Autophagy Markers Are Altered in Alzheimer's Disease, Dementia with Lewy Bodies and Frontotemporal Dementia.

The accumulation of protein aggregates defines distinct, yet overlapping pathologies such as Alzheimer's disease (AD), dementia with Lewy bodies (DLB), and frontotemporal dementia (FTD). In this study, we investigated ATG5, UBQLN2, ULK1, and LC3 concentrations in 66 brain specimens and 120 plasma samples from AD, DLB, FTD, and control subjects (CTRL). Protein concentration was measured with ELISA kits in temporal, frontal, and occipital cortex specimens of 32 AD, 10 DLB, 10 FTD, and 14 CTRL, and in plasma samples of 30 AD, 30 DLB, 30 FTD, and 30 CTRL. We found alterations in ATG5, UBQLN2, ULK1, and LC3 levels in patients; ATG5 and UBQLN2 levels were decreased in both brain specimens and plasma samples of patients compared to those of the CTRL, while LC3 levels were increased in the frontal cortex of DLB and FTD patients. In this study, we demonstrate alterations in different steps related to ATG5, UBQLN2, and LC3 autophagy pathways in DLB and FTD patients. Molecular alterations in the autophagic processes could play a role in a shared pathway involved in the pathogenesis of neurodegeneration, supporting the hypothesis of a common molecular mechanism underlying major neurodegenerative dementias and suggesting different potential therapeutic targets in the autophagy pathway for these disorders.

Imbalance of Essential Metals in Traumatic Brain Injury and Its Possible Link with Disorders of Consciousness.

Dysfunction of the complex cerebral networks underlying wakefulness and awareness is responsible for Disorders of Consciousness (DoC). Traumatic Brain Injury (TBI) is a common cause of DoC, and it is responsible for a multi-dimensional pathological cascade that affects the proper functioning of the brainstem and brain consciousness pathways. Iron (Fe), Zinc (Zn), and Copper (Cu) have a role in the neurophysiology of both the ascending reticular activating system, a multi-neurotransmitter network located in the brainstem that is crucial for consciousness, and several brain regions. We aimed to summarize the role of these essential metals in TBI and its possible link with consciousness alterations. We found that TBI alters many neuronal molecular mechanisms involving essential metals, causing neurodegeneration, neural apoptosis, synaptic dysfunction, oxidative stress, and inflammation. This final pattern resembles that described for Alzheimer's disease (AD) and other neurological and psychiatric diseases. Furthermore, we found that amantadine, zolpidem, and transcranial direct current stimulation (tDCS)-the most used treatments for DoC recovery-seem to have an effect on essential metals-related pathways and that Zn might be a promising new therapeutic approach. This review summarizes the neurophysiology of essential metals in the brain structures of consciousness and focuses on the mechanisms underlying their imbalance following TBI, suggesting their possible role in DoC. The scenario supports further studies aimed at getting a deeper insight into metals' role in DoC, in order to evaluate metal-based drugs, such as metal complexes and metal chelating agents, as potential therapeutic options.

The Impact of Hydroxytyrosol on the Metallomic-Profile in an Animal Model of Alzheimer's Disease.

It is undeniable that as people get older, they become progressively more susceptible to neurodegenerative illnesses such as Alzheimer's disease (AD). Memory loss is a prominent symptom of this condition and can be exacerbated by uneven levels of certain metals. This study used inductively coupled plasma mass spectrometry (ICP-MS) to examine the levels of metals in the blood plasma, frontal cortex, and hippocampus of Wistar rats with AD induced by streptozotocin (STZ). It also tested the effects of the antioxidant hydroxytyrosol (HT) on metal levels. The Barnes maze behavior test was used, and the STZ group showed less certainty and greater distance when exploring the Barnes maze than the control group. The results also indicated that the control group and the STZ + HT group exhibited enhanced learning curves during the Barnes maze training as compared to the STZ group. The ICP-MS analysis showed that the STZ group had lower levels of cobalt in their blood plasma than the control group, while the calcium levels in the frontal cortex of the STZ + HT group were higher than in the control group. The most important finding was that copper levels in the frontal cortex from STZ-treated animals were higher than in the control group, and that the STZ + HT group returned to equivalent levels to the control group. The antioxidant HT can restore copper levels to their basal physiological state. This finding may help explain HT's potential beneficial effect in AD-patients.

