Modulation of neuronal nicotinic receptor by quinolizidine alkaloids causes neuroprotection on a...

Unc

orre

cted

Aut

hor P

roof

Journal of Alzheimer’s Disease xx (20xx) x–xxDOI 10.3233/JAD-132045IOS Press

1

Modulation of Neuronal Nicotinic Receptorby Quinolizidine Alkaloids CausesNeuroprotection on a Cellular AlzheimerModel

1

2

3

4

Juan A. Arayaa, Alejandra E. Ramıreza, Daniela Figueroa-Arocaa, Gaston J. Sotesb, Claudia Perezb,Jose Becerrab, Francisco Saez-Orellanaa, Leonardo Guzmanc, Luis G. Aguayod and Jorge Fuentealbaa,∗

5

6

aLaboratorio de Screening de Compuestos Neuroactivos, Departamento de Fisiologıa, Facultad de CienciasBiologicas, Universidad de Concepcion, Concepcion, Chile

7

8

bLaboratorio de Quımica de Productos Naturales, Departamento de Botanica, Facultad de Ciencias Naturales yOceanograficas, Universidad de Concepcion, Concepcion, Chile

9

10

cLaboratorio de Neurobiologıa Molecular, Departamento de Fisiologıa, Facultad de Ciencias Biologicas, Univer-sidad de Concepcion, Concepcion, Chile

11

12

dLaboratorio de Neurofisiologıa, Departamento de Fisiologıa, Facultad de Ciencias Biologicas, Universidad deConcepcion, Concepcion, Chile

13

14

Accepted 19 March 2014

Abstract. Alzheimer’s disease (AD) is a progressive and neurodegenerative disorder and one of the current therapies involvesstrengthening the cholinergic tone in central synapses. Neuroprotective properties for nicotine have been described in AD, throughits actions on nicotinic receptors and the further activation of the PI3K/Akt/Bcl-2 survival pathway. We have tested a quino-lizidine alkaloid extract (TM0112) obtained from Teline monspessulanna (L.) K. Koch seeds to evaluate its action on nicotinicacetylcholine receptor (nAChR) in a neuronal AD model. Our data show that PC-12 cells pretreated with amyloid-� (A�) peptidefor 24 h in presence of TM0112 modified A�-reduction on cellular viability (A� = 80 ± 3%; +TM0112 = 113 ± 4%, n = 15). Inaddition, this effect was blocked with atropine, MLA, and �-BTX (+TM0112+atropine = 87 ± 4%; +TM0112+MLA = 86 ± 4%;+TM0112+�-BTX = 92 ± 3%). Furthermore, similar protective effects were observed in rat cortical neurons (A� = 63 ± 6%;+TM0112 = 114 ± 8%), but not in HEK293T cells (A� = 61.4 ± 6.1%; +TM0112 = 62.8 ± 5.2) that do no express �7 nAChR.Moreover, the frequency of synaptic activity in the neuronal network (A� = 51.6 ± 16.9%; +TM0112 = 210.8 ± 47.9%, n > 10),as well as the intracellular Ca2+ transients were recovered by TM0112 (A� = 61.4 ± 6.9%; +TM0112 = 112.0 ± 5.7%; n = 3)in rat hippocampal neurons. TM0112 increased P-Akt, up to 250% with respect to control, and elevated Bcl-2/Bax percentage(A� = 61.0 ± 8.2%; +TM0112 = 105.4 ± 19.5%, n = 4), suggesting a coupling between nAChR activation and an intracellularneuroprotective pathway. Our results suggest that TM0112 could be a new potential source for anti-AD drugs.

15

16

17

18

19

20

21

22

23

24

25

26

27

28

Keywords: Alkaloids, Alzheimer’s disease, neuroprotection, nicotinic receptor, Teline monspessulana29

∗Correspondence to: Jorge Fuentealba Arcos, PhD, Screening ofNeuroactive Compounds Unit, Department of Physiology, Faculty ofBiological Sciences, University of Concepcion, Barrio Universitarios/n, Concepcion, POBOX 160-C, Chile. Tel.: +56 41 2661082; Fax:+56 41 2204557; E-mail: [email protected].

ISSN 1387-2877/14/$27.50 © 2014 – IOS Press and the authors. All rights reserved

mailto:[email protected]

