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HOME ABOUT US STAFF FAD REGISTRY RESEARCH PUBLICATIONS	RESEARCH PARTICIPATION AND CLINICAL TRIALS People interested in participating in research on the mechanisms and causes of Alzheimer Disease, Parkinson Disease, Fronto-temporal dementia, Pick Disease and other neurodegenerative diseases (e.g. people interested in participating in genetic studies, brain donation, etc) can send enquiries to crnd.secr@utoronto.ca or to crnd.admin@utoronto.ca. People interested in participating in future clinical trials of AZD-103 (scyllo-cyclohexanehexol) should contact Soraya Centeno at Transition Therapeutics (scenteno@transitiontherapeutics.com 416-263-1213). PRINCIPAL INVESTIGATORS AND AREAS OF RESEARCH DR. CATHERINE BERGERON DR. PAUL FRASER DR. ANTHONY LANG DR. JOANNE MCLAURIN DR. HOWARD MOUNT DR. EKATERINA ROGAEVA DR. PETER ST GEORGE-HYSLOP DR. ANURAG TANDON DR. DAVID WESTAWAY DR. GEROLD SCHMITT-ULMS DR. JANICE ROBERTSON DR. CATHERINE BERGERON My laboratory focuses on the neuropathology of human neurodegenerative diseases at both the basic and clinical levels. Over the past year my lab has concentrated on the pathological diagnosis and molecular classification of 61 Canadians who have died of suspect Creutzfeldt Jakob disease (CJD). Each case is examined using traditional neurohistological methods as well as immunohistochemistry for the prion protein. Frozen brain tissue was available for molecular characterization on 18 cases. CJD was found in 30 patients or 50% of suspect cases, a figure consistent over the past 8 years of our surveillance program. The most frequent cause of misdiagnosis was AD in 10 cases or 16%. Other conditions encountered include frontotemporal degeneration (3), diffuse Lewy body disease (3), metabolic encephalopathy (6), encephalitis (2), cerebellar degeneration (2), dentato-rubro-pallido-luysian degeneration (l), argyrophilic grain disease (1) and lymphoma (1). The remaining two cases were labeled as “not CJD”. One case consisted of brain tissue from a living patient suspect of suffering from CJD, obtained at craniotomy for a head injury 2 years prior to the onset of dementia. The other case showed no evidence of CJD, but no other significant pathological changes. The presence of some immune cells raises the possibility of Hashimoto’s encephalopathy. Frozen tissue was available for molecular testing on 18 cases, a combination of codon 129 analysis and prion banding pattern. The following CJD phenotypes were identified: 12 patients were found to have the MM1 phenotype characterized by rapid progressive dementia with myoclonus. Cerebellar phenotypes of longer durations were also represented (VV2 x 2 and MV2 x 1). A very unusual phenomenon this year was the detection of 3 patients (16% of the cases analyzed) with the MM2 phenotype. This phenotype is only rarely observed and accounts for only 4% of all sporadic CJD. All three patients had a long disease duration of over one year. DR. PAUL FRASER Research in my laboratory focuses on the biochemistry and biophysics of amyloid plaques and their relationship to sporadic and familial forms of Alzheimer’s disease. Plaques are a principal pathological feature of Alzheimer’s disease and appear as abnormal accumulations of fibrous or thread-like structures within the brain. These plaques are assembled by the misfolding and aggregation of the amyloid-b (Ab) protein. We have been studying its properties with an emphasis on the factors responsible for the transition from the normal to diseased fibrous state, the ability of aggregated Ab to kill nerve cells in culture, and the mechanism by which this is accomplished. Considerable advances have been made in this area and we are expanding our efforts to look for modulators of plaque formation as well as the cellular receptors which we feel are responsible for amyloid’s “killer” action. Our ultimate goal is to understand the events that culminate in these abnormal and detrimental proteins and the development of drugs capable of controlling these processes. These investigations are relevant to both the sporadic and familial forms of Alzheimer’s disease which exhibit identical amyloid pathology but differ only in their rate of progression. In conjunction with our amyloid research and drug development, my group has been concentrating its efforts on understanding the biochemistry and structural biology of the presenilin family of proteins. Mutations in the presenilin genes are the major cause of inherited forms of Alzheimer’s disease and we have been examining the location and expression of these proteins in neuronal cells and their relationship to the pathology of Alzheimer’s disease. This biochemical and molecular biological work is complemented by our examination of the three-dimensional organization of particular regions of the presenilin protein using a variety of biophysical techniques. Through these two approaches, we will be able to provide details on presenilin function and the molecular mechanism by which this is achieved. The importance of these studies is that they enable us to understand the earliest events in Alzheimer’s disease and allow us to develop novel approaches to the treatment of a principal cause of Alzheimer’s disease. Over the course of the past year a new line of investigation has also been established to examine an enzyme called PTEN-induced kinase-1 (PINK1) which has recently been linked to familial Parkinson’s disease. In collaboration with several other investigators at the CRND, we are investigating the molecular structure of PINK1 as well as identifying interacting proteins in an effort to expand our understanding of the cellular pathways that it regulates. The major objective of this study is to determine how PINK1 causes this particular form of Parkinson’s disease and potentially reveal unique biological avenues for the development of new therapies. DR. ANTHONY LANG I am a clinician investigator recruited to the CRND to help develop a basic science program directed towards gaining a better understanding of the etiology and pathogenesis of Parkinson’s disease, which, it is hoped, will be the basis for developing more effective therapies having an impact on the progressive nature of the illness. My own research involves multiple aspects of “movement disorders”, a subspecialty of neurology dealing with diseases dominated by parkinsonian loss of normal movement or excessive abnormal involuntary movements (e.g. chorea, dystonia, tics, tremor). I have been involved in studying and more explicitly defining neurodegenerative diseases such as corticobasal degeneration (e.g., determining that dementia may be the most common presentation of this disorder), and in developing, implementing and reporting a large number experimental therapeutic trials, particularly in Parkinson’s disease. Some of these have been carried out in collaboration with the Parkinson Study Group, others with industry, and still others have been conducted exclusively in our own unit at the Toronto Western Hospital. I direct the neurological component of an internationally renowned neurosurgical program for Parkinson’s disease and other movement disorders. We have published both clinical and basic research from this program in a number of top-level journals (clinical: e.g. clinical outcomes of patients undergoing pallidotomy and deep brain stimulation; basic: e.g. results of microrecording in various regions of the basic ganglia at the time surgery and impact of various interventions at this time). In collaboration with several other colleagues, I have been involved in studies assessing the molecular biology of various movement disorders (e.g. dope-responsive dystonia, myoclonus dystonia, Parkinson’s disease), electrophysiological aspects of movement disorders (e.g. orthostatic tremor, stimulus sensitive myoclonus), imaging of movement disorders (e.g. pre- and post-synaptic dopaminergic SPECT scanning in parkinsonian disorders). Overall, my research is designed to better understand the neurological diseases causing movement disorders, to define more effective methods of diagnosing and evaluating patients with these conditions, and to develop more effective therapies to treat the significant disability that they cause. Over the past year I have continued to direct a very active clinical research program in the field of movement disorders specializing particularly in Parkinson’s disease (PD) and to provide active collaborative support for the basic laboratories