Characterization of Interacting Partners of ABC Lipid Transporters

This thesis outlines a series of experimental approaches that aim to investigate the roles of the co-factor Alix (apoptosis-linked gene 2-interacting protein X), the deubiquitinase USP9X (Ubiquitin-specific protease 9, X-linked), and sterols, including cholesterol synthesis intermediates and oxysterols, in the post-translational regulation of ATP-binding cassette (ABC) lipid transporters, ABCG1 and ABCG4. Alix and USP9X were identified in our laboratory using peptide mass spectrometry analysis as binding partners of ABCG1. The experiments presented in the following chapters characterize the effects of Alix, USP9X as well as sterols on the ABC transporters’ protein expression. Chapter 1 provides a general introduction about atherosclerosis and the important roles of ABC lipid transporters, including ABCA1 and ABCG1, in mediating cellular cholesterol balance through the reverse cholesterol transport (RCT) pathway. It presents also a general overview of Alzheimer’s disease (AD), and the suggested roles of the ABCA1, ABCG1 and ABCG4 transporters on AD progression. In addition, it describes the transcriptional and post-translational regulatory pathways of ABCA1, ABCG1 and ABCG4. This Chapter also outlines the roles of oxidized metabolites of cholesterol, i.e.‘oxysterols’, in atherosclerosis and AD. As an additional topic, it describes the roles of E3-ubiquitin ligases as well as deubiquitinases on the stability of proteins, such as the ABC lipid transporters, through the ubiquitin proteasomal pathway (UPS). Finally, this Chapter highlights the potential for therapeutic targeting of the UPS, and describes the hypotheses and aims of this thesis. In Chapter 2, materials and methods that are common to all the data chapters as described, including cell culture, optimization of siRNA transfection, preparation of cell lysates, SDS-PAGE and Western blotting, immunoprecipitations and cholesterol efflux experiments. Chapter 3 describes experiments investigating the role of the adaptor protein, Alix, as a potential cofactor between the E3-ubiquitin ligase NEDD4-1 (Neural precursor cell-expressed developmentally down regulated gene 4) and its target, ABCG1. NEDD4-1 is a HECT-domain E3-ubiquitin ligase that was previously identified in our laboratory as a regulator of ABCG1/ABCG4 protein expression (Aleidi, Howe et al. 2015). The experiments presented in this Chapter demonstrate that Alix and NEDD4-1 are co-expressed in a number of brain cell lines including astrocytes and neurons where ABCG1 is expressed. ABCG1 protein levels were significantly increased after knockdown of Alix when ABCG1 overexpressing CHOK1 cells were incubated only with full serum media (i.e. high cholesterol content). Interaction of Alix, NEDD4-1 and ABCG1 was established using a co-immunoprecipitation (co-IP) approach. These observations confirm that Alix is a novel player in regulating ABCG1 expression and facilitates the interaction between NEDD4-1 with ABCG1. Chapter 4 describes experiments investigating the role of Alix in the NEDD4-1 mediated regulation of ABCG4 protein, a close relative of ABCG1. Unlike ABCG1 that was increased after Alix knockdown only when the cells were maintained in full serum media (Chapter 3), Alix knockdown resulted in an upregulation of ABCG4 expression when ABCG4 overexpressing CHOK1 cells were incubated with full as well as low serum media (i.e. high and low cholesterol content). Like ABCG1, Alix and NEDD4-1 were co-immunoprecipitated with ABCG4 using a co-IP approach. These observations confirm that Alix regulates ABCG4 expression by being a potential co-factor between NEDD4-1 and ABCG4. Chapter 5 demonstrates experiments examining the effect of deubiquitinases (DUBs) and specifically USP9X, on ABCG1 and ABCG4 protein levels. Firstly, experiments presented indicated that the use of the general DUB inhibitor PR-619 produced toxic side effects on our cell models. However, treatment of these cells with WP1130, which is a more selective inhibitor for specific DUBs, including USP9X, significantly reduced ABCG1 protein levels in ABCG1 overexpressing CHOK1 cells. Further experiments described the effect of USP9X knockdown on ABCG1 protein levels, however these experiments were complicated by the fact that inconsistent results were found between three independent USP9X siRNA primers, each being effective in reducing USP9X protein expression. Overall, these experiments indicated a role for deubiquitinases in the regulation of ABCG1 protein expression, but were not able to confirm that this was solely due to USP9X. Chapter 6 outlines experiments investigating the role of sterols, including cholesterol, oxysterols and cholesterol synthesis intermediates in the regulation of ABCG1 and ABCG4. Incubating ABCG1 overexpressing CHOK1 cells with cholesterol, oxysterols and cholesterol synthesis intermediates enhanced ABCG1 protein levels, with the highest increase after the addition of Liver X Receptor (LXR) ligands, specifically 24S-OH. However, ABCG4 stabilization was enhanced when ABCG4 overexpressing CHOK1 cells were incubated with cholesterol as well as cholesterol synthesis intermediates, including desmosterol, lathosterol and lanosterol. Consequently, ABCG1 and ABCG4 are regulated differently at the post-translational levels by oxysterols and cholesterol synthesis intermediates. The toxicity of sterols on cells, assessed using a variety of methods, made overall conclusions complicated and indicated that further experiments are warranted. In conclusion, the work presented in this thesis shows promising regulators of cholesterol homeostasis in cells. It describes newly discovered roles for the cofactor Alix, inhibition of deubiquitinases, and sterols in the regulation of ABCG1 and ABCG4 protein expression. Unravelling these pathways by which cells can fine-tune their lipid transporter expression and hence cellular lipid levels provides avenues in future to find novel therapeutic targets for treatments in the setting of atherosclerosis and AD

