Melatonin ameliorates Aβ42‐induced alteration of βAPP‐processing secretases via the melatonin receptor through the Pin1/GSK3β/NF‐κB pathway in SH‐SY5Y cells

Melatonin is involved in the physiological regulation of the β‐amyloid precursor protein (βAPP)‐cleaving secretases which are responsible for generation of the neurotoxic amyloid beta (Aβ) peptide, one of the hallmarks of Alzheimer's disease (AD) pathology. In this study, we aimed to determine the underlying mechanisms of this regulation under pathological conditions. We establish that melatonin prevents Aβ₄₂‐induced downregulation of a disintegrin and metalloproteinase domain‐containing protein 10 (ADAM10) as well as upregulation of β‐site APP‐cleaving enzyme 1 (BACE1) and presenilin 1 (PS1) in SH‐SY5Y cell cultures. We also demonstrate that the intrinsic mechanisms of the observed effects occurred via regulation of nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) and glycogen synthase kinase (GSK)‐3β as melatonin reversed Aβ₄₂‐induced upregulation and nuclear translocation of NF‐κBp65 as well as activation of GSK3β via its receptor activation. Furthermore, specific blocking of the NF‐κB and GSK3β pathways partially abrogated the Aβ₄₂‐induced reduction in the BACE1 and PS1 levels. In addition, GSK3β blockage affected α‐secretase cleavage and modulated nuclear translocation of NF‐κB. Importantly, our study for the first time shows that peptidyl‐prolyl cis‐trans isomerase NIMA‐interacting 1 (Pin1) is a crucial target of melatonin. The compromised levels and/or genetic variation of Pin1 are associated with age‐dependent tau and Aβ pathologies and neuronal degeneration. Interestingly, melatonin alleviated the Aβ₄₂‐induced reduction of nuclear Pin1 levels and preserved the functional integrity of this isomerase. Our findings illustrate that melatonin attenuates Aβ₄₂‐induced alterations of βAPP‐cleaving secretases possibly via the Pin1/GSK3β/NF‐κB pathway.     

Melatonin stimulates the nonamyloidogenic processing of βAPP through the positive transcriptional regulation of ADAM10 and ADAM17

Melatonin controls many physiological functions including regulation of the circadian rhythm and clearance of free radicals and neuroprotection. Importantly, melatonin levels strongly decrease as we age and patients with Alzheimer's disease (AD) display lower melatonin than age‐matched controls. Several studies have reported that melatonin can reduce aggregation and toxicity of amyloid‐β peptides that are produced from the β‐amyloid precursor protein (βAPP). However, whether melatonin can directly regulate the βAPP‐cleaving proteases (‘secretases’) has not been investigated so far. In this study, we establish that melatonin stimulates the α‐secretase cleavage of βAPP in cultured neuronal and non‐neuronal cells. This effect is fully reversed by ADAM10‐ and ADAM17‐specific inhibitors and requires both plasma membrane‐located melatonin receptor activation, and ERK1/2 phosphorylation. Moreover, we demonstrate that melatonin upregulates both ADAM10 and ADAM17 catalytic activities and endogenous protein levels. Importantly, genetic depletion of one or the other protease in mouse embryonic fibroblasts prevents melatonin stimulating constitutive and PKC‐regulated sAPPα secretion and ADAM10/ADAM17 catalytic activities. Furthermore, we show that melatonin induces ADAM10 and ADAM17 promoter transactivation, and we identify the targeted promoter regions. Finally, we correlate melatonin‐dependent sAPPα production with a protection against staurosporine‐induced apoptosis. Altogether, our results provide the first demonstration that melatonin upregulates the nonamyloidogenic ADAM10 and ADAM17 proteases through melatonin receptor activation, ERK phosphorylation and the transactivation of some specific regions of their promoters and further underline the preventive rather than curative nature of melatonin regarding AD treatment.     

Melatonin ameliorates amyloid beta‐induced memory deficits,tau hyperphosphorylation and neurodegeneration via PI3/Akt/GSk3β pathway in the mouse hippocampus

Alzheimer's disease (AD) is the most prevalent age‐related neurodegenerative disease, pathologically characterized by the accumulation of amyloid beta (Aβ) aggregation in the brain, and is considered to be the primary cause of cognitive dysfunction. Aβ aggregates lead to synaptic disorder, tau hyperphosphorylation, and neurodegeneration. In this study, the underlying neuroprotective mechanism of melatonin against Aβ_1‐42‐induced neurotoxicity was investigated in the mice hippocampus. Intracerebroventricular (i.c.v.) Aβ_1‐42‐injection triggered memory impairment, synaptic disorder, hyperphosphorylation of tau protein, and neurodegeneration in the mice hippocampus. After 24 hr of Aβ_1‐42 injection, the mice were treated with melatonin (10 mg/kg, intraperitonially) for 3 wks, reversed the Aβ_1‐42‐induced synaptic disorder via increasing the level of presyanptic (Synaptophysin and SNAP‐25) and postsynaptic protein [PSD95, p‐GluR1 (Ser845), SNAP23, and p‐CREB (Ser133)], respectively, and attenuated the Aβ_1‐42‐induced memory impairment. Chronic melatonin treatment attenuated the hyperphosphorylation of tau protein via PI3K/Akt/GSK3β signaling by activating the p‐PI3K, p‐Akt (Ser 473) and p‐GSK3β (Ser9) in the Aβ_1‐42‐treated mice. Furthermore, melatonin decreased Aβ_1‐42‐induced apoptosis through decreasing the overexpression of caspase‐9, caspase‐3, and PARP‐1 level. Additionally, the evaluation of immunohistochemical analysis of caspase‐3, Fluorojade‐B, and Nissl staining indicated that melatonin prevented neurodegeneration in Aβ_1‐42‐treated mice. Our results demonstrated that melatonin has neuroprotective effect against Aβ_1‐42‐induced neurotoxicity through decreasing memory impairment, synaptic disorder, tau hyperphosphorylation, and neurodegeneration via PI3K/Akt/GSK3β signaling in the Aβ_1‐42‐treated mouse model of AD. On the basis of these results, we suggest that melatonin could be an effective, promising, and safe neuroprotective candidate for the treatment of progressive neurodegenerative disorders, such as AD.     

Melatonin regulates the transcription of βAPP‐cleaving secretases mediated through melatonin receptors in human neuroblastoma SH‐SY5Y cells

Melatonin is involved in the control of various physiological functions, such as sleep, cell growth and free radical scavenging. The ability of melatonin to behave as an antioxidant, together with the fact that the Alzheimer‐related amyloid β‐peptide (Aβ) triggers oxidative stress through hydroxyl radical‐induced cell death, suggests that melatonin could reduce Alzheimer's pathology. Although the exact etiology of Alzheimer's disease (AD) remains to be established, excess Aβ is believed to be the primary contributor to the dysfunction and degeneration of neurons that occurs in AD. Aβ peptides are produced via the sequential cleavage of β‐secretase β‐site APP‐cleaving enzyme 1 (BACE1) and γ‐secretase (PS1/PS2), while α‐secretase (ADAM10) prevents the production of Aβ peptides. We hypothesized that melatonin could inhibit BACE1 and PS1/PS2 and enhance ADAM10 expression. Using the human neuronal SH‐SY5Y cell line, we found that melatonin inhibited BACE1 and PS1 and activated ADAM10 mRNA level and protein expression in a concentration‐dependent manner and mediated via melatonin G protein‐coupled receptors. Melatonin inhibits BACE1 and PS1 protein expressions through the attenuation of nuclear factor‐κB phosphorylation (pNF‐κB). Moreover, melatonin reduced BACE1 promoter transactivation and consequently downregulated β‐secretase catalytic activity. The present data show that melatonin is not only a potential regulator of β/γ‐secretase but also an activator of α‐secretase expression through the activation of protein kinase C, thereby favoring the nonamyloidogenic pathway over the amyloidogenic pathway. Altogether, our findings suggest that melatonin may be a potential therapeutic agent for reducing the risk of AD in humans.     

