Versatile Endogenous Editing of GluRIIA in Drosophila melanogaster.

Glutamate receptors at the postsynaptic side translate neurotransmitter release from presynapses into postsynaptic excitation. They play a role in many forms of synaptic plasticity, e.g., homeostatic scaling of the receptor field, activity-dependent synaptic plasticity and the induction of presynaptic homeostatic potentiation (PHP). The latter process has been extensively studied at Drosophila melanogaster neuromuscular junctions (NMJs). The genetic removal of the glutamate receptor subunit IIA (GluRIIA) leads to an induction of PHP at the synapse. So far, mostly imprecise knockouts of the GluRIIA gene have been utilized. Furthermore, mutated and tagged versions of GluRIIA have been examined in the past, but most of these constructs were not expressed under endogenous regulatory control or involved the mentioned imprecise GluRIIA knockouts. We performed CRISPR/Cas9-assisted gene editing at the endogenous locus of GluRIIA. This enabled the investigation of the endogenous expression pattern of GluRIIA using tagged constructs with an EGFP and an ALFA tag for super-resolution immunofluorescence imaging, including structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). All GluRIIA constructs exhibited full functionality and PHP could be induced by philanthotoxin at control levels. By applying hierarchical clustering algorithms to analyze the dSTORM data, we detected postsynaptic receptor cluster areas of ~0.15 µm2. Consequently, our constructs are suitable for ultrastructural analyses of GluRIIA.

Nanoscaled RIM clustering at presynaptic active zones revealed by endogenous tagging.

Chemical synaptic transmission involves neurotransmitter release from presynaptic active zones (AZs). The AZ protein Rab-3-interacting molecule (RIM) is important for normal Ca2+-triggered release. However, its precise localization within AZs of the glutamatergic neuromuscular junctions of Drosophila melanogaster remains elusive. We used CRISPR/Cas9-assisted genome engineering of the rim locus to incorporate small epitope tags for targeted super-resolution imaging. A V5-tag, derived from simian virus 5, and an HA-tag, derived from human influenza virus, were N-terminally fused to the RIM Zinc finger. Whereas both variants are expressed in co-localization with the core AZ scaffold Bruchpilot, electrophysiological characterization reveals that AP-evoked synaptic release is disturbed in rimV5-Znf but not in rimHA-Znf In addition, rimHA-Znf synapses show intact presynaptic homeostatic potentiation. Combining super-resolution localization microscopy and hierarchical clustering, we detect ∼10 RIMHA-Znf subclusters with ∼13 nm diameter per AZ that are compacted and increased in numbers in presynaptic homeostatic potentiation.

Single-Molecule Localization Microscopy of Presynaptic Active Zones in Drosophila melanogaster after Rapid Cryofixation.

Single-molecule localization microscopy (SMLM) greatly advances structural studies of diverse biological tissues. For example, presynaptic active zone (AZ) nanotopology is resolved in increasing detail. Immunofluorescence imaging of AZ proteins usually relies on epitope preservation using aldehyde-based immunocompetent fixation. Cryofixation techniques, such as high-pressure freezing (HPF) and freeze substitution (FS), are widely used for ultrastructural studies of presynaptic architecture in electron microscopy (EM). HPF/FS demonstrated nearer-to-native preservation of AZ ultrastructure, e.g., by facilitating single filamentous structures. Here, we present a protocol combining the advantages of HPF/FS and direct stochastic optical reconstruction microscopy (dSTORM) to quantify nanotopology of the AZ scaffold protein Bruchpilot (Brp) at neuromuscular junctions (NMJs) of Drosophila melanogaster. Using this standardized model, we tested for preservation of Brp clusters in different FS protocols compared to classical aldehyde fixation. In HPF/FS samples, presynaptic boutons were structurally well preserved with ~22% smaller Brp clusters that allowed quantification of subcluster topology. In summary, we established a standardized near-to-native preparation and immunohistochemistry protocol for SMLM analyses of AZ protein clusters in a defined model synapse. Our protocol could be adapted to study protein arrangements at single-molecule resolution in other intact tissue preparations.

Does diabetes mellitus affect the safety profile of valproic acid for the treatment of status epilepticus? A retrospective cohort study.

