Transthyretin as a potential CSF biomarker for AlzheimerÕs disease and dementia with Lewy bodies: effects of treatment with cholinesterase inhibitors

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1 European Journal of Neurology 2009 doi: /j x Transthyretin as a potential CSF biomarker for AlzheimerÕs disease and dementia with Lewy bodies: effects of treatment with cholinesterase inhibitors K. Schultz a, K. Nilsson b, J. E. Nielsen c,d, S. G. Lindquist d, L. E. Hjermind c,d, B. B. Andersen d, A. Wallin e, C. Nilsson b and Å. Petersén a a Department of Experimental Medical Science, Translational Neuroendocrine Research Unit, Lund University, Lund; b Division of Geriatric Psychiatry, Department of Clinical Sciences, Lund University, Lund, Sweden; c Department of Medical Genetics, University of Copenhagen, Copenhagen; d Department of Neurology, Memory Disorders Research Unit, Rigshospitalet, Copenhagen, Denmark; and e Institute of Neuroscience and Physiology, Sahlgrenska Academy, Go teborg University, Mo lndal, Sweden Keywords: AlzheimerÕs disease, cerebrospinal fluid, dementia with Lewy bodies, transthyretin Received 25 August 2009 Accepted 23 September 2009 Background: Previous studies have indicated that transthyretin (TTR) levels in cerebrospinal fluid (CSF) are altered in depression and dementia. The present study aimed to investigate whether CSF TTR can be used to discriminate between patients with AlzheimerÕs disease (AD) and patients with dementia with Lewy bodies (DLB) with or without medication, as well as to reveal whether CSF TTR correlates with depression in dementia. Methods: CSF samples from 59 patients with AD, 13 patients with DLB and 13 healthy controls were collected, and biochemical analysis was performed. Subjects were assessed for the presence of depression. Results: No significant differences in CSF TTR were found between AD, DLB, and control subjects or between depressed and non-depressed dementia patients. Interestingly, we found a significant reduction in CSF TTR (14%) in AD patients who were medicated with cholinesterase inhibitors compared to those AD patients who were not. Conclusions: Significant reductions in CSF TTR were found after cholinesterase inhibitor treatment in patients with AD compared to untreated individuals. CSF TTR was unaltered in patients with DLB and had no relationship to depression in the present cohort with dementias. Introduction Clinical and neuropathological criteria exist for the diagnoses of both AlzheimerÕs disease (AD) and dementia with Lewy bodies (DLB), but there is an overlap of the symptoms. Biomarkers for AD and DLB are indeed essential to facilitate disease diagnosis, to monitor disease progression, and for assessment of responses to existing and future treatments. A combination of decreased levels of amyloid beta peptide (Ab), increased levels of total tau (t-tau) and phosphorylated tau (P-Tau) are increasingly used as biomarkers in CSF in AD, but no specific CSF biomarkers for DLB exist at present [1 4]. Although cognitive decline is the central Correspondence: K. Schultz, Translational Neuroendocrine Research Unit, BMC D11, Lund, Sweden (tel.: ; fax: ; kristofer.schultz@med.lu.se). characteristic of dementia, depression affects around 50% of all AD and DLB patients [5]. The underlying neurobiological mechanisms of psychiatric symptoms in DLB or AD are not fully understood. In AD, where comorbid depression has been studied to a greater extent than in DLB, it has been suggested that depression may exist as both a predisposing factor and a prodromal symptom of disease [6]. Interestingly, a meta-analysis of case control studies found that a history of depression approximately doubles the risk of developing AD [7]. Transthyretin (TTR), a protein involved in the transport of thyroxin across the blood-brain barrier, has been found to be reduced in CSF from patients with depression as well as AD [8,9]. Also, one recent study showed that CSF TTR was further reduced in AD patients compared to DLB patients [10]. The role of TTR in the pathogenesis of AD has been hypothesized Journal compilation Ó 2009 EFNS 1

