Recent Submissions

  • Effect of enzymatic hyaluronan depletion on the structure and function of the brain’s extracellular space

    Naik, Aditi (2024-06-21)
    Hyaluronic acid (HA) is a large, abundant and unique matrix molecule situated in the brain’s extracellular space (ECS). In this project, I aimed to study the role of HA in the ECS and hypothesized that HA plays a significant role in: (1) diffusion-based transport through the ECS, and (2) maintaining ECS structural properties like volume and ultrastructural geometry. HA was cleaved and depleted by treating the brain tissue with hyaluronidase (Hyal) enzyme that was highly selective for HA. For treatment of acute brain slices with this expensive enzyme, we designed BubbleDrive, a 3D printed incubation chamber, that reduced the overall cost of the project by successfully maintaining the health of slices in a very low volume of incubation solution. Health of the BubbleDrive-incubated acute brain slices was validated through quantification of neuronal, glial and ECS-biophysical functions, and comparison with slices that were incubated in a conventional incubation chamber. Using immunohistochemistry, I confirmed that Hyal treatment depleted HA in the ECS. To quantify the transport through ECS in control and Hyal treated tissue, I used diffusion studies with ECS probe molecules (0.5-12 nm in diameter) that were excellent size-surrogates for many physiologically important molecules that are transported through the ECS, like neuroactive substances, metabolic and therapeutic proteins. I found that the extracellular diffusion of molecules was up to 25% more hindered in the absence of HA in the somatosensory cortex, both in acute brain slices and in vivo. Interestingly, the HA-depleted ECS was 50% larger in that same region. This means that the molecules were getting more hindered even when the space available for their diffusion was increasing. This seemingly counterintuitive result can be explained by formation and/or enlargement of residual wide spaces at junctions of cellular components, called dead-space microdomains. When molecules enter such microdomains, they become transiently trapped within, increasing their dwell-time, which delays their diffusive spread within the tissue. ECS ultrastructure analysis using electron microscopy showed a higher number of ECS expansions that can potentially act as dead-space microdomains in the Hyal treated tissue as compared to the control tissue. In addition, HA-depleted tissue had significantly more astrocyte-associated expansions, suggesting that some of these expansions could have formed because of astrocyte remodeling. Together, these results lead me to infer that: (1) HA promotes the extracellular diffusion of molecules with 0.5 -12 nm diameter, and (2) Presence of HA affects the ECS volume and ECS ultrastructure, possibly through astrocyte remodeling. I also found that HA depletion led to a partial loss of other matrix molecules, confirming its scaffold-like function within the extracellular matrix meshwork. These results signify HA’s role in maintaining ECS structure and function to support cellular communication, toxic metabolite clearance and drug delivery, all of which depend on diffusion-based transport through ECS. These results are also relevant in pathologies that involve increased cleavage of HA, like tumor tissue and neuroinflammation, and treatment strategies that involve complete HA synthesis inhibition, like 4-methylumbelliferone administration.
  • Physiological and clinical insights on autonomic modulation of the inflammatory reflex

    Rodriguez Alvarez, Milena (2023-04-07)
    Stimulation of the inflammatory reflex (IR) is a promising strategy to treat systemic inflammatory disorders. However, this strategy is hindered by the cost and side effects of traditional IR activators. Recently, oral intake of sodium bicarbonate (NaHCO3) has been suggested to activate the IR, providing a safe and inexpensive alternative. Critically, the mechanisms whereby NaHCO3 might achieve this effect and more broadly the pathways underlying the IR remain poorly understood. Here, we argue that the recognition of NaHCO3 as a potential IR activator presents exciting clinical and research opportunities. To aid this quest, we provide an integrative review of our current knowledge of the neural and cellular pathways mediating the IR and discuss the status of physiological models of IR activation. From this vantage point, we derive testable hypotheses on potential mechanisms whereby NaHCO3 might stimulate the IR and compare NaHCO3 with classic IR activators. Elucidation of these mechanisms will help determine the therapeutic value of NaHCO3 as an IR activator and provide new insights into the IR circuitry.
  • The Role of ribosomal RNA in Autism spectrum disorder (ASD)

