Monoubiquitination of histone H2B is a crucial regulator of the transcriptome during memory formation.

Posttranslational modification of histone proteins is critical for memory formation. Recently, we showed that monoubiquitination of histone H2B at lysine 120 (H2Bub) is critical for memory formation in the hippocampus. However, the transcriptome controlled by H2Bub remains unknown. Here, we found that fear conditioning in male rats increased or decreased the expression of 86 genes in the hippocampus but, surprisingly, siRNA-mediated knockdown of the H2Bub ligase, Rnf20, abolished changes in all but one of these genes. These findings suggest that monoubiquitination of histone H2B is a crucial regulator of the transcriptome during memory formation.

Phosphorylation of RPT6 Controls Its Ability to Bind DNA and Regulate Gene Expression in the Hippocampus of Male Rats during Memory Formation.

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.

Sex-differences in proteasome-dependent K48-polyubiquitin signaling in the amygdala are developmentally regulated in rats.

BACKGROUND: Sex differences have been observed in several brain regions for the molecular mechanisms involved in baseline (resting) and memory-related processes. The ubiquitin proteasome system (UPS) is a major protein degradation pathway in cells. Sex differences have been observed in lysine-48 (K48)-polyubiquitination, the canonical degradation mark of the UPS, both at baseline and during fear memory formation within the amygdala. Here, we investigated when, how, and why these baseline sex differences arise and whether both sexes require the K48-polyubiquitin mark for memory formation in the amygdala. METHODS: We used a combination of molecular, biochemical and proteomic approaches to examine global and protein-specific K48-polyubiquitination and DNA methylation levels at a major ubiquitin coding gene (Uba52) at baseline in the amygdala of male and female rats before and after puberty to determine if sex differences were developmentally regulated. We then used behavioral and genetic approaches to test the necessity of K48-polyubiquitination in the amygdala for fear memory formation. RESULTS: We observed developmentally regulated baseline differences in Uba52 methylation and total K48-polyubiquitination, with sexual maturity altering levels specifically in female rats. K48-polyubiquitination at specific proteins changed across development in both male and female rats, but sex differences were present regardless of age. Lastly, we found that genetic inhibition of K48-polyubiquitination in the amygdala of female, but not male, rats impaired fear memory formation. CONCLUSIONS: These results suggest that K48-polyubiquitination differentially targets proteins in the amygdala in a sex-specific manner regardless of age. However, sexual maturity is important in the developmental regulation of K48-polyubiquitination levels in female rats. Consistent with these data, K48-polyubiquitin signaling in the amygdala is selectively required to form fear memories in female rats. Together, these data indicate that sex-differences in baseline K48-polyubiquitination within the amygdala are developmentally regulated, which could have important implications for better understanding sex-differences in molecular mechanisms involved in processes relevant to anxiety-related disorders such as post-traumatic stress disorder (PTSD).

Male and female brains have differences in size, development, and cellular processes. Further, males and females have differences in likelihood of developing certain anxiety-related disorders, such as post-traumatic stress disorder (PTSD). We previously observed sex differences in a cellular mechanism that controls the destruction of proteins via tagging by the protein modifier ubiquitin in resting and behaviorally trained animals. We found that adult female rats "ubiquitinated" different proteins during learning and had more ubiquitin than male rats at rest in the amygdala, the brain region that controls emotional regulation. This study investigated if the sex difference in ubiquitin at rest changed as animals age, including the proteins being ubiquitinated and how the amount of ubiquitin was controlled. We also investigated if male and female rats need ubiquitin for memory formation. We found that males and females ubiquitinate different proteins, but that aging also contributes to changes in this, suggesting that sexual maturity may be important for controlling the amount of ubiquitin in females. Lastly, we found that only female rats needed ubiquitin in the amygdala for forming a fear memory. These results are important for understanding the role of ubiquitin activity at different developmental stages and for forming fear-based memories in both sexes. Since females are more likely to develop PTSD than males, these data could help understand how different cellular processes work together in PTSD development to create better treatment options.

Hypothalamic DNA 5-hydroxymethylation levels are altered by diet-induced weight gain during the development of obesity in a sex-specific manner.

Obesity is a major health concern that is associated with altered gene transcription in the hypothalamus. However, the mechanisms controlling this gene expression dysregulation remain largely unknown. DNA 5-hydroxymethylation (5-hmC) is a potent transcriptional activator that is expressed at 10 times higher levels in the brain than the periphery. Despite this, no study has examined if DNA 5-hmC is altered in the brain following exposure to obesogenic diets or contributes to abnormal weight gain over time. 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 DNA 5-hmC in abnormal weight gain in male and female rats. 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 directly correlate with increased 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, though the extent of this is diet-dependent. Importantly, CRISPR-dCas9-mediated upregulation of DNA 5-hmC enzymes in the male, but not female, ventromedial nucleus of the hypothalamus significantly reduced the percentage of weight gained on the high fat diet relative to controls. These results suggest that hypothalamic DNA 5-hmC is an important sex-specific regulator of abnormal weight gain following exposure to high fat diets.

Proteasome-independent K63 polyubiquitination selectively regulates ATP levels and proteasome activity during fear memory formation in the female amygdala.

Females are more likely than males to develop post-traumatic stress disorder (PTSD). However, the neurobiological mechanisms responsible for these sex differences remain elusive. The ubiquitin proteasome system (UPS) is involved in fear memory formation and implicated in PTSD development. Despite this, proteasome-independent functions of the UPS have rarely been studied in the brain. Here, using a combination of molecular, biochemical, proteomic, behavioral, and novel genetic approaches, we investigated the role of proteasome-independent lysine-63 (K63)-polyubiquitination, the second most abundant ubiquitin modification in cells, in the amygdala during fear memory formation in male and female rats. Only females had increased levels of K63-polyubiquitination targeting in the amygdala following fear conditioning, which targeted proteins involved in ATP synthesis and proteasome function. CRISPR-dCas13b-mediated knockdown of K63-polyubiquitination in the amygdala via editing of the K63 codon in the major ubiquitin gene, Ubc, impaired fear memory in females, but not males, and caused a reduction in learning-related increases in ATP levels and proteasome activity in the female amygdala. These results suggest that proteasome-independent K63-polyubiquitination is selectively involved in fear memory formation in the female amygdala, where it is involved in the regulation of ATP synthesis and proteasome activity following learning. This indicates the first link between proteasome-independent and proteasome-dependent UPS functions in the brain during fear memory formation. Importantly, these data are congruent with reported sex differences in PTSD development and may contribute to our understanding of why females are more likely to develop PTSD than males.

Controlling hypothalamic DNA methylation at the Pomc promoter does not regulate weight gain during the development of obesity.

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.

Neuronal and astrocytic protein degradation are critical for fear memory formation.

Strong evidence has implicated proteasome-mediated protein degradation in the memory consolidation process. However, due to the use of pharmacological approaches, the cell type specificity of this remains unknown. Here, we used neuron-specific and novel astrocyte-specific CRISPR-dCas9-KRAB-MECP2 plasmids to inhibit protein degradation in a cell type-specific manner in the amygdala of male rats. We found that while inhibition of neuronal, but not astrocytic, protein degradation impaired performance during the training session, both resulted in impaired contextual fear memory retention. Together, these data provide the first evidence of a cell type-specific role for protein degradation in the memory consolidation process.

Short-term exposure to an obesogenic diet causes dynamic dysregulation of proteasome-mediated protein degradation in the hypothalamus of female rats.

OBJECTIVES: Previous work has shown that exposure to a high fat diet dysregulates the protein degradation process in the hypothalamus of male rodents. However, whether this occurs in a sex-independent manner is unknown. The objective of this study was to determine the effects of a short-term obesogenic diet on the ubiquitin-proteasome mediated protein degradation process in the hypothalamus of female rats. METHODS: We fed young adult female rats a high fat diet or standard rat chow for 7 weeks. At the end of the 7th week, animals were euthanized and hypothalamus nuclear and cytoplasmic fractions were collected. Proteasome activity and degradation-specific (K48) ubiquitin signaling were assessed. Additionally, we transfected female rats with CRISPR-dCas9-VP64 plasmids in the hypothalamus prior to exposure to the high fat diet in order to increase proteasome activity and determine the role of reduced proteasome function on weight gain from the obesogenic diet. RESULTS: We found that across the diet period, females gained weight significantly faster on the high fat diet than controls and showed dynamic downregulation of proteasome activity, decreases in proteasome subunit expression and an accumulation of degradation-specific K48 polyubiquitinated proteins in the hypothalamus. Notably, while our CRISPR-dCas9 manipulation was able to selectively increase some forms of proteasome activity, it was unable to prevent diet-induced proteasome downregulation or abnormal weight gain. CONCLUSIONS: Collectively, these results reveal that acute exposure to an obesogenic diet causes reductions in the protein degradation process in the hypothalamus of females.

The epigenetic role of proteasome subunit RPT6 during memory formation in female rats.

Reports of sex differences in the neurobiology of memory formation are becoming more prevalent. Despite this, much remains unknown about the role of sex in this process. We previously reported the first evidence of a novel epigenetic role for proteasome subunit RPT6 during memory formation in the hippocampus of male rodents whereby it associated with monoubiquitinated histone H2B (H2Bubi). Here, we used molecular, biochemical, and behavioral approaches to investigate whether RPT6 has a similar epigenetic role during memory formation in female rats. Following contextual fear conditioning, we found that RPT6 levels and DNA binding at regions coding for c-fos, the previously identified target of RPT6 in males, were unchanged in the hippocampus of females and that loss of RPT6 did not alter learning-induced increases in c-fos However, RPT6 was in complex with H2Bubi in the female hippocampus and this association increased with fear conditioning, suggesting that it could still retain an epigenetic function. Consistent with this, hippocampal siRNA-mediated knockdown of the RPT6-coding gene, Psmc5, impaired memory in females. These results suggest that while RPT6 does associate with epigenetic H2Bubi during memory formation in both males and females, it has sex-specific gene targets during the memory consolidation process.

Passive exposure to e-cigarette emissions is associated with worsened mental health.

BACKGROUND: Cigarette smoking, secondhand cigarette smoke (SHS) exposure, and e-cigarette use ("vaping") are each associated with increased rates of depressive symptoms and other internalizing mental health disorders. The prevalence of vaping has increased greatly, yet the mental health correlates of secondhand exposure to e-cigarette emissions are as yet to be investigated. This study examined the potential adverse mental health outcomes associated with different tobacco exposures (direct and passive), with a particular focus on the mental health correlates of secondhand exposure to e-cigarette emissions. METHODS: The Population Assessment of Tobacco and Health Study data collected from a sample of 16,173 Wave 4 adults were used to test the hypothesis that secondhand e-cigarette emissions exposure is associated with increased odds of internalizing mental health disorders. Individuals were categorized as exclusive cigarette smokers, exclusive e-cigarette users, cigarette and e-cigarette dual users, exclusive noncombustible tobacco users, secondhand smoke exposed non-users, secondhand e-cigarette emissions exposed non-users, and non-users with no current SHS/secondhand e-cigarette aerosol exposure. Adjusted weighted logistic regression analysis was used to investigate the association between exposure type and internalizing problems as assessed by scores on the Global Appraisal of Individual Needs-Short Screener (GAIN-SS), a widely used instrument for assessing mental health problems. RESULTS: Cigarette smokers (AOR = 2.53, 95% CI: 2.19-2.92), e-cigarette users (AOR = 3.14, 2.41-4.09), dual users (AOR = 3.37, 2.85-4.00), noncombustible tobacco users (AOR = 1.48, 1.01-2.17), SHS exposed non-users (AOR = 1.63, 1.37-1.94), and secondhand e-cigarette emissions exposed non-users (AOR = 1.43, 1.03-1.99) were each associated with increased odds of moderate to severe internalizing mental health problems as compared to unexposed non-users. Odds of internalizing problems among SHS and secondhand e-cigarette emissions exposed non-users did not differ (p = 0.46). CONCLUSIONS: This is the first study, to our knowledge, to identify an association between recent secondhand exposure to e-cigarette emissions and mental health problems, and the risk is comparable to that of SHS. Corroboration of this relationship needs further research to explicate directionality and mechanisms underlying this association.

Protein SUMOylation is a sex-specific regulator of fear memory formation in the amygdala.

