Caryl E. Sortwell, Ph.D. - US grants

Critical to clinical success of transplantation for Parkinson's disease (PD) is the development of methods whereby grafted DA neuron viability and reinnervation of the host striatum are markedly increased. The survival rate of DA neurons in grafts to young rats is 5-10 percent, and is even poorer in grafts to the aged striatum. The focus of this revised KO1 Mentored Research Award application is to pursue previous work by the candidate which demonstrated that striatal target cells can increase the survival of simultaneously grafted DA neurons by supplying diffusible trophic factors. The studies proposed in this application seek to define some of the cellular and molecular mediators of enhanced DA neuron survival, through close collaboration with the mentor's laboratory in the use of cell culture techniques, and apply findings in cell culture to therapeutic intervention in the aged nervous system. Based on suggestions in the literature, it is likely that the striatal oligodendrocyte type 2-astrocyte (0-2A) progenitor cell is one cellular source of striatal-derived trophic activity DA neurons. One goal of this application will be to develop expertise in cell culture techniques and growth factor treatments in culture to selectively enrich striatal 0-2A cells for investigation of their role in neurotrophic effects on DA neuron survival and apoptotic death rates. A second goal of the proposed training will be to develop experience in the neurobiology of aging animals, another focus of the mentor's laboratory. Recent evidence in the mentor's laboratory indicates that morphological and behavioral effects of DA grafts in aged rats with long-term lesions is greatly diminished as compared to grafts in young rats. Information learned from the initial set of culture experiments will be applied to transplantation studies in young and aged rats, in order to determine whether DA neurons co-grafted with 0-2A enriched striatal cells can provide superior morphological and behavioral outcomes in aged subjects. Should these studies indicate that enhanced DA neuron survival is related to the inhibition of apoptosis, then a direct apoptosis inhibition approach also will be studied. Therefore, the final goal of this training proposal will be to gain experience in utilizing viral vectors in cell culture to overexpress bcl-2, an identified "survival" gene, in DA neurons and to examine the effect of this transduction on apoptosis in both cultures and in grafts to young and aged rats. Tissue culture aspects of this study will be learned in the mentor's laboratory, and viral vector will-be provided by Dr. Howard Federoff, Univ. of Rochester School of Medicine. In addition, the candidate will visit the Federoff laboratory learn viral vector transduction techniques. The overall hypothesis of this proposal is that by inhibiting apoptotic cell death of grafted DA neurons, both indirectly by supplying trophic factors and directly by interfering with the genetic death program, it will be possible to increase DA graft viability and subsequent reinnervation of the aged striatum. This training will promote the candidate's development into a fully independent investigator in the fields of neural cell culture, aging in the DA system, apoptosis and neural grafting for PD.

DESCRIPTION (provided by applicant) The strategy of augmenting striatal dopamine (DA) levels via grafts of DA-producing cells holds great promise for the treatment of Parkinson's disease (PD). Critical to clinical success is the development of methods whereby grafted DA neuron viability and reinnervation of the host striatum are markedly increased. This objective holds true for both primary mesencephalic tissue grafts as well as for alternative tissue sources such as stem- and progenitor-derived DA neurons. The survival rate of DA neurons in fetal mesencephalic grafts to young adult rats is 5-10%. Our laboratory has recently demonstrated that this survival is even poorer in grafts to the aged striatum. Our research indicates that massive apoptotic cell death occurs within the graft during the first few days after transplantation and then sharply diminishes. Not surprisingly, this timecourse closely parallels the delay in host vascularization of the grafted cells. The lack of blood-borne nutrients, including oxygen, experienced by the grafted cells during the immediate post-grafting interval is a likely candidate to trigger apoptosis. The overall hypothesis of this proposal is that grafted DA neuron survival is severely limited by lack of blood-borne nutrients during the early post-transplantation interval when grafted cells are not adequately vascularized. Therefore augmentation of graft vascularization via gene transfer of vascular endothelial growth factor (VEGF) will significantly augment graft viability and subsequent reinnervation of the host striaturn. This strategy will be evaluated using primary mesencephalic tissue. However, results from these studies will be applicable to alternative sources of DA neurons, including stem- and progenitor-derived cells.