Unc

orre

cted

Aut

hor P

roof

2 J.A. Araya et al. / nAChR Activation by Alkaloids Induces Neuroprotection

INTRODUCTION30

Alzheimer’s disease (AD) is a disorder character-31

ized by the loss of brain cognitive functions [1] and32

abundant evidence suggest that soluble oligomers of33

amyloid-� (SO-A�) peptides cause the main neuronal34

alterations associated to AD [2]. Lesne et al. described35

that injection of 56 KDa A� oligomers into the brain of36

young rats, without the presence of senile plaques, was37

able to negatively affect memory [3]. Also, SO-A�s38

induced cell death and diminished electrical activity39

of neuronal networks in vitro [4]. In addition, SO-A�40

affected neurotransmission and caused chronic synap-41

tic failure, which could be one of the first events leading42

to AD development [5]. Furthermore, A� causes a43

series of noxious actions involving oxidative damage,44

Ca2+ dyshomeostasis, mitochondrial stress, release of45

cytochrome-C, and activation of cellular apoptosis [6,46

7].47

The main drugs approved by the FDA and used for48

the treatment of mild and moderate AD are donopezil,49

rivastigmine, and galantamine [8]. Clinical trials with50

these acetylcholinesterase (AChE) inhibitors showed51

limited improvement in cognitive capacities and global52

daily functions and even after two years of treat-53

ment, the beneficial effects were not permanent and the54

patient’s cognitive functioning continued worsening55

with time [9, 10].56

Notwithstanding the failure of AChE therapies,57

there is strong evidence supporting the role cholin-58

ergic transmission in AD physiopathology, involving59

the activation of neuronal acetylcholine receptors60

(nAChR) and that could lead to a more effective AD61

treatment. Also, there is strong evidence about the par-62

ticipation of nAChR in neuroprotection. This notion63

began by observing that chronic tobacco smokers suf-64

fered less neurodegenerative conditions, including AD65

[11]. It was also recognized that patients in early stages66

of AD present a reduction in nAChR density in cortex67

and hippocampus [12, 13] and that chronic nicotine68

use was able to produce overexpression of �7 receptors69

in temporal cortex, compared with aged non-smokers70

[14]. In addition, chronic nicotine increases TrkA71

level, a high affinity NGF receptor, which is dimin-72

ished in brains of AD patients [15, 16]. The protective73

effects of nicotine depend on nAChR �7 activation74

and �7 receptors can inhibit A� toxicity and promote75

cellular survival [11, 17]. In addition, nicotine activa-76

tion of �7 receptors can inhibit glutamatergic-induced77

neurotoxicity, protective effect that was inhibited by78

PI3K inhibitors and �-BTX [18]. Nicotine can also79

induce Akt phosphorylation, a PI3K effector, and Bcl-80

2 expression, effects that were also blocked by the PI3K 81

inhibitors [11, 18, 19]. 82

The interaction between �7 receptors and sig- 83

naling pathways was corroborated through co- 84

immunoprecipitation with Fyn protein, demonstrating 85

an actual physical association [18]. Therefore, �7 acti- 86

vation appears to trigger entry of Ca2+ through �7 87

receptors followed by Fyn and JAK2 activation, which 88

then activates the PI3K/Akt/Bcl-2 pathway, thus lead- 89

ing to cellular survival [20]. 90

Many highly pharmacological important nicotinic 91

drugs have been obtained from plants and several are 92

alkaloids. Some natural alkaloids such as nicotine, cyti- 93

sine, anatoxin, and epibatidine are still in use [21]. 94

Teline monspessulana (L) K. Koch is specie member 95

of the Fabacea family, one of the twenty most impor- 96

tant alkaloid-containing plant families [22]. Similar 97

to other plants from its genus, it is characterized by 98

accumulating quinolizidinic alkaloids, mainly cytisine 99

and its metylated derivatives [23, 24], which are high 100

affinity nAChR modulators (KAF = 260 nM). There- 101

fore, Teline monspessulana is an excellent candidate as 102

a source of compounds with possible neuroprotective 103

actions [21]. 104

Cytisine has been used with relative success in sev- 105

eral neuroprotective assays [17, 25, 26]; therefore we 106

reasoned that a quinolizidinic alkaloid extract obtained 107

from Teline monspessulana could have neuroprotec- 108

tive activity against A� toxicity. If this is the case, 109

there is a good opportunity to obtain vast natural and 110

complex structures that could modulate the neuronal 111

nicotinic receptors, representing a potential source of 112

new molecules with neuroprotective properties for AD 113

treatment. 114

MATERIALS AND METHODS 115

TM0112 extract obtention and characterization 116

The extract was obtained from seeds of T. mon- 117

spessulana that were collected in March 2012 in the 118

metropolitan area of Concepcion (VIII Region, Chile, 119

36◦49’S, 73◦01’W). Voucher specimens (CONC- 120

167614) were deposited in the Herbarium of the 121

University of Concepcion. The seeds (25 g) under- 122

went liquid extraction three times with MeOH at 123

room temperature for 24 h. The solvent was evapo- 124

rated under reduced pressure and the residue dissolved 125

in 10 ml HCl (2%). After removing the neutral com- 126

pounds with diethyl ether, the extract was basified with 127

ammonia solution (25%) and the alkaloids extracted 128

with CHCl3. The organic solvent was evaporated and 129

Unc

orre

cted

Aut

hor P

roof

J.A. Araya et al. / nAChR Activation by Alkaloids Induces Neuroprotection 3

105 mg of alkaloid extract was obtained. The extract130

was dissolved in MeOH to a final concentration of131

10 mg/ml for GC/MS analysis. GC/MS analysis of132

underivatized extract components were carried out133

on gas chromatograph Agilent 6890 A with an FID134

detector and a chromatograph Hewlett Packard Mod.135

5890 Series II (California, USA) with a mass detector136

HP model 5972. HP-5MS capillary column (Agilent137

Technologies Santa Clara, CA, USA). Relative per-138

centages of the alkaloid components were obtained139

using helium gas (constant flow 1 mL/min) for sep-140

aration. The alkaloids present in the extract were141

identified by comparison of their MS with those in142

the NIST library, with those reported in the literature.143

The presence of five main compounds was confirmed144

by GC/MS analysis. The identification of the alkaloid145

components was accomplished by matching their mass146

spectra with those recorded in the NIST 05 (NIST147

/EPA/NIH MASS 2005 Spectral Library). The main148

components in the mixture were cytisine (66.33%) and149

epiaphylline (10.99%). Furthermore, the structurally150

related alkaloids N-methyl cytosine (4.91%), angus-151

tifoline (6.99%), and 11,12-dehydrolupanine (6.45%)152

were present in small quantities.153

Rat hippocampal and cortical cultures154

18-19 days pregnant Sprague-Dawley rats were155

treated in accordance with regulations established156

by NIH and the Ethics Committee at the University157

of Concepcion. Primary cultures of embryonic hip-158

pocampi were plated at 250,000 cells/ml on coverslips159

coated with poly-L-lysine (Sigma Aldrich). Cortical160

neurons were plated in poly-L-lysine treated plates161

of 96 wells at 250,000 cells/ml. Cultures were main-162

tained at 37◦C with 5% CO2. Culture medium was163

replaced every 3 days and consisted of 90% Dulbecco’s164

minimal essential medium (DMEM; Gibco), 5% heat-165

inactivated horse serum, 5% fetal bovine serum and166

N3 (mg/ml: BSA 1, Putrescine 3.2, Insulin 1, Apo-167

transferrin 5, Corticosterone 0.5; (�g/ml: sodium168

selenite 5, TH3 0.5, Progesterone 0.6)). Experiments169

were performed at 10–12 DIV in control and treated170

neurons.171

PC12 CELLS172

PC12 cells from ATCC (Manassas, VA, USA) were173

utilized in experiments with a 96-well plate reader. The174

