Gene Expression Responses in a Cellular Model of Parkinson's Disease Louis Beverly Brill II Manassas, Virginia B.A., Johns Hopkins University, 1995 A Dissertation presented to the Graduate Faculty of the University of Virginia in Candidacy for the Degree of Doctor of Philosophy Department of Cell Biology University of Virginia May, 2004 Table of Contents Chapter 1 . 1 Chapter 2 . 48 Chapter 3 . 87 Chapter 4 . 123 Chapter 5 . 133 References . 137 Appendix A . 163 Appendix B . 209 Appendix C . 216 Appendix D . 223 Appendix E . 232 Appendix F . 234 Appendix G . 283 Appendix H . 318 Appendix I . 324 Abstract This research represents initial steps towards understanding the relation between changes in gene expression, mitochondrial function and cell death in cell-based models of Parkinson’s disease. The main hypothesis is that rapid gene expression changes in cells exposed to parkinsonian neurotoxins occur, are dependent on mitochondrial status, and directly impact intracellular signaling pathways that determine whether a cell lives or dies. Our cellular model is comprised of SH-SY5Y neuroblastoma cells exposed to the parkinsonian neurotoxin methylpyridinium ion. Transcriptomic changes are evaluated with nylon and glass-based cDNA microarray technology. Cardinal symptoms of Parkinson’s disease, characteristic pathological changes, therapeutic modalities, and current theories on the etiology of the disorder are discussed. Our results verify the existence of mitochondrial-nuclear signaling in the context of electron transport chain deficits, as well as suggesting the vital roles played in this process by previously described intracellular signaling pathways. These results will serve to direct future investigations into gene expression changes relevant to the processes of cell death and cell survival in our cellular model of Parkinson’s disease, and may provide important insights into the pathophysiology of the in vivo disease process. 1 Chapter 1 My research in the Center for the Study of Neurodegenerative Disease has explored the relation between changes in gene expression, mitochondrial function and cell death in cell-based models of Parkinson’s disease. The main hypothesis behind my work is that rapid gene expression changes in cells exposed to parkinsonian neurotoxins occur, are dependent on mitochondrial status, and directly impact intracellular signaling pathways that determine whether a cell lives or dies. I have attempted to bring two novel experimental techniques to bear upon this problem: cDNA microarrays and RNA interference (RNAi). This introductory chapter will serve as a review of the background information necessary to place the dissertation research in its proper context. The cardinal symptoms and pathology of Parkinson’s disease will be discussed, as will the primary therapeutic modalities, and major hypotheses regarding the etiology of the disorder. The most recent developments in cDNA microarrays and RNA interference (RNAi) will be reviewed. Subsequent chapters will document my experimental techniques and results, along with my suggestions for future directions in cellular and molecular Parkinson’s disease research. 2 Background Information Parkinson’s Disease Parkinson’s disease is a progressive neurodegenerative disorder that can cause profound motor impairment in its victims. It is among the most prevalent neurological disorders, with approximately 50,000 new cases diagnosed each year in the United States alone. The four cardinal symptoms of PD are bradykinesia, tremor at rest, postural instability, and rigidity. Tremor is the symptom that is often first to prompt a patient to seek medical attention; the tremor of PD is described as a “pill-rolling” tremor, referring to the stereotyped motion of the fingers and hands. It is of low frequency (about 4-5 cycles per second) and occurs at rest. The characteristic Parkinsonian tremor tends to reduce in severity or even disappear when the patient initiates a voluntary movement. Bradykinesia, a slowing of movements in general, is often present early in the clinical course of PD and can become highly debilitating. Inability to execute sequential motor tasks as a result of bradykinesia frequently has severe impacts on the patient’s ability to perform activities of daily living. Rigidity is generally manifested in movements of the extremities (cogwheel rigidity) and, in moderate to severe cases of PD, episodes of “freezing,” or the inability to initiate any voluntary movements. In addition to the obvious reduction in ability to perform activities of daily living, such episodes often provoke intense feelings of fear and helplessness, with negative impacts on the patient’s mental and emotional well- 3 being. Some reports have also detailed the phenomenon of akinesia paradoxica, referring to temporary periods of normal or near normal motor activity under stressful situations. Postural abnormalities manifest in the PD patient with unusual degrees of flexion in the limbs. The trunk is often stooped or hunched forward, and when combined with flexion of the knees and elbows, the patient’s center of gravity is frequently displaced forward to a sufficient degree to cause the characteristic shuffling gait of PD. As the disease progresses, cerebellar reflexes required to maintain balance are lost, with the frequent onset of episodes of falling. Falls are a major cause of morbidity and mortality in PD patients, as for the elderly population at large. Other non-motor symptoms frequently associated with the disease process, particularly in its early stages, include a generalized loss of manual dexterity, manifesting as difficulty in performing simple motor tasks, mild depression, a reduction of facial expressions (mask facies) and micrographia, a reduction in size of handwriting. On the order of forty percent of PD patients present with non-specific sensory complaints, including numbness, tingling, aching or burning sensations, particularly in the extremities. A high percentage of patients present with anosmia, loss of the sense of smell. Anosmia may occur years before onset of the cardinal motor symptoms of PD. As the olfactory tissues are directly exposed to the environment, this phenomenon has been invoked by some researchers as an endorsement of the environmental hypothesis of PD, i.e., that a neurotoxic agent responsible for PD present in the 4 environment either gains entry to the brain via the olfactory nerves, or that the olfactory receptors alternatively represent a highly vulnerable neuronal population whose demise is linked to chronic exposure to a Parkinsonian neurotoxin. Dementia is frequently reported in PD, but there exists considerable variability in reports of its rate of occurrence (10-40%). Some investigators have reported a higher prevalence of dementia in PD in males, and in those patients of more advanced age. Likewise, depression is reported in a variable but high portion of the PD population (37-90%). As depression often occurs early in the clinical course, It is unclear whether the majority of depressive symptoms are related to the etiology of PD, or rather a reaction to symptoms on the part of the patient. Historical Context Parkinson’s Disease (PD) is generally referred to as the archetype of the hypokinetic movement disorders. The symptoms of this condition were first described in the 1817 "Essay on the Shaking Palsy" by James Parkinson, a general practitioner in the suburbs of London (Parkinson 1817). The impetus for the essay was Parkinson's keen observations of Londoners with characteristic gait abnormalties, tremors and muscle weakness that tended to increase in severity with advancing age: 5 "The patient can [rarely] form any recollection of the precise period of its commencement. The first symptoms perceived are, a slight sense of weakness, with a proneness to trembling in some particular part; sometimes in the head, but most commonly in one of the hands and arms…. The propensity to lean forward becomes invincible…. As the debility increases and the influence of the will over the muscles fades away, the tremulous agitation becomes more vehement." Parkinson and his contemporaries were hamstrung by the lack of a standardized system of neurological examination, a problem that continues in part to the present day. PD remains a diagnosis that is based primarily on clinical observation by the physician, with autopsy examinations of brain tissue and quantitative assessments of symptoms playing secondary roles. It was not until half a century following the publication of the Essay that Parkinson's name became permanently associated with the condition, formerly referred to as paralysis agitans, following acceptance by preeminent neuroscientists of the time such as Charcot and Gowers (Charcot 1861; Gowers 1893). Following the devastating epidemics of influenza in 1918-1919, a population of patients exhibiting peculiar parkinsonian symptoms began to present at neurological clinics throughout Europe, Australia and the Americas. In addition to parkinsonian motor symptoms, cranial nerve palsies, oculogyric crisis and severe somnolence were common components of this disease. It was initially described as a form of “chronic encephalitis,” and was only later to be described as postencephalitic parkinsonism, or encephalitis lethargica. One of the eminent neurologists of the period, Dr. Constantin von Economo, presented 6 his description of the condition to the Vienna Psychiatric Society in April of 1917. As early as 1916, he described the typical syndromes in a monograph: