The Brain Injury Biomarker VLP-1 Is Increased in the Cerebrospinal Fluid of Alzheimer Disease Patients

Clinical Chemistry 54:10 1617 1623 (2008) Proteomics and Protein Markers The Brain Injury Biomarker VLP-1 Is Increased in the Cerebrospinal Fluid of Alzheimer Disease Patients Jin-Moo Lee, 2 Kaj Blennow, 4 Niels Andreasen, 5 Omar Laterza, 1 Vijay Modur, 1 Jitka Olander, 1 Feng Gao, 3 Matt Ohlendorf, 1 and Jack H. Ladenson 1* BACKGROUND: Definitive diagnosis of Alzheimer disease (AD) can be made only by histopathological examination of brain tissue, prompting the search for premortem disease biomarkers. We sought to determine if the novel brain injury biomarker, visinin-like protein 1 (VLP-1), is altered in the CSF of AD patients compared with controls, and to compare its values to the other well-studied CSF biomarkers 42-amino acid amyloid- peptide (A 1 42 ), total Tau (ttau), and hyperphosphorylated Tau (ptau). METHODS: Using ELISA, we measured concentrations of A 1 42, ttau, ptau, and VLP-1 in CSF samples from 33 AD patients and 24 controls. We compared the diagnostic performance of these biomarkers using ROC curves. RESULTS: CSF VLP-1 concentrations were significantly higher in AD patients [median (interquartile range) 365 (166) ng/l] compared with controls [244 (142.5) ng/l]. Although the diagnostic performance of VLP-1 alone was comparable to that of A, ttau, or ptau alone, the combination of the 4 biomarkers demonstrated better performance than each individually. VLP-1 concentrations were higher in AD subjects with APOE 4/ 4 genotype [599 (240) ng/l] compared with 3/ 4 [376 (127) ng/l] and 3/ 3 [280 (115.5) ng/l] genotypes. Furthermore, VLP-1 values demonstrated a high degree of correlation with ptau (r 0.809) and ttau (r 0.635) but not A 1 42 (r 0.233). VLP-1 was the only biomarker that correlated with MMSE score (r 0.384, P 0.030). CONCLUSIONS: These results suggest that neuronal injury markers such as VLP-1 may have utility as biomarkers for AD. 2008 American Association for Clinical Chemistry The diagnosis of Alzheimer disease (AD), 6 the most common form of dementia in Western countries, is largely based on historical and clinical criteria. Although many studies report a reasonably high degree of diagnostic accuracy (80% 90%), these studies often include patients with advanced disease evaluated at specialized centers (1). At present, postmortem examination of brain tissue is the only tool for definitive diagnosis. Therefore, the development of a biomarker for AD would aid greatly in the diagnosis of this disease. In addition, such a marker could potentially be used to measure efficacy in future therapeutic trials. Most studies of AD biomarkers have focused on known pathological substrates for the disease. Amyloid plaques and neurofibrillary tangles are pathological hallmarks of AD (2) and primarily comprise abnormally aggregated endogenous proteins. Amyloid plaques (extracellular proteinaceous aggregates) are principally composed of the amyloid- peptide (A ), a 38 to 42 amino acid peptide fragment of the amyloid precursor protein (APP). The major species, the 42 amino acid peptide (A 1 42 ) (3, 4), is significantly decreased in the cerebrospinal fluid (CSF) of patients with AD (5 8). Neurofibrillary tangles are intraneuronal protein aggregates found mainly in neurites and primarily composed of hyperphosphorylated Tau (ptau), a microtubule-associated protein. Several studies have 1 Division of Laboratory and Genomic Medicine, Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO; 2 The Hope Center for Neurological Disorders and the Department of Neurology, Washington University School of Medicine, St. Louis, MO; 3 Division of Biostatistics, Washington University School of Medicine, St. Louis, MO; 4 Clinical Neurochemistry Laboratory, Department of Neuroscience and Physiology, Sahlgrenska University Hospital, Mölndal, Sweden; 5 Memory Clinic, Department of Geriatric Medicine, Karolinska University Hospital, Huddinge, Sweden. * Address correspondence to this author at: Department of Pathology and Immunology, Washington University School of Medicine, 660 S. Euclid Ave., Campus Box 8118, St. Louis, MO 63110. Fax # 314-454-5208; e-mail ladenson@wustl.edu. Received February 5, 2008; accepted July 2, 2008. Previously published online at DOI: 10.1373/clinchem.2008.104497 6 Nonstandard abbreviations: AD, Alzheimer disease; A 1 42, 42 amino acid amyloid- peptide; CSF, cerebrospinal fluid; ptau, hyperphosphorylated Tau; ttau, total Tau; VLP-1, visinin-like protein 1; MMSE, Mini-Mental Status Examination; AUC, area under the curve; NINCDS-ADRDA, National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer s Disease and Related Disorders Association. 1617