[unreadable] DESCRIPTION (provided by applicant): This R13 application seeks funding to support graduate student/postdoctoral fellow travel awards to the annual meeting of the American Society for Neural Transplantation and Repair (ASNTR). Since its founding in 1994, the ASNTR has convened yearly each April/May in Clearwater, Florida. At the meeting various research advances in the fields of transplantation and gene therapy applied to neurodegenerative diseases such as Parkinson's disease, Huntington's disease, Alzheimer's disease, stroke, spinal cord injury and traumatic brain injury are discussed via platform and poster presentations. Attendance of this meeting ranges between 150-250 registrants. Each year the ASNTR Education Committee encourages students/postdocs to submit their abstracts in a competition and the top rated students/postdocs receive travel awards to cover the cost of their meeting attendance and travel. ASNTR has always placed a high priority on funding travel awards for students and post-doctoral fellows presenting their scientific findings at the yearly conference. As the field of neural transplantation and repair continues to expand we feel that it is critical to support students and postdocs so that they may attend this meeting and share their recent scientific findings with leaders in their field of research. In the past ASNTR has received donations from biotech companies and non-profit organizations to cover these travel awards. However, in recent years ASNTR has seen these contributions dwindle and although ASNTR will continue to seek funding from the private sector, we do not anticipate any increase in contributions above recent levels in the coming years. This application requests $15,000 in total direct costs to support travel awards for the top 15 graduate students/postdocs from US institutions that apply (awards of $1000 per student/postdoc). [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Intracerebral neural grafting strategies for neurological disorders are limited by the poor survival rate of grafted cells. For example, the survival rate of dopamine (DA) neurons grafted in parkinsonian animal models and in clinical trials with Parkinson s patients is merely 5-20%. Critical to clinical success is the development of methods whereby grafted DA neuron viability and reinnervation of the host striatum are markedly increased. The focus of this R03 application is to directly examine the role of hypoxia in intracerebral grafts utilizing a well-established paradigm, grafts of mesencephalic DA neurons. Our research indicates that massive apoptosis of grafted mesencephalic cells occurs within the first few days after transplantation and then sharply diminishes. This time course of grafted DA neuron death closely parallels the delay in host vascularization of the grafted cells. The lack of blood-borne oxygen, or hypoxia, experienced by the grafted cells during the immediate post-grafting interval is a likely candidate to trigger apoptotic cell death. However, the role of hypoxia in limiting graft survival has never been directly assessed. The overall hypothesis of this proposal is that grafted DA neuron survival is severely limited by hypoxia during the early post-transplantation interval when grafted cells are not adequately vascularized. Identification of hypoxia as a significant constraint on graft survival would direct future strategies aimed at enhancing intracerebral grafts of numerous cell types (primary cells, stem cells) implanted to treat a wide range of neurological disorders.

