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MECHANISMS PROMOTING PHOSPHORYLATION OF THE NF2 TUMOR SUPPRESSOR AND ITS EFFECTS ON SCHWANN CELL DEVELOPMENT
Title
MECHANISMS PROMOTING PHOSPHORYLATION OF THE NF2 TUMOR SUPPRESSOR AND ITS EFFECTS ON SCHWANN CELL DEVELOPMENT.
Creator
Thaxton, Courtney, Fernandez-Valle, Cristina, University of Central Florida
Abstract / Description
Neurofibromatosis type 2 is an autosomal dominant disease characterized by the formation of schwannomas and other peripheral neuropathies. The nf2 gene encodes the protein Schwannomin, or merlin. Schwannomin (Sch) is a membrane-cytoskeletal linking protein that suppresses cell proliferation at high cell density and modulates cell shape. Sch's tumor suppressive activity is regulated by its localization, conformation, and phosphorylation at serine 518 (S518). Sch's localization is...
Show moreNeurofibromatosis type 2 is an autosomal dominant disease characterized by the formation of schwannomas and other peripheral neuropathies. The nf2 gene encodes the protein Schwannomin, or merlin. Schwannomin (Sch) is a membrane-cytoskeletal linking protein that suppresses cell proliferation at high cell density and modulates cell shape. Sch's tumor suppressive activity is regulated by its localization, conformation, and phosphorylation at serine 518 (S518). Sch's localization is dependent on binding the scaffold protein, paxillin. Phosphorylation of Sch at S518 regulates its conformation and tumor suppressor function. In a negative feedback loop, unphosphorylated Sch restricts cell proliferation downstream of Rac and p21-activated kinase (Pak), whereas Pak-induced phosphorylation inactivates Sch's ability to inhibit Pak and cell proliferation. Little is known about the function of the phosphorylated form of Sch, or the molecular mechanisms leading to its phosphorylation. Here we demonstrate that Sch-S518 phosphorylation is dependent on paxillin-binding and plasma membrane localization in SCs. Phosphorylation of Sch at the plasma membrane is mediated by Cdc42-Pak and results in altered SC morphology and polarity. Moreover, we have identified two extracellular stimuli that trigger Sch-S518 phosphorylation; these are neuregulin (NRG) and laminin, two potent activators of SC proliferation and myelination. NRG promotes Sch-S518 phosphorylation downstream of ErbB2/ErbB3 through PKA, whereas laminin-1 stimulation of β1 integrin promotes Pak- dependent phosphorylation of Sch-S518. Additionally, we find that Sch promotes process formation and elongation in primary and myelinating SCs, independent of Sch S518 phosphorylation. However, Sch phosphorylation was found to influence SC differentiation, as expression of an unphosphorylatable variant, Sch-S518A, facilitated SC myelination, whereas expression of a phospho-mimicking variant, Sch-S518D, reduced the SC's ability to myelinate. Together, these findings have identified receptor-mediated and paxillin-dependent pathways that regulate phosphorylation and inactivation of Sch's tumor suppressor function. Additionally, these results have elucidated novel normal functions for Sch during peripheral nerve development and myelination, and identify novel therapeutic targets for treatment of NF2 and other peripheral neuropathies.
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Date Issued
2007
Identifier
CFE0001898, ucf:47393
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0001898
A NOVEL BINDING INTERACTION FOR THE PAXILLIN LD3 MOTIF: PAXILLIN LD3 MEDIATES MERLIN-PAXILLIN BINDING AT PAXILLIN BINDING DOMAIN 1
Title
A NOVEL BINDING INTERACTION FOR THE PAXILLIN LD3 MOTIF: PAXILLIN LD3 MEDIATES MERLIN-PAXILLIN BINDING AT PAXILLIN BINDING DOMAIN 1.
Creator
Geden, Sandra, Fernandez-Valle, Cristina, University of Central Florida
Abstract / Description
Neurofibromatosis type 2, an autosomal dominant genetic disorder, causes predisposed individuals to develop various benign central and peripheral nervous system tumors. The characteristic tumors of this disease are schwannomas, which are tumors of the Schwann cells, typically on the vestibular nerve. These and the other associated tumors slowly compress nervous system structures causing deafness and loss of balance, resulting in an average life-span of less than 40 years. The product of the...
Show moreNeurofibromatosis type 2, an autosomal dominant genetic disorder, causes predisposed individuals to develop various benign central and peripheral nervous system tumors. The characteristic tumors of this disease are schwannomas, which are tumors of the Schwann cells, typically on the vestibular nerve. These and the other associated tumors slowly compress nervous system structures causing deafness and loss of balance, resulting in an average life-span of less than 40 years. The product of the Nf2 gene is the protein named merlin or schwannomin. In individuals diagnosed with NF2, merlin is either absent or mutated to the point of inactivation. As such, merlin functions as a negative growth regulator in that it suppresses tumor growth. Being that NF2 is predominately a disease of the Schwann cells, merlin's functional role within the signal transduction pathways of Schwann cell growth and differentiation are being investigated. This thesis explores the molecular relationships between merlin and its various interactors within Schwann cells, and illuminates one step in elucidating merlin's functional mechanism of action. Merlin has been shown to associate with paxillin in a density-dependant manner and to bind directly to paxillin through two specific paxillin binding domains. Individual paxillin LD domain fusion proteins were produced, as well as recombinant merlin lacking the paxillin binding domains. Direct binding assays were performed in order to determine which specific paxillin domains merlin might interact with directly. The results indicate that, in vitro, merlin binds, through its PBD1 domain, to the paxillin LD3 motif. Supporting this data, the results also demonstrate that when the merlin PBD1 domain is deleted, merlin binding to paxillin LD3 is abrogated. The direct binding shown here between paxillin and merlin, coupled with research demonstrating that merlin is present in â1 integrin immuno-precipitations, leads to the question of whether merlin binds directly to â1 integrin or associates with â1 integrin through paxillin. Using direct binding assays, this research shows that the merlin C-terminus binds directly to the cytoplasmic domain of â1 integrin, in vitro. Lastly, since merlin is an ERM family protein and has been shown to dimerize with ezrin (another ERM family member), and because merlin has been shown to bind directly to paxillin, the question asked is whether paxillin can interact directly with ezrin. The results indicate that paxillin can bind directly to the N-terminus of ezrin, in vitro. The findings presented here, when examined together, provide a framework for the proposal of a model in which paxillin LD3 mediates the localization of merlin to the plasma membrane, where it associates with the â1 integrin cytoplasmic domain and ezrin. These results and the proposed model offer additional insight into the mechanism of action of merlin's negative growth regulating function in Schwann cells.
