AMPK: Potential Therapeutic Target for Ischemic Stroke

The brain is an organ that serves as the central nervous system among all vertebrate. Stroke is caused by occulusion of cerebral vessels or angiorhagia 1 - 4 . It is reported that stroke is the primary cause of disability and affects approximately 795,000 people annually. Stroke is characterized by '3H', namely high mortality, high morbidity, and high disability, it causes immense health and economic burdens to families and society 5 . Stroke may contribute to brain dysfunction due to a disturbance in the cerebral blood supply, which is caused by occlusion of vessels due to a blood clot or a bleeding vessel 6 . Mild or chronic cerebral ischemia might be nonfatal and cause mild somatic symptoms, whereas acute stroke may cause hemiplegia, cardiac dysfunction, and even death. Ischemic stroke is more common than hemorrhagic stroke in all cases, we pay attention to the former throughout this review. Previous studies have demonstrated that the dysfunction of energy metabolism plays a significant role in ischemic stroke. The underlying mechanism appears to involve an energy sensor called AMPK, a member of the serine/threonine (Ser/Thr) kinase group 7 - 11 . The historical background of AMPK may date back to two independent works by Gibon and Carbson in 1973. Later, it was extracted and sequenced by Carling et al until 1994 and was identified as a peripheral 'energy sensor' under conditions of energy deprivation, ischemic stress, and heavy exercise 12 . Studies have demonstrated that AMPK plays an important role in various organs, including the brain 13 , heart 14 , liver 15 , kidney 16 , gastrointestinal tract 17 , lung 18 , and pancreas 19 . AMPK, which functions as the cellular energy sensor, can resist cerebral ischemic injury by switching on catabolic pathways and switching off ATP-consuming processes 20 , 21 . Over the past 40 years, the annual number of publications on AMPK has surged from several to nearly 2000. Our laboratory has discovered that AMPK is a protective molecule in myocardial ischemia 22 , vasorelaxation 23 , angiogenesis 24 , fluid shear stress 25 , 26 , and diabetic cardiac injury 27 . A positive effect of AMPK has been reviewed in the heart 28 , liver 29 , and kidney 30 , etc., whereas its relationship with ischemic stroke has not been well discussed. Thus, further understanding of AMPK and ischemic stroke may aid in experimental studies and clinical therapeutics. Herein, we first introduce the background of AMPK and ischemic stroke, followed by a review of its upstream agonists and antagonists. Second, we summarize the regulatory mechanism of AMPK in ischemic stroke, including oxidative stress, autophagy, apoptosis, mitochondrial dysfunction, glutamate excitotoxicity, neuroinflammation, and angiogenesis. AMPK stands at the crossroads of diverse regulatory mechanism to main energy homeostasis during stroke. Third, therapeutic methods and benefits beyond ischemia are discussed. Finally, we bring attention to various controversial opinions, ethical issues, and potential directions. This review highlights recent advances and provides a comprehensive picture of AMPK, which may be helpful in drug design and clinical therapy for treatment of ischemic stroke.

AMPK is widely but distinctly distributed in different tissues and organs, which might explain the susceptibility of different organs to ischemic stimuli. The early work of Turnley and colleagues reported a systematic study on AMPK that provides a comprehensive understanding of the distribution of AMPK in specific types of cells 20 . The α1 subunit mainly exists in the brain, heart, lung, kidney, and liver. The α2 subunit mainly exists in the liver, heart, skeletal muscle, and cerebral neurons. The α1 subunit is present in the cytoplasm whereas the α2 subunit is present in both the cytoplasm and nucleus 38 . The α2 subunit can regulate transcription in the nucleus, which allows neurons that contain more AMPK α2 subunit to regulate energy metabolism and to survive metabolic stress during ischemia. The β1 subunit has a low content in skeletal muscle, while the β2 subunit is highly expressed in skeletal muscle. The γ3 subunit is almost exclusively restricted to fast twitch skeletal muscle 39 , whereas the γ1 and subunit are widely expressed in almost all tissues. The γ2 subunit is not expressed in high concentration in all tissues 20 . AMPK is expressed in the central nervous system (CNS) from at least the time of neuroepithelial formation. Gao and colleagues detected the expression of AMPK in the adult brain in 1995 40 . The brain mRNA levels of the α2 subunit are increased from the 10th to 14th day of the embryonic period, whereas the α1, β1, and γ1 subunits are expressed consistently at all ages. Moreover, the α2 catalytic subunit is highly expressed in neurons and activated astrocytes, whereas the α1 catalytic subunit exhibits only low expression in neuropil. Some neurons, such as granule cells of the olfactory bulb, do not express detectable levels of the β subunit 20 . The disparate expression and nuclear localization of the α, β, and γ subunits suggest specific functions and physiological roles of AMPK in different nerve cells.

Compounds C and C75 are two important antagonists of AMPK. Zhou and colleagues studied more than 10,000 hydrones and identified that Compound C is a reversible inhibitor of AMPK under various levels of ATP 55 . Initially, studies on Compound C were still limited to in vitro experiments of the CNS, despite in vivo experiments began to be carried out a few years ago 47 , 56 . Zhu et al discovered that inhibition of AMPK by Compound C reduces the viability and inhibits the energy metabolism of cultured primary cortical neurons under physiological conditions 57 . Moreover, atorvastatin treatment enhances the phosphorylation of AMPK and the expression of vascular endothelial growth factor (VEGF) in cultured human umbilical vein endothelial cells (HUVEC), whereas these effects are abolished by Compound C 34 . The inhibition of AMPK by Compound C was also reported by Li's 58 and Izumi's groups 59 . C75 is a synthetic FA synthase inhibitor or carnitine palmitoyltransferase (CPT-1) stimulator that indirectly restrains the phosphorylation of AMPK 60 . C75 rapidly reduces the level of the phosphorylated AMPK α subunit (pAMPKα) in the hypothalamus, even in fasted mice with elevated hypothalamic pAMPKα levels. C75 reduces the levels of pAMPK α and phosphorylated cAMP response element-binding protein (pCREB) in the arcuate nucleus neurons of the hypothalamus 61 . Moreover, Landree and colleagues discovered that C75 can decrease food intake and induce weight loss by inhibiting AMPK in primary cultures of cortical neurons 62 . McCullough and colleagues reported that the suppression of AMPK by C75 plays a role in not only the ischemic area but also the non-ischemic area. The inhibition of AMPK by C75 in the non-ischemic area is delayed and secondary to the ischemic area 56 . It was reported that C75 injections in mice induce rapid reduction in phosphorylated-AMPK (pAMPK) levels 61 and ATP levels in cultured hypothalamic neurons 63 . Strictly speaking, compound C and antimycin A cannot be regarded as specific inhibitors of AMPK, indicating the pleiotropic effects of them should be observed.

Oxidative stress Oxidative stress is a pathological process manifesting overproduction of reactive oxygen species (ROS) and reactive nitrogen species (RNS) under harmful stimuli 74 - 79 . These alterations result in an imbalance of antioxidants and ROS/RNS and cause immense injury to neurons. The brain is a highly energy-consuming organ that almost utilizes 50% of the total energy. However, the energy storage of brain can sustain only a few minutes without energy influx, which determines the high liability of the brain to ischemic stimuli. Compelling evidence has suggested that oxidative stress involving AMPK plays a pivotal role in the brain. Trans-caryophyllene (TC), a bicyclic sesquiterpene, is a selective agonist of type 2 cannabinoid receptor (CB2R). Choi and colleagues studied cortical neuron/glia-mixed cultures from Sprague-Dawley rats and found that aftertreatment of TC enhances AMPK/cAMP response element-binding protein (CREB) signaling and reduces OGD/reoxygenation-evoked neuronal oxidative stress. Exposure to hypoxia results in a significant increase in ROS that is closely related to neurological pathologies. Extracellular superoxide dismutase transgenic mice exposed to hypoxia for 10 days display increased pAMPK activity and decreased malondialdehyde (MDA) and lactic acid levels compared to the controls. This revealed that activation of cortical CB2R can ameliorate oxidative stress via activating AMPK/CREB signaling 80 . Pathophysiologically, quiescent astrocytes convert to the activated state and protect neurons from harmful stimuli, therefore rapidly alleviating injury to NVUs. AICAR, a cell-permeable activator of AMPK, significantly promotes the production of ketone bodies in rat cortical astrocytes under hypoxic conditions, thereby attenuating the oxidative injury of NVUs 81 . These data demonstrate that AMPK has potential prophylactic roles in diseases with chronic ischemia/hypoxia-induced cerebral injury, suggesting that AMPK is a pivotal and protective molecule in cerebral ischemic/hypoxic diseases.

Apoptosis is a process of programmed cell death that occurs in multicellular organisms. It is a dynamic process of blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, chromosomal DNA fragmentation, and global mRNA decay 97 - 101 . Apoptosis represents the execution of an ATP-dependent death program, which is often initiated by death ligand/death receptor interactions, such as Fas ligand and Bax/Bcl-2 102 . Neuron apoptosis might be induced and even worsen under pathological situations, including ischemia and hyperglycemia 103 . Neuron apoptosis is usually secondary to ischemic injury and serves as an index of ischemic extent, whereas this process may be ameliorated by AMPK. IPC can activate AMPK and ameliorate cell apoptosis in the peri-infarct region, as evidenced by the reduced percentage of TUNEL-positive cells, suggesting the ability of AMPK to reduce apoptosis in ischemic stroke 90 . Acute preconditioning of metformin ameliorates neuron apoptosis via activating AMPK, corresponding to a reduced percentage of TUNEL-positive cells and endoplasmic reticulum stress (ERS) during subsequent ischemic stimuli 93 , 104 . Chikusetsu Saponin Iva preconditioning activates AMPK/GSK-3β pathway and inhibits apoptosis of PC12 cells, as evidenced by reduced amounts of caspase-3 and Bax/Bcl-2. Activated AMPK and suppressed apoptosis also reduce infarct size and improve neurological outcomes after ischemia reperfusion injury (IRI) in MCAO mice. This indicates that Chikusetsu Saponin Iva protects against cerebral ischemia through the AMPK/GSK-3β-restrained apoptosis pathway 105 . Moreover, Yang et al discovered that preconditioning of Apelin-13 promotes the phosphorylation of AMPK and suppresses apoptosis after cerebral IR, while Apelin-13 has minimal effect on attenuating apoptosis when Compound C is added. This study provides evidence that Apelin-13 upregulates the levels of AMPK to perform neuroprotective effects by restraining apoptosis 106 . Estrogen deficiency induced by ovariectomy aggravates brain infarction in experimentally induced cerebral ischemic rats, while estrogen pretreatment reduces ischemia-induced cerebral injuries. In OGD neurons, aftertreatment of estrogen promotes AMPK activation and suppresses apoptosis through estrogen receptor α while neuroprotection of estrogen is prevented by AMPK inhibition 107 . These evidences suggest that inhibiting apoptosis by AMPK is a potentially valuable therapeutic strategy for ischemic stroke in the future.

Glutamate, the derivative of glutamic acid, is the most common excitatory neurotransmitter in the CNS and plays a pivotal role in neuronal activity 116 . Glutamate mediates synaptic transmission and is necessary for neuronal growth, maturation, and synaptic plasticity. Cerebral ischemia induces excessive release of extra-synaptic glutamate and the activation of glutamate receptors 117 , the process of which is also known as glutamate excitotoxicity. It may trigger disproportional calcium and sodium into neurons and serious neuronal death 118 . Compelling evidence has exhibited that activation of AMPK is able to protect neurons from glutamate excitotoxicity 119 . Curcumin, a natural polyphenolic compound from Curcuma longa, has been proven to exhibit beneficial effects on neuronal protection by activating AMPK. Li and colleagues reported that pretreatment of curcumin-induced production of AMPK protects neurons from glutamate neurotoxicity in the hippocampus 120 . GABA(B) receptors are heterodimeric G protein-coupled receptors composed of R1 and R2 subunits. AMPK binds directly to GABA(B) receptors, phosphorylates site S783 of the R2 subunits and suppresses the release of glutamate, which enhances the coupling of GABA(B) receptors to G-protein-gated inward rectifier K + (GIRK) channels, promoting energy storage and neuronal survival after hypoxic injury. This reveals that AMPK may decrease synaptic activity and limit neuronal excitotoxic injury to conserve cellular energy levels 121 . Intriguingly, it is worth noting that injury-induced phosphorylation of GABA was observed mainly in the hippocampal region, which usually disaccords with the location of MCAO 88 . These findings suggest a probable link between glutamate excitotoxicity and AMPK involving neuroprotection, which paves a new avenue for targeting AMPK in the treatment of cerebral ischemia.

Endothelial cells are epithelium that line the interior surface of blood vessels and lymphatic vessels. Among them, endothelial cells that line vessel walls are called vascular endothelial cells. Clinically, stroke patients with a higher density of blood vessels usually have less morbidity and longer survival, indicating that angiogenesis is important for neurological repair in the ischemic region 129 . Venna and colleague used C57BL/6N male mice subjected to 60-min MCAO and discovered that aftertreatment of metformin significantly promotes the expression of VEGF, concomitant with decreased infarct size and glial scaring. When tested in AMPKα2 knockout mice, they found that metformin did not have the same beneficial angiogenic effects, suggesting that metformin-induced protective effects are mediated by AMPK. Notably, they also reported that a 30-day treatment of metformin may improve neurological outcomes, further supporting the pharmacological value of metformin in ischemic stroke 130 . Jin et al reported that chronic metformin treatment after stroke activates AMPK and significantly enhances angiogenesis, as revealed by elevated vessel density and increased CD31, the endothelial marker, in the ischemic striatum 131 . Therefore, AMPK has an important role in upregulating angiogenic factor VEGF and promoting angiogenesis to ameliorate ischemic injury. To sum up, we discussed the regulatory mechanisms of AMPK in ischemic stroke, including oxidative stress, autophagy, apoptosis, mitochondrial dysfunction, glutamate excitotoxicity, neuroinflammation, and angiogenesis. AMPK stands at the crossroads of diverse regulatory mechanism to main energy homeostasis during stroke. The findings above demonstrate that pretreatment of estrogen (by inhibiting apoptosis) 107 , metformin (by promoting mitochondrial biogenesis and inhibiting neuroinflammation) 66 , curcumin (by inhibiting glutamate excitotoxicity) 120 , may ameliorates the ischemic or hypoxic symptoms. Moreover, aftertreatment of estrogen (by inhibiting apoptosis) 107 , trans-caryophyllene (by inhibiting oxidative stress) 80 , metformin (by inhibiting neuroinflammation) 127 , and sinomenine (by inhibiting neuroinflammation) also helps to resist against stroke in vivo and in vitro 128 . Moreover, AMPK attenuates ischemic injury in the neurovascular unit, such as neurons, astrocytes, microglial cells, to maintain the neurological homeostasis.

Electroacupuncture, also known as acuelectrostimulation pretreatment, is a special form of acupuncture that has shown therapeutic effects in animal experiments and stroke patients 139 . In 2003, Xiong and colleagues proposed that electroacupuncture preconditioning is able to induce cerebral ischemic tolerance 140 . Electroacupuncture, which combines the traditional advantages of acupuncture treatment and acuelectrostimulation, has great advantages, as it can be readily controlled, standardized, and objectively measured 141 . Electroacupuncture preconditioning significantly enhances the expressions of AMPKα and pAMPKα, concomitant with reduced TUNEL-positive cells and attenuated neuron apoptosis after stroke. However, intraperitoneal injections of Compound C suppress this phenomenon and exacerbate ischemic injury. This finding suggests that electroacupuncture preconditioning alleviates cerebral ischemic injury via AMPK signaling 142 . Exercise can be viewed as a chronic metabolic stress that leads to an increase in regional cerebral blood flow and metabolic functioning 143 . Adult male Sprague-Dawley rats were subjected to 1-3 weeks of exercise and displayed high expression of AMPK, concomitant with decreased infarct volume and enhanced glucose uptake and ATP production after stroke. Notably, they discovered that exercise attenuates neurological deficits and improves neurological outcomes in the long-term 144 . This study revealed that an increased expression of AMPK following exercise may enhance cerebral ischemic tolerance. Collectively, physical therapy targeting AMPK is an important type of non-pharmacotherapy compliant to stroke patients.

