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, effects 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 the mitochondrial program for apoptosis. Mitochondrial dysfunction has been recently reported in Alzheimers' and Parkinson's diseases and it is not clear if similar mitochondrial abnormalities occur in bipolar disorders.The purpose of this ongoing study is to investigate the effects of mood stabilizers on various aspects of mitochondrial function. Rat primary cortical neurons and human neuroblastoma SH-SY5Y cells were treated chronically with valproate and lithium at therapeutically relevant concentrations. Western Blot analysis was used to determine mitochondrial protein levels. The Oxygraph system was used to measure the respiratory activity. 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 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 overexpressing 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 dtsabiliziers. SiRNAs are small double-strand RNAs, 21-22 nucleotides in length. They trigger the degradation of cognate RNA and induce the shut-down of a specific gene expression in mammalian cells. 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. The knockdown of Bcl-2 gene in SH-SY5Y cells was accompanied by clear reductions in mitochondrial oxidation. These results demonstrate the utility of the siRNA strategy to regulate the expression of critical proteins in a graded manner. 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.
