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Abstract 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.

Abstract 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. Graphical Abstract

Abstract Background Anorexia nervosa (AN) is a potentially fatal psychiatric condition, associated with structural brain changes such as gray matter volume loss. The pathophysiological mechanisms for these changes are not yet fully understood. Iron is a crucial element in the development and function of the brain. Considering the systemic alterations in iron homeostasis in AN, we hypothesized that brain iron would be altered as a possible factor associated with structural brain changes in AN. Methods In this study, we used quantitative susceptibility mapping (QSM) magnetic resonance imaging to investigate brain iron in current AN (c-AN) and weight-restored AN compared with healthy individuals. Whole-brain voxel wise comparison was used to probe areas with possible group differences. Further, the thalamus, caudate nucleus, putamen, nucleus accumbens, hippocampus, and amygdala were selected as the regions of interest (ROIs) for ROI-based comparison of mean QSM values. Results Whole-brain voxel-wise and ROI-based comparison of QSM did not reveal any differences between groups. Exploratory analyses revealed a correlation between higher regional QSM (higher iron) and lower body mass index, higher illness severity, longer illness duration, and younger age at onset in the c-AN group. Conclusions This study did not find evidence of altered brain iron in AN compared to healthy individuals. However, the correlations between clinical variables and QSM suggest a link between brain iron and weight status or biological processes in AN, which warrants further investigation.

Abstract Background Despite overall improvement in breast cancer patient outcomes from earlier diagnosis and personalised treatment approaches, some patients continue to experience recurrence and incurable metastases. It is therefore imperative to understand the molecular changes that allow transition from a non-aggressive state to a more aggressive phenotype. This transition is governed by a number of factors. Methods As crosstalk with extracellular matrix (ECM) is critical for tumour cell growth and survival, we applied high throughput shRNA screening on a validated ‘3D on-top cellular assay’ to identify novel growth suppressive mechanisms. Results A number of novel candidate genes were identified. We focused on COMMD3, a previously poorly characterised gene that suppressed invasive growth of ER + breast cancer cells in the cellular assay. Analysis of published expression data suggested that COMMD3 is normally expressed in the mammary ducts and lobules, that expression is lost in some tumours and that loss is associated with lower survival probability. We performed immunohistochemical analysis of an independent tumour cohort to investigate relationships between COMMD3 protein expression, phenotypic markers and disease-specific survival. This revealed an association between COMMD3 loss and shorter survival in hormone-dependent breast cancers and in particularly luminal-A-like tumours (ER+/Ki67-low; 10-year survival probability 0.83 vs. 0.73 for COMMD3-positive and -negative cases, respectively). Expression of COMMD3 in luminal-A-like tumours was directly associated with markers of luminal differentiation: c-KIT, ELF5, androgen receptor and tubule formation (the extent of normal glandular architecture; p

Situated at the intersection of technology and medicine, the Internet of Things (IoT) holds the promise of addressing some of healthcare's most pressing challenges, from medical error, to chronic drug shortages, to overburdened hospital systems, to dealing with the COVID-19 pandemic. However, despite considerable recent technological advances, the pace of successful implementation of promising IoT healthcare initiatives has been slow. To inspire more productive collaboration, we present here a simple—but surprisingly underrated—problem-oriented approach to developing healthcare technologies. To further assist in this effort, we reviewed the various commercial, regulatory, social/cultural, and technological factors in the development of the IoT. We propose that fog computing—a technological paradigm wherein the burden of computing is shifted from a centralized cloud server closer to the data source—offers the greatest promise for building a robust and scalable healthcare IoT ecosystem. To this end, we explore the key enabling technologies that underpin the fog architecture, from the sensing layer all the way up to the cloud. It is our hope that ongoing advances in sensing, communications, cryptography, storage, machine learning, and artificial intelligence will be leveraged in meaningful ways to generate unprecedented medical intelligence and thus drive improvements in the health of many people.

Vitamin A (retinol) is a lipid-soluble vitamin that acts as a precursor for several bioactive compounds, such as retinaldehyde (retinal) and isomers of retinoic acid. Retinol and all-trans-retinoic acid (atRA) penetrate the blood-brain barrier and are reported to be neuroprotective in several animal models. We characterised the impact of retinol and its metabolites, all-trans-retinal (atRAL) and atRA, on ferroptosis—a programmed cell death caused by iron-dependent phospholipid peroxidation. Ferroptosis was induced by erastin, buthionine sulfoximine or RSL3 in neuronal and non-neuronal cell lines. We found that retinol, atRAL and atRA inhibited ferroptosis with a potency superior to α-tocopherol, the canonical anti-ferroptotic vitamin. In contrast, we found that antagonism of endogenous retinol with anhydroretinol sensitises ferroptosis induced in neuronal and non-neuronal cell lines. Retinol and its metabolites atRAL and atRA directly interdict lipid radicals in ferroptosis since these compounds displayed radical trapping properties in a cell-free assay. Vitamin A, therefore, complements other anti-ferroptotic vitamins, E and K; metabolites of vitamin A, or agents that alter their levels, may be potential therapeutics for diseases where ferroptosis is implicated.

Abstract 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

Dysregulation of lipid metabolism is associated with coronary artery disease (CAD). Here, the authors perform GWAS of the serum lipidome to identify variants associated with lipid species that are putatively in the mechanistic pathway to CAD.

