Expression of GABAA α2-, ß1- and ε-receptors are altered significantly in the lateral cerebellum of subjects with schizophrenia, major depression and bipolar disorder.

There is abundant evidence that dysfunction of the γ-aminobutyric acid (GABA)ergic signaling system is implicated in the pathology of schizophrenia and mood disorders. Less is known about the alterations in protein expression of GABA receptor subunits in brains of subjects with schizophrenia and mood disorders. We have previously demonstrated reduced expression of GABA(B) receptor subunits 1 and 2 (GABBR1 and GABBR2) in the lateral cerebella of subjects with schizophrenia, bipolar disorder and major depressive disorder. In the current study, we have expanded these studies to examine the mRNA and protein expression of 12 GABA(A) subunit proteins (α1, α2, α3, α5, α6, ß1, ß2, ß3, δ, ε, γ2 and γ3) in the lateral cerebella from the same set of subjects with schizophrenia (N=9-15), bipolar disorder (N=10-15) and major depression (N=12-15) versus healthy controls (N=10-15). We found significant group effects for protein levels of the α2-, ß1- and ε-subunits across treatment groups. We also found a significant group effect for mRNA levels of the α1-subunit across treatment groups. New avenues for treatment, such as the use of neurosteroids to promote GABA modulation, could potentially ameliorate GABAergic dysfunction in these disorders.

mRNA and protein expression for novel GABAA receptors θ and ρ2 are altered in schizophrenia and mood disorders; relevance to FMRP-mGluR5 signaling pathway.

Fragile X mental retardation protein (FMRP) is an RNA-binding protein that targets ∼5% of all mRNAs expressed in the brain. Previous work by our laboratory demonstrated significantly lower protein levels for FMRP in lateral cerebella of subjects with schizophrenia, bipolar disorder and major depression when compared with controls. Absence of FMRP expression in animal models of fragile X syndrome (FXS) has been shown to reduce expression of gamma-aminobutyric acid A (GABAA) receptor mRNAs. Previous work by our laboratory has found reduced expression of FMRP, as well as multiple GABAA and GABAB receptor subunits in subjects with autism. Less is known about levels for GABAA subunit protein expression in brains of subjects with schizophrenia and mood disorders. In the current study, we have expanded our previous studies to examine the protein and mRNA expression of two novel GABAA receptors, theta (GABRθ) and rho 2 (GABRρ2) as well as FMRP, and metabotropic glutamate receptor 5 (mGluR5) in lateral cerebella of subjects with schizophrenia, bipolar disorder, major depression and healthy controls, and in superior frontal cortex (Brodmann Area 9 (BA9)) of subjects with schizophrenia, bipolar disorder and healthy controls. We observed multiple statistically significant mRNA and protein changes in levels of GABRθ, GABRρ2, mGluR5 and FMRP molecules including concordant reductions in mRNA and proteins for GABRθ and mGluR5 in lateral cerebella of subjects with schizophrenia; for increased mRNA and protein for GABRρ2 in lateral cerebella of subjects with bipolar disorder; and for reduced mRNA and protein for mGluR5 in BA9 of subjects with bipolar disorder. There were no significant effects of confounds on any of the results.

Disruption of actin-myosin interactions results in the inhibition of focal adhesion assembly in Xenopus XR1 glial cells.

In the present study we have investigated the role of actin-myosin interactions in regulating focal adhesion assembly in Xenopus XR1 glial cells. Actin-myosin interactions, stress fiber formation, and focal adhesion assembly are thought to allow cells to exert tension in the surrounding extracellular matrix, a process essential during morphogenesis and wound healing. Immunocytochemical analysis has revealed that myosin heavy chain-A (MHC-A), the predominant isoform in XR1 cells, was distributed in a filamentous pattern in the central region but was more diffuse towards the cell periphery. Myosin heavy chain-A-like immunoreactivity (IR) partially colocalized with phalloidin stained F-actin microfilaments in XR1 cells but not with microtubules. Furthermore, MHC-A-IR colocalized with immunoreactivity for beta1 integrin receptors and vinculin at focal adhesions located more centrally along the ventral surface of the cells. The partial colocalization of MHC-A with the F-actin cytoskeleton, as well as at focal adhesions, provides evidence that actin-myosin interactions may be involved in regulating focal adhesion assembly and stabilization. To examine this possibility, we have used drugs shown to inhibit cell contractility: the kinase inhibitors H7 and HA100, and 2,3-butanedione 2-monoxime (BDM), which inhibits muscle and nonmuscle ATPase activity. Compared to control cultures, those treated with the inhibitors exhibited a dose-dependent decrease in the percentage of cells that displayed focal adhesions. In addition, these cells also displayed disrupted actin cytoskeletons and a similar disruption in myosin filaments. Taken together, these results provide evidence for an important role of actin-myosin generated forces during focal adhesion assembly in glial cells.

Characterization of focal adhesion assembly in XR1 glial cells.

In the present communication, we have characterized focal adhesions in cultured glial cells derived from the Xenopus retina. Using antibodies directed against focal adhesion proteins we found that beta1 integrin immunoreactivity colocalized with talin, vinculin, and phosphotyrosine immunoreactivities in glial cells from primary cultures of Xenopus retina, as well as in the XR1 glial cell line, an immortal cell line derived from Xenopus retinal neuroepithelium. beta1 integrin immunoreactivity also colocalized with the termini of rhodamine phalloidin-labeled filamentous-actin at focal adhesions. The regulation of focal adhesion assembly was examined in XR1 glial cells using inhibitors against actin polymerization (cytochalasins) or tyrosine kinase activity (genistein). Compared to control cultures, those treated with the inhibitors exhibited a dose-dependent decrease in the proportion of cells displaying focal adhesions. Treatment with cytochalasin B also resulted in a dose-dependent decrease in cell area. Mature focal adhesions in XR1 cells with a flattened, spread morphology also were disrupted by the presence of these inhibitors. These results provide strong evidence that an intact actin cytoskeleton and tyrosine kinase activity regulate focal adhesion assembly and also play important roles in the maintenance of the integrity of focal adhesions in glial cells.