Neurological Sciences

, Volume 34, Issue 4, pp 445–448

Cerebrospinal fluid cathepsin B and S

Authors

  • Elin Nilsson
    • Department of Medical Sciences, Clinical Chemistry Uppsala University Hospital
  • Constantin Bodolea
    • Department of Anaesthesia and Intensive CareUniversity of Cluj Napoca
  • Torsten Gordh
    • Department of Surgical SciencesUppsala University Hospital
    • Department of Medical Sciences, Clinical Chemistry Uppsala University Hospital
Original Article

DOI: 10.1007/s10072-012-1022-0

Cite this article as:
Nilsson, E., Bodolea, C., Gordh, T. et al. Neurol Sci (2013) 34: 445. doi:10.1007/s10072-012-1022-0

Abstract

Cathepsins are increased in the brain of elderly animals. We investigate the presence of cathepsin B and S in human cerebrospinal fluid (CSF) plasma and the associations with cystatin C, age and sex. We measured cathepsin B and S concentrations in CSFs from 118 persons, undergoing elective surgical procedures, with ELISA. Both cathepsin B and cathepsin S were positively correlated with age. No correlation was observed between cathepsin B or S and length, height or body mass index. Both cathepsin B and S were positively correlated to the cystatin C concentration in CSF. Calculated reference intervals were 4,893–17,636 pg/mL for cathepsin B and 2,681–11,459 pg/mL for cathepsin S. Elderly individuals had significantly higher levels of both cathepsin B (rs = 0.38, p = 0.00002) and cathepsin S (rs = 0.35, p = 0.0001) in CSF.

Keywords

Cathepsin BCathepsin SCerebrospinal fluidAge

Introduction

The papain protease family includes a group of human lysosomal cysteine peptidases known as cathepsins (B, C, F, H, K, L, O, S, V, W and X). Cathepsins are the most abundant of all cystein proteases and they have a broad tissue distribution [12]. Cathepsins were originally identified as lysosomal proteases but recent work has shown that cathepsins play important roles also in the extracellular space as well as in the cytosol and nucleus.

Cathepsin B (EC 3.4.22.1) possesses both endopeptidase and exopeptidase activities [7]. It participates in intracellular degradation and turnover of proteins. It has also extracellular effects degrading extracellular matrix-components such as laminin, type IV collagen and fibronectin [4, 8].

Cathepsin S (EC 3.4.22.27) is the key protease responsible for the removal of the invariant chain chaperone molecules, thus controlling antigen presentation to CD4 T-cells by MHC II molecules [5]. Human Cathepsin S is synthesised as a preproenzyme of 331 amino-acid residues consisting a signal peptide (residues 1–16), a pro region (residues 17–114), and the mature enzyme (residues 115–331).

The levels of cathepsin B has been shown to be increased in the neostriatum of aged rats [16]. In old mice, cathepsin S expression was increased in microglial cells and a larger number of neuronal cells stained positive for cathepsin S [20]. Cathepsins are involved in many normal and pathological processes. Inhibition of cathepsin B in transgenic mice resulted in significantly decreased levels of amyloid-β in the brain and CSF and improved memory [10, 11]. Brain tissue from both Alzheimer’s disease (AD) and Down’s syndrome patients have increased cathepsin S immunoreactivity in neocortical and hippocampal neurons and glia [13]. Cathepsin S has been shown to cause an increased production of amyloid beta-peptides indicating a role for cathepsin S in Alzheimer’s disease [14]. Cathepsin S is also considered to play a role in chronic pain. Cathepsin S is up-regulated in rat dorsal root ganglia following peripheral nerve injury [1]. Injection of cathepsin S induce pain reactions [2] while administration of inhibitors to cathepsin S can reduce neuropathic pain [13]. Cathepsin S expression is up-regulated in astrocytoma cells [6]. Several of these diseases are age related. This, in combination with increased levels of these cathepsins with age in animal models, indicates that cathepsin levels may be influenced by age also in humans.

