Browsing by Subject "Astrocyte"

Chronically implanted recording electrode arrays linked to prosthetics have the potential to make positive impacts on patients suffering from full or partial paralysis [1;2]. Such arrays are implanted into the patient's cortical tissue and record extracellular potentials from nearby neurons, allowing the information encoded by the neuronal discharges to control external devices. While such systems perform well during acute recordings, they often fail to function reliably in clinically relevant chronic settings [3]. Available evidence suggests that a major failure mode of electrode arrays is the brain tissue reaction against these implants (termed the glial scar), making the biocompatibility of implanted electrodes a primary concern in device design. Previous studies have focused on modifying the form factor of recording arrays, implanting such arrays in experimental animals, and, upon explantation, evaluating the glial scarring in response to the implant after several weeks in vivo. Because of a lack of information regarding the mechanisms involved in the tissue reaction to implanted biomaterials in the brain, it is not surprising that these in vivo studies have met with limited success. This dissertation describes the development of a simple, controlled in vitro model of glial scarring and the utilization of that model to probe the cellular and molecular mechanisms behind glial scarring.

A novel in vitro model of glial scarring was developed by adapting a primary cell-based system previously used for studying neuroinflammatory processes in neurodegenerative disease [4]. Midbrains from embryonic day 14 Fischer 344 rats were mechanically dissociated and grown on poly-D-lysine coated 24 well plates to a confluent layer of neurons, astrocytes, and microglia. The culture was injured with either a mechanical scrape or foreign-body placement (segments of 50 mm diameter stainless steel microwire), fixed at time points from 6 h to 10 days, and assessed by immunocytochemistry. Microglia invaded the scraped wound area at early time points and hypertrophied activated astrocytes repopulated the wound after 7 days. The chronic presence of microwire resulted in a glial scar forming at 10 days, with microglia forming an inner layer of cells coating the microwire, while astrocytes surrounded the microglial core with a network of cellular processes containing upregulated GFAP. Neurons within the culture did not repopulate the scrape wound and did not respond to the microwire, although they were determined to be electrically active through patch clamp recording.

This initial model recreated many of the hallmarks of glial scarring around electrodes used for recording in the brain; however, the model lacked the reproducibility necessary to establish a useful characterization tool. After the protocol was amended to resemble protocols typically used to culture neural stem/precursor cells, an intense scarring reaction was consistently seen [5]. To further optimize and characterize the reaction, six independent cell culture variables (growth media, seeding density, bFGF addition day, serum concentration in treatment media, treatment day, and duration of culture) were varied systematically and the resulting scars were quantified. The following conditions were found to give the highest level of scarring: Neurobasal medium supplemented with B27, 10% fetal bovine serum at treatment, 10 ng/ml b-FGF addition at seeding and at treatment, treatment at least 6 days after seeding and scar growth of at least 5 days. Seeding density did not affect scarring as long as at least 500,000 cells were seeded per well, but appropriate media, bFGF, and serum were essential for significant scar formation.

The optimized in vitro model was then used to help uncover the underlying molecular and cellular mechanisms behind glial scarring. A microwire coating that mimics the basal lamina present within glial scars was developed that allows cells responding to the coated microwire to be isolated and evaluated (i.e. through cell counting or cell staining). A panel of soluble factors known to be involved in glial scar formation was added to the media and the cellular response was recorded. The extent of cell accumulation on the coated microwires was significantly increased by titration of the culture with serum, the pleotropic growth factor bFGF, the inflammatory cytokines IL-1α and IL-1β, and the growth factors PDGF and BMP-2. The other fourteen soluble factors tested had little to no effect on the number of cells that attached to the coated microwires, although a specific blocker of the bFGF receptor was able to abrogate the effect of bFGF. This study proposes essential roles in glial scarring of serum, which infiltrates brain tissue upon disruption of the blood-brain barrier, and bFGF, which is a necessary growth and survival factor for the neural precursor cells that respond to injury. These insights suggest repeated rounds of implant micromotion-induced cellular damage, with the resultant neuronal death, serum release, and bFGF deposition may thicken the glial scar and lead to recording signal loss.

