2008 Grants

Share

Funding from The Parkinson Alliance helped to finance the following Parkinson's research. Grantees were selected by scientific review committees of participating organizations. Updates will be posted, when available.


Project Title: PPAR Receptors: A New Target for the Treatment of Levodopa-Induced Dyskinesias

Grant Awarded to:  Andrea Giuffrida, PhD – Assistant Professor, Pharmacology

Objectives:  To identify the PPAR receptor subtypes responsible for the anti-dyskinetic effects of FAAH inhibitors and evaluate the efficacy of selective PPAR agonists as anti-dyskinetic agents.

Background: Levodopa-induced dyskinesias (LID) can be modeled via chronic administration of levodopa to rats with unilateral denervation of the nigrostriatal pathway produced by the neurotoxin 6-OHDA. In these rats, chronic levodopa induces increasingly severe axial limb and oro-facial abnormal involuntary movements (AIMs) that have been extensively characterized and pharmacologically validated. Previous studies in our lab showed that activation of CB1 cannabinoid receptors by WIN55212-2 (WIN) significantly alleviates AIMs in 6-OHDA-treated rats. However, pharmacological blockade of the endocannabinoid inactivating enzyme FAAH by URB597 (URB), results in the elevation of the endocannabinoid anandamide in the brain and decreases levodopa-induced AIMs in a CB1-independent fashion and only when, co-administered with the TRPV1 antagonist capsazepine (CPZ). These data indicate that endocannabinoids differ from exogenous cannabinoid agonists, like WIN, for their ability to modulate LID, and suggest the involvement of alternative endocannabinoid-sensitive pharmacological targets.

Methods/Design: The investigators characterized the neurochemical effects of subchronic WIN rats with unilateral 6-OHDA lesions.

Project Update:

Results: WIN prevents levodopa-induced glutamate unbalances across the two brain hemispheres. The severity of dyskinesias is inversely correlated to the glutamate levels in the denervated striatum. WIN exerts its anti-dyskinetic effects not only via activation of CB1 receptors, but also by desensitizing TRPV1 receptors and reversing levodopa-induced PKA over-activity in the striatum of dyskinetic rats. The investigators were able to provide experimental evidence that the PPAR? receptor, a transcription factor activated by anandamide, represents a new pharmacological target for the treatment of LID.

Conclusion/Relevance to Parkinson’s Disease: The research has identified new pharmacological targets (CB1, TRPV1 and PPAR? receptors) for the treatment of levodopa-induced dyskinesias, which are complications experienced by the majority of PD patients as consequence of their long-term use of levodopa. These data open new avenues to investigate the role played by these receptors, and their related signaling cascades, in the development and/or prevention of levodopa associated motor disturbances and side effects. The discovery that the PPAR? agonists Rosiglitazone and Piogiitazone have antidyskinetic properties is of particular relevance since these drugs are: 1) already available on the market as anti-diabetic agents; 2) do not interfere with the anti-parkinsonian activity of levodopa nor produce general motor-suppression, making them particularly for clinical studies in PD patients.


Project Title:  Creating dopaminergic neurons with authentic Parkinson's disease - A new path to drug discovery and understanding mechanisms of neurodegeneration.

Investigator:  Dr. J. William Langston, Founder, CEO, and Scientific Director of the Parkinson's Institute and Dr. Birgitt Schuele, MD, Director of Neurogenetics, Parkinson’s Institute.

Study Goal:  The goal of this study is to derive induced stem cell lines, known as human-induced pluripotent stem (iPS) cells from skin biopsies taken from patients with Parkinson's disease (PD).  More specifically, we wish to derive iPS cells from patients with parkinsonism due to genetic causes, including mutations in the genes that code for a number of different genetic forms of parkinsonism (PARK 8 or LRRK2, PARK 1 or SNCA [alpha-synuclein], PARK 2 or PARKIN, PARK 6 or PINK1, and late-onset Gaucher disease or GBA). We will then use established technologies to differentiate these cells into dopamine neurons, one of the key neuronal populations that degenerate in PD.  This work also includes collaborations with academic medical centers such as Stanford University as well as industry partners. 

