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FTIR studies of the similarities between pathology induced protein aggregation in vivo and chemically induced protein aggregation ex vivo
Accepted Manuscript Title: FTIR Studies of the Similarities between Pathology Induced Protein Aggregation in vivo and Chemically Induced Protein Aggregation ex vivo Author: Rebecca J. Tidy Virginie Lam Nicholas Fimognari John C. Mamo Mark J. Hackett PII: DOI: Reference:
S0924-2031(16)30263-6 http://dx.doi.org/doi:10.1016/j.vibspec.2016.09.016 VIBSPE 2630
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3-6-2016 10-9-2016 20-9-2016
Please cite this article as: Rebecca J.Tidy, Virginie Lam, Nicholas Fimognari, John C.Mamo, Mark J.Hackett, FTIR Studies of the Similarities between Pathology Induced Protein Aggregation in vivo and Chemically Induced Protein Aggregation ex vivo, Vibrational Spectroscopy http://dx.doi.org/10.1016/j.vibspec.2016.09.016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
FTIR Studies of the Similarities between Pathology Induced Protein Aggregation in vivo and Chemically Induced Protein Aggregation ex vivo Rebecca J. Tidy,1,2,5 Virginie Lam,2,3 Nicholas Fimognari,2,4 John C. Mamo,2,3 Mark J. Hackett1,2,5*
1
Department of Chemistry, Curtin University, GPOBox U1987, Bentley WA, 6845 Curtin Health Innovation Research Institute, Curtin University, Bentley, WA, 6102 3 School of Public Health, Curtin University, Bentley, WA, 6102 4 School of Biomedical Sciences, Curtin University, Bentley, WA, 6102 5 Nanochemistry Research Institute, Curtin University, Bentley WA, 6845 2
*Corresponding author, email: [email protected]
ABSTRACT Fourier transform infrared (FTIR) spectroscopy has been well documented to discriminate between protein secondary structures, at the micron scale. This capability has enabled in situ localization of β-sheet aggregate accumulation within the central nervous system during pathological protein misfolding associated with Prion disease, Amyotrophic Lateral Sclerosis, Huntington’s Disease, Alzheimer’s’ Disease, and Parkinson’s Disease. In addition to the above diseases, similar spectral alterations occurring over the range ~1625 – 1630 cm-1 have been reported in other biological systems, including inclusion body formation within bacteria and during the formation of high molecular weight protein aggregates via protein oxidation and denaturation. Thus, the characteristic spectral alterations to the amide- I band observed during protein misfolding in neurological disorders are likely not specific to these diseases, but rather, reflect an aggregated protein end point, which can result from a range of biochemical events. For example, a common pathogenic component of many neurological conditions is oxidative stress, protein oxidation and altered ion homeostasis, which have the potential to denature proteins and promote the formation of high molecular weight aggregates. Oxidative stress is a gener i c feature of neurodegenerative diseases and also occurs during neurodegenerative conditions, such as stroke, multiple sclerosis, epilepsy and cerebral malaria. The above mentioned neuropathological disorders (stroke, cerebral malaria, epilepsy, multiple sclerosis) do not have an established protein misfolding pathology, in contrast to Prion disease, Amyotrophic Lateral Sclerosis, Huntington’s Disease, Alzheimer’s’ Disease, which do have an established protein misfolding pathology. Interestingly, recent studies using FTIR have confirmed the presence of protein aggregates within the central nervous system during stroke, cerebral malaria, epilepsy, multiple sclerosis. Such reports suggest FTIR spectroscopy may be a highly valuable research tool to study protein aggregation as a marker of oxidative stress and neurodegeneration in many diseases, not just those with a characteristic pathology for protein misfolding. This manuscript extends the recent literature and reports further characterization of the alterations to the amide I band that result from ubiquitous ex vivo protein aggregation in cerebral tissue. The new data presented highlights that the spectroscopic alterations to the amide I band often reported for amyloid-β plaques in Alzheimer’s disease, are spectroscopically very similar to spectroscopic alterations observed during ischemia induced neurodegeneration (stroke) and ex vivo induced protein aggregation. As such, this study further validates FTIR as a useful platform to study protein aggregation in neurological disorders, including those not characterized by protein misfolding pathology.
KEYWORDS: FTIR, protein aggregation, oxidative stress, β-sheet, brain, central nervous system
1.0 INTRODUCTON X-ray diffraction and X-ray scattering techniques are the “gold standards” to identify the structure of a purified protein. However, Fourier transform infrared (FTIR) spectroscopy, particularly when applied at the micron level, in a mapping or imaging modality, is superior for in situ analysis of protein secondary structure, within chemically complex biological samples. The sensitivity of FTIR spectra to protein secondary structure arises from the hydrogen bonding network across the amide bond of proteins, which correlates with the secondary structure of the poly-peptide chain.[1] The fundamental physics and chemistry underlying the ability of FTIR to detect alterations in protein secondary structure has been discussed in detail elsewhere,[2, 3] however, briefly, the hydrogen bonding network across the poly-peptide chain influences strain through the amide bond and manifests in altered vibrational frequency.[1-3] Thus, the position of the amide I band observed in
FTIR spectra, or changes in the relative position or intensity of the amide I band, can be used as a diagnostic marker for specific protein secondary structures.[4-11] Due to multiple protein secondary structures that exist within the many individual proteins interrogated by a micro-infrared beam in a mapping or imaging experiment, the amide I band observed in a biological sample is typically considered a linear combination of discrete underlying bands unique to individual secondary structures.[2, 12-17] Numerous studies have investigated in depth the sensitivity of the amide I band to protein secondary structure, using various spectral deconvolution methods such as curve fitting, Fourier self-deconvolution and second-derivative analysis, to resolve the underlying components.[4-11] Predicated on existing literature, it would seem that relative alterations in protein secondary structure can be identified with deconvolution methods, or analysis of absorbance intensity ratios across the amide I band.[13, 18-20] However, the analysis methods should consider the fundamental physics and chemistry behind the spectroscopic measurements.[3] The assumption that the amide I band is a linear combination of several underlying bands unique to individual secondary structures simplifies the ease at which relative alterations in secondary structure can be identified in data sets. However, in reality, the underlying features have greater complexity.[3] Unfortunately, this renders absolute quantitative determination of protein secondary structure impractical for complex biological samples. Nonetheless, the identification of relative changes in protein secondary structure, in situ at micron spatial resolution, can be achieved,[13, 18, 20-23] and this feature may be important to increase understanding of the role(s) of protein misfolding and protein aggregation during neurodegenerative processes. A large volume of research in the field has applied FTIR spectroscopic mapping or imaging to localize β-sheet proteins in the central nervous system (CNS) for disease/disorders with characteristic misfolding protein pathology, such as: prion disease;[24-27] Huntington’s disease;[28] amyotrophic lateral sclerosis;[13] Parkinson’s disease;[29-30] and Alzheimer’s disease.[18-20, 23, 31-33] In such disorders, misfolding is typically limited to one or few proteins (Table 1), forming β–sheet aggregates or fibrils, reported by the above studies to exhibit amide I absorbance at ~1630 - 1625 cm-1, and thus, this is now a commonly used spectral marker to detect the presence of β–sheet plaques or fibrils in situ within ex vivo tissue sections.[13, 18-20, 23-32] Recently, rapid diffraction limited spatial resolution imaging of neurons, with multiple synchrotron beams and a focal plane array detector has been developed,[18-21, 34-38] and made possible real time studies of the formation of protein aggregates in living cells.[13] The increased amide I absorbance between ~1630 – 1625 cm-1 reported in studies of protein misfolding diseases produces a distinct second-derivative peak, which has also been reported for other biological systems, such as in the formation of aggregated inclusion bodies in recombinant bacteria.[39-42] Similar features are also observed during thermal or solvent induced protein denaturation and irreversible aggregation, although often at a lower wavenumber ~1620 - 1615 cm-1 if measurements are performed in aqueous solution.[43,44] In these cases the band has been assigned to insoluble high molecular weight protein aggregates, with strong intermolecular hydrogen bonding (thus the red shift of the amide I band).[39-44] In light of this, the spectroscopic alterations to the amide I band observed when studying the protein misfolding diseases listed in Table 1, do not appear to be unique to a specific protein misfolding pathology, but rather reflect an end point of protein aggregation, which could be produced under a variety of biochemical conditions, such as oxidative stress, altered pH, altered ion concentrations. Recently, increased absorbance at ~1630 - 1625 cm-1, and particularly the appearance of a separate peak between ~1630 - 1625 cm-1 and increased intensity of this peak in second-
derivative spectra has been reported in FTIR studies of multiple sclerosis,[45] epilepsy,[22] haemorrhagic stroke,[17] ischemic stroke,[15, 21] and cerebral malaria.[16] None of these neurodegenerative conditions have a known pathology for specific protein misfolding, however, all contain a pathological component of altered ion homeostasis, oxidative stress and protein oxidation. These conditions have the potential to disrupt the hydrogen bonding network of poly- peptide chains, potentially promoting aggregate formation. Therefore, in addition to studying diseases with a characteristic pathology for protein misfolding, FTIR has emerged as a novel tool to study more generalized protein aggregation induced by multiple pathways of disease pathology. As specific brain regions often show enhanced vulnerability to certain diseases, or altered biochemical conditions, FTIR presents additional opportunities to explore/delineate protein aggregation in physiological and complex pathological states In this study we extend from the recent literature to compare features of the amide I band when protein aggregation is observed in diseases with a characteristic protein misfolding pathology (Alzheimer’s disease) and in diseases/conditions without a characteristic protein misfolding pathology (brain ischemia/stroke). We also compare the spectroscopic features of the amide I band in brain tissue manipulated ex vivo to induce protein aggregation using a classical alcohol induced protein precipitation protocol or ex vivo protein oxidation via incubation in solutions of free metal ions. The indicated comparisons reveal that the appearance of a distinct peak at ~1630 – 1625 cm-1 in secondderivative spectra, which is commonly reported in CNS disorders with a protein misfolding pathology,[2,46] and recently for diseases without a protein misfolding pathology, is also observed following ex vivo chemically induced protein aggregation (protein precipitation and protein oxidation). The new data further validates FTIR as a useful platform to study protein aggregation in neurological disorders, including those not characterized by a specific protein misfolding pathology.
