Structural aging of human neurons is opposite of the changes in schizophrenia.

Human mentality develops with age and is altered in psychiatric disorders, though their underlying mechanism is unknown. In this study, we analyzed nanometer-scale three-dimensional structures of brain tissues of the anterior cingulate cortex from eight schizophrenia and eight control cases. The distribution profiles of neurite curvature of the control cases showed a trend depending on their age, resulting in an age-correlated decrease in the standard deviation of neurite curvature (Pearson's r = -0.80, p = 0.018). In contrast to the control cases, the schizophrenia cases deviate upward from this correlation, exhibiting a 60% higher neurite curvature compared with the controls (p = 7.8 × 10-4). The neurite curvature also showed a correlation with a hallucination score (Pearson's r = 0.80, p = 1.8 × 10-4), indicating that neurite structure is relevant to brain function. This report is based on our 3D analysis of human brain tissues over a decade and is unprecedented in terms of the number of cases. We suggest that neurite curvature plays a pivotal role in brain aging and can be used as a hallmark to exploit a novel treatment of schizophrenia.

Schizophrenia-Mimicking Layers Outperform Conventional Neural Network Layers.

We have reported nanometer-scale three-dimensional studies of brain networks of schizophrenia cases and found that their neurites are thin and tortuous when compared to healthy controls. This suggests that connections between distal neurons are suppressed in microcircuits of schizophrenia cases. In this study, we applied these biological findings to the design of a schizophrenia-mimicking artificial neural network to simulate the observed connection alteration in the disorder. Neural networks that have a "schizophrenia connection layer" in place of a fully connected layer were subjected to image classification tasks using the MNIST and CIFAR-10 datasets. The results revealed that the schizophrenia connection layer is tolerant to overfitting and outperforms a fully connected layer. The outperformance was observed only for networks using band matrices as weight windows, indicating that the shape of the weight matrix is relevant to the network performance. A schizophrenia convolution layer was also tested using the VGG configuration, showing that 60% of the kernel weights of the last three convolution layers can be eliminated without loss of accuracy. The schizophrenia layers can be used instead of conventional layers without any change in the network configuration and training procedures; hence, neural networks can easily take advantage of these layers. The results of this study suggest that the connection alteration found in schizophrenia is not a burden to the brain, but has functional roles in brain performance.

Brain capillary structures of schizophrenia cases and controls show a correlation with their neuron structures.

Brain blood vessels constitute a micrometer-scale vascular network responsible for supply of oxygen and nutrition. In this study, we analyzed cerebral tissues of the anterior cingulate cortex and superior temporal gyrus of schizophrenia cases and age/gender-matched controls by using synchrotron radiation microtomography or micro-CT in order to examine the three-dimensional structure of cerebral vessels. Over 1 m of cerebral blood vessels was traced to build Cartesian-coordinate models, which were then used for calculating structural parameters including the diameter and curvature of the vessels. The distribution of vessel outer diameters showed a peak at 7-9 µm, corresponding to the diameter of the capillaries. Mean curvatures of the capillary vessels showed a significant correlation to the mean curvatures of neurites, while the mean capillary diameter was almost constant, independent of the cases. Our previous studies indicated that the neurites of schizophrenia cases are thin and tortuous compared to controls. The curved capillaries with a constant diameter should occupy a nearly constant volume, while neurons suffering from neurite thinning should have reduced volumes, resulting in a volumetric imbalance between the neurons and the vessels. We suggest that the observed structural correlation between neurons and blood vessels is related to neurovascular abnormalities in schizophrenia.

Structural diverseness of neurons between brain areas and between cases.

