European Journal of Neurology 2014, 21: 486–491
doi:10.1111/ene.12336
Cerebrospinal fluid biomarkers of b-amyloid metabolism and neuronal damage in epileptic seizures P. Shahima, R. Rejdakb,c, P. Ksiazekd, K. Blennowa, H. Zetterberga,e, N. Mattssona,f and K. Rejdakc,g a
Clinical Neurochemistry Laboratory, Department of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology, University of Gothenburg, Sahlgrenska University Hospital/M€ olndal, M€ olndal, Sweden; bDepartment of General Ophthalmology, Medical University of Lublin, Lublin; cMedical Research Center, Polish Academy of Sciences, Warsaw; dDepartment of Public Health, Medical University of Lublin, Lublin, Poland; eDepartment of Molecular Neuroscience and Reta Lilla Weston Laboratories, UCL Institute of Neurology, London, UK; fSan Francisco VA Medical Center, Center for Imaging of Neurodegenerative Diseases (CIND), University of California San Francisco, San Francisco, CA, USA; and gDepartment of Neurology, Medical University of Lublin, Lublin, Poland
Keywords:
amyloid, heart-type fatty acid binding protein, neuronal injury, seizures, amyloid precursor protein, tau Received 16 June 2013 Accepted 18 November 2013
Background and purpose: The main objectives of this study were to investigate if epileptic seizures have effects on brain metabolism of b-amyloid (Ab), as reflected by cerebrospinal fluid (CSF) levels of different isoforms of Ab peptides and soluble amyloid precursor protein (APP), and neuronal degeneration, as reflected by CSF biomarker signs of acute neuronal injury. Methods: Forty-five patients were included, 21 of whom had single generalized tonic-clonic seizures sGTCS), 11 had repetitive GTCS, 7 had repetitive partial seizures (rPS), 6 had single partial seizure (sPS) and 4 fulfilled the criterion for nonconvulsive status epilepticus (nSE). CSF was analyzed for Abx-38, Abx-40, Abx-42, Ab1-42, soluble APP fragments (sAPP-a/b), total-tau (T-tau) and phosphorylated tau (P-tau), as well as heart-type fatty acid binding protein (H-FABP). Results: Patients with seizures had decreased levels of T-tau (P = 0.0016) and Ptau (P = 0.0028) compared with controls, but no differences in H-FABP (P = 0.67). There were no overall differences in Ab or sAPP peptides between seizure patients and controls. In patients with rPS, the levels of Abx-38 and Abx-40 were elevated compared with nSE (P < 0.01), sPS (P < 0.05) and controls (P < 0.05), and Abx-42 was elevated in rPS relative to nSE (P < 0.05). Conclusions: The findings of this study argue against acute neuronal injury following medically treated seizures but suggest that seizures may reduce CSF levels of tau. Although seizures generally did not affect CSF levels of Ab or sAPP peptides, our findings suggest that different types of seizures may have different effects on APP metabolism.
Introduction There is growing interest in cerebrospinal fluid (CSF) biomarkers to assess neuronal damage [1,2] or detect other subtle pathological processes induced by seizures. There is some evidence of overlapping mechanisms in Alzheimer’s disease (AD) and epilepsy, including increased susceptibility for seizure in AD patients [3] and presence of amyloid plaque and tau pathology in epileptic tissue [4,5]. One of the key Correspondence: P. Shahim, Clinical Neurochemistry Laboratory, Sahlgrenska University Hospital/M€ olndal, S-431 80 M€ olndal, Sweden (tel.: +46 76 27045 84; fax: +46 (0) 31 34324 26; e-mail: [email protected]).
486
features of AD is accumulation of amyloid b (Ab) in plaques [6]. Ab is produced through proteolytic processing of amyloid precursor protein (APP) by b- and c-secretases [7]. Due to variability in the c-secretase cleavage site, APP processing may yield peptides with different C-terminal amino acids, such as Abx-38, Abx-40 and Abx-42. Interestingly, the release of Ab peptides depends on synaptic activity [8]. One postmortem study showed increased prevalence of Ab plaques in patients with epilepsy relative to controls, although the plaques were less prevalent than in AD [9]. Numerous studies suggest that peptides and proteases involved in Ab synthesis influence excitability and do so by diverse mechanisms [10,11]. Together, these © 2013 The Author(s) European Journal of Neurology © 2013 EFNS
CSF biomarkers in seizures
studies suggest a possible link between disturbed APP processing pathways and epilepsy. Examination of CSF levels of APP degradation products in patients with seizures may further elucidate the consequences of seizure on neuronal APP metabolism in humans. To our knowledge, no previous study has tested CSF Ab and APP peptides in patients with seizures. Another key feature of AD is the hyperphosphorylation and accumulation of tau proteins in neurofibrillary tangles [6]. It has been suggested that tau, which normally stabilizes cytoskeleton, may play a critical role in excitability [12]. A recent study found that release of tau is related to neuronal activity [13]. In neurodegenerative diseases, increased CSF total-tau (T-tau) is often considered to reflect axonal damage [14]. Increased CSF phosphorylated tau (P-tau) is mainly found in AD, where levels are elevated and correlate to the number of tangles [15]. One case report described a transient increase in CSF T-tau after seizure [16]. To our knowledge, only one previous study has systematically tested CSF tau levels after seizures [17]. That study reported increased Ttau, but not P-tau, in patients with seizures of known etiology but not in patients with unknown etiology, suggesting that seizures per se do not increase tau levels. Here our aim was to confirm this finding, extend it to several subgroups of epilepsy and also test a novel marker of axonal degeneration, heart-type fatty acid binding protein (H-FABP). Thus, the main objectives of this study were to determine in patients with different types of seizure (i) alterations of APP metabolism and (ii) signs of acute neuronal damage. CSF biomarkers related to APP metabolism and neuronal damage were therefore measured in patients with different types of seizures as well as in controls.
