Abstract
The molecular mechanisms of alpha7 nicotinic acetylcholine receptor (nAChR)-mediated neuroprotection remain unclear. In this study we provide evidence that nicotine stimulation of alpha7 nAChR transduces signals to phosphatidylinositol 3-kinase and Akt via Janus kinase 2 (JAK2) in a cascade, which results in neuroprotection. Exposure to beta-amyloid results in the activation of the apoptotic enzyme caspase-3 and cleavage of the DNA-repairing enzyme poly-(ADP-ribose) polymerase. This cascade is inhibited by nicotine through JAK2 activation, and these effects are blocked by preincubation with the JAK2-specific inhibitor AG-490. We also found that pretreatment of cells with angiotensin II blocks the nicotine-induced activation of JAK2 via the AT(2) receptor and completely prevents alpha7 nAChR-mediated neuroprotective effects further suggesting a pivotal role for JAK2. These findings identify novel mechanisms of receptor interactions relevant to neuronal viability and suggest novel therapeutic strategies to optimize neuroprotection.
Highlights
The cholinergic deficit in Alzheimer’s disease (AD)[1] has been clearly established and is the basis for the current symptomatic strategy
We provide evidence that nicotine interaction with the ␣7 nicotinic acetylcholine receptor (nAChR) is “dominant” over A-(1– 42) interaction with the receptor and that the A-(1– 42)-induced apoptosis is prevented through the nicotine-induced activation of Janus kinase 2 (JAK2)
We found that the nicotine neuroprotective effects can be neutralized through activation of the angiotensin II AT2 receptor as evidenced by the reversal of JAK2 phosphorylation and inhibition of nicotine-induced neuroprotection
Summary
Materials—Molecular weight standards, acrylamide, SDS, N,NЈmethylenebisacrylamide, N,N,NЈ,NЈ-tetramethylenediamine, protein assay reagents, and nitrocellulose membranes were purchased from Bio-Rad. Samples were resolved by 10% SDS-PAGE, transferred to a nitrocellulose membrane, and blocked by a 60-min incubation at 22 °C in TTBS (Tris-buffered saline with 0.05% Tween 20, pH 7.4) plus 5% skimmed milk powder. The nitrocellulose membrane was incubated overnight at 4 °C with affinity-purified anti-phospho-specific JAK2 and Akt antibodies. Immunoprecipitation Studies of PI-3-K—The cell lysate, prepared as described above, was incubated with 10 g/ml anti-PI-3-K monoclonal antibodies at 4 °C for 2 h and precipitated by addition of 50 l of protein A/G-agarose at 4 °C overnight. The nitrocellulose membrane was incubated overnight at 4 °C with 10 g/ml affinity-purified anti-phosphotyrosine antibodies, and the bound antibodies were visualized using a Pierce Supersignal chemiluminescence detection kit.
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