ß-Adrenergic Signaling Promotes Morphological Maturation of Astrocytes in Female Mice.

Astrocytes play essential roles in the developing nervous system, including supporting synapse function. These astrocyte support functions emerge coincident with brain maturation and may be tailored in a region-specific manner. For example, gray matter astrocytes have elaborate synapse-associated processes and are morphologically and molecularly distinct from white matter astrocytes. This raises the question of whether there are unique environmental cues that promote gray matter astrocyte identity and synaptogenic function. We previously identified adrenergic receptors as preferentially enriched in developing gray versus white matter astrocytes, suggesting that noradrenergic signaling could be a cue that promotes the functional maturation of gray matter astrocytes. We first characterized noradrenergic projections during postnatal brain development in mouse and human, finding that process density was higher in the gray matter and increased concurrently with astrocyte maturation. RNA sequencing revealed that astrocytes in both species expressed α- and ß-adrenergic receptors. We found that stimulation of ß-adrenergic receptors increased primary branching of rodent astrocytes in vitro Conversely, astrocyte-conditional knockout of the ß1-adrenergic receptor reduced the size of gray matter astrocytes and led to dysregulated sensorimotor integration in female mice. These studies suggest that adrenergic signaling to developing astrocytes impacts their morphology and has implications for adult behavior, particularly in female animals. More broadly, they demonstrate a mechanism through which environmental cues impact astrocyte development. Given the key roles of norepinephrine in brain states, such as arousal, stress, and learning, these findings could prompt further inquiry into how developmental stressors impact astrocyte development and adult brain function.SIGNIFICANCE STATEMENT This study demonstrates a role for noradrenergic signaling in the development of gray matter astrocytes. We provide new evidence that the ß1-adrenergic receptor is robustly expressed by both mouse and human astrocytes, and that conditional KO of the ß1-adrenergic receptor from female mouse astrocytes impairs gray matter astrocyte maturation. Moreover, female conditional KO mice exhibit behavioral deficits in two paradigms that test sensorimotor function. Given the emerging interest in moving beyond RNA sequencing to probe specific pathways that underlie astrocyte heterogeneity, this study provides a foundation for future investigation into the effect of noradrenergic signaling on astrocyte functions in conditions where noradrenergic signaling is altered, such as stress, arousal, and learning.

Loss of Serpin E2 alters antimicrobial gene expression by microglia but not astrocytes.

Microglia are the brain-resident immune cells responsible for surveilling and protecting the central nervous system. These cells can express a wide array of immune genes, and that expression can become highly dynamic in response to changes in the environment, such as traumatic injury or neurological disease. Though microglial immune responses are well studied, we still do not know many mechanisms and regulators underlying all the varied microglial responses. Serpin E2 is a serine protease inhibitor that acts on a wide variety of serine proteases, with particularly potent affinity for the blood clotting enzyme thrombin. In the brain, Serpin E2 is highly expressed by many cell types, especially glia, and loss of Serpin E2 leads to behavioral changes as well as deficits in synaptic plasticity. To determine whether Serpin E2 is important for maintaining homeostasis in glia, we performed RNA sequencing of microglia and astrocytes from Serpin E2-deficient mice in a healthy state or under immune activation due to lipopolysaccharide (LPS) injection. We found that microglia in Serpin E2-deficient mice had higher expression of antimicrobial genes, while astrocytes did not display any robust changes in transcription. Furthermore, the lack of Serpin E2 did not affect transcriptional responses to LPS in either microglia or astrocytes. Overall, we find that Serpin E2 is a regulator of antimicrobial genes in microglia.

Prdm16 and Vcam1 regulate the postnatal disappearance of embryonic radial glia and the ending of cortical neurogenesis.

