Neurochemical Research

, Volume 33, Issue 6, pp 1138–1144

Effect of Melatonin and Melatonylvalpromide on β-amyloid and Neurofilaments in N2a Cells

Authors

    • Department of Pathophysiology, Hubei Provincial Key Laboratory in Neurological Diseases, Tongji Medical CollegeHuazhong University of Science and Technology
  • Yin-Chun Zhang
    • Department of Pathophysiology, Hubei Provincial Key Laboratory in Neurological Diseases, Tongji Medical CollegeHuazhong University of Science and Technology
  • Nithiananda Chatterjie
    • Department of NeurochemistryNYS Institute for Basic Research
  • Inge Grundke-Iqbal
    • Department of NeurochemistryNYS Institute for Basic Research
  • Khalid Iqbal
    • Department of NeurochemistryNYS Institute for Basic Research
    • Department of Pathophysiology, Hubei Provincial Key Laboratory in Neurological Diseases, Tongji Medical CollegeHuazhong University of Science and Technology
Original Paper

DOI: 10.1007/s11064-007-9563-y

Cite this article as:
Wang, X., Zhang, Y., Chatterjie, N. et al. Neurochem Res (2008) 33: 1138. doi:10.1007/s11064-007-9563-y

Abstract

In the present study, we have studied the effect of melatonin (Mt) and melatonin derivative, i.e., melatonylvalpromide (Mtv), on cell viability, β-amyloid (Aβ) production, cell morphology, and expression and phosphorylation of neurofilament proteins in wild-type murine neuroblastoma N2a (N2a/wt) and N2a stably transfected with amyloid precursor protein (N2a/APP) cell lines. The study used MTT assay, Sandwich ELISA, immunocytochemistry and Western blots techniques. The results showed that both Mt and Mtv could increase cell viability, but Mtv did so more effectively. The N2a/APP showed shorter and less amount of cell processes than N2a/wt, and Mtv but not Mt slightly improved the morphological changes in N2A/APP. Both Mt and Mtv suppressed the Aβ level in cell lysates, but the effect of Mtv was stronger than Mt. The immunoreaction to the non-phosphorylated neurofilament proteins probed by SMI32 and SMI33 were remarkably weaker in N2a/APP than N2a/wt, while the immunoreaction to the phosphorylated neurofilament proteins at SMI34 epitopes was slightly stronger in N2a/APP than N2a/wt, suggesting higher phosphorylation level of neurofilament proteins in N2a/APP. Treatment of the cells with Mt and Mtv increased the immunoreaction at SMI32 and SMI33 epitopes, while only Mtv but not Mt decreased the staining at SMI34 epitope, suggesting both Mt and Mtv promote dephosphorylation of neurofilament at SMI32 and SMI33 epitopes, while Mtv stimulates dephosphorylation of neurofilament at SMI34 epitope. These results suggest that Mtv may be a better candidate in arresting the intracellular accumulation of Aβ and protecting the cells from Aβ-related toxicity.

Keywords

Alzheimer’s diseaseβ-AmyloidNeurofilamentMelatoninMelatonylvalproylamide

Introduction

Alzheimer’s disease (AD) is the most common neurodegenerative disease that causes progressive cognitive and behavioral deterioration in the elderly. Although the pathology of AD is complex, two of the histopathological features that characterize the brain of AD are the formation of extracellular senile plaques and intracellular neurofibrillary tangles (NFTs) [1, 2]. The main component of senile plaques is made up of β-amyloid peptide (Aβ), generated from proteolysis of amyloid precursor protein (APP) [3, 4]. Mutation in APP has been shown to increase the production of Aβ [57]. In patients affected with pathogenic APP mutations, neurofibrillary tangles are also observed in addition to abundant Aβ deposits, suggesting that this mutation might also sensitize neurons to the formation of neurofibrillary tangles. According to amyloid cascade hypothesis, the Aβ deposits might also be directly responsible for the generation of neurofibrillary tangles [8]. Neurofibrillary tangles consist of bundles of paired helical filaments (PHFs). The subunit protein of PHF is cytoskeleton protein tau [9]. Tau, as microtubule associated protein, promotes the assembly and maintains the stability of microtubules [10]. In this process, another cytoskeleton protein, neurofilament (NF) also plays important role. Neurofilament belongs to the intermediate filament of neurons, contributes to maintenance of cytoskeleton and stabilizes the cell morphology and axonal transport [11]. It is also hyperphosphorylated and accumulated in neurons and elevated in cerebrospinal fluid of AD patients [1214]. Until now, the effect of Aβ on neurofilament is still not reported.

