uncorrected proof - University of Minnesota Duluth

Behavioural Brain Research xxx (2005) xxx–xxx

Research report 3

4

5

Increased anxiety-like behaviors and mitochondrial dysfunction in mice with targeted mutation of the Bcl-2 gene: Further support for the involvement of mitochondrial function in anxiety disorders Haim Einat ∗ , Peixiong Yuan 1 , Husseini K. Manji 2

6

Laboratory of Molecular Pathophysiology, National Institute of Mental Health, National Institutes of Health, Department of Health and Human Services, 35 Convent Drive, Room 1C 912, Bethesda, MD 20892-3711, USA

7 8

Received 24 August 2004; received in revised form 24 June 2005; accepted 25 June 2005

12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

There is growing evidence that anxiety disorders are associated with impairments of cellular plasticity and resilience. Paralleling these advances in our understanding of the neurobiologic underpinnings of anxiety disorders is the growing appreciation of the diverse functions that mitochondria play in regulating integrated CNS function. The emerging data suggest that mitochondrial Ca2+ sequestration has a key role in modulating the tone of synaptic plasticity in a variety of neuroanatomical regions, including those implicated in the pathophysiology of anxiety disorders. Furthermore, activation of peripheral mitochondrial benzodiazepine receptors resulted in reduced anxiety in rats. One of the major modulators of mitochondrial function is Bcl-2 proteins imbedded in the inner mitochondrial membrane. Bcl-2 overexpression increases mitochondria Ca2+ uptake capacity and resistance to Ca2+ -inhibition of respiration and upregulation of Bcl-2 increases maximal uptake capacity of mitochondria. We have, therefore, explored the significance of Bcl-2 in the association between mitochondrial function and affective disorders testing Bcl-2 heterozygote mice in models of affective and anxiety disorders. Mutant mice have reduced mitochondrial Bcl-2 levels, and although they have no gross behavioral abnormalities, they demonstrate a significant increase of anxiety-like behaviors. Bcl-2 heterozygote mice spent less time in the center of an open field, spent less time outside an enclosure in the “emergence test”, were less likely to explore the transparent part of a black/white box or the open arms of an elevated plus maze compared with WT controls. Mutant mice did not differ from WT in measures of locomotion or in the forced swim test for depression-like behavior suggesting a specific effect on anxiety-like behaviors. Our study, therefore demonstrates that Bcl-2 may be a key factor in anxiety disorders and that its effects may possibly originate from its role in the mitochondria. © 2005 Published by Elsevier B.V.

PR

11

Abstract

TE D

10

OO F

9

28

32 33 34

EC

31

1. Introduction

Anxiety disorders are common, severe, chronic, and often life-threatening illnesses [29,35]. There is a growing appreciation that, far from being diseases with purely psychological manifestations, anxiety disorders are systemic diseases

RR

30

∗

0166-4328/$ – see front matter © 2005 Published by Elsevier B.V. doi:10.1016/j.bbr.2005.06.012

UN

1 2

CO

Corresponding author at: College of Pharmacy, University of Minnesota, Duluth, 376 Kirby Plaza, 1208 Kirby Dr., Duluth, MN 55812, USA. Tel.: +1 218 726 6029; fax: +1 218 726 6500. E-mail addresses: [email protected] (H. Einat), [email protected] (P. Yuan), [email protected] (H.K. Manji). 1 Tel.: +1 301 451 8466; fax: +1 301 480 0123. 2 Tel.: +1 301 451 8441; fax: +1 301 480 0123.

with deleterious effects on multiple organ systems. Although genetic factors play a major, unquestionable role in etiology of these disorders, the biochemical abnormalities underlying the predisposition to, and the pathophysiology remain to be fully elucidated. The brain systems which have heretofore received the greatest attention in neurobiologic studies of anxiety disorders have been the monoaminergic neurotransmitter systems, which are extensively distributed throughout the network of limbic, striatal and prefrontal cortical neuronal circuits thought to support the behavioral and visceral manifestations of anxiety disorders [13,35,47]. Thus, clinical studies over the past 40 years have attempted to uncover the specific deficits or excess in these neurotransmitter systems

BBR 4358 1–9

35 36 37 38 39 40 41 42 43 44 45 46 47

54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103

pressants, improve mitochondrial efficiency [21,38,62] and protect mitochondrial function against a variety of insults [1,7,10,38,48,58]. Moreover, in vivo animal studies demonstrated that the activation of peripheral mitochondrial benzodiazepine receptors resulted in reduced stress and anxiety in rats [4,51]. Additionally, mitochondria have specific binding sites for neurosteroids that are major modulators of Ca2+ efflux [24] and were shown to protect mitichondria from a variety of insults (e.g. [27,44]. Neurosteroids are well known for their anxiolytic properties in animal models (e.g. [2,52]) and it is, therefore conceivable that their properties as anxiolytics may be related to their actions in the mitochondria. These studies suggest that targeting mitochondrial function may represent a novel avenue for the development of therapeutics for the treatment of anxiety disorders. In this context, one of the major modulators of mitochondrial function is the protein Bcl-2 (originally identified from b cell leukemias). Bcl-2 is the first gene shown to be involved in apoptosis. The Bcl-2 family consists of both pro- and anti-apoptotic proteins. The expression of bcl-2 was localized to a large population of neurons and some glial cells in CNS and PNS [19,32]. It is present in the outer mitochondrial membrane, endoplasmic reticulum and nuclear membrane interacting with other Bcl-2 family members (such as Bax and Bad). A number of mitochondrial related mechanisms have been proposed for the action of Bcl-2, including increasing mitochondrial calcium buffering capacity and protecting mitochondrial membrane integrity, so preventing the release of cytochrome c and the formation of the apoptosome and activation of caspases. It can prevent mitochondrial dysfunction such as membrane potential loss and the permeability transition (PT) precedes cell death. In studies of isolated mitochondria, Murphy et al have shown that Bcl-2 overexpression increases mitochondria Ca2+ uptake capacity, increasing the resistance of mitochondria to Ca2+ -inhibition of respiration [45]. Thus, Bcl-2 overexpressing cells appear to be particularly resistant to the destructive influence of elevated intracellular Ca2+ . Bcl-2 likely exerts significant effects on cellular Ca2+ buffering not only under the extreme conditions of Ca2+ -induced apoptosis, but potentially even during normal synaptic activity. Thus, Murphy et al. [45] demonstrated that upregulation of Bcl-2 increases the maximal uptake capacity of mitochondria, effects which likely maintain mitochondrial activity during very high frequency stimulation. Recent studies suggest that Bcl-2 may have a more general role in regulating mitochondrial metabolism and function [34], and that its protective effect may not be limited to an antiapoptotic role. Its overexpression reduces the rate of mitochondrial ATP consumption under conditions in which ATP hydrolysis is stimulated [25]. Overexpression of bcl-2 in the desmin null heart results in correction of mitochondrial defects [65]. Interestingly, in the context of anxiety disorders, it was demonstrated that mice with overexpression of the Bcl2 gene exhibited reduced anxiety-like (fear) behavior [54]. Inactivation of bcl-2 results in progressive degeneration of

