In the current study, to explore the effects and the molecular mechanism of TREM2 on Aβ pathology, neuroinflammatory responses, neurodegenerative changes, and behavioral deficits, we used a lentiviral-mediated strategy to downregulation of TREM2 expression in the brain of APP/PS1 mice and BV2 cells. Our results demonstrated that TREM2 was uniquely expressed by microglia, and the expression of TREM2 was significantly increased with aging in the cortex and hippocampus of APP/PS1 mice. Furthermore, downregulation of TREM2 expression exacerbated neuropathologies including Aβ deposition, microgliosis, astrogliosis, as well as neuronal and synaptic loss, which was accompanied by a decline in cognitive function in APP/PS1 mice. Moreover, the mechanistic study revealed that downregulation of TREM2 expression increases pro-inflammatory cytokines production via TLR4-mediated MAPK signaling pathway in the brain of APP/PS1 mice and BV2 cells. These data coupled with the previous findings strongly suggested that TREM2 is involved in AD pathogenesis, and it may represent a potential therapeutic target against AD [20, 24, 29].
Neuroinflammation is a critical pathological feature and considered a major contributor to AD pathogenesis [1]. Activated microglia and astrocytes are both involved in the initialization and progression of neuroinflammation in AD. There is increasing evidence to suggest that neuroinflammatory responses were closely associated with the several AD-related pathological processes, including Aβ accumulation, neuronal and synaptic loss, tau pathology, as well as cognitive deficits [39–41]. In the present study, after downregulation of TREM2 expression, the number of Iba1-positive microglia and GFAP-positive astrocytes were markedly increased, and they clustered around Aβ plaques. Furthermore, the protein levels of Iba1 and GFAP were effectively elevated in the cortex and hippocampus of APP/PS1 mice. The current study indicated that TREM2 downregulation leads to more obvious microgliosis and astrogliosis in the brain of APP/PS1 mice, which are closely associated with neuroinflammation. However, the molecular mechanisms by which TREM2 downregulation aggravated neuroinflammatory responses remains unclear. TLR4, a pattern recognition receptor for Aβ, is highly expressed on microglia and astrocytes surfaces. TLR4 ligation by Aβ can activate multiple downstream signaling pathways, including MAPK. In line with this notion, the expression of TLR4/MAPK signaling pathway molecules was determined. The present study revealed that TLR4, MyD88, and its adapter protein TRAF6 were markedly upregulated in the brain of APP/PS1 mice after downregulation of TREM2 expression. Furthermore, the phosphorylation of p38, JNK, and ERK1/2 was significantly increased in the cortex and hippocampus of APP/PS1 mice. In Aβ1−42-induced BV2 cells, we found that TREM2 downregulation significantly elevated the levels of TLR4, MyD88, and TRAF6. Meanwhile, a significant increase of phospho-p38, JNK, and ERK1/2 were also observed after knockdown of TREM2 expression. Taken together with previous findings, our results suggested that TREM2 downregulation aggravated neuroinflammatory responses through TLR4/MAPK signaling pathway in AD [46].
Mounting evidence suggests that Aβ induces TLR4/MAPK signaling pathway activation, which subsequently stimulates the activated microglia and astrocytes to produce and release pro-inflammatory cytokines and other inflammatory mediators.
TNF-α can stimulate β- and γ-secretase enzyme activity, which results in increased synthesis of Aβ peptides and a further increase in TNF-α release [10, 47]. This auto-amplified loop in the AD brain can contribute to the maintenance of excessive levels of TNF-α, which then stimulate Aβ synthesis and neuronal loss, also suppressing microglia phagocytosis of Aβ [47, 48]. Besides, TNF-α could contribute to promoting insulin resistance and finally lead to cognitive decline in AD [49, 50]. IL-1β activates p38 pathway, which could lead to tau hyperphosphorylation and further exacerbate synaptic and neuronal cell dysfunction [9, 51]. Similarly to TNF-α, IL-1β can promote Aβ production by modulating γ-secretase enzyme activity in neurons [10]. IL-6 stimulates and promotes the recruitment of microglia and astrocytes to release pro-inflammatory cytokines, and it also promotes β-secretase enzyme activity and tau phosphorylation in AD [11, 52, 53]. Our results showed that pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 were increased in the cortex and hippocampus of APP/PS1 mice after downregulation of TREM2 expression. Furthermore, knockdown of TREM2 expression also elevated the production of TNF-α, IL-1β, and IL-6 in BV2 cells, similar to the results of in vivo studies. The current findings were coincident with previous reports, which identified that downregulation of TREM2 expression may contribute to the development of neuroinflammation in AD [27, 28]. Afterward, we attempted to elucidate whether knockdown of TREM2 expression could regulate Aβ1−42-induced neuroinflammatory responses via the MAPK signaling pathway. We treated TREM2-knocked down BV2 cells with the p38 inhibitor SB202190, JNK inhibitor SP600125, and ERK1/2 inhibitor U0126. The current results showed that the higher levels of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 seen in TREM2 knockdown BV2 cells were significantly suppressed by SB202190, SP600125, and U0126. Collectively, these findings demonstrated that TREM2 regulates pro-inflammatory cytokines production by the MAPK signaling pathway.