    Phatarpekar, Shwetha S. (2023-03-03)
    Autism spectrum disorder (ASD) is a group of disorders characterized by social communication deficits, increased repetitive behaviors, and cognitive deficits. [1, 2]. Post-mortem brain studies of ASD patients show smaller hippocampus size, and the volume and the size of both the nucleus and the cytoplasm are smaller [3, 4, 5, 6]. Ribosome biogenesis is one of the cellular functions that influence a cell's growth and volume [7] and is essential for learning and memory. The above observation suggests ribosome biogenesis might be dysregulated, leading to social and cognitive impairment. Using male BTBR T+ Itpr3tf/J (BTBR) mice, a model that reproduces most of the core behavioral phenotypes of ASD, we determined the role of ribosomal RNA synthesis, required for ribosome biogenesis and its impact on cognition, social and repetitive behavior. Presented here, data show that ribosomal RNA expression is decreased in the dorsal hippocampus of BTBR mice compared with B6 mice. Further 3BDO treatment leads to pharmacological up-regulation of ribosomal RNA synthesis, improved learning, and reduced repetitive behavior in BTBR mice. Suggesting that newly synthesized ribosomal RNA plays an essential role in learning and memory in ASD mice model (BTBR). We also found that there is a reduction of learning-induced ribosomal RNA variant expression in BTBR mice, and 3BDO treatment led to differential expression of the ribosomal variant in BTBR. This might imply that newly synthesized ribosomal RNA variants are essential for producing qualitatively different ribosomes, which might be required to improve behavioral deficits in the ASD mouse model. Overall, this thesis provides a new perspective on how ribosome biogenesis plays an essential role in ASD, and this data might help pave a road toward therapeutic intervention.
  • The Role of Tau Isoforms in Sub-Acute and Chronic Closed Head Injury

    Furhang, Rachel (2024-03-25)
    Traumatic brain injury (TBI) is a leading cause of death and disability in the United States and increases the risk of neurodegenerative diseases and dementia [4, 5]. There are no treatments available to prevent neurodegeneration after TBI. Hyperphosphorylation of the microtubule-associated protein tau (pTau) is a key feature of neurodegeneration triggered by TBI. The tau gene can be alternatively spliced to generate tau with 3 or 4 repeated microtubule-binding sequences (3R and 4R). Adult mice express only 4R tau, while the adult human brain expresses 3R and 4R tau. This raises the possibility that tau isoform expression may influence chronic closed-head injury pathophysiology. The central hypothesis of this work is that tau isoform expression modulates neurodegeneration following a single closed-head injury. Two specific aims (SA) test this hypothesis. SA1: What is the time course and distribution of pathological protein aggregates after a single moderate closed-head injury? SA2: Does the expression of 3R tau aggravate the development of pathological protein aggregates and memory deficits after a single moderate closed-head injury? The studies in this work show that a single closedhead injury initially induces pTau+ cells and memory deficits in wild-type (WT) mice, which chronically develop protein aggregates in the thalamus. In contrast, MAPTKI mice expressing both 3R and 4R tau initially have a greater density of pTau+ cells but no memory deficits. However, chronically injured MAPTKI mice have more protein aggregates in the corpus callosum than WT mice and develop chronic spatial memory deficits. These data suggest that a single TBI in the context of 3R tau may drive a more severe early tauopathy that develops into chronic protein aggregate accumulation with functional deficits. Further evaluation of this model could identify targets for treating neurodegenerative diseases and dementia after TBI.
  • The architecture of claustrum and related limbic cortical regions in Carollia perspicillata revealed by latexin and calcium-binding proteins

