2009 Grants

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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.


Grant Awarded to:

Giselle Petzinger, MD Assistant Professor Department of Neurology, USC Keck School of Medicine, Division for Movement Disorders. University of Southern California

Michael Jakowec, PhD, Assistant Professor Department of Neurology, USC Keck School of Medicine, Division for Movement Disorders, University of Southern California

Beth Fisher, PhD, PT, USC Keck School of Medicine, Division for Movement Disorders, University of Southern California

The following two sections describe allocation of funds to support two important research programs in our labs. Both research programs focus on understanding the underlying mechanisms by which exercise, in the form of intensive treadmill running, leads to improvement in motor behavior in both the MPTP rodent model for Parkinson’s disease and in patients with Parkinson’s disease. Together these projects are integrated in a translational research program where findings from the lab impact clinical studies in patients with PD, and vice versa, resulting in improved treatment for patients in the community.

(1) From The laboratories of Giselle Petzinger, MD and Michael Jakowec, PhD:

        A fundamental aspect of studies in the mouse model of PD is to elucidate changes in protein expression within the basal ganglia, the region of the brain responsible for aspects of motor control affected in PD. Ongoing studies in our lab have shown that the electrophysiological properties of medium spiny neurons within the basal ganglia, a region affected in PD, are altered in a way consistent with our molecular studies showing that subunits of the AMPA receptor subtype of the glutamate receptor family, specifically the subunit GluR2, as well as the dopamine D2 receptor, are elevated with intensive treadmill running in animals rendered parkinsonian with MPTP. Currently we are investigating important aspects of this observation including (i) to determine if these changes are due to elevated expression of the genes and proteins encoding these receptors, (ii) what are the molecules that are responsible for trafficking these receptors to the synapses where they exert their effects, and (iii) are their morphological changes within the dendritic spines of medium spiny neurons that underlie these changes. To achieve many of the goals necessary to understand the mechanisms of motor behavior improvement we have purchased a spinning-disc confocal microscope from Olympus, Inc. This microscope will allow us to image and quantify changes in protein and genes expression at a high level of resolution within dendrites of neurons within the basal ganglia.

(2)     From the laboratory of Beth Fisher, PhD, PT.

        Ongoing studies in the Transcranial Magnetic Stimulation (TMS) Lab called the Neuroplasticity and Imaging Laboratory (NAIL) have shown beneficial changes in the brains of newly diagnosed individuals with Parkinson’s disease not yet on dopamine replacement therapy that had participated in intensive exercise.  Specifically, we showed that exercise normalized the abnormal cortical excitability state consistently seen in Parkinson’s disease.  We are the first group to demonstrate a brain effect in Parkinson’s disease from physical therapy, suggesting that exercise may have an important role in influencing the brain and potentially modifying disease progression. Findings from these studies are having direct impact on patient care as demonstrated by their influence on clinical practice within rehabilitation units for Parkinson’s disease now being established at sites such as Rancho Los Amigos National Rehabilitation Center here in Southern California, and at others around the country. To further our research studies we wish to purchase additional equipment from Jali Medical Inc., which will allow us to utilize an additional electrophysiological parameter called Paired-Pulse, and will enable us to demonstrate more robust changes in the brain in response to intensive exercise and provide insight into the mechanisms responsible for the beneficial effects of exercise on brain and behavior in individuals with Parkinson’s disease.

2010 Project Update (1):

Project Title:  Exercise and PD:  The role of exercise in modifying synaptic strength within medium spiny neurons of the basal ganglia

Investigators/Authors:  Michael Jakowec, PhD;  Giselle Petzinger, MD; John Walsh, PhD

Name of Organization:  University of Southern California, Keck School of Medicine, PD Neuroscience Program

Objective:  To examine whether intensive exercise leads to alterations in glutamatergic synaptic connections (dendritic spine density) and function ( AMPA receptor subunits, and long term plasticity).  We also examined whether synaptic changes were specific to the D2 (indirect) and/or D1 (direct) pathways

Background:   Although exercise has been shown to be beneficial in PD, its potential for modifying disease is poorly understood.  One effect of exercise may be through modulating glutamatergic synaptic connections in the basal ganglia, at the level of the medium spiny neurons through altering both the number and strength of synapses.  AMPA receptors (a subtype of the glutamate receptor family) are critical for dictating the intensity or “strength” of synaptic connections and long-term plasticity, including long-term depression (LTD) and long-term potentiation (LTP).  LTD is the most common type of synaptic strength expressed in the striatum, and is absent with dopamine depletion.   Loss of LTD may be due to changes in AMPA receptor subunit expression.  Effects of exercise ion AMPA receptor subunit expression may account for behavioral improvement and long-term disease modification. 
 
