Research reportIncreased 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
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 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 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 antidepressants, 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 Bcl-2 gene exhibited reduced anxiety-like (fear) behavior [54]. Inactivation of bcl-2 results in progressive degeneration of 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.
Section snippets
Animals
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
Bcl-2 heterozygous mice have reduced mitochondrial Bcl-2 levels
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).
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 session (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.
Bcl-2 heterozygous mice spend less time outside the sheltered area in the emergence test
Bcl-2 heterozygote mice spent less time outside the
Discussion
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
Acknowledgments
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.
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