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Cells have developed a highly regulated system for the uptake, transport, utilization, storage, and export of metals, ensuring the maintenance of cellular homeostasis. Small extracellular vesicles (sEVs) function as a mechanism through which a cell can export its cargo and transfer it to recipient cells. However, in contrast to the other molecular cargo associated with sEVs, the metal content of sEVs is not well characterized. To address this gap in knowledge, we measured the levels of nine essential metals (copper, iron, zinc, manganese, magnesium, potassium, calcium, chromium, cobalt) and six non-essential metals (nickel, rubidium, titanium, aluminium, lithium, lead) in sEVs originating from multiple in vitro and ex vivo sources. Our findings reveal that, beyond containing redox-active essential metals and those involved in redox reactions, sEVs also exhibit the capability to export and transfer non-physiological, potentially toxic metals., Competing Interests: The authors report no conflict of interest., (© 2024 The Author(s). Journal of Extracellular Biology published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

The Alzheimer's disease (AD)-affected brain purges K with concurrently increasing serum K, suggesting brain-blood K transferal. Here, natural stable K isotope ratios-δ41K-of human serum samples were characterized in an AD biomarker pilot study (plus two paired Li-heparin and potassium ethylenediaminetetraacetic acid [K-EDTA] plasma samples). AD serum was found to have a significantly lower mean δ41K relative to controls. To mechanistically explore this change, novel ab initio calculations (density functional theory) of relative K isotope compositions between hydrated K+ and organically bound K were performed, identifying hydrated K+ as isotopically light (lower δ41K) compared to organically bound K. Taken together with literature, serum δ41K and density functional theory results are consistent with efflux of hydrated K+ from the brain to the bloodstream, manifesting a measurable decrease in serum δ41K. These data introduce serum δ41K for further investigation as a minimally invasive AD biomarker, with cost, scalability, and stability advantages over current techniques., (© The Author(s) 2024. Published by Oxford University Press.)

The strongest genetic risk factor for late-onset Alzheimer's disease (AD) is allelic variation of the APOE gene, with the following risk structure: ε4 > ε3 > ε2. The biochemical basis for this risk profile is unclear. Here, we reveal a new role for the APOE gene product, apolipoprotein E (ApoE) in regulating cellular copper homeostasis, which is perturbed in the AD brain. Exposure of ApoE target replacement (TR) astrocytes (immortalised astrocytes from APOE knock-in mice) to elevated copper concentrations resulted in exacerbated copper accumulation in ApoE4- compared to ApoE2- and ApoE3-TR astrocytes. This effect was also observed in SH-SY5Y neuroblastoma cells treated with conditioned medium from ApoE4-TR astrocytes. Increased intracellular copper levels in the presence of ApoE4 may be explained by reduced levels and delayed trafficking of the copper transport protein, copper-transporting ATPase 1 (ATP7A/Atp7a), potentially leading to impaired cellular copper export. This new role for ApoE in copper regulation lends further biochemical insight into how APOE genotype confers risk for AD and reveals a potential contribution of ApoE4 to the copper dysregulation that is a characteristic pathological feature of the AD brain., (© 2024 The Author(s). Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.)

Multiple sclerosis (MS) is a debilitating affliction of the central nervous system (CNS) that involves demyelination of neuronal axons and neurodegeneration resulting in disability that becomes more pronounced in progressive forms of the disease. The involvement of neurodegeneration in MS underscores the need for effective neuroprotective approaches necessitating identification of new therapeutic targets. Herein, we applied an integrated elemental analysis workflow to human MS-affected spinal cord tissue utilising multiple inductively coupled plasma-mass spectrometry methodologies. These analyses revealed shifts in atomic copper as a notable aspect of disease. Complementary gene expression and biochemical analyses demonstrated that changes in copper levels coincided with altered expression of copper handling genes and downstream functionality of cuproenzymes. Copper-related problems observed in the human MS spinal cord were largely reproduced in the experimental autoimmune encephalomyelitis (EAE) mouse model during the acute phase of disease characterised by axonal demyelination, lesion formation, and motor neuron loss. Treatment of EAE mice with the CNS-permeant copper modulating compound Cu II (atsm) resulted in recovery of cuproenzyme function, improved myelination and lesion volume, and neuroprotection. These findings support targeting copper perturbations as a therapeutic strategy for MS with Cu II (atsm) showing initial promise., Competing Interests: Declaration of competing interest Collaborative Medicinal Development has licensed intellectual property pertaining to Cu(II)(atsm) from the University of Melbourne where the inventors include ARW and PSD. AIB is a paid consultant for Collaborative Medicinal Development LLC and has a profit share interest in Collaborative Medicinal Development Pty Ltd. PJC and JSB are unpaid consultants for Collaborative Medicinal Development LLC. DJH received research and material support from Agilent Technologies and ESI Ltd., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: The associations between mood disorders (anxiety and depression) and mild cognitive impairment (MCI) or Alzheimer's dementia (AD) remain unclear., Methods: Data from the Australian Imaging, Biomarker & Lifestyle (AIBL) study were subjected to logistic regression to determine both cross-sectional and longitudinal associations between anxiety/depression and MCI/AD. Effect modification by selected covariates was analysed using the likelihood ratio test., Results: Cross-sectional analysis was performed to explore the association between anxiety/depression and MCI/AD among 2,209 participants with a mean [SD] age of 72.3 [7.4] years, of whom 55.4% were female. After adjusting for confounding variables, we found a significant increase in the odds of AD among participants with two mood disorders (anxiety: OR 1.65 [95% CI 1.04-2.60]; depression: OR 1.73 [1.12-2.69]). Longitudinal analysis was conducted to explore the target associations among 1,379 participants with a mean age of 71.2 [6.6] years, of whom 56.3% were female. During a mean follow-up of 5.0 [4.2] years, 163 participants who developed MCI/AD (refer to as PRO) were identified. Only anxiety was associated with higher odds of PRO after adjusting for covariates (OR 1.56 [1.03-2.39]). However, after additional adjustment for depression, the association became insignificant. Additionally, age, sex, and marital status were identified as effect modifiers for the target associations., Conclusion: Our study provides supportive evidence that anxiety and depression impact on the evolution of MCI/AD, which provides valuable epidemiological insights that can inform clinical practice, guiding clinicians in offering targeted dementia prevention and surveillance programs to the at-risk populations., (© 2024. The Author(s).)