Melatonin enhances the human mesenchymal stem cells motility via melatonin receptor 2 coupling with Gαq in skin wound healing

Melatonin, a circadian rhythm–promoting molecule, has a variety of biological functions, but the functional role of melatonin in the motility of mesenchymal stem cells (MSCs) has yet to be studied. In a mouse skin excisional wound model, we found that transplantation of umbilical cord blood (UCB)‐MSCs pretreated with melatonin enhanced wound closure, granulation, and re‐epithelialization at mouse skin wound sites, where relatively more UCB‐MSCs which were engrafted onto the wound site were detected. Thus, we identified the signaling pathway of melatonin, which affects the motility of UCB‐MSCs. Melatonin (1 μm ) significantly increased the motility of UCB‐MSCs, which had been inhibited by the knockdown of melatonin receptor 2 (MT₂). We found that Gαq coupled with MT₂ and that the binding of Gαq to MT₂ uniquely stimulated an atypical PKC isoform, PKCζ. Melatonin induced the phosphorylation of FAK and paxillin, which were concurrently downregulated by blocking of the PKC activity. Melatonin increased the levels of active Cdc42 and Arp2/3, and it has the ability to stimulate cytoskeletal reorganization‐related proteins such as profilin‐1, cofilin‐1, and F‐actin in UCB‐MSCs. Finally, a lack of MT₂ expression in UCB‐MSCs during a mouse skin transplantation experiment resulted in impaired wound healing and less engraftment of stem cells at the wound site. These results demonstrate that melatonin signaling via MT₂ triggers FAK/paxillin phosphorylation to stimulate reorganization of the actin cytoskeleton, which is responsible for Cdc42/Arp2/3 activation to promote UCB‐MSCs motility.     

Melatonin MT1 and MT2 receptor ERK signaling is differentially dependent on Gi/o and Gq/11 proteins

G protein-coupled receptors (GPCRs) transmit extracellular signals into cells by activating G protein- and β-arrestin-dependent pathways. Extracellular signal-regulated kinases (ERKs) play a central role in integrating these different linear inputs coming from a variety of GPCRs to regulate cellular functions. Here, we investigated human melatonin MT₁ and MT₂ receptors signaling through the ERK1/2 cascade by employing different biochemical techniques together with pharmacological inhibitors and siRNA molecules. We show that ERK1/2 activation by both receptors is exclusively G protein-dependent, without any participation of β-arrestin1/2 in HEK293 cells. ERK1/2 activation by MT₁ is only mediated though G_i/o proteins, while MT₂ is dependent on the cooperative activation of G_i/o and G_q/11 proteins. In the absence of G_q/11 proteins, however, MT₂-induced ERK1/2 activation switches to a β-arrestin1/2-dependent mode. The signaling cascade downstream of G proteins is the same for both receptors and involves activation of the PI3K/PKCζ/c-Raf/MEK/ERK cascade. The differential G protein dependency of MT₁- and MT₂-mediated ERK activation was confirmed at the level of EGR1 and FOS gene expression, two ERK1/2 target genes. G_i/o/G_q/11 cooperativity was also observed in Neuroscreen-1 cells expressing endogenous MT₂, whereas in the mouse retina, where MT₂ is engaged into MT₁/MT₂ heterodimers, ERK1/2 signaling is exclusively G_i/o-dependent. Collectively, our data reveal differential signaling modes of MT₁ and MT₂ in terms of ERK1/2 activation, with an unexpected G_i/o/G_q/11 cooperativity exclusively for MT₂. The plasticity of ERK activation by MT₂ is highlighted by the switch to a β-arrestin1/2-dependent mode in the absence of G_q/11 proteins and by the switch to a G_i/o mode when engaged into MT₁/MT₂ heterodimers, revealing a new mechanism underlying tissue-specific responses to melatonin.     

