Deborah Downs

β-Secretase inhibitor GRL-8234 rescues age-related cognitive decline in APP transgenic mice

Alzheimer disease is intimately linked to an excess amount of amyloid‐β (Aβ) in the brain. Thus, therapeutic inhibition of Aβ production is an attractive clinical approach to treat this disease. Here we provide the first direct experimental evidence that the treatment of Tg2576 transgenic mice with an inhibitor of β‐secretase, GRL‐8234, rescues the age‐related cognitive decline. We demonstrated that the injected GRL‐8234 effectively enters the brain and rapidly decreases soluble Aβ in the brain of Tg2576 mice. The rescue of cognition, which was observed only after long‐term inhibitor treatment ranging from 5 to 7.5 mo, was associated with a decrease of brain amyloid‐β plaque load. We also found no accumulation of amyloid‐β precursor protein after several months of inhibitor treatment. These observations substantiate the idea that Aβ accumulation plays a major role in the cognitive decline of Tg2576 mice and support the concept of Aβ reduction therapy as a treatment of AD.—Chang, W.‐P., Huang, X., Downs, D., Cirrito, J. R., Koelsch, G., Holtzman, D. M. Ghosh, A. K., Tang, J. β‐Secretase inhibitor GRL‐8234 rescues age‐related cognitive decline in APP transgenic mice. FASEB J. 25, 775–784 (2011). www.fasebj.org

In vivo inhibition of Aβ production by memapsin 2 (β-secretase) inhibitors

We have previously reported structure‐based design of memapsin 2 (β‐secretase) inhibitors with high potency. Here we show that two such inhibitors covalently linked to a ‘carrier peptide’ penetrated the plasma membrane in cultured cells and inhibited the production of β‐amyloid (Aβ). Intraperitoneal injection of the conjugated inhibitors in transgenic Alzheimers mice (Tg2576) resulted in a significant decrease of Aβ level in the plasma and brain. These observations verified that memapsin 2 is a therapeutic target for Aβ reduction and also establish that transgenic mice are suitable in vivo models for the study of memapsin 2 inhibition.

In vivo inhibition of Abeta production by memapsin 2 (beta-secretase) inhibitors.

We have previously reported structure‐based design of memapsin 2 (β‐secretase) inhibitors with high potency. Here we show that two such inhibitors covalently linked to a ‘carrier peptide’ penetrated the plasma membrane in cultured cells and inhibited the production of β‐amyloid (Aβ). Intraperitoneal injection of the conjugated inhibitors in transgenic Alzheimers mice (Tg2576) resulted in a significant decrease of Aβ level in the plasma and brain. These observations verified that memapsin 2 is a therapeutic target for Aβ reduction and also establish that transgenic mice are suitable in vivo models for the study of memapsin 2 inhibition.

Amyloid-beta reduction by memapsin 2 (beta-secretase) immunization

Memapsin 2 (β‐secretase, BACE1) is the protease that initiates cleavage of β‐amyloid precursor protein leading to the production of amyloid‐β (Aβ) and the onset of Alzheimers disease (AD). Reducing Aβ by targeting memapsin 2 is a major strategy in developing new AD therapy. Here, in a proof‐of‐con‐cept study, we show that immunization of transgenic AD mice (Tg2576) with memapsin 2 resulted in Aβ reduction and cognitive improvement. To study the basis of this therapy, we demonstrated that anti‐memap‐sin 2 (anti‐M2) antibodies were rapidly internalized and reduced Aβ production in cultured cells. These antibodies also effectively crossed the blood‐brain barrier to reach the brain. Two‐ and 10‐month Tg2576 mice were immunized and monitored over 10 and 6 months, respectively. We observed a significant decrease of plasma and brain Aβ40 and Aβ42 (~35%) in the immunized mice as compared to controls. Immunized mice also showed better cognitive performance than controls in both cohorts. Brain histological analyses found no evidence of T cell/microglia/astrocyte activation in the immunized mice, suggesting the absence of inflammatory responses. These results suggest that memapsin 2 immunization in Tg2576 was effective in reducing Aβ production and improving cognitive function and that the current approach warrants further investigation as a therapy for AD.—Chang, W.‐P., Downs, D., Huang, X.‐P., Da, H., Fung, K. M., Tang, J. Amyloid‐beta reduction by memepsin 2 (beta‐secretase) immunization. FASEB J. 21, 3184–3196 (2007)

Candida albicans secreted aspartic proteases 4–6 induce apoptosis of epithelial cells by a novel Trojan horse mechanism

Systemic infection by the pathogenic yeast Candida albicans produces high mortality in immune‐compromised people. Such infection starts with the penetration of the organism at the mucosal surfaces, facilitated by the secreted aspartic proteases (Saps) 4, 5, and 6. The functional mechanism of these virulence factors is unclear. We discovered that Saps 4–6 each contains amino acid motifs RGD/KGD to bind integrins on epithelial cell A549 and are internalized to endosomes and lysosomes. These processes are inhibited by RGD‐containing peptides or by substituting RGD motifs of these Saps. The internalization of Saps 4–6 results in partial permeabilization of lysosomal membranes, measured by the redistribution of the lysosomal tropic dye acridine orange to the cytosol, and the triggering of apoptosis via caspase activation. Sap 2 and mutated Saps 4–6 contain no RGD motif, are ineffective in these processes, and a proteolytic inhibitor abolished Sap 4 activity in lysosome permeabilization. Same results were also seen for human tongue keratinocyte SCC‐15 cells. Mucosal lesions from this fundamental new mechanism may permit C. albicans to enter the body and may be used to attack cells in immune defense during systemic infections. RGD‐motif may also be incorporated in Sap inhibitors for Candidiasis drugs targeting to lysosomes.—Wu, H., Downs, D., Ghosh, K., Ghosh, A. K., Staib, P., Monod, M., and Tang, J. Candida albicans secreted aspartic proteases 4–6 induce apoptosis of epithelial cells by a novel Trojan horse mechanism. FASEB J. 27, 2132–2144 (2013). www.fasebj.org

