Novel beta-glucocerebrosidase chaperone compounds identified from cell-based screening reduce pathologically accumulated glucosylsphingosine in iPS-derived neuronal cells.

The beta-glucocerebrosidase (GBA1) gene encodes the lysosomal beta-glucocerebrosidase (GCase) that metabolizes the lipids glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph). Biallelic loss-of-function mutations in GBA1 such as L444P cause Gaucher disease (GD), which is the most prevalent lysosomal storage disease and is histopathologically characterized by abnormal accumulation of the GCase substrates GlcCer and GlcSph. GD with neurological symptoms is associated with severe mutations in the GBA1 gene, most of which cause impairment in the process of GCase trafficking to lysosomes. Given that recombinant GCase protein cannot cross the blood-brain barrier due to its high molecular weight, it is invaluable to develop a brain-penetrant small-molecule pharmacological chaperone as a viable therapeutic strategy to boost GCase activity in the central nervous system. Despite considerable efforts to screen potent GCase activators/chaperones, cell-free assays using recombinant GCase protein have yielded compounds with only marginal efficacy and micromolar EC50 that would not have sufficient clinical efficacy or an acceptable safety margin. Therefore, we utilized a fluorescence-labeled GCase suicide inhibitor, MDW933, to directly monitor lysosomal GCase activity and performed a cell-based screening in fibroblasts from a GD patient with homozygotic L444P mutations. Here, we identified novel compounds that increase the fluorescence signal from labeled GCase with L444P mutations in a dose-dependent manner. Secondary assays using an artificial cell-permeable lysosomal GCase substrate also demonstrated that the identified compounds augment lysosomal GCase L444P in the fibroblast. Moreover, those compounds increased the total GCase L444P protein levels, suggesting the pharmacological chaperone-like mechanism of action. To further elucidate the effect of the compounds on the endogenous GCase substrate GlcSph, we generated iPSC-derived dopaminergic neurons with a GBA1 L444P mutation that exhibit GlcSph accumulation in vitro. Importantly, the identified compounds reduce GlcSph in iPSC-derived dopaminergic neurons with a GBA1 L444P mutation, indicating that the increase in lysosomal GCase resulting from application of the compounds leads to the clearance of pathologically-accumulated GlcSph. Together, our findings pave the way for developing potent and efficacious GCase chaperone compounds as a potential therapeutic approach for neurological GD.

Phosphorylation of α-synuclein at T64 results in distinct oligomers and exerts toxicity in models of Parkinson's disease.

α-Synuclein accumulates in Lewy bodies, and this accumulation is a pathological hallmark of Parkinson's disease (PD). Previous studies have indicated a causal role of α-synuclein in the pathogenesis of PD. However, the molecular and cellular mechanisms of α-synuclein toxicity remain elusive. Here, we describe a novel phosphorylation site of α-synuclein at T64 and the detailed characteristics of this post-translational modification. T64 phosphorylation was enhanced in both PD models and human PD brains. T64D phosphomimetic mutation led to distinct oligomer formation, and the structure of the oligomer was similar to that of α-synuclein oligomer with A53T mutation. Such phosphomimetic mutation induced mitochondrial dysfunction, lysosomal disorder, and cell death in cells and neurodegeneration in vivo, indicating a pathogenic role of α-synuclein phosphorylation at T64 in PD.

Antidepressant-like effect of male mating behavior through oxytocin-induced CREB signaling.

Male sexual activity reduces the level of depression through oxytocin (OT) release within the brain. In this study, we showed that male mating behavior reduces depression-like behavior through OT-induced cAMP response element binding protein (CREB) signaling in the hippocampus. Moreover, we showed that mating behavior in wild-type (WT) male mice increased CREB phosphorylation in hippocampus, whereas that OT receptor knockout (OTR KO) male mice had no effect on CREB phosphorylation. CREB phosphorylation in hippocampus was also increased after OT induction in hippocampal slices prepared from WT mice. In addition, male mating behavior induced the expression of brain-derived neurotrophic factor (BDNF), which was not observed in OTR KO mice. Antidepressant-like effect of mating behavior had no effect in OTR KO mice. These findings suggest that male sexual activity has antidepressant effects through OT-induced CREB signaling in the hippocampus.

