Biocompatibility and proteomic profiling of DMSA-coated iron nanocubes in a human glioblastoma cell line.

Background: Superparamagnetic iron core iron oxide shell nanocubes have previously shown superior performance in magnetic resonance imaging T2 contrast enhancement compared with spherical nanoparticles. Methods: Iron core iron oxide shell nanocubes were synthesized, stabilized with dimercaptosuccinic acid (DMSA-NC) and physicochemically characterized. MRI contrast enhancement and biocompatibility were assessed in vitro. Results: DMSA-NC showed a transverse relaxivity of 122.59 mM-1·s-1 Fe. Treatment with DMSA-NC did not induce cytotoxicity or oxidative stress in U-251 cells, and electron microscopy demonstrated DMSA-NC localization within endosomes and lysosomes in cells following internalization. Global proteomics revealed dysregulation of iron storage, transport, transcription and mRNA processing proteins. Conclusion: DMSA-NC is a promising T2 MRI contrast agent which, in this preliminary investigation, demonstrates favorable biocompatibility with an astrocyte cell model.

MRI is a powerful tool used in the diagnosis of cancer, strokes and other injuries. An MRI scan can be improved with the use of iron oxide nanoparticles, which enhance the contrast of the image. In this study we have developed cube-shaped iron nanoparticles (nanocubes), which have been previously shown to be more effective at inducing contrast. We demonstrated that iron-based nanocubes do not damage or induce stress in cells and work effectively as an MRI contrast agent. We further analyzed how the nanocubes may affect cell functioning by investigating changes to protein levels in the cells. The results of this study are promising steps towards using iron-based nanocubes as a tool to improve the clarity of MRI scans for medical imaging and diagnosis. Future work must determine whether these nanocubes work effectively and safely in an animal model, which is a critical step in progressing to their use in clinical settings.

Evaluation of Dimercaptosuccinic Acid-Coated Iron Nanoparticles Immunotargeted to Amyloid Beta as MRI Contrast Agents for the Diagnosis of Alzheimer's Disease.

Nanoparticle-based magnetic contrast agents have opened the potential for magnetic resonance imaging (MRI) to be used for early non-invasive diagnosis of Alzheimer's disease (AD). Accumulation of amyloid pathology in the brain has shown association with cognitive decline and tauopathy; hence, it is an effective biomarker for the early detection of AD. The aim of this study was to develop a biocompatible magnetic nanoparticle targeted to amyloid beta (Aß) plaques to increase the sensitivity of T2-weighted MRI for imaging of amyloid pathology in AD. We presented novel iron core-iron oxide nanoparticles stabilized with a dimercaptosuccinic acid coating and functionalized with an anti-Aß antibody. Nanoparticle biocompatibility and cellular internalization were evaluated in vitro in U-251 glioblastoma cells using cellular assays, proteomics, and transmission electron microscopy. Iron nanoparticles demonstrated no significant in vitro cytotoxicity, and electron microscopy results showed their movement through the endocytic cycle within the cell over a 24 h period. In addition, immunostaining and bio-layer interferometry confirmed the targeted nanoparticle's binding affinity to amyloid species. The iron nanoparticles demonstrated favourable MRI contrast enhancement; however, the addition of the antibody resulted in a reduction in the relaxivity of the particles. The present work shows promising preliminary results in the development of a targeted non-invasive method of early AD diagnosis using contrast-enhanced MRI.

The association between dietary patterns, plasma lipid profiles, and inflammatory potential in a vascular dementia cohort.

Background: Inflammation and altered lipid dyshomeostasis have been implicated in the pathogenesis of Alzheimer's disease and vascular dementia. Objective: To determine if there are any associations between dietary patterns, plasma lipid profiles, and inflammatory potential in a vascular dementia cohort. Methods: One hundred fifty participants (36 subjects with Vascular Dementia and 114 healthy controls) from two Australian teaching hospitals completed a cross-sectional survey examining their dietary and lifestyle patterns. Each participant's diet was further evaluated using the Empirical Dietary Inflammatory Index. Some participants also donated blood samples for lipidomic analysis. Results: After adjusting for age, education, and socioeconomic status, participants with vascular dementia tend to have higher lipid profiles, do less exercise, and engage less frequently in social interaction, educational, or reading activities. They also tend to consume more deep-fried food and full-fat dairy compared to control subjects. However, there was no difference in Empirical Dietary Inflammatory Index between the two groups after adjusting for age, education, and socioeconomic status. Conclusion: Our findings suggest a graded inverse association between healthy lifestyle factors and vascular dementia.