cells were cultured in DMEM with 5% fetal bovine175

serum, 100 U/ml penicillin, 100 �g/ml streptomycin,176

and 2 mM L-glutamine. The cells were incubated under177

standard conditions (37◦C, 5% CO2) and when 80% 178

confluence was achieved, the cells were treated with 179

0.25% trypsin for 10 min, washed and resuspended 180

in HyQ DMEM/High-Glucose (Hyclone, Logan, UT, 181

USA) with 5% fetal bovine serum (Hyclone), 2 mM 182

L-glutamine (Gibco, Grand Island, NY) and 1% 183

penicillin-streptomycin (Gibco). The cells were then 184

plated at a concentration of 50,000 cells/well for exper- 185

iments using the plate reader (96 wells, NOVOstar 186

Labtech) and used 24 h after plating under experimen- 187

tal conditions similar for neurons. 188

HEK293T cells 189

HEK293T cells were used in experiments with a 190

96-well plate reader (Novostar). Cells were cultured in 191

DMEM supplemented with fetal bovine serum (5%). 192

Cells were kept in a thermo-regulated incubator at 193

37◦C and 5% CO2 and had 80% confluence when 194

used. In a series of experiments, we used HEK293T 195

cells without transfection and found that they did not 196

show any response to full or partial agonists (data not 197

shown). 198

Aβ1-40 oligomer preparation 199

The human A�1-40 (rPeptide, USA) was recon- 200

stituted in DMSO at a concentration of 80 mM and 201

stored at –20◦C. The soluble oligomer (SO-A�) solu- 202

tion was freshly prepared from a stock external solution 203

and aggregated under standard conditions (200 rpm at 204

37◦C, 2 h). The aggregation process and the formation 205

of SO species (3–56 kDa) were checked by turbidity at 206

405 nm (pH 7.4) and electron microscopy, according 207

to our previous characterization [27, 28]. The turbidity 208

measurements were done on a SmartSpec Plus spec- 209

trophotometer (Bio-Rad, Hercules, CA) using a 5-nm 210

broad band and 10-s measurements. The final concen- 211

trations obtained for SO-A� was 0.5 �M. 212

MTT assay 213

Cell cultures were incubated with MTT solu- 214

tion (1 mg/ml) for 30 min, and precipitated MTT 215

was dissolved using isopropanol cooled for 15 min. 216

Absorbance was measured in a multiplate reader 217

(NovoStar, LabTech BMG, Germany) at two wave- 218

lengths: 560 nm and 620 nm, and the difference was 219

quantified using NovoStar Software for the different 220

experimental conditions [29–31]. 221

Unc

orre

cted

Aut

hor P

roof


Live/dead determination222

To asses if the TM0112 extract induced prolifera-223

tive effects, we evaluated the number of cells using the224

Live/Dead cell kit from Invitrogen (Invitrogen, Carls-225

bad, CA, USA), either in PC12 cells or primary cultures226

of hippocampus according to manufacturer instruc-227

tions. Briefly, the assay consists of two probes: Calcein228

(2 �M; ex: 495 nm; em: 515 nm) to label live cells and229

Ethidium Homodimer (5 �M; ex: 530 nm; em: 617 nm)230

to label dead cells. The cells were incubated for 30 min231

at 37◦C with both probes and then were visualized232

on an epifluorescence microscope (Nikon TE 2000,233

Japan), 5 random fields were acquired and analyzed234

using image J (NIH, Bethesda, MD, USA).235

Electrophysiology recordings236

Spontaneous postsynaptic currents were recorded237

in hippocampal neurons during 2–4 min, using patch238

clamp techniques (Axon amplifier 200B, Molecular239

Devices) in the voltage clamp mode (−60 mV holding240

potential) and whole cell configuration. These record-241

ings were made in neurons pre-incubated during 24 h in242

the presence or absence of TM0112 extracts (81 ng/ml)243

and A� (0.5 �M). Frequency was determined and ana-244

lyzed with MiniAnalysis 5.0 software (Synaptosoft,245

Inc, USA). For electrophysiological recordings, the246

pipette solution contained (in mM): 140 KCl, 10247

BAPTA, 10 HEPES (pH 7.4), 4 MgCl2, 0.3 GTP and248

2 ATP-Na2, 300 mOSM.249

Spontaneous Ca2+ transients250

Spontaneous Ca2+ transients were measured after251

24 h of incubation with A� (0.5 �M) and TM0112252

(81 ng/ml) using the calcium probe Fluo4-AM®253

at 3 �M (Invitrogen, USA). Hippocampal neurons254

were placed on coverslips coated with poly-L-lysine255

(Sigma-Aldrich, USA) and incubated with the dye for256

30 min at 37◦C. The neurons were then washed twice257

and mounted on a perfusion chamber on a microscope258

(Nikon, TE 2000, Japan). The Fluo4-AM® was excited259

with a band pass filter at 480 nm wavelength and the260

emission recovered at 535 nm. Changes in cytosolic261

Ca2+ were registered with an EM-CCD camera (iXon262

ANDOR, USA) and a Lambda 10-B (Sutter Instru-263

ments, USA) interface. Each image was recorded every264

0.5–1 s with a time exposure of 100 ms. Image analy-265

sis was made with an Imaging Workbench 6.0 software266

(Indec System, USA). Differential Ca2+ increase in the267

cell was measured in the cytosolic regions.268

Immunofluorescence 269

Hippocampal neurons were incubated for 24 h with 270

A�1-40 and TM0112 81 ng/ml. Samples were later 271

washed with phosphate-buffered saline (PBS) 1X 272

and fixed for 10 min with methanol −20◦C. Then, 273

the samples were washed with PBS 1X and per- 274

meabilized with 0.1% Triton X-100 and nonspecific 275

immunoreactivity was blocked with 10% horse serum 276

for 30 min at room temperature. Monoclonal anti-SV2 277

antibody (1:100, Developmental Studies Hybridoma 278

Bank, Iowa City, IA) and polyclonal anti MAP2 279

(1:400, Santa Cruz Biotechnology, CA, USA) antibody 280

were incubated overnight followed by incubation with 281

an anti-mouse secondary antibody conjugated with 282

FITC (1:400; Jackson ImmunoResearch Laboratories) 283

and anti-rabbit secondary antibody conjugated with 284

Cy3 (1:400; Jackson ImmunoResearch Laboratories), 285

respectively. The samples were mounted in fluorescent 286

mounting medium (DAKO) for microscopic analysis. 287

Confocal microscopy 288

FITC and Cy3 immunofluorescence were visualized 289

using a Zeiss LSM 780 Confocal Microscope (63X, 290

NA 1.4, oil immersion, on CMA BioBio) with lasers of 291

Argon (488 nm) and He-Ne (543 nm) for FITC and Cy3 292

respectively. After acquisition, images were processed 293

with ImageJ (NIH, Maryland, USA). 294

Western blot 295

Protein lysates of PC-12 cells were separated 296

by electrophoresis in 10% acrylamide gel (100 V, 297

100 min) and transferred to a nitrocellulose mem- 298

brane. Membranes were later blocked with 5% milk 299

in TBS. The primary antibodies used were anti p- 300

Akt/Akt (mouse, 1:500, SantaCruz Biotechnology), 301

anti Bcl-2/Bax (mouse, 1:200, SigmaAldrich) and �- 302

actin (mouse, 1:3000, Santa Cruz Biotechnology). Anti 303

mouse-HRP (1:5000, Santa Cruz Biotechnology) was 304

used as secondary antibodies. Immunoreactive marks 305

were visualized and quantified with Li-Cor (Odyssey) 306

detection system. 307

Statistical analysis 308

Data in graphics is expressed as mean ± SEM. Sta- 309

tistical analysis of the results was performed using 310

Student´s t test and ANOVA. It was considered sta- 311

tistically significant results: *p < 0.05, **p < 0.01, and 312

***p < 0.001 versus Control; +p < 0.05, ++p < 0.01, 313

Unc

orre

cted

Aut

hor P

roof


and +++p < 0.001 versus A�; and ‡p < 0.05, ‡‡p < 0.01314

versus A�+TM0112.315

RESULTS316

TM0112 modified SO-Aβ neurotoxic effect without317

altering cellular viability318

To eliminate the possibility of having an intrin-319

sic TM0112 toxicity that could affect cell viability,320

PC-12 cells were incubated during 24 h with increas-321

ing concentrations of TM0112 extract (0.81 ng/ml to322

81 �g/ml) and viability was evaluated by MTT assays.323

We observed that none of the concentrations reduced324

the viability with respect to the control levels, indi-325

cating that TM0112 did not produce toxic effects in326