2 2 K. Schultz et al. to be as a scaffolding protein binding to Ab [11] and thereby possibly protecting against neurodegeneration [12] by its clearance of Ab [13,14]. A reduction in TTR in CSF would thereby supposedly lead to increased Ab-plaque formation, a core neuropathological characteristic of AD. In depression, the thyroid hormone transporter function of TTR has been emphasized, as reduced levels of TTR have been found in CSF from patients with major depressive disorder, which could potentially lead to lower levels of thyroid hormone in the brain [8]. The fact that CSF TTR has been found to be reduced in patients with both dementia and depression suggests that TTR may provide a potential common link between these two conditions. Furthermore, initial observations indicate that it can also be used to distinguish between AD and DLB. Therefore, the aim of this study was to evaluate whether CSF TTR indeed can be used for differentiating between AD and DLB patients with or without medication and to assess whether CSF TTR correlates to depression in these two disorders. Methods Subjects In this retrospective study, clinical data and CSF samples from 85 subjects at the Memory Disorders Clinic at Rigshospitalet in Copenhagen, Denmark, the Psychogeriatric Clinic at Lund University Hospital in Lund, and the Neuropsychiatric Clinic, Sahlgrenska University Hospital, Gothenburg, Sweden were analyzed. The groups consisted of 34 women and 25 men with AD, 6 women and 7 men with DLB, and 8 women and 5 men in the control group. The 85 subjects were chosen out of a cohort originally consisting of 99 subjects to create age- and sex-matched groups. The study was approved by the medical ethics committees at the three hospitals. Patients fulfilled the consensus criteria for DLB [15] or AD (NINCDS- ADRDA) [16], respectively. Depression was diagnosed according to the ICD-10 [17], and memory function was assessed with the mini mental state examination (MMSE). Controls were recruited from senior citizen organizations and via information meetings on dementia. Inclusion criteria were that the individuals should be in good physical and mental health and not experience or exhibit any cognitive impairment. Minor vascular or other disorders in a stable phase did not lead to exclusion. All controls were thoroughly interviewed about their somatic and mental health by a research nurse before inclusion in the study. Biochemical analyses TTR levels in CSF were assessed using the enzymeenhanced Mancini method [18,19]. The Mancini method is an immunochemical precipitation method that allows quantitation of antigens by single radial immunodiffusion on an agar gel labeled with the primary antibody [19]. The diffusion period of the CSF antigen was 7 days at 4 C in the present study. The enzyme enhancement was obtained with the horse radish peroxidase (HRP)-conjugated secondary antibody using carbazole as the chromogen [18]. The primary anti-ttr antibody (made in rabbit) was used at 1:800, and the secondary swine anti-rabbit antibody was used at 1:100 (both from Siemens Healthcare Diagnostics, Deerfield, IL, USA). Statistical analysis The Statistical Package for the Social Sciences (SPSS; SPSS Inc., Chicago, IL, USA) program version for Windows was used for all statistical analyses. For multiple comparisons, one-way Analyses of Variance (ANOVA) were used. For comparisons between two groups, StudentÕs t-test was used. Tests of non-parametric correlations were performed using SpearmanÕs rho. Results There were no significant differences in age between the three groups of controls, patients with AD or DLB (Table 1). MMSE scores were significantly reduced in the groups with DLB and AD compared to controls (ScheffeÕs post hoc test P < 0.001; Table 1). We did not Table 1 Demographic characteristics Controls AD DLB ANOVA Gender (female/male) 8/5 34/25 6/7 n.a. Age (years) 70 ± ± ± 4.1 n.s. TTR (mg/l) 21.8 ± ± ± 3.8 n.s. MMSE (points) 29.4 ± ± ± 4.7 P < 0.01 Data stated as mean ± standard deviation. AD, AlzheimerÕs disease; DLB, dementia with Lewy bodies; TTR, transthyretin, MMSE, mini mental state examination.

