Soluble ANPEP Released From Human Astrocytes as a Positive Regulator of Microglial Activation and Neuroinflammation: Brain Renin-Angiotensin System in Astrocyte-Microglia Crosstalk.

Astrocytes are major supportive glia and immune modulators in the brain; they are highly secretory in nature and interact with other cell types via their secreted proteomes. To understand how astrocytes communicate during neuroinflammation, we profiled the secretome of human astrocytes following stimulation with proinflammatory factors. A total of 149 proteins were significantly upregulated in stimulated astrocytes, and a bioinformatics analysis of the astrocyte secretome revealed that the brain renin-angiotensin system (RAS) is an important mechanism of astrocyte communication. We observed that the levels of soluble form of aminopeptidase N (sANPEP), an RAS component that converts angiotensin (Ang) III to Ang IV in a neuroinflammatory milieu, significantly increased in the astrocyte secretome. To elucidate the role of sANPEP and Ang IV in neuroinflammation, we first evaluated the expression of Ang IV receptors in human glial cells because Ang IV mediates biological effects through its receptors. The expression of angiotensin type 1 receptor was considerably upregulated in activated human microglial cells but not in human astrocytes. Moreover, interleukin-1ß release from human microglial cells was synergistically increased by cotreatment with sANPEP and its substrate, Ang III, suggesting the proinflammatory action of Ang IV generated by sANPEP. In a mouse neuroinflammation model, brain microglial activation and proinflammatory cytokine expression levels were increased by intracerebroventricular injection of sANPEP and attenuated by an enzymatic inhibitor and neutralizing antibody against sANPEP. Collectively, our results indicate that astrocytic sANPEP-induced increase in Ang IV exacerbates neuroinflammation by interacting with microglial proinflammatory receptor angiotensin type 1 receptor, highlighting an important role of indirect crosstalk between astrocytes and microglia through the brain RAS in neuroinflammation.

Analyzing the glial proteome in Alzheimer's disease.

INTRODUCTION: Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive cognitive decline, memory loss, and changes in behavior. Accumulating evidence indicates that dysfunction of glial cells, including astrocytes, microglia, and oligodendrocytes, may contribute to the development and progression of AD. Large-scale analysis of glial proteins sheds light on their roles in cellular processes and diseases. In AD, glial proteomics has been utilized to understand glia-based pathophysiology and identify potential biomarkers and therapeutic targets. AREA COVERED: In this review, we provide an updated overview of proteomic analysis of glia in the context of AD. Additionally, we discuss current challenges in the field, involving glial complexity and heterogeneity, and describe some cutting-edge proteomic technologies to address them. EXPERT OPINION: Unbiased comprehensive analysis of glial proteomes aids our understanding of the molecular and cellular mechanisms of AD pathogenesis. These investigations highlight the crucial role of glial cells and provide novel insights into the mechanisms of AD pathology. A deeper understanding of the AD-related glial proteome could offer a repertoire of potential biomarkers and therapeutics. Further technical advancement of glial proteomics will enable us to identify proteins within individual cells and specific cell types, thus significantly enhancing our comprehension of AD pathogenesis.

Utilizing databases for astrocyte secretome research.

INTRODUCTION: Astrocytes are the most abundant cell type in the central nervous system (CNS). They play a pivotal role in supporting neuronal function and maintaining homeostasis by releasing a variety of bioactive proteins, collectively known as the astrocyte secretome. Investigating secretome provides insights into the molecular mechanisms underlying astrocyte function and dysfunction, as well as novel strategies to prevent and treat diseases affecting the CNS. AREAS COVERED: Proteomics databases are a valuable resource for studying the role of astrocytes in healthy and diseased brain function, as they provide information about gene expression, protein expression, and cellular function. In this review, we discuss existing databases that are useful for astrocyte secretome research. EXPERT OPINION: Astrocyte secretomics is a field that is rapidly progressing, yet the availability of dedicated databases is currently limited. To meet the increasing demand for comprehensive omics data in glia research, developing databases specifically focused on astrocyte secretome is crucial. Such databases would allow researchers to investigate the intricate molecular landscape of astrocytes and comprehend their involvement in diverse physiological and pathological processes. Expanding resources through the development of databases dedicated to the astrocyte secretome may facilitate further advancements in this field.

