Investigators/Authors: Robert E. Burke, MD, Departments of Neurology and Pathology, Director of the Udall Parkinson's Disease Research Center at Columbia University
Objective/Rationale: Based on modern data, it is now estimated that only 30% of dopamine neurons are lost at the time of first diagnosis of PD (Ann Neurol, 2010). Thus, at disease onset, and throughout its course, there is an opportunity to re-establish function by restoring axons of surviving dopamine neurons. It has been believed that axons, the long fibers that connect one neuron to another, cannot be re-grown in the mature brain. However, we have shown that they can be induced to grow by using a gene therapy approach to re-invigorate the mechanisms that are active during development (Ann Neurol, 2011). The object of this proposal is to further develop these gene therapy approaches.
Project Description/Methods/Design: We have shown that two molecules that normally induce axon growth during development, a kinase called Akt and a GTPase called Rheb, can induce robust re-growth of axons in the mature brain (Ann Neurol, 2011). These molecules function in just one of the major pathways for new axon growth. In another pathway, a molecule called Rap1B is especially abundant in dopamine neurons. We propose to investigate the ability of Rap1B to induce re-growth of dopamine axons. We will first make an adeno-associated viral (AAV) vector that will contain a highly active form of Rap1B. We will make a lesion of the dopaminergic axons in mice by use of a neurotoxin, 6OHDA. After 3 weeks most of the axons have been destroyed. At that time, we inject AAV Rap1b into the dopaminergic neurons and wait 12 weeks for the AAV to take effect. We then study the behavioral recovery of the mice, and following these tests, we will study the brains, to see if there has been axon re-growth.
Relevance to Treatment of Parkinson’s Disease: While there are now many medical treatments and deep brain stimulation (DBS) therapy for the symptoms of PD, these approaches only treat the symptoms; they do not restore the axon circuitry damaged by the disease. Not surprisingly, these treatments lose efficacy over time, and they begin to cause adverse effects. A more lasting and complication-free treatment can be achieved by restoring the normal anatomical circuitry of the brain by inducing the endogenous surviving neurons to re-grow their axons and restore this circuitry. We have had preliminary success in achieving this goal by stimulating intrinsic neuronal mechanisms of growth. We anticipate that this approach will make restoration of neural circuitry and robust, lasting clinical benefit a reality.
Expected Outcome: We expect that AAV Rap1b will induce re-growth of dopaminergic axons following their destruction in a neurotoxin model. We further anticipate that this anatomical restoration will lead to a functional, behavioral improvement in motor deficits. These results will represent a first step towards the development of a gene therapy for patients with PD, in which the intended therapeutic goal is the restoration of the axonal circuitry destroyed by the disease. In the future, we plan to optimize this new approach to therapy by seeking the most effective stimulators of axon growth and by designing vectors that will minimize the possibility of adverse effects.
Cheng HC, Ulane CM, Burke RE (2010) Clinical progression in Parkinson disease and the neurobiology of axons. Ann Neurol 67:715-725.
Kim SR, Chen X, Oo TF, Kareva T, Yarygina O, Wang C, During MJ, Kholodilov N, Burke RE (2011) Dopaminergic pathway reconstruction by Akt/Rheb-induced axon regeneration Ann Neurol 70 110-120.
September 2013 Project Update:
We have previously shown that, contrary to the long-held belief that the adult central nervous system is incapable of long-range axon re-growth, lesioned dopamine neurons can be induced to grow new axons when they are transduced by adeno-associated viral (AAV) vector to express highly active forms of the kinase Akt or the GTPase Rheb These molecules are known to play a role in the growth of axons during development. Our work has shown that even in maturity dopamine neurons remain responsive to these genes and generate new axons. In order to investigate the promise of this approach for the treatment of PD patients, we sought to determine how long after axon destruction the dopaminergic neurons remain capable of growing new axons. In order to more accurately model chronic human PD, we assessed the axon growth response following AAV treatment at six weeks following lesion. In the rodent lesion model, about 70% of dopamine neurons have degenerated at that time, so this would be comparable to PD of about ten years duration. We found in the lesion model that even after this long delay in AAV treatment there was a significant axon growth response (published in Molecular Therapy, 2012).
