Abstract
INTRODUCTION
Alzheimer’s disease (AD), which is the most common dementia and the major cause of senile dementia, is a global health challenge. The pathological changes of AD are characterized by the deposition of extracellular amyloid-β (Aβ) and the presence of neurofibrillary tangles. As one of the most convincing hypothesis of AD pathophysiological mechanism, the increase of Aβ in the brain starts the pathogenic cascade, and ultimately leads to neuronal death. Recently, increasing evidences suggest that ceramides are a key mediator of thisprocess [1].
Ceramides, which are core constituents of sphingolipids, play a crucial role in various cell physio-logical functions, including apoptosis, growth arrest, senescence, migration, and adhesion [2]. Until now, in both cell and animal AD models, studies have suggested the associations between ceramides and Aβ [3]. Exposure of hippocampal neurons to Aβ induces an increase of ceramide species, and treatment with an inhibitor to prevent this ceramide accumulation can protect the neurons against Aβ-induced death [4]. A significant increase of ceramides occurred in the early stage, prior to neuronal loss, in the cortex of APP(SL)/PS1Ki mice [5]. A remarkable increase of ceramides was also observed in the brain tissues of AD patients, occurring with very mild dementia [6].
Consistent with these laboratory studies, in longitudinal clinical studies, high plasma levels of ceramides were associated with the increased risk of AD [7], and were predictive of the cognitive decline and hippocampal volume atrophy in amnestic mild cognitive impairment (MCI) [8]. Although AD is usually characterized by cognitive impairments, neuropsychiatric symptoms, as another important clinical manifestation of AD, affect most of patients with AD. Furthermore, ceramides could be associated with multiple psychiatric disorders, including schizophrenia, bipolar disorder, depression, and anxiety [9–11]. However, studies on the association between ceramide levels and neuropsychiatric symptoms of AD remain insufficient. Only one study with a small sample size found elevated ceramides in AD patients with depression [9]. In the present study, we systematically investigated the association between plasma ceramide levels and multiple neuropsychiatric symptoms in AD. Previous studies have suggested that ceramide levels in brain vary by disease severity; ceramide levels and associated gene expressions increase in the early stage of AD, but decrease in moderate to severe stage [3, 12]. Thus, considering the influences of dementia severity on both ceramide levels and neuropsychiatric symptoms, a subgroup analysis was conducted by dementia severity.
MATERIALS AND METHODS
Subjects
All AD patients and cognitive normal controls were selected from the baseline stage of China Cognition and Aging Study (China COAST), which is a national study on MCI and dementia based on hospital population. The detailed protocol of China COAST was described in our previous studies [13, 14]. The diagnosis of dementia was according to the Diagnostic and Statistical Manual of Mental Disorders, 4th edition (DSM-IV) criteria. The diagnosis of AD was based on the criteria of the National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer’s Disease and Related Disorders Association (NINCDS-ADRDA) for probable AD [15]. All controls had normal cognitive abilities, as assessed by the Mini-Mental State Examination (MMSE) and the Clinical Dementia Rating (CDR), and none of them had a previous history of neurological disorders. Written informed consent was obtained from all participants or their relatives. This study was approved by the Institutional Review Board of Xuan Wu Hospital.
Assessments
All the participants in the present study underwent testing with the MMSE [16] and CDR scale [17] to assess global cognitive ability and dementia severity. In AD patients, the Neuropsychiatric Inventory (NPI) was used to determine neuropsychiatric symptoms [18]. The scoring of NPI was according to the information from the patients’ caregivers. The NPI evaluates various neuropsychiatric symptoms, including delusions, hallucinations, agitation/aggression, apathy, anxiety, depression, euphoria, irritability, disinhibition, aberrant motor behavior, sleep behavior disturbances, and appetite abnormalities. If a patient did not have any of these symptoms in the previous month, the NPI was scored as 0. If the answer was “yes”, then the frequency and severity were asked. The score of each symptom was calculated as the product of the frequency and severity (maximum score = 12).
