Fredric P. Manfredsson, Ph.D. - US grants

PROJECT SUMMARY Our current understanding of the molecular etiology of Parkinson's disease (PD) is incomplete and likely the product of multiple interacting factors. The best-validated participant in the molecular pathology of PD is alpha-synuclein (?-syn). Mutations in, and multiplication of, the gene encoding ?-syn result in inherited forms of PD. In addition, the presence of ?-syn in Lewy bodies and neurites provides evidence for its association with idiopathic PD. A common hypothesis states that excess ?-syn and consequent aggregation causes neurotoxicity in a direct toxic gain-of-function event. Conversely, it has also been proposed that ?-syn aggregation may endanger neurons by removing the protein from its normal cellular location and diminishing its function in a toxic loss-of-function event. This issue remains a topic of debate. Despite this ambiguity, approaches that reduce ?-syn in the central nervous system represent an active area of research as a strategy for treating PD. Our published data in rats and nonhuman primates, and preliminary data in mice, supports the dissenting viewpoint, showing that eliminating ?-syn from mature dopamine (DA) neurons is not protective, but instead is toxic. Utilizing our newly developed conditional ?-syn knock-out mouse, our proposed studies will define the consequences that result from ablated/decreased expression of ?-syn in the nigrostriatal system. The use of rAAV to deliver CRE recombinase (iCRE) to dopamine neurons of adult mice carrying the floxed ?- syn allele will allow us to efficiently abolish ?-syn expression in mature neurons. This approach addresses the limitations of current germ-line mouse models by preventing the hypothesized compensatory adaptations that occur when ?-syn expression is abolished during development. The following proposal will utilize this new mouse model to: 1) Characterize the pattern, timing, and ?-syn dose-dependence of neurodegenerative effects on the nigrostriatal system and resultant impairment in motor behavior. 2) Define the characteristics of degeneration in its pattern and association with markers of cellular events previously implicated as participating in degeneration in PD (DA mishandling, oxidative/nitrative stress, proteasome/lysosome dysfunction, and microglial activation) at early and late stages of degeneration following removal of ?-syn. This proposal will answer the lingering question of whether ?-syn is crucial for the function and survival of mature DA neurons while also providing a new murine-based tool to study the importance of ?-syn induced DA neurodegeneration for the study of synucleinopathies. The successful completion of these aims will advance our knowledge and ability to develop therapeutic strategies aimed at halting neurodegeneration due to ?-syn dysfunction.

Project Summary/Abstract The standard treatment for Parkinson's disease patients is the use of levodopa (L-DOPA), which restores dopaminergic tone to the striatum and provides subsequent symptomatic relief. However, the higher doses required with PD progression result in levodopa-induced dyskinesias (LID) which dramatically reduce the quality of life for afflicted individuals. Despite its public health importance, the etiology of LID remains unknown, thus, no uniformly effective therapies exist. To study the molecular etiology of LID we utilized a genome array comparing mRNAs in the striata of LID+ rats to those that remained refractory to LID development. One validated protein that was induced in LID+ animals but absent in LID- animals was the orphan nuclear receptor Nurr1. We therefore hypothesized that Nurr1 is a molecular trigger of LID. To test this, we delivered adeno- associated viruses (AAV) expressing, or silencing, Nurr1 to the striatum of parkinsonian rats. Following L- DOPA treatment, Nurr1 overexpressing rats had significantly higher LID scores whereas Nurr1 silencing significantly attenuated LID scores. Moreover, we found that Nurr1 overexpression in the parkinsonian, L- DOPA naïve, striatum potentiates corticostriatal neurotransmission, identical to that seen in a LID+ parkinsonian rat. Finally, we have shown that LID is associated with changes in medium spiny neuron (MSN) dendrite distribution and morphology. Based on these findings we developed the central hypothesis of this proposal: Striatal Nurr1 activity is the key central component in developing LID by promoting structural and functional changes in the striatum. We further propose that Nurr1 facilitates the formation of this ?dyskinetic state? via its well defined role in plasticity and dendrite reorganization. To test this hypothesis we propose 2 distinct Aims where we will measure the same outcomes within the striatum: MSN morphology, function, and LID behavior. (1) Following dopamine denervation and L-DOPA administration roughly 30% of Sprague- Dawley rats do not develop LID. These LID- subjects also do not express striatal Nurr1. To test our central hypothesis we will thus utilize AAV to express Nurr1 in LID- subjects and observe the effect on striatal neurons and LID behavior. (2) Finally, as we posit that induction of striatal Nurr1 activity is a core event in the development of LID we ask the critical question: Does reversing Nurr1 induction normalize striatal function, reverse structural adaptions, and thus reverse LID? Together, the aims in this proposal will provide for an extensive understanding of the molecular events that underlies the formation of the dyskinetic striatum, including the novel role for striatal Nurr1 in an array of established molecular events linked to LID. The successful identification of Nurr1 as a central factor in LID formation will open a completely unexplored area for dyskinesia treatment development whereby Nurr1 itself, or downstream Nurr1 targets, can be exploited for therapeutic development.