    Morello, Timothy (2022-05-11)
    Claustrum is a region of grey matter between striatum and cerebral cortex that is among the most well-connected structures in the brain. It is hypothesized to function as a high-level coordinator of brain-wide activities like sensory integration, attention, sleep, and consciousness. The exact anatomical boundaries of claustrum have been controversial, and claustral subregions have not been well-defined. This may be in part due to its compact structure in rodents and other commonly studied species. In contrast, Seba’s short-tailed fruit (Carollia perspicillata) bat has a remarkably large claustrum, lending itself as a model and magnified view for investigating claustrum. We studied the distributions of the claustral marker latexin and the calcium-binding proteins calbindin, calretinin, and parvalbumin in claustrum. Using these markers, we defined clear claustral boundaries and several distinct subregions. The calcium-binding proteins, which mark different subtypes of inhibitory neurons, were differentially distributed among subregions, suggesting that these regions are under the control of different inhibitory systems. In addition to having a large claustrum, Carollia is a relatively long-lived species, lending itself as a model for the neurobiology of aging and neurodegeneration. Two brain regions highly affected in the aging process are retrosplenial cortex (Brodmann areas 29 and 30) and hippocampus. In the course of this work, we found latexin was present in retrosplenial cortex, a region involved in memory and navigation, but only in Brodmann areas 29a and 29b. This distinct division of retrosplenial cortex differs from cytoarchitecturally-defined divisions but aligns with connectivity evidence that supports grouping areas 29a and 29b separate from areas 29c and 30. Finally, we found, several features of Carollia hippocampus, including a compacted CA3 cell layer and a prosubiculum, that are also present in primate but not rodent hippocampus. Due to these unique neuroanatomical features, Carollia may offer advantages in studying claustrum and other limbic cortical structures, especially in the context of aging, that are not present in more commonly studied model species.
  • Unraveling the Mystery of Non-Coding Genomic Content: Evolution, Regulation, and Functional Significance

    velez, Carlos (2023-12-22)
    Abstract This comprehensive review explores the diverse realm of non-coding genomic content, shedding light on its crucial functions in intricate organisms. Once considered mere "junk DNA”, non-coding genomic elements have now proven to be pivotal regulators in genetic coordination. The primary focus of this review is centered around understanding the indispensability of noncoding genomic content for complex organisms. To further unpack this, an in depth look of key non-coding elements, including long non-coding RNAs (lncRNAs), microRNAs (miRNAs), small cytoplasmic RNAs (scRNAs), nucleolar RNAs (noRNAs) and transposable elements (TEs) was done. The possible evolutionary and regulatory role of non-coding genomic content was also explored. Specifically, gene regulatory network formation, and cell specific regulation. Understanding these non-coding elements is pivotal not only for the understanding of evolutionary biology, but for the development of our own precision medicine and innovative strategies in fields like conservation and agriculture. The multifaceted functions of non-coding DNA in complex organisms emphasizes its central significance in the intricate genetic framework. Ultimately this genomic content serves as a fundamental and dynamic component of the genomic landscape. This article intends to encourage additional research and allow for a deeper appreciation for the role of non-coding genomic content in the realm of complex life forms.
  • Exploring Sphingolipid Metabolism: Focus on Serine Palmitoyltransferase (SPT) and Therapeutic Implications of Targeting SPTLC3

    Markopoulou, Eftychia (2024-03-28)
    Sphingolipids perform vital functions in diverse cellular processes and contribute significantly to the development and functionality of organs. This scientific review provides an overview of sphingolipid metabolism, focusing on the synthesis, degradation, and the role of key enzymes such as serine palmitoyltransferase (SPT). The synthetic pathway, which includes enzymes such as 3-ketosphinganine reductase and dihydroceramide desaturase, facilitates the generation of intricate sphingolipids from ceramide. Sphingolipid degradation, particularly within the lysosomal compartment, is essential for cellular homeostasis. The scientific review emphasizes the importance of SPT in regulating sphingolipid levels, with specific insights into its activity in different tissues. Furthermore, it delves into the role of SPT in the liver and provides an understanding of the architecture and function of SPTLC3. The paper concludes by highlighting the potential therapeutic implications of targeting SPTLC3 and the need for further research in this area.
  • Oral/gut microbiome and inflammatory markers in African American colon cancer patients