In order to make this approach safe for clinical use in the treatment of PD patients, we need to be able to control where the genes are expressed. Ideally it would be best to limit expression to the dopamine neurons where they are needed. One approach to achieve this specificity is to put the gene that induces axon growth under the control of a promoter that is expressed exclusively in dopamine neurons. One such promoter is the tyrosine hydroxylase (TH) promoter, which is active exclusively in catecholaminergic neurons such as dopamine neurons. This year we have successfully evaluated the ability of the rat TH promoter to express genes in dopaminergic neurons. In addition, we have cloned three candidate human TH promoter constructs and have finished an initial evaluation of strength and specificity of expression.
This year we have demonstrated that the rat TH promoter does show regional specificity of staining; it mediates expression in the SN, but not in regions that do not contain dopamine neurons, such as striatum and cortex. Within the SN, it drives expression equally well in two subtypes of dopamine neuron that make up the dorsal and ventral tier of the SNpc. In addition, we have shown that transgene expression driven by the rTHp is stable over time; there was no change in expression patterns between 6 weeks and 6 months. We have begun to assess three published components of the human TH promoter: a 522bp sequence in the 5’ flanking region, a 1.5 kb sequence that contains 5’ and 3’ elements, and a 3.3 kb 5’ flanking sequence. Using AAV vectors to examine the strength of these promoters in vivo (in mice), we find that the 3.3 kb hTHp is the strongest. We are currently evaluating its specificity.
September 2014 Project Update:
We have shown that lesioned dopamine neurons can be induced to grow new axons when they are transduced by adeno-associated viral (AAV) vectors to express highly active forms of molecules that ordinarily induce axon growth during development, including the kinase Akt and the GTPase Rheb.
In order to make this approach safe for clinical use in the treatment of PD patients, we need to be able to control where the genes are expressed. Ideally it would be best to limit expression to the dopamine neurons where they are needed. One approach to achieve this specificity is to put the gene that induces axon growth under the control of a promoter that is expressed exclusively in dopamine neurons. One such promoter is the tyrosine hydroxylase (TH) promoter, which is active exclusively in catecholaminergic neurons such as dopamine neurons. Last year we successfully evaluated the ability of the rat TH promoter to express genes in dopaminergic neurons.
This year we have cloned three candidate human TH promoter constructs and have finished an evaluation of their specificity of expression. We have worked with three published components of the human TH promoter: a 522bp sequence in the 5’ flanking region, a 1.5 kb sequence that contains 5’ and 3’ elements, and a 3.3 kb 5’ flanking sequence. For each of these candidate promoters we have demonstrated that expression is achieved in dopamine neurons of the SN. Like the rat TH promoter, previously studied, they each show regional specificity of staining; they mediate expression in the SN, but not in regions that do not contain dopamine neurons, such as striatum and cortex. Within the SN, each drives expression equally well in two subtypes of dopamine neuron that make up the dorsal and ventral tier of the SNpc. In addition, within the SN, none of them transduce glia, indicating that the combination of an AAV vector and the TH promoter restricts expression to neurons, thereby avoiding the risk of oncogenic transformation of glia. Thus all three candidate promoters look promising, and our next steps will be to quantitatively evaluate their specificity and efficiency of expression in SN dopamine neurons.
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|Investigators/Authors: Nader Pouratian, MD, PhD, UCLA Neurosurgery, Jeff Bronstein,
MD, PhD, Director of Movement Disorders, UCLA
Objective/Rationale: While DBS has revolutionized advanced Parkinson’s disease management, the therapy remains imperfect and time-consuming, requiring physicians to evaluate countless combinations of stimulation parameters to achieve “best” therapy. Ideally, patient-specific biomarkers could help optimize individualized therapy by identifying the optimal site and parameters for stimulation. Local field potentials (LFP), which are a measure of population-level neuronal activity, can easily be measured with DBS electrodes and hold great promise as such a biomarker. Our objective is to evaluate LFP across time, activity states, and therapeutic states to elucidate their role in developing self-programming DBS systems that improve therapeutic efficacy and efficiency.
Project Description/Methods/Design: Electrophysiological signals (LFP) will be recorded from patients’ brains who are electively undergoing clinically indicated DBS surgery. LFP will be recorded using two mechanisms. (1) Our laboratory has already established a program to record LFP during surgical implantation of DBS electrodes. After surgical implantation but before closing the wounds, LFP signals are recorded from deep brain electrodes and an electrode placed on the brain surface while the patient performs various tasks and with various stimulation parameters. (2) Ten patients will be implanted with a specially designed generator that not only stimulates like standard generators, but also records LFP chronically (Activa PC+S) for one year. Biosignals will be compared to the clinical effect of stimulation at each contact (as determined by a movement disorders neurologist) to identify biomarkers associated with the site of optimal stimulation and ideal stimulation parameters. Signals will be evaluated for changes with activity, medication, and time.
