Neuroscience Letters 570 (2014) 81–85
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Elevated levels of cerebrospinal fluid neuron-specific enolase (NSE) in Alzheimer’s disease Frank Martin Schmidt a,∗ , Roland Mergl a , Barbara Stach b , Ina Jahn a , Hermann-Josef Gertz a , Peter Schönknecht a a b
University Hospital Leipzig, Department of Psychiatry and Psychotherapy, Semmelweisstr. 10, 04103 Leipzig, Germany Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Liebigstr. 27, D-04103 Leipzig, Germany
h i g h l i g h t s • • • • •
CSF-NSE was significantly elevated in patients with AD compared to HS. Diagnostic sensitivity and specificity of CSF-NSE for AD vs. HS were ≥91%. Accuracy rates of CSF-NSE further increased in combination with T-tau and P-tau. High correlations between CSF-NSE and T-Tau, P-Tau in AD and HS. Range of biomarkers in AD may be extended with CSF-NSE.
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Article history: Received 3 February 2014 Accepted 7 April 2014 Available online 16 April 2014 Keywords: Alzheimer’s disease Cerebrospinal fluid Major depressive disorder Neuron-specific enolase NSE Tau
a b s t r a c t Neuron-specific enolase (NSE) is a neuronal glycolytic enzyme of which cerebrospinal fluid (CSF) levels are found altered following acute or prolonged neuronal damage. Investigations concerning the role of NSE in Alzheimer’s disease (AD) are conflicting with both elevated and reduced levels. We measured CSFlevels of NSE in 32 patients with AD and 32 healthy subjects (HS) using an electrochemiluminescence immunoassay (ECLIA). Mean levels of adjusted NSE were significantly elevated in AD (18.12 ng/mL (95% CI 15.63–20.60), HS 8.46 ng/mL (95% CI 5.98–10.94), p = 0.00002) and effect size for mean group differences high (1.84). NSE alone (cut-off score 15.80 ng/mL, 94% sensitivity, 97% specificity) and in combination with T-tau and P-Tau had high diagnostic accuracy to differentiate AD from HS. NSE correlated significantly with T-tau (r ≥ 0.87, p < 0.000001) and P-tau (r ≥ 0.88, p < 0.000001) in both AD and HS. Our results indicate elevated CSF-NSE levels to reflect altered neuronal metabolism in AD that may be used in supporting AD diagnostics. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction In Alzheimer’s disease (AD), amyloid-beta 1–42 (A42 ), total protein Tau (T-tau) and hyperphosphorylated Tau (P-tau) are the clinically solely applied biomarkers [1]. As the formation of neurofibrillary tangles leading to elevations of CSF-levels of T-tau and P-tau and the formation of plaques resulting in reduced CSF-levels of A42 are regarded as ‘hallmarks of AD pathology’, CSF examinations are diagnostically relevant [2] in the discrimination between
∗ Corresponding author at: University Hospital Leipzig, Department of Psychiatry and Psychotherapy, Semmelweisstr. 10, 04103 Leipzig, Germany. Tel.: +49 341 9718963; fax: +49 341 9724448. E-mail address: [email protected] (F.M. Schmidt). http://dx.doi.org/10.1016/j.neulet.2014.04.007 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
different types of dementia [3] and to other psychiatric diseases like major depressive disorder (MDD) [4,5]. A protein derived from neurons and neuroendocrine cells and involved in neuronal energy metabolism, axoplasmatic transport, neuroplastic pathways and cell survival, and used as a marker for neuronal density is the cytoplasmatic ␥-isoenzyme of the glycolytic enzyme neuron-specific enolase (NSE) [6–9]. Physiologically not secreted into the extracellular space, increased levels in CSF are found after neuronal damage in stroke, haemorrhage and trauma, and regarded to depict leakage or metabolic upregulation that follows increased energy demand [10–12]. Reduced levels as in multiple sclerosis, on the other hand, are regarded to depict reduced neuronal metabolic activity [13]. Investigations on NSE in the CSF in AD, however, revealed inconsistent findings with elevated levels suggesting NSE as biomarker for AD [14], elevated levels in both AD and vascular dementia as
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non-disease-specific marker for neuronal degeneration in dementia [15], severity-dependent levels [16], but also unaffected [17,18] or even reduced levels [19]. Therefore, in order to investigate the potential of NSE to serve as clinical marker for neuronal turnover in AD, CSF-levels of NSE were compared between patients with AD and healthy subjects (HS) together with CSF T-tau and P-tau concentrations. We hypothesized levels of CSF-NSE to be significantly higher in AD compared to HS. Further, to the authors’ knowledge for the first time, the accuracy of CSF-NSE alone and in combination with T-tau and P-tau to distinguish AD from HS was calculated exploratively. 2. Materials and methods 2.1. Subjects Thirty-two patients with mild to severe AD and 32 HS without any psychiatric or neurological disorder were consecutively recruited to participate in the study. All patients and HS were admitted to hospital as inpatients. Patients with AD were recruited from the Department of Psychiatry, University Hospital Leipzig, where they were admitted for diagnosis and treatment. All HS and patients or, when appointed, proxy provided formal consent to the appropriation in this clinical study. Patients or HS showing any indication of limitation to provide full consent and lacking a proxy were excluded from the study. Patients were diagnosed with AD under the supervision of a senior specialist in geriatric psychiatry according to Dubois criteria [20]. HS were recruited from the Department of Anaesthesiology and Intensive Care Medicine, University Hospital Leipzig, prior to elective abdominal or urological surgery. HS showing any signs of neurological or psychiatric disorder in clinical and laboratory investigation were excluded from the study. In order to further survey exclusion criteria, the Structured Clinical Interview Axis I Disorder (SCID) [21] and the Mini Mental Status Examination (MMSE) [22] were performed when applicable. Participants showing medical conditions reported to potentially affect levels of NSE were excluded from statistical analyses. These included suspected or confirmed pulmonary microcytoma, neuroblastoma, neuroendocrine tumours, leukoencephalopathy and present or history of head trauma. This study was approved by Leipzig University and Saxony Medical Ethics Committee. 