Acute Treatment with the M-Channel (Kv7, KCNQ) Opener Retigabine Reduces the Long-Term Effects of Repetitive Blast Traumatic Brain Injuries.

We investigated whether pharmacological increase of "M-type" (KCNQ, Kv7) K + channel currents by the M-channel opener, retigabine (RTG), acutely after repetitive traumatic brain injuries (rTBIs), prevents or reduces their long-term detrimental effects. rTBIs were studied using a blast shock air wave mouse model. Animals were monitored by video and electroencephalogram (EEG) records for nine months after the last injury to assess the occurrence of post-traumatic seizures (PTS), post-traumatic epilepsy (PTE), sleep-wake cycle architecture alterations, and the power of the EEG signals. We evaluated the development of long-term changes in the brain associated with various neurodegenerative diseases in mice by examining transactive response DNA-binding protein 43 (TDP-43) expression and nerve fiber damage ~ 2 years after the rTBIs. We observed acute RTG treatment to reduce the duration of PTS and impair the development of PTE. Acute RTG treatment also prevented post-injury hypersomnia, nerve fiber damage, and cortical TDP-43 accumulation and translocation from the nucleus to the cytoplasm. Mice that developed PTE displayed impaired rapid eye movement (REM) sleep, and there were significant correlations between seizure duration and time spent in the different stages of the sleep-wake cycle. We observed acute RTG treatment to impair injury-induced reduction of age-related increase in gamma frequency power of the EGG, which has been suggested to be necessary for a healthy aged brain. The data show that RTG, administered acutely post-TBI, is a promising, novel therapeutic option to blunt/prevent several long-term effects of rTBIs. Furthermore, our results show a direct relationship between sleep architecture and PTE.

Dequalinium chloride is an antagonists of α7 nicotinic acetylcholine receptors.

Dequalinium chloride has been used primarily as antiseptic compounds, but recently has been investigated for its effects on specific targets, including muscarinic acetylcholine receptors. Here we investigated dequalinium chloride as an antagonist to α7 nicotinic acetylcholine receptors. The pharmacological properties of dequalinium were established using cell lines stably co-transfected with the calcium-permeable human α7 nicotinic acetylcholine receptors and its chaperone NACHO, calcium dye fluorescent measurements or a calcium-sensitive protein reporter, and patch clamp recording of ionic currents. Using calcium dye fluorescence plate reader measurements, we find dequalinium chloride is an antagonist of α7 nicotinic acetylcholine receptors with an IC50 of 672 nM in response to activation with 500 µM acetylcholine chloride and positive allosteric modulator PNU-120596. However, using a membrane-tethered GCAMP7s calcium reporter allowed detection of α7-mediated calcium flux in the absence of PNU-120596. Using this approach revealed an IC50 of 157 nM for dequalinium on 300 µM acetylcholine-evoked currents. Using patch clamp recordings with 300 µM acetylcholine chloride and 10 µM PNU-120596, we find lower concentrations are sufficient to block ionic currents, with IC50 of 120 nM for dequalinium chloride and 54 nM for the related UCL 1684 compound. In summary, we find that dequalinium chloride and UCL1684, which are generally used to block SK-type potassium channels, are also highly effective antagonists of α7 nicotinic acetylcholine receptors. This finding, in combination with previous studies of muscarinic acetylcholine receptors, clearly establishes dequalinium compounds within the class of general anti-cholinergic antagonists.

Effects of Sublethal Organophosphate Toxicity and Anti-cholinergics on Electroencephalogram and Respiratory Mechanics in Mice.

