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Keywords:

  • Alzheimer's disease;
  • amyloid;
  • glycosaminoglycan;
  • sulfation

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Alzheimer's disease is associated with abnormal accumulation of Aβ, which is produced from the β-amyloid precursor protein (APP) by the β-site APP-cleaving enzyme (BACE1) and γ-secretase. Our previous studies showed that heparin can decrease APP processing by decreasing the levels of BACE1 and ADAM10. In this study, we examined the effects of glycosaminoglycans (GAGs) on APP processing and Aβ production with the aim of understanding the specificity of the effects. Various GAG analogs were incubated with primary cortical cells derived from APP (SW)Tg2576 mice and the level of APP, proteolytic products of APP and APP-cleavage enzymes were measured. The effect of GAGs on APP processing was both size- and sulfation-dependent. 6-O-Sulfation was important for the effect on APP processing as heparin lacking 6-O sulfate were less potent than native heparin. However, deletion of carboxyl groups on heparin had no significant effect on APP processing. Our studies suggest that there is structural specificity to the effect of GAGs on APP processing and that certain GAGs have a greater effect on Aβ production than others. This suggests that it might be possible to alter the structure of GAGs to achieve more specific inhibitors of APP processing that can cross the blood–brain barrier.

Abbreviations used
AD

Alzheimer's disease

APP

amyloid precursor protein

BACE1

β-site APP-cleaving enzyme-1

BBB

blood–brain barrier

GAGs

glycosaminoglycans

HS

heparan sulfate

LH

lung heparin

LMW

low molecular weight

MH

mucosal heparin

PPS

pentosan polysulfate

Alzheimer's disease (AD) is an irreversible neurodegenerative disease (Ferri et al. 2005) that is characterized by the deposition of amyloid plaques and neurofibrillary tangles in the brain (Kidd 1964; Terry et al. 1964). The major component of the amyloid plaque is a 40–42 amino acid residue polypeptide (Glenner and Wong 1984; Masters et al. 1985) called the β-amyloid protein (Aβ). Aβ is generated from the β-amyloid precursor protein (APP) (Kang et al. 1987) by cleavage by the β-site APP-cleaving enzyme-1 (BACE1) (Sinha et al. 1999; Vassar et al. 1999; Yan et al. 1999; Cai et al. 2001) to liberate a C-terminally truncated fragment (C99), which is subsequently cleaved by γ-secretase (Haass et al. 1992). Both ADAM10 and ADAM17, also known as tumor necrosis factor-α-converting enzyme or tumor necrosis factor-α-converting enzyme (TACE), are α-secretases that can cleave APP within the Aβ sequence to produce an 83-amino acid fragment known as C83 (Sisodia et al. 1990; Sisodia 1992; Buxbaum et al. 1998). Cleavage by either of these α-secretases precludes formation of Aβ (Esch et al. 1990).

Oligomeric forms of Aβ are now thought to be the major toxic species in the initiation and development of AD (Lambert et al. 1998; Hartley et al. 1999; Kim et al. 2003; Haass and Selkoe 2007). Considering the central role of Aβ in the pathogenesis of AD, one of the main therapeutic strategies is to decrease the production of Aβ (Small et al. 2004).

Our studies (Cui et al. 2011) and those of other groups (Snow et al. 1988; Kisilevsky et al. 1995; Dudas et al. 2002; Scholefield et al. 2003; Bergamaschini et al. 2004) raise the possibility that glycosaminoglycans (GAGs) or GAG analogs may be effective in the treatment of AD. Peripheral administration of enoxaparin, a low molecular weight (LMW) heparin, has been reported to reduce Aβ load in the brain (Bergamaschini et al. 2004). Heparin can potentially influence Aβ production by disrupting β-secretase processing of APP. Leveugle et al. (1997) first reported that heparin stimulates β-secretase cleavage of APP in a cultured cell line. Heparin binds close to the prodomain of the BACE1 zymogen (proBACE1), and this binding stimulates proBACE1 activity (Beckman et al. 2006; Klaver et al. 2010). In contrast, Scholefield et al. (2003) reported that heparan sulfate and its more highly sulfated analog, heparin, can inhibit BACE1 activity and decrease Aβ production in cell culture. Our more recent studies have shown that treatment with heparin can lower Aβ secretion from primary cortical cells (Cui et al. 2011). However, although heparin can bind directly to BACE1, decreased secretion of Aβ in cell culture is because of a decrease in the level of BACE1, rather than direct inhibition of the enzyme.

The development of GAG analogs which can be used for the treatment of AD will require the identification of high-potency compounds that have the ability to cross the blood–brain barrier (BBB). Several reports indicate that low molecular weight GAGs can penetrate the BBB (Leveugle et al. 1998; Ma et al. 2002) and this idea is further supported by the observation that peripheral administration of enoxaparin can lower brain amyloid load (Bergamaschini et al. 2004). However, developing high-affinity compounds that can inhibit Aβ production may be problematic. Our studies showed that the most potent GAG heparin inhibits Aβ production in cell culture at micromolar concentrations (Cui et al. 2011). However, the development of GAG analogs which can be used for the treatment of AD may require high-potency compounds acting in the nanomolar concentration range.

The pattern of sulfation of heparan sulfate (HS) may provide specificity for binding to certain proteins. For example, studies by Nurcombe et al. (1993) have shown that the specificity of HS for binding to fibroblast growth factor receptors is controlled by the sulfation pattern. Similarly, the fine structure of HS may regulate syndecan-1 function (Sanderson et al. 1994), whereas a specific HS sulfation pattern regulates retinal axon targeting (Irie et al. 2002). Studies by Patey et al. (2006) show that specific sulfation patterns on heparin derivatives can result in high-affinity compounds with great selectivity for inhibition of BACE1 activity and reduced activity against Factor Xa and other proteases. Indeed, the different sulfation patterns of different HS species may reflect the need to bind specifically to different ligands. On this basis, then, it may be possible to alter the sulfate pattern of GAGs to achieve high affinity and specific effects on APP metabolism and Aβ secretion.

In view of the relationship of GAG size to BBB permeability and of sulfation pattern to binding specificity, the aim of this study was to examine the role of molecular size and sulfation of GAGs on APP processing and Aβ production in primary cell cultures. We tested the effects of various GAGs and sulfated polysaccharides on APP processing in cortical cells derived from transgenic mice expressing human APP695 with the Swedish familial AD mutant (Tg2576 mouse). These mice were used for the study because we wished to examine effects on human APP processing and because human APP and its fragments can be more easily detected by existing anti-human antibodies than rodent APP and Aβ. Our study shows that LMW heparin species can alter APP processing and that the effect of heparin on APP processing is dependent upon the degree of sulfation. Although no high-potency GAG analogs were identified in this study, the results demonstrate that there is structural specificity to the effect of GAGs on APP, raising the possibility that high-affinity BBB-permeable GAGs may eventually be identified.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Materials

Heparan sulfate (HO-10595) and 12.5-kDa heparin were purchased from Celsus Laboratories, Inc. (Cincinnati, OH, USA). Bovine lung heparin (LH) was from Calbiochem (Melbourne, Australia). Porcine mucosal heparin (MH), pentosan polysulfate (PPS), 6-kDa heparin (H2149), 3-kDa heparin (H3400), chondroitin sulfate A (ChS A), chondroitin sulfate B (ChS B), chondroitin sulfate C (ChS C), fucoidan, monoclonal anti-β-actin antibody, rabbit anti-BACE1 (EE-17) antibody, and polyclonal anti-APP C-terminal antibody (APP-CT) were purchased from Sigma-Aldrich Pty. Ltd. (Sydney, Australia). Rabbit anti-ADAM10 (ab1997) and rabbit anti-ADAM17 (ab2051) were purchased from Sapphire Bioscience Pty. Ltd. (Waterloo, Australia). Monoclonal anti-Aβ antibody 6E10 was from Covance Pty. Ltd. (North Ryde, Australia). Neurobasal medium and B27 supplement were purchased from Invitrogen (Mulgrave, Australia). Mouse and rabbit horseradish peroxidase-conjugated secondary antibodies were purchased from DAKO (Campbellfield, Australia). Complete mini protease inhibitor cocktail tablet was from Roche Diagnostics (Castle Hill, Australia).

Mucosal heparin (5-kDa MH) was prepared by treatment of periodate-oxidized mucosal heparin with sodium hydroxide, followed by reduction with sodium borohydride and acid hydrolysis (Islam et al. 2002). The resulting heparin fragments had an average degree of polymerization of 16, corresponding to an average molecular weight of 5 kDa. Mucosal heparin lacking the 2-O-sulfate group (MH de 2S), N-sulfate group (MH de NS), all sulfate groups (MH de S), or MH with the carboxyl group removed (MH CR) were prepared as described previously (Klaver et al. 2010). Mucosal heparin lacking 6-O sulfate (MH de 6S) was prepared by the treatment of the pyridinium salt of heparin with N,O-bis(trimethylsilyl)acetamide in pyridine for 2 h at 60°C (Matsuo et al. 1993). This procedure resulted in specific 6-O-desulfation of MH without depolymerization or other chemical changes.

Primary cortical cell culture

Cortical cells were prepared from brains of newborn (P0) Tg2576 mice (Taconic Farms Inc., Hudson, New York, USA). All animal experiments were approved by the University of Tasmania Animal Ethics Committee. Cerebral cortices were dissected in Neurobasal medium and incubated with 0.25% (w/v) papain and 0.06% (w/v) deoxyribonuclease I (DNase I) mixture for 20 min at 37°C, followed by three washes with Neurobasal medium. Cells were then separated by gentle mechanical dissociation and 3 × 105 cells/well were plated onto poly-d-lysine-coated 12-well culture plates, and maintained in 1.2 mL complete Neurobasal medium containing 2% B27 supplement, 1 mM glutamine, and 1% penicillin/streptomycin (10 000 units of penicillin and 10 000 μg of streptomycin stock) in an atmosphere containing 5% CO2 at 37°C. After 3 days in vitro (DIV), half of the culture medium was replaced with fresh complete Neurobasal medium. All experiments were performed at 7 DIV. Primary cortical cells were incubated with heparin derivatives or other compounds for 24 h prior to analysis.

SDS–polyacrylamide gel electrophoresis and western blotting

Culture medium was removed from cells for determination of Aβ and sAPPα. The cells were incubated with cold ristocetin-induced platelet agglutination buffer (150 mM NaCl, 50 mM Tris, 0.5% w/v Na-deoxycholate, 1% v/v Nonidet P-40, 0.1% sodium dodecyl sulfate, pH 7.4) containing protease inhibitor cocktail on ice for 10 min and the cell lysates were then harvested for determination of C99, BACE1, ADAM10, ADAM17, and total APP. The protein concentration of cell lysate was measured using the Bio-Rad DC protein assay kit (Bio-Rad Laboratories Pty. Ltd., Gladesville, Australia) with bovine serum albumin as standard.

The amount of Aβ40 or Aβ42 secreted into cell medium was determined using 15% Tris-bicine-urea sodium dodecyl sulfate–polyacrylamide gel electrophoresis as described previously (Klafki et al. 1996). Tris-glycine sodium dodecyl sulfate–polyacrylamide gel electrophoresis was used to determine sAPPα, BACE1, ADAM10, ADAM17, and total APP (Cui et al. 2011). To determine the level of C99 and C83 in cells, cell lysates were applied to 16.5% Tris-tricine gels (Cui et al. 2011). Briefly, cell lysates containing 12 μg of protein were applied to gels. After electrophoretic transfer of proteins onto a polyvinylidene difluoride membrane, the membrane was stained for C99 and C83 using a polyclonal anti-APP C-terminal antibody (1 : 2,000 dilution), which was raised against residues 676–695 of the APP695 sequence. The density of protein staining was quantified using Image J software (Research Service Branch; National Institute of Health, http://rsbweb.nih.gov/ij/index.html). The ratio of staining intensity of each protein to the staining intensity of β-actin was determined, and then each ratio was used to calculate a percentage relative to mean value for control incubations. The statistical tests were performed using SigmaPlot software (10.0v; Systat Software, Inc., San Jose, CA, USA). Statistical comparisons were made using one-way analysis of variance followed by Tukey's test for post hoc comparisons. Values of < 0.05 were considered statistically significant.

