Nanoparticle encapsulated oxytocin increases resistance to induced seizures and restores social behavior in Scn1a-derived epilepsy.

Oxytocin (OT) has broad effects in the brain and plays an important role in cognitive, social, and neuroendocrine function. OT has also been identified as potentially therapeutic in neuropsychiatric disorders such as autism and depression, which are often comorbid with epilepsy, raising the possibility that it might confer protection against the behavioral and seizure phenotypes in epilepsy. Dravet syndrome (DS) is an early-life encephalopathy associated with prolonged and recurrent early-life febrile seizures (FSs), treatment-resistant afebrile epilepsy, and cognitive and behavioral deficits. De novo loss-of-function mutations in the voltage-gated sodium channel SCN1A are the main cause of DS, while genetic epilepsy with febrile seizures plus (GEFS+), also characterized by early-life FSs and afebrile epilepsy, is typically caused by inherited mutations that alter the biophysical properties of SCN1A. Despite the wide range of available antiepileptic drugs, many patients with SCN1A mutations do not achieve adequate seizure control or the amelioration of associated behavioral comorbidities. In the current study, we demonstrate that nanoparticle encapsulation of OT conferred robust and sustained protection against induced seizures and restored more normal social behavior in a mouse model of Scn1a-derived epilepsy. These results demonstrate the ability of a nanotechnology formulation to significantly enhance the efficacy of OT. This approach will provide a general strategy to enhance the therapeutic potential of additional neuropeptides in epilepsy and other neurological disorders.

The RNA-binding protein, ZC3H14, is required for proper poly(A) tail length control, expression of synaptic proteins, and brain function in mice.

A number of mutations in genes that encode ubiquitously expressed RNA-binding proteins cause tissue specific disease. Many of these diseases are neurological in nature revealing critical roles for this class of proteins in the brain. We recently identified mutations in a gene that encodes a ubiquitously expressed polyadenosine RNA-binding protein, ZC3H14 (Zinc finger CysCysCysHis domain-containing protein 14), that cause a nonsyndromic, autosomal recessive form of intellectual disability. This finding reveals the molecular basis for disease and provides evidence that ZC3H14 is essential for proper brain function. To investigate the role of ZC3H14 in the mammalian brain, we generated a mouse in which the first common exon of the ZC3H14 gene, exon 13 is removed (Zc3h14Δex13/Δex13) leading to a truncated ZC3H14 protein. We report here that, as in the patients, Zc3h14 is not essential in mice. Utilizing these Zc3h14Δex13/Δex13mice, we provide the first in vivo functional characterization of ZC3H14 as a regulator of RNA poly(A) tail length. The Zc3h14Δex13/Δex13 mice show enlarged lateral ventricles in the brain as well as impaired working memory. Proteomic analysis comparing the hippocampi of Zc3h14+/+ and Zc3h14Δex13/Δex13 mice reveals dysregulation of several pathways that are important for proper brain function and thus sheds light onto which pathways are most affected by the loss of ZC3H14. Among the proteins increased in the hippocampi of Zc3h14Δex13/Δex13 mice compared to control are key synaptic proteins including CaMK2a. This newly generated mouse serves as a tool to study the function of ZC3H14 in vivo.

Reduced cannabinoid 2 receptor activity increases susceptibility to induced seizures in mice.

OBJECTIVE: The endocannabinoid system (ECS) is comprised of cannabinoid receptors 1 and 2 (CB1R and CB2R), endogenous ligands, and regulatory enzymes, and serves to regulate several important physiological functions throughout the brain and body. Recent evidence suggests that the ECS may be a promising target for the treatment of epilepsy, including epilepsy subtypes that arise from mutations in the voltage-gated sodium channel SCN1A. The objective of this study was to explore the effects of modulating CB2R activity on seizure susceptibility. METHODS: We examined susceptibility to induced seizures using a number of paradigms in CB2R knockout mice (Cnr2-/- ), and determined the effects of the CB2R agonist, JWH-133, and the CB2R antagonist, SR144528, on seizure susceptibility in wild-type mice. We also examined seizure susceptibility in Cnr2 mutants harboring the human SCN1A R1648H (RH) epilepsy mutation and performed Electroencephalography (EEG) analysis to determine whether the loss of CB2Rs would increase spontaneous seizure frequency in Scn1a RH mutant mice. RESULTS: Both heterozygous (Cnr2+/- ) and homozygous (Cnr2-/- ) knockout mice exhibited increased susceptibility to pentylenetetrazole (PTZ)-induced seizures. The CB2R agonist JWH-133 did not significantly alter seizure susceptibility in wild-type mice; however, administration of the CB2R antagonist SR144528 resulted in increased susceptibility to PTZ-induced seizures. In offspring from a cross between the Cnr2 × RH lines, both Cnr2 and RH mutants were susceptible to PTZ-induced seizures; however, seizure susceptibility was not significantly increased in mutants expressing both mutations. No spontaneous seizures were observed in either RH or Cnr2/RH mutants during 336-504 hours of continuous EEG recordings. SIGNIFICANCE: Our results demonstrate that reduced CB2R activity is associated with increased seizure susceptibility. CB2Rs might therefore provide a therapeutic target for the treatment of some forms of epilepsy.

In vitro skin expansion: Wound healing assessment.