Non-Ceruloplasmin Copper Identifies a Subtype of Alzheimer's Disease (CuAD): Characterization of the Cognitive Profile and Case of a CuAD Patient Carrying an RGS7 Stop-Loss Variant.

Alzheimer's disease (AD) is a type of dementia whose cause is incompletely defined. Copper (Cu) involvement in AD etiology was confirmed by a meta-analysis on about 6000 participants, showing that Cu levels were decreased in AD brain specimens, while Cu and non-bound ceruloplasmin Cu (non-Cp Cu) levels were increased in serum/plasma samples. Non-Cp Cu was advocated as a stratification add-on biomarker of a Cu subtype of AD (CuAD subtype). To further circumstantiate this concept, we evaluated non-Cp Cu reliability in classifying subtypes of AD based on the characterization of the cognitive profile. The stratification of the AD patients into normal AD (non-Cp Cu ≤ 1.6 µmol/L) and CuAD (non-Cp Cu > 1.6 µmol/L) showed a significant difference in executive function outcomes, even though patients did not differ in disease duration and severity. Among the Cu-AD patients, a 76-year-old woman showed significantly abnormal levels in the Cu panel and underwent whole exome sequencing. The CuAD patient was detected with possessing the homozygous (c.1486T > C; p.(Ter496Argext*19) stop-loss variant in the RGS7 gene (MIM*602517), which encodes for Regulator of G Protein Signaling 7. Non-Cp Cu as an add-on test in the AD diagnostic pathway can provide relevant information about the underlying pathological processes in subtypes of AD and suggest specific therapeutic options.

Regulatory miRNAs in Cardiovascular and Alzheimer's Disease: A Focus on Copper.

Non-coding RNAs (ncRNAs), including microRNAs (miRNAs), are key regulators of differentiation and development. In the cell, transcription factors regulate the production of miRNA in response to different external stimuli. Copper (Cu) is a heavy metal and an essential micronutrient with widespread industrial applications. It is involved in a number of vital biological processes encompassing respiration, blood cell line maturation, and immune responses. In recent years, the link between deregulation of miRNAs' functionality and the development of various pathologies as well as cardiovascular diseases (CVDs) has been extensively studied. Alzheimer's disease (AD) is the most common cause of dementia in the elderly with a complex disease etiology, and its link with Cu abnormalities is being increasingly studied. A direct interaction between COMMD1, a regulator of the Cu pathway, and hypoxia-inducible factor (HIF) HIF-1a does exist in ischemic injury, but little information has been collected on the role of Cu in hypoxia associated with AD thus far. The current review deals with this matter in an attempt to structurally discuss the link between miRNA expression and Cu dysregulation in AD and CVDs.

Copper in Glucose Intolerance, Cognitive Decline, and Alzheimer Disease.

Trace metal dyshomeostasis has been linked to loss of cognitive performance. In particular, a disturbance in the regulation of copper (Cu), characterized by an increase in circulating Cu not bound to ceruloplasmin (non-Cp Cu), is thought to play a role in the development of Alzheimer disease (AD) and other neurodegenerative diseases in the aging population. Non-Cp Cu is redox active and its toxicity is thought to result from its ability to accelerate oxidative stress and advanced glycation endproduct (AGE) formation, leading to extracellular matrix damage in tissues including the brain. Cognitive loss is increasingly recognized to be a feature of type 2 diabetes and the increased AGE formation characteristic of diabetes may play a role in the development of this complication. There also is evidence for copper dyshomeostasis in type 2 diabetes, and therefore this could contribute to the cognitive deterioration associated with this disease. Demonstrating that disturbances of copper homeostasis correlate with an increased rate of cognitive decline in type 2 diabetes patients, and that they correlate with an increased rate of conversion from prediabetes to diabetes would bring almost immediate benefits in the clinical community in terms of treatment efficacy, AD prevention, and cost savings.

An exploratory study of BDNF and oxidative stress marker alterations in subacute and chronic stroke patients affected by neuropathic pain.

