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Neuromuscular junction defects in a mouse model of Charcot-Marie-Tooth disease type 2O
Title
Neuromuscular junction defects in a mouse model of Charcot-Marie-Tooth disease type 2O.
Creator
Sabblah, Thywill, Kim, Yoon-Seong, King, Stephen, Bossy-Wetzel, Ella, Altomare, Deborah, University of Central Florida
Abstract / Description
Charcot Marie Tooth disease (CMT) represents the most common inheritable peripheral group of motor and sensory disorders; affecting 1 in 2500 people worldwide. Individuals with CMT experience slow progressing weakness of the muscle, atrophy, mild loss of motor coordination and in some cases loss of sensory function in the hands and feet which could ultimately affect mobility. Dynein is an essential molecular motor that functions to transport cargos in all cells. A point mutation in the dynein...
Show moreCharcot Marie Tooth disease (CMT) represents the most common inheritable peripheral group of motor and sensory disorders; affecting 1 in 2500 people worldwide. Individuals with CMT experience slow progressing weakness of the muscle, atrophy, mild loss of motor coordination and in some cases loss of sensory function in the hands and feet which could ultimately affect mobility. Dynein is an essential molecular motor that functions to transport cargos in all cells. A point mutation in the dynein heavy chain was discovered to cause CMT disease in humans, specifically CMT type 2O. We generated a knock-in mouse model bearing the same mutation(H304R) in the dynein heavy chain to study the disease. We utilized behavioral assays to determine whether our mutant mice had a phenotype linked to CMT disease. The mutant mice had motor coordination defects and reduced muscle strength compared to normal mice. To better understand the disease pathway, we obtained homozygous mutants from a heterozygous cross, and the homozygotes show even more severe deficits compared to heterozygotes. They also developed an abnormal gait which separates them from heterozygous mice. In view of the locomotor deficits observed in mutants, we examined the neuromuscular junction (NMJ) for possible impairments. We identified defects in innervation at the later stages of the study and abnormal NMJ architecture in the muscle as well. The dysmorphology of the NMJ was again worse in the homozygous mutants with reduced complexity and denervation at all the timepoints assessed. Our homozygous dynein mutants can live up to two years and therefore make the design of longitudinal studies possible. Altogether, this mouse model provides dynein researchers an opportunity to work towards establishing the link between dynein mutations, dynein dysfunction and the onset and progression of disease.
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Date Issued
2018
Identifier
CFE0007088, ucf:51956
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0007088
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 Role of SOD1 Acetylation in Neurodegeneration
Title
The Role of SOD1 Acetylation in Neurodegeneration.
Creator
Kaliszewski, Michael, Bossy-Wetzel, Ella, Estevez, Alvaro, Kim, Yoon-Seong, Tatulian, Suren, University of Central Florida
Abstract / Description
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.
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Date Issued
2016
Identifier
CFE0006467, ucf:51409
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0006467
Cerium oxide nanoparticles act as a unique catalyst and scavenge nitric oxide and peroxynitrite and decrease RNS in vitro and in vivo
Title
Cerium oxide nanoparticles act as a unique catalyst and scavenge nitric oxide and peroxynitrite and decrease RNS in vitro and in vivo.
Creator
Dowding, Janet, Self, William, Bossy-Wetzel, Ella, Zervos, Antonis, Seal, Sudipta, Santra, Swadeshmukul, University of Central Florida
Abstract / Description
Cerium oxide nanoparticles (CeO2 NPs)(nanoceria) have been shown to possess a substantial oxygen storage capacity via the interchangeable surface reduction and oxidation of cerium atoms, cycling between the Ce4+ and Ce3+ redox states. Reduction of Ce4+ to Ce3+ causes oxygen vacancies or defects on the surface of the crystalline lattice structure of the particles, generating a cage for redox reactions to occur. The study of the chemical and biological properties of CeO2 NPs has expanded...