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Date Issued
2005
Identifier
CFE0000654, ucf:46499
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0000654
THE PRESENCE OF PAIN RELATED CYTOKINES AND CHEMOKINES IN SCHWANNOMAS AND THEIR POTENTIAL ASSOCIATION WITH CHRONIC PAIN IN SCHWANNOMATOSIS
Title
THE PRESENCE OF PAIN RELATED CYTOKINES AND CHEMOKINES IN SCHWANNOMAS AND THEIR POTENTIAL ASSOCIATION WITH CHRONIC PAIN IN SCHWANNOMATOSIS.
Creator
Nagamoto, Jackson D, Fernandez-Valle, Cristina, University of Central Florida
Abstract / Description
Schwannomatosis (SWN) is a genetic disorder that predisposes affected individuals to develop multiple Schwannomas anywhere in the peripheral nervous system. This can be due to a mutation in the LZTR1 or SMARCB1 genes on chromosome 22. SWN has the defining clinical symptom of chronic pain and a lack of vestibular schwannomas, which sets it apart from other, related disorders such as Neurofibromatosis Type II (NF2). Currently, it is unknown what causes the chronic pain of SWN patients but it is...
Show moreSchwannomatosis (SWN) is a genetic disorder that predisposes affected individuals to develop multiple Schwannomas anywhere in the peripheral nervous system. This can be due to a mutation in the LZTR1 or SMARCB1 genes on chromosome 22. SWN has the defining clinical symptom of chronic pain and a lack of vestibular schwannomas, which sets it apart from other, related disorders such as Neurofibromatosis Type II (NF2). Currently, it is unknown what causes the chronic pain of SWN patients but it is hypothesized that cytokines may have promote the neuropathic pain experienced by patients. This study investigates the presence of the chemokine CCL2 and the cytokine IL6 in human SWN schwannomas and non-SWN schwannomas to determine if there is a difference in the presence of these cytokines between the two tumor types. It was demonstrated that all of the SWN schwannomas expressed both CCL2 and IL6 whereas the non-SWN schwannomas expressed only one or the other protein if either. These results indicate that the presence of these cytokines within the SWN schwannomas is different from non-SWN schwannomas and could be a potential contributing factor in the occurrence of neuropathic pain experienced by SWN which is part of the differential diagnosis for NF2 and SWN.
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Date Issued
2019
Identifier
CFH2000525, ucf:45627
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFH2000525
Genetic Interactions Between the Guanine Nucleotide Exchange Factor GEfmeso and GTPase signaling components in the drosophila wing reveal microenvironment dependent variation within gtpase signaling n
Title
Genetic Interactions Between the Guanine Nucleotide Exchange Factor GEfmeso and GTPase signaling components in the drosophila wing reveal microenvironment dependent variation within gtpase signaling n.
Creator
Iketani, Ashley, Vonkalm, Laurence, Stern, Michael, Fernandez-Valle, Cristina, University of Central Florida
Abstract / Description
The Ras superfamily of GTPases are important regulators of morphogenesis involved in control of cytoskeletal dynamics, intracellular trafficking, apical-basal polarity and cell migration. Mis-regulation of GTPase signaling interferes with development and is linked to pathogenesis. Traditionally, GTPase signaling has been depicted as a series of independent linear pathways. However, recently it has become apparent that multiple GTPases can interact to regulate a single cellular process,...
Show moreThe Ras superfamily of GTPases are important regulators of morphogenesis involved in control of cytoskeletal dynamics, intracellular trafficking, apical-basal polarity and cell migration. Mis-regulation of GTPase signaling interferes with development and is linked to pathogenesis. Traditionally, GTPase signaling has been depicted as a series of independent linear pathways. However, recently it has become apparent that multiple GTPases can interact to regulate a single cellular process, functioning in poorly understood networks of cross talk between pathways during development. Jim Fristrom (unpublished data) identified a mutation (18-5) that interacts with components of the GTPases Rho1, Rala, and Cdc42 signaling in multiple developmental contexts. Genetic analysis, physical mapping studies, and sequencing of the mutant allele have indicated that the gene was an allele of GEFmeso (CG30115), which encodes guanine nucleotide exchange factor. To show that 18-5 is an allele of GEFmeso, I demonstrated that a GEFmeso transgene could functionally rescue developmental defects associated with the 18-5 mutation. I also investigated cross talk and network variation in signaling interactions between GEFmeso and other GTPase signaling components in the Drosophila wing. My data provide evidence for microenvironment-dependent variation in GTPase signaling networks in specific domains of the wing, and reveal intercellular variation in GTPase signaling within an otherwise uniform epithelium.
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Date Issued
2012
Identifier
CFE0004252, ucf:49541
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0004252
Dissecting the Components of Neuropathic Pain
Title
Dissecting the Components of Neuropathic Pain.
Creator
George, Dale, Lambert, Stephen, Kim, Yoon-Seong, Fernandez-Valle, Cristina, Ebert, Steven, University of Central Florida
Abstract / Description
Pain is a public health issue affecting the lives of nearly 116 million adults in the US, annually. Understanding the physiological and phenotypic changes that occur in response to painful stimuli is of tremendous clinical interest, but, the complexity of pain and the lack of a representative in vitro model hinders the development of new therapeutics. Pain stimuli are first perceived and transmitted by the neurons within the dorsal root ganglia (DRG) which become hyperexcitable under these...