Organophosphates are used in agriculture as insecticides but are potentially toxic to humans when exposed at high concentrations. The mechanism of toxicity is through antagonism of acetylcholinesterase, which secondarily causes excess activation of cholinergic receptors leading to seizures, tremors, respiratory depression, and other physiological consequences. Here we investigated two of the major pathophysiological effects, seizures and respiratory depression, using subcutaneous injection into mice of the organophosphate diisopropylfluorophosphate (DFP) at sublethal concentrations (2.1 mg/Kg) alone and co-injected with current therapeutics atropine (50 mg/Kg) or acetylcholinesterase reactivator HI6 (3 mg/Kg). We also tested a non-specific cholinergic antagonist dequalinium chloride (2 mg/Kg) as a novel treatment for organophosphate toxicity. Electroencephalogram (EEG) recordings revealed that DFP causes focal delta frequency (average 1.4 Hz) tonic spikes in the parietal region that occur transiently (lasting an average of 171 ± 33 min) and a more sustained generalized theta frequency depression in both parietal and frontal electrode that did not recover the following 24 h. DFP also caused behavioral tremors that partially recovered the following 24 h. Using whole body plethysmography, DFP revealed acute respiratory depression, including reduced breathing rates and tidal volumes, that partially recover the following day. Among therapeutic treatments, dequalinium chloride had the most potent effect on all physiological parameters by reducing acute EEG abnormalities and promoting a full recovery after 24 h from tremors and respiratory depression. Atropine and HI6 had distinct effects on EEGs. Co-treatment with atropine converted the acute 1.4 Hz tonic spikes to 3 Hz tonic spikes in the parietal electrode and promoted a partial recovery after 24 h from theta frequency and respiratory depression. HI6 fully removed the parietal delta spike increase and promoted a full recovery in theta frequency and respiratory depression. In summary, while all anticholinergic treatments promoted survival and moderated symptoms of DFP toxicity, the non-selective anti-cholinergic dequalinium chloride had the most potent therapeutic effects in reducing EEG abnormalities, moderating tremors and reducing respiratory depression.

Neuroprotective Roles of the Adenosine A3 Receptor Agonist AST-004 in Mouse Model of Traumatic Brain Injury.

Traumatic brain injury (TBI) remains one of the greatest public health concerns with increasing morbidity and mortality rates worldwide. Our group reported that stimulation of astrocyte mitochondrial metabolism by P2Y1 receptor agonists significantly reduced cerebral edema and reactive gliosis in a TBI model. Subsequent data on the pharmacokinetics (PK) and rapid metabolism of these compounds suggested that neuroprotection was likely mediated by a metabolite, AST-004, which binding data indicated was an adenosine A3 receptor (A3R) agonist. The neuroprotective efficacy of AST-004 was tested in a control closed cortical injury (CCCI) model of TBI in mice. Twenty-four (24) hours post-injury, mice subjected to CCCI and treated with AST-004 (0.22 mg/kg, injected 30 min post-trauma) exhibited significantly less secondary brain injury. These effects were quantified with less cell death (PSVue794 fluorescence) and loss of blood brain barrier breakdown (Evans blue extravasation assay), compared to vehicle-treated TBI mice. TBI-treated mice also exhibited significantly reduced neuroinflammatory markers, glial-fibrillary acidic protein (GFAP, astrogliosis) and ionized Ca2+-binding adaptor molecule 1 (Iba1, microgliosis), both at the mRNA (qRT-PCR) and protein (Western blot and immunofluorescence) levels, respectively. Four (4) weeks post-injury, both male and female TBI mice presented a significant reduction in freezing behavior during contextual fear conditioning (after foot shock). AST-004 treatment prevented this TBI-induced impairment in male mice, but did not significantly affect impairment in female mice. Impairment of spatial memory, assessed 24 and 48 h after the initial fear conditioning, was also reduced in AST-004-treated TBI-male mice. Female TBI mice did not exhibit memory impairment 24 and 48 h after contextual fear conditioning and similarly, AST-004-treated female TBI mice were comparable to sham mice. Finally, AST-004 treatments were found to increase in vivo ATP production in astrocytes (GFAP-targeted luciferase activity), consistent with the proposed mechanism of action. These data reveal AST-004 as a novel A3R agonist that increases astrocyte energy production and enhances their neuroprotective efficacy after brain injury.

Bis-Quinolinium Cyclophane Blockers of SK Potassium Channels Are Antagonists of M3 Muscarinic Acetylcholine Receptors.

