KCC2 neuromodulation remodels metabolic and morphofunctional signatures across the neuromuscular axis in mdx mice

Duchenne muscular dystrophy is a hereditary neuromuscular disorder caused by dystrophin deficiency, leading to progressive muscle degeneration and weakness. Beyond muscle pathology, this condition is systemic and also affects central and peripheral nervous system function, promoting synaptic imbalance, metabolic alterations, and neuronal hyperexcitability. In this study, we investigated whether pharmacological modulation of chloride homeostasis using the CLP290, a carbamate prodrug of CLP257, could restore synaptic, metabolic, and functional balance under dystrophic conditions. Four-week-old dystrophic mice and non-dystrophic controls received CLP290 10 mg·kg⁻¹ via an intradermal biomembrane implant with controlled release for seven consecutive days. Motor performance was assessed using automated gait analysis, while molecular and cellular changes were evaluated by immunohistochemistry of lumbar spinal cord segments and protein expression analysis in skeletal muscle. Metabolic adaptations were examined using nuclear magnetic resonance-based metabolomics of neural tissues combined with liquid chromatography-mass spectrometry-based metabolomics of skeletal muscle. Treatment with significantly improved gait stability, balance, and plantar contact dynamics without detectable toxic effects. Functional improvements were associated with increased expression of the neuronal potassium-chloride cotransporter and glutamate decarboxylase in presynaptic inputs to spinal alpha motor neurons, suggesting enhanced chloride regulation, strengthened inhibitory neurotransmission, and reduced neuronal excitability. Metabolomic analyses revealed coordinated metabolic remodeling in central and peripheral tissues, including bioenergetic adaptations in the spinal cord and partial normalization of dystrophic muscle metabolic profiles. Together, these findings support an integrated neural and metabolic mechanism underlying the functional benefits of the treatment and highlight its potential as an adjuvant strategy for early intervention in Duchenne muscular dystrophy.