Lipolytic degradation of human very low density lipoproteins by human milk lipoprotein lipase: The identification of lipoprotein B as the main lipoprotein degradation product☆

Although the direct conversion of very low density lipoproteins (VLDL) into low density (LDL) and high density (HDL) lipoproteins only requires lipoprotein lipase (LPL) as a catalyst and albumin as the fatty acid acceptor, the in vitro-formed LDL and HDL differ chemically from their native counterparts. To investigate the reason(s) for these differences, VLDL were treated with human milk LPL in the presence of albumin, and the LPL-generated LDL1-, LDL2-, and HDL-like particles were characterized by lipid and apolipoprotein composition. Results showed that the removal of apolipoproteins B, C, and E from VLDL was proportional to the degree of triglyceride hydrolysis with LDL2 particles as the major and LDL1 and HDL + VHDL particles as the minor products of a complete in vitro lipolysis of VLDL. In comparison with native counterparts, the in vitro-formed LDL2 and HDL + VHDL were characterized by lower levels of triglyceride and cholesterol ester and higher levels of free cholesterol and lipid phosphorus. The characterization of lipoprotein particles present in the in vitro-produced LDL2 showed that, as in plasma LDL2, lipoprotein B (LP-B) was the major apolipoprotein B-containing lipoprotein accounting for over 90% of the total apolipoprotein B. Other, minor species of apolipoprotein B-containing lipoproteins included LP-B:C-I:E and LP-B:C-I:C-II:C-III. The lipid composition of in vitro-formed LP-B closely resembled that of plasma LP-B. The major parts of apolipoproteins C and E present in VLDL were released to HDL + VHDL as simple, cholesterol/phospholipid-rich lipoproteins including LP-C-I, LP-C-II, LP-C-III, and LP-E. However, some of these same simple lipoprotein particles were present after ultracentrifugation in the LDL2 density segment because of their hydrated density and/or because they formed, in the absence of naturally occurring acceptors (LP-A-I:A-II), weak associations with LP-B. Thus, the presence of varying amounts of these cholesterol/phospholipid-rich lipoproteins in the in vitro-formed LDL2 appears to be the main reason for their compositional difference from native LDL2. These results demonstrate that the formation of LP-B as the major apolipoprotein B-containing product of VLDL lipolysis only requires LPL as a catalyst and albumin as the fatty acid acceptor. However, under physiological circumstances, other modulating agents are necessary to prevent the accumulation and interaction of phospholipid/cholesterol-rich apolipoprotein C- and E-containing particles.

Isolation and characterization of recombinant human apolipoprotein C-II expressed in Escherichia coli

A full-length recombinant human apolipoprotein C-II (ApoC-II) has been successfully expressed in Escherichia coli using the T7 expression system. The recombinant ApoC-II. which was expressed intracellularly in the inclusion bodies, was solubilized with 8 M urea and purified using Sephadex G-75 gel permeation chromatography. Four liters of the bacterial culture yielded 16-20 mg of purified recombinant ApoC-II. Sequencing and mass spectrometric analyses indicated that the isolated recombinant ApoC-II contained predominantly (64%) the native form with threonine as the N-terminus, but also contained a minor (36%) molecular form of ApoC-II with an additional methionine at the N-terminus (Met-ApoC-II). Analysis of the recombinant ApoC-II by tryptic digestion and high performance liquid chromatography-electrospray mass spectrometry provides additional conclusive evidence that, with the exception of the N-terminus of Met-ApoC-II, the expressed ApoC-II has the expected peptide sequence. However, this extra N-terminal methionine residue can be excised by further in vitro treatment with methionine aminopeptidase. The purified recombinant ApoC-II was found to be competent in the activation of bovine milk lipoprotein lipase. Thus, the recombinant ApoC-II prepared from E. coli may have a pharmacological application for the treatment of patients with genetic hypertriglyceridemia caused by ApoC-II deficiency.

In vivo inhibition of Aβ production by memapsin 2 (β-secretase) inhibitors: Inhibition of Aβ production in vivo

We have previously reported structure‐based design of memapsin 2 (β‐secretase) inhibitors with high potency. Here we show that two such inhibitors covalently linked to a ‘carrier peptide’ penetrated the plasma membrane in cultured cells and inhibited the production of β‐amyloid (Aβ). Intraperitoneal injection of the conjugated inhibitors in transgenic Alzheimers mice (Tg2576) resulted in a significant decrease of Aβ level in the plasma and brain. These observations verified that memapsin 2 is a therapeutic target for Aβ reduction and also establish that transgenic mice are suitable in vivo models for the study of memapsin 2 inhibition.

Design of potent and highly selective inhibitors for human β-secretase 2 (memapsin 1), a target for type 2 diabetes

Structure-based design and syntheses of potent and highly selective BACE2 inhibitors are described.