Diagnostic Value of Cerebrospinal Fluid Biomarkers (Phospho-Tau 181,total-Tau,Aβ 42,and Aβ 40 ) in Prodromal Stage of Alzheimer’s Disease and Dementia with Lewy Bodies

Abstract

Background:

Dementia with Lewy bodies (DLB) symptoms are close to those of Alzheimer’s disease (AD), and the differential diagnosis is difficult especially early in the disease. Unfortunately, AD biomarkers in cerebrospinal fluid (CSF), and more particularly Aβ_1 - 42, appear to be altered in dementia with Lewy bodies (DLB). However, the level of these biomarkers has never been studied in the prodromal stage of the disease.

Objective:

To compare these biomarkers between DLB and AD, with a particular focus on the prodromal stage.

Methods:

A total of 166 CSF samples were collected at the memory clinic of Strasbourg. They were obtained from prodromal DLB (pro-DLB), DLB dementia, prodromal AD (pro-AD), and AD dementia patients, and elderly controls. Phospho-Tau₁₈₁, total-Tau, Aβ₄₂, and Aβ₄₀ were measured in the CSF.

Results:

At the prodromal stage, contrary to AD patients, DLB patients’ biomarker levels in the CSF were not altered. At the demented stage of DLB, Aβ₄₂ levels were reduced as well as Aβ₄₀ levels. Thus, the Aβ₄₂/Aβ₄₀ ratio remained unchanged between the prodromal and demented stages, contrary to what was observed in AD. Tau and Phospho-Tau₁₈₁ levels were unaltered in DLB patients.

Conclusions:

We have shown that at the prodromal stage the DLB patients had no pathological profile. Consequently, CSF AD biomarkers are extremely useful for differentiating AD from DLB patients particularly at this stage when the clinical diagnosis is difficult. Thus, these results open up new perspectives on the interpretation of AD biomarkers in DLB.

Keywords

Alzheimer’s disease cerebrospinal fluid biomarkers dementia with Lewy bodies dementia phospho-Tau181 prodromal total-Tau

INTRODUCTION

Dementia with Lewy bodies (DLB) is the second most common dementia in the elderly after Alzheimer’s disease (AD) [1], and it represents 20% of demented patients [2]. The concept of mild cognitive impairment (MCI) is more recent in DLB than in AD [3]. DLB symptoms are close to those of AD, especially at an early stage in the disease, with common symptoms that affect cognition (episodic memory, working memory, executive functions). However, the differential diagnosis between AD and DLB remains difficult, particularly at an early stage in the disease [4]. DLB is also close to Parkinson’s disease (PD) due to the presence of parkinsonism (akinesia, rigidity, and resting tremor), which is often discrete especially at the beginning of the disease [5, 6]. This dementia is also close to PD because of its pathophysiology, with the presence of positive synuclein aggregates in the brain, forming Lewy bodies [7]. However, DLB patients display some specific behavioral disorders, such as illusions or visual hallucinations, fluctuating cognitive impairment and neuroleptic sensitivity.

Worthy of note, AD and DLB are frequently associated in patients: Thus, Jellinger and Attems [8] have shown that among 1,100 autopsied patients, 8.5% had a pure DLB and 8.9% an AD with Lewy pathology. Such a pathological overlap seems to be more frequent with oldest old people and, in such patients with DLB, clinical aspects are correlated to neurofibrillary pathology more than to Lewy-related pathology [9].

The clinical diagnostic criteria for DLB defined by McKeith and collaborators [10] have excellent specificity (96 to 98%) but on the other hand weak sensitivity (32%) in the case of pure DLB, and their sensitivity was even lower (12%) when DLB was associated with AD [11]. In other words, the majority of DLB patients are not diagnosed. Furthermore, various imaging systems (e.g., MRI, brain perfusion SPECT, [¹²³I]FP-CIT SPECT) do not allow an early diagnosis [3, 12]. There is currently a need for more sensitive diagnostic tools for use in patients with cognitive impairment, to avoid underestimating the diagnosis of DLB. Improving the accuracy of DLB diagnosis, particularly at the beginning of the disease, by finding new biomarkers, for example, is of great interest. Due to its aggregation in the brain of DLB patients, alpha-synuclein would appear to be a cerebrospinal fluid (CSF) biomarker of interest, and it would enable AD patients to be discriminated from DLB patients [13].

