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Parkinson’s Disease (PD) is a neurodegenerative disorder characterized in part by the selective loss of dopaminergic neurons in the substantia nigra pars compacta. Although the precise cause of PD is not yet fully understood, environmental factors are known to contribute to the etiology of a vast number of cases. Rotenone, a pesticide that inhibits Complex 1 of the Mitochondrial Electron Transport Chain, is one such toxin. Importantly, there is no known cure for PD and effective treatment options are severely limited both in number and efficacy. We are interested in measuring mitochondrial oxygen consumption rate (OCR) using the Seahorse Analyzer in our in vitro and ex vivo model exposed to mitochondrial stressors (Oligomycin, FCCP, and Rotenone/Antimycin). Once mitochondrial respiration measurements have been optimized, we aim to test whether treatment with dacarbazine, a possible neuroprotective agent, can ameliorate the effects of mitochondrial stress. Here, we present preliminary data on the effect of mitochondrial stressors in fly heads and Schneider cells (S2 cells) and the effect of dacarbazine on OCR in fly heads and S2 cells. In sum, our report shows the effects of toxins in mitochondrial metabolism and the utility of a potential neuroprotective chemical against a model of PD and suggests a possible neuroprotective chemical against the disease.

Delaware Neuroscience Symposium 2021

Optimizing the measurement of mitochondrial respiration in Drosophila melanogaster adult brains and late-stage embryonic cells

poster

This annual event showcases and celebrates the neuroscience research in Delaware.  Posters and talks highlight neuroscience research carried out by graduate students, undergraduate students, and research staff at all of Delaware’s research and education institutions. The Lounge section of the symposium allows real-time interactions and networking among attendees.  New this year is an expo of research core facilities in Delaware.  Check them out in the Research Cores section of our poster session, or see the logos below.

Most animals, including humans appear symmetrical externally, but display visceral lateral asymmetry with a bias for one enantiomeric body plane over the other. We are interested in understanding functional effects of bilateral asymmetry in the nervous system. The left and right cerebral hemispheres appear symmetrical in gross anatomy but are known to be functionally asymmetrical. For example, language dominance has an asymmetrical presence on the left side of the brain in 90% of the population classified as right handed. Hemispheric laterality plays an important role in neuropsychological models of neuro-psychiatric disorders, including autism spectrum disorder (ASD), bipolar disorder and schizophrenia. Typically developing individuals more commonly have a right dominant laterality when it comes to handedness, footedness and language laterality. These reflect a leftward asymmetry in the brain. Previous studies have found higher rates of atypical modalities such as handedness and footedness in the ASD community. However, many of these modalities have been studied separately. We propose to observe handedness, footedness, language laterality, eye dominance and hair whorl in individuals in the ASD community to observe whether or not an occurrence of atypical asymmetry is observed. Samples have been collected from the typical population as well as the ASD community. To begin with, we have tested university students and faculty to standardize the WatHand Box, The Measurement of Foot Preference, Distance in Hole Card Test and iDichotic listening app. Our preliminary data on individual modalities is in line with previous studies. We will continue this testing with children in the ASD community. In a separate line of experiments, the C. elegans model will be used to test laterality phenotypes of genes implicated in ASD, schizophrenia, bipolar disorder and left handedness. Effects of these genes on laterality are being observed in early embryonic development as well as in the neurons of the adult organism. Effects on behavior as a result of functional laterality will be observed through associative and nonassociative learning assays.