487
Materials and methods Study participants
Consecutive patients admitted to a neurological ward due to seizures were studied. Forty-five patients with focal onset seizures were included, 21 of whom had single secondary generalized tonic-clonic seizure (sGTCS), 11 had repetitive secondary generalized tonic-clonic seizure (rGTCS), 7 had repetitive partial seizure (rPS), 6 had single partial seizure (sPS) and 4 non-convulsive status epilepticus (nSE) [18] (Table 1). Patients in whom seizures were determined to be caused by a systemic disease were not included (N = 10) and three patients refused to participate in the study. For control values, CSF samples collected by lumbar puncture from 17 persons (Table 1) were obtained from clinical routine samples at Clinical Neurochemistry Laboratory, Sahlgrenska University Hospital, M€ olndal, Sweden. These control samples were selected from patients with normal CSF cell counts, normal CSF/serum albumin ratio and no signs of intrathecal immunoglobulin production. The indication for lumbar puncture in controls was suspicion of neurological disease, mainly headache or central nervous system infection. The controls had no medical history of seizure. Study procedures
On admission, all patients underwent neurological examination, blood biochemical assessment and magnetic resonance imaging (MRI) of the brain to exclude acute symptomatic etiology. Eight patients had remote structural focal changes of ischaemic lesions, four had remote post-traumatic lesions, one had demyelinating
Table 1 Demographics and disease characteristics of study subjects
N F/M (N) Age (years, median [range]) Time since last seizure (days, median [range]) Etiology (N, %) Genetic Structural Unknown History of epilepsy (N, %) AED therapy (%) Monotherapy Polytherapy
All
sGTCS
rGTCS
rPS
nSE
sPS
Controls
45 12/33* 35 [19–80] 4 [0.12–10]
19 3/16 36 [19–73] 4 [0.2–10]
9 0/9 35 [22–80] 0.2 [0.12–10]
7 4/3 34 [19–78] 10 [0.5–10]
4 1/3 39 [30–56] 3.5 [1–6]
6 4/2 29 [22–49] 5 [1.25–10]
17 6/11 49 [19–65] NA
0 4 (9) 41 (91) 33 (73)
0 2 (10) 17 (90) 10 (52)
0 1 (11) 8 (89) 7 (78)
0 1 (14) 6 (86) 6 (86)
0 0 4 (100) 4 (100)
0 0 6 (100) 6 (100)
NA NA NA NA
17 (38) 22 (49)
8 (42) 6 (31)
3 (33) 5 (55)
3 (43) 4 (57)
0 4 (100)
3 (50) 3 (50)
NA NA
sGTCS, single generalized tonic-clonic seizure; rGTCS, repetitive generalized tonic-clonic seizure; rPS, repetitive partial seizure; nSE, non-convulsive status epilepticus; sPS, single partial seizure; AED, anti-epileptic drug; NA, not applicable. *P > 0.05 vs. controls.
© 2013 The Author(s) European Journal of Neurology © 2013 EFNS
488
P. Shahim et al.
lesions consistent with multiple sclerosis, one had a subarachnoid cyst and one had mild brain atrophy. All patients with repetitive seizures received diazepam treatment (5–30 mg, iv). This stopped ictal activity in all patients except for four who were diagnosed with nSE and immediately taken to the neurological intensive care unit for further management. Electroencephalographic (EEG) monitoring was applied in order to confirm the diagnosis and they received sodium valproate (30 mg/kg, iv) as a second line treatment, which led to complete clinical and EEG confirmed cessation of seizures. EEG recording performed intra-ictally and inter-ictally confirmed focal onset of seizures in all patients. Lumbar puncture was performed in all seizure patients within 10 days after seizure cessation (median 4 days; range 3 h to 10 days), between 8 a.m. and 2 p.m. for CSF collection. There were no significant differences between groups in the interval between seizure cessation and CSF collection except between the subgroup of patients with rGTCS and rPS (Table 1). All CSF samples were centrifuged at 2000 g at 4°C for 10 min and stored at 80°C pending analysis. Biochemical analyses
The xMAP assay INNO-BIA AlzBio3 (Innogenetics, Ghent, Belgium) was used for quantification of Ab142, T-tau and P-tau, as described previously [19]. The MSD Human/Rodent Abeta Triplex assay (Meso Scale Discovery, Gaithersburg, MD, USA) was used for quantification of Abx-38, Abx-40, Abx-42 and Ab1-40/ 42 and the MSD sAPPa/sAPPb Multiplex Assay for quantification of the soluble APP fragments sAPP-a and sAPP-b, as described previously [20]. The H-FABP concentration was assessed using an ELISA from Hycult Biotech (Hycult Biotech, Uden, the Netherlands) according to the manufacturer’s instructions [21]. The analyses were done by experienced and boardcertified laboratory technicians who were blinded to all clinical data. Statistical analysis
The distribution of quantitative data for skewness was tested by the D’Agostino and Pearson omnibus test of normality. For data that were significantly skewed, nonparametric tests were conducted using the Kruskal– Wallis method with Dunn’s post hoc test for multiple group comparisons and the Mann–Whitney U test for pairwise group comparisons. When indicated, the t test was used for comparisons of normally distributed data. The linear relation between parameters was assessed by the Spearman rank correlation coefficient (r). v2 statis-
tics with the Fisher exact test were used for group comparison of categorical variables. A two-sided P < 0.05 was considered significant. Statistical analyses were performed using SPSS 20.0 (SPSS Inc., Chicago, IL, USA) or GraphPad Prism 5.0 (GraphPad Inc., San Diego, CA, USA). Ethics
The study protocol was approved by the local ethics committee at the University of Lublin. Informed consent was obtained from each patient or the next of kin if the patient was incapable.
Results There was no significant difference in age or gender between the diagnostic groups (Table 1, Fig. S1). Seventeen seizure patients were on monotherapy with an anti-epileptic drug (AED), 22 were on polytherapy and 6 had no treatment prior to the study inclusion. Drugs used in monotherapy or combinations included carbamazepine, valproate, levetyracetam, topiramate, gabapentine and oxcarbazepine. There were no significant differences between the diagnostic groups regarding the distribution of AED therapy at the time of study inclusion (data not shown). Ab and sAPP-a/b species
There were no significant differences in levels of Abx38/40/42, Ab1-42 or sAPP-a/b between all seizure patients and the controls. When the subgroups were analyzed separately, Abx-38 was higher in rPS (median 1847 pg/ml; range 1635–2683) than in nSE (883 pg/ml; 760–1287, P < 0.01), sPS (1095 pg/ml; 854–2212, P < 0.05) and controls (1366 pg/ml; 743–1918, P < 0.05) (Fig. 1a). Similarly, Abx-40 was higher in rPS (8695 pg/ml; 7456–13294) than in SE (4548 pg/ml; 3894–6318, P < 0.01), sPS (6020 pg/ml; 4376–9571, P < 0.05) and controls (6711 pg/ml; 4048–9343, P < 0.05) (Fig. 1b). Abx-42 was also higher in patients with rPS (1327 pg/ml; 755–2309) than in nSE (682 pg/ml; 658–1015, P < 0.05) (Fig. 1c). Ab1-42 and sAPP-a/b did not differ between subgroups (Fig. S2a–c). CSF biomarkers of neuronal injury
The seizure patients had lower T-tau (22 pg/ml; 10– 446) and P-tau (19 pg/ml; 6–81) concentrations than the controls (51 pg/ml; 24–89, P = 0.0016; 29 pg/ml; 15–51, P = 0.0028). There was no significant difference in the concentrations of H-FABP between the seizure patients (480 pg/ml; 102–2761) and the controls © 2013 The Author(s) European Journal of Neurology © 2013 EFNS
CSF biomarkers in seizures
(a)
Figure 1 CSF concentrations of Ab and sAPP peptides. In patients with rPS, the concentrations of Abx-38 (a) and Abx-40 (b) were elevated compared with nSE, sPS and controls. Abx-42 (c) was also elevated in rPS relative to nSE. Horizontal lines represent median values. sGTCS, single generalized tonic-clonic seizure; rGTCS, repetitive generalized tonic-clonic seizure; rPS, repetitive partial seizure; sPS, single partial seizure; nSE, non-convulsive status epilepticus. *P < 0.05; **P < 0.01.
