LRP1 knockdown aggravates Aß1-42-stimulated microglial and astrocytic neuroinflammatory responses by modulating TLR4/NF-κB/MAPKs signaling pathways.

Neuroinflammation is an important pathological feature and an early event in the pathogenesis of Alzheimer's disease (AD), which is characterized by activation of microglia and astrocytes. Low-density lipoprotein receptor-related protein 1 (LRP1) is an endocytic receptor that is abundantly expressed in neurons, microglia, and astrocytes, and plays a critical role in AD pathogenesis. There is increasing evidence to show that LRP1 regulates inflammatory responses by modulating the release of pro-inflammatory cytokines and phagocytosis. However, the effects of LRP1 on ß-amyloid protein (Aß)-induced microglial and astrocytic neuroinflammatory responses and its underlying mechanisms have not been studied in detail. In the present study, knockdown of LRP1 significantly enhanced Aß1-42-stimulated neuroinflammation by increasing the production of pro-inflammatory cytokines in both BV2 microglial cells and mouse primary astrocytes. Furthermore, it is revealed that LRP1 knockdown further led to the activation of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) signaling pathways. The phosphorylation of IκBα, p38, and JNK was significantly up-regulated in LRP1 knockdown BV2 microglial cells and primary astrocytes. Meanwhile, LRP1 knockdown increased expression of the NF-κB p65 subunit in the nucleus while decreased its expression in the cytoplasm. Besides, the upstream signaling adaptor molecules such as toll-like receptor 4 (TLR4), myeloid differentiation primary response protein 88 (MyD88), and tumor necrosis factor receptor-associated factor 6 (TRAF6) were also further increased. Moreover, blockade of NF-κB, p38, and JNK inhibited the production of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6) induced by the knockdown of LRP1. Taken together, these findings indicated that LRP1 as an effective therapeutic target against AD and other neuroinflammation related diseases.

Tanshinone IIA attenuates neuroinflammation via inhibiting RAGE/NF-κB signaling pathway in vivo and in vitro.

BACKGROUND: Glial activation and neuroinflammation play a crucial role in the pathogenesis and development of Alzheimer's disease (AD). The receptor for advanced glycation end products (RAGE)-mediated signaling pathway is related to amyloid beta (Aß)-induced neuroinflammation. This study aimed to investigate the neuroprotective effects of tanshinone IIA (tan IIA), a natural product isolated from traditional Chinese herbal Salvia miltiorrhiza Bunge, against Aß-induced neuroinflammation, cognitive impairment, and neurotoxicity as well as the underlying mechanisms in vivo and in vitro. METHODS: Open-field test, Y-maze test, and Morris water maze test were conducted to assess the cognitive function in APP/PS1 mice. Immunohistochemistry, immunofluorescence, thioflavin S (Th-S) staining, enzyme-linked immunosorbent assay (ELISA), real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR), and western blotting were performed to explore Aß deposition, synaptic and neuronal loss, microglial and astrocytic activation, RAGE-dependent signaling, and the production of pro-inflammatory cytokines in APP/PS1 mice and cultured BV2 and U87 cells. RESULTS: Tan IIA treatment prevented spatial learning and memory deficits in APP/PS1 mice. Additionally, tan IIA attenuated Aß accumulation, synapse-associated proteins (Syn and PSD-95) and neuronal loss, as well as peri-plaque microgliosis and astrocytosis in the cortex and hippocampus of APP/PS1 mice. Furthermore, tan IIA significantly suppressed RAGE/nuclear factor-κB (NF-κB) signaling pathway and the production of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1ß) in APP/PS1 mice and cultured BV2 and U87 cells. CONCLUSIONS: Taken together, the present results indicated that tan IIA improves cognitive decline and neuroinflammation partly via inhibiting RAGE/NF-κB signaling pathway in vivo and in vitro. Thus, tan IIA might be a promising therapeutic drug for halting and preventing AD progression.

TREM2 Inhibits Tau Hyperphosphorylation and Neuronal Apoptosis via the PI3K/Akt/GSK-3ß Signaling Pathway In vivo and In vitro.

