Evaluation of an a6ßy2 GABA Receptor-Specific Drug as Potential Therapy

Subtitle: Evaluation of an α6βγ2 GABA Receptor-Specific Drug as Potential Therapy

Adrian Handforth, MD

Progress Report Feb 27, 2020

Background
Evidence suggests that in essential tremor the oscillatory circuit includes cerebellar granule cells, and that these cells are too active in this condition. Their activity is controlled by mossy fiber excitatory inputs and by GABA locally released by Golgi neurons. The inhibitory action of GABA is mediated by synaptic α6βγ2 and extra-synaptic α6βδ GABA_A receptors. The cerebellar granule cell is the main site within brain that expresses α6 receptors of either class. We previously found that THIP, low-dose ethanol, and the neurosteroid ganaxolone suppress tremor in the mouse harmaline model in an α6- and a δ-dependent way, suggesting they act on the cerebellar granule cell. Unfortunately, they also act on extra-synaptic α4βδ GABA_A receptors that are dominant in the brain outside the cerebellum, causing side effects. We are not aware of any α6βδ-selective drug. However, an alternative strategy for inhibiting cerebellar granule cells relatively specifically is to employ drugs selective for synaptic α6βγ2 GABA_A receptors, as these are also mainly expressed on cerebellar granule cells. We therefore examined Compound 6 (PZ-II-029), which has been shown in vitro to act as a positive allosteric modulator of α6β3γ2 receptors, and less potently of synaptic α6β2γ2 receptors. Little in vivo work has been performed with Compound 6. Doses of 3 and 10 mg/kg have been reported to affect pre-pulse inhibition in mice, while the deuterated form has been found to exert analgesia in a rat model of trigeminal neuralgia. Based on its receptor action, we anticipated that Compound 6 would suppress tremor in doses that do not cause psychomotor impairment.

Aims
Aim 1: Effect of genotype on straight wire test response to Compound 6. The goal is to find the highest dose of Compound at which 6/6 mice pass the straight wire test, a sensitive test of psychomotor impairment, in α6 wild-type (WT) and in α6 knockout (KO) mice.

Aim 2:  Effect of Compound 6 on harmaline tremor in mice with or without GABA_A receptor α6 subunit. The goal is to assess whether Compound 6, in doses that were found in Aim 1 not to cause failures on the straight wire test, suppresses tremor in the harmaline mouse model.

Methods
Mice in our α6 colony have been back-crossed with C57BL6 mice for 10 generations. For experiments, littermate WT and KO offspring of heterozygote parents are utilized. Compound 6, synthetized in Belgrade by Dr. Miroslav Savic, and provided by Dr Margot Ernst of Austria, was dissolved in 85% saline, 14% propylene glycol, and 1% Tween 80, and injected intraperitoneally.

Straight wire test. Compound 6 in various doses was injected and mice tested every 10 minutes for up to 1 hour. The mouse is suspended by its front paws from a 2-mm diameter wire and the time taken to bring a hind paw up to the wire noted. Mice normally do so within 10 seconds, whereas sedated or ataxic mice fail to do this or fall off. The doses at which 6/6 pass all tests are determined. Only doses at which 6/6 pass the test or lower doses are to be used in harmaline experiments. This is a sensitive test of psychomotor impairment.

Harmaline tremor. The mouse is placed on an elevated platform on a cylinder that rests on a chamber floor fitted with a seismic detector and allowed to habituate before collecting motion power data. The mouse is free to move within the confines of the platform. After subcutaneous injection of harmaline, 20 mg/kg, tremor is observed to develop and stabilize, then motion power again accessed for 15 minutes. Compound 6 or vehicle is then administered. Motion power then is collected for five more 15-minute epochs separated by 5-minute rests in the home cage. Harmaline tremor is associated with an increase in motion power at 10-16 Hz. The tremor measure is motion power at 10-16 Hz as a percentage of overall motion power at 0.5-32 Hz to control for activity level. Percentages of 20% to 35% are seen with normal activity whereas higher values indicate tremor at 10-16 Hz.

Results
Aim 1: We determined that doses of 10 mg/kg and 20 mg/kg were associated with all 6/6 mice passing the straight wire test. We did not try higher doses in order to preserve the Compound 6 supply, which was provided as a 100 mg initial shipment.

Aim 2: Compound 6 was found to suppress harmaline tremor in doses of 10 mg/kg and 20 mg/kg. The pattern of tremor suppression was unusual in that it was brief, lasting one or two 15-minute epochs, but the onset of this suppression varied, so that it could occur in the first post-drug 15-minute epoch, or in the second or third post-injection 15-minute epoch. Therefore, we used as a measure the minimum motion power percentage (MPP) among the first three post-drug epochs. As shown in the table below, α6 WT mice among the vehicle-, 10 mg/kg-, and 20 mg/kg-treated groups displayed comparable MPP values during pre-harmaline baseline and harmaline pre-drug epochs, but the groups receiving 10 mg/kg or 20 mg/kg showed a reduction of MPP compared to the vehicle-treated group, reflecting tremor suppression. No such reduction occurred in α6 KO mice.

Table 1: Effect of Compound 6, a α6βγ2-selective positive modulator, on harmaline tremor

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Genotype                    Sample            Baseline                      Harmaline       Post-drug least            p value

& Treatment                 size                pre-harmaline              pre-drug          motion power

—————————————————————————————————————————————–

α6-WT

Vehicle                       8                    24.4 ± 2.4                    81.4 ± 4.1        78.5 ± 2.9

10 mg/kg                    9                    26.3 ± 3.1                    77.9 ± 4.1        53.6 ± 7.5                    0.001

20 mg/kg                  10                    33.6 ± 6.5                    79.9 ± 3.1        52.9 ± 4.8                    0.0005

α6-KO

Vehicle                     9                      27.0 ± 1.7                    82.7 ± 3.9        80.0 ± 1.6

10 mg/kg                  9                      27.2 ± 2.4                    82.9 ± 4.0        86.7 ± 2.9                    NS

20 mg/kg                  10                    32.0 ± 1.9                    90.4 ± 2.9        84.1 ± 2.9                    NS

—————————————————————————————————————————————–

Data represent motion power percentage of the tremor bandwidth power divided by overall motion power

(10-16 Hz)/(0.5-32 Hz) x 100. Means and SEM are shown. Post-drug least motion power refers to least motion power during any of three post-drug 15-minute epochs. Comparisons with vehicle, Student’s t test.

Discussion
The initial results shown in Table 1 indicate that Compound 6, a selective positive allosteric modulator of α6βγ2 GABA_A receptors, suppresses tremor in the harmaline model of essential tremor. Doses of 10 and 20 mg/kg exerted similar efficacy, suggesting that doses higher than 10 m/kg do not add to efficacy. The variable timing of tremor suppression and brief action likely reflect variable absorption, as the compound comes out of solution easily, and rapid degradation or re-distribution to fat stores. Nonetheless, the findings importantly indicate proof of principle that drugs designed to activate this receptor selectively can suppress tremor, so that this strategy may be fruitful for developing new therapies for essential tremor.

Plans
 We wish to examine the effect of a lower dose on tremor in order to understand better the relationship between dose and efficacy. The collaborators are preparing another 100 mg shipment of Compound 6, which will enable us to complete both the harmaline and straight wire testing. Higher doses in the straight wire test will allow us to compare the highest dose tolerated in the straight wire test with those that suppress tremor. The PI is grateful to the IETF for supporting this work.

