Biology subjects

Kelly, R. D.

Publications and source records attributed to Kelly, R. D..

7 recordsLinked to original sources

bioRxiv2025

Ku suppresses RNA-mediated innate immune responses in human cells to accommodate primate-specific Alu expansion.

Ku70 and Ku80 form Ku, a ring-shaped protein that initiates the non-homologous end-joining (NHEJ) DNA repair pathway.1 Specifically, Ku binds to double-stranded DNA (dsDNA) ends and recruits other NHEJ factors (e.g., DNA-PKcs and LIG4). While Ku binds to double-stranded RNA (dsRNA)2 and traps mutated-DNA-PKcs on ribosomal RNA in vivo,3,4 the physiological significance of Ku-dsRNA interactions in otherwise wild-type cells remains elusive. Intriguingly, while dispensable for murine development,5,6 Ku is essential in human cells.7 Despite similar genome sizes, human cells express [~]100-fold more Ku than mouse cells, implying functions beyond NHEJ, possibly through a dose-sensitive interaction with dsRNA, which is [~]100 times weaker than with dsDNA.2,8 While investigating the essentiality of Ku in human cells, we found that depletion of Ku - unlike LIG4 - induces profound interferon (IFN) and NF-kB responses reliant on the dsRNA-sensor MDA5/RIG-I and adaptor MAVS. Prolonged Ku-degradation also activates other dsRNA-sensors, e.g. PKR that suppresses protein translation, and OAS/RNaseL that cleaves rRNAs and eventually induces growth arrest and cell death. MAVS, RIG-I, or MDA5 knockouts suppressed IFN signaling and, together with PKR knockouts, partially rescued Ku-depleted human cells. Ku-irCLIP analyses revealed that Ku binds to diverse dsRNA, predominantly stem-loops in primate-specific Alu elements9 at anti-sense orientation in introns and 3-UTRs. Ku expression rose sharply in higher primates tightly correlating with Alu-expansion (r = 0.94/0.95). Together, our study identified a vital role of Ku in accommodating Alu-expansion in primates by mitigating a dsRNA-induced innate immune response, explaining the rise of Ku levels and its essentiality in human cells.

genetics↗

bioRxiv2024

The histone demethylase KDM5 has insulator activity in the brain

KDM5 family proteins are best known for their demethylation of the promoter proximal chromatin mark H3K4me3. KDM5-regulated transcription is critical in the brain, with variants in the X-linked paralog KDM5C causing the intellectual disability (ID) disorder Claes-Jensen syndrome. Although the demethylase activity of KDM5C is known to be important for neuronal function, the contribution of non-enzymatic activities remain less characterized. We therefore used Drosophila to model the ID variant Kdm5L854F, which disrupts a C5HC2 zinc finger adjacent to the enzymatic JmjC domain. Kdm5L854F causes similar transcriptional changes in the brain to a demethylase dead strain, Kdm5J1310C*, despite having little effect on enzymatic activity. KDM5L854F is also distinct from KDM5J1310C* in its reduced interactions with insulator proteins and enhancement of position effect variegation. Instead, the common transcriptional deficits likely result from both the JmjC and C5HC2 domains driving proper genomic organization through their activity in promoting proper loop architecture.

genetics↗

bioRxiv2024

Effects of High Fat Diet on Metabolic Health Vary by Age of Menopause Onset

Menopause accelerates metabolic dysfunction, including (pre-)diabetes, obesity and visceral adiposity. However, the effects of endocrine vs. chronological aging in this progression are poorly understood. We hypothesize that menopause, especially in the context of middle-age, will exacerbate the metabolic effects of a high fat diet. Using young-adult and middle-aged C57BL/6J female mice, we modeled diet-induce obesity via chronic administration of high fat (HF) diet vs. control diet. We modeled peri-menopause/menopause via injections of 4-vinylcyclohexene diepoxide, which accelerates ovarian failure vs. vehicle. We performed glucose tolerance tests 2.5 and 7 months after diet onset, during the peri-menopausal and menopausal phases, respectively. Peri-menopause increased the severity of glucose intolerance and weight gain in middle-aged, HF-fed mice. Menopause increased weight gain in all mice regardless of age and diet, while chronological aging drove changes in adipose tissue distribution towards more visceral vs. subcutaneous adiposity. These data are in line with clinical data showing that post-menopausal women are more susceptible to metabolic dysfunction and suggest that greater chorological age exacerbates the effects of endocrine aging (menopause). This work highlights the importance of considering both chronological and endocrine aging in studies of metabolic health.

neuroscience↗

bioRxiv2023

Effects of menopause and high fat diet on metabolic outcomes in a mouse model of Alzheimer's disease.

