Sirtuin 3 regulates astrocyte activation by reducing Notch1 signaling after status epilepticus.

Sirtuin3 (Sirt3) is a nicotinamide adenine dinucleotide enzyme that contributes to aging, cancer, and neurodegenerative diseases. Recent studies have reported that Sirt3 exerts anti-inflammatory effects in several neuropathophysiological disorders. As epilepsy is a common neurological disease, in the present study, we investigated the role of Sirt3 in astrocyte activation and inflammatory processes after epileptic seizures. We found the elevated expression of Sirt3 within reactive astrocytes as well as in the surrounding cells in the hippocampus of patients with temporal lobe epilepsy and a mouse model of pilocarpine-induced status epilepticus (SE). The upregulation of Sirt3 by treatment with adjudin, a potential Sirt3 activator, alleviated SE-induced astrocyte activation; whereas, Sirt3 deficiency exacerbated astrocyte activation in the hippocampus after SE. In addition, our results showed that Sirt3 upregulation attenuated the activation of Notch1 signaling, nuclear factor kappa B (NF-κB) activity, and the production of interleukin-1ß (IL1ß) in the hippocampus after SE. By contrast, Sirt3 deficiency enhanced the activity of Notch1/NF-κB signaling and the production of IL1ß. These findings suggest that Sirt3 regulates astrocyte activation by affecting the Notch1/NF-κB signaling pathway, which contributes to the inflammatory response after SE. Therefore, therapies targeting Sirt3 may be a worthy direction for limiting inflammatory responses following epileptic brain injury.

Vascular endothelial growth factor receptor-3 regulates astroglial glutamate transporter-1 expression via mTOR activation in reactive astrocytes following pilocarpine-induced status epilepticus.

Recent evidence has shown that the vascular endothelial growth factor (VEGF) system plays a crucial role in several neuropathological processes. We previously reported an upregulation of VEGF-C and its receptor, VEGFR-3, in reactive astrocytes after the onset of status epilepticus (SE). However, it remains unknown, which molecules act as downstream signals following VEGFR-3 upregulation, and are involved in reactive astrogliosis after SE. Therefore, we investigated whether VEGFR-3 upregulation within reactive astrocytes is associated with the activation of mammalian target of rapamycin (mTOR) signaling, which we confirmed by assaying for the phosphorylated form of S6 protein (pS6), and whether VEGFR-3-mediated mTOR activation induces astroglial glutamate transporter-1 (GLT-1) expression in the hippocampus after pilocarpine-induced SE. We found that spatiotemporal expression of pS6 was consistent with VEGFR-3 expression in the hippocampus after SE, and that both pS6 and VEGFR-3 were highly expressed in SE-induced reactive astrocytes. Treatment with the mTOR inhibitor rapamycin decreased astroglial VEGFR-3 expression and GLT-1 expression after SE. Treatment with a selective inhibitor for VEGFR-3 attenuated astroglial pS6 expression as well as suppressed GLT-1 expression and astroglial reactivity in the hippocampus after SE. These findings demonstrate that VEGFR-3-mediated mTOR activation could contribute to the regulation of GLT-1 expression in reactive astrocytes during the subacute phase of epilepsy. In conclusion, the present study suggests that VEGFR-3 upregulation in reactive astrocytes may play a role in preventing hyperexcitability induced by continued seizure activity.

Spatiotemporal expression of RCAN1 and its isoform RCAN1-4 in the mouse hippocampus after pilocarpine-induced status epilepticus.

Regulator of calcineurin 1 (RCAN1) can be induced by an intracellular calcium increase and oxidative stress, which are characteristic features of temporal lobe epilepsy. Thus, we investigated the spatiotemporal expression and cellular localization of RCAN1 protein and mRNA in the mouse hippocampus after pilocarpine-induced status epilepticus (SE). Male C57BL/6 mice were given pilocarpine hydrochloride (280 mg/kg, i.p.) and allowed to develop 2 h of SE. Then the animals were given diazepam (10 mg/kg, i.p.) to stop the seizures and sacrificed at 1, 3, 7, 14, or 28 day after SE. Cresyl violet staining showed that pilocarpine-induced SE resulted in cell death in the CA1 and CA3 subfields of the hippocampus from 3 day after SE. RCAN1 immunoreactivity showed that RCAN1 was mainly expressed in neurons in the shammanipulated hippocampi. At 1 day after SE, RCAN1 expression became detected in hippocampal neuropils. However, RCAN1 signals were markedly enhanced in cells with stellate morphology at 3 and 7 day after SE, which were confirmed to be reactive astrocytes, but not microglia by double immunofluorescence. In addition, real-time reverse transcriptase-polymerase chain reaction showed a significant upregulation of RCAN1 isoform 4 (RCAN1-4) mRNA in the SE-induced hippocampi. Finally, in situ hybridization with immunohistochemistry revealed astrocytic expression of RCAN1-4 after SE. These results demonstrate astrocytic upregulation of RCAN1 and RCAN1-4 in the mouse hippocampus in the acute and subacute phases of epileptogenesis, providing foundational information for the potential role of RCAN1 in reactive astrocytes during epileptogenesis.