The sex-dependent response to psychosocial stress and ischaemic heart disease.

Stress is an important risk factor for modern chronic diseases, with distinct influences in males and females. The sex specificity of the mammalian stress response contributes to the sex-dependent development and impacts of coronary artery disease (CAD). Compared to men, women appear to have greater susceptibility to chronic forms of psychosocial stress, extending beyond an increased incidence of mood disorders to include a 2- to 4-fold higher risk of stress-dependent myocardial infarction in women, and up to 10-fold higher risk of Takotsubo syndrome-a stress-dependent coronary-myocardial disorder most prevalent in post-menopausal women. Sex differences arise at all levels of the stress response: from initial perception of stress to behavioural, cognitive, and affective responses and longer-term disease outcomes. These fundamental differences involve interactions between chromosomal and gonadal determinants, (mal)adaptive epigenetic modulation across the lifespan (particularly in early life), and the extrinsic influences of socio-cultural, economic, and environmental factors. Pre-clinical investigations of biological mechanisms support distinct early life programming and a heightened corticolimbic-noradrenaline-neuroinflammatory reactivity in females vs. males, among implicated determinants of the chronic stress response. Unravelling the intrinsic molecular, cellular and systems biological basis of these differences, and their interactions with external lifestyle/socio-cultural determinants, can guide preventative and therapeutic strategies to better target coronary heart disease in a tailored sex-specific manner.

Importance of NAD+ Anabolism in Metabolic, Cardiovascular and Neurodegenerative Disorders.

The role of nicotinamide adenine dinucleotide (NAD+) in ageing has emerged as a critical factor in understanding links to a wide range of chronic diseases. Depletion of NAD+, a central redox cofactor and substrate of numerous metabolic enzymes, has been detected in many major age-related diseases. However, the mechanisms behind age-associated NAD+ decline remains poorly understood. Despite limited conclusive evidence, supplements aimed at increasing NAD+ levels are becoming increasingly popular. This review provides renewed insights regarding the clinical utility and benefits of NAD+ precursors, namely nicotinamide (NAM), nicotinic acid (NA), nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN), in attenuating NAD+ decline and phenotypic characterization of age-related disorders, including metabolic, cardiovascular and neurodegenerative diseases. While it is anticipated that NAD+ precursors can play beneficial protective roles in several conditions, they vary in their ability to promote NAD+ anabolism with differing adverse effects. Careful evaluation of the role of NAD+, whether friend or foe in ageing, should be considered.

Involvement of single nucleotide polymorphisms of junction adhesion molecule with small vessel vascular dementia.

Objectives: It is now recognized that blood brain barrier (BBB) leakage occurs in cerebral small vascular disease (CSVD) and plays a significant role in the pathophysiology of vascular dementia. We hypothesized that genetic polymorphisms of junctional adhesion molecule-A (JAM-A) (which may result in compromised structure of tight junction proteins that form the BBB) in combination with cerebrovascular risk factors hypertension, lipid disorders, and type 2 diabetes may result in BBB leakage and increase the individual's risk of CSVD-related dementia. Methods: In this case-control study, 97 controls with a mean Mini-Mental State Exam (MMSE) score of 29 and 38 CSVD-related vascular dementia participants (mean MMSE score of 19) were recruited. Bloods were collected for the analysis of two common single nucleotide polymorphisms (SNPs) of the JAM-A genotypes rs790056 and rs2481084 using real-time polymerase chain reaction (PCR) assay. Medical history of hypertension, hyperlipidemia, and diabetes was collected for all participants. Results: Polymorphisms of genotype JAM-A SNP rs790056 showed statistically significant result when the subgroup with hyperlipidemia was analyzed (OR = 3.130, p = 0.042 for TC + CC genotypes with hyperlipidaemia vs controls). Similar result was found with diabetes (OR = 4.670, p = 0.031 for TC + CC genotypes vs controls). No significant result was found with hypertension. Borderline results of statistical significance were found for JAM-A SNP rs2481084 with hyperlipidemia (OR = 3.210, p = 0.054 for TC + CC genotypes vs controls) and with diabetes (OR = 3.620, p = 0.069 for TC + CC genotypes vs controls) but not for hypertension. The borderline results might have been due to lack of statistical power because of small sample size. Conclusions: These results lend further support that cerebrovascular risk factors interact with genetic polymorphisms of BBB proteins to increase the risk of vascular dementia.

