A Brief Description of Currently Funded Research Grants
2011-2012
Protective effects of docosahexaenoic acid in a mouse model of neuroinflammation
Dr. Richard Bazinet
University of Toronto
Toronto, ON
Purpose of the research: Brain inflammation is associated with many brain disorders including Alzheimer’s disease. Omega-3fatty acids from fish are known to decrease inflammation in the body. However, if they can also decrease inflammation in the brain is not known. We have used several chronic genetic and dietaryapproaches in mice to increase brain levels of omega-3 fatty acids. Furthermore, we have found that asthe level of one omega-3 fatty acid in the brain (known as DHA) increases, levels of inflammation decrease in the brain. Because it takes a long time to change brain DHA levels with genetic or dietaryapproaches, we then added DHA back to the brain acutely to see if it would be directly protective against inflammation. Indeed, we found that acute administration of DHA to the brain was protective.However, we also found that a metabolite of DHA was protective. This is interesting, but adds a level of complexity to our project. It is possible that DHA is converted to this metabolite and that is what isprotective and not DHA per se. In this study we want to administer DHA or its metabolite directly tothe brain of mice lacking the enzyme that converts DHA to the metabolite. This will tell us if the metabolism of DHA is critical for its ability to decrease neuroinflammation.
Proposed research plan: We are proposing three studies. In study one we want to test another metabolite of DHA (called PD1) tosee if it is also protective in the brain. Our study design will be the same as our previous “acute” studies. At 12 weeks of age, we will inject the brain of mice with an agent that causes inflammationthen we will administer PD1 directly to the brain to see if it decreases inflammation 24 hours later.The second study will be very similar to the first, except we will administer DHA or one of theprotective metabolites in mice that are missing the key enzyme (commonly called knockout mice) forproduction of the metabolites. This will tell us if the metabolite is critical for the protective effect ofDHA.In the third study we will crossbreed our genetic mice (that produce high brain levels of DHA)with mice missing the key enzyme. In the past we showed that these genetic mice were highly protectedagainst brain inflammation. This study will tell us if the protection in these mice was due to the enzymethat metabolizes DHA.
Anticipated outcome: We will identify if DHA or its metabolites are protective against inflammation. Right now we canconfidently say that brain DHA is protective against neuroinflammation, but we do not yet know if itneeds to be converted to specific metabolites to be protective.
Projected benefits and applications of findings: If we find that DHA itself is protective people could consume more fish oil to prevent diseases in whichbrain inflammation is problematic. Conversely, if we find that a metabolite of DHA is protective wecould develop drugs to increase the level of this metabolite in the brain or make more stable analoguesof it. So far the one metabolite we have found to be protective worked at a dose of one micro gram (1millionth of a gram) delivered over 24 hours. These are highly potent compounds that could stimulate alot of new approaches to treating neuroinflammation.
Evaluation of the neuroprotective effects of pyruvate dehydrogenase kinase-1 in an Alzheimer's disease mouse model
Dr. Robert Cummings
University of Western Ontario
London, ON
Purpose of the research: Alzheimer’s Disease (AD) is an irreversible, progressive brain disorder that slowly destroys memory,verbal and thinking skills in the elderly. An estimated 300,000 Canadians over the age of 65 presentlysuffer from AD; a number that is expected to double over the next 25 years. Currently there is no cure for the disease and treatment remains limited. AD is characterized by the accumulation of abnormalproteins and widespread nerve cell death within the brain of affected individuals. In particular, the presence of plaques containing an aggregated form of a small protein called amyloid β-peptide (Aβ) isa hallmark feature of AD. Aβ accumulation is strongly believed to be one of the principle factorscausing nerve cell loss in the brains of AD patients. However, up to 30% of elderly individuals havewidespread Aβ containing plaques within their brains but show no overt symptoms of dementia. One possible explanation for this observation is that some individuals have an innate resistance to thetoxic effects of Aβ. However, this hypothesis has never formally been examined. Recent studies in my lab have shown that cultured nerve cells selected for resistance to Aβ undergo a switch inmetabolism. Specifically, Aβ-resistant nerve cells break down sugar (glucose) in a manner reminiscentof cancer cells. By using this unique metabolism, nerve cells repress the activity of mitochondria (anenergy producing organelle) and prevent the formation of harmful oxygen molecules (free radicals);toxins known to be elevated in AD brains. One of the key enzymes that promotes this protectivemetabolic effect is pyruvate dehydrogenase kinase 1 (PDK1). Interestingly, Aβ-resistant nerve cellsand mice that are genetically engineered to develop Aβ-containing plaques have elevated levels of thePDK1 enzyme. High levels of PDK1 may explain why these mice have limited nerve cell death, eventhough they accumulate pronounced levels of plaques. In contrast, we have found that levels of PDK1are lower in brain tissue from AD patients compared to normal patients. These findings suggest thatincreased levels of PDK1 may play a key role in protecting the brain from the highly toxic effects ofAβ. In this study we will test the hypothesis that elevated PDK1 expression and activity in an ADmouse model is required to protect brain cells from the deadly effects of Aβ accumulation.
Proposed research plan: The central hypothesis will be evaluated by the following specific aims:i) To determine if elevated levels of the PDK1 protein are found in surviving neurons of AD mice.Immunological staining of AD mouse brain sections will be performed to determine if elevated PDK1levels are specifically found in nerve cells that are in close proximity to Aβ containing plaques.ii) To determine if chemically inhibiting PDK1 renders brain cells in AD mice more susceptibleto mitochondrial dysfunction and Aβ-induced death. Inhibition of PDK1 will be achieved byadding the PDK specific inhibitor dichloroacetate (DCA) to the drinking water of AD mice followedby the analysis of brain tissue over time to detect the features of cell death. Comparisons will be madeto mice not fed DCA.iii) To determine if chemical inhibition of PDK1 further impairs memory in AD mice. DCA fedmice will also be examined for memory loss by standard visual perception of spatial relationships test.
Anticipated Outcome: It is anticipated that elevated PDK1 activity strongly correlates with increased survival of nerve cells inAD mice. In contrast, chemical inhibition of PDK1 in AD mice will lead to a suppression of thisprotective effect and hasten both nerve cell loss and memory decline.
Projected benefits and applications of findings: This study will reveal for the first time that elevated PDK1 expression confers an innately protective response in the brain that counters the toxic effect of Aβ. Loss of this protective effect may renderelderly individuals susceptible to AD. Future studies using advanced brain imaging techniques tomeasure the loss of PDK1 controlled metabolism associated with Aβ-resistance may provide apredictive indicator of disease progression. Moreover, investigation of compounds that either mimic orpromote PDK1 activity in the brain and may offer a new therapeutic avenue for the treatment of AD.
Genetics of Cryptogenic Infantile Spasms
Dr. Else Rossignol
Hôpital Ste. Justine
Montreal, PQ
Introduction: Infantile spasms (IS) is one of the catastrophic epilepsies of childhood and affects 2.5 in 10 000 children.IS ischaracterized by the apparition of a specific type of seizures (spasms), usually during the first year oflife, accompanied by characteristic abnormal brain wave activities on electroencephalograms (EEG).They can appear in the context of severe diffuse brain injuries (ex birth asphyxia, strokes) or diffuse brainmalformations. When such a cause is identified, IS are considered symptomatic. By contrast, when noapparent cause is found on brain imaging or biochemical testing, IS are considered cryptogenic. Bothsymptomatic and cryptogenic IS often lead to severe neurological and cognitive impairment, includingmental retardation and autism. They are resistant to usual anticonvulsant medications, but they respondrelatively well to vigabatrin or ACTH. Early seizure control (within a month) without relapse tends tolead to a better prognosis in term of development and mental abilities later on. However, it is impossibleat this point to predict which patient will respond better to which drug, or how they will evolve over time.These are critical questions for the treating physicians and families. The identification of some of theunderlying mechanisms that cause IS might help us predict how given subsets of patients will respond totreatment and foster the development of new drugs more specific for these underlying defects.
Objective: We hypothesize that genetic mutations affecting the development or function of specific brain cells (neurons) and their circuits underlie a majority of cryoptogenic cases of IS. Therefore, identifyingthese genetic causes will clarify some of the underlying mechanisms involved in IS and help usunderstand why some patients respond better to one treatment than another. To test this hypothesis, we propose to study the genetic causes of IS in a wide pan-Canadian cohort of patients using state-of-the-art genome-wide sequencing techniques. Our objective is to identify new genetic mutations causing IS.3. Outline:Our collaborators (L. Carmant and CPEN) recently conducted a pan-Canadian study to treat and followchildren with IS to assess the efficiency of a new neuroprotective drug (flunarizine) on the outcome of IS. This study identified 69 children with IS between 2003-2006, including 28 with cryptogenic IS. We are currently recruiting these families for genetic analysis, and already have collected DNA samples from 8 families (8 patients and their parents). We will perform whole-exome sequencing of these 8 trios to lookfor new mutations in patients that are not inherited from their unaffected parents and are not found incontrol subjects. We will assess if these new variants are indeed disease-causing mutations by their nature, the way they are predicted to affect gene function (i.e. protein truncation is likely pathogenic), andthe particular genes involved (i.e. mutations that disrupt genes known to be important for neuronal development will be considered likely pathogenic). This technique will enable us to look at all the codingregions of all the genes of the human genome in each of these individuals, allowing us to identify raremutations in different genes. Such techniques have revolutionized the study of rare geneticall yheterogeneous diseases and have already allowed one of us to identify new mutations in patients withintellectual disability (J. Michaud). Our center has considerable expertise in neurogenetics (E. Rossignoland J. Michaud) and works closely with the Réseau de Médecine Génétique Appliquée (RMGA, G.Rouleau), which puts us in a good position to complete the project described here. Furthermore, we have access to a large well-characterized group of children with IS (L. Carmant and CPEN).
Projected benefits and applications: The proposed experiments will allow us to find new genetic causes of IS. This will help clarify some ofthe mechanisms underlying IS. In turn, this might trigger the development of new drugs to treat IS andimprove neurological outcome for this severe form of epilepsy. For instance, finding new mutations ingenes that are important for the function of specific inhibitory brain cells might promote the developmentof new drugs aimed at reinforcing this specific type of inhibition. Furthermore, we have developed anexpertise in the neurobiology of epilepsy using genetically engineered mouse models of human disease(E. Rossignol). This expertise will prove useful in the future to investigate the role of these new mutationsin causing IS. Finally, although IS is a rare disease, some of these findings might become relevant forother more common forms of epilepsy which might also affect similar genetic pathways.
Structural and Functional Correlates
Dr. Christine Till
York University
Toronto, ON
Introduction: Multiple Sclerosis (MS) is achronic demyelinating neurological disease that is increasingly being diagnosed in children. Canada has aprevalence rate of MS that is among the highest worldwide, affecting 250 per 100,000, with 5-15% of these individuals diagnosed prior to age 18. Cognitive impairment occurs in 30-50% of youth with MS, and can negatively impact academic and intellectual abilities as skills fail to develop at an appropriate rate. While severity of deficits progress over time, it is difficult to predict impairment on the basis of clinical factors alone, such as disease duration, frequency of relapses, or physical disability. Studies on childhood MS show minimal correlation between cognitive deficits and these factors, suggesting that cognitive dysfunction can be present early in the disease and in the absence of physical disability. The proposed study will examine factors associated with cognitive performance in childhood-onset MS patients with aparticular focus on whether functional reorganization of brain networks preserves cognition in some individuals, despite the accrual of multiple lesions during the course of the disease.
i)To investigate whether childhood-onset MS patients produce aJess efficient pattern of cerebral activation compared with healthy controls. We will examine whether cerebral activation is increased amongst patients with preserved cognition compared with patients identified as cognitively impaired. ii)To determine whether cognitively impaired MS patients who were older at disease onset show greater recruitment of prefrontal cortical regions owing to the potential for more available compensatory mechanisms relative to patients with ayounger age of MS onset
Outline of research: We will study 35 patients recruited from SickKids hospital with pediatric-onset relapsing-remitting MS and 15 healthy controls, aged 14-25 years. All participants will complete aneuropsychological test battery, as well as measures assessing demographics, mood, and fatigue. On the same day, participants will undergo magnetic resonance imaging (MRI) in ahigh-strength 3 Tesla scanner. The structural MRI indices will include: T1 and T2 lesion volume, measures of white matter integrity and regional and global brain volumes, using established protocols as used in our prior work. Functional MRI (fMRI) techniques will assess cerebral activity and behavioural performance (reaction time, accuracy) on paradigms designed to assess executive control. To achieve Aim 1, cognitive performance will first be evaluated. Based on our prior evaluations of these patients, we expect approximately one third of the MS group to have cognitive impairment. We will then compare the extent of cerebral activation (blood oxygen level dependent, or BOLD signal) among the cognitively impaired and non-impaired groups to assess whether the non-impaired group produces an expanded neural network (considered to be less efficient), particularly during tasks with high cognitive demand. These findings would support the hypothesis that compensatory strategies (or possible cortical reorganization) contribute to preserved cognition in MS. For Aim 2, we will compare extent of cerebral activation in cognitively impaired individuals with young vs. older disease onset to determine whether compensatory strategies (defined as increased recruitment of cerebral resources) are more likely in the older disease-onset group. Analyses will consider additional factors, such as IQ, socioeconomic status, lesion burden, and regional and global brain volume, as potential moderators of cerebral activity.
Projected benefits and application of findings: This comprehensive neuroimaging evaluation will expand upon our prior work by providing new insights into the neural substrate of cognitive impairment in patients with childhood-onset MS. If functional reorganization of the brain occurs in these patients, this may explain why some individuals are able to withstand more severe neuropathology than others before showing cognitive impairment -crucial to advancing the development and monitoring of neurorehabilitation strategies in these individuals.