Saskia M. van der Vies

Amyloid Beta Induces Oxidative Stress-Mediated Blood-Brain Barrier Changes in Capillary Amyloid Angiopathy

Cerebral amyloid angiopathy (CAA) is frequently observed in Alzheimers disease (AD) and is characterized by deposition of amyloid beta (Aβ) in leptomeningeal and cortical brain vasculature. In 40% of AD cases, Aβ mainly accumulates in cortical capillaries, a phenomenon referred to as capillary CAA (capCAA). The aim of this study was to investigate blood-brain barrier (BBB) alterations in CAA-affected capillaries with the emphasis on tight junction (TJ) changes. First, capCAA brain tissue was analyzed for the distribution of TJs. Here, we show for the first time a dramatic loss of occludin, claudin-5, and ZO-1 in Aβ-laden capillaries surrounded by NADPH oxidase-2 (NOX-2)-positive activated microglia. Importantly, we observed abundant vascular expression of the Aβ transporter receptor for advanced glycation endproducts (RAGE). To unravel the underlying mechanism, a human brain endothelial cell line was stimulated with Aβ1-42 to analyze the effects of Aβ. We observed a dose-dependent cytotoxicity and increased ROS generation, which interestingly was reversed by administration of exogenous antioxidants, NOX-2 inhibitors, and by blocking RAGE. Taken together, our data evidently show that Aβ is toxic to brain endothelial cells via binding to RAGE and induction of ROS production, which ultimately leads to disruption of TJs and loss of BBB integrity.

Monitoring macromolecular complexes involved in the chaperonin-assisted protein folding cycle by mass spectrometry

We have used native mass spectrometry to analyze macromolecular complexes involved in the chaperonin-assisted refolding of gp23, the major capsid protein of bacteriophage T4. Adapting the instrumental methods allowed us to monitor all intermediate complexes involved in the chaperonin folding cycle. We found that GroEL can bind up to two unfolded gp23 substrate molecules. Notably, when GroEL is in complex with the cochaperonin gp31, it binds exclusively one gp23. We also demonstrated that the folding and assembly of gp23 into 336-kDa hexamers by GroEL-gp31 can be monitored directly by electrospray ionization mass spectrometry (ESI-MS). These data reinforce the great potential of ESI-MS as a technique to investigate structure-function relationships of protein assemblies in general and the chaperonin-protein folding machinery in particular. A major advantage of native mass spectrometry is that, given sufficient resolution, it allows the analysis at the picomole level of sensitivity of heterogeneous protein complexes with molecular masses up to several million daltons.

Cdc37p Is Required for Stress-Induced High-Osmolarity Glycerol and Protein Kinase C Mitogen-Activated Protein Kinase Pathway Functionality by Interaction with Hog1p and Slt2p (Mpk1p)

ABSTRACT The yeast Saccharomyces cerevisiae utilizes rapidly responding mitogen-activated protein kinase (MAPK) signaling cascades to adapt efficiently to a changing environment. Here we report that phosphorylation of Cdc37p, an Hsp90 cochaperone, by casein kinase 2 controls the functionality of two MAPK cascades in yeast. These pathways, the high-osmolarity glycerol (HOG) pathway and the cell integrity (protein kinase C) MAPK pathway, mediate adaptive responses to high osmotic and cell wall stresses, respectively. Mutation of the phosphorylation site Ser14 in Cdc37p renders cells sensitive to osmotic stress and cell wall perturbation by calcofluor white. We found that levels of the MAPKs Hog1p and Slt2p (Mpk1p) in cells are reduced in a cdc37-S14A mutant, and consequently downstream responses mediated by Hog1p and Slt2p are compromised. Furthermore, we present evidence that Hog1p and Slt2p both interact in a complex with Cdc37p in vivo, something that has not been reported previously. The interaction of Hsp90, Slt2p, and Hog1p with Cdc37p depends on the phosphorylation status of Cdc37p. In fact, our biochemical data show that the osmosensitive phenotype of the cdc37-S14A mutant is due to the loss of the interaction between Cdc37p, Hog1p, and Hsp90. Likewise, during cell wall stress, the interaction of Slt2p with Cdc37p and Hsp90 is crucial for Slt2p-dependent downstream responses, such as the activation of the transcription factor Rlm1p. Interestingly, phosphorylated Slt2p, but not phosphorylated Hog1p, has an increased affinity for Cdc37p. Together these observations suggest that Cdc37p acts as a regulator of MAPK signaling.