Table 1. Demographics of study subjects. a n Median age, years (SE) Female, n (%) Median duration, years (interquartile range) APOE 4 b MMSE score (SE) Mean A 1 42, ng/l (SE) Mean ttau, ng/l (SE) Mean ptau, ng/l (SE) Controls 24 68.5 (1.3) 11 (46) NA ND 29.8 (0.1) 698 (47) 395 (42) 61 (6) AD patients 33 67.0 (1.8) 18 (55) 4 (2) 25 23.0 (1.1) 471 (30) 735 (77) 100 (7) P 0.544 0.516 0.001 0.001 0.001 0.001 a NA, not applicable; ND, not determined. b Patients with at least 1 4 allele. shown that total Tau (ttau) and ptau are increased in CSF from AD patients (9 12). Still, substantial overlap in values for these biomarkers between cases and controls limits their utility as diagnostic biomarkers. Another class of biomarkers that may have utility in the diagnosis of AD are those that reflect neuronal death rather than specific markers of disease pathogenesis. Such markers may provide information about disease progression related to functional outcome and may have utility in future clinical trials testing therapeutic efficacy. Several reports have demonstrated the lack of correlation between amyloid plaque load and degree of dementia, suggesting that the former may not directly relate to the latter (13, 14). Therefore, a neuronal death biomarker might have greater correlation with dementia severity than the well-studied pathological biomarkers. We recently identified several potential biomarkers for brain injury and have characterized one of these markers in acute ischemic stroke patients (15). This biomarker, visinin-like protein 1 (VLP-1), is a calcium sensor protein expressed in high abundance in neurons of the central nervous system (16, 17). VLP-1 is increased in the CSF of rats following transient focal ischemia and is detectable in increased concentrations in the plasma of ischemic stroke patients (15). In this study, we examined the possibility that this novel biomarker of brain injury might be altered in AD. Materials and Methods STUDY SUBJECTS All patients underwent a thorough clinical evaluation, which included medical and family history, physical, neurologic, psychiatric, and Mini-Mental Status Examination (MMSE), performed by a dementia specialist (N. Andreasen). Electrocardiogram, electroencephalogram, and head computed tomography were also performed. We included in the study 33 patients with a clinical diagnosis of AD [National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer s Disease and Related Disorders Association (NINCDS-ADRDA) criteria for probable AD] (18); 24 healthy controls (free of neurological or psychiatric disorders) also participated. APOE (apolipoprotein E) genotyping was performed by minisequencing, as described in detail (19). All patients (or their nearest relatives) and controls gave informed consent to participate in the study, which was conducted in accordance with the provisions of the Helsinki Declaration. The Ethics Committees at Karolinska Institute and Göteborg University approved the study. The mean age and sex distribution of subjects in each group did not differ significantly (Table 1). The mean duration of disease in the AD group was 3.9 years. As expected, MMSE scores were significantly lower in the AD group (23.0 vs 29.8, P 0.001). CSF SAMPLES CSF samples were collected into polypropylene tubes by lumbar puncture at the L3/L4 interspace. Immediately after collection, a cell count was performed by light microscopy in Bürker chambers, and all samples had 500 erythrocytes/ L. The remaining CSF was centrifuged at 2000g for 10 min (to eliminate cells) and frozen in aliquots at 80 C. ELISAS We assayed CSF samples