3 CSF transthyretin as a biomarker 3 (a) (b) (n = 7) compared to the non-cholinesterase inhibitortreated group (n = 6) was found (16%; P = 0.098). Discussion Figure 1 CSF levels of transthyretin. (a) There were no significant differences in CSF transthyretin (TTR) between the different groups. AlzheimerÕs disease (AD): n = 59, CV = 0.149; dementia with Lewy bodies (DLB): n = 13, CV = 0.186; controls: n = 12, CV = (b). A significant difference was found between AD patients with or without treatment with choline esterase inhibitors (ChEI) (***ScheffeÕs post hoc test P < 0.005); AD ChEI: n = 37, CV = 0.161; AD non-chei: n = 22, CV = detect any significant differences in CSF TTR between AD and DLB patients or between these two disorders and controls (Fig. 1a; Table 1). Also, no significant difference in CSF TTR could be seen between women and men or between depressed and non-depressed patients, neither in the diagnostic groups nor in the cohort as a whole. There were also no significant correlations between TTR and MMSE in the different groups (AD: SpearmanÕs rho )0.06, P = n.s.; DLB: SpearmanÕs rho )0.23, P = n.s.; Controls: SpearmanÕs rho 0.11, P = n.s.) or in the whole cohort (SpearmanÕs rho 0.033, P = n.s.). However, in the AD patient group, there was a significant reduction (14%; P < 0.001) in CSF TTR in patients medicated with cholinesterase inhibitors (n = 22) compared to those who were not (n = 37) (Fig. 1b). In the DLB group, a trend to reduction in TTR in the cholinesterase inhibitor-treated group In our study of clinically well-defined and age-matched AD and DLB patients, we found reductions in CSF TTR in the two patient groups with ongoing treatment with cholinesterase inhibitors. Although a protective role of TTR in Ab-pathology has been suggested from studies performed in animal models [11,12,20], a recent study showed that TTR accelerates vascular Ab deposition in a mouse model of AD [21], which would be in line with our finding of reduced TTR levels after treatment with cholinesterase inhibitors. We could not detect any significant differences in CSF TTR levels between AD and DLB patients, or compared to control subjects. However, conflicting results on CSF TTR in dementia exist in the literature. Although the majority of the reports have presented results of decreased levels of TTR in AD, one study has shown increased levels of CSF TTR (Table 2). Studies on CSF TTR in DLB patients have also shown different results. Significantly increased levels of CSF TTR in DLB patients in comparison with controls have been shown by Abdi et al. [22], but no significant differences in CSF TTR between DLB patients and controls have been reported by Gloeckner et al. [10] (Table 3). Taken together, CSF TTR does not appear to be a robust biomarker for differentiating AD from DLB and controls in all cohorts. We found no differences in CSF TTR between dementia patients with or without depression. It has previously been shown that CSF TTR levels are reduced in depressed patients compared to controls [8,23,24] (Table 4). Therefore, it was postulated that the low levels of TTR resulted in reduced levels of thyroid hormones being distributed throughout the Table 2 Studies on CSF TTR in AD Author/reference Method Subjects TTR change Change in percent Schultz et al., this study Enzyme-enhanced Mancini 59 AD, 13 controls fi No significant change Hansson et al. [30] Immunonephelometric assay 35 AD, 29 controls fl Not stated Gloeckner et al. [10] Nephelometry using N-antisera 23 AD, 19 controls fl 21% against human prealbumin Biroccio et al. [27] MALDI-TOF MS analysis 39 AD, 27 controls fl Not applicable Castan o et al. [28] Two-dimensional gel electrophoresis 43 AD, 43 controls fl Not stated Puchades et al. [31] Two-dimensional gel electrophoresis 7 AD, 7 controls fl 69% Davidsson et al. [29] Two-dimensional gel electrophoresis 15 AD, 12 controls Not stated Merched et al. [9] Array protein system 20 AD, 10 controls fl 36% Riisoen et al. [32] Rocket immunoelectrophoresis 24 AD, 47 controls fl Negative correlation between CSF TTR and AD TTR, transthyretin; CSF, cerebrospinal fluid; AD, AlzheimerÕs disease.

