Background: How N-terminal acetylation affects the structure and function of ␣-syn remains unknown. Results: N-terminally acetylated and WT ␣-syn are unfolded monomers and exhibit similar aggregation and cellular properties. Conclusion: ␣-syn N-terminal acetylation does not dramatically affect its structure or oligomerization state in vitro and in intact cells. Significance: Recombinant nonacetylated or N ␣ -acetylated ␣-syn remains suitable for ␣-syn biophysical studies.

Although trace levels of phosphorylated ␣-synuclein (␣-syn) are detectable in normal brains, nearly all ␣-syn accumulated within Lewy bodies in Parkinson disease brains is phosphorylated on serine 129 (Ser-129). The role of the phosphoserine residue and its effects on ␣-syn structure, function, and intracellular accumulation are poorly understood. Here, co-expression of ␣-syn and polo-like kinase 2 (PLK2), a kinase that targets Ser-129, was used to generate phosphorylated ␣-syn for biophysical and biological characterization. Misfolding and fibril formation of phosphorylated ␣-syn isoforms were detected earlier, although the fibrils remained phosphatase-and protease-sensitive. Membrane binding of ␣-syn monomers was differentially affected by phosphorylation depending on the Parkinson disease-linked mutation. WT ␣-syn binding to presynaptic membranes was not affected by phosphorylation, whereas A30P ␣-syn binding was greatly increased, and A53T ␣-syn was slightly lower, implicating distal effects of the carboxyl-on amino-terminal membrane binding. Endocytic vesicle-mediated internalization of pre-formed fibrils into non-neuronal cells and dopaminergic neurons matched the efficacy of ␣-syn membrane binding. Finally, the disruption of internalized vesicle membranes was enhanced by the phosphorylated ␣-syn isoforms, a potential means for misfolded extracellular or lumenal ␣-syn to access cytosolic ␣-syn. Our results suggest that the threshold for vesicle permeabilization is evident even at low levels of ␣-syn internalization and are relevant to therapeutic strategies to reduce intercellular propagation of ␣-syn misfolding.

Background: ␣-Synuclein is known to undergo exchange between membrane and cytosolic compartments. Results: ␣-Synuclein interacts with GTP-bound Rab3a on synaptic vesicles, and its dissociation is mediated by GDI/Hsp90. Conclusion: ␣-Synuclein's membrane association and dissociation cycle is linked to synaptic activity by the Rab3a recycling machinery. Significance: Significance: Impairments to ␣-synuclein interactions with vesicles and with the Rab3a recycling machinery may affect neurodegeneration.

Many lines of evidence indicate the importance of the Rho family guanine nucleotide triphosphatases (GTPases) in directing axon extension and guidance. The signaling networks that involve these proteins regulate actin cytoskeletal dynamics in navigating neuronal growth cones. However, the intricate patterns that regulate Rho GTPase activation and signaling are not yet fully defined. Activity and subcellular localization of the Rho GTPases are regulated by post-translational modification. The addition of a geranylgeranyl group to the carboxy (C-) terminus targets Rho GTPases to the plasma membrane and promotes their activation by facilitating interaction with guanine nucleotide exchange factors and allowing sequestering by association with guanine dissociation inhibitors. However, it is unclear how these modifications affect neurite extension or how subcellular localization alters signaling from the classical Rho GTPases (RhoA, Rac1, and Cdc42). Here, we review recent data addressing this issue and propose that Rho GTPase geranylgeranylation regulates outgrowth.

Inhibitors of the mevalonate pathway, including the highly prescribed statins, reduce the production of cholesterol and isoprenoids such as geranylgeranyl pyrophosphates. The Rho family of small guanine triphosphatases (GTPases) requires isoprenylation, specifically geranylgeranylation, for activation. Because Rho GTPases are primary regulators of actin filament rearrangements required for process extension, neurite arborization and synaptic plasticity, statins may affect cognition or recovery from nervous system injury. Here, we assessed how manipulating geranylgeranylation affects neurite initiation, elongation and branching in neuroblastoma growth cones. Treatment with the statin, lovastatin (20 μM) decreased measures of neurite initiation by 17.0% to 19.0% when a source of cholesterol was present and increased neurite branching by 4.03 to 9.54 fold (regardless of exogenous cholesterol). Neurite elongation was increased by treatment with lovastatin only in cholesterol-free culture conditions. Treatment with lovastatin decreased growth cone actin filament content by up to 24.3%. In all cases, co-treatment with the prenylation precursor, geranylgeraniol (10 μM), reversed the effect of lovastatin. In prior work, statin effects on outgrowth were linked to modulating the actin depolymerizing factor, cofilin. In our assays, treatment with lovastatin or geranylgeraniol decreased cofilin phosphorylation in whole cell lysates. However, lovastatin increased cofilin phosphorylation in cell bodies and decreased it in growth cones, indicating differential regulation in specific cell regions. Together, we interpret these data to suggest that protein geranylgeranylation likely regulates growth cone actin filament content and subsequent neurite outgrowth through mechanisms that also affect actin nucleation and polymerization.

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