Characterization of the novel HLA allele: B*14:58.

HLA-B*14:58 differs from B*14:02:01 by a polymorphism at position 506 a C instead a G.

Characterization of a new HLA-DRB1 allele: DRB1*14:172.

DRB1*14:172 homologous to DRB1*14:01 has been found with a variation in 314 A > G.

Characterization of a new HLA allele: A*02:548.

HLA-A*02:548 differs from A*02:01:01:01 by one nucleotide at position 367T > C resulting in histidine at codon 99.

Characterisation of a new HLA-DRB1 allele: DRB1*03:85.

HLA-DRB1*03:85 differs from HLA-DRB1*03:06 by two nucleotides, position 257 A>T and position 258T>C, resulting in Valine at codon 57.

Turnover of amyloid beta-protein in mouse brain and acute reduction of its level by phorbol ester.

Fibrillar amyloid deposits are defining pathological lesions in Alzheimer's disease brain and are thought to mediate neuronal death. Amyloid is composed primarily of a 39-42 amino acid protein fragment of the amyloid precursor protein (APP), called amyloid beta-protein (Abeta). Because deposition of fibrillar amyloid in vitro has been shown to be highly dependent on Abeta concentration, reducing the proteolytic release of Abeta is an attractive, potentially therapeutic target. Here, the turnover rate of brain Abeta has been determined to define treatment intervals over which a change in steady-state concentration of Abeta could be measured. Mice producing elevated levels of human Abeta were used to determine approximate turnover rates for Abeta and two of its precursors, C99 and APP. The t1/2 for brain Abeta was between 1.0 and 2.5 hr, whereas for C99, immature, and fully glycosylated forms of APP695 the approximate t1/2 values were 3, 3, and 7 hr, respectively. Given the rapid Abeta turnover rate, acute studies were designed using phorbol 12-myristate 13-acetate (PMA), which had been demonstrated previously to reduce Abeta secretion from cells in vitro via induction of protein kinase C (PKC) activity. Six hours after intracortical injection of PMA, Abeta levels were significantly reduced, as measured by both Abeta40- and Abeta42-selective ELISAs, returning to normal by 12 hr. An inactive structural analog of PMA, 4alpha-PMA, had no effect on brain Abeta levels. Among the secreted N-terminal APP fragments, APPbeta levels were significantly reduced by PMA treatment, whereas APPalpha levels were unchanged, in contrast to most cell culture studies. These results indicate that Abeta is rapidly turned over under normal conditions and support the therapeutic potential of elevating PKC activity for reduction of brain Abeta.

Heparin promotes beta-secretase cleavage of the Alzheimer's amyloid precursor protein.

In Alzheimer's disease, abnormal processing of the amyloid precursor protein (APP) is thought to play an important role in amyloid deposition. We investigated the effect of heparin, a highly sulfated glycosaminoglycan related to heparan sulfate, on the secretion of the beta-secretase cleavage product of APP (sAPP beta) in a human neuroblastoma cell line. Heparin induced an increase in the secretion of total APP, and an even greater relative increase in the secretion of sAPP beta. The effect on sAPP beta was specific to heparin. These data support the hypothesis that highly sulfated heparan sulfate proteoglycans may promote amyloidogenic pathways of APP metabolism.

Processing of the beta-amyloid precursor. Multiple proteases generate and degrade potentially amyloidogenic fragments.

Proteolytic processing of the beta-amyloid precursor proteins (APP) is required for release of the beta/A4 protein and its deposition into the amyloid plaques characteristic of aging and Alzheimer's disease. We have examined the involvement of acidic intracellular compartments in APP processing in cultured human cells. The use of acidotropic agents and inhibitors to a specific class of lysosomal protease, coupled with metabolic labeling and immunoprecipitation, revealed that APP is degraded within an acidic compartment to produce at least 12 COOH-terminal fragments. Nine likely contain the entire beta/A4 domain and, therefore, are potentially amyloidogenic. Treatment with E64 or Z-Phe-Ala-CHN2 irreversibly blocked activities of the lysosomal cysteine proteases cathepsins B and L but did not inhibit the lysosomal aspartic protease cathepsin D and did not alter the production of potentially amyloidogenic fragments. Instead, the inhibitors prevented further degradation of the fragments. Thus, large numbers of potentially amyloidogenic fragments of APP are routinely generated in an acidic compartment by noncysteine proteases and then are eliminated within lysosomes by cysteine proteases. Immunoblot and immunohistochemical analyses confirmed that chronic cysteine protease inhibition leads to accumulation of potentially amyloidogenic APP fragments in lysosomes. The results provide further support for the hypothesis that an acidic compartment may be involved in amyloid formation and begin to define the proteolytic events that may be important for amyloidogenesis.