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Understanding Neuronal Network Activity in Developmental and Epileptic Encephalopathy 9

Date of Publication: October 18, 2024

Developmental and epileptic encephalopathy 9 is a neurodevelopmental disorder caused by a mutation in PCDH19. Because disease only occurs in heterozygous PCDH19 individuals, scientists developed a mosaic mouse model where some cells express PCDH19 and others do not. This model helped them understand that PCDH19-negative neurons are hyperexcitable in a DEE9 mouse model and how the brain responds as a result.

Developmental and Epileptic Encephalopathies (DEE) is a group of neurodevelopmental disorders that’s characterized by early-onset seizures and developmental delays. Most cases of DEEs are due to genetic mutations, with many mutations linked to the X chromosome1. One of these X-linked DEEs is DEE9. People with DEE9 have varied symptoms including seizures, autism spectrum disorder (ASD), late-onset schizophrenia and degrees of intellectual disability.

The PCDH19 Protein

The gene behind DEE9 is Pcdh19 which codes for protocadherin-19. This gene is widely expressed across the central nervous system but is particularly highly expressed in the limbic system, which helps regulate behavior and emotion. Its expression peaks during periods where neuronal networks undergo significant remodeling, such as during embryonic development, although it continues to be expressed after birth and into adulthood. While previous work has shown the involvement of PCDH19 on synaptic transmission, it’s still unclear how PCDH19 mutations lead to the development of DEE9. Recent research led by scientists from the Institute of Neuroscience at CNR thus investigated what happens in brains containing PCDH19 mutations2.

Mimicking Pcdh19 mosaicism in mouse model

While DEE9 is an X-linked disease, it only affects those with a heterozygous PCDH19 mutation. Scientists think that only individuals with only one mutant PCDH19 gene get disease based on the cellular interference hypothesis3. In this hypothesis, cells with two X chromosomes undergo random X inactivation, meaning that some cells express PCDH19 while others do not. This mosaicism can lead to abnormalities when PCDH19 positive and PCDH19 negative neurons interact. Males with a PCDH19 mutation are unaffected carriers.

This mosaicism lends itself well to an animal model where one Pcdh19 allele can be turned on and off using Cre-lox. In this model, Pcdh19 exon 3 is flanked by loxP sites. Then, either by breeding or through intracerebroventricular injectipron, Cre expression triggers excision of the exon, producing a frameshift mutation. This generates a premature stop codon that targets the protein for mRNA nonsense-mediated decay.

Building and validating the Pcdh19 mosaic mouse model

To build their mouse model the researchers first confirmed whether excision of exon 3 eliminated expression of Pcdh19. They used primary cortical and hippocampal neurons from Pcdh19 floxed mice (Pcdh19fl/fl and Pcdh19fl/y) infected with GFP-Cre AAVs from Vector Biolabs, a Fortis Life Sciences® company. They found that these neurons did not produce Pcdh19 transcript when they expressed Cre. The researchers verified these results at the protein level with immunofluorescence and Western blot using an anti-PCDH19 antibody from Bethyl Laboratories, a Fortis Life Sciences company.

Taking this in vivo, the scientists created conditional knockout mice which are heterozygous for the Pcdh19 mutation (one floxed Pcdh19 and one wild-type Pcdh19). When they cross male mice expressing Cre, they generate the Pcdh19 mosaic mice (Pcdh19fl/x Syn1-Cre). Using RT-PCR and western blot, they found that these mice had about half the expression of the PCDH19 transcript and protein in the hippocampus and the cortex. While this data showed what was expected, assuming half of the cells inactivated the chromosome containing floxed Pcdh19 and half of the cells inactivated the chromosome containing the wild-type Pcdh19, it didn’t show what happened on the single-cell level. Therefore, the researchers performed immunofluorescence staining with the anti-PCDH19 antibody and found that some cells expressed PCDH19 while others did not (Figure 1).

