Janus Kinase 2, an Early Target of (cid:1) 7 Nicotinic Acetylcholine Receptor-mediated Neuroprotection against A (cid:2) -(1– 42) Amyloid*

The molecular mechanisms of (cid:1) 7 nicotinic acetylcholine receptor (nAChR)-mediated neuroprotection remain unclear. In this study we provide evidence that nicotine stimulation of (cid:1) 7 nAChR transduces signals to phosphati-dylinositol 3-kinase and Akt via Janus kinase 2 (JAK2) in a cascade, which results in neuroprotection. Exposure to (cid:2) -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-spe-cific 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 pre- vents (cid:1) 7 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 strate-gies to optimize neuroprotection. The cholinergic deficit in Alzheimer’s disease (AD) 1 has been clearly established and is the basis with anti-PI-3-K The PI-3-K immunoprecipitated proteins are then immunoblotted with anti-phosphotyrosine and anti-PI-3-K of three experiments. cleavage endogenous substrate is a fragment

The molecular mechanisms of ␣7 nicotinic acetylcholine receptor (nAChR)-mediated neuroprotection remain unclear. In this study we provide evidence that nicotine stimulation of ␣7 nAChR transduces signals to phosphatidylinositol 3-kinase and Akt via Janus kinase 2 (JAK2) in a cascade, which results in neuroprotection. Exposure to ␤-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 ␣7 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.
The cholinergic deficit in Alzheimer's disease (AD) 1 has been clearly established and is the basis for the current symptomatic strategy. There is an early and significant depletion of high affinity nicotinic receptors in the brains of Alzheimer's patients (1), and a number of studies have shown cognitive improvement in rodent and primates including humans following administration of ligands targeting nicotinic acetylcholine receptor (nAChR) (2). In addition to their known symptomatic effects, neuronal nicotinic ligands have shown neuroprotective activity in vitro (3) and in vivo (4) suggesting an additional potential for disease modification.
In comparison to the findings above, another study (9) has shown that whereas nicotine activates the PI-3-K neuroprotective cascade, A␤-(1-42) chronically activates the mitogen-activated protein kinase (MAPK) cascade via the hippocampal ␣7 nAChR. The investigators suggest that this chronic activation of the MAPK pathway by A␤-(1-42) eventually leads to the down-regulation of MAPK which then sets up a positive feedback for A␤ accumulation and decreased phosphorylation of the cAMP-regulatory protein which is a necessary component for hippocampus-dependent memory formation in mammals (9).
The angiotensin-converting enzyme density is also increased in the temporal cortex from patients with AD (10), and the angiotensin-converting enzyme genotype is associated with AD in some populations (11). The angiotensin II (Ang II) AT 2 receptor exerts growth inhibitory effects or apoptosis both in cultured cells and in vivo (12) are expressed in PC12 cells, and have been shown to inhibit the Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling cascade (13).
In this study we show that nicotine-induced neuroprotection against A␤-(1-42) is mediated through tyrosine phosphorylation of JAK2, subsequent activation of PI-3-K and Akt, and inhibition of both caspase-3 activity and cleavage of the DNArepairing enzyme poly(ADP-ribose) polymerase (PARP). In contrast, pretreatment of cells with Ang II blocks the nicotineinduced activation of JAK2 via the AT 2 receptor and completely prevents ␣7 nAChR-mediated neuroprotective effects.
Isolation and Culture of PC12 Cells-PC12, rat pheochromocytoma cells, were maintained in proliferative growth phase in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% horse serum, * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Western Blotting Studies of JAK2 and Akt-The phosphorylation of JAK2 and Akt proteins was determined in serum-starved PC12 cells stimulated with 10 M nicotine (0 -120 min) in the presence or absence of 10 M (1 h preincubation) of the JAK2-specific inhibitor AG-490 (15,16). Whereas many tyrosine kinase inhibitors are often promiscuous in the enzyme they target, AG-490 is unique in that it does not inhibit other tyrosine kinases such as Lck, Lyn, Btk, Syk, Src, JAK1, or Tyk2 (15). At the end of stimulation, cells were washed twice with ice-cold PBSV (phosphate-buffered saline with 1 mmol/liter Na 3 VO 4 ). Each dish was then treated for 60 min with ice-cold lysis buffer (20 mmol/liter Tris-HCl, pH 7.4, 2.5 mmol/liter EDTA, 1% Triton X-100, 10% glycerol, 10 mmol/liter Na 4 P 2 O 7 , 50 mmol/liter NaF, 1 mmol/liter Na 3 VO 4 , and 1 mmol/liter phenylmethylsulfonyl fluoride), and the supernatant fraction was obtained as cell lysate by centrifugation at 58,000 ϫ g for 25 min at 4°C. 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. The nitrocellulose membranes were washed twice for 10 min with TTBS and incubated with goat anti-rabbit IgG horseradish peroxidase conjugate. After extensive washing, the bound antibody was visualized on a Kodak Biomax film using a Pierce Supersignal substrate chemiluminescence detection kit.
