NG2 Cells Research Articles

Stress-induced c-fos expression in the medial prefrontal cortex differentially affects the main residing cell phenotypes

Stress poses a challenge to the body's equilibrium and triggers a series of responses that enable organisms to adapt to stressful stimuli. The medial prefrontal cortex (mPFC), particularly in acute stress conditions, undergoes significant physiological changes to cope with the demands associated with cellular activation. The proto-oncogene c-fos and its protein product c-Fos have long been utilized to investigate the effects of external factors on the central nervous system (CNS). While c-Fos expression has traditionally been attributed to neurons, emerging evidence suggests its potential expression in glial cells. In this study, our main objective was to explore the expression of c-Fos in glial cells and examine how acute stress influences these activity patterns. We conducted our experiments on male Wistar rats, subjecting them to acute stress and sacrificing them 2 h after the stressor initiation. Using double-labelling fluorescent immunohistochemistry targeting c-Fos, along with markers such as GFAP, Iba-1, Olig2, NG2, and NeuN, we analyzed 35 μm brain slices obtained from the mPFC. Our findings compellingly demonstrate that c-Fos expression extends beyond neurons and is present in astrocytes, oligodendrocytes, microglia, and NG2 cells—the diverse population of glial cells. Moreover, we observed distinct regulation of c-Fos expression in response to stress across different subregions of the mPFC. These results emphasize the importance of considering glial cells and their perspective in studies investigating brain activity, highlighting c-Fos as a response marker in glial cells. By shedding light on the differential regulation of c-Fos expression in response to stress, our study contributes to the understanding of glial cell involvement in stress-related processes. This underscores the significance of including glial cells in investigations of brain activity and expands our knowledge of c-Fos as a potential marker for glial cell responses.

Neuronal conversion from glia to replenish the lost neurons.

Neuronal injury, aging, and cerebrovascular and neurodegenerative diseases such as cerebral infarction, Alzheimer's disease, Parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis, and Huntington's disease are characterized by significant neuronal loss. Unfortunately, the neurons of most mammals including humans do not possess the ability to self-regenerate. Replenishment of lost neurons becomes an appealing therapeutic strategy to reverse the disease phenotype. Transplantation of pluripotent neural stem cells can supplement the missing neurons in the brain, but it carries the risk of causing gene mutation, tumorigenesis, severe inflammation, and obstructive hydrocephalus induced by brain edema. Conversion of neural or non-neural lineage cells into functional neurons is a promising strategy for the diseases involving neuron loss, which may overcome the above-mentioned disadvantages of neural stem cell therapy. Thus far, many strategies to transform astrocytes, fibroblasts, microglia, Müller glia, NG2 cells, and other glial cells to mature and functional neurons, or for the conversion between neuronal subtypes have been developed through the regulation of transcription factors, polypyrimidine tract binding protein 1 (PTBP1), and small chemical molecules or are based on a combination of several factors and the location in the central nervous system. However, some recent papers did not obtain expected results, and discrepancies exist. Therefore, in this review, we discuss the history of neuronal transdifferentiation, summarize the strategies for neuronal replenishment and conversion from glia, especially astrocytes, and point out that biosafety, new strategies, and the accurate origin of the truly converted neurons in vivo should be focused upon in future studies. It also arises the attention of replenishing the lost neurons from glia by gene therapies such as up-regulation of some transcription factors or down-regulation of PTBP1 or drug interference therapies.

Neurod1 mediates the reprogramming of NG2 glial into neurons in vitro

Neuronal defect and loss are the main pathological processes of many central nervous system diseases. Cellular reprogramming is a promising method to supplement lost neurons. However, study on cellular reprogramming is still limited and its mechanism remains unclear. Herein, the effect of Neurod1 expression on differentiation of NG2 glia into neurons was investigated. In this study, we successfully isolated NG2 glial cells from mice prior to identification with immunofluorescence. Afterwards, AAV-Neurod1 virus was used to construct Neurod1 overexpression vectors in NG2 glia. Later, we detected neuronal markers expression with immunofluorescence and real time quantitative polymerase-chain reaction (qRT-PCR). Besides, expression of MAPK-signaling-pathway-related proteins were detected by western blotting technique. Through immunofluorescence and qRT-PCR techniques, we observed that Neurod1 overexpression contributed to NG2 cells differentiated into neurons. Further experiments also showed that Neurod1 overexpression induced the activation of MAPK pathway, but PD98059 (a selective inhibitor of MAPK pathway) partly inhibited the neuronal differentiation induced by Neurod1 overexpression. These findings suggest that Neurod1 could promote NG2 glia cells differentiating into neurons, wherein the mechanism under the differentiation is related to activation of MAPK pathway.

Dynamic Involvement of Striatal NG2-glia in L-DOPA Induced Dyskinesia in Parkinsonian Rats: Effects of Doxycycline.

NG2-glia alters its dynamics in response to L-DOPA-induced dyskinesia. In these animals, striatal NG2-glia density was reduced with cells presenting activated phenotype while doxycycline antidyskinetic therapy promotes a return to NG2-glia cell density and protein to a not activated state.

Application and prospects of somatic cell reprogramming technology for spinal cord injury treatment.

Spinal cord injury (SCI) is a serious neurological trauma that is challenging to treat. After SCI, many neurons in the injured area die due to necrosis or apoptosis, and astrocytes, oligodendrocytes, microglia and other non-neuronal cells become dysfunctional, hindering the repair of the injured spinal cord. Corrective surgery and biological, physical and pharmacological therapies are commonly used treatment modalities for SCI; however, no current therapeutic strategies can achieve complete recovery. Somatic cell reprogramming is a promising technology that has gradually become a feasible therapeutic approach for repairing the injured spinal cord. This revolutionary technology can reprogram fibroblasts, astrocytes, NG2 cells and neural progenitor cells into neurons or oligodendrocytes for spinal cord repair. In this review, we provide an overview of the transcription factors, genes, microRNAs (miRNAs), small molecules and combinations of these factors that can mediate somatic cell reprogramming to repair the injured spinal cord. Although many challenges and questions related to this technique remain, we believe that the beneficial effect of somatic cell reprogramming provides new ideas for achieving functional recovery after SCI and a direction for the development of treatments for SCI.

Cells of the Central Nervous System: An Overview of Their Structure and Function.

The central nervous system is the last major organ system in the vertebrate body to yield its cellular structure, due to the complexity of its cells and their interactions. The fundamental unit of the nervous system is the neuron, which forms complex circuits that receive and integrate information and generate adaptive responses. Each neuron is composed of an input domain consisting of multiple dendrites along with the cell body, which is also responsible for the majority of macromolecule synthesis for the cell. The output domain is the axon which is a singular extension from the cell body that propagates the action potential to the synapse, where signals pass from one neuron to another. Facilitating these functions are cohorts of supporting cells consisting of astrocytes, oligodendrocytes and microglia along with NG2 cells and ependymal cells. Astrocytes have a dazzling array of functions including physical support, maintenance of homeostasis, development and integration of synaptic activity. Oligodendrocytes form the myelin sheath which surrounds axons and enables rapid conduction of the nerve impulse. Microglia are the resident immune cells, providing immune surveillance and remodeling of neuronal circuits during development and trauma. All these cells function in concert with each other, producing the remarkably diverse functions of the nervous system.

The 5-HT and PLC Signaling Pathways Regulate the Secretion of IL-1β, TNF-α and BDNF from NG2 Cells

The present study was clarified the relationship between NG2 glial cells and 5-hydroxytryptamine (5-HT) to further revealed a role in the regulation of cortical excitability. The co-localization of NG2 cells and 5-HT in rat prefrontal cortex was determined using immunofluorescence. Different concentrations of 5-HT were applied to cultured NG2 cells. Real-time PCR measured the expression of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and brain-derived neurotrophic factor (BDNF). Changes in the expression of IL-1β, TNF-α, and BDNF in NG2 cells were detected after the addition of 5-HT receptor specific blockers and phospholipase C (PLC) specific activators and inhibitors. The results confirmed that the NG2 protein and 5-HT co-localized in the prefrontal cortex. 5-HT treatment of NG2 cells significantly reduced the expression of IL-1β and BDNF mRNA and increased the expression of TNF-α. The 5-HT receptor specific inhibitors alverine citrate, ketanserin, ondansetron and SB-399885 blocked the regulatory effects of 5-HT on NG2 cells. The PLC signal was linked to the secretion of IL-1β, TNF-α and BDNF in NG2 cells. These results indicated that 5-HT affected IL-1β, TNF-α, and BDNF secretion from NG2 cells via the 5-HT1A, 5-HT2A, 5-HT3, 5-HT6 receptors and the PLC signaling pathway.

Lineage Relationships Between Subpallial Progenitors and Glial Cells in the Piriform Cortex

The piriform cortex is a paleocortical area, located in the ventrolateral surface of the rodent forebrain, receiving direct input from the olfactory bulb. The three layers of the PC are defined by the diversity of glial and neuronal cells, marker expression, connections, and functions. However, the glial layering, ontogeny, and sibling cell relationship along the PC is an unresolved question in the field. Here, using multi-color genetic lineage tracing approaches with different StarTrack strategies, we performed a rigorous analysis of the derived cell progenies from progenitors located at the subpallium ventricular surface. First, we specifically targeted E12-progenitors with UbC-StarTrack to analyze their adult derived-cell progeny and their location within the piriform cortex layers. The vast majority of the cell progeny derived from targeted progenitors were identified as neurons, but also astrocytes and NG2 cells. Further, to specifically target single Gsx-2 subpallial progenitors and their derived cell-progeny in the piriform cortex, we used the UbC-(Gsx-2-hyPB)-StarTrack to perform an accurate analysis of their clonal relationships. Our results quantitatively delineate the adult clonal cell pattern from single subpallial E12-progenitors, focusing on glial cells. In summary, there is a temporal pattern in the assembly of the glial cell diversity in the piriform cortex, which also reveals spatio-temporal progenitor heterogeneity.

Spinal Cord Injury Inhibits the Differentiation and Maturation of NG2 Cells in the Cerebellum in Mice

Neuroimaging studies have shown that spinal cord injury (SCI) may lead to significant brain changes that are the key factors affecting functional recovery. However, little is known about the molecular and cellular biological mechanisms of these brain changes. The aim of this study was to investigate the molecular and cellular biological changes in the cerebellum after SCI. A total of 72 mice were randomly divided into 2 groups: sham group and SCI group. A mouse model of SCI was established by an aneurysm clip. Pathological examinations of the injured site were performed by hematoxylin and eosin staining and immunohistochemical. Western blot and immunohistochemical were used to determine the effect of SCI on the differentiation and maturation of NG2 cells. Compared with the sham group, the spinal cord tissue structure was disrupted and the motor function decreased significantly in the SCI group; the number of NG2 cells in the ansiform lobule crus Ⅰ increased on the 7th and 14th days, whereas the expression of oligodendrocyte transcription factor 2, myelin basic protein, and proteolipid protein decreased on the 7th and 14th days after SCI. These results showed that the differentiation and maturation of NG2 cells in the ansiform lobule crus Ⅰ were inhibited after SCI, resulting in the decrease of the formation of mature oligodendrocytes. These results indicate that SCI can lead to secondary changes in the cerebellum, which may affect the functional recovery. These findings may be used as biomarkers to evaluate the secondary changes in the brain after SCI.

Traumatic Brain Injury: Mechanisms of Glial Response.

Traumatic brain injury (TBI) is a heterogeneous disorder that involves brain damage due to external forces. TBI is the main factor of death and morbidity in young males with a high incidence worldwide. TBI causes central nervous system (CNS) damage under a variety of mechanisms, including synaptic dysfunction, protein aggregation, mitochondrial dysfunction, oxidative stress, and neuroinflammation. Glial cells comprise most cells in CNS, which are mediators in the brain’s response to TBI. In the CNS are present astrocytes, microglia, oligodendrocytes, and polydendrocytes (NG2 cells). Astrocytes play critical roles in brain’s ion and water homeostasis, energy metabolism, blood-brain barrier, and immune response. In response to TBI, astrocytes change their morphology and protein expression. Microglia are the primary immune cells in the CNS with phagocytic activity. After TBI, microglia also change their morphology and release both pro and anti-inflammatory mediators. Oligodendrocytes are the myelin producers of the CNS, promoting axonal support. TBI causes oligodendrocyte apoptosis, demyelination, and axonal transport disruption. There are also various interactions between these glial cells and neurons in response to TBI that contribute to the pathophysiology of TBI. In this review, we summarize several glial hallmarks relevant for understanding the brain injury and neuronal damage under TBI conditions.

Sera from Patients with NMOSD Reduce the Differentiation Capacity of Precursor Cells in the Central Nervous System.

