RESUMEN
Excitotoxicity arises from unusually excessive activation of excitatory amino acid receptors such as glutamate receptors. Following an energy crisis, excitotoxicity is a major cause for neuronal death in neurological disorders. Many glutamate antagonists have been examined for their efficacy in mitigating excitotoxicity, but failed to generate beneficial outcome due to their side effects on healthy neurons where glutamate receptors are also blocked. In this study, we found that during chronic hypoxia there is upregulation and activation of a nonselective cation channel TRPM4 that contributes to the depolarized neuronal membrane potential and enhanced glutamate-induced calcium entry. TRPM4 is involved in modulating neuronal membrane excitability and calcium signaling, with a complex and multifaceted role in the brain. Here, we inhibited TRPM4 using a newly developed blocking antibody M4P, which could repolarize the resting membrane potential and ameliorate calcium influx upon glutamate stimulation. Importantly, M4P did not affect the functions of healthy neurons as the activity of TRPM4 channel is not upregulated under normoxia. Using a rat model of chronic hypoxia with both common carotid arteries occluded, we found that M4P treatment could reduce apoptosis in the neurons within the hippocampus, attenuate long-term potentiation impairment and improve the functions of learning and memory in this rat model. With specificity to hypoxic neurons, TRPM4 blocking antibody can be a novel way of controlling excitotoxicity with minimal side effects that are common among direct blockers of glutamate receptors.
Asunto(s)
Ácido Glutámico , Canales Catiónicos TRPM , Ratas , Animales , Ácido Glutámico/metabolismo , Calcio/metabolismo , Receptores de Glutamato/metabolismo , Neuronas/metabolismo , Hipoxia/metabolismo , Canales Catiónicos TRPM/metabolismoRESUMEN
Reperfusion therapy is currently the gold standard treatment for acute ischemic stroke. However, reperfusion injuries such as oedema and haemorrhagic transformation largely limit the use of this potent treatment to a narrow time window. Recently, transient receptor potential melastatin 4 (TRPM4) channel has emerged as a potential target for vascular protection in stroke management. Non-specificity and side effects are major concerns for current TRPM4 blockers. The present study was undertaken to develop a novel TRPM4 blocker for stroke management. We report the generation of a TRPM4-specific antibody M4P which binds to a region close to the channel pore. M4P could inhibit TRPM4 current and downregulate TRPM4 surface expression, therefore prevent hypoxia-induced cell swelling. In the rat model of 3-h stroke reperfusion, application of M4P at 2 h after occlusion ameliorated reperfusion injury by improving blood-brain barrier integrity, and enhanced functional recovery. Our results demonstrate that TRPM4 blockade could attenuate reperfusion injury in stroke recanalization. When applied together with reperfusion treatments, TRPM4 blocking antibody has the potential to extend the therapeutic time window for acute ischemic stroke.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Daño por Reperfusión/tratamiento farmacológico , Accidente Cerebrovascular/tratamiento farmacológico , Canales Catiónicos TRPM/antagonistas & inhibidores , Animales , Barrera Hematoencefálica/metabolismo , Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/metabolismo , Modelos Animales de Enfermedad , Femenino , Masculino , Ratas , Ratas Wistar , Daño por Reperfusión/metabolismo , Accidente Cerebrovascular/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
BACKGROUND: Blocking Transient Receptor Potential Melastatin 4 (TRPM4) in rodents by our antibody M4P has shown to attenuate cerebral ischaemia-reperfusion injury. Since M4P does not interact with human TRPM4, the therapeutic potential of blocking human TRPM4 remains unclear. We developed a monoclonal antibody M4M that inhibited human TRPM4 in cultured cells. However, M4M has no effect on stroke outcome in wild-type rats. Therefore, M4M needs to be evaluated on animal models expressing human TRPM4. METHODS: We generated a humanised rat model using the CRISPR/Cas technique to knock-in (KI) the human TRPM4 antigen sequence. RESULTS: In primary neurons from human TRPM4 KI rats, M4M binds to hypoxic neurons, but not normoxic nor wild-type neurons. Electrophysiological studies showed that M4M blocked ATP depletion-induced activation of TRPM4 and inhibited hypoxia-associated cell volume increase. In a stroke model, administration of M4M reduced infarct volume in KI rats. Rotarod test and Neurological deficit score revealed improvement following M4M treatment. CONCLUSION: M4M selectively binds and inhibits hypoxia-induced human TRPM4 channel activation in neurons from the humanised rat model, with no effect on healthy neurons. Use of M4M in stroke rats showed functional improvements, suggesting the potential for anti-human TRPM4 antibodies in treating acute ischaemic stroke patients.
Asunto(s)
Isquemia Encefálica , Daño por Reperfusión , Accidente Cerebrovascular , Canales Catiónicos TRPM , Canales de Potencial de Receptor Transitorio , Ratas , Humanos , Animales , Accidente Cerebrovascular/tratamiento farmacológico , Canales de Potencial de Receptor Transitorio/uso terapéutico , Anticuerpos Monoclonales/farmacología , Isquemia Encefálica/tratamiento farmacológico , Canales Catiónicos TRPM/metabolismo , Daño por Reperfusión/tratamiento farmacológico , HipoxiaRESUMEN
Neuronal swelling is a pathological feature of stroke which contributes to the formation of cytotoxic edema. Under hypoxic condition, aberrant accumulation of sodium and chloride ions inside neurons increases osmotic pressure, leading to cell volume increase. Sodium entry pathway in neurons has been studied extensively. Here, we determine whether SLC26A11 is the major chloride entry pathway under hypoxia and could be the target for protection against ischemic stroke. In this study, electrophysiological properties of chloride current in primary cultured neurons were characterized using low chloride solution, 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid, and SLC26A11-specific siRNA under physiological conditions or ATP-depleted conditions. In vivo effect of SLC26A11 was evaluated on a rat stroke reperfusion model. We found that SLC26A11 mRNA in primary cultured neurons was upregulated as early as 6 h after oxygen glucose deprivation, and later, the protein level was elevated accordingly. Blockade of SLC26A11 activity could reduce chloride entry and attenuate hypoxia-induced neuronal swelling. In the animal stroke model, SLC26A11 upregulation was mainly located in surviving neurons close to the infarct core. SLC26A11 inhibition ameliorates infarct formation and improves functional recovery. These findings demonstrate that SLC26A11 is a major pathway for chloride entry in stroke, contributing to neuronal swelling. Inhibition of SLC26A11 could be a novel therapeutic strategy for stroke.
Asunto(s)
Isquemia Encefálica , Daño por Reperfusión , Accidente Cerebrovascular , Ratas , Animales , Cloruros , Accidente Cerebrovascular/complicaciones , Accidente Cerebrovascular/tratamiento farmacológico , Accidente Cerebrovascular/metabolismo , Hipoxia/patología , Edema , Daño por Reperfusión/complicaciones , Daño por Reperfusión/tratamiento farmacológico , Infarto , Sodio/metabolismo , Glucosa , Isquemia Encefálica/patologíaRESUMEN
Reperfusion therapy for acute ischemic stroke aims to restore the blood flow of occluded blood vessels. However, successful recanalization is often associated with disruption of the blood-brain barrier, leading to reperfusion injury. Delayed recanalization increases the risk of severe reperfusion injury, including severe cerebral edema and hemorrhagic transformation. The TRPM4-blocking antibody M4P has been shown to alleviate reperfusion injury and improve functional outcomes in animal models of early stroke reperfusion. In this study, we examined the role of M4P in a clinically relevant rat model of delayed stroke reperfusion in which the left middle cerebral artery was occluded for 7 h. To mimic the clinical scenario, M4P or control IgG was administered 1 h before recanalization. Immunostaining showed that M4P treatment improved vascular morphology after stroke. Evans blue extravasation demonstrated attenuated vascular leakage following M4P treatment. With better vascular integrity, cerebral perfusion was improved, leading to a reduction of infarct volume and animal mortality rate. Functional outcome was evaluated by the Rotarod test. As more animals with severe injuries died during the test in the control IgG group, we observed no difference in functional outcomes in the surviving animals. In conclusion, we identified the potential of TRPM4 blocking antibody M4P to ameliorate vascular injury during delayed stroke reperfusion. If combined with reperfusion therapy, M4P has the potential to improve current stroke management.
RESUMEN
Mouse monoclonal antibody M4M was recently designed to block human TRPM4 channel. The polypeptide for generating M4M is composed of peptide A1 between the transmembrane segment 5 (S5) and the pore, and a second peptide A2 between the pore and the transmembrane segment 6 (S6). Using peptide microarray, a 4-amino acid sequence EPGF within the A2 was identified to be the binding epitope for M4M. Substitution of EPGF with other amino acids greatly reduced binding affinity. Structural analysis of human TRPM4 structure indicates that EPGF is located externally to the channel pore. A1 is close to the EPGF binding epitope in space, albeit separated by a 37-amino acid peptide. Electrophysiological study reveals that M4M could block human TRPM4, but with no effect on rodent TRPM4 which shares a different amino acid sequence ERGS for the binding motif. Our results demonstrate that M4M is a specific inhibitor for human TRPM4.