Dequalinium is used as an antimicrobial compound for oral health and other microbial infections. Derivatives of dequalinium, the bis-quinolinium cyclophanes UCL 1684 and UCL 1848, are high affinity SK potassium channel antagonists. Here we investigated these compounds as M3 muscarinic receptor (mACHR) antagonists. We used the R-CEPIAer endoplasmic reticulum calcium reporter to functionally assay for Gq-coupled receptor signaling, and investigated the bis-quinolinium cyclophanes as antagonists of M3 mACHR activation in transfected CHO cells. Given mACHR roles in airway smooth muscle (ASM) contractility, we also tested the ability of UCL 1684 to relax ASM. We find that these compounds antagonized M3 mACHRs with an IC50 of 0.27 µM for dequalinium chloride, 1.5 µM for UCL 1684 and 1.0 µM for UCL 1848. UCL 1684 also antagonized M1 (IC50 0.12 µM) and M5 (IC50 0.52 µM) mACHR responses. UCL 1684 was determined to be a competitive antagonist at M3 receptors as it increased the EC50 for carbachol without a reduction in the maximum response. The Ki for UCL1684 determined from competition binding experiments was 909 nM. UCL 1684 reduced carbachol-evoked ASM contractions (>90%, IC50 0.43 µM), and calcium mobilization in rodent and human lung ASM cells. We conclude that dequalinium and bis-quinolinium cyclophanes antagonized M3 mACHR activation at sub- to low micromolar concentrations, with UCL 1684 acting as an ASM relaxant. Caution should be taken when using these compounds to block SK potassium channels, as inhibition of mACHRs may be a side-effect if excessive concentrations are used.

Mechanisms associated with the antidepressant-like effects of L-655,708.

Previous research has demonstrated that selective modulation of hippocampal transmission by systemic administration of an α5-GABAA receptor negative allosteric modulator, L-655,708, reproduces the sustained antidepressant-like (AD-like) effect of R,S-ketamine in the absence of any psychotomimetic or abuse-related effects. Pharmacological, electrophysiological (whole-cell patch clamp), and behavioral approaches were used to examine the mechanisms by which L-655,708 produces plasticity within the hippocampus that accounts for its sustained AD-like effect in rats. Inhibitors of either transcription or translation prevented the sustained AD-like effect of L-655,708. Unlike R,S-ketamine, L-655,708 did not cause an increase in the phosphorylation of the receptor for BDNF, TrkB, in the ventral hippocampus (vHipp) 30 or 60 min after its administration nor did administration of the TrkB inhibitor, K252a, directly into the vHipp, block the sustained AD-like effect of L-655,708. Similar to previous results with R,S-ketamine, administration of L-655,709 increased levels of GluA1 in the mPFC and, blockade of such receptors by direct administration of NBQX into the mPFC blocked the sustained AD-like effect of L-655,708. Patch-clamp recordings of ventral CA1 pyramidal cells 24 h after a single systemic administration of L-655,708 revealed a significant increase in input resistance, which resulted in an approximately two-fold increase in action potential frequency. These experiments indicate that the sustained AD-like effects of L-655,708 require protein synthesis and plasticity of GluA1 glutamate receptors in the mPFC. The drug also caused changes in GABAA receptor gating properties in the vHipp with resultant changes in ventral CA1 that indirectly increases neuronal excitability. Such effects likely contribute to its sustained AD-like activity.

Prevention of brain damage after traumatic brain injury by pharmacological enhancement of KCNQ (Kv7, "M-type") K+ currents in neurons.

Nearly three million people in the USA suffer traumatic brain injury (TBI) yearly; however, there are no pre- or post-TBI treatment options available. KCNQ2-5 voltage-gated K+ channels underlie the neuronal "M current", which plays a dominant role in the regulation of neuronal excitability. Our strategy towards prevention of TBI-induced brain damage is predicated on the suggested hyper-excitability of neurons induced by TBIs, and the decrease in neuronal excitation upon pharmacological augmentation of M/KCNQ K+ currents. Seizures are very common after a TBI, making further seizures and development of epilepsy disease more likely. Our hypothesis is that TBI-induced hyperexcitability and ischemia/hypoxia lead to metabolic stress, cell death and a maladaptive inflammatory response that causes further downstream morbidity. Using the mouse controlled closed-cortical impact blunt TBI model, we found that systemic administration of the prototype M-channel "opener", retigabine (RTG), 30 min after TBI, reduces the post-TBI cascade of events, including spontaneous seizures, enhanced susceptibility to chemo-convulsants, metabolic stress, inflammatory responses, blood-brain barrier breakdown, and cell death. This work suggests that acutely reducing neuronal excitability and energy demand via M-current enhancement may be a novel model of therapeutic intervention against post-TBI brain damage and dysfunction.

A Mouse Model of Repetitive Blast Traumatic Brain Injury Reveals Post-Trauma Seizures and Increased Neuronal Excitability.

