Histone Deacetylases Enzyme, Copper, and IL-8 Levels in Patients With Alzheimer’s Disease

Abstract

Background:

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive loss of cognitive abilities. Epigenetic modification, oxidative stress, and inflammation play an important role in the pathogenesis of the disease. We aimed to detect noninvasive peripheral biomarkers with a high degree of sensitivity and specificity in diagnosis and progression of AD.

Methods:

A total of 25 elderly patients with AD and 25 healthy control participants were selected and subjected to cognitive assessment and laboratory measures including histone deacetylases (HDACs), copper, and interleukin 8 (IL-8) levels.

Results:

The levels of HDACs, copper, and IL-8 were significantly higher in patients with AD (P < .001) and had a significant negative effect on all cognitive assessment tests. Receiver–operating curve (ROC) analysis revealed that HDACs and copper levels had higher sensitivity and specificity.

Conclusions:

Plasma levels of HDACs and copper may be used as peripheral biomarkers in diagnosis of AD, while IL-8 level could be a useful biomarker in following AD progression.

Introduction

Alzheimer’s disease (AD) is the leading cause of dementia that accounts for up to 75% of all dementia cases, ¹ and the risk of dementia increases with advanced age. ²

The sporadic nature of 90% of the patients with AD, the differential susceptibility and course of illness, and the late onset (in terms of age) of the disease suggest that the epigenetic and environmental components play a role in the etiology of late-onset AD. ³

Patients with AD lose their memory and their cognitive abilities, and their personalities may change dramatically. These changes are due to the progressive dysfunction and death of nerve cells that are responsible for the storage and processing of information. Although drugs can temporarily improve memory, there are no treatments that can stop or reverse the inexorable neurodegenerative process. The actual cause of AD is not clear. It may be a combination of many processes such as inflammation, oxidative stress, and epigenetics. ⁴

The hallmark neuropathological features of AD include extracellular plaques and intracellular tangles. Endoproteolytic cleavage of the transmembrane amyloid precursor protein (APP) generates β-amyloid (Aβ) peptides that aggregate to form plaques. Such plaques interfere with transcription and cause deficits in plasticity and cognition. ⁵

Epigenetic is the study of heritable changes in gene expression or cellular phenotype caused by mechanisms other than changes in the underlying DNA sequence; hence the name epi (Greek: ∊πi = over, above, outer). Such changes may include DNA methylation and histone deacetylation, both serve to suppress gene expression without altering the sequence of the silenced genes. ⁶

Epigenetic remodeling is crucial to cellular differentiation, development, and behavior. Epigenetic dysregulation is a common theme in many neurodegenerative disorders such as Huntington’s disease, Parkinson’s disease and AD, and mood disorders (depression and anxiety). ⁵

Histone acetylation has received much attention in the nervous system, and acetylation of core histones is catalyzed by transcriptional coactivators, such as CREB (cAMP response element-binding protein)-binding protein (CBP), which possess histone acetyltransferase (HAT) activity. Histone acetylation remodels chromatin structure, thereby modulating transcription. ⁷

Histone deactylases (HDACs) remove acetyl groups from lysine/arginine residues in the amino terminal tails of core histones and other proteins, thus reversing the effects of the HATs. Deacetylation of histone proteins shifts the balance toward chromatin condensation and thereby silences gene expression. ⁸

Altogether, mammalian HDACs fall into 4 main classes, classes 1 to 4, with class 1 and class 2 HDACs receiving the most attention in the nervous system, as the anticonvulsant and mood-stabilizing drug valproic acid was identified as an inhibitor of HDAC1, thereby linking its antiepileptic effects to changes in histone acetylation. Several studies have revealed that inhibitors of class 1 and 2 HDACs represent novel therapeutic approaches to treat neurodegenerative disorders, depression, and anxiety and the cognitive deficits that accompany many neurodevelopmental disorders. ⁹

Thus, despite controversy, the epigenetic modification constitutes a basic molecular mechanism and contributes to AD pathogenesis. Therefore, understanding of its role in AD will provide important clues in solving the disease. ¹⁰

