68 69 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 0 / 2 1 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 0 / 2 1 Direct chemical imaging of neuromelanin in themost vulnerable brain cells in Parkinson’s disease Related publication: Brooks J., Everett J., Lermyte F.,TjhinV.T., Banerjee S., O’Connor P. B., Morris C. M., Sadler P. J.,Telling N. D. & Collingwood J. F. Label-Free Nanoimaging of Neuromelanin in the Brain by Soft X-ray Spectromicroscopy. Angew. Chemie - Int. Ed. 59 , 11984–11991 (2020). DOI: 10.1002/anie.202000239 Publication keywords: Neuromelanin; Parkinson’s Disease; Spectromicroscopy P arkinson’s disease causes the loss of a particular group of brain cells, the neurons that produce the neurotransmitter dopamine. As these cells contain a dark pigment, neuromelanin, the change is evident from the loss of pigment in this brain region. Characterisation of neuromelanin in tissue remains dependent on visible pigmentation. Faint pigmentationmay be interpreted as cell loss, and so contrast- enhancing stains are commonly used. However, this staining constrains further chemical analysis of the tissue. Researchers explored the use of synchrotron X-ray microscopy to visualise neuromelanin without relying on visible pigmentation or chemical staining. They performed combined imaging and spectroscopy (spectromicroscopy) on Diamond Light Source’s Scanning X-ray Microscopy beamline(I08),allowingthecreationofimagesfromdistinctX-rayabsorptionfeatures.Nanoscalespatialresolutionusingsoft(lowenergy)X-rays allowed the researchers to probe the organic structure of neuromelanin to seek distinguishing spectral features. This revealed a characteristic feature in the absorption spectrumfor neuromelanin. The teamused this feature to createmaps of neuromelanin distributions, whichmatched those observed in stained tissue sections. The teamalso used nanoscale X-ray Fluorescence (XRF) with hard (high energy) X-rays on the Hard X-ray Nanoprobe beamline (I14) to discover a signatureforidentifyingneuromelanin.Thisshowedthatneuromelanincouldbeidentifiedbyitselevatedsulfurcontent.However,thisapproach is not as specific toneuromelaninas the soft X-raymethod.Thediscovery of the soft X-rayneuromelanin signatureoffers significant potential for non-destructive studies of the relationships between depigmentation, metal binding and neurodegeneration in Parkinson’s disease. Dopamine-producing neurons within the substantia nigra region of the brain normally show black/brown pigmentation with advancing age as granules of neuromelanin, a biological polymer formed by dopamine oxidation. In Parkinson’s disease, loss of pigmentation is particularly evident in the substantia nigra pars compacta region and is directly associated with progressive neurodegeneration. Evaluating the loss of visible pigmentation in affected areas of brain tissue is fundamental to post-mortem staging of Parkinson’s disease, yet is constrained by conventional methods of analysis. Emerging synchrotron techniques at Diamond Light Source may shed new light on neuromelanin and its long-debated role in Parkinson’s disease. Parkinson’s disease is the second most common neurodegenerative disease globally (after Alzheimer’s disease), and remains incurable, whilst the underlying causes are still unknown. Recognised clinical symptoms of Parkinson’s, including tremor, rigidity and bradykinesia (slowness of movement), arise due to a characteristic depletion of neurotransmitter dopamine in the brain, caused by the death of dopamine-producing neurons. By the time a patient’s symptoms become apparent,80%oftheseneuromelanin-pigmentedneuronsmayhavealreadydied. Whilst the role of neuromelanin in neurodegeneration is still unclear, the strong affinityofneuromelaninformetal ions isafactorreceivingconsiderableattention 1 . Despite the importance of recognising pigmentation loss in Parkinson’s tissue, conventional analysis relies on visible light, restricting neuromelanin visualisation to only the biggest and darkest clusters and excluding those too faint to view using an optical microscope 2 . Enhancing neuromelanin contrast by staining is effective but prohibits chemical analysis of neuromelanin-associated metal ions. Although the need for a neuromelanin-specific marker was recognised over three decades ago 2 , it has not yet beenmet. Meeting this analytical challenge requires a unique combination of measurement sensitivity, specificity and spatial resolution; synchrotron tools may hold the key. The aim of this work was to use synchrotron X-ray microscopy in the hard and soft X-ray regimes to identify new means for mapping neuromelanin in situ without requirement for chemically-disruptive labelling. At beamline I14, neuromelanin-rich regions of Parkinson’s substantia nigra tissue were mapped for multiple elements simultaneously using nanoscale hard XRF imaging. Sulfur was shown to be unambiguously elevated in neuromelanin granules relative to surrounding tissue (Fig. 1a). Sulfur is also ubiquitously distributed throughout human brain tissue, for example in sulfates and in various amino acids. Hard XRF imaging is an exceptional tool for visualising the variety of elements associating with neuromelanin, but a more direct marker was still required to map neuromelanin with complete independence from surrounding tissue material (Fig. 1b). This was achieved using a soft X-ray approach (Fig. 1d) as described below. Scanning Transmission X-ray Microscopy (STXM) was employed at beamline I08, with additional data obtained at beamline 11.0.2 at the Advanced Light Source, USA, to obtain images and spectral data fromultra-thin sections of human substantia nigra. Image sequences were acquired over a specified energy range known as a stack, enabling X-ray absorption spectra to be derived from target regions within the tissue. This approach was optimised in prior work to provide detailed compositional analysis of amyloid plaque cores 3 . Stacks were collected over the carbon K-edge at 100-200 nm spatial resolution to reveal organic compositionalfeaturesassociatedwithneuromelanin-richregionswithinneurons, as well as tissue areas adjacent to the neurons known as the neuropil. Neuromelanin-rich regions were identified using STXM in unstained tissue sections, guided by correlative silver nitrate staining of neuromelanin in adjacent tissue sections (Fig. 2a). X-ray absorption spectra acquired from neuromelanin granules displayed a different shape to spectra acquired from tissue regions, including a reproducible peak at 287.4 eV that was not present in the tissue spectra (Fig. 2e,f). Speciationmaps, created by subtracting an off-peak image (not associatedwith any spectral features) froman image taken at 287.4 eV, revealed a detailed depiction of neuromelanin distribution (Fig. 2c). Imaging at nanoscale resolution enabled individual neuromelanin granules to be clearly resolved, and the correlation between the neuromelanin distributions in stained and STXM-mapped tissue sections was verified. Iron mapping using STXM, and subsequently confirmed using XRF mapping, also demonstrated some neurons to be heavily loaded with iron. This is consistent with neuromelanin’s strong affinity for iron. The same spectral features observed for biological neuromelanin were reproduced in spectra acquired from synthetic neuromelanin analogues (with and without iron loading), confirming that the peak originated fromthe neuromelanin polymer (Fig. 3). The overall neuromelanin spectrum was distinct from those of its precursor materials, dopamine and L-cysteine. However, a sharp absorption feature was observed at 287.4 eV in the spectrum for benzothiazine, a constituent functional group of the melanin polymer (Fig. 3). The characteristic absorption peak at 287.4 eV was ultimately attributed to 1s → σ* (C-S) electron transitions in the benzothiazine spectrum, reported to occur around this energy in a range of similar organic, sulfur-containingmolecules 4 . Using this novel approach to neuromelanin visualisation, neuromelanin contrast was verified for substantia nigra tissue from multiple Parkinson’s cases, as well as for cases of Alzheimer’s disease, Neurodegeneration with