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Keywords:

  • Alzheimer’s disease;
  • microglia;
  • neuron;
  • neurosin

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Abstract Neurosin, a novel type of trypsin-like serine protease, has been shown to be preferentially expressed in human brain by northern blotting. We examined neurosin immunolabeling in the brains of neurologically normal persons and patients with Alzheimer's disease (AD) and with Parkinson's disease. We also identified the expression of the mRNA for neurosin by in situ hybridization histochemistry and reverse transcription–polymerase chain reaction (RT-PCR). The neurosin antibody stained all of the nuclei of various cell types. In neurons, there was also staining of neuronal cytoplasm, nucleoli and their processes. In AD, staining of neurons with processes was rare in the damaged areas. Some senile plaques, extracellular tangles and Lewy bodies were also positive for neurosin. Expression of the mRNA for neurosin was seen in neurons in the gray matter, and in microglial cells in the white matter. In AD, the intensity of the signal for neurosin mRNA in the gray matter was decreased compared with normal control brains. The relative levels of neurosin mRNA in AD brains, measured by RT-PCR, were lower than those in controls. These results suggest that in human brain neurosin plays various physiological roles, and that in AD this molecule, like other serine proteases, may have a role in the degradation of such substances as β-amyloid protein.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Brain serine proteases are implicated in developmental processes, synaptic plasticity, and in neurological disorders including Alzheimer's disease (AD). Recently, Davies et al. reported high levels of expression of tissue plasminogen activator, 30 kDa serine protease from rat natural killer cell line (designated RNK-Met-1), brain serine protease 1 (BSP1) and 2 (BSP2) from rat hippocampus. 1 Both BSP1 and BSP2 are newly discovered trypsin-like proteases, and BSP1 has been identified as a mouse serine protease ‘neuropsin’. 2 Recently, we cloned a novel trypsin-like serine protease preferentially expressed in brain and designated it ‘neurosin’. 3 The predicted protein consists of 244 amino acids and shows some similarity to other members of serine protease family. There is 28.4% homology with human trypsinogen I, 26.3% with human trypsinogen II, 22.9% with human kallikrein, 13.8% with human Factor X and 12.5% with human chymotrypsinogen, but it does not cleave substrates for thrombin, chymotrypsin and plasmin. This indicates that neurosin acts like trypsin in the interstitial space of the brain. Northern blot analysis has clearly shown the relatively specific expression of neurosin mRNA in brain, and a weak expression in the spleen. However, its physiological function in human brain has not been elucidated.

In the present study, using the brains of AD, Parkinson's disease (PD) and non-neurological patients, we have examined the localization of neurosin as well as the expression of its mRNA. We found nuclear localization of neurosin in all the cell types, and in other neuronal components, as well as positive immunolabeling to senile plaques (SP), neurofibrillary tangles (NFT) and Lewy bodies.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Antibody

A monoclonal antibody against neurosin was raised as follows. A bALB/c mouse was immunized with human recombinant neurosin purified from the conditioned medium of CHO cell lines transfected with neurosin cDNA. Spleen cells were removed from the mouse and fused with P3U1 myeloma cells. Hybridomas were screened for secretion of antibody, and the anti-neurosin antibody (α-hNS, IgG1) was prepared from one of these hybridomas.

Brain

All investigations were performed after approval by the Ethics Committee, Fukushimura Hospital. The brains from seven neurologically normal control patients (two patients died with pneumonia, three with lung cancer and two with liver cancer), where AD-type changes were lacking, six patients with AD and five with PD were examined. The diagnosis of AD was established using the criteria recommended by the National Institute on Aging. 4 The diagnosis of PD was established using the criteria by Calne et al.5 The ages of the three male and four female neurologically normal controls ranged from 60 to 82 years, of the two male and four female patients with AD from 67 to 82 years, and of the two male and three female patients with PD from 70 to 75 years. Brains in all instances were obtained 2–12 h after death.

Immunohistochemistry

Small blocks were dissected from the parietal lobes, hippocampus and midbrain, and fixed for 2 days in phosphate-buffered 4% paraformaldehyde. They were then transferred to a maintenance solution of 15% sucrose in 0.1 mol/L phosphate buffer, pH 7.4, and kept in the cold (4°C) until used. Sections were cut on a freezing microtome at 20 μm in thickness and stained by immunohistochemical procedures. 6 The dilution of anti-neurosin antibody was 1 : 1000. The sections were treated with the primary antibody for 48 h in the cold. They were then treated for 2 h at room temperature with biotinylated secondary antibody (Vector), followed by incubation in the avidin-biotinylated horseradish peroxidase (HRP) complex (Vector). Washing between steps was done with 0.1 mol/L phosphate-buffered saline containing 0.3% Triton X-100 (PBST). Peroxidase labeling was visualized by incubating with a solution containing 0.001% 3.3′-diaminobenzidine, 0.6% nickel ammonium sulfate, 0.05% imidazole and 0.0003% H2O2. When a dark purple product formed, the reaction was terminated. Sections were washed, mounted on glass slides, dehydrated with graded alcohol and cover-slipped with Entellan.