Embryonic neural stem cells (NSCs, i.e., radial glia) in the ventricular-subventricular zone (V-SVZ) generate the majority of neurons and glia in the forebrain. Postnatally, embryonic radial glia disappear and a subpopulation of radial glia transition into adult NSCs. As this transition occurs, widespread neurogenesis in brain regions such as the cerebral cortex ends. The mechanisms that regulate the postnatal disappearance of radial glia and the ending of embryonic neurogenesis remain poorly understood. Here, we show that PR domain-containing 16 (Prdm16) promotes the disappearance of radial glia and the ending of neurogenesis in the cerebral cortex. Genetic deletion of Prdm16 from NSCs leads to the persistence of radial glia in the adult V-SVZ and prolonged postnatal cortical neurogenesis. Mechanistically, Prdm16 induces the postnatal reduction in Vascular Cell Adhesion Molecule 1 (Vcam1). The postnatal disappearance of radial glia and the ending of cortical neurogenesis occur normally in Prdm16-Vcam1 double conditional knockout mice. These observations reveal novel molecular regulators of the postnatal disappearance of radial glia and the ending of embryonic neurogenesis, filling a key knowledge gap in NSC biology.

Transplantation of human neural progenitor cells secreting GDNF into the spinal cord of patients with ALS: a phase 1/2a trial.

Amyotrophic lateral sclerosis (ALS) involves progressive motor neuron loss, leading to paralysis and death typically within 3-5 years of diagnosis. Dysfunctional astrocytes may contribute to disease and glial cell line-derived neurotrophic factor (GDNF) can be protective. Here we show that human neural progenitor cells transduced with GDNF (CNS10-NPC-GDNF) differentiated to astrocytes protected spinal motor neurons and were safe in animal models. CNS10-NPC-GDNF were transplanted unilaterally into the lumbar spinal cord of 18 ALS participants in a phase 1/2a study (NCT02943850). The primary endpoint of safety at 1 year was met, with no negative effect of the transplant on motor function in the treated leg compared with the untreated leg. Tissue analysis of 13 participants who died of disease progression showed graft survival and GDNF production. Benign neuromas near delivery sites were common incidental findings at post-mortem. This study shows that one administration of engineered neural progenitors can provide new support cells and GDNF delivery to the ALS patient spinal cord for up to 42 months post-transplantation.

Conservation and divergence of vulnerability and responses to stressors between human and mouse astrocytes.

Astrocytes play important roles in neurological disorders such as stroke, injury, and neurodegeneration. Most knowledge on astrocyte biology is based on studies of mouse models and the similarities and differences between human and mouse astrocytes are insufficiently characterized, presenting a barrier in translational research. Based on analyses of acutely purified astrocytes, serum-free cultures of primary astrocytes, and xenografted chimeric mice, we find extensive conservation in astrocytic gene expression between human and mouse samples. However, the genes involved in defense response and metabolism show species-specific differences. Human astrocytes exhibit greater susceptibility to oxidative stress than mouse astrocytes, due to differences in mitochondrial physiology and detoxification pathways. In addition, we find that mouse but not human astrocytes activate a molecular program for neural repair under hypoxia, whereas human but not mouse astrocytes activate the antigen presentation pathway under inflammatory conditions. Here, we show species-dependent properties of astrocytes, which can be informative for improving translation from mouse models to humans.

Astrocyte-to-astrocyte contact and a positive feedback loop of growth factor signaling regulate astrocyte maturation.

Astrocytes are critical for the development and function of the central nervous system. In developing brains, immature astrocytes undergo morphological, molecular, cellular, and functional changes as they mature. Although the mechanisms that regulate the maturation of other major cell types in the central nervous system such as neurons and oligodendrocytes have been extensively studied, little is known about the cellular and molecular mechanisms that control astrocyte maturation. Here, we identified molecular markers of astrocyte maturation and established an in vitro assay for studying the mechanisms of astrocyte maturation. Maturing astrocytes in vitro exhibit similar molecular changes and represent multiple molecular subtypes of astrocytes found in vivo. Using this system, we found that astrocyte-to-astrocyte contact strongly promotes astrocyte maturation. In addition, secreted signals from microglia, oligodendrocyte precursor cells, or endothelial cells affect a small subset of astrocyte genes but do not consistently change astrocyte maturation. To identify molecular mechanisms underlying astrocyte maturation, we treated maturing astrocytes with molecules that affect the function of tumor-associated genes. We found that a positive feedback loop of heparin-binding epidermal growth factor-like growth factor (HBEGF) and epidermal growth factor receptor (EGFR) signaling regulates astrocytes maturation. Furthermore, HBEGF, EGFR, and tumor protein 53 (TP53) affect the expression of genes important for cilium development, the circadian clock, and synapse function. These results revealed cellular and molecular mechanisms underlying astrocytes maturation with implications for the understanding of glioblastoma.

