Browsing by Author "Ploski, Jonathan E."
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Item Adeno-associated viral serotypes produce differing titers and differentially transduce neurons within the rat basal and lateral amygdala(BioMed Central) Holehonnur, Roopashri; Luong, Jonathan A.; Chaturvedi, Dushyant; Ho, Anthony; Lella, Srihari K.; Hosek, Matthew P.; Ploski, Jonathan E.Background: In recent years, there has been an increased interest in using recombinant adeno-associated viruses (AAV) to make localized genetic manipulations within the rodent brain. Differing serotypes of AAV possess divergent capsid protein sequences and these variations greatly influence each serotype's ability to transduce particular cell types and brain regions. We therefore aimed to determine the AAV serotype that is optimal for targeting neurons within the Basal and Lateral Amygdala (BLA) since the transduction efficiency of AAV has not been previously examined within the BLA. This region is desirable to genetically manipulate due to its role in emotion, learning & memory, and numerous psychiatric disorders. We accomplished this by screening 9 different AAV serotypes (AAV2/1, AAV2/2, AAV2/5, AAV2/7, AAV2/8, AAV2/9, AAV2/rh10, AAV2/DJ and AAV2/DJ8) designed to express red fluorescent protein (RFP) under the regulation of an alpha Ca2+/calmodulin-dependent protein kinase II promoter (aCaMKII).; Results: We determined that these serotypes produce differing amounts of virus under standard laboratory production. Notably AAV2/2 consistently produced the lowest titers compared to the other serotypes examined. These nine serotypes were bilaterally infused into the rat BLA at the highest titers achieved for each serotype and at a normalized titer of 7.8E + 11 GC/ml. Twenty one days following viral infusion the degree of transduction was quantitated throughout the amygdala. These viruses exhibited differential transduction of neurons within the BLA. AAV2/7 exhibited a trend toward having the highest efficiency of transduction and AAV2/5 exhibited significantly lower transduction efficiency as compared to the serotypes examined. AAV2/5's decreased ability to transduce BLA neurons correlates with its significantly different capsid protein sequences as compared to the other serotypes examined.; Conclusions: For laboratories producing their own recombinant adeno-associated viruses, the use of AAV2/2 is likely less desirable since AAV2/2 produces significantly lower titers than many other serotypes of AAV. Numerous AAV serotypes appear to efficiently transduce BLA neurons, with the exception of AAV2/5. Taking into consideration the ability of certain serotypes to achieve high titers and transduce BLA neurons well, in our hands AAV2/DJ8 and AAV2/9 appear to be ideal serotypes to use when targeting neurons within the BLA.;Item Characterizing the Role of Heme in Alzheimer’s Disease Pathogenesis(2021-05-01T05:00:00.000Z) Vidal, Chantal; Zhang, Li; Pantano, Paul; Burr, John G.; Ploski, Jonathan E.; Delk, NikkiIn humans, heme accounts for 97% of functional iron. With a porphyrin ring and an iron ion, heme possesses structural and chemical features fitting for electron transfer, oxidation/reduction, and interaction with oxygen. Three oxidative phosphorylation (OXPHOS) complexes, II, III, and IV, require heme for proper functioning. Cells that require high levels of adenosine triphosphate (ATP) and OXPHOS require elevated levels of heme. Heme also serves as a powerful antioxidant for cells because it can be degraded to biliverdin and reduced to bilirubin. The reduction of biliverdin to bilirubin helps relieve reactive oxygen species (ROS) in cells. Neuronal cells are known to be high-energy demanding cells that depend on mitochondrial respiration to function. Specifically, in neurodegenerative diseases such as Alzheimer’s Disease (AD), mitochondrial dysfunction is one of the key characteristics associated with the progression of this disease. Oxidative stress has also been implicated in the pathogenesis of AD. Due to the role of heme in both of these cellular functions, it is important to understand how heme contributes to neuronal function and how it may play a pivotal role in developing this neurodegenerative disease. The objective of this research is to dissect the role of heme in the pathogenesis of AD. Utilizing immunocytochemistry and Western blot techniques, I have depicted the importance of heme in neuronal development. Heme uptake, synthesis, and degradation are significantly increased in developing and differentiating neurons. This increase in heme flux coincides with an increase in mitochondrial proteins. Using the APPPS1 mouse model and microarray expression data of human patients, I detected specific heme-related enzymes that are downregulated in AD. These alterations can lead to a decrease in the availability of heme for cellular functions. The decrease in heme availability can lead to disturbed mitochondrial function and an increase in oxidative stress. Furthermore, to glean whether heme