(b)
(c)
(438 pg/ml; 110–988, P = 0.67). There were no significant differences in the concentrations of T-tau, P-tau or H-FABP within the seizure subgroups (Fig. 2a–c). CSF biomarkers did not correlate with the time between seizure cessation and CSF collection (Fig. S3) except H-FABP (r = 0.030; P = 0.045, n = 45; Fig. S3c). Previous studies have found correlations between H-FABP and T-tau/P-tau [22]. Since H-FABP and tau proteins seemed to be differentially regulated here, an exploratory analysis was done to test if diagnosis (all seizure patients versus controls) affected the relationship between tau and H-FABP. This was tested by linear regression, where T-tau and P-tau were used © 2013 The Author(s) European Journal of Neurology © 2013 EFNS
(b)
(c)
(a)
Figure 2 CSF levels of neuronal injury biomarkers. CSF concentrations of T-tau (a), P-tau (b) and H-FABP (c) did not differ between the subgroups of seizure patients and controls. Horizontal lines represent median values. sGTCS, single generalized tonic-clonic seizure; rGTCS, repetitive GTCS; rPS, repetitive partial seizure; sPS, single partial seizure; nSE, non-convulsive status epilepticus.
489
as dependent variables and H-FABP, diagnosis and an interaction term (H-FABP 9 diagnosis) were used as independent variables. The interaction terms were non-significant, arguing against an effect of diagnosis on the relationship between T-tau or P-tau and H-FABP (P > 0.05). Pathological MRI versus normal MRI
One of the obstacles in interpreting the changes in biomarkers in epileptic seizure is whether the changes measured in the biomarkers are due to the structural damage or solely the effect of the seizure. Patients
490
P. Shahim et al.
with pathological MRI were therefore tested versus those with normal MRI, but there were no differences in biomarker levels between these groups (Fig. S4).
Discussion The major findings of this study were that (i) Ab or sAPP peptides did not differ between patients with seizures and controls, (ii) patients with rPS had significantly increased concentrations of Ab peptides compared with patients with nSE or sPS and controls, (iii) patients with seizures had reduced T-tau and P-tau compared with controls and (iv) H-FABP did not differ between patients and controls. The first finding was that CSF Ab and sAPP levels were similar between the overall group of seizure patients and controls. This argues against a major effect of seizures in general on acute Ab and sAPP release, at least during the time frame of this study. The second finding was that Ab levels (but not sAPP levels) differed between the epilepsy subgroups. Specifically, significantly increased concentrations of Abx-38, Abx-40 and Abx-42 were found in patients with rPS compared with SE, sPS and controls, with the strongest effect for Abx-38. This suggests that APP metabolism may be differentially affected in different types of seizures. The exact relationship between seizures and Ab levels cannot be determined in this cross-sectional study, but one possible interpretation of our findings is that increased neuronal activity in rPS seizures led to increased neuronal Ab release. Experimental studies have shown that Ab production is related to synaptic activity [23] and a previous CSF study found correlations between Ab peptides and synaptic proteins [24]. However, it is at present unclear why the rPS patients differed from the other groups. The third finding of this study was that concentrations of T-tau and P-tau were lower in patients with seizures compared with controls, with no significant differences between seizure subgroups. A previous study [17] found increased CSF T-tau only in patients with seizures of known etiology, but not in all such patients and not in patients with seizures of unknown etiology. No difference in tau levels was found between patients with or without MRI abnormalities. Thus, our results support the notion that seizures in general do not cause increased CSF tau levels. On the contrary, the reduced CSF tau levels found in seizure patients in this study suggest that seizure activities may disturb the normal turnover and release of tau proteins. Reducing tau in mouse models of epilepsy reduces the seizure frequency and duration and normalizes hippocampal hyperexcitability in vitro [25].
Decreased tau in epileptic seizure may therefore have protective mechanisms. However, from the present study it is not possible to determine the mechanism underlying the reduced tau levels in these patients. The fourth finding was that H-FABP was unchanged in epilepsy patients compared with controls. H-FABP has been suggested as a marker of neuronal injury, mainly in glial cells, and is elevated in a number of disorders [26], especially in Creutzfeldt Jakob disease [27]. The unchanged H-FABP levels in this study argue against major acute neuronal damage in these seizure patients. The main limitation of this study was the small number of patients, mainly in the nSE group, which reduces the power to detect significant effects. Another possible limitation was the difference in interval between seizure cessation and CSF collection, especially between rPS and rGTCS. However, there were no correlations between the biomarkers and time since last seizure. In conclusion, no overall effects of seizures on Ab or sAPP peptides was found, arguing against major effects of seizures on the CSF levels of these molecules, although our results suggest that there may be differences in APP metabolism between different types of seizures. Patients with seizures had decreased levels of T-tau and P-tau, and normal levels of H-FABP, which speaks against acute axonal damage in this patient cohort.
Acknowledgements We thank Asa K€ allen, Monica Christiansson, Sara Hullberg and Dzemila Secic for skillful technical assistance.
