Exosomes: mediators of communication in eukaryotes

In addition to the established mechanisms of intercellular signaling, a new way of communication has gained much attention in the last decade: communication mediated by exosomes. Exosomes are nanovesicles (with a diameter of 40-120 nm) secreted into the extracellular space by the multivesicular endosome after its outer membrane fuses with the plasma membrane. Once released, exosomes modulate the response of the recipient cells that recognize them. This indicates that exosomes operate in a specific manner and participate in the regulation of the target cell. Remarkably, exosomes occur from unicellular organisms to mammals, suggesting an evolutionarily conserved mechanism of communication. In this review we describe the cascade of exosome formation, intracellular traffic, secretion, and internalization by recipient cells, and review their most relevant effects. We also highlight important steps that are still poorly understood

Tyrosine hydroxylase is short-term regulated by the ubiquitin-proteasome system in PC12 cells and hypothalamic and brainstem neurons from spontaneously hypertensive rats: possible implications in hypertension.

Aberrations in the ubiquitin-proteasome system (UPS) are implicated in the pathogenesis of various diseases. Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamines biosynthesis, is involved in hypertension development. In this study we investigated whether UPS regulated TH turnover in PC12 cells and hypothalamic and brainstem neurons from spontaneously hypertensive rats (SHR) and whether this system was impaired in hypertension. PC12 cells were exposed to proteasome or lysosome inhibitors and TH protein level evaluated by Western blot. Lactacystin, a proteasome inhibitor, induced an increase of 86 ± 15% in TH levels after 30 min of incubation, then it started to decrease up to 6 h to reach control levels and finally it rose up to 35.2 ± 8.5% after 24 h. Bafilomycin, a lysosome inhibitor, did not alter TH protein levels during short times, but it increased TH by 92 ± 22% above basal after 6 h treatment. Before degradation proteasome substrates are labeled by conjugation with ubiquitin. Efficacy of proteasome inhibition on TH turnover was evidenced by accumulation of ubiquitinylated TH after 30 min. Further, the inhibition of proteasome increased the quantity of TH phosphorylated at Ser40, which is essential for TH activity, by 2.7 ± 0.3 fold above basal. TH protein level was upregulated in neurons from hypothalami and brainstem of SHR when the proteasome was inhibited during 30 min, supporting that neuronal TH is also short-term regulated by the proteasome. Since the increased TH levels reported in hypertension may result from proteasome dysfunction, we evaluate proteasome activity. Proteasome activity was significantly reduced by 67 ± 4% in hypothalamic and brainstem neurons from SHR while its protein levels did not change. Present findings show that TH is regulated by the UPS. The impairment in proteasome activity observed in SHR neurons may be one of the causes of the increased TH protein levels reported in hypertension

Colocalization of TH with ubiquitin or proteasome in PC12 cells.

<p>Cells were incubated with either mouse anti-TH plus rabbit anti-ubiquitin antibodies (upper panel) or rabbit anti-TH plus mouse anti-proteasome antibodies (lower panel) followed by incubation with a anti-rabbit antibody coupled to Alexa 488 and anti-mouse coupled to AlexaFluor 594. See text for details. Scale bar = 5 μm. Three separate cell culture preparations were performed and 10 different images were taken and analyzed for each experiment. One representative image is presented.</p

Effect of proteasome and lysosome inhibition on TH protein level.

<p>PC12 cells were treated with 1 μmol/L lactacystin (A), a specific proteasome inhibitor, or 5 nmol/L bafilomycin (B), a H⁺-ATPase inhibitor, at different times and TH protein level assessed by Western-blot as described under Methods. The blots were stripped and reprobed for GAPDH to assess protein loading. Signals from TH protein level were normalized to GAPDH levels. Each bar represents the mean ± SEM of 3 determinations from four separate cell culture preparations. * P < 0.05 as compared to basal.</p

Colocalization of phospho-Ser40-TH (P-Ser 40 TH) or phospho-Ser19-TH (P-Ser 19 TH) with proteasome in PC12 cells.

<p>Cells were incubated with either rabbit phospho-Ser40-TH plus mouse proteasome antibodies (upper panel) or rabbit phospho-Ser19-TH plus mouse proteasome antibodies (lower panel) followed by incubation with an anti-rabbit antibody coupled to Alexa 488 and anti-mouse coupled to AlexaFluor 594. See text for details. Scale bar = 5 μm. Three separate cell culture preparations were performed and 10 different images were taken and analyzed for each experiment. One representative image is presented.</p

A, Proteasome activity in the hypothalamus and brainstem of WKY and SHR rats.

<p>Values are mean ±SEM (n = 7). * P < 0.05 as compared with WKY. B, Proteasome protein level in the hypothalamus and brainstem, respectively, from WKY and SHR. Proteasome protein level was measured by Western-blot as described under Methods. Blots were stripped and reprobed for GAPDH to assess protein loading. Signals from proteasome protein level were normalized to GAPDH levels. Values are mean ±SEM (n = 7). * P < 0.05 as compared to WKY. C, Proteasome protein level and activity in neuronal cultures from the hypothalamus and brainstem of WKY and SHR rats. Values are mean ±SEM of 3 determinations from three separate cell culture preparations. * P < 0.05 as compared with WKY.</p

TH ubiquitination.

<p>PC12 cells were incubated for 30 min in the absence (basal) or presence of lactacystin (Lacta) (1 μmol/L). Ub-TH was immunoprecipitated with an ubiquitinated protein enrichment kit (IP: anti-Ub). Proteins were resolved in a 4–12% SDS-PAGE and Ub-TH protein level assessed by Western-blot as described under Methods. The same membrane was stripped and reprobed with an antibody against ubiquitin (Ub) to further corroborate that Ub-proteins were isolated with the kit. Three separate experiments were performed. A representative Western blot immunoblotted with antibodies directed against TH or Ub is shown.</p

TH protein level in the hypothalamus and brainstem of WKY or SHR rats.

<p>A, TH protein level was assessed by Western-blot as described under Methods. Blots were stripped and reprobed for GAPDH to assess protein loading. Signals from TH protein level were normalized to GAPDH levels. Each bar represents the mean ± SEM (n = 7). * P < 0.05 as compared to WKY. B, Change in mean arterial pressure (MAP) in WKY and SHR after intracerebroventricular injection of aCSF (control) or the proteasome inhibitor MG-132. Values are mean ±SEM (n = 7). * P < 0.05 as compared with basal.</p