RhoGTPase Regulators Orchestrate Distinct Stages of Synaptic Development

Small RhoGTPases regulate changes in post-synaptic spine morphology and density that support learning and memory. They are also major targets of synaptic disorders, including Autism. Here we sought to determine whether upstream RhoGTPase regulators, including GEFs, GAPs, and GDIs, sculpt specific stages of synaptic development. The majority of examined molecules uniquely regulate either early spine precursor formation or later matura- tion. Specifically, an activator of actin polymerization, the Rac1 GEF β-PIX, drives spine pre- cursor formation, whereas both FRABIN, a Cdc42 GEF, and OLIGOPHRENIN-1, a RhoA GAP, regulate spine precursor elongation. However, in later development, a novel Rac1 GAP, ARHGAP23, and RhoGDIs inactivate actomyosin dynamics to stabilize mature synap- ses. Our observations demonstrate that specific combinations of RhoGTPase regulatory pro- teins temporally balance RhoGTPase activity during post-synaptic spine development

Gamma-ray and radio properties of six pulsars detected by the fermi large area telescope

We report the detection of pulsed γ-rays for PSRs J0631+1036, J0659+1414, J0742-2822, J1420-6048, J1509-5850, and J1718-3825 using the Large Area Telescope on board the Fermi Gamma-ray Space Telescope (formerly known as GLAST). Although these six pulsars are diverse in terms of their spin parameters, they share an important feature: their γ-ray light curves are (at least given the current count statistics) single peaked. For two pulsars, there are hints for a double-peaked structure in the light curves. The shapes of the observed light curves of this group of pulsars are discussed in the light of models for which the emission originates from high up in the magnetosphere. The observed phases of the γ-ray light curves are, in general, consistent with those predicted by high-altitude models, although we speculate that the γ-ray emission of PSR J0659+1414, possibly featuring the softest spectrum of all Fermi pulsars coupled with a very low efficiency, arises from relatively low down in the magnetosphere. High-quality radio polarization data are available showing that all but one have a high degree of linear polarization. This allows us to place some constraints on the viewing geometry and aids the comparison of the γ-ray light curves with high-energy beam models

α-Actinin-2 mediates spine morphology and assembly of the post-synaptic density in hippocampal neurons.

Dendritic spines are micron-sized protrusions that constitute the primary post-synaptic sites of excitatory neurotransmission in the brain. Spines mature from a filopodia-like protrusion into a mushroom-shaped morphology with a post-synaptic density (PSD) at its tip. Modulation of the actin cytoskeleton drives these morphological changes as well as the spine dynamics that underlie learning and memory. Several PSD molecules respond to glutamate receptor activation and relay signals to the underlying actin cytoskeleton to regulate the structural changes in spine and PSD morphology. α-Actinin-2 is an actin filament cross-linker, which localizes to dendritic spines, enriched within the post-synaptic density, and implicated in actin organization. We show that loss of α-actinin-2 in rat hippocampal neurons creates an increased density of immature, filopodia-like protrusions that fail to mature into a mushroom-shaped spine during development. α-Actinin-2 knockdown also prevents the recruitment and stabilization of the PSD in the spine, resulting in failure of synapse formation, and an inability to structurally respond to chemical stimulation of the N-methyl-D-aspartate (NMDA)-type glutamate receptor. The Ca2+-insensitive EF-hand motif in α-actinin-2 is necessary for the molecule's function in regulating spine morphology and PSD assembly, since exchanging it for the similar but Ca2+-sensitive domain from α-actinin-4, another α-actinin isoform, inhibits its function. Furthermore, when the Ca2+-insensitive domain from α-actinin-2 is inserted into α-actinin-4 and expressed in neurons, it creates mature spines. These observations support a model whereby α-actinin-2, partially through its Ca2+-insensitive EF-hand motif, nucleates PSD formation via F-actin organization and modulates spine maturation to mediate synaptogenesis

