Youngbuhm Huh

Mulberry fruit protects dopaminergic neurons in toxin-induced Parkinson's disease models

Parkinsons disease (PD), one of the most common neurodegenerative disorders, is characterised by the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) to the striatum (ST), and involves oxidative stress. Mulberry fruit from Morus alba L. (Moraceae) is commonly eaten, and has long been used in traditional oriental medicine. It contains well-known antioxidant agents such as anthocyanins. The present study examined the protective effects of 70 % ethanol extract of mulberry fruit (ME) against neurotoxicity in in vitro and in vivo PD models. In SH-SY5Y cells stressed with 6-hydroxydopamine (6-OHDA), ME significantly protected the cells from neurotoxicity in a dose-dependent manner. Other assays demonstrated that the protective effect of ME was mediated by its antioxidant and anti-apoptotic effects, regulating reactive oxygen species and NO generation, Bcl-2 and Bax proteins, mitochondrial membrane depolarisation and caspase-3 activation. In mesencephalic primary cells stressed with 6-OHDA or 1-methyl-4-phenylpyridinium (MPP+), pre-treatment with ME also protected dopamine neurons, showing a wide range of effective concentrations in MPP+-induced toxicity. In the sub-acute mouse PD model induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), ME showed a preventative effect against PD-like symptoms (bradykinesia) in the behavioural test and prevented MPTP-induced dopaminergic neuronal damage in an immunocytochemical analysis of the SNpc and ST. These results indicate that ME has neuroprotective effects in in vitro and in vivo PD models, and that it may be useful in preventing or treating PD.

Hwangryun-Hae-Dok-tang (Huanglian-Jie-Du-Tang) extract and its constituents reduce ischemia-reperfusion brain injury and neutrophil infiltration in rats

The preventive effect of Hwangryun-Hae-Dok-tang (HHDT, Huanglian-Jie-Du-Tang), a Chinese herbal medicine, and its ingredients on ischemia/reperfusion-induced brain injury was evaluated in the rat brain. HHDT consists of four herbs, namely, Coptidis rhizoma, Scutellariae radix, Phellodendri cortex, and Gardeniae fructus. Ischemia was induced by intraluminal occlusion of the right middle cerebral artery for 120 min and reperfusion was continued for 22 h. HHDT (200 mg/kg), Coptidis rhizoma (100 mg/kg), Scutellariae radix (100 mg/kg), Phellodendri cortex (100 mg/kg), and Gardeniae fructus (100 mg/kg) were orally administered, promptly prior to reperfusion and 2 h after reperfusion. Baicalein, a component of Scutellariae radix, was also examined at a dosage of 50 mg/kg given 2 h apart, promptly prior to and 2 h after reperfusion. Total infarction volume in the ipsilateral hemisphere of ischemia/reperfusion rats was significantly lowered by treatment with HHDT, Scutellariae radix, and balicalein. However, the other ingredient of HHDT did not show any ameliorating effects on total infarction volume. The inhibiting effect of Scutellariae radix on total infarction volume was much higher than that of the others. In addition, HHDT, Scutellariae radix, and baicalein significantly inhibited myeloperoxidase (MPO) activity, an index of neutrophil infiltration in ischemic brain tissue at about the same rate (30%). There was marked mismatch between total infarction volume and MPO activity in the Scutellariae radix-treated rats but not in the HHDT- and baicalein-treated groups. Our findings suggest that Scutellariae radix as an ingredient of HHDT plays a crucial protective role in ischemia-induced brain injury. In addition, it is apparent that the effect of Scutellariae radix is the result, in part, of baicalein, a compound contained in Scutellariae radix.

Bee Venom Reduces Neuroinflammation in the MPTP-Induced Model of Parkinson's Disease

ABSTRACT Aim: This study was designed to investigate the anti-inflammatory effects of bee venom (BV) in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of Parkinsons disease (PD). Method: MPTP was administered by intraperitoneal (IP) injection at 2-hr intervals over an 8-hr period. Mice were then subjected to BV subcutaneous injection and sacrificed on days 1 and 3 following the final MPTP injection. The loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) was assessed by tyrosine hydroxylase (TH) immunohistochemistry. Microglial activation was measured by immunohistochemistry for macrophage antigen complex-1 (MAC-1) and inducible nitric oxide synthase (iNOS). The staining intensities of MAC-1 and iNOS were quantified with respect to optical density. Result: In animals treated with MPTP, the survival percentages of TH+ cells in the SNpc were 32% on day 1 and 46% on day 3 compared with normal mice. In BV-treated mice, the survival percentages of TH+ cells improved to 70% on day 1 and 78% on day 3 compared with normal mice. BV treatment also resulted in reduced expression of the inflammation markers MAC-1 and iNOS in the SNpc. Conclusion: These data suggest that BV injection may have a neuroprotective effect that attenuates the activation of the microglial response, which has implications for the treatment of PD.

