Deletion of Class II ADP-Ribosylation Factors in Mice Causes Tremor by the Nav1.6 Loss in Cerebellar Purkinje Cell Axon Initial Segments.

ADP-ribosylation factors (ARFs) are a family of small monomeric GTPases comprising six members categorized into three classes: class I (ARF1, 2, and 3), class II (ARF4 and 5), and class III (ARF6). In contrast to class I and III ARFs, which are the key regulators in vesicular membrane trafficking, the cellular function of class II ARFs remains unclear. In the present study, we generated class II ARF-deficient mice and found that ARF4+/-/ARF5-/- mice exhibited essential tremor (ET)-like behaviors. In vivo electrophysiological recordings revealed that ARF4+/-/ARF5-/- mice of both sexes exhibited abnormal brain activity when moving, raising the possibility of abnormal cerebellar excitability. Slice patch-clamp experiments demonstrated the reduced excitability of the cerebellar Purkinje cells (PCs) in ARF4+/-/ARF5-/- mice. Immunohistochemical and electrophysiological analyses revealed a severe and selective decrease of pore-forming voltage-dependent Na+ channel subunit Nav1.6, important for maintaining repetitive action potential firing, in the axon initial segment (AIS) of PCs. Importantly, this decrease in Nav1.6 protein localized in the AIS and the consequent tremors in ARF4+/-/ARF5-/- mice could be alleviated by the PC-specific expression of ARF5 using adeno-associated virus vectors. Together, our data demonstrate that the decreased expression of the class II ARF proteins in ARF4+/-/ARF5-/- mice, leading to a haploinsufficiency of ARF4 in the absence of ARF5, impairs the localization of Nav1.6 to the AIS and hence reduces the membrane excitability in PCs, resulting in the ET-like movement disorder. We suggest that class II ARFs function in localizing specific proteins, such as Nav1.6, to the AIS.SIGNIFICANCE STATEMENT We found that decreasing the expression of class II ARF proteins, through the generation of ARF4+/-/ARF5-/- mice, impairs Nav1.6 distribution to the axon initial segment (AIS) of cerebellar Purkinje cells (PCs), thereby resulting in the impairment of action potential firing of PCs. The ARF4+/-/ARF5-/- mutant mice exhibited movement-associated essential tremor (ET)-like behavior with pharmacological profiles similar to those in ET patients. The exogenous expression of ARF5 reduced the tremor phenotype and restored the localization of Nav1.6 immunoreactivity to the AIS in ARF4+/-/ARF5-/- mice. Thus, our results suggest that class II ARFs are involved in the localization of Nav1.6 to the AISs in cerebellar PCs and that the reduction of class II ARF activity leads to ET-like movement disorder.

CAPS1 deficiency perturbs dense-core vesicle trafficking and Golgi structure and reduces presynaptic release probability in the mouse brain.

Ca(2+)-dependent activator protein for secretion 1 (CAPS1) plays a regulatory role in the dense-core vesicle (DCV) exocytosis pathway, but its functions at the cellular and synaptic levels in the brain are essentially unknown because of neonatal death soon after birth in Caps1 knock-out mice. To clarify the functions of the protein in the brain, we generated two conditional knock-out (cKO) mouse lines: 1) one lacking Caps1 in the forebrain; and 2) the other lacking Caps1 in the cerebellum. Both cKO mouse lines were born normally and grew to adulthood, although they showed subcellular and synaptic abnormalities. Forebrain-specific Caps1 cKO mice showed reduced immunoreactivity for the DCV marker secretogranin II (SgII) and the trans-Golgi network (TGN) marker syntaxin 6, a reduced number of presynaptic DCVs, and dilated trans-Golgi cisternae in the CA3 region. Cerebellum-specific Caps1 cKO mice had decreased immunoreactivity for SgII and brain-derived neurotrophic factor (BDNF) along the climbing fibers. At climbing fiber-Purkinje cell synapses, the number of DCVs was markedly lower and the number of synaptic vesicles was also reduced. Correspondingly, the mean amplitude of EPSCs was decreased, whereas paired-pulse depression was significantly increased. Our results suggest that loss of CAPS1 disrupts the TGN-DCV pathway, which possibly impairs synaptic transmission by reducing the presynaptic release probability.

