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(1 - 9 of 9)
- Title
- Motor and sensory characterization of a mouse model of Charcot-Marie-Tooth type 2O disease.
- Creator
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Nandini, Swaran, King, Stephen, Kim, Yoon-Seong, Estevez, Alvaro, University of Central Florida
- Abstract / Description
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Dynein is an essential motor protein required for the maintenance and survival of cells. Dynein forms a motor complex to carry intracellular cargoes like organelles, growth factors, peptides, and hormones along the microtubules inside the cells. In neurons, the dynein is the retrograde motor protein that moves cargoes from the neuronal tip to the neuronal soma along the length of an axon. Dynein has an established role in neuronal nuclear migration, transport of neuronal survival signals and...
Show moreDynein is an essential motor protein required for the maintenance and survival of cells. Dynein forms a motor complex to carry intracellular cargoes like organelles, growth factors, peptides, and hormones along the microtubules inside the cells. In neurons, the dynein is the retrograde motor protein that moves cargoes from the neuronal tip to the neuronal soma along the length of an axon. Dynein has an established role in neuronal nuclear migration, transport of neuronal survival signals and growth factors, organelle positioning inside neurons etc. Hence, it is not very surprising that numerous mutations in dynein have been reported in association with neurodegenerative diseases in humans. The first human mutation (H306R) in dynein heavy chain was reported to cause Charcot-Marie-Tooth Type 2O disease (CMT2O) in humans. CMT2O patients display motor-sensory neuropathy symptoms such as muscle weaknesses and wasting in legs, skeletal deformities like pes cavus (high foot arching), difficulty in walking, and a loss of sensation.We developed a novel knock-in H304R mouse model with the corresponding CMT2O linked dynein mutation to understand the disease's molecular mechanism. We investigated and characterized the motor-sensory phenotype of the H304R mouse model (wildtype, heterozygous (H304R/+) and homozygous (H304R/R) mice). First, we started with testing mice on motor skills behavior tests such as tail suspension reflex, grip strength test, and rotarod test at 3, 6, 9 and 12 months of age. Both male and female groups of heterozygous (H304R/+) mice displayed mild defects in tail suspension reflex, grip strength, and rotarod performance. In contrast, homozygous (H304R/R) mice exhibited severe defects in the tail suspension reflex, grip strength, and rotarod performance right from an early age. Next, I analyzed the sensory phenotype of the H304R mouse model. Homozygous H304R/R mice appeared to have thinner sciatic nerves, reduced total fascicular area of the sciatic nerve, and significantly quicker latency to tail withdrawal from a pain stimulus than the wildtype and heterozygous H304R/+ mice. Collectively, our motor and sensory characterization studies reveal that H304R dynein mouse model recapitulates many of the phenotypes associated with CMT symptoms. Hence, the H304R model is a useful tool in understanding the dynein function in the onset and progression of CMT2O in humans.
Show less - Date Issued
- 2019
- Identifier
- CFE0007508, ucf:52651
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0007508
- Title
- CHARACTERIZATION OF MOTILITY ALTERATIONS CAUSED BY THE IMPAIRMENT OF DYNEIN/DYNACTIN MOTOR PROTEIN COMPLEX.
- Creator
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Nandini, Swaran, King, Stephen, Kim, Yoon-Seong, Estevez, Alvaro, University of Central Florida
- Abstract / Description
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Transport of intracellular cargo is an important and dynamic process required for cell maintenance and survival. Dynein is the motor protein that carries organelles and vesicles from the cell periphery to the cell center along the microtubule network. Dynactin is a protein that activates dynein for this transport process. Together, dynein and dynactin forms a motor protein complex that is essential for transport processes in all the vertebrate cells. Using fluorescent microscope based live...
Show moreTransport of intracellular cargo is an important and dynamic process required for cell maintenance and survival. Dynein is the motor protein that carries organelles and vesicles from the cell periphery to the cell center along the microtubule network. Dynactin is a protein that activates dynein for this transport process. Together, dynein and dynactin forms a motor protein complex that is essential for transport processes in all the vertebrate cells. Using fluorescent microscope based live cell imaging techniques and kymograph analyses, I studied dynein/dynactin disruptions on the intracellular transport in two different cell systems. In one set of experiments, effects of dynein heavy chain (DHC) mutations on the vesicular motility were characterized in the fungus model system Neurospora crassa. I found that many DHC mutations had a severe transport defect, while one mutation linked to neurodegeneration in mice had a subtle effect on intracellular transport of vesicles. In a different set of experiments in mammalian tissue culture CAD cells, I studied the effects of dynactin knockdown and dynein inhibition on mitochondrial motility. My results indicated that reductions in dynactin levels decrease the average number of mitochondrial movements and surprisingly, increase the mitochondrial run lengths. Also, I determined that the dynein inhibitory drug Ciliobrevin causes changes in mitochondrial morphology and decreases the number of mitochondrial movements inside cells. Overall, my research shows that distinct disruptions in the dynein and dynactin motor complex alters intracellular motility, but in different ways. So far, my studies have set the ground work for future experiments to analyze the motility mechanism of motor proteins having mutations that lead to neurodegenerative disorders.
