Subhojit Roy, MD, PhD - US grants

A common feature in all dementias associated with Lewy Bodies is the accumulation and aggregation of the small 14kD protein a-synuclein in the perikaryon and proximal neurites. This proximal accumulation of ct- synuclein in diseased states is very different from the physiologic situation, where the protein is predominantly localized to distant presynaptic sites. Thus pathologic conditions lead to a mis-localization of a-synuclein into proximal neuronal compartments, in addition to the accumulation/aggregation of the protein. While many previous studies have focused on the biochemical processes leading to the aggregation of o> synuclein into the insoluble fibrils that are seen in the end-stage Lewy bodies, much less is known about the initial mechanisms that lead to the proximal mis-localization of the protein. As a-synuclein is synthesized in the neuronal perikarya and is transported into axons, eventually targeting to synapses, our working hypothesis is that defects in the mechanisms of axonal transport and/or presynaptic targeting of a-synuclein is the basis for its mis-localization in pathologic states. To test this hypothesis, we have developed novel model-systems and imaging tools that allow us to directly visualize and precisely quantify axonal transport and presynaptic targeting of a-synuclein in axons and boutons of living neurons. Indeed defects in transport/targeting of pathologic forms of a-synuclein are seen in this system, supporting our hypothesis. Completion of the proposed project will provide insights into initial pathologic mechanisms in these dementias, and may also lead to novel early therapeutic targets. RELEVANCE (See instructions): Dementias associated with Lewy bodies is a common cause of dementia among the elderly, second only to Alzheimer's. To date, there is no known cure. Our best chance of treating this disease is to attack it at an early stage, however, early mechanisms leading to these dementias is poorly understood. In this project we will unravel such early events by determining how a key protein gets misplaced in neurons, causing disease.

Project Summary The vast majority of proteins in a neuron are synthesized in the cell bodies and transported along axons and up-to synapses by a process called axonal transport. Defects in slow axonal transport of proteins such as tau and ¿-synuclein have long been implicated in many neurodegenerative diseases including Alzheimer's and Parkinson's disease, however mechanisms of slow axonal transport of these (and other) cytosolic proteins is very poorly understood. We developed a model-system in cultured neurons to directly visualize the transport of cytosolic proteins (including ¿-synuclein) and found that these cargoes move coherently with a slow, motor-dependent anterograde bias. This type of movement has not been reported before and likely represents a new form of trafficking/transport within cells. Based on these and other in-vivo data from brains, we propose a new model where individual cytosolic protein monomers cluster and assemble into multi-protein complexes that are carried in neurons by molecular motors, a process we call 'dynamic clustering'. Here we propose a series of experiments to test predictions and hypotheses generated by this model. Upon completion, these studies would answer long-standing questions about the transport of these proteins and also open the door for investigation of their transport in pathologic states.

The overall goal of this application is to clarify the molecular organization and function of enigmatic axonal actin assemblies that my lab recently discovered. Actin organization along axon shafts has been relatively ignored for decades, probably because routine staining ? using the classic actin-filament marker phalloidin ? only shows a patchy, uninteresting pattern. However, recent studies from us and others' using super-resolution microscopy and low-light imaging have revealed a hidden world of actin in axon shafts ? replete with elaborate circumferential rings underneath the plasma membrane, and rapidly elongating linear filaments along the axis of the axon shaft. We have been fortunate to be at the forefront of these discoveries, and our studies have revealed fascinating actin dynamics in axons. Specifically, we found that axons have focal ?hotspots? of actin ? spaced ~ 3-4 µm apart ? where actin continuously polymerizes and depolymerizes. These hotspots give rise to long actin filaments that rapidly (and bidirectionally) elongate along the axon shaft (we named these ?actin trails?). Actin rings, trails and hotspots are seen in axons in vivo, and we will use in vivo model systems in this proposal as well. Although the overall assemblies have been seen, molecular events underlying the generation and maintenance of these structures is unclear. Importantly, their functional roles in axons are unknown. Here we propose three specific aims to clarify the organization and function of these newly-discovered actin assemblies: Aim #1: Identify mechanisms initiating actin trails in axons. Aim #2: Identify mechanisms elongating actin trails in axons. Aim #3: Determine functions of axonal actin assemblies.