Gamma subunit of complement component 8 is a neuroinflammation inhibitor.

The complement system is part of the innate immune system that comprises several small proteins activated by sequential cleavages. The majority of these complement components, such as components 3a (C3a) and C5a, are chemotactic and pro-inflammatory. However, in this study, we revealed an inhibitory role of complement component 8 gamma (C8G) in neuroinflammation. In patients with Alzheimer's disease, who exhibit strong neuroinflammation, we found higher C8G levels in brain tissue, CSF, and plasma. Our novel findings also showed that the expression level of C8G increases in the inflamed mouse brain, and that C8G is mainly localized to brain astrocytes. Experiments using recombinant C8G protein and shRNA-mediated knockdown showed that C8G inhibits glial hyperactivation, neuroinflammation, and cognitive decline in acute and chronic animal models of Alzheimer's disease. Additionally, we identified sphingosine-1-phosphate receptor 2 (S1PR2) as a novel interaction protein of C8G and demonstrated that astrocyte-derived C8G interacts with S1PR2 to antagonize the pro-inflammatory action of S1P in microglia. Taken together, our results reveal the previously unrecognized role of C8G as a neuroinflammation inhibitor. Our findings pave the way towards therapeutic containment of neuroinflammation in Alzheimer's disease and related neurological diseases.

No Differences In Clinical Outcomes Between Rectangular and Round Tunnel Techniques For Anterior Crucial Ligament Reconstruction.

PURPOSE: To compare the clinical outcomes between conventional round tunnel and rectangular tunnel in anatomic anterior cruciate ligament (ACL) reconstruction. METHODS: This was a retrospective comparative cohort study between March 2015 and September 2018. The primary ACL reconstructions using anteromedial portal technique with minimum of 2 years follow-up were enrolled for this study. The exclusion criteria were patients with revision ACL reconstruction, high tibial osteotomy, multiligament injuries, and associated fractures around the knee. Outcome measures included the subjective International Knee Documentation Committee score, Tegner activity score, knee laxity testing, and measurement of the centers of the femoral and tibial tunnels on postoperative computed tomography (CT) images. RESULTS: Forty-seven patients with ACL reconstruction with rectangular tunnel (group 1) and 108 patients with ACL reconstructions with conventional rounded tunnel (group 2) were included consecutively. There were no significant differences between groups in terms of clinical scores or knee laxity, as well as femoral and tibial tunnel positions on CT. One patient in group 2 had ACL failure because of trauma and was treated with revision surgery. Two patients had incomplete tibial fracture, but they healed spontaneously and showed no residual laxity at final follow-up. The intraobserver and interobserver reliability for the radiological measurements ranged from 0.78 to 0.86. CONCLUSIONS: There were no differences in radiological and clinical results between rectangular tunnel group and conventional round tunnel group for arthroscopic ACL reconstruction. ACL reconstruction with a rectangular tunnel could be considered as a reliable technique, but care should be taken during tunnel establishment because of risk of fractures and malposition of rectangular tunnel.

Clinical outcomes of rectangular tunnel technique in posterior cruciate ligament reconstruction were comparable to the results of conventional round tunnel technique.

PURPOSE: To compare clinical outcomes between the conventional round and rectangular tunnel techniques in single-bundle posterior cruciate ligament (PCL) reconstruction. METHODS: Twenty-seven and 108 patients who underwent PCL reconstructions using a rectangular dilator (Group 1) and rounded tunnel reamer (Group 2), respectively, were included. The exclusion criteria were having a concomitant fracture, osteotomy, subtotal or total meniscectomy, and no remnant PCL tissue. A 4:1 propensity score matching was performed. The knee laxity on stress radiography, International Knee Documentation Committee Subjective Knee Evaluation score, Tegner activity score and Orthopädische Arbeitsgruppe Knie score were evaluated. RESULTS: No significant differences were found between the groups in terms of clinical scores. (n.s.) The mean posterior translations were also not significantly different between the Group 1 and 2 (3.6 ± 2.8 and 3.8. ± 3.1 mm, respectively; n.s.). However, 3 patients (11.1%) in Group 1 and 15 patients (13.8%) in Group 2 showed posterior translation of > 5 mm. The combined posterolateral corner sling technique was performed for 27 patients (100%) in Group 1 and for 96 patients (88.9%) in Group 2. We found no significant difference in rotational stability at the final follow-up. One patient was found to have a femoral condyle fracture during rectangular femoral tunnel establishment, which was healed after screw fixation, without laxity, during follow-up. The intra- and inter-observer reliabilities of the radiological measurements ranged from 0.81 to 0.89. CONCLUSION: Arthroscopic anatomical remnant-preserving PCL reconstruction using a rectangular dilator showed satisfactory clinical results and stability as compared with PCL reconstruction using a conventional rounded reamer. Rectangular tunnel technique in PCL reconstruction could be a good treatment option with theoretical advantage to be anatomic. LEVEL OF EVIDENCE: Level IV.

