Jeffrey H. Kordower - US grants

In the last decade, cognitive deficits displayed by rodents in animal models of Alzheimer's disease have been ameliorated following the neural grafting of fetal cholinergic cells and differentiated neuroblastoma cells. These data have led to the suggestion that neural implants may be a useful procedure for the treatment of neurodegenerative diseases such as Alzheimer's disease. However, a data base for such clinical trials has not been establish in non-human primates. A major obstacle in gathering such a data base is the lack of an adequate non-human primate model of cognitive dysfunction. The first goal of this proposal is to establish such a model. Neurotoxic lesions will be made in the nucleus basalis of Meynert of young and aged Cebus apella monkeys. This brain sites provides the major cholinergic input to the amygdala and neocortex and degenerate in certain diseases of cognitive dysfunction including Alzheimer's disease. Aged monkeys display cognitive dysfunction including Alzheimer's disease. Aged monkeys display cognitive deficits that are moderated by cholinomimetics suggesting that the memory impairments they display are partly mediated through cholinergic mechanisms. Creating lesions in aged monkeys may induce more robust cognitive deficits from which the effects of neural implants can better be evaluated. For clinical trials, the source of donor cells is a critical issue. Most experimental studies employ fetal primordia as donor material. Our research group has extensively investigated the potential of non-fetal differentiated neuroblastoma cells to serve as donor material. This proposal will directly compare the ability of differentiated neuroblastoma cells and fetal cholinergic primordia to survive transplantation into telencephalic brain sites and innervate the primate brain whose hose cholinergic system(s) have been impaired through neurotoxic lesions of the nucleus basalis of Meynert or the aging process. The final goal of this proposal is to determine whether grafts of cholinergic cells can ameliorate lesion-induced or age-related cognitive dysfunction in the non-human primate. These studies will be a major step determining the potential of neural implants to serve as an innovative therapeutic strategy for the treatment of neurodrogenerative diseases such as Alzheimer's disease. It will determine the ability of the age primate to serve as host for neural implants and will also create a non-human primate model of Alzheimer's disease for which other innovative strategies can be evaluated.

Converging lines of evidence have suggested that cholinergic basal forebrain [CBF] neurons are sensitive to the trophic effects of a number of growth factors including nerve growth factor [NGF] and fibroblast growth factor [FGF]. Furthermore, these neurons require NGF for their survival and phenotypic differentiation. CBF neurons consistently degenerate in Alzheimer's disease (AD). These data, along with the substantial clinical and experimental literature implicating CBF systems in normal cognitive processes, has led to the hypothesis that administration of growth factors such as NGF may prevent the degeneration of CBF neurons in AD. One shortcoming of this contention is the paucity of supporting data gathered in nonhuman primates. This proposal aims to examine the structural and functional consequences of peripheral nerve grafts upon nonhuman primate CBF neurons. Following peripheral nerve transection, Schwann cells which ensheath peripheral nerves begin de novo synthesis of a number of growth factors and neurite promoting molecules including NGF, ciliary neuronotropic factor, brain derived neuronotrophic factor, laminin, and possibly FGF. We have previously demonstrated that peripheral nerve provides trophic and neurite promoting effects upon primate adrenal chromaffin cells following grafting into parkinsonian monkeys. Recently we have demonstrated that axotomized monkey CBF neurons can be rescued following peripheral nerve grafts if the implant precedes neuronal degeneration. Peripheral nerve grafts also invoke a cholinergic sprouting response. The proposed experiments intend to expand upon these latter observations. The long-term trophic and neurite promoting effects of peripheral nerve implants will be assessed and the dependence of these effects upon the continued presence of the graft will be determined. NGF levels produced by the implant will be quantified over time. Possible detrimental effects of peripheral nerve implantation will be determined via in situ hybridization for the beta amyloid precursor protein and with an immunocytochemical analysis of abnormally synthesized cytoskeletal proteins. The ability of aged primate transected nerve to manifest trophic influences upon NGF sensitive cells will also be determine. NGF production in severed peripheral nerves from young and aged monkeys will be compared in vitro and following grafting. The ability of autologous peripheral nerve grafts to prevent experimentally induced CBF neural degeneration in aged monkeys and the ability of such grafts to reverse age-related cognitive dysfunction will also be assessed. These studies will establish whether peripheral nerve grafts are a good donor source for reversing the consequences of basal forebrain degeneration in primates and will shed light on whether this approach is relevant for the treatment of AD.