PR

53

TE D

52

EC

51

RR

50

in mood disorders by utilizing a variety of biochemical and neuroendocrine strategies. While such investigations have been heuristic over the years, they have been of limited value in elucidating the unique biology of anxiety disorders. The recognition of the clear need for better treatments and the lack of significant advances in our ability to develop novel, improved therapeutics for these devastating illnesses has led to the investigation of the putative roles of intracellular signaling cascades and non-aminergic systems in the pathophysiology and treatment of anxiety disorders. Consequently, recent evidence demonstrating that impairments of cellular plasticity and resilience may underlie the pathophysiology of anxiety disorders, and that effective treatments (notably antidepressants) exert major effects on signaling pathways which regulate neuroplasticity and cell survival [8,14,70], have generated considerable excitement among the clinical neuroscience community, and are reshaping views about the neurobiological underpinnings of these disorders. Paralleling these advances in our understanding of the neurobiologic underpinnings of anxiety disorders is the growing appreciation of the diverse functions that mitochondria play in regulating integrated CNS function. Thus, mitochondria are intracellular organelles best known for their critical roles in regulating energy production via oxidative phosphorylation, the regulation of intracellular calcium (Ca2+ ), and critical mediators of apoptosis; however, increasing evidence suggests mitochondria may be integrally involved in the general processes of synaptic plasticity. In a detailed investigation of the relative roles of mitochondrial and ER Ca2+ buffering, it was found that the dendritic mitochondrion rapidly accumulates Ca2+ , while the endoplasmic reticulum displays a more delayed increase in Ca2+ during high frequency stimulation [49]. Furthermore, increased synaptic activity has been shown to induce the expression of mitochondrial-encoded genes, suggesting that the regulation of metabolism is an important component in the long-term regulation of synaptic strength [66]. This regulation occurred even with stimulations that were under the threshold for long-term potentiation induction, suggesting that a sort of ‘metabolic priming’ might take place. All in all, these findings suggest that mitochondrial Ca2+ sequestration has a key role in modulating the tone of synaptic plasticity in a variety of neuroanatomical regions, including those implicated in the pathophysiology of anxiety disorders. In total, these observations suggest that regulation of mitochondrial function is likely to play important roles in regulating synaptic strength neuronal circuitry mediating complex behaviors [56]. For further discussion of recent research into the mitochondrion’s role in synaptic plasticity, the reader is referred to Mattson & Liu’s recent review [39]. For the purposes of the present discussion, it is noteworthy that there has already been indirect evidence accumulating that suggests that mitochondrial function may be related to the pathophysiology and treatment of behavioral disorders. Thus, monoamine oxidase inhibitors (MAO-I) which are arguably amongst the most potent anxiolytics and antide-

CO

49

UN

48

H. Einat et al. / Behavioural Brain Research xxx (2005) xxx–xxx

OO F

2

BBR 4358 1–9

104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159


162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186

motoneurons, sympathetic and sensory neurons during early postnatal development, and enhanced oxidative stress and susceptibility to oxidants as well as altered levels of antioxidant enzymes have been observed in brains of Bcl-2-deficient mice [22]. Most of the studies that have contributed to elucidation of the roles of Bcl-2 have been performed using overexpression or deficient null systems. Nevertheless, it is conceivable that the study of animal behaviour when Bcl-2 is down-regulated may contribute further to the understanding of its functions. In many neurological disease models, altered Bcl-2 family member mRNA and protein expression has been observed. These findings provide hope that development of targeted pharmacological agents that enhance anti-apoptotic Bcl-2 family function or inhibit pro-apoptotic Bcl-2 family function will prove useful in the treatment of human neuropathological conditions. To further explore the involvement of mitochondrial function in affective disorders and particularly in anxiety disorders, and the importance of Bcl-2 in this context, the present study was designed to explore the behavioral outcome of deletion of the Bcl-2 gene in mice in behavioral models of psychiatric disorders in mice with targeted mutation of the Bcl-2 gene (heterozygote mice). We found that these mutant mice – which have reduced mitochondrial Bcl-2 levels – do not show gross behavioral abnormalities but demonstrate significant increase in a number of animal models of anxiety-like behavior.

187

2. Methods

188

2.1. Animals

210

2.2. Sequence of experiments

194 195 196 197 198 199 200 201 202 203 204 205 206 207 208

211 212

EC

193

RR

192

CO

191

The sequence of experiments was designed from less to more intrusive in order to minimize the effects of previous experiments

UN

190

2.3. Equipments and procedures

213 214 215 216 217 218

219

2.3.1. Large open ﬁeld A 120 cm × 120 cm transparent Plexiglas platform with 35 cm high transparent walls, elevated 35 cm above the floor served to study locomotor and exploratory behavior. Such large open fields have previously been demonstrated to be much more effective in the study of behavior compared with the more commonly used, smaller activity monitors [11]. As done previously [16] the center square of the open field, measuring 40 X 40 cm was defined as the “Center” area of the field. A video camera was placed 2.3 m above the center the open field and interfaced with a computer and a VCR. Data were collected by the Noldus Ethovision system, a video tracking system designed to study spatial behavior and simultaneously videotaped for further analysis. The Ethovision system follows the center of the body of the mouse and records its position according to the location of this point. Each mouse was placed individually in the center of the open field and its behavior tracked for a 45 min session. At the end of the session mice were returned to their home cages. Locomotor activity measures as well as time and frequency measures for center visits were collected. The open field was wiped clean between trials with a 10% alcohol solution. 2.3.2. Elevated plus-maze A Plexiglas + shaped maze with two dark, enclosed arms and two open and lit arms, without walls, elevated 50 cm above ground served to examine anxiety-like behavior. The size of the arms was 30 cm × 5 cm with a 5 cm × 5 cm center area and the walls of the closed arms were 40 cm high. A video camera tracking system (Ethovision, Noldus VA) was used to track mice activity in the maze as described above. Each mouse was placed in the center of the maze for a 5 min session and then placed back in its home cage. Time and frequency of visits to the different zones of the maze, as well as locomotion measures were collected for later analysis. The plus-maze was wiped clean between trials with a 10% alcohol solution.

