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.

Trends in clinical characteristics, medication use, and glycemic control in insulin-treated patients with type 1 and type 2 diabetes in Finland in 2012-2019: Nationwide real-world evidence study.

AIMS: To describe the clinical characteristics and medication purchases of insulin-treated adults in Finland at index (January 1, 2012 or first insulin purchase) and December 31, 2019. Additionally, to describe basal insulin (BI) treatment patterns and associated changes in hemoglobin A1c (HbA1c) values. MATERIALS AND METHODS: In this descriptive study using nationwide registries, we included adults with at least two reimbursed insulin purchases within 12 months of the first purchase between January 1, 2012 and December 31, 2019. We formed four study groups: type 1 diabetes (T1D) and type 2 diabetes (T2D)-diagnosed people who were further divided into prevalent or naïve users (start of insulin use before or after January 1, 2012). Insulin treatment patterns were estimated from medication purchase data and glycemic control from HbA1c results. RESULTS: Out of 145 020 people included, 34 359 had T1D and 110 661 T2D. By 2019, in parallel with the adaptation of new noninsulin medications, second-generation basal insulin (BI) analogues were adopted by 45.9% and 21.1% of prevalent T1D and T2D users. At index, HbA1c target (≤53 mmol/mol) was reached by 17% and 35% of T2D naïve and prevalent users, respectively, and by 17% of T1D prevalent users. At study end, the target was reached respectively by 41%, 34%, and 22% of insulin users. Insulin initiation improved and discontinuation worsened glycemic control in T2D, with lesser effects seen after treatment gaps or switches between BIs. CONCLUSIONS: Our study showed that glycemic control in insulin users has remained stable or improved between 2012 and 2019 despite aging population and in parallel with introduction of new treatment options, providing valuable insight into Finnish national diabetes care.

Transdermal Application of Soluble CuII(atsm) Increases Brain and Spinal Cord Uptake Compared to Gavage with an Insoluble Suspension.

CuII(atsm) is a blood-brain barrier permeant copper(II) compound that is under investigation in human clinical trials for the treatment of neurodegenerative diseases of the central nervous system (CNS). Imaging in humans by positron emission tomography shows the compound accumulates in affected regions of the CNS in patients. Most therapeutic studies to date have utilised oral administration of CuII(atsm) in an insoluble form, as either solid tablets or a liquid suspension. However, two pre-clinical studies have demonstrated disease-modifying outcomes following transdermal application of soluble CuII(atsm) prepared in dimethyl sulphoxide. Whether differences in the method of administration lead to different degrees of tissue accumulation of the compound has never been examined. Here, we compare the two methods of administration in wild-type mice by assessing changes in tissue concentrations of copper. Both administration methods resulted in elevated copper concentrations in numerous tissues, with the largest increases evident in the liver, brain and spinal cord. In all instances where treatment with CuII(atsm) resulted in elevated tissue copper, transdermal application of soluble CuII(atsm) led to higher concentrations of copper. In contrast to CuII(atsm), an equivalent dose of copper(II) chloride resulted in minimal changes to tissue copper concentrations, regardless of the administration method. Data presented herein provide quantitative insight to transdermal application of soluble CuII(atsm) as a potential alternative to oral administration of the compound in an insoluble formulation.

Comorbidity characteristics of multiple myeloma patients diagnosed in Finland 2005-2016.

Multiple myeloma (MM) patients are predominantly elderly with comorbidities that have an impact on patient mortality and treatment decisions. We previously reported the patient characteristics and overall survival outcomes of the Finnish MM cohort diagnosed between 2005 and 2016 in a nationwide retrospective registry study comprising 3,851 adults. Here, we report detailed comorbidity characteristics for this real-world Finnish MM population at cohort entry and during follow-up. Data on diagnoses and causes of death were obtained from Finnish healthcare data registries and interrogated using various multistate time-to-event models. In the year preceding MM diagnosis, comorbidities (as per Charlson Comorbidity Index definition) were recorded in 38.0% of the cohort, of which 27.9% presented with pre-existing cardiovascular disease (CVD) and 4.8% had suffered a major adverse cardiac event (MACE). At 2 years post-MM diagnosis, cumulative incidence for CVD and MACE more than doubled to 57.1% and 11.4%, respectively, and only 31.9% of the cohort remained CVD-free. Prevalent secondary malignancies were recorded in 16.8% of the patient population at MM diagnosis, with cumulative incidence increasing steadily to 27.5% at 2 years and 33% at 5 years post-diagnosis. The main cause of mortality attributed to MM, CVD, secondary malignancy, or other causes remained stable throughout the follow-up, at an average of 74.2%, 9.4%, 9.8%, and 6.5%, respectively. Prevalence of CVDs and secondary malignancies is high in Finnish patients at MM diagnosis, with older male patients suffering from higher MACE and mortality risk. Proper recording and management of comorbidities alongside novel treatments remain crucial for optimal MM management.