Proteomic examination of the neuroglial secretome: lessons for the clinic.

Introduction: Glial cells are closely associated with neurons located throughout the nervous system and regulate neuronal activity and function through various mechanisms including the secretion of proteins and other signaling molecules. Glia-secreted proteins play crucial roles in modulating neuronal function in physiological and pathological conditions. Aberrant activation of glial cells leading to neuroinflammation is a common phenomenon observed in various neurological disorders. Aberrantly activated glial cells secrete proteins in disease-specific manner and can be exploited as a repository for novel biomarker discovery.Areas covered: In this review, we describe the recent advances in proteomic techniques, highlighting the need for their application to the secretomic field. Studies regarding the secretome profile of glial cells published within the last 5 years are discussed in detail. The use of glia-based biomarkers in various neuroinflammatory and neurodegenerative diseases is also discussed.Expert opinion: Precise diagnosis and timely treatment of neurological disorders remains a challenge and glia-focused research to identify specific biomarkers appears to be a promising approach to combat these disorders. Recent technological advancement in proteomic research would open new frontiers for more rigorous analysis of glial secretome variations over time and the discovery/development of novel biomarkers for neurological disorders.

Development of 3D-printed myoelectric hand orthosis for patients with spinal cord injury.

BACKGROUND: Spinal cord injury (SCI) is a severe medical condition affecting the hand and locomotor function. New medical technologies, including various wearable devices, as well as rehabilitation treatments are being developed to enhance hand function in patients with SCI. As three-dimensional (3D) printing has the advantage of being able to produce low-cost personalized devices, there is a growing appeal to apply this technology to rehabilitation equipment in conjunction with scientific advances. In this study, we proposed a novel 3D-printed hand orthosis that is controlled by electromyography (EMG) signals. The orthosis was designed to aid the grasping function for patients with cervical SCI. We applied this hand exoskeleton system to individuals with tetraplegia due to SCI and validated its effectiveness. METHODS: The 3D architecture of the device was designed using computer-aided design software and printed with a polylactic acid filament. The dynamic hand orthosis enhanced the tenodesis grip to provide sufficient grasping function. The root mean square of the EMG signal was used as the input for controlling the device. Ten subjects with hand weakness due to chronic cervical SCI were enrolled in this study, and their hand function was assessed before and after wearing the orthosis. The Toronto Rehabilitation Institute Hand Function Test (TRI-HFT) was used as the primary outcome measure. Furthermore, improvements in functional independence in daily living and device usability were evaluated. RESULTS: The newly developed orthosis improved hand function of subjects, as determined using the TRI-HFT (p < 0.05). Furthermore, participants obtained immediate functionality on eating after wearing the orthosis. Moreover, most participants were satisfied with the device as determined by the usability test. There were no side effects associated with the experiment. CONCLUSIONS: The 3D-printed myoelectric hand orthosis was intuitive, easy to use, and showed positive effects in its ability to handle objects encountered in daily life. This study proved that combining simple EMG-based control strategies and 3D printing techniques was feasible and promising in rehabilitation engineering. TRIAL REGISTRATION: Clinical Research Information Service (CRiS), Republic of Korea. KCT0003995. Registered 2 May 2019 - Retrospectively registered.

Astrocytic Orosomucoid-2 Modulates Microglial Activation and Neuroinflammation.

