CHAPTER 2
Plasma β-amyloid peptides levels: A pilot study in bipolar depressed patients (Journal of
Affective Disorders 2012; 138(1-2):160-164)
Authors: Piccinni A, Origlia N, Veltri A, Vizzaccaro C, Marazziti D, Dell’Osso MC,
Conversano C, Moroni I, Domenici L, Dell’Osso L.
ABSTRACT
Background: Patients with mood disorders present a great risk for dementia and generally for
cognitive decline. Low levels of β-amyloid peptide 1–42 (Aβ42) and high Aβ40/Aβ42 ratio have been associated with this risk and have been reported also in geriatric patients suffering from depression. The aim of the present study was to compare the plasma levels of Aβ40 and Aβ42 in patients with bipolar depression and healthy subjects, and to correlate them with the characteristics of clinical course. Methods: Levels of Aβ40 and Aβ42 were measured by using specific ELISA kits in 16 patients with bipolar depression and in 16 control subjects with a negative history for somatic, psychiatric, neurological and substance abuse disorders. Results: Patients presented significantly lower plasma Aβ42 levels and higher Aβ40/Aβ42 ratio, as compared with control subjects. Moreover, a significant negative correlation was found between Aβ42 plasma levels and the duration of the illness, while a positive correlation was detected between the Aβ40/Aβ42 ratio and the number of affective episodes. Limitations: The major limitations of the study are the small sample size, the scanty characterization of the illness episodes and the fact that all the patients were under psychopharmacological treatment.
Conclusion: Although further research is necessary to establish firm conclusions, the present
data would suggest that changes in plasma levels of different Aβ peptides might represent a useful tool to identify the risk for cognitive decline in bipolar patients.
INTRODUCTION
Bipolar disorder is characterized by alternating mood phases, which often result in disturbances of cognitive functions such as attention, verbal and non-verbal learning, memory and executive functions (Bulbena and Berrios, 1993; McGrath et al., 1997; Waddington et al., 1989;Wolfe et al., 1987).More recently, such deficits have been reported also in euthymic phases (Ferrier et al., 1999; Torrent et al., 2006; Zubieta et al., 2001). The risk for dementia and cognitive deterioration is greater in patients affected by mood disorders (Geerlings et al., 2008; Gualtieri and Johnson, 2008) than in the general population, and correlates with the
number of affective episodes, manic polarity and the presence of psychotic symptoms (Kessing and Andersen, 2004; Robinson et al., 2006; Torres et al., 2007). Neuroimaging studies in bipolar patients have shown early neuroanatomical alterations, such as an increased volume of the amygdala, atrophy of the anterior cingulate cortex and white matter hyperintensity (Brambilla et al., 2005). On the other hand, other brain areas (cerebellar vermis, inferior prefrontal cortex and lateral ventricles) seem to degenerate only after recurrent affective episodes (Strakowski et al., 2005). A four year follow-up of bipolar patients showed a progressive impairment of the memory associated with atrophy of the medial temporal cortex in severely progressive forms of disease (Moorhead et al., 2007). The relationship between mood disorders and cognitive decline has been interpreted as the result of a common neuropathological mechanism, or as the expression of a greater vulnerability to the triggering of neurodegenerative phenomena (Aznar and Knudsen, 2011).
