A Tightly Controlled Conditional Knockdown System Using the Tol2 Transposon-Mediated Technique

Background: Gene knockdown analyses using the in utero electroporation method have helped reveal functional aspects of genes of interest in cortical development. However, the application of this method to analyses in later stages of brain development or in the adult brain is still difficult because the amount of injected plasmids in a cell decreases along with development due to dilution by cell proliferation and the degradation of the plasmids. Furthermore, it is difficult to exclude the influence of earlier knockdown effects. Methodology/Principal Findings: We developed a tightly controlled conditional knockdown system using a newly constructed vector, pT2K-TBI-shRNAmir, based on a Tol2 transposon-mediated gene transfer methodology with the tetracycline-inducible gene expression technique, which allows us to maintain a transgene for a long period of time and induce the knockdown of the gene of interest. We showed that expression of the endogenous amyloid precursor protein (APP) was sharply decreased by our inducible, stably integrated knockdown system in PC12 cells. Moreover, we induced an acute insufficiency of Dab1 with our system and observed that radial migration was impaired in the developing cerebral cortex. Such inhibitory effects on radial migration were not observed without induction, indicating that our system tightly controlled the knockdown, without any expression leakage in vivo. Conclusions/Significance: Our system enables us to investigate the brain at any of the later stages of development or in th

PIP3-Phldb2 is crucial for LTP regulating synaptic NMDA and AMPA receptor density and PSD95 turnover

The essential involvement of phosphoinositides in synaptic plasticity is well-established, but incomplete knowledge of the downstream molecular entities prevents us from understanding their signalling cascades completely. Here, we determined that Phldb2, of which pleckstrin-homology domain is highly sensitive to PIP3, functions as a phosphoinositide-signalling mediator for synaptic plasticity. BDNF application caused Phldb2 recruitment toward postsynaptic membrane in dendritic spines, whereas PI3K inhibition resulted in its reduced accumulation. Phldb2 bound to postsynaptic scaffolding molecule PSD-95 and was crucial for localization and turnover of PSD-95 in the spine. Phldb2 also bound to GluA1 and GluA2. Phldb2 was indispensable for the interaction between NMDA receptors and CaMKII, and the synaptic density of AMPA receptors. Therefore, PIP3-responsive Phldb2 is pivotal for induction and maintenance of LTP. Memory formation was impaired in our Phldb2−/− mice

<i>Tol2</i> knockdown vectors were genomically integrated and the inducible expression was tightly controlled <i>in vivo</i>.

<p>(A) Representative neocortical sections showing the basal expression and induced expression of EGFP derived from the pT2K-BI-shRNAmir or pT2K-TBI-shRNAmir vectors. (B) The retention and expression of inducible knockdown vector in glial cells. In the presence of <i>Tol2</i> transposase, EGFP was observed in the glial cells (arrowheads). The right panels are higher magnification views of the boxed regions in the left panels. (C) Representative neocortical sections showing the retention and expression of the inducible knockdown vector in the adult cortex. CP, cortical plate; VZ, ventricular zone. Scale bars, 100 µm.</p

Macrophage-Colony Stimulating Factor Derived from Injured Primary Afferent Induces Proliferation of Spinal Microglia and Neuropathic Pain in Rats.

Peripheral nerve injury induces proliferation of microglia in the spinal cord, which can contribute to neuropathic pain conditions. However, candidate molecules for proliferation of spinal microglia after injury in rats remain unclear. We focused on the colony-stimulating factors (CSFs) and interleukin-34 (IL-34) that are involved in the proliferation of the mononuclear phagocyte lineage. We examined the expression of mRNAs for macrophage-CSF (M-CSF), granulocyte macrophage-CSF (GM-CSF), granulocyte-CSF (G-CSF) and IL-34 in the dorsal root ganglion (DRG) and spinal cord after spared nerve injury (SNI) in rats. RT-PCR and in situ hybridization revealed that M-CSF and IL-34, but not GM- or G-CSF, mRNAs were constitutively expressed in the DRG, and M-CSF robustly increased in injured-DRG neurons. M-CSF receptor mRNA was expressed in naive rats and increased in spinal microglia following SNI. Intrathecal injection of M-CSF receptor inhibitor partially but significantly reversed the proliferation of spinal microglia and in early phase of neuropathic pain induced by SNI. Furthermore, intrathecal injection of recombinant M-CSF induced microglial proliferation and mechanical allodynia. Here, we demonstrate that M-CSF is a candidate molecule derived from primary afferents that induces proliferation of microglia in the spinal cord and leads to induction of neuropathic pain after peripheral nerve injury in rats

