- Open Access
Heparan sulfate proteoglycan induces the production of NO and TNF-α by murine microglia
© Bussini et al; licensee BioMed Central Ltd. 2005
- Received: 22 April 2005
- Accepted: 16 July 2005
- Published: 16 July 2005
A common feature of Alzheimer's disease (AD) pathology is the abundance of activated microglia in neuritic plaques containing amyloid-beta protein (Aβ) and associated molecules including heparan sulfate proteoglycan (HSPG). Besides the role as pathological chaperone favouring amyloidogenesis, little is known about whether or not HSPG can induce microglial activation. Cultures of primary murine microglia were used to assess the effect of HSPG on production of proinflammatory molecules that are known to be present in neuritic plaques of AD.
HSPG stimulated up-regulation of tumor necrosis factor-alpha (TNF-α), production of inducible nitric oxide synthase (iNOS) mRNA and accumulation of TNF-α protein and nitrite (NO2-) in a time- and concentration-dependent manner. The effects of HSPG were primarily due to the property of the protein core as indicated by the lack of microglial accumulation of TNF-α and NO2- in response to denaturated HSPG or heparan sulfate GAG chains (HS).
These data demonstrate that HSPG may contribute to chronic microglial activation and neurodegeneration seen in neuritic plaques of AD.
- Nitric Oxide
- Alzheimer Disease
- Microglial Cell
- Heparan Sulfate
- Microglial Activation
Senile plaques in the Alzheimer disease (AD) brain are characterized by the presence of an amyloid core consisting of fibrillar Aβ, surrounded by a wreath of dystrophic neurites and activated microglial cells. Reactive microglia release a variety of potentially neurotoxic compounds, including cytokines and free radicals. Many research groups have provided evidence that deposition of aggregated Aβ is centrally involved in the chronic inflammatory process occurring in senile plaques of AD brain [1, 2].
Aβ is a 39- to 42-amino-acid peptide that arises from proteolytic processing of the amyloid precursor protein (APP) [3, 4]. The Aβ peptide that is found in the senile plaques and cerebrovascular deposits exists as a multimeric aggregate with a fibrillar appearance . Several other molecules have also been shown to be associated with Aβ deposits, and in vitro studies of fibrillogenesis suggest they may be important in the aggregation and persistence of the Aβ fibrils in vivo. These include apolipoprotein E, laminin, acetylcholinesterase, α1-antichymotrypsin and heparan sulfate proteoglycan (HSPG) [6–11].
HSPG is a multifunctional macromolecule characterized by a core polypeptide to which glycosaminoglycans (GAGs) are covalently attached. There are at least four different classes of HSPG present in AD, which are either associated with the cell membrane or with the extracellular matrix . HSPG has been consistently associated with both diffuse and neuritic plaques [13, 14]. Its early presence in AD pathological alterations as well as its immunohistochemical colocalization with all varieties of Aβ plaques, irrespective of their stage of maturation, have suggested that HSPG could play an active role in plaque formation. In this regard it has been proposed that HSPG facilitates Aβ deposition and/or promotes Aβ persistence by inhibiting clearance mechanisms, thus augmenting the formation of Aβ deposits in AD . Consistent with this hypothesis, in vitro studies have shown that HSPG can bind with high affinity to Aβ as well as to APP and it protects Aβ from protease degradation [16–19].
Besides its function in amyloidogenic pathways, HSPG might contribute to AD pathogenesis also through activation of microglial cells. This possibility has never been investigated. To study this we have assessed in "in vitro" cultures of mouse microglial cells, two known markers of their activation, i.e. production of the proinflammatory cytokine tumor necrosis factor-α (TNF-α) and expression of the mRNA for the inducible nitric oxide (NO) synthase (iNOS). This enzyme is generated in microglia in response to a variety of pro-inflammatory cytokines and bacterial products, such as lipopolysaccharide (LPS). In addition, we have measured in the culture media supernatants the accumulation of NO2-, a good proxy for generation of NO by iNOS. Our results show that HSPG might contribute to neurodegeneration in neuritic plaques of AD also through activation of microglial cells and the ensuing increased inflammatory response.
Heparan sulfate proteoglycan (HSPG), heparan sulfate (HS), lipopolysaccharide (LPS, from Escherichia coli 026.B6) were purchased from Sigma (St Louis, MO, USA) and dissolved in clinical pyrogen-free H2O. Levels of endotoxin in HSPG and HS stocks were measured by E-TOXATE (Limulus Amebocyte Lysate, LAL) kit purchased from Sigma (St Louis, MO, USA).
