Mood disorders have traditionally been considered to be neurochemical disorders, but new evidence demonstrates impairments of structural plasticity and cellular resilience in the central nervous system. Recent preclinical and clinical studies have shown that signaling pathways involved in regulating cell survival and death are long-term targets for the actions of mood stabilizers. Lithium and valproate are common mood stabilizers for bipolar disorder and are shown to indirectly regulate cell pathways, including CREB, BDNF, Bcl-2, and MAP kinases, which may stimulate some of their delayed long-term beneficial effects. Specifically, lithium and valproate upregulate Bcl-2 in vivo and in vitro, preventing neuronal cell death through blocking mitochondrial program for apoptosis. In this study, rat primary cortical neurons and human neuroblastoma SH-SY5Y cells were treated chronically with valproate and lithium at therapeutically relevant concentrations. Mitochondrial protein levels were determined with western blot analysis; respiratory activity was measured using the Oxygraph system. Western blot analysis showed upregulation of porin (a voltage dependent anion selective channel) and anti-apoptotic proteins Bcl-2 and Bcl-x/L in the mitochondrial fraction of cells treated with lithium and valproate. Oxygraph measurements demonstrated that lithium and valproate increased respiratory rate both time- and dose-dependently. It is postulated that Bcl-xL and Bcl-2 may act to influence the properties of other outer membrane proteins to maintain their ability to pass complex anions. Recently it was reported that Bcl-xL can interact with porin and regulate its gating properties in vitro. It has also been shown that Bcl-2 over-expressing cells result in an increased mitochondrial volume and structural complexity. ? Since Bcl-2 is a key regulator of cell survival, we have used SiRNAs (small interfering RNAs) to knock down bcl-2 levels and determine the consequences of this manipulation on the biochemical effects of mood stabiliziers. In this study, we investigated the role of Bcl-2 as a regulator of mitochondrial function, through the use of siRNA knockdown techniques. Bcl-2 siRNA/pSilencer significantly knocked down Bcl-2 gene expression, resulting in 40% reductions in Bcl-2 protein levels in human neuroblastoma SH-SY5Y cells. 3 different dyes were used to study the effects of lithium and valproate (VPA) on quantity of mitochondria, mitochondrial membrane potential, mitochondrial oxidation and generation of free radicals (ROS) in neuronal cells. Mitotracker green becomes fluorescent in the lipid environment of mitochondria regardless of the membrane potential; JC-1 accumulates in proportion to the mitochondrial membrane potential; Mitotracker red oxidizes to a fluorescent product once inside the mitochondria and 2',7'-dichlorofluorescin diacetate (H2DCFDA) for assessing oxidative stress by staining ROS. Confocal microscope study showed that chronic lithium treatment increased mitochondrial membrane potential of the SH-SY5Y cells. The knockdown of Bcl-2 gene in SH-SY5Y cells was accompanied by clear reductions in mitochondrial oxidation and significantly reduced Bcl-2-protein levels. Previous studies from our lab showed that lithium and VPA enhanced Bcl-2 protein expression in vivo and in vitro. There is some evidence that mood stabilizers might stabilize mitochondrial function by enhancing their bioenergetic capacity and avoiding apoptosis through Bcl-2 dependent mechanisms. In order to elucidate how mood-stabilizers exert its neuroprotective effects, we investigated mitochondrial function after lithium and valproate treatment in vitro. Both, lithium- and VPA- treatments enhanced membrane potential and mitochondrial oxidation in SH-SY5Y human neuroblastoma cells in a time dependent manner. In addition, Bcl-2 siRNA significantly knocked down Bcl-2 gene expression and caused clear reductions in mitochondrial membrane potential and mitochondrial oxidation in lithium- or valproate- treated SH-SY5Y neuroblastoma cells. These findings indicate that mood stabilizers regulated mitochondrial function partially through the enhanced Bcl-2-gene expression in mitochondria. Bcl-2 mediated increase of mitochondrial function after lithium- or valproate- treatment may have utility in the long-term treatment of a variety of disorders where mitochondrial dysfunction contributes to the diseases pathophysiology/progression, it might be a novel therapeutics for the treatment of mitochondrial disorders. ? To further investigate the chronic effects of mood-stabilizers on mitochondrial function in vivo, adult Wistar rats were treated with lithium or VPA chow for 4 weeks, then challenged with neurotoxin methamphetamine (5mg/kg, i.p, 4 times in 8h). We found that chronic lithium and VPA treatment attenuated methamphetamine-induced decrease of Bcl-2 and increase of Bax in rat frontal cortex mitochondrial fraction. VPA treatment preserved rat frontal cortex mitochondrial electron transport complex IV-- cytochrome C oxidase activity, while lithium pretreatment significantly prevented methamphetamine-induced hyperthermia and high-dose toxic mortality. These novel results suggest that lithium and valproate may exert some of their long-term effects on neuroplasticity and cellular resilience via hitherto underappreciated effects on mitochondrial proteins and mitochondrial function.? In addition, calcium dysfunction in BPD is considered the most reproducible biological abnormality described in BPD, and recent data support a role for calcium dysregulation in the mitochondrial and endoplasmic reticulum (ER) of individuals with BPD. Mitochondria and the ER critically regulate intracellular calcium signaling through several different mechanisms. Furthermore, the anti-apoptotic protein Bcl-2 has been shown to play an important role in regulating calcium kinetics within the ER and mitochondria. This role of Bcl-2 in intracellular calcium kinetics contributes to the modulation of many cellular functions, including gene expression, synaptic plasticity and cellular resilience. In addition, recent human genetic data suggest that single nucleotide polymorphism (SNPs) in the Bcl-2 gene may be associated with BPD. We thus sought to evaluate the functional role of Bcl-2 in intracellular calcium regulation in lymphoblasts from eighteen BPD patients with the SNP rs956572 (variants AA, AG, or GG, six subjects per group). We found that the Bcl-2 gene variant AA (associated with increased risk for BPD) decreased Bcl-2 levels, and significantly increased cell apoptosis. Using different calcium ratiometric dyes (Fura-2 and Fura-2FF), we observed increased baseline cytosolic calcium levels Reinforcing the role of Bcl-2 in calcium dynamics, baseline cytosolic calcium levels showed a significant inverse correlation with Bcl-2 levels in the total sample (r=-0.59). Furthermore, electron microscopy showed mitochondrial morphological alterations in some BPD samples. The results demonstrate for the first time that the BPD-associated SNP variations of Bcl-2 play a critical role in cytosolic calcium dynamics. Overall, these findings may provide a better understanding of the genetic association between BPD SNPs and the pathophysiology of this devastating illness.