Nogo receptor impairs the clearance of fibril amyloid-ß by microglia and accelerates Alzheimer's-like disease progression.

Alzheimer's disease (AD) is characterized by the progressive accumulation of ß-amyloid (Aß)-containing amyloid plaques, and microglia play a critical role in mediating Aß clearance. Mounting evidence has confirmed that the ability of microglia in clearing Aß decreased with aging and AD progress, but the underlying mechanisms are unclear. Previously, we have demonstrated that Nogo receptor (NgR), a receptor for three axon growth inhibitors associated with myelin, can decrease adhesion and migration of microglia to fibrils Aß with aging. However, whether NgR expressed on microglia affect microglia phagocytosis of fibrils Aß with aging remains unclear. Here, we found that aged but not young microglia showed increased NgR expression and decreased Aß phagocytosis in APP/PS1 transgenic mice. NgR knockdown APP/PS1 mice showed simultaneous reduced amyloid burden and improved spatial learning and memory, which were associated with increased Aß clearance. Importantly, Nogo-P4, an agonist of NgR, enhanced the protein level of p-Smad2/3, leading to a significant transcriptional inhibition of CD36 gene expression, which in turn decreased the microglial phagocytosis of Aß. Moreover, ROCK accounted for Nogo-P4-induced activation of Smad2/3 signaling. Finally, the decreasing effect of NgR on microglial Aß uptake was confirmed in a mouse model of intra-hippocampal fAß injection. Our findings suggest that NgR may play an important role in the regulation of Aß homeostasis, and has potential as a therapeutic target for AD.

Anti-apoptotic efficacy of Qingnao Yizhi formula in hypoxia/reoxygenation primary cortical neurons through the promotion of synaptic plasticity by modulating the NogoA-Nogo receptor/Rho-Rho kinase signaling pathway.

OBJECTIVE: To evaluate the anti-apoptotic efficacy of Qingnao Yizhi formula (，QNYZ) in cultured cerebral cortical neuronal cells (CNCs) and the regulation of the NogoA-Nogo receptor (NgR)/Rho-Rho kinase (ROCK) signaling pathway. METHODS: Primary cultured CNCs were randomly divided into the following groups: normal control group (N-C), hypoxia-reoxygenation group (H/R), high-dose QNYZ group (Q-H), low-dose QNYZ group (Q-L) butylphthalide (NBP) group, and Y-27632 (a selective ROCK transduction pathway inhibiter) group. Except those in the N-C group, CNCs were placed in hypoxic conditions for 24 h and then in reoxygenation conditions for 24 h. Cell media was changed every 48 h, and various assays were performed on the 7th day. Cell viability was evaluated by measuring mitochondrial dehydrogenase activity, using a CCK-8 assay, in triplicate. Synapsin (SYN) protein concentrations were evaluated by enzyme-linked immunosorbent assay. NogoA and RhoA protein expression were evaluated through Western blotting. The gene expression of NogoA, NgR, RhoA, and ROCK was evaluated by reverse transcription-polymerase chain reaction. Cell apoptosis was measured using a terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assay. RESULTS: Compared with the N-C group, the cell viability of the H/R group decreased significantly (P < 0.05). The cell viability values for the Q-H and Q-L groups increased compared with that for the H/R group, and the difference was significant for the Q-H group (P < 0.05). The NogoA and RhoA protein levels and the NogoA, NgR, RhoA, and ROCK mRNA expression levels increased in the H/R group, compared with the N-C group, and decreased significantly in the Q-H and Q-L groups (P < 0.05) and in the Y-27632 group (P < 0.05) compared with the H/R group. The SYN levels in the Q-H, Q-L, and NBP groups significantly increased compared with that in the H/R group (P < 0.05). Compared with the H/R group, the numbers of apoptotic cells in the Q-H, Q-L, and NBP groups significantly decreased (P < 0.05). CONCLUSION: The presented study demonstrated that QNYZ exerted anti-apoptotic effects on H/R-induced CNCs, possibly through the modulation of the NogoA-NgR/Rho-ROCK signaling pathway and the promotion of synaptic plasticity in H/R CNCs.

Single-nucleus RNA sequencing of mouse auditory cortex reveals critical period triggers and brakes.