Western blot analysis

The specificity of the anti-neurosin antibody was tested by immunoblot analysis using brain tissues from two non-neurological and two AD patients. The parietal lobe samples were homogenized in buffer (five volumes of 20 mmol/L Tris-HCl, pH 7.4, 1 mmol/L EGTA, 1 mmol/L EDTA, 10 μmol/L leupeptin, 1 μmol/L pepstatin and 0.3 μmol/L aprotinin), the homogenates were centrifuged at 15 000 g for 30 min at 4°C, and the supernatants were collected as crude cytosolic fractions. The pellets were re-dissolved in the homogenization buffer and used as the membranous fractions. Protein measurement was done using BCA protein assay kit (Pierce, Rockford, USA). Aliquots of both fractions containing 50 μg of protein were electrophoresed on sodium dodecylsulfate–polyacrylamide gel (15% polyacrylamide gel; reducing conditions) and then transferred to a nitrocellulose membrane (25 mmol/L Tris-glycine buffer, pH 8.3, containing 20% methanol). The membrane was then pretreated with 5% skim milk powder in 25 mmol/L Tris-HCl (pH 7.4) containing 150 mmol/L NaCl (TBS), then incubated with anti-neurosin antibody (1 : 1000) in 2% skim milk/TBS, for 18 h at 4°C. The membrane was extensively washed in TBS + 0.1% Tween 20 (TBST), and then reacted with alkaline phosphatase-conjugated antimouse antibody (BRL, 1 : 5000, 2 h at room temperature) in TBST containing 1% skim milk. Following further washing, the membrane was developed in alkaline phosphatase substrate buffer (0.33 mg/mL nitroblue tetrazolium (BRL), 0.44 mg/mL 5-bromo-4-chloro-3-indolyl phosphate (BRL), 0.1 mol/L NaCl and 50 mmol/L MgCl2 in 0.1 mol/L Tris-HCl (pH 9.5)).

In situ hybridization histochemistry

For in situ hybridization histochemistry, a cDNA probe for human neurosin was constructed by polymerase chain reaction (PCR) using the oligonucleotides 5′-GCCCAGCCAAACTCTCTG-3′ and 5′-TGTTACCCCATGACACAAGG-3′ as primers. The primers span nucleotides 341–646 of the neurosin sequence. 3 Amplifications were done in 100 μL PCR buffer containing 10 pmol/L primer, 2 nmol/L dNTPs, 200 pmol/L digoxigenin-11-dUTP (Boehringer Mannheim Biochemica, Mannheim, Germany), 10 ng human neurosin cDNA and 5 U ampli-Taq DNA polymerase using a thermal cycler (Perkin-Elmer GeneAmp PCR System 9600; Wellesley, USA). After an initial PCR cycle of steps at 94°C for 5 min, 50°C for 30 s, and 72°C for 30 s, the remaining 29 cycles were the same except that the 94°C incubation was shortened to 30 s. During the last cycle, the 72°C step was extended to 5 min.

Frozen parietal lobe samples from three non-neurological and three AD cases were thawed and fixed in 4% paraformaldehyde for 2 days, then stored in 0.1 mol/L phosphate-buffered saline (PBS) containing 15% sucrose and 0.1% sodium azide. Sections were cut on a cryostat at 20 μm and washed in PBS. Sections were hybridized at 37°C for 2 days in a buffer containing 50% formamide, 4 × SSC, 0.2 × Denhardt's solution, 21 ng/mL of salmon sperm DNA and 250 ng/mL of the digoxigenin-11dUTP-labeled PCR DNA probe. After hybridization, sections were rinsed three times in 1 × SSC. Hybridization was detected by an enzyme-catalyzed color reaction using the DIG Nucleic Acid Detection Kit (Boehringer Mannheim Biochemica) according to the supplier's instructions. Negative controls were prepared by the same procedure but after pretreatment with RNase. Other control experiments were done using mixtures of either 10 : 1 or 1 : 1 of the digoxigenin-11dUTP-labeled and non-labeled PCR DNA probes, respectively.