Discovery of Novel HCN4 Blockers with Unique Blocking Kinetics and Binding Properties.

The hyperpolarization-activated cyclic nucleotide-gated 4 (HCN4) channel underlies the pacemaker currents, called "If," in sinoatrial nodes (SANs), which regulate heart rhythm. Some HCN4 blockers such as ivabradine have been extensively studied for treating various heart diseases. Studies have shown that these blockers have diverse state dependencies and binding sites, suggesting the existence of potential chemical and functional diversity among HCN4 blockers. Here we report approaches for the identification of novel HCN4 blockers through a random screening campaign among 16,000 small-molecule compounds using an automated patch-clamp system. These molecules exhibited various blockade profiles, and their blocking kinetics and associating amino acids were determined by electrophysiological studies and site-directed mutagenesis analysis, respectively. The profiles of these blockers were distinct from those of the previously reported HCN channel blockers ivabradine and ZD7288. Notably, the mutagenesis analysis showed that blockers with potencies that were increased when the channel was open involved a C478 residue, located at the pore cavity region near the cellular surface of the plasma membrane, while those with potencies that were decreased when the channel was open involved residues Y506 and I510, located at the intracellular region of the pore gate. Thus, this study reported for the first time the discovery of novel HCN4 blockers by screening, and their profiling analysis using an automated patch-clamp system provided chemical tools that will be useful to obtain unique molecular insights into the drug-binding modes of HCN4 and may contribute to the expansion of therapeutic options in the future.

Self-assembling A6K peptide nanotubes as a mercaptoundecahydrododecaborate (BSH) delivery system for boron neutron capture therapy (BNCT).

Boron neutron capture therapy (BNCT) is a tumor selective therapy, the effectiveness of which depends on sufficient 10B delivery to and accumulation in tumors. In this study, we used self-assembling A6K peptide nanotubes as boron carriers and prepared new boron agents by simple mixing of A6K and BSH. BSH has been used to treat malignant glioma patients in clinical trials and its drug safety and availability have been confirmed; however, its contribution to BNCT efficacy is low. A6K nanotube delivery improved two major limitations of BSH, including absence of intracellular transduction and non-specific drug delivery to tumor tissue. Varying the A6K peptide and BSH mixture ratio produced materials with different morphologies-determined by electron microscopy-and intracellular transduction efficiencies. We investigated the A6K/BSH 1:10 mixture ratio and found high intracellular boron uptake with no toxicity. Microscopy observation showed intracellular localization of A6K/BSH in the perinuclear region and endosome in human glioma cells. The intracellular boron concentration using A6K/BSH was almost 10 times higher than that of BSH. The systematic administration of A6K/BSH via mouse tail vein showed tumor specific accumulation in a mouse brain tumor model with immunohistochemistry and pharmacokinetic study. Neutron irradiation of glioma cells treated with A6K/BSH showed the inhibition of cell proliferation in a colony formation assay. Boron delivery using A6K peptide provides a unique and simple strategy for next generation BNCT drugs.

A Novel Inhibition Modality for Phosphodiesterase 2A.

