Abstract
Meta-analyses show copper dyshomeostasis in Alzheimer’s disease. However, a study evaluating copper changes in other neurodegenerative forms of dementia has not yet been performed. In this study, we assessed copper, ceruloplasmin, copper not bound to ceruloplasmin, and copper to ceruloplasmin ratio in 85 patients affected by frontotemporal lobar degeneration (FTLD) and 55 healthy controls. Data were analyzed through multivariate ANOVA models taking into account age and sex as covariates and the stratification for FTLD variants, after calculating power analysis to ensure the reliability of the conclusions drawn. The study revealed no difference between the groups.
INTRODUCTION
Frontotemporal lobar degeneration (FTLD) is one of the most common cause of early onset dementia and is characterized by progressive degeneration of the frontal and anterior temporal lobes of the brain, with a wide and heterogeneous clinical presentation.
The most frequent subtypes that have been identified are the behavioral variant frontotemporal dementia (bvFTD), characterized by progressive deterioration of personality and social conduct [1], and the primary progressive aphasia (PPA), a phenotype with predominant language disorders like nonfluent/agrammatic presentation, logopenic variant, and semantic dementia [2]. Moreover, FTLD can clinically overlap with motor disorders: corticobasal syndrome, progressive supranuclear palsy (PSP), and
motor neuron disease (MND) [3–5]. Most FTLD cases are sporadic but up to a half has a family history and 15% to 40% are caused by single gene mutations [6].
Alzheimer’s disease (AD) is a leading cause of dementia caused by neurodegenerative processes. A number of meta-analyses focusing on the alterations of trace elements in AD have been published in the latest five years [7–11] showing a picture of copper dyshomeostasis. Specifically, patients with AD suffer of a copper deficiency in the brain [9], and of a copper increase in the periphery [8], due to the expansion of low molecular weight component of plasma copper not structurally bound to proteins, and primarily, not bound to ceruloplasmin (nCp-Cu, also known
as ‘free’ copper) [10, 12]. Studies evaluating copper changes in other forms of neurodegenerative diseases leading to dementia are still scanty. Herein we reported the results from the analysis of an extensive panel of copper markers (i.e., Copper, Cu; Ceruloplasmin, Cp; nCp-Cu; Copper:Ceruloplasmin ratio, Cu:Cp ratio) in serum of patients affected by FTLD. Either the effects of age and sex were taken in account along with the stratification of FTLD cases on the basis of the disease variants.
SUBJECTS AND METHODS
Subjects
Patients were recruited at the Memory Clinic of the IRCCS Fatebenefratelli in Brescia, Italy. The inclusion criteria for the present study were the diagnosis of FTLD according the criteria reported in [1, 13]. Moreover, a description of the FTLD population afferent to Memory Clinic of the IRCCS Fatebenefratelli in Brescia, Italy is detailed in [14]. FTLD is an umbrella term for different clinical variants which can been distinguished on the basis of predominant symptoms. In current study, we included patients with bvFTD and PPA, which represented the most frequent subtypes in our center. A few patients presented PSP and a patient came to our attention in an advanced phase, thus it was not possible to define the pattern of signs and symptoms at the onset which allowed classification variant. This classification yielded then four subgroups but just two (bvFTD and PPA) were representative of homogeneous subtypes in a number sufficient to run statistical analyses for comparisons (Table 1). Healthy controls underwent an extensive neuropsychological battery to exclude neurological disorders and memory complaints. A total of 85 FTLD (45 bvFTD, 35 PPA, 4 PSP, and 1 undefined FTD) and 55 Controls were analyzed in this study.
Demographic and clinical characteristics of the sample
Demographic and clinical characteristics of the sample
F, F-statistic of ANOVA test; K-W, Kruskal-Wallis statistic test; Me, median; IQR, interquartile range; χ2, chi-squared statistic test; IADL, Instrumental Activity of Daily Living; BADL, Basic Activity of Daily Living; CDR, Clinical Dementia Rating; sd, standard deviation.
Biological samples, isolated according to standard procedures, were stored at Fatebenefratelli Biobank (Brescia, Italy). Patients signed an informed consent (approval number 2/1992; 26/2014). The present study was approved by the local ethics committee (approval number 53/2016). Demographic and clinical features in the two groups (age, sex, APOE, and neuropsychological assessment) are reported in Table 1.
