Mika Takeshima

Neuroprotective effects of zonisamide target astrocyte.

Recent double‐blind, controlled trials in Japan showed that the antiepileptic agent zonisamide (ZNS) improves the cardinal symptoms of Parkinsons disease. Glutathione (GSH) exerts antioxidative activity through quenching reactive oxygen species and dopamine quinone. GSH depletion within dopaminergic neurons impairs mitochondrial complex I activity, followed by age‐dependent nigrostriatal neurodegeneration. This study examined changes in GSH and GSH synthesis‐related molecules, and the neuroprotective effects of ZNS on dopaminergic neurodegeneration using 6‐hydroxydopamine–injected hemiparkinsonian mice brain and cultured neurons or astrocytes.

Dopamine-Induced Behavioral Changes and Oxidative Stress in Methamphetamine-Induced Neurotoxicity

High-dose administration of amphetamine-like compounds is associated with acute behavioral toxicity (including stereotypic and self-injurious behavior and schizophrenic-like psychoses) as well as long-lasting damage to dopaminergic neurons. Several mechanisms are thought to be responsible for methamphetamine-induced neurotoxicity including the formation of reactive oxygen species, dopamine quinones, glutamatergic activity, apoptosis, etc. Recently, new factors regarding glial cell line-derived neurotorophic factor, tumor necrosis factor-alpha, and interferon-gamma have also been associated with methamphetamine-induced neurotoxicity. The objective of this review is to link the behavioral and neurotoxic responses of the amphetamines, emphasizing their common underlying mechanism of monoaminergic release together with inhibition of monoamine oxidase activity. The amphetamine-induced release of dopamine and inhibition of monoamine oxidase increases both cytosolic and synaptic levels of dopamine leading to the acute manifestation of stereotypic and self-injurious behavior. In turn, the enhanced extravesicular levels of dopamine lead to oxidative stress through the generation of reactive oxygen species and dopamine quinones, and cause the long-lasting neuronal damage. Thus, we propose that acute behavioral observation of subjects immediately following methamphetamine administration may provide insight into the long-lasting toxicity to dopaminergic neurons.

Astrocyte-derived metallothionein protects dopaminergic neurons from dopamine quinone toxicity

Our previous studies demonstrated the involvement of quinone formation in dopaminergic neuron dysfunction in the L‐DOPA‐treated parkinsonian model and in methamphetamine (METH) neurotoxicity. We further reported that the cysteine‐rich metal‐binding metallothionein (MT) family of proteins protects dopaminergic neurons against dopamine (DA) quinone neurotoxicity by its quinone‐quenching property. The aim of this study was to examine MT induction in astrocytes in response to excess DA and the potential neuroprotective effects of astrocyte‐derived MTs against DA quinone toxicity. DA exposure significantly upregulated MT‐1/‐2 in cultured striatal astrocytes, but not in mesencephalic neurons. This DA‐induced MT upregulation in astrocytes was blocked by treatment with a DA‐transporter (DAT) inhibitor, but not by DA‐receptor antagonists. Expression of nuclear factor erythroid 2‐related factor (Nrf2) and its binding activity to antioxidant response element of MT‐1 gene were significantly increased in the astrocytes after DA exposure. Nuclear translocation of Nrf2 was suppressed by the DAT inhibitor. Quinone formation and reduction of mesencephalic DA neurons after DA exposure were ameliorated by preincubation with conditioned media from DA‐treated astrocytes. These protective effects were abrogated by MT‐1/‐2‐specific antibody. Adding exogenous MT‐1 to glial conditioned media also showed similar neuroprotective effects. Furthermore, MT‐1/‐2 expression was markedly elevated specifically in reactive astrocytes in the striatum of L‐DOPA‐treated hemi‐parkinsonian mice or METH‐injected mice. These results suggested that excess DA taken up by astrocytes via DAT upregulates MT‐1/‐2 expression specifically in astrocytes, and that MTs or related molecules secreted specifically by astrocytes protect dopaminergic neurons from damage through quinone quenching and/or scavenging of free radicals.

Targeting 5-HT1A receptors in astrocytes to protect dopaminergic neurons in parkinsonian models

Astrocytes are abundant neuron-supporting glial cells that harbor a powerful arsenal of neuroprotective antioxidative molecules and neurotrophic factors. Here we examined whether enrichment with healthy striatal astrocytes can provide neuroprotection against progressive dopaminergic neurodegeneration. Serotonin 1A (5-HT1A) agonist 8-OH-DPAT induced astrocyte proliferation and increased metallothionein-1/-2 (MT-1/-2), antioxidative molecules, in cultured astrocytes and the striatum of mice. Primary cultured mesencephalic dopamine neurons were protected against oxidative stress by preincubation with conditioned media from 8-OH-DPAT-treated astrocytes. These protective effects were canceled by 5-HT1A antagonist or MT-1/-2-specific antibody. Furthermore, reduction of nigrostriatal dopaminergic neurons in 6-hydroxydopamine-lesioned parkinsonian model mice was significantly abrogated by repeated injections of 8-OH-DPAT. Treatment with 8-OH-DPAT markedly increased the expression of MT in striatal astrocytes in the hemi-parkinsonian mice. Our study provides a promising therapeutic strategy of neuroprotection against oxidative stress and progressive dopaminergic neurodegeneration by demonstrating the efficacy of targeting 5-HT1A receptors in astrocytes.

