Neonatal Fc Receptor Inhibition Enables Adeno-Associated Virus Gene Therapy Despite Pre-Existing Humoral Immunity.

Advances in adeno-associated virus (AAV)-based gene therapy are transforming our ability to treat rare genetic disorders and address other unmet medical needs. However, the natural prevalence of anti-AAV neutralizing antibodies (NAbs) in humans currently limits the population who can benefit from AAV-based gene therapies. Neonatal Fc receptor (FcRn) plays an essential role in the long half-life of IgG, a key NAb. Researchers have developed several FcRn-inhibiting monoclonal antibodies to treat autoimmune diseases, as inhibiting the interaction between FcRn and IgG Fc can reduce circulating IgG levels to 20-30% of the baseline. We evaluated the utility of one such monoclonal antibody, M281, to reduce pre-existing NAb levels and to permit gene delivery to the liver and heart via systemic AAV gene therapy in mice and nonhuman primates. M281 successfully reduced NAb titers along with total IgG levels; it also enhanced gene delivery to the liver and other organs after intravenous administration of AAV in NAb-positive animals. These results indicate that mitigating pre-existing humoral immunity via disruption of the FcRn-IgG interaction may make AAV-based gene therapies effective in NAb-positive patients.

Selective Elimination of NRF2-Activated Cells by Competition With Neighboring Cells in the Esophageal Epithelium.

BACKGROUND & AIMS: NF-E2-related factor 2 (NRF2) is a transcription factor that regulates cytoprotective gene expression in response to oxidative and electrophilic stresses. NRF2 activity is mainly controlled by Kelch-like ECH-associated protein 1 (KEAP1). Constitutive NRF2 activation by NRF2 mutations or KEAP1 dysfunction results in a poor prognosis for esophageal squamous cell carcinoma (ESCC) through the activation of cytoprotective functions. However, the detailed contributions of NRF2 to ESCC initiation or promotion have not been clarified. Here, we investigated the fate of NRF2-activated cells in the esophageal epithelium. METHODS: We generated tamoxifen-inducible, squamous epithelium-specific Keap1 conditional knockout (Keap1-cKO) mice in which NRF2 was inducibly activated in a subset of cells at the adult stage. Histologic, quantitative reverse-transcription polymerase chain reaction, single-cell RNA-sequencing, and carcinogen experiments were conducted to analyze the Keap1-cKO esophagus. RESULTS: KEAP1-deleted/NRF2-activated cells and cells with normal NRF2 expression (KEAP1-normal cells) coexisted in the Keap1-cKO esophageal epithelium in approximately equal numbers, and NRF2-activated cells formed dysplastic lesions. NRF2-activated cells exhibited weaker attachment to the basement membrane and gradually disappeared from the epithelium. In contrast, neighboring KEAP1-normal cells exhibited accelerated proliferation and started dominating the epithelium but accumulated DNA damage that triggered carcinogenesis upon carcinogen exposure. CONCLUSIONS: Constitutive NRF2 activation promotes the selective elimination of epithelial cells via cell competition, but this competition induces DNA damage in neighboring KEAP1-normal cells, which predisposes them to chemical-induced ESCC.

Intravenous immunoglobulin prevents peripheral liver transduction of intrathecally delivered AAV vectors.

Gene therapy using neurotropic adeno-associated virus vectors represents an emerging solution for genetic disorders affecting the central nervous system. The first approved central nervous system-targeting adeno-associated virus gene therapy, Zolgensma®, for treating spinal muscular atrophy is administered intravenously at high doses that cause liver-associated adverse events in 20%-30% of patients. Intrathecal routes of vector administration, such as the intra-cisterna magna route, provide efficient gene transduction to central nervous system cells while reducing off-target liver transduction. However, significant levels of liver transduction often occur upon intra-cisterna magna vector delivery in preclinical studies. Using vectors expressing monoclonal antibody transgenes, we examined whether passive transfer of adeno-associated virus-neutralizing antibodies as intravenous immunoglobulin before intrathecal adeno-associated virus delivery improved the safety of viral gene therapy targeting the central nervous system in mice and nonhuman primates. We used intracerebroventricular and intra-cisterna magna routes for vector administration to mice and nonhuman primates, respectively, and evaluated transgene expression and vector genome distribution. Our data indicate that pretreatment with intravenous immunoglobulin significantly reduced gene transduction to the liver and other peripheral organs but not to the central nervous system in both species. With further refinement, this method may improve the safety of adeno-associated virus-based, central nervous system-targeting gene therapies in clinical settings.

