The focus of the Laboratory for Molecular Neurobiology and Neurochemical Dementia Diagnostics, Department of Psychiatry and Psychotherapy, University of Erlangen-Nuremberg headed by Professor Dr. Jens Wiltfang is clinical neuroproteomics of human body fluids, and neurochemichal diagnostics of dementias. Professor Dr. Jens Wiltfang (neurologist and psychiatrist) is deputy director of the Department of Psychiatry and Psychotherapy, he leads the Molecular Neurobiology Lab, and is in charge of the Outpatient Unit of the Department. Priv.-Doz. Dr. Piotr Lewczuk (neurologist and clinical neurochemist certified by German Society for CSF Analysis and Clinical Neurochemistry) is a senior researcher at Molecular Neurobiology Lab of the Department, being responsible for neurochemical dementia diagnostics and clinical neuroproteomics. Our group is the nationwide reference neurochemical laboratory of the German Federal Ministry of Education and Research (BMBF)-funded Competence Net Dementia, and simultaneously we participate as a co-reference laboratory in the first national network for rapidly progressing dementias with a focus on CJD. Currently, we have collected and stored approximately 1000 human body fluid (cerebrospinal fluid, plasma, serm, blood) samples from patients with mild cognitive impairment (MCI), and early dementias, which is, to our best knowledge, the largest prospective MCI/early dementias sample collection world-wide. Our group is also a member of the EU-funded consortium 'Abnormal proteins in the pathogenesis of neurodegenerative disorders' (APOPIS).
Currently, on behalf of German Society of Clinical Neurochemistry (DGLN), JW and PL are coordinating the first nationwide inter-laboratory quality survey of neurochemical dementia diagnostics (manuscript in preparation).
On behalf of The World Federation of Societies of Biological Psychiatry, we have recently coordinated preparation of a consensus paper on biological markers of dementias (Wiltfang et al., 2005).
In NGFN2, our subproject focuses on clinical neuroproteomics of human body fluids. This approach needs a synergistic cooperation of large clinically-oriented networks with a strong proteomic and bioinformatic platform. These clinical requirements are met by the Competence Network Dementia and the Creutzfeldt-Jakob Disease Network, which guarantee expert clinical dementia diagnosis, highly standardized multicenter pre-analytical sample handling and large-scale sample banking. Our subproject cross-bridges these clinical networks to advanced proteomic core facilities of NGFN2.
Project Status
Current and forthcoming therapies for Alzheimer's disease (AD) and other dementias, like dementia with Lewy bodies (LBD) will be most effective if the treatments start as soon as possible, in as much as diseases, like AD and LBD have a subclinical course of years and decades preceding the dementia state (Wiltfang et al., 2001). Correspondingly, molecular markers which are closely correlated to the pathophysiological key events of AD or LBD should improve their early and differential diagnosis. However, so far only proteins and peptides from the cerebrospinal fluid (CSF) have consistently and successfully been identified as neurochemical dementia markers. Only a few years ago, CSF neuro¬chemical dementia diagnostics (CSF-NDD) served merely to exclude dementia syndromes due to other causes than primary progressive dementia, e.g. neuroinflammation (Wiltfang et al., 2002). Currently, CSF-NDD is a rapidly developing field of clinical neurochemistry offering a positive diagnosis of dementias and has already entered neurochemical routine diagnostics in specialised gerontopsychiatric centres (Wiltfang et al., 2002).
Recently, the first promising set of neurochemical dementia markers emerged for the AD diagnosis, i.e. the amloid # peptides (A# peptides) 1-40/42, total Tau and phospho-Tau proteins. Using these parameters, we demonstrated that AD could be differentiated from all other patients, i.e. other dementias, non-demented patients, and controls with a sensitivity and specificity approaching 85% (Lewczuk et al., 2004a; Lewczuk et al., 2004d). Regarding specificity, the A# peptide ratio x-40/x-42 seems to be superior to A#1-42, and phospho-Tau is clearly superior to total-Tau (Lewczuk et al., 2004a). Moreover, we developed a novel one- and two-dimensional urea-based attomolar-sensitive A#-SDS-PAGE/immunoblot, and demonstrated that in addition to A#1-40/42 the carboxyterminally truncated A# peptides 1-37/38/39 are present in CSF (Wiltfang et al., 2001; Wiltfang et al., 2002). This A# peptide quintet is constitutively expressed and regulated within surprisingly narrow limits. In contrast to A#1-42, the CSF concentration of the carboxyterminally truncated A# species was correlated to the severity of AD.
