Heterogeneity of astrocytes: Electrophysiological properties of juxtavascular astrocytes before and after brain injury.

Astrocyte heterogeneity is increasingly recognized, but still little is known about juxtavascular astrocytes with their somata directly adjacent to blood vessels, despite their importance after brain injury. As juxtavascular astrocytes originate from common progenitor cells, that is, have a clonal origin, they may intrinsically differ from other, non-juxtavascular astrocytes. To explore this, we examined the electrophysiological properties of these groups of astrocytes and the underlying ion channels. Using brain slices of BAC Aldh1l1-eGFP transgenic mice with astrocytes labeled by GFP expression, we compared juxtavascular and non-juxtavascular astrocytes in the somatosensory cortex by means of whole-cell patch-clamp recordings and immunohistochemical staining. Prior to injury, juxta- and non-juxtavascular astrocytes exhibit comparable electrophysiological properties with characteristic mostly passive conductance and a typical negative resting membrane potential. Immunohistochemical analysis of K+ channels showed that all astrocytes were Kir 4.1+ , but revealed an intriguing difference for Kv 4.3. The expression of Kv 4.3 in sibling astrocytes (non-juxtavascular, juxtavascular and pial) was dependent on their ontogenetic origin with lowest levels in juxtavascular astrocytes located in upper cortical layers. After traumatic brain injury (TBI), we found profound changes in the electrophysiological type of astrocytes with a predominance of non-passive properties and this pattern was significantly enriched in juxtavascular astrocytes. This was accompanied by pronounced down-regulation of Kir 4.1 in proliferating astrocytes, which was significantly more in juxtavascular compared to non-juxtavascular astrocytes. Taken together, TBI induces profound differences in electrophysiological properties between juxtavascular and non-juxtavascular astrocytes that might be related to the preponderance of juxtavascular astrocyte proliferation.

Secretome Analysis of Mesenchymal Stem Cell Factors Fostering Oligodendroglial Differentiation of Neural Stem Cells In Vivo.

Mesenchymal stem cell (MSC)-secreted factors have been shown to significantly promote oligodendrogenesis from cultured primary adult neural stem cells (aNSCs) and oligodendroglial precursor cells (OPCs). Revealing underlying mechanisms of how aNSCs can be fostered to differentiate into a specific cell lineage could provide important insights for the establishment of novel neuroregenerative treatment approaches aiming at myelin repair. However, the nature of MSC-derived differentiation and maturation factors acting on the oligodendroglial lineage has not been identified thus far. In addition to missing information on active ingredients, the degree to which MSC-dependent lineage instruction is functional in vivo also remains to be established. We here demonstrate that MSC-derived factors can indeed stimulate oligodendrogenesis and myelin sheath generation of aNSCs transplanted into different rodent central nervous system (CNS) regions, and furthermore, we provide insights into the underlying mechanism on the basis of a comparative mass spectrometry secretome analysis. We identified a number of secreted proteins known to act on oligodendroglia lineage differentiation. Among them, the tissue inhibitor of metalloproteinase type 1 (TIMP-1) was revealed to be an active component of the MSC-conditioned medium, thus validating our chosen secretome approach.

The role of clonal communication and heterogeneity in breast cancer.

BACKGROUND: Cancer is a rapidly evolving, multifactorial disease that accumulates numerous genetic and epigenetic alterations. This results in molecular and phenotypic heterogeneity within the tumor, the complexity of which is further amplified through specific interactions between cancer cells. We aimed to dissect the molecular mechanisms underlying the cooperation between different clones. METHODS: We produced clonal cell lines derived from the MDA-MB-231 breast cancer cell line, using the UbC-StarTrack system, which allowed tracking of multiple clones by color: GFP C3, mKO E10 and Sapphire D7. Characterization of these clones was performed by growth rate, cell metabolic activity, wound healing, invasion assays and genetic and epigenetic arrays. Tumorigenicity was tested by orthotopic and intravenous injections. Clonal cooperation was evaluated by medium complementation, co-culture and co-injection assays. RESULTS: Characterization of these clones in vitro revealed clear genetic and epigenetic differences that affected growth rate, cell metabolic activity, morphology and cytokine expression among cell lines. In vivo, all clonal cell lines were able to form tumors; however, injection of an equal mix of the different clones led to tumors with very few mKO E10 cells. Additionally, the mKO E10 clonal cell line showed a significant inability to form lung metastases. These results confirm that even in stable cell lines heterogeneity is present. In vitro, the complementation of growth medium with medium or exosomes from parental or clonal cell lines increased the growth rate of the other clones. Complementation assays, co-growth and co-injection of mKO E10 and GFP C3 clonal cell lines increased the efficiency of invasion and migration. CONCLUSIONS: These findings support a model where interplay between clones confers aggressiveness, and which may allow identification of the factors involved in cellular communication that could play a role in clonal cooperation and thus represent new targets for preventing tumor progression.