2.0 MATERIALS & METHODS 2.1 Animal Models and Tissue Collection – Tissue used in this study, was excess material from studies hosted within the Curtin Health Innovation Research Institute (CHIRI), under approved animal ethics at Curtin University. Biological specimens for FTIR spectroscopic analysis of the amide I profile in amyloid-β plaques was obtained from a 9 month old amyloid precursor protein (APP) transgenic mouse, with spectra of “control” non-plaque tissue taken from the cortex, in a region absent of amyloid enriched plaques. The APP mouse used was anaesthetised with isoflurane and killed via transcardial perfusion with phosphate buffered saline, containing heparin. Following dissection, brain tissue was embedded in optimal cutting temperature medium (OCT) and flash frozen in a liquid nitrogen cooled iso-pentane slurry. Tissue sections for ex vivo induced protein aggregation were taken from a 6 month old control Sprague-Dawley Rat. The animal was sacrificed as described above, and the brain tissue was immediately flash frozen in liquid nitrogen. Tissue was stored at -80 0C until required for analysis. Chemical fixation was not performed to minimise biochemical alterations previously reported for rodent brain tissue.[47] The time from animal death to flash freezing was less than 2 minutes, and not expected to affected protein aggregate levels, as previously reported for rodent brain tissue.[48] 2.2 Sample Preparation – 10-µm-thick tissue sections were cut at -17 0C using a cryo-microtome, and melted onto CaF2 substrate and air-dried. Tissue sections were analysed by FTIR on the same day of tissue sectioning, to minimise chemical alterations due to long term storage.[50] Coronal tissue
sections were cut from the amyloid transgenic APP mouse, and sagittal tissue sections cut from the Sprague-Dawley rat. 2.3 Ex vivo Protein Aggregation – Protein aggregation was induced ex vivo in tissue form the Sprague- Dawley rat, by incubation of tissue sections in for 2 minutes in methanol or ethanol (Sigma), or a 40 mM solution of FeCl2 or CuSO4 (Sigma), which has previously been reported in the literature to induce protein aggregation.[44,50,51] Following incubation the tissue sections were briefly rinsed in deionised water, including the control tissue section (no incubation). Aggregated protein “infarcts” (Figure 4) were induced ex vivo by application of a 5 µL drop of ethanol or FeCl2 solution onto the cortex. 2.4 FTIR Spectroscopic Data Collection – FTIR spectroscopic images of a control rat brain, sagittal section (Figure 1), were collected with a Nicolet iN 10MX FTIR microscope, with a 8x2 pixel array detector, 25 µm pixel size. Spectra were at 8 cm-1 resolution with the co-addition of 8 scans. A background image was collected under the same conditions from blank substrate. Spectra from an amyloid plaque and surrounding plaque free cortical tissue and tissue sections incubated in alcohol, or metal ion solutions, were collected with a single point Mercury Cadmium Telluride (MCT) detector, with a 50 x 50 µm aperture at 4 cm-1 resolution, with the co-addition of 32 scans. A background spectrum was collected with identical parameters from blank substrate. FTIR spectroscopic images of a control rat brain, with a drop of ethanol or FeCl2 added to mimic protein aggregation observed during ischemic stroke were collected with a Nicolet iN 10MX FTIR microscope, with an 8x2 pixel array detector, 25 µm pixel size. Spectra were at 4 cm-1 resolution with the co-addition of 16 scans. A background image was collected under the same conditions from blank substrate. Spectra from ischemic tissue were reproduced from reference 15, with permission from Elsevier. Synchrotron FTIR mapping of individual amyloid-β plaques in APP tissue was performed at the Australian Synchrotron, infrared microspectroscopy beamline. Data was collected with a Bruker Vertex 80v FTIR spectrometer, with a 36× objective and a liquid nitrogen cooled mercury cadmium telluride detector. Maps were collected with an apertured beam spot of ~6 μm and a step size of 5 μm. A background spectrum was collected from the blank CaF2 substrate using 128 co-added scans, and sample spectra recorded with 32 co-added scans. 2.5 FTIR Spectroscopic Data Analysis – FTIR spectra were analysed with Cytospec v2.00.03 and OPUS v7.0. Second-derivative spectra were calculated from vector normalized raw spectra (1600 – 1700 cm- 1), using a 9 smoothing point Savitzky-Golay function. This study aimed to demonstrate that protein aggregation, a characteristic spectral alteration to the amide I band, regardless of mechanistic origin. For example, a protein misfolding disease, a neurodegenerative disorder without a characteristic protein misfolding component, or ex vivo chemically induced protein aggregation all produce a characteristic spectral alteration to the amide I band between 1630 – 1625 cm-1. In this regard, the analysis was qualitative, and thus, second-derivatives were used. Likewise, secondderivatives were used to highlight that differences in the underlying components of the amide I band correlate with anatomic features within the brain. It is acknowledged that second-derivative analysis does not necessarily conserve quantitative information from the original spectrum, as secondderivative intensity varies as a function of band-width. Thus, this paper does not infer quantitative conclusions from the second-derivative data. 2.6 Histology – 10-µm-thick tissue sections, adjacent to those analysed with FTIR were melted onto regular glass microscope slides, post-fixed in 10% buffered formalin (Sigma) and stained with a routine haematoxylin and eosin (H&E) protocol.
