Evidence for disrupted copper availability in human spinal cord supports CuII(atsm) as a treatment option for sporadic cases of ALS.

The copper compound CuII(atsm) has progressed to phase 2/3 testing for treatment of the neurodegenerative disease amyotrophic lateral sclerosis (ALS). CuII(atsm) is neuroprotective in mutant SOD1 mouse models of ALS where its activity is ascribed in part to improving availability of essential copper. However, SOD1 mutations cause only ~ 2% of ALS cases and therapeutic relevance of copper availability in sporadic ALS is unresolved. Herein we assessed spinal cord tissue from human cases of sporadic ALS for copper-related changes. We found that when compared to control cases the natural distribution of spinal cord copper was disrupted in sporadic ALS. A standout feature was decreased copper levels in the ventral grey matter, the primary anatomical site of neuronal loss in ALS. Altered expression of genes involved in copper handling indicated disrupted copper availability, and this was evident in decreased copper-dependent ferroxidase activity despite increased abundance of the ferroxidases ceruloplasmin and hephaestin. Mice expressing mutant SOD1 recapitulate salient features of ALS and the unsatiated requirement for copper in these mice is a biochemical target for CuII(atsm). Our results from human spinal cord indicate a therapeutic mechanism of action for CuII(atsm) involving copper availability may also be pertinent to sporadic cases of ALS.

Microglial ferroptotic stress causes non-cell autonomous neuronal death.

BACKGROUND: Ferroptosis is a form of regulated cell death characterised by lipid peroxidation as the terminal endpoint and a requirement for iron. Although it protects against cancer and infection, ferroptosis is also implicated in causing neuronal death in degenerative diseases of the central nervous system (CNS). The precise role for ferroptosis in causing neuronal death is yet to be fully resolved. METHODS: To elucidate the role of ferroptosis in neuronal death we utilised co-culture and conditioned medium transfer experiments involving microglia, astrocytes and neurones. We ratified clinical significance of our cell culture findings via assessment of human CNS tissue from cases of the fatal, paralysing neurodegenerative condition of amyotrophic lateral sclerosis (ALS). We utilised the SOD1G37R mouse model of ALS and a CNS-permeant ferroptosis inhibitor to verify pharmacological significance in vivo. RESULTS: We found that sublethal ferroptotic stress selectively affecting microglia triggers an inflammatory cascade that results in non-cell autonomous neuronal death. Central to this cascade is the conversion of astrocytes to a neurotoxic state. We show that spinal cord tissue from human cases of ALS exhibits a signature of ferroptosis that encompasses atomic, molecular and biochemical features. Further, we show the molecular correlation between ferroptosis and neurotoxic astrocytes evident in human ALS-affected spinal cord is recapitulated in the SOD1G37R mouse model where treatment with a CNS-permeant ferroptosis inhibitor, CuII(atsm), ameliorated these markers and was neuroprotective. CONCLUSIONS: By showing that microglia responding to sublethal ferroptotic stress culminates in non-cell autonomous neuronal death, our results implicate microglial ferroptotic stress as a rectifiable cause of neuronal death in neurodegenerative disease. As ferroptosis is currently primarily regarded as an intrinsic cell death phenomenon, these results introduce an entirely new pathophysiological role for ferroptosis in disease.

Evidence for decreased copper associated with demyelination in the corpus callosum of cuprizone-treated mice.

Demyelination within the central nervous system (CNS) is a significant feature of debilitating neurological diseases such as multiple sclerosis and administering the copper-selective chelatorcuprizone to mice is widely used to model demyelination in vivo. Conspicuous demyelination within the corpus callosum is generally attributed to cuprizone's ability to restrict copper availability in this vulnerable brain region. However, the small number of studies that have assessed copper in brain tissue from cuprizone-treated mice have produced seemingly conflicting outcomes, leaving the role of CNS copper availability in demyelination unresolved. Herein we describe our assessment of copper concentrations in brain samples from mice treated with cuprizone for 40 d. Importantly, we applied an inductively coupled plasma mass spectrometry methodology that enabled assessment of copper partitioned into soluble and insoluble fractions within distinct brain regions, including the corpus callosum. Our results show that cuprizone-induced demyelination in the corpus callosum was associated with decreased soluble copper in this brain region. Insoluble copper in the corpus callosum was unaffected, as were pools of soluble and insoluble copper in other brain regions. Treatment with the blood-brain barrier permeant copper compound CuII(atsm) increased brain copper levels and this was most pronounced in the soluble fraction of the corpus callosum. This effect was associated with significant mitigation of cuprizone-induced demyelination. These results provide support for the involvement of decreased CNS copper availability in demyelination in the cuprizone model. Relevance to human demyelinating disease is discussed.