After detection of the mRNA signal for neurosin by in situ hybridization, immunohistochemistry was used to characterize the labeled cells. Antibody against leukocyte common antigen (LCA; DAKO, 1 : 100 dilution) was used in the immunohistochemical procedures outlined above. The sections were mounted on glass slides and coverslipped with liquid paraffin.

Semiquantitative mRNA analysis by reverse transcription–polymerase chain reaction

Total RNA was isolated from freshly frozen human parietal lobes (two neurologically normal and three AD brains) by an acid guanidinium thiocyanate-phenol-chloroform procedure. The mRNA in the extracted total RNA was then converted to cDNA by reverse transcriptase (Superscript II; Life Tech-nologies). The resultant cDNA was subjected to PCR analysis for glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and human neurosin mRNA. The specific primer pair for GAPDH consisted of a sense primer, 5′-TGGTATCGTGGAAGGACTCATGAC-3′ and an antisense primer, 5′-AGTCCAGTGAGCTTCCCGTTCAGC-3′. The specific primer pair for neurosin was the same as that used in in situ hybridization histochemistry. The reaction mixture for PCR amplification consisted of 200 ng of cDNA, 0.5 μmol/L of each primer, 0.2 mmol/L of each of the four deoxynucleotide triphosphates and 1.25 U Taq polymerase (Takara Co., Kusatsu, Japan) in 50 μL 10 × PCR buffer (Takara Co.). The thermal cycle protocol used was denaturation at 94°C for 5 min, followed by cycles of 94°C, 1 min, 59°C, 1 min, 72°C, 1 min, with the reaction terminated by a final 7 min incubation at 72°C. A preliminary experiment demonstrated that the amount of PCR products increased exponentially from the 26th cycle with the GAPDH primers and with the neurosin primers, and then reached a plateau after the 35th cycle, respectively, due to the plateau effect. Therefore, we performed the PCR procedure for 35 cycles with the GAPDH primers, and with the neurosin primers. Controls were run without reverse transcriptase or without template cDNA to ensure that results were not due to amplification of any genomic or contaminating DNA. Each reaction mixture (8 μL) was mixed with 2 μL of 2% bromophenol blue and electrophoresed through 1.5% NuSieve 3 : 1 (FMC BioProducts, Rockland, USA) agarose gel for 30 min. Visualization was by incubation for 15 min in a solution containing 100 ng of ethidium bromide per milliliter.

For the cloning of the PCR product obtained by using the neurosin primer pair, an Original TA Cloning Kit (Invitrogen, Carlsbad, USA) was used. The ligation and transformation procedures were carried out strictly in accordance with the manufacturer’s instructions. The amplified plasmid DNA was extracted and purified using a Plasmid Maxi Kit (Qiagen GmbH, Hilden, Germany) and finally applied to a DNA sequencer (ALF, Pharmacia).

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

On western blotting, only one band of 29 kDa was detected in the whole homogenates and the membranous fractions from either AD or control brains ( Fig. 1). No band was detected in the cytosolic fractions.

image

Figure 1. Immunoblot analysis using non-neurological control brain tissue. A single band at approximately 29 kDa was recognized in the whole homogenate (W) and in the membranous fraction (M) by the antibody to neurosin. No band appeared in the cytosolic fraction (C).

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Immunohistochemistry using the anti-neurosin antibody gave staining of all the nuclei ( Fig. 2) and other components of neurons, such as nucleoli, processes and cytoplasm in controls ( Fig. 2a,c). The nuclear staining was similar in pattern in all brains studied ( Fig. 2).

image

Figure 2. (a,b) Immunostaining with the antibody to neurosin in neurologically normal control and (c–f) Alzheimer's disease (AD) brain tissues. (a) The neurosin-like staining in the parietal cortex gray matter is in neurons with processes as well as in the nuclei of various cells. (b) The staining in the parietal cortex white matter showed nuclei of glial cells. (c,d) Only a few neurons with processes were stained in the parietal cortex in AD. Nuclei and nucleoli of neurons were stained (arrowheads). Some senile plaques (arrows) were also positive to neurosin. (e) In the CA4 of the hippocampus, pyramidal neurons with processes were positively stained. (f) In the CA1 of the hippocampus, extracellular tangles were positive to neurosin. (a,b,d,e,f) Original magnification × 343, (c) original magnification × 172.

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In AD, neuronal staining with processes was rarely seen in the severely damaged areas such as parietal cortices ( Fig. 2c) and CA1 ( Fig. 2f). Only nuclear staining of neurons was shown in these sections. In CA4 of the hippocampus, however, staining was clearly visible in all neuronal components ( Fig. 2e). Some senile plaques were stained ( Fig. 2c,d) and extracellular neurofibrillary tangles were also positive to neurosin ( Fig. 2f). However, intracellular tangles were not stained.

In midbrain tissues, all cases showed positive immunolabeling for neurosin in all neurons in the oculomotor nuclei ( Fig. 3a). In the substantia nigra, some of the melanin-containing neurons were positive to neurosin in control brains ( Fig. 3b). In PD brain, neurosin-positive/melanin-containing neurons were rarely seen ( Fig. 3c). Lewy bodies were positive by the antibody to neurosin ( Fig. 3d).

image

Figure 3. (a,b) Immunostaining with the antibody to neurosin in neurologically normal control and (c,d) Parkinson's disease (PD) midbrain tissues. (a) In the oculomotor nuclei, many neurons were positive to neurosin as well as nuclei of glial cells. (b) Some melanin-containing neurons were positively stained. (c) In PD, the number of melanin-containing neurons was extremely decreased, and a few remaining neurons had positive immunolabeling for neurosin. (d) A Lewy body was stained by the antibody to neurosin. (a–c) Original magnification × 172, (d) original magnification × 343.

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In situ hybridization histochemistry revealed that the mRNA of neurosin was located in neurons in the gray matter ( Fig. 4a,b). The intensity of the mRNA signal for neurosin was less in AD than in non-neurological controls. In the white matter, some cells showed strong mRNA signals in both AD and control brains ( Fig. 4c). They were LCA-positive microglial cells ( Fig. 4d). The intensity of the signals was almost the same between AD and control brains. Control hybridization sections, which were incubated without the DNA probe or with a 1 : 1 mixture of the digoxigenin-11dUTP-labeled and non-labeled PCR DNA probe, were negative. When a 10 : 1 mixture of the digoxigenin-11dUTP-labeled and non-labeled PCR DNA probe was used, only a weak signal could be detected.

image

Figure 4. In situ hybridization histochemistry using polymerase chain reaction DNA probes for neurosin in (a) neurologically normal control and (b,c,d) AD brain tissues. (a) Positive signals for neurosin mRNA were seen in neurons in the parietal gray matter. (b) In Alzheimer's disease, a few weak signals were seen in the parietal cortex. (c) In the white matter, positive signals for neurosin mRNA were seen in some glial cells. (d) In situ hybridization histochemistry for neurosin (blue black) followed by immunohistochemistry with anti-leukocyte common antigen (LCA) antibody (brown). Cells in the white matter positive for neurosin mRNA were also positive for LCA. (a,b,d) Original magnification × 343, (c) original magnification × 172.

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The PCR products amplified by primers for GAPDH and neurosin are shown in Fig. 5. All five specimens used showed a single band of similar size for each pair of primers employed. There was a clear difference in the intensity of neurosin PCR products between the AD and control groups. In contrast, the intensity of GAPDH PCR products was almost the same in all cases, showing that this mRNA was a suitable internal standard.

image

Figure 5. Reverse transcription–polymerase chain reaction products amplified by the primers for neurosin and GAPDH. AD, Alzheimer's disease; NC, non-neurological control.

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Highly expressed PCR products obtained with neurosin primers from one normal control individual and one AD patient were subjected to sequencing analysis. Sequences derived from the PCR product confirmed that the target fragments corresponded to the same sequence in the human neurosin gene. 3

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

This present study clearly demonstrated that various cells in control brain contain neurosin. In contrast to the specific nuclear localization of neurosin in glial cell, neurons have positive immunolabeling in the cytoplasm, nuclei, nucleoli and their processes. Neurons in the gray matter and microglial cells in the white matter preferentially expressed the signals for the neurosin mRNA. In the damaged areas of AD as well as PD brains, few neurons contained neurosin. By in situ hybridization histochemistry and RT-PCR analysis, decreased expression of neurosin mRNA was also noted in AD. These findings suggest that this molecule may have various physiological functions in the human brain, but these are still unknown.

