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2018 Research Showcase
MR Abstracts
Advisor : ETHAN BIER
Abstract Title : A Mechanistic Understanding of the Elusive Black Eschars in Cutaneous Anthrax
Abstract : The severe and often lethal Anthrax disease has been known since antiquity, and is caused by Bacillus anthracis (B. a), a spore-forming gram-positive bacterium. Anthrax in Greek translates to coal, because of the black skin lesions seen by those with cutaneous anthrax. The cause of these black eschars is not understood nor has been adequately investigated. B.a secretes Edema toxin (ET), which paralyze the immune response in the early phase of infection and produce symptoms in later stages of the disease. ET is a potent Adenylate Cyclase, that causes an uncontrolled rise in cAMP concentrations, and is responsible for edema, and other symptoms. Interestingly, activation of Protein Kinase A (PKA) by cAMP, leads to phosphorylation and activation of Tyrosine Hydroxylase (TH) a rate-limiting enzyme in melanin and dopamine synthesis. Exploitation of TH in Drosophila melanogaster established a direct relationship with pigmentation, allowing us to further hypothesize EF’s direct effect on pigmentation through TH.
Advisor : LARS BODE
Abstract Title : Novel Approach to Investigate the Role of Human Milk Oligosaccharides on Neglected Infectious Diseases
Abstract : Neglected tropical diseases (NTDs) cause over one million deaths each year; the majority of which are caused by diseases such as Chagas disease, African trypanosomiasis, intestinal and nematode infections, such as giardiasis, Schistosomiases and many others. They are very common in developing countries and produce an economical and social burden that exceeds malaria and tuberculosis. Many of the treatment options are not effective in treating all stages of the parasitic life cycle or the phase of the disease progression. Therefore, a novel approach was used to investigate human milk oligosaccharides (HMOs), which are complex carbohydrate found in breast milk, on neglected diseases. Further, HMOs are known to act as anti-adhesive antimicrobials that can lower the risk of pathogenic diseases. This study was designed to gain insight on the effect of structurally different HMOs on different parasites such as Naegleria fowleri, Giardia lamblia, Trypanosoma cruzi, Trypanosoma brucei, and Schistosoma. Through whole organism screenings, the experimental results initially showed that HMOs incubation enhanced the T. brucei growth, inhabited the T. cruzi and Giardia, caused phenotypic changes in Schistosoma mansoni and had no effect on Naegleria fowleri. After experimental design optimization, the faciliatory and inhibitory results obtained from T. brucei, T. cruzi and G. lamblia, were not reproducible. An alternative experimental approach was further investigated to understand the antiadhesive mechanism of HMOs on G. lamblia.
Abstract Title : Circadian Regulation of Defense Responses via NPR1 Expression in Arabidopsis
Abstract : The circadian clock is an endogenous mechanism that allows organisms to coordinate the temporal organization of biological processes to specific times of the day or night. It is known that the circadian clock controls immune responses such that plant susceptibility to a pathogen attack depends on the time of day at which the infection occurs. However, the molecular mechanisms underlying this response as related to the expression of clock-controlled genes like NONEXPRESSER OF PATHOGENESIS RELATED GENES 1 (NPR1) remain largely unknown. Therefore, we set out to investigate if NPR1, a known master immune regulator in plants, mediates clock regulation of defense responses and the underlying mechanisms involved in the circadian regulation of NPR1 expression. We provide evidence that NPR1 mediates the circadian regulation of immune responses in plants given that time-dependent NPR1 expression affects plant susceptibility to pathogen attacks, and NPR1 expression oscillations mediate circadian defense responses. We expect that miss-expression of transcription factors involved in NPR1 expression could abolish the circadian regulation of defense responses. These findings are important for the establishment of circadian-regulated pathways in plants. By identifying the sophisticated mechanisms involved host-pathogen interactions, we may transform our understanding of plant immune responses and how to regulate them to increase crop yield around the world.