Hypothesis to be tested:  The underlying hypothesis of this proposal is that these "re-born" dopaminergic neurons that started out as skin cells from patients with PD will recapitulate one or more of key molecular and/or morphological aspects of neural degeneration associated with PD.  If successful, we will for the first time ever have truly parkinsonian dopamine nerve cells in a tissue culture dish (“PD in a dish”).  We believe in a high probability of success since these cells will have been obtained from patients with parkinsonism of a known cause, and that cause will have been carried with the iPS cells in the form of a known mutation in their DNA.

Significance: These proposed studies have the potential to provide an entirely new tool for investigating disease mechanisms of PD.   Furthermore, these "parkinsonian cells" could provide a transformative way to screen large numbers of drugs to see if they can prevent or even reverse the disease process.  The power of this approach is that it could, for the first time, provide “authentic Parkinson's disease cells” that are not a “best guess” model, but neuronal populations with actual PD pathology.  In fact, there is a general consensus that the reason why clinical trials aimed slowing or halting disease progression have not had more success is that to date we have not had an truly predictive model in which to test drugs pre-clinically.  This is important because studies in our iPS cell system would be directly relevant to humans with the disease. 

Project Update:

Progress in specific areas: Over the last nine months we have made a exciting progress towards the goal of this study that is modeling pathology of Parkinson’s disease in a culture dish.

* Isolation of human fibroblasts: Human skin cells were isolated from biopsies of 25 patients with both sporadic and genetic forms PD and controls individuals from the Parkinson’s Institute and we have established several collaborations with other clinical centers to ascertain skin cells from different mutation carriers.

* iPS cell derivation: Our first focus has been on fibroblasts derived from a patient with an SNCA triplication and an unaffected sibling. Primary skin cells were reprogrammed through infection with four viruses that expressed specific proteins for reprogramming named Oct3/4, Sox2, Klf4, and c-Myc. Approximately 20-25 days after infection, potential iPS cell colonies were mechanically picked based upon morphological similarity to human embryonic stem cell (hESC) colonies and were expanded on feeder plates. These clones survived in culture for more than 30 passages and were similar to undifferentiated hESCs in morphology and show all characteristics of hESCs.

* Derivation of midbrain dopaminergic neurons: To determine the ability of our genetically defined SNCA triplication hiPSC line and the sibling control, we differentiated them into midbrain DA cells using mechanical isolation and temporal application of growth and signaling factors. To confirm the enrichment of DA neurons, mRNA and protein markers were assessed that were indicative of immature neurons and midbrain DA neurons.

* Phenotypic differences in hiPSC-derived dopaminergic cells: To determine specific protein overexpression in derived neurons, cells were analyzed by rt-PCR and immunocytochemistry, and Western blot analysis. We consistently observed an overexpression/accumulation of specific proteins known to be involved in the pathology of PD.

* New Grant: We are very excited to announce that this research can now be greatly expanded and accelerated with a recently awarded $3.7 million grant for the California Institute for Regenerative Medicine.  Thank you Parkinson’s Alliance for playing such an important role in helping this research get started!


Conclusion: We have demonstrated, for the first time, the ability to generate DA neurons from a PD patient with genetically defined PD through cellular reprogramming and directed differentiation. These iPS cell lines are similar in morphology and molecular signatures to hESCs. Importantly, the SNCA triplication line shows early signs of a cellular PD phenotype with an increase of specific protein levels compared to control cell lines.  These critical first steps provide the foundation for further refining a pathological phenotype that can be study disease mechanisms and be applied to the discovery of drugs aimed at disease modification.


Lay Description Of The Burke Laboratory

The Laboratory is under the direction of Dr. Robert Burke. He is the Alfred and Minnie Bressler Professor of Neurology and Pathology at Columbia.  He has served as the Director of Laboratory Research in Parkinson’s Disease and Related Disorders in the Department of Neurology at Columbia since 1997, and as the Director of the Morris K. Udall Center of Excellence for Parkinson’s Disease Research at Columbia University since 2003.