Show morePain is a public health issue affecting the lives of nearly 116 million adults in the US, annually. Understanding the physiological and phenotypic changes that occur in response to painful stimuli is of tremendous clinical interest, but, the complexity of pain and the lack of a representative in vitro model hinders the development of new therapeutics. Pain stimuli are first perceived and transmitted by the neurons within the dorsal root ganglia (DRG) which become hyperexcitable under these conditions. It has now been established that satellite glial cells (SGCs) that ensheathe the DRG cell body actively contribute to this neuronal dysregulation. To understand the role of SGCs in this pain circuit, first, we looked at the development of SGCs within the DRG of rats, and we showed that SGCs developed postnatally, and appeared morphologically, transcriptionally and functionally similar to Schwann cells precursors (SCs), supporting the idea that these cells may exhibit multipotent behavior. Secondly, we describe here, a three-dimensional in vitro model of the DRG which is functionally characterized on a microelectrode array (MEA). This model can be used to assess the long-term recording of spontaneous activity from bundles of axons while preserving the neuronal-SGC interactions similar to those observed in vivo. Furthermore, using capsaicin, an agonist of the TRPV1 nociceptive receptor, we show that this model can be used as an in vitro assay to acquire evoked responses from nociceptive neurons. Overall, this study advances our knowledge on the development and differentiation of SGCs and establishes a novel functional three-dimensional model for the study of SGCs. This model can now be used as a tool to study the underlying basis of neuronal dysregulation in pain.
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Date Issued
2018
Identifier
CFE0007002, ucf:52053
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0007002
Characterization of Novel Borrelia burgdorferi Transcripts Expressed during Tick and Mammalian Infection
Title
Characterization of Novel Borrelia burgdorferi Transcripts Expressed during Tick and Mammalian Infection.
Creator
Adams, Philip, Jewett, Mollie, Rohde, Kyle, Moore, Sean, Fernandez-Valle, Cristina, University of Central Florida
Abstract / Description
The purpose of this dissertation is to characterize the transcriptome of Borrelia (Borreliella) burgdorferi to discover novel transcripts, important for pathogenesis. As a spirochete and the etiological agent of Lyme disease, the foremost vector-borne bacterial infection in the world, B. burgdorferi fulfills a distinctive niche among bacterial pathogens. Persisting in the disparate environments of a tick vector and mammalian reservoirs, it is absolutely dependent on its hosts for transmission...
Show moreThe purpose of this dissertation is to characterize the transcriptome of Borrelia (Borreliella) burgdorferi to discover novel transcripts, important for pathogenesis. As a spirochete and the etiological agent of Lyme disease, the foremost vector-borne bacterial infection in the world, B. burgdorferi fulfills a distinctive niche among bacterial pathogens. Persisting in the disparate environments of a tick vector and mammalian reservoirs, it is absolutely dependent on its hosts for transmission and nutrient acquisition. B. burgdorferi harbors a complex fragmented genome which is largely linear, unlike that of most prokaryotes, lacks an array of classically described metabolic genes, and contains an unusually large percentage of unique genomic sequences specific to Borrelia (Borreliella) species. To date, few regulatory mechanisms have been identified which contribute to the ability of the spirochete to sense and respond to its environment. Efforts to use global transcript analysis to elucidate the molecular mechanisms of B. burgdorferi host adaptation have proven challenging due to the low numbers of the pathogen present during infection. Previously, our laboratory successfully developed an in vivo expression technology based approach for B. burgdorferi (BbIVET) to identify spirochete promoter sequences that are active during a murine infection. This screen identified 233 unique putative promoters which mapped to locations across the entire genome. These putative infection-active B. burgdorferi promoters were not only located at the 5' end of annotated open reading frames (ORFs), but also mapped to unannotated locations antisense, intergenic, and intragenic to ORFs. Given the limited characterization of the B. burgdorferi transcriptome, this dissertation applies an RNA sequencing approach (5'RNA-seq) to globally annotate the transcriptional start sites (TSSs) and 5' processed ends of the spirochete's RNA during in vitro cultivation. This resulted in the discovery of numerous novel internal, intergenic, and antisense transcripts. Synergistic analysis combining Northern blotting techniques, alignments of these transcripts to BbIVET proposed promoters, and interrogation of promoter activity via in vivo live imaging of mice, confirmed the expression of a variety of RNAs during laboratory culture and mammalian infection. Further, as a means to improve quantitation of the expression of these transcripts, a new methodology was developed and applied to measure B. burgdorferi promoter activity during tick-pathogen interactions, in a strand specific manner. Finally, because the Lyme disease spirochete harbors many unclassified and unique genomic sequences, the mammalian infection-expressed gene bb0562, identified through BbIVET and 5'RNA-seq, was selected for targeted deletion and evaluation throughout B. burgdorferi's infectious cycle. This demonstrated that gene bb0562 encodes a membrane associated protein, whose presence is critical for establishing murine infection through the bite of an infected tick. In sum, this work contributes significant insight into the transcriptome of B. burgdorferi, provides an innovative approach for the analysis of RNA transcripts at the tick-pathogen interface, and identifies a novel gene critical for Lyme disease pathogenesis.
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Date Issued
2017
Identifier
CFE0006707, ucf:51915
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0006707
Chaperonin Containing TCP1 (CCT) as a Target for Cancer Therapy
Title
Chaperonin Containing TCP1 (CCT) as a Target for Cancer Therapy.
Creator
Carr, Ana, Khaled, Annette, Altomare, Deborah, Tigno-Aranjuez, Justine, Fernandez-Valle, Cristina, University of Central Florida
Abstract / Description
Treatments for aggressive cancers like triple negative breast cancer (TNBC) and small-cell lung cancer (SCLC) have not improved and remain associated with debilitating side effects. There is an unmet medical need for better, druggable targets and improved therapeutics. To this end, we investigated the role of Chaperonin-Containing TCP1 (CCT), an evolutionarily conserved protein-folding complex composed of eight subunits (CCT1-8), in oncogenesis. Our laboratory was the first to report that the...