    Tortora-Morel, Sofia (2024-03-12)
    Background: Colorectal cancer (CRC) is the third leading cause of cancer deaths in the US, with African American (AA) patients having the highest incidence and mortality rate. Periodontitis, a chronic oral inflammatory condition, increases the risk of CRC. Fusobacterium nucleatum (Fn), a periodontal pathogen, has been implicated in CRC pathogenesis by altering gut microbiota composition and promoting inflammation. Our study explored the complex interplay between the immune response and the microbiome as it relates to colon cancer in AA patients. Methods: Our study included qPCR analysis, 16S rRNA gene sequencing, and RNA sequencing of tumor vs. non-tumor tissues, analysis of inflammatory mediators by using a quantitative slidebased array and antibody responses to specific bacteria by novel protein microarray NAPPA in serum, and immunohistochemistry staining to quantify T cell subset densities in tumors. Results: Our analysis revealed a distinct microbial profile in colon tumor tissues, characterized by reduced microbial diversity and increased oral-origin bacterial abundance. Right-sided tumors revealed distinct inflammation and immune responses, in contrast to metabolic and signaling expression pathways in left tumors. Fn was significantly increased in tumors (p= 0.0003) and was associated with gut microbiota changes by co-aggregating with other oral pathogens and reducing probiotic and CRC-associated bacteria. Upregulated pathways in Fn-positive tumors were neutrophil chemotaxis, NF-κB, TLR signaling and Wnt signaling, and T cell differentiation with increased CD8+ T cell density within the tumor (p=0.036). Additionally, patients with early-stage and right-sided tumors exhibited higher anti-Fn seropositivity (p< 0.00001). Conclusions: Our study reveals unique microbial and immunological profiles in AAs with colon cancer. These findings offer insights into genetic pathways and possible microbial contributions across different colon tumor locations and cancer stages, highlighting the need for further investigation into underlying mechanisms and potential new therapeutic targets.
  • Multiscale computer modeling of brain excitability: applications to spreading depression and neuronal impedance

    Kelley, Craig (2023-06-26)
    Multiscale modeling of biological systems integrates disparate experimental results into unified theoretical frameworks. We used multiscale modeling to investigate excitability in neural systems at the scales of dendrites, single neurons, and tissue-scale activation patterns. We employed impedance analysis to study subthreshold excitability in morphologically and biophysically detailed models of neocortical layer 5b pyramidal neurons, which predicted that the interaction of hyperpolarizationactivated cyclic nucleotide-gated (HCN) and Twik-associated acid-sensitive K+ (TASK) channels are integral to producing their observed impedance profiles. Impedance analysis is properly limited to studying neuronal responses to small, subthreshold stimuli, but this excludes a great deal of neuronal function. To overcome the limitations of impedance analysis, we developed an analog to impedance phase to characterize high amplitude signals, both sub- and suprathreshold. For high amplitude stimuli, we found different phase shifts during hyperpolarizing and depolarizing halfcycles. We also found two nonstationary phase relationships between spiking and stimulus: phase retreat, where action potentials occurred progressively later in cycles of the input stimulus resulting from adaptation, and phase advance, where action potentials occurred progressively earlier. In a separate study, we developed a computer model of spreading depolarization (SD) in brain slices using the NEURON simulator: 36,000+ neurons in the extracellular space (ECS) of a slice with ion and O2 diffusion and equilibration with a surrounding bath. Simulations reproduced key features of SD, including its speed moving across the tissue and firing properties of individual neurons, and led to a number of experimentally-testable predictions. We have also developed a model of neocortex in vivo with realistic distributions of O2 sources based on histology from human subjects. This model can be used to investigate the role of connectivity in SD propagation and how ischemic insults lead to SD initiation.
  • Type 1 diabetes and lung diseases: Investigating the effects of hyperglycemia in the lung

