Jae K. Lee, PhD - US grants

7. Project Summary The purpose of this proposal is to conduct a rigorous in vivo efficacy study to test the activity of our optimized lead molecule, NOVO-118, in an animal model of spinal cord injury (SCI). SCI prevalence ranges between 205-906 cases per million worldwide, and is characterized by a catastrophic and irreversible loss of motor and sensory function below the level of injury. Currently, no treatments exist to address the fundamental cause of dysfunction, which is the disruption of neuronal connectivity and failed restoration of neuronal pathways after damage. We have discovered that low-density lipoprotein receptor-related protein 1 (LRP1) is a novel master regulator of the diverse signaling pathways that converge onto the pathological hyperactivation of RhoA, which is the necessary and sufficient signal for neuroregenerative failure. In animal models, directly targeting RhoA, or its downstream effector Rho-associated kinase (ROCK), has been well-validated as a means of restoring neuronal regeneration and functional recovery after SCI. However, current approaches which target the RhoA/ROCK pathway do not discriminate between the pathological hyperactivation responsible for regenerative arrest and endogenous Rho activity, which is needed for normal cellular function. In targeting LRP1, we have demonstrated that we can overcome pathological hyperactivation of RhoA while leaving endogenous function intact, greatly reducing the potential of toxicity via our approach. To this end, we have shown that both pharmacologic antagonism and genetic silencing of LRP1 results not only in abrogation of pathological RhoA activity, but also coincides with a robust restoration of neuronal growth in the presence of a diverse array of inhibitory molecules. To therapeutically target LRP1 in SCI, we have developed a novel biologic antagonist of LRP1, NOVO-118, to be used as a therapeutic to promote neuronal regeneration. In this application, we look to assess the ability of NOVO-118 to restore behavioral deficits after SCI in a long-term in vivo model of SCI. Successful demonstration of the long-term in vivo efficacy of NOVO-118 will warrant formal pre-clinical development of our lead molecule for the treatment of SCI.

PROJECT SUMMARY/ABSTRACT After spinal cord injury (SCI), the injury site is filled with cellular debris, especially myelin debris that creates a very unique lipid-dense environment. Macrophages are the predominant phagocyte that are responsible for debris-clearance, but this process is not only inefficient, it is also maladaptive. The excessive amount of myelin debris present at the injury site leads to formation lipid-laden macrophages (a.k.a. foamy macrophages) that become pro-inflammatory and contribute to tissue regeneration failure. In addition to macrophages, microglia and fibroblasts also become foam cells. Therefore, understanding the mechanisms of myelin debris uptake and catabolism after SCI may lead to novel therapeutic targets to promote repair after SCI. In this application, we will investigate the mechanism of myelin debris uptake as well as the export of its catabolic byproduct in macrophages, microglia, and fibroblasts after SCI. In addition, we will test the therapeutic potential of novel nanoparticles that can target the uptake and efflux mechanisms.

SUMMARY Aging-related inflammation and metabolic disorders, including Alzheimer's disease (AD), Lewy body dementia (LBD), and Dementia with Lewy bodies (DLB), constitute serious threats to human health as they are risk factors for dementia. Microglia play a critical role in immune surveillance in the CNS, clearing abnormal protein aggregates, and maintaining energy balance and metabolism. However, microglia undergo phenotypic changes during neurodegenerative disorders and contribute to neurodegenerative diseases. Therefore, we may be able to harness the activity of microglia and restore metabolic homeostasis as an effective therapeutic for age-related neurodegenerative disorders. The objective of this proposal is to develop a microglia-specific nanotherapeutic for amyloid fibrils-induced neurodegeneration composed of an antibody targeting to microglia (Tmem119) and plasmid encoding regulator of G-protein signaling 10 (pRGS10). RGS10 is a homeostatic protein in microglia and its level is significantly decreased with chronic inflammation and aging. Our preliminary study demonstrated that RGS10 enhances phagocytosis of abnormally aggregated proteins including fibrillar ?- amyloid (fA?) and ?-synuclein (?-syn). We hypothesize that enriching RGS10 levels through microglia-specific nanoparticles carrying pRGS10 may restore microglial homeostasis, enhance amyloid fibril clearance, and provide neuroprotection against amyloid-fibril-induced neuronal death. We will utilize two innovative approaches: a novel cationic amphiphilic co-polymer, poly (lactide-co-glycolide)-graft-polyethylenimine (PgP): and a preformed fibril (PFF) ?-syn mouse model of Lewy body diseases (PFF mouse model) to determine if microglial RGS10 is neuroprotective. In Aim 1, we will conjugate anti-Tmem119 mAb to the surface of PgP (Tmem-PgP), formulate Tmem-PgP/pRGS10 nanoparticles and evaluate target-specificity and neuroprotection in a primary neuron/microglia co-culture system. In Aim 2, we will demonstrate the therapeutic efficacy of Tmem-PgP/pRGS10 in the PFF mouse model. The completion of this study will elucidate the role of RGS10 in maintaining microglia homeostatic conditions and how we may utilize RGS10 as a therapeutic target for amyloid fibril-associated neurodegenerative diseases.

