Abstract
Glycine-proline-glutamate (GPE) and cycloprolylglycine (cPG) are naturally cleaved peptides of the insulinlike growth factor I (IGF-I) in the central nervous system. The neuroprotective actions of IGF-I have been widely studied in experimental models of Alzheimer´s disease (AD) and AD patients. However, there is less data about the molecular mechanisms involved in the protective effects of both IGF-I derived peptides in murine models of this disease. Here, we have analyzed the key issues of our study on the effects of GPE and cPG in a murine model of amyloid-β peptide infusion and revised the research progress on the effects of both compounds and new analogues of these molecules against inflammation, its relationship with the expression and synthesis of hormones implicated in memory processes and the intracellular signaling pathways related to these protective effects. Understanding the molecular mechanisms involved in the action of these molecules in experimental models of AD will help to develop strategies to fight one of the most common neurological diseases.
Keywords
Alzheimer’s disease, β-amyloid, GPE, Cycloprolylglycine, Inflammation, Neuroprotection, Signaling, Somatostatin
Abbreviations
Aβ: Amyloid-β peptide; AD: Alzheimer´s disease; AMPA: α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; cPG: cycloprolylglycine; CREB: cAMP response element-binding protein; ERK: Extracellular signal-regulated kinase; GABA: γ-aminobutyric acid; GFAP: Glial fibrillary acidic protein; GPE: Glycine-proline-glutamate; GRK: G-protein-coupled receptor kinase; GSK3β: Glycogen synthase kinase-3β; ICV: Intracerebroventricular; IGF-I: Insulin-like growth factor I; JNK: c-Jun N-terminal kinase; mTOR: mechanistic target of rapamycin; NMDA: N-methyl-D-aspartate; PI3K: Phosphatidylinositol 3-kinase; p70S6K: p70 ribosomal S6 kinase; SRIF: Somatostatin.
Commentary
Alzheimer´s disease (AD) is a devastating disease caused by the accumulation and interaction of tau-containing neurofibrillary tangles and amyloid beta peptide (Aβ) plaques in the brain [1,2]. The increased production and deposition of Aβ peptides results in microglial activation, and the production of inflammatory cytokines further increases Aβ synthesis [3], leading to neuronal death and pathological changes in astrocytes that impair Aβ clearance [4].
This disease is the most common cause of cognitive impairment, being associated with a decrease in numerous neurotransmitters in the brain and cerebrospinal fluid of patients with AD [5]. One of the most affected is somatostatin [6], which is widely distributed in the hippocampus and is involved in the control of cognitive functions. Furthermore, the reduction of SRIF levels in the cerebrospinal fluid is directly related to the severity of this disease [7]. Intracerebroventricular (ICV) infusion of Aβ is an experimental approach to this disease, since it increases Aβ deposition in the hippocampus, along with an increase in neuronal death and alterations in synaptic plasticity, neurotransmitter levels, learning and memory [8]. It also reduces SRIF levels in the brain [9], with the Aβ25-35 fragment having a greater deleterious effect on the cerebral somatostatinergic system than Aβ1-42 peptide [10]. In this regard, a decrease in the number of receptors for this neuropeptide has been described in AD patients, along with a decrease in SRIF levels [11].
Various approaches have been tried to block Aβ toxicity and reduce the progression of AD. Among them, the administration of insulin-like growth factor I (IGF-I) in experimental models of the disease, which acts as a neurotrophic and survival factor by activating the phosphatidylinositol 3-kinase (PI3K) pathway [12]. We have reported that co-administration of IGF-I with Aβ25-35 reduced cell death in the hippocampus, associated with an increase in Aβ-degrading enzymes [13]. This increase appeared to be associated with the restoration of hippocampal SRIF levels, as this neuropeptide stimulates neprilysin and insulin-degrading-enzyme (IDE) activities [14,15]. However, although brain levels of IGF-I are reduced in AD patients [16], its limited ability to cross the blood-brain barrier and possible mitogenic effects limits its clinical use [17,18].
Enzymatic degradation of IGF-I at its N-terminal portion generates the tripeptide glycine-proline-glutamate (GPE), which is present in the circulation and brain [19]. A dipeptide, cycloprolylglycine (cGP) is formed from GPE, by cyclization after enzymatic cleavage of glutamate [20]. These small peptides exhibit pharmacological effects similar to those of IGF-I showing anti-inflammatory properties and protective effects after ischemic injury or Aβ infusion in the brain [21-23]. Similarly, GPE reduces cell death and microglial activation, and the effects of cGP are mediated by stabilizing IGF-I bioavailability, and consequently its function [24,25]. It is also important to note that, like IGF-I, GPE has a protective effect on the SRIF receptor-effector system in the temporal cortex in experimental models of AD [26].