Introduction: AQP4 (aquaporin-4)–immunoglobulin G (IgG)-mediated neuromyelitis optica spectrum disorder (NMOSD) is an inflammatory demyelinating disease that affects the central nervous system, particularly the spinal cord and optic nerve; remyelination capacity in neuromyelitis optica is yet to be determined, as is the role of AQP4–IgG in cell differentiation. Material and Methods: We included three groups—a group of patients with AQP4–IgG-positive neuromyelitis optica, a healthy group, and a sham group. We analyzed differentiation capacity in cultures of neurospheres from the subventricular zone of mice by adding serum at two different times: early and advanced stages of differentiation. We also analyzed differentiation into different cell lines. Results and Conclusions: The effect of sera from patients with NMOSD on precursor cells differs according to the degree of differentiation, and probably affects oligodendrocyte progenitor cells from NG2 cells to a lesser extent than cells from the subventricular zone; however, the resulting oligodendrocytes may be compromised in terms of maturation and possibly limited in their ability to generate myelin. Furthermore, these cells decrease in number with age. It is very unlikely that the use of drugs favoring the migration and differentiation of oligodendrocyte progenitor cells in multiple sclerosis would be effective in the context of neuromyelitis optica, but cell therapy with oligodendrocyte progenitor cells seems to be a potential alternative.

Identifying the functions of two biomarkers in human oligodendrocyte progenitor cell development

BackgroundHuman oligodendrocyte precursor cells (hOPCs) are an important source of myelinating cells for cell transplantation to treat demyelinating diseases. Myelin oligodendrocytes develop from migratory and proliferative hOPCs. It is well known that NG2 and A2B5 are important biological markers of hOPCs. However, the functional differences between the cell populations represented by these two biomarkers have not been well studied in depth.ObjectiveTo study the difference between NG2 and A2B5 cells in the development of human oligodendrocyte progenitor cells.MethodsUsing cell sorting technology, we obtained NG2+/−, A2B5+/− cells. Further research was then conducted via in vitro cell proliferation and migration assays, single-cell sequencing, mRNA sequencing, and cell transplantation into shiverer mice.ResultsThe proportion of PDGFR-α + cells in the negative cell population was higher than that in the positive cell population. The migration ability of the NG2+/−, A2B5+/− cells was inversely proportional to their myelination ability. The migration, proliferation, and myelination capacities of the negative cell population were stronger than those of the positive cell population. The ability of cell migration and proliferation of the four groups of cells from high to low was: A2B5− > NG2− > NG2+ > A2B5+. The content of PDGFR-α+ cells and the ability of cell differentiation from high to low was: NG2− > A2B5− > A2B5+ > NG2+.ConclusionIn summary, NG2+ and A2B5+ cells have poor myelination ability due to low levels of PDGFR-α+ cells. Therefore, hOPCs with a higher content of PDGFR-α+ cells may have a better effect in the cell transplantation treatment of demyelinating diseases.

The Notch Signaling Pathway Regulates Differentiation of NG2 Cells into Oligodendrocytes in Demyelinating Diseases.

NG2 cells are highly proliferative glial cells that can self-renew or differentiate into oligodendrocytes, promoting remyelination. Following demyelination, the proliferative and differentiation potentials of NG2 cells increase rapidly, enhancing their differentiation into functional myelinating cells. Levels of the transcription factors Olig1 and Olig2 increase during the differentiation of NG2 cells and play important roles in the development and repair of oligodendrocytes. However, the ability to generate new oligodendrocytes is hampered by injury-related factors (e.g., myelin fragments, Wnt and Notch signaling components), leading to failed differentiation and maturation of NG2 cells into oligodendrocytes. Here, we review Notch signaling as a negative regulator of oligodendrocyte differentiation and discuss the extracellular ligands, intracellular pathways, and key transcription factors involved.

Age-related changes in NG2-expressing telocytes of rat stomach.

NG2 immunoreactive cells (NG2 cells) are found in the brain and peripheral tissues including the skin, intestinal tracts, and bladder. In a previous study, we observed the presence of NG2 cells in the stomach using bioluminescence imaging techniques in NG2-firefly luciferase (fLuc) transgenic (Tg) rats. Here, we aimed to identify and characterize NG2 cells in the adult rat stomach. Immunohistochemical studies showed that NG2 cells were mainly present in the lamina propria and most of the cells were gastric telocytes, co-expressing CD34, and platelet-derived growth factor receptor alpha (PDGFRα), with a small oval-shaped cell body and extremely long and thin cellular prolongations. In the rat stomach, NG2-expressing telocytes comprised two subpopulations: NG2+/CD34+/PDGFRα+ and NG2+/CD34+/PDGFRα-. Furthermore, we showed that the expression of NG2 gene in the aged rat stomach decreased relative to that of the young rat stomach and the decline of NG2 expression in aged rats was mainly observed in NG2+/CD34+/PDGFRα+ telocytes. These findings suggested age-related alterations in NG2+/CD34+/PDGFRα+ telocytes of rat stomach.

