Peter Brechlin

Beta-Amlyoid 1-42 and Tau-Protein in Cerebrospinal Fluid of Patients with Parkinson's Disease Dementia

Measurement of τ-protein and β-amyloid1–42 (Aβ42) in cerebrospinal fluid (CSF) has gained increasing acceptance in the differential diagnosis of Alzheimer’s disease. We investigated CSF τ-protein and Aβ42 concentrations in 73 patients with advanced idiopathic Parkinson’s disease with dementia (PDD) and 23 patients with idiopathic Parkinson’s disease without dementia (PD) and in a comparison group of 41 non-demented neurological patients (CG) using commercially available enzyme-linked-immunoabsorbant-assay (ELISA). τ-Protein levels were statistically significantly higher and Aβ42 lower in the PDD patients compared to PD patients and the CG. This observation was most marked (p < 0.05) in a subgroup of patients with PDD carrying the apolipoprotein genotype Ε3/Ε3. The distribution of the apolipoprotein genotypes in PDD and PD patients was similar to that of the CG. Although a significant difference in τ-protein values was observed between PDD and CG, no diagnostic cut-off value was established. These findings suggest that such protein CSF changes may help to support the clinical diagnosis of cognitive decline in PD and that there may be apolipoprotein-E-isoform-specific differences in CSF protein regulation in advanced PDD.

Cerebrospinal fluid‐optimized two‐dimensional difference gel electrophoresis (2‐D DIGE) facilitates the differential diagnosis of Creutzfeldt–Jakob disease

So far only the detection of 14‐3‐3 proteins in cerebrospinal fluid (CSF) is included in the diagnostic criteria for sporadic Creutzfeldt–Jakob disease (sCJD). However, this assay cannot be used for screening because of the high rate of false positive results in sCJD, and often negative results in variant CJD. To facilitate the differential diagnosis of CJD, we applied 2‐D differential gel‐electrophoresis (2‐D DIGE) as a quantitative proteomic screening system for CSF proteins. We compared 36 patients suffering from sCJD with 30 patients suffering from other neurodegenerative diseases. Sample preparation was optimized in consideration of the fact that CSF is composed of blood‐ and brain‐derived proteins, and an improved 2‐D DIGE protocol was established. Using this method in combination with protein identification by MALDI‐TOF‐MS, several known surrogate markers of sCJD like 14‐3‐3 protein, neuron‐specific enolase, and lactate dehydrogenase were readily identified. Moreover, a not yet identified protein with an approximate molecular mass of 85 kDa was found as marker for sCJD with high diagnostic specificity and sensitivity. We conclude that our proteomic approach is useful to differentiate CJD from other neurodegenerative diseases and expect that CSF‐optimized 2‐D DIGE will find broad application in the search for other brain derived proteins in CSF.

Heart fatty acid binding protein as a potential diagnostic marker for neurodegenerative diseases

The diagnosis of neurodegenerative diseases with dementias requires several different test approaches and often remains uncertain. Using a proteomic approach it was shown in nine patients that heart fatty acid binding protein (H-FABP) might be a biomarker for Creutzfeldt-Jakob disease (CJD). The aim of our study was to evaluate whether H-FABP is a biomarker for the differential diagnosis of dementias. Therefore we measured H-FABP in cerebrospinal fluid (CSF) and serum of patients having CJD, dementia with Lewy-bodies (DLB), Alzheimers disease (AD) and in non-demented control (NDC) patients. H-FABP levels in CSF and serum of CJD patients are increased compared to non-demented controls. Levels of H-FABP were significantly higher in CJD patients compared to AD and DLB in CSF. However, discrimination between CJD and AD was not possible in serum. Interestingly, highest levels of H-FABP were found in serum of DLB patients. Our results suggest that H-FABP might be a useful biomarker for the differentiation between the dementias examined if levels in CSF and serum are determined in parallel.

CSF diagnosis of Alzheimer’s disease and dementia with Lewy bodies

Summary.Differential diagnosis of Alzheimer’s disease (AD) and dementia with Lewy bodies (DLB) is often crucial. CSF Tau protein and Amyloid-beta (Aβ) peptides have shown diagnostic value for the diagnosis of AD, but discrimination from DLB was poor.Herein, we investigate CSF of 18 patients with probable AD, 25 with probable DLB and 14 non-demented disease controls (NDC) by Aβ-SDS-PAGE/immunoblot and commercially available ELISAs for Aβ1-42 and tau. CSF Aβ peptide patterns and tau exhibited disease specific alterations among AD and DLB. The ratio of Aβ1-42 to Aβ1-38 and Aβ1-42 to Aβ1-37, respectively, in combination with absolute tau, yielded a sensitivity and specificity of 100 and 92%, respectively. We conclude that CSF Aβ peptide patterns and tau levels reflect disease-specific pathophysiological pathways of these dementias as distinct neurochemical phenotypes. Combined evaluation of these biomarkers provides a reasonable accuracy for differential diagnosis of AD and DLB.

Unchanged Survival Rates of 14-3-3γ Knockout Mice after Inoculation with Pathological Prion Protein

ABSTRACT The diagnosis of sporadic Creutzfeldt-Jakob disease (CJD) is based on typical clinical findings and is supported by a positive 14-3-3 Western blot of cerebrospinal fluid. However, it is not clear whether 14-3-3 indicates general neuronal damage or is of pathophysiological relevance in CJD. The fact that the 14-3-3 isoform spectrum in cerebrospinal fluid does not correspond to that found in the brain points to a regulated process. To investigate a possible role of 14-3-3 proteins in transmissible spongiform diseases, we generated a 14-3-3γ-deficient mutant mouse line by using a classical knockout strategy. The anatomy and cage behavior of the mutant mice were normal. Western blot analyses of brain homogenates revealed no changes in the protein expression of other 14-3-3 isoforms (ε, β, ζ, and η). Proteomic analyses of mouse brains by two-dimensional differential gel electrophoresis showed that several proteins, including growth hormone, 1-Cys peroxiredoxin, CCT-zeta, glucose-6-phosphate isomerase, GRP170 precursor, and α-SNAP, were differentially expressed. Mutant and wild-type mice were inoculated either intracerebrally or intraperitoneally with the Rocky Mountain Laboratory strain of scrapie, but no differences were detected in the postinoculation survival rates. These results indicate that 14-3-3γ is unlikely to play a causal role in CJD and related diseases.

Serum heart-type fatty acid-binding protein and cerebrospinal fluid tau: Marker candidates for dementia with Lewy bodies

Background: The measurement of biomarkers in cerebrospinal fluid (CSF) has gained increasing acceptance in establishing the diagnosis of some neurodegenerative diseases. Heart-type fatty acid-binding protein (H-FABP) was recently discovered in CSF and serum of patients with neurodegenerative diseases. Objective: We investigated H-FABP in CSF and serum alone and in combination with CSF tau protein to evaluate these as potential biomarkers for the differentiation between dementia with Lewy bodies (DLB) and Alzheimer’s disease (AD). Methods: We established H-FABP and tau protein values in a set of 144 persons with DLB (n = 33), Parkinson disease with dementia (PDD; n = 25), AD (n = 35) and nondemented neurological controls (NNC; n = 51). Additionally, serum H-FABP levels were analyzed in idiopathic Parkinson disease patients without evidence of cognitive decline (n = 45) using commercially available enzyme-linked immunosorbent assays. We calculated absolute values of H-FABP and tau protein in CSF and serum and established relative ratios between the two to obtain the best possible match for the clinical working diagnosis. Results: Serum H-FABP levels were elevated in DLB and PDD patients compared with NNC and AD subjects. To better discriminate between DLB and AD, we calculated the ratio of serum H-FABP to CSF tau protein levels. At the arbitrary chosen cutoff ratio ≧8 this quotient reached a sensitivity of 91% and a specificity of 66%. Conclusion: Our results suggest that the measurement of CSF tau protein, together with H-FABP quantification in serum and CSF, and the ratio of serum H-FABP to CSF tau protein represent marker candidates for the differentiation between AD and DLB.

Proteome profiling in murine models of multiple sclerosis: identification of stage specific markers and culprits for tissue damage.

The identification of new biomarkers is of high interest for the prediction of the disease course and also for the identification of pathomechanisms in multiple sclerosis (MS). To specify markers of the chronic disease phase, we performed proteome profiling during the later phase of myelin oligodendrocyte glycoprotein induced experimental autoimmune encephalomyelitis (MOG-EAE, day 35 after immunization) as a model disease mimicking many aspects of secondary progressive MS. In comparison to healthy controls, high resolution 2 dimensional gel electrophoresis revealed a number of regulated proteins, among them glial fibrilary acidic protein (GFAP). Phase specific up-regulation of GFAP in chronic EAE was confirmed by western blotting and immunohistochemistry. Protein levels of GFAP were also increased in the cerebrospinal fluid of MS patients with specificity for the secondary progressive disease phase. In a next step, proteome profiling of an EAE model with enhanced degenerative mechanisms revealed regulation of alpha-internexin, syntaxin binding protein 1, annexin V and glutamate decarboxylase in the ciliary neurotrophic factor (CNTF) knockout mouse. The identification of these proteins implicate an increased apoptosis and enhanced axonal disintegration and correlate well the described pattern of tissue injury in CNTF −/− mice which involve oligodendrocyte (OL) apoptosis and axonal injury. In summary, our findings underscore the value of proteome analyses as screening method for stage specific biomarkers and for the identification of new culprits for tissue damage in chronic autoimmune demyelination.

Hamster pancreatic beta cell lines with altered sensitivity towards apoptotic signalling by phosphatase inhibitors.

