Treatment with AAV1-Rheb(S16H) provides neuroprotection in a mouse model of photothrombosis-induced ischemic stroke.

We recently reported that upregulation of the constitutively active ras homolog enriched in brain [Rheb(S16H)], which induces the activation of the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway, can protect adult neurons, mediated by the induction of neurotrophic factors, such as brain-derived neurotrophic factor (BDNF), in animal models of neurodegenerative diseases. Here we show that neuronal transduction of Rheb(S16H) using adeno-associated virus serotype 1 provides neuroprotection in a mouse model of photothrombosis-induced ischemic stroke. Rheb(S16H)-expressing neurons exhibited neurotrophic effects, such as mTORC1 activation, increases in neuronal size, and BDNF production, in mouse cerebral cortex. Moreover, the upregulation of neuronal Rheb(S16H) significantly attenuated ischemic damage and behavioral impairments as compared to untreated mice, suggesting that Rheb(S16H) upregulation in cortical neurons may be a useful strategy to treat ischemic stroke.

Induction of GDNF and GFRα-1 Following AAV1-Rheb(S16H) Administration in the Hippocampus in vivo.

The activation of neurotrophic signaling pathways following the upregulation of glial cell line-derived neurotrophic factor (GDNF), a member of the transforming growth factor-ß family, has a potential neuroprotective effect in the adult brain. Herein, we report that hippocampal transduction of adeno-associated virus serotype 1 (AAV1) with a constitutively active form of ras homolog enriched in brain [Rheb(S16H)], which can stimulate the production of brain-derived neurotrophic factor (BDNF) in hippocampal neurons, induces the increases in expression of GDNF and GDNF family receptor α-1 (GFRα-1), in neurons and astrocytes in the hippocampus of rat brain in vivo. Moreover, upregulation of GDNF and GFRα-1 contributes to neuroprotection against thrombin-induced neurotoxicity in the hippocampus. These results suggest that AAV1-Rheb(S16H) transduction of hippocampal neurons, resulting in neurotrophic interactions between neurons and astrocytes, may be useful for neuroprotection in the adult hippocampus.

Therapeutic Potential of AAV1-Rheb(S16H) Transduction Against Alzheimer's Disease.

We recently reported that adeno-associated virus serotype 1-constitutively active Ras homolog enriched in brain [AAV1-Rheb(S16H)] transduction of hippocampal neurons could induce neuron-astroglia interactions in the rat hippocampus in vivo, resulting in neuroprotection. However, it remains uncertain whether AAV1-Rheb(S16H) transduction induces neurotrophic effects and preserves the cognitive memory in an animal model of Alzheimer's disease (AD) with characteristic phenotypic features, such as ß-amyloid (Aß) accumulation and cognitive impairments. To assess the therapeutic potential of Rheb(S16H) in AD, we have examined the beneficial effects of AAV1-Rheb(S16H) administration in the 5XFAD mouse model. Rheb(S16H) transduction of hippocampal neurons in the 5XFAD mice increased the levels of neurotrophic signaling molecules, including brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF), and their corresponding receptors, tropomyosin receptor kinase B (TrkB) and CNTF receptor α subunit (CNTFRα), respectively. In addition, Rheb(S16H) transduction inhibited Aß production and accumulation in the hippocampus of 5XFAD mice and protected the decline of long-term potentiation (LTP), resulting in the prevention of cognitive impairments, which was demonstrated using novel object recognition testing. These results indicate that Rheb(S16H) transduction of hippocampal neurons may have therapeutic potential in AD by inhibiting Aß accumulation and preserving LTP associated with cognitive memory.

Perspective: Therapeutic Potential of Flavonoids as Alternative Medicines in Epilepsy.

Epilepsy is a chronic neurological disorder that affects many people worldwide. Temporal lobe epilepsy is the most common and most studied type of epilepsy, but the pathological mechanisms underlying this condition are poorly understood. More than 20 antiepileptic drugs (AEDs) have been developed and used for the treatment of epilepsy; however, 30% of patients still experience uncontrolled epilepsy and associated comorbidities, which impair their quality of life. In addition, various side effects have been reported for AEDs, such as drowsiness, unsteadiness, dizziness, blurred or double vision, tremor (shakiness), greater risk of infections, bruising, and bleeding. Thus, critical medical needs remain unmet for patients with uncontrolled epilepsy. Flavonoids belong to a subclass of polyphenols that are widely present in fruits, vegetables, and certain beverages. Recently, many studies have reported that some flavonoids elicit various beneficial effects in patients with epilepsy without causing the side effects associated with conventional medical therapies. Moreover, flavonoids may have a property of regulating microRNA expression associated with inflammation and cell survival. These findings suggest that flavonoids, which are more effective but impose fewer adverse effects than conventional AEDs, could be used in the treatment of epilepsy.

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.

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.

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.

Silibinin attenuates MPP⁺-induced neurotoxicity in the substantia nigra in vivo.

Parkinson's disease (PD) is characterized by degeneration of the nigrostriatal dopaminergic (DA) pathway. The cause of neuronal death in PD is largely unknown, but it is becoming clear that inflammation plays a significant role in the pathophysiology of PD. Silibinin is a major flavonoid in milk thistle which has an anti-inflammatory activity. We investigated whether silibinin could have neuroprotective effects on DA neurons in the 1-methyl-4-phenylpyridinium ion (MPP(+))-treated animal model of PD in vivo. To address this question, animals received intraperitoneal (i.p.) injections 10, 50, or 100 mg/kg of silibinin, starting 1 day before MPP(+) injection and continued daily until 6 days post-lesion for tyrosine hydroxylase (TH) staining, or until 1 hour prior to the MPP(+) injection to examine the expression levels of inflammatory proteins. Finally, their brains were harvested at the indicated time points for the analyses. Silibinin treatment with 10 mg/kg had no significantly neuroprotective effects in the substantia nigra (SN). However, 50 and 100 mg/kg of silibinin ameliorated the MPP(+)-induced neurotoxicity in the SN in a dose-dependent manner, and the increased levels of inflammatory molecules such as tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß) and inducible nitric oxide synthase (iNOS) by MPP(+) treatment were attenuated by treatment with 100 mg/kg of silibinin. These results indicate that silibinin could be a useful and beneficial natural product offering promise for the prevention of DA neuronal degeneration involved in PD.

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.