Differential expression of sema3A and sema7A in a murine model of multiple sclerosis: Implications for a therapeutic design.

We characterised the expression of semaphorin (sema)3A, sema7A and their receptors in the immune and the central nervous system (CNS) at different stages of experimental autoimmune encephalomyelitis (EAE). We also studied their expression in neonatal and adult oligodendrocyte progenitor cell (OPC) and in mature oligodendrocyte cultures. Our results show that sema3A is increased in the CNS and decreased in the immune system upon EAE induction. However, sema7A expression is increased in both the CNS and the immune system during EAE. We also detected sema3A, sema7A and their receptors in neonatal and adult OPCs and in mature oligodendrocytes. These data suggest that sema3A and sema7A are involved in the pathogenesis of EAE, in the modulation of the immune response and in the neurodegeneration that take place in the CNS. Sema7A may represent an intriguing potential therapeutic target for the treatment of both the neurodegenerative and immune-mediated disease processes in MS.

ERK1/2 signaling is essential for the chemoattraction exerted by human FGF2 and human anosmin-1 on newborn rat and mouse OPCs via FGFR1.

Signaling through fibroblast growth factor receptors (FGFRs) is essential for many cellular processes including proliferation and migration, as well as differentiation events such as myelination. Anosmin-1 is an extracellular matrix (ECM) glycoprotein that interacts with the fibroblast growth factor receptor 1 (FGFR1) to exert its biological actions through this receptor, although the intracellular pathways underlying anosmin-1 signaling remain largely unknown. This protein is defective in the X-linked form of Kallmann syndrome (KS) and has a prominent role in the migration of neuronal and oligodendroglial precursors. We have shown that anosmin-1 exerts a chemotactic effect via FGFR1 on neuronal precursors from the subventricular zone (SVZ) and the essential role of the ERK1/2 signaling. We report here the positive chemotactic effect of FGF2 and anosmin-1 on rat and mouse postnatal OPCs via FGFR1. The same effect was observed with the truncated N-terminal region of anosmin-1 (A1Nt). The introduction in anosmin-1 of the missense mutation F517L found in patients suffering from KS annulled the chemotactic activity; however, the mutant form carrying the disease-causing mutation E514K also found in KS patients, behaved as the wild-type protein. The chemoattraction exhibited by FGF2 and anosmin-1 on OPCs was blocked by the mitogen-activated protein kinase (MAPK) inhibitor U0126, suggesting that the activation of the ERK1/2 MAPK signaling pathway following interaction with the FGFR1 is necessary for FGF2 and anosmin-1 to exert their chemotactic effect. In fact, both proteins were able to induce the phosphorylation of the ERK1/2 kinases after the activation of the FGFR1 receptor.

Regulation of oligodendrocyte precursor migration during development, in adulthood and in pathology.

Oligodendrocytes are the myelin-forming cells in the central nervous system (CNS). These cells originate from oligodendrocyte precursor cells (OPCs) during development, and they migrate extensively from oligodendrogliogenic niches along the neural tube to colonise the entire CNS. Like many other such events, this migratory process is precisely regulated by a battery of positional and signalling cues that act via their corresponding receptors and that are expressed dynamically by OPCs. Here, we will review the cellular and molecular basis of this important event during embryonic and postnatal development, and we will discuss the relevance of the substantial number of OPCs existing in the adult CNS. Similarly, we will consider the behaviour of OPCs in normal and pathological conditions, especially in animal models of demyelination and of the demyelinating disease, multiple sclerosis. The spontaneous remyelination observed after damage in demyelinating pathologies has a limited effect. Understanding the cellular and molecular mechanisms underlying the biology of OPCs, particularly adult OPCs, should help in the design of neuroregenerative strategies to combat multiple sclerosis and other demyelinating diseases.

