Unconjugated
BACKGROUND:
Neuroinflammation mediated by overactivated microglia plays a key role in many neurodegenerative diseases, including Alzheimer's disease (AD). In this study, we investigated for the first time the anti-neuroinflammatory effects and possible mechanisms of SCM-198 (an alkaloid extracted from Herbaleonuri), which was previously found highly cardioprotective, both in vitro and in vivo.
METHODS:
For in vitro experiments, lipopolysaccharide (LPS) or β-amyloid(1-40) (Aβ(1-40)) was applied to induce microglial overactivation. Proinflammatory mediators were measured and activations of NF-κB and mitogen-activated protein kinases' (MAPKs) pathways were investigated. Further protective effect of SCM-198 was evaluated in microglia-neuron co-culture assay and Sprague-Dawley (SD) rats intrahippocampally-injected with Aβ(1-40).
RESULTS:
SCM-198 reduced expressions of nitric oxide (NO), TNF-α, IL-1β and IL-6 possibly via, at least partially, inhibiting c-Jun N-terminal kinase (JNK) and NF-κB signaling pathways in microglia. Co-culture assay showed that activated microglia pretreated with SCM-198 led to less neuron loss and decreased phosphorylation of tau and extracellular signal-regulated kinase (ERK) in neurons. Besides, SCM-198 also directly protected against Aβ(1-40)-induced neuronal death and lactate dehydrogenase (LDH) release in primary cortical neurons. For in vivo studies, SCM-198 significantly enhanced cognitive performances of rats 12 days after intrahippocampal injections of aged Aβ(1-40) peptides in the Morris water maze (MWM), accompanied by less hippocampal microglial activation, decreased synaptophysin loss and phosphorylation of ERK and tau. Co-administration of donepezil and SCM-198 resulted in a slight cognitive improvement in SD rats 50 days after intrahippocampal injections of aged Aβ(1-40) peptides as compared to only donepezil or SCM-198 treated group.
CONCLUSIONS:
Our findings are the first to report that SCM-198 has considerable anti-neuroinflammatory effects on inhibiting microglial overactivation and might become a new potential drug candidate for AD therapy in the future.
Ubiquitin C-terminal hydrolase L1 (UCH-L1) is critical for protein degradation and free ubiquitin recycling. In Alzheimer's disease brains, UCH-L1 is negatively related to neurofibrillary tangles whose major component is hyperphosphorylated tau protein, but the direct action of UCH-L1 on tau has not been reported. In the current study, mouse neuroblastoma Neuro2a (N2a) cells were treated by the different concentrations of UCH-L1 inhibitor LDN (2.5, 5 and 10 μM) to inhibit the hydrolase activity of UCH-L1. In addition, we also used UCH-L1 siRNA to treat the HEK293/tau441 cells to decrease the expression of UCH-L1. After LDN and UCH-L1 siRNA treatment, we used immunofluorescence, immunoprecipitation, and tau-microtubule binding assay to measure the microtubule-binding ability and post-translational modifications of tau protein. All the results presented that both inhibition of the activity and expression of UCH-L1 induced the decreased microtubule-binding ability and increased phosphorylation of tau protein. Abnormal aggregation and ubiquitination of tau protein was also observed after UCH-L1 inhibition. The above results suggested that aggregation of tau protein might be devoted to the abnormal post-translational modifications of tau protein. Our study first indicates that dysfunction of UCH-L1 most likely affected normal biological function of tau protein through decreasing degradation of ubiquitinated and hyperphosphorylated tau.