Alzheimer's disease brain is marked by severe neuronal death that has been partly attributed to increased oxidative stress that constitutes a hallmark of AD. Recent studies also point to redox active metals such as copper in mediating oxidative stress in AD. ¹¹ A possible role of copper in AD has remained a contentious topic during the past 15 years, as has been the question whether extracellular amyloid deposited in plaques is the causative agent in AD. The scientific community was divided as to whether copper has a role at all, and—if yes—whether it is a friend or foe. ¹²

The importance of inflammation in AD is suggested by data showing that nonsteroidal anti-inflammatory drugs (NSAIDs) diminish the risk of developing AD. The effects of NSAID are largely attributed to the inhibition of the enzymatic activity of cyclo-oxygenase (COX)-1 and cyclo-oxygenase-2, leading to the suppression of prostaglandin synthesis with a subsequent anti-inflammatory effect. On the other hand, NSAID may affect Aβ deposition and metabolism. So the effect on AD may be related to lowering Aβ42 and not to their anti-inflammatory properties. ¹³

The role of the chemokines interleukine 8 (IL-8) in AD progression is not understood, but it probably represents an additional recruitment mechanism for the migration of microglia to Aβ deposits associated with senile plaques, followed by a subsequent and persistent activation of microglial cells. ¹⁴

Our aim was to detect noninvasive, readily accessible peripheral biomarkers with a high degree of sensitivity and specificity in diagnosis and progression of AD. These markers play a role in epigenetic modification as HDACs, oxidative stress as copper, and inflammation as IL-8.

Methods

Study Population

A total of 50 elderly patients (age ≥60 years) were included in this study. Informed consent was taken from all participants (consent was taken from the family in case of incompetent patients with AD). The participants were divided into 2 groups.

1. Patients group included 25 patients with AD, their age ranged from 72 to 84 years (mean ± standard deviation [SD] = 72.2 ± 5.9). They were recruited from Geriatrics department and the Geriatrics outpatient’s clinic, Ain Shams University hospital. Diagnosis of dementia was done according to Diagnostic and Statistical Manual of Mental Disorders (Fourth Edition, Text Tevision [DSM-IV-TR] criteria, ¹⁵ and diagnosis of probable AD was done according to the National Institute of Neurological and Communicable Disease and Stroke–AD and Related Disorders Association criteria(NINCDS-ADRDA). ¹⁶ The NINCDS-ADRDA is valid, sensitive, and had been used in many studies to diagnose AD. ¹⁷ Those patients with AD were subdivided into mild, moderate, and severe, according to their Mini-Mental State Examination (MMSE) score; mild (MMSE: 26-20), moderate (MMSE 10-20), and severe (MMSE <10).

2. Control group included 25 cognitively normal, healthy elderly participants, with no evidence of any neurological, psychiatric, or medical illnesses that could affect cognition. Their age ranged from 76 to 83 years (mean ± SD = 72.8 ± 4.1).

Exclusion Criteria

The participants with any of the following criteria were excluded from our study:(a) Patients with major depressive disorder or other psychiatric disorders, (b) patients with a history of traumatic brain injury or other neurological disorder (eg, cerebrovascular strokes, etc), (c) patients with abnormalities in thyroid hormones levels, (d) patients with advanced medical problems including advanced diabetes mellitus ([DM] those with severe neuropathy and/or severe nephropathy (macroalbuminuria >300 mg/d and or creatinine >250 mol/L), advanced hepatic disease (those with clinical signs of liver cell failure eg, jaundice, ascites, encephalopathy), advanced chronic kidney disease (patients with chronic kidney disease [CKD] stage 4 or stage 5), advanced pulmonary disease (patients who need long-term oxygen therapy), advanced cardiac disease (patients with New York Heart Association [NYHA] heart failure grade III or more), anemia with hemoglobin <10 g), (e) patients with recent infections or myocardial infarction, (f) patients who received any of these medications: antiplatelets, systemic anti-inflammatory drugs, centrally acting antihypertensive drugs, antineoplastic, or immunosuppressive drugs at least 8 weeks prior to their assessments, and (g) finally, patients with possible AD were also excluded.