Show moreCerium oxide nanoparticles (CeO2 NPs)(nanoceria) have been shown to possess a substantial oxygen storage capacity via the interchangeable surface reduction and oxidation of cerium atoms, cycling between the Ce4+ and Ce3+ redox states. Reduction of Ce4+ to Ce3+ causes oxygen vacancies or defects on the surface of the crystalline lattice structure of the particles, generating a cage for redox reactions to occur. The study of the chemical and biological properties of CeO2 NPs has expanded recently, and the methods used to synthesize these materials are also quite diverse. This has led to a plethora of studies describing various preparations of CeO2 NPs for potential use in both industry and for biomedical research. Our own work has centered on studies that measure the ability of water-based CeO2 NPs materials to reduce reactive oxygen and nitrogen species in biological systems, and correlating changes in surface chemistry and charge to the catalytic nature of the particles. The application in experimental and biomedical research of CeO2 NPs began with the discovery that water-based cerium oxide nanoparticles could act as superoxide dismutase mimetics followed by their ability to reduce hydrogen dioxide similar to catalase. While their ROS scavenging ability was well established, their ability to interact with specific RNS species, specifically nitric oxide (NO) or peroxynitrite (ONOO-) was not known. The studies described in this dissertation focus on the study of RNS and cerium oxide nanoparticles.Our in vitro work revealed that CeO2 NPs that have higher levels of reduced cerium sites (3+) at the surface (which are effective SOD mimetics) are also capable of accelerating the decay of peroxynitrite in vitro. In contrast, CeO2 NPs that have fewer reduced cerium sites at the particle surface (which also exhibit better catalase mimetic activity) have NO scavenging capabilities as well as some reactivity with peroxynitrite. Our studies and many others have shown cerium oxide nanoparticles can reduce ROS and RNS in cell culture or animal models. The accumulation of ROS and RNS is a common feature of many diseases including Alzheimer's disease (AD). Testing our CeO2 NPS in cortical neurons, we used addition of A? peptide as an AD model system. CeO2 NPs delayed A?-induced mitochondrial fragmentation and neuronal cell death. When mitochondrial ROS levels are increased, mitochondrial fission is activated by DRP1 S616 phosphorylation. Specifically, our studies showed the reduction of phosphorylated DRP1 S616 in the presence of CeO2 NPs. Results from our studies have begun to unravel the molecule mechanism behind the catalytic nature of how CeO2 NPs reduce ROS/RNS in biological systems and represents an important step forward to test the potential neuroprotective effects of CeO2 NPs in model systems of AD.A plethora of studies describing various preparations of CeO2 NPs for potential use in both industry and for biomedical research have been described in the past five years. It has become apparent that the outcomes of CeO2 NPs exposure can vary as much as the synthesis methods and cell types tested. In an effort to understand the disparity in reports describing the toxicity or protective effects of exposure to CeO2 NPs, we compared CeO2 NPs synthesized by three different methods; H2O2 (CNP1), NH4OH (CNP2) or hexamethylenetetramine (HMT-CNP1). Exposure to HMT-CNP1 led to reduced metabolic activity (MTT) at a 10-fold lower concentration than CNP1 or CNP2 and surprisingly, exposure to HMT-CNP1 led to substantial decreases in the ATP levels. Mechanistic studies revealed that HMT-CNP1 and CNP2 exhibited robust ATPase (phosphatase) activity, whereas CNP1 lacked ATPase activity. HMT-CNP1 were taken up into HUVECs far more efficiently than the other preparations of CeO2 NPs. Taken together, these results suggest the combination of increased uptake and ATPase activity of HMT-CNP1 may underlie the mechanism of the toxicity of this preparation of CeO2 NPs, and may suggest ATPase activity should be considered when synthesizing CeO2 NPs for use in biomedical applications. Overall the studies have uncovered two new catalytic activities for water-based CeO2 NPs (NO scavenging and accelerated decay of peroxynitrite), demonstrated their ability to reduce RNS in an AD cell culture model as well as identifying a catalytic activity (phosphatase) that may underlie the observed toxicity of CeO2 NPs reported in other studies.
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Date Issued
2012
Identifier
CFE0004782, ucf:49783
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0004782
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
Role of Cardiac Catecholamines in Embryos and Adults Under Stress
Title
Role of Cardiac Catecholamines in Embryos and Adults Under Stress.
Creator
Baker, Candice, Ebert, Steven, Bossy-Wetzel, Ella, Siddiqi, Shadab, Lambert, Stephen, University of Central Florida
Abstract / Description
Cardiovascular disease is responsible for the loss of one life every 38 seconds and accounts for 26.6 percent of all infants that die of congenital birth defects. Adrenergic hormones are critically important regulators of cardiovascular physiology in embryos and adults. They are key mediators of stress responses and have profound stimulatory effects on cardiovascular function, and dysregulation of adrenergic function has been associated with many adverse cardiac conditions, including...
Show moreCardiovascular disease is responsible for the loss of one life every 38 seconds and accounts for 26.6 percent of all infants that die of congenital birth defects. Adrenergic hormones are critically important regulators of cardiovascular physiology in embryos and adults. They are key mediators of stress responses and have profound stimulatory effects on cardiovascular function, and dysregulation of adrenergic function has been associated with many adverse cardiac conditions, including congenital malformations, arrhythmias, ischemic heart disease, heart failure, and sudden cardiac death. Despite intensive study, the specific roles these hormones play in the developing heart is not well-understood. Further, there is little information available regarding how these important hormones mediate stress responses in adult females (before and after menopause) in comparison to males. My thesis thus has two major foci: (1) What role(s) do catecholamines play in the embryonic heart?, and (2) Do catecholamines differentially influence cardiac function in aging male and female hearts? Initially, we sought to uncover the roles of adrenergic hormones in the embryonic heart by utilizing an adrenergic-deficient (Dbh-/-) mouse model. We found that adrenergic hormones influence heart development by stimulating expression of the gap junction protein, connexin 43, facilitating atrioventricular conduction, and helping to maintain cardiac rhythm. As development progresses, cardiac energy demands increase substantially, and oxidative phosphorylation becomes vital. Adrenergic hormones regulate metabolism in adults, thus we hypothesized they may stimulate energy metabolism during the embryonic/fetal transition period. We examined ATP, ADP, oxygen consumption rate, and extracellular acidification rates and found these metabolic indices were significantly decreased in Dbh-/- hearts compared to Dbh+/+ controls. We employed transmission electron microscopy of embryonic cardiomyocytes and found the mitochondria were significantly larger in Dbh-/- hearts compared to controls, and had more branch points. Taken together, these results suggest adrenergic hormones play a major role mediating the shift from predominantly anaerobic to aerobic metabolism during the embryonic/fetal transition period.Since there are known differential cardiac responses due to sex, age, and menopause to stress, we used echocardiography to measure left ventricular (LV) function in adult (9, 18 and 21 month) male and female mice (pre and postmenopausal) in response to epinephrine, and immobilization stress to investigate the roles of these factors. My results show 9-month premenopausal female mice display significantly decreased LV responsiveness to epinephrine compared to males, and an increased response to epinephrine due to age, especially in the premenopausal females. Similar LV function was also observed between postmenopausal females and males, and this pattern persisted after immobilization stress. I also investigated anatomical differences in the distribution of adrenergic cells within the heart comparing age, sex, and menopausal status. Notably, the density of cells derived from an adrenergic lineage in the heart was significantly increased in postmenopausal mice compared to age-matched males and cycling females. The selective re-appearance of adrenergic cells in the heart following menopause may provide an explanation for the differential stress responses observed in our system, and could have important clinical ramifications for stress-induced cardiomyopathies.
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Date Issued
2014
Identifier
CFE0005458, ucf:50373
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0005458