Summary

Background: Considerable work has identified the cerebellum as a state of physiological abnormality in essential tremor (ET). In previous work funded by IETF we explored the potential role of certain inhibitory receptors that respond to GABA. GABA_A receptors are not uniform, but differ according to their subunit composition. They all contain 2 α (alpha) subunits, of which there are 6 types, 2 β (beta, 3 types), and either a δ (delta, only 1 type) or a γ (gamma, 3 types) subunit. We have been interested in GABA_A receptors containing both δ and α6 subunits, as such receptors are found mainly on cerebellar granule cells, the most abundant type of cell in the cerebellum. In previous work we found that low-dose alcohol, ganaxolone, and gaboxadol each suppresses tremor in a mouse model of ET in which tremor is induced with the drug harmaline, but not if the mice lack the δ or the α6 GABA_A receptor subunit. Unfortunately, there is no drug that specifically activates only δ GABA_A receptors containing α6 subunits. Therefore, given that cerebellar granule cells are also the main brain site expressing α6βγ2 GABA_A receptors, we turned out attention to the idea that a drug that activates such receptors will suppress tremor. The candidate compound was a drug known as Compound 6, reported to selectively activate α6βγ2 receptors. This drug had been shown to affect cerebellar granule cell function when injected systemically. We predicted that Compound 6 would suppress tremor at doses much lower than those that cause psychomotor impairment, and that this action would depend on the presence of α6 subunits.

Methods: The straight wire test was used to assess what doses of Compound 6 do or do not interfere with the ability of mice to hang on to a wire for 10 seconds at 10-minute intervals over an hour. This is a sensitive test of psychomotor performance. In the harmaline tremor experiment, mice received harmaline to induce tremor, then received either vehicle or Compound 6 in various doses intraperitoneally. The tremor measure was motion power in the 9-16 Hz tremor bandwidth divided by background 0.25-32 Hz motion power, as accessed with a seismic detector and digitized by software. Wild-type (WT) and littermate α6 knockout (KO) mice were assessed.

Results: Due to low solubility, the highest dose that was tested in the straight wire test was 20 mg/kg, at which 6/6 mice passed. In harmaline experiments with WT mice, 10 mg/kg strongly suppressed tremor, with 2 mg/kg exerting mild tremor suppression, and 5 mg/kg moderate tremor suppression. Similarly, Compound 6 caused dose-dependent tremor suppression in α6 KO mice.

Conclusions: Compound 6 exerts strong tremor suppression in well-tolerated doses. Our results indicate that, contrary to our prediction, this action does not depend on the presence of the GABA_A receptor α6 subunit. The mechanism of anti-tremor action is thus uncertain. Nonetheless, the results suggest that this compound, and potentially others in its class, may hold promise as therapy for ET.

1. Rationale and Relevance to Essential Tremor

During tremor an oscillatory circuit involves prefrontal and frontal cortex, pons, cerebellum, and thalamus (1) As the sole output from cerebellar cortex; Purkinje cells receive a massive excitatory input via parallel fibers from granule cells, which in turn receive massive excitatory inputs from ascending mossy fibers. Evidence that this entire cerebellar cortical system is excessively active in ET is provided by imaging scans that demonstrated increased blood flow (2). Because cerebellar metabolism is dominated by the vast cerebellar granule cell population, this finding suggests these cells are more active in ET. It was also found that when subjects were given a non-intoxicating dose of alcohol sufficient to suppress tremor, with a blood level of 0.04 g/dl, the cerebellar hyper-metabolism was reduced towards normal. (2). The findings were interpreted as indicating that alcohol reduces excitatory drive by granule cells on Purkinje cells, leading to reduction of tremor. In support of this interpretation, high-density EEG has indicated that alcohol reduces tremor by acting in the cerebellum (3). Infarcts of the pons, which destroy mossy fibers and remove the main excitatory input to granule cells, abolishes tremor (4). The Golgi cell, as the main source of inhibition to cerebellar granule cells, is positioned to play a critical role in controlling cerebellar activity, and hence tremor, by releasing GABA. This brain site is unique in its intense expresses α6βδ and α6βγ2 GABA_A receptors. GABA_A receptors form two main classes. Synaptic receptors are pentameric structures containing 2 α, 2 β, and a γ subunit, and mediate fast signaling. There are 6 types of α and 3 types of β subunits. Extrasynaptic receptors are also pentameric, but utilize a δ rather than a γ subunit, usually contain α4 or α6 subunits, are sensitive to very low GABA levels, and induce tonic inhibitory currents. In contrast to δ GABA_A receptors elsewhere in brain that usually contain α4, those of cerebellar granule cells utilize α6 subunits (5). GABA_A receptor subunit mapping reveals very intense α6 expression that is almost restricted to cerebellum, with only light staining in certain other CNS areas, such as sensory brainstem nuclei (6). The α6 KO state results in 50% loss of cerebellar GABA_A receptors, with a severe loss of δ, and 40% depletion of γ2 (7). As expected, tonic inhibitory currents are lost in cerebellar granule cells (8). Despite these considerable changes, α6 KO mice are agile, show no motor deficits, and exhibit normal harmaline-induced tremor. This is explained by the finding that in α6 KO mice there is a compensatory increase in voltage-independent potassium conductance, so that granule cells are normally excitable (8).