Abstract Title : Observing Horizontal Gene Transfer of iRon Update Siderophore Transport Island (RUSTI) in Native Cheese Bacteria and Creating a Better Understanding RUSTI’s Role in Species Specific Growth in the Cheese Microbial Community
Abstract : Horizontal Gene Transfer (HGT) is an intricate way for microbes to interact with one another and is a vital tool that allows various microbes to adapt rapidly to new environments by exchanging potentially beneficial genes. We now know that most microbes live in microbiomes, and these communities play an intricate role in the health of our bodies, the ocean, the soil, as well as a variety of other environments. However, to study these communities in vitro is difficult because most microbiomes are extremely complex and include many microbes that cannot be traditionally cultured. Because of these restraints most microbiome research to date is focused on community composition and not on the specific interactions inside the community. To address this, our lab pioneered using the biofilm of the cheese rind as a model to study microbiomes. The cheese rinds’ limited complexity and the culturability of cheese-associated microbes makes it an ideal model microbiome. Previously, our lab identified a large mobile genetic region putatively involved in iron acquisition (termed RUSTI for iRon Update Siderophore Transport Island) which we believe is horizontally transferred in the cheese community. The RUSTI region is on a much larger mobile genetic region called an integrative and conjugative element (ICE). To test if RUSTI is indeed horizontally transferred we paired a Vibrio casei carrying the RUSTI-containing ICE with three cheese-rind isolated Proteobacteria. Neither of them were found to carry a RUSTI-containing ICE. In addition, to better characterize the function RUSTI plays on its host as well as on other members of the community a series of pairwise growth experiments was performed by pairing a cheese-rind isolate that does not carry a RUSTI-containing ICE with either wild-type Vibrio casei carrying the RUSTI-containing ICE or a Vibrio casei strain carrying the ICE but in which RUSTI was deleted. These findings are important for the development of our model because they highlight the characteristics of RUSTI in various ways. Creating a better understanding of how mobile genetic elements such as RUSTI behave in species-species interactions provides us insight into how the microbes or their genes in more complex microbial communities may interact with one another.
Advisor : DR. STANLEY LO
Abstract Title : Building on Bloom’s: Understanding the Structure of Bloom’s Revised Taxonomy in Biology
Abstract : Bloom’s taxonomy is a classification system of learning objectives. Since its original publication, Bloom’s taxonomy has been widely used across all subject matter and class level, serving as a basis for lesson plans, evaluation, and course development. However, Bloom’s taxonomy is not strictly hierarchical as originally proposed, as demonstrated by subsequent statistical analyses of the underlying structure to the taxonomy. That is, mastering one objective is not necessary to accomplish higher order objectives. This inconsistency lead to the revision of the taxonomy into two dimensions that intersect in learning: Knowledge and Cognitive Processes. Since its revision, the relationships among these two dimensions and the subcategories within each dimension are not yet understood. To quantify these relationship in the context of undergraduate biology education, a sample of over 1700 exam questions from standardized tests and college biology courses were classified using the revised taxonomy. Our data reveal connections among certain subcategories. For example, Factual knowledge is highly associated with the Cognitive process Remember, while Conceptual knowledge is highly associated with Understand and Analyze. Moreover, prompt words, such as “describe” or “explain,” are not good predictors of the types of knowledge or cognitive processes deployed in the taxonomy. Together, these results provide a framework for students to think about how they would approach exams questions and, conversely, how instructors could think about writing questions.
Abstract Title : Quantification and Epigenetic Analysis of Brain-Derived Neurotrophic Factor in Huntington’s Disease
Abstract : Huntington’s disease (HD) is a autosomal dominant neurodegenerative disorder that affects motor coordination and leads to cognitive decline. HD is caused by a trinucleotide repeat mutation in the Huntingtin gene (HTT) leading to a polyglutamine expansion in the huntingtin (htt) protein, which results in toxic aggregation that disrupts several neuronal pathways. HD neuropathology is characterized by substantial loss of medium spiny neurons in the striatum. Brain derived neurotrophic factor (BDNF) plays fundamental roles in the survival and activity of neurons, including striatal cells that die in HD. Previous reports shown substantial alterations in BDNF levels in the brains of HD cases; however, limited studies have explored the changes in BDNF across clinical stages of HD and levels present in peripheral fluids. We hypothesized that BDNF is increasingly deregulated during HD progression, and could be detected in plasma and saliva. Moreover, we propose that changes in BDNF expression may result from altered methylation on the BDNF promoter, as previously reported for other neurodegenerative disorders. Here, we examined the potential use of BDNF as a peripheral biomarker. We optimized an enzyme-linked immunosorbent assay to measure BDNF levels in plasma and saliva of patients in different HD clinical stages and control patients. We tested samples from n=14 pre-manifest patients, n=12 patients with full onset HD, and n=15 normal controls. We found significantly lower levels of BDNF in saliva samples from subjects that tested positive for the HTT gene mutation, regardless of HD clinical disease status in comparison to non-carriers. No significant changes in BDNF were found in plasma. In addition, our results corroborate previous findings suggesting gender and age based differences in BDNF. Further analysis of BDNF levels with clinical data showed no correlations of protein abundance with mental and physical states of subjects. Altogether, our findings suggest that the HTT gene mutation, gender, and age may give rise to fluctuations in BDNF, which may be detected in saliva. A larger sample and further studies will be needed to determine whether changes in BDNF levels may predict the onset of clinical symptoms in mutation carriers.