Altered SOD1 maturation and post-translational modification in amyotrophic lateral sclerosis spinal cord.

Aberrant self-assembly and toxicity of wild-type and mutant superoxide dismutase 1 (SOD1) has been widely examined in silico, in vitro and in transgenic animal models of amyotrophic lateral sclerosis. Detailed examination of the protein in disease-affected tissues from amyotrophic lateral sclerosis patients, however, remains scarce. We used histological, biochemical and analytical techniques to profile alterations to SOD1 protein deposition, subcellular localization, maturation and post-translational modification in post-mortem spinal cord tissues from amyotrophic lateral sclerosis cases and controls. Tissues were dissected into ventral and dorsal spinal cord grey matter to assess the specificity of alterations within regions of motor neuron degeneration. We provide evidence of the mislocalization and accumulation of structurally disordered, immature SOD1 protein conformers in spinal cord motor neurons of SOD1-linked and non-SOD1-linked familial amyotrophic lateral sclerosis cases, and sporadic amyotrophic lateral sclerosis cases, compared with control motor neurons. These changes were collectively associated with instability and mismetallation of enzymatically active SOD1 dimers, as well as alterations to SOD1 post-translational modifications and molecular chaperones governing SOD1 maturation. Atypical changes to SOD1 protein were largely restricted to regions of neurodegeneration in amyotrophic lateral sclerosis cases, and clearly differentiated all forms of amyotrophic lateral sclerosis from controls. Substantial heterogeneity in the presence of these changes was also observed between amyotrophic lateral sclerosis cases. Our data demonstrate that varying forms of SOD1 proteinopathy are a common feature of all forms of amyotrophic lateral sclerosis, and support the presence of one or more convergent biochemical pathways leading to SOD1 proteinopathy in amyotrophic lateral sclerosis. Most of these alterations are specific to regions of neurodegeneration, and may therefore constitute valid targets for therapeutic development.

Iron overload and impaired iron handling contribute to the dystrophic pathology in models of Duchenne muscular dystrophy.

BACKGROUND: Oxidative stress is implicated in the pathophysiology of Duchenne muscular dystrophy (DMD, caused by mutations in the dystrophin gene), which is the most common and severe of the muscular dystrophies. To our knowledge, the distribution of iron, an important modulator of oxidative stress, has not been assessed in DMD. We tested the hypotheses that iron accumulation occurs in mouse models of DMD and that modulation of iron through the diet or chelation could modify disease severity. METHODS: We assessed iron distribution and total elemental iron using LA-ICP-MS on skeletal muscle cross-sections of 8-week-old Bl10 control mice and dystrophic mdx mice (with moderate dystrophy) and dystrophin/utrophin-null mice (dko, with severe dystrophy). In addition, mdx mice (4 weeks) were treated with either an iron chelator (deferiprone 150 mg/kg/day) or iron-enriched feed (containing 1% added iron as carbonyl iron). Immunoblotting was used to determine the abundance of iron- and mitochondria-related proteins. (Immuno)histochemical and mRNA assessments of fibrosis and inflammation were also performed. RESULTS: We observed a significant increase in total elemental iron in hindlimb muscles of dko mice (+50%, P < 0.05) and in the diaphragm of mdx mice (+80%, P < 0.05), with both tissues exhibiting severe pathology. Iron dyshomeostasis was further evidenced by an increase in the storage protein ferritin (dko: +39%, P < 0.05) and ferroportin compared with Bl10 control mice (mdx: +152% and dko: +175%, P < 0.05). Despite having features of iron overload, dystrophic muscles had lower protein expression of ALAS-1, the rate-limiting enzyme for haem synthesis (dko -44%, P < 0.05), and the haem-containing protein myoglobin (dko -54%, P < 0.05). Deferiprone treatment tended to decrease muscle iron levels in mdx mice (-30%, P < 0.1), which was associated with lower oxidative stress and fibrosis, but suppressed haem-containing proteins and mitochondrial content. Increasing iron via dietary intervention elevated total muscle iron (+25%, P < 0.05) but did not aggravate the pathology. CONCLUSIONS: Muscles from dystrophic mice have increased iron levels and dysregulated iron-related proteins that are associated with dystrophic pathology. Muscle iron levels were manipulated by iron chelation and iron enriched feed. Iron chelation reduced fibrosis and reactive oxygen species (ROS) but also suppressed haem-containing proteins and mitochondrial activity. Conversely, iron supplementation increased ferritin and haem-containing proteins but did not alter ROS, fibrosis, or mitochondrial activity. Further studies are required to investigate the contribution of impaired ferritin breakdown in the dysregulation of iron homeostasis in DMD.