Show moreTreatments for aggressive cancers like triple negative breast cancer (TNBC) and small-cell lung cancer (SCLC) have not improved and remain associated with debilitating side effects. There is an unmet medical need for better, druggable targets and improved therapeutics. To this end, we investigated the role of Chaperonin-Containing TCP1 (CCT), an evolutionarily conserved protein-folding complex composed of eight subunits (CCT1-8), in oncogenesis. Our laboratory was the first to report that the CCT2 subunit is highly expressed in breast cancer and could be therapeutically targeted. To determine whether CCT is a marker of disease progression in other cancers, we analyzed CCT2 gene expression in liver, prostate and lung cancer, using publicly available genetic databases, and confirmed findings by assessing CCT2 and client proteins, like STAT3, in tumor tissues by immunohistochemistry. We found that CCT2 was high in all cancers, especially SCLC, and correlated with decreased patient survival. We tested CT20p, the peptide therapeutic developed by our laboratory to inhibit CCT, on SCLC and primary lung cells, finding that CT20p was only cytotoxic to SCLC cells. Since SCLC currently lacks targeted therapeutics, our work yielded a new targeted agent that could improve lung cancer mortality. To establish a mechanism of action for CT20p, we partially knocked out CCT2 in TNBC cells, which decreased tumorigenicity in mice and reduced levels of essential proteins like STAT3. To confirm, we overexpressed CCT2 in non-tumorigenic cells and conferred tumor-like characteristics such as increased migration and elevated STAT3. These studies positioned us to develop and validate a strategy for discovery of new small molecule inhibitors of CCT. We thus advanced the field of cancer research by demonstrating that CCT could have diagnostic potential for cancers, such as SCLC and TNBC, that are a significant cause of human death and showed that targeting CCT is a promising therapeutic approach.
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Date Issued
2017
Identifier
CFE0007280, ucf:52191
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0007280
Development of a Functional In Vitro 3D Model of the Peripheral Nerve
Title
Development of a Functional In Vitro 3D Model of the Peripheral Nerve.
Creator
Anderson, Wesley, Lambert, Stephen, Hickman, James, Fernandez-Valle, Cristina, Willenberg, Bradley, University of Central Florida
Abstract / Description
Peripheral neuropathies, affect approximately 20 million people in the United States and are often a complication of conditions such as diabetes that can result in amputation of affected areas such as the feet and toes. In vitro methodologies to facilitate the understanding and treatment of these disorders often lack the cellular and functional complexity required to accurately model peripheral neuropathies. In particular, they are often 2-D and functional readouts, such as electrical...
Show morePeripheral neuropathies, affect approximately 20 million people in the United States and are often a complication of conditions such as diabetes that can result in amputation of affected areas such as the feet and toes. In vitro methodologies to facilitate the understanding and treatment of these disorders often lack the cellular and functional complexity required to accurately model peripheral neuropathies. In particular, they are often 2-D and functional readouts, such as electrical activity, are limited to cell bodies thereby limiting the understanding of axonopathy which often characterizes these disorders. We have developed a functional 3-D model of peripheral nerves using a capillary alginate gel (Capgel(TM)), as a scaffold. We hypothesize that: 1) The unique microcapillary structure of Capgel(TM) allows for the modeling of the 3-D microstructure of the peripheral nerve, and 2) That axon bundling in the capillary allows for the detection of axonal electrical activity. In our initial studies, we demonstrate that culturing embryonic dorsal root ganglia (DRG) within the Capgel(TM) environment allows for the separation of cell bodies from axons and recreates many of the features of an in vivo peripheral nerve fascicle including myelinated axons and the formation of a rudimentary perineurium. To develop functionality for this model we have integrated the DRG Capgel(TM) culture with a microelectrode array to examine spontaneous activity in axon bundles, which we find demonstrates superiority to other widely used 2-D models of the same tissue. Furthermore, by analyzing the activity on individual electrodes, we were able to record action potentials from multiple axons within the same bundle indicating a functional complexity comparable to that observed in fascicles in vivo. This 3D model of the peripheral nerve can be used to study the functional complexities of peripheral neuropathies and nerve regeneration as well as being utilized in the development of novel therapeutics.
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Date Issued
2018
Identifier
CFE0007150, ucf:52303
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0007150
Defective Dynamics of Mitochondria in Amyotrophic Lateral Sclerosis and Huntington's Disease
Title
Defective Dynamics of Mitochondria in Amyotrophic Lateral Sclerosis and Huntington's Disease.
Creator
Song, Wenjun, Bossy-Wetzel, Ella, Fernandez-Valle, Cristina, Cheng, Zixi, Self, William, University of Central Florida
Abstract / Description
Mitochondria play important roles in neuronal function and survival, including ATP production, Ca2+ buffering, and apoptosis. Mitochondrial dysfunction is a common event in the pathogenesis of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and Huntington's disease (HD); however, what causes the mitochondrial dysfunction remains unclear. Mitochondrial fission is mediated by dynamin-related protein 1 (DRP1) and fusion by mitofusin 1/2 (MFN1/2) and optic atrophy 1 (OPA1),...
Show moreMitochondria play important roles in neuronal function and survival, including ATP production, Ca2+ buffering, and apoptosis. Mitochondrial dysfunction is a common event in the pathogenesis of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and Huntington's disease (HD); however, what causes the mitochondrial dysfunction remains unclear. Mitochondrial fission is mediated by dynamin-related protein 1 (DRP1) and fusion by mitofusin 1/2 (MFN1/2) and optic atrophy 1 (OPA1), which are essential for mitochondrial function. Mutations in the mitochondrial fission and fusion machinery lead to neurodegeneration. Thus, whether defective mitochondrial dynamics participates in ALS and HD requires further investigation.ALS is a fatal neurodegenerative disease characterized by upper and lower motor neuron loss. Mutations in Cu/Zn superoxide dismutase (SOD1) cause the most common familiar form of ALS by mechanisms not fully understood. Here, a new motor neuron-astrocyte co-culture system was created and live-cell imaging was used to evaluate mitochondrial dynamics. Excessive mitochondrial fission was observed in mutant SOD1G93A motor neurons, correlating with impaired axonal transport and neuronal cell death. Inhibition of mitochondrial fission restored mitochondrial dynamics and protected neurons against SOD1G93A-induced mitochondrial fragmentation and neuronal cell death, implicating defects in mitochondrial dynamics in ALS pathogenesis.HD is an inherited neurodegenerative disorder caused by glutamine (Q) expansion in the polyQ region of the huntingtin (HTT) protein. In the current work, mutant HTT caused mitochondrial fragmentation in a polyQ-dependent manner in both primary cortical neurons and fibroblasts from human patients. An abnormal interaction between DRP1 and HTT was observed in mutant HTT mice and inhibition of mitochondrial fission or promotion of mitochondrial fusion restored mitochondrial dynamics and protected neurons against mutant HTT-induced cell death. Thus, mutant HTT may increase mitochondrial fission by elevating DRP1 GTPase activity, suggesting that mitochondrial dynamics plays a causal role in HD.In summary, rebalanced mitochondrial fission and fusion rescues neuronal cell death in ALS and HD, suggesting that mitochondrial dynamics could be the molecular mechanism underlying these diseases. Furthermore, DRP1 might be a therapeutic target to delay or prevent neurodegeneration.