Myelin-associated proteins block the migration of olfactory ensheathing cells: an in vitro study using single-cell tracking and traction force microscopy.

Newly generated olfactory receptor axons grow from the peripheral to the central nervous system aided by olfactory ensheathing cells (OECs). Thus, OEC transplantation has emerged as a promising therapy for spinal cord injuries and for other neural diseases. However, these cells do not present a uniform population, but instead a functionally heterogeneous population that exhibits a variety of responses including adhesion, repulsion, and crossover during cell-cell and cell-matrix interactions. Some studies report that the migratory properties of OECs are compromised by inhibitory molecules and potentiated by chemical gradients. Here, we demonstrated that rodent OECs express all the components of the Nogo receptor complex and that their migration is blocked by myelin. Next, we used cell tracking and traction force microscopy to analyze OEC migration and its mechanical properties over myelin. Our data relate the decrease of traction force of OEC with lower migratory capacity over myelin, which correlates with changes in the F-actin cytoskeleton and focal adhesion distribution. Lastly, OEC traction force and migratory capacity is enhanced after cell incubation with the Nogo receptor inhibitor NEP1-40.

Expression of Semaphorin 4F in neurons and brain oligodendrocytes and the regulation of oligodendrocyte precursor migration in the optic nerve.

Semaphorins are secreted or membrane-anchored proteins that play critical roles in neural development and adult brain plasticity. Sema4F is a transmembrane semaphorin found on glutamatergic synapses, in which it is attached to the PSD-95-scaffolding protein. Here we further examined the expression of Sema4F by raising specific antibodies. We show that Sema4F protein is widely expressed by neurons during neural development and in the adult brain. We also demonstrate a preferential localization of this protein in postsynaptic dendrites. Moreover, Sema4F is expressed not only by neurons but also by oligodendrocyte precursors in the optic nerve and along the migratory pathways of oligodendroglial cells, and also by subsets of postnatal oligodendroglial cells in the brain. Finally, in vitro experiments demonstrate that endogenous Sema4F expressed by brain cells of oligodendroglial lineage regulates the outgrowth migration of oligodendrocyte precursors and promotes their differentiation. The present data extend our knowledge about the expression of Sema4F and uncover a novel function in the control of oligodendrocyte precursor migration in the developing brain.

Developmental expression of the oligodendrocyte myelin glycoprotein in the mouse telencephalon.

The oligodendrocyte myelin glycoprotein is a glycosylphosphatidylinositol-anchored protein expressed by neurons and oligodendrocytes in the central nervous system. Attempts have been made to identify the functions of the myelin-associated inhibitory proteins (MAIPs) after axonal lesion or in neurodegeneration. However, the developmental roles of some of these proteins and their receptors remain elusive. Recent studies indicate that NgR1 and the recently discovered receptor PirB restrict cortical synaptic plasticity. However, the putative factors that trigger these effects are unknown. Because Nogo-A is mostly associated with the endoplasmic reticulum and myelin associated glycoprotein appears late during development, the putative participation of OMgp should be considered. Here, we examine the pattern of development of OMgp immunoreactive elements during mouse telencephalic development. OMgp immunoreactivity in the developing cortex follows the establishment of the thalamo-cortical barrel field. At the cellular level, we located OMgp neuronal membranes in dendrites and axons as well as in brain synaptosome fractions and axon varicosities. Lastly, the analysis of the barrel field in OMgp-deficient mice revealed that although thalamo-cortical connections were formed, their targeting in layer IV was altered, and numerous axons ectopically invaded layers II-III. Our data support the idea that early expressed MAIPs play an active role during development and point to OMgp participating in thalamo-cortical connections.

Cell Fate Potential of NG2 Progenitors.