Erratum: Intravenous immunoglobulin prevents peripheral liver transduction of intrathecally delivered AAV vectors.

[This corrects the article DOI: 10.1016/j.omtm.2022.09.017.].

The ß-TrCP-Mediated Pathway Cooperates with the Keap1-Mediated Pathway in Nrf2 Degradation In Vivo.

Nrf2 activates cytoprotective gene expression, and Nrf2 activity is regulated through at least two protein degradation pathways: the Keap1-mediated and ß-TrCP-mediated pathways. To address the relative contributions of these pathways, we generated knock-in mouse lines expressing an Nrf2SA mutant that harbored two substitution mutations of serine residues interacting with ß-TrCP. The homozygous (Nrf2SA/SA) mice grew normally, with Nrf2 levels comparable to those of wild-type (WT) mice under unstressed conditions. However, when Keap1 activity was suppressed, high levels of Nrf2 accumulated in Nrf2SA/SA macrophages compared with that in WT macrophages. We crossed Nrf2SA/SA mice with mice in which Keap1 was knocked down to two different levels. We found that the Nrf2SA/SA mutation induced higher Nrf2 activity when the Keap1 level was strongly reduced, and these mice showed severe growth retardation. However, activation and growth retardation were not evident when Keap1 was moderately suppressed. These increases in Nrf2 activity induced by the Nrf2SA mutation caused severe hyperplasia and hyperkeratosis in the esophageal epithelium but did not cause abnormalities in the other tissues/organs examined. These results indicate that the ß-TrCP-mediated pathway cooperates with the Keap1-mediated pathway to regulate Nrf2 activity, which is apparent when the Keap1-mediated pathway is profoundly suppressed.

Thoracoscopic esophagectomy for esophageal carcinoma after peroral endoscopic myotomy for esophageal achalasia: a case report.

BACKGROUND: Esophageal achalasia causes dysphagia following impaired relaxation of the lower esophageal sphincter due to the degeneration of Auerbach's plexus in the esophageal smooth muscle. Recently, peroral endoscopic myotomy (POEM) has become one of the preferred treatment options for esophageal achalasia. However, pathomorphological changes after POEM have not been well examined. CASE PRESENTATION: A 65-year-old man with a history of POEM for esophageal achalasia was diagnosed with clinical stage II (cT2-N0-M0) thoracic esophageal squamous cell carcinoma and was consequently treated with neoadjuvant chemotherapy followed by thoracoscopic esophagectomy. Intraoperatively, the esophagus appeared dilated, reflecting esophageal achalasia; however, fairly slight fibrous adhesions were observed between the esophagus and the pericardial surface despite previously performed POEM via an anterior incision. Histopathological examination revealed esophageal wall thickening, edema, and fibrosis extending from the lamina propria to the submucosa. Besides, the majority of the inner layer and some proportion of the outer layer of the muscularis propria were found to be missing or atrophic at the esophagogastric junction (EGJ). No ganglion cells could be detected at the Auerbach's plexus. CONCLUSIONS: The previous history of POEM did not affect circumferential mediastinal periesophageal dissection during thoracoscopic esophagectomy. Nevertheless, a large proportion of the inner layer of the muscularis propria at the EGJ level seemed to have become lost or atrophic because of the POEM procedure.

Intranasal gene therapy to prevent infection by SARS-CoV-2 variants.

SARS-CoV-2 variants have emerged with enhanced pathogenicity and transmissibility, and escape from pre-existing immunity, suggesting first-generation vaccines and monoclonal antibodies may now be less effective. Here we present an approach for preventing clinical sequelae and the spread of SARS-CoV-2 variants. First, we affinity matured an angiotensin-converting enzyme 2 (ACE2) decoy protein, achieving 1000-fold binding improvements that extend across a wide range of SARS-CoV-2 variants and distantly related, ACE2-dependent coronaviruses. Next, we demonstrated the expression of this decoy in proximal airway when delivered via intranasal administration of an AAV vector. This intervention significantly diminished clinical and pathologic consequences of SARS-CoV-2 challenge in a mouse model and achieved therapeutic levels of decoy expression at the surface of proximal airways when delivered intranasally to nonhuman primates. Importantly, this long-lasting, passive protection approach is applicable in vulnerable populations such as the elderly and immune-compromised that do not respond well to traditional vaccination. This approach could be useful in combating COVID-19 surges caused by SARS-CoV-2 variants and should be considered as a countermeasure to future pandemics caused by one of the many pre-emergent, ACE2-dependent CoVs that are poised for zoonosis.

