mRNA expression of transient receptor potential melastatin (TRPM) channels 2 and 7 in perinatal brain development.

TRPM7 and TRPM2 are non-specific cation channels of the Transient Receptor Potential channel superfamily. Each channel has gained attention for their potential to mediate oxidative and anoxic cell death (Rama and García, 2016; Naziroglu, 2011a; Abiria et al., 2017; Sun, 2017), however their physiological expression and roles in the developing brain remain poorly defined. We employed real-time reverse transcription PCR to examine mRNA expression of TRPM7 and TRPM2 in the developing rat brain and brain-specific cell types. We determined the temporal and spatial expression patterns at four developmental time points (postnatal day 7, 14, 21, and 90) in four critical regions of the brain (cortex, hippocampus, striatum, and cerebellum) and examined gene expression in neuronal, astrocytic, and microglial primary cell cultures. Our results revealed that TRPM7 mRNA expression peaks in the cortex at 2-weeks after birth, and thus correlates most closely with a period of rat brain development associated with neurite outgrowth, which is heightened at 2-weeks after birth. Our cell-specific gene expression assays revealed that TRPM7 was expressed at equivalent levels in neurons, astrocytes, and microglia. Conversely, TRPM2 was most highly expressed in microglia with little expression in neurons and astrocytes. In the hippocampus and striatum, the expression profile of TRPM2 parallels the perinatal expression timeline for microglial infiltration and maturation in the rat brain. Microglial maturation is highest from the time of birth, up to 7-days, but subsequently declines. The latter developmental expression profiles indicate a role for TRPM2 in microglial activation.

Uncoupling PSD-95 interactions leads to rapid recovery of cortical function after focal stroke.

Since the most significant ischemic sequelae occur within hours of stroke, it is necessary to understand how neuronal function changes during this time. While histologic and behavioral models show the extent of stroke-related damage, only in vivo recordings can illustrate changes in brain activity during stroke and validate effectiveness of neuroprotective compounds. Spontaneous and evoked field potentials (fEPs) were recorded in the deep layers of the cortex with a linear microelectrode array for 3 hours after focal stroke in anesthetized rats. Tat-NR2B9c peptide, which confers neuroprotection by uncoupling the PSD-95 protein from N-methyl-D-aspartate receptor (NMDAR), was administered 5 minutes before ischemia. Evoked field potentials were completely suppressed within 3 minutes of infarct in all ischemic groups. Evoked field potential recovery after stroke in rats treated with Tat-NR2B9c (83% of baseline) was greater compared with stroke-only (61% of baseline) or control peptide (Tat-NR2B-AA; 67% of baseline) groups (P<0.001). Electroencephalography (EEG) power was higher in Tat-NR2B9c-treated animals at both 20 minutes and 1 hour (50% and 73% of baseline, respectively) compared with stroke-only and Tat-NR2B-AA-treated rats (P<0.05). Tat-NR2B9c significantly reduces stroke-related cortical dysfunction as evidenced by greater recovery of fEPs and EEG power; illustrating the immediate effects of the compound on poststroke brain function.

Calmodulin kinase IV-dependent CREB activation is required for neuroprotection via NMDA receptor-PSD95 disruption.

NMDA-type glutamate receptors mediate both trophic and excitotoxic signalling in CNS neurons. We have previously shown that blocking NMDAR- post-synaptic density-95 (PSD95) interactions provides significant protection from excitotoxicity and in vivo ischaemia; however, the mechanism of neuroprotection is unclear. Here, we report that blocking PSD-95 interactions with the Tat-NR2B9c peptide enhances a Ca²âº-dependent protective pathway converging on cAMP Response Element binding protein (CREB) activation. We provide evidence that Tat-NR2B9c neuroprotection from oxygen glucose deprivation and NMDA toxicity occurs in parallel with the activation of calmodulin kinase signalling and is dependent on a sustained phosphorylation of the CREB transcription factor and its activator CaMKIV. Tat-NR2B9c-dependent neuroprotection and CREB phosphorylation are blocked by coapplication of CaM kinase (KN93 and STO-609) or CREB (KG-501) inhibitors, and by siRNA knockdown of CaMKIV. These results are mirrored in vivo in a rat model of permanent focal ischaemia. Tat-NR2B9c application significantly reduces infarct size and causes a selective and sustained elevation in CaMKIV phosphorylation; effects which are blocked by coadministration of KN93. Thus, calcium-dependent nuclear signalling via CaMKIV and CREB is critical for neuroprotection via NMDAR-PSD95 blockade, both in vitro and in vivo. This study highlights the importance of maintaining neuronal function following ischaemic injury. Future stroke research should target neurotrophic and pro-survival signal pathways in the development of novel neuroprotective strategies.