Synapse formation and elimination are two developmental processes that concurrently take place in the neonatal brain. Dysregulation of these two processes have been implicated in the etiology and progression of neurodevelopmental and neurodegenerative diseases. Previous work has found that in mice, the first three postnatal weeks are highly active periods of synapse remodeling throughout the entire brain. Glial cells called astrocytes are highly complex neural derived cells that are born and mature during this period. As they mature, astrocytes instruct the formation of synapses through contact with synaptic components and through the secretion of various synaptogenic factors. Microglia by contrast are the tissue resident macrophages of the central nervous system (CNS). During the first three postnatal weeks, microglia sculpt developing synaptic circuits by engulfment of synaptic components through various phagocytic mechanisms. While the field has steadily grown our understanding of the importance of these two cell types in synapse formation and elimination separately, few studies have addressed the possibility of communication between these two cell types to regulate their respective functions at synapses. Here I used the developing visual thalamocortical circuit as a model system to investigate the molecular cross talk between astrocytes and microglia. To address the impact of this communication on synapse development and function, I focused on one factor called Hevin/Sparcl1 which has previously been shown to be necessary and sufficient for thalamocortical synapse formation and plasticity. Previous studies have shown that Hevin induces thalamocortical synapse formation during the second postnatal week in mouse visual cortex. Hevin orchestrates this process by bridging pre-and post-synaptic cell adhesion molecules, Nrxn1α and Nlgn1B. Curiously, I found that despite high levels of Hevin in the maturing primary visual cortex, thalamocortical synapse numbers decrease even during the time when Hevin expression is at its peak. This refinement process, I determined, was dependent on microglia. Using super resolution microscopy, I found that only a subset thalamocortical synapses have Hevin at their cleft and that loss of Hevin aberrantly enhances microglia phagocytic activity. These initial findings suggested that Hevin likely functioned to spare only specific synapses from microglia mediated elimination. To interrogate this possibility, I used an in vitro microglia culture system to assess the transcriptional responses of microglia to Hevin treatment. Surprisingly, this treatment led to robust transcriptional changes in microglia that were distinct from well described immunological stimulation. This screen implicated Toll-like receptors (TLR) 2 and 4 in this transcriptional response. Further studies using our in vitro culture system showed that proteolytic cleavage of Hevin was required to upregulate TLR2 expression in microglia and that its C-terminus alone was sufficient to upregulate TLR2. Moving in vivo, I found that TLR2 expression is strongly developmentally regulated and highly heterogeneously expressed by microglia in the mouse primary visual cortex. Using overexpression studies in vivo, I also found that microglia strongly upregulate TLR2 in response to Hevin or Hevin’s C-terminus and that these TLR2 high microglia have enhanced phagocytic activity both in normal development and after Hevin/Hevin C-terminal overexpression. These findings indicate that Hevin function is regulated by proteolytic cleavage and suggest that Hevin is a dual signal in synaptic development: both to stimulate synapse formation by neurons and enhance synapse elimination by microglia. I next sought to test the functional relevance of the microglia specific response to Hevin. To do this, I used co-immunoprecipitation studies to identify candidate receptors for Hevin on microglia. I found that Hevin and its C-terminus interacted with both TLR2 and TLR4 but seemed to have a stronger affinity for TLR4. Therefore, I used TLR4 KO mice to test if microglia could still be stimulated by Hevin in vivo. I found that TLR4 KO microglia were no longer responsive to Hevin overexpression and had reduced phagocytic capacity compared to WT microglia. Ultimately, I found that TLR4 KO mice had impaired thalamocortical synapse refinement and impaired circuit plasticity. Taken together, my results identify astrocyte-derived Hevin as a synaptogenic molecule that links thalamocortical synapse formation with synaptic refinement mediated by microglia.