Abstract Title : Salivary huntingtin protein levels are elevated in Huntington's disease patients
Abstract : Huntington’s disease (HD), a polyglutamine disease, is an autosomal dominant disorder caused by a repeat expansion of the CAG sequence in exon 1 of the huntington gene which encodes the ubiquitously expressed Huntingtin (Htt) protein. It affects 10 in every 100,000 individuals in the US, with symptoms showing substantial variability in age-of-symptom onset, symptom severity and course of illness, thus warranting the need for reliable biomarkers to anticipate the onset of the disease and track its progression. As HD progresses, patients exhibit motor impairment symptoms - primarily chorea, diminished cognitive ability, and reduced functional capacity. The protein associated with the disease, mutant huntingtin (mHtt), is released into the extracellular fluid as neurons undergo degeneration in HD, and is thus a potentially useful biomarker that can indicate levels of neurodegeneration. It has been previously measured in cerebrospinal fluid (CSF) and blood plasma in HD. In this study, we measured total Htt protein, mutant and normal, in human saliva, a body fluid that is less invasive than CSF or plasma to collect, and determined whether Htt protein levels are associated with clinical symptoms of HD. We assayed 98 saliva samples from manifest HD, premanifest HD, and age-matched normal control participants using a total Htt (tHtt) ELISA. We found that tHtt protein levels were significantly increased in saliva from manifest HD individuals compared to normal controls. Salivary tHtt showed no gender effects. Salivary tHtt was correlated to have statistical significance in the positive direction with age, but not with age of symptom onset or CAG repeat length in the manifest HD group. As expected, tHtt levels had a positive correlation with motor symptoms, a negative correlation with cognitive ability, and a trend towards a negative correlation with functional capacity in manifest HD subjects. tHtt protein can be measured in saliva relatively easily and can potentially be developed as a non-invasive disease biomarker for HD in the future.
Abstract Title : Nicotine-induced Neurotransmitter Plasticity in the Substantia Nigra
Abstract : Cigarette smoking is generally known for its detrimental effects on health; however, extensive epidemiological studies have indicated inverse correlation between smoking and Parkinson's Disease (PD), a progressive neurodegenerative disorder characterized by loss of dopaminergic (DA) neurons in the substantia nigra (SN). Subsequent studies have shown that nicotine protects DA neurons against nigrostriatal damage in PD primate and rodent models. Nicotine became the focus of these studies due to its well-known ability to modulate function and activity of midbrain DA neurons. Because altered circuit activation can induce neurons to acquire a DA phenotype in the mature brain (Dulcis et al. 2013 Science), we hypothesized that chronic nicotine treatment in adult mice contributes to neuroprotection against nigrostriatal damage in an animal model of PD via a mechanism of neurotransmitter (NT) plasticity. Nicotine was given to adult (P60) mice in drinking water for two weeks. Brains of various transgenic reporter mouse lines were subsequently processed for immunohistochemistry and retrobead tracing for detection of NT-expressing cell types, such as tyrosine hydroxylase (TH) and VGAT, in the SN and their connectivity. Selective overexpression of human alfa-synuclein in midbrain DA neurons was used as a PD mouse model (Lin et al., 2012 JN). Our findings showed that chronic nicotine treatment significantly increased DA (TH+) expression within a pool of SNr (pars reticulata) GABAergic neurons that express transcription factors associated with DA differentiation, such as Nurr1 and Foxa2, prior to nicotine exposure. Importantly, our retrograde labelling experiments showed that this GABAergic neuronal pool in the SNr projects to the caudate nucleus, the same target of SNc (pars compacta) DA neurons. Ongoing behavioral experiments on a PD mouse model will reveal whether nicotine-induced neurotransmitter plasticity ameliorates any motor deficits in these mice. Our findings indicate that neurotransmitter plasticity occurs in the SN in response to chronic nicotine treatment. Understanding its role in neuroprotection against nigrostriatal damages in PD could reveal insights that might lead to the development of new treatments for Parkinson's Disease.