Cellular Nrf2 Levels Determine Cell Fate during Chemical Carcinogenesis in Esophageal Epithelium.

Nrf2 is essential for cytoprotection against carcinogens, and through systemic Nrf2 knockout mice, Nrf2-deficient cells were shown to be susceptible to chemical carcinogens and prone to developing cancers. However, the oncogenic potential of Nrf2-deficient epithelial cells surrounded by normal cells in the esophagus could not be assessed by previous models, and the fate of Nrf2-deficient cells in such situations remains elusive. In this study, therefore, we generated mice that harbor almost equal levels of cells with Nrf2 deleted and those with Nrf2 intact in the basal layer of the esophageal epithelium, utilizing inducible Cre-mediated recombination of Nrf2 alleles in adults through moderate use of tamoxifen. In this mouse model, epithelial cells with Nrf2 deleted were maintained with no obvious decrease or phenotypic changes for 12 weeks under unstressed conditions. Upon exposure to the carcinogen 4-nitroquinoline-1-oxide (4NQO), the cells with Nrf2 deleted accumulated DNA damage and selectively disappeared from the epithelium, so almost all 4NQO-induced tumors originated from cells with Nrf2 intact and not from those with Nrf2 deleted. We propose that cells with Nrf2 deleted do not undergo carcinogenesis due to selective elimination upon exposure to 4NQO, indicating that cellular Nrf2 abundance and the epithelial environment determine the cell fate or oncogenic potential of esophageal epithelial cells in 4NQO-induced carcinogenesis.

Adeno-Associated Virus-Induced Dorsal Root Ganglion Pathology.

The administration of adeno-associated virus (AAV) vectors to nonhuman primates (NHP) via the blood or cerebrospinal fluid (CSF) can lead to dorsal root ganglion (DRG) pathology. The pathology is minimal to moderate in most cases; clinically silent in affected animals; and characterized by mononuclear cell infiltrates, neuronal degeneration, and secondary axonopathy of central and peripheral axons on histopathological analysis. We aggregated data from 33 nonclinical studies in 256 NHP and performed a meta-analysis of the severity of DRG pathology to compare different routes of administration, dose, time course, study conduct, age of the animals, sex, capsid, promoter, capsid purification method, and transgene. DRG pathology was observed in 83% of NHP that were administered AAV through the CSF, and 32% of NHP that received an intravenous (IV) injection. We show that dose and age at injection significantly affected the severity whereas sex had no impact. DRG pathology was minimal at acute time points (i.e., <14 days), similar from one to 5 months post-injection, and was less severe after 6 months. Vector purification method had no impact, and all capsids and promoters that we tested resulted in some DRG pathology. The data presented here from five different capsids, five different promoters, and 20 different transgenes suggest that DRG pathology is almost universal after AAV gene therapy in nonclinical studies using NHP. None of the animals receiving a therapeutic transgene displayed any clinical signs. Incorporation of sensitive techniques such as nerve-conduction velocity testing can show alterations in a minority of animals that correlate with the severity of peripheral nerve axonopathy. Monitoring sensory neuropathies in human central nervous system and high-dose IV clinical studies seems prudent to determine the functional consequences of DRG pathology.

Promoting Axon Regeneration in Adult CNS by Targeting Liver Kinase B1.

Liver kinase B1 (LKB1), a downstream effector of cyclic AMP (cAMP)/PKA and phosphatidylinositol 3-kinase (PI3K) pathways, is a determinant for migration and differentiation of many cells, but its role in CNS axon regeneration is unknown. Therefore, LKB1 was overexpressed in sensorimotor cortex of adult mice five days after mid-thoracic spinal cord injury, using an AAV2 vector. Regeneration of corticospinal axons was dramatically enhanced. Next, systemic injection of a mutant-AAV9 vector was used to upregulate LKB1 specifically in neurons. This promoted long-distance regeneration of injured corticospinal fibers into caudal spinal cord in adult mice and regrowth of descending serotonergic and tyrosine hydroxylase immunoreactive axons. Either intracortical or systemic viral delivery of LKB1 significantly improved recovery of locomotor functions in adult mice with spinal cord injury. Moreover, we demonstrated that LKB1 used AMPKα, NUAK1, and ERK as the downstream effectors in the cortex of adult mice. Thus, LKB1 may be a critical factor for enhancing the growth capacity of mature neurons and may be an important molecular target in the treatment of CNS injuries.

Protein tyrosine phosphatase σ regulates autoimmune encephalomyelitis development.

Protein tyrosine phosphatases (PTPs) play essential roles in regulating signaling events in multiple cells by tyrosine dephosphorylation. One of them, PTPσ, appears important in regulating function of plasmacytoid dendritic cells (pDC). Here we report that PTPσ deletion in knockout mice and inhibition with a selective antagonist peptide exacerbated symptoms of experimental autoimmune encephalomyelitis (EAE) by enhancing axon and myelin damage in the spinal cord. PTPσ-/- mice displayed pro-inflammatory profiles in the spinal cord and lymphoid organs following MOG peptide immunization. PTPσ deletion promoted a pro-inflammatory phenotype in conventional DCs and directly regulated differentiation of CD4+ T cells. It also facilitated infiltration of T lymphocytes, activation of macrophages in the CNS and development of EAE. Therefore, PTPσ is a key negative regulator in EAE initiation and progression, which acts by regulating functions of DCs, T cells, and other immune cells. PTPσ may become an important molecular target for treating autoimmune disorders.

Differing intrinsic biological properties between forebrain and spinal oligodendroglial lineage cells.

Differentiation of oligodendroglial progenitor cells (OPCs) into myelinating oligodendrocytes is known to be regulated by the microenvironment where they differentiate. However, current research has not verified whether or not oligodendroglial lineage cells (OLCs) derived from different anatomical regions of the central nervous system (CNS) respond to microenvironmental cues in the same manner. Here, we isolated pure OPCs from rat neonatal forebrain (FB) and spinal cord (SC) and compared their phenotypes in the same in vitro conditions. We found that although FB and SC OLCs responded differently to the same external factors; they were distinct in proliferation response to mitogens, oligodendrocyte phenotype after differentiation, and cytotoxic responses to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-type glutamate receptor-mediated excitotoxicity at immature stages of differentiation in a cell-intrinsic manner. Moreover, transcriptome analysis identified genes differentially expressed between these OPC populations, including those encoding transcription factors (TFs), cell surface molecules, and signaling molecules. Particularly, FB and SC OPCs retained the expression of FB- or SC-specific TFs, such as Foxg1 and Hoxc8, respectively, even after serial passaging in vitro. Given the essential role of these TFs in the regional identities of CNS cells along the rostrocaudal axis, our results suggest that CNS region-specific gene regulation by these TFs may cause cell-intrinsic differences in cellular responses between FB and SC OLCs to extracellular molecules. Further understanding of the regional differences among OPC populations will help to improve treatments for demyelination in different CNS regions and to facilitate the development of stem cell-derived OPCs for cell transplantation therapies for demyelination. Cover Image for this issue: doi. 10.1111/jnc.13809.

CX3CR1 ablation ameliorates motor and respiratory dysfunctions and improves survival of a Rett syndrome mouse model.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by loss-of-function mutations in the gene encoding MeCP2, an epigenetic modulator that binds the methyl CpG dinucleotide in target genes to regulate transcription. Previously we and others reported a role of microglia in the pathophysiology of RTT. Because microglia in the Mecp2 knockout (Mecp2KO) mouse model of RTT over-produce neurotoxic mediators glutamate and reactive oxygen species, we hypothesize that blocking neuron-microglia interaction by ablation of CX3CR1, a chemokine receptor expressed in microglia/myeloid cells mediating such interaction by pairing with its neuronal ligand CX3CL1, would ameliorate the RTT-like phenotype in Mecp2KO mice. Here we report that CX3CR1 ablation prolonged the lifespan of Mecp2KO mice from a median survival of 54.5-74days, and significantly improved the body weight gain, symptomatic scores, major respiratory parameters, and motor coordination and performance. CX3CR1 ablation rectified previously identified histological abnormalities in the Mecp2KO brain such as neuronal soma size in hippocampal CA2, and the number, soma size, and process complexity of microglia. Moreover, CX3CR1 ablation enhanced the neurotrophic action of microglia in Mecp2KO mice by producing higher amount of insulin-like growth factor 1. Our data support a role of myeloid cells/microglia in RTT and suggest a novel therapeutic approach for RTT by targeting CX3CR1 with specific antagonists or genetic downregulation.

