Inhibition of Granule Cell Dispersion and Seizure Development by Astrocyte Elevated Gene-1 in a Mouse Model of Temporal Lobe Epilepsy.

Although granule cell dispersion (GCD) in the hippocampus is known to be an important feature associated with epileptic seizures in temporal lobe epilepsy (TLE), the endogenous molecules that regulate GCD are largely unknown. In the present study, we have examined whether there is any change in AEG-1 expression in the hippocampus of a kainic acid (KA)-induced mouse model of TLE. In addition, we have investigated whether the modulation of astrocyte elevated gene-1 (AEG-1) expression in the dentate gyrus (DG) by intracranial injection of adeno-associated virus 1 (AAV1) influences pathological phenotypes such as GCD formation and seizure susceptibility in a KA-treated mouse. We have identified that the protein expression of AEG-1 is upregulated in the DG of a KA-induced mouse model of TLE. We further demonstrated that AEG-1 upregulation by AAV1 delivery in the DG-induced anticonvulsant activities such as the delay of seizure onset and inhibition of spontaneous recurrent seizures (SRS) through GCD suppression in the mouse model of TLE, while the inhibition of AEG-1 expression increased susceptibility to seizures. The present observations suggest that AEG-1 is a potent regulator of GCD formation and seizure development associated with TLE, and the significant induction of AEG-1 in the DG may have therapeutic potential against epilepsy.

Control of hippocampal prothrombin kringle-2 (pKr-2) expression reduces neurotoxic symptoms in five familial Alzheimer's disease mice.

BACKGROUND AND PURPOSE: There is a scarcity of information regarding the role of prothrombin kringle-2 (pKr-2), which can be generated by active thrombin, in hippocampal neurodegeneration and Alzheimer's disease (AD). EXPERIMENTAL APPROACH: To assess the role of pKr-2 in association with the neurotoxic symptoms of AD, we determined pKr-2 protein levels in post-mortem hippocampal tissues of patients with AD and the hippocampi of five familial AD (5XFAD) mice compared with those of age-matched controls and wild-type (WT) mice, respectively. In addition, we investigated whether the hippocampal neurodegeneration and object memory impairments shown in 5XFAD mice were mediated by changes to pKr-2 up-regulation. KEY RESULTS: Our results demonstrated that pKr-2 was up-regulated in the hippocampi of patients with AD and 5XFAD mice, but was not associated with amyloid-ß aggregation in 5XFAD mice. The up-regulation of pKr-2 expression was inhibited by preservation of the blood-brain barrier (BBB) via addition of caffeine to their water supply or by treatment with rivaroxaban, an inhibitor of factor Xa that is associated with thrombin production. Moreover, the prevention of up-regulation of pKr-2 expression reduced neurotoxic symptoms, such as hippocampal neurodegeneration and object recognition decline due to neurotoxic inflammatory responses in 5XFAD mice. CONCLUSION AND IMPLICATIONS: We identified a novel pathological mechanism of AD mediated by abnormal accumulation of pKr-2, which functions as an important pathogenic factor in the adult brain via blood brain barrier (BBB) breakdown. Thus, pKr-2 represents a novel target for AD therapeutic strategies and those for related conditions.

Role of the Lipid Membrane and Membrane Proteins in Tau Pathology.

Abnormal accumulation of misfolded tau aggregates is a pathological hallmark of various tauopathies including Alzheimer's disease (AD). Although tau is a cytosolic microtubule-associated protein enriched in neurons, it is also found in extracellular milieu, such as interstitial fluid, cerebrospinal fluid, and blood. Accumulating evidence showed that pathological tau spreads along anatomically connected areas in the brain through intercellular transmission and templated misfolding, thereby inducing neurodegeneration and cognitive dysfunction. In line with this, the spatiotemporal spreading of tau pathology is closely correlated with cognitive decline in AD patients. Although the secretion and uptake of tau involve multiple different pathways depending on tau species and cell types, a growing body of evidence suggested that tau is largely secreted in a vesicle-free forms. In this regard, the interaction of vesicle-free tau with membrane is gaining growing attention due to its importance for both of tau secretion and uptake as well as aggregation. Here, we review the recent literature on the mechanisms of the tau-membrane interaction and highlights the roles of lipids and proteins at the membrane in the tau-membrane interaction as well as tau aggregation.

Control of Reactive Oxygen Species for the Prevention of Parkinson's Disease: The Possible Application of Flavonoids.

