Striatal astrocytes act as a reservoir for L-DOPA.

L-DOPA is therapeutically efficacious in patients with Parkinson's disease (PD), although dopamine (DA) neurons are severely degenerated. Since cortical astrocytes express neutral amino acid transporter (LAT) and DA transporter (DAT), the uptake and metabolism of L-DOPA and DA in striatal astrocytes may influence their availability in the dopaminergic system of PD. To assess possible L-DOPA- and DA-uptake and metabolic properties of striatal astrocytes, we examined the expression of L-DOPA, DA and DAT in striatal astrocytes of hemi-parkinsonian model rats after repeated L-DOPA administration, and measured the contents of L-DOPA, DA and their metabolite in primary cultured striatal astrocytes after L-DOPA/DA treatment. Repeated injections of L-DOPA induced apparent L-DOPA- and DA-immunoreactivities and marked expression of DAT in reactive astrocytes on the lesioned side of the striatum in hemi-parkinsonian rats. Exposure to DA for 4h significantly increased the levels of DA and its metabolite DOPAC in cultured striatal astrocytes. L-DOPA was also markedly increased in cultured striatal astrocytes after 4-h L-DOPA exposure, but DA was not detected 4 or 8h after L-DOPA treatment, despite the expression of aromatic amino acid decarboxylase in astrocytes. Furthermore, the intracellular level of L-DOPA in cultured striatal astrocytes decreased rapidly after removal of extracellular L-DOPA. The results suggest that DA uptaken into striatal astrocytes is rapidly metabolized and that striatal astrocytes act as a reservoir of L-DOPA that govern the uptake or release of L-DOPA depending on extracellular L-DOPA concentration, but are less capable of converting L-DOPA to DA.

Transplantation of melanocytes obtained from the skin ameliorates apomorphine-induced abnormal behavior in rodent hemi-parkinsonian models.

Tyrosinase, which catalyzes both the hydroxylation of tyrosine and consequent oxidation of L-DOPA to form melanin in melanocytes, is also expressed in the brain, and oxidizes L-DOPA and dopamine. Replacement of dopamine synthesis by tyrosinase was reported in tyrosine hydroxylase null mice. To examine the potential benefits of autograft cell transplantation for patients with Parkinson's disease, tyrosinase-producing cells including melanocytes, were transplanted into the striatum of hemi-parkinsonian model rats or mice lesioned with 6-hydroxydopamine. Marked improvement in apomorphine-induced rotation was noted at day 40 after intrastriatal melanoma cell transplantation. Transplantation of tyrosinase cDNA-transfected hepatoma cells, which constitutively produce L-DOPA, resulted in marked amelioration of the asymmetric apomorphine-induced rotation in hemi-parkinsonian mice and the effect was present up to 2 months. Moreover, parkinsonian mice transplanted with melanocytes from the back skin of black newborn mice, but not from albino mice, showed marked improvement in the apomorphine-induced rotation behavior up to 3 months after the transplantation. Dopamine-positive signals were seen around the surviving transplants in these experiments. Taken together with previous studies showing dopamine synthesis and metabolism by tyrosinase, these results highlight therapeutic potential of intrastriatal autograft cell transplantation of melanocytes in patients with Parkinson's disease.

Dipeptidyl compounds ameliorate the serum-deprivation-induced reduction in cell viability via the neurotrophin-activating effect in SH-SY5Y cells.

OBJECTIVES AND METHODS: We have searched for low-molecular-weight compounds as potent new non-immunosuppressive immunophilin ligands (NI-IPLs) that are stronger than existent NI-IPLs such as GPI1046 and/or V10367 from the perspective of neuroprotective efficacy. We selected six dipeptidyl compounds as new NI-IPL candidates, and first examined the effects of each of these compounds on the serum-deprivation-induced reduction in the viability of SH-SY5Y cells. In addition, we clarified the effects of these compounds on neurotrophin release into medium in SH-SY5Y cells. RESULTS: Pre-treatment with Leu-Ile and Ile-Ile prevented the serum deprivation-induced reduction in cell viability in SH-SY5Y cells. In naive SH-SY5Y cells, treatment with Leu-Ile and Ile-Ile for 24 hours significantly increased both brain-derived neurotrophic factor and glial cell-line-derived neurotrophic factor releases in comparison with relative vehicle treatments. Moreover, none of the dipeptidyl compounds could prevent the concanavalin A-induced enhancement in interleukin-2 and interleukin-4 release in mouse spleen cells. DISCUSSION: The immunosuppressive effect is not essential to the neuroprotective properties of dipeptidyl compounds, and Leu-Ile and Ile-Ile have neurotrophin-activating effects, like FK506 and its existing non-immunosuppressive derivatives.