Effects of insulin and amyloid β1–42 oligomers on glucose incorporation and mitochondrial function in cultured rat hippocampal neurons

Aim: The molecular basis for impaired glucose metabolism in patients with Alzheimer's disease (AD) has not been fully clarified. We tested whether insulin and amyloid (A)β_1–42 oligomers would regulate glucose metabolism and energy homeostasis directly in cultured rat hippocampal neurons and evaluated possible interactions between insulin signaling and Aβ_1–42 oligomers. Methods: Dissociated hippocampal neurons were prepared from Wistar rat embryos at day 21 of gestation and cultured for 14 days. Cultured neurons were exposed to insulin (1 µM) for 30 min, and Aβ_1–42 oligomers (1 µM) were added to culture media for 10–30 min. The glucose uptake of cultured neurons was measured by enzymatic fluorescence assay using 2‐deoxy‐d‐glucose (2DG), and adenosine triphosphate (ATP) contents were quantified using a luciferin/luciferase luminescence assay. Results: Aβ_1–42 oligomers did not suppress 2DG uptake, reflecting the activities of glucose transporters and/or hexokinase, but led to disrupted ATP contents in the presence and absence of monocarboxylates (lactate/pyruvate). Insulin and C‐peptide did not change glucose uptake or ATP concentrations. Conclusion: The primary target of Aβ_1–42 oligomers might be mitochondria, which could explain the reduced cerebral glucose levels in patients with AD. Moreover, insulin signaling was not directly linked to glucose metabolism or energy homeostasis in cultured rat hippocampal neurons. Geriatr Gerontol Int 2011; 11: 517–524.     

Rosiglitazone induces interleukin‐1 receptor antagonist in interleukin‐1β–stimulated rat synovial fibroblasts via a peroxisome proliferator–activated receptor β/δ–dependent mechanism

Objective

To study the potency of 2 peroxisome proliferator–activated receptor γ (PPARγ) agonists, 15‐deoxy‐Δ^12,14‐prostaglandin J₂ (15‐deoxy‐PGJ₂) and rosiglitazone, to modulate the expression of interleukin‐1 receptor antagonist (IL‐1Ra) in rat synovial fibroblasts.

Methods

Levels of messenger RNA for IL‐1Ra and PPAR isotypes (α, β/δ, γ) were assessed by real‐time polymerase chain reaction in rat synovial fibroblasts exposed to 10 ng/ml of IL‐1β. PPAR levels were assessed by Western blotting and secreted IL‐1Ra levels by immunoassay. The potency of PPARγ agonists and the PPARβ/δ agonist GW‐501516 on IL‐1Ra levels was tested in the range of 1–10 μM and at 100 pM, respectively. The contribution of PPARγ to the effects of rosiglitazone on IL‐1Ra secretion was examined either by its overexpression or by inhibition using wild‐type or dominant‐negative constructs and the antagonist GW‐9662 (10 μM), respectively. The dominant‐negative strategy was also performed to investigate the possible contribution of PPARβ/δ and NF‐κB activation.

Results

IL‐1β–induced IL‐1Ra production was increased by 10 μM rosiglitazone but was reduced dose‐dependently by 15‐deoxy‐PGJ₂. Both agonists lowered IL‐1β secretion, but rosiglitazone alone reduced the imbalance of IL‐1β/IL‐1Ra toward basal levels. Enhancement of IL‐1β–induced IL‐1Ra production by rosiglitazone was not affected by PPARγ overexpression or by its inhibition with dominant‐negative PPARγ or GW‐9662. Inhibition of NF‐κB was also ineffective against rosiglitazone but abolished the stimulating effect of IL‐1β on IL‐1Ra. All PPAR isotypes were expressed constitutively in rat synoviocytes, but PPARγ decreased dramatically upon IL‐1β exposure, whereas PPARβ/δ remained stable. Dominant‐negative PPARβ/δ abolished the enhancement of IL‐1Ra by rosiglitazone, whereas GW‐501516 reproduced the effect of rosiglitazone on IL‐1Ra secretion.

Conclusion

Rosiglitazone stimulates IL‐1Ra production by a PPARβ/δ mechanism in activated rat synovial fibroblasts, further contributing to its potential antiarthritic properties and opening new perspectives for the modulation of inflammatory genes by specific PPAR agonists in articular cells.

