Reelin Mobilizes a VAMP7-Dependent Synaptic Vesicle Pool and Selectively Augments Spontaneous Neurotransmission

SummaryReelin is a glycoprotein that is critical for proper layering of neocortex during development as well as dynamic regulation of glutamatergic postsynaptic signaling in mature synapses. Here, we show that Reelin also acts presynaptically, resulting in robust rapid enhancement of spontaneous neurotransmitter release without affecting properties of evoked neurotransmission. This effect of Reelin requires a modest but significant increase in presynaptic Ca2+ initiated via ApoER2 signaling. The specificity of Reelin action on spontaneous neurotransmitter release is encoded at the level of vesicular SNARE machinery as it requires VAMP7 and SNAP-25 but not synaptobrevin2, VAMP4, or vti1a. These results uncover a presynaptic regulatory pathway that utilizes the heterogeneity of synaptic vesicle-associated SNAREs and selectively augments action potential-independent neurotransmission

Visualization and Quantification of Post-Stroke Neural Connectivity and Neuroinflammation Using Serial Two-Photon Tomography in the Whole Mouse Brain

Whole-brain volumetric microscopy techniques such as serial two-photon tomography (STPT) can provide detailed information on the roles of neuroinflammation and neuroplasticity throughout the whole brain post-stroke. STPT automatically generates high-resolution images of coronal sections of the entire mouse brain that can be readily visualized in three dimensions. We developed a pipeline for whole brain image analysis that includes supervised machine learning (pixel-wise random forest models via the “ilastik” software package) followed by registration to a standardized 3-D atlas of the adult mouse brain (Common Coordinate Framework v3.0; Allen Institute for Brain Science). These procedures allow the detection of cellular fluorescent signals throughout the brain in an unbiased manner. To illustrate our imaging techniques and automated image quantification, we examined long-term post-stroke motor circuit connectivity in mice that received a motor cortex photothrombotic stroke. Two weeks post-stroke, mice received intramuscular injections of pseudorabies virus (PRV-152), a trans-synaptic retrograde herpes virus driving expression of green fluorescent protein (GFP), into the affected contralesional forelimb to label neurons in descending tracts to the forelimb musculature. Mice were sacrificed 3 weeks post-stroke. We also quantified sub-acute neuroinflammation in the post-stroke brain in a separate cohort of mice following a 60 min transient middle cerebral artery occlusion (tMCAo). Naive e450+-labeled splenic CD8+ cytotoxic T cells were intravenously injected at 7, 24, 48, and 72 h post-tMCAo. Mice were sacrificed 4 days after stroke. Detailed quantification of post-stroke neural connectivity and neuroinflammation indicates a role for remote brain regions in stroke pathology and recovery. The workflow described herein, incorporating STPT and automated quantification of fluorescently labeled features of interest, provides a framework by which one can objectively evaluate labeled neuronal or lymphocyte populations in healthy and injured brains. The results provide region-specific quantification of neural connectivity and neuroinflammation, which could be a critical tool for investigating mechanisms of not only stroke recovery, but also a wide variety of brain injuries or diseases

B Cells Migrate into Remote Brain Areas and Support Neurogenesis and Functional Recovery after Focal Stroke in Mice

Lymphocytes infiltrate the stroke core and penumbra and often exacerbate cellular injury. B cells, however, are lymphocytes that do not contribute to acute pathology but can support recovery. B cell adoptive transfer to mice reduced infarct volumes 3 and 7 d after transient middle cerebral artery occlusion (tMCAo), independent of changing immune populations in recipient mice. Testing a direct neurotrophic effect, B cells cocultured with mixed cortical cells protected neurons and maintained dendritic arborization after oxygen-glucose deprivation. Whole-brain volumetric serial two-photon tomography (STPT) and a custom-developed image analysis pipeline visualized and quantified poststroke B cell diapedesis throughout the brain, including remote areas supporting functional recovery. Stroke induced significant bilateral B cell diapedesis into remote brain regions regulating motor and cognitive functions and neurogenesis (e.g., dentate gyrus, hypothalamus, olfactory areas, cerebellum) in the whole-brain datasets. To confirm a mechanistic role for B cells in functional recovery, rituximab was given to human CD20+ (hCD20+) transgenic mice to continuously deplete hCD20+-expressing B cells following tMCAo. These mice experienced delayed motor recovery, impaired spatial memory, and increased anxiety through 8 wk poststroke compared to wild type (WT) littermates also receiving rituximab. B cell depletion reduced stroke-induced hippocampal neurogenesis and cell survival. Thus, B cell diapedesis occurred in areas remote to the infarct that mediated motor and cognitive recovery. Understanding the role of B cells in neuronal health and disease-based plasticity is critical for developing effective immune-based therapies for protection against diseases that involve recruitment of peripheral immune cells into the injured brain

