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Keywords:

  • α-synuclein;
  • Alzheimer's disease;
  • biomarkers;
  • cerebrospinal fluid;
  • dementia with Lewy bodies;
  • VILIP-1

Abstract

  1. Top of page
  2. Abstract
  3. Subjects and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. Authorship credit
  9. Disclosure statement
  10. References
Thumbnail image of graphical abstract

The overlapping clinical features of Alzheimer's disease (AD) and Dementia with Lewy bodies (DLB) make differentiation difficult in the clinical environment. Evaluating the CSF levels of biomarkers in AD and DLB patients could facilitate clinical diagnosis. CSF Visinin-like protein-1 (VILIP-1), a calcium-mediated neuronal injury biomarker, has been described as a novel biomarker for AD. The aim of this study was to investigate the diagnostic utility of CSF VILIP-1 and VILIP-1/Aβ1–42 ratio to distinguish AD from DLB. Levels of CSF VILIP-1, t-tau, p-tau181P, Aβ1–42, and α-synuclein were measured in 61 AD patients, 32 DLB patients, and 40 normal controls using commercial ELISA kits. The results showed that the CSF VILIP-1 level had significantly increased in AD patients compared with both normal controls and DLB patients. The CSF VILIP-1 and VILIP-1/Aβ1–42 levels had enough diagnostic accuracy to allow the detection and differential diagnosis of AD. Additionally, CSF VILIP-1 levels were positively correlated with t-tau and p-tau181P within each group and with α-synuclein in the AD and control groups. We conclude that CSF VILIP-1 could be a diagnostic marker for AD, differentiating it from DLB. The analysis of biomarkers, representing different neuropathologies, is an important approach reflecting the heterogeneous features of AD and DLB.

Neuronal Ca2+-sensor protein VILIP-1 has been implicated in the calcium-mediated neuronal injury and pathological change of AD. The CSF VILIP-1 and VILIP-1/Aβ1-42 levels had enough diagnostic accuracy to allow the detection and differential diagnosis of AD. CSF VILIP-1 is a useful biomarker for AD. Evaluating the CSF levels of VILIP-1 in AD and DLB patients could facilitate clinical diagnosis.

Abbreviations used
AD

Alzheimer's disease

APOE

apolipoprotein E

AUC

area under the curve

CDR

Clinical Dementia Rating

DLB

dementia with Lewy bodies

MMSE

Mini Mental State Examination

NFTs

neurofibrillary tangles

VILIP-1

visinin-like Protein-1

Dementia with Lewy bodies (DLB) is the second most common type of degenerative dementia in elderly people after Alzheimer's disease (AD) (McKeith et al. 2005). Currently, the diagnoses of AD and DLB are based on clinical criteria. However, the overlap in clinical manifestations of AD and DLB patients means that differential diagnosis is often difficult (Nelson et al. 2010).

Abnormal aggregation of proteins is a neuropathological feature of both AD and DLB. β-Amyloid (Aβ), representative of cerebral amyloid plaques, and the phosphorylated and aggregated form of tau, for neurofibrillary tangles (NFTs), have been identified as specific pathological biomarkers of Alzheimer-type pathology (Querfurth and LaFerla 2010). The biomarker signature of higher levels of t-tau and p-tau181P, and lower levels Aβ1–42 in the CSF has good sensitivity and specificity for discriminating AD patients from normal controls. Because of this, the revised National Institute of Neurological Disorders and Stroke–Alzheimer Disease and Related Disorders (NINCDS–ADRDA) and the National Institute on Aging (NIA) Alzheimer's Association diagnostic criteria have emphasized the critical roles of CSF Alzheimer-type biomarkers as diagnostic tools, on which further studies would be necessary (McKhann et al. 1984; Dubois et al. 2007; Jack et al. 2011). Lewy bodies and Lewy neurites as Lewy-related pathological features of DLB are mainly composed of α-synuclein (Scherzer et al. 2008). Decreasing levels of α-synuclein in the CSF of DLB patients indicate the accumulation of α-synuclein in the brain, making CSF α-synuclein levels a potential biomarker for Lewy-related pathologies and discriminating marker between AD and DLB (Schulz-Schaeffer 2010).

