The Neuroprotective Effect of 2-(3-Pyridyl)-1-azabicyclo[3.2.2]nonane (TC-1698), a Novel α7 Ligand, Is Prevented through Angiotensin II Activation of a Tyrosine Phosphatase

Article abstract of DOI:10.1124/jpet.103.061655

We have recently provided evidence for nicotine-induced complex formation between the α7 nicotinic acetylcholine receptor (nAChR) and the tyrosine-phosphorylated enzyme Janus kinase 2 (JAK2) that results in subsequent activation of phosphatidylinositol-3-kinase (PI-3-K) and Akt. Nicotine interaction with the α7 nAChR inhibits Aβ (1-42) interaction with the same receptor, and the Aβ (1-42)-induced apoptosis is prevented through nicotine-induced activation of JAK2. These effects can be shown by measuring markers of cytotoxicity, including the cleavage of the nuclear protein poly(ADP-ribose) polymerase (PARP), the induction of caspase 3, or cell viability. In this study, we found that 2-(3-pyridyl)-1-azabicyclo[3.2.2]nonane (TC-1698), a novel α7-selective agonist, exerts neuroprotective effects via activation of the JAK2/PI-3K cascade, which can be neutralized through activation of the angiotensin II (Ang II) AT₂ receptor. Vanadate not only augmented the TC-1698-induced tyrosine phosphorylation of JAK2 but also blocked the Ang II neutralization of TC-1698-induced neuroprotection against Aβ (1-42)-induced cleavage of PARP. Furthermore, when SHP-1 was neutralized via antisense transfection, the Ang II inhibition of TC-1698-induced neuroprotection against Aβ (1-42) was prevented. These results support the main hypothesis that states that JAK2 plays a central role in the nicotinic α7 receptor-induced activation of the JAK2-PI-3K cascade in PC12 cells, which ultimately contribute to nAChR-mediated neuroprotection. Ang II inhibits this pathway through the AT₂ receptor activation of the protein tyrosine phosphatase SHP-1. This study supports central and opposite roles for JAK2 and SHP-1 in the control of apoptosis and α7-mediated neuroprotection in PC12 cells.

Full text of DOI:10.1124/jpet.103.061655

0
022-3565/03/3091-16–27$20.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2003 by The American Society for Pharmacology and Experimental Therapeutics
JPET 309:16–27, 2003
Vol. 309, No. 1
61655/1133905
Printed in U.S.A.
The Neuroprotective Effect of 2-(3-Pyridyl)-1-
azabicyclo[3.2.2]nonane (TC-1698), a Novel ␣7 Ligand, Is
Prevented through Angiotensin II Activation of a Tyrosine
Phosphatase
Mario B. Marrero, Roger L. Papke, Balwinder S. Bhatti, Se a´ n Shaw, and
Merouane Bencherif
Department of Pharmacology and Toxicology (M.B.M.), Vascular Biology Center, Medical College of Georgia, Augusta, Georgia
(
M.B.M., S.S.); Department of Physiology and Pharmacology, University of Florida, Gainesville, Florida (R.L.P.); and Department
Preclinical Research (M.B.) and Drug Discovery and Development (B.S.B.), Targacept Inc., Winston-Salem, North Carolina
Received October 16, 2003; accepted December 10, 2003
ABSTRACT
We have recently provided evidence for nicotine-induced com- Vanadate not only augmented the TC-1698-induced tyrosine
plex formation between the ␣7 nicotinic acetylcholine receptor phosphorylation of JAK2 but also blocked the Ang II neutral-
(
2
nAChR) and the tyrosine-phosphorylated enzyme Janus kinase ization of TC-1698-induced neuroprotection against A␤ (1-42)-
(JAK2) that results in subsequent activation of phosphatidyl- induced cleavage of PARP. Furthermore, when SHP-1 was
inositol-3-kinase (PI-3-K) and Akt. Nicotine interaction with the neutralized via antisense transfection, the Ang II inhibition of
7 nAChR inhibits A␤ (1-42) interaction with the same receptor, TC-1698-induced neuroprotection against A␤ (1-42) was pre-
␣
and the A␤ (1-42)-induced apoptosis is prevented through nic- vented. These results support the main hypothesis that states
otine-induced activation of JAK2. These effects can be shown that JAK2 plays a central role in the nicotinic ␣7 receptor-
by measuring markers of cytotoxicity, including the cleavage of induced activation of the JAK2-PI-3K cascade in PC12 cells,
the nuclear protein poly(ADP-ribose) polymerase (PARP), the which ultimately contribute to nAChR-mediated neuroprotec-
induction of caspase 3, or cell viability. In this study, we found tion. Ang II inhibits this pathway through the AT2 receptor
that 2-(3-pyridyl)-1-azabicyclo[3.2.2]nonane (TC-1698), a novel activation of the protein tyrosine phosphatase SHP-1. This
␣7-selective agonist, exerts neuroprotective effects via activa- study supports central and opposite roles for JAK2 and SHP-1
tion of the JAK2/PI-3K cascade, which can be neutralized in the control of apoptosis and ␣7-mediated neuroprotection in
through activation of the angiotensin II (Ang II) AT2 receptor. PC12 cells.
Neuronal nicotinic acetylcholine receptors (nAChRs) are and neuroprotection (Kem, 2000; Bencherif and Schmitt,
composed of various combinations of ␣-subunits (␣2–␣10) 2002; Kitagawa et al., 2003; Wang et al., 2003). The cholin-
and ␤-subunits (␤2–␤4) that form homo- or heteropentamers. ergic deficit in neurodegenerative diseases has been clearly
The ␣7 nAChR forms functional homomeric ligand-gated ion established and is the basis for current therapeutic strate-
2
ϩ
channels that promote rapidly desensitizing Ca influx, is
widely expressed throughout the mammalian brain, and has
been implicated in sensory gating, cognition, inflammation,
gies. There is an early and significant depletion of high-
affinity nicotinic receptors in the brains of Alzheimer’s pa-
tient’s (Breese et al., 1997; Court et al., 2001), with a selective
loss of nAChR predominating in brain regions with ␤-amy-
loid deposition. Several studies have shown cognitive im-
provement in rodents and primates, including humans, after
administration of ligands targeting nicotinic acetylcholine
receptors (Newhouse et al., 2001). In addition to their known
symptomatic effects, neuronal nicotinic ligands have shown
This work was supported by Targacept Inc. (to M.B.M., R.L.P., S.S.), and in
part by National Institutes of Health Grants HL58139 and DK50268 (to
M.B.M.), the American Heart Association Established Investigator Award (to
M.B.M.), and National Institutes of Health Grant PO1 AG10485 (to R.L.P.).
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
DOI: 10.1124/jpet.103.061655.
ABBREVIATIONS: nAChR, nicotinic acetylcholine receptor; PI-3-K, phosphatidylinositol-3-kinase; MAPK, mitogen-activated protein kinase;
JAK2, Janus kinase 2; PARP, poly(ADP-ribose) polymerase; Ang II, angiotensin II; PTPase, protein tyrosine phosphatase; PMSF, phenylmethyl-
sulfonyl fluoride; PBS, phosphate-buffered saline; BSA, bovine serum albumin; MLA, methyllycaconitine; ACh, acetylcholine; PAGE, polyacryl-
amide gel electrophoresis; TTBS, Tris-Tween 20-buffered saline; IL, interleukin; PD 123,177, S(ϩ)-1-[(4-amino-3-methylphenyl)methyl]-5-(diphe-
nylacetyl)-4,5,6,7-tetrahydro-1H-imidazo-(4,5-c)]pyridine-6-carboxylic acid; AG-490, ␣-cyano-(3,4-dihydroxy)-N-benzylcinnamide.
1
6