Abstract Ischemic stroke represents a significant danger to human beings, especially the elderly. Interventions are only available to remove the clot, and the mechanism of neuronal death during ischemic stroke is still in debate. Ferroptosis is increasingly appreciated as a mechanism of cell death after ischemia in various organs. Here we report that the serine protease, thrombin, instigates ferroptotic signaling by promoting arachidonic acid mobilization and subsequent esterification by the ferroptotic gene, acyl-CoA synthetase long-chain family member 4 (ACSL4). An unbiased multi-omics approach identified thrombin and ACSL4 genes/proteins, and their pro-ferroptotic phosphatidylethanolamine lipid products, as prominently altered upon the middle cerebral artery occlusion in rodents. Genetically or pharmacologically inhibiting multiple points in this pathway attenuated outcomes of models of ischemia in vitro and in vivo. Therefore, the thrombin-ACSL4 axis may be a key therapeutic target to ameliorate ferroptotic neuronal injury during ischemic stroke.

Abstract Background The Australian Imaging and Biomarker Lifestyle (AIBL) study of aging is designed to aid the discovery of biomarkers. The current study aimed to discover differentially expressed plasma proteins that could yield a blood-based screening tool for Alzheimer’s disease. Methods The concentration of proteins in plasma covers a vast range of 12 orders of magnitude. Therefore, to search for medium to low abundant biomarkers and elucidate mechanisms of AD, we immuno-depleted the most abundant plasma proteins and pre-fractionated the remaining proteins by HPLC, prior to two-dimensional gel electrophoresis. The relative levels of approximately 3400 protein species resolved on the 2D gels were compared using in-gel differential analysis with spectrally resolved fluorescent protein detection dyes (Zdyes™). Here we report on analysis of pooled plasma samples from an initial screen of a sex-matched cohort of 72 probable AD patients and 72 healthy controls from the baseline time point of AIBL. Results We report significant changes in variants of apolipoprotein E, haptoglobin, α1 anti-trypsin, inter-α trypsin inhibitor, histidine-rich glycoprotein, and a protein of unknown identity. α1 anti-trypsin and α1 anti-chymotrypsin demonstrated plasma concentrations that were dependent on APOE ε4 allele dose. Our analysis also identified an association with the level of Vitamin D binding protein fragments and complement factor I with sex. We then conducted a preliminary validation study, on unique individual samples compared to the discovery cohort, using a targeted LC-MS/MS assay on a subset of discovered biomarkers. We found that targets that displayed a high degree of isoform specific changes in the 2D gels were not changed in the targeted MS assay which reports on the total level of the biomarker. Conclusions This demonstrates that further development of mass spectrometry assays is needed to capture the isoform complexity that exists in theses biological samples. However, this study indicates that a peripheral protein signature has potential to aid in the characterization of AD.

P-glycoprotein (P-gp), expressed at the blood-brain barrier (BBB), is critical in preventing brain access to substrate drugs and effluxing amyloid beta (Aβ), a contributor to Alzheimer’s disease (AD). Strategies to regulate P-gp expression therefore may impact central nervous system (CNS) drug delivery and brain Aβ levels. As we have demonstrated that the copper complex copper diacetyl bis(4-methyl-3-thiosemicarbazone) (Cu(ATSM)) increases P-gp expression and function in human brain endothelial cells, the present study assessed the impact of Cu(ATSM) on expression and function of P-gp in mouse brain endothelial cells (mBECs) and capillaries in vivo, as well as in peripheral organs. Isolated mBECs treated with Cu(ATSM) (100 nM for 24 h) exhibited a 1.6-fold increase in P-gp expression and a 20% reduction in accumulation of the P-gp substrate rhodamine 123. Oral administration of Cu(ATSM) (30 mg/kg/day) for 28 days led to a 1.5 & 1.3-fold increase in brain microvascular and hepatic expression of P-gp, respectively, and a 20% reduction in BBB transport of [3H]-digoxin. A metallomic analysis showed a 3.5 and 19.9-fold increase in Cu levels in brain microvessels and livers of Cu(ATSM)-treated mice. Our findings demonstrate that Cu(ATSM) increases P-gp expression and function at the BBB in vivo, with implications for CNS drug delivery and clearance of Aβ in AD.

Abstract Biological applications of nanomaterials as delivery carriers have been embedded in traditional biomedical research for decades. Despite lagging behind, recent significant breakthroughs in the use of nanocarriers as tools for plant biotechnology have created great interest. In this Perspective, we review the outstanding recent works in nanocarrier‐mediated plant transformation and its agricultural applications. We analyze the chemical and physical properties of nanocarriers determining their uptake efficiency and transport throughout the plant body.

Metals such as calcium and potassium have long been known to regulate the diameter of arteries that control blood flow. Here, we report that zinc causes relaxation of blood vessels and reduces blood pressure by its coordinated action in sensory nerves, endothelium and smooth muscle cells.

Abstract 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

The onset and pathology of Alzheimer’s disease (AD) is associated with changes to lipid metabolism. Here, the authors analysed 569 lipids from 32 classes and subclasses in two independent patient cohorts to identify key lipid pathways to link the plasma lipidome with AD and the future onset of AD.

Multiple System Atrophy (MSA) is a rare neurodegenerative synucleinopathy which leads to severe disability followed by death within 6–9 years of symptom onset. There is compelling evidence suggesting that biological trace metals like iron and copper play an important role in synucleinopathies like Parkinson's disease and removing excess brain iron using chelators could slow down the disease progression. In human MSA, there is evidence of increased iron in affected brain regions, but role of iron and therapeutic efficacy of iron-lowering drugs in pre-clinical models of MSA have not been studied.We studied age-related changes in iron metabolism in different brain regions of the PLP-αsyn mice and tested whether iron-lowering drugs could alleviate disease phenotype in aged PLP-αsyn mice. Iron content, iron-ferritin association, ferritin protein levels and copper-ceruloplasmin association were measured in prefrontal cortex, putamen, substantia nigra and cerebellum of 3, 8, and 20-month-old PLP-αsyn and age-matched non-transgenic mice. Moreover, 12-month-old PLP-αsyn mice were administered deferiprone or ceruloplasmin or vehicle for 2 months. At the end of treatment period, motor testing and stereological analyses were performed.We found iron accumulation and perturbed iron-ferritin interaction in substantia nigra, putamen and cerebellum of aged PLP-αsyn mice. Furthermore, we found significant reduction in ceruloplasmin-bound copper in substantia nigra and cerebellum of the PLP-αsyn mice. Both deferiprone and ceruloplasmin prevented decline in motor performance in aged PLP-αsyn mice and were associated with higher neuronal survival and reduced density of α-synuclein aggregates in substantia nigra.This is the first study to report brain iron accumulation in a mouse model of MSA. Our results indicate that elevated iron in MSA mice may result from ceruloplasmin dysfunction and provide evidence that targeting iron in MSA could be a viable therapeutic option.

Dysregulated brain-derived neurotrophic factor (BDNF)/tropomyosin receptor kinase B (TrkB) signalling is implicated in several neurodegenerative diseases, including Alzheimer’s disease. A failure of neurotrophic support may participate in neurodegenerative mechanisms, such as ferroptosis, which has likewise been implicated in this disease class. The current study investigated whether modulators of TrkB signalling affect ferroptosis. Cell viability, C11 BODIPY, and cell-free oxidation assays were used to observe the impact of TrkB modulators, and an immunoblot assay was used to detect TrkB expression. TrkB modulators such as agonist BDNF, antagonist ANA-12, and inhibitor K252a did not affect RSL3-induced ferroptosis sensitivity in primary cortical neurons expressing detectable TrkB receptors. Several other modulators of the TrkB receptor, including agonist 7,8-DHF, activator phenelzine sulphate, and inhibitor GNF-5837, conferred protection against a range of ferroptosis inducers in several immortalised neuronal and non-neuronal cell lines, such as N27 and HT-1080 cells. We found these immortalised cell lines lack detectable TrkB receptor expression, so the anti-ferroptotic activity of these TrkB modulators was most likely due to their inherent radical-trapping antioxidant properties, which should be considered when interpreting their experimental findings. These modulators or their variants could be potential anti-ferroptotic therapeutics for various diseases.

Abstract The blood–brain barrier (BBB) is a highly specialized neurovascular unit, initially described as an intact barrier to prevent toxins, pathogens, and potentially harmful substances from entering the brain. An intact BBB is also critical for the maintenance of normal neuronal function. In cerebral vascular diseases and neurological disorders, the BBB can be disrupted, contributing to disease progression. While restoration of BBB integrity serves as a robust biomarker of better clinical outcomes, the restrictive nature of the intact BBB presents a major hurdle for delivery of therapeutics into the brain. Recent studies show that the BBB is actively engaged in crosstalk between neuronal and the circulatory systems, which defines another important role of the BBB: as an interfacing conduit that mediates communication between two sides of the BBB. This role has been subject to extensive investigation for brain‐targeted drug delivery and shows promising results. The dual roles of the BBB make it a unique target for drug development. Here, recent developments and novel strategies to target the BBB for therapeutic purposes are reviewed, from both barrier and carrier perspectives.

β-Amyloid pathology is elevated in ~30% of cognitively normal people over 65, and is associated with accelerated neurodegeneration in the pre-clinical stages of Alzheimer's disease. Recent findings reveal that brain iron might also act to propel neurodegeneration in people with underlying amyloid pathology. Here, repeated PET scans of fluorodeoxyglucose (FDG) were used as a biomarker for brain hypometabolism and a downstream biomarker of neurodegeneration to investigate whether levels of ferritin in the cerebrospinal fluid (CSF; a reporter of brain iron load) are associated with prodromal disease progression of people with high β-amyloid pathology determined by established cut-off values in CSF t-tau/Aβ42 ratio. Nineteen cognitively normal participants with low t-tau/Aβ42, and 71 participants with high t-tau/Aβ42 who were cognitively normal or had mild cognitive impairment were included as participants from the Alzheimer's Disease Neuroimaging Initiative (ADNI) study. These subjects had repeated FDG-PET scans at 6-month intervals for 2 years, and yearly intervals for up to a further 3 years. In mixed-effects linear models of FDG signal, baseline CSF ferritin was associated with an accelerated decline in FDG PET in high t-tau/Aβ42 participants (β[SE] = −0.066 [0.017]; P = .0002), but not in people with low t-tau/Aβ42 (−0.029 [0.049]; P = .554). These data implicate iron as a contributing factor to neurodegeneration associated with β-amyloid pathology, and highlight CSF ferritin as a complementary prognostic biomarker to the t-tau/Aβ42 ratio that predicts near-term risk for disease progression.

Background: Biomarkers that are indicative of early biochemical aberrations are needed to predict the risk of dementia onset and progression in Alzheimer’s disease (AD). We assessed the utility of cerebrospinal fluid (CSF) neurofilament light (NfL) chain for screening preclinical AD, predicting dementia onset among cognitively healthy (CH) individuals, and the rate of cognitive decline amongst individuals with mild cognitive impairment (MCI). Methods: Neurofilament light levels were measured in CSF samples of participants (CH, n = 154 and MCI, n = 32) from the Australian Imaging, Biomarkers and Lifestyle study of ageing (AIBL). Cases of preclinical AD were identified using biomarker-guided classification (CH, amyloid-β [Aβ]+, phosphorylated-tau [P-tau]+ and total-tau [T-tau]±; A+T+/N±). The prediction of dementia onset (questionable dementia) among CH participants was assessed as the risk of conversion from Clinical Dementia Rating [CDR = 0] to CDR ≥ 0.5 over 6 years. Mixed linear models were used to assess the utility of baseline CSF NfL levels for predicting the rate of cognitive decline among participants with MCI over 4.5 years. Results: Neurofilament light levels were significantly higher in preclinical AD participants (CH, A+T+/N±) as compared to A-T-N- (p < 0.001). Baseline levels of CSF NfL were higher in CH participants who converted to CDR ≥ 0.5 over 6 years (p = 0.045) and the risk of conversion to CDR ≥ 0.5 was predicted (hazard ratio [HR] 1.60, CI 1.03–2.48, p = 0.038). CH participants with CSF NfL > cut-off were at a higher risk of developing dementia (HR 4.77, CI 1.31–17.29, p = 0.018). Participants with MCI and with higher baseline levels of CSF NfL (>median) had a higher rate of decline in cognition over 4.5 years. Conclusion: An assessment of CSF NfL levels can help to predict dementia onset among CH vulnerable individuals and cognitive decline among those with MCI.

Iron has been increasingly implicated in the pathology of neurodegenerative diseases. In the past decade, development of the new magnetic resonance imaging technique, quantitative susceptibility mapping (QSM), has enabled for the more comprehensive investigation of iron distribution in the brain. The aim of this systematic review was to provide a synthesis of the findings from existing QSM studies in neurodegenerative diseases. We identified 80 records by searching MEDLINE, Embase, Scopus, and PsycInfo databases. The disorders investigated in these studies included Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Wilson's disease, Huntington's disease, Friedreich's ataxia, spinocerebellar ataxia, Fabry disease, myotonic dystrophy, pantothenate-kinase-associated neurodegeneration, and mitochondrial membrane protein-associated neurodegeneration. As a general pattern, QSM revealed increased magnetic susceptibility (suggestive of increased iron content) in the brain regions associated with the pathology of each disorder, such as the amygdala and caudate nucleus in Alzheimer's disease, the substantia nigra in Parkinson's disease, motor cortex in amyotrophic lateral sclerosis, basal ganglia in Huntington's disease, and cerebellar dentate nucleus in Friedreich's ataxia. Furthermore, the increased magnetic susceptibility correlated with disease duration and severity of clinical features in some disorders. Although the number of studies is still limited in most of the neurodegenerative diseases, the existing evidence suggests that QSM can be a promising tool in the investigation of neurodegeneration.

Objectives: The mechanisms leading to neurodegeneration in Alzheimer's disease (AD) may involve oxidative stress and neuroinflammation. Ceruloplasmin (Cp) is a circulating protein that intersects both these pathways, since its expression is increased during the acute phase response, and the protein acts to lower pro-oxidant iron in cells. Since the role of Cp in AD, and its potential for use as a biomarker is not established, we investigated CSF Cp and its association with longitudinal outcome measures related to AD. Methods: This was an observational study of 268 people from the Alzheimer's Disease Neuroimaging (ADNI) cohort. Subjects were classified clinically as having AD, mild cognitive impairment (MCI) or were cognitively normal (CN), and were also classified as being positive for β-amyloid using established thresholds in the CSF t-tau/Aβ42 ratio. Subjects underwent cognitive tests and MRI studies every 6 months for 2 years, then yearly thereafter for up to 6 years. Results: At baseline, CSF Cp was not associated with clinical or pathological diagnosis, but we found an unexpected association between CSF Cp and levels of CSF apolipoprotein E. In longitudinal analysis, high level of CSF Cp was associated with accelerated cognitive decline (as assessed by ADAS-Cog, CDR-SB, and MMSE) and ventricular volume enlargement in people classified as MCI and who had underlying β-amyloid pathology. Conclusion: These results raise new questions into the role of Cp in neuroinflammation, oxidative stress, and APOE pathways involved in AD, and reveal the potential for this protein to be used as a biomarker in disease prognostication.

Abstract Introduction This study assessed the utility of cerebrospinal fluid (CSF) neurofilament light (NfL) in Alzheimer's disease (AD) diagnosis, its association with amyloid and tau pathology, as well as its potential to predict brain atrophy, cognition, and amyloid accumulation. Methods CSF NfL concentration was measured in 221 participants from the Australian Imaging, Biomarkers & Lifestyle Flagship Study of Ageing (AIBL). Results CSF NfL levels as well as NfL/amyloid β (Aβ42) were significantly elevated in AD compared to healthy controls (HC; P < .001), and in mild cognitive impairment (MCI) compared to HC (P = .008 NfL; P < .001 NfL/Aβ42). CSF NfL and NfL/Aβ42 differentiated AD from HC with an area under the receiver operating characteristic (ROC) curve (AUC) of 0.84 and 0.90, respectively. CSF NfL and NfL/Aβ42 predicted cortical amyloid load, brain atrophy, and cognition. Discussion CSF NfL is a biomarker of neurodegeneration, correlating with cognitive impairment and brain neuropathology.

Abstract Parkinson’s disease is diagnosed upon the presentation of motor symptoms, resulting from substantial degeneration of dopaminergic neurons in the midbrain. Prior to diagnosis, there is a lengthy prodromal stage in which non-motor symptoms, including olfactory deficits (hyposmia), develop. There is limited information about non-motor impairments and there is a need for directed research into these early pathogenic cellular pathways that precede extensive dopaminergic death in the midbrain. The protein tau has been identified as a genetic risk factor in the development of sporadic PD. Tau knockout mice have been reported as an age-dependent model of PD, and this study has demonstrated that they develop motor deficits at 15-months-old. We have shown that at 7-month-old tau knockout mice present with an overt hyposmic phenotype. This olfactory deficit correlates with an accumulation of α-synuclein, as well as autophagic impairment, in the olfactory bulb. This pathological feature becomes apparent in the striatum and substantia nigra of 15-month-old tau knockout mice, suggesting the potential for a spread of disease. Initial primary cell culture experiments have demonstrated that ablation of tau results in the release of α-synuclein enriched exosomes, providing a potential mechanism for disease spread. These alterations in α-synuclein level as well as a marked autophagy impairment in the tau knockout primary cells recapitulate results seen in the animal model. These data implicate a pathological role for tau in early Parkinson’s disease.

Cellular senescence is characterised by the irreversible arrest of proliferation, a pro-inflammatory secretory phenotype and evasion of programmed cell death mechanisms. We report that senescence alters cellular iron acquisition and storage and also impedes iron-mediated cell death pathways. Senescent cells, regardless of stimuli (irradiation, replicative or oncogenic), accumulate vast amounts of intracellular iron (up to 30-fold) with concomitant changes in the levels of iron homeostasis proteins. For instance, ferritin (iron storage) levels provided a robust biomarker of cellular senescence, for associated iron accumulation and for resistance to iron-induced toxicity. Cellular senescence preceded iron accumulation and was not perturbed by sustained iron chelation (deferiprone). Iron accumulation in senescent cells was driven by impaired ferritinophagy, a lysosomal process that promotes ferritin degradation and ferroptosis. Lysosomal dysfunction in senescent cells was confirmed through several markers, including the build-up of microtubule-associated protein light chain 3 (LC3-II) in autophagosomes. Impaired ferritin degradation explains the iron accumulation phenotype of senescent cells, whereby iron is effectively trapped in ferritin creating a perceived cellular deficiency. Accordingly, senescent cells were highly resistant to ferroptosis. Promoting ferritin degradation by using the autophagy activator rapamycin averted the iron accumulation phenotype of senescent cells, preventing the increase of TfR1, ferritin and intracellular iron, but failed to re-sensitize these cells to ferroptosis. Finally, the enrichment of senescent cells in mouse ageing hepatic tissue was found to accompany iron accumulation, an elevation in ferritin and mirrored our observations using cultured senescent cells. Keywords: Senescence, Iron, Ferritinophagy, Ferroptosis, Ferritin, Autophagy, Ageing

Abstract Alzheimer’s Disease (AD) is the most common form of dementia, characterised by extracellular amyloid deposition as plaques and intracellular neurofibrillary tangles of tau protein. As no current clinical test can diagnose individuals at risk of developing AD, the aim of this project is to evaluate a blood-based biomarker panel to identify individuals who carry this risk. We analysed the levels of 22 biomarkers in clinically classified healthy controls (HC), mild cognitive impairment (MCI) and Alzheimer’s participants from the well characterised Australian Imaging, Biomarker and Lifestyle (AIBL) study of aging. High levels of IL-10 and IL-12/23p40 were significantly associated with amyloid deposition in HC, suggesting that these two biomarkers might be used to detect at risk individuals. Additionally, other biomarkers (Eotaxin-3, Leptin, PYY) exhibited altered levels in AD participants possessing the APOE ε4 allele. This suggests that the physiology of some potential biomarkers may be altered in AD due to the APOE ε4 allele, a major risk factor for AD. Taken together, these data highlight several potential biomarkers that can be used in a blood-based panel to allow earlier identification of individuals at risk of developing AD and/or early stage AD for which current therapies may be more beneficial.

Acid fibroblast growth factor (aFGF) has shown neuroprotection in Alzheimer’s disease (AD) models in previous studies, yet its mechanism is still uncertain. Here we report that the efficacy of Tat-haFGF14–154 is markedly increased when loaded cationic liposomes for intranasal delivery are intranasally administered to APP/PS1 mice. Our results demonstrated that liposomal Tat-haFGF14–154 treatment significantly ameliorated behavioral deficits, relieved brain Aβ burden, and increased the expression and activity of disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) in the brain. Tat-haFGF14–154 antagonized Aβ1–42-induced cell death and structural damage in rat primary neurons in an ADAM10-dependent manner, which, in turn, was promoted by the activation of XBP1 splicing and modulated by the PI3K-CREB pathway. Both knockdown of ADAM10 and inhibition of PI3K (LY294002) negated Tat-haFGF14–154 rescue. Thus, Tat-haFGF14–154 activates the IRE1α/XBP1 pathway of the unfolded protein response (UPR) against the endoplasmic reticulum (ER) stress induced by Aβ, and, subsequently, the nuclear translocation of spliced XBP1 (XBP1s) promotes transcription of ADAM10. These results highlight the important role of ADAM10 and its activation through the PI3K-CREB-IRE1α/XBP1 pathway as a key factor in the mechanism of neuroprotection for Tat-haFGF14–154.

Abstract Elevated iron in the SNpc may play a key role in Parkinson’s disease (PD) neurodegeneration since drug candidates with high iron affinity rescue PD animal models, and one candidate, deferirpone, has shown efficacy recently in a phase two clinical trial. However, strong iron chelators may perturb essential iron metabolism, and it is not yet known whether the damage associated with iron is mediated by a tightly bound (eg ferritin) or lower-affinity, labile, iron pool. Here we report the preclinical characterization of PBT434, a novel quinazolinone compound bearing a moderate affinity metal-binding motif, which is in development for Parkinsonian conditions. In vitro, PBT434 was far less potent than deferiprone or deferoxamine at lowering cellular iron levels, yet was found to inhibit iron-mediated redox activity and iron-mediated aggregation of α-synuclein, a protein that aggregates in the neuropathology. In vivo, PBT434 did not deplete tissue iron stores in normal rodents, yet prevented loss of substantia nigra pars compacta neurons (SNpc), lowered nigral α-synuclein accumulation, and rescued motor performance in mice exposed to the Parkinsonian toxins 6-OHDA and MPTP, and in a transgenic animal model (hA53T α-synuclein) of PD. These improvements were associated with reduced markers of oxidative damage, and increased levels of ferroportin (an iron exporter) and DJ-1. We conclude that compounds designed to target a pool of pathological iron that is not held in high-affinity complexes in the tissue can maintain the survival of SNpc neurons and could be disease-modifying in PD.

Abstract Introduction A blood‐based biomarker panel to identify individuals with preclinical Alzheimer's disease (AD) would be an inexpensive and accessible first step for routine testing. Methods We analyzed 14 biomarkers that have previously been linked to AD in the Australian Imaging Biomarkers lifestyle longitudinal study of aging cohort. Results Levels of apolipoprotein J (apoJ) were higher in AD individuals compared with healthy controls at baseline and 18 months (P = .0003) and chemokine‐309 (I‐309) were increased in AD patients compared to mild cognitive impaired individuals over 36 months (P = .0008). Discussion These data suggest that apoJ may have potential in the context of use (COU) of AD diagnostics, I‐309 may be specifically useful in the COU of identifying individuals at greatest risk for progressing toward AD. This work takes an initial step toward identifying blood biomarkers with potential use in the diagnosis and prognosis of AD and should be validated across other prospective cohorts.

Abstract Introduction For early detection of Alzheimer's disease (AD), the field needs biomarkers that can be used to detect disease status with high sensitivity and specificity. Apolipoprotein J (ApoJ, also known as clusterin) has long been associated with AD pathogenesis through various pathways. The aim of this study was to investigate the potential of plasma apoJ as a blood biomarker for AD. Methods Using the Australian Imaging, Biomarkers and Lifestyle (AIBL) study of aging, the present study assayed plasma apoJ levels over baseline and 18 months in 833 individuals. Plasma ApoJ levels were analyzed with respect to clinical classification, age, gender, apolipoprotein E (APOE) ε4 allele status, mini‐mental state examination score, plasma amyloid beta (Aβ), neocortical Aβ burden (as measured by Pittsburgh compound B‐positron emission tomography), and total adjusted hippocampus volume. Results ApoJ was significantly higher in both mild cognitive impairment (MCI) and AD groups as compared with healthy controls (HC; P

Metals are critical cellular elements that are involved in a variety of cellular processes, with recent literature demonstrating that zinc, and the synaptic zinc transporter (ZnT3), are specifically involved in learning and memory and may also be key players in age-related neurodegenerative disorders such as Alzheimer’s disease. Whilst the cellular content and location of metals is critical, recent data has demonstrated that the metalation state of proteins is a determinant of protein function and potential toxicity. As we have previously reported that ZnT3 knockout (KO) mice have deficits in total zinc levels at both 3 and 6 months of age, we were interested in whether there might be changes in the metalloproteomic profile in these animals. To do this, we utilised size exclusion chromatography-inductively coupled plasma mass spectrometry (SEC-ICP-MS) and examined hippocampal homogenates from ZnT3 KO and age-matched wild-type mice at 3, 6 and 18 months of age. Our data suggest that there are alterations in specific metal binding proteins, for zinc, copper and iron all being modulated in the ZnT3 KO mice compared to wild-type (WT). These data suggest that ZnT3 KO mice may have impairments in the levels or localisation of multiple transition metals, and that copper- and iron-dependent cellular pathways may also be impacted in these mice.

Zinc transporter-3 (ZnT3) protein is responsible for loading zinc into presynaptic vesicles and consequently controls the availability of zinc at the glutamatergic synapse. ZnT3 has been shown to decline with age and in Alzheimer's disease (AD) and is crucially involved in learning and memory. In this study, we utilised whole animal behavioural analyses in the ZnT3 KO mouse line, together with electrophysiological analysis of long-term potentiation in brain slices from ZnT3 KO mice, to show that metal chaperones (clioquinol, 30 mg/kg/day for 6 weeks) can prevent the age-dependent cognitive phenotype that characterises these animals. This likely occurs as a result of a homeostatic restoration of synaptic protein expression, as clioquinol significantly restored levels of various pre- and postsynaptic proteins that are critical for normal cognition, including PSD-95; AMPAR and NMDAR2b. We hypothesised that this clioquinol-mediated restoration of synaptic health resulted from a selective increase in synaptic zinc content within the hippocampus. While we demonstrated a small regional increase in hippocampal zinc content using synchrotron x-ray fluorescence microscopy, further sub-region analyses are required to determine whether this effect is seen in other regions of the hippocampal formation that are more closely linked to the synaptic plasticity effects observed in this study. These data support our recent report on the use of a different metal chaperone (PBT2) to prevent normal age-related cognitive decline and demonstrate that metal chaperones are efficacious in preventing the zinc-mediated cognitive decline that characterises ageing and disease.

Iron accumulation and tau protein deposition are pathological features of Alzheimer's (AD) and Parkinson's diseases (PD). Soluble tau protein is lower in affected regions of these diseases, and we previously reported that tau knockout mice display motor and cognitive behavioral abnormities, brain atrophy, neuronal death in substantia nigra, and iron accumulation in the brain that all emerged between 6 and 12 months of age. This argues for a loss of tau function in AD and PD. We also showed that treatment with the moderate iron chelator, clioquinol (CQ) restored iron levels and prevented neuronal atrophy and attendant behavioral decline in 12-month old tau KO mice when commenced prior to the onset of deterioration (6 months). However, therapies for AD and PD will need to treat the disease once it is already manifest. So, in the current study, we tested whether CQ could also rescue the phenotype of mice with a developed phenotype. We found that 5-month treatment of symptomatic (13 months old) tau KO mice with CQ increased nigral tyrosine hydroxylase phosphorylation (which induces activity) and reversed the motor deficits. Treatment also reversed cognitive deficits and raised BDNF levels in the hippocampus, which was accompanied by attenuated brain atrophy, and reduced iron content in the brain. These data raise the possibility that lowering brain iron levels in symptomatic patients could reverse neuronal atrophy and improve brain function, possibly by elevating neurotrophins.

The pathological role of zinc in Alzheimer's disease (AD) is not yet fully elucidated, but there is strong evidence that zinc homeostasis is impaired in the AD brain and that this contributes to disease pathogenesis. In this study we examined the effects of zinc on the proteolysis of synthetic Apolipoprotein E (ApoE), a protein whose allelic variants differentially contribute to the onset/progression of disease. We have demonstrated that zinc promotes the proteolysis (using plasma kallikrein, thrombin and chymotrypsin) of synthetic ApoE in an isoform-specific way (E4>E2 and E3), resulting in more ApoE fragments, particularly for ApoE4. In the absence of exogenous proteases there was no effect of metal modulation on either lipidated or non-lipidated ApoE isoforms. Thus, increased zinc in the complex milieu of the ageing and AD brain could reduce the level of normal full-length ApoE and increase other forms that are involved in neurodegeneration. We further examined human plasma samples from people with different ApoE genotypes. Consistent with previous studies, plasma ApoE levels varied according to different genotypes, with ApoE2 carriers showing the highest total ApoE levels and ApoE4 carriers the lowest. The levels of plasma ApoE were not affected by either the addition of exogenous metals (copper, zinc or iron) or by chelation. Taken together, our study reveals that zinc may contribute to the pathogenesis of AD by affecting the proteolysis of ApoE, which to some extent explains why APOE4 carriers are more susceptible to AD.

Huntington’s disease (HD) is a progressive neurodegenerative disorder caused by a CAG repeat expansion that encodes a polyglutamine tract in huntingtin (htt) protein. Dysregulation of brain iron homeostasis, oxidative stress and neurodegeneration are consistent features of the HD phenotype. Therefore, environmental factors that exacerbate oxidative stress and iron dysregulation may potentiate HD. Iron supplementation in the human population is common during infant and adult-life stages. In this study, iron supplementation in neonatal HD mice resulted in deterioration of spontaneous motor running activity, elevated levels of brain lactate and oxidized glutathione consistent with increased energetic dysfunction and oxidative stress, and increased striatal and motor cortical neuronal atrophy, collectively demonstrating potentiation of the disease phenotype. Oxidative stress, energetic, and anatomic markers of degeneration were not affected in wild-type littermate iron-supplemented mice. Further, there was no effect of elevated iron intake on disease outcomes in adult HD mice. We have demonstrated an interaction between the mutant huntingtin gene and iron supplementation in neonatal HD mice. Findings indicate that elevated neonatal iron intake potentiates mouse HD and promotes oxidative stress and energetic dysfunction in brain. Neonatal-infant dietary iron intake level may be an environmental modifier of human HD.

Disruption of redox homeostasis is a prominent feature in the pathogenesis of Huntington's disease (HD). Selenium an essential element nutrient that modulates redox pathways and has been reported to provide protection against both acute neurotoxicity (e.g. methamphetamine) and chronic neurodegeneration (e.g. tauopathy) in mice. The objective of our study was to investigate the effect of sodium selenite, an inorganic form of selenium, on behavioral, brain degeneration and biochemical outcomes in the N171-82Q Huntington's disease mouse model. HD mice, which were supplemented with sodium selenite from 6 to 14 weeks of age, demonstrated increased motor endurance, decreased loss of brain weight, decreased mutant huntingtin aggregate burden and decreased brain oxidized glutathione levels. Biochemical studies revealed that selenite treatment reverted HD-associated changes in liver selenium and plasma glutathione in N171-82Q mice and had effects on brain selenoprotein transcript expression. Further, we found decreased brain selenium content in human autopsy brain. Taken together, we demonstrate a decreased selenium phenotype in human and mouse HD and additionally show some protective effects of selenite in N171-82Q HD mice. Modification of selenium metabolism results in beneficial effects in mouse HD and thus may represent a therapeutic strategy.

Iron dyshomeostasis is a feature of Alzheimer’s disease (AD). The impact of iron on AD is attributed to its interactions with the central proteins of AD pathology (amyloid precursor protein and tau) and/or through the iron-mediated generation of prooxidant molecules (e.g., hydroxyl radicals). However, the source of iron accumulation in pathologically relevant regions of the brain and its contribution to AD remains unclear. One likely contributor to iron accumulation is the age-associated increase in tissue-resident senescent cells that drive inflammation and contribute to various pathologies associated with advanced age. Iron accumulation predisposes ageing tissue to oxidative stress that can lead to cellular dysfunction and to iron-dependent cell death modalities (e.g., ferroptosis). Further, elevated brain iron is associated with the progression of AD and cognitive decline. Elevated brain iron presents a feature of AD that may be modified pharmacologically to mitigate the effects of age/senescence-associated iron dyshomeostasis and improve disease outcome.

Acrodermatitis enteropathica (AE) is a rare disease characterised by a failure in intestinal zinc absorption, which results in a host of symptoms that can ultimately lead to death if left untreated. Current clinical treatment involves life-long high-dose zinc supplements, which can introduce complications for overall nutrient balance in the body. Previous studies have therefore explored the pharmacological treatment of AE utilising metal ionophore/transport compounds in an animal model of the disease (conditional knockout (KO) of the zinc transporter, Zip4), with the perspective of finding an alternative to zinc supplementation. In this study we have assessed the utility of a different class of zinc ionophore compound (zinc diethyl bis(N4-methylthiosemicarbazone), Zn-DTSM; Collaborative Medicinal Development, Sausalito, CA, USA) to the one we have previously described (clioquinol), to determine whether it is effective at preventing the stereotypical weight loss present in the animal model of disease. We first utilised an in vitro assay to assess the ionophore capacity of the compound, and then assessed the effect of the compound in three in vivo animal studies (in 1.5-month-old mice at 30 mg/kg/day, and in 5-month old mice at 3 mg/kg/day and 30 mg/kg/day). Our data demonstrate that Zn-DTSM has a pronounced effect on preventing weight loss when administered daily at 30 mg/kg/day; this was apparent in the absence of any added exogenous zinc. This compound had little overall effect on zinc content in various tissues that were assessed, although further characterisation is required to more fully explore the cellular changes underlying the physiological benefit of this compound. These data suggest that Zn-DTSM, or similar compounds, should be further explored as potential therapeutic options for the long-term treatment of AE.

Iron is the most abundant transition metal within the brain, and is vital for a number of cellular processes including neurotransmitter synthesis, myelination of neurons, and mitochondrial function. Redox cycling between ferrous and ferric iron is utilized in biology for various electron transfer reactions essential to life, yet this same chemistry mediates deleterious reactions with oxygen that induce oxidative stress. Consequently, there is a precise and tightly controlled mechanism to regulate iron in the brain. When iron is dysregulated, both conditions of iron overload and iron deficiencies are harmful to the brain. This review focuses on how iron metabolism is maintained in the brain, and how an alteration to iron and iron metabolism adversely affects neurological function.

Altered copper homeostasis and hypercholesterolemia have been identified independently as risk factors for Alzheimer’s disease (AD). Abnormal copper and cholesterol metabolism are implicated in the genesis of amyloid plaques and neurofibrillary tangles (NFT), which are two key pathological signatures of AD. Amyloidogenic processing of a sub-population of amyloid precursor protein (APP) that produces Aβ occurs in cholesterol-rich lipid rafts in copper deficient AD brains. Co-localization of Aβ and a paradoxical high concentration of copper in lipid rafts fosters the formation of neurotoxic Aβ:copper complexes. These complexes can catalytically oxidize cholesterol to generate H2O2, oxysterols and other lipid peroxidation products that accumulate in brains of AD cases and transgenic mouse models. Tau, the core protein component of NFTs, is sensitive to interactions with copper and cholesterol, which trigger a cascade of hyperphosphorylation and aggregation preceding the generation of NFTs. Here we present an overview of copper and cholesterol metabolism in the brain, and how their integrated failure contributes to development of AD.

Alzheimer’s disease (AD) is the leading cause of dementia and represents a significant burden on the global economy and society. The role of transition metals, in particular copper (Cu), in AD has become of significant interest due to the dyshomeostasis of these essential elements, which can impart profound effects on cell viability and neuronal function. We tested the hypothesis that there is a systemic perturbation in Cu compartmentalization in AD, within the brain as well as in the periphery, specifically within erythrocytes. Our results showed that the previously reported decrease in Cu within the human frontal cortex was confined to the soluble (P

The role of metals in the pathophysiology of Alzheimer's disease (AD) has gained considerable support in recent years, with both in vitro and in vivo data demonstrating that a mis-metabolism of metal ions, such as copper and zinc, may affect various cellular cascades that ultimately leads to the development and/or potentiation of AD. In this paper, we will provide an overview of the preclinical and clinical literature that specifically relates to attempts to affect the AD cascade by the modulation of brain copper levels. We will also detail our own novel animal data, where we treated APP/PS1 (7-8 months old) mice with either high copper (20 ppm in the drinking water), high cholesterol (2% supplement in the food) or a combination of both and then assessed β-amyloid (Aβ) burden (soluble and insoluble Aβ), APP levels and behavioural performance in the Morris water maze. These data support an interaction between copper/cholesterol and both Aβ and APP and further highlight the potential role of metal ion dyshomeostasis in AD.

Glycogen synthase kinase-3 (GSK-3) regulates multiple cellular processes, and its dysregulation is implicated in the pathogenesis of diverse diseases. In this paper we will focus on the dysfunction of GSK-3 in Alzheimer’s disease and Parkinson’s disease. Specifically, GSK-3 is known to interact with tau, β-amyloid (Aβ), and α-synuclein, and as such may be crucially involved in both diseases. Aβ production, for example, is regulated by GSK-3, and its toxicity is mediated by GSK-induced tau phosphorylation and degeneration. α-synuclein is a substrate for GSK-3 and GSK-3 inhibition protects against Parkinsonian toxins. Lithium, a GSK-3 inhibitor, has also been shown to affect tau, Aβ, and α-synuclein in cell culture, and transgenic animal models. Thus, understanding the role of GSK-3 in neurodegenerative diseases will enhance our understanding of the basic mechanisms underlying the pathogenesis of these disorders and also facilitate the identification of new therapeutic avenues.

Recent data from in vitro, animal, and human studies have shed new light on the positive roles of copper in many aspects of AD. Copper promotes the non-amyloidogenic processing of APP and thereby lowers the Aβ production in cell culture systems, and it increases lifetime and decreases soluble amyloid production in APP transgenic mice. In a clinical trial with Alzheimer patients, the decline of Aβ levels in CSF, which is a diagnostic marker, is diminished in the verum group (8 mg copper/day), indicating a beneficial effect of the copper treatment. These observations are in line with the benefit of treatment with compounds aimed at normalizing metal levels in the brain, such as PBT2. The data reviewed here demonstrate that there is an apparent disturbance in metal homeostasis in AD. More research is urgently needed to understand how this disturbance can be addressed therapeutically.