CSF biomarkers are under intensive investigation to increase the diagnostic accuracy of differentiating AD from DLB and reflect the pathogenic processes. Perturbation of cellular calcium homeostasis is an important mechanism involved in the progression of neurodegeneration and cognitive impairment (Tymianski and Tator 1996; Mattson 2007). Visinin-like protein-1 (VILIP-1) is a member of the neuronal calcium sensor protein family, and has been implicated in calcium-mediated neuronal injury (Spilker and Braunewell 2003; Laterza et al. 2006; Stejskal et al. 2011). Furthermore, VILIP-1 plays a critical role in linking calcium-mediated neurotoxicity and Alzheimer-type pathological changes (Bernstein et al. 1999; Schnurra et al. 2001). The disruption of Ca2+ homeostasis caused by Alzheimer-type pathologies may damage the VILIP-1-containing neurons in the brain, giving rise to increased CSF levels of VILIP-1 (Blandini et al. 2004; Braunewell 2012). Previous studies showed that CSF VILIP-1 levels increased in patients with AD compared with controls, and could therefore act as a novel CSF biomarker for AD (Lee et al. 2008).

In this study, we further investigate the diagnostic utility of CSF VILIP-1 (reflective of Ca2+-mediated neurodegeneration) alone or in combination with the well-established CSF biomarkers t-tau and p-tau181P (reflective of NFTs/degeneration), Aβ1–42 (reflective of senile plaque formation), and α-synuclein (altered in DLB because of increased deposition in the brain) for discriminating patients with AD from normal controls and patients with DLB. For this purpose, the CSF levels of VILIP-1, t-tau, p-tau181P, Aβ1–42, and α-synuclein in patients with AD, DLB, and normal controls were measured by ELISA.

Subjects and methods

  1. Top of page
  2. Abstract
  3. Subjects and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. Authorship credit
  9. Disclosure statement
  10. References

Participants

A total of 133 participants, including 61 patients with AD (25 men and 36 women, aged 55 to 85 years), 32 patients with DLB (15 men and 17 women, aged 54 to 87 years) and 40 normal controls (14 men and 26 women, aged 55 to 82 years), were enrolled from the Guangzhou Brain Hospital, Guangzhou, Guangdong Province, China between 2009 to 2012. The ethics committee of the Guangzhou Brain Hospital approved the study, and informed consent was obtained from all subjects, or the guardians of patients with severe cognitive impairment.

All participants underwent clinical evaluation consisting of medical history, physical and neurological examinations, neuroimaging (CT and/or MRI), and laboratory testing. To improve diagnostic accuracy, polysomnogram and dopamine transporter single photon emission computed tomography were performed in a small group of AD and DLB patients without contraindications. Neuropsychological assessments Mini Mental State Examination (MMSE) and Clinical Dementia Rating (CDR) scale were used to estimate cognitive function and the severity of dementia. MMSE scores are known to vary with both age and education level within any given population (Crum et al. 1993; Bravo and Hebert 1997). Considering the relatively low education levels (average 8.3 years) of our participants, the MMSE cut-off scores were determined using previously described education levels (Zhang et al. 2005). All normal controls who scored below the MMSE cut-off score for their educational level were excluded from the study. The CDR score of all patients was 1 to 3, indicating a mild to severe degree of dementia.

The inclusion criteria for normal controls were that the individuals should be in good physical and mental conditions, without experiencing or complaining of any cognitive impairment. AD and DLB patients fulfilled the DSM-IV criteria for dementia. Patients with AD met the NNINCDS-ADRDA criteria for a clinical diagnosis of probable AD (Dubois et al. 2007), and patients with DLB fulfilled the McKeith criteria for probable DLB (McKeith et al. 2005). All the patients with DLB had core features including fluctuating cognition and consciousness, spontaneous features of parkinsonism, and visual hallucinations. A senior neurologist with subspecialty training in neurodegenerative disorders reviewed all diagnoses.

Sample preparation and analysis

All participants fasted overnight until blood and CSF samples were drawn in the morning. CSF was obtained by lumbar puncture at the L3/L4 or L4/L5 interspace. Samples were collected in polypropylene tubes, and then centrifuged at 3000 g at 4°C for 15 min. Aliquots of 250 μL were stored at −80°C until analysis, and were not subject to additional freeze-thaw. All procedures were performed within an hour of CSF withdrawal. The first 2 mL of CSF was collected separately to perform a cell count. Because the α-synuclein level in whole blood is approximately 5000 times higher than in CSF (Scherzer et al. 2008), samples with erythrocyte counts greater than 500 cells/μL were not included in all analyses, based on the previously described standardized operating procedure (Mollenhauer et al. 2010).

ELISA kits were used to quantify the levels of CSF VILIP-1 (BioVendor®, Brno, CZE), α-synuclein (Invitrogen®, Camarillo, CA, USA), t-tau, p-tau181P, and Aβ1–42 (all Innogenetics®, Gent, Belgium). All ELISAs were performed following the manufacturer's instructions, and all samples were analyzed in duplicate. Genomic DNA was extracted from whole blood, and the Apolipoprotein E (APOE) genotype was determined using previously described procedures (Wenham et al. 1991).

Statistical analysis

The gender and APOE ε4 allele distributions of the AD and DLB patients and normal controls were analyzed using the chi-squared test. Differences between the three groups of subjects were compared using anova, followed by Tukey's post hoc test for multiple comparisons. The results are shown as the mean ± SD. The overall diagnostic accuracy of CSF biomarkers or ratios was compared by the area under the curve (AUC) of the receiver operating characteristic curve analysis, and the optimum cut-off values at which the sum of the sensitivity and specificity was maximal were determined by the Youden index. An AUC estimate of 0.5 indicates no discrimination, while an AUC estimate of 1.0 indicates a perfect diagnostic test. The Pearson correlation was used for assessing the correlation between variables. All statistical analyses were performed using SPSS 16.0 (SPSS Inc., Chicago, IL, USA), and p < 0.05 were considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Subjects and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. Authorship credit
  9. Disclosure statement
  10. References

Demographic and clinical characteristics

The demographic and clinical information for the participants are listed in Table 1. There was no statistically significant difference in the mean age, gender, and education level between the three groups (> 0.05). Normal controls had higher mean MMSE scores than the AD (< 0.001) and DLB groups (< 0.001). The mean duration of disease and MMSE scores did not significantly differ between AD and DLB patients (> 0.05). The prevalence of the APOE ε4 allele in patients with AD was significantly higher than in patients with DLB (< 0.05) and controls (< 0.05), but no significant difference was observed between the DLB and control groups (> 0.05).

Table 1. Demographic, genotype, and clinical data for all participants
VariableAD (n = 61)DLB (n = 32)Controls (n = 40)p-value
  1. Numbers are the mean ± SD or n (%). Significance was set at a p < 0.05 for analysis.

  2. AD, Alzheimer's disease; DLB, dementia with Lewy bodies; MMSE, Mini-Mental State Examination; NA, not applicable.

Gender (M/F)25/3615/1714/260.593
Age (years)68.1 ± 7.667.8 ± 8.465.1 ± 6.00.118
Education (years)7.9 ± 3.89.4 ± 4.68.3 ± 3.30.225
Duration (months)53.4 ± 40.741.4 ± 28.9NA0.143
MMSE score12.1 ± 3.913.5 ± 5.826.3 ± 3.2< 0.0001
APOE ε4 carrier (%)27 (44.3%)4 (12.5%)7 (17.5%)0.001

Levels of CSF biomarkers

The CSF levels and ratios of the individual biomarkers in the patients are listed in Table 2. Significantly higher CSF levels of VILIP-1 (Fig. 1), t-tau, and p-tau181P were observed for patients with AD (all < 0.0001) compared with DLB and controls (all < 0.0001). No significant difference was observed between the DLB and control groups (all > 0.05). For Aβ1–42, significantly lower CSF levels were observed in the AD group compared with the control group (< 0.0001), and in the DLB group compared with normal controls (< 0.0001). No significant difference was observed between the DLB and AD groups (> 0.05). Significantly lower CSF levels of α-synuclein were observed in the DLB group compared with either AD (< 0.0001) or normal controls (< 0.0001), while no differences were seen between AD and normal controls (> 0.05).

Table 2. Levels of CSF biomarkers and ratios for all participants
VariableAD (n = 61)DLB (n = 32)Controls (n = 40)p-value
  1. Numbers are the mean ± SD. Significance was set at a < 0.05 for analysis.

  2. AD, Alzheimer's disease; DLB, dementia with Lewy bodies.

CSF VILIP-1 (pg/mL)72.1 ± 21.246.7 ± 8.843.0 ± 9.5< 0.0001
CSF t-tau (pg/mL)526.3 ± 190.0265.7 ± 81.7208.8 ± 53.0< 0.0001
CSF p-tau181P (pg/mL)74.5 ± 23.451.9 ± 13.248.1 ± 14.5< 0.0001
CSF Aβ1–42 (pg/mL)478.3 ± 179.8568.6 ± 198.0996.6 ± 193.9< 0.0001
CSF α-synuclein (pg/mL)433.0 ± 78.3323.1 ± 80.0445.6 ± 72.5< 0.0001
VILIP-1/Aβ1–420.19 ± 0.150.10 ± 0.060.04 ± 0.01< 0.0001
t-tau/Aβ1–421.36 ± 0.950.56 ± 0.350.21 ± 0.04< 0.0001
p-tau181P/Aβ1–420.20 ± 0.140.11 ± 0.060.05 ± 0.01< 0.0001
image

Figure 1. CSF levels of Visinin-like protein-1 in Alzheimer's disease, dementia with Lewy bodies and normal controls.

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Significantly higher ratios of VILIP-1/Aβ1–42, t-tau/Aβ1–42, and p-tau181P/Aβ1–42 were observed in patients with AD (all < 0.0001) compared with either DLB or control groups (all > 0.05). The ratios of VILIP-1/Aβ1–42 and t-tau/Aβ1–42 were not different between DLB and control groups (> 0.05). The ratio of p-tau181P/Aβ1–42 in the DLB group was slightly higher than in the normal control groups, but no significant difference was obtained after adjustment for multiple comparisons.

Receiver operating characteristic curve: diagnostic performances of CSF biomarkers

The AUC and the optimum cut-off values of CSF biomarkers that displayed sensitivity and specificity for discrimination among the AD, DLB, and control groups are listed in Table 3.

Table 3. Results of the ROC analyses
VariablesAUCp-value95% CICut-offSensitivity (%)Specificity (%)
Lower boundUpper bound
  1. Significant discriminations (< 0.05) indicate a significant AUC value.

  2. AD, Alzheimer's disease; AUC, area under the curve; CI, confidence interval; DLB, dementia with Lewy bodies; ROC, receiver operating characteristic curve.

AD versus DLB
VILIP-1 (pg/mL)0.864< 0.00010.7920.93559.0568.993.8
CSF-t-tau (pg/mL)0.894< 0.00010.8320.956427.168.9100.0
CSF-p-tau181P (pg/mL)0.788< 0.00010.6980.87962.9568.984.4
CSF-Aβ1–42 (pg/mL)0.630.0410.5090.751454.049.275.0
CSF-α-synuclein (pg/mL)0.845< 0.00010.7580.932366.080.378.1
VILIP-1/Aβ1–420.805< 0.00010.7080.9020.1080.375.0
t-tau/Aβ1–420.849< 0.00010.7660.9330.6285.275.0
p-tau181P/Aβ1–420.752< 0.00010.6520.8530.1452.587.5
AD versus controls
VILIP-1 (pg/mL)0.902< 0.00010.8460.95953.3578.787.5
CSF-t-tau (pg/mL)0.959< 0.00010.9230.995311.586.9100.0
CSF-p-tau181P (pg/mL)0.824< 0.00010.7460.90164.1067.285.0
CSF-Aβ1–42 (pg/mL)0.971< 0.00010.9440.997768.695.187.5
CSF-α-synuclein (pg/mL)0.5460.4350.4330.66419.049.265.0
VILIP-1/Aβ1–420.997< 0.00010.9921.0020.0698.497.5
t-tau/Aβ1–420.999< 0.00010.9961.0020.3498.4100.0
p-tau181P/Aβ1–420.987< 0.00010.9711.0040.0695.197.5
DLB versus controls
VILIP-1 (pg/mL)0.6170.090.4870.74746.8553.170.0
CSF-t-tau (pg/mL)0.7050.0030.5830.827262.050.082.5
CSF-p-tau181P (pg/mL)0.570.3130.4370.70241.2081.240.0
CSF-Aβ1–42 (pg/mL)0.933< 0.00010.8790.987859.996.980.0
CSF-α-synuclein (pg/mL)0.884< 0.00010.8030.966357.571.995.0
VILIP-1/Aβ1–420.941< 0.00010.8840.9990.0587.595.0
t-tau/Aβ1–420.947< 0.00010.8871.0070.2890.695.0
p-tau181P/Aβ1–420.921< 0.00010.8450.9970.0684.495.0

For distinguishing patients with AD from normal controls, the AUC values representing overall diagnositic accuracy of CSF VILIP-1 and the ratio of VILIP-1/Aβ1–42 were 0.902 and 0.997, respectively. At the optimum cut-off value, calculated by the Youden index, the sensitivity and specificity were 78.7% and 87.5%, respectively, for CSF VILIP-1, and 98.4% and 97.5%, respectively, for the VILIP-1/Aβ1–42 ratio. The sensitivity and specificity were 98.4% and 100.0% versus 95.1% and 97.5% for the ratios of t-tau/Aβ1–42 and p-tau181P/Aβ1–42, respectively, and both AUC values were over 0.9 (0.999 and 0.987, respectively).

As shown in Table 3 and Fig. 2, for differential diagnosis between patients with AD and DLB, the AUC values of CSF VILIP-1 and the ratio of VILIP-1/Aβ1–42 were 0.864 and 0.805, respectively. The optimum sensitivity and specificity were 68.9% and 93.8% versus 80.3% and 75% for VILIP-1 and VILIP-1/Aβ1–42, respectively. The AUC values of the CSF ratios of t-tau/Aβ1–42 and p-tau181P/Aβ1–42 were 0.849 and 0.752, respectively, while the sensitivity and specificity were 85.2% and 75.0% versus 52.5% and 87.5% for CSF t-tau/Aβ1–42 and p-tau181P/Aβ1–42 ratios, respectively.

image

Figure 2. Receiver operating characteristic curve for the differential accuracy of levels of CSF biomarkers and ratios in patients with Alzheimer's disease from dementia with Lewy bodies. (a) Visinin-like protein-1 (VILIP-1), t-tau, p-tau181P, and α-synuclein, (b) VILIP-1/Aβ1–42, t-tau/Aβ1–42, and p-tau181P/Aβ1–42.

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Correlations

The CSF levels of VILIP-1 were not associated with disease duration or MMSE within each group. Positive correlations were found between CSF VILIP-1 and t-tau or p-tau181P levels in patients with AD (= 0.856 and = 0.844, respectively), patients with DLB (= 0.849 and 0.831, respectively), and controls (= 0.911 and 0.874, respectively) (all < 0.0001) (Fig. 3a and b). CSF VILIP-1 was also positively correlated with CSF α-synuclein in patients with AD (= 0.706; < 0.0001) and controls (= 0.849; < 0.0001), but the relationship between VILIP-1 and α-synuclein was not found in patients with DLB (= 0.116; > 0.05) (Fig. 3c). CSF levels of t-tau and p-tau181P were positively correlated in patients with AD (= 0.877; < 0.0001), DLB (= 0.864; < 0.0001) and controls (= 0.927; < 0.0001) (Fig. 3d).

image

Figure 3. Correlations between CSF levels of biomarkers. (a) Visinin-like protein-1 (VILIP-1) and t-tau, (b) VILIP-1 and p-tau181P, (c) VILIP-1 and α-synuclein and (d) t-tau and p-tau181P.

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Discussion

  1. Top of page
  2. Abstract
  3. Subjects and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. Authorship credit
  9. Disclosure statement
  10. References

Several important advances have been made in this study. First, we confirmed and characterized the levels of CSF biomarkers that represent neuronal injury (VILIP-1) and specific pathologies (Aβ1–42, t-tau, p-tau181P, and α-synuclein) in patients with AD, DLB, and normal controls. Second, we investigated the diagnostic effectiveness of VILIP-1 alone, or in combination with Aβ1–42 for differentiation between AD, DLB, and normal controls. Finally, we identified and characterized correlations between VILIP-1 and well-established biomarkers in each group.

Our study confirmed a significantly elevated CSF level of VILIP-1 in patients with AD compared with normal controls. To distinguish patients with AD from normal controls, the optimum sensitivity and specificity of CSF VILIP-1 levels were 78.7% and 87.5%. This is consistent with a previous study with good diagnostic accuracy, which showed increased CSF levels of VILIP-1 in patients with AD with modest sensitivity of 67.9% and good specificity of 90.5% (Lee et al. 2008). Further, CSF VILIP-1 levels increased even in the early stages of AD, with a diagnostic accuracy of 0.75, to differentiate from normal controls; however, CSF VILIP-1 did not increase in a group of non-AD dementia patients including those with frontotemporal lobar degeneration, progressive supranuclear (PSP), and DLB (Tarawneh et al. 2012), increasing its diagnostic potential for AD. The CSF levels of VILIP-1 have not been thoroughly assessed in a single group of patients with DLB in any previous study. Our results showed that the CSF VILIP-1 levels in patients with DLB did not increase as they did in AD patients. For differential diagnosis between AD and DLB, the CSF level of VILIP-1 had modest diagnositic accuracy with an AUC over 0.7, similar to tau and p-tau181P. These findings suggest that CSF VILIP-1 is a potential biomarker for discriminating AD from normal controls and patients with subtypes of dementia.

VILIP-1 is closely associated with neurofibrillary tangles and amyloid plaques in the brain (Schnurra et al. 2001). In addition, the expression of VILIP-1 has previously been shown to enhance the hyperphosphorylation of tau proteins in cultured cells (Braunewell et al. 2001). The association of CSF levels of VILIP-1 and CSF and p-tau181P in patients with AD has been previously reported (Lee et al. 2008). In our study, similarly correlated features of CSF VILIP-1 and t-tau or p-tau181P in patients with dementia and cognitively normal individuals are a strong indication of the close relationship between VILIP-1 and tau proteins. Although the levels of CSF VILIP-1 and Aβ1–42 did not correlate in either this or previous studies (Lee et al. 2008), amyloid imaging with Pittsburgh Compound-B showed that CSF VILIP-1 levels are positively correlated with amyloid load in pre-clinical AD (Tarawneh et al. 2012). The neuronal injury biomarker VILIP-1 is therefore an important biomarker for the pathogenesis of AD. By using more than one marker of neuronal injury progress in AD, the level of CSF VILIP-1 could have important utility for identifying specific Alzheimer-type pathological changes (especially NFTs) in the brain.

In our study, the patients with AD showed the characteristic Alzheimer-type features of increased levels of t-tau and p-tau181P, and decreased Aβ1–42 in CSF, which is consistent with previous studies (Sunderland et al. 2003; Hort et al. 2010; Engelborghs and Le Bastard 2012). CSF t-tau, p-tau181P, and Aβ1–42 are commonly used as specific biomarkers to identify and characterize patients with AD. Genetic studies have shown that tau mutation alone is sufficient to cause the formation of NFTs (Bertram et al. 2010), and it has been linked directly to the neurodegeneration of a collective group of diseases termed as tauopathies (Lee et al. 2001). Increased CSF levels of t-tau and p-tau181P correlate with cognitive impairment (Wallin et al. 2006) and also show utility in predicting cognitive decline (Buerger et al. 2005; Snider et al. 2009; Stomrud et al. 2010). Therefore, CSF t-tau and p-tau181P seem to be specific markers for neurodegeneration caused by abnormal functions of t-tau and p-tau181P. The combination of CSF Aβ (amyloid plaques) and tau (NFTs/neurodegeneration) can predict the presence of Alzheimer-type pathological changes in the brain (Tapiola et al. 2009). The combination of CSF Aβ1–42 and tau proteins can improve the diagnositic utility for discriminating AD from normal controls compared with the use of biomarkers alone in clinical diagnosis studies (Shaw et al. 2009; Kasuga et al. 2010). The ratio of VILIP-1/Aβ1–42 also showed a higher diagnostic accuracy than biomarkers alone for use in differentiating AD from normal controls, in agreement with a previous study (Tarawneh et al. 2012). Importantly, combined analysis of CSF VILIP-1 and Aβ1–42 could enhance both the sensitivity and specificity to greater than 90%, which is comparable with CSF t-tau/Aβ1–42 and p-tau181P/Aβ1–42 ratios.

Combined analysis of different biomarkers could only enhance the sensitivity, with relatively low specificity, compared to using biomarkers alone to discriminate AD from DLB. Since there is a substantial pathological overlap between AD and DLB patients, the sensitivity and specificity of using CSF VILIP-1 alone or in combination with Aβ1–42 did not fulfill the proposed criteria for the validity of an AD biomarker, both of which should be over 80% (The Ronald and Nancy Reagan Research Institute of the Alzheimer's Association and the National Institute on Aging Working Group 1998). Combined analysis of t-tau and Aβ1–42, which has a modest sensitivity of 80%, but a low specificity of 53%, has been reported to distinguish AD from DLB (Bibl et al. 2010). DLB patients often have concomitant Alzheimer-type pathologies, predominantly amyloid plaques, and less NFTs in the brain (Hansen and Samuel 1997; Dickson 2002). The presence of amyloid plaque may contribute to the decreased level of CSF Aβ1–42 in DLB patients (Mollenhauer et al. 2005; Bibl et al. 2006). Although Aβ accumulation has previously been observed in patients with DLB, given the disease-specific neurochemical role of α-synuclein, these patients may have distinct metabolisms of Aβ peptide from AD (Bibl et al. 2006). When trying to differentiate patients with AD from those with DLB, overlaps between Alzheimer-type and Lewy-related pathologies in the brain limit the sensitivity and specificity of CSF VILIP-1, as well as well-established biomarkers. Since AD and DLB are heterogeneous diseases, combining VILIP-1 with other specific CSF biomarkers representing different pathologies could be a powerful tool to differentiate patients with AD from those with DLB.

Importantly, the positive correlations between CSF levels of VILIP-1 and α-synuclein were found in both patients with AD and normal controls, but not in patients with DLB. α-Synuclein is a highly expressed protein localized predominantly in pre-synaptic terminals (Maroteaux et al. 1988), and has been implicated in vesicle mobilization and fusion in vivo and in vitro (Gitler et al. 2008; Burre et al. 2010). The intraneuronal accumulation and aggregation of α-synuclein plays an important role in the pathogenesis of DLB and other neurodegenerative diseases, classified as synucleinopathy (Trojanowski and Lee 2002). Significantly, a lower level of CSF α-synuclein in patients with DLB is a unique representation of the aggregation of Lewy-related pathology in brain (Ohrfelt et al. 2009), which has been confirmed in a study of autopsy-confirmed patients with DLB (Mollenhauer et al. 2011). Although the pathophysiological mechanism of interactions between VILIP-1 and α-synuclein in the brain has not been fully described, α-synuclein is also involved in Ca2+ homeostasis (Martinez et al. 2003; Hettiarachchi et al. 2009), giving a potential link between α-synuclein and VILIP-1 in the brain (reviewed by (Nejatbakhsh and Feng 2011)). The association between CSF VILIP-1 and α-synuclein may be disrupted with the initiation of α-synuclein deposition in the DLB, which may explain why CSF levels of VILIP-1 and α-synuclein are not correlated in the CSF of DLB patients. Interactions between α-synuclein and tau proteins have been demonstrated both in vitro and in vivo (Giasson et al. 2003; Mandal et al. 2006), which may aggravate the overlapping clinical and pathological features of AD and DLB (Lee et al. 2004). Synergistic relationships between α-synuclein, tau and Aβ1–42 could enhance the accumulation and aggregation of the corresponding proteins, accelerating cognitive impairment (Clinton et al. 2010). The CSF levels of VILIP-1 correlated with CSF t-tau, p-tau181P, and α-synuclein in AD and normal controls, which could be reflective of the complex interactions and neuropathological features involved in the neurodegenerative process. The overlapping levels of CSF VILIP-1, tau, Aβ, and α-synuclein caused by interactions in the brain may contribute to the relatively lower sensitivity and specificity of biomarkers for differentiating AD from DLB.