The Role of Kynurenine Pathway and NAD+ Metabolism in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a serious, complex, and highly debilitating long-term illness. People with ME/CFS are typically unable to carry out their routine activities. Key hallmarks of the disease are neurological and gastrointestinal impairments accompanied by pervasive malaise that is exacerbated after physical and/or mental activity. Currently, there is no validated cure of biomarker signature for this illness. Impaired tryptophan (TRYP) metabolism is thought to play significant role in the pathobiology of ME/CFS. TRYP is an important precursor for serotonin and the essential pyridine nucleotide nicotinamide adenine dinucleotide (NAD+). TRYP has been associated with the development of some parts of the brain responsible for behavioural functions. The main catabolic route for TRYP is the kynurenine pathway (KP). The KP produces NAD+ and several neuroactive metabolites with neuroprotective (i.e., kynurenic acid (KYNA)) and neurotoxic (i.e., quinolinic acid (QUIN)) activities. Hyperactivation of the KP, whether compensatory or a driving mechanism of degeneration can limit the availability of NAD+ and exacerbate the symptoms of ME/CFS. This review discusses the potential association of altered KP metabolism in ME/CFS. The review also evaluates the role of the patient's gut microbiota on TRYP availability and KP activation. We propose that strategies aimed at raising the levels of NAD+ (e.g., using nicotinamide mononucleotide and nicotinamide riboside) may be a promising intervention to overcome symptoms of fatigue and to improve the quality of life in patients with ME/CFS. Future clinical trials should further assess the potential benefits of NAD+ supplements for reducing some of the clinical features of ME/CFS.

Recent Neurotherapeutic Strategies to Promote Healthy Brain Aging: Are we there yet?

Owing to the global exponential increase in population ageing, there is an urgent unmet need to develop reliable strategies to slow down and delay the ageing process. Age-related neurodegenerative diseases are among the main causes of morbidity and mortality in our contemporary society and represent a major socio-economic burden. There are several controversial factors that are thought to play a causal role in brain ageing which are continuously being examined in experimental models. Among them are oxidative stress and brain inflammation which are empirical to brain ageing. Although some candidate drugs have been developed which reduce the ageing phenotype, their clinical translation is limited. There are several strategies currently in development to improve brain ageing. These include strategies such as caloric restriction, ketogenic diet, promotion of cellular nicotinamide adenine dinucleotide (NAD+) levels, removal of senescent cells, 'young blood' transfusions, enhancement of adult neurogenesis, stem cell therapy, vascular risk reduction, and non-pharmacological lifestyle strategies. Several studies have shown that these strategies can not only improve brain ageing by attenuating age-related neurodegenerative disease mechanisms, but also maintain cognitive function in a variety of pre-clinical experimental murine models. However, clinical evidence is limited and many of these strategies are awaiting findings from large-scale clinical trials which are nascent in the current literature. Further studies are needed to determine their long-term efficacy and lack of adverse effects in various tissues and organs to gain a greater understanding of their potential beneficial effects on brain ageing and health span in humans.

Strong Predictive Algorithm of Pathogenesis-Based Biomarkers Improves Parkinson's Disease Diagnosis.