Phosphodiesterase type 2A (PDE2A) has received considerable interest as a molecular target for treating central nervous system diseases that affect memory, learning, and cognition. In this paper, the authors present the discovery of small molecules that have a novel modality of PDE2A inhibition. PDE2A possesses GAF-A and GAF-B domains and is a dual-substrate enzyme capable of hydrolyzing both cGMP and cAMP, and activation occurs through cGMP binding to the GAF-B domain. Thus, positive feedback of the catalytic activity to hydrolyze cyclic nucleotides occurs in the presence of appropriate concentrations of cGMP, which binds to the GAF-B domain, resulting in a "brake" that attenuates downstream cyclic nucleotide signaling. Here, we studied the inhibitory effects of some previously reported PDE2A inhibitors, all of which showed impaired inhibitory effects at a lower concentration of cGMP (70 nM) than a concentration effective for the positive feedback (4 µM). This impairment depended on the presence of the GAF domains but was not attributed to binding of the inhibitors to these domains. Notably, we identified PDE2A inhibitors that did not exhibit this behavior; that is, the inhibitory effects of these inhibitors were as strong at the lower concentration of cGMP (70 nM) as they were at the higher concentration (4 µM). This suggests that such inhibitors are likely to be more effective than previously reported PDE2A inhibitors in tissues of patients with lower cGMP concentrations.

Oxytocin and Stress: Neural Mechanisms, Stress-Related Disorders, and Therapeutic Approaches.

Clinical reports show that oxytocin (OT) is related to stress-related disorders such as depression, anxiety disorder, and post-traumatic stress disorder. Two key structures in the brain should be paid special attention with regard to stress regulation, namely, the hypothalamus and the hippocampus. The former is the region for central command for most, if not all, of the major endocrine systems, and the latter takes a key position in the regulation of mood and anxiety. There are extensive neural projections between the two structures, and both are functionally intertwined. The hypothalamus projects OTergic neurons to the hippocampus, and the latter possesses high levels of OT receptors. The hippocampus also regulates the secretion of glucocorticoids, a major group of stress hormones. Excessive levels of glucocorticoids in chronic stress cause atrophy of the hippocampus, whereas OT has been shown to protect hippocampal neurons from the toxic effects of glucocorticoids. In this article, we discuss how neural and endocrine mechanisms interplay in stress regulation, with an emphasis on the role of OT, as well as its therapeutic potential in the treatment of stress-related disorders.

Neutralization of negative charges of siRNA results in improved safety and efficient gene silencing activity of lipid nanoparticles loaded with high levels of siRNA.

Lipid nanoparticles (LNPs) are one of the leading technologies for the in vivo delivery of short interfering RNA (siRNA). Cationic lipids are an important component for efficient endosomal escape via membrane fusion followed by release of siRNAs in cytosol where the site of action is located. A high cationic lipid/siRNA charge ratio is usually necessary for maximizing the gene silencing activity of the siRNA-loaded LNPs. However, high levels of cationic lipids are known to cause cytotoxicity through interactions with negatively charged biocomponents. A strategy for solving this dilemma is important, in terms of producing clinically applicable LNPs with a wide therapeutic window. We herein report on the development of LNPs with a high gene silencing activity and a low cationic lipid/siRNA charge ratio, which we refer to as low lipid core-nanoparticles (LLC-NPs). The negative charges of the siRNAs were neutralized by protamines, cationic proteins, to reduce the net dose of cationic lipid, YSK05, which was developed in our laboratory, for endosomal escape, resulting in preserved fusogenic activity and gene silencing activity, both in vitro and in vivo factor VII mouse model. In addition, the LLC-NPs showed an improved hepatotoxicity compared to conventional LNPs, which have a relatively higher cationic lipid/siRNA charge ratio. The concept of the LLC-NPs helps to realize clinically applicable LNPs with a wide therapeutic window and has the potential for use in various applications and for the delivery of different classes of nucleic acid.

Oxytocin Inhibits Corticosterone-induced Apoptosis in Primary Hippocampal Neurons.

