Background
Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family and plays essential roles in differentiation and survival of central and peripheral neurons [
1]. Thus, BDNF has been implicated in neurodegenerative diseases and has been tried as a therapeutic intervention for such diseases [
2]. In addition to these classic neurotrophic actions, BDNF regulates synaptic plasticity in the brain [
3,
4]. Because of its effects on functional alterations in the central nervous system, it was implicated in psychiatric diseases such as depression [
5]. Another face of BDNF has been emerging, which is the regulation of food intake and energy metabolism [
6,
7]. The involvement of BDNF in diabetes has been studied from those aspects [
8].
Diabetes mellitus (DM) is associated with macrovascular complications, including increased risks of coronary heart disease, stroke and amputation, and microvascular complications, such as nephropathy, retinopathy and neuropathy. In addition to its beneficial effects on neuropathies [
9] and retinopathies [
10], the cardiovascular protective effects of BDNF were reported. BDNF was shown to prevent or ameliorate myocardial infraction [
11] and to promote revascularization after ischemic injury [
12]. Furthermore, the association of blood BDNF levels with cardiovascular diseases, such as angina pectoris [
13] and heart failure [
14,
15] were reported, with lower levels of BDNF being associated with the risk of cardiovascular diseases.
These effects must come from outside of the CNS, presumably from BDNF in blood. Both increased [
16,
17] and decreased [
18‐
20] BDNF were reported in blood of patients with DM. The mechanism of this inconsistency remains unclear although blood glucose levels, duration of DM, medications, gender, etc., may play a role.
Platelets are the major source of BDNF in human blood, at least under physiological conditions [
21,
22], which may indicate that serum BDNF levels reflect the amount released from platelets. Although the causality and mechanisms remain unclear, it is possible that the dynamics of BDNF release are dysregulated in diabetes patients, thus altering serum levels. Therefore, we investigated the acute BDNF release from platelets from healthy individuals. BDNF is known to be released from platelets in response to certain stimulants such as thrombin [
22]. Thus, we examined the effects of molecules that are abundant in the blood of DM patients and whose receptors are expressed in platelets.
We focused on advanced glycation end products (AGEs). AGEs are a group of carbonyl compounds produced by the non-enzymatic reaction of reducing sugars and amino groups of proteins, lipids and nucleic acids, which is called the Maillard reaction [
23]. AGE levels are high in blood of patients with diabetes because of their chronic hyperglycemia [
23], and AGE accumulation induces adverse effects on endothelial cells [
24] and cardiovascular systems [
25]. Their receptors, such as receptors for AGE (RAGE) [
26], CD36 [
27] and 5HT2A/C receptor [
28], are expressed in human platelets.
To analyze the dynamics of acute BDNF release from platelets under physiological conditions, we examined the effect of AGEs on BDNF release from platelets of healthy control participants (non-diabetic) and analyzed their signaling mechanisms.
Discussion
Reports of serum BDNF levels in patients with DM have been controversial. In comparison with non-diabetic individuals, some studies found that serum BDNF levels in patients with DM were lower [
18‐
20] while others found higher values [
16,
17].
Because the major origin of BDNF in blood is platelets [21, 22 also see Additional file
1: Figure S1, Additional file
2: Table S1], alterations in BDNF levels may be the result of dysregulation of BDNF release from platelets. Therefore, we examined the acute effects of high glucose, a primary indication of DM, and AGE, which are resultant products of chronic hyperglycemia.
Here, we revealed that AGE, a glycated-BSA in this study, induced BDNF release from human platelets through intracellular Ca2+ elevation possibly via the Src-Syk pathway.
AGEs are heterogeneous carbonyl compounds that are formed by the Maillard reaction between reducing sugars and the amino groups of protein, lipids and nucleic acids. Because of the chronic hyperglycemia in diabetic patients, AGE accumulation in blood is accelerated in those individual [
42]. AGE accumulation correlated with microvascular lesions that cause diabetic retinopathy [
43] or nephropathy [
44]. In addition, AGE was reported to enhance aggregation and activation of platelets [
35]. These abnormal platelets may be related to the risk of the development of cardiovascular complications [
45]. Results of a clinical study also supported the concept that high AGE in diabetes mellitus is related to the risk of peripheral arterial diseases [
46].
The deleterious action on endothelial cells by AGEs is mediated by their receptors. While many receptors for AGEs have been identified, the receptor for AGEs (RAGE) is the most characterized. Upon binding of AGEs to the RAGE, a variety of downstream responses occur, such as production of reactive oxygen species and expression of cytokines, cell adhesion molecules, etc. that lead to cellular insults [
47]. While the expression of RAGE [
26] as well as CD36 [
27] and 5HT2A/C receptor [
28] were reported on the platelet surface, it remains which (or all) receptors are responsive to AGE for BDNF release.
