Icariin ameliorates the cuprizone-induced demyelination associated with antioxidation and anti-inflammation.

The treatment of immunomodulation in multiple sclerosis (MS) can alleviate the severity and relapses. However, it cannot improve the neurological disability of patients due to a lack of myelin protection and regeneration. Therefore, remyelinating therapies may be one of the feasible strategies that can prevent axonal degeneration and restore neurological disability. Natural product icariin (ICA) is a flavonol compound extracted from epimedium flavonoids, which has neuroprotective effects in several models of neurological diseases. Here, we attempt to explore whether ICA has the potential to treat demyelination and its possible mechanisms of action using lipopolysaccharide-treated BV2 microglia, primary microglia, bone marrow-derived macrophages, and cuprizone-induced demyelination model. The indicators of oxidative stress and inflammatory response were evaluated using commercial kits. The results showed that ICA significantly reduced the levels of oxidative intermediates nitric oxide, hydrogen peroxide, malondialdehyde, and inflammatory cytokines TNF-α, IL-1ß, and increased the levels of antioxidants superoxide dismutase, catalase, glutathione peroxidase, and anti-inflammatory cytokines IL-10 and TGF-ß in vitro cell experiments. In vivo demyelination model, ICA significantly alleviated the behavioral abnormalities and enhanced the integrated optical density/mm2 of Black Gold II and myelin basic protein myelin staining, accompanied by the inhibition of oxidative stress/inflammatory response. Immunohistochemical staining showed that ICA significantly induced the expression of nuclear factor erythroid derived 2/heme oxygenase-1 (Nrf2/HO-1) and inhibited the expression of toll-like receptor 4/ nuclear factor kappa B (TLR4/NF-κB), which are two key signaling pathways in antioxidant and anti-inflammatory processes. Our results strongly suggest that ICA may be used as a potential agent to treat demyelination via regulating Nrf2/HO-1-mediated antioxidative stress and TLR4/NF-κB-mediated inflammatory responses.

[Tetramethylpyrazine regulates angiogenesis of endothelial cells in cerebral ischemic stroke injury via SIRT1/VEGFA signaling pathway].

This study aims to investigate whether tetramethylpyrazine(TMP) can stimulate angiogenesis in cerebral microvascular endothelial cells and alleviate cerebral ischemic stroke(CIS) and to explore the underlying mechanisms. In the animal study, adult Sprague-Dawley rats(n=15) were assigned into sham surgery(sham), middle cerebral artery occlusion/reperfusion(MCAO/R), and MCAO/R+TMP(intraperitoneal injection of 20 mg·kg~(-1)) groups. The neurological function was evaluated by the Z-Longa method. The cerebral infarction volume was detected by TTC staining. Enzyme-linked immunosorbent assay(ELISA) was employed to detect the expression of vascular endothelial growth factor(VEGF), angiopoietin(Ang), and platelet-derived growth factor(PDGF). Immunofluorescence staining was employed to detect Ki67 and the expression of vascular endothelial growth factor A(VEGFA) and slient information regulator 1(SIRT1). Western blot was employed to determine the expression levels of VEGFA, SIRT1, angiopoietin-2(Ang-2), and platelet-derived growth factor B(PDGFB). In the cell study, mouse brain-derived endothelial cells(Bend.3) were cultured, and the optimal concentration of TMP was determined. Then, VEGF, Ang, and PDGF were detected by ELISA after the addition of cabozantinib. Western blot was employed to measure the expression of VEGFA, Ang-2, and PDGFB. Immunofluorescence staining was used to detect CD31, CD34, and Ki67, and the proliferation, migration, and tube formation ability of Bend.3 cells were observed in vitro. Western blot and immunofluorescence staining were performed to measure the expression of SIRT1 and VEGFA after addition of the SIRT1-specific inhibitor selisistat(EX-527). The results showed that compared with the sham group, the MCAO/R group had severe neurological function damage, increased infarction volume, up-regulated expression of VEGF, VEGFA, Ang, Ang-2, PDGF, and PDGFB, and down-regulated expression of Ki67 and SIRT1(P<0.01). Compared with the MCAO/R group, the MCAO/R+TMP group presented alleviated neurological function damage, reduced infarction volume, and activated expression of VEGF, VEGFA, Ang, Ang-2, PDGF, PDGFB, Ki67, and SIRT1(P<0.01). The cell experiments showed that compared with the normal group, Bend.3 cells were activated by oxygen glucose deprivation/reoxygenation(OGD/R) treatment(P<0.05, P<0.01). Compared with the OGD/R group, the OGD/R+TMP group upregulated the expression levels of VEGF, VEGFA, Ang, Ang-2, PDGF, PDGFB, SIRT1, Ki67, CD31, and CD34, enhanced the angiogenic ability of Bend.3 cells without being inhibited by BMS or EX-527(P<0.05, P<0.01, P<0.001). The results suggest that TMP can activate the SIRT1/VEGFA signaling pathway to stimulate angiogenesis and alleviate CIS injury.