The <i>Tol2</i> transposable vector enables inducible knockdown from a stably integrated knockdown cassette.

<p>(A) A schematic diagram of the pT2K-TBI-shRNAmir vector. The shRNAmir cassette was inserted into the pT2K-BI-TRE-EGFP vector, and TRE-BI was replaced with TRE-TBI. The shRNAmir cassette consisted of the hairpin stem, which is composed of siRNA sense and antisense strands designed for the knockdown of the target gene, a loop derived from human mir30, and mir30 flanking sequences on the 3′ and 5′ sides of the hairpin. (B) A schematic diagram showing the principle of induction of knockdown from the genomically integrated shRNAmir cassette. The <i>Tol2</i>-flanked region of the plasmids were excised and integrated into the chromosome using <i>Tol2</i> transposase. In the presence of Doxycycline (Dox), rtTA-M2 bound to TRE-TBI, and the expression of both EGFP and the mir30-based knockdown cassette were induced under the control of TRE-TBI. (C) Expression of EGFP, induced from the each of pT2K-TBI-shRNAmir vectors (mir-empty, mir-APP#2 and mir-APP#3), was observed in almost all PC12 cells following Dox administration. The upper panels show the bright-field images. Scale bar, 100 µm. (D) Immunoblot analyses for evaluating the knockdown efficiency against APP. Actin was used as a loading control. (E) The basal expression (−Dox) and the induced expression (+Dox) of EGFP from the pT2K-BI-shRNAmir and pT2K-TBI-shRNAmir vectors in HEK293T cells. pCAGGS-tdTomato was co-transfected as a transfection control. Inset shows a higher magnification. Scale bars: 100 µm, inset 20 µm. (F) Ratio of the number of EGFP-positive cells to tdTomato-positive cells between the cells expressing pT2K-BI-shRNAmir and pT2K-TBI-shRNAmir with or without Dox. (mean ± SEM, n = 3). Abbreviations of the vector name and their components are listed in the table (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033380#pone.0033380.s002" target="_blank">Table S1</a>).</p

Subcellular distribution of non-muscle myosin IIb is controlled by FILIP through Hsc70

<div><p>The neuronal spine is a small, actin-rich dendritic or somatic protrusion that serves as the postsynaptic compartment of the excitatory synapse. The morphology of the spine reflects the activity of the synapse and is regulated by the dynamics of the actin cytoskeleton inside, which is controlled by actin binding proteins such as non-muscle myosin. Previously, we demonstrated that the subcellular localization and function of myosin IIb are regulated by its binding partner, filamin-A interacting protein (FILIP). However, how the subcellular distribution of myosin IIb is controlled by FILIP is not yet known. The objective of this study was to identify potential binding partners of FILIP that contribute to its regulation of non-muscle myosin IIb. Pull-down assays detected a 70-kDa protein that was identified by mass spectrometry to be the chaperone protein Hsc70. The binding of Hsc70 to FILIP was controlled by the adenosine triphosphatase (ATPase) activity of Hsc70. Further, FILIP bound to Hsc70 via a domain that was not required for binding non-muscle myosin IIb. Inhibition of ATPase activity of Hsc70 impaired the effect of FILIP on the subcellular distribution of non-muscle myosin IIb. Further, in primary cultured neurons, an inhibitor of Hsc70 impeded the morphological change in spines induced by FILIP. Collectively, these results demonstrate that Hsc70 interacts with FILIP to mediate its effects on non-muscle myosin IIb and to regulate spine morphology.</p></div

Inhibition of Hsc70 results in the suppression of the effects of FILIP on the subcellular distribution of non-muscle myosin IIb.