Previous studies reported that neuroinflammation is related to the aggregation of Aβ in the brain of AD [54, 55]. Abnormal Aβ deposition can stimulate nuclear factor-κB (NF-κB) and MAPK signaling pathways, which are associated with the transcription of pro-inflammatory cytokines and mediators. In turn, pro-inflammatory cytokines and mediators cause the initiation of APP cleavage through the MAPK and NF-κB signaling pathways [10, 56]. Therefore, Aβ can be viewed both as a cause and consequence of neuroinflammation in AD. TREM2 facilitates microglia clustering around Aβ plaques and promotes Aβ phagocytosis and degradation. However, TREM2 deletion decreased the microglial phagocytic clearance of Aβ, resulting in exacerbated brain Aβ deposition and cognitive deficits in the AD animal model [22, 57]. In this study, after downregulation of TREM2 expression, more Aβ plaques in the cortex and hippocampus of APP/PS1 mice were observed. Likewise, the protein levels of both soluble/insoluble Aβ1−40 and Aβ1−42 were higher in the brain of APP/PS1 mice after TREM2 downregulation. Thus, combined with previous reports, downregulation of TREM2 expression may lead to Aβ aggregation and accumulation in the brain, and further exacerbating spatial cognitive impairment [27, 28, 58].
Both activated microglia and astrocytes may gradually release pro-inflammatory cytokines TNF-α, IL-1β, and IL-6. These cytokines are destructive to neurons by altering synaptic proteins and finally lead to cognitive deficits [59, 60]. The synapse-associated proteins, especially pre-synaptic Syn and post-synaptic PSD-95, play important roles in synaptic plasticity and memory formation [61, 62]. It has been shown that deficits in Syn and PSD-95 correlate with cognitive impairment in AD and aging [63, 64]. Previous studies reported that there is a direct interaction between neuroinflammation and Syn as well as PSD-95 in newborn neurons [65, 66]. In this study, TREM2 downregulation remarkably increased neuronal loss in the cortex and hippocampal CA1 region of APP/PS1 mice. Additionally, the levels of Syn and PSD-95 markedly decreased in the cortex and hippocampus of APP/PS1 mice after downregulation of TREM2 expression. These data showed that TREM2 downregulation led to the exacerbation of neurodegenerative changes, as the more severe neuronal and synaptic loss was observed. Cognitive dysfunction is associated with neuronal and synaptic loss in the cortex and hippocampus in AD [27, 67, 68]. Thus, we assessed the learning and memory capacity of APP/PS1 mice using established behavioral tests. In the NOR test, APP/PS1 mice exhibited a significantly lower preference for exploring the novel object in the testing phase following TREM2 downregulation, indicating the recognition memory impairment. Furthermore, short-term working memory impairment was evaluated by the Y-maze test. APP/PS1 mice showed a markedly reduced percentage of spontaneous alteration behavior after downregulation of TREM2 expression. At last, in the hidden platform test, the escape latency was increased in APP/PS1 mice that received TREM2 lentivirus. When removing the hidden platform, APP/PS1 mice had a significant decrease in the number of target crossing and the time spent in the target quadrant, suggesting an obvious spatial memory disorder. Our study coupled with the work of others strongly revealed that exacerbation of neuronal and synaptic loss may contribute to worsening cognitive deficits after downregulation of TREM2 expression [22, 27, 57].
In conclusion, this study revealed that downregulation of TREM2 expression in the brain of APP/PS1 mice markedly exacerbated AD-related neuropathology including Aβ deposition, gliosis, neuroinflammation, as well as neuronal and synaptic loss, which was accompanied by a decline in cognitive function in APP/PS1 mice. Furthermore, in vivo and in vitro evidence demonstrated that TREM2 downregulation increases the production of pro-inflammatory cytokines through TLR4-mediated MAPK signaling pathway activation. Taken together, our results indicated that TREM2 could be a promising treatment to improve cognitive deficits and neuroinflammation in AD and other central nervous system diseases.