Tomohiko Irie

Mutant PKCγ in spinocerebellar ataxia type 14 disrupts synapse elimination and long-term depression in Purkinje cells in vivo

Cerebellar Purkinje cells (PCs) express a large amount of the γ isoform of protein kinase C (PKCγ) and a modest level of PKCα. The PKCγ is involved in the pruning of climbing fiber (CF) synapses from developing PCs, and PKCα plays a critical role in long-term depression (LTD) at parallel fiber (PF)-PC synapses. Moreover, the PKC signaling in PCs negatively modulates the nonselective transient receptor potential cation channel type 3 (TRPC3), the opening of which elicits slow EPSCs at PF-PC synapses. Autosomal dominant spinocerebellar ataxia type 14 (SCA14) is caused by mutations in PKCγ. To clarify the pathology of this disorder, mutant (S119P) PKCγ tagged with GFP was lentivirally expressed in developing and mature mouse PCs in vivo, and the effects were assessed 3 weeks after the injection. Mutant PKCγ-GFP aggregated in PCs without signs of degeneration. Electrophysiology results showed impaired pruning of CF synapses from developing PCs, failure of LTD expression, and increases in slow EPSC amplitude. We also found that mutant PKCγ colocalized with wild-type PKCγ, which suggests that mutant PKCγ acts in a dominant-negative manner on wild-type PKCγ. In contrast, PKCα did not colocalize with mutant PKCγ. The membrane residence time of PKCα after depolarization-induced translocation, however, was significantly decreased when it was present with the mutant PKCγ construct. These results suggest that mutant PKCγ in PCs of SCA14 patients could differentially impair the membrane translocation kinetics of wild-type γ and α PKCs, which would disrupt synapse pruning, synaptic plasticity, and synaptic transmission.

Kv3.3 channels harbouring a mutation of spinocerebellar ataxia type 13 alter excitability and induce cell death in cultured cerebellar Purkinje cells

•  The cerebellum plays crucial roles in controlling sensorimotor functions, and patients with spinocerebellar ataxia type 13 exhibit cerebellar atrophy and cerebellar symptoms. •  The disease is an autosomal dominant disorder caused by missense mutations in the voltage‐gated K+ channel Kv3.3, which is expressed intensely in the cerebellar Purkinje cells, the sole output neurons from the cerebellar cortex. •  Here, we examined how these mutations cause the cerebellar disease by lentiviral expression of the mutant Kv3.3 in mouse cultured Purkinje cells. •  Expression of the mutant Kv3.3 suppressed outward currents, broadened action potentials and elevated basal intracellular calcium concentration in Purkinje cells. Moreover, the mutant‐expressing Purkinje cells showed impaired dendrites and extensive cell death, both of which were significantly rescued by blockade of P/Q‐type Ca2+ channels. •  These results suggest that Purkinje cells in the patients also exhibit similar abnormalities, which may account for the pathology of the disease.

MAM-2201, a synthetic cannabinoid drug of abuse, suppresses the synaptic input to cerebellar Purkinje cells via activation of presynaptic CB1 receptors.

Herbal products containing synthetic cannabinoids-initially sold as legal alternatives to marijuana-have become major drugs of abuse. Among the synthetic cannabinoids, [1-(5-fluoropentyl)-1H-indol-3-yl](4-methyl-1-naphthalenyl)-methanone (MAM-2201) has been recently detected in herbal products and has psychoactive and intoxicating effects in humans, suggesting that MAM-2201 alters brain function. Nevertheless, the pharmacological actions of MAM-2201 on cannabinoid receptor type 1 (CB1R) and neuronal functions have not been elucidated. We found that MAM-2201 acted as an agonist of human CB1Rs expressed in AtT-20 cells. In whole-cell patch-clamp recordings made from Purkinje cells (PCs) in slice preparations of the mouse cerebellum, we also found that MAM-2201 inhibited glutamate release at parallel fiber-PC synapses via activation of presynaptic CB1Rs. MAM-2201 inhibited neurotransmitter release with an inhibitory concentration 50% of 0.36 μM. MAM-2201 caused greater inhibition of neurotransmitter release than Δ(9)-tetrahydrocannabinol within the range of 0.1-30 μM and JWH-018, one of the most popular and potent synthetic cannabinoids detected in the herbal products, within the range of 0.03-3 μM. MAM-2201 caused a concentration-dependent suppression of GABA release onto PCs. Furthermore, MAM-2201 induced suppression of glutamate release at climbing fiber-PC synapses, leading to reduced dendritic Ca(2+) transients in PCs. These results suggest that MAM-2201 is likely to suppress neurotransmitter release at CB1R-expressing synapses in humans. The reduction of neurotransmitter release from CB1R-containing synapses could contribute to some of the symptoms of synthetic cannabinoid intoxication including impairments in cerebellum-dependent motor coordination and motor learning.