Advisor : JING YANG
Abstract Title : Establishing Twist1-Inducible Breast Cancer Mouse Model to Investigate Tumor Dormancy and Metastasis
Abstract : Most breast cancer-related deaths are due to distant metastasis that occur many years after successful tumor removal and treatment. Tumor metastasis is a multi-step process in which primary tumors lose cell junctions and undergo dissemination by epithelial-mesenchymal- transition (EMT). The disseminated tumor cells enter blood circulation and lodge at distant organs and remain in a dormant state. Once the microenvironment becomes suitable, the cells will exit dormancy by mesenchymal-epithelial transition (MET), which results in macrometastasis. However, the molecular mechanisms of metastasis are not well understood due to the lack of an in vivo mouse model that can monitor disseminated tumor cells. Previous studies have shown that a transcription factor Twist1 plays a key role in inducing EMT and promoting tumor metastasis. Therefore, we established a Twist1-inducible breast cancer mouse model that enables us to utilize fluorescent markers to visualize and characterize disseminated tumor cells during various stages of metastasis.
Advisor : NAN HAO
Abstract Title : Stress signaling regulates mating decision in Saccharomyces cerevisiae
Abstract : Cells living in constantly changing environments are required to respond to a variety of extracellular signals to better survive. However, how different signaling pathways coordinate to help cells adapt and make decisions remains unclear. In budding yeast, Protein Kinase A(PKA) and Mitogen Activated Protein Kinase(MAPK) signaling pathway are two important pathways which regulate many cellular processes such as proliferation, differentiation, and apoptosis. It has been shown that glucose starvation could delay MAPK driven mating response, which implies that stress-mediating PKA and MAPK pathway could crosstalk and help cells to make decisions. In this study, we mimic stress condition by directly inhibiting PKA activity. When PKA-inhibited yeast cells are exposed to mating pheromone, the mating response is significantly inhibited. Cells showed significantly reduced MAPK activity, complete inhibition of mating-specific gene expression, and reduced mating-induced shmoo formation. Our results indicate that there is crosstalk between PKA signaling pathway and mating MAPK pathway, as PKA might directly control MAPK, but the mechanism is still under investigation. Our data identified a new mechanism to regulate MAPK activity and could contribute to clinical interventions of human diseases.
Abstract Title : Identification of SARAH Domain-Specific E3 Ligases Targeting RASSF2 for Degradation
Abstract : Tumor suppressor RASSF2 (RAS association domain family 2) is a key regulator of the Hippo pathway, which controls the cell cycle by balancing cell proliferation and apoptosis (Harvey et al., 2013). Disruption of this cell cycle regulation leads to uncontrolled cell growth which contributes to malignancy. RASSF2 expression is often suppressed in human cancers but little is known about its regulation. Protein turnover is highly regulated by the ubiquitin-mediated proteasomal degradation pathway, in which specific E3 ligases attach ubiquitin on its targeted proteins, tagging the proteins for degradation by proteasomes (Schrader et al., 2009). We assessed RASSF2 stability by monitoring protein levels over time following cyclohexamide treatment. We found that RASSF2 was rapidly degraded with a half-life of approximately three hours. In contrast, we have found that RASSF2 with a deletion of the SARAH domain (Sav, RASSF, Hpo) resulted in a protein that was much more stable, with a half-life of >24 hours, suggesting that the SARAH domain might be a target for negative regulation. Therefore, we hypothesize that E3 ligase(s) specific to the SARAH domain of RASSF2 promote its degradation. In this study, we used shRNAs to knockdown E3 ligases potentially specific to the SARAH domain of RASSF2 and monitored the changes in RASSF2 protein stability. HEK 293T cells were co-transfected with plasmids expressing the shRNAs and plasmids expressing either RASSF2 full length or RASSF2 with SARAH domain deletion. Protein expression of RASSF2 full length increased upon shRNA knockdown of E3 ligase PIAS2 and ITCH. Upon identification of E3 ligase(s) specific to RASSF2 SARAH domain, small molecule inhibitors can be designed to disrupt the E3 ligase-RASSF2 interaction. Preventing E3 ligase(s) from binding RASSF2 would likely increase the expression of RASSF2 to normal levels in cancer cells, proposing an approach for cancer therapy.