Patients affected by stroke, particularly subacute stroke patients, often complain of neuropathic pain (NP), which may severely impair their quality of life. The aim of this exploratory study was to characterize NP and to investigate whether there is a correlation between NP and serum brain-derived neurotrophic factor (BDNF) and serum markers of oxidative stress. We enrolled 50 patients divided in subacute (< 90 days from stroke onset) and chronic (> 90 and 180 < days from stroke onset). The Barthel Index, Deambulation Index, and Short Form 36 were used to assess the patients' degree of disability and quality of life. Pain-specific tools, namely the Numeric Rating Scale (NRS), Neuropathic Pain Diagnostic questionnaire (DN4), and Neuropathic Pain Symptom Inventory (NPSI), were also used. Serum levels of BDNF and markers of oxidative stress and of metal status were evaluated: copper, iron, transferrin, ferritin, ceruloplasmin concentration (iCp) and activity (eCp), Total Antioxidant status (TAS), Cp/Tf ratio, eCp/iCp ratio, and non-ceruloplasmin bound copper (Non-Cp Cu). We found the highest value of BDNF in subacute with NP (DN4 score ≥ 4). The TAS, Cp/Tf ratio, and eCp/iCp not only fell outside the normal reference range in a high percentage of subacute and chronic patients, but were also found to be related to specific NP symptoms. These preliminary results reveal altered BDNF and oxidative stress indices in subacute stroke patients who complain of NP. These investigative findings may shed more light on the mechanisms underlying NP and consequently lead to a more tailored therapeutic and/or rehabilitation procedure of subacute stroke patients.

Measurements of serum non-ceruloplasmin copper by a direct fluorescent method specific to Cu(II).

BACKGROUND: Meta-analyses indicated the breakdown of copper homeostasis in the sporadic form of Alzheimer's disease (AD), comprising copper decreases within the brain and copper increases in the blood and the pool not bound to ceruloplasmin (non-Cp Cu, also known in the literature as "free" copper). The calculated non-Cp Cu (Walshe's) index has many limitations. METHODS: A direct fluorescent method for non-Cp Cu detection has been developed and data are presented herein. The study included samples from 147 healthy subjects, 36 stable mild cognitive impairment (MCI) and 89 AD patients, who were tested for non-Cp Cu through the direct method, total serum copper, ceruloplasmin concentration and o-dianisidine ceruloplasmin activity. The indirect non-Cp Cu Walshe's index was also calculated. RESULTS: The direct method was linear (0.9-5.9 µM), precise (within-laboratory coefficient variation of 9.7% for low and 7.1% for high measurements), and had a good recovery. A reference interval (0-1.9 µM) was determined parametrically in 147 healthy controls (27-84 years old). The variation of non-Cp Cu was evaluated according to age and sex. Non-Cp Cu was 1.5 times higher in AD patients (regarding the upper value of the reference interval) than in healthy controls. Healthy, MCI and AD subjects were differentiated through the direct non-Cp Cu method [areas under the curve (AUC)=0.755]. Considering a 95% specificity and a 1.91 µmol/L cut-off, the sensitivity was 48.3% (confidence interval 95%: 38%-58%). The likelihood ratio (LR) was 9.94 for positive test results (LR+) and 0.54 for negative test result (LR-). CONCLUSIONS: The direct fluorescent test reliably and accurately measures non-Cp Cu, thereby determining the probability of having AD.

Biological factors and age-dependence of primary motor cortex experimental plasticity.

To evaluate whether the age-dependence of brain plasticity correlates with the levels of proteins involved in hormone and brain functions we executed a paired associative stimulation (PAS) protocol and blood tests. We measured the PAS-induced plasticity in the primary motor cortex. Blood levels of the brain-derived neurotrophic factor (BDNF), estradiol, the insulin-like growth factor (IGF)-1, the insulin-like growth factor binding protein (IGFBP)-3, progesterone, sex hormone-binding globulin (SHBG), testosterone, and the transforming growth factor beta 1 (TGF-ß1) were determined in 15 healthy men and 20 healthy women. We observed an age-related reduction of PAS-induced plasticity in females that it is not present in males. In females, PAS-induced plasticity displayed a correlation with testosterone (p = 0.006) that became a trend after the adjustment for the age effect (p = 0.078). In males, IGF-1 showed a nominally significant correlation with the PAS-induced plasticity (p = 0.043). In conclusion, we observed that hormone blood levels (testosterone in females and IGF-1 in males) may be involved in the age-dependence of brain plasticity.

