Agomelatine Prevents Amyloid Plaque Deposition, Tau Phosphorylation, and Neuroinflammation in APP/PS1 Mice.

Agomelatine, an agonist of melatonergic MT1 and MT2 receptors and a selective 5-hydroxytryptamine 2C receptor antagonist, is widely applied in treating depression and insomnia symptoms in several neurogenerative diseases. However, the neuroprotective effect of agomelatine in Alzheimer's disease (AD) is less known. In this study, a total of 30 mice were randomly divided into three groups, namely, wild type (WT), APP/PS1, and agomelatine (50 mg/kg). After 30 days, the Morris water maze was performed to test the cognitive ability of mice. Then, all mice were sacrificed, and the hippocampus tissues were collected for ELISA, Western blot, and immunofluorescence analysis. In this study, we found that agomelatine attenuated spatial memory deficit, amyloid-ß (Aß) deposition, tau phosphorylation, and neuroinflammation in the hippocampus of APP/PS1 mice. Further study demonstrated that agomelatine treatment upregulated the protein expression of DHCR24 and downregulated P-Akt, P-mTOR, p-p70s6k, Hes1, and Notch1 expression. In summary, our results identified that agomelatine could improve cognitive impairment and ameliorate AD-like pathology in APP/PS1 mice via activating DHCR24 signaling and inhibiting Akt/mTOR and Hes1/Notch1 signaling pathway. Agomelatine may become a promising drug candidate in the therapy of AD.

Melatonin receptor stimulation by agomelatine prevents Aß-induced tau phosphorylation and oxidative damage in PC12 cells.

PURPOSE: As a novel antidepressant drug, agomelatine has good therapeutic effect on the mood disorder and insomnia in Alzheimer's disease (AD). Recent studies have shown the neuroprotective function of agomelatine, including anti-oxidative and anti-apoptosis effect. However, it remains unclear whether agomelatine exerts neuroprotection in AD. Thus, the neuroprotective effect of agomelatine against amyloid beta 25-35 (Aß25-35)-induced toxicity in PC12 cells was evaluated in this study. METHODS: The concentration of malondialdehyde (MDA), LDH, and ROS was investigated to evaluate oxidative damage. The expression of P-tau, tau, PTEN, P-Akt, Akt, P-GSK3ß, and GSK3ß proteins was assessed by Western blotting. Our results demonstrated that Aß25-35 significantly increased the content of MDA, LDH, and ROS. Meanwhile, Aß25-35 upregulated the expression of P-tau and PTEN as well as downregulated P-Akt and P-GSK3ß expression. These effects could be blocked by agomelatine pretreatment. Furthermore, luzindole, the melatonin receptor (MT) antagonist, could reverse the neuroprotective effect of agomelatine. CONCLUSION: The results demonstrated that antidepressant agomelatine might prevent the tau protein phosphorylation and oxidative damage induced by Aß25-35 in PC12 cells by activating MT-PTEN/Akt/GSK3ß signaling. This study provided a novel therapeutic target for AD in the future.

The Synergy of Aging and LPS Exposure in a Mouse Model of Parkinson's Disease.

Aging is an inevitable physiological challenge occurring in organisms over time, and is also the most important risk factor of neurodegenerative diseases. In this study, we observed cellular and molecular changes of different age mice and LPS-induced Parkinson disease (PD) model. The results showed that behavioral performance and dopaminergic (DA) neurons were declined, accompanied by increased expression of pro-inflammatory factors (TLR2, p-NF-kB-p65, IL-1ß and TNF-α), as well as pro-oxidative stress factor gp91phox in aged mice compared with young mice. Aging exaggerated inflammatory M1 microglia, and destroyed the balance between oxidation and anti-oxidation. The intranasal LPS instillation induced PD model in both young and aged mice. The poor behavioral performance and the loss of DA neurons as well as TLR2, p-NF-kB-p65, IL-1ß, TNF-α, iNOS and gp91phox were further aggravated in LPS-aged mice. Interestingly, the expression of Nrf2 and HO-1 was up-regulated by LPS only in young LPS-PD mice, but not in aged mice. The results indicate that the synergy of aging process and LPS exposure may prominently aggravate the DA neurons loss caused by more serious neuroinflammation and oxidative stress in the brain.