Preventing development of post-stroke hyperexcitability by optogenetic or pharmacological stimulation of cortical excitatory activity.

Stroke is the most common cause of acquired epilepsy, but treatment for preventing the development of post-stroke epilepsy is still unavailable. Since stroke results in neuronal damage and death as well as initial loss of activity in the affected brain region, homeostatic plasticity may be trigged and contribute to an increase in network hyperexcitability that underlies epileptogenesis. Correspondingly, enhancing brain activity may inhibit hyperexcitability from enhanced homeostatic plasticity and prevent post-stroke epileptogenesis. To test these hypotheses, we first used in vivo two-photon and mesoscopic imaging of activity of cortical pyramidal neurons in Thy1-GCaMP6 transgenic mice to determine longitudinal changes in excitatory activity after a photothrombotic ischemic stroke. At 3-days post-stroke, there was a significant loss of neuronal activity in the peri-injury area as indicated by reductions in the frequency of calcium spikes and percentage of active neurons, which recovered to baseline level at day 7, supporting a homeostatic activity regulation of the surviving neurons in the peri-injury area. We further used optogenetic stimulation to specifically stimulate activity of pyramidal neurons in the peri-injury area of Thy-1 channelrhodopsin transgenic mice from day 5 to day 15 after stroke. Using pentylenetetrazole test to evaluate seizure susceptibility, we showed that stroke mice are more susceptible to Racine stage V seizures (time latency 54.3 ± 12.9 min) compared to sham mice (107.1 ± 13.6 min), but optogenetic stimulation reversed the increase in seizure susceptibility (114.0 ± 9.2 min) in mice with stroke. Similarly, administration of D-cycloserine, a partial N-methyl-d-aspartate (NMDA) receptor agonist that can mildly enhance neuronal activity without causing post-stroke seizure, from day 5 to day 15 after a stroke significantly reversed the increase in seizure susceptibility. The treatment also resulted in an increased survival of glutamic acid decarboxylase 67 (GAD67) positive interneurons and a reduced activation of glial fibrillary acidic protein (GFAP) positive reactive astrocytes. Thus, this study supports the involvement of homeostatic activity regulation in the development of post-stroke hyperexcitability and potential application of activity enhancement as a novel strategy to prevent post-stroke late-onset seizure and epilepsy through regulating cortical homeostatic plasticity.

Drug-induced microglial phagocytosis in multiple sclerosis and experimental autoimmune encephalomyelitis and the underlying mechanisms.