The Neuroprotective Effect of Hericium erinaceus Extracts in Mouse Hippocampus after Pilocarpine-Induced Status Epilepticus.

Hericium erinaceus (HE), a culinary-medicinal mushroom, has shown therapeutic potential in many brain diseases. However, the role of HE in status epilepticus (SE)-mediated neuronal death and its underlying mechanisms remain unclear. We investigated the neuroprotective effects of HE using a pilocarpine-induced SE model. Male C57BL/6 mice received crude extracts of HE (60 mg/kg, 120 mg/kg, or 300 mg/kg, p.o.) for 21 d from 14 d before SE to 6 d after SE. At 7 d after SE, cresyl violet and immunohistochemistry of neuronal nuclei revealed improved hippocampal neuronal survival in animals treated with 60 mg/kg and 120 mg/kg of HE, whereas those treated with 300 mg/kg of HE showed similar neuronal death to that of vehicle-treated controls. While seizure-induced reactive gliosis, assessed by immunohistochemistry, was not altered by HE, the number of hippocampal cyclooxygenase 2 (COX2)-expressing cells was significantly reduced by 60 and 120 mg/kg of HE. Triple immunohistochemistry demonstrated no overlap of COX2 labeling with Ox42, in addition to a decrease in COX2/GFAP-co-immunoreactivity in the group treated with 60 mg/kg HE, suggesting that the reduction of COX2 by HE promotes neuroprotection after SE. Our findings highlight the potential application of HE for preventing neuronal death after seizures.

Increased expression of vascular endothelial growth factor-C and vascular endothelial growth factor receptor-3 after pilocarpine-induced status epilepticus in mice.

Vascular endothelial growth factor (VEGF)-C and its receptor, vascular endothelial growth factor receptor (VEGFR)-3, are responsible for lymphangiogenesis in both embryos and adults. In epilepsy, the expression of VEGF-C and VEGFR-3 was significantly upregulated in the human brains affected with temporal lobe epilepsy. Moreover, pharmacologic inhibition of VEGF receptors after acute seizures could suppress the generation of spontaneous recurrent seizures, suggesting a critical role of VEGF-related signaling in epilepsy. Therefore, in the present study, the spatiotemporal expression of VEGF-C and VEGFR-3 against pilocarpine-induced status epilepticus (SE) was investigated in C57BL/6N mice using immunohistochemistry. At 1 day after SE, hippocampal astrocytes and microglia were activated. Pyramidal neuronal death was observed at 4 days after SE. In the subpyramidal zone, VEGF-C expression gradually increased and peaked at 7 days after SE, while VEGFR-3 was significantly upregulated at 4 days after SE and began to decrease at 7 days after SE. Most VEGF-C/VEGFR-3-expressing cells were pyramidal neurons, but VEGF-C was also observed in some astrocytes in sham-manipulated animals. However, at 4 days and 7 days after SE, both VEGFR-3 and VEGF-C immunoreactivities were observed mainly in astrocytes and in some microglia of the stratum radiatum and lacunosum-moleculare of the hippocampus, respectively. These data indicate that VEGF-C and VEGFR-3 can be upregulated in hippocampal astrocytes and microglia after pilocarpine-induced SE, providing basic information about VEGF-C and VEGFR-3 expression patterns following acute seizures.

Pathogenic Upregulation of Glial Lipocalin-2 in the Parkinsonian Dopaminergic System.

