A Role for GABAA Receptor ß3 Subunits in Mediating Harmaline Tremor Suppression by Alcohol: Implications for Essential Tremor Therapy.

Background: Essential tremor patients may find that low alcohol amounts suppress tremor. A candidate mechanism is modulation of α6ß3δ extra-synaptic GABAA receptors, that in vitro respond to non-intoxicating alcohol levels. We previously found that low-dose alcohol reduces harmaline tremor in wild-type mice, but not in littermates lacking δ or α6 subunits. Here we addressed whether low-dose alcohol requires the ß3 subunit for tremor suppression. Methods: We tested whether low-dose alcohol suppresses tremor in cre-negative mice with intact ß3 exon 3 flanked by loxP, and in littermates in which this region was excised by cre expressed under the α6 subunit promotor. Tremor in the harmaline model was measured as a percentage of motion power in the tremor bandwidth divided by overall motion power. Results: Alcohol, 0.500 and 0.575 g/kg, reduced harmaline tremor compared to vehicle-treated controls in floxed ß3 cre- mice, but had no effect on tremor in floxed ß3 cre+ littermates that have ß3 knocked out. This was not due to potential interference of α6 expression by the insertion of the cre gene into the α6 gene since non-floxed ß3 cre+ and cre- littermates exhibited similar tremor suppression by alcohol. Discussion: As α6ß3δ GABAA receptors are sensitive to low-dose alcohol, and cerebellar granule cells express ß3 and are the predominant brain site for α6 and δ expression together, our overall findings suggest alcohol acts to suppress tremor by modulating α6ß3δ GABAA receptors on these cells. Novel drugs that target this receptor may potentially be effective and well-tolerated for essential tremor. Highlights: We previously found with the harmaline essential tremor model that GABAA receptors containing α6 and δ subunits mediate tremor suppression by alcohol. We now show that ß3 subunits in α6-expressing cells, likely cerebellar granule cells, are also required, indicating that alcohol suppresses tremor by modulating α6ß3δ extra-synaptic GABAA receptors.

Alcohol and Ganaxolone Suppress Tremor via Extra-Synaptic GABAA Receptors in the Harmaline Model of Essential Tremor.

Background: A long-standing question is why essential tremor often responds to non-intoxicating amounts of alcohol. Blood flow imaging and high-density electroencephalography have indicated that alcohol acts on tremor within the cerebellum. As extra-synaptic δ-subunit-containing GABAA receptors are sensitive to low alcohol levels, we wondered whether these receptors mediate alcohol's anti-tremor effect and, moreover, whether the δ-associated GABAA receptor α6 subunit, found abundantly in the cerebellum, is required. Methods: We tested the hypotheses that low-dose alcohol will suppress harmaline-induced tremor in wild-type mice, but not in littermates lacking GABAA receptor δ subunits, nor in littermates lacking α6 subunits. As the neurosteroid ganaxolone also activates extra-synaptic GABAA receptors, we similarly assessed this compound. The harmaline mouse model of essential tremor was utilized to generate tremor, measured as a percentage of motion power in the tremor bandwidth (9-16 Hz) divided by background motion power at 0.25-32 Hz. Results: Ethanol, 0.500 and 0.575 g/kg, and ganaxolone, 7 and 10 mg/kg, doses that do not impair performance in a sensitive psychomotor task, reduced harmaline tremor compared to vehicle-treated controls in wild-type mice but failed to suppress tremor in littermates lacking the δ or the α6 GABAA receptor subunit. Discussion: As cerebellar granule cells are the predominant brain site intensely expressing GABAA receptors containing both α6 and δ subunits, these findings suggest that this is where alcohol acts to suppress tremor. It is anticipated that medications designed specifically to target α6ßδ-containing GABAA receptors may be effective and well-tolerated for treating essential tremor. Highlights: How does alcohol temporarily ameliorate essential tremor? This study with a mouse model found that two specific kinds of GABA receptor subunits were needed for alcohol to work. As receptors with both these subunits are found mainly in cerebellum, this work suggests this is where alcohol acts to suppress tremor.

Suppression of Harmaline Tremor by Activation of an Extrasynaptic GABAA Receptor: Implications for Essential Tremor.

Background: Metabolic imaging has revealed excessive cerebellar activity in essential tremor patients. Golgi cells control cerebellar activity by releasing gamma-aminobutyric acid (GABA) onto synaptic and extrasynaptic receptors on cerebellar granule cells. We postulated that the extrasynaptic GABAA receptor-specific agonist THIP (gaboxadol; 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) would suppress tremor in the harmaline model of essential tremor and, since cerebellar extrasynaptic receptors contain α6 and δ subunits, would fail to do so in mice lacking either subunit. Methods: Digitally measured motion power, expressed as 10-16 Hz power (the tremor bandwidth) divided by background 8-32 Hz motion power, was accessed during pre-harmaline baseline, pre-THIP harmaline exposure, and after THIP administration (0, 2, or 3 mg/kg). These low doses were chosen as they did not impair performance on the straight wire test, a sensitive test for psychomotor impairment. Littermate δ wild-type and knockout (Gabrd+/+, Gabrd-/-) and littermate α6 wild-type and knockout (Gabra6+/+, Gabra6-/- ) mice were tested. Results: Gabrd+/+ mice displayed tremor reduction at 3 mg/kg THIP but not 2 mg/kg, and Gabra6+/+ mice showed tremor reduction at 2 and 3 mg/kg. Their respective subunit knockout littermates displayed no tremor reduction compared with vehicle controls at either dose. Discussion: The loss of anti-tremor efficacy with deletion of either δ or α6 GABAA receptor subunits indicates that extrasynaptic receptors containing both subunits, most likely located on cerebellar granule cells where they are highly expressed, mediate tremor suppression by THIP. A medication designed to activate only these receptors may display a favorable profile for treating essential tremor.

Expression of CCL5 (RANTES) and CCR5 in prostate cancer.

BACKGROUND: Expression of the inflammatory chemokine CCL5 (RANTES) by tumor cells is thought to correlate with the progression of several cancers. CCL5 was shown to induce breast cancer cell migration, mediated by the receptor CCR5. A CCR5 antagonist was demonstrated to inhibit experimental breast tumor growth. Recently, CCL5 and CCR5 mRNA expression was reported in prostate cancer (PCa) tissues. Herein, we characterized CCL5 and CCR5 expression in cultures of PCa cells and explored possible functions of CCL5 in PCa progression. METHODS: Quantitative RT-PCR, ELISA, and immunohistochemical staining were performed to examine CCL5 expression in prostate cell lines. CCR5 expression was measured by flow cytometry. Proliferation and invasion assays were performed to determine potential functions of CCL5 and CCR5 in PCa. RESULTS: Expression of CCL5 mRNA and protein was found in human PCa cell lines (PC-3; DU-145; LNCaP) and primary prostate adenocarcinoma cells. CCL5 and CCR5 were also detected in human PCa tissues. CCR5 expression was demonstrated on the cell surface of PCa cells, as well as in intracellular pools. Incubation with CCL5 (10-100 ng/ml) induced PCa cell proliferation, and the CCR5 antagonist TAK-779 inhibited CCL5-induced proliferation. CCL5 was found to stimulate PCa cell invasion, and TAK-779 blocked the effects of CCL5. CONCLUSIONS: In light of evidence that inflammation influences the pathogenesis of PCa, these results suggest that inflammatory chemokines, such as CCL5, expressed by prostate cells may act directly on the growth and survival of PCa cells. Chemokine receptor antagonists may thus block autocrine mechanisms of PCa progression.