Herpetosiphon Secondary Metabolites Inhibit Amyloid-β Toxicity in Human Primary Astrocytes

Abstract

Background:

The accumulation of extracellular plaques containing amyloid-β protein (Aβ) in the brain is one of the main pathological hallmarks of Alzheimer’s disease (AD). Aβ peptide can promote the production of highly volatile free radicals and reactive oxygen species (ROS) that can induce oxidative damage to neurons and astrocytes. At present, numerous studies have investigated the neuroprotective and glioprotective effects of natural products derived from plants, animals, and microorganisms.

Objective:

We investigated the glioprotective effect of secondary metabolites obtained from Herpetosiphon sp. HM 1988 against Aβ₄₀-induced toxicity in human primary astrocytes.

Methods:

The protective effect of bacterial secondary metabolites against Aβ₄₀-induced inducible nitric oxide synthase (iNOS) activity was evaluated using the citrulline assay. To confirm the iNOS activity, nitrite production was assessed using the fluorometric Griess diazotization assay. Intracellular NAD⁺ depletion and lactate dehydrogenase (LDH) release in human primary astrocytes were also examined using well-established spectrophotometric assays.

Results:

Our results indicate that Aβ₄₀ can induce elevation in iNOS and LDH activities, nitrite production, and cellular energy depletion. Importantly, extract of Herpetosiphon sp. HM 1988 decreased iNOS activity, nitrite production, and LDH release. In addition, metabolites of the strain were able to restore cellular energy deficits through inhibition of NAD⁺ depletion mediated by Aβ₄₀.

Conclusion:

These findings suggest that Herpetosiphon metabolites may represent a promising, novel source for the prevention of Aβ toxicity in AD.

Keywords

Alzheimer’s disease amyloid-β inducible nitric oxide synthase natural products oxidative stress

INTRODUCTION

Alzheimer’s disease (AD) is a progressive neurodegenerative disease, leading to the degeneration and the loss of cholinergic neurons in the brain. This deleterious disease was first identified in 1906, and is considered one of the leading causes of dementia and death since 1976 [1 –3]. According to recent reports, 5.7 million people suffer from AD in the United States alone, and this number is estimated to rise to 16 million by 2050 [4]. In 2015, more than 110,000 deaths were attributed to AD in the United States alone, making AD the fifth leading cause of death in American people older than 65 years [4]. Unlike the downward trend in mortality numbers of prostate cancer, stroke, and heart diseases in 2000-2015, the number of deaths associated with AD remarkably increased in the same period. In 2018, the total health care burden for AD patients older than 65 years was estimated to be $277 billion [4].

Amyloid-β (Aβ) plaques and intracellular neurofibrillary tangles composed of hyperphosphorylated tau protein are two important hallmarks of AD. Aβ peptide contains 38 to 43 amino acids resulting from enzymatic cleavage of amyloid-β protein precursor (AβPP) by β- and γ-secretases. AβPP is a transmembrane protein highly expressed in the brain and plays significant roles in synaptic plasticity [5 –7]. Among various Aβ species, Aβ₄₀ is proposed to be the most abundant (80% –90%) type followed by Aβ₄₂ [8, 9]. While the soluble form of Aβ peptide can be found in the brain of healthy individuals, its aggregation initiates pathological symptoms related to AD. It is well known that Aβ peptide can promote free radical generation, i.e., a phenomenon known as “oxidative stress” that leads to protein oxidation and lipid peroxidation [7 , 11].