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.

Project Summary/Abstract Parkinson's disease (PD) is typically recognized as a somatic motor disorder. However, gastrointestinal motor dysfunction resulting in constipation is often described as having a greater negative impact on the quality of life than somatic motor dysfunction. Moreover, GI dysfunction complicates the clinical management of PD as it interferes with the absorption of levodopa, the standard treatment for PD motor symptoms. The protein alpha- synuclein (?-syn) is linked to CNS degeneration and is present in aggregated forms in neurons throughout the enteric nervous system (ENS). Overexpression of ?-syn in specific populations of DA neurons in the CNS results in ?-syn aggregation, altered neurotransmission, and neurodegeneration; providing a model with high face validity for PD. However, to date, no attempts have been made linking pathological enteric ?-syn to GI dysfunction. To that end, we developed a novel gene delivery method whereby we can direct transgene expression specifically to enteric neurons. Importantly, this approach limits all transgene expression to the ENS with no expression seen in central neurons, muscle, or other organs. In other words, we have developed a novel platform by which to study ENS function without adding confound of using systemically applied agents such as toxicants or the use of transgenic animals with ubiquitous transgene expression. Using this approach we delivered adeno-associated virus (AAV) expressing ?-syn to the descending colon. Within one month of vector delivery the animals displayed a significant decrease in fecal output, decreased inhibitory neuromuscular transmission, and decreased basal levels of calcium in neurons and enteric glia, without neuronal loss in the treated ENS. ?-syn has a role in synaptic vesicle exocytosis, recycling, and docking, and aggregation of ?-syn results in impaired neurotransmission. We therefore hypothesize that ?-syn contributes to GI dysfunction, and that this effect is mediated via impaired synaptic transmission, resulting in a net increase of the inhibitory tone of the ENS. Herein we propose to (1) Determine whether ?-syn aggregation is required for the observed colonic dysmotility. (2) Determine whether overexpression of ?-syn results in a net increase in the inhibitory tone of ENS neurotransmission and whether this impairs propulsive motility. (3). Determine whether ?-syn overexpression impairs enteric ganglionic neurotransmission. We will perform these analyses by using vectors that encode different isoforms of ?-syn (wildtype, hyper-aggregatable, and non-aggregatable). We will compare the data observed in treated rats and mice with transgenic ?-syn overexpressing animals. We will also determine whether ?-syn overexpression in inhibitory motor neurons (the population of ENS neurons most often associated with ?-syn pathology in PD) per se is sufficient to elicit GI dysfunction. Together the aims in this proposal intend to demonstrate that aggregated ?-syn in the ENS causes GI dysfunction, and to define the molecular etiology underlying GI dysfunction in PD. This work will thus provide a basis for future research efforts focused on investigating new treatments of this devastating comorbidity in PD.

Neurodegenerative disorders such as Alzheimer?s disease (AD) and Parkinson?s disease (PD), acute disorders such as traumatic brain injury, spinal cord injury, and stroke, are amongst a plethora of devastating neurological conditions for which there are no treatment. As the incidence of many of these disorders increase with age, the number of patients with these disorders is expected to dramatically increase as the US population continues to age. The financial and emotional burden to patients and their families is indescribable; in addition, the cost of care and lost wages is an enormous financial liability for the US economy. The American Society for Neural Therapy and Repair (ASNTR) is a society focused on basic, translational, and clinical aspects of neurological disorders. The ASNTR membership uses cell transplantation, gene therapy, tissue engineering, neuropharmacology, and other cutting-edge technologies to study, and treat, such disorders. The 2017 annual meeting of ASNTR will be held at Sheraton Sand Key, Clearwater Beach, FL. This locale provides for a very unique and intimate setting which promotes interactions between junior trainees and accomplished investigators, as well as fostering unique collaborations. The 2017 meeting will be focused on challenges facing neural repair and therapy in the coming decade. As such, planned sessions will cover biomarker discovery, disease etiology, imaging, pediatrics (Zika), amongst others. Moreover, ASNTR is tremendously committed to trainees. The society encourages trainee participation in the governance of ASNTR and focuses on the development of trainees. The purpose of this R13 is to garner travel support for top trainees (selected from submitted abstracts) to offset their costs to attend the 2017 meeting. Moreover, in 2017 we have several trainee-activities planned. This includes a workshop (Navigating the NIH and Private Foundations Funding Opportunities) with confirmed speakers from the NIH, DOD, and private foundations such as the Michael J. Fox Foundation. We will also hold a data blitz for trainees as well as meet-the-speaker luncheons. ASNTR has a strong track record of fostering young investigators, and we aim to continue in this trajectory. Although we always pursue all methods available (i.e. approach the biotech industry and private foundations) in order to provide travel support for trainees, support from the NIH is crucial in order for us to continue on our strong training mission.

Neurodegenerative disorders such as Alzheimer?s disease (AD) and Parkinson?s disease (PD), acute disorders such as traumatic brain injury, spinal cord injury, and stroke, are amongst a plethora of devastating neurological conditions for which there are no treatment. As the incidence of many of these disorders increase with age, the number of patients with these disorders is expected to dramatically increase as the US population continues to age. The financial and emotional burden to patients and their families is indescribable; in addition, the cost of care and lost wages is an enormous financial liability for the US economy. The American Society for Neural Therapy and Repair (ASNTR) is a society focused on basic, translational, and clinical aspects of neurological disorders. The ASNTR membership uses cell transplantation, gene therapy, tissue engineering, neuropharmacology, and other cutting-edge technologies to study, and treat, such disorders. The 2018 annual meeting of ASNTR will be held at Sheraton Sand Key, Clearwater Beach, FL. This locale provides for a very unique and intimate setting which promotes interactions between junior trainees and accomplished investigators, as well as fostering unique collaborations. The 2018 meeting will be focused on new directions in neural repair and therapy in the coming decade, and the challenges of translating new therapies. As such, planned sessions will cover the role of aging, biomaterials, imaging, delivery, amongst others. A special session (open to patients) describing the role of dance and physical therapy with standard PD treatments will be held. Moreover, ASNTR is tremendously committed to trainees. The society encourages trainee participation in the governance of ASNTR and focuses on the development of trainees. The purpose of this R13 is to garner travel support for top trainees (selected from submitted abstracts) to offset their costs to attend the 2018 meeting. Moreover, in 2018 we have several trainee-activities planned. This includes a workshop ?techniques in neural repair - know how do how? with confirmed speakers with expertise in gene technology, stem cell technology, and electrophysiology. We will also hold a data blitz for trainees as well as meet-the-editor luncheons. Moreover, we have planned social events, aimed at furthering the interaction between faculty and trainees. ASNTR has a strong track record of fostering young investigators, and we aim to continue in this trajectory. Although we always pursue all methods available (i.e. approach the biotech industry and private foundations) in order to provide travel support for trainees, support from the NIH is crucial in order for us to continue on our strong training mission.

Project Summary/Abstract The parent R01 of this supplement is investigating the role of Parkinson?s disease (PD) pathology in the periphery, specifically in the enteric nervous system (ENS). The rationale for these studies is the fact that peripheral pathology (in the form of aggregated alpha-synuclein (?-syn)) is hypothesized to be linked both to gastrointestinal (GI) dysfunction as well as being the source of pathology that eventually leads to neurodegeneration in the brain. Our initial findings have confirmed that ENS ?-syn pathology does cause GI symptoms; however, we were unable to find evidence of a direct transfer of pathology to the brain (i.e. via ?prion-like? transfer). However, we are now investigating whether the ENS pathology can afflict the brain in a ?paracrine? fashion; for instance, via enhanced inflammation originating from the ENS. In this supplement we propose that the same premise holds true for AD-related pathologies of the periphery (e.g. tau, amyloid precursor protein(APP)). To that end, our central hypothesis states that induction of enteric AD pathology will result in systemic inflammation that exacerbates neurotoxicity in rodent models of AD neurodegeneration. Interestingly, investigations into AD-related pathologies of the ENS are extremely rare. The reason for this is likely stemming from the fact that GI dysfunction is not a reported prodromal symptom in AD. This does, however, not necessitate the absence of ENS pathology in AD. In fact, A?-immunoreactive plaques have been reported in human AD patients, and ENS pathology is found in transgenic animals. We propose that any such peripheral pathology will induce systemic inflammation, a condition which can predispose or sensitize central neurons to degeneration. Inflammation, once thought to be a consequence of neurodegeneration, is becoming increasingly thought of as having a causative role in AD neurodegeneration. However, we still do not understand what exactly triggers neuroinflammation, and have yet to understand the temporal relationship between inflammation and neurodegeneration. Herein we will utilize a novel gene therapy method developed for the parent R01 whereby we will overexpress AD-related proteins specifically in neurons of the ENS. A crucial aspect of this approach is that transgene expression is spatially restricted to enteric neurons and temporally controlled. This will allow us to specifically study the role of ENS pathology in disease, without the confound of ubiquitous expression that can be found in transgenic animals, or following systemic delivery of AAV. At the same time, we will also utilize AAV to overexpress disease related proteins in the CNS. With this approach we expect to find 1) An increase in markers of systemic inflammation, 2) An increase in indices of neuroinflammation, and 3) Increased susceptibility to disease proteins such as mutant tau or APP in central neurons. If any of these outcomes are proven true, it will demonstrate that changes in the PNS can contribute to CNS disease, laying the foundation for a novel area of AD research. Moreover, if we can demonstrate that non-cell autonomous factors contribute, or even trigger, neurodegeneration, a significant focus moving forward will be to better understand exactly where in the molecular process of neuronal loss that neuroinflammation plays a contributory role.

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.