Director: Timothy J. Collier, Ph.D.
Title: Aging and Parkinson's Disease: Models of Therapeutics and Neurologic Comorbidity
The Udall Center at Michigan State University and the University of Cincinnati focuses on studies of two aspects of Parkinson’s disease (PD): neural mechanisms associated with development of adverse consequences of disease and treatment, and mechanisms associated with translational therapeutics. In addition, it long has been appreciated that advancing age is a primary risk factor for PD, yet aging rarely is incorporated into experimental studies. Thus, our Center groups these topics under the rubric of “adaptive and maladaptive plasticity” and examines their expression in the context of advancing chronological age. The proposed studies examine such themes as (1) the roles of altered dendritic morphology in projection neurons of the dopamine (DA) depleted striatum in the expression of therapy-induced dyskinesias; (2) exploration of mechanisms associated with electrical stimulation of the subthalamic nucleus (STN) that may promote neuroprotection of the nigrostriatal system; (3) examination of the hypothesis that grafted undifferentiated neural progenitor cells protect and repair the nigrostriatal system not by replacing lost DA neurons but by stimulating plasticity in the host brain; and (4) begin to study the known association of depression with PD to determine whether stress, chronic anxiety and depression exacerbate neurodegeneration and whether manipulation of these states influences the efficacy of therapeutic interventions. A critical aspect of all of the proposed projects will be to incorporate the recurring factor of advancing chronological age on the expression of mechanisms and outcomes derived from therapeutic interventions.
This Udall Center assembles six principal investigators at two institutions to provide a team-based approach to our studies: Timothy Collier, Ph.D, (Director, Michigan State University), James Herman, Ph.D. (University of Cincinnati), Jack Lipton, Ph.D. (Michigan State University), Kim Seroogy, Ph.D. (University of Cincinnati), Caryl Sortwell, Ph.D. (Michigan State University), and Kathy Steece-Collier, Ph.D. (Michigan State University). The Center includes an Administrative Core to coordinate activities and communications associated with the projects, and an Analytical Chemistry, Gene Expression, and Surgical Core to provide the animal model and analytical endpoints common to all projects.
We are examining remodeling that occurs in the parkinsonian brain and its impact on therapeutics, with a particular focus on loss of dendritic spines and altered synaptic contacts on the striatal medium spiny neurons. As part of Aim 2, we hypothesized that regardless of whether dendritic spine loss influenced levodopa induced dyskinesias (LIDs), dyskinesia indices would be correlated with abnormal dendrite and spine morphology and/or aberrant synapse formation on medium spiny neurons in striatum (MSNs) in animals that display these behaviors. Our hypothesis was based on the fact that previous studies in animal models have suggested that changes in glutamate function lead to aberrant synaptic plasticity. Indeed, there is a loss of bidirectional plasticity, indicating the presence of sustained long term potentiation (LTP). In other brain regions, LTP is accompanied by increases in synapses, synaptic remodeling and/or sprouting. Thus we questioned whether an increase in corticostriatal or thalamostriatal glutamatergic synapses could underlie LIDs in a rat model.
We used stereology to count synapses in the dorsal striatum of adult male rats that received sham or 6-OHDA lesions, and were treated with L-dopa or saline for 3 weeks. Abnormal involuntary movements (AIMs) were rated following an injection of L-dopa, 3 times/week and rats were categorized into severe or mild AIMs groups. We examined the ultrastructural characteristics of corticostriatal (immunoreacted with VGlut1 antisera) and thalamostriatal (immunoreacted with VGlut2 antisera) terminals.
Our data show that dopamine depletion significantly reduced the total number of asymmetrical (excitatory) inputs from the cortex (p<0.01), especially the number of axospinous contacts (p<0.01), but not the axodendritic contacts. In contrast, dopamine depletion significantly decreased the number of excitatory axodendritic asymmetric inputs from the thalamus compared to the sham/saline group, and there was only a trend toward a reduction in axospinous synapses from the thalamus. Treatment with L-dopa alone in the sham group did not alter the total number of excitatory synapses compared to saline-treated controls, which was the case for both corticostriatal and thalamostriatal contacts. The parkinsonian rats treated with chronic l-dopa that developed severe AIMs showed a significant increase in the number of VGlut1-labeled corticostriatal synapses (p<0.005); this included both axospinous (p<0.01) and axodendritic contacts (p<0.005). The post-synaptic targets of these synapses also changed. Notably, the animals with severe AIMs showed a large increase in the proportion of axodendritic to axospinous synapses, suggesting an aberrant redistribution of these terminals. Interestingly, there was a significant decrease for VGlut1 multisynaptic boutons (MSBs) in the severe AIMs versus control groups (p<0.05). Results show that corticostriatal contacts are increased in the dyskinetic striatum, and that these changes are due to sprouting onto dendrites and spines, rather than a remodeling of existing boutons, as suggested by the significant decrease in MSBs. In contrast, there was no difference in the number of thalamostriatal synapses between the mild AIMs and the lesion group and likewise between the severe AIMs and the lesion group suggesting that the development of LIDs is not associated with a change of thalamostriatal contacts after the changes accompanying the lesion. Thus, overall these results suggest that while both corticostriatal and thalamostriatal contacts onto dendrites are altered with dopamine depletion only corticostriatal synapses appear to be involved with LIDs.
We have made initial progress on Specific Aim 4 where we have investigated whether the loss of dendritic spines, which occurs secondary to striatal dopamine depletion, can be reversed once this structural component is lost in the parkinsonian striatum. We have found that dendritic spine loss on medium spiny neurons within the striatum can be both prevented and reversed in both young and aged rats. In the next year we will be examining whether these spared or “re-grown” dendritic spines are associated with normal circuitry within the parkinsonian striatum. These studies will be done with electron microscopy and labeling for vGLut1, serotonin and tyrosine hydroxylase.
Kathy Steece-Collier, David J. Rademacher, Katherine E. Soderstrom (2012). Anatomy of graft-induced dyskinesias: Circuit remodeling in the parkinsonian striatum. Basal Ganglia 2(1):15-30.
In the third year of this award we have made considerable progress on Project 2. We have expanded our Aim 1 examination of the neuroprotective effects of subthalamic nucleus deep brain stimulation (STN DBS) (Spieles-Engemann et al., 2010) by the completion of three new studies and the establishment of an additional rat model of PD. First, we examined whether the neuroprotection against intrastriatal 6-OHDA that is mediated by 2 weeks of STN DBS is maintained long-term. Our results reveal that STN DBS-mediated neuroprotection of nigral dopamine (DA) neurons and improvements in forelimb akinesia persist for up to 5 months, the duration of the study. Second, given that DBS of the internal globus pallidus (GPi DBS) produces equivalent efficacy for motor symptoms as STN DBS in PD patients, we investigated whether GPi DBS is similarly neuroprotective against 6-OHDA. We found that in contrast to STN DBS, GPi DBS does not halt nigral DA neuron degeneration induced by 6-OHDA and further, GPi DBS does not ameliorate 6-OHDA induced deficits in contralateral forelimb akinesia. As we previously have demonstrated that STN DBS increases the expression of brain derived neurotrophic factor (BDNF) in the nigrostriatal system (Spieles-Engemann et al., 2011), which may mediate neuroprotection, we are presently determining the impact of GPi DBS on BDNF expression levels in an additional cohort of rats. Third, based on evidence linking both the 6-OHDA model and BDNF expression to the expression of tumor necrosis factor-alpha (TNF-α) we initiated a study to characterize the precise time course of nigrostriatal TNF-α expression after intrastriatal 6-OHDA injection (or vehicle control). We intended to use this information to evaluate whether STN DBS impacts TNF-α expression. Our results revealed that nigrostriatal upregulation of TNF-α mRNA and protein can be solely attributable to the surgical injection procedure and that DA neuron degeneration per se does not contribute to TNF-α expression in this model. The fact that previous studies had not properly controlled for the injection procedure has mistakenly perpetuated the concept that the 6-OHDA model can be used to model the role of TNF-α in PD.
Based on these results and other limitations of the 6-OHDA model we have turned our attention to the establishment and characterization of a different preclinical model of PD, viral vector mediated over-expression of alpha-synuclein (α-syn) targeted to the nigrostriatal system. During this past year we have carefully characterized the relationship between titer level of adenoassociated virus (AAV) expressing human wildtype α-syn and the resulting magnitude of nigrostriatal degeneration in both young and aged rats. We are now able to tailor the nigrostriatal degeneration induced by AAV α-syn to specific levels of loss ranging between 20-95% at 2 months after transduction. We have recently initiated our first study to examine the ability of STN DBS to provide neuroprotection against nigrostriatal α-syn overexpression.
As mentioned, in previous studies under the framework of Aim 2 we determined that STN DBS upregulates the expression (protein and mRNA) of BDNF within the nigrostriatal system, M1 cortex, and the internal globus pallidus (GPi). Our findings suggest that this frequently practiced therapy has the potential to produce pronounced and underappreciated effects on plasticity in basal ganglia circuitry. This suggests that STN DBS-mediated BDNF expression may play a role in symptomatic efficacy or disease progression. To explore these possibilities in the clinical setting we have collaborated with Dr. P. David Charles at Vanderbilt University. Dr. Charles leads a clinical trial that investigates the safety and efficacy of STN DBS in early stage PD patients (NCT00282152). We are presently evaluating blood samples from the 30 patients enrolled in this trial in order to genotype their BDNF status. Within the general human population there exists a single-nucleotide polymorphism of the BDNF gene (val66met at codon 66) that results in a decreased ability to package and release BDNF. To date, Core B has genotyped 18 of the patients with results indicating that 7 patients (39%) possess either 1 (val66met) or 2 (met66met) met alleles. Ultimately we will determine whether BDNF polymorphism status impacts the efficacy of STN-DBS (UPDRS Stim On), PD progression (UPDRS Stim Off) as well as time to levodopa induced dyskinesias (LIDS).
Future Studies.In Year 04 of this award we will conduct studies using Taqman Low Density Arrays (TLDA) to expand our investigation of whether trophic factors beyond BDNF are impacted by long term STN DBS. We will also initiate our first studies examining the effects of STN DBS in aged rats.
We continue to study mechanisms associated with neuroprotection of the nigrostriatal dopamine (DA) system associated with grafting undifferentiated neural progenitor cells proximal to the substantia nigra. Our previous work revealed the unexpected finding that NP cell grafts stimulate proliferation of endogenous NP cells in the host brain subventricular zone and that these host cells migrate to surround the graft. Inhibition of this host response by administering a mitotic inhibitor blunts the neuroprotective effect of grafted cells. Furthermore, absence of the host response alters grafted cell phenotype as well as the response of host cells to the microenvironment. Thus, we demonstrate that collaboration exists between grafted cells and host cells to yield neuroprotection. Our most recent study examines the effects of using shRNA to silence expression of two factors expressed by grafted cells – glial cell line-derived neurotrophic factor (GDNF) and sonic hedgehog (SHH) – prior to transplantation. Histological and behavioral analyses indicate that GDNF silencing significantly reduced NP cell-mediated neuroprotection but not endogenous neurogenesis. On the other hand, knock-down of SHH, or a combination of GDNF and SHH caused a profound decrease in both graft-mediated neuroprotection and endogenous neurogenesis. Further, no migration of endogenous NP cells was observed in the animals that had received SHH silenced cell grafts, suggesting that SHH was a key molecule contributing to NP cell mediated therapeutic effects. Overall, the studies help determine some of the micro-environmental signals fundamental to neural precursor based neuroprotection, and propose SHH as a plausible molecular target to develop novel therapeutic approaches for PD.
We have completed our studies of rat NP cells and our focus now shifts to human-derived NP cells. We have performed our first set of grafting experiments, comparing neuroprotective effects of human-derived midbrain NP cells and spinal cord NP cells (provided by Neuralstem Inc.). We have collected parallel samples from grafted animals to examine gene expression analysis in the graft microenvironment.
Madhavan L, Daley BF, Sortwell CE, Collier TJ. (2012) “Endogenous neural precursors influence grafted neural stem cells and contribute to neuroprotection in the parkinsonian rat.” Eur J Neurosci 35(6): 883-95. PMID 22417168.
Paumier KL, Siderowf AD, Auinger P, Oakes D, Madhavan L, Espay AJ, Revilla FJ, Collier TJ, for the Parkinson Study Group Genetics Epidemiology Working Group. (2012) “Tricyclic antidepressants delay the need for dopaminergic therapy in early Parkinson’s disease.” Mov Disord 27(7):880-7. PMID 22555881.