At present, the biomarkers routinely used are those searched for in the CSF for the diagnosis of AD (i.e., Aβ₄₂, t-Tau, phospho-Tau₁₈₁, and to a lesser extent Aβ₄₀). Many publications have focused on the study of these biomarkers in dementias other than AD, and in particular DLB. It appears likely that Tau phosphorylated and more particularly phospho-Tau₁₈₁ could represent a useful biomarker to discriminate AD from DLB [14 –17]. The majority of studies indicate, as in AD, a decrease of Aβ_1 - 42 in the CSF of DLB patients [15 , 18–21]. Regarding t-Tau levels in the CSF of DLB patients, the majority of these studies showed either a normal level or a less increased level than in AD, whereas some indicated that t-Tau levels overlap between AD and DLB [18 , 22]. Only a few studies have investigated Aβ₄₀ [22 –24], but overall this peptide appears to decrease compared to AD and controls patients.

It should be noted that all these studies were conducted in patients with dementia. No study focusing on CSF biomarkers for the differential diagnosis between AD and DLB in MCI patients has been published so far. Yet, early diagnosis would help in the early recognition of the disease-specific symptoms and allow faster management of patients, such as determining the appropriate therapeutic strategies (i.e., use of cholinesterase inhibitor, avoidance of neuroleptics), or to develop clinical trials with new drugs. We analyzed the conventional AD biomarkers in CSF of prodromal stage (MCI) and demented patients as well as AD and DLB patients selected on clinical criteria.

METHODS

Patients

All patients were recruited from the tertiary Memory Clinic (CMRR) of Strasbourg University Hospital by an experienced team of neurologists, geriatricians, and neuropsychologists between January 2013 and January 2015. Patients underwent detailed clinical evaluation, a large neuropsychological evaluation (including the Mini-Mental State Examination [MMSE] for general cognitive functions; the French version of the Free and Cued Selective Reminding Test [FCSRT], the Delayed Matching-to-Sample - 48 items [DMS48] test and the digit-span test for memory functions; the Dénomination Orale - 80 items [DO80] [oral naming test] for language; the Frontal Assessment Battery [FAB], the Trail Making Test [TMT] A and B, the Digit Symbol Substitution Test [DSST], and ‘formal and semantic lexical evocation’ for executive functions; the ‘praxis set of Mahieux’ and the Rey-Osterrieth complex figure test for praxis; and the number localization and cube analysis of the Visual Object and Space Perception [VOSP] battery for visuo-perceptive functions), blood examination, brain MRI (3 Tesla), and lumbar puncture for CSF biomarkers. Nineteen patients also had an amyloid PET scan. We retrospectively included 55 patients with DLB according to McKeith’s and DSM-V criteria (probable DLB; based on the existence of two core symptoms in addition to the cognitive decline) for demented DLB patients (DLB-d, n = 20) [10]. Prodromal-DLB (Pro-DLB) patients (n = 35) were defined as patients with MCI (Petersen criteria) [25] and according to the DSM-V criteria (mild neurocognitive disorder of Lewy body disease). Their CSF biomarker levels were compared to a group of AD patients and to a control group without neurodegenerative disease selected retrospectively. AD patients were selected according to Albert’s and Dubois’ criteria [26, 27] for prodromal-AD patients (pro-AD, n = 39) and McKhann’s criteria [28] and Dubois’ criteria [27] for demented AD patients (AD-d, n = 31) with exclusion of the CSF criteria. Indeed, the classification was done double-blind, the clinicians having classified patients without knowing the levels of biomarkers and the biologist having established the biomarker results without knowing the diagnosis of patients. Table 1 summarizes the main clinical information of the patients at the time of lumbar puncture. Several features of the DLB patients were taken into account: Hallucinations, fluctuations, parkinsonism, and rapid eye movement sleep behavior disorder; and for the AD patients: Hippocampal atrophy, PET amyloid (when available), as well as the memory profile using the FCSRT, with a reduced sensitivity to cueing indicating hippocampal dysfunction [27]. The association of these two pathologies being frequent, data were analyzed separately for patients presenting with a pure AD or pure DLB, and for patients with mixed pathology, meeting both criteria for DLB and AD (pro-AD/DLB [n = 5] and AD/DLB-d [n = 21], see flowchart in Fig. 1). We excluded patients who disclosed a history of major depression, severe traumatic brain injury, neurovascular disease, or other possible causes of neurocognitive disorder, such as frontotemporal lobar degeneration.