Age-related change in the neuropeptide Y and NADPH-diaphorase-positive neurons in the cerebral cortex and striatum of aged rats

Age-related changes of neuropeptide Y (NPY) and nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) were examined in the rat cerebral cortex and striatum by immunohistochemical and histochemical methods. Double labeling for NPY and NADPH-d showed that about 30-70% of NPY-immunoreactive (NPY-IR) neurons in the cerebral cortex of the control (4-month-old) rats contained NADPH-d and that 50-75% in the aged (24-month-old) rats. The aged rats showed a significant increase in percentage of colocalization of NPY and NADPH-d in comparison with the control rats in the temporal cortex, occipital cortex, cingulate cortex, insular cortex, retrosplenial cortex and caudatoputamen. However, colocalization percentage between control and aged rats in the frontal cortex, parietal cortex, perirhinal cortex, entorhinal cortex and nucleus accumbens were practically identical. In the aged group, the number of NPY-IR/NADPH-d-positive neurons was not significantly decreased in the cerebral cortex and striatum compared to the control group. However, the number of NPY-IR/NADPH-d-negative neurons was significantly decreased in all cerebral cortical areas and caudatoputamen in the aged group except in the nucleus accumbens. Major loss of NPY-IR/NADPH-d-negative neurons in the aged group were observed in the neurons of layer II/III and V/VI. These results demonstrate that the NADPH-d containing NPY-IR neurons are less influenced by aging than the control group in the cerebral cortex and striatum of rats.

Sinapic acid attenuates kainic acid-induced hippocampal neuronal damage in mice

Excitotoxin induces neurodegeneration via glutamatergic activation or oxidative stress, which means that the blockade of glutamate receptors and the scavenging of free radicals are potential therapeutic targets in neurodegenerative diseases. Sinapic acid (SA) has a GABA(A) receptor agonistic property and free radical scavenging activity. We investigated the neuroprotective effects of SA on kainic acid (KA)-induced hippocampal brain damage in mice. SA (10 mg/kg) by oral administration has an anticonvulsant effect on KA-induced seizure-like behavior. Moreover, SA (10 mg/kg) significantly attenuated KA-induced neuronal cell death in the CA1 and CA3 hippocampal regions when administered as late as 6 h after KA. In addition, flumazenil, a GABA(A) antagonist, blocked the effect of SA administered immediately after KA but not the effect of SA administered 6 h after KA. This late protective effect of SA was accompanied by reduced levels of reactive gliosis, inducible nitric oxide synthase expression, and nitrotyrosine formation in the hippocampus. In the passive avoidance task, KA-induced memory impairments were ameliorated by SA. These results suggest that the potential therapeutic effect of SA is due to its attenuation of KA-induced neuronal damage in the brain via its anti-convulsive activity through GABA(A) receptor activation and radical scavenging activity.

6-Shogaol, an active constituent of ginger, attenuates neuroinflammation and cognitive deficits in animal models of dementia.

Recently, increased attention has been directed towards medicinal extracts as potential new drug candidates for dementia. Ginger has long been used as an important ingredient in cooking and traditional herbal medicine. In particular, ginger has been known to have disease-modifying effects in Alzheimers disease (AD). However, there is no evidence of which constituents of ginger exhibit therapeutic effects against AD. A growing number of experimental studies suggest that 6-shogaol, a bioactive component of ginger, may play an important role as a memory-enhancing and anti-oxidant agent against neurological diseases. 6-Shogaol has also recently been shown to have anti-neuroinflammatory effects in lipopolysaccharide (LPS)-treated astrocytes and animal models of Parkinsons disease, LPS-induced inflammation and transient global ischemia. However, it is still unknown whether 6-shogaol has anti-inflammatory effects against oligomeric forms of the Aβ (AβO) in animal brains. Furthermore, the effects of 6-shogaol against memory impairment in dementia models are also yet to be investigated. In this study, we found that administration of 6-shogaol significantly reduced microgliosis and astrogliosis in intrahippocampal AβO-injected mice, ameliorated AβO and scopolamine-induced memory impairment, and elevated NGF levels and pre- and post-synaptic marker in the hippocampus. All these results suggest that 6-shogaol may play a role in inhibiting glial cell activation and reducing memory impairment in animal models of dementia.