Sub-toxic concentrations of nano-ZnO and nano-TiO 2 suppress neurite outgrowth in differentiated PC12 cells

Nanomaterials have been extensively used in our daily life, and may also induce health effects and toxicity. Nanomaterials can translocate from the outside to internal organs, including the brain. For example, both nano-ZnO and nano-TiO2 translocate into the brain via the olfactory pathway in rodents, possibly leading to toxic effects on the brain. Although the effects of nano-ZnO and nano-TiO2 on neuronal viability or neuronal excitability have been studied, no work has focused on how these nanomaterials affect neuronal differentiation and development. In this study, we investigated the effects of nano-ZnO and nano-TiO2 on neurite outgrowth of PC12 cells, a useful model system for neuronal differentiation. Surprisingly, the number, length, and branching of differentiated PC12 neurites were significantly suppressed by the 7-day exposure to nano-ZnO (in the range of 1.0 × 10-4 to 1.0 × 10-1 µg/mL), at which the cell viability was not affected. The number and length were also significantly inhibited by the 7-day exposure to nano-TiO2 (1.0 × 10-3 to 1.0 µg/mL), which did not have cytotoxic effects. These results demonstrate that the neurite outgrowth in differentiated PC12 cells was suppressed by sub-cytotoxic concentrations of nano-ZnO or nano-TiO2.

Simple in vitro migration assay for neural crest cells and the opposite effects of all-trans-retinoic acid on cephalic- and trunk-derived cells

Here, we describe a simple in vitro neural crest cell (NCC) migration assay and the effects of all‐trans‐retinoic acid (RA) on NCCs. Neural tubes excised from the rhombencephalic or trunk region of day 10.5 rat embryos were cultured for 48 h to allow emigration and migration of NCCs. Migration of NCCs was measured as the change in the radius (radius ratio) calculated from the circular spread of NCCs between 24 and 48 h of culture. RA was added to the culture medium after 24 h at embryotoxic concentrations determined by rat whole embryo culture. RA (10 μM) reduced the migration of cephalic NCCs, whereas it enhanced the migration of trunk NCCs, indicating that RA has opposite effects on these two types of NCCs.

Effects of 13 developmentally toxic chemicals on the migration of rat cephalic neural crest cells in vitro

The inhibition of neural crest cell (NCC) migration has been considered as a possible pathogenic mechanism underlying chemical developmental toxicity. In this study, we examined the effects of 13 developmentally toxic chemicals on the migration of rat cephalic NCCs (cNCCs) by using a simple in vitro assay. cNCCs were cultured for 48 h as emigrants from rhombencephalic neural tubes explanted from rat embryos at day 10.5 of gestation. The chemicals were added to the culture medium at 24 h of culture. Migration of cNCCs was measured as the change in the radius (radius ratio) calculated from the circular spread of cNCCs between 24 and 48 h of culture. Of the chemicals examined, 13‐cis‐retinoic acid, ethanol, ibuprofen, lead acetate, salicylic acid, and selenate inhibited the migration of cNCCs at their embryotoxic concentrations; no effects were observed for acetaminophen, caffeine, indium, phenytoin, selenite, tributyltin, and valproic acid. In a cNCC proliferation assay, ethanol, ibuprofen, salicylic acid, selenate, and tributyltin inhibited cell proliferation, suggesting the contribution of the reduced cell number to the inhibited migration of cNCCs. It was determined that several developmentally toxic chemicals inhibited the migration of cNCCs, the effects of which were manifested as various craniofacial abnormalities.