Methods/Design: Studies in our lab use a combination of molecular biology, morphology (imaging) and neurophysiology to characterize both the number of synaptic connections (e.g. dendritic spine density), and the expression of glutatmatergic AMPA receptor subtype.   For these studies we employed both the C57BL6 and BAC-D2-EGFP mouse lines and administered MPTP.   Intensive treadmill exercise was started 5 days after lesioning, when cell death is complete.  Golgi staining, biocytin labeling and electron microscopy were used to examine spine morphology and density at the completion of 28 days of exercise.  Post-synaptic density (PSD) complexes were isolated from striatal tissues to examine alterations in AMPA receptor subunits and their accessory proteins.  Neurophysiology in slice cultures were used to examine exercise-induced alterations in synaptic plasticity.    The recent acquisition of the spinning-disc confocal microscope now allows us to determine the pattern of expression of genes and proteins involved in synaptogenesis and modifying synaptic strength, including AMPA and NMDA receptors, transcription factors (delta fos B, Creb) and structural proteins (synaptophysin).  

Results:  Our results showed  (i) improved motor learning; (ii) an exercise-induced increase in spine density and synapse number, including mushroom and thin spines; (ii) an increase in GluR2 containing AMPA receptors, and (iii) a restoration of synaptic function as based on Long-term depression (LTD).   Exercise induced changes were noted in both the indirect (D2R) and direct (D1R) pathways. 

Conclusion/Relevance to Parkinson’s disease:  These findings support the potential role of exercise in facilitating synaptic strength in PD, and hence modifying disease progression through the alterations of AMPA receptor expression.    By understanding the role of glutamate receptor modulation in PD, these studies begin to delineate potential new therapeutic targets for treatment. For example, findings with transcranial magnetic stimulation have shown the importance of suppressing hyper-excitability of cortical paths through exercise as part of the beneficial mechanisms of exercise. In addition, our recent report of increased expression of dopamine D2 receptors and the link between dopamine and glutamate neurotransmission leads us to investigate the relationship between these two systems and again may represent a paradigm shift in our understanding and treatment of PD.

2011 Project Update (1):

Results: We have continued to elucidate the molecular and physiological changes that take place in neurons within the basal ganglia that are responsible for mediating motor behavior under conditions of dopamine depletion. In addition, we are beginning to understand the mechanisms that take place during exercise-induced reversal of motor behavior deficits in our model of parkinsonism, specifically the MPTP-lesioned mouse, following intensive treadmill exercise. A newly acquired confocal microscope is allowing us to visualize changes in proteins expressed in neurons, specifically those involved in dopamine and glutamate neurotransmission. These changes, which are termed experience-dependent neuroplasticity, represent an important new potential therapeutic target in Parkinson’s disease treatment. Changes in a specific set of proteins involved in neurotransmission with the chemical glutamate show changes that alter the strength and connections between neurons within the basal ganglia. These studies were published in VanLeeuwen et al 2010 Journal of Neuroscience Research 88: 650-668.

Relevance to Parkinson’s Disease: Studies in animal models and in patients with Parkinson’s disease have shown beneficial effects of exercise. Our labs have begun to determine the molecules important for mediating alterations in the connections within the basal ganglia that are responsible for reversal of parkinsonian motor deficits. These studies have identified a previously unrecognized role of receptors involved in glutamate neurotransmission in serving as a template for which experience (exercise for example) can mediate its beneficial effects. We have begun to test several novel therapeutic strategies including viral vectors that can promote the expression of proteins we have identified to be beneficial and new drugs, which influence biochemical pathways involved in synaptic plasticity and altering the strength of connections within the brain.