Lithium administered to pregnant, lactating and neonatal rats: entry into developing brain.

BACKGROUND: Little is known about the extent of drug entry into developing brain, when administered to pregnant and lactating women. Lithium is commonly prescribed for bipolar disorder. Here we studied transfer of lithium given to dams, into blood, brain and cerebrospinal fluid (CSF) in embryonic and postnatal animals as well as adults. METHODS: Lithium chloride in a clinically relevant dose (3.2 mg/kg body weight) was injected intraperitoneally into pregnant (E15-18) and lactating dams (birth-P16/17) or directly into postnatal pups (P0-P16/17). Acute treatment involved a single injection; long-term treatment involved twice daily injections for the duration of the experiment. Following terminal anaesthesia blood plasma, CSF and brains were collected. Lithium levels and brain distribution were measured using Laser Ablation Inductively Coupled Plasma-Mass Spectrometry and total lithium levels were confirmed by Inductively Coupled Plasma-Mass Spectrometry. RESULTS: Lithium was detected in blood, CSF and brain of all fetal and postnatal pups following lithium treatment of dams. Its concentration in pups' blood was consistently below that in maternal blood (30-35%) indicating significant protection by the placenta and breast tissue. However, much of the lithium that reached the fetus entered its brain. Levels of lithium in plasma fluctuated in different treatment groups but its concentration in CSF was stable at all ages, in agreement with known stable levels of endogenous ions in CSF. There was no significant increase of lithium transfer into CSF following application of Na+/K+ ATPase inhibitor (digoxin) in vivo, indicating that lithium transfer across choroid plexus epithelium is not likely to be via the Na+/K+ ATPase mechanism, at least early in development. Comparison with passive permeability markers suggested that in acute experiments lithium permeability was less than expected for diffusion but similar in long-term experiments at P2. CONCLUSIONS: Information obtained on the distribution of lithium in developing brain provides a basis for studying possible deleterious effects on brain development and behaviour in offspring of mothers undergoing lithium therapy.

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.

Regular Physical Exercise Modulates Iron Homeostasis in the 5xFAD Mouse Model of Alzheimer's Disease.

Dysregulation of brain iron metabolism is one of the pathological features of aging and Alzheimer's disease (AD), a neurodegenerative disease characterized by progressive memory loss and cognitive impairment. While physical inactivity is one of the risk factors for AD and regular exercise improves cognitive function and reduces pathology associated with AD, the underlying mechanisms remain unclear. The purpose of the study is to explore the effect of regular physical exercise on modulation of iron homeostasis in the brain and periphery of the 5xFAD mouse model of AD. By using inductively coupled plasma mass spectrometry and a variety of biochemical techniques, we measured total iron content and level of proteins essential in iron homeostasis in the brain and skeletal muscles of sedentary and exercised mice. Long-term voluntary running induced redistribution of iron resulted in altered iron metabolism and trafficking in the brain and increased iron content in skeletal muscle. Exercise reduced levels of cortical hepcidin, a key regulator of iron homeostasis, coupled with interleukin-6 (IL-6) decrease in cortex and plasma. We propose that regular exercise induces a reduction of hepcidin in the brain, possibly via the IL-6/STAT3/JAK1 pathway. These findings indicate that regular exercise modulates iron homeostasis in both wild-type and AD mice.

SLN124, a GalNac-siRNA targeting transmembrane serine protease 6, in combination with deferiprone therapy reduces ineffective erythropoiesis and hepatic iron-overload in a mouse model of ß-thalassaemia.

