Heparan sulfate proteoglycan induces the production of NO and TNF-α by murine microglia

Background 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. Results 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). Conclusion These data demonstrate that HSPG may contribute to chronic microglial activation and neurodegeneration seen in neuritic plaques of AD.


Introduction
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 aggre-gated 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 [5]. 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, α 1 -antichymotrypsin and heparan sulfate proteoglycan (HSPG) [6][7][8][9][10][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 [12]. 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 [15]. 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][17][18][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 NO 2 -, 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.  , 1996). Primary murine microglial cultures were prepared as previously described [20]. Briefly, cerebral cortical cells from day 1old mice were dissociated with 0.25% trypsin and 0.1% DNAse (Sigma) and plated in 75 cm 2 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% CO 2 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 × 10 4 cells per 100 µl per well, or in 48-well tissue plates at a concentration of 25 × 10 4 per 500 µl per well, and maintained at 37°C with 5% CO 2 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% CO 2 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 [21], 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 (NO 2 -). 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 NO 2accumulation and TNF-α release as described below.

TNF-α assay
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 assay
Nitrite (NO 2 -) is a stable end-product used extensively as an indicator of NO production by cultured cells. In our experimental conditions, NO 2accumulation was assayed by the Griess reaction, according to the method previously described [22]. 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. NO 2concentrations were calculated in accordance with a sodium nitrite standard curve.

RT-PCR
One µg total RNA quantitated spectrophotometrically and isolated from each condition was reverse transcribed using oligo(dT) 20 -primers and Superscript II-Reverse Transcriptase according to the manifacturer's protocol (Invitrogen, Carlsbad, CA, USA). c-DNA equivalent to 20 ng of total RNA was subjected to subsequent PCR analysis in a total volume of 30 µl containing 25 pmol of primers specific for TNF-α, iNOS and glyceraldehyde phosphate dehydrogenase (GAPDH; used as an internal control) (Table I)

Statistical analysis
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.

HSPG triggers production of NO 2 and TNF-α in cultured microglia
To test whether the interaction of HSPG with microglia could induce the production of proinflammatory and potentially cytotoxic mediators, we assayed the accumulation of NO 2as an indirect measure of NO production from mouse primary microglia stimulated with HSPG and, for comparison, with LPS. As shown in Fig. 1, microglia in resting conditions did not release detectable NO 2 even after a 48-h incubation, whereas HSPG induced significant accumulation of NO 2in culture media supernatants in the range of that observed with LPS. The effect of HSPG on NO 2production was time and concentration-dependent, with maximal accumulation observed at 48 h (7.5. ± 0.5-fold increase over control at 30 µg/ml HSPG; P < 0.05, n = 9) (Fig. 1A) and production already significantly increased after a 24 h exposure to 15 µg/ml (5.2 ± 0.4-fold increase over control; P < 0.05, n = 9) (Fig.  1B). We also investigated whether or not HSPG was able to induce the production of TNF-α by microglia. Untreated cells constitutively produced very small amounts of TNF-α, whereas their stimulation with HSPG or LPS resulted in the release of significant levels of TNFα, with maximal accumulation observed at 4 h (25 ± 1.8fold increase over control; P < 0.05, n = 9), followed by a decrease at later times ( Fig. 2A). Concentration-response studies demonstrated that the amount of TNF-α released into the culture media supernatants increased with increasing concentrations of HSPG (Fig. 2B). TNF-α release was already significantly increased after a 24 h exposure to 15 µg/ml HSPG (11.6 ± 0.85.-fold increase over control; P < 0.05, n = 9). Specificity of the effects of Effect of HSPG on the accumulation of NO 2 from murine microglia http://www.immunityageing.com/content/2/1/11 HSPG on microglial activation was confirmed by LAL test that excluded trace levels of endotoxin in our HSPG stocks.

HSPG induces expression of iNOS and TNF-α mRNA in cultured microglia
To determine whether the production of NO 2and TNF-α triggered by HSPG reflected induction of iNOS and TNFα mRNA, RT-PCR analysis was performed on microglia total RNA, using probes complementary to the mouse macrophage iNOS and TNF-α coding sequences. In resting conditions the mRNA expression for iNOS and TNF-α in microglia was absent. On the contrary, mRNA levels for iNOS and TNF-α were clearly induced after stimulation of the cells for 4 h with HSPG at the concentration of 15 µg/ ml or 100 ng/ml LPS (Fig. 3).

Microglial activation induced by HSPG is mediated primarily by the protein core
To examine whether the protein core or the GAGs of HSPG were involved in mediating microglial activation, the effects of HSPG were compared with those obtained using HSPG denaturated at 90°C for 10 min (HSPG-hd) or HS-GAG chains (HS). As shown in Fig. 4, exposure of microglia to 30 µg/ml HSPG-hd almost completely abolished the production of NO 2 -(1 ± 0.2 µM; P < 0.05, n = 9) and particularly of TNF-α (98 ± 10 pg/ml; n = 9). Stimulation with 30 µg/ml HS only slightly affected release of TNF-α (200 ± 18 pg/ml; P < 0.05, n = 9) but not accumulation of NO 2 -(0.8 ± 0.1 µM; n = 9). The lack of ability of HSPG-hd and HS to maintain NO 2 accumulation and TNF-α release appeared to be mediated at the transcriptional level since HSPG-hd and HS failed to increase both iNOS and TNF-α mRNA transcription (Fig.  5).

Discussion
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][24][25][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 NO 2 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 TNFα 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][30][31]. Even though physiological levels of NO may influence synaptic efficacy by regulating neurotrasmitter release [32], excess NO may cause neuronal degeneration by combining with oxygen radicals such as superoxide anion to form the highly toxic peroxynitrite ion [33]. Similarly, TNF-α has been reported to be trophic to rat hippocampal neurons [34]. However, transgenic mice that overexpress TNF-α exibit severe inflammation and neurodegeneration [35]. 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 Effect of heat denaturated HSPG and HS-GAG chains on NO 2 and TNF-α release by murine microglia Figure 4 Effect of heat denaturated HSPG and HS-GAG chains on NO 2and TNF-α release by murine microglia. Microglial cells were cultured in 96-well plates and stimulated with 15 µg/ml HSPG, 15 µg/ml heat denaturated HSPG (HSPG-hd) or 15 µg/ml HS-GAG chains (HS). After 24 h, culture media supernatants were assayed for NO 2 accumulation (A) and TNF-α release (B). Mean values ± SD of assays performed with culture media supernatants collected and pooled from triplicate wells for each condition are shown (n = 9). Both panels depict a representative experiment out of three performed with similar results. *p < 0.05, **p < 0.01. 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 activa-tion of microglial cells by HSPG is concentration-dependent, both in terms of NO 2 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 [42]. Thus, we cannot establish at present whether the threshold for microglial activation we found can Effect of heat denaturated HSPG and HS-GAG chains on iNOS and TNF-α mRNA expression in murine microglia 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 [45]. Therefore, the stimulation of microglial cells by HSPG, followed by increased production of proinflammatory cytokines could stimulate further HSPG formation in an autocrine, feedforward 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 [46]. 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 [47]. 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.