Reduction of nuclear peroxisome proliferator-activated receptor gamma expression in methamphetamine-induced neurotoxicity and neuroprotective effects of ibuprofen.

We examined changes in nuclear peroxisome proliferator-activated receptor gamma (PPAR gamma) in the striatum in methamphetamine (METH)-induced dopaminergic neurotoxicity, and also examined effects of treatment with drugs possessing PPAR gamma agonistic properties. The marked reduction of nuclear PPAR gamma-expressed cells was seen in the striatum 3 days after METH injections (4 mg/kg x 4, i.p. with 2-h interval). The reduction of dopamine transporter (DAT)-positive signals and PPAR gamma expression, and accumulation of activated microglial cells were significantly and dose-dependently attenuated by four injections of a nonsteroidal anti-inflammatory drug and a PPAR gamma ligand, ibuprofen (10 or 20 mg/kg x 4, s.c.) given 30 min prior to each METH injection, but not by either a low or high dose of aspirin. Either treatment of ibuprofen or aspirin, that showed no effects on METH-induced hyperthermia, significantly blocked the METH-induced striatal cyclooxygenase (COX) expression. Furthermore, the treatment of an intrinsic PPAR gamma ligand 15d-PG J2 also attenuated METH injections-induced reduction of striatal DAT. Therefore, the present study suggests the involvement of reduction of PPAR gamma expression in METH-induced neurotoxicity. Taken together with the previous report showing protective effects of other PPAR gamma ligand, these results imply that the protective effects of ibuprofen against METH-induced neurotoxicity may be based, in part, on its anti-inflammatory PPAR gamma agonistic properties, but not on its COX-inhibiting property or hypothermic effect.

Dopamine induces supernumerary centrosomes and subsequent cell death through Cdk2 up-regulation in dopaminergic neuronal cells.

Aggregation of proteins in the centrosome is implicated in the pathophysiology of Parkinson's disease. However, the relevance of the centrosome in neurodegeneration is still obscure. Centrosome duplication is initiated by the cyclin E/cyclin-dependent kinase 2 (Cdk2) complex. The present study determined changes in cyclin E or Cdk2 expression and in the centrosomal structure in dopaminergic neuronal CATH.a cells exposed to 50, 100 and 150 micromolar dopamine (DA) for 24 h. DA induced significant increase in Cdk2 protein and cyclin E protein, but not cyclin e mRNA. In DA-treated cells, the intense cyclin E- and Cdk2-immunofluorescence signals were co-localized around large and supernumerary centrosomes, and these two parameters of centrosome amplification were significantly increased compared with the control. Simultaneous co-treatment with DA and a Cdk2 inhibitor blocked centrosome amplification and enhanced cell viability. Our results demonstrated that DA could lead to cyclin E accumulation and Cdk2 up-regulation triggering supernumerary centrosomes and apoptotic cell death.

Specific induction of PAG608 in cranial and spinal motor neurons of L-DOPA-treated parkinsonian rats.

We identified p53-activated gene 608 (PAG608) as a specifically induced gene in striatal tissue of L-DOPA (100mg/kg)-injected hemi-parkinsonian rats using differential display assay. In the present study, we further examined morphological distribution of PAG608 in the central nervous system of L-DOPA-treated hemi-parkinsonian rats. PAG608 expression was markedly induced in fibers and neuronal cells of the lateral globus pallidus and reticular thalamic nucleus adjacent to internal capsule, specifically in the parkinsonian side of L-DOPA-treated models. The protein was also constitutively expressed in motor neurons specifically in either side of the pontine nucleus and motor nuclei of trigeminal and facial nerves. Furthermore, L-DOPA-induced PAG608 expression on motor neurons in the contralateral side of the ventral horn of the spinal cord and the lateral corticospinal tract without cell loss. The specific induction of PAG608 6-48h after L-DOPA injection in the extrapyramidal tracts, pyramidal tracts and corresponding lower motor neurons of the spinal cords suggests its involvement in molecular events in stimulated motor neurons. Taken together with the constitutive expression of PAG608 in the motor nuclei of cranial nerves, PAG608 may be a useful marker of stressed or activated lower motor neurons.

