Genome-wide microarray analysis of the differential neuroprotective effects of antioxidants in neuroblastoma cells overexpressing the familial Parkinson's disease alpha-synuclein A53T mutation.

In Parkinson's disease substantia nigra neurons degenerate likely due to oxidative damage interacting with genetic risk factors. Here, SH-SY5Y cells expressing wild-type or A53T alpha-synuclein had increased sensitivity to methyl-4-phenylpyridinium iodide (MPP(+)), which induces mitochondrial dysfunction, and 6-hydroxydopamine (6-OHDA), which causes oxidative stress. Edaravone protected only against MPP(+), and EGCG ((-)-epigallocatechin-3-O-gallate) protected only against 6-OHDA. Thus genomic responses to MPP(+) and 6-OHDA in the presence of these antioxidants were analyzed using microarrays. Pathway analysis indicated that MPP(+) activated p53 (P < 0.001) while 6-OHDA induced the Nrf2 antioxidative stress response (P < 0.0001). EGCG was more effective at blocking 6-OHDA-mediated genomic responses, while edaravone was more effective against MPP(+). We identified 32 genes that responded to both toxins except in the presence of an effective anti-oxidant; eight are transcription factors and potentially constitute a stress-response transcriptional network. These data provide insights into the mechanisms of neurotoxicity and identifies genes that might mediate antioxidant efficacy.

Increased nuclear factor-erythroid 2 p45-related factor 2 activity protects SH-SY5Y cells against oxidative damage.

The ability of cells to control the balance between the generation and quenching of reactive oxygen species is important in combating potentially damaging effects of oxidative stress. One mechanism that cells use to maintain redox homeostasis is the antioxidant response pathway. Antioxidant response elements (AREs) are cis-acting elements located in regulatory regions of antioxidant and phase II detoxification genes. Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) is a member of the Cap 'n' Collar family of transcription factors that binds to the ARE and regulates the transcription of specific ARE-containing genes such as NAD(P)H:quinone oxidoreductase 1, glutamylcysteine synthetase and heme oxygenase. Activation of Nrf2 results in release from its negative repressor, Kelch-like ECH-associated protein 1 (Keap1), and allows Nrf2 to translocate into the nucleus to induce gene expression. In this study, we demonstrate that increasing Nrf2 activity by various methods, including chemical induction, Nrf2 overexpression or Keap1 siRNA knockdown, protects cells against specific types of oxidative damage. Cells were protected against 6-hydroxydopamine- and 3-morpholinosydnonimine-mediated toxicity but not against 1-methyl-1-4-phenylpyridinium toxicity. As oxidative stress is a hallmark of several neurodegenerative disorders, including Parkinson's disease, pharmacological agents that selectively target the Keap1-Nrf2 pathway may provide a novel neuroprotective strategy for the treatment of these diseases.

The composition of the beta-2 adrenergic receptor oligomer affects its membrane trafficking after ligand-induced endocytosis.

The beta-2 adrenergic receptor (B2AR) is well known to form oligomeric complexes in vivo, but the functional significance of this process is not fully understood. The present results identify an effect of oligomerization of the human B2AR on the membrane trafficking of receptors after agonist-induced endocytosis in stably transfected human embryonic kidney 293 cells. A sequence present in the cytoplasmic tail of the B2AR has been shown previously to be required for efficient recycling of internalized receptors. Mutation of this sequence was observed to inhibit recycling not only of the receptor containing the mutation but also of the coexpressed wild-type B2AR. Coexpression of recycling-defective mutant B2ARs also enhanced proteolytic degradation of the wild-type B2AR after agonist-induced endocytosis, consistent with trafficking of both receptors to lysosomes in an oligomeric complex. Coexpression of the delta opioid receptor (DOR) at similar levels produced a much smaller effect on endocytic trafficking of the B2AR, even though DOR traverses a similar membrane pathway as recycling-defective mutant B2ARs. Biochemical studies confirmed that B2AR/B2AR-ala homomeric complexes form more readily than DOR/B2AR heteromers in expression-matched cell clones and support the hypothesis that B2AR/B2AR-ala complexes are not disrupted by agonist. These results suggest that a significant fraction of B2ARs exists in oligomeric complexes after ligand-induced endocytosis and that the composition of the oligomeric complex influences the sorting of endocytosed receptors between functionally distinct recycling and degradative membrane pathways.

Myosin-Va binds to and mechanochemically couples microtubules to actin filaments.

Myosin-Va was identified as a microtubule binding protein by cosedimentation analysis in the presence of microtubules. Native myosin-Va purified from chick brain, as well as the expressed globular tail domain of this myosin, but not head domain bound to microtubule-associated protein-free microtubules. Binding of myosin-Va to microtubules was saturable and of moderately high affinity (approximately 1:24 Myosin-Va:tubulin; Kd = 70 nM). Myosin-Va may bind to microtubules via its tail domain because microtubule-bound myosin-Va retained the ability to bind actin filaments resulting in the formation of cross-linked gels of microtubules and actin, as assessed by fluorescence and electron microscopy. In low Ca2+, ATP addition induced dissolution of these gels, but not release of myosin-Va from MTs. However, in 10 microM Ca2+, ATP addition resulted in the contraction of the gels into aster-like arrays. These results demonstrate that myosin-Va is a microtubule binding protein that cross-links and mechanochemically couples microtubules to actin filaments.