Quantitative real-time PCR

Total RNA was extracted from control or treatment cells using an SV total RNA isolation system from Promega (Sydney, Australia) following the manufacturer's instructions. The concentration and purity of the RNA were assessed spectrophotometrically at a wavelength of 260 and 280 nm using a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, MA, USA). RNA (250 ng) was reverse transcribed to form cDNA in a 20-μL reaction volume using a Fastlane cell cDNA kit from Qiagen Pty. Ltd. (Doncaster, Australia) following the manufacturer's instructions. PCR was performed using a QuantiFast SYBR Green PCR kit (Qiagen Pty. Ltd., Doncaster, Australia). Each reaction contained 10 μL 2x QuantiFast SYBR Green PCR Master Mix, 1 μL forward and reverse primers (both from 10 μM stock), 2 μL template cDNA, and 6 μL RNase-free water to a total volume of 20 μL. The reactions were carried out on a Rotor-Gene 6000 PCR cycler (Qiagen Pty. Ltd., Doncaster, Australia) according to the following protocol: pre-heat at 95°C for 5 min to activate DNA polymerase, followed by 50 cycles of 10 s at 95°C, and 30 s at 60°C. Primers with the following sequences were chosen: BACE1: Fw, 5′-CAGTGGGACCACCAACCTTC-3′, Rev, 5′-GCTGCCTTGATGGACTTGAC-3′; ADAM10: Fw, 5′-TAAGGAATTATGCC ATGTTTGC TGC-3′, Rev, 5′-ACTGAACTGCTTGCTCCACTGCA-3′; actin: Fw, 5′-ATGCTCCCCGGGCTGTAT-3′, Rev, 5′-CATAGGAGTCCTTCTGACCCATTC-3′; GAPDH: Fw, 5′-TGTGTCCGTCGTGGATCTGA-3′, Rev, 5′-TTGCTGTTGAAGTCGCAGGA G-3′. All primers were obtained from GeneWorks Pty. Ltd. (Hindmarsh, Australia). Fluorescence data were acquired at the end of each cycle, and a melt curve was determined at the end of cycling. Comparative concentration of target genes was normalized to the comparative concentration of housekeeping gene actin and the results were expressed as ratio of target gene and actin expression. Expression of additional housekeeping (e.g. glyceraldehyde-3-phosphate dehydrogenase, GAPDH) genes was also analyzed to verify the reliability of normalization relative to actin.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Effect of heparin fragments on APP processing

Initially, the relationship between the size of heparin fragments and the effect on APP processing was examined. Cortical cells derived from brains of newborn Tg2576 mice were treated with heparin fragments of different size (18 kDa, 12.5 kDa, 6 kDa, 5 kDa, and 3 kDa) for 24 h, and the level of total APP, sAPPα, C99, and Aβ was measured. For the measurement of Aβ, only the level of Aβ40 was quantified, because, as previously reported (Cui et al. 2011), very little Aβ42 was produced by the cells and the level could not be measured accurately (Fig. 1).

image

Figure 1. Effects of mucosal heparin (MH) fragments on amyloid precursor protein (APP) processing. Tg2576 mouse cortical cells were treated with different heparin fragments (100 μg/mL) for 24 h. Aβ40, C99, sAPPα, and total APP were measured using western blotting. (a) Typical western blots showing the effects of heparin fragments on the level of Aβ40, C99, sAPPα, and total APP. Figure also shows quantification of Aβ40 immunoreactivity (b), sAPPα immunoreactivity (c), C99 immunoreactivity (d), and total APP immunoreactivity (e) on the western blots. Asterisks show values that are significantly different from control incubations (p < 0.05, n = 8).

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In agreement with our previous report (Cui et al. 2011), treatment of cells with 100 μg/mL 18 kDa MH (MH 18) reduced the level of Aβ secretion to approximately 60% of control values (Fig. 1a and b). MH fragments ranging in molecular mass from 3 kDa to 12.5 kDa also decreased the level of Aβ secretion when incubated at the same concentration. However, 3-kDa and 5-kDa MH were less potent in decreasing Aβ secretion than 6- and 12.5-kDa MH (Fig. 1a and b). Associated with the decrease in Aβ secretion, there was also a decrease in the level of sAPPα secretion in the presence of 6-, 12.5-, and 18-kDa MH, although levels of sAPPα were not significantly reduced by 3-kDa and 5-kDa MH (Fig. 1a and c). The effect of 6-, 12.5-, and 18-kDa MH on the level of sAPPα was greater than the effect of 3 kDa MH. All MH fragments decreased the level of C99 in the cells, but did not decrease the level of total APP (Fig. 1a, c and d).

The results showed that MH with molecular masses between 3 kDa and 12.5 kDa can inhibit APP processing to Aβ. As our previous studies demonstrated that the decrease in α- and β-secretase processing of APP was because of a specific decrease in the level of BACE1 (β-secretase) and ADAM10 (α-secretase) (Cui et al. 2011), we also examined the effects of different-sized MH fragments on the level of β-secretase (BACE1) and two major α-secretases (ADAM10 and ADAM17) (Fig. 2). The level of BACE1 was significantly reduced to approximately 30-50% of control values by treatment with 3-, 5-, 6-, 12.5-, and 18-kDa MH fragments (Fig. 2a and b). The large MH fragments (6, 12.5, and 18 kDa) reduced the level of ADAM10 to approximately 10–20% of the control values (Fig. 2a and c). MH (5 kDa) also decreased the level of ADAM10, but to a lesser extent than the large MH fragments, and 3-kDa MH did not have any significant effect on ADAM10 levels (Fig. 2a and c). In contrast to ADAM10, the level of ADAM17 was not affected by any of the MH fragments tested (Fig. 2a and d).