Naturally-occurring cell death is a fundamental developmental mechanism for regulating cell numbers and sculpting developing organs. This is particularly true in the central nervous system, where large numbers of neurons and oligodendrocytes are eliminated via apoptosis during normal development. Given the profound impact of death upon these two major cell populations, it is surprising that developmental death of another major cell type – the astrocyte – has rarely been studied. It is presently unclear whether astrocytes are subject to significant amounts of developmental death, or how it occurs. Here we address these questions using mouse retinal astrocytes as our model system. We show that the total number of retinal astrocytes declines by over 3-fold during a death period spanning postnatal days 5-14. Surprisingly, these astrocytes do not die by apoptosis, the canonical mechanism underlying the vast majority of developmental cell death. Instead, we find that microglia kill and engulf astrocytes to mediate their developmental removal. Genetic ablation of microglia inhibits astrocyte death, leading to a larger astrocyte population size at the end of the death period. However, astrocyte death is not completely blocked in the absence of microglia, apparently due to the ability of astrocytes to engulf each other. Nevertheless, mice lacking microglia showed significant anatomical changes to the retinal astrocyte network, with functional consequences for the astrocyte-associated vasculature leading to retinal hemorrhage. These results establish a novel modality for naturally-occurring cell death, and demonstrate its importance for formation and integrity of the retinal gliovascular network.

The brain, the source of human cognition, is composed of numerous cell types and a staggering number (over 1 quadrillion) of specialized connections called synapses. Each central nervous system (CNS) synapse is a complex entity organized as a presynaptic axon terminal and a postsynaptic dendritic structure, which pass chemical signals from one neuron to the next. How does each synapse form and how do synapses assemble into organized networks that permit cognition?

Research from the past two decades have identified that glial cells, predominantly astrocytes, regulate the formation and function of CNS synapses. Astrocytes are morphologically complex cells that interact with and ensheathe synapses through fine perisynaptic astrocyte processes. However, the molecular mechanisms of astrocyte development lay largely unknown. Furthermore, it is unclear how the morphology of the astrocyte is linked to its function as a regulator of synapse formation and function. Notwithstanding, more than a dozen astrocyte-secreted factors have been discovered that govern synaptogenesis, including proteins such as thrombospondins, glypicans, and hevin.

Here, I examine the molecular mechanism of astrocyte-synapse interactions and how excitatory synapses are assembled in the CNS using the mouse as a model system. First, I investigated the mechanism of hevin-induced thalamocortical excitatory synapse formation. I find that hevin trans-synaptically binds to two neuronal cell adhesion molecules, neurexin-1 and neuroligin-1B and that this molecular bridge is critical for the formation and plasticity of thalamocortical synapses in the cerebral cortex. Second, I uncover that astrocytes express a family of cell adhesion molecules, the neuroligins, which are critical for governing the developmental morphogenesis of astrocytes. Furthermore, I discover that astrocytic neuroligins are essential to establish the proper balance between synaptic excitation and inhibition in the brain.

Taken together, the data presented here detail two mechanisms of how astrocytes control the assembly of CNS synapses. Moreover, they highlight how bidirectional signals between astrocytes and neurons properly form the brain and its specialized connections. The research performed herein is of high importance to the clinical neuroscience community, because the genes investigated (neuroligins, neurexins, and hevin) are linked to neurological disorders such as autism and schizophrenia. By understanding of how these cells communicate in development and disease, the neuroscience field will hopefully be able to design therapeutics aimed at restoring cognition for patients afflicted with neurological impairments.

Developmental cell death is essential for nervous system development, sculpting the developing tissue by controlling cell numbers. While developmental neuron death has been studied extensively, the most abundant cell type of the nervous system – the astrocyte – has often been overlooked. Our lab recently showed that astrocytes in the developing retina undergo an unusual non-apoptotic form of death that eliminates a vast proportion of the original population. Further, we found that microglia are the major effectors of astrocyte death. However, the mechanisms that induce microglia to kill astrocytes remain mysterious. It is important to understand these astrocyte death mechanisms because astrocytes play a crucial role in patterning the retinal blood vessel network. Developmental perturbations to astrocyte number have large effects on their patterning, and in turn cause severe vascular patterning defects – some of which resemble vasculopathies typical of human blinding disorders. Because death has such a major impact on astrocyte number, it presumably has an outsized impact on this critical patterning process. We therefore sought to identify the non-apoptotic mechanisms that drive astrocyte death. Previously, we showed that astrocyte numbers modulate microglial phagocytic activity – increasing this activity as astrocyte numbers rise and decreasing it as astrocyte numbers decline. This observation suggested that astrocytes themselves are the source of cues that drive their own death via recruitment of phagocytic microglia. Here we identify the membrane lipid phosphatidylserine (PtdSer) as one such astrocyte-derived “eat-me” cue. PtdSer is best known as an “eat-me” signal expressed on the surface of apoptotic cells. We show that PtdSer is also externalized on the cell surface of apparently normal astrocytes during the developmental death period. Moreover, using a genetic approach to increase cell-surface PtdSer, we show that it is sufficient to drive astrocyte death. For these studies, we used an astrocyte-specific mouse knockout of Tmem30a, an obligate subunit of the flippase enzymes that normally remove PtdSer from the cell surface. In these knockout animals, microglia are recruited to Tmem30a mutant astrocytes, engulf them, and cause a significant acceleration of cell number decline. This excess astrocyte loss has functional consequences for the development of the vasculature: The astrocytic template for angiogenesis is overly sparse, which leads to vascular patterning defects and delayed angiogenesis. Interestingly, these defects can be rescued by blocking the function of a phagocytic signaling pathway that can recognize PtdSer exposure, suggesting that the excess PtdSer exposure in the Tmem30a knockout animals is responsible for the increase in astrocyte death. Altogether our findings highlight the broad impact of dysregulated astrocyte death. Understanding how astrocyte population size is controlled will provide new insights into death mechanisms that are crucial for development not only in the retina but may also sculpt glial populations elsewhere in the central nervous system.