3.0 RESULTS 3.1 Protein aggregation induced ex vivo produces a characteristic amide I feature between 1625 – 1627 cm-1 second-derivative spectra collected from air-dried rodent brain tissue sections It is well established that short chain alcohols, such as methanol and ethanol induce protein aggregation via disruption of the poly-peptide hydrogen bond network,[44] and this forms the basis of standard protein precipitation protocols in the field of biochemistry. Additionally, incubation of proteins in aqueous solution with metal ions, which catalyse free radicals through classic Fenton chemistry pathways, has been established to induce protein oxidation and the formation of high molecular weight protein aggregates.[50,51] In this investigation we have incubated rodent brain tissue sections in either methanol, ethanol, or aqueous solution of Cu 2+ or Fe2+, and subsequently airdried the tissue section prior to spectroscopic analysis. The result were compared the amide I band in the second-derivative FTIR spectra collected from the control air-dried tissue section without incubation (Figure 1). Figure 1 shows a range of spectroscopic alterations occurs across the amide I region in the second-derivative spectrum, and not unexpectedly, all four treatments (methanol, ethanol, Cu2+, and Fe2+) induced the appearance of a distinct peak in the second-derivative spectrum, at approximately 1627 – 1625 cm-1. It has been established in the literature that a distinct and well defined β-sheet secondary structure, with intra-molecular hydrogen bonding, typically results in an amide I peak between 1640 – 1625 cm-1.[43,44] In contrast, β-sheet structures in aggregated denatured proteins contain greater intermolecular hydrogen bonding, and display a red-shift in the amide I band position to approximately 1620 – 1610 cm-1.[43,44] These previous assignments were determined from proteins recorded in aqueous solution, with variation expected to be observed in air-dried dehydrated tissue sections. Given that alcohol solvents and metal catalysed protein oxidation are well established to induce irreversible protein aggregation, we assign the distinct second-derivative feature observed between 1627 – 1625 cm-1 in air-dried brain tissue to denatured aggregated protein with intermolecular β-sheets, and the second-derivative feature at ~ 1637 cm-1 is assigned to nonaggregated intra-molecular β-sheet structures. 3.2 Amyloid-β plaques within air-dried brain tissue, from APP mice, display a pronounced amide I feature at 1628 cm-1 in the FTIR second-derivative spectrum Several groups have well documented the prominent features observed in FTIR spectra collected from amyloid-β plaques in brain tissue.[18-20,23,31-33] Typically a strong shoulder or separate peak is observed in the amide I region, approximately between 1620 – 1640 cm-1, with second-derivative spectra revealing a distinct feature at approximately 1625 – 1630 cm-1. A lipid rich halo surrounding the plaque has also been reported.[20,23,31] The results from this study are in agreement with the previous published work, and we demonstrate, using synchrotron radiation FTIR microscopy, a distinct lipid halo around a β-sheet enriched plaque, with maximum second-derivative intensity observed at 1628 cm-1 (Figure 2). Analysis of the same tissue section using a thermal infrared source and linear array detector also revealed an intense second-derivative feature at 1628 cm-1 (Figure 3). This spectroscopic feature was less intense in comparison to the synchrotron data, and the shoulder less pronounced in the raw spectra, attributed to spectral blending associated with the poorer spatial resolution of the thermal
source (25 µm for thermal source, diffraction limited for synchrotron source, ~5 µm). Nonetheless, both the synchrotron data and data collected with thermal source and linear array demonstrate a distinct second-derivative feature at 1628 cm-1 in amyloid-β plaques. Interestingly, as shown in Figure 1 and Figure 3, spectra collected from brain tissue incubated ex vivo with ethanol, show a distinct shoulder between 1620 – 1640 cm-1 in the raw spectra, and a corresponding second-derivative feature at 1626 cm-1, which appears similar to the spectra collected from amyloid-β plaques. 3.3 FTIR spectra collected from air-dried brain tissue within the ischemic infarct in a murine stroke model contain a pronounced amide I feature at 1626 cm-1 in second-derivative spectra Interestingly, recent publications have reported the appearance of a distinct second-derivative feature between 1625 – 1630 cm-1 in several brain diseases or disorders, such as cerebral malaria,[16] multiple sclerosis,[46] epilepsy,[22] and stroke (ischemic and haemorrhagic stroke).[15,17,21] There is no specified protein misfolding pathology for these diseases/conditions. As can be seen in Figure 4, in the case of ischemic stroke, spectra within the ischemic infarct contain a pronounce secondderivative feature at ~1626 cm-1, similar to spectra from tissue sections incubated in alcohol solutions or with solutions of metal ions. 3.4 Specific anatomic structures within the brain give rise to regional specific variation in secondderivative intensity across the amide I band In addition to the ability to image the location of protein aggregates during neurodegenerative diseases and disorders, FTIR spectroscopy has the potential to image the distribution of specific protein secondary structures in non-pathological conditions. Figure 5 highlights an average spectrum of brain tissue (raw and second-derivative), as well as the amide I region in second-derivative spectra, from three distinct tissue regions of the brain (white matter, grey matter, and molecular layer) collected at 4 cm-1 and 8 cm-1 spectral resolution. From the second derivative spectra, collected at both 4 cm-1 and 8 cm-1 resolution, it is apparent the increased second-derivative intensity is observed at 1656 cm-1 in white matter (WM), increased intensity at 1691 cm-1 is observed in grey matter (GM), and increased intensity at 1637 cm-1 is observed in the molecular layer (ML). Although 8 cm-1 spectral resolution is not ideal for investigation of protein secondary structures, it significantly reduces file data sizes, and was used to allow imaging of an entire sagittal section of the rat brain (Figure 6). The results demonstrate a strong correlation between neuro-anatomy and the variation in second-derivative intensity at different spectral locations within the amide I band, described above.
4.0 DISCUSSION The results of this study demonstrate that protein aggregation during a disease with a specific protein misfolding pathology (amyloid-β in Alzheimer’s disease), protein aggregation during neurodegenerative condition without a specific protein misfolding pathology (brain ischemia), and protein aggregation induced ex vivo (alcohol solvent or metal catalyse oxidation), result in similar characteristic spectral alterations in the amide I region. Specifically, all of these events result in appearance of a distinct spectral feature between 1625 – 1628 cm-1, in the second-derivative FTIR spectrum. The consistency of this observation, strongly supports the assignment of this spectral feature to denatured aggregated protein with intermolecular β-sheets.[43, 44] The comparisons presented in this study are important findings to the field, as it demonstrates the potential of FTIR
spectroscopy to not only study protein aggregation in diseases with a characteristic protein misfolding pathology, but highlights that protein aggregation may be studied for a wide-range of pathological conditions with the potential to induce protein aggregation (i.e., ischemia, oxidative stress, altered ion homeostasis). With the exception of specific stains developed for amyloid-β, direct detection of protein aggregates within specific tissues, or at the sub-cellular level is difficult with traditional methods. As regional or cell-specific vulnerability to pathological conditions is established to occur across the brain, it is important to demonstrate the capability of FTIR to image protein aggregation at the micron level, for a range of diseases, and not only those with a classical established protein misfolding pathology, which has been provided by this study. To demonstrate the ability to correlate spectroscopic information with neuroanatomic location, we have compared false colour images generated from spectral intensity in second-derivative FTIR spectra, at specific locations across the amide I band (1691, 1656 and 1637 cm-1) The results show a striking correlation between second-derivative intensity at these spectral location and specific anatomic structures within the brain. The results may reflect an increased α-helix protein (1656 cm-1) content in the white matter, increased non-aggregated intra-molecular β-sheets (1637 cm-1) in the molecular layer, and increased anti-parallel β-sheets or β-turns (1691 cm-1) in the grey matter. If so, such information would be highly valuable for many applications in the field of neuroscience. However, the complex underlying biochemistry of brain tissue is an important consideration. Galactocerebrosides form a large component of tissue, and the amide link in these molecules has been reported to absorb between 1640 – 1620 cm-1.[52] Indeed, white matter which is rich in galactocerebrosides displays increased second-derivative intensity at ~1625 cm-1 (Figure 5C,D), possibly due to galactocerebroside content. This finding highlights that care must be taken not to assign the increased intensity at ~1625 cm-1 in healthy white matter relative to grey matter to increased aggregated β-sheet protein, when it more likely reflects the underlying high galactocerebroside content of this tissue structure. Similarly, grey matter, rich in neuron bodies and their nuclei, present strong DNA and RNA absorbance, particular across the region 1690 – 1720 cm1 ,[53,54] which may account for the increased second-derivative intensity observed at 1691 cm-1 in the grey matter. This may in fact be a more likely explanation as anti-parallel β-sheets have been shown difficult to positively identify for finite structures.[55] As such, this highlights the importance of interpretation of FTIR spectra in context of other biochemical measurements and current knowledge of the underlying biochemical composition of the tissue been investigated. Nonetheless the images presented in Figure 6 highlight the immense potential for continued integration of FTIR into the field of neuroscience to study numerous anatomical specific biochemical pathways.