  PROJECT SUMMARY/ABSTRACT Lewy body diseases including Parkinson's disease (PD), Lewy body dementia (LBD), and multiple system atrophy are characterized by the accumulation of aggregated alpha-synuclein (?-syn) protein, which is the principal component of Lewy bodies (LBs). Neuroinflammation is the hallmark of Lewy body diseases and the role of the immune system has been implicated in the progression of synuclein pathology and neuropathology. However, the role of cellular immunity in promoting neurodegeneration or exerting neuroprotection remains unclear. We established a preclinical mouse model of ?-synucleinopathy and showed a robust increase in immune responses in the CNS and the periphery. We noted significantly increased peripheral leukocytes including natural killer (NK) cells in the mouse model of ?-synucleinopathy. As innate immune cells, NK cells are of particular interest for neurological disorders because they are modified in the periphery and/or travel into the CNS. It has been shown that NK cell numbers are increased in the blood of PD patients compared to age- matched controls and their activity is associated with disease severity. However, the role of NK cells in the context of PD has never been explored. We recently reported NK cells are present in the substantia nigra of post mortem brains of PD and LBD patients. Based on our experimental data, NK cells efficiently clear ?-syn aggregates and in vivo depletion of NK cells results in exacerbated synuclein pathology, neuroinflammation, and striatal degeneration in a preclinical mouse model of ?-syn aggregation. The proposed study will investigate the mechanism (s) by which NK cells reduces ?-syn burden, modulate inflammation, and exert neuroprotection in the CNS and periphery. We will use both in vitro and in vivo mouse model of synucleinopathies to address the physiological role of NK cells in the context of Lewy body diseases. To test our central hypothesis and achieve our objective we propose three specific aims as follows: In Aim 1, we will investigate the mechanism by which NK cells scavenge ?-syn and modulate ?-syn-induced inflammation and neurotoxicity. In Aim 2, we will determine whether NK cells are neuroprotective in a mouse model of ?-syn aggregations. In Aim 3, we will determine if peripheral NK cell infiltration into the CNS is essential for neuroprotection. To date, there are no therapies available to slow or stop disease progression of synucleinopathies. Our study will provide a comprehensive understanding of the role of NK cells in the context of Lewy body diseases. Therefore, this work can have a positive impact by providing a scientific basis for pursuing NK cells as a potential immunotherapeutic target for aged-related neurodegenerative diseases.    

ABSTRACT Limited regeneration of central nervous system (CNS) axons is a major barrier to recovery after CNS injury. Major advances have been made in identifying neuron-intrinsic mechanisms to promote axonal growth, but regenerating axons still require an environment that is growth-permissive. Although the astroglial scar has been considered to be a major inhibitory barrier to axon regeneration, there is mounting evidence that in certain conditions, reactive astrocytes may aid, rather than inhibit, regeneration of axons across the injury site. One possible explanation that might reconcile these conflicting roles of the astroglial scar is that there are astrocyte subpopulations that can inhibit, and other subpopulations that can permit, axon regeneration. One potential source of these regeneration-permissive astrocytes is oligodendrocyte progenitor cells (OPCs), which we and others have shown can differentiate into astrocytes after spinal cord injury (SCI). Since this differentiation capacity is limited to about 10-20% of OPCs in the glial scar region, we hypothesize that enhancing the number of OPC-derived astrocytes can enhance axon regeneration by increasing the amount of regeneration-permissive substrate across the injured spinal cord.