Ethanol enhances GABA currents at concentrations as low as 3 mM in α6β3δ receptors in recombinant systems (9). Slices of cerebellar granule cells respond with enhanced tonic currents to ethanol at 10 mM, but not if taken from δ KO mice (10) or from α6 KO mice (8). This level is below the legal driving limit of 0.08 g/dL (17.3 mM). Reasoning that this may be the mechanism by which alcohol suppresses tremor in ET, we found that low-dose alcohol suppresses tremor in the harmaline model in WT mice but not in mice lacking the α6 or the δ GABA_A receptor subunit (unpublished). Similarly, we found that gaboxadol, which activates α6βδ receptors at sub-micromolar levels (11), and induces tonic currents in slices of cerebellar granule cells in δ- and α6-subunit dependent fashion, also suppresses harmaline tremor in mice and requires δ and α6 GABA_A (12). Neurosteroids are activate tonic currents (13). The neurosteroid ganaxolone exerts anxiolytic effects in mice, but not if they lack the δ subunit (14). We found that ganaxolone reduces harmaline tremor in WT mice but not in littermate mice lacking the α6 or the δ GABA_A receptor subunit (unpublished).

Collectively, these results indicate that the inhibition of cerebellar granule cells, as by activating α6βδ receptors, is a valid strategy to treat tremor. However, a drawback is that neither alcohol, ganaxolone, nor gaboxadol are α6βδ-receptor selective, as they each also activate α4βδ GABA_A receptors. That is a major problem, because the concurrent activation of the later receptors is prone to cause adverse effects such as somnolence, imbalance, addiction, disinhibition, and other effects. Selective activation of α6βδ GABA_A receptors to inhibit overactive cerebellar granule cells represents a promising strategy for treating tremor but unfortunately is so far not feasible, as no compounds are available that activate α6βδ GABA_A receptors with high selectively.

An alternative strategy for selective inhibition of cerebellar granule cells is to stimulate GABA_A α6βγ2 receptors, as these are intensely expressed by these cells. The pyrazoloquinolinones are a class of compounds suggested to hold promise as anxiolytics as they exert exceedingly low sedation (15).  Studies with derivatives on recombinant GABA_A receptors expressed on oocytes showed that Compound 6 (PZ-II-029) displays high affinity for the α6βγ2 receptor, and acts as a positive allosteric modulator, so that it does not exert intrinsic activating activity, but enhanced GABA-induced currents (16, 17). The α6βγ2 GABA_A synaptic receptor is expressed mainly, but not solely, in cerebellar granule cells, activated by GABA released by Golgi neurons. Compound 6 has over 1000 times higher affinity for α6βγ2 GABA_A receptors than for any other GABA_A receptor, but does have weak affinity for α3βγ2 receptors (17).

Methamphetamine causes disruption of prepulse inhibition of the acoustic startle response, an effect that is blocked by intraperitoneal (i.p.) injection of Compound 6 in doses of 3 or 10 mg/kg. This effect of Compound 6 is itself blocked by intracerebellar injection of furosemide, which blocks α6 GABA_A receptors (18). Compound 6 thus appears to affect α6βγ2 receptors in the cerebellum when given systemically.