Parkinson’s disease (PD) is the most common neurodegenerative disorder of the basal ganglia. Although  many effective treatments for the motor symptoms of PD exist, their benefits are limited in degree and duration.   The greatest challenge posed by this disease is to develop therapies that address the underlying degenerative process.  The goal of the Burke Laboratory is to address this challenge. Our efforts have two guiding principles. The first is that development of such therapies ultimately depends on a better understanding of mechanisms of disease. The second is that patients cannot wait for a full understanding of this disease to be in hand before efforts are made to translate new knowledge into treatments.

While there are many good approaches to developing neuroprotective therapies, we focus on targeting the pathways of programmed cell death (PCD), also known as apoptosis. There is a growing consensus that the degeneration of neurons in PD is not a passive event, but rather it is due to the activation of genes which destroy the cell. These genes are the mediators of PCD, and we believe that It may be possible to prevent neuron death by blocking these pathways.

We utilize information about the basic mechanisms of PCD to attempt to develop translational approaches that may ultimately prove useful in the treatment of patients.  Most recently, we have focused our efforts on the development of gene therapy as a way of blocking PCD in dopamine neurons in the living brain.  For example, in one of our recent publications, we show that it is possible to use a viral vector to block important death-mediator molecules in the dopamine neurons and thereby prevent their death in a neurotoxin model (Journal of Neuroscience, 2008).

This award from the Parkinson Alliance will help support our work in the creation of animal models and in the production of novel viral vectors.


Project Title:  Effects of Deep Brain Stimulation (DBS) on Balance Control

Investigators:  Fay B. Horak, PhD, Patricia Carlson-Kuhta, PhD, Penny Hogarth, MD

Objective:  To compare the effects of DBS in the Subthalamic Nucleus (STN) and the Globus Pallidus, internus (GPi) for Parkinson’s Disease on balance control. We will determine which aspects of balance and gait initiation are improved, and which are worsened, by DBS and whether one site has better outcome for balance.

Background: Although DBS ameliorates many symptoms of PD, how much these clinical benefits extend to the disabling problem of poor balance is still controversial. The few studies of DBS on axial motor function have been limited to STN stimulation and suggest improvement in some tasks such as walking and quiet stance but with worsening in other tasks, such as balance responses and gait initiation. No previous studies have directly compared the effects of DBS in STN and GPi on balance control.

We hypothesize that stimulation in STN and in GPi will affect different types of balance control.  Specifically, we predict that the STN is involved in anticipatory postural adjustments prior to step initiation, whereas the GPi may be more involved in balance responses to slips and trips.  If this is true, then patients with start-hesitation freezing should target STN, whereas patients with falls due to poor balance responses should target GPi for their DBS surgery.  We also predict that some types of balance control will be improved, but other aspects will be worsened by DBS.

Methods/Design:
  Thirty subjects with PD have been randomized into DBS surgery either in the STN or GPi. They will be tested a few days before DBS surgery, while on levodopa and off levodopa, as well as 6-months after surgery under 4 conditions: off both DBS and levodopa, on levodopa alone, on DBS alone, and on both levodopa and DBS.  Two control groups will also be tested at baseline and 6 months later: 1) subjects with PD who choose not to get DBS surgery and 2) age-matched controls without PD.  Step initiation and balance strategies in response to computerized surface perturbations will be quantified with forces under the feet, body motion and muscle activation patterns.  We will determine if changes in gait and balance after surgery, compared to before surgery, differ compared to gait and balance changes across 6 months in subjects with PD who have not had surgery and compare effects of DBS in the two sites.

Relevance to Parkinson’s Disease:  This study will help clinicians and patients better understand the effects of DBS on balance and gait initiation so they can make informed decisions about treatment options.

Project Update:

Our research indicates that deep brain stimulation in people with Parkinson’s disease does not improve their ability to initiate voluntary movements or to quickly react to balance disturbances.

