Mutations in CEP78 Cause Cone-Rod Dystrophy and Hearing Loss Associated with Primary-Cilia Defects.

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

Increased α-synuclein phosphorylation and nitration in the aging primate substantia nigra

Post-translational modifications of α-synuclein occur in the brain of patients affected by Parkinson's disease and other α-synucleinopathies, as indicated by the accumulation of Lewy inclusions containing phosphorylated (at serine 129) and nitrated α-synuclein. Here we found that phospho-Ser 129 and nitrated α-synuclein are also formed within dopaminergic neurons of the monkey substantia nigra as a result of normal aging. Dopaminergic cell bodies immunoreactive for phospho-Ser 129 and nitrated α-synuclein were rarely seen in adult mature animals but became significantly more frequent in the substantia nigra of old primates. Dual labeling with antibodies against phospho-Ser 129 and nitrated α-synuclein revealed only limited colocalization and mostly stained distinct sub-populations of dopaminergic neurons. Age-related elevations of modified protein paralleled an increase in the number of neurons immunoreactive for unmodified α-synuclein, supporting a relationship between higher levels of normal protein and enhanced phosphorylation/nitration. Other mechanisms were also identified that likely contribute to α-synuclein modifications. In particular, increased expression of Polo-like kinase 2 within neurons of older animals could contribute to phospho-Ser 129 α-synuclein production. Data also indicate that a pro-oxidant environment characterizes older neurons and favors α-synuclein nitration. Aging is an unequivocal risk factor for human α-synucleinopathies. These findings are consistent with a mechanistic link between aging, α-synuclein abnormalities and enhanced vulnerability to neurodegenerative processes

α-Synuclein in central nervous system and from erythrocytes, mammalian cells, and Escherichia coli exists predominantly as disordered monomer

Since the discovery and isolation of α-synuclein (α-syn) from human brains, it has been widely accepted that it exists as an intrinsically disordered monomeric protein. Two recent studies suggested that α-syn produced in Escherichia coli or isolated from mammalian cells and red blood cells exists predominantly as a tetramer that is rich in α-helical structure (Bartels, T., Choi, J. G., and Selkoe, D. J. (2011) Nature 477, 107-110; Wang, W., Perovic, I., Chittuluru, J., Kaganovich, A., Nguyen, L. T. T., Liao, J., Auclair, J. R., Johnson, D., Landeru, A., Simorellis, A. K., Ju, S., Cookson, M. R., Asturias, F. J., Agar, J. N., Webb, B. N., Kang, C., Ringe, D., Petsko, G. A., Pochapsky, T. C., and Hoang, Q. Q. (2011) Proc. Natl. Acad. Sci. 108, 17797-17802). However, it remains unknown whether or not this putative tetramer is the main physiological form of α-syn in the brain. In this study, we investigated the oligomeric state of α-syn in mouse, rat, and human brains. To assess the conformational and oligomeric state of native α-syn in complex mixtures, we generated α-syn standards of known quaternary structure and conformational properties and compared the behavior of endogenously expressed α-syn to these standards using native and denaturing gel electrophoresis techniques, size-exclusion chromatography, and an oligomer-specific ELISA. Our findings demonstrate that both human and rodent α-syn expressed in the central nervous system exist predominantly as an unfolded monomer. Similar results were observed when human α-syn was expressed in mouse and rat brains as well as mammalian cell lines (HEK293, HeLa, and SH-SY5Y). Furthermore, we show that α-syn expressed in E. coli and purified under denaturing or nondenaturing conditions, whether as a free protein or as a fusion construct with GST, is monomeric and adopts a disordered conformation after GST removal. These results do not rule out the possibility that α-syn becomes structured upon interaction with other proteins and/or biological membranes

Post translational changes to α-synuclein control iron and dopamine trafficking : a concept for neuron vulnerability in Parkinson's disease

