Original researchNlrc3-like is required for microglia maintenance in zebrafish
Introduction
Microglia are tissue-resident macrophages that reside in the central nervous system (CNS) (Barron, 1995; Hanisch and Kettenmann, 2007; Davoust et al., 2008). Recent studies have suggested that in addition to functioning as scavengers to remove cellular debris and infectious agents in the CNS, microglia are involved in many other biological processes including neurotrophic factor synthesis, synaptic pruning, and neuronal activity modulation (Trang et al., 2011; Li et al., 2012b; Nayak et al., 2014). Despite the extensive studies in the past decades, the molecular basis governing the development of microglia and their homeostasis remain largely undefined.
The NOD-like receptors (nucleotide-binding oligomerization domain like receptors, NLRs) are a class of intracellular receptors that recognize pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs) (Kawai and Akira, 2009). Most of these receptors contain three function domains: an N-terminal effector domain mediating the activation of downstream signaling; a central NOD or NACHT domain (neuronal apoptosis inhibitor protein (NAIP), major histocompatibility complex class II transactivator (CIITA), incompatibility locus protein from Podospora anserina (HET-E), and telomerase-associated protein (TP1) domain) necessary for self-oligomerization; and a C-terminal leucine rich repeat (LRR) domain responsible for sensing upstream stimuli (Chen et al., 2009). Based on the type of N-terminal effector domain, NLR family is divided into 4 subfamilies, namely NLRA (A for acidic activation domain), NLRB (B for BIR – baculovirus inhibitory repeat), NLRC (C for CARD – caspase-activating and recruitment domain) and NLRP (P for PYD – pyrin domain) (Chen et al., 2009). Upon recognition of DAMPs and PAMPs, the NLR proteins oligomerize and recruit apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and caspase-1 to form cytoplasmic multi-protein complexes known as inflammasomes, which leads to the activation of caspase-1, release of proinflammatory cytokines and pyroptotic cell death (Miao et al., 2011; Latz et al., 2013; Lamkanfi and Dixit, 2014). Unlike other NLR proteins, NLRP10 contains a PYD and a NACHT domain but lacks the C-terminal LRR domain (Wang et al., 2004; Damm et al., 2013), suggesting it has a distinctive role in the inflammasome activation. Indeed, both in vitro and in vivo ectopic overexpression assay has indicated that NLRP10 likely functions as a negative regulator during inflammasome activation through sequestering ASC in the cytoplasm (Wang et al., 2004; Imamura et al., 2010). Consistent with this notion, a recent study in zebrafish has shown that the inactivation of NOD-like receptor Nlrc3-like, which, like NLRP10, consists of a PYD and a NACHT but lacks the LRR domain, causes a systemic inflammatory activation of peripheral macrophages, thereby preventing peripheral macrophages from migrating into the brain (Shiau et al., 2013). However, whether Nlrc3-like is required for the maintenance of microglia is unclear.
In this study, we identified and characterized a temperature sensitive mutant zebrafish puer, which harbors a loss-of-function mutation in the NOD-like receptor nlrc3-like. When raised under the permissive temperature, puer mutants contained normal number of microglia but the number of microglia was drastically reduced 24 h after switched to the restricted temperature. Time-lapse imaging revealed that under the restricted temperature, nlrc3-like deficient microglia exhibited excessive cell death with characteristics similar to that of pyroptosis, including cell swelling, plasma-membrane rupture and pro-inflammatory cytokine production. The microglia cell death in nlrc3-like deficient mutants could be partially rescued by the suppression of Asc function, a key regulator of inflammasome cascade.
Section snippets
Microglia defect in puer mutants is temperature sensitive
To uncover new regulators in microglia development, we conducted an N-ethyl-N-nitrosourea (ENU) based forward genetic screening in zebrafish. By using Neutral Red (NR) staining of microglia (Herbomel et al., 2001), we identified a mutant line puer (puer represents a kind of Chinese tea) (nlrc3hkz6 allele), in which the NR staining signal in the brains of the homozygous embryos was reduced at 3 days post-fertilization (dpf). We noticed that the degree of the reduction of NR signal varied among
Discussion
Here, we report the identification and characterization of a zebrafish mutant puer, which harbors a point mutation (I to N change at the position of amino acid 495) in the Nlrc3-like, resulting in microglia cell death in a temperature sensitive manner. We further show that loss of Nlrc3-like function results in aberrant activation of inflammasome pathway in an Asc-dependent manner. Interestingly, similar to human NLRP10 (Wang et al., 2004; Damm et al., 2013; Shiau et al., 2013), zebrafish
Fish lines
AB, WIK, puer (nlcr3lhkz6 allele), ascΔ31(aschkz7), Tg(coro1a:eGFP; lyz:DsRed2)hkz05t; nz50t (Hall et al., 2007; Li et al., 2012a), Tg(coro1a:DsRedx;lyz:eGFP)hkz011t;nz117t (Hall et al., 2007; Xu et al., 2016), Tg(coro1a:nlrc3-like)hkz22Tg and Tg(mpeg1:loxP-DsRedx-loxP-GFP) hkz015t (Xu et al., 2016) strains were used in this study.
ENU mutagenesis and NR staining
ENU (Sigma, USA) mutagenesis was carried out as described (Mullins et al., 1994; Solnica-Krezel et al., 1994). NR staining was performed as reported (Herbomel et al.,
Acknowledgments
This work was supported by the National Natural Science Foundation of China (81801977, 31761163008), Shanghai Sailing Program (18YF1420400), the Outstanding Youth Training Program of Shanghai Municipal Health Commission (2018YQ54), the Research Grants Council of the HKSAR (16102414; HKUST5/CRF/12R; AoE/M-09/12, and T13-607/12R), and the Innovation and Technology Commission of the HKSAR (ITCPD/17-9).
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These authors contributed equally to this work.