Although these peptides were discovered more than three decades ago, their use has been limited and confined to experimental models of neurodegenerative diseases. This is partly due to the relatively short half-life of these compounds in serum [27], although the activity of proteases is lower in the central nervous system, and therefore, the half-life is longer [28]. Nevertheless, to maintain effective plasma levels of GPE, continuous intravenous administration may improve neuroprotection after brain damage [29]. There are also limitations in our knowledge of the neuroprotective mechanisms of these molecules; for example, although it interacts with glutamate receptors, the prevention of neuronal death by GPE and its analogues is not directly related to its affinity for these receptors [30]. Controversial actions have also been described, and thus, although GPE can bind to NMDA receptors and act as an antagonist, protecting hippocampal neurons from NMDA-induced toxicity “in vitro” [31], it can also be a weak agonist of these receptors [32]. Another problem with the use of these molecules is a selective protective effect on certain neurons; thus, after brain damage the infusion of these peptides prevents the loss of γ-aminobutyric acid (GABA)- and SRIFergic neurons, but not of those containing parvalbumin [21].
To avoid such instability problems, conjugates of these compounds with different functional groups have been created. Data in the literature indicate that in most cases GPE is an NMDA receptor antagonist, alleviating the harmful effects of glutamate overload, but with low affinity for this receptor [33], so new compounds with a higher affinity and a longer half-life have been designed. In this way, both compounds regulate IGF-I bioavailability by modifying the IGF-I binding to the IGF-I binding protein-3 [34] and new pseudopeptides show longer half-lives in plasma and good water solubility, modifying inflammatory state after Aβ exposure [35]. However, it has been reported that some analogs show biases in neuroprotection levels, depending on the design of the conjugates [36], so more work is needed to research new compounds for a greater efficacy in reducing the causes of AD. Another problem is that systemic administration of certain molecules may cause peripheral side effects in other tissues. Therefore, intranasal administration of nanoparticles is a strategy to be considered in this disease, given their ease of crossing the blood-brain barrier [37].
The use of these peptides is also limited by the lack of studies that have analyzed in depth the molecular mechanisms associated with their anti-inflammatory properties [35] and their association with neurotransmitter receptor-effector systems and signaling pathways involved in these protective actions. On this way, the few studies that have been carried out show that that GPE interacts with N-methyl-D-aspartate (NMDA) and cPG excites α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors [38], albeit usually with lower affinity at physiological brain levels. Therefore, higher concentrations of both molecules must be achieved locally, usually after treatments, in order to improve synaptic transmission [39]. The phosphatidylinositol 3-kinase (PI3-K)/Akt pathway is one of the keys signaling cascades that can modulate NMDA receptor-mediated synaptic plasticity and contribute to long-term potentiation [40].
Although both peptides mimic many of the effects of IGF-I, they do not bind to its receptor. Using autoradiographic techniques, it has been shown "in vivo" that GPE binds to glial cells [41] and that the effects of GPE and des-IGF-I, the molecule derived from the tripeptide rem differ in their actions in glia, since blockade of NMDA receptors prevented the effects of GPE in these cells, whereas the additive effects of both molecules indicate that des-IGF-I acts through IGF-I receptors [42]. Data on the actions of both peptides on neurons mostly refer to “in vitro” studies. In the studies of neuroprotective and proliferative effects in neural stem cell cultures, they have been described as being related to the activation of survival pathways and neuroprotective factors [43,44]. However, there are data suggesting that the neuroprotective actions of GPE are not as evident, as it binds weakly to the dentate gyrus, where NMDA receptors are highly expressed [45].
Therefore, to gain insight into the mechanisms of both peptides involved in the protection/restoration of the SRIF system, involved in learning and memory processes, we had studied the hippocampal effects of co-administration of GPE or cPG in an experimental model of AD, based on chronic ICV infusion of Aβ25-35 [46]. Intraperitoneal injections of 300 µg of GPE or cPG at 0, 6 and 12 days were used, based on previous reports with similar concentrations and routes of administration [22,41,47]. In these previous publications both peptides were shown to exert numerous benefits after neuronal damage caused by Aβ or ischemia. These include their protective role on neurotransmitters involved in learning and memory processes through cytosolic calcium modulation, increased neuronal survival mediated by glial cells and the prevention of sensorimotor impairments stand out. In a first set of studies, we analyzed the effect of both peptides on the β-amyloid-induced cell death and depletion of SRIFergic system in the hippocampus. Both IGF-I metabolites reduced Aβ-induced cell death and depletion of the hippocampal SRIFergic system, with GPE being more effective than cPG and related to the reduction of c-Jun N-terminal kinase (JNK) activation.