image

Figure 2. Effects of mucosal heparin (MH) fragments on the level of β-site APP-cleaving enzyme-1 (BACE1), ADAM10, and ADAM17. Cells were treated with different heparin fragments (100 μg/mL) for 24 h and the level of BACE1, ADAM10, and ADAM17 were measured using western blotting. (a) Typical western blots showing the level of BACE1, ADAM10, and ADAM17 after MH fragments treatment. Figure shows quantification of BACE1 immunoreactivity (b), ADAM10 immunoreactivity (c), and ADAM17 immunoreactivity (d) on the western blots. Figure also shows relative mRNA level of BACE1 (e) and ADAM10 (f) determined by RT-PCR after 100 μg/mL MH treatment. Asterisks show values that are significantly different from control treatments (p < 0.05, n = 8).

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As the level of BACE1 and ADAM10 was reduced by treatment with heparin, we speculated that heparin might affect the expression of BACE1 and ADAM10 mRNA. The level of BACE1 and ADAM10 mRNA was measured by real-time PCR. However, we were unable to find any evidence that heparin treatment alters the expression of either BACE1 or ADAM10 (Fig. 2e and f). This suggested that GAGs probably decrease BACE1 or ADAM10 at a post-translational level.

Effects of different classes of GAGs on APP processing and Aβ production

To examine the specificity of the effect of MH on APP processing further, we compared the ability of different classes of GAG and other polysulfated compounds to alter Aβ production from APP. Primary cortical cells were treated with MH, LH, HS, chondroitin sulfate A (ChS A), chondroitin sulfate B (ChS B), chondroitin sulfate C (ChS C), and sulfated polysaccharides (PPS and fucoidan). The relative amount of sAPPα, C99, Aβ, BACE1, ADAM10, and ADAM17 was then measured by western blotting.

Initially, the effect of MH was compared with that of LH and HS. LH is a more highly sulfated form of heparin than MH, whereas HS from porcine mucosal tissue is less highly sulfated (Hileman et al. 1998). Incubation with LH led to a similar decrease in the level of Aβ as was observed with MH (Fig. 3a and b), whereas HS had no effect on Aβ levels in the culture medium. The chondroitin sulfates (ChS A, ChS B, and ChS C) did not reduce secretion of Aβ when incubated at a concentration of 100 μg/mL. Polysulfates, both PPS and fucoidan, at a concentration of 100 μg/mL, decreased Aβ secretion to a similar extent as MH and LH (Fig. 3a and b). Incubation with MH, LH, HS, PPS, and fucoidan caused a significant decrease in sAPPα secretion. However, sAPPα secretion was not affected by treatment with ChS A, ChS B, and ChS C (Fig. 3a and c). Several GAGs (MH, LH, and HS), PPS, and fucoidan also significantly decreased the level of C99, although levels of C99 were not altered by ChS A, ChS B, or ChS C treatment (Fig. 3a and d). All of these different GAGs had no significant effect on the level of total APP (Fig. 3a and e).

image

Figure 3. Effects of different classes of glycosaminoglycans (GAGs) and sulfated polysaccharides on amyloid precursor protein (APP) processing and Aβ production. Primary cortical cells from Tg2576 were treated with different GAGs or sulfated polysaccharides (100 μg/mL) for 24 h. Levels of Aβ40, C99, sAPPα, and total APP were measured using western blotting. (a) Western blots showing the effects of different GAGs and sulfated polysaccharides on the level of Aβ40, C99, sAPPα, and total APP. Figure also shows quantification of Aβ40 immunoreactivity (b), sAPPα immunoreactivity (c), C99 immunoreactivity (d), and total APP immunoreactivity (e) on the western blots. Asterisks show values that are significantly different from control incubations (p < 0.05, n = 8).

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Next, the effects of GAG analogs on BACE1, ADAM10, and ADAM17 were examined. MH, LH, PPS, and fucoidan (100 μg/mL) decreased the level of BACE1 and ADAM10 to approximately 50% and 20% of the control values, respectively (Fig. 4a, b, and c). HS had a small but significant effect on ADAM10, decreasing the enzyme by approximately 20%, but HS had no effect on the level of BACE1. ChS A, ChS B, and ChS C did not lower the levels of either BACE1 or ADAM10. None of the GAGs tested had any significant effect on the level of ADAM17 (Fig. 4a and d).

image

Figure 4. Effects of different classes of glycosaminoglycans (GAGs) and sulfated polysaccharides on the level of β-site APP-cleaving enzyme-1 (BACE1), ADAM10, and ADAM17. Primary cortical cells were treated with different GAGs and sulfated polysaccharides (100 μg/mL) for 24 h and the level of BACE1, ADAM10, and ADAM17 were measured by western blotting. (a) Typical western blots showing the level of BACE1, ADAM10, and ADAM17 after different types of GAGs and sulfated polysaccharides treatment. Figure also shows quantification of BACE1 immunoreactivity (b), ADAM10 immunoreactivity (c), and ADAM17 immunoreactivity (d) on the western blots. Asterisks show values that are significantly different from control treatments (p < 0.05, n = 8).