High-frequency cortical activity associated with postischemic epileptiform discharges in an in vivo rat focal stroke model.

OBJECT: The postischemic brain has greater susceptibility to epileptogenic activity than physiologically healthy tissue. Epileptiform discharges are thought to exacerbate postischemic brain function. The aim of this study was to develop an in vivo focal stroke model in rats to characterize epileptiform activity. METHODS: The authors developed a parasagittal 8-channel intracortical microelectrode array to obtain recordings of cortical oscillations of local field potentials following partial middle and anterior cerebral artery occlusion. All experiments were done in urethane-anesthetized Sprague-Dawley rats. RESULTS: Theta runs (TRs), ranging in duration from 5 seconds to 5 minutes, were observed in 62% of animals within 1 hour of occlusion. High-frequency oscillations (HFOs) in the high gamma range (80-120 Hz) were observed 5-15 seconds before each TR and terminated at the onset of the discharge. Periodic epileptiform discharges (PEDs) were detected in 54% of rats following ischemia. The PEDs consisted of an early negative slow wave, a high-amplitude positive spike, and a short negative slow wave. Transient HFOs in the low gamma range (30-70 Hz) occurred during the first negative wave and the rising phase of the positive spike of the PED. CONCLUSIONS: These recordings provide the first intracortical evidence of a high-frequency component that could be an important element for diagnosis and intervention in postischemic epileptogenic activity. The early onset also suggests that HFOs could serve as a reliable method of detecting small epileptiform events and could be used as a consideration in deciding whether antiepileptic medications are appropriate as part of a patient's poststroke care.

TRPMs and neuronal cell death.

Death of CNS neurons during acute injury occurs as a result of a complex combination of excitotoxicity, necrosis, apoptosis, oedema and inflammatory reactions. Neuroprotection via glutamate receptor blockade or antioxidant or anti-inflammatory therapy have not proven effective in the clinical treatment of brain damage due to narrow therapeutic windows, poor pharmacokinetics or blockade of the signalling essential for normal excitatory neurotransmission and neuronal survival. Recent work in neuronal biochemistry, genomics and proteomics has increased understanding of the molecular organization of the excitatory synapse and the neuronal postsynaptic density. Transient receptor potential (TRP) channels are an exciting new family of cation channels that are highly expressed in the brain. Several members can be induced by oxidative stress and oxygen free radicals, both of which play important roles in neurodegeneration. Recent work has indicated that members of the melastatin subfamily (TRPM) of TRP proteins, particularly TRPM7 and TRPM2, may play key roles in neuronal death that is activated by oxidative stress and downstream from excitotoxic signal pathways. This discovery provides an exiting new avenue for research into the pathophysiology and treatment of acute neurodegeneration.

TRPM7 and ischemic CNS injury.

Ischemic brain damage represents a major source of morbidity and mortality in westernized society and poses a significant financial burden on the health care system. To date, few effective therapies have been realized to treat stroke and once promising avenues such as antiexcitotoxic therapy with NMDA receptor antagonists have not proven clinically useful. Thus, we need to identify new targets for research and therapeutic intervention of the neurodegeneration caused by stroke. Transient receptor potential (TRP) channels are an exciting new family of cation channels that respond to intracellular and extracellular stimuli. Indeed, several members can be induced by oxidative stress and oxygen free radicals. We have recently demonstrated that one member, TRPM7, is an essential mediator of anoxic neuronal death that is activated by oxidative stress, in parallel to excitotoxic signal pathways. Thus, future treatment of ischemic brain injury may need to include strategies that inhibit or modulate TRPM7 activity. Further investigation of the physiology and pathophysiology of TRPM7 and other TRP family members is needed to provide both pharmacological targets and a better understanding of ischemic brain disorders.

Molecular mechanisms underlying specificity of excitotoxic signaling in neurons.

The central role of glutamate receptors in mediating excitotoxic neuronal death in stroke, epilepsy and trauma has been well established. Glutamate is the major excitatory amino acid transmitter within the CNS and it's signaling is mediated by a number of postsynaptic ionotropic and metabotropic receptors. Although calcium ions are considered key regulators of excitotoxicity, new evidence suggests that specific second messenger pathways rather than total Ca(2+) load, are responsible for mediating neuronal degeneration. Glutamate receptors are found localized at the synapse within electron dense structures known as the postsynaptic density (PSD). Localization at the PSD is mediated by binding of glutamate receptors to submembrane proteins such as actin and PDZ containing proteins. PDZ domains are conserved motifs that mediate protein-protein interactions and self-association. In addition to glutamate receptors PDZ-containing proteins bind a multitude of intracellular signal molecules including nitric oxide synthase. In this way PDZ proteins provide a mechanism for clustering glutamate receptors at the synapse together with their corresponding signal transduction proteins. PSD organization may thus facilitate the individual neurotoxic signal mechanisms downstream of receptors during glutamate overactivity. Evidence exists showing that inhibiting signals downstream of glutamate receptors, such as nitric oxide and PARP-1 can reduce excitotoxic insult. Furthermore we have shown that uncoupling the interaction between specific glutamate receptors from their PDZ proteins protects neurons against glutamate-mediated excitotoxicity. These findings have significant implications for the treatment of neurodegenerative diseases using therapeutics that specifically target intracellular protein-protein interactions.

Peptide action in stroke therapy.

Glutamate receptor antagonists, although effective in preventing in vitro excitotoxic death, also block the glutamatergic signalling that is essential for normal excitatory neurotransmission and neuronal survival. This has contributed to the failure of clinical trials employing glutamate receptor antagonists as stroke therapeutics. However, recent years have seen an increased understanding of the molecular organisation of glutamate receptors in the neuronal postsynaptic density. This and a dissection of their associated intracellular signalling cascades has allowed the identification of distinct pathways responsible for excitotoxicity. It has become possible to uncouple toxic signalling cascades from glutamate receptors by targeting the interactions of membrane receptors with downstream proteins. Toxic signalling can be effectively uncoupled from glutamate receptors using targeted, cell-permeable peptides to disrupt specific protein-protein interactions. This approach does not block essential excitatory neurotransmission, but attenuates neurotoxic signals specifically and reduces stroke damage. This novel approach to blocking excitotoxic signalling in cerebral ischaemia may constitute a practical approach to stroke therapy.

Novel treatment of excitotoxicity: targeted disruption of intracellular signalling from glutamate receptors.

Glutamate signalling plays key physiological roles in excitatory neurotransmission and CNS plasticity, but also mediates excitotoxicity, the process responsible for triggering neurodegeneration through glutamate receptor overactivation. Excitotoxicity is thought to be a key neurotoxic mechanism in neurological disorders, including brain ischemia, CNS trauma and epilepsy. However, treating excitotoxicity using glutamate receptor antagonists has not proven clinically viable, necessitating more sophisticated approaches. Increasing knowledge of the composition of the postsynaptic density at glutamatergic synapses has allowed us to extend our understanding of the molecular mechanisms of excitotoxicity and to dissect out the distinct signalling pathways responsible for excitotoxic damage. Key molecules in these pathways are physically linked to the cytoplasmic face of glutamate receptors by scaffolding proteins that exhibit binding specificity for some receptors over others. This imparts specificity to physiological and pathological glutamatergic signalling. Recently, we have capitalized on this knowledge and, using targeted peptides to selectively disrupt intracellular interactions linked to glutamate receptors, have blocked excitotoxic signalling in neurones. This therapeutic approach circumvents the negative consequences of blocking glutamate receptors, and may be a practical strategy for treating neurological disorders that involve excitotoxicity.