Polybrominated diphenyl ether (PBDE) flame retardants are ubiquitous contaminants in the environment due to their heavy usage in plastics, foam, and textiles to comply with flammability standards from the 1970s through the late 2000s. Due to their toxicity and persistence in the environment, two of the three PBDE commercial mixtures (PentaBDE and OctaBDE) were banned by the Stockholm Convention on Persistent Organic Pollutants in 2009. The DecaBDE commercial mixture, which consists primarily of the fully brominated congener BDE-209, has been banned or phased out in the United States and Europe but is still in use in other parts of the world. Human exposure to PBDEs persists via environmental reservoirs of PBDEs and products produced before the bans/phase-outs. PBDEs disrupt thyroid hormone levels and neurodevelopment in fish and rodents and are associated with altered thyroid hormone levels and neurodevelopmental impairments in humans. However, the mechanism by which PBDEs alter neurodevelopment remains unclear. Knowledge of the mechanisms and molecular targets of PBDEs is necessary for a causal link to be established between PBDEs and neurodevelopmental impairments. The hypothesis of this thesis research is that PBDEs alter thyroid hormone levels in the brain by interfering with the activity of PBDE-metabolizing deiodinase enzymes in brain cells, which may result in decreased levels of thyroid hormones in the brain and impaired neurodevelopment.

In the first aim of this thesis research, the biotransformation of PBDEs was examined to determine whether hydroxylated PBDEs (OH-BDEs) are formed in the human brain. In biotransformation assays performed with human astrocytes, which are cells located at the blood brain barrier, no debrominated or OH-BDE metabolites were identified. The results indicate that the enzyme responsible for PBDE hydroxylation (CYP2B6) was not expressed in sufficient quantities to metabolize PBDEs in the astrocyte cells used in this study, but future studies should analyze the potential for PBDE hydroxylation in other brain cells.

In the second aim of this thesis research, the effects of PBDEs on the thyroid-activating enzyme Type 2 deiodinase (DIO2) were determined in human astrocyte cells. DIO2 converts thyroxine (T4) into triiodothyronine (T3), which is the primary ligand that binds to the thyroid nuclear receptors, and is a very important signaling molecule during neurodevelopment. Cultured primary astrocytes and a human glioma cell line (H4 cells) were exposed to PBDEs and OH-BDEs, and changes in DIO2 activity were measured using liquid chromatography with tandem mass spectrometry (LC/MS/MS). Exposure to BDE-99, -153, and -209, 3-OH-BDE-47, and 5'-OH-BDE-99 all resulted in significant decreases in DIO2 activity in the H4 cells by up to 80% at doses of 500-1,000 nM. Further experiments deduced that the primary mechanism responsible for this decrease in activity was attributed to decreased DIO2 mRNA expression, increased post-translational degradation of DIO2, and competitive inhibition of DIO2. The reduction in DIO2 activity by PBDE and OH-BDE exposures could potentially reduce the concentration of T3 in the brain, which may be responsible for the neurodevelopmental impairments produced by exposure to this class of compounds and needs to be further explored.