Development of a comprehensive approach for performance evaluation of a quantitative multi-attribute method as a quality control method.

The multi-attribute method has been recognized as an elegant quantification tool for post-translational modifications (PTMs) of therapeutic proteins, since it can evaluate several attributes spontaneously and site-specifically. Here, the abundance of PTMs calculated by three different types of formula were compared and there was little difference among the results. For the method evaluation, two different kinds of peptides were used as internal standards (ISs) and one of the IS was used as the "standard peak" to define the signal strength of MS. They are also used for system suitability testing to verify whether the condition or sensitivity of mass spectrometry are high enough to evaluate the minor components by confirming the recovery rate of one IS to the another. This system is beneficial that since we have defined the limit of quantification as a certain ratio to IS, consistent MS intensity is applied as the threshold across all detected peaks.

Establishment of a primary human hepatocyte spheroid system for evaluating metabolic toxicity using dacarbazine under conditions of CYP1A2 induction.

Some drugs induce cytochrome P450s (CYPs), and thus may cause increased metabolic toxicity from concomitantly administered agents. Hence, we need a means of evaluating the potential of compounds to cause drug-induced liver injury (DILI) under conditions where inducers of CYP1A2 are present. Here, we present a system for evaluating CYP1A2-mediated metabolic toxicity using three-dimensional (3D) cultures of primary human hepatocyte spheroids treated with the CYP1A2 inducer omeprazole (OPZ). As a test substrate, we employed dacarbazine (DTIC), which causes toxicity during the metabolic process. We measured cell viability, CYP1A2 mRNA expression level and metabolism of DTIC, as well as several markers of hepatic function, i.e. albumin secretion, urea secretion, and aspartate aminotransferase (AST) leakage. Markers of hepatic function were significantly decreased by addition of OPZ and DTIC even under conditions where the cell viability was largely unchanged. This experimental system sensitively detected CYP1A2-mediated metabolic toxicity. Therefore, the developed system should be helpful for evaluating the potential of compounds to cause DILI under conditions where inducers of CYP1A2 are present.

[Further investigation of 3D culture spheroid models of human hepatocytes].

Three-dimensional (3D) cultured hepatocyte capable of maintaining liver-specific function in an in vivo state over a relatively long period of time have drawn attention as a new method for evaluating the metabolic process, hepatotoxicity and enzyme induction potential of drugs. When human hepatocytes were seeded on a plate for spheroid formation, and cell morphology and albumin secretion were examined, hepatocyte spheroid was stably maintained for at least 21 days after seeding. As a result of drug exposure to this spheroid, sequential metabolic reactions by Phase I and Phase II enzymes and metabolic reactions peculiar to only humans were observed. Moreover, when several drugs were exposed to spheroids and hepatotoxicity was evaluated, stable values were obtained for the 50% inhibitory concentration (IC50) of albumin secretion at 14 and 21 days. The IC50 values of most of the tested drugs were lower than in conventional assays, suggesting that the reported evaluation methods might underestimate hepatotoxicity. Furthermore, examination of mRNA expression level and activity of various cytochrome P450 (CYP) after exposure of typical inducers of CYPs to hepatocyte spheroid resulted in a significant increase in the expression level and activity of each. From these results, it was shown that this 3D hepatocyte spheroid system is suitable for follow-up of metabolic processes, long-term tests of hepatotoxicity and enzyme activity induction potential of drugs.

Isohumulones modulate blood lipid status through the activation of PPAR alpha.

Isohumulones derived from hops are the major bitter compounds in beer. It was recently reported that isohumulones activated peroxisome proliferator-activated receptors (PPARs) alpha and gamma in vitro and modulated glucose and lipid metabolism in vivo. In this study, we examined the effects of isomerized hop extract (IHE) primarily containing isohumulones in C57BL/6N male mice and found that such treatment increased their liver weight and reduced their plasma triglyceride and free fatty acid levels. Microarray analysis and quantitative real time PCR (QPCR) showed that IHE dose-dependently upregulated the expression of a battery of hepatic genes that are involved in microsomal omega-oxidation and peroxisomal and mitochondrial beta-oxidation. These effects were common in both genders and very similar to those found with the PPARalpha agonist, fenofibrate (FF). Moreover, these effects were not found in PPARalpha-deficient mice. Thus, our results strongly suggest that IHE intake upregulates the expression of key genes that are involved in hepatic fatty acid oxidation, and that it ameliorates the blood lipid profile by activating PPARalpha.

