Kaylene M. Young

PDGFRA/NG2 glia generate myelinating oligodendrocytes and piriform projection neurons in adult mice

Platelet-derived growth factor α receptor (PDGFRA)/NG2–expressing glia are distributed throughout the adult CNS. They are descended from oligodendrocyte precursors (OLPs) in the perinatal CNS, but it is not clear whether they continue to generate myelinating oligodendrocytes or other differentiated cells during normal adult life. We followed the fates of adult OLPs in Pdgfra-creERT2/Rosa26-YFP double-transgenic mice and found that they generated many myelinating oligodendrocytes during adulthood; >20% of all oligodendrocytes in the adult mouse corpus callosum were generated after 7 weeks of age, raising questions about the function of the late-myelinating axons. OLPs also produced some myelinating cells in the cortex, but the majority of adult-born cortical cells did not appear to myelinate. We found no evidence for astrocyte production in gray or white matter. However, small numbers of projection neurons were generated in the forebrain, especially in the piriform cortex, which is the main target of the olfactory bulb.

Oligodendrocyte Dynamics in the Healthy Adult CNS: Evidence for Myelin Remodeling

Summary Oligodendrocyte precursors (OPs) continue to proliferate and generate myelinating oligodendrocytes (OLs) well into adulthood. It is not known whether adult-born OLs ensheath previously unmyelinated axons or remodel existing myelin. We quantified OP division and OL production in different regions of the adult mouse CNS including the 4-month-old optic nerve, in which practically all axons are already myelinated. Even there, all OPs were dividing and generating new OLs and myelin at a rate higher than can be explained by first-time myelination of naked axons. We conclude that adult-born OLs in the optic nerve are engaged in myelin remodeling, either replacing OLs that die in service or intercalating among existing myelin sheaths. The latter would predict that average internode length should decrease with age. Consistent with that, we found that adult-born OLs elaborated much shorter but many more internodes than OLs generated during early postnatal life.

CNS-resident glial progenitor/stem cells produce Schwann cells as well as oligodendrocytes during repair of CNS demyelination.

After central nervous system (CNS) demyelination-such as occurs during multiple sclerosis-there is often spontaneous regeneration of myelin sheaths, mainly by oligodendrocytes but also by Schwann cells. The origins of the remyelinating cells have not previously been established. We have used Cre-lox fate mapping in transgenic mice to show that PDGFRA/NG2-expressing glia, a distributed population of stem/progenitor cells in the adult CNS, produce the remyelinating oligodendrocytes and almost all of the Schwann cells in chemically induced demyelinated lesions. In contrast, the great majority of reactive astrocytes in the vicinity of the lesions are derived from preexisting FGFR3-expressing cells, likely to be astrocytes. These data resolve a long-running debate about the origins of the main players in CNS remyelination and reveal a surprising capacity of CNS precursors to generate Schwann cells, which normally develop from the embryonic neural crest and are restricted to the peripheral nervous system.

Subventricular Zone Stem Cells Are Heterogeneous with Respect to Their Embryonic Origins and Neurogenic Fates in the Adult Olfactory Bulb

We determined the embryonic origins of adult forebrain subventricular zone (SVZ) stem cells by Cre-lox fate mapping in transgenic mice. We found that all parts of the telencephalic neuroepithelium, including the medial ganglionic eminence and lateral ganglionic eminence (LGE) and the cerebral cortex, contribute multipotent, self-renewing stem cells to the adult SVZ. Descendants of the embryonic LGE and cortex settle in ventral and dorsal aspects of the dorsolateral SVZ, respectively. Both populations contribute new (5-bromo-2′-deoxyuridine-labeled) tyrosine hydroxylase- and calretinin-positive interneurons to the adult olfactory bulb. However, calbindin-positive interneurons in the olfactory glomeruli were generated exclusively by LGE-derived stem cells. Thus, different SVZ stem cells have different embryonic origins, colonize different parts of the SVZ, and generate different neuronal progeny, suggesting that some aspects of embryonic patterning are preserved in the adult SVZ. This could have important implications for the design of endogenous stem cell-based therapies in the future.