Preventing effects of a novel anti-parkinsonian agent zonisamide on dopamine quinone formation.

The neurotoxicity of dopamine (DA) quinones as dopaminergic neuron-specific oxidative stress is considered to play a role in the pathogenesis and/or progression of Parkinson's disease (PD), since DA quinones conjugate with several key PD pathogenic molecules (e.g., tyrosine hydroxylase, alpha-synuclein and parkin) to form protein-bound quinone (quinoprotein) and consequently inhibit their functions. Zonisamide (ZNS) is used as an anti-epileptic agent but also improved the cardinal symptoms of PD in recent clinical trials in Japan. To evaluate the effects of ZNS on excess cytosolic free DA-induced quinone toxicity, we examined changes in DA quinone-related indices after ZNS treatment both in in vitro cell-free system and in cultured cells. Co-incubation of DA and ZNS in a cell-free system caused conversion of DA to stable melanin via formation of DA-semiquinone radicals and DA chrome. Long-term (5 days) treatment with ZNS decreased quinoprotein and increased DA/DOPA chromes in dopaminergic CATH.a cells. ZNS significantly inhibited quinoprotein formation induced by treatment with tetrahydrobiopterin and ketanserin that elevate cytosolic free DA in the cells. Our results suggest that the novel anti-parkinsonian agent ZNS possesses preventing effects against DA quinone formation induced by excess amount of cytosolic DA outside the synaptic vesicles.

Suppression of p53-activated gene, PAG608, attenuates methamphetamine-induced neurotoxicity.

The p53-activated gene 608 (PAG608) is a proapoptotic gene activated and regulated by p53 expression in oxidative stress-induced apoptosis of neuronal cells. In this study, we determined the role of PAG608 in methamphetamine-induced neurotoxicity. Treatment of mouse dopaminergic CATH.a cells with 2 mM methamphetamine increased PAG608 expression at 3h followed by increase in phosphorylated p53 expression. Transient transfection of PAG608 antisense cDNA or RNA interference using PAG608 small interfering RNA significantly attenuated the dose-dependent decrease in cell viability of CATH.a cells by methamphetamine (1-4 mM) exposure. In monoaminergic neuronal B65 cells, which contain serotonin rather than dopamine, methamphetamine-induced cell death was also significantly but partially protected by transient transfection of PAG608 antisense cDNA. Furthermore, cell death of PC12 cells produced by methamphetamine (1-5 mM) was almost completely prevented by stable expression of PAG608 antisense cDNA, compared with significant reduction of cell viability in control PC12 cells. Our results showed that suppression of PAG608 using transient and stable transfection with PAG608 antisense cDNA or small interfering RNA attenuates methamphetamine-induced death of various monoaminergic neuronal cells, suggesting that methamphetamine neurotoxicity in monoaminergic cells is related, at least in part, to induction of PAG608 expression.

Involvement of STAT3 in bladder smooth muscle hypertrophy following bladder outlet obstruction.

We examined the involvement of the signal transducer and activator of transcription 3 (STAT3) in bladder outlet obstruction (BOO)-induced bladder smooth muscle hypertrophy using a rat in vivo and in vitro study. BOO induced increases in bladder weight and bladder smooth muscle thickness 1 week after the operation. By using antibody microarrays, 64 of 389 proteins blotted on the array met our selection criteria of an INR value between > or = 2.0 and < or = 0.5. This result revealed up-regulation of transcription factors, cell cycle regulatory proteins, apoptosis-associated proteins and so on. On the other hand, down-regulation (INR value < or = 0.5) of proteins was not found. In a profiling study, we found an increase in the expression of STAT3. A significant increase in nuclear phosphorylated STAT3 expression was confirmed in bladder smooth muscle tissue by immunohistochemistry and Western blot analysis. Cyclical stretch-relaxation (1 Hz) at 120% elongation significantly increased the expression of STAT3 and of alpha-smooth muscle actin in primary cultured bladder smooth muscle cells. Furthermore, the blockade of STAT3 expression by the transfection of STAT3 small interfering RNA (siRNA) significantly prevented the stretch-induced increase in alpha-smooth muscle actin expression. These results suggest that STAT3 has an important role in the induction of bladder smooth muscle hypertrophy.