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Date Issued
2012
Identifier
CFE0004444, ucf:49356
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0004444
The Actin-Severing Protein Cofilin Is Downstream Of Neuregulin Signaling, Is Regulated By The Tumor Suppressor Merlin, And Is Essential For Schwann Cell Myelination
Title
The Actin-Severing Protein Cofilin Is Downstream Of Neuregulin Signaling, Is Regulated By The Tumor Suppressor Merlin, And Is Essential For Schwann Cell Myelination.
Creator
Sparrow, Nicklaus, Fernandez-Valle, Cristina, Lambert, Stephen, Ebert, Steven, Altomare, Deborah, University of Central Florida
Abstract / Description
Myelination is a complex process requiring coordination of directional motility and an increase in Schwann cell (SC) size to generate a multi-lamellar myelin sheath. Regulation of actin dynamics during myelination is poorly understood. However, it is known that myelin thickness is related to the abundance of neuregulin1-type III (NRG) expressed on the axon surface. NRG binding to ErbB2/3 receptors on the Schwann cell surface initiates signaling cascades necessary for myelination. We identify...
Show moreMyelination is a complex process requiring coordination of directional motility and an increase in Schwann cell (SC) size to generate a multi-lamellar myelin sheath. Regulation of actin dynamics during myelination is poorly understood. However, it is known that myelin thickness is related to the abundance of neuregulin1-type III (NRG) expressed on the axon surface. NRG binding to ErbB2/3 receptors on the Schwann cell surface initiates signaling cascades necessary for myelination. We identify cofilin1, an actin depolymerizing and severing protein, as a downstream target of NRG-ErbB2/3 signaling in rat SC. A five minute exposure of SCs to NRG triggers phosphorylation of ErbB2 with concomitant dephosphorylation, and activation, of cofilin, and its upstream regulators, LIM domain kinase (LIMK) and Slingshot-1 phosphatase (SSH). This leads to cofilin activation and recruitment to the leading edge of the SC plasma membrane. These changes are associated with rapid plasma membrane expansion yielding a 35(-)50% increase in SC size within 30 minutes of NRG1 exposure. Cofilin1-deficient SCs increase phosphorylation of ErbB2, ERK, focal adhesion kinase, and paxillin in response to NRG, but fail to increase in size possibly due to stabilization of unusually long focal adhesions. Cofilin1-deficient SCs co-cultured with sensory neurons fail to elaborate myelin. Ultrastructural analysis reveals that they unsuccessfully segregate or engage axons and form only patchy basal lamina. After 48 hours of co-culturing with neurons, cofilin-deficient SCs fail to align and elongate on axons and often adhere to the underlying substrate rather than to axons. We show that the Neurofibromatosis Type II (NF2) tumor suppressor, merlin, is an upstream regulator of cofilin1, and that merlin knockdown in Schwann cells inhibits their elaboration of normal myelin sheaths in vitro. Merlin-deficient SCs form shorter myelin segments in DRG neuron/SC co-cultures. Merlin-deficient Schwann cells have increased levels of both active Rac (Rac-GTP) and F-actin indicative of a stable actin cytoskeleton. Surprisingly merlin-deficient Schwann cells fail to dephosphorylate and activate cofilin1 in response to NRG stimulation. Inhibition of LIMK restores the ability of merlin-deficient SCs to activate cofilin in response to NRG. In developing rat sciatic nerve, merlin becomes hyper-phosphorylated at S518 during the time of peak myelin formation. During this time, cofilin is localized to the inner mesaxon, and subsequently to Schmidt-Lanterman incisures in mature myelin. This study: 1) identifies cofilin and its upstream regulators, LIMK and SSH, as end targets of a NRG-ErbB2/3 signaling pathway in Schwann cells, 2) demonstrates that cofilin modulates actin dynamics in Schwann cells allowing for motility needed to effectively engage and myelinate axons, 3) shows that merlin regulates NRG-ErbB2/3-cofilin-actin signaling during SC myelination to determine the myelin segment length.
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Date Issued
2017
Identifier
CFE0006664, ucf:51217
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0006664
Targeted Therapy Development for Neurofibromatosis Type 2
Title
Targeted Therapy Development for Neurofibromatosis Type 2.
Creator
Fuse, Marisa, Fernandez-Valle, Cristina, Lambert, Stephen, Altomare, Deborah, Khaled, Annette, University of Central Florida
Abstract / Description
Neurofibromatosis type 2 (NF2) is a debilitating disease characterized by the formation of bilateral vestibular schwannomas, which compress the vestibulocochlear nerve and cause deafness. Additional peripheral schwannomas, meningiomas and ependymomas may also form. NF2 is caused by mutations in the NF2 gene, resulting in the loss of function of the merlin tumor suppressor. Merlin functions in multiple signaling pathways and its absence in Schwann cells yields increased cell survival and...
