Patrik Brundin - US grants

DESCRIPTION (provided by applicant): Grand Challenges in Parkinson's Disease - From Molecules to People will highlight current efforts to develop novel interventions to stop or retar the progression of Parkinson's disease (PD). The development of disease-modifying therapies for PD is urgently needed to improve the quality of life of people with PD and to reduce the financial burden of PD. Grand Challenges in Parkinson's Disease - From Molecules to People gathers experts both from basic and clinical research and will include world-class speakers who will give overviews covering every step in the development of effective drug therapies, from the identification of unmet medical needs; target identification and validation; drug screening and discovery, to clinical trial design. We aim to (i) Educate the audience regarding the multiple steps required to move laboratory discoveries to patients; (ii) Offer a cross-disciplinary forum fo discussions on the limitations of current approaches to the development of disease-modifying therapies for PD; (iii) Establish a solid interaction with people with PD, including their active participation as lecturers, discussants, attendees and organizers; (iv) Create opportunities to network and exchange ideas for potential collaborations; and (v) Promote relevant discussions among the participants. The conference has been designed to cover as many aspects as possible of a selected topic in a focused program, gathering leading scientists in that particular topic. This year the focus will be on the challenges that have been identified in the development of therapies against PD and to novel discoveries of therapeutic relevance. The program consists of 5 sessions, (i) Unmet Medical Needs; (ii) Animal Models of Parkinson's Disease; (iii) Target Discovery And Validation; (iv) High Throughput Screening and Drug Development; (v) Clinical Trial Design And Outcome Measures. This focused approach allows us to encompass experts from different fields in a format that facilitates interactions; these experts would normally not gather unless the conference format is large enough (e.g., World Parkinson's Congress). Our approach has ensured the participation of researchers, clinicians, postdocs, graduate students, as well as of people with PD and their family members, in a welcoming environment that guarantees high degree of interaction.

? DESCRIPTION (provided by applicant): Neuron-to-neuron propagation of ?-synuclein (?-syn) aggregates is thought to contribute to the pathogenesis of Parkinson's disease (PD) and underlie the stereotypical progression pattern of ?-syn neuropathology. This postulate suggests that aggregated ?-syn transfers from one neuron to another where it seeds further ?-syn aggregation. However, it is not known how microglia influence this process, and how specific microglia activation states that occur upon inflammation affect ?-syn transfer. To fill this gap i knowledge we developed a unique mouse model that allows us to monitor ?-syn prion-like propagation between neurons. Our in vivo paradigm involves transplantation of embryonic midbrain neurons into the striatum of a mouse overexpressing human ?- syn and allows the manipulation of microglia (i.e. ablation or specific activation). In this novel model, the presence of human ?-syn within the grafted mouse cells (initially devoid of human ?-syn) is used as a read-out for ?-syn transfer. Based on our preliminary data we hypothesize that under normal conditions, microglia take up ?-syn from the extracellular space, resulting in reduced ?-syn transfer from neuron to neuron. We also hypothesize that ?-syn accumulates in microglia following lipopolysaccharide treatment, as lipopolysaccharide -activated microglia have reduced proteolytic capacity whereas Interleukin 4-induced microglia effectively reduce the pool of extracellular ?-syn, and thereby mitigate ?-syn transfer from neuron to neuron. Two specific aims will be pursued to test this hypothesis: 1) Determine how the absence of microglia affects neuron-to-neuron transfer of ?-syn; and 2) Determine how the presence of lipopolysaccharide-induced vs Interleukin 4-induced activated microglia affects the rate of neuron-to-neuron transfer of ?-syn. First, we will monitor if the absence of microglia results in a different degre of ?-syn cell-to-cell transfer using our unique in vivo model of cell-to-cell transfer. Second, we wil assess the transfer of ?-syn into grafted neurons in the context of distinct microglial phenotypes lipopolysaccharide or Interleukin 4 injection will be used to stimulate these differential phenotypes. Our approach is innovative because it allows us to assess the interaction between two factors (inflammation and ?-syn propagation) both considered to play key roles in PD pathogenesis in a single animal model, and we can define outcomes using unbiased, automated and quantitative measures of neuropathology. We predict that the absence of microglia will translate to increased neuron-to-neuron transfer of ?-syn and that the nature of microglial activation will affect the accumulation of ?-syn within microglia. Ultimately, the proposed research will result in an innovative and valid model of ?-syn pathology propagation with the potential to facilitate the development of disease-modifying therapies based on treatments that modulate inflammation.

