In vivo CRISPRa decreases seizures and rescues cognitive deficits in a rodent model of epilepsy.

Epilepsy is a major health burden, calling for new mechanistic insights and therapies. CRISPR-mediated gene editing shows promise to cure genetic pathologies, although hitherto it has mostly been applied ex vivo. Its translational potential for treating non-genetic pathologies is still unexplored. Furthermore, neurological diseases represent an important challenge for the application of CRISPR, because of the need in many cases to manipulate gene function of neurons in situ. A variant of CRISPR, CRISPRa, offers the possibility to modulate the expression of endogenous genes by directly targeting their promoters. We asked if this strategy can effectively treat acquired focal epilepsy, focusing on ion channels because their manipulation is known be effective in changing network hyperactivity and hypersynchronziation. We applied a doxycycline-inducible CRISPRa technology to increase the expression of the potassium channel gene Kcna1 (encoding Kv1.1) in mouse hippocampal excitatory neurons. CRISPRa-mediated Kv1.1 upregulation led to a substantial decrease in neuronal excitability. Continuous video-EEG telemetry showed that AAV9-mediated delivery of CRISPRa, upon doxycycline administration, decreased spontaneous generalized tonic-clonic seizures in a model of temporal lobe epilepsy, and rescued cognitive impairment and transcriptomic alterations associated with chronic epilepsy. The focal treatment minimizes concerns about off-target effects in other organs and brain areas. This study provides the proof-of-principle for a translational CRISPR-based approach to treat neurological diseases characterized by abnormal circuit excitability.

Designer receptor technology for the treatment of epilepsy.

Epilepsy remains refractory to medical treatment in ~30% of patients despite decades of new drug development. Neurosurgery to remove or disconnect the seizure focus is often curative but frequently contraindicated by risks of irreversible impairment to brain function. Novel therapies are therefore required that better balance seizure suppression against the risks of side effects. Among experimental gene therapies, chemogenetics has the major advantage that the action on the epileptogenic zone can be modulated on demand. Two broad approaches are to use a designer G-protein-coupled receptor or a modified ligand gated ion channel, targeted to specific neurons in the epileptogenic zone using viral vectors and cell-type selective promoters. The receptor can be activated on demand by either an exogenous compound or by pathological levels of extracellular glutamate that occur in epileptogenic tissue. We review the principal designer receptor technologies and their modes of action. We compare the drawbacks and benefits of each designer receptor with particular focus on the drug activators and the potential for clinical translation in epilepsy.

Olanzapine: A potent agonist at the hM4D(Gi) DREADD amenable to clinical translation of chemogenetics.

Designer receptors exclusively activated by designer drugs (DREADDs) derived from muscarinic receptors not only are a powerful tool to test causality in basic neuroscience but also are potentially amenable to clinical translation. A major obstacle, however, is that the widely used agonist clozapine N-oxide undergoes conversion to clozapine, which penetrates the blood-brain barrier but has an unfavorable side effect profile. Perlapine has been reported to activate DREADDs at nanomolar concentrations but is not approved for use in humans by the Food and Drug Administration or the European Medicines Agency, limiting its translational potential. Here, we report that the atypical antipsychotic drug olanzapine, widely available in various formulations, is a potent agonist of the human M4 muscarinic receptor-based DREADD, facilitating clinical translation of chemogenetics to treat central nervous system diseases.

An ex vivo model to quantitatively analyze cell migration in tissue.

BACKGROUND: Within the developing central nervous system, the ability of cells to migrate throughout the tissue parenchyma to reach their target destination and undergo terminal differentiation is vital to normal central nervous system (CNS) development. To develop novel therapies to treat the injured CNS, it is essential that the migratory behavior of cell populations is understood. Many studies have examined the ability of individual neurons to migrate through the developing CNS, describing specific modes of migration including locomotion and somal translocation. Few studies have investigated the mass migration of large populations of neural progenitors, particularly in the developing the spinal cord. Here, we describe a method to robustly analyze large numbers of migrating cells using a co-culture assay. RESULTS: The ex vivo tissue model promotes the survival and differentiation of co-cultured progenitor cells. Using this assay, we demonstrate that migrating neuroepithelial progenitor cells display region specific migration patterns within the dorsal and ventral spinal cord at defined developmental time points. CONCLUSIONS: The technique described here is a viable ex vivo model to quantitatively analyze cell migration and differentiation. We demonstrate the ability to detect changes in cell migration within distinct tissue region across tissue samples using the technique described here. Developmental Dynamics 247:201-211, 2018. © 2017 Wiley Periodicals, Inc.

What role does tobacco smoking play in multiple sclerosis disability and mortality? A review of the evidence.

There is increasing evidence of tobacco smoking as an associative factor in multiple sclerosis (MS). Numerous studies have been conducted investigating the effects of smoking before the onset of MS as well as its impact on disease course. This special report reviews the available evidence and summarizes the contribution of smoking to increased mortality in patients with MS. It also explores some putative mechanisms for the involvement of tobacco constituents in the pathology of MS and the effects of smoking on disease-modifying treatments.

Tobacco smoking and excess mortality in multiple sclerosis: a cohort study.

OBJECTIVE: As patients with multiple sclerosis (MS) have more than 2.5-fold increased mortality risk, we sought to investigate the impact of tobacco smoking on the risk of premature death and its contribution to the excess mortality in MS patients. METHODS: We studied 1032 patients during the period 1994-2013 in a UK-based register. Cox regression model was used to investigate the impact of smoking on the risk of premature death, controlling for confounders. Smoking-specific mortality rates were compared with the UK general population. RESULTS: Of 923 patients with clinically definite MS, 80 (46 males and 34 females) had died by December 2012. HRs for death in current smokers and ex-smokers relative to never smokers were 2.70 (95% CI 1.59 to 4.58, p<0.001) and 1.30 (95% CI 0.72 to 2.32; p = 0.37). The standardised mortality ratio, compared with the UK general population, when stratified by smoking status was 3.83 (95% CI 2.71 to 5.42) in current smokers, 1.96 (95% CI 1.27 to 3.0) in ex-smokers and 1.27 (95% CI 0.87 to 1.86) in non-smokers. Never smokers and ex-smokers with MS had similar mortality rates compared with never smokers and ex-smokers without MS in the male British doctors cohort (1.12 (95% CI 0.63 to 1.97) and 0.54 (95% CI 0.26 to 1.14), respectively), while current smokers with MS had 84% higher rate of death compared with current smokers without MS (95% CI 1.24 to 2.72). CONCLUSIONS: Tobacco smoking can account for some of the excess mortality associated with MS and is a risk determinant for all-cause and MS-related death.