Melatonin (Mt), a pineal gland secreted hormone, declines progressively with age [15, 16], and the level of Mt in the cerebrospinal fluid of AD patients is lower than that of the age-matched population [17, 18]. Recent studies also show that Mt protects neuronal cells from Aβ-mediated oxidative damage `and inhibits the formation of amyloid fibrils in vitro [19, 20]. These observations suggest an intrinsic link between the deficits of Mt and development of AD. Meanwhile, it is also reported that valproic acid, a widely used anticonvulsant agent is a potent anti-epileptic and effective mood stabilizer. It also upregulates melatonin receptors and neurotrophic factors [21]. In an effort to maximize such effects, a substituted amidic derivative was prepared by replacing the acetyl group of melatonin with a valproyl group derived from valproic acid. Thus, melatonylvalpromide (Mtv) was synthesized with the expectation that the 5-methoxytryptamine moiety with its intact indole nucleus, neuroprotective properties antioxidant effects, and low toxicity when coupled with the valproyl moiety would yield an interesting substance with less toxic potential than valproic acid and possible beneficial neuroprotective properties different from Mt [22]. The role of melatonylvalpromide (Mtv) in Alzheimer-related alterations has not been previously reported.

In the present study, we compared the effect of Mt and Mtv on Aβ production, cell viability and morphology, as well as neurofilament expression and phosphorylation in N2a cell lines. We observed that Mtv was more effective than Mt in arresting the Aβ production and preserving the cell viability and morphology; it also decreased the phosphorylation level of SMI34 epitope phosphorylated neurofilament in N2a cells stably transfected with Aβ precursor protein (APP).

Materials and Methods

Cell culture

Wild-type murine neuroblastoma N2a (N2a/wt) and N2a stably transfected with human APP695 (N2a/APP) were obtained from Dr. H Xu (The Burnham Institute, San Diego, CA, USA). The cells were maintained in a medium containing 50% Dulbecco’ modified Eagle’s medium (DMEM) and 50% Opti-MEM, supplemented with 5% fetal bovine serum (GibcoBRL, Grand Island, NY, USA) in 5% CO2 at 37°C, and stably transfected cells were selected in medium in the presence of 0.2 g/l G418 (GibcoBRL, Grand Island, NY, USA).

MTT assay

Mitochondrial respiration, an indicator of cell viability, was assessed by the mitochondrial-dependent reduction of 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) to formazan as described previously [23]. In brief, N2a/wt and N2a/APP cells were seeded in 96-well plates at a density of 104 cells per well. After growing for 24 h, the cells were incubated in serum-free DMEM-Opti-MEM for 12 h. Then Mt or Mtv with final concentration of 5 μM, 10 μM or 50 μM, respectively, or 0.01% of dimethyl sulfoxide (DMSO, used as vehicle control), were added to the culture medium and incubated with the cells for 12 h. About 10 μl MTT (5 mg/ml) in phosphate buffered saline (PBS) solution was added to the plates and the cells were allowed to incubate for 4 h at 37°C. Then, the medium was replaced with 100 μl DMSO and the absorption was measured at 570 nm with DG3022-micro-plate reader (TECAN, Austria). Results were expressed as the percentage of MTT reduction, assuming that the absorbance of control cells was 100%.

ELISA assay

For quantitative analysis of Aβ in medium or cell lysates, a Sandwich enzyme linked immunosorbent assay (ELISA) was performed as described previously [24, 25]. In brief, Aβ1–40 and Aβ1–42 in samples were captured with G2–10, a mAb specific for Aβ1–40, or G2–11, a mAb specific for Aβ1–42 (Aβeta GmBH, Deutsch), respectively. The presence of Aβ was then detected specifically by antibody Biotin-WO2 (Aβeta GmBH, Deutsch), and further developed with horseradish peroxidase (HRP)-NeutrAvidin (Pierce Rockford, IL, USA). HRP activity was assayed by color development using TMP microwell peroxidase system (Kirkegaard and Perry Laboratories, Gaithersburg, MD, USA).