Abstract Title : Investigating the Role of the Adult Maize Leaf Cuticle in Providing Pathogen Resistance
Abstract : The plant cuticle is a waxy, hydrophobic layer found on non-woody plant tissue and serves as a physical barrier protecting the plant from environmental stresses including fungal infection, dehydration and UV radiation. Little functional analysis to characterize the cuticle’s relationship to pathogen resistance has been performed in adult Zea mays, the highest grossing industrial and agricultural crop in the United States, thus leaving the agronomic impact of the cuticle on the adult-stage plant largely unknown. Maize glossy mutants have been identified to show defects in cuticle production as a result of impaired levels of lipid biosynthesis or wax transport proteins, thus making them effective tools to study the cuticle’s impact on pathogen resistance. In this study, we take glossy mutants and observe their differences in the early stages of pathogen resistance to Cochliobolus heterostrophus, a necrotrophic fungus that causes Southern Leaf Blight. Our objective is to establish methods to compare differences in C. heterostrophus infection stages, including fungal adhesion, germination, hyphal penetration into leaf epidermal tissue, and long-term lesion development. We show adherence progression of GFP-labelled C. heterostrophus to the leaf surface, initial penetration into the leaf, varying levels of visual lesion formation and fungal growth via GFP quantification amongst glossy mutants, suggesting that differences in C. heterostrophus infection might be attributed to changes in cuticle components. We are currently engaged in ongoing collaboration to further analyze the cuticle composition of our mutants with gas chromatography/mass spectrophotometry and their transcriptome for cuticle-related gene expression.
Abstract Title : Circadian Entrainment of Bipolar Patient Fibroblasts
Abstract : Bipolar disorder (BD) is a psychiatric disorder characterized by recurrent periods of depression and mania, accompanied by major disruptions in activity and sleep patterns. The circadian clock controls these behavioral rhythms however; genome-wide association studies have failed to identify any of the essential "clock genes" that regulate rhythms as major genetic contributors in BD, but have associated the genes encoding L-type calcium channels (LTCCs) as important risk factors. CACNA1C encodes Cav1.2, a LTCC essential for entrainment of the circadian clock. In post mortem brain studies, those with BD risk-associated variants in CACNA1C show alterations in gene expression. However, it is not known if abnormal expression of calcium channels mediates the circadian disruptions observed in BD patients. We utilized a viral per2::luciferase reporter to measure circadian rhythms in vitro and evaluate the role of a BD-associated risk allele in CACNA1C (rs4765913) and its role in human fibroblasts during phase-shifting. CACNA1C genotype (rs4765913) predicted the ability of human fibroblasts from BD patients to entrain to temperature cycles. These findings give insight as to the role of CACNA1C genotype in the ability of fibroblasts to entrain to daily stimuli, and how the abnormal entrainment displayed in BD-associated risk allele carriers could contribute to the circadian abnormalities observed in BD patients.
Abstract Title : Auditory Discrimination Learning in a Rodent Model of Human Targeted Cognitive Training
Abstract : Introduction: After 50 hours of Targeted Cognitive Training (TCT) schizophrenia patients show large gains in neurocognition. However, durable changes may require extended training, functional gains are modest, and individual responses are variable. We describe a novel paradigm for studying mechanisms of learning in TCT, in advance of testing interventions to enhance its clinical impact. Methods: Male Long Evans rats (n=15) were trained to respond for reward in 3 distinct auditory discrimination tasks (ADT). Auditory stimuli were either a 500 ms pure tone of high (7 kHz) or low frequency (4 kHz) (ADT#1); a 200 ms sound sweep ranging from low to high frequency ("upsweep") or high to low frequency ("downsweep") (ADT#2); or a 500 ms upsweep or downsweep (ADT#3). The primary outcome variable was accuracy in choosing the lit aperture associated with the stimulus. Results: Learning was reflected in a main effect of session on accuracy (F(16,160)=3.467, p<0.05). A session x group interaction (F(32,160)=1.522, p<0.05) revealed that the 500 ms sweeps group (ADT#3) learned faster than the other two groups. By training day 6 of 17, rats in ADT#3 exhibited greater accuracy vs. day 1. Performance superior to day 1 was never achieved in ADT#1, and required 13 days in ADT#2. Conclusions: Long Evans rats: 1) can discriminate auditory frequency upsweeps vs. downsweeps; 2) learn faster from sweeps than pure tones; and 3) learn faster from long (500 ms) vs. short (200 ms) sweep duration. Studies will now assess the impact of pharmacologic agents on this model of TCT learning. Funding Sources: R01MH059803 (NRS)
Advisor : TOM HNASKO
Abstract Title : Expression of VGLUT2 is upregulated after injury to dopaminergic neurons in mouse substantia nigra pars compacta