Strong evidence has implicated ubiquitin signaling in the process of fear memory formation. While less abundant than ubiquitination, evidence suggests that protein SUMOylation may also be involved in fear memory formation in neurons. However, the importance of amygdala protein SUMOylation in fear memory formation has never been directly examined. Furthermore, while recent evidence indicates that males and females differ significantly in the requirement for ubiquitin signaling during fear memory formation, whether sex differences also exist in the importance of protein SUMOylation to this process remains unknown. Here we found that males and females differ in the requirement for protein SUMOylation in the amygdala during fear memory formation. Western blot analysis revealed that while females had higher resting levels of SUMOylation, both sexes showed global increases following fear conditioning. However, SUMOylation-specific proteomic analysis revealed that only females have increased targeting of individual proteins by SUMOylation following fear conditioning, some of which were heat shock proteins. This suggests that protein SUMOylation is more robustly engaged in the amygdala of females following fear conditioning. In vivo siRNA mediated knockdown of Ube2i, the coding gene for the essential E2 ligase for SUMOylation conjugation, in the amygdala impaired fear memory in males without any effect in females. Importantly, higher siRNA concentrations than what was needed to impair memory in males reduced Ube2i levels in the amygdala of females but resulted in an increase in SUMOylation levels, suggesting a compensatory effect in females that was not observed in males. Collectively, these data reveal a novel, sex-specific role for protein SUMOylation in the amygdala during fear memory formation and expand our understanding of how ubiquitin-like signaling regulates memory formation.

Exhalation of alternative tobacco product aerosols differs from cigarette smoke-and may lead to alternative health risks.

BACKGROUND: Variation in alternative tobacco product (ATP) constituents, heating potential, and consumer behaviors have made it difficult to characterize their health risks. To date, most toxicity studies of ATPs have used established cigarette endpoints to inform study design. Furthermore, to assess where ATPs fall on the tobacco harm continuum, with cigarettes representing maximum potential risk, studies have tended to compare the relative biological responses to ATPs against those due to cigarettes. OBJECTIVES: 1) To characterize the exhalation profiles of two popular ATPs: electronic cigarettes (e-cigarettes) and hookah waterpipes (hookah) and 2) to determine if ATP exhalation patterns were representative of cigarette exhalation patterns. METHODS: Exhalation patterns were recorded (mouth only, nose only, or both mouth and nose) among individuals observed in the New York City tri-state area using a recognizable tobacco product (cigarette, e-cigarette, or hookah). Cigarette smokers and e-cigarette vapers were observed on city streets; water-pipe smokers were observed inside Manhattan hookah bars. RESULTS: E-cigarette vapers practiced exclusive nasal exhalation at far higher rates than did cigarette smokers (19.5% vs 4.9%). Among vapers, e-cigarette device type was also significantly associated with exhalation profile. Overall, cigarette smokers exhaled from their nose approximately half to one-third as often as ATP users (hookah and e-cigarettes, respectively). CONCLUSIONS: Nasal exhalation of tobacco emissions appears to be a shared characteristic across several types of ATPs. It is therefore plausible that ATP-specific consumer behaviors may foster unique upper respiratory health consequences that have not been observed in smokers. Thus, product-specific behaviors should inform the prioritization of biological endpoints used in studies evaluating ATP toxicity and health effects.

Use of Genome Editing Techniques to Produce Transgenic Farm Animals.

Recombinant proteins are essential for the treatment and diagnosis of clinical human ailments. The availability and biological activity of recombinant proteins is heavily influenced by production platforms. Conventional production platforms such as yeast, bacteria, and mammalian cells have biological and economical challenges. Transgenic livestock species have been explored as an alternative production platform for recombinant proteins, predominantly through milk secretion; the strategy has been demonstrated to produce large quantities of biologically active proteins. The major limitation of utilizing livestock species as bioreactors has been efforts required to alter the genome of livestock. Advancements in the genome editing field have drastically improved the ability to genetically engineer livestock species. Specifically, genome editing tools such as the CRISPR/Cas9 system have lowered efforts required to generate genetically engineered livestock, thus minimizing restrictions on the type of genetic modification in livestock. In this review, we discuss characteristics of transgenic animal bioreactors and how the use of genome editing systems enhances design and availability of the animal models.

Epigenetic Mechanisms in Memory and Cognitive Decline Associated with Aging and Alzheimer's Disease.

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.

E-Cigarette Use, Systemic Inflammation, and Depression.