RhoGTPase Regulators Orchestrate Distinct Stages of Synaptic Development

Small RhoGTPases regulate changes in post-synaptic spine morphology and density that support learning and memory. They are also major targets of synaptic disorders, including Autism. Here we sought to determine whether upstream RhoGTPase regulators, including GEFs, GAPs, and GDIs, sculpt specific stages of synaptic development. The majority of examined molecules uniquely regulate either early spine precursor formation or later matura- tion. Specifically, an activator of actin polymerization, the Rac1 GEF β-PIX, drives spine pre- cursor formation, whereas both FRABIN, a Cdc42 GEF, and OLIGOPHRENIN-1, a RhoA GAP, regulate spine precursor elongation. However, in later development, a novel Rac1 GAP, ARHGAP23, and RhoGDIs inactivate actomyosin dynamics to stabilize mature synap- ses. Our observations demonstrate that specific combinations of RhoGTPase regulatory pro- teins temporally balance RhoGTPase activity during post-synaptic spine development

Knockdown of α-actinin-2 prevents spine maturation in response to NMDA receptor activation.

<p><b>A)</b> When α-actinin-2 is knocked down, spines do not shorten or assume a “mushroom” morphology in response to glycine. Hippocampal neurons were co-transfected at DIV 6 with GFP and either pSUPER or pSUPER-α-actinin-2. Neurons were chronically treated with AP-5, an NMDA receptor antagonist, to inhibit spine maturation. At DIV 19–21, neurons were acutely stimulated by the addition of 200 µM glycine and AP-5 withdrawal, while control neurons were continuously treated with AP-5. <b>B</b>–<b>C)</b> Quantification of spine morphology in response to α-actinin-2 inhibition and glycine stimulation. Fraction of spines with a large head, spine tip width >0.4 µm, increases in response to glycine stimulation but is prevented by α-actinin-2 knockdown, B. In contrast to stimulated controls, inhibition of α-actinin-2 does not increase the fraction of mushroom-shaped spines and decrease filopodia-like spines, C. For each condition, 556–1721 spines of 16–24 neurons from 2 separate cultures were analyzed. <b>D</b>–<b>E)</b> α-Actinin-2 knockdown prevents enrichment of actin filaments in spines. Hippocampal neurons were co-transfected at DIV 6 with GFP and either pSUPER or pSUPER-α-actinin-2, fixed on DIV 21, and stained for rhodamine-phalloidin. Arrowheads mark actin enrichment in spines of control neurons and arrows point to the lack of actin in spines of neurons with α-actinin-2 knocked down, D. The fluorescent intensity of rhodamine-phalloidin is reduced in spines with α-actinin-2 knocked down, E. For each condition, 87–97 spines from 5 neurons were analyzed. Error bars represent SEM. p-values were derived using the paired t-test. Scale = 5 µm.</p

α-actinin-2 localizes to post-synaptic sites in dendritic spines on hippocampal neurons.

<p><b>A)</b> An anti-α-actinin antibody (ab68167) recognizes α-actinin-2 and not α-actinin-1 or α-actinin-4. CHO-K1 cells were transfected with human α-actinin-1-GFP, α-actinin-2-GFP, or α-actinin-4-GFP. Cells were lysed and immunoblotted for α-actinin-2 and GFP. <b>B</b>) α-Actinin-2 is enriched in hippocampal neurons but not in glia cells or COS-7 cells, which lacks α-actinin-2. Cells were lysed and immunoblotted for α-actinin-2. Actin is the loading control. <b>C)</b> α-Actinin-2 localizes to dendritic spines. Hippocampal neurons were transfected at DIV 17 with GFP (green), and fixed, and immunostained for endogenous α-actinin-2 (magenta) at DIV 21. <b>D)</b> α-Actinin-2 co-localizes with post-synaptic markers, but not with a pre-synaptic marker. Hippocampal neurons were fixed at DIV 16 or 21 and immunostained for endogenous α-actinin-2 (green) and either endogenous synaptophysin, PSD-95, or the NR1 subunit of the NMDA receptor (magenta). <b>E</b>–<b>G)</b> The siRNA is specific for α-actinin-2. Hippocampal neurons were co-transfected at DIV 17 with GFP and either a control empty vector (pSUPER), or a vector containing siRNA against α-actinin-2 (pSUPER-α-actinin-2), or the α-actinin-2 siRNA-containing vector plus a α-actinin-2 vector conferring resistance to RNAi (pSUPER-α-actinin-2+ α-actinin-2-SS). The cells were fixed at DIV 21 and immunostained for endogenous α-actinin-2. Arrows point to the neurons co-expressing GFP and its immunostaining for α-actinin-2. For each condition (55 control cells and 46 α-actinin-2 knockdown cells), the integrated density of the cell body and dendrites were measured from the transfected neuron and adjacent untransfected neuron of the same image and the percent change was plotted, F. Error bars represent SEM. p-values were derived using the paired t-test. <b>G)</b> CHO-K1 cells were co-transfected with GFP, pSUPER or pSUPER-α-actinin-2, plus either α-actinin-2-Flag or α-actinin-2-SS-Flag. Transfection efficiency was close to 100% as >95% of the cells in each condition exhibited GFP fluorescence (data not shown). Cells were lysed 72 hours after transfection and immunoblotted for α-actinin-2 and α-actinin-4. Tubulin is the loading control.</p