Characterization of pericentrin isoforms in vivo.

Pericentrin was first identified as a mouse centrosomal protein and is now referred to as pericentrin A. A larger homologous protein in humans with a C-terminal calmodulin-binding domain was later identified as pericentrin B. Pericentrin has been shown to be one of the key components in ciliogenesis, but in vivo pericentrin products have remained ambiguous. Here we characterized pericentrin isoforms in mice. Two pericentrin transcripts of 9.5 and 6.9 kb were recognized on the mouse tissue Northern blots, while a cRNA probe for a 5'-terminal sequence shared by pericentrin A and B failed to hybridize to the 6.9-kb message. Two pericentrin cDNAs were identified, which encoded pericentrin B and a novel isoform, pericentrin S, sharing with pericentrin B a C-terminal calmodulin-binding motif. Three pericentrin proteins of 360, 255, and 250 kDa revealed by immunoprecipitation analysis were thought to correspond to pericentrin B, pericentrin S, and an unknown N-terminal product.

Molecular basis of 6-hydroxydopamine-induced caspase activations due to increases in oxidative stress in the mouse striatum.

To clarify the possible role of in the in vivo toxic effects of 6-hydroxydopamine (6-OHDA), especially caspase activations, we examined its effects on striatal lipid peroxidation (LPO) and caspase activations in 6-OHDA-lesioned mice. Both dopamine (DA) levels and DA turnover were significantly changed by the 6-OHDA i.c.v. injection compared with the pre-injection level in the striatum. In addition, the striatal glutathione (GSH) content fluctuated and was significantly decreased both at 3 and 14 days after 6-OHDA i.c.v. injection. Moreover, superoxide dismutase (SOD) activity at 7 days after 6-OHDA i.c.v. injection was transiently and significantly increased compared with the pre-injection level. The levels of thiobarbituric acid-reactive substances (TBA-RS) were significantly increased at 1, 3 and 14 days. 6-OHDA significantly increased the activities of all three caspases, except for the caspase-3 activity at 7 days throughout the experimental period compared with the pre-injection level. In conclusion, 6-OHDA-induced dopaminergic dysfunction is mainly due to caspase activations by increases in oxidative stress in the mouse striatum.

Embryonic expression of pericentrin suggests universal roles in ciliogenesis.

Pericentrin (Pcnt) is a giant coiled-coil protein known to mediate microtubule organization. It has been recently reported that mitosis-specific centrosomal anchoring of gamma tubulin complexes by Pcnt acts to control mitotic spindle organization, though little is known about the in vivo expression of Pcnt. In this study, we investigated Pcnt expression in mouse embryos. In situ hybridization analysis revealed preferential expression of Pcnt in quiescent G(0) phase cells throughout the embryo with an unexpectedly low expression level in proliferating cells, suggesting that Pcnt might not play an important role in mitotic proliferation. Immunofluorescence analysis confirmed preferential expression of the Pcnt protein in G(0) phase cells. Moreover, Pcnt was shown to be localized to the base of primary cilia in multiple embryonic tissues, in agreement with a recent study demonstrating the involvement of Pcnt in primary cilia formation using cultured mammalian cells.

Methamphetamine-induced dopaminergic neurotoxicity is regulated by quinone-formation-related molecules.