Thermal activation of the co-chaperonins GroES and gp31 probed by mass spectrometry

Many biological active proteins are assembled in protein complexes. Understanding the (dis)assembly of such complexes is therefore of major interest. Here we use mass spectrometry to monitor the disassembly induced by thermal activation of the heptameric co-chaperonins GroES and gp31. We use native electrospray ionization mass spectrometry (ESI-MS) on a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer to monitor the stoichiometry of the chaperonins. A thermally controlled electrospray setup was employed to analyze conformational and stoichiometric changes of the chaperonins at varying temperature. The native ESI-MS data agreed well with data obtained from fluorescence spectroscopy as the measured thermal dissociation temperatures of the complexes were in good agreement. Furthermore, we observed that thermal denaturing of GroES and gp31 proceeds via intermediate steps of all oligomeric forms, with no evidence of a transiently stable unfolded heptamer. We also evaluated the thermal dissociation of the chaperonins in the gas phase using infrared multiphoton dissociation (IRMPD) for thermal activation. Using gas-phase activation the smaller (2-4) oligomers were not detected, only down to the pentamer, whereafter the complex seemed to dissociate completely. These results demonstrate clearly that conformational changes of GroES and gp31 due to heating in solution and in the gas phase are significantly different.

Protein Kinase Activity Decreases with Higher Braak Stages of Alzheimer's Disease Pathology.

Alzheimer’s disease (AD) is characterized by a long pre-clinical phase (20–30 years), during which significant brain pathology manifests itself. Disease mechanisms associated with pathological hallmarks remain elusive. Most processes associated with AD pathogenesis, such as inflammation, synaptic dysfunction, and hyper-phosphorylation of tau are dependent on protein kinase activity. The objective of this study was to determine the involvement of protein kinases in AD pathogenesis. Protein kinase activity was determined in postmortem hippocampal brain tissue of 60 patients at various stages of AD and 40 non-demented controls (Braak stages 0-VI) using a peptide-based microarray platform. We observed an overall decrease of protein kinase activity that correlated with disease progression. The phosphorylation of 96.7% of the serine/threonine peptides and 37.5% of the tyrosine peptides on the microarray decreased significantly with increased Braak stage (p-value <0.01). Decreased activity was evident at pre-clinical stages of AD pathology (Braak I-II). Increased phosphorylation was not observed for any peptide. STRING analysis in combination with pathway analysis and identification of kinases responsible for peptide phosphorylation showed the interactions between well-known proteins in AD pathology, including the Ephrin-receptor A1 (EphA1), a risk gene for AD, and sarcoma tyrosine kinase (Src), which is involved in memory formation. Additionally, kinases that have not previously been associated with AD were identified, e.g., protein tyrosine kinase 6 (PTK6/BRK), feline sarcoma oncogene kinase (FES), and fyn-associated tyrosine kinase (FRK). The identified protein kinases are new biomarkers and potential drug targets for early (pre-clinical) intervention.

Dissociation Kinetics of the GroEL−gp31 Chaperonin Complex Studied with Förster Resonance Energy Transfer

Propagation of bacteriophage T4 in its host Escherichia coli involves the folding of the major capsid protein gp23, which is facilitated by a hybrid chaperone complex consisting of the bacterial chaperonin GroEL and the phage-encoded co-chaperonin, gp31. It has been well established that the GroEL-gp31 complex is capable of folding gp23 whereas the homologous GroEL-GroES complex cannot perform this function. To assess whether this is a consequence of differences in the interactions of the proteins within the chaperonin complex, we have investigated the dissociation kinetics of GroEL-gp31 and GroEL-GroES complexes using Forster resonance energy transfer. Here we report that the dissociation of gp31 from GroEL is slightly faster than that of GroES from GroEL and is further accelerated by the binding of gp23. In contrast to what had been observed previously, we found that gp23 is able to interact with the GroEL-GroES complex, which might explain how bacteriophage T4 redirects the folding machinery of Escherichia coli during morphogenesis.