Microglia are resident macrophages of the central nervous system (CNS). It plays a significant role in immune surveillance under physiological conditions. On stimulation by pathogens, microglia change their phenotypes, phagocytize toxic molecules, secrete pro-inflammatory/anti-inflammatory factors, promotes tissue repair, and maintain the homeostasis in CNS. Accumulation of myelin debris in multiple sclerosis (MS)/experimental autoimmune encephalomyelitis (EAE) inhibits remyelination by decreasing the phagocytosis by microglia and prevent the recovery of MS/EAE. Drug induced microglia phagocytosis could be a novel therapeutic intervention for the treatment of MS/EAE. But the abnormal phagocytosis of neurons and synapses by activated microglia will lead to neuronal damage and degeneration. It indicates that the phagocytosis of microglia has many beneficial and harmful effects in central neurodegenerative diseases. Therefore, simply promoting or inhibiting the phagocytic activity of microglia may not achieve ideal therapeutic results. However, limited reports are available to elucidate the microglia mediated phagocytosis and its underlying molecular mechanisms. On this basis, the present review describes microglia-mediated phagocytosis, drug-induced microglia phagocytosis, molecular mechanism, and novel approach for MS/EAE treatment.

Wuzi Yanzong Pill relieves MPTP-induced motor dysfunction and neuron loss by inhibiting NLRP3 inflammasome-mediated neuroinflammation.

Parkinson disease (PD) is an age-related neurodegenerative disease, which is associated with the loss of dopaminergic neurons (DA neurons) in the substantia nigra pars compacta (SNpc), and neuroinflammation may lead to the occurrence of PD. Wuzi Yanzong Pill (WYP) has demonstrated neuroprotective and anti-inflammatory properties, but its molecular mechanism of action is still unclear. In this study, we used 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mice and LPS-mediated BV2 microglia to explore WYP intervention, anti-inflammatory effect and molecular mechanism in vivo and in vitro. The results showed that oral administration of WYP in MPTP-induced PD mice for 2 weeks ameliorated abnormal motor dysfunction, attenuated the loss of TH + neurons in SNpc, protected dopaminergic neurons, and inhibited the activation of microglia in MPTP-induced PD mice and LPS-stimulated BV2 cell. Meanwhile, WYP intervention inhibited the expression of IL-6, TNF-α, Pro-IL-1ß, IL-1ß, Pro-IL-18, IL-18 and enhanced the expression of IL-10 in the SNpc of PD mice. Simultaneously, WYP intervention inhibited the expression of NLRP3 inflammasome, accompanied by the decrease of the TLR4/MyD88/NF-κB pathway. However, the exact target and interaction of WYP on NLRP3 inflammasome and TLR4/MyD88/NF-κB pathway still needs to be further investigated.

Neuroprotective effect of hyperoside in MPP+/MPTP -induced dopaminergic neurodegeneration.

Parkinson's disease (PD) is a neurodegenerative disease characterized by the pathological loss of nigrostriatal dopaminergic neurons, which causes an insufficient release of dopamine (DA) and then induces motor and nonmotor symptoms. Hyperoside (HYP) is a lignan component with anti-inflammatory, antioxidant, and neuroprotective effects. In this study, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its active neurotoxic metabolite 1-methyl-4-phenylpyridinium ion (MPP+) were used to induce dopaminergic neurodegeneration. The results showed that HYP (100 µg/mL) reduced MPTP-mediated cytotoxicity of SH-SY5Y cells in vitro, and HYP [25 mg/(kg d)] alleviated MPTP-induced motor symptoms in vivo. HYP treatment reduced the contents of nitric oxide (NO), H2O2, and malondialdehyde (MDA), as well as the mitochondrial damage of dopaminergic neurons, both in vitro and in vivo. Meanwhile, HYP treatment elevated the levels of neurotrophic factors such as glial cell line-derived neurotrophic factor, brain-derived neurotrophic factor, and recombinant cerebral dopamine neurotrophic factor in vivo, but not in vitro. Finally, Akt signaling was activated after the administration of HYP in MPP+/MPTP-induced dopaminergic neurodegeneration. However, the blockage of the Akt pathway with Akt inhibitor did not abolish the neuroprotective effect of HYP on DA neurons. These results showed that HYP protected the dopaminergic neurons from the MPP+- and MPTP-induced injuries, which did not rely on the Akt pathway.