BACKGROUND: E-cigarette use (vaping) is an emerging public health problem. Depression has been found to be associated with e-cigarette use, and vaping and depression are each associated with elevated systemic inflammation. To date, the role of inflammation in the relationship between vaping and depression has not been explored. OBJECTIVE: To assess the independent associations between e-cigarette use, depression, and inflammation, and to investigate whether the likelihood of depression among current e-cigarette users is associated with systemic inflammation. METHODS: Nationally representative NHANES data from 2015-2018 were used (n = 4961). Systemic inflammation was defined as serum C-reactive protein (CRP) ≥ 8.0 mg/L. Depressed individuals were characterized by a score ≥ 10 on the Patient Health Questionnaire-9 (PHQ-9). Current e-cigarette users were defined as individuals who vaped at least once in the past 30 days and these individuals were stratified by use: exclusive users (reported smoking less than 100 combustible cigarettes in their lifetime), dual users (reported current use of electronic and combustible cigarettes), and e-cigarette users who were previous smokers. Bivariate analyses were used to assess independent associations between vaping, depression, and inflammation; and weighted logistic regression analyses adjusting for BMI, sex, and economic status were used to determine the odds ratios (ORs) for depression by e-cigarette category stratified by differential CRP levels. RESULTS: Depression occurred in 16.7% of all e-cigarette users vs. 5.0% of those who never used e-cigarettes (p < 0.001). In adjusted analyses, the following elevated ORs were found: all current e-cigarette users with CRP <8 = 3.37 (95% CI: 2.06, 5.51) vs. CRP ≥8 = 6.70 (2.48, 18.11); exclusive e-cigarette users with CRP <8 = 1.91 (0.78, 4.69) vs. those with CRP ≥8 = 5.09 (1.44, 18.02); and dual users with CRP <8 = 4.31 (2.35, 7.89) vs. those with CRP ≥8 = 7.37 (1.85, 29.41). These ORs indicate that depression is associated with each category of e-cigarette use; however, we found this association did not vary by systemic inflammation level (interaction p-values > 0.05). CONCLUSION: While a pattern of greater ORs for depression among e-cigarette users with elevated CRP provides provocative findings that might suggest a potential role of inflammation in the association between vaping and depression, we failed to find evidence that inflammation clearly moderates this association. While it is possible that depression among e-cigarette users may be influenced by systemic inflammation, a reproduction of the current study is necessary among a larger cohort to elucidate the effect of inflammation on depression among e-cigarette users.

Proteomic Analysis Reveals Sex-Specific Protein Degradation Targets in the Amygdala During Fear Memory Formation.

Ubiquitin-proteasome mediated protein degradation has been widely implicated in fear memory formation in the amygdala. However, to date, the protein targets of the proteasome remain largely unknown, limiting our understanding of the functional significance for protein degradation in fear memory formation. Additionally, whether similar proteins are targeted by the proteasome between sexes has yet to be explored. Here, we combined a degradation-specific K48 Tandem Ubiquitin Binding Entity (TUBE) with liquid chromatography mass spectrometry (LC/MS) to identify the target substrates of the protein degradation process in the amygdala of male and female rats following contextual fear conditioning. We found that males (43) and females (77) differed in the total number of proteins that had significant changes in K48 polyubiquitin targeting in the amygdala following fear conditioning. Many of the identified proteins (106) had significantly reduced levels in the K48-purified samples 1 h after fear conditioning, suggesting active degradation of the substrate due to learning. Interestingly, only 3 proteins overlapped between sexes, suggesting that targets of the protein degradation process may be sex-specific. In females, many proteins with altered abundance in the K48-purified samples were involved in vesicle transport or are associated with microtubules. Conversely, in males, proteins involved in the cytoskeleton, ATP synthesis and cell signaling were found to have significantly altered abundance. Only 1 protein had an opposite directional change in abundance between sexes, LENG1, which was significantly enhanced in males while lower in females. This suggests a more rapid degradation of this protein in females during fear memory formation. Interestingly, GFAP, a critical component of astrocyte structure, was a target of K48 polyubiquitination in both males and females, indicating that protein degradation is likely occurring in astrocytes following fear conditioning. Western blot assays revealed reduced levels of these target substrates following fear conditioning in both sexes, confirming that the K48 polyubiquitin was targeting these proteins for degradation. Collectively, this study provides strong evidence that sex differences exist in the protein targets of the degradation process in the amygdala following fear conditioning and critical information regarding how ubiquitin-proteasome mediated protein degradation may contribute to fear memory formation in the brain.

Sex-Specific Linear Polyubiquitination Is a Critical Regulator of Contextual Fear Memory Formation.