<p>A-D: The graphs show the ratio of the COS-7 cells exhibiting a stress fiber-like distribution versus a granular distribution of non-muscle myosin IIb (A) after treatment with 2 mM clofibric acid (numbers of cells containing a stress fiber-like distribution/total cells: 543/625 control cells treated with vehicle; 571/615 control cells treated with clofibric acid; 182/628 FILIP-expressing cells treated with vehicle; and 243/620 FILIP-expressing cells treated with clofibric acid. *p < 0.01 (Fisher’s exact test)); (B) under the expression of FILIP d687-960 with or without the application of clofibric acid (numbers of stress fiber-like distributed cells/total cells: 166/318 FILIP d687-960-expressing cells treated with vehicle and 158/307 FILIP d687-960-expressing cells treated with clofibric acid); (C) under the expression of FILIP d872-1111 with or without the application of clofibric acid (numbers of stress fiber-like distributed cells/total cells: 178/325 FILIP d872-1111-expressing cells treated with vehicle and 185/331 FILIP d872-1111-expressing cells treated with clofibric acid); and (D) under the expression of Hsc70K71M and FILIP (numbers of non-muscle myosin IIb stress fiber-like distributed cells 273/320 control/Hsc70-expressing cells; 282/320 control/Hsc70K71M-expressing cells, 131/340 FILIP/Hsc70-expressing cells, and 151/318 FILIP/Hsc70K71M-expressing cells. (E) Mutation of Hsc70 does not influence the binding of FILIP and non-muscle myosin IIb. Immunoprecipitation was performed using an anti-FLAG antibody, and blots were probed with an antibody against non-muscle myosin IIb.</p

The regulation of spine length by FILIP in primary neurons requires Hsc70.

<p>(A) Treatment with clofibric acid resulted in shorter spines in piriform neurons. Scale bar = 2 μm. The graph shows summary data from 554 spines from 6 neurons treated with vehicle and 571 spines from 4 neurons treated with clofibric acid. *p < 0.01 (B) Expression of FILIP d872-1111 in hippocampal neurons did not result in elongated spines. Scale bar = 1 μm. The graph shows summary data from 882 spines from 5 neurons (control), 851 spines from 5 neurons (FILIP d872-1111), and 696 spines from 5 neurons (FILIP). *p < 0.01 (C) Inhibition of the function of Hsc70 reverses the effects of FILIP on spine length. Scale bar = 1 μm. The graph shows summary data from 863 spines from 6 neurons (control/vehicle), 821 spines from 6 neurons (control/VER-155008), 603 spines from 6 neurons (FILIP/vehicle), and 741 spines from 6 neurons (FILIP/VER-155008). **p < 0.01, *p < 0.05.</p

Exogenous expression of FILIP influences the subcellular distribution of non-muscle myosin IIb.

<p>(A) Non-muscle myosin IIb was visualized (red) using an anti-NMHCIIb antibody in COS-7 cells. Green, bicistronic GFP expression vector for FILIP; blue, cell nuclei labeled with Hoescht dye. Scale bar = 10 μm. (B) The graph shows the ratio of the cells exhibiting a stress fiber-like distribution of non-muscle myosin IIb. The numbers of cells containing stress fiber-like distributions of non-muscle myosin IIb/total cells were: 287/330 control cells, 74/318 FILIP-expressing cells, 58/312 FILIP d248-685-expressing cells, 186/323 FILIP d872-1111-expressing cells, and 195/310 FILIP d687-960-expressing cells. *p < 0.01 (Fisher’s exact test) (C) The graph shows the effects of jasplakinolide. The numbers of cells containing a stress fiber-like distribution of non-muscle myosin IIb/total cells were: 285/312 control cells treated with vehicle, 286/314 control cells treated with jasplakinolide, 76/313 FILIP-expressing cells treated with vehicle, and 180/310 FILIP-expressing cells treated with jasplakinolide. *p < 0.01 (Fisher’s exact test).</p