Amyloid beta-degrading cryptidases: insulin degrading enzyme, presequence peptidase, and neprilysin.

The accumulation of aggregates of amyloidogenic peptides is associated with numerous human diseases. One well studied example is the association between deposition of amyloid beta (Abeta) and Alzheimer's disease. Insulin degrading enzyme and neprilysin are involved in the clearance of Abeta, and presequence peptidase is suggested to play a role in the degradation of mitochondrial Abeta. Recent structural analyses reveal that these three peptidases contain a catalytic chamber (crypt) that selectively encapsulates and cleaves amyloidogenic peptides, hence the name cryptidase. The substrate selectivity of these cryptidases is determined by the size and charge distribution of their crypt as well as the conformational flexibility of substrates. The interaction of Abeta with the catalytic core of these cryptidases is controlled by conformational changes that make the catalytic chambers accessible for Abeta binding. These new structural and biochemical insights into cryptidases provide potential therapeutic strategies for the control of Abeta clearance.

Optimization of multiplexed bead-based cytokine immunoassays for rat serum and brain tissue.

The ability to simultaneously quantify multiple signaling molecule protein levels from microscopic neural tissue samples would be of great benefit to deciphering how they affect brain function. This follows from evidence that indicates signaling molecules can be pleiotropic and can have complex interactive behavior that is regionally and cellularly heterogeneous. Multiplexed examination of tissue proteins has been exceedingly difficult because of the absence of available techniques. This void now has been removed by the commercial availability of bead-based immunoassays for targeted proteins that allow analyses of up to 100 (6-150 kDa) proteins from as little as 12 microl. Thus far used only for sera (human and mouse) and culture media, we demonstrate here that sensitive (as low as 2 pg/ml), wide-ranging (up to 2-32 000 pg/ml), accurate (8% intra-assay covariance) and reliable (4-7% inter-assay covariance) measurements can be made of nine exemplary cytokines (e.g., IL-1alpha, IL-1beta, IL-2, IL-4, IL-6, IL-10, GM-CSF, IFN-gamma, TNF-alpha) simultaneously not only from rat serum but, for the first time, also brain tissue. Furthermore, we describe animal handling procedures that minimize stress as determined by serum glucocorticoid levels since they can influence cytokine expression.

Astrocytic clasmatodendrosis in hippocampal organ culture.

Mechanisms by which astrocytes are irreversibly injured from ischemic brain injury remain incompletely defined. More than 90 years ago Alzheimer showed that astrocytes lose their distal processes (i.e., undergo "clasmatodendrosis") when irreversibly injured by a reduction in blood flow, a process shown by Friede and van Houten (1961) to be due to energy failure and acidosis. Such alterations in astrocytic morphology can relate directly to changes in cell function. However, astrocytic clasmatodendrosis has largely been lost to the modern literature, perhaps because of a inability to study it under controlled conditions. In the present study, novel four-dimensional (4D)and digital deblurring imaging of glial fibrillary acidic protein (GFAP) immunostaining changes in hippocampal organ cultures (HOTCs) were used to establish an in vitro model of astrocytic clasmatodendrosis. Also, astrocytes in primary culture were transfected with green fluorescent protein (GFP) to show the occurrence of clasmatodendrosis via a parallel and separate means. In HOTCs, a significant reduction in astrocytic process length occurred 15 min (and remained for 60 min) after exposure to acidic Ringer's and mitochondrial inhibition in the pyramidal cell body layer. Time-lapsed images of primary cultures showed thinning of cell processes within 15 min of exposure to acidic Ringer's and mitochondrial inhibition. Distal processes subsequently broke away but retained their fluorescence for minutes before disintegrating along with their parent cell bodies. This report shows the spatiotemporal occurrence of clasmatodendrosis in astrocytes of HOTCs closely parallels that seen in vivo. Thus, HOTCs, where microenvironmental conditions can be controlled and single, identified cells can be followed in space and time, can be applied to study the interrelations between energy metabolism and pH that result in clasmatodendrosis.