Various definitions of reproductive indices: a proposal for combined use of brief definitions.

Several reproductive indices, such as live birth index, are calculated as endpoints to be evaluated in toxicity tests concerning reproductive effects of chemicals. These indices are useful to correct for variations resulting from infertility and multiple pregnancy, for example, the varied numbers of pups, among treatment groups and dams, respectively. In the toxicity test reports, the reproductive indices are used with their definitions, usually expressed as calculation formulae, to describe what they mean. Despite their frequent use, however, the definitions of the reproductive indices have not been standardized; that is, they are different among laboratories, and are confusing. For example, the live birth index is “number of live newborns/number of implantation sites × 100” in some laboratories, but is “number of live newborns/ number of total newborns × 100” in others, as listed in Table 1. These two definitions are quite different from each other in that the latter does not involve postimplantation loss, but the former does, though the live birth index is one of the most important reproductive indices. In most toxicity test laboratories, on the other hand, the definitions of reproductive indices cannot be changed even for standardization because they are defined as a part of laboratory computer systems. In the database era, the confusion of reproductive indices has become more serious than ever, because data from various laboratories in the toxicity databases are frequently consulted at a time as in meta-analyses for building quantitative structure-activity relationship models. In the meta-analysis of reproductive toxicity data, reproductive indices cannot be used as toxicological endpoints to be evaluated unless their definitions, usually not found in the abstract because of their lengthiness, are clearly identified. As a solution to this issue, we here propose combined use of brief definitions that describe the meaning of the reproductive indices with simpler words than the calculation formulae, for example, “live newborn/nidation rate” for “number of live newborns/number of implantation sites × 100.” Explanatory descriptions of the reproductive indices with their brief definitions, for example, “the live birth index (live newborn/nidation rate)” at their first appearance in the abstract and main text would be most helpful. In this letter, we show various definitions of representative reproductive indices and propose their brief definitions. We found 14 reproductive indices with 23 definitions by a brief survey of toxicological reference books (Manson and Kang 1989; Mizutani 1992; Saikikeisei ni kansuru dejitaruka sagyogruupu iinkai 1994; Econbichon 1995; Parker 2012) and contract research organizations’ reports in a toxicological database (Japan Existing Chemical Data Base, http://dra4.nihs.go.jp/mhlw_data/jsp/SearchPage.jsp). From these indices, we show seven representative indices and 12 brief definitions as examples (Table 1), but it is not intended that the brief definitions presented here should be used as they are. Makoto Usami, Katsuyoshi Mitsunaga, Tomohiko Irie, and Mikio Nakajima Division of Pharmacology, National Institute of Health Sciences, Tokyo, School of Pharmaceutical Sciences, Toho University, Chiba, and Pharmaceuticals Research Center, Asahi Kasei Pharma Corporation, Shizuoka, Japan

Presynaptic and postsynaptic inhibition by GABAB receptor in fusiform cells in mice dorsal cochlear nucleus

The small ubiquitin-like modifier protein (SUMO) regulates various cellular events inside the nucleus. The identification of SUMO substrates outside the nucleus is progressing but little is yet known about the neuronal role of protein SUMOylation. Here we report that in neurons multiple SUMOylation targets are present at synapses and we show that the kainate receptor subunit GluR6 exhibits low levels of SUMOylation under resting conditions and is rapidly SUMOylated in response to a kainate treatment. Reducing GluR6 SUMOylation using the SUMOspecific isopeptidase prevents kainate-evoked endocytosis of the kainate receptor. Furthermore, a mutated non-SUMOylatable form of GluR6 is not endocytosed in response to kainate. Consistent with this, recordings in hippocampal slices demonstrate that kainate receptor mediated EPSCs are diminished by SUMOylation and augmented by deSUMOylation. These data reveal a previously unsuspected role for SUMO in the regulation of synaptic function.