Parkinson's disease-associated alpha-synuclein is more fibrillogenic than beta- and gamma-synuclein and cannot cross-seed its homologs.

Parkinson's disease (PD) is a neurodegenerative disorder that is pathologically characterized by the presence of intracytoplasmic Lewy bodies. Recently, two point mutations in alpha-synuclein were found to be associated with familial PD, but as of yet no mutations have been described in the homologous genes beta- and gamma-synuclein. alpha-Synuclein forms the major fibrillar component of Lewy bodies, but these do not stain for beta- or gamma-synuclein. This result is very surprising, given the extent of sequence conservation and the high similarity in expression and subcellular localization, in particular between alpha- and beta-synuclein. Here we compare in vitro fibrillogenesis of all three purified synucleins. We show that fresh solutions of alpha-, beta-, and gamma- synuclein show the same natively unfolded structure. While over time alpha-synuclein forms the previously described fibrils, no fibrils could be detected for beta- and gamma-synuclein under the same conditions. Most importantly, beta- and gamma-synuclein could not be cross-seeded with alpha-synuclein fibrils. However, under conditions that drastically accelerate aggregation, gamma-synuclein can form fibrils with a lag phase roughly three times longer than alpha-synuclein. These results indicate that beta- and gamma-synuclein are intrinsically less fibrillogenic than alpha-synuclein and cannot form mixed fibrils with alpha-synuclein, which may explain why they do not appear in the pathological hallmarks of PD, although they are closely related to alpha-synuclein and are also abundant in brain.

Both familial Parkinson's disease mutations accelerate alpha-synuclein aggregation.

Parkinson's disease (PD) is a neurodegenerative disorder that is pathologically characterized by the presence of intracytoplasmic Lewy bodies, the major component of which are filaments consisting of alpha-synuclein. Two recently identified point mutations in alpha-synuclein are the only known genetic causes of PD, but their pathogenic mechanism is not understood. Here we show that both wild type and mutant alpha-synuclein form insoluble fibrillar aggregates with antiparallel beta-sheet structure upon incubation at physiological temperature in vitro. Importantly, aggregate formation is accelerated by both PD-linked mutations. Under the experimental conditions, the lag time for the formation of precipitable aggregates is about 280 h for the wild type protein, 180 h for the A30P mutant, and only 100 h for the A53T mutant protein. These data suggest that the formation of alpha-synuclein aggregates could be a critical step in PD pathogenesis, which is accelerated by the PD-linked mutations.

The native and the heat-induced denatured states of alpha-chymotrypsinogen A: thermodynamic and spectroscopic studies.

We report the first protein phase-diagram characterized by a combination of volumetric, calorimetric, and spectroscopic techniques. More specifically, we use ultrasonic velocimetry, densimetry, and differential scanning calorimetry, in conjunction with UV absorbance and CD spectroscopy to detect and to characterize the conformational transitions of alpha-chymotrypsinogen A as a function of both pH and temperature. As judged by the CD spectra, we find that, at room temperature, the protein remains in the native state over the entire pH range investigated (pH 1 to 10). The melting profiles of the native state reveal three distinct pH domains in which protein denaturation produces different final states. Below pH 3.1, we find the heat-induced denatured state of the protein to be molten globule (MG), lacking the native-like tertiary structure, while exhibiting significant secondary structural elements. At neutral and alkaline pH, we find the heat-induced denatured state to be unfolded (U), lacking both tertiary and secondary structures, while being structurally similar to the urea-unfolded state. At intermediate pH values (between pH 3.1 and 7), we find the heat-induced denatured state to exhibit properties characteristic of both the MG and U states. Although at room temperature the protein remains native within the whole pH range studied (pH 1 to 10), our volumetric data reveal that the native state slightly "softens" at low pH, probably, due to pH-induced alterations in electrostatic forces causing the packing of the protein interior at low pH and room temperature to become less "tight". This softening of the protein at low pH is reflected in an 8% increase in the intrinsic compressibility, kM, of the protein "native" state. Our volumetric data also allow us to conclude that the heat-induced MG state retains a liquid-like, water-inaccessible core, with a volume that corresponds to about 40% of the solvent-inaccessible core of the native state. By contrast, our volumetric data are consistent with the U state of the protein being essentially unfolded, with the majority of its constituent atomic groups being solvent exposed and, therefore, strongly hydrated.

Intracavity adaptive optics. 3: Hsuria performance.

An experimental study has been conducted on the ability of intracavity adaptive optics to improve the performance of a half-symmetric unstable resonator with intracavity axicon (HSURIA) by correcting for aberrations from misfigure or misalignment of the intracavity conical optics. When the deformable mirror had adequate spatial frequency correction capability, near diffraction limited far-field performance was achieved, but secondary maxima lock-on by the multidither servo control severely limited the fully adaptive correction capability. A key observation from these experiments was the extreme sensitivity of the HSURIA to perturbations near the resonator optic axis. Variations as small as lambda/140 had a significant effect. This perturbation sensitivity is the principal cause of the secondary maxima lock-on problem since the open loop deformable mirror figure must be extremely close to the ideal figure to assure global maximum lock-on by the multidither servo control.

Intracavity adaptive optics. 1: Astigmatism correction performance.

A detailed experimental study has been conducted on adaptive optical control methodologies inside a laser resonator. A comparison is presented of several optimization techniques using a multidither zonal coherent optical adaptive technique system within a laser resonator for the correction of astigmatism. A dramatic performance difference is observed when optimizing on beam quality compared with optimizing on power-in-the-bucket. Experimental data are also presented on proper selection criteria for dither frequencies when controlling phase front errors. The effects of hardware limitations and design considerations on the performance of the system are presented, and general conclusions and physical interpretations on the results are made when possible.

Intracavity adaptive optics. 2: Tilt correction performance.

This paper continues the discussion of the detailed experimental study conducted on adaptive optical control methodologies inside a laser resonator.(1) In this paper we discuss the experimental results for the correction of tilt within a laser resonator utilizing a multidither zonal COAT system. The tilt aberration was introduced in three different ways: (1) static tilt was introduced while the COAT system was open loop and then the control loop closed, (2) static tilt was incrementally introduced while the COAT system control loop was closed loop (denoted as continuously closed loop), and (3) dynamic tilt was applied while the COAT system's control loop was closed. Experimental data are presented on the correction capability of the COAT system for each method of introduction of tilt over a large range of tilt angles and control loop configurations (open/ closed and continuously closed). Limitations of the system are discussed, and design recommendations and conclusions are provided.