UNLABELLED: Lipocalin-2 (LCN2) is a member of the highly heterogeneous secretory protein family of lipocalins and increases in its levels can contribute to neurodegeneration in the adult brain. However, there are no reports on the role of LCN2 in Parkinson's disease (PD). Here, we report for the first time that LCN2 expression is increased in the substantia nigra (SN) of patients with PD. In mouse brains, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment for a neurotoxin model of PD significantly upregulated LCN2 expression, mainly in reactive astrocytes in both the SN and striatum. The increased LCN2 levels contributed to neurotoxicity and neuroinflammation, resulting in disruption of the nigrostriatal dopaminergic (DA) projection and abnormal locomotor behaviors, which were ameliorated in LCN2-deficient mice. Similar to the effects of MPTP treatment, LCN2-induced neurotoxicity was also observed in the 6-hydroxydopamine (6-OHDA)-treated animal model of PD. Moreover, treatment with the iron donor ferric citrate (FC) and the iron chelator deferoxamine mesylate (DFO) increased and decreased, respectively, the LCN2-induced neurotoxicity in vivo In addition to the in vivo results, 1-methyl-4-phenylpyridinium (MPP(+))-induced neurotoxicity in cocultures of mesencephalic neurons and astrocytes was reduced by LCN2 gene deficiency in the astrocytes and conditioned media derived from MPP(+)-treated SH-SY5Y neuronal enhanced glial expression of LCN2 in vitro Therefore, our results demonstrate that astrocytic LCN2 upregulation in the lesioned DA system may play a role as a potential pathogenic factor in PD and suggest that inhibition of LCN2 expression or activity may be useful in protecting the nigrostriatal DA system in the adult brain. SIGNIFICANCE STATEMENT: Lipocalin-2 (LCN2), a member of the highly heterogeneous secretory protein family of lipocalins, may contribute to neuroinflammation and neurotoxicity in the brain. However, LCN2 expression and its role in Parkinson's disease (PD) are largely unknown. Here, we report that LCN2 is upregulated in the substantia nigra of patients with PD and neurotoxin-treated animal models of PD. Our results suggest that LCN2 upregulation might be a potential pathogenic mechanism of PD, which would result in disruption of the nigrostriatal dopaminergic system through neurotoxic iron accumulation and neuroinflammation. Therefore, inhibition of LCN2 expression or activity may be useful in protecting the nigrostriatal dopaminergic projection in PD.

In vivo AAV1 transduction with hRheb(S16H) protects hippocampal neurons by BDNF production.

Recent evidence has shown that Ras homolog enriched in brain (Rheb) is dysregulated in Alzheimer's disease (AD) brains. However, it is still unclear whether Rheb activation contributes to the survival and protection of hippocampal neurons in the adult brain. To assess the effects of active Rheb in hippocampal neurons in vivo, we transfected neurons in the cornu ammonis 1 (CA1) region in normal adult rats with an adeno-associated virus containing the constitutively active human Rheb (hRheb(S16H)) and evaluated the effects on thrombin-induced neurotoxicity. Transduction with hRheb(S16H) significantly induced neurotrophic effects in hippocampal neurons through activation of mammalian target of rapamycin complex 1 (mTORC1) without side effects such as long-term potentiation impairment and seizures from the alteration of cytoarchitecture, and the expression of hRheb(S16H) prevented thrombin-induced neurodegeneration in vivo, an effect that was diminished by treatment with specific neutralizing antibodies against brain-derived neurotrophic factor (BDNF). In addition, our results showed that the basal mTORC1 activity might be insufficient to mediate the level of BDNF expression, but hRheb(S16H)-activated mTORC1 stimulated BDNF production in hippocampal neurons. These results suggest that viral vector transduction with hRheb(S16H) may have therapeutic value in the treatment of neurodegenerative diseases such as AD.

Influence of aspirin on pilocarpine-induced epilepsy in mice.

Aspirin (acetylsalicylic acid) is one of the most widely used therapeutic agents based on its pharmacological actions, including anti-inflammatory, analgesic, anti-pyretic, and anti-thrombotic effects. In this study, we investigated the effects of aspirin on seizure susceptibility and hippocampal neuropathology following pilocarpine-induced status epilepticus (SE). SE was induced by pilocarpine hydrochloride (280 mg/kg, i.p.) administration in C57BL/6 mice (aged 8 weeks). Aspirin was administered daily (15 mg/kg or 150 mg/kg, i.p.) for 10 days starting 3 days before SE, continuing until 6 days after SE. After pilocarpine injection, SE onset time and mortality were recorded. Neuronal cell death was examined using cresyl violet and Fluoro-Jade staining, and glial responses were observed 7 days post SE using immunohistochemistry. In the aspirin-treated group, the onset time of SE was significantly shortened and mortality was markedly increased compared to the control group. However, in this study, aspirin treatment did not affect SE-induced neuronal cell death or astroglial and microglial responses in the hippocampus. In conclusion, these results suggest that the safety of aspirin should be reevaluated in some patients, especially with neurological disorders such as temporal lobe epilepsy.