Preparation of Microglial Cultures
Mice were obtained from Charles River Laboratories, Inc. (Wilmington, MA, USA) and were used according to institutional guidelines that are in compliance with national (D.I. no. 116, G.U. suppl. 40, Feb. 18, 1992, Circolare No.8, G.U., 14 Luglio 1994) and international law and policies (EEC Council Directive 86/609, OJ L358, 1 Dec. 12, 1987; Guide for the Care and Use of Laboratory Animals, U.S. National Research Council, 1996). Primary murine microglial cultures were prepared as previously described . Briefly, cerebral cortical cells from day 1-old mice were dissociated with 0.25% trypsin and 0.1% DNAse (Sigma) and plated in 75 cm2 culture flasks (Corning, Acton, MA) in Dulbecco's Modified Eagle's Medium (DMEM) (Invitrogen Corporation Grand Island, NY, USA) containing 10% heat-inactivated FBS (Invitrogen Corporation Grand Island, NY, USA) and 100 μg/ml gentamicin (Invitrogen Corporation Grand Island, NY, USA). Dissociated glial cultures were maintained at 37°C with 5% CO2 and medium was replenished 4 days after plating. On day twelve of culture, flasks were shaken for 2 hours. The culture media supernatants (containing predominantly microglia and oligodendrocytes) were then collected and the cells were plated in either 96-well tissue plates (Nunc, Roskild, DK) at a concentration of 4 × 104 cells per 100 μl per well, or in 48-well tissue plates at a concentration of 25 × 104 per 500 μl per well, and maintained at 37°C with 5% CO2 for 1 hour. Loosely adherent oligodendrocytes were then removed from the cultures by gentle shaking of the culture plates by hand. After pouring off the culture media supernatants (containing oligodendrocytes), the adherent microglia were maintained at 37°C with 5% CO2 for subsequent treatment. The purity of microglial cultures was routinely assessed by staining with the F4/80-antibody (Serotec, Oxford, UK), which recognizes a glycoprotein expressed predominantly in microglia/macrophages cells , and found to be in the range of 96–98% in all cell preparations.
Exposure of microglia to HSPG
Microglial cells were incubated with HSPG at a concentration of 5, 15, 30, 40 μg/ml for 2, 4, 12, 24 and 48 h. After the indicated times, culture supernatants were harvested, and frozen at -70°C until assayed for levels of secreted TNF-α and nitrite (NO2-). After 4 h total RNA for each conditions was isolated from cells plated in 48-well tissue culture plates, using the TRIzol reagent protocol (Invitrogen; Carlsbad, CA, USA). In some experiments cells were exposed to 15 μg/ml of HS or HSPG denaturated at 90°C for 10 min. After treatment at indicated times, culture media supernatants were assayed for NO2- accumulation and TNF-α release as described below.
Antigenic mouse TNF-α was detected by ELISA system from Biosurce (Camarillo, CA, USA), and based on the quantitative "sandwich" enzyme immunoassay technique. The sensitivity of the assay was 10 pg/ml.
Nitrite (NO2-) is a stable end-product used extensively as an indicator of NO production by cultured cells. In our experimental conditions, NO2- accumulation was assayed by the Griess reaction, according to the method previously described . Briefly, culture media supernatants were mixed with equal amounts of Griess reagent (p-aminobenzene sulfonamide 1%, naphtylethylenediamide 0.1% in phosphoric acid 2.5%) in 96-well plates: samples were incubated at room temperature for 10 min, and subsequently absorbance was read at 540 nm using a microplate reader. NO2- concentrations were calculated in accordance with a sodium nitrite standard curve.
Oligonucleotide Primers Used for cDNA amplification
Length of PCR Fragments (bp)
Data are expressed as means ± standard deviations (SD). Statistical evaluation was performed by repeated measures ANOVA (analysis of variance) followed by Dunnet's test for specific comparisons. Statistical significance was set at P < 0.05.
1. HSPG triggers production of NO2- and TNF-α in cultured microglia
2. HSPG induces expression of iNOS and TNF-α mRNA in cultured microglia
3. Microglial activation induced by HSPG is mediated primarily by the protein core
The identification in senile plaques of pathologic stimuli that can lead to microglial activation represents one of the important issues of immunologic research in AD. Previous studies have shown that microglia upon stimulation with Aβ can produce proinflammatory and cytotoxic mediators, and that these mediators play a role in the pathogenesis of AD [23–26]. The demonstration of the indirect neuronal injury prompted us to investigate if, in addition to Aβ, others molecules present in senile plaques could be involved in similar mechanisms of microglial activation. We have identified HSPG as a potential candidate in this process on the basis that this molecule has been found to be associated with Aβ peptide-containing deposits and suggested to act as pathological chaperone, increasing β-pleated structure within Aβ [27, 28].