Oxidative stress reflects an imbalance between the production of reactive oxygen species (ROS) and antioxidant defense systems, and it can be associated with the pathogenesis and progression of neurodegenerative diseases such as multiple sclerosis, stroke, and Parkinson's disease (PD). The application of antioxidants, which can defend against oxidative stress, is able to detoxify the reactive intermediates and prevent neurodegeneration resulting from excessive ROS production. There are many reports showing that numerous flavonoids, a large group of natural phenolic compounds, can act as antioxidants and the application of flavonoids has beneficial effects in the adult brain. For instance, it is well known that the long-term consumption of the green tea-derived flavonoids catechin and epigallocatechin gallate (EGCG) can attenuate the onset of PD. Also, flavonoids such as ampelopsin and pinocembrin can inhibit mitochondrial dysfunction and neuronal death through the regulation of gene expression of the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. Additionally, it is well established that many flavonoids exhibit anti-apoptosis and anti-inflammatory effects through cellular signaling pathways, such as those involving (ERK), glycogen synthase kinase-3ß (GSK-3ß), and (Akt), resulting in neuroprotection. In this review article, we have described the oxidative stress involved in PD and explained the therapeutic potential of flavonoids to protect the nigrostriatal DA system, which may be useful to prevent PD.

Effects of Silibinin Against Prothrombin Kringle-2-Induced Neurotoxicity in the Nigrostriatal Dopaminergic System In Vivo.

Parkinson's disease (PD) and Alzheimer's disease exhibit common features of neurodegenerative diseases and can be caused by numerous factors. A common feature of these diseases is neurotoxic inflammation by activated microglia, indicating that regulation of microglial activation is a potential mechanism for preserving neurons in the adult brain. Recently, we reported that upregulation of prothrombin kringle-2 (pKr-2), one of the domains that make up prothrombin and which is cleaved and generated by active thrombin, induces nigral dopaminergic (DA) neuronal death through neurotoxic microglial activation in the adult brain. In this study, we show that silibinin, a flavonoid found in milk thistle, can suppress the production of inducible nitric oxide synthase and neurotoxic inflammatory cytokines, such as interleukin-1ß and tumor necrosis factor-α, after pKr-2 treatment by downregulating the extracellular signal-regulated kinase signaling pathway in the mouse substantia nigra. Moreover, as demonstrated by immunohistochemical staining, measurements of the dopamine and metabolite levels, and open-field behavioral tests, silibinin treatment protected the nigrostriatal DA system resulting from the occurrence of pKr-2-triggered neurotoxic inflammation in vivo. Thus, we conclude that silibinin may be beneficial as a natural compound with anti-inflammatory effects against pKr-2-triggered neurotoxicity to protect the nigrostriatal DA pathway and its properties, and thus, may be applicable for PD therapy.

Upregulation of neuronal astrocyte elevated gene-1 protects nigral dopaminergic neurons in vivo.

The role of astrocyte elevated gene-1 (AEG-1) in nigral dopaminergic (DA) neurons has not been studied. Here we report that the expression of AEG-1 was significantly lower in DA neurons in the postmortem substantia nigra of patients with Parkinson's disease (PD) compared to age-matched controls. Similarly, decreased AEG-1 levels were found in the 6-hydroxydopamine (6-OHDA) mouse model of PD. An adeno-associated virus-induced increase in the expression of AEG-1 attenuated the 6-OHDA-triggered apoptotic death of nigral DA neurons. Moreover, the neuroprotection conferred by the AEG-1 upregulation significantly intensified the neurorestorative effects of the constitutively active ras homolog enriched in the brain [Rheb(S16H)]. Collectively, these results demonstrated that the sustained level of AEG-1 as an important anti-apoptotic factor in nigral DA neurons might potentiate the therapeutic effects of treatments, such as Rheb(S16H) administration, on the degeneration of the DA pathway that characterizes 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.

Induction of microglial toll-like receptor 4 by prothrombin kringle-2: a potential pathogenic mechanism in Parkinson's disease.

Microglia-mediated neuroinflammation may play an important role in the initiation and progression of dopaminergic (DA) neurodegeneration in Parkinson's disease (PD), and toll-like receptor 4 (TLR4) is essential for the activation of microglia in the adult brain. However, it is still unclear whether patients with PD exhibit an increase in TLR4 expression in the brain, and whether there is a correlation between the levels of prothrombin kringle-2 (pKr-2) and microglial TLR4. In the present study, we first observed that the levels of pKr-2 and microglial TLR4 were increased in the substantia nigra (SN) of patients with PD. In rat and mouse brains, intranigral injection of pKr-2, which is not directly toxic to neurons, led to the disruption of nigrostriatal DA projections. Moreover, microglial TLR4 was upregulated in the rat SN and in cultures of the BV-2 microglial cell line after pKr-2 treatment. In TLR4-deficient mice, pKr-2-induced microglial activation was suppressed compared with wild-type mice, resulting in attenuated neurotoxicity. Therefore, our results suggest that pKr-2 may be a pathogenic factor in PD, and that the inhibition of pKr-2-induced microglial TLR4 may be protective against degeneration of the nigrostriatal DA system in vivo.

Induction of GDNF and BDNF by hRheb(S16H) transduction of SNpc neurons: neuroprotective mechanisms of hRheb(S16H) in a model of Parkinson's disease.