There are limitations to the evidence supporting the notion that Compound 6 may exert in vivo selectivity for α6βγ2 GABA_A receptors as is seen in recombinant systems. Validation with cerebellar granule cell slices has not been performed.  In the prepulse inhibition studies, α6 knockout (KO) mice were not tested. The effect of furosemide on blocking Compound 6’s effect on prepulse inhibition may have been downstream from the site of Compound 6’s action. The extent to which Compound 6 affects non-GABA receptors is not known.

Nonetheless, given the extraordinary selectivity for recombinant α6βγ2 GABA_A receptors, we chose to study Compound 6 for anti-tremor efficacy, as a vehicle for evaluating this attractive therapeutic target. In addition, we assessed whether the α6 GABA_A receptor subunit is required for its anti-tremor action, if any.

To induce tremor in mice, we used the harmaline model, which is not a model of ET the disease, but a symptom-based model of action tremor. Harmaline tremor has considerable physiological, anatomic, and pharmacological overlap with tremor of ET. Harmaline tremor in rodents responds to drugs that suppress tremor in ET (propranolol, primidone, alcohol, benzodiazepines, gabapentin, zonisamide, 1-octanol, γ-hydroxybutyrate), while being exacerbated by drugs that worsen ET (tricyclics, caffeine). Like ET, harmaline causes cerebellar hyper-metabolism, including Purkinje cells, the cortex, and deep cerebellar nuclei, all implicated in ET. Cerebellar granule cells are also activated during harmaline tremor, as indicated by c-fos labelling (19, 20). Thalamic deep brain stimulation suppresses harmaline tremor (21), as in ET. False positives have occurred, in which drugs that suppress harmaline tremor do not reduce tremor in ET patients (22). Many of these disparities were likely from the use of doses that caused non-specific psychomotor impairment in animals.

Our first aim was to assess the effect of Compound 6 on psychomotor impairment, utilizing the straight wire test, a highly sensitive test for psychomotor compromise in mice (23). The purpose of this experiment was to determine the highest dose of Compound 6 at which 6/6 mice passed all straight wire tests. Only doses at which all mice pass were to be used in subsequent harmaline experiments.

The second aim was to assess whether Compound 6 suppresses harmaline-induced tremor in doses not associated with impairment on the straight wire test, and whether any anti-tremor effect depended on the presence of the α6 GABA_A receptor subunit.

2. Research Methods and Procedures

Breeding: Our α6 colony, on a C57BL6 background, has been backcrossed for 10 generations with C57BL6 mice in our laboratory. To generate mice for experiments, littermate α6 WT and α6 KO mice were bred from α6 heterozygote parents. For polymerase chain reaction (PCR) genotyping, ear snips were collected before 3 weeks of age and sent to Transnetyx, Inc. (Memphis, TN). Mice were housed with ad libitum access to food and water on a 12:12 h light:dark cycle; temperature 22-24 degrees Celsius. Both males and females were used.

Drugs: Harmaline was obtained from Sigma-Aldrich, and Compound 6 was donated by Dr. Margot Ernst (Center for Brain Research, Medical University Vienna, Vienna, Austria). Harmaline was dissolved in saline and administered subcutaneously as 20 mg/kg in 4 ml/kg. Compound 6 was dissolved by sonicating and warming in a mixture of 85% distilled water, 14% propylene glycol, and 1% Tween 80 (Sigma-Aldrich), and was given i.p. in various doses in a volume of 10 ml/kg, except for the dose 20 mg/kg, which was given as 20 ml/kg, as the maximum solubility of the drug in this solution is approximately 1 mg/ml.

Straight wire test: In this test for impairment, Compound 6 in various doses was injected and mice tested every 10 minutes for up to 1 hour. Each mouse was suspended by its front paws from a 2-mm diameter wire and the time taken to bring a hind paw up to the wire noted. Mice normally do so within 10 seconds, whereas sedated or ataxic mice fail to do this or fall off. The dosage at which 6/6 pass all tests was be determined. Only doses at which 6/6 pass the straight wire test or lower doses were used in harmaline experiments. This is a sensitive test of psychomotor impairment. For example, we found that 3 mg/kg gaboxadol is the maximum dose at which 6/6 pass (12); doses of 4-6 mg/kg are known to affect the EEG in mice (24).