Stress is an adaptive and coordinated response to endogenous or exogenous stressors that pose an unpleasant and aversive threat to an individual's homeostasis and wellbeing. Glucocorticoids, corticosterone (CORT) in rodents and cortisol in humans, are adrenal steroids which are released in response to stressful stimuli. Although they help individuals to cope with stress, their overexposure in animals has been implicated in hippocampal dysfunction and neuronal loss. Oxytocin (OT) plays an active role in adaptive stress-related responses and protects hippocampal synaptic plasticity and memory during stress. In this study, we showed that OT protects primary mouse hippocampal neurons from CORT-induced apoptosis. OT receptors (OTR) were expressed in primary mouse hippocampal neurons and glial cells. CORT induced apoptosis in hippocampal neurons, but had no effect on apoptosis in glial cells. OT inhibited CORT-induced apoptosis in primary hippocampal neurons. OT was unable to protect primary hippocampal neurons prepared from OTR KO mice from CORT-induced apoptosis. These results indicate that OT has inhibitory effects on CORT-induced neuronal death in primary hippocampal neurons via acting on OTR. The findings suggest a therapeutic potential of OT in the treatment of stress-related disorders.

Control of molecular weight distribution in synthesis of poly(2-hydroxyethyl methacrylate) using ultrasonic irradiation.

Poly(2-hydroxyethyl methacrylate) (PHEMA) was synthesized using ultrasonic irradiation without any chemical initiator. The effect of the ultrasonic power intensity on the time course of the conversion to polymer, the number average molecular weight, and the polydispersity were investigated in order to synthesize a polymer with a low molecular weight distribution (i.e., low polydispersity). The conversion to polymer increased with time. A higher ultrasonic power intensity resulted in a faster reaction rate. The number average molecular weight increased during the early stage of the reaction and then gradually decreased with time. A higher ultrasonic intensity resulted in a faster degradation rate of the polymer. The polydispersity decreased with time. This was because the degradation rate of a polymer with a higher molecular weight was faster than that of a polymer with a lower molecular weight. A polydispersity below 1.3 was obtained under ultrasonic irradiation. By changing the ultrasonic power intensity during the reaction, the number average molecular weight can be controlled while maintaining low polydispersity. When the ultrasonic irradiation was halted, the reactions stopped and the number average molecular weight and polydispersity did not change. On the basis of the experimental results, a kinetic model for synthesis of PHEMA under ultrasonic irradiation was constructed considering both polymerization and polymer degradation. The kinetic model was in good agreement with the experimental results for the time courses of the conversion to polymer, the number average molecular weight, and the polydispersity for various ultrasonic power intensities.

Highly specific delivery of siRNA to hepatocytes circumvents endothelial cell-mediated lipid nanoparticle-associated toxicity leading to the safe and efficacious decrease in the hepatitis B virus.

Lipid nanoparticles (LNPs) are the leading technology for delivering short interfering RNA (siRNA) in vivo. While numerous attempts to improve the efficiency of siRNA delivery have been reported, only a few studies of the mechanism of LNP-mediated toxicity and attempts to develop safe LNPs in vivo have been reported, in spite of the significance of such systems, in the light of treatment and clinical applications. We herein report on the elucidation of the mechanism of hepatotoxicity following the intravenous injection of a high dose of hepatotropic LNPs. The LNPs accumulated in liver sinusoidal endothelial cells (LSECs), resulting in their activation and the induction of several cytokines related to neutrophils, followed by neutrophilic inflammation. To circumvent this toxic cascade, the LNPs were modified with a hepatocyte-specific ligand, N-acetyl-d-galactosamine (GalNAc), which resulted in a substantial improvement of hepatocyte-specificity and in a dramatic reduction in toxicity. Moreover, modification of the GalNAc-LNPs with polyethyleneglycol abrogated the LNP-associated toxicity without any detectable loss of gene silencing activity in hepatocytes. Finally, we observed that a single injection of the LNPs resulted in a significant reduction of hepatitis B virus (HBV) genomic DNA and their antigens without any sign of toxicity in chimeric mice with humanized livers that had been persistently infected with HBV. These lines of the fact suggest that the newly designed siRNA-loaded LNPs promise to be a useful technology for the treatment of liver diseases.

Fluvoxamine, an anti-depressant, inhibits human glioblastoma invasion by disrupting actin polymerization.