Angiotensin II Blocks TC-1698 Neuroprotection via SHP-1
17
neuroprotective activity in vitro and in vivo, suggesting an prevented through the nicotine-induced activation of JAK2.
additional potential for disease modification (Donnelly-Rob- These effects can be shown by measuring markers of cytotoxic-
erts et al., 1996; Kihara et al., 2001; Nordberg et al., 2002). A ity such as the cleavage of the nuclear protein PARP, the in-
direct interaction of the ␤-amyloid peptide with the ␣7 duction of caspase 3, or cell viability. Finally, we reported that
nAChR is suggested by recent findings. ␤-Amyloid peptide neuroprotective effects of nicotine could be neutralized through
interacts with high affinity to the ␣7 nAChR and results in activation of the angiotensin II AT receptor as evidenced by the
2
functional noncompetitive blockade in hippocampal neurons reversal of JAK2 phosphorylation and inhibition of nicotine-
(
Wang et al., 2000; Liu et al., 2001). In addition, nicotinic- induced neuroprotection (Shaw et al., 2002).
induced neuroprotection against ␤-amyloid induced toxicity In this study, we report that 2-(3-pyridyl)-1-azabicyclo
is suppressed by ␣-bungarotoxin, and selective ␣7 nAChR [3.2.2]nonane (TC-1698) is a highly selective nicotinic ␣7
agonists exert cytoprotective effects (Kem, 2000; Shaw et al., receptor agonist that it activates JAK2 in PC12 cells and that
2
002).
this activation and downstream activation of PI-3-K and Akt
Recent studies have reported that ␣7-mediated effects are are blocked by the specific inhibitor AG490. TC-1698-induced
mediated through phosphorylation of specific kinases such as phosphorylation of JAK2 can be neutralized through angio-
Akt and subsequent activation of phosphatidylinositol 3-ki- tensin II (Ang II)-activation of the AT2 receptor and these
nase (Kihara et al., 2001). Another study has shown that effects are mediated through the protein tyrosine phospha-
whereas nicotine activates the PI-3-K neuroprotective cas- tase (PTPase) SHP-1. Furthermore, usage of the PTPase
cade, A␤ (1-42) chronically activates the mitogen-activated SHP-1 antisense identified central and opposite roles for
protein kinase (MAPK) cascade via the hippocampal ␣7 Jak2 and SHP-1 in the control of ␣7 nAChR-mediated PC12
nAChR (Dineley et al., 2001). These findings were inter- cell survival and apoptosis.
preted as evidence that chronic activation of the MAPK path-
way by A␤ (1-42) eventually leads to the down-regulation of
MAPK, which then sets up a positive feedback for A␤ accu-
mulation and decreased phosphorylation of the cAMP regu-
latory protein (cAMP response element-binding protein),
Materials and Methods
Synthetic Procedures
The compound TC-1698 was prepared by the alkylation of the
which is a necessary component for hippocampus-dependent imine derived from 3-acetylpyridine and isopropylamine. Thus, the
memory formation in mammals. Nonetheless, these findings sequential treatment of imine with lithium diisopropyl amide and
4
2
-(bromomethyl)oxane provided the key intermediate 1-(3-pyridyl)-
-(4-oxanyl)propan-1-one, which was readily elaborated into the
suggest that the ␣7 nAChR transduces signals to PI-3-K in a
cascade, which ultimately contributes to a neuroprotective
effect against A␤ (1-42).
There is recent evidence for the nicotine-induced complex
formation between the ␣7 nAChR and the tyrosine-phosphory-
lated enzyme JAK2 that results in subsequent activation of
PI-3-K and Akt (Shaw et al., 2002). In addition, nicotine inter-
higher homolog of 2-(3-pyridyl)-quinuclidines. The construction of
the [3.2.2] ring was accomplished by the transformation of the inter-
mediate into oxime (1-pyridin-3-yl-3-(tetrahydropyran-4-yl)-propan-
1
-one oxime) and then to the amine 1-pyridin-3-yl-3-(tetrahydro-
pyran-4-yl)-propylamine. The amine upon heating with concentrated
HBr in a sealed tube, followed by removal of the acid, and then
action with the ␣7 nAChR is “dominant” over A␤ (1-42) inter- refluxing with dilute ethanolic potassium carbonate yielded TC-1698
action with the receptor, and the A␤ (1-42)-induced apoptosis is (Fig. 1). The structure was confirmed by H an C NMR, gas chro-
1
13
Fig. 1. Synthetic scheme for TC-1698.

1
8
Marrero et al.
matography-mass spectrometry, and elemental analysis as 2-(3-pyri- ng) each of the appropriate subunit cRNAs. Recordings were made 1
dyl)-1-azabicyclo[3.2.2]nonane dihydrochloride (TC-1698) as 99.9% to 15 days after injection.
pure.
Electrophysiology
Binding Studies
Experiments were conducted using a beta version of OpusXpress
000A (Axon Instruments, Union City, CA). OpusXpress is an inte-
6
Tissue Preparation. Rats were killed by decapitation after an-
grated system that provides automated impalement and voltage
clamp of up to eight oocytes in parallel. The beta unit used for these
studies recorded from four cells simultaneously. Cells were automat-
ically perfused with bath solution, and agonist solutions were deliv-
ered from a 96-well plate. Both the voltage and current electrodes
were filled with 3 M KCl. The agonist solutions were applied via
disposable tips, which eliminated any possibility of cross-contamina-
tion. Drug applications alternated between ACh controls and exper-
imental applications. Flow rates were set at 1 ml/min. Cells were
voltage-clamped at a holding potential of Ϫ60 mV. Data were col-
lected at 50 Hz and filtered at 20 Hz. Drug applications were 20 s in
duration followed by 383-s washout periods for ␣7 receptors and 10 s
with 383-s wash periods for other subtypes.
esthesia with 70% CO . The brain was rapidly removed and placed
2
on an ice-cold platform. The cerebral cortex, cerebellum, hippocam-
pus, and striatum regions were dissected and stored at –20°C until
use for membrane preparation.
Preparation of Membranes from Rat Tissues. Tissue was
homogenized in 10 vol (w/v) of ice-cold preparative buffer (11 mM
KCl, 6 mM KH PO , 137 mM NaCl, 8 mM Na HPO , 20 mM HEPES,
2
4
2
4
5
mM iodoacetamide, 1.5 mM EDTA, and 0.1 mM PMSF, pH 7.4,
using a Polytron (Brinkmann Instruments, Westbury, NY) at setting
for 15 s. The homogenate was then be centrifuged at 40,000g for 20
6
min at 4°C, the pellet was resuspended in 20 vol of ice-cold water,
and incubated for 20 min at 4°C. The final pellet (40,000g for 20 min
at 4°C) was then be resuspended in preparative buffer and stored at
Ϫ20°C. On the day of assay, tissue was thawed, centrifuged at
Experimental Protocols and Data Analysis
40,000g for 20 min at 4°C, and then resuspended in Dulbecco’s
phosphate-buffered saline (PBS, #21300; Invitrogen, Carlsbad, CA),
Each oocyte received two initial control applications of ACh and an
pH 7.4, to a final concentration of 2 to 3 mg/ml total protein. PBS experimental drug application, and then a follow-up control applica-
with 0.05% BSA was used to resuspend hippocampal membranes. tion of 300 ␮M ACh. The control ACh concentrations for ␣1␤1⑀␦,
Protein concentration was determined by the Bradford method using ␣3␤4, ␣4␤2, ␣3␤2, and ␣7 receptors were 30, 100, 10, 30, and 300 ␮M,
BSA as the standard.
respectively. These concentrations were determined to be the EC74
,
3
3
[
H]Methyllycaconitine (MLA) and [ H]Nicotine Binding EC15, EC22, EC18, and EC100, respectively. Responses to TC-1698
3
Assays. The [ H]MLA binding assay was used to detect and quantify applications were calculated relative to the preceding ACh control
the ␣7 nAChRs in cerebral cortex, cerebellum, hippocampus, and responses to normalize the data, compensating for the varying levels
striatum as described previously (Davies et al., 1999). Briefly, each of channel expression among the oocytes. Drug responses were ini-
sample (150 ␮l of total volume) consisted of membrane suspension tially normalized to the ACh control response values and then ad-
3
(
ϳ150 ␮g of protein), 5 nM [ H]MLA for single-point screening, or 0.5 justed to reflect the TC-1698 responses relative to the ACh maxi-
to 20 nM for the saturation analysis. Nonspecific binding was deter- mums. Responses for ␣7 receptors were calculated as net charge over
mined in the presence of 10 ␮M cold MLA. Binding reactions were a 90-s interval, beginning with the drug application (Papke and
conducted for 2 h at room temperature in 96-well microtiter plates in Papke, 2002). For subtypes other than ␣7, responses were calculated
triplicate. The binding reaction was terminated by rapid filtration from the peak current amplitudes. Means and S.E.M. were calcu-
onto Whatman GF/B glass fiber filters, presoaked in 0.3% polyeth- lated from the normalized responses of at least three oocytes for each
yleneimine, using a tissue harvester (Brandel Inc., Gaithersburg, experimental concentration. The application of some experimental
MD). After washing five times with ϳ350 ␮l of the ice-cold PBS, the drugs caused the subsequent ACh control responses to be reduced,
filter plate was dried at 49°C for approx. 2 h. MeltiLex A melt-on suggesting some form of residual inhibition (or prolonged desensiti-
scintillator sheets (PerkinElmer Life Sciences, Boston, MA) were zation). To measure the residual inhibitory effects, this subsequent
then be applied to the dry filters, and radioactivity bound to the control response was compared with the preapplication control ACh
membranes was determined by liquid scintillation counting. The response.
3
[
H]nicotine binding assay used the same procedure to detect and
For concentration-response relations, data derived from net
charge analyses were plotted using KaleidaGraph 3.0.2 (Abelbeck
Software, Reading, PA), and curves were generated from the Hill
equation as follows:
quantify ␣4␤2 nAChRs (Romano and Goldstein, 1980).
Preparation of RNA
The human nAChR clones were obtained from Dr. Jon Lindstrom
University of Pennsylvania, Philadelphia, PA) and the mouse mus-
n
I
max͓agonist͔
(
Response ϭ
agonist͔ ϩ ͑EC50͒n
n
͓
cle subunit clones were from Dr. Jim Boulter (University of Califor-
nia, Los Angeles, Los Angeles, CA); the mouse epsilon clone was
provided by Dr. Paul Gardener (University of Massachusetts Medi-
cal School, Worcester, MA). After linearization and purification of
cloned cDNAs, RNA transcripts were prepared in vitro using the
appropriate mMessage mMachine kit from Ambion (Austin, TX).
where Imax denotes the maximal response for a particular agonist/
subunit combination, and n represents the Hill coefficient. Imax, n,
and the EC50 were all unconstrained for the fitting procedures.
Negative Hill slopes were applied for the calculation of IC50 values.
Materials and Chemicals
Expression in Xenopus Oocytes
Chemicals for electrophysiology were obtained from Sigma-Al-
Mature (Ͼ9 cm) female X. laevis African toads (Nasco, Ft. Atkin- drich (St. Louis, MO) with the exception of TC-1698, which was
son, WI) were used as a source of oocytes. Before surgery, frogs were synthesized. Other chemicals were purchased from Aldrich Chemical
anesthetized by placing the animal in a 1.5 g/l solution of 3-amino- Co. (Milwaukee, WI). These were used without further purification,
benzoic acid ethyl ester for 30 min. Oocytes were removed from an
incision made in the abdomen. To remove the follicular cell layer, from sodium and benzophenone. Merck silica gel 60 (70–230 mesh)
harvested oocytes were treated with 1.25 mg/ml collagenase from was used for all the chromatographic purifications. Molecular weight
except in the case of tetrahydrofuran, which was dried by distillation
Worthington Biochemicals (Freehold, NJ) for 2 h at room tempera- standards, SDS, N-NЈ-methylene-bisacrylamide, N,N,NЈ,NЈ-tetram-
ture in calcium-free Barth’s solution (88 mM NaCl, 10 mM HEPES, ethylenediamine, protein assay reagents, and nitrocellulose mem-
pH 7.6, 0.33 mM MgSO , and 0.1 mg/ml gentamicin sulfate). Subse- branes were purchased from Bio-Rad (Hercules, CA). Protein A/G-
4
quently, stage 5 oocytes were isolated and injected with 50 nl (5–20 agarose was obtained from Santa Cruz Biotechnology, Inc. (Santa

