The Role Of NLRP3 Inflammasome Mediated Microglia Inflammation In Manganese-induced Neurotoxicity

BackgroundManganese(Mn) is an essential trace element that is distributed in the earth crust. It not only plays a key role in numerous biochemical reactions, including immune response, ATP generation, bone growth, digestion and reproduction, but also serves as a cofactor for various enzymes. However, due to the susceptibility of the brain(especially for the striatum and substantia nigra) to an excess Mn, accumulation of Mn from either environment or occupational sources leads to a neuron-degenerative disorder referred to as manganism. Patients with manganism present with cognitive and motor dysfunction, which are similar to PD. In the past two decade, there has been a growing interest in understanding the neurotoxic effect of Mn and their influence on various neurodegenerative diseases, including manganism, Wilson’s disease(WD), Parkinson’s disease(PD), and Alzheimer’s disease(AD).In the central nerve system(CNS), microglia are the chief innate immune cells which are mainly located in in the meninges, choroid plexus and perivascular space. These cells constantly survey the proximal environment through the pattern-recognition receptors(PRRs) that they express(for example, Toll-like receptors and NOD-like receptors(NLRs)). When these cells sense tissue injury or a foreign(infectious) agent, a network of activation pathways is induced in microglia, resulting in an altered microglial morphology, intense respiratory metabolism and the expression and release of immune molecules. This response promotes the recruitment of peripheral innate immune cells(macrophages and neutrophils) and adaptive immune cells(T cells and B cells) to the site of CNS injury as well as further activation of nearby glial cells. However, overactivated microglia can induce significant and highly detrimental neurotoxic effects by the excess production of a large number of pro-inflammatory cytokines, such as IL-1β, IL-18 and TNF-α.Inflammasomes are molecular platforms that trigger the maturation of proinflammatory cytokines. Recently, increasing studies have focused on the inflammasomes associated with human inflammation diseases. Belonging to the NLRs inflammasome family, NLRP3 inflammasome play an important role in regulating the release of proinflammatory cytokines, including IL-1β, IL-18. In the process of neuroinflammation, the NLRP3 inflammasome activation in microglia and macrophages within CNS can be induced by misfolded protein, such as Aβ, α-synuclein and prion protein(Pr P). The active NLRP3 can oligomerize and recruit the adaptor protein ASC(apoptosis-associated speck-like protein containing a caspase recruitment domain [CARD]) by using pyrin-domain interaction. Subsequently, caspase-1, which is synthesized as a 45 k Da pro-caspase-1(p45), is recruited by ASC through CARD-CARD interactions. Upon cleavage pro-caspase-1 generates two proteins of 10 and 20 k Da(p10 and p20) that form a heterotetrameric complex with enzymatic activity, thus processing and releasing pro-inflammatory cytokines IL-1β.It has been reported that multiple factors can lead to NLRP3 inflammasome activation, mainly including extracellular ATP, reactive oxygen species(ROS), as well as the release of cathepsin B from lysosomal damage. Autophagy is a highly conserved homeostatic process by which cytoplasmic macromolecules, excess or damaged organelles, and some pathogens are delivered to lysosomes for degradation. Autophagy consists of several steps, including initiation, elongation, fusion of autophagosome and lysosome, and degradation. Disruption each step of them can contribute to the dysfunctional autophagy. It has been reported that autophagy dysfunction plays a vital role in a range of neurodegenerative diseases, including Parkinson’s disease(PD), Huntington’s disease(HD) and Alzheimer’s disease(AD). Recent reports reveal a complex interplay between NLRP3 inflammasome and autophagic pathways. Activation of NLRP3 inflammasome promotes the autophagy pathway. However, over-reaction of autophagy pathway prohibits the NLRP3 inflammasome activation. Further research is required to clarify the complex reciprocal regulation of inflammasome and autophagic pathway in neurodegenerative diseases. Aims1. Investigation on the role of Mn-induced microglial activation, thus releasing pro-inflammatory cytokines in the Mn mediated