Thymosin ß4 reverses phenotypic polarization of glial cells and cognitive impairment via negative regulation of NF-κB signaling axis in APP/PS1 mice.

BACKGROUND: Thymosin ß4 (Tß4) is the most abundant member of the ß-thymosins and plays an important role in the control of actin polymerization in eukaryotic cells. While its effects in multiple organs and diseases are being widely investigated, the safety profile has been established in animals and humans, currently, little is known about its influence on Alzheimer's disease (AD) and the possible mechanisms. Thus, we aimed to evaluate the effects and mechanisms of Tß4 on glial polarization and cognitive performance in APP/PS1 transgenic mice. METHODS: Behavior tests were conducted to assess the learning and memory, anxiety and depression in APP/PS1 mice. Thioflavin S staining, Nissl staining, immunohistochemistry/immunofluorescence, ELISA, qRT-PCR, and immunoblotting were performed to explore Aß accumulation, phenotypic polarization of glial cells, neuronal loss and function, and TLR4/NF-κB axis in APP/PS1 mice. RESULTS: We demonstrated that Tß4 protein level elevated in all APP/PS1 mice. Over-expression of Tß4 alone alleviated AD-like phenotypes of APP/PS1 mice, showed less brain Aß accumulation and more Insulin-degrading enzyme (IDE), reversed phenotypic polarization of microglia and astrocyte to a healthy state, improved neuronal function and cognitive behavior performance, and accidentally displayed antidepressant-like effect. Besides, Tß4 could downregulate both TLR4/MyD88/NF-κB p65 and p52-dependent inflammatory pathways in the APP/PS1 mice. While combination drug of TLR4 antagonist TAK242 or NF-κB p65 inhibitor PDTC exerted no further effects. CONCLUSIONS: These results suggest that Tß4 may exert its function by regulating both classical and non-canonical NF-κB signaling and is restoring its function as a potential therapeutic target against AD.

PNU282987 alleviates Aß-induced anxiety and depressive-like behaviors through upregulation of α7nAChR by ERK-serotonin receptors pathway.

Patients with Alzheimer's disease often undergo anxiety and depression. Our previous studies have shown that α7nAChR protects against Aß-induced neurotoxicity via downregulation of p38 and JNK MAPKs, but the role of α7nAChR on Aß-induced anxiety and depressive-like behaviors and the effect of α7nAChR on the regulation of MAPKs pathways remain unknown. To examine the effects of α7nAChR and MAPKs pathways on Aß-induced anxiety and depression-like behaviors and to explore their relationships between them, elevated plus maze, open field and forced swim tests were performed. Protein levels of 5-HT1A receptor, 5-HT2C receptor, α7nAChR, t-ERK1/2 and p-ERK1/2 in the amygdala were analyzed by western blotting and immunostaining. Our study found out that Aß oligomers induced anxiety and depression-like behaviors in C56BL/6 mice with open field, elevated plus maze and forced swim tests. However, activation of α7nAChR or inhibition of ERK pathways showed significant antidepressant and anxiolytic-like effects on Aß-injected mice. Moreover, Aß significantly decreased the level of 5-HT1A receptor but increased the level of 5-HT2C receptor in the basolateral amygdala. Treatment with α7nAChR agonist PNU282987 or ERK inhibitor U0126 reversed Aß-induced 5-HT1A and 5-HT2C receptor changes. Moreover, activation of α7nAChR inhibited ERK pathway in the amygdala of Aß1-42-injected mice. Our study provides a new insight into the mechanism of α7nAChR in Aß-induced depression and anxiety-related symptoms through the regulation of ERK1/2 pathway and the potential association with serotonin receptors. Together, our data suggests that α7nAChR is protective against Aß-induced anxiety and depression-like behaviors in mice.

5-HT1AR alleviates Aß-induced cognitive decline and neuroinflammation through crosstalk with NF-κB pathway in mice.