Disclosure of conflicts of interest The study was supported by the National Science Center in Poland with grant no. UMO-2011/01/M/ NZ4/04916 (Dr Konrad Rejdak), the Swedish Research Council (Dr Niklas Mattsson and Dr Henrik Zetterberg), Goteborgs Lakaresallskap, Svenska Lakaresallskapet, Sahlgrenska Universitetssjukhuset, Carl-Bertil Laurells fund, and Klinisk Biokemi i Norden (Dr Niklas Mattsson). Dr Kaj Blennow has served on the advisory board for Innogenetics, Belgium. The remaining authors have nothing to disclose.
Supporting Information Additional Supporting Information may be found in the online version of this article: © 2013 The Author(s) European Journal of Neurology © 2013 EFNS
CSF biomarkers in seizures
Figure S1. Age in seizure subgroups and controls. Figure S2. CSF concentrations of Ab and sAPP peptides in different subgroups of epileptic seizures. Figure S3. Correlation between concentrations of CSF biomarkers and time since last seizure. Figure S4. Comparison between seizure patients with pathological MRI versus normal MRI.
14.
15.
16.
References 1. Correale J, Rabinowicz AL, Heck CN, Smith TD, Loskota WJ, DeGiorgio CM. Status epilepticus increases CSF levels of neuron-specific enolase and alters the blood brain barrier. Neurology 1998; 50: 1388–1391. 2. DeGiorgio CM, Correale JD, Gott PS, et al. Serum neuron-specific enolase in human status epilepticus. Neurology 1995; 45: 1134–1137. 3. Amatniek JC, Hauser WA, DelCastillo-Castaneda C, et al. Incidence and predictors of seizures in patients with Alzheimer’s disease. Epilepsia 2006; 47: 867–872. 4. Gouras GK, Relkin NR, Sweeney D, Munoz DG, Mackenzie IR, Gandy S. Increased apolipoprotein E epsilon 4 in epilepsy with senile plaques. Ann Neurol 1997; 41: 402–404. 5. Pollard H, Khrestchatisky M, Moreau J, Ben-Ari Y, Represa A. Correlation between reactive sprouting and microtubule protein expression in epileptic hippocampus. Neuroscience 1994; 61: 773–787. 6. Blennow K, de Leon MJ, Zetterberg H. Alzheimer’s disease. Lancet 2006; 368: 387–403. 7. Hardy J, Selkoe DJ. The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science 2002; 297: 353–356. 8. Cirrito JR, Kang JE, Lee J, et al. Endocytosis is required for synaptic activity-dependent release of amyloid-beta in vivo. Neuron 2008; 58: 42–51. 9. Thom M, Liu JY, Thompson P, et al. Neurofibrillary tangle pathology and Braak staging in chronic epilepsy in relation to traumatic brain injury and hippocampal sclerosis: a post-mortem study. Brain 2011; 134: 2969–2981. 10. Minkeviciene R, Rheims S, Dobszay MB, et al. Amyloid beta-induced neuronal hyperexcitability triggers progressive epilepsy. J Neurosci 2009; 29: 3453–3462. 11. Scharfman HE. ‘Untangling’ Alzheimer’s disease and epilepsy. Epilepsy Curr 2012; 12: 178–183. 12. Roberson ED, Halabisky B, Yoo JW, et al. Amyloidbeta/Fyn-induced synaptic, network, and cognitive impairments depend on tau levels in multiple mouse models of Alzheimer’s disease. J Neurosci 2011; 31: 700–711. 13. Pooler AM, Phillips EC, Lau DH, Noble W, Hanger DP. Physiological release of endogenous tau is
© 2013 The Author(s) European Journal of Neurology © 2013 EFNS
17.
18. 19.
20.
21.
22.
23.
24.
25.
26.
27.
491
stimulated by neuronal activity. EMBO Rep 2013; 14: 389–394. Zetterberg H, Hietala MA, Jonsson M, et al. Neurochemical aftermath of amateur boxing. Arch Neurol 2006; 63: 1277–1280. Buerger K, Ewers M, Pirttila T, et al. CSF phosphorylated tau protein correlates with neocortical neurofibrillary pathology in Alzheimer’s disease. Brain 2006; 129: 3035–3041. Matsui T, Maruyama M, Matsushita S, Arai H, Higuchi S, Maruyama K. A transient increase in cerebrospinal fluid tau level after epileptic seizure in an elderly patient. J Am Geriatr Soc 2007; 55: 2096–2097. Palmio J, Suhonen J, Keranen T, Hulkkonen J, Peltola J, Pirttila T. Cerebrospinal fluid tau as a marker of neuronal damage after epileptic seizure. Seizure 2009; 18: 474–477. Shorvon S. Clinical trials in acute repetitive seizures and status epilepticus. Epileptic Disord 2012; 14: 138–147. Olsson A, Vanderstichele H, Andreasen N, et al. Simultaneous measurement of beta-amyloid(1-42), total tau, and phosphorylated tau (Thr181) in cerebrospinal fluid by the xMAP technology. Clin Chem 2005; 51: 336–345. Mattsson N, Olsson M, Gustavsson MK, et al. Amyloid-beta metabolism in Niemann Pick C disease models and patients. Metab Brain Dis 2012; 27: 573–585. Wodzig KW, Pelsers MM, van der Vusse GJ, Roos W, Glatz JF. One-step enzyme-linked immunosorbent assay (ELISA) for plasma fatty acid-binding protein. Ann Clin Biochem 1997; 34(Pt 3): 263–268. Bjerke M, Zetterberg H, Edman A, Blennow K, Wallin A, Andreasson U. Cerebrospinal fluid matrix metalloproteinases and tissue inhibitor of metalloproteinases in combination with subcortical and cortical biomarkers in vascular dementia and Alzheimer’s disease. J Alzheimers Dis 2011; 27: 665–676. Cirrito JR, Yamada KA, Finn MB, et al. Synaptic activity regulates interstitial fluid amyloid-beta levels in vivo. Neuron 2005; 48: 913–922. Mattsson N, Johansson P, Hansson O, et al. Converging pathways of chromogranin and amyloid metabolism in the brain. J Alzheimers Dis 2010; 20: 1039–1049. Holth JK, Bomben VC, Reed JG, et al. Tau loss attenuates neuronal network hyperexcitability in mouse and Drosophila genetic models of epilepsy. J Neurosci 2013; 33: 1651–1659. Ohrfelt A, Andreasson U, Simon A, et al. Screening for new biomarkers for subcortical vascular dementia and Alzheimer’s disease. Dement Geriatr Cogn Dis Extra 2011; 1: 31–42. Steinacker P, Mollenhauer B, Bibl M, et al. Heart fatty acid binding protein as a potential diagnostic marker for neurodegenerative diseases. Neurosci Lett 2004; 370: 36–39.