Postmortem evaluation of AD brains indicated that Aβ₄₀ oligomers could represent a more significant biomarker for early prognosis of AD, compared to Aβ₄₂ [12]. The high lipid and poly-unsaturated fatty acid content of the brain makes this organ remarkably vulnerable to oxidative stress, particularly lipid peroxidation. Although several lines of evidence have demonstrated the neurotoxic effect of Aβ₄₂ [13 –15], few studies are available on the effects of Aβ₄₀ in human astrocytes. Astrocytes are among the important cells of the central nervous system involved in homeostatic balance preservation and several other processes such as maintenance of blood-brain barrier integrity, neurotransmitter uptake, and inflammatory/immune responses of the central nervous system [16 –18]. These cells could be negatively impacted by oxidative stress induced by Aβ oligomers through a series of processes. For example, binding Aβ₄₂ oligomers to astrocytes through receptor for advanced glycation end products activates NADPH oxidase complex leading to reactive oxygen species (ROS) production [19]. Moreover, astrocytes can mediate cytosolic phospholipase A2 phosphorylation and ERK1/2 pathways leading to mitochondrial dysfunction [20]. It is known that oligomeric Aβ₄₀ triggers cytotoxicity and inflammation in human astrocytes by downregulating superoxide dismutase and glutathione peroxidase [9]. In contrast, the oxidation of DNA, proteins, and lipids, and release of proinflammatory cytokines are significantly induced in the presence of oligomeric Aβ₄₀ [9]. Interestingly, Aβ₄₀ oligomers are the only type of amyloid able to induce the activity of the matrix metalloproteinase 2 in rat astrocytes [21].

Microorganisms are potent producers of biochemicals that can be exploited in many industrial sectors such as food, energy, and pharmacy [22 –25]. The application of natural products in the treatment of various human diseases has a long history [26, 27]. Natural products possess diverse chemical structures and are a rich source for drug discovery [28 –30]. Animals, plants, and microorganisms are able to produce valuable natural products with potential therapeutic applications. Among all the natural product producers, microorganisms represent a plentiful source of medicinally important compounds with a broad range of biological activities [28, 29]. The genome analysis of Herpetosiphon spp. (family Herpetosiphonaceae, order Herpetosiphonales, class Chloroflexia, and phylum Chloroflexi) has allowed to identify an untapped source of natural products with various potential bioactive properties [31 –33]. These bacteria are Gram negative, and filamentous aerobes that commonly reside in freshwater, soil, rotting wood, dung of herbivorous, and to lesser extent, in activated sludge of sewage plants [34]. Herpetosiphon spp. can develop colonies on the surface of the medium via gliding movement, resembling that of myxobacterial swarm colonies [35]. Another interesting characteristic of Herpetosiphon spp. is their ability to feed on other bacteria through a “wolf pack” mechanism.

In the present study, we examined the glioprotective effect of secondary metabolites produced by Herpetosiphon sp. HM 1988 against Aβ₄₀-induced oxidative stress in primary human astrocytes. The strain was partially characterized, and its extracted metabolites were subjected to iNOS and LDH activities, nitrite production and intracellular NAD⁺ assay. Importantly, the current research is among the very few studies investigating Herpetosiphon spp. in respect to their potential natural product metabolites, and antioxidant and anti-inflammatory activities.

MATERIALS AND METHODS

Isolation of bacterial strain

The bacterial strain was isolated from Golden Mystery Snail (Pomacea bridgesii) collected from a freshwater tank in Semnan province, Iran. Snail shell was wiped using 70% ethanol; thereafter, the digestive tract of the snail was aseptically removed and homogenized in a sterile microcentrifuge tube with a pestle. The homogenate was cultured on WAT agar medium (CaCl₂.2H₂O, 0.1%; agar, 1.5%; pH, 7.2) supplemented with cycloheximide (25μg/mL) and baited with strikes of living Escherichia coli [36]. Following incubation (30°C, 7 d), swarm colonies with long flares were transferred onto the VY2 agar (bakers’ yeast, 0.5%; CaCl₂.2H₂O, 0.1%; cyanocobalamin, 0.5 mg/L; agar, 1.5%; pH, 7.2) for purification of Herpetosiphon spp. To achieve this goal, 0.5×0.5 mm agar pieces from little areas of the swarms were picked up with sharp, sterile needles and transferred to VY2 medium. This step was repeated until the pure strain was obtained.

Characterization of strain and phylogenetic analysis

The purified isolate was inoculated into MD1 liquid medium (casitone, 0.3%; CaCl₂.2H₂O, 0.07%; MgSO₄.7H₂O, 0.2%; cyanocobalamin, 0.5 mg/L; pH, 7) and incubated at 150 rpm at 30°C for 5–7 d. Microscopic morphology of the isolate was observed under a light microscope. Following morphological assessment, DNA extraction was performed using phenol-chloroform method [37]. Polymerase chain reaction was employed to amplify the bacterial 16S rRNA gene via universal primers (9F, 1541R). The PCR products were purified and sequenced using Sanger method. The similarity of 16S rRNA gene sequences was analyzed using EzTaxon-e database, and a phylogenetic tree was constructed using MEGA software.