Transient Receptor Potential Vanilloid 6 Modulates Aberrant Axonal Sprouting in a Mouse Model of Pilocarpine-Induced Epilepsy.

Transient receptor potential vanilloid 6 (TRPV6) is a highly selective calcium-ion channel that belongs to the TRPV family. TRPV6 is widely distributed in the brain, but its role in neurological diseases such as epilepsy remains unknown. Here, we report for the first time that TRPV6 expression is upregulated in the hippocampus of a pilocarpine-induced status epilepticus model, mainly in the suprapyramidal bundle of the mossy fiber (MF) projection of the hippocampal CA3 regions. We found that TRPV6 overexpression via viral vector transduction attenuated abnormal MF sprouting (MFS), whereas TRPV6 knockdown aggravated the development of MFS and the incidence of recurrent seizures during epileptogenic progression. In the in vitro experiments, our results showed that modulation of TRPV6 expression resulted in a change in axonal formation in cultured hippocampal neurons. In addition, we found that TRPV6 was implicated in the regulation of Akt-glycogen synthase kinase-3-ß activity, which is closely related to the cellular mechanism of axonal outgrowth. Therefore, these findings suggest that TRPV6 may regulate the formation of aberrant synaptic circuits during epileptogenesis.

Eugenol alleviates neuronal damage via inhibiting inflammatory process against pilocarpine-induced status epilepticus.

Neuroinflammation is one of the most common pathological outcomes in various neurological diseases. A growing body of evidence suggests that neuroinflammation plays a pivotal role in the pathogenesis of epileptic seizures. Eugenol is the major phytoconstituent of essential oils extracted from several plants and possesses protective and anticonvulsant properties. However, it remains unclear whether eugenol exerts an anti-inflammatory effect to protect against severe neuronal damage induced by epileptic seizures. In this study, we investigated the anti-inflammatory action of eugenol in an experimental epilepsy model of pilocarpine-induced status epilepticus (SE). To examine the protective effect of eugenol via anti-inflammatory mechanisms, eugenol (200 mg/kg) was administrated daily for three days after pilocarpine-induced SE onset. The anti-inflammatory action of eugenol was evaluated by examining the expression of reactive gliosis, pro-inflammatory cytokines, nuclear factor-κB (NF-κB), and the nucleotide-binding domain leucine-rich repeat with a pyrin-domain containing 3 (NLRP3) inflammasome. Our results showed that eugenol reduced SE-induced apoptotic neuronal cell death, mitigated the activation of astrocytes and microglia, and attenuated the expression of interleukin-1ß and tumor necrosis factor α in the hippocampus after SE onset. Furthermore, eugenol inhibited NF-κB activation and the formation of the NLRP3 inflammasome in the hippocampus after SE. These results suggest that eugenol is a potential phytoconstituent that suppresses the neuroinflammatory processes induced by epileptic seizures. Therefore, these findings provide evidence that eugenol has therapeutic potential for epileptic seizures.

Decreased vulnerability of hippocampal neurons after neonatal hypoxia-ischemia in bis-deficient mice.