The major symptoms of Parkinson's disease, bradykinesia, postural instability, rigidity, and tremor, result principally from a dopaminergic insufficiency within the striatum. Levo-dopa (Sinemet) pharmacology remains the standard treatment strategy which is often preceded by and/or augmented with monoamine oxidase inhibitors, dopamine receptor agonists and cholinergic antagonists. This multipharmacy approach is extremely effective for many years. However, the benefits of levodopa and adjunctive agents wane after 6-7 years and there is an increase in drug- related disabling side effects. In recent years, the transplantation of dopaminergic neurons has been tested as a novel therapeutic strategy for the treatment of Parkinson's disease. Among the donor tissues employed, experimental and clinical evidence favor fetal dopaminergic neurons as the cell type of choice for neural grafting. However, there are a number of practical considerations make widespread fetal grafting difficult and potentially impractical. Principal among these issues is the availability of sufficient numbers of embryonic donors between the ages of 6-10 weeks gestation. The present proposal will employ embryonic dopaminergic nigral neurons which have been genetically modified by transfection with the temperature sensitive SV40 large T antigen. These cells are immortal at 33degreesC but become permanently amitotic when shifted to 37degreesC-39degreesC regardless of any subsequent temperature change. This provides for a virtually limitless supply of clonal dopaminergic neurons which can then easily be rendered amitotic prior to transplantation. We have recently demonstrated that these cells survive grafting, express dopaminergic markers, and reverse functional deficits following transplantation for up to one month in unilaterally nigrostriatal lesioned rats. Furthermore, these cells survive grafting and express dopaminergic markers for up to one month in hemiparkinsonian monkeys. This proposal aims to determine the long-term structural and functional consequence of grafting SV40 transfected dopaminergic cells in rodent and nonhuman primate models of Parkinson's disease. Furthermore, attempts will be made to augment the viability and functional improvement mediated by these implants by cografting these cells with astrocytes or fibroblasts which have been genetically modified to synthesize brain derived neurotrophic factor (BDNF), a neurotrophin which enhances viability of fetal nigral neurons in culture. Lastly, we will determine the ability of a human SV40 transfected dopaminergic cells to survive grafting and mediate functional recovery in unilateral nigrostriatal lesioned rats. These data will determine whether genetically modified neurons can survive grafting long-term and reverse functional deficits in rodent and nonhuman primate models of Parkinson's disease and serve as the foundation for evaluating the potential for this unique source of donor neurons to be employed in future clinical transplantation studies.