People with Parkinson’s disease have difficulties initiating voluntary movements and in quickly reacting with the required force to balance disturbances, such as recovering from a trip. Parkinson’s disease results in more falls than any other neurological disease. Frequent falls affect over 60% of people with Parkinson’s disease.  Finding a way to decrease falls is important to the health of people with Parkinson’s disease.  

The typical treatment for Parkinson’s disease is medication, but its effectiveness in alleviating the symptoms tends to last for less time as the years progress. In recent years some people have had surgery to implant stimulating electrodes in the area of the brain affected by Parkinson’s disease (deep brain stimulation, DBS).  This surgery has shown to be beneficial to reduce tremor, slowness of movement, and muscle stiffness symptoms of the disease, but it was unclear how the DBS would affect balance control.  Our study sought to test whether this treatment would improve balance and posture control in Parkinson’s disease.  

 We tested people with Parkinson’s disease before and six months after DBS surgery.  Subjects stood on a specialized platform that could move forward, backward or rotate upwards to challenge the person’s balance ability.  Information about body motion, muscle activity, and forces produced were recorded.  During some trials the subjects had to regain their balance following platform movement, while during other trials the subjects were asked to start walking or rise up on their toes.

Our results suggest that balance control of both voluntary and reactive movements is not helped by DBS.  Although medication significantly improves balance before surgery, the balance responses are worse after surgery.  Patients also had more difficulty after surgery, initiating the movements required to stand on their toes or start walking. This may leave people with Parkinson’s disease more vulnerable to falls following DBS surgery.
     
Further study is underway to determine if the worsened balance is due solely to the DBS surgery or to the natural progression of the disease.


Project Title:  Non-Cardiac Surgery and PD:  A “Proof of Principle” Study.  Investigating the Frequency and Severity of Post-Operative Cognitive Decline Among PD Patients.

Investigators:  Catherine Price, Ph.D., Assistant Professor in Clinical and Health Psychology, as Principal Investigator, with Hubert Fernandez, MD as co-Principal Investigator and Michael Okun, MD as Collaborator, all from the University of Florida, Movement Disorders Center, Gainesville, FL.

Project Description: The effect of surgery on cognitive and bodily function among individuals with PD is currently unknown, despite recent research showing cognitive declines in 10 to 14% of “healthy” older adults three-months post-surgery. In this preliminary “proof of principle” study we will assess whether PD orthopedic surgery patients experience greater cognitive change relative to 1) non-surgical PD patients and 2) a group of non-PD “healthy” surgery patients. We will obtain preliminary data examining the hypothesis that pre-surgical neuroanatomical variables, measured with brain MRI (i.e., white matter integrity, basal ganglia and whole brain volume), contribute to PD post-surgery cognitive decline. The study will include 42 participants (14 PD surgery, 14 PD non-surgery, 14 “healthy” surgery). Cognitive function will be assessed with neuropsychological measures administered at pre-surgery and post-surgery at two-weeks and three months. PD surgery participants will also complete a pre-surgery brain MRI. A post-surgery MRI will rule out surgery related cerebrovascular events. 

Objective:

 

  • To investigate the frequency and severity of post-operative cognitive dysfunction among individuals with PD having non-cardiac surgery, while controlling for the effect of the disease process with a comparison group of PD non-surgery patients.
  • To investigate the hypothesis that PD surgery patients will experience a greater decline in cognitive function relative to non-PD ‘healthy’ surgery patients.
  • To investigate the hypothesis that reduced pre-surgical neuroanatomical factors, measured with Magnetic Resonance Imaging (MRI) Diffusion Tensor Imaging and gray matter volumetric methods, is associated with greater post-operative dysfunction.

    Background: The term “Post-Operative Cognitive Dysfunction” – or POCD – denotes post-operative memory and/or thinking problems that have been corroborated by neuropsychological testing. Typically these problems include reduced attention and concentration, word finding problems and difficulty learning information relative to pre-surgery abilities. Increasing age is the least controversial risk factor for POCD among cardiac and non-cardiac surgeries. In large studies of patients undergoing non-cardiac surgery the prevalence of cognitive decline among adults 60 years of age and older was between 20 to 30% at one week post-surgery and 10 to 15% at three months. We know little about POCD beyond one year, although one study reports that only one percent of elderly adults appear to experience persistent effects of cognitive decline after surgery. This may indicate that POCD is often reversible even among the elderly.
    Methods/Design: This was a prospective longitudinal pilot study with control arms involving a baseline (pre-surgery) neuropsychological assessment with repeat assessments at 2-weeks and 3-months post-surgery. PD surgery individuals had a pre-operative MRI conduced within 2-weeks prior to surgery and a post-operative MRI within 48 hours after surgery.

    Project Update:

    Results: Reliable change analyses indicate that two of five PD patients electing surgery experienced changes in executive dysfunction at three months after surgery. These changes were significantly greater than mean changes from PD non-surgery peers and ‘healthy’ surgery peers. Changes in executive dysfunction would be expected over that of other cognitive domains due to the impact of the disease process, but more research is certainly needed to clarify and confirm this hypothesis. Group mean comparisons did not suggest that there is an overall group change in executive dysfunction or any domain for the PD patients.

    Conclusion/Relevance to Parkinson’s Disease
    : The study preliminarily suggests that individuals with idiopathic non-demented PD are at risk for cognitive changes after major non-cardiac surgery – and specifically more executive changes.  This is re relevant given our increase in patient population and desire for more ‘quality of life’ surgeries such as hip and knee replacement surgeries.

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

Objective & Background:  The Parkinson Alliance funding has supported research on the impact of  different types of sleep disturbances and symptoms on health- related  quality of life in Parkinson's Disease.  A set of sleep measures were  added on to an ongoing observational cohort study of patients with  movement disorder-confirmed PD from central California.  The Medical  Outcomes Study sleep scales and the Berlin Questionnaire for sleep  apnea have been administered to study participants in two rounds of  data collection, about two years apart, along with a broad range of  clinical measures at each of those rounds.  Data from the first round  have been analyzed.

Project Update:


Methods/Design:  Study participants included in analyses on average were 72 years old,  60% female, and had a mean duration of diagnosed Parkinson's disease of 5 years.   As anticipated, health-related quality of life was markedly lower (worse) than age- and gender-adjusted norms on  seven of eight scales (physical function, limitations in work or daily  activities due to physical problems, limitations in work or daily  activities due to emotional problems, emotional well-being, energy,  general health, and social function);  only pain scores were the same  as the general population.

The Medical Outcomes Study Sleep Scales measure six different types of  sleep disturbance:  disturbances in the initiation of sleep,  disturbances in the maintenance of sleep, snoring, awakening short of  breath or with a headache, sleep inadequacy, and daytime somnolence. When we analyzed which different types of sleep disturbances most  strongly affected health-related quality of life, we found that  awakening short of breath or with a headache, initiation of sleep, and  daytime somnolence were the biggest factors - all of which negatively  impacted health-related quality of life - out of the six.  (This is  after taking into account the stage of Parkinson's disease).  We also  found that these three types of sleep disturbances were highly  correlated with extent of depression symptoms.

Results:   These results confirmed that sleep disturbances are common in PD, but  the new knowledge we found was that three specific types of sleep  disturbances/symptoms are strongly associated with all aspects of  health-related quality of life:  physical, mental, and social health.  Healthcare teams should make sure that people with PD are periodically  screened for these specific sleep disturbances in routine care. 

Relevance to Parkinson's disease:  Our next step is to analyze the second round of follow-up data to assess  the impact of these sleep symptoms on change in health-related quality  of life over time.



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 Parkinson 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. 