this cell type (see Fig. 1A). We then chose 81 ng/ml 327

to use for our study. Additionally, to evaluate potential 328

proliferative effects, experiments were made using a 329

Live/Dead assay (see materials and methods) on PC12 330

cells (Fig. 1B) and hippocampal neurons (Fig. 1C). 331

TM0112 did not show significant differences on cell 332

viability as compared to control conditions, suggesting 333

that it did not stimulate the proliferation in either PC12 334

or hippocampal cell cultures. 335

To determine potential protective effects of TM0112 336

against SO-A� on a neuronal model, viability assays 337

were made in PC-12 cells after 24 h of incubation 338

with SO-A� (0.5 �M) and TM0112 (81 ng/ml). As 339

expected, we found a significant reduction in viabil- 340

ity (20 ± 3%) in cells incubated with SO-A� which 341

was consistent with previous reports from our research 342

group [32] for this cell type. Interestingly, when co- 343

A

B

C

D

Fig. 1. Effects of TM0112 on cellular viability and A�1-40 oligomer toxicity on PC-12 cells. A) PC-12 cells were incubated during 24 h withseveral concentrations of the TM0112 extract (from 0.81 ng/ml to 81 �g/ml) to evaluate cell viability with the MTT method and represented as% compared to cell treated only with the vehicle (external solution, n = 9). B) data show that at 24 h incubations with TM0112, the percent oflive and dead PC12 cells did not change as compared with the control conditions (n = 3). C) The same protocol as shown in B for hippocampalneurons (n = 3), where TM0112 did not show differences with control condition on the live and dead cell ratio. D) Reduction on cell viabilityinduced by A� (0.5 �M) was prevented by co-incubation with TM0112 (81 ng/ml). FCCP (100 �M) was used as a positive control for cell death(n = 29) * versus control.

Unc

orre

cted

Aut

hor P

roof


Fig. 2. Effect of A�1-40 oligomers and TM0112 on the viability ofrat cortical neurons. Using the same experimental protocol as Fig.1D, TM0112 (81 ng/ml) inhibited A�1-40 (0.5 �M) neurotoxicity byinducing a recovery on neuronal viability similar to that observedin PC-12 cells (see Fig. 1). FCCP (100 �M) was used as a positivecontrol. (n = 6) * versus control, + versus A�.

incubated with TM0112, the SO-A� cytotoxicity was344

significantly attenuated, reaching values near control345

(113 ± 5%, see Fig. 1D) and without statistical differ-346

ences, suggesting an intrinsic positive effect related347

with a high mitochondrial redox status, corroborated348

by 10–15% of the cells being resistant to a high FCCP349

concentration (100 �M). Additionally, we proceeded350

to test the viability assay in a primary culture of rat cor-351

tical neurons to evaluate the effect of TM0112 in a more352

relevant AD model. The obtained results were similar353

to the PC-12 assay, i.e., cell viability reduction induced354

by A� showed a more intense effect (37 ± 6%), while355

co-incubation with TM0112 demonstrated a reversion356

in the toxic effects with values nearer to the control con- 357

dition (114 ± 8, see Fig. 2). Finally, to discard a direct 358

effect of TM0112 on SO-A�, we used a Thioflavin T 359

assay, and no differences were observed on the aggre- 360

gation kinetics between A� alone or in the presence 361

of TM0112 (data not shown), suggesting a different 362

mechanism to induce neuroprotection in the cellular 363

models used. 364

TM0112 modified the reduction on neuronal 365

network activity induced by chronic SO-Aβ 366

It has been described that acute incubations with 367

A� peptide provokes neuronal excitoxicity [5]. This 368

effect at chronic times (24 h) induces a synaptic silenc- 369

ing reflected as reduction in the frequency of neuronal 370

synaptic electrical activity and intracellular calcium 371

transients, both parameters indicative of basal net- 372

work activity. To evaluate the effect of TM0112 on 373

synaptic alterations induced by chronic incubation 374

(24 h) with SO-A� (0.5 �M), we treated rat hippocam- 375

pal neurons with SO-A� plus TM0112 (81 ng/mL). 376

Chronic exposure of the neurons to SO-A� showed a 377

significant reduction in the frequency of intracellular 378

calcium transients (39 ± 7%, see Fig. 3A). However, 379

this reduction was modified when neurons were co- 380

incubated with TM0112 (112 ± 6%, see Fig. 3B). 381

Furthermore, the recorded synaptic currents correlated 382

the results obtained with calcium microfluorimetry; 383

where SO-A� significantly reduced the frequency of 384

spontaneous synaptic activity (48 ± 17%, see Fig. 4A), 385

and co-incubation with TM0112 showed a significant 386

recovery with respect to A� alone (210.8 ± 47.9%, 387

Fig. 4B). These results indicate that TM0112 both 388

A B

Fig. 3. TM0112 prevented the reduction on the frequency of intracellular Ca2+ transients induced by A�1-40 oligomers. A) The traces areoriginal microfluorimetric recordings of intracellular Ca2+ transients in rat hippocampal neurons after 24 h of treatment with A� (0.5 �M) aloneor with TM0112 (81 ng/ml). B) Shows the quantification Ca2+ transients in different conditions. TM0112 recovered the frequency with valuesslightly above control, promoting by itself an increase in Ca2+ transient frequency. (n = 3; N > 16 * versus control, + versus A�).

Unc

orre

cted

Aut

hor P

roof


A B

Fig. 4. A�1-40-induced synaptic silencing was prevented by TM0112. A) The traces are current recordings of total synaptic activity measuredby patch-clamp (whole cell/voltage clamp) in hippocampal neurons in different experimental conditions. B) Quantification of the frequency ofspontaneous synaptic currents in the neuronal network after incubation with A�1-40 (0.5 �M) alone or with TM0112 (81 ng/ml) for 24 h (n > 10,* versus control, + versus A�).

A

B

Fig. 5. Chronic treatment with TM0112 prevented the loss of synapses induced by A�. Levels of the synaptic protein SV-2 (Green) werequantified through immunocytochemistry in neurons (MAP positive stain, red). A) Confocal images were obtained from equatorial plane indifferent experimental conditions: control, SO-A� (0.5 �M), SO-A� plus TM0112 (81 mg/ml), TM0112 alone. Note that the lower panel showsa magnification of primary neuronal processes (calibration bar: 20 �m). B) Quantification of SV-2 puncta shown in lower panel in A. The graphshows the number of SV2 positive puncta for each 20 �m of primary process. (*, *** versus control, +++ versus A�, n ≥ 3).

inhibits synaptic toxicity of A� and foments sponta-389

neous neuronal network activity, perhaps by increasing390

the number of active synapses or the neuronal network391

functionality.392

To confirm these results, we evaluated the effect 393

of TM0112 on the level of SV2, a protein present 394

in synaptic vesicles where acts as a positive modu- 395

lator of calcium-dependent exocytosis [33–35]. Also, 396

Unc

orre

cted

Aut

hor P

roof


AB

C

D

Fig. 6. Nicotinic antagonists block TM0112 protective effect on A�1-40 in PC-12 cells. A) 24 h incubations of PC-12 cells with A�1-40 (0.5 �M),TM0112 (81 ng/ml), and nAChR antagonists. TM0112 cytoprotective effect on A�1-40 induced cell death was partly blocked by atropine (1 �M;n > 7), �-BTX (3 �M; n > 3), and MLA (100 nM; n > 3). Results are presented as viability percentage with respect to untreated cells. B) Effectsof MLA on the PC12 cell viability co-treated (24 h) with TM0112 (n = 4). C) Effects of MLA and �-BTX on cortical neuron viability with thesame protocol used in B. Note that the blockers did not affect viability or the effects of TM0112 (n = 4). D) The current traces were obtained froma single hippocampal neuron stimulated with TM0112, nicotine (100 nM) and glutamate (50 �M) as control. Note that application of TM0112,unlike nicotine and glutamate, was unable to induce any detectable membrane current by itself.