Specific inhibitors of serine/threonine phosphatases like okadaic acid can induce apoptotic cell death in the pancreatic beta cell line HIT. Cultivation in stepwise increased concentrations of okadaic acid enabled the isolation of HIT100R cells which proliferate at 100 nM okadaic acid (8–10 times the initially lethal concentration). These two cell lines were used to characterize the events triggered by okadaic acid that led to apoptosis. Biochemical markers, e.g. cytochrome c release from mitochondria and increase of caspase‐3‐like activity, revealed that induction of apoptosis by 100 nM okadaic acid in parental HIT cells started with the release of cytochrome c. In HIT100R cells 500 nM okadaic acid were necessary to induce alterations comparable to those observed with 100 nM okadaic acid in non‐resistant HIT cells. In contrast to okadaic acid, the potency of the structurally different phosphatase inhibitor cantharidic acid to induce cytochrome c release, increase of caspase‐3‐like activity and DNA fragmentation was comparable in HIT and HIT100R cells. Thus, no cross‐resistance between these phosphatase inhibitors seemed to exist. Phosphatase activity in extracts from HIT and HIT100R cells did not differ in its total amount or in its sensitivity for okadaic acid. Since higher concentrations of okadaic acid were needed to induce apoptosis in HIT100R cells, a compromised intracellular accumulation of the toxin appeared likely. Functional and structural analysis revealed that this was achieved by the development of the multidrug resistance phenotype in HIT100R cells. The underlying mechanism appeared to be the enhanced expression of the pgp1 but not the pgp2 gene.

Proteomic analysis of the cerebrospinal fluid of patients with Creutzfeldt-Jakob disease.

So far, only the detection of 14-3-3 proteins in cerebrospinal fluid (CSF) has been accepted as diagnostic criterion for Creutzfeldt-Jakob disease (CJD). However, this assay cannot be used for screening because of the high rate of false-positive results, whereas patients with variant CJD are often negative for 14-3-3 proteins. The aim of this study was to compare the spot patterns of CSF by 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) to search for a CJD-specific spot pattern. We analyzed the CSF of 28 patients [11 CJD, 9 Alzheimer’s disease (AD), 8 nondemented controls (NDC)] employing 2D-PAGE which was optimized for minimal volumes of CSF (0.1 ml; 7-cm strips). All samples were run at least three times, gels were silver stained and analyzed by an analysis software and manually revised. We could consistently match 268 spots which were then compared between all groups. By the use of 5 spots, we were able to differentiate CJD from AD or NDC with a sensitivity of 100%. CJD could also be distinguished from both groups by using a heuristic clustering algorithm of 2 spots. We conclude that this proteomic approach can differentiate CJD from other diseases and may serve as a model for other neurodegenerative diseases.

Upregulation of CRABP1 in human neuroblastoma cells overproducing the Alzheimer-typical Aβ42 reduces their differentiation potential

BackgroundAlzheimers disease (AD) is characterized by neurodegeneration and changes in cellular processes, including neurogenesis. Proteolytic processing of the amyloid precursor protein (APP) plays a central role in AD. Owing to varying APP processing, several β-amyloid peptides (Aβ) are generated. In contrast to the form with 40 amino acids (Aβ40), the variant with 42 amino acids (Aβ42) is thought to be the pathogenic form triggering the pathological cascade in AD. While total-Aβ effects have been studied extensively, little is known about specific genome-wide effects triggered by Aβ42 or Aβ40 derived from their direct precursor C99.MethodsA combined transcriptomics/proteomics analysis was performed to measure the effects of intracellularly generated Aβ peptides in human neuroblastoma cells. Data was validated by real-time polymerase chain reaction (real-time PCR) and a functional validation was carried out using RNA interference.ResultsHere we studied the transcriptomic and proteomic responses to increased or decreased Aβ42 and Aβ40 levels generated in human neuroblastoma cells. Genome-wide expression profiles (Affymetrix) and proteomic approaches were combined to analyze the cellular response to the changed Aβ42- and Aβ40-levels. The cells responded to this challenge with significant changes in their expression pattern. We identified several dysregulated genes and proteins, but only the cellular retinoic acid binding protein 1 (CRABP1) was up-regulated exclusively in cells expressing an increased Aβ42/Aβ40 ratio. This consequently reduced all-trans retinoic acid (RA)-induced differentiation, validated by CRABP1 knock down, which led to recovery of the cellular response to RA treatment and cellular sprouting under physiological RA concentrations. Importantly, this effect was specific to the AD typical increase in the Aβ42/Aβ40 ratio, whereas a decreased ratio did not result in up-regulation of CRABP1.ConclusionWe conclude that increasing the Aβ42/Aβ40 ratio up-regulates CRABP1, which in turn reduces the differentiation potential of the human neuroblastoma cell line SH-SY5Y, but increases cell proliferation. This work might contribute to the better understanding of AD neurogenesis, currently a controversial topic.