MAB345 Sigma-AldrichAnti-O4 Antibody, clone 81

Mutations in GPR56, a member of the adhesion G protein-coupled receptor family, cause a human brain malformation called bilateral frontoparietal polymicrogyria (BFPP). Magnetic resonance imaging (MRI) of BFPP brains reveals myelination defects in addition to brain malformation. However, the cellular role of GPR56 in oligodendrocyte development remains unknown. Here, we demonstrate that loss of Gpr56 leads to hypomyelination of the central nervous system in mice. GPR56 levels are abundant throughout early stages of oligodendrocyte development, but are downregulated in myelinating oligodendrocytes. Gpr56-knockout mice manifest with decreased oligodendrocyte precursor cell (OPC) proliferation and diminished levels of active RhoA, leading to fewer mature oligodendrocytes and a reduced number of myelinated axons in the corpus callosum and optic nerves. Conditional ablation of Gpr56 in OPCs leads to a reduced number of mature oligodendrocytes as seen in constitutive knockout of Gpr56. Together, our data define GPR56 as a cell-autonomous regulator of oligodendrocyte development.

In the vertebrate central nervous system, myelinating oligodendrocytes are postmitotic and derive from proliferative oligodendrocyte precursor cells (OPCs). The molecular mechanisms that govern oligodendrocyte development are incompletely understood, but recent studies implicate the adhesion class of G protein-coupled receptors (aGPCRs) as important regulators of myelination. Here, we use zebrafish and mouse models to dissect the function of the aGPCR Gpr56 in oligodendrocyte development. We show that gpr56 is expressed during early stages of oligodendrocyte development. In addition, we observe a significant reduction of mature oligodendrocyte number and myelinated axons in gpr56 zebrafish mutants. This reduction results from decreased OPC proliferation, rather than increased cell death or altered neural precursor differentiation potential. Finally, we show that these functions are mediated by Gα12/13 proteins and Rho activation. Together, our data establish Gpr56 as a regulator of oligodendrocyte development.

Decoding heterogeneity of pluripotent stem cell (PSC)-derived neural progeny is fundamental for revealing the origin of diverse progenitors, for defining their lineages, and for identifying fate determinants driving transition through distinct potencies. Here we have prospectively isolated consecutively appearing PSC-derived primary progenitors based on their Notch activation state. We first isolate early neuroepithelial cells and show their broad Notch-dependent developmental and proliferative potential. Neuroepithelial cells further yield successive Notch-dependent functional primary progenitors, from early and midneurogenic radial glia and their derived basal progenitors, to gliogenic radial glia and adult-like neural progenitors, together recapitulating hallmarks of neural stem cell (NSC) ontogeny. Gene expression profiling reveals dynamic stage-specific transcriptional patterns that may link development of distinct progenitor identities through Notch activation. Our observations provide a platform for characterization and manipulation of distinct progenitor cell types amenable for developing streamlined neural lineage specification paradigms for modelling development in health and disease.

Mechanisms underlying motor neuron degeneration in spinal muscular atrophy (SMA), the leading inherited cause of infant mortality, remain largely unknown. Many studies have established the importance of hyperphosphorylation of the microtubule-associated protein tau in various neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. However, tau phosphorylation in SMA pathogenesis has yet to be investigated. Here we show that tau phosphorylation on serine 202 (S202) and threonine 205 (T205) is increased significantly in SMA motor neurons using two SMA mouse models and human SMA patient spinal cord samples. Interestingly, phosphorylated tau does not form aggregates in motor neurons or neuromuscular junctions (NMJs), even at late stages of SMA disease, distinguishing it from other tauopathies. Hyperphosphorylation of tau on S202 and T205 is mediated by cyclin-dependent kinase 5 (Cdk5) in SMA disease condition, because tau phosphorylation at these sites is significantly reduced in Cdk5 knock-out mice; genetic knock-out of Cdk5 activating subunit p35 in an SMA mouse model also leads to reduced tau phosphorylation on S202 and T205 in the SMA;p35(-/-) compound mutant mice. In addition, expression of the phosphorylation-deficient tauS202A,T205A mutant alleviates motor neuron defects in a zebrafish SMA model in vivo and mouse motor neuron degeneration in culture, whereas expression of phosphorylation-mimetic tauS202E,T205E promotes motor neuron defects. More importantly, genetic knock-out of tau in SMA mice rescues synapse stripping on motor neurons, NMJ denervation, and motor neuron degeneration in vivo. Altogether, our findings suggest a novel mechanism for SMA pathogenesis in which hyperphosphorylation of non-aggregating tau by Cdk5 contributes to motor neuron degeneration.