Explorative genetic association study of GSTT2B copy number variant in complex disease risks.

BACKGROUND: Glutathione S-transferases (GSTs) are the main phase II enzymes involved in cellular detoxification. Through phase I and phase II detoxification reactions, the cell is able to detoxify endogenous and exogenous toxic compounds. AIMS: This study focused attention on the GSTT2B copy number variant (CNV) in order to explore its involvement in the genetic pre-disposition to asthma, Alzheimer's disease (AD), allergic rhinitis (AR), essential hypertension (EH), hypothyroidism and recurrent miscarriage (RM). METHODS: The study population consists of 1225 individuals divided into six case-control groups. The genotyping of the GSTT2B CNV was performed by using a duplex-PCR. Odds Ratios (ORs) were calculated, adjusting for the confounding variables, to estimate the association between GSTT2B CNV and the disease status. RESULTS: The χ(2)-test and ORs did not show any association between this genetic marker and pathological phenotypes. CONCLUSION: The data highlights that GSTT2B CNV is not associated with the investigated complex diseases in Italian patients. However, further investigations are necessary to replicate these findings in larger sample sizes and to explore other health-related phenotypes.

Value of serum nonceruloplasmin copper for prediction of mild cognitive impairment conversion to Alzheimer disease.

OBJECTIVE: Meta-analyses show that nonbound ceruloplasmin (non-Cp) copper (also known as free or labile copper) in serum is higher in patients with Alzheimer disease (AD). It differentiates subjects with mild cognitive impairment (MCI) from healthy controls. However, a longitudinal study on an MCI cohort has not yet been performed to assess the accuracy of non-Cp copper for the prediction of conversion from MCI to AD during a long-term follow-up. METHODS: The study included 42 MCI converters and 99 stable MCI subjects. We assessed levels of copper, ceruloplasmin, non-Cp copper, iron, transferrin, ferritin, and APOE genotype. A multiple Cox regression analysis-with age, sex, baseline Mini-Mental State Examination, APOE4, iron, non-Cp copper, transferrin, ferritin, hypercholesterolemia, and hypertension as covariates-was applied to predict the conversion from MCI to AD. RESULTS: Among the evaluated parameters, the only significant predictor of conversion to AD was non-Cp copper (hazard ratio = 1.23, 95% confidence interval = 1.03-1.47, p = 0.022); for each additional micromole per liter unit (µmol/l) of non-Cp copper, the hazard increased by ~20%. Subjects with non-Cp copper levels >1.6 µmol/l had a hazard conversion rate (50% of conversion in 4 years) that was ~3× higher than those with values ≤1.6 µmol/l (<20% in 4 years). The rate of conversion was similar between APOE4 carriers and noncarriers (p = 0.321), indicating that the non-Cp copper association was independent of APOE4. INTERPRETATION: Non-Cp copper appears to predict conversion from MCI to AD. These results encourage healthy life style choices and dietary intervention to modify this risk.

Altered metal metabolism in patients with HCV-related cirrhosis and hepatic encephalopathy.

Dysfunctional metal homeostasis contributes to oxidative stress and neuronal damage. These have been implicated in hepatic encephalopathy pathogenesis. To investigate whether altered metal metabolism is associated with hepatic encephalopathy. Twenty-one controls and 34 HCV-cirrhotic patients (ENC/NEC patients according to presence/absence of previous overt episodes of hepatic encephalopathy) and a control group were studied. Serum iron, copper, ceruloplasmin, ceruloplasmin activity, transferrin, and ceruloplasmin/transferrin ratio were determined. Neuropsychological tests were performed by the repeatable battery of neuropsychological status. Magnetic resonance assessed basal ganglia volumes and metal deposition (pallidal index and T2*). Cirrhotic patients performed worse than controls at cognitive tests, especially ENC patients,. At biochemical analysis copper concentrations, ceruloplasmin activity and transferrin levels were lower in ENC than in NEC patients and controls (p < 0.05 and p < 0.01, respectively). Ceruloplasmin/transferrin ratio was higher in ENC compared to NEC patients (p < 0.05), and controls (p < 0.01). By brain magnetic resonance, ENC patients showed reduced caudate and globus pallidus volumes compared to controls (p < 0.05), and ENC and NEC patients an increased pallidal index compared to controls (p < 0.01). In ENC patients, ceruloplasmin activity correlated with caudate volume and pallidal index (ρ = 0.773 and ρ = -0.683, p < 0.05). Altered metal metabolism likely contributes to cirrhotic hepatic encephalopathy.