The Bcl-2-interacting death suppressor (Bis) protein is involved in antiapoptosis and antistress pathways. However, its roles after neonatal hypoxia-ischemia remain obscure. Therefore, we investigated the effects of Bis deletion on hippocampal cell death following neonatal hypoxia-ischemia. We transected the right common carotid artery of bis(+/+) and bis(-/-) mice at postnatal Day 7 and subjected them to hypoxia for 35 min. Cresyl violet staining showed that hypoxia-ischemia induced progressive cell death in the hippocampi of bis(+/+) mice. Moreover, Bis was expressed in astrocytes, not microglia, in sham-manipulated hippocampi of bis(+/+) mice, and was markedly enhanced after hypoxia-ischemia. Immunoblotting showed that Bis expression significantly increased 3 and 7 days following hypoxia-ischemia. Unexpectedly, 7 days after hypoxia-ischemia, the number of hippocampal NeuN-positive cells was higher in the bis(-/-) mice than in the bis(+/+) mice. We subsequently performed transcriptomic analysis and quantitative real time polymerase chain reaction to search for the underlying genes responsible for resistance to hypoxia-ischemia in the bis(-/-) hippocampus. These studies showed that 6 h after hypoxia-ischemia, galectin 3 and filamin C levels increased to a lesser extent in the bis(-/-) hippocampi compared with the bis(+/+) hippocampi. Finally, our in vitro hypoxia-ischemia model, using A172 glioma cells and primary astrocytes, showed that downregulation of Bis blocked the enhanced expression of galectin 3 after oxygen-glucose deprivation. This study demonstrated that Bis was upregulated in the astrocytes after hypoxia-ischemia. In addition, we showed that hippocampal neurons are less vulnerable to hypoxia-ischemia in mice lacking Bis, possibly because of the modulation of galectin 3 induction.

Adjudin prevents neuronal damage and neuroinflammation via inhibiting mTOR activation against pilocarpine-induced status epilepticus.

Inflammatory responses in the brain play an etiological role in the development of epilepsy, suggesting that finding novel molecules for controlling neuroinflammation may have clinical value in developing the disease-modifying strategies for epileptogenesis. Adjudin, a multi-functional small molecule compound, has pleiotropic effects, including anti-inflammatory properties. In the present study, we aimed to investigate the effects of adjudin on pilocarpine-induced status epilepticus (SE) and its role in the regulation of reactive gliosis and neuroinflammation. SE was induced in male C57BL/6 mice that were then treated with adjudin (50 mg/kg) for 3 days after SE onset. Immunofluorescence staining, terminal deoxynucleotidyl transferase dUTP nick end labeling staining, and western blot analysis were used to evaluate the effects of adjudin treatment in the hippocampus after SE. Our results showed that adjudin treatment significantly mitigated apoptotic cell death in the hippocampus after SE onset. Moreover, adjudin treatment suppressed SE-induced glial activation and activation of mammalian target of rapamycin signaling in the hippocampus. Concomitantly, adjudin treatment significantly reduced SE-induced inflammatory processes, as confirmed by changes in the expression of inflammatory mediators such as tumor necrosis factor-α, interleukin-1ß, and arginase-1. In conclusion, these findings suggest that adjudin may serve as a potential neuroprotective agent for preventing pathological mechanisms implicated in epileptogenesis.

Withanolide-A treatment exerts a neuroprotective effect via inhibiting neuroinflammation in the hippocampus after pilocarpine-induced status epilepticus.

Status epilepticus (SE) is a medical emergency with high mortality and a risk factor for the development of chronic epilepsy. Given that effective treatments for the pathophysiology following SE are still lacking, suppressing pathophysiological mechanisms of SE may be important to inhibit epileptogenesis. Withanolide-A (WA), a major bioactive component of Withania somnifera, is a potential medicinal natural compound showing improvement of some neurological diseases, such as cerebral ischemia. In the present study, we examined whether administration of WA can exert the beneficial effects involved in neuroprotection and anti-inflammatory effects in a mouse model of pilocarpine-induced SE. Our results showed that WA treatment ameliorated SE-induced apoptotic neuronal cell death in the hippocampus. Moreover, WA treatment reduced immunoreactivity of both ionized calcium binding adapter molecule 1-positive microglia/macrophage and glial fibrillary acidic protein-positive reactive astrocytes, and the SE-induced increase in both interleukin-1 ß and tumor necrosis factor in the hippocampus, suggesting that inhibiting pro-inflammatory factors by WA treatment might induce neuroprotection after SE. These results suggest that WA may be useful in improving the treatment efficacy for pathophysiology following SE.

Immunohistochemical localization of Krüppel-like factor 6 in the mouse forebrain.