2.2. Neuropeptide assays After lumbar puncture was performed, samples were immediately aliquoted in non-absorbing polypropylen-tubes of 300 l and probes were shock-frozen in fluid N2 and stored in freezers at −80 ◦ C until further measurements or were instantly measured. Storage duration ranged from 3 to 58 months. For the measurement of NSE we used an electrochemiluminescence immunoassay (ECLIA) with a linear measuring range between 0.050 and 370 ng/mL (Elecsys, Roche Diagnostics). For the measurement of T-tau and P-tau we used a commercially available enzyme-linked immunosorbent assays (ELISA) (Innotest, Innogenetics) with a linear measuring range between 75 and 1200 pg/mL (T-tau) and 15.6 and 500 pg/mL (P-tau). 2.3. Statistical analysis A one-way analysis of covariance (ANCOVA) was performed with CSF-NSE as the dependent variable, group and sex as independent variables and age as well as duration of storage as the covariates. Preliminary analyses revealed that several requirements for the ANCOVA were fulfilled. The level of measurement of the dependent variable was metric, the sample size sufficient (cell n ≥ 11); the relationship between CSF-NSE and the covariates
was linear, at a statistically significant level (age: F(1,26) = 38.46; p = 0.000001; duration of storage: F(1,47) = 85.60; p < 0.000001). Evaluation of the assumption regarding the homogeneity of regression slopes indicated that the association between the afore-mentioned covariates and CSF-NSE concentrations did not significantly differ between patients with AD and HS (F(3,56) = 1.44; p = 0.24). Whereas NSE-CSF and age were normally distributed in both samples (Kolmogorov–Smirnov test: Z ≥ 0.39; p ≥ 0.42), this was not the case for the covariate “duration of storage” (Z ≥ 1.85; p ≤ 0.002). However, this variable was not transformed in view of the fact that the ANCOVA is quite robust regarding violations of the normal distribution assumption. To assess the accuracy rates of the marker, receiver operating characteristic (ROC) analyses resulting in area under the curve values (AUC) were performed. In this context, the diagnosis of AD was the gold standard. Larger AUCs represent higher sensitivity and specificity, with AUCs above 0.80 indicating good diagnostic accuracy [23]. Sensitivity and specificity were computed for different cut-off scores. The Youden index was used to select optimal cut-off scores [24]. Adjustments for the effects of covariables were performed by linear regression with age, sex and duration of storage (NSE) as well as age and duration of storage (T-tau and P-tau) as independent variables, respectively. Multicollinearity of the predictor variables in the linear regression model was not given since the variance inflation factors which have been computed for each of the predictors (age, sex and duration of storage) did not exceed 1.88 and thus were clearly below the critical threshold being 4. Distribution of sex between groups was compared using the Chi2 -test. Means of age, levels of T-tau and P-tau between groups were compared using independent samples t-test, duration of storage and MMSE sum scores were compared using Mann–Whitney-U-test. Correlation between CSF-NSE and age was performed using Pearson’s correlation, correlation between CSFNSE and duration of storage using Spearman rank correlation. The IBM Statistical Package for the Social Sciences (SPSS) programme version 20.0 for Windows was used for all statistical analyses. The significance level was set at p < 0.05.
3. Results Subjects’ characteristics are depicted in Table 1. The ANCOVA was found to be significant for diagnosis (F(1,58) = 21.61, p = 0.00002). Men and women did not significantly differ in NSE-CSF concentrations (F(1,58) = 0.22; p = 0.64). There was no significant interaction between the factors “group” and “sex” (F(1,58) = 0.63; p = 0.43). 27.1% of the total variance in CSF-NSE values were explainable by the predictor “group” while controlling for the effects of age, sex and duration of storage (partial 2 = 0.271). The post hoc comparisons demonstrated significantly higher CSFNSE values in patients with AD than HS (p = 0.00002). The effect size for this significant adjusted mean difference was high (1.84) (see Fig. 1 and Table 2). Levels of CSF-NSE and T-tau and P-tau correlated significantly in AD (r = 0.59, p < 0.001 for T-tau; r = 0.68, p < 0.0001 for P-tau) and HS (r = 0.60, p < 0.001 for T-tau, r = 0.59, p < 0.001 for P-tau). Adjusted levels of CSF-NSE and T-tau and P-tau correlated in the AD (r = 0.87, p < 0.000001 for T-tau; r = 0.88, p < 0.000001 for P-tau), as well as the HS (r = 0.97, p < 0.000001 for T-tau, r = 0.97, p < 0.000001 for P-tau). Correlations were also found significant for non-adjusted original values within the two groups (see figure 2). For the differentiation between AD and HC, ROC analyses for original and adjusted values resulted in AUC ≥ 0.945. Sensitivities and specificities for NSE alone and in combination with T-Tau and P-Tau both in original and adjusted levels were found high (see Table 3).
F.M. Schmidt et al. / Neuroscience Letters 570 (2014) 81–85
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Table 1 Subjects’ characteristics.
Gender [male/female] Age [years] MMSE [score] Duration of storage NSE [months] CSF-T-tau [pg/mL] CSF-P-tau [pg/mL] Psychotropic medication [no/yes] Nootropics Antidepressants Mood stabilizer Neuroleptics a b c
AD (N = 32) (mean ± SD)
HS (N = 32) (mean ± SD)
Test statistics
11/21 74.37 (±6.64) 19.22 (±6.69) 23.78 (±17.51) 470.53 (±260.44) 79.19 (±36.20) 24/8 8 – – 1
18/14 50.75 (±16.50) 29.88 (±0.35) 17.91 (±13.94) 137.10 (±53.33) 33.93 (±13.79) 32/0 – – – –
p = 0.079a p < 0.001b p < 0.001c p = 0.143c p < 0.001b p < 0.001b p = 0.005a
Chi2 -test. Independent samples t-test. Mann–Whitney-U-test.