Western blots

After treatment, the cells were washed with PBS and then lysed in sample buffer containing 50 mM Tris–Cl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1 mM phenylmethyl sulfonylfluoride (PMSF), 1 mM ethylenediamine tetraacetic acid (EDTA), and 10 μg/ml each of protease inhibitors leupeptin, aprotinin and pepstatin followed by sonication for 60 s on ice. The lysates were then centrifuged at 12,000g for 5 min at 4°C, and the supernatants (discarded the pellets) were mixed with Laemmli buffer (60 mM Tris, pH 6.8, 10% glycerol, 2% SDS, 10% β-mercaptoethanol and 0.005% bromophenol blue) and boiled for 10 min, then the supernatants were stored at −70°C or used immediately for Western blot analysis. To collect any possible aggregated neurofilament proteins in the cells, the cell lysates without centrifugation were extracted directly with modified Laemmli buffer (60 mM Tris, pH 6.8, 10% glycerol and 2% SDS) and boiled for 10 min. The extracts were used for Western blot analysis. Protein concentrations were determined with BCA Protein Assay Reagent (Pierce, Rockford, IL, USA). Proteins were separated by 10% SDS-polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride (PVDF) membranes (Amersham Pharmacia Biotech, NJ, USA). To determine the expression and phosphorylation state of neurofilaments, the blots were incubated with primary antibodies, i.e., SMI33 and SMI34 (Sternberger Monoclonals, Baltimore, MD, USA; 1:5000 diluted in 3% BSA in PBS, containing 0.02% NaN3) for overnight at 4°C, and then the blots were further incubated with peroxidase-conjugated goat anti-rabbit or anti-mouse antibody (Sigma Chemical Co. St. Louis, MO, USA) and developed with 5-bromo-4-chloro-3-indolyl-phosphate/nitroblue tetrazolium (BCIP/NBT, Amersham Pharmacia Biotech, Piscataway, NJ, USA). The blots were scanned and the sum optical density was quantitatively analyzed by Kodak Digital Science 1D software (Eastman Kodak Company, New Haven, CT, USA).

Immunocytochemistry

Cells were cultured in the presence or absence of 10 μM Mt or 10 μM Mtv for 24 h. The cells on the coverslip were fixed for 7 min in ice-cold acetone after rinse with PBS, and then permeabilized in 0.3% Triton X-100 in PBS. Endogenous peroxidase was blocked with 0.3% H2O2 for 10 min and non-specific binding sites were blocked with 10% normal goat serum for 10 min at room temperature. The cells were exposed to primary antibodies SMI32, SMI33, and SMI34 (1:5000 diluted in 3% BSA in PBS, containing 0.02% NaN3) for overnight at 4°C, then to peroxidase-labeled goat-anti-mouse secondary antibody for 2 h at 37°C and developed with diaminobenzidine tetrachloride (5 g/l). The optical density of the cells was quantitatively analyzed by Image Pro Plus software.

Results

Effect of Mt and Mtv on cell viability and cell morphology in N2a/wt and N2a/APP

After treatment of the cells with Mt or Mtv at final concentrations of 5 μM, 10 μM or 50 μ, for 12 h, we measured the cell viability by MTT assay. Compared with vehicle control (0.01% DMSO), no difference in cell viability was observed in N2a/wt upon Mt or Mtv treatment (Fig. 1a). In N2a/APP, the cell viability was significantly improved by 10 μM Mt, and this improvement was seen by 5, 10 and 50 μM of Mtv (Fig. 1b). Based on these results, we used 10 μM Mt and Mtv for the rest of the study.
https://static-content.springer.com/image/art%3A10.1007%2Fs11064-007-9563-y/MediaObjects/11064_2007_9563_Fig1_HTML.gif
Fig. 1

Effect of Mt and Mtv on cell viability and morphology in N2a/wt and N2a/APP. Cell viability was measured by MTT assay in differentiated cells N2a/wt (a) and N2a/APP (b). Mt at 10 μM and Mtv at 5, 10 and 50 μM increased cell viability in N2a/APP. * < 0.05, ** < 0.01 vs untreated cells (mean ± S.D, N = 6). Cell morphology was assessed by phase-contrast microscopy (c). Abundant and long neurites were seen in N2a/wt while much less and shorter neurites were shown in N2a/APP. Slight improvement of cell morphology was seen in N2a/APP by Mtv. Bar: 30 μm

The morphology of N2a/wt was presented mostly as fusiform in shape with sharp edge, long neurites and exiguous dendrites, while much less and shorter neurites were shown in N2a/APP (Fig. 1c). A slightly improved morphology of N2a/APP was observed in Mtv-treated samples, but no obvious change was seen in Mt-treated cells (Fig. 1c). These results together suggest that Mtv may be more efficient in preserving cell viability and cell morphology.