Krüppel-like factor 6 (KLF6) is a transcriptional regulator that shows widespread distribution in the peripheral organs of the body. However, it remains uncertain where KLF6 is expressed in the adult forebrain under physiological conditions. Therefore, the present study investigated the spatial patterns of KLF6 expression and identified cell types expressing KLF6 in the forebrain. KLF6 immunoreactivity was widely seen throughout the forebrain including the olfactory bulb, cerebral cortex, hippocampus, septum, amygdala, basal ganglia, thalamus, and hypothalamus. Moreover, KLF6-positive cells were also detected in the radial migratory stream (RMS) and subventricular zone. Immunofluorescent double-labeling revealed that KLF6-immunoreactive cells were co-localized with neuronal nuclei or platelet endothelial cell adhesion molecule-1, a mature neuronal and endothelial marker, respectively, in most forebrain regions. In the RMS, KLF6 was co-expressed with polysialic neural cell adhesion molecule, a marker of neuronal progenitor cells. This is the first report showing that KLF6 protein is expressed in various regions of the adult forebrain and KLF6-positive cells manifest neuronal or endothelial phenotypes under physiological conditions.

Sirtuin3 Protected Against Neuronal Damage and Cycled into Nucleus in Status Epilepticus Model.

In pathological conditions such as status epilepticus (SE), neuronal cell death can occur due to oxidative stress that is caused by an excessive production and accumulation of reactive oxygen species (ROS). Sirtuin3 (Sirt3) plays an important role in maintaining appropriate ROS levels by regulating manganese superoxide dismutase (MnSOD), which scavenges ROS in mitochondria. Using a SE model, we demonstrated that Sirt3 directly regulated MnSOD activity by deacetylation, which protects hippocampal cells against damage from ROS. Furthermore, we showed that after formation in the nucleus, Sirt3 is primarily located in the mitochondria, where it is activated and exerts its major function. Sirt3 then completed its pathway and moved back into the nucleus. Our data indicate that Sirt3 has an important function in regulating MnSOD, which results in decreased ROS in hippocampal cells. Sirt3 may have potential as an effective therapeutic target in SE conditions that would delay the progression of epileptogenesis.

Increased expression of WNK3 in dispersed granule cells in hippocampal sclerosis of mesial temporal lobe epilepsy patients.

Granule cell dispersion (GCD) is a common neuropathological feature of hippocampal sclerosis (HS) in patients with temporal lobe epilepsy (TLE). However, the underlying molecular mechanism of GCD formation remains unclear. The present study aimed to investigate the expressional changes of With No Lysine protein kinase subtype 3 (WNK3), a molecule upstream of cation-chloride cotransporters with reciprocal expression in sclerosed hippocampus of TLE patients. Using immunofluorescence staining, we analyzed WNK3 immunoreactivity in hippocampal specimens from histologically normal controls and TLE patients with HS. Our results showed that WNK3 expression was significantly increased in dispersed granule neurons in hippocampal tissues from patients with TLE compared with histologically normal hippocampus. These findings demonstrate a potential association between an increased expression of WNK3 and GCD formation during the chronic phase of epilepsy. Controlling WNK3 expression may thus be a novel therapeutic target in epileptogenesis.

Naringin attenuates granule cell dispersion in the dentate gyrus in a mouse model of temporal lobe epilepsy.

Morphological abnormalities of the dentate gyrus (DG) are an important phenotype in the hippocampus of patients with temporal lobe epilepsy. We recently reported that naringin, a bioflavonoid in grapefruit and citrus fruits, exerts beneficial effects in the kainic acid (KA) mouse model of epilepsy. We found that naringin treatment reduced seizure activities and decreased autophagic stress and neuroinflammation in the hippocampus following in vivo lesion with KA. However, it remains unclear whether naringin may also attenuate seizure-induced morphological changes in the DG, collectively known as granule cell dispersion (GCD). To clarify whether naringin treatment reduces GCD, we evaluated the effects of intraperitoneal injection of naringin on GCD and activation of mammalian target of rapamycin complex 1 (mTORC1), an important regulator of GCD, following intrahippocampal injection of KA. Our results showed that naringin treatment significantly reduced KA-induced GCD and mTORC1 activation, which was confirmed by assessing the phosphorylated form of the mTORC1 substrate, 4E-BP1, in the hippocampus. These results suggest that naringin treatment may help prevent epilepsy-induced hippocampal injury by inhibiting mTORC1 activation and thereby reducing GCD in the hippocampus in vivo.

Myricitrin Ameliorates 6-Hydroxydopamine-Induced Dopaminergic Neuronal Loss in the Substantia Nigra of Mouse Brain.