Fermentation and metabolite extraction

A seeding culture was prepared by inoculating the isolate in H medium (soy meal, 0.2%; glucose, 0.2%; starch, 0.8%; yeast extract, 0.2%; MgSO₄.7H₂O, 0.1%; CaCl₂.2H₂O, 0.1%; Fe-EDTA, 8 mg/L; pH, 7.2), followed by incubation at 30°C for 48 h. Thereafter, 5% of the seeding culture was inoculated into fermentation medium (i.e., H medium supplemented with 1% Amberlite XAD-16 resin, Sigma–Aldrich) and incubated at 180 rpm and 30°C for 7 d. Biomass and resins were collected and rinsed with distilled water, then repeatedly washed with methanol to extract secondary metabolites. The obtained bacterial extract was concentrated by evaporating the extraction solvent under vacuum at 38°C. The dried extract was kept under anoxia condition at –20°C in airtight container for further experiments [36].

Cell cultures

Human brain cell cultures were generated from human fetal brain tissue, which was obtained from therapeutically terminated 17–20 weeks old fetuses. Written informed consent was obtained from all participants, and the study was approved by the Human Research Ethics Committee of Macquarie University (#5201200411). Astrocyte cell cultures were prepared according to the protocol previously described [38, 39]. Briefly, cerebral portions were washed with phosphate-buffered saline (PBS) and passed through a 100μm nylon mesh. Thereafter, the cell suspension was centrifuged at 500×g for 5 min and the pellet was resuspended in RPMI-1640 medium (heat-inactivated fetal calf serum, 10%; glucose, 0.5%; glutamine, 2 mg; penicillin G, 200 IU/mL; streptomycin sulphate, 200μg/mL). The suspension was plated onto 75 cm² culture flasks (Corning, USA) and incubated at 37°C in 5% humidified CO₂ atmosphere for 3 d.

Immunocytochemistry

Astrocytes grown for 2–3 d on Permanox chamber slides were fixed in 0.1 M PBS containing 4% paraformaldehyde at room temperature for 10 min. After washing with PBS, the slides were incubated with PBS containing 0.1% Triton X-100 for 10 min at room temperature. Next, the slides were incubated with glial-associated fibrillary protein (mouse GFAP, 1 : 500, Sigma-Aldrich, USA) in the blocking solution (Tween 20, 0.1%; bovine serum albumin, 1%; glycine, 22.52 mg/mL) for 30 min at room temperature. The slides were washed again with PBS and incubated with secondary antibodies (1 : 2000, Thermo Fisher Scientific, USA) coupled to Alexa 488 in the dark for 1 h at room temperature. After further washing, coverslips were mounted on the slides before observing under a fluorescence microscope (Zeiss, Germany) equipped with a digital camera.

Toxicity assay

The toxicity of bacterial extract against astrocytes was assessed using CellTiter 96^® AQueous One Solution Reagent (Promega, USA). Cell cultures were incubated with different extract concentrations (6.25, 12.5, 25, and 50μg/mL) at 37°C for 24 and 48 h. Then, 20μL of reagent [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) was added to each well and incubated at 37°C for 2 h. The absorbance of samples was read at 490 nm in a microplate reader (PHERAstar FS, Germany). Solvent controls (i.e., DMSO) corresponding to its concentrations that were used for the preparation of the extract dilutions (0.025μL/mL–0.2μL/mL) were also included to eliminate the possible solvent effects on human primary astrocytes.

Recombinant Aβ₄₀ peptide preparation

Aβ₄₀ peptide was purchased from Recombinant Peptide Technologies (Athens, GA, USA). Recombinant Aβ₄₀ peptide were prepared as previously described 9]. After solubilization, recombinant oligomeric Aβ₄₀ were characterized using atomic force microscopy (AFM) and western immunoblotting techniques according to the protocol described previously [9]. The prepared peptides were characterized using a Nanoscope III Scanning Probe Microscope (Digital Instruments Extended Dimension 3000, Santa Barbara, CA, USA) under ambient conditions operating with an Olympus tapping mode etched silicon probe (OTESPA, Veeco, Camarillo, CA, USA).

