A growing body of evidence suggests that soluble oligomeric forms of the amyloid beta peptide known as amyloid-derived diffusible ligands (ADDLs) are the toxic species responsible for neurodegeneration associated with Alzheimer's disease. Accurate biophysical characterization of ADDL preparations is hampered by the peptide's strong tendency to self-associate and the effect of factors such as ionic strength, temperature, and pH on its behavior. In addition, amyloid peptides are known to interact with common laboratory excipients, specifically detergents, further complicating the results from standard analytical methods such as denaturing polyacrylamide gel electrophoresis. We have studied the solution behavior of various amyloid peptide preparations using analytical ultracentrifugation and size exclusion chromatography coupled with multiangle laser light scattering. Our results indicate that ADDL preparations exist in solution primarily as a binary mixture of a monomeric peptide and high-molecular mass oligomers. We relate our findings to previously described characterizations utilizing atomic force microscopy and electrophoretic methods and demonstrate that low-molecular mass oligomers identified by gel electrophoresis likely represent artifacts induced by the peptide's interaction with detergent, while atomic force microscopy results are likely skewed by differential binding of monomeric and oligomeric peptide species. Finally, we confirm that only the high-molecular mass oligomeric components of an ADDL preparation are capable of binding to subpopulations of primary hippocampal neurons in vitro.

Mast cells generated from Rac2-deficient (-/-) mice demonstrated defective actin-based functions, including adhesion, migration, and degranulation. Rac2(-/-) mast cells generated lower numbers and less mast cell colonies in response to growth factors and were deficient in vivo. Rac2(-/-) mast cells demonstrated a significant reduction in growth factor-induced survival, which correlated with the lack of activation of Akt and significant changes in the expression of the Bcl-2 family members BAD and Bcl-XL, in spite of a 3-fold induction of Rac1 protein. These results suggest that Rac2 plays a unique role in multiple cellular functions and describe an essential role for Rac2 in growth factor-dependent survival and expression of BAD/Bcl-XL.

Synaptic degeneration, including impairment of synaptic plasticity and loss of synapses, is an important feature of Alzheimer disease pathogenesis. Increasing evidence suggests that these degenerative synaptic changes are associated with an accumulation of soluble oligomeric assemblies of amyloid ␤ (A␤) known as ADDLs. In primary hippocampal cultures ADDLs bind to a subpopulation of neurons. However the molecular basis of this cell type-selective interaction is not understood. Here, using siRNA screening technology, we identified ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits and calcineurin as candidate genes potentially involved in ADDL-neuron interactions. Immunocolocalization experiments confirmed that ADDL binding occurs in dendritic spines that express surface AMPA receptors, particularly the calcium-impermeable type II AMPA receptor subunit (GluR2). Pharmacological removal of the surface AMPA receptors or inhibition of AMPA receptors with antagonists reduces ADDL binding. Furthermore, using co-immunoprecipitation and photoreactive amino acid cross-linking, we found that ADDLs interact preferentially with GluR2-containing complexes. We demonstrate that calcineurin mediates an endocytotic process that is responsible for the rapid internalization of bound ADDLs along with surface AMPA receptor subunits, which then both colocalize with cpg2, a molecule localized specifically at the postsynaptic endocytic zone of excitatory synapses that plays an important role in activity-dependent glutamate receptor endocytosis. Both AMPA receptor and calcineurin inhibitors prevent oligomer-induced surface AMPAR and spine loss. These results support a model of disease pathogenesis in which A␤ oligomers interact selectively with neurotransmission pathways at excitatory synapses, resulting in synaptic loss via facilitated endocytosis. Validation of this model in human disease would identify therapeutic targets for Alzheimer disease. Alzheimer disease (AD)2 likely begins with deficits in synaptic transmission in brain regions that are critical for higher cognitive function (1), as stereological analyses of post-mortem samples show that synaptic loss correlates with cognitive dysfunction better than amyloid plaque or neurofibrillary tangle load (2-5). One of the triggers for such synaptic impairments is thought to be soluble oligomers of amyloid ␤ (A␤) peptides that accumulate in the brain and are potently toxic to synapses (6 -8). A␤ synaptotoxicity is consistently observed with A␤ oligomers from synthetic preparations (9 -11), cell-derived forms (7,8,12), and the brains of AD patients (13) and APPswe transgenic mice (14), such that these oligomers disrupt synaptic structure (9, 10, 15-18), inhibit long term potentiation (LTP) (7,8,19), and induce memory deficits (13,20).Although the mechanisms by which A␤ oligomers disrupt synaptic function are not known, A␤ oligomers bind to the dendritic processes of specific subtypes of hippocampal neurons with high affinity (9,15,21), leading to the hypothesi...

Differences in engraftment potential of hematopoietic stem cells (HSCs) in distinct phases of cell cycle may result from the inability of cycling cells to home to the bone marrow (BM) and may be influenced by the rate of entry of BM-homed HSCs into cell cycle. Alternatively, preferential apoptosis of cycling cells may contribute to their low engraftment potential. This study examined homing, cell cycle progression, and survival of human hematopoietic cells transplanted into nonobese diabetic severe combined immunodeficient (NOD/SCID) recipients. At 40 hours after transplantation (AT), only 1% of CD34(+) cells, or their G(0) (G(0)CD34(+)) or G(1) (G(1)CD34(+)) subfractions, was detected in the BM of recipient mice, suggesting that homing of engrafting cells to the BM was not specific. BM of NOD/SCID mice receiving grafts containing approximately 50% CD34(+) cells harbored similar numbers of CD34(+) and CD34(-) cells, indicating that CD34(+) cells did not preferentially traffic to the BM. Although more than 64% of human hematopoietic cells cycled in culture at 40 hours, more than 92% of cells recovered from NOD/SCID marrow were quiescent. Interestingly, more apoptotic human cells were detected at 40 hours AT in the BM of mice that received xenografts of expanded cells in S/G(2)+M than in recipients of G(0)/G(1) cells (34.6% +/- 5.9% and 17.1% +/- 6.3%, respectively; P <.01). These results suggest that active proliferation inhibition in the BM of irradiated recipients maintains mitotic quiescence of transplanted HSCs early AT and may trigger apoptosis of cycling cells. These data also illustrate that trafficking of transplanted cells to the BM is not selective, but lodgment of BM-homed cells may be specific.