Brain Iron Accumulation, and neurologically healthy controls. Use of soft x-ray STXM enabled neuromelanin to be distinguished from surroundingtissue,aswellasfromotherintracellularproteinsincludingferritinand alpha-synuclein, without chemical labelling or prerequisite identification of cells. Application of multi-modal synchrotron techniques across different beamlines, including the nanoscale XRF imaging illustrated here in tandem with STXM, presents further opportunities to investigate neuromelanin’s interactions with metal ions.Thismayhelptounderstandbettertheenigmaticroleofneuromelanin in Parkinson’s disease and related disorders. References: 1. Zucca F. A. et al. Interactions of iron, dopamine and neuromelanin pathways in brain aging and Parkinson’s disease. Prog. Neurobiol. 155 , 96–119 (2017). DOI: 10.1016/j.pneurobio.2015.09.012 2. Hirsch E. et al. Melanized dopaminergic neurons are differentially susceptible to degeneration in Parkinson’s disease. Nature 334 , 345–348 (1988). DOI: 10.1038/334345a0 3. Everett J. et al. Nanoscale synchrotron X-ray speciation of iron and calcium compounds in amyloid plaque cores fromAlzheimer’s disease subjects. Nanoscale 10 , 11782–11796 (2018). DOI: 10.1039/c7nr06794a 4. Myneni S. C. B. Soft X-ray spectroscopy and spectromicroscopy studies of organic molecules in the environment. Rev. Mineral. Geochemistry 49 , 485–579 (2002). DOI: 10.2138/gsrmg.49.1.485 Funding acknowledgement: This work was supported by EPSRC grants EP/N033191/1, EP/N033140/1, EP/ K035193/1, an EPSRC DoctoralTraining Award to Jake Brooks (EP/N509796/1), and a Royal Society Newton-Bhahba International Fellowship for Samya Banerjee. We also thank Diamond Light Source, where themajority of the synchrotron data presented in this study were collected, for access to beamlines I08 (proposals SP15230, SP15854, SP20809, MG24526) and I14 (proposal MG24531). Corresponding authors: Dr Jake Brooks, School of Engineering, University ofWarwick,
[email protected]; Prof. Joanna Collingwood, School of Engineering, University ofWarwick,
[email protected] Imaging andMicroscopy Group Beamlines I08 and I14 Figure 1: Enabling correlative analysis of neuromelanin in human substantia nigra using optical microscopy, Scanning Transmission X-ray Microscopy (STXM) and X-ray Fluorescence imaging (XRF). (a) XRF map of sulfur concentration distribution in a melanised neuron from Parkinson’s substantia nigra, collected at beamline I14; (b) Optical image of melanised neurons in an unstained tissue section of approx. 30 µm thickness; (c) Axial view of the human brain, highlighting the region incorporating neuromelanin-pigmented substantia nigra; (d) STXM composite map showing neuromelanin (green) and surrounding tissue (grey) in control substantia nigra, for data collected at beamline I08. Part (c) was adapted, under license CCBY4.0, from Blausen.com staff, WikiJournal of Medicine 2014, 1, DOI: 10.15347/ wjm/2014.010. Figure 2: Melanised neuron identified in neurologically-healthy control substantia nigra using optical microscopy, then mapped using STXM at the carbon K-edge in an adjacent unstained tissue section. Data collected at beamline I08. (a) Silver nitrate stained cell showing neuromelanin (NM) distribution; (b) Protein map showing tissue structure; (c) Neuromelanin map generated using absorption peak at 287.4 eV; (d) Composite map showing tissue- derived protein (grey), neuromelanin (green); (e) Carbon K-edge spectrum from neuropil region;. (f) Carbon K-edge spectrum from neuromelanin-rich region. A dashed line marks the position of the absorption feature distinguishing neuromelanin at 287.4 eV. Figure 3: (a) carbon K-edge X-ray absorption spectra from intracellular neuromelanin (NM), synthetic NM analogue, and neuromelanin constituent group benzothiazine. Data collected at beamline I08. Intracellular and synthetic neuromelanin display the same spectral features. The peak at 287.4 eV (marked by a dashed line) is also shared with benzothiazine; (b) Benzothiazine group shown in red in the region of the neuromelanin polymer where it is incorporated.