Orosomucoid (ORM) is an acute-phase protein that belongs to the immunocalin subfamily, a group of small-molecule-binding proteins with immunomodulatory functions. Little is known about the role of ORM proteins in the CNS. The aim of the present study was to investigate the brain expression of ORM and its role in neuroinflammation. Expression of Orm2, but not Orm1 or Orm3, was highly induced in the mouse brain after systemic injection of lipopolysaccharide (LPS). Plasma levels of ORM2 were also significantly higher in patients with cognitive impairment than in normal subjects. RT-PCR, Western blot, and immunofluorescence analyses revealed that astrocytes are the major cellular sources of ORM2 in the inflamed mouse brain. Recombinant ORM2 protein treatment decreased microglial production of proinflammatory mediators and reduced microglia-mediated neurotoxicity in vitro LPS-induced microglial activation, proinflammatory cytokines in hippocampus, and neuroinflammation-associated cognitive deficits also decreased as a result of intracerebroventricular injection of recombinant ORM2 protein in vivo Moreover, lentiviral shRNA-mediated Orm2 knockdown enhanced LPS-induced proinflammatory cytokine gene expression and microglial activation in the hippocampus. Mechanistically, ORM2 inhibited C-C chemokine ligand 4 (CCL4)-induced microglial migration and activation by blocking the interaction of CCL4 with C-C chemokine receptor type 5. Together, the results from our cultured glial cells, mouse neuroinflammation model, and patient studies suggest that ORM2 is a novel mediator of astrocyte-microglial interaction. We also report that ORM2 exerts anti-inflammatory effects by modulating microglial activation and migration during brain inflammation. ORM2 can be exploited therapeutically for the treatment of neuroinflammatory diseases.SIGNIFICANCE STATEMENT Neural cell interactions are important for brain physiology and pathology. Particularly, the interaction between non-neuronal cells plays a central role in regulating brain inflammation, which is closely linked to many brain disorders. Here, we newly identified orosomucoid-2 (ORM2) as an endogenous protein that mediates such non-neuronal glial cell interactions. Based on the critical role of astrocyte-derived ORM2 in modulating microglia-mediated neuroinflammation, ORM2 can be exploited for the diagnosis, prevention, or treatment of devastating brain disorders that have a strong neuroinflammatory component, such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and multiple sclerosis.

Pathogenic Upregulation of Glial Lipocalin-2 in the Parkinsonian Dopaminergic System.

UNLABELLED: Lipocalin-2 (LCN2) is a member of the highly heterogeneous secretory protein family of lipocalins and increases in its levels can contribute to neurodegeneration in the adult brain. However, there are no reports on the role of LCN2 in Parkinson's disease (PD). Here, we report for the first time that LCN2 expression is increased in the substantia nigra (SN) of patients with PD. In mouse brains, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment for a neurotoxin model of PD significantly upregulated LCN2 expression, mainly in reactive astrocytes in both the SN and striatum. The increased LCN2 levels contributed to neurotoxicity and neuroinflammation, resulting in disruption of the nigrostriatal dopaminergic (DA) projection and abnormal locomotor behaviors, which were ameliorated in LCN2-deficient mice. Similar to the effects of MPTP treatment, LCN2-induced neurotoxicity was also observed in the 6-hydroxydopamine (6-OHDA)-treated animal model of PD. Moreover, treatment with the iron donor ferric citrate (FC) and the iron chelator deferoxamine mesylate (DFO) increased and decreased, respectively, the LCN2-induced neurotoxicity in vivo In addition to the in vivo results, 1-methyl-4-phenylpyridinium (MPP(+))-induced neurotoxicity in cocultures of mesencephalic neurons and astrocytes was reduced by LCN2 gene deficiency in the astrocytes and conditioned media derived from MPP(+)-treated SH-SY5Y neuronal enhanced glial expression of LCN2 in vitro Therefore, our results demonstrate that astrocytic LCN2 upregulation in the lesioned DA system may play a role as a potential pathogenic factor in PD and suggest that inhibition of LCN2 expression or activity may be useful in protecting the nigrostriatal DA system in the adult brain. SIGNIFICANCE STATEMENT: Lipocalin-2 (LCN2), a member of the highly heterogeneous secretory protein family of lipocalins, may contribute to neuroinflammation and neurotoxicity in the brain. However, LCN2 expression and its role in Parkinson's disease (PD) are largely unknown. Here, we report that LCN2 is upregulated in the substantia nigra of patients with PD and neurotoxin-treated animal models of PD. Our results suggest that LCN2 upregulation might be a potential pathogenic mechanism of PD, which would result in disruption of the nigrostriatal dopaminergic system through neurotoxic iron accumulation and neuroinflammation. Therefore, inhibition of LCN2 expression or activity may be useful in protecting the nigrostriatal dopaminergic projection in PD.