Most recently, using surface enhanced laser desorption/ionisation time of flight mass spectrometry (SELDI-TOF MS) we identified further novel A# peptide fragments in the brain and CSF of AD patients, which may support the neurochemical diagnosis of AD (Lewczuk et al., 2003; Lewczuk et al., 2004c). We demonstrated also that CSF-NDD can be successfully applied for the diagnosis of Creutzfeldt-Jakob dementia, measuring total-Tau, phospho-Tau, total 14-3-3, 14-3-3 isoforms, S100B and beta-Amyloid peptides. Surprisingly, the selective drop of CSF A#1-42 which characterizes AD was also observed in CJD, however, in the latter case we did not observe A# plaques at autopsy (Wiltfang et al., 2003).
Accordingly, the textbook explanation for the CSF drop of A#1-42 in AD that is A# plaque-associated precipitation of soluble A#1-42 has to be questioned. Alternative mechanisms, like a common chaperone with high-avidity binding for misfolded proteins/peptides, and formation of highly neurotoxic soluble detergent-stable A# peptide oligomers, have to be considered. Due to the rapid-progressive course of CJD with massive neurogeneration and strong glial activation, we showed that increased concentrations of brain-derived proteins may even be measured in the blood. Accordingly, we speculate that rapid-progressive dementias will allow identification of further brain-derived proteins/peptides in the blood, which subsequently may be used to monitor disease progression and treatment efficacy in the periphery.
A set of neurochemical dementia markers may even identify non-demented patients with high-risk for incipient AD, e.g. patients with MCI of AD-type (MCI AD-type), who could benefit from prophylactic therapies.
This becomes increasingly important, since several potentially reversible long-term dysmetabolic conditions have recently been identified also as risk factors for AD. Moreover, protective treatment strategies like aerobic exercise, cognitive training, and drug therapy (acethylcholine esterase inhibitors and/or memantine) may be employed. However, due to high costs and potential side effects, drug therapy should only be offered to MCI patients at risk to develop dementia, e.g. MCI of AD-type.
Last but not least, recently we have applied our method of A#-SDS-PAGE/immunoblot to reveal a complex A# peptides' pattern in blood plasma (Lewczuk et al., 2004b).
To achieve the goals we planned in NGFN2, a close cooperation has been undertaken with Brain-Net in Munich (Prof. Dr. H. Kretzschmar), and recently a detailed planning has been discussed regarding harvesting of the plaques from human post mortem brain tissue for amyloid # peptides analysis. In parallel, to adjust two-dimensional differential gel electrophoresis (DIGETM) for cerebrospinal fluid analysis, we started optimization experiments with immunodepletion of high-abundance proteins (albumin, immunoglobulin G). We have also launched the cooperation with University of Kassel (Prof. Dr. F. Herberg) aiming at the detection of A# peptides in human body fluids with a Biacore system. In cooperation with the group of Prof. Dr. J. Beckers (GSF, Munich), we are currently optimizing the protocols for expression analysis in human full blood.
Outlook
Our tasks and goals in the NGFN2 Project are:
- providing state-of-the-art clinical dementia phenotyping:
# as a BMBF reference center for clinical diagnosis of dementias (Modul E1, Competence Network Dementia) and a center of the Creutzfeldt-Jakob Network for rapid-progressive dementias (CJD network) we will provide the state-of-the-art clinical phenotyping for large cohorts of patients with mild cognitive impairment, dementias, and non-demented disease controls. - providing optimized preanalytical sample handling and a large-scale sample bank
# our group will provide the consortium with a large-scale CSF/plasma sample bank and optimized standard operation procedures (SOPs) for multi-center pre-analytical sample handling of human body fluids as a prerequisite for successful neuroproteomic dementia diagnostics. - establishing a close cooperation with the BMBF-funded German Brain-Net (Professor Dr. H. Kretzschmar, Munich) to validate novel neuroproteomic dementia markers by autopsy-controlled dementia diagnosis.
- searching for novel neuroproteomic dementia markers in CSF for the improved differential diagnosis of early stages of Alzheimer's disease, Lewy-body dementia, and vascular dementias.
- identifying novel neuroproteomic dementia markers in the CSF, which allow preclinical neurochemical dementia diagnosis of incipient dementia (AD&LBD) at the stage of MCI.
- identifying brain-derived proteins in blood plasma, which allow to monitor the progress of neurodegeneration and the efficacy of treatment intervention for the group of primary progressive dementias.
# pairs of CSF and plasma samples from patients with rapid-progressive dementias will be studied after removal of high-abundance proteins. For identified proteins/peptides we will create attomolar-sensitive assays to investigate if these parameters can also be used to monitor primary progressive dementias with less acute neurodegeneration (e.g. AD, LBD, VD). - identifying brain-derived proteins in blood (plasma), which allow the diagnosis of early AD and LBD.
# in this part of the project, pooled plasma samples of patients with early dementia and the typical CSF neuroproteomic dementia marker panel will be investigated. Following removal of high-abundance proteins promising subfractions of proteins will be enriched for further expression pattern profiling by DIGETM and SELDI-TOF-MS.
# in parallel, we will develop fluorescence-based proteomic methods, which allow complex pattern expression analysis of highly diluted plasma proteins (low attomolar sensitivity). - developing HTS-capable dementia biochips for the multiparameter assessment of several neuroproteomic dementia marker, each reflecting a pathophysiological key feature of a distinct dementia and possibly different states of dementia severity.
# this will be realized by multiplexing technology (LuminexTM platform) and a SELDI-based template. - identifying of distinct neuroproteomic phenotypes for a given dementia (AD, LBD, and VD each).
# We will investigate if these neuroproteomic phenotypes are correlated to new clinical dementia subtypes. Moreover, using proteomic/genomic backward strategy, this approach will be used to identify novel disease-related genes.
Lit.: 1. Lewczuk P., Esselmann H., Meyer M., Wollscheid V., Neumann M., Otto M., Maler J.M., Rüther E., Kornhuber J. & Wiltfang J. (2003) The amyloid-b (Ab) peptide pattern in cerebrospinal fluid in Alzheimer's disease: evidence of a novel carboxyterminally elongated Ab peptide. Rapid Commun Mass Spectrom 17, 1291-6. 2. Lewczuk P., Esselmann H., Bibl M., Beck G., Maler J.M., Otto M., Kornhuber J. & Wiltfang J. (2004a) Tau Protein Phosphorylated at Threonine 181 in CSF as a Neurochemical Biomarker in Alzheimer's Disease: Original Data and Review of the Literature. J Mol Neurosci 23, 115-22. 3. Lewczuk P., Esselmann H., Bibl M., Paul S., Svitek J., Miertschischk J., Meyrer R., Smirnov A., Maler J.M., Klein C., Otto M., Bleich S., Sperling W., Kornhuber J., Ruther E. & Wiltfang J. (2004b) Electrophoretic separation of amyloid beta peptides in plasma. Electrophoresis 25, 3336-3343. 4. Lewczuk P., Esselmann H., Groemer T.W., Bibl M., Maler J.M., Steinacker P., Otto M., Kornhuber J. & Wiltfang J. (2004c) Amyloid b peptides in cerebrospinal fluid as profiled with surface enhanced laser desorption/ionization time-of-flight mass spectrometry: evidence of novel biomarkers in Alzheimer's disease. Biol Psychiatry 55, 524-30. 5. Lewczuk P., Esselmann H., Otto M., Maler J.M., Henkel A.W., Henkel M.K., Eikenberg O., Antz C., Krause W.R., Reulbach U., Kornhuber J. & Wiltfang J. (2004d) Neurochemical diagnosis of Alzheimer's dementia by CSF Ab42, Ab42/Ab40 ratio and total tau. Neurobiol Aging 25, 273-81. 6. Wiltfang J., Esselmann H., Cupers P., Neumann M., Kretzschmar H., Beyermann M., Schleuder D., Jahn H., Rüther E., Kornhuber J., Annaert W., De Strooper B. & Saftig P. (2001) Elevation of b-amyloid peptide 2-42 in sporadic and familial Alzheimer's disease and its generation in PS1 knockout cells. J Biol Chem 276, 42645-57. 7. Wiltfang J., Esselmann H., Bibl M., Smirnov A., Otto M., Paul S., Schmidt B., Klafki H.W., Maler M., Dyrks T., Bienert M., Beyermann M., Rüther E. & Kornhuber J. (2002) Highly conserved and disease-specific patterns of carboxyterminally truncated Ab peptides 1-37/38/39 in addition to 1-40/42 in Alzheimer's disease and in patients with chronic neuroinflammation. J Neurochem 81, 481-96. 8. Wiltfang J., Esselmann H., Smirnov A., Bibl M., Cepek L., Steinacker P., Mollenhauer B., Buerger K., Hampel H., Paul S., Neumann M., Maler M., Zerr I., Kornhuber J., Kretzschmar H.A., Poser S. & Otto M. (2003) b-amyloid peptides in cerebrospinal fluid of patients with Creutzfeldt-Jakob disease. Ann Neurol 54, 263-7. 9. Wiltfang J., Lewczuk P., Riederer P., Grünblatt E., Hock C., Scheltens P., Hampel H., Vanderstichele H., Iqbal K., Galasko D., Lannfelt L., Otto M., Esselmann H., Henkel A.W., Kornhuber J. & Blennow K. (2005) Consensus Paper of the WFSBP Task Force on Biological Markers of Dementia: The role of CSF and blood analysis in the early and differential diagnosis of dementia. World J Biol Psychiatry 6, 69-84.