Repetitive blast traumatic brain injury (TBI) affects numerous soldiers on the battlefield. Mild TBI has been shown to have long-lasting effects with repeated injury. We have investigated effects on neuronal excitability after repetitive, mild TBI in a mouse model of blast-induced brain injury. We exposed mice to mild blast trauma of an average peak overpressure of 14.6 psi, repeated across three consecutive days. While a single exposure did not reveal trauma as indicated by the glial fibrillary acidic protein indicator, three repetitive blasts did show significant increases. As well, mice had an increased indicator of inflammation (Iba-1) and increased tau, tau phosphorylation, and altered cytokine levels in the spleen. Video-electroencephalographic monitoring 48 h after the final blast exposure demonstrated seizures in 50% (12/24) of the mice, most of which were non-convulsive seizures. Long-term monitoring revealed that spontaneous seizures developed in at least 46% (6/13) of the mice. Patch clamp recording of dentate gyrus hippocampus neurons 48 h post-blast TBI demonstrated a shortened latency to the first spike and hyperpolarization of action potential threshold. We also found that evoked excitatory postsynaptic current amplitudes were significantly increased. These findings indicate that mild, repetitive blast exposures cause increases in neuronal excitability and seizures and eventual epilepsy development in some animals. The non-convulsive nature of the seizures suggests that subclinical seizures may occur in individuals experiencing even mild blast events, if repeated.

Cataract-associated connexin 46 mutation alters its interaction with calmodulin and function of hemichannels.

Connexin channels help maintain eye lens homeostasis and transparency. The G143R missense substitution in connexin (Cx) 46 is associated with congenital Coppock cataracts; however, the underlying molecular mechanism is largely unknown. Here, we report that compared with WT Cx46, the G143R substitution abolishes hemichannel conductance in Xenopus oocytes and in HeLa cells. Moreover, this substitution is dominant-negative and inhibits conductance of WT Cx46. CD analysis indicated that the substitution greatly reduces the α-helical structure of the intracellular Cx46 loop domain. Protein pulldown assays and isothermal titration calorimetry revealed that this Cx46 domain directly interacts with calmodulin (CaM) in a Ca2+-dependent fashion, an observation confirmed by immunofluorescent co-localization of Cx46 with CaM. Interestingly, the G143R substitution enhanced the Cx46-CaM interaction and attenuated its abolishment by Ca2+ depletion. Moreover, Cx46 increased dye influx, and the G143R substitution augmented this effect. Inhibition of Ca2+-mediated CaM activation blocked hemichannel permeability. The membrane potential plays a crucial role in Cx46 membrane permeability. We found that the activity of hemichannels is detectable under rest and hyperpolarization conditions but is eliminated with depolarization. These results suggested that the G143R substitution impairs voltage-dependent electrical conductance and alters membrane permeability mediated by Cx46 hemichannels. The latter likely is caused by the substitution-induced structural changes of the intracellular loop domain associated with the increased interaction with CaM and reduced Ca2+ sensitivity. The data suggest that the G143R-induced enhancement of the CaM-Cx46 interaction results in altered hemichannel activities and might be related to cataract formation.

Nanoparticle delivery of CRISPR into the brain rescues a mouse model of fragile X syndrome from exaggerated repetitive behaviours.

Technologies that can safely edit genes in the brains of adult animals may revolutionize the treatment of neurological diseases and the understanding of brain function. Here, we demonstrate that intracranial injection of CRISPR-Gold, a nonviral delivery vehicle for the CRISPR-Cas9 ribonucleoprotein, can edit genes in the brains of adult mice in multiple mouse models. CRISPR-Gold can deliver both Cas9 and Cpf1 ribonucleoproteins, and can edit all of the major cell types in the brain, including neurons, astrocytes and microglia, with undetectable levels of toxicity at the doses used. We also show that CRISPR-Gold designed to target the metabotropic glutamate receptor 5 (mGluR5) gene can efficiently reduce local mGluR5 levels in the striatum after an intracranial injection. The effect can also rescue mice from the exaggerated repetitive behaviours caused by fragile X syndrome, a common single-gene form of autism spectrum disorders. CRISPR-Gold may significantly accelerate the development of brain-targeted therapeutics and enable the rapid development of focal brain-knockout animal models.

Knockout of the BK ß4-subunit promotes a functional coupling of BK channels and ryanodine receptors that mediate a fAHP-induced increase in excitability.