Pyruvate Dehydrogenase Kinase-mediated Glycolytic Metabolic Shift in the Dorsal Root Ganglion Drives Painful Diabetic Neuropathy.

The dorsal root ganglion (DRG) is a highly vulnerable site in diabetic neuropathy. Under diabetic conditions, the DRG is subjected to tissue ischemia or lower ambient oxygen tension that leads to aberrant metabolic functions. Metabolic dysfunctions have been documented to play a crucial role in the pathogenesis of diverse pain hypersensitivities. However, the contribution of diabetes-induced metabolic dysfunctions in the DRG to the pathogenesis of painful diabetic neuropathy remains ill-explored. In this study, we report that pyruvate dehydrogenase kinases (PDK2 and PDK4), key regulatory enzymes in glucose metabolism, mediate glycolytic metabolic shift in the DRG leading to painful diabetic neuropathy. Streptozotocin-induced diabetes substantially enhanced the expression and activity of the PDKs in the DRG, and the genetic ablation of Pdk2 and Pdk4 attenuated the hyperglycemia-induced pain hypersensitivity. Mechanistically, Pdk2/4 deficiency inhibited the diabetes-induced lactate surge, expression of pain-related ion channels, activation of satellite glial cells, and infiltration of macrophages in the DRG, in addition to reducing central sensitization and neuroinflammation hallmarks in the spinal cord, which probably accounts for the attenuated pain hypersensitivity. Pdk2/4-deficient mice were partly resistant to the diabetes-induced loss of peripheral nerve structure and function. Furthermore, in the experiments using DRG neuron cultures, lactic acid treatment enhanced the expression of the ion channels and compromised cell viability. Finally, the pharmacological inhibition of DRG PDKs or lactic acid production substantially attenuated diabetes-induced pain hypersensitivity. Taken together, PDK2/4 induction and the subsequent lactate surge induce the metabolic shift in the diabetic DRG, thereby contributing to the pathogenesis of painful diabetic neuropathy.

Astrocyte-derived lipocalin-2 mediates hippocampal damage and cognitive deficits in experimental models of vascular dementia.

Lipocalin-2 (LCN2) has diverse functions in multiple pathophysiological conditions; however, its pathogenic role in vascular dementia (VaD) is unknown. Here, we investigated the role of LCN2 in VaD using rodent models of global cerebral ischemia and hypoperfusion with cognitive impairment and neuroinflammation. Mice subjected to transient bilateral common carotid artery occlusion (tBCCAo) for 50 min showed neuronal death and gliosis in the hippocampus at 7 days post-tBCCAo. LCN2 expression was observed predominantly in the hippocampal astrocytes, whereas its receptor was mainly detected in neurons, microglia, and astrocytes. Furthermore, Lcn2-deficient mice, compared with wild-type animals, showed significantly weaker CA1 neuronal loss, cognitive decline, white matter damage, blood-brain barrier permeability, glial activation, and proinflammatory cytokine production in the hippocampus after tBCCAo. Lcn2 deficiency also attenuated hippocampal neuronal death and cognitive decline at 30 days after unilateral common carotid artery occlusion (UCCAo). Furthermore, intracerebroventricular (i.c.v) injection of recombinant LCN2 protein elicited CA1-neuronal death and a cognitive deficit. Our studies using cultured glia and hippocampal neurons supported the decisive role of LCN2 in hippocampal neurotoxicity and microglial activation, and the role of the HIF-1α-LCN2-VEGFA axis of astrocytes in vascular injury. Additionally, plasma levels of LCN2 were significantly higher in patients with VaD than in the healthy control subjects. These results indicate that hippocampal damage and cognitive impairment are mediated by LCN2 secreted from reactive astrocytes in VaD.

