High-resolution phase-shifting Ronchi test.

A method adding phase-shifting capacity in two mutually perpendicular axes to the Ronchi test is presented in this work. The phase of the object with the position of the reflected ray on the grating was identified and used to solve the equation of reflection in two orthogonal directions. In this manner, the test-surface figure was obtained. The procedure was demonstrated with an inverse qualitative test and a direct, quantitative test. Both tests give results comparable to Fizeau interferometry, with the precision of the order of 25 nm peak to valley. This technique is a good alternative to interferometry because, in addition to its inherent high-resolution, it is possible to obtain the radius of curvature and conic constant, which interferometers, requiring auxiliary optics, cannot provide. This method also has a high dynamic range and is not as susceptible to vibrations or turbulence. The setup can be built with low-cost, readily available components, is easily aligned, uses a white light source, and can be made very lightweight and compact, which makes it ideal for mounting onto existing polishing machines in any optical fabrication workshop, to perform in situ surface metrology.

α-Synuclein antisense oligonucleotides as a disease-modifying therapy for Parkinson's disease.

Parkinson's disease (PD) is a prevalent neurodegenerative disease with no approved disease-modifying therapies. Multiplications, mutations, and single nucleotide polymorphisms in the SNCA gene, encoding α-synuclein (aSyn) protein, either cause or increase risk for PD. Intracellular accumulations of aSyn are pathological hallmarks of PD. Taken together, reduction of aSyn production may provide a disease-modifying therapy for PD. We show that antisense oligonucleotides (ASOs) reduce production of aSyn in rodent preformed fibril (PFF) models of PD. Reduced aSyn production leads to prevention and removal of established aSyn pathology and prevents dopaminergic cell dysfunction. In addition, we address the translational potential of the approach through characterization of human SNCA-targeting ASOs that efficiently suppress the human SNCA transcript in vivo. We demonstrate broad activity and distribution of the human SNCA ASOs throughout the nonhuman primate brain and a corresponding decrease in aSyn cerebral spinal fluid (CSF) levels. Taken together, these data suggest that, by inhibiting production of aSyn, it may be possible to reverse established pathology; thus, these data support the development of SNCA ASOs as a potential disease-modifying therapy for PD and related synucleinopathies.

TRIM11 Prevents and Reverses Protein Aggregation and Rescues a Mouse Model of Parkinson's Disease.

Neurodegenerative diseases are characterized by the formation and propagation of protein aggregates, especially amyloid fibrils. However, what normally suppresses protein misfolding and aggregation in metazoan cells remains incompletely understood. Here, we show that TRIM11, a member of the metazoan tripartite motif (TRIM) family, both prevents the formation of protein aggregates and dissolves pre-existing protein deposits, including amyloid fibrils. These molecular chaperone and disaggregase activities are ATP independent. They enhance folding and solubility of normal proteins and cooperate with TRIM11 SUMO ligase activity to degrade aberrant proteins. TRIM11 abrogates α-synuclein fibrillization and restores viability in cell models of Parkinson's disease (PD). Intracranial adeno-associated viral delivery of TRIM11 mitigates α-synuclein-mediated pathology, neurodegeneration, and motor impairments in a PD mouse model. Other TRIMs can also function as ATP-independent molecular chaperones and disaggregases. Thus, we define TRIMs as a potent and multifunctional protein quality-control system in metazoa, which might be applied to treat neurodegenerative diseases.

Alpha-synuclein is a DNA binding protein that modulates DNA repair with implications for Lewy body disorders.

Alpha-synuclein is a presynaptic protein that forms abnormal cytoplasmic aggregates in Lewy body disorders. Although nuclear alpha-synuclein localization has been described, its function in the nucleus is not well understood. We demonstrate that alpha-synuclein modulates DNA repair. First, alpha-synuclein colocalizes with DNA damage response components within discrete foci in human cells and mouse brain. Removal of alpha-synuclein in human cells leads to increased DNA double-strand break (DSB) levels after bleomycin treatment and a reduced ability to repair these DSBs. Similarly, alpha-synuclein knock-out mice show increased neuronal DSBs that can be rescued by transgenic reintroduction of human alpha-synuclein. Alpha-synuclein binds double-stranded DNA and helps to facilitate the non-homologous end-joining reaction. Using a new, in vivo imaging approach that we developed, we find that serine-129-phosphorylated alpha-synuclein is rapidly recruited to DNA damage sites in living mouse cortex. We find that Lewy inclusion-containing neurons in both mouse model and human-derived patient tissue demonstrate increased DSB levels. Based on these data, we propose a model whereby cytoplasmic aggregation of alpha-synuclein reduces its nuclear levels, increases DSBs, and may contribute to programmed cell death via nuclear loss-of-function. This model could inform development of new treatments for Lewy body disorders by targeting alpha-synuclein-mediated DNA repair mechanisms.

Induction of the Immunoproteasome Subunit Lmp7 Links Proteostasis and Immunity in α-Synuclein Aggregation Disorders.

Accumulation of aggregated α-synuclein into Lewy bodies is thought to contribute to the onset and progression of dopaminergic neuron degeneration in Parkinson's disease (PD) and related disorders. Although protein aggregation is associated with perturbation of proteostasis, how α-synuclein aggregation affects the brain proteome and signaling remains uncertain. In a mouse model of α-synuclein aggregation, 6% of 6215 proteins and 1.6% of 8183 phosphopeptides changed in abundance, indicating conservation of proteostasis and phosphorylation signaling. The proteomic analysis confirmed changes in abundance of proteins that regulate dopamine synthesis and transport, synaptic activity and integrity, and unearthed changes in mRNA binding, processing and protein translation. Phosphorylation signaling changes centered on axonal and synaptic cytoskeletal organization and structural integrity. Proteostatic responses included a significant increase in the levels of Lmp7, a component of the immunoproteasome. Increased Lmp7 levels and activity were also quantified in postmortem human brains with PD and dementia with Lewy bodies. Functionally, the immunoproteasome degrades α-synuclein aggregates and generates potentially antigenic peptides. Expression and activity of the immunoproteasome may represent testable targets to induce adaptive responses that maintain proteome integrity and modulate immune responses in protein aggregation disorders.

Differential α-synuclein expression contributes to selective vulnerability of hippocampal neuron subpopulations to fibril-induced toxicity.

The accumulation of misfolded α-synuclein (aSyn) and neuron loss define several neurodegenerative disorders including Parkinson's disease (PD) and dementia with Lewy bodies (DLB). However, the precise relationship between pathology and neurotoxicity and why these processes disproportionately affect certain neuron subpopulations are poorly understood. We show here that Math2-expressing neurons in the hippocampal Cornu ammonis (CA), a region significantly affected by aSyn pathology in advanced PD and DLB, are highly susceptible to pathological seeding with pre-formed fibrils (PFFs), in contrast to dentate gyrus neurons, which are relatively spared. Math2+ neurons also exhibited more rapid and severe cell loss in both in vitro and in vivo models of synucleinopathy. Toxicity resulting from PFF exposure was dependent on endogenous aSyn and could be attenuated by N-acetyl-cysteine through a glutathione-dependent process. Moreover, aSyn expression levels strongly correlate with relative vulnerability among hippocampal neuron subtypes of which Math2+ neurons contained the highest amount. Consistent with this, antisense oligonucleotide (ASO)-mediated knockdown of aSyn reduced the neuronal pathology in a time-dependent manner. However, significant neuroprotection was observed only with early ASO intervention and a substantial reduction of aSyn pathology, indicating toxicity occurs after a critical threshold of pathological burden is exceeded in vulnerable neurons. Together, our findings reveal considerable heterogeneity in endogenous aSyn levels among hippocampal neurons and suggest that this may contribute to the selective vulnerability observed in the context of synucleinopathies.

Bilaterian Giant Ankyrins Have a Common Evolutionary Origin and Play a Conserved Role in Patterning the Axon Initial Segment.

In vertebrate neurons, the axon initial segment (AIS) is specialized for action potential initiation. It is organized by a giant 480 Kd variant of ankyrin G (AnkG) that serves as an anchor for ion channels and is required for a plasma membrane diffusion barrier that excludes somatodendritic proteins from the axon. An unusually long exon required to encode this 480Kd variant is thought to have been inserted only recently during vertebrate evolution, so the giant ankyrin-based AIS scaffold has been viewed as a vertebrate adaptation for fast, precise signaling. We re-examined AIS evolution through phylogenomic analysis of ankyrins and by testing the role of ankyrins in proximal axon organization in a model multipolar Drosophila neuron (ddaE). We find giant isoforms of ankyrin in all major bilaterian phyla, and present evidence in favor of a single common origin for giant ankyrins and the corresponding long exon in a bilaterian ancestor. This finding raises the question of whether giant ankyrin isoforms play a conserved role in AIS organization throughout the Bilateria. We examined this possibility by looking for conserved ankyrin-dependent AIS features in Drosophila ddaE neurons via live imaging. We found that ddaE neurons have an axonal diffusion barrier proximal to the cell body that requires a giant isoform of the neuronal ankyrin Ank2. Furthermore, the potassium channel shal concentrates in the proximal axon in an Ank2-dependent manner. Our results indicate that the giant ankyrin-based cytoskeleton of the AIS may have evolved prior to the radiation of extant bilaterian lineages, much earlier than previously thought.

