TREM2 regulates microglial phagocytosis of synapses in innate immune tolerance.

Increasing evidence indicates that innate immune cells also possess immunological memory. Microglia are brain-resident innate immune cells and execute inflammatory and phagocytic functions upon environmental stimulation, during which processes triggering receptor expressed on myeloid cells 2 (TREM2) plays an important regulatory role. However, although microglia are known to exhibit innate immune memory related to inflammation when subjected to continuous inflammatory stimuli, whether microglia exhibit innate immune memory related to phagocytosis and whether TREM2 participates in innate immune memory of microglia remain unknown. Herein, we treated WT and Trem2 KO mice with peripheral injection of lipopolysaccharides (LPS) to induce microglial activation or microglial immune tolerance. We found that Tnfα and Il-1ß expression levels in the hippocampi were significantly elevated after 1xLPS and then dramatically decreased after 4xLPS in both WT and Trem2 KO mice; and their level changes were indistinguishable between WT and Trem2 KO mice. Moreover, 1xLPS significantly promoted microglial phagocytosis of synapses and caused microglial morphology changes resembling activated status in both WT and Trem2 KO mice. However, 4xLPS significantly reduced synapse phagocytosis and largely reversed morphology changes in WT microglia. While 4xLPS had no effect on reducing synapse phagocytosis in Trem2 KO microglia. RNA-seq analysis revealed that TREM2 deficiency reprogrammed complement and phagosome-related transcriptional landscape during immune tolerance. Our results demonstrate that microglia also exhibit immune tolerance related to phagocytosis of synapses and that TREM2 plays a crucial role in this process possibly through regulating complement system and phagosome-related gene expressions.

Tau reduction attenuates autism-like features in Fmr1 knockout mice.

BACKGROUND: Fragile X syndrome (FXS) is a leading cause of autism spectrum disorder (ASD) and resulted from a loss of the FMR1-encoded fragile X messenger ribonucleoprotein 1 (FMRP) protein due to large CGG repeat expansions in the promoter region of the FMR1 gene. The microtubule-associated protein Tau is a promising target for Tauopathic diseases and our preliminary study found that Tau protein levels were increased in the brain of Fmr1 knockout (KO) mice, a model of FXS. However, whether Tau reduction can prevent autism-like features in Fmr1 KO mice and become a novel strategy for FXS treatment remain unknown. METHODS: Tau was genetically reduced in Fmr1 KO mice through crossing Fmr1± female mice with Mapt± male mice. The male offspring with different genotypes were subjected to various autism-related behavioral tests, RNA sequencing, and biochemical analysis. Fmr1 KO male mice were treated with Tau-targeting antisense oligonucleotide (ASO) and then subjected to behavioral tests and biochemical analysis. RESULTS: Tau expression was increased in the cortex of Fmr1 KO mice. Genetically reducing Tau prevented social defects, stereotyped and repetitive behavior, and spine abnormality in Fmr1 KO mice. Tau reduction also reversed increased periodic activity and partially rescued Per1 expression reduction in Fmr1 KO mice. Moreover, Tau reduction reversed compromised P38/MAPK signaling in Fmr1 KO mice. Finally, Tau-targeting ASO also effectively alleviated autism-like phenotypes and promoted P38/MAPK signaling in Fmr1 KO mice. LIMITATIONS: Our study is limited to male mice, in agreement with the higher incidence of FXS in males than females. Whether Tau reduction also exerts protection in females deserves further scrutiny. Moreover, although Tau reduction rescues impaired P38/MAPK signaling in Fmr1 KO mice, whether this is the responsible molecular mechanism requires further determination. CONCLUSION: Our data indicate that Tau reduction prevents autism-like phenotypes in Fmr1 KO mice. Tau may become a new target for FXS treatment.

Long-term potentiation-based screening identifies neuronal PYGM as a synaptic plasticity regulator participating in Alzheimer's disease.