DESCRIPTION: (Applicant's Abstract) It has been demonstrated previously that fetal cholinergic basal forebrain grafts survive and improve memory function in a variety of animal models of Alzheimer's disease including lesioned rodents, aged rodents, and lesioned nonhuman primates. This suggests that fetal cholinergic grafting may be an innovative strategy for the treatment of the cholinergic deficit consistently observed in Alzheimer's disease. The present proposal aims to establish the structural and functional efficacy of fetal cholinergic grafting in aged monkeys, an essential step prior to initiating clinical trials of cholinergic grafting for the treatment of alzheimer's disease. In this regard, we have recently demonstrated that fetal nigral grafts survive in a patient with Parkinson's disease in a manner indistinguishable from that seen in animal models, indicating that data gathered in animal models have high predictive validity for grafting studies in humans. These findings increase our enthusiasm for the use of neural transplantation for the treatment of neurological diseases such as Alzheimer's disease. In the previous funding period, we demonstrated that fetal cholinergic allografts survive long-term following grafting into nonhuman primates. Further, we demonstrated that grafts of cells genetically modified to secrete human NGF can prevent the degeneration of cholinergic basal forebrain neurons in young adult and aged monkeys. Based upon these and other data, the present proposal aims to establish the optimal fetal donor tissue for transplantation and establish the structural and functional efficacy of fetal cholinergic grafts and fetal cholinergic/NGF cografts in aged monkeys. Xenografts extensively recircuit the damaged mammalian brain and have great clinical potential as a source of donor tissue for transplantation. In this regard, clinical trials employing porcine and bovine cells have recently been initialed for the treatment of Parkinson's disease and pain control, respectively. We will first attempt to identify an optimal source of fetal cells donor by comparing the ability of fetal cholinergic neurons derived from porcine, monkey, and human sources to survive and reinnervate the aged monkey hippocampus in aged nonhuman primates. All evidence gathered in animal models and human autopsy cases demonstrate that only 5-10 percent of grafted neurons survive transplantation. therefor, we will attempt to increase cholinergic cell survival and innervation by cografting fetal basal forebrain neurons with cells genetically modified to secrete human NGF. Lastly, we will determine whether fetal cholinergic grafts and/or fetal cholinergic/NGF cografts can ameliorate the cognitive decline observed in aged monkeys. These studies will determine the value of grafting of fetal cholinergic neurons as a treatment strategy for the cholinergic deficit in dementing illnesses such as Alzheimer's disease.

One of the most consistent pathological features in Alzheimer's disease (AD) is a cortical cholinergic hypofunction which results from degeneration of cholinergic neurons located within the basal forebrain. The cholinergic deficit occurs early in the disease process and correlates with disease severity and duration. Additionally, there is a large experimental and clinical literature demonstrating that the cholinergic basal forebrain system is involved in normal and pathological memory processes. These data indicate the contribution of this system tot eh major symptomatology of AD. Converging lines of evidence indicate that the development and maintenance of cholinergic basal forebrain neurons is dependent upon the trophic factor nerve growth factor (NGF). Based upon these data, it was suggested that impaired NGF trophism underlies the degeneration of basal forebrain neurons in AD. Initial studies demonstrating normal NGF synthesis and expression of its low affinity NGF receptor in AD diminished enthusiasm for this view. However, NGF trophism occurs through a series of molecular events. We have recently demonstrated that NGF transport mechanisms are defective in AD and hypothesize this event may mediate the degenerative events seen in the AD basal forebrain. In AD, we found a reduced expression of NGF- immunoreactivity within the cholinergic basal forebrain and an accumulation of NGF within the cerebral cortex. In the present series of studies. Specific Aims 1 and 2 will establish the extent to which diminished NGF- immunoreactivity within the basal forebrain and accumulated NGF protein within the cerebral cortex is associated with the clinical progression of AD. Well characterized patients with mild, moderate, and severe dementia will be compared to age-matched controls. The immunohistochemical expression of NGF within the basal forebrain and NGF protein within different regions of the cerebral cortex will be quantified and compared between patient populations. Specific Aims 3 will utilize the fact that subfields of the cholinergic basal forebrain are selectively vulnerable in AD to assess the degree to which reduced transported NGF is associated specifically with neural degeneration. NGF-immunoreactivity will be quantified and the expression of NGF-ir will be compared between cholinergic septal.diagonal neurons which are sustained in AD and neurons of the nucleus basalis which extensively degenerate in AD. Specific Aim 4 will focus upon the mechanisms responsible for the diminished NGF- immunoreactivity in the basal forebrain and accumulated NGF protein within the cortex in AD. We hypothesize that these events are due to an impairment in the high affinity trkA receptor. This is the receptor which transduces the NGF signal. In situ hybridization will be used to determine whether trkA mRNA is altered in AD as a function of disease progression. These studies will help establish the role of impaired NGF trophism in CBF degeneration and the progression of clinical dementia in AD. The data derived from these studies will be integrated with others in the program project which are also investigating radiologic and pathologic parameters of disease progression in AD.