for ttau, ptau (at Thr-181), and A 1 42 using sandwich ELISA as described (11, 20, 21). We measured CSF VLP-1 using a sandwich ELISA (monoclonal antibody for capture and rabbit polyclonal antibody for detection) as described (15). STATISTICAL ANALYSIS Differences in patient characteristics and biomarkers between 2 groups were compared using 2 test or Student t test as appropriate. We used ANOVA with posthoc Tukey test for comparisons between multiple groups. We also assessed correlations between CSF VLP-1 and other markers with partial correlation coefficients, which measure the strength of a relationship between 2 variables, while controlling for the effect of 1618 Clinical Chemistry 54:10 (2008)

VLP-1 Is Elevated in Alzheimer Disease was observed (similar to A and Tau). To see if VLP-1 provides utility to the diagnosis of AD beyond the contribution of A, ttau, or ptau alone, we performed a ROC analysis for each individual biomarker alone compared to the combination of all biomarkers. The AUCs for VLP-1, A, ttau, ptau, and an optimum linear combination of all biomarkers are shown in Fig. 2. AUCs were similar between all biomarkers individually; however, the linear combination of all biomarkers resulted in an approximately 5% improvement (Fig. 2). Fig. 1. CSF VLP-1 values in AD patients and controls. Scatter plot of CSF VLP-1 values in control vs AD patients. The line within the box represents the median value, the box encompasses 25th to 75th percentiles, and the error bars encompass the 10th to 90th percentiles. A significant difference was found in control vs AD patients (P 0.001, Student t-test). AD status. We evaluated the diagnostic ability of these biomarkers using ROC curves, which plot true-positive rates (sensitivity) vs false-positive rates (1 minus specificity) across all possible thresholds. As a global measure for the accuracy of diagnosis, we also calculated the area under ROC curve (AUC) for each individual biomarker (22). All statistical comparisons were performed using the statistical package SAS (version 9), whereas all ROC analyses were performed with ROC- KIT, a widely used freeware available from the Kurt Rossman Laboratories at the University of Chicago. A P value 0.05 was considered significant, and all statistical tests were 2-sided. Results CONCENTRATIONS OF CSF ttau, ptau, A 1 42, AND VLP-1 CSF ttau and ptau values were significantly higher in AD patients than in controls (P 0.001 for both) (Table 1). In addition, A 1 42 values were lower in AD patients than in controls (P 0.001) (Table 1), as in numerous other studies (9 12). VLP-1 concentrations in the CSF were significantly higher in AD patients than in controls [median (interquartile range) 365 (166) vs 244 (142.5) ng/l; P 0.001] (Fig. 1). DIAGNOSTIC PERFORMANCE OF THE BIOMARKERS Despite the significant difference between VLP-1 values in the CSF of controls vs AD patients, considerable overlap CORRELATIONS BETWEEN VLP-1 VALUES AND PATIENT CHARACTERISTICS To examine possible relationships between CSF VLP-1 values and patient characteristics, we performed correlation analyses between VLP-1 and age, disease duration, MMSE, and the number of APOE 4 alleles. VLP-1 correlated with MMSE and the number of APOE 4 alleles (Fig. 3A). None of the other biomarkers correlated with MMSE in this patient population (A 1 42, r 0.350, P 0.497; ttau, r 0.295, P 0.100; ptau, r 0.202, P 0.264). To further examine the relationship between APOE genotype and CSF VLP-1 concentrations, we calculated mean CSF VLP-1 values by different genotypes. APOE 4/ 4 individuals had the highest concentrations, followed by 3/ 4 and 3/ 3 individuals (Fig. 3B). CORRELATION OF VLP-1 WITH OTHER PATHOLOGICAL BIOMARKERS To examine if VLP-1 concentrations in the CSF were related to values of the other biomarkers studied, we performed correlations between VLP-1 and ttau, ptau, or A 1 42 using data from both AD patients and controls (Fig. 4). VLP-1 and ptau showed the greatest correlation (r 0.809) (Fig. 4C), whereas A 1 42 did not correlate with VLP-1 (Fig. 4A, r 0.233). Individual correlations for AD patients analyzed separately from controls were also performed, and