Robert A Rissman - US grants

In previous grant cycles, the ADCS has not played a major role in biomarker measurement; assays for Apolipoprotein E (ApoE) genotype, and for plasma and CSF measurement of amyloid peptides and tau proteins have been subcontracted to companies or external academic labs. However, the importance of biomarker measurement has grown steadily, in part due to the success of the Alzheimer's Disease Neuroimaging Initiative. There have been an increasing number of requests for stored ADCS samples, and increasing requests for biomarker measurement from ADCS investigators. To meet these needs, in late 2008, we established a Biomarker Core which is located on the 3rd floor of the Medical Teaching Facility building at UCSD School of Medicine.

Lewy body disease (LBD) is a group of disorders characterized by alpha-synuclein (alpha-syn) accumulation and parkinsonism. During the previous period, the objective was to understand the mechanisms by which (3- synuclein ((3-syn)?a close homologue to alpha-syn?blocks a-syn aggregation and might have a role in the treatment of degenerative disorders such as Alzheimer's disease (AD) and LBD. Both p-syn and antibodies against alpha-syn target alpha-syn aggregates for clearance probably via autophagy, a process of degradation and recycling of cellular constituents. Alterations in autophagy might play a role in the pathogenesis of AD and LBD, and might represent a target for treatment development. The objectives of this renewal application are: i) to gain new knowledge as to the involvement of the autophagy pathways in the mechanisms of neurodegeneration in LBD, ii) to develop new experimental therapies for LBD by targeting the autophagy pathways and iii) to better understand the involvement of the autophagy pathways in the mechanisms of asyn clearance mediated by immunotherapy. We propose the following aims: AIM 1. Characterize in vivo the contribution of selected molecular components of the autophagy pathway to the pathogenesis of LBD. AIM 2. Investigate in in vivo models of alpha-synucleinopathy the therapeutic and neuroprotective effects of activators of the autophagy pathway. AIM 3. Better understand the cellular mechanisms involved in the clearance of toxic alpha-syn aggregates via specific antibodies. AIM 4. Determine in immunized animals, the contribution of autophagy to the molecular mechanisms involved in alpha-syn clearance. alpha-Syn transgenic mice will be crossed with mice either deficient in or transgenic for components of the autophagy pathway (e.g. Beclinl, LAMP2, mTor). Mice will be treated with stimulators of autophagy (rapamycin, immunotherapy) and analyzed behaviorally, biochemically and neuropathologically. Studies will be complemented with primary neuronal cultures treated with lentiviral vectors and analyzed for markers of autophagy. Better understanding the autophagic pathways involved in alpha-syn clearance is of central importance toward elucidating the pathogenesis of LBD and developing new treatments for these conditions. Thus, enhancing autophagy and lysosomal degradation of alpha-syn may represent a promising therapeutical strategy for the treatment not only for LBD but also for AD.

PROJECT SUMMARY/ABSTRACT This application directly responds to the objective of RFA-AG-17-051. We have recently reported a positive correlation of the levels of pathogenic phosphorylated microtubule-associated protein tau (p-tau) in plasma- derived neuronal exosomes in Alzheimer's disease (AD) patients, suggesting their potential application for biomarkers and their implication as a novel machinery of spreading pathogenic molecules in the brain. However, the exact mechanisms regulating exosome biogenesis and secretion, and their contribution to the propagation of pathogenic molecules are poorly understood. Here we propose comprehensive approaches to delineate the molecular mechanisms that regulate biogenesis, secretion and trafficking of exosomes in different brain cell types (neurons, astrocytes and microglia) in vitro and in vivo. This project will (1) analyze the roles of the ESCRT (endosomal sorting complexes required for transport)-dependent pathway, combined with the ESCRT-independent (lipid- and tetraspanin-dependent pathways), for exosome biogenesis and secretion in murine and human neuronal cell types, (2) define the molecular machinery components responsible for exosome biogenesis and secretion in human induced pluripotent stem cells (hiPSCs) derived from AD patients, (3) characterize the exosome protein interactions with target cells for propagation using AD hiPSCs, and (4) characterize the trafficking of exosomes originated from specific neuronal and glial cell types in the central nervous system to periphery. We have assembled a group of collaborative investigators with established programs in cell biology, exosome biology, proteomics and animal models of neurodegenerative disorders. The proposed research work will develop a new understanding of exosomal biology and detailed functions of exosomes in progression of AD pathology. The findings gained from this research project will have a potential to discover new molecular targets for suppression of the disease spread via exosomes, and also address the National Alzheimer's Project Act plan to accelerate basic research toward development of AD therapeutics.