Angiotensin II Blocks TC-1698 Neuroprotection via SHP-1
19
Cruz, CA), whereas Dulbecco’s modified Eagle’s medium (DMEM; SHP-1 Tyrosine Phosphatase Activity Assay
Invitrogen), fetal bovine serum (Atlanta Biologicals, Norcross, GA),
SHP-1 activity was determined as described previously (Mar-
rero et al., 1998). Briefly, SHP-1 proteins were immunoprecipi-
tated with anti-SHP-1 antibodies from PC12 cell lysates, and the
immunocomplexes were washed three times with ice-cold wash
buffer and then three times with phosphatase buffer (50 mM
HEPES, 60 mM NaCl, 60 mM KCl, 0.1 mM PMSF, 10 ␮g/ml
aprotinin, and 10 ␮g/ml leupeptin, pH 7.4). Phosphatase activity
was measured by monitoring the rate of formation of p-nitrophe-
and trypsin and all medium additives were obtained from Mediatech
Herndon, VA). Monoclonal antibody to phosphotyrosine (PY20) and
(
SHP-2 were procured from BD Biosciences Transduction Laborato-
ries (Lexington, KY). PARP antibodies were purchased from New
England Biolabs (Beverly, MA). Anti-phosphotyrosine JAK2 and
JAK2 antibodies were obtained from BioSource International (Cam-
arillo, CA). The Supersignal substrate chemiluminescence detection
kit was obtained from Pierce Chemical (Rockford, IL). Goat anti-
mouse IgG and anti-rabbit IgG were acquired from Amersham Bio- nol by dephosphorylation of p-nitrophenyl phosphate. Immuno-
sciences Inc. (Princeton, NJ), and Tween 20, A␤ (1-42) peptide, complex pellets were thus resuspended in 100 ␮l of phosphatase
anti-A␤ (1-42), and anti-␣7 nAChR and all other chemicals were buffer containing 1 mg/ml BSA, 5 mM EDTA, and 10 mM dithio-
purchased from Sigma-Aldrich.
threitol. The reaction was initiated by the addition of p-nitrophe-
nyl phosphate (10 mM final concentration). After a 30-min incu-
bation at room temperature, the reaction was stopped by the
addition of 1 M NaOH, and absorbance of the sample was deter-
mined at 410 nm in a spectrophotometer.
Isolation and Culture of PC12 Cells
PC12, rat pheochromocytoma cells, were maintained in prolifera-
tive growth phase in Dulbecco’s modified Eagle’s medium supple-
mented with 10% horse serum, 5% fetal calf serum, and antibiotics
(
penicillin/streptomycin) according to routine protocols (Bencherif et
Antisense against SHP-1
al., 1996).
An antisense oligonucleotide that targets the translational start
site of the murine SHP-1 coding sequence (5Ј-ACCTCACCATCCTT-
Western Blotting Studies of JAK2
The tyrosine phosphorylation of JAK2 was determined in serum- GGGGT-3Ј) has been found to significantly reduce SHP-1 expression
starved PC12 cells stimulated with 10 ␮M TC-1698 (0–60 min) in in human erythroleukemic SKT6 cells (Sharlow et al., 1997). There-
the presence or absence of 10 ␮M (1-h preincubation) of the JAK2 fore, we have tested the effect of SHP-1 antisense phophorothiorate
specific inhibitor AG-490 (Meydan et al., 1996; Dicou et al., 2001). oligonucleotide on SHP-1 expression in PC12 cells. Cells were
Although 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 (Meydan et al., 1996). At the end of stimulation, cells were
washed twice with ice-cold phosphate-buffered saline with 1 mM
Na VO . Each dish was then treated for 60 min with ice-cold lysis
treated with the sense or antisense oligonucleotides (10 ␮M) in
LipofectAMINE for various times, SHP-1 was immunoprecipitated,
and the immunoprecipitates were immunoblotted with anti-SHP-1
antibody.
3
4
buffer (20 mM Tris-HCl, pH 7.4, 2.5 mM EDTA, 1% Triton X-100,
0% glycerol, 10 mM Na P O , 50 mM NaF, 1 mM Na VO , and 1
mM PMSF), and the supernatant fraction was obtained as cell lysate
Assessment of PC12 Cell Apoptosis
1
4
2
7
3
4
Apoptosis was determined by assessing the cleavage of the DNA-
by centrifugation at 58,000g for 25 min at 4°C. Samples were re- repairing enzyme PARP using a Western blot assay. PARP (116 kDa)
solved by 10% SDS-PAGE, transferred to a nitrocellulose membrane, is an endogenous substrate for caspase-3, which is cleaved to a
and blocked by 60-min incubation at 22°C in TTBS (Tris-buffered typical 85-kDa fragment during various forms of apoptosis. PC12
saline with 0.05% Tween 20, pH 7.4) plus 5% skimmed milk powder. cells were treated with 0.1 ␮M A␤ for 8 h in the presence or absence
The nitrocellulose membrane was incubated overnight at 4°C with of TC-1698 and/or AG-490. The cells were collected, washed with
affinity-purified anti-phospho specific JAK2 antibodies. The nitrocel-
lulose membranes were washed 10 min twice with TTBS and incu-
bated with goat anti-rabbit IgG horseradish peroxidase conjugate.
After extensive washing, the bound antibody was visualized on a
Kodak Biomax film using a Supersignal substrate chemilumines-
cence detection kit (Pierce Chemical).
PBS, 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, and 0.05% Tween 20). Membranes
were incubated with primary PARP antibody specific for the 85-kDa
fragments for 2 to 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 enhanced chemiluminescence
system (Amersham Biosciences Inc.).
Immunoprecipitation Studies of SHP-1
The cell lysate prepared as described above was incubated with
1
0 ␮g/ml anti-SHP-1 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 was recovered by centrifuga-
tion and washed three times with ice-cold wash buffer (Tris-
buffered saline, 0.1% Triton X-100, 1 mM PMSF, and 1 mM
Na VO ). Immunoprecipitated proteins were dissolved in 100 ␮l of
Laemmli sample buffer, and 80 ␮l of each sample was resolved by
SDS-PAGE. Samples were transferred to a nitrocellulose mem-
brane and blocked by 60-min incubation at room temperature
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) con-
tains the specific caspase 3 cleavage sequence (DEVD) coupled at
the C-terminal to the fluorochrome 7-amino-4-methyl coumarin.
The substrate emits a blue fluorescence when excited at a wave-
length of 360 nm. When cleaved from the peptide by the caspase 3
enzyme activity in the cell lysate, free 7-amino-4-methyl coumarin
is released and can be detected by its yellow/green emission at 460
nm. Appropriate controls included a reversible aldehyde inhibitor
of caspase 3 to assess the specific contribution of the caspase 3
3
4
(
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. The nitrocellu-
lose membranes were washed for 10 min twice with TTBS and
incubated with goat anti-mouse IgG horseradish peroxidase con- enzyme activity (data not shown). Fluorescence units were nor-
jugate. After extensive washing, the bound antibody was visual- malized relative to total protein concentration of the cell extract.
ized on a Kodak Biomax film using a Supersignal substrate chemi- We performed the assays in triplicate and repeated the experi-
luminescence detection kit (Pierce Chemical).
ments six times.

2
0
Marrero et al.
Fig. 2. Effects of TC-1698 on
nAChR. Responses of oocytes ex-
pressing human ␣4␤2 receptors (A),
human ␣7 receptors oocytes (B), hu-
man ␣3␤2 (C), mouse muscle-type
receptors ␣1␤1⑀␦ (D), or human
␣
3␤4 (E). Representative raw data
traces are shown on the left, and
concentration-response curves to the
application of either ACh or TC-1698
are shown on the right. In the con-
centration-response curves, each
point is the mean response of at
least three cells (ϮS.E.M.). Each
measurement was initially normal-
ized to the ACh control response
measured in the same cell. These
values were subsequently scaled by
the ratio of the ACh controls to the
ACh maximum response.