Aging modulates microglia phenotypes in neuroinflammation of MPTP-PD mice.

As the crucial etiological factor, aging-related microglia activation promotes the development of Parkinson's disease (PD). However, the molecular and functional changes of aged-microglia and their contribution to neurodegeneration in PD are only partially understood, which was investigated in our study. Female C57BL/6 mice were randomly divided into four groups, included young-control group, young-MPTP group, aged-control group and aged-MPTP group. Pole test and adhesive removal test were firstly performed. ELISA assay was used to detect the content of interleukin-1ß (IL-1ß) and tumor necrosis factor alpha (TNF-α) in brain tissue. Then we tested the expression of tyrosine hydroxylase (TH), p-nuclear transcription factor (NF-κB), toll-like receptor2 (TLR2), arginase-1 (arg-1), inducible nitric oxide synthase (iNOS) by western blot and immunofluorescence analysis. Our results showed that aging promoted M1 microglia activation and inhibited M2 microglia activation in SN in MPTP-PD model, accompanied by the elevation of proinflammatory cytokine (TNF-α and IL-1ß). Consequently, aging significantly aggravated motor dysfunction and dopaminergic neuron loss in SN. Besides, compared with young-MPTP group, the protein expression of TLR2 and p-NF-κB-p65 increased obviously in aged-MPTP group. The results revealed that aging aggravated inflammatory response by modulated microglia phenotypes transition in SN in PD, and contributed to further understand the pathogenesis of PD.

The inhibition of CB1 receptor accelerates the onset and development of EAE possibly by regulating microglia/macrophages polarization.

Cannabinoid 1 receptor (CB1R) regulates the neuro-inflammatory and neurodegenerative damages of experimental autoimmune encephalomyelitis (EAE) and of multiple sclerosis (MS). The mechanism by which CB1R inhibition exerts inflammatory effects is still unclear. Here, we explored the cellular and molecular mechanisms of CB1R in the treatment of EAE by using a specific and selective CB1R antagonist SR141716A. Our study demonstrated that SR141716A accelerated the clinical onset and development of EAE, accompanied by body weight loss. SR141716A significantly up-regulated the expression of toll like receptor-4 (TLR-4) and nuclear factor-kappaB/p65 (NF-κB/p65) on microglia/macrophages of EAE mice as well as levels of inflammatory factors (TNF-α, IL-1ß, IL-6) and chemokines (MCP-1, CX3CL1), accompanied by the shifts of cytokines from Th2 (IL-4, IL-10) to Th1 (IFN-γ)/Th17 (IL-17) in the spinal cords of EAE mice. Similar changes happened on splenic mononuclear cells (MNCs) except chemokine CX3CL1. Consistently, SR141716A promoted BV-2 microglia to release inflammatory factors (TNF-α, IL-1ß, IL-6) while inhibited the production of IL-10 and chemokines (MCP-1, CX3CL1). Furthermore, when splenic CD4+ T cells co-cultured with SR141716A-administered BV-2 microglia, the levels of IL-4 and IL-10 were decreased while production of IL-17 and IFN-γ increased significantly. Our research indicated that inhibition of CB1R induced M1 phenotype-Th17 axis changed of microglia/macrophages through TLR-4 and NF-κB/p65 which accelerated the onset and development of EAE. Therefore, CB1R may be a promising target for the treatment of MS/EAE, but its complexity remains to be carefully considered and studied in further clinical application.

Rho kinase II interference by small hairpin RNA ameliorates 1­methyl­4­phenyl­1,2,3,6­tetrahydropyridine­induced parkinsonism in mice.