doi:10.1111/ene.12336
Cerebrospinal fluid biomarkers of b-amyloid metabolism and neuronal damage in epileptic seizures P. Shahima, R. Rejdakb,c, P. Ksiazekd, K. Blennowa, H. Zetterberga,e, N. Mattssona,f and K. Rejdakc,g a
Clinical Neurochemistry Laboratory, Department of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology, University of Gothenburg, Sahlgrenska University Hospital/M€ olndal, M€ olndal, Sweden; bDepartment of General Ophthalmology, Medical University of Lublin, Lublin; cMedical Research Center, Polish Academy of Sciences, Warsaw; dDepartment of Public Health, Medical University of Lublin, Lublin, Poland; eDepartment of Molecular Neuroscience and Reta Lilla Weston Laboratories, UCL Institute of Neurology, London, UK; fSan Francisco VA Medical Center, Center for Imaging of Neurodegenerative Diseases (CIND), University of California San Francisco, San Francisco, CA, USA; and gDepartment of Neurology, Medical University of Lublin, Lublin, Poland
Keywords:
amyloid, heart-type fatty acid binding protein, neuronal injury, seizures, amyloid precursor protein, tau Received 16 June 2013 Accepted 18 November 2013
Background and purpose: The main objectives of this study were to investigate if epileptic seizures have effects on brain metabolism of b-amyloid (Ab), as reflected by cerebrospinal fluid (CSF) levels of different isoforms of Ab peptides and soluble amyloid precursor protein (APP), and neuronal degeneration, as reflected by CSF biomarker signs of acute neuronal injury. Methods: Forty-five patients were included, 21 of whom had single generalized tonic-clonic seizures sGTCS), 11 had repetitive GTCS, 7 had repetitive partial seizures (rPS), 6 had single partial seizure (sPS) and 4 fulfilled the criterion for nonconvulsive status epilepticus (nSE). CSF was analyzed for Abx-38, Abx-40, Abx-42, Ab1-42, soluble APP fragments (sAPP-a/b), total-tau (T-tau) and phosphorylated tau (P-tau), as well as heart-type fatty acid binding protein (H-FABP). Results: Patients with seizures had decreased levels of T-tau (P = 0.0016) and Ptau (P = 0.0028) compared with controls, but no differences in H-FABP (P = 0.67). There were no overall differences in Ab or sAPP peptides between seizure patients and controls. In patients with rPS, the levels of Abx-38 and Abx-40 were elevated compared with nSE (P < 0.01), sPS (P < 0.05) and controls (P < 0.05), and Abx-42 was elevated in rPS relative to nSE (P < 0.05). Conclusions: The findings of this study argue against acute neuronal injury following medically treated seizures but suggest that seizures may reduce CSF levels of tau. Although seizures generally did not affect CSF levels of Ab or sAPP peptides, our findings suggest that different types of seizures may have different effects on APP metabolism.
Introduction There is growing interest in cerebrospinal fluid (CSF) biomarkers to assess neuronal damage [1,2] or detect other subtle pathological processes induced by seizures. There is some evidence of overlapping mechanisms in Alzheimer’s disease (AD) and epilepsy, including increased susceptibility for seizure in AD patients [3] and presence of amyloid plaque and tau pathology in epileptic tissue [4,5]. One of the key Correspondence: P. Shahim, Clinical Neurochemistry Laboratory, Sahlgrenska University Hospital/M€ olndal, S-431 80 M€ olndal, Sweden (tel.: +46 76 27045 84; fax: +46 (0) 31 34324 26; e-mail: [email protected]).
486
features of AD is accumulation of amyloid b (Ab) in plaques [6]. Ab is produced through proteolytic processing of amyloid precursor protein (APP) by b- and c-secretases [7]. Due to variability in the c-secretase cleavage site, APP processing may yield peptides with different C-terminal amino acids, such as Abx-38, Abx-40 and Abx-42. Interestingly, the release of Ab peptides depends on synaptic activity [8]. One postmortem study showed increased prevalence of Ab plaques in patients with epilepsy relative to controls, although the plaques were less prevalent than in AD [9]. Numerous studies suggest that peptides and proteases involved in Ab synthesis influence excitability and do so by diverse mechanisms [10,11]. Together, these © 2013 The Author(s) European Journal of Neurology © 2013 EFNS
CSF biomarkers in seizures
studies suggest a possible link between disturbed APP processing pathways and epilepsy. Examination of CSF levels of APP degradation products in patients with seizures may further elucidate the consequences of seizure on neuronal APP metabolism in humans. To our knowledge, no previous study has tested CSF Ab and APP peptides in patients with seizures. Another key feature of AD is the hyperphosphorylation and accumulation of tau proteins in neurofibrillary tangles [6]. It has been suggested that tau, which normally stabilizes cytoskeleton, may play a critical role in excitability [12]. A recent study found that release of tau is related to neuronal activity [13]. In neurodegenerative diseases, increased CSF total-tau (T-tau) is often considered to reflect axonal damage [14]. Increased CSF phosphorylated tau (P-tau) is mainly found in AD, where levels are elevated and correlate to the number of tangles [15]. One case report described a transient increase in CSF T-tau after seizure [16]. To our knowledge, only one previous study has systematically tested CSF tau levels after seizures [17]. That study reported increased Ttau, but not P-tau, in patients with seizures of known etiology but not in patients with unknown etiology, suggesting that seizures per se do not increase tau levels. Here our aim was to confirm this finding, extend it to several subgroups of epilepsy and also test a novel marker of axonal degeneration, heart-type fatty acid binding protein (H-FABP). Thus, the main objectives of this study were to determine in patients with different types of seizure (i) alterations of APP metabolism and (ii) signs of acute neuronal damage. CSF biomarkers related to APP metabolism and neuronal damage were therefore measured in patients with different types of seizures as well as in controls.