Recently, the neurotoxicity of dopamine (DA) quinone formation by auto-oxidation of DA has focused on dopaminergic neuron-specific oxidative stress. In the present study, we examined DA quinone formation in methamphetamine (METH)-induced dopaminergic neuronal cell death using METH-treated dopaminergic cultured CATH.a cells and METH-injected mouse brain. In CATH.a cells, METH treatment dose-dependently increased the levels of quinoprotein (protein-bound quinone) and the expression of quinone reductase in parallel with neurotoxicity. A similar increase in quinoprotein levels was seen in the striatum of METH (4 mg/kg X4, i.p., 2 h interval)-injected BALB/c mice, coinciding with reduction of DA transporters. Furthermore, pretreatment of CATH.a cells with quinone reductase inducer, butylated hydroxyanisole, significantly and dose-dependently blocked METH-induced elevation of quinoprotein, and ameliorated METH-induced cell death. We also showed the protective effect of tyrosinase, which rapidly oxidizes DA and DA quinone to form stable melanin, against METH-induced dopaminergic neurotoxicity in vitro and in vivo using tyrosinase null mice. Our results indicate that DA quinone formation plays an important role, as a dopaminergic neuron-specific neurotoxic factor, in METH-induced neurotoxicity, which is regulated by quinone formation-related molecules.

L-DOPA treatment from the viewpoint of neuroprotection. Possible mechanism of specific and progressive dopaminergic neuronal death in Parkinson's disease.

With regard to the mechanism of selective dopaminergic neuronal death, experimental results of studies on the neurotoxicity of MPTP and rotenone indicate that degeneration of dopamine neurons is closely related to mitochondrial dysfunction, inflammatory process and oxidative stress, particularly with regard to the generation of quinones as dopamine neuron-specific oxidative stress. Thus, it is now clear that the presence of high levels of discompartmentalized free dopamine in dopaminergic neurons may explain the specific vulnerability of dopaminergic neurons through the generation of highly toxic quinones.

Gene delivery of Tim44 reduces mitochondrial superoxide production and ameliorates neointimal proliferation of injured carotid artery in diabetic rats.

Hyperglycemia induces the production of reactive oxygen species (ROS) from mitochondria, which is closely related to diabetic vascular complications. Mammalian translocase of inner mitochondrial membrane (Tim)44 was identified by upregulation in streptozotocin (STZ)-induced diabetic mouse kidneys; Tim44 functions as a membrane anchor of mtHsp70 to TIM23 complex and is involved in the import of preproteins with mitochondria-targeted presequence into mitochondrial matrix. The process is dependent on inner membrane potential (Delta psi) and ATP hydrolysis on ATPase domain of mtHsp70. Here, we show that the gene delivery of Tim44 using pcDNA3.1 vector (pcDNA3.1/TIM44) into the balloon injury model of STZ-induced diabetic rats ameliorated neointimal proliferation. ROS production, inflammatory responses, and cell proliferation in injured carotid artery were diminished by delivery of pcDNA3.1/TIM44. In vitro experiments using human aortic smooth muscle cells (HASMCs) revealed that the gene delivery of Tim44 normalized high-glucose-induced enhanced ROS production and increased ATP production, alterations in inner membrane potential, and cell proliferation. Transfection of siRNA and pcDNA3.1/TIM44 using HASMC culture clarified that import of antioxidative enzymes such as superoxide dismutase and glutathione peroxidase was facilitated by Tim44. Tim44 and its related molecules in mitochondrial import machinery complex are novel targets in the therapeutic interventions for diabetes and its vascular complications.

Dopamine agonist pergolide prevents levodopa-induced quinoprotein formation in parkinsonian striatum and shows quenching effects on dopamine-semiquinone generated in vitro.

The neurotoxicity of dopamine (DA) quinones that appears in dopaminergic neuron-specific oxidative stress has recently been shown to play a role in the pathogenesis and/or progression of Parkinson disease. To clarify the effects of a DA agonist, pergolide, on the levodopa-induced elevation of quinones, the authors examined striatal changes in quinoprotein using a hemi-parkinsonian mouse model. The level of striatal quinoprotein was significantly elevated specifically on the parkinsonian side, but not on the control side, after repeated levodopa administration. This levodopa-induced increase in striatal quinoprotein was almost completely suppressed by adjunctive administration with pergolide on the lesioned side. Furthermore, it was clarified that pergolide scavenged DA-semiquinones generated in vitro in a dose-dependent manner. These suppressive and quenching effects of pergolide against cytotoxic DA quinones may play a key role in its neuroprotective mechanism in the parkinsonian brain.

Pramipexole has ameliorating effects on levodopa-induced abnormal dopamine turnover in parkinsonian striatum and quenching effects on dopamine-semiquinone generated in vitro.

OBJECTIVES AND METHODS: To clarify the effects of a non-ergot dopamine agonist pramipexole on levodopa-induced abnormal dopamine metabolism in the parkinsonian model, we examined striatal changes in dopamine and its metabolites after repeated administration of pramipexole and/or levodopa using 6-hydroxydopamine-lesioned hemi-parkinsonian mice. Moreover, the effects of pramipexole on dopamine-semiquinones were also accessed using an in vitro dopamine-semiquinone generating system to elucidate its neuroprotective property against dopamine quinone-induced neurotoxicity that appears as dopamine neuron-specific oxidative stress. RESULTS: Combined administration of pramipexole (0.5 or 1 mg/kg/day, 7 days) selectively suppressed the levodopa-induced (50 mg/kg/day) increase of striatal dopamine turnover in the parkinsonian side, but not in the non-lesioned side. In addition to the antioxidant properties previously reported, it was clarified that pramipexole scavenged dopamine-semiquinones generated in a dose-dependent manner either in simultaneous incubation or post-incubation. DISCUSSION: The neurotoxicity of dopamine quinones that appear as dopaminergic neuron-specific oxidative stress has recently been known to play a role in the pathogenesis of Parkinson's disease and neurotoxin-induced parkinsonism. Therefore, the present results revealed that pramipexole possesses neuroprotective effects against abnormal dopamine metabolism in excessively levodopa-administered parkinsonian brains and against cytotoxic dopamine quinones generated from excess dopamine, preventing consequently dopaminergic neuronal damage induced by excess dopamine or levodopa.

Common anti-apoptotic roles of parkin and alpha-synuclein in human dopaminergic cells.

Parkin, a product of the gene responsible for autosomal recessive juvenile parkinsonism (AR-JP), is an important player in the pathogenic process of Parkinson's disease (PD). Despite numerous studies including search for the substrate of parkin as an E3 ubiquitin-protein ligase, the mechanism by which loss-of-function of parkin induces selective dopaminergic neuronal death remains unclear. Related to this issue, here we show that antisense knockdown of parkin causes apoptotic cell death of human dopaminergic SH-SY5Y cells associated with caspase activation and accompanied by accumulation of oxidative dopamine (DA) metabolites due to auto-oxidation of DOPA and DA. Forced expression of alpha-synuclein (alpha-SN), another familial PD gene product, prevented accumulation of oxidative DOPA/DA metabolites and cell death caused by parkin loss. Our findings indicate that both parkin and alpha-SN share a common pathway in DA metabolism whose abnormality leads to accumulation of oxidative DA metabolites and subsequent cell death.

The production of CXCR3-agonistic chemokines by synovial fibroblasts from patients with rheumatoid arthritis.

The inflamed synovial tissue of rheumatoid arthritis (RA) is characterized by an infiltration with Th1 cells that predominantly express the chemokine receptors CXCR3 and CCR5. In this study, we investigated the production of the CXCR3-agonistic chemokines CXCL9, CXCL10, and CXCL11 by synovial tissue cells and synovial fibroblast-cell lines (fourth or fifth passage) from RA patients. Concentrations of all CXCR3 ligands in synovial fluids were markedly higher in RA patients than in osteoarthritis (OA) patients. Synovial tissue cells from RA patients more strongly expressed mRNAs for CXCR3 ligands and spontaneously secreted larger amounts of these chemokine proteins than the cells from OA patients. The mRNA expression of all CXCR3 ligands was induced in synovial fibroblasts from RA patients after stimulation with interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), or interleukin-1 beta (IL-1beta). However, synovial fibroblasts significantly secreted CXCL9 and CXCL10 proteins, but not CXCL11 protein, after IFN-gamma stimulation and secreted only CXCL10 protein after TNF-alpha or IL-1beta stimulation. When stimulated with a combination of IFN-gamma and TNF-alpha, these cells were able to secrete large amounts of all three chemokines. These results indicate that synovial fibroblasts may be involved in perpetuating the Th1 immune response by producing the Th1-associated CXCR3 ligands, and the synergistic effect of IFN-gamma and TNF-alpha may be important for their chemokine production in RA joints.