An integrated mass spectrometry imaging and digital pathology workflow for objective detection of colorectal tumours by unique atomic signatures.

Tumours are abnormal growths of cells that reproduce by redirecting essential nutrients and resources from surrounding tissue. Changes to cell metabolism that trigger the growth of tumours are reflected in subtle differences between the chemical composition of healthy and malignant cells. We used LA-ICP-MS imaging to investigate whether these chemical differences can be used to spatially identify tumours and support detection of primary colorectal tumours in anatomical pathology. First, we generated quantitative LA-ICP-MS images of three colorectal surgical resections with case-matched normal intestinal wall tissue and used this data in a Monte Carlo optimisation experiment to develop an algorithm that can classify pixels as tumour positive or negative. Blinded testing and interrogation of LA-ICP-MS images with micrographs of haematoxylin and eosin stained and Ki67 immunolabelled sections revealed Monte Carlo optimisation accurately identified primary tumour cells, as well as returning false positive pixels in areas of high cell proliferation. We analysed an additional 11 surgical resections of primary colorectal tumours and re-developed our image processing method to include a random forest regression machine learning model to correctly identify heterogenous tumours and exclude false positive pixels in images of non-malignant tissue. Our final model used over 1.6 billion calculations to correctly discern healthy cells from various types and stages of invasive colorectal tumours. The imaging mass spectrometry and data analysis methods described, developed in partnership with clinical cancer researchers, have the potential to further support cancer detection as part of a comprehensive digital pathology approach to cancer care through validation of a new chemical biomarker of tumour cells.

U-Pb-dated flowstones restrict South African early hominin record to dry climate phases.

The Cradle of Humankind (Cradle) in South Africa preserves a rich collection of fossil hominins representing Australopithecus, Paranthropus and Homo1. The ages of these fossils are contentious2-4 and have compromised the degree to which the South African hominin record can be used to test hypotheses of human evolution. However, uranium-lead (U-Pb) analyses of horizontally bedded layers of calcium carbonate (flowstone) provide a potential opportunity to obtain a robust chronology5. Flowstones are ubiquitous cave features and provide a palaeoclimatic context, because they grow only during phases of increased effective precipitation6,7, ideally in closed caves. Here we show that flowstones from eight Cradle caves date to six narrow time intervals between 3.2 and 1.3 million years ago. We use a kernel density estimate to combine 29 U-Pb ages into a single record of flowstone growth intervals. We interpret these as major wet phases, when an increased water supply, more extensive vegetation cover and at least partially closed caves allowed for undisturbed, semi-continuous growth of the flowstones. The intervening times represent substantially drier phases, during which fossils of hominins and other fossils accumulated in open caves. Fossil preservation, restricted to drier intervals, thus biases the view of hominin evolutionary history and behaviour, and places the hominins in a community of comparatively dry-adapted fauna. Although the periods of cave closure leave temporal gaps in the South African fossil record, the flowstones themselves provide valuable insights into both local and pan-African climate variability.

A versatile quantitative microdroplet elemental imaging method optimised for integration in biochemical workflows for low-volume samples.

Laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) analysis of µ-droplets is becoming an attractive alternative for detecting and quantifying elements in biological samples. With minimal sample preparation required and detection limits comparable to solution nebulisation ICP-MS, µ-droplets have substantial advantages over traditional elemental detection, particularly for low volumes, such as aliquots taken from samples required for multiple independent biochemical assays, or fluids and tissues where elements of interest exist at native concentrations not suited to the necessary dilution steps required for solution nebulisation ICP-MS. However, the characteristics of µ-droplet residue deposition are heavily dependent on the matrix, and potential effects on signal suppression or enhancement have not been fully characterised. We present a validated and flexible high-throughput method for quantification of elements in µ-droplets using LA-ICP-MS imaging and matrix-matched external calibrants. Imaging the entire µ-droplet area removes analytical uncertainty arising from the often-heterogenous distribution when compared to radial or bisecting line scans that capture only a small portion of the droplet residue. We examined the effects of common matrices found in a standard biochemistry workflow, including native protein and salt contents, as well as reagents used in typical preparation steps for concurrent biochemical assays, such as total protein quantification and enzyme activity assays. We found that matrix composition results in systemic, concentration-dependent signal enhancement and suppression for carbon, whereas high sodium content has a specific space-charge-like suppression effect on high masses. We confirmed the accuracy of our method using both a certified serum standard (Seronorm™ L1) and independent measurements of analysed samples by solution nebulisation ICP-MS, then tested the specificity and reproducibility by examining spinal cord tissue homogenates from SOD1-G93A transgenic mice with a known molecular phenotype of increased copper- and zinc-binding superoxide dismutase-1 expression and altered copper-to-zinc stoichiometry. The method presented is rapid and transferable to multiple other biological matrices and allows high-throughput analysis of low-volume samples with sensitivity comparable to standard solution nebulisation ICP-MS protocols. Graphical Abstract ᅟ.

Imaging Metals in Brain Tissue by Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry (LA-ICP-MS).

Metals are found ubiquitously throughout an organism, with their biological role dictated by both their chemical reactivity and abundance within a specific anatomical region. Within the brain, metals have a highly compartmentalized distribution, depending on the primary function they play within the central nervous system. Imaging the spatial distribution of metals has provided unique insight into the biochemical architecture of the brain, allowing direct correlation between neuroanatomical regions and their known function with regard to metal-dependent processes. In addition, several age-related neurological disorders feature disrupted metal homeostasis, which is often confined to small regions of the brain that are otherwise difficult to analyze. Here, we describe a comprehensive method for quantitatively imaging metals in the mouse brain, using laser ablation - inductively coupled plasma - mass spectrometry (LA-ICP-MS) and specially designed image processing software. Focusing on iron, copper and zinc, which are three of the most abundant and disease-relevant metals within the brain, we describe the essential steps in sample preparation, analysis, quantitative measurements and image processing to produce maps of metal distribution within the low micrometer resolution range. This technique, applicable to any cut tissue section, is capable of demonstrating the highly variable distribution of metals within an organ or system, and can be used to identify changes in metal homeostasis and absolute levels within fine anatomical structures.

Correction: Visualising mouse neuroanatomy and function by metal distribution using laser ablation-inductively coupled plasma-mass spectrometry imaging.

[This corrects the article DOI: 10.1039/C5SC02231B.].

Visualising mouse neuroanatomy and function by metal distribution using laser ablation-inductively coupled plasma-mass spectrometry imaging.

Metals have a number of important roles within the brain. We used laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) to map the three-dimensional concentrations and distributions of transition metals, in particular iron (Fe), copper (Cu) and zinc (Zn) within the murine brain. LA-ICP-MS is one of the leading analytical tools for measuring metals in tissue samples. Here, we present a complete data reduction protocol for measuring metals in biological samples, including the application of a pyramidal voxel registration technique to reproducibly align tissue sections. We used gold (Au) nanoparticle and ytterbium (Yb)-tagged tyrosine hydroxylase antibodies to assess the co-localisation of Fe and dopamine throughout the entire mouse brain. We also examined the natural clustering of metal concentrations within the murine brain to elucidate areas of similar composition. This clustering technique uses a mathematical approach to identify multiple 'elemental clusters', avoiding user bias and showing that metal composition follows a hierarchical organisation of neuroanatomical structures. This work provides new insight into the distinct compartmentalisation of metals in the brain, and presents new avenues of exploration with regard to region-specific, metal-associated neurodegeneration observed in several chronic neurodegenerative diseases.

Correction: Visualising mouse neuroanatomy and function by metal distribution using laser ablation-inductively coupled plasma-mass spectrometry imaging.

[This corrects the article DOI: 10.1039/C5SC02231B.].