DESCRIPTION (provided by applicant): This R13 application seeks funding to support travel awards for students and postdoctoral fellows to attend the annual meeting of the American Society for Neural Therapy and Repair (ASNTR) 2009. Since its founding in 1994, the ASNTR has convened yearly each April/May in Clearwater, Florida. At the meeting various research advances in the fields of transplantation, neuroengineering and gene therapy applied to neurological diseases such as Parkinson's disease, Huntington's disease, Alzheimer's disease, stroke, spinal cord injury and traumatic brain injury are discussed via platform and poster presentations. ASNTR focuses on treatment strategies throughout all its sessions. Thus, students and postdoctoral fellows not only learn the basic sciences, they also learn translational issues discussed between basic scientists and clinicians. Therefore, ASNTR is truly a unique gathering. Attendance of this meeting ranges between 125-200 registrants. Each year the ASNTR Education and Training Committee encourages students/postdoctoral fellows to submit their abstracts in a competition and the top rated applicants receive travel awards to cover the cost of their meeting attendance and travel. ASNTR has always placed a high priority on funding travel awards for students and post-doctoral fellows. In the past ASNTR has received donations from biotech companies and non-profit organizations to cover these travel awards, However, in recent years ASNTR has seen these contributions dwindle and although ASNTR will continue to seek funding from the private sector, we do not anticipate any increase in contributions above recent levels in the coming years. Therefore NIH funding for this activity is critical. This application requests $20,000 in total direct costs to support travel awards for the top 20 graduate students/postdoctoral fellows from US institutions that apply (awards of $1000 per student/postdoc). ASNTR and the Education and Training Committee includes and encourages participation of women, minorities and persons with disabilities in all its activities. PUBLIC HEALTH RELEVANCE: This application seeks funding for travel awards for graduate students and postdocs to attend the annual meeting of the American Society for Neural Therapy and Repair. This meeting will allow young and senior scientists and doctors working to discover new treatments for neurological disorders to come together and discuss their new research findings. This meeting will potentially allow new research collaborations to occur that could lead to improved treatments.

DESCRIPTION (provided by applicant): Neurotrophic factors are normally involved in the development and maintenance of the nigrostriatal system, yet they also possess incredible potential to repair it. Such trophic factors may have great potential as therapeutics in Parkinson's disease (PD) by providing long-term, lasting efficacy without the disabling side effects associated with current treatments. Our laboratory and others have revealed that the novel growth factor pleiotrophin (PTN) participates in the development and maintenance of the nigrostriatal system in a manner similar to glial cell line-derived neurotrophic factor (GDNF): 1) Both PTN and GDNF receptors are expressed by mesencephalic DA neurons;2) PTN and GDNF expression levels in the striatum peak during early postnatal development and decrease to low levels in adulthood;3) PTN and GDNF protein, mRNA and receptor expression in the striatum are upregulated in response to striatal denervation and;4) The addition of either PTN or GDNF to embryonic mesencephalic cultures specifically promotes tyrosine hydroxylase immunoreactive (THir) neuronal survival and neurite outgrowth. Such parallels suggest that PTN is an intrinsic factor critical to the development, maintenance and repair of the nigrostriatal DA system and point to its potential to provide neuroprotection and promote reconstruction of nigrostriatal circuitry when used as an exogenous therapeutic. This proposal will study the potential of PTN to provide neurorestoration in a rat model of PD, testing the hypothesis that overexpression of PTN in the adult rat nigrostriatal system can facilitate long-term functional recovery in a rat model of parkinsonism. Additional studies will examine whether PTN overexpression can improve dopamine graft survival and innervation of the denervated host striatum. Initially we will determine the peak levels of PTN expression in the nigrostriatal system of rats during development in order to identify the levels of PTN overexpression to be mediated by subsequent viral vector gene transfer. Our hypothesis then will be tested utilizing two separate approaches. First, we determine whether gene transfer of PTN to the striatum of rats with partial lesions can promote neurorestoration of the nigrostriatal system dopamine (DA) levels. Second, we will assess whether striatal PTN gene transfer can provide superior DA graft-mediated recovery in rats with complete unilateral lesions of the nigrostriatal system, including a decrease in the severity of levodopa-induced dyskinesias (LIDs). These studies will determine whether PTN gene transfer can be used as a therapeutic strategy to treat PD.

