Investigating roles of Topoisomerase 3b (TOP3B) in LPS-induced microglia-mediated neuroinflammation in mouse

National Institute On Aging

Investigators

Abstract

Topoisomerase 3b (TOP3B) is a dual-activity enzyme that can alter the topology of both RNA and DNA. TOP3B associates with TDRD3 and can interact with Fragile X Mental Retardation Protein (FMRP). Our recent research indicates that Top3b-TDRD3 controls stimulus-induced transcription, mRNA translation and turnover, and siRNA-guided heterochromatin formation. Top3b mutations in humans has been linked to neurological disorders such as schizophrenia and autism. We have also revealed that Top3b-KO mice have behavioral and neurodevelopmental defects. Interestingly, TOP3B-KO mice have a shorter lifespan and immune-dysfunction, as evidenced by higher serum autoantibodies and an increase in numerical chromosomal abnormalities in splenocytes and bone marrow cells. However, the mechanism of how Top3B mutations cause various neurological disorders remain unclear. Here, we hypothesize that one mechanism could be neuroinflammation due to abnormal microglia activation. We plan to investigate this hypothesis by studying whether Top3b mutant mice display neuroinflammation and abnormal microglia activation. Neuroinflammation is a characteristic of many age-associated neurological diseases (NDs), including AD and age-related dementia, and plays an important role in disease initiation and progression. In particular, neuroinflammation has been observed in schizophrenia and autism, which are known to be associated with Top3b mutations, raising a possibility that Top3b mutations can cause neuroinflammation, which leads to these neurological disorders. The main contributors of neuroinflammation are microglia, which serve as the first line of the immunological response for the CNS. Microglia are immunological guardians of the brain that play an important role in providing host defense against infections and CNS diseases. The maintenance of CNS homeostasis during development, adulthood, and aging is another important housekeeping task carried out by microglia. Notably, abnormal activation of microglia has been reported in schizophrenia, and autism, both of which are associated with Top3b mutations. Thus, one mechanism by which Top3b mutations may cause these neurological disorders could be through aberrant microglia activation and neuroinflammation. We have already published that Top3β knockout mice show transcriptional and behavioral impairments associated with neurogenesis and synaptic plasticity. We have also generated Top3b-Y336F-Knockin mutant mice line, which expresses a Top3b mutant that lacks topoisomerase activity but retains RNA binding activity. Here, we plan to use our established Top3b mutant mouse models to investigate whether Top3b inactivation can lead to abnormal microglia activation in 3 Aims. Aim1: Investigate the roles of TOP3B in untreated and LPS (lipopolysaccharide)-induced-microglia activation, and phenotypic transition in developing mouse brain. Aim2: Investigate the roles of TOP3B in untreated and LPS-induced- microglia functional alteration and consequences in developing mouse brain. Aim3: Investigate the molecular mechanism how TOP3B regulates the microglia activation and functional alteration in developing mouse brain. The following preliminary data have been obtained. 1. Top3b mutant mice show increased microglia activation and impulse towards a M1 phenotype. We observed that microglia activation marker, CD68, and M1 phenotypic marker, CD86, were expressed higher in Top3b-Y336F mutant mice, as compared to WT mice at PND22 under untreated conditions (postnatal day 22). On the contrary, the levels of M2 phenotypic markers Arg1 and CD206 were lower in the mutant mice. The data suggest that Top3b mutations can alter the transition of microglia towards the M1 phenotype. M1 microglia can release inflammatory cytokines, consistent with our hypothesis that Top3b mutations may cause increased neuroinflammation, leading to neurological defects. 2. Top3b mutant mice show increased levels of activated microglia markers and decreased levels of microglia phagocytosis markers. We observed higher expression of both TLR4 and CD36 mRNA in mutant mice as compared to WT mice under untreated conditions, suggesting the abnormal activation of microglia in the absence of Top3b. Because the activated microglia make the local milieu hostile for neurons, and the activated microglial phenotype, M1, is associated with phagocytosis, we examined the phagocytic potential of microglia as well as interactions between microglia and neurons. Sirp-α and CD47 are two markers present on microglia and neuron membrane, respectively. By their interactions, neurons provide the signal to microglia “do not eat me” (higher CD47 inhibits microglial phagocytosis by interacting with Sirp-a). We observed that Top3b-mutant mice showed decreased expression of sirp-α and CD47 mRNAs in brain lysate, as compared to WT mice, suggesting increased microglia phagocytosis when Top3b is mutated. 3. Top3b mutant mouse brains show increased levels of proinflammatory and decreased levels of anti-inflammatory cytokines. In addition, the levels of proinflammatory cytokine IL6 and TNFα were increased, whereas those of anti-inflammatory cytokines IL4 and IL10 were decreased in Top3b-mutant mice. The increased expression of proinflammatory cytokines in Top3b mutant mice support our hypothesis that Top3b inactivation results in elevated neuroinflammation. 4. Top3b mutant mouse brains show decreased expression levels of synaptic markers We evaluated the expression levels of two synapse-associated markers, SNAP25 and PSD95. We found that mRNA levels of SNAP25 and PSD95 were unchanged, but their protein levels were decreased in Top3b mutant mice, as compared to WT mice under untreated conditions. The data are in accord with the notion that Top3b mutant mice have aberrant synaptic pruning by microglia in developing brain, and they are also consistent with our early findings that neurons from Top3b-KO mice have a reduced number of synapses (Xu et al., Nat. Neurosci, 2013). 5. We have performed RNA-seq analysis to compare gene expression level changes in Top3b-KO mice. We observed changes in some inflammation genes, and are in the process of analyzing the data.