HPLC-MS/MS analysis
A non-fasting blood sample of each subject was collected and maintained at –80°C until analyzed. Ceramide assays were conducted at the Institute of Materia Medica, Peking Union Medical College(Beijing, China). A three-phase solvent system was used for preparing samples. The detail procedures for standard curves and result precisions were as previously described [19]. HPLC-MS/MS was performed on an Agilent 6410B Triple Quad mass spectrometer (QQQ; Agilent Technologies, Santa Clara, CA, USA), comprising a triple quadrupole MS analyzer with an electrospray ionization (ESI) interface and an Agilent 1200 RRLC system. A Spectra C8SR Column (150×3.0 mm; 3 μm particle size; Peeke Scientific, Redwood City, CA) was used to perform chromatographic separation. The parameters for electrospray ionization tandem mass spectrometry (ESI-MS/MS) were as follows: polarity = positive, gas temperature = 350°C, gas flow = 6 L/min, nebulizer = 15 psi, capillary = 4000 V.
Statistical analysis
To explore subject characteristics, we used independent sample t-tests for continuous data and χ2 tests for dichotomous variables (Fisher’s exact tests if needed). The Kolmogorov-Smirnov test was used to determine normality. The correlations between plasma ceramide levels and neuropsychiatric symptom scores were assessed by Spearman tests. The ceramide levels were divided into quartiles. Logistic regression analyses were performed to examine the associations between ceramide species and individual neuropsychiatric symptom. The levels and the quartiles of each ceramide species were separately added to the regression models as independent variables. For all AD patients, logistic regression analyses were adjusted for age, gender, anti-dementia medications, CDR score, diabetes status, and ApoE ɛ4 status. For the subgroup analysis by dementia severity, regression models were controlled for age, gender, anti-dementia medications, diabetes status and ApoE ɛ4 status. A p value <0.05 was regarded as statistically significant.
RESULTS
Patients’ characteristics and plasma ceramide levels
A total of 98 AD patients and 92 cognitively normal control subjects participated in this study, including 56 mild AD and 42 moderate to severe AD. All the mild AD patients had a CDR score of 1; in moderate to severe AD, 32 patients had a CDR score of 2, and 10 patients had a CDR score of 3. Because the China COAST was a large screening study and a majority of our patients underwent cognitive examinations for the first time, only a small proportion of our patients (22.4%, including 19.6% in mild AD and 23.8% moderate to severe AD) had been diagnosed with AD and received the anti-dementia drugs prior to our examinations. Cholinesterase inhibitors were used in all of these patients, and 2 patients with mild AD and 4 patients with moderate to severe AD were receiving memantine simultaneously. No patient was receiving antipsychotics. In AD patients, 27 had at least one ApoE ɛ4 allele (5 were homozygotes). Subject characteristics and plasma ceramide levels are presented in the Table 1. Compared with controls, AD patients had significantly less education and lower MMSE scores. Except for C24 : 0, all ceramide species were significantly higher in AD patients. In subgroup analyses by dementia severity and ApoE ɛ4 status, no significant differences were observed in ceramide levels.
The frequencies and scores of neuropsychiatric symptoms of our patients are shown in the Table 2. In our patients, 77.6% (73.2% in mild AD and 83.3% in moderate to severe AD) reported at least one neuropsychiatric symptom. Multiple symptoms, including hallucinations, depression, disinhibition, and sleep behavior were more common and more severe in moderate to severe AD than those in mild AD (Table 2).
Correlations between ceramide levels and neuropsychiatric symptoms scores
Correlations between ceramide species and neuropsychiatric symptoms were observed for delusions and depression. The levels of C16 : 0 (r =0.252, p = 0.012), C20 : 0 (r = 0.329, p = 0.001), C22 : 0 (r = 0.212, p = 0.036), and C24 : 1 (r = 0.215, p = 0.033) were significantly correlated with delusions; C14 : 0 (r = 0.214, p = 0.0340), C22 : 0 (r =0.219, p = 0.030), and C24 : 0 (r = 0.220, p = 0.030) were correlated with depression. Interestingly, in subgroup analysis by dementia severity, correlations between ceramides and delusions only existed in mild AD (for C16 : 0: r = 0.283, p = 0.035; for C20 : 0: r = 0.367, p = 0.005), while the correlations between ceramides and depression were only noted in moderate to severe AD (for C14 : 0: r = 0.345, p = 0.025; for C22 : 0: r = 0.447, p = 0.003, and for C24 : 0: r = 0.495, p = 0.001).