revealed results similar to that of the total patient population: VLP-1 vs A 1 42 was not statistically significant (r 0.29671 and 0.1698 in AD and controls, respectively), whereas VLP-1 vs ttau (r 0.6221 and 0.7247 in AD and controls) and ptau (r 0.8747 and 0.6227 in AD and controls) were significantly correlated in the AD and control populations analyzed separately. Discussion In this study, we demonstrated that CSF values of VLP-1 are significantly higher in AD patients than in control subjects. Like other well-studied biomarkers for AD (A or Tau), however, there is substantial overlap in values between cases and controls. By ROC analysis (AUC), the individual biomarkers alone (A, ttau, ptau, and VLP-1) were roughly equivalent with regard Clinical Chemistry 54:10 (2008) 1619

Fig. 2. ROC curves, AUCs, and 95% CIs for A 1 42 (A); ttau (B); ptau (C); VLP-1 (D); and combined markers (E). 1620 Clinical Chemistry 54:10 (2008)

VLP-1 Is Elevated in Alzheimer Disease Fig. 3. (A), Correlation of VLP-1 values and patient characteristics, expressed as the correlation coefficient (r) and P value (n sample size). (B), Mean CSF VLP-1 values graphed according to APOE genotypes ( 3/ 3, 3/ 4, 4/ 4). Mean VLP-1 levels in APOE 4/ 4 were more than twice that in 3/ 3 individuals [*P 0.001 (3/3 vs 4/4), **P 0.05 (3/4 vs 4/4), using ANOVA with post-hoc Tukey test]. to their diagnostic accuracy. When combined, however, their diagnostic accuracy increased, suggesting added benefit of multiple biomarkers. Clearly, VLP-1 is not a biomarker that is specific for AD. Indeed, its utility was pioneered in brain injury caused by acute ischemic stroke (15). It is likely that measures of VLP-1 reflect neuronal injury with subsequent release of this intracellular protein into the CSF. Thus, there may be utility in combining biomarkers that reflect different aspects of disease pathogenesis. Both A and Tau reflect different pathological features of AD, whereas VLP-1 may reflect the end result of the disease neuronal cell death. VLP-1 is a cytoplasmic calcium-sensor protein found almost exclusively in neurons of the central nervous system and is widely expressed throughout the brain (16, 17, 23) but undetectable in other peripheral tissues (15, 24). We have previously found that VLP-1 concentrations increased in the CSF of rats subjected to middle cerebral artery occlusion but were undetectable in sham-operated controls. Moreover, VLP-1 was detected in the serum of acute ischemic stroke patients Fig. 4. Correlation of VLP-1 and other CSF biomarkers. CSF VLP-1 values were plotted against A (A), ttau (B), or ptau (C) values for all subjects to assess the degree of correlation. Correlation was most striking between VLP-1 and ptau (r 0.809) and absent with A 1 42 (r 0.233), where r is the partial correlation coefficient after controlling for the potential effect of AD status. Open circles represent values from AD patients; closed circles are values from controls. but not in normal blood donors (15). The presumed mechanism of appearance is via leakage from injured or dying neurons into the CSF and peripheral blood. Thus, in acute brain injury, high concentrations (up to 1000 g/l) are detectable in plasma samples (15). In neurodegenerative disorders, however, VLP-1 concen- Clinical Chemistry 54:10 (2008) 1621

trations are likely to reflect the equilibrium between the protein released from dying neurons and clearance of the protein from CSF. The lower values detected in the CSF of AD patients support this contention. If VLP-1 is a biomarker of neuronal loss, one might expect that its concentration may correlate with dementia severity. Indeed, in this cohort of AD patients, VLP-1 was the only biomarker that did correlate with MMSE (albeit weakly). Others have reported weak correlations between A 1 42 or Tau and a variety of dementia severity scores (25 27). It is likely that we were unable to find similar correlations because of our relatively small sample size. Future studies in larger and more well-characterized populations using more sensitive dementia scores will be needed to better determine any relationship between neuronal injury biomarkers and