Parkinson's disease (PD) is a chronic and progressive movement disorder, resulting from the degeneration of the nigrostriatal dopaminergic (DA) pathway. The cause of DA neuronal loss in PD is still unclear; however, accumulating evidence suggests that treatment with certain flavonoids can induce neuroprotective properties, such as activation of mammalian target of rapamycin complex 1 (mTORC1) and anti-inflammatory activities in animal models of PD. The bioflavonoid myricitrin is well known for its anti-inflammatory and antioxidant properties. However, it is unclear whether systemic treatment with myricitrin can protect neurons against neurotoxin-induced DA degeneration in vivo via the preservation of tyrosine hydroxylase (TH) activity and the induction of mTORC1 activation. Our results found no significant neuroprotective effect of 30 mg/kg myricitrin on 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in the substantia nigra (SN) of mice. However, myricitrin treatment with 60 mg/kg protected DA neurons against 6-OHDA-induced neurotoxicity. Moreover, myricitrin treatment preserved TH enzyme activity and mTORC1 activation in nigral DA neurons in the SN of 6-OHDA-treated mice, and its treatment suppressed an increase in tumor necrosis factor-α expression in activated microglia. These results suggest that myricitrin may have neuroprotective properties linked to mTORC1 activation, preservation of TH enzyme activity, and anti-neuroinflammation for preventing DA neuronal degeneration in vivo.

Naringin treatment induces neuroprotective effects in a mouse model of Parkinson's disease in vivo, but not enough to restore the lesioned dopaminergic system.

We recently reported that treatment with naringin, a major flavonoid found in grapefruit and citrus fruits, attenuated neurodegeneration in a rat model of Parkinson's disease (PD) in vivo. In order to investigate whether its effects are universally applied to a different model of PD and whether its treatment induces restorative effects on the lesioned nigrostriatal dopaminergic (DA) projection, we observed the effects of pre-treatment or post-treatment with naringin in a mouse model of PD. For neuroprotective effects, 6-hydroxydopamine (6-OHDA) was unilaterally injected into the striatum of mouse brains for a neurotoxin model of PD in the presence or absence of naringin by daily intraperitoneal injection. Our results showed that naringin protected the nigrostriatal DA projection from 6-OHDA-induced neurotoxicity. Moreover, similar to the effects in rat brains, this treatment induced the activation of mammalian target of rapamycin complex 1 (mTORC1), which is well known as an important survival factor for DA neurons, and inhibited microglial activation in the substantia nigra (SN) of mouse brains treated with 6-OHDA. However, there was no significant change of DA phenotypes in the SN and striatum post-treated with naringin compared with 6-OHDA-lesioned mice, despite the treatment being continued for 12 weeks. These results suggest that post-treatment with naringin alone may not be enough to restore the nigrostriatal DA projection in a mouse model of PD. However, our results apparently suggest that naringin is a beneficial natural product to prevent DA degeneration, which is involved in PD.

Prothrombin Kringle-2: A Potential Inflammatory Pathogen in the Parkinsonian Dopaminergic System.

Although accumulating evidence suggests that microglia-mediated neuroinflammation may be crucial for the initiation and progression of Parkinson's disease (PD), and that the control of neuroinflammation may be a useful strategy for preventing the degeneration of nigrostriatal dopaminergic (DA) projections in the adult brain, it is still unclear what kinds of endogenous biomolecules initiate microglial activation, consequently resulting in neurodegeneration. Recently, we reported that the increase in the levels of prothrombin kringle-2 (pKr-2), which is a domain of prothrombin that is generated by active thrombin, can lead to disruption of the nigrostriatal DA projection. This disruption is mediated by neurotoxic inflammatory events via the induction of microglial Toll-like receptor 4 (TLR4) in vivo , thereby resulting in less neurotoxicity in TLR4-deficient mice. Moreover, inhibition of microglial activation following minocycline treatment, which has anti-inflammatory activity, protects DA neurons from pKr-2-induced neurotoxicity in the substantia nigra (SN) in vivo. We also found that the levels of pKr-2 and microglial TLR4 were significantly increased in the SN of PD patients compared to those of age-matched controls. These observations suggest that there may be a correlation between pKr-2 and microglial TLR4 in the initiation and progression of PD, and that inhibition of pKr-2-induced microglial activation may be protective against the degeneration of the nigrostriatal DA system in vivo. To describe the significance of pKr-2 overexpression, which may have a role in the pathogenesis of PD, we have reviewed the mechanisms of pKr-2-induced microglial activation, which results in neurodegeneration in the SN of the adult brain.