hippocampal-dependent impairment of learning and memory.2. Investigation on the mechanism of Mn-induced microglial activation involved in neuroinflammation via NLRP3 inflammasome.3. Investigation on the relationship between autophagy dysfunction and NLRP3 inflammasome activation in Mn-induced neurodegenerative diseases.4. Investigation on the molecular mechanism of autophagy dysfunction in regulating the activation of NLRP3 inflammasome. Methods1. A sub-acute Mn exposure in murine model was established by subcutaneously injected with manganese(Ⅱ) chloride tetrahydrate(Mn Cl2 4H2O) based on a published protocol. Mn levels in blood and brain tissue in various groups of mice were tested by atomic fluorescence spectrophotometer. Contextual fear conditioning and patch clamp were used to evaluate the learning and memory ability of mice.2. Immunofluorescence staining was carried out to investigate whether Mn induced the activation of NLRP3 inflammasome in hippocampal microglia. The protein expression of NLRP3, caspase-1 and IL-1β was analysed in response to Mn, LPS and Na Cl by western blotting.3. The protein expression of IL-1β, IL-18 and TNF-α was analysed by ELISA. The gene expression of NLRP3, IL-1β and IL-18 was analysed by q PCR.4. Immunofluorescence staining was carried out to evaluate the protein expression of LC3. The protein expression of Beclin, Atg5, LC3, p62 and cathepsin B was analysed by western blotting. Morphological change of BV2 associated with autophagy were analyzed by Transmission electron microscopy(TEM).5. NLRP3 and Atg5 in BV2 were knocked-down by si RNA and two types of pharmacological inhibitors(including NH4 Cl and Baf A1) were used, to evaluated the protein expressions of NLRP3 inflammasome as well as the pro-inflammatory cytokine(including IL-1β and IL-18) in response to Mn by western blot and ELISA, respectively. Results1. Compared with Na Cl-injected group, the concentration of Mn in blood and brain tissue was significantly increased after 7 days of Mn injection. The accumulation of Mn in the hippocampal region significantly reduced contextual fear conditioning, indicating that Mn selectively influences the learning and memory ability of mice.2. The protein analysis by western blotting and immunofluorescence staining demonstrated that the microglia can be activated by Mn both in vivo and in vitro, thus leading to the up-regulation of NLRP3 inflammasome.3. The release of pro-inflammatory cytokine(including IL-1β and IL-18) was increased both in gene and protein level in response to Mn demonstrated by q PCR and ELISA, respectively.4. After 6h Mn treatment, BV2 were positively stained for LC3 in the cytoplasm compared with the untreated cells in vitro study. And the protein expression of autophagy related genes(including Beclin 1, Atg5 and LC3II) was significantly increased. Additionally, the up-regulation of p62 and cathepsin B, as well as the increased number of dysfunctional swollen lysosomes containing high dense particulate materials and mitochondrial vacuolus as evident by TEM, indicated that Mn could induce lysosomal dysfunction in BV2.5. Blocking autophagy pathway by Atg5 si RNA and Baf A1 could not prohibit the activation of NLRP3 inflammasome pathway in BV2 in response to Mn induction. However, the release of cathepsin B could be down-regulated by NH4 Cl in BV2, thus further prohibiting the activation of NLRP3 inflammasome, as well as the release of pro-inflammatory cytokine, including IL-1β and IL-18. Conclusion1. Excessive accumulation of Mn in the hippocampus of mice leads to impairment of learning and memory ability.2. The microglia activated by Mn leads to the activation of NLRP3 inflammasome pathway and subsequently the release of pro-inflammatory cytokine, including IL-1β and IL-18, which may be related to impairment of learning and memory ability in hippocampal region.3. Mn-induced the activation NLRP3 inflammation pathway in microglia plays an important role in triggering the release of pro-inflammatory cytokine, including IL-1β and IL-18.4. Autophagy-lysosomal dysfunction is involved in the Mn-induced NLRP3 inflammation activation. However, disruption the steps of initiation, elongation, fusion of autophagosome and lysosome exerts little effect on the activation of NLRP3 inflammation.5. The release of lysosomal cathpsin B from Mn-induced dysfunctional lysosomal plays an important role in Mn-induced NLRP3 inflammasome activation.