Site-specific perturbations of alpha-synuclein fibril structure by the Parkinson's disease associated mutations A53T and E46K.

PMCID: PMC3591419This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Parkinson's disease (PD) is pathologically characterized by the presence of Lewy bodies (LBs) in dopaminergic neurons of the substantia nigra. These intracellular inclusions are largely composed of misfolded α-synuclein (AS), a neuronal protein that is abundant in the vertebrate brain. Point mutations in AS are associated with rare, early-onset forms of PD, although aggregation of the wild-type (WT) protein is observed in the more common sporadic forms of the disease. Here, we employed multidimensional solid-state NMR experiments to assess A53T and E46K mutant fibrils, in comparison to our recent description of WT AS fibrils. We made de novo chemical shift assignments for the mutants, and used these chemical shifts to empirically determine secondary structures. We observe significant perturbations in secondary structure throughout the fibril core for the E46K fibril, while the A53T fibril exhibits more localized perturbations near the mutation site. Overall, these results demonstrate that the secondary structure of A53T has some small differences from the WT and the secondary structure of E46K has significant differences, which may alter the overall structural arrangement of the fibrils

Plectin as a prognostic marker in non-metastatic oral squamous cell carcinoma

Background: Oral squamous cell carcinoma (OSCC) is associated with a poor 5-year survival rate. In general, patients diagnosed with small tumors have a fairly good prognosis, but some small tumors have an aggressive behavior leading to early death. There are at present no reliable prognostic biomarkers for oral cancers. Thus, to optimize treatment for the individual patient, there is a need for biomarkers that can predict tumor behavior. Method: In the present study the potential prognostic value of plectin was evaluated by a tissue microarray (TMA) based immunohistochemical analysis of primary tumor tissue obtained from a North Norwegian cohort of 115 patients diagnosed with OSCC. The expression of plectin was compared with clinicopathological variables and 5 year survival. Results: The statistical analysis revealed that low expression of plectin in the tumor cells predicted a favorable outcome for patients with non-metastatic disease (p = 0.008). Furthermore, the expression of plectin was found to correlate (p = 0.01) with the expression of uPAR, which we have previously found to be a potential prognostic marker for T1N0 tumors. Conclusions: Our results indicate that low expression of plectin predicts a favorable outcome for patients with non-metastatic OSCC and the expression level of plectin may therefore be used in the treatment stratification for patients with early stage disease

Marine migration and habitat use of anadromous brown trout Salmo trutta

The biology and ecology of anadromous brown trout Salmo trutta at sea is poorly understood. This study provided information on spatial and temporal distribution of sea trout in the ocean. The behaviour of 115 individuals (veteran migrants, 270-700 mm) was tracked by using acoustic telemetry in a fjord system during April-September in 2012-2013. Overall, fish spent 68% of their marine residence time close to river mouths (< 4 km). Most fish registrations (75%) were in near shore habitats, but pelagic areas were also used. The maximum migration distance of tagged fish was categorized as short (< 4 km from river mouth, 40% of fish), medium (4-~13 km, 18% of fish) or long (> ~13 km, 42% of fish). Long distance migrants had poorer body condition in spring prior to migration, used pelagic areas more often and returned earlier to freshwater than short and medium distance migrants. Marine residence time was 7-183 days, and was positively correlated to body length and smolt age, but negatively correlated to the date of sea entry

Sequential backbone-walk used to obtain the chemical shift assignments of A53T.

<p>Illustration of backbone connectivity through the NCACX (red), NCOCX (blue) and CAN(co)CX (black) spectra of residues A90-E83. In all cases the homonuclear mixing was achieved with 50 ms DARR.</p

TALOS+ predicted backbone dihedral angles ψ and φ as a function of residue number.

<p>(A) E46K and (B) A53T AS fibrils. Error bars based on the 10 best TALOS+ database matches. Representation of the secondary structure for WT (black), E46K (blue) and A53T (red) AS fibrils based on TALOS+ analysis (β-strands, arrows; turn or loop curved lines; not predicted, dashed lines). WT TALOS+ results based on those from Comellas <i>et al</i>.</p

Fibrils of mutant AS proteins prepared <i>in vitro</i> have a highly homogeneity and morphology similar to WT fibrils.

<p>(A) AS fibrils formation of (blue circles) E46K, (red triangles) A53T and (black squares) WT monitored by the Thioflavin T fluorescence assay. Error bars were determined from seven replicates for each. Measurements were normalized to the highest fluorescence intensity obtained across all samples. (B) Comparison of the electron micrographs of (top) E46K, (middle) A53T and (bottom) WT AS fibrils. ¹³C-¹³C 2D with 50 ms DARR mixing of (C) E46K and (D) A53T AS fibrils.</p

The A53T mutation causes minor perturbations throughout the AS fibril sequence.

<p>(A) Expansions of ¹³C-¹³C 2D spectral overlays (50 ms DARR mixing, 600 MHz ¹H frequency and 13.3 kHz MAS) of WT (black) and A53T (red) AS fibril samples. (B) Plot of the chemical shift perturbations between WT and A53T chemical shifts versus residue number. Residues labeled as (*) correspond to perturbations above 1 ppm. Residues labeled as (#) correspond to glycines. The mutation is indicated with (†). Error bars correspond to the chemical shift variations from one WT batch to another. WT chemical shift assignments were obtained from the BMRB #16939.</p

The E46K mutation causes major chemical shift perturbations throughout the AS fibril sequence.

<p>(A) Expansions of ¹³C-¹³C 2D spectral overlays (50 ms DARR mixing) of WT (black) and E46K (blue) AS fibril samples. (B) Plot of chemical shift perturbations between WT and E46K chemical shifts versus residue number. Residues labeled as (*) correspond to perturbations greater than 5 ppm (¹⁵N) or 3 ppm (¹³C). Residues labeled as (#) correspond to glycines. The mutation is indicated with (†). Error bars correspond to the chemical shift variations from one WT batch to another. WT chemical shift assignments were obtained from the BMRB #16939.</p