Glial cells: More than just a silent disciple of neural system

The dominant focus of neuroscience has long been neurons and synapses; thus, under the neuronal doctrine, the pathophysiology of psychiatric disorders has come to be recognized, ignoring other types of CNS cells. Many cells, including neurons and glial cells, make up our brain: astrocytes, oligodendrocytes, NG2 cells, microglia and ependyma. Neurons were known to be the brain's basic information processing unit, so much of the neurological research was based solely on neurons. Recent research, however, show that glial cells are far more than just the "glue" binding the CNS neurons together. Glial cells provide neurons with support functions and they are much more numerous than neurons. A novel theory has shown that glial and neurons can talk and understand the same chemical language, so glial cell dysfunction results in abnormal neuro - glial interactions, which in turn impairs neuronal cell functionality. It can shed new light on the explanation of several mysterious aspects by digging up the glial functions and further comprehension of these vital cells, and the interaction between neurons and glial.

Characterization of cell type-specific S100B expression in the mouse olfactory bulb

S100B is an EF-hand type Ca2+-binding protein of the S100 family, known to support neurogenesis and to promote the interactions between brain’s nervous and immune systems. Here, we characterized the expression of S100B in the mouse olfactory bulb, a neurogenic niche comprising mature and adult-born neurons, astrocytes, oligodendrocytes and microglia. Besides astrocytes, for which S100B is a classical marker, S100B was also expressed in NG2 cells and, surprisingly, in APC-positive myelinating oligodendrocytes but not in mature/adult-born neurons or microglia. Various layers of the bulb differed substantially in the composition of S100B-positive cells, with the highest fraction of the APC-positive oligodendrocytes found in the granule cell layer. Across all layers, ∼50 % of NG2 cells were S100B-negative. Finally, our data revealed a strong correlation between the fraction of myelinating oligodendrocytes among the S100B-positive cells and the oligodendrocyte density in different brain areas, underscoring the importance of S100B for the establishment and maintenance of myelin sheaths.

Rab6A as a Pan-Astrocytic Marker in Mouse and Human Brain, and Comparison with Other Glial Markers (GFAP, GS, Aldh1L1, SOX9).

Astrocytes contribute to many higher brain functions. A key mechanism in glia-to-neuron signalling is vesicular exocytosis; however, the identity of exocytosis organelles remains a matter of debate. Since vesicles derived from the trans-Golgi network (TGN) are not considered in this context, we studied the astrocyte TGN by immunocytochemistry applying anti-Rab6A. In mouse brain, Rab6A immunostaining is found to be unexpectedly massive, diffuse in all regions, and is detected preferentially and abundantly in the peripheral astrocyte processes, which is hardly evident without glial fibrillary acid protein (GFAP) co-staining. All cells positive for the astrocytic markers glutamine synthetase (GS), GFAP, aldehyde dehydrogenase 1 family member L1 (Aldh1L1), or SRY (sex determining region Y)-box 9 (SOX9) were Rab6A+. Rab6A is excluded from microglia, oligodendrocytes, and NG2 cells using cell type-specific markers. In human cortex, Rab6A labelling is very similar and associated with GFAP+ astrocytes. The mouse data also confirm the specific astrocytic labelling by Aldh1L1 or SOX9; the astrocyte-specific labelling by GS sometimes debated is replicated again. In mouse and human brain, individual astrocytes display high variability in Rab6A+ structures, suggesting dynamic regulation of the glial TGN. In summary, Rab6A expression is an additional, global descriptor of astrocyte identity. Rab6A might constitute an organelle system with a potential role of Rab6A in neuropathological and physiological processes.

Reactivity and increased proliferation of NG2 cells following central nervous system infection with Theiler\u2019s murine encephalomyelitis virus

