VTechWorks Repository :: Browsing by Author "Jarome, Timothy J."

Browsing by Author "Jarome, Timothy J."

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Activity Dependent Protein Degradation Is Critical for the Formation and Stability of Fear Memory in the Amygdala
Jarome, Timothy J.; Werner, Craig T.; Kwapis, Janine L.; Helmstetter, Fred J. (PLOS, 2011-09)
Protein degradation through the ubiquitin-proteasome system [UPS] plays a critical role in some forms of synaptic plasticity. However, its role in memory formation in the amygdala, a site critical for the formation of fear memories, currently remains unknown. Here we provide the first evidence that protein degradation through the UPS is critically engaged at amygdala synapses during memory formation and retrieval. Fear conditioning results in NMDA-dependent increases in degradationspecific polyubiquitination in the amygdala, targeting proteins involved in translational control and synaptic structure and blocking the degradation of these proteins significantly impairs long-term memory. Furthermore, retrieval of fear memory results in a second wave of NMDA-dependent polyubiquitination that targets proteins involved in translational silencing and synaptic structure and is critical for memory updating following recall. These results indicate that UPS-mediated protein degradation is a major regulator of synaptic plasticity necessary for the formation and stability of long-term memories at amygdala synapses.
Controlling hypothalamic DNA methylation at the Pomc promoter does not regulate weight gain during the development of obesity
McFadden, Taylor; Gaito, Natasha; Carucci, Isabella; Fletchall, Everett; Farrell, Kayla; Jarome, Timothy J. (Public Library of Science, 2023-04)
Obesity is a complex medical condition that is linked to various health complications such as infertility, stroke, and osteoarthritis. Understanding the neurobiology of obesity is crucial for responding to the etiology of this disease. The hypothalamus coordinates many integral activities such as hormone regulation and feed intake and numerous studies have observed altered hypothalamic gene regulation in obesity models. Previously, it was reported that the promoter region of the satiety gene, Pomc, has increased DNA methylation in the hypothalamus following short-term exposure to a high fat diet, suggesting that epigenetic-mediated repression of hypothalamic Pomc might contribute to the development of obesity. However, due to technical limitations, this has never been directly tested. Here, we used the CRISPR-dCas9-TET1 and dCas9-DNMT3a systems to test the role of Pomc DNA methylation in the hypothalamus in abnormal weight gain following acute exposure to a high fat diet in male rats. We found that exposure to a high fat diet increases Pomc DNA methylation and reduces gene expression in the hypothalamus. Despite this, we found that CRISPR-dCas9-TET1-mediated demethylation of Pomc was not sufficient to prevent abnormal weight gain following exposure to a high fat diet. Furthermore, CRISPR-dCas9-DNMT3a-mediated methylation of Pomc did not alter weight gain following exposure to standard or high fat diets. Collectively, these results suggest that high fat diet induced changes in Pomc DNA methylation are a consequence of, but do not directly contribute to, abnormal weight gain during the development of obesity.
DNA Double-Strand Breaks Are a Critical Regulator of Fear Memory Reconsolidation
Navabpour, Shaghayegh; Rogers, Jessie; McFadden, Taylor; Jarome, Timothy J. (MDPI, 2020-11-26)
Numerous studies have shown that following retrieval, a previously consolidated memory requires increased transcriptional regulation in order to be reconsolidated. Previously, it was reported that histone H3 lysine-4 trimethylation (H3K4me3), a marker of active transcription, is increased in the hippocampus after the retrieval of contextual fear memory. However, it is currently unknown how this epigenetic mark is regulated during the reconsolidation process. Furthermore, though recent evidence suggests that neuronal activity triggers DNA double-strand breaks (DSBs) in some early-response genes, it is currently unknown if DSBs contribute to the reconsolidation of a memory following retrieval. Here, using chromatin immunoprecipitation (ChIP) analyses, we report a significant overlap between DSBs and H3K4me3 in area CA1 of the hippocampus during the reconsolidation process. We found an increase in phosphorylation of histone H2A.X at serine 139 (H2A.XpS139), a marker of DSB, in the Npas4, but not c-fos, promoter region 5 min after retrieval, which correlated with increased H3K4me3 levels, suggesting that the two epigenetic marks may work in concert during the reconsolidation process. Consistent with this, in vivo siRNA-mediated knockdown of topoisomerase II β, the enzyme responsible for DSB, prior to retrieval, reduced Npas4 promoter-specific H2A.XpS139 and H3K4me3 levels and impaired long-term memory, indicating an indispensable role of DSBs in the memory reconsolidation process. Collectively, our data propose a novel mechanism for memory reconsolidation through increases in epigenetic-mediated transcriptional control via DNA double-strand breaks.