Differential Kv1.3, KCa3.1, and Kir2.1 expression in "classically" and "alternatively" activated microglia.

Microglia are highly plastic cells that can assume different phenotypes in response to microenvironmental signals. Lipopolysaccharide (LPS) and interferon-γ (IFN-γ) promote differentiation into classically activated M1-like microglia, which produce high levels of pro-inflammatory cytokines and nitric oxide and are thought to contribute to neurological damage in ischemic stroke and Alzheimer's disease. IL-4 in contrast induces a phenotype associated with anti-inflammatory effects and tissue repair. We here investigated whether these microglia subsets vary in their K+ channel expression by differentiating neonatal mouse microglia into M(LPS) and M(IL-4) microglia and studying their K+ channel expression by whole-cell patch-clamp, quantitative PCR and immunohistochemistry. We identified three major types of K+ channels based on their biophysical and pharmacological fingerprints: a use-dependent, outwardly rectifying current sensitive to the KV 1.3 blockers PAP-1 and ShK-186, an inwardly rectifying Ba2+ -sensitive Kir 2.1 current, and a Ca2+ -activated, TRAM-34-sensitive KCa 3.1 current. Both KV 1.3 and KCa 3.1 blockers inhibited pro-inflammatory cytokine production and iNOS and COX2 expression demonstrating that KV 1.3 and KCa 3.1 play important roles in microglia activation. Following differentiation with LPS or a combination of LPS and IFN-γ microglia exhibited high KV 1.3 current densities (∼50 pA/pF at 40 mV) and virtually no KCa 3.1 and Kir currents, while microglia differentiated with IL-4 exhibited large Kir 2.1 currents (∼ 10 pA/pF at -120 mV). KCa 3.1 currents were generally low but moderately increased following stimulation with IFN-γ or ATP (∼10 pS/pF). This differential K+ channel expression pattern suggests that KV 1.3 and KCa 3.1 inhibitors could be used to inhibit detrimental neuroinflammatory microglia functions. GLIA 2016;65:106-121.

The Wnt effector transcription factor 7-like 2 positively regulates oligodendrocyte differentiation in a manner independent of Wnt/ß-catenin signaling.

Genetic or pharmacological activation of canonical Wnt/ß-catenin signaling inhibits oligodendrocyte differentiation. Transcription factor 7-like 2 (TCF7l2), also known as TCF4, is a Wnt effector induced transiently in the oligodendroglial lineage. A well accepted dogma is that TCF7l2 inhibits oligodendrocyte differentiation through activation of Wnt/ß-catenin signaling. We report that TCF7l2 is upregulated transiently in postmitotic, newly differentiated oligodendrocytes. Using in vivo gene conditional ablation, we found surprisingly that TCF7l2 positively regulates neonatal and postnatal mouse oligodendrocyte differentiation during developmental myelination and remyelination in a manner independent of the Wnt/ß-catenin signaling pathway. We also reveal a novel role of TCF7l2 in repressing a bone morphogenetic protein signaling pathway that is known to inhibit oligodendrocyte differentiation. Thus, our study provides novel data justifying therapeutic attempts to enhance, rather than inhibit, TCF7l2 signaling to overcome arrested oligodendroglial differentiation in multiple sclerosis and other demyelinating diseases.

Dysregulation of glutamine transporter SNAT1 in Rett syndrome microglia: a mechanism for mitochondrial dysfunction and neurotoxicity.

Rett syndrome (RTT) is an autism spectrum disorder caused by loss-of-function mutations in the gene encoding MeCP2, an epigenetic modulator that binds the methyl CpG dinucleotide in target genes to regulate transcription. Previously, we and others reported a role of microglia in the pathophysiology of RTT. To understand the mechanism of microglia dysfunction in RTT, we identified a MeCP2 target gene, SLC38A1, which encodes a major glutamine transporter (SNAT1), and characterized its role in microglia. We found that MeCP2 acts as a microglia-specific transcriptional repressor of SNAT1. Because glutamine is mainly metabolized in the mitochondria, where it is used as an energy substrate and a precursor for glutamate production, we hypothesize that SNAT1 overexpression in MeCP2-deficient microglia would impair the glutamine homeostasis, resulting in mitochondrial dysfunction as well as microglial neurotoxicity because of glutamate overproduction. Supporting this hypothesis, we found that MeCP2 downregulation or SNAT1 overexpression in microglia resulted in (1) glutamine-dependent decrease in microglial viability, which was corroborated by reduced microglia counts in the brains of MECP2 knock-out mice; (2) proliferation of mitochondria and enhanced mitochondrial production of reactive oxygen species; (3) increased oxygen consumption but decreased ATP production (an energy-wasting state); and (4) overproduction of glutamate that caused NMDA receptor-dependent neurotoxicity. The abnormalities could be rectified by mitochondria-targeted expression of catalase and a mitochondria-targeted peptide antioxidant, Szeto-Schiller 31. Our results reveal a novel mechanism via which MeCP2 regulates bioenergetic pathways in microglia and suggest a therapeutic potential of mitochondria-targeted antioxidants for RTT.