NG2 Glia Generate New Oligodendrocytes But Few Astrocytes in a Murine Experimental Autoimmune Encephalomyelitis Model of Demyelinating Disease

The adult mammalian brain and spinal cord contain glial precursors that express platelet-derived growth factor receptor α subunit (PDGFRA) and the NG2 proteoglycan. These “NG2 cells” descend from oligodendrocyte precursors in the perinatal CNS and continue to generate myelinating oligodendrocytes in the gray and white matter of the postnatal brain. It has been proposed that NG2 cells can also generate reactive astrocytes at sites of CNS injury or demyelination. To test this we examined the fates of PDGFRA/NG2 cells in the mouse spinal cord during experimental autoimmune encephalomyelitis (EAE)—a demyelinating condition that models some aspects of multiple sclerosis in humans. We administered tamoxifen to Pdgfra-CreERT2:Rosa26R-YFP mice to induce yellow fluorescent protein (YFP) expression in PDGFRA/NG2 cells and their differentiated progeny. We subsequently induced EAE and observed a large (>4-fold) increase in the local density of YFP+ cells, >90% of which were oligodendrocyte lineage cells. Many of these became CC1-positive, NG2-negative differentiated oligodendrocytes that expressed myelin markers CNP and Tmem10/Opalin. PDGFRA/NG2 cells generated very few GFAP+-reactive astrocytes (1–2% of all YFP+ cells) or NeuN+ neurons (<0.02%). Thus, PDGFRA/NG2 cells act predominantly as a reservoir of new oligodendrocytes in the demyelinated spinal cord.

p75 Neurotrophin Receptor Expression Defines a Population of BDNF-Responsive Neurogenic Precursor Cells

Although our understanding of adult neurogenesis has increased dramatically over the last decade, confusion still exists regarding both the identity of the stem cell responsible for neuron production and the mechanisms that regulate its activity. Here we show, using flow cytometry, that a small population of cells (0.3%) within the stem cell niche of the rat subventricular zone (SVZ) expresses the p75 neurotrophin receptor (p75NTR) and that these cells are responsible for neuron production in both newborn and adult animals. In the adult, the p75NTR-positive population contains all of the neurosphere-producing precursor cells, whereas in the newborn many of the precursor cells are p75NTR negative. However, at both ages, only the neurospheres derived from p75NTR-positive cells are neurogenic. We also show that neuron production from p75NTR-positive but not p75NTR-negative precursors is greatly enhanced after treatment with brain-derived neurotrophic factor (BDNF) or nerve growth factor. This effect appears to be mediated specifically by p75NTR, because precursor cells from p75NTR-deficient mice show a 70% reduction in their neurogenic potential in vitro and fail to respond to BDNF treatment. Furthermore, adult p75NTR-deficient mice have significantly reduced numbers of PSA-NCAM (polysialylated neural cell adhesion molecule)-positive SVZ neuroblasts in vivo and a lower olfactory bulb weight. Thus, p75NTR defines a discrete population of highly proliferative SVZ precursor cells that are able to respond to neurotrophin activation by increasing neuroblast generation, making this pathway the most likely mechanism for the increased neurogenesis that accompanies raised BDNF levels in a variety of disease and behavioral situations.

Properties and Fate of Oligodendrocyte Progenitor Cells in the Corpus Callosum, Motor Cortex, and Piriform Cortex of the Mouse

Oligodendrocyte progenitor cells (OPCs) in the postnatal mouse corpus callosum (CC) and motor cortex (Ctx) reportedly generate only oligodendrocytes (OLs), whereas those in the piriform cortex may also generate neurons. OPCs have also been subdivided based on their expression of voltage-gated ion channels, ability to respond to neuronal activity, and proliferative state. To determine whether OPCs in the piriform cortex have inherently different physiological properties from those in the CC and Ctx, we studied acute brain slices from postnatal transgenic mice in which GFP expression identifies OL lineage cells. We whole-cell patch clamped GFP-expressing (GFP+) cells within the CC, Ctx, and anterior piriform cortex (aPC) and used prelabeling with 5-ethynyl-2′-deoxyuridine (EdU) to assess cell proliferation. After recording, slices were immunolabeled and OPCs were defined by strong expression of NG2. NG2+ OPCs in the white and gray matter proliferated and coexpressed PDGFRα and voltage-gated Na+ channels (INa). Approximately 70% of OPCs were capable of generating regenerative depolarizations. In addition to OLIG2+ NG2+ INa+ OPCs and OLIG2+ NG2neg INaneg OLs, we identified cells with low levels of NG2 limited to the soma or the base of some processes. These cells had a significantly reduced INa and a reduced ability to incorporate EdU when compared with OPCs and probably correspond to early differentiating OLs. By combining EdU labeling and lineage tracing using Pdgfrα–CreERT2 : R26R–YFP transgenic mice, we double labeled OPCs and traced their fate in the postnatal brain. These OPCs generated OLs but did not generate neurons in the aPC or elsewhere at any time that we examined.

An Fgfr3-iCreER T2 Transgenic Mouse Line for Studies of Neural Stem Cells and Astrocytes

The lack of markers for astrocytes, particularly gray matter astrocytes, significantly hinders research into their development and physiological properties. We previously reported that fibroblast growth factor receptor 3 (Fgfr3) is expressed by radial precursors in the ventricular zone of the embryonic neural tube and subsequently by differentiated astrocytes in gray and white matter. Here, we describe an Fgfr3‐iCreERT2 phage artificial chromosome transgenic mouse line that allows efficient tamoxifen‐induced Cre recombination in Fgfr3‐expressing cells, including radial glial cells in the embryonic neural tube and both fibrous and protoplasmic astrocytes in the mature central nervous system. This mouse strain will therefore be useful for studies of normal astrocyte biology and their responses to CNS injury or disease. In addition, Fgfr3‐iCreERT2 drives Cre recombination in all neurosphere‐forming stem cells in the adult spinal cord and at least 90% of those in the adult forebrain subventricular zone. We made use of this to show that there is continuous accumulation of all major interneuron subtypes in the olfactory bulb (OB) from postnatal day 50 (P50) until at least P230 (∼8 months of age). It therefore seems likely that adult‐born interneurons integrate into existing circuitry and perform long‐term functions in the adult OB.

SOCS3 negatively regulates LIF signaling in neural precursor cells.

Cytokines that signal through the LIFRbeta/gp130 receptor complex, including LIF and CNTF, promote the self-renewal of embryonic and adult neural precursor cells (NPCs). In non-CNS tissues, the protein suppressor of cytokine signaling-3 (SOCS3) negatively regulates signaling through gp130. Here, we analyze the role of SOCS3 in inhibiting LIF signaling in NPCs in vitro. SOCS3 is rapidly expressed by NPCs in response to LIF stimulation, with this expression largely dependent on recruitment of STAT proteins to the activated gp130 receptor. Proliferating NPC cultures can be generated from SOCS3 knockout (SOCS3KO/KO) embryos and display prolonged STAT3 phosphorylation and induction of the GFAP gene in response to LIF. In comparison with SOCS3 wild-type (SOCS3WT/WT) NPCs, SOCS3KO/KO cultures display enhanced self-renewal capacity. However, the clonal potential of SOCS3WT/WT but not SOCS3KO/KO NPCs is enhanced by exogenous LIF. Thus, SOCS3 acts as a negative regulator of LIF signaling in NPCs.

Role of cystatin C in amyloid precursor protein-induced proliferation of neural stem/progenitor cells

Background: The role of the amyloid precursor protein (APP) in neural stem/progenitor cell (NSPC) proliferation is poorly understood. Results: Immunodepletion of cystatin C from NSPC conditioned medium abrogated an effect of APP on NSPC proliferation. Conclusion: Cystatin C mediates APP-induced NSPC proliferation. Significance: The results increase understanding of mechanisms promoting NSPC survival and differentiation. The amyloid precursor protein (APP) is well studied for its role in Alzheimer disease. However, little is known about its normal function. In this study, we examined the role of APP in neural stem/progenitor cell (NSPC) proliferation. NSPCs derived from APP-overexpressing Tg2576 transgenic mice proliferated more rapidly than NSPCs from the corresponding background strain (C57Bl/6xSJL) wild-type mice. In contrast, NSPCs from APP knock-out (APP-KO) mice had reduced proliferation rates when compared with NSPCs from the corresponding background strain (C57Bl/6). A secreted factor, identified as cystatin C, was found to be responsible for this effect. Levels of cystatin C were higher in the Tg2576 conditioned medium and lower in the APP-KO conditioned medium. Furthermore, immunodepletion of cystatin C from the conditioned medium completely removed the ability of the conditioned medium to increase NSPC proliferation. The results demonstrate that APP expression stimulates NSPC proliferation and that this effect is mediated via an increase in cystatin C secretion.