Adan Aguirre

Postnatal NG2 proteoglycan–expressing progenitor cells are intrinsically multipotent and generate functional neurons

Neurogenesis is known to persist in the adult mammalian central nervous system (CNS). The identity of the cells that generate new neurons in the postnatal CNS has become a crucial but elusive issue. Using a transgenic mouse, we show that NG2 proteoglycan–positive progenitor cells that express the 2′,3′-cyclic nucleotide 3′-phosphodiesterase gene display a multipotent phenotype in vitro and generate electrically excitable neurons, as well as astrocytes and oligodendrocytes. The fast kinetics and the high rate of multipotent fate of these NG2+ progenitors in vitro reflect an intrinsic property, rather than reprogramming. We demonstrate in the hippocampus in vivo that a sizeable fraction of postnatal NG2+ progenitor cells are proliferative precursors whose progeny appears to differentiate into GABAergic neurons capable of propagating action potentials and displaying functional synaptic inputs. These data show that at least a subpopulation of postnatal NG2-expressing cells are CNS multipotent precursors that may underlie adult hippocampal neurogenesis.

Notch and EGFR pathway interaction regulates neural stem cell number and self-renewal

Specialized cellular microenvironments, or ‘niches’, modulate stem cell properties, including cell number, self-renewal and fate decisions. In the adult brain, niches that maintain a source of neural stem cells (NSCs) and neural progenitor cells (NPCs) are the subventricular zone (SVZ) of the lateral ventricle and the dentate gyrus of the hippocampus. The size of the NSC population of the SVZ at any time is the result of several ongoing processes, including self-renewal, cell differentiation, and cell death. Maintaining the balance between NSCs and NPCs in the SVZ niche is critical to supply the brain with specific neural populations, both under normal conditions or after injury. A fundamental question relevant to both normal development and to cell-based repair strategies in the central nervous system is how the balance of different NSC and NPC populations is maintained in the niche. EGFR (epidermal growth factor receptor) and Notch signalling pathways have fundamental roles during development of multicellular organisms. In Drosophila and in Caenorhabditis elegans these pathways may have either cooperative or antagonistic functions. In the SVZ, Notch regulates NSC identity and self-renewal, whereas EGFR specifically affects NPC proliferation and migration. This suggests that interplay of these two pathways may maintain the balance between NSC and NPC numbers. Here we show that functional cell–cell interaction between NPCs and NSCs through EGFR and Notch signalling has a crucial role in maintaining the balance between these cell populations in the SVZ. Enhanced EGFR signalling in vivo results in the expansion of the NPC pool, and reduces NSC number and self-renewal. This occurs through a non-cell-autonomous mechanism involving EGFR-mediated regulation of Notch signalling. Our findings define a novel interaction between EGFR and Notch pathways in the adult SVZ, and thus provide a mechanism for NSC and NPC pool maintenance.

A functional role for EGFR signaling in myelination and remyelination.

Cellular strategies for oligodendrocyte regeneration and remyelination involve characterizing endogenous neural progenitors that are capable of generating oligodendrocytes during normal development and after demyelination, and identifying the molecular signals that enhance oligodendrogenesis from these progenitors. Using both gain- and loss-of-function approaches, we explored the role of epidermal growth factor receptor (EGFR) signaling in adult myelin repair and in oligodendrogenesis. We show that 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP) promoter–driven overexpression of human EGFR (hEGFR) accelerated remyelination and functional recovery following focal demyelination of mouse corpus callosum. Lesion repopulation by Cspg4+ (also known as NG2) Ascl1+ (also known as Mash1) Olig2+ progenitors and functional remyelination were accelerated in CNP-hEGFR mice compared with wild-type mice. EGFR overexpression in subventricular zone (SVZ) and corpus callosum during early postnatal development also expanded this NG2+Mash1+Olig2+ progenitor population and promoted SVZ-to-lesion migration, enhancing oligodendrocyte generation and axonal myelination. Analysis of hypomorphic EGFR-mutant mice confirmed that EGFR signaling regulates oligodendrogenesis and remyelination by NG2+Mash1+Olig2+ progenitors. EGFR targeting holds promise for enhancing oligodendrocyte regeneration and myelin repair.

