In vivo evaluation of monoclonal antibody M4M using a humanised rat model of stroke demonstrates attenuation of reperfusion injury via blocking human TRPM4 channel.

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.

SLC26A11 Inhibition Reduces Oncotic Neuronal Death and Attenuates Stroke Reperfusion Injury.

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.

TRPM4 Blocking Antibody Protects Cerebral Vasculature in Delayed Stroke Reperfusion.

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.

Binding epitope for recognition of human TRPM4 channel by monoclonal antibody M4M.

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.

Comparison of Anti-oncotic Effect of TRPM4 Blocking Antibody in Neuron, Astrocyte and Vascular Endothelial Cell Under Hypoxia.

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.

Curcumin Ameliorates Neuroinflammation, Neurodegeneration, and Memory Deficits in p25 Transgenic Mouse Model that Bears Hallmarks of Alzheimer's Disease.

Several studies have indicated that neuroinflammation is indeed associated with neurodegenerative disease pathology. However, failures of recent clinical trials of anti-inflammatory agents in neurodegenerative disorders have emphasized the need to better understand the complexity of the neuroinflammatory process in order to unravel its link with neurodegeneration. Deregulation of Cyclin-dependent kinase 5 (Cdk5) activity by production of its hyperactivator p25 is involved in the formation of tau and amyloid pathology reminiscent of Alzheimer's disease (AD). Recent studies show an association between p25/Cdk5 hyperactivation and robust neuroinflammation. In addition, we recently reported the novel link between the p25/Cdk5 hyperactivation-induced inflammatory responses and neurodegenerative changes using a transgenic mouse that overexpresses p25 (p25Tg). In this study, we aimed to understand the effects of early intervention with a potent natural anti-inflammatory agent, curcumin, on p25-mediated neuroinflammation and the progression of neurodegeneration in p25Tg mice. The results from this study showed that curcumin effectively counteracted the p25-mediated glial activation and pro-inflammatory chemokines/cytokines production in p25Tg mice. Moreover, this curcumin-mediated suppression of neuroinflammation reduced the progression of p25-induced tau/amyloid pathology and in turn ameliorated the p25-induced cognitive impairments. It is widely acknowledged that to treat AD, one must target the early-stage of pathological changes to protect neurons from irreversible damage. In line with this, our results demonstrated that early intervention of inflammation could reduce the progression of AD-like pathological outcomes. Moreover, our data provide a rationale for the potential use of curcuminoids in the treatment of inflammation associated neurodegenerative diseases.

Specific inhibition of p25/Cdk5 activity by the Cdk5 inhibitory peptide reduces neurodegeneration in vivo.

The aberrant hyperactivation of Cyclin-dependent kinase 5 (Cdk5), by the production of its truncated activator p25, results in the formation of hyperphosphorylated tau, neuroinflammation, amyloid deposition, and neuronal death in vitro and in vivo. Mechanistically, this occurs as a result of a neurotoxic insult that invokes the intracellular elevation of calcium to activate calpain, which cleaves the Cdk5 activator p35 into p25. It has been shown previously that the p25 transgenic mouse as a model to investigate the mechanistic implications of p25 production in the brain, which recapitulates deregulated Cdk5-mediated neuropathological changes, such as hyperphosphorylated tau and neuronal death. To date, strategies to inhibit Cdk5 activity have not been successful in targeting selectively aberrant activity without affecting normal Cdk5 activity. Here we show that the selective inhibition of p25/Cdk5 hyperactivation in vivo, through overexpression of the Cdk5 inhibitory peptide (CIP), rescues against the neurodegenerative pathologies caused by p25/Cdk5 hyperactivation without affecting normal neurodevelopment afforded by normal p35/Cdk5 activity. Tau and amyloid pathologies as well as neuroinflammation are significantly reduced in the CIP-p25 tetra transgenic mice, whereas brain atrophy and subsequent cognitive decline are reversed in these mice. The findings reported here represent an important breakthrough in elucidating approaches to selectively inhibit the p25/Cdk5 hyperactivation as a potential therapeutic target to reduce neurodegeneration.