 

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 here 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 described 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 (see statements below) 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:  Development of a Model Curriculum for the Training of Interdisciplinary Rehabilitation Teams in Parkinson's Disease

Principal Investigators are:
Terry Ellis, Ph.D., PT, NCS
Clinical Associate Professor, Department of Physical Therapy & Athletic Training.
Boston University

Cathi A. Thomas, MS, RN
Assistant Clinical Professor of Neurology and Program Director of the Parkinson’s
Disease and Movement Disorders Center Coordinator – APDA Information & Referral
Center.
Boston University Medical Center

Tami Rork, PT, MSPT
Senior Physical Therapist and Lecturer
Center for Neurorehabilitation and Department of Physical Therapy & Athletic Training.
Boston University

Marie Saint Hilaire, MD FRCPC
Associate Professor of Neurology
Medical Director of Parkinson’s Disease and Movement Disorders Center and APDA
Center for Advanced Research
Boston University Medical Center

Donna Diaz, MS, RN
Coordinator – APDA Information & Referral Center
Hospital of Saint Raphael
New Haven, CT

The check transmitted to APDA national office on March 7, 2008 will be used to partially support a grant which APDA made to Boston University to train healthcare providers who have been identified as having the skills, interest, resources and commitment to caring for and impacting the lives of people living with Parkinson’s disease in their communities.

The Parkinson’s Disease & Movement Disorders Center at Boston Medical Center has been designated a Center for Advance Research in Parkinson’s Disease and a Parkinson Disease Information and Referral Center by the American Parkinson Disease Association. It offers the most current and innovative medical and surgical treatments available to people with Parkinson’s disease.  The Center for Neurorehabilitation at Sargent College of Health & Rehabilitation Sciences, Boston University works with the Parkinson’s Disease Center at Boston Medical Center to offer the most current, innovative, evidence-based rehabilitation services available to people with Parkinson’s disease.

The research conducted in the Center for Neurorehabilitation focuses on investigating the efficacy of rehabilitation approaches to the advancement of the rehabilitation sciences in the area of Parkinson’s disease.  These advances assist people with Parkinson’s disease to reach their maximum functional potential and level of independence in order to improve their quality of life.  The purpose of the program is to disseminate this emerging knowledge in the field of rehabilitation science directly to practicing health care professionals, students, individuals with Parkinson’s disease and their families via clinical and educational activities.

Project Update:

Train the Trainer Model

Researchers and Clinicians at Boston university developed a 2.5 day curriculum to educate interdisciplinary rehabilitation teams in the following areas:

  • Interdisciplinary rehabilitation of patients with Parkinson’s diseas
  • The most recent, evidence-based medical, pharmacological, surgical advances
  • Current research findings supporting the benefits of rehabilitation and exercise for people with Parkinson’s disease
  • Recommended exercises (both speech and mobility) that are evidence-based, appropriate and safe for people with Parkinson’s disease
  • Instruction on how to implement a Community Wellness Programs within participants’ local communities

Three annual Seminars titled “A Parkinson’s Disease Seminar for Healthcare Professionals: Evidence Based Concepts and Current Treatment Approaches” have been presented by the Boston University Interdisciplinary Team. 

  • Over 150 Physical Therapists, Occupational Therapists, Speech Language Pathologists, nurses and social workers who practice in settings including acute rehabilitation, home care, outpatient, long-term care and academics have attended.
  • Interdisciplinary teams represented over 20 facilities in the New England area
  • Program evaluation revealed a rating of 4.95 out of 5 on the summative evaluation form for the question: “This course increased my knowledge and skills in the care of individuals diagnosed with Parkinson’s Disease”.

Through the Train-the-Trainer program, expansion of Community Wellness Programs offered to people with Parkinson’s disease has resulted: 

  • Over 500 people with Parkinson’s disease have participated in Community Wellness Programs in 14 facilities in the New England


This Train-the-Trainer Model is a successful model to train health care teams in the rehabilitation management of people with Parkinson’s disease.  As a result, greater numbers of people with Parkinson’s disease are able to access exercise programs in their local communities with the goal of reducing disability and improving quality of life.