Triggering receptor expressed on myeloid cells-2 (TREM2), a cell surface receptor mainly expressed on microglia, has been shown to play a critical role in Alzheimer's disease (AD) pathogenesis and progression. Our recent results showed that overexpression of TREM2 inhibited inflammatory response in APP/PS1 mice and BV2 cells. Several studies indicated that TREM2 ameliorated tau hyperphosphorylation might be ascribed to the inhibition of neuroinflammation. However, the precise signaling pathways underlying the effect of TREM2 on tau pathology and neuronal apoptosis have not been fully elucidated. In the present study, upregulation of TREM2 significantly inhibited tau hyperphosphorylation at Ser199, Ser396, and Thr205, respectively, as well as prevented neuronal loss and apoptosis. We also found that upregulation of TREM2 alleviated behavioral deficits and improved the spatial cognitive ability of APP/PS1 mice. Further study revealed that TREM2 could activate phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway, resulting in an inhibitory effect on glycogen synthase kinase-3ß (GSK-3ß), which is a major kinase responsible for tau hyperphosphorylation in AD. In line with in vivo findings, TREM2-overexpressing BV2 microglia following ß-amyloid (Aß) stimulation led to a significant increase in the phosphorylation of PI3K, Akt, and GSK-3ß, accompanied by a decrease in tau hyperphosphorylation and apoptosis in co-cultured SH-SY5Y cells. Furthermore, LY294002, a specific PI3K inhibitor, was observed to abolish the beneficial effects of TREM2 on tau hyperphosphorylation, neuronal apoptosis, and spatial cognitive impairments in vivo and in vitro. Thus, our findings indicated that TREM2 inhibits tau hyperphosphorylation and neuronal apoptosis, at least in part, by the activation of the PI3K/Akt/GSK-3ß signaling pathway. Taken together, the above results allow us to better understand how TREM2 protects against tau pathology and suggest that upregulation of TREM2 may provide new ideas and therapeutic targets for AD.

Tanshinone IIA regulates glycogen synthase kinase-3ß-related signaling pathway and ameliorates memory impairment in APP/PS1 transgenic mice.

Our previous findings indicated that tanshinone IIA (tan IIA), a natural component extracted from the root and rhizome of danshen, significantly attenuated ß-amyloid accumulation, neuroinflammation, and endoplasmic reticulum stress, as well as improved learning and memory deficits in APP/PS1 transgenic mouse model of Alzheimer's disease (AD). However, whether tan IIA can ameliorate tau pathology and the underlying mechanism in APP/PS1 mice remains unclear. In the current study, tan IIA (15 mg/kg and 30 mg/kg) or saline was intraperitoneally administered to the 5-month-old APP/PS1 mice once daily for 4 weeks. The open-field test, novel object recognition test, Y-maze test, and Morris water maze test were performed to assess the cognitive function. Nissl staining, immunohistochemistry, TUNEL, and western blotting were conducted to explore tau hyperphosphorylation, neuronal injury, and phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt)/glycogen synthase kinase-3ß (GSK-3ß) signaling pathway. The activity of GSK-3ß, acetylcholinesterase (AChE), choline acetyltransferase (ChAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px), and the level of malondialdehyde (MDA) were measured using commercial kits. Our results revealed that tan IIA treatment significantly ameliorated behavioral deficits and improved spatial learning and memory ability of APP/PS1 mice. Additionally, tan IIA markedly attenuated tau hyperphosphorylation and prevented neuronal loss and apoptosis in the parietal cortex and hippocampus. Simultaneously, tan IIA reversed cholinergic dysfunction and reduced oxidative stress. Furthermore, tan IIA activated the PI3K/Akt signaling pathway and suppressed GSK-3ß. Taken together, the above findings suggested that tan IIA improves cognitive decline and tau pathology may through modulation of PI3K/Akt/GSK-3ß signaling pathway.

Downregulation of TREM2 expression exacerbates neuroinflammatory responses through TLR4-mediated MAPK signaling pathway in a transgenic mouse model of Alzheimer's disease.