Novel therapeutic targets are required for the treatment of Parkinson's disease (PD). Previous studies suggest that the Rho/Rho­associated, coiled­coil­containing protein kinases (ROCKs) signaling pathway may be a promising therapeutic target in PD. To elucidate the importance of ROCKII in the pathogenesis of dopaminergic (DA) neuron loss and to investigate the efficacy of ROCK inhibitors in PD, ROCKII expression in the substantia nigra (SN) of mice was silenced through the injection of a lentivirus­based small hairpin RNA system. Empty lentivirus vectors served as controls. Mice were subsequently challenged with 1­methyl­4­phenyl­1,2,3,6­tetrahydropyridine (MPTP). The expression levels and activity of ROCKII were elevated in tyrosine hydroxylase­positive neurons and in cluster of differentiation (CD) 11b­positive microglia within the SN of MPTP­treated mice, which was accompanied by an increased level of expression of inducible nitric oxide synthase (iNOS) and activation of the Toll­like receptor (TLR)2/nuclear factor (NF)­κB signaling pathway in M1 microglia. ROCKII interference (RI) significantly improved movement disorder and attenuated DA neuron loss induced by MPTP. In addition, RI inhibited the activation of M1 microglia in the SN, exhibiting reduced activity of the TLR2/NF­κB signaling pathway and decreased expression levels of iNOS and inflammatory factors, including interleukin (IL)­1ß and IL­6. The results of the present study verify that ROCKII participates in the loss of DA neurons induced by MPTP and suggest that ROCKII inhibition may be a promising therapeutic target for PD.

Changes of synapses in experimental autoimmune encephalomyelitis by using Fasudil.

The ROCK signaling pathway is involved in numerous fundamental cellular functions such as cell migration, apoptosis, inflammatory responses, and neurite outgrowth. Previous studies demonstrate that Fasudil exhibited therapeutic potential of experimental autoimmune encephalomyelitis (EAE) possibly through immune-modulation and anti-inflammation. In this study, we observed the effect of Fasudil on synaptic protection of EAE mice. Fasudil ameliorated the clinical severity of EAE and inhibited Rho kinase (ROCK), especially ROCK II, in brain and spinal cord of EAE mice. Protein extracts from spinal cord of Fasudil-treated EAE mice promoted the formation of neurite outgrowth when co-cultured with primary neurons, indicating that peripheral administration of Fasudil can enter the central nervous system (CNS) and exhibited its biological effect on the formation of neurite outgrowth. Synapse-related molecule synaptophysin was enhanced, and CRMP-2, AMPA receptor, and GSK-3ß were declined in spinal cord of Fasudil-treated mice. Neurotrophic factor BDNF and GDNF as well as immunomodulatory cytokine IL-10 in spinal cord were elevated in Fasudil-treated mice, while inflammatory cytokine IL-17, IL-1ß, IL-6, and TNF-α were obviously inhibited, accompanied by the decrease of inflammatory M1 iNOS and the increase of anti-inflammatory M2 Arg-1, providing a microenvironment that contributes to synaptic protection. Our results indicate that Fasudil treatment protected against synaptic damage and promoted synaptic formation, which may be related with increased neurotrophic factors as well as decreased inflammatory microenvironment in the CNS of EAE mice.

Safflower Yellow regulates microglial polarization and inhibits inflammatory response in LPS-stimulated Bv2 cells.

Activated microglia, especially polarized M1 cells, produce pro-inflammatory cytokines and free radicals, thereby contributing directly to neuroinflammation and various brain disorders. Given that excessive or chronic neuroinflammation within the central nervous system (CNS) exacerbates neuronal damage, molecules that modulate neuroinflammation are candidates as neuroprotective agents. In this study, we provide evidence that Safflor yellow (SY), the main active component in the traditional Chinese medicine safflower, modulates inflammatory responses by acting directly on BV2 microglia. LPS stimulated BV2 cells to upregulate expression of TLR4-Myd88 and MAPK-NF-κB signaling pathways and to release IL-1ß, IL-6, TNF-α, and COX-2. However, SY treatment inhibited expression of TLR4-Myd88 and p-38/p-JNK-NF-κB, downregulated expression of iNOS, CD16/32, and IL-12, and upregulated CD206 and IL-10. In conclusion, our results demonstrate that SY exerts an anti-inflammatory effect on BV2 microglia, possibly through TLR-4/p-38/p-JNK/NF-κB signaling pathways and the conversion of microglia from inflammatory M1 to an anti-inflammatory M2 phenotype.