Fetal nigral grafts can cause "runaway" dyskinesias in patients with Parkinson's disease (PD;Freed et al., 2001). These dyskinesias are severe, debilitating and strongly indicate that 1) novel dopaminergic surgical therapeutic strategy planned for clinical trials need to be tested preclinically for their effects upon dyskinesias and 2) the mechanisms underlying these dyskinesias need to be elucidated. We have recently demonstrated that lentiviral gene delivery of glial cell-derived neurotrophic factor (GDNF) potently prevents motor dysfunction and prevents nigrostriatal degeneration in nonhuman primate models of PD (Kordower et al., 2000). Prior to initiating clinical trials with lenti-GDNF, it effects upon dyskinesias need to be evaluated in parkinsonian monkeys. Freed, Fahn and coworkers (2001) have hypothesized that grafted-mediated dyskinesias result from graft overgrowth. However, their own PET and post-mortem data, as well as the data from others (Kordower et al., 1995, Lee et al 1999), do not support this view. We propose an alternative hypothesis that these dyskinesias result from local "hot spots" of hyperdopaminergic function interacting with the levodopa primed brain. We plan to test this hypothesis by comparing gene therapies that induce either a) widespread or b) local hyperdopaminergic function upon dopa-induced dyskinesias and the role of dopa priming. This application will have three Specific Aims. Specific Aim 1 will test the hypothesis that lenti-GDNF treatment to non-levodopa primed MPTP-treated monkeys will prevent, or diminish the intensity of dyskinesias when they are later treated with levodopa. Specific Aim 2 will test the hypothesis that lenti-GDNF will diminish the dyskinesia profile in dyskinesic MPTP-treated monkeys previously primed with levodopa. Specific Aim 3 will test the hypothesis that "hot- spot" hyperdopaminergic function, but not homogenous hyperdopaminergic innervation, will enhance the dyskinesia profile of parkinsonian monkeys and that elimination of GDNF will reverse the functional and dyskinesic effects established previously by this trophic factor. The study of dyskinesias has become a compelling area of PD research. Exciting therapeutic strategies such as gene therapy need to be evaluated for their effects on dyskinesias so that they are both safe and effective. This application will determine whether potent dopaminergic gene therapies influence dyskinesias in the best animal model of PD.

This proposal brings together three experienced groups proposing an integrated series of experiments using adeno-associated virus (Gainesville) in rodent (Lund) and nonhuman primate (Rush) models of Parkinson's disease (PD). A concensus is emerging that site-specific rAAV-mediated striatal L-dopa delivery might be a useful strategy for treating PD. However, clinical trials using this approach cannot even begin to be considered before critical efficacy and safety studies need to be performed. Towards this end, three research themes will be explored in this application. First we will perform studies in monkeys designed at vector optimization. We will determine the optimal AAV serotype in nonhuman primates and establish the optimal ratio of TH to GTPCH1.Currently, a 1:1 ratio is utilized. However, we believe that a higher ratio will be more effective due to the kinestics of GTPCH1 and thus we will be able to deliver more TH, and ultimately more LDOPA. These studies will also provide critical "scaling-up" data in primates that will be relevant for futures clinical trials. The second research theme is efficacy. The TH/GTPCH1 gene delivery approach has already been shown to be effective in reversing drug-induced and spontaneous motor deficits in rodent models of PD. The present proposal will establish efficacy in MPTP treated monkeys, the best animal model available for PD. The third aim is safety. With regards to functional safety, our main concern is dyskinesias. We have already demonstrated that rAAV-Ldopa reverses already manifest dyskinesias in 6-OHDA lesioned rats and have new data demonstrating that rAAV-LDOPA prevents the emergence of dyskinesias. IN this new application, we will evaluate the role of serotonin in the expression of dyskinesias in rats. Further, the effect of "hot spot" versus "widespread" delivery of TH/GTPCH1 upon efficacy and dyskinesias will be evaluated. The monkey model of dyskinesias is recognized as the best available for the study of dyskinesias. In the present study we will test the hypothesis that gene delivered levodopa can both reverse already manifest Idopa dyskinesias and delay the emergence of new dyskinesias. These studies will determine the safety and efficacy of gene delivery of LDOPA and determine whether this approach is appropriate for clinical trials.