Automation of o-dianisidine assay for ceruloplasmin activity analyses: usefulness of investigation in Wilson's disease and in hepatic encephalopathy.

Ceruloplasmin (Cp) is a serum ferroxidase that plays an essential role in iron metabolism. It is routinely tested by immunoturbidimetric assays that quantify the concentration of the protein both in its active and inactive forms. Cp activity is generally analyzed manually; the process is time-consuming, has a limited repeatability, and is not suitable for a clinical setting. To overcome these inconveniences, we have set the automation of the o-dianisidine Cp activity assay on a Cobas Mira Plus apparatus. The automation was rapid and repeatable, and the data were provided in terms of IU/L. The assay was adapted for human sera and showed a good precision [coefficient of variation (CV) 3.7 %] and low limit of detection (LoD 11.58 IU/L). The simultaneous analysis of Cp concentration and activity in the same run allowed us to calculate the Cp-specific activity that provides a better index of the overall Cp status. To test the usefulness of this automation, we tested this assay on 104 healthy volunteers and 36 patients with Wilson's disease, hepatic encephalopathy, and chronic liver disease. Cp activity and specific activity distinguished better patients between groups with respect to Cp concentration alone, and providing support for the clinical investigation of neurological diseases in which liver failure is one of the clinical hallmarks.

In silico investigation of the ATP7B gene: insights from functional prediction of non-synonymous substitution to protein structure.

ATP7B is a copper-transporting ATPase that plays a key role in the regulation of copper homeostasis. Mutations in the ATP7B gene are causative for Wilson's disease, and recent reports have suggested that genetic variants are associated with susceptibility to Alzheimer's disease. Unfortunately, it is difficult to profile experimentally novel genetic variants in the ATP7B gene, because the human protein X-ray structure is not yet entirely understood. In order to investigate ATP7B non-synonymous substitutions, we used an in silico amino acid sequence-based approach. Specifically, we analyzed 337 ATP7B non-synonymous substitutions, which included Wilson's disease-causing mutations (DVs) and non Wilson's disease-causing variants (NDVs), with an algorithm that estimated a combined probability (cPdel) of an amino acidic change to be deleterious for the protein function. This approach appeared to reliably indentify the probability of DVs and NDVs to be deleterious and to profile still unknown gene variants. Specifically, after analyzing ATP7B protein domains with the cPdel method, we found results in line with the predicted-modeled domains and some new suggestions. In conclusion, a functional survey of amino acid changes in the ATP7B protein is provided herein, and we suggest that this bioinformatic method can furnish information about novel ATP7B mutations. Furthermore, the same approach can be applied to other uncharacterized proteins.

Copper excess in psychiatric disorders: a focus on mood spectrum disorders and sex.

BACKGROUND: Meta-analyses show increased copper (Cu) levels in major depression disorder. However, the association of Cu biomarkers with clinical classification in other mental health disorders has not been fully explored. METHODS: To this aim, we compared an extensive panel of Cu biomarkers, composed of Cu, ceruloplasmin (Cp) Cp activity, Cp specific activity, Cu not bound to ceruloplasmin (non-Cp Cu, also known as 'free' copper) in 171 consecutive patients affected by psychiatric disorders and in 61 healthy controls (HC) using MANOVA adjusting for the effect of sex and age, and studied their association with the clinical scale outcomes at psychiatric examination, namely Global Assessment of Functioning, Clinical Global Impression, and Brief Psychiatric Rating Scale. RESULTS: individuals with psychiatric disorders were classified as 109 patients affected by mood spectrum disorders (MSD), 20 patients with schizophrenia spectrum disorders (SSD), and 42 with personality disorders (PD). Cu and non-Cp Cu were increased in psychiatric individuals than in HC, which also differed among the patients stratified per the clinical classification, being higher in the MSD individuals. The analysis stratified for sex revealed that women from the patient group, and specifically from the MSD group, had increased levels of Cu and non-Cp Cu than healthy women, while no difference was revealed in men. A logistic regression model considering the effect of sex and age revealed that non-Cp Cu could explain 26 % increased odds of having MSD per µmol/L unit increase (OR = 1.26; p = 0.0008; 95 % CI 1.099-1.436), that reached 40 % when considering only women. This result was driven by non-Cp Cu that correctly classified 64.1 % MSD (70 % in women) individuals vs. HC in a decision tree model, with values higher than 2.1 µmol/L which could distinguish the majority of MSD patients (86.3 % MSD vs. 13.7 % HC in women). None of the biological variables under study correlated with outcomes of the clinical scales, substances, or alcohol abuse. CONCLUSION: Current results suggest mild Cu toxicity in women with MSD, as revealed by a value of non-Cp Cu higher than 2.1 µmol/L, which can be further investigated to assess its potential diagnostic accuracy in bigger and longitudinal cohorts.

