Additional file 2: of Impairment of social behaviors in Arhgef10 knockout mice

Impulsive and Non-impulsive behavior. (PDF 15155 kb

Additional file 1: of Impairment of social behaviors in Arhgef10 knockout mice

Generation of ARHGEF10 -/- mice. (PPTX 46 kb

hEPO+MBs/FUS inhibits the ischemia/reperfusion-induced neuronal death and inflammation in rat experiments.

<p>Immunohistochemical staining of NeuN, CD-11b, and GFAP was performed 24 h after I/R. (A) illustrated the position of FUS sonication. In NeuN staining (B, E, and H), the I/R group showed a marked loss of neurons, whereas, neurons were intact in the sham and I/R+hEPO+MBs/FUS groups. In CD-11b staining (C, F, and I), the I/R group showed microglia activation (condensed nuclei), whereas the sham and I/R+hEPO+MBs/FUS groups showed ramified microglia. In GFAP staining (D, G, and J), there was an increase of GFAP in the I/R group but not in the sham and I/R+hEPO+MBs/FUS groups. (Scale bar  =  200 µm).</p

Improvement in catwalk automated gait analysis test by hEPO plus MBs/FUS in chronic phase of 3VO.

<p>Two gait analysis parameters, paw intensity (A) and paw angle (B), were assessed from Day-7 to Day-28 after I/R. In both paw intensity and paw angle, treatment with hEPO+MBs/FUS significantly improved the performance of impaired limb. Data were shown as mean ± SEM (n = 5 for each group), * p<0.05 as compared with the sham (control group), # p<0.05 as compared with the I/R group.</p

Enhancement of hEPO delivery into the brain tissues by MBs/FUS.

<p>hEPO and MBs were intravenously administered and FUS was transcranially applied. (A) Rat brains were perfused and sliced into sections at 3 h after hEPO injection. Sonicated region of the brain was dissected and quantified. The hEPO levels in sections 3 and 4 were significantly higher in the I/R+hEPO+MBs/FUS group compared to the I/R+hEPO group (n = 3 for each). Data were given as means ± S.E.M., * p<0.05, as compared with the I/R+hEPO group. (B) CSF was sampled at 3 h after hEPO injection. The hEPO concentration of CSF in the I/R+hEPO+MBs/FUS group showed significant enhancement compared with the I/R+hEPO group. (C) The serum hEPO was also sampled at 3 h after hEPO injection. No hEPO was found in the sham and I/R groups without hEPO injection.</p

Antagonism of proteasome inhibitor-induced heme oxygenase-1 expression by PINK1 mutation

<div><p>PTEN-induced putative kinase 1 (PINK1) is an integral protein in the mitochondrial membrane and maintains mitochondrial fidelity. Pathogenic mutations in <i>PINK1</i> have been identified as a cause of early-onset autosomal recessive familial Parkinson’s disease (PD). The ubiquitin proteasome pathway is associated with neurodegenerative diseases. In this study, we investigated whether mutations of PINK1 affects the cellular stress response following proteasome inhibition. Administration of MG132, a peptide aldehyde proteasome inhibitor, significantly increased the expression of heme oxygenase-1 (HO-1) in rat dopaminergic neurons in the substantia nigra and in the SH-SY5Y neuronal cell line. The induction of HO-1 expression by proteasome inhibition was reduced in PINK1 G309D mutant cells. MG132 increased the levels of HO-1 through the Akt, p38, and Nrf2 signaling pathways. Compared with the cells expressing WT-PINK1, the phosphorylation of Akt and p38 was lower in those cells expressing the PINK1 G309D mutant, which resulted in the inhibition of the nuclear translocation of Nrf2. Furthermore, MG132-induced neuronal death was enhanced by the PINK1 G309D mutation. In this study, we demonstrated that the G309D mutation impairs the neuroprotective function of PINK1 following proteasome inhibition, which may be related to the pathogenesis of PD.</p></div

PINK1 G309D mutation inhibits MG132-induced Nrf2 expression and nuclear translocation of Nrf2 in SH-SY5Y cells.

<p>Empty vector-transfected cells and PINK1 G309D mutant cells were treated with MG132 (1 μM) for various time intervals. (A) MG132 time-dependently increased the expression of Nrf2, which was inhibited by the expression of the recombinant PINK1 G309D mutant. (B) The Nrf2 mRNA levels were analyzed by real time-PCR. Note that PINK1 G309D mutant cells inhibited Nrf2 mRNA upregulation following MG132 treatment. (C) Empty vector-transfected cells and PINK1 G309D mutant cells were treated with MG132 (1 μM) for 3 and 6 h, and the nuclear extracts were then obtained to evaluate the Nrf2 expression in nucleus. It showed MG132 time-dependently increased the nuclear translocation of Nrf2, which was inhibited by the PINK1 G309D mutation. C23 served as a nuclear internal control in the nucleus. (D) Cells were transfected with scramble or siRNA against Nrf2 and then treated with vehicle or MG132. Note that MG132-induced upregulation of HO-1 protein was antagonized by the knockdown of Nrf2. Data are presented as multiples of respective controls (mean ± S.E.M) (n = 4). *p < 0.05 compared with the empty vector or scramble RNA -transfected control cells; #p < 0.05 compared with the corresponding MG132-treated control.</p

Upregulation of heme oxygenase-1 (HO-1) following proteasome inhibition by MG132 in the substantia nigra of rat.

<p>(A) MG132 (9.5 ng) was locally injected into the substantia nigra of rats, and the rats were sacrificed 24 h later. Immunofluorescent staining shows that HO-1 expression increased following MG132 treatment and HO-1 occurred mostly in TH (+) neurons. Scale bar: 100 μm. (B) Treatment with MG132 (1 μM) in cultured SH-SY5Y cells. Levels of ROS were analyzed using a DCF fluorescence assay. Note that the generation of ROS increased time-dependently after MG132-treatment. Results are expressed as percentage of wild-type control (mean ± S.E.M.) (n = 4).</p

Schematic depiction of the effect of PINK1 G309D mutation on proteasome inhibition-induced cellular response.

<p>Under the stress of proteasome inhibition, p38 and Akt are phosphorylated and they activate Nrf2, which then dissociates from Keap1. Activated Nrf2 subsequently translocates into the nucleus and induces the expression of HO-1, which provides a `neuroprotective effect. However, this pathway is abrogated by the PINK1 G309D mutation.</p

Enhancement of MG132-induced neuronal death by PINK1 G309D mutation.

<p>Empty vector-transfected cells and PINK1 G309D mutant cells were treated with different concentrations of MG132 for 24 h. Subsequently, the cell viability was evaluated by MTT assay. Note that transfection of WT-PINK1 or G309D PINK1 alone did not affect cell viability. MG132 concentration-dependently induced cell death, which was enhanced by the PINK1 G309D mutation. Results are presented as percentage of control (mean ± S.E.M) for three experiments performed in triplicate. *p < 0.05 compared with the empty vector-transfected control cells (con); #p < 0.05 compared with the respective empty vector cells in the presence of various concentrations of MG132.</p