Measurements

1. Comprehensive geriatric assessment (CGA), including screening for dementia using the Arabic version ¹⁸ of MMSE. ¹⁹

2. For demented participants, diagnosis of probable AD was done according to the NINCDS-ADRDA criteria. ¹⁶

3. Cognitive assessment for all participants was done using MMSE ¹⁹ plus 7 tests from the neuropsychological battery of Arabic version of Consortium to Establish a Registry for Alzheimer's disease (CERAD). ²⁰

4. Routine laboratory workup including complete blood count (CBC), kidney function tests, liver function tests, thyroid stimulating hormone (TSH) (for all participants), and computed tomography (CT) of the brain (only for patients with dementia) to exclude other causes of dementia.

5. Specific laboratory workup including HDAC concentration IL-8, and copper level in plasma were measured for all participants. Heparinized blood samples were collected and were stored at −80°C until laboratory analysis. The activity of HDACs was measured in peripheral blood mononuclear cells by layering onto Ficoll-Hypaque gradient and separating them by density centrifugation. ²¹ The HDAC assay was performed according to BioVision Colorimetric HDAC Activity BioVision Research Products Catalog #K331). ²² Interleukin 8 was measured by Enzyme-linked immunosorbent assay (ELISA) technique using a kit provided by Orgenium Laboratories, Finland (catalogue no: IL 08070319) ²³ and copper level in plasma was measured by atomic absorption, according to Makino and Takaha. ²⁴ Previous biochemical assessments were performed for all participants.

Cognitive assessment tests

Seven tests of the neuropsychological assessment battery of CERAD were administered after the clinical assessment was completed. The CERAD has developed valid and reliable procedure and assessment form in the evaluation of AD. ²⁵ This cognitive assessment battery included the following tests.

1. Verbal fluency test. ²⁶ This test measures verbal production, semantic memory, and language. It requires the subject to name as many examples of the category “animal” as possible in 1 minute.

2. Mini-Mental State Examination. ¹⁹ This is a well-known brief general cognitive battery that measures orientation, language, concentration, constructional praxis, and memory.

3. Word list memory. ²⁷ This is a free-recall memory test that assesses learning ability for new verbal information. The maximum score on each trial is 10.

4. Constructional praxis. ²⁸ It measures visuospatial and constructional abilities and requires the subject to copy 4 line drawings presented in order of increasing complexity. The total possible score is 11.

5. Word list recall. ²⁰ This test assesses the ability to recall, after a 5-minute delay, the 10 words given in the word list memory test.

6. Word list recognition. ²⁹ This test assesses recognition of the words presented in the word list memory task when presented among 10 distractor words.

7. Recall of constructional praxis. ³⁰ In this test, the participants were asked to draw the 4 figures (circle, diamond, rectangles, and cube) from their memories. The total score is 11.

Statistical Analyses

All analyses were performed using SPSS version 16 statistical software. Data were presented as mean ± SD or as number and percentage. Chi-square test was used for categorical variables. Student t test or Mann-Whitney test was used when appropriate in quantitative data. The correlation was done by Spearman rho test. Receiver–operating curve (ROC) analysis was done to detect sensitivity, specificity, area under the curve, and cutoff value. P value was considered significant when ≤.05.

Results

Our participants were age and gender matched, no significant difference in their educational level was found, only 3 patients with AD had positive family history of AD, and the mean value of disease duration was 2.8 years. Concerning severity of the disease, our patients were subdivided according to their MMSE score into mild (n = 2), moderate (n = 10), and severe (n = 13). In all, 68% of patients withAD versus 56% of control subjects were free from comorbid diseases Table 1.

Table 1.

Demographic Data of the Study Participants.

	AD patients (n = 25)	Control group (n = 25)	P value a
Gender: male	11 (44%)	12 (48%)	>.05
Age (mean ± SD)	72.2 ± 5.9	72.8 ± 4.1	>.05
Educational level
Illiterate	11 (44%)	11 (44%)	>.05
Can read and write	8 (32%)	6 (24%)
≥6 years formal education	3 (12%)	5 (20%)
University level or higher	3 (12%)	3 (12%)
Duration of AD, years	2.8 ± 1.9
Positive family history of AD	3 (12%)
Severity of AD
Mild AD	2 (8%)
Moderate AD	10 (40%)
Severe AD	13 (52%)
Comorbid diseases
Diabetes mellitus	3 (12%)	5 (20%)
COPD	2 (8%)	1 (4%)
CHF	2 (8%)	1 (4%)
Hypertension	1 (4%)	4 (16%)

Abbreviations: COPD,chronic obstructive pulmonary disease; CHF, congestive heart failure; AD, Alzheimer’s disease.