Epigenetic and Ubiquitin-Proteasome Mechanisms of Obesity Development
McFadden, Taylor Marie (Virginia Tech, 2023-04-14)
Obesity is a major health condition in which little is known about the molecular mechanisms that drive it. The hypothalamus is the primary control center for controlling both food intake and energy expenditure in order to maintain the body's energy balance and dysregulation of molecular processes in this region have been implicated in the development and progression of obesity. Recently, several studies have shown altered DNA methylation of critical appetite genes, including the satiety gene Pomc, in the hypothalamus of rodents fed a high fat obesogenic diet. However, it has not previously been studied whether diet-induced changes in DNA methylation of critical appetite genes in the hypothalamus contributes to the development and persistence of the obesity phenotype. Further, DNA 5-hydroxymethylation (5-hmC) is one type of DNA methylation that is 10 times more abundant in the brain than peripheral tissues. However, to date, no study has been conducted examining whether DNA 5-hmC becomes altered in the brain following weight gain and/or contributes to the obesity phenotype. Additionally, there is also evidence to support that exposure to a high fat diet dysregulates the activity of the ubiquitin-proteasome system, the master regulator of protein degradation in cells, in the hypothalamus of male rodents. Despite this, whether this can occur in both sexes and directly contributes to abnormal weight gain has not been investigated. Here, we used a rodent diet-induced obesity model in combination with quantitative molecular assays and CRISPR-dCas9 manipulations to test the role of hypothalamic 1) DNA 5-hmC levels, 2) Pomc methylation, and 3) dysregulated ubiquitin-proteasome signaling in abnormal weight gain following exposure to obesogenic diets. We found that males, but not females, have decreased levels of DNA 5-hmC in the hypothalamus following exposure to a high fat diet, which tracked body weight. Short-term exposure to a high fat diet, which does not result in significant weight gain, resulted in decreased hypothalamic DNA 5-hmC levels, suggesting these changes occur prior to obesity development. Moreover, decreases in DNA 5-hmC persist even after the high fat diet is removed. Importantly, CRISPR-dCas9 mediated upregulation of DNA 5-hmC enzymes in the male, but not female, hypothalamus significantly reduced the percentage of weight gained on the high fat diet relative to controls. Next, we used the CRISPR-dCas9-TET1 and dCas9-DNMT3a systems to test the role of Pomc DNA methylation in the hypothalamus in abnormal weight gain following acute exposure to a high fat diet in male rats. We found that exposure to a high fat diet increases Pomc DNA methylation and reduces gene expression in the hypothalamus. Despite this, we found that CRISPR-dCas9-TET1-mediated demethylation of Pomc was not sufficient to prevent abnormal weight gain following exposure to a high fat diet. Moreover, CRISPR-dCas9-DNMT3a-mediated methylation of Pomc did not alter weight gain following exposure to standard or high fat diets. Finally, we found that both males and females showed dynamic downregulation of proteasome activity, decreases in proteasome subunit expression and an accumulation of degradation-specific K48 polyubiquitinated proteins in the hypothalamus. However, while the CRISPR-dCas9 system was able to selectively increase some forms of proteasome activity, it was unable to prevent diet-induced proteasome downregulation or abnormal weight gain. Collectively, this data reveals novel, sex-specific differences in the engagement of the ubiquitin proteasome system and role of DNA 5-hydroxymethylation in the hypothalamus during the development of the obesity phenotype.
Epigenetic Mechanisms in Memory and Cognitive Decline Associated with Aging and Alzheimer's Disease
Maity, Sabyasachi; Farrell, Kayla; Navabpour, Shaghayegh; Narayanan, Sareesh Naduvil; Jarome, Timothy J. (MDPI, 2021-11-13)
Epigenetic mechanisms, which include DNA methylation, a variety of post-translational modifications of histone proteins (acetylation, phosphorylation, methylation, ubiquitination, sumoylation, serotonylation, dopaminylation), chromatin remodeling enzymes, and long non-coding RNAs, are robust regulators of activity-dependent changes in gene transcription. In the brain, many of these epigenetic modifications have been widely implicated in synaptic plasticity and memory formation. Dysregulation of epigenetic mechanisms has been reported in the aged brain and is associated with or contributes to memory decline across the lifespan. Furthermore, alterations in the epigenome have been reported in neurodegenerative disorders, including Alzheimer’s disease. Here, we review the diverse types of epigenetic modifications and their role in activity- and learning-dependent synaptic plasticity. We then discuss how these mechanisms become dysregulated across the lifespan and contribute to memory loss with age and in Alzheimer’s disease. Collectively, the evidence reviewed here strongly supports a role for diverse epigenetic mechanisms in memory formation, aging, and neurodegeneration in the brain.
Evaluation of Sex Differences in the Hippocampus and Pituitary of Egr1 conditional knockout mice mediated by Nestin-Cre
Swilley, Cody Lynn (Virginia Tech, 2023-08-29)
Early growth response 1 (Egr1) is a transcription factor critical for learning and memory in the hippocampus and pituitary cell differentiation. Egr1 has been shown to extend continuation of the long-term potentiation in the hippocampus and is credited for forming long-term memories. The somatotrophs in the pituitary produce growth hormone and are found to be decreased in Egr1KO mice. These animals are also found to be sterile due to a decrease in LHB, which blocks ovulation. All previous studies have evaluated these physiological processes with complete Egr1KO research strains or antisense oligonucleotides, up until now, no data specific to individual type of cells has been generated. In an attempt to focus on the understanding of the functions of Egr1 gene in neural cell lineage, we are using an Egr1cKO Nestin-Cre model. Nestin allows for targeting neuronal lineage specific cells. In Chapter 1, we provide a systemic view of Egr1 gene and Nestin-Cre as a system for generating conditional knockout mouse strains. The Chapter begins with the identification of Egr1 gene and its protein structure, then proceeds to grasp its link to memory with behavior testing. The critical role of Egr1 in the pituitary and what cell populations are affected is also described. The same goes for Nestin-Cre, along with its limitations and understanding how to account for them in a study. The Egr1cKO Nestin-Cre system is the best form to understand neurological cell populations with Egr1 removal. In Chapter 2 and Chapter 3, we employ the Egr1cKO Nestin-Cre mouse model to understand cell-specific knockout of Egr1 in the nervous system by evaluating the hippocampus and pituitary. We explore learning and memory through behavioral tests and ribonucleic acid sequencing (RNA-seq) analysis to understand gene expression changes with Egr1 removal. Females showed higher activity during behavior tests, with more movement in the elevated plus maze and lower freezing times during the contextual fear conditioning. RNA-seq had higher changes in females than males but was not affected by the Nestin-Cre system overall. The same RNA-seq changes in the pituitary gland were present, with females having higher genomic differentiation. Females had growth-specific pathways altered by Nestin-Cre.
Females, but not males, require protein degradation in the hippocampus for contextual fear memory formation
Martin, Kiley; Musaus, Madeline; Navabpour, Shaghayegh; Gustin, Aspen; Ray, W. Keith; Helm, Richard F.; Jarome, Timothy J. (2021-08)
Strong evidence supports a role for protein degradation in fear memory formation. However, these data have been largely done in only male animals. Here, we found that following contextual fear conditioning, females, but not males, had increased levels of proteasome activity and K48 polyubiquitin protein targeting in the dorsal hippocampus, the latter of which occurred at chaperones or RNA processing proteins. In vivo CRISPR-dCas9-mediated repression of protein degradation in the dorsal hippocampus impaired contextual fear memory in females, but not males. These results suggest a sex-specific role for protein degradation in the hippocampus during the consolidation of a contextual fear memory.
GluR2 endocytosis-dependent protein degradation in the amygdala mediates memory updating
Ferrara, Nicole C.; Jarome, Timothy J.; Cullen, Patrick K.; Orsi, Sabrina A.; Kwapis, Janine L.; Trask, Sydney; Pullins, Shane E.; Helmstetter, Fred J. (Springer Nature, 2019-03-26)
Associations learned during Pavlovian fear conditioning are rapidly acquired and long lasting, providing an ideal model for studying long-term memory formation, storage, and retrieval. During retrieval, these memories can "destabilize" and become labile, allowing a transient "reconsolidation" window during which the memory can be updated, suggesting that reconsolidation could be an attractive target for the modification of memories related to past traumatic experiences. This memory destabilization process is regulated by protein degradation and GluR2-endocytosis in the amygdala. However, it is currently unknown if retrieval-dependent GluR2-endocytosis in the amygdala is critical for incorporation of new information into the memory trace. We examined whether the addition of new information during memory retrieval required GluR2-endocytosis to modify the original memory. The presentation of two foot shocks of weaker intensity during retrieval resulted in GluR2 endocytosis-dependent increase in fear responding on a later test, suggesting modification of the original memory. This increase in fear expression was associated with increased protein degradation and zif268 expression in the same population of cells in the amygdala, indicating increased destabilization processes and cellular activity, and both were lost following blockade of GluR2-endocytosis. These data suggest that the endocytosis of GluR2-containing AMPA receptors in the amygdala regulates retrieval-induced strengthening of memories for traumatic events by modulating cellular destabilization and activity.