The novel resveratrol derivative 3,5-diethoxy-3',4'-dihydroxy-trans-stilbene induces mitochondrial ROS-mediated ER stress and cell death in human hepatoma cells in vitro.

Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a well-known polyphenol that is present in grapes, peanuts, pine seeds, and several other plants. Resveratrol exerts deleterious effects on various types of human cancer cells. Here, we analyzed the cell death-inducing mechanisms of resveratrol-006 (Res-006), a novel resveratrol derivative in human liver cancer cells in vitro. Res-006 was more effectively suppressed the viability of HepG2 human hepatoma cells than resveratrol (the IC50 values were 67.2 and 354.8 µmol/L, respectively). Co-treatment with the ER stress regulator 4-phenylbutyrate (0.5 mmol/L) or the ROS inhibitor N-acetyl-L-cysteine (NAC, 1 mmol/L) significantly attenuated Res-006-induced HepG2 cell death, suggesting that pro-apoptotic ER stress and/or ROS may govern the Res-006-induced HepG2 cell death. We further revealed that treatment of HepG2 cells with Res-006 (65 µmol/L) immediately elicited the dysregulation of mitochondrial dynamics and the accumulation of mitochondrial ROS. It also collapsed the mitochondrial membrane potential and further induced ER stress and cell death. These events, except for the change in mitochondrial morphology, were prevented by the exposure of the HepG2 cells to the mitochondrial ROS scavenger, Mito-TEMPO (300-1000 µmol/L). The results suggest that Res-006 may kill HepG2 cells through cell death pathways, including the ER stress initiated by mitochondrial ROS accumulation. The cell death induced by this novel resveratrol derivative involves crosstalk between the mitochondria and ER stress mechanisms.

A novel role for protein tyrosine phosphatase 1B as a positive regulator of neuroinflammation.

BACKGROUND: Protein tyrosine phosphatase 1B (PTP1B) is a member of the non-transmembrane phosphotyrosine phosphatase family. Recently, PTP1B has been proposed to be a novel target of anti-cancer and anti-diabetic drugs. However, the role of PTP1B in the central nervous system is not clearly understood. Therefore, in this study, we sought to define PTP1B's role in brain inflammation. METHODS: PTP1B messenger RNA (mRNA) and protein expression levels were examined in mouse brain and microglial cells after LPS treatment using RT-PCR and western blotting. Pharmacological inhibitors of PTP1B, NF-κB, and Src kinase were used to analyze these signal transduction pathways in microglia. A Griess reaction protocol was used to determine nitric oxide (NO) concentrations in primary microglia cultures and microglial cell lines. Proinflammatory cytokine production was measured by RT-PCR. Western blotting was used to assess Src phosphorylation levels. Immunostaining for Iba-1 was used to determine microglial activation in the mouse brain. RESULTS: PTP1B expression levels were significantly increased in the brain 24 h after LPS injection, suggesting a functional role for PTP1B in brain inflammation. Microglial cells overexpressing PTP1B exhibited an enhanced production of NO and gene expression levels of TNF-α, iNOS, and IL-6 following LPS exposure, suggesting that PTP1B potentiates the microglial proinflammatory response. To confirm the role of PTP1B in neuroinflammation, we employed a highly potent and selective inhibitor of PTP1B (PTP1Bi). In LPS- or TNF-α-stimulated microglial cells, in vitro blockade of PTP1B activity using PTP1Bi markedly attenuated NO production. PTP1Bi also suppressed the expression levels of iNOS, COX-2, TNF-α, and IL-1ß. PTP1B activated Src by dephosphorylating the Src protein at a negative regulatory site. PTP1B-mediated Src activation led to an enhanced proinflammatory response in the microglial cells. An intracerebroventricular injection of PTP1Bi significantly attenuated microglial activation in the hippocampus and cortex of LPS-injected mice compared to vehicle-injected mice. The gene expression levels of proinflammatory cytokines were also significantly suppressed in the brain by a PTP1Bi injection. Together, these data suggest that PTP1Bi has an anti-inflammatory effect in a mouse model of neuroinflammation. CONCLUSIONS: This study demonstrates that PTP1B is an important positive regulator of neuroinflammation and is a promising therapeutic target for neuroinflammatory and neurodegenerative diseases.