Equitable evaluation of AMPK Existing contradictions Although numerous studies have shown that AMPK is a protective molecule in ischemic stroke, we would like to discuss existing discrepancies to provide comprehensive information to readers. Some studies have revealed that restrained AMPK is capable of exerting protective effects. Nam et al discovered that increased AMPK immunoreactivity may enhance the ischemic injury in the hippocampal CA1 region 1-2 days after IR, whereas Compound C reverses the effects above, correlated with decreased reactive gliosis, ATP depletion, and lactate accumulation, suggesting that inhibition of AMPK has protective effects against ischemic stroke 147 . Suppression of AMPK by Compound C reduces infarct size and improves neurological outcome at 24 h after reperfusion, which is correlated with restrained release of microglial pro-inflammatory factors 148 . Hypothermia is one of the few therapies that has been translated into the clinic for global cerebral ischemia 149 . Hypothermia-related inhibition of pAMPK reduces infarct size, edema, and cerebral metabolic rate after MCAO, and this protection is enhanced after administration of Compound C. This study provides evidence that hypothermia exerts its neuroprotective effects by inhibiting AMPK in stroke 150 . Activated AMPK or elevated expressions of AMPK have been shown to exert harmful effects. One high dose of metformin remarkably increases the levels of pAMPK and lactic acid, promotes the consumption of energy, and exacerbates cerebral metabolic crisis and neuron death 47 , 56 . Moreover, long-term treatment with metformin for treatment of stroke may increase the deposition of β-amyloid, which possibly results from chronic metformin treatment 151 . These findings all suggest that AMPK is harmful, and its downregulation might be a potent therapy for stroke. Additionally, some studies maintain that AMPK is a double-edged sword in ischemic injury. AMPK overexpression downregulates the levels of CREB by promoting the SIRT1/miR-134 pathway. However, phosphorylated CREB is decreased in AMPK knockout primary hippocampal neurons. Therefore, AMPK plays a dual role in the regulation of CREB in OGD models, exerting a negative effect on total CREB expression by elevating the levels of SIRT1/miR-134 and a positive effect by phosphorylating CREB 152 . In primary neurons of hypoxia/ischemia, AMPK activation results in elevated cell death, whereas inhibition of AMPK may protect neurons from harmful stimuli, implicating that activated AMPK is involved in the injury process. Conversely, activation of AMPK prior to injury enhances the tolerance of neurons to subsequent insult. These data reveal that the dual roles of AMPK likely depend on whether it is activated prior to or during the ischemic/hypoxic insult 153 . In summary, AMPK indeed has dual actions on ischemic stroke, and its internal role needs further research.

As discussed above, stroke is a major global disease that threatens human health. It is characterized by high mortality and disability, and there are no ideal therapeutic methods. Currently, thrombolysis via the AMPK pathway is almost the only available treatment for acute ischemic stroke 159 . The ideal thrombolytic time of ischemic stroke is 3-6 hours, which is also known as the window phase. During this period, energy deprivation is the 'blasting fuse' that leads to a series of secondary injuries, including oxidative stress, acidosis, apoptosis, and excitotoxicity, and thus proper thrombolysis during this period is effective and might reduce post-stroke complications. However, only 3%-5% of stroke patients can receive thrombolytic therapy as a result of the strict time window, low precaution consciousness of cerebrovascular accidents, and reperfusion injury after thrombolysis 160 . Thereby, clinical and basic research require additional efforts to solve this problem. Clinically, anesthetists and surgeons are committed to cerebral resuscitation and protection during operations 161 . Cerebral injury during the perioperative period is a significant adverse event that results from the relative cerebral ischemia, usually including coronary artery bypass grafts (CABG) and intracranial tumor surgeries 162 . Glucose-insulin-potassium (GIK) is an important method for upregulating the levels of AMPK. In a previous study, 217 patients who underwent aortic valve replacement were assigned to GIK or placebo over a 4-year period. The result revealed that GIK treatment induces a substantial increase in pAMPK and its downstream molecule Akt compared to controls, concomitant with a significant reduction in the incidence of low cardiac output 163 . According to the conditions of systemic circulation, the blood flow of the internal carotid artery and the vertebral artery depends largely on cardiac output, and thus, we presume that GIK infusion during the perioperative period upregulates the levels of AMPK, which is helpful for restoring brain blood flow and ameliorating cerebral injury. Moreover, Kim et al recruited 1609 subjects (aged from 60 to 80) and performed a multivariate logistic regression analyses. They discovered that the polymorphism of PRKAG2-26C/T gene has a significant association with cognitive impairment in stroke patients (OR, 1.6; 95% CI, 1.1—2.3) 164 , suggesting that AMPK has a role not only in metabolic function but also in cognitive function of post-stroke patients. Notably, Campbell's group recruited 70 patients and randomly assigned patients into two groups. The results showed that patients who undergo stent retriever thrombectomy and receive alteplase (an AMPK activator) display improved reperfusion, early neurologic recovery, and improved functional outcomes compared to those who receive alteplase alone. Of the experimental group, 89% of patients exhibited restored the cerebral perfusion compared to 34% of patients in the control group. In addition, 71% of experimental patients achieved functional independence compared to 40% of control patients 165 . This trial demonstrated that an integrated combination of thrombolysis and interventional therapy may achieve profit maximization for ischemic stroke rather than AMPK-related thrombolysis alone.