2011 Project Update (2):

Project Title:  Determining the Optimal Method for Measuring Exercise-Induced Changes in Corticomotor Excitability in Individuals with Parkinson’s Disease

Investigators/Authors:  Beth Fisher, PhD, PT; Giselle Petzinger, MD

Name of Organization:  Division of Biokinesiology and Physical Therapy – University of Southern California

Objective:  The funding provided from Parkinson Alliance for 2009 was used to purchase additional Transcranial Magnetic Stimulation (TMS) to determine the optimal method for measuring corticomotor excitability (CE) in individuals with Parkinson’s disease (PD).  Specifically the goal was to compare measures of CE in individuals with PD using two different TMS methods namely, Single pulse TMS which generates Cortical Silent Period (CSP) duration and Paired-Pulse which generates Long-latency Cortical Inhibition

Background: 
It is now understood that Parkinson’s disease is a problem of dopamine neurotransmission that results in corticostriatal glutamatergic hyperexcitability. There is compelling evidence that this hyperexcitable state underlies the very motor dysfunction associated with Parkinson’s disease including gait and balance impairments, slowness and stiffness.  Transcranial Magnetic Stimulation is a noninvasive, painless method for measuring corticomotor excitability.  TMS studies in PD have shown higher motor system excitability in patients with PD at rest compared to controls. Importantly surgical and pharmacological interventions can change excitability back toward those levels seen in control subjects. These changes in excitability often correlate with clinical improvement. In accordance with higher corticomotor excitability at rest in PD, silent period (SP) durations are shortened.  In fact, shortened silent periods are among the most consistent and widely reproduced TMS finding among PD patients. We previously demonstrated a significant relationship between a decrease in CSP and an increase in the Unified Parkinson’s Disease Rating Scale (Wu et al., 2007). Our group was the first to use TMS measures of CSP to assess changes in corticomotor excitability in subjects with PD following intense treadmill training. In our published study (Fisher et al, 2008) subjects showed ‘normalization’ of TMS measures. Specifically, silent period duration was consistently lengthened.  These changes were not consistently observed in subjects that participated in low-intensity physical therapy or no-exercise. Our group was the first to capture intensive exercise-induced changes in TMS measures.  In 2007, Cantello et al., advanced the study of altered CE in patient’s with PD.  The TMS studies mainly focused on the motor cortical inhibitory phenomena previously identified in PD. However, differences in patients and methods caused discrepancies related specifically to long-latency cortical inhibition (LICI). Cantello et al., confirmed LICI as a pathologic marker in PD and suggested that it could act as a candidate physiological hallmark of the disease, to be tested in various circumstances such as therapeutic interventions or differential diagnosis.

Methods/Design: 
Three individuals with PD were studied with both single and paired-pulse TMS techniques.  Motor-evoked potentials and CSP were recorded from surface electrodes placed on the first dorsal interosseous muscle (FDI) of the more affected hand.  All TMS variables were studied during FDI activation at 20% maximum voluntary contraction. The TMS was delivered through a focal figure-of-eight coil at the “hot spot” for the FDI on primary motor cortex of the more affected hemisphere.

Once the hot spot was located we determined active motor threshold (AMT).  We measured the duration of the CSP, i.e. the period of suppression of the EMG activity produced by a magnetic stimulation of 1.5 X AMT in the pre-activated FDI. The CSP length went from the TMS pulse to the return of baseline EMG activity after a period of EMG silence.  To study LICI, we used the paired-pulse technique, with two stimulators coupled with a BiStim device (Magstim Co.) A first (conditioning) stimulus was followed by a second (test) stimulus. The intensity of both was 150% AMT, and they were delivered through the same coil at the following inter-stimulus intervals (ISIs): 50, 100, 150, 200, 250 and 300 ms. For each ISI, we recorded 10 unconditioned and 10 conditioned MEPs in a random order. The effect of the conditioning was the ratio of the averaged conditioned MEP to the averaged control MEP (peak-to-peak size). Inhibition occurred when the ratio was less than 1, facilitation when it was greater than 1.

Results: 
Subjects demonstrating short silent period also had a shorter LICI.
The study reinforced previous TMS findings of decreased inhibition or increased CE in PD as a feature of the disease pathophysiology.

Conclusion/Relevance to Parkinson’s disease: 
As Cantello proposed LICI and CSP as two expressions of the same inhibitory phenomenon, both measures represent pathophysiologic central features of PD and as such can be used in future studies to test the effects of skill training on brain repair and behavioral recovery in Parkinson’s disease.


Project Title:  Novel, small-molecule inhibitors of  a-synuclein assembly and toxicity for disease-modifying therapy of Parkinson's disease.

Grant Awarded to:  Dr. Gal Bitan, Ph.D.

