Browsing by Author "Eroglu, Cagla"

Astrocytes, the perisynaptic glial cells of the brain, display a complex morphology that is strongly linked to their functions at the synapse. Primary processes radiating from the astrocyte cell soma branch out to secondary and tertiary processes, which further ramify into tiny perisynaptic astrocyte processes, giving a mature astrocyte its characteristic arborized structure. Astrocyte processes dynamically ensheath the pre- and post-synapse to provide instructive cues for synapse formation, maturation, and function. Perturbations in astrocyte-synapse interactions result in synaptic deficits, leading to excitation/inhibition imbalance and aberrant neural circuitry. However, the mechanisms linking astrocyte morphology and function to neuronal contact and synaptic adhesion are poorly understood. In a candidate-based reverse genetic screen utilizing rodent cortical neurons and astrocytes, I identified two genes, HepaCAM and CTNND2, as regulators of astrocyte morphogenesis in response to neuronal adhesion.HepaCAM is an astrocyte-enriched cell adhesion molecule that participates in cell-cell and cell-ECM interactions to regulate cell migration and proliferation. shRNA-mediated silencing of hepaCAM expression in astrocytes resulted in decreased astrocyte complexity in vitro and in vivo. HepaCAM stabilizes the gap junction protein connexin 43 (Cx43) at cell-cell junctions. We used stimulated emission depletion (STED) microscopy to show that hepaCAM and Cx43 colocalize at astrocyte processes in the mouse cortex and performed native affinity purifications followed by liquid chromatography-coupled high-resolution mass spectrometry (AP-MS) to demonstrate that Cx43 binds to hepaCAM. Finally, utilizing the same shRNA silencing approach, we found that hepaCAM and Cx43 were epistatic to each other in the regulation of astrocyte morphogenesis. Through mosaic analysis with double markers (MADM), we found that hepaCAM knockout astrocytes lost their ability to tile and had mislocalized Cx43. Consequently, gap junction coupling is impaired in astrocytes without hepaCAM. Additionally, we found decreased colocalization of hepaCAM puncta with synapses, a marked decrease in inhibitory synapses density, and a significant decrease in amplitude of miniature inhibitory postsynaptic currents, suggesting that loss of astrocytc hepaCAM disrupts the balance between synaptic excitation and inhibition. During development, astrocytes need to form non-overlapping territories within which they dynamically ensheathe synapses within discrete regions of neuropil. Taken together, our findings suggest that hepaCAM and Cx43 are critical proteins at the intersection of these two processes to ensure the proper molecular regulation of astrocyte self-organization and territory formation for normal circuit formation and function. Next, we identified Ctnnd2 (protein: δ-catenin) as another key regulator of astrocyte morphological complexity. δ-catenin was previously thought to be a neuron-specific protein that regulates dendrite morphology. Utilizing RNA fluorescence in situ hybridization (RNA-FISH) and immunohistochemistry, we found Ctnnd2 mRNA and δ-catenin is also highly expressed by astrocytes during the critical period of astrocyte morphological maturation and synapse formation during cortical development. shRNA-mediated silencing of Ctnnd2 expression in astrocytes resulted in decreased astrocyte complexity in vitro and in vivo. δ-catenin is hypothesized to mediate transcellular interactions through the cadherin family of cell adhesion proteins. We used structural modeling and surface biotinylation assays in both HEK293T and purified astrocyte cultures to reveal that δ-catenin interacts with N-cadherin juxtamembrane domain to promote N-cadherin surface expression. An autism-linked δ-catenin point mutation impaired N-cadherin cell surface expression and reduced astrocyte complexity. In the developing mouse cortex, only lower-layer cortical neurons express N-cadherin. Remarkably, when we silenced astrocytic N-cadherin throughout the cortex, only lower-layer astrocyte morphology was disrupted. These findings show that δ-catenin controls astrocyte-neuron cadherin interactions that regulate layer-specific astrocyte morphogenesis.

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.

Huntington’s disease (HD) is an adult-onset, neurodegenerative disease caused by an autosomal dominant mutation in the huntingtin (HTT) gene. HD patients suffer from motor, cognitive, and psychiatric symptoms. The pathogenic mutation of HD is expansion of a CAG repeat in the first exon of the HTT that encodes for a polyglutamine (poly-Q) repeat in the huntingtin protein (Htt). HD results in neurodegeneration of the striatum and cortex, which is thought to underlie the development of HD symptoms, but recent evidence has shown that there are alterations to the connectivity of patients’ brains preceding degeneration. This study focuses on how wild type Htt contributes to establishing and maintaining synaptic connectivity and how a loss of normal Htt function may contribute to the synaptic alterations in HD.

In this study, I examined the role of wild type Htt in synapse formation and maturation synapses in the basal ganglia circuit, and I examined how loss of wild type Htt function may affect the pathogenesis of HD. To do so, I created conditional deletions of Htt in the mouse brain by crossing a floxed allele of Huntingtin to transgenic Cre lines. I conditionally deleted Htt from the cortex and the indirect pathway spiny neurons (iSPNs) of the striatum using Emx1-Cre and Adora2A-Cre, respectively. I also used a knock-in mouse model of HD, the zQ175 mouse, to examine alterations caused by the HD mutation. I used imaging and electrophysiological techniques to determine how loss of Huntingtin affected synapse number, function, and morphology in the cortex, striatum, and basal ganglia.

In the cortex and striatum, loss of Htt leads to disruptions in synaptic connectivity followed by neuronal stress and death. Htt is critical for moderating the formation of excitatory synapse formation in both the cortex and striatum, and that in the cortex this function is lost in HD. In the striatum, Htt is required for stabilizing striatopallidal synapses, and for proper basal ganglia function.

In order to explore the molecular mechanisms behind Htt’s control of excitatory synapse formation, I investigated its interaction with α2δ-1. α2δ-1 is a genetic modifier of mutant Htt toxicity that our lab had previously identified as the neuronal receptor of the synaptogenic Thrombospondin family (TSP) of proteins. I used in vitro neuronal cultures and biochemical analysis to determine how Htt interacts with α2δ-1 and how Htt affects TSP/α2δ-1 excitatory synapse formation. I characterized α2δ-1’s biochemical interaction with Htt and discovered that Htt postsynaptically suppresses excitatory synapses.

Taken together, these results suggest that wild type Htt functions to moderate excitatory activity in the brain. It slows the formation of excitatory connections and stabilized inhibitory ones, which may protect the brain from excitotoxic damage. These results show that Htt plays an important role in maintaining neuronal health and the establishment of synaptic connectivity in cortical and striatal circuits.

Zebrafish possess the remarkable capacity to regenerate from spinal cord injuries that would leave mammals such as humans permanently paralyzed. Much research into zebrafish spinal cord regeneration has focused on identifying extracellular growth factors and matrix components which create a pro-regenerative environment; however, it is just as important to identify and understand the transcription factors which control pro-regenerative transcriptional responses within the resident stem cell population of the spinal cord, and the signaling cascades which translate the known extracellular ligands into cellular responses. Using CRISPR/Cas9, we generated two novel transcription factor knockout zebrafish lines which we tested for spinal cord regeneration defects and found no difference in regenerative capacity. Using a chemical screen, we identify JNK signaling as a necessary regulator of glial cell cycling and tissue bridging during spinal cord regeneration in larval zebrafish. With a kinase translocation reporter, we visualize and quantify JNK signaling dynamics at single-cell resolution in glial cell populations in developing larvae and during injury-induced regeneration. Glial JNK signaling is patterned in time and space during development and regeneration, decreasing globally as the tissue matures and increasing in the rostral cord stump upon transection injury. Thus, we present a tool to visualize signaling activity in the larval zebrafish spinal cord and demonstrate that dynamic JNK activity after spinal cord injury directs a proliferative response of glial cells during spinal cord regeneration.

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.