Table 2 Pairwise comparisons and effect sizes of NSE concentrations by group. Group
Original values means in ng/mL (±SD)
Adjusted values means in ng/mL (95% CI)
Adjusted mean differences (95% CI) [effect sizes] AD
AD HS
20.45 (7.21) 6.43 (4.10)
18.12 (15.63–20.60) 8.46 (5.98–10.94)
– 9.66* (5.50–13.82) [1.84]
*
p = 0.00002.
CSF-NSE significantly correlated with both age (r = 0.428; p = 0.015) and duration of storage (rho = −0.697; p < 0.00001) in the HS but not the AD (age: r = −0.138; p = 0.450; duration of storage: rho = −0.228; p = 0.210). CSF-NSE did not correlate with MMSE sum scores in the AD (rho = −0.212; p = 0.245). 4. Discussion
Fig. 1. Levels of adjusted CSF-NSE in the two groups investigated. Notes: Levels of adjusted CSF-NSE were found elevated in AD (mean 18.12 ng/mL, 95% CI 15.63–20.60) compared to HS (8.46 ng/mL, 5.98–10.94, p = 0.00002).
In the hereby presented investigation on levels of CSF-NSE in patients with AD compared to HS, we could demonstrate NSE to be significantly elevated in AD, yielding high effect size for the group comparison. Alone and for the first time in combination with T-tau and P-tau, NSE further showed both high sensitivity and specificity to distinguish AD from HS, suggesting a clinical application of this potential biomarker. Positive correlations between NSE, T-tau and P-tau in both groups thirdly indicate a relationship of the markers both in physiological neuronal regulation as well as pathological conditions. Not actively secreted into the extracellular space but localized in neuronal cytoplasm [9], elevated CSF-levels of NSE, found for original and adjusted values in AD, may follow cortical and subcortical neuronal death during the course of AD [25]. To investigate this
Table 3 Diagnostic accuracy for NSE, T-tau and P-tau in the differentiation between groups of AD, MDD and HS. Variables
AUC (95% CI)
Cut-off
Sensitivity (%) (95% CI)
Specificity (%) (95% CI)
Youden index
(a) NSE [pg/mL] T-tau [pg/mL] P-tau [pg/mL] NSE + T-tau NSE + P-tau NSE + T-tau + P-tau
0.945 (0.884–1.000)* 0.935 (0.876–0.994)* 0.884 (0.801–0.968)* 0.940 (0.885–0.996)* 0.914 (0.842–0.986)* 0.937 (0.879–0.994)*
12.05 242.0 64.0 252.48 60.76 303.48
91 (76–97) 81 (64–91) 69 (53–83) 81 (65–91) 84 (68–93) 81 (65–91)
91 (76–97) 97 (84–99) 97 (84–99) 97 (84–99) 91 (76–97) 97 (84–99)
0.81 0.78 0.66 0.78 0.75 0.78
(b) NSE [pg/mL] T-tau [pg/mL] P-tau [pg/mL] NSE + T-tau NSE + P-tau NSE + T-tau + P-tau
0.975 (0.931–1.000)* 0.977 (0.933–1.000)* 0.977 (0.933–1.000)* 0.977 (0.933–1.000)* 0.977 (0.933–1.000)* 0.977 (0.933–1.000)*
15.80 353.49 63.77 368.05 78.42 431.83
94 (77–98) 97 (82–99) 97 (82–99) 97 (82–99) 97 (82–99) 97 (82–99)
96.9 (82–99) 96.9 (82–99) 96.9 (82–99) 96.9 (82–99) 96.9 (82–99) 96.9 (82–99)
0.91 0.94 0.94 0.94 0.94 0.94
Notes: (a) Non-adjusted NSE, T-tau and P-tau. (b) Values after controlling for the effects of age, sex and duration of storage (NSE) and age and duration of storage (T-tau and P-tau), respectively. 95% CI for sensitivities and specificities were computed by using an online calculator (http://vassarstats.net/clin1.html). * p < 0.001.
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Fig. 2. Correlation of NSE and T-tau (a) and NSE and P-tau (b) in original and adjusted values. Notes: Non-adjusted as well as adjusted levels of NSE and T-tau and NSE and P-tau correlated in AD and HS. Lines display correlations in the total study group.
assumption, combined imaging and CSF-NSE in AD, as performed for infarct volume in stroke showing high correlations [10], should be performed in the future. In post mortem brains of AD patients however, a regional disparity with selectively reduced NSE-mRNA in the frontal but not in temporal cortex and thalamus [26] and reduced protein levels in homogenates of temporal lobe cortices [27] were found. The finding of high positive correlations between NSE and Tau that corresponds to the investigation of Palumbo et al. [14] further suggests both markers not only to be liberated after traumatic axonal damage but also possibly follow an interrelated regulation. Alterations of NSE-levels in AD that are missing in primary tauopathy, as in fronto-temporal dementia [14], could suggest a differential regulation of NSE and Tau within primary and secondary tauopathies. Investigations of CSF-NSE in other primary tauopathies like progressive supranuclear palsy and corticobasal degeneration [28] and studies on the molecular mechanisms should be carried out to further elucidate this assumption. Tau-transgenic (Tg-NSE/htau23) mice expressing human tau23 under the control of the NSE promoter showing Alzheimer-associated pathological features like an increase in activated microglia and astrocytes [29], as well as presenilin 2 protein transgenic mice under NSE promotor regulation (NSE/hPS2m) showing accumulation of A42 and
increase of Tau hyperphosphorylation [30] could give first evidence for a NSE-depending Tau-regulation. This relation between the markers could further explain the high diagnostic accuracies of NSE, resembling sensitivities and specificities for T-tau and P-tau found here and elsewhere [4,5]. Accuracies for NSE were high in original values and adjusted values, suggesting a clinical use of the CSF-NSE-levels under consideration of several factors. Though age was not a significant predictor for differences in NSE levels between groups, an influence of age on the differences reported here may not fully be ruled out, making it the main limitation of our study. In other studies, findings for an influence of age, sex and storage conditions were contradictory [31–33]. Regarding the heterogeneous literature reporting on CSF-NSE in AD [14–19], several potential confounders should be noted. None of the previous studies accounted for storage conditions, although NSE concentrations decrease within 1 month at −20 ◦ C and 6 months at −80 ◦ C [34], making such information, alike for Tau [34], essential. Consequently, the investigations with a pre-analytic storage at −20 ◦ C both reported of missing group differences [17,18]. Further, screening of patients with somatic conditions possibly affecting NSE levels was reported in one of the investigations only [19] and patients with other psychiatric syndromes, like possible depressive episodes, were included in the AD group [14]. Therefore, in
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order to minimize possible influence of covariates, our analyses were performed on original and adjusted values with both resulting in elevated NSE levels in AD, significant AUC’s and correlations with Tau. 5. Conclusions Our results demonstrating an increase in CSF-NSE in AD patients compared to HS point towards NSE as a diagnostic marker in AD associated with CSF Tau protein concentration that rewards further research. References [1] C.M. Roe, A.M. Fagan, M.M. Williams, N. Ghoshal, M. Aeschleman, E.A. Grant, D.S. Marcus, M.A. Mintun, D.M. Holtzman, J.C. Morris, Improving CSF biomarker accuracy in predicting prevalent and incident Alzheimer disease, Neurology 76 (2011) 501–510. [2] J. Hertze, L. Minthon, H. Zetterberg, E. Vanmechelen, K. Blennow, O. Hansson, Evaluation of CSF biomarkers as predictors of Alzheimer’s disease: a clinical follow-up study of 4.7 years, J. Alzheimer’s Dis. 21 (2010) 1119–1128. [3] H. Hampel, K. Bürger, R. Zinkowski, S.J. Teipel, A. Goernitz, N. Andreasen, M. Sjoegren, J. DeBernardis, D. Kerkman, K. Ishiguro, H. Ohno, E. Vanmechelen, H. Vanderstichele, C. McCulloch, H.J. Möller, P. Davies, K. Blennow, Measurement of phosphorylated tau epitopes in the differential diagnosis of Alzheimer disease: a comparative cerebrospinal fluid study, Arch. Gen. Psychiatry 61 (2004) 95–102. [4] P. Schönknecht, J. Pantel, E. Kaiser, P. Thomann, J. Schröder, Increased tau protein differentiates mild cognitive impairment from geriatric depression and predicts conversion to dementia, Neurosci. Lett. 416 (2007) 39–42. [5] P. Schönknecht, J. Pantel, E. Werle, T. Hartmann, M. Essig, K. Baudendistel, K. Beyreuther, J. Schröder, Cerebrospinal fluid protein tau levels in the differential diagnosis of Alzheimer’s disease, Fortschr. Neurol. Psychiatry 68 (2000) 439–446. [6] E.H. Cooper, Neuron-specific enolase, Int. J. Biol. Markers 9 (1999) 205–210. [7] E. Kaiser, R. Kuzmits, P. Pregant, O. Burghuber, W. Worofka, Clinical biochemistry of neuron specific enolase, Clin. Chim. Acta 183 (1989) 13–31. [8] D. Pollak, N. Cairns, G. Lubec, Cytoskeleton derangement in brain of patients with Down syndrome, Alzheimer’s disease and Pick’s disease, J. Neural. Transm. (Suppl.) (2003) 149–158. [9] P. Duan, Y. Zhang, X. Han, J. Liu, W. Yan, Y. Xing, Effect of neuronal induction on NSE, Tau, and Oct4 promoter methylation in bone marrow mesenchymal stem cells, In Vitro Cell. Dev. Biol. Anim. 48 (2012) 251–258. [10] O. Ahmad, J. Wardlaw, W.N. Whiteley, Correlation of levels of neuronal and glial markers with radiological measures of infarct volume in ischaemic stroke: a systematic review, Cerebrovasc. Dis. 33 (2012) 47–54. [11] S. Moritz, J. Warnat, S. Bele, B.M. Graf, C. Woertgen, The prognostic value of NSE and S100B from serum and cerebrospinal fluid in patients with spontaneous subarachnoid hemorrhage, J. Neurosurg. Anesthesiol. 22 (2010) 21–31. [12] A.E. Böhmer, J.P. Oses, A.P. Schmidt, C.S. Perón, C.L. Krebs, P.P. Oppitz, T.T. D‘Avila, D.O. Souza, L.V. Portela, M.A. Stefani, Neuron-specific enolase, S100B, and glial fibrillary acidic protein levels as outcome predictors in patients with severe traumatic brain injury, Neurosurgery 68 (2011) 1624–1630. [13] K. Hein Née Maier, A. Köhler, R. Diem, M.B. Sättler, I. Demmer, P. Lange, M. Bähr, M. Otto, Biological markers for axonal degeneration in CSF and blood of patients with the first event indicative for multiple sclerosis, Neurosci. Lett. 436 (2008) 72–76. [14] B. Palumbo, D. Siepi, I. Sabalich, C. Tranfaglia, L. Parnetti, Cerebrospinal fluid neuron-specific enolase: a further marker of Alzheimer’s disease? Funct. Neurol. 23 (2008) 93–96.
