Biology subjects

MacKenzie, A.

Publications and source records attributed to MacKenzie, A..

8 recordsLinked to original sources

bioRxiv2026

The obesity associated BDNF intron-3 cis-regulatory element, BE5.1, modulates the effects of early-life stress on weight gain, anxiety and gene expression in a sex dependent manner.

Early-life stress (ELS) has been associated with increases in obesity and anxiety and affects males and females in different ways. Animal and human studies indicate that ELS has maladaptive effects on brain development due, in part, to disruptions in the expression of genes that include brain-derived neurotrophic factor (BDNF). Our previous studies identified a cis-regulatory element (CRE, BE5.1) within BDNF intron-3 that harbours a SNP associated with obesity and anxiety. We used maternal separation (MS) to examine the role of BE5.1 in controlling resilience to the maladaptive effects of ELS. At 9-11 weeks, BE5.1KO mice experience a delay in weight gain compared to WT mice, a phenotype partly reversed by MS in BE5.1KO females. The effects of either MS or BE5.1KO decreased anxiety-like behaviour in males whereas MS had little effect in females who displayed increased anxiety-like behaviour in animals lacking BE5.1. We also found that social behaviours are influenced by BE5.1 deletion in females but are more influenced by MS in males. Furthermore, deletion of BE5.1 and MS together significantly decreased marble burying behaviour in male mice, an effect negated by diazepam. RNA-seq analyses suggest that, in hypothalamus, BE5.1 is required to stabilize or down-regulate the transcription of 26 key genes, with diverse neuroregulatory roles, in response to MS as these 26 genes are strongly upregulated in the absence of BE5.1. We also provide evidence that three of these genes; Prkcg, Foxg1 and Mef2c, modulate BE5.1 activity in an allele-specific manner. Taken together, these results suggest that the BE5.1CRE acts as a buffer element that protects against the maladaptive effects of MS on gene expression and later life behaviours in a sex-dependent manner.

molecular biology↗

bioRxiv2025

Acute AMPK activation does not adequately stimulate insulin signaling in skeletal muscle models of Myotonic Dystrophy Type 1

Myotonic Dystrophy Type 1 (DM1) is a multisystemic neuromuscular disorder characterized by skeletal muscle weakness, muscle atrophy, myotonia, cognitive impairments, gastrointestinal complications, and insulin resistance. While insulin resistance is well characterized in type 2 diabetes, its pathomechanism in DM1 remains unclear. Our study aims to elucidate the pathomechanism of insulin resistance in DM1 and how the pathway responds to AMPK stimulation. Proteomic analysis from sedentary wildtype and sedentary HSA-LR mice, a common DM1 mouse model, revealed downregulation of the AMPK-PGC-1 axis. Analysis of sedentary HSA-LR mice and exercised HSA-LR mice revealed activation of the AMPK-PGC-1 axis in exercised animals. To investigate this pathway, we treated WT and HSA-LR mice with the AMPK activator AICAR to examine the impact of AMPK stimulation on insulin signaling in DM1. This revealed impaired responses in the insulin pathway activation in the HSA-LR mice. Next, we examined whether these differences extended to a human model by treating control and DM1 myotubes with insulin and/or AICAR. In DM1 myotubes, both treatments produced dampened responses of key insulin signaling intermediates compared to controls. Taken together, these results suggest impaired activation of insulin signaling pathways in DM1 models and confirm the presence of insulin resistance with an impaired response to acute AMPK stimulation.

molecular biology↗

bioRxiv2025

An automated metabolite extraction workflow for global metabolomics analysis using the Agilent Bravo liquid handling platform

Metabolomics is the comprehensive study of small molecules that provides a snapshot of an organisms physiological state. Reflecting phenotype more closely than genes or proteins, metabolites reveal changes linked to diseases, mutations, genetic interventions, and environmental stimuli. Recent technological advancements in metabolomics analysis through the application of ion mobility mass spectrometry have enhanced the comprehensive analysis of complex metabolic mixtures. However, pre-analytical bottlenecks in throughput and consistent extraction persist. We developed an automated, sample-agnostic metabolite extraction workflow for diverse liquid samples using an Agilent Bravo liquid handling platform. Here, we provide device, protocol, and form files for efficient sample processing to extract metabolites for global metabolomics analysis using liquid chromatography - mass spectrometry techniques.

biochemistry↗

bioRxiv2023

Meiotic Cells Escape Prolonged Spindle Checkpoint Activity Through Premature Silencing and Slippage