at CRND. The latter includes provision of financial support for basic research projects in the Tandon laboratory through the University’s Clark Chair and Parkinson Research funds. I am also actively collaborating with the Rogaeva laboratory in the evaluation of the genetic basis of Parkinson’s disease. This collaboration has been extremely productive recently in studies of all genes involved in familial parkinsonism including the initiation of clinical epidemiological studies involving at risk individuals who have inherited the most common gene defined to cause familial PD, LRRK2. Our clinical research program has recently recruited Dr. Connie Marras MD PhD to direct a clinical epidemiology program in movement disorders and she is assisting in the development of a multinational project (in collaboration with groups in Spain and the United Kingdom) evaluating LRRK2 families. The program at Toronto Western Hospital remains one of the most productive clinical research units in the world. We continue to evaluate the pathogenesis of Parkinson’s disease, complications of its treatment and mechanisms of action of interventions such as deep brain stimulation using electrophysiological techniques such as transcranial magnetic stimulation under the direction of Dr. Robert Chen. Dr. Elena Moro continues to direct the neurological component of our renowned functional neurosurgery program and we continue to make important research contributions to this field with current studies evaluating new indications for deep brain stimulation and new targets in PD. We have recently received Center of Excellence funding through the National Parkinson Foundation (USA) for the development of a unique comprehensive palliative care program (directed by Dr. Janis Miyasaki) and a videoconferencing patient outreach program (directed by Dr. Connie Marras). Dr. Susan Fox is directing our neuropharmacology program studying animal models of movement disorders and their management in early phase clinical trials in humans (e.g. levodopa induced dyskinesias). She has recently been working on an interesting animal model of levodopa-induced psychosis in the MPTP marmoset. Both Dr. Fox and myself have active collaborative research projects ongoing with the laboratory of Dr. Jonathan Brotchie at TWRI. In addition to the large number of clinical research studies outlined in the list of peer-reviewed scientific research publications, in the past year I have also been involved in coordinating a multinational effort to review brain stimulation for Parkinson’s disease (and edit a full Supplement of the journal Movement Disorders which will contain the results of this effort) and I have chaired the publications committee of the trial of direct intra-putamenal infusion of glial-derived neurotrophic factor (GDNF) for Parkinson’s disease and am the first author of the final paper from this trial which will be published early in 2006. Finally, I have been successful in obtaining funding from the Edmund J. Safra Philanthropic Foundation for the support of two new research faculty positions at the University of Toronto, a movement disorder neuroimager to be based at UHN and CAMH and an invertebrate modeler to be based at CRND. Searches for these two positions are actively underway. DR. JOANNE MCLAURIN I am investigating mechanisms that render the central nervous system susceptible to neurodegenerative processes during normal aging. During the past 12 months my laboratory, in collaboration with the laboratories of Paul Fraser, David Westaway and Peter St George-Hyslop, has continued to develop and characterize potential small molecule therapies for the treatment of Alzheimer’s disease, to understand the mechanism of action and potentially develop more potent compounds. We have used a multi-component approach including in vitro screening and in vivo animal models to identify a lead candidate that will be advanced to early clinical trials in 2006. This compound has been shown to be effective as both a prophylactic but also as a treatment once disease has already progressed in two separate animal models. We are now investigating the biophysical interaction of this compound with the amyloid-beta peptide to further understand the mechanism of action and identify sites on the molecule that can be modified to enhance it’s action. Reports in the literature demonstrated that molecules that target amyloid-beta peptide might also be effective at inhibiting Tau aggregation. The molecules reported to date, have similar properties to the family of compounds that we have identified for inhibiting amyloid-beta aggregation and therefore are testing effectiveness in mouse models of Tauopathies In addition, we have continued to investigate novel vaccination strategies to treat Alzheimer’s disease, focusing primarily on different modes of administration that may circumvent the menigo-encephalitis seen in the first clinical trials. These studies have indicated that indeed the immune system can be directed to exhibit primarily a helpful antibody response our animal model. Continuing with attempts to bias the immune system, we have now started to investigate the potential of a DNA vaccine to develop an effective antibody response. In conjunction with studies above that remove the toxic amyloid-beta peptide from the CNS, we have investigated the inflammatory response within the central nervous system with an aim at identifying signals that can be targeted in cells that naturally degrade amyloid-beta peptide. DR. HOWARD MOUNT In the 2004-05 academic year, my laboratory initiated two new programs of research to test putative relationships between the accumulation of toxic protein species, cellular energy deficits, mitochondrial dysfunction and progressive behavioral impairments in Alzheimer's- and Parkinson's-like pathologies. The shared goals of these programs are to determine whether depletion of cellular energy stores can be observed in animal models of these diseases, whether any such deficits occur early, or late in disease progression and whether they arise through hyperactivation of cellular enzymatic defenses (e.g. NAD depletion secondary to constitutive PARP activation) and/or failure of the mitochondrial oxidative phosphorylation machinery. With new grant support from the Ontario Mental Health Foundation and in collaboration with Dr. Marc Marien of the Instituut de Recherche Pierre Fabre (Castres, France), we produced an extensive resource of regionally dissected, microwave-fixed brain tissue from the TgCRND8 APP-transgenic mouse for HPLC analysis of high energy phosphate donors. We also characterized an expanded range of behavioral phenotypes that will enable us to better relate changes regional brain biochemistry to relevant functional endpoints in this model. For example, we developed an object recognition memory task to test entorhinal cortical function and discovered robust changes in this area of brain that is known to be targeted early in the course of the human disease. In a parallel effort, funded by the Parkinson's Society Canada and the Parkinson Disease Foundation (USA), we began an analysis of bioenergetic and neurochemical deficits in alpha-synuclein transgenic mouse models of familial Parkinson's disease, generated in-house by Dr. David Westaway. Alpha-synuclein accumulation in the amygdala and entorhinal cortex have been correlated with the occurrence and severity of a "Parkinson's Disease Dementia" that occurs in ~50% of Parkinson's patients. In the mutant alpha-synuclein-transgenic mice, we discerned deficits in both fear conditioning (a test of amygdala function) and object recognition memory, that developed in tandem with progressive neuromotor deficits. To test for relationships between these behavioral phenotypes, alpha-synuclein accumulation, bioenergetic status and dopaminergic function, we generated microwave-fixed regional samples throughout the brains of these animals so as to enable tandem analyses of monoamine turnover and high energy phosphates by HPLC. In collaboration with Dr. Brian Robinson (Hospital for Sick Children) we also began the first systematic survey of brain mitochondrial enzyme activities in these models, using regional fresh samples at time points associated with progressive stages of behavioral impairment. DR. EKATERINA ROGAEVA Research in my laboratory focuses on investigating the functional and pathological significance of the presenilin isoforms. Mutations in the presenilin genes are the major cause of inherited