Parkinson's disease is a multifactorial neurodegenerative disorder, the aetiology of which remains elusive. The primary clinical feature of progressively impaired motor control is caused by a loss of midbrain substantia nigra dopamine neurons that have a high α-synuclein (α-syn) and iron content. α-Syn is a neuronal protein that is highly modified post-translationally and central to the Lewy body neuropathology of the disease. This review provides an overview of findings on the role post translational modifications to α-syn have in membrane binding and intracellular vesicle trafficking. Furthermore, we propose a concept in which acetylation and phosphorylation of α-syn modulate endocytic import of iron and vesicle transport of dopamine during normal physiology. Disregulated phosphorylation and oxidation of α-syn mediate iron and dopamine dependent oxidative stress through impaired cellular location and increase propensity for α-syn aggregation. The proposition highlights a connection between α-syn, iron and dopamine, three pathological components associated with disease progression in sporadic Parkinson's disease

Evaluating the relationship between amyloid-β and α-synuclein phosphorylated at Ser129 in dementia with Lewy bodies and Parkinson’s disease

INTRODUCTION: Lewy body and Alzheimer-type pathologies often co-exist. Several studies suggest a synergistic relationship between amyloid-β (Aβ) and α-synuclein (α-syn) accumulation. We have explored the relationship between Aβ accumulation and the phosphorylation of α-syn at serine-129 (pSer129 α-syn), in post-mortem human brain tissue and in SH-SY5Y neuroblastoma cells transfected to overexpress human α-syn. METHODS: We measured levels of Aβ40, Aβ42, α-syn and pSer129 α-syn by sandwich enzyme-linked immunosorbent assay, in soluble and insoluble fractions of midfrontal, cingulate and parahippocampal cortex and thalamus, from cases of Parkinson’s disease (PD) with (PDD; n = 12) and without dementia (PDND; n = 23), dementia with Lewy bodies (DLB; n = 10) and age-matched controls (n = 17). We also examined the relationship of these measurements to cognitive decline, as measured by time-to-dementia and the mini-mental state examination (MMSE) score in the PD patients, and to Braak tangle stage. RESULTS: In most brain regions, the concentration of insoluble pSer129 α-syn correlated positively, and soluble pSer129 α-syn negatively, with the levels of soluble and insoluble Aβ. Insoluble pSer129 α-syn also correlated positively with Braak stage. In most regions, the levels of insoluble and soluble Aβ and the proportion of insoluble α-syn that was phosphorylated at Ser129 were significantly higher in the PD and DLB groups than the controls, and higher in the PDD and DLB groups than the PDND brains. In PD, the MMSE score correlated negatively with the level of insoluble pSer129 α-syn. Exposure of SH-SY5Y cells to aggregated Aβ42 significantly increased the proportion of α-syn that was phosphorylated at Ser129 (aggregated Aβ40 exposure had a smaller, non-significant effect). CONCLUSIONS: Together, these data show that the concentration of pSer129 α-syn in brain tissue homogenates is directly related to the level of Aβ and Braak tangle stage, and predicts cognitive status in Lewy body diseases. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13195-014-0077-y) contains supplementary material, which is available to authorized users

Parkinson disease mutant E46K enhances alpha-synuclein phosphorylation in mammalian cell lines, in yeast, and in vivo.

Although alpha-synuclein (alpha-syn) phosphorylation has been considered as a hallmark of sporadic and familial Parkinson disease (PD), little is known about the effect of PD-linked mutations on alpha-syn phosphorylation. In this study, we investigated the effects of the A30P, E46K, and A53T PD-linked mutations on alpha-syn phosphorylation at residues Ser-87 and Ser-129. Although the A30P and A53T mutants slightly affected Ser(P)-129 levels compared with WT alpha-syn, the E46K mutation significantly enhanced Ser-129 phosphorylation in yeast and mammalian cell lines. This effect was not due to the E46K mutant being a better kinase substrate nor due to alterations in endogenous kinase levels, but was mostly linked with enhanced nuclear and endoplasmic reticulum accumulation. Importantly, lentivirus-mediated overexpression in mice also showed enhanced Ser-129 phosphorylation of the E46K mutant compared to WT alpha-syn, thus providing in vivo validation of our findings. Altogether, our findings suggest that the different PD-linked mutations may contribute to PD pathogenesis via different mechanisms

An in vitro Model of Human Retinal Detachment Reveals Successive Death Pathway Activations.

was to create an in vitro model of human retinal detachment (RD) to study the mechanisms of photoreceptor death. Human 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. The 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. AIF 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