Inducible nitric oxide synthase activity

Inducible nitric oxide synthase (iNOS) activity was assessed by measuring the conversion of L-[³H]arginine to L-[³H] citrulline as previously described [40]. The effect of bacterial extract on iNOS activity was determined by treating human astrocytes with the analyte (12.5μg/mL) for 30 min. Next, 0.3 M HClO₄ (pH 5.5) containing EDTA (4 mM) was added and L-[3H] citrulline content was measured using a Beckman LS6500 scintillation counter and iNOS activity was determined as ng L-citrulline/500μg protein/30 min.

The effect of bacterial extract on Aβ₄₀–induced iNOS activity was also determined by exposing the pre-treated astrocytes with bacterial extract (12.5μg/mL, 30 min) to the Aβ₄₀ oligomers (10μg/mL) for 30 min. Thereafter, iNOS activity was measured as explained above.

Nitrite production assessment

The amount of nitrite in the astrocytes was measured using fluorometric Griess diazotization assay. In the first set of experiments, the human astrocytes were only pre-treated with bacterial extract (12.5μg/mL, 30 min) and 100μL of culture supernatant was transferred into a 96-well microplate. Thereafter, 100μL of diaminonaphthalene (DAN) solution (10 mM DAN + 1% HCl) was added to the wells and the samples were incubated at room temperature for 10 min. The fluorescence intensity was then measured at an excitation wavelength of 355 nm and an emission wavelength of 460 nm. In the second set of experiments, the astrocytes were pre-treated with bacterial extract (12.5μg/mL, 30 min), incubated with Aβ₄₀ oligomers (10μg/mL) for 30 min, and subsequently the nitrite content was determined as described above.

Lactate dehydrogenase assay

In the first set of experiments, the primary human astrocytes were only pre-treated with bacterial extract (12.5μg/mL) at 37°C. After 24 h, supernatants were collected and diluted by 100-time factor with cell culture medium. Fifty micro-liter of diluted solution was added to a new 96-well microplate with three replications. Thereafter, NADH (0.7 mM in 0.1 M potassium phosphate buffer) and sodium pyruvate (11.5 mM) were added to the wells and the absorbance was recorded at 340 nm. In the second set of experiments, cell cultures were pre-treated with bacterial extract (12.5μg/mL, 30 min), followed by the addition of Aβ₄₀ oligomers (10μg/mL). LDH activity in supernatant was measured as described above.

Assessment of intracellular NAD⁺ levels

The effect of bacterial extract on intracellular NAD⁺ level was determined according to the method described by [41]. In the first set of experiments, human astrocytes were pre-treated with bacterial extract (12.5μg/mL). After 30 min of incubation, the cells were washed with PBS and extracted using 0.5 M HClO₄ (100μL, 15 min), neutralized with 1 M KOH, and centrifuged at 10,000×g for 5 min. Thereafter, the reaction solution [alcohol dehydrogenase, 13 U/mL; bicine, 100 mM; EDTA, 5 mM; ethanol, 600 mM; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 0.5 mM; phenazine ethosulfate, 2 mM; pH, 7.5] was added to the supernatant and the absorbance was recorded at 570 nm. The second set of experiments involved the sequential pre-treatment of astrocytes with bacterial extract (12.5μg/mL, 30 min) and Aβ₄₀ oligomers (10μg/mL, 30 min), followed by the determination of intracellular NAD⁺ level.

Statistical analysis

Data were analyzed for normality using the Shapiro-Wilk test. Variance was evaluated using Levene’s test. The results were presented as means±standard error of measurement and analyzed by one-way analysis of variance (ANOVA). Post-hoc, pairwise comparisons were carried out using Dunnett’s test. Due to small within group sample size, the Benjamini and Hochberg procedure was carried out to evaluate risk of Type I error. All analyses were evaluated using an α-value of 0.05. Statistical analyses were carried out using SPSS software version 21.

