Mamoru Shibata

An AAV-derived Apaf-1 dominant negative inhibitor prevents MPTP toxicity as antiapoptotic gene therapy for Parkinson's disease

Adeno-associated virus (AAV) vector delivery of an Apaf-1-dominant negative inhibitor was tested for its antiapoptotic effect on degenerating nigrostriatal neurons in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinsons disease. The wild-type caspase recruitment domain of Apaf-1 was used as a dominant negative inhibitor of Apaf-1 (rAAV-Apaf-1-DN-EGFP). An AAV virus vector was used to deliver it into the striatum of C57 black mice, and the animals were treated with MPTP. The number of tyrosine hydroxylase-positive neurons in the substantia nigra was not changed on the rAAV-Apaf-1-DN-EGFP injected side compared with the noninjected side. We also examined the effect of a caspase 1 C285G mutant as a dominant negative inhibitor of caspase 1 (rAAV-caspase-1-DN-EGFP) in the same model. However, there was no difference in the number of tyrosine hydroxylase-positive neurons between the rAAV-caspase-1-DN-EGFP injected side and the noninjected side. These results indicate that delivery of Apaf-1-DN by using an AAV vector system can prevent nigrostriatal degeneration in MPTP mice, suggesting that it could be a promising therapeutic strategy for patients with Parkinsons disease. The major mechanism of dopaminergic neuronal death triggered by MPTP seems to be the mitochondrial apoptotic pathway.

Activation of caspase-12 by endoplasmic reticulum stress induced by transient middle cerebral artery occlusion in mice.

We sought to clarify the involvement of caspase-12, a representative molecule related to endoplasmic reticulum (ER) stress-induced cell-death signaling pathways, in neuronal death resulting from ischemia/reperfusion in mice. Transient focal cerebral ischemia (1 h) was produced by intraluminal occlusion of the middle cerebral artery (MCA). We assessed the expression patterns of caspase-12, Bip/GRP78, an ER-resident molecular chaperone whose expression serves as a good marker of ER stress, and caspase-7 by Western blotting and/or immunohistochemistry. Double-fluorescent staining of caspase-12 immunohistochemistry and the terminal deoxynucleotidyl transferase-mediated DNA nick-end labeling (TUNEL) method was performed to clarify the involvement of caspase-12 in cell death. We confirmed that ER stress was induced during reperfusion in our model, as witnessed by up-regulated Bip/GRP78 expression in the MCA territory. Western blot analysis revealed that caspase-12 activation occurred at 5-23 h of reperfusion, and immunoreactivity for caspase-12 was enhanced mainly in striatal neurons on the ischemic side at the same time points. We found the co-localization of caspase-12 immunoreactivity and DNA fragmentation detectable by the TUNEL method. We did not detect the presence of caspase-7 in the ER fraction at the period of caspase-12 cleavage. Our results imply that cerebral ischemia/reperfusion induces ER stress and that caspase-12 activation concurred with ER stress. Caspase-12 seems to be involved in neuronal death induced by ischemia/reperfusion. Caspase-7 is not likely to contribute to the cleavage of caspase-12 in our experimental model.

A novel in vivo assay system for consecutive measurement of brain nitric oxide production combined with the microdialysis technique

A novel spectrophotometric nitrite (NO2-)/nitrate (NO3-) assay system for a small quantity (5 microliter) of dialysate sample obtained by in vivo brain microdialysis was developed based on the diazotization reaction. The system has the advantage of in vivo consecutive measurement, high precision, good reproducibility, technical simplicity, relatively short resolution time (2.5-20 min), and wide availability. The NO3- level in the rat striatum was found to be 3 times higher than the NO2- level. A nitric oxide (NO) synthase inhibitor, NG-nitro-L-arginine methyl ester, reduced striatal NO2-/citrulline formation in a dose-related manner and increased arginine, indicating that the tissue NO2- level detected by this assay system adequately reflects the striatal NO synthase activity.