DESCRIPTION (provided by applicant): Huntington's disease is a devastating, universally fatal, untreatable neurodegenerative disorder, which is debilitating and inflicts terrible suffering on the patient's mind and body. Human neural stem cells have been identified as a potential alternative cellular substrate for treatment, but to date, have not been tested for long- term therapeutic efficacy in disease relevant animal models (transgenic HD-mice). Our long-term goal is to assess the safety and temporal efficacy of hNSC transplantation in HD patients. The current objective is to determine if hNSC grafts are a potent long-lasting therapy that structurally protect neurons and reverse functional deficits associated with HD in transgenic mice. Our central hypothesis is that intrastriatal delivery of hNSC into previously "desensitized" transgenic HD mice will provide neuroprotection, improving measures in structural neuroanatomy, as well as reduce motor and cognitive deficits. The rationale for the proposed research is that once it is known if fetal hNSC grafts ameliorate progressive deficits in HD mice, we can pursue safety and tolerability studies in this most relevant animal model and subsequently employ a non-human primate model for HD as a logical next-step before clinical translation. Guided by preliminary data, this hypothesis will test the following two Specific Aims, that: 1) intrastriatal administration of hNSC will prevent HD pathology and behavioral deficits and 2) prolong the survival of HD transgenic mice. Utilizing a novel "desensitization" paradigm that allows grafted hNSC to bypass immunorejection, we will determine the effects of grafted hNSC on host anatomical structure and function and correlate these results to overall lifespan. In aim#1, immunohistochemical analysis and behavioral testing will be used to complement survival studies in Aim#2, in an effort to bridge a critical gap in knowledge for HD therapeutics. This research is innovative, as it 1) focuses on "desensitization" as a novel means to bypass graft rejection, 2) advances our knowledge of the functional efficacy of this approach by utilizing a true disease specific genetic model, and 3) will shed valuable insight for the future advancement of hNSC therapy in HD. This proposal is significant as rigorously testing hNSC therapies in transgenic mice more accurately depicts human HD, and therefore, the proposed experiments are more likely to provide quality translatable results to the clinic. The outcomes are expected to vertically advance the field of HD therapy through the blending of neurosurgery and stem cell biology. The knowledge obtained here has the potential to provide a therapeutic option that will reduce the terrible symptoms and prevent certain death associated with HD. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health and the NIH-NINDS's mission because the discovery of human stem cell based treatments that successfully slow or reverse progressive neurodegeneration and behavioral deficits will help develop a desperately needed treatment for HD patients. Furthermore, development of human stem cell therapies are needed now more than ever with the advent of exciting new technologies centered on patient-derived induced neural cells. Thus, the proposed research is relevant to the part of NIH's mission that pertains to the application of knowledge that enhances health and reduces the overall burden of illness.