Objective: To study a novel inhibitor of a-synuclein self-assembly and toxicity in vitro and in vivo as a drug candidate for Parkinson’s disease.

2010 Project Update:

Background:
Several lines of evidence implicate self-assembly of a-synuclein as a key causative event in PD. In particular, formation of neurotoxic a-synuclein oligomers is believed to be a major cause for the neurodegeneration observed in multiple brain regions. a-Synuclein is a ubiquitous, naturally unstructured protein whose function is not well understood. Fibrillar a-synuclein is the main component of Lewy Bodies and Lewy neurites, the pathological hallmarks of PD. Mutations in, or multiplication of, the a-synuclein encoding gene cause familial PD. Recent genome wide association studies have repeatedly found a-synuclein as a major risk factor for PD. CLR01 is a novel compound we have discovered using a rational approach. The mechanism of action of CLR01 is novel and unique. The compound binds potently to Lys residues in proteins (Kd ~20 uM), with 10-times lower affinity to Arg, and with little, if any, affinity to most other cationic biomolecules. By binding to Lys residues, CLR01 inhibits a combination of hydrophobic and electrostatic interactions that are key to nucleation and aggregation by most amyloidogenic proteins. During the nucleation events, intermolecular contacts among protein monomers are fairly weak and therefore the moderate binding affinity of CLR01 to Lys is sufficient to interrupt them. At the same time, the uM binding of CLR01 to Lys does not affect the structure or function of “normal,” natively folded proteins unless substantially higher MT concentrations are present.

Methods/Design: 
We tested:
1. The capability of CLR01 to inhibit a-synuclein aggregation in vitro using thioflavin T fluorescence and electron microscopy.
2. Inhibition of a-synuclein-induced toxicity in cell culture.
3. Rescue of a-synuclein-induced toxicity leading to deformation and mortality in a zebrafish model.
4. Rescue of a-synuclein-induced motor deficits in a transgenic mouse model expressing human, wild-type a-synuclein under the control of the Thy1 promoter.
These experiments included extensive collaboration with Drs. Jeff Bronstein and Marie-Françoise Chesselet, Department of Neurology, UCLA.

Results:
1. CLR01 inhibited a-synuclein aggregation completely at a 1:1 concentration ratio and partially at a 1:10 concentration ratio, respectively.
2. CLR01 inhibited the toxicity of both exogenous a-synuclein oligomers and endogenously expressed in cell culture.
3. CLR01 showed significant rescue of the phenotype and survival of zebrafish embryos expressing human, wild-type a-synuclein. The mechanism of rescue was shown to be maintaining a-synuclein in a soluble form, preventing formation of toxic aggregates, and enabling degradation of a-synuclein via the proteasome.
4. CLR01 was found to significantly ameliorate motor deficits in transgenic mice expressing human, wild-type a-synuclein in the brain when administered subcutaneously for 28 days at a low dose (0.4 mg/kg/day). The mechanism is still under study but initial results suggest that it involves reduction in the levels of soluble, likely oligomeric, a-synuclein.
5. Importantly, in cell culture and zebrafish experiments, the dose of CLR01 showing toxicity was substantially higher than the dose needed for the beneficial effects. In the transgenic mice, no side effects were observed.

Conclusion/Relevance to Parkinson’s disease:
The data accumulated so far suggest that CLR01 is a novel and promising therapeutic candidate for Parkinson’s disease and support its further development for human clinical trials.

2011 Project Update:

These experiments included extensive collaboration with Drs. Jeff Bronstein and Marie-Françoise Chesselet, Department of Neurology, UCLA.

Results:

1. CLR01 inhibited ?-synuclein aggregation completely at a 1:1 concentration ratio and partially at a 1:10 concentration ratio, respectively.
2. CLR01 inhibited the toxicity of both exogenous a-synuclein oligomers and endogenously expressed in cell culture.
3. CLR01 showed significant rescue of the phenotype and survival of zebrafish embryos expressing human, wild-type a-synuclein. The mechanism of rescue was shown to be maintaining a-synuclein in a soluble form, preventing formation of toxic aggregates, and enabling degradation of a-synuclein via the proteasome.
4. CLR01 was found to significantly ameliorate motor deficits in transgenic mice expressing human, wild-type a-synuclein in the brain when administered subcutaneously for 28 days at a low dose (0.4 mg/kg/day). The mechanism is still under study but initial results suggest that it involves reduction in the levels of soluble, likely oligomeric, a-synuclein.
5. Upon ICV administration for 28 days, the effect of CLR01 was particularly significant in the challenge beam test, where is was consistent in all 5 trials and also increased by 2.2-fold relative to subcutaneous administration. The data demonstrate the efficacy of CLR01 and suggest that blood–brain barrier penetration likely needs to be optimized in future medicinal chemistry and/or formulation studies.