The gut microbiome: a contributing mechanism to the anti-seizure effect of topiramate
Thai, K'Ehleyr Asia Puanani (Virginia Tech, 2023-07-28)
Epilepsy is one of the most common neurological disorders worldwide. This neurological disorder is characterized by spontaneous recurrent seizures and impacts about 65 million people globally. As there is no cure for epilepsy, the treatment goal for patients is seizure management, and ultimately seizure freedom. The first line of defense in seizure management is anti-epileptic drugs, which aim to restore the excitatory and inhibitory balance in the brain. Unfortunately, about 30% of people with epilepsy are drug resistant, a number which has remained unchanged despite the increasing amount of anti-epileptic drugs. This leads patients to seek alternative treatments, which include surgery, vagus nerve stimulation, or diet alterations such as the ketogenic diet. Due to the invasiveness of surgeries, difficulty to maintain specialty diets, or lack of effectiveness of these treatments in some patients, additional therapies are needed. The gut-brain axis is a bidirectional communication network connecting the central and enteric nervous systems. Part of this network includes communication via the gut microbiota. The gut microbiota consists of all the microorganisms living in the gut, including bacteria, viruses, and fungi. It is involved in aiding nutrient absorption, promoting the maturation of immune cells and functions, and protection against pathogens. There is growing interest in the role of the gut microbiome in human health and disease. Studies have shown that patients with epilepsy have altered gut microbiomes compared to healthy controls, and that gut microbiome alteration can impact seizure frequencies. These exciting findings have ignited research on the potential therapeutic role of the gut microbiome in epilepsy. Although studies have explored the impact of alterations in the gut microbiome on seizure activity, they have not studied how anti-epileptic drugs may contribute to this relationship. Thus, this dissertation explores the role of the commonly prescribed anti-epileptic drug topiramate on the gut microbiome. Fecal samples of mice treated with topiramate were analyzed using 16S ribosomal RNA gene sequencing. Analysis revealed that topiramate ingestion increased the probiotic bacteria Lactobacillus johnsonii in the gut microbiome. In addition, cotreatment of topiramate and Lactobacillus johnsonii reduced seizure susceptibility in a pentylenetetrazol-kindling seizure model. Moreover, cotreatment increased the butyrate producing family Lachnospiraceae and subsequently increased the neuroprotective SCFA, butyrate in the gut microbiome. Importantly, cotreatment also resulted in an increased GABA/glutamate ratio in the cortex of mice that underwent pentylenetetrazol-kindling. These results are the first to demonstrate that the anti-seizure effect of topiramate may be facilitated by the modulation of the gut microbiota via increasing butyrate and altering the GABA/glutamate ratio in the cortex. Lastly, this work highlights the potential for probiotics as an adjuvant therapy in seizure management.
Happy Chickens: Novel Physiological and Behavioral Measures of Cumulative Experience in Broilers and Laying Hens
Campbell, Andrew Michael (Virginia Tech, 2023-04-03)
Conventional housing environments for broiler chickens and commercial laying hens are often barren, high-density environments with an emphasis on production efficiency. These housing conditions limit birds' ability to display species-specific behaviors, can negatively impact health, and may contribute to negative cumulative experience. Cumulative experience is the culmination of all positive and negative experienced during an animal's lifetime. However, cumulative experience is difficult to quantify, as no validated measures of cumulative experience exist. Additionally, existing measures of negative animal experience mostly rely on interpretations of animal behavior which can be subjective, time consuming, and difficult to interpret. Therefore, there is scientific need for objective measures that can detect cumulative experience in poultry. Secretory and plasma Immunoglobulin A (IgA), telomere length, feather corticosterone concentrations, and attention bias testing all seem to respond to positive and negative experiences in humans or other non-human animal species, indicating that they may be useful as measures for poultry. Therefore, the objective of this thesis was to determine if these novel measures could be used as indicators of cumulative experience in broiler chickens and laying hens. In chapter 3, secretory and plasma IgA concentrations were measured in broilers raised in either high-complexity or low-complexity environments under either high or low stocking density over three replicated experiments. Birds housed in highly complex environments showed higher concentrations of plasma IgA compared to birds housed in low-complexity environments at day 48 of age, indicating reduced chronic stress in the former. Additionally, day 48 secretory IgA concentrations were decreased in birds housed in high-density environments compared to birds housed in low density environments, indicating birds from high-density environments were more chronically stressed. In chapter 4, gonad and kidney telomere length was measured to determine cumulative experience in broilers raised in the same housing conditions and replicated experiments of chapter 3. Treatment did not impact gonad telomere length, in line with expectations as gonads contain stem cells which produce high concentrations of telomerase. Birds housed in high-complexity pens had longer kidney telomeres compared to birds in low-complexity pens, indicating high-complexity birds had more positive cumulative experience. Stocking density did not impact kidney telomere length. In chapter 5, attention bias, tonic immobility, plasma and secretory IgA concentrations, and feather corticosterone concentrations were determined in laying hens raised in conventional cages or enriched floor pens. Birds in enriched floor pens showed increased attention bias, decreased tonic immobility, increased secretory IgA concentrations at week 22 of age, and decreased feather corticosterone concentrations compared to caged hens. These results indicate that compared to conventional cages, enriched pens in this study improved immune systems, reduced chronic stress, reduced fear, but increased anxiety in hens. In conclusion, secretory and plasma IgA and telomere length show appropriate contrast in response to broiler chicken housing conditions. However, additional work needs to be done before these measures can be widely used as measures of cumulative experience in poultry. Furthermore, attention bias, secretory IgA, and feather corticosterone showed an appropriate contrast between chronic stress responses in laying hens, but confirmation is needed in other contexts. Overall, the results indicate a beneficial relationship between environmental complexity and poultry welfare physiology and affective state, with the exception for anxiety in laying hens. Thus, providing an enriched environment can improve the welfare of commercial poultry and result in positive cumulative experience in most situations. Additionally, these results indicate that stocking density is a negative environment in broilers but potentially less intense than previously thought under experimental conditions. The assessment of behavioral and physiological measures of cumulative and positive animal experience should be included in experiments seeking to determine the impacts of environmental or management conditions to determine the broader impacts on poultry welfare.