Interaction of Ca(2+)-dependent activator protein for secretion 1 (CAPS1) with septin family proteins in mouse brain.

The Ca(2+)-dependent activator protein for secretion 1 (CAPS1) protein plays a regulatory role in the dense-core vesicle exocytosis pathway. To clarify the functions of this protein in the brain, we searched for novel interaction partners of CAPS1 by mass spectrometry. We identified a specific interaction of CAPS1 with septin family proteins. We also demonstrated that the C-terminal region of the CAPS1 protein binds to part of the deduced GTP-binding domain of septin proteins. It is possible that a tertiary complex of septin, CAPS, and syntaxin contributes to dense-core vesicle trafficking and exocytosis in neurons.

Axonal localization of Ca2+-dependent activator protein for secretion 2 is critical for subcellular locality of brain-derived neurotrophic factor and neurotrophin-3 release affecting proper development of postnatal mouse cerebellum.

Ca2+-dependent activator protein for secretion 2 (CAPS2) is a protein that is essential for enhanced release of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) from cerebellar granule cells. We previously identified dex3, a rare alternative splice variant of CAPS2, which is overrepresented in patients with autism and is missing an exon 3 critical for axonal localization. We recently reported that a mouse model CAPS2Δex3/Δex3 expressing dex3 showed autistic-like behavioral phenotypes including impaired social interaction and cognition and increased anxiety in an unfamiliar environment. Here, we verified impairment in axonal, but not somato-dendritic, localization of dex3 protein in cerebellar granule cells and demonstrated cellular and physiological phenotypes in postnatal cerebellum of CAPS2Δex3/Δex3 mice. Interestingly, both BDNF and NT-3 were markedly reduced in axons of cerebellar granule cells, resulting in a significant decrease in their release. As a result, dex3 mice showed developmental deficits in dendritic arborization of Purkinje cells, vermian lobulation and fissurization, and granule cell precursor proliferation. Paired-pulse facilitation at parallel fiber-Purkinje cell synapses was also impaired. Together, our results indicate that CAPS2 plays an important role in subcellular locality (axonal vs. somato-dendritic) of enhanced BDNF and NT-3 release, which is indispensable for proper development of postnatal cerebellum.

Dietary isohumulones, the bitter components of beer, raise plasma HDL-cholesterol levels and reduce liver cholesterol and triacylglycerol contents similar to PPARalpha activations in C57BL/6 mice.

The effects of dietary isohumulones, the main components accounting for the bitter taste of beer, on lipid metabolism were examined. Young female C57BL/6N mice were fed diets containing isomerized hop extract (IHE), which consists mainly of isohumulones. Administration of IHE with an atherogenic (high-fat and high-cholesterol) diet for 2 weeks resulted in a significant increase in plasma HDL-cholesterol (P<0.01), along with a concomitant reduction in the atherosclerosis index, an increase in liver weight and a decrease in body weight gain in a dose-dependent manner. When animals received IHE with either a cholesterol or a basal diet for 1 week, significant decreases in the liver content of cholesterol (P<0.01) and triacylglycerol (cholesterol diet, P<0.01) were observed. Quantitative analyses of hepatic mRNA levels revealed that IHE administration resulted in up-regulation of mRNA for acyl-CoA oxidase, acyl-CoA synthetase, hydroxymethylglutaryl-CoA synthetase, lipoprotein lipase and fatty acid transport protein, and down-regulation of mRNA for Apo CIII and Apo AI. Administration of purified isohumulones effectively resulted in the same changes as IHE. Administration of fenofibrate, an agonist for PPARalpha, with a cholesterol diet caused marked hepatomegaly, an increase in plasma HDL-cholesterol, a decrease in hepatic cholesterol content, and alterations in hepatic mRNA levels similar to those observed in mice given IHE. Taken together, these results suggest that the modulation of lipid metabolism observed in mice fed diets containing isohumulones is, at least in part, mediated by activation of PPARalpha.