     

Genetic deletion of MT1 and MT2 melatonin receptors differentially abrogates the development and expression of methamphetamine‐induced locomotor sensitization during the day and the night in C3H/HeN mice

Abstract: This study explored the role of the melatonin receptors in methamphetamine (METH)‐induced locomotor sensitization during the light and dark phases in C3H/HeN mice with genetic deletion of the MT₁ and/or MT₂ melatonin receptors. Six daily treatments with METH (1.2 mg/kg, i.p.) in a novel environment during the light phase led to the development of locomotor sensitization in wild‐type (WT), MT₁KO and MT₂KO mice. Following four full days of abstinence, METH challenge (1.2 mg/kg, i.p.) triggered the expression of locomotor sensitization in METH‐pretreated but not in vehicle (VEH)‐pretreated mice. In MT₁/MT₂KO mice, the development of sensitization during the light phase was significantly reduced and the expression of sensitization was completely abrogated upon METH challenge. During the dark phase the development of locomotor sensitization in METH‐pretreated WT, MT₁KO and MT₂KO mice was statistically different from VEH‐treated controls. However, WT and MT₂KO, but not MT₁KO mice receiving repeated VEH pretreatments during the dark phase expressed a sensitized response to METH challenge that is of an identical magnitude to that observed upon 6 days of METH pretreatment. We conclude that exposure to a novel environment during the dark phase, but not during the light phase, facilitated the expression of sensitization to a METH challenge in a manner dependent on MT₁ melatonin receptor activation by endogenous melatonin. We suggest that MT₁ and MT₂ melatonin receptors are potential targets for pharmacotherapeutic intervention in METH abusers.     

Alteration of articular cartilage frictional properties by transforming growth factor β, interleukin‐1β, and oncostatin M

Objective

To evaluate the functional effects of transforming growth factor β1 (TGFβ1), interleukin‐1β (IL‐1β), and oncostatin M (OSM) on the frictional properties of articular cartilage and to determine the role of cytokine‐mediated changes in cartilage frictional properties by extracting and redepositing lubricin on the surface of cartilage explants.

Methods

Neonatal bovine cartilage explants were cultured in the presence or absence of 10 ng/ml of TGFβ1, IL‐1β, or OSM over 48 hours. Boundary lubrication tests were conducted to determine the effects of endogenously produced surface localized lubricin and of exogenous lubricin at the tissue surface and in the lubricant solution. The initial friction coefficient (μ₀), equilibrium friction coefficient (μ_eq), and Young's modulus (E_Y) were determined from the temporal load data.

Results

IL‐1β and OSM decreased tissue glycosaminoglycan (GAG) content by ∼20% over 48 hours and decreased E_Y to a similar extent (11–17%), but TGFβ did not alter GAG content or E_Y. Alterations in proteoglycan content corresponded to changes in μ₀, but endogenous lubricin decreased boundary mode μ_eq. The addition of exogenous lubricin, either localized at the tissue surface or in the lubricating solution, did not modulate μ₀, but it did lower μ_eq in cytokine‐treated cartilage.

Conclusion

This study provides new insight into the functional consequences of cytokine‐mediated changes in friction coefficient. In combination with established pathways of cytokine‐mediated lubricin metabolism, these data provide evidence of distinct biochemical origins of boundary and biphasic pressure‐mediated lubrication mechanisms in cartilage, with boundary lubrication regulated by surface accumulation of lubricants and biphasic lubrication controlled by factors such as GAG content that affect water movement through the tissue.

     

Amyloid‐β neurotoxicity in organotypic culture is attenuated by melatonin: involvement of GSK‐3β, tau and neuroinflammation

Abstract: Alzheimer’s disease (AD) is a progressive neurodegenerative disorder marked by accumulation of extracellular deposits of amyloid‐β (Aβ) peptide in brain regions that are important for memory and cognition. The buildup of Aβ aggregates in the AD is followed by the formation of intracellular neurofibrillary tangles and activation of neuroinflammatory reactions. The present study investigated whether melatonin possesses a neuroprotective effect against Aβ‐induced toxicity. For this purpose, organotypic hippocampal slices were cultured and exposed to 25 μm of Aβ_25–35 in the absence or in the presence of melatonin (25, 50, or 100 μm ). In addition, the authors have investigated the involvement of GSK‐3β, tau protein, astroglial, and microglial activation, and cytokine levels in the melatonin protection against Aβ‐induced neurotoxicity. Melatonin prevented the cell damage in hippocampus induced by the exposure to Aβ_25–35. In addition, melatonin significantly reduced the activation of GSK‐3β, the phosphorylation of tau protein, the glial activation and the Aβ‐induced increase of TNF‐α and IL‐6 levels. On the basis of these findings, we speculate that melatonin may provide an effective therapeutic strategy for AD, by attenuating Aβ‐induced phosphorylation of tau protein, and preventing GSK‐3β activation and neuroinflammation.     

