Neuronal Roles of the Multifunctional Protein Dipeptidyl Peptidase-like 6 (DPP6).

The concerted action of voltage-gated ion channels in the brain is fundamental in controlling neuronal physiology and circuit function. Ion channels often associate in multi-protein complexes together with auxiliary subunits, which can strongly influence channel expression and function and, therefore, neuronal computation. One such auxiliary subunit that displays prominent expression in multiple brain regions is the Dipeptidyl aminopeptidase-like protein 6 (DPP6). This protein associates with A-type K+ channels to control their cellular distribution and gating properties. Intriguingly, DPP6 has been found to be multifunctional with an additional, independent role in synapse formation and maintenance. Here, we feature the role of DPP6 in regulating neuronal function in the context of its modulation of A-type K+ channels as well as its independent involvement in synaptic development. The prevalence of DPP6 in these processes underscores its importance in brain function, and recent work has identified that its dysfunction is associated with host of neurological disorders. We provide a brief overview of these and discuss research directions currently underway to advance our understanding of the contribution of DPP6 to their etiology.

Cushing Syndrome in a Pediatric Patient With a KCNJ5 Variant and Successful Treatment With Low-dose Ketoconazole.

CONTEXT: Pathogenic variants in KCNJ5, encoding the GIRK4 (Kir3.4) potassium channel, have been implicated in the pathogenesis of familial hyperaldosteronism type-III (FH-III) and sporadic primary aldosteronism (PA). In addition to aldosterone, glucocorticoids are often found elevated in PA in association with KCNJ5 pathogenic variants, albeit at subclinical levels. However, to date no GIRK4 defects have been linked to Cushing syndrome (CS). PATIENT: We present the case of a 10-year-old child who presented with CS at an early age due to bilateral adrenocortical hyperplasia (BAH). The patient was placed on low-dose ketoconazole (KZL), which controlled hypercortisolemia and CS-related signs. Discontinuation of KZL for even 6 weeks led to recurrent CS. RESULTS: Screening for known genes causing cortisol-producing BAHs (PRKAR1A, PRKACA, PRKACB, PDE11A, PDE8B, ARMC5) failed to identify any gene defects. Whole-exome sequencing showed a novel KCNJ5 pathogenic variant (c.506T>C, p.L169S) inherited from her father. In vitro studies showed that the p.L169S variant affects conductance of the Kir3.4 channel without affecting its expression or membrane localization. Although there were no effects on steroidogenesis in vitro, there were modest changes in protein kinase A activity. In silico analysis of the mutant channel proposed mechanisms for the altered conductance. CONCLUSION: We present a pediatric patient with CS due to BAH and a germline defect in KCNJ5. Molecular investigations of this KCNJ5 variant failed to show a definite cause of her CS. However, this KCNJ5 variant differed in its function from KCNJ5 defects leading to PA. We speculate that GIRK4 (Kir3.4) may play a role in early human adrenocortical development and zonation and participate in the pathogenesis of pediatric BAH.

P38 Regulates Kainic Acid-Induced Seizure and Neuronal Firing via Kv4.2 Phosphorylation.

The subthreshold, transient A-type K+ current is a vital regulator of the excitability of neurons throughout the brain. In mammalian hippocampal pyramidal neurons, this current is carried primarily by ion channels comprising Kv4.2 α-subunits. These channels occupy the somatodendritic domains of these principle excitatory neurons and thus regulate membrane voltage relevant to the input-output efficacy of these cells. Owing to their robust control of membrane excitability and ubiquitous expression in the hippocampus, their dysfunction can alter network stability in a manner that manifests in recurrent seizures. Indeed, growing evidence implicates these channels in intractable epilepsies of the temporal lobe, which underscores the importance of determining the molecular mechanisms underlying their regulation and contribution to pathologies. Here, we describe the role of p38 kinase phosphorylation of a C-terminal motif in Kv4.2 in modulating hippocampal neuