Glia Are Required For Sensory Neuron Morphology And Function In Caenorhabditis elegans

The nervous system emerges from the coordinated development of neurons and glia. To better understand the processes that enable nervous system development and function we have studied the sensory organs of Caenorhabditis elegans because their anatomy and function are well-characterized. Specifically, we have focused on two aspects of sensory organs: how do glia interact with neurons to enable proper development and function and how are sensory cilia generated. To uncover any glial roles, we ablated the major glial cell of the amphid sensilla. Embryonic glial ablation did not affect neuronal survival and resulted in sensory neuron dendrites that were far too short, revealing a glial role in anchoring sensory neuron dendrites. To examine post-developmental glial roles, we ablated glia after the amphid sensory organ was fully formed. These glia-ablated animals exhibited profound sensory deficits as determined by behavioral assays, failed to maintain the proper morphology of some modified sensory cilia, and had defects in neuronal uptake of lipophilic dyes. Further, animals lacking glia showed no Ca2+ responses in the ASH sensory neuron after stimulation with a high osmolarity solution. To understand the molecular bases of these glial activities, we characterized a sheath glia expressed gene, fig-1, that encodes a protein with thrombospondin type I domains. FIG-1 likely functions extracellularly, is essential for neuronal dye uptake, and also affects behavior. To characterize the molecular basis of cilia morphogenesis and function, we cloned the che-12 and dyf-11 mutants which have chemotaxis and dye uptake defects. CHE-12 and DYF-11 are conserved ciliary proteins required for maintenance of cilium morphology and function. Furthermore, DYF-11 undergoes intraflagellar transport (IFT) and may function at an early stage of IFT-B particle assembly. Our results suggest that glia are required for multiple aspects of sensory organ function. Moreover, as thrombospondin 1 is a glial-secreted protein required for synapse formation in mice, these results suggest that some of the molecular components underlying glia-neuron interactions in C. elegans might be conserved

Lipid-Anchored SNAREs Lacking Transmembrane Regions Fully Support Membrane Fusion during Neurotransmitter Release

SummarySynaptic vesicle fusion during neurotransmitter release is mediated by assembly of SNARE- and SM-protein complexes composed of syntaxin-1, SNAP-25, synaptobrevin-2/VAMP2, and Munc18-1. Current models suggest that SNARE-complex assembly catalyzes membrane fusion by pulling the transmembrane regions (TMRs) of SNARE proteins together, thus allowing their TMRs to form a fusion pore. These models are consistent with the requirement for TMRs in viral fusion proteins. However, the role of the SNARE TMRs in synaptic vesicle fusion has not yet been tested physiologically. Here, we examined whether synaptic SNAREs require TMRs for catalysis of synaptic vesicle fusion, which was monitored electrophysiologically at millisecond time resolution. Surprisingly, we find that both lipid-anchored syntaxin-1 and lipid-anchored synaptobrevin-2 lacking TMRs efficiently promoted spontaneous and Ca2+-triggered membrane fusion. Our data suggest that SNARE proteins function during fusion primarily as force generators, consistent with the notion that forcing lipid membranes close together suffices to induce membrane fusion

Correction: Extended Synaptotagmin (ESyt) Triple Knock-Out Mice Are Viable and Fertile without Obvious Endoplasmic Reticulum Dysfunction.

[This corrects the article DOI: 10.1371/journal.pone.0158295.]

Extended Synaptotagmin (ESyt) Triple Knock-Out Mice Are Viable and Fertile without Obvious Endoplasmic Reticulum Dysfunction.