VAMP4 directs synaptic vesicles to a pool that selectively maintains asynchronous neurotransmission

Synaptic vesicles in the brain harbor several soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) proteins. With the exception of synaptobrevin2, or VAMP2 (syb2), which is directly involved in vesicle fusion, the role of these SNAREs in neurotransmission is unclear. Here we show that in mice syb2 drives rapid Ca2+-dependent synchronous neurotransmission, whereas the structurally homologous SNARE protein VAMP4 selectively maintains bulk Ca2+-dependent asynchronous release. At inhibitory nerve terminals, up- or downregulation of VAMP4 causes a correlated change in asynchronous release. Biochemically, VAMP4 forms a stable complex with SNAREs syntaxin-1 and SNAP-25 that does not interact with complexins or synaptotagmin-1, proteins essential for synchronous neurotransmission. Optical imaging of individual synapses indicates that trafficking of VAMP4 and syb2 show minimal overlap. Taken together, these findings suggest that VAMP4 and syb2 diverge functionally, traffic independently and support distinct forms of neurotransmission. These results provide molecular insight into how synapses diversify their release properties by taking advantage of distinct synaptic vesicle–associated SNAREs

Erratum: Author Correction: Identification of genes required for eye development by high-throughput screening of mouse knockouts.

[This corrects the article DOI: 10.1038/s42003-018-0226-0.]

BMD loci contribute to ethnic and developmental differences in skeletal fragility across populations: Assessment of evolutionary selection pressures

Bone mineral density (BMD) is a highly heritable trait used both for the diagnosis of osteoporosis in adults and to assess bone health in children. Ethnic differences in BMD have been documented, with markedly higher levels in individuals of African descent, which partially explain disparity in osteoporosis risk across populations. To date, 63 independent genetic variants have been associated with BMD in adults of Northern-European ancestry. Here, we demonstrate that at least 61 of these variants are predictive of BMD early in life by studying their compound effect within two multiethnic pediatric cohorts. Furthermore, we show that within these cohorts and across populations worldwide the frequency of those alleles associated with increased BMD is systematically elevated in individuals of Sub-Saharan African ancestry. The amount of differentiation in the BMD genetic scores among Sub-Saharan and non-Sub-Saharan populations together with neutrality tests, suggest that these allelic differences are compatible with the hypothesis of selective pressures acting on the genetic determinants of BMD. These findings constitute an explorative contribution to the role of selection on ethnic BMD differences and likely a new example of polygenic adaptation acting on a human trait

Genomic, Pathway Network, and Immunologic Features Distinguishing Squamous Carcinomas

This integrated, multiplatform PanCancer Atlas study co-mapped and identified distinguishing molecular features of squamous cell carcinomas (SCCs) from five sites associated with smokin

Pan-Cancer Analysis of lncRNA Regulation Supports Their Targeting of Cancer Genes in Each Tumor Context

Long noncoding RNAs (lncRNAs) are commonly dys-regulated in tumors, but only a handful are known toplay pathophysiological roles in cancer. We inferredlncRNAs that dysregulate cancer pathways, onco-genes, and tumor suppressors (cancer genes) bymodeling their effects on the activity of transcriptionfactors, RNA-binding proteins, and microRNAs in5,185 TCGA tumors and 1,019 ENCODE assays.Our predictions included hundreds of candidateonco- and tumor-suppressor lncRNAs (cancerlncRNAs) whose somatic alterations account for thedysregulation of dozens of cancer genes and path-ways in each of 14 tumor contexts. To demonstrateproof of concept, we showed that perturbations tar-geting OIP5-AS1 (an inferred tumor suppressor) andTUG1 and WT1-AS (inferred onco-lncRNAs) dysre-gulated cancer genes and altered proliferation ofbreast and gynecologic cancer cells. Our analysis in-dicates that, although most lncRNAs are dysregu-lated in a tumor-specific manner, some, includingOIP5-AS1, TUG1, NEAT1, MEG3, and TSIX, synergis-tically dysregulate cancer pathways in multiple tumorcontexts