Understanding astrocyte differentiation: Clinical relevance, technical challenges, and new opportunities in the omics era.

Astrocytes are a major type of glial cells that have essential functions in development and homeostasis of the central nervous system (CNS). Immature astrocytes in the developing CNS support neuronal maturation and possess neural-stem-cell-like properties. Mature astrocytes partially lose these functions but gain new functions essential for adult CNS homeostasis. In pathological conditions, astrocytes become "reactive", which disrupts their mature homeostatic functions and reactivates some immature astrocyte-like properties, suggesting a partial reversal of astrocyte maturation. The loss of homeostatic astrocyte functions contributes to the pathogenesis of various neurological conditions, and therefore activating maturation-promoting mechanisms may be a promising therapeutic strategy to restore homeostasis. Manipulating the mechanisms underlying astrocyte maturation might also allow to facilitate CNS regeneration by enhancing developmental functions of adult astrocytes. However, such therapeutic strategies are still some distance away because of our limited understanding of astrocyte differentiation and maturation, due to biological and technical challenges, including the high degree of similarity of astrocytes with neural stem cells and the shortcomings of astrocyte markers. Current advances in systems biology have a huge potential to overcome these challenges. Recent transcriptomic analyses have already revealed new astrocyte markers and new regulators of astrocyte differentiation. However, the epigenomic changes that presumably occur during astrocyte differentiation remain an important, largely unexplored area for future research. Emerging technologies such as CRISPR/Cas9-based functional screens will further improve our understanding of the mechanisms underlying astrocyte differentiation. This may open up new clinical approaches to restore homeostasis in neurological disorders and/or promote CNS regeneration. This article is categorized under: Neurological Diseases > Genetics/Genomics/Epigenetics Neurological Diseases > Stem Cells and Development Neurological Diseases > Molecular and Cellular Physiology.

IKK2/NF-κB Activation in Astrocytes Reduces amyloid ß Deposition: A Process Associated with Specific Microglia Polarization.

Alzheimer's disease (AD) is a common neurodegenerative disease that is accompanied by pronounced neuroinflammatory responses mainly characterized by marked microgliosis and astrogliosis. However, it remains open as to how different aspects of astrocytic and microglial activation affect disease progression. Previously, we found that microglia expansion in the spinal cord, initiated by IKK2/NF-κB activation in astrocytes, exhibits stage-dependent beneficial effects on the progression of amyotrophic lateral sclerosis. Here, we investigated the impact of NF-κB-initiated neuroinflammation on AD pathogenesis using the APP23 mouse model of AD in combination with conditional activation of IKK2/NF-κB signaling in astrocytes. We show that NF-κB activation in astrocytes triggers a distinct neuroinflammatory response characterized by striking astrogliosis as well as prominent microglial reactivity. Immunohistochemistry and Congo red staining revealed an overall reduction in the size and number of amyloid plaques in the cerebral cortex and hippocampus. Interestingly, isolated primary astrocytes and microglia cells exhibit specific marker gene profiles which, in the case of microglia, point to an enhanced plaque clearance capacity. In contrast, direct IKK2/NF-κB activation in microglia results in a pro-inflammatory polarization program. Our findings suggest that IKK2/NF-κB signaling in astrocytes may activate paracrine mechanisms acting on microglia function but also on APP processing in neurons.

Extensive transcriptional and chromatin changes underlie astrocyte maturation in vivo and in culture.

Astrocytes have essential functions in brain homeostasis that are established late in differentiation, but the mechanisms underlying the functional maturation of astrocytes are not well understood. Here we identify extensive transcriptional changes that occur during murine astrocyte maturation in vivo that are accompanied by chromatin remodelling at enhancer elements. Investigating astrocyte maturation in a cell culture model revealed that in vitro-differentiated astrocytes lack expression of many mature astrocyte-specific genes, including genes for the transcription factors Rorb, Dbx2, Lhx2 and Fezf2. Forced expression of these factors in vitro induces distinct sets of mature astrocyte-specific transcripts. Culturing astrocytes in a three-dimensional matrix containing FGF2 induces expression of Rorb, Dbx2 and Lhx2 and improves astrocyte maturity based on transcriptional and chromatin profiles. Therefore, extrinsic signals orchestrate the expression of multiple intrinsic regulators, which in turn induce in a modular manner the transcriptional and chromatin changes underlying astrocyte maturation.

IKK2/NF-κB signaling protects neurons after traumatic brain injury.