PROTEIN KINASE ACTIVITY DECREASES WITH BRAAK STAGE IN HIPPOCAMPAL POSTMORTEM BRAIN TISSUE AS REVEALED BY USING A PEPTIDE-BASED MICROARRAY PLATFORM

tissue. Methods: Frozen brain tissue from healthy controls (HCs) and neuropathologically confirmed cases of AD (CERAD criteria) were obtained from the Douglas-Bell Canada Brain Bank (Douglas Mental Health University Institute, Montreal, Canada). In total, 26 samples from the hippocampus (HIPP) (HC1⁄419, AD1⁄47), 18 from the prefrontal cortex (PFC) (HC1⁄414, AD1⁄44), 20 from the posterior cingulate cortex (PCC) (HC 1⁄4 11, AD 1⁄4 9) and 22 from the inferior parietal cortex (IPC) (HC1⁄415, A1⁄47)were included.[18 F]T807 autoradiography (specific activity, > 71,000 mCi/micromol) was carried out following 2.5-hour incubation of frozen tissue slices (20 mm thick). Imaging plates were scanned using BAS5000 Phosphoimager (Fuji-Film), with total binding obtained for all regions of interest. Results: No significant differences between control and AD groups were noted in terms of sex distribution, age and postmortem delay (P < 0.05). Binding of [18 F]T807 was significantly higher in AD tissue, as compared to CN [HIPP (P < 0.01), PFC (P < 0.01), IPC (P < 0.05), PCC (P < 0.05], with the magnitude of difference highest in the IPC. Conclusions: [18 F]T807 successfully differentiated CN and AD post-mortem tissue, with binding substantially higher in AD, particularly in the IPC. Our findings support the current perspective on [18 F]T807 as a promising tau molecular imaging agent.

A RE-EVALUATION OF EARLY ALZHEIMER'S DISEASE BIOMARKERS ACCOUNTING FOR INACCURACY OF THE CLINICAL DIAGNOSIS

the timing of symptom onset in ADAD. Methods: We have collected data on ages of symptom onset from 390 ADAD pedigrees, compiled from 138 peer-reviewed publications, the Dominantly Inherited Alzheimer Network (DIAN) database, and two large kindreds of Colombian (PSEN1 E280A) and Volga German (PSEN2 N141I) ancestry. Our combined dataset includes 3282 individuals, of whom 1314 were affected by ADAD with known age of symptom onset. We assessed the relative contributions of several factors in influencing age of onset, including the affected gene and amino acid location of the ADAD mutation, parental age of onset, age of onset by mutation type and family, and APOE genotype and gender. We additionally performed a confirmatory survival analysis using data from 183 ADAD mutation carriers followed longitudinally in the DIAN study. Results: We report summary statistics on age of onset and disease course for 176 ADAD mutations, and discover strong and highly significant (p 0.38) correlations between individual age of symptom onset and predicted values based on parental age of onset and mean ages of onset by mutation type and family, which persist after controlling for APOE genotype and gender. Conclusions: Significant proportions of the observed variance in age of symptom onset in ADAD can be explained by the gene and amino acid location of ADAD mutations, providing empirical support for use of these data to estimate onset in clinical research.

Protein kinase activity profiling on cerebrospinal fluid to find diagnostic biomarkers for Alzheimer's disease

Background: Humoral immunity related to neuronand disease-specific cytoskeletal proteins in patients with Alzheimer’s disease (AD) is largely unknown. Methods: Antibodies against three targets [tau, light (NFL) and heavy (NFH) neurofilaments] were measured using ELISA in 80 paired serum and CSF samples from 25 patients with AD fulfilling NINCDSADRDA criteria, 13 patients with other dementias (OD), 25 cognitively normal elderly controls (NC), 17 patients with neuroinflammatory diseases (IC). We estimated intrathecal synthesis of all antibodies according to formula (CSF anti-neurocytoskeletal IgG /serum anti-neurocytoskeletal IgG)/ (CSF total IgG /serum total IgG).Results:TheAD patients had significantly higher intrathecal anti-tau antibodies than in the OD group (p 1⁄4 0.005) or the IC group (P 1⁄4 0.0003) and higher intrathecal anti-NFH antibodies than in the OD group (P 1⁄4 0.046), the NC group (p 1⁄4 0.04) or the IC group (P 1⁄4 0.0007), but not intrathecally synthesized anti-NFL antibodies. All three anti-neurocytoskeletal antibodies in serum or CSF did not differ among four groups with the exceptions for the IC group having them elevated in CSF. Conclusions: These new findings suggest some hints of specific alterations in humoral anti-neurocytoskeletal immunity and selectivity in AD. It may have diagnostic and immunotherapeutic implications.