α6GABAA RECEPTOR-SELECTIVE POSITIVE ALLOSTERIC MODULATOR AS A POTENTIAL NOVEL THERAPY FOR AUTISM: A PRECLINICAL STUDY IN OFFSPRING OF VALPROIC ACID-TREATED RATS

Abstract Background Autism Spectrum Disorder (ASD) represents a group of developmental disorders characterized by social impairment and repetitive behaviors with the prevalence around 2.7% [1]. ASD treatment remains an unmet medical need. The pathogenesis of ASD is not clear but is believed to be due the existence of an excitatory-inhibitory (E-I) imbalance in the brain, particularly in the prefrontal cortex (PFC), a region renowned for behavioral regulation and social functioning [2, 3]. Besides, structural and functional aberrations in the cerebellum were found in ASD subjects [4, 5]. Consequently, the interplay between the cerebellar-PFC circuits may constitute a significant element of the underlying pathophysiology of ASD [6]. Aims and Objectives We explored the therapeutic potential of pyrazoloquinolinone (PQ) compounds, the first series of positive allosteric modulators (PAMs) selectively targeting the α6 subunit-containing α 6GABAA receptors (α6GABAARs), which are densely expressed in cerebellar granule cells [7]. Our hypothesis focuses on PQ compound’ s ability to alter the PFC activity by increasing GABAergic transmission in cerebellar granule cells, and ultimately modulating the output from the deep cerebellar nucleus that send projections to the PFC [8] and reducing ASD symptoms. To validate this hypothesis, we examined effects of PQ compounds in a well-recognized animal model of ASD, the prenatal valproic acid (VPA)-exposure juvenile rats. Method Pregnant rats received a single injection of VPA (500 mg/kg, i.p.) or saline on E12.5. Their male and female offspring from P21 were daily treated with DK-I-56-1 (10 mg/kg, i.p.), a druggable PQ compound, or its vehicle (Veh). Social behaviors, quantified by the time spent with the social object (sociability) and the novel stranger (social preference), along with repetitive behaviors measured by the number of marbles buried in the marble burying test, were compared among four offspring groups: VPA+DK (n = 9), VPA+Veh (n = 9), Saline+DK (n = 12), and Saline+Veh (n = 11). Results Two-way ANOVA followed by Tukey's multiple comparisons test determined the VPA+Veh group had significant social withdrawal behaviors compared to the Saline+Veh group, evidenced by significant lower sociability (44±18 vs. 234±18 sec, p <0.001) and social preference (65±25 vs. 229±13 sec, p <0.001). The VPA+DK group receiving DK-I-56-1 daily treatment for five days increased sociability to the level as in Saline+Veh group (227±15 vs. 238±11 sec, p = 0.9448), but not the VPA+Veh group (113±19 vs. 238±11 sec, p <0.001). Similar results were observed in social preference, where the VPA+DK group showed preference for novel strangers comparable to Saline+Veh group (195±16 vs. 227±13 sec, p = 0.9469), while the VPA+Veh group didn’ t exhibit this preference (86±17 vs. 227±13 sec, p <0.001). Discussion & Conclusion These results suggested that daily treatment with DK-I-56-1 for five days significantly restored the sociability that was impaired by prenatal VPA exposure. The half-life of DK-I- 56-1 is about 12-14 hr [7]. We will reduce the treatment days to explore the minimum required dosages of DK-I-56-1. While the precise mechanistic require detailed exploration, these results underscore the potential therapeutic promise of PQ compounds, by modulating the cerebellum output, in mitigating key features of ASD pathology. References 1.Baio, J., et al., Prevalence of Autism Spectrum Disorder Among Children Aged 8 Years - Autism and Developmental Disabilities Monitoring Network, 11 Sites, United States, 2014. MMWR Surveill Summ, 2018. 67(6): p. 1-23. 2.Yizhar, O., et al., Neocortical excitation/inhibition balance in information processing and social dysfunction. Nature, 2011. 477(7363): p. 171-178. 3.Trakoshis, S., et al., Intrinsic excitation-inhibition imbalance affects medial prefrontal cortex differently in autistic men versus women. Elife, 2020. 9. 4.Palmen, S.J., et al., Neuropathological findings in autism. Brain, 2004. 127(Pt 12): p. 2572-83. 5.Tsai, P.T., et al., Autistic-like behaviour and cerebellar dysfunction in Purkinje cell Tsc1 mutant mice. Nature, 2012. 488(7413): p. 647-51. 6.Kelly, E., et al., Regulation of autism-relevant behaviors by cerebellar-prefrontal cortical circuits. Nat Neurosci, 2020. 23(9): p. 1102-1110. 7.Knutson, D.E., et al., Design and Synthesis of Novel Deuterated Ligands Functionally Selective for the γ-Aminobutyric Acid Type A Receptor (GABAAR) α6 Subtype with Improved Metabolic Stability and Enhanced Bioavailability. Journal of Medicinal Chemistry, 2018. 61(6): p. 2422-2446. 8.Watson, T.C., et al., Back to front: cerebellar connections and interactions with the prefrontal cortex. Front Syst Neurosci, 2014. 8: p. 4.