Young bone marrow transplantation preserves learning and memory in old mice.

Restoration of cognitive function in old mice by transfer of blood or plasma from young mice has been attributed to reduced C-C motif chemokine ligand 11 (CCL11) and ß2-microglobulin, which are thought to suppress neurogenesis in the aging brain. However, the specific role of the hematopoietic system in this rejuvenation has not been defined and the importance of neurogenesis in old mice is unclear. Here we report that transplantation of young bone marrow to rejuvenate the hematopoietic system preserved cognitive function in old recipient mice, despite irradiation-induced suppression of neurogenesis, and without reducing ß2-microglobulin. Instead, young bone marrow transplantation preserved synaptic connections and reduced microglial activation in the hippocampus. Circulating CCL11 levels were lower in young bone marrow recipients, and CCL11 administration in young mice had the opposite effect, reducing synapses and increasing microglial activation. In conclusion, young blood or bone marrow may represent a future therapeutic strategy for neurodegenerative disease.

Transplantation of Neural Progenitor Cells Expressing Glial Cell Line-Derived Neurotrophic Factor into the Motor Cortex as a Strategy to Treat Amyotrophic Lateral Sclerosis.

Early dysfunction of cortical motor neurons may underlie the initiation of amyotrophic lateral sclerosis (ALS). As such, the cortex represents a critical area of ALS research and a promising therapeutic target. In the current study, human cortical-derived neural progenitor cells engineered to secrete glial cell line-derived neurotrophic factor (GDNF) were transplanted into the SOD1G93A ALS rat cortex, where they migrated, matured into astrocytes, and released GDNF. This protected motor neurons, delayed disease pathology and extended survival of the animals. These same cells injected into the cortex of cynomolgus macaques survived and showed robust GDNF expression without adverse effects. Together this data suggests that introducing cortical astrocytes releasing GDNF represents a novel promising approach to treating ALS. Stem Cells 2018;36:1122-1131.

Human iPSC-Derived Endothelial Cells and Microengineered Organ-Chip Enhance Neuronal Development.

Human stem cell-derived models of development and neurodegenerative diseases are challenged by cellular immaturity in vitro. Microengineered organ-on-chip (or Organ-Chip) systems are designed to emulate microvolume cytoarchitecture and enable co-culture of distinct cell types. Brain microvascular endothelial cells (BMECs) share common signaling pathways with neurons early in development, but their contribution to human neuronal maturation is largely unknown. To study this interaction and influence of microculture, we derived both spinal motor neurons and BMECs from human induced pluripotent stem cells and observed increased calcium transient function and Chip-specific gene expression in Organ-Chips compared with 96-well plates. Seeding BMECs in the Organ-Chip led to vascular-neural interaction and specific gene activation that further enhanced neuronal function and in vivo-like signatures. The results show that the vascular system has specific maturation effects on spinal cord neural tissue, and the use of Organ-Chips can move stem cell models closer to an in vivo condition.

Survival of iPSC-derived grafts within the striatum of immunodeficient mice: Importance of developmental stage of both transplant and host recipient.

