SummaryAge-related macular degeneration (AMD) is a common cause of central visual loss in the elderly. Retinal pigment epithelial (RPE) cell loss occurs early in the course of AMD and RPE cell transplantation holds promise to slow disease progression. We report that subretinal transplantation of RPE stem cell (RPESC)-derived RPE cells (RPESC-RPE) preserved vision in a rat model of RPE cell dysfunction. Importantly, the stage of differentiation that RPESC-RPE acquired prior to transplantation influenced the efficacy of vision rescue. Whereas cells at all stages of differentiation tested rescued photoreceptor layer morphology, an intermediate stage of RPESC-RPE differentiation obtained after 4 weeks of culture was more consistent at vision rescue than progeny that were differentiated for 2 weeks or 8 weeks of culture. Our results indicate that the developmental stage of RPESC-RPE significantly influences the efficacy of RPE cell replacement, which affects the therapeutic application of these cells for AMD.

In Parkinson's disease (PD), misfolded and aggregated α-synuclein protein accumulates in degenerating midbrain dopaminergic neurons. The amino acid alanine-76 in α-synuclein and phosphorylation at serine-87 and serine-129 are thought to regulate its aggregation and toxicity. However, their exact contributions to α-synuclein membrane association are less clear. We found that α-synuclein is indeed phosphorylated in fission yeast and budding yeast, the two models that we employed for assessing α-synuclein aggregation and membrane association properties, respectively. Surprisingly, blocking serine phosphorylation (S87A, S129A, and S87A/S129A) or mimicking it (S87D, S129D) altered α-synuclein aggregation in fission yeast. Either blocking or mimicking this phosphorylation increased endomembrane association in fission yeast, but only mimicking it decreased plasma membrane association in budding yeast. Polar substitution mutations of alanine-76 (A76E and A76R) decreased α-synuclein membrane association in budding yeast and decreased aggregation in fission yeast. These yeast studies extend our understanding of serine phosphorylation and alanine-76 contributions to α-synuclein aggregation and are the first to detail their impact on α-synuclein's plasma membrane and endomembrane association.

In Parkinson's disease (PD), midbrain dopaminergic neuronal death is linked to the accumulation of aggregated α-synuclein. The familial PD mutant form of α-synuclein, E46K, has not been thoroughly evaluated yet in an organismal model system. Here, we report that E46K resembled wild-type (WT) α-synuclein in Saccharomyces cerevisiae in that it predominantly localized to the plasma membrane, and it did not induce significant toxicity or accumulation. In contrast, in Schizosaccharomyces pombe, E46K did not associate with the plasma membrane. Instead, in one strain, it extensively aggregated in the cytoplasm and was as toxic as WT. Remarkably, in another strain, E46K extensively associated with the endomembrane system and was more toxic than WT. Our studies recapitulate and extend aggregation and phospholipid membrane association properties of E46K previously observed in vitro and cell culture. Furthermore, it supports the notion that E46K generates toxicity partly due to increased association with endomembrane systems within cells.

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Parkinson's disease (PD) is an incurable neurodegenerative disorder linked to the misfolded protein α‐synuclein, which is often phosphorylated and membrane‐lipid associated within the Lewy bodies in the dying substantia nigra neurons. Exactly how α‐synuclein phosphorylation, aggregation, and membrane association contribute to toxicity is unresolved, as is the mechanism of toxicity linked with newer familial mutant E46K. We recently created two yeast models that recapitulate α‐synuclein properties: budding yeast for membrane association and fission yeast for aggregation. Here, first, we report that E46K resembled wildtype and A53T α‐synuclein by associating with the plasma membrane in budding yeast and forming aggregates in fission yeast. Next, we demonstrate that serine phosphorylation promoted α‐synuclein aggregation, and also, unexpectedly, membrane association. In fission yeast, S87A & S129A mutants redistributed to diverse intracellular compartments, while in budding yeast both mutants reduced plasma membrane localization. Lastly, we provide evidence that the domains N and NAC (and alanine‐76 within NAC), but not domain C, contributed to membrane association in budding yeast. Furthermore, in fission yeast, A76E was less aggregated. Thus, both yeast models of α‐synuclein further illuminate mechanisms that underlie familial and sporadic PD. (Supported by NSF‐MRI, NSF‐CCLI & NIH R15)