Glioblastoma multiforme (GBM) is the most common malignant brain tumor with a median survival time about one year. Invasion of GBM cells into normal brain is the major cause of poor prognosis and requires dynamic reorganization of the actin cytoskeleton, which includes lamellipodial protrusions, focal adhesions, and stress fibers at the leading edge of GBM. Therefore, we hypothesized that inhibitors of actin polymerization can suppress GBM migration and invasion. First, we adopted a drug repositioning system for screening with a pyrene-actin-based actin polymerization assay and identified fluvoxamine, a clinically used antidepressant. Fluvoxamine, selective serotonin reuptake inhibitor, was a potent inhibitor of actin polymerization and confirmed as drug penetration through the blood-brain barrier (BBB) and accumulation of whole brain including brain tumor with no drug toxicity. Fluvoxamine inhibited serum-induced ruffle formation, cell migration, and invasion of human GBM and glioma stem cells in vitro by suppressing both FAK and Akt/mammalian target of rapamycin signaling. Daily treatment of athymic mice bearing human glioma-initiating cells with fluvoxamine blocked tumor cell invasion and prolonged the survival with almost same dose of anti-depressant effect. In conclusion, fluvoxamine is a promising anti-invasive treatment against GBM with reliable approach.

Size-dependent specific targeting and efficient gene silencing in peritoneal macrophages using a pH-sensitive cationic liposomal siRNA carrier.

Macrophages are key contributors to various inflammatory diseases. Therefore, the development of an efficient in vivo short interference RNA (siRNA) system that can be delivered to macrophages represents a novel treatment strategy for addressing these disorders. It was recently revealed that peritoneal macrophages (PEMs) are involved in several diseases including ovarian cancer, and are now recognized as a promising drug target. We report herein on the use of pH-sensitive cationic YSK05-MENDs as siRNA carriers and on the impact of both the size of the YSK05-MENDs and their administration routes for the efficient targeting PEMs to achieve a high level of gene silencing activity. The size of the YSK05-MENDs had a dramatic effect on their specificity for PEMs when administered intravenously, but not for intraperitoneal injection. Also, significant gene silencing was achieved by an intraperitoneal administration of the YSK05-MEND at a dose in the single digit µg/kg range. To our knowledge, this is the most efficacious method for siRNA delivery for gene silencing in PEMs in vivo reported to date. These findings enabled us to investigate the complex function of PEMs through several gene silencing simultaneously.

Biochemical characterization of smoothened receptor antagonists by binding kinetics against drug-resistant mutant.

Hedgehog (Hh) signaling critical for development, differentiation, and cell growth is involved in several cancers, including medulloblastoma and basal cell carcinoma. Although antagonism of the smoothened receptor (SMO), which mediates Hh signaling, is an attractive therapeutic target, a drug-resistant mutation in SMO (SMO-D473H) was identified in a clinical trial of the approved drug vismodegib. TAK-441 potently inhibits SMO-D473H, unlike vismodegib and another SMO antagonist, cyclopamine, whereas the differences in binding modes between these antagonists remain unknown. Here we report the biochemical characterization of TAK-441, vismodegib, and cyclopamine by binding kinetics. The association (kon) and dissociation (koff) rates were determined by kinetic binding studies using [(3)H]TAK-441, and dissociation was confirmed by label-free affinity selection-mass spectrometry (AS-MS). In the [(3)H]TAK-441 competition assay, TAK-441 but not vismodegib and cyclopamine showed time-dependent inhibition. Quantitative kinetic binding analysis revealed that koff of TAK-441 was >10-fold smaller than those of vismodegib and cyclopamine. To further assess the binding mode of antagonists, kinetic binding analysis was performed against SMO-D473H. The D473H mutation affected koff of TAK-441 but not kon. In contrast, only kon was changed by the D473H mutation in the case of vismodegib and cyclopamine. These results suggest that the difference in antagonist efficacy against D473H is associated with the binding mode of antagonists. These findings provide a new insight into the drug action of SMO antagonists and help develop potential therapeutics for drug-resistant mutants.

Possible role of cortactin phosphorylation by protein kinase Cα in actin-bundle formation at growth cone.

BACKGROUND INFORMATION: Cortactin contributes to growth cone morphogenesis by forming with dynamin, ring-shaped complexes that mechanically bundle and stabilise F-actin. However, the regulatory mechanism of cortactin action is poorly understood. RESULTS: Immunofluorescence microscopy revealed that protein kinase C (PKC) α colocalises with cortactin at growth cone filopodia in SH-SY5Y neuroblastoma cells. PKC activation by phorbol 12-myristate 13-acetate causes cortactin phosphorylation, filopodial retraction and F-actin-bundle loss. Moreover, PKCα directly phosphorylates cortactin in vitro at S135/T145/S172, mitigating both cortactin's actin-binding and actin-crosslinking activity, whereas cellular expression of a phosphorylation-mimetic cortactin mutant hinders filopodial formation with a significant decrease of actin bundles. CONCLUSIONS: Our results indicate that PKC-mediated cortactin phosphorylation might be implicated in the maintenance of growth cone.

Tumor-specific delivery of BSH-3R for boron neutron capture therapy and positron emission tomography imaging in a mouse brain tumor model.

Glioblastoma, a malignant brain tumor with poor disease outcomes, is managed in modern medicine by multimodality therapy. Boron neutron capture therapy (BNCT) is an encouraging treatment under clinical investigation. In malignant cells, BNCT consists of two major factors: neutron radiation and boron uptake. To increase boron uptake in cells, we created a mercapto-closo-undecahydrododecaborate ([B12HnSH](2-)2Na(+), BSH) fused with a short arginine peptide (1R, 2R, 3R) and checked cellular uptake in vitro and in vivo. In a mouse brain tumor model, only BSH with at least three arginine domains could penetrate cell membranes of glioma cells in vitro and in vivo. Furthermore, to monitor the pharmacokinetic properties of these agents in vivo, we fused BSH and BSH-3R with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA); DOTA is a metal chelating agent for labeling positron emission tomography (PET) probe with (64)Cu. We administered BSH-DOTA-(64)Cu and BSH-3R-DOTA-(64)Cu to the tumor model through a mouse tail vein and determined the drugs' pharmacokinetics by PET imaging. BSH-3R showed a high uptake in the tumor area on PET imaging. We concluded that BSH-3R is the ideal boron compound for clinical use during BNCT and that in developing this compound for clinical use, the BSH-3R PET probe is essential for pharmacokinetic imaging.

[Safe treatment of lung squamous cell carcinoma with nanoparticle albumin-bound Paclitaxel in a patient with a previous hypersensitivity reaction after docetaxel administration].

A 61-year-old man was diagnosed with lung squamous cell carcinoma in the lower lobe of the right lung. He had received first-line chemotherapy consisting of cisplatin and docetaxel (DTX); however, an allergic/hypersensitivity reaction occurred shortly after administration of the second course of DTX. Thirty-nine months later, he received nanoparticle albumin-bound paclitaxel (nab-PTX) as sixth-line chemotherapy, which did not produce a hypersensitivity reaction. Hypersensitivity after DTX administration may have been due to the DTX vehicle. Therefore, nab-PTX administered under close supervision is a valid therapeutic option in similar cases.

Cdk5rap1-mediated 2-methylthio modification of mitochondrial tRNAs governs protein translation and contributes to myopathy in mice and humans.