We have previously shown that there are higher cystatin C levels in cerebrospinal fluid (CSF) than in plasma [17]. Cystatin C is the main endogenous inhibitor of cathepsins. Thus, it seems likely that increased concentrations of cathepsins could be balanced by high levels of cystatin C. The aim of this study was thus to study the effects of age and cystatin C concentrations on the levels of the cathepsins B and S in human CSF.

Materials and methods

Participants and samples

Cerebrospinal fluid samples (n = 118) were collected from patients undergoing surgical procedures under spinal anesthesia at the University Hospital, Cluj Napoca, Romania. The spinal punctures were performed using a 25 G spinal needle. The position of the needle tip in the subarachnoid space was confirmed prior to sampling. The CSF sample was prelevated by free flow into a sterile tube. The samples were stored at −70 °C prior to analysis. All patients gave informed consent prior to sampling. The collection of the samples was approved by the local ethical board of the University of Medicine Cluj Napoca, Romania.

Cathepsin B and S ELISA

Serum cathepsin B and S were analyzed by commercial sandwich ELISAs (DY2176 and DY1183, R&D Systems, Minneapolis, MN, USA), according to the recommendations of the manufacturer. Briefly, the microtitre plates were first coated with specific monoclonal antibodies and then blocked with bovine serum albumin. After blocking and washing, standards and samples (diluted 1:10 for cathepsin B and diluted 1:20 for cathepsin S in bovine serum albumin) were added to the wells. During the following incubation period, cathepsin present in standards and samples were bound to the immobilized antibody. After a thorough wash, a biotinylated antibody was pipetted into the wells. After incubation with the biotinylated antibodies and washing a streptavidin-HRP conjugate was added, and following an additional incubation-and wash step, a substrate solution was added and color developed in proportion to the amount of bound cathepsin. The color development was subsequently stopped and the intensity of color was measured by photospectrometry. Calculation of results was performed according to the manufacturer’s recommendations. The assays had a total coefficient of variation (CV) of approximately 7 %.

Cystatin C assay

Serum cystatin C (reagent: 1014, Gentian, Moss, Norway) were analyzed on an Architect Ci8200® analyzer (Abbott, Abbot Park, IL, USA). The total analytical imprecision of the cystatin C method was 1.1 % at 1.25 mg/L and 1.4 % at 5.45 mg/L.

Statistical calculations

Figures were prepared in Excel 2003 (Microsoft Corporation, Seattle, WA, USA) and statistical analysis was performed with Statistica 7.1 (StatSoft, Tulsa, OK, USA). Comparisons between cathepsins and age and sex were performed with Spearman rank correlation. Calculations of reference intervals were performed by bootstrap estimation using RefVal 4.0 (Department of Clinical Chemistry, Rikshospitalet, Oslo, Norway). The statistical program performs log transformations of the values to achieve normal distribution. When normal distribution of the material is achieved, the program then randomly selects subsets from this population and calculates reference intervals. This calculation is performed 500 times and this forms the basis for the calculation of the reference intervals.

Results

Patient characteristics

One-hundred and eighteen individuals were included (13 females, 105 males) in the study. The median age of the patients was 65 years (range 21–82 years). The median weight was 76 kg (range 49–104 kg) and the median height was 170 cm (range 150–189 cm).

CSF cathepsin B

The median CSF cathepsin B concentration was 8,399 pg/mL with an interquartile range of 7,007–9,896 pg/mL. There were significant positive correlation between cathepsin B and age (rs = 0.38, p = 0.00002, Fig. 1) and between cathepsin B and cystatin C (rs = 0.46, p ≤ 0.000001). There were no significant correlations between cathepsin B levels and sex, body weight or height.
https://static-content.springer.com/image/art%3A10.1007%2Fs10072-012-1022-0/MediaObjects/10072_2012_1022_Fig1_HTML.gif
Fig. 1

Correlation between cathepsin B values and age in individual patients. The age of the patient (x-axis) was plotted against the cathepsin B concentration (y-axis)