The cerebral cortex is composed of multiple cortical areas that exert a wide variety of brain functions. Although human brain neurons are genetically and areally mosaic, the three-dimensional structural differences between neurons in different brain areas or between the neurons of different individuals have not been delineated. Here we report a nanometer-scale geometric analysis of brain tissues of the superior temporal gyrus of schizophrenia and control cases. The results of the analysis and a comparison with results for the anterior cingulate cortex indicated that (1) neuron structures are significantly dissimilar between brain areas and that (2) the dissimilarity varies from case to case. The structural diverseness was mainly observed in terms of the neurite curvature that inversely correlates with the diameters of the neurites and spines. The analysis also revealed the geometric differences between the neurons of the schizophrenia and control cases. The schizophrenia cases showed a thin and tortuous neuronal network compared with the controls, suggesting that the neuron structure is associated with the disorder. The area dependency of the neuron structure and its diverseness between individuals should represent the individuality of brain functions.

Cutting-edge morphological studies of post-mortem brains of patients with schizophrenia and potential applications of X-ray nanotomography (nano-CT).

Kraepelin expected that the neuropathological hallmark of schizophrenia would be identified when he proposed the concept of dementia praecox 120 years ago. Although a variety of neuropathological findings have been reported since then, a consensus regarding the pathology of schizophrenia has not been established. The discrepancies have mainly been ascribed to limitations in the disease definition of schizophrenia that accompanies etiological heterogeneity and to the incompleteness of the visualization methodology and technology for biochemical analyses. However, macroscopic structural changes in the schizophrenia brain, such as volumetric changes of brain regions, must entail structural changes to cells composing the brain. This paper overviews neuropathology of schizophrenia and also summarizes recent application of synchrotron radiation nanotomography (nano-CT) to schizophrenia brain tissues. Geometric parameters of neurites determined from the 3-D nano-CT images of brain tissues indicated that the curvature of neurites in schizophrenia cases is significantly higher than that of controls. The schizophrenia case with the highest curvature carried a frameshift mutation in the glyoxalase 1 gene and exhibited treatment resistance. Controversies in the neuropathology of schizophrenia are mainly due to the difficulty in reproducing histological findings reported for schizophrenia. Nano-CT visualization using synchrotron radiation and subsequent geometric analysis should shed light on this long-standing question about the neuropathology of schizophrenia.

Structural and biochemical basis of the formation of isoaspartate in the complementarity-determining region of antibody 64M-5 Fab.

The formation of the isoaspartate (isoAsp) is one of spontaneous degradation processes of proteins, affecting their stability and activity. Here, we report for the first time the crystal structures of an antibody Fab that contains isoAsp in the complementarity-determining region (CDR), along with biochemical studies to detect isoAsp. By comparing the elution profiles of cation-exchange chromatography, it was clarified that the antibody 64M-5 Fab is converted from the normal form to isoAsp form spontaneously and time-dependently under physiological conditions. The isoAsp residue was identified with tryptic peptide mapping, N-terminal sequencing, and the protein isoaspartyl methyltransferase assay. Based on the fluorescence quenching method, the isoAsp form of 64M-5 Fab shows a one order of magnitude lower binding constant for its dinucleotide ligand dT(6-4)T than the normal form. According to the structure of the isoAsp form, the conformation of CDR L1 is changed from the normal form to isoAsp form; the loss of hydrogen bonds involving the Asn28L side-chain, and structural conversion of the ß-turn from type I to type II'. The formation of isoAsp leads to a large displacement of the side chain of His27dL, and decreased electrostatic interactions with the phosphate group of dT(6-4)T. Such structural changes should be responsible for the lower affinity of the isoAsp form for dT(6-4)T than the normal form. These findings may provide insight into neurodegenerative diseases (NDDs) and related diseases caused by misfolded proteins.

Structures of the antibody 64M-5 Fab and its complex with dT(6-4)T indicate induced-fit and high-affinity mechanisms.

