Modeling midbrain dopaminergic neurobiology and neuropathology using human ES and iPS cells [electronic resource]

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by tremor, rigidity, bradykinesia, and akinesia which affects approximately 1% of the population over the age of 60. These motor symptoms result from the selective destruction of midbrain dopamine (DA) neurons which form the nigrostriatal pathway. While there is likely to be a diverse range of inciting factors across patients, common pathological pathways mediating the selective loss of substantia nigral dopaminergic neurons are predicted. Only a small fraction of all PD cases can be ascribed to monogenic causes, however, these mutations provide our best chance for making progress in understanding PD pathophysiology. The accumulation and aggregation of alpha-synuclein (SNCA) in the form of Lewy bodies and Lewy neurites are defining neurocytological hallmarks for PD, suggesting that SNCA may be a linchpin to PD pathology. Duplications and triplications of SNCA are autosomal dominant causes of PD, and overexpression of SNCA has also recapitulated certain disease characteristics in animal and cell models. Mutations in the leucine rich repeat kinase 2 (LRRK2) are also implicated in PD pathogenesis. LRRK2 is predicted to interact either directly or indirectly with SNCA. Indeed, LRRK2 mutations are predicted to cause increased SNCA expression. As PD is only known to naturally occur in humans, and selectively targets midbrain DA neurons, a human midbrain DA neuron model involving known monogentic mutations would be a powerful platform for examining disease pathology. Here we describe the generation of induced pluripotent stem cells (iPSCs) created from patients with PD stemming from either an SNCA triplication (Trpl) or a G2019S mutation in LRRK2 (Lrkm). Human dopamine neuron cultures from these genetically-defined PD patients demonstrated overt pathological phenotypes. Both Trpl and Lrkm DA neuronal cultures contained an accumulation of monomeric SNCA, and markers of oxidative stress as compared to control embryonic stem cell (ESC) or iPSC lines. Trpl DA neuronal cultures possessed a marked increase in cells laden with ubiquitin puncta as compared to sibling control lines. Lrkm DA neuronal cultures were more susceptible to both the general oxidative stressor H2O2 and the selective DA neurotoxin 6-OHDA. These and other similarly derived lines will provide a powerful platform for investigating the molecular events in PD, and will enable large-scale screening for new therapeutic compounds to prevent and halt the progression of PD. While a fraction of all PD cases have known monogenic correlates, the majority cases are sporadic, with no recognized cause. In these cases, PD cannot be predicted and diagnosis is only made following the onset of symptoms, so even with significant advances in halting the progression of DA neuronal loss, there is and will continue to be a need for late-stage treatment options. Transplantation of human DA neurons is one such option. While most prior transplantation models for PD therapy graft cells directly into the target striatum, it should be remembered that DA neurons are part of a circuit, so ideal transplantation would graft DA neurons into the striatum and coax them to innervate the striatum thus rebuilding the native circuit. A major challenge to this type of advanced transplantation is the ability to accurately control the outgrowth and guidance of DA neurites. This requires detailed knowledge of the axon guidance cues that these particular neurons normally respond to, and how these responses may change in the context of severe neuroinflammation present in the diseased and post-op brain. Utilizing an in-vitro COS-cell aggregate outgrowth assay, we demonstrate that ESC-derived human midbrain DA neurons are attracted to gradients of netrin-1 and repelled by gradients of slit-2 in a manner that is similar to primary rat midbrain DA neurons. While these responses were not abolished in the presence of inflammatory cytokines, the response to netrin-1 was modified. These findings suggest that the outgrowth and innervation of transplanted human ESC/iPSC-derived DA neurons may be targeted by application of exogenous guidance factors, or manipulation of endogenous guidance factors and inflammatory cytokines in order to achieve better therapeutic outcomes. Further development these of human ESC and iPSC derived midbrain DA neuron models are likely to lead to major advances in our understanding of the basic developmental and functional neurobiology of DA neurons, and how these features are disrupted in disease processes like PD.

Advisor: Palmer Theo primary advisor. Thesis advisor
Heilshorn Sarah advisor. Thesis advisor
McConnell Susan K. advisor. Thesis advisor
Reijo Pera Renee advisor. Thesis advisor