Recently, the Jeremy M. Sullivan/Charlotte J. Sumner team at Johns Hopkins University published a study. They found that mutations in the transient receptor potential vanilloid receptor 4 (Trpv4) gene in neurovascular endothelial cells can destroy the integrity of the blood-spinal cord barrier and drive the degeneration of motor neurons in a non-cell autonomous manner. Administration of TRPV4-specific antagonists can restore the integrity of the blood-spinal cord barrier and improve the motor neuron degeneration phenotype of Trpv4 mutant mice. The relevant research was published in Science Translational Medicine.
TRPV4 is a member of the transient receptor potential ion channel family. Recent studies have shown that dominant missense mutations in Trpv4 can cause distal spinal muscular atrophy (dSMA) and Charcot-Marie-Tooth disease type 2 (CMT2). The typical manifestations of these diseases are weakness of the neck, diaphragm, limbs, and vocal cord muscles innervated by spinal and brainstem motor neurons.
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Given that the blood-spinal cord barrier is a powerful weapon to protect spinal cord neurons from neurotoxic substances. Therefore, the research team speculated that dominant missense mutations in Trpv4 may have destroyed the blood-spinal cord barrier, thereby causing damage to spinal cord neurons and ultimately causing motor neuron diseases. To verify this hypothesis, the research team introduced two mutations (R269C or R232C, corresponding to dSMA and CMT2) into the mouse Trpv4 gene and generated two Trpv4 mutant mouse models. The results showed that Trpv4R269C/R269C mice showed motor behavior defects within three weeks after birth and died before weaning age.
The initial manifestation of motor behavior defects is mild impairment of motor ability (stage A). Then, within 24 to 36 hours before death, it gradually progresses to forelimb paralysis, kyphosis, and difficulty in keeping the head upright (this is stage B). Finally, within a few hours before death, the ability to walk is lost and breathing becomes difficult (this is stage C and D). These behaviors can also be observed in Trpv4R232C/+ mice (so the following uses Trpv4R269C/R269C mice as an example).
Figure 1. Mutant mice show motor behavior disorders. (Sullivan J M, et al., 2024)
Since motor behavior defects mainly occurred in the neck and forelimbs, the research team conducted a histopathological evaluation of the cervical spinal cord of Trpv4R269C/R269C mutant mice in stage C. The results showed that obvious motor neuron loss and proximal axonal lesions occurred in the upper cervical spinal cord segments, especially in the C1 segment. Proximal axonal lesions can also be observed in the C2 and C3 segments, but there is no neuronal loss. The above results indicate that the motor behavior defects of Trpv4 mutant mice are related to the loss and degeneration of motor neurons.
Figure 2. Motor neuron loss. (Sullivan J M, et al., 2024)
Next, the research team deleted the Trpv4 mutant gene in some cell types related to motor nerves, such as neurons, glial cells, muscles, and neural endothelial cells, trying to see if this could improve the behavioral defects of mice. The results showed that the survival rate of mice was significantly improved only after the Trpv4 mutant gene in neural endothelial cells was deleted. More importantly, the specific deletion of the Trpv4 mutant gene from neural endothelial cells also eliminated the motor behavior defects of weaned mice, as well as the loss of motor neurons in the C1 segment of the cervical spinal cord and proximal axonal lesions.
Further, the research team wanted to know the effect of the Trpv4 mutation on neural endothelial cells, so they isolated primary neural endothelial cells from the brain and spinal cord of Trpv4R269C/R269C mice. Using fluorescent calcium imaging technology and electrophysiological experiments, the research team observed that under the action of the TRPV4-specific agonist GSK101, the isolated neural endothelial cells showed an increase in current density. This shows that the mutant TRPV4 channel is abnormally active and the channel activity is enhanced.
Analysis of the transendothelial resistance of neural endothelial cells showed that this value dropped rapidly after the activity of the TRPV4 channel increased. Since this value is an indicator for evaluating the integrity of the blood-spinal cord barrier (a decrease means that the barrier is damaged), this also means that the increase in TRPV4 channel activity will lead to the loss of blood-spinal cord barrier integrity. Subsequently, the research team verified this phenomenon in mice. By injecting a tracer (EZ-biotin, which normally cannot penetrate the intact blood-spinal cord barrier), the research team found that Trpv4 mutant mice with obvious motor behavior disorders (stage B) had obvious tracer leakage in the upper cervical spinal cord, while there was no obvious tracer leakage in the lower cervical spinal cord and lumbar region.
Figure 3. The integrity of the blood-spinal cord barrier is disrupted (tracer leakage). (Sullivan J M, et al., 2024)
This shows that the mutation of Trpv4 in neural endothelial cells activates its channels, further destroying the integrity of the blood-spinal cord barrier, causing local damage to the blood-spinal cord barrier, driving the degeneration of motor neurons in a non-cell autonomous manner, and thus leading to the occurrence of neurodegenerative diseases.
Finally, the research team also found that when the TRPV4-specific antagonist GSK219 was intraperitoneally injected into Trpv4 mutant mice, the survival rate of the mice was significantly improved, and the motor behavior disorder was also significantly improved. When the highest dose was given, the motor behavior disorder of Trpv4 mutant mice could be reversed. In addition, GSK219 treatment also restored the integrity of the blood-spinal cord barrier, as well as motor neuron loss and axonal lesions. In conclusion, this study found that Trpv4 mutation is a driving factor for blood-spinal cord barrier damage and motor neuron degeneration. TRPV4-specific antagonists may be an effective means of treating motor neuron diseases mediated by Trpv4 mutations.
Reference
Sullivan J M, et al. Gain-of-function mutations of TRPV4 acting in endothelial cells drive blood-CNS barrier breakdown and motor neuron degeneration in mice. Science translational medicine, 2024, 16(748): eadk1358.