Immunofluorescence staining of cortex brain slices.
Immunofluorescence staining with anti-PCDH19 antibody shows that some cells are PCDH19-negative while others are PCDH19-positive. Image from Giansante et al., 2024. CC BY 4.0
With the genetics confirmed, the researchers next examined whether the mosaic mice modeled DEE9 symptoms. Pcdh19 mosaic mice were more susceptible to drug-induced seizures than their Cre-negative counterparts. In behavioral experiments, they found that Pcdh19 mosaic mice, when compared to control mice, had difficulty with the Morris water maze, which tests spatial learning and short-term memory4, and the fear conditioning test, which measures learning and memory associated with fear5. Pcdh19 mosaic mice also had patterns in self-grooming activity associated with ASD in mouse models6. Based on these and other tests, the authors concluded that their mosaic mouse model replicated the symptoms of DEE9. The researchers found that short-term plasticity of CA1 neurons were impaired and identified other structural changes in CA1 neurons that could play a role in these behavioral changes.

Neuronal alterations in mosaic mice

To gain a better understanding of the neuronal changes that occur with DEE9, the researchers ran a series of experiments focusing on regions of the limbic system, as this region normally expresses high levels of PCDH19.

They began by examining individual PCDH19-positive and PCDH19-negative dentate gyrus granule cells in hippocampal slices because they previously found that decreased PCDH19 expression leads to increased excitability in the hippocampus7. In the current study, they saw that PCDH19-negative cells had increased firing frequencies compared to PCDH19-positive cells. When considering both PCDH19-negative and PCDH19-positive neurons from cultured neurons and ex vivo hippocampal brain slices from mosaic mice, these samples had reduced firing rates, burst rates, and increased neuronal synchronization. In hippocampal slices, they saw these traits persist from birth to adulthood.

Since the previous studies were done in vitro and ex vivo, the team next sought to see whether these results could be replicated in vivo. To do this, they recorded neuronal activity in mosaic mice in various areas of the limbic system using microelectrodes. This time, they used male mice with Cre recombinase expression at less than 100% to generate the mosaicism because these mice recovered better after surgery. They found that the mean firing rate was reduced in mosaic mice: 16% reduction in CA1 neurons, 47% reduction in the amygdala, 44% reduction in the entorhinal cortex, and 31% in the perirhinal cortex. The researchers examined the role of inhibitory and excitatory neurons in the overall activity and found an increase in inhibitory neuron activity and reduction in excitatory neuron activity in PCDH19 mosaic mice, possibily in an attempt to correct neuronal hyperexcitability and explaining the reduced network activity. They also noticed that the synapses rearranged to increase inhibitory signals towards PCDH19-negative hyperexcitable neurons.

Conclusions

The results from this study show that PCDH19-negative neurons in the mosaic brain are hyperexcitable. Perhaps in attempts to compensate for this activity or possibly a result of synaptic dysfunction, the limbic region has reduced its overall activity and increased inhibitory neuron activity in the DEE9 mouse model. This work adds further insights to the mechanism behind DEE9 and could help identify targets for future therapies for DEE9.

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References

  1. Happ, H. C., & Carvill, G. L. (2020). A 2020 View on the Genetics of Developmental and Epileptic Encephalopathies. Epilepsy currents, 20(2), 90–96. https://doi.org/10.1177/1535759720906118
  2. Giansante, G., et al. (2024). Neuronal network activity and connectivity are impaired in a conditional knockout mouse model with PCDH19 mosaic expression. Molecular psychiatry, 29(6), 1710–1725. https://doi.org/10.1038/s41380-023-02022-1
  3. Depienne, C., et al. (2011). Mutations and deletions in PCDH19 account for various familial or isolated epilepsies in females. Human mutation, 32(1), E1959–E1975. https://doi.org/10.1002/humu.21373
  4. Vorhees, C. V., & Williams, M. T. (2006). Morris water maze: procedures for assessing spatial and related forms of learning and memory. Nature protocols, 1(2), 848–858. https://doi.org/10.1038/nprot.2006.116
  5. Shoji, H., et al. (2014). Contextual and cued fear conditioning test using a video analyzing system in mice. Journal of visualized experiments : JoVE, (85), 50871. https://doi.org/10.3791/50871
  6. Liu, H., et al. (2021). Dissection of the relationship between anxiety and stereotyped self-grooming using the Shank3B mutant autistic model, acute stress model and chronic pain model. Neurobiology of stress, 15, 100417. https://doi.org/10.1016/j.ynstr.2021.100417
  7. Serratto, G. M., et al. (2020). The Epilepsy-Related Protein PCDH19 Regulates Tonic Inhibition, GABAAR Kinetics, and the Intrinsic Excitability of Hippocampal Neurons. Molecular neurobiology, 57(12), 5336–5351. https://doi.org/10.1007/s12035-020-02099-7
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