NEUROPATHOLOGY CORE - SUMMARY/ ABSTRACT For the past 35 years, the UCSD-ADRC Neuropathology Core has been instrumental in providing support for establishing the accuracy of clinical diagnosis of Alzheimer's Disease (AD) and dementia with Lewy bodies (DLB), delineating structural and clinico-pathological correlates of dementia in AD, identifying new neuropathological entities causing dementia, providing tissues to investigators and helping to better understand the mechanisms of synaptic and other aspects of neurodegeneration in AD. For the renewal there will be 6 Aims of the Neuropathology Core: 1) perform rapid autopsies and procure brains from the ADRC participants, using a standardized protocol; 2) perform standardized neuropathological diagnoses and immunocytochemical analysis of demented and normal aged (control) patients clinically evaluated by the UCSD ADRC following the new NIA criteria; 3) perform clinical neuropathology assessments on brain, spinal cords and eyes from postmortem material from ADRC participants using best practice (NIA-AA) guidelines. Perform immunochemical analysis relevant to neurodegeneration and synapse loss in MCI and early AD cases; 4) maintain a state of the art brain repository to provide the ADRC projects and other investigators with well characterized including early AD and MCI cases; 5) foster the utilization of the ADRC Neuropathology tissue repository for new research and inter-center collaborations. Approximately 50 to 60 cases and over 100 tissue requests are processed a year. The neuropathological results will be submitted to the National Alzheimer?s Coordinating Committee (NACC) in compliance with NIA requirements. As part of the mission of the Core we will also continue to support extensive collaborations with national and international investigators and train fellows, residents, graduate and undergraduate students in neuropathology and microscopy techniques. With the ADRC Outreach, Recruitment and Education (ORE) Core organize meetings to encourage the use of the neuropathology core; 6) Mentor young and minority investigators in neuropathology and neuropathological research. We will foster the next generation of scientists by encouraging them to conduct research with the postmortem tissues. We will continue to provide tissues to local and national investigators and for multi-center initiatives such as the Genome-Wide Analysis Studies organized by NIA.

PROJECT SUMMARY/ABSTRACT Neurodegenerative disorders of the aging population are characterized by the progressive accumulation of proteins such as ?-synuclein (?-syn), amyloid beta (Aß) and microtubule associate protein (tau). Misfolded and aggregated ?-syn has been implicated in neurological disorders with Parkinsonism including Dementia with Lewy Body, Parkinson?s disease (PD), and Multiple Systems Atrophy. Accumulation of ?-syn has even been confirmed in over 50% of Alzheimer?s disease (AD). Recent evidence points to a role of ?-syn accumulation in the aggregation of tau and Aß in AD. Thus, regulation of ?-syn expression may be crucial to the therapeutic control of numerous neurodegenerative diseases. Short interfering RNA molecules (siRNA) can bind specifically to target RNAs and deliver them for degradation; however, RNA molecules do not cross the blood- brain barrier so the only method for delivery is repeat intra-thecal injections. We recently developed a peptide (ApoB11) that binds oligonucleotides for transport across the blood-brain barrier following systemic administration. Using this peptide, we showed that we can deliver a si ?-syn to reduce expression of ?- synuclein in a mouse. We recently converted the ribonucleotide backbone of this siRNA to a 2?-MOe anti-sense oligonucleotide to increase half-life and affinity to the mRNA target. We plan to examine the pharmacokinetics and toxicology of systemic ApoB11:2?-MOe si ?-syn following intra-peritoneal delivery in an ?-syn tg mouse model of DLB. Then we will examine the ability of the ApoB11:2?-MOe si ?-syn to reduce ?-syn and improve survival of neurons and improve cognitive ability and motor coordination in an ?-syn tg mouse model of DLB. Finally, we will examine the ability of the ApoB11:2?-MOe si ?-syn to reduce the accumulation of ?-syn in an in vitro model of human DLB neurons derived from iPSC cells in a blood-brain barrier model. We believe this may represent a new method of therapeutic delivery for DLB and other neurological disorders.