Biochemical and immunocytochemical analyses were performed to evaluate the composition of the amyloid beta protein (A beta) deposited in the brains of patients with Alzheimer's disease (AD). To quantitate all A beta s present, cerebral cortex was homogenized in 70% formic acid, and the supernatant was analyzed by sandwich enzyme-linked immunoabsorbent assays specific for various forms of A beta. In 9 of 27 AD brains examined, there was minimal congophilic angiopathy and virtually all A beta (96%) ended at A beta 42(43). The other 18 AD brains contained increasing amounts of A beta ending at A beta 40. From this set, 6 brains with substantial congophilic angiopathy were separately analyzed. In these brains, the amount of A beta ending at A beta 42(43) was much the same as in brains with minimal congophilic angiopathy, but a large amount of A beta ending at A beta 40 (76% of total A beta) was also present. Immunocytochemical analysis with monoclonal antibodies selective for A beta s ending at A beta 42(43) or A beta 40 confirmed that, in brains with minimal congophilic angiopathy, virtually all A beta is A beta ending at A beta 42(43) and showed that this A beta is deposited in senile plaques of all types. In the remaining AD brains, A beta 42(43) was deposited in a similar fashion in plaques, but, in addition, widely varying amounts of A beta ending at A beta 40 were deposited, primarily in blood vessel walls, where some A beta ending at A beta 42(43) was also present. These observations indicate that A beta s ending at A beta 42(43), which are a minor component of the A beta in human cerebrospinal fluid and plasma, are critically important in AD where they deposit selectively in plaques of all kinds.

Cell-cell interactions mediated via cell adhesion molecules (CAMs) are dynamically regulated during nervous system development. One mechanism to control the amount of cell surface CAMs is to regulate their recycling from the plasma membrane. The L1 subfamily of CAMs has a highly conserved cytoplasmic domain that contains a tyrosine, followed by the alternatively spliced RSLE (Arg-Ser-Leu-Glu) sequence. The resulting sequence of YRSL conforms to a tyrosine-based sorting signal that mediates clathrin-dependent endocytosis of signal-bearing proteins. The present study shows that L1 associates in rat brain with AP-2, a clathrin adaptor that captures plasma membrane proteins with tyrosine-based signals for endocytosis by coated pits. In vitro assays demonstrate that this interaction occurs via the YRSL sequence of L1 and the mu 2 chain of AP-2. In L1-transfected 3T3 cells, L1 endocytosis is blocked by dominant-negative dynamin that specifically disrupts clathrin-mediated internalization. Furthermore, endocytosed L1 colocalizes with the transferrin receptor (TfR), a marker for clathrin-mediated internalization. Mutant forms of L1 that lack the YRSL do not colocalize with TfR, indicating that the YRSL mediates endocytosis of L1. In neurons, L1 is endocytosed preferentially at the rear of axonal growth cones, colocalizing with Eps15, another marker for the clathrin endocytic pathway. These results establish a mechanism by which L1 can be internalized from the cell surface and suggest that an active region of L1 endocytosis at the rear of growth cones is important in L1-dependent axon growth.

L1 is a neural cell adhesion molecule critical for neural development. Full-length L1 (L1 FL ) contains an alternatively spliced cytoplasmic sequence, RSLE, which is absent in L1 expressed in nonneuronal cells. The RSLE sequence follows a tyrosine, creating an endocytic motif that allows rapid internalization via clathrin-mediated endocytosis. We hypothesized that L1 FL would internalize more rapidly than L1 lacking the RSLE sequence (L1 ⌬RSLE ) and that internalization might regulate L1-mediated adhesion. L1 internalization was measured by immunofluorescence microscopy and by uptake of 125 Ianti-rat-L1 antibody, demonstrating that L1 FL is internalized 2-3 times faster than L1 ⌬RSLE . Inhibition of clathrin-mediated endocytosis slowed internalization of L1 FL but did not affect initial uptake of L1 ⌬RSLE . To test whether L1 endocytosis regulates L1 adhesion, cell aggregation rates were tested. L1 ⌬RSLE cells aggregated two times faster than L1 FL cells. Inhibition of clathrinmediated endocytosis increases the aggregation rate of the L1 FL cells to that of L1 ⌬RSLE cells. Our results demonstrate that rapid internalization of L1 dramatically affects L1 adhesion.Development of the nervous system is a complex process that requires coordination of many cellular events including cell migration, axon outgrowth, and synapse formation. Many cell adhesion molecules (CAMs) 1 participate in these events. Although CAM expression appears to be rather static in the adult nervous system, CAM expression is both spatially and temporally dynamic during development. An important question in developmental neurobiology is how expression of CAMs is regulated to allow for the precise adhesive events necessary for proper neural development. The CAMs are responsible for mediating homophilic and heterophilic binding events and, in turn, cell-cell or cell-substrate interactions (1). Many CAMs interact with signaling molecules (2) and the cytoskeleton (3-5). Therefore, CAM-CAM binding can produce intracellular signals that regulate adhesion and trigger other events such as migration, proliferation, and synapse formation (6 -11).L1 is an immunoglobulin superfamily CAM important in vertebrate neural development. L1 participates in neurite outgrowth (12) as well as neuronal migration (13,14). It binds both homophilically and heterophilically with a number of CAMs including axonin-1 (15), CD9 (16), and integrins (17-19). The importance of proper L1 function is demonstrated by the severe complications resulting from mutations in the human L1 molecule (20, 21), causing X-linked hydrocephalus which is characterized by varying degrees of corticospinal tract and corpus callosum agenesis, retardation, adducted thumbs, spastic paraplegia, and hydrocephalus (20). Mutations in L1 can disrupt proper L1 adhesive function, which in turn causes mistakes in cell migration and axon extension.Alternative RNA splicing results in two L1 isoforms (22). Neurons express the full-length form, L1 FL . Non-neuronal L1-positive cells express L1 lacking the cytoplas...

The survival of transfused red cells (RBCs) diminishes with time of in vitro storage in blood banks, but the molecular mechanisms underlying the slow but incessant deterioration are incompletely understood. To investigate the possibility that impaired resistance to autologous complement attack could play a role in this phenomenon, packed RBCs stored for variable periods were assayed for decay-accelerating factor (DAF) and CD59, two glycoinositol-phospholipid (GPI)-anchored, membrane-associated complement regulatory proteins that function physiologically to protect blood cells from autologous complement activation on their surfaces. Immunoradiometric and flow cytometric assays employing DAF and CD59 monoclonal antibodies showed that levels of both surface proteins gradually declined over 6 weeks. Digestion analyses with phosphatidylinositol-specific phospholipase C, an enzyme that releases GPI-anchored proteins from cell surfaces, showed that DAF and CD59 molecules with GPI anchors containing unacylated inositol were preferentially lost. These findings suggest: 1) that DAF and CD59 molecules with acylated GPI anchors are more stable in RBC membranes than are molecules with unacylated GPI anchors, and 2) that DAF and CD59 loss may participate with other membrane alterations that occur during in vitro storage in compromising the survival of transfused cells.

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