TE D

209

Male Bcl-2 heterozygote mice and colony littermates Wild Type controls (Strain Name: 129S1/SvImJ-Bcl2tm1Mpin /J) originally developed by Dr. S.J. Korsmeyer [64] were purchased from Jackson Laboratories (Jackson Laboratories, ME). We chose not to use null mutants because they were reported to have retarded growth, a variety of peripheral disorders and they die at young age [64] and accordingly are not appropriate for behavioral studies whereas clear pathologies were not reported for the heterozygote animals [41,64]. Breeding was not performed in our lab and the source of all mice was Jackson Labs and all details are available at their web site at www.jax.org. Mice were transported to our laboratory and experimentation started no less than one week later, to allow for appropriate acclimatization time. At the beginning of experiments mice weighed 25–30 g and were housed, two per cage in an animal room with constant temperature (22 ± 1 ◦ C) and 12 h light/dark cycle (lights on/off at 6:00 a.m/p.m.), with free access to food and water. All experiments were performed in the early phase of the light cycle under standard room fluorescent lights. All experimental procedures were approved by the Animal Use Committee of the National Institute of Mental Health (Protocol number 1171) and were conducted according to NIH guidelines.

189

on future behaviors [9]. Accordingly, mice were tested in the following sequence: emergence test; open field test; black/white box test; elevated plus-maze test; forced swim test. Mice had at least 3 days separating between consecutive tests. Approximately, a week after the end of all behavioral testing, mice were decapitated, their brains extracted and used for the mitochondria assay.

OO F

161

PR

160

3

2.3.3. Emergence test A 65 cm × 45 cm gray metal table served as the platform for the emergence test designed to examine anxiety-like behaviors. A white (opaque) cup, 6 cm in diameter and 10 cm deep, was attached to the corner of the table with its opening facing the center of the table. At the beginning of the test session each mice was placed inside the cup and its behavior videotaped for 5 min. The latency to exit the cup, number of exits from the cup to the open arena and time spent outside the cup were manually scored from videotapes for later analysis. At the end of the session mice were returned to their home cages and the cup and area wiped clean with a 10% alcohol solution. 2.3.4. Black/white box The black white box test is another model for anxiety-like behavior. A Plexiglas box (50 cm × 25 cm with 30 cm walls) was divided into two compartments: black (one-third of the box) and transparent (two-thirds of the box) with a separating door that can be open to

BBR 4358 1–9

220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239

240 241 242 243 244 245 246 247 248 249 250 251

252 253 254 255 256 257 258 259 260 261 262

263 264 265 266 267

268 269 270 271 272 273 274


allow free transitions between the compartments. Each mouse was placed in the transparent part of the box, with the separation door open, and allowed to freely locomote for a 5 min session. Behavior was tracked (as described above) and measures of time and frequency of visits to the transparent part of the box were collected. At the end of the session mice were returned to their home cages and the area was wiped clean with a 10% alcohol solution.

288

2.3.5. Forced swim test Transparent Plexiglas cylinders, 50 cm high with a diameter of 20 cm, served for the forced swim test. The cylinders were filled with tap water at 22–23 ◦ C to about 25 cm in a way that mice, when placed in the water, were not able to touch the floor or escape. Each mouse was placed individually into the water for a single 6 min session and behavior was videotaped for later analysis. At the end of each session, mice were taken out of the water, dried with a paper towel and re-placed in its home cage. Water was replaced after each mouse. As done before (e.g. [15]), the measures of immobility versus activity during the last 4 minutes of the session (first minutes excluded) were collected for later analysis where immobility was defined as lack of activity except for small movements needed to keep the body afloat.

289

2.4. Biochemistry

281 282 283 284 285 286 287

290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313

314 315 316 317 318 319 320

321 322 323

2.4.1. Isolation of mitochondria Mice were decapitated and their brains quickly extracted and placed in beakers containing chilled (0–4 ◦ C) isolation buffer (0.25 M sucrose containing 10 mM Tris–HCl, pH 7.4, 1 mM EDTA, and 0.25 mg BSA/ml). The tissue (forebrain) was washed and minced in the isolation buffer and 10% (w/v) homogenate was prepared using a glass Dounce homogenizer. Three hundred microliters of the homogenate was put on dry ice for protein assay and Western blot. The nuclei and cell debris were sedimented by centrifugation at 650 × g for 10 min and discarded. The supernatant was subjected to a further centrifugation at 12,000 × g for 8 min. From the sedimented fraction the loosely packed synaptosomal–myelin fraction was discarded after gentle swirling, taking care not to disturb the tightly packed mitochondrial pellet. The mitochondria were washed by gently suspending them in the isolation buffer and resedimenting at 10,000 × g for 10 min. Finally, the mitochondria were suspended in the isolation buffer. It has previously been shown that this procedure yields pure mitochondrial preparations which are practically free from cytosolic contamination. Protein concentration was measured using Bio-Rad Protein Assay Reagent. The forebrain region was chosen because it provides enough tissue to isolate enough high-quality mitochondria fraction. We assume there is an overall reduction in Bcl-2 levels in all brain regions because this transgenic KO is not conditional or region-specific.

2.5. Data analysis Data for all tests were analyzed using a Student s t-test. Significance level was set at p < 0.05. Since some of the behavioral experiments were run in two batches, results for these experiments were first tested for homogeneity of variance across trials. Since variance was not significantly different (Levene’s test) data from the two replications were pooled.

