Implications of the serine protease HtrA1 in amyloid precursor protein processing

Abstract

The defining features of the widely conserved HtrA (high temperature requirement) family of serine proteases are the combination of a catalytic protease domain with one or more C-terminal PDZ domains and reversible zymogen activation. Even though HtrAs have previously been implicated in protein quality control and various diseases, including cancer, arthritis, and neuromuscular disorder, the biology of the human family members is not well understood. Our data suggest that HtrA1 is directly involved in the beta-amyloid pathway as it degrades various fragments of amyloid precursor protein while an HtrA1 inhibitor causes accumulation of Abeta in astrocyte cell culture supernatants. Furthermore, HtrA1 colocalizes with beta-amyloid deposits in human brain samples. Potential implications in Alzheimer's disease are discussed.

Fig. 2.

Identification of cleavage sites of HtrA1 in C99 by MALDI-FTICR-MS. The detected cleavage sites of HtrA1 in recombinant C99 are shown. The transmembrane segment of C99 is shown in bold and underlined. Digestion was performed at an enzyme/substrate ratio of 1:50. Proteolytic fragment mixture was desalted and analyzed by MALDI-FTICR-MS as described in Methods.(A) MALDI-FTICR spectrum of HtrA1-digested C99. Monoisotopic masses were selected manually and used for fragment identification by means of the gpmaw program (Lighthouse Data, Odense, Denmark). (B) Fragments of C99 and sequences are identified.

Fig. 1.

Degradation of C99. (A) Purified recombinant C99 (2 μg) was incubated with increasing amounts of purified recombinant HtrA1 for 16 h at 37°C. Samples were loaded on a SDS/PAGE, immunoblotted, and stained with either monoclonal α-HtrA1 antibody or polyclonal α-C99 antiserum. The asterisk indicates a proteolytic product of C99. (B) To monitor the efficiency of the HtrA1 inhibitor in vitro, purified HtrA1 (5 μg) was preincubated for 20 min at room temperature with various inhibitor concentrations before adding 60 μg of resorufin-labeled casein. Data represent the mean of four experiments. (C) Purified recombinant HtrA1 (2.5 μg) was preincubated with various concentrations of HtrA1 inhibitor for 20 min before adding purified C99 (2 μg). Samples were loaded on a SDS/PAGE, immunoblotted, and stained with either monoclonal α-HtrA1 or α-C99 antibody. (D) Degradation of membrane-bound C99 was analyzed by using cytoplasmic membrane fractions of strain KU98 overproducing C99. Membranes were incubated with or without 10 μg of purified HtrA1. Samples were analyzed by Western blotting using α-C99 antibody. (E) Degradation of Aβ42. One microgram of Aβ42 (preincubated in PBS to allow multimerization) was incubated with 5 μg of HtrA1 for 14 h at 37°C. Samples were immunoblotted and stained with α-Aβ42 antibody.

Fig. 3.

Aβ40 accumulation in supernatants of astrocytes after HtrA1 inhibition. The astrocytoma cell line U373 was grown in six-well plates to 80% confluence in DMEM. (A) After 24 h, supernatants were harvested and assayed for HtrA1. (B)Aβ40 levels in culture supernatants were determined from cells grown with or without HtrA1 inhibitor (1-5 μM final concentration). Data represent the mean of four different treatments, each measured in duplicates. ^*, P < 0.05; ^**, P < 0.01; ^***, P < 0.001 as determined by Student's t test. ELISAs in A and B were done as described in Methods. (C) Equal concentrations of protein from supernatants and cell lysates were analyzed by Western blotting to detect full-length APP, APPs, and β-actin by using antibodies against APP and β-actin, respectively.

Fig. 4.

Histopathological and immunohistochemical analysis of AD's autoptic brains. For histopathological analysis, AD brains were analyzed by hematoxylin/eosin staining, Bielschowsky's silver staining, and Congo red staining. (A-C) Typical lesions shown are: an amyloid plaque positive for Congo red staining (A); a neurofibrillar tangle evidenced by Bielschowsky's silver staining (B); and a dystrophic neuron positive for β-amyloid immunohistochemistry (C). (D-F) Analysis of HtrA1 expression by immunohistochemistry revealed that the protein was present both in cortical neurons (D and F) and astrocytes (E). (G-L) For colocalization of HtrA1 and amyloid deposits, HtrA1 was expressed in the brain's areas where amyloid deposits were evident, such as in amyloid plaques (G), dystrophic neurons (H), or peri-vascular level (I-L). The same small arteriole of a cortical region evidenced by hematoxylin/eosin (I) is positive for Congo red staining (J), HtrA1 (K), and β-amyloid immunohistochemistry (L). (Magnifications: ×500, A, B, D, E, and G-L; ×1,000, C and F.)