CSF cathepsin S

The median CSF cathepsin S concentration was 5,911 pg/mL with an interquartile range of 4,616–7,070 pg/mL. There were significant positive correlation between cathepsin s and age (rs = 0.35, p = 0.0001, Fig. 2) and between cathepsin S and cystatin C (rs = 0.56, p ≤ 0.000001). There were no significant correlations between cathepsin S levels and sex, body weight or height. A significant correlation was observed between cathepsin B and cathepsin S levels (rs = 0.64, p ≤ 0.000001, Fig. 3).
https://static-content.springer.com/image/art%3A10.1007%2Fs10072-012-1022-0/MediaObjects/10072_2012_1022_Fig2_HTML.gif
Fig. 2

Correlation between cathepsin S values and age in individual patients. The age of the patient (x-axis) was plotted against the cathepsin S concentration (y-axis)

https://static-content.springer.com/image/art%3A10.1007%2Fs10072-012-1022-0/MediaObjects/10072_2012_1022_Fig3_HTML.gif
Fig. 3

Correlation between age and cathepsin B and cathepsin S values in individual patients

Reference intervals for cathepsin B and S

Reference intervals (0.025–0.975 fractions) calculated for the whole population was 4,893–17,636 pg cathepsin B/mL with bootstrap estimates. 0.90-Confidence intervals for the estimates were 3,884–5,902 and 10,545–24,727, respectively.

The corresponding reference interval for cathepsin S was 2,681–11,459 pg/mL (0.90-confidence intervals 1,975–3,388 and 7,587–15,332).

Discussion

Cathepsins have been implicated in many types of normal and pathological processes in the central nervous system [15] and both cathepsin B and S have been shown to influence the survival of neurological cells. Cathepsin S deficient mice had significantly shorter survival of facial motor neurons after nerve injury [9] while cathepsin B has been implicated in three major pathways of cell death; apoptosis, necrosis and autophagic cell death [18]. Cathepsin B is suggested to be involved in amyloid-β (Aβ) processing and Alzheimer’s disease [19] while cathepsin S has been shown to be important for the maintenance of neuropathic pain and spinal microglia activation [1]. Neuropathic pain becomes manifested when nerves normally transmitting pain signals are damaged or dysfunctional, disturbing the normal signaling processes. This impairment may results in tingling, burning or painful sensations. Neuropathic pain is a serious and debilitating condition that affects large number of patients. Current therapies are only partially effective so there is a large unmet medical need for new therapeutics to treat neuropathic pain. To be able to develop new treatment, we need to increase our knowledge on the mechanisms involved in chronic pain.

Cathepsin S inhibitors are a novel therapeutic approach for the treatment of neuropathic pain which has gained considerable interest. The molecule is activated at the site of injury in models of neuropathic pain induced by induced nerve damage. Intrathecal delivery of recombinant cathepsin S induced hyperalgesia and allodynia [2]. Pharmacological inhibition of cathepsin S, either centrally or peripherally, reverses hyperalgesia in these models.

Both cathepsin B and S are inhibited by the endogenous inhibitor cystatin C. The concentrations of cystatin C is approximately five to tenfold higher in CSF than in plasma. The cathepsin assays are not affected by the presence of cystatin C in the samples, according to the manufacturer of the ELISA kits. This is supported by the positive correlation between cathepsins and cystatin C concentrations in this study. Thus, an increase in the enzyme in CSF is in this study is usually accompanied by an increase in the inhibitor, thus balancing the activity of the enzyme.

There were significant positive correlations between the studied cathepsins and age. This is in agreement with previous studies showing increased levels of cathepsin B and S in the brain and CSF of elderly animals. Both Alzheimer’s disease and chronic pain increases with age. A change in the CSF concentrations of cathepsins could contribute to the development of these diseases. Further studies are warranted to explore cathepsin and cystatin C levels in CSF and their associations with diseases such as chronic pain and dementia.

Acknowledgments

This study was financially supported by the Uppsala University Hospital Research Fund and Uppsala Berzelii center.

Copyright information

© Springer-Verlag 2012