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Effects of selectively desulfated and decarboxylated heparin on APP processing

The structure specificity studies indicated that the highly sulfated MH and LH are more potent in their ability to disrupt APP processing than HS, which is less sulfated. The studies also showed that ChS A, ChS B, and ChS C, all of which are less sulfated than MH and LH (Hileman et al. 1998), had no significant effect. Furthermore, the highly sulfated polysaccharides PPS and fucoidan had similar effects to the highly sulfated GAGs (MH and LH) on APP processing. These results suggested that the degree of sulfation could be important for the ability of GAGs to regulate APP processing.

Therefore, to examine the role of specific negatively charged sulfate and carboxyl groups of heparin on APP processing, MH derivatives were generated without 2-O sulfate (MH de 2S), or 6-O sulfate (MH de 6S), or N-sulfate (MH de NS), or without any sulfates (MH de S), or with the carboxyl group removed (MH CR). Primary cortical cells were then treated with these MH derivatives and APP fragments and APP-cleavage enzymes were examined by western blotting.

Compared with native MH, which reduced Aβ secretion to approximately 50% of control values, both MH derivatives lacking one sulfate residue (MH de 2S and MH de NS) were less potent in lowering Aβ (Fig. 5a and b). MH derivatives lacking 6-O sulfate (MH de 6S) or those lacking all sulfate groups were unable to decrease Aβ secretion (Fig. 5a and b). Βy contrast, MH lacking the carboxyl group (MH CR) had a similar effect to native MH on Aβ secretion (Fig. 5a and b). MH CR decreased sAPPα and C99 similarly to native MH; however, desulfated MH had no significant effect on either the level of sAPPα or C99 (Fig. 5a, c and d). In addition, all of these MH derivatives had no effect on the level of total APP (Fig. 5a and e).

image

Figure 5. Effects of selectively desulfated and decarboxylated mucosal heparin (MH) on amyloid precursor protein (APP) processing and Aβ production. Primary cortical cells from Tg2576 were treated with MH derivatives (100 μg/mL) lacking 2-O sulfate (MH de 2S), 6-O sulfate (MH de 6S), N-sulfate (MH de NS), all sulfates (MH de S), or carboxyl group (MH CR) for 24 h. The levels of Aβ40, C99, sAPPα, and total APP were measured using western blotting. (a) Western blots showing the effects of MH derivatives on the level of Aβ40, C99, sAPPα, and total APP. Figure also shows quantification of Aβ40 immunoreactivity (b), sAPPα immunoreactivity (c), C99 immunoreactivity (d), and total APP immunoreactivity (e) on the western blots. Asterisks show values that are significantly different from control incubations (p < 0.05, n = 8).

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We also examined the role of sulfate and carboxyl groups of MH on the level of BACE1, ADAM10, and ADAM17. In contrast to MH, MH de 2S and MH de NS only weakly decreased BACE1 (Fig. 6a and b). Neither MH de 6S nor MH de S lowered the level of BACE1. However, MH CR was similar to native MH in its ability to lower the level of BACE1 (Fig. 6a and b). Native MH and MH CR possessed similar potency in decreasing ADAM10. MH de 2S significantly reduced ADAM10, but to a lesser extent than MH. By contrast, MH de 6S, MH de NS, and MH de S did not affect the level of ADAM10 (Fig. 6a and c). None of these MH derivatives had a significant effect on the level of ADAM17 (Fig. 6a and d).

image

Figure 6. Effects of selectively desulfated and decarboxylated mucosal heparin (MH) on the level of β-site APP-cleaving enzyme-1 (BACE1), ADAM10, and ADAM17. Tg2576 cortical cells were treated with 100 μg/mL MH derivatives lacking 2-O sulfate (MH de 2S), 6-O sulfate (MH de 6S), N-sulfate (MH de NS), all sulfates (MH de S), or carboxyl group (MH CR) and the level of BACE1, ADAM10, and ADAM17 were measured using western blotting. (a) Western blots showing the level of BACE1, ADAM10, and ADAM17 after treatment of different MH derivatives. Figure also shows quantification of BACE1 immunoreactivity (b), ADAM10 immunoreactivity (c), and ADAM17 immunoreactivity (d) on the western blots. Asterisks show values that are significantly different from control treatments (p < 0.05, n = 8).

