The role of reciprocal activation of cAbl and Mst1 in the oxidative death of cultured astrocytes.

The protein kinase Mst1 (mammalian Sterile 20-like kinase 1) likely plays a role in oxidative neuronal cell death as a target of its activator, cAbl. We previously found that H2O2-induced death of astrocytes is mediated by cAbl in a metallothionein-3 (Mt3)-dependent manner. In the present study, we examined a possible role for Mst1 in the oxidative death of astrocytes. Treatment of cortical astrocytes with 170 µM H2O2 activated Mst1. Knockdown of Mst1 reduced H2O2-induced cell death, indicating that Mst1 activation contributes to astrocytic cell death. STI571, an inhibitor of cAbl, blocked induction/activation of Mst1 and H2O2-induced cell death. However, Mst1 silencing also inhibited induction/activation of cAbl, suggesting that the two kinases are regulated by a reciprocal activating mechanism. The zinc chelator TPEN blocked induction/activation of cAbl and Mst1, indicating that these phenomena are dependent on the rise of intracellular zinc. Moreover, H2O2 exposure did not increase free zinc levels in Mt3-null astrocytes, suggesting that the increased levels of free zinc were largely from Mt3. Consistent with the involvement of FoxO1/3, which may play a role in the Mst1-cell death cascade, we found an increase in the level of phosphorylated FoxO1/3 in H2O2-treated astrocytes. Moreover, inhibition of cAbl or Mst1 reversed this effect. The present results suggest the interesting possibility that cAbl and Mst1 are reciprocally activated under oxidative stress conditions in astrocytes. Both kinases appear to be regulated by changes in the levels of free zinc originating from Mt3 and contribute to oxidative cell death through a FoxO-dependent mechanism.

Mitochondrial inhibitor models of Huntington's disease and Parkinson's disease induce zinc accumulation and are attenuated by inhibition of zinc neurotoxicity in vitro or in vivo.

BACKGROUND: Inhibition of mitochondrial function occurs in many neurodegenerative diseases, and inhibitors of mitochondrial complexes I and II are used to model them. The complex II inhibitor, 3-nitroproprionic acid (3-NPA), kills the striatal neurons susceptible in Huntington's disease. The complex I inhibitor N-methyl-4-phenylpyridium (MPP(+)) and 6-hydroxydopamine (6-OHDA) are used to model Parkinson's disease. Zinc (Zn(2+)) accumulates after 3-NPA, 6-OHDA and MPP(+) in situ or in vivo. OBJECTIVE: We will investigate the role of Zn(2+) neurotoxicity in 3-NPA, 6-OHDA and MPP(+). METHODS: Murine striatal/midbrain tyrosine hydroxylase positive, or near-pure cortical neuronal cultures, or animals were exposed to 3-NPA or MPP(+) and 6-OHDA with or without neuroprotective compounds. Intracellular zinc ([Zn(2+)](i)), nicotinamide adenine dinucleotide (NAD(+)), NADH, glycolytic intermediates and neurotoxicity were measured. RESULTS: We showed that compounds or genetics which restore NAD(+) and attenuate Zn(2+) neurotoxicity (pyruvate, nicotinamide, NAD(+), increased NAD(+) synthesis, sirtuin inhibition or Zn(2+) chelation) attenuated the neuronal death induced by these toxins. The increase in [Zn(2+)](i) preceded a reduction in the NAD(+)/NADH ratio that caused a reversible glycolytic inhibition. Pyruvate, nicotinamide and NAD(+) reversed the reductions in the NAD(+)/NADH ratio, glycolysis and neuronal death after challenge with 3-NPA, 6-OHDA or MPP(+), as was previously shown for exogenous Zn(2+). To test efficacy in vivo, we injected 3-NPA into the striatum of rats and systemically into mice, with or without pyruvate. We observed early striatal Zn(2+) fluorescence, and pyruvate significantly attenuated the 3-NPA-induced lesion and restored behavioral scores. CONCLUSIONS: Together, these studies suggest that Zn(2+) accumulation caused by MPP(+) and 3-NPA is a novel preventable mechanism of the resultant neurotoxicity.