Laura A. Volpicelli-Daley, Ph.D. - US grants

DESCRIPTION (provided by applicant): One of the primary characteristics of Alzheimer's disease (AD) is senile plaques containing Ab which is formed by the processing of amyloid precursor protein (APP). Intracellular transport plays a crucial role in APP processing, but the mechanisms involved in controlling APP intracellular traffic remain insufficiently characterized. Two proteins, Mints and FE65 play opposite roles in APP processing: Mints inhibit and FE65 increases the production of Ab. Recent data suggest that Mints may function as a vesicular coat protein and thus Mints may regulate APP processing by controlling APP traffic. This proposal will establish biochemical assays that will define the molecular components involved APP vesicular trafficking. Membrane recruitment assays will be used to test the hypothesis that APP acts as a cargo receptor that recruits Mints to the membrane in an ARF-dependent manner that is prevented by FE65. In vitro budding assays will be performed to determine if the budding of APP-containing vesicles requires ARF and Mints. Because APP processing depends on APP trafficking, these data will elucidate the mechanisms by which APP is targeted to a nonpathogenic pathway and consequently identify novel therapeutic targets for AD.

Parkinson?s Disease (PD) and Dementia with Lewy Bodies (DLB) are characterized by cytoplasmic inclusions called Lewy Bodies and Lewy Neurites which are composed primarily of ?-synuclein (?-syn). Multiple lines of evidence suggest that these inclusions contribute to disease pathogenesis. Thus, preventing ?-syn aggregation is a critical therapeutic target for preventing disease progression. Normally, ?-syn resides at the presynaptic terminal, preferentially associates with synaptic vesicles, and interaction of ?-syn with membranes inhibit its aggregation. Our proposed study seeks to determine how presynaptic targeting of ?-syn prevents it from converting to a pathologic, aggregation prone form and how PD risk factors disrupt the normal localization of ?-syn and increase its propensity to forming inclusions. Mutations in leucine rich repeat kinase (LRRK2) are the most common cause of familial Parkinson?s disease. We showed that the G2019S-LRRK2 mutations increase the formation of ?-syn inclusions and that LRRK2 kinase inhibitors reduce inclusion formation, but the mechanisms by which LRRK2 controls the propensity of ?-syn to form aggregates is unknown. Although current research focuses on the role of LRRK2 in degradative organelles, LRRK2 has also been functionally implicated at the presynaptic terminal and may play a role in ?-syn presynaptic membrane targeting. Recently, a subset of Rab GTPases was identified as LRRK2 substrates. Rabs control distinct steps in membrane trafficking pathways in the cell. Ten years ago, the Lindquist lab identified the same Rabs (that were recently shown to be phosphorylated by LRRK2) as protective in models of ?-syn toxicity. We will determine if Rabs phosphorylated by LRRK2 enhance presynaptic targeting of ?-syn and prevent ?-syn inclusion formation. Because inclusions localize to multiple brain regions in PD and DLB, we will inject fibrils into the mouse striatum which produces inclusions in the substantia nigra pars compacta (SNpc), cortex, and amygdala. In Aim 1, we will determine the extent to which select Rab isoforms influence ?-syn presynaptic targeting using novel AAV vectors to increase Rab expression in the brain or use antisense oligonucleotides (ASOs) to reduce Rab levels. Immunohistochemistry and biochemistry will be used to determine if expression of these Rabs in vivo prevents formation of fibril-induced inclusions in multiple brain regions, and loss of dopamine neurons in the SNpc. In Aim 2, we will identify the Rab effectors in neurons that specify targeting of vesicles containing ?- syn cargo to the presynaptic terminal. Finally, in Aim 3, we will determine if LRRK2 kinase activity prevents presynaptic targeting of ?-syn and if the mislocalization of ?-syn in the cell increases its propensity to form inclusions. We will also determine the extent to which LRRK2 kinase inhibitors and ASOs in preclinical development enhance presynaptic targeting of ?-syn. These studies will open up new research avenues for understanding how factors and genes that cause PD and DLB increase the propensity of ?-syn to aggregate, and how to therapeutically target LRRK2 and downstream effectors to prevent the progression of PD.

Project Summary: Core C, Animal Models Core Current treatments for Parkinson's disease (PD) alleviate the symptoms, but do not prevent the progression of the disease. Finding novel therapeutic targets to prevent the progression of the disease requires animal model that recapitulate the features of PD. The ?-syn fibril model of Parkinson Disease replicates features of the disease such as inclusions in the substantia nigra pars compacta, dopamine neuron loss and motor defects. The goal of the Core C: Animal Models Core is to standardize this models to generate consistent results across the projects. Animal Models Core will purify and generate low endotoxin recombinant ?-syn, generate fibrils and perform quality control assays such as sedimentation and Thioflavin T assays, dynamic light scattering and transmission electron microscopy. The Animal Models Core will also perform all of the stereotactic injections to minimize variability in stereotactic technique. Perfusions, tissue sections and immunohistological techniques and unbiased stereology will also be performed. Having highly trained personnel from the Core perform these techniques will produce high quality data and will minimize potential variability.

Dementia with Lewy bodies (DLB) and Parkinson's disease dementia (PDD), collectively called Lewy body dementias (LBDs), are the second most common cognitive disorder after Alzheimer's disease (AD). Patients with LBDs suffer from PD-related motor defects and deficits in executive dysfunction, attention, and visuospatial processing, reflective of cortical dysfunction. Pathologically, LBDs are characterized by abundant cortical aggregates of ?-synuclein (?-syn) called Lewy pathology (LP). The cognitive impairments in LBDs are the main cause for institutionalization and mortality. There are no treatments that halt the progression of LBDs. Outstanding scientists in the field of LBDs have provided data supporting that the combination of Lewy pathology and aggregates of tau, similar to those found in Alzheimer's disease, together are strongly associated with reduced performance on cognitive tasks. In Alzheimer's disease, loss of synapses is the strongest correlate of cognitive decline. Recently evidence has emerged from several labs, including ours,that synapse loss in the cortex may contribute to cognitive changes in LBDs. We propose that the presence of both alpha-synuclein and tau aggregates at the synapse synergistically contributes to synapse degeneration in LBDs. We propose the interaction of ?-syn and tau at the presynaptic terminal induces synapse degeneration. This proposal will combine the expertise of multiple investigators to test whether 1) the interaction of alpha- synuclein and tau facilitates synapse degeneration and 2) whether presynaptic terminals with small aggregates of both alpha-synuclein and tau show enhanced degeneration in the prefrontal cortex of patients that suffered from Lewy body dementias. We will use novel antibodies that selectively detect oligomeric and pathologic conformation of alpha-synuclein and tau. We will use a novel super-resolution technique called Expansion Microscopy (ExM) that can rapidly and quantitatively provide nanoscale resolution of synapses. We will also use a novel antibody multiplexing technique that can amplify low-abundance synaptic signals and allow imaging of >5 proteins at once. The results of this proposal will move the field of LBDs forward by 1) beginning to elucidate the mechanisms that contribute to synapse loss and cognitive changes in LBDs, and 2) identify if targeting the alpha-synuclein/tau interaction prevents synapse loss and cognitive decline.