Dual kinase inhibition provides early clues to restoration of neuroimmune homeostasis in an AAV-haSyn model of Parkinsons disease

Abstract

Background Previous studies of drugs targeting mixed linage kinase type 3 (MLK3) and/or leucine rich repeat kinase type 2 (LRRK2) in sporadic Parkinsons disease (PD) have failed to meet clinical endpoints despite multiple lines of evidence implicating these kinases in pathogenesis. This has reduced enthusiasm for MLK3 and LRRK2 as druggable targets. Largely overlooked is the evidence that these targets remain valid but must be considered in combination to achieve a therapeutic outcome. This provides the rationale for this study. Methods We conducted a pilot study modeling early, pre-symptomatic PD with subsequent evaluation of therapeutic potential of a multi-kinase inhibitor (URMC-099) in alleviating disease pathology. Sprague Dawley rats received unilateral intranigral injections of low dose AAV-haSyn. After a two-week recovery period, all animals were administered either once-daily intraperitoneal (i.p.) injection of URMC-099 or its vehicle control until their terminal endpoint. Induction of early PD, and its alleviation with URMC-099, was evaluated by lateralized motor impairment using the cylinder test and immunohistochemical analysis of neuropathology in the substantia nigra (SN) and striatum. Finally, an in vitro microglial PD model exposed to aSyn pre-formed fibrils and treated with URMC-099 was used to further evaluate the mechanism underlying therapeutic activity. Results A unilateral model of early and progressive AAV-haSyn model for PD was used to demonstrate the therapeutic potential of a targeted multi-kinase inhibitor to attenuate PD-associated neuropathology and prevent the manifestation of lateral motor deficits. Immunohistochemical analysis revealed protection of neuronal cellular integrity along the nigral-striatal pathway with once-daily i.p. URMC-099. The mitigation of degenerative neuropathology was linked to targeted inhibition of MLK3 and LRRK2 and complemented by reduced levels of gliosis in astrocytes and microglia in the SN. Single-microglia segmentation analysis suggested URMC-099 treatment preserved dynamic effector state switching in microglia to enhance autophagic flux to reduce the overall burden of pathologic aSyn. Transcriptomic analysis of a complementary in vitro microglia model corroborated the retention of a homeostatic-like effector state with URMC-099 treatment that was unique to MLK3/LRRK2 dual inhibition. Conclusions Unilateral injection of low dose AAV-haSyn successfully resulted in a lateralized model of early, progressive PD. Dual inhibition of aberrant MLK3 and LRRK2 activity with once-daily i.p. URMC-099 therapy significantly mitigated the development of PD pathology and progression in an early manifest phenotype. Our results present a potential mechanism of therapeutic action dependent on the dual inhibition of MLK3 and LRRK2. This presents a novel therapeutic modality that may be beneficial to a broad population of etiologic PD patients.