The overall goal of this proposal is to clarify mechanistic pathobiological events underlying Lewy body (LB) dementias ? a dementing illness with cognitive impairment that affects more than a million Americans. An established molecular player in LB dementia is the small presynaptic protein ?-synuclein. Amongst a plethora of incriminating evidence, genomic multiplications and mutations of ?-synuclein are seen in families harboring these diseases; and it has been long recognized that understanding the mechanistic events that lead to ?-synuclein-mediated toxicity in LB dementia is of utmost importance. For over a decade, a primary focus in the field has been to decipher the normal function of ?-synuclein, with the ultimate goal of understanding transition to pathologic states. However, despite considerable effort, the precise mechanisms underlying the normal function of ?-synuclein, and early triggers leading to pathologic aggregation remain elusive. The basis of our proposal is a series of pilot experiments, where we uncovered novel roles for two functional partners of ?-synuclein, and we hypothesize that abnormalities in these associations are the initial pathologic triggers for LB dementias. Previous work from us and others has helped shape a consensus that ?-synuclein is a physiologic attenuator of neurotransmitter release, though underlying mechanistic events are unclear. In these previous studies, we proposed a model where ?-syn organizes into higher-order multimers that physiologically tether synaptic vesicles (SVs) ? leading to a diminution in SV-mobilization, SV-recycling, and consequently, neurotransmitter release. In new pilot experiments, we discovered novel roles for two other presynaptic proteins ? VAMP2 and synapsin ? in helping ?-synuclein attenuate neurotransmission. Eventually, our data led us to a working model where synapsin and VAMP2 play sequential roles in executing ?-synuclein function. Tenets of this model will be tested in Aims 1/2. Additionally, an emerging idea in the field is that disruption of physiologic associations might allow free ?-synuclein monomers to aggregate ? triggering pathology ? and that this might be one of the earliest pathologic events in disease; however, in vivo evidence is lacking. Leveraging our discoveries on functional ?-synuclein partners, Aims 2/3 will ask if a disruption of these associations might also accelerate pathology in cellular and animal models of LB dementias. Our aims are: Aim #1: Identify the role of VAMP2 in ?-synuclein mediated synaptic attenuation. Aim #2: Identify the role of synapsin in ?-synuclein mediated synaptic attenuation and pathology. Aim #3: Test the hypothesis that disrupting physiologic associations can trigger ?-synuclein pathology in vivo. Upon completion, our studies should reveal vital clues into the normal function of ?-synuclein, as well as events that trigger dementia and cognitive impairment in these devastating illnesses.

Project Summary: CRISPR/Cas9 is a revolutionary and versatile genome editing technique with wide-ranging utility. In vivo genome editing is anticipated to be the next wave of therapeutics for various major health threats, including neurode- generative diseases. However, there is an urgent need to develop efficient, non-viral delivery vehicles for safe and efficient in vivo CRISPR genome editing. Furthermore, delivering CRISPR genome editing machinery to the brain/neuron represents a major hurdle due to its dense structure and the blood?brain barrier (BBB). The objective of this project is to engineer a family of versatile, novel, non-viral Cas9-gRNA ribonucleoprotein (RNP) delivery nanocapsules (NCs) that can robustly and safely generate targeted gene edits in neurons within the brain. We envision that our robust and universal RNP delivery nanoplatforms will enable innovative treatments for devastating neurodegenerative diseases. Towards this goal, we will evaluate the feasibility of our approach, in a demonstration, by targeting the amyloid precursor protein (APP) ? relevant to Alzheimer's disease (AD) in healthy mice and monkey models. During our preliminary studies, we developed a PEGylated NC with a high RNP loading content (68 wt.%), versatile surface chemistry, ultrasmall size (dH~13 nm), controllable stoichiometry, excellent biocompatibility, and high genome editing efficiency in vitro and in vivo. In UG3 Aim 1, we will further optimize the design of the NC for brain/neuron-targeted genome editing. In particular, we will investigate the synergistic effects of hybrid targeting ligands, including (1) glucose+RVG peptide for intravenous (i.v.) injection to enhance the crossing of the BBB and neuron-specific editing, and (2) CPP+RVG peptide for intracerebral injection to enhance uptake and neuron-specific genome editing. The effects of different types/amounts of targeting ligands on the cellular uptake, biocompatibility, genome editing efficiency, and functional consequences of the NCs in both Neuro2a and primary neuron cells will be investigated. In UG3 Aim 2, we will evaluate the brain/neuron targeting specificity, genome editing efficiency, and potential immune response and systemic toxicity of the i.v. or intracerebrally administered NCs conjugated with various targeting ligands in healthy mice. In UH3 Aim 1, we will develop the set up and synthesis process to scale up the production of NCs. In UH3 Aim 2, we will further evaluate the genome editing efficiency and biocompatibility of the brain/neuron-targeted NCs in healthy rhesus macaques. Our uniquely designed NCs are expected to achieve high brain accumulation, high penetration depth, and high neuron-specific genome editing efficiency due to their desirable characteristics. Given the modularity and ease of targeting different genes by the CRISPR system, we anticipate that the resulting NCs will be useful for a wide range of human diseases, including debilitating neurodegenerative diseases for which there are no cures.