Asunto(s)
Anticuerpos Monoclonales , Canales Catiónicos TRPM , Ratones , Animales , Humanos , Epítopos , Anticuerpos Monoclonales/metabolismo , Secuencia de Aminoácidos , Péptidos/metabolismo , Mutación Missense , Canales Catiónicos TRPM/genética , Canales Catiónicos TRPM/metabolismoRESUMEN
TRPM4 is a calcium-activated non-selective monovalent cation channel implicated in diseases such as stroke. Lack of potent and selective inhibitors remains a major challenge for studying TRPM4. Using a polypeptide from rat TRPM4, we have generated a polyclonal antibody M4P which could alleviate reperfusion injury in a rat model of stroke. Here, we aim to develop a monoclonal antibody that could block human TRPM4 channel. Two mouse monoclonal antibodies M4M and M4M1 were developed to target an extracellular epitope of human TRPM4. Immunohistochemistry and western blot were used to characterize the binding of these antibodies to human TRPM4. Potency of inhibition was compared using electrophysiological methods. We further evaluated the therapeutic potential on a rat model of middle cerebral artery occlusion. Both M4M and M4M1 could bind to human TRPM4 channel on the surface of live cells. Prolonged incubation with TRPM4 blocking antibody internalized surface TRPM4. Comparing to M4M1, M4M is more effective in blocking human TRPM4 channel. In human brain microvascular endothelial cells, M4M successfully inhibited TRPM4 current and ameliorated hypoxia-induced cell swelling. Using wild type rats, neither antibody demonstrated therapeutic potential on stroke. Human TRPM4 channel can be blocked by a monoclonal antibody M4M targeting a key antigenic sequence. For future clinical translation, the antibody needs to be humanized and a transgenic animal carrying human TRPM4 sequence is required for in vivo characterizing its therapeutic potential.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Daño por Reperfusión/prevención & control , Canales Catiónicos TRPM/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales/uso terapéutico , Encéfalo/citología , Encéfalo/efectos de los fármacos , Encéfalo/patología , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/patología , Células HEK293 , Humanos , Infarto de la Arteria Cerebral Media/complicaciones , Masculino , Técnicas de Placa-Clamp , Ratas , Daño por Reperfusión/etiología , Daño por Reperfusión/patología , Canales Catiónicos TRPM/metabolismoRESUMEN
In stroke and other neurological diseases, Transient Receptor Potential Melastatin 4 (TRPM4) has been reported to cause oncotic cell death which is due to an excessive influx of sodium ions. Following stroke, hypoxia condition activates TRPM4 channel, and the sodium influx via TRPM4 is further enhanced by an increased TRPM4 expression. However, the effect of TRPM4 inhibition on oncotic cell death, particularly during the acute stage, remains largely unknown. Recently, we have developed a polyclonal antibody M4P that specifically inhibits TRPM4 channel. M4P blocks the channel via binding to a region close to the channel pore from extracellular space. Using M4P, we evaluated the acute effect of blocking TRPM4 in neurons, astrocytes, and vascular endothelial cells. In a rat stroke model, M4P co-localized with neuronal marker NeuN and endothelial marker vWF, whereas few GFAP positive astrocytes were stained by M4P in the ipsilateral hemisphere. When ATP was acutely depleted in cultured cortical neurons and microvascular endothelial cells, cell swelling was induced. Application of M4P significantly blocked TRPM4 current and attenuated oncosis. TUNEL assay, PI staining and western blot on cleaved Caspase-3 revealed that M4P could ameliorate apoptosis after 24 h hypoxia exposure. In contrast, acute ATP depletion in cultured astrocytes failed to demonstrate an increase of cell volume, and application of M4P or control IgG had no effect on cell volume change. When TRPM4 was overexpressed in astrocytes, acute ATP depletion successfully induced oncosis which could be suppressed by M4P treatment. Our results demonstrate that comparing to astrocytes, neurons, and vascular endothelial cells are more vulnerable to hypoxic injury. During the acute stage of stroke, blocking TRPM4 channel could protect neurons and vascular endothelial cells from oncotic cell death.
RESUMEN
The original version of the article unfortunately contained an error.
RESUMEN
The transient receptor potential melastatin 4 (TRPM4) channel has been suggested to play a key role in the treatment of ischemic stroke. However, in vivo evaluation of TRPM4 channel, in particular by direct channel suppression, is lacking. In this study, we used multimodal imaging to assess edema formation and quantify the amount of metabolically functional brain salvaged after a rat model of stroke reperfusion. TRPM4 upregulation in endothelium emerges as early as 2 h post-stroke induction. Expression of TRPM4 channel was suppressed directly in vivo by treatment with siRNA; scrambled siRNA was used as a control. T2-weighted MRI suggests that TRPM4 inhibition successfully reduces edema by 30% and concomitantly salvages functionally active brain, measured by 18F-FDG-PET. These in vivo imaging results correlate well with post-mortem 2,3,5-triphenyltetrazolium chloride (TTC) staining which exhibits a 34.9% reduction in infarct volume after siRNA treatment. Furthermore, in a permanent stroke model, large areas of brain tissue displayed both edema and significant reductions in metabolic activity which was not shown in transient models with or without TRPM4 inhibition, indicating that tissue salvaged by TRPM4 inhibition during stroke reperfusion may survive. Evans Blue extravasation and hemoglobin quantification in the ipsilateral hemisphere were greatly reduced, suggesting that TRPM4 inhibition can improve BBB integrity after ischemic stroke reperfusion. Our results support the use of TRPM4 blocker for early stroke reperfusion.