Ferroptosis, an intricately regulated form of cell death characterized by uncontrolled lipid peroxidation, has garnered substantial interest since this term was first coined in 2012. Recent years have witnessed remarkable progress in elucidating the detailed molecular mechanisms that govern ferroptosis induction and defence, with particular emphasis on the roles of heterogeneity and plasticity. In this Review, we discuss the molecular ecosystem of ferroptosis, with implications that may inform and enable safe and effective therapeutic strategies across a broad spectrum of diseases., (© 2024. Springer Nature Limited.)

Lipids play a significant role in maintaining central nervous system (CNS) structure and function, and the dysregulation of lipid metabolism is known to occur in many neurological disorders, including Alzheimer's disease. Here we review what is currently known about lipid dyshomeostasis in Alzheimer's disease. We propose that small extracellular vesicle (sEV) lipids may provide insight into the pathophysiology and progression of Alzheimer's disease. This stems from the recognition that sEV likely contributes to disease pathogenesis, but also an understanding that sEV can serve as a source of potential biomarkers. While the protein and RNA content of sEV in the CNS diseases have been studied extensively, our understanding of the lipidome of sEV in the CNS is still in its infancy., (© 2023 The Authors. PROTEOMICS published by Wiley‐VCH GmbH.)

Background: Current antidepressants have limitations due to insufficient efficacy and delay before improvement in symptoms. Polymorphisms of the serotonin transporter (5-HTT) gene have been linked to depression (when combined with stressful life events) and altered response to selective serotonergic reuptake inhibitors. We have previously revealed the antidepressant-like properties of the iron chelator deferiprone in the 5-HTT knock-out (KO) mouse model of depression. Furthermore, deferiprone was found to alter neural activity in the prefrontal cortex of both wild-type (WT) and 5-HTT KO mice., Methods: In the current study, we examined the molecular effects of acute deferiprone treatment in the prefrontal cortex of both genotypes via phosphoproteomics analysis., Results: In WT mice treated with deferiprone, there were 22 differentially expressed phosphosites, with gene ontology analysis implicating cytoskeletal proteins. In 5-HTT KO mice treated with deferiprone, we found 33 differentially expressed phosphosites. Gene ontology analyses revealed phosphoproteins that were predominantly involved in synaptic and glutamatergic signalling. In a drug-naïve cohort (without deferiprone administration), the analysis revealed 21 differentially expressed phosphosites in 5-HTT KO compared to WT mice. We confirmed the deferiprone-induced increase in tyrosine hydroxylase serine 40 residue phosphorylation (pTH-Ser40) (initially revealed in our phosphoproteomics study) by Western blot analysis, with deferiprone increasing pTH-Ser40 expression in WT and 5-HTT KO mice., Conclusion: As glutamatergic and synaptic signalling are dysfunctional in 5-HTT KO mice (and are the target of fast-acting antidepressant drugs such as ketamine), these molecular effects may underpin deferiprone's antidepressant-like properties. Furthermore, dopaminergic signalling may also be involved in deferiprone's antidepressant-like properties., Competing Interests: Declaration of competing interest The authors have no conflict of interest to report., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The copper compound Cu II (atsm) has progressed to phase 2/3 testing for treatment of the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Cu II (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 Cu II (atsm). Our results from human spinal cord indicate a therapeutic mechanism of action for Cu II (atsm) involving copper availability may also be pertinent to sporadic cases of ALS., (© 2024. The Author(s).)