ZPK/DLK and MKK4 form the critical gateway to axotomy-induced motoneuron death in neonates.

Motoneuron death after transection of the axons (axotomy) in neonates is believed to share the same mechanistic bases as naturally occurring programmed cell death during development. The c-Jun N-terminal kinase pathway is activated in both forms of motoneuron death, but it remains unknown to what extent these two forms of motoneuron death depend on this pathway and which upstream kinases are involved. We found that numbers of facial motoneurons are doubled in neonatal mice deficient in either ZPK/DLK (zipper protein kinase, also known as dual leucine zipper kinase), a mitogen-activated protein kinase kinase kinase, or in MKK4/MAP2K4, a mitogen-activated protein kinase kinase directly downstream of ZPK/DLK, and that the facial motoneurons in those mutant mice are completely resistant to axotomy-induced death. Conditional deletion of MKK4/MAP2K4 in neurons further suggested that ZPK/DLK and MKK4/MAP2K4-dependent mechanisms underlying axotomy-induced death are motoneuron autonomous. Nevertheless, quantitative analysis of facial motoneurons during embryogenesis revealed that both ZPK/DLK and MKK4/MAP2K4-dependent and -independent mechanisms contribute to developmental elimination of excess motoneurons. In contrast to MKK4/MAP2K4, mice lacking MKK7/MAP2K7, another mitogen-activated protein kinase kinase directly downstream of ZPK/DLK, conditionally in neurons did not have excess facial motoneurons. However, some MKK7/MAP2K7-deficient facial motoneurons were resistant to axotomy-induced death, indicating a synergistic effect of MKK7/MAP2K7 on axotomy-induced death of these facial motoneurons. Together, our study provides compelling evidence for the pivotal roles of the ZPK/DLK and MKK4/MAP2K4-dependent mechanism in axotomy-induced motoneuron death in neonates and also demonstrates that axotomy-induced motoneuron death is not identical to developmental motoneuron death with respect to the involvement of ZPK/DLK, MKK4/MAP2K4 and MKK7/MAP2K7.

Adenomatous polyposis coli regulates oligodendroglial development.

The expression of the gut tumor suppressor gene adenomatous polyposis coli (Apc) and its role in the oligodendroglial lineage are poorly understood. We found that immunoreactive APC is transiently induced in the oligodendroglial lineage during both normal myelination and remyelination following toxin-induced, genetic, or autoimmune demyelination murine models. Using the Cre/loxP system to conditionally ablate APC from the oligodendroglial lineage, we determined that APC enhances proliferation of oligodendroglial progenitor cells (OPCs) and is essential for oligodendrocyte differentiation in a cell-autonomous manner. Biallelic Apc disruption caused translocation of ß-catenin into the nucleus and upregulated ß-catenin-mediated Wnt signaling in early postnatal but not adult oligodendroglial lineage cells. The results of conditional ablation of Apc or Ctnnb1 (the gene encoding ß-catenin) and of simultaneous conditional ablation of Apc and Ctnnb1 revealed that ß-catenin is dispensable for postnatal oligodendroglial differentiation, that Apc one-allele deficiency is not sufficient to dysregulate ß-catenin-mediated Wnt signaling in oligodendroglial lineage cells, and that APC regulates oligodendrocyte differentiation through ß-catenin-independent, as well as ß-catenin-dependent, mechanisms. Gene ontology analysis of microarray data suggested that the ß-catenin-independent mechanism involves APC regulation of the cytoskeleton, a result compatible with established APC functions in neural precursors and with our observation that Apc-deleted OPCs develop fewer, shorter processes in vivo. Together, our data support the hypothesis that APC regulates oligodendrocyte differentiation through both ß-catenin-dependent and additional ß-catenin-independent mechanisms.

