Background:The oligomeric state of ␣-syn in vivo remains unknown. Results: ␣-syn in the CNS and produced by erythrocytes, mammalian cells, and Escherichia coli exists predominantly as a disordered monomer. Conclusion: Native ␣-syn from various sources behaves as unstructured and monomeric. Significance: Stabilizing monomeric ␣-syn, lowering its levels, and/or inhibiting its fibrillization remain viable therapeutic strategies for Parkinson disease.

Increasing evidence suggests that the c-Abl protein tyrosine kinase could play a role in the pathogenesis of Parkinson's disease (PD) and other neurodegenerative disorders. c-Abl has been shown to regulate the degradation of two proteins implicated in the pathogenesis of PD, parkin and α-synuclein (α-syn). The inhibition of parkin's neuroprotective functions is regulated by c-Abl-mediated phosphorylation of parkin. However, the molecular mechanisms by which c-Abl activity regulates α-syn toxicity and clearance remain unknown. Herein, using NMR spectroscopy, mass spectrometry, in vitro enzymatic assays and cell-based studies, we established that α-syn is a bona fide substrate for c-Abl. In vitro studies demonstrate that c-Abl directly interacts with α-syn and catalyzes its phosphorylation mainly at tyrosine 39 (pY39) and to a lesser extent at tyrosine 125 (pY125). Analysis of human brain tissues showed that pY39 α-syn is detected in the brains of healthy individuals and those with PD. However, only c-Abl protein levels were found to be upregulated in PD brains. Interestingly, nilotinib, a specific inhibitor of c-Abl kinase activity, induces α-syn protein degradation via the autophagy and proteasome pathways, whereas the overexpression of α-syn in the rat midbrains enhances c-Abl expression. Together, these data suggest that changes in c-Abl expression, activation and/or c-Abl-mediated phosphorylation of Y39 play a role in regulating α-syn clearance and contribute to the pathogenesis of PD.

Background:A new SNCA mutation, H50Q, has been linked to familial Parkinson disease (PD). Results: The H50Q mutation does not affect the structure, membrane binding, or subcellular localization of ␣-Syn but alters its pathogenic properties. Conclusion: The H50Q mutation increases ␣-Syn aggregation, secretion, and extracellular toxicity. Significance: ␣-Syn mutations contribute to the pathogenesis of PD via multiple mechanisms.

Cortical dynamics can be imaged at high spatiotemporal resolution with voltage-sensitive dyes (VSDs) and calcium-sensitive dyes (CaSDs). We combined these two imaging techniques using epifluorescence optics together with whole cell recordings to measure the spatiotemporal dynamics of activity in the mouse somatosensory barrel cortex in vitro and in the supragranular layers in vivo. The two optical signals reported distinct aspects of cortical function. VSD fluorescence varied linearly with membrane potential and was dominated by subthreshold postsynaptic potentials, whereas the CaSD signal predominantly reflected local action potential firing. Combining VSDs and CaSDs allowed us to monitor the synaptic drive and the spiking activity of a given area at the same time in the same preparation. The spatial extent of the two dye signals was different, with VSD signals spreading further than CaSD signals, reflecting broad subthreshold and narrow suprathreshold receptive fields. Importantly, the signals from the dyes were differentially affected by pharmacological manipulations, stimulation strength, and depth of isoflurane anesthesia. Combined VSD and CaSD measurements can therefore be used to specify the temporal and spatial relationships between subthreshold and suprathreshold activity of the neocortex.

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.

Sirtuin genes have been associated with aging and are known to affect multiple cellular pathways. Sirtuin 2 was previously shown to modulate proteotoxicity associated with age-associated neurodegenerative disorders such as Alzheimer and Parkinson disease (PD). However, the precise molecular mechanisms involved remain unclear. Here, we provide mechanistic insight into the interplay between sirtuin 2 and α-synuclein, the major component of the pathognomonic protein inclusions in PD and other synucleinopathies. We found that α-synuclein is acetylated on lysines 6 and 10 and that these residues are deacetylated by sirtuin 2. Genetic manipulation of sirtuin 2 levels in vitro and in vivo modulates the levels of α-synuclein acetylation, its aggregation, and autophagy. Strikingly, mutants blocking acetylation exacerbate α-synuclein toxicity in vivo, in the substantia nigra of rats. Our study identifies α-synuclein acetylation as a key regulatory mechanism governing α-synuclein aggregation and toxicity, demonstrating the potential therapeutic value of sirtuin 2 inhibition in synucleinopathies.

Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, is considered an attractive therapeutic target in multiple inflammatory and autoimmune disorders. In addition to its known biologic activities, MIF can also function as a tautomerase. Several small molecules have been reported to be effective inhibitors of MIF tautomerase activity in vitro. Herein we employed a robust activity-based assay to identify different classes of novel inhibitors of the catalytic and biological activities of MIF. Several novel chemical classes of inhibitors of the catalytic activity of MIF with IC 50 values in the range of 0.2-15.5 M were identified and validated. The interaction site and mechanism of action of these inhibitors were defined using structure-activity studies and a battery of biochemical and biophysical methods. MIF inhibitors emerging from these studies could be divided into three categories based on their mechanism of action: 1) molecules that covalently modify the catalytic site at the N-terminal proline residue, Pro 1 ; 2) a novel class of catalytic site inhibitors; and finally 3) molecules that disrupt the trimeric structure of MIF. Importantly, all inhibitors demonstrated total inhibition of MIF-mediated glucocorticoid overriding and AKT phosphorylation, whereas ebselen, a trimer-disrupting inhibitor, additionally acted as a potent hyperagonist in MIF-mediated chemotactic migration. The identification of biologically active compounds with known toxicity, pharmacokinetic properties, and biological activities in vivo should accelerate the development of clinically relevant MIF inhibitors. Furthermore, the diversity of chemical structures and mechanisms of action of our inhibitors makes them ideal mechanistic probes for elucidating the structurefunction relationships of MIF and to further determine the role of the oligomerization state and catalytic activity of MIF in regulating the function(s) of MIF in health and disease. Macrophage migration inhibitory factor (MIF)2 was discovered in the 1960's as a T-lymphocyte product that inhibits the random migration of macrophages during delayed-type hypersensitivity responses (1, 2). Two decades later, a human MIF gene was cloned (3). Yet, the biological activity of MIF remained ambiguous until the production of bioactive MIF and anti-MIF antibodies (4). Various biological activities have been attributed to MIF, which is recognized as a major regulator of inflammation and a central upstream mediator of innate immune responses (5, 6). MIF has broad regulatory properties and is considered as a critical mediator of multiple disorders including inflammatory and autoimmune diseases such as rheumatoid arthritis (7, 8), glomerulonephritis (9, 10), diabetes (11), atherosclerosis (12), sepsis (13-15), asthma (16,17), and acute respiratory distress syndrome (18). Furthermore, recent studies have highlighted a role for MIF in tumorigenesis. Human cancer tissues, including skin, brain, breast, colon, prostate, and lung-derived tumors were observed to overexpress MIF, ...

Sorting, transport, and autophagic degradation of proteins in endosomes and lysosomes, as well as the division of these organelles, depend on scission of membrane-bound tubulo-vesicular carriers. How scission occurs is poorly understood, but family proteins bind these membranes. Here, we show that the yeast PROPPIN Atg18 carries membrane scission activity. Purified Atg18 drives tubulation and scission of giant unilamellar vesicles. Upon membrane contact, Atg18 folds its unstructured CD loop into an amphipathic α-helix that inserts into the bilayer. This allows the protein to engage its two lipid binding sites for PI3P and PI(3,5)P PI(3,5)P induces Atg18 oligomerization, which should concentrate lipid-inserted α-helices in the outer membrane leaflet and drive membrane tubulation and scission. The scission activity of Atg18 is compatible with its known roles in endo-lysosomal protein trafficking, autophagosome biogenesis, and vacuole fission. Key features required for membrane tubulation and scission by Atg18 are shared by other PROPPINs, suggesting that membrane scission may be a generic function of this protein family.