We also included 15 control subjects (CS) with cognitive complaint but who showed no clinical features of neurodegenerative disease. The group included patients suffering from psychiatric disorder (i.e., depression) (n = 6), traumatic brain injury and depression (n = 1), sleep apnea syndrome (n = 1), sleep apnea syndrome and traumatic brain injury and alcoholism (n = 1), vascular MCI and sleep apnea syndrome (n = 1), neuroborreliosis (n = 1), traumatic brain injury and cavernous angioma (n = 1), pre-fragile X syndrome (n = 1), Sneddon syndrome and cerebrovascular lesions (n = 1), encephalopathy (vitamin B12 deficiency, n = 1; autoimmune n = 1).

CSF analyses

CSF samples were obtained by lumbar puncture in the context of a diagnostic workup of presumed cognitive deterioration and underwent a standard protocol (i.e. they were collected in polypropylene tubes [Sarstedt, ref.: 62.610.201] to decrease adsorption of Aβ into the test tubes. All samples were transported to the laboratory on ice in less than 4 hours. Each sample was homogenized upon receipt at the laboratory and then centrifuged for 10 minutes (1,000 g at 4°C). All samples were free of blood contamination (the samples were checked visually; if a stain in the sample was detected, the sample was not measured). Samples were then transferred in polypropylene tubes in 0.5-mL aliquots (Dutscher ref.: 033283) and stored at –80°C until analysis. Aβ₄₂, Aβ₄₀, t-tau, and phospho-tau₁₈₁ were measured by sandwich enzyme-linked immunosorbent assay (ELISA) using commercially available kits (INNOTEST^®; Fujirebio Europe, Ghent, Belgium). All assays were performed according to the manufacturer’s instructions and the methodology did not change during the period in which the analyses were performed. All CSF measurements were performed by technicians trained in CSF analysis at the biochemistry laboratory of University Hospital of Strasbourg. Assays of t-Tau, Aβ₄₂ and phospho-Tau₁₈₁ were run as routine clinical neurochemical analyses. Furthermore, the laboratory participates in the quality control (QC) worldwide program organized by the Alzheimer’s Association QC program for CSF biomarkers. Of note, our results are acceptable in comparison with the other laboratories. Moreover, two internal QC samples per parameter were included in ELISA tests to control for interassay variation. Interassay coefficients of variations were 5.4% –6.4% for Aβ₄₂, 8.8% –7.9% for t-Tau, and 10.2% –16.4% for phospho-Tau₁₈₁, 3.7% –2.8% for Aβ₄₀. The analysis and interpretation of biomarkers are based on data provided by the manufacturer. Thus the cut-offs used are for Aβ₄₂: 500 ng/L (reduced levels are considered pathological); for t-Tau (depending on age): 300ng/L (< 50 years old), 450 ng/L (50–70 years old), 500 ng/L (> 70 years old); for phospho-Tau₁₈₁: 60 ng/L; for t-Tau and phospho-Tau₁₈₁ increased levels are considered pathological. Note that for some patients insufficient CSF was available to perform an additional Aβ₄₀ assay. For this parameter, the groups were as follows: n = 8 for CS, n = 24 for pro-DLB, n = 9 for DLB-d, n = 18 for pro-AD, n = 13 for AD-d, n = 5 for pro-AD/DLB and n = 10 for AD/DLB-d.

Statistical analysis

Statistical analyses were carried out using GraphPadPRISM, version 5 (GraphPad, Inc., San Diego, CA). Normally distributed data were analyzed using one-way analysis of variance (ANOVA) with Tukey’s post hoc analyses to determine between-group differences. In the case of non-Gaussian-distributed parameters we used the Kruskal-Wallis test. In the case of two independent variables, a chi-square test was used. Receiver-operator curve (ROC) analysis was employed to evaluate the diagnostic value of CSF parameters. ROC curve comparisons were performed using MedCalc, version 12.7.0 (MedCalcSoftware, Ostend, Belgium).

RESULTS

Individual analysis of each CSF biomarker for each diagnostic group

Once the patients had been characterized based upon clinical and imaging data, we analyzed the results for the AD biomarkers (Aβ₄₂, t-Tau, and phospho-Tau₁₈₁). For each parameter studied, the pro-AD/DLB group was excluded due to the small number of patients; however, for information purposes, the results of this group are shown in Fig. 2. As shown in Fig. 2A, mean CSF analyses of t-Tau levels for the DLB groups were not statistically different, either between groups (pro-DLB and DLB-d) or from the CS. As expected, the AD groups (pro-AD, AD-d, and AD/DLB-d groups) presented an increase of t-Tau levels compared to CS (p < 0.001 for pro-AD and AD-d and p < 0.05 for AD/DLB-d). Additionally, it was also the case when we compared the AD groups to the DLB groups (pro-DLB and DLB-d; p < 0.001 compared to pro-AD or AD-d groups). Thus, both the pro-DLB and DLB-d groups were distinguishable from any of these AD groups (pro-AD, AD-d, and AD/DLB-d groups) on the basis of the CSF t-Tau level.

The profiles of CSF phospho-Tau₁₈₁ levels (Fig. 2B) were similar to those of t-Tau. It should be noted that, on the one hand, the levels were not statistically different between the CS, pro-DLB, and DLB-d groups, and on the other hand the levels were not statistically different between the pro-AD, AD-d, and AD/DLB-d groups. Moreover, the CS, pro-DLB, and DLB-d groups were statistically different from the pro-AD, AD-d and AD/DLB-d groups (p < 0.001 for CS versus AD groups; DLB groups versus AD groups). Overall, in the pure DLB groups, t-Tau and phospho-Tau₁₈₁ levels were not affected by the pathology whatever the stage.

Concerning CSF Aβ₄₂ levels (Fig. 2C), the pro-DLB group was not statistically different from the CS group, whereas the DLB-d group showed a low mean level of CSF Aβ₄₂ compared to the CS and pro-DLB groups (p < 0.001 in each case), similarly to what was expected and observed in the AD groups (pro-AD and AD-d) as compared to the CS (p < 0.001 in each case), with no statistical difference between the DLB-d and AD groups, though, with a significant difference between the pro-DLB group and AD groups (p < 0.001 for each comparison). In other words, DLB patients were distinguishable from AD patients on the basis of CSF Aβ₄₂, only in the prodromal stage of the disease.

Finally, to further characterize the existence of amyloid deposition, we also assayed CSF Aβ₄₀ (Fig. 2D). Although little variation of Aβ₄₀ has been reported in neurodegenerative diseases, we found that the levels of Aβ₄₀ appeared to be disturbed in two groups: With a lower Aβ₄₀ production (< 7,000 ng/L) for the DLB-d group and with a higherAβ₄₀ production (> 12,000 ng/L) for the pro-AD group (Aβ₄₀ cut-offs are given according to the ELISA kit manufacturer Fujirebio). Thus, Aβ₄₀ levels appeared to be statistically higher in the pro-AD group compared to the DLB groups (p < 0.001 for DLB-d versus pro-AD; p < 0.01 for pro-DLB versus pro-AD). Overall, CSF Aβ₄₀ levels seemed to decrease with the severity of the disease both in AD and DLB, with a higher level in the prodromal compared to the demented groups, though the difference was significant in the AD patients (p < 0.05 for pro-AD versus AD-d) but not in the DLB patients.

Regarding the Aβ₄₂/Aβ₄₀ ratio (Fig. 2E), it appeared to be normal in DLB patients at both the prodromal and demented stage (no significant difference was found between the DLB-d, pro-DLB and CS groups) whereas it was decreased in the AD groups (p < 0.001 for pro-AD versus pro-DLB and DLB-d; p < 0.01 for pro-AD versus CS; p < 0.05 for AD-d versus pro-DLB and DLB-d).