Mixed lineage leukemia-1 (Mll1) epigenetically regulates gene expression patterns that specify cellular identity in both embryonic development and adult stem cell populations. In the adult mouse brain, multipotent neural stem cells (NSCs) in the subventricular zone generate new neurons throughout life, and Mll1 is required for this postnatal neurogenesis but not for glial cell differentiation. Analysis of Mll1-dependent transcription may identify neurogenic genes useful for the direct reprogramming of astrocytes into neurons.To identify Mll1-dependent transcriptional modules and to determine whether genes in the neurogenic modules can be used to directly reprogram astrocytes into neurons.We performed gene coexpression module analysis on microarray data from differentiating wild-type and Mll1-deleted subventricular zone NSCs. Key developmental regulators belonging to the neurogenic modules were overexpressed in Mll1-deleted cells and cultured cortical astrocytes, and cell phenotypes were analyzed by immunocytochemistry and electrophysiology.Transcriptional modules that correspond to neurogenesis were identified in wild-type NSCs. Modules related to astrocytes and oligodendrocytes were enriched in Mll1-deleted NSCs, consistent with their gliogenic potential. Overexpression of genes selected from the neurogenic modules enhanced the production of neurons from Mll1-deleted cells, and overexpression of Brn4 (Pou3f4) in nonneurogenic cortical astroglia induced their transdifferentiation into electrophysiologically active neurons.Our results demonstrate that Mll1 is required for the expression of neurogenic but not gliogenic transcriptional modules in a multipotent NSC population and further indicate that specific Mll1-dependent genes may be useful for direct reprogramming strategies.

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative neurology. This directive has arisen from the extensive evidence that brain repair is achieved after focal or systemic NPC transplantation in several preclinical models of neurological diseases. These experimental data have identified the cell delivery route as one of the main hurdles of restorative stem cell therapies for brain diseases that requires urgent assessment. Intraparenchymal stem cell grafting represents a logical approach to those pathologies characterized by isolated and accessible brain lesions such as spinal cord injuries and Parkinson's disease. Unfortunately, this principle is poorly applicable to conditions characterized by a multifocal, inflammatory and disseminated (both in time and space) nature, including multiple sclerosis (MS). As such, brain targeting by systemic NPC delivery has become a low invasive and therapeutically efficacious protocol to deliver cells to the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system (CNS). This alternative method of cell delivery relies on the NPC pathotropism, specifically their innate capacity to (i) sense the environment via functional cell adhesion molecules and inflammatory cytokine and chemokine receptors; (ii) cross the leaking anatomical barriers after intravenous (i.v.) or intracerebroventricular (i.c.v.) injection; (iii) accumulate at the level of multiple perivascular site(s) of inflammatory brain and spinal cord damage; and (i.v.) exert remarkable tissue trophic and immune regulatory effects onto different host target cells in vivo. Here we describe the methods that we have developed for the i.v. and i.c.v. delivery of syngeneic NPCs in mice with experimental autoimmune encephalomyelitis (EAE), as model of chronic CNS inflammatory demyelination, and envisage the systemic stem cell delivery as a valuable technique for the selective targeting of the inflamed brain in regenerative neurology.

Background. The neural stem cells (NSCs) migrate to the damaged sites in multiple sclerosis (MS) and in experimental autoimmune encephalomyelitis (EAE). However, the differentiation into neurons or oligodendrocytes is blocked. Epidermal growth factor (EGF) stimulates NSC proliferation and mobilization to demyelinated lesions but also induces astrogenesis and glial scar. Objective. To examine the clinical and histopathological effects of EGF neutralization on EAE. Methods. EAE-induced SJL mice were intravenously treated with either anti-EGF neutralizing antibody (Ab) or isotype control or PBS. On day 9 after immunization, 3 mice of each group were daily treated for 9 days with BrdU and then sacrificed for immunohistochemical analysis. Results. Treatment with anti-EGF Ab significantly ameliorated EAE symptoms during the second relapse. Anti-EGF Ab induced a shift from BrdU(+)GFAP(+) NSCs to BrdU(+)DCX(+) neuroblasts in the subventricular zone (SVZ), increased BrdU(+)NeuN(+) neurons in the granular cell layer of the dentate gyrus, and increased BrdU(+)O4(+) oligodendrocytes in the SVZ. There was no change in the inflammatory infiltrates in response to anti-EGF Ab. Conclusions. Therapy with anti-EGF Ab ameliorates EAE via induction of neurogenesis and oligodendrogenesis. No immunosuppressive effect was found. Further investigation is needed to support these notions of beneficial effect of anti-EGF Ab in MS.

Studies of central nervous system myelination lack defined in vitro models which would effectively dissect molecular mechanisms of myelination that contain cells of the correct phenotype. Here we describe a co-culture of purified motoneurons and oligodendrocyte progenitor cells, isolated from rat embryonic spinal cord using a combination of immunopanning techniques. This model illustrates differentiation of oligodendrocyte progenitors into fully functional mature oligodendrocytes that myelinate axons. It also illustrates a contribution of axons to the rate of oligodendrocyte maturation and myelin gene expression. The defined conditions used allow molecular analysis of distinct stages of myelination and precise manipulation of inductive cues affecting axonal-oligodendrocyte interactions. This phenotypic in vitro myelination model can provide valuable insight into our understanding of demyelinating disorders, such as multiple sclerosis and traumatic diseases such as spinal cord injury where demyelination represents a contributing factor to the pathology of the disorder.

Purifying and culturing cells from the central nervous system (CNS) has proved to be an incredibly powerful tool for dissecting fundamental neuron and glial properties, and especially powerful in understanding neuronal-glial interactions. In a series of detailed protocols, we have provided step-by-step instructions for purifying and culturing specific types of neurons, glia, and vascular cells from the CNS by immunopanning. This article discusses common pitfalls and errors as well as important design considerations for the immunopanning procedure.

Objectives. Human breast milk contains a heterogeneous population of cells that have the potential to provide a noninvasive source of cells for cell therapy in many neurodegenerative diseases without any ethical concern. The objectives of this study were to differentiate the breast milk-derived stem cells (BMDSC) toward neural stem cells and then into the neurons and neuroglia. Materials and Methods. To do this, the BMDSC were isolated from human breast milk and cultured in Dulbecco's modified Eagle medium/F12 (DMEM/F12) containing fibroblast growth factor (bFGF). The cells were then characterized by evaluation of the embryonic and stem cell markers. Then, the cells were exposed to culture medium containing 1% B27 and 2% N2 for 7-10 days followed by medium supplemented with B27, N2, bFGF 10 µg/mL, and endothelial growth factor (EGF) 20 µg/mL. Then, the sphere-forming assay was performed. The spheres were then differentiated into three neural lineages by withdrawing growth factor in the presence of 5% FBS (fetal bovine serum). The immunofluorescence was done for β-tubulin III, O4, and GFAP (glial fibrillary acidic protein). Results. The results indicated that the cells expressed both embryonic and mesenchymal stem cell (MSC) markers. They also showed neurospheres formation that was nestin-positive. The cells were also differentiated into all three neural lineages. Conclusion. The BMDSC can behave in the same way with neural stem cells. They were differentiated into oligodendrocytes, and astrocytes as well as neurons.