DNA photoproducts with (6-4) pyrimidine-pyrimidone adducts produced by ultraviolet light are mutagenic and carcinogenic. The crystal structures of the anti-(6-4) photoproduct antibody 64M-5 Fab and of its complex with dT(6-4)T were determined at 2.5 and 2.0 Šresolution, respectively. A comparison between the dT(6-4)T-liganded and unliganded structures indicates that the side chain of His93L is greatly rotated and shifted on binding to dT(6-4)T, leading to the formation of an electrostatic interaction with the phosphate moiety of dT(6-4)T, which shows a remarkable induced fit. Based on a comparison of the dT(6-4)T-liganded structures of the 64M-5 and 64M-2 Fabs, the electrostatic interaction between the side chain of His93L in 64M-5 and the phosphate moiety of dT(6-4)T is lost for Leu93L in 64M-2, while Arg90L in 64M-5 instead of Gln90L in 64M-2 stabilizes the conformation of complementarity-determining region (CDR) L3. These differences contribute to the higher affinity of 64M-5 for dT(6-4)T compared with that of 64M-2.

Three-dimensional alteration of neurites in schizophrenia.

Psychiatric symptoms of schizophrenia suggest alteration of cerebral neurons. However, the physical basis of the schizophrenia symptoms has not been delineated at the cellular level. Here, we report nanometer-scale three-dimensional analysis of brain tissues of schizophrenia and control cases. Structures of cerebral tissues of the anterior cingulate cortex were visualized with synchrotron radiation nanotomography. Tissue constituents visualized in the three-dimensional images were traced to build Cartesian coordinate models of tissue constituents, such as neurons and blood vessels. The obtained Cartesian coordinates were used for calculating curvature and torsion of neurites in order to analyze their geometry. Results of the geometric analyses indicated that the curvature of neurites is significantly different between schizophrenia and control cases. The mean curvature of distal neurites of the schizophrenia cases was ~1.5 times higher than that of the controls. The schizophrenia case with the highest neurite curvature carried a frame shift mutation in the GLO1 gene, suggesting that oxidative stress due to the GLO1 mutation caused the structural alteration of the neurites. The differences in the neurite curvature result in differences in the spatial trajectory and hence alter neuronal circuits. It has been shown that the anterior cingulate cortex analyzed in this study has emotional and cognitive functions. We suggest that the structural alteration of neurons in the schizophrenia cases should reflect psychiatric symptoms of schizophrenia.

Synchrotron radiation microtomography of brain hemisphere and spinal cord of a mouse model of multiple sclerosis revealed a correlation between capillary dilation and clinical score.

Multiple sclerosis is a neurological disorder in which the myelin sheaths of axons are damaged by the immune response. We report here a three-dimensional structural analysis of brain and spinal cord tissues of a mouse model of multiple sclerosis, known as experimental autoimmune encephalomyelitis (EAE). EAE-induced mice were raised with or without administration of fingolimod, which is used in the treatment of multiple sclerosis. Brains and spinal cords dissected from the EAE mice were lyophilized so as to reconstitute the intrinsic contrast of tissue elements, such as axons, in X-ray images. Three-dimensional structures of the brain hemispheres and spinal cords of the EAE mice were visualized with synchrotron radiation microtomography. Microtomographic cross sections reconstructed from the X-ray images revealed dilation of capillary vessels and vacuolation in the spinal cord of the EAE mice. Vacuolation was also observed in the cerebellum, suggesting that the neuroinflammatory response progressed in the brain. The vessel networks and vacuolation lesions in the spinal cords were modelled by automatically tracing the three-dimensional image in order to analyze the tissue structures quantitatively. The results of the analysis indicated that the distribution of vacuolations was not uniform but three-dimensionally localized. The mean vessel diameter showed a linear correlation with the clinical score, indicating that vasodilation is relevant to paralysis severity in the disease model. We suggest that vasodilation and vacuolation are related with neurological symptoms of multiple sclerosis.

Method for estimating modulation transfer function from sample images.