Show less - Date Issued
- 2013
- Identifier
- CFE0004897, ucf:49664
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0004897
- Title
- Development of Cytotoxic Natural Killer Cells for Ovarian Cancer Treatment.
- Creator
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Pandey, Veethika, Altomare, Deborah, Zhao, Jihe, Khaled, Annette, Estevez, Alvaro, University of Central Florida
- Abstract / Description
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Ovarian cancer is a leading cause of gynecological malignancy. Cytoreductive surgery and frontline platinum/taxane-based chemotherapy provides good initial efficacy in the treatment, but poor long-term patient survival. This is mainly caused by tumor relapse due to intraperitoneal spreading and ineffective alternate therapies to treat these resistant tumors. The challenge in the field is to develop strategies that would prove effective in these patients and extend overall survival.Over the...
Show moreOvarian cancer is a leading cause of gynecological malignancy. Cytoreductive surgery and frontline platinum/taxane-based chemotherapy provides good initial efficacy in the treatment, but poor long-term patient survival. This is mainly caused by tumor relapse due to intraperitoneal spreading and ineffective alternate therapies to treat these resistant tumors. The challenge in the field is to develop strategies that would prove effective in these patients and extend overall survival.Over the years, various treatments have been developed for the treatment of cancer amongst which, adoptive cell immunotherapy has shown promising results. But despite the efficacy seen in the clinic, there are concerns with the complexity of treatment and associated side effects. Therefore, there is still a need for better understanding of how different components of the immune system react to the presence of tumor. In this study, healthy human peripheral blood mononuclear cells (PBMCs) were used to examine the immune response in a mouse model with residual human ovarian tumor, where natural killer (NK) cells were found to be the effector cells that elicited an anti-tumor response. Presence of tumor was found to stimulate NK cell expansion and cytotoxicity in mice treated intraperitoneally (IP) with PBMCs+Interleukin-2 (IL- 2). Intravenous (IV) adoptive transfer of isolated NK cells has been attempted in ovarian cancer patients before, but showed lack of persistence in patients resulting in lack of anti-tumor efficacy. Experiments in this study highlight the significance of NK cell-cytotoxic response to tumor, which may be attributed to interacting immune cell types in the PBMC population (when treated IP), as opposed to clinically used isolated NK cells showing lack of anti-tumor efficacy in ovarian cancer patients (when treated IV).iiiNK cell immunotherapy is mainly limited by insufficient numbers generated for adoptive transfer, limited in vivo life span after adoptive transfer, lack of cytotoxicity and some logistical concerns that impede its widespread implementation. Therefore there is a need to develop methods of NK cell expansion that provide stimulation similar to other immune cell types in the PBMC population. The second part of this study utilizes a method of in vivo NK cell expansion using a particle-based approach in which plasma membranes of K562-MB21-41BBL cells (K562 cells expressing membrane-bound IL-21 and 41BB ligand) are used for specific NK cell expansion from PBMCs. NK cells expanded with this method were cytotoxic, showed in vivo persistence and biodistribution in different organs.Collectively, these studies show that NK cells are a major innate immune component that can recognize and kill the tumor. Their cytotoxic ability, using particle-based stimulation, can be enhanced for a second-line treatment of relapsed tumors such as in ovarian cancer as well as other cancer types.
Show less - Date Issued
- 2015
- Identifier
- CFE0006369, ucf:51531
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0006369
- Title
- The CT20 peptide as an agent for cancer treatment.
- Creator
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Bassiouni, Rania, Khaled, Annette, Altomare, Deborah, Zhao, Jihe, Estevez, Alvaro, University of Central Florida
- Abstract / Description
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Due to cancer recurrence and the development of drug resistance, metastatic breast cancer is a leading cause of death in women. In the search for a new therapeutic to treat metastatic disease, we discovered CT20p, an amphipathic peptide based on the C-terminus of Bax. Due to inherent properties of its sequence and similarity to antimicrobial peptides, CT20p is a promising cytotoxic agent whose activity is distinct from the parent protein (e.g. does not cause apoptosis). CT20p is not membrane...