Angiotensin II Blocks TC-1698 Neuroprotection via SHP-1
21
Fig. 3. ACh and TC-1698 coapplication responses of ␣4␤2 nAChR. A, effect of coapplication of 1 ␮M TC-1698 on the response inhibition of an oocyte
expressing human ␣4␤2 receptors. On the left is shown the response to 30 ␮M ACh alone. On the right is the response of the same oocyte to 30 ␮M
ACh plus 1 ␮M TC-1698. B, inhibition curve for the effect of increasing concentrations of TC-1698 on the responses of ␣4␤2-expressing oocytes to the
application of 30 ␮M ACh coapplied with TC-1698. Each point is the mean response of at least three cells (ϮS.E.M.). Each measurement is expressed
relative to the ACh control response measured in the same cell before the coapplication of ACh and TC-01698. C, peak responses to high concentrations
of ACh are relatively unaffected by coapplication of TC-1698. On the left is shown the response to 1 mM ACh alone. On the right is the response of
the same oocyte to 1 mM ACh plus 1 ␮M TC-1698. D, effect of 1 ␮M TC-1698 on the inhibition of responses to varying concentrations of ACh coapplied
to oocytes expressing human ␣4␤2 receptors. Data are normalized to the responses of the same oocytes to ACh alone applied at the indicated
concentrations. Each bar is the mean response of at least three cells (ϮS.E.M.).
Data Analysis
expressing ␣4␤2 receptors (Ͻ3% ACh maximum; Fig. 2).
However, subsequent to the application of TC-1698, we noted
that subsequent ACh control responses were progressively
inhibited (IC50 Ͼ 30 ␮M, Fig. 3). Because we noted an inhi-
bition of ␣4␤2 control ACh responses after the application of
TC-1698, we further investigated whether TC-1698 might
function as an antagonist of ␣4␤2 nAChR. When TC-1698
was coapplied at increasing concentration with 30 ␮M ACh,
All statistical comparisons were made using Student’s t test for
paired data and analysis of variance. Significance was p Ͻ 0.05.
Results
Electrophysiological Studies Indicate That TC-1698 Is a
Selective Agonist to the ␣7 nAChR
␣
4␤2 nAChR. TC-1698 had little or no agonist activity we noted a concentration-dependent inhibition of the ACh
when applied alone at concentrations up to 100 ␮M to oocytes response (IC50 Ϸ 300 nM; Fig. 3). To further investigate the

2
2
Marrero et al.
TABLE 1
Binding selectivity profile of TC-1698
Data are mean percent inhibition of control binding (or activity) for duplicate determinations. No data denotes less than 25% change in binding at 10 ␮M TC-1698. For all
non-nicotinic receptors tested, TC-1698 had an IC50 Ͼ 1 ␮M.
Binding Site
Radioligand
Inhibition (Percentage at 10 ␮M)
Acetylcholinesterase
Acetylthiocholine
3
Adenosine, nonselective
[ H]NECA
3
Adrenergic ␣ (rat cortex)
[ H]MeOxy-prazocin
1
3
Adrenergic ␣ (rat cortex)
[ H]RX-821002
2
3
Adrenergic ␤ nonselective
[ H]DHA
1
1
3
25
25
Angiotensin AT (human)
[
[
I]-(Sar1-Ile8) angiotensin
I]Tyr4-angiotensin II
1
Angiotensin AT2
Calcium channel, type L
Bradykinin, BK2
[ H]Nitrendipine
3
[ H]Bradykinin
3
Calcium channel, type N
Cholecystokinin, CCK1 (CCKA)
Cholecystokinin, CCK2 (CCKB)
Choline acetyltransferase
Glutamic acid decarboxylase
Corticotropin-releasing factor
Dopamine D3 (rat recombinant)
Dopamine transporter
[ H]conotoxin GVIA
1
1
1
1
3
25
25
4
[
[
[
[
I]CCK-8
I]CCK-8
C]-Acetyl Coenzyme
C]-Glutamic Acid
4
[ H]-Tyr0-oCRF
3
[ H]Spiperone
3
[ H]-WIN 35,428
1
1
3
25
25
Endothelin ET-A (Human)
Endothelin ET-B (Human)
GABA (rat cortex)
[
[
I]-Endothelin1
I]-Endothelin1
[ H]-GABA
1
3
25
Estrogen
[
I] 3,17B-Estradiol, 16a
GABA-A, BDZ, ␣1, central
GABA-B transporter (rat cortex)
Galanin, nonselective
[ H]-Flunitrazepam
3
[ H]-CGP 5462A
1
3
25
[
I] Galanin
Glycine (rat spinal cord)
Histamine H1 periph. (guinea pig lung)
Histamine H1
Histamine H2
Histamine H3
Leukotriene B4, LTB4
Leukotriene D4, LTD4
Melatonin ML1 (chicken brain)
Monoamine oxidase A, peripheral
Monoamine oxidase B, peripheral
Muscarinic, M1 (human recombinant)
Muscarinic, M2 (human recombinant)
Muscarinic, nonselective central
Muscarinic, nonselective, peripheral
Neurokinin, NK1
Neurokinin, NK2 (human recombinant)
Neurokinin, NK3 (NKB)
Nicotinic (rat cortex)
Norepinephrine transporter (rat cortex)
NOS (neuronal-binding)
Opioids (rat cortex)
Oxytocin (rat uterus)
Platelet-activating factor, PAF
Potassium KATP channels (rat cortex)
Potassium KVI channels (rat cortex)
Potassium KVS channels (rat cortex)
Serotonin transporter
Serotonin, nonselective
Sigma (rat cortex)
Sodium channels site 2 (rat cortex)
Testosterone (cytosolic)
[ H]-Strychnine
3
[ H]-Pyrilamine
3
[ H]-Pyrilamine
3
[ H]-Pyrilamine
39
60
3
[ H]-Pyrilamine
3
[ H]-LTB4
3
[ H]-LTD4
1
1
1
3
25
4
[
[
[
I]-Iodomelatonin
C]-5HT
4
C]phenylethylamine
[ H]-QNB
3
[ H]-QNB
46
3
[ H]-QNB
3
[ H]-QNB
3
[ H]-SP
3
[ H]-NKA
3
[ H]-Eledoisin
3
[ H]-Cytisine
104
3
[ H]-Nisoxetine
3
[ H]-NOARG
3
[ H]-Naloxone
3
[ H]-oxytocin
3
Hexadecyl-[ H]-acetyl-PAF
3
[ H]-Glibenclamide
1
1
3
25
25
[
[
I]-Apamin
I]-Charybdotoxin
[ H]-Citalopram, N-methyl
1
3
25
[
I]-LSD
[ H]-DTG
35
3
[ H]-Batrachotoxin
3
[ H]-Methyltrienolone
3
Thromboxane A2 (Human)
Thyrotropin-releasing hormone, TRH
Vasoactive intestinal peptide
[ H]-SQ 29,548
3
[ H]-(3MeHis2)TRH
1
3
25
[
I]-VIP
Vasopressin V (rat aortic A7r5 cells)
[ H]-AVP
1
nature of the TC-1698 inhibition of ␣4␤2 ACh responses, we expressing ␣3␤2 receptors (Ͻ5% ACh maximum; Fig. 2; Table
conducted some competition experiments. We noted that 1). However, subsequent ACh responses were decreased after
when TC-1698 at a fixed concentration of 1 ␮M was coapplied TC-1698 was applied alone (IC50 Ϸ 25 ␮M; data not shown).
with increasing concentrations of ACh, there was inhibition
of the response to low concentrations of ACh but not to high pressing ␣3␤4 receptors, very small currents were observed
concentrations of ACh, consistent with competitive inhibition at very high concentrations (Imax Ϸ5% that of ACh, EC50
Fig. 3; Table 1). 1600 ␮M; Fig. 2). After the application of TC-1698 to oocytes
3␤2 nAChR. TC-1698 also had relatively little activity expressing ␣3␤4 receptors, little or no inhibition of subse-
when applied alone at concentrations up to 300 ␮M to oocytes quent ACh control responses was observed (data not shown).
␣3␤4 nAChR. When TC-1698 was applied to oocytes ex-
ϭ
(
␣