Hypothalamic mechanisms of appetite regulation involve stress response and epigenetic modification
Cao, Chang (Virginia Tech, 2021-06-03)
Appetite regulation is primarily mediated by the hypothalamus, within which many neurotransmitters that regulate feeding are shared by the stress response circuitry. Stressors, especially those occur during critical periods of life, influence epigenetic programming and gene expression in the long-term. Therefore, the aim of this dissertation was to elucidate how hypothalamic mechanisms of appetite regulation correlate with the stress response and epigenetic modifications, using avian models and intracerebroventricular administration of various appetite-regulating factors. We first administered two methylation modifiers, S-adenosylmethionine (SAM), a methyl donor, and 5-azacytidine (AZA), a methylation inhibitor, to determine their effects on appetite. When measuring food intake immediately post-injection, SAM didn't affect fed or fasted chickens from a line selected for low bodyweight (LWS, individuals with anorexia), but suppressed feeding in fed and fasted broilers. In Japanese quail, SAM transiently induced satiety in fed but not fasted chicks. Intriguingly, AZA increased feeding in fasted LWS but decreased it in fed chicks. While it didn't affect either fed or fasted broilers, AZA induced satiety in both fed and fasted quail. These results suggests that SAM/AZA can directly affect appetite depending on genetics and nutritional state. The LWS chickens, when injected with SAM or AZA on day of hatch, didn't show increased feeding to the orexigenic stimulation of neuropeptide Y central injection on day 5 post-hatch. This suggests that epigenetic modifications occurred following SAM/AZA injection and affect appetite regulation that persisted. In other studies, we injected broilers with prostaglandin E2 (PGE2) or β-melanocyte-stimulating hormone (β-MSH) since their effects on appetite are unknown in meat-type chicks. We found that they both potently induced satiety, but the effective duration was longer in β-MSH-injected birds (up to 9 hours) than in PGE2-injected chicks (lasted for 1.5 hours). They both activated the paraventricular nucleus of the hypothalamus. The satiety induced by β-MSH mainly involved corticotropin-releasing factor and mesotocin, while the effect of PGE2 included ghrelin and brain-derived neurotropic factor. Nevertheless, all affected appetite-related factors have connections with the stress response. Thus, our results demonstrate that the hypothalamic mechanisms underlying anorexia induced by different neuroactive molecules involve the stress response and epigenetic modifications.
Impact of Training Method on Behavioral, Physiological, and Relationship Measures in Horses
Isernia, Lindsay Taylor (Virginia Tech, 2021-01-07)
With a rise in concern for animal welfare, the equine world has started using positive reinforcement (R+); as such, horses often experience a combination of negative reinforcement (R-) and R+. I compared the effects of R- to a combination of positive and negative reinforcement (R-/R+) training. Horses were trained to walk across two visually discriminable liverpools (striped, Experiment 1; colored water, Experiment 2), each associated with either R- or R-/R+, and training type alternating across six days. I measured highest training criteria reached, prevalence of undesirable behaviors, salivary cortisol (pre- and post-training), time spent by the trainer in motionless human tests (pre- and post-training), and horses' preference for the two liverpools using concurrent choice. Across both experiments, I found no significant difference in the proportions of criteria reached between training types; horses engaged in mugging for longer periods of time in R-/R+ than R-; no significant difference between training types for the pre- to post-change of cortisol; a greater proportion of horses increased time spent with R-/R+ trainer than the R- trainer; and no difference between first choice in the preference test or time horses spent in proximity to the liverpool, based on the training type with which the liverpool was associated. Overall, I found few differences between R-/R+ and R-, which could be due to horses only having 30 min total training contact with either training, or my use of relatively low intensities of R- and R+.
The Interaction of Early Growth Response Gene 1 and Myocyte Enhancer Factor 2C in the Murine Brain Cortex
Murray, Alexander James (Virginia Tech, 2021-09-16)
Early growth response gene – 1 (Egr1) encodes a protein widely present in mammalian body, such as connective tissue, cardiac tissue, the liver, and the brain. As a transcription factor (TF), it is involved in processes that take place in the endocrine, digestive, immune, musculo-skeletal and central nervous systems, for instance, B cell maturation upon B cell receptor activation, tendon repair upon mechano-stimulation, and long-term spatial memory formation. In mammalian brains, EGR1 controls the responses to environmental stimuli such as chronic stress and physical contact. It also participates in processes such as long-term memory consolidation and synapse re-structuring. It plays a role in enacting responses and qualities of gene transcription cascades upon neuronal stimulation. Inside the epigenetic realm, EGR1 recruits Ten-eleven translocation methylcytosine dioxygenase 1 (TET1) to remove DNA methylation at target loci. Due to its critical functions during brain development and upon neuronal activation, mis-regulation of EGR1 is associated with neuropsychological disorders such as post-traumatic stress disorder (PTSD) and schizophrenia (SCZ) in humans. In this study, we performed bioinformatics analysis with brain methylomes and predicted EGR1 may interact with myocyte enhancer factor 2C (MEF2C), which is known to be involved in many similar processes as EGR1, such as synapse architecture, cell migration, and learning and memory. EGR1 and MEF2C ChIP-seq data derived from mouse frontal cortex suggest these two proteins may regulate a common set of downstream genes. To begin, co-immunoprecipitation experiments were performed with HEK293T cells co-transfected with EGR1-FLAG and MEF2C-HA tagged constructs, allowing for specific interaction identification without endogenous protein expression interference. Furthermore, co-immunoprecipitation experiments performed with brain tissues additionally indicated the two proteins interact with each other endogenously. Altogether, this study provides protein-protein interaction evidence for EGR1 and MEF2C in cultured HEK293 cells and in the cortices of adult male mice. This information provides a foundation for future examinations of how these two TFs interact to initiate cascading events following neuronal stimulation.