Strong evidence supports that protein ubiquitination is a critical regulator of fear memory formation. However, as this work has focused on protein degradation, it is currently unknown whether polyubiquitin modifications that are independent of the proteasome are involved in learning-dependent synaptic plasticity. Here, we present the first evidence that atypical linear (M1) polyubiquitination, the only ubiquitin chain that does not occur at a lysine site and is largely independent of the proteasome, is critically involved in contextual fear memory formation in the amygdala in a sex-specific manner. Using immunoblot and unbiased proteomic analyses, we found that male (49) and female (14) rats both had increased levels of linear polyubiquitinated substrates following fear conditioning, though none of these protein targets overlapped between sexes. In males, target protein functions involved cell junction and axonal guidance signaling, while in females the primary target was Adiponectin A, a critical regulator of neuroinflammation, synaptic plasticity, and memory, suggesting sex-dependent functional roles for linear polyubiquitination during fear memory formation. Consistent with these increases, in vivo siRNA-mediated knockdown of Rnf31, an essential component of the linear polyubiquitin E3 complex LUBAC, in the amygdala impaired contextual fear memory in both sexes without affecting memory retrieval. Collectively, these results provide the first evidence that proteasome-independent linear polyubiquitination is a critical regulator of fear memory formation, expanding the potential roles of ubiquitin-signaling in learning-dependent synaptic plasticity. Importantly, our data identify a novel sex difference in the functional role of, but not a requirement for, linear polyubiquitination in fear memory formation.

Ubiquitination of Histone H2B by Proteasome Subunit RPT6 Controls Histone Methylation Chromatin Dynamics During Memory Formation.

BACKGROUND: Posttranslational histone modifications play a critical role in the regulation of gene transcription underlying synaptic plasticity and memory formation. One such epigenetic change is histone ubiquitination, a process that is mediated by the ubiquitin-proteasome system in a manner similar to that by which proteins are normally targeted for degradation. However, histone ubiquitination mechanisms are poorly understood in the brain and in learning. In this article, we describe a new role for the ubiquitin-proteasome system in histone crosstalk, showing that learning-induced monoubiquitination of histone H2B (H2Bubi) is required for increases in the transcriptionally active H3 lysine 4 trimethylation (H3K4me3) mark at learning-related genes in the hippocampus. METHODS: Using a series of molecular, biochemical, electrophysiological, and behavioral experiments, we interrogated the effects of short interfering RNA-mediated knockdown and CRISPR (clustered regularly interspaced short palindromic repeats)-mediated upregulation of ubiquitin ligases, deubiquitinating enzymes and histone methyltransferases in the rat dorsal hippocampus during memory consolidation. RESULTS: We show that H2Bubi recruits H3K4me3 through a process that is dependent on the 19S proteasome subunit RPT6 and that a loss of H2Bubi in the hippocampus prevents learning-induced increases in H3K4me3, gene transcription, synaptic plasticity, and memory formation. Furthermore, we show that CRISPR-dCas9-mediated increases in H2Bubi promote H3K4me3 and memory formation under weak training conditions and that promoting histone methylation does not rescue memory impairments resulting from loss of H2Bubi. CONCLUSIONS: These results suggest that H2B ubiquitination regulates histone crosstalk in learning by way of nonproteolytic proteasome function, demonstrating a novel mechanism by which histone modifications are coordinated in response to learning.

Establishing an immunocompromised porcine model of human cancer for novel therapy development with pancreatic adenocarcinoma and irreversible electroporation.

New therapies to treat pancreatic cancer are direly needed. However, efficacious interventions lack a strong preclinical model that can recapitulate patients' anatomy and physiology. Likewise, the availability of human primary malignant tissue for ex vivo studies is limited. These are significant limitations in the biomedical device field. We have developed RAG2/IL2RG deficient pigs using CRISPR/Cas9 as a large animal model with the novel application of cancer xenograft studies of human pancreatic adenocarcinoma. In this proof-of-concept study, these pigs were successfully generated using on-demand genetic modifications in embryos, circumventing the need for breeding and husbandry. Human Panc01 cells injected subcutaneously into the ears of RAG2/IL2RG deficient pigs demonstrated 100% engraftment with growth rates similar to those typically observed in mouse models. Histopathology revealed no immune cell infiltration and tumor morphology was highly consistent with the mouse models. The electrical properties and response to irreversible electroporation of the tumor tissue were found to be similar to excised human pancreatic cancer tumors. The ample tumor tissue produced enabled improved accuracy and modeling of the electrical properties of tumor tissue. Together, this suggests that this model will be useful and capable of bridging the gap of translating therapies from the bench to clinical application.