We demonstrate that HSPG is able to induce the release of NO2- and TNF-α by cultured primary murine microglia. By assessing iNOS and TNF-α mRNA expression with RT-PCR we have also shown that the release of NO and TNF-α in HSPG-stimulated microglial cells is due to the induction of iNOS and TNF-α gene expression. These findings suggest that, in addition to Aβ, also HSPG is able to activate microglia with production of proinflammatory molecules known to be present in the brain of AD patients.
The precise mechanism by which microglia mediate neuronal cell injury in AD is incompletely understood and several mediators have been proposed, among them NO and TNF-α [29–31]. Even though physiological levels of NO may influence synaptic efficacy by regulating neurotrasmitter release , excess NO may cause neuronal degeneration by combining with oxygen radicals such as superoxide anion to form the highly toxic peroxynitrite ion . Similarly, TNF-α has been reported to be trophic to rat hippocampal neurons . However, transgenic mice that overexpress TNF-α exibit severe inflammation and neurodegeneration . Moreover, in vitro studies have shown that some Aβ-induced microglial activities, including neurotoxicity and chemokine production, are mediated through release of endogenous TNF-α [23, 36, 37]. That TNF-α is presumably involved in AD pathology is also supported by its elevated levels in the serum, CSF and cerebral cortex of AD patients [38, 39]. In view of the information summarized above, it is conceivable that generation of NO and TNF-α from microglial cells is one means by which HSPG may enhance the inflammatory reaction in neuritic plaques, and thus pathogenesis of AD. However, it must be pointed out that astrocytes represent the main source of NO in the plaques whereas release of neurotoxic levels of NO by microglia has been shown in vitro [40, 23, 24, 41]. Future studies will be needed in order to determine whether HSPG also activates astrocytes to produce NO and TNF-α.
The lack of inflammation and microglial activation described in diffuse plaques opens the question about the actual proinflammatory role played by HSPG at early stages of plaque evolution. Our results show that activation of microglial cells by HSPG is concentration-dependent, both in terms of NO2- accumulation and TNF-α release in the culture medium, and that 15 μg/ml of the compound is sufficient to trigger the activation process. Although HSPG has been immunohistologically localized to senile plaques its biochemical isolation from these structures, and thus its quantification, have yet to be performed . Thus, we cannot establish at present whether the threshold for microglial activation we found can explain the discrepancies in immunohistochemical localization of HSPG in diffuse plaques lacking inflammation.
The cellular source of HSPG in mammalian brain is represented by microglia and astrocytes which have been shown by immunofluorescence and/or western blotting to express HSPG both in vitro and in vivo [43, 44]. Nevertheless, the factors implicated in HSPG biosynthesis and deposition have been poorly investigated. Recently regulation of HSPG by injury and IL-1α has been demonstrated in astrocytes and microglia . Therefore, the stimulation of microglial cells by HSPG, followed by increased production of proinflammatory cytokines could stimulate further HSPG formation in an autocrine, feed-forward manner.
The importance of HSPG deposition in AD pathogenesis is supported by the fact that HSPG binds Aβ, accelerates Aβ fibril formation and maintains Aβ fibril stability . Most of these effects of HSPG have been shown to be due to its associated HSGAG side-chains, as also suggested by the lack of extracellular Aβ deposits in transgenic mice overexpressing HSPG protein core . This contrasts with our results showing that the proinflammatory role of HSPG is primarily mediated by its protein core. However, in these transgenic mice HSPG was detected only inside the cells, i.e. without accumulation of the compound in the extracellular environment, which instead is a hallmark of AD neurodegeneration. Studies in these animals, therefore, do not allow drawing conclusions about the role of extracellular HSPG in AD.
In conclusion, the potential participation of HSPG in NO and TNF-α-mediated neuronal injury induced by microglia adds a novel biological role of this molecule in the pathogenesis of AD. These data indicate that HSPG plays an immunomodulatory role in the activation of microglia, in addition to that proposed in amyloidogenic pathways and demonstrate another mechanism by which immune responses may be triggered in AD brain. Therefore, a further understanding of the role of HSPG in the pathogenesis of AD would assist in the development of rational, targeted therapeutic strategies to combat this neurodegenerative disorder.
We are grateful to financial support from the MANAD project of the European Community and from "Ricerca Finalizzata Alzheimer 2000" of the Italian Ministery of Health.
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