DESCRIPTION (provided by applicant): Parkinson's disease is a devastating, universally fatal, incurable neurodegenerative disorder, which is debilitating and inflicts terrible suffering on the patient's mind and body. Human pluripotent cells, including embryonic stem cells, have been identified as a potential alternative cellular substrate for treatment, but to date, have not been tested for long-term therapeutic efficacy in disease relevant animal models (MPTP lesioned monkeys). Our long-term goal is to assess the safety and temporal efficacy of HESC-DA cell therapy in PD patients. The current objective is to determine if grafted HESC-DA cells are a potent long-lasting therapy that structurally integrate within the dopamine depleted primate brain, as well as reverse functional deficits associated with PD. Our central hypothesis is that intrastriatal delivery of HESC-DA cells will reverse motor disability in MPTP treated monkeys and this effect will be potentiated by co-treating with AAV-neurturin. The rationale for the proposed research is if HESC-DA grafts ameliorate functional deficits in parkinsonian primates, we can pursue large-scale, long-term safety and tolerability studies as the next logical step for clinical translation. Guided by preliminary data, this hypothesis will test the following Specific Aim, that intrastriatal grafting of HESC-DA cells, or HESC-DA cells + AAV2-Neurturin will provide structural and functional recovery in MPTP monkeys. Utilizing a novel floor-plate based, dopaminergic neuralization paradigm that allows HESC to be efficiently differentiated into midbrain specific dopaminergic neurons, and we will determine the effects of grafted HESC-DA cells on host anatomical structure and function and correlate these results to overall quality of life (clinical rating scale). In the proposed Aim, immunohistochemical analysis and behavioral testing will be used to complement in vivo imaging in an effort to bridge a critical gap in knowledge for PD therapeutics. This research is innovative, as it 1) focuses on well characterized, functionally active, midbrain specific HESC- DA neurons as an alternative renewable source for cellular transplantation 2) advances our knowledge of the functional efficacy of this approach by utilizing a clinically relevant non-human primate model of PD, and 3) vertically advances and bridges our present studies with the addition of AAV-2 neurturin neurotrophic support. This proposal is significant as rigorously testing HESC-DA therapies in parkinsonian monkeys more accurately depicts human PD, and therefore, the proposed experiments are more likely to provide quality translatable results to the clinic. The outcomes are expected to vertically advance the field of PD therapy through the blending of neurosurgery and stem cell biology. The knowledge obtained here has the potential to provide a therapeutic option that will reduce the terrible symptoms and enhance overall quality of life in PD. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health and the NIH-NINDS's mission because the discovery of human pluripotent stem cell based treatments that successfully slow or reverse progressive neurodegeneration and behavioral deficits will help develop a desperately needed treatment for PD patients. Furthermore, pre-clinical development of human stem cell therapies are needed now more than ever with the advent of exciting new technologies centered on patient-derived induced neural cells. Thus, the proposed research is relevant to the part of NIH's mission that pertains to the application of knowledge that enhances health and reduces the overall burden of illness.

? DESCRIPTION (provided by applicant): Parkinson's disease (PD) pathology is characterized by the formation of intraneuronal inclusions called Lewy bodies (LBs) and Lewy Neurites (LNs), that are comprised primarily of misfolded, fibrillar ?-synuclein (?-syn). One therapeutic strateg to slow disease progression is to reduce these toxic aggregates by preventing the native/monomeric form of ?-syn from aggregating. There is substantial need for new, efficacious disease-modifying therapies in PD. Despite the fact that antidepressants have already been shown to be safe and efficacious for depression in PD, the effects of these drugs on disease progression remain unknown. However, previous work from our laboratory suggests tricyclic antidepressants (TCAs) slow disease progression in both preclinical toxin models (Paumier et al., 2014) and in a retrospective analysis of data from an early cohort of patients with PD (Paumier et al., 2012). Together these findings, and others (Jeannotte et al., 2009a, Jeannotte et al., 2009b, Trushina et al., 2009, Chung et al., 2010, Chadwick et al., 2011, Zschocke et al., 2011, Valera et al., 2014), support the notion that antidepressants have disease-modifying potential within an existing framework of established safety. The objective of the proposed studies is to determine whether NOR can be a disease- modifying treatment for PD. We will test our central hypothesis that NOR attenuates the accumulation/aggregation of ?-syn that occurs in PD, resulting in nigrostriatal preservation. Our hypothesis has been formulated on the basis of our own preliminary findings that NOR is a potent inhibitor of ?-syn aggregation in vitro and in vivo. Rationale for the proposed studies is related to the inability to assess engagement of the ?-syn target in the clinic and subsequently link neurobiological changes directly to improvement. Absent a clinical biomarker for target engagement desirable for a prospective clinical trial, we propose to further develop the case for clinical use of NOR by: 1.) testing in nonhuman primates, and 2.) mining data from subjects enrolled in the ongoing Parkinson's Progressive Marker Initiative (PPMI) clinical trial. We predict that the capacity of NOR to reduce the rate of?-syn aggregation will prevent the spread and resulting dysfunction associated with LB-like pathology and this prevention of aggregation will be correlated with neurobiological benefit.