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The relative potency of MH, MH CR, and fucoidan on the level of BACE1 was investigated. Cells were treated with 0, 1, 10, 100 μg/mL MH, MH CR, or fucoidan for 24 h, and the level of BACE1 was then measured by western blotting. Treatment with 1 μg/mL MH, MH CR, or fucoidan had no significant effect on the level of BACE1. However, incubation with 10 or 100 μg/mL MH or fucoidan significantly decreased the level of BACE1 to approximately 85% and 55% of control values, respectively (Fig. 7a and b). MH CR was less effective than MH and fucoidan, and significantly decreased the level of BACE1 to 85% and 70% of the control values at 10 and 100 μg/mL concentrations, respectively.

image

Figure 7. Concentration dependence of the effect of sulfated polysaccharides on the level of β-site APP-cleaving enzyme-1 (BACE1). Cells were treated with 0, 1, 10, 100 μg/mL mucosal heparin (MH), carboxyl group-removed mucosal heparin (MH CR), or fucoidan for 24 h. The level of BACE1 was measured by western blotting. Figure shows representative western blots of the effect of different concentrations of MH, MH CR, or fucoidan on the level of BACE1 (a). Figure also shows quantification of BACE1 immunoreactivity after the treatment with different concentrations of MH, MH CR, and fucoidan. (b) Asterisks show values that are significantly different from control treatments (p < 0.05, n = 6).

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

In this study, the effects of different sulfated carbohydrates on APP processing and Aβ production was examined in primary cortical cells derived from APP (SW) Tg2576 mice. Our results showed that both size and structure of GAGs are important for effects on APP processing. Indeed, the sulfation of MH is essential for an effect on APP processing. However, the carboxyl group on MH was not important as deletion of the carboxyl group did not block the effect of MH on APP processing.

Lung heparin, which contains more highly sulfated disaccharide units than MH (Hileman et al. 1998), was similar to MH in its effects on APP processing. PPS and fucoidan, which are highly sulfated polysaccharides (Degenhardt et al. 2001; Li et al. 2008), also reduced APP proteolytic processing. As PPS and fucoidan have a similar degree of sulfation to MH and LH, the results support the view that sulfation is important for the effect on APP processing. The results also suggest that the backbone structure of the carbohydrate may not be as important for the effect on APP processing, as PPS and fucoidan, which do not belong to the GAG family, were as potent as MH and LH. The fact that polysulfated compounds that are not GAGs also altered APP processing suggests that other sulfated polysaccharides could be candidates for the screening of therapeutic agents for AD. Compared with the highly sulfated GAGs and polysaccharides, HS and chondroitin sulfate A, B, and C, which are less highly sulfated (Hileman et al. 1998), were less potent in lowering BACE1 and ADAM10. Furthermore, they did not lower Aβ levels. Taken together, these results indicate that sulfation is vital for the effect on the level of BACE1, ADAM10, and on APP processing.

To confirm the role of sulfation on APP processing further, individual sulfate groups were removed from MH and the effect of removal was determined. Our results indicated that the removal of any sulfate group decreased the potency of MH on APP processing. The 6-O sulfate was the most important of all the sulfate groups, as MH de 6S had no effect on the level of BACE1 and ADAM10. This finding is consistent with a previous study that reported that removal of 6-O sulfate of MH reduced the inhibitory potency of MH on BACE1 (Scholefield et al. 2003; Patey et al. 2006). Although our study indicated that the decreased β-secretase cleavage of APP is because of a decrease in the level of BACE1, rather than inhibition of the enzyme activity, these findings indicate the 6-O sulfate of MH is critical for the activity of MH. MH derivatives lacking all sulfate groups lacked the ability to disrupt APP processing. In contrast, removal of the carboxyl groups on MH did not attenuate the effects of MH on APP cleavage, suggesting that while sulfation of GAGs is important for inhibition of APP processing, the effect was not solely because of the negative charge on the carbohydrate. A concentration-dependence study indicated that MH lacking carboxyl groups had a similar potency to MH in its ability to decrease BACE1 (Fig. 7a and b). The results of this experiment suggest that while it may be possible to modify the structure of GAG to achieve more specific GAG derivatives for the treatment of AD, whether there are modified GAGs that possess a higher potency than MH for inhibiting APP processing is unclear.