    Park, Sangmi Sarah (2023-04-10)
    Type 1 diabetes (T1D) is a metabolic disease characterized by hyperglycemia, resulting from decreased insulin secretion by the pancreatic b cells. In the long term, T1D can affect multiple organs, and lead to life-threatening complications. Increased prevalence of pulmonary abnormalities and respiratory diseases is observed in T1D patients. However, the effects of T1D on the lung are not well defined and the exact mechanisms underlying diabetic complications in the lung remain elusive. The objective of the study was to determine whether T1D exacerbates the progression of lung damage in the presence of chronic lung diseases and to delineate the mechanisms leading to lung pathologies. We performed pulmonary function test (PFT) in streptozotocin (STZ)-induced T1D mouse model, which demonstrated a restrictive pulmonary pattern in the lungs of STZ-injected mice, corresponding to fibrotic changes in the lung. In line with this, lungs from STZ-injected mice showed an increase in collagen accumulation as well as in gene expression of the fibrotic markers Acta2 and Fn1. We studied the effect of T1D in two mouse models of emphysema: a cigarette smoke (CS) exposure model and an alpha-1 antitrypsin (AAT) deficiency model. CS exposure alone did not impair pulmonary function but increased collagen accumulation in the lung. CS-exposed STZ mice showed similar pulmonary function patterns and collagen deposition in the lung as room air (RA)-exposed STZ mice. STZ injection in AAT-deficient mice accelerated the accumulation of collagen and the development of emphysema in the lung. To determine the molecular mechanisms for fibrotic remodeling in STZ-injected mouse lungs, we performed RNA-sequencing of primary human bronchial epithelial cells (HBECs) cultured in physiologically normal (5 mM) and high (12.5 mM) glucose conditions. Pathways associated with metabolism, oxidative stress and cellular senescence were overexpressed in primary HBECs response to high glucose. We investigated these pathways to define mechanisms that may lead to high glucose-induced fibrotic remodeling in the lungs using HBE cell line cultured in normal (5 mM) and high (12.5 mM) glucose media. HBECs increased the uptake of glucose in response to high glucose treatment for 24 hours and showed a metabolic shift in which the excess glucose was routed towards the pentose phosphate pathway, lactate synthesis and glycogen synthesis. These metabolic shifts were not associated with changes in the cell proliferation rate of HBECs. In contrast to our RNA-seq data, oxidative stress and cellular senescence pathways were not altered in HBECs cultured in high glucose media. To explore whether HBECs cultured in high glucose media can directly activate fibroblasts, primary human lung fibroblasts were cultured in normal and high glucose media conditioned by HBECs. Fibroblasts cultured in high glucose media conditioned media showed an increase in the gene expression of COL1A1 and COL1A2 along with increased protein expression of a-SMA and COL1A1. Protein array using media conditioned by HBECs demonstrated high glucose treatment increased secretion of proteins associated with pulmonary fibrosis and ECM remodeling into the media by HBECs, suggesting a potential mechanism of high glucoseinduced pulmonary fibrosis. In this study, we demonstrated that STZ-injected mice exhibit functional, histological and gene expression changes in the lung that correspond to the restrictive pulmonary pattern. Our findings from the study suggest that T1D exacerbates the progression of lung damage in AAT deficiency by increasing collagen accumulation in the lung and accelerating the development of emphysema. Our in vitro study also showed that HBECs undergo metabolic reprogramming in high glucose conditions and that high glucose-exposed HBECs increase the secretion of profibrotic mediators to the media and are capable of activating fibroblasts. These data expand primary HBECs response to high glucose. We investigated these pathways to define mechanisms the existing knowledge on the mechanisms for hyperglycemia exerting deleterious effects on pulmonary function and reveal new comorbidities for AAT deficiency. In addition, this work presents new knowledge on the metabolic use of glucose by pulmonary epithelium and suggests potential mechanisms of high glucose-induced pathogenesis of pulmonary fibrosis. that may lead to high glucose-induced fibrotic remodeling in the lungs using HBE cell line cultured in normal (5 mM) and high (12.5 mM) glucose media. HBECs increased the uptake of glucose in response to high glucose treatment for 24 hours and showed a metabolic shift in which the excess glucose was routed towards the pentose phosphate pathway, lactate synthesis and glycogen synthesis. These metabolic shifts were not associated with changes in the cell proliferation rate of HBECs. In contrast to our RNA-seq data, oxidative stress and cellular senescence pathways were not altered in HBECs cultured in high glucose media. To explore whether HBECs cultured in high glucose media can directly activate fibroblasts, primary human lung fibroblasts were cultured in normal and high glucose media conditioned by HBECs. Fibroblasts cultured in high glucose media conditioned media showed an increase in the gene expression of COL1A1 and COL1A2 along with increased protein expression of a-SMA and COL1A1. Protein array using media conditioned by HBECs demonstrated high glucose treatment increased secretion of proteins associated with pulmonary fibrosis and ECM remodeling into the media by HBECs, suggesting a potential mechanism of high glucoseinduced pulmonary fibrosis.
  • Inhibiting phosphatidylcholine remodeling in adipose tissue increases insulin sensitivity

    He, Mulin (2023-08-24)
    Cell membrane phosphatidylcholine composition is regulated by lysophosphatidylcholine acyltransferase (LPCAT); changes in membrane phosphatidylcholine saturation are implicated in metabolic disorders. Here, we identified LPCAT3 as the major isoform of LPCAT in adipose tissues and created adipocyte-specific Lpcat3-knockout mice to study adipose tissue lipid metabolism. Transcriptome sequencing and plasma adipokine profiling were used to investigate how LPCAT3 regulates adipose tissue insulin signaling. LPCAT3 deficiency reduced polyunsaturated phosphatidylcholines in adipocyte plasma membranes, increasing insulin sensitivity. LPCAT3 deficiency influenced membrane lipid rafts, which activated insulin receptors and AKT in adipose tissue, and attenuated diet-induced insulin resistance. Conversely, higher LPCAT3 activity in adipose tissues from ob/ob, db/db, and high-fat diet-fed mice reduced insulin signaling. Adding polyunsaturated phosphatidylcholines to mature human or mouse adipocytes in vitro worsened insulin signaling. We suggest that targeting LPCAT3 in adipose tissues to manipulate membrane phospholipid saturation is a new strategy to treat insulin resistance.
  • Impacts of Early Life Stress on Adult Emotive Behavior and the PFC-Amygdala Circuit

