Mouse Study Suggests Need to Test Therapeutic Approaches Across Rett Models

Treatment with a protein called CNF1 produced different results in two different mouse models of Rett syndrome, demonstrating a need to test therapeutic approaches across disease models, according to a study.

Specifically, the CNF1 protein was able to rescue cognitive deficits, but not motor difficulties, in a mouse model of Rett syndrome with a mutation in the Mecp2 gene associated with severe disease.

In contrast, CNF1 rescued both cognitive and motor impairments in a Rett mouse model with an Mecp2 mutation linked to less severe disease.

“These results demonstrate that CNF1 efficacy depends on the mutation [carried] by MeCP2-mutated mice, stressing the need of testing potential therapeutic approaches across [Rett] models,” the researchers wrote.

The study, “Treatment with the Bacterial Toxin CNF1 Selectively Rescues Cognitive and Brain Mitochondrial Deficits in a Female Mouse Model of Rett Syndrome Carrying a MeCP2-Null Mutation,” was published in the International Journal of Molecular Sciences.

Most cases of Rett syndrome are caused by mutations in the MECP2 gene, resulting in a deficiency in working MECP2 protein, which plays a vital role in the activity of other genes essential for brain development and function.

These mutations are associated with difficulties in cognition, motor, emotional, and sensory skills, leading to learning, speech, movement, mood, and breathing problems.

The development and severity of these symptoms can depend on the type and location of the mutation in the MECP2 gene. Some mutations that result in a deletion at the end the amino acid chain of MECP2 are characterized by a less severe form of the disease, whereas other mutations can generate a shorter (truncated) protein, causing more severe disease.

Based on these findings, various Rett mouse models with different mutation types have been created to mimic the multiple degrees of disease severity.

Recently, researchers in Italy used a bacterial toxin protein called CNF1 to rescue cognitive and motor impairments in a Rett mouse model that carried an MECP2 mutation (MeCP2-308) associated with milder disease. They further showed that CNF1 restored alterations in the activity of mitochondria, the machinery responsible for energy production in the cell.

The same team has now tested the general CNF1 efficacy in a female mouse model with a mutation (MeCP2-Bird) that results in no protein being produced, mirroring what is found in about 10% of Rett patients and associated with more severe disease.

First, the mice were subjected to a fear conditioning memory test, which assesses cognitive ability. Mice were placed in a soundproof chamber and allowed to acclimatize (baseline), then exposed to sound that accompanied electric foot-shock.

The next day, they were returned to the chamber, and time spent without body movement (freezing) was measured either without sound (contextual fear) or after sound exposure (cued fear). Either CNF1 or a control was injected into the fluid surrounding the brain in each animal.

MeCP2-Bird mice showed less freezing with contextual fear than control animals, “suggesting the presence of defective fear memory,” the team wrote. CNF1 did not rescue this characteristic in Rett mice in this context. In contrast, CNF1 treatment did show an increased freezing response of MeCP2-Bird mice to normal levels in the cued fear memory test (after sound exposure).

Mice were then given a pre-pulse inhibition (PPI) test to measure if the brain can filter out irrelevant sensory stimulation by exposing them to sound that startles the animals. This test has been used in people with neuropsychiatric disorders.

Results showed no differences in MeCP2-Bird mice compared with controls regarding the percentage of PPI. CNF1 tended toward increased PPI percentage in both normal and MeCP2-Bird mice, with treatment showing a significant improvement of MeCP2-Bird mice performance at the loudest sound intensity. These results suggest that CNF1 treatment improved cognitive ability in Rett mice.

Next, motor function was measured using a nest-building test that required coordinated and goal-directed use of the front limbs. Nesting capacity was impaired in MeCP2-Bird mice compared with controls. CNF1 treatment did not rescue the quality of the nests built by Rett mice.

The rotarod test for motor coordination showed that, as expected, MeCP2-Bird mice spent less time on the rod than controls. Again, CNF1 treatment did not improve the motor coordination skills of MeCP2-Bird mice. A general health assessment confirmed that these mice displayed worse health. CNF1 treatment did not affect general health.

CNF1 was able to previously rescue impaired mitochondrial function in MeCP2-308 mouse brains with a shorter MECP2 protein. In MeCP2-Bird mice, there was a significant reduction in brain mitochondria activity. Consistent with MeCP2-308 mice, CNF1 treatment increased the activity of mitochondria to normal levels.

To further understand the molecular mechanisms, the team assessed the activity of the mTOR protein, which is a central regulator of many cell processes, including cell metabolism and growth, and is involved in Rett disease development.

The total levels of mTOR did not differ between MeCP2-308 and MeCP2-Bird mice, relative to controls. Notably, CNF1 treatment significantly increased mTOR levels in MeCP2-308 mice but not in MeCP2-Bird animals.

“In conclusion, the present study provides evidence that the CNF1 treatment may provide differential outcomes for RTT depending on the MeCP2 mutation,” the researchers wrote. “Taken together these results underline the importance of exploiting the available RTT mouse models carrying different MeCP2 mutations for testing the therapeutic efficacy of novel therapeutic approaches.”