Molecular diversity of diencephalic astrocytes reveals adult astrogenesis regulated by Smad4.

Astrocytes regulate brain-wide functions and also show region-specific differences, but little is known about how general and region-specific functions are aligned at the single-cell level. To explore this, we isolated adult mouse diencephalic astrocytes by ACSA-2-mediated magnetic-activated cell sorting (MACS). Single-cell RNA-seq revealed 7 gene expression clusters of astrocytes, with 4 forming a supercluster. Within the supercluster, cells differed by gene expression related to ion homeostasis or metabolism, with the former sharing gene expression with other regions and the latter being restricted to specific regions. All clusters showed expression of proliferation-related genes, and proliferation of diencephalic astrocytes was confirmed by immunostaining. Clonal analysis demonstrated low level of astrogenesis in the adult diencephalon, but not in cerebral cortex grey matter. This led to the identification of Smad4 as a key regulator of diencephalic astrocyte in vivo proliferation and in vitro neurosphere formation. Thus, astrocytes show diverse gene expression states related to distinct functions with some subsets being more widespread while others are more regionally restricted. However, all share low-level proliferation revealing the novel concept of adult astrogenesis in the diencephalon.

Repetitive injury and absence of monocytes promote astrocyte self-renewal and neurological recovery.

Unlike microglia and NG2 glia, astrocytes are incapable of migrating to sites of injury in the posttraumatic cerebral cortex, instead relying on proliferation to replenish their numbers and distribution in the affected region. However, neither the spectrum of their proliferative repertoire nor their postinjury distribution has been examined in vivo. Using a combination of different thymidine analogs and clonal analysis in a model of repetitive traumatic brain injury, we show for the first time that astrocytes that are quiescent following an initial injury can be coerced to proliferate after a repeated insult in the cerebral cortex grey matter. Interestingly, this process is promoted by invasion of monocytes to the injury site, as their genetic ablation (using CCR2-/- mice) increased the number of repetitively dividing astrocytes at the expense of newly proliferating astrocytes in repeatedly injured parenchyma. These differences profoundly affected both the distribution of astrocytes and recovery period for posttraumatic behavior deficits suggesting key roles of astrocyte self-renewal in brain repair after injury.

Lipoprotein receptor loss in forebrain radial glia results in neurological deficits and severe seizures.

The Alzheimer disease-associated multifunctional low-density lipoprotein receptor-related protein-1 is expressed in the brain. Recent studies uncovered a role of this receptor for the appropriate functioning of neural stem cells, oligodendrocytes, and neurons. The constitutive knock-out (KO) of the receptor is embryonically lethal. To unravel the receptors' role in the developing brain we generated a mouse mutant by specifically targeting radial glia stem cells of the dorsal telencephalon. The low-density lipoprotein receptor-related protein-1 lineage-restricted KO female and male mice, in contrast to available models, developed a severe neurological phenotype with generalized seizures during early postnatal development. The mechanism leading to a buildup of hyperexcitability and emergence of seizures was traced to a failure in adequate astrocyte development and deteriorated postsynaptic density integrity. The detected impairments in the astrocytic lineage: precocious maturation, reactive gliosis, abolished tissue plasminogen activator uptake, and loss of functionality emphasize the importance of this glial cell type for synaptic signaling in the developing brain. Together, the obtained results highlight the relevance of astrocytic low-density lipoprotein receptor-related protein-1 for glutamatergic signaling in the context of neuron-glia interactions and stage this receptor as a contributing factor for epilepsy.

Cross-talk between monocyte invasion and astrocyte proliferation regulates scarring in brain injury.

Scar formation after brain injury is still poorly understood. To further elucidate such processes, here, we examine the interplay between astrocyte proliferation taking place predominantly at the vascular interface and monocyte invasion. Using genetic mouse models that decrease or increase reactive astrocyte proliferation, we demonstrate inverse effects on monocyte numbers in the injury site. Conversely, reducing monocyte invasion using CCR2-/- mice causes a strong increase in astrocyte proliferation, demonstrating an intriguing negative cross-regulation between these cell types at the vascular interface. CCR2-/- mice show reduced scar formation with less extracellular matrix deposition, smaller lesion site and increased neuronal coverage. Surprisingly, the GFAP+ scar area in these mice is also significantly decreased despite increased astrocyte proliferation. Proteomic analysis at the peak of increased astrocyte proliferation reveals a decrease in extracellular matrix synthesizing enzymes in the injury sites of CCR2-/- mice, highlighting how early key aspects of scar formation are initiated. Taken together, we provide novel insights into the cross-regulation of juxtavascular proliferating astrocytes and invading monocytes as a crucial mechanism of scar formation upon brain injury.