Extended synaptotagmins (ESyts) are endoplasmic reticulum (ER) proteins composed of an N-terminal transmembrane region, a central SMP-domain, and five (ESyt1) or three C-terminal cytoplasmic C2-domains (ESyt2 and ESyt3). ESyts bind phospholipids in a Ca2+-dependent manner via their C2-domains, are localized to ER-plasma membrane contact sites, and may catalyze lipid exchange between the plasma membrane and the ER via their SMP-domains. However, the overall function of ESyts has remained enigmatic. Here, we generated triple constitutive and conditional knock-out mice that lack all three ESyt isoforms; in addition, we produced knock-in mice that express mutant ESyt1 or ESyt2 carrying inactivating substitutions in the Ca2+-binding sites of their C2A-domains. Strikingly, all ESyt mutant mice, even those lacking all ESyts, were apparently normal and survived and bred in a manner indistinguishable from control mice. ESyt mutant mice displayed no major changes in brain morphology or synaptic protein composition, and exhibited no large alterations in stress responses. Thus, in mice ESyts do not perform an essential role in basic cellular functions, suggesting that these highly conserved proteins may perform a specialized role that may manifest only during specific, as yet untested challenges

Synaptic function of Rab11Fip5: selective requirement for hippocampal long-term depression

Postsynaptic AMPA-type glutamate receptors (AMPARs) are among the major determinants of synaptic strength and can be trafficked into and out of synapses. Neuronal activity regulates AMPAR trafficking during synaptic plasticity to induce long-term changes in synaptic strength, including long-term potentiation (LTP) and long-term depression (LTD). Rab family GTPases regulate most membrane trafficking in eukaryotic cells; particularly, Rab11 and its effectors are implicated in mediating postsynaptic AMPAR insertion during LTP. To explore the synaptic function of Rab11Fip5, a neuronal Rab11 effector and a candidate autism-spectrum disorder gene, we performed shRNA-mediated knock-down and genetic knock-out (KO) studies. Surprisingly, we observed robust shRNA-induced synaptic phenotypes that were rescued by a Rab11Fip5 cDNA but that were nevertheless not observed in conditional KO neurons. Both in cultured neurons and acute slices, KO of Rab11Fip5 had no significant effect on basic parameters of synaptic transmission, indicating that Rab11Fip5 is not required for fundamental synaptic operations, such as neurotransmitter release or postsynaptic AMPAR insertion. KO of Rab11Fip5 did, however, abolish hippocampal LTD as measured both in acute slices or using a chemical LTD protocol in cultured neurons but did not affect hippocampal LTP. The Rab11Fip5 KO mice performed normally in several behavioral tasks, including fear conditioning, but showed enhanced contextual fear extinction. These are the first findings to suggest a requirement for Rab11Fip5, and presumably Rab11, during LTD.This work was supported by National Institute of Neurological Disorders and Stroke fellowships 1F32NS067896 and 1K99NS087086 to T.B. and by National Institutes of Health Grants P50MH086403 and R01NS077906 to R.C.M. and T.C.SPeer reviewe

Constitutive ESyt123 triple KO mice are viable, fertile, develop apparently normally, and do not show major phenotypic changes.

<p>(A) Graph showing the distribution of the offspring obtained by crossing heterozygous 123EC KO mice. Black bars represent the expected ratio considering a Mendelian segregation of the alleles. Blue bars show the observed distribution. Chi-square test, p = 0.9432, n = 158. (B) Survival plot showing that homozygous 123EC KO mice are viable and have a normal lifespan (n = 8). (C) Both 123EC KO male and female mice do not show alterations in body weight if compared to WT mice. Data are shown as mean± SEM, One way ANOVA for repetitive measurements, p>0.05, n = 13 WT, n = 12 123EC. (D) Dapi staining of brain sections (top) confirming that 123EC KO mice do not show major alterations in the brain architecture. Magnification of the hippocampus (left) and cerebellum (right) confirming normal organizations of these structures in ESyt123 KO mice. Abbreviation: Cx, cortex; Hp, hippocampus; Cb, cerebellum; Bs, brainstem; St, striatum; Th, thalamus; DG, dentate gyrus; GCL, granule cell layer; WM, white matter.</p

Triple ESyt123 KO does not increase the susceptibility of neurons to stress, induce obvious changes in the ER, or affect calcium dynamics in neurons.