Angiotensin II Blocks TC-1698 Neuroprotection via SHP-1
23
␣1␤1⑀␦ nAChR. TC-1698 was also a relatively potent and Effects of the JAK2 Inhibitor AG-490 on TC-1698-Induced
modestly efficacious agonist of mouse muscle nAChR ex- Tyrosine Phosphorylation of JAK2 and PI-3-Kinase and
pressed in Xenopus oocytes. The maximum current was 28 Ϯ Serine Phosphorylation of Akt in PC12 Cells
1
% of the maximum response to ACh. TC-1698 had an EC50
value of 20 Ϯ 1.3 ␮M, 50 times less potent than for ␣7, rosine phosphorylation. The tyrosine phosphorylated JAK2
compared with 82 ␮M for ACh (Fig. 2).
sometimes shows up as a doublet or shifts in its migration in
7 nAChR. Whereas TC-1698 had very little agonist ac- the SDS-PAGE gel. These anomalies may be due to either
Enzymatic activation of JAK2 was determined via its ty-
␣
tivity with ␤-subunit-containing neuronal nAChR, it seemed proteolysis of JAK2 or different levels of serine phosphoryla-
to be a potent and efficacious agonist of ␣7-type receptors. It tion of the enzyme (Marrero et al., 1998; Shaw et al., 2002).
In this study, we found that JAK2 is tyrosine phosphorylated
in response to the ␣7 receptor specific ligand TC-1698 (10
produced maximum responses equivalent to those produced
by ACh (i.e., Imax Ն 100% compared to ACh). TC-1698 was
approximately 30-fold more potent than ACh, with an EC50
value of 440 Ϯ 14 nM, compared with 30 ␮M for ACh (Fig. 2).
TC-1698 application to ␣7-expressing oocytes produced no
significant residual inhibition of subsequent ACh responses
␮
M) within 5 min, and this activation remained above basal
levels even after longer exposure (60 min) to compound TC-
698 (Fig. 4). We also found that preincubation for 1 h with
1
the JAK2 inhibitor AG-490 (10 ␮M) inhibited the TC-1698-
induced JAK2 tyrosine phosphorylation, the tyrosine phos-
phorylation of PI-3-K, and the serine phosphorylation of Akt
(
data not shown). In Fig. 2A, note that because TC-1698 is a
far more potent agonist than ACh, although the TC-1698
peak current is larger than that of the ACh controls, the net
charge is roughly equivalent to that evoked by the 300 ␮m
ACh control.
(
Fig. 4). These results are similar in their kinetics of JAK2
activation to our previous reported results when we used
nicotine (Shaw et al., 2002).
Effects of Ang II Pretreatment with or without Ang II
Receptor Antagonists on TC-1698-Induced Tyrosine
Activity Profile for TC1698 in Clonal Cells
The affinity of TC-1698 for brain nAChR was determined Phosphorylation of JAK2
by radioligand binding studies. Membranes prepared from
Preincubation of PC12 cells with Ang II blocked TC-1698-
rat hippocampus, a brain region enriched in ␣7 nAChR, were induced tyrosine phosphorylation of JAK2 (Fig. 5A). This
3
used along with [ H]MLA as labeling agent and TC-1698 inhibition was completely prevented by preincubation with
exhibited a K of 11 Ϯ 1.7 nM in this preparation (n ϭ 4). The an AT antagonist (PD 123177 at 100 nM) but not by an AT
i
2
1
potency and efficacy of TC-1698 at peripheral nAChR was antagonist (candesartan at 100 nM) (Fig. 5A), consistent
assessed using radioactive rubidium efflux assays in rat and with the Ang II receptor phenotype expressed in PC12 cells.
human cell lines expressing muscle- and ganglion-type Consistent with our data indicating that TC-1698 is a potent
selective agonist of ␣7-type receptors, TC-1698-induced acti-
vation of JAK2 was blocked by the ␣7 antagonist ␣-bungora-
toxin (Fig. 5C).
nAChR (Lukas and Cullen, 1988). TC-1698 is a poor activator
of rat and human ganglion-type nAChR and human muscle
type receptors expressed in clonal cell lines. At 100 ␮M,
activation of either human muscle receptor (human TE671/
RD) or ganglion-type receptors (rat PC12 and human SH-
SY5Y cells) were below 20% of that of nicotine. The intrinsic
activity of TC-1698 at brain nAChR was assessed using
3
[
H]dopamine release from rat striatal synaptosomes. TC-
698 resulted in no significant activation of dopamine re-
1
lease, suggesting a lack of agonist activity at ␣4␤2 nAChR.
A binding profile was conducted to evaluate the interaction
of TC-1698 with other receptors, transporters, enzymes, or
ion channels. With the exception of nicotinic receptors at
which binding was totally displaced, 10 ␮M TC-1698 had no
or very low affinity for all binding sites examined (Table 2).
TABLE 2
Activation parameters for TC-1698
I
max
n
EC50
a
a
a
␣
␣
␣
␣
␣
4␤2
7
3␤2
1␤1⑀␦
3␤4
Ͻ0.05
1.0
Յ0.06
0.28
Ͻ0.05
NA
0.9
NA
2.0
NA
(.03 ACh Imax @ 100 ␮M)
345 Ϯ 110 nM
(0.05–0.02 ACh Imax 30–300 ␮M)
20 Ϯ 1.3 ␮M
(.02 ACh Imax @ 1 mM)
a
Fig. 4. TC-1698-induced activation of JAK2, Akt, and PI-3-kinase in
PC12 cells in the presence or absence of AG-490. PC12 cells preincubated
in the presence or absence of the JAK2 inhibitor AG-490 (10 ␮M) were
stimulated with 10 ␮M TC-1698 for the time indicated. Cells were im-
munoblotted with phospho-specific and nonphosphospecific anti-JAK2
and anti-Akt antibodies or with anti-PI-3-kinase antibody. The PI-3-
kinase immunoprecipitated proteins were then immunoblotted with anti-
phosphotyrosine and anti-PI-3-kinase antibodies. Results shown for each
immunoblot is representative of three immunoblots.
a
NA, not available, that is, the responses were too low for the data to give a
meaningful curve fit.
b
Between 30 and 300 ␮M there were detectable responses that varied between
0
␮
.05 and 0.02 of the ACh maximum response; however, responses to 30 ␮M and 100
M were not statistically different and responses to 300 ␮M were, if anything, less
than the responses to 100 and 30 ␮M.