Show moreDue to cancer recurrence and the development of drug resistance, metastatic breast cancer is a leading cause of death in women. In the search for a new therapeutic to treat metastatic disease, we discovered CT20p, an amphipathic peptide based on the C-terminus of Bax. Due to inherent properties of its sequence and similarity to antimicrobial peptides, CT20p is a promising cytotoxic agent whose activity is distinct from the parent protein (e.g. does not cause apoptosis). CT20p is not membrane permeable but can be introduced to cells using polymeric nanoparticles, a method that promotes efficient delivery of the peptide into the intracellular environment.We demonstrated that CT20p was cytotoxic using triple negative breast cancer (TNBC) cell lines, primary breast tumor tissue, and breast tumor murine xenografts. Importantly, normal breast epithelial cells and normal primary breast cells were resistant to the lethal effects of the peptide. Examination of multiple cellular processes showed that CT20p causes cell death by promoting cytoskeletal disruption, cell detachment, and loss of substrate-mediated survival signals.In order to identify the intracellular target of CT20p, we performed pull-down experiments using a biotinylated peptide and found that CT20p binds directly to a type II chaperonin called chaperonin containing T-complex (CCT), which is essential for the folding of actin and tubulin into their native forms. The resulting effect of CT20p upon the cytoskeleton of cancer cells is disruption of vital cellular processes such as migration and adhesion. CCT gene expression and protein levels were examined across several breast cancer cell lines, and we found that susceptibility to CT20p correlated with higher CCT levels. Using human cancer tissue microarrays, we determined that CCT was present in significantly higher amounts in tumor tissues compared to normal tissues and that expression often increased with advanced cancer stage. These results indicate that CCT is a promising therapeutic target for the treatment of metastatic breast cancer and suggest that the use of cancer-targeted nanoparticles loaded with CT20p is a novel and effective therapeutic strategy for cancers, such as TNBC, that recur and are refractory to current treatments.
Show less - Date Issued
- 2015
- Identifier
- CFE0006207, ucf:51095
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0006207
- Title
- The contribution of visceral fat to positive insulin signaling in Ames dwarf mice.
- Creator
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Menon, Vinal, Masternak, Michal, Khaled, Annette, Altomare, Deborah, Estevez, Alvaro, University of Central Florida
- Abstract / Description
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Ames dwarf (df/df) mice are homozygous for a spontaneous mutation in the prop1 gene due to which there is no development of anterior pituitary cells (-) somatotrophs, lactotrophs and thyrotrophs, leading to a deficiency of growth hormone (GH), prolactin (PRL) and thyrotropin (TSH). They tend to become obese as they age, but still live longer and healthier lives compared to their wild-type littermates, being very insulin sensitive, showing no signs of diabetes and cancer. These mutant mice...
Show moreAmes dwarf (df/df) mice are homozygous for a spontaneous mutation in the prop1 gene due to which there is no development of anterior pituitary cells (-) somatotrophs, lactotrophs and thyrotrophs, leading to a deficiency of growth hormone (GH), prolactin (PRL) and thyrotropin (TSH). They tend to become obese as they age, but still live longer and healthier lives compared to their wild-type littermates, being very insulin sensitive, showing no signs of diabetes and cancer. These mutant mice also have high circulating levels of anti-inflammatory and anti-diabetic adiponectin. Plasma levels of this adipokine usually decrease with an increase in accumulation of visceral fat (VF). We thus believe that VF in df/df mice, developed in the absence of GH signaling, may be functionally different from the same fat depots in normal (N) mice and may be beneficial, rather than detrimental, to the overall health of the animal. We performed surgeries involving removal of VF depots (epididymal and perirenal fat) in both groups of mice and hypothesize that the beneficial effects of visceral fat removal (VFR) will be present exclusively in N mice as VF in df/df mice contributes to enhanced insulin sensitivity by producing decreased levels of pro-inflammatory adipokines like TNF? and IL-6. We found that VFR improved insulin sensitivity only in N mice but not in the df/df mice. This intervention led to an upregulation of certain players of the insulin signaling pathway in the skeletal muscle of N mice only, with no alteration in df/df mice. The subcutaneous fat of df/df mice showed a downregulation of these insulin signaling genes upon VFR. Compared to N mice, epididymal fat of df/df mice (sham-operated) had increased gene expression of some of the players involved in insulin signaling and a decrease in transcript levels of TNFa. Ames dwarf mice had decreased levels of IL-6 protein in EF and in circulation. High circulating levels of adiponectin and decreased levels of IL-6 in circulation could contribute to the high insulin sensitivity observed in the Ames dwarf mice. Understanding the mechanisms responsible for VF having positive effects on insulin signaling in df/df mice would be important for future treatment of obese diabetic patients.
Show less - Date Issued
- 2013
- Identifier
- CFE0004890, ucf:49654
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0004890
- Title
- The Role of SOD1 Acetylation in Neurodegeneration.
- Creator
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Kaliszewski, Michael, Bossy-Wetzel, Ella, Estevez, Alvaro, Kim, Yoon-Seong, Tatulian, Suren, University of Central Florida
- Abstract / Description
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Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder affecting motor neurons. Cu, Zn superoxide dismutase (SOD1), a cytoplasmic free radical scavenging enzyme, is mutated in familial ALS (fALS) and post-translational modification of the wild-type protein has been associated with sporadic ALS (sALS). Proteomic studies indicate that SOD1 is acetylated at Lys123; however, the role of this modification remains unknown. To investigate its function, we generated antibodies for...
Show moreAmyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder affecting motor neurons. Cu, Zn superoxide dismutase (SOD1), a cytoplasmic free radical scavenging enzyme, is mutated in familial ALS (fALS) and post-translational modification of the wild-type protein has been associated with sporadic ALS (sALS). Proteomic studies indicate that SOD1 is acetylated at Lys123; however, the role of this modification remains unknown. To investigate its function, we generated antibodies for Lys123-acetylated SOD1 (Ac-K123 SOD1). Sod1 deletion in Sod1-/- mice, K123 mutation, or preabsorption with Ac-K123 peptide suppressed immunoreactivity, confirming antibody specificity. In the normal central nervous system, Ac-K123 SOD1 maps to glutamatergic neurons of the cerebellar cortex, dentate gyrus, hippocampus, olfactory bulb, and retina. In cultured neurons, Ac-K123 SOD1 localized to defined regions of axons and dendrites. Previous studies have suggested a role for SOD1 in cell cycle regulation. Therefore, we tested the distribution of Ac-K123 SOD1 during the cell cycle of astrocytes. In G1 Ac-K123 SOD1 localized to the nucleus, in G0 to the primary cilium, in metaphase and anaphase to chromosomes, and in telophase to the midbody. The deacetylase HDAC6 and acetyl-transferase ?-TAT1 are associated with the primary cilium. Therefore, we tested whether they regulate reversible acetylation of SOD1. HDAC6 knockdown or pharmacological inhibition markedly increased, while HDAC6 overexpression decreased, SOD1 Lys123 acetylation. By contrast, SOD1 Lys123 acetylation was decreased by ?-TAT1 knockdown and increased by ?-TAT1 overexpression. These results suggest that HDAC6 and ?-TAT1 regulate SOD1 Lys123 acetylation. Next, we examined Lys123 acetylation in fALS SOD1 mutants. Remarkably, Lys123 acetylation was dramatically increased in fALS mutants including SOD1 A4V. The acetyl-Lys123 mimetic of wild-type SOD1 caused axonal transport deficits similar to those observed in SOD1 pathogenic mutants such as A4V. Interestingly, HDAC6 deacetylation or acetylation resistance by Lys123 mutation, abolished A4V protein misfolding, axonal transport defects, and neuronal cell death. These results suggest that Lys123 acetylation plays a key role in the neurotoxicity of fALS mutants and may have implications in sALS. Because Ac-K123 SOD1 maps to the primary cilium, we examined whether ciliogenesis is altered in fALS mutant SOD1 astrocytes. Strikingly, fALS mutants caused centriole and primary cilia proliferation with ciliary ectosome secretion. Notably, multiciliated ependymal cells in the brain ventricles and spinal cord central canal, which are critical for cerebral spinal fluid circulation, stained strongly for Ac-K123 SOD1. Thus, we speculate that ciliary ectosome shedding from ependymal cells accounts for the presence of misfolded SOD1 in the CSF in fALS and perhaps sALS. In summary, we identified SOD1 Lys123 acetylation as a novel mechanism underlying protein misfolding and neurodegeneration in ALS. Ac-K123 SOD1 may emerge as novel target for the diagnosis and treatment of ALS.