The transduction of dopaminergic (DA) neurons with human ras homolog enriched in brain, which has a S16H mutation [hRheb(S16H)] protects the nigrostriatal DA projection in the 6-hydroxydopamine (6-OHDA)-treated animal model of Parkinson's disease (PD). However, it is still unclear whether the expression of active hRheb induces the production of neurotrophic factors such as glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF), which are involved in neuroprotection, in mature neurons. Here, we show that transduction of nigral DA neurons with hRheb(S16H) significantly increases the levels of phospho-cyclic adenosine monophosphate (cAMP) response element-binding protein (p-CREB), GDNF, and BDNF in neurons, which are attenuated by rapamycin, a specific inhibitor of mammalian target of rapamycin complex 1 (mTORC1). Moreover, treatment with specific neutralizing antibodies for GDNF and BDNF reduced the protective effects of hRheb(S16H) against 1-methyl-4-phenylpyridinium (MPP(+))-induced neurotoxicity. These results show that activation of hRheb/mTORC1 signaling pathway could impart to DA neurons the important ability to continuously produce GDNF and BDNF as therapeutic agents against 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.

Naringin: a protector of the nigrostriatal dopaminergic projection.

Parkinson's disease is the second most common neurodegenerative disorder characterized by the progressive degeneration of dopaminergic neurons and a biochemical reduction of striatal dopamine levels. Despite the lack of fully understanding of the etiology of Parkinson's disease, accumulating evidences suggest that Parkinson's disease may be caused by the insufficient support of neurotrophic factors, and by microglial activation, resident immune cells in the brain. Naringin, a major flavonone glycoside in grapefruits and citrus fruits, is considered as a protective agent against neurodegenerative diseases because it can induce not only anti-oxidant effects but also neuroprotective effects by the activation of anti-apoptotic pathways and the induction of neurotrophic factors such as brain-derived neurotrophic factor and vascular endothelial growth factor. We have recently reported that naringin has neuroprotective effects in a neurotoxin model of Parkinson's disease. Our observations show that intraperitoneal injection of naringin induces increases in glial cell line-derived neurotrophic factor expression and mammalian target of rapamycin complex 1 activity in dopaminergic neurons of rat brains with anti-inflammatory effects. Moreover, the production of glial cell line-derived neurotrophic factor by naringin treatment contributes to the protection of the nigrostriatal dopaminergic projection in a neurotoxin model of Parkinson's disease. Although the effects of naringin on the nigrostriatal dopaminergic system in human brains are largely unknown, these results suggest that naringin may be a beneficial natural product for the prevention of dopaminergic degeneration in the adult brain.

Naringin protects the nigrostriatal dopaminergic projection through induction of GDNF in a neurotoxin model of Parkinson's disease.

This study investigated the effect of naringin, a major flavonoid in grapefruit and citrus fruits, on the degeneration of the nigrostriatal dopaminergic (DA) projection in a neurotoxin model of Parkinson's disease (PD) in vivo and the potential underlying mechanisms focusing on the induction of glia-derived neurotrophic factor (GDNF), well known as an important neurotrophic factor involved in the survival of adult DA neurons. 1-Methyl-4-phenylpyridinium (MPP(+)) was unilaterally injected into the medial forebrain bundle of rat brains for a neurotoxin model of PD in the presence or absence of naringin by daily intraperitoneal injection. To ascertain whether naringin-induced GDNF contributes to neuroprotection, we further investigated the effects of intranigral injection of neutralizing antibodies against GDNF in the MPP(+) rat model of PD. Our observations demonstrate that naringin could increase the level of GDNF in DA neurons, contributing to neuroprotection in the MPP(+) rat model of PD, with activation of mammalian target of rapamycin complex 1. Moreover, naringin could attenuate the level of tumor necrosis factor-α in microglia increased by MPP(+)-induced neurotoxicity in the substantia nigra. These results indicate that naringin could impart to DA neurons the important ability to produce GDNF as a therapeutic agent against PD with anti-inflammatory effects, suggesting that naringin is a beneficial natural product for the prevention of DA degeneration in the adult brain.

Inhibition of prothrombin kringle-2-induced inflammation by minocycline protects dopaminergic neurons in the substantia nigra in vivo.

Prothrombin kringle-2 (pKr-2), a domain of prothrombin, can cause the degeneration of mesencephalic dopaminergic neurons through microglial activation. However, the chemical products that inhibit pKr-2-induced inflammatory activities in the brain are still not well known. The present study investigated whether minocycline, a semisynthetic tetracycline derivative, could inhibit pKr-2-induced microglial activation and prevent the loss of nigral dopaminergic (DA) neurons in vivo. To address this question, rats were administered a unilateral injection of pKr-2 in the substantia nigra in the presence or absence of minocycline. Our results show that pKr-2 induces the production of proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß), and inducible nitric oxide synthase from the activated microglia. In parallel, 7 days after pKr-2 injection, tyrosine hydroxylase immunocytochemical analysis and western blot analysis showed a significant loss of nigral DA neurons. This neurotoxicity was antagonized by minocycline and the observed neuroprotective effects were associated with the ability of minocycline to suppress the expression of tumor necrosis factor-α, interleukin-1ß, and nitric oxide synthase. These results suggest that minocycline may be promising as a potential therapeutic agent for the prevention of DA neuronal degeneration associated with pKr-2-induced microglial activation.