2
4
Marrero et al.
Fig. 5. A, effects of Ang II pretreatment with or
without Ang II receptor antagonists on the TC-
1
698-induced activation of JAK2 in PC12 cells.
Cells preincubated with Ang II for 8 h in the
presence or absence of AT antagonist candesar-
1
tan or AT2 antagonist PD 123177 were stimu-
lated with TC-1698 for the time indicated. Cells
were immunoblotted with phospho-specific and
nonphosphospecific anti-JAK2. Results shown for
each immunoblot is representative of three im-
munoblots. B, angiotensin II-induced phosphory-
lation of SHP-1 in PC12 cells. PC12 cells were
incubated for 24 h in serum-free medium before
exposure to Ang II (100 nM) for the times indi-
cated. Cells were lysed, and SHP-1 was immuno-
precipitated from lysates with 10 ␮g/ml anti-
SHP-1 monoclonal antibodies and immuno-blotted
with anti-phosphotyrosine antibody. Results shown
for each immunoblot are representative of three
immunoblots. C, effects of ␣-bungarotoxin on TC-
1698-stimulated JAK2 phosphorylation. Cells pre-
incubated with 0.1 ␮M ␣-bungarotoxin or vehicle
followed by addition of 0.1 ␮M TC-1698 for the
times indicated. Results shown for each immuno-
blot is representative of three immunoblots.
Ang II-Induced Activation and Tyrosine Phosphorylation
of SHP-1 and Its Effects on TC-1698-Induced Tyrosine
Phosphorylation of JAK2
antisense oligos could be used to regulate the TC-1698-in-
duced activation JAK2 in PC12 cells. PC12 cells were stim-
ulated with Ang II and lysed. JAK2 was then immunopre-
cipitated from lysates with anti-JAK2 antibody.
Immunoprecipitated proteins were separated by gel electro-
phoresis, transferred to nitrocellulose, and then immunoblot-
ted with anti-phosphotyrosine antibody. As a control, cells
were exposed to SHP-1 sense oligonucleotide. As shown in
Fig. 7B, when cells were exposed to the SHP-1 antisense form
The AT receptor exerts growth-inhibitory effects in cul-
2
tured cells and in vivo, one of which has been proposed to be
programmed cell death (Horiuchi et al., 1998; Lehtonen et
al., 1999). Despite growing interest in AT receptor-mediated
2
apoptosis, relatively little is known about the molecular basis
of this process. Recently growth-inhibitory effects of the AT2
receptor have been reported to be mediated by the activation
of PTPases, AT2 receptor stimulation is associated with a
rapid activation of SHP-1 in rat pheochromocytoma PC12
cells (Horiuchi et al., 1998). However, at present, no func-
tional role has been demonstrated for SHP-1 activation by
12 h, JAK2 tyrosine phosphorylation was augmented. These
results suggest that SHP-1 is the PTPase that dephosphory-
lates JAK2 after TC-1698-induced JAK2 phosphorylation in
PC12 cells.
Assessment of PC12 Cell Apoptosis
the AT receptor, and it is interesting to note that SHP-1 has
2
been shown to function as a negative regulator of JAK2
Apoptosis was determined by assessing the cleavage of the
signaling (Marrero et al., 1998). Therefore, the potential bi- DNA-repairing enzyme PARP using a Western blot assay.
ological significance of AT2 receptor-induced programmed PC12 cells were treated with 0.1 ␮M A␤ for 8 h in the
cell death led us to investigate whether SHP-1 activation presence or absence of TC-1698 (10 ␮M). As shown in Fig. 8,
could be involved in this process. We found that Ang II PARP (116 kDa) was cleaved to its 85-kDa fragment after A␤
induced both the tyrosine phosphorylation and activation of (1-42) treatment. The A␤ (1-42)-induced cleavage of PARP
SHP-1 (Fig. 5B) and that vanadate, a specific inhibitor of was blocked by TC-1698, which was prevented by preincuba-
PTPases (Marrero et al., 1996), blocked the activation of tion with AG-490 or Ang II (Fig. 8). Further 12-h pretreat-
SHP-1 directly (Fig. 6). Furthermore, vanadate also aug- ment with SHP-1 antisense, but not sense, completely pre-
mented TC-1698-induced tyrosine phosphorylation of JAK2 vented the cleavage of PARP (compare lanes 8 and 10). These
(
Fig. 6).
results support our main hypothesis, which states that JAK2
plays a central role in the nicotinic ␣7 receptor-induced neu-
Antisense against SHP-1 and Its Effects on TC-1698-
Induced Tyrosine Phosphorylation of JAK2 in PC12 Cells
roprotection, which Ang II blocks through the AT receptor
2
activation of the PTPase SHP-1.
Because vanadate is not a specific inhibitor of SHP-1, we
Apoptosis was also determined by activation of caspase 3.
also tested the effect of SHP-1 antisense phophorothiorate Caspase 3 is expressed in PC12 cells and is known to be
oligonucleotide on SHP-1 expression in PC12 cells. Cells were involved in apoptosis (Shaw et al., 2002). Therefore, we ex-
treated with the sense, or antisense oligonucleotides (10 ␮M) amined caspase 3 activity after Ang II-induced apoptosis. We
for various times, SHP-1 was immunoprecipitated, and the used the fluorescent peptide substrate Ac-DEVD-7AMC to
immunoprecipitates were immunoblotted with anti-SHP-1 measure caspase 3-like activity in cell lysates. As shown in
antibody. As shown in Fig. 7A, the antisense (but not the Fig. 9, the caspase 3-like activity that resulted in the cleav-
sense) oligonucleotide was effective in completely inhibiting age of the peptide substrate Ac-DEVD-7AMC is evident after
SHP-1 expression within 12 h. We then tested whether these 2 h of Ang II treatment and increased over time until it

