α6GABAA RECEPTOR-SELECTIVE POSITIVE ALLOSTERIC MODULATOR AS A NOVEL THERAPY FOR ADHD: A PRECLINICAL STUDY IN MK-801- TREATED JUVENILE MICE

Abstract Background The α6 subunit-containing GABAA receptors (α6GABAARs) are almost exclusively expressed in granule cells of the cerebellum in the whole brain. Their pathophysiological roles were understudied without selective ligands before. We identified a series of pyrazoloquinolinones (PQ) as α6GABAAR- selective positive allosteric modulators (PAMs) [1]. PQs display high affinity at the α+/β- interface, distinct from the GABA binding site, of GABAARs. They selectively increase the GABA current mediated by α6GABAARs among other α subunits. The cerebellum is recently implicated in several neuropsychiatric disorders, including attention-deficit hyperactivity disorder (ADHD) [2], a disorder affecting 5-12% of school-aged children. Clinical studies showed impaired cerebellar-striatal-frontal circuits and reduced cerebellar volume in ADHD patients. Animal models of ADHD also exhibited cerebellar deficits, like cerebellar Purkinje cell (PC) loss in spontaneous hypertensive rats. Mice lacking the ADHD-associated gene, GIT1, showed three core symptoms of ADHD and impaired tonic inhibition on cerebellar granule cells [3]. This tonic inhibition, interestingly, has been known to be mediated by α6GABAARs [4]. Aims & Objectives We, therefore, hypothesized that α6GABAAR-selective PAMs may restore tonic inhibition on cerebellar granule cells and ultimately rescue the functions of the prefrontal cortex (PFC) and striatum to alleviate ADHD symptoms. To validate this hypothesis, we employed an animal model of ADHD induced by MK-801, an NMDA channel blocker, at P28, a critical neurodevelopment period. Method ICR mice receiving MK-801 (0.2 mg/kg i.p.) at P28 showed significant behavioral phenotypes mimicking the three core symptoms of ADHD: hyperlocomotion, impulsivity, and inattention. They were evidenced, respectively, by increased locomotor activity in the open-field test, shorter latency and higher frequency of jumping events in the cliff avoidance reaction test, and decreased exploratory preference in the novel object recognition (NOR) test. Results PQ Compound 6 at 3 mg/kg (i.p.), the optimal effective dose in animal models of schizophrenia [5], migraine [6], and essential tremor [7], significantly prevented MK-801-induced hyperlocomotion, impulsivity, and impaired NOR performance in juvenile mice with an efficacy comparable to atomoxetine, an ADHD medication. The rescue effects of Compound 6 were prevented by furosemide, an α6GABAAR- selective antagonist, given by intra-cerebellar microinjection, and were nullified in mice lacking Gabra6, the gene encoding the α6 subunit. SCH23390, a dopamine D1 receptor antagonist, when given by bilateral intra-PFC microinjection (0.1 µg/side), mimicked the rescue effects of Compound 6 on three core symptoms. Interestingly, when intra-PFC pre-administered before Compound 6, SCH23390 occluded the rescue effects of Compound 6 on hyperlocomotion and impulsivity but not inattention. Discussion and Conclusion: These results suggest Compound 6 exerts its rescue effects in this ADHD model by acting as a PAM of cerebellar α6GABAARs, ultimately decreasing PFC dopaminergic transmission via the mechanism(s) more than blocking PFC D1 receptor-mediated transmission. Thus, α6GABAAR-selective PAMs, like PQ compounds, are potential novel drug candidates for ADHD therapy. References [1]Knutson DE, et al., 2018. Design and synthesis of novel deuterated ligands functionally selective for the gamma-aminobutyric acid type A receptor (GABA(A)R) alpha6 subtype with improved metabolic stability and enhanced bioavailability. J Med Chem, 61, 2422-2446. [2]Bruchhage MMK, et al., 2018. Cerebellar involvement in autism and ADHD. Handb Clin Neurol, 155, 61-72. [3]Kim YS, et al., 2017. Decreased glial GABA and tonic inhibition in cerebellum of mouse model for attention-deficit/hyperactivity disorder (ADHD). Exp Neurobiol, 26, 206-212. [4]Sieghart W, et al., 2022. alpha6-containing GABA(A) receptors: Functional roles and therapeutic potentials. Pharmacol Rev, 74, 238-270. [5]Chiou LC, et al., 2018. Cerebellar alpha6 -subunit-containing GABAA receptors: a novel therapeutic target for disrupted prepulse inhibition in neuropsychiatric disorders. Br J Pharmacol, 175, 2414-2427. [6]Tzeng HR, et al., 2021. alpha6GABA(A) receptor positive modulators alleviate migraine-like grimaces in mice via compensating GABAergic deficits in trigeminal ganglia. Neurotherapeutics, 18, 569-585. [7]Huang YH, et al., 2023. Cerebellar alpha6GABA(A) receptors as a therapeutic target for essential tremor: Proof-of-concept study with ethanol and pyrazoloquinolinones. Neurotherapeutics, 20, 399-418.