^a Significant P value <.05.

As expected, all cognitive assessment tests scores were significantly lower in AD patients in comparison to control participants (P < .001; Table 2). Comparison between moderate and severe patients with AD as regard their cognitive assessment tests were significant (Figure 1). The mean rank of word list memory for moderate compared to severe AD was 16.6 versus 8.5. In construction praxis, the mean rank for moderate compared to severe AD was 17 versus 9. In word list recognition, the mean rank for moderate compared to severe AD was 15.2 versus 9.5. While, in construction praxis, the mean rank for moderate AD was 14 versus 10.5 for severe AD. Patients with mild AD were not included because of their small number (n = 2).

Table 2.

Comparison Between Patients With AD and Control Participants as Regard Cognitive Assessments.

	AD patients (n = 25)	Control group (n = 25)	P value
MMSE	12.56 ± 5.8	27.8 ± 4.4	<.001 a
Verbal fluency test	2.6 ± 2.7	12.9 ± 3.6	<.001 a
Word list memory	5.7 ± 4.7	8.2 ± 5.8	<.001 a
Constructional praxis	1.9 ± 3.3	7.6 ± 2.7	<.001 a
Word list recognition	1.2 ± 1.7	6.8 ± 1.5	<.001 a
Word list recall	1.7 ± 0.9	9 ± 1.3	<.001 a
Recall of construction praxis	0.3 ± 0.7	6 ± 2.2	<.001 a

Abbreviations: MMSE, Mini-Mental State Examination; AD, Alzheimer’s disease.

^a Significant P value <.05.

Figure 1.

Comparison in cognitive assessment between moderate and severe AD patients.

All laboratory measurements in patients with AD, namely HDACs, copper, and IL-8 levels were significantly higher in comparison to control participants (P < .001; Table 3). The IL-8 level was significantly correlated with AD severity as it was higher in patients with severe AD compared to those with moderate stage (14.3 ± 0.16 versus 10.6 ± 3.2), while copper and HDAC levels were not significantly differing between patients with moderate and severe AD (Figure 2).

Table 3.

Comparison in Laboratory Measurements Between Patients With AD and Control Group.

	AD patients (n = 25)	Control group (n = 25)	P value
HDACs, ug/mg protein	25.9 ± 2.3	14.1 ± 3.5	<.001 a
Copper level, mg/L	1.1 ± 0.12	0.78 ± 0.13	<.001 a
IL-8, ng/mL	12.6 ± 3.4	11.6 ± 1.6	<.001 a

Abbreviations: AD, Alzheimer’s disease; HDACs, histone deacetylases; IL-8, interleukin 8.

^a Significant P value <.05.

Figure 2.

Comparison in laboratory measures between moderate and severe AD patients.

Table 4 and Figure 3 show ROC analysis of HDACs, copper, and IL-8 levels. All laboratory measures, HDACs, copper, and IL-8 had a negative significant correlation with cognitive assessments (P < .05; Table 5).

Table 4.

Receiver–Operating Curve (ROC) Analysis for Laboratory Measures.

	Sensitivity	Specificity	AUC	Cutoff value	95% CI	PPV	NPV	LR+	LR−
HDACs	100%	96%	0.966	16.7 ug/mg protein	12.8-25.6	96%	100%	25	0
Copper	100%	92%	0.956	0.85 mg/L	0.89-1	92.6%	100%	12.5	0
IL-8	56%	80%	0.642	12.95 ng/mL)	10.9-13.8	73.7%	63%	2.7	0.6

Abbreviations: AUC, area under the curve; 95% CI,95% confidence interval; PPV, positive predictive value; NPV,negative predictive value; LR+,positive likelihood ratio; LR−, negative likelihood ratio; HDACs, histone deacetylases; IL-8, interleukin 8.

Figure 3.

ROC (Receiver operating curve) analysis of Laboratory measures.

Table 5.

Correlation Between Laboratory Data and Cognitive Tests.