About two-thirds of those with Alzheimers disease (AD) are women, most of whom are post-menopausal. Menopause accelerates the risk for dementia by increasing the risk for metabolic, cardiovascular, and cerebrovascular diseases. Mid-life metabolic disease (e.g. obesity, diabetes, or prediabetes) is a well-known risk factor for dementia. A high fat diet can lead to poor metabolic health in both humans and rodents. The goal of this study was to determine the effects of menopause and high fat diet on metabolic outcomes in the AppNL-F knock-in mouse model of Alzheimers disease. To model menopause, we used an accelerated ovarian failure model (4-vinylcyclohexene diepoxide, VCD). This ovary-intact model is more clinically relevant than an ovariectomy model, as mice go through a perimenopausal period. At 3 months of age, AppNL-F mice were administered VCD or vehicle (oil) and then placed on either a control diet (10% fat) or a high fat diet (HF; 60% fat) and maintained on the diets until 10 months of age. Menopause led to metabolic impairment (weight gain and glucose intolerance) and further exacerbated obesity in response to a high fat diet. Menopause had independent effects on some serum metabolic health biomarkers (insulin) and synergic effects with HF diet on other markers (glucagon). Some metabolic effects of menopause may be centrally mediated, as menopause altered the expression of hypothalamic genes related to energy balance and increased microgliosis in the lateral hypothalamic nucleus. This work highlights the need to model endocrine aging in animal models of dementia and will contribute to further understanding the interaction between menopause and metabolic health in the context of AD. HighlightsO_LIIn a mouse model of AD, menopause, modeled by accelerated ovarian failure, leads to metabolic impairment. C_LIO_LIMenopause has independent effects on some serum metabolic health biomarkers (insulin) and synergic effects with HF diet on other markers (glucagon). C_LIO_LIMenopause alters the expression of hypothalamic energy balance related genes. C_LIO_LIMenopause leads to increased microgliosis in the lateral hypothalamic nucleus. C_LI

neuroscience↗

bioRxiv2023

Brain Specific Estrogen Ameliorates Cognitive Effects of Surgical Menopause in Mice

Menopause is a major endocrinological shift that leads to an increased vulnerability to the risk factors for cognitive impairment and dementia. This is thought to be due to the loss of circulating estrogens, which exert many potent neuroprotective effects in the brain. Systemic replacement of estrogen post-menopause has many limitations, including increased risk for estrogen-sensitive cancers. A more promising therapeutic approach therefore might be to deliver estrogen only to the brain thus limiting adverse peripheral side effects. We examined whether we could enhance cognitive performance by delivering estrogen exclusively to the brain in post-menopausal mice. We modeled surgical menopause via bilateral ovariectomy (OVX). We treated mice with the pro-drug 10{beta},17{beta}-dihydroxyestra-1,4-dien-3-one (DHED), which can be administered systemically but is converted to 17{beta}-estradiol only in the brain. Young (2.5-month) and middle-aged (11-month-old) female C57BL/6J mice received ovariectomy and a subcutaneous implant containing vehicle (cholesterol) or DHED. At 3.5 months old (young group) and 14.5 months old (middle-aged group), mice underwent behavior testing to assess memory. DHED did not significantly alter metabolic status in middle-aged, post-menopausal mice. In both young and middle-aged mice, the brain-specific estrogen DHED improved spatial memory. Additional testing in middle-aged mice also showed that DHED improved working and recognition memory. These promising results lay the foundation for future studies aimed at determining if this intervention is as efficacious in models of dementia that have comorbid risk factors.