Deep brain Stimulation (DBS) of the subthalamic nucleus (STN) has become the most often practiced neurosurgery for treatment of the cardinal motor features of Parkinson's disease (PD). Preclinical evidence indicates that high frequency stimulation (HFS) of the STN protects substantia nigra pars compacta (SNc) dopamine (DA) neurons from DA-depleting lesions. However, none of these studies examined the impact of STN HFS on DA terminal density or neurochemistry in the striatum and thus our understanding of the entire scope of protection is incomplete. Further, these studies did not examine the ability of STN HFS to provide neuroprotection in the face of previous large scale DA neuron loss. Growing evidence suggests that STN HFS drives and synchronizes the STN making it unlikely that decreased excitotoxicity is involved in STN HFS-mediated neuroprotection. We therefore propose to systematically examine the magnitude and mechanism of neuroprotection conferred by STN HFS in rats made parkinsonian by intrastriatal 6-hydroxydopamine (6-OHDA). We will examine the impact of STN HFS on nigrostriatal morphology and dopamine biochemistry. Our findings suggest that initiation of long-term HFS of the STN two weeks after lesion completely halts nigral DA neuron degeneration. We will test the hypothesis that HFS of the STN elicits upregulation of brain-derived neurotrophic factor (BDNF) mRNA and protein in primary and secondary STN target structures. Indeed, our findings demonstrate that long-term unilateral STN DBS upregulates the expression of BDNF protein and mRNA in the striatum and external pallidum, respectively. Lastly, we will examine the impact of STN HFS on the aged nigrostriatal system and test the hypothesis that STN HFS will provide little to no neuroprotection in the aged brain. We speculate that STN HFS may not be neuroprotective due to limited trophic plasticity of the aged brain. The proposed studies will determine whether STN DBS can slow the progression of DA neuron degeneration and whether this outcome is compromised by advancing age. The results of these studies may inform optimal therapeutic timing for intervention with STN DBS as well as identify a neuroprotective mechanism to be harnessed via this therapy. RELEVANCE (See Instmctions): The purpose of this project is to determine whether the surgical treatment for Parkinson's disease, deep brain stimulation, can potentially slow the progression of the disease. The mechanism of action of deep brain stimulation will also be investigated.

DESCRIPTION (provided by applicant): This R13 application seeks funding to support travel awards for students and postdoctoral fellows to attend the joint meeting of the American Society for Neural Therapy and Repair (ASNTR) and the International Conference on Neural Therapy and Repair (INTR) to be held May 4 - May 8, 2011. Since its founding in 1994, the ASNTR has convened yearly each April/May in Clearwater, Florida. This year the meeting has merged with the 11th convening of the INTR which meets every three years alternating between the US, Asia and Europe. Both societies have a strong tradition of focusing on research advances in the fields of transplantation, neuroengineering and gene therapy applied to neurological diseases such as Parkinson's disease, Huntington's disease, Alzheimer's disease, stroke, spinal cord injury and traumatic brain injury that may lead to novel treatment strategies. Clinical trials that have emerged from basic science research reported on at previous meetings are often presented. Thus, students and postdoctoral fellows not only learn the basic sciences, they also learn translational issues discussed between basic scientists and clinicians. Therefore, ASNTR/INTR are truly unique gatherings. Attendance of this meeting is projected to be approximately 250- 300 registrants, based on previous participation levels. Each year the ASNTR Education and Training Committee encourages students/postdoctoral fellows to submit their abstracts in a competition and the top rated applicants receive travel awards to cover the cost of their meeting attendance and travel. ASNTR has always placed a high priority on funding travel awards for students and post-doctoral fellows. In the past ASNTR has received donations from biotech companies and non-profit organizations to cover these travel awards. However, in recent years ASNTR has seen these contributions dwindle and although ASNTR will continue to seek funding from the private sector, we do not anticipate any increase in contributions above recent levels in the coming years. This application requests $20,000 in total direct costs to support travel awards for the top 20 graduate students/postdoctoral fellows from US institutions that apply (awards of $1000 per student/postdoc). The ASNTR and the Education and Training Committee include and encourage participation of women, minorities and persons with disabilities in all its activities. PUBLIC HEALTH RELEVANCE: This application seeks funding for travel awards for graduate students and postdocs to attend the upcoming combined meeting of the American Society for Neural Therapy and Repair and the International Conference on Neural Therapy and Repair. This meeting will allow junior and senior scientists and doctors working to discover new treatments for neurological disorders to come together and discuss their new research findings. This meeting will potentially allow new research collaborations to occur that could lead to improved treatments for neurological disorders.