RESULTS

Bacterial strain

According to the results of 16S rRNA gene sequences comparison, the strain HM was characterized as a species of genus Herpetosiphon, closely related to the Herpetosiphon aurantiacus DSM 785 with a 16S rRNA gene similarity of 97.7%, hence named as Herpetosiphon sp. HM 1988 (GenBank: MG251450.1). The strain is a Gram-negative bacterium with ability to form long, unbranched, multicellular filaments consisting of cylindrical cells. The swarms with a swirled appearance are spread quickly on the plate during 5–6 d with predatory behavior on E. coli. Figure 1 illustrates the phylogenetic position of Herpetosiphon sp. HM 1988 relative to the strains of the genus Herpetosiphon and other representatives of the phylum Chloroflexi.

Fig. 1

Phylogenetic tree derived from 16S rRNA gene sequences, showing the phylogenetic positions of strain HM 1988 relative to species of the genus Herpetosiphon and some candidates of the phylum Chloroflexi. The evolutionary tree was inferred using the Neighbor-Joining method. Bar, 0.01 substitutions per site.

Immunocytochemistry

Immunostaining of fetal brain cells with astrocytic marker GFAP showed cell culture purity of 90–95% after 7 d of incubation (Fig. 2). Moreover, no cells were stained with antibodies examined against CD68 (microglia), MAP2 (neurons), and factor VIII (endothelial cells) after 15 or 30 d (data not shown), showing the purity of astrocytes after 3–5 weeks.

Fig. 2

Immunostaining of human primary astrocytes. Cell culture were stained using anti-GFAP antibody and DAPI after 7 d of incubation to determine the purity of human fetal astrocytes.

Toxicity assessment of Herpetosiphon sp. HM 1988 extract on human astrocytes

The effect of strain HM 1988 metabolites on human astrocytes showed no significant toxicity (p > 0.05) at 6.25–25μg/mL concentration after 24 h of incubation. However, pre-treatment of the cells with higher concentration of extract (i.e., 50μg/mL) significantly reduced the cell viability by 19.78% (p < 0.05) (Fig. 3A). After 48 h of incubation, cell viability decreased in the presence of 25 and 50μg/mL of the bacterial extract by 29.58% and 31.59%, respectively (p < 0.05) (Fig. 3B). The solvent controls confirmed that DMSO has no positive or negative effects on the human primary astrocytes at the concentrations that have been used for the preparation of different extract dilutions (Fig. 3).

Fig. 3

Toxicity assessment of Herpetosiphon sp. HM 1988 extract on human primary astrocytes. Effect of Herpetosiphon sp. HM 1988 extract at concentrations of 6.25, 12.5, 25, and 50μg/ml on viability of astrocytes after 24 h (A) and 48 h (B) incubation using MTS solution as reagent (*p < 0.05 compared with DMSO-treated cells, i.e., solvent controls, n = 3).

Effect of Herpetosiphon sp. HM 1988 extract on iNOS activity and nitrite level

Pre-treatment of primary astrocytes with the extract of strain HM 1988 (12.5μg/mL) significantly reduced (36.6%) iNOS activity, compared to the untreated cells (p < 0.05) (Fig. 4). In addition, the incubation of primary astrocytes with Aβ₄₀ oligomers (10μg/mL) significantly increased the iNOS activity by 56%, compared to the control (p < 0.05). Interestingly, pre-treatment of the cells with extract of Herpetosiphon sp. HM 1988 (12.5μg/mL) could remarkably reduce the iNOS activity (40.86%) under oxidative stress (p < 0.05) (Fig. 4).

Fig. 4

Effect of Herpetosiphon sp. HM 1988 extract (12.5μg/mL) on iNOS activity in human primary astrocytes (*p < 0.05, n = 3).

Similar to iNOS, the extract of Herpetosiphon sp. HM 1988 had significant effect on nitrite production in astrocytes alone (Fig. 5). Accordingly, pre-treatment of astrocyte culture with strain HM 1988 extract (12.5μg/mL) suppressed the amount of nitrite released by 31.7%. Moreover, the production of Aβ₄₀-induced nitrite was decreased by 1.9 times in the presence of the bacterial extract, compared to the control, i.e., Aβ₄₀-treated astrocytes alone (Fig. 5).

Fig. 5

Effect of Herpetosiphon sp. HM 1988 extract (12.5μg/mL) on nitrite production in human primary astrocytes (*p < 0.05, n = 3).