Synaptic dysfunction is an important pathological hallmark and cause of Alzheimer's disease (AD). High-frequency stimulation (HFS)-induced long-term potentiation (LTP) has been widely used to study synaptic plasticity, with impaired LTP found to be associated with AD. However, the exact molecular mechanism underlying synaptic plasticity has yet to be completely elucidated. Whether genes regulating synaptic plasticity are altered in AD and contribute to disease onset also remains unclear. Herein, we induced LTP in the hippocampal CA1 region of wild-type (WT) and AD model mice by administering HFS to the CA3 region and then studied transcriptome changes in the CA1 region. We identified 89 genes that may participate in normal synaptic plasticity by screening HFS-induced differentially expressed genes (DEGs) in mice with normal LTP, and 43 genes that may contribute to synaptic dysfunction in AD by comparing HFS-induced DEGs in mice with normal LTP and AD mice with impaired LTP. We further refined the 43 genes down to 14 by screening for genes with altered expression in pathological-stage AD mice without HFS induction. Among them, we found that the expression of Pygm, which catabolizes glycogen, was also decreased in AD patients. We further demonstrated that down-regulation of PYGM in neurons impaired synaptic plasticity and cognition in WT mice, while its overexpression attenuated synaptic dysfunction and cognitive deficits in AD mice. Moreover, we showed that PYGM directly regulated energy generation in neurons. Our study not only indicates that PYGM-mediated energy production in neurons plays an important role in synaptic function, but also provides a novel LTP-based strategy to systematically identify genes regulating synaptic plasticity under physiological and pathological conditions.

Gene therapy using human FMRP isoforms driven by the human FMR1 promoter rescues fragile X syndrome mouse deficits.

Fragile X syndrome (FXS) is caused by the loss of the fragile X messenger ribonucleoprotein 1 (FMRP) encoded by the FMR1 gene. Gene therapy using adeno-associated virus (AAV) to restore FMRP expression is a promising therapeutic strategy. However, so far AAV gene therapy tests for FXS only utilized rodent FMRPs driven by promoters other than the human FMR1 promoter. Restoration of human FMRP in appropriate cell types and at physiological levels, preferably driven by the human FMR1 promoter, would be more suitable for its clinical use. Herein, we generated two human FMR1 promoter subdomains that effectively drive gene expression. When AAVs expressing two different human FMRP isoforms under the control of a human FMR1 promoter subdomain were administered into bilateral ventricles of neonatal Fmr1 -/y and wild-type (WT) mice, both human FMRP isoforms were expressed throughout the brain in a pattern reminiscent to that of mouse FMRP. Importantly, human FMRP expression attenuated social behavior deficits and stereotyped and repetitive behavior, and reversed dysmorphological dendritic spines in Fmr1 -/y mice, without affecting WT mouse behaviors. Our results demonstrate that human FMR1 promoter can effectively drive human FMRP expression in the brain to attenuate Fmr1 -/y mouse deficits, strengthening the notion of using AAV gene therapy for FXS treatment.

Microglial Tmem59 Deficiency Impairs Phagocytosis of Synapse and Leads to Autism-Like Behaviors in Mice.

Synaptic abnormality is an important pathologic feature of autism spectrum disorders (ASDs) and responsible for various behavioral defects in these neurodevelopmental disorders. Microglia are the major immune cells in the brain and also play an important role in synapse refinement. Although dysregulated synaptic pruning by microglia during the brain development has been associated with ASDs, the underlying mechanism has yet to be fully elucidated. Herein, we observed that expression of Transmembrane protein 59 (TMEM59), a protein recently shown to regulate microglial function, was decreased in autistic patients. Furthermore, we found that both male and female mice with either complete or microglia-specific loss of Tmem59 developed ASD-like behaviors. Microglial TMEM59-deficient mice also exhibited enhanced excitatory synaptic transmission, increased dendritic spine density, and elevated levels of excitatory synaptic proteins in synaptosomes. TMEM59-deficient microglia had impaired capacity for synapse engulfment both in vivo and in vitro. Moreover, we demonstrated that TMEM59 interacted with the C1q receptor CD93 and TMEM59 deficiency promoted CD93 protein degradation in microglia. Downregulation of CD93 in microglia also impaired synapse engulfment. These findings identify a crucial role of TMEM59 in modulating microglial function on synapse refinement during brain development and suggest that TMEM59 deficiency may contribute to ASDs through disrupting phagocytosis of excitatory synapse and thus distorting the excitatory-inhibitory (E/I) neuronal activity balance.SIGNIFICANCE STATEMENT Microglia play an important role in synapse refinement. Dysregulated synaptic pruning by microglia during brain development has been associated with autism spectrum disorders (ASDs). However, the underlying mechanism has yet to be fully elucidated. Herein, we observe that the expression of Transmembrane protein 59 (TMEM59), an autophagy-related protein, is decreased in autistic patients. Moreover, we find ASD-like behaviors in mice with complete loss and with microglia-specific loss of Tmem59 Mechanistic studies reveal that TMEM59 deficiency in microglia impairs their synapse engulfment ability likely through destabilizing the C1q receptor CD93, thereby leading to enhanced excitatory neurotransmission and increased dendritic spine density. Our findings demonstrate a crucial role of microglial TMEM59 in early neuronal development and provide new insight into the etiology of ASDs.