neuroscience↗

bioRxiv2022

Noncannonical functions of Ku may underlie essentiality in human cells

The Ku70/80 heterodimer is a key player in non-homologous end-joining DNA repair but has also been involved in other cellular functions like telomere regulation and maintenance, in which Kus role is not fully characterized. It was previously reported that knockout of Ku80 in a human cell line results in lethality, but the underlying cause of Ku essentiality in human cells has yet to be fully explored. Here, we established conditional Ku70 knockout cells to study the essentiality of Ku70 function. Endogenous Ku70 knockout was achieved using CRISPR/Cas9 editing in cells where Ku70 expression was maintained through integration of an HA-tagged Ku70 cDNA under the control of a doxycycline-inducible promoter. Ku70 conditional knockout cell lines were identified via western blotting, and edits were validated by Sanger sequencing. We visually observed cell death in Ku70 knockout cells 8-10 days post Ku70-HA depletion, and loss of viability following Ku depletion was quantified using crystal violet assays. Interestingly, assessment of telomere length in Ku70 knockout cells using telomere restriction fragment analyses did not reveal any changes in average telomere length following Ku70-HA depletion. Immunofluorescence analysis used to assess {gamma}H2AX foci accumulation as a measure of double-stranded DNA breaks following Ku70-HA depletion allowed us to conclude that increased DNA damage is not the driving cause of loss of cell viability. Finally, quantitative proteome analysis of Ku70 knockout cells following Ku70-HA depletion identified a number of pathways and proteins that are significantly dysregulated following the loss of Ku70, including processes which Ku function has been previously associated with such as cell cycle/mitosis, RNA related processes, and translation/ribosome biogenesis. Overall, this conditional Ku70 knockout system reveals that loss of Ku affects multiple cellular processes and pathways and suggests that Ku plays critical roles in other cellular processes beyond DNA repair and telomere maintenance to maintain cell viability. Author SummaryThe Ku70/80 heterodimer is a key player in non-homologous end-joining DNA repair, where it acts as a scaffold for other repair factors needed to process double-stranded DNA breaks. Ku has also been involved in other cellular functions like telomere regulation and maintenance, in which Kus role is not fully characterized. Previous data suggest that while loss of Ku70/80 can be tolerated in other species, Ku is essential to humans. We have established a conditional Ku70 knockout in HEK293 cells to evaluate the basis of Ku essentiality in human cells. While we observed loss of cell viability upon Ku depletion, we did not observe significant changes in telomere length nor did we record lethal levels of DNA damage upon loss of Ku, suggesting that the reasons for the loss of viability is not linked to the functions of Ku in DNA repair or at telomeres. Analysis of global proteome changes following Ku70 depletion revealed dysregulations of several cellular pathways including cell cycle/mitosis, RNA related processes, and translation/ribosome biogenesis. Our study reveals that loss of Ku affects multiple cellular processes and pathways and suggests that Ku plays critical roles in cellular processes beyond DNA repair and telomere maintenance to maintain cell viability.

molecular biology↗

bioRxiv2020

Utility of a high-resolution mouse single nucleotide polymorphism microarray assessed for rodent comparative genomics

In the study of genetic diversity in non-model species there is a notable lack of the low-cost, high resolution tools that are readily available for model organisms. Genotyping microarray technology for model organisms is well-developed, affordable, and potentially adaptable for cross-species hybridization. The Mouse Diversity Genotyping Array (MDGA), a single nucleotide polymorphism (SNP) genotyping tool designed for Mus musculus, was tested as a tool to survey genomic diversity of wild species for inter-order, inter-genus, and intra-genus comparisons. Application of the MDGA cross-species provides genetic distance information that reflects known taxonomic relationships reported previously between non-model species, but there is an underestimation of genetic diversity for non-Mus samples, indicated by a plateau in loci genotyped beginning 10-15 millions of years divergence from the house mouse. The number and types of samples included in datasets genotyped together must be considered in cross-species hybridization studies. The number of loci with heterozygous genotypes mapped to published genome sequences indicates potential for cross-species MDGA utility. A case study of seven deer mice yielded 159,797 loci (32% of loci queried by the MDGA) that were genotyped in these rodents. For one species, Peromyscus maniculatus, 6,075 potential polymorphic loci were identified. Cross-species utility of the MDGA provides needed genetic information for non-model species that are lacking genomic resources. Genotyping arrays are widely available, developed tools that are capable of capturing large amounts of genetic information in a single application, and represent a unique opportunity to identify genomic variation in closely related species that currently have a paucity of genomic information available. A candidate list of MDGA loci that can be utilized in cross-species hybridization studies was identified and may prove to be informative for rodent species that are known as environmental sentinels. Future studies may evaluate the utility of candidate SNP loci in populations of non-model rodents. Author SummaryThere is a need for a tool that can assay DNA sequence differences in species for which there is little or no DNA information available. One method of analyzing differences in DNA sequences in species with well-understood genomes is through a genotyping microarray, which has demonstrated utility cross-species. The Mouse Diversity Genotyping Array (MDGA) is a tool designed to examine known differences across the genome of the house mouse, Mus musculus. Given that related organisms share genetic similarity, the MDGA was tested for utility in identifying genome variation in other wild mice and rodents. Variation identified from distantly related species that were not of the same genus as the house mouse was an underestimate of the true amount of variation present in the genomes of wild species. Utility of the MDGA for wild species is best suited to mice from the same genus as the house mouse, and candidate variation identified can be tested in rodent populations in future studies. Identifying changes in genetic variation within populations of wild rodents can help researchers understand the links between specific genome changes and the ability to adapt to pressures in the environment, as well as better understand the evolution of rodents.

genomics↗