Heterogeneity of endocytic proteins: distribution of clathrin adaptor proteins in neurons and glia.

Clathrin adaptor protein (AP)180 is a synaptic protein that regulates the assembly of clathrin-coated vesicles. Several endocytic proteins including AP2, CALM, and epsin 1 have functions or molecular structures similar to AP180. We determined if AP180 associates with functional synapses in cultured hippocampal neurons. We also compared the expression pattern of AP180 with the other endocytic proteins. The distribution of AP180 corresponded with the synaptic vesicle-associated protein synapsin I, and with functional presynaptic terminals labeled with the styryl dye FM1-43. Synaptic AP2 colocalized with AP180, but the distribution of AP2 was not limited to synapses of neurons and it was also expressed in glia. CLAM and epsin 1 immunoreactivities were also detected in both neurons and glia. Unlike AP180, the neuronal immunoreactivity of CALM was not intense in the synaptic puncta. Epsin 1 immunoreactivity was found in both synaptic and extrasynaptic sites, and its synaptic distribution only partially overlapped with that of AP180. These results support roles for AP180 in synaptic function in neurons. The findings also provide information on the distribution of AP2, CALM, and epsin 1 in cells of the nervous system that suggest different roles for these endocytic proteins in the biology of these cells.

[The effect of temperature on the enantiomeric resolutions on albumin and beta-cyclodextrin chiral stationary phases].

The effect of column temperature on enantiomeric resolutions of tryptophan, warfarin and ketoprofen was investigated on bovine and human serum albumin (BSA and HSA) stationary phases, which were synthesized with s-triazine as the activator. It was observed that the entropy change made a great contribution to the separation of those enantiomers on albumin chiral stationary phases. The column temperature for the maximal resolution of tryptophan on the BSA stationary phase prepared by this method was about 35 degrees C, which was not 24 degrees C as reported for the BSA stationary phase synthesized with glutaric dialdehyde as the activator. This results may come from the different conformation of the immobilized BSA and HSA due to the different coupling methods used. On the other hand, it was indicated that the resolution of enantiomers on beta-CD chiral stationary phase was mainly contributed from the change of enthalpy, which means the resolution of chiral solutes on albumin and beta-CD stationary phases has different thermodynamic behaviors.

Clathrin assembly protein AP-2 is detected in both neurons and glia, and its reduction is prominent in layer II of frontal cortex in Alzheimer's disease.

There are several adaptor proteins associated with clathrin coated vesicles. Among them are AP180 and AP-2. We and others have previously described synaptic localization of AP180. AP180 immunoreactivity is altered in both the superior frontal gyrus and hippocampus in Alzheimer's disease (AD). We here investigate the location and alteration of another adaptor protein, AP-2. In contrast to AP180, we have found that AP-2 is expressed by both neurons and glia. Furthermore, the only noticeable change of AP-2 in AD is a loss of its immunoreactivity in layer II of the superior frontal gyrus.

Changes in synaptic expression of clathrin assembly protein AP180 in Alzheimer's disease analysed by immunohistochemistry.

Clathrin assembly protein AP180 plays a regulatory role in clathrin-mediated synaptic vesicle recycling in synapses. Previously, using immunoblot analysis, we observed a significant reduction of AP180 protein in Alzheimer's disease neocortex. In this study, we examined immunohistochemically the expression of AP180 in post mortem brains with Alzheimer's disease (n = 5) in comparison with neurologically normal controls (n = 5). Overall, AP180 was revealed as immunoreactive punctate granules located in the neuropil, and around neuronal cell bodies and their processes, consistent with the typical expression of synaptic proteins. Reduced density of AP180 immunoreactive puncta was seen throughout all layers of the superior frontal gyrus in Alzheimer's disease, but the loss of AP180 immunoreactivity was not as prominent in the cerebellum. This regional difference is in agreement with our previous results from immunoblot analyses. In the hippocampus, cell body AP180 immunoreactivity normally seen in the hilus and the CA3 regions of control brains was completely lost in Alzheimer's disease. In addition, AP180 immunoreactivity in the molecular layer of the dentate gyrus showed several changes in Alzheimer's disease. These appeared to be expansion of the inner molecular layer and relative changes in immunoreactivity that resulted in clearer delineation of the inner and outer molecular layers. These results provide anatomical and spatial information on AP180 expression in Alzheimer's disease brains. The variations in altered AP180 immunoreactivity in different brain regions of Alzheimer's disease may underlie the dysfunction of the corresponding synapses.

Reduced O-glycosylated clathrin assembly protein AP180: implication for synaptic vesicle recycling dysfunction in Alzheimer's disease.

Synapse loss is one of the neuropathologies in Alzheimer's disease (AD) that may play a crucial role in the mechanism of its distinct cognitive impairment and dementia. In a previous study [18], a significant reduction of O-glycosylated clathrin assembly protein AP180 was observed in neocortex of AD. The reduction correlated with the density of neurofibrillary tangles. In this study we further determine that the O-GlcNAc/AP180 ratio is not changed, but the level of AP180 protein decreases in AD. Furthermore, whereas the level of neurofilament (NF-M) remains relatively unchanged, another clathrin assembly protein, AP-2, is also reduced in AD along with a small loss of synaptophysin. Our findings suggest that synaptic vesicle recycling dysfunction may be involved in the pathology of synapse loss in AD.

Reduction of O-linked N-acetylglucosamine-modified assembly protein-3 in Alzheimer's disease.

Abnormal protein processing and modification is associated with Alzheimer's disease (AD) pathology. The role of phosphorylation in AD has been studied extensively because the presumed abnormal phosphorylation of tau protein is believed to play a role in the formation of paired helical filaments. Glycosylation with O-linked N-acetylglucosamine (O-GlcNAc) to serine and threonine residues is a dynamic protein modification of intracellular proteins, and it shares similar features with protein phosphorylation. In this study, O-GlcNAc glycosylation of proteins from autopsied human brains with confirmed AD and non-AD age-matched controls was examined. O-GlcNAcylation was demonstrated by labeling protein extracts with [3H]galactose in the presence of galactosyltransferase and subsequent analyses of saccharide-protein linkage and saccharide structure. The number of O-GlcNAc-containing proteins and the overall O-GlcNAc level do not appear to be different between AD and control brain tissues. The only significant change observed is a marked reduction of O-GlcNAcylated clathrin assembly protein-3 (AP-3) in AD. The reduction is more evident in brain neocortical regions, and there appears to be a negative correlation between O-glycosylated AP-3 and the density of neurofibrillary tangles. These data suggest a possible association between the O-glycosylated AP-3 and AD pathology.