Loss of α-actinin-2 prevents synapse formation.

<p><b>A)</b> Actively firing pre-synaptic boutons do not synapse with spines on neurons lacking α-actinin-2. Hippocampal neurons were co-transfected at DIV 6 with GFP and either pSUPER or pSUPER-α-actinin-2, treated with FM4-64 for 5 min on DIV 19 and observed live. Arrows mark FM4-64 juxtaposition to spines or the dendrite. The fraction of spines juxtaposed to FM4-64 is reduced in neurons lacking α-actinin-2 (see results). For each condition, 14 neurons from 2 separate cultures were analyzed. <b>B)</b> Loss of α-actinin-2 prevents synapse formation with excitatory axon boutons. Hippocampal neurons were co-transfected at DIV 6 with GFP and either pSUPER or pSUPER-α-actinin-2, fixed on DIV 21, and immunostained for VGLUT1. Arrows mark VGLUT1 juxtaposition to spines or the dendrite. The fraction of spines juxtaposed to VGLUT1 is reduced in neurons lacking α-actinin-2 (see results). For each condition 23–26 neurons from 2 separate cultures were analyzed.</p

α-Actinin-2 contributes to development of dendritic arbors and spine morphology in early cultures.

<p><b>A–C)</b> Hippocampal neurons were co-transfected at DIV 6 with GFP and either pSUPER or pSUPER-α-actinin-2 and fixed on DIV 22. Note the reduced size and number of dendrite arbors on neurons lacking α-actinin-2. While there is no difference in the number of primary dendrites, the number of secondary and tertiary dendrites on neurons lacking α-actinin-2 is reduced, B. The length of primary and secondary dendrites is smaller in neurons with α-actinin-2 knocked down, C. The branching complexity is reduced in neurons lacking α-actinin-2, D. Dendrites from 17 control neurons and 21 α-actinin-2 knockdown neurons from 2 different cultures were analyzed. <b>E)</b> Hippocampal neurons were co-transfected at DIV 6 with GFP and either pSUPER or pSUPER-α-actinin-2. Neurons were fixed on DIV 21 and scored for <b>(E</b>–<b>I)</b> changes in spine density, length, head width, and morphology. Two examples of control neurons and α-actinin-2 knockdown neurons are shown. Arrows point to irregularly shaped protrusions containing numerous filopodia, which is observed in several α-actinin-2 knockdowns. Inhibition of α-actinin-2 increases the number of spines per µm length of the dendrite (spine density), F. Spine density was quantified from 73 control neurons and 65 α-actinin-2 knockdown neurons from more than 3 cultures. The fraction of spine head widths >0.4 µm is significantly reduced in neurons with α-actinin-2 knocked down, G. Spine length is shifted to the right (longer) in neurons lacking α-actinin-2, H. α-Actinin-2 knockdown creates an increase in the fraction of filopodia-like spines (long protrusions without a spine head) and a concomitant decrease in the fraction of mushroom-shaped spines, I. For quantification of spine width, length, and morphology, 1875–2245 spines from 29 control neurons and 35 α-actinin-2 knockdown neurons from more than 3 cultures were analyzed. Error bars represent standard error of the mean (SEM). p-values were derived using the paired t-test (B, C, D, H, I) and Mann-Whitney test (F, G).</p

Loss of α-actinin-2 prevents assembly of the post-synaptic density.