it has been reported that presynaptic proteins such as397

SV2, SNAP-25, synaptophysin, and synaptotagmin are398

reduced in AD brains and after A� treatment [36].399

The results obtained by immunofluorescence (Fig. 5)400

showed that the decrease in immunoreactive puncta401

of SV2 caused by A� aggregates (A� = 45 ± 4%)402

was modified by TM0112 (188 ± 13% of control).403

These results confirm the protective effect of TM0112404

against the toxicity of A� aggregates on neuronal405

functionality, suggesting a positive effect on synaptic406

activity. This is in agreement with previous evidence407

related to the positive modulation of nicotinic receptors408

by agonists-enhanced Ca2+ transients and increased409

action potential firing [37].410

Neuroprotective effect of TM0112 is mediated by 411

the activation of nAChR 412

It has been discussed that nicotine exerts neuro- 413

protection through nAChR, primarily mediated by �7 414

activation. To determine nAChR participation in the 415

neuroprotective effect of TM0112, we treated PC-12 416

cells during 24 h with SO-A� (0.5 �M) plus TM0112. 417

Additionally, atropine (1�M), �-BTX (3 �M), and 418

MLA (100 nM) were added to inhibit nAChRs, par- 419

ticularly �7. The data shows that atropine was able to 420

partially antagonize the protective effect of TM0112 421

on cellular viability (87 ± 4% versus 105.1 ± 3.8% 422

of TM0112 plus SO-A�, see Fig. 6A), suggesting 423

Unc

orre

cted

Aut

hor P

roof


Fig. 7. Absence of TM0112 effects in a cell model devoid of nAChR.Chronic incubations (24 h) with A�1-40 (0.5 �M) and TM0112(81 ng/ml) in HEK293T cells did not show the preventive effectof TM0112 on this cellular model, maintaining the same percentageof viability (68% versus 72%, n = 3). FCCP (100 �M) was used aspositive control. (*** versus control, ++ versus A�).

the involvement of these receptors. Furthermore, the424

use of �7 nicotinic selective antagonists MLA (100425

nM) and �-BTX (3 �M) also partly reverted the426

TM0112 neuroprotective effect (86 ± 4% and 92 ± 3%427

of TM0112 plus SO-A�, respectively). In summary,428

atropine, MLA and �-BTX (3 �M) blocked the pro-429

tective effect of TM0112 on cell viability induced430

by the A� toxic effect. In parallel studies, we found431

Fig. 9. TM0112 prevented the decrease in the Bcl-2/Bax ratioinduced by A�1-40. A) 24 h incubation with A�1-40 (0.5 �M) andTM0112 (81 ng/ml) in PC-12 cells and evaluation of Bcl-2 and Baxexpression by western blot technique B) Data is plotted as Bcl-2/Baxratio (n = 4). A� decreased the Bcl-2/Bax ratio, an effect that wascounteracted by co-incubation with TM0112. Values are presentedas percentage of untreated control.

Fig. 8. TM0112 induced Akt activation at 3 h incubation. Show the effect of incubation with A�1-40 (0.5 �M) and TM0112 (81 ng/ml) in PC-12cells. Akt phosphorylation was measured by Western blot. Data represent p-Akt/Akt ratio whose increase indicates an anti-apoptotic effectand vice versa. A) 3 h treatment showed that A� did not produce changes in Akt phosphorylation, while the presence of TM0112 significantlyincreased Akt phosphorylation despite the presence of A� (n = 7). B) After 24 h treatment there was no significant differences between thedifferent conditions (n = 6). All values are presented as percentage of untreated control.

Unc

orre

cted

Aut

hor P

roof


Fig. 10. Scheme of proposed neuroprotection mechanism ofTM0112 against A�1-40 toxicity. A� exerts several and noxiouseffects on cellular processes leading to apoptosis. TM0112 modu-late �7 AChR, activating a calcium dependent survival pathway thatinvolves Akt phosphorylation and Bcl-2 expression. Thus, TM0112protects neuronal viability and performance, promoting synapticactivity.