Easily accessible and accurate biomarkers can aid Parkinson's disease diagnosis. We investigated whether combining plasma levels of α-synuclein, anti-α-synuclein, and/or their ratios to amyloid beta-40 correlated with clinical diagnosis. The inclusion of amyloid beta-40 (Aß40) is novel. Plasma levels of biomarkers were quantified with ELISA. Using receiver operating characteristic (ROC) curve analysis, levels of α-synuclein, anti-α-synuclein, and their ratios with Aß40 were analyzed in an initial training set of cases and controls. Promising biomarkers were then used to build a diagnostic algorithm. Verification of the results of biomarkers and the algorithm was performed in an independent set. The training set consisted of 50 cases (age 65.2±9.3, range 44-83, female:male=21:29) with 50 age- and gender-matched controls (67.1±10.0, range 45-96 years; female:male=21:29). ROC curve analysis yielded the following area under the curve results: anti-α-synuclein=0.835, α-synuclein=0.738, anti-α-synuclein/Aß40=0.737, and α-synuclein/Aß40=0.663. A 2-step diagnostic algorithm was built: either α-synuclein or anti-α-synuclein was ≥2 times the means of controls (step-1), resulting in 74% sensitivity; and adding α-synuclein/Aß40 or anti-α-synuclein/Aß40 (step-2) yielded better sensitivity (82%) while using step-2 alone yielded good specificity in controls (98%). The results were verified in an independent sample of 46 cases and 126 controls, with sensitivity reaching 91.3% and specificity 90.5%. The algorithm was equally sensitive in Parkinson's disease of ≤5-year duration with 92.6% correctly identified in the training set and 90% in the verification set. With two independent samples totaling 272 subjects, our study showed that combination of biomarkers of α-synuclein, anti-α-synuclein, and their ratios to Aß40 showed promising sensitivity and specificity.

Macrophage- and Microglia-Related Chemokines Are Associated with Small Vessel (White Matter) Vascular Dementia: A Case-Control Study.

INTRODUCTION: Little is known about the role of inflammation in the process of small vessel vascular dementia (VaD). Recently, the notion that small vessel VaD is caused solely by vascular pathology has been challenged by new evidence of concomitant breakdown of the blood-brain barrier and dysregulation of neuroinflammation in the white matter. METHODS: We examined selected inflammatory cytokines and chemokines in the plasma from patients with small vessel VaD (n = 41) and from age-matched controls (n = 131) using multiplex bead-based assays. Participants were recruited from a memory disorder clinic and from a hospital or community. RESULTS: When compared to controls, patients with small vessel VaD had a highly significant increase in the plasma interferon-γ-inducible protein 10 (IP-10) level (p < 0.0001) and a highly significant decrease in plasma macrophage inflammatory protein 1-beta (MIP-1ß) level (p < 0.0001). We also observed a significant increase in patients' levels of interleukin-10 (IL-10) (p = 0.022) as well as decreases in interleukin-8 (IL-8) (p = 0.004) and interleukin-7 (IL-7) (p = 0.011) when compared to age-matched controls. CONCLUSION: Both IP-10 and MIP-1ß are macrophage-related chemokines. The significant differences between cases and controls suggest a potential role for macrophages in small vessel VaD neuroinflammation. Although it remains unclear whether there is a causal effect of their alteration for small vessel VaD, a better understanding of these molecules in the pathogenesis of small vessel VaD may lead to improved diagnosis and future treatment outcomes against this disease.

The parasite-derived peptide FhHDM-1 activates the PI3K/Akt pathway to prevent cytokine-induced apoptosis of ß-cells.

Type 1 diabetes (T1D) is an autoimmune disease characterised by the destruction of the insulin-producing beta (ß)-cells within the pancreatic islets. We have previously identified a novel parasite-derived molecule, termed Fasciola hepatica helminth defence molecule 1 (FhHDM-1), that prevents T1D development in non-obese diabetic (NOD) mice. In this study, proteomic analyses of pancreas tissue from NOD mice suggested that FhHDM-1 activated the PI3K/Akt signalling pathway, which is associated with ß-cell metabolism, survival and proliferation. Consistent with this finding, FhHDM-1 preserved ß-cell mass in NOD mice. Examination of the biodistribution of FhHDM-1 after intraperitoneal administration in NOD mice revealed that the parasite peptide localised to the pancreas, suggesting that it exerted a direct effect on the survival/function of ß-cells. This was confirmed in vitro, as the interaction of FhHDM-1 with the NOD-derived ß-cell line, NIT-1, resulted in increased levels of phosphorylated Akt, increased NADH and NADPH and reduced activity of the NAD-dependent DNA nick sensor, poly(ADP-ribose) polymerase (PARP-1). As a consequence, ß-cell survival was enhanced and apoptosis was prevented in the presence of the pro-inflammatory cytokines that destroy ß-cells during T1D pathogenesis. Similarly, FhHDM-1 protected primary human islets from cytokine-induced apoptosis. Importantly, while FhHDM-1 promoted ß-cell survival, it did not induce proliferation. Collectively, these data indicate that FhHDM-1 has significant therapeutic applications to promote ß-cell survival, which is required for T1D and T2D prevention and islet transplantation. KEY MESSAGES: FhHDM-1 preserves ß-cell mass in NOD mice and prevents the development of T1D. FhHDM-1 enhances phosphorylation of Akt in mouse ß-cell lines. FhHDM-1 increases levels of NADH/NADPH in mouse ß-cell lines in vitro. FhHDM-1 prevents cytokine-induced cell death of mouse ß-cell lines and primary human ß-cells in vitro via activation of the PI3K/Akt pathway.