Determining the origin of different glial subtypes is crucial to understand glial heterogeneity, and to enhance our knowledge of glial and progenitor cell behavior in embryos and adults. NG2-glia are homogenously distributed in a grid-like manner in both, gray and white matter of the adult brain. While some NG2-glia in the CNS are responsible for the generation of mature oligodendrocytes (OPCs), most of them do not differentiate and they can proliferate outside of adult neurogenic niches. Thus, NG2-glia constitute a heterogeneous population containing different subpopulations with distinct functions. We hypothesized that their diversity emerges from specific progenitors during development, as occurs with other glial cell subtypes. To specifically target NG2-pallial progenitors and to define the NG2-glia lineage, as well as the NG2-progenitor potential, we designed two new StarTrack strategies using the NG2 promoter. These approaches label NG2 expressing progenitor cells, permitting the cell fates of these NG2 progenitors to be tracked in vivo. StarTrack labelled cells producing different neural phenotypes in different regions depending on the age targeted, and the strategy selected. This specific genetic targeting of neural progenitors in vivo has provided new data on the heterogeneous pool of NG2 progenitors at both embryonic and postnatal ages.

A Clonal NG2-Glia Cell Response in a Mouse Model of Multiple Sclerosis.

NG2-glia, also known as oligodendrocyte precursor cells (OPCs), have the potential to generate new mature oligodendrocytes and thus, to contribute to tissue repair in demyelinating diseases like multiple sclerosis (MS). Once activated in response to brain damage, NG2-glial cells proliferate, and they acquire a reactive phenotype and a heterogeneous appearance. Here, we set out to investigate the distribution and phenotypic diversity of NG2-glia relative to their ontogenic origin, and whether there is a clonal NG2-glial response to lesion in an experimental autoimmune encephalomyelitis (EAE) murine model of MS. As such, we performed in utero electroporation of the genomic lineage tracer, StarTrack, to follow the fate of NG2-glia derived from single progenitors and to evaluate their response to brain damage after EAE induction. We then analyzed the dispersion of the NG2-glia derived clonally from single pallial progenitors in the brain of EAE mice. In addition, we examined several morphological parameters to assess the degree of NG2-glia reactivity in clonally-related cells. Our results reveal the heterogeneity of these progenitors and their cell progeny in a scenario of autoimmune demyelination, revealing the ontogenic phenomena at play in these processes.

Functional Heterogeneity of Mouse and Human Brain OPCs: Relevance for Preclinical Studies in Multiple Sclerosis.

Besides giving rise to oligodendrocytes (the only myelin-forming cell in the Central Nervous System (CNS) in physiological conditions), Oligodendrocyte Precursor Cells (OPCs) are responsible for spontaneous remyelination after a demyelinating lesion. They are present along the mouse and human CNS, both during development and in adulthood, yet how OPC physiological behavior is modified throughout life is not fully understood. The activity of adult human OPCs is still particularly unexplored. Significantly, most of the molecules involved in OPC-mediated remyelination are also involved in their development, a phenomenon that may be clinically relevant. In the present article, we have compared the intrinsic properties of OPCs isolated from the cerebral cortex of neonatal, postnatal and adult mice, as well as those recovered from neurosurgical adult human cerebral cortex tissue. By analyzing intact OPCs for the first time with 1H High Resolution Magic Angle Spinning Nuclear Magnetic Resonance (1H HR-MAS NMR) spectroscopy, we show that these cells behave distinctly and that they have different metabolic patterns in function for their stage of maturity. Moreover, their response to Fibroblast Growth Gactor-2 (FGF-2) and anosmin-1 (two molecules that have known effects on OPC biology during development and that are overexpressed in individuals with Multiple Sclerosis (MS)) differs in relation to their developmental stage and in the function of the species. Our data reveal that the behavior of adult human and mouse OPCs differs in a very dynamic way that should be very relevant when testing drugs and for the proper design of effective pharmacological and/or cell therapies for MS.

Clonal Glial Response in a Multiple Sclerosis Mouse Model.