To prevent chromosome mis-segregation, a surveillance mechanism known as the spindle checkpoint delays the cell cycle if kinetochores are not attached to spindle microtubules, allowing the cell additional time to correct improper attachments. During spindle checkpoint activation, checkpoint proteins bind the unattached kinetochore and send a diffusible signal to inhibit the anaphase promoting complex/cyclosome (APC/C). Previous work has shown that mitotic cells with depolymerized microtubules can escape prolonged spindle checkpoint activation in a process called mitotic slippage. During slippage, spindle checkpoint proteins bind unattached kinetochores, but the cells cannot maintain the checkpoint arrest. We asked if meiotic cells had as robust of a spindle checkpoint response as mitotic cells and whether they also undergo slippage after prolonged spindle checkpoint activity. We performed a direct comparison between mitotic and meiotic budding yeast cells that signal the spindle checkpoint due to a lack of either kinetochore-microtubule attachments or due to a loss of tension-bearing attachments. We find that the spindle checkpoint is not as robust in meiosis I or meiosis II compared to mitosis, overcoming a checkpoint arrest approximately 150 minutes earlier in meiosis. In addition, cells in meiosis I escape spindle checkpoint signaling using two mechanisms, silencing the checkpoint at the kinetochore and through slippage. We propose that meiotic cells undertake developmentally-regulated mechanisms to prevent persistent spindle checkpoint activity to ensure the production of gametes. AUTHOR SUMMARYMitosis and meiosis are the two major types of cell divisions. Mitosis gives rise to genetically identical daughter cells, while meiosis is a reductional division that gives rise to gametes. Cell cycle checkpoints are highly regulated surveillance mechanisms that prevent cell cycle progression when circumstances are unfavorable. The spindle checkpoint promotes faithful chromosome segregation to safeguard against aneuploidy, in which cells have too many or too few chromosomes. The spindle checkpoint is activated at the kinetochore and then diffuses to inhibit cell cycle progression. Although the checkpoint is active in both mitosis and meiosis, most studies involving checkpoint regulation have been performed in mitosis. By activating the spindle checkpoint in both mitosis and meiosis in budding yeast, we show that cells in meiosis elicit a less persistent checkpoint signal compared to cells in mitosis. Further, we show that cells use distinct mechanisms to escape the checkpoint in mitosis and meiosis I. While cells in mitosis and meiosis II undergo anaphase onset while retaining checkpoint proteins at the kinetochore, cells in meiosis I prematurely lose checkpoint protein localization at the kinetochore. If the mechanism to remove the checkpoint components from the kinetochore is disrupted, meiosis I cells can still escape checkpoint activity. Together, these results highlight that cell cycle checkpoints are differentially regulated during meiosis to avoid long delays and to allow gametogenesis.

cell biology↗

bioRxiv2021

Leptin-mediated suppression of food intake by conserved Glp1r-expressing neurons prevents obesity

The adipose-derived hormone leptin acts via its receptor (LepRb) in the brain to control energy balance. A previously unidentified population of GABAergic hypothalamic LepRb neurons plays key roles in the restraint of food intake and body weight by leptin. To identify markers for candidate populations of LepRb neurons in an unbiased manner, we performed single-nucleus RNA-sequencing of enriched mouse hypothalamic LepRb cells, as well as with total hypothalamic cells from multiple mammalian species. In addition to identifying known LepRb neuron types, this analysis identified several previously unrecognized populations of hypothalamic LepRb neurons. Many of these populations display strong conservation across species, including GABAergic Glp1r-expressing LepRb (LepRbGlp1r) neurons that express more Lepr and respond more robustly to exogenous leptin than other LepRb populations. Ablating LepRb from these cells provoked hyperphagic obesity without impairing energy expenditure. Conversely, reactivating LepRb in Glp1r-expressing cells decreased food intake and body weight in otherwise LepRb-null mice. Furthermore, LepRb reactivation in GABA neurons improved energy balance in LepRb-null mice, and this effect required the expression of LepRb in GABAergic Glp1r-expressing neurons. Thus, the conserved GABAergic LepRbGlp1r neuron population plays crucial roles in the control of food intake and body weight by leptin.

neuroscience↗

bioRxiv2021

An ancient polymorphic regulatory region within the BDNF gene associated with obesity modulates anxiety-like behaviour in mice and humans.

