Background: Soluble A␤ oligomers (A␤Os) have been increasingly proposed as the cause of synapse failure and cognitive dysfunction in Alzheimer disease. Results: Sublethal A␤O concentrations induce changes in gene expression in adult human brain slices. Conclusion: A␤Os impact transcription in important neuronal pathways preceding neurodegeneration. Significance: Results establish early mechanisms involved in A␤O-triggered neuronal dysfunction in a novel human-derived experimental model.

No abstract

Prion diseases are fatal neurodegenerative disorders associated with conformational conversion of the cellular prion protein, PrP C , into a misfolded, protease-resistant form, PrP Sc . Here we show, for the first time, the oligomerization and fibrillization of the C-terminal domain of murine PrP, mPrP-(121-231), which lacks the entire unstructured N-terminal domain of the protein. In particular, the construct we used lacks amino acid residues 106 -120 from the so-called amyloidogenic core of PrP (residues 106 -126). Amyloid formation was accompanied by acquisition of resistance to proteinase K digestion. Aggregation of mPrP-(121-231) was investigated using a combination of biophysical and biochemical techniques at pH 4.0, 5.5, and 7.0 and at 37 and 65°C. Under partially denaturing conditions (65°C), aggregates of different morphologies ranging from soluble oligomers to mature amyloid fibrils of mPrP-(121-231) were formed. Transmission electron microscopy analysis showed that roughly spherical aggregates were readily formed when the protein was incubated at pH 5.5 and 65°C for 1 h, whereas prolonged incubation led to the formation of mature amyloid fibrils. Samples incubated at 65°C at pH 4.0 or 7.0 presented an initial mixture of oligomers and protofibrils or fibrils. Electrophoretic analysis of samples incubated at 65°C revealed formation of sodium dodecyl sulfate-resistant oligomers (dimers, trimers, and tetramers) and higher molecular weight aggregates of mPrP-(121-231). These results demonstrate that formation of an amyloid form with physical properties of PrP Sc can be achieved in the absence of the flexible N-terminal domain and, in particular, of residues 106 -120 of PrP and does not require other cellular factors or a PrP Sc template.The conformational conversion of the normal cellular isoform of the prion protein, PrP C , 4 into an abnormal pathological isoform, PrP Sc , underlies a group of fatal neurodegenerative disorders known as transmissible spongiform encephalopathies or prion diseases, which includes Creutzfeldt-Jakob disease in humans, scrapie in sheep and goats, and bovine spongiform encephalopathy in cattle (1). Neuropathologically, prion diseases are characterized by neuronal loss and astrogliosis and often by spongiform degeneration of the brain and deposition of amyloid plaques (1). The unique feature of these diseases is that, in addition to sporadic and inherited forms, they may be acquired by transmission of an infectious agent. The "proteinonly" hypothesis of prion propagation postulates that the abnormal isoform, PrP Sc , acts as a transmissible agent of the disease and self-propagates its pathological conformation using PrP C as a substrate (1).PrP Sc is defined as an aggregated form of PrP that is largely resistant to proteinase K (PK) digestion under conditions in which PrP C and most other proteins are readily degraded (2). In addition to their different stabilities to proteolytic degradation, the secondary, tertiary, and quaternary structures of PrP C and PrP Sc also differ (3-7...

ATPdiphosphohydrolases (ATPDases) are ubiquitous enzymes capable of hydrolyzing nucleoside di- and triphosphates. Although a number of possible physiological roles have been proposed for ATPDases, detailed studies on structure-function relationships have generally been hampered by the lack of specific inhibitors of these enzymes. We have previously characterized a Ca2+-activated ATPDase on the external surface of the tegument of Schistosoma mansoni, the etiologic agent of human schistosomiasis. In the present work, we have examined the effects of thapsigargin, a sesquiterpene lactone known as a high affinity inhibitor of sarco-endoplasmic reticulum calcium transport (SERCA) ATPase, on ATPDase activity. Whereas other lactones tested had little or no inhibitory action, thapsigargin inhibited ATP hydrolysis by the ATPDase (K(i) approximately 20 microM). Interestingly, hydrolysis of ADP was not inhibited by thapsigargin. The lack of inhibition of ATPase activity by orthovanadate, a specific inhibitor of P-type ATPases, and the inhibition of the Mg2+-stimulated ATP hydrolysis by thapsigargin ruled out the possibility that the observed inhibition of the ATPDase by thapsigargin could be due to the presence of contaminating SERCA ATPases in our preparation. Kinetic analysis indicated that a single active site in the ATPDase is responsible for hydrolysis of both ATP and ADP. Thapsigargin caused changes in both Vmax and Km for ATP, indicating a mixed type of inhibition. Inhibition by thapsigargin was little or not affected by changes in free Ca2+ or Mg2+ concentrations. These results suggest that interaction of thapsigargin with the S. mansoni ATPDase prevents binding of ATP or its hydrolysis at the active site, while ADP can still undergo catalysis.

The structures and stabilities of recombinant chicken muscle troponin I (TnI) and T (TnT) were investigated by a combination of bis-ANS binding and equilibrium unfolding studies. Unlike most folded proteins, isolated TnI and TnT bind the hydrophobic fluorescent probe bis-ANS, indicating the existence of solvent-exposed hydrophobic domains in their structures. Bis-ANS binding to binary or ternary mixtures of TnI, TnT and troponin C (TnC) in solution is significantly lower than binding to the isolated subunits, which can be explained by burial of previously exposed hydrophobic domains upon association of the subunits to form the native troponin complex. Equilibrium unfolding studies of TnT and TnI by guanidine hydrochloride and urea monitored by changes in far-UV CD and bis-ANS fluorescence revealed noncooperative folding transitions for both proteins and the existence of partially folded intermediate states.Taken together, these results indicate that isolated TnI and TnT are partially unstructured proteins, and suggest that conformational plasticity of the isolated subunits may play an important role in macromolecular recognition for the assembly of the troponin complex.