Project Summary/Abstract Hyposmia, the reduced ability to smell, is very common in Parkinson?s disease (PD). Almost 90% of PD patients have hyposmia, which often develops about a decade before motor symptoms manifest. The pathology of PD is characterized by the presence of aggregated ?-synuclein in neurons across the brain; ?-Synuclein aggregation is believed to start in the olfactory brain regions, especially the olfactory bulb, and then spreads to other structures in the brain. The manifestation of the symptoms in PD is therefore believed to reflect the spreading of the pathology, explaining why olfactory deficits would manifest before other symptoms. In addition to ?-synuclein aggregation, there are other key processes that normally associate with PD ? neuronal death and neuroinflammation. There is, however, a fundamental gap in knowledge regarding the pathogenic mechanisms which cause hyposmia in PD. Thus, the objective of this multi-PI project is to establish how the progressive spreading of aggregated ?-synuclein from the olfactory bulb to other olfactory structures, and the associated neural cell death and neuroinflammation, trigger hyposmia. To this end, we will perform sophisticated measures of olfactory function (Wesson) in an experimental paradigm that we recently developed and which recreates spreading of ?-synuclein pathology across olfactory structures associated with olfactory deficits (Brundin). With this approach we will define the links between olfactory dysfunction and key underlying mechanisms of early PD, testing the hypothesis that ?- synuclein pathology progression from the olfactory bulb induces widespread neurodegeneration, protein aggregation, and neuroinflammation in the olfactory system, resulting in impaired olfaction. Specifically, we aim to demonstrate that ?-synuclein pathology affects odor information processing and to identify neuropathological underpinnings of these olfactory deficits. Further, we will test innovative approaches to modulate pathogenesis and to determine whether these interventions can improve olfactory function and/or stop the spreading of the pathology. These findings will provide fundamental information on the olfactory system and on how olfaction is impacted by specific neurodegenerative processes. We expect that our findings will eventually facilitate the development of therapeutic approaches to prevent the development of olfactory deficits associated with the spreading of ?-synuclein pathology across olfactory structures. Since these therapies should also prevent the spreading of ?- synuclein pathology to other brain regions, they have the potential to become disease-modifying interventions against PD.

Abstract Attempts to protect dopamine neurons, the main target of neuronal loss in Parkinson?s disease (PD), have focused on single mechanisms or cell signaling pathways, and have not been successful in clinical trials. Thus, it is possible that to develop effective therapies the coordinated activation of multiple neuroprotective pathways might be required. Recent research has shown that several transcription factors responsible for the development of dopamine neurons also protect adult dopamine neurons against toxic insults that induce their death. These include Foxa2, Lmx1a, Lmx1b, Nurr1 and En1. Our preliminary data show that a novel modified protein (PM-Nato3) promotes expression of these neuroprotective transcription factors. We propose that activation of all of these transcription factors via the delivery of PM-Nato3 will protect DA neurons from toxicity related to PD. To test this hypothesis, we will use cellular and animal systems in which PD-like loss of dopamine neurons occurs, and define the effects of PM-Nato3 expression. We will focus on (1) MPP+ and ?-synuclein toxicity models in human cultured dopamine neurons; and (2) a new model in which En1 haploinsufficiency exacerbates the development of an induced PD-like ?-synuclein aggregate pathology. First, we will express PM-Nato3 in human dopamine neurons before exposing them to either MPP+ or ?-synuclein and monitor survival, shape, respiration and oxidative stress; when using ?-synuclein we will also monitor presence of ?-synuclein accumulation in these neurons. Second, we will specifically express PM-Nato3 in nigral neurons in mice that display PD-like features and that are exposed to fibrillar ?-synuclein (to trigger the development of ?-synuclein pathology). We will also monitor motor function, cell survival and dopamine production. This is an innovative approach as the coordinated expression of multiple neuroprotective factors has never been tested before, and it is now possible due to the discovery of PM-Nato3. We predict that PM-Nato3 will prevent cytotoxic changes and loss of motor function in these established PD models. Once we define how expression of PM-Nato3 promotes dopamine neuron survival upon PD-like neurotoxicity, we can apply this knowledge towards the development of novel treatments to prevent the loss of dopamine neurons in PD.