Obesity and anxiety are morbidities notable for their increased impact on society during the recent COVID-19 pandemic. Understanding the mechanisms governing susceptibility to these conditions will increase quality of life and our resilience to future pandemics. In the current study we explored the function of a highly conserved regulatory region (BE5.1) within the BDNF gene that harbours a polymorphism strongly associated with obesity (rs10767664; p=4.69x10-26). Analysis in primary cells suggested that the major T-allele of BE5.1 was an enhancer whereas the obesity associated A-allele was not. However, CRISPR/CAS9 deletion of BE5.1 from the mouse genome (BE5.1KO) produced no significant effect on the expression of BDNF transcripts in the hypothalamus, no change in weight gain after 28 days and only a marginally significant increase in food intake. Nevertheless, transcripts were significantly increased in the amygdala of female mice and elevated zero maze and marble burying tests demonstrated a significant increase in anxiety-like behaviour that could be reversed by diazepam. Consistent with these observations, human GWAS cohort analysis demonstrated a significant association between rs10767664 and anxiousness in human populations. Intriguingly, interrogation of the human GTEx eQTL database demonstrated no effect on BDNF mRNA levels associated with rs10767664 but a highly significant effect on BDNF-antisense (BDNF-AS) gene expression and splicing suggesting a possible mechanism. We discuss our findings within the context of the known function and regulation of BDNF in obesity and anxiety whilst exploring the validity of interrogating GWAS data using comparative genomics and functional analysis using CRISPR genome editing in mice.

genetics↗

bioRxiv2019

CRISPR disruption and UK Biobank analysis of a highly conserved polymorphic enhancer suggests its role in anxiety and male alcohol intake.

Excessive alcohol intake is associated with 5.9% of global deaths. However, this figure is especially acute in men such that 7.6% of deaths can be attributed to alcohol intake. Previous studies identified a significant interaction between genotypes of the galanin (GAL) gene with anxiety and alcohol abuse in different male populations but were unable to define a mechanism. To address these issues the current study analysed the human UK Biobank cohort and identified a significant interaction (n=115,865; p=0.0007) between allelic variation (GG or CA genotypes) in the highly conserved human GAL5.1 enhancer, alcohol intake (AUDIT questionnaire scores) and anxiety in men that was consistent with these previous studies. Critically, disruption of GAL5.1 in mice using CRISPR genome editing significantly reduced GAL expression in the amygdala and hypothalamus whilst producing a corresponding reduction in ethanol intake in KO mice. Intriguingly, we also found evidence of reduced anxiety-like behaviour in male GAL5.1KO animals mirroring that seen in humans. Using bioinformatic analysis and co-transfection studies we further identified the EGR1 transcription factor, that is co expressed with GAL in amygdala and hypothalamus, as being important in the protein kinase C (PKC) supported activity of the GG genotype of GAL5.1 but less so in the CA genotype. Our unique study uses a novel combination of human association analysis, CRISPR genome editing in mice, animal behavioural analysis and cell culture studies to identify a highly conserved regulatory mechanism linking anxiety and alcohol intake that might contribute to increased susceptibility to anxiety and alcohol abuse in men.

genetics↗

bioRxiv2019

Genetic, epigenetic and pharmacological influences modulating tissue specific regulation of the cannabinoid receptor -1 (CB1) gene; implications for cannabinoid pharmacogenetics.

Cannabinoid receptor-1 (CB1) represents a potential drug target against conditions that include obesity and substance abuse. However, drug trials targeting CB1 (encoded by the CNR1 gene) have been compromised by differences in patient response. Towards addressing the hypothesis that genetic and epigenetic changes within the regulatory regions controlling CNR1 expression contribute to these differences, we isolated the human CNR1 promotor (CNR1prom) and demonstrate its activity in primary cells and transgenic mice. We also provide evidence of CNR1prom in CB1 autoregulation and its repression by DNA-methylation. We further characterised a conserved regulatory sequence (ECR1) in CNR1 intron 2 that contained a polymorphism in linkage disequilibrium with disease associated SNPs. Deletion of ECR1 from mice using CRISPR genome editing significantly reduced CNR1 expression in the hippocampus. These mice also displayed reduced ethanol intake and hypothermia response to CB1 agonism. Moreover, human specific C-allele variants of ECR1 (ECR1(C)) drove higher levels of CNR1prom activity in hippocampal cells than did the ancestral T-allele. We further demonstrate a role for the AP-1 transcription factor in driving higher ECR1(C) activity. In the context of the known roles of CB1 the current study suggests a mechanism through which ECR1(C) may be neuroprotective in the hippocampus against stress. The cell-specific approaches used in our study to determine the functional effects of genetic and epigenetic changes on the activity of tissue-specific regulatory elements at the CNR1 locus represent an important step in gaining a mechanistic understanding of cannabinoid pharmacogenetics.

genetics↗