Yongmei Pu

Minocycline prevents glutamate-induced apoptosis of cerebellar granule neurons by differential regulation of p38 and Akt pathways

Minocycline has been shown to have remarkably neuroprotective qualities, but underlying mechanisms remain elusive. We reported here the robust neuroprotection by minocycline against glutamate‐induced apoptosis through regulations of p38 and Akt pathways. Pre‐treatment of cerebellar granule neurons (CGNs) with minocycline (10–100 µm) elicited a dose‐dependent reduction of glutamate excitotoxicity and blocked glutamate‐induced nuclear condensation and DNA fragmentations. Using patch‐clamping and fluorescence Ca2+ imaging techniques, it was found that minocycline neither blocked NMDA receptors, nor reduced glutamate‐caused rises in intracellular Ca2+. Instead, confirmed by immunoblots, minocycline in vivo and in vitro was shown to directly inhibit the activation of p38 caused by glutamate. A p38‐specific inhibitor, SB203580, also attenuated glutamate excitotoxicity. Furthermore, the neuroprotective effects of minocycline were blocked by phosphatidylinositol 3‐kinase (PI3‐K) inhibitors LY294002 and wortmannin, while pharmacologic inhibition of glycogen synthase kinase 3β (GSK3β) attenuated glutamate‐induced apoptosis. In addition, immunoblots revealed that minocycline reversed the suppression of phosphorylated Akt and GSK3β caused by glutamate, as were abolished by PI3‐K inhibitors. These results demonstrate that minocycline prevents glutamate‐induced apoptosis in CGNs by directly inhibiting p38 activity and maintaining the activation of PI3‐K/Akt pathway, which offers a novel modality as to how the drug exerts protective effects.

Measuring dynamics of caspase-8 activation in a single living HeLa cell during TNFα-induced apoptosis

In this study, we reported the first measurement of the dynamics of activation of caspase-8 in a single living cell. This measurement was conducted using a specially developed molecular sensor based on the FRET (fluorescence resonance energy transfer) technique. This sensor was constructed by fusing a CFP (cyan fluorescent protein) and a YFP (yellow fluorescent protein) with a linker containing a tandem caspase-8-specific cleavage site. The change of the FRET ratio upon cleavage was larger than 4-fold. Using this sensor, we found that during TNFalpha-induced apoptosis, the activation of caspase-8 was a slower process than that of caspase-3, and it was initiated much earlier than the caspase-3 activation. Inhibition of caspase-9 delayed the full activation of caspase-3 but did not affect the dynamics of caspase-8. Results of these single-cell measurements suggested that caspase-3 was activated by caspase-8 through two parallel pathways during TNFalpha-induced apoptosis in HeLa cells.

A Ca2+ signal is found upstream of cytochrome c release during apoptosis in HeLa cells

We showed previously that a cytosolic Ca(2+) signal is involved in regulating UV-induced apoptosis in HeLa cells. In this study, we found evidence that this Ca(2+) signal occurs upstream of the release of cytochrome c from mitochondria. First, when we abolished [Ca(2+)](i) increases by injecting BAPTA or heparin into UV-treated HeLa cells, cytochrome c release was either blocked or severely delayed. Second, using a living cell imaging technique, we observed a series of transient [Ca(2+)](i) increases (typically lasting about 40-60s) in many apoptotic cells induced by either UV- or TNFalpha-treatment. Third, using GFP-tagged cytochrome c, we found that the Ca(2+) spikes appear in a time window before cytochrome c was released. Finally, by fixing the TNFalpha-treated cell at the time when it started to display Ca(2+) spikes, we examined the distribution of its endogenous cytochrome c using immunostaining. We found that cytochrome c was not yet released from mitochondria. These findings suggest the existence of certain apoptotic pathways, in which an early Ca(2+) signal is activated upstream of cytochrome c release.