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 immunoprecipitates were recovered by centrifugation and washed three times with ice-cold wash buffer (Tris-buffered saline, 0.1% Triton X-100, 1 mmol/liter phenylmethylsulfonyl fluoride, and 1 mmol/liter Na 3 VO 4 ). Immunoprecipitated proteins were dissolved in 100 l of Laemmli sample buffer, and 80 l of each sample were resolved by SDS-PAGE. Samples were transferred to a nitrocellulose membrane and blocked by 60-min incubation at room temperature (22°C) in TTBS plus 5% skimmed milk powder. The nitrocellulose membrane was then 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.
Assessment of PC12 Cell Apoptosis-Apoptosis was determined by as- sessing the cleavage of the DNA-repairing enzyme PARP using a Western blot assay. PARP (116-kDa) is an endogenous substrate for caspase-3, which is cleaved to a typical 85-kDa fragment during various forms of apoptosis. PC12 cells were treated with 0.1 M A␤ for 8 h in the presence or absence of nicotine and/or AG-490. The cells were collected, washed with phosphate-buffered saline, and lysed in 1 ml of SDS-PAGE sample buffer boiled for 10 min. Total cell lysates (30 g of protein) were separated by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked for 1 h at 25°C with 5% nonfat dry milk in TBST (25 mM Tris-HCl, pH 7.5, 0.5 M NaCl, 0.05% Tween 20). Membranes were incubated with primary PARP antibody specific for the 85-kDa fragment for 2-3 h at 25°C, rinsed with TBST, and incubated with secondary antibody for 1 h at 25°C. Immunodetection was performed with appropriate antibody using an ECL system (Amersham Biosciences).
Caspase-3 enzyme activity was determined with a fluorogenic substrate for caspase-3 in crude PC12 cell extracts. The caspase-3 fluorogenic peptide Ac-DEVD-AMC (Promega, Madison, WI) contained the specific caspase-3 cleavage sequence (DEVD) coupled at the C terminus to the fluorochrome 7-amino-4-methylcoumarin. The substrate emitted a blue fluorescence when excited at a wavelength of 360 nm. When cleaved from the peptide by the caspase-3 enzyme activity in the cell lysate, free 7-amino-4-methylcoumarin was released and was detected by its yellow/green emission at 460 nm. Appropriate controls included a reversible aldehyde inhibitor of caspase-3 to assess the specific contri-bution of the caspase-3 enzyme activity (data not shown). Fluorescence units were normalized relative to total protein concentration of the cell extract. We performed the assays in triplicate and repeated the experiments three times. In addition we measured the decrease in PC12 cell number using a Coulter counter (model ZM, Coulter, Hialeah, FL).
Data Analysis-All statistical comparisons were made using Student's t test for paired data and analysis of variance. Significance was p Ͻ 0.05.

Effects of the JAK2 Inhibitor AG-490 on the Nicotine-induced Tyrosine Phosphorylation of JAK2 and PI-3-K and Serine
Phosphorylation of Akt in PC12 Cells-JAK2 was tyrosine-phosphorylated in response to nicotine within 5-10 min, and this activation remained above basal levels even after longer exposure (120 min) to nicotine (Fig. 1). The JAK2 inhibitor AG-490 inhibited the basal and nicotine-stimulated JAK2 tyrosine phosphorylation, the tyrosine phosphorylation of PI-3-K, and the serine phosphorylation of Akt (Fig. 1). Similar results were observed in the human cell line SH-SY5Y (data not shown). These results suggest that JAK2 activation by nicotine precedes the activation of PI-3-K and its effector Akt. JAK2 activation is completely prevented by preincubation of ␣-Bgt indicating a receptor-mediated effect ( Fig. 2A).

Effects of Nicotine on the JAK2 Complex Formation with the
␣7 nAChR-To test the hypothesis that JAK2 interacts directly with ␣7 nAChR we conducted co-immunoprecipitation studies using a rabbit polyclonal anti-JAK2 antibody. We stimulated cultured PC12 cells with nicotine (10 M) for various times, lysed the cells, and immunoprecipitated JAK2 with anti-JAK2 antibody. Immunoprecipitated proteins were separated by gel electrophoresis, transferred to nitrocellulose, and immunoblotted with anti-␣7 nAChR antibodies. As shown in Fig. 2B, nicotine induced a rapid association of JAK2 with the ␣7 nAChR within 5 min. This time course of ␣7 nAChR association with JAK2 was similar to that of the nicotine-induced activation of JAK2 (Fig. 1). Similar results were also obtained when the experiments were repeated using ␣7 nAChR receptor antibody to immunoprecipitate the receptor and to probe the Western blot with the anti-JAK2 antibody (Fig. 2B).
Effects of Nicotine on the A␤-(1-42)-induced Apoptosis and the Role of JAK2-Caspase-3 is expressed in PC12 cells and is known to be involved in apoptosis. We examined caspase-3 activity following A␤-(1-42)-induced apoptosis. We used the fluorescent peptide substrate Ac-DEVD-7AMC to measure caspase-3-like activity in cell lysates. As shown in Fig. 5, the caspase-3-like activity that resulted in the cleavage of the peptide substrate Ac-DEVD-7AMC is evident after 4 h of A␤-(1-42) treatment and increased over time until it reached a peak after 8 h of treatment. The A␤-(1-42)-induced activation of caspase-3 was blocked by nicotine (p Ͻ 0.01), and this inhibition was prevented by AG-490 (Fig. 5).
We explored further the activation of caspase-3 following A␤-(1-42) treatment by measuring the cleavage of the DNArepairing enzyme PARP using Western blot assay. PARP is an endogenous substrate for caspase-3, which is cleaved to a typical 85-kDa fragment during various forms of apoptosis. As shown in Fig. 6, PARP (116-kDa) was cleaved to its 85-kDa fragment following A␤-(1-42) treatment. Again, just like the activation of caspase-3, the A␤-(1-42)-induced cleavage of PARP was blocked by nicotine, and this inhibition was prevented by AG-490 and Ang II (Fig. 6). This PARP cleavage further indicates that caspase-3 or caspase-3-like proteases are activated in A␤-(1-42)-induced cells death.
We tested the involvement of JAK2 in nicotine-induced neuroprotection in the presence or absence of A␤-(1-42). We measured the decrease in PC12 cell number using a Coulter counter following A␤-(1-42) and Ang II treatments in the presence or absence of nicotine and AG-490. As shown in Fig. 7, cell death induced by A␤-(1-42) treatment was significantly reduced in the presence of nicotine (p Ͻ 0.01). Nicotine had no effect on A␤-(1-42)-induced cell death when co-incubated with AG-490  (Fig. 7). These results suggest that JAK2 plays an essential role in the nicotine-induced neuroprotection against A␤-(1-42)induced cell death. In contrast, Ang II-induced apoptosis was not affected by nicotine (Fig. 7).

Effects of Ang II Pretreatment with or without Ang II Receptor Antagonists on Nicotine-induced Activation of JAK2-Pre-
incubation of PC12 cells with Ang II blocked the nicotineinduced tyrosine phosphorylation of JAK2 via the AT 2 receptor (Fig. 8). This inhibition was completely prevented by preincubation with an AT 2 antagonist (PD 123177 at 100 nM) but not by an AT 1 antagonist (candesartan at 100 nM) consistent with the receptor phenotype expressed in PC12 cells. This inhibition of nicotine-induced JAK2 phosphorylation was accompanied by a complete reversal of nicotine-induced neuroprotection as shown by the nicotine-insensitive PARP cleavage and cell viability ( Fig. 6 and Fig. 7). DISCUSSION In this study we provide evidence for the nicotine-induced complex formation between the ␣7 nAChR and the tyrosinephosphorylated enzyme JAK2 that results in subsequent activation of PI-3-K and Akt. We also provide evidence that nicotine interaction with the ␣7 nAChR is "dominant" over A␤-  interaction with the receptor and that the A␤-(1-42)-induced apoptosis is prevented through the nicotine-induced activation of JAK2. Finally, we also found that the nicotine neuroprotective effects can be neutralized through activation of the angiotensin II AT 2 receptor as evidenced by the reversal of JAK2 phosphorylation and inhibition of nicotine-induced neuroprotection.