	HDACs	IL-8	Copper
MMSE	−0.80 a	−0.41 a	−0.65 a
Verbal fluency test	−0.75 a	−0.33 a	−0.77 a
Word list memory	−0.76 a	−0.41 a	−0.81 a
Construction praxis	−0.73 a	−0.42 a	−0.78 a
Word list recall	−0.74 a	−0.34 a	−0.80 a
Word list recognition	−0.78 a	−0.34 a	−0.78 a
Recall of construction praxis	−0.76 a	−0.37 a	−0.79 a
Copper	0.69 a	0.28

Abbreviations: MMSE, Mini-Mental State Examination; HDACs, histone deacetylases; IL-8, interleukin 8.

^a Significant P value <.05.

Discussion

Alzheimer’s disease is the most common age-dependent neurodegenerative disorder, which shows progressive memory loss and cognitive decline. Recent studies bolster that aberrant change in epigenetic modification within the genome of the aging brain cause gene misregulation that drives cognitive decline. Such epigenetic modification as histone acetylation that facilitates gene expression profiles in the brain. ¹⁰

Our study was conducted on 50 Egyptian participants; 25 patients who were diagnosed as probable AD according to the NINCDS-ADRDA (17) and 25 healthy participants as control. Age and gender were matched. In all, 68% of patients with AD versus 56% of control participants had no associated comorbid diseases. Small percentage of our participants had DM or, chronic obstructive pulmonary disease (COPD), congestive heart failure, or hypertension. In this study there were newly diagnosed patients with AD, and they did not receive any medications for AD before participating in this study. Patients with AD were subdivided into mild (n = 2), moderate (n = 10), and severe (n = 13) according to their MMSE score. Due to the small number of Patients with mild AD, they were not included in further statistical analysis.

In our work, we found that HDACs level was significantly higher in patients with AD in comparison to control participants. In addition, HDACs had negative impact on all cognitive assessments. This high level of HDACs may lead to chromatin condensation and thereby silences gene expression. The disruption of transcriptional homeostasis may trigger signaling cascades linked with a number of pathological mechanisms in AD. The abnormal signaling cascades during aging may ultimately promote neuronal dysfunction and subsequent neuronal damage. ⁹

The negative effect of HDACs on cognitive function may be due to repression of HAT activity by the high level of HDACs. Activity of HAT is essential for neuronal functions linked to cognition, including learning and memory, axonal outgrowth, axonal transport, and synaptic plasticity. ²²

In the same context, Guan et al ³¹ found that enhancement of memory formation was found in mice treated with HDACs inhibitors or subjected to genetic knockout of the HDAC2 gene. So HDACs inhibitors could be considered for clinical application to patients with AD. So correction of histone modification abnormalities may be of therapeutic benefit in AD. The HDACs inhibitors may improve phenotypes by either upregulating survival genes that are repressed in AD or repressing prodeath genes that are elevated in AD. ^{32 , 33}

It is important to realize that epigenetic modification is reversible while genetic mutation is not. Therefore, drug compounds can dynamically modulate the status of histone hypoacetylation. ³²

There is a conflict regarding plasma copper level in patients with AD compared to control participants. A significant elevation in plasma copper level was reported by Squitti et al, ^{34 , 35} although others found decreased level of total copper in AD brain compared to controls. ^{36 , 37} The elevated copper level in blood and its depletion in the brain of patients with AD may reflect the imbalance in copper metabolism. In addition, we found that copper had negative impact on cognitive assessments. Similarly, Mueller et al ¹¹ found that increase in copper level in participants with mild cognitive impairment would lead to dementia.

Interestingly, copper may affect histone acetylation through its direct inhibition of HAT. ³⁸ It was found that exposure of hepatoma cell line to copper resulted in a significant decrease in histone acetylation, as indicated by the decrease in the overall histone acetylation, especially H3 and H4 histone acetylation. This negative effect of copper on histone acetylation was through direct inhibition of histone acetylase activity. In addition, when the hepatoma cell line was exposed to chelator of copper, histone acetylation was detected. ³⁸

In the current results, impaired cognitive function in our patients with AD could be explained by histone hypoacetylation due to the suppression of HAT activity. This suppression may be due to the high levels of HDACs and copper.

Recently, Kaden et al ¹² found that copper promotes the nonamyloidogenic processing of APP and thereby lowers the Aβ production, the diagnostic marker in cell culture systems. In a clinical trial with patients having AD, the decline in Aβ levels in cerebrospinal fluid (CSF) is diminished in the verum group, who received copper supplementation (8 mg copper/d), indicating a beneficial effect of the copper treatment. This conflicting role of copper in AD either benefits or not, may need a lot of further researches to be clarified.

