Multimodal Synchrotron Radiation Microscopy of Intact Astrocytes from

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Multimodal synchrotron radiation microscopy of intact astrocytes from the hSOD1 G93A rat model of amyotrophic lateral sclerosis Tanja Ducic, Stefan Stamenkovic, Barry Lai, Pavle Andjus, and Vladan Lucic Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04273 • Publication Date (Web): 20 Dec 2018 Downloaded from http://pubs.acs.org on December 21, 2018

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Analytical Chemistry

Multimodal synchrotron radiation microscopy of intact astrocytes from the hSOD1 G93A rat model of amyotrophic lateral sclerosis

Tanja Dučić1*, Stefan Stamenković2, Barry Lai3, Pavle Andjus2, Vladan Lučić4

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CELLS – ALBA, Carrer de la Llum 2-26, 08290 Cerdanyola del Vallès, Barcelona, Spain

Faculty of Biology, University of Belgrade, Center for laser microscopy – CLM, Studentski Trg 3, 11000 Belgrade, Serbia 3

Advanced Photon Source, Argonne National Laboratory, 9700 S. Cass Avenue, Argonne, IL 60439, USA 4

Max Planck Institute of Biochemistry, Am Klopferspitz 1, 82152, Martinsried, Germany

ACS Paragon Plus Environment

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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT

Amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease, is the most common adult onset neurodegenerative disorder affecting motor neurons. Disruptions in metal ion homeostasis have been described in association with ALS, but the pathological mechanisms are still poorly understood. One of the familial ALS cases is caused by mutations in the metallo-enzyme copper-zinc superoxide dismutase (SOD1). In this study, we employed orthogonal cellular synchrotron radiation based spectro-microscopies to investigate the astrocytes of an ALS animal model: the rat hSOD1 G93A that overexpresses human mutated SOD1, which is known to increase the susceptibility of the SOD1 protein to form insoluble intracellular aggregates. Specifically, we applied soft X-ray transmission tomography and hard X-ray fluorescence microscopy in situ, and the Fourier transform infrared spectro-microscopy to detect and analyze aggregates, as well as to determine the alterations in the cellular ultrastructure, and in the elemental and the organic composition of ALS model astrocytes in respect to the control astrocytes isolated from nontransgenic littermates (NTg). The present study demonstrates that large aggregates in the form of multivesicular inclusions form exclusively in the ALS model astrocytes and not in the NTg counterpart. Furthermore, the number of mitochondria, the cellular copper concentration and the amount of anti-parallel β-sheet structures were significantly changed within the cells of the ALS model, as well as the lipid localization and composition. Also, our data indicate that choline was decreased in the ALS model astrocytes, which could explain their higher sensitivity to oxidative stress that we observed. These results show that the hG93A SOD1 mutation causes metabolic and ultrastructural cellular changes and point to a link between an increased copper concentration and aggregation: the most probably that the aggregation of G93A hSOD1 may perturb its binding to Cu, thus directly or indirectly affecting Cu homeostasis.


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Introduction

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disorder characterized by dysfunction and death of neurons in the motor pathways of the cerebral cortex, brainstem and spinal cord 1. Most ALS cases are sporadic, while one tenth occurs in familial forms (fALS), of which 20-25% are caused by mutations in the sod1 gene. This gene encodes Cu-Znsuperoxide dismutase (SOD1), a 32 kDa cytosolic protein that catalyzes the detoxification of the superoxide anion by dismutation into oxygen and hydrogen peroxide 2 and is thus involved in the cellular defense against oxidative stress 3. SOD1 is a metalloenzyme that binds one zinc ion per monomer in order to dimerize, and one catalytic copper ion per monomer for catalytic activity 4. More than 150 mutations in the sod1 gene have been associated with fALS. These mutations dramatically alter the biochemical properties of SOD1, such as its metal binding affinity, and its antioxidant activity levels5. Also, there is strong evidence from in vitro and in vivo studies pointing out that mutated forms of SOD1 are recruited to the mitochondrial intermembrane space, leading to mitochondrial dysfunction and increased production of reactive oxygen species, another wellknown pathological hallmark of ALS6–8. However, it is still unknown how these mutations lead to a toxic gain of function of SOD1 that eventually leads to death of motor neurons5. Human G93A SOD1 mutation causes destabilization of the protein structure and increases its oligomerization and formation of intracellular protein aggregates9. The intracellular accumulation of misfolded and abnormal proteins is a hallmark of ALS, while altered protein degradation due to autophagy dysregulation has been proposed to contribute to ALS pathogenesis 10