The kynurenine pathway in chronic diseases: a compensatory mechanism or a driving force?

The kynurenine (KYN) pathway (KP) of tryptophan (TRP) metabolism is dysregulated in inflammation-driven pathologies including oncological and brain diseases [e.g., multiple sclerosis (MS), depression] and thus is a promising therapeutic target. Both pathological and compensatory mechanisms underlie disease-associated KP activation. There is growing evidence for bioenergetic roles of certain KP metabolites such as kynurenic acid (KA), or quinolinic acid (QA) as an NAD+ precursor, which may explain its frequently observed 'pathological' overactivation. Disease- and tissue-specific aspects, negative feedback on inflammatory signals, and the balance of downstream metabolites are likely to be decisive factors in the interpretation of an imbalanced KP. Therapeutic strategies should consider the compensatory actions and bioenergetic roles of KP metabolites to successfully design future theragnostic approaches aimed at attenuating disease progression.

Mechanisms of impaired mitochondrial homeostasis and NAD+ metabolism in a model of mitochondrial heart disease exhibiting redox active iron accumulation.

Due to the high redox activity of the mitochondrion, this organelle can suffer oxidative stress. To manage energy demands while minimizing redox stress, mitochondrial homeostasis is maintained by the dynamic processes of mitochondrial biogenesis, mitochondrial network dynamics (fusion/fission), and mitochondrial clearance by mitophagy. Friedreich's ataxia (FA) is a mitochondrial disease resulting in a fatal hypertrophic cardiomyopathy due to the deficiency of the mitochondrial protein, frataxin. Our previous studies identified defective mitochondrial iron metabolism and oxidative stress potentiating cardiac pathology in FA. However, how these factors alter mitochondrial homeostasis remains uncharacterized in FA cardiomyopathy. This investigation examined the muscle creatine kinase conditional frataxin knockout mouse, which closely mimics FA cardiomyopathy, to dissect the mechanisms of dysfunctional mitochondrial homeostasis. Dysfunction of key mitochondrial homeostatic mechanisms were elucidated in the knockout hearts relative to wild-type littermates, namely: (1) mitochondrial proliferation with condensed cristae; (2) impaired NAD+ metabolism due to perturbations in Sirt1 activity and NAD+ salvage; (3) increased mitochondrial biogenesis, fusion and fission; and (4) mitochondrial accumulation of Pink1/Parkin with increased autophagic/mitophagic flux. Immunohistochemistry of FA patients' heart confirmed significantly enhanced expression of markers of mitochondrial biogenesis, fusion/fission and autophagy. These novel findings demonstrate cardiac frataxin-deficiency results in significant changes to metabolic mechanisms critical for mitochondrial homeostasis. This mechanistic dissection provides critical insight, offering the potential for maintaining mitochondrial homeostasis in FA and potentially other cardio-degenerative diseases by implementing innovative treatments targeting mitochondrial homeostasis and NAD+ metabolism.

NADomics: Measuring NAD+ and Related Metabolites Using Liquid Chromatography Mass Spectrometry.