<p>(A) Hippocampal cultures obtained from ESyt123 conditional KO mice and infected with lentiviruses expressing GFP-Cre and GFP-ΔCre. Images show GFP fluorescence merged with brightfield signal to illustrate the high efficiency of infection of the virus for both Cre and ΔCre conditions. (B) Neuronal cell death in response to mild stress (DTT, Tunicamycin, Thapsigargin and Paraquat) was assessed by the MTT assay. The stress conditions induced partial neuronal cell death, which was unchanged in neurons infected with Cre or ΔCre, suggesting that loss of ESyts does not increase susceptibility to toxic agents. Data were normalized to the control ΔCre condition, and are expressed as means ± SEM. Two-way ANOVA, Bonferroni post-hoc test, *p<0.05 vs CTR, **p<0.01 vs CTR, ***p<0.001 vs control, n = 8. (C) Confocal images showing hippocampal neurons transfected with Sec61β-EGFP in green and mCherry signal in red. Insert shows a magnification of the soma to better visualize the ER associated with the nucleus. (D) Quantification of the total neuronal area (mCherry signal) and the fraction of the nuclear-associated ER (Sec61β area normalized to mCherry area) showing no difference between ΔCre and Cre treated neurons. Data are expressed as means ± SEM. Student t-test, p>0.05, n = 18 ΔCre, n = 15 Cre. (E) Representative images of Sec61β-EGFP signal (green) acquired at higher exposure, showing that cytoplasmic ER is present in dendrites as well as in the neck of a subset of spines (see arrowheads) in both ΔCre and Cre treated neurons. The mCherry signal (red) allows visualization of dendrites and dendritic spines. (F) Representative images of GCaMP6M-expressing hippocampal neurons at resting (left panel-absence of Ca2+-signal) or during network activity (right panel-presence of a Ca2+-signal) for ΔCre (top panel) and Cre (low panel) treated neurons. Calcium activity is present in both ΔCre and Cre treated neurons. (G) Quantification of the number of calcium peaks, the integrated peak area, as well as the rise time and decay time kinetics, during 5min recording of neuronal activity. No significant differences has been observed between ΔCre and Cre treated neurons. Data are expressed as means ± SEM. Student t-test, p>0.05, n = 5 pups (total of analyzed neurons = 184) ΔCre, n = 5 pups (total of analyzed neurons = 129) Cre.</p

Generation of ESyt1, ESyt2 and ESyt3 triple KO mice.

<p>(A) ESyt1, ESyt2 and ESyt3 gene structures (exons are numbered). (B) ESyts domain organization (TM, transmembrane domain; SMP, synaptotagmin-like mitochondrial-lipid-binding protein). (C) Strategy used to generate conditional ESyt1 KO alleles. (D) Strategy used to generate conditional ESyt2 KO alleles. (E) Strategy for generating a knock-in line expressing mutantESyt1/ESyt2 unable to bind calcium in the C2A domain. Using Flp recombinase the WT exon 10 was removed in favor of expression of a mutated exon 10 (D4A), in which 4 Asp residues have been mutated into Ala to prevent calcium binding. (F) Strategy to additional modify the targeting vector by using recombinase-mediated cassette exchange method. This manipulation is possible only for ESyt1 mice. (G) Distribution of ESyts mRNA transcripts in different tissues of WT mice, measured by RT-PCR. In the insert, enlargement of the scale for ESyts mRNA level in the brain.</p

Loss of ESyts does not affect the level of synaptic and ER markers in the brain.

<p>(A and B) Immunoblots and relative quantifications of major pre-synaptic proteins in WT and constitutive ESyt123 triple KO mice. Data are shown as means ± SEM, Student’s t-test, p>0.05, n = 4. (C and D) Western blot and relative quantification of ER proteins, showing no differences between WT and 123EC KO mice. Data are shown as means ± SEM, Student’s t-test, p>0.05, n = 4.</p