Project Summary Oral levodopa (L-dopa) has become the mainstay pharmacotherapy for Parkinson's disease (PD). Although generally effective in treating the motor symptoms of PD, the clinical response is highly variable. We contend that the efficacy of oral L-dopa may be influenced by subject genotype. Indeed, response to antidepressant and antipsychotic pharmacotherapy is influenced by the Bdnf gene coding for brain-derived neurotrophic factor (BDNF), specifically the single nucleotide polymorphism (SNP) rs6265. The Bdnf SNP rs6265 is relatively common with a 40.6% prevalence of carrying the minor Met66 allele (Val/Met or Major/Minor = 35.4%, Met/Met or Minor/Minor = 5.2%, allelic frequency assuming Hardy-Weinberg). Presence of the Met allele disrupts packaging and release of activity-dependent BDNF. We recently genotyped early-stage PD patients who were treated with either deep brain stimulation of the subthalamic nucleus (DBS) or optimized drug therapy (ODT, predominantly oral L-dopa) and enrolled in the Vanderbilt DBS in Early Stage PD clinical trial (NCT00282152). The trial occurred over a period of 24 months. Five of 15 subjects (33%) and 6 of 13 subjects (46%) in the DBS and ODT treatment arms, respectively, carried the Met allele of the Bdnf SNP rs6265. At baseline, all clinical endpoints were statistically similar across Bdnf genotype (p > 0.05). However, Met allele carriers in the ODT arm exhibited significantly higher (worse) UPDRS scores ON medications at 18 (p = 0.017) and 24 months (p = 0.019) and significantly higher PDQ-39 scores at 12 (p = 0.033) and 24 months (p = 0.018, compared to ODT subjects with the most common genotype Val/Val). In contrast, no significant differences were observed due to Met allele status in subjects receiving DBS at any time point with any clinical metric (p > 0.05). Our discovery cohort results suggest that possession of the Met66 allele of the SNP rs6265 confers a treatment-specific, suboptimal response to dopaminergic PD medication that emerges over long treatment intervals. Validation in a larger cohort of early PD subjects treated with dopaminergic medication is warranted to establish whether our phenomenon is truly generalizable to the PD population as a whole. Our ultimate goal is to determine whether genotyping for the Bdnf SNP rs6265 could be used as a precision medicine approach for the treatment of PD by either medical or surgical interventions as well as a method for stratification of subjects enrolled in clinical trials for more efficient and effective clinical trial design.

Project Summary/Abstract Approximately 6.4 million Americans suffer from neurodegenerative diseases of aging. Clinical trials utilizing viral vector-mediated gene therapy for treating age-related neurodegenerative diseases such as Parkinson's disease (PD) are ongoing. This approach requires efficient gene transfer to the aged brain. Preclinical studies in rodents and non-human primates have informed optimal viral vector design for specific target structures and cellular populations, however these studies almost exclusively use young adult animals and therefore fail to recapitulate the aged brain environment. Our laboratories use viral vectors to model PD or test the therapeutic effects of trophic factors in young adult and aged rats. Recently, we found that the transduction efficiency mediated by various viral constructs is markedly compromised in the aged rat. Specifically, we utilized three different pseudotypes of recombinant adeno-associated virus (rAAV2/2, rAAV2/5 and rAAV2/9) or lentivirus to express green fluorescent protein (GFP). Injections were made into either the substantia nigra pars compacta (SNpc) or the striatum of young adult or aged rats. The efficiency of nigrostriatal or striatonigral transduction was evaluated utilizing a variety of methods (stereology, immunofluorescence, in situ hybridization, western blot, qPCR). Following injection to the striatum, all vector constructs exhibited significant transduction deficiencies in aged rats. Following injection into the SNpc, rAAV2/2, rAAV2/5 and lentivirus exhibited deficient transduction associated with aging whereas equivalent transduction efficiency was observed in young and aged rats using rAAV2/9. Our results thus far suggest that, in general, the aged brain is strikingly resistant to transduction. The proposed studies seek to identify the causes of age-related transduction deficiencies in order to optimize future gene therapy clinical trials for Parkinson's and other age-related diseases.