Beta-thalassaemia is an inherited blood disorder characterised by ineffective erythropoiesis and anaemia. Consequently, hepcidin expression is reduced resulting in increased iron absorption and primary iron overload. Hepcidin is under the negative control of transmembrane serine protease 6 (TMPRSS6) via cleavage of haemojuvelin (HJV), a co-receptor for the bone morphogenetic protein (BMP)-mothers against decapentaplegic homologue (SMAD) signalling pathway. Considering the central role of the TMPRSS6/HJV/hepcidin axis in iron homeostasis, the inhibition of TMPRSS6 expression represents a promising therapeutic strategy to increase hepcidin production and ameliorate anaemia and iron overload in ß-thalassaemia. In the present study, we investigated a small interfering RNA (siRNA) conjugate optimised for hepatic targeting of Tmprss6 (SLN124) in ß-thalassaemia mice (Hbbth3/+ ). Two subcutaneous injections of SLN124 (3 mg/kg) were sufficient to normalise hepcidin expression and reduce anaemia. We also observed a significant improvement in erythroid maturation, which was associated with a significant reduction in splenomegaly. Treatment with the iron chelator, deferiprone (DFP), did not impact any of the erythroid parameters. However, the combination of SLN124 with DFP was more effective in reducing hepatic iron overload than either treatment alone. Collectively, we show that the combination therapy can ameliorate several disease symptoms associated with chronic anaemia and iron overload, and therefore represents a promising pharmacological modality for the treatment of ß-thalassaemia and related disorders.

Iron accumulation in skeletal muscles of old mice is associated with impaired regeneration after ischaemia-reperfusion damage.

BACKGROUND: Oxidative stress is implicated in the insidious loss of muscle mass and strength that occurs with age. However, few studies have investigated the role of iron, which is elevated during ageing, in age-related muscle wasting and blunted repair after injury. We hypothesized that iron accumulation leads to membrane lipid peroxidation, muscle wasting, increased susceptibility to injury, and impaired muscle regeneration. METHODS: To examine the role of iron in age-related muscle atrophy, we compared the skeletal muscles of 3-month-old with 22- to 24-month-old 129SvEv FVBM mice. We assessed iron distribution and total elemental iron using laser ablation inductively coupled plasma mass spectrometry and Perls' stain on skeletal muscle cross-sections. In addition, old mice underwent ischaemia-reperfusion (IR) injury (90 min ischaemia), and muscle regeneration was assessed 14 days after injury. Immunoblotting was used to determine lipid peroxidation (4HNE) and iron-related proteins. To determine whether muscle iron content can be altered, old mice were treated with deferiprone (DFP) in the drinking water, and we assessed its effects on muscle regeneration after injury. RESULTS: We observed a significant increase in total elemental iron (+43%, P < 0.05) and lipid peroxidation (4HNE: +76%, P < 0.05) in tibialis anterior muscles of old mice. Iron was further increased after injury (adult: +81%, old: +135%, P < 0.05) and associated with increased lipid peroxidation (+41%, P < 0.05). Administration of DFP did not impact iron or measures of lipid peroxidation in skeletal muscle or modulate muscle mass. Increased muscle iron concentration and lipid peroxidation were associated with less efficient regeneration, evident from the smaller fibres in cross-sections of tibialis anterior muscles (-24%, P < 0.05) and an increased percentage of fibres with centralized nuclei (+4124%, P < 0.05) in muscles of old compared with adult mice. Administration of DFP lowered iron after IR injury (PRE: -32%, P < 0.05 and POST: -41%, P < 0.05), but did not translate to structural improvements. CONCLUSIONS: Muscles from old mice have increased iron levels, which are associated with increased lipid peroxidation, increased susceptibility to IR injury, and impaired muscle regeneration. Our results suggest that iron is involved in effective muscle regeneration, highlighting the importance of iron homeostasis in muscle atrophy and muscle repair.

A potential role for zinc in restless legs syndrome.

STUDY OBJECTIVES: Evaluate serum and brain noniron metals in the pathology and genetics of restless legs syndrome (RLS). METHODS: In two independent studies (cohorts 1 and 2), in which subjects either remained on medications or tapered off medications, we analyzed serum levels of iron, calcium, magnesium, manganese, copper, and zinc both in RLS patients and controls, and assessed the prevalence of the MEIS1 and BTBD9 risk alleles previously established through genome-wide association studies. Human brain sections and a nematode genetic model were also quantified for metal levels using mass spectrometry. RESULTS: We found a significant enrichment for the BTBD9 risk genotype in the RLS affected group compared to control (p = 0.0252), consistent with previous literature. Serum (p = 0.0458 and p = 0.0139 for study cohorts 1 and 2, respectively) and brain (p = 0.0413) zinc levels were significantly elevated in the RLS patients versus control subjects. CONCLUSION: We show for the first time that serum and brain levels of zinc are elevated in RLS. Further, we confirm the BTBD9 genetic risk factor in a new population, although the zinc changes were not significantly associated with risk genotypes. Zinc and iron homeostasis are interrelated, and zinc biology impacts neurotransmitter systems previously linked to RLS. Given the modest albeit statistically significant increase in serum zinc of ~20%, and the lack of association with two known genetic risk factors, zinc may not represent a primary etiology for the syndrome. Further investigation into the pathogenetic role that zinc may play in restless legs syndrome is needed.

Copper-ATSM as a Treatment for ALS: Support from Mutant SOD1 Models and Beyond.

The blood-brain barrier permeant, copper-containing compound, CuII(atsm), has successfully progressed from fundamental research outcomes in the laboratory through to phase 2/3 clinical assessment in patients with the highly aggressive and fatal neurodegenerative condition of amyotrophic lateral sclerosis (ALS). The most compelling outcomes to date to indicate potential for disease-modification have come from pre-clinical studies utilising mouse models that involve transgenic expression of mutated superoxide dismutase 1 (SOD1). Mutant SOD1 mice provide a very robust mammalian model of ALS with high validity, but mutations in SOD1 account for only a small percentage of ALS cases in the clinic, with the preponderant amount of cases being sporadic and of unknown aetiology. As per other putative drugs for ALS developed and tested primarily in mutant SOD1 mice, this raises important questions about the pertinence of CuII(atsm) to broader clinical translation. This review highlights some of the challenges associated with the clinical translation of new treatment options for ALS. It then provides a brief account of pre-clinical outcomes for CuII(atsm) in SOD1 mouse models of ALS, followed by an outline of additional studies which report positive outcomes for CuII(atsm) when assessed in cell and mouse models of neurodegeneration which do not involve mutant SOD1. Clinical evidence for CuII(atsm) selectively targeting affected regions of the CNS in patients is also presented. Overall, this review summarises the existing evidence which indicates why clinical relevance of CuII(atsm) likely extends beyond the context of cases of ALS caused by mutant SOD1.

Effects of paracetamol (acetaminophen) on gene expression and permeability properties of the rat placenta and fetal brain.

Background: Paracetamol (acetaminophen) is widely used in pregnancy and generally regarded as "safe" by regulatory authorities. Methods: Clinically relevant doses of paracetamol were administered intraperitoneally to pregnant rats twice daily from embryonic day E15 to 19 (chronic) or as a single dose at E19 (acute). Control samples were from un-treated age-matched animals. At E19, rats were anaesthetised, administered a final paracetamol dose, uteruses were opened and fetuses exposed for sample collection. For RNA sequencing, placentas and fetal brains were removed and flash frozen. Fetal and maternal plasma and cerebrospinal fluid were assayed for ⍺-fetoprotein and interleukin 1ß (IL1ß). Brains were fixed and examined (immunohistochemistry) for plasma protein distribution. Placental permeability to a small molecule ( 14C-sucrose) was tested by injection into either mother or individual fetuses; fetal and maternal blood was sampled at regular intervals to 90 minutes. Results: RNA sequencing revealed a large number of genes up- or down-regulated in placentas from acutely or chronically treated animals compared to controls. Most notable was down-regulation of three acute phase plasma proteins (⍺-fetoprotein, transferrin, transthyretin) in acute and especially chronic experiments and marked up-regulation of immune-related genes, particularly cytokines, again especially in chronically treated dams. IL1ß increased in plasma of most fetuses from treated dams but to variable levels and no IL1ß was detectable in plasma of control fetuses or any of the dams. Increased placental permeability appeared to be only from fetus to mother for both 14C-sucrose and ⍺-fetoprotein, but not in the reverse direction. In the fetal brain, gene regulatory changes were less prominent than in the placenta of treated fetuses and did not involve inflammatory-related genes; there was no evidence of increased blood-brain barrier permeability. Conclusion: Results suggest that paracetamol may induce an immune-inflammatory-like response in placenta and more caution should be exercised in use of paracetamol in pregnancy.

Potential Diagnostic Imaging of Alzheimer's Disease with Copper-64 Complexes That Bind to Amyloid-ß Plaques.