The 5-hydroxytryptamine (5-HT) receptor is significant for the regulation of mood and memory. However, the role of 5-HT1AR in ß-Amyloid protein (Aß)-induced cognitive decline, neuroinflammation and the possible mechanism remains elusive. Thus, we aimed to evaluate the effects of 5-HT1AR on Aß-induced learning and memory decline and neuroinflammation in mice. Novel object recognition and Morris water maze tests were performed to observe learning and memory behavior in mice. Protein levels of Iba1, GFAP, MAP2, TNF-α, Tß4, C-fos, IKK-ß, IKB-α, NF-κBp65, phospho-NF-κBp65 in the hippocampus were examined by immunostaining or western blotting. Aß1-42-treatment inducing learning and memory decline was shown in novel object recognition and Morris water maze tests; neuroinflammation shown in immunostaining. Our study found out that 5-HT1AR inhibitor WAY100635 showed significant improvement in Aß-induced learning and memory decline. Moreover, WAY100635 decreases levels of Iba1, GFAP, and TNF-α in the hippocampus, which were related to neuroinflammation. While treatment with 5-HT1AR agonist 8-OH-DPAT or ERK inhibitor U0126 exerted no effects or even aggravated Aß-induced learning and memory decline. In addition, WAY100635 could downregulate phospho-NF-κB in the hippocampus of Aß1-42-injected mice. These results provide new insight into the mechanism, for 5-HT1AR in Aß-induced cognitive impairments through crosstalk with the NF-κB signaling pathway. Our data indicated that WAY100635 was involved in the protective effects against neuroinflammation and improvement of learning and memory in Alzheimer's disease.

Modulation of the MAPKs pathways affects Aß-induced cognitive deficits in Alzheimer's disease via activation of α7nAChR.

Cognitive impairment in Alzheimer's disease (AD) is characterized by being deficient at learning and memory. Aß1-42 oligomers have been shown to impair rodent cognitive function. We previously demonstrated that activation of α7nAChR, inhibition of p38 or JNK could alleviate Aß-induced memory deficits in Y maze test. In this study, we investigated whether the effects of α7nAChR and MAPKs on Y maze test is reproducible with a hippocampus-dependent spatial memory test such as Morris water maze. We also assessed the possible co-existence of hippocampus-independent recognition memory dysfunction using a novel object recognition test and an alternative and stress free hippocampus-dependent recognition memory test such as the novel place recognition. Besides, previous research from our lab has shown that MAPKs pathways regulate Aß internalization through mediating α7nAChR. In our study, whether MAPKs pathways exert their functions in cognition by modulating α7nAChR through regulating glutamate receptors and synaptic protein, remain little known. Our results showed that activation of α7nAChR restored spatial memory, novel place recognition memory, and short-term and long-term memory in novel object recognition. Inhibition of p38 restored spatial memory and short-term and long-term memory in novel object recognition. Inhibition of ERK restored short-term memory in novel object recognition and novel place recognition memory. Inhibition of JNK restored spatial memory, short-term memory in novel object recognition and novel place recognition memory. Beside this, the activation of α7nAChR, inhibition of p38 or JNK restored Aß-induced levels of NMDAR1, NMDAR2A, NMDAR2B, GluR1, GluR2 and PSD95 in Aß-injected mice without influencing synapsin 1. In addition, these treatments also recovered the expression of acetylcholinesterase (AChE). Finally, we found that the inhibition of p38 or JNK resulted in the upregulation of α7nAChR mRNA levels in the hippocampus. Our results indicated that inhibition of p38 or JNK MAPKs could alleviate Aß-induced spatial memory deficits through regulating activation of α7nAChR via recovering memory-related proteins. Moreover, p38, ERK and JNK MAPKs exert different functions in spatial and recognition memory.

Activation of α7 nicotinic acetylcholine receptor alleviates Aß1-42-induced neurotoxicity via downregulation of p38 and JNK MAPK signaling pathways.

Amyloid ß peptide 1-42 (Aß1-42) could induce cognitive deficits through oxidative stress, inflammation, and neuron death in Alzheimer's disease (AD). MAPK pathways have been thought to mediate Aß1-42-induced neuroinflammation responses, neuron death and cognitive decline in AD. The α7 nicotinic acetylcholine receptor (α7nAChR) exerts a neuroprotective effect. However, whether α7nAChR alleviates Aß1-42-induced neurotoxicity through MAPKs (p38, ERK, JNK) in vivo remains unclear. In our study, memory was assessed in C57BL/6 mice using a Y-maze test. Cell death was assessed by Nissl and Hoechst staining and Bax, Bcl-2, Caspase 3, and Cytochrome C levels using Western blotting. Oxidative stress was assayed by superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) levels. Inflammation was examined with GFAP and Iba1 using immunohistochemistry. The Aß degrading enzymes insulin degrading enzyme (IDE) and neprilysin (NEP) were tested using Western blotting. We found that activating α7nAChR or inhibiting p38 or JNK pathway alleviated Aß1-42-induced cognitive deficits and neuron loss and death by reducing oxidative stress. In addition, activating α7nAChR or inhibiting p38 or JNK pathway also reduced inflammation, which was observed as reduced GFAP and Iba1 levels with different effects on Aß degrading enzymes. Finally, we found that the activation of α7nAChR led to the downregulation of pp38 and pJNK levels. Conversely, the inhibition of p38 or JNK resulted in the upregulation of α7nAChR levels in the hippocampus and cortex. Our data indicate that the activation of α7nAChR alleviates Aß1-42-induced neurotoxicity, and this protective effect might act through the downregulation of p38 and JNK MAPKs.

The p38 mitogen activated protein kinase regulates ß-amyloid protein internalization through the α7 nicotinic acetylcholine receptor in mouse brain.

Alzheimer's disease (AD) is one of the most devastating neurodegenerative disorders. Intracellular ß-amyloid protein (Aß) is an early event in AD. It induces the formation of amyloid plaques and neuron damage. The α7 nicotinic acetylcholine receptor (α7nAChR) has been suggested to play an important role in Aß caused cognition. It has high affinity with Aß and could mediate Aß internalization in vitro. However, whether in mouse brain the p38 MAPK signaling pathway is involved in the regulation of the α7nAChR mediated Aß internalization and their role in mitochondria remains little known. Therefore, in this study, we revealed that Aß is internalized by cholinergic and GABAergic neurons. The internalized Aß were found deposits in lysosomes/endosomes and mitochondria. Aß could form Aß-α7nAChR complex with α7nAChR, activates the p38 mitogen activated protein kinase (MAPK). And the increasing of α7nAChR could in return mediate Aß internalization in the cortex and hippocampus. In addition, by using the α7nAChR agonist PNU282987, the p38 phosphorylation level decreases, rescues the biochemical changes which are tightly associated with Aß-induced apoptosis, such as Bcl2/Bax level, cytochrome c (Cyt c) release. Collectively, the p38 MAPK signaling pathway could regulate the α7nAChR-mediated internalization of Aß. The activation of α7nAChR or the inhibition of p38 MAPK signaling pathway may be a beneficial therapy to AD.

The p38 mitogen-activated protein kinase signaling pathway is involved in regulating low-density lipoprotein receptor-related protein 1-mediated ß-amyloid protein internalization in mouse brain.

Alzheimer's disease (AD) is one of the most common neurodegenerative diseases. Recently, increasing evidence suggests that intracellular ß-amyloid protein (Aß) alone plays a pivotal role in the progression of AD. Therefore, understanding the signaling pathway and proteins that control Aß internalization may provide new insight for regulating Aß levels. In the present study, the regulation of Aß internalization by p38 mitogen-activated protein kinases (MAPK) through low-density lipoprotein receptor-related protein 1 (LRP1) was analyzed in vivo. The data derived from this investigation revealed that Aß1-42 were internalized by neurons and astrocytes in mouse brain, and were largely deposited in mitochondria and lysosomes, with some also being found in the endoplasmic reticulum. Aß1-42-LRP1 complex was formed during Aß1-42 internalization, and the p38 MAPK signaling pathway was activated by Aß1-42 via LRP1. Aß1-42 and LRP1 were co- localized in the cells of parietal cortex and hippocampus. Furthermore, the level of LRP1-mRNA and LRP1 protein involved in Aß1-42 internalization in mouse brain. The results of this investigation demonstrated that Aß1-42 induced an LRP1-dependent pathway that related to the activation of p38 MAPK resulting in internalization of Aß1-42. These results provide evidence supporting a key role for the p38 MAPK signaling pathway which is involved in the regulation of Aß1-42 internalization in the parietal cortex and hippocampus of mouse through LRP1 in vivo.