ORIGINAL RESEARCH published: 24 May 2018 doi: 10.3389/fnagi.2018.00152 A Specific Reduction in Aβ1−42 vs. a Universal Loss of Aβ Peptides in CSF Differentiates Alzheimer’s Disease From Meningitis and Multiple Sclerosis Philipp Spitzer 1*, Roland Lang 2, Timo J. Oberstein 1, Piotr Lewczuk 1,3, Natalia Ermann 1, Hagen B. Huttner 4, Ilias Masouris 5, Johannes Kornhuber 1, Uwe Ködel 5 and Juan M. Maler 1 1 Department of Psychiatry and Psychotherapy, Friedrich-Alexander-University Erlangen-Nuremberg, University Hospital Erlangen, Erlangen, Germany, 2 Institute of Clinical Microbiology, Immunology and Hygiene, Friedrich-Alexander-University Erlangen-Nuremberg, University Hospital Erlangen, Erlangen, Germany, 3 Department of Neurodegeneration Diagnostics, Medical University of Bialystok, Bialystok, Poland, 4 Department of Neurology, Friedrich-Alexander-University Erlangen-Nuremberg, University Hospital Erlangen, Erlangen, Germany, 5 Department of Neurology, Ludwig-Maximilian-University, Munich, Germany Edited by: A reduced concentration of Aβ1−42 in CSF is one of the established biomarkers of Robert David Moir, Alzheimer’s disease. Reduced CSF concentrations of Aβ1−42 have also been shown Massachusetts General Hospital, Harvard Medical School, in multiple sclerosis, viral encephalitis and bacterial meningitis. As neuroinflammation is United States one of the neuropathological hallmarks of Alzheimer’s disease, an infectious origin of Reviewed by: the disease has been proposed. According to this hypothesis, amyloid pathology is a Miguel Calero, Instituto de Salud Carlos III, Spain consequence of a microbial infection and the resulting immune defense. Accordingly, Adelaide Fernandes, changes in CSF levels of amyloid-β peptides should be similar in AD and inflammatory Faculdade de Farmácia, Universidade brain diseases. Aβ1−42 and Aβ1−40 levels were measured in cerebrospinal fluid by ELISA de Lisboa, Portugal and Western blotting in 34 patients with bacterial meningitis (n = 9), multiple sclerosis *Correspondence: Philipp Spitzer (n = 5) or Alzheimer’s disease (n = 9) and in suitable controls (n = 11). Reduced [email protected] concentrations of Aβ1−42 were detected in patients with bacterial meningitis, multiple sclerosis and Alzheimer’s disease. However, due to a concurrent reduction in Aβ1−40 Received: 31 January 2018 Accepted: 04 May 2018 in multiple sclerosis and meningitis patients, the ratio of Aβ1−42/Aβ1−40 was reduced Published: 24 May 2018 only in the CSF of Alzheimer’s disease patients. Urea-SDS-PAGE followed by Western Citation: blotting revealed that all Aβ peptide variants are reduced in bacterial meningitis, whereas Spitzer P, Lang R, Oberstein TJ, in Alzheimer’s disease, only Aβ is reduced. These results have two implications. First, Lewczuk P, Ermann N, Huttner HB, 1−42 Masouris I, Kornhuber J, Ködel U and they confirm the discriminatory diagnostic power of the Aβ1−42/Aβ1−40 ratio. Second, Maler JM (2018) A Specific Reduction the differential pattern of Aβ peptide reductions suggests that the amyloid pathology in in Aβ1-42 vs. a Universal Loss of Aβ Peptides in CSF Differentiates meningitis and multiple sclerosis differs from that in AD and does not support the notion Alzheimer’s Disease From Meningitis of AD as an infection-triggered immunopathology. and Multiple Sclerosis Front. Aging Neurosci. 10:152. Keywords: meningitis, amyloid, Alzheimer’s disease, multiple sclerosis, biomarker, dementia, neuroinflammation, doi: 10.3389/fnagi.2018.00152 neurodegeneration Frontiers in Aging Neuroscience | www.frontiersin.org 1 May 2018 | Volume 10 | Article 152 Spitzer et al. Reduced Aβ Peptides in Meningitis INTRODUCTION laboratory examinations. Additionally, an MRI scan and a CSF analysis were performed. The CSF of patients with meningitis In addition to amyloid plaque deposition, neuroinflammation is was drawn under emergency conditions, so oligoclonal bands one of the neuropathological hallmarks of Alzheimer’s disease were not routinely determined. The diagnosis of AD was (AD) (Heneka et al., 2015). Viral, bacterial and even fungal made according to the National Institute of Neurological and antigens have been found in association with the pathognomonic Communicative Disorders and Stroke and the Alzheimer’s amyloid-beta (Aβ) depositions (Itzhaki, 2014; Little et al., 2014; Disease and Related Disorders Association (NINCDS-ADRDA) Piacentini et al., 2015; Pisa et al., 2015; Zhan et al., 2016). criteria, taking into account the Aβ42/Aβ40 ratio and the total tau The secretion of Aβ peptides during inflammation and the and phospho-tau levels in the CSF (Albert et al., 2011; McKhann observation of anti-infective properties of Aβ peptides support et al., 2011). The Erlangen Score (ES) algorithm (Lewczuk et al., the idea that