Showing papers by "Lin Mei published in 2019"

Autism candidate gene DIP2A regulates spine morphogenesis via acetylation of cortactin.

Abstract: Dendritic spine development is crucial for the establishment of excitatory synaptic connectivity and functional neural circuits. Alterations in spine morphology and density have been associated with multiple neurological disorders. Autism candidate gene disconnected-interacting protein homolog 2 A (DIP2A) is known to be involved in acetylated coenzyme A (Ac-CoA) synthesis and is primarily expressed in the brain regions with abundant pyramidal neurons. However, the role of DIP2A in the brain remains largely unknown. In this study, we found that deletion of Dip2a in mice induced defects in spine morphogenesis along with thin postsynaptic density (PSD), and reduced synaptic transmission of pyramidal neurons. We further identified that DIP2A interacted with cortactin, an activity-dependent spine remodeling protein. The binding activity of DIP2A-PXXP motifs (P, proline; X, any residue) with the cortactin-Src homology 3 (SH3) domain was critical for maintaining the level of acetylated cortactin. Furthermore, Dip2a knockout (KO) mice exhibited autism-like behaviors, including excessive repetitive behaviors and defects in social novelty. Importantly, acetylation mimetic cortactin restored the impaired synaptic transmission and ameliorated repetitive behaviors in these mice. Altogether, our findings establish an initial link between DIP2A gene variations in autism spectrum disorder (ASD) and highlight the contribution of synaptic protein acetylation to synaptic processing.

Agrin-Lrp4-Ror2 signaling regulates adult hippocampal neurogenesis in mice

Abstract: Adult neurogenesis in the hippocampus may represent a form of plasticity in brain functions including mood, learning and memory. However, mechanisms underlying neural stem/progenitor cells (NSPCs) proliferation are not well understood. We found that Agrin, a factor critical for neuromuscular junction formation, is elevated in the hippocampus of mice that are stimulated by enriched environment (EE). Genetic deletion of the Agrn gene in excitatory neurons decreases NSPCs proliferation and increases depressive-like behavior. Low-density lipoprotein receptor-related protein 4 (Lrp4), a receptor for Agrin, is expressed in hippocampal NSPCs and its mutation blocked basal as well as EE-induced NSPCs proliferation and maturation of newborn neurons. Finally, we show that Lrp4 interacts with and activates receptor tyrosine kinase-like orphan receptor 2 (Ror2); and Ror2 mutation impairs NSPCs proliferation. Together, these observations identify a role of Agrin-Lrp4-Ror2 signaling for adult neurogenesis, uncovering previously unexpected functions of Agrin and Lrp4 in the brain.

A mechanism in agrin signaling revealed by a prevalent Rapsyn mutation in congenital myasthenic syndrome.

Abstract: Neuromuscular junction is a synapse between motoneurons and skeletal muscles, where acetylcholine receptors (AChRs) are concentrated to control muscle contraction. Studies of this synapse have contributed to our understanding of synapse assembly and pathological mechanisms of neuromuscular disorders. Nevertheless, underlying mechanisms of NMJ formation was not well understood. To this end, we took a novel approach - studying mutant genes implicated in congenital myasthenic syndrome (CMS). We showed that knock-in mice carrying N88K, a prevalent CMS mutation of Rapsyn (Rapsn), died soon after birth with profound NMJ deficits. Rapsn is an adapter protein that bridges AChRs to the cytoskeleton and possesses E3 ligase activity. In investigating how N88K impairs the NMJ, we uncovered a novel signaling pathway by which Agrin-LRP4-MuSK induces tyrosine phosphorylation of Rapsn, which is required for its self-association and E3 ligase activity. Our results also provide insight into pathological mechanisms of CMS.

Microglial VPS35 deficiency regulates microglial polarization and decreases ischemic stroke-induced damage in the cortex.