Role of Rho Kinase and Fasudil on Synaptic Plasticity in Multiple Sclerosis.

In addition to myelin loss and oligodendrocyte injury, axonal damage is a major cause of irreversible neurological disability in multiple sclerosis (MS). A series of studies have demonstrated that Rho kinase (ROCK) is involved in synaptic plasticity of neurons. Here, we found that ROCK activity in MS serum was elevated compared with serum from healthy controls. In experimental autoimmune encephalomyelitis (EAE), ROCK activity was also increased in serum, spleen, brain and spinal cord. Neuron injury with scratch and TNF-α stimulation induced the up-regulation of ROCK activity. When serum of MS patients was co-cultured with mouse cortical neurons in vitro, MS serum caused neurite shortening and reduction of cell viability, while the addition of Fasudil partially restored synaptic morphology of neurons, revealing that MS sera inhibited neurite outgrowth and synapse formation. The expression of synaptophysin was decreased in MS serum-neurons, and elevated in the presence of Fasudil. In contrast, the expression of phosphorylated collapsin response mediator protein-2 (CRMP-2) was elevated in MS serum-neurons and decreased in the presence of Fasudil. However, the addition of anti-ROCK I/II mixed antibodies in MS serum partially declined ROCK activity, but did not improve neurite outgrowth of neurons, revealing that Fasudil should prevent synaptic damage possibly through inhibiting intracellular ROCK activation mediated with MS serum. Our results indicate that axonal loss in MS may be related to increased ROCK activity. Fasudil could promote synaptogenesis and thus may contribute to preventing irreversible neurological disability associated with MS.

Multitarget intervention of Fasudil in the neuroprotection of dopaminergic neurons in MPTP-mouse model of Parkinson's disease.

Recent studies have demonstrated that activation of the Rho-associated kinase (ROCK) pathway participates in the dopaminergic neuron degeneration and possibly in Parkinson's disease (PD). In the current study, we tried to observe the therapeutic potential of ROCK inhibitor Fasudil against dopaminergic neuron injury in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-mouse model of PD, and explore possible molecular mechanisms by enzyme-linked immunosorbent assay (ELISA), western blot and immunofluorescent assays. The results showed that MPTP-PD mice presented motor deficits, dopaminergic neuron loss, activation of inflammatory response and oxidative stress as well as ROCK and glycogen synthase kinase 3ß (GSK-3ß) signaling pathways. The administration of Fasudil exhibited neuroprotective effects against the dopaminergic neurons and improved the motor function recovery in the MPTP-PD mice, accompanied by the suppression of inflammatory responses (IL-1ß, TNF-α, NF-κB-p65 and TLR-2), and oxidative stress (iNOS and gp91Phox), which might be associated with the inhibition of ROCK and GSK-3ß activity. Simultaneously, the administration of Fasudil resulted in the shift from inflammatory M1 to anti-inflammatory/neurorepair M2 microglia. Additionally, Fasudil intervention enhanced the expression of anti-oxidative factors such as NF-E2-related factor 2 (Nrf2), Hmox as well as neurotrophic factor including GDNF. Our observations defined the neuroprotective effects of Fasudil in MPTP-PD mice, and we found a series of novel effector molecules and pathways for explaining the neuroprotective effects against dopaminergic neurons. However, a lot of investigations are warranted to further elucidate the crosstalk among Fasudil, oxidative stress, inflammatory response, GDNF and ROCK/NF-kB/Nrf2 pathways in the therapeutic potential of PD.