that MLA and �-BTx did not affect cell viability or432

on the TM0112 effect in PC12 and cortical neurons433

(Fig. 6B-C). These results strongly suggest the role434

of nicotinic receptors, mainly the �7 subtype, in the435

neuroprotective mechanism of TM0112 against A�436

oligomer toxicity. However, TM0112 was unable to437

induce a current by itself on hippocampal neurons438

indicating that it is not an agonist of nicotinic recep-439

tors (Fig. 6D). To support that the effects of TM0112440

are mediated by nicotinic receptors, a cellular model441

that lacked the �7 nAChR was used [38]. HEK293Y442

cells, that previously did not show any response to full443

or partial nicotinic agonists (not shown), were treated444

with the same experimental protocol and cellular via-445

bility was determined. Co-incubation of SO-A� with446

TM0112 did not show any recovery in cell viability as447

observed in PC12 and neurons (A� = 61.4 ± 6.1% of448

control and A�+TM0112 = 62.8 ± 5.2 of control), sug-449

gesting that the protective effect of TM0112 required450

the presence of �7 nAChR. The idea that �7 could451

be involved in these effects tightly correlates with the452

observations made with the pharmacological inhibitors453

(Fig. 7).454

Activation of nAChR by TM0112 stimulated a455

survival pathway associated to Akt and Bcl-2456

signaling457

It has been described that �7 nAChR activates a458

survival signaling pathway involving Akt activation459

through phosphorylation and increase of Bcl-2 [18,460

20]. To evaluate if nAChR activation by TM0112 is461

coupled to this pathway, we performed 3 and 24 h incu- 462

bation protocols of PC-12 cells with A� and TM0112; 463

and measured the activity of p-Akt/Akt. 464

PC-12 cells were treated with TM0112 during 3 h, 465

and changes on p-Akt and AkT levels were mea- 466

sured with antibodies, considering that this is an early 467

step in the signaling pathway activation. The western 468

blots showed a significant increase in p-Akt/Akt ratio 469

(150 ± 62% over the control) at 3 h of TM0112 incu- 470

bation (Fig. 8A), suggesting a coupling between this 471

survival pathway and nAChR (�7 subtype). The find- 472

ing that the presence of SO-A� alone did not induce 473

changes on P-Akt/Akt levels as compared to control 474

(A� = 118 ± 16%, not significant) support the idea that 475

TM0112 neuroprotective effect could be related with 476

the activation of this pathway through �7 nAChR. On 477

the other hand, treatments during 24 h of incubation did 478

not show any significant changes on P-Akt/Akt ratio 479

with respect to control condition (Fig. 8B). Therefore, 480

Akt phosphorylation appears to be an early event in 481

the TM0112 action mechanism. Additionally, using a 482

similar approach used for Akt experiments, Bcl-2/Bax 483

levels were measured at 6, 12, and 24 h of incuba- 484

tion. The data showed that the only clear decrease in 485

Bcl-2/Bax ratio on SO-A� treated cells with respect 486

to control conditions (38 ± 8%) was observed at 24 h 487

(Fig. 9); while the presence of TM0112 induced a sig- 488

nificant increase in Bcl-2/Bax percentages reaching 489

values similar to control (105.4 ± 19.5% of control). 490

Considering that Bcl-2/Bax is a later step (down- 491

stream) in the pathway, these results confirm the 492

participation of Akt/Bcl-2 pathways on the neuropro- 493

tective effects of TM0112. 494

DISCUSSION 495

In recent years, there have been great efforts to 496

establish a deeper understanding of the etiology, neu- 497

ropathology, and neurochemical mechanisms involved 498

in AD in order to develop new therapeutic strategies. 499

Considering the strong evidence of the neuroprotective 500

properties of nicotine, an additional interest in develop- 501

ing new nicotinic agonists as emerged on the potential 502

therapeutic efficacy of these drugs against AD [25]. In 503

our study, the alkaloid-rich extract modified the toxic 504

effects of A� oligomers. Additionally, these results 505

suggest a coupled mechanism between nAChR and 506

a survival signaling pathway (Akt and Bcl-2), espe- 507

cially through the �7 nAChR subtype. We found that 508

TM0112 alone showed no changes in PC-12 viability; 509

therefore, a concentration of 81 ng/ml was chosen for 510

Unc

orre

cted

Aut

hor P

roof


further assays in neurons finding that it modified the511

A� cytotoxic effect. Because nicotine disturbs cellular512

cycles and promotes hyperplasia [39, 40] in a, PI3K and513

Akt pathway dependent [41], we examined its poten-514

tial mitogenic action. However, this potential effect515

was discarded by various approaches in PC12, cortical,516

and hippocampal neurons. Thus, TM0112 mediates a517

protective mechanism, via nAChR. Additionally, we518

found that TM0112 was able to inhibit the toxic effect519

of A� without affecting cell proliferation.520

We previously reported the strong synaptotoxic521

action caused by 24 h incubation with A� oligomers522

(3–56 kDa) [4, 34]. Here, using calcium microfluo-523

rimetry and electrophysiology, we found that TM0112524

modified neuronal network function weakening, as525

expressed in parameters of frequency of calcium526

transients and overall synaptic activity, respectively.527

Interestingly, neurons treated with TM0112 alone528

showed an increase in activity when compared529

to untreated neurons, also supporting the idea of530

enhanced connectivity. These results were in agree-531

ment with the increase of SV2 immunostaining.532

Therefore, TM0112 not only induced neuronal survival533

in presence of A�, but also protected and enhanced its534

connectivity.535

TM0112 modified A� neurotoxicity viability536

through activation of the �7 nAChR subtype and the537

survival signaling pathway mediated by Akt and Bcl-2.538

For this, we used HEK293T cells, which did not exhibit539

cholinergic responses mediated by nAChR [38, 42].540

This cell line, although distant from neuronal models541

previously used, allowed us to obtain a good indication542

about the mechanism of action of TM0112 on cell via-543

bility. Here, the extract was unable to reverse the toxic544

effects induced by A� and these results are consistent545

with our working hypothesis that TM0112 neuropro-546

tective action is nAChR dependent. Additionally, the547

use of cholinergic blockers; atropine (1 �M), �-BTX548

(3 �M), and MLA (100 nM) allowed us to demon-549

strate that TM0112 protective effect on cell viability550

was dependent on nAChR, probably the �7 subtype.551

Atropine is considered a classic selective antagonist552

of muscarinic acetylcholine receptors, however other553

studies have described actions on nicotinic receptors,554

for example, inhibiting up to 56% (at 1 �M) of cur-555

rents induced by 1 mM acetylcholine in nAChR �2�2,556

�2�4, �3�2, �3�4, �4�2, �4�4, and �7 expressed in557

Xenopus laevi oocytes [43]. Nanomolar concentrations558

of atropine can also competitively block catecholamine559

release induced by DMPP and nicotinic-induced elec-560

trophysiological currents in bovine chromaffin cells561

[44]. The usual concentration of atropine used to block562

muscarinic receptors is 100 nM [43]. We used a work- 563