The goal of the studies in Project 4 of the Udall grant is to test the hypothesis that stress-induced corticosterone (CORT) hypersecretion is a causal link between chronic stress-induced depression and enhanced dysfunction and dopaminergic neurotoxicity in the nigrostriatal system. We have previously found that chronic variable stress (CVS) hastened midbrain neurodegeneration and exacerbated associated motor deficits. These data indicate that chronic stress accelerates dopaminergic neuronal loss in the injured nigrostriatal system, and suggests that life stress may accelerate the progression of Parkinson’s disease. Experiments 1b and 1c of Specific Aim 1 addressed possible mechanisms involving glucocorticoid (corticosterone; CORT) substitution for CVS and the requirement of glucocorticoid receptor (GR) binding (using the GR antagonist RU486). Both results were negative, suggesting glucocorticoids are not critical for chronic-stress induced exacerbation of experimental parkinsonism. However, upon re-examination of the data, we noticed that the 6-OHDA lesions were much more extensive than in our initial studies, raising the possibility that a “floor effect” was reached in these experiments which obviated further worsening of behavior and cell degeneration. We have therefore re-calibrated our 6-OHDA dosing regimen to establish a more limited loss of nigral TH neurons (targeting 50% loss). Experiment 1a, which is analyzing the necessity of glucocorticoids for stress exacerbation of experimental parkinsonian symptoms using blockade of steroid production (adrenalectomy in combination with physiological corticosterone replacement) is currently underway. Tissue continues to be collected for the gene expression analyses (Experiment 1d), and will be sent to Udall Core B for analysis upon completion of Experiment 1a.
Hemmerle, A.M., Herman, J.P. and Seroogy, K.B. (2012) Stress, depression and Parkinson’s disease. Experimental Neurology 233: 79-86. PMID: 22001159; PMCID: PMC3268878 [Available on 2013/1/1]
Core B is well underway in the first phase of developing a “consensus array” for the subprojects of MSU Udall Center. The primary goal of the consensus array development is to: (1) Employ a comprehensive microarray to identify gene expression changes in the acute, mid- and long-term phases of degeneration (1 to 16 weeks post-lesion) from intrastriatal 6-OHDA infusion in the rat and then; (2) Construct a “consensus” Taqman Low Density Array (TLDA) of the significant genes for each structure of interest (striatum and nigra). These focused consensus arrays will then be used to explore the impact of each project’s treatment (stem cell implantation, deep-brain stimulation, nimodipine, levodopa induced dyskinesias, chronic variable stress (CVS)) on the gene-expression signature observed from the 6-OHDA lesion alone. These data will then be subjected to pathway analysis to determine common pathways affected by treatments across projects. In this way, the projects have specific overlapping data sets on which project leaders can move forward together in developing complementary experiments associated with these matching data sets (for example: Are the genes upregulated by experimentally-induced depression in the consensus array down-regulated by deep brain stimulation?) If so, we can begin to piece together the overlapping critical players in gene expression that may exacerbate or reduce cell loss across projects.
To date we have finished the collection of nigral and striatal tissue from rats through the acute and mid-term post-lesion phases. Tissue dissection and RNA isolation and quality assessment have also been completed on these tissues. The tissue will be subjected to the Affymetrix GeneChip Gene 1.0 ST Array System. This system was chosen because it is not based on the 3’-based expression design that has probes localized to the extreme 3’ end of the gene. Such systems may not accurately display changes when there is alternative splicing at the 3’ end of the gene, alternative polyadenylation sites, nonpolyadenylated messages, truncated transcripts or samples have some degradation. By utilizing a random priming strategy, we should end up with a more robust and reproducible data set.
We are continuing to characterize the time course and features of the AAV alpha-synuclein over-expression rat model of PD in both young adult and aged animals.
Two aspects of Parkinson’s disease (PD) that have received relatively little study are the nervous system mechanisms associated with development of adverse consequences of disease and treatment (stress, depression and medication-induced side-effects) and mechanisms associated with experimental therapies (deep brain stimulation, cell transplantation). In addition, it long has been appreciated that advancing age is a primary risk factor for PD, yet aging rarely is incorporated into experimental studies. Our Center studies these adaptive and maladaptive changes associated with PD and its’ treatments in model systems that incorporate the factor of advancing chronological age.
Our findings suggest that many negative side-effects of disease and treatment can be avoided or improved, and that experimental therapies currently in development may possess previously unrecognized additional benefits. Our goal is that through continued study of these issues our work can provide for development of optimal therapeutics for PD, inform their use in the clinical setting, and ultimately improve the quality of life for those living with PD.
Last updated March 20, 2013