Nicotinic neurotransmission is compromised in the brains of AD patients and selective loss of nAChR predominates in brain regions with ␤-amyloid deposition (1). A direct interaction of the ␤-amyloid peptide with the ␣7 nAChR is suggested by recent findings. ␤-Amyloid peptide interacts with high affinity to the ␣7 nAChR and results in functional non-competitive blockade in hippocampal neurons (17,18). In addition, a recent study (9) has also shown that A␤-(1-42) chronically activates the MAPK cascade via the hippocampal ␣7 nAChR and that this chronic activation leads to derangement of hippocampus signal transduction in the AD brain. On the other hand, neuroprotective mechanisms mediated by nicotine in clonal cells have implicated the tyrosine phosphorylation of PI-3-K (8), an enzyme involved in phosphoinositide metabolism and linked to cell survival and apoptosis. Anti-apoptotic signals transduced via JAK2 have also been reported. For example, in hematopoietic cells, the kinase domain of JAK2 mediates the induction of Bcl-2 and inhibits cell death (19), and treatment with the JAK2 inhibitor AG-490 reduces the phosphorylation of PI-3-K (20) and STAT3 resulting in an increase in caspase-3 activity and Bax protein in acute myocardial infarction (21). In addition, activation of neuronal erythropoietin receptors prevents apoptosis by triggering cross-talk between the signaling pathways of JAK2 and the nuclear factor-B (22).
Our findings indicate that ␣7 nAChR activation induces JAK2 activation via tyrosine phosphorylation and that this initial event is followed by tyrosine phosphorylation of PI-3-K and Akt serine phosphorylation as indicated by the inhibitory effect of AG-490 on the phosphorylation of both proteins. The JAK2 phosphorylation in the presence of nicotine is completely inhibited by ␣-Bgt, an antagonist to ␣7 nAChR. Our findings indicate that nicotine-stimulated ␣7 nAChR results in the formation of a complex between the ␣7 nAChR protein and JAK2. Because interaction between ␣7 nAChR and A␤-(1-42) has been reported based on ligand binding and functional studies, we tested the possibility that ␤-amyloid could also induce an ␣7 nAChR-JAK2 complex. Our results confirm the association of ␤-amyloid and ␣7 nAChR but indicate no detectable levels of JAK2. In the presence of nicotine, no A␤ immunoreactivity can be detected in the lysate suggesting that nicotine has "displaced" A␤ from ␣7 nAChR. This effect is independent of JAK2 phosphorylation as shown by the lack of any reversal of this effect in the presence of AG-490.
It is well established that nicotine inhibits A␤ toxicity, but the mechanism is unclear. Our results demonstrate a central role for JAK2 in the ␣7 nAChR activation of key cellular enzymes involved in cell survival and in inhibition of pro-apoptotic pathways. Nicotine inhibits ␤-amyloid cytotoxicity, and this effect is completely prevented by inhibition of the tyrosine phosphorylation of JAK2. These effects can be shown by measuring markers of cytotoxicity like the cleavage of the nuclear protein PARP, the induction of caspase-3, or cell viability.
Several reports have documented the apoptotic effects of Ang II through AT 2 receptors. AT 2 receptors are expressed in PC12 and have been shown to inhibit the JAK/STAT signaling cascade (23). In contrast to nicotine-induced neuroprotection against ␤-amyloid-(1-42), pretreatment of cells with Ang II blocks the nicotine-induced activation of JAK2 via the AT 2 receptor and completely prevents ␣7 nAChR-mediated neuroprotective effects further suggesting a pivotal role for JAK2 phosphorylation. Our findings are consistent with opposite roles on cell viability between ␣7 nAChR and AT 2 receptor, activation of the latter overriding the potential benefit through the former (Fig. 9). These results and the convergence of these pathways on phosphorylated JAK2 suggest that recruitment of ␣7 receptor-mediated neuroprotection against A␤-(1-42) may be optimized under conditions where AT 2 -mediated inhibition is minimized. These findings identify novel molecular mechanisms that are fully consistent with the role attributed to ␣7 nAChR, AT 2 , and ␤-amyloid on the pathophysiology observed in the brains of Alzheimer's patients.