Harmaline tremor: Motion activity was measured with a Convuls-1 Sensing Platform (Columbus Instruments, Columbus, OH), that has a load sensor, connected to a Grass amplifier (Grass Instruments, West Warwick RI) with 1 and 70 Hz filter settings. Motion power was digitally analyzed using Spike2 software (Cambridge Electronic Design UK) to transform data into frequency spectra. The motion power percentage (MPP) is the tremor bandwidth divided by overall motion: (9-16 Hz power)/(0.25-32 Hz power) x 100.

Each mouse was placed on a 9.5-inch high 3.2-inch wide cylinder situated on the tremor platform, allowed 20 minutes to habituate, then 15 minutes of pre-harmaline baseline motion power data collected. The exposed location has an alerting effect, encouraging mice to move about, eliciting action tremor. Harmaline was then injected, and motion power measurement initiated 10 minutes later when full tremor had stabilized. Harmaline tremor was then recorded for 15 minutes to verify an adequate tremor response, defined as an MPP increase of at least 30% over pre-harmaline baseline. MPP is usually 25-35% during baseline, then rises to 60-90% after harmaline injection, and remains high for at least an hour. Compound 6 or vehicle was then injected after the end of the harmaline epoch, then motion power collection initiated 10 minutes later, and measured in 5 successive epochs of 15 minutes each, separated by 5-minute rest periods in the home cage (E1 to E5). Data from mice that failed to show an adequate harmaline tremor response were not used (approximately 10-20% of mice). Animals were exposed to harmaline only once.

3. Results

Straight wire testing. The highest dose tested was 20 mg/kg, at which 6/6 WT mice passed. Higher doses could not be assessed as Compound 6’s limited solubility, a maximum of 1 mg/ml, meant that higher doses would have exceeded our institution’s permitted maximum volume limit of 25 ml/kg i.p. in mice. Thus the maximal tolerated dose was not determined but is at least 20 mg/kg, given that 6/6 WT mice passed at this dose.

Harmaline tremor. In the table below, motion power data, expressed motion power in the tremor bandwidth (9-16 Hz) as a percentage of overall motion power (0.25-32 Hz) are provided during the pre-harmaline baseline epoch, during the pre-treatment harmaline tremor epoch, and at 10-45 minutes after vehicle or Compound 6 administration in various doses (combining data from post-injections epochs E1 and E2), and at 50-85 minutes post-injection (combining epochs E3 and E4).

Table 1: Harmaline tremor in GABA_A receptor α6 subunit wild-type and α6 knockout mice

after vehicle or Compound 6.

————————————————————————————————————

Treatment       n      Pre-Harmaline    Pre-Treatment            Post-injection

Baseline            Harmaline     10-45 min        50-85 min

————————————————————————————————————

GABA_A receptor α6 subunit wild-type (α6+/+)

Vehicle          16        31.8 ± 2.3        73.7 ± 2.4        71.3 ± 3.6        77.0 ± 2.1

10 mg/kg       11        32.9 ± 2.2        76.3 ± 3.3        34.2 ± 5.5***   52.6 ± 5.9***

5 mg/kg        11        27.0 ± 3.1        75.3 ± 3.1        49.4 ± 4.5***   58.3 ± 4.2***

2 mg/kg        15        33.0 ± 3.0        75.2 ± 2.5        63.0 ± 4.7        63.6 ± 4.3**

GABA_A receptor α6 subunit knock-out (α6-/-)

Vehicle          11        36.4 ± 1.3        68.6 ± 2.0        69.1 ± 2.1        71.7 ± 2.7

10 mg/kg       11        31.8 ± 3.1        74.6 ± 2.9        40.4 ± 6.3***   56.9 ± 6.1*

5 mg/kg        11        36.6 ± 1.7        79.7 ± 2.3        46.7 ± 5.2***   58.1 ± 7.1

2 mg/kg        15        33.3 ± 2.3        75.7 ± 1.6        58.0 ± 4.9        61.6 ± 4.9

————————————————————————————————————

Means ± S.E.M are shown. * P<0.05 ** P<0.01 *** P <0.001, Student’s t-test.

Comparisons are with vehicle-treated group.

These results indicate that at doses well below 20 mg/kg that was found to be tolerated on the straight wire test, a sensitive test of psychomotor impairment, Compound 6 reduces harmaline-induced tremor. The effect is seen at both 10-45 and at 50-85 minutes after injection, but the drug is more effective during the earlier post-injection period. Tremor suppression was dose-related, with 10 mg/kg exerting marked tremor suppression, 2 mg/kg minimal suppression, and 5 mg/kg acting with moderate efficacy. Contrary to prediction, tremor suppression occurred in  α6 subunit KO mice, and to a degree comparable to that seen in WT mice.

4. Discussion

We found that Compound 6 effectively suppresses harmaline tremor at doses in the well-tolerated range, below the 20 mg/kg dose at which 6/6 mice passed the straight wire test, suggesting that Compound 6 may exert clinical efficacy for essential tremor at well-tolerated doses. This is in keeping with the reputation of pyrazoloquinolinones, of which Compound 6 is a member, for lacking sedative effects (15). The results also imply that the Compound enters the brain and exerts efficacy within minutes, suppresses tremor for up to an hour in mice.

Surprisingly, given Compound 6’s reported very high and selective affinity for the α6βγ2 GABA_A receptor compared to other GABA_A receptors (16, 17), our α6 KO mice responded comparably to WT littermates with tremor suppression on administration of Compound 6. This result might be due to allosteric activation of α3βγ2 receptors, as Compound 6 does bind to these receptors and enhances GABA currents at high concentrations (17). A recent publication has reported that compounds that selectively activate α3 GABA­_A receptors suppress harmaline tremor (25). Compound 6 might be another example of a compound that suppresses tremor by activating α3 GABA_A­ receptors. As these receptors exert anxiolytic effects, this mechanism could be attractive for ET patients.  On the other hand, the binding profile of Compound 6 has been explored only to a limited degree, and it is possible it suppresses tremor through an unidentified non-GABA receptor mechanism.

We believe the present results indicate that Compound 6 (and structurally related compounds) hold promise in the treatment of essential tremor. These findings represent an early step. More needs to be known about the safety and binding profile of this interesting compound and others in its class.

References

1.Schnitzler A, Monks C, Butz M, et al. Synchronized brain network associated with essential tremor as revealed by magnetoencephalography. Mov Disord 2009;24:1629‑35.

2. Boecker H, Wills AJ, Ceballos‑Baumann A, et al. The effect of ethanol on alcohol‑responsive essential tremor: a positron emission tomography study. Ann Neurol 1996;39:650‑8.

3. Pedrosa DJ, Nelles C, Brown P, et al. The differentiated networks related to essential tremor onset and its amplitude modulation after alcohol intake. Exp Neurol 2017;297:50-61.

4. Dupuis MJ, Evrard FL, Jacquerye PG, et al. Disappearance of essential tremor after stroke. Mov Disord 2010;25:2884‑7.

5. Nusser Z, Sieghart W, Somogyi P. Segregation of different GABA_A receptors to synaptic and extrasynaptic membranes of cerebellar granule cells. J Neurosci 1998;18:1693‑703.

6. Pirker S, Schwarzer C, Wieselthaler A, et al. GABA_A receptors: immunocytochemical distribution of 13 subunits in the adult rat brain. Neuroscience 2000;101:815‑50.

7. Nusser Z, Ahmad Z, Tretter V, et al. Alterations in the expression of GABA_A receptor subunits in cerebellar granule cells after the disruption of the alpha6 subunit gene. Eur J Neurosci 1999;11:1685‑97.