ing concentration that was 10 times higher in order 564

to guarantee a global blockade of cholinergic trans- 565

mission. Thus, reverting the TM0112 protective effect 566

using atropine indicates that its effect was by one of 567

these two pathways activated by acetylcholine. 568

At a concentration of 3 �M, �-BTX, a selective 569

�7 subtype antagonist, produced a partial blockade 570

of the TM0112 effect suggesting that the effect of 571

TM0112 is produced by activation of nAChR. Addi- 572

tionally, MLA, another �7 selective inhibitor, was also 573

able to block TM0112 effects. Taken together, these 574

experimental data support our hypothesis that TM0112 575

actions depend on �7 nAChR activation. 576

The results showed a significant increase in Akt 577

phosphorylation at 3 h of incubation with TM0112, 578

even in the absence of A�. On the other hand, this 579

increase in p-Akt was no detected at 24 h of incubation. 580

It is known that nicotine can induce Akt phospho- 581

rylation quite rapidly. In bronchial epithelial cells, 582

for example, serine 473 phosphorylation occurs after 583

30–60 min and can persist for 24 h. [41]. Diverse 584

effects of A� have been reported for Akt phosphory- 585

lation [45, 46]. These divergent actions are likely due 586

to the experimental models and the type of A� species 587

used. In our study, we did not expect increases in Akt 588

phosphorylation by A� because this pathway should 589

be already activated when the neurons were exposed 590

to TM0112. These findings reported here are consistent 591

with other studies [47–49], and support the hypothesis 592

of nicotinic modulation by TM0112, where Akt acti- 593

vation is an early event in the �7 dependent survival 594

pathway. 595

Finally, TM0112 was also able to increase the Bcl- 596

2/Bax ratio in the presence of A�, and although this 597

ratio seemed to decrease in cells treated only with 598

TM0112, there were no significant differences as com- 599

pared with control. These experiments were performed 600

after 6, 12, and 24 h of incubation, but only evident 601

effects were observed at 24 h. Therefore, it is possi- 602

ble that the increase in the Bcl-2/Bax ratio will be a 603

later step (downstream) in the pathway, suggesting that 604

Akt activation is necessary to activate Bcl-2/Bax on 605

the nicotinic mediated survival pathway. This is con- 606

sistent with the working hypothesis and indicates that 607

TM0112 activates a signaling pathway associated to 608

nicotine action in neuronal cells. 609

Altogether, these results show that the alkaloid 610

extract from exerts a neuroprotective effect through 611

activation of a signaling pathway that depends on 612

�7 nAChR and can interfere A� toxicity improv- 613

ing cellular viability and functionality (Fig. 10), and 614

Unc

orre

cted

Aut

hor P

roof


could therefore be a source for new potential anti-AD615

drugs.616

ACKNOWLEDGMENTS617

We thank Mrs. L. J. Aguayo for technical assis-618

tance and editing the manuscript; and Carolina Castillo619

for technical assistance. This work was supported by620

Fondecyt Iniciacion 11090091 (JF); Fondecyt 1130747621

(JF), Fondecyt 1100502(LG); and INNOVABIOBIO622

12.118-EM.TES (12.226).623

Authors’ disclosures available online (http://www.j-624

alz.com/disclosures/view.php?id=2222).625

REFERENCES626

[1] Price BH, Gurvit H, Weintraub S, Geula C, Leimkuhler E,627

Mesulam M (1993) Neuropsychological patterns and lan-628

guage deficits in 20 consecutive cases of autopsy-confirmed629

Alzheimer’s disease. Arch Neurol 50, 931-937.630

[2] Glabe CG (2008) Structural classification of toxic amyloid631

oligomers. J Biol Chem 283, 29639-29643.632

[3] Lesne S, Koh MT, Kotilinek L, Kayed R, Glabe CG, Yang A,633

Gallagher M, Ashe KH (2005) A specific amyloid- protein634

assembly in the brain impairs memory. Nature 440, 352-357.635

[4] Fuentealba J, Dibarrart AJ, Fuentes-Fuentes MC, Saez-636

Orellana F, Quinones K, Guzman L, Perez C, Becerra J,637

Aguayo LG (2011) Synaptic failure and adenosine triphos-638

phate imbalance induced by amyloid-� aggregates are639

prevented by blueberry-enriched polyphenols extract. J Neu-640

rosci Res 89, 1499-1508.641

[5] Aguayo LG, Parodi J, Sepulveda FJ, Opazo C (2009)642

Pore-forming neurotoxin-like mechanism for A� oligomer-643

induced synaptic failure. In Current Hypotheses and Research644

Milestones in Alzheimer’s Disease, Maccioni RB, Perry G,645

eds. Springer Press, pp. 13-21.646

[6] Camandola S, Mattson MP (2010) Aberrant subcellular neu-647

ronal calcium regulation in aging and Alzheimer’s disease.648

Biochim Biophys Acta 1813, 965-973.649

[7] Bezprozvanny I, Mattson MP (2008) Neuronal calcium mis-650

handling and the pathogenesis of Alzheimer’s disease. Trends651

Neurosci 31, 454-463.652

[8] Bartus RT (2000) On neurodegenerative diseases, models, and653

treatment strategies: Lessons learned and lessons forgotten a654

generation following the cholinergic hypothesis. Exp Neurol655

163, 495-529.656

[9] Craig LA, Hong NS, McDonald RJ (2011) Revisiting the657

cholinergic hypothesis in the development of Alzheimer’s658

disease. Neurosci Biobehav Rev 35, 1397-1409.659

[10] Suh Y-H, Checler F (2002) Amyloid precursor protein,660

presenilins, and �-synuclein: Molecular pathogenesis and661

pharmacological applications in Alzheimer’s disease. Phar-662

macol Rev 54, 469-525.663

[11] Kawamata J, Shimohama S (2011) Stimulating nicotinic664

receptors trigger multiple pathways attenuating cytotoxic-665

ity in models of Alzheimer’s and Parkinson’s diseases. J666

Alzheimers Dis 24, 95-109.667

[12] Pappas BA, Bayley PJ, Bui BK, Hansen LA, Thal LJ (2000)668

Choline acetyltransferase activity and cognitive domain669

scores of Alzheimer’s patients. Neurobiol Aging 21, 11-17.670

[13] Perry EK, Tomlinson BE, Blessed G, Bergmann K, Gibson 671

PH, Perry RH (1978) Correlation of cholinergic abnormalities 672

with senile plaques and mental test scores in senile dementia. 673

BMJ 2, 1457-1459. 674

[14] Mousavi M, Hellstrom-Lindahl E, Guan ZZ, Shan KR, Ravid 675

R, Nordberg A (2003) Protein and mRNA levels of nicotinic 676

receptors in brain of tobacco using controls and patients with 677

Alzheimer’s disease. Neuroscience 122, 515-520. 678

[15] Jonnala RR, Terry AV, Jr., Buccafusco JJ (2002) Nicotine 679

increases the expression of high affinity nerve growth fac- 680

tor receptors in both in vitro and in vivo. Life Sci 70, 681

1543-1554. 682

[16] Li X, Arias E, Jonnala R, Mruthinti S, Buccafusco J (2005) 683

Effect of amyloid peptides on the increase in TrkA receptor 684

expression induced by nicotine in vitro and in vivo. J Mol 685

Neurosci 27, 325-336. 686

[17] Kihara T, Shimohama S, Urushitani M, Sawada H, Kimura 687

J, Kume T, Maeda T, Akaike A (1998) Stimulation of �4�2 688

nicotinic acetylcholine receptors inhibits �-amyloid toxicity. 689

Brain Res 792, 331-334. 690

[18] Kihara T, Shimohama S, Sawada H, Honda K, Nakamizo T, 691

Shibasaki H, Kume T, Akaike A (2001) �7 nicotinic receptor 692

transduces signals to phosphatidylinositol 3-kinase to block 693

A�-amyloid-induced neurotoxicity. J Biol Chem 276, 13541- 694

13546. 695