Diffraction-limited step-zoom telescope by image restoration.

The design of a step-zoom telescope and its ability to achieve a diffraction-limited performance is explored. The basic idea is to include digital postprocessing to compensate for changes in the modulation transfer function of the system, assuming the knowledge of the range to the object. The instrument is conformed of a two-mirror telescope, two lenses, and a detector. High-quality images and a zoom telescope that ranges from 22 to 61 f-number is achieved by moving the primary mirror and two lenses. The preliminary calculations for the design process and a simulation that shows the performance of the step-zoom telescope are described.

Bent out of shape: α-Synuclein misfolding and the convergence of pathogenic pathways in Parkinson's disease.

Protein inclusions made up primarily of misfolded α-synuclein (α-Syn) are the hallmark of a set of disorders known as synucleinopathies, most notably Parkinson's disease (PD). It is becoming increasingly appreciated that α-Syn misfolding can spread to anatomically connected regions in a prion-like manner. The protein aggregates that ensue are correlated with neurodegeneration in the various yet select neuronal populations that are affected. Recent advances have begun to shed light on the spreading and toxicity mechanisms that may be occurring in PD. Several key emerging themes are arising from this work suggesting that α-Syn mediated neurodegeneration is due to a combination of relative α-Syn expression level, connectivity to affected brain regions, and intrinsic vulnerability to pathology.

HE3286 reduces axonal loss and preserves retinal ganglion cell function in experimental optic neuritis.

PURPOSE: Optic nerve inflammation, demyelination, and axonal loss are all prominent features of optic neuritis. While corticosteroids hasten visual recovery in optic neuritis, no treatment improves final visual outcomes. HE3286 (17α-ethynyl-5-androstene-3ß,7ß,17ß-triol), a synthetic derivative of a natural steroid, ß-AET (5-androstene-3ß,7ß,17ß-triol), exerts anti-inflammatory effects in several disease models and has purported neuroprotective effects as well. HE3286's ability to suppress optic neuritis was examined in experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. METHODS: Experimental autoimmune encephalomyelitis was induced in C57/BL6 mice. Mice were treated daily with intraperitoneal vehicle or 40 mg/kg HE3286. Visual function was assessed by optokinetic responses (OKR) at baseline and every 10 days until euthanasia at 40 days post immunization. Retinas and optic nerves were isolated. Inflammation (hematoxylin and eosin and Iba1 staining), demyelination (Luxol fast blue staining), and axonal loss (neurofilament staining) were assessed in optic nerve sections. Retinal ganglion cells (RGCs) were immunolabeled with Brn3a antibodies to quantify RGC survival. RESULTS: Progressive decreases in OKR occurred in vehicle-treated EAE mice, and HE3286 treatment reduced the level of this vision loss. HE3286 also attenuated the degree of inflammation, demyelination, and axonal loss in EAE optic nerves as compared to nerves from vehicle-treated EAE mice. Retinal ganglion cell loss that occurred in both vehicle- and HE3286-treated EAE mice was reduced in the temporal retinal quadrant of HE3286-treated mice. CONCLUSIONS: HE3286 suppresses inflammation, reduces demyelination and axonal loss, and promotes RGC survival during experimental optic neuritis. Importantly, HE3286 treatment also preserves some RGC function. Results suggest that HE3286 is a potential novel treatment for optic neuritis.

Common-pull, multiple-push, vacuum-activated telescope mirror cell.

A new concept for push-pull active optics is presented, where the push-force is provided by means of individual airbag type actuators and a common force in the form of a vacuum is applied to the entire back of the mirror. The vacuum provides the pull-component of the system, in addition to gravity. Vacuum is controlled as a function of the zenithal angle, providing correction for the axial component of the mirror's weight. In this way, the push actuators are only responsible for correcting mirror deformations, as well as for supporting the axial mirror weight at the zenith, allowing for a uniform, full dynamic-range behavior of the system along the telescope's pointing range. This can result in the ability to perform corrections of up to a few microns for low-order aberrations. This mirror support concept was simulated using a finite element model and was tested experimentally at the 2.12 m San Pedro Mártir telescope. Advantages such as stress-free attachments, lighter weight, large actuator area, lower system complexity, and lower required mirror-cell stiffness could make this a method to consider for future large telescopes.