5.0 CONCLUSION The results of this study highlight that protein aggregation induced ex vivo by alcohol mediated protein precipitation, or metal catalysed protein oxidation, is spectroscopically similar to protein aggregation in vivo. Further, protein aggregation associated with misfolding of a specific folding (βamyloid) in an animal model of Alzheimer’s disease appear spectroscopically similar to non-specific protein aggregation under conditions of oxidative stress following ischemic insult to the brain (stroke). In all of the above cases, the characteristic spectral alterations to the amide I band in FTIR spectra is the appearance of a distinct peak at ~1630 - 1625 cm-1. Although difficult, or impossible to identify the mechanistic cause of protein aggregation from analysis of the amide I band alone, the
opportunity to image the location and identify the time-line of generalized protein aggregation may be highly beneficial in the study of diseases and disorders in the future. The results of this study suggest that there is significant potential to expand the application of FTIR spectroscopy to the field of neuroscience to study protein aggregation as a consequence of a range of pathological conditions.
6.0 ACKNOWLEDGEMENTS The authors acknowledge and are grateful of support from the Curtin Health Innovation Research Institute and the Curtin Nanochemistry Research Institute. MJH acknowledges previous early career support from an Australian Postgraduate Award, ANSTO-PGRA, Lamberton fellowship, SHRF post-doctoral fellowship, CIHR post-doctoral fellowship, the SMI team (HSFC and CIHR funded) and a CIHR-THRUST postdoctoral fellowship. JM is supported by the Australian National Health and Medical Research Council. We acknowledge travel funding provided by the International Synchrotron Access Program (ISAP) managed by the Australian Synchrotron and funded by the Australian Government. A component of this research was undertaken on the infrared microspectroscopy beamline at the Australian Synchrotron, Victoria, Australia
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30. Araki, K., et al., Synchrotron FTIR micro-spectroscopy for structural analysis of Lewy bodies in the brain of Parkinson’s disease patients. Scientific Reports, 2015. 5, 17625. 31. Kuzyk, A., et al., Association among amyloid plaque, lipid, and creatine in hippocampus of TgCRND8 mouse model for Alzheimer disease. Journal of Biological Chemistry, 2010. 285, 31202-31207. 32. Miller, L.M., et al., Synchrotron-based infrared and X-ray imaging shows focalized accumulation of Cu and Zn co-localized with beta-amyloid deposits in Alzheimer's disease. Journal of Structural Biology, 2006. 155, 3037. 33. Choo, L.P., et al., In Situ Characterization of β-Amyloid in Alzheimer’s Diseased Tissue by Synchrotron Fourier Transform Infrared Microspectroscopy. Biophysical Journal, 1996. 71, 1672-1679. 34. Hackett, M.J., et al., Development of single-beam wide-field infrared imaging to study sub- cellular neuron biochemistry. Vibrational Spectroscopy, 2015. 77, 51-59. 35. Barabas, M.E., et al., Chemical Structure and Morphology of Dorsal Root Ganglion Neurons from Naive and Inflamed Mice. Journal of Biological Chemistry, 2014. 289, 34241-34249. 36. Hackett, M.J., et al., A Sub-Cellular Biochemical Investigation of Purkinje Neurons Using Synchrotron Radiation Fourier Transform Infrared Spectroscopic Imaging with a Focal Plane Array Detector. ACS Chemical Neuroscience 2013. 4, 1071-1080. 37. Mattson, E.C., et al., Opportunities for Live Cell FT-Infrared Imaging: Macromolecule Identification with 2D and 3D Localization. International Journal of Molecular Sciences, 2013. 14, 22753-22781. 38. Nasse, M.J., et al., High-resolution Fourier-transform infrared chemical imaging with multiple synchrotron beams. Nat Meth, 2011. 8, 413-416. 39. Seshadri, S., R. Khurana, and A.L. Fink, Fourier transform infrared spectroscopy in analysis of protein deposits, in Methods in Enzymology. 1999, 309, 559-576. 40. Ami, D., et al., FT-IR study of heterologous protein expression in recombinant Escherichia coli strains. Biochimica et Biophysica Acta (BBA) - General Subjects, 2003. 1624, 6-10. 41. Ami, D., et al., Kinetics of inclusion body formation studied in intact cells by FT-IR spectroscopy. FEBS letters, 2005. 579, 3433-3436. 42. Ami, D., et al., Structural analysis of protein inclusion bodies by Fourier transform infrared microspectroscopy. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2006. 1764, 793-799. 43. Casal, H.L., Kohler, U., Mantsch, H.H. Structural and conformational changes of β-lactoglobulin B: an infrared spectroscopic study of the effect of pH and temperature. Biochimica et Biophysica Acta (BBA) Protein Structure and Molecular Enzymology, 1988. 957, 11-20. 44. Purcell, J.M., and Susi, H. Solvent denaturation of proteins as observed by resolution-enhanced Fourier transform infrared spectroscopy. Journal of Biochemical and Biophysical Methods, 1984. 9, 193-199. 45. Caine, S., et al., The application of Fourier transform infrared microspectroscopy for the study of diseased central nervous system tissue. NeuroImage, 2012. 59(4): p. 3624-3640. 46. Heraud, P., et al., Early Dectection of the Chemical Changes Occurring During the Induction and Prevention of Autoimmune-mediated Demyelination Detected by FT-IR Imaging. NeuroImage, 2009. 49, 1180-1189. 47. Hackett, M.J., et al., Chemical alterations to murine brain tissue induced by formalin fixation: implications for biospectroscopic imaging and mapping studies of disease pathogenesis. Analyst, 2011. 136, 2941-2952. 48. Hackett, M.J., et al., In situ Bio-Spectroscopic Investigation of Rapid Ischemic and Post- mortem Induced Biochemical Alterations in the Rat Brain. ACS Chemical Neuroscience, 2015. 6, 226-238. 49. Stitt, D.M., et al., Tissue acquisition and storage associated oxidation considerations for FTIR microspectroscopic imaging of polyunsaturated fatty acids. Vibrational Spectroscopy, 2012. 60, 16-22. 50. Mirzaei, H., and Regnier, F. Protein:protein aggregation induced by protein oxidation. Journal of Chromatography B, 2008. 873, 8-14. 51. Stadtman, E.R. Metal ion-catalyzed oxidation of proteins: biochemical mechanism and biological consequences. Free Radicals in Biology and Medicine, 1990. 9, 315-325. 52. Kahlil, M.B., Carrier, D., Wong, P.T.T., and Tanphaichitr, N. Polymorphic phases of galactocerebrosides: spectroscopic evidence of lamellar crystalline structures. Biochimica et Biophysica Acta (BBA) – Biomembranes, 2011. 1512, 158-170.
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Figure Captions Figure 1: Comparison second-derivative FTIR spectra showing protein aggregation in brain tissue sections induced ex vivo, via incubation of control Sprague-Dawley rat tissue in (A) ethanol, (B) methanol, (C) aqueous solution of Cu2+, and (D) aqueous solution of Fe2+. (A-B) highlight protein aggregation induced via alcohol induced hydrogen bond disruption and protein precipitation, (C-D) highlight oxidative protein damage inducing aggregation. Arrow indicdates location of distinct spectral feature assigned to aggregated denatured protein with intermolecular β-sheet structure. Figure 2: Synchrotron radiation FTIR microspectroscopic analysis of amyloid-β plaques in APP mouse brain tissue. (A) Visible light microscope image of unstained tissue section on CaF2, showing location of dense core plaque (white arrow). (B) FTIR false-colour functional group image of lipid esters (integrated area under the curve, 1755 – 1715 cm-1), showing lipid rich halo surrounding plaque. (C) FTIR false-colour image of relative aggregated β-sheet content of the plaque generated from second-derivative intensity at 1628 cm-1. (D) Non-derivative and (E) second-derviative FTIR spectrum of the amide I band, showing spectra from the center of the plaque and non-plaque tissue. Spectra in D and E were vector normalized from 1600 – 1700 cm-1. Scale bar = 15 µm. Figure 3: Comparison of (A-B) FTIR spectra and (C-D) their corresponding second-derivative spectrum from (A, C) control and plaque cortical brain tissue from an APP transgenic mouse, and (BD) brain tissue from a healthy control Sprague-Dawley rat, with protein aggregation induced ex vivo via incubation of the tissue section in ethanol. Arrows in A-B indicate broadening of the amide I band in response to increased content of proteins with an aggregated β-sheet secondary structure, which becomes more visually apparent as a distinct peak in the second-derivative spectrum (dashed line indicates 1625 cm-1). Spectra in Figure 3 collected with a thermal source and linear array detector 25 µm pixel size. Figure 4: A comparison of imaging aggregated protein “infarct” created (A, D) in vivo in a mouse model of stroke and (B, C, E, F) ex vivo via (B, E) protein precipitation and (C-F) protein oxidation. (A-C) H&E Histology. (D-F) Relative aggregated protein content measured from second-derivative intensity at 1625 cm-1. (G) Representative second-derivative spectra from D, E, F, indicating the appearance of a distinct peak at ~1625 cm-1 (arrow) following both in vivo and ex vivo protein aggregation. Scale bar = 250 µm. Panels A and D and black trace in panel G, reproduced from reference 15, with permission. Figure 5: (A) Average non-derivative and (B) second-derivative FTIR spectrum of brain tissue, and (C) representative second-derivative spectra of different tissue layers within the brain, white matter (WM), grey matter (GM), and molecular layer (ML), collected at 4 cm-1 and (D) 8 cm-1 spectra resolution. The spectroscopic differences observed between tissue layers in spectra collected at 4 cm-1 resolution are evident in spectra collected at 8 cm-1 resolution. Figure 6: FTIR spectroscopic imaging of a sagittal tissue section of rat brain. (A-C) False-colour FTIR images were generated from second-derivative intensity to highlight regional variation in intensity across the amide I band co-localised with specific anatomic structures (A) second-derivative intensity at 1691 cm-1, (B) second-derivative intensity at 1658 cm-1 (C) second-derivative intensity at 1637 cm-1 (D) Tri-colour overlay reveals distinct anatomical structure, similar to that revealed by the distribution of (E) lipid-esters (integrated area under the curve, 1755 – 1715 cm-1), and (F) H&E histology. Scale bar = 1000 µm. The individual colours used in the tri-colour image are denoted in Figures A-C.
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Table 1: Neurodegenerative diseases and disorders that present with specific protein misfolding pathology and diseases without a pathology for misfolding of a specific protein Specific Protein Misfolding Pathology Disease Alzheimer’s disease Parkinson’s disease Amyotrophic lateral sclerosis Prion Disease Huntington’s disease
Protein Amyloid-β, tau-protein α-Synuclein Superoxide dismutase Prion protein Huntington’s protein
No Specific Protein Misfolding Pathology Disease / Condition Stroke (ischemia) Intracerebral haemorrhage Multiple Sclerosis Cerebral Malaria Epilepsy
S0924-2031(16)30263-6 http://dx.doi.org/doi:10.1016/j.vibspec.2016.09.016 VIBSPE 2630
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Please cite this article as: Rebecca J.Tidy, Virginie Lam, Nicholas Fimognari, John C.Mamo, Mark J.Hackett, FTIR Studies of the Similarities between Pathology Induced Protein Aggregation in vivo and Chemically Induced Protein Aggregation ex vivo, Vibrational Spectroscopy http://dx.doi.org/10.1016/j.vibspec.2016.09.016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
FTIR Studies of the Similarities between Pathology Induced Protein Aggregation in vivo and Chemically Induced Protein Aggregation ex vivo Rebecca J. Tidy,1,2,5 Virginie Lam,2,3 Nicholas Fimognari,2,4 John C. Mamo,2,3 Mark J. Hackett1,2,5*
1
Department of Chemistry, Curtin University, GPOBox U1987, Bentley WA, 6845 Curtin Health Innovation Research Institute, Curtin University, Bentley, WA, 6102 3 School of Public Health, Curtin University, Bentley, WA, 6102 4 School of Biomedical Sciences, Curtin University, Bentley, WA, 6102 5 Nanochemistry Research Institute, Curtin University, Bentley WA, 6845 2
*Corresponding author, email: [email protected]
ABSTRACT Fourier transform infrared (FTIR) spectroscopy has been well documented to discriminate between protein secondary structures, at the micron scale. This capability has enabled in situ localization of β-sheet aggregate accumulation within the central nervous system during pathological protein misfolding associated with Prion disease, Amyotrophic Lateral Sclerosis, Huntington’s Disease, Alzheimer’s’ Disease, and Parkinson’s Disease. In addition to the above diseases, similar spectral alterations occurring over the range ~1625 – 1630 cm-1 have been reported in other biological systems, including inclusion body formation within bacteria and during the formation of high molecular weight protein aggregates via protein oxidation and denaturation. Thus, the characteristic spectral alterations to the amide- I band observed during protein misfolding in neurological disorders are likely not specific to these diseases, but rather, reflect an aggregated protein end point, which can result from a range of biochemical events. For example, a common pathogenic component of many neurological conditions is oxidative stress, protein oxidation and altered ion homeostasis, which have the potential to denature proteins and promote the formation of high molecular weight aggregates. Oxidative stress is a gener i c feature of neurodegenerative diseases and also occurs during neurodegenerative conditions, such as stroke, multiple sclerosis, epilepsy and cerebral malaria. The above mentioned neuropathological disorders (stroke, cerebral malaria, epilepsy, multiple sclerosis) do not have an established protein misfolding pathology, in contrast to Prion disease, Amyotrophic Lateral Sclerosis, Huntington’s Disease, Alzheimer’s’ Disease, which do have an established protein misfolding pathology. Interestingly, recent studies using FTIR have confirmed the presence of protein aggregates within the central nervous system during stroke, cerebral malaria, epilepsy, multiple sclerosis. Such reports suggest FTIR spectroscopy may be a highly valuable research tool to study protein aggregation as a marker of oxidative stress and neurodegeneration in many diseases, not just those with a characteristic pathology for protein misfolding. This manuscript extends the recent literature and reports further characterization of the alterations to the amide I band that result from ubiquitous ex vivo protein aggregation in cerebral tissue. The new data presented highlights that the spectroscopic alterations to the amide I band often reported for amyloid-β plaques in Alzheimer’s disease, are spectroscopically very similar to spectroscopic alterations observed during ischemia induced neurodegeneration (stroke) and ex vivo induced protein aggregation. As such, this study further validates FTIR as a useful platform to study protein aggregation in neurological disorders, including those not characterized by protein misfolding pathology.
KEYWORDS: FTIR, protein aggregation, oxidative stress, β-sheet, brain, central nervous system
1.0 INTRODUCTON X-ray diffraction and X-ray scattering techniques are the “gold standards” to identify the structure of a purified protein. However, Fourier transform infrared (FTIR) spectroscopy, particularly when applied at the micron level, in a mapping or imaging modality, is superior for in situ analysis of protein secondary structure, within chemically complex biological samples. The sensitivity of FTIR spectra to protein secondary structure arises from the hydrogen bonding network across the amide bond of proteins, which correlates with the secondary structure of the poly-peptide chain.[1] The fundamental physics and chemistry underlying the ability of FTIR to detect alterations in protein secondary structure has been discussed in detail elsewhere,[2, 3] however, briefly, the hydrogen bonding network across the poly-peptide chain influences strain through the amide bond and manifests in altered vibrational frequency.[1-3] Thus, the position of the amide I band observed in
FTIR spectra, or changes in the relative position or intensity of the amide I band, can be used as a diagnostic marker for specific protein secondary structures.[4-11] Due to multiple protein secondary structures that exist within the many individual proteins interrogated by a micro-infrared beam in a mapping or imaging experiment, the amide I band observed in a biological sample is typically considered a linear combination of discrete underlying bands unique to individual secondary structures.[2, 12-17] Numerous studies have investigated in depth the sensitivity of the amide I band to protein secondary structure, using various spectral deconvolution methods such as curve fitting, Fourier self-deconvolution and second-derivative analysis, to resolve the underlying components.[4-11] Predicated on existing literature, it would seem that relative alterations in protein secondary structure can be identified with deconvolution methods, or analysis of absorbance intensity ratios across the amide I band.[13, 18-20] However, the analysis methods should consider the fundamental physics and chemistry behind the spectroscopic measurements.[3] The assumption that the amide I band is a linear combination of several underlying bands unique to individual secondary structures simplifies the ease at which relative alterations in secondary structure can be identified in data sets. However, in reality, the underlying features have greater complexity.[3] Unfortunately, this renders absolute quantitative determination of protein secondary structure impractical for complex biological samples. Nonetheless, the identification of relative changes in protein secondary structure, in situ at micron spatial resolution, can be achieved,[13, 18, 20-23] and this feature may be important to increase understanding of the role(s) of protein misfolding and protein aggregation during neurodegenerative processes. A large volume of research in the field has applied FTIR spectroscopic mapping or imaging to localize β-sheet proteins in the central nervous system (CNS) for disease/disorders with characteristic misfolding protein pathology, such as: prion disease;[24-27] Huntington’s disease;[28] amyotrophic lateral sclerosis;[13] Parkinson’s disease;[29-30] and Alzheimer’s disease.[18-20, 23, 31-33] In such disorders, misfolding is typically limited to one or few proteins (Table 1), forming β–sheet aggregates or fibrils, reported by the above studies to exhibit amide I absorbance at ~1630 - 1625 cm-1, and thus, this is now a commonly used spectral marker to detect the presence of β–sheet plaques or fibrils in situ within ex vivo tissue sections.[13, 18-20, 23-32] Recently, rapid diffraction limited spatial resolution imaging of neurons, with multiple synchrotron beams and a focal plane array detector has been developed,[18-21, 34-38] and made possible real time studies of the formation of protein aggregates in living cells.[13] The increased amide I absorbance between ~1630 – 1625 cm-1 reported in studies of protein misfolding diseases produces a distinct second-derivative peak, which has also been reported for other biological systems, such as in the formation of aggregated inclusion bodies in recombinant bacteria.[39-42] Similar features are also observed during thermal or solvent induced protein denaturation and irreversible aggregation, although often at a lower wavenumber ~1620 - 1615 cm-1 if measurements are performed in aqueous solution.[43,44] In these cases the band has been assigned to insoluble high molecular weight protein aggregates, with strong intermolecular hydrogen bonding (thus the red shift of the amide I band).[39-44] In light of this, the spectroscopic alterations to the amide I band observed when studying the protein misfolding diseases listed in Table 1, do not appear to be unique to a specific protein misfolding pathology, but rather reflect an end point of protein aggregation, which could be produced under a variety of biochemical conditions, such as oxidative stress, altered pH, altered ion concentrations. Recently, increased absorbance at ~1630 - 1625 cm-1, and particularly the appearance of a separate peak between ~1630 - 1625 cm-1 and increased intensity of this peak in second-
derivative spectra has been reported in FTIR studies of multiple sclerosis,[45] epilepsy,[22] haemorrhagic stroke,[17] ischemic stroke,[15, 21] and cerebral malaria.[16] None of these neurodegenerative conditions have a known pathology for specific protein misfolding, however, all contain a pathological component of altered ion homeostasis, oxidative stress and protein oxidation. These conditions have the potential to disrupt the hydrogen bonding network of poly- peptide chains, potentially promoting aggregate formation. Therefore, in addition to studying diseases with a characteristic pathology for protein misfolding, FTIR has emerged as a novel tool to study more generalized protein aggregation induced by multiple pathways of disease pathology. As specific brain regions often show enhanced vulnerability to certain diseases, or altered biochemical conditions, FTIR presents additional opportunities to explore/delineate protein aggregation in physiological and complex pathological states In this study we extend from the recent literature to compare features of the amide I band when protein aggregation is observed in diseases with a characteristic protein misfolding pathology (Alzheimer’s disease) and in diseases/conditions without a characteristic protein misfolding pathology (brain ischemia/stroke). We also compare the spectroscopic features of the amide I band in brain tissue manipulated ex vivo to induce protein aggregation using a classical alcohol induced protein precipitation protocol or ex vivo protein oxidation via incubation in solutions of free metal ions. The indicated comparisons reveal that the appearance of a distinct peak at ~1630 – 1625 cm-1 in secondderivative spectra, which is commonly reported in CNS disorders with a protein misfolding pathology,[2,46] and recently for diseases without a protein misfolding pathology, is also observed following ex vivo chemically induced protein aggregation (protein precipitation and protein oxidation). The new data further validates FTIR as a useful platform to study protein aggregation in neurological disorders, including those not characterized by a specific protein misfolding pathology.
2.0 MATERIALS & METHODS 2.1 Animal Models and Tissue Collection – Tissue used in this study, was excess material from studies hosted within the Curtin Health Innovation Research Institute (CHIRI), under approved animal ethics at Curtin University. Biological specimens for FTIR spectroscopic analysis of the amide I profile in amyloid-β plaques was obtained from a 9 month old amyloid precursor protein (APP) transgenic mouse, with spectra of “control” non-plaque tissue taken from the cortex, in a region absent of amyloid enriched plaques. The APP mouse used was anaesthetised with isoflurane and killed via transcardial perfusion with phosphate buffered saline, containing heparin. Following dissection, brain tissue was embedded in optimal cutting temperature medium (OCT) and flash frozen in a liquid nitrogen cooled iso-pentane slurry. Tissue sections for ex vivo induced protein aggregation were taken from a 6 month old control Sprague-Dawley Rat. The animal was sacrificed as described above, and the brain tissue was immediately flash frozen in liquid nitrogen. Tissue was stored at -80 0C until required for analysis. Chemical fixation was not performed to minimise biochemical alterations previously reported for rodent brain tissue.[47] The time from animal death to flash freezing was less than 2 minutes, and not expected to affected protein aggregate levels, as previously reported for rodent brain tissue.[48] 2.2 Sample Preparation – 10-µm-thick tissue sections were cut at -17 0C using a cryo-microtome, and melted onto CaF2 substrate and air-dried. Tissue sections were analysed by FTIR on the same day of tissue sectioning, to minimise chemical alterations due to long term storage.[50] Coronal tissue
sections were cut from the amyloid transgenic APP mouse, and sagittal tissue sections cut from the Sprague-Dawley rat. 2.3 Ex vivo Protein Aggregation – Protein aggregation was induced ex vivo in tissue form the Sprague- Dawley rat, by incubation of tissue sections in for 2 minutes in methanol or ethanol (Sigma), or a 40 mM solution of FeCl2 or CuSO4 (Sigma), which has previously been reported in the literature to induce protein aggregation.[44,50,51] Following incubation the tissue sections were briefly rinsed in deionised water, including the control tissue section (no incubation). Aggregated protein “infarcts” (Figure 4) were induced ex vivo by application of a 5 µL drop of ethanol or FeCl2 solution onto the cortex. 2.4 FTIR Spectroscopic Data Collection – FTIR spectroscopic images of a control rat brain, sagittal section (Figure 1), were collected with a Nicolet iN 10MX FTIR microscope, with a 8x2 pixel array detector, 25 µm pixel size. Spectra were at 8 cm-1 resolution with the co-addition of 8 scans. A background image was collected under the same conditions from blank substrate. Spectra from an amyloid plaque and surrounding plaque free cortical tissue and tissue sections incubated in alcohol, or metal ion solutions, were collected with a single point Mercury Cadmium Telluride (MCT) detector, with a 50 x 50 µm aperture at 4 cm-1 resolution, with the co-addition of 32 scans. A background spectrum was collected with identical parameters from blank substrate. FTIR spectroscopic images of a control rat brain, with a drop of ethanol or FeCl2 added to mimic protein aggregation observed during ischemic stroke were collected with a Nicolet iN 10MX FTIR microscope, with an 8x2 pixel array detector, 25 µm pixel size. Spectra were at 4 cm-1 resolution with the co-addition of 16 scans. A background image was collected under the same conditions from blank substrate. Spectra from ischemic tissue were reproduced from reference 15, with permission from Elsevier. Synchrotron FTIR mapping of individual amyloid-β plaques in APP tissue was performed at the Australian Synchrotron, infrared microspectroscopy beamline. Data was collected with a Bruker Vertex 80v FTIR spectrometer, with a 36× objective and a liquid nitrogen cooled mercury cadmium telluride detector. Maps were collected with an apertured beam spot of ~6 μm and a step size of 5 μm. A background spectrum was collected from the blank CaF2 substrate using 128 co-added scans, and sample spectra recorded with 32 co-added scans. 2.5 FTIR Spectroscopic Data Analysis – FTIR spectra were analysed with Cytospec v2.00.03 and OPUS v7.0. Second-derivative spectra were calculated from vector normalized raw spectra (1600 – 1700 cm- 1), using a 9 smoothing point Savitzky-Golay function. This study aimed to demonstrate that protein aggregation, a characteristic spectral alteration to the amide I band, regardless of mechanistic origin. For example, a protein misfolding disease, a neurodegenerative disorder without a characteristic protein misfolding component, or ex vivo chemically induced protein aggregation all produce a characteristic spectral alteration to the amide I band between 1630 – 1625 cm-1. In this regard, the analysis was qualitative, and thus, second-derivatives were used. Likewise, secondderivatives were used to highlight that differences in the underlying components of the amide I band correlate with anatomic features within the brain. It is acknowledged that second-derivative analysis does not necessarily conserve quantitative information from the original spectrum, as secondderivative intensity varies as a function of band-width. Thus, this paper does not infer quantitative conclusions from the second-derivative data. 2.6 Histology – 10-µm-thick tissue sections, adjacent to those analysed with FTIR were melted onto regular glass microscope slides, post-fixed in 10% buffered formalin (Sigma) and stained with a routine haematoxylin and eosin (H&E) protocol.
