Kathleen M. Gough

The Creatine Kinase/Creatine Connection to Alzheimer's Disease: CK Inactivation, APP-CK Complexes and Focal Creatine Deposits

Cytosolic brain-type creatine kinase (BB-CK), which is coexpressed with ubiquitous mitochondrial uMtCK, is significantly inactivated by oxidation in Alzheimers disease (AD) patients. Since CK has been shown to play a fundamental role in cellular energetics of the brain, any disturbance of this enzyme may exasperate the AD disease process. Mutations in amyloid precursor protein (APP) are associated with early onset AD and result in abnormal processing of APP, and accumulation of Aβ peptide, the main constituent of amyloid plaques in AD brain. Recent data on a direct interaction between APP and the precursor of uMtCK support an emerging relationship between AD, cellular energy levels, and mitochondrial function. In addition, recently discovered creatine (Cr) deposits in the brain of transgenic AD mice, as well as in the hippocampus from AD patients, indicate a direct link between perturbed energy state, Cr metabolism, and AD. Here, we review the roles of Cr and Cr-related enzymes and consider the potential value of supplementation with Cr, a potent neuroprotective substance. As a hypothesis, we consider whether Cr, if given at an early time point of the disease, may prevent or delay the course of AD-related neurodegeneration.

Fourier transform infrared spectrochemical imaging: review of design and applications with a focal plane array and multiple beam synchrotron radiation source.

The beamline design, microscope specifications, and initial results from the new mid-infrared beamline (IRENI) are reviewed. Synchrotron-based spectrochemical imaging, as recently implemented at the Synchrotron Radiation Center in Stoughton, Wisconsin, demonstrates the new capability to achieve diffraction limited chemical imaging across the entire mid-infrared region, simultaneously, with high signal-to-noise ratio. IRENI extracts a large swath of radiation (320 hor. × 25 vert. mrads 2 ) to homogeneously illuminate a commercial infrared (IR) microscope equipped with an IR focal plane array (FPA) detector. Wide-field images are collected, in contrast to single-pixel imaging from the confocal geometry with raster scanning, commonly used at most synchrotron beamlines. IRENI rapidly generates high quality, high spatial resolution data. The relevant advantages (spatial oversampling, speed, sensitivity, and signal-to-noise ratio) are discussed in detail and demonstrated with examples from a variety of disciplines, including formalin-fixed and flash-frozen tissue samples, live cells, fixed cells, paint cross-sections, polymer fibers, and novel nanomaterials. The impact of Mie scattering corrections on this high quality data is shown, and first results with a grazing angle objective are presented, along with future enhancements and plans for implementation of similar, small-scale instruments.

Focally Elevated Creatine Detected in Amyloid Precursor Protein (APP) Transgenic Mice and Alzheimer Disease Brain Tissue

The creatine/phosphocreatine system, regulated by creatine kinase, plays an important role in maintaining energy balance in the brain. Energy metabolism and the function of creatine kinase are known to be affected in Alzheimer diseased brain and in cells exposed to the β-amyloid peptide. We used infrared microspectroscopy to examine hippocampal, cortical, and caudal tissue from 21–89-week-old transgenic mice expressing doubly mutant (K670N/M671L and V717F) amyloid precursor protein and displaying robust pathology from an early age. Microcrystalline deposits of creatine, suggestive of perturbed energetic status, were detected by infrared microspectroscopy in all animals with advanced plaque pathology. Relatively large creatine deposits were also found in hippocampal sections from post-mortem Alzheimer diseased human brain, compared with hippocampus from non-demented brain. We therefore speculate that this molecule is a marker of the disease process.

Fourier transform infrared evaluation of microscopic scarring in the cardiomyopathic heart: effect of chronic AT1 suppression.