Traumatic brain injury (TBI) is the leading cause of death in young adults. After the initial injury, a poorly understood secondary phase, including a strong inflammatory response determines the final outcome of TBI. The inhibitor of NF-κB kinase (IKK)/NF-κB signaling system is the key regulator of inflammation and also critically involved in regulation of neuronal survival and synaptic plasticity. We addressed the neuron-specific function of IKK2/NF-κB signaling pathway in TBI using an experimental model of closed-head injury (CHI) in combination with mouse models allowing conditional regulation of IKK/NF-κB signaling in excitatory forebrain neurons. We found that repression of IKK2/NF-κB signaling in neurons increases the acute posttraumatic mortality rate, worsens the neurological outcome, and promotes neuronal cell death by apoptosis, thus resulting in enhanced proinflammatory gene expression. As a potential mechanism, we identified elevated levels of the proapoptotic mediators Bax and Bad and enhanced expression of stress response genes. This phenotype is also observed when neuronal IKK/NF-κB activity is inhibited just before CHI. In contrast, neuron-specific activation of IKK/NF-κB signaling does not alter the TBI outcome. Thus, this study demonstrates that physiological neuronal IKK/NF-κB signaling is necessary and sufficient to protect neurons from trauma consequences.-Mettang, M., Reichel, S. N., Lattke, M., Palmer, A., Abaei, A., Rasche, V., Huber-Lang, M., Baumann, B., Wirth, T. IKK2/NF-κB signaling protects neurons after traumatic brain injury.

Transient IKK2 activation in astrocytes initiates selective non-cell-autonomous neurodegeneration.

BACKGROUND: Neuroinflammation is associated with a wide range of neurodegenerative disorders, however the specific contribution to individual disease pathogenesis and selective neuronal cell death is not well understood. Inflammatory cerebellar ataxias are neurodegenerative diseases occurring in various autoimmune/inflammatory conditions, e.g. paraneoplastic syndromes. However, how inflammatory insults can cause selective cerebellar neurodegeneration in the context of these diseases remains open, and appropriate animal models are lacking. A key regulator of neuroinflammatory processes is the NF-κB signalling pathway, which is activated by the IκB kinase 2 (IKK2) in response to various pathological conditions. Importantly, its activation is sufficient to initiate neuroinflammation on its own. METHODS: To investigate the contribution of IKK/NF-κB-mediated neuroinflammation to neurodegeneration, we established conditional mouse models of cerebellar neuroinflammation, which depend either on the tetracycline-regulated expression of IKK2 in astrocytes or Cre-recombination based IKK2 activation in Bergmann glia. RESULTS: We demonstrate that IKK2 activation for a limited time interval in astrocytes is sufficient to induce neuroinflammation, astrogliosis and loss of Purkinje neurons, resembling the pathogenesis of inflammatory cerebellar ataxias. We identified IKK2-driven irreversible dysfunction of Bergmann glia as critical pathogenic event resulting in Purkinje cell loss. This was independent of Lipocalin 2, an acute phase protein secreted by reactive astrocytes and well known to mediate neurotoxicity. Instead, downregulation of the glutamate transporters EAAT1 and EAAT2 and ultrastructural alterations suggest an excitotoxic mechanism of Purkinje cell degeneration. CONCLUSIONS: Our results suggest a novel pathogenic mechanism how diverse inflammatory insults can cause inflammation/autoimmune-associated cerebellar ataxias. Disease-mediated elevation of danger signals like TLR ligands and inflammatory cytokines in the cerebellum activates IKK2/NF-κB signalling in astrocytes, which as a consequence triggers astrogliosis-like activation of Bergmann glia and subsequent non-cell-autonomous Purkinje cell degeneration. Notably, the identified hit and run mechanism indicates only an early window for therapeutic interventions.

Sustained, neuron-specific IKK/NF-κB activation generates a selective neuroinflammatory response promoting local neurodegeneration with aging.

BACKGROUND: Increasing evidence indicates that neuroinflammation is a critical factor contributing to the progression of various neurodegenerative diseases. The IKK/NF-κB signalling system is a central regulator of inflammation, but it also affects neuronal survival and differentiation. A complex interplay between different CNS resident cells and infiltrating immune cells, which produce and respond to various inflammatory mediators, determines whether neuroinflammation is beneficial or detrimental. The IKK/NF-κB system is involved in both production of and responses to these mediators, although the precise contribution depends on the cell type as well as the cellular context, and is only partially understood. Here we investigated the specific contribution of neuronal IKK/NF-κB signalling on the regulation of neuroinflammatory processes and its consequences. To address this issue, we established and analysed a conditional gain-of-function mouse model that expresses a constitutively active allele of IKK2 in principal forebrain neurons (IKK2nCA). Proinflammatory gene and growth factor expression, histopathology, microgliosis, astrogliosis, immune cell infiltration and spatial learning were assessed at different timepoints after persistent canonical IKK2/NF-κB activation. RESULTS: In contrast to other cell types and organ systems, chronic IKK2/NF-κB signalling in forebrain neurons of adult IKK2nCA animals did not cause a full-blown inflammatory response including infiltration of immune cells. Instead, we found a selective inflammatory response in the dentate gyrus characterized by astrogliosis, microgliosis and Tnf-α upregulation. Furthermore, downregulation of the neurotrophic factor Bdnf correlated with a selective and progressive atrophy of the dentate gyrus and a decline in hippocampus-dependent spatial learning. Neuronal degeneration was associated with increased Fluoro-jade staining, but lacked activation of apoptosis. Remarkably, neuronal loss could be partially reversed when chronic IKK2/NF-κB signalling was turned off and Bdnf expression was restored. CONCLUSION: Our results demonstrate that persistent IKK2/NF-κB signalling in forebrain neurons does not induce overall neuroinflammation, but elicits a selective inflammatory response in the dentate gyrus accompanied by decreased neuronal survival and impaired learning and memory. Our findings further suggest that chronic activation of neuronal IKK2/NF-κB signalling, possibly as a consequence of neuroinflammatory conditions, is able to induce apoptosis-independent neurodegeneration via paracrine suppression of Bdnf synthesis.