BACKGROUND: In the treatment of status epilepticus less is known about the influence of comorbidities on the safety profile of anticonvulsive drugs. Especially patients with diabetes mellitus may be predisposed to certain adverse events that have been related to therapy with valproic acid. In this single-center retrospective cohort study we examined if the complications of the intravenous treatment with valproic acid is different in patients with or without diabetes. METHODS: Patients who were treated for status epilepticus with intravenous valproic acid between 2008 and 2020 were identified. Primary endpoint was the discontinuation of therapy with valproic acid due to adverse events. Relevant secondary endpoints were the functional status at the time of discharge from hospital in comparison to the premorbid state and the in-hospital mortality. Both groups (patients with or without diabetes) were compared by Mann-Whitney U-Test or Pearson´s Chi2 test. To identify therapy with valproic acid as a risk factor of in-hospital mortality, a binary regression model was used. RESULTS: During the study period 408 patients and 482 episodes of status epilepticus were treated with intravenous valproic acid. Group comparisons did not reveal a significant difference in the rates of discontinuation of therapy. A difference was found in the rate of thrombocytopenia (p = 0.015), which occurred more often in patients with diabetes. In total, 36 hypoglycemic episodes could be identified, two occurred spontaneously under intravenous valproic acid. After correction for potential confounders, continuous therapy with valproic acid could not be confirmed as an independent risk factor for in-hospital mortality (p = 0.079). In patients with diabetes, the proportion of patients with a good functional state, indicated by the modified Rankin Scale, was significantly lower in both times (premorbid: 55% vs. 69%, p = 0.008; at discharge: 22% vs. 36%, p = 0.004). CONCLUSIONS: Tolerability of the treatment with valproic acid was similar in patients with or without diabetes. Diabetes as a relevant comorbidity can signal a potentially increased risk of a poor outcome after status epilepticus. TRIAL REGISTRATION: The study was registered at the German Clinical Trials Register on 8 April 2022 (DRKS 00,027,836).

Altered gene expression profiles impair the nervous system development in individuals with 15q13.3 microdeletion.

The 15q13.3 microdeletion has pleiotropic effects ranging from apparently healthy to severely affected individuals. The underlying basis of the variable phenotype remains elusive. We analyzed gene expression using blood from three individuals with 15q13.3 microdeletion and brain cortex tissue from ten mice Df[h15q13]/+. We assessed differentially expressed genes (DEGs), protein-protein interaction (PPI) functional modules, and gene expression in brain developmental stages. The deleted genes' haploinsufficiency was not transcriptionally compensated, suggesting a dosage effect may contribute to the pathomechanism. DEGs shared between tested individuals and a corresponding mouse model show a significant overlap including genes involved in monogenic neurodevelopmental disorders. Yet, network-wide dysregulatory effects suggest the phenotype is not caused by a single critical gene. A significant proportion of blood DEGs, silenced in adult brain, have maximum expression during the prenatal brain development. Based on DEGs and their PPI partners we identified altered functional modules related to developmental processes, including nervous system development. We show that the 15q13.3 microdeletion has a ubiquitous impact on the transcriptome pattern, especially dysregulation of genes involved in brain development. The high phenotypic variability seen in 15q13.3 microdeletion could stem from an increased vulnerability during brain development, instead of a specific pathomechanism.

Improving one-step scarless genome editing in Drosophila melanogaster by combining ovoD co-CRISPR selection with sgRNA target site masking.

The precise and rapid construction of alleles through CRISPR/Cas9-mediated genome engineering renders Drosophila melanogaster a powerful animal system for molecular structure-function analyses and human disease models. Application of the ovoD co-selection method offers expedited generation and enrichment of scarlessly edited alleles without the need for linked transformation markers, which specifically in the case of exon editing can impact allele usability. However, we found that knockin procedures by homology-directed repair (HDR) under ovoD co-selection resulted in low transformation efficiency. This is likely due to repeated rounds of Cas9 cleavage of HDR donor and/or engineered genomic locus DNA, as noted for other CRISPR/Cas9 editing strategies before, impeding the recovery of correctly edited alleles. Here we provide a one-step protocol to improve the generation of scarless alleles by ovoD -co-selection with single-guide RNA (sgRNA) binding site masking. Using this workflow, we constructed human disease alleles for two Drosophila genes, unc-13/CG2999 and armadillo/CG11579. We show and quantify how a known countermeasure, the insertion of silent point mutations into protospacer adjacent motif (PAM) or sgRNA homology regions, can potently suppress unintended sequence modifications during CRISPR/Cas9 genome editing of D. melanogaster under ovoD co-selection. This strongly increased the recovery frequency of disease alleles.

The human cognition-enhancing CORD7 mutation increases active zone number and synaptic release.