    Cote, Ernest (2023-12-14)
    Fear responses in mammals are ancient and adaptive responses. Specialized neural systems have evolved to support adaptive responses, and these have critical developmental time points. Disrupting development of these systems with environmental stress impacts mood and conduct, and as adults, stressed individuals demonstrate depressive or anxiety -like behavior. This project investigates impacts of early life stress (ELS) on behavior and the synaptic physiology of the basolateral amygdala and prelimbic (PL) and infralimbic (IL) areas of the prefrontal cortex (PFC) in rodents. C57/BL6 male and female mouse pups were subjected to maternal separation (with concurrent maternal stress) and isolation during the critical neurodevelopmental period of postnatal day 10 to 17 (PND10-17) and then housed normally until adulthood (ELS group). Another cohort of male and female mice grew under normal conditions but underwent forced swim stress in adulthood (Adult stress group). Both groups of animals plus a home-cage control group (male and female) were assessed with a battery of tests to measure depressive and anxiety- like behaviors, and subsequently sacrificed for electrophysiological experiments. Behavioral analyses show ELS mice exhibited phenotypes consistent with depressive and anxiety- like behaviors that appear to match those observed in adult stressed mice. Analysis for sex differences revealed performance-specific differences between groups. Electrophysiological analyses indicate ELS mice show increased postsynaptic excitability in the basolateral amygdala and increased presynaptic drive in PL and IL PFC compared with control animals, as well as sex differences. Our findings indicate that ELS causes behavioral disruptions and dysregulation of synaptic function in amygdala and PFC that lasts into adulthood.

    Bhattacharya, Aparajita (2023-08-23)
    ABSTRACT ENGINEERING SELF-ASSEMBLING BIOMATERIALS USING PROTEIN BLOCK COPOLYMERS FOR DRUG DELIVERY AND MOLECULAR IMAGING IN GLIOBLASTOMA By Aparajita Bhattacharya Thesis Advisor: Jin Kim Montclare, PhD A thesis submitted to the faculty of The School of Graduate Studies State University of New York Downstate Health Sciences University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy in Molecular and Cellular Biology Aug 22, 2023 In this thesis, we discuss the engineering a single protein-based system combining drug delivery and diagnostic capabilities in addition to its variants. Chapter 2 covers the engineering and characterization of a protein-based theranostic nanomaterial, TRAP2 that performs drug delivery and near infrared fluorescence imaging in a preclinical model of glioblastoma. Though NIR imaging has limited application in the clinical setting except its role in surgical guidance, it has vast potential within preclinical research due to its tissue penetration. To enhance the targeting ability of TRAP2 to glioblastoma, Chapter 3 explores the recombinant conjugation of a transferrin receptor-targeting short peptide tag to the protein and its characterization. Chapter 4 addresses the development of protein engineered PET agent, 18F-TRAP3, for bestowing it with a high LOD and resulting sensitivity. We employ residue-specific incorporation of an azide-bearing methionine analog, azidohomoalanine to give rise to clickable azide-functionalized TRAP3AHA that can be subsequently conjugated to a 18F bearing alkyne analog of boron-dipyrromethene (BDP FL DBCO) dye, imparting it an ability to be used as a dual modality PET probe capable of fluorescence as well as 18F-PET imaging. Together, these proteins represent an endeavor directed at the development of theranostic agents for enhanced drug delivery and preclinical imaging in glioblastoma.
  • Circuit Breakers: α4βδ GABA-A Receptors Drive Adolescent Refinement of Neural Circuits in Prefrontal Cortex

