An Epilepsy-Associated Mutation of Salt-Inducible Kinase 1 Increases the Susceptibility to Epileptic Seizures and Interferes with Adrenocorticotropic Hormone Therapy for Infantile Spasms in Mice.

Six mutations in the salt-inducible kinase 1 (SIK1) have been identified in developmental and epileptic encephalopathy (DEE-30) patients, and two of the mutations are nonsense mutations that truncate the C-terminal region of SIK1. In a previous study, we generated SIK1 mutant (SIK1-MT) mice recapitulating the C-terminal truncated mutations using CRISPR/Cas9-mediated genome editing and found an increase in excitatory synaptic transmission and enhancement of neural excitability in neocortical neurons in SIK1-MT mice. NMDA was injected into SIK1-MT males to induce epileptic seizures in the mice. The severity of the NMDA-induced seizures was estimated by the latency and the number of tail flickering and hyperflexion. Activated brain regions were evaluated by immunohistochemistry against c-fos, Iba1, and GFAP. As another epilepsy model, pentylenetetrazol was injected into the adult SIK1 mutant mice. Seizure susceptibility induced by both NMDA and PTZ was enhanced in SIK1-MT mice. Brain regions including the thalamus and hypothalamus were strongly activated in NMDA-induced seizures. The epilepsy-associated mutation of SIK1 canceled the pharmacological effects of the ACTH treatment on NMDA-induced seizures. These results suggest that SIK1 may be involved in the neuropathological mechanisms of NMDA-induced spasms and the pharmacological mechanism of ACTH treatment.

Risperidone Mitigates Enhanced Excitatory Neuronal Function and Repetitive Behavior Caused by an ASD-Associated Mutation of SIK1.

Six mutations in the salt-inducible kinase 1 (SIK1)-coding gene have been identified in patients with early infantile epileptic encephalopathy (EIEE-30) accompanied by autistic symptoms. Two of the mutations are non-sense mutations that truncate the C-terminal region of SIK1. It has been shown that the C-terminal-truncated form of SIK1 protein affects the subcellular distribution of SIK1 protein, tempting to speculate the relevance to the pathophysiology of the disorders. We generated SIK1-mutant (SIK1-MT) mice recapitulating the C-terminal-truncated mutations using CRISPR/Cas9-mediated genome editing. SIK1-MT protein was distributed in the nucleus and cytoplasm, whereas the distribution of wild-type SIK1 was restricted to the nucleus. We found the disruption of excitatory and inhibitory (E/I) synaptic balance due to an increase in excitatory synaptic transmission and enhancement of neural excitability in the pyramidal neurons in layer 5 of the medial prefrontal cortex in SIK1-MT mice. We also found the increased repetitive behavior and social behavioral deficits in SIK1-MT mice. The risperidone administration attenuated the neural excitability and excitatory synaptic transmission, but the disrupted E/I synaptic balance was unchanged, because it also reduced the inhibitory synaptic transmission. Risperidone also eliminated the repetitive behavior but not social behavioral deficits. These results indicate that risperidone has a role in decreasing neuronal excitability and excitatory synapses, ameliorating repetitive behavior in the SIK1-truncated mice.

DNA repair protein RAD51 enhances the CRISPR/Cas9-mediated knock-in efficiency in brain neurons.

Gene knock-in using the CRISPR/Cas9 system can be achieved in a specific population of neurons in the mouse brain, by using in utero electroporation to introduce DNA fragments into neural progenitor cells. Using this strategy, we previously knocked-in the EGFP coding sequence into the N-terminal region of the ß-actin gene specifically in the pyramidal neurons in layer 2/3 of the somatosensory cortex. However, the knock-in efficiency was less than 2% of the transfected neurons. In this study, we sought to improve the knock-in efficiency using this system. First, we varied the length of the homology arms of the ß-actin donor template DNA, and found that the knock-in efficiency was increased to ∼14% by extending the length of the 5' and 3' homology arms to 1.6 kb and 2.0 kb, respectively. We then tested the effect of the DNA repair protein RAD51 and the knock-in efficiency was increased up to 2.5-fold when co-transfecting with two different ß-actin and a camk2a targeting EGFP knock-in modules. The RAD51 overexpression did not alter the migration of developing neurons, density or morphology of the dendritic spines compared to those in neurons not transfected with RAD51. RAD51 expression will be useful for increasing the knock-in efficiency in neurons in vivo by CRISPR/Cas9-mediated homology directed repair (HDR).