Role of TRPM7 in cerebral ischaemia and hypoxia

Abstract

Transient receptor potential melastatin 7 (TRPM7) channel, a calcium‐permeable non‐selective divalent cation channel, is broadly expressed in various cells and tissues, including the brain. TRPM7 is thought to be coupled to the metabolic state and regulate calcium homeostasis in the cell. TRPM7 takes part in a wide range of cell biology processes that affect cell growth and proliferation, as well as in embryonic development and skeleton formation. TRPM7 plays a significant role in ischaemic and hypoxic brain injury and neuronal cell death. TRPM7, as a key non‐glutamate mechanism of cerebral ischaemia, also triggers an intracellular ionic imbalance and neuronal cell death in ischaemia and hypoxia. We have reported that TRPM7 is expressed in neurons of the hippocampus and cortex and activation of TRPM7 induced ischaemic neuronal cell death; suppression of TRPM7 with virally mediated gene silencing using siRNA reduced ischaemic neuronal cell death and improved neurobehavioural outcomes in vivo. Recently, we also demonstrated that inhibition of TRPM7 using pharmacological means promoted neuronal outgrowth in vitro and provided neuroprotection against brain injury to hypoxia in vivo. Thus, we have shown the contributions of TRPM7 in many physiological and pathophysiological processes, including hypoxia and ischaemia.

Abbreviations

AAV

adeno‐associated virus

AET

anti‐excitotoxic therapies

AMPA

α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid

ASIC

acid‐sensing ion channel

HIE

hypoxic–ischaemic encephalopathy

K_ATP

ATP‐sensitive potassium channel

MCAO

middle cerebral artery occlusion

NCX

sodium–calcium exchanger

NMDA

N‐methyl‐d‐aspartate

OGD

oxygen‐glucose deprivation

STAIR

Stroke Therapy Academic Industry Roundtable

tPA

tissue plasminogen activator

TRP

transient receptor potential

TRPM7

transient receptor potential melastatin 7

VRAC

volume‐regulated anion channel

Introduction

Cerebral ischaemia and stroke (Dirnagl et al. 1999; Lipton, 1999) is a leading cause of mortality and a major cause of long‐term immobility in the world based on statistics from the World Health Organization (WHO) (WHO, 2014; Mozaffarian et al. 2016). Stroke has a high mortality rate, and stroke prevalence is projected to increase. There is no effective treatment for stroke, except for the tissue plasminogen activator (tPA), which has a limited therapeutic window (Zivin, 2009). Stroke has already shown significant social and economic impacts worldwide (American Stroke Association, 2016). In addition, hypoxia could cause neonatal hypoxic–ischaemic brain injury and subsequent early‐onset brain and behavioural disorders in children, termed hypoxic–ischaemic encephalopathy (HIE) (Vannucci, 2000; Nelson & Lynch, 2004). HIE is characterized by neurodevelopmental delay, motor and cognitive impairments, and epilepsy. Neonatal hypoxic–ischaemic brain injury and its related HIE have also caused noticeable burdens worldwide.

Stroke triggers intracellular calcium overload and ionic imbalance, eventually leading to neuronal cell death (Dirnagl et al. 1999; Lipton, 1999). The time course and sequence of events occurring in cerebral ischaemia includes (1) anoxic depolarization in seconds to minutes, (2) peri‐infarct depolarization in minutes, (3) excitotoxicity in minutes, to later cause (4) apoptosis and (5) inflammation in days. During cerebral ischaemia, the excitatory neurotransmitter glutamate is released from the brain, acts on the glutamate receptor channels, and triggers a calcium overload and neuronal cell death (Dirnagl et al. 1999; Lipton, 1999). Excitotoxicity, mediated through NMDA and AMPA receptor channels (Besancon et al. 2008; Tymianski, 2011), has been the central focus of stroke research for decades. Preventing the calcium overload is theoretically considered to be neuroprotective. Blocking calcium‐mediated glutamate receptor channels in vitro and in vivo inhibits intracellular calcium overload and prevents ischaemic brain damage. Even experimental studies have shown hopeful data; however, the subsequent clinical trials of anti‐excitotoxic therapies (AET) could not support AET as a further therapeutic development (Davis et al. 2000). As a result, stroke researchers began searching for unconventional mechanism(s) outside the traditional glutamate mechanism. In addition to the traditional glutamate excitotoxicity mediated through NMDA and AMPA receptor channels (Besancon et al. 2008; Tymianski, 2011), new data indicates that a non‐glutamate mechanism in cerebral ischaemia also causes intracellular ionic imbalance and neuronal cell death (Besancon et al. 2008; Tymianski, 2011). Ischaemic neuronal death is now accepted as a result of both glutamate‐mediated excitotoxicity and the newly discovered non‐glutamate mechanisms (Besancon et al. 2008; Tymianski, 2011). The newly accepted non‐glutamate mechanism undeniably contributes to the disappointing results of the AET clinical trials. Thus, we may need to consider both glutamate and non‐glutamate mechanisms in new drug development for stroke, as well as using multiple in vivo animal models of human disease based on the Stroke Therapy Academic Industry Roundtable (STAIR) Protocol (Stroke Therapy Academic Industry Roundtable (STAIR), 1999), which emphasizes the necessity for testing potential stroke drugs using multiple animal stroke models in multiple species. The non‐glutamate mechanism includes transient receptor potential (TRP) channels (Sun et al. 2009; Alim et al. 2013; Chen et al. 2015a), ATP‐sensitive potassium (K_ATP) channels (Sun et al. 2006, 2007, 2015; Liu et al. 2016), acid‐sensing ion channels (ASICs) (Xiong et al. 2004), hemichannels (Thompson et al. 2006), volume‐regulated anion channels (VRACs) (Alibrahim et al. 2013), sodium–calcium exchangers (NCXs) (Pignataro et al. 2004), and other non‐selective cation channels (Simard et al. 2006). Activation of these ion channels could be at the initial stages of cerebral ischaemia and/or later in the ischaemic events. For example, K_ATP channels are activated at the initiation of the ischaemic event during the anoxic depolarization; TRPM7 may be activated at the same time or shortly after the excitotoxicity; and TRPM2 may be activated in the later stages as it is involved in inflammation. Ion channels are the third largest target in drug development (Dabrowski et al. 2008). Thus, the non‐glutamate mechanism is a target for neuroprotection.

Transient receptor potential channels

Ion channels play many fundamental roles in physiological and pathophysiological functions in the brain. We have been working on the non‐glutamate mechanism in cerebral ischaemia and hypoxia, including TRP channels, K_ATP channels and VRAC channels. Here, the focus is on the role of TRP channels in cerebral ischaemia and hypoxia.

Transient receptor potential channels, also termed TRP channels, are a group of non‐selective cation channels located on the cell membrane of various cell types (Clapham, 2003; Pedersen et al. 2005; Wu et al. 2010). TRP channels were originally discovered in fruit fly (Drosophila) (Minke et al. 1975) photoreceptors where they participated in phototransduction (Monteilh‐Zoller et al. 2003). The channel received its name, transient receptor potential, because the Drosophila photoreceptors in the fly carrying the mutant trp gene initiated a transient response to light (Minke et al. 1975). The TRP channel superfamily now has approximately 30 mammalian TRP channels (Wu et al. 2010). The TRP channels, classified based on their homologous sequences, are divided into six sub‐families: (1) TRPC (canonical), (2) TRPV (vanilloid), (3) TRPM (melastatin), (4) TRPA (ankyrin), (5) TRPML (mucolipin), and (6) TRPP (polycystin). TRP channels can be activated by different physical and chemical stimuli and play many physiological and pathological functions in various cells (Clapham, 2003; Pedersen et al. 2005; Wu et al. 2010).

The TRPM family has eight members, TRPM 1 to 8. TRPM7 (Clapham, 2003), the seventh member of the TRPM channel sub‐family, is widely expressed in many tissues and cells including the brain and neurons. TRPM7 is a calcium‐permeable non‐selective divalent cation channel and is also permeable to other trace metal ions, i.e. Zn²⁺ ≈ Ni²⁺ >> Ba²⁺ > Co²⁺ > Mg²⁺ ≥ Mn²⁺ ≥ Sr²⁺ ≥ Cd²⁺ ≥ Ca²⁺ ions (Monteilh‐Zoller et al. 2003).

