Abstracts

Rationale:
Many studies have examined the redundant functionality of Doublecortin (DCX) and Doublecortin-like kinase (DCLK1) and how their concurrent absence results in a severe epileptic phenotype. However, while the loss of DCLK1 results in a host of synaptic and neuropathological changes, its role in acquired epilepsy has never been explored in adult animals. In this study we examined the effect of DCLK1 loss in a pilocarpine model of epilepsy in mice and how disease both begins and progresses.

Methods:
In this study we utilized numerous molecular and pathological methods. We used Western blotting for protein quantification and immunocytochemistry for neuronal culture staining. We also did antibody staining, fluorojade staining, and Timm staining on fixed brain tissue. Our electrophysiological methods include EEG for live mouse brain activity tracking as well local field potential (LFP) recording in live brain slices for circuit activity measurement.

Results:
This study has found numerous lines of evidence establishing an important connection between DCLK1 epilepsy. Firstly, we recapitulated this result showing DCLK1 is also strongly upregulated in mice with epilepsy, and showed strong expression of DCLK1 throughout the hippocampus. This increase in expression is also apparent through IHC in relevant areas of the hippocampus as well as in primary hippocampal cultures. When we knocked out DCLK1 we found increased mortality from pilocarpine administration at two different concentrations. We also saw increased fluorojade positive cells in the hippocampus of knockout mice after pilocarpine injection. Pathology of these knockout epileptic mice revealed hallmarks of increased epileptic severity. This includes a far greater density of mossy fiber sprouting in the hippocampus and decreases in PV and SST interneurons. Interestingly, we also found an increase in calretinin positive cells in the dentate gyrus.
Upon quantifying the neurological activity of the hippocampus and cortex using EEG we found a greater number of sleeping spikes in the knockout epileptic animals compared to all other genetic and treatment groups. This was also apparent in increase in the number of sleeping spike series we found along with the number of spikes within each series. When examining the sleeping patterns of the knockout epileptic mice we found that they spent a greater time asleep and less time awake than all other groups. The delta values during both sleeping and waking periods were greater in the knockout epileptic mice as well.

Conclusions:
Based on this research there are numerous conclusions we can draw. While DCLK1/DCX double knockouts show the most fundamental defects, DCLK1 itself still plays an important role in modulation of the epileptic phenotype. Loss of DCLK1 can cause a more severe reaction to status epilepticus, revealing DCLK1 plays an important role in the brain’s ability to survive status epilepticus. Loss of DCLK1 in the epileptic animals also reveals changes on numerous electrical, behavioral, and neuropathological axes, implicating loss of DCLK1 as a fundamental disruption affecting essentially every aspect of the disease.

Funding: 5R01NS104428-03