3. Results

325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340

342 343 344 345 346 347

348

3.1. Bcl-2 heterozygous mice have reduced mitochondrial Bcl-2 levels

349 350

Mice heterozygote for the Bcl-2 gene had less than 40% Bcl-2 mitochondrial protein compared with wild type controls (Fig. 1; t(6) = 4.44, p < 0.005). 3.2. Bcl-2 heterozygous mice spend less time in the center of the open ﬁeld Bcl-2 heterozygote mice did not differ from their colony WT controls in measures of locomotor activity across the ses-

2.4.2. Measurement of oxygen consumption Measurement of oxygen consumption was carried out at 28 ◦ C using Clark type oxygen electrodes (Hansatech Instruments, UK). Fifty microliters of fresh homogenate sample was added to the oxygraph chamber which containing 300 ␮l respiration mesium (same as isolation buffer). Oxygen consumption was recorded for 5 min, and the rate was calculated on the graph automatically. 2.4.3. Western blotting 5× loading buffer (0.3 M Tris, pH 6.8, 50% glycerol, 10% sodium dodecyl sulfate, and 12.5% 2-mercaptoethanol,) was added

324

341

PR

280

TE D

279

EC

278

RR

277

CO

276

UN

275

to the sample aliquot, and heated at 95 ◦ C for 5 min. One micrograms of sample was loaded onto 4–20% SDS-polyacrylamide gels and resolved by standard electrophoresis. Gels were blotted electrophoretically to nitrocellulose membrane in a transfer tank using a constant current of 350 mA for 3 h. The membrane blots were blocked for 1 h at room temperature in 5% dry milk dissolved in Tris-buffered saline (TBS-T; 50 mM Tris pH 7.5, 150 mM NaCl, 0.1% Tween 20). Then blots were incubated with an anti-Bcl-2 (Santa Cruz Biotechnology, Inc., diluted 1:500) antibody in blocking buffer. The blots were washed four times with TBS-T for 10 min each wash and then incubated with an HRP-conjugated anti-rabbit secondary antibody (Amersham Biosciences, diluted 1:3000). After four more washes, the blots were developed with Enhanced Chemiluminescence (Amersham Biosciences, Arlington Heights, IL) and visualized with Kodak Biomax MR imaging film. Densitometric analysis of immunoreactivity was conducted with a Kodak Image Station 440CF.

OO F

4

Fig. 1. Bcl-2 mitochondrial levels: Heterozygote mice had a significantly reduced Bcl-2 mitochondrial protein level than WT controls. Measures reflect densitometric analysis of immunoreactivity. N = 4/group, (*) denotes p < 0.05, N = 10/group.

BBR 4358 1–9

351 352 353

354 355

356 357


5

Table 1 Behaviors not affected by mutation Test

Behavior

Results (mean ± S.E.)

Black/white box

Frequency of visits to white

Forced swim test

Immobility time (s)

WT: 24.7 ± 2.2 Het: 21.0 ± 2.2 WT: 141 ± 14.9 Het: 137 ± 19.1

Elevated plus-maze

Entries to closed arms

WT: 20.1 ± 1.3 Het: 20.2 ± 1.7 WT: 172.6 ± 12.0 Het: 190.4 ± 14.3 WT: 15.0 ± 0.7 Het: 13.5 ± 0.7

Distance (m)

364 365 366 367

Bcl-2 heterozygote mice spent less time outside the “sheltered” area compared with the WT controls (Fig. 3; t(18) = 2.48, p < 0.03). No differences between the groups were observed in the latency for the first exit out of the shel-

t(18)=1.6, p = 0.13

ter or the distance traveled outside of the shelter (Table 1). The Emergence test data, therefore, supports an anxiety-like behavior in the mutant mice. 3.4. Bcl-2 heterozygous mice spend less time in the transparent part of the box in the black-white box As in the previous tests, Bcl-2 heterozygote mice demonstrated anxiety-like behavior spending less time in the transparent part of the box compared with the WT controls (Fig. 4; t(10) = 2.56, p < 0.03) but there was no effect on the number of transitions between the parts of the box (Table 1). Inter-

PR

363

3.3. Bcl-2 heterozygous mice spend less time in the sheltered area in the emergence test

t(18)=0.96, p = 0.35

TE D

362

t(18)=0.05, p = 0.96

Fig. 2. Behavior in the open field: heterozygote mice spent significantly less time in the center of a large novel open field (t(18) = 2.26, p < 0.04; A), without changes in total activity levels (t(18) = 0.7, p = 0.49; B), behavior that is representative of increased anxiety levels. (*) denotes p < 0.05, N = 10/group.

EC

361

t(14)=0.8, p = 0.87

RR

360

sion (Table 1), however, the mutant mice spent significantly less time in the center of the open field (Fig. 2, t(18) = 2.26, p < 0.04), a measure reflecting increased anxiety-like behavior.

10 10 10 10 10 10

t(10)=1.2, p = 0.25

CO

359

Fig. 3. Behavior in the emergence test: the time spent outside of an enclosed shelter, and on an unfamiliar open arena was significantly shorter for heterozygote Bcl-2 mice compared with their wild type counterparts (A t(18) = 2.48, p < 0.03) without difference in the amount of locomotion; (B t(18) = 0.03, p = 0.97). (*) denotes p < 0.05, N = 10/group.

UN

358

6 6 8 8

Statistics

OO F

Time in closed arms (s)

N

BBR 4358 1–9

368 369 370

371 372

373 374 375 376 377

6


the initial open-field results indicating no effects of Bcl-2 mutation on activity levels. 3.6. Bcl-2 heterozygous mice show no differences in measures of immobility time in the forced swim test Bcl-2 heterozygote mice did not differ from WT’s in measures of immobility time in the forced swim test (Table 1) indicating no apparent depression-like behavior.

4. Discussion

384 385 386

387 388

389 390 391 392 393 394 395 396 397

3.5. Bcl-2 heterozygous mice have less entries into the open arms of the elevated plus maze

The results of the present study clearly demonstrate an increase in anxiety-like behaviors in mice with reduced mitochondrial Bcl-2 levels. Whereas, these mice did not show any changes in gross neurological and motor function and did not differ from the WT controls in behaviors that model mania (locomotion levels) or depression (the forced swim test), they demonstrated anxiety-like behaviors in four separate models: the elevated plus-maze, the black/white box, emergence test and time spent in the center of a large open field. Together, these results support the possibility that mitochondrial function, modulated by Bcl-2, may be related to the regulation of anxiety behaviors and may be critical in the etiology of anxiety disorders. A general problem with the study of mutant mice is that it is not always simple to relate a behavioral deficit to a specific mechanism rather than more generalized abnormalities in physiology and behavior [9]. Accordingly, we have chosen to test the heterozygote Bcl-2 mice and not the null mutants. The null mutants were repeatedly reported to have major physiological abnormalities [46,59,64] that would have made any specific mechanistic interpretation of the behavior impossible. However, unlike the null mutants, the heterozygote mice were reported to have normal organs development [46,50,59], they were not significantly different than WT in neurological measurements [20] and displayed regular rhythm of body temperature [61]. The anxiety-like models measures presented here are all of the exploratory nature, related to approach/avoidance conflict and accordingly can be significantly influenced by any sensory or motor impairment. Yet, the behavior of the mutant mice in locomotor tasks as well as the forced swim test were not different than the WT mice suggesting that the increased anxiety-like behaviors are not the consequences of such general impairments. Previous studies demonstrated that transgenic mice, overexpressing Bcl-2, have learning deficits [54,55]. It may be interesting to test the effects of Bcl-2 gene deletion on learning but since the anxiety models used in the present study do not include a significant learning component the lack of current knowledge about possible subtle changes in learning should not confound the conclusions regarding anxiety-like behaviors. Accordingly, whereas we cannot completely negate the possibility that the anxiety-like changes reported here are the consequences of some gross changes in the animals’

PR

383

Results of the elevated plus-maze test re-confirm the previous results as heterozygote Bcl-2 mice entered the open arms significantly less times than WT controls (Fig. 5; t(18) = 2.27, p < 0.04) and had similar trends in all other plus-maze related measures of anxiety: time in open arms (t(18) = 1.9, p = 0.07) and entries (t(18) = 2.0, p = 0.067) ratio. Mutant mice did not differ from controls in the total distance traveled in the plusmaze during the test session or in the number of entries and time spent in the closed arms (Table 1), thereby supporting

TE D

382

EC

381

RR

380

CO

379

estingly, the WT mice treated the different compartments of the box nearly at random (the light compartment was 2/3 of the entire are so random division of time would be 200 s in the light and 100 in the dark whereas the WT animals spent a mean of 183 s in the light part). Since no freezing was observed it may be possible that considering the short time of the test the exploratory drive of these mice was still strong enough to partially overcome the aversion from the lighted area.

Fig. 5. Elevated plus-maze: number of entries into the open (threatening) arms of the elevated plus maze was significantly lower in the heterozygote Bcl-2 mice compared with wild type controls. (*) denotes p < 0.05, N = 10/group.

UN

378

399

400 401

402 403 404

405

OO F

Fig. 4. Black/white box: time spent in the transparent part of the black/white box was significantly shorter for the heterozygote Bcl-2 mice compared with wild type controls. (*) denotes p < 0.05, N = 10/group.

398

BBR 4358 1–9

406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448


456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504

OO F

455

PR

454

susceptible to insults compared to neurons from WT animals [18,60] offering additional support to the possible involvement of the gene in neurodegenerative (including psychiatric) disorders although no data is available at this time regarding neurodegeneration in the brain of the heterozygote mice used in the present experiments. Furthermore, in the context of anxiety it was recently reported that Bcl-2 transgenic mice with overexpression of Bcl-2 demonstrate less fear-like behavior and neophobia in the elevated plus-maze and the emergence test models than their WT controls [54]. Our present study further demonstrates that Bcl-2 may be a factor in anxiety disorders and that its effects in that context originate from its role in the mitochondria. It is still possible that the effects of Bcl-2 manipulations on anxiety levels may be related to the activity of the molecule outside the mitochondria as Bcl-2 is also expressed in other intracellular areas, however, the crucial role of Bcl-2 in the mitochondria (e.g. [12,30]) and the increasing support for the relationship between mitochondrial function and affective disorders (e.g. [17,28]) substantiate the possibility that the currently demonstrated increased anxiety in mice with reduced Bcl-2 levels (as probably the decreased anxiety in mice overexpressing Bcl-2 [54]) are related to changes in the mitochondria. Attention to potential drugs that can regulate the activity of Bcl-2 is already given in relation to other disorders. For example, the neuroprotective activity of propargylamines based drugs developed in the context of dementia-related disorders has been attributed to the ability of propargylaminesinducing the antiapoptotic family proteins Bcl-2 and Bclxl, while decreasing Bad and Bax and preventing opening of mitochondrial permeability transition pore [68]. Yet, the future development of treatments that more directly target molecules involved in critical CNS cell survival may be a promising avenue for the development of improved long-term treatments for anxiety and affective disorders [36].

TE D

453

EC

452

RR

451

well being, the previous reports as well as the specificity of changes reported here do suggest that the increased anxietylike behavior is indeed directly related to the genetic mutation. Notably, mitochondrial function has been recently associated with the pathophysiology and treatment of affective and anxiety disorders. Thus, decreases in mitochondrial production rate (MAPR) and enzyme ratios were found in patients with major depression compared with healthy controls with a correlation between MAPR and anxiety (Karolinska Scale of Personality) [17] and polymorphisms in genes coding for mitochondrial DNA were found to be a risk factor in bipolar disorder [28]. Furthermore, MAO-I’s, known for their anxiolytic (as well as antidepressant) properties, improve mitochondrial function [62]; activation of mitochondrial benzodiazepine receptors reduced stress and anxiety in rats [4,51] and neurosteroids that had been extensively documented as having anxiolytic properties [2,52] have specific binding sites on mitochondria and were demonstrated to modulate mitochondrial Ca2+ efflux [24] and increase mitochondrial resilience [27,44]. Finally, it is noteworthy that very recent studies have shown robust anxiolyitc effects of a novel mitochondrial benzodiazepine receptor (MBR) ligand, in various animal models [31]. The bcl-2 gene produces two transcripts as a result of alternative splicing [63]. A 6.0-kb transcript, bcl-2a, contains sequence that encodes the hydrophobic, COOH-terminal signal anchor sequence, while the failure to splice intron 2 results in a 2.4-kb transcript, bcl-2b, that lacks a part of the BH2 domain and the hydrophobic tail. 25 kDa; 205 amino acids (BCL2b) or 239 amino acids (BCL2a) Bcl-2 protein (Bcl2b, encoded by the bcl-2b transcript has not been detected in any tissue. Overexpression of Bcl-2b predominantly resides in the cytoplasm and lacks survival activity, or may exert partial survival activity [23], while Bcl-2a is membrane bound and capable of conferring cell survival [5]. Bcl-2 expression varies and can be regulated at the transcriptional level [57,69], although it is not markedly upregulated by activation events. It has been proposed that transcription may be negatively regulated, at least in part, by the tumor suppressor p53 [42,43]. The half-life of bcl-2 mRNA is approximately 2.5 h [57], whereas the Bcl-2 protein demonstrates considerable stability, with a half-life of 10 h or better [40,53]. Bcl-2 is a key modulator of mitochondrial function. Bcl-2 overexpression was demonstrated to increase the resistance of mitochondria to the destructive influence of elevated intracellular Ca2+ and enhance the maximal uptake capacity of mitochondria [45]. Bcl-2 had been implicated in a number of psychiatric disorders. For example, the Bax/Bcl-2 ration was found to be higher in the temporal lobe of schizophrenic patients [26], repeated stress was shown to decrease brain expression of Bcl-2 in rat hippocampus [33] and a variety of psychiatric drugs including atypical antipsychotics [3], mood stabilizers [6,37] and antidepressants [67] were found to increase Bcl-2 expression and levels. Tissue culture studies showed that neurons from Bcl-2 deficient mice are more

CO

450

UN

449

7

Acknowledgments

505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539

540

This study was supported by an intramural grant from the National Institute of Mental Health, by a Stanley Medical research Institute Grant to HKM and by a National Alliance for Research on Schizophrenia and Depression 2005 Young Investigator Award to HE.

References

541 542 543 544 545

546

[1] Adipudi V, Reddy VK. Effect of chronic lithium chloride on membrane adenosine triphosphatases in certain postural muscles of rats. Eur J Pharmacol 1994;259(1):7–13. [2] Aikey JL, Nyby JG, Anmuth DM, James PJ. Testosterone rapidly reduces anxiety in male house mice (Mus musculus). Horm Behav 2002;42(4):448–60. [3] Bai O, Zhang H, Li XM. Antipsychotic drugs clozapine and olanzapine upregulate bcl-2 mRNA and protein in rat frontal cortex and hippocampus. Brain Res 2004;1010(1–2):81–6.

BBR 4358 1–9

547 548 549 550 551 552 553 554 555

562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622

[25] Imahashi K, Schneider MD, Steenbergen C, Murphy E. Transgenic expression of Bcl-2 modulates energy metabolism, prevents cytosolic acidification during ischemia, and reduces ischemia/reperfusion injury. Circ Res 2004;95(7):734–41. Epub 2004 Sep 2. [26] Jarskog LF, Selinger ES, Lieberman JA, Gilmore JH. Apoptotic proteins in the temporal cortex in schizophrenia: high Bax/Bcl-2 ratio without caspase-3 activation. Am J Psychiat 2004;161(1):109–15. [27] Kaasik A, Safiulina D, Kalda A, Zharkovsky A. Dehydroepiandrosterone with other neurosteroids preserve neuronal mitochondria from calcium overload. J Steroid Biochem Mol Biol 2003;87(1):97–103. [28] Kato T, Kato N. Mitochondrial dysfunction in bipolar disorder. Bipolar Disord 2000;2(3 Pt 1):180–90. [29] Kaufman J, Charney D. Comorbidity of mood and anxiety disorders. Depress Anxiety 2000;12(Suppl. 1):69–76. [30] Kirkin V, Joos S, Zornig M. The role of Bcl-2 family members in tumorigenesis. Biochim Biophys Acta 2004;16–44(23):229–49. [31] Kita A, Kohayakawa H, Kinoshita T, Ochi Y, Nakamichi K, Kurumiya S, et al. Antianxiety and antidepressant-like effects of AC-5216, a novel mitochondrial benzodiazepine receptor ligand. Br J Pharmacol 2004;142(7):1059–72. Epub 2004 Jul 12. [32] Kramer BC, Mytilineou C. Alterations in the cellular distribution of bcl-2, bcl-x and bax in the adult rat substantia nigra following striatal 6-hydroxydopamine lesions. J Neurocytol 2004;33(2):213–23. [33] Luo C, Xu H, Li XM. Post-stress changes in BDNF and Bcl-2 immunoreactivities in hippocampal neurons: effect of chronic administration of olanzapine. Brain Res 2004;1025(1–2):194–202. [34] Manfredi G, Kwong JQ, Oca-Cossio JA, Woischnik M, Gajewski CD, Martushova K, et al. BCL-2 improves oxidative phosphorylation and modulates adenine nucleotide translocation in mitochondria of cells harboring mutant mtDNA. J Biol Chem 2003;278(8):5639–45. Epub 2002 Nov 12. [35] Manji HK, Drevets WC, Charney DS. The cellular neurobiology of depression. Nat Med 2001;7(5):541–7. [36] Manji HK, Duman RS. Impairments of neuroplasticity and cellular resilience in severe mood disorders: implications for the development of novel therapeutics. Psychopharmacol Bull 2001;35(2):5–49. [37] Manji HK, Moore GJ, Chen G. Bipolar disorder: leads from the molecular and cellular mechanisms of action of mood stabilizers. Br J Psychiat Suppl 2001;41:s107–19. [38] Marcocci L, Marchi U, Salvi M, Milella ZG, Nocera S, Agostinelli E, et al. Tyramine and monoamine oxidase inhibitors as modulators of the mitochondrial membrane permeability transition. J Membr Biol 2002;188(1):23–31. [39] Mattson MP, Liu D. Mitochondrial potassium channels and uncoupling proteins in synaptic plasticity and neuronal cell death. Biochem Biophys Res Commun 2003;304(3):539–49. [40] Merino R, Ding L, Veis DJ, Korsmeyer SJ, Nunez G. Developmental regulation of the Bcl-2 protein and susceptibility to cell death in B lymphocytes. Embo J 1994;13(3):683–91. [41] Michaelidis TM, Sendtner M, Cooper JD, Airaksinen MS, Holtmann B, Meyer M, et al. Inactivation of bcl-2 results in progressive degeneration of motoneurons, sympathetic and sensory neurons during early postnatal development. Neuron 1996;17(1):75–89. [42] Miyashita T, Harigai M, Hanada M, Reed JC. Identification of a p53-dependent negative response element in the bcl-2 gene. Cancer Res 1994;54(12):3131–5. [43] Miyashita T, Krajewski S, Krajewska M, Wang HG, Lin HK, Liebermann DA, et al. Tumor suppressor p53 is a regulator of bcl-2 and bax gene expression in vitro and in vivo. Oncogene 1994;9(6):1799– 805. [44] Morin C, Zini R, Simon N, Tillement JP. Dehydroepiandrosterone and alpha-estradiol limit the functional alterations of rat brain mitochondria submitted to different experimental stresses. Neuroscience 2002;115(2):415–24. [45] Murphy AN, Bredesen DE, Cortopassi G, Wang E, Fiskum G. Bcl2 potentiates the maximal calcium uptake capacity of neural cell mitochondria. Proc Natl Acad Sci USA 1996;93(18):9893–8.