Novel concepts in excitotoxic neurodegeneration after stroke.

Brain injury following cerebral ischaemia (stroke) involves a complex combination of pathological processes, including excitotoxicity and inflammation leading to necrotic and apoptotic forms of cell death. At the cellular level, excitotoxicity is mediated by glutamate and its cognate receptors, resulting in increased intracellular calcium and free radical production, and eventual cell death. Recent evidence suggests that scaffolding molecules that associate with glutamate receptors at the postsynaptic density allow coupling of receptor activity to specific second messengers capable of mediating excitotoxicity. These findings have important implications in the search for effective neuroprotective therapies in treating stroke.

Enhanced vulnerability to NMDA toxicity in sublethal traumatic neuronal injury in vitro.

Traumatic brain injury causes neuronal disruption and triggers secondary events leading to additional neuronal death. To study injuries triggered by secondary events, we exposed cultured cortical neurons to sublethal mechanical stretch, thus eliminating confounding death from primary trauma. Sublethally stretched neurons maintained cell membrane integrity, viability, and electrophysiological function. However, stretching induced in the cells a heightened vulnerability to subsequent challenges with L-glutamate or NMDA. This heightened vulnerability was specifically mediated by NMDA receptors (NMDARs), as stretched neurons did not become more vulnerable to either kainate toxicity or to that induced by the Ca(2+) ionophore A23187. Stretch-enhanced vulnerability to NMDA occurred independently of endogenous glutamate release, but required Ca(2+) and Na(+) influx through NMDARs. Stretch did not affect the electrophysiological properties of NMDARs nor excitatory synaptic activity, indicating that specificity of enhanced vulnerability to NMDA involves postsynaptic mechanisms downstream from NMDARs. To test whether this specificity requires physical interactions between NMDARs and cytoskeletal elements, we perturbed actin filaments and microtubules, both of which are linked to NMDARs. This had no effect on the stretch-induced vulnerability to NMDA, suggesting that sublethal stretch does not affect cell survival through the cytoskeleton. Our data illustrate that sublethal in vitro stretch injury triggers distinct signaling pathways that lead to secondary injury, rather than causing a generalized increase in vulnerability to secondary insults.

1Alpha,25-dihydroxyvitamin D3 promotes vascularization of the chondro-osseous junction by stimulating expression of vascular endothelial growth factor and matrix metalloproteinase 9.

Vitamin D deficiency results in defects in endochondral bone development characteristic of rickets, which include elongation of the cartilaginous growth plates and disorganization of the primary spongiosa. These defects are caused in part by impaired cartilage mineralization and vascularization of the chondro-osseous junction. Blood vessel invasion of mineralized cartilage is an essential step in endochondral ossification, providing access for cells that degrade cartilage as well as those that form bone. Vascular endothelial growth factor (VEGF) was shown to be a key regulator of this process when infusion of a dominant negative VEGF receptor effectively blocked vascular invasion and endochondral ossification in the growth plates of juvenile mice. Here, we show that the active metabolite of vitamin D 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] directly stimulates transcription of mRNAs encoding VEGF121 and -165 isoforms in the CFK2 chondrogenic cell line. Enhanced VEGF expression also was evident in growth plate chondrocytes and osteoblasts in the tibia of juvenile mice treated systemically with 1alpha,25(OH)2D3. This was seen in conjunction with enhanced expression of matrix metalloproteinase (MMP) 9, which activates VEGF stored in the cartilage matrix, in osteoclastic cells adjacent to the chondro-osseous junction. The alterations in VEGF and MMP-9 expression were accompanied by enhanced vascular invasion of mineralized cartilage, as assessed by CD31 immunoreactivity. These results provide evidence that 1alpha,25(OH)2D3 signaling stimulates VEGF and MMP-9 gene expression and promotes neovascularization of the epiphyseal growth plate in vivo through increased availability of active growth factor.