The lack of animal models recreating the progressive pathology characteristic of Parkinson?s disease (PD) hinders the development of effective disease-modifying therapies. Thus, the goal of this project is to generate a new animal PD model that supports the development of such therapies. Clinical data revealed that axon terminal failure and ?dying back? of dopaminergic neurons likely precede loss of substantia nigra cell bodies by many years in PD. This protracted process is not replicated in the acute toxin-induced animal models of PD, providing one possible explanation for the low predictive power of these models. The heterozygous deletion of the engrailed 1 gene in mice (En1+/?) results in axon terminal dysfunction and degeneration eventually leading to protracted loss of nigral dopaminergic neurons. This process causes striatal dopamine deficiency that leads to motor impairment. Furthermore, these changes are associated with mitochondrial deficits akin to those observed in some PD patients. Despite all the advantages that the En1+/? mouse model represents, it lacks ?-syn aggregation. Thus, we hypothesize that mitochondrial deficits (due to heterozygous loss of En1) combined with bilateral injections of pathogenic ?-syn fibrils (PFFs) will synergistically generate a highly relevant PD model ? En1/SYN. We further predict that the En1/SYN model will exhibit a comprehensive set of PD-relevant behavioral deficits (both motor and non-motor) and will mimic PD neuropathology. This approach is innovative in combining, in mice, key aspects of PD, the susceptibility of the dopaminergic system, ?-syn and the multifactorial nature of the etiology of PD. Supporting our hypothesis, our preliminary data show that PFFs-induced ?-syn pathology is significantly exacerbated by the loss of En1. There are two major goals in this project: (1) We will trigger PFFs-induced ?-syn pathology bilaterally by injecting pathogenic ?-synuclein into both striata. By triggering the pathology on both sides of the brain the new mouse model is expected to induce robust motor, and more importantly cognitive deficits. We will bilaterally inject recombinant fibrillar ?-syn (provided by Dr. Jiyan Ma), measure the development of ?-syn-associated pathology, and then assess motor and non-motor function of mice at different time points; (2) We will validate the En1/SYN model as a model of PD, by comparing the features of our model to human PD in long-term experiments, as well as by testing if the gold-standard PD treatment L-DOPA reverses motor deficits induced by loss of nigral dopamine neurons and widespread ?-syn aggregation. Our research is expected to generate a powerful tool, which can accelerate the development of symptomatic and/or disease-modifying therapies to treat motor and non-motor PD.

Parkinson's disease (PD) is a devastating neurodegenerative disease characterized by motor and non-motor symptoms. The hallmark pathology of PD in the brain is the presence of alpha-synuclein (?-syn) aggregates, along with the loss of dopaminergic neurons in the substantia nigra. The brain ?-syn pathology is thought to start in the gastrointestinal (GI) tract, since both GI dysfunction and the presence of ?-syn aggregates in the GI tract usually precede motor symptoms by many years. In addition, experimental models show that ?-syn aggregates can reach the brain from the gut via the vagal nerve. To understand GI tract contributions to PD, we performed a study that identified the human appendix as a key GI tissue that impacts the risk for PD. This study demonstrated that ?-syn aggregates are abundant in the appendix and that removal of the appendix was associated with a reduced risk of PD. It also showed that the appendix contains aberrant truncated forms of ?-syn, analogous to those in the PD brain, and that these were more abundant in the appendix of PD patients than in healthy individuals. This innovative work provides the basis for a unique opportunity to understand how the appendix contributes to PD. The proposed study will determine how the appendix, and the ?-syn aggregates within, can impact the development of PD. Specifically, this project aims to establish: 1) what gene regulatory changes are prominent in the PD appendix compared to that of healthy controls; 2) the specific truncated forms of ?-syn enriched in the PD appendix and their capacity to seed further aggregation; 3) the consequences of initiating ?-syn pathology in the appendix on the subsequent development of PD-like pathology in the brain, in vivo. This study will generate detailed genome-wide maps of epigenetic abnormalities in the PD appendix ? a resource for understanding gene regulatory and biological pathway changes in the PD GI tract. Profiling the epigenetic mark DNA methylation will be accompanied by an integrative network analysis with transcriptomic data to determine key genes dysregulated in the PD appendix. Next, the identification of the truncated ?-syn proteoforms elevated in the PD appendix, will be carried out using top-down mass spectrometry, the gold-standard for intact protein identification. Furthermore, seeding activity for pathogenic ?-syn in the appendix of PD cases, will be determined and compared to controls, using powerful biochemical assays. Finally, the capacity for ?-syn pathology triggered in the mouse cecal patch ? aka the appendix ? to lead to the development of PD-like neuropathology in the brain, will be determined. We will also determine if intestinal inflammation in combination with ?-syn pathology in the cecal patch augments brain pathology relevant to PD. Together, this study will provide new insights on the mechanisms underlying ?-syn abnormalities in the PD gut and its capacity to induce brain neuropathology. Moreover, this work can help shape the development of early PD diagnostics and treatments directed to the accessible GI tract.