PR

561

TE D

560

EC

559

RR

558

[4] Bitran D, Foley M, Audette D, Leslie N, Frye CA. Activation of peripheral mitochondrial benzodiazepine receptors in the hippocampus stimulates allopregnanolone synthesis and produces anxiolytic-like effects in the rat. Psychopharmacology (Berl) 2000;151(1):64–71. [5] Borner C, Martinou I, Mattmann C, Irmler M, Schaerer E, Martinou JC, et al. The protein bcl-2 alpha does not require membrane attachment, but two conserved domains to suppress apoptosis. J Cell Biol 1994;126(4):1059–68. [6] Brunello N. Mood stabilizers: protecting the mood.protecting the brain. J Affect Disord 2004;79(Suppl. 1):15–20. [7] Cohen G, Kesler N. Monoamine oxidase inhibits mitochondrial respiration. Ann NY Acad Sci 1999;893:273–8. [8] Coyle JT, Duman RS. Finding the intracellular signaling pathways affected by mood disorder treatments. Neuron 2003;38(2):157–60. [9] Crawley JN. What’s wrong with my mouse? behavioral phenotyping of transgenic and knockout Mice. 1st ed. New York: Wiley-Liss; 2000, 376. [10] De Marchi U, Pietrangeli P, Marcocci L, Mondovi B, Toninello A. l-Deprenyl as an inhibitor of menadione-induced permeability transition in liver mitochondria. Biochem Pharmacol 2003;66(9):1749– 54. [11] Decker S, Grider G, Cobb M, Li XP, Huff MO, El-Mallakh RS, et al. Open field is more sensitive than automated activity monitor in documenting ouabain-induced hyperlocomotion in the development of an animal model for bipolar illness. Prog Neuropsychopharmacol Biol Psychiat 2000;24(3):455–62. [12] Donovan M, Cotter TG. Control of mitochondrial integrity by Bcl-2 family members and caspase-independent cell death. Biochim Biophys Acta 2004;1644(2–3):133–47. [13] Drevets WC. Neuroimaging and neuropathological studies of depression: implications for the cognitive-emotional features of mood disorders. Curr Opin Neurobiol 2001;11(2):240–9. [14] Duman RS. Pathophysiology of depression: the concept of synaptic plasticity. Eur Psychiat 2002;17(Suppl. 3):306–10. [15] Einat H, Clenet F, Shaldubina A, Belmaker RH, Bourin M. The antidepressant activity of inositol in the forced swim test involves 5-HT(2) receptors, Behav. Brain Res 2001;118(1):77–83. [16] Einat H, Yuan P, Gould TD, Li J, Du J, Zhang L, et al. The role of the extracellular signal-regulated kinase signaling pathway in mood modulation. J Neurosci 2003;23(19):7311–6. [17] Gardner A, Johansson A, Wibom R, Nennesmo I, von Dobeln U, Hagenfeldt L, et al. Alterations of mitochondrial function and correlations with personality traits in selected major depressive disorder patients. J Affect Disord 2003;76(1–3):55–68. [18] Greenlund LJ, Korsmeyer SJ, Johnson Jr EM. Role of BCL-2 in the survival and function of developing and mature sympathetic neurons. Neuron 1995;15(3):649–61. [19] Hara A, Hirose Y, Wang A, Yoshimi N, Tanaka T, Mori H. Localization of Bax and Bcl-2 proteins, regulators of programmed cell death, in the human central nervous system. Virchows Arch 1996;429(4–5):249–53. [20] Hata R, Gillardon F, Michaelidis TM, Hossmann KA. Targeted disruption of the bcl-2 gene in mice exacerbates focal ischemic brain injury. Metab Brain Dis 1999;14(2):117–24. [21] Hauptmann N, Grimsby J, Shih JC, Cadenas E. The metabolism of tyramine by monoamine oxidase A/B causes oxidative damage to mitochondrial DNA. Arch Biochem Biophys 1996;335(2):295–304. [22] Hochman A, Sternin H, Gorodin S, Korsmeyer S, Ziv I, Melamed E, et al. Enhanced oxidative stress and altered antioxidants in brains of Bcl-2-deficient mice. J Neurochem 1998;71(2):741–8. [23] Hockenbery DM, Oltvai ZN, Yin XM, Milliman CL, Korsmeyer SJ. Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell 1993;75(2):241–51. [24] Horvat A, Nikezic G, Petrovic S, Kanazir DT. Binding of estradiol to synaptosomal mitochondria: physiological significance. Cell Mol Life Sci 2001;58(4):636–44.