Project Title:  Low frequency deep brain stimulation for gait dysfunction in Parkinson’s disease

Investigator:  Isabelle Barnaure, Michele Tagliati, MD, Ron Alterman, MD, Hana Brozova, MD

Objective: A prospective, randomized study of subthalamic deep brain stimulation (STN DBS) settings for the treatment of gait impairment in Parkinson’s disease (PD), comparing the effects of low (60 Hz) and high frequency stimulation (130-185 Hz).

Background: Current knowledge of the effects of STN DBS on gait impairment is mostly based on short-term results obtained in small patient cohorts. However, the benefit from STN DBS on PD gait and balance dysfunction appears to decline over time, as compared to a sustained improvement for tremor, bradykinesia and rigidity. As the current therapeutic options seem insufficient for the treatment of gait dysfunction, there has been a search for new treatment modalities, including new patterns of stimulation (low frequency).

Project Update:

Methods: Patients with residual gait difficulty on otherwise therapeutic DBS settings being followed at the PI’s clinic will initially be randomized into one of two groups: one in which the stimulation frequency will be switched to 60 Hz, and a control group that will undergo sham setting adjustments, ending with the maintenance of the current stimulation parameters. After one month, subjects will be moved to the other group. Neither the study subject nor the evaluator will know to which group they were assigned. Outcome measures will be collected at two data points by a blinded evaluator:  1) Acute: UPDRS scores and the stand-walk-sit test will be recorded on the day of the intervention.  2) Chronic: One month after therapeutic intervention, subjective patients’ reports (including information on the number of falls and freezing episodes) will be collected, in addition to the objective scores (UPDRS, stand-walk-sit test). 

Methods/Design: PD patients with residual gait difficulty on otherwise therapeutic DBS settings were randomized into one of two groups: one in which the stimulation frequency was switched to 60 Hz, and a control group that underwent sham setting adjustments. After one month, subjects were moved to the other group. Outcome measures were collected at two data points by a blinded evaluator: 1) Acute: UPDRS scores and the stand-walk-sit test on the day of the intervention. 2) Chronic: One month after therapeutic intervention, subjective patients’ reports (including information on the number of falls and freezing episodes) and objective scores (UPDRS, stand-walk-sit test). After the crossover, the evaluator in the same fashion recorded acute and chronic clinical changes.

Results: The randomized study resulted extremely difficult to complete due to early terminations. Five of the first seven patients enrolled did not complete the original design, due to difficulty tolerating the rigorous protocol and/or the experimental settings. We were however able to study “open label” 14 patients with persistent gait difficulties using 60 Hz stimulation after a mean 4.3 years (range 2–8) of bilateral high frequency STN-DBS. Seven patients had freezing of gait, six had postural instability, and seven experienced falls. Five patients could not acutely tolerate the change in settings because of increased tremor (1), worse rigidity (1), gait worsening (1) and general worsening of PD (2). The remaining 9 subjects (3 women, 6 men) were followed open label. For 8-12 weeeks. These 9 subjects had a mean age of 66.8 (9.6 SD) years and a mean PD duration of 16.6 (5.0 SD) years. ON-medication UPDRS scores after switching to 60 Hz were compared to ON-medication scores obtained at higher stimulation frequencies. Significant average improvements were observed for the UPDRS-II subscale (3.9 points; p <0.05), UPDRS-II sub-items relative to speech, falling, and walking (p _ 0.05), and UPDRS-III subitems relative to speech and gait (p < 0.05). Postural instability and gait (items 27–30) worsened in two patients. An average voltage increase of 1.3 volts (range 0.7–2.5) was required bilaterally in seven patients for beneficial maintenance of other PD symptoms.

Conclusion/Relevance to Parkinson’s disease:
The conflicting responses observed in this study suggest that lower frequency STN stimulation may not be the solution to all gait problems in advanced PD. In our limited experience, we failed to find distinguishing features between responders and non-responders. The selection of proper candidates will be a challenge to the widespread use of 60 Hz STNDBS in PD patients with chronic gait difficulties.

Click for a printer friendly version