Caspases determine the vulnerability of oligodendrocytes in the ischemic brain

Although oligodendrocytes (OLGs) are thought to be vulnerable to hypoxia and ischemia, little is known about the detailed mechanism by which these insults induce OLG death. From the clinical viewpoint, it is imperative to protect OLGs as well as neurons against ischemic injury (stroke), because they are the only myelin-forming cells of the central nervous system. Using the Cre/loxP system, we have established a transgenic mouse line that selectively expresses p35, a broad-spectrum caspase inhibitor, in OLGs. After hypoxia, cultured OLGs derived from wild-type mice exhibited significant upregulation of caspase-11 and substantial activation of caspase-3, which led to cell loss. Expression of p35 or elimination of caspase-11 suppressed the caspase-3 activation and conferred significant protection against hypoxic injury. Expression of p35 in OLGs in vivo resulted in significant protection from ischemia-induced cell injury, thus indicating that caspases are involved in the ischemia-induced cell death of OLGs. Furthermore, the induction of caspase-11 was evident in the ischemic brains of wild-type mice, and OLGs exhibited resistance to brain ischemia in mice deficient in caspase-11, suggesting that caspase-11 is critically implicated in the mechanism(s) underlying ischemia-induced OLG death. Caspases may therefore offer a good therapeutic target for reducing ischemia-induced damage to OLGs.

Modification of striatal arginine and citrulline metabolism by nitric oxide synthase inhibitors.

The effects of NG-substituted L-arginine (ARG) analogues on striatal ARG and citrulline (CIT) levels were investigated using in vivo microdialysis technique. A microdialysis probe was implanted into the striatum of anaesthesized Sprague-Dawley rats. Direct intrastriatal perfusion with 1 mM NG-nitro-L-arginine methyl ester (n = 8) increased striatal ARG release and decreased CIT release, suggesting suppressed NO synthase activity in the tissue. On the other hand, 1 mM NG-monomethyl-L-arginine (L-NMMA) (n = 6) evoked a persistent increase in both ARG and CIT. Considering that 4-320 microM L-ARG (n = 8) failed to increase CIT formation, CIT seems to be synthesized in the striatal tissue from L-NMMA by the enzyme that has been demonstrated in the kidney and aortic endothelium (NG,NG-dimethylarginine dimethyl-aminohydrolase).

Upregulation of Akt phosphorylation at the early stage of middle cerebral artery occlusion in mice

Akt is a serine/threonine kinase that is believed to promote cell viability in many different cell types, including neurons. Here, we observed the state of Akt phosphorylation at several time points (1, 3, 6, 12, and 24 h) during permanent occlusion of the middle cerebral artery (MCA) in mice. We detected a transient upregulation of Akt phosphorylation at 1 h of MCA occlusion (MCAO) by Western blot analysis. Double immunostaining revealed that the enhanced phosphorylation of Akt occurred mainly in neurons located in the outer area of the MCA territory (ischemic penumbra). This phenomenon was accompanied by the nuclear translocation of Akt. We confirmed that Akt enzymatic activity is elevated in both the nuclear and cytosolic fractions of brain tissue subjected to 1 h of ischemia. cAMP-response-element-binding protein (CREB), an intranuclear target molecule of Akt, exhibited increased phosphorylation after 1 h of MCAO. In our ischemia model, caspase-3 was activated in the central part of the MCA territory as little as 1 h after MCAO. However, caspase-3 activation was not recognized at this time in the outer area of the MCA territory, where Akt activity was upregulated. These results suggest that prosurvival cell signaling is initiated in an active fashion before cell death pathways are activated in neurons situated in the ischemic penumbra at the early stage of ischemia.

Reduction of TRPV1 expression in the trigeminal system by botulinum neurotoxin type-A.

Botulinum neurotoxin type-A (BoNT-A) is clinically used for patients with pain disorders and dystonia. The precise mechanism whereby BoNT-A controls pain remains elusive. Here, we studied how BoNT-A affects the expression of the transient receptor potential vanilloid subfamily member 1 (TRPV1), a cation channel critically implicated in nociception, in the trigeminal system. Histological studies revealed that subcutaneous BoNT-A injection (0.25, 0.5, or 5 ng/kg) into the face targeted the ophthalmic division of trigeminal ganglion (TG) neurons and decreased TRPV1-immunoreactive neurons in the TG and TRPV1-immunoreactive fibers in rat trigeminal terminals. Of note, TG neurons that received projections from the dura mater, a principal site of headache generation, had reduced TRPV1 expression. BoNT-A-induced cleavage of SNAP25 (synaptosomal-associated protein of 25-kDa) in the TG became obvious 2 days after BoNT-A administration and persisted for at least 14 days. Quantitative real-time RT-PCR (reverse transcription-polymerase chain reaction) data indicated that the TRPV1-decreasing effects of BoNT-A were not mediated by transcriptional downregulation. By employing a surface protein biotin-labeling assay, we demonstrated that BoNT-A inhibited TRPV1 trafficking to the plasma membrane in primary TG neurons. Moreover, Y200F-mutated TRPV1, which is incapable of trafficking to the plasma membrane, was expressed in PC12 cells by transfection, and pharmacological studies revealed that TRPV1 in the cytoplasm was more predisposed to proteasome-mediated proteolysis than plasma membrane-located TRPV1. We conclude that the mechanism by which BoNT-A reduces TRPV1 expression involves the inhibition of TRPV1 plasma membrane trafficking and proteasome-mediated degradation in the cytoplasm. This paradigm seems to explain how BoNT-A alleviates TRPV1-mediated pain. Our data reveal a likely molecular mechanism whereby BoNT-A treatment reduces TRPV1 expression in the trigeminal system and provide important clues to novel therapeutic measures for ameliorating craniofacial pain.