Relevance to Treatment of Parkinson’s Disease: Current DBS practice requires patients to follow-up for months postoperatively to optimize therapy. This process is time-consuming, varies based on programmer experience, and places geographical constraints on DBS eligibility. Moreover, generator power consumption is not necessarily optimized, potentially leading to early generator replacement. The electrophysiological biomarkers that we will characterize aim to guide programming, making therapy more effective, efficient, and therefore more accessible to those who are remote from implanting centers. In the future, such signals will ideally be integrated into closed-loop stimulation systems that rapidly respond to real-time patient needs and obviate the need for human programming.
Expected Outcome: Preliminary work in our laboratory has identified two critical biomarkers in the LFP signals of the globus pallidus (one of the principal DBS targets for Parkinson’s disease). These LFP biomarkers are specific to the DBS target (i.e., not seen in other places) and they respond to movement (results submitted to Journal of Neuroscience). Through the proposed work, we will further characterize these LFP biosignals. We will demonstrate the stability of these signals over time to ensure their long-term reliability. Moreover, we expect LFP signals to change with clinical condition, providing a biomarker of effective therapy.
September 2013 Project Update:
Ongoing research in this field has extensively characterized electrophysiological biomarkers of disease in Parkinson’s disease that could potentially be used as signals to automatically control deep brain stimulation system. Although most studies have focused on the subthalamic nucleus (STN), the the globus pallidus (GPi) is considered an equally efficacious site for therapy. Our preliminary analysis of electrical signals in the GPi suggested very high frequency activity that may be a viable biomarker for PD. Using invasive recordings in patients undergoing DBS, we have identified and characterized a previously undescribed electrical activity centered at approximately 235Hz that responds to patient movement. We propose that this newly identified activity in the GPi could have a functional role in the basal ganglia and could be contributing to abnormal signal processing within the basal ganglia and contribute to disease in patients with PD.
In addition to analyzing signals limited to the GPi, the next step in our work has focused on characterizing electrical signals across multiple points in the motor network in patients with PD. We have shown for the first time that electrical activity with distinct frequencies within the GPi are interacting with one another and that this interaction is also modulated by patient arm movement. We have found a similar pattern of electrical activity and regulation in the motor cortex of PD patients. Finally, we have found that the motor cortex and GPi signals are intimately linked to one another through a phenomenon known as coherence. This information once again provides a critical understanding into the electrophysiological underpinning of network pathophysiology in PD and provides critical biomarkers that can be used for self-programmed deep brain stimulation.
These biomarkers will be used in our initial investigations of closed-loop neuromodulation in patients with PD when such patients are implanted with the planned Activa PC+S systems.
September 2014 Project Update:
Last year, we reported having identified a novel electrical signal in the globus pallidus internus (GPi) of patients with Parkinson’s disease. We found that this signal was only detected when the deep brain stimulation electrode was optimally positioned within the GPi and that this signal could be further confirmed by observing changes in its strength when the patient moves.
In the last year, we have further investigated the electrical activity of the GPi in order to better understand how we can use these signals to automatically program and control a deep brain stimulator system. As planned last year, we have begun to characterize how the electrical activity across different parts of the motor control network relate to one another. In these studies, we have found additional novel “signatures” that confirm the optimal DBS position in GPi. Specifically, we have found that electrical activity within the GPi is synchronized with activity seen in the motor cortex (the part of the brain that specifically and directly controls voluntary movement). The synchronization is quite complex, and can be identified using multiple sophisticated analysis techniques. The synchronization of these signals is decreased when the patient moves their hands. Even more interesting, we have explored what happens to these complex relationships across the motor network when the patient is placed under anesthesia, knowing that anesthesia abolishes the abnormal movements associated with Parkinson’s disease. Anesthesia decreases the synchronization across the network, suggesting that this synchronization is a potentially important source of abnormal brain activity in Parkinson’s disease and contributing to disease characteristics. This information once again provides a critical understanding into the electrophysiological underpinning of network pathophysiology in PD and provides critical biomarkers that can be used for self-programmed deep brain stimulation.
While the original intent of the proposed research was to study signals using the Activa PC+S system, regulatory approval for this device is still pending. Nevertheless, using opportunities during deep brain stimulation surgery, we have made great progress in understanding the signals that will be critical for studies in patients who ultimately receive the Activa PC+S system.
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