Degeneration of the striatum can occur in multiple disorders with devastating consequences for the patients. Infantile infections with streptococcus, measles, or herpes can cause striatal necrosis associated with dystonia or dyskinesia; and in patients with Huntington's disease the striatum undergoes massive degeneration, leading to behavioral, psychological and movement issues, ultimately resulting in death. Currently, only supportive therapies are available for striatal degeneration. Clinical trials have shown some efficacy using transplantation of fetal-derived primary striatal progenitors. Large banks of fetal progenitors that give rise to medium spiny neurons (MSNs), the primary neuron of the striatum, are needed to make transplantation therapy a reality. However, fetal tissue is of limited supply, has ethical concerns, and is at risk of graft immunorejection. An alternative potential source of MSNs is induced pluripotent stem cells (iPSCs), adult somatic tissues reprogrammed back to a stem cell fate. Multiple publications have demonstrated the ability to differentiate striatal MSNs from iPSCs. Previous publications have demonstrated that the efficacy of fetal progenitor transplants is critically dependent upon the age of the donor embryo/fetus as well as the age of the transplant recipient. With the advent of iPSC technology, a question that remains unanswered concerns the graft's "age," which is crucial since transplanting pluripotent cells has an inherent risk of over proliferation and teratoma formation. Therefore, in order to also determine the effect of transplant recipient age on the graft, iPSCs were differentiated to three stages along a striatal differentiation paradigm and transplanted into the striatum of both neonatal and adult immunodeficient mice. This study demonstrated that increased murine transplant-recipient age (adult vs neonate) resulted in decreased graft survival and volume/rostro-caudal spread after six weeks in vivo, regardless of "age" of the cells transplanted. Importantly, this study implicates that the in vivo setting may provide a better neurogenic niche for iPSC-based modeling as compared to the in vitro setting. Together, these results recapitulate findings from fetal striatal progenitor transplantation studies and further demonstrate the influence of the host environment on cellular survival and maturation.

Human neural progenitors differentiate into astrocytes and protect motor neurons in aging rats.

Age-associated health decline presents a significant challenge to healthcare, although there are few animal models that can be used to test potential treatments. Here, we show that there is a significant reduction in both spinal cord motor neurons and motor function over time in the aging rat. One explanation for this motor neuron loss could be reduced support from surrounding aging astrocytes. Indeed, we have previously shown using in vitro models that aging rat astrocytes are less supportive to rat motor neuron function and survival over time. Here, we test whether rejuvenating the astrocyte niche can improve the survival of motor neurons in an aging spinal cord. We transplanted fetal-derived human neural progenitor cells (hNPCs) into the aging rat spinal cord and found that the cells survive and differentiate into astrocytes with a much higher efficiency than when transplanted into younger animals, suggesting that the aging environment stimulates astrocyte maturation. Importantly, the engrafted astrocytes were able to protect against motor neuron loss associated with aging, although this did not result in an increase in motor function based on behavioral assays. We also transplanted hNPCs genetically modified to secrete glial cell line-derived neurotrophic factor (GDNF) into the aging rat spinal cord, as this combination of cell and protein delivery can protect motor neurons in animal models of ALS. During aging, GDNF-expressing hNPCs protected motor neurons, though to the same extent as hNPCs alone, and again had no effect on motor function. We conclude that hNPCs can survive well in the aging spinal cord, protect motor neurons and mature faster into astrocytes when compared to transplantation into the young spinal cord. While there was no functional improvement, there were no functional deficits either, further supporting a good safety profile of hNPC transplantation even into the older patient population.

Axon initial segment-associated microglia.

Microglia are the brain's resident immune cells and function as the main defense against pathogens or injury. However, in the absence of disease, microglia have other functions in the normal brain. For example, previous studies showed that microglia contribute to circuit refinement and synaptic plasticity in the developing and adult brain, respectively. Thus, microglia actively participate in regulating neuronal excitability and function. Here, we report that in the cortex, but not other brain regions, a subset of microglia extend a single process that specifically associates and overlaps with the axon initial segment (AIS), the site where action potentials are generated. Similar associations were not observed with dendrites or distal axons. Microglia-AIS interactions appear early in development, persist throughout adulthood, and are conserved across species including mice, rats, and primates. However, these interactions are lost after microglial activation following brain injury, suggesting that such interactions may be part of healthy brain function. Loss of microglial CX3CR1 receptors, or the specialized extracellular matrix surrounding the AIS, did not disrupt the interaction. However, loss of AIS proteins by the neuron-specific deletion of the master AIS scaffold AnkyrinG disrupted microglia-AIS interactions. These results reveal a unique population of microglia that specifically interact with the AIS in the adult cortex.