HyDRa: polishing with a vortex.

We present a hydrodynamic, deterministic polishing tool (HyDRa) based on the fluid-jet polishing (FJP) principle. In contrast to other FJP methods, the polishing flux is accelerated with pressurized air and then expelled at high velocity, forming a radial, grazing abrasive pattern that exerts no net force of the tool on the surface to be polished, since the vacuum and thrust forces that are created at the tool's output balance each other out. The grazing effect minimizes microroughness, making it appropriate for finishing high-quality surfaces. The principle of operation as well as polishing results of a series of small etalon plates are presented.

HyDRa: polishing process convergence rate optimization.

In an effort to optimize the hydrodynamic radial (HyDRa) polishing process for applications where the amount of material that has to be removed implies long polishing times, we have developed a method to determine the optimum correction fraction that has to be made for a given error map, in terms of the level of determinism of the process, the number of iterations, and their associated polishing runs as well as run times.

Low-cost, phase-shifting mechanisms for Newton-type interferometers.

Using standard optical shop equipment, it is possible to implement simple, low-cost, phase-shifting Newton interferometers sufficiently accurate for surface evaluation. The simplification of the phase-shifting mechanism is compensated with image-processing algorithms that can deal with vibrations and uneven, nonsequential steps. The results are cross-compared with a Fizeau phase-shifting interferometer to verify the effectiveness of the method.

Deterministic convergence in iterative phase shifting.

Previous implementations of the iterative phase shifting method, in which the phase of a test object is computed from measurements using a phase shifting interferometer with unknown positions of the reference, do not provide an accurate way of knowing when convergence has been attained. We present a new approach to this method that allows us to deterministically identify convergence. The method is tested with a home-built Fizeau interferometer that measures optical surfaces polished to lambda/100 using the Hydra tool. The intrinsic quality of the measurements is better than 0.5 nm. Other possible applications for this technique include fringe projection or any problem where phase shifting is involved.

Geometric method to measure astigmatism aberration at astronomical telescopes.

A simple geometrical method to measure the aberration of astigmatism present in the wavefront that emerges from a telescope is presented. The method is based on the analysis of the external contour of the image of a slightly defocused star. An expression elliptical edge is obtained, which links the rms value of Z22 to the geometric parameters of the ellipse. This expression is tested as a function of introduced defocus and astigmatism aberration in telescopes of the San Pedro Martir Observatory. It is shown that the method gives comparable results to wavefront tests, being capable of measuring astigmatism values of approximately 60 nm and larger without the need for auxiliary optics.

Experimental results on piston detection by using the classical Ronchi test.

We present some experimental results of piston detection by using a classical Ronchi test in a segmented spherical surface. We compare the Ronchi fringes frequency of both segments (reference and under test). When the piston term is present at the segment under test, it is moved iteratively until we obtain a minimum piston error. When the surface is almost cophased, both segments have the same fringe frequency. The fringes of each segment are compared by correlating the intensity versus position in each segment. The Ronchi test is used at the same time as Shack interferometry; this second method is used as a reference to avoid segments tilts. With the Ronchi test we have detected experimentally a piston of the order of 45 nm (lambda/14 with lambda=632.8 nm). This result was compared with images of simulated Ronchigrams.

Water jet: a promising method for cutting optical glass.

We present an alternative method for cutting optical glass. It works with a high-pressure fluid, carring abrasive powder. This technique offers some advantages over conventional methods that use diamond abrasive covered wires or disks. We make a critical comparison between those two techniques, characterizing cuts with interferometric, polarimetric, and Ronchi testing. The main feature of the water-jet technique is that it allows surface of any shape, already polished, to be cut safely.

Optimal defocus distance for testing the 2.1 m telescope at San Pedro Mártir.

Within the preliminary development of a low-cost active optics system (AOS), computer simulations have been performed. The general purpose of simulations is to find an optimal scheme for the wavefront control of the primary mirror. We present results for the wavefront sensor of the AOS proposed for the 2.1 m telescope at San Pedro Mártir. The method presented here can be used for any other telescopes as well.

Static and dynamic removal rates of a new hydrodynamic polishing tool.

A new tool for hydrodynamic radial polishing, HyDra, allows for the local polishing of optical surfaces with a controllable wear rate. The results of the removal rate for different polisher types and sizes, applied air pressures for slurry expulsion, and tool height with respect to the working surface, are reported. We present a numerical analysis of the volumetric removal rate for the dynamic experiments as well as a comparison with a similar technique.