ABSTRACT Parkinson’s disease (PD) is a progressive neurodegenerative disease that affects 10 million people worldwide. Its motor symptoms result from selective degeneration of dopaminergic neurons in the substantia nigra pars compacta, leading to a loss of their long-projecting axonal inputs to the striatum. Conventional cell therapy involves implanting dopaminergic neurons into the striatum; however, this strategy disregards the important systems-level implications of the native neuroanatomy. Pathway reconstruction strategies aim to address this limitation by replacing both neurons and axonal fibers in a manner that restores the anatomy – and hence circuit function – of the lost pathway. We have developed a reconstruction strategy whereby tissue-engineered nigrostriatal pathways (TE-NSPs) are pre-fabricated in vitro featuring a population of human stem cell-derived dopaminergic neurons and their long-projecting axonal tracts encased within a biocompatible tubular hydrogel. TE-NSPs may be implanted to directly replace the pathway, supplying both dopaminergic neurons to the nigra and providing axonal inputs to the striatum, thereby restoring crucial interconnectivity of the basal ganglia. In this proposal, we will answer a fundamental and neglected question in cell therapy for PD by characterizing whether pathway reconstruction with the TE-NSPs enables improved restoration of motor function compared to conventional striatal grafts in a rat model of PD. Our overarching hypothesis is that TE-NSPs will lead to more robust motor recovery than striatal grafts through a mechanism involving the reestablishment of physiological innervation and striatal dopamine regulation patterns more closely matching those of native basal ganglia. This hypothesis will be tested over three Aims: (1) Establish the ability of TE-NSPs to reconstruct basal ganglia circuitry via axonal-dendritic synaptic integration; (2) Demonstrate real-time efficacy of TE-NSPs in restoring nigrostriatal functionality; (3) Assess the influence of TE-NSP activity on motor recovery. TE-NSP mechanisms and efficacy will be compared to hydrogel-encased nigral or striatal grafts, acellular hydrogel implants, as well as non-implant and non-lesioned animals out to 24 weeks post-implantation. Motor function will be evaluated with rotational, forelimb asymmetry and adhesive removal tests. Innervation and connectivity patterns will be assessed with immunohistochemistry and monosynaptic rabies tracing, while ex vivo and in vivo voltammetry and [18F]F-DOPA positron emission tomography will be used to analyze real-time dopamine release and uptake in the striatum. We will also employ chemogenetics to silence neural activity in TE-NSPs to test the effects on motor function. Overall, TE-NSPs address a crucial gap in clinical treatment by providing a means to directly replace the nigrostriatal pathway, which may yield significant benefits over other methods by providing properly-regulated dopamine in the stri...