Our primary aim was to investigate the use of Fourier transform infrared (FTIR) spectromicroscopy as an accurate assay of cardiac extracellular matrix remodeling. Abnormal rearrangement or remodeling of the cardiac extracellular matrix is known to contribute to cardiac dysfunction. The microscopic multifocal necrosis and scarring are modulated by chronic AT(1) receptor blockade in experimental cardiomyopathy; thus, we also wished to rationalize the spectromicroscopic differences among control, untreated cardiomyopathic (CMP), and losartan-treated cardiomyopathic (LOS) hearts according to the pathogenesis of experimental cardiomyopathy. Male UM-X7.1 cardiomyopathic Syrian hamsters at early and late (65 and 200 days) stages of cardiomyopathy were subjected to 4-week losartan (15 mg/kg/day continuous infusion) treatment. Focal collagen microdomain distribution was confirmed spectroscopically by observation of the collagen IR fingerprint in the 1000-1800 cm(-1) region. Synchrotron FTIR spectromicroscopic map data were obtained from control (F1-beta strain) hamsters, nontreated cardiomyopathic, and losartan-treated CMP animals and imaged with mapping software, according to intensity of collagen fingerprint. Compared to controls, untreated late-stage CMP myocardium was characterized by elevated levels of fibrillar collagens and this was partially normalized with a 4-week losartan treatment. FTIR spectromicroscopy revealed that elevated collagen expression in focal microdomains is present in late-stage cardiomyopathy, and 4-week AT(1) blockade is associated with attenuation of collagen absorption in these lesions.

Biochemical label-free tissue imaging with subcellular-resolution synchrotron FTIR with focal plane array detector.

The critical questions into the cause of neural degeneration, in Alzheimer disease and other neurodegenerative disorders, are closely related to the question of why certain neurons survive. Answers require detailed understanding of biochemical changes in single cells. Fourier transform infrared microspectroscopy is an excellent tool for biomolecular imaging in situ, but resolution is limited. The mid-infrared beamline IRENI (InfraRed ENvironmental Imaging) at the Synchrotron Radiation Center, University of Wisconsin-Madison, enables label-free subcellular imaging and biochemical analysis of neurons with an increase of two orders of magnitude in pixel spacing over current systems. With IRENIs capabilities, it is now possible to study changes in individual neurons in situ, and to characterize their surroundings, using only the biochemical signatures of naturally-occurring components in unstained, unfixed tissue. We present examples of analyses of brain from two transgenic mouse models of Alzheimer disease (TgCRND8 and 3xTg) that exhibit different features of pathogenesis. Data processing on spectral features for nuclei reveals individual hippocampal neurons, and neurons located in the proximity of amyloid plaque in TgCRND8 mouse. Elevated lipids are detected surrounding and, for the first time, within the dense core of amyloid plaques, offering support for inflammatory and aggregation roles. Analysis of saturated and unsaturated fatty acid ester content in retina allows characterization of neuronal layers. IRENI images also reveal spatially-resolved data with unprecedented clarity and distinct spectral variation, from sub-regions including photoreceptors, neuronal cell bodies and synapses in sections of mouse retina. Biochemical composition of retinal layers can be used to study changes related to disease processes and dietary modification.

Synchrotron FTIR reveals lipid around and within amyloid plaques in transgenic mice and Alzheimer's disease brain

While the basis of neuronal degeneration in Alzheimers disease (AD) continues to be debated, the amyloid cascade hypothesis remains central. Amyloid plaques are a required pathological marker for post mortem diagnosis, and Aβ peptide is regarded by most as a critical trigger at the very least. We present spectrochemical image analysis of brain tissue sections obtained with the mid-infrared beamline IRENI (InfraRed ENvironmental Imaging, Synchrotron Radiation Center, U Wisconsin-Madison), where the pixel resolution of 0.54 × 0.54 µm(2) permits analysis at sub-cellular dimensions. Spectrochemical images of dense core plaque found in hippocampus and cortex sections of two transgenic mouse models of AD (TgCRND8 and 3×Tg) are compared with plaque images from a 91 year old apoE43 human AD case. Spectral analysis was done in conjunction with histochemical stains of serial sections. A lipid membrane-like spectral signature surrounded and infiltrated the dense core plaques in all cases. Remarkable compositional similarities in early stage plaques suggest similar routes to plaque formation, regardless of genetic predisposition or mammalian origin.