flux alterations are early and potentially initiating causes of AD, I utilized patient-derived neurons generated from human-induced pluripotent stem cells (iPSCs). These studies revealed a distinct role of heme in the development of familial (FAD) and sporadic AD (SAD). SAD neurons have downregulated heme synthetic and degradation enzymes, while FAD neurons only had a slight yet significant reduction in the biliverdin reductase B (BLVRB) enzyme involved in heme degradation. Moreover, analysis of the tricarboxylic acid (TCA) cycle enzymes and intermediates revealed a significant alteration in enzymes and intermediates within both SAD and FAD neurons relative to gender-matched controls. This study revealed that although heme flux alterations are likely an early event in SAD pathogenesis, perturbations in the TCA cycle are probably a common characteristic of both SAD and FAD.Item Emotional Modulation of Synapses, Circuits and Memory(Frontiers Research Foundation) Ploski, Jonathan E.; McIntyre, Christa K.Item Is Arc mRNA Unique: A Search For mRNAs That Localize to the Distal Dendrites of Dentate Gyrus Granule Cells Following Neural Activity(Frontiers Media Sa) de Solis, Christopher A.; Morales, Anna A.; Hosek, Matthew P.; Partin, Alex C.; Ploski, Jonathan E.; de Solis, Christopher A.; Morales, Anna A.; Hosek, Matthew P.; Ploski, Jonathan E.There have been several attempts to identify which RNAs are localized to dendrites; however, no study has determined which RNAs localize to the dendrites following the induction of synaptic activity. We sought to identify all RNA transcripts that localize to the distal dendrites of dentate gyrus granule cells following unilateral high frequency stimulation of the perforant pathway (pp-HFS) using Sprague Dawley rats. We then utilized laser microdissection (LMD) to very accurately dissect out the distal 2/3rds of the molecular layer (ML), which contains these dendrites, without contamination from the granule cell layer, 2 and 4 h post pp-HFS. Next, we purified and amplified RNA from the ML and performed an unbiased screen for 27,000 RNA transcripts using Affymetrix microarrays. We determined that Activity Regulated Cytoskeletal Protein (Arc/Arg3.1) mRNA, exhibited the greatest fold increase in the ML at both timepoints 2 and 4 h). In total, we identified 31 transcripts that increased their levels within the ML following pp-HFS across the two timepoints. Of particular interest is that one of these identified transcripts was an unprocessed micro-RNA pri-miR132). Fluorescent in situ hybridization and qRT-PCR were used to confirm some of these candidate transcripts. Our data indicate Arc is a unique activity dependent gene, due to the magnitude that its activity dependent transcript localizes to the dendrites. Our study determined other activity dependent transcripts likely localize to the dendrites following neural activity, but do so with lower efficiency compared to Arc.Item Viral Tool Development for Investigations of Learning and Memory(2018-08) de Solis, Christopher A; Ploski, Jonathan E.Developing new viral tools to investigate biological and behavioral phenomena is important to push neuroscience research to new avenues that were once limited by expensive and time consuming transgenic and knockout rodent models. This dissertation is a collection of experiments that outline the development and optimization of several viral vector systems for application of studying a gene’s function in the brain and the role it plays in behavioral phenomena. First, I show that knockdown of a gene’s transcript using short hairpin RNAs (shRNAs) delivered to neurons using adeno associated virus (AAV), is toxic to neurons and results in an impairment of Pavlovian auditory fear conditioning. Next, I outline the development of a doxycycline dependent CRISPR/Cas genome editing system for knockdown of genes in the brain using AAV as an alternative to gene knockdown with shRNAs. I then examine the efficiency of several AAV serotypes for their ability to function in a specific cell type, inhibitory neurons. Next, I sought to look for important genes that might be potentially important for memory consolidation in the hippocampus, with respect to transport of mRNA transcripts to the distal ends of dendrites of dentate gyrus granule cells following the induction of long-term potentiation (LTP). I found that Arc (Activity related cytoskeletal protein) mRNA was the most present in the dendrites following neuronal activation compared to all other known transcripts in the rat genome. Interestingly, another gene that was highly upregulated was an unprocessed microRNA (pri-miRNA), pri-miRNA-132. Finally, I use lentiviral vectors to investigate the role of GluN2B, an NMDA receptor subunit, in the updating of strong memories and found that increasing the level of GluN2B within the mouse basal and lateral complex of the amygdala (BLA) allows for the modification of an existing strong fear memory via reconsolidation, a process typically not initiated in stronger memories.