[19] Yu W, Mechawar N, Krantic S, Quirion R (2011) �7 Nicotinic 696

receptor activation reduces �-amyloid-induced apoptosis by 697

inhibiting caspase-independent death through phosphatidyli- 698

nositol 3-kinase signaling. J Neurochem 119, 848-858. 699

[20] Akaike A, Takada-Takatori Y, Kume T, Izumi Y (2010) 700

Mechanisms of neuroprotective effects of nicotine and acetyl- 701

cholinesterase inhibitors: Role of �4 and �7 receptors in 702

neuroprotection. J Mol Neurosci 40, 211-216. 703

[21] Daly JW (2005) Nicotinic agonists, antagonists, and modula- 704

tors from natural sources. Cell Mol Neurobiol 25, 513-552. 705

[22] Cordell GA, Quinn-Beattie ML, Farnsworth NR (2001) The 706

potential of alkaloids in drug discovery. Phytother Res 15, 707

183-205. 708

[23] Barlow RB, McLeod LJ (1969) Some studies on cytisine and 709

its methylated derivatives. Br J Pharmacol 35, 161-174. 710

[24] Fitch RW, Kaneko Y, Klaperski P, Daly JW, Seitz G, Gundisch 711

D (2005) Halogenated and isosteric cytisine derivatives with 712

increased affinity and functional activity at nicotinic acetyl- 713

choline receptors. Bioorg Med Chem Lett 15, 1221-1224. 714

[25] Cassels BK, Bermudez I, Dajas F, Abin-Carriquiry JA, Won- 715

nacott S (2005) From ligand design to therapeutic efficacy: 716

The challenge for nicotinic receptor research. Drug Discov 717

Today 10, 1657-1665. 718

[26] del Barrio L, Martın-de-Saavedra MD, Romero A, Parada 719

E, Egea J, Avila J, McIntosh JM, Wonnacott S, Lopez MG 720

(2011) Neurotoxicity induced by okadaic acid in the human 721

neuroblastoma SH-SY5Y line can be differentially prevented 722

by �7 and �2* nicotinic stimulation. Toxicol Sci 123, 193-205. 723

[27] Parodi J, Sepulveda FJ, Roa J, Opazo C, Inestrosa NC, Aguayo 724

LG (2010) Beta-amyloid causes depletion of synaptic vesicles 725

leading to neurotransmission failure. J Biol Chem 285, 2506- 726

2514. 727

[28] Sepulveda FJ, Parodi J, Peoples RW, Opazo C, Aguayo LG 728

(2010) Synaptotoxicity of Alzheimer beta amyloid can be 729

explained by its membrane perforating property. PLoS One 730

5, e11820. 731

[29] Ferreiro E, Eufrasio A, Pereira C, Oliveira CR, Rego AC 732

(2007) Bcl-2 overexpression protects against amyloid-beta 733

and prion toxicity in GT1-7 neural cells. J Alzheimers Dis 734

12, 223-228. 735

http://www.j-alz.com/disclosures/view.php?id=2222

Unc

orre

cted

Aut

hor P

roof


[30] Latini A, da Silva CG, Ferreira GC, Schuck PF, Scussi-736

ato K, Sarkis JJ, Dutra Filho CS, Wyse AT, Wannmacher737

CM, Wajner M (2005) Mitochondrial energy metabolism is738

markedly impaired by D-2-hydroxyglutaric acid in rat tissues.739

Mol Genet Metab 86, 188-199.740

[31] Xia YP, Dai RL, Li YN, Mao L, Xue YM, He QW, Huang741

M, Huang Y, Mei YW, Hu B (2012) The protective effect742

of sonic hedgehog is mediated by the propidium iodide 3-743

kinase/AKT/BCL-2 pathway in cultured rat astrocytes under744

oxidative stress. Neuroscience 209, 1-11.745

[32] Fuentealba J, Dibarrart A, Saez-Orellana F, Fuentes-Fuentes746

MC, Oyanedel CN, Guzman J, Perez C, Becerra J, Aguayo LG747

(2012) Synaptic silencing and plasma membrane dyshome-748

ostasis induced by amyloid-� peptide are prevented by749

Aristotelia chilensis enriched extract. J Alzheimers Dis 31,750

879-889.751

[33] Alabi AA, Tsien RW (2013) Perspectives on kiss-and-run:752

Role in exocytosis, endocytosis, and neurotransmission. Ann753

Rev Physiol 75, 393-422.754

[34] Parodi J, Sepulveda FJ, Roa J, Opazo C, Inestrosa NC, Aguayo755

LG (2010) Beta-amyloid causes depletion of synaptic vesicles756

leading to neurotransmission failure. J Biol Chem 285, 2506-757

2514.758

[35] Sepulveda FJ, Parodi J, Peoples RW, Opazo C, Aguayo LG759

(2010) Synaptotoxicity of Alzheimer beta amyloid can be760

explained by its membrane perforating property. PLoS One761

5, e11820.762

[36] Chauhan NB, Siegel GJ (2002) Reversal of amyloid � toxic-763

ity in Alzheimer’s disease model Tg2576 by intraventricular764

antiamyloid � antibody. J Neurosci Res 69, 10-23.765

[37] Fuentealba J, Olivares R, Ales E, Tapia L, Rojo J, Arroyo766

G, Aldea M, Criado M, Gandia L, Garcia AG (2004) A767

choline-evoked [Ca2+]c signal causes catecholamine release768

and hyperpolarization of chromaffin cells. FASEB J 18, 1468-769

1470.770

[38] Buisson B, Gopalakrishnan M, Arneric SP, Sullivan JP,771

Bertrand D (1996) Human �4�2 neuronal nicotinic acetyl-772

choline receptor in HEK 293 cells: A patch-clamp study. J773

Neurosci 16, 7880-7891.774

[39] Chu M, Guo J, Chen C-Y (2005) Long-term exposure to nico-775

tine, via Ras pathway, induces cyclin D1 to stimulate G1 cell776

cycle transition. J Biol Chem 280, 6369-6379.777

[40] Dasgupta P, Rastogi S, Pillai S, Ordonez-Ercan D, Morris M, 778

Haura E, Chellappan S (2006) Nicotine induces cell prolifer- 779

ation by �-arrestin–mediated activation of Src and Rb–Raf-1 780

pathways. J Clin Invest 116, 2208-2217. 781

[41] West KA, Brognard J, Clark AS, Linnoila IR, Yang X, Swain 782

SM, Harris C, Belinsky S, Dennis PA (2003) Rapid Akt acti- 783

vation by nicotine and a tobacco carcinogen modulates the 784

phenotype of normal human airway epithelial cells. J Clin 785

Invest 111, 81-90. 786

[42] Shaw G, Morse S, Ararat M, Graham FL (2002) Prefer- 787

ential transformation of human neuronal cells by human 788

adenoviruses and the origin of HEK 293 cells. FASEB J 16, 789

869-871. 790

[43] Zwart R, Vijverberg HPM (1997) Potentiation and Inhibition 791

of neuronal nicotinic receptors by atropine: Competitive and 792

noncompetitive effects. Mol Pharmacol 52, 886-895. 793

[44] Gonzalez-Rubio JM, Garcıa de Diego AM, Egea J, Olivares 794

R, Rojo J, Gandıa L, Garcıa AG, Hernandez-Guijo JM (2006) 795

Blockade of nicotinic receptors of bovine adrenal chromaffin 796

cells by nanomolar concentrations of atropine. Eur J Phar- 797

macol 535, 13-24. 798

[45] Abbott JJ, Howlett DR, Francis PT, Williams RJ (2008) 799

Abeta(1-42) modulation of Akt phosphorylation via alpha7 800

nAChR and NMDA receptors. Neurobiol Aging 29, 801

992-1001. 802

[46] Magrane J, Rosen KM, Smith RC, Walsh K, Gouras GK, 803

Querfurth HW (2005) Intraneuronal beta-amyloid expression 804

downregulates the Akt survival pathway and blunts the stress 805

response. J Neurosci 25, 10960-10969. 806

[47] Akaike A, Takada-Takatori Y, Kume T, Izumi Y (2010) 807

Mechanisms of neuroprotective effects of nicotine and acetyl- 808

cholinesterase inhibitors: Role of alpha4 and alpha7 receptors 809

in neuroprotection. J Mol Neurosci 40, 211-216. 810

[48] Kawamata J, Shimohama S (2011) Stimulating nicotinic 811

receptors trigger multiple pathways attenuating cytotoxic- 812

ity in models of Alzheimer’s and Parkinson’s diseases. J 813

Alzheimers Dis 24(Suppl 2), 95-109. 814

[49] Kihara T, Shimohama S, Sawada H, Honda K, Nakamizo T, 815

Shibasaki H, Kume T, Akaike A (2001) alpha 7 nicotinic 816

receptor transduces signals to phosphatidylinositol 3-kinase 817

to block A beta-amyloid-induced neurotoxicity. J Biol Chem 818

276, 13541-13546. 819

Modulation of neuronal nicotinic receptor by quinolizidine alkaloids causes neuroprotection on a...

Documents

Transcript of Modulation of neuronal nicotinic receptor by quinolizidine alkaloids causes neuroprotection on a...