<p><b>A–C)</b> α-Actinin-2 knockdown inhibits PSD assembly in spines. Hippocampal neurons were co-transfected at DIV 6 with GFP and either pSUPER or pSUPER-α-actinin-2, fixed on DIV 21, and immunostained for PSD-95. Arrows mark localization of PSD-95 in spines or in the dendrite shaft, A. The fraction of spines with localized PSD-95 is reduced in neurons with α-actinin-2 knocked down, B. PSD-95 localizes to the dendrite shaft with increased frequency in neurons lacking α-actinin-2, C. For each condition, 37-45 neurons from 5 separate cultures were analyzed. <b>D</b>–<b>F)</b> Hippocampal neurons were co-transfected at DIV 17 with GFP and either pSUPER, pSUPER-α-actinin-2, or pSUPER-α-actinin-2 plus α-actinin-2-SS, D. The fraction of spines with PSD-95 is rescued in neurons expressing exogenous α-actinin-2-SS, E. The area of PSD-95 is significantly reduced in spines lacking α-actinin-2 and rescued in neurons expressing exogenous α-actinin-2-SS, F. For each condition, PSD-95 area was measured from 543–991 spines of 23–27 neurons from 3 separate cultures. <b>G</b>–<b>I)</b> α-Actinin-2 knockdown prevents the recruitment of the NMDA receptor to the spine. Hippocampal neurons were co-transfected at DIV 6 with SEP-NR1 and either pSUPER or pSUPER-α-actinin-2, fixed on DIV 22, and immunostained for GFP and rhodamine-phalloidin. Arrows mark SEP-NR1 localization in either spines or in the dendrite, G. The fraction of spines co-localized with SEP-NR1 is reduced in neurons with α-actinin-2 knocked down, H. For each condition, 12–19 neurons from 3 separate cultures were analyzed. <b>I)</b> Hippocampal neurons were co-transfected at DIV 17 with GFP and either pSUPER-α-actinin-2 or pSUPER-α-actinin-2 plus α-actinin-2-SS, fixed on DIV 21, and immunostained for NMDAR1. Error bars represent SEM. p-values were derived using the paired t-test.</p

Knockdown of α-actinin-2 increases spine number and inhibits spine maturation.

<p><b>A)</b> Hippocampal neurons were co-transfected at DIV 17 with GFP and either pSUPER, pSUPER-α-actinin-2, or pSUPER-α-actinin-2 plus α-actinin-2-SS. Neurons were fixed on DIV 21 and scored for <b>(B</b>–<b>D)</b> changes in spine density, head width, and morphology. Inhibition of α-actinin-2 at DIV 17 increases spine density, B. For each condition, spine density was analyzed on 35–42 dendrites from 3 separate cultures. The fraction of spine head widths >0.4 µm is significantly reduced in neurons with α-actinin-2 knocked down at DIV 17, C. α-Actinin-2 knockdown creates an increase in the fraction of filopodia-like spines and a decrease in the fraction of mushroom-shaped spines, thin spines (long protrusions with small head at tip), and stubby spines, D. For quantification of spine head width and morphology, 1444–2081 spines from 51 control neurons, 40 α-actinin-2 knockdown neurons, and 35 rescue neurons of 3 separate cultures were analyzed. <b>E)</b> Hippocampal neurons were transfected at DIV 6 with either GFP alone or GFP + α-actinin-2-SS. Neurons were fixed on DIV 22 and scored for <b>(F</b>–<b>H)</b> changes in spine length, head width, and morphology. Overexpression of α-actinin-2-SS increases spine length (F) and reduces spine head width (G). α-Actinin-2-SS overexpression creates an increase in the fraction of filopodia-like spines and a decrease in the fraction of stubby spines, H. For quantification of spine length, head width, and morphology, 311–610 spines from 15 control neurons and 21 α-actinin-2-SS overexpression neurons of 3 separate cultures were analyzed. Error bars represent SEM. p-values were derived using the paired t-test. Scale = 5 µm.</p