Show less - Date Issued
- 2016
- Identifier
- CFE0006467, ucf:51409
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0006467
- Title
- Consequences of Altered Short-Chain Carbon Metabolism in Heart Failure.
- Creator
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Horton, Julie, Estevez, Alvaro, Kelly, Daniel, Parthasarathy, Sampath, Crawford, Peter, University of Central Florida
- Abstract / Description
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Cardiovascular disease is currently the foremost cause of death within the United States. Heart failure (HF) is a syndrome defined by the inability of the heart to adequately execute requisite pump function in order to deliver nutrients and oxygen to peripheral tissues, irrespective of etiology. One of the most common causes of HF is chronic pressure overload due to hypertension. Ischemic heart disease is also a common driver of HF, often in conjunction with hypertension. Pressure overload...
Show moreCardiovascular disease is currently the foremost cause of death within the United States. Heart failure (HF) is a syndrome defined by the inability of the heart to adequately execute requisite pump function in order to deliver nutrients and oxygen to peripheral tissues, irrespective of etiology. One of the most common causes of HF is chronic pressure overload due to hypertension. Ischemic heart disease is also a common driver of HF, often in conjunction with hypertension. Pressure overload initially causes compensatory metabolic changes. Structural changes follow shortly thereafter typically resulting in left ventricular hypertrophy. Eventually, the heart loses the ability to compensate for the aberrant hemodynamic load and begins failing. The failing heart is unable to supply adequate adenosine triphosphate (ATP) for contractile function as evidenced by falling phosphocreatine (PCr) levels. This energy deficit occurs concurrently with a metabolic re-programming that results in a fuel utilization pattern resembling the fetal heart. Notably, enzymes involved in catabolism of fatty acids, the chief fuel substrate for ATP generation in the normal adult heart, are downregulated in the failing heart. However, the extent to which alternative fuels compensate for decreased fatty acid oxidation (FAO) is not well-known. Furthermore, consequences of the fuel substrate switches that occur in heart failure are not well established. In this work, we discover a new paradigm for alternate fuel utilization in the failing heart and define consequences of altered fuel metabolism in HF. We discovered a post-translational modification resultant from an accumulation of acetyl groups (C2) present in a mouse model of early-stage HF and human HF. Mitochondrial proteins were found to be hyperacetylated in the failing heart, and at least some of these alterations result in diminished electron-transport chain (ETC) capacity as shown by mutagenesis studies on succinate dehydrogenase A (SDHA). We also found an accumulation of C4-OH carnitine, a by-product of ketone oxidation in HF. This metabolite aggregation occurred alongside an increase in b-hydroxybutyrate dehydrogenase 1 (BDH1) transcript and protein levels. This signature suggested that the failing heart shifted to ketone bodies as a fuel. Subsequent experiments confirmed increased capacity for myocardial ketone oxidation in compensated cardiac hypertrophy and in HF. The consequences of increased ketone oxidation were then assessed using a cardiac-specific BDH1 knockout (BDH1 KO) mouse. Despite not having any apparent defect at baseline, we found BDH1 KO mouse hearts are completely unable to oxidize 3-hydroxybutyrate. The deficit for ketone oxidation capacity became consequential upon subjugation to transverse aortic constriction with a small apical myocardial infarction (TAC/MI). The BDH1 KO mice exhibit altered pathological cardiac remodeling compared to wild-type controls. These latter data suggest the increased reliance on ketone oxidation in HF, mediated by BDH1, is an adaptive response. Together the results of these studies provide important information regarding the consequences of altered fuel metabolism in HF. Recent reports of reduced HF mortality and elevated circulating ketone levels in patients prescribed Empagliflozin make cardiac ketone metabolism research in this dissertation particularly apropos.
Show less - Date Issued
- 2017
- Identifier
- CFE0006948, ucf:51663
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0006948
- Title
- Implication of alpha-synuclein transcriptional regulation and mutagenesis in the pathogenesis of sporadic Parkinson's disease.
- Creator
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Basu, Sambuddha, Kim, Yoon-Seong, King, Stephen, Estevez, Alvaro, Altomare, Deborah, University of Central Florida
- Abstract / Description
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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.
Show less - Date Issued
- 2017
- Identifier
- CFE0006719, ucf:51882
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0006719
- Title
- Amyotrophic Lateral Sclerosis: mechanism behind mutant SOD toxicity and improving current therapeutic strategies.
- Creator
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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.
Show less - Date Issued
- 2015
- Identifier
- CFE0005785, ucf:50069
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0005785