BackgroundNeuron-glial antigen 2 (NG2) cells are a glial cell type tiled throughout the gray and white matter of the central nervous system (CNS). NG2 cells are known for their ability to differentiate into oligodendrocytes and are commonly referred to as oligodendrocyte precursor cells. However, recent investigations have begun to identify additional functions of NG2 cells in CNS health and pathology. NG2 cells form physical and functional connections with neurons and other glial cell types throughout the CNS, allowing them to monitor and respond to the neural environment. Growing evidence indicates that NG2 cells become reactive under pathological conditions, though their specific roles are only beginning to be elucidated. While reactive microglia and astrocytes are well-established contributors to neuroinflammation and the development of epilepsy following CNS infection, the dynamics of NG2 cells remain unclear. Therefore, we investigated NG2 cell reactivity in a viral-induced mouse model of temporal lobe epilepsy.MethodsC57BL6/J mice were injected intracortically with Theiler’s murine encephalomyelitis virus (TMEV) or PBS. Mice were graded twice daily for seizures between 3 and 7 days post-injection (dpi). At 4 and 14 dpi, brains were fixed and stained for NG2, the microglia/macrophage marker IBA1, and the proliferation marker Ki-67. Confocal z stacks were acquired in both the hippocampus and the overlying cortex. Total field areas stained by each cell marker and total field area of colocalized pixels between NG2 and Ki67 were compared between groups.ResultsBoth NG2 cells and microglia/macrophages displayed increased immunoreactivity and reactive morphologies in the hippocampus of TMEV-injected mice. While increased immunoreactivity for IBA1 was also present in the cortex, there was no significant change in NG2 immunoreactivity in the cortex following TMEV infection. Colocalization analysis for NG2 and Ki-67 revealed a significant increase in overlap between NG2 and Ki-67 in the hippocampus of TMEV-injected mice at both time points, but no significant differences in cortex.ConclusionsNG2 cells acquire a reactive phenotype and proliferate in response to TMEV infection. These results suggest that NG2 cells alter their function in response to viral encephalopathy, making them potential targets to prevent the development of epilepsy following viral infection.

Novel guanidine compounds inhibit platelet-derived growth factor receptor alpha transcription and oligodendrocyte precursor cell proliferation.

Oligodendrocyte precursor cells (OPCs), also known as NG2 cells or polydendrocytes, are distributed widely throughout the developing and mature central nervous system. They remain proliferative throughout life and are an important source of myelinating cells in normal and demyelinating brain as well as a source of glioma, the most common type of primary brain tumor with a poor prognosis. OPC proliferation is dependent on signaling mediated by platelet-derived growth factor (PDGF) AA binding to its alpha receptor (PDGFRα). Here, we describe a group of structurally related compounds characterized by the presence of a basic guanidine group appended to an aromatic core that is effective in specifically repressing the transcription of Pdgfra but not the related beta receptor (Pdgfrb) in OPCs. These compounds specifically and dramatically reduced proliferation of OPCs but not that of astrocytes and did not affect signal transduction by PDGFRα. These findings suggest that the compounds could be further developed for potential use in combinatorial treatment strategies for neoplasms with dysregulated PDGFRα function.

Inactivation of HIF-prolyl 4-hydroxylases 1, 2 and 3 in NG2-expressing cells induces HIF2-mediated neurovascular expansion independent of erythropoietin.

AimNG2 cells in the brain are comprised of pericytes and NG2 glia and play an important role in the execution of cerebral hypoxia responses, including the induction of erythropoietin (EPO) in pericytes. Oxygen‐dependent angiogenic responses are regulated by hypoxia‐inducible factor (HIF), the activity of which is controlled by prolyl 4‐hydroxylase domain (PHD) dioxygenases and the von Hippel‐Lindau (VHL) tumour suppressor. However, the role of NG2 cells in HIF‐regulated cerebral vascular homeostasis is incompletely understood.MethodsTo examine the HIF/PHD/VHL axis in neurovascular homeostasis, we used a Cre‐loxP‐based genetic approach in mice and targeted Vhl, Epo, Phd1, Phd2, Phd3 and Hif2a in NG2 cells. Cerebral vasculature was assessed by immunofluorescence, RNA in situ hybridization, gene and protein expression analysis, gel zymography and in situ zymography.Results Vhl inactivation led to a significant increase in angiogenic gene and Epo expression. This was associated with EPO‐independent expansion of capillary networks in cortex, striatum and hypothalamus, as well as pericyte proliferation. A comparable phenotype resulted from the combined inactivation of Phd2 and Phd3, but not from Phd2 inactivation alone. Concomitant PHD1 function loss led to further expansion of the neurovasculature. Genetic inactivation of Hif2a in Phd1/Phd2/Phd3 triple mutant mice resulted in normal cerebral vasculature.ConclusionOur studies establish (a) that HIF2 activation in NG2 cells promotes neurovascular expansion and remodelling independently of EPO, (b) that HIF2 activity in NG2 cells is co‐controlled by PHD2 and PHD3 and (c) that PHD1 modulates HIF2 transcriptional responses when PHD2 and PHD3 are inactive.