Nuclear factor κB activation impairs ependymal ciliogenesis and links neuroinflammation to hydrocephalus formation.

Hydrocephalus formation is a frequent complication of neuropathological insults associated with neuroinflammation. However, the mechanistic role of neuroinflammation in hydrocephalus development is unclear. We have investigated the function of the proinflammatory acting inhibitor of κB kinase (IKK)/nuclear factor κB (NF-κB) signaling system in neuroinflammatory processes and generated a novel mouse model that allows conditional activation of the IKK/NF-κB system in astrocytes. Remarkably, NF-κB activation in astrocytes during early postnatal life results in hydrocephalus formation and additional defects in brain development. NF-κB activation causes global neuroinflammation characterized by a strong, astrocyte-specific expression of proinflammatory NF-κB target genes as well as a massive infiltration and activation of macrophages. In this animal model, hydrocephalus formation is specifically induced during a critical time period of early postnatal development, in which IKK/NF-κB-induced neuroinflammation interferes with ependymal ciliogenesis. Our findings demonstrate for the first time that IKK/NF-κB activation is sufficient to induce hydrocephalus formation and provides a potential mechanistic explanation for the frequent association of neuroinflammation and hydrocephalus formation during brain development, namely impairment of ependymal cilia formation. Therefore, our study might open up new perspectives for the treatment of certain types of neonatal and childhood hydrocephalus associated with hemorrhages and infections.

Astrocyte-specific IKK2 activation in mice is sufficient to induce neuroinflammation but does not increase susceptibility to MPTP.

A key regulator of inflammatory gene expression is the transcription factor NF-κB that is controlled by the IκB proteins. We used a transgenic mouse model expressing a constitutively active IκB-kinase-2 (IKK2-CA) in astrocytes under control of the human glial fibrillary acidic protein promotor (IKK2-mice) to investigate neuroinflammation, proinflammatory cytokine expression, microglial activation and a potential enhanced susceptibility to the neurotoxin MPTP (4×10 mg/kg). Readouts included the determination of cytokines, striatal dopamine (DA), nigral tyrosine hydroxylase (TH) positive neurons, microglial activation and motor activity. IKK2-CA expression in astrocytes conditionally induced by the tet-off system resulted in a widespread neuroinflammation indicated by the increased expression of inflammatory cytokines and the presence of activated microglia and astrogliosis. Additionally, striatal DA concentrations but not nigral TH-positive neurons were reduced in IKK2-mice by 20%. Motor activity of IKK2-mice was not affected. Surprisingly, there was a similar reduction in striatal DA concentrations and the number of nigral TH-positive neurons in IKK2 and control mice after MPTP treatment. In conclusion, although naïve IKK2-mice showed reduced striatal DA concentrations and an increase in inflammatory markers in the brain, a higher susceptibility to MPTP was not observed. This finding argues against a prominent role of astrocyte specific, IKK2-mediated neuroinflammation in MPTP-induced neurodegeneration.

IκB kinase/nuclear factor κB-dependent insulin-like growth factor 2 (Igf2) expression regulates synapse formation and spine maturation via Igf2 receptor signaling.

Alterations of learning and memory in mice with deregulated neuron-specific nuclear factor κB (NF-κB) activity support the idea that plastic changes of synaptic contacts may depend at least in part on IκB kinase (IKK)/NF-κB-related synapse-to-nucleus signaling. There is, however, little information on the molecular requirements and mechanisms regulating this IKK/NF-κB-dependent synapse development and remodeling. Here, we report that the NF-κB inducing IKK kinase complex is localized at the postsynaptic density (PSD) and activated under basal conditions in the adult mouse brain. Using different models of conditional genetic inactivation of IKK2 function in mouse principal neurons, we show that IKK/NF-κB signaling is critically involved in synapse formation and spine maturation in the adult brain. IKK/NF-κB blockade in the forebrain of mutant animals is associated with reduced levels of mature spines and postsynaptic proteins PSD95, SAP97, GluA1, AMPAR-mediated basal synaptic transmission and a spatial learning impairment. Synaptic deficits can be restored in adult animals within 1 week by IKK/NF-κB reactivation, indicating a highly dynamic IKK/NF-κB-dependent regulation process. We further identified the insulin-like growth factor 2 gene (Igf2) as a novel IKK/NF-κB target. Exogenous Igf2 was able to restore synapse density and promoted spine maturation in IKK/NF-κB signaling-deficient neurons within 24 h. This process depends on Igf2/Igf2R-mediated MEK/ERK activation. Our findings illustrate a fundamental role of IKK/NF-κB-Igf2-Igf2R signaling in synapse formation and maturation in adult mice, thus providing an intriguing link between the molecular actions of IKK/NF-κB in neurons and the memory enhancement factor Igf2.