Table 1.
Protein phosphorylation in synaptoneurosomal preps from rat PFC as a result of chronic lithium, valproate, and paliperidone treatment.
Figure 1.
Actin and tubulin were phosphorylated after mood stabilizer treatment.
A. 2D-DIGE gel image stained with Sypro Ruby stain showing phosphorylation of synaptoneurosomal preparations of saline treated animals (controls). B. location of actin identified spots (green spot) and tubulin (red spot) in saline treated preparations. C. Spots identified with actin antibodies during Western blots (circled) of synaptoneurosomal preparations after mood stabilizer treatment. D. Western blot identification of phosphorylated actin (circled). E. 2D-DIGE gel image showing phosphorylation after mood stabilizer treatment and the location of tubulin identified spots as indicated by circle. F. Spots identified with tubulin antibodies (circled).
Figure 2.
Mood stabilizer treatment resulted in increased mitochondrial transport to the synapse in cell culture as indicated by increased staining of neuronal processes and rat PFC tissue.
From left to right: MitoTracker (red), SYN1 (green), and superimposed images (red and green). A. Saline control, B. Lithium (1 mM) treated cells, C. Paliperidone (10 µM) treated cells, D. Saline treated rat PFC tissue slice (left to right: MitoTracker red, DAPI nuclear stain and superimposed image), E. Lithium (22 mg/Kg) treated rat PFC tissue slice (left to right: MitoTracker red, DAPI and superimposed image). F. A close-up of localization of Mito Tracker red (red, left) and SYN1 (green) in paliperidone (0.1 µM) treated cells. Nuclei shown in blue (Secondary antibody: Alexafluor488 Mouse 1∶1000, Mitotracker CFX-Ros 50 nM, Hoecsht nuclear stain 1∶1000).
Figure 3.
Mitochondrial staining with MitoTracker® green (total mitochondria) and MitoTracker red® (active function mitochondria) shows different morphology in cells at a given time indicative of rapid changes in movement and mitochondrial function as exemplified by the saline controls (A, B, C and D).
Anterograde mitochondrial movement (away from the cell nuclei) was observed after treatment with 20 µM lithium (E) and 50 µM Paliperidone (F) as indicated by long filamentous mitochondria. Ballooning and concentration around cell nuclei (blue) was observed after treatment with 50 µM Clozapine (G). Detrimental effects were observed in mitochondria after treatment with 50 µM Haloperidol (H). Superimposed images shown in yellow.
Figure 4.
Simplified scheme to illustrate the effects of lithium, valproate and paliperidone treatment on mitochondrial and cytoskeletal protein phosphorylation with effects on mitochondrial transport towards the synapse.
A. Cytoskeletal proteins (desmoplakin homolog, myosin, actin, tubulin and alpha internexin) are phosphorylated and involved in the mechanism of anterograde mitochondrial transport. Actin fiber formation is promoted by phosphorylation of actin isoforms. Actin binding domains indicated as purple ovals contribute to network formation through binding of desmoplakin homolog to actin. Phosphorylation of tubulin isoforms promotes microtubule formation and binding to desmoplakin homolog is facilitated through tubulin binding domains (green ovals). Tubulin in turn binds alpha internexin. Anterograde mitochondrial movement is possible through Myosin interaction with actin. Inside the mitochondria (insert), UQCRC1 from complex III (red) was phosphorylated after treatment with all three drugs with effects on mitochondrial function. B. Schematic representation of the effects of anterograde mitochondrial movement on synaptic plasticity and neurotransmission. Each step where the drugs have effects is indicated by a yellow square and a letter inside. These events may or may not happen simultaneously. A. Anterograde mitochondrial movement to the pre-synapse is promoted by all three drugs. B. Mitochondrial function near SNARE complexes provides necessary ATP for neurotransmitter release as well as regulation of Ca2+ levels. Myosin heavy chain also plays a role on synaptic vesicle opening. Ca2+ affects calcineurin which in turns affects DRP-1. Phosphorylation of DRP-1 influences mitochondria. C. Serotonin and dopamine release is regulated by 5HT2A and D2 receptors. The ratio of serotonin (5HT) and dopamine (DA) available regulates mitochondrial movement to the post-synapse as serotonin promotes anterograde movement while dopamine inhibits it. D. Mitochondrial function at post-synaptic terminals contributes to Ca2+ homeostasis influencing CAMKII and GSK3β E. Mitochondrial transport to the post-synapse results in increased levels of ATP necessary for proper functioning of post-synaptic machinery. The number of mitochondria is directly and reciprocally related to the number of active synapses (Li et al., 2004).