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Active coping is an adaptive stress response that improves outcomes in medical and neuropsychiatric diseases. To date, most research into coping style has focused on neurotransmitter activity and little is known about the intrinsic excitability of neurons in the associated brain regions that facilitate coping. Previous studies have shown that HCN channels regulate neuronal excitability in pyramidal cells and that HCN channel current (I ) in the CA1 area increases with chronic mild stress. Reduction of I in the CA1 area leads to antidepressant-like behavior, and this region has been implicated in the regulation of coping style. We hypothesized that the antidepressant-like behavior achieved with CA1 knockdown of I is accompanied by increases in active coping. In this report, we found that global loss of TRIP8b, a necessary subunit for proper HCN channel localization in pyramidal cells, led to active coping behavior in numerous assays specific to coping style. We next employed a viral strategy using a dominant negative TRIP8b isoform to alter coping behavior by reducing HCN channel expression. This approach led to a robust reduction in I in CA1 pyramidal neurons and an increase in active coping. Together, these results establish that changes in HCN channel function in CA1 influences coping style.
© 2018 International Society for Neurochemistry.
Voltage-gated ion channels are critical for neuronal integration. Some of these channels, however, are misregulated in several neurological disorders, causing both gain- and loss-of-function channelopathies in neurons. Using several transgenic mouse models of Alzheimer's disease (AD), we find that sub-threshold voltage signals strongly influenced by hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels progressively deteriorate over chronological aging in hippocampal CA1 pyramidal neurons. The degraded signaling via HCN channels in the transgenic mice is accompanied by an age-related global loss of their non-uniform dendritic expression. Both the aberrant signaling via HCN channels and their mislocalization could be restored using a variety of pharmacological agents that target the endoplasmic reticulum (ER). Our rescue of the HCN channelopathy helps provide molecular details into the favorable outcomes of ER-targeting drugs on the pathogenesis and synaptic/cognitive deficits in AD mouse models, and implies that they might have beneficial effects on neurological disorders linked to HCN channelopathies.
Copyright © 2018. Published by Elsevier Inc.
Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels are important regulators of excitability in neural, cardiac, and other pacemaking cells, which are often altered in disease. In mice, loss of HCN2 leads to cardiac dysrhythmias, persistent spike-wave discharges similar to those seen in absence epilepsy, ataxia, tremor, reduced neuropathic and inflammatory pain, antidepressant-like behavior, infertility, and severely restricted growth. While many of these phenotypes have tissue-specific mechanisms, the cause of restricted growth in HCN2 knockout animals remains unknown. Here, we characterize a novel, 3kb insertion mutation of Hcn2 in the Tremor and Reduced Lifespan 2 (TRLS/2J) mouse that leads to complete loss of HCN2 protein, and we show that this mutation causes many phenotypes similar to other mice lacking HCN2 expression. We then demonstrate that while TRLS/2J mice have low blood glucose levels and impaired growth, dysfunction in hormonal secretion from the pancreas, pituitary, and thyroid are unlikely to lead to this phenotype. Instead, we find that homozygous TRLS/2J mice have abnormal gastrointestinal function that is characterized by less food consumption and delayed gastrointestinal transit as compared to wildtype mice. In summary, a novel mutation in HCN2 likely leads to impaired GI motility, causing the severe growth restriction seen in mice with mutations that eliminate HCN2 expression.
Neuroinflammation is consistently found in many neurological disorders, but whether or not the inflammatory response independently affects neuronal network properties is poorly understood. Here, we report that intracerebroventricular injection of the prototypical inflammatory molecule lipopolysaccharide (LPS) in rats triggered a strong and long-lasting inflammatory response in hippocampal microglia associated with a concomitant upregulation of Toll-like receptor (TLR4) in pyramidal and hilar neurons. This, in turn, was associated with a significant reduction of the dendritic hyperpolarization-activated cyclic AMP-gated channel type 1 (HCN1) protein level while Kv4.2 channels were unaltered as assessed by western blot. Immunohistochemistry confirmed the HCN1 decrease in CA1 pyramidal neurons and showed that these changes were associated with a reduction of TRIP8b, an auxiliary subunit for HCN channels implicated in channel subcellular localization and trafficking. At the physiological level, this effect translated into a 50% decrease in HCN1-mediated currents (I) measured in the distal dendrites of hippocampal CA1 pyramidal cells. At the functional level, the band-pass-filtering properties of dendrites in the theta frequency range (4-12 Hz) and their temporal summation properties were compromised. We conclude that neuroinflammation can independently trigger an acquired channelopathy in CA1 pyramidal cell dendrites that alters their integrative properties. By directly changing cellular function, this phenomenon may participate in the phenotypic expression of various brain diseases.
Hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels have important functions in controlling neuronal excitability and generating rhythmic oscillatory activity. The role of tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b) in regulation of hyperpolarization-activated inward current, I , in the thalamocortical system and its functional relevance for the physiological thalamocortical oscillations were investigated. A significant decrease in I current density, in both thalamocortical relay (TC) and cortical pyramidal neurons was found in TRIP8b-deficient mice (TRIP8b). In addition basal cAMP levels in the brain were found to be decreased while the availability of the fast transient A-type K current, I , in TC neurons was increased. These changes were associated with alterations in intrinsic properties and firing patterns of TC neurons, as well as intrathalamic and thalamocortical network oscillations, revealing a significant increase in slow oscillations in the delta frequency range (0.5-4 Hz) during episodes of active-wakefulness. In addition, absence of TRIP8b suppresses the normal desynchronization response of the EEG during the switch from slow-wave sleep to wakefulness. It is concluded that TRIP8b is necessary for the modulation of physiological thalamocortical oscillations due to its direct effect on HCN channel expression in thalamus and cortex and that mechanisms related to reduced cAMP signaling may contribute to the present findings.