Presynaptic ionotropic glutamate receptors modulate in vivo release and metabolism of striatal dopamine, noradrenaline, and 5-hydroxytryptamine: involvement of both NMDA and AMPA/kainate subtypes

In order to explore further the presynaptic modulation of monoamine release by glutamatergic nerve fibers, we investigated the effects of selective agonists for ionotropic glutamate (GLU) receptors on striatal release of dopamine (DA), noradrenaline (NA) and 5-hydroxytryptamine (5-HT). In the striatum of anesthetized Sprague-Dawley rats, in vivo microdialysis was performed to measure the release of monoamines and metabolities, and also to administer GLU agonists locally in the tissue. L-GLU and its selective agonists (N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) and kainate (KA)) evoked simultaneous release of striatal DA, NA and 5-HT in a dose-dependent manner. Pretreatment with MK-801 (5 mg/kg i.p.), a noncompetitive NMDA receptor antagonist, selectively suppressed NMDA-evoked monoamine release. The rank order of GLU agonist efficacy in releasing monoamines was different among DA, NA, and 5-HTergic terminals: AMPA = KA > NMDA for DA release, AMPA > NMDA = KA for NA release, and NMDA = AMPA = KA for 5-HT release. In conclusion, presynaptic ionotropic GLU receptors exist extensively on monoaminergic terminals including not only catecholaminergic (DA and NA) but also indoleaminergic (5-HT) terminals in the rat striatum. Their subtypes include both NMDA subtype and AMPA/KA subtype, and show a differential distribution among these three monoaminergic terminals and a differential contribution to facilitating monoamine release.

Cardiac parasympathetic dysfunction concurrent with cardiac sympathetic denervation in Parkinson's disease

We aimed to characterize the relationship between cardiac sympathetic and parasympathetic dysfunction employing cardiac (123)I-meta-iodobenzylguanidine (MIBG) uptake and other autonomic function parameters in Parkinsons disease (PD). 79 PD patients were studied. We performed (123)I-MIBG myocardial scintigraphy to assess the extent of cardiac sympathetic denervation. Electrocardiogram readings at rest and postural change in blood pressure were also examined. Coefficient variation of RR intervals (CVR-R) was used as an index for cardiac parasympathetic activity. Cardiac (123)I-MIBG uptake did not vary significantly among the Hoehn-Yahr (H-Y) stages. There was a significant correlation between cardiac (123)I-MIBG uptake and CVR-R (early, r=0.457, p<0.001; late, r=0.442, p<0.001). While the correlation was present among the patients who had had the disease less than two years (early, r=0.558, p<0.001; late, r=0.530, p<0.001), the patients with the disease duration longer than two years did not have such a significant correlation. Age, disease duration, corrected QT interval, or postural blood pressure change did not correlate with cardiac (123)I-MIBG uptake. Orthostatic hypotension was observed in 13 out of 72 subjects, and reduced CVR-R was a major determinant for the development of orthostatic hypotension. We conclude that cardiac parasympathetic dysfunction occurs concurrent with sympathetic denervation as revealed by (123)I-MIBG myocardial scintigraphy in PD and contributes to the development of orthostatic hypotension.

Brain nitrite production during global ischemia and reperfusion: an in vivo microdialysis study

Nitric oxide (NO) is considered to be associated with the pathogenesis of cerebral ischemic injury. In the present study, NO production was continuously monitored employing in vivo microdialysis. A microdialysis probe was inserted into the stratum. Levels of the major NO metabolite, NO-2, in the dialysate were determined using the Griess reaction. Rats were subjected to global cerebral ischemia produced by occlusion of both common carotid arteries together with induced hypotension. Cerebral ischemia induced a decrease in NO production, which was interrupted by a transient increase in NO synthesis. This increment was abolished in the presence of a NO synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), suggesting that NO synthase activity is transiently activated during ischemia. Following reperfusion, NO synthesis was enhanced. To our knowledge, this is the first report concerning the continuous temporal profile of NO production during global cerebral ischemia and reperfusion.