. We investigate the impact of different levels of anisotropy on network structure and volume. The empirically observed level of anisotropy suggests a good trade-off between volume reduction and maintenance of both network efficiency and robustness. Future work will test the model's predictions for connectivity in larger cortices to gain insight into how the regulation of axonal outgrowth may have evolved to achieve efficient and economical connectivity in larger brains

Data from: Mutational analysis of Rab3 function for controlling active zone protein composition at the Drosophila neuromuscular junction

At synapses, the release of neurotransmitter is regulated by molecular machinery that aggregates at specialized presynaptic release sites termed active zones. The complement of active zone proteins at each site is a determinant of release efficacy and can be remodeled to alter synapse function. The small GTPase Rab3 was previously identified as playing a novel role that controls the distribution of active zone proteins to individual release sites at the Drosophila neuromuscular junction. Rab3 has been extensively studied for its role in the synaptic vesicle cycle; however, the mechanism by which Rab3 controls active zone development remains unknown. To explore this mechanism, we conducted a mutational analysis to determine the molecular and structural requirements of Rab3 function at Drosophila synapses. We find that GTP-binding is required for Rab3 to traffick to synapses and distribute active zone components across release sites. Conversely, the hydrolytic activity of Rab3 is unnecessary for this function. Through a structure-function analysis we identify specific residues within the effector-binding switch regions that are required for Rab3 function and determine that membrane attachment is essential. Our findings suggest that Rab3 controls the distribution of active zone components via a vesicle docking mechanism that is consistent with standard Rab protein function

CDR Region Mutation Brp Distribution Rescue

Zipped folder contains Multichannel TIFFs. Explanation of files included as .txt file in Zipped Folde

Q80L Rab3 Intensity

Zipped folder contains Multichannel TIFFs. Explanation of files included as .txt file in Zipped Folde

Q80L Brp Distribution Rescue

Zipped folder contains Multichannel TIFFs. Explanation of files included as .txt file in Zipped Folde

Traces for Facilitation Index Quantification

Zipped folder contains .ABF Files. Explanation of files included as .txt file in Zipped Folde