4 4 K. Schultz et al. Table 3 Studies on CSF TTR in DLB Author/reference Method Subjects TTR change Change in percent Schultz et al., this study Enzyme-enhanced Mancini 13 DLB, 13 controls fi No significant change Gloeckner et al. [10] Nephelometry using N-antisera against human prealbumin 23 DLB, 19 controls fi No significant change Abdi et al. [22] Quantitative proteomic itraq 5 DLB, 10 controls >50% TTR, transthyretin; CSF, cerebrospinal fluid; DLB, dementia with Lewy bodies. Table 4 Studies on CSF TTR in depression Author/reference Method Subjects TTR change Change in percent Schultz et al., this study Enzyme-enhanced Mancini 59 AD, 13 DLB, 13 controls fi No significant change Schultz et al. [25] Enzyme-enhanced Mancini 108 suicide attempters/15 controls fi No significant change Sullivan et al. [24] Radio immunoassay 17 MDD, 15 controls fl 10% Sullivan et al. [23] Dot-blot immunoassay 18 MDE /1 BPD, 24 controls fl 39% Hatterer et al. [8] Rocket immunoelectro-phoretic procedure 8 MDD, 9 neurologic patients fl 28% TTR, transthyretin; AD, AlzheimerÕs disease; MDD, major depressive disorder; MDE, major depressive episode; BPD, bipolar disorder. brain and CSF, which could lead to thyroid hormone-related depression [8]. In a previous study using CSF from depressed patients after an actual suicide attempt, we found no differences in CSF TTR compared to other psychiatric diagnoses or controls [25]. However, in the same study, we found a significant correlation between CSF TTR and the item Ôfailing memoryõ assessed using the Comprehensive Psychopathology Rating Scale [26], which could be interpreted as a relationship between CSF TTR and cognitive impairment [25]. However, in the present study, no relationship was found between CSF TTR and severity of cognitive impairment in AD and DLB as assessed with the MMSE. In conclusion, although a number of reports have shown a modest decrease in CSF TTR in AD patients, this has not been reproduced in all studies, including the present report. A clear difference between CSF levels of TTR in AD compared to DLB patients has not been established, and there is no apparent relationship of CSF TTR to depression in dementia. We suggest that further studies are required before CSF TTR can be established as a biomarker for AD and as a tool for differential diagnosis to DLB. Acknowledgements We are grateful to Eva Bringman, Ewa Styrud and other members of the staff at the Memory Clinic and the Neurochemical Department at the Sahlgrenska University Hospital/Mo lndal for technical assistance. Technical expertise on transthyretin measurements were provided by Professor Anders Grubb and Veronica Lindstro m. This work was supported by grants from the Åhle n foundation, the Stohnes foundation, the So derstro m-ko nigska foundation, the Lindhaga foundation, the Bror Gadelius foundation, the Alzheimer foundation, the Sahlgrenska University Hospital, the Go teborg Medical Society, Swedish Brain Power, Hjalmar Svenssons forskningsfond, Stiftelsen fo r Gamla Tja narinnor, the province of Ska ne state grants (ALF), the Swedish Research Council (09946 to AW; M to A P) and The Danish Alzheimer Research Foundation and the Novo Nordisk Foundation (JEN). References 1. Hampel H, Teipel SJ, Fuchsberger T, et al. Value of CSF beta-amyloid1-42 and tau as predictors of AlzheimerÕs disease in patients with mild cognitive impairment. Mol Psychiatry 2004; 9: Hampel H, Burger K, Teipel SJ, Bokde AL, Zetterberg H, Blennow K. Core candidate neurochemical and imaging biomarkers of AlzheimerÕs disease. Alzheimers Dement 2008; 4: Lewczuk P, Esselmann H, Otto M, et al. Neurochemical diagnosis of AlzheimerÕs dementia by CSF Abeta42, Abeta42/Abeta40 ratio and total tau. Neurobiol Aging 2004; 25: Otto M, Lewczuk P, Wiltfang J. Neurochemical approaches of cerebrospinal fluid diagnostics in neurodegenerative diseases. Methods 2008; 44: Lyketsos CG, Lee HB. Diagnosis and treatment of depression in AlzheimerÕs disease. A practical update for the clinician. Dement Geriatr Cogn Disord 2004; 17: Thomas A. Clinical aspects of dementia: AlzheimerÕs disease. In: Jacoby R, Oppenheimer C, Dening T, Thomas A eds. Oxford Textbook of Old Age Psychiatry, 1st edn. Oxford: Oxford University Press, 2008: Jorm AF. History of depression as a risk factor for dementia: an updated review. Aust N Z J Psychiatry 2001; 35:

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LUP. Lund University Publications Institutional Repository of Lund University LUP Lund University Publications Institutional Repository of Lund University This is an author produced version of a paper published in Neuroscience letters. This paper has been peer-reviewed but does