Conclusion/Relevance to Parkinson’s disease:  The data accumulated so far suggest that CLR01 is a novel and promising therapeutic candidate for Parkinson’s disease and support its further development for human clinical trials.
A manuscript describing the data summarized in points 1–3 above has been submitted for publication and is now being revised to address reviewers’ comments. The mouse experiments are still being analyzed and will be published in the near future.


Project Title:  Is there abnormal network activity in the motor cortex of 6-OHDA lesioned mice.

Grant Awarded to:  Dr. Carlos Portera, M.D., PhD

Project Description:  The current treatments for Parkinson disease (PD) offer some symptomatic relief, but often at the cost of serious side effects, including dyskinesias. The introduction of deep brain stimulation (DBS) in the treatment of PD two decades ago has arguably been the most effective treatment strategy since the discovery of levodopa. Understanding the exact mechanisms of how DBS helps PD patients will help improve this therapeutic strategy. A recent study using the 6-hydroxydopamine (6-OHDA) rodent model of PD (Gradinaru et al., 2009) suggests that DBS may help by reducing the activity of the subthalamic nucleus (STN) through its effects on the firing of neurons in motor cortex, which is the part of the brain that controls movement. This raises the possibility that neurons in the motor cortex of PD patients fire less than normal, leading to an overactive STN that produces some of the symptoms of PD. I propose to test the hypothesis that neurons are hypoactive in the motor cortex of mice that had been rendered parkinsonian by injection of the neurotoxin 6-OHDA into the substantia nigra. Specifically, I intend to examine the spontaneous activity of neurons in the motor cortex of mice before and after administering 6-OHDA. We will use the cutting-edge technique of two-photon calcium imaging to record the activity of large numbers of cortical neurons non-invasively.

These experiments will shed light into the mechanisms of circuit dysfunction in PD and may lead to improved treatments for this devastating disorder.

2010 Project Update:

We originally proposed to test the hypothesis that neurons in the motor cortex of Parkinsonian mice are hypoactive. In a way, this is expected in patients with Parkinson disease (PD), because the low levels of dopamine lead to reduce activity in a part of the brain called the thalamus, which normally stimulates the motor cortex to initiate movement. Specifically, we proposed to render mice Parkinsonian by injecting the neurotoxin 6-hydroxy-dopamine (6-OHDA) into the substantia nigra. Next, we planned to examine the spontaneous activity of neurons in the motor cortex of mice before and after the 6-OHDA injection. However, after discussions with UCLA investigators and PD research experts Marie-Francoise Chesselet and Michael Levine, we decided instead to examine neuronal activity in motor cortex of the Alphasynuclein over-expressing transgenic mice (the so-called ASO mice). Drs. Chesselet and Levine considered the ASO mice to be a better model of PD.

We have been using the cutting-edge technique of two-photon microscopy to record the activity of large numbers of cortical neurons non-invasively, with calcium imaging. So far, we find that the average firing rate of neurons in the motor cortex is lower in ASO mice than in normal controls, meaning that the motor cortex of mutant mice is less active. Although the data are very preliminary because we have only imaged 2 ASO mice and 4 control mice, the result is what we expected. Next, we intend to image more mice to confirm these results.  Eventually, we will attempt to stimulate neurons in motor cortex of ASO mice with light or with electrodes to restore their activity levels back to normal. The hope is that this will have a therapeutic benefit in the Parkinsonian mice. Our goal is to shed light into the mechanisms of circuit dysfunction in PD that may lead to improved treatments for this devastating disorder. The funds from the Parkinson Alliance will hopefully allow us to generate sufficient preliminary data for other grants, including Federal funding.

2011 Project Update:

In addition to acute two-photon calcium imaging experiments using the synthetic calcium indicator dye Oregon Green BAPTA-1, we also intend to pursue chronic recordings of neuronal activity in the neocortex of ASO mice with the genetically encoded calcium indicator GCaMP3. Using viral mediated transfections we will be able to record the firing of cortical neurons over periods of several weeks.

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