Extracellular α-synuclein leads to microtubule destabilization via GSK-3β-dependent Tau phosphorylation in PC12 cells.

α-Synuclein (ASN) plays an important role in pathogenesis of Parkinson's disease (PD) and other neurodegenerative disorders. Novel and most interesting data showed elevated tauopathy in PD and suggested relationship between ASN and Tau protein. However, the mechanism of ASN-evoked Tau protein modification is not fully elucidated. In this study we investigated the role of extracellular ASN in Tau hyperphosphorylation in rat pheochromocytoma (PC12) cells and the involvement of glycogen synthase kinase-3β (GSK-3β) and cyclin-dependent kinase 5 (CDK5) in ASN-dependent Tau modification. Our results indicated that exogenously added ASN increases Tau phosphorylation at Ser396. Accordingly, the GSK-3β inhibitor (SB-216763) prevented ASN-evoked Tau hyperphosphorylation, but the CDK5 inhibitor had no effect. Moreover, western blot analysis showed that ASN affected GSK-3β via increasing of protein level and activation of this enzyme. GSK-3β activity evaluated by its phosphorylation status assay showed that ASN significantly increased the phosphorylation of this enzyme at Tyr216 with parallel decrease in phosphorylation at Ser9, indicative of stimulation of GSK-3β activity. Moreover, the effect of ASN on microtubule (MT) destabilization and cell death with simultaneous the involvement of GSK-3β in these processes were analyzed. ASN treatment increased the amount of free tubulin and concomitantly reduced the amount of polymerized tubulin and SB-216763 suppressed these ASN-induced changes in tubulin, indicating that GSK-3β is involved in ASN-evoked MT destabilization. ASN-induced apoptotic processes lead to decrease in PC12 cells viability and SB-216763 protected those cells against ASN-evoked cytotoxicity. Concluding, extracellular ASN is involved in GSK-3β-dependent Tau hyperphosphorylation, which leads to microtubule destabilization. GSK-3β inhibition may be an effective strategy for protecting against ASN-induced cytotoxicity

The role of Gsk-3β in apoptosis induced by ASN after 48 h treatment.

<p>(<b>A</b>) The effect of the exogenous ASN and GSK-3β inhibitor, SB-216763, on PC12 cells viability was spectrophotometrically determined using the MTT assay. ** p<0.01 <i>versus</i> control; ## p<0.01 <i>versus</i> ASN-treated cells using a one-way ANOVA followed by Dunnett's Multiple Comparison test. (<b>B1</b>) The effect of 10 μM ASN and 10 μM SB-216763 on PC12 cells apoptosis was determined <i>via</i> Hoechst 33258 fluorescent staining. The arrows indicate nuclei with typical apoptotic features. (<b>B2</b>) Apoptosis expressed as the percentage of apoptotic cells on the coverslips after 48 h treatment with 10 μM ASN. Data represent the mean value ± S.E.M. for 3 independent experiments. ** p<0.01 <i>versus</i> control; ## p<0.01 <i>versus</i> ASN-treated cells using a one-way ANOVA followed by Dunnett's Multiple Comparison test. (<b>C1</b>) The effect of 10 μM ASN and 10 μM SB-216763 on PC12 cells apoptosis was determined <i>via</i> TUNEL assay. Red colour indicates FITC-negative cells (all fixed cells stain with Propidium Iodide), green and yellow colours indicates FITC-positive cells (respectively, early and later stages of apoptotic cells characterized by specific DNA fragmentation). The arrows indicate nuclei with typical apoptotic features (FITC-positive cells). (<b>C2</b>) The number of apoptotic (FITC-stained) cells and the total number (PI-stained) of cells were counted, and apoptosis expressed as percentages of apoptotic cells (FITC-positive cells) determined within total population (PI-stained) cells on the coverslips after 48 h treatment with 10 μM ASN. Data represent the mean value ± S.E.M. for 3 independent experiments. ** p<0.01 <i>versus</i> control; ## p<0.01 <i>versus</i> ASN-treated cells using a one-way ANOVA followed by Dunnett's Multiple Comparison test.</p

The effect of ASN on tubulin polymerization.

