Characterisation of the effect of genetic deletion ofMicroRNA-22 on brain structure and functional outcomes in experimental epilepsy.pdf (15.88 MB)
Characterisation of the effect of genetic deletion of MicroRNA-22 on brain structure and functional outcomes in experimental epilepsy
thesis
posted on 2020-08-12, 11:19 authored by Luiz Fernando Almeida SilvaEpilepsy is a serious chronic neurological disease that affects over 65 million
people worldwide. Temporal lobe epilepsy is the most common form of epilepsy in
adults and patients are often refractory to available pharmacotherapy.
Unfortunately, antiepileptic drugs only treat the symptoms and do not exert
disease-modifying effects in patients. It is crucial to develop new pharmacological
approaches to aid patients with drug-resistant epilepsy as well as the development
of pharmacotherapy with disease-modifying capability. The pathomechanisms
involved in the development and maintenance of the epileptic state remain
incompletely understood. However, gene profiling and other “omics” techniques
have revealed large-scale gene expression changes accompany brain insults that
cause epilepsy and the gene expression landscape remains altered in established
epilepsy. Therefore, identifying key regulators of this gene expression landscape
is critical if we are to develop novel disease-modifying therapies. In line with this
view, miRNAs have emerged as important contributors to the gene expression
landscape in health and disease through post-transcriptional mechanisms.
MiRNAs are small non-coding RNA that function as an additional regulatory layer
of gene expression. There is now extensive evidence that multiple miRNAs are
altered in experimental and human epilepsy and that targeting individual miRNAs
can influence seizures and neuropathological processes including inflammatory
signalling and gliosis.
MiRNA-22 was recently identified as a novel miRNA involved in opposing
hyperexcitability in experimental epilepsy, mainly by suppressing the P2rx7
receptor which drives microgliosis. As with most other work to date, however, the
functional evidence is based entirely on oligonucleotide targeting of the miRNA.
These offer incomplete silencing of variable duration and have potential off-target
effects. Genetic evidence for miRNA involvement in epilepsy remains scarce.
Therefore, the present thesis focused on obtaining genetic evidence for the role of
miR-22 in experimental status epilepticus and epilepsy using mice engineered to
lack miR-22. These mice were developed on a 129 background strain and
therefore the modelling of epilepsy in this strain was also a focus of studies here.
In the first of three Results Chapters, we identified a number of phenotypes in the
intra-amygdala kainic acid model of status epilepticus (SE) and epilepsy that
differed in 129 mice compared to the standard C57BL/6 strain used. These
included subdued convulsive behaviour and minor variations in the seizure
features on the electroencephalogram (EEG). However, neuropathology was
similar between strains. Importantly, studies characterised for the first time the
emergence of epilepsy after intra-amygdala KA-induced SE in a mouse strain
besides the C57BL/6. Overall, the results in this chapter showed that 129 mice are
suitable for use in subsequent studies.
The second Results Chapter featured a comprehensive analysis of the brains of
the miR-22 mice. Overall, the mice show little to no difference, with normal
appearing brain structures and neuron and glial populations. A subtle molecular
phenotype was noted, however, with increased levels of a miR-22 target. The
mice also appear more susceptible to seizures induced by the GABA antagonist
PTZ.
In the final results Chapter, studies investigated the response of the mice to SE in
the intra-amygdala kainic acid model. A key finding here was that female miR-22-
deficient mice had altered responses to SE the first sex difference observed for a
miRNA in an epilepsy study.. As a result, epilepsy-monitoring studies were
performed on male mice. Long-term telemetry-based EEG monitoring studies
demonstrated that male miR-22-deficient mice develop a more severe form of
epilepsy compared to wildtype mice, suggesting miR-22 is involved in adapting to
brain injuries, likely by suppressing inflammatory signalling. Studies involving the
reintroduction of miR-22 into the knockout mice failed to rescue the phenotype,
pointing to limitations in mimic-based miRNA therapies. Finally, gene expression
studies identified a number of novel genes and pathways disrupted in miR-22-
deficient mice that could explain the phenotype and point to novel targets for the
future.
Taken together, the studies in this thesis provide important genetic evidence for a
role for miR-22 in the pathomechanisms of epilepsy and point to opportunities as
well as challenges in miRNA-based therapies for the treatment of epilepsy.
Funding
Funded by National Council for Scientific and Technological Development (Cnpq), Brazil.
History
First Supervisor
Prof. David C. HenshallComments
A thesis submitted for the degree of Doctor of Philosophy from the Royal College of Surgeons in Ireland in 2019.Published Citation
Luiz Fernando AS. Characterisation of the effect of genetic deletion of MicroRNA-22 on brain structure and functional outcomes in experimental epilepsy. [PhD Thesis]. Dublin: Royal College of Surgeons in Ireland; 2019.Degree Name
- Doctor of Philosophy (PhD)
Date of award
2019-06-30Programme
- Doctor of Philosophy (PhD)
