ABSTRACT / PROJECT SUMMARY Tourette syndrome (TS) is a disabling neurodevelopmental disorder characterized by motor and phonic tics. Available treatment strategies remain unsatisfactory, due to our limited knowledge of the biological foundations of this disorder. The studies proposed in this application will explore the mechanisms underlying two of the least well-understood biological characteristics of TS, namely its marked male predominance and stress sus- ceptibility. Studies from our group suggest that these features of TS are contributed by neuroactive steroids, a family of mediators implicated in sex and stress regulation. The typical age of onset of TS is 6-7 years, coinciding with adrenarche, a phase of adrenal maturation charac- terized by an upsurge in adrenal neuroactive steroids, such as dehydroepiandrosterone (DHEA) and its sulfate (DHEAS). In preliminary studies, we found that DHEA exacerbated tic-like responses in animal models of TS. Interestingly, the dose of DHEA needed to elicit TS-like responses in females is higher than those needed in males, possibly pointing to a mechanism of sex differences in TS. Stress reduces the ability of TS patients to suppress tics, but the underlying mechanisms remain unknown. Our studies in animal models indicate that this process may be due to the elevation of the neuroactive steroid allo- pregnanolone (AP) in the prefrontal cortex. By inhibiting the ability of the prefrontal cortex to suppress tics, AP promotes tic execution. In a pilot study, we found that tic suppression, a well-known stressful task in TS pa- tients, increases AP salivary levels. Furthermore, in another proof-of-concept study, we found that inhibiting AP synthesis led to a reduction in tic severity and facilitated voluntary control of tics in stressful situations. These findings lead us to hypothesize that TS patients exhibit alterations of the composition of their neuroac- tive steroid profiles, including: 1) an increase in baseline DHEA(S) levels in male TS patients, in correlation with lifetime severity; and 2) an exaggerated elevation in AP in response to acute stress. The two Aims of this proposal will test this hypothesis by: 1) comparing the baseline urinary steroidomic profile of TS-affected boys and girls with non-affected sex- and age-matched controls; and 2) charting the dynamic alterations in steroidomic salivary profiles in response to tic suppression. These studies will advance our un- derstanding of the endocrine mechanisms in TS and lead to the identification of potential biomarkers for the severity of tics and comorbid symptoms. In the long run, the results of these studies may open the way for the development of new therapies for TS that may reduce tic severity and increase patients' responsiveness to be- havioral interventions.