Changes in the Proliferative Program Limit Astrocyte Homeostasis in the Aged Post-Traumatic Murine Cerebral Cortex.

Aging leads to adverse outcomes after traumatic brain injury. The mechanisms underlying these defects, however, are not yet clear. In this study, we found that astrocytes in the aged post-traumatic cerebral cortex develop a significantly reduced proliferative response, resulting in reduced astrocyte numbers in the penumbra. Moreover, experiments of reactive astrocytes in vitro reveal that their diminished proliferation is due to an age-related switch in the division mode with reduced cell-cycle re-entry rather than changes in cell-cycle length. Notably, reactive astrocytes in vivo and in vitro become refractory to stimuli increasing their proliferation during aging, such as Sonic hedgehog signaling. These data demonstrate for the first time that age-dependent, most likely intrinsic changes in the proliferative program of reactive astrocytes result in their severely hampered proliferative response to traumatic injury thereby affecting astrocyte homeostasis.

Reactive astrocytes as neural stem or progenitor cells: In vivo lineage, In vitro potential, and Genome-wide expression analysis.

Here, we review the stem cell hallmarks of endogenous neural stem cells (NSCs) during development and in some niches of the adult mammalian brain to then compare these with reactive astrocytes acquiring stem cell hallmarks after traumatic and ischemic brain injury. Notably, even endogenous NSCs including the earliest NSCs, the neuroepithelial cells, generate in most cases only a single type of progeny and self-renew only for a rather short time in vivo. In vitro, however, especially cells cultured under neurosphere conditions reveal a larger potential and long-term self-renewal under the influence of growth factors. This is rather well comparable to reactive astrocytes in the traumatic or ischemic brain some of which acquire neurosphere-forming capacity including multipotency and long-term self-renewal in vitro, while they remain within their astrocyte lineage in vivo. Both reactive astrocytes and endogenous NSCs exhibit stem cell hallmarks largely in vitro, but their lineage differs in vivo. Both populations generate largely a single cell type in vivo, but endogenous NSCs generate neurons and reactive astrocytes remain in the astrocyte lineage. However, at some early postnatal stages or in some brain regions reactive astrocytes can be released from this fate restriction, demonstrating that they can also enact neurogenesis. Thus, reactive astrocytes and NSCs share many characteristic hallmarks, but also exhibit key differences. This conclusion is further substantiated by genome-wide expression analysis comparing NSCs at different stages with astrocytes from the intact and injured brain parenchyma.

Astrocyte reactivity after brain injury-: The role of galectins 1 and 3.

Astrocytes react to brain injury in a heterogeneous manner with only a subset resuming proliferation and acquiring stem cell properties in vitro. In order to identify novel regulators of this subset, we performed genomewide expression analysis of reactive astrocytes isolated 5 days after stab wound injury from the gray matter of adult mouse cerebral cortex. The expression pattern was compared with astrocytes from intact cortex and adult neural stem cells (NSCs) isolated from the subependymal zone (SEZ). These comparisons revealed a set of genes expressed at higher levels in both endogenous NSCs and reactive astrocytes, including two lectins-Galectins 1 and 3. These results and the pattern of Galectin expression in the lesioned brain led us to examine the functional significance of these lectins in brains of mice lacking Galectins 1 and 3. Following stab wound injury, astrocyte reactivity including glial fibrillary acidic protein expression, proliferation and neurosphere-forming capacity were found significantly reduced in mutant animals. This phenotype could be recapitulated in vitro and was fully rescued by addition of Galectin 3, but not of Galectin 1. Thus, Galectins 1 and 3 play key roles in regulating the proliferative and NSC potential of a subset of reactive astrocytes.

Shared inflammatory glial cell signature after stab wound injury, revealed by spatial, temporal, and cell-type-specific profiling of the murine cerebral cortex.

Traumatic brain injury leads to a highly orchestrated immune- and glial cell response partially responsible for long-lasting disability and the development of secondary neurodegenerative diseases. A holistic understanding of the mechanisms controlling the responses of specific cell types and their crosstalk is required to develop an efficient strategy for better regeneration. Here, we combine spatial and single-cell transcriptomics to chart the transcriptomic signature of the injured male murine cerebral cortex, and identify specific states of different glial cells contributing to this signature. Interestingly, distinct glial cells share a large fraction of injury-regulated genes, including inflammatory programs downstream of the innate immune-associated pathways Cxcr3 and Tlr1/2. Systemic manipulation of these pathways decreases the reactivity state of glial cells associated with poor regeneration. The functional relevance of the discovered shared signature of glial cells highlights the importance of our resource enabling comprehensive analysis of early events after brain injury.