Restoration and Spectral Recovery of Mid-Infrared Chemical Images

Fourier transform infrared (FTIR) microspectroscopy is a powerful technique for label-free chemical imaging that has supplied important chemical information about heterogeneous samples for many problems across a variety of disciplines. State-of-the-art synchrotron based infrared (IR) microspectrometers can yield high-resolution images, but are truly diffraction limited for only a small spectral range. Furthermore, a fundamental trade-off exists between the number of pixels, acquisition time and the signal-to-noise ratio, limiting the applicability of the technique. The recently commissioned infrared synchrotron beamline, infrared environmental imaging (IRENI), overcomes this trade off and delivers 4096-pixel diffraction limited IR images with high signal-to-noise ratio in under a minute. The spatial oversampling for all mid-IR wavelengths makes the IRENI data ideal for spatial image restoration techniques. Here, we measured and fitted wavelength-dependent point-spread-functions (PSFs) at IRENI for a 74× objective between the sample plane and detector. Noise-free wavelength-dependent theoretical PSFs are deconvoluted from images generated from narrow bandwidths (4 cm(-1)) over the entire mid-infrared range (4000-900 cm(-1)). The stack of restored images is used to reconstruct the spectra. Restored images of metallic test samples with features that are 2.5 μm and smaller are clearly improved in comparison to the raw data images for frequencies above 2000 cm(-1). Importantly, these spatial image restoration methods also work for samples with vibrational bands in the recorded mid-IR fingerprint region (900-1800 cm(-1)). Improved signal-to-noise spectra are reconstructed from the restored images as demonstrated for a mixture of spherical polystyrene beads in a polyurethane matrix. Finally, a freshly thawed retina tissue section is used to demonstrate the success of deconvolution achievable with a heterogeneous, irregularly shaped, biologically relevant sample with distinguishing spectroscopic features across the entire mid-IR spectral range.

Association among Amyloid Plaque, Lipid, and Creatine in Hippocampus of TgCRND8 Mouse Model for Alzheimer Disease

Amyloid peptide (Aβ) aggregation in the brain is a characteristic feature of Alzheimer disease (AD). Previously, we reported the discovery of focally elevated creatine deposits in brain tissue from TgCRND8 mice, which express double mutant (K670N/M671L and V717F) amyloid protein precursor. In this study, frozen hippocampal tissue sections from 5-, 8-, 11-, 14-, and 17-month old TgCRND8 and littermate control mice were examined with Fourier transform infrared microspectroscopy to explore the distribution of lipid, creatine, and dense core plaque deposits. Lipid distribution throughout the hippocampus was similar in transgenic (Tg) and non-Tg littermates at all ages. Dense core plaques were always found to lie within a thin (30–50 μm) lipid envelope, confirmed by imaging through serial sections. Creatine deposits were found in all TgCRND8 mice; the extent of deposition increased with age. Minor creatine deposits appeared in the oldest littermate controls. Distribution in the serial sections showed moderate correlation between layers, slightly disturbed by the freeze/thaw process. Creatine deposits in Tg mice were not specifically co-localized with plaques or lipid halos. The dimension of the lipid envelope is comparable with that of the diffuse halo of nonaggregated amyloid, implying a dynamic association in vivo, postulated to have a significant role in the evolving neurotoxicity.

Conformational preferences and internal rotation in toluene, o-xylene and hexamethylbenzene☆

Abstract Barriers to methyl internal rotation have been investigated through ab initio MO calculations with geometry optimization on toluene (STO-3G and 4-31G), o -xylene (STO-3G), and hexamethylbenzene (STO-3G), and compared with experimental results. Toluene is found to prefer a conformation in which one methyl CH bond makes a dihedral angle of 90° with the ring plane. However, the sixfold barrier to internal rotation is only 17 J mol −1 at the 4-31G level. The potential energy surface for coupled methyl group rotation in o -xylene is displayed and the preferred conformer is that known from microwave spectroscopy. Neglect of coupling and the sixfold component are partially responsible for over-estimates of the barrier from experiments. The most stable conformer of hexamethylbenzene has D 3d symmetry with slight puckering of the ring and tilting of the methyl groups to reduce steric crowding. The barrier to geared internal rotation is 2.42 kJ mol −1 .

High spatial resolution analysis of fungal cell biochemistry – bridging the analytical gap using synchrotron FTIR spectromicroscopy

Fungi impact humans and the environment in many ways, for good and ill. Some fungi support the growth of terrestrial plants or are used in biotechnology, and yet others are established or emerging pathogens. In some cases, the same organism may play different roles depending on the context or the circumstance. A better understanding of the relationship between fungal biochemical composition as related to the fungal growth environment is essential if we are to support or control their activities. Synchrotron FTIR (sFTIR) spectromicroscopy of fungal hyphae is a major new tool for exploring cell composition at a high spatial resolution. Brilliant synchrotron light is essential for this analysis due to the small size of fungal hyphae. sFTIR biochemical characterization of subcellular variation in hyphal composition will allow detailed exploration of fungal responses to experimental treatments and to environmental factors.