Goal-directed actions (GDAs), the optimal set of actions to achieve an outcome, are voluntary behaviors requiring complex cognitive processes. Cortico-striatal circuits, particularly neuronal projections from the prefrontal cortex to the dorsomedial striatum (DMS), are critical for the establishment and performance of goal-directed behaviors. Synaptogenesis underlies the formation and remodeling of neural circuits that control cognition and behavior. Astrocytes, a major glial-cell type in the central nervous system (CNS), promote synapse formation and remodeling. During development, astrocytes secrete synaptogenic proteins called thrombospondins (TSPs), which act through the neuronal receptor α2δ-1 to induce excitatory synaptogenesis. The role of astrocytes in synapse formation during development is well established. However, it is less clear whether astrocytes promote synaptogenesis in the adult brain to control complex learned voluntary behaviors. Here we used instrumental operant conditioning to investigate the role of adult synaptogenesis in brain regions engaged during GDA training. We discovered that during the establishment of GDAs in adult mice, new excitatory synapses are formed in the Anterior Cingulate Cortex (ACC). The loss of α2δ-1 reduces this training-induced excitatory synapse formation in the ACC and causes a profound persistence of GDA performance, even when the effort required for the reward is drastically increased. Ablation of α2δ-1 only in ACC neurons projecting to the DMS (ACC->DMS) recapitulated the synaptic and behavioral phenotype observed with constitutive loss of α2δ-1. Based on the findings from the loss of α2δ-1, we predicted that astrocyte-secreted TSPs, which bind to α2δ-1, are also required for the proper performance of GDAs. Surprisingly, we found that constitutive loss of TSP isoforms 1 and 2 (TSP1/2) significantly reduced GDA performance as the effort required for reward increased. Interestingly, the absence of TSP1/2 did not impair GDA training-induced synaptogenesis. Ablation of TSP1/2 in developing but not adult astrocytes was able to reduce GDA performance during high-effort tasks. Furthermore, transcriptomic analysis suggested that constitutive loss of TSP1/2 alters the expression of a group of genes associated with inhibitory and cholinergic neurons. Additionally, we found that mice lacking TSP1/2 have increased inhibitory synapse density in the ACC, which is diminished with GDA training. Altogether, my doctoral dissertation research demonstrates that new synapses are formed in adult animals to control complex voluntary behaviors such as GDAs. These findings also reveal novel roles for astrocyte-secreted thrombospondins in controlling brain circuit formation and adult plasticity, which involve a developmental process that controls inhibition in the mouse ACC. Taken together, my findings reveal that synaptogenic signaling between astrocytes and neurons is critical for modulating GDA performance.

Huntington’s Disease (HD) is a fatal, autosomal dominant disorder caused by a polyglutamine expansion mutation near the N-terminus of the Huntingtin (Htt) gene. Patients with HD experience severe and progressive motor, cognitive, and psychiatric symptoms before succumbing to the disease approximately 15-20 years after disease onset. At the neuropathological level, HD causes the extensive and selective degeneration of striatal projection neurons (SPNs) with the striatum, which are intimately involved in the regulation of voluntary movements. Long thought of as solely a neurodegenerative disease, more recent evidence suggests that both HD symptoms and neurodegeneration are preceded by defects in synaptic connectivity and function. However, how synaptic pathology and neurodegeneration arise in HD remain poorly understood.

The presence of the HD-causing polyglutamine expansion mutation in Htt, along with the well-documented toxicity of mutant Htt (mHtt) to neurons, has led to the widely accepted but not yet proven hypothesis that HD is caused by a toxic gain-of-function of mHtt. However, wildtype Htt is known to play important roles in several cellular processes, suggesting that loss of Htt function, due to a dominant-negative interaction with mHtt, may also contribute to HD pathology. Here, I used the Cre/Lox system to conditionally delete Htt from murine SPNs to investigate the role of Htt in SPNs and to determine whether loss of Htt function can recapitulate aspects of HD. I discovered that loss of Htt in SPNs leads to dysregulated motor function and causes SPNs to degenerate with aging, similar to observations in HD. I also found indications for impaired neuronal health in SPNs lacking Htt long before degeneration occurred. For instance, loss of Htt in SPNs disrupts SPN synaptic connectivity and gene expression, similar to findings in HD patients and mouse models. In sum, my results strongly suggest that Htt is required for SPN health, function, and longevity.

With the importance of Htt in regulating synaptic connectivity well-established, I next investigated how Htt regulates synapse formation at the molecular level, and how this function is impaired when mHtt is present. Specifically, I sought to determine whether mHtt interacts with its pro-synaptogenic binding partner, calcium channel subunit 2-1, to regulate synapse formation. As mice expressing mHtt form too many synapses early in development, I asked whether deleting a copy of 2-1 from mHtt-expressing mice would restore their synapse development to wildtype levels. Indeed, I found that reducing 2-1 in mice expressing mHtt rescued their early synapse development. Taken together, my doctoral dissertation research demonstrates that loss of Htt is sufficient to recapitulate several key features of HD, suggesting that Htt loss-of-function may contribute to HD pathology. My research shows that Htt plays critical roles in maintaining the cellular health and viability of neurons most vulnerable in HD and introduces a potential molecular mechanism underlying Htt’s role in regulating synapse formation and function.