Amyloid-ß plaques, consisting of aggregated amyloid-ß peptides, are one of the pathological hallmarks of Alzheimer's disease. Copper complexes formed using positron-emitting copper radionuclides that cross the blood-brain barrier and bind to specific molecular targets offer the possibility of noninvasive diagnostic imaging using positron emission tomography. New thiosemicarbazone-pyridylhydrazone based ligands that incorporate pyridyl-benzofuran functional groups designed to bind amyloid-ß plaques have been synthesized. The ligands form stable complexes with copper(II) ( Kd = 10-18 M) and can be radiolabeled with copper-64 at room temperature. Subtle changes to the periphery of the ligand backbone alter the metabolic stability of the complexes in mouse and human liver microsomes, and influenced the ability of the complexes to cross the blood-brain barrier in mice. A lead complex was selected based on possessing the best metabolic stability and brain uptake in mice. Synthesis of this lead complex with isotopically enriched copper-65 allowed us to show that the complex bound to amyloid-ß plaques present in post-mortem human brain tissue using laser ablation-inductively coupled plasma-mass spectrometry. This work provides insight into strategies to target metal complexes to amyloid-ß plaques, and how small modifications to ligands can dramatically alter the metabolic stability of metal complexes as well as their ability to cross the blood-brain barrier.

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.

CuII(atsm) Attenuates Neuroinflammation.

Background: Neuroinflammation and biometal dyshomeostasis are key pathological features of several neurodegenerative diseases, including Alzheimer's disease (AD). Inflammation and biometals are linked at the molecular level through regulation of metal buffering proteins such as the metallothioneins. Even though the molecular connections between metals and inflammation have been demonstrated, little information exists on the effect of copper modulation on brain inflammation. Methods: We demonstrate the immunomodulatory potential of the copper bis(thiosemicarbazone) complex CuII(atsm) in an neuroinflammatory model in vivo and describe its anti-inflammatory effects on microglia and astrocytes in vitro. Results: By using a sophisticated in vivo magnetic resonance imaging (MRI) approach, we report the efficacy of CuII(atsm) in reducing acute cerebrovascular inflammation caused by peripheral administration of bacterial lipopolysaccharide (LPS). CuII(atsm) also induced anti-inflammatory outcomes in primary microglia [significant reductions in nitric oxide (NO), monocyte chemoattractant protein 1 (MCP-1), and tumor necrosis factor (TNF)] and astrocytes [significantly reduced NO, MCP-1, and interleukin 6 (IL-6)] in vitro. These anti-inflammatory actions were associated with increased cellular copper levels and increased the neuroprotective protein metallothionein-1 (MT1) in microglia and astrocytes. Conclusion: The beneficial effects of CuII(atsm) on the neuroimmune system suggest copper complexes are potential therapeutics for the treatment of neuroinflammatory conditions.

The accumulation of enzymatically inactive cuproenzymes is a CNS-specific phenomenon of the SOD1G37R mouse model of ALS and can be restored by overexpressing the human copper transporter hCTR1.

Mutations to the copper-dependent enzyme Cu/Zn-superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS) in humans, and transgenic overexpression of mutant SOD1 represents a robust murine model of the disease. We have previously shown that the copper-containing compound CuII(atsm) phenotypically improves mutant SOD1 mice and delivers copper to copper-deficient SOD1 in the CNS to restore its physiological function. CuII(atsm) is now in clinical trials for the treatment of ALS. In this study, we demonstrate that cuproenzyme dysfunction extends beyond SOD1 in SOD1G37R mice to also affect the endogenous copper-dependent ferroxidase ceruloplasmin. We show that SOD1 and ceruloplasmin both accumulate progressively in the SOD1G37R mouse spinal cord as the animals' ALS-like symptoms progress, yet the biochemical activity of the two cuproenzymes does not increase commensurately, indicating that, as per mutant SOD1, ceruloplasmin accumulates in a copper-deficient form. Consistent with this finding, we show that expression of the human copper transporter 1 (hCTR1) in SOD1G37R mice increases copper levels in the spinal cord and concurrently restores SOD1 and ceruloplasmin activity. Soluble misfolded SOD1, a proposed driver of pathology in this model, is readily detectable in the SOD1G37R mouse spinal cord. However, misfolded SOD1G37R levels do not change in abundance with disease progression and are less abundant than misfolded SOD1 in the spinal cords of age-matched transgenic SOD1WT mice which do not exhibit an evident ALS-like phenotype. Collectively, these outcomes support a copper malfunction phenomenon in mutant SOD1 mouse models of ALS and a copper-related mechanism of action for the therapeutic agent CuII(atsm).