Project Summary One often debilitating side-effect of standard pharmacotherapy for Parkinson's disease (PD), levodopa administration, are unwanted involuntary movements known as levodopa-induced dyskinesia (LID). Eliminating LID remains a significant unmet need in PD therapy. There are currently no FDA approved drug treatments for LID, yet up to 90% of individuals with PD develop this side-effect. The L-type calcium channel CaV1.3 is a target of interest for LID prevention. Loss of striatal dopamine (DA) in PD results in dysregulation and overactivity of striatal CaV1.3 channels leading to synaptic pathology, including the loss of dendritic spines on striatal spiny projection neurons that appears to be involved in LID. While initial studies delivering pharmacological CaV1.3 channel antagonists reduced LID dose-dependently, the effects were partial and transient, with potential liability for cardiovascular side-effects due to the lack of specificity of existing drugs for the CaV1.3 channel. To provide unequivocal target validation, free of pharmacological limitations, we developed a rAAV-CaV1.3-shRNA to provide continuous, high potency, target-selective, mRNA-level silencing of striatal CaV1.3 channels. We examined whether genetic silencing of these dysregulated calcium channels could prevent LID induction in previously levodopa naïve parkinsonian rats and/or whether it could reverse these abnormal behaviors in parkinsonian rats already expressing a severe LID phenotype. In our `LID prevention studies' we found that gene level silencing of striatal CaV1.3 channels in severely parkinsonian rats, prior to the introduction of levodopa provides uniform and complete protection against the induction of LID, and that the antidyskinetic benefit is sustained over time even with high doses of daily levodopa. In our `LID reversal studies' we observed that rAAV-mediated CaV1.3 silencing in parkinsonian rats with already established LID could ameliorate these behaviors, with a one-week drug withdrawal 'drug holiday' appearing to be beneficial and/or necessary. Importantly this approach did NOT interfere with motor benefit of levodopa and showed a tendency to enhance motoric response to low dose levodopa. Gene delivery resulting in striatal CaV1.3 silencing provides some of the most profound antidyskinetic benefit reported to date. If these findings can be translated into a clinical application with a similar magnitude, this would provide a much-needed breakthrough in treatment of individuals with PD and would allow the most powerful antiparkinsonian therapy ever identified to work unabated through the duration of the disease. In the current application we propose a series of translational studies in rats and nonhuman primates that will allow us to expand upon these initial proof-of-principle studies and test specific hypotheses of safety and efficacy that will be required for the clinical development of genetic silencing of striatal CaV1.3 channels for LID.

