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.

Phosphorylation of ␣-synuclein (␣-syn) at Ser-129 is a hallmark of Parkinson disease and related synucleinopathies. However, the identity of the natural kinases and phosphatases responsible for regulating ␣-syn phosphorylation remain unknown. Here we demonstrate that three closely related members of the human Polo-like kinase (PLK) family (PLK1, PLK2, and PLK3) phosphorylate ␣-syn and ␤-syn specifically at Ser-129 and Ser-118, respectively. Unlike other kinases reported to partially phosphorylate ␣-syn at Ser-129 in vitro, phosphorylation by PLK2 and PLK3 is quantitative (>95% conversion). Only PLK1 and PLK3 phosphorylate ␤-syn at Ser-118, whereas no phosphorylation of ␥-syn was detected by any of the four PLKs (PLK1 to -4). PLK-mediated phosphorylation was greatly reduced in an isolated C-terminal fragment (residues 103-140) of ␣-syn, suggesting substrate recognition via the N-terminal repeats and/or the non-amyloid component domain of ␣-syn. PLKs specifically co-localized with phosphorylated Ser-129 (Ser(P)-129) ␣-syn in various subcellular compartments (cytoplasm, nucleus, and membranes) of mammalian cell lines and primary neurons as well as in ␣-syn transgenic mice, especially cortical brain areas involved in synaptic plasticity. Furthermore, we report that the levels of PLK2 are significantly increased in brains of Alzheimer disease and Lewy body disease patients. Taken together, these results provide biochemical and in vivo evidence of ␣-syn and ␤-syn phosphorylation by specific PLKs. Our results suggest a need for further studies to elucidate the potential role of PLK-syn interactions in the normal biology of these proteins as well as their involvement in the pathogenesis of Parkinson disease and other synucleinopathies.Increasing evidence suggests that phosphorylation may play an important role in the oligomerization and fibrillogenesis (1), Lewy body formation (1, 2) and neurotoxicity of ␣-synuclein (␣-syn) 5 in vivo (3). The majority of ␣-syn within Lewy bodies (LBs) in diseased human brains and animal models of Parkinson disease (PD) and related synucleinopathies is phosphorylated at Ser-129 (Ser(P)-129) (1, 2, 4 -7). Although recent studies support the notion that phosphorylation at Ser-129 is related to pathology and blocks ␣-syn fibrillization in vitro (8, 9), the exact mechanisms by which phosphorylation at Ser-129 modulates ␣-syn aggregation and toxicity in vivo remain elusive. Unraveling the role of phosphorylation in modulating the physiological and pathogenic activities of ␣-syn requires identification of the kinases and phosphatases involved in regulating its phosphorylation in vivo.Several kinases that phosphorylate ␣-syn at serine and tyrosine residues, primarily in its C-terminal region, have been identified using in vitro kinase assays and co-transfection studies. Casein kinase I and II, G-protein-coupled receptor kinases (GRK1, GRK2, GRK5, and GRK6), and calmodulin-dependent kinase II (10 -12) phosphorylate ␣-syn at Ser-129. Ser-87 is the only residue outside the C-terminal region report...

A novel mutation in the α-Synuclein (α-Syn) gene "G51D" was recently identified in two familial cases exhibiting features of Parkinson's disease (PD) and multiple system atrophy (MSA). In this study, we explored the impact of this novel mutation on the aggregation, cellular and biophysical properties of α-Syn, in an attempt to unravel how this mutant contributes to PD/MSA. Our results show that the G51D mutation significantly attenuates α-Syn aggregation in vitro. Moreover, it disrupts local helix formation in the presence of SDS, decreases binding to lipid vesicles C-terminal to the site of mutation and severely inhibits helical folding in the presence of acidic vesicles. When expressed in yeast, α-Syn(G51D) behaves similarly to α-Syn(A30P), as both exhibit impaired membrane association, form few inclusions and are non-toxic. In contrast, enhanced secreted and nuclear levels of the G51D mutant were observed in mammalian cells, as well as in primary neurons, where α-Syn(G51D) was enriched in the nuclear compartment, was hyper-phosphorylated at S129 and exacerbated α-Syn-induced mitochondrial fragmentation. Finally, post-mortem human brain tissues of α-Syn(G51D) cases were examined, and revealed only partial colocalization with nuclear membrane markers, probably due to post-mortem tissue delay and fixation. These findings suggest that the PD-linked mutations may cause neurodegeneration via different mechanisms, some of which may be independent of α-Syn aggregation.

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.

Cone-rod degeneration (CRD) belongs to the disease spectrum of retinal degenerations, a group of hereditary disorders characterized by an extreme clinical and genetic heterogeneity. It mainly differentiates from other retinal dystrophies, and in particular from the more frequent disease retinitis pigmentosa, because cone photoreceptors degenerate at a higher rate than rod photoreceptors, causing severe deficiency of central vision. After exome analysis of a cohort of individuals with CRD, we identified biallelic mutations in the orphan gene CEP78 in three subjects from two families: one from Greece and another from Sweden. The Greek subject, from the island of Crete, was homozygous for the c.499+1G>T (IVS3+1G>T) mutation in intron 3. The Swedish subjects, two siblings, were compound heterozygotes for the nearby mutation c.499+5G>A (IVS3+5G>A) and for the frameshift-causing variant c.633delC (p.Trp212Glyfs(∗)18). In addition to CRD, these three individuals had hearing loss or hearing deficit. Immunostaining highlighted the presence of CEP78 in the inner segments of retinal photoreceptors, predominantly of cones, and at the base of the primary cilium of fibroblasts. Interaction studies also showed that CEP78 binds to FAM161A, another ciliary protein associated with retinal degeneration. Finally, analysis of skin fibroblasts derived from affected individuals revealed abnormal ciliary morphology, as compared to that of control cells. Altogether, our data strongly suggest that mutations in CEP78 cause a previously undescribed clinical entity of a ciliary nature characterized by blindness and deafness but clearly distinct from Usher syndrome, a condition for which visual impairment is due to retinitis pigmentosa.

Background:Little is known about the effect of PD-linked mutations on ␣-synuclein (␣-syn) phosphorylation. Results: The E46K mutation increases ␣-syn phosphorylation at Ser-129 and influences its subcellular localization in vivo. Conclusion: The E46K mutation alters ␣-syn localization and post-translational modifications. Significance: PD-linked ␣-syn mutations may contribute to PD via different mechanisms.

Purposewas to create an in vitro model of human retinal detachment (RD) to study the mechanisms of photoreceptor death.MethodsHuman retinas were obtained through eye globe donations for research purposes and cultivated as explants. Cell death was investigated in retinas with (control) and without retinal pigment epithelium (RPE) cells to mimic RD. Tissues were studied at different time points and immunohistological analyses for TUNEL, Cleaved caspase3, AIF, CDK4 and the epigenetic mark H3K27me3 were performed. Human and monkey eye globes with retinal detachment served as controls.ResultsThe number of TUNEL-positive cells, compared between 1 and 7 days, increased with time in both retinas with RPE (from 1.2 ± 0.46 to 8 ± 0.89, n = 4) and without RPE (from 2.6 ± 0.73 to 16.3 ± 1.27, p < 0.014). In the group without RPE, cell death peaked at day 3 (p = 0.014) and was high until day 7. Almost no Cleaved-Caspase3 signal was observed, whereas a transient augmentation at day 3 of AIF-positive cells was observed to be about 10-fold in comparison to the control group (n = 2). Few CDK4-positive cells were found in both groups, but significantly more in the RD group at day 7 (1.8 ± 0.24 vs. 4.7 ± 0.58, p = 0.014). The H3K27me3 mark increased by 7-fold after 5 days in the RD group (p = 0.014) and slightly decreased at day 7 and was also observed to be markedly increased in human and monkey detached retina samples.ConclusionAIF expression coincides with the first peak of cell death, whereas the H3K27me3 mark increases during the cell death plateau, suggesting that photoreceptor death is induced by different successive pathways after RD. This in vitro model should permit the identification of neuroprotective drugs with clinical relevance.

Inherited retinal dystrophies (IRD) are due to various gene mutations. Each mutated gene instigates a specific cell homeostasis disruption, leading to a modification in gene expression and retinal degeneration. We previously demonstrated that the polycomb-repressive complex-1 (PRC1) markedly contributes to the cell death process. To better understand these mechanisms, we herein study the role of PRC2, specifically EZH2, which often initiates the gene inhibition by PRC1. We observed that the epigenetic mark H3K27me3 generated by EZH2 was progressively and strongly expressed in some individual photoreceptors and that the H3K27me3-positive cell number increased before cell death. H3K27me3 accumulation occurs between early (accumulation of cGMP) and late (CDK4 expression) events of retinal degeneration. EZH2 hyperactivity was observed in four recessive and two dominant mouse models of retinal degeneration, as well as two dog models and one IRD patient. Acute pharmacological EZH2 inhibition by intravitreal injection decreased the appearance of H3K27me3 marks and the number of TUNEL-positive cells revealing that EZH2 contributes to the cell death process. Finally, we observed that the absence of the H3K27me3 mark is a biomarker of gene therapy treatment efficacy in XLRPA2 dog model. PRC2 and PRC1 are therefore important actors in the degenerative process of multiple forms of IRD.