Multiple sclerosis (MS) is an autoimmune disease causing central nervous system (CNS) demyelination and axonal injury. In the last years the importance of astrocytes in MS is rapidly increasing, recognizing astrocytes as highly active players in MS pathogenesis. Usually the role assigned to astrocytes in MS lesions has been the formation of the glial scar, but now their implication during lesion formation and the immune response increasingly recognized. Since astrocytes are a heterogeneous cell population with diverse roles in the CNS, the aim of this study was to analyze the putative clonal response of astrocytes in a demyelinating scenario. To undertake this aim, we used the induced experimental autoimmune encephalomyelitis (EAE) as a murine model for MS in previously electroporated mice with in vivo multicolor lineage tracing system, the StarTrack methodology. Our data revealed a variety of morphological changes that were different among distinct clones. In many cases, cells of the same clone responded equally to the injury, while in other cases clonally-related cells responded differently to the injury. Therefore, whereas some clones exhibited a strong morphological alteration, other clones located at similar distances to the lesion were apparently unresponsive. Thus, at present there is no compelling evidences that clonal relationship influences the position or function of astrocytes in the EAE model. Further, the coexistence of different astroglial clonal responses to the bran injury reveals the significance of development to determine the astrocyte features that respond to brain injuries.

Promoting in vivo remyelination with small molecules: a neuroreparative pharmacological treatment for Multiple Sclerosis.

Multiple Sclerosis (MS) is a neurodegenerative disease where immune-driven demyelination occurs with inefficient remyelination, but therapies are limited, especially those to enhance repair. Here, we show that the dual phosphodiesterase (PDE)7- glycogen synthase kinase (GSK)3 inhibitor, VP3.15, a heterocyclic small molecule with good pharmacokinetic properties and safety profile, improves in vivo remyelination in mouse and increases both adult mouse and adult human oligodendrocyte progenitor cell (OPC) differentiation, in addition to its immune regulatory action. The dual inhibition is synergistic, as increasing intracellular levels of cAMP by cyclic nucleotide PDE inhibition both suppresses the immune response and increases remyelination, and in addition, inhibition of GSK3 limits experimental autoimmune encephalomyelitis in mice. This combination of an advantageous effect on the immune response and an enhancement of repair, plus demonstration of its activity on adult human OPCs, leads us to propose dual PDE7-GSK3 inhibition, and specifically VP3.15, as a neuroprotective and neuroreparative disease-modifying treatment for MS.

Foxg1-Cre Mediated Lrp2 Inactivation in the Developing Mouse Neural Retina, Ciliary and Retinal Pigment Epithelia Models Congenital High Myopia.

Myopia is a common ocular disorder generally due to increased axial length of the eye-globe. Its extreme form high myopia (HM) is a multifactorial disease leading to retinal and scleral damage, visual impairment or loss and is an important health issue. Mutations in the endocytic receptor LRP2 gene result in Donnai-Barrow (DBS) and Stickler syndromes, both characterized by HM. To clearly establish the link between Lrp2 and congenital HM we inactivated Lrp2 in the mouse forebrain including the neural retina and the retinal and ciliary pigment epithelia. High resolution in vivo MRI imaging and ophthalmological analyses showed that the adult Lrp2-deficient eyes were 40% longer than the control ones mainly due to an excessive elongation of the vitreal chamber. They had an apparently normal intraocular pressure and developed chorioretinal atrophy and posterior scleral staphyloma features reminiscent of human myopic retinopathy. Immunomorphological and ultrastructural analyses showed that increased eye lengthening was first observed by post-natal day 5 (P5) and that it was accompanied by a rapid decrease of the bipolar, photoreceptor and retinal ganglion cells, and eventually the optic nerve axons. It was followed by scleral thinning and collagen fiber disorganization, essentially in the posterior pole. We conclude that the function of LRP2 in the ocular tissues is necessary for normal eye growth and that the Lrp2-deficient eyes provide a unique tool to further study human HM.

The adhesion molecule anosmin-1 in neurology: Kallmann syndrome and beyond.

Anosmin-1 is the glycoprotein encoded by the KAL1 gene and part of the extracellular matrix, which was first identified as defective in human Kallmann syndrome (KS, characterised by hypogonadotropic hypogonadism and anosmia); biochemically it is a cell adhesion protein. The meticulous biochemical dissection of the anosmin-1 domains has identified which domains are necessary for the protein to bind its different partners to display its biological effects. Research in the last decade has unravelled different roles of anosmin-1 during CNS development (axon pathfinding, axonal collateralisation, cell motility and migration), some of them intimately related with the cited KS but not only with this. More recently, anosmin-1 has been identified in other pathological scenarios both within (multiple sclerosis) and outside (cancer, atopic dermatitis) the CNS.