Effect of Mt and Mtv on Aβ in N2a/wt and N2a/APP cells

The levels of Aβ1–40 and Aβ1–42 in medium and cell lysates were determined by sandwich ELISA as described in Materials and Methods. It was shown that the levels of Aβ1–40 and Aβ1–42 in culture medium and cell lysates of N2a/APP were markedly higher than that of N2a/wt (Fig. 2a, b). After treatment of the cells with 10 μM Mt or Mtv for 24 h, the elevation of Aβ in cell lysates of N2a/APP was significantly attenuated, and more prominent protection was seen in Mtv treated cells (Fig. 2d, f). No significant effect of Mt and Mtv on Aβ1–40 and Aβ1–42 was detected in culture medium of N2a/wt and N2a/APP cell lines (Fig. 2c, e). These results suggest that both Mt and Mtv can effectively attenuate intracellular Aβ overproduction in N2/APP, and Mtv is more effective than Mt.
https://static-content.springer.com/image/art%3A10.1007%2Fs11064-007-9563-y/MediaObjects/11064_2007_9563_Fig2_HTML.gif
Fig. 2

Overproduction of Aβ in N2a/APP and the effect of Mt and Mtv. The levels of Aβ1–40 and Aβ1–42 in medium and cell lysates of N2a/APP were markedly higher than those in N2a/wt (a, b). Mt and Mtv significantly suppressed the production of Aβ in the cell lysate (d, f) but not in medium (c, e) of N2a/APP. < 0.05, ** < 0.01 vs N2a/wt (mean ± S.D, N = 3)

Effect of Mt and Mtv on neurofilament proteins in N2a/wt and N2a/APP

The alterations of the phosphorylated (SMI34) and non-phosphorylated (SMI32 and SMI33) neurofilament proteins were studied by immunocytochemistry and by Western blots. By immunocytochemistry, the reaction to SMI32 and SMI33 was significantly weaker, while the binding to SMI34 was stronger in N2a/APP than N2a/wt (Fig. 3a–f and m), suggesting higher phosphorylation level of neurofilament proteins in N2a/APP cell line. Upon treatment with Mt and Mtv, the immunoreaction of the non-phosphorylated neurofilament at SMI32 and SMI33 epitopes was significantly increased, suggesting both Mt and Mtv promote dephosphorylation of neurofilament at these epitopes (Fig. 3, compare d with g and j, and e with h and k, Fig. 3m). A significantly decreased staining at SMI34 epitope was observed in Mtv but not Mt treated samples (Fig. 3, compare l with f and i, Fig. 3m), suggesting the role of Mtv in dephosphorylating neurofilament at SMI34 epitope. By Western blots, an obviously increased staining at SMI33 (P < 0.01) and a slightly decreased staining at SMI34 (P < 0.05) were also observed in N2a/APP upon Mtv but not Mt treatment (Fig. 4). These results suggest that both Mt and Mtv promote dephosphorylation of neurofilament proteins, and only Mtv can dephosphorylate neurofilament at SMI34 epitope.
https://static-content.springer.com/image/art%3A10.1007%2Fs11064-007-9563-y/MediaObjects/11064_2007_9563_Fig3_HTML.gif
Fig. 3

Immunocytochemical staining showing the effect of Mt and Mtv on neurofilament proteins. Phosphorylated neurofilament proteins were probed by SMI34 and non-phosphorylated neurofilament proteins were measured by SMI32 and SMI33. The images were visualized by DAB system. The level of the non-phosphorylated neurofilament was significantly lower in N2a/APP than N2a/wt, while the phosphorylated neurofilament was slightly higher in N2a/APP than N2a/wt (compare a, b and c with d, e and f, see panel m for quantitative analysis). Both Mt and Mtv enhanced the staining in non-phosphorylated neurofilament, especially at SMI33 epitope. Mtv decreased significantly the staining at SMI34 epitope (compare g, h and i and j, k and l with d, e and f, also see panel m for quantitative analysis). Bar: 30 μm. * < 0.05, ** < 0.01 vs N2a/wt; Δ < 0.05, ΔΔ < 0.01 vs N2a/APP; #< 0.05, ##< 0.01 vs Mt

https://static-content.springer.com/image/art%3A10.1007%2Fs11064-007-9563-y/MediaObjects/11064_2007_9563_Fig4_HTML.gif
Fig. 4

Western blots showing the effect of Mt and Mtv on neurofilament proteins in N2a/APP. The cells were treated with 10 μM Mt or Mtv. The blots were probed with the antibodies as indicated and developed with BCIP/NBT system. The sum intensity was expressed. The immunoreaction of the non-phosphorylated neurofilament (SMI33) was increased, but that of the phosphorylated neurofilament (SMI34) was decreased in N2a/APP upon Mtv treatment. The data were from 3 to 4 separate experiments. * P < 0.05 or ** P < 0.01 vs untreated cells