Injury-specific factors in the cerebrospinal fluid regulate astrocyte plasticity in the human brain.

The glial environment influences neurological disease progression, yet much of our knowledge still relies on preclinical animal studies, especially regarding astrocyte heterogeneity. In murine models of traumatic brain injury, beneficial functions of proliferating reactive astrocytes on disease outcome have been unraveled, but little is known regarding if and when they are present in human brain pathology. Here we examined a broad spectrum of pathologies with and without intracerebral hemorrhage and found a striking correlation between lesions involving blood-brain barrier rupture and astrocyte proliferation that was further corroborated in an assay probing for neural stem cell potential. Most importantly, proteomic analysis unraveled a crucial signaling pathway regulating this astrocyte plasticity with GALECTIN3 as a novel marker for proliferating astrocytes and the GALECTIN3-binding protein LGALS3BP as a functional hub mediating astrocyte proliferation and neurosphere formation. Taken together, this work identifies a therapeutically relevant astrocyte response and their molecular regulators in different pathologies affecting the human cerebral cortex.

TDP-43 condensates and lipid droplets regulate the reactivity of microglia and regeneration after traumatic brain injury.

Decreasing the activation of pathology-activated microglia is crucial to prevent chronic inflammation and tissue scarring. In this study, we used a stab wound injury model in zebrafish and identified an injury-induced microglial state characterized by the accumulation of lipid droplets and TAR DNA-binding protein of 43 kDa (TDP-43)+ condensates. Granulin-mediated clearance of both lipid droplets and TDP-43+ condensates was necessary and sufficient to promote the return of microglia back to the basal state and achieve scarless regeneration. Moreover, in postmortem cortical brain tissues from patients with traumatic brain injury, the extent of microglial activation correlated with the accumulation of lipid droplets and TDP-43+ condensates. Together, our results reveal a mechanism required for restoring microglia to a nonactivated state after injury, which has potential for new therapeutic applications in humans.

Chondroitin sulfates are required for fibroblast growth factor-2-dependent proliferation and maintenance in neural stem cells and for epidermal growth factor-dependent migration of their progeny.

The neural stem cell niche of the embryonic and adult forebrain is rich in chondroitin sulfate glycosaminoglycans (CS-GAGs) that represent complex linear carbohydrate structures on the cell surface of neural stem/progenitor cells or in their intimate environment. We reported earlier that the removal of CS-GAGs with the bacterial enzyme chondroitinase ABC (ChABC) reduced neural stem/progenitor cell proliferation and self-renewal, whereas this treatment favored astroglia formation at the expense of neurogenesis. Here, we studied the consequences of CS-deglycanation further and revealed that CS-GAGs are selectively required for neurosphere formation, proliferation, and self-renewal of embryonic cortical neural stem/progenitor cells in response to fibroblast growth factor (FGF)-2. Consistently, the FGF-2-dependent activation of the MAPKinase in neural stem/progenitor cells was diminished after ChABC treatment, but unaltered after epidermal growth factor (EGF) stimulation. Upon EGF treatment, fewer radial glia were brain lipid-binding protein (BLBP)-positive, whereas more were glutamate aspartate transporter (GLAST)-positive after CS-GAG removal. Only in this latter situation, GLAST-positive radial glia cells extended processes that supported neuronal migration from differentiating neurospheres. CS-deglycanation also selectively increased astrocyte numbers and their migration in response to EGF. Thus, our approach revealed that CS-GAGs are essential for FGF-2-mediated proliferation and maintenance of neuron-generating neural stem/progenitor cells. Simultaneously, CS-GAGs act as a brake on the EGF-dependent maturation, migration, and gliogenesis of neural stem/progenitor cells. We conclude that neural stem/progenitor cell subpopulations reside in neurospheres that are distinguishable by their responsiveness to FGF-2 and EGF which is differentially regulated by CS-carbohydrate structures.

Focal laser-lesions activate an endogenous population of neural stem/progenitor cells in the adult visual cortex.