Logistic regressions between ceramide species levels and individual neuropsychiatric symptom
In all AD patients, after controlling for age, gender, anti-dementia medications, CDR score, diabetes status, and ApoE ɛ4 status, we found C16 : 0 and C20 : 0 were positively associated with delusions, and the quartiles of C22 : 0 and C24 : 0 were associated with depression. Logistic regression model results for delusions and depression were shown in the Table 3. Consistent with correlation results, in subgroup analysis, the associations between ceramide species and delusions were only identified in mild AD, and the associations between ceramides and depression were only in moderate to severe AD. In mild AD, after controlling for age, gender, anti-dementia medications, diabetes status, and ApoE ɛ4 status, C16 : 0, C20 : 0, and quartiles of C24 : 1 were positively associated with delusions. In moderate to severe AD, there were significantly positive associations between depression and C22 : 0 and C24 : 0 (Table 3).
DISCUSSION
Although numerous studies have suggested the pathogenic effects of ceramides on AD and the influences of ceramides on cognitive decline, the associations between ceramides and neuropsychiatric symptoms of AD remain unclear. The present study systematically investigated the associations between ceramide species and multiple neuropsychiatric symptoms in different severities of AD. The results suggested a stage-specific effect of ceramides on neuropsychiatric symptoms in AD. Ceramides were positively associated with delusions anddepression in mild stage and moderate to severe stage of AD, respectively.
In our study, nearly all plasma ceramide species were significantly higher in AD patients compared with controls. This was consistent with a previousstudy showing higher ceramides were associatedwith an increased risk of AD [7]. To our best knowledge, this study is the first one to demonstrate the association between ceramides and delusions in AD. However, in schizophrenia, in which delusions are prominent, altered ceramide levels have been recorded [10]. While the role of ceramides in the pathogenesis of delusions in AD needs further investigation, we speculate that the abnormal accumulation of ceramides in brain white matter could be a contributing factor. White matter abnormalities were suggested as a crucial factor to the development of delusions in AD [20, 21]. In neuropsychiatric disorders (schizophrenia and bipolar disorder), ceramides were significantly increased in white matter compared with controls [10]. Similarly, ceramides were elevated more than three-fold in white matter of AD patients, and more interestingly, this change peaked in mild dementia stage [6]. This finding is consistent with our study that the association between ceramides and delusions were observed in mild AD patients.
Multiple studies have suggested the association between ceramides and depression [22, 23], and ceramide system was even considered to be a potential anti-depression target. Ceramide exposure increased striatal 5-HT synaptosomal uptake, and the increase of ceramides influences the ligand binding activity of serotonin1A receptor [9, 25]. In AD, one previous study investigated the association between plasma ceramides and depression. It showed plasma ceramides were elevated in patients with major depression [9]. However, in this previous study, all participants had a CDR score of 1; while in our study, we only found the association between ceramides and depression in moderate to severe AD, but not in mild AD. We thought there may be at least two reasons to explain these findings: 1) the previous study and our study used different evaluation methods for depression, and 2) the majority of the patients in this previous study were taking anti-depressants, which would influence ceramide levels [9, 26].
There are some limitations in our study. First, the sample size is relatively small compared with the multiple symptoms of the NPI. Second, although the NPI is a validated and widely used instrument, it relies on the information from caregivers instead of patients. Furthermore, potential confounding factors were controlled when we analyzed the data; however, the influences of unknown or unmeasured factorscannot be excluded.
In conclusions, our study is valuable for further observation and intervention studies on ceramide species and neuropsychiatric symptoms in AD. The stage-specific associations between ceramides and neuropsychiatric symptoms of AD need confirmation in larger samples, and the potential mechanisms deserve further investigations.
Footnotes
ACKNOWLEDGMENTS
This article was supported by the National 973 Project Grant of China (2011CB504104), Key Medical Professional Development Plan of Beijing Municipal Administration of Hospitals (ZYLX201301), Beijing Municipal Science & Technology Commission (Z151100004015078), BeiJing Talents Fund (2014000021223ZK31) and the Specialized Research Fund for the Doctoral Program of Higher Education (20131107120002).