Abstract: Vacuolar sorting protein 35 (VPS35), a critical component of retromer, is essential for selective endosome-to-Golgi retrieval of membrane proteins. It is highly expressed in microglial cells, in addition to neurons. We have previously demonstrated microglial VPS35’s functions in preventing hippocampal, but not cortical, microglial activation, and in promoting adult hippocampal neurogenesis. However, microglial VPS35’s role in the cortex in response to ischemic stroke remains largely unclear. We used mice with VPS35 cKO (conditional knockout) in microglial cells and examined and compared their responses to ischemic stroke with control mice. The brain damage, cell death, changes in glial cells and gene expression, and sensorimotor deficits were assessed by a combination of immunohistochemical and immunofluorescence staining, RT-PCR, Western blot, and neurological functional behavior tests. We found that microglial VPS35 loss results in an increase of anti-inflammatory microglia in mouse cortex after ischemic stroke. The ischemic stroke-induced brain injury phenotypes, including brain damage, neuronal death, and sensorimotor deficits, were all attenuated by microglial VPS35-deficiency. Further analysis of protein expression changes revealed a reduction in CX3CR1 (CX3C chemokine receptor 1) in microglial VPS35-deficient cortex after ischemic stroke, implicating CX3CR1 as a potential cargo of VPS35 in this event. Together, these results reveal an unrecognized function of microglial VPS35 in enhancing ischemic brain injury-induced inflammatory microglia, but suppressing the injury-induced anti-inflammatory microglia. Consequently, microglial VPS35 cKO mice exhibit attenuation of ischemic brain injury response.

Lack of Myosin X Enhances Osteoclastogenesis and Increases Cell Surface Unc5b in Osteoclast‐Lineage Cells

Abstract: Normal bone mass is maintained by balanced bone formation and resorption. Myosin X (Myo10), an unconventional "myosin tail homology 4-band 4.1, ezrin, radixin, moesin" (MyTH4-FERM) domain containing myosin, is implicated in regulating osteoclast (OC) adhesion, podosome positioning, and differentiation in vitro. However, evidence is lacking for Myo10 in vivo function. Here we show that mice with Myo10 loss of function, Myo10m/m , exhibit osteoporotic deficits, which are likely due to the increased OC genesis and bone resorption because bone formation is unchanged. Similar deficits are detected in OC-selective Myo10 conditional knockout (cko) mice, indicating a cell autonomous function of Myo10. Further mechanistic studies suggest that Unc-5 Netrin receptor B (Unc5b) protein levels, in particular its cell surface level, are higher in the mutant OCs, but lower in RAW264.7 cells or HEK293 cells expressing Myo10. Suppressing Unc5b expression in bone marrow macrophages (BMMs) from Myo10m/m mice by infection with lentivirus of Unc5b shRNA markedly impaired RANKL-induced OC genesis. Netrin-1, a ligand of Unc5b, increased RANKL-induced OC formation in BMMs from both wild-type and Myo10m/m mice. Taken together, these results suggest that Myo10 plays a negative role in OC formation, likely by inhibiting Unc5b cell-surface targeting, and suppressing Netrin-1 promoted OC genesis. © 2019 American Society for Bone and Mineral Research.

pHluorin-BACE1-mCherry Acts as a Reporter for the Intracellular Distribution of Active BACE1 In Vitro and In Vivo

Abstract: β-site APP-cleaving enzyme 1 (BACE1) initiates amyloid precursor protein (APP) cleavage and β-amyloid (Aβ) production, a critical step in the pathogenesis of Alzheimer's disease (AD). It is thus of considerable interest to investigate how BACE1 activity is regulated. BACE1 has its maximal activity at acidic pH and GFP variant-pHluorin-displays pH dependence. In light of these observations, we generated three tandem fluorescence-tagged BACE1 fusion proteins, named pHluorin-BACE1-mCherry, BACE1-mCherry-pHluorin and BACE1-mCherry-EGFP. Comparing the fluorescence characteristics of these proteins in response to intracellular pH changes induced by chloroquine or bafilomycin A1, we found that pHluorin-BACE1-mCherry is a better pH sensor for BACE1 because its fluorescence intensity responds to pH changes more dramatically and more quickly. Additionally, we found that (pro)renin receptor (PRR), a subunit of the v-ATPase complex, which is critical for maintaining vesicular pH, regulates pHluorin's fluorescence and BACE1 activity in pHluorin-BACE1-mCherry expressing cells. Finally, we found that the expression of Swedish mutant APP (APPswe) suppresses pHluorin fluorescence in pHluorin-BACE1-mCherry expressing cells in culture and in vivo, implicating APPswe not only as a substrate but also as an activator of BACE1. Taken together, these results suggest that the pHluorin-BACE1-mCherry fusion protein may serve as a useful tool for visualizing active/inactive BACE1 in culture and in vivo.