RPS23RG1 modulates tau phosphorylation and axon outgrowth through regulating p35 proteasomal degradation.

Tauopathies are a group of neurodegenerative diseases characterized by hyperphosphorylation of the microtubule-binding protein, tau, and typically feature axon impairment and synaptic dysfunction. Cyclin-dependent kinase5 (Cdk5) is a major tau kinase and its activity requires p35 or p25 regulatory subunits. P35 is subjected to rapid proteasomal degradation in its membrane-bound form and is cleaved by calpain under stress to a stable p25 form, leading to aberrant Cdk5 activation and tau hyperphosphorylation. The type Ib transmembrane protein RPS23RG1 has been implicated in Alzheimer's disease (AD). However, physiological and pathological roles for RPS23RG1 in AD and other tauopathies are largely unclear. Herein, we observed retarded axon outgrowth, elevated p35 and p25 protein levels, and increased tau phosphorylation at major Cdk5 phosphorylation sites in Rps23rg1 knockout (KO) mice. Both downregulation of p35 and the Cdk5 inhibitor roscovitine attenuated tau hyperphosphorylation and axon outgrowth impairment in Rps23rg1 KO neurons. Interestingly, interactions between the RPS23RG1 carboxyl-terminus and p35 amino-terminus promoted p35 membrane distribution and proteasomal degradation. Moreover, P301L tau transgenic (Tg) mice showed increased tau hyperphosphorylation with reduced RPS23RG1 levels and impaired axon outgrowth. Overexpression of RPS23RG1 markedly attenuated tau hyperphosphorylation and axon outgrowth defects in P301L tau Tg neurons. Our results demonstrate the involvement of RPS23RG1 in tauopathy disorders, and implicate a role for RPS23RG1 in inhibiting tau hyperphosphorylation through homeostatic p35 degradation and suppression of Cdk5 activation. Reduced RPS23RG1 levels in tauopathy trigger aberrant Cdk5-p35 activation, consequent tau hyperphosphorylation, and axon outgrowth impairment, suggesting that RPS23RG1 may be a potential therapeutic target in tauopathy disorders.

TMEM59 Haploinsufficiency Ameliorates the Pathology and Cognitive Impairment in the 5xFAD Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disease associated with cognitive deficits and synaptic impairments. Amyloid-ß (Aß) plaque deposition, dystrophic neurite accumulation and neurofibrillary tangles are pathological hallmarks of AD. TMEM59 has been implicated to play a role in AD pathogenesis; however, the underlying mechanism remains unknown. Herein, we found that overexpression of TMEM59 in the hippocampal region led to memory impairment in wild type mice, suggesting its neurotoxic role. Interestingly, while TMEM59 overexpression had no effect on worsening synaptic defects and impaired memory in the 5xFAD mouse model of AD, it significantly exacerbated AD-like pathologies by increasing levels of detergent-insoluble Aß and Aß plaques, as well as dystrophic neurites. Importantly, haploinsufficiency of TMEM59 reduced insoluble Aß levels, Aß plaques, and neurite dystrophy, thereby rescuing synaptic plasticity and memory deficits in 5xFAD mice. Moreover, the level of TMEM59 in the brain of 5xFAD mice increased compared to wild type mice during aging, further corroborating its detrimental functions during neurodegeneration. Together, these results demonstrate a novel function of TMEM59 in AD pathogenesis and provide a potential therapeutic strategy by downregulating TMEM59.

TMEM59 interacts with TREM2 and modulates TREM2-dependent microglial activities.

The surface receptor triggering receptor expressed on myeloid cells 2 (TREM2) plays a crucial role in maintaining a multitude of microglial activities, such as survival, proliferation, migration, metabolism, inflammation, and phagocytosis. However, the molecular mechanisms underlying TREM2-mediated microglial activities remain largely elusive. Herein, we found that TREM2 interacted with the type I transmembrane protein TMEM59, whose expression could facilitate autophagic flux through its carboxyl-terminus. TMEM59 expression was decreased upon lipopolysaccharide treatment. While downregulation of TMEM59 promoted anti-inflammatory factor expression and attenuated lipopolysaccharide treatment-induced inflammation. Importantly, we found that overexpression of TREM2 reduced TMEM59 protein levels through promoting its degradation, whereas TMEM59 levels were elevated in Trem2-deficient microglia. Finally, impaired survival, proliferation, migration, and phagocytosis, as well as dysregulated autophagy and metabolism in Trem2-deficient microglia were attenuated upon TMEM59 silencing. Together, our findings reveal a novel function of TREM2 in mediating TMEM59 protein degradation and demonstrate the importance of TMEM59 homeostasis in maintaining TREM2-mediated microglial activities.