    Evrard, Matthew (23-12-12)
    Adolescence is a time when synaptic connections are sculpted to prepare for the cognitive challenges of adulthood, a process known as synaptic pruning. Although this process was first reported over 30 years ago, the initial trigger and functional reason for pruning remain unknown. This thesis provides a multifaceted investigation of α4βδ GABA-A receptor regulation of dendritic spine pruning within pyramidal neurons of the prelimbic prefrontal cortex across adolescent development in mice. The prelimbic cortex projects to the amygdala and drives anxiety states, making precise pruning of juvenile connections critical for proper maturation. Using highresolution microscopic analysis of Golgi-stained samples, I report a dramatic developmental decrease in basilar dendritic spine density on layer 5 pyramidal neurons between early puberty (16.39 ± 1.55 spines/10μm) and young adulthood (6.10 ± 0.58 spines/10μm), reflecting a 63% reduction (p < 0.0001). This remodeling coincides with a transient 10-fold increase in α4 subunit expression precisely at puberty onset (p < 0.00001) within layer 5 pyramidal neuron dendrites, revealed by immunohistochemistry and amplified electrophysiological responses to a δ subunit-selective agonist (p = 0.00125). Convergent pharmacological, local knockdown (using viral shRNA knockdown and Cre-loxP deletion), and global knockout of α4βδ GABAARs prevented adolescent pruning, while augmenting α4βδ signaling via the selective agonist gaboxadol during early puberty significantly enhanced spine elimination (p<0.05). This demonstrates the causal role of this signaling pathway in mediating the extensive remodeling. The mechanism likely involves α4βδ receptor-mediated suppression of NMDA receptor activation of Kalirin-7 pathways which maintain the dendritic cytoskeleton. α4 knockout prevents the typical 50% decrease in Kalirin-7 levels at puberty (p<0.0001), suggesting dependence on α4βδ signaling. In contrast, increasing NMDAR expression prevents pruning. Critically, preventing pubertal pruning through localized α4βδ knockdown in the prelimbic cortex using AAV-Cre administration increases anxiety-like avoidance behaviors on the elevated plus maze test after an aversive stimulus by 60% in late adolescence (p<0.0001) and 40% in adulthood (p<0.05). This causally links excess prelimbic connectivity from disrupted juvenile synapse elimination to anxiety-related behavioral phenotypes. Furthermore, a similar developmental decrease in spine density occurs in layer 2/3 pyramidal neurons in female mice, aligned with a key role for α4-containing receptors as evidenced by a lack of pruning in α4 knockout mice. Taken together, these results suggest a role for the extra-synaptic α4βδ GABAARs in triggering synaptic pruning and further demonstrate one pathological outcome which can result from dysregulated pruning.
  • Modeling exposure to folate receptor antibodies during neural development to understand its clinical significance.

    Bobrowski-Khoury, Natasha
    Autism spectrum disorder (ASD) is a multifactorial neurodevelopmental disorder with defined core symptoms of impaired social communication, stereotyped or repetitive behaviors, and cognitive deficits. These core symptoms present early in childhood, which distinguishes this disorder from other neurological disorders that share similar characteristics. To our knowledge, there is currently no pharmacologic treatment for the core symptoms of ASD. A possible therapeutic strategy is supplementing folate, more specifically the reduced form, folinic acid, during pregnancy. Folate is the umbrella term for biological forms of vitamin B9. The well recognized role of folates as precursors of DNA synthesis, single carbon exchange reactions and in methylation reactions is a priori evidence of its requirement for cellular replication, metabolism, and epigenetic control of gene expression. This is especially important during the early stages of neural development. While the role of folate in cellular metabolism has been clearly defined, there has been no study, to our knowledge, that demonstrates the characteristics of folate uptake and distribution during gestation and early development. This is critical to understand because of the emerging findings that folate receptor alpha autoantibodies (FRAuAb) are linked to disruptions of neural development leading to ASD. Our lab has shown that a rat model of exposure to IgG antibodies specific to rat folate receptor alpha during gestation develops a behavioral phenotype comparable to the human ASD. This phenotype appears to be preventable with folinic acid and dexamethasone treatment. Preliminary observations in this model also suggest that the phenotype seen in rats directly exposed to FRAb is preserved in subsequent generations. Overall, this thesis provides new data on the uptake and distribution of folate and folate receptor antibodies in utero (Study 1, Part 1) and in juvenile stages of development (Study 1, Part 2) and observes an appearance of deficits to a subsequent generation of animals previously exposed to FRAb directly (Study 2)