Essential tremor affects 0.5-0.9% of the general population and about 4.5% of those above age 65. Medications for tremor are re-purposed drugs found empirically rather than through laboratory research. As such they lack potency and specificity, and are often not well tolerated. In our program we seek to identify molecular targets to which anti-tremor drugs could be designed, offering greater on-target specificity and potency. A clue is that patients often report that as little as 1-2 glasses of wine can suppress tremor. Brain mapping studies in essential tremor indicate that hyper-oscillatory activity within circuits of the cerebellum underlies tremor. When patients are given low doses of alcohol, not only does the tremor diminish, but excessive activity in the cerebellar cortex also diminishes. We postulate that low-dose alcohol works on tremor by activating GABAA receptors that contain alpha6, beta3 and delta subunits, found only in the cerebellar cortex. If we can show that this receptor can mediate tremor suppression, it could represent an important target for anti-tremor therapy. The GABAA receptor is a pentamer made of alpha, beta, and either gamma or delta subunits. Delta GABAA receptors exert tonic inhibition, and are much more sensitive to GABA. Studies with receptors expressed in cells, and with brain slices, have shown that THIP, muscimol and neurosteroids such as ganaxolone selectively activate delta GABAA receptors rather than gamma GABAA receptors. In addition, ethanol in the range of 1-2 glasses of wine has been found to activate those delta GABAA receptors that use beta3 subunits. We hypothesize that activation of delta GABAA receptors by THIP, muscimol, ganaxolone, and low-dose alcohol will suppress tremor in the harmaline model of essential tremor in wild-type but not littermate delta knockout mice. In addition, the tremor-suppressing effect of alcohol in wild-type mice should be reversed by the alcohol antagonist Ro15-4513, providing further evidence that alcohol is suppressing tremor via a GABAA receptor. We use the harmaline tremor model, which has many similarities to tremor of essential tremor. Motion power is digitally analyzed to provide measures of motion in the tremor bandwidth as a ratio of overall motion power. Our pilot data indicate that THIP, muscimol, ganaxolone, and low-dose alcohol all suppress harmaline tremor in wild-type but not in delta knockout mice. The effect of low-dose alcohol was blocked by Ro15-4513. We propose to replicate these findings (Aim 1). The delta subunit is associated with either alpha 4 or with alpha 6 in GABAA receptors. The alpha4- delta receptors mediate sedation and motor impairment when activated, whereas no behavior has previously been found associated with alpha6-delta receptor activation. Our pilot experiments showed that THIP and low-dose alcohol suppress harmaline tremor in wild-type but not in alpha6 knockout mice, indicating the alpha6-delta receptor mediates tremor suppression. We propose to replicate this finding and test ganaxolone as well (Aim 2). GABAA receptors containing alpha6-delta are found only in the cerebellar cortex, where they associate with beta3, beta2 or beta1. The next step is to investigate whether cerebellar beta3 is important for tremor suppression by these delta-receptor acting drugs by breeding mice with beta3 missing in the part of the cerebellum where alpha6 is expressed (Aim 3). It is anticipated that beta3 will be critical for low-dose alcohol's anti-tremor action, but itmay not be critical for THIP or ganaxolone. Should appropriate strains of knockout mice become available, we will test whether beta2 or beta1 are important for mediating the anti-tremor action of THIP and ganaxolone. These experiments are expected to lead to the identification of a GABAA receptor subtype, alpha6-beta2/3- delta, found only in the cerebellar cortex that will constitute a molecular target to which anti-tremor therapies can be designed to activate. Such a therapy, through its high selectivity, is anticipated to be potent and well tolerated. As the first drug to be developed specifically for essential tremor from laboratory research, it would be expected to confer greater efficacy than existing drugs, preventing disability and loss of occupation in veterans with essential tremor.
Essential tremor is a common neurological disorder affecting millions of Americans. It is often disabling, interfering with daily tasks or occupation, and forcing early retirement. Current therapies are often unsatisfactory. Molecular targets need to be identified for drug development. GABAA receptors contain alpha and beta subunits and are divided into those that use gamma or delta subunits. Based on pilot findings that drugs and low-dose alcohol known to activate delta GABAA receptors suppress tremor in the harmaline model of essential tremor in mice, we wish to first test the hypothesis that activation of delta GABAA receptors suppresses tremor, using wild- type and delta-knockout mice. Next, through the use of alpha and beta subunit knockout strains, we wish to identify more precisely which subtype of delta GABAA receptor mediates tremor suppression. This will provide a molecular target to which selective and potent drugs can be developed for subsequent testing in clinical trials. This work is intended to facilitae the development of better therapies for disabling tremor in veterans.