3.0 RESULTS 3.1 Protein aggregation induced ex vivo produces a characteristic amide I feature between 1625 – 1627 cm-1 second-derivative spectra collected from air-dried rodent brain tissue sections It is well established that short chain alcohols, such as methanol and ethanol induce protein aggregation via disruption of the poly-peptide hydrogen bond network,[44] and this forms the basis of standard protein precipitation protocols in the field of biochemistry. Additionally, incubation of proteins in aqueous solution with metal ions, which catalyse free radicals through classic Fenton chemistry pathways, has been established to induce protein oxidation and the formation of high molecular weight protein aggregates.[50,51] In this investigation we have incubated rodent brain tissue sections in either methanol, ethanol, or aqueous solution of Cu 2+ or Fe2+, and subsequently airdried the tissue section prior to spectroscopic analysis. The result were compared the amide I band in the second-derivative FTIR spectra collected from the control air-dried tissue section without incubation (Figure 1). Figure 1 shows a range of spectroscopic alterations occurs across the amide I region in the second-derivative spectrum, and not unexpectedly, all four treatments (methanol, ethanol, Cu2+, and Fe2+) induced the appearance of a distinct peak in the second-derivative spectrum, at approximately 1627 – 1625 cm-1. It has been established in the literature that a distinct and well defined β-sheet secondary structure, with intra-molecular hydrogen bonding, typically results in an amide I peak between 1640 – 1625 cm-1.[43,44] In contrast, β-sheet structures in aggregated denatured proteins contain greater intermolecular hydrogen bonding, and display a red-shift in the amide I band position to approximately 1620 – 1610 cm-1.[43,44] These previous assignments were determined from proteins recorded in aqueous solution, with variation expected to be observed in air-dried dehydrated tissue sections. Given that alcohol solvents and metal catalysed protein oxidation are well established to induce irreversible protein aggregation, we assign the distinct second-derivative feature observed between 1627 – 1625 cm-1 in air-dried brain tissue to denatured aggregated protein with intermolecular β-sheets, and the second-derivative feature at ~ 1637 cm-1 is assigned to nonaggregated intra-molecular β-sheet structures. 3.2 Amyloid-β plaques within air-dried brain tissue, from APP mice, display a pronounced amide I feature at 1628 cm-1 in the FTIR second-derivative spectrum Several groups have well documented the prominent features observed in FTIR spectra collected from amyloid-β plaques in brain tissue.[18-20,23,31-33] Typically a strong shoulder or separate peak is observed in the amide I region, approximately between 1620 – 1640 cm-1, with second-derivative spectra revealing a distinct feature at approximately 1625 – 1630 cm-1. A lipid rich halo surrounding the plaque has also been reported.[20,23,31] The results from this study are in agreement with the previous published work, and we demonstrate, using synchrotron radiation FTIR microscopy, a distinct lipid halo around a β-sheet enriched plaque, with maximum second-derivative intensity observed at 1628 cm-1 (Figure 2). Analysis of the same tissue section using a thermal infrared source and linear array detector also revealed an intense second-derivative feature at 1628 cm-1 (Figure 3). This spectroscopic feature was less intense in comparison to the synchrotron data, and the shoulder less pronounced in the raw spectra, attributed to spectral blending associated with the poorer spatial resolution of the thermal
source (25 µm for thermal source, diffraction limited for synchrotron source, ~5 µm). Nonetheless, both the synchrotron data and data collected with thermal source and linear array demonstrate a distinct second-derivative feature at 1628 cm-1 in amyloid-β plaques. Interestingly, as shown in Figure 1 and Figure 3, spectra collected from brain tissue incubated ex vivo with ethanol, show a distinct shoulder between 1620 – 1640 cm-1 in the raw spectra, and a corresponding second-derivative feature at 1626 cm-1, which appears similar to the spectra collected from amyloid-β plaques. 3.3 FTIR spectra collected from air-dried brain tissue within the ischemic infarct in a murine stroke model contain a pronounced amide I feature at 1626 cm-1 in second-derivative spectra Interestingly, recent publications have reported the appearance of a distinct second-derivative feature between 1625 – 1630 cm-1 in several brain diseases or disorders, such as cerebral malaria,[16] multiple sclerosis,[46] epilepsy,[22] and stroke (ischemic and haemorrhagic stroke).[15,17,21] There is no specified protein misfolding pathology for these diseases/conditions. As can be seen in Figure 4, in the case of ischemic stroke, spectra within the ischemic infarct contain a pronounce secondderivative feature at ~1626 cm-1, similar to spectra from tissue sections incubated in alcohol solutions or with solutions of metal ions. 3.4 Specific anatomic structures within the brain give rise to regional specific variation in secondderivative intensity across the amide I band In addition to the ability to image the location of protein aggregates during neurodegenerative diseases and disorders, FTIR spectroscopy has the potential to image the distribution of specific protein secondary structures in non-pathological conditions. Figure 5 highlights an average spectrum of brain tissue (raw and second-derivative), as well as the amide I region in second-derivative spectra, from three distinct tissue regions of the brain (white matter, grey matter, and molecular layer) collected at 4 cm-1 and 8 cm-1 spectral resolution. From the second derivative spectra, collected at both 4 cm-1 and 8 cm-1 resolution, it is apparent the increased second-derivative intensity is observed at 1656 cm-1 in white matter (WM), increased intensity at 1691 cm-1 is observed in grey matter (GM), and increased intensity at 1637 cm-1 is observed in the molecular layer (ML). Although 8 cm-1 spectral resolution is not ideal for investigation of protein secondary structures, it significantly reduces file data sizes, and was used to allow imaging of an entire sagittal section of the rat brain (Figure 6). The results demonstrate a strong correlation between neuro-anatomy and the variation in second-derivative intensity at different spectral locations within the amide I band, described above.