Localization of neurosin in some SP and ghost NFT in the AD brain may provide some clues to AD pathogenesis. Amyloid precursor protein (APP) exists in at least three different forms that differ in the number of amino acids and designated APP695, APP751 and APP770. The latter two contain a 56-amino acid domain that functions as a Kunitz-type serine protease inhibitor (KPI). To date, no clear consensus has emerged as to whether the levels of transcripts for isoforms containing a KPI-encoded region are increased or decreased in AD. However, very recently, Moir et al. reported that, by their detailed analysis, the brains of AD patients had a significantly higher proportion of a KPI-containing species of APP than the brains of unaffected individuals. 7 They suggested that the increase of these more amyloidogenic species may be one possible mechanism for amyloid deposition in sporadic AD. The secreted forms of 751/770 APP containing the KPI domain is identical to protease nexin-2 (PN-2). This PN-2 has been recognized as a potent inhibitor of several serine proteases such as trypsin, 8,9 chymotrypsin, 8,9 factor IXa, 10 Xa 11 and XIa. 12 Trypsin, 13,14 factor Xa, 15 XIa 16 and the other serine proteases, thrombin 17 and a novel chymotrypsione-like enzyme 18 have been suggested as enzymes for processing of APP. Furthermore, thrombin 19 and trypsin 13 have been shown to be localized on β-amyloid (Aβ) deposits in AD brain.

Three kinds of trypsin-like molecules other than neurosin have been identified in brain tissues. They are neuropsin (BSP1), BSP2 1 and trypsinogen IV. 14 However, their exact localization and the relationship with AD pathology have not been demonstrated. Here, we have shown that a novel trypsin-like protease, neurosin, may have a function in Aβ deposi-tion because of its localization in SP. Several studies suggest that Aβ may have to be membrane bound to be cleaved by unknown secretases. 20,21 Because neurosin is associated with a membrane-rich fraction, neurosin may also have a function for Aβ degradation. Our preliminary in vitro study showed Aβ degradation by neurosin (N.Y., unpubl. data, 1999). Further study to identify whether neurosin is α-secretase is in progress in the Research Institute for Neurological Diseases and Geriatrics. Positive staining of extracellular tangles by the antibody to neurosin may indicate that neurosin secreted from living neurons attaches to these pathological structures for proteolysis. Trypsin binding to NFT has also been reported. 13 Localization in Lewy bodies of neurosin might reflect a proteolytic function for the aggregated structures.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES
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    Yamashiro K, Tsuruoka N, Kodama S et al. Molecular cloning of a novel trypsin-like serine protease (neurosin) preferentially expressed in brain. Biochem. Biophys. Acta 1997; 1350: 11 14.
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    Van Nostrand WE, Wagner SL, Farrow JS, Cunningham DD. Immunoprecipitation and protease inhibitory properties of protease Nexin-2/amyloid β-protein precursor. J. Biol. Chem. 1990; 265: 9591 9594.
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    Wiegan U, Corbach S, Minn A, Kang J, Müller-Hill B. Cloning of the cDNA encoding human brain trypsinogen and characterization of its product. Gene 1993; 136: 167 175.
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    Haas C, Aldudo J, Cazorla P et al. Proteolysis of Alzheimer's disease β-amyloid precursor protein by factor Xa. Biochem. Biophys. Acta 1997; 1343: 85 94.
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    Saportito-Irwin SM & Van Nostrand WE. Coagulation factor XIa cleaves the RHDS sequence and abolishes the cell adhesive properties of the amyloid β-protein. J. Biol. Chem. 1995; 270: 26 265 26 269.
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    Igarashi K, Murai H, Asaka J. Proteolytic processing of amyloid β protein precursor (APP) by thrombin. Biochem. Biophysic. Res. Com. 1992; 185: 1000 1004.
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    Little SP, Dixon EP, Norris F et al. Zyme, a novel and potentially amyloidogenic enzyme cDNA isolated from Alzheimer’s disease brain. J. Biol. Chem. 1998; 272: 25 135 25 142.
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    Akiyama H, Ikeda K, Kondo H, McGeer PL. Thrombin accumulation in brains of patients with Alzheimer's disease. Neurosci. Lett. 1992; 146: 152 154.
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    Roberts SB, Ripellino JA, Ingalls KM, Robakis NK, Felsenstein KM. Non-amyloidogenic cleavage of the β-amyloid precursor protein by an integral membrane metalloendopeptidase. J. Biol. Chem. 1994; 269: 3111 3116.
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    Vassilacopoulou D, Ripellino JA, Tezapsidis N, Hook YYH, Robakis NK. Full-length and truncated Alzheimer amyloid precursors in chromaffin granules: Solubilization of membrane amyloid precursor is mediated by an enzymatic mechanism. J. Neurochem. 1995; 64: 2140 2146.