Show moreNeurofibromatosis type 2 (NF2) is a debilitating disease characterized by the formation of bilateral vestibular schwannomas, which compress the vestibulocochlear nerve and cause deafness. Additional peripheral schwannomas, meningiomas and ependymomas may also form. NF2 is caused by mutations in the NF2 gene, resulting in the loss of function of the merlin tumor suppressor. Merlin functions in multiple signaling pathways and its absence in Schwann cells yields increased cell survival and proliferation, thereby causing schwannoma formation. First line treatment for NF2 is watchful waiting and surgical removal of tumors, potentially resulting in facial paralysis and deafness. To date, there are no pharmacological options for patients with NF2. Since the first clinical trials were completed in 2012, only 5 drugs have been investigated in NF2 patients. Few drugs have elicited a measurable radiographic tumor response and most only result in temporary hearing improvement in a small subset of patients. Development of novel therapeutic compounds is a slow, expensive process. However, re-purposing FDA-approved drugs for NF2 accelerates the transfer of efficacious drugs to the clinic. This dissertation used a systematic approach to identify drugs capable of reducing NF2-associated schwannoma growth. An initial screen revealed drugs that reduced viability of mouse and human merlin-deficient Schwann cells. Efficacious drugs were then advanced to an allograft mouse model of NF2 to identify those that reduced tumor growth in vivo. Drug efficacy was also examined in human primary schwannoma cells. We showed that Src, c-MET and MEK inhibitors reduced viability of merlin-deficient Schwann cells both in vitro and in vivo. We also identified a combination treatment of Src and c-MET inhibitors that induced apoptosis, suggesting the potential for preventing tumor recurrence after completion of drug treatment. The work presented here provides valuable pre-clinical evidence for the advancement of several approved drugs to clinical trials for NF2-associated schwannomas.
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Date Issued
2017
Identifier
CFE0006877, ucf:51707
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0006877
Target validation for Neurofibromatosis Type 2 therapeutics.
Title
Target validation for Neurofibromatosis Type 2 therapeutics.
Creator
Petrilli Guinart, Alejandra, Fernandez-Valle, Cristina, Altomare, Deborah, Khaled, Annette, Lambert, Stephen, University of Central Florida
Abstract / Description
Neurofibromatosis type 2 (NF2) is a benign tumor disease of the nervous system. Development of bilateral vestibular schwannomas is characteristic of NF2; however patients frequently present schwannomas on other nerves, as well as meningiomas and ependymomas. Currently, there are no drug therapies for NF2. There is an urgent need for development of NF2 therapeutics and this dissertation presents two independent potential therapeutic targets.The disease is caused by mutations in the NF2 gene...
Show moreNeurofibromatosis type 2 (NF2) is a benign tumor disease of the nervous system. Development of bilateral vestibular schwannomas is characteristic of NF2; however patients frequently present schwannomas on other nerves, as well as meningiomas and ependymomas. Currently, there are no drug therapies for NF2. There is an urgent need for development of NF2 therapeutics and this dissertation presents two independent potential therapeutic targets.The disease is caused by mutations in the NF2 gene that encodes a tumor suppressor called merlin. Loss of merlin function is associated with increased activity of Rac and p21-activated kinases (PAK) and deregulation of cytoskeletal organization. LIM domain kinases (LIMK1 and 2) are substrates for Cdc42/Rac-PAK, and modulate actin dynamics by phosphorylating cofilin, an actin severing and depolymerizing agent. LIMKs also translocate into the nucleus and regulate cell cycle progression. Here we report that mouse Schwann cells (MSCs) in which merlin function is lost as a result of Nf2 exon2 deletion (Nf2 delta Ex2) exhibited increased levels of LIMK1, LIMK2, and active phospho-Thr508/505-LIMK1/2, as well as phospho-Ser3-cofilin, compared to wild-type normal MSCs. Similarly, levels of LIMK1 and 2 total protein and active phosphorylated forms were elevated in human vestibular schwannomas compared to normal human Schwann cells (SCs). Reintroduction of wild-type NF2 into Nf2?delta Ex2 MSC reduced LIMK1 and LIMK2 levels. Pharmacological inhibition of LIMK with BMS-5, decreased the viability of Nf2?delta Ex2 MSCs in a dose-dependent manner, but did not affect viability of control MSCs. Similarly, LIMK knockdown decreased viability of Nf2?delta Ex2 MSCs. The decreased viability of Nf2?delta Ex2 MSCs was due to inhibition of cell cycle progression as evidenced by accumulation of cells in G2/M phase. Inhibition of LIMKs arrest cells in early mitosis by decreasing Aurora A activation and cofilin phosphorylation.To increase the search for NF2 therapeutics, we applied an alternative approach to drug discovery with an unbiased pilot high-throughput screen of the Library of Pharmacologically Active Compounds. We assayed for compounds capable of reducing viability of Nf2?delta Ex2 MSC as a cellular model for human NF2 schwannomas. AGK2, a SIRT2 (sirtuin 2) inhibitor, was identified as a candidate compound. SIRT2, a mammalian sirtuin, is a NAD+-dependent protein deacetylase. We show that Nf2?delta Ex2 MSC have higher expression levels of SIRT2 and lower levels of overall lysine acetylation than wild-type control MSC. Pharmacological inhibition of SIRT2 decreases Nf2?delta Ex2 MSC viability in a dose dependent manner without substantially reducing wild-type MSC viability. Inhibition of SIRT2 activity in Nf2?delta Ex2 MSC causes cell death accompanied by release of the necrotic markers lactate dehydrogenase and high mobility group box 1 protein into the medium in the absence of significant apoptosis, autophagy, or cell cycle arrest.Overall this work uncovered two novel potential therapeutic targets, LIMK and SIRT2 for NF2 and tumors associated with merlin deficiency.
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Date Issued
2013
Identifier
CFE0005398, ucf:50453
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0005398
Amyotrophic Lateral Sclerosis: mechanism behind mutant SOD toxicity and improving current therapeutic strategies
Title
Amyotrophic Lateral Sclerosis: mechanism behind mutant SOD toxicity and improving current therapeutic strategies.