BDNF is a neurotrophic factor promoting differentiation and survival and modulating synaptic plasticity in central and peripheral neurons through its cognate receptor TrkB. Outside of the nervous system, TrkB expression was reported in several cell types such as immune [
48], pancreatic alpha [
49], endothelial [
50] and myocardial [
51] cells. In contrast to AGEs, BDNF was shown to exert a protective action on these cells [
12,
52]. Exogenous BDNF induced vasodilatation and protected against vascular injury and thrombus formation in the walls of cerebral arteries [
53] and also was noted to act on revascularization [
12]. Clinical observations showed that low blood BDNF levels are associated with a high risk of heart failure [
14,
15,
54]. Thus, regulated release of BDNF from platelets may be an important mechanism for maintaining cardiovascular homeostasis.
Mechanism of BDNF release has been well characterized in neuronal cells. BDNF is released in an activity‒dependent manner; in other words, depolarization-induced Ca
2+ influx triggers its release [
55]. Other studies showed that an increase in cytosolic Ca
2+ derived from intracellular Ca
2+ stores is sufficient for BDNF release [
56]. These results indicate that the increase in intracellular Ca
2+, whether it comes from an extracellular space or intracellular stores, induces BDNF release. Thus, Ca
2+-dependency of BDNF release from platelets was examined. In the present study, Ca
2+ ions were not included in the assay buffer to avoid basal platelet activation. Ca
2+-dependency was analyzed by using BAPTA-AM, a cell permeable Ca
2+-chelator. BAPTA-AM completely inhibited AGE-induced BDNF release, suggesting that the BDNF release mechanism is rather common among different cell types.
Similar to other growth factors, BDNF is contained in α-granules of platelets [
57]. To determine the action of AGE on alpha-granules, release of another molecule in these granules was examined. AGE induced the release of PF4, one of the contents of alpha-granules. Although AGE significantly increased the release of 5-HT in dense granules, the ratio to its total content was quite low (Additional file
3: Figure S2). This suggests that under extracellular Ca
2+-free conditions, 5-HT release is not sufficiently driven by a particular stimulation.
How is Ca
2+ released from intracellular stores? We examined the roles of SFKs because a previous study indicated that there were high levels of Src in platelets [
38] and that SFKs were involved in in AGE-induced signaling. In addition, activation of SFKs was shown to lead to an increase in intracellular Ca
2+ [
41,
58]. AGE was reported to induce Src activation in vascular endothelial cells through RAGE [
39]. CD36, another receptor for AGE, interacted with Fyn, Lyn and Yes, members of SFKs in platelets [
40]. Thus, whether the inhibitor of kinases suppresses the effect of AGE was analyzed. PP2, a potent Src family kinase inhibitor, completely blocked the AGE-induced BDNF release. The results suggest that Src and/or other members of the family kinases are activated by AGE in human platelets. Indeed, western blot analysis using phospho-Src antibody showed that AGE increased the phosphorylation of Src (Fig.
5). Although a comprehensive investigation is awaited, other members of SFKs may be involved in the process of BDNF release. In B-cells, Lyn, a member of SFKs, phosphorylates and activates Syk, which induces Ca
2+ release from the stores through phospholipase Cγ (PLCγ)-IP3 [
41]. Syk was shown to be activated in platelets and increase intracellular Ca
2+ in response to stimuli [
58]. AGE actually induced phosphorylation of Syk (Fig.
6). It was inhibited by PP2, suggesting that it occurred downstream of SFKs after AGE stimulation. Thus, the results obtained in this study suggest that AGE activates SFKs, at least Src, and that downstream Syk then increases intracellular Ca
2+ possibly through PLCγ-IP3 activation.
Considering the toxic action of AGEs and protective roles of BDNF, it can be hypothesized that AGE-induced BDNF release is a biological defense system in the early phase of diabetes when the levels of AGEs are becoming higher. To protect against AGE-induced damage to, for example, vascular endothelial cells, platelets release BDNF to protect these cells in an early phase of the disease. However, a chronic elevation of AGEs may induce depletion or downregulation of BDNF in platelets during the progression of DM. Actually, a recent report supported this idea. While the serum BDNF levels are higher in prediabetic or early diabetic individuals than in normoglycemic persons, the serum BDNF levels are lower in patients with longstanding DM than in normal controls [
16]. Thus, at least one causative factor in the breakdown of serum BDNF homeostasis in DM patients seems to be due to the accumulation of AGEs in the blood.
Authors’ contributions
KF, IF, OH, HN, HS and NT designed the research. KF, YI, HS and NT performed the experiments. KF and NT wrote the paper. All authors read and approved the final manuscript.