Abstract Lewy body disorders are a family of fatal neurodegenerative conditions with α-synucleinopathic inclusions spreading across brain regions involved in smell, affect, movement, and cognition. There are ~1.3M patients living in the US with dementia with Lewy bodies (DLB) and another ~4M patients living with Parkinson's disease (PD). In addition, an estimated ~50% of Alzheimer's disease patients display coexisting Lewy pathology (ADLB). If therapies could be developed that slow the onset and spread of α-synucleinopathy, neurodegeneration might be mitigated and deterioration of function would be delayed in millions of patients. Recent studies reveal that α-synuclein is a novel microtubule (MT) dynamase, disrupting axonal transport across microtubules (MTs) in its aggregated form. Thus, tipping MT dynamicity towards stabilization has emerged as a potential new treatment for neurodegenerative disorders. Accordingly, we observed significant loss of detyrosinated (stable) α-tubulin in the olfactory bulb of men with Lewy body disorders. The olfactory bulb/anterior olfactory nucleus (OB/AON) complex is closely connected with the limbic system and is one of the earliest regions to display α-synucleinopathic inclusions in this family of conditions. We have also recently discovered that two novel MT stabilizers (AG161-41 & 47) reduce the emergence of α-synuclein aggregates (up to a remarkable 48%) in primary hippocampal cultures exposed to preformed α-synuclein fibrils. To our knowledge, this observation is unique, as other MT stabilizers have not been demonstrated to mitigate Lewy pathology. Our human tissue and primary culture pilot data make a compelling argument for further proof-of-concept studies. Thus, we will synthesize improved analogs and test the hypothesis that MT stabilization tempers the pathological sequelae of exposure to α-synuclein fibrils in cellular and animal models of limbic Lewy pathology. In Aim 1, we will synthesize additional AG161-41 & 47 and 12 novel analogs of AG161-47. In Aims 2A-C, we will determine if tipping the balance towards MT stabilization with these 14 compounds reduces inclusion density, cell loss, protein aggregation, and markers of MT destabilization in primary limbic neuron cultures treated with α-synuclein fibrils. In Aim 2D, we will evaluate the compounds in assays of tubulin assembly and displacement with vincristine. In Aim 3A, we will characterize the pharmacokinetic properties of AG161- 47 and 4 of the most promising analogs from Aim 2. In Aim 3B, we will test the neuroprotective potential of 5 doses of the single most effective, safe analog from Aims 2-3A, in mice infused with α-synuclein fibrils in the OB/AON. Cognitive, anxiety, olfactory, and motor behavior will be assessed at baseline and monthly intervals. α-synuclein inclusions and cell counts will be measured with unbiased stereological tools. Aim 3B will identify a pharmacologically active dose (PAD) that mitigates inclusions, cell loss, protein aggregation, and MT destabilization. In Aim 3C, we will use this PAD in a longer-term, interventional study designed to test if the most promising candidate can rescue neurons after behavioral deficits emerge (e.g., smell loss emerges within 3 months). The assays of Aim 2B will be repeated. These proof-of-concept studies are designed to 1) pave the way for future SAR studies, 2) generate new hypotheses about the role of cytoskeletal stabilization in Lewy body disorders in vivo, and 3) help accelerate the translation of MT-modifying agents to the clinic.

Project Narrative The cytoskeleton is severely compromised in neurodegenerative diseases, including the fatal conditions known as Lewy body disorders. To our knowledge, we have designed the first cytoskeletal stabilizers that mitigate proteinopathic Lewy pathology to a significant extent (48%), without cellular toxicity. We propose to test additional derivatives of the lead compounds in our new animal model of limbic Lewy body disorders, in which aggregations of damaged proteins fill key regions of the brain involved in olfaction, emotion, and memory. We expect this body of work to accelerate the future translation of a novel treatment modality for patients suffering from Lewy body disorders.