Autonomic brain functioning and age-related health concerns

The autonomic nervous system (ANS) regulates involuntary bodily functions such as blood pressure, heart rate, breathing, and digestion, in addition to controlling motivation and behavior. In older adults, the ANS is dysregulated, which changes the ability of the ANS to respond to physiological signals, regulate cardiovascular autonomic functionality, diminish gastric motility, and exacerbate sleep problems. For example, a decrease in heart rate variability, or the variation in the interval between heartbeats, is one of the most well-known alterations in the ANS associated with health issues, including cardiovascular diseases and cognitive decline. The inability to perform fundamental activities of daily living and compromising the physiological reactivity or motivational responses of older adults to moving toward or away from specific environmental stimuli are significant negative consequences of chronic and geriatric conditions that pose grave threats to autonomy, health, and well-being. The most updated research has investigated the associations between the action responsiveness of older adults and the maintenance of their physiological and physical health or the development of mental and physical health problems. Once autonomic dysfunction may significantly influence the development of different age-related diseases, including ischemic stroke, cardiovascular disease, and autoimmune diseases, this review aimed to assess the relationship between aging and autonomic functions. The review explored how motivational responses, physiological reactivity, cognitive processes, and lifelong developmental changes associated with aging impact the ANS and contribute to the emergence of health problems

The Adaptor Protein Alix is Involved in the Interaction Between the Ubiquitin Ligase NEDD4-1 and its Targets, ABCG1 and ABCG4

Several ATP-Binding Cassette (ABC) transporters, including ABCG1 and the related ABCG4, are essential regulators of cellular lipid homeostasis. ABCG1 is expressed ubiquitously and is functional in the context of atherosclerosis. However, ABCG4 is expressed almost exclusively in brain and has been linked to Alzheimer’s disease (AD). These transporters are highly regulated post-translationally by E3 ubiquitin ligases, with the ligase NEDD4-1 (Neural precursor cell-expressed developmentally downregulated gene 4) implicated in their protein stability. In this study, we investigated interacting partners of ABCG1 using peptide-mass spectrometry and identified the potential adaptor protein, Alix (apoptosis-linked gene 2-interacting protein X). In this paper, we hypothesized and investigated whether Alix could facilitate the interaction between NEDD4-1 and the ABC transporters. We showed that Alix and NEDD4-1 proteins were co-expressed in several commonly used cell lines. Knockdown of Alix in cells overexpressing ABCG1 or ABCG4 increased transporter protein expression while co-immunoprecipitation experiments showed interaction between NEDD4-1, Alix, and ABC transporters. In summary, we provide evidence that Alix serves as a co-factor for the interaction between the E3-ubiquitin ligase NEDD4-1 and the ABC transporter targets, ABCG1 and ABCG4

Regulation of ABCG4 transporter expression by sterols and LXR ligands

Background: Oxysterols, which are derivatives of cholesterol produced by enzymic or non-enzymic pathways, are potent regulators of cellular lipid homeostasis. Sterol homeostasis in the brain is an important area of interest with regards to neurodegenerative conditions like Alzheimer's disease (AD). Brain cells including neurons and astrocytes express sterol transporters belonging to the ABC transporter family of proteins, including ABCA1, ABCG1 and ABCG4, and these transporters are considered of interest as therapeutic targets. Although regulation of ABCA1 and ABCG1 is well established, regulation of ABCG4 is still controversial, in particular whether the transporter is an Liver X receptor (LXR) target. ABCG4 is thought to transport cholesterol, oxysterols and cholesterol synthesis intermediates, and was recently found on the blood brain barrier (BBB), implicated in amyloid-beta export. In this study, we investigate the regulation of ABCG4 by oxysterols, cholesterol-synthesis intermediates and cholesterol itself. Methods: ABC transporter expression was measured in neuroblastoma and gliablastoma cell lines and cells overexpressing ABCG4 in response to synthetic LXR ligands, oxysterols and cholesterol-synthesis intermediates. Results: In contrast to previous reports, ABCG4 expression was induced by a synthetic LXR ligand in U87-MG astrocytes but not in neuroblastoma and BBB endothelial cell lines. In addition, ABCG4 protein was stabilized by cholesterol as was previously shown for ABCG1. ABCG4 protein was furthermore stabilized by cholesterol-synthesis intermediates, desmosterol, lathosterol and lanosterol. Conclusions: These results identify new aspects of the post-translational control of ABCG4 that warrant further exploration into the role of this transporter in the maintenance of sterol homeostasis in the brain

Isorhamnetin Reduces Glucose Level, Inflammation, and Oxidative Stress in High-Fat Diet/Streptozotocin Diabetic Mice Model

Background: Isorhamnetin is a flavonoid that is found in medical plants. Several studies showed that isorhamnetin has anti-inflammatory and anti-obesity effects. This study aims to investigate the anti-diabetic effects of isorhamnetin in a high-fat diet and Streptozotocin-(HFD/STZ)-induced mice model of type 2 diabetes. Materials and Methods: Mice were fed with HFD followed by two consecutive low doses of STZ (40 mg/kg). HFD/STZ diabetic mice were treated orally with isorhamnetin (10 mg/kg) or (200 mg/kg) metformin for 10 days before sacrificing the mice and collecting plasma and soleus muscle for further analysis. Results: Isorhamnetin reduced the elevated levels of serum glucose compared to the vehicle control group (p p p p > 0.01). LDL, triglyceride, and cholesterol were reduced in diabetic mice treated with isorhamnetin compared to vehicle control (p p p p < 0.05) in diabetic mice compared to vehicle control. Conclusions: Isorhamnetin ameliorates insulin resistance, oxidative stress, and inflammation. Isorhamnetin could represent a promising therapeutic agent to treat T2D

Perspective of biopharmaceutics knowledge and practice of pharmacy personnel toward the effect of medication route and medical procedure on nullifying fasting

Background: In Ramadan, most of the dosing schedules for the patients are changed, and to ensure patient compliance to medications and to healthy life among patients, appropriate guidelines and educations are needed. This can be achieved by pharmacy personnel in all clinical settings who are recognized as biopharmaceutical experts and integral educators of medications. Aims: This study aimed to identify the perspective knowledge of pharmacy personnel about effect of medication route and medical procedure on nullifying fasting in Ramadan and to determine the predictors of this knowledge. Methods: A cross-sectional study was conducted in Jordan during March-April 2022. An internet-based self-administrated questionnaire on knowledge, and views was distributed using social media groups to the pharmacy personnel among different geographical areas in Jordan. A descriptive and univariate analysis were performed. Binary logistic regression was conducted to determine the predictors of knowledge including all variables with p < 0.20 on univariate analysis. Results: A total of 1003 responses to the study questionnaire were collected and included in the analysis. The most common source that pharmacy personnel used to get information on medication intake and medical procedures during fasting in Ramadan was Fatwa (57.8%) followed by Islamic materials “books and brochures” (47.1%). The majority of respondents were knowledgeable about the effect of administration route of medication and medical procedures on nullifying fasting in Ramadan (greater than70%). The univariate analysis showed that more than half of respondents (56.1%) were considered knowledgeable, and the binary logistic regression analysis identified that both professional degree type and confidence of respondents to modify the patient's medication schedule as predictors for knowledge (OR = 1.791, 95% CI = 1.035–3.098, p = 0.037), (OR = 1.375, 95% CI = 1.04–1.817, p = 0.025), respectively. Conclusions: Most of pharmacy personnel in Jordan are knowledgeable in biopharmaceutics principles and practice toward effect of medication route and medical procedure on nullifying fasting, and the identified predictors for this knowledge, can provide an opportunity to improve safe and effective use of medications and medical procedures during the holy month of Ramadan