BK channels are large-conductance calcium- and voltage-activated potassium channels with diverse properties. Knockout of the accessory BK ß4-subunit in hippocampus dentate gyrus granule neurons causes BK channels to change properties from slow-gated type II channels to fast-gated type I channels that sharpen the action potential, increase the fast afterhyperpolarization (fAHP) amplitude, and increase spike frequency. Here we studied the calcium channels that contribute to fast-gated BK channel activation and increased excitability of ß4 knockout neurons. By using pharmacological blockers during current-clamp recording, we find that BK channel activation during the fAHP is dependent on ryanodine receptor activation. In contrast, L-type calcium channel blocker (nifedipine) affects the BK channel-dependent repolarization phase of the action potential but has no effect on the fAHP. Reducing BK channel activation during the repolarization phase with nifedipine, or during the fAHP with ryanodine, indicated that it is the BK-mediated increase of the fAHP that confers proexcitatory effects. The proexcitatory role of the fAHP was corroborated using dynamic current clamp. Increase or decrease of the fAHP amplitude during spiking revealed an inverse relationship between fAHP amplitude and interspike interval. Finally, we show that the seizure-prone ryanodine receptor gain-of-function (R2474S) knockin mice have an unaltered repolarization phase but larger fAHP and increased AP frequency compared with their control littermates. In summary, these results indicate that an important role of the ß4-subunit is to reduce ryanodine receptor-BK channel functional coupling during the fAHP component of the action potential, thereby decreasing excitability of dentate gyrus neurons.

Renal tubular epithelial cell prorenin receptor regulates blood pressure and sodium transport.

The physiological significance of the renal tubular prorenin receptor (PRR) has been difficult to elucidate due to developmental abnormalities associated with global or renal-specific PRR knockout (KO). We recently developed an inducible renal tubule-wide PRR KO using the Pax8/LC1 transgenes and demonstrated that disruption of renal tubular PRR at 1 mo of age caused no renal histological abnormalities. Here, we examined the role of renal tubular PRR in blood pressure (BP) regulation and Na(+) excretion and investigated the signaling mechanisms by which PRR regulates Na(+) balance. No detectable differences in BP were observed between control and PRR KO mice fed normal- or low-Na(+) diets. However, compared with controls, PRR KO mice had elevated plasma renin concentration and lower cumulative Na(+) balance with normal- and low-Na(+) intake. PRR KO mice had an attenuated hypertensive response and reduced Na(+) retention following angiotensin II (ANG II) infusion. Furthermore, PRR KO mice had significantly lower epithelial Na(+) channel (ENaC-α) expression. Treatment with mouse prorenin increased, while PRR antagonism decreased, ENaC activity in isolated split-open collecting ducts (CD). The prorenin effect was prevented by protein kinase A and Akt inhibition, but unaffected by blockade of AT1, ERK1/2, or p38 MAPK pathways. Taken together, these data indicate that renal tubular PRR, likely via direct prorenin/renin stimulation of PKA/Akt-dependent pathways, stimulates CD ENaC activity. Absence of renal tubular PRR promotes Na(+) wasting and reduces the hypertensive response to ANG II.

Recording ion channels in isolated, split-opened tubules.

Ion channels play key roles in physiology. They function as protein transducers able to transform stimuli and chemical gradients into electrical signals. They also are critical for cell signaling and play a particularly important role in epithelial transport acting as gateways for the movement of electrolytes across epithelial cell membranes. Experimental limitations, though, have hampered the recording of ion channel activity in many types of tissue. This has slowed progress in understanding the cellular and physiological function of these channels with most function inferred from in vitro systems and cell culture models. In many cases, such inferences have clouded rather than clarified the picture. Here, we describe a contemporary method for isolating and patch-clamping renal tubules for ex vivo analysis of ion channel function in native tissue. Focus is placed on quantifying the activity of the epithelial Na(+) channel (ENaC) in the aldosterone--sensitive distal nephron (ASDN). This isolated, split-open tubule preparation enables recording of renal ion channels in the close-to-native environment under the control of native cell signaling pathways and receptors. When combined with complementary measurements of organ and system function, and contemporary molecular genetics and pharmacology used to manipulate function and regulation, patch-clamping renal channels in the isolated, split-open tubule enables understanding to emerge about the physiological function of these key proteins from the molecule to the whole animal.