Sema3E/PlexinD1 regulates the migration of hem-derived Cajal-Retzius cells in developing cerebral cortex.

During the development of the cerebral cortex, Cajal-Retzius (CR) cells settle in the preplate and coordinate the precise growth of the neocortex. Indeed, CR cells migrate tangentially from specific proliferative regions of the telencephalon (for example, the cortical hem (CH)) to populate the entire cortical surface. This is a very finely tuned process regulated by an emerging number of factors that has been sequentially revealed in recent years. However, the putative participation of one of the major families of axon guidance molecules in this process, the Semaphorins, was not explored. Here we show that Semaphorin-3E (Sema3E) is a natural negative regulator of the migration of PlexinD1-positive CR cells originating in the CH. Our results also indicate that Sema3E/PlexinD1 signalling controls the motogenic potential of CR cells in vitro and in vivo. Indeed, absence of Sema3E/PlexinD1 signalling increased the migratory properties of CR cells. This modulation implies negative effects on CXCL12/CXCR4 signalling and increased ADF/Cofilin activity.

Protocol to isolate a large amount of functional oligodendrocyte precursor cells from the cerebral cortex of adult mice and humans.

During development, oligodendrocytes are generated from oligodendrocyte precursor cells (OPCs), a cell type that is a significant proportion of the total cells (3-8%) in the adult central nervous system (CNS) of both rodents and humans. Adult OPCs are responsible for the spontaneous remyelination that occurs in demyelinating diseases like Multiple Sclerosis (MS) and they constitute an interesting source of cells for regenerative therapy in such conditions. However, there is little data regarding the neurobiology of adult OPCs isolated from mice since an efficient method to isolate them has yet to be established. We have designed a protocol to obtain viable adult OPCs from the cerebral cortex of different mouse strains and we have compared its efficiency with other well-known methods. In addition, we show that this protocol is also useful to isolate functional OPCs from human brain biopsies. Using this method we can isolate primary cortical OPCs in sufficient quantities so as to be able to study their survival, maturation and function, and to facilitate an evaluation of their utility in myelin repair.

Neuregulin-1/ErbB4 signaling controls the migration of oligodendrocyte precursor cells during development.

During embryonic development, the oligodendrocyte precursors (OPCs) are generated in specific oligodendrogliogenic sites within the neural tube and migrate to colonize the entire CNS. Different factors have been shown to influence the OPC migration and differentiation, including morphogens, growth factors, chemotropic molecules, and extracellular matrix proteins. Neuregulins have been shown to influence the migration of neuronal precursors as well as the movement and differentiation of Schwann cells for peripheral myelination, but their role in the motility of OPCs has not been explored. In the present study, we have used the optic nerve as an experimental model to examine the function of Nrg1 and its ErbB4 receptor in the migration of OPCs in the developing embryo. In vitro experiments revealed that Nrg1 is a potent chemoattractant for the first wave of OPCs, and that this effect is mediated via ErbB4 receptor. In contrast, OPCs colonizing the optic nerve at postnatal stages (PDGFRα(+)-OPCs) does not respond to Nrg1-chemoattraction. We also found that mouse embryos lacking ErbB4 display deficits in early OPC migration away from different oligodendrogliogenic regions in vivo. The present findings reveal a new role for Nrg1/ErbB4 signaling in regulating OPC migration selectively during early stages of CNS development.

Role of the cellular prion protein in oligodendrocyte precursor cell proliferation and differentiation in the developing and adult mouse CNS.

There are numerous studies describing the signaling mechanisms that mediate oligodendrocyte precursor cell (OPC) proliferation and differentiation, although the contribution of the cellular prion protein (PrP(c)) to this process remains unclear. PrP(c) is a glycosyl-phosphatidylinositol (GPI)-anchored glycoprotein involved in diverse cellular processes during the development and maturation of the mammalian central nervous system (CNS). Here we describe how PrP(c) influences oligodendrocyte proliferation in the developing and adult CNS. OPCs that lack PrP(c) proliferate more vigorously at the expense of a delay in differentiation, which correlates with changes in the expression of oligodendrocyte lineage markers. In addition, numerous NG2-positive cells were observed in cortical regions of adult PrP(c) knockout mice, although no significant changes in myelination can be seen, probably due to the death of surplus cells.