Interferon regulatory factor 8/interferon consensus sequence binding protein is a critical transcription factor for the physiological phenotype of microglia.

BACKGROUND: Recent fate-mapping studies establish that microglia, the resident mononuclear phagocytes of the CNS, are distinct in origin from the bone marrow-derived myeloid lineage. Interferon regulatory factor 8 (IRF8, also known as interferon consensus sequence binding protein) plays essential roles in development and function of the bone marrow-derived myeloid lineage. However, little is known about its roles in microglia. METHODS: The CNS tissues of IRF8-deficient mice were immunohistochemically analyzed. Pure microglia isolated from wild-type and IRF8-deficient mice were studied in vitro by proliferation, immunocytochemical and phagocytosis assays. Microglial response in vivo was compared between wild-type and IRF8-deficient mice in the cuprizon-induced demyelination model. RESULTS: Our analysis of IRF8-deficient mice revealed that, in contrast to compromised development of IRF8-deficient bone marrow myeloid lineage cells, development and colonization of microglia are not obviously affected by loss of IRF8. However, IRF8-deficient microglia demonstrate several defective phenotypes. In vivo, IRF8-deficient microglia have fewer elaborated processes with reduced expression of IBA1/AIF1 compared with wild-type microglia, suggesting a defective phenotype. IRF8-deficient microglia are significantly less proliferative in mixed glial cultures than wild-type microglia. Unlike IRF8-deficient bone marrow myeloid progenitors, exogenous macrophage colony stimulating factor (colony stimulating factor 1) (M-CSF (CSF1)) restores their proliferation in mixed glial cultures. In addition, IRF8-deficient microglia exhibit an exaggerated growth response to exogenous granulocyte-macrophage colony stimulating factor (colony stimulating factor 2) (GM-CSF (CSF2)) in the presence of other glial cells. IRF8-deficient microglia also demonstrate altered cytokine expressions in response to interferon-gamma and lipopolysaccharide in vitro. Moreover, the maximum phagocytic capacity of IRF8-deficient microglia is reduced, although their engulfment of zymosan particles is not overtly impaired. Defective scavenging activity of IRF8-deficient microglia was further confirmed in vivo in the cuprizone-induced demyelination model in mice. CONCLUSIONS: This study is the first to demonstrate the essential contribution of IRF8-mediated transcription to a broad range of microglial phenotype. Microglia are distinct from the bone marrow myeloid lineage with respect to their dependence on IRF8-mediated transcription.

PEDF is a novel oligodendrogenic morphogen acting on the adult SVZ and corpus callosum.

Pigment epithelium-derived factor (PEDF) is a serine protease inhibitor (serpin) protein with well established neuroprotective and anti-angiogenic properties. Recent studies have also shown that PEDF enhances renewal of adult subventricular zone (SVZ) neural precursors. In neurosphere cultures prepared from the SVZ of adult mice, we found that addition of recombinant PEDF to the medium enhanced expressions of oligodendroglial lineage markers (NG2 and PDGFrα) and transcription factors (Olig1, Olig2, and Sox10). Similarly, continuous PEDF administration into the lateral ventricles of adult glial fibrillary acidic protein:green fluorescent protein (GFAP:GFP) transgenic mice increased the proportions of GFAP:GFP+ and GFAP:GFP- SVZ neural precursors coexpressing oligodendroglial lineage markers and transcription factors. Notably, PEDF infusion also resulted in an induction of doublecortin- and Sox10 double-positive cells in the adult SVZ. Immunoreactive PEDF receptor was detectable in multiple cell types in both adult SVZ and corpus callosum. Furthermore, PEDF intracerebral infusion enhanced survival and maturation of newly born oligodendroglial progenitor cells in the normal corpus callosum, and accelerated oligodendroglial regeneration in lysolecithin-induced corpus callosum demyelinative lesions. Western blot analysis showed a robust upregulation of endogenous PEDF in the corpus callosum upon lysolecithin-induced demyelination. Our results document previously unrecognized oligodendrotrophic effects of recombinant PEDF on the adult SVZ and corpus callosum, demonstrate induction of endogenous CNS PEDF production following demyelination, and make PEDF a strong candidate for pharmacological intervention in demyelinative diseases.