Lipocalin-2 protein deficiency ameliorates experimental autoimmune encephalomyelitis: the pathogenic role of lipocalin-2 in the central nervous system and peripheral lymphoid tissues.

Lipocalin-2 (LCN2) plays an important role in cellular processes as diverse as cell growth, migration/invasion, differentiation, and death/survival. Furthermore, recent studies indicate that LCN2 expression and secretion by glial cells are induced by inflammatory stimuli in the central nervous system. The present study was undertaken to examine the regulation of LCN2 expression in experimental autoimmune encephalomyelitis (EAE) and to determine the role of LCN2 in the disease process. LCN2 expression was found to be strongly increased in spinal cord and secondary lymphoid tissues after EAE induction. In spinal cords astrocytes and microglia were the major cell types expressing LCN2 and its receptor 24p3R, respectively, whereas in spleens, LCN2 and 24p3R were highly expressed in neutrophils and dendritic cells, respectively. Furthermore, disease severity, inflammatory infiltration, demyelination, glial activation, the expression of inflammatory mediators, and the proliferation of MOG-specific T cells were significantly attenuated in Lcn2-deficient mice as compared with wild-type animals. Myelin oligodendrocyte glycoprotein-specific T cells in culture exhibited an increased expression of Il17a, Ifng, Rorc, and Tbet after treatment with recombinant LCN2 protein. Moreover, LCN2-treated glial cells expressed higher levels of proinflammatory cytokines, chemokines, and MMP-9. Adoptive transfer and recombinant LCN2 protein injection experiments suggested that LCN2 expression in spinal cord and peripheral immune organs contributes to EAE development. Taken together, these results imply LCN2 is a critical mediator of autoimmune inflammation and disease development in EAE and suggest that LCN2 be regarded a potential therapeutic target in multiple sclerosis.

Phenotypic polarization of activated astrocytes: the critical role of lipocalin-2 in the classical inflammatory activation of astrocytes.

Astrocytes provide structural and functional support for neurons, as well as display neurotoxic or neuroprotective phenotypes depending upon the presence of an immune or inflammatory microenvironment. This study was undertaken to characterize multiple phenotypes of activated astrocytes and to investigate the regulatory mechanisms involved. We report that activated astrocytes in culture exhibit two functional phenotypes with respect to pro- or anti-inflammatory gene expression, glial fibrillary acidic protein expression, and neurotoxic or neuroprotective activities. The two distinct functional phenotypes of astrocytes were also demonstrated in a mouse neuroinflammation model, which showed pro- or anti-inflammatory gene expression in astrocytes following challenge with classical or alternative activation stimuli; similar results were obtained in the absence of microglia. Subsequent studies involving recombinant lipocalin-2 (LCN2) protein treatment or Lcn2-deficient mice indicated that the pro- or anti-inflammatory functionally polarized phenotypes of astrocytes and their intracellular signaling pathway were critically regulated by LCN2 under in vitro and in vivo conditions. Astrocyte-derived LCN2 promoted classical proinflammatory activation of astrocytes but inhibited IL-4-STAT6 signaling, a canonical pathway involved in alternative anti-inflammatory activation. Our results suggest that the secreted protein LCN2 is an autocrine modulator of the functional polarization of astrocytes in the presence of immune or inflammatory stimuli and that LCN2 could be targeted therapeutically to dampen proinflammatory astrocytic activation and related pathologies in the CNS.

Sensor-based auto-focusing system using multi-scale feature extraction and phase correlation matching.

This paper presents a novel auto-focusing system based on a CMOS sensor containing pixels with different phases. Robust extraction of features in a severely defocused image is the fundamental problem of a phase-difference auto-focusing system. In order to solve this problem, a multi-resolution feature extraction algorithm is proposed. Given the extracted features, the proposed auto-focusing system can provide the ideal focusing position using phase correlation matching. The proposed auto-focusing (AF) algorithm consists of four steps: (i) acquisition of left and right images using AF points in the region-of-interest; (ii) feature extraction in the left image under low illumination and out-of-focus blur; (iii) the generation of two feature images using the phase difference between the left and right images; and (iv) estimation of the phase shifting vector using phase correlation matching. Since the proposed system accurately estimates the phase difference in the out-of-focus blurred image under low illumination, it can provide faster, more robust auto focusing than existing systems.