So far as the status, stroke is still the fifth leading cause of death but the first cause of disability among all diseases, which produces in the United States and globally. Annually, about 795,000 people experience ischemic or hemorrhagic stroke. Approximately 610,000 of them are first events and 185,000 are recurrent events in the United States 170 . Metabolic therapy for treatment of ischemic stroke via AMPK signaling is almost the most important finding and of great potential for future research. The greatest discovery in this field may be the establishment of the MCAO model and the application of thrombolytic drugs. The MCAO model includes a relatively simple extracranial microsurgical dissection procedure that interrupts cerebral blood flow, which paves a way for subsequent scientific research. Clinically, both statins and metformin are used clinically, which is helpful to high-risk population and reduces the stroke incidence 135 , 171 . Thrombolytic therapy is applied in acute cerebral ischemia, especially acute cerebral infarction. However, there still exist some problems for the treatment of AMPK. First, whether AMPK plays a protective role in ischemic stroke is elusive, and some reports have identified negative effects 147 - 149 . Second, only a few of the AMPK activators have been applied in the clinic, and there are very few clinical trials of AMPK. Third, pre-stroke patients are almost asymptomatic, and most strokes are acute and may contribute to secondary injury. Finally, the thrombolytic time of stroke is short, and there are no sufficient effective therapeutic methods based on AMPK signaling after the window phase. New directions of AMPK studies should 1) evaluate the actions of AMPK in acute/chronic cerebral ischemia, or cerebral hemorrhage, 2) choose a more selective AMPK activator, 3) target NVUs rather than the neuron alone in ischemic stroke, 4) provide more reliable clinical evidence (e.g. randomized controlled trials) based on AMPK signaling, and 5) explore a cerebral drug delivery system involving AMPK signaling 172 . This review is committed to describing the beneficial roles of AMPK following ischemic stroke. AMPK is a Ser/Thr kinase that maintains cerebral metabolic homeostasis by enhancing catabolic pathways and restraining ATP-consuming processes. Initially, we provide a general introduction of AMPK and ischemic stroke, including descriptions of its structure, expression, localization, and upstream regulators. The high expression of AMPK in the cerebrum determines its protective biological actions under ischemic stimuli, such as inhibiting oxidative stress, apoptosis, mitochondrial dysfunction, glutamate excitotoxicity, and neuroinflammation and promoting autophagy. The NVU is the basic elementary unit of the brain tissues, and therefore, targeting NVUs via AMPK may induce better effects than targeting neurons alone. Next, we introduce the roles of AMPK in different phases, in commonly used animal models, and in various therapeutic methods, including medical treatment, receptor-targeted therapy, and physical therapy. However, the results of some studies question the protective role of AMPK in ischemic stroke, and we have discussed these discrepancies in detail. Moreover, additional benefits of AMPK beyond those for cerebral ischemic stroke have also been found for other cerebral diseases, such as neurological improvements and neurogenesis, the attenuation cerebral hemorrhage, benefits in NDD, and amelioration of neural tumors. Lastly, we discuss controversial opinions, ethical issues, and potential directions of future AMPK studies based on the existing evidence. Although some scholars suggest that activating AMPK is harmful and can aggravate ischemic injury, most studies demonstrate that it is a protective molecule in ischemic stroke. However, few studies have identified the role of AMPK in the clinic, which leads to an incomplete picture of AMPK. Therefore, more clinical studies are required to elucidate the roles of AMPK, which may aid in the prevention and treatment of stroke in the future.