CO

557

UN

556


OO F

8

BBR 4358 1–9

623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689


697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731

OO F

696

PR

695

TE D

694

EC

693

[59] Sorenson CM, Rogers SA, Korsmeyer SJ, Hammerman MR. Fulminant metanephric apoptosis and abnormal kidney development in bcl-2-deficient mice. Am J Physiol 1995;268(1 Pt 2):F73–81. [60] Tanabe H, Eguchi Y, Kamada S, Martinou JC, Tsujimoto Y. Susceptibility of cerebellar granule neurons derived from Bcl-2-deficient and transgenic mice to cell death. Eur J Neurosci 1997;9(4):848– 56. [61] Tesfaigzi Y, Rudolph K, Fischer MJ, Conn CA. Bcl-2 mediates sexspecific differences in recovery of mice from LPS-induced signs of sickness independent of IL-6. J Appl Physiol 2001;91(5):2182–9. [62] Thiffault C, Quirion R, Poirier J. The effect of l-deprenyl, d-deprenyl and MDL72974 on mitochondrial respiration: a possible mechanism leading to an adaptive increase in superoxide dismutase activity. Brain Res Mol Brain Res 1997;49(1–2):127–36. [63] Tsujimoto Y, Croce CM. Analysis of the structure, transcripts, and protein products of bcl-2, the gene involved in human follicular lymphoma. Proc Natl Acad Sci USA 1986;83(14):5214–8. [64] Veis DJ, Sorenson CM, Shutter JR, Korsmeyer SJ. Bcl-2-deficient mice demonstrate fulminant lymphoid apoptosis, polycystic kidneys, and hypopigmented hair. Cell 1993;75(2):229–40. [65] Weisleder N, Taffet GE, Capetanaki Y. Bcl-2 overexpression corrects mitochondrial defects and ameliorates inherited desmin null cardiomyopathy. Proc Natl Acad Sci USA 2004;101(3):769–74. Epub 2004 Jan 8. [66] Williams JM, Thompson VL, Mason-Parker SE, Abraham WC, Tate WP. Synaptic activity-dependent modulation of mitochondrial gene expression in the rat hippocampus. Brain Res Mol Brain Res 1998;60(1):50–6. [67] Xu H, Steven Richardson J, Li XM. Dose-related effects of chronic antidepressants on neuroprotective proteins BDNF, Bcl-2 and Cu/Zn-SOD in rat hippocampus. Neuropsychopharmacology 2003;28(1):53–62. [68] Youdim MB, Fridkin M, Zheng H. Bifunctional drug derivatives of MAO-B inhibitor rasagiline and iron chelator VK-28 as a more effective approach to treatment of brain ageing and ageing neurodegenerative diseases. Mech Ageing Dev 2005;126(2):317–26. [69] Young RL, Korsmeyer SJ. A negative regulatory element in the bcl-2 5 -untranslated region inhibits expression from an upstream promoter. Mol Cell Biol 1993;13(6):3686–97. [70] Yuan PX, Zhou R, Farzad N, Gould TD, Gray NA, Du J et al. Enhancing resilience to stress: the role of signaling cascades. In: Steckler N, Kalin JM, Reul HM, editors. Handbook on stress, immunology and behaviour, in press.

RR

692

[46] Nagata M, Nakauchi H, Nakayama K, Nakayama K, Loh D, Watanabe T. Apoptosis during an early stage of nephrogenesis induces renal hypoplasia in bcl-2-deficient mice. Am J Pathol 1996;148(5):1601–11. [47] Nestler EJ, Barrot M, DiLeone RJ, Eisch AJ, Gold SJ, Monteggia LM. Neurobiology of depression. Neuron 2002;34(1):13–25. [48] Nicklas WJ, Youngster SK, Kindt MV, Heikkila RE. MPTP, MPP+ and mitochondrial function. Life Sci 1987;40(8):721–9. [49] Pivovarova NB, Pozzo-Miller LD, Hongpaisan J, Andrews SB. Correlated calcium uptake and release by mitochondria and endoplasmic reticulum of CA3 hippocampal dendrites after afferent synaptic stimulation. J Neurosci 2002;22(24):10653–61. [50] Ratts VS, Flaws JA, Kolp R, Sorenson CM, Tilly JL. Ablation of bcl-2 gene expression decreases the numbers of oocytes and primordial follicles established in the post-natal female mouse gonad. Endocrinology 1995;136(8):3665–8. [51] Reddy DS, Kulkarni SK. Role of GABA-A and mitochondrial diazepam binding inhibitor receptors in the anti-stress activity of neurosteroids in mice. Psychopharmacology (Berl) 1996;128(3):280–92. [52] Reddy DS, Kulkarni SK. Differential anxiolytic effects of neurosteroids in the mirrored chamber behavior test in mice. Brain Res 1997;752(1–2):61–71. [53] Reed JC, Tsujimoto Y, Epstein SF, Cuddy M, Slabiak T, Nowell PC, et al. Regulation of bcl-2 gene expression in lymphoid cell lines containing normal #18 or t(14;18) chromosomes. Oncogene Res 1989;4(4):271–82. [54] Rondi-Reig L, Lemaigre Dubreuil Y, Martinou JC, DelhayeBouchaud N, Caston J, Mariani J. Fear decrease in transgenic mice overexpressing bcl-2 in neurons. Neuroreport 1997;8(11):2429–32. [55] Rondi-Reig L, Mariani J. To die or not to die, does it change the function? Behavior of transgenic mice reveals a role for developmental cell death. Brain Res Bull 2002;57(1):85–91. [56] Schweitzer N. Pegging pathology on mitochondrial dysfunction. Scientist 2004;18(10):28. [57] Seto M, Jaeger U, Hockett RD, Graninger W, Bennett S, Goldman P, et al. Alternative promoters and exons, somatic mutation and deregulation of the Bcl-2-Ig fusion gene in lymphoma. Embo J 1988;7(1):123–31. [58] Sharma SK, Carlson EC, Ebadi M. Neuroprotective actions of Selegiline in inhibiting 1-methyl, 4-phenyl, pyridinium ion (MPP+)induced apoptosis in SK-N-SH neurons Peroxynitrite and mitochondrial dysfunction in the pathogenesis of Parkinson’s disease. J Neurocytol 2003;32(4):329–43.

CO

691

UN

690

9

BBR 4358 1–9

732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774