CNS lesions stimulate adult neurogenic niches. Endogenous neural stem/progenitor cells represent a potential resource for CNS regeneration. Here, we investigate the response to unilateral focal laser-lesions applied to the visual cortex of juvenile rats. Within 3 days post-lesion, an ipsilateral increase of actively cycling cells was observed in cortical layer one and in the callosal white matter within the lesion penumbra. The cells expressed the neural stem/progenitor cell marker Nestin and the 473HD-epitope. Tissue prepared from the lesion area by micro-dissection generated self-renewing, multipotent neurospheres, while cells from the contralateral visual cortex did not. The newly formed neural stem/progenitor cells in the lesion zone might support neurogenesis, as suggested by the expression of Pax6 and Doublecortin, a marker of newborn neurons. We propose that focal laser-lesions may induce the emergence of stem/progenitor cells with neurogenic potential. This could underlie the beneficial effects of laser application in neurosurgery.

Investigating Age-Related Changes in Proliferation and the Cell Division Repertoire of Parenchymal Reactive Astrocytes.

Reactive gliosis is a complicated process involving all types of glial cells and is the therapeutic target of efforts to treat several types of neuropathologies. Parenchymal astrocytes continuously survey their microenvironment to identify even tiny abnormalities in the central nervous system (CNS) homeostasis and react rapidly to brain damage, such as following ischemia, trauma, or neurodegenerative diseases, to prevent propagation of tissue damage. Aging can play causal roles in certain astroglial dysfunctions, however, still little is known to what extent the heterogeneous reaction of astrocytes at the injury site might be impaired over the course of aging. Based on our experience with both in vitro and in vivo experimental paradigms, we describe here in detail the analysis of age-related changes in (1) proliferative response of parenchymal astrocytes within the posttraumatic cerebral cortex grey matter (GM), and (2) repertoire of their cell divisions in adherent cell culture prepared from the injured GM of young and old double transgenic GFAP-mRFP1/(FUCCI)-S/G2/M-mAG-hGeminin mice by single cell time-lapse imaging.

Traumatic Brain Injury: At the Crossroads of Neuropathology and Common Metabolic Endocrinopathies.

Building on the seminal work by Geoffrey Harris in the 1970s, the neuroendocrinology field, having undergone spectacular growth, has endeavored to understand the mechanisms of hormonal connectivity between the brain and the rest of the body. Given the fundamental role of the brain in the orchestration of endocrine processes through interactions among neurohormones, it is thus not surprising that the structural and/or functional alterations following traumatic brain injury (TBI) can lead to endocrine changes affecting the whole organism. Taking into account that systemic hormones also act on the brain, modifying its structure and biochemistry, and can acutely and chronically affect several neurophysiological endpoints, the question is to what extent preexisting endocrine dysfunction may set the stage for an adverse outcome after TBI. In this review, we provide an overview of some aspects of three common metabolic endocrinopathies, e.g., diabetes mellitus, obesity, and thyroid dysfunction, and how these could be triggered by TBI. In addition, we discuss how the complex endocrine networks are woven into the responses to sudden changes after TBI, as well as some of the potential mechanisms that, separately or synergistically, can influence outcomes after TBI.

Re-evaluation of neuronal P2X7 expression using novel mouse models and a P2X7-specific nanobody.

The P2X7 channel is involved in the pathogenesis of various CNS diseases. An increasing number of studies suggest its presence in neurons where its putative functions remain controversial for more than a decade. To resolve this issue and to provide a model for analysis of P2X7 functions, we generated P2X7 BAC transgenic mice that allow visualization of functional EGFP-tagged P2X7 receptors in vivo. Extensive characterization of these mice revealed dominant P2X7-EGFP protein expression in microglia, Bergmann glia, and oligodendrocytes, but not in neurons. These findings were further validated by microglia- and oligodendrocyte-specific P2X7 deletion and a novel P2X7-specific nanobody. In addition to the first quantitative analysis of P2X7 protein expression in the CNS, we show potential consequences of its overexpression in ischemic retina and post-traumatic cerebral cortex grey matter. This novel mouse model overcomes previous limitations in P2X7 research and will help to determine its physiological roles and contribution to diseases.

The unique 473HD-Chondroitinsulfate epitope is expressed by radial glia and involved in neural precursor cell proliferation.

Neural stem cells have been documented in both the developing and the mature adult CNSs of mammals. This cell population holds a considerable promise for therapeutical applications in a wide array of CNS diseases. Therefore, universally applicable strategies for the purification of this population to further its cell biological characterization are sought. Here, we report that the unique chondroitin sulfate epitope recognized by the monoclonal antibody 473HD is surface expressed on actively cycling, multipotent progenitor cells of the developing telencephalon with radial glia-like properties. When used for immunopanning, the antibody enriched at least threefold for neural stem/progenitor cells characterized by the ability to self-renew as neurospheres that generated all major neural lineages in differentiation assays. In contrast, the 473HD-depleted cell fraction was mostly devoid of neurosphere-forming cells. The isolation of 473HD-positive adult multipotent progenitors from the subependymal zone of the lateral ventricle wall revealed a substantial overlap with the known adult neural stem cell marker LewisX. When the chondroitin sulfates were removed from immunoselected 473HD-positive neural stem/progenitor cell surfaces by chondroitinase ABC treatment or perturbed by the monoclonal antibody 473HD that recognizes the unique DSD-1 chondroitin sulfate epitope, the generation of neurospheres was significantly reduced. Thus, the 473HD epitope could not only be used for the isolation of multipotent neural progenitors during forebrain development as well as from the adult neurogenic niche but may also constitute a functionally important entity of the neural stem cell niche.

Reactive glia in the injured brain acquire stem cell properties in response to sonic hedgehog. [corrected].

As a result of brain injury, astrocytes become activated and start to proliferate in the vicinity of the injury site. Recently, we had demonstrated that these reactive astrocytes, or glia, can form self-renewing and multipotent neurospheres in vitro. In the present study, we demonstrate that it is only invasive injury, such as stab wounding or cerebral ischemia, and not noninvasive injury conditions, such as chronic amyloidosis or induced neuronal death, that can elicit this increase in plasticity. Furthermore, we find that Sonic hedgehog (SHH) is the signal that acts directly on the astrocytes and is necessary and sufficient to elicit the stem cell response both in vitro and in vivo. These findings provide a molecular basis for how cells with neural stem cell lineage emerge at sites of brain injury and imply that the high levels of SHH known to enter the brain from extraneural sources after invasive injury can trigger this response.

Structural and functional analysis of chondroitin sulfate proteoglycans in the neural stem cell niche.

The stem cell niche plays an important role for the maintenance and differentiation of neural stem/progenitor cells (NSPCs). It is composed of distinct cell types that influence NSCPs by the release of paracrine factors, and a specialized extracellular matrix that structures the NSPC environment. During the past years, several components of the neural stem cell (NSC) niche could be deciphered on the molecular level. One prominent constituent is the tenascin-C (Tnc) glycoprotein and its isoforms that intervene in NSPC proliferation and differentiation. Distinct chondroitin sulfate proteoglycans (CSPGs) associate with Tnc in the niche territory and we could show that these have functional connotations in the stem cell compartment in their own rights. In this chapter, we give an account of the tools and methods we developed to unravel the structures and functions of CSPGs in the NSC niche.

Chondroitin sulfate glycosaminoglycans control proliferation, radial glia cell differentiation and neurogenesis in neural stem/progenitor cells.

Although the local environment is known to regulate neural stem cell (NSC) maintenance in the central nervous system, little is known about the molecular identity of the signals involved. Chondroitin sulfate proteoglycans (CSPGs) are enriched in the growth environment of NSCs both during development and in the adult NSC niche. In order to gather insight into potential biological roles of CSPGs for NSCs, the enzyme chondroitinase ABC (ChABC) was used to selectively degrade the CSPG glycosaminoglycans. When NSCs from mouse E13 telencephalon were cultivated as neurospheres, treatment with ChABC resulted in diminished cell proliferation and impaired neuronal differentiation, with a converse increase in astrocytes. The intrauterine injection of ChABC into the telencephalic ventricle at midneurogenesis caused a reduction in cell proliferation in the ventricular zone and a diminution of self-renewing radial glia, as revealed by the neurosphere-formation assay, and a reduction in neurogenesis. These observations suggest that CSPGs regulate neural stem/progenitor cell proliferation and intervene in fate decisions between the neuronal and glial lineage.

Evidence for distinct leptomeningeal cell-dependent paracrine and EGF-linked autocrine regulatory pathways for suppression of fibrillar collagens in astrocytes.

A unique and unresolved property of the central nervous system is that its extracellular matrix lacks fibrillar elements. In the present report, we show that astrocytes secrete triple helices of fibrillar collagens type I, III and V in culture, while no astroglial collagen expression could be detected in vivo. We discovered two inhibitory mechanisms that could underlie this apparent discrepancy. Thus, we uncover a strong inhibitory effect of meningeal cells on astrocytic collagen expression in coculture assays. Furthermore, we present evidence that EGF-receptor activation downregulates collagen expression in astrocytes via an autocrine loop. These investigations provide a rational framework to explain why the brain is devoid of collagen fibers, which is a unique feature that characterizes the structure of the neural extracellular matrix. Moreover, fibrillar collagens were found transiently upregulated in a laser-induced cortical lesion, suggesting that these could contribute to the glial scar that inhibits axonal regeneration.