The modulation transfer function (MTF) represents the frequency domain response of imaging modalities. Here, we report a method for estimating the MTF from sample images. Test images were generated from a number of images, including those taken with an electron microscope and with an observation satellite. These original images were convolved with point spread functions (PSFs) including those of circular apertures. The resultant test images were subjected to a Fourier transformation. The logarithm of the squared norm of the Fourier transform was plotted against the squared distance from the origin. Linear correlations were observed in the logarithmic plots, indicating that the PSF of the test images can be approximated with a Gaussian. The MTF was then calculated from the Gaussian-approximated PSF. The obtained MTF closely coincided with the MTF predicted from the original PSF. The MTF of an x-ray microtomographic section of a fly brain was also estimated with this method. The obtained MTF showed good agreement with the MTF determined from an edge profile of an aluminum test object. We suggest that this approach is an alternative way of estimating the MTF, independently of the image type.

Three-dimensional X-ray visualization of axonal tracts in mouse brain hemisphere.

Neurons transmit active potentials through axons, which are essential for the brain to function. In this study, the axonal networks of the murine brain were visualized with X-ray tomographic microscopy, also known as X-ray microtomography or micro-CT. Murine brain samples were freeze-dried to reconstitute the intrinsic contrast of tissue constituents and subjected to X-ray visualization. A whole brain hemisphere visualized by absorption contrast illustrated three-dimensional structures including those of the striatum, corpus callosum, and anterior commissure. Axonal tracts observed in the striatum start from the basal surface of the cerebral cortex and end at various positions in the basal ganglia. The distribution of X-ray attenuation coefficients indicated that differences in water and phospholipid content between the myelin sheath and surrounding tissue constituents account for the observed contrast. A rod-shaped cutout of brain tissue was also analyzed with a phase retrieval method, wherein tissue microstructures could be resolved with up to 2.7 µm resolution. Structures of axonal networks of the striatum were reconstructed by tracing axonal tracts. Such an analysis should be able to delineate the functional relationships of the brain regions involved in the observed network.

A method for estimating spatial resolution of real image in the Fourier domain.

Spatial resolution is a fundamental parameter in structural sciences. In crystallography, the resolution is determined from the detection limit of high-angle diffraction in reciprocal space. In electron microscopy, correlation in the Fourier domain is used for estimating the resolution. In this paper, we report a method for estimating the spatial resolution of real images from a logarithmic intensity plot in the Fourier domain. The logarithmic intensity plots of test images indicated that the full width at half maximum of a Gaussian point spread function can be estimated from the images. The spatial resolution of imaging X-ray microtomography using Fresnel zone-plate optics was also estimated with this method. A cross section of a test object visualized with the imaging microtomography indicated that square-wave patterns up to 120-nm pitch were resolved. The logarithmic intensity plot was calculated from a tomographic cross section of brain tissue. The full width at half maximum of the point spread function estimated from the plot coincided with the resolution determined from the test object. These results indicated that the logarithmic intensity plot in the Fourier domain provides an alternative measure of the spatial resolution without explicitly defining a noise criterion.

One-step generation of multiple transgenic mouse lines using an improved Pronuclear Injection-based Targeted Transgenesis (i-PITT).

BACKGROUND: The pronuclear injection (PI) is the simplest and widely used method to generate transgenic (Tg) mice. Unfortunately, PI-based Tg mice show uncertain transgene expression due to random transgene insertion in the genome, usually with multiple copies. Thus, typically at least three or more Tg lines are produced by injecting over 200 zygotes and the best line/s among them are selected through laborious screening steps. Recently, we developed technologies using Cre-loxP system that allow targeted insertion of single-copy transgene into a predetermined locus through PI. We termed the method as PI-based Targeted Transgenesis (PITT). A similar method using PhiC31-attP/B system was reported subsequently. RESULTS: Here, we developed an improved-PITT (i-PITT) method by combining Cre-loxP, PhiC31-attP/B and FLP-FRT systems directly under C57BL/6N inbred strain, unlike the mixed strain used in previous reports. The targeted Tg efficiency in the i-PITT typically ranged from 10 to 30%, with 47 and 62% in two of the sessions, which is by-far the best Tg rate reported. Furthermore, the system could generate multiple Tg mice simultaneously. We demonstrate that injection of up to three different Tg cassettes in a single injection session into as less as 181 zygotes resulted in production of all three separate Tg DNA containing targeted Tg mice. CONCLUSIONS: The i-PITT system offers several advantages compared to previous methods: multiplexing capability (i-PITT is the only targeted-transgenic method that is proven to generate multiple different transgenic lines simultaneously), very high efficiency of targeted-transgenesis (up to 62%), significantly reduces animal numbers in mouse-transgenesis and the system is developed under C57BL/6N strain, the most commonly used pure genetic background. Further, the i-PITT system is freely accessible to scientific community.