487
Materials and methods Study participants
Consecutive patients admitted to a neurological ward due to seizures were studied. Forty-five patients with focal onset seizures were included, 21 of whom had single secondary generalized tonic-clonic seizure (sGTCS), 11 had repetitive secondary generalized tonic-clonic seizure (rGTCS), 7 had repetitive partial seizure (rPS), 6 had single partial seizure (sPS) and 4 non-convulsive status epilepticus (nSE) [18] (Table 1). Patients in whom seizures were determined to be caused by a systemic disease were not included (N = 10) and three patients refused to participate in the study. For control values, CSF samples collected by lumbar puncture from 17 persons (Table 1) were obtained from clinical routine samples at Clinical Neurochemistry Laboratory, Sahlgrenska University Hospital, M€ olndal, Sweden. These control samples were selected from patients with normal CSF cell counts, normal CSF/serum albumin ratio and no signs of intrathecal immunoglobulin production. The indication for lumbar puncture in controls was suspicion of neurological disease, mainly headache or central nervous system infection. The controls had no medical history of seizure. Study procedures
On admission, all patients underwent neurological examination, blood biochemical assessment and magnetic resonance imaging (MRI) of the brain to exclude acute symptomatic etiology. Eight patients had remote structural focal changes of ischaemic lesions, four had remote post-traumatic lesions, one had demyelinating
Table 1 Demographics and disease characteristics of study subjects
N F/M (N) Age (years, median [range]) Time since last seizure (days, median [range]) Etiology (N, %) Genetic Structural Unknown History of epilepsy (N, %) AED therapy (%) Monotherapy Polytherapy
All
sGTCS
rGTCS
rPS
nSE
sPS
Controls
45 12/33* 35 [19–80] 4 [0.12–10]
19 3/16 36 [19–73] 4 [0.2–10]
9 0/9 35 [22–80] 0.2 [0.12–10]
7 4/3 34 [19–78] 10 [0.5–10]
4 1/3 39 [30–56] 3.5 [1–6]
6 4/2 29 [22–49] 5 [1.25–10]
17 6/11 49 [19–65] NA
0 4 (9) 41 (91) 33 (73)
0 2 (10) 17 (90) 10 (52)
0 1 (11) 8 (89) 7 (78)
0 1 (14) 6 (86) 6 (86)
0 0 4 (100) 4 (100)
0 0 6 (100) 6 (100)
NA NA NA NA
17 (38) 22 (49)
8 (42) 6 (31)
3 (33) 5 (55)
3 (43) 4 (57)
0 4 (100)
3 (50) 3 (50)
NA NA
sGTCS, single generalized tonic-clonic seizure; rGTCS, repetitive generalized tonic-clonic seizure; rPS, repetitive partial seizure; nSE, non-convulsive status epilepticus; sPS, single partial seizure; AED, anti-epileptic drug; NA, not applicable. *P > 0.05 vs. controls.
© 2013 The Author(s) European Journal of Neurology © 2013 EFNS
488
P. Shahim et al.
lesions consistent with multiple sclerosis, one had a subarachnoid cyst and one had mild brain atrophy. All patients with repetitive seizures received diazepam treatment (5–30 mg, iv). This stopped ictal activity in all patients except for four who were diagnosed with nSE and immediately taken to the neurological intensive care unit for further management. Electroencephalographic (EEG) monitoring was applied in order to confirm the diagnosis and they received sodium valproate (30 mg/kg, iv) as a second line treatment, which led to complete clinical and EEG confirmed cessation of seizures. EEG recording performed intra-ictally and inter-ictally confirmed focal onset of seizures in all patients. Lumbar puncture was performed in all seizure patients within 10 days after seizure cessation (median 4 days; range 3 h to 10 days), between 8 a.m. and 2 p.m. for CSF collection. There were no significant differences between groups in the interval between seizure cessation and CSF collection except between the subgroup of patients with rGTCS and rPS (Table 1). All CSF samples were centrifuged at 2000 g at 4°C for 10 min and stored at 80°C pending analysis. Biochemical analyses
The xMAP assay INNO-BIA AlzBio3 (Innogenetics, Ghent, Belgium) was used for quantification of Ab142, T-tau and P-tau, as described previously [19]. The MSD Human/Rodent Abeta Triplex assay (Meso Scale Discovery, Gaithersburg, MD, USA) was used for quantification of Abx-38, Abx-40, Abx-42 and Ab1-40/ 42 and the MSD sAPPa/sAPPb Multiplex Assay for quantification of the soluble APP fragments sAPP-a and sAPP-b, as described previously [20]. The H-FABP concentration was assessed using an ELISA from Hycult Biotech (Hycult Biotech, Uden, the Netherlands) according to the manufacturer’s instructions [21]. The analyses were done by experienced and boardcertified laboratory technicians who were blinded to all clinical data. Statistical analysis
The distribution of quantitative data for skewness was tested by the D’Agostino and Pearson omnibus test of normality. For data that were significantly skewed, nonparametric tests were conducted using the Kruskal– Wallis method with Dunn’s post hoc test for multiple group comparisons and the Mann–Whitney U test for pairwise group comparisons. When indicated, the t test was used for comparisons of normally distributed data. The linear relation between parameters was assessed by the Spearman rank correlation coefficient (r). v2 statis-
tics with the Fisher exact test were used for group comparison of categorical variables. A two-sided P < 0.05 was considered significant. Statistical analyses were performed using SPSS 20.0 (SPSS Inc., Chicago, IL, USA) or GraphPad Prism 5.0 (GraphPad Inc., San Diego, CA, USA). Ethics
The study protocol was approved by the local ethics committee at the University of Lublin. Informed consent was obtained from each patient or the next of kin if the patient was incapable.
Results There was no significant difference in age or gender between the diagnostic groups (Table 1, Fig. S1). Seventeen seizure patients were on monotherapy with an anti-epileptic drug (AED), 22 were on polytherapy and 6 had no treatment prior to the study inclusion. Drugs used in monotherapy or combinations included carbamazepine, valproate, levetyracetam, topiramate, gabapentine and oxcarbazepine. There were no significant differences between the diagnostic groups regarding the distribution of AED therapy at the time of study inclusion (data not shown). Ab and sAPP-a/b species
There were no significant differences in levels of Abx38/40/42, Ab1-42 or sAPP-a/b between all seizure patients and the controls. When the subgroups were analyzed separately, Abx-38 was higher in rPS (median 1847 pg/ml; range 1635–2683) than in nSE (883 pg/ml; 760–1287, P < 0.01), sPS (1095 pg/ml; 854–2212, P < 0.05) and controls (1366 pg/ml; 743–1918, P < 0.05) (Fig. 1a). Similarly, Abx-40 was higher in rPS (8695 pg/ml; 7456–13294) than in SE (4548 pg/ml; 3894–6318, P < 0.01), sPS (6020 pg/ml; 4376–9571, P < 0.05) and controls (6711 pg/ml; 4048–9343, P < 0.05) (Fig. 1b). Abx-42 was also higher in patients with rPS (1327 pg/ml; 755–2309) than in nSE (682 pg/ml; 658–1015, P < 0.05) (Fig. 1c). Ab1-42 and sAPP-a/b did not differ between subgroups (Fig. S2a–c). CSF biomarkers of neuronal injury
The seizure patients had lower T-tau (22 pg/ml; 10– 446) and P-tau (19 pg/ml; 6–81) concentrations than the controls (51 pg/ml; 24–89, P = 0.0016; 29 pg/ml; 15–51, P = 0.0028). There was no significant difference in the concentrations of H-FABP between the seizure patients (480 pg/ml; 102–2761) and the controls © 2013 The Author(s) European Journal of Neurology © 2013 EFNS
CSF biomarkers in seizures
(a)
Figure 1 CSF concentrations of Ab and sAPP peptides. In patients with rPS, the concentrations of Abx-38 (a) and Abx-40 (b) were elevated compared with nSE, sPS and controls. Abx-42 (c) was also elevated in rPS relative to nSE. Horizontal lines represent median values. sGTCS, single generalized tonic-clonic seizure; rGTCS, repetitive generalized tonic-clonic seizure; rPS, repetitive partial seizure; sPS, single partial seizure; nSE, non-convulsive status epilepticus. *P < 0.05; **P < 0.01.