These results provide evidence that it may be possible to selectively modify the structure of GAGs and reduce the unwanted side effects without changing the potency of GAGs on APP processing. For instance, the carboxyl group is essential for the anticoagulant and vasodilatory activity of heparin (Agarwal and Danishefsky 1986; Paredes-Gamero et al. 2012). Chemical removal of the carboxyl group of heparin could reduce these unwanted effects, but still potently inhibit APP processing.

Our results also show that the ability of GAGs to inhibit APP processing is dependent on chain length. A minimum GAG size of 6 kDa, which is equivalent to a length of 17 saccharide monomers, was necessary to achieve a similar effect on APP processing as that obtained with MH. While 6-kDa MH was similar to native 18-kDa MH in its effect on BACE1 and ADAM10, small heparin fragments (e.g. 3 kDa) only weakly reduced the level of BACE1 and ADAM10 and only weakly inhibited APP processing. Scholefield et al. (2003) previously reported that the inhibitory effect of MH on BACE1 activity is size dependent. Together with our results, this suggests that the size of MH is important for action on BACE1 activity.

Heparin fragment size may influence its activity in vivo. It has been reported that a chain length of 17 saccharides (approximately 6 kDa) is required for efficient thrombin inhibition (Petitou et al. 1999). The chain length of heparin is also important for its binding to fibroblast growth factor-2, as the tetrasaccharides or longer oligosaccharides of heparin are required to bind to fibroblast growth factor-2 and induce proliferation of chlorate-treated rat mammary fibroblasts (Delehedde et al. 2002). These data suggest that it may be possible to design GAGs, which can alter APP processing but which have fewer unwanted side effects.

It may also be possible to reduce the size of GAGs so that they can cross the BBB and still retain the ability to decrease Aβ production. Previous studies have shown that full-length MH cannot cross the BBB, whereas 3 kDa or smaller heparins can cross (Leveugle et al. 1998; Ma et al. 2002). In our study, the 3-kDa MH derivative decreased APP processing, albeit weakly, raising the possibility that small MH derivatives, without hemorrhagic side effects, could pass through the BBB and inhibit APP processing.

The mechanism by which polysulfated carbohydrates decrease BACE1 and ADAM10 and alter APP processing is unclear. The decreased level of BACE1 and ADAM10 was probably not caused by a decrease in BACE1 or ADAM10 mRNA, because BACE1 and ADAM10 mRNA levels were not significantly changed after MH treatment. This suggests that the effect was on some post-translational event such as BACE1 and ADAM10 turnover. However, further studies will be needed to delineate this mechanism.

Whether GAGs will have therapeutic value for the treatment of AD is still unclear. While it may be possible to design compounds which are more limited in their actions (i.e., affect APP processing and Aβ production without unwanted side effects) and which cross the BBB, it is still unclear whether high-potency compounds will be identified. In this regard, it was of particular interest to note that fucoidan was as potent as MH in lowering BACE1 and Aβ levels (Fig. 5a and b and Fig. 7a and b). Fucoidans are a group of polysaccharides derived from algae and seaweed. Because of their considerable structural diversity, it seems logical to investigate further the effect of other fucoidans on Aβ production.

Another consideration with regard to the suitability of GAGs as drugs for the treatment of AD is their action on sAPPα. GAGs also reduced the secretion of sAPPα in our study. However, some studies suggest that sAPPα may have neurotrophic actions (Saitoh et al. 1989; Milward et al. 1992; Mattson 1994; Smith-Swintosky et al. 1994; Meziane et al. 1998). To date, the side effects of decreasing sAPPα production are unknown. GAG derivatives which act specifically on the β-secretase cleavage pathway of APP may be needed. In our study, MH de 2S and MH de NS treatment decreased the secretion of Aβ but had no significant, or had only a small effect on the level of sAPPα and ADAM10. These findings suggest that it is possible to design GAG derivatives that selectively target the β-secretase cleavage of APP.

In summary, this study shows that there is structural specificity to the effects of GAG on APP processing. LMW heparins can cross the BBB (Leveugle et al. 1998; Ma et al. 2002) and potentially may attenuate Aβ-induced inflammation (Kisilevsky et al. 1995; Zhu et al. 2001), decrease Aβ aggregation (Kisilevsky et al. 1995), lower Aβ generation, and improve cognition (Bergamaschini et al. 2004; Timmer et al. 2010). Ultimately, it may be possible to design more potent GAG derivatives which act specifically to inhibit β-secretase cleavage of APP that can be used for the treatment of AD.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The authors have no conflicts of interest to declare. This work was funded by project grants the National Health and Medical Research Council of Australia.

References

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  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
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