Discussion

AD is the most common neurodegenerative disorder in the elderly. The etiology and pathogenesis of AD is not fully elucidated until now. Studies have shown that abnormal modification and accumulation of cytoskeletal proteins, such as microtubule associated protein tau [9] and neurofilament [26, 27], and overproduction and deposition of Aβ [2830] are key events in the development of AD. However, the mechanisms leading to these alterations are still not understood, and thus there is still no effective method to arrest the pathological changes. In the present study, we observed that the cells overproducing Aβ showed retraction and decrease of cell processes and increased phosphorylation of neurofilament proteins. Both Mt and Mtv suppressed Aβ production, preserved cell viability and arrested neurofilament phosphorylation at SMI32 and SMI33 epitopes. However, Mtv presented stronger effect than Mt in maintaining cell viability and arresting neurofilament phosphorylation. Mtv also preserved cell processes and attenuated neurofilament phosphorylation at SMI34 epitope while Mt did not show these effects. These results suggest that Mtv may be a better candidate in preserving cell viability and attenuating Aβ-related cell toxicities in N2a cells.

It was shown in the present study that the cells overexpressing APP and high level of Aβ had obviously shorter and less processes when compared to the wild-type parental cells. Neurite outgrowth is associated closely with cytoskeletal proteins, such as neurofilament. Therefore, we studied the expression and phosphorylation level of neurofilament proteins in this cell line. Neurofilament is composed of triplet proteins, namely neurofilament light chain (NF-L), middle chain (NF-M), and heavy chain (NF-H). The normal function of neurofilament proteins is to maintain and stabilize cellular structure, and thus to ensure axonal diameter and transportation, neurite outgrowth and repair [31]. In AD brain, neurofilaments are abnormally hyperphosphorylated and these abnormally modified neurofilaments are also detected in neurofibrillary tangles [26]. However, the relationship between Aβ overproduction and neurofilament phosphorylation was not reported. We observed in the present study that the level of the non-phosphorylated neurofilament was significantly decreased in the cells bearing high level of Aβ, suggesting an intrinsic link between Aβ overproduction and neurofilament phosphorylation.

Increasing studies support the fact that deficiency of Mt may be critical for the development of AD. Mt is a pineal hormone participating in biologic modulation of mood, sleep and circadian rhythm. It is involved in aging and age-related diseases [1518]. A selective impairment of the nocturnal Mt secretion has been observed in elder subjects, being significantly related either to the age or to the severity of dementia [32]. In AD patients, a dramatic decrease of Mt level is found, and sleep disruption, nightly restlessness, sun downing, and other circadian disturbances are also frequently seen [17]. Results from initial therapeutic trials of Mt in AD patients have demonstrated a significant slowing of pathological progression [33]. We reported previously that, in SH-SY5Y cells, Mt could effectively attenuate tau and neurofilament hyperphosphorylation induced by inhibition of protein phosphatase (PP)-2A and PP-1 [34, 35], and activation of glycogen synthase kinase-3 [36] or protein kinase A [27, 37]. Mtv is a Mt derivative that shows low toxicity. It possesses anticonvulsive properties and is now expected to be submitted in trials for control of subclinical seizures. It may also exhibit improvement over melatonin in the enhancement of cognition, behavior modification and alleviation of symptoms caused by neuronal damage [22]. Our present study of the effect of Mtv on cell viability and the Aβ-related toxicity in N2a cell, and comparison of the effect of Mtv with Mt and the results demonstrate that Mtv is more efficient than Mt in preserving the cell viability, arresting the production of Aβ and neurofilament phosphorylation. These data suggest that Mtv may play neuroprotective role through regulating the production of Aβ. Early studies suggested that the neuroprotective properties of Mt and Mtv are probably related to its direct and/or indirect antioxidant activity [19, 22, 33]. We conclude that Mtv is more efficient in preserving the cell viability, inhibiting Aβ production and neurofilament phosphorylation in N2a cell.

Acknowledgements

We thank Dr. Xu H (The Burnham Institute, CA, USA) for the generous gift of the N2a cell lines. Dr. Gong CX (NYS Institute for Basic Research, Staten Island, NY, USA) for scientific discussion and reagents. This work was supported in part by grants from the National Natural Science Foundation of China (30400103, 32328007 and 30430270) and National Major Grant for Basic Research (2006CB500703).

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