Classification of patients according to the number of pathologic CSF biomarkers for each diagnostic group

We have seen in each group, each biomarker was analyzed individually. However, the diagnosis of a patient requires the interpretation of all his or her biomarkers. Thus, for each patient, it is interesting to determine the number of pathological biomarkers and take stock for each diagnostic group. Thus, we analyzed each diagnostic group according to the profile of each of its individuals using cut-offs provided by the manufacturer (described in 2.2 CSF analyses). As shown in Fig. 3, the majority of patients in the pro-DLB group had no pathological biomarker (30 of 35 patients). Very few patients had just one pathological biomarker (1 for Aβ₄₂ and 4 for phospho-Tau₁₈₁) and none had more than one pathological biomarker. In the DLB-d group a majority of patients (12 of 20 patients) also had no pathological biomarker, a quarter of patients had one pathological biomarker (3 for Aβ₄₂ and 2 for phospho-Tau₁₈₁), and a few had 2 pathological biomarkers (2 for t-Tau & phospho-Tau₁₈₁ and 1 for Aβ₄₂ & phospho-Tau₁₈₁). Of note, the proportion of patients with an Aβ₄₂ pathological biomarker in this group was only 20% (4 patients) although we observed an Aβ₄₂ decrease in this group compared to the CS and pro-DLB groups (Fig. 2C). This could be explained by the fact that the majority of patients were just above the 500 ng/L cut-off.

For the AD groups, it is interesting to note that the pie charts are radically different from DLB groups. In these groups, we found that more than 80% of patients had 2 or 3 pathological biomarkers, whereas patients with 3 pathological biomarkers represented a quarter of pro-AD patients; this type of profile was observed in almost half of the AD-d patients (Fig. 3).

For pro-AD/DLB patients, the results are not representative due to the small number of patients in this group. For the demented form we might have expected to have more or less the same profile as the AD-d group but this was not the case: We observed an intermediate profile between DLB-d and AD-d (Fig. 3).

Diagnostic accuracy of CSF parameters

The ROC curves indicate that all biomarkers, including Aβ₄₀, can discriminate pure AD and pure DLB at the prodromal stage, independently from one another, with a good specificity and sensitivity (see Table 2). Individually, t-Tau (AUC = 0.93) and phospho-Tau₁₈₁ (AUC = 0.94) were the most efficient biomarkers, as opposed to Aβ₄₂ (AUC = 0.84) and Aβ₄₀ (AUC = 0.76). However, the Aβ₄₂/Aβ₄₀ ratio also appeared to have a good diagnostic accuracy (AUC = 0.95). Conversely, the combination of t-Tau_ phospho-Tau_Aβ₄₂/Aβ₄₀ (AUC = 0.95) or t-Tau_ phospho-Tau_Aβ₄₂ (AUC = 0.95) did not improve the diagnostic value when compared to the most efficient parameters taken individually.

For the demented stages, t-Tau (AUC = 0.94) and phospho-Tau₁₈₁ (AUC = 0.92) remained more efficient than Aβ₄₂ (AUC = 0.77) and Aβ₄₀ (AUC = 0.84) to discriminate pure AD from pure DLB. The ratio Aβ₄₂/Aβ₄₀ slightly improved the diagnostic value (AUC = 0.86) compared to the two biomarkers individually. In the case of demented patients, the combination of t-Tau_phospho-Tau_Aβ₄₂ (AUC = 0.95) or t-Tau_phospho-Tau_Aβ₄₂/Aβ₄₀ (AUC = 0.92) further improved discrimination between the two diseases (Table 2).

Concerning the mixed pathology AD/DLB at the demented stage, we observed that the biomarkers were a little less discriminating as regards DLB-d than when comparing pure pathologies (Table 2: DLB-D versus DLB/AD-d). As expected, the biomarkers were not discriminating when we compared the AD-d and DLB/AD-d groups (Table 2).

Correlation between amyloid peptides and MMSE

Both in DLB and in AD patients, we observed that the CSF levels of Aβ₄₂ and Aβ₄₀ seemed to decrease with disease progression (prodromal versus demented groups in both diseases, Fig. 2). We therefore investigated whether there was a correlation between these biomarkers and the MMSE in pure pathologies. Thus, we found a positive correlation between the MMSE and Aβ₄₂ (Fig. 4A, p < 0.01 for DLB; Fig. 4D p < 0.001 for AD) and Aβ₄₀ (Fig. 4B, p < 0.01 for DLB; Fig. 4E p < 0.01 for AD), i.e., the reduction of CSF amyloid peptide levels was associated with a decreased global cognitive efficiency. As levels of amyloid peptides 1–42 and 1–40 seemed to decrease in parallel, we performed a correlation study and de facto the decrease of these two peptides appeared to be closely correlated both in AD and DLB (Fig. 4C, p < 0.001 for DLB; Fig. 4F p≈0.001 for AD).