the production of Aβ peptides provides an immune 2009, 2015a) was also used to classify patients: all AD patients defense (Spitzer et al., 2010, 2016; Condic et al., 2014; Kumar had an ES ≥ 3, and all controls had an ES ≤ 1. In reference to et al., 2016). These and other findings have culminated in the criteria suggested by Jack et al, the patients within the AD the formulation of the infection hypothesis of AD (Miklossy, group were classified according to the presence (+) or absence 2011). According to this hypothesis, amyloid deposition is (–) of Aβ (A), neurofibrillary tangles (T) and neurodegeneration the consequence of ongoing neuroinflammation evoked by a (N) as A+/T+/N+, whereas those in the control group were A– pathogen that evades the immune system. /T–/N– (Jack et al., 2016). Amyloid pathology was evaluated by In the diagnosis of AD, decreased levels of soluble Aβ the Aβ42/Aβ40 ratio, tau pathology was evaluated by phospho- peptide 1-42 (Aβ1−42) in the cerebrospinal fluid (CSF) are widely tau levels, and neurodegeneration was evaluated by total-tau accepted as a surrogate for brain amyloidosis (McKhann et al., levels and temporo-parietal atrophy in the MRI scan. Patients 2011; Blennow and Zetterberg, 2015; Jack et al., 2016; Lewczuk with intermediate signs of AD pathophysiology were excluded. et al., 2017). Fitting perfectly into the infection hypothesis, MS was diagnosed according to the revised McDonald criteria reduced levels of Aβ42 are also found in patients with brain (Polman et al., 2011), and meningitis was diagnosed according infections, such as bacterial meningitis, herpes encephalitis to the European Society of Clinical Microbiology and Infectious or human immunodeficiency virus (HIV)-associated dementia Diseases (ESCMID) guidelines (van de Beek et al., 2016). The (Sjögren et al., 2001; Krut et al., 2013).Aβ1−42 is generated during control group comprised five patients with tension headache, one cleavage of amyloid precursor protein (APP). In addition to with schizophrenia, two with idiopathic epilepsy and one with a Aβ42, several other Aβ peptide variants with different C- and N- depressive episode. CSF samples were collected in polypropylene termini are generated during this process (Wiltfang et al., 2002). tubes, centrifuged within 24 h after sampling and stored in It is hypothesized that it is not the overproduction of Aβ42 that aliquots at −80◦C until further use. leads to its deposition but rather an imbalance of the different Aβ peptide variants (Hasegawa et al., 1999; Jan et al., 2008). ELISA Consequently, the concentration of Aβ1−42 in relation to Aβ1−40, Aβ1−40 and Aβ1−42 levels were quantified with certified ELISA the most abundant Aβ peptide variant, was found to be superior tests (IBL international GmbH, Hamburg, Germany) according to Aβ1−42 alone as a biomarker for AD (Lewczuk et al., 2004, to the manufacturer’s instructions. Samples were thawed 2015a,b; Janelidze et al., 2016; Niemantsverdriet et al., 2017). If immediately before the analysis and diluted 1:20 in reagent this imbalance between Aβ42 and Aβ40 that is observed in AD diluent. Then, 100 µl of each sample, standard, positive control could also be found in inflammatory brain diseases, those data or blank were added in duplicate to a pre-coated microtiter plate. would further support the infection hypothesis of AD. After incubation for 2 h, the plate was washed, and horseradish Therefore, this study investigates whether the changes in peroxidase (HRP)-conjugated detection antibody (clone 82E1) Aβ1−42 and Aβ1−40 levels in CSF during AD resemble those was added for another 1 h. After washing, the chromogenic ′ ′ observed in multiple sclerosis (MS) and bacterial meningitis substrate 3,3 ,5,5 -tetramethylbenzidine (TMB) was added, and and whether the Aβ-ratio (Aβ1−42/Aβ1−40) differs between these the plate was read. The analyses were performed under careful diseases. quality control, and a measurement was regarded as valid if the range-to-average coefficient of the duplicate measurements was MATERIALS AND METHODS below 20%. Patients SDS-PAGE/Western Blotting Samples were collected in the memory clinic of the Department The immunoprecipitation, urea-sodium dodecyl sulfate- of Psychiatry, Erlangen, the Neurological Department, Erlangen, polyacrylamide gel electrophoresis (SDS-PAGE) and Western and the Neurological Department of Ludwig-Maximilian- blotting procedures have been described in detail before University (LMU), Munich. Patients or their legal representatives (Wiltfang et al., 2002; Oberstein et al., 2015). In short, provided their informed consent, and the study protocol was immunoprecipitation was carried out with mouse anti- approved by the ethics committee of the University Hospital amyloid antibody 6E10 (Biolegend,