A conditional Vrk1 cholinergic knockout mouse recapitulates severe human motor neuron disease

Mutations in the VRK1 (Vaccinia-related kinase 1) gene have been implicated in neurodegenerative disease, primarily affecting motor neurons (MNs) and brain development. However, VRK1’s role in MN function and underlying disease mechanisms remain poorly understood. In this study, we investigated VRK1’s role in motor neuron disease (MND) by generating a conditional knockout (cKO) mouse model lacking VRK1 in cholinergic neurons. We created a Vrk1 -targeted Cre-lox MND mouse model, in which Cre recombination resulted in a null mutation of Vrk1 . Cre-recombinase was expressed in cholinergic neurons, including MNs, generating a cholinergic neuron-specific Vrk1 KO mouse ( Vrk1 ChAT - cKO). The phenotype was analyzed by observation and motor testing; MN degeneration, neuromuscular junction (NMJ) denervation, and muscle atrophy were assessed using histological and immunostaining techniques. Vrk1 ChAT-cKO mice showed progressive motor degeneration, including weight loss, reduced grip strength, tail dragging, decreased mobility, tremors, kyphosis, and respiratory distress, leading to premature death by approximately 27 days. Histological analysis of spinal cord sections from postnatal day 24-27 cKO mice revealed significant MN degeneration, including vacuolation, chromatolysis, apoptosis and neuroinflammation, with a 40-50% loss of ChAT positive MNs. Remaining MNs exhibited a 36-44% reduction in mean soma size. NMJ analysis showed 60% denervation and abnormal postsynaptic AChR cluster maturation. Muscle histology revealed nearly 79% reduction in myofiber area. The Vrk1 ChAT - cKO mouse model recapitulates the severe MN phenotype observed in human VRK1 -related neurodegeneration, underscoring VRK1's critical role in MN integrity. The progressive motor dysfunction, NMJ abnormalities and muscle atrophy, suggest that VRK1 is essential for MN survival and neuromuscular functions. This model offers a valuable tool for studying VRK1-associated MND and could contribute to the development and assessment of therapeutic strategies targeting VRK1 pathways. Additionally, it may provide insights into shared mechanisms between other MNDs, such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA).