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Team Parkinson is using its distribution to fund the following:


In the following proposal for The Parkinson Alliance and Team Parkinson we outline important research needs from several investigators within the Parkinson’s Disease Research Group at the University of Southern California. The grant to USC will be divided up equally in the labs of the following investigators including Project 1, Giselle Petzinger, MD; Project 2, Michael Jakowec, PhD; Project 3, John Walsh, PhD; Project 4, Jennifer Hui, MD; and Project 5, Mickie Welsh, PhD. The following sections describe the details of the use of the funds. For the first 3 Projects funds will be used for specific equipment needs while in Projects 4 and 5 funds will be used in part for data collection and analysis, and hence have larger descriptions of the projects.

 

PROJECT 1:
Giselle Petzinger, MD Assistant Professor
Department of Neurology, USC Keck School of Medicine, Division for Movement Disorders.

There are 2 major equipment needs in the laboratory that are necessary to support ongoing studies. One project involves the examination of changes in connections between neurons occurring within the medium spiny neurons of the basal ganglia. The structure of communication between neurons is at a specific anatomical feature called the spine. We are examining how the structure and number of spines changes in animal models of Parkinson’s disease and how changes that are detrimental can in fact be reversed to result in improvement in behavior and in fact may help to understand the molecular mechanisms for developing new therapeutic treatments for PD. To make these studies possible we are in need of a specialized objective lens for our Olympus BX-51 system. This lens is a 60X water immersion lends with a depth of field greater than other lenses allowing us to trace neuronal processes to allow for their tracing and reconstruction in our computer assisted image analysis system.

PROJECT 2:
Michael Jakowec, PhD, Assistant Professor
Department of Neurology, USC Keck School of Medicine, Division for Movement Disorders.

An important technical aspect of our studies in the lab is the analysis of neurochemicals using High performance liquid chromatography. Our present system from ESA Instruments (Chelmsford, MA) is a state of the art unit. However, there are a number of components that are in need of replacement including aspects of the detector system and column separation. In addition, we are in need of obtaining a new precision balance necessary for weighing chemical reagents in the low mg range, which is used in conjunction with our HPLC system.

Items to be purchased include Ohaus precision balance, ESA electrochemical cell replacement for HPLC Coularray electrochemical detector, Agilent separation columns for ESA HPLC system, maintenance kits for autosampler, pumps, and fittings, chemicals, reagents, tubing, and vials for HPLC system.

PROJECT 3:
John Walsh, PhD, Associate Professor
Andrus Gerontology Center, University of Southern California

One of the strengths of our research program in PD here at USC is the application of electrophysiological analysis to examine neuronal function. Despite our ability to obtain exciting and important findings, some of which has led to 2 Journal of Neuroscience manuscripts we have some technical limitations that can be overcome with the addition of some key components to our current equipment. These items will make possible electrophysiological studies in conjunction with fluorescence to examine and identify important changes within the basal ganglia of unique animal models of PD we have available now in our lab. These items include U-P106, U-DICTS transmitted DIC prism slider, shift type, 3-U806, U-DPTS; modular dual port for use with trinocular obs tube; 5-UR710A, BX-RFAA fluorescence illumin, 6-cube turret, GFP specific florescent cube.

PROJECT 4:
Jennifer Hui, MD, and Deborah Won, PhD
Department of Neurology, USC Keck School of Medicine, Division for Movement Disorders.

Use of quantitative EEG to assess cognitive effects of STN-DBS in Parkinson’s disease

Specific Aim 1: To assess the cognitive effects of STN DBS in moderate-advanced Parkinson’s disease

Hypothesis: STN DBS will result in cognitive decline in a subset of Parkinson’s disease patients

Specific Aim 2: To correlate measures of cognitive decline with quantitative EEG recordings

Hypothesis: Quantitative EEG recordings will show changes in synchronization and frequency in the frontal lobes correlating to cognitive impairment on frontal lobe tasks

Background and Significance:
Parkinson’s disease (PD), a neurodegenerative disease of the basal ganglia (BG), causes patients to exhibit resting tremor, bradykinesia, rigidity, postural instability, depression, and cognitive impairment, all of which are disabling and can be detrimental to the patients’ quality of life [1]. Deep brain stimulation (DBS) has been used to treat the motor deficits in over 30,000 PD patients since its FDA approval. While DBS has demonstrated great success in suppressing tremor and relieving other motor symptoms [2-4], its effect on cognition and behavior has been variable [5-12]. Studies reveal declines in executive function, verbal memory, verbal fluency, mood, and other non-motor symptoms after DBS, but few systematic efforts to understand the cause or minimize these effects have been documented.

Specific changes in quantitative EEG recordings have been documented in Parkinson’s disease with and without dementia [13]. These include abnormal slow rhythm frequencies (delta, theta) and changes in dominant posterior background rhythm frequency. Specifically, changes in dominant posterior background activity and global relative EEG power in the delta and alpha frequencies have been shown to correlate with decline in the Mimi-Mental Status Exam in PD, and to differentiate between PD with normal cognition, mild cognitive impairment, and dementia [13].

DBS involves the placement of a 4-contact electrode lead in the subthalamic nucleus in the basal ganglia (BG), inserted through a tract that passes through the frontal lobe. The lead is connected to an implantable pulse generator (IPG) which is programmed to control the stimulus parameters. Patients can receive unilateral or bilateral implantation depending on their symptomatology.

DBS surgery can influence cognition in several potential ways. Most directly, the passage of an electrode through the frontal lobe, often after several attempts, may physically disrupt thalamocortical circuits involved in cognition. In addition, the STN sends synaptic projections to the anterior cingulate gyrus and neighboring areas in the prefrontal cortex, areas implicated in cognitive behavior [24, 25]. A limited number of studies have shown that stimulation of STN causes significant increases in synaptic activity in these regions. Hence, stimulation of STN may influence cognition and behavior through the existing anatomical infrastructure. Although one group has implicated that electrode placement and stimulation parameters are possible factors in the appearance of cognitive changes, the physiological correlates mediating these changes are not known [9].

Oscillatory activity in the frontal cortex may provide the desired metric for neuropsychiatric effects. It has been shown that the levels of activity in associative frontal areas increase with DBS in STN [26], and animal studies have demonstrated that connections between medial frontal cortex and STN play a significant role in attention and perseveration [27, 34, 35]. LFP synchronous oscillations have been found to correlate with clinical motor symptoms [36-41]. Such pathological activity is hypothesized to be paralleled in the circuits related to emotion and cognition, since the striatum participates in the limbic and associative thalamocortical loops, analogous to its participation in the sensorimotor loop.

Methods: Subjects with PD, who are appropriate candidates for STN DBS, as assessed by a Movement disorders specialist, will be recruited from the Neurology Clinic at the University of Southern California. Subjects routinely undergo a standardized pre-operative evaluation process, including assessment of severity of PD using the UPDRS, formal neuropsychological testing, and MRI of the brain. Neuropsychological testing includes measures of verbal fluency (animal recall), semantic memory, and frontal lobe tasks and assessment of executive function (Stroop task, Trails A and B). In addition to the standard pre-operative assessment, subjects will undergo a scalp EEG recording prior to DBS surgery. 6 months post-operatively, subjects will undergo repeat neuropsycholoigcal testing and scalp EEG recording. Changes in specific areas of cognition will be correlated to quantitative changes on the EEG.

Data analysis: Because of the difficulty of keeping a patient in testing for long periods of time and therefore of collecting large amounts of data, a computational model will be used to help formulate hypotheses about possible mechanisms of the interaction between the electrical stimulation in STN and the function of the limbic thalamocortical loop. Computational models of DBS stimulation have only recently been produced as have ones of the oscillatory activity in the basal ganglia [42-45]. Only two studies have been published which incorporate the effects of DBS on BG network activity, and none of these models include the limbic or associative circuits[43, 44].

Significance: DBS is an important clinical tool in the treatment of Parkinson’s disease, significantly improving motor functioning. However, post-operative changes in non-motor symptoms such as cognition can also impact quality of life. The pathophysiology of these changes is not clear, and there are currently no clear predictors of cognitive decline for patients who are considering DBS surgery. This study hopes to better elucidate the striatal-frontal connections that may underlie cognitive decline after DBS surgery, and potentially provide a clinically relevant screening tool for patients considering the procedure.

Budget: Patients for this study have already been enrolled and these dollars will be used for the collection of data from these patients, the analysis of data including statistical methods.

PROJECT 5:
Group Exercise in Persons with Parkinson’s Disease
George Salem PhD., Abbie Ferris, Mickie Welsh RN, DNSc.

Background: Although the prescription of exercise is standard care for patients with Parkinson’s Disease (PD), well-designed exercise studies that have assessed the safety & efficacy of activity-intervention modalities, are sparse for this cohort. Therefore, it is unclear which types of programs are most beneficial for addressing disease related symptoms associated with PD such as decreased strength, increased fall risk and depression. A recent report suggests that the inclusion of cueing strategies is one effective modality for improving walking performance in patients with PD (Suteerawattananon, 2004). Yoga is an exercise activity ideally suited to provide visual, auditory, and sensory cues in addition to “…combine(ing) balance, flexibility, and strengthening benefits.” (The National Institute of Diabetes & Digestive & Kidney Diseases Publication). Yoga is also being promoted as a safe and effective exercise program, capable of increasing the strength, flexibility, and functional capacity of older & younger adults including those in robust physical condition as well as those with musculoskeletal disorders (Kolasinski, 2005; Raub, 2002; Tran, 2001). Additionally it has been shown as an effective therapy for decreasing depressive symptoms in young adults (Shapiro 2007).

Additionally, other exercise approaches, such as combined resistance/endurance training programs, may also be suitable for PD patients. Circuit-training programs, such as those promoted by Curves? and Cuts?, are widely popular in the US as they emphasize both muscular strength and cardiovascular endurance. Moreover, these programs have been proven effective and safe for older adults (Takeshima 2004). It is reasonable to believe these positive results may transfer to PD patients.

To this end, both modalities of exercise target large and small muscle groups, which are vital in sustaining balance and challenging the cardiovascular system. Because both Yoga and circuit-training programs are likely to improve the physical and mental health, the goal of this proposal is to compare the safety and efficacy of circuit-training and Yoga programs, in improving physical performance and overall quality of life in persons with PD.

Our research group has begun a feasibility study into the efficacy and safety of Yoga and Circuit training in persons with Parkinson’s Disease. (IRB: HS-07-00517). This is an eight-week study involving seven participants randomized in to two groups: Yoga (n = 3) and Circuit Training (n = 4). Classes are held biweekly in the Division of Biokinesiology and Physical Therapy. Measurements of depression, strength, quality of life, UPDRS, fear of falling, self perception, activity scale, and biomechanical analysis are measured at three time points: 8 weeks prior to the start of the study, one week prior to the study, and upon completion of the study. Participants also fill out a weekly questionnaire regarding pain/discomfort, enjoyment of the class, and personal comments. Upon completion of the class, a follow-up questionnaire will also be filled out by the participants regarding willingness to continue in future classes, self-perception of balance, PD symptoms, and strength compared to before the start of the classes.

Specific aims for the current study include:

Specific Aim 1: To quantify the change (baseline, 8-weeks, 17-weeks) in balance as determined by the Fullerton Advanced Balance (FAB) test, Activities-specific Balance Confidence Scale (ABC), and biomechanical variables (walking, standing, turning tasks), following completion of the Yoga and circuit training programs.

Specific Aim 2: To quantify the change (baseline, 8-weeks, 17-weeks) in depression using the Center for Epidemiological Studies-Depression Scale (CES-D), following the Yoga or circuit training interventions.

Specific Aim 3: To quantify changes (baseline, 8-weeks, 17-weeks) in, walking speed, lower extremity mechanics, and upper and lower extremity strength, following completion of the Yoga or circuit training program.

Specific Aim 4: To characterize changes (baseline, 8-weeks, 17-weeks) in the UPDRS score following completion of the Yoga or circuit training program.

Currently we are in our 6th week of classes for both groups. Thus far, participants have enjoyed their classes and ask if the classes can continue after the study is over. Attendance rate is extremely high 97% and self reported pain/discomfort levels are low (0-3 on a 10 point scale) and occur most frequently in the Circuit group. Data from this feasibility study will be used to power a more long term study (16 weeks) and evaluate the safety and effectiveness of Circuit or Yoga training for people with PD. Additionally, results from this study will guide the 16-week study development and direction. We hope to refine our current data collection methods/outcomes and begin the new study very soon after completion of this feasibility study.

Budget: Patients for this study have already been enrolled and these dollars will be used for the collection of data from these patients, and the analysis of data including statistical methods.




Project Title:
Sleep Apnea and Other Sleep Disturbances as Predictors of Progression and Health-Related Quality of Life in Parkinson’s Disease

Investigator: Dr. Barbara Vickrey, UCLA School of Medicine, Dept. of Neurology

A range of sleep disturbances are relatively common in patients with Parkinson’s Disease. These include insomnia (difficulty initiating or maintaining sleep), parasomnias, and excessive daytime sleepiness (hypersomnolence). Causes are likely multifactorial but include sleep apnea, medication effects, depression, and comorbidities including pain. Sleep apnea in particular has been linked to increased mortality among individuals with stroke, and appears to have a higher occurrence in Parkinson’s disease than the general population. In addition, manifestations of sleep apnea include excessive daytime sleepiness, which is likely to have substantial impacts on health-related quality of life.

Our goal with this request is to tease apart the extent of occurrence and the unique impact of sleep apnea and other sleep disturbances (i.e., sleep quality, insomnia) on progression of motor and non-motor manifestations of Parkinson’s disease, as well as on health-related quality of life. To do this, we will add on two self-report sleep questionnaires, the Berlin Questionnaire for sleep apnea, and the Medical Outcomes Study sleep scale, to an ongoing observational cohort study of over 300 patients with movement disorder specialist-confirmation of Parkinson’s disease. This cohort is a population-based, representative sample of persons with recently-diagnosed Parkinson’s disease, now being followed in an NIH-funded study through the UCLA Udall Center to measure how the development and rate of progression of motor and key non-motor manifestations in PD - cognitive impairment and depression - are influenced by environmental, behavioral, and social factors, including pesticide exposure, physical activity, stress, coping, and social support.

The grant from Team Parkinson and The Parkinson Alliance will allow us to support the collection of the additional sleep measures and to analyze these data to determine the impact of sleep apnea and other sleep disturbances on motor progression, non-motor progression, and changes in health-related quality of life over a 2-year follow-up interval in this population-based cohort of persons with Parkinson’s disease.


Project Title:
Benomyl Exposure as a Risk for Developing Parkinson’s Disease; Studies in Primary Neuronal Culture

Investigator: Dr. Nigel Maidment, UCLA School of Medicine, Dept. of Neurology

The causes of Parkinson’s disease (PD) remain elusive but epidemiology studies have implicated pesticide exposure as a risk factor for developing the disease. Recent studies from our group have identified benomyl, a commonly used pesticide, as one of a few agents that may contribute to the pathogenesis of PD. We have also determined that benomyl is an inhibitor of a degradative pathway (the ubiquitin proteasome system, UPS) that has been implicated in the pathogenesis of PD. Furthermore, benomyl inhibits microtubule formation and aldehyde dehydrogenase activity, cellular processes that have both been implicated in PD. Taken together, benomyl is an excellent candidate toxicant that might increase one’s risk of developing the disease.

Few studies have investigated benomyl toxicity to neurons. We propose in these studies to isolate primary neurons (dopaminergic and non-dopaminergic) from the midbrains of neonatal rats. Cultures will be exposed to different concentrations of benomyl and related analogues to determine cell toxicity and the mechanisms of this toxicity. We will not only measure cell death, but also dopamine and dopamine metabolites using high performance liquid chromatography. Furthermore, we will study benomyl’s effects on a-synuclein metabolism, a potential mediator of toxicity.

The grant from Team Parkinson and Parkinson Alliance will allow us to obtain preliminary data necessary for obtaining a larger grant from the NIH to study the role of benomyl and related compounds in the pathogenesis of PD.

Project Update:

We proposed to isolate primary neurons (dopaminergic and non-dopaminergic) from the midbrains of neonatal rats.  Cultures were exposed to different concentrations of benomyl and related analogues to determine cell toxicity and the mechanisms of this toxicity.  We will not only measure cell death, but also dopamine and dopamine metabolites using high performance liquid chromatography. Furthermore, we will study benomyl’s effects on a-synuclein metabolism, a potential mediator of toxicity.

Specific achievements:

Benomyl’s toxicity was tested in primary ventral mesencephalic cultures (VMCs). Exposure to 1 μM benomyl (n=45) for 48 hours resulted in a 35% selective loss of dopaminergic (TH+) neurons as determined by immunohistochemistry. This effect was recapitulated by exposure to a benomyl metabolite shown to inhibit ALDH, S-methyl-N-butylthiocarbamate (MBT). 1 μM MBT (n=14) resulted in a 31% loss of TH+ neurons, whereas exposure to another metabolite, the MT and UPS inhibitor carbendazim (1 μM, n=14), did not significantly affect viability treatment with the MAO inhibitor pargyline prevented the loss of TH+ neurons exposed to 1 μM benomyl, presumably due to the prevention of DOPAL accumulation that can result from ALDH inhibition.
       
Further support for ALDH inhibition playing a role in benomyl’s toxicity comes from measurement of dopamine metabolites. Compared to vehicle controls (n=8), brain homogenates from adult zebrafish injected with benomyl (300 mg/kg, n=4) exhibited 19%, 38%, and 16% respective decreases of DOPAC/DA, HVA/DA and 5-HIAA/5-HT ratios; a 39% increase of 3-MT/DA; and 16% and 33% increases of DA and 5-HT concentrations, consistent with ALDH inhibition.
    
The results from these studies are very exciting because it supports a novel biochemical pathway that appears to be involved in how some pesticides increase the risk of PD.  These data not only have become the basis of a large NIH Center, but has also led to investigating the genetics of ALDH.  Preliminary studies are extremely exciting.




Project Title:
Studying Genetic and Environmental Causes of Parkinson’s Disease Using Zebrafish

Investigator: Jeff Bronstein MD, PhD, UCLA School of Medicine, Dept. of Neurology

The cause of Parkinson’s disease (PD) remains elusive but almost certainly involves gene-environmental interactions. A number of potential risk factor genes and environmental toxins (especially pesticides and proteasome inhibitors) have been implicated but it is still not known if these factors actually cause PD. Current animal models to test causality of these interactions are inadequate. We propose to use zebrafish to test potential gene-environment interactions because they have several advantages over current animal models. Zebrafish are vertebrates, small, have a short life cycle, are relatively easy to insert transgenes, and the larvae are transparent enabling imaging of molecular and cellular processes in living animals. Behavior can also be readily measured. Preliminary data suggests that several pesticides are proteasome inhibitors and administration of a proteasome inhibitor alters swimming behavior. In this study, we propose to expose transgenic zebrafish, that express a fluorescent protein in dopamine cells, to pesticides and proteasome inhibitors and determine whether they cause a PD-like condition. We will also make transgenic fish that express gene mutations that are believed to cause or increase one's risk of developing PD in humans. If successful, a large number of potential interactions implicated in causing PD can be readily tested.

The grant from Team Parkinsons will be used to primarily to construct equipment to evaluate different behaviors associated with PD (altered motor behavior and smell). Studies are being performed in Dr. Bronstein’s lab and in the lab of Dr. Carlos Portera, a close collaborator.

Project Update:

We proposed to develop and use a novel zebrafish (ZF) model to study genetic and environmental causes of Parkinson’s disease (PD).  I am happy to report that we have made great progress towards completing our goals.

Specific achievements:


Tyrosine-Hydroxylase Green Fluorescent ZF:   We have successfully created a transgenic ZF in which their dopamine neurons turn a fluorescent green color (referred to as TH-GFP ZF).   This allows us to visualize the health of dopamine neurons in living ZF as well as determine their function using a high power laser.  We have systematically killed dopamine cells and have determined that a few dopamine neurons are essential for normal swimming behavior and a few others are essential for life.

Pesticide exposure is associated with an increased risk of developing PD and through a number of studies in humans and in cells, we have identified a number of specific toxins that maybe responsible for this increased risk.  We have begun exposing the TH-GFP ZF to pesticides and measured their ability to swim and determined the health of their dopamine neurons.  Very low concentrations of a commonly used fungicide called ziram (1-10 nM) caused the ZF to swim in an abnormal fashion and practically wiped out their dopamine cells.  We are now testing the toxicity of other putative pesticides in this model.
       
Alpha-Synuclein (a-syn) Neurotoxicity in ZF
:  It is clear that increased a-syn in neurons is involved in the pathogenesis of PD but few models are available to study its toxicity. We are studying a-syn neurotoxicity by genetically altering ZF to make high levels of human a-syn.  These ZF showed many of the key aspects seen in human PD.  We found that human a-syn tended to clump up in the neurons (aggregates) and caused the neurons (and eventually the ZF) to die.  We also found that theses clumps caused the machinery that normally removes a-syn to dysfunction further exacerbating the problem.  These a-syn ZF appear to be an excellent model for testing potential therapies.  For example, CLR01 is a small molecule designed by a UCLA researcher (Dr. Gal Bitan) to break up a-syn clumps.  We treated our a-syn ZF with CLR01 and found that it reversed or prevented almost all of the defects caused by the overexpression of a-syn.  We are now testing other potential treatments.

In summary, we have made important progress in our studies using ZF to study PD.  Importantly, these studies funded by the Parkinson Alliance have resulted in obtaining a NIH R21 and a Michael J. Fox Foundation Award to Dr. Gal Bitan to further the CRL01 studies to rodents based on the results described here.

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