[Mechanism of bilobalide promoting neuroprotection of macrophages].

This study aims to explore the neuroprotective effect of bilobalide(BB) and the mechanisms such as inhibiting inflammatory response in macrophage/microglia, promoting neurotrophic factor secretion, and interfering with the activation and differentiation of peripheral CD4~+ T cells. BB of different concentration(12.5, 25, 50, 100 µg·mL~(-1)) was used to treat the RAW264.7 and BV2 cells for 24 h. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay and cell counting kit-8(CCK-8) were employed to detect the cytotoxicity of BB and appropriate concentration was selected for further experiment. Lipopolysaccharide(LPS) was applied to elicit inflammation in RAW264.7 and BV2 cells, mouse bone marrow-derived macrophages(BMDMs), and primary microglia, respectively. The effect of BB on cell proliferation and secretion of inflammatory cytokines and neurotrophic factors was detected by enzyme-linked immunosorbent assay(ELISA). Spleen monocytes of C57BL/6 female mice(7-8 weeks old) were isolated, and CD4~+ T cells were separated by magnetic beads under sterile conditions. Th17 cells were induced by CD3/CD28 and the conditioned medium for eliciting the inflammation in BMDMs. The content of IL-17 cytokines in the supernatant was detected by ELISA to determine the effect on the activation and differentiation of CD4~+ T cells. In addition, PC12 cells were incubated with the conditioned medium for eliciting inflammation in BMDMs and primary microglia and the count and morphology of cells were observed. The cytoto-xicity was determined by lactate dehydrogenase(LDH) assay. The result showed that BB with the concentration of 12.5-100 µg·mL~(-1) had no toxicity to RAW264.7 and BV2 cells, and had no significant effect on the activity of cell model with low inflammation. The 50 µg·mL~(-1) BB was selected for further experiment, and the results indicated that BB inhibited LPS-induced secretion of inflammatory cytokines. The experiment on CD4~+ T cells showed that the conditioned medium for LPS-induced inflammation in BMDMs promoted the activation and differentiation of CD4~+ T cells, while the conditioned medium of the experimental group with BB intervention reduced the activation and differentiation of CD4~+ T cells. In addition, BB also enhanced the release of neurotrophic factors from BMDMs and primary microglia. The conditioned medium after BB intervention can significantly reduce the death of PC12 neurons, inhibit neuronal damage, and protect neurons. To sum up, BB plays a neuroprotective role by inhibiting macrophage and microglia-mediated inflammatory response and promoting neurotrophic factors.

Wuzi Yanzong pill attenuates MPTP-induced Parkinson's Disease via PI3K/Akt signaling pathway.

Wuzi Yanzong Pill (WYP) was found to play a protective role on nerve cells and neurological diseases, however the molecular mechanism is unclear. To understand the molecular mechanisms that underly the neuroprotective effect of WYP on dopaminergic neurons in Parkinson's disease (PD). PD mouse model was induced by the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Gait and hanging tests were used to assess motor behavioral function. Immunofluorescence assay was used to determine TH-positive neurons in substantia nigra (SN). Apoptosis, dopamine and neurotrophic factors as well as expression of PI3K/Akt pathway were detected by TUNEL staining, ELISA and western blotting, respectively. First, it was observed that WYP intervention improved abnormal motor function in MPTP-induced PD model, alleviated the loss of TH+ neurons in SN, and increased dopamine content in brain, revealing a potential protective effect. Second, network pharmacology was used to analyze the possible targets and pathways of WYP action in the treatment of PD. A total of 126 active components related to PD were screened in WYP, and the related core targets included ALB, GAPDH, Akt1, TP53, IL6 and TNF. Particularly, the effect of WYP on PD may be medicate through PI3K/Akt signaling pathway and apoptotic regulation. The WYP treated PD mice had higher expression of p-PI3K, p-Akt and Bcl-2 but lower expression of Bax and cleaved caspase-3 than the non-WYP treated PD mice. Secretion of brain-derived neurotrophic factor (BDNF) and cerebral dopamine neurotrophic factor (CDNF) were also increased in the treated mice. WYP may inhibit apoptosis and increase the secretion of neurotrophic factor via activating PI3K/ Akt signaling pathway, thus protecting the loss of dopamine neurons in MPTP-induced PD mice.

Targeting the differentiation of astrocytes by Bilobalide in the treatment of Parkinson's disease model.

Background: The etiology of Parkinson's disease (PD), a chronic and progressive neurodegenerative disease, is multifactorial but not fully unknown. Until now, no drug has been proven to have neuroprotective or neuroregenerative effects in patients with PD.Objectives: To observe the therapeutic potential of Bilobalide (BB), a constituent of ginkgo biloba, in MPTP-induced PD model, and explore its possible mechanisms of action.Material and Methods: Mice were randomly divided into three groups: healthy group, MPTP group and MPTP + BB group. PD-related phenotypes were induced by intraperitoneal injection of MPTP into male C57BL/6 mice, and BB (40 mg/kg/day) was intraperitoneally given for 7 consecutive days at the end of modeling. The injection of saline was set up as the control in a similar manner.Results: BB induced M2 polarization of microglia, accompanied by inhibition of neuroinflammation in the brain. Simultaneously, BB promoted the expression of BDNF in astrocytes and neurons, and expression of GDNF in neurons. Most interestingly, BB enhanced the formation of GFAP+ astrocytes expressing nestin, Brn2 and Ki67, as well as the transformation of GFAP+ astrocytes expressing tyrosine hydroxylase around subventricular zone, providing experimental evidence that BB could promote the conversion of astrocytes into TH+ dopamine neurons in vivo and in vitro.Conclusions: These results suggest the natural product BB may utilize multiple pathways to modify degenerative process of TH+ neurons, revealing an exciting opportunity for novel neuroprotective therapeutics. However, its multi-target and important mechanisms need to be further explored.

Temporal and spatial evolution of various functional neurons during demyelination induced by cuprizone.

Multiple sclerosis (MS) is an inflammatory, demyelinating, and neurodegenerative disease of the central nervous system (CNS). Here we report the temporal and spatial evolution of various functional neurons during demyelination in a cuprizone (CPZ)-induced mouse model. CPZ did not significantly induce the damage of axons and neurons after 2 wk of feeding. However, after 4-6 wk of CPZ feeding, axons and neurons were markedly reduced in the cortex, posterior thalamic nuclear group, and hippocampus. Simultaneously, the expression of TPH+ tryptophan neurons and VGLUT1+ glutamate neurons was obviously decreased, and the expression of TH+ dopaminergic neurons was slightly decreased in the tail part of the substantia nigra striatum, whereas the number of ChAT+ cholinergic neurons was not significantly different in the brain. In the second week of feeding, CPZ caused a higher level of glutamate secretion and upregulated the expression of EAAT2 on astrocytes, which should contribute to rapid and sufficient glutamate uptake and removal. This finding reveals that astrocyte-driven glutamate reuptake protected the CNS from excitotoxicity by rapid reuptake of glutamate in 4-6 wk of CPZ feeding. At this stage, although NG2+ oligodendroglia progenitor cells (OPCs) were enhanced in the demyelination foci, the myelin sheath was still absent. In conclusion, we comprehensively observed the temporal and spatial evolution of various functional neurons. Our results will assist with understanding how demyelination affects neurons during CPZ-induced demyelination and provide novel information for neuroprotection in myelin regeneration and demyelinating diseases.NEW & NOTEWORTHY Our results further indicate temporal and spatial evolution of various functional neurons during the demyelination in a cuprizone (CPZ)-induced mouse model, which mainly occur 4-6 wk after CPZ feeding. At the same time, the axonal compartment is damaged and, consequently, neuronal death occurs, while glutamate neurons are lost obviously. The astrocyte-mediated glutamate reuptake could protect the neurons from the excitatory effects of glutamate.