(b)
(c)
(438 pg/ml; 110–988, P = 0.67). There were no significant differences in the concentrations of T-tau, P-tau or H-FABP within the seizure subgroups (Fig. 2a–c). CSF biomarkers did not correlate with the time between seizure cessation and CSF collection (Fig. S3) except H-FABP (r = 0.030; P = 0.045, n = 45; Fig. S3c). Previous studies have found correlations between H-FABP and T-tau/P-tau [22]. Since H-FABP and tau proteins seemed to be differentially regulated here, an exploratory analysis was done to test if diagnosis (all seizure patients versus controls) affected the relationship between tau and H-FABP. This was tested by linear regression, where T-tau and P-tau were used © 2013 The Author(s) European Journal of Neurology © 2013 EFNS
(b)
(c)
(a)
Figure 2 CSF levels of neuronal injury biomarkers. CSF concentrations of T-tau (a), P-tau (b) and H-FABP (c) did not differ between the subgroups of seizure patients and controls. Horizontal lines represent median values. sGTCS, single generalized tonic-clonic seizure; rGTCS, repetitive GTCS; rPS, repetitive partial seizure; sPS, single partial seizure; nSE, non-convulsive status epilepticus.
489
as dependent variables and H-FABP, diagnosis and an interaction term (H-FABP 9 diagnosis) were used as independent variables. The interaction terms were non-significant, arguing against an effect of diagnosis on the relationship between T-tau or P-tau and H-FABP (P > 0.05). Pathological MRI versus normal MRI
One of the obstacles in interpreting the changes in biomarkers in epileptic seizure is whether the changes measured in the biomarkers are due to the structural damage or solely the effect of the seizure. Patients
490
P. Shahim et al.
with pathological MRI were therefore tested versus those with normal MRI, but there were no differences in biomarker levels between these groups (Fig. S4).
Discussion The major findings of this study were that (i) Ab or sAPP peptides did not differ between patients with seizures and controls, (ii) patients with rPS had significantly increased concentrations of Ab peptides compared with patients with nSE or sPS and controls, (iii) patients with seizures had reduced T-tau and P-tau compared with controls and (iv) H-FABP did not differ between patients and controls. The first finding was that CSF Ab and sAPP levels were similar between the overall group of seizure patients and controls. This argues against a major effect of seizures in general on acute Ab and sAPP release, at least during the time frame of this study. The second finding was that Ab levels (but not sAPP levels) differed between the epilepsy subgroups. Specifically, significantly increased concentrations of Abx-38, Abx-40 and Abx-42 were found in patients with rPS compared with SE, sPS and controls, with the strongest effect for Abx-38. This suggests that APP metabolism may be differentially affected in different types of seizures. The exact relationship between seizures and Ab levels cannot be determined in this cross-sectional study, but one possible interpretation of our findings is that increased neuronal activity in rPS seizures led to increased neuronal Ab release. Experimental studies have shown that Ab production is related to synaptic activity [23] and a previous CSF study found correlations between Ab peptides and synaptic proteins [24]. However, it is at present unclear why the rPS patients differed from the other groups. The third finding of this study was that concentrations of T-tau and P-tau were lower in patients with seizures compared with controls, with no significant differences between seizure subgroups. A previous study [17] found increased CSF T-tau only in patients with seizures of known etiology, but not in all such patients and not in patients with seizures of unknown etiology. No difference in tau levels was found between patients with or without MRI abnormalities. Thus, our results support the notion that seizures in general do not cause increased CSF tau levels. On the contrary, the reduced CSF tau levels found in seizure patients in this study suggest that seizure activities may disturb the normal turnover and release of tau proteins. Reducing tau in mouse models of epilepsy reduces the seizure frequency and duration and normalizes hippocampal hyperexcitability in vitro [25].
Decreased tau in epileptic seizure may therefore have protective mechanisms. However, from the present study it is not possible to determine the mechanism underlying the reduced tau levels in these patients. The fourth finding was that H-FABP was unchanged in epilepsy patients compared with controls. H-FABP has been suggested as a marker of neuronal injury, mainly in glial cells, and is elevated in a number of disorders [26], especially in Creutzfeldt Jakob disease [27]. The unchanged H-FABP levels in this study argue against major acute neuronal damage in these seizure patients. The main limitation of this study was the small number of patients, mainly in the nSE group, which reduces the power to detect significant effects. Another possible limitation was the difference in interval between seizure cessation and CSF collection, especially between rPS and rGTCS. However, there were no correlations between the biomarkers and time since last seizure. In conclusion, no overall effects of seizures on Ab or sAPP peptides was found, arguing against major effects of seizures on the CSF levels of these molecules, although our results suggest that there may be differences in APP metabolism between different types of seizures. Patients with seizures had decreased levels of T-tau and P-tau, and normal levels of H-FABP, which speaks against acute axonal damage in this patient cohort.
Acknowledgements We thank Asa K€ allen, Monica Christiansson, Sara Hullberg and Dzemila Secic for skillful technical assistance.
Disclosure of conflicts of interest The study was supported by the National Science Center in Poland with grant no. UMO-2011/01/M/ NZ4/04916 (Dr Konrad Rejdak), the Swedish Research Council (Dr Niklas Mattsson and Dr Henrik Zetterberg), Goteborgs Lakaresallskap, Svenska Lakaresallskapet, Sahlgrenska Universitetssjukhuset, Carl-Bertil Laurells fund, and Klinisk Biokemi i Norden (Dr Niklas Mattsson). Dr Kaj Blennow has served on the advisory board for Innogenetics, Belgium. The remaining authors have nothing to disclose.