Lack of dopaminergic inputs elongates the primary cilia of striatal neurons.

In the rodent brain, certain G protein-coupled receptors and adenylyl cyclase type 3 are known to localize to the neuronal primary cilium, a primitive sensory organelle protruding singly from almost all neurons. A recent chemical screening study demonstrated that many compounds targeting dopamine receptors regulate the assembly of Chlamydomonas reinhardtii flagella, structures which are analogous to vertebrate cilia. Here we investigated the effects of dopaminergic inputs loss on the architecture of neuronal primary cilia in the rodent striatum, a brain region that receives major dopaminergic projections from the midbrain. We first analyzed the lengths of neuronal cilia in the dorsolateral striatum of hemi-parkinsonian rats with unilateral lesions of the nigrostriatal dopamine pathway. In these rats, the striatal neuronal cilia were significantly longer on the lesioned side than on the non-lesioned side. In mice, the repeated injection of reserpine, a dopamine-depleting agent, elongated neuronal cilia in the striatum. The combined administration of agonists for dopamine receptor type 2 (D2) with reserpine attenuated the elongation of striatal neuronal cilia. Repeated treatment with an antagonist of D2, but not of dopamine receptor type 1 (D1), elongated the striatal neuronal cilia. In addition, D2-null mice displayed longer neuronal cilia in the striatum compared to wild-type controls. Reserpine treatment elongated the striatal neuronal cilia in D1-null mice but not in D2-null mice. Repeated treatment with a D2 agonist suppressed the elongation of striatal neuronal cilia on the lesioned side of hemi-parkinsonian rats. These results suggest that the elongation of striatal neuronal cilia following the lack of dopaminergic inputs is attributable to the absence of dopaminergic transmission via D2 receptors. Our results provide the first evidence that the length of neuronal cilia can be modified by the lack of a neurotransmitters input.

Protective effects of baicalein against excess L-DOPA-induced dopamine quinone neurotoxicity

Abstract Objectives: Baicalein, a flavonoid derived from the root of Scutelaria baicalensis Georgi, possesses anti-oxidative properties including reactive oxygen species scavenging and lipid peroxidation inhibiting activities. The present study was undertaken to investigate the neuroprotective effect of baicalein against dopamine (DA) neurotoxicity induced by exposure to a synthetic DA precursor, L-3,4-dihydroxyphenylalanine (L-DOPA), in cultured dopaminergic CATH.a cells. Methods and results: Exposure to L-DOPA for 24 hours reduced the number of viable cells and enhanced protein-bound quinone (quinoprotein) formation in the cell. Both effects were prevented by simultaneous treatment with baicalein. In addition, baicalein prevented the formation of DA semiquinone radicals from DA in an in vitro cell-free system. Long-term baicalein treatment for 96 hours also protected against excess L-DOPA-induced cell death, and also increased glutathione (GSH) levels in CATH.a cells. Discussion: Our results indicate that baicalein has neuroprotective properties against excess L-DOPA-induced DA neurotoxicity through the suppression of DA quinone formation. Furthermore, the long-term treatment of baicalein upregulates intracellular GSH contents, which may also exert neuroprotective effects against oxidative stress-induced neuronal damage.

Cyclooxygenase-Independent Neuroprotective Effects of Aspirin Against Dopamine Quinone-Induced Neurotoxicity

Prostaglandin H synthase exerts not only cyclooxygenase activity but also peroxidase activity. The latter activity of the enzyme is thought to couple with oxidation of dopamine to dopamine quinone. Therefore, it has been proposed that cyclooxygenase inhibitors could suppress dopamine quinone formation. In the present study, we examined effects of various cyclooxygenase inhibitors against excess methyl L-3,4-dihydroxyphenylalanine (L-DOPA)-induced quinoprotein (protein-bound quinone) formation and neurotoxicity using dopaminergic CATH.a cells. The treatment with aspirin inhibited excess methyl L-DOPA-induced quinoprotein formation and cell death. However, acetaminophen did not show protective effects, and indomethacin and meloxicam rather aggravated these methyl L-DOPA-induced changes. Aspirin and indomethacin did not affect the level of glutathione that exerts quenching dopamine quinone in dopaminergic cells. In contrast with inhibiting effects of higher dose in the previous reports, relatively lower dose of aspirin that affected methyl L-DOPA-induced quinoprotein formation and cell death failed to prevent cyclooxygenase-induced dopamine chrome generation in cell-free system. Furthermore, aspirin but not acetaminophen or meloxicam showed direct dopamine quinone-scavenging effects in dopamine-semiquinone generating systems. The present results suggest that cyclooxygenase shows little contribution to dopamine oxidation in dopaminergic cells and that protective effects of aspirin against methyl L-DOPA-induced dopamine quinone neurotoxicity are based on its cyclooxygenase-independent property.