Markers of functional laterality of the nervous system

Parkinson’s Disease (PD) is a progressive neurodegenerative disorder characterized in part by dopaminergic neuronal loss in the substantia nigra pars compacta and includes symptoms as such as: tremor, muscle rigidity, unsteady gait, as well as dementia in severe cases. Over 10 million people around the world suffer from this debilitating motor disorder, with this number climbing by about 60,000 new diagnoses every year. Epidemiological studies by several different groups indicate that exposure to certain pesticides and herbicides increase an individual’s likelihood of developing PD. The objective of this study was to determine whether different combinations of pesticides and herbicides have a synergistic effect and further increase an individual’s risk of developing PD. We exposed Drosophila to different combinations of the following insecticide (i) and herbicides (h) for a total of eight weeks: Acephate (i), Atrazine (h), Diuron (h) and Alachor (h). Locomotor abilities were assessed using the Multi-Worm Tracker once per week, and the survival assay was run twice per week. Our preliminary data suggest synergistic interactions involving Atrazine and Diuron as well as Atrazine and Acephate with respect to increase organismal lethality. We also present ongoing studies on locomotion assays involving single and combinations of the pesticides we tested. Together, our data suggest that exposure to some combinations of commonly-used pesticides may render increased susceptibility to environment toxins and their deleterious effects.

Pesticide and Herbicide Interactions may cause Parkinson's Disease in a Drosophila Model

Amyotrophic Lateral Sclerosis (ALS) is characterized by the death of both upper and lower motor neurons. After diagnosis, the average lifespan of the patient is ~5 years, most commonly due to respiratory failure and pneumonia. There are currently no effective treatments for ALS. In addition, 97% of cases are pathologically characterized by cytoplasmic accumulation of hyperphosphorylated and ubiquitinated transactive response DNA binding protein 43 (TDP-43). Familial mutations in TDP-43 account for ~5-10% of all ALS cases. In order to develop better drug treatments, we must have effective animal and cellular models to represent the disease. To this end, we have inserted three familiar mutations (A315T, M337V, S379P) in a TDP-43 construct (3X-TDP-43) in order to create a new cellular pathological model. Cells transfected with 3X-TDP-43 into HEK293T and SH-SY5Y have many of the hallmark characteristics found in ALS. This cellular model induces cytoplasmic accumulation of phosphorylated TDP-43 and a distinct change in solubility that has only been observed in human cases of TDP-43 proteinopathy. Based on these findings, we hope to develop a better understanding of the disease process. This cellular model may provide a better avenue in the development of therapeutics targeting aggregation.

A cellular model of TDP-43 proteinopathy

Acetylcholine (ACh) is one of the most abundant neurotransmitters in a human. It works in the central and peripheral nervous systems. In addition to its major role in neuromuscular junctions, ACh functions in memory and cognition in the brain. It is known that many Alzheimer’s disease patients have lower levels of acetylcholine. RNase H2 is a ribonuclease that removes ribonucleotides from DNA. It is also involved in gene expression. Although the full scope of this enzyme’s role in vivo is not fully known, it is a well-known fact that mutations in the RNase H2 genes in humans causes Aicardi- Goutières syndrome (AGS), a severe neurological disorder characterised by abnormal neurology and different types of encephalopathies. C. elegans is a nematode and model organism that has been widely used in Biomedical Research. Not only are they easy to work with and have quick generation times that allow for efficiency in research, but the organism also share more than 80% of their genes with humans. The purpose of this study was to determine, using the organism C. elegans, the effects of a mutation in the RNase H2 gene on cholinergic neurotransmission and the associated behaviours. RNase H2 S114F mutant worms were examined in time course experiments in the presence of ACh in the medium and their behaviours were compared to those of wild-type worms. The specific behaviour I observed were locomotion, pharyngeal pumping, and defecation. I found that the rnh-2 S114F mutation significantly affected all these behaviours in which cholinergic neurotransmission is involved. These results suggest that RNase H2 plays an important role in regulation of cholinergic neurotransmission and associated behaviours in C. elegans.

