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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
Implication of alpha-synuclein transcriptional regulation and mutagenesis in the pathogenesis of sporadic Parkinson's disease
Title
Implication of alpha-synuclein transcriptional regulation and mutagenesis in the pathogenesis of sporadic Parkinson's disease.
Creator
Basu, Sambuddha, Kim, Yoon-Seong, King, Stephen, Estevez, Alvaro, Altomare, Deborah, University of Central Florida
Abstract / Description
Parkinson's disease (PD) is an age-related neurodegenerative disorder characterized by selective loss of dopaminergic neurons (DA neurons) from the substantia nigra (SN) of the mid-brain. PD is classically associated with cytoplasmic inclusion of aggregated proteins called Lewy bodies. alpha-synuclein (?-SYN) coded by the gene SNCA, is one of the major components of Lewy body and neurite along with several other proteins like ubiquitin, neurofilament to name a few. PD is broadly categorized...
Show moreParkinson's disease (PD) is an age-related neurodegenerative disorder characterized by selective loss of dopaminergic neurons (DA neurons) from the substantia nigra (SN) of the mid-brain. PD is classically associated with cytoplasmic inclusion of aggregated proteins called Lewy bodies. alpha-synuclein (?-SYN) coded by the gene SNCA, is one of the major components of Lewy body and neurite along with several other proteins like ubiquitin, neurofilament to name a few. PD is broadly categorized into two groups based on their incidence of occurrence. First is the familial form that occurs due to known genetic aberrations like mutation, gene duplication/triplication in important PD associated gene like SNCA which in turn leads to early-onset PD (EOPD). Second is the late-onset idiopathic or sporadic form, whose origin of occurrence is often unknown. Interestingly, more than 90%-95% of reported PD cases belong to the latter category. Although, the familial and the idiopathic form of PD are different in their respective cause of occurrence, aggregation of ?-SYN into Lewy body is a common pathologic hallmark seen in both. Aggregation of ?-SYN in turn is strongly implicated by the transcriptional upregulation of the gene as seen in both familial forms as well as idiopathic forms. In this thesis, we first describe the designing and functioning of a novel tool to monitor real-time SNCA transcription in Human Embryonic Kidney (HEK) 293T cells. In the next part, we shed light into a novel transcriptional deregulation phenomenon called transcriptional mutagenesis, which leads to accelerated aggregation of ?-SYN as seen in sporadic PD. In brief, the focus of this work is to highlight the importance of transcriptional regulation of SNCA gene, through development of a tool and a mechanism affecting the fidelity of transcription under pathologic condition. In the first study, we developed a stable cell line in HEK293T cells in which ?-SYN was tagged with Nanoluc luciferase reporter using CRISPR/Cas9-mediated genome editing. Nanoluc is a small stable reporter of 19KDa size, which is 150 fold brighter compared to firefly and Renilla luciferase, thus making it a very good candidate for endogenous monitoring of gene regulations. We successfully integrated the Nanoluc at the 3'end of the SNCA before the stop codon. Successful integration of the Nanoluc was demonstrated by the fusion ?-SYN protein containing the Nanoluc. This allowed efficient monitoring of ?-SYN transcription keeping its native epigenetic landscape unperturbed which was otherwise difficult using exogenous luciferase reporter assays. The Nanoluc activity monitored by a simple two-step assay faithfully reflected the endogenous deregulation of SNCA following treatment with different drugs including epigenetic modulators and dopamine which were already known to up-regulate SNCA transcription. Interestingly, use of exogenous promoter-reporter assays (firefly luciferase assays) failed to reproduce the similar outcomes. In fact, exogenous system showed contradictory results in terms of the ?-SYN regulation which aroused from spurious effects of the drug on the reporter system. To our knowledge, this is the first report showing endogenous monitoring of ?-SYN transcription, thus making it an efficient drug screening tool that can be very effectively used for therapeutic intervention in PD. In the next study, we investigated the effect of oxidative DNA damage in the form of 8-hydroxy-2-deoxyguanosine (8-oxodG, oxidized guanine) on aggregation of ?-SYN through a novel phenomenon called transcriptional mutagenesis. It is already known that 8-oxodG is repaired by a specific component of the base excision repair machinery of the cell called 8-oxodG-DNA glycosylase 1 (OGG1). If left unrepaired, 8-oxodG can lead to misincorporation of adenine instead of cytosine (C?A transversion) in the synthesized mRNA during transcription for post-mitotic cells like neurons. This phenomenon is called transcriptional mutagenesis (TM) and can generate novel mutant variants of any functional protein. ?-SYN, which is implicated very strongly in the pathogenesis of PD, has been shown to become aggregation prone by specific point mutation. Previous studies have shown that certain point mutations can make ?-SYN more prone to aggregation and can affect the aggregation of the parental protein as a template directed misfolding mechanism. We used SNCA as a model gene and predicted the generation of forty-three different positions that can be mutated by the TM event. We investigated the generation of three out of the forty-three possible TM mutants from the SN of post-mortem PD and age-matched control brain cohorts based on their potential to aggregate as predicted by aggregation prediction software TANGO. The three mutants were Serine42Tyrosine (S42Y), Alanine53Glutamate (A53E) and Serine129Tyrosine (S129Y). We confirmed the presence of all the three mutant ?-SYN (S42Y, A53E and S129Y) in SNCA mRNA from the SN of human post-mortem PD brain using a PCR-based detection technique. As expected, analysis of the overall distribution of the three mutants showed a higher rate of occurance in the PD cohort compared to the age-matched controls. Sequencing genomic DNA of the same PD sample from the same region of ?-SYN revealed no mutations at the genomic DNA level, thus implying its generation during transcription. Although we could detect the presence of S42Y, A53E and S129Y ?-SYN in the cohort of PD patients, we focused to analyse the contribution of S42Y towards the aggregation of wild-type (WT) ?-SYN parental protein based on its higher potential to aggregate. By using cell-based biochemical and recombinant protein assays, we saw that S42Y-?-SYN can accelerate the aggregation process involving the WT protein even when present in significantly lower proportion (100 times less compared to the WT). Importantly, we developed antibody to specifically detect the S42Y ?-SYN in human PD cohort. Immunohistochemical analysis of serial post-mortem PD brain sections with Hematoxylin and Eosin staining (H(&)E), anti-ubiquitin staining and anti-S42Y ?-SYN staining, showed Lewy bodies that stained positively with S42Y ? -SYN. To our knowledge, this is the first report about TM related mutations of ?-SYN in Parkinson's disease and their role in the pathogenesis.
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Date Issued
2017
Identifier
CFE0006719, ucf:51882
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0006719
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

Pages