Effect of Herpetosiphon sp. HM 1988 extract on lactate dehydrogenase activity

Astrocytes pre-treated with the extract of Herpetosiphon sp. HM 1988 (12.5μg/mL) showed no significant change in extracellular LDH activity after 24 h (p > 0.05) (Fig. 6). As shown in Fig. 6, Aβ₄₀ (10μg/mL) significantly increased LDH activity (55.9%) that was measured in cell supernatant. However, pre-treatment of astrocytes with the bacterial extract (12.5μg/mL) led to 40.9% decrease in LDH activity (p < 0.05).

Fig. 6

Effect of Herpetosiphon sp. HM 1988 extract (12.5μg/mL) on LDH activity in human primary astrocytes (*p < 0.05, n = 3).

Effect of Herpetosiphon sp. HM 1988 extract on intracellular NAD⁺

No significant change in NAD⁺ content in primary astrocytes pre-treated with the extract of strain HM 1988 alone was observed (Fig. 7). In contrast, the bacterial extract protected astrocytes against Aβ₄₀-treated cells by significantly increasing NAD⁺ concentrations. More specifically, the pre-treatment of astrocytes with 12μg/mL of bacterial extract for 30 min before treating the cells with Aβ₄₀ alleviated Aβ₄₀-induced toxicity by restoring the intracellular NAD⁺ content (37%) (Fig. 7).

Fig. 7

Effect of Herpetosiphon sp. HM 1988 extract (12.5μg/mL) on intracellular NAD⁺ concentration in human primary astrocytes (*p < 0.05, n = 3)

DISCUSSION

Accumulation of Aβ plaques in the brain is a well-known hallmark of AD. Aβ peptides can induce oxidative stress and inflammation in the brain, leading to protein oxidation and lipid peroxidation. Herpetosiphon is a chemoheterotrophic, filamentous gliding bacterium with predatory behavior associated with the excretion of several hydrolytic enzymes. Complete genome sequencing of H. aurantiacus strain 114-95T revealed that the secondary metabolism capacity of this bacterium is comparable with other strong secondary metabolite producers such as actinobacteria and myxobacteria [28 , 42–45], suggesting an unexplored source for the production of natural products. Despite this, the potential of the genus Herpetosiphon for the production of lead compounds has not been adequately investigated. Several studies focusing on the secondary metabolites of this genus have previously identified two antibacterial compounds, i.e., siphonazole and auriculamide [33, 46]. In the present study, we examined the glioprotective effects of the natural products extracted from Herpetosiphon sp. HM 1988 as a novel resource for therapeutic agents against oxidative stress induced by Aβ₄₀ in human primary astrocytes.

Accumulation of fibrillar form of Aβ in the brain is associated with inflammation in microglial cells which initiates signal transduction cascades related to the release of inflammatory molecules [47, 48]. Aβ-dependent reactive astrocytes and microglia have been observed surrounding the Aβ plaques, producing ROS, inflammatory mediators, and neurotoxins [49]. Aβ₄₀ is the most abundant species of Aβ in human biological fluids [50]. Aggregation of Aβ₄₀ oligomers in cerebral cortex is associated with cognitive and spatial memory impairment. Expression of Aβ₄₀ and neuronal cell damage significantly increased in patients with AD and other dementia-associated diseases [51]. Analysis of senile plaques in AD brains have indicated that one third of the plaques contained Aβ₄₀ and others were Aβ₄₂ positive, but significant association has been found between Aβ₄₀ positivity and mature plaques [52, 53]. Some pathological processes in the brain such as loss of cholinergic neurons, alteration of fluidity of cell membranes, cortical neurons apoptosis, and memory impairment have been linked to Aβ₄₀ [54 –56].

Nitric oxide, a key signaling molecule, is involved in the pathogenesis of AD [59]. Our results are consistent with other findings that support the role of Aβ peptide in inducing iNOS activity, and hence increased nitric oxide concentrations [57 –60]. High nitric oxide levels could be associated with neuronal apoptosis, damaging the brain in AD patients [61 –63]. Aβ-induced iNOS activity could be modulated through the transcription factor kappa-light-chain-enhancer of activated B cells (NF-κB) [64]. This process has regulatory roles in iNOS transcription by binding to the regulatory site of the iNOS gene [57, 65]. In the present study, we demonstrated that the pre-treatment of astrocytes with the metabolites-derived from Herpetosiphon sp. HM 1988 could effectively protect the cells from Aβ₄₀-induced oxidative stress by reducing iNOS activity. This protective mechanism may be attributed to the inhibition of Aβ accumulation, followed by the suppression of Aβ-mediated NF-κB activation, and the subsequent decrease in iNOS expression.