Enhancement of high glucose‐induced PINK1 expression by melatonin stimulates neuronal cell survival: Involvement of MT2/Akt/NF‐κB pathway

Hyperglycemia is a representative hallmark and risk factor for diabetes mellitus (DM) and is closely linked to DM‐associated neuronal cell death. Previous investigators reported on a genome‐wide association study and showed relationships between DM and melatonin receptor (MT), highlighting the role of MT signaling by assessing melatonin in DM. However, the role of MT signaling in DM pathogenesis is unclear. Therefore, we investigated the role of mitophagy regulators in high glucose‐induced neuronal cell death and the effect of melatonin against high glucose‐induced mitophagy regulators in neuronal cells. In our results, high glucose significantly increased PTEN‐induced putative kinase 1 (PINK1) and LC‐3B expressions; as well it decreased cytochrome c oxidase subunit 4 expression and Mitotracker? fluorescence intensity. Silencing of PINK1 induced mitochondrial reactive oxygen species (ROS) accumulation and mitochondrial membrane potential impairment, increased expressions of cleaved caspases, and increased the number of annexin V‐positive cells. In addition, high glucose‐stimulated melatonin receptor 1B (MTNR1B) mRNA and PINK1 expressions were reversed by ROS scavenger N‐acetyl cysteine pretreatment. Upregulation of PINK1 expression in neuronal cells is suppressed by pretreatment with MT₂ receptor‐specific inhibitor 4‐P‐PDOT. We further showed melatonin stimulated Akt phosphorylation, which was followed by nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) phosphorylation and nuclear translocation. Silencing of PINK1 expression abolished melatonin‐regulated mitochondrial ROS production, cleaved caspase‐3 and caspase‐9 expressions, and the number of annexin V‐positive cells. In conclusion, we have demonstrated the melatonin stimulates PINK1 expression via an MT₂/Akt/NF‐κB pathway, and such stimulation is important for the prevention of neuronal cell apoptosis under high glucose conditions.     

Selection of a new generation of orally active α-ketohydroxypyridine iron chelators intended for use in the treatment of iron overload

The prospect of selecting oral α-ketohydroxypyridine chelators intended for clinical use in iron overload has been examined using several animal models of efficacy and toxicity. Studies using iron dextran-loaded mice labelled with ⁵⁹Fe have shown that only the 1-substituted methyl, ethyl, (n)propyl, allyl, cyclopropyl, 2′-methoxyethyl, 3′-ethoxypropyl, or 2-methyl- or 2-ethyl- 3-hydroxypyrid-4-one chelators were orally effective in increasing iron (⁵⁹Fe) excretion by comparison to intraperitoneally administered desferrioxamine at the same dose (250 mg/kg). In contrast, chelators containing -H, mono- or dihydroxyalkyl and diethoxyethyl 1-substituents caused very little or no increase in iron (⁵⁹Fe) excretion by the oral or intraperitoneal routes. In vitro studies using ferritin and haemosiderin have shown that equivalent iron release took place with both groups of chelators irrespective of their in vivo effects. In most cases there was no correlation between the n-octanol/water partition coefficient (K_par) and iron removal efficacy but positive correlation between the lipophilicity and acute or subacute toxicity of these chelators in rats. The most toxic chelator in the chronic toxicity studies in rats was the lipophilic 1, 2-diethyl-3-hydroxypyrid-4-one (EL1NEt). The most effective chelator in increasing iron excretion in mice and rabbits was 1-allyl-2-methyl-3-hydroxypyrid-4-one (L1NAll), and the chelator with the highest safety margin in mice and rats was 1, 2-dimethyl-3-hydroxypyrid-4-one (L1). Overall the oral effectiveness in increasing iron excretion by these chelators in animals does not appear to be related to their lipophilicity or their ability to mobilise polynuclear iron in vitro but rather to other properties possibly related to their rate of biotransformation and excretion. © 1993 Wiley-Liss, Inc.