Until recently, no animal model of Parkinson's disease (PD) has adequately incorporated both widespread alpha-synuclein (?-syn) pathology and protracted significant nigrostriatal degeneration. In 2012, Luk and colleagues described how intrastriatal injection of synthetic ?-syn preformed fibrils (PFFs) into wildtype (WT) mice seeded endogenous accumulation of Lewy Body (LB)-like intracellular ?-syn inclusions and ultimate nigrostriatal degeneration. In light of the fact that the rat model system offers distinct advantages over mice (fine motor behaviors, greater synaptic complexity, genetics and pharmacokinetics more similar to humans, larger brain and body size more amenable to neurosurgical interventions and sample collection), we recently characterized the results of unilateral injection of mouse ?-syn PFFs into the striatum of rats. Similar to the mouse, we observed phosphorylated ?-syn intraneuronal accumulations in several areas that innervate the striatum, most prominently the frontal and insular cortices, the amygdala, and the substantia nigra pars compacta (SNpc). ?-Syn accumulations co-localized with ubiquitin, p62, and were thioflavin-S-positive and proteinase-k resistant. Although ?-syn inclusions within the SNpc remained ipsilateral to striatal injection, we observed bilateral reductions in nigral dopamine neurons 6 months following ?-syn PFF injection. Further, PFF injected rats exhibited reductions in striatal dopaminergic innervation as well as deficits in striatal dopamine and metabolites. This initial study demonstrates that ?-syn PFFs are sufficient to seed the pathological conversion and propagation of endogenous ?-syn to induce a progressive, neurodegenerative model of ?-synucleinopathy in rats. In the present IGNITE application we seek to identify which ?-syn species (human, mouse, rat) results in consistent loss of 60% nigral dopamine neurons over the course of 4 months after intrastriatal injection into rats (Aim 1). Once identified, we will conduct internal validation studies to systematically characterize the time course and magnitude of ?-syn pathology, striatal dopamine and metabolite loss, levels of striatal dopaminergic innervation (tyrosine hydroxylase, vesicular monoamine transporter, and dopamine transporter) and deficits in motor behaviors (Aim 2). Lastly, we will conduct PD-relevant external validation studies including: 1) investigating whether PFF-induced motor deficits are reversible with levodopa and 2) conduct non-invasive longitudinal in vivo imaging of nigrostriatal DA synthesis, storage and turnover using positron emission tomography (PET) (Aim 3). We propose that optimization, internal validation and external validation studies in the progressive rat ?-syn PFF PD model will allow for direct comparison to clinical metrics in PD patients and facilitate the development of novel disease modifying therapies.

PROJECT SUMMARY Parkinson's disease (PD) is a culmination of aging-related changes, genetic predispositions, and environmental insults that result in accumulation of alpha-synuclein (?-syn)-containing Lewy bodies and degeneration of the nigrostriatal system. Neuroinflammation has been proposed to be involved in PD pathophysiology, supported by observations of inflammatory microglia in post mortem PD brains and longitudinal PET imaging that reveals early and sustained microglial activation in the basal ganglia of PD patients. Not only has increased MHC-II expression been observed, it correlates positively with ?-syn burden. Chronic microglial activation can result in a feed-forward cycle of proinflammatory cytokine secretion, production of reactive oxygen species, and subsequent neuronal damage. Activated microglia also have the potential to convert astrocytes to a toxic A1 phenotype that can induce the death of neurons. Both microglia and neurons can signal to peripheral T-cells to invade from the periphery, providing another source of toxic damage to neurons. However, whether neuroinflammation contributes to, or is simply a consequence of nigrostriatal degeneration, remains unclear. Neurotoxicant, ?-syn transgenic and ?-syn vector models of PD have been used to investigate the role of neuroinflammation in nigrostriatal degeneration. Similar to the PD brain, these models reveal neuroinflammation associated with either ?-syn inclusions or nigral degeneration. However, none of these models exhibit the complete sequence of events associated with nigrostriatal pathology in PD: prolonged ?- syn accumulation resembling Lewy bodies followed by protracted degeneration. Recently, our lab has characterized a new rat PD model that recapitulates these features, a model in which nigrostriatal synucleinopathy is induced by intrastriatal injection of preformed ?-syn fibrils (?-syn PFFS). We have observed that peak reactive microglial morphology is evident in the SN at 2 months, corresponding to the time point in which the greatest number of phosphorylated ?-syn (pSyn) inclusions are observed. Further, we observe a pSyn-triggered increase in MHC-II immunoreactive microglia at this same 2-month time point that significantly correlates with pSyn inclusion load. Importantly, we observe reactive microglia and increased microglial MHC-II expression in association with peak load of SNc pSyn inclusions months prior to degeneration, suggesting that neuroinflammation may!contribute to nigrostriatal degeneration. In the present exploratory R21 application we propose to extend these observations regarding the response of microglia to ?-syn inclusions to understanding the role that astrocytes may play in this degenerative cascade. We also will directly test the involvement of microglia in degeneration. The overarching hypothesis of this proposal is that microglial activation, triggered by accumulation of pathological ?-syn, contributes to nigral degeneration.