Most of the studies in Alzheimer's disease (AD) and mild cognitive impairment (MCI) reported a reduction of plasma β-amyloid 42 (Aβ42), and an increase of Aβ40 and Aβ40/Aβ42 ratio (Graff-Radford et al., 2007; Van Oijen et al., 2006; Xu et al., 2008). Moreover, follow-up studies in elderly patients detected a positive correlation between the Aβ40/Aβ42 ratio and subsequent cognitive decline (Okereke et al., 2009; Seppälä et al., 2010; Yaffe et al., 2011). Preclinical studies showed that the infusion of soluble Aβ in animal models mimics the development of a depressive phenotype at least during an acute phase, that is associated with decreased serotonin and norepinephrine content in the prefrontal cortex and brain derived neurotrophic factor (BDNF) expression (Arancio and Chao, 2007; Colaianna et al., 2010). However, only a few data are available regarding the relationship between depression and peripheral levels of Aβ peptides. Higher levels of Aβ40, lower levels of Aβ42 and a greater Aβ40/Aβ42 ratio have been reported in elderly patients with depression, compared with healthy controls (Qiu et al., 2007; Sun et al., 2007; Sun et al., 2008). However, in another study (Kita et al., 2009) depressed patients showed an enhanced Aβ40/Aβ42 ratio, but no differences in Aβ42 levels, when compared with age-matched control subjects. Given the paucity of the available information, the aim of the present study was to investigate potential changes of Aβ40 and Aβ42 plasma levels and/or of Aβ40/Aβ42 ratio in a group of bipolar patients, as well as to explore the possible relationship between the clinical course of the disorder and Aβ levels.
SUBJECTS/MATERIALS AND METHODS
Sixteen patients (8, 50%, men and 8, 50%, women) suffering from bipolar disorder of type I or II, major depressive episode with or without psychotic symptoms according to DSM-IV-TR criteria, were selected at the outpatients' ward and Day Hospital of the Department of Psychiatry, at the University of Pisa. Exclusion criteria included patients younger than 18 years, diagnoses of substance abuse in the last 6 months, pregnancy, patients unable to sign an informed consent, presence of neurological disorders or clinical disorders in acute phase or phase of instability. Diagnosis was confirmed by means of the Mini International Neuropsychiatric Interview (MINI) (Sheehan et al., 1998). Sixteen subjects (5, 31.2%, men and 11, 68.8%, women), with no personal or family history of neurological or psychiatric disorders, were selected as control group. They were free of physical illness, were neither heavy cigarette smokers, nor belonged to groups of high-risk HIV individuals, nor did take any regular medication or abuse drug. All these information were derived from the medical history collected by one of the authors (DM). A written informed consent was obtained from all subjects to participate in the study, which was approved by the Ethics Committee of the University of Pisa, after procedure and effects were fully explained.
Clinical assessment
The severity of depression was assessed by means of the 21-item Hamilton Rating Scale for Depression (HRSD-21) (Hamilton, 1960) and the Clinical Global Impressions — Severity of Illness scale (CGI-S) (Guy, 1976). The sample was screened for significant cognitive impairment by means of the Mini Mental State Examination (MMSE) (Folstein et al., 1975). Two patients had a CGI score of 5 (considerably ill), the remaining 14 had a score of 6 (severely ill). The HRSD total score (mean±SD) was 31.1±3.3: two patients suffered from a moderate depression (HRSD score between 18–24), while the remaining 14 were severely depressed (HRSD score≥25). The MMSE total score (mean±SD) was 23.6±1.50. Five patients were taking lithium plus SSRIs; two, lithium plus lamotrigine; five, lithium plus valproate or carbamazepine and tryciclic antidepressants; four, valproate plus SSRIs. In order to measure the amount of time patients spent being ill, the parameter “Duration of illness” was assessed according to the following equation (current age−onset age)×100/current age, developed by us. In the control group, axis I psychiatric diagnoses were excluded by means of MINI administration. The HRSD total score (mean±SD) was 1.2±1.2, and that of the MMSE (mean±SD) was 29±0.8.
Aβ assay
All participants in the study were subjected to a blood draw (16 ml test-tube) for the measurements of the Aβ plasma levels. The sample was centrifuged at 2500 rpm×10 min. The plasma was divided into aliquots and preserved at −80 °C. Analyses of the levels of Aβ40 and Aβ42 were carried out using specific ELISA kits (Invitrogen, Cammarillo, CA, USA). Briefly, samples were incubated with the monoclonal antibody specific for the NH2-terminus region of human Aβ that has been coated onto the microplate wells. During the first incubation, standards, controls and unknown samples were pipetted into the wells and co-incubated with a rabbit antibody specific for the COOH-terminus of the 1–42 or 1–40 Aβ sequence. Bound rabbit antibody was detected by the use of a horseradish peroxidase-labeled anti-rabbit antibody. After washing, horseradish peroxidase-labeled antirabbit antibody (enzyme) was added. After a second incubation and washing to remove all unbound enzyme, a substrate solution was added, which is acted upon by the bound enzyme to produce color. The intensity of this colored product is directly proportional to the concentration of human Aβ42 or Aβ40 present in the original specimen. Absorbance was read at 450 nm using a microplate reader (iMARK, Biorad Laboratories). Samples were quantitated by the standard curve generated from the recombinant Aβ42 or Aβ40 included with the kit.