Chronic sleep deprivation-induced proteome changes in astrocytes of the rat hypothalamus.

Sleep deprivation (SD) can influence cognition, memory, and sleep/wake homeostasis and can cause impairments in many physiological processes. Because the homeostatic control of the sleep/wake cycle is closely associated with the hypothalamus, the current study was undertaken to examine proteomic changes occurring in hypothalamic astrocytes following chronic partial SD. After chronic partial SD for 7 days, astrocytes were prepared from rat hypothalamus using a Percoll gradient method, and their proteome profiles were determined by LC-MS/MS. Comparisons of the proteome profiles of hypothalamic astrocytes revealed that chronic partial SD increased (≥1.5-fold) 89 proteins and decreased (≤0.7-fold) 50 proteins; these changes in protein expression were validated by western blot or immunohistochemistry. DAVID and IPA analyses of these proteins suggested that SD may influence gliotransmission and astrocyte activation. PPP2R1A, RTN4, VAMP-2, LGI-1, and SLC17A7 were identified and validated as the main targets of SD in astrocytes. Our results suggest that SD may modulate gliotransmission in the hypothalamus, thereby disturbing sleep/wake homeostasis and increasing susceptibility to neurological disease; however, further studies are required to confirm whether the proteome changes are specific to SD.

The secretome signature of reactive glial cells and its pathological implications.

Glial cells are non-neuronal components of the central nervous system (CNS). They are endowed with diverse functions and are provided with tools to detect their own activities and those of neighboring neurons. Glia and neurons are in continuous reciprocal communication under both physiological and neuropathological conditions, and glia secrete various guidance factors or proteinaceous signals that service vital neuronal-glial interactions in health and disease. Analysis and profiling of glial secretome, especially of microglia and astrocytes, have raised new expectations for the diagnosis and treatment of CNS disorders, and the availability of a catalog of glia-secreted proteins might provide an origin for further research on the complex extracellular signaling mediated by glial cells. Components of the glial secretome play important roles as mediators and modulators of brain structure and function during neuroprotection and neurodegeneration. Therapeutic hypothermia has been acclaimed an effective modulator of brain injury via its substantial effect on the protein expression profiles of glia. Furthermore, emerging proteomic tools and methodologies make feasible the documentation of the reactive glial secretome signature. This review focuses on reactive glial cells and the uniqueness of their secretome during diverse neuropathological conditions. This article is part of a Special Issue entitled: An Updated Secretome.

Secreted protein lipocalin-2 promotes microglial M1 polarization.

Activated macrophages are classified into two different forms: classically activated (M1) or alternatively activated (M2) macrophages. The presence of M1/M2 phenotypic polarization has also been suggested for microglia. Here, we report that the secreted protein lipocalin 2 (LCN2) amplifies M1 polarization of activated microglia. LCN2 protein (EC 1 µg/ml), but not glutathione S-transferase used as a control, increased the M1-related gene expression in cultured mouse microglial cells after 8-24 h. LCN2 was secreted from M1-polarized, but not M2-polarized, microglia. LCN2 inhibited phosphorylation of STAT6 in IL-4-stimulated microglia, suggesting LCN2 suppression of the canonical M2 signaling. In the lipopolysaccharide (LPS)-induced mouse neuroinflammation model, the expression of LCN2 was notably increased in microglia. Primary microglial cultures derived from LCN2-deficient mice showed a suppressed M1 response and enhanced M2 response. Mice lacking LCN2 showed a markedly reduced M1-related gene expression in microglia after LPS injection, which was consistent with the results of histological analysis. Neuroinflammation-associated impairment in motor behavior and cognitive function was also attenuated in the LCN2-deficient mice, as determined by the rotarod performance test, fatigue test, open-field test, and object recognition task. These findings suggest that LCN2 is an M1-amplifier in brain microglial cells.