Interleukin-6 and its Contribution to Embryogenesis in Cattle
Speckhart, Savannah Laurel (Virginia Tech, 2023-05-10)
In vitro systems like those used for in vitro embryo production are invaluable for our understanding of embryogenesis and the processes that regulate it. However, extensive research has also highlighted that in vitro produced embryos negatively differ from their in vivo counterparts in various ways. Not surprisingly, there is ~20% decrease in pregnancy success from pregnancies established using in vitro produced embryos. Therefore, much research has relied on attempting to produce a better in vitro embryo that more closely resembles their in vivo counterparts. Our laboratory has investigated this by supplementing a cytokine, interleukin-6 (IL6), during in vitro embryo culture. My dissertation work expands upon those initial efforts by answering more detailed questions related to the biological role of IL6 during cattle embryogenesis. In the work presented herein, IL6 supplementation during in vitro culture was able to transform the transcriptome of resulting conceptuses post embryo transfer. The transcriptome of these conceptuses included an abundance of genes associated with survival. Indeed, we witnessed IL6-treated conceptuses resulted in a 20% increased survival rate and were longer than their non-treated counterparts. In the second research project, we employed CRISPR-Cas9 genome editing technology to understand the embryo phenotype after part of the IL6 receptor responsible for signal transduction, interleukin-6 signal transducer (IL6ST), is disrupted. We discovered that IL6ST is required for development before the blastocyst stage. In addition, IL6ST disrupted blastocysts, presumed to contain wildtype, presented with severe, abnormal morphology. Not only did this group of embryos have decreased ICM and TE cell numbers, but they also had an increased occurrence of cells within the TE region that were negative for its traditional marker, CDX2. This suggests IL6ST is likely involved in a pathway responsible for determining cell fate identity at the blastocyst stage. Collectively, IL6 in cooperation with IL6ST, is a key controller of embryogenesis in cattle.
Investigation of somatomotor-sympathetic brain circuit abnormalities in two rat models featuring inborn differences in emotional behavior
Shupe, Elizabeth Anne (Virginia Tech, 2023-07-27)
Major depressive disorder (MDD) features symptoms spanning cognitive, affective, behavioral, and physiological domains. While many of the neural circuit disruptions mediating emotional and cognitive disturbances in depression have been described, far fewer studies have explored neurobiological mechanisms underlying its associated motor or physiological impairments. Emotionally motivated behaviors, including responses to stress, are characterized by concomitant somatomotor actions and autonomic changes that require intricate coordination of the motor and autonomic systems. Prior investigations by our group used a pseudorabies virus (PRV)-mediated retrograde tract-tracing approach to identify brain regions with parallel descending premotor and presympathetic efferents that play a role in integrating somatomotor and sympathetic functions. Several nodes of this circuitry, including the hypothalamic paraventricular nucleus (PVN), locus coeruleus (LC), and periaqueductal gray (PAG), are implicated in responses to stressful and emotionally salient stimuli. Based on this observation, it was hypothesized that these parallel descending circuits shape responses to diverse stressors and are altered in clinical depression and comorbid anxiety disorders. To explore this possibility, the experiments in this dissertation used two recombinant PRV strains to trace polysynaptic premotor and presympathetic pathways innervating sympathectomized skeletal muscle and adrenal gland, respectively, in two rat models with heritable differences in emotionality and stress reactivity: the Wistar-Kyoto (WKY) rat and the selectively bred Low Novelty Responder (bLR) rat. During our initial neuroanatomical investigations in the PVN, we observed that both WKY and bLR rats displayed significant decreases in the quantity of PVN neurons with premotor projections to skeletal muscle compared to their respective control strains. Labeling of neurons with presympathetic projections to adrenal gland or dual-labeled polysynaptic projections to both motor and sympathetic targets was not altered in either model. Our subsequent neuroanatomical studies focused on comparing premotor efferent projections from LC and PAG. In LC, fewer premotor efferent projections to skeletal muscle were observed in both models. There were also reductions in the number of premotor efferents in the four subdivisions of the PAG. WKY rats had significantly fewer premotor projections in the dorsomedial (DMPAG), lateral (LPAG), and ventrolateral (VLPAG) subdivisions, while bLR rats had significantly fewer premotor efferents in dorsolateral (DL)PAG. The final experiments in this dissertation sought to determine whether one potential therapeutic intervention, environmental enrichment during late childhood and adolescence, can improve emotional behavior disturbances and reverse premotor circuit alterations in bLR rats. Rearing young bLR rats in conditions with increased environmental complexity partially but incompletely improved aspects of depression- and anxiety-relevant behaviors and their corresponding PVN premotor circuit abnormalities. Cumulatively, these findings highlight somatomotor circuits in several brain structures involved in responses to stress and emotional stimuli that could be implicated in mediating motor-related impairments in clinical depression.
Memory formation for trace fear conditioning requires ubiquitin-proteasome mediated protein degradation in the prefrontal cortex
Reis, David S.; Jarome, Timothy J.; Helmstetter, Fred J. (Frontiers, 2012-10-23)
The cellular mechanisms supporting plasticity during memory consolidation have been a subject of considerable interest. De novo protein and mRNA synthesis in several brain areas are critical, and more recently protein degradation, mediated by the ubiquitin-proteasome system (UPS), has been shown to be important. Previous work clearly establishes a relationship between protein synthesis and protein degradation in the amygdala, but it is unclear whether cortical mechanisms of memory consolidation are similar to those in the amygdala. Recent work demonstrating a critical role for prefrontal cortex (PFC) in the acquisition and consolidation of fear memory allows us to address this question. Here we use a PFC-dependent fear conditioning protocol to determine whether UPS mediated protein degradation is necessary for memory consolidation in PFC. Groups of rats were trained with auditory delay or trace fear conditioning and sacrificed 60 min after training. PFC tissue was then analyzed to quantify the amount of polyubiquibated protein. Other animals were trained with similar procedures but were infused with either a proteasome inhibitor (clasto-lactacystin β-lactone) or a translation inhibitor (anisomycin) in the PFC immediately after training. Our results show increased UPS-mediated protein degradation in the PFC following trace but not delay fear conditioning. Additionally, post-training proteasome or translation inhibition significantly impaired trace but not delay fear memory when tested the next day. Our results further support the idea that the PFC is critical for trace but not delay fear conditioning and highlight the role of UPS-mediated degradation as critical for synaptic plasticity.
Molecular and epigenetic mechanisms of fear memory
Valajannavabpour, Shaghayegh (Virginia Tech, 2023-07-25)
Numerous memory studies have demonstrated that epigenetic-mediated transcriptional regulation, such as post-translational histone modifications, is essential to memory formation and maintenance. Moreover, many studies on the mechanisms of memory have focused on fear memories underlying traumatic events, which helps to understand post-traumatic stress disorder (PTSD). However, these mainly focus on individuals directly experiencing the event, while different species have shown the ability to learn fear indirectly by observing a conspecific experiencing a trauma. Thus, our understanding of indirect fear learning (IFL)'s characteristics is very limited. The trimethylation of histone 3 lysine 4 (H3K4me3) is an essential regulator of active gene transcription in cells and has been shown to be critical for memory formation in the hippocampus, a major site of memory storage. However, it is unknown how H3K4me3 is coordinated to target genes during memory formation. Monoubiquitination of histone H2B (H2Bubi) is critical for recruiting H3K4me3 to DNA in a gene-specific manner during memory formation in the hippocampus. Furthermore, there is a great overlap between H3K4me3 and phosphorylation of histone H2A.X at serine 139 (H2A.XpS139), a marker to study DNA double-strand break (DSB) loci. DSB is a critical mechanism for solving DNA-related topological issues during transcription and replication, which could be triggered in some immediate early genes (IEGs) by neuronal activity, such as memory consolidation.Here, we used rat fear conditioning paradigms in combination with quantitative molecular assays, such as chromatin immunoprecipitation (ChIP), and gene editing techniques, like siRNAs and CRISPR-dCas9 manipulations, to study the role of hippocampal 1) H2Bubi and 2) DSBs in contextual fear memory consolidation and reconsolidation, respectively. Additionally, we behaviorally and molecularly characterized IFL and compared it to directly acquired fear subjects. We found that contextual fear conditioning changed the expression of 86 genes in the hippocampus one hour after training. Remarkably, siRNA knockdown of the H2Bubi ligase, Rnf20, abolished changes in all but one of these genes, Per1. Additionally, we report that the loss of Rnf20 in neurons, but not astrocytes, of the hippocampus impaired long-term memory formation. We next found an increase in H2A.XpS139 and H3K4me3 levels in the Npas4, an IEG important for contextual fear memory, promoter region 5 minutes after retrieval. In vivo siRNAmediated knockdown of the enzyme responsible for DSB, topoisomerase II β, prior to retrieval, decreased Npas4 promoter-specific H3K4me3 and H2A.XpS139 levels and impaired long-term memory. Lastly, our data show that both sexes can indirectly acquire fear from either sex using the auditory-cued IFL model. Moreover, our data show that molecular profiles in the amygdala are largely unique to direct or indirect fear learning and vary by sex. Collectively, this data reveals novel roles for histone phosphorylation and ubiquitination in regulating H3K4me3 and memory formation and shows behavioral and molecular differences in each sex based on the way they acquire fear.