Abstract : Parkinson Disease (PD) is known as one of the most common neurodegenerative diseases and affects 1%-2% of the population. Previous research suggests that PD is caused by the loss of dopaminergic (DA) neurons in midbrain areas, especially in substantia nigra pars compacta (SNc). In mammalian and Drosophila melanogaster PD models, the expression of vesicular glutamate transporter 2 (Vglut2) is markedly upregulated. However, the upregulation of VGLUT2 expression in DA neurons is unexpected and its function remains unclear in PD. To explore the mechanism of VGLUT2 regulation in DA neurons in PD, DA neurons can be reversibly injured to simulate the damaged DA neurons in PD. Here, we reversibly injure DA neurons with amphetamine and visualize the Vglut2 expression by RNA in situ hybridization staining for VGLUT2 messenger RNA (mRNA) and tyrosine hydroxylase (Th) mRNA as a marker for DA neurons. We analyze the Vglut2 expression level by counting for the number of puncta of Vglut2 mRNA signal co-localizing with Th mRNA signal. We hypothesize that with injury to DA neurons in SNc, VGLUT2 expression level is markedly upregulated.
Abstract Title : Molecular and behavioral characteristics of epileptogenesis in a rat model of medial temporal lobe epilepsy
Abstract : The hippocampus is critical to the consolidation of experiences from short-term memory into long-term memory. One consolidation process is known as pattern separation. Pattern separation allows memory circuits in the brain to differentiate similar experiences from each other in order to facilitate accurate retrieval of distinct memories. This process is observed by the activity of neurons in the dentate gyrus, a sub-region of the hippocampus. Additionally, the dentate gyrus is especially vulnerable to neuronal death and synaptic reorganization in diseases such as medial temporal lobe epilepsy (MTLE). MTLE is the most common seizure disorder in adults and involves a slow development period known as epileptogenesis. Due to the slow nature of epileptogenesis, research has yet to identify any diagnostic markers for MTLE before the onset of chronic and recurrent seizures. The purpose of this study was to investigate if molecular changes in the dentate gyrus caused by epileptogenesis result in a deficit of dentate-dependent pattern separation. We used kainate-acid, a chemotoxin with potent excitatory effects on the hippocampus, to induce epileptogenesis in Long Evans rats. Then, both control and kainate-induced rats were trained using a discrimination task involving the adjacent arms of an 8-radial arm maze – discriminating between the adjacent arms requires the dentate gyrus. We observed that the kainate-induced rats required more trials to learn the task, indicating a deficit in dentate dependent behavior thought to be supported by pattern separation processes. In order to investigate if the behavioral performance was a result of the molecular changes in the dentate gyrus, behavioral performance was correlated with the degree of mossy fiber sprouting, somatostatin neuron loss, and the progression of epilepsy. Mossy fiber sprouting was measured and scored based on severity, the number of somatostatin neurons was quantified using stereology, and the presence or number of seizures in kainate-induced rats was tracked through continuous monitoring and scored using the Racine scale. By identifying early markers of epileptogenesis, a better guide can be created for diagnosing and treating MTLE.
Abstract Title : Dual overexpression of SIRT1 and knockout of GCN5 in adult skeletal muscle does not alter exercise capacity or mitochondrial function in mice
Abstract : The acetyltransferase (KAT), general control of amino acid synthesis 5 (GCN5), and the NAD+-dependent lysine deacetylase, sirtuin 1 (SIRT,1) regulate the activity of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) by modulating its acetylation status. GCN5 acetylates and deactivates PGC-1α, while SIRT1 deacetylates and increase its activity. PGC-1α is a transcriptional co-activator that controls mitochondrial biogenesis and skeletal muscle metabolism. We have previously found that neither germline or inducible muscle-specific overexpression (OX) of SIRT1 (mOX), or germline muscle-specific knockout (KO) of GCN5 (mKO) impact skeletal muscle mitochondrial biogenesis. To better define the combined role of GCN5 and SIRT1 to muscle metabolism and oxidative capacity we used CreLoxP methodology to generate transgenic (TG) mice with inducible, muscle-specific KO of GCN5 and OX of SIRT1 (referred to as, dTG). All mice were ‘floxed’, and were either iHSA-Cre (tamoxifen [TMX]-inducible human skeletal actin Cre) negative (Cre⎯; i.e. wildtype [WT]) or positive (Cre+; i.e. dTG). We hypothesized that dTG mice would have enhanced glucose tolerance, and improved exercise capacity and muscle oxidative capacity, as compared to WT littermates. At 6 weeks of age, all mice were orally gavaged with TMX for 5 consecutive days and studies were conducted at 10-12 weeks of age. There were no genotype differences in muscle mass, body weight or glucose tolerance. Moreover, there were no genotype differences in mitochondrial electron transport chain protein abundance or function, or the abundance of proteins related to glucose or fatty acid metabolism in skeletal muscle. In conclusion, concurrent OX of SIRT1 and KO of GCN5 in skeletal muscle of adult mice does not modify exercise capacity, glucose tolerance, markers of mitochondrial biogenesis.