Structural biology of DNA (6-4) photoproducts formed by ultraviolet radiation and interactions with their binding proteins.

Exposure to the ultraviolet component of sunlight causes DNA damage, which subsequently leads to mutations, cellular transformation, and cell death. DNA photoproducts with (6-4) pyrimidine-pyrimidone adducts are more mutagenic than cyclobutane pyrimidine dimers. These lesions must be repaired because of the high mutagenic potential of (6-4) photoproducts. We here reviewed the structures of (6-4) photoproducts, particularly the detailed structures of the (6-4) lesion and (6-4) lesion-containing double-stranded DNA. We also focused on interactions with their binding proteins such as antibody Fabs, (6-4) photolyase, and nucleotide excision repair protein. The (6-4) photoproducts that bound to these proteins had common structural features: The 5'-side thymine and 3'-side pyrimidone bases of the T(6-4)T segment were in half-chair and planar conformations, respectively, and both bases were positioned nearly perpendicularly to each other. Interactions with binding proteins showed that the DNA helices flanking the T(6-4)T segment were largely kinked, and the flipped-out T(6-4)T segment was recognized by these proteins. These proteins had distinctive binding-site structures that were appropriate for their functions.

Spatiotemporal development of soaked protein crystal.

Crystal soaking is widely performed in biological crystallography. This paper reports time-resolved X-ray crystallographic and microtomographic analyses of tetragonal crystals of chicken egg-white lysozyme soaked in mother liquor containing potassium hexachloroplatinate. The microtomographic analysis showed that X-ray attenuation spread from the superficial layer of the crystal and then to the crystal core. The crystallographic analyses indicated that platinum sites can be classified into two groups from the temporal development of the electron densities. A soaking process consisting of binding-rate-driven and equilibrium-driven layers is proposed to describe these results. This study suggests that the composition of chemical and structural species resulting from the soaking process varies depending on the position in the crystal.

Three-dimensional network of Drosophila brain hemisphere.

The first step to understanding brain function is to determine the brain's network structure. We report a three-dimensional analysis of the brain network of the fruit fly Drosophila melanogaster by synchrotron-radiation tomographic microscopy. A skeletonized wire model of the left half of the brain network was built by tracing the three-dimensional distribution of X-ray absorption coefficients. The obtained models of neuronal processes were classified into groups on the basis of their three-dimensional structures. These classified groups correspond to neuronal tracts that send long-range projections or repeated structures of the optic lobe. The skeletonized model is also composed of neuronal processes that could not be classified into the groups. The distribution of these unclassified structures correlates with the distribution of contacts between neuronal processes. This suggests that neurons that cannot be classified into typical structures should play important roles in brain functions. The quantitative description of the brain network provides a basis for structural and statistical analyses of the Drosophila brain. The challenge is to establish a methodology for reconstructing the brain network in a higher-resolution image, leading to a comprehensive understanding of the brain structure.

X-ray microtomographic visualization of Escherichia coli by metalloprotein overexpression.