L-Theanine protects against excess dopamine-induced neurotoxicity in the presence of astrocytes

l-Theanine (γ-glutamylethylamide), a component of green tea, is considered to have regulatory and neuroprotective roles in the brain. The present study was designed to determine the effect of l-theanine on excess dopamine-induced neurotoxicity in both cell culture and animal experiments. The primary cultured mesencephalic neurons or co-cultures of mesencephalic neurons and striatal astrocytes were pretreated with l-theanine for 72 h, and then treated with excess dopamine for further 24 h. The cell viability of dopamine neurons and levels of glutathione were evaluated. Excess dopamine-induced neurotoxicity was significantly attenuated by 72 h preincubation with l-theanine in neuron-astrocyte co-cultures but not in neuron-rich cultures. Exposure to l-theanine increased the levels of glutathione in both astrocytes and glial conditioned medium. The glial conditioned medium from l-theanine-pretreated striatal astrocytes attenuated dopamine-induced neurotoxicity and quinoprotein formation in mesencephalic neurons. In addition, replacement of l-glutamate with l-theanine in an in vitro cell-free glutathione-synthesis system produced glutathione-like thiol compounds. Furthermore, l-theanine administration (4 mg/kg, p.o.) for 14 days significantly increased glutathione levels in the striatum of mice. The results suggest that l-theanine provides neuroprotection against oxidative stress-induced neuronal damage by humoral molecules released from astrocytes, probably including glutathione.

Deep sequencing of the prothoracic gland transcriptome reveals new players in insect ecdysteroidogenesis.

Ecdysteroids are steroid hormones that induce molting and determine developmental timing in arthropods. In insect larva, the prothoracic gland (PG) is a major organ for ecdysone synthesis and release. Released ecdysone is converted into the active form, 20-hydroxyecdysone (20E) in the peripheral tissues. All processes from ecdysone synthesis and release from the PG to its conversion to 20E are called ecdysteroidogenesis and are under the regulation of numerous factors expressed in the PG and peripheral tissues. Classical genetic approaches and recent transcriptomic screening in the PG identified several genes responsible for ecdysone synthesis and release, whereas the regulatory mechanism remains largely unknown. We analyzed RNA-seq data of the silkworm Bombyx mori PG and employed the fruit fly Drosophila melanogaster GAL4/UAS binary RNAi system to comprehensively screen for genes involved in ecdysone synthesis and/or release. We found that the genes encoding δ-aminolevulinic acid synthase (CG3017/alas) and putative NAD kinase (CG33156) were highly expressed in the PG of both B. mori and D. melanogaster. Neither alas nor CG33156 RNAi-induced larvae could enter into the pupal stage, and they had a lower abundance of the active form ecdysteroids in their prolonged larval stage. These results demonstrated that alas and CG33156 are indispensable for ecdysteroidogenesis.

Effects of rotenone exposure on primary cultured enteric neuronal or glial cells

G-CYPMPO as the stable crystals having gauche conformation was successfully synthesized as a novel 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO)-type spin trap agent. However, the function of GCYPMPO in vivo is still unclear. Thus, the purpose of this study was to evaluate the effects of G-CYPMPO in an in vivo model of Parkinson’s disease (PD). Rats were microinjected with 6-hydroxydopamine (6-OHDA, 32 nmol) in the presence or absence of G-CYPMPO (0.4, 1.2, 4 nmol). We investigated behavioral and histochemical parameters in this rat model of PD. In addition, to examine the effects of G-CYPMPO against oxidative stress, we used electron spin resonance (ESR) spectrometry. Intranigral injection of 6OHDA alone induced a massive loss of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra pars compacta (SNpc). Co-microinjection of G-CYPMPO significantly prevented 6-OHDA-induced dopaminergic neurodegeneration and behavioral impairments. Immunoreactivities for glial markers, such as cluster of differentiation antigen-11b (CD11b) and glial fibrillary acidic protein (GFAP), were notably detected in the SNpc of rats injected with 6-OHDA alone. These immunoreactivities were markedly suppressed by the co-microinjection of G-CYPMPO, similar to the results in vehicle-treated rats. In addition, G-CYPMPO directly trapped hydroxyl radical in a concentration-dependent manner. These results suggest that G-CYPMPO attenuates 6-OHDA-induced dopaminergic neurodegeneration in a rat model of PD, and is a useful tool for biological research.