NG2-expressing cells in the subventricular zone are type C–like cells and contribute to interneuron generation in the postnatal hippocampus

The subventricular zone (SVZ) is a source of neural progenitors throughout brain development. The identification and purification of these progenitors and the analysis of their lineage potential are fundamental issues for future brain repair therapies. We demonstrate that early postnatal NG2-expressing (NG2+) progenitor cells located in the SVZ self-renew in vitro and display phenotypic features of transit-amplifier type C–like multipotent cells. NG2+ cells in the SVZ are highly proliferative and express the epidermal growth factor receptor, the transcription factors Dlx, Mash1, and Olig2, and the Lewis X (LeX) antigen. We show that grafted early postnatal NG2+ cells generate hippocampal GABAergic interneurons that propagate action potentials and receive functional glutamatergic synaptic inputs. Our work identifies Dlx+/Mash1+/LeX+/NG2+/GFAP-negative cells of the SVZ as a new class of postnatal multipotent progenitor cells that may represent a specific cellular reservoir for renewal of postnatal and adult inhibitory interneurons in the hippocampus.

Postnatal Neurogenesis and Gliogenesis in the Olfactory Bulb from NG2-Expressing Progenitors of the Subventricular Zone

We used a 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP)-enhanced green fluorescent protein (EGFP) transgenic mouse to study postnatal subventricular zone (SVZ) progenitor fate, with a focus on the olfactory bulb (OB). The postnatal OB of the CNP-EGFP mouse contained EGFP+ interneurons and oligodendrocytes. In the anterior SVZ, the majority of EGFP+ progenitors were NG2+. These NG2+/EGFP+ progenitors expressed the OB interneuron marker Er81, the neuroblast markers doublecortin (DC) and Distalless-related homeobox (DLX), or the oligodendrocyte progenitor marker Nkx2.2. In the rostral migratory stream (RMS), EGFP+ cells displayed a migrating phenotype. A fraction of these cells were either NG2-/Er81+/DC+/DLX+ or NG2+/Nkx2.2+. DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate) injection into the lateral ventricle (LV) of early postnatal mice demonstrated that NG2+/EGFP+ progenitors migrate from the SVZ through the RMS into the OB. Moreover, fluorescence-activated cell-sorting-purified NG2+/CNP-EGFP+ or NG2+/β-actin-enhanced yellow fluorescent protein-positive (EYFP+) progenitors transplanted into the early postnatal LV displayed extensive rostral and caudal migration. EYFP+ or EGFP+ graft-derived cells within the RMS were DLX+/Er81+ or Nkx2.2+, migrated to the OB, and differentiated to interneurons and oligodendrocytes. In the subcortical white matter (SCWM), grafted cells differentiated to either oligodendrocytes or astrocytes. Transplantation of NG2+/EYFP+ progenitors selectively purified from the SVZ showed that these cells were migratory and generated glia and neurons in the OB, hippocampus, and striatum. In contrast, cortical, OB, or cerebellar NG2+ cells had a very limited migratory potential and gave rise to glia in the SCWM and striatum. Our findings indicate region-specific differences between NG2+ progenitor cells and show that NG2+ cells can migrate throughout the RMS and contribute to both gliogenesis and neurogenesis in the postnatal OB.

NG2‐positive cells in the mouse white and grey matter display distinct physiological properties

Cells that express the NG2 proteoglycan are the largest proliferative progenitor population in the postnatal central nervous system (CNS). Although this entire population has long been considered to be oligodendrocyte progenitors, numerous NG2+ cells are present in the cerebral cortex, where relatively little myelination occurs, and also persist long after myelination is complete in the CNS. Several studies have alluded to the presence of distinct NG2+ cell subtypes based on marker expression, but no experimentally derived hypotheses about the physiological role of these subtypes has been proposed. In the current study, whole‐cell patch‐clamp data from acutely isolated slices demonstrate that subcortical white matter and cortical NG2+ cells display distinct membrane properties in addition to possessing differing K+‐ and Na+‐channel expression profiles. A striking observation is that a subpopulation of cortical, but not white matter NG2+ cells, elicit depolarization‐induced spikes that are akin to immature action potentials. Our data demonstrate that a population of cortical NG2+ cells display physiological properties that differ from their white matter counterparts.