forms of Alzheimer Disease; however, little is known about biological function of their alternative splicing events. Presenilins (PSs) have a complex functional profile as integrators of several signaling pathways, in which the roles of the presenilins may be functionally separated. For example, the expression of a PS splice variant that lacks exon 8 does not reduce Abeta production, although it does prevent Notch1 cleavage. This alternative splicing event may help to fine-tune the functional activities of the PSs. We are evaluating the abundance of each splicing transcript in Alzheimer Disease versus normal tissues and characterizing the circumstances under which splicing occurs (i.e., whether sequence polymorphisms surrounding the intron/exon boundaries alter alternative splicing efficiency). During the past year my laboratory has continued the intense investigation into the genetics underlying different neurodegenerative disorders (e.g. Frontal Temporal Dementia, familial prion disease, and Spastic Paraplegia) with the main focus being on Parkinson Disease (PD) and Alzheimer Disease (AD). The result of this year of research is presented in 11 peer-reviewed papers. We have continued to develop a DNA collection of Canadian PD patients with early-onset PD and/or positive family history to determine genetic factors responsible for PD or influencing the course/treatment of illness. We are systematically analyzing five PD-linked genes (parkin, a-synuclein, DJ-1, PINK1 and LRRK2). Our results indicate that a novel PD gene (PINK1) is a relatively common cause of early-onset PD; we detected PINK1 mutations in four Canadian PD families and in a large consanguineous Saudi PD family. In addition, we conducted a mutation analysis and the first case-control study of another novel PD gene (LRRK2) and identified three Canadian families with LRRK2 mutations. The investigation of eight mutation carries demonstrated that mutations in LRRK2 cause typical PD with a very variable age-at-onset that is not explained by APOE genotypes. The value of discovering such PD kindreds is the possibility of longitudinal studies initiated by Dr. Lang (e.g. investigation of pre-symptomatic carries could be important in testing the predictive value of neuroimaging techniques). Families without pathological mutations will serve as a basis for our future genetic analysis in order to search for novel PD genes. Currently our dataset consists of ~200 PD samples, which is now sufficient to perform independent case-control association studies to search for PD risk factors. We have continued to be tightly involved in the analysis of known AD genes and the identification of novel genetic risk factors for late-onset AD. We did not support the notion that genetic variability in the set of cholesterol metabolism genes (CH25H, ABCA1 and CH24H) influence the development of AD. We are continuing to work on the identification of a novel AD risk factor on chromosome 12 by performing linkage analyses of 34 microsatellite markers and association studies with 103 polymorphisms. In two independent late-onset AD datasets, linkage analysis generated significant evidence for an AD locus at a ~5 cM interval near position 20 cM that was prioritized for a case-control association study. We observed statistically significant results for 14 SNPs clustered in two distinct areas (~2.4 Mb apart). DR. PETER ST GEORGE-HYSLOP Work in my laboratory focuses on two areas. The first area is a continuation of previous efforts to understand how mutations in the presenilin proteins lead to neurodegeneration in Alzheimer Disease (AD). Recently, we have shown that the presenilin proteins interact with a number of other cellular proteins to form a high molecular weight protein complex. We have also shown that mutations in the presenilin proteins alter the function of these high molecular weight protein complexes, clearly indicating that dysfunction of this complex is probably central to the mechanism by which presenilin mutations cause AD. Future work in this area will focus on trying to isolate the other protein components of this complex, and on trying to understand how defects in the function of this complex lead to abnormal processing of the amyloid precursor protein and ultimately to Alzheimer-type neurodegeneration. The other major area of research focus is the discovery of additional genes causing susceptibility to AD. Following cloning of the presenilin protein genes, we came to the realization that mutations and/or naturally-occurring variants in the four known Alzheimer susceptibility genes (ß-amyloid precursor protein gene on chromosome 21, presenilin 1 on chromosome 14, presenilin 2 on chromosome 1, and apolipoprotein E on chromosome 19) accounted for only about 40-60% of the genetic risk factors for AD. Consequently, we set out to identify additional genes in families which had familial AD, but did not have mutations and/or variants in any of the four known genes. This work has recently come to fruition by the demonstration that a novel Alzheimer susceptibility gene is located on chromosome 12. Additional studies will have to be undertaken during the next couple of years in order to isolate and characterize the precise gene. However, identification of this gene will provide further clues to the biochemical pathogenesis of AD, which will ultimately be very useful in our attempts to generate effective treatments for this disease. A secondary conclusion derived from the genetic linkage studies is that at least one other Alzheimer gene must exist because the chromosome 12 gene does not appear to account for all of the remaining cases. Ultimately, further genetic linkage studies will be required to search for this sixth Alzheimer susceptibility gene, which is presumably on chromosomes other than 1, 12, 14, 19, or 21. In addition to these areas of investigation, my laboratory has also been collaborating with the laboratory of Dr. David Westaway in order to generate better laboratory models of AD, which can be used not only to understand the disease mechanism but also for pre-clinical testing of potential therapies. We have initiated attempts to generate transgenic models in the laboratory mouse for AD, Fronto-Temporal Dementia and Parkinson's disease. DR. ANURAG TANDON The primary focus of our research is Parkinson’s disease (PD), a progressive neurodegenerative disorder that is influenced by genetic and environmental factors. In recent years, 6 independent genes that cause PD have been identified, and another 4 genetic loci/risk factors are known. Although the genetic cases account for less than 5% of the incidence of PD, the shared pathological features between familial and sporadic PD suggest that there is a common pathogenic pathway. Therefore, we can address the complexity of PD by better understanding the biology of the individual causative factors. Currently, we are investigating the normal biology of a-synuclein and the pathobiology induced by genetic mutations. A key hallmark of PD and other synucleinopathies is the accrual of detergent-insoluble a-synuclein. Therefore, a change in a-synuclein solubility is a valuable pre-clinical marker of the earliest pathogenetic events and provides an important starting point for investigation. To characterize a-synuclein interactions with membranes and with other proteins, we have developed semi-intact cell preparations that are composed of cells or isolated nerve terminals whose outer membranes are rendered permeable to macromolecules. This approach, combined with the spectrum of cell and molecular biological techniques, is being exploited to define a-synuclein interactions. An essential component of these studies is our new transgenic mouse lines which express human a-synuclein but not the endogenous murine a-synuclein. These lines permit analysis of wild-type and mutant a-synuclein interactions without interference from the endogenous murine protein. A second avenue of investigation is the development of a pathobiological model of PD based on the ubiquitin-proteasome degradation pathway. Mutations in UPS components, such as the parkin gene, account for a majority of autosomal recessive PD cases and ubiquitin is a major constituent of Lewy bodies. We are using pharmacological inhibitors and genetic mutations in the catalytic subunits to induce impairments in proteasome function as a means to assess the long-term biochemical changes that precede the formation of insoluble a-synuclein in cell culture and transgenic mice. Additional investigations in the laboratory are examining the role of PD genes, DJ-1 and PINK1, in regulating mitochondrial function and the impact of the loss of function mutations on cell viability. DR. DAVID WESTAWAY Genetic techniques such as transgenic mice are used to decipher the biochemical events and pathways which underlie neurodegenerative diseases. Two types of disease are under study in my laboratory at this time. The first is Alzheimer’s Disease (AD), the fourth leading cause of death in Canada, and the second is prion disease, exemplified by sporadic Creutzfeldt-Jakob Disease (CJD) of humans and mad cow disease (BSE) of livestock. With regards to AD we have been working for several years with our collaborators in the CRND to create a robust mouse model for early-onset AD. These studies involve expressing a number of proteins implicated in AD in the brains of transgenic mice: the proteins include the presenilin I, presenilin 2, the Alzheimer precursor protein (APP), alpha-synuclein, and tau. These studies use a pan-neuronal prion protein gene expression vector. We have attained a success in the construction of the TgCRND8 line of transgenic mice, which exhibits the fastest rate of deposition of Alzheimer b-amyloid of any animal model of AD and shows a robust impairment in a standard test of cognitive performance. The TgCRND8 mice therefore comprise a useful platform for applied research into AD, and are being used to test candidate therapies from four major pharmaceutical companies. In a separate series of studies we have created a line of transgenic mice denoted TgTau(P301L)23027 that develop florid neuronal tau pathologies such as neurofibrillary tangles, and glial pathologies remarkably similar to those seen in corticobasal degeneration (CBD) and progressive supranuclear palsy (PSP), two other AD-like diseases. Besides offering insights into disease pathogenesis, both of these transgenic disease models are being used in systematic studies to find very early diagnostic markers of disease. A second avenue of AD research seeks to make explicit the molecular deficits that initiate the disease process. This focuses upon the composition and action of a protein “machine”, the membrane-associated endoprotease gamma-secretase. The studies are carried out with Drs. St George-Hyslop, Schmitt-Ulms and Fraser. These studies have already defined unexpected divergence in the actions of PS1 and PS2 in the mammalian central nervous system. Ultimately, with Dr. Charlie Boone, we intend to use the simple nucleated organism yeast to identify all the genes that interact with gamma-secretase Research into prion disease seeks to decipher the physiology and mis-folding of these small yet enigmatic membrane-linked proteins. Based upon discovery of a second PrP-like protein denoted doppel (Dpl) we have pioneered a genetic assay for the activity of the cellular prion protein in CNS neurons. Notably, this genetic push-pull between PrP and Dpl is now being exploited by a number of prion labs. In other studies we are pursuing a panel of PrP-interacting proteins identified via chemical cross-linking (by Dr. Gerold-Schmitt-Ulms). Using additional genetic information from PrP transgenic mice, and the aforementioned cell biological assay, we intend to triangulate upon the true in vivo interactors. Progress on both fronts has been excellent, to the extent we have (i) unearthed new activity determinants in cellular PrP that “explain” results in transgenic mice, and (ii) defined evolutionarily conserved protein-protein interactions. DR. GEROLD SCHMITT-ULMS The CRND Proteomics Unit occupies three rooms on the second floor of the Tanz Neuroscience research building at the southern end of Queen’s Park. The laboratory offers new facilities with state of the art equipment and is embedded in a highly stimulating research environment at the CRND. Our work contributes to two strands of research at the interface of proteomics and neurodegenerative disease research: the development of strategies for the study of protein interactions and the application of these strategies to address questions in the context of neurodegenerative disease research. The laboratory is involved in collaborative activities with the objectives to (i) understand the molecular assembly and activation of the presenilin complex in Alzheimer’s disease; (ii) map the molecular environment that hosts the conversion of the prion protein in Creutzfeldt-Jacob disease; and (iii) elucidate molecular events that underlie the intracellular deposition of Lewy bodies and cell death in Parkinson’s Disease. During the past 12 months the laboratory has continued to grow. We have hired two postdoctoral fellows with expertise in proteomics / mass spectrometry and target validation applications. We have used the CRND’s new mass spectrometer to assemble data from a presenilin-1 (PS1) interactome mapping study and have described a novel negative regulator of PS1 that intriguingly selectively inhibits γ-secretase mediated Aβ production without affecting ε-site cleavage of APP or ε-site cleavage of other established γ-secretase substrates. We have combined tcTPC with iTRAQ labeling to generate quantitative interactome data of APP and its two mammalian homologues APLP-1 and APLP-2. We also collected comprehensive tcTPC interactome data for nicastrin, DJ-1, PrP, α-synuclein. Each datasets revealed interesting candidate interactors currently being followed-up on in CRND collaborations with Dr. A. Tandon, Dr. P. Fraser, Dr. D. Westaway, Dr. P. St. George-Hyslop. Finally, with Drs Fraser, St George-Hyslop, and Rogaeva we have worked on the biological characterization of a new candidate gene for late onset forms of Alzheimer Disease. DR. JANICE ROBERTSON Our laboratory is dedicated to the study of the disease mechanism(s) causing amyotrophic lateral sclerosis (ALS). We use a multidisciplinary approach that includes live cell imaging, animal modeling, molecular-cell biology techniques and biochemical/neuropathological analyses of human ALS brain and spinal cord material. Currently we are following three lines of investigations: (1) Understanding how neurofilament and peripherin abnormalities occur in ALS. Cytoplasmic inclusions comprised of neurofilaments and peripherin are a common pathological finding in ALS. How they are formed and their relationship to the disease mechanism is not known. We have been studying a process called alternative splicing, whereby several mRNA transcripts can be derived from a single gene (Robertson et al, JCB 160:939-49, 2003). In this regard we have identified four alternatively spliced variants of peripherin and two of the neurofilament subunit, NF-L. We have shown that deregulated expression of these variants occurs in ALS and can cause neurofilament and peripherin aggregation. We are currently studying the functional properties of these splice variants. (2) Studies of the neurotoxicity of mutant superoxide dismutase-1 (SOD1). Over 100 different mutations in the SOD1 gene are causative of about 2-3% of ALS cases. In collaboration with Dr Avijit Chakrabartty we have been studying disease relevant folding intermediates of SOD1using an epitope specific antibody. We are also studying the proteins that interact with mutant SOD1 using mass spectrometry approaches and testing various therapeutic strategies in mutant SOD1 transgenic mice. (3) Neuroinflammation and ALS. Activated microglia are associated with degenerating motor neurons in ALS. We have shown that the proinflammatory cytokine TNF-alpha released by activated microglia promotes apoptosis of neurons containing peripherin aggregates (Robertson et al, JCB 155:217-26, 2001). Subsequently it has been demonstrated that motor neuron death in mutant SOD1 transgenic mice is non-cell autonomous and that factor(s) released by activated microglia are important to the disease mechanism. We are investigating this using a co-culture paradigm of primary motor neurons and microglia. We have established a core group of clinician/scientists that have a common interest in ALS. As such, we have initiated a program of blood collection (for genetic analysis) and of tissue collection (for biochemistry and neuropathology) from ALS cases seen by our team member, Dr Lorne Zinman, Medical Director of the ALS Clinic at Sunnybrook Health Sciences Centre. With over 200 new cases diagnosed at the clinic every year, it is expected that we will build a sizeable resource in which to undertake our ongoing studies.

People interested in participating in research on the mechanisms and causes of Alzheimer Disease, Parkinson Disease, Fronto-temporal dementia, Pick Disease and other neurodegenerative diseases (e.g. people interested in participating in genetic studies, brain donation, etc) can send enquiries to crnd.secr@utoronto.ca or to crnd.admin@utoronto.ca.

People interested in participating in future clinical trials of AZD-103 (scyllo-cyclohexanehexol) should contact Soraya Centeno at Transition Therapeutics
(scenteno@transitiontherapeutics.com 416-263-1213).

My laboratory focuses on the neuropathology of human neurodegenerative diseases at both the basic and clinical levels.

Over the past year my lab has concentrated on the pathological diagnosis and molecular classification of 61 Canadians who have died of suspect Creutzfeldt Jakob disease (CJD).

Each case is examined using traditional neurohistological methods as well as immunohistochemistry for the prion protein. Frozen brain tissue was available for molecular characterization on 18 cases. CJD was found in 30 patients or 50% of suspect cases, a figure consistent over the past 8 years of our surveillance program. The most frequent cause of misdiagnosis was AD in 10 cases or 16%. Other conditions encountered include frontotemporal degeneration (3), diffuse Lewy body disease (3), metabolic encephalopathy (6), encephalitis (2), cerebellar degeneration (2), dentato-rubro-pallido-luysian degeneration (l), argyrophilic grain disease (1) and lymphoma (1). The remaining two cases were labeled as “not CJD”. One case consisted of brain tissue from a living patient suspect of suffering from CJD, obtained at craniotomy for a head injury 2 years prior to the onset of dementia. The other case showed no evidence of CJD, but no other significant pathological changes. The presence of some immune cells raises the possibility of Hashimoto’s encephalopathy. Frozen tissue was available for molecular testing on 18 cases, a combination of codon 129 analysis and prion banding pattern. The following CJD phenotypes were identified: 12 patients were found to have the MM1 phenotype characterized by rapid progressive dementia with myoclonus. Cerebellar phenotypes of longer durations were also represented (VV2 x 2 and MV2 x 1). A very unusual phenomenon this year was the detection of 3 patients (16% of the cases analyzed) with the MM2 phenotype. This phenotype is only rarely observed and accounts for only 4% of all sporadic CJD. All three patients had a long disease duration of over one year.

Research in my laboratory focuses on the biochemistry and biophysics of amyloid plaques and their relationship to sporadic and familial forms of Alzheimer’s disease. Plaques are a principal pathological feature of Alzheimer’s disease and appear as abnormal accumulations of fibrous or thread-like structures within the brain. These plaques are assembled by the misfolding and aggregation of the amyloid-b (Ab) protein. We have been studying its properties with an emphasis on the factors responsible for the transition from the normal to diseased fibrous state, the ability of aggregated Ab to kill nerve cells in culture, and the mechanism by which this is accomplished. Considerable advances have been made in this area and we are expanding our efforts to look for modulators of plaque formation as well as the cellular receptors which we feel are responsible for amyloid’s “killer” action. Our ultimate goal is to understand the events that culminate in these abnormal and detrimental proteins and the development of drugs capable of controlling these processes. These investigations are relevant to both the sporadic and familial forms of Alzheimer’s disease which exhibit identical amyloid pathology but differ only in their rate of progression.

In conjunction with our amyloid research and drug development, my group has been concentrating its efforts on understanding the biochemistry and structural biology of the presenilin family of proteins. Mutations in the presenilin genes are the major cause of inherited forms of Alzheimer’s disease and we have been examining the location and expression of these proteins in neuronal cells and their relationship to the pathology of Alzheimer’s disease. This biochemical and molecular biological work is complemented by our examination of the three-dimensional organization of particular regions of the presenilin protein using a variety of biophysical techniques. Through these two approaches, we will be able to provide details on presenilin function and the molecular mechanism by which this is achieved. The importance of these studies is that they enable us to understand the earliest events in Alzheimer’s disease and allow us to develop novel approaches to the treatment of a principal cause of Alzheimer’s disease.

Over the course of the past year a new line of investigation has also been established to examine an enzyme called PTEN-induced kinase-1 (PINK1) which has recently been linked to familial Parkinson’s disease. In collaboration with several other investigators at the CRND, we are investigating the molecular structure of PINK1 as well as identifying interacting proteins in an effort to expand our understanding of the cellular pathways that it regulates. The major objective of this study is to determine how PINK1 causes this particular form of Parkinson’s disease and potentially reveal unique biological avenues for the development of new therapies.

I am a clinician investigator recruited to the CRND to help develop a basic science program directed towards gaining a better understanding of the etiology and pathogenesis of Parkinson’s disease, which, it is hoped, will be the basis for developing more effective therapies having an impact on the progressive nature of the illness.

My own research involves multiple aspects of “movement disorders”, a subspecialty of neurology dealing with diseases dominated by parkinsonian loss of normal movement or excessive abnormal involuntary movements (e.g. chorea, dystonia, tics, tremor). I have been involved in studying and more explicitly defining neurodegenerative diseases such as corticobasal degeneration (e.g., determining that dementia may be the most common presentation of this disorder), and in developing, implementing and reporting a large number experimental therapeutic trials, particularly in Parkinson’s disease. Some of these have been carried out in collaboration with the Parkinson Study Group, others with industry, and still others have been conducted exclusively in our own unit at the Toronto Western Hospital. I direct the neurological component of an internationally renowned neurosurgical program for Parkinson’s disease and other movement disorders. We have published both clinical and basic research from this program in a number of top-level journals (clinical: e.g. clinical outcomes of patients undergoing pallidotomy and deep brain stimulation; basic: e.g. results of microrecording in various regions of the basic ganglia at the time surgery and impact of various interventions at this time). In collaboration with several other colleagues, I have been involved in studies assessing the molecular biology of various movement disorders (e.g. dope-responsive dystonia, myoclonus dystonia, Parkinson’s disease), electrophysiological aspects of movement disorders (e.g. orthostatic tremor, stimulus sensitive myoclonus), imaging of movement disorders (e.g. pre- and post-synaptic dopaminergic SPECT scanning in parkinsonian disorders).

Overall, my research is designed to better understand the neurological diseases causing movement disorders, to define more effective methods of diagnosing and evaluating patients with these conditions, and to develop more effective therapies to treat the significant disability that they cause.