All patients underwent screening for GNR mutations, for expansion of the chromosome 9 open reading frame 72 (C9orf72), as well as for APOE4 genotype. The presence of null mutations in GRN gene was first investigated by plasma dosage of progranulin, as detailed in previous papers of ours [14–16], using ELISA assay (Human Progranulin ELISA Kit, Adipo-Gen Inc., Liestal, Switzerland): for values <61.55 ng/ml, the presence of the common Italian Leu271LeufsX10 mutation was assessed; if this mutation was not found, all other exons were sequenced as described in [16]. The GGGGCC repeat in the C9orf72 was amplified by PCR and allele identification was performed by agarose gel electrophoresis. A value equal or higher than 30 repeat units was classified as pathological expansion [17].
APOE4 genotype was assessed in patients and controls according to established methods [18].
Biochemical investigations
Fasting blood samples were collected in the morning and sera samples were separated by centrifugation (3000 rpm, 10 min, and 4 °C). They were then divided into 0.5 mL aliquots and rapidly stored at – 80°C. The subjects’ samples were shipped to Fatebenefratelli ‘San Giovanni Calibita’, Rome, Italy for blinded biochemical analyses. The aliquots were thawed just before the assay.
Concentration of Cp was measured with immunoturbidimetric assay (Futura System SRL, Rome, Italy) and was calibrated against the international reference preparation (ERM 470) [19], performed in duplicate on the multiple biochemical analyzer Horiba Pentra 400 (ABX Diagnostic, Montpellier, France). Serum copper (Cu) concentration was measured by atomic absorption spectrophotometry (A Analyst 600, Perkin-Elmer, Norwalk, CT, USA, equipped with graphite furnace).
Serum copper concentration was measured with Graphite Furnace Atomic Absorption GF-AAS equipped with a Longitudinal Zeeman Background correction (THGA AAnalyst 600 Perkin Elmer Instruments), following a specific method suggested from the company (wavelength 324.8 nm, slit width 0.7 mm, thermic program with an Atomization temperature of 2100°C). A standard calibration were prepared with 0; 0.5; 1.0; 1.5 mg/L of Cu, diluting a 1000 mg/L Cu calibration standards (Perkin Elmer) in HNO3 0.2 % (v/v) (ultrapure grade from J.T.Baker ®). 0.1% Triton (wetting agent, Perkin Elmer) and 0.1% Pd+ 0.1% Mg(NO3)2 modifier (GF-AAS Modifier Perkin-Elmer) were added to the serum samples and references solutions, with a 1 + 19 (v/v) dilution. nCp-Cu was calculated from the equation provided by Walshe (appendix of [20]), based on the measures of concentration of total copper and Cp in serum. Equivalent data can be obtained calculating nCp-Cu from mg/L of ceruloplasmin and considering the conversion of 3.15μg/Cu for mg of ceruloplasmin [20].
Copper:Ceruloplasmin ratio (Cu:Cp) [10] was calculated as reported in Twomey et al. [21]. These authors provided the equation as follows:
[copper μmol/L]* [132000 (g/mol)] / [Cp (mg/dL)*104].
Statistical analyses
Summary statistics of socio-demographic and clinical characteristics were carried out through means and standard deviations (SDs) for continuous variables and frequencies and percentages for discrete variables. Comparison of socio-demographic and clinical variables were carried out by ANOVA, multivariate ANOVA model (MANOVA)—in order to take into account correlation among metal variables all measured for whole study sample—Kruskal-Wallis and χ2 test based on type or variable distribution (continuous or categorical and Gaussian or non-Gaussian). Gaussianity was tested by Shapiro-Wilk e Kolmogorov-Smirnov test. Goodness of fit of MANOVA models was evaluated by the analysis of residuals [22–24]. Based on these studies, we evaluated the copper mean and standard deviation (SD) values in serum measured in a group of healthy volunteers equal around 13.0μmol/L with a SD of about 2.3; and the corresponding values for AD patients around 15.6μmol/L for the mean and 3.4 for the SD. We assumed for FTLD, in a precautionary scenario, an effect size lightly smaller than the one found for AD in the previous cited literature, i.e., a change in copper mean of 15% between healthy group and FTLD group instead of 20% (found between healthy group and AD group). So, the minimum sample size necessary for this variation to be significant (considering the above SD, with a two-tail independent student t-test with alpha equal to 0.05 and 80% power) is about n = 35 subjects per each group. Our sample is thus enough to ensure enough power.