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Elevated levels of cerebrospinal fluid neuron-specific enolase (NSE) in Alzheimer’s disease Frank Martin Schmidt a,∗ , Roland Mergl a , Barbara Stach b , Ina Jahn a , Hermann-Josef Gertz a , Peter Schönknecht a a b
University Hospital Leipzig, Department of Psychiatry and Psychotherapy, Semmelweisstr. 10, 04103 Leipzig, Germany Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Liebigstr. 27, D-04103 Leipzig, Germany
h i g h l i g h t s • • • • •
CSF-NSE was significantly elevated in patients with AD compared to HS. Diagnostic sensitivity and specificity of CSF-NSE for AD vs. HS were ≥91%. Accuracy rates of CSF-NSE further increased in combination with T-tau and P-tau. High correlations between CSF-NSE and T-Tau, P-Tau in AD and HS. Range of biomarkers in AD may be extended with CSF-NSE.
a r t i c l e
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Article history: Received 3 February 2014 Accepted 7 April 2014 Available online 16 April 2014 Keywords: Alzheimer’s disease Cerebrospinal fluid Major depressive disorder Neuron-specific enolase NSE Tau
a b s t r a c t Neuron-specific enolase (NSE) is a neuronal glycolytic enzyme of which cerebrospinal fluid (CSF) levels are found altered following acute or prolonged neuronal damage. Investigations concerning the role of NSE in Alzheimer’s disease (AD) are conflicting with both elevated and reduced levels. We measured CSFlevels of NSE in 32 patients with AD and 32 healthy subjects (HS) using an electrochemiluminescence immunoassay (ECLIA). Mean levels of adjusted NSE were significantly elevated in AD (18.12 ng/mL (95% CI 15.63–20.60), HS 8.46 ng/mL (95% CI 5.98–10.94), p = 0.00002) and effect size for mean group differences high (1.84). NSE alone (cut-off score 15.80 ng/mL, 94% sensitivity, 97% specificity) and in combination with T-tau and P-Tau had high diagnostic accuracy to differentiate AD from HS. NSE correlated significantly with T-tau (r ≥ 0.87, p < 0.000001) and P-tau (r ≥ 0.88, p < 0.000001) in both AD and HS. Our results indicate elevated CSF-NSE levels to reflect altered neuronal metabolism in AD that may be used in supporting AD diagnostics. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction In Alzheimer’s disease (AD), amyloid-beta 1–42 (A42 ), total protein Tau (T-tau) and hyperphosphorylated Tau (P-tau) are the clinically solely applied biomarkers [1]. As the formation of neurofibrillary tangles leading to elevations of CSF-levels of T-tau and P-tau and the formation of plaques resulting in reduced CSF-levels of A42 are regarded as ‘hallmarks of AD pathology’, CSF examinations are diagnostically relevant [2] in the discrimination between
∗ Corresponding author at: University Hospital Leipzig, Department of Psychiatry and Psychotherapy, Semmelweisstr. 10, 04103 Leipzig, Germany. Tel.: +49 341 9718963; fax: +49 341 9724448. E-mail address: [email protected] (F.M. Schmidt). http://dx.doi.org/10.1016/j.neulet.2014.04.007 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
different types of dementia [3] and to other psychiatric diseases like major depressive disorder (MDD) [4,5]. A protein derived from neurons and neuroendocrine cells and involved in neuronal energy metabolism, axoplasmatic transport, neuroplastic pathways and cell survival, and used as a marker for neuronal density is the cytoplasmatic ␥-isoenzyme of the glycolytic enzyme neuron-specific enolase (NSE) [6–9]. Physiologically not secreted into the extracellular space, increased levels in CSF are found after neuronal damage in stroke, haemorrhage and trauma, and regarded to depict leakage or metabolic upregulation that follows increased energy demand [10–12]. Reduced levels as in multiple sclerosis, on the other hand, are regarded to depict reduced neuronal metabolic activity [13]. Investigations on NSE in the CSF in AD, however, revealed inconsistent findings with elevated levels suggesting NSE as biomarker for AD [14], elevated levels in both AD and vascular dementia as
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non-disease-specific marker for neuronal degeneration in dementia [15], severity-dependent levels [16], but also unaffected [17,18] or even reduced levels [19]. Therefore, in order to investigate the potential of NSE to serve as clinical marker for neuronal turnover in AD, CSF-levels of NSE were compared between patients with AD and healthy subjects (HS) together with CSF T-tau and P-tau concentrations. We hypothesized levels of CSF-NSE to be significantly higher in AD compared to HS. Further, to the authors’ knowledge for the first time, the accuracy of CSF-NSE alone and in combination with T-tau and P-tau to distinguish AD from HS was calculated exploratively. 2. Materials and methods 2.1. Subjects Thirty-two patients with mild to severe AD and 32 HS without any psychiatric or neurological disorder were consecutively recruited to participate in the study. All patients and HS were admitted to hospital as inpatients. Patients with AD were recruited from the Department of Psychiatry, University Hospital Leipzig, where they were admitted for diagnosis and treatment. All HS and patients or, when appointed, proxy provided formal consent to the appropriation in this clinical study. Patients or HS showing any indication of limitation to provide full consent and lacking a proxy were excluded from the study. Patients were diagnosed with AD under the supervision of a senior specialist in geriatric psychiatry according to Dubois criteria [20]. HS were recruited from the Department of Anaesthesiology and Intensive Care Medicine, University Hospital Leipzig, prior to elective abdominal or urological surgery. HS showing any signs of neurological or psychiatric disorder in clinical and laboratory investigation were excluded from the study. In order to further survey exclusion criteria, the Structured Clinical Interview Axis I Disorder (SCID) [21] and the Mini Mental Status Examination (MMSE) [22] were performed when applicable. Participants showing medical conditions reported to potentially affect levels of NSE were excluded from statistical analyses. These included suspected or confirmed pulmonary microcytoma, neuroblastoma, neuroendocrine tumours, leukoencephalopathy and present or history of head trauma. This study was approved by Leipzig University and Saxony Medical Ethics Committee. 2.2. Neuropeptide assays After lumbar puncture was performed, samples were immediately aliquoted in non-absorbing polypropylen-tubes of 300 l and probes were shock-frozen in fluid N2 and stored in freezers at −80 ◦ C until further measurements or were instantly measured. Storage duration ranged from 3 to 58 months. For the measurement of NSE we used an electrochemiluminescence immunoassay (ECLIA) with a linear measuring range between 0.050 and 370 ng/mL (Elecsys, Roche Diagnostics). For the measurement of T-tau and P-tau we used a commercially available enzyme-linked immunosorbent assays (ELISA) (Innotest, Innogenetics) with a linear measuring range between 75 and 1200 pg/mL (T-tau) and 15.6 and 500 pg/mL (P-tau). 2.3. Statistical analysis A one-way analysis of covariance (ANCOVA) was performed with CSF-NSE as the dependent variable, group and sex as independent variables and age as well as duration of storage as the covariates. Preliminary analyses revealed that several requirements for the ANCOVA were fulfilled. The level of measurement of the dependent variable was metric, the sample size sufficient (cell n ≥ 11); the relationship between CSF-NSE and the covariates
was linear, at a statistically significant level (age: F(1,26) = 38.46; p = 0.000001; duration of storage: F(1,47) = 85.60; p < 0.000001). Evaluation of the assumption regarding the homogeneity of regression slopes indicated that the association between the afore-mentioned covariates and CSF-NSE concentrations did not significantly differ between patients with AD and HS (F(3,56) = 1.44; p = 0.24). Whereas NSE-CSF and age were normally distributed in both samples (Kolmogorov–Smirnov test: Z ≥ 0.39; p ≥ 0.42), this was not the case for the covariate “duration of storage” (Z ≥ 1.85; p ≤ 0.002). However, this variable was not transformed in view of the fact that the ANCOVA is quite robust regarding violations of the normal distribution assumption. To assess the accuracy rates of the marker, receiver operating characteristic (ROC) analyses resulting in area under the curve values (AUC) were performed. In this context, the diagnosis of AD was the gold standard. Larger AUCs represent higher sensitivity and specificity, with AUCs above 0.80 indicating good diagnostic accuracy [23]. Sensitivity and specificity were computed for different cut-off scores. The Youden index was used to select optimal cut-off scores [24]. Adjustments for the effects of covariables were performed by linear regression with age, sex and duration of storage (NSE) as well as age and duration of storage (T-tau and P-tau) as independent variables, respectively. Multicollinearity of the predictor variables in the linear regression model was not given since the variance inflation factors which have been computed for each of the predictors (age, sex and duration of storage) did not exceed 1.88 and thus were clearly below the critical threshold being 4. Distribution of sex between groups was compared using the Chi2 -test. Means of age, levels of T-tau and P-tau between groups were compared using independent samples t-test, duration of storage and MMSE sum scores were compared using Mann–Whitney-U-test. Correlation between CSF-NSE and age was performed using Pearson’s correlation, correlation between CSFNSE and duration of storage using Spearman rank correlation. The IBM Statistical Package for the Social Sciences (SPSS) programme version 20.0 for Windows was used for all statistical analyses. The significance level was set at p < 0.05.
3. Results Subjects’ characteristics are depicted in Table 1. The ANCOVA was found to be significant for diagnosis (F(1,58) = 21.61, p = 0.00002). Men and women did not significantly differ in NSE-CSF concentrations (F(1,58) = 0.22; p = 0.64). There was no significant interaction between the factors “group” and “sex” (F(1,58) = 0.63; p = 0.43). 27.1% of the total variance in CSF-NSE values were explainable by the predictor “group” while controlling for the effects of age, sex and duration of storage (partial 2 = 0.271). The post hoc comparisons demonstrated significantly higher CSFNSE values in patients with AD than HS (p = 0.00002). The effect size for this significant adjusted mean difference was high (1.84) (see Fig. 1 and Table 2). Levels of CSF-NSE and T-tau and P-tau correlated significantly in AD (r = 0.59, p < 0.001 for T-tau; r = 0.68, p < 0.0001 for P-tau) and HS (r = 0.60, p < 0.001 for T-tau, r = 0.59, p < 0.001 for P-tau). Adjusted levels of CSF-NSE and T-tau and P-tau correlated in the AD (r = 0.87, p < 0.000001 for T-tau; r = 0.88, p < 0.000001 for P-tau), as well as the HS (r = 0.97, p < 0.000001 for T-tau, r = 0.97, p < 0.000001 for P-tau). Correlations were also found significant for non-adjusted original values within the two groups (see figure 2). For the differentiation between AD and HC, ROC analyses for original and adjusted values resulted in AUC ≥ 0.945. Sensitivities and specificities for NSE alone and in combination with T-Tau and P-Tau both in original and adjusted levels were found high (see Table 3).
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Table 1 Subjects’ characteristics.
Gender [male/female] Age [years] MMSE [score] Duration of storage NSE [months] CSF-T-tau [pg/mL] CSF-P-tau [pg/mL] Psychotropic medication [no/yes] Nootropics Antidepressants Mood stabilizer Neuroleptics a b c
AD (N = 32) (mean ± SD)
HS (N = 32) (mean ± SD)
Test statistics
11/21 74.37 (±6.64) 19.22 (±6.69) 23.78 (±17.51) 470.53 (±260.44) 79.19 (±36.20) 24/8 8 – – 1
18/14 50.75 (±16.50) 29.88 (±0.35) 17.91 (±13.94) 137.10 (±53.33) 33.93 (±13.79) 32/0 – – – –
p = 0.079a p < 0.001b p < 0.001c p = 0.143c p < 0.001b p < 0.001b p = 0.005a
Chi2 -test. Independent samples t-test. Mann–Whitney-U-test.
Table 2 Pairwise comparisons and effect sizes of NSE concentrations by group. Group
Original values means in ng/mL (±SD)
Adjusted values means in ng/mL (95% CI)
Adjusted mean differences (95% CI) [effect sizes] AD
AD HS
20.45 (7.21) 6.43 (4.10)
18.12 (15.63–20.60) 8.46 (5.98–10.94)
– 9.66* (5.50–13.82) [1.84]
*
p = 0.00002.
CSF-NSE significantly correlated with both age (r = 0.428; p = 0.015) and duration of storage (rho = −0.697; p < 0.00001) in the HS but not the AD (age: r = −0.138; p = 0.450; duration of storage: rho = −0.228; p = 0.210). CSF-NSE did not correlate with MMSE sum scores in the AD (rho = −0.212; p = 0.245). 4. Discussion
Fig. 1. Levels of adjusted CSF-NSE in the two groups investigated. Notes: Levels of adjusted CSF-NSE were found elevated in AD (mean 18.12 ng/mL, 95% CI 15.63–20.60) compared to HS (8.46 ng/mL, 5.98–10.94, p = 0.00002).
In the hereby presented investigation on levels of CSF-NSE in patients with AD compared to HS, we could demonstrate NSE to be significantly elevated in AD, yielding high effect size for the group comparison. Alone and for the first time in combination with T-tau and P-tau, NSE further showed both high sensitivity and specificity to distinguish AD from HS, suggesting a clinical application of this potential biomarker. Positive correlations between NSE, T-tau and P-tau in both groups thirdly indicate a relationship of the markers both in physiological neuronal regulation as well as pathological conditions. Not actively secreted into the extracellular space but localized in neuronal cytoplasm [9], elevated CSF-levels of NSE, found for original and adjusted values in AD, may follow cortical and subcortical neuronal death during the course of AD [25]. To investigate this
Table 3 Diagnostic accuracy for NSE, T-tau and P-tau in the differentiation between groups of AD, MDD and HS. Variables
AUC (95% CI)
Cut-off
Sensitivity (%) (95% CI)
Specificity (%) (95% CI)
Youden index
(a) NSE [pg/mL] T-tau [pg/mL] P-tau [pg/mL] NSE + T-tau NSE + P-tau NSE + T-tau + P-tau
0.945 (0.884–1.000)* 0.935 (0.876–0.994)* 0.884 (0.801–0.968)* 0.940 (0.885–0.996)* 0.914 (0.842–0.986)* 0.937 (0.879–0.994)*
12.05 242.0 64.0 252.48 60.76 303.48
91 (76–97) 81 (64–91) 69 (53–83) 81 (65–91) 84 (68–93) 81 (65–91)
91 (76–97) 97 (84–99) 97 (84–99) 97 (84–99) 91 (76–97) 97 (84–99)
0.81 0.78 0.66 0.78 0.75 0.78
(b) NSE [pg/mL] T-tau [pg/mL] P-tau [pg/mL] NSE + T-tau NSE + P-tau NSE + T-tau + P-tau
0.975 (0.931–1.000)* 0.977 (0.933–1.000)* 0.977 (0.933–1.000)* 0.977 (0.933–1.000)* 0.977 (0.933–1.000)* 0.977 (0.933–1.000)*
15.80 353.49 63.77 368.05 78.42 431.83
94 (77–98) 97 (82–99) 97 (82–99) 97 (82–99) 97 (82–99) 97 (82–99)
96.9 (82–99) 96.9 (82–99) 96.9 (82–99) 96.9 (82–99) 96.9 (82–99) 96.9 (82–99)
0.91 0.94 0.94 0.94 0.94 0.94
Notes: (a) Non-adjusted NSE, T-tau and P-tau. (b) Values after controlling for the effects of age, sex and duration of storage (NSE) and age and duration of storage (T-tau and P-tau), respectively. 95% CI for sensitivities and specificities were computed by using an online calculator (http://vassarstats.net/clin1.html). * p < 0.001.
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Fig. 2. Correlation of NSE and T-tau (a) and NSE and P-tau (b) in original and adjusted values. Notes: Non-adjusted as well as adjusted levels of NSE and T-tau and NSE and P-tau correlated in AD and HS. Lines display correlations in the total study group.