Statistical analysis
Data were recorded in a specifically designed database and elaborated by using SPSS software (version no. 17). The comparison between quantitative variables of non-gaussian distribution was performed with non-parametric statistical tests, in particular, for the comparison of independent samples, the Mann–Whitney test was used. Correlations between the variables were explored by means of Spearman's coefficient. The chi-squared test was used to compare categorical variables (gender), while the variables with Gaussian distribution (age) were compared with Student's T test for independent samples.
RESULTS
The group of bipolar patients was not different from healthy control subjects in terms of age (mean+SD: 47±12.4 years vs 38±13.3 years, t=−1.91, p=0.07) and gender (χ2=0.518, p=0.47). Only three patients were suffering from bipolar I disorder, the remaining 13 from bipolar II disorder: The age of onset (mean±SD) was 31±13.4 years. The number (mean±SD) of previous affective episodes was 4±2, and the duration (mean±SD) of the ongoing depressive episode was 9.3±1.8 months, with a maximum of 12 and a minimum of 6 months.
The Aβ42 mean plasma levels in the patients were significantly lower, as compared with those of the control subjects (z=−3.75, p<0.001), while no significant difference was detected in Aβ40 level. As a consequence, a significant higher Aβ40/Aβ42 ratio was found in bipolar patients respect to healthy subjects (z=−2.07, p=0.039) (Table 1).
A statistically significant negative correlation was observed between the mean Aβ42 plasma levels and the duration of illness (rs=−0.61, p=0.012) (Fig. 1).
Figure 1 - Correlation between Aβ42 plasma levels and illness duration. For each patient Aβ42 (pg/ml) plasma level is plotted against illness duration expressed as the amount of time a patient spent being ill and calculated as: (current age−onset age)×100/ current age. A statistically significant negative correlation was observed between the mean Aβ42 plasma levels and the duration of illness parameter (rs=−0.61, p=0.012).
Moreover, a statistically significant and positive correlation emerged between the number of affective episodes and the Aβ40/Aβ42 ratio (rs=0.76, p=0.001) (Fig. 2). No significant correlations were found between the mean Aβ40 and Aβ42 plasma levels, the Aβ40/Aβ42 ratio and the HRSD total score or single item.
Figure 2 - Correlation between Aβ40/Aβ42 ratio and the number of affective episodes. For each patient the ratio between Aβ40 and Aβ42 plasma levels was calculated and plotted against the number of affective episodes during the course of bipolar illness. A statistically significant positive correlation emerged between the number of affective episodes and the Aβ40/Aβ42 ratio (rs=0.76, p=0.001).