Previous studies have already demonstrated the protective effects of natural products to limit iNOS activity and nitric oxide content [18 , 67] For instance, Huang et al. showed that spatial memory in rats was improved in the presence of resveratrol, a naturally occurring polyphenol, that could effectively reduce cellular levels of iNOS and lipid peroxidation in a response to Aβ-induced toxicity [57]. Caniferolide A, a macrolide from Streptomyces caniferus, also suppressed iNOS activity in microglia by decreasing the release of proinflammatory cytokines and nitric oxide. Moreover, this compound inhibited the Aβ-induced microglia activation through reducing ROS level [68].

Interestingly, the metabolites derived from Herpetosiphon sp. HM 1988 drastically restored Aβ-induced NAD⁺ depletion in human astrocytes. A large number of studies have demonstrated that NAD⁺ contributes to several essential cellular processes such as regulation of calcium concentration, energy metabolism, mitochondrial function, aging and cell death [69, 70]. It has been previously shown that NAD⁺ can protect brain cells from oxidative stress inducers [71]. Moreover, NAD⁺ concentrations are inversely correlated with Aβ toxicity in AD patient [72, 73]. This can be attributed to the fact that oxidative stress induces poly (ADP-ribosyl) polymerase 1 (PARP-1) hyperactivation, leading to the depletion of cytosolic NAD⁺ due to higher glycolysis rate. Apart from that, PARP-1 is a DNA repair enzyme, which is overexpressed under oxidative stress-induced DNA strand breaks. The results of the present study showed that the extract obtained from Herpetosiphon sp. HM 1988 was able to protect human primary astrocytes against Aβ₄₀ oligomer-induced toxicity by improving the intracellular levels of NAD⁺ and maintaining cellular energy.

Overall, the extract of Herpetosiphon sp. HM 1988 was able to protect human primary astrocytes against Aβ₄₀-induced toxicity by 1) decreasing the iNOS activity and nitrite production, 2) restoring the intracellular NAD⁺ supply, and 3) decreasing the LDH activity. This study provides a new insight for further studies to investigate the potential of Herpetosiphon secondary metabolites as novel candidates for promoting the pipeline of drug discovery for AD and neurodegenerative diseases.

Footnotes

ACKNOWLEDGMENTS

MD and HK are supported by international scholarships from Macquarie University. Prof Guillemin’s is funded by the National Health and Medical Research Council (NHMRC) and Macquarie University.

Authors’ disclosures available online ().

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Herpetosiphon Secondary Metabolites Inhibit Amyloid-β Toxicity in Human Primary Astrocytes

Abstract

Background:

Objective:

Methods:

Results:

Conclusion:

Keywords

INTRODUCTION

MATERIALS AND METHODS

Isolation of bacterial strain

Characterization of strain and phylogenetic analysis

Fermentation and metabolite extraction

Cell cultures

Immunocytochemistry

Toxicity assay

Recombinant Aβ40 peptide preparation

Inducible nitric oxide synthase activity

Nitrite production assessment

Lactate dehydrogenase assay

Assessment of intracellular NAD+ levels

Statistical analysis

RESULTS

Bacterial strain

Immunocytochemistry

Toxicity assessment of Herpetosiphon sp. HM 1988 extract on human astrocytes

Effect of Herpetosiphon sp. HM 1988 extract on iNOS activity and nitrite level

Effect of Herpetosiphon sp. HM 1988 extract on lactate dehydrogenase activity

Effect of Herpetosiphon sp. HM 1988 extract on intracellular NAD+

DISCUSSION

Footnotes

ACKNOWLEDGMENTS

References

Recombinant Aβ₄₀ peptide preparation

Assessment of intracellular NAD⁺ levels

Effect of Herpetosiphon sp. HM 1988 extract on intracellular NAD⁺