<p>PC12 cells were incubated with 10 μM ASN for 48 h. SB-216763, Colchicine and Taxol were used as GSK-3β inhibitor, microtubule destabilizer and microtubule stabilizer, respectively. The level of free and polymerized α/β-tubulin was determined using the Western blotting method. (<b>A</b>) Immunoreactivity of free and polymerized α/β-tubulin (<b>B</b>) Densitometric analysis of free and polymerized α/β-tubulin immunoreactivity. Microtubule instability expressed as a ratio of free to the polymerized tubulin. Data represent the mean value ± S.E.M. for 3 independent experiments. ** p<0.01 <i>versus</i> control; # p<0.01 <i>versus</i> ASN-treated cells using a one-way ANOVA followed by Dunnett's Multiple Comparison test.</p

The effect of ASN on the protein level and gene expression for GSK-3β.

<p>PC12 cells were incubated in the presence of 10 μM ASN for 48 h. The total level of GSK-3β was determined using Western blot analysis. (<b>A</b>) Immunoreactivity of GSK-3β and GAPDH protein, which is presented as a loading control. (<b>B</b>) Densitometric analysis of GSK-3β immunoreactivity. Results were normalized to GAPDH levels. (<b>C</b>) The gene expression for total GSK-3β was measured with real-time PCR. Data represent the mean value ± S.E.M. for 5 independent experiments. * p<0.05 <i>versus</i> control using a Student's <i>t</i>-test.</p

Post embedment immunostaining of p-Tau (Ser396) with 18 nm particles gold-conjugated antibody.

<p>(<b>A</b>) Control PC12 cells. (<b>B1–B3</b>) PC12 cells after 48 h treatment with 10 μM ASN. (<b>C</b>) Bar charts representing the number of gold particles representing p-Tau (Ser396) protein. Gold particles are indicated by arrows. ASN treatment resulted in significantly elevated expression of p-Tau protein in comparison to the control PC12 cells. Data represent the mean value ± S.E.M. for 3 independent experiments. *** p<0.001 <i>versus</i> control using a Student's <i>t</i>-test. Bar 1 μm. (N - nucleus, M - mitochondria, ER - endoplasmic reticulum, E - endosomes).</p

The effect of CDK5 and GSK-3β inhibitors on ASN-evoked increase in Tau phosphorylation.

<p>PC12 cells were incubated with 10 μM ASN in the presence of 10 μM inhibitors for 48 h. BML-259 and SB-216763 were used as CDK5 and GSK-3β inhibitor, respectively. The level of Tau phosphorylation at Ser396 was determined using the Western blotting method. (<b>A</b>) Immunoreactivity of p-Tau (Ser396) and GAPDH protein, which is presented as a loading control. (<b>B</b>) Densitometric analysis of p-Tau (Ser396) immunoreactivity. Results were normalized to GAPDH levels. Data represent the mean value ± S.E.M. for 3 independent experiments. ** p<0.01 <i>versus</i> control; ## p<0.01 <i>versus</i> ASN-treated cells, using a one-way ANOVA followed by Dunnett's Multiple Comparison test.</p

Maternal Immune Activation Induces Neuroinflammation and Cortical Synaptic Deficits in the Adolescent Rat Offspring

Maternal immune activation (MIA), induced by infection during pregnancy, is an important risk factor for neuro-developmental disorders, such as autism. Abnormal maternal cytokine signaling may affect fetal brain development and contribute to neurobiological and behavioral changes in the offspring. Here, we examined the effect of lipopolysaccharide-induced MIA on neuro-inflammatory changes, as well as synaptic morphology and key synaptic protein level in cerebral cortex of adolescent male rat offspring. Adolescent MIA offspring showed elevated blood cytokine levels, microglial activation, increased pro-inflammatory cytokines expression and increased oxidative stress in the cerebral cortex. Moreover, pathological changes in synaptic ultrastructure of MIA offspring was detected, along with presynaptic protein deficits and down-regulation of postsynaptic scaffolding proteins. Consequently, ability to unveil MIA-induced long-term alterations in synapses structure and protein level may have consequences on postnatal behavioral changes, associated with, and predisposed to, the development of neuropsychiatric disorders