Decreased concentration of human kallikrein 6 in brain extracts of Alzheimer’s disease patients

Abstract

Background: The human kallikrein 6 gene (KLK6) encodes for a secreted serine protease, hK6, which is highly expressed in brain. Previous reports have associated hK6 with the pathogenesis of Alzheimer’s disease. Our objective was to develop a highly sensitive immunoassay for hK6 and use it to examine the levels of hK6 in brain tissue extracts from Alzheimer’s disease patients and control subjects.

Methods: We developed antibodies against hK6 and constructed a ’sandwich’ type immunoassay. We then assessed levels of hK6 in brain extracts from normal individuals and patients with Alzheimer’s disease.

Results: The hK6 assay was developed using a combination of two antibodies (a mouse monoclonal and a rabbit polyclonal). Purified recombinant hK6 was used as a calibrator. The detection limit of the assay was 0.05 μg/L. The intra and inter-assay coefficient of variation was less than 6.5%. We found no detectable cross-reactivity by the homologous proteins hK2, hK3, hK8, hK10, hK11, hK13 and hK14. The hK6 concentration in human brain tissue extracts from healthy (n = 24) and Alzheimer’s patients (n = 55) were 10.1 ± 1.0 and 3.39 ± 0.26 μg/g of total protein (mean ± SE), respectively (p < 0.001). Similar differences were seen when the tissues were stratified by brain region (occipital, parietal, frontal and temporal cortex).

Conclusions: We conclude that the newly developed hK6 immunoassay is suitable for quantification of hK6 protein in biologic fluids and tissue extracts. The brain of Alzheimer’s disease patients contains significantly less hK6 than the brain of nonaffected individuals. The possible connection of hK6 with the pathogenesis of Alzheimer’s disease merits further investigation.

Introduction

Mammalian serine proteases are involved in many biologic functions including coagulation and fibrinolysis, digestion, activation or inactivation of hormones, receptors, cytokines, etc. The human kallikrein gene family consists of 15 serine protease genes (designated KLK1-KLK15) which share significant sequence homologies at the DNA and amino acid level (40–80%). Many of these genes are regulated by steroid hormones [1]. The human kallikrein 6 gene (KLK6), encodes for a secreted serine protease (hK6) [2]. hK6 has been identified by several laboratories and designated as zyme [2], protease M [3] or neurosin [4]. New nomenclature has now been adopted for human kallikrein genes [5]. The presence of aspartate in the binding pocket of hK6 predicts that this protein will produce trypsin-like cleavage. hK6 is synthesized as an inactive zymogen and is converted to an active enzyme by cleavage between Lys 21 and Leu 22 [4].

Messenger RNA encoding hK6 can be detected in some mammalian species but not in mice, rats or hamsters [2]. This gene is down regulated at metastatic breast cancer sites and is up-regulated in a subset of primary breast and ovarian tumors [3]. Serum hK6 concentration has been proposed as a biomarker for ovarian carcinoma [6]. hK6 is up-regulated in the breast carcinoma cell line BT474 by estrogens and progestins, and to a lesser extent by androgens [7]. This gene is highly expressed in brain tissue, including cerebellum and spinal cord and also, in kidney, spleen, mammary tissue and salivary gland [7].

Brain serine proteases are implicated in synaptic plasticity, developmental processes, neurite outgrowth and in neurologic disorders including Alzheimer’s disease (AD) [8], [9]. These actions may be mediated by the proteolytic cleavage of zymogen precursors and propeptides, the activation of specific cell surface receptors and/or by the degradation of extracellular matrix proteins [10], [11], [12]. hK6 has been proposed to have amyloidogenic potential in the brain and may play a role in the development and progression of AD by cleaving APP (amyloid precursor protein), along the amyloidogenic pathway [2].

Four genes have been implicated to date in familial forms of AD. Three of them, when mutant, cause autosomal dominant forms of the disease (β APP, presenilin 1, and presenilin 2) and one in which a naturally occurring polymorphism (ApoE4) represents a major risk factor. ApoE is associated with late onset AD, while the 3 other genes are associated with highly penetrant early onset AD. Despite the genetic heterogeneity, all four genes have been shown to increase the production and/or deposition of amyloid beta peptide in brain, triggering AD-related neuronal degeneration [13]. The pathologic hallmark of AD is the deposition of amyloid as cerebrovascular, diffuse and neuritic plaques(within the brain extracellular space) and neurofibrillary tangles (within neurons). The regions that are most affected are the hippocampus and cerebral cortex. The pathogenesis of AD is thought to involve the disregulated expression or abnormal processing of APP [14], [15].

In recent years two cerebrospinal fluid (CSF) biochemical markers have emerged. Increased levels of CSF-tau and decreased CSF levels of Aβ_42, are good markers for AD [16]. Direct measurements of Aβ isoforms in postmortem brain tissue of patients dying with presenilin1-linked familial form of AD, also show marked increases in the amount of Aβ₄₂ compared to control brain tissue and to brain tissue from subjects with sporadic AD [17]. In the recent Consensus Report of the Working Group on ‘Molecular and Biochemical Markers of Alzheimer’s Disease’, it was recognized that although many molecular and biochemical markers for AD have been proposed, none has achieved universal acceptance or, for that matter, met the proposed criteria for an ideal biomarker [18].

Given the tremendous interest on proteases in the development of Alzheimer’s disease [14] and the recent demonstration of highly expressed levels of hK6 in various parts of human brain [7], we set out to develop an immunoassay and to examine and compare the levels of this protease in brain tissue of Alzheimer’s disease patients and nonaffected controls. We hypothesize that hK6 and other serine proteases of the kallikrein family may play a role in the development and progression of Alzheimer’s disease.