Auditory experience drives neural circuit refinement during windows of heightened brain plasticity, but little is known about the genetic regulation of this developmental process. The primary auditory cortex (A1) of mice exhibits a critical period for thalamocortical connectivity between postnatal days P12 and P15, during which tone exposure alters the tonotopic topography of A1. We hypothesized that a coordinated, multicellular transcriptional program governs this window for patterning of the auditory cortex. To generate a robust multicellular map of gene expression, we performed droplet-based, single-nucleus RNA sequencing (snRNA-seq) of A1 across three developmental time points (P10, P15, and P20) spanning the tonotopic critical period. We also tone-reared mice (7 kHz pips) during the 3-d critical period and collected A1 at P15 and P20. We identified and profiled both neuronal (glutamatergic and GABAergic) and nonneuronal (oligodendrocytes, microglia, astrocytes, and endothelial) cell types. By comparing normal- and tone-reared mice, we found hundreds of genes across cell types showing altered expression as a result of sensory manipulation during the critical period. Functional voltage-sensitive dye imaging confirmed GABA circuit function determines critical period onset, while Nogo receptor signaling is required for its closure. We further uncovered previously unknown effects of developmental tone exposure on trajectories of gene expression in interneurons, as well as candidate genes that might execute tonotopic plasticity. Our single-nucleus transcriptomic resource of developing auditory cortex is thus a powerful discovery platform with which to identify mediators of tonotopic plasticity.

Static Leukoencephalopathy Associated with 17p13.3 Microdeletion Syndrome: A Case Report.

BACKGROUND: Leukoencephalopathy associated with dysmorphic features may be attributed to chromosomal abnormalities such as 17p13.3 microdeletion syndrome. CASE: A 19-year-old female patient was referred to our hospital for diagnostic evaluation of her leukoencephalopathy. She demonstrated moderate intellectual disability, minor dysmorphic features, and short stature. Serial brain magnetic resonance images obtained within a 16-year interval revealed prolonged T2 signals in the deep cerebral white matter with enlarged Virchow-Robin spaces. A nonsymptomatic atlas anomaly was also noted. Using microarray-based comparative genomic hybridization, we identified a 2.2-Mb terminal deletion at 17p13.3, encompassing YWHAE, CRK, and RTN4RL1 but not PAFAH1B1. CONCLUSION: Except for atlas anomaly, the patient's clinical and imaging findings were compatible with the diagnosis of 17p13.3 microdeletion syndrome. The white matter abnormality was static and nonprogressive. The association between the atlas abnormality and this deletion remains elusive. We note the importance of exploring submicroscopic chromosomal imbalance when patients show prominent but static white matter abnormalities with discrepantly mild and stable neurological signs.

Effects of Bu Shen Yi sui capsule on NogoA/NgR and its signaling pathways RhoA/ROCK in mice with experimental autoimmune encephalomyelitis.

BACKGROUND: Axon growth inhibitory factors NogoA/Nogo receptor (NgR) and its signaling pathways RhoA/Rho kinase (ROCK) play a critical role in the repair of nerve damage in multiple sclerosis (MS). Bu Shen Yi Sui Capsule (BSYSC) is an effective Chinese formula utilized to treat MS in clinical setting and noted for its potent neuroprotective effects. In this study, we focus on the effects of BSYSC on promoting nerve repair and the underlying mechanisms in mice with experimental autoimmune encephalomyelitis (EAE), an animal model of MS. METHODS: The EAE mouse model was induced by injecting subcutaneously with myelin oligodendrocyte glycoprotein (MOG) 35-55 supplemented with pertussis toxin. BSYSC was orally administrated at dose of 3.0 g/kg once a day for 40 days. The levels of protein gene product (PGP) 9.5, p-Tau, growth associated protein (GAP) -43, KI67 and Nestin in the brain or spinal cord on 20 and 40 day post-induction (dpi) were detected via immunofluorescence and Western blot analysis. Furthermore, NogoA/NgR and RhoA/ROCK signaling molecules were studied by qRT-PCR and Western blot analysis. RESULTS: Twenty or 40 days of treatment with BSYSC increased markedly PGP9.5 and GAP-43 levels, reduced p-Tau in the brain or spinal cord of mice with EAE. In addition, BSYSC elevated significantly the expression of KI67 and Nestin in the spinal cord 40 dpi. Further study showed that the activation of NogoA/NgR and RhoA/ROCK were suppressed by the presence of BSYSC. CONCLUSIONS: BSYSC could attenuate axonal injury and promote repair of axonal damage in EAE mice in part through the down-regulation of NogoA/NgR and RhoA/ROCK signaling pathways.

The Nogo receptor inhibits proliferation, migration and axonal extension by transcriptionally regulating WNK1 in PC12 cells.