dementia. A variety of other brain-injury biomarkers have been examined in the CSF of patients with dementia, including neuron-specific enolase (28, 29), S100 protein (30), and glial fibrillary acidic protein (GFAP) (31), all with variable diagnostic specificity and sensitivity. More recently, proteomic profiling has resulted in the identification of several candidate biomarkers (32), including heart-fatty acid binding protein (33, 34), Park 7, and nucleoside diphosphate kinase A (35). The effectiveness of a fluid biomarker is dependent on a multitude of factors, including organ specificity, accumulation in accessible body fluids, stability, clearance, and detectability. Direct comparisons between biomarker candidates will be important to identify such an ideal biomarker. Although we did not perform direct comparisons to other candidate biomarkers in the current study, it will be important to do so in the future. APOE genotype is the strongest known genetic risk factor for the development of late-onset AD, with the 4 allele incurring greatest risk (36 38). The molecular mechanism for this risk is not known; however, it is believed that ApoE protein may play a role in A transport/clearance (39), and that the genotype may also impart increased vulnerability to a variety of central nervous system injuries (40). Consistent with the latter contention, we have found that AD patients with APOE 4/ 4 genotypes had the highest concentrations of CSF VLP-1 compared with 3/ 4 and 3/ 3 genotypes. Indeed, 4/ 4 individuals had more than twice the concentration of CSF VLP-1 compared with 3/ 3 individuals. At face value, these results suggest that the 4 genotype increases vulnerability to neuronal death; however, it is also possible that APOE genotype influences plaque load, which may also influence neurodegeneration. Our finding that CSF VLP-1 values correlated highly with ptau but not A values in our patient population is very interesting in light of the relationship between A and Tau. Dementia severity appears to correlate with the number of neurofibrillary tangles, but not to the degree of plaque deposition (13). The close correlation between VLP-1 and ptau concentrations in the CSF of AD patients is consistent with these findings, as is the lack of correlation with A. There are several limitations to this study. First, the number of patients in both control and disease groups is limited. Further studies will be needed to confirm our findings in larger, more well-characterized populations. Second, because the diagnosis of AD was made by clinical criteria, there will undoubtedly be a small but significant group of patients that were misdiagnosed (10% 20%) (1). This may account for some of the overlap in values for CSF biomarkers. ApoE genotyping in the control group might help with this diagnostic uncertainty. A much more rigorous study would require autopsy confirmation of diagnosis. Third, our study is limited to a comparison of VLP-1 concentrations in AD patients vs controls, a situation that is unlikely to occur clinically. A more relevant comparison should be made across patients carrying the differential diagnosis of dementia. Finally, our CSF samples represent a single snapshot in AD pathogenesis; further studies will be required to understand the time course or biomarker evolution with disease pathogenesis. Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article. Authors Disclosures of Potential Conflicts of Interest: Upon submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest: Employment or Leadership: None declared. Consultant or Advisory Role: J. Ladenson, Siemens Healthcare Diagnostics. Stock Ownership: None declared. Honoraria: None declared. Research Funding: J. Ladenson, NIH; J. Lee, American Health Assistance Foundation, Sahlgrenska Hospital, and the Swedish Research Council. Expert Testimony: None declared. Other: O. Laterza, V. Modur, and J. Ladenson are named as coinventors on pending patents filed by Washington University concerning brain biomarkers. Role of Sponsor: The funding organizations played no 1622 Clinical Chemistry 54:10 (2008)

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