The toxic effects of Aβ peptides, as well as the protective effects of GPE and cPG are modulated by opposing changes in common signaling pathways, that ultimately regulate, among other actions, cell death and the implementation of neurotransmitter systems [43,48]. We therefore examined the activation of different signaling targets that can be grouped into two different pathways, Akt/cAMP response element-binding protein (CREB) and extracellular signal-regulated kinase (ERK)/ G-protein-coupled receptor kinase (GRK), which converge on the mechanistic target of rapamycin (mTOR)/p70 ribosomal S6 kinase (p70S6K). Our results showed restoration of Akt phosphorylation in Aβ-treated rats when GPE was co-administered, together with blockade of JNK and glycogen synthase kinase-3β (GSK3β) and an increase in CREB activation. Blockade of these targets is associated with a reduction in cell death, and probably with a protective effect on the SRIFergic system [49]. In addition, the increase in CREB phosphorylation can directly increase SRIF and SRIF receptor 2, as both genes have CRE sites [50,51] and we detected an increase in both mRNA levels after co-administration of GPE [46].
Co-administration of cPG did not modify either GSK3β or CREB phosphorylation, but increased ERK1/2 and GRK2 activation. ERK phosphorylation upregulates the transcription of GRK2 [52], and this protein phosphorylates SRIF receptor 2, potentiating the SRIF receptor-effector system [53]. It is noteworthy that both IGF-I metabolites activate, through different pathways, the phosphorylation of p70S6K, a crucial factor in protein synthesis, with translational control at synapses required for the development of long-term memory [54].
This neurodegenerative disease is associated with increased brain inflammation and astrogliosis [55,56]. In our study [46], we had found an increase in glial fibrillary acidic protein (GFAP) and vimentin levels in Aβ-treated rats, together with an increase in the inflammatory environment, as reported [57]. Co-administration of these peptides favored the generation of an anti-inflammatory profile, i.e., GPE binds to astrocytes and promote their survival, which is associated with reduced brain inflammation in models of neurodegenerative disorders [58].
In turn, anti-inflammatory interleukins (IL) may play a relevant role in altering survival signaling pathways and the functionality of brain neurotransmitter systems [59,60]. Consequently, we had chosen IL-4, which was increased after co-administration of GPE or cPG and analyzed its effect on Akt phosphorylation and SRIF levels “in vitro”. IL-4 increased Akt activation and SRIF levels in neuronal cultures. Furthermore, Aβ25-35 plus GPE or cPG increased IL-4 levels in glial cell cultures. These data show that at least some of the effects of both small peptides are mediated by changes in the inflammatory environment that activate survival pathways. The most remarkable feature of this study is that both peptides in most cases prevented Aβ-induced changes in signaling pathways modulating anti-inflammatory processes (Figure 1).
Figure 1: Proposed model for reduction in hippocampal Aβ levels after GPE or cPG administration to Aβ25-35 treated rats. Infusion of Aβ reduces Akt signaling, whereas both peptides activate it. After binding them to NMDA receptors or to a possible unknow receptor in astrocytes, Akt activation may produce a rise in mTOR hosphorylation, increasing insulin-degrading enzyme and anti-inflammatory cytokines. These cytokines may bind to receptors in neurons, enhancing Akt-related ignaling, and subsequently; IDE and SRIF synthesis, that in turn reduce Aβ content. Thus, these events can improve neuroprotection by inhibiting Aβ-induced excitotoxicity. Aβ: β-amyloid 25-35 peptide; AI-ILs: Anti-inflammatory interleukins; Akt: Protein kinase B; cPG: cycloprolylglycine; ERK: Extracellular signal-regulated kinase; GPE: Gly-Pro-Glu; glu: glutamate; IDE: Insulin-degrading-enzyme; IL-R: IL-receptor; mTOR: mechanistic target of rapamycin; NMDA-R: N-methyl-D-aspartate receptor; SRIF: somatostatin.
In summary, our study demonstrated that GPE and cPG attenuation increased cell death and inflammation in an experimental model of AD disease. The intracellular signaling mechanisms involved in the protective effects of the two IGF-I-derived peptides are different, although their protective effects were mediated by the activation of targets associated with protein synthesis and memory consolidation, coinciding with the restoration of a neurotransmitter system involved in cognitive processes. Recent reports have shown that cPG normalizes IGF-I function in age-related neurological situations [61] and reduces amyloid plaque burden, thereby, improving memory in a transgenic model of AD [62]. It should also be noted that different peptidomimetics of GPE modulate oxidative stress and inactivate α-secretase, thereby reducing cell death by apoptosis and necrosis [63]. In fact, the development of analogs of these small peptides is providing compounds with longer half-lives and lack of cytotoxicity [64], which may allow their future use in the treatment of neurodegenerative diseases.
Funding
This work received no external funding.
Conflict of Interests
The author declares no conflict of interest.
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