Cloning, Yeast Expression, and Characterization of a ß-Amyrin C-28 Oxidase (CYP716A249) Involved in Triterpenoid Biosynthesis in Polygala tenuifolia.

Polygala tenuifolia Willd. is a traditional Chinese herbal medicine that is widely used in treating nervous system disorders. Triterpene saponins in P. tenuifolia (polygala saponins) have excellent biological activity. As a precursor for the synthesis of presenegin, oleanolic acid (OA) plays an important role in the biosynthesis of polygala saponins. However, the mechanism behind the biosynthesis of polygala saponins remains to be elucidated. In this study, we found that CYP716A249 (GenBank: ASB17946) oxidized the C-28 position of ß-amyrin to produce OA. Using quantitative real-time PCR, we observed that CYP716A249 had the highest expression in the roots of 2-year-old P. tenuifolia, which provided a basis for the selection of samples for gene cloning. To identify the function of CYP716A249, the strain R-BE-20 was constructed by expressing ß-amyrin synthase in yeast. Then, CYP716A249 was co-expressed with ß-amyrin synthase to construct the strain R-BPE-20 by using the lithium acetate method. Finally, we detected ß-amyrin and OA by ultra-HPLC-Q Exactive hybrid quadrupole-Orbitrap high-resolution accurate mass spectrometry and GC-MS. The results of this study provide insights into the biosynthesis pathway of polygala saponins.

The therapeutic potential of bilobalide on experimental autoimmune encephalomyelitis (EAE) mice.

Inflammatory demyelination in the central nervous system (CNS) is a hallmark of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Besides MS disease-modifying therapy, targeting myelin sheath protection/regeneration is currently a hot spot in the treatment of MS. Here, we attempt to explore the therapeutic potential of Bilobalide (BB) for the myelin protection/regeneration in EAE model. The results showed that BB treatment effectively prevented worsening and demyelination of EAE, accompanied by the inhibition of neuroinflammation that should be closely related to T cell tolerance and M2 macrophages/microglia polarization. BB treatment substantially inhibited the infiltration of T cells and macrophages, thereby alleviating the enlargement of neuroinflammation and the apoptosis of oligodendrocytes in CNS. The accurate mechanism of BB action and the feasibility of clinical application in the prevention and treatment of demyelination remain to be further explored.

Fasudil reduces ß-amyloid levels and neuronal apoptosis in APP/PS1 transgenic mice via inhibition of the Nogo-A/NgR/RhoA signaling axis.

Recent studies have shown that Nogo-A and the Nogo-A receptor affect ß-amyloid metabolism and the downstream Rho GTP enzyme signaling pathway, which may affect the levels of ß-amyloid and tau. Nogo-A may play a key role in the pathogenesis of Alzheimer's disease. However, the underlying molecular mechanisms of Fasudil treatment in Alzheimer's disease are not yet clear. Our results have found that Fasudil treatment for two months substantially ameliorated behavioral deficits, diminished ß-amyloid plaque and tau protein pathology, and alleviated neuronal apoptosis in APP/PS1 transgenic mice. More importantly, two well-established markers for synaptic function, growth-associated protein 43 and synaptophysin, were upregulated after Fasudil treatment. Finally, the levels of Nogo-A, Nogo-A receptor complex NgR/p75NTR/LINGO-1 and the downstream Rho/Rho kinase signaling pathway were significantly reduced. These findings suggest that Fasudil exerts its neuroprotective function in Alzheimer's disease by inhibiting the Nogo-A/NgR1/RhoA signaling pathway.

Hydroxyfasudil alleviates demyelination through the inhibition of MOG antibody and microglia activation in cuprizone mouse model.

Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system characterized by oligodendrocyte loss and progressive neurodegeneration. The cuprizone (CPZ)-induced demyelination is widely used to investigate the demyelination/remyelination. Here, we explored the therapeutic effects of Hydroxyfasudil (HF), an active metabolite of Fasudil, in CPZ model. HF improved behavioral abnormality and reduced myelin damage in the corpus callosum. Splenic atrophy and myelin oligodendrocyte glycoprotein (MOG) antibody were observed in CPZ model, which were partially restored and obviously inhibited by HF, therefore reducing pathogenic binding of MOG antibody to oligodendrocytes. HF inhibited the percentages of CD4+IL-17+ T cells from splenocytes and infiltration of CD4+ T cells and CD68+ macrophages in the brain. HF also declined microglia-mediated neuroinflammation, and promoted the production of astrocyte-derived brain derived neurotrophic factor (BDNF) and regeneration of NG2+ oligodendrocyte precursor cells. These results provide potent evidence for the therapeutic effects of HF in CPZ-induced demyelination.

Mdivi-1, a mitochondrial fission inhibitor, modulates T helper cells and suppresses the development of experimental autoimmune encephalomyelitis.

BACKGROUND: Unrestrained activation of Th1 and Th17 cells is associated with the pathogenesis of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). While inactivation of dynamin-related protein 1 (Drp1), a GTPase that regulates mitochondrial fission, can reduce EAE severity by protecting myelin from demyelination, its effect on immune responses in EAE has not yet been studied. METHODS: We investigated the effect of Mdivi-1, a small molecule inhibitor of Drp1, on EAE. Clinical scores, inflammation, demyelination and Drp1 activation in the central nervous system (CNS), and T cell responses in both CNS and periphery were determined. RESULTS: Mdivi-1 effectively suppressed EAE severity by reducing demyelination and cellular infiltration in the CNS. Mdivi-1 treatment decreased the phosphorylation of Drp1 (ser616) on CD4+ T cells, reduced the numbers of Th1 and Th17 cells, and increased Foxp3+ regulatory T cells in the CNS. Moreover, Mdivi-1 treatment effectively inhibited IFN-γ+, IL-17+, and GM-CSF+ CD4+ T cells, while it induced CD4+ Foxp3+ regulatory T cells in splenocytes by flow cytometry. CONCLUSIONS: Together, our results demonstrate that Mdivi-1 has therapeutic potential in EAE by modulating the balance between Th1/Th17 and regulatory T cells.

The Rho kinase inhibitor fasudil attenuates Aß1-42-induced apoptosis via the ASK1/JNK signal pathway in primary cultures of hippocampal neurons.

Alzheimer's disease (AD), a chronic, progressive, neurodegenerative disorder, is the most common type of dementia. Beta amyloid (Aß) peptide aggregation and phosphorylated tau protein accumulation are considered as one of the causes for AD. Our previous studies have demonstrated the neuroprotective effect of the Rho kinase inhibitor fasudil, but the mechanism remains elucidated. In the present study, we examined the effects of fasudil on Aß1-42 aggregation and apoptosis and identified the intracellular signaling pathways involved in these actions in primary cultures of mouse hippocampal neurons. The results showed that fasudil increased neurite outgrowth (52.84%), decreased Aß burden (46.65%), Tau phosphorylation (96.84%), and ROCK-II expression. In addition, fasudil reversed Aß1-42-induced decreased expression of Bcl-2 and increases in caspase-3, cleaved-PARP, phospho-JNK(Thr183/Tyr185), and phospho-ASK1(Ser966). Further, fasudil decreased mitochondrial membrane potential and intracellular calcium overload in the neurons treated with Aß1-42. These results suggest that inhibition of Rho kinase by fasudil reverses Aß1-42-induced neuronal apoptosis via the ASK1/JNK signal pathway, calcium ions, and mitochondrial membrane potential. Fasudil could be a drug of choice for treatment of Alzheimer's disease.

Therapeutic potentials of the Rho kinase inhibitor Fasudil in experimental autoimmune encephalomyelitis and the related mechanisms.