Parkinson disease (PD) is a neurodegenerative disease affecting at least 1 million people in the U.S. and it has been estimated that 14.6 million people will be affected worldwide by 2040. Pathologic intraneuronal inclusions composed of misfolded ?-synuclein accumulate in Lewy bodies and Lewy neurites, resulting in progressive degeneration within the periphery and across the neuraxis including the of the nigrostriatal system which mediates the cardinal symptoms of the disease. Because of disease heterogeneity, before therapeutic strategies can be fairly tested, it is critical to identify and examine specific subtypes of PD, as different forms of PD are likely to have both distinct and overlapping pathogenic mechanisms. Mutations in the glucocerebrosidase (GBA) gene are the most common genetic risk factor for PD. Clinically, GBA mutation carriers with PD have more aggressive motor decline and develop dementia faster than non-mutation carriers with PD. In parallel, PD subjects with GBA mutations also have a more rapid accumulation and spread of ?- synuclein. The relationship between GBA mutations, rapid and both motor and non-motor decline, and this wide-spread ?-synuclein accumulation, remains to be clearly elucidated and its understanding will likely related to sporadic PD as well. This application aims to define whether the aggressive pathologic and motor phenotype of GBA mutation carriers with PD is due to: 1) reduced host GCase enzymatic activity and/or 2) specific strains of aggregated ?-synuclein unique to GBA mutation carriers result in enhanced ?-synuclein propagation and functional motor progression in a GBA mouse model. In aim 1, we will determine whether there is more aggressive alpha-syn propagation and more aggressive functional decline in a Gba1D409V/D409V mouse model compared with wild-type mice. This mutant mouse model, like GBA mutation carriers with PD, has reduced GCase enzymatic activity. We hypothesize that wild-type HuPFF injection into the OB in Gba1D409V/D409V mice will have increased ?-synuclein propagation and functional motor and cognitive progression compared with wild-type mice. In aim 2, we will compare the effect of GBA vs. WT PFFs on structural and functional progression of alpha-syn pathology in wild-type mice. We hypothesize that GBA PFF injection into the OB in wild-type mice will result in increased ?-synuclein propagation and functional motor and cognitive progression compared with wild-type PFF injections. We are uniquely equipped to test this hypothesis as our lab has done critical work in demonstrating transneuronal ?-synuclein propagation. The rationale for the proposed research is that once we determine the mechanism of ?-synuclein propagation and functional motor and cognitive progression in our model, we will apply this knowledge toward novel treatments to prevent ?-synuclein propagation for GBA associated PD. The proposed research will also open the door to new research aimed at understanding how ?-synuclein strains contribute to the diverse pathological and clinical presentations of a variety of ?-synucleinopathies, including PD, MSA, and DLB.

Alzheimer?s disease (AD) is a devastating condition that affects more than 5 million Americans, with a total annual cost of more than $300 billion predicted in 2020. Currently there are no effective treatments to counteract or slow the progression of AD, with promising findings in rodents failing to translate into successful therapies for patients. Monkey models may provide a more powerful translational model. The goal of this proposal is to characterize a monkey model of tau pathology in AD. This is responsive to RFA-AG-21-003 requesting proposals that target the ?development, characterization, and validation of suitable new or unconventional mammalian non-murine models of AD that may represent improved translational potential by better replicating pathological features of the disease?. With respect to nonhuman primate (NHP) models of AD, the RFA states explicitly that ?NHP have a very high translational value because of their close relationship to humans in terms of phylogeny, genetics, physiology, cognition, emotion, and social behavior?. In this proposal we describe initial findings in a tau-based monkey model of AD and propose a program to fully develop and validate the model by three PIs who have decades of experience on aging and neurodegeneration in NHP models. We have targeted the highly vulnerable entorhinal cortex (ERC) for unilateral infusions of an adeno-associated virus expressing a double tau mutation known to cause tau-related dementia in humans (AAV-P301L/S320F) and characterized neuropathology at 3 and 6 months after viral injection in NHPs. This causes extensive and progressive neuroinflammation and tau-based neuropathology, including end-stage neurofibrillary tangles, in ERC and in hippocampal and neocortical targets of ERC. Preliminary PET imaging in these monkeys displays robust phospho-tau accumulation in the hippocampus. The progressive time course relative to the time of vector injection is a great strength in terms of using this model for therapeutic development. These early studies demonstrate the potential for this model to replicate pathological features of AD in the monkey brain and to capture aspects of pathology that have not been well-modeled in rodents. We propose to do a full, rigorous characterization of this model, including long-term behavioral assessment, in vivo imaging, fluid biomarker assessment, and microscopic analyses. Full characterization of this model, will provide a platform to test therapeutic agents at different points in the disease process.