Project Summary Subthalamic nucleus deep brain stimulation (STN DBS) to treat the cardinal motor symptoms of Parkinson?s disease (PD) has increased dramatically since its first use was reported in 1994. DBS is a vetted, safe and efficacious neurosurgical therapy for PD. Once considered a treatment of last-resort with patients undergoing neurosurgery approximately 10-16 years post diagnosis, STN DBS now is FDA approved for use as early as four years after diagnosis and symptomatic efficacy may be superior to medical therapy at that time. Questions remain as to whom will best benefit from additional and earlier years of stimulation treatment. Specifically, the question of whether early STN DBS can modify the progression of PD has yet to be examined in an appropriately designed clinical trial. Dopaminergic denervation of the putamen is nearly complete within four years of PD diagnosis and precedes loss of nigral neurons. Thus, neuroprotective therapies that seek to protect the nigrostriatal system cannot be adequately evaluated in subjects with disease duration longer than this four-year timeframe. Several clinical studies have investigated whether STN DBS has the ability to slow or halt the progression of PD. However, the common thread in all of these studies is that the subjects enrolled were mid to late-stage PD when STN DBS was initiated. Most recently, a pilot trial has shown that STN DBS was applied within 2 years of PD diagnosis is safe and efficacious with subjects receiving STN DBS exhibiting a slower worsening of rest tremor. This suggests that early DBS may slow some aspects of PD progression. Preclinical studies by our group and others have demonstrated that STN DBS can protect against degeneration of nigrostriatal dopamine (DA) neurons induced by neurotoxicant insult in both rats and nonhuman primates. In our laboratory we have previously shown that STN DBS significantly increases brain- derived neurotrophic factor (BDNF) in the nigrostriatal system and the primary motor cortex (M1). Further, we have directly linked the neuroprotective effect of STN DBS to BDNF-tropomyosin receptor kinase type B (trkB) signaling in substantia nigra pars compacta (SNpc) neurons as trkB blockade prevents this neuroprotection. In contrast to results in neurotoxicant models, STN DBS applied in the alpha-synuclein (?-syn) overexpression models has yielded mixed neuroprotection results. Whether STN DBS can protect the nigrostriatal system in the context of synucleinopathy therefore remains an open question. In the present proposal we employ an alternative synucleinopathy model: the ?-syn preformed fibril (PFF) model. The ?-syn PFF model shares key features of idiopathic PD and may be more disease-relevant to idiopathic PD than ?-syn overexpression models, potentially providing greater predictive validity. Using the ?-syn PFF model we will determine whether STN DBS can provide neuroprotection of SNpc cell bodies, SNpc nigrostriatal terminals and M1 corticostriatal neurons. We will further investigate the impact of long-term STN DBS on potential arbiters of neuroprotection and disease-modification: neuroinflammation and BDNF.