For treatments requiring split-thickness skin grafts, it is preferable to mesh the grafts. This reduces the amount of excised skin and covers more wound area. The mesh technique, however, destroys surface continuity, which results in scarring. Strain-based bioreactors, on the other hand, have successfully expanded split-thickness skin grafts in vitro within a 7-day period, increasing graft coverage. After in vitro expansion, the expanded skin grafts were tested in a porcine full-thickness excisional wound model. Expanded graft take rate was 100%. Volumetric, histologic, and mechanical assessments indicated that expanded grafts were comparable to unexpanded grafts (positive control). While there was considerable variation in expansion (31% to -3.1%), this technique has the potential to enhance the coverage area of skin grafts while reducing or eliminating scarring.

Cannabidiol Increases Seizure Resistance and Improves Behavior in an Scn8a Mouse Model.

Voltage-gated sodium channel genes are an important family of human epilepsy genes. De novo missense mutations in SCN8A (encoding Nav1.6) are associated with a spectrum of clinical presentation, including multiple seizure types, movement disorders, intellectual disability, and behavioral abnormalities such as autism. Patients with SCN8A mutations are often treated with multiple antiepileptic drugs, the most common being sodium channel blockers. Cannabidiol (CBD) has been included as a component of treatment regimens for some SCN8A patients; however, to date, there are no clinical trials that have evaluated the therapeutic potential of CBD in patients with SCN8A mutations. In the current manuscript, we demonstrated a dose-dependent increase in seizure resistance following CBD treatment in mice expressing the human SCN8A mutation R1620L (RL/+). We also found that CBD treatment improved social behavior and reduced hyperactivity in the RL/+ mutants. Our findings suggest that CBD may be beneficial in patients with SCN8A-associated disease.

Allosteric modulation of the cannabinoid 2 receptor confers seizure resistance in mice.

Mounting evidence suggests that modulation of cannabinoid 2 receptors (CB2Rs) is therapeutic in mouse models of neurological disorders, including neuropathic pain, neurodegenerative disease, and stroke. We previously showed that reducing CB2R activity increases seizure susceptibility in mice. In the present study, we evaluated the therapeutic potential of the CB2R positive allosteric modulator, Ec21a, against induced seizures in mice. The pharmacokinetic profile of Ec21 demonstrated a similar distribution in brain and plasma, with detection up to 12 h following injection. Ec21a increased resistance to induced seizures in CF1 wild-type mice and mice harboring the SCN1A R1648H human epilepsy mutation. A rotarod test provided evidence that Ec21a does not cause neurotoxicity-induced motor deficits at its therapeutic dose, and seizure protection was maintained with repeated drug administration. The selectivity of Ec21a for CB2R was supported by the ability of the CB2R antagonist AM630, but not the CB1R antagonist AM251, to block Ec21a-conferred seizure protection in mice, and a lack of significant binding of Ec21a to 34 brain-expressed receptors and transporters in vitro. These results identify allosteric modulation of CB2Rs as a promising therapeutic approach for the treatment of epilepsy.

Pathogenic in-Frame Variants in SCN8A: Expanding the Genetic Landscape of SCN8A-Associated Disease.

Numerous SCN8A mutations have been identified, of which, the majority are de novo missense variants. Most mutations result in epileptic encephalopathy; however, some are associated with less severe phenotypes. Mouse models generated by knock-in of human missense SCN8A mutations exhibit seizures and a range of behavioral abnormalities. To date, there are only a few Scn8a mouse models with in-frame deletions or insertions, and notably, none of these mouse lines exhibit increased seizure susceptibility. In the current study, we report the generation and characterization of two Scn8a mouse models (ΔIRL/+ and ΔVIR/+) carrying overlapping in-frame deletions within the voltage sensor of domain 4 (DIVS4). Both mouse lines show increased seizure susceptibility and infrequent spontaneous seizures. We also describe two unrelated patients with the same in-frame SCN8A deletion in the DIV S5-S6 pore region, highlighting the clinical relevance of this class of mutations.

In vitro evaluation of functionalized decellularized muscle scaffold for in situ skeletal muscle regeneration.

Current treatment options for repairing volumetric muscle loss injury involve the use of existing host tissue like muscular flaps or grafts. However, host muscle tissue may not be available and donor site morbidity, such as functional loss and volume deficiency, is often present. In this study, we developed a biofunctionalized muscle-derived decellularized extracellular matrix scaffolding system to utilize endogenous stem/progenitor cells for in situ muscle tissue regeneration. We optimized the decellularization process to enhance cellular infiltration and fabricated an insulin-like growth factor-binding protein 3 (IGFBP-3)-conjugated scaffold for controlled delivery of IGF-I. We then tested in vitro characterization including IGF-I release kinetics and cellular infiltration. In addition, we have analyzed the bioactivities of skeletal muscle cells (C2C12) to assess the indirect effect of released IGF-1 from the scaffold. The IGFBP-3 conjugated scaffolds demonstrated showed sustained release of IGF-1 and 1% SDS decellularized scaffold with IGF-1 showed higher cellular infiltration compared to control scaffolds (no conjugation). In indirect bioactivity assay, IGF-1 conjugated scaffold showed 2.1-fold increased cell activity compared to control (fresh media). Our results indicate that IGFBP-3/IGF-I conjugated scaffold has the potential to be used for in situ muscle tissue regeneration.