assumption, combined imaging and CSF-NSE in AD, as performed for infarct volume in stroke showing high correlations [10], should be performed in the future. In post mortem brains of AD patients however, a regional disparity with selectively reduced NSE-mRNA in the frontal but not in temporal cortex and thalamus [26] and reduced protein levels in homogenates of temporal lobe cortices [27] were found. The finding of high positive correlations between NSE and Tau that corresponds to the investigation of Palumbo et al. [14] further suggests both markers not only to be liberated after traumatic axonal damage but also possibly follow an interrelated regulation. Alterations of NSE-levels in AD that are missing in primary tauopathy, as in fronto-temporal dementia [14], could suggest a differential regulation of NSE and Tau within primary and secondary tauopathies. Investigations of CSF-NSE in other primary tauopathies like progressive supranuclear palsy and corticobasal degeneration [28] and studies on the molecular mechanisms should be carried out to further elucidate this assumption. Tau-transgenic (Tg-NSE/htau23) mice expressing human tau23 under the control of the NSE promoter showing Alzheimer-associated pathological features like an increase in activated microglia and astrocytes [29], as well as presenilin 2 protein transgenic mice under NSE promotor regulation (NSE/hPS2m) showing accumulation of A42 and
increase of Tau hyperphosphorylation [30] could give first evidence for a NSE-depending Tau-regulation. This relation between the markers could further explain the high diagnostic accuracies of NSE, resembling sensitivities and specificities for T-tau and P-tau found here and elsewhere [4,5]. Accuracies for NSE were high in original values and adjusted values, suggesting a clinical use of the CSF-NSE-levels under consideration of several factors. Though age was not a significant predictor for differences in NSE levels between groups, an influence of age on the differences reported here may not fully be ruled out, making it the main limitation of our study. In other studies, findings for an influence of age, sex and storage conditions were contradictory [31–33]. Regarding the heterogeneous literature reporting on CSF-NSE in AD [14–19], several potential confounders should be noted. None of the previous studies accounted for storage conditions, although NSE concentrations decrease within 1 month at −20 ◦ C and 6 months at −80 ◦ C [34], making such information, alike for Tau [34], essential. Consequently, the investigations with a pre-analytic storage at −20 ◦ C both reported of missing group differences [17,18]. Further, screening of patients with somatic conditions possibly affecting NSE levels was reported in one of the investigations only [19] and patients with other psychiatric syndromes, like possible depressive episodes, were included in the AD group [14]. Therefore, in
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order to minimize possible influence of covariates, our analyses were performed on original and adjusted values with both resulting in elevated NSE levels in AD, significant AUC’s and correlations with Tau. 5. Conclusions Our results demonstrating an increase in CSF-NSE in AD patients compared to HS point towards NSE as a diagnostic marker in AD associated with CSF Tau protein concentration that rewards further research. References [1] C.M. Roe, A.M. Fagan, M.M. Williams, N. Ghoshal, M. Aeschleman, E.A. Grant, D.S. Marcus, M.A. Mintun, D.M. Holtzman, J.C. Morris, Improving CSF biomarker accuracy in predicting prevalent and incident Alzheimer disease, Neurology 76 (2011) 501–510. [2] J. Hertze, L. Minthon, H. Zetterberg, E. Vanmechelen, K. Blennow, O. Hansson, Evaluation of CSF biomarkers as predictors of Alzheimer’s disease: a clinical follow-up study of 4.7 years, J. Alzheimer’s Dis. 21 (2010) 1119–1128. [3] H. Hampel, K. Bürger, R. Zinkowski, S.J. Teipel, A. Goernitz, N. Andreasen, M. Sjoegren, J. DeBernardis, D. Kerkman, K. Ishiguro, H. Ohno, E. Vanmechelen, H. Vanderstichele, C. McCulloch, H.J. Möller, P. Davies, K. Blennow, Measurement of phosphorylated tau epitopes in the differential diagnosis of Alzheimer disease: a comparative cerebrospinal fluid study, Arch. Gen. Psychiatry 61 (2004) 95–102. [4] P. Schönknecht, J. Pantel, E. Kaiser, P. Thomann, J. Schröder, Increased tau protein differentiates mild cognitive impairment from geriatric depression and predicts conversion to dementia, Neurosci. Lett. 416 (2007) 39–42. [5] P. Schönknecht, J. Pantel, E. Werle, T. Hartmann, M. Essig, K. Baudendistel, K. Beyreuther, J. Schröder, Cerebrospinal fluid protein tau levels in the differential diagnosis of Alzheimer’s disease, Fortschr. Neurol. Psychiatry 68 (2000) 439–446. [6] E.H. Cooper, Neuron-specific enolase, Int. J. Biol. Markers 9 (1999) 205–210. [7] E. Kaiser, R. Kuzmits, P. Pregant, O. Burghuber, W. Worofka, Clinical biochemistry of neuron specific enolase, Clin. Chim. Acta 183 (1989) 13–31. [8] D. Pollak, N. Cairns, G. Lubec, Cytoskeleton derangement in brain of patients with Down syndrome, Alzheimer’s disease and Pick’s disease, J. Neural. Transm. (Suppl.) (2003) 149–158. [9] P. Duan, Y. Zhang, X. Han, J. Liu, W. Yan, Y. Xing, Effect of neuronal induction on NSE, Tau, and Oct4 promoter methylation in bone marrow mesenchymal stem cells, In Vitro Cell. Dev. Biol. Anim. 48 (2012) 251–258. [10] O. Ahmad, J. Wardlaw, W.N. Whiteley, Correlation of levels of neuronal and glial markers with radiological measures of infarct volume in ischaemic stroke: a systematic review, Cerebrovasc. Dis. 33 (2012) 47–54. [11] S. Moritz, J. Warnat, S. Bele, B.M. Graf, C. Woertgen, The prognostic value of NSE and S100B from serum and cerebrospinal fluid in patients with spontaneous subarachnoid hemorrhage, J. Neurosurg. Anesthesiol. 22 (2010) 21–31. [12] A.E. Böhmer, J.P. Oses, A.P. Schmidt, C.S. Perón, C.L. Krebs, P.P. Oppitz, T.T. D‘Avila, D.O. Souza, L.V. Portela, M.A. Stefani, Neuron-specific enolase, S100B, and glial fibrillary acidic protein levels as outcome predictors in patients with severe traumatic brain injury, Neurosurgery 68 (2011) 1624–1630. [13] K. Hein Née Maier, A. Köhler, R. Diem, M.B. Sättler, I. Demmer, P. Lange, M. Bähr, M. Otto, Biological markers for axonal degeneration in CSF and blood of patients with the first event indicative for multiple sclerosis, Neurosci. Lett. 436 (2008) 72–76. [14] B. Palumbo, D. Siepi, I. Sabalich, C. Tranfaglia, L. Parnetti, Cerebrospinal fluid neuron-specific enolase: a further marker of Alzheimer’s disease? Funct. Neurol. 23 (2008) 93–96.
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