Background: Chronic anxiety is common, disabling, and often refractory to current therapies. Mounting evidence implicates mitochondrial abnormalities in anxiety-related phenotypes. Urolithin A (UA), a gut microbiota-derived metabolite known to enhance mitochondrial health, has shown neuroprotective effects. However, its potential to alleviate anxiety remains unexplored.

Methods: UA was administered chronically to two validated rodent models of high anxiety: outbred animals displaying natural variation in trait anxiety and rats selectively bred for high stress-reactivity, while low-anxiety animals served as controls. Anxiety-like behaviors were assessed across multiple tasks. Molecular profiling of nucleus accumbens (NAc) medium spiny neurons (MSNs) was performed using single-nucleus RNA sequencing, with MitoPathway analysis to evaluate mitochondria-related transcriptomic signatures. Electrophysiological, immunohistochemical, and morphological approaches were used to assess synaptic and structural correlates.

Results: UA produced a robust anxiolytic effect in both high-anxiety models, in both sexes, without altering behavior in low-anxiety animals. High-anxiety MSNs displayed coupled dysregulation of mitochondrial and synaptic gene pathways that UA normalized to low-anxiety levels across MSN subtypes. These changes were accompanied by structural and functional rescue of MSN dendritic architecture, spine density, and excitatory synaptic transmission. Notably, UA also restored expression of mitofusin-2 (Mfn2), a mitochondrial protein causally involved in the regulation of anxiety-related behavior and circuit dysfunction in the NAc, further supporting a mechanistic link between mitochondrial remodeling and UA's anxiolytic efficacy.

Conclusions: These findings position UA as a mechanistically informed intervention in preclinical models of heightened anxiety and provide systems-level insights into how mitochondrial pathways interface with synaptic function and circuit regulation in anxiety states.