Serum copper status of patients with colorectal cancer: A systematic review and meta-analysis.

BACKGROUND: Colorectal cancer (CRC) is the third most commonly diagnosed cancer worldwide and a public health problem. Several clinical studies have shown that copper (Cu) is involved in carcinogenesis, possibly via cuproptosis, a new form of programmed cell death, but the conclusions from published reports are inconsistent. This study aimed at evaluating the potential of Cu dysregulation as a CRC susceptibility factor. METHODS: In this systematic review and meta-analysis, we searched Cochrane Library, EBSCOhost, EMBASE, ProQuest, PubMed/MEDLINE, Scopus, and Web of Science for studies reporting serum Cu concentrations in CRC patients and controls from articles published till June 2023. The studies included reported measurements of serum/plasma/blood Cu levels. Meta-analyses were performed as well as study quality, heterogeneity, and small study effects were assessed. Based on a random effects model, summary standardized mean differences (SMDs) and the corresponding 95% confidence intervals (95% CIs) were applied to compare the levels of Cu between CRC patients and controls. RESULTS: 26 studies with a pooled total of9628 participants and 2578 CRC cases were included. The pooled SMD was equal to 0.85 (95% CIs -0.44; 2.14) showing that the CRC patients had higher mean Cu levels than the control subjects, but the difference was not significant (p = 0.185) and the heterogeneity was very high, I2 = 97.9% (95% CIs: 97.5-98.3%; p < 0.001). CONCLUSION: The pooled results were inconclusive, likely due to discordant results and inaccuracy in reporting data of some studies; further research is needed to establish whether Cu dysregulation might contribute to the CRC risk and whether it might reflect different CRC grades.

The Debated Issue on Tissue Copper Levels in Colorectal Cancer Patients: A Meta-analysis and Replication Study.

Colorectal cancer (CRC) is a growing public health problem. Several clinical studies have shown a potentially oncogenic role of copper in CRC progression, but the reports are inconsistent. To examine published evidence on the association between tissue copper status and CRC, we carried out a systematic review and meta-analysis, searching Cochrane Library, EBSCOhost, Embase, ProQuest, PubMed/Medline, Scopus, and Web of Science for studies reporting colon tumor and matched non-cancerous tissue copper concentrations in CRC patients for articles published till June 2023. Based on a random effects model, standardized mean differences (SMD) were assessed. We also completed a replication study on 17 CRC patients that analyzed copper levels in both cancer tissue specimens and healthy mucosa dissected from the same patient. Thirteen studies investigating copper levels (including the replication study) in colorectal specimens from a pooled total of 312 CRC and 298 healthy mucosa were selected. Our meta-analysis estimated a high between-study heterogeneity (I2 = 96%) and lower levels of copper in CRC tissue cancer specimens than in matched healthy mucosa: the decrease was equal to - 0.74 (95% CI, - 2.18; 0.71) but was not significant. The replication study showed a significant decrease in tissue cancer specimens. Sensitivity analyses of the meta-analysis revealed that pre-analytical methodology for tissue preparation significantly reduced the between-study heterogeneity strongly influencing copper levels (p < 0.01), indicating a copper decrease in the cytoplasmic copper pool of the tumor tissue suggesting a rapid turnover of the metal in cancer cells.