8. Brickley SG, Revilla V, Cull-Candy SG, et al. Adaptive regulation of neuronal excitability by a voltage-independent potassium conductance. Nature 2001; 409: 88-92

9. Wallner M, Hanchar HJ, Olsen RW. Ethanol enhances α₄β₃δ and α₆β₃δ γ‑aminobutyric acid type A receptors at low concentrations known to affect humans. Proc Natl Acad Sci U S A 2003; 100: 15218-15223.

10. Santhakumar V, Meera P, Karakossian MH, et al. A reinforcing circuit action of extrasynaptic GABA_A receptor modulators on cerebellar granule cell inhibition. PLoS One 2013; 8: e72976.

11. Meera P, Wallner M, Otis TS. Molecular basis for the high THIP/gaboxadol sensitivity of extrasynaptic GABA_A receptors. J Neurophysiol 2011;106:2057‑2064.

12. Handforth A, Kadam PA, Kosoyan HP, et al. Suppression of harmaline tremor by activation of an extrasynaptic GABA_A receptor: Implications for essential tremor. Tremor Other Hyperkinet Mov (N Y). 2018;8:546.

13. Stell BM, Brickley SG, Tang CY, et al. Neuroactive steroids reduce neuronal excitability by selectively enhancing tonic inhibition mediated by delta subunit‑containing GABA_A receptors. Proc Natl Acad Sci U S A 2003;100:14439‑444.

14. Mihalek RM, Banerjee PK, Korpi ER, et al. Attenuated sensitivity to neuroactive steroids in gamma‑aminobutyrate type A receptor delta subunit knockout mice. Proc Natl Acad Sci USA 1999;96:12905‑10.

15. Williams M, Bennett DA, Loo PS, Braunwalder AF, Amrick CL, Wilson DE, Wasley JW. CGS 20625, a novel pyrazolopyridine anxiolytic. J Pharmacol Exp Ther 1989;248:89-96.

16. Treven M, Siebert DCB, Holzinger R, et al. Towards functional selectivity for α6β3γ2 GABA_A receptors: a series of novel pyrazoloquinolinones. Br J Pharmacol 2018;175:419-428.

17. Varagic Z, Ramerstorfer J, Huang S, et al.. Subtype selectivity of α+β- site ligands of GABA_A receptors: identification of the first highly specific positive modulators at α6β2/3γ2 receptors. Br J Pharmacol 2013;169:384-399.

18. Chiou LC, Lee HJ, Ernst M, et al. Cerebellar α6 -subunit-containing GABA_A receptors: a novel therapeutic target for disrupted prepulse inhibition in neuropsychiatric disorders. Br J Pharmacol 2018;175:2414-2427.

19. Batini C, Buisseret-Delmas C, Conrath-Verrier M. Harmaline-induced tremor. I. Regional metabolic activity as revealed by [¹⁴C]2-deoxyglucose in cat. Exp Brain Res. 1981;42:371-382.

20. Tian JB, Bishop GA. Stimulus-dependent activation of c-Fos in neurons and glia in the rat cerebellum. J Chem Neuroanat 2002;23:157-170.

21. Bekar L, Libionka W, Tian GF et al., Adenosine is crucial for deep brain stimulation-mediated attenuation of tremor. Nat Med 2008;14:75-80.

22. Handforth A. Harmaline tremor: underlying mechanisms in a potential animal model of essential tremor. Tremor Other Hyperkinet Mov (N Y) 2012;2. pii:02-92-769-1.

23. Vanover KE, Suruki M, Robledo S, et al. Positive allosteric modulators of the GABA_A receptor: differentialinteraction of benzodiazepines and neuroactive steroids with ethanol. Psychopharmacology (Berl) 1999;141:77-82.

24. Winsky‑Sommerer R, Vyazovskiy VV, Homanics GE, et al. The EEG effects of THIP (Gaboxadol) on sleep and waking are mediated by the GABA_A delta‑subunit‑containing receptors. Eur J Neurosci 2007;25:1893‑9.

25. Amrutkar DV, Dyhring T, Jacobsen TA, Larsen JS, Sandager-Nielsen K. Anti-tremor action of subtype selective positive allosteric modulators of GABA_A receptors in a rat model of essential tremors. Cerebellum 2020;19:265-274.