Chordin-induced lineage plasticity of adult SVZ neuroblasts after demyelination

The mechanisms that regulate the developmental potential of adult neural progenitor populations under physiological and pathological conditions remain poorly defined. Glutamic acid decarboxylase 65 (GAD65)- and Doublecortin (Dcx)-expressing cells constitute major progenitor populations in the adult mouse subventricular zone (SVZ). Under normal physiological conditions, SVZ-derived GAD65-positive and Dcx-positive cells expressed the transcription factor Pax6 and migrated along the rostral migratory stream to the olfactory bulb to generate interneurons. After lysolecithin-induced demyelination of corpus callosum, however, these cells altered their molecular and cellular properties and migratory path. Demyelination upregulated chordin in the SVZ, which redirected GAD65-positive and Dcx-positive progenitors from neuronal to glial fates, generating new oligodendrocytes in the corpus callosum. Our findings suggest that the lineage plasticity of SVZ progenitor cells could be a potential therapeutic strategy for diseased or injured brain.

Adult-born SVZ progenitors receive transient synapses during remyelination in corpus callosum.

We found that demyelinated axons formed functional glutamatergic synapses onto adult-born NG2+ oligodendrocyte progenitor cells (OPCs) migrating from the subventricular zone after focal demyelination of adult mice corpus callosum. This glutamatergic input was substantially reduced compared with endogenous callosal OPCs 1 week after lesion and was lost on differentiation into oligodendrocytes. Therefore, axon–oligodendrocyte progenitor synapse formation is a transient and regulated step that occurs during remyelination of callosal axons.

Spatial and temporal expression of S100B in cells of oligodendrocyte lineage.

The analysis of oligodendrocyte (OL) lineage development has been facilitated by the immunocytochemical characterization of OL‐specific antigens and definition of the phenotypes sequentially acquired by differentiating OLs. The purpose of the present study was to address an enduring discrepancy between several reported cases of S100B immunodetection in CNS myelin and myelinating OLs on the one hand, and the systematic use of the S100B protein as an alleged astrocytic marker in studies of the mammalian CNS on the other. To resolve this discrepancy, we have compared the developmental distribution of EGFP+ cells in the CNS of s100b‐enhanced green fluorescent protein (EGFP) (Vives et al., 2003) and cnp‐EGFP (Yuan et al., 2002) mice, and examined the degree of overlap between EGFP expression and that of stage‐specific markers of OL differentiation during the embryonic and postnatal phases of development. We demonstrate that the S100B protein is expressed in postnatal and adult populations of NG2+ progenitors of mouse brain, as well as in immature and mature myelinating OLs present in the brain and spinal cord of embryonic and adult mice, respectively. Comparison between EGFP and endogenous S100B expression in the s100b‐EGFP and cnp‐EGFP mice indicates that S100B protein expression is upregulated in immature and mature OLs. These results argue against the current view that S100B expression is restricted to the astrocytic lineage in the CNS, and indicate that the use of S100B in combination with other molecular markers will help discriminate oligodendrocytes from astrocytes.

Identification of Sox17 as a transcription factor that regulates oligodendrocyte development.

Microarray analysis of oligodendrocyte lineage cells purified by fluorescence-activated cell sorting (FACS) from 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNP)–enhanced green fluorescent protein (EGFP) transgenic mice revealed Sox17 (SRY-box containing gene 17) gene expression to be coordinately regulated with that of four myelin genes during postnatal development. In CNP–EGFP-positive (CNP–EGFP+) cells, Sox17 mRNA and protein levels transiently increased between postnatal days 2 and 15, with white matter O4+ preoligodendrocytes expressing greater Sox17 levels than Nkx2.2+ (NK2 transcription factor related, locus 2) NG2+, or GalC+ (galactocerebroside) cells. In spinal cord, Sox17 protein expression was undetectable in the primary motor neuron domain between embryonic days 12.5 and 15.5 but was evident in Nkx2.2+ and CC1+ cells. In cultured oligodendrocyte progenitor cells (OPCs), Sox17 levels were maximal in O4+ cells and peaked during the phenotypic conversion from bipolar to multipolar. Parallel increases in Sox17 and p27 occurred before MBP protein expression, and Sox17 upregulation was prevented by conditions inhibiting differentiation. Sox17 downregulation with small interfering RNAs increased OPC proliferation and decreased lineage progression after mitogen withdrawal, whereas Sox17 overexpression in the presence of mitogen had opposite effects. Sox17 overexpression enhanced myelin gene expression in OPCs and directly stimulated MBP gene promoter activity. These findings support important roles for Sox17 in controlling both oligodendrocyte progenitor cell cycle exit and differentiation.