Tetratricopeptide repeat (TPR) domains are ubiquitous structural motifs that mediate protein-protein interactions. For example, the TPR domains in the peroxisomal import receptor PEX5 enable binding to a range of type 1 peroxisomal targeting signal motifs. A homolog of PEX5, tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b), binds to and functions as an auxiliary subunit of hyperpolarization-activated cyclic nucleotide (HCN)-gated channels. Given the similarity between TRIP8b and PEX5, this difference in function raises the question of what mechanism accounts for their binding specificity. In this report, we found that the cyclic nucleotide-binding domain and the C terminus of the HCN channel are critical for conferring specificity to TRIP8b binding. We show that TRIP8b binds the HCN cyclic nucleotide-binding domain through a 37-residue domain and the HCN C terminus through the TPR domains. Using a combination of fluorescence polarization- and co-immunoprecipitation-based assays, we establish that binding at either site increases affinity at the other. Thus, allosteric coupling of the TRIP8b TPR domains both promotes binding to HCN channels and limits binding to type 1 peroxisomal targeting signal substrates. These results raise the possibility that other TPR domains may be similarly influenced by allosteric mechanisms as a general feature of protein-protein interactions.
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the excitability of neurons. The subcellular distribution of these channels in pyramidal neurons of hippocampal area CA1 is regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit. Genetic knockout of HCN pore forming subunits or TRIP8b, both lead to an increase in antidepressant-like behavior, suggesting that limiting the function of HCN channels may be useful as a treatment for Major Depressive Disorder (MDD). Despite significant therapeutic interest, HCN channels are also expressed in the heart, where they regulate rhythmicity. To circumvent off-target issues associated with blocking cardiac HCN channels, our lab has recently proposed targeting the protein-protein interaction between HCN and TRIP8b in order to specifically disrupt HCN channel function in the brain. TRIP8b binds to HCN pore forming subunits at two distinct interaction sites, although here the focus is on the interaction between the tetratricopeptide repeat (TPR) domains of TRIP8b and the C terminal tail of HCN1. In this protocol, an expanded description of a method for purifying TRIP8b and executing a high throughput screen to identify small molecule inhibitors of the interaction between HCN and TRIP8b, is described. The method for high throughput screening utilizes a Fluorescence Polarization (FP) -based assay to monitor the binding of a large TRIP8b fragment to a fluorophore-tagged eleven amino acid peptide corresponding to the HCN1 C terminal tail. This method allows 'hit' compounds to be identified based on the change in the polarization of emitted light. Validation assays are then performed to ensure that 'hit' compounds are not artifactual.
Major depressive disorder (MDD) is a prevalent psychiatric condition with limited therapeutic options beyond monoaminergic therapies. Although effective in some individuals, many patients fail to respond adequately to existing treatments, and new pharmacologic targets are needed. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels regulate excitability in neurons, and blocking HCN channel function has been proposed as a novel antidepressant strategy. However, systemic blockade of HCN channels produces cardiac effects that limit this approach. Knockout (KO) of the brain-specific HCN-channel auxiliary subunit tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b) also produces antidepressant-like behavioral effects and suggests that inhibiting TRIP8b function could produce antidepressant-like effects without affecting the heart. We examined the structural basis of TRIP8b-mediated HCN-channel trafficking and its relationship with antidepressant-like behavior using a viral rescue approach in TRIP8b KO mice. We found that restoring TRIP8b to the hippocampus was sufficient to reverse the impaired HCN-channel trafficking and antidepressant-like behavioral effects caused by TRIP8b KO. Moreover, we found that hippocampal expression of a mutated version of TRIP8b further impaired HCN-channel trafficking and increased the antidepressant-like behavioral phenotype of TRIP8b KO mice. Thus, modulating the TRIP8b-HCN interaction bidirectionally influences channel trafficking and antidepressant-like behavior. Overall, our work suggests that small-molecule inhibitors of the interaction between TRIP8b and HCN should produce antidepressant-like behaviors and could represent a new paradigm for the treatment of MDD.
Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels function in the brain to limit neuronal excitability. Limiting the activity of these channels has been proposed as a therapy for major depressive disorder, but the critical role of HCN channels in cardiac pacemaking has limited efforts to develop therapies directed at the channel. Previous studies indicated that the function of HCN is tightly regulated by its auxiliary subunit, tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), which is not expressed in the heart. To target the function of the HCN channel in the brain without affecting the channel's function in the heart, we propose disrupting the interaction between HCN and TRIP8b. We developed a high-throughput fluorescence polarization (FP) assay to identify small molecules capable of disrupting this interaction. We used this FP assay to screen a 20,000-compound library and identified a number of active compounds. The active compounds were validated using an orthogonal AlphaScreen assay to identify one compound (0.005%) as the first confirmed hit for inhibiting the HCN-TRIP8b interaction. Identifying small molecules capable of disrupting the interaction between HCN and TRIP8b should enable the development of new research tools and small-molecule therapies that could benefit patients with depression.
© 2015 Society for Laboratory Automation and Screening.