Activation of glial cells and neuroinflammation play an important role in the onset and development of Alzheimer's disease (AD). Triggering receptor expressed on myeloid cells 2 (TREM2) is a microglia-specific receptor in the brain that is involved in regulating neuroinflammation. However, the precise effects of TREM2 on neuroinflammatory responses and its underlying molecular mechanisms in AD have not been studied in detail. Here, we employed a lentiviral-mediated strategy to downregulation of TREM2 expression on microglia in the brain of APPswe/PS1dE9 (APP/PS1) transgenic mice and BV2 cells. Our results showed that downregulation of TREM2 significantly aggravated AD-related neuropathology including Aß accumulation, peri-plaque microgliosis and astrocytosis, as well as neuronal and synapse-associated proteins loss, which was accompanied by a decline in cognitive ability. The further mechanistic study revealed that downregulation of TREM2 expression initiated neuroinflammatory responses through toll-like receptor 4 (TLR4)-mediated mitogen-activated protein kinase (MAPK) signaling pathway and subsequent stimulating the production of pro-inflammatory cytokines in vivo and in vitro. Moreover, blockade of p38, JNK, and ERK1/2 inhibited the release of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6) induced by Aß1-42 in TREM2-knocked down BV2 cells. Taken together, these findings indicated that TREM2 might be a potential therapeutic target for AD and other neuroinflammation-related diseases.

TREM2 overexpression rescues cognitive deficits in APP/PS1 transgenic mice by reducing neuroinflammation via the JAK/STAT/SOCS signaling pathway.

Overactivated microglia and neuroinflammation are considered to play a crucial role in the progression of Alzheimer's disease (AD). Triggering receptor expressed on myeloid cells-2 (TREM2), a type I transmembrane receptor, expressed uniquely by microglia in the brain, is involved in the neuroinflammatory responses of AD. In this study, to further explore the precise effects of TREM2 on neuroinflammation and the underlying mechanisms in AD, we employed a lentiviral-mediated strategy to overexpress TREM2 in the brain of APPswe/PS1dE9 (APP/PS1) transgenic mice and cultured BV2 cells. Our results showed that TREM2 overexpression rescued cognitive deficits, decreased ß-amyloid (Aß) plaques deposition, reduced synaptic and neuronal loss, as well as ameliorated neuroinflammation. The mechanistic study revealed that these protective effects were likely attributed to inhibition of neuroinflammatory responses through the JAK/STAT/SOCS signaling pathway and subsequent attenuation of pro-inflammatory cytokines. Furthermore, suppression of neuroinflammation might be ascribed to activation of the M2 microglia, as the levels of M2 phenotype markers Arg-1, IL-10 and Ym1 were markedly increased. Similarly, overexpression of TREM2 in BV2 cells also promoted M2 polarization and led to the alleviation of M1 microglial inflammatory responses through JAK/STAT/SOCS signaling pathway, suggesting that TREM2 is an important factor in shifting the microglia from M1 to M2 phenotype. Taken together, our results further provide insights into the role of TREM2 in AD pathogenesis and highlight TREM2 as a potential target against AD.

Tanshinone IIA suppresses lipopolysaccharide-induced neuroinflammatory responses through NF-κB/MAPKs signaling pathways in human U87 astrocytoma cells.

Tanshinone IIA (tan IIA), a key component of Salvia miltiorrhiza Bunge (Danshen), has been proven to play a significant role in suppressing inflammation. However, the molecular mechanisms underlying the anti-inflammatory properties of tan IIA against lipopolysaccharide (LPS)-induced neuroinflammation and neurotoxicity in human U87 astrocytoma cells have not been well justified. Therefore, in this study, U87 cells were pretreated with tan IIA (1, 5 and 10 µM) for 30 min, followed by stimulation with LPS for 24 h. Immunofluorescence, reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and western blotting were performed to investigate the effects of tan IIA on neuroinflammatory responses. The findings demonstrated that tan IIA prevented LPS-induced cell viability decrease, inhibited U87 cells activation, and suppressed the expression of glial fibrillary acidic protein (GFAP). Furthermore, tan IIA significantly reduced the mRNA expression of interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in LPS-stimulated U87 cells. Meanwhile, the increased protein levels of IL-1ß, TNF-α, and IL-6 in cell culture supernatants were also markedly inhibited by tan IIA. Moreover, tan IIA significantly alleviated the phosphorylation of IκBα, nuclear factor-kappa B (NF-κB), p38, and JNK induced by LPS. Additionally, tan IIA suppressed the upstream signaling adaptor molecules toll-like receptor 4 (TLR4), myeloid differentiation primary response protein 88 (MyD88), and tumor necrosis factor receptor-associated factor 6 (TRAF6). Blockade of NF-κB, p38, and JNK obviously attenuated IL-1ß, TNF-α, and IL-6 in U87 cells. In conclusion, the present results suggested that tan IIA can attenuate LPS-induced neurotoxicity and neuroinflammation partly by inhibiting TLR4/NF-κB/MAPKs signaling pathways in U87 cells.

Tanshinone IIA ameliorates cognitive deficits by inhibiting endoplasmic reticulum stress-induced apoptosis in APP/PS1 transgenic mice.

Our previous data indicated that tanshinone IIA (tan IIA) improves learning and memory in a mouse model of Alzheimer's disease (AD) induced by streptozotocin via restoring cholinergic function, attenuating oxidative stress and blocking p38 MAPK signal pathway activation. This study aims to estimate whether tan IIA inhibits endoplasmic reticulum (ER) stress-induced apoptosis to prevent cognitive decline in APP/PS1 transgenic mice. Tan IIA (10 mg/kg and 30 mg/kg) was intraperitoneally administered to the six-month-old APP/PS1 mice for 30 consecutive days. ß-amyloid (Aß) plaques were measured by immunohistochemisty and Thioflavin S staining, apoptotic cells were observed by TUNEL, ER stress markers and apoptosis signaling proteins were investigated by western blotting and RT-PCR. Our results showed that tan IIA significantly ameliorates cognitive deficits and improves spatial learning ability of APP/PS1 mice in the nest-building test, novel object recognition test and Morris water maze test. Furthermore, tan IIA significantly reduced the deposition of Aß plaques and neuronal apoptosis, and markedly prevented abnormal expression of glucose regulated protein 78 (GRP78), initiation factor 2α (eIF2α), inositol-requiring enzyme 1α (IRE1α), activating transcription factor 6 (ATF6), as well as suppressed the activation of C/EBP homologous protein (CHOP) and c-Jun N-terminal kinase (JNK) pathways in the parietal cortex and hippocampus. Moreover, tan IIA induced an up-regulation of the Bcl-2/Bax ratio and down-regulation of caspase-3 protein activity. Taken together, the above findings indicated that tan IIA improves learning and memory through attenuating Aß plaques deposition and inhibiting ER stress-induced apoptosis. These results suggested that tan IIA might become a promising therapeutic candidate drug against AD.

Silencing of LRP1 Exacerbates Inflammatory Response Via TLR4/NF-κB/MAPKs Signaling Pathways in APP/PS1 Transgenic Mice.

Activation of glial cells (including microglia and astrocytes) appears central to the initiation and progression of neuroinflammation in Alzheimer's disease (AD). The low-density lipoprotein receptor-related protein 1 (LRP1) is a major receptor for amyloid-ß (Aß), which plays a critical role in AD pathogenesis. LRP1 regulates inflammatory response by modulating the release of pro-inflammatory cytokines and phagocytosis. However, the effects of LRP1 on microglia- and astrocytic cell-mediated neuroinflammation and their underlying mechanisms in AD remain unclear. Therefore, using APP/PS1 transgenic mice, we found that LRP1 is downregulated during disease progression. Silencing of brain LRP1 markedly exacerbated AD-related neuropathology including Aß deposition, neuroinflammation, and synaptic and neuronal loss, which was accompanied by a decline in spatial cognitive ability. Further mechanistic study revealed that silencing of LRP1 initiated neuroinflammation by increasing microgliosis and astrogliosis, enhancing pro-inflammatory cytokine production, and regulating toll-like receptor 4 (TLR4)-mediated activation of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways. Taken together, these findings indicated that LRP1 suppresses microglia and astrocytic cell activation by modulating TLR4/NF-κB/MAPK signaling pathways. Our results further provide insights into the role of LRP1 in AD pathogenesis and highlight LRP1 as a potential therapeutic target for the treatment of AD.