Creator
Dennys, Cassandra, Estevez, Alvaro, Kim, Yoon-Seong, Fernandez-Valle, Cristina, Khaled, Annette, University of Central Florida
Abstract / Description
Amyotrophic Lateral Sclerosis (ALS) is an always lethal motor neuron disease with unknown pathogenesis. Inhibitors of the molecular chaperone heat shock protein 90 (Hsp90) have limited neuroprotection in some models of motor neuron degeneration. However the direct effect of Hsp90 inhibition on motor neurons is unknown. Here we show that Hsp90 inhibition induced motor neuron death through activation of the P2X7 receptor. Motor neuron death required phosphatase and tensein homolog (PTEN)...
Show moreAmyotrophic Lateral Sclerosis (ALS) is an always lethal motor neuron disease with unknown pathogenesis. Inhibitors of the molecular chaperone heat shock protein 90 (Hsp90) have limited neuroprotection in some models of motor neuron degeneration. However the direct effect of Hsp90 inhibition on motor neurons is unknown. Here we show that Hsp90 inhibition induced motor neuron death through activation of the P2X7 receptor. Motor neuron death required phosphatase and tensein homolog (PTEN)-mediated inhibition of the PI3K/AKT pathway leading to Fas receptor activation and caspase dependent death. The relevance of Hsp90 for motor neuron survival was investigated in mutant Cu/Zn superoxide dismutase (SOD) transgenic animal models for ALS. Nitrated Hsp90, a posttranslational modification known to induce cell death (Franco, Ye et al. 2013), was present in motor neurons after intracellular release of zinc deficient (Zn, D83S) and the SOD in which copper binding site was genetically ablated (Q) but not after copper deficient (Cu) wild type SOD. Zn deficient and Q mutant SOD induced motor neuron death in a peroxynitrite mediated and copper dependent mechanism. Nitrated Hsp90 was not detected in the spinal cord of transgenic animals for ALS-mutant SOD animal models until disease onset. Increased nitrated Hsp90 concentrations correlated with disease progression. Addition of Zn or Q SOD to nontransgenic brain homogenate treated with peroxynitrite led to an increase level of nitrotyrosine in comparison to wild type controls. However, in the same samples there was a 2 to 10 time increase in Hsp90 nitration as compared to nitrotyrosine. The selective increase is likely due to the binding of Hsp90 to Zn deficient and Q SOD as oppose to wild type SOD. These results suggest that Hsp90 nitration facilitated by mutant SOD may cause motor neuron degeneration in ALS. Targeted inhibition of nitrated Hsp90 may be a novel therapeutic approach for ALS. An alternative therapeutic strategy is to target the production of survival factors by glial cells. Riluzole is the only FDA approved drug for the treatment of ALS and it shows a small but significant increase in patient lifespan. Our results show that acute riluzole treatment stimulated trophic factor production by astrocytes and Schwann cells. However long-term exposure reversed and even inhibited the production of trophic factors, an observation that may explain the modest increase in patient survival in clinical trials. Discontinuous riluzole treatment can maintain elevated trophic factor levels and prevent trophic factor reduction in spinal cords of nontransgenic animals. These results suggest that discontinuous riluzole administration may improve ALS patient survival. In summary, we demonstrated that Hsp90 has an essential function in the regulation of motor neuron survival. We have also shown that Hsp90 was nitrated in the presence of mutant SOD and was present during symptom onset and increases as disease progresses, which may explain the toxic gain of function of mutant SOD. Finally we demonstrate a biphasic effect of riluzole on trophic factor production and propose changes in administration to improve effects in ALS patients.
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Date Issued
2015
Identifier
CFE0005785, ucf:50069
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0005785
Overexpression of human Cu/Zn Superoxide Dismutase in Mice: A Model to Study the Effect of Increased Superoxide Scavenging on the Autonomic Control of the Heart.
Title
Overexpression of human Cu/Zn Superoxide Dismutase in Mice: A Model to Study the Effect of Increased Superoxide Scavenging on the Autonomic Control of the Heart.
Creator
Hatcher, Jeffrey, Cheng, Zixi, Bossy-Wetzel, Ella, Fernandez-Valle, Cristina, Belfield, Kevin, University of Central Florida
Abstract / Description
Dysregulation of the autonomic cardiovascular control is a complication of diseases including diabetes, hypertension, sleep apnea, and aging. A common factor in these conditions is an increase in reactive oxygen species (ROS) in neural, cardiac, and endothelial tissues. Cu/Zn superoxide dismutase (SOD1) is an intracellular anti-oxidant enzyme that catalyzes dismutation of the superoxide anion (O2.-) to hydrogen peroxide (H2O2). Expression and function of this enzyme are diminished in...