Nicotinamide adenine dinucleotide (NAD+) and its metabolome (NADome) play important roles in preserving cellular homeostasis. Altered levels of the NADome may represent a likely indicator of poor metabolic function. Accurate measurement of the NADome is crucial for biochemical research and developing interventions for ageing and neurodegenerative diseases. In this mini review, traditional methods used to quantify various metabolites in the NADome are discussed. Owing to the auto-oxidation properties of most pyridine nucleotides and their differential chemical stability in various biological matrices, accurate assessment of the concentrations of the NADome is an analytical challenge. Recent liquid chromatography mass spectrometry (LC-MS) techniques which overcome some of these technical challenges for quantitative assessment of the NADome in the blood, CSF, and urine are described. Specialised HPLC-UV, NMR, capillary zone electrophoresis, or colorimetric enzymatic assays are inexpensive and readily available in most laboratories but lack the required specificity and sensitivity for quantification of human biological samples. LC-MS represents an alternative means of quantifying the concentrations of the NADome in clinically relevant biological specimens after careful consideration of analyte extraction procedures, selection of internal standards, analyte stability, and LC assays. LC-MS represents a rapid, robust, simple, and reliable assay for the measurement of the NADome between control and test samples, and for identifying biological correlations between the NADome and various biochemical processes and testing the efficacy of strategies aimed at raising NAD+ levels during physiological ageing and disease states.

Plasma lipidome is dysregulated in Alzheimer's disease and is associated with disease risk genes.

Lipidomics research could provide insights of pathobiological mechanisms in Alzheimer's disease. This study explores a battery of plasma lipids that can differentiate Alzheimer's disease (AD) patients from healthy controls and determines whether lipid profiles correlate with genetic risk for AD. AD plasma samples were collected from the Sydney Memory and Ageing Study (MAS) Sydney, Australia (aged range 75-97 years; 51.2% male). Untargeted lipidomics analysis was performed by liquid chromatography coupled-mass spectrometry (LC-MS/MS). We found that several lipid species from nine lipid classes, particularly sphingomyelins (SMs), cholesterol esters (ChEs), phosphatidylcholines (PCs), phosphatidylethanolamines (PIs), phosphatidylinositols (PIs), and triglycerides (TGs) are dysregulated in AD patients and may help discriminate them from healthy controls. However, when the lipid species were grouped together into lipid subgroups, only the DG group was significantly higher in AD. ChEs, SMs, and TGs resulted in good classification accuracy using the Glmnet algorithm (elastic net penalization for the generalized linear model [glm]) with more than 80% AUC. In general, group lipids and the lipid subclasses LPC and PE had less classification accuracy compared to the other subclasses. We also found significant increases in SMs, PIs, and the LPE/PE ratio in human U251 astroglioma cell lines exposed to pathophysiological concentrations of oligomeric Aß42. This suggests that oligomeric Aß42 plays a contributory, if not causal role, in mediating changes in lipid profiles in AD that can be detected in the periphery. In addition, we evaluated the association of plasma lipid profiles with AD-related single nucleotide polymorphisms (SNPs) and polygenic risk scores (PRS) of AD. We found that FERMT2 and MS4A6A showed a significantly differential association with lipids in all lipid classes across disease and control groups. ABCA7 had a differential association with more than half of the DG lipids (52.63%) and PI lipids (57.14%), respectively. Additionally, 43.4% of lipids in the SM class were differentially associated with CLU. More than 30% of lipids in ChE, PE, and TG classes had differential associations with separate genes (ChE-PICALM, SLC24A4, and SORL1; PE-CLU and CR1; TG-BINI) between AD and control group. These data may provide renewed insights into the pathobiology of AD and the feasibility of identifying individuals with greater AD risk.

Quantum dots as a theranostic approach in Alzheimer's disease: a systematic review.

Aim: Quantum dots (QDs) are nanoparticles that have an emerging application as theranostic agents in several neurodegenerative diseases. The advantage of QDs as nanomedicine is due to their unique optical properties that provide high sensitivity, stability and selectivity at a nanoscale range. Objective: To offer renewed insight into current QD research and elucidate its promising application in Alzheimer's disease (AD) diagnosis and therapy. Methods: A comprehensive literature search was conducted in PubMed and Google Scholar databases that included the following search terms: 'quantum dots', 'blood-brain barrier', 'cytotoxicity', 'toxicity' and 'Alzheimer's disease'; PRISMA guidelines were adhered to. Results: Thirty-four publications were selected to evaluate the ability of QDs to cross the blood-brain barrier, potential toxicity and current AD diagnostic and therapeutic applications. Conclusion: QD's unique optical properties and versatility to conjugate to various biomolecules, while maintaining a nanoscale size, render them a promising theranostic tool in AD.