Neuronal regeneration and axonal regrowth mechanisms in the injured mammalian central nervous system are largely unknown. As part of a major pathway for inhibiting axonal regeneration, activated neuronal glycosylphosphatidylinositol-anchored Nogo receptor (NgR) interacts with LINGO-1 and p75NTR to form a complex at the cell surface. However, it was found in our previous report that upregulation of NgR stimulated by injury plays a key role in neuronal regeneration in the neonatal cortex freeze-lesion model, but its downstream signalling remains elusive. In the present study, the novel regulatory role of NgR in a serine-threonine kinase WNK1 was identified. NgR's transcriptional regulation of WNK1 was identified by RT-qPCR and semiquantitative western blot after the overexpression or knockdown of NgR, and the regulation is specific to WNK1, which is not the same for its family members, WNK2, WNK3 and WNK4. Furthermore, NgR inhibition by NEP fails to affect WNK1, which indicates that WNK1 functions outside of the Nogo-A/NgR pathway. By performing a proliferation, migration and axonal extension assay, we also identified that overexpressed NgR critically regulated these processes and impairment by overexpressing NgR was rescued with coexpression of WNK1, indicating the partial role of WNK1 in NgR-mediated morphological regulation. Our study identifies a separation of functions for the NgR-regulated WNK1 in mediating proliferation, migration and axonal extension in PC12 cells as well as a specific regulatory role between NgR and WNK1 that is important for recovery from central nervous system injury.

Lipocalin-2 Promotes Endoplasmic Reticulum Stress and Proliferation by Augmenting Intracellular Iron in Human Pulmonary Arterial Smooth Muscle Cells.

Endoplasmic reticulum (ER) stress, a feature of many conditions associated with pulmonary hypertension (PH), is increasingly recognized as a common response to promote proliferation in the walls of pulmonary arteries. Increased expression of Lipocalin-2 in PH led us to test the hypothesis that Lipocalin-2, a protein known to sequester iron and regulate it intracellularly, might facilitate the ER stress and proliferation in pulmonary arterial smooth muscle cells (PASMCs). In this study, we observed greatly increased Lcn2 expression accompanied with increased ATF6 cleavage in a standard rat model of pulmonary hypertension induced by monocrotaline. In cultured human PASMCs, Lcn2 significantly promoted ER stress (determined by augmented cleavage and nuclear localization of ATF6, up-regulated transcription of GRP78 and NOGO, increased expression of SOD2, and mild augmented mitochondrial membrane potential) and proliferation (assessed by Ki67 staining and BrdU incorporation). Lcn2 promoted ER stress accompanied with augmented intracellular iron levels in human PASMCs. Treatment human PASMCs with FeSO4 induced the similar ER stress and proliferation response and iron chelator (deferoxamine) abrogated the ER stress and proliferation induced by Lcn2 in cultured human PASMCs. In conclusion, Lcn2 significantly promoted human PASMC ER stress and proliferation by augmenting intracellular iron. The up-regulation of Lcn2 probably involved in the pathogenesis and progression of PH.

Nogo receptor complex expression dynamics in the inflammatory foci of central nervous system experimental autoimmune demyelination.

BACKGROUND: Nogo-A and its putative receptor NgR are considered to be among the inhibitors of axonal regeneration in the CNS. However, few studies so far have addressed the issue of local NgR complex multilateral localization within inflammation in an MS mouse model of autoimmune demyelination. METHODS: Chronic experimental autoimmune encephalomyelitis (EAE) was induced in C57BL/6 mice. Analyses were performed on acute (days 18-22) and chronic (day 50) time points and compared to controls. The temporal and spatial expression of the Nogo receptor complex (NgR and coreceptors) was studied at the spinal cord using epifluorescent and confocal microscopy or real-time PCR. Data are expressed as cells/mm2, as mean % ± SEM, or as arbitrary units of integrated density. RESULTS: Animals developed a moderate to severe EAE without mortality, followed by a progressive, chronic clinical course. NgR complex spatial expression varied during the main time points of EAE. NgR with coreceptors LINGO-1 and TROY was increased in the spinal cord in the acute phase whereas LINGO-1 and p75 signal seemed to be dominant in the chronic phase, respectively. NgR was detected on gray matter NeuN+ neurons of the spinal cord, within the white matter inflammatory foci (14.2 ± 4.3 % NgR+ inflammatory cells), and found to be colocalized with GAP-43+ axonal growth cones while no ß-TubIII+, SMI-32+, or APP+ axons were found as NgR+. Among the NgR+ inflammatory cells, 75.6 ± 9.0 % were microglial/macrophages (lectin+), 49.6 ± 14.2 % expressed CD68 (phagocytic ED1+ cells), and no cells were Mac-3+. Of these macrophages/monocytes, only Arginase-1+/NgR+ but not iNOS+/NgR+ were present in lesions both in acute and chronic phases. CONCLUSIONS: Our data describe in detail the expression of the Nogo receptor complex within the autoimmune inflammatory foci and suggest a possible immune action for NgR apart from the established inhibitory one on axonal growth. Its expression by inflammatory macrophages/monocytes could signify a possible role of these cells on axonal guidance and clearance of the lesioned area during inflammatory demyelination.