Multiple sclerosis (MS), Parkinson's disease (PD), Alzheimer's disease (AD), and other neurodegenerative diseases of central nervous system (CNS) disorders are serious human health problems. Rho-kinase (ROCK) is emerging as a potentially important therapeutic target relevant to inflammatory neurodegeneration diseases. This is supported by studies showing the beneficial effects of fasudil, a ROCK inhibitor, in inflammatory neurodegeneration diseases. MS is an autoimmune disease resulting from inflammation and demyelination in the white matter of the CNS. It has been postulated that activation of Rho/ROCK causes neuropathological changes accompanied with related clinical symptoms, which are improved by treatment with ROCK inhibitors. Therefore, inhibition of abnormal activation of the Rho/ROCK signaling pathway appears to be a new mechanism for treating CNS diseases. In this review, we extensively discussed the role of ROCK inhibitors, summarized the efficacy of fasudil in the MS conventional animal model of experimental autoimmune encephalomyelitis (EAE), both in vivo and in vitro, and highlighted the mechanism involved. Overall, the findings collected in this review support the role of the ROCK signaling pathway in neurodegenerative diseases. Hence, ROCK inhibitors such as fasudil can be novel, and efficacious treatment for inflammatory neurodegenerative diseases.

Effect of Fingolimod on Neural Stem Cells: A Novel Mechanism and Broadened Application for Neural Repair.

Inflammatory demyelination and axonal damage of the CNS are hallmarks of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Fingolimod (FTY720), the first FDA-approved oral medication for MS, suppresses acute disease but is less effective at the chronic stage, and whether it has a direct effect on neuroregeneration in MS and EAE remains unclear. Here we show that FTY720, at nanomolar concentrations, effectively protected survival of neural stem cells (NSCs) and enhanced their development into mature oligodendrocytes (OLGs) in vitro, primarily through the S1P3 and S1P5 receptors. In vivo, treatment with either FTY720 or NSCs alone had no effect on the secondary progressive stage of remitting-relapsing EAE, but a combination therapy with FTY720 and NSCs promoted significant recovery, including ameliorated clinical signs and CNS inflammatory demyelination, enhanced MBP synthesis and remyelination, inhibited axonal degeneration, and reduced astrogliosis. Moreover, FTY720 significantly improved incorporation and survival of transplanted NSCs in the CNS and drove their differentiation into more OLGs but fewer astrocytes, thus promoting remyelination and CNS repair processes in situ. Our data demonstrate a novel effect of FTY720 on NSC differentiation and remyelination, broadening its possible application to NSC-based therapy in the secondary progressive stage of MS.

RETRACTED: Neural Stem Cells Engineered to Express Three Therapeutic Factors Mediate Recovery from Chronic Stage CNS Autoimmunity

Treatment of chronic neurodegenerative diseases such as multiple sclerosis (MS) remains a major challenge. Here we genetically engineer neural stem cells (NSCs) to produce a triply therapeutic cocktail comprising IL-10, NT-3, and LINGO-1-Fc, thus simultaneously targeting all mechanisms underlie chronicity of MS in the central nervous system (CNS): persistent inflammation, loss of trophic support for oligodendrocytes and neurons, and accumulation of neuroregeneration inhibitors. After transplantation, NSCs migrated into the CNS inflamed foci and delivered these therapeutic molecules in situ. NSCs transduced with one, two, or none of these molecules had no or limited effect when injected at the chronic stage of experimental autoimmune encephalomyelitis; cocktail-producing NSCs, in contrast, mediated the most effective recovery through inducing M2 macrophages/microglia, reducing astrogliosis, and promoting axonal integrity and endogenous oligodendrocyte/neuron differentiation. These engineered NSCs simultaneously target major mechanisms underlying chronicity of multiple sclerosis (MS) and encephalomyelitis (EAE), thus representing a novel and potentially effective therapy for the chronic stage of MS, for which there is currently no treatment available.