Supporting Information Additional Supporting Information may be found in the online version of this article: © 2013 The Author(s) European Journal of Neurology © 2013 EFNS
CSF biomarkers in seizures
Figure S1. Age in seizure subgroups and controls. Figure S2. CSF concentrations of Ab and sAPP peptides in different subgroups of epileptic seizures. Figure S3. Correlation between concentrations of CSF biomarkers and time since last seizure. Figure S4. Comparison between seizure patients with pathological MRI versus normal MRI.
14.
15.
16.
References 1. Correale J, Rabinowicz AL, Heck CN, Smith TD, Loskota WJ, DeGiorgio CM. Status epilepticus increases CSF levels of neuron-specific enolase and alters the blood brain barrier. Neurology 1998; 50: 1388–1391. 2. DeGiorgio CM, Correale JD, Gott PS, et al. Serum neuron-specific enolase in human status epilepticus. Neurology 1995; 45: 1134–1137. 3. Amatniek JC, Hauser WA, DelCastillo-Castaneda C, et al. Incidence and predictors of seizures in patients with Alzheimer’s disease. Epilepsia 2006; 47: 867–872. 4. Gouras GK, Relkin NR, Sweeney D, Munoz DG, Mackenzie IR, Gandy S. Increased apolipoprotein E epsilon 4 in epilepsy with senile plaques. Ann Neurol 1997; 41: 402–404. 5. Pollard H, Khrestchatisky M, Moreau J, Ben-Ari Y, Represa A. Correlation between reactive sprouting and microtubule protein expression in epileptic hippocampus. Neuroscience 1994; 61: 773–787. 6. Blennow K, de Leon MJ, Zetterberg H. Alzheimer’s disease. Lancet 2006; 368: 387–403. 7. Hardy J, Selkoe DJ. The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science 2002; 297: 353–356. 8. Cirrito JR, Kang JE, Lee J, et al. Endocytosis is required for synaptic activity-dependent release of amyloid-beta in vivo. Neuron 2008; 58: 42–51. 9. Thom M, Liu JY, Thompson P, et al. Neurofibrillary tangle pathology and Braak staging in chronic epilepsy in relation to traumatic brain injury and hippocampal sclerosis: a post-mortem study. Brain 2011; 134: 2969–2981. 10. Minkeviciene R, Rheims S, Dobszay MB, et al. Amyloid beta-induced neuronal hyperexcitability triggers progressive epilepsy. J Neurosci 2009; 29: 3453–3462. 11. Scharfman HE. ‘Untangling’ Alzheimer’s disease and epilepsy. Epilepsy Curr 2012; 12: 178–183. 12. Roberson ED, Halabisky B, Yoo JW, et al. Amyloidbeta/Fyn-induced synaptic, network, and cognitive impairments depend on tau levels in multiple mouse models of Alzheimer’s disease. J Neurosci 2011; 31: 700–711. 13. Pooler AM, Phillips EC, Lau DH, Noble W, Hanger DP. Physiological release of endogenous tau is
© 2013 The Author(s) European Journal of Neurology © 2013 EFNS
17.
18. 19.
20.
21.
22.
23.
24.
25.
26.
27.
491
stimulated by neuronal activity. EMBO Rep 2013; 14: 389–394. Zetterberg H, Hietala MA, Jonsson M, et al. Neurochemical aftermath of amateur boxing. Arch Neurol 2006; 63: 1277–1280. Buerger K, Ewers M, Pirttila T, et al. CSF phosphorylated tau protein correlates with neocortical neurofibrillary pathology in Alzheimer’s disease. Brain 2006; 129: 3035–3041. Matsui T, Maruyama M, Matsushita S, Arai H, Higuchi S, Maruyama K. A transient increase in cerebrospinal fluid tau level after epileptic seizure in an elderly patient. J Am Geriatr Soc 2007; 55: 2096–2097. Palmio J, Suhonen J, Keranen T, Hulkkonen J, Peltola J, Pirttila T. Cerebrospinal fluid tau as a marker of neuronal damage after epileptic seizure. Seizure 2009; 18: 474–477. Shorvon S. Clinical trials in acute repetitive seizures and status epilepticus. Epileptic Disord 2012; 14: 138–147. Olsson A, Vanderstichele H, Andreasen N, et al. Simultaneous measurement of beta-amyloid(1-42), total tau, and phosphorylated tau (Thr181) in cerebrospinal fluid by the xMAP technology. Clin Chem 2005; 51: 336–345. Mattsson N, Olsson M, Gustavsson MK, et al. Amyloid-beta metabolism in Niemann Pick C disease models and patients. Metab Brain Dis 2012; 27: 573–585. Wodzig KW, Pelsers MM, van der Vusse GJ, Roos W, Glatz JF. One-step enzyme-linked immunosorbent assay (ELISA) for plasma fatty acid-binding protein. Ann Clin Biochem 1997; 34(Pt 3): 263–268. Bjerke M, Zetterberg H, Edman A, Blennow K, Wallin A, Andreasson U. Cerebrospinal fluid matrix metalloproteinases and tissue inhibitor of metalloproteinases in combination with subcortical and cortical biomarkers in vascular dementia and Alzheimer’s disease. J Alzheimers Dis 2011; 27: 665–676. Cirrito JR, Yamada KA, Finn MB, et al. Synaptic activity regulates interstitial fluid amyloid-beta levels in vivo. Neuron 2005; 48: 913–922. Mattsson N, Johansson P, Hansson O, et al. Converging pathways of chromogranin and amyloid metabolism in the brain. J Alzheimers Dis 2010; 20: 1039–1049. Holth JK, Bomben VC, Reed JG, et al. Tau loss attenuates neuronal network hyperexcitability in mouse and Drosophila genetic models of epilepsy. J Neurosci 2013; 33: 1651–1659. Ohrfelt A, Andreasson U, Simon A, et al. Screening for new biomarkers for subcortical vascular dementia and Alzheimer’s disease. Dement Geriatr Cogn Dis Extra 2011; 1: 31–42. Steinacker P, Mollenhauer B, Bibl M, et al. Heart fatty acid binding protein as a potential diagnostic marker for neurodegenerative diseases. Neurosci Lett 2004; 370: 36–39.