In the third aim of this thesis research, the effects of PBDEs and OH-BDEs were examined in the H4 cells and in a mixed culture containing a human neuroblastoma cell line (SK-N-AS cells). The SK-N-AS cells express the thyroid hormone-inactivating enzyme Type 3 deiodinase (DIO3), which works in concert with DIO2 to buffer the concentration of T3 in the brain. Exposure to BDE-99 decreased the concentration of T3 and the inactive thyroid hormone rT3 in the cell culture medium of co-cultured cells by 59-76%. 3-OH-BDE-47 competitively inhibited DIO3 with an IC50 of 19 uM. 5'-OH-BDE-99 increased the rT3 concentrations in cell culture medium by 400%, increased DIO3 activity in exposed cells by 50%, and increased DIO3 catalytic activity in cellular homogenates by over 500%. Further effects on the mRNA expression of several thyroid-regulated genes (DIO3, TR-a, TR-b, MCT8, and ENPP2) and oxidative respiration were also assessed in the SK-N-AS cells. DIO3 mRNA expression increased by 9 fold in cells exposed to 400 nM BDE-99, and ENPP2 mRNA expression increased by 2 fold in cells exposed to 500 nM BDE-99 and a mixture of the three congeners, but no other significant effects on mRNA expression were observed. The basal respiration rates and other parameters of oxidative respiration were also not significantly altered by exposure to PBDEs or OH-BDEs, but proton leak was increased by over 400% in cells exposed to 2 uM 5'-OH-BDE-99.

This was the first study to examine the effects of an environmental contaminant on human DIO2 and DIO3 in cultured cells. The results indicated that BDE-99 and OH-BDEs decreased the activity of DIO2 and 5'-OH-BDE-99 increased the activity of DIO3, which combined would lead to decreased levels of T3 exported from the cells into the extracellular environment. These results provide more evidence that disruption of DIO2 and DIO3 by PBDEs during development may mediate the neurodevelopment effects associated with PBDEs.

The inflammasomes are a group of pattern recognition receptors (PRRs) with unique characteristics and critical to innate immunity by translating microbial and damage signals into inflammation. While early investigation into inflammasomes focused on their ability to respond to invading pathogens, now inflammasomes are known to collectively respond to a broad range of sterile damage signals. Indeed, several inflammasomes, and most notably the NLRP3 inflammasome, are known to promote pathogenesis of several autoinflammatory conditions, including the autoimmune neuroinflammation which is modeled in the mouse model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE). While this association of inflammasomes with EAE is well established, the timing, tissue localization, and cell-specificity of inflammasome activation in the central nervous system (CNS) remains poorly understood during disease. Thus, this dissertation details our investigation into the specific sites and timing of inflammasome activation during EAE, as well as determining which inflammasome is activated in the CNS during disease. The interrogation of inflammasome activation in vivo during disease states required our use of genetically modified mice with fluorescent-protein tagged inflammasome adaptor ASC (ASC-Citrine), which serves as a reporter of active inflammasomes. We identified in situ inflammasome activation in specific CNS cell types of these mice using antibody-based immunofluorescent staining techniques and confocal microscopy, and confirmed the result by genetically modified mice, which allowed cell-specific expression of ASC-Citrine. Further, we used mice genetically deficient in inflammasome components ASC and AIM2 to investigate the involvement of these proteins in disease development. Our study concluded with several insights. Firstly, inflammasome activation occurs in the antigen draining lymph nodes prior to symptom onset during EAE, but inflammasome activation occurs more significantly in the spinal cord at 30 days post induction (dpi) of EAE. Spinal cord inflammasome activation during EAE occurs selectively in radioresistent cells, mainly in astrocytes. Further, this astrocyte inflammasome is dependent on AIM2, and does not result in outcomes, which are canonically observed in myeloid cells, such as release of the inflammatory cytokine, IL-1β. Indeed, AIM2 limits EAE development. Lastly, astrocyte inflammasomes differ morphologically from the traditional structure (known as the “ASC speck”) as seen in macrophages. This dissertation thus expands our understanding of inflammasome activation during EAE, and introduces new areas of investigation into the AIM2 inflammasome during EAE and inflammasome activation in astrocytes.