A cytoskeleton is a central component of all cells, and is made of protein filaments that assemble into networks. These networks allows cells to divide, change shape as needed and perform a multitude of other vital functions. Microtubules (MTs), are essential cytoskeletal components composed of an elementary protein called tubulin. To fulfill its cellular function, the activity and level of tubulin must be maintained optimally by a process known as homeostasis. This process is not well understood, but is known to be particularly important for nervous system function. In fact, disruption of tubulin homeostasis can lead to neurological problems such as Huntington’s disease. Furthermore, because MTs are important in the uncontrolled division of tumour cells, tubulin represents an important target for cancer treatment. To improve our understanding of the fundamental principles guiding tubulin homeostasis, Dr. Melissa Frederic has undertaken research to identify and characterize proteins associated with the function, organization and maintenance of tubulin, using mainly C. elegans, a tiny worm, and mammalian tissue culture cells as model systems. One protein that will be characterized at the molecular and cellular levels, termed HECTD1, has been identified in her lab as a likely factor influencing tubulin homeostasis; importantly, it has also been linked to neural tube defects in a mouse system where the protein was removed. At the same time, Dr. Frederic is doing genetic screens to identify proteins that effect tubulin homeostasis, including one that utilizes the anticancer drug taxol or benzyl isothiocyanate. Together, the characterization of HECTD1 and the discovery and subsequent characterization of additional proteins implicated in tubulin homeostasis, are expected to shed new light on nervous system disorders such as neurodegeneration and neural tube defects, the most common congenital malformation in humans, as well as cancer.
Research Pillar: Biomedical
Influence of aging on candidate neuropsychiatric disease genes measured using differential coexpression
Aging and developmental change represent body wide changes in genes. Because many genes change as people age, the relationships between genes also often change, a phenomenon called differential coexpression (of RNA levels). Studying differential coexpression has uncovered changes that cause disease. However, knowledge gaps remain with respect to relationships between disease and aging in neurological diseases, for example. Many diseases have a specific age of onset, schizophrenia for example, typically strikes in early adulthood. This suggests that in multi-gene disorders, where interactions between genes play a role, rewiring may occur between susceptibility genes at the age of disease onset. Dr. Gillis’s current research project builds on his earlier work which showed that aging is associated with numerous changes in coexpression, and that genes known to be associated with specific diseases change their relationships with age in healthy individuals. His current project involves studying how the relationships between candidate genes – differential coexpression – in schizophrenia and Alzheimer’s Disease, change as a function of age. By understanding how networks of gene interactions might be rewired in diseases, we can identify candidate genes that would be missed otherwise, and beneficially influence the design of treatments and diagnostics.
Developing New Non-Invasive Optical Techniques for Detecting and Diagnosing Cancer
While cancer continues to affect thousands of Canadians, when detected at an early stage patients have a better chance of survival. Therefore, the development of sensitive diagnostic tools to enable early cancer detection and diagnosis is important. Dr. Anthony Lee is focusing his research efforts on the design and development of high resolution, non-invasive, in vivo optical imaging tools that will allow clinicians to perform so called ‘optical biopsies’ to detect and diagnose lung and skin cancers while the patient is being examined. Lung cancer is the leading cause of cancer mortality. The only reliable way to definitively diagnose the disease is to perform a lung biopsy for histological inspection by a pathologist. This technique is invasive and is associated with numerous problems. Dr. Lee’s Optical Coherence Tomography (OCT), is a technique that shows promise as a non-invasive diagnostic tool for lung cancer. Part of his project will be dedicated to developing a new OCT instrument designed specifically for use in patients’ lungs. OCT is similar in principle to ultrasound except that it uses light rather than sound as the imaging signal. It has higher resolution than ultrasound and sufficient penetration into tissue to examine the lung epithelial lining, where most cancers originate. The endoscopic probe being designed can image large segments of the bronchial tree in high resolution. Additionally, Dr. Lee is developing a Multiphoton Microscopy (MPM), instrument for use in diagnosing skin cancer, the most commonly diagnosed form of cancer. MPM has microscopic resolution and will be able to create 3-dimensional volumetric images of tissue. The results of Dr. Lee’s work will provide improved diagnostic tools to replace traditional biopsies which are time and resource intensive. Moreover, if cancer diagnoses can be confirmed in situ, immediate treatment becomes a possibility and may eliminate the need for subsequent patient visits.
Developing an innovative antibody-based nanopharmaceutical for treating cancer
Rituximab is an anti-CD20 monoclonal antibody (mAb), approved for use in combination with standard chemotherapeutic agents for treatment of patients with CD20-positive B cell lymphomas. Although it provides significant benefits for lymphoma patients, it is not curative, and for several specific forms of lymphoma, rituximab offers little or no benefit. To date, the mechanism(s) underlying the anti-tumour activity of this mAb in vivo are not clear. However, one area of particular interest is in activities that involve clustering of the CD20 molecule on the cell surface. Clustering of CD20 has been shown to elicit changes in cell signalling pathways that promote cell death, while enhancing sensitivity of lymphoma cell lines to cytotoxic agents. By better understanding this mechanism of antibody-induced tumour death it will be possible to determine the clinical basis for insensitivity to rituximab. Jesse Popov’s research is exploring this mechanism of activity by comparing a novel, highly active multivalent form of rituximab that he has developed, to the activity of rituximab. The results of his research will provide for improvements on the novel mAb he has developed and may also provide a possible therapeutic alternative to rituximab. Importantly, this novel agent can be made with any therapeutic antibody, not just rituximab, which means that it has the potential to be used for treating virtually any type of cancer. Such improvements over current therapies translate directly into a higher quality of life for cancer patients.
The Relationship Between the Immune System and the Normal Gut Microflora in Salmonella Typhimurium Infection: A Two Sided Tale
Understanding the role of the microbiota in the development and progression of diseases has received a great deal of attention in recent years. The microbiota is defined as the group of microorganisms, such as bacteria, which normally inhabit the human body. These microorganisms, also known as microflora, are composed of a variety of species of bacteria, each having a different function, and there are some bacteria whose functions remain unknown. Several studies have shown that patients with inflammatory bowel diseases, such as Crohn’s Disease, have a microbiota composition that is different from healthy individuals, suggesting that certain species of bacteria might be important in causing some common gut inflammatory disorders. Dr. Navkiran Gill is investigating how the human immune system regulates the microbiota and how our microbiota may direct our immune responses to various pathogens. Specifically, she is doing a series of experiments involving antibiotic use in specially bred mice infected with Salmonella. The results will provide important information regarding the effect of antibiotics on the microflora, and allow her to correlate changes in our microflora to changes in our ability to mount an immune response against a pathogen such as Salmonella. The results of Dr. Gill’s research will provide information that may be used to design new therapeutics that take into consideration the important role of our microflora.
The role of Raf-1 in pancreatic beta cell survival and insulin signaling
While we know that Type 1 diabetes is caused by the destruction of insulin-secreting beta-cells in the pancreatic islets, the processes that regulate beta-cell death remain unclear. This has hindered the development of strategies to halt or prevent the development of diabetes. One possible new treatment, islet transplantation, was initially heralded as a promising therapy for Type 1 diabetes because it removed the need of daily insulin injections. However, the transplanted beta-cells were found to gradually die, which resulted in transplant recipients having to resume the use of insulin injections. In order for islet transplantation to be effective, new approaches to promote islet survival are required. In earlier work, Dr. Gareth Lim’s colleagues identified insulin as a critical pro-survival factor for beta-cells. Their findings suggest that secreted insulin from beta-cells may promote self-survival. However, the mechanisms that lead to the beneficial effects of insulin need to be clarified. Consequently, Gareth Lim is currently investigating the mechanisms by which insulin acts on the beta- cell. Specifically, he is doing a series of experiments to show that the protein Raf-1 kinase, which is activated by insulin and has been shown to have an important role in regulating cell death, is essential for beta-cell survival. The results of his studies will improve our understanding of the mechanisms of beta-cell death. They may also lead to novel therapeutic strategies for preventing beta-cell destruction in Type 1 diabetics and their at-risk relatives. Furthermore, an understanding of the pathways involved in beta-cell survival may also lead to new methods to increase the survival of beta-cells after islet transplantation, thereby increasing the effectiveness of this treatment.
Defining the Transcription Factors Capable of Forming Pancreatic Beta-Cells from Human Embryonic Stem Cells
In Type 1 diabetes the body’s immune system attacks key cells of the pancreas known as beta-cells. The loss of these cells results in a loss of the protein hormone insulin which is secreted by the beta-cells in response to high blood sugar levels. Recently, advances in the field of pancreatic islet cell transplantation have shown that the replacement of beta-cells represents a possible cure for diabetes. Unfortunately, the poor availability of donor organs to provide the transplantation cell source greatly limits the use of this treatment. One promising possible source of new beta-cells for transplantation is human embryonic stem cells (hESCs,) and a number of researchers have shown that these cells can form pancreatic tissue including beta-cells. Blair Gage is currently exploring how proteins known as transcription factors (TFs), control the formation of beta-cells from hESCs. Specifically, he is investigating whether adding TFs, which help in the formation of beta-cells, and removing the TFs, which block the formation of beta-cells, will help in understanding how to control hESC growth and development. The results of Mr. Gage’s research will enhance ongoing work with industry and the Canadian Stem Cell Network that is focused on stimulating hESCs to form beta-cells for transplantation. The ultimate goals is to apply this technology to the treatment of patients with diabetes in a similar way to that of islet tissue transplantation, using a theoretically limitless supply of beta-cells.
The role of Apical Junction Complex in airway epithelial repair and differentiation in asthma
Asthma is a serious global health problem, affecting over 300 million people worldwide. The disease is predominantly an inflammatory disorder of the conducting airways, and can be treated or controlled using current therapies. However, un-controlled asthma leads to continual inflammation and damage, resulting in permanent scaring which is termed airway remodeling. Airway remodeling can be defined as changes in the composition, content and organization of cellular and molecular constituents of the conducting airways. One of the structural changes that occurs as a result of airway remodeling is detachment of the cells that line the surface of the airways, called the epithelium. In normal airways, the epithelium forms a barrier against the inhaled external environment which includes aero allergens, viruses and particulate matter, through the formation of apical junction complexes (AJCs). In asthma, part of the abnormal response to inhaled allergens is thought to be due to impaired barrier function caused by damage to the airway epithelium and loss of AJCs. Emerging evidence suggests that AJCs are able to influence other aspects of epithelial function such as release of inflammatory mediators and mechanisms of epithelial repair. Building on earlier work in this area, Dr. Tillie-Louise Hackett’s current research is designed to determine whether AJCs play an important role in normal airway epithelial repair and if the mechanisms involved are altered in asthmatic patients. The results of her research will provide scientists and clinicians with a better understanding of the pathological mechanisms that contribute to multiple respiratory diseases. In addition, Dr. Hackett’s findings will open avenues for the development of new therapeutics to improve the lung health of Canadians living with obstructive lung diseases, such as asthma and Chronic Obstructive Pulmonary Disorder.
Identification of alternative BACE1 and BACE2 substrates and affected pathways in neuroinflammation and Alzheimer's disease
Alzheimer’s disease (AD) is the most common neurodegenerative disease in humans, affecting millions of people worldwide. Currently, there is no cure for AD or treatment that can mitigate the disease process. However, recent research has revealed beta-site amyloid precursor cleaving enzyme 1 (BACE1), as a promising therapeutic target for AD. BACE1 is the protease that cuts Amyloid Precursor Protein (APP) at the beta-site. This cleavage of APP triggers a second cleavage, which releases the Amyloid-beta fragment. Accumulation of Amyloid-beta is believed to initiate the catastrophic cascade of events that lead to the onset of AD. Animal data suggest that BACE1 inhibition prevents Amyloid-beta formation and may be well tolerated, and therefore, BACE1 is considered one of the most promising drug targets for preventing and mitigating AD. However, recent work has revealed that APP is not the only substrate of BACE1. Consequently, drug-targeting strategies will modulate processing of both known and unknown substrates, any one of which may lead to undesirable or deleterious side effects. Therefore, the identification of all BACE1 substrates is necessary to predict and understand side effects of BACE1 inhibitors. Pitter Huesgen is working to identify new substrates and pathways modified by BCAE1 and the related enzyme, BACE2. His results will provide essential information on the complex physiological functions of BACE1 and BACE2 and their roles in the pathogenesis of AD, and help to predict side effects of BACE1 inhibitors, which will ultimately decide if BACE1 inhibition is a viable treatment strategy for AD.
Mapping phosphorylation pathways to discover host signaling events induced by Salmonella.
Responding and adapting to environmental changes is crucial to the survival of all living organisms, including cells. Cells use signaling cascades to detect stimuli in their environment and respond by altering the expression and turnover of specific genes and proteins. Since many signaling events are regulated by the addition of a phosphate to serine, threonine, or tyrosine residues on proteins within these cascades, identifying and characterizing these modifications is crucial to understanding how signalling pathways function. Until very recently, studying protein phosphorylation has been a slow and laborious process, as existing techniques limited researchers to studying only a few phosphsphorylation sites in isolation. However, the recent emergence of highly sensitive techniques in liquid chromatography-tandem mass spectrometry (LC-MS/MS), has enabled scientists to analyze thousands of phosphorylated proteins simultaneously. Lindsay Roger’s research utilizes LC-MS/MS to analyze thousands of protein phosphorylation events simultaneously in cells infected by Salmonella. Salmonella is an intracellular bacterial pathogen which, in humans, causes gastroenteritis and typhoid fever and is one of the most common and widely distributed food borne illnesses. During infection, Salmonella use a needle like complex to transport bacterial proteins, termed effectors, into host cells where they mimic host proteins and influence signalling. Currently, little is known about the host targets of the majority of Salmonella effectors and how they cause disease. Using these LC-MS/MS experiments, Ms. Rogers’s research is identifying a myriad of novel host targets of these proteins. It is expected that this research will provide a considerable leap in our understanding of how Salmonella infects its host.