Lipoprotein lipase (LPL) is a key regulator of triglyceride clearance. Its coordinated regulation during feeding and fasting is critical for maintaining lipid homeostasis and energy supply. Angiopoietin-like (Angptl)3 and Angptl4 are secreted proteins that have been demonstrated to regulate triglyceride metabolism by inhibiting LPL. We have taken a targeted genetic approach to generate Angptl4- and Angptl3-deficient mice as well as transgenic mice overexpressing human Angptl4 in the liver. The Angptl4 transgenic mice displayed elevated plasma triglycerides and reduced postheparin plasma (PHP) LPL activity. A purified recombinant Angptl4 protein inhibited mouse LPL and recombinant human LPL activity in vitro. In contrast to the transgenic mice, Angptl4-deficient mice displayed hypotriglyceridemia and increased PHP LPL activity, with greater effects in the fasted compared with the fed state. Angptl3-deficient mice also displayed hypotriglyceridemia with elevated PHP LPL activity, but these mice showed a greater effect in the fed state. Mice deficient in both Angptl proteins showed an additive effect on plasma triglycerides and did not survive past 2 months of age. Our results show that Angptl3 and Angptl4 function to regulate circulating triglyceride levels during different nutritional states and therefore play a role in lipid metabolism during feeding/fasting through differential inhibition of LPL.

The amyloid proteins isolated from neuritic plaques and the cerebrovasculature of Alzheimer's disease are self-aggregating moieties termed A4 protein and beta-protein, respectively. A putative A4 amyloid precursor (herein termed A4(695] has been characterized by analysis of a human brain complementary DNA. We report here the sequence of a closely related amyloid cDNA, A4(751), distinguished from A4(695) by the presence of a 168 base-pair (bp) sequence which adds 57 amino acids to, and removes one residue from, the predicted A4(695) protein. The peptide predicted from this insert is very similar to the Kunitz family of serine proteinase inhibitors. The two A4-specific messenger RNAs are differentially expressed: in a limited survey, A4(751) mRNA appears to be ubiquitous, whereas A4(695) mRNA has a restricted pattern of expression which includes cells from neuronal tissue. These data may have significant implications for understanding amyloid deposition in Alzheimer's disease.

A subset of nonsteroidal anti-inflammatory drugs (NSAIDs) has been shown to preferentially reduce the secretion of the highly amyloidogenic, 42-residue amyloid-beta peptide Abeta42. We found that Rho and its effector, Rho-associated kinase, preferentially regulated the amount of Abeta42 produced in vitro and that only those NSAIDs effective as Rho inhibitors lowered Abeta42. Administration of Y-27632, a selective Rock inhibitor, also preferentially lowered brain levels of Abeta42 in a transgenic mouse model of Alzheimer's disease. Thus, the Rho-Rock pathway may regulate amyloid precursor protein processing, and a subset of NSAIDs can reduce Abeta42 through inhibition of Rho activity.

The phosphorylation status of amyloid precursor protein (APP) at Thr668 is suggested to play a critical role in the proteolytic cleavage of APP, which generates either soluble APP L (sAPP L ) and L L-amyloid peptide (AL L), the major component of senile plaques in patient brains in£icted with Alzheimer's disease (AD), or soluble APP K (sAPP K ) and a peptide smaller than AL L. One of the protein kinases known to phosphorylate APP Thr668 is cyclin-dependent kinase 5 (Cdk5). Cdk5 is activated by the association with its regulatory partner p35 or its truncated form, p25, which is elevated in AD brains. The comparative e¡ects of p35 and p25 on APP Thr668 phosphorylation and APP processing, however, have not been reported. In this study, we investigated APP Thr668 phosphorylation and APP processing mediated by p35/Cdk5 and p25/Cdk5 in the human neuroblastoma cell line SH-SY5Y. Transient overexpression of p35 and p25 elicited distinct patterns of APP Thr668 phosphorylation, speci¢cally, p35 increasing the phosphorylation of both mature and immature APP, whereas p25 primarily elevated the phosphorylation of immature APP. Despite these di¡erential e¡ects on APP phosphorylation, both p35 and p25 overexpression enhanced the secretion of AL L, sAPP L , as well as sAPP K . These results con¢rm the involvement of Cdk5 in APP processing, and suggest that p35-and p25-mediated Cdk5 activities lead to discrete APP phosphorylation. ß

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The kinetics of amyloid fibril formation by f3-amyloid peptide (A18) are typical of a nucleation-dependent polymerization mechanism.

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b-amyloid peptides (Ab) are produced by a sequential cleavage of amyloid precursor protein (APP) by b-and c-secretases. The lack of Ab production in beta-APP cleaving enzyme (BACE1) -/mice suggests that BACE1 is the principal b-secretase in mammalian neurons. Transfection of human APP and BACE1 into neurons derived from wild-type and BACE1 -/mice supports cleavage of APP at the canonical b-secretase site. However, these studies also revealed an alternative BACE1 cleavage site in APP, designated as b¢, resulting in Ab peptides starting at Glu11. The apparent inability of human BACE1 to make this b¢-cleavage in murine APP, and vice versa, led to the hypothesis that this alternative cleavage was species-specific. In contrast, the results from human BACE1 transgenic mice demonstrated that the human BACE1 is able to cleave the endogenous murine APP at the b¢-cleavage site. To address this discrepancy, we designed fluorescent resonance energy transfer peptide substrates containing the b-and b¢-cleavage sites within human and murine APP to compare: (i) the enzymatic efficiency; (ii) binding kinetics of a BACE1 active site inhibitor LY2039911; and (iii) the pharmacological profiles for human and murine recombinant BACE1. Both BACE1 orthologs were able to cleave APP at the b-and b¢-sites, although with different efficiencies. Moreover, the inhibitory potency of LY2039911 toward recombinant human and native BACE1 from mouse or guinea pig was indistinguishable. In summary, we have demonstrated, for the first time, that recombinant BACE1 can recognize and cleave APP peptide substrates at the postulated b¢-cleavage site. It does not appear to be a significant species specificity to this cleavage. Keywords: amyloid precursor protein, b-amyloid precursor protein cleaving enzyme, b-cleavage, b¢-cleavage, speciesspecificity.