Limitation

In a case-control study, such as ours, with a relatively small sample size of demented patients, CSF biomarkers are not sufficient for evaluating the relationship between pathological changes and disease progression. Indeed, the level of CSF VILIP-1 was previously assumed to more directly reflect the degree of neuronal injury in the brain, and was shown to increase proportionally with the severity of cognitive impairment (Lee et al. 2008). Moreover, the ratio of CSF VILIP-1/Aβ1–42 showed similar utility to the t-tau/Aβ1–42 or p-tau181P/Aβ1–42 ratios for predicting cognitive decline in early AD patients (Tarawneh et al. 2012) and normal controls (Tarawneh et al. 2011) over a 2- to 3-year follow-up period. Longitudinal follow-up studies with confirmed AD and DLB patients are needed to investigate the diagnositic utility and relationship between CSF VILIP-1 and well-established pathological biomarkers.

The diagnosis of AD and DLB patients in our study was based on clinical assessment without post-mortem confirmation. However, standardized diagnostic procedures were an important approach to the confirmation of the diagnosis in our study. Furthermore, to confirm our diagnostic accuracy, DLB patients demonstrated lower CSF levels of α-synuclein, and AD patients showed typical Alzheimer-type patterns of increased CSF t-tau, and p-tau181P, decreased CSF Aβ1–42, and a higher frequency of APOE ε4.

Conclusion

  1. Top of page
  2. Abstract
  3. Subjects and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. Authorship credit
  9. Disclosure statement
  10. References

In summary, this study has demonstrated that VILIP-1 is a potential biomarker for AD, and that it also shows modest diagnostic accuracy for the differential diagnosis of AD and DLB patients. CSF t-tau, p-tau181P, and Aβ1–42 are used as specific well-established biomarkers to identify and characterize patients with AD. The decreased levels of CSF α-synuclein in DLB patients enhanced the diagnostic accuracy for Lewy-related neuropathology. The combination of CSF levels of VILIP-1 and Aβ1–42 could enhance the sensitivity and specificity for distinguishing AD patients from normal controls. Furthermore, because of the substantial overlap in CSF levels of VILIP-1 and Aβ1–42, the sensitivity and specificity of CSF VILIP-1 and VILIP-1/Aβ1–42 are lower than the proposed criteria. In conclusion, investigating the CSF levels of VILIP-1, t-tau, p-tau, Aβ1–42, and α-synuclein alone or in combination is crucial to revealing the multifaceted neuropathological features of these diseases.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Subjects and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. Authorship credit
  9. Disclosure statement
  10. References

The study was supported by the Chinese National Science Foundation (No. 30970902), Guangdong Science and Technology Program (No. 2010B031600018) and the Guangzhou Science and Technology Program (No. 2010Y1-C631).

The authors are grateful for assistance from the Department of Neurology, the Department of Geriatrics, the Clinical Laboratory and the Department of Clinical Studies of Guangzhou Brain Hospital; helpful discussions with Dr. Hongbo He and Professor Li Guo; and skilled technical support from Minlin Zhang and Xini Huang.

Authorship credit

  1. Top of page
  2. Abstract
  3. Subjects and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. Authorship credit
  9. Disclosure statement
  10. References

XNL designed the study, analyzed the data, drafted and revised the manuscript. LH, HSS, and XMZ designed the study, analyzed the data. GYH performed the ELISA and gene analysis, and analyzed the data. YFZ, DZ, YT, NM, and JPC recruited patients and performed clinical assessments. XRC and YXF performed neuropsychological assessments and analyzed the data. FCW and HBH revised the manuscript. YPN obtained funding, designed and supervised the study, and revised the manuscript. The corresponding author (YPN) had full access to all the study data and takes responsibility for the accuracy of the data and analysis.

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  2. Abstract
  3. Subjects and methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. Authorship credit
  9. Disclosure statement
  10. References
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