DISCUSSION

For AD diagnosis, lumbar puncture and dosage of biomarkers (Aβ₄₂, Tau, and Phospho-Tau₁₈₁) in CSF has become widespread. Unfortunately, these biomarkers appear to be altered in some other neurodegenerative diseases, such as DLB. Classically, Aβ₄₂ is decreased in the CSF of DLB demented patients with little or no modification of other biomarkers. Until now these biomarkers had never been investigated in patients at early stages of DLB; for the first time we have shown that for these patients the AD biomarkers were not pathological. Consequently, at a time when the differential diagnosis is difficult, these biomarkers can provide interesting information to discriminate between the two pathologies.

CSF biomarker levels can help to differentiate DLB from AD

One of the most interesting results of our analysis is that the Aβ₄₂ decrease, described in the literature in DLB patients at the demented stage, was absent in the prodromal stage of our DLB patients. However, the lower level of Aβ₄₂ noted for DLB-d patients was equivalent to the level in pro-AD patients, but this reduction was not as marked as that in the AD-d group. Similar results have already been reported by Bibl and colleagues [20], who showed a decrease in Aβ₄₂ levels in the AD and DLB dementia groups compared to controls, but this decrease measured by Aβ-SDS-PAGE/immunoblot was significant for AD patients but not for DLB patients. Likewise, Kasuga and colleagues reported, though not significantly higher Aβ₄₂ level in DLB patients than in AD patients and a lower Aβ₄₂ level than patients with other forms of dementia [13]. Thus, the evolution of Aβ₄₂ in DLB patients seems to follow a similar evolution to that in AD patients but with a shift in the severity of the disease: The decrease is later in DLB patients.

Our results in AD and in DLB patients highlight a correlation between MMSE and amyloid peptides (Aβ₄₂ and Aβ₄₀). The correlation between CSF amyloid peptides and the severity of cognitive decline is a controversial point. Most of the studies, as described in the recent review by Blennow et al., report no relation between Aβ₄₂ levels and the progress of the cognitive decline [29], supporting the idea that the change from normal to pathologic levels most likely occurs during the preclinical asymptomatic phase of the disease. However, this observation is not shared by all. For example, Ganzer and colleagues found a weak correlation between CSF-Aβ_1–42 and MMSE score [30]; one explanation for this weak correlation could be the fact that the cognitive performance appeared to be associated with CSF Aβ₄₂ exclusively in AD for patients who were homozygous for the ɛ3 allele [31]. Kaerst and colleagues, using a slightly different approach, showed in three groups of patients with DLB dementia classified according to disease severity (mild, medium, and severe) that the greater the DLB progression the lower the Aβ₄₂ levels, which implies an evolution of the levels of Aβ₄₂ throughout the pathology [21]. More obviously, Mizoi and collaborators [32] found, similarly to our results, a correlation of the MMSE with Aβ₄₂ as well as with Aβ₄₀. Interestingly, in PD, another pathology involving alpha-synuclein aggregation, some studies have reported that Aβ₄₂ decreases are associated with accelerated cognitive decline [33], and a higher risk of developing PD-related dementia in more advanced PD [34, 35]. Taken together with these previous studies in PD and DLB, our results suggest that the progression to dementia is associated with the change in Aβ₄₂ level, whereas the other biomarkers (t-Tau and phospho-Tau₁₈₁) are little or not affected. Several studies have shown that Aβ exacerbates alpha-synuclein aggregation and related neurologic deficits in Lewy body diseases [36, 37], as is the case for Tau aggregation in AD [38]. These studies and those on Aβ₄₂ in CSF in these pathologies therefore seem to reinforce the link between synucleinopathy and amyloidopathy.

The CSF Aβ₄₀ level decreased (though not significantly) in DLB dementia patients in comparison to that in pro-DLB patients (and all other groups in our study). Similarly, these levels decreased between pro-AD and AD-d patients, with the difference that Aβ₄₀ level in pro-AD was very high and consequently, in AD-d patients, this level was similar to control subjects (Fig. 2D).

The level of Aβ₄₀ in AD patients at the prodromal stage is a controversial point. While most studies showed no change in Aβ₄₀ level in MCI (e.g., [39, 40]) or perhaps even a decrease of Aβ₄₀ concentrations in CSF of AD patients [41], our study highlighted an increase in Aβ₄₀ level in the CSF of the pro-AD group. However, three recent studies have highlighted a significant Aβ₄₀ increase in AD patients, either, in corroboration of our results, in AD-MCI patients [32, 42], or more globally among AD patients (MCI excluded) [43]. Moreover, it has been shown that total amyloid Aβ peptides are markedly increased in MCI patients [44], which is consistent if we take into account the fact that Aβ₄₀ reflects total amyloid peptides [23, 45].

Concerning DLB patients, this Aβ₄₀ decrease did not reach statistical significance but this was likely due to the small number of patients in the group. Indeed, some studies have shown that Aβ₄₀ levels are significantly lower in DLB demented patients compared to AD demented patients and controls [22 –24]. Therefore, we hypothesized that the CSF Aβ₄₀ level decreases with the severity of the disease as is the case for Aβ₄₂, which was confirmed by the correlation analyses performed with the MMSE scores. Given that the CSF Aβ₄₀ level represents quite well the level of amyloid burden [45], we suggest that the decrease of Aβ₄₂ in the CSF of demented patients with DLB is consecutive to the changes in global amyloid burden. Yet, amyloid deposits were shown to occur late after the alpha-synuclein aggregation in DLB patients [46, 47], which explains why the decrease of these amyloid peptides occurs only at the dementia stage.

Contrary to what emerges mainly from the literature, it seems likely that in AD and DLB, the Aβ₄₀ level (and more globally the amyloid burden) in the CSF of an individual is altered and progresses during the course of the pathology.

Our results show that the Aβ₄₂/Aβ₄₀ ratio clearly differentiated AD from DLB patients whatever the stage of the disease. In a similar way, other studies have shown that the Aβ₄₂/Aβ₄₀ ratio improved the ability to differentiate AD patients from vascular dementia, DLB, and non-AD dementia patients, in comparison to Aβ₄₂ alone [13, 23]. With regard to the prodromal stage and in corroboration of our results, the Aβ₄₂/Aβ₄₀ ratio was shown to be significantly decreased in the MCI patients who developed AD as compared to cognitively stable MCI patients and MCI patients developing other forms of dementia [39, 48].

In terms of the CSF t-Tau and phospho-Tau₁₈₁ levels in DLB patients, we found no change of these biomarkers between the prodromal and dementia stages, and also no statistical difference compared to control subjects. As described in the introduction, these findings are fully in line with previous reports in the literature.

Quality of patient selection

Our study has limitations, notably because the diagnosis of patients was based on clinical criteria, not on autopsy verification. However, the analysis of the results obtained in the CSF of our patients seemed globally to confirm the clinical diagnosis.

Concerning the AD diagnosis, the CSF analysis in AD patients at either the prodromal or demented stage is in line with the expected results and so confirms the quality of the clinical diagnosis. Indeed, the results obtained in the CSF of AD patients are consistent with the results described in the literature: i.e., t-Tau and phospho-Tau₁₈₁ increase, Aβ₄₂ decrease, and a decrease of the Aβ₄₂/Aβ₄₀ ratio, whatever the stage (prodromal or demented). These results in AD, in agreement with the expected results, suggest that the selection of AD patients was performed correctly. Interestingly, the fact that biomarker levels are abnormal from the early stage of the disease confirms that these biomarkers may become abnormal before any cognitive complaint of patients, making them very useful for early diagnosis of AD.