Angiotensin II Blocks TC-1698 Neuroprotection via SHP-1
25
Fig. 6. A, TC-1698-induced activation of JAK2 in
PC12 cells in the absence or presence of vana-
date. PC12 cells preincubated in the presence or
absence of the tyrosine phosphatase vanadate
were stimulated with TC-1698 for the times indi-
cated. Cells were immunoblotted with phospho-
specific and nonphospho-specific anti-JAK2. B,
time-dependent increase in SHP-1 activity in the
absence or presence of vanadate. Results shown
for each immunoblot is representative of three
immunoblots.
reached a peak after 8 h of treatment. However, the Ang TC-1698 should predominantly activate ␣7 and inhibit ␣4␤2
II-induced activation of caspase 3 was blocked significantly with relatively little effect on other receptors. TC-1698 seems
in the presence of SHP-1 antisense (ϩ, P Ͻ 0.01) or vanadate to be a weak partial agonist/antagonist of beta subunit-con-
(
*, P Ͻ 0.01) (Fig. 9). Coincubation with the SHP-1 sense had taining neuronal receptors. The studies were conducted in
no effect on the Ang II-induced activation of caspase 3. In PC12 cells with similar effects to those observed in human
addition, incubation with TC-1698 had also no effect caspase SH-SY5Y cells. Both of these cell lines exhibit ␣7- and ␣3␤4-
3
activation (Fig. 9).
containing receptors and TC-1698 interacts with the former
but not the latter.
Discussion
Several reports have documented the apoptotic effects of
Ang II through AT receptors. AT receptors are expressed in
PC12 and have been shown to inhibit the JAK/STAT signal-
ing cascade (Kunioku et al., 2001). In contrast to nicotine-
induced neuroprotection against ␤-amyloid (1-42), pretreat-
ment of cells with Ang II blocks nicotine-induced activation of
2
2
In this study, we found that TC-1698, a novel ␣7-selective
ligand, exerted neuroprotective effects via activation of the
JAK2/PI-3K cascade, which can be neutralized through acti-
vation of the Ang II AT receptor. The Ang II AT receptor
2
2
effects are reversed by nullifying a PTPase as evidenced by
the usage of the PTPase-specific inhibitor vanadate (Marrero
et al., 1996). Vanadate not only augmented the TC-1698-
induced tyrosine phosphorylation of JAK2 but also blocked
the Ang II neutralization of TC-1698-induced neuroprotec-
tion against A␤ (1-42)-induced cleavage of PARP. Further-
more, when we also neutralized SHP-1 via antisense trans-
fection the Ang II neutralization of TC-1698-induced
neuroprotection against A␤ (1-42) was again blocked. These
results support our main hypothesis, which states that JAK2
plays a central role in the nicotinic ␣7 receptor-induced acti-
vation of the JAK2-PI-3K cascade in PC12 cells, which ulti-
mately contribute to nAChR-mediated neuroprotection. Fur-
thermore, we also found that Ang II blocked this pathway
JAK2 via the AT receptor and completely prevents nicotine-
2
mediated neuroprotective effects, further suggesting a piv-
otal role for JAK2 phosphorylation (Shaw et al., 2002). Our
findings in this study are consistent with the opposite roles
on cell viability that exist between the ␣7 nAChR and the
AT receptor with activation of the AT receptor overriding
2
2
the potential benefit through the ␣7 nAChR. These results
and the convergence of these pathways on phosphorylated
JAK2 suggest that recruitment of nicotinic ␣7 nAChR recep-
tor-mediated neuroprotection against A␤ (1-42) may be opti-
mized under conditions where the AT -mediated inhibition is
2
minimized by blocking the AT -induced activation of the
2
PTPase SHP-1. Therefore, the findings in this study identify
novel molecular mechanisms, which are fully consistent with
through the AT receptor activation of SHP-1 (Fig. 10).
2
TC-1698 was relatively potent as an antagonist of the ACh the role attributed to ␣7 nAChR-induced activation of JAK2
responses of ␣4␤2 receptors, apparently working through a and subsequent neuroprotective effect, AT
competitive mechanism. TC-1698 also blocked subsequent tion of SHP-1, and its purported role in apoptotic events.
ACh control responses of ␣4␤2 and ␣3␤2 receptors after it
SHP-1 is a soluble tyrosine phosphatase that participates
-induced activa-
2
was applied in the absence of ACh. Interestingly, TC-1698 in the negative regulation of the tyrosine kinase JAK2 (Mar-
seems to be a full potent agonist only for the ␣7 receptors. rero et al., 1998), and it has been recently reported that

2
6
Marrero et al.
Fig. 8. Effects of SHP-1 antisense on TC-1698-induced protection against
A␤- and Ang II-induced apoptosis. PARP expression was measured from
lysates of cells treated with A␤ (1-42) peptide and/or Ang II in the
presence or absence of TC-1698 and/or SHP-1 antisense. Results shown
for each immunoblot are representative of three immunoblots.
Fig. 7. A, effects of SHP-1 sense and antisense oligonucleotides on SHP-1
expression in PC12 cells. PC12 cells were treated with SHP-1 sense and
antisense oligonucleotides for the times indicated and lysed. SHP-1 was
immunoprecipitated from the lysates with anti-SHP-1 antibody. Precip-
itated SHP-1 proteins were then immunoblotted with specific anti-SHP-1
antibody. Results shown for each immunoblot are representative of three
immunoblots. B, effects of SHP-1 antisense on the TC-1698-induced ac-
tivation of JAK2 in PC12 cells. Cells preincubated in the presence or
absence of SHP-1 antisense or sense oligonucleotides were stimulated
with TC-1698 for the times indicated. Cells were immunoblotted with
phospho-specific and nonphospho-specific anti-JAK2. Results shown for
each immunoblot are representative of three immunoblots.
stimulation of AT receptors rapidly activates SHP-1 in N1E-
2
1
15 and AT -transfected Chinese hamster ovary cells (Horiu-
2
chi et al., 1998; Lehtonen et al., 1999). In the present study,
we document that the Ang II AT receptor activates SHP-1 in
2
PC12 and that the TC-1698-induced activation of JAK2 is
augmented by SHP-1 antisense transfection. These results
suggest that both SHP-1 activation and JAK2 deactivation
constitute sequential events in the same signaling pathway.
Nicotinic neurotransmission is compromised in the brains
of Alzheimer’s disease patients and accumulating evidence Ang II in the presence of either SHP-1 antisense or SHP-1 sense or
suggests that nAChR-selective ligands can offer neuroprotec- vanadate. Caspase-3 activities are shown as the mean Ϯ S.E. of six
tive effects in several in vitro models, including neuronal
Fig. 9. Effects of SHP-1 antisense on the angiotensin II-induced activa-
tion of caspase-3. PC12 cells were incubated for the duration shown with
independent cultures.
death resulting from ␤-amyloid toxicity, N-methyl-D-aspar-
tate-mediated cytotoxicity, or growth factor deprivation, and that the ␣7 nAChR is also an essential regulator of inflam-
in in vivo models, including chemically induced neurotoxicity mation and is required for inhibition of cytokine release
(
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine models and (Wang et al., 2003). The physiological mechanism coined “the
systemic kainic acid-induced excitotoxic effects). Nicotinic cholinergic anti-inflammatory pathway”, which has been pro-
ligands reduce ␤-amyloid aggregation and toxicity and in- posed to have major implications in immunology and thera-
hibit amyloid deposition in transgenic mice with APPsw peutics, remains unknown. The induction and resolution of
(
Nordberg et al., 2002). A recent report has demonstrated inflammatory processes are the complex outcome of interplay

Angiotensin II Blocks TC-1698 Neuroprotection via SHP-1
27
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rodegenerative diseases can be recruited through a single
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2
lective up-regulation of AT receptor density (Ge and Barnes,
2
1
996) and biosynthetic enzymes (Narain et al., 2000; Savas-
kan et al., 2001) concurrent with down-regulation of nAChR
in the temporal cortex of some Alzheimer’s disease patients
(
Court et al., 2001) are consistent with the opposite effects on
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AT and ␣7-nAChR.
2
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Acknowledgments
We thank Julia Porter Papke, Irena Garic, Bernadette Schoneb-
urg, and Clare Stokes for technical assistance. We are very grateful
to Axon Instruments for the use of an OpusXpress 6000A and
pClamp 9. We particularly thank Dr. Cathy Smith-Maxwell for sup-
port and help with OpusXpress.
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Address correspondence to: Dr. Merouane Bencherif, Targacept Inc., 200
East First St., Suite 300, Winston-Salem, NC 27101-4165. E-mail:
merouane.bencherif@targacept.com
184.