In order to evaluate our results in the population stratified for the clinical phenotype, three groups were yielded (bvFTD, PPA, and PSP). However, only two (bvFTD and PPA) were representative of homogeneous disease subtypes in a number sufficient to run statistical analyses for comparisons with group size equal to n = 45 and n = 35, respectively.
Data of the bvFTD and PPA were described in terms of mean±standard deviation. After clinical phenotype stratification, two outliers, detected by Inter-quantile-range (IQR), were deleted (one in PPA and one in bvFTD).
Statistical analyses were performed using SPSS (version 21.0, IBM SPSS Statistics for Windows. Armonk, NY: IBM Corp). Statistical significance was set at p < 0.05.
RESULTS
Markers of copper metabolism were analyzed in 85 FTLD and 55 Controls (Table 1). These groups did not differ for age and sex. Patients differed from healthy controls in terms of neuropsychological performance and ability in daily living (Table 1). MANOVA was applied for the comparison of the biological variables under study in order to take into account the correlation among all the measured copper variables for the whole study sample. Table 2 reports these comparisons: no statistically significant differences were found between the two groups. Table 3 reports the data as classified on the basis of the main representative FTLD variants. When considering this stratification the analysis revealed that the PPA group had higher values of nCp-Cu and of Cu:Cp. Nevertheless, these differences resulted not significant, confirming data of the whole FTLD population. Either the patient carrying the C9orf72 expansion or the two carrying the GRN mutations received a diagnosis of bvFTD and had a value of copper of 14μmol/L, very close to the mean values of healthy controls.
Serum copper profile of the sample: multivariate ANOVA (MANOVA)
Serum copper profile of the patients stratified on the basis of the FTLD disease variants
We checked for possible association between copper markers profile and progranulin levels, but no correlation was found (all p > 0.2; data not shown).
DISCUSSION
Essential metal perturbation has been extensively reported in AD [25]. The picture gleaned from meta-analysis studies showing deficiency of copper in the brain [9] and excess of copper in the periphery [8] pertains to a process of copper dyshomeostasis. The same process can account for the expansion of the labile component of copper not bound to proteins in the disease [10, 27]. A shift from bound metal ions in proteins (typical Kd< ∼10–7 M) to loosely bound, chelatable metal ions (typical Kd> ∼10–6 M) [28] either in the brain [26, 27] or in the periphery [10] has been proposed as a key of interpretation of the failure of copper control in AD [28, 29]. On this basis, we explored copper trend in FTLD, one of the most common form of early onset dementia.
Recently iron accumulation in the brain, and specifically in the basal ganglia, of FTLD patients has been described [30, 31]. Differently from AD, copper homeostasis does not appear altered in FTLD. FTLD is heterogeneous in terms of clinical symptoms, genetics, or neuropathological features, and may coexist with amyotrophic lateral sclerosis (ALS), recently discovered to be associated to C9orf72 expansion. The stratification in terms of disease variant confirmed data from the general FTLD population: copper and ceruloplasmin did not differ, even though PPA had increased values of nCp-Cu and Cu:Cp. However, both the nCp-Cu and Cu:Cp values were not significantly different from healthy controls and far from the values found in other neurodegenerative diseases, as for example AD [12, 32].
Our study has a number of limitations which include primarily the lack of cases exhibiting motor symptoms, including rare forms as MND and CBD. Another limitation consists in the small number of patients exhibiting PSP, not enough to run statistical analyses. We found a C9orf72 mutated patient, as well additional two with GRN mutations, which did not appear different from the healthy controls in terms of copper profile. Nevertheless, the remarkable overlap between ALS and non-tau FTLD recently discovered suggests potential common molecular pathways that can involve copper abnormalities. Thus, future studies in bigger populations enriched with patients exhibiting motor symptoms in less common forms of FTLD, as, for example, PSP, CBD, and MND, are certainly needed.
Footnotes
ACKNOWLEDGMENTS
This work was supported by Italian Ministry of Health [5XMille project ‘Un metodo sensibile, diretto e preciso per misurare il rame Non-legato alla Ceruloplasmina nel siero per applicazione in ambiente clinico’ 02/09/2013 al 31/08/2015]; Italian Ministry of Health, Ricerca Corrente.