Concerning DLB diagnosis, given the excellent specificity (96–98%) of the McKeith criteria [10], it seems likely that the patients in our DLB group were indeed correctly diagnosed. Furthermore, biomarker differences between the AD and DLB groups, including t-Tau and phospho-Tau₁₈₁ levels and Aβ₄₂/Aβ₄₀ ratio, and the difference in profile between the two groups, as shown in Fig. 3, may suggest that these groups really did not have the same disease and so reinforce the relevance of all of our findings. However, another limitation is the sensitivity of the McKeith criteria (32%, and only 12% if DLB associated with AD). This raises the question of whether the biochemical profiles highlighted in our patients are similar to those of DLB patients not meeting the McKeith criteria. In addition, the concomitant decrease of Aβ₄₂ and Aβ₄₀ in DLB-d resulting in a normal Aβ₄₂/Aβ₄₀ ratio certainly provides good discrimination between AD and DLB; however, the decrease of CSF Aβ₄₂ levels in the wake of an overall drop of CSF Aβ peptides has been described in other dementias [49]. For example, in normal-pressure hydrocephalus, CSF Aβ₄₂ concentration is low [50] as well as in a percentage of patients with frontotemporal dementia and vascular dementia [51, 52], with multiple sclerosis [53], with Creutzfeldt-Jakob disease [54], or even in patients with CNS infections such as bacterial meningitis, HSV-1 encephalitis, HIV associated dementia and neuroborreliosis [55, 56]. Thus, the low sensitivity of clinical criteria and the reduction in amyloid peptides in the CSF occurring in other demented disorders make the diagnosis of DLB more complicated in current practice. Consequently, to improve early diagnosis of DLB, it would be interesting to find specific biomarkers of DLB capable of affording more precise differentiation between DLB and other forms of dementia. Alpha-synuclein is a protein that specifically aggregates in DLB; therefore, it is likely that the latter is a potentially interesting biomarker. However, a few studies are rather contradictory when analyzing the alpha-synuclein levels results in the CSF of DLB patients: Decreased, unchanged, and even elevated levels have all been reported [57]. Furthermore, to date no major study has evaluated correctly the interest of alpha-synuclein in addition to CSF biomarkers (other than conventional markers including Aβ₄₂, the Tau protein and its phosphorylated form) in patients with MCI or mild dementia in order to distinguish AD and DLB. This point should be further investigated.

Conclusion

Thus, globally at the prodromal stage, AD biomarker values of DLB patients are unchanged compared to controls, whatever the biomarkers. These results indicate that, at an early stage when the differential diagnosis is the most difficult, conventional AD biomarkers (t-Tau, phospho-Tau₁₈₁, Aβ₄₂, Aβ₄₂/Aβ₄₀, and even Aβ₄₀) are able to discriminate pure AD and pure DLB.

At the demented stage, the Aβ₄₂ level seems to be a less discriminating biomarker between AD and DLB; however, the levels of total-Tau and phospho-Tau₁₈₁ and the Aβ₄₂/Aβ₄₀ ratio are still excellent parameters for differentiating between the two diseases.

In AD and DLB there is a correlation between cognitive impairment (MMSE) and the levels of Aβ₄₂ and Aβ₄₀ in the CSF. So these peptides appear to be good indicators to assess the level of dementia in these two pathologies. This information can be important in cases where the cognitive assessment of a patient is difficult (non-native language speaker, delirium, psychiatric disorders, etc.).

These results open up new perspectives on the interpretation of AD biomarkers in DLB. However, to improve early diagnosis of DLB, it would be interesting to find specific biomarkers capable of affording more precise differentiation between DLB and other forms of dementia.

Footnotes

ACKNOWLEDGMENTS

The authors thank Mélanie Stackfleth and Laetitia Berly for help with organization; Timothée Albasser, Mathias Bilger, Laure Di Bitonto, Emmanuelle Ehrhard, Jennifer Kemp, Géraldine Heim, Catherine Kleitz, Nadine Longato, Laetitia Monjoin and Natacha Vogt for neuropsychological assessment; Paulo Loureiro de Sousa, Corinne Marrer, Stéphane Kremer and Daniel Gounot for MRI. We also thank Nick Barton for language editing.

This study was funded by Appel á Projet Interne (API) of the University Hospital of Strasbourg, Alsace Alzheimer 67, Fondation Université de Strasbourg and famille Jean Amrhein, and Projet Hospitalier de Recherche Clinique (PHRC) inter-régional (IDRCB 2012-A00992-41).

Authors’ disclosures available online ().

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