4.0 DISCUSSION The results of this study demonstrate that protein aggregation during a disease with a specific protein misfolding pathology (amyloid-β in Alzheimer’s disease), protein aggregation during neurodegenerative condition without a specific protein misfolding pathology (brain ischemia), and protein aggregation induced ex vivo (alcohol solvent or metal catalyse oxidation), result in similar characteristic spectral alterations in the amide I region. Specifically, all of these events result in appearance of a distinct spectral feature between 1625 – 1628 cm-1, in the second-derivative FTIR spectrum. The consistency of this observation, strongly supports the assignment of this spectral feature to denatured aggregated protein with intermolecular β-sheets.[43, 44] The comparisons presented in this study are important findings to the field, as it demonstrates the potential of FTIR
spectroscopy to not only study protein aggregation in diseases with a characteristic protein misfolding pathology, but highlights that protein aggregation may be studied for a wide-range of pathological conditions with the potential to induce protein aggregation (i.e., ischemia, oxidative stress, altered ion homeostasis). With the exception of specific stains developed for amyloid-β, direct detection of protein aggregates within specific tissues, or at the sub-cellular level is difficult with traditional methods. As regional or cell-specific vulnerability to pathological conditions is established to occur across the brain, it is important to demonstrate the capability of FTIR to image protein aggregation at the micron level, for a range of diseases, and not only those with a classical established protein misfolding pathology, which has been provided by this study. To demonstrate the ability to correlate spectroscopic information with neuroanatomic location, we have compared false colour images generated from spectral intensity in second-derivative FTIR spectra, at specific locations across the amide I band (1691, 1656 and 1637 cm-1) The results show a striking correlation between second-derivative intensity at these spectral location and specific anatomic structures within the brain. The results may reflect an increased α-helix protein (1656 cm-1) content in the white matter, increased non-aggregated intra-molecular β-sheets (1637 cm-1) in the molecular layer, and increased anti-parallel β-sheets or β-turns (1691 cm-1) in the grey matter. If so, such information would be highly valuable for many applications in the field of neuroscience. However, the complex underlying biochemistry of brain tissue is an important consideration. Galactocerebrosides form a large component of tissue, and the amide link in these molecules has been reported to absorb between 1640 – 1620 cm-1.[52] Indeed, white matter which is rich in galactocerebrosides displays increased second-derivative intensity at ~1625 cm-1 (Figure 5C,D), possibly due to galactocerebroside content. This finding highlights that care must be taken not to assign the increased intensity at ~1625 cm-1 in healthy white matter relative to grey matter to increased aggregated β-sheet protein, when it more likely reflects the underlying high galactocerebroside content of this tissue structure. Similarly, grey matter, rich in neuron bodies and their nuclei, present strong DNA and RNA absorbance, particular across the region 1690 – 1720 cm1 ,[53,54] which may account for the increased second-derivative intensity observed at 1691 cm-1 in the grey matter. This may in fact be a more likely explanation as anti-parallel β-sheets have been shown difficult to positively identify for finite structures.[55] As such, this highlights the importance of interpretation of FTIR spectra in context of other biochemical measurements and current knowledge of the underlying biochemical composition of the tissue been investigated. Nonetheless the images presented in Figure 6 highlight the immense potential for continued integration of FTIR into the field of neuroscience to study numerous anatomical specific biochemical pathways.
5.0 CONCLUSION The results of this study highlight that protein aggregation induced ex vivo by alcohol mediated protein precipitation, or metal catalysed protein oxidation, is spectroscopically similar to protein aggregation in vivo. Further, protein aggregation associated with misfolding of a specific folding (βamyloid) in an animal model of Alzheimer’s disease appear spectroscopically similar to non-specific protein aggregation under conditions of oxidative stress following ischemic insult to the brain (stroke). In all of the above cases, the characteristic spectral alterations to the amide I band in FTIR spectra is the appearance of a distinct peak at ~1630 - 1625 cm-1. Although difficult, or impossible to identify the mechanistic cause of protein aggregation from analysis of the amide I band alone, the
opportunity to image the location and identify the time-line of generalized protein aggregation may be highly beneficial in the study of diseases and disorders in the future. The results of this study suggest that there is significant potential to expand the application of FTIR spectroscopy to the field of neuroscience to study protein aggregation as a consequence of a range of pathological conditions.
6.0 ACKNOWLEDGEMENTS The authors acknowledge and are grateful of support from the Curtin Health Innovation Research Institute and the Curtin Nanochemistry Research Institute. MJH acknowledges previous early career support from an Australian Postgraduate Award, ANSTO-PGRA, Lamberton fellowship, SHRF post-doctoral fellowship, CIHR post-doctoral fellowship, the SMI team (HSFC and CIHR funded) and a CIHR-THRUST postdoctoral fellowship. JM is supported by the Australian National Health and Medical Research Council. We acknowledge travel funding provided by the International Synchrotron Access Program (ISAP) managed by the Australian Synchrotron and funded by the Australian Government. A component of this research was undertaken on the infrared microspectroscopy beamline at the Australian Synchrotron, Victoria, Australia
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Figure Captions Figure 1: Comparison second-derivative FTIR spectra showing protein aggregation in brain tissue sections induced ex vivo, via incubation of control Sprague-Dawley rat tissue in (A) ethanol, (B) methanol, (C) aqueous solution of Cu2+, and (D) aqueous solution of Fe2+. (A-B) highlight protein aggregation induced via alcohol induced hydrogen bond disruption and protein precipitation, (C-D) highlight oxidative protein damage inducing aggregation. Arrow indicdates location of distinct spectral feature assigned to aggregated denatured protein with intermolecular β-sheet structure. Figure 2: Synchrotron radiation FTIR microspectroscopic analysis of amyloid-β plaques in APP mouse brain tissue. (A) Visible light microscope image of unstained tissue section on CaF2, showing location of dense core plaque (white arrow). (B) FTIR false-colour functional group image of lipid esters (integrated area under the curve, 1755 – 1715 cm-1), showing lipid rich halo surrounding plaque. (C) FTIR false-colour image of relative aggregated β-sheet content of the plaque generated from second-derivative intensity at 1628 cm-1. (D) Non-derivative and (E) second-derviative FTIR spectrum of the amide I band, showing spectra from the center of the plaque and non-plaque tissue. Spectra in D and E were vector normalized from 1600 – 1700 cm-1. Scale bar = 15 µm. Figure 3: Comparison of (A-B) FTIR spectra and (C-D) their corresponding second-derivative spectrum from (A, C) control and plaque cortical brain tissue from an APP transgenic mouse, and (BD) brain tissue from a healthy control Sprague-Dawley rat, with protein aggregation induced ex vivo via incubation of the tissue section in ethanol. Arrows in A-B indicate broadening of the amide I band in response to increased content of proteins with an aggregated β-sheet secondary structure, which becomes more visually apparent as a distinct peak in the second-derivative spectrum (dashed line indicates 1625 cm-1). Spectra in Figure 3 collected with a thermal source and linear array detector 25 µm pixel size. Figure 4: A comparison of imaging aggregated protein “infarct” created (A, D) in vivo in a mouse model of stroke and (B, C, E, F) ex vivo via (B, E) protein precipitation and (C-F) protein oxidation. (A-C) H&E Histology. (D-F) Relative aggregated protein content measured from second-derivative intensity at 1625 cm-1. (G) Representative second-derivative spectra from D, E, F, indicating the appearance of a distinct peak at ~1625 cm-1 (arrow) following both in vivo and ex vivo protein aggregation. Scale bar = 250 µm. Panels A and D and black trace in panel G, reproduced from reference 15, with permission. Figure 5: (A) Average non-derivative and (B) second-derivative FTIR spectrum of brain tissue, and (C) representative second-derivative spectra of different tissue layers within the brain, white matter (WM), grey matter (GM), and molecular layer (ML), collected at 4 cm-1 and (D) 8 cm-1 spectra resolution. The spectroscopic differences observed between tissue layers in spectra collected at 4 cm-1 resolution are evident in spectra collected at 8 cm-1 resolution. Figure 6: FTIR spectroscopic imaging of a sagittal tissue section of rat brain. (A-C) False-colour FTIR images were generated from second-derivative intensity to highlight regional variation in intensity across the amide I band co-localised with specific anatomic structures (A) second-derivative intensity at 1691 cm-1, (B) second-derivative intensity at 1658 cm-1 (C) second-derivative intensity at 1637 cm-1 (D) Tri-colour overlay reveals distinct anatomical structure, similar to that revealed by the distribution of (E) lipid-esters (integrated area under the curve, 1755 – 1715 cm-1), and (F) H&E histology. Scale bar = 1000 µm. The individual colours used in the tri-colour image are denoted in Figures A-C.
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Table 1: Neurodegenerative diseases and disorders that present with specific protein misfolding pathology and diseases without a pathology for misfolding of a specific protein Specific Protein Misfolding Pathology Disease Alzheimer’s disease Parkinson’s disease Amyotrophic lateral sclerosis Prion Disease Huntington’s disease
Protein Amyloid-β, tau-protein α-Synuclein Superoxide dismutase Prion protein Huntington’s protein
No Specific Protein Misfolding Pathology Disease / Condition Stroke (ischemia) Intracerebral haemorrhage Multiple Sclerosis Cerebral Malaria Epilepsy