Abstract Title : Caspase-4 and -5 mediate non-canonical NLRP3 inflammasome activation to induce interleukin-1β in human primary macrophages exposed to human immunodeficiency virus type-1 GU-rich single ssRNA.
Abstract : Human immunodeficiency virus type-1 (HIV) pathogenesis is associated with immune activation and chronic inflammation during active viral replication as well as during antiretroviral viral suppression. Despite effective treatment, HIV-infected persons display inflammatory markers leading to accelerated immunosenescence, or aging of the immune system. Processing of the pro-inflammatory marker interleukin-1β (IL-1) is associated with nod-like receptor protein-3 (NLRP3) inflammasome and caspase activation in many inflammatory diseases. The conventional NLRP3 inflammasome pathway requires caspase-1 and is modulated by a two-step process: an initial and secondary stimulus are necessary to trigger inflammasome complex assembly. In contrast, studies show that human primary monocytes may undergo unconventional NLRP3 inflammasome activation by LPS through toll-like receptor (TLR)-4 signaling; however, the mechanism of IL-1 induction in human primary macrophages exposed to HIV is unknown. To elucidate the mechanism of IL-1 activation by HIV, we exposed human primary macrophages to ssRNA40 (a GU-rich ssRNA within the HIV long-terminal repeat region) which signals through TLR-7/8 and found a significant induction of IL-1β. To examine the mechanism of inflammasome activation, macrophages were exposed to caspase inhibitors in the presence or absence of ssRNA40. We discovered that ssRNA40 triggers caspase-4 and -5 activation to mediate IL-1 release in a one-step event. Additionally, we have identified that ssRNA40 promotes caspase-5 cleavage in a time-dependent manner suggesting the effector role of the non-canonical inflammasome may occur early after stimulation with ssRNA40. In summary, human primary macrophages exposed to ssRNA40 trigger the NLRP3 inflammasome through a non-canonical pathway involving caspase-4 and caspase-5 to process IL-1. This mechanism provides further insight into HIV pathogenesis, and suggests that targeting caspase-4 and -5 could be useful in controlling HIV-associated chronic inflammation.
Abstract Title : Potential Mechanisms of the Influence of Cholesterol on Pathogenic Amyloid Precursor Protein Processing
Abstract : Alzheimer’s Disease (AD) is a chronic neurodegenerative disease that begins with short-term memory loss and slowly progresses to dementia and ultimately leads to death. AD is characterized by the formation of Amyloid-beta (Aβ) plaques and hyperphosphorylated tau protein neurofibrillary tangles in the brain. Interestingly, when we treat wild-type with cholesterol-lowering drugs, we witness a decrease in Aβ levels, indicating that cholesterol levels influence amyloid precursor protein (APP) processing. Recently, it was found that APP FL has a transmembrane cholesterol binding site. In order to determine if the cholesterol binding site plays a role in regulating APP processing, we used CRISPR-CAS9 to induce point mutations that abolish cholesterol binding in human induced pluripotent stem cells (hiPSCs). We have observed that Aβ secretion is decreased in neurons derived from the cholesterol mutant cells indicating that pathogenic APP processing is downregulated. Previously it has been shown that APP dimerization can alter APP processing. The hypothesis that I will test is that cholesterol binding site mutations affect APP processing by altering APP dimerization levels. I am testing this hypothesis by using a crosslinking agent to stabilize interacting proteins followed by western blotting. We observe an APP band that corresponds to the expected dimer size and are currently using mass spectrometry to validate the identity of this band. In the future, I will use co-immunoprecipitation to analyze expression of various enzymes interacting with APP in our mutants in comparison to wild types to determine the mechanism of how cholesterol levels influence the generation of Aβ.
Abstract Title : Using the CRISPR/Cas9 System and Human Isogenic Neurons to Elucidate the Genotype-Phenotype Relationship of Niemann Pick Type C1
Abstract : Niemann-Pick disease type C1 (NPC1) is a rare lysosome storage disease that results in neurodegeneration, cognitive impairment, and death. NPC1 is caused by a mutation in the NPC1 gene and is characterized by the accumulation of cholesterol in lysosomes. Over the past several years, there has been an increase in the documented incidence of NPC1, coinciding with improved genetic tools that allow earlier diagnosis. With over 250 described mutations, NPC1 has been shown to have a wide variation of biochemical and clinical phenotypes, even among patients with similar mutations. As our knowledge of the genetic heterogeneity of NPC1 increases, the importance of studying this disease in an isogenic human model is becoming more evident. For this reason, we are using the CRISPR/Cas9 system to engineer isogenic human induced pluripotent stem cell (hiPSC)-derived NPC1 knock-out neuronal lines in a common donor background to study the implications of genetic disruption to NPC1. Prior studies by our group using hiPSC-derived NPC1 patient lines have identified a novel pathological mechanism that is responsible for the neurodegeneration associated to mutations in NPC1. We found that neurons lacking NPC1 function have cholesterol sequestration in the lysosome, which triggers strong activation of the autophagy pathway. Persistent autophagy leads to mitochondrial dysfunction and accumulation of fragmented and depolarized mitochondria, eventually leading to neuronal death. We have observed these phenotypes in three independent patient lines with different mutations of NPC1, however due to the complex genetics underlying NPC1, we seek to explore this mechanism in isogenic NPC1 knock-out lines. As expected, isogenic NPC1 knockout lines exhibit aberrant cholesterol distribution, replicating the classic biochemical abnormality of NPC1. Our observations thus far also suggest that NPC1 knockout lines replicate cellular phenotypes downstream of cholesterol sequestration, specifically impaired autophagy flow and increased mitochondrial fragmentation. Despite these promising early observations, further studies are needed to fully confirm the pathogenic mechanism observed in our patient-derived and isogenic NPC1 lines. Our studies illustrate the value of using isogenic lines to study the genotype-phenotype relationship of complex genetic diseases that have a wide spectrum of biochemical and clinical abnormalities such as NPC1.
Advisor : DR. YISHI JIN
Abstract Title : Modulation of aberrant neural circuit activity by neuropeptides in the C. elegans locomotor circuit
Abstract : Neuropeptides are modulatory proteins that maintain homeostasis and typical behavior. Neuropeptide signaling may serve to dampen the effects of aberrant neural circuit activity, a feature of many neurological diseases, including epilepsy and schizophrenia. For example, the neuropeptide galanin in mice was found to regulate epileptic activity. Understanding these important modulators of circuit activity, then, is critical for the development of potential treatments for dysfunctional circuits. The nematode C. elegans has long been a useful in vivo system to study neurobiology. Using RNA-seq, we found that several neuropeptides were up-regulated in a genetic C. elegans epilepsy model, acr-2 gain-of-function [acr-2(gf)]. acr-2 encodes a conserved neuronal nicotinic acetylcholine receptor subunit, and mutations in human receptor subunits are associated with epilepsy. acr-2(gf) animals exhibit impaired locomotion and stochastic convulsions, due to increased locomotion circuit activity. We combined mutants for several up-regulated peptides with acr-2(gf) to understand how they affect locomotion. These include: ins-25, ins-29, flp-12, and flp-24. These are members of the insulin-like (ins) and FMRF-amide (flp) families, respectively. These C. elegans neuropeptide families are structurally similar to human neuropeptides. We found that a loss-of-function mutation in flp-12 [flp-12(lf)] combined with either a null mutation in ins-29 and ins-25 [ins-29 ins-25(0)] or flp-24(lf) resulted in a reduction of the acr-2(gf) convulsion rate. Studies are ongoing to elucidate the role of flp-12 and the features of synaptic transmission it affects in the C. elegans locomotor circuit. Furthermore, we aim to describe the expression pattern of these neuropeptides to determine in which cells they function to affect behavior. Shedding light on the connections between neuropeptides, neural circuit activity, and behavior may advance the development of new strategies to target and treat circuit imbalances that have been implicated in many neurological diseases.