Humans carrying the CORD7 (cone-rod dystrophy 7) mutation possess increased verbal IQ and working memory. This autosomal dominant syndrome is caused by the single-amino acid R844H exchange (human numbering) located in the 310 helix of the C2A domain of RIMS1/RIM1 (Rab3-interacting molecule 1). RIM is an evolutionarily conserved multi-domain protein and essential component of presynaptic active zones, which is centrally involved in fast, Ca2+-triggered neurotransmitter release. How the CORD7 mutation affects synaptic function has remained unclear thus far. Here, we established Drosophila melanogaster as a disease model for clarifying the effects of the CORD7 mutation on RIM function and synaptic vesicle release. To this end, using protein expression and X-ray crystallography, we solved the molecular structure of the Drosophila C2A domain at 1.92 Šresolution and by comparison to its mammalian homologue ascertained that the location of the CORD7 mutation is structurally conserved in fly RIM. Further, CRISPR/Cas9-assisted genomic engineering was employed for the generation of rim alleles encoding the R915H CORD7 exchange or R915E, R916E substitutions (fly numbering) to effect local charge reversal at the 310 helix. Through electrophysiological characterization by two-electrode voltage clamp and focal recordings we determined that the CORD7 mutation exerts a semi-dominant rather than a dominant effect on synaptic transmission resulting in faster, more efficient synaptic release and increased size of the readily releasable pool but decreased sensitivity for the fast calcium chelator BAPTA. In addition, the rim CORD7 allele increased the number of presynaptic active zones but left their nanoscopic organization unperturbed as revealed by super-resolution microscopy of the presynaptic scaffold protein Bruchpilot/ELKS/CAST. We conclude that the CORD7 mutation leads to tighter release coupling, an increased readily releasable pool size and more release sites thereby promoting more efficient synaptic transmitter release. These results strongly suggest that similar mechanisms may underlie the CORD7 disease phenotype in patients and that enhanced synaptic transmission may contribute to their increased cognitive abilities.

Endogenous tagging of Unc-13 reveals nanoscale reorganization at active zones during presynaptic homeostatic potentiation.

Introduction: Neurotransmitter release at presynaptic active zones (AZs) requires concerted protein interactions within a dense 3D nano-hemisphere. Among the complex protein meshwork the (M)unc-13 family member Unc-13 of Drosophila melanogaster is essential for docking of synaptic vesicles and transmitter release. Methods: We employ minos-mediated integration cassette (MiMIC)-based gene editing using GFSTF (EGFP-FlAsH-StrepII-TEV-3xFlag) to endogenously tag all annotated Drosophila Unc-13 isoforms enabling visualization of endogenous Unc-13 expression within the central and peripheral nervous system. Results and discussion: Electrophysiological characterization using two-electrode voltage clamp (TEVC) reveals that evoked and spontaneous synaptic transmission remain unaffected in unc-13 GFSTF 3rd instar larvae and acute presynaptic homeostatic potentiation (PHP) can be induced at control levels. Furthermore, multi-color structured-illumination shows precise co-localization of Unc-13GFSTF, Bruchpilot, and GluRIIA-receptor subunits within the synaptic mesoscale. Localization microscopy in combination with HDBSCAN algorithms detect Unc-13GFSTF subclusters that move toward the AZ center during PHP with unaltered Unc-13GFSTF protein levels.

Active zone compaction correlates with presynaptic homeostatic potentiation.

Neurotransmitter release is stabilized by homeostatic plasticity. Presynaptic homeostatic potentiation (PHP) operates on timescales ranging from minute- to life-long adaptations and likely involves reorganization of presynaptic active zones (AZs). At Drosophila melanogaster neuromuscular junctions, earlier work ascribed AZ enlargement by incorporating more Bruchpilot (Brp) scaffold protein a role in PHP. We use localization microscopy (direct stochastic optical reconstruction microscopy [dSTORM]) and hierarchical density-based spatial clustering of applications with noise (HDBSCAN) to study AZ plasticity during PHP at the synaptic mesoscale. We find compaction of individual AZs in acute philanthotoxin-induced and chronic genetically induced PHP but unchanged copy numbers of AZ proteins. Compaction even occurs at the level of Brp subclusters, which move toward AZ centers, and in Rab3 interacting molecule (RIM)-binding protein (RBP) subclusters. Furthermore, correlative confocal and dSTORM imaging reveals how AZ compaction in PHP translates into apparent increases in AZ area and Brp protein content, as implied earlier.

Targeted volumetric single-molecule localization microscopy of defined presynaptic structures in brain sections.

Revealing the molecular organization of anatomically precisely defined brain regions is necessary for refined understanding of synaptic plasticity. Although three-dimensional (3D) single-molecule localization microscopy can provide the required resolution, imaging more than a few micrometers deep into tissue remains challenging. To quantify presynaptic active zones (AZ) of entire, large, conditional detonator hippocampal mossy fiber (MF) boutons with diameters as large as 10 µm, we developed a method for targeted volumetric direct stochastic optical reconstruction microscopy (dSTORM). An optimized protocol for fast repeated axial scanning and efficient sequential labeling of the AZ scaffold Bassoon and membrane bound GFP with Alexa Fluor 647 enabled 3D-dSTORM imaging of 25 µm thick mouse brain sections and assignment of AZs to specific neuronal substructures. Quantitative data analysis revealed large differences in Bassoon cluster size and density for distinct hippocampal regions with largest clusters in MF boutons.