Expression of transgenes in midbrain dopamine neurons using the tyrosine hydroxylase promoter

Billions of neurons are interconnected in the central nervous system (CNS). Identification of specific neuronal circuit is indispensable for understanding the relationship between structure and function in the CNS. The midbrain dopamine (DA) neuron system consists of the retrorubral area (A8), the substantia nigra (SN; A9) and the ventral tegmental area (VTA; A10). We hypothesized that genetic methods using cell-type-specific promoters may offer the possibility to express tracer molecules in DA neurons to facilitate neuronal tracing. To address this, we used the 2.5 kb rat tyrosine hydroxylase (TH) promoter in adenovirus or adeno-associated virus (AAV) to express tracers specifically in DA neurons. We found that stereotaxic injection of TH promoter containing adenoviral construct resulted in cell-type-specific transgene expression in the noradrenaline (NA) neurons of the locus coeruleus (LC). However, it caused a significant toxicity to DA neurons in the SN. In contrast, stereotaxic injection of TH promoter containing AAV to the SN resulted in cell-type-specific transgene expression in DA neurons with no detectable toxicity. Taken together, our results demonstrate that it is possible to selectively trace DA neuronal circuits in rodent brains using the TH promoter in the context of AAV.

GATA‐3 regulates the transcriptional activity of tyrosine hydroxylase by interacting with CREB

The zinc finger transcription factor GATA‐3 is a master regulator of type 2 T‐helper cell development. Interestingly, in GATA‐3–/– mice, noradrenaline (NA) deficiency is a proximal cause of embryonic lethality. However, neither the role of GATA‐3 nor its target gene(s) in the nervous system were known. Here, we report that forced expression of GATA‐3 resulted in an increased number of tyrosine hydroxylase (TH) expressing neurons in primary neural crest stem cell (NCSC) culture. We also found that GATA‐3 transactivates the promoter function of TH via specific upstream sequences, a domain of the TH promoter residing at −61 to −39 bp. Surprisingly, this domain does not contain GATA‐3 binding sites but possesses a binding motif, a cAMP response element (CRE), for the transcription factor, CREB. In addition, we found that site‐directed mutation of this CRE almost completely abolished transactivation of the TH promoter by GATA‐3. Furthermore, protein–protein interaction assays showed that GATA‐3 is able to physically interact with CREB in vitro as well as in vivo. Based on these results, we propose that GATA‐3 may regulate TH gene transcription via a novel and distinct protein–protein interaction, and directly contributes to NA phenotype specification.

Microglial activation and tyrosine hydroxylase immunoreactivity in the substantia nigral region following transient focal ischemia in rats.

The temporal profiles of the changes of dopaminergic cells and microglial activation induced by transient cerebral ischemia were investigated in the substantia nigra pars compacta (SNc) located outside ischemic areas of rat brain. Transient cerebral ischemia was induced by intraluminal occlusion of the right middle cerebral artery for 2 h and reperfusion was continued for 1, 2, 3, 4, 7, 10, 14, 28, 60, and 120 days. Dopaminergic cells immunostained with tyrosine hydroxylase (TH)-antibody in the ipsilateral SNc were significantly decreased at 7 days post-ischemia compared with those in the contralateral side (P<0.05). However, at 60 and 120 days, there were no significant differences between ipsilateral and contralateral side of the SNc. Unlike the TH immunoreactivity, activated microglial cells immunostained with OX-42 antibody were significantly increased at 2 and 3 days and then decreased gradually until 10 days post-ischemia. Activated microglial cells were increased at 2 weeks post-ischemia, and this pattern remained until 60 days. These results suggest that the transient changes of TH-immunoreactive cells in the SNc caused by transient focal ischemia are correlated with a biphasic microglial cell activation response.

Inhibition of neuronal nitric oxide synthase enhances cell proliferation in the dentate gyrus of the adrenalectomized rat

Recent studies have demonstrated that the elimination of adrenal steroids by an adrenalectomy (ADX) increases the expression of neuronal nitric oxide synthase (NOS), and that it increases cell proliferation in the rat dentate gyrus. However, no evidence has been presented to date which indicates that NO regulates cell proliferation in the dentate gyrus of the adult rats. In this study, the effect of blocking NO production on ADX-induced increase of cell proliferation and serotonergic innervation was examined in the rat dentate gyrus. 7-nitroindazole (7-NI; 30 mg/kg, intraperitoneally), a selective inhibitor of neuronal NOS, was injected 1 day before an ADX and then once every 24 h for 4 days after the ADX subsequently. The proliferating cells were identified with 5-bromo-2-deoxyuridine (BrdU) immunostaining. Long-term inhibition of the neuronal NOS by 7-NI markedly increased the BrdU-labeled cell population density 4-18-fold in the dentate gyrus of the adrenalectomized rats compared to that in the vehicle-injected adrenalectomized rats. Immunoreactivity of serotonin, known as a mediator of granule cell genesis, was detected only in the dentate gyrus of 7-NI-injected adrenalectomized rats. These results indicate that NO may be involved in the cell proliferation in the dentate gyrus of the adrenalecomized rat and that serotonin may mediate the regulatory effect of NO on the cell proliferation in rat dentate gyrus.