Over the past year I have continued to direct a very active clinical research program in the field of movement disorders specializing particularly in Parkinson’s disease (PD) and to provide active collaborative support for the basic laboratories at CRND. The latter includes provision of financial support for basic research projects in the Tandon laboratory through the University’s Clark Chair and Parkinson Research funds. I am also actively collaborating with the Rogaeva laboratory in the evaluation of the genetic basis of Parkinson’s disease. This collaboration has been extremely productive recently in studies of all genes involved in familial parkinsonism including the initiation of clinical epidemiological studies involving at risk individuals who have inherited the most common gene defined to cause familial PD, LRRK2. Our clinical research program has recently recruited Dr. Connie Marras MD PhD to direct a clinical epidemiology program in movement disorders and she is assisting in the development of a multinational project (in collaboration with groups in Spain and the United Kingdom) evaluating LRRK2 families.

The program at Toronto Western Hospital remains one of the most productive clinical research units in the world. We continue to evaluate the pathogenesis of Parkinson’s disease, complications of its treatment and mechanisms of action of interventions such as deep brain stimulation using electrophysiological techniques such as transcranial magnetic stimulation under the direction of Dr. Robert Chen. Dr. Elena Moro continues to direct the neurological component of our renowned functional neurosurgery program and we continue to make important research contributions to this field with current studies evaluating new indications for deep brain stimulation and new targets in PD. We have recently received Center of Excellence funding through the National Parkinson Foundation (USA) for the development of a unique comprehensive palliative care program (directed by Dr. Janis Miyasaki) and a videoconferencing patient outreach program (directed by Dr. Connie Marras). Dr. Susan Fox is directing our neuropharmacology program studying animal models of movement disorders and their management in early phase clinical trials in humans (e.g. levodopa induced dyskinesias). She has recently been working on an interesting animal model of levodopa-induced psychosis in the MPTP marmoset. Both Dr. Fox and myself have active collaborative research projects ongoing with the laboratory of Dr. Jonathan Brotchie at TWRI. In addition to the large number of clinical research studies outlined in the list of peer-reviewed scientific research publications, in the past year I have also been involved in coordinating a multinational effort to review brain stimulation for Parkinson’s disease (and edit a full Supplement of the journal Movement Disorders which will contain the results of this effort) and I have chaired the publications committee of the trial of direct intra-putamenal infusion of glial-derived neurotrophic factor (GDNF) for Parkinson’s disease and am the first author of the final paper from this trial which will be published early in 2006. Finally, I have been successful in obtaining funding from the Edmund J. Safra Philanthropic Foundation for the support of two new research faculty positions at the University of Toronto, a movement disorder neuroimager to be based at UHN and CAMH and an invertebrate modeler to be based at CRND. Searches for these two positions are actively underway.

I am investigating mechanisms that render the central nervous system susceptible to neurodegenerative processes during normal aging.

During the past 12 months my laboratory, in collaboration with the laboratories of Paul Fraser, David Westaway and Peter St George-Hyslop, has continued to develop and characterize potential small molecule therapies for the treatment of Alzheimer’s disease, to understand the mechanism of action and potentially develop more potent compounds. We have used a multi-component approach including in vitro screening and in vivo animal models to identify a lead candidate that will be advanced to early clinical trials in 2006. This compound has been shown to be effective as both a prophylactic but also as a treatment once disease has already progressed in two separate animal models. We are now investigating the biophysical interaction of this compound with the amyloid-beta peptide to further understand the mechanism of action and identify sites on the molecule that can be modified to enhance it’s action. Reports in the literature demonstrated that molecules that target amyloid-beta peptide might also be effective at inhibiting Tau aggregation. The molecules reported to date, have similar properties to the family of compounds that we have identified for inhibiting amyloid-beta aggregation and therefore are testing effectiveness in mouse models of Tauopathies

In addition, we have continued to investigate novel vaccination strategies to treat Alzheimer’s disease, focusing primarily on different modes of administration that may circumvent the menigo-encephalitis seen in the first clinical trials. These studies have indicated that indeed the immune system can be directed to exhibit primarily a helpful antibody response our animal model. Continuing with attempts to bias the immune system, we have now started to investigate the potential of a DNA vaccine to develop an effective antibody response. In conjunction with studies above that remove the toxic amyloid-beta peptide from the CNS, we have investigated the inflammatory response within the central nervous system with an aim at identifying signals that can be targeted in cells that naturally degrade amyloid-beta peptide.

In the 2004-05 academic year, my laboratory initiated two new programs of research to test putative relationships between the accumulation of toxic protein species, cellular energy deficits, mitochondrial dysfunction and progressive behavioral impairments in Alzheimer's- and Parkinson's-like pathologies. The shared goals of these programs are to determine whether depletion of cellular energy stores can be observed in animal models of these diseases, whether any such deficits occur early, or late in disease progression and whether they arise through hyperactivation of cellular enzymatic defenses (e.g. NAD depletion secondary to constitutive PARP activation) and/or failure of the mitochondrial oxidative phosphorylation machinery.

With new grant support from the Ontario Mental Health Foundation and in collaboration with Dr. Marc Marien of the Instituut de Recherche Pierre Fabre (Castres, France), we produced an extensive resource of regionally dissected, microwave-fixed brain tissue from the TgCRND8 APP-transgenic mouse for HPLC analysis of high energy phosphate donors. We also characterized an expanded range of behavioral phenotypes that will enable us to better relate changes regional brain biochemistry to relevant functional endpoints in this model. For example, we developed an object recognition memory task to test entorhinal cortical function and discovered robust changes in this area of brain that is known to be targeted early in the course of the human disease.

In a parallel effort, funded by the Parkinson's Society Canada and the Parkinson Disease Foundation (USA), we began an analysis of bioenergetic and neurochemical deficits in alpha-synuclein transgenic mouse models of familial Parkinson's disease, generated in-house by Dr. David Westaway. Alpha-synuclein accumulation in the amygdala and entorhinal cortex have been correlated with the occurrence and severity of a "Parkinson's Disease Dementia" that occurs in ~50% of Parkinson's patients. In the mutant alpha-synuclein-transgenic mice, we discerned deficits in both fear conditioning (a test of amygdala function) and object recognition memory, that developed in tandem with progressive neuromotor deficits. To test for relationships between these behavioral phenotypes, alpha-synuclein accumulation, bioenergetic status and dopaminergic function, we generated microwave-fixed regional samples throughout the brains of these animals so as to enable tandem analyses of monoamine turnover and high energy phosphates by HPLC. In collaboration with Dr. Brian Robinson (Hospital for Sick Children) we also began the first systematic survey of brain mitochondrial enzyme activities in these models, using regional fresh samples at time points associated with progressive stages of behavioral impairment.

Research in my laboratory focuses on investigating the functional and pathological significance of the presenilin isoforms. Mutations in the presenilin genes are the major cause of inherited forms of Alzheimer Disease; however, little is known about biological function of their alternative splicing events. Presenilins (PSs) have a complex functional profile as integrators of several signaling pathways, in which the roles of the presenilins may be functionally separated. For example, the expression of a PS splice variant that lacks exon 8 does not reduce Abeta production, although it does prevent Notch1 cleavage. This alternative splicing event may help to fine-tune the functional activities of the PSs. We are evaluating the abundance of each splicing transcript in Alzheimer Disease versus normal tissues and characterizing the circumstances under which splicing occurs (i.e., whether sequence polymorphisms surrounding the intron/exon boundaries alter alternative splicing efficiency).

During the past year my laboratory has continued the intense investigation into the genetics underlying different neurodegenerative disorders (e.g. Frontal Temporal Dementia, familial prion disease, and Spastic Paraplegia) with the main focus being on Parkinson Disease (PD) and Alzheimer Disease (AD). The result of this year of research is presented in 11 peer-reviewed papers.

We have continued to develop a DNA collection of Canadian PD patients with early-onset PD and/or positive family history to determine genetic factors responsible for PD or influencing the course/treatment of illness. We are systematically analyzing five PD-linked genes (parkin, a-synuclein, DJ-1, PINK1 and LRRK2). Our results indicate that a novel PD gene (PINK1) is a relatively common cause of early-onset PD; we detected PINK1 mutations in four Canadian PD families and in a large consanguineous Saudi PD family. In addition, we conducted a mutation analysis and the first case-control study of another novel PD gene (LRRK2) and identified three Canadian families with LRRK2 mutations. The investigation of eight mutation carries demonstrated that mutations in LRRK2 cause typical PD with a very variable age-at-onset that is not explained by APOE genotypes. The value of discovering such PD kindreds is the possibility of longitudinal studies initiated by Dr. Lang (e.g. investigation of pre-symptomatic carries could be important in testing the predictive value of neuroimaging techniques). Families without pathological mutations will serve as a basis for our future genetic analysis in order to search for novel PD genes. Currently our dataset consists of ~200 PD samples, which is now sufficient to perform independent case-control association studies to search for PD risk factors.

We have continued to be tightly involved in the analysis of known AD genes and the identification of novel genetic risk factors for late-onset AD. We did not support the notion that genetic variability in the set of cholesterol metabolism genes (CH25H, ABCA1 and CH24H) influence the development of AD. We are continuing to work on the identification of a novel AD risk factor on chromosome 12 by performing linkage analyses of 34 microsatellite markers and association studies with 103 polymorphisms. In two independent late-onset AD datasets, linkage analysis generated significant evidence for an AD locus at a ~5 cM interval near position 20 cM that was prioritized for a case-control association study. We observed statistically significant results for 14 SNPs clustered in two distinct areas (~2.4 Mb apart).

Work in my laboratory focuses on two areas. The first area is a continuation of previous efforts to understand how mutations in the presenilin proteins lead to neurodegeneration in Alzheimer Disease (AD). Recently, we have shown that the presenilin proteins interact with a number of other cellular proteins to form a high molecular weight protein complex. We have also shown that mutations in the presenilin proteins alter the function of these high molecular weight protein complexes, clearly indicating that dysfunction of this complex is probably central to the mechanism by which presenilin mutations cause AD. Future work in this area will focus on trying to isolate the other protein components of this complex, and on trying to understand how defects in the function of this complex lead to abnormal processing of the amyloid precursor protein and ultimately to Alzheimer-type neurodegeneration.

The other major area of research focus is the discovery of additional genes causing susceptibility to AD. Following cloning of the presenilin protein genes, we came to the realization that mutations and/or naturally-occurring variants in the four known Alzheimer susceptibility genes (ß-amyloid precursor protein gene on chromosome 21, presenilin 1 on chromosome 14, presenilin 2 on chromosome 1, and apolipoprotein E on chromosome 19) accounted for only about 40-60% of the genetic risk factors for AD. Consequently, we set out to identify additional genes in families which had familial AD, but did not have mutations and/or variants in any of the four known genes. This work has recently come to fruition by the demonstration that a novel Alzheimer susceptibility gene is located on chromosome 12. Additional studies will have to be undertaken during the next couple of years in order to isolate and characterize the precise gene. However, identification of this gene will provide further clues to the biochemical pathogenesis of AD, which will ultimately be very useful in our attempts to generate effective treatments for this disease. A secondary conclusion derived from the genetic linkage studies is that at least one other Alzheimer gene must exist because the chromosome 12 gene does not appear to account for all of the remaining cases. Ultimately, further genetic linkage studies will be required to search for this sixth Alzheimer susceptibility gene, which is presumably on chromosomes other than 1, 12, 14, 19, or 21.

In addition to these areas of investigation, my laboratory has also been collaborating with the laboratory of Dr. David Westaway in order to generate better laboratory models of AD, which can be used not only to understand the disease mechanism but also for pre-clinical testing of potential therapies. We have initiated attempts to generate transgenic models in the laboratory mouse for AD, Fronto-Temporal Dementia and Parkinson's disease.

The primary focus of our research is Parkinson’s disease (PD), a progressive neurodegenerative disorder that is influenced by genetic and environmental factors. In recent years, 6 independent genes that cause PD have been identified, and another 4 genetic loci/risk factors are known. Although the genetic cases account for less than 5% of the incidence of PD, the shared pathological features between familial and sporadic PD suggest that there is a common pathogenic pathway. Therefore, we can address the complexity of PD by better understanding the biology of the individual causative factors.

Currently, we are investigating the normal biology of a-synuclein and the pathobiology induced by genetic mutations. A key hallmark of PD and other synucleinopathies is the accrual of detergent-insoluble a-synuclein. Therefore, a change in a-synuclein solubility is a valuable pre-clinical marker of the earliest pathogenetic events and provides an important starting point for investigation. To characterize a-synuclein interactions with membranes and with other proteins, we have developed semi-intact cell preparations that are composed of cells or isolated nerve terminals whose outer membranes are rendered permeable to macromolecules. This approach, combined with the spectrum of cell and molecular biological techniques, is being exploited to define a-synuclein interactions. An essential component of these studies is our new transgenic mouse lines which express human a-synuclein but not the endogenous murine a-synuclein. These lines permit analysis of wild-type and mutant a-synuclein interactions without interference from the endogenous murine protein.

A second avenue of investigation is the development of a pathobiological model of PD based on the ubiquitin-proteasome degradation pathway. Mutations in UPS components, such as the parkin gene, account for a majority of autosomal recessive PD cases and ubiquitin is a major constituent of Lewy bodies. We are using pharmacological inhibitors and genetic mutations in the catalytic subunits to induce impairments in proteasome function as a means to assess the long-term biochemical changes that precede the formation of insoluble a-synuclein in cell culture and transgenic mice.

Additional investigations in the laboratory are examining the role of PD genes, DJ-1 and PINK1, in regulating mitochondrial function and the impact of the loss of function mutations on cell viability.

Genetic techniques such as transgenic mice are used to decipher the biochemical events and pathways which underlie neurodegenerative diseases. Two types of disease are under study in my laboratory at this time. The first is Alzheimer’s Disease (AD), the fourth leading cause of death in Canada, and the second is prion disease, exemplified by sporadic Creutzfeldt-Jakob Disease (CJD) of humans and mad cow disease (BSE) of livestock.

With regards to AD we have been working for several years with our collaborators in the CRND to create a robust mouse model for early-onset AD. These studies involve expressing a number of proteins implicated in AD in the brains of transgenic mice: the proteins include the presenilin I, presenilin 2, the Alzheimer precursor protein (APP), alpha-synuclein, and tau. These studies use a pan-neuronal prion protein gene expression vector. We have attained a success in the construction of the TgCRND8 line of transgenic mice, which exhibits the fastest rate of deposition of Alzheimer b-amyloid of any animal model of AD and shows a robust impairment in a standard test of cognitive performance. The TgCRND8 mice therefore comprise a useful platform for applied research into AD, and are being used to test candidate therapies from four major pharmaceutical companies. In a separate series of studies we have created a line of transgenic mice denoted TgTau(P301L)23027 that develop florid neuronal tau pathologies such as neurofibrillary tangles, and glial pathologies remarkably similar to those seen in corticobasal degeneration (CBD) and progressive supranuclear palsy (PSP), two other AD-like diseases. Besides offering insights into disease pathogenesis, both of these transgenic disease models are being used in systematic studies to find very early diagnostic markers of disease.

A second avenue of AD research seeks to make explicit the molecular deficits that initiate the disease process. This focuses upon the composition and action of a protein “machine”, the membrane-associated endoprotease gamma-secretase. The studies are carried out with Drs. St George-Hyslop, Schmitt-Ulms and Fraser. These studies have already defined unexpected divergence in the actions of PS1 and PS2 in the mammalian central nervous system. Ultimately, with Dr. Charlie Boone, we intend to use the simple nucleated organism yeast to identify all the genes that interact with gamma-secretase

Research into prion disease seeks to decipher the physiology and mis-folding of these small yet enigmatic membrane-linked proteins. Based upon discovery of a second PrP-like protein denoted doppel (Dpl) we have pioneered a genetic assay for the activity of the cellular prion protein in CNS neurons. Notably, this genetic push-pull between PrP and Dpl is now being exploited by a number of prion labs. In other studies we are pursuing a panel of PrP-interacting proteins identified via chemical cross-linking (by Dr. Gerold-Schmitt-Ulms). Using additional genetic information from PrP transgenic mice, and the aforementioned cell biological assay, we intend to triangulate upon the true in vivo interactors. Progress on both fronts has been excellent, to the extent we have (i) unearthed new activity determinants in cellular PrP that “explain” results in transgenic mice, and (ii) defined evolutionarily conserved protein-protein interactions.

The CRND Proteomics Unit occupies three rooms on the second floor of the Tanz Neuroscience research building at the southern end of Queen’s Park. The laboratory offers new facilities with state of the art equipment and is embedded in a highly stimulating research environment at the CRND. Our work contributes to two strands of research at the interface of proteomics and neurodegenerative disease research: the development of strategies for the study of protein interactions and the application of these strategies to address questions in the context of neurodegenerative disease research.

The laboratory is involved in collaborative activities with the objectives to (i) understand the molecular assembly and activation of the presenilin complex in Alzheimer’s disease; (ii) map the molecular environment that hosts the conversion of the prion protein in Creutzfeldt-Jacob disease; and (iii) elucidate molecular events that underlie the intracellular deposition of Lewy bodies and cell death in Parkinson’s Disease.

During the past 12 months the laboratory has continued to grow. We have hired two postdoctoral fellows with expertise in proteomics / mass spectrometry and target validation applications. We have used the CRND’s new mass spectrometer to assemble data from a presenilin-1 (PS1) interactome mapping study and have described a novel negative regulator of PS1 that intriguingly selectively inhibits γ-secretase mediated Aβ production without affecting ε-site cleavage of APP or ε-site cleavage of other established γ-secretase substrates. We have combined tcTPC with iTRAQ labeling to generate quantitative interactome data of APP and its two mammalian homologues APLP-1 and APLP-2. We also collected comprehensive tcTPC interactome data for nicastrin, DJ-1, PrP, α-synuclein. Each datasets revealed interesting candidate interactors currently being followed-up on in CRND collaborations with Dr. A. Tandon, Dr. P. Fraser, Dr. D. Westaway, Dr. P. St. George-Hyslop. Finally, with Drs Fraser, St George-Hyslop, and Rogaeva we have worked on the biological characterization of a new candidate gene for late onset forms of Alzheimer Disease.

DR. JANICE ROBERTSON

Our laboratory is dedicated to the study of the disease mechanism(s) causing amyotrophic lateral sclerosis (ALS). We use a multidisciplinary approach that includes live cell imaging, animal modeling, molecular-cell biology techniques and biochemical/neuropathological analyses of human ALS brain and spinal cord material. Currently we are following three lines of investigations:

(1) Understanding how neurofilament and peripherin abnormalities occur in ALS. Cytoplasmic inclusions comprised of neurofilaments and peripherin are a common pathological finding in ALS. How they are formed and their relationship to the disease mechanism is not known. We have been studying a process called alternative splicing, whereby several mRNA transcripts can be derived from a single gene (Robertson et al, JCB 160:939-49, 2003). In this regard we have identified four alternatively spliced variants of peripherin and two of the neurofilament subunit, NF-L. We have shown that deregulated expression of these variants occurs in ALS and can cause neurofilament and peripherin aggregation. We are currently studying the functional properties of these splice variants.

(2) Studies of the neurotoxicity of mutant superoxide dismutase-1 (SOD1). Over 100 different mutations in the SOD1 gene are causative of about 2-3% of ALS cases. In collaboration with Dr Avijit Chakrabartty we have been studying disease relevant folding intermediates of SOD1using an epitope specific antibody. We are also studying the proteins that interact with mutant SOD1 using mass spectrometry approaches and testing various therapeutic strategies in mutant SOD1 transgenic mice.

(3) Neuroinflammation and ALS. Activated microglia are associated with degenerating motor neurons in ALS. We have shown that the proinflammatory cytokine TNF-alpha released by activated microglia promotes apoptosis of neurons containing peripherin aggregates (Robertson et al, JCB 155:217-26, 2001). Subsequently it has been demonstrated that motor neuron death in mutant SOD1 transgenic mice is non-cell autonomous and that factor(s) released by activated microglia are important to the disease mechanism. We are investigating this using a co-culture paradigm of primary motor neurons and microglia.

We have established a core group of clinician/scientists that have a common interest in ALS. As such, we have initiated a program of blood collection (for genetic analysis) and of tissue collection (for biochemistry and neuropathology) from ALS cases seen by our team member, Dr Lorne Zinman, Medical Director of the ALS Clinic at Sunnybrook Health Sciences Centre. With over 200 new cases diagnosed at the clinic every year, it is expected that we will build a sizeable resource in which to undertake our ongoing studies.