Transfer RNAs (tRNAs) contain a wide variety of posttranscriptional modifications that are important for accurate decoding. Mammalian mitochondrial tRNAs (mt-tRNAs) are modified by nuclear-encoded tRNA-modifying enzymes; however, the physiological roles of these modifications remain largely unknown. In this study, we report that Cdk5 regulatory subunit-associated protein 1 (Cdk5rap1) is responsible for 2-methylthio (ms(2)) modifications of mammalian mt-tRNAs for Ser(UCN), Phe, Tyr, and Trp codons. Deficiency in ms(2) modification markedly impaired mitochondrial protein synthesis, which resulted in respiratory defects in Cdk5rap1 knockout (KO) mice. The KO mice were highly susceptive to stress-induced mitochondrial remodeling and exhibited accelerated myopathy and cardiac dysfunction under stressed conditions. Furthermore, we demonstrate that the ms(2) modifications of mt-tRNAs were sensitive to oxidative stress and were reduced in patients with mitochondrial disease. These findings highlight the fundamental role of ms(2) modifications of mt-tRNAs in mitochondrial protein synthesis and their pathological consequences in mitochondrial disease.

Discovery of 6-[5-(4-fluorophenyl)-3-methyl-pyrazol-4-yl]-benzoxazin-3-one derivatives as novel selective nonsteroidal mineralocorticoid receptor antagonists.

In the course of our study on selective nonsteroidal mineralocorticoid receptor (MR) antagonists, a series of novel benzoxazine derivatives possessing an azole ring as the core scaffold was designed for the purpose of attenuating the partial agonistic activity of the previously reported dihydropyrrol-2-one derivatives. Screening of alternative azole rings identified 1,3-dimethyl pyrazole 6a as a lead compound with reduced partial agonistic activity. Subsequent replacement of the 1-methyl group of the pyrazole ring with larger lipophilic side chains or polar side chains targeting Arg817 and Gln776 increased MR binding activity while maintaining the agonistic response at the lower level. Among these compounds, 6-[1-(2,2-difluoro-3-hydroxypropyl)-5-(4-fluorophenyl)-3-methyl-1H-pyrazol-4-yl]-2H-1,4-benzoxazin-3(4H)-one (37a) showed highly potent in vitro activity, high selectivity versus other steroid hormone receptors, and good pharmacokinetic profiles. Oral administration of 37a in deoxycorticosterone acetate-salt hypertensive rats showed a significant blood pressure-lowering effect with no signs of antiandrogenic effects.

Methylphenidate improves learning impairments and hyperthermia-induced seizures caused by an Scn1a mutation.

OBJECTIVE: Developmental disorders including cognitive deficit, hyperkinetic disorder, and autistic behaviors are frequently comorbid in epileptic patients with SCN1A mutations. However, the mechanisms underlying these developmental disorders are poorly understood and treatments are currently unavailable. Using a rodent model with an Scn1a mutation, we aimed to elucidate the pathophysiologic basis and potential therapeutic treatments for developmental disorders stemming from Scn1a mutations. METHODS: We conducted behavioral analyses on rats with the N1417H-Scn1a mutation. With high-performance liquid chromatography, we measured dopamine and its metabolites in the frontal cortex, striatum, nucleus accumbens, and midbrain. Methylphenidate was administered intraperitoneally to examine its effects on developmental disorder-like behaviors and hyperthermia-induced seizures. RESULTS: Behavioral studies revealed that Scn1a-mutant rats had repetitive behavior, hyperactivity, anxiety-like behavior, spatial learning impairments, and motor imbalance. Dopamine levels in the striatum and nucleus accumbens in Scn1a-mutant rats were significantly lower than those in wild-type rats. In Scn1a-mutant rats, methylphenidate, by increasing dopamine levels in the synaptic cleft, improved hyperactivity, anxiety-like behavior, and spatial learning impairments. Surprisingly, methylphenidate also strongly suppressed hyperthermia-induced seizures. SIGNIFICANCE: Dysfunction of the mesolimbic dopamine reward pathway may contribute to the hyperactivity and learning impairments in Scn1a-mutant rats. Methylphenidate was effective for treating hyperactivity, learning impairments, and hyperthermia-induced seizures. We propose that methylphenidate treatment may ameliorate not only developmental disorders but also epileptic seizures in patients with SCN1A mutations.