The role of inflammation in AD is well evident. The IL-1beta, IL-6, and tumor necrosis factor-alpha (TNF-α) play a role in complex cognitive processes at the molecular level, such as synaptic plasticity, neurogenesis, and neuromodulation. Interleukin 8, a chemokine produced by macrophage response to proinflammatory mediators, such as amyloid, could be important for recruiting activated microglia into sites of the brain damaged by AD. The IL-8 receptor, chemokine (C-X-C motif) receptor 2 (CXCR2), has been localized to dystrophic neurites, suggesting that IL-8 mediates glial interactions with neurons and thereby contributes to neuronal damage. ³⁹

So, in our study we measured IL-8 in peripheral blood as one of the cytokines that was not well studied in the literature. We found that IL-8 was significantly higher in patients with AD in comparison to the control participants. This significant elevation was, also, reported either in blood ⁴⁰ or in CSF ⁴¹ of patients with AD. No significant difference in IL-8 level either in CSF or in peripheral blood in patients with AD was found by others. ⁴²

Some authors found that plasma level of IL-8 in late-onset AD and vascular dementia did not differ from the controls in the European participants. ⁴³ On the other hand, Kim et al ⁴⁴ found that the IL-8 level was lower in Asian patients with AD.

This controversy might partly be explained by differences in the study populations, such as inclusion criteria or the number of participants. In addition, circulating cytokines have short half-lives, may reach high concentrations at the sites of release and much lower concentrations after dilution in blood, and may circulate bound to molecules that can prevent their detection by immunological methods. All of these may contribute to the great variability in the reported data. ⁴⁵

Nevertheless, the identification of an inflammatory biomarker of AD, in combination with other biological markers, is required to improve the accuracy of diagnosis and monitor disease progression. ⁴⁴

Interestingly, it was found that the most highly induced chemokine genes was IL-8 gene in postmortem brain of patients with AD. ⁴⁶ Moreover, upregulation of IL-8 is likely to be a very early event in the AD pathogenesis. ⁴⁷

The role of IL-8 in AD progression is not understood, but it probably represents an additional recruitment mechanism for the migration of microglia to Aβ deposits associated with senile plaques, followed by a subsequent and persistent activation of microglial cells. Moreover, the IL-8 chemokine activity could potentiate Aβ-induced proinflammatory responses mediated by activated microglia in the AD brain. The incubation of human microglia with Aβ in the presence of IL-8 led to enhanced expression and production of IL-6, IL-1β, TNF-α, and COX-2 as compared to the levels with Aβ applied alone. Thus, these studies have provided strong support for the hypothesis that IL-8 overexpression, and a consequent neuroinflammatory response, is of pathogenetic importance in AD. ¹⁴

The effect of IL-8 on cognitive function is our concern. We found that the high level of IL-8 was negatively correlated with all cognitive assessments. The same finding was, also, reported by Zhang et al, ⁴¹ but the significant correlation was absent in IL-1β, IL-6, and IL8, although their levels were elevated. ⁴⁸

The identification of IL-8 role in various brain activities may facilitate the understanding of specific biological mechanisms involved in neuropsychiatric diseases, such as dementia and depression. Future research is required to investigate the physiological effects of other cytokines on cognitive function. ⁴⁹

Finally, no correlation was found between laboratory markers measured and comorbid diseases or medications received by our participants, as the number of participants with comorbid diseases was statistically invalid. Plus, our participants did not receive any medications for AD before starting the study. So the association between these markers and AD was independent. In the ROC analysis of our laboratory measures, we found that both HDACs and copper levels had higher sensitivity, specificity, and area under the curve. Although the IL-8 level was the lowest in the measured parameters, it was significantly higher in patients with AD. Concerning the severity of AD, we find that IL-8 was the only laboratory measure that was significantly higher in severe stage. So we suggest that the estimation of HDACs and copper level may be one of the biological markers in the detection and diagnosis of AD. While IL-8 level may be used to follow progression of the disease.

Limitation of this study included its small sample size because we used highly restricted criteria to select our participants. We need to conduct more studies by including more number of patients and other dementia types beside AD.