Background and Purpose: Magnetic resonance spectroscopy (MRS) measures neurochemicals in vivo. Glutathione (GSH) is a neuroprotective chemical shown to vary significantly in patients with Alzheimer's disease (AD). This work investigates the reproducibility of GSH measures in the mesial temporal lobe (MTL) to identify its potential clinical utility., Methods: MRS data were acquired from eight healthy volunteers (31.1 ± 5.2 years; 4 male/female) using Mescher-Garwood-Point Resolved Spectroscopy (MEGA-PRESS) from the MTL in the left hemisphere across two scan sessions in the same visit. Total N-acetylaspartate (tNAA), choline (tCho), creatine (tCr), and GSH were quantified. Reproducibility of quantifications of these neurochemicals were tested using coefficient of variance (CV) between scan sessions. Reproducibility of voxel placement on the left MTL was calculated by measuring the tissue overlap and percent of hippocampus within that voxel. CV measured across different scan sessions in each individual, with a CV<15% was accepted as "good" reproducibility. Paired t-tests were carried out to establish the significant differences between the two scans across each individual with p<.05 as significant., Results: TNAA (%CV = 7.2; p = .5), tCr (%CV = 7.8; p = .6) and tCho (%CV = 9.3; p = .4), and GSH (%CV = 22; p = .1). The dice coefficient that reflects the level of overlap of hippocampal tissue in the voxel was shown to be 0.8 ± 0.1. Voxel tissue composition were: Scan 1 (cerebrospinal fluid [CSF]: 5 ± 1%, white matter [WM]: 52 ± 3%, gray matter [GM]: 43 ± 3%); Scan 2 (CSF: 5 ± 1%, WM: 52 ± 4%, GM: 44 ± 4%)., Conclusion: The data suggest measures of abundant metabolites in the MTL using the MEGA-PRESS sequence has a high reproducibility. Reproducibility of GSH in this area was poorer requiring care when interpreting measures of GSH in the MTL for clinical translational purposes., (© 2024 The Authors. Journal of Neuroimaging published by Wiley Periodicals LLC on behalf of American Society of Neuroimaging.)

A great deal of nanocarriers have been applied to induce ferroptosis in cancer research, yet there are limited examples of nanocarrier formulations to rescue ferroptosis, which can be applied to neurodegeneration, inflammation, liver damage, kidney disease, and more. Here, we present the synthesis, characterization, and in vitro evaluation of pH-responsive, core-cross-linked micelle (CCM) ferrostatin-1 (Fer-1) conjugates with amine, valproic acid, and biotin surface chemistries. Fer-1 release from stable and defined CCM Fer-1 conjugates was quantified, highlighting the sustained release for 24 h. CCM Fer-1 conjugates demonstrated excellent ferroptosis rescue by their antilipid peroxidation activity in a diverse set of cell lines in vitro. Additionally, CCMs showed tunable cell association in SH-SY5Y and translocation across an in vitro blood-brain barrier (BBB) model, highlighting potential brain disease applications. Overall, here, we present a polymeric Fer-1 delivery system to enhance Fer-1 action, which could help in improving Fer-1 action in the treatment of ferroptosis-related diseases.

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 SOD1 G37R 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 SOD1 G37R mouse model where treatment with a CNS-permeant ferroptosis inhibitor, Cu II (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., (© 2024. The Author(s).)

Allelic variation to the APOE gene confers the greatest genetic risk for sporadic Alzheimer's disease (AD). Independent of genotype, low abundance of apolipoprotein E (apoE), is characteristic of AD CSF, and predicts cognitive decline. The mechanisms underlying the genotype and apoE level risks are uncertain. Recent fluid and imaging biomarker studies have revealed an unexpected link between apoE and brain iron, which also forecasts disease progression, possibly through ferroptosis, an iron-dependent regulated cell death pathway. Here, we report that apoE is a potent inhibitor of ferroptosis (EC 50  ≈ 10 nM; N27 neurons). We demonstrate that apoE signals to activate the PI3K/AKT pathway that then inhibits the autophagic degradation of ferritin (ferritinophagy), thus averting iron-dependent lipid peroxidation. Using postmortem inferior temporal brain cortex tissue from deceased subjects from the Rush Memory and Aging Project (MAP) (N = 608), we found that the association of iron with pathologically confirmed clinical Alzheimer's disease was stronger among those with the adverse APOE-ε4 allele. While protection against ferroptosis did not differ between apoE isoforms in vitro, other features of ε4 carriers, such as low abundance of apoE protein and higher levels of polyunsaturated fatty acids (which fuel ferroptosis) could mediate the ε4 allele's heighted risk of AD. These data support ferroptosis as a putative pathway to explain the major genetic risk associated with late onset AD., (© 2022. The Author(s).)