NF-κB mediates Gadd45β expression and DNA demethylation in the hippocampus during fear memory formation
Jarome, Timothy J.; Butler, Anderson A.; Nichols, Jessica N.; Pacheco, Natasha L.; Lubin, Farah D. (Frontiers, 2015-09-16)
Gadd45-mediated DNA demethylation mechanisms have been implicated in the process of memory formation. However, the transcriptional mechanisms involved in the regulation of Gadd45 gene expression during memory formation remain unexplored. NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) controls transcription of genes in neurons and is a critical regulator of synaptic plasticity and memory formation. In silico analysis revealed several NF-κB (p65/RelA and cRel) consensus sequences within the Gadd45β gene promoter. Whether NF-κB activity regulates Gadd45 expression and associated DNA demethylation in neurons during memory formation is unknown. Here, we found that learning in a fear conditioning paradigm increased Gadd45β gene expression and brain-derived neurotrophic factor (BDNF) DNA demethylation in area CA1 of the hippocampus, both of which were prevented with pharmacological inhibition of NF-κB activity. Further experiments found that conditional mutations in p65/RelA impaired fear memory formation but did not alter changes in Gadd45β expression. The learning-induced increases in Gadd45β mRNA levels, Gadd45β binding at the BDNF gene and BDNF DNA demethylation were blocked in area CA1 of the c-rel knockout mice. Additionally, local siRNA-mediated knockdown of c-rel in area CA1 prevented fear conditioning-induced increases in Gadd45β expression and BDNF DNA demethylation, suggesting that c-Rel containing NF-κB transcription factor complex is responsible for Gadd45β regulation during memory formation. Together, these results support a novel transcriptional role for NF-κB in regulation of Gadd45β expression and DNA demethylation in hippocampal neurons during fear memory.
Phosphorylation of RPT6 Controls Its Ability to Bind DNA and Regulate Gene Expression in the Hippocampus of Male Rats during Memory Formation
Farrell, Kayla; Auerbach, Aubrey; Musaus, Madeline; Navabpour, Shaghayegh; Liu, Catherine; Lin, Yu; Xie, Hehuang; Jarome, Timothy J. (Society for Neuroscience, 2024-01)
Memory formation requires coordinated control of gene expression, protein synthesis, and ubiquitin–proteasome system (UPS)-mediated protein degradation. The catalytic component of the UPS, the 26S proteasome, contains a 20S catalytic core surrounded by two 19S regulatory caps, and phosphorylation of the 19S cap regulatory subunit RPT6 at serine 120 (pRPT6-S120) has been widely implicated in controlling activity-dependent increases in proteasome activity. Recently, RPT6 was also shown to act outside the proteasome where it has a transcription factor-like role in the hippocampus during memory formation. However, little is known about the proteasome-independent function of “free” RPT6 in the brain or during memory formation and whether phosphorylation of S120 is required for this transcriptional control function. Here, we used RNA-sequencing along with novel genetic approaches and biochemical, molecular, and behavioral assays to test the hypothesis that pRPT6-S120 functions independently of the proteasome to bind DNA and regulate gene expression during memory formation. RNA-sequencing following siRNA-mediated knockdown of free RPT6 revealed 46 gene targets in the dorsal hippocampus of male rats following fear conditioning, where RPT6 was involved in transcriptional activation and repression. Through CRISPR-dCas9-mediated artificial placement of RPT6 at a target gene, we found that RPT6 DNA binding alone may be important for altering gene expression following learning. Further, CRISPR-dCas13-mediated conversion of S120 to glycine on RPT6 revealed that phosphorylation at S120 is necessary for RPT6 to bind DNA and properly regulate transcription during memory formation. Together, we reveal a novel function for phosphorylation of RPT6 in controlling gene transcription during memory formation.
Protein degradation and protein synthesis in long-term memory formation
Jarome, Timothy J.; Helmstetter, Fred J. (Frontiers, 2014-06-26)
Long-term memory (LTM) formation requires transient changes in the activity of intracellular signaling cascades that are thought to regulate new gene transcription and de novo protein synthesis in the brain. Consistent with this, protein synthesis inhibitors impair LTM for a variety of behavioral tasks when infused into the brain around the time of training or following memory retrieval, suggesting that protein synthesis is a critical step in LTM storage in the brain. However, evidence suggests that protein degradation mediated by the ubiquitin-proteasome system (UPS) may also be a critical regulator of LTM formation and stability following retrieval. This requirement for increased protein degradation has been shown in the same brain regions in which protein synthesis is required for LTM storage. Additionally, increases in the phosphorylation of proteins involved in translational control parallel increases in protein polyubiquitination and the increased demand for protein degradation is regulated by intracellular signaling molecules thought to regulate protein synthesis during LTM formation. In some cases inhibiting proteasome activity can rescue memory impairments that result from pharmacological blockade of protein synthesis, suggesting that protein degradation may control the requirement for protein synthesis during the memory storage process. Results such as these suggest that protein degradation and synthesis are both critical for LTM formation and may interact to properly “consolidate” and store memories in the brain. Here, we review the evidence implicating protein synthesis and degradation in LTM storage and highlight the areas of overlap between these two opposing processes. We also discuss evidence suggesting these two processes may interact to properly form and store memories. LTM storage likely requires a coordinated regulation between protein degradation and synthesis at multiple sites in the mammalian brain.

Browsing by Author "Jarome, Timothy J."

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