TRPM7 plays an important role in a wide range of physiological and pathophysiological functions, and its channel activities can be modulated by diverse intracellular and extracellular factors (Penner & Fleig, 2007), such as Mg²⁺ and Mg²⁺‐complexed nucleotides (such as MgATP and MgGTP) (Takezawa et al. 2004; Demeuse et al. 2006), extracellular pH (Jiang et al. 2005; Li et al. 2007), shear stress (Oancea et al. 2006), etc. TRPM7 participates in a wide scope of cell biology processes ranging from cell proliferation, cell growth and cell adhesion (Nadler et al. 2001; Inoue & Xiong, 2009); TRPM7 overexpression reduces cell viability (Nadler et al. 2001; Su et al. 2006; Chen et al. 2010). TRPM7 also regulates embryonic development (Jin et al. 2008) and skeleton formation (Elizondo et al. 2005). Globally knocking out TRPM7 has been shown to be embryonically lethal in mice (Jin et al. 2008). Thus, TRPM7 is essential for development. TRPM7 channels are thought to be activated during ischaemia based on the metabolic state of the cell. Therefore, the favourable condition for TRPM7 activation during ischaemia would be low concentrations of Mg²⁺‐nucleotides (Demeuse et al. 2006) and acidic conditions (Rehncrona, 1985; Li et al. 2007).

In the brain, TRPM7 plays key roles both under physiological conditions, e.g. cell growth (Nadler et al. 2001; Inoue & Xiong, 2009; Turlova et al. 2016), and under pathophysiological conditions, e.g. hypoxia‐ and ischaemia‐induced neuronal cell death (Aarts et al. 2003; Sun et al. 2009; Chen et al. 2015a) and survival of brain tumour cells (Chen et al. 2015b,c). In addition to showing the role of TRPM7 in ischaemia (Sun et al. 2009), we have recently also demonstrated the following: (1) inhibition of TRPM7 in vitro enhances neurite outgrowth and maturation in mouse culture hippocampal cells (Turlova et al. 2016); (2) TRPM7 plays a role in neonatal hypoxic–ischaemic brain injury in mice in vivo (Chen et al. 2015a); and (3) TRPM7 also plays an important role in cell survival in glioma cell lines in vitro (Chen et al. 2015b,c). Here, the role of TRPM7 in neuronal cell death and brain damage during ischaemia and hypoxia in vivo (Sun et al. 2009; Chen et al. 2015a), its pharmacology and its potential in drug development for stroke will be further discussed.

Molecular and pharmacological reagents are available for TRPM7; these include channel activators and blockers (Zierler et al. 2011; Chubanov et al. 2014; Chen et al. 2015a; Turlova et al. 2016), antibodies (Sun et al. 2009; Chen et al. 2015a,; Turlova et al. 2016), and siRNA (Sun et al. 2009; Turlova et al. 2016). These will be beneficial for studying TRPM7, and many studies have used various TRPM7 reagents.

TRPM7 plays a key role in anoxic cell death in cultured neurons (Aarts et al. 2003). We have demonstrated that TRPM7 plays a significant role in ischaemic brain damage and neuronal cell death in vivo (Sun et al. 2009). The study showed that virally mediated gene silencing of TRPM7 in vivo with siRNA increased neuronal cell survival and improved neurobehavioural outcomes after cerebral ischaemia (Sun et al. 2009). Later, we also showed that TRPM7 plays many important roles both in vitro and in vivo: inhibiting TRPM7 in vitro enhances hippocampal neuronal cell outgrowth (Turlova et al. 2016); blocking TRPM7 in vivo reduces brain damage in hypoxia (Chen et al. 2015a); and suppressing TRPM7 in vitro decreases glioma cell survival in vitro (Chen et al. 2015b,c).

TRPM7 in cerebral ischaemia and hypoxia

In the event of cerebral ischaemia and/or hypoxia, calcium overload and ionic imbalance inside the neuronal cells have been the accepted cellular and molecular mechanisms for ischaemic and/or hypoxic neuronal cell death and brain damage (Besancon et al. 2008; Tymianski, 2011). In addition to the traditional glutamate mechanism, which is mainly focused on glutamate receptor channel‐mediated excitotoxicity, the non‐glutamate mechanism also triggers intracellular ionic imbalance and initiates ischaemic and/or hypoxic neuronal cell death and brain damage in stroke (Besancon et al. 2008; Tymianski, 2011). The non‐glutamate mechanism contains many other ion channels and newly described calcium‐mediated non‐selective cation channels, e.g. ATP‐sensitive potassium channels (K_ATP) (Sun et al. 2006, 2007, 2015; Liu et al. 2016), transient receptor potential (TRP) channels (Sun et al. 2009; Alim et al. 2013; Chen et al. 2015a), volume‐regulated anion channels (VRACs) (Alibrahim et al. 2013), hemichannels (Thompson et al. 2006), acid‐sensing ion channels (ASICs) (Xiong et al. 2004), ion exchangers (Pignataro et al. 2004) and other non‐selective cation channels (Simard et al. 2006). In this report, the focus is on the neuronal cell death and brain damage mediated by TRPM7 in stroke and hypoxia in vivo.

An early in vitro study reported that TRPM7 plays a key role in anoxic‐induced neuronal cell death in culture (Aarts et al. 2003). This study originally described the so‐called I _OGD currents in response to prolonged in vitro oxygen–glucose deprivation (OGD) challenge in primary culture cortical neurons, which eventually triggered a secondary neuronal cell death through a secondary increase in Ca²⁺ influx through the later identified TRPM7 (Aarts et al. 2003). The effects of the prolonged anoxic cell death were eventually unmasked with treatment of a cocktail of blockers aimed at blocking the glutamate NMDA and AMPA receptors and L‐type calcium channels using MK‐801, CNQX and nimodipine in the in vitro OGD experiment (Aarts et al. 2003). The study was facilitated by calcium imaging and electrophysiological approaches, as well as additional molecular biology verification. The in vitro study further used the TRPM7 siRNA directly against the TRPM7 in the mouse primary culture cortical neurons and showed that both the expression level of TRPM7 mRNA and the subsequent prolonged anoxia‐mediated neuronal cell death were reduced. Thus, this was the first study that revealed the key role of TRPM7 in mediating Ca²⁺ influx and subsequent anoxic neuronal cell death in vitro during prolonged OGD. As OGD is a simplified in vitro anoxia model for studying neuronal cell death, it is not a good representation of ‘ischaemia’ because it lacks other factors in vivo, such as blood, neuronal circuitry, network connectivity and the involvement of other brain cells. An unrelated in vivo study using the middle cerebral artery occlusion (MCAO) model reported that TRPM7 expression levels in mRNA and protein were increased after MCAO (Jiang et al. 2008), indicating that TRPM7 may be implicated in cerebral ischaemia. As a result, we still need to confirm the study in vivo and use animal models to further investigate the pathophysiological role of TRPM7 in ischaemia and/or hypoxia.

We have later confirmed that TRPM7 also plays an important role in cerebral ischaemia in vivo (Sun et al. 2009). In the report, we showed that virally mediated gene silencing of TRPM7 in hippocampal CA1 neurons in vivo suppressed mRNA and protein TRPM7 expression levels, reduced CA1 neuronal death and preserved behavioural outcomes after cerebral ischaemia. TRPM7 pharmacology was not clear, and selective TRPM7 blockers were not available at that time; the study utilized a virally mediated gene silencing shRNA approach to suppress TRPM7 in adult rat hippocampal CA1 neurons. The adeno‐associated viral vector (AAV) was used to package the TRPM7 shRNA, and a stereotaxic microinjection was used to deliver the AAV to the hippocampal CA1 area in vivo. We confirmed that the AAV infected the adult hippocampal CA1 neurons in vivo. We also showed suppression of TRPM7 in the infected hippocampal CA1 neurons at the mRNA level with RT‐PCR, at the protein level with Western Blot and immunohistochemistry, and at the functional level with electrophysiology. We verified that transient suppression of TRPM7 in the adult hippocampal CA1 neurons showed no noticeable effects on neuronal cell survival, fine structures and electrophysiological properties. We then revealed that TRPM7 suppression significantly reduced hippocampal CA1 cell death in vivo and preserved behavioural outcomes using a global ischaemia model. We demonstrated that the surviving hippocampal CA1 neuronal cells were also healthy with intact morphology and fine cell structures. We also proved that the surviving hippocampal CA1 neurons had well maintained electrophysiological properties. Lastly, we confirmed that suppressed TRPM7 in vivo not only reduced hippocampal cell death but also preserved the hippocampal‐associated behavioural tasks, e.g. fear‐associated and spatial‐navigation memory (fear conditioning (Cheng et al. 2006; Sun et al. 2009) and Morris water maze (Morris et al. 1982; Sun et al. 2009)). This study was the first detailed in vivo study showing the important role of TRPM7 in cerebral ischaemia.

We also recently showed that TRPM7 plays a significant role in brain damage in hypoxic–ischaemic brain injury in vivo (Chen et al. 2015a). We reported that the non‐selective TRPM7 inhibitor carvacrol significantly reduced the brain damage in a mouse hypoxic–ischaemic brain injury model (Chen et al. 2015a). The inhibition of TRPM7 also preserved behavioural outcomes after hypoxic–ischaemic brain injury (Chen et al. 2015a), and these included the geotaxic reflex (Ten et al. 2003; Sun et al. 2015; Chen et al. 2015a), which tests the vestibular and/or proprioceptive functions; the cliff avoidance reaction (Ten et al. 2003; Sun et al. 2015; Chen et al. 2015a), which assesses maladaptive impulsive behaviour; and the grip test (Liu et al. 2013; Sun et al. 2015; Chen et al. 2015a), which evaluates force and fatigability. Neuroprotective effects in reducing brain damage by TRPM7 inhibition in the hypoxic–ischaemic brain injury were arbitrated partly by promoting pro‐survival signalling (e.g. Akt signalling) and inhibiting pro‐apoptotic signalling (e.g. caspase‐3 and Bcl/Bax signalling) (Chen et al. 2015a).

Conclusions and future direction

TRPM7, a calcium‐mediated non‐selective divalent cation channel, is one of the newly described non‐glutamate mechanisms of neuronal cell death and brain damage in cerebral ischaemia and hypoxia. Both in vitro and in vivo studies have shown that TRPM7 plays a critical role in ischaemic and hypoxic neuronal cell death and brain damage. Thus, TRPM7 is a potential therapeutic target for drug development for stroke and hypoxic–ischaemic brain injury. With the new development of selective TRPM7 inhibitors (Zierler et al. 2011; Turlova et al. 2016), we could further study TRPM7 drug development in stroke and hypoxia following the Stroke STAIR protocol (Stroke Therapy Academic Industry Roundtable (STAIR), 1999).

Additional information

Competing interests

The author declares no conflicts of interest.

Funding

Supported by grants to H.‐S.S from the Heart and Stroke Foundation of Canada (G‐13‐0003069), the Canadian Institutes of Health Research (CIHR) China‐Canada Joint Health Research Initiative (CIHR, FRN no. 132571) and Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grants (RGPIN‐2016‐04574).

Biography

Hong‐Shuo Sun is a neuroscientist with expertise in ion channels, neuroprotection and drug development for stroke and other brain diseases. He first received his MD degree from Zhongshan Medical College, Sun Yat‐Sen University, Guangzhou, China. He then received his MSc degree in Pharmaceutical Sciences (Pharmacology) from the University of Alberta, Canada, and his PhD degree in Neuroscience from the University of Calgary, Canada. He later did his postdoctoral training at the University Health Network, Toronto, Canada. Currently, he is a tenured Associate Professor and PhD Supervisor in the Departments of Surgery, Physiology, and Pharmacology and the Institute of Medical Science, Faculty of Medicine at the University of Toronto, Canada.