Show moreDysregulation of the autonomic cardiovascular control is a complication of diseases including diabetes, hypertension, sleep apnea, and aging. A common factor in these conditions is an increase in reactive oxygen species (ROS) in neural, cardiac, and endothelial tissues. Cu/Zn superoxide dismutase (SOD1) is an intracellular anti-oxidant enzyme that catalyzes dismutation of the superoxide anion (O2.-) to hydrogen peroxide (H2O2). Expression and function of this enzyme are diminished in pathologies that impair cardiovascular autonomic control. This study employed mice overexpressing a transgene for human SOD1 (hSOD1) to determine if its overexpression would alter autonomic regulation of BP, HR, and BRS in healthy animals, and if this animal line (C57B6SJL-Tg (SOD1)2 Gur/J) could be used in future studies to determine if hSOD1 overexpression can preserve cardiac autonomic function in disease models. To accomplish this aim, using anesthetized SOD1 and C57 (control) mice, we recorded HR, and aortic depressor nerve (ADN) activity changes in response to pharmacologically-induced BP changes in order to measure baroreflex and baroreceptor sensitivity, respectively. In order to identify any alterations in central, efferent, and cardiac components of the baroreflex arc, we electrically stimulated the left ADN and left cervical vagus and compared the reductions in BP and HR between the C57 and SOD1 mice. Time- and frequency-domain analysis of heart rate variability (HRV) was performed using pulse pressure recordings prior to pharmacologic or surgical procedures. We found that hSOD1 overexpression in the SOD1 mouse line, in comparison to C57 controls did not significantly affect resting HR (C57: 558 (&)#177; 8 vs. SOD1:553 (&)#177; 13 beats-per-minute) or blood pressure (C57: 88.8 (&)#177; 2.9 vs.SOD1: 85.8 (&)#177; 2.1 mmHg). hSOD1 overexpression did not affect the decrease in average mean arterial pressure (MAP) following injection of sodium nitroprusside (SNP) (C57: 38.7 (&)#177; 1.4 vs. SOD1: 39.5 (&)#177; 1.3 mmHg) or increase in average MAP (C57: 135.8 (&)#177; 3.1 vs. SOD1: 136.6 (&)#177; 3.5 mmHg) following injection of phenylephrine (PE). BRS, as measured by the averaged regression lines for ?HR/?MAP for the SNP-induced tachycardic baroreflex (C57: 0.57 (&)#177; 0.06 bpm/mmHg, SOD1: 0.61 (&)#177; 0.08 bpm/mmHg)) and the PE-induced bradycardic baroreflex (C57: -2.9 (&)#177; 0.57 bmp/mmHg, SOD1: -4.3 (&)#177; 0.84 bpm/mmHg) are not significantly different between C57 and SOD1. Baroreceptor activation showed a significant increase in gain (C57: 5.4 (&)#177; 0.3 vs. SOD1: 7.4 (&)#177; 0.5 %/mmHg, P (<) 0.01) in the SOD1 transgenic mice. Heart rate depression in response to electrical stimulation of the left ADN and cervical vagus was comparable between C57 and SOD1, though MAP reduction in response to ADN stimulation is slightly, but significantly increased at 50 Hz in SOD1 animals. Time- domain analysis of HRV did not reveal any significant difference in beat-to-beat variability between SOD1 and C57 (SDNN: C57: 2.78 (&)#177; 0.20, SOD1: 2.89 (&)#177; 0.27), although frequency-domain analysis uncovered a significant reduction in the low-frequency power component of the HRV power spectral distribution (C57: 1.19 (&)#177; 0.11, SOD1: 0.35 (&)#177; 0.06, P (<) 0.001). This study shows that although hSOD1 overexpression does not affect overall baroreflex function, it does potentiate baroreceptor sensitivity and brain stem control of arterial pressure, and reduces low-frequency beat-to-beat variations in HR, without affecting total HRV.
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Date Issued
2015
Identifier
CFE0005803, ucf:50025
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0005803
Characterization of composite broadband absorbing conjugated polymer nanoparticles by steady-state, time-resolved and single particle spectroscopy
Title
Characterization of composite broadband absorbing conjugated polymer nanoparticles by steady-state, time-resolved and single particle spectroscopy.
Creator
Bonner, Maxwell, Gesquiere, Andre, Campiglia, Andres, Santra, Swadeshmukul, Hernandez, Florencio, Perez Figueroa, Jesus, Ye, Jingdong, Fernandez-Valle, Cristina, University of...
Show moreBonner, Maxwell, Gesquiere, Andre, Campiglia, Andres, Santra, Swadeshmukul, Hernandez, Florencio, Perez Figueroa, Jesus, Ye, Jingdong, Fernandez-Valle, Cristina, University of Central Florida
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Abstract / Description
As the global economy searches for reliable, inexpensive and environmentally friendly renewable energy resources, energy conservation by means of photovoltaics has seen near exponential growth in the last decade. Compared to state-of-the-art inorganic solar cells, organic photovoltaics (OPVs) composed of conjugated polymers are particularly interesting because of their processability, flexibility and the potential for large area devices at a reduced fabrication cost. It has been extensively...
Show moreAs the global economy searches for reliable, inexpensive and environmentally friendly renewable energy resources, energy conservation by means of photovoltaics has seen near exponential growth in the last decade. Compared to state-of-the-art inorganic solar cells, organic photovoltaics (OPVs) composed of conjugated polymers are particularly interesting because of their processability, flexibility and the potential for large area devices at a reduced fabrication cost. It has been extensively documented that the interchain and intrachain interactions of conjugated polymers complicate the fundamental understanding of the optical and electronic properties in the solid-state (i.e. thin film active layer). These interactions are highly dependent on the nanoscale morphology of the solid-state material, leading to a heterogeneous morphology where individual conjugated polymer molecules obtain a variety of different optoelectronic properties. Therefore, it is of the utmost importance to fundamentally study conjugated polymer systems at the single molecule or nanoparticle level instead of the complex macroscopic bulk level.This dissertation research aims to develop simplified nanoparticle models that are representation of the nanodomains found in the solid-state material, while fundamentally addressing light harvesting, energy transfer and interfacial charge transfer mechanisms and their relationship to the electronic structure, material composition and morphology of the nanoparticle system. In preceding work, monofunctional doped nanoparticles (polymer-polymer) were fabricated with enhanced light harvesting and F?rster energy transfer properties by blending Poly[(o-phenylenevinylene)-alt-(2-methoxy-5-(2-ethylhexyloxy)-p-phenylenevinylene)] (BPPV) and Poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene] (MEH-PPV) at various MEH-PPV doping ratios. While single particle spectroscopy (SPS) reveals a broad distribution of optoelectronic and photophysical properties, time-correlated single photon counting (TC-SPC) spectroscopy displays multiple fluorescence lifetime components for each nanoparticle composition, resulting from changing polymer chain morphologies and polymer-polymer aggregation. In addition, difunctional doped nanoparticles were fabricated by doping the monofunctional doped nanoparticles with PC60BM ([6,6]-phenyl-C61-butyric acid methyl ester) to investigate competition between intermolecular energy transfer and interfacial charge transfer. Specifically, the difunctional SPS data illustrated enhanced and reduced energy transfer mechanisms that are dependent on the material composition of MEH-PPV and PC60BM. These data are indicative of changes in inter- and intrachain interactions of BPPV and MEH-PPV and their respective nanoscale morphologies. Together, these fundamental studies provide a thorough understanding of monofunctional and difunctional doped nanoparticle photophysics, necessary for understanding the morphological, optoelectronic and photophysical processes that can limit the efficiency of OPVs and provide insight for strategies aimed at improving device efficiencies.
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Date Issued
2011
Identifier
CFE0004089, ucf:49143
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0004089