The role of RNase H2 in cholinergic neurotransmission in Caenorhabditis elegans

The inability to determine corresponding ages between humans and model organisms has inhibited progress in mapping findings from model systems to humans. We build upon the translating time project (http://www.translatingtime.org), which utilizes neural development models from 18 mammalian species including humans, macaques, and other rodent species. To extend the translating time project from up to 2 years after birth in humans and its equivalent in model organisms to span postnatal ages, we utilize transcriptional, behavioral, and anatomical variation data across humans, mice, and non-human primates. We capture temporal changes in gene expression from RNA sequencing extracted from bulk samples of the frontal cortex in humans and chimpanzees. To find corresponding time points across species, we dynamically time warped trajectories and collected other time points to generate in 137 time points from prenatal to approximately 55 years in humans and their equivalent in other species (chimpanzees). We find consistency in results of the time points from the different scales of organization (i.e transcriptional, anatomical, behavioral), and conservation in the timing of developmental programs across humans and other model organisms. This work will be an important resource in finding corresponding ages across species and identifying conserved developmental programs across species.

Translating time in humans and other species from brain transcriptional and structural variation.

Synchronous neuronal activity is a hallmark of developing as well as fully developed neuronal networks and is necessary for the functional organization of the brain. However, the mechanisms of the development of these synchronous oscillations are poorly understood. While existing models assume it as a process intrinsic to neurons, recent evidence suggests that astrocytes have an important role in the development of synchronous activity. We established pure neuron only and mixed (astrocyte and neuron) cultures on multielectrode arrays (MEAs) from the embryonic chick optic tectum. Our preliminary results indicate that astrocytes are necessary for synchronous activity of neurons in culture. Mixed neuron and astrocyte cultures show random spiking activity which synchronizes over time whereas astrocyte-free neurons only show random activity without synchronization. Our model predicts that glutamate sensing at tripartite synapses via mGluRs elevates local calcium stores within astrocyte processes. With sufficient activation, the localized calcium elevation crosses a threshold causing a calcium induced calcium release (CICR) within the astrocyte leading to glutamate exocytosis. We targeted the SNARE protein Synaptobrevin (Vamp2) within astrocytes as crucial for communication with neurons via exocytotic release of glutamate. We proposed to test this model by expressing a truncated Vamp2 subunit (Vamp2 DN) which acts as a dominant negative to block exocytotic release. Astrocytes expressing the Vamp2 DN are expected to release significantly less glutamate upon calcium elevation thereby reducing synchrony of neuronal activity. We have generated astrocyte lines expressing the Synaptobrevin dominant negative (Vamp2 DN) along with the glutamate sensor iGluSnFR. We demonstrate that Vamp2 DN expressing astrocytes have significantly reduced glutamate exocytosis when CICR is induced with Ionomycin. With these tools a more comprehensive molecular model for astrocyte involvement in the generation of neuronal synchrony can be developed.

Molecular Role of Synaptobrevin & Glutamate in astrocyte-neuron interactions in the development of synchronized activity in neuronal networks

Neurons must fire together in networks to perform a variety of functions but the mechanisms by which they accomplish this synchronization are not entirely understood. In some cases, this phenomenon does not function as intended, leading to states like epileptic seizures. Existing models of synchronous activity assume that it is intrinsic to neurons. Astrocytes have been shown to modulate oscillatory activity in networks of neurons possibly by releasing gliotransmitters like glutamate and ATP. Using the mitotic inhibitor 5-fluorodeoxyuridine (FUdR) we established pure neuron only cultures from chick embryonic day 7 (E7) optic tectum and recorded neuronal network activity on multielectrode arrays (MEAs). Our preliminary results indicate that astrocytes are necessary for synchronous activity of neurons in culture. Mixed neuron and astrocyte cultures on multi-electrode arrays (MEAs) show random spiking activity which synchronizes over time whereas astrocyte-free neurons only show random activity without synchronization. Our results validated that the physical presence of astrocytes is required to properly establish synchronized activity in neuronal networks. We implicate calcium mobilization within astrocytes via metabotropic g-protein coupled receptor (GPCR) pathways leading to gliotransmitter release as a possible mechanism for neuronal synchronization. To test this hypothesis, we used an optogenetic approach – using a lentiviral vector we expressed the photopigment melanopsin, a Gaq coupled GPCR with an absorption peak of around 470nm, in chick optic tectum astrocytes. Our preliminary optogenetic activation experiments indicate Ca++ flux in astrocytes upon optogenetic stimulation with 470nm blue light. This powerful tool allows us to combine live calcium imaging with multielectrode array experiments to clearly define the role of astrocytic calcium in the development of network activity of neurons, particularly synchronization.

An Optogenetic Approach To Understand Molecular Mechanisms of Astrocyte-Neuron Interactions in the Development of Neuronal Synchrony

The mission of the Bioinformatics Core Facility at UD’s Center for Bioinformatics and Computational Biology (CBCB) is to provide scientific expertise, training, and infrastructure support in bioinformatics and data science for the Delaware life science and biomedical research and education community. Drawing on the combined resources of CBCB, the Delaware Biotechnology Institute (DBI), the Data Science Institute (DSI), and the Protein Information Resource (PIR), the Core provides both computing infrastructure and knowledgeable staff with significant expertise across various specialties in bioinformatics, data science, and related fields. The Core is further supported by the Delaware Biotechnology Institute’s BioIT team providing support for systems administration and high performance computing support. A special focus of the Core has been omics data analysis, and pipelines and support are available under fee-for-service models to support investigators. The facility also has substantial capabilities built upon its close relationship with the Protein Information Resource (PIR) that have been successfully applied to a number of research projects including proteomic analysis using tools such as iProXpress and literature and data mining capabilities of iProLINK. These combined omics analysis and data integration pipelines form the nucleus of a bioinformatics framework to provide a user portal for dynamic analysis and visualization of genomic, transcriptomic, and proteomic data to its users at UD. Consultation, advice, and training offerings are also available to help support researchers in analysis and interpretation of data. The Core also works with investigators to design and host web portals and databases to store, organize, and share scientific data and findings. The Center’s in-house High-Performance Computational hardware include the Biomix cluster (freely accessible to members of Delaware INBRE partner institutions), as well as development and production web/database servers and data storage options including a triple redundancy Hadoop data archiving system.

CBCB Bioinformatics Core Facility

The Center for Biomedical and Brain Imaging is a core facility at the University of Delaware. We provide instruments for structural and functional imaging. We have a research-dedicated Siemens Prisma 3T MRI scanner for humans, and a Bruker 9.4T MRI scanner for small animals. Our instruments are capable of acquiring images in multiple modalities, including structural MRI, functional MRI (fMRI), DTI, spectroscopy and X-nuclei imaging. We have multiple MRI coils specialized for body and brain imaging, and a variety of peripheral devices for physiological monitoring, stimulus presentation and subject responses. In addition to the MRI scanners, we also have instruments for Transcranial Magnetic Stimulation (TMS), both inside and outside the MRI scanner, and an EEG system for simultaneous EEG/fMRI. We have three full-time staff, including a certified MRI technologist and an MRI physicist. We are available for consultation to help design your MRI experiments.

Center for Biomedical and Brain Imaging at the University of Delaware

The Nemours Biomedical Research Informatics Center (BRIC) (formerly Bioinformatics Core facility) provides consultation, training and computational resources (both hardware and software). Services offered by BRIC include experimental design of parallel, crossover, longitudinal and cross-section studies, generalized statistical analysis, EHR and PEDSnet data requests and analysis and development of special purpose software and applications. BRIC serves as the interface between PEDSnet and the Nemours Children’s Healthcare System. This includes maintaining relationships with the partner institutions, facilitating data transfer from our institution to the national pediatric database, and aiding other entities from within Nemours in participating in the PEDSnet community. As a member of the National COVID Cohort Collaborative, BRIC and the DE-CTR aid in providing COVID-related patient data to an open-source national database and serve as an ambassador for the program to the entire Nemours and DE-CTR community. BRIC maintains the Nemours distribution of REDCap. Responsibilities include aiding in project setup and management as well as maintaining hosting and compute resources for Nemours sponsored projects.

Optimizing the measurement of mitochondrial respiration in Drosophila melanogaster adult brains and late-stage embryonic cells

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