DISCUSSION
The results of the present study showed that a group of bipolar patients present lower plasma levels of Aβ42 and higher Aβ40/Aβ42 ratio respect to a control group. These data are consistent with previous findings in depressed patients showing lower plasma levels of Aβ42, no changes in Aβ40 and a greater Aβ40/Aβ42 ratio respect to healthy subjects (Qiu et al., 2007; Sun et al., 2007; Sun et al., 2008). Moreover, the study by Sun et al. (Sun et al., 2008) highlighted how the so-called “amyloid-associated” depression (higher Aβ40/Aβ42 ratio) correlates with a more severe deficit in memory, visual-spatial skills and in the executive functions. However, the above mentioned studies were carried out in geriatric patients, where depressive symptoms, associated with cognitive deficits and with a dysfunction of the Aβ system, could be interpreted as a possible prodromal manifestation of AD. The effect of age on Aβ peripheral levels has been previously studied by Kita et al. (2009) who measured serum levels of Aβ peptides in samples from young and elderly depressed patients (cut-off=60 years); they found a significantly higher Aβ40/Aβ42 ratio compared to two age-matched control groups. In particular, Aβ40 levels were significantly higher in the young patients respect to control subjects while, interestingly, Aβ42 levels were substantially unchanged amongst patients and control subjects regardless of age. Therefore, our study point out for the first time that lower Aβ42 values and higher Aβ40/Aβ42 ratio may characterize depressed bipolar
AD patients suggested that the decreased Aβ42 levels in the cerebrospinal fluid (CSF) may be the result of the intracerebral accumulation of Aβ starting during a prodromal phase, i.e., before the formation of senile plaques and the appearance of marked cognitive deficits. Indeed, early synaptic dysfunction/neurotoxicity and mild cognitive deficits depend on brain accumulation/aggregation of Aβ peptides and consequent development of neurodegenerative processes in AD (Origlia et al., 2009). Peripheral plasma levels of Aβ peptides in AD have lesser degree of predictability respect to CSF. However, an alteration of the blood–brain barrier with an impaired Aβ clearance has been consistently reported in AD especially during an advanced phase (Deane et al., 2009). Particularly related to this issue is the evidence that in AD there is an increased expression of the receptor for advanced glycation end products (RAGE) in neurons, perycites and endothelial cells, which regulates trafficking of Aβ between brain and blood. In addition, the dysfunction of a protein associated with the receptor for low-density lipoproteins (LRP1) has been previously reported in AD (Zlokovic et al., 2010); remarkably, LRP1 plays a role in the release of Aβ from the brain and at the same time its hepatic re-uptake (Sagare et al., 2011). Given these findings it would be interesting to study whether and how the blood–brain barrier functionality is also affected during the illness course in bipolar patients. Beyond the complex mechanisms underlying Aβ exchange between brain and blood, increasing evidence suggests that changes of Aβ plasma levels represent a blood protein signature to predict development of neurodegeneration in different diseases (Thambisetty and Lovestone, 2010).
Our study showed a negative correlation between plasma levels of Aβ42 and the duration of illness and a positive correlation between the Aβ40/Aβ42 ratio and the number of recurrent affective episodes; this would suggest that different changes occurring in different Aβ peptides may also be used to monitor disease progression and worsening of the illness in bipolar patients.
Further, the present results are in agreement with previous reports showing that patients suffering from mood disorders with multiple affective episodes present a greater risk for developing cognitive deficit and overt demential forms (Kessing and Andersen, 2004; Robinson et al., 2006; Torres et al., 2007), with neuroanatomical alterations (Moorhead et al., 2007; Strakowski et al., 2005). Although the small sample size prevented us to perform reliable statistical analyses, we observed a trend towards higher Aβ40 levels and Aβ40/Aβ42 ratio in those patients (n=6) with a MMSE total score<24, i.e. with a cognitive decline. This is in line with Sun's hypothesis of a cognitive impaired “amyloid-associated” depression (Sun et al., 2008). The small sample size is the major bias of the study. Another important limitation is
the scanty characterization of the illness episodes: while we had no problem in collecting the personal history data, as well as the number and/or chronology of episodes, it was difficult to define with reasonable certainty the polarity of the episodes. Last, the fact that all the patients were taking psychotropic drugs may constitute a possible bias, although others found no differences in plasma levels of Aβ42 between treated and not treated depressed patients (Sun et al., 2007). Further examination of drug-free patients in the near future will help to understand whether changes in plasma Aβ peptides, possibly together with other biomarkers of depressed patients, such as the BDNF (Piccinni et al., 2008), represent a trait or a state marker of mood disorders. In conclusion, our results focus on different plasma Aβ peptides not only as possible biomarker in the pathophysiology of mood disorders, but also as a parameter whose change might identify subgroups of patients with an increased risk for cognitive decline.
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