. Furthermore, SOD1 aggregation was shown not only to change the activity of the enzyme, but

also to lead to inhibition of axonal transport, excitotoxicity, oxidative stress, endoplasmic-reticulum stress, mitochondrial dysfunction, prion-like propagation, and non-cell autonomous toxicity of neuro-glia (reviewed in 11). Trace metal imbalances are implicated in a number of neurodegenerative diseases including ALS 12,13. When improperly regulated, metal ions, such as copper and zinc, can be highly toxic to neural cells. Copper is especially dangerous due to its catalytic activity and redox potential, which


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could cause the production of damaging reactive oxygen species (ROS), and lead to the propagation of oxidative stress found in patients with ALS 1,14. In order to elucidate cellular changes caused by the G93A mutation of hSOD1, we investigated the transgenic rat model of ALS that has multiple copies of the human sod1 gene containing the G93A mutation and shows the same pathological changes as those seen in human patients15. We focused on astrocytes, because our earlier investigations of brain slices of the same ALS rat animal model showed a pronounced astroglial activation 16–18. Also astrocytes expressing mutated SOD1 induce apoptosis in motor neuron cultures19 , thus implicating astrocytes as one of the key players in pathological processes present in this disease. We used a combination of multiple cellular synchrotron methods: imaging of rapidly frozen intact cells in a fully hydrated, vitrified state allows visualization of the unperturbed cellular interior where only minimal rearrangement of diffusible ions may occur

20

. Previously, SOD1 G93A

aggregates were imaged in chemically fixed, dehydrated mouse thin sectioned tissue of spinal cord by electron microscopy

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or by using several indirect methods as bulk cell fluorescence, flow

cytometry, fluorescence microscopy, enzymatic activity, solid-state NMR or FTIR of whole cells (reviewed in 22). Among the most employed methods to study protein aggregation in situ, some are based on the fluorescence detection of genetically encoded fusion tags, or of conformationalsensitive fluorescent dyes. Cryo X-ray tomography (cryo-XT) also allows direct imaging of intact cellular structures in situ, but differs from cryo-electron tomography in that tomograms of entire large cells can be obtained without sectioning and staining of cells at the expense of lower resolution. Here, we applied cryo-XT to investigate the cellular localization and organization of aggregates as well as the ultrastructural alterations in astrocytes of the G93A ALS model. Furthermore, we determined alterations in the sub-cellular levels of relevant chemical elements in intact astrocytes by cryo X-ray fluorescence (cryo-XRF). Finally, we used synchrotron-based Fourier transform infrared spectro-microscopy (SR-FTIR) to detect alterations in the proteins secondary structure and the distribution of biological relevant organic material23 in rapidly frozen and freeze-dried ALS model astrocytes. This technique was previously used to reveal that, contrary to fibrils, oligomers adopt an anti-parallel β-sheet conformation 24 and to confirm the correlation of the anti-parallel β sheet structure and amyloid protein toxicity

9,25

. Together, these results allowed us to associate

changes in ultrastructure with the levels of chemical elements, distribution of organic structures, and


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an increased susceptibility to oxidative stress, which are expected to promote better understanding of this disease.