This paper reports X-ray microtomographic visualization of the microorganism Escherichia coli overexpressing a metalloprotein ferritin. The three-dimensional distribution of linear absorption coefficients determined using a synchrotron radiation microtomograph with a simple projection geometry revealed that the X-ray absorption was homogeneously distributed, suggesting that every E. coli cell was labeled with the ferritin. The ferritin-expressing E. coli exhibited linear absorption coefficients comparable with those of phosphotungstic acid stained cells. The submicrometer structure of the ferritin-expressing E. coli cells was visualized by Zernike phase contrast using an imaging microtomograph equipped with a Fresnel zone plate. The obtained images revealed curved columnar or bunching oval structures corresponding to the E. coli cells. These results indicate that the metalloprotein overexpression facilitates X-ray visualization of three-dimensional cellular structures of biological objects.

Structure of a double-stranded DNA (6-4) photoproduct in complex with the 64M-5 antibody Fab.

DNA photoproducts with (6-4) pyrimidine-pyrimidone adducts formed by ultraviolet radiation have been implicated in mutagenesis and cancer. The crystal structure of double-stranded DNA containing the (6-4) photoproduct in complex with the anti-(6-4)-photoproduct antibody 64M-5 Fab was determined at 2.5 Šresolution. The T(6-4)T segment and the 5'-side adjacent adenosine are flipped out of the duplex and are accommodated in the concave antigen-binding pocket composed of six complementarity-determining regions (CDRs). A loop comprised of CDR L1 residues is inserted between the flipped-out T(6-4)T segment and the complementary DNA. The separation of strands by the insertion of the loop facilitates extensive and specific recognition of the photoproduct. The DNA helices flanking the T(6-4)T segment are kinked by 87°. The 64M-5 Fab recognizes the T(6-4)T segment dissociated from the complementary strand, indicating that the (6-4) photoproduct can be detected in double-stranded DNA as well as in single-stranded DNA using the 64M-5 antibody. The structure and recognition mode of the 64M-5 antibody were compared with those of the DNA (6-4) photolyase and nucleotide-excision repair protein DDB1-DDB2. These proteins have distinctive binding-site structures that are appropriate for their functions, and the flipping out of the photolesion and the kinking of the DNA are common to mutagenic (6-4) photoproducts recognized by proteins.

Crystal structure of 6-guanidinohexanoyl trypsin near the optimum pH reveals the acyl-enzyme intermediate to be deacylated.

The force driving the conversion from the acyl intermediate to the tetrahedral intermediate in the deacylation reaction of serine proteases remains unclear. The crystal structure of 6-guanidinohexanoyl trypsin was determined at pH 7.0, near the optimum reaction pH, at 1.94 Å resolution. In this structure, three water molecules are observed around the catalytic site. One acts as a nucleophile to attack the acyl carbonyl carbon while the other two waters fix the position of the catalytic water through a hydrogen bond. When the acyl carbonyl oxygen oscillates thermally, the water assumes an appropriate angle to catalyze the deacylation.

Structure of the DNA (6-4) photoproduct dTT(6-4)TT in complex with the 64M-2 antibody Fab fragment implies increased antibody-binding affinity by the flanking nucleotides.

Pyrimidine (6-4) pyrimidone DNA photoproducts produced by ultraviolet light are highly mutagenic and carcinogenic. The crystal structure of the dTT(6-4)TT photoproduct in complex with the Fab fragment of the antibody 64M-2 that is specific for (6-4) photoproducts was determined at 2.4 Šresolution. The dT(6-4)T segment is fully accommodated in the concave binding pocket of the Fab, as observed in the complex of dT(6-4)T with the Fab. The pyrimidine and pyrimidone bases of the dT(6-4)T segment are positioned nearly perpendicularly to each other. The thymidine segments flanking both ends extend away from the dT(6-4)T segment. The 5'-side thymine base is parallel to the side chain of Tyr100iH of the antibody heavy chain and is also involved in electrostatic interactions with Asn30L, Tyr32L and Lys50L of the antibody light chain. The 5'-side and 3'-side phosphate groups exhibit electrostatic interactions with Asn28L and Ser58H, respectively. These interactions with the flanking nucleotides explain why longer oligonucleotides containing dT(6-4)T segments in the centre show higher antibody-binding affinities than the dT(6-4)T ligand.