EXPERIMENTAL SECTION

Primary cultures of cerebral cortical astrocytes were prepared from 2 days old transgenic hSOD1 G93A (NTac: SD-TgN(SOD1G93A)L26H) rats, Taconic (Albany, NY, USA), and from their non-transgenic (wild-type) NTg littermate Sprague–Dawley rats. Animals were sacrificed according to the guidelines for animal care proposed by the European Communities Council Directive (86/609/EEC) and Serbian Laboratory Animal Science Association and approved by the Ethics Committee of the Faculty of Biology, University of Belgrade. Authorization reference number EK-BF-2016/03. Astrocytes were isolated from the fresh brain by a previously described protocol 17. Cells were cultured on gold quantifoil R 2/2 holey film microscopy grids Au-G200F1 (Quantifoil, Jena, Germany) or silicon-nitride 200 µm thin membrane (Si3N4). At DIV2, cells were thoroughly washed with fresh PBS buffer pH=7.4 and rapidly frozen in liquid ethane chilled with liquid nitrogen by using the Leica EM GP Automatic Plunge Freezer for the Bare Grid Technique (Leica Microsystems, Germany). Subsequently, samples were inspected by using cryo- visible light microscope setup consisting of Zeiss Axio Scope (Zeiss, Germany) microscope equipped with a LINKAM CMS196 cryo-stage (Linkam Scientific, UK). Samples were kept under cryogenic conditions at all times and imaged by cryo-XRF and cryo-XT microscopy in the fully-hydrated, vitrified state. Cryo-XRF was performed at the Advanced Photon Source (APS) at Argonne National Laboratory at the X-ray fluorescence microprobe at 2-ID-D beamline and the Bionanoprobe. We collected scanning images of whole cells that were 60 μm × 40 μm, and 80 x 40 μm in size, having a pixel size of 150 and 300 nm and dwell time 200 ms per pixel. To determine the distribution of trace metals within intact cells with the best possible resolution, we used a novel Bionanoprobe (BNP) instrument at 21-ID-D beamline. Currently, the best spatial resolution of these instruments for two-dimensional fluorescence imaging are 200nm and 30 nm and spectral resolution of 0.5 eV and 0.2 eV at 10 keV, at the 2-ID-D and the Bionanoprobe, respectively 26.


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X-ray tomograms of vitrified samples were recorded on the full field transmission X-ray microscope at the Mistral beamline at the ALBA-Light Source, Spain. Imaging was done using soft X-rays (520 eV) and the zone plate objective that had the outermost zone width of 40 nm. The zone plate objectives relies on diffraction rather than refraction to focus the light, thus limiting the resolution to 40 nm27 (detailed protocols are in Supplemental material).

For the SR-FTIR analysis, vitrified samples were freeze-dried in the home built apparatus, where freeze drying was performed starting with temperature of 77K, with controlled increase of temperature for two days until 200K was reached. Finally, over the next day the temperature was increased slowly to 296K, and samples were stored in a desiccator over silica gel at room temperature. The SR-FTIR measurements were carried out at the FTIR spectro-microscopy station of MIRAS beamline at the synchrotron ALBA, Spain. The IR micro-spectroscopic absorption maps were collected in transmission mode using an Infrared microscope coupled to a SR-FTIR Hyperion 3000 spectrometer and microscope (Bruker, Germany). IR microscope was equipped with a motorized sample stage. Positions of the peaks in spectral analysis were determined using the second derivative method by the OriginLab 9-1 Pro software (Northampton, MA, USA). We used the OPUS 7.5 (Bruker) software package for data analysis and UNSCRAMBLER X (CAMO Software, Oslo, Norway) to perform the Principal component analysis (PCA). PCA analysis was applied on baseline corrected and unit vector normalized spectra, without further corrections. Principal components were calculated using the Nonlinear Iterative Partial Least Squares (NIPALS) algorithm on mean centered data, without weighting. The deconvolution of the amide I area (1700 cm-1 to 1600 cm-1) of SR-FTIR spectra was performed by using least-squares iterative curve fitting to Gaussian line shapes in OriginLab 9-1 Pro software (Northampton, MA, USA). Statistical test for antiparallel βsheet structure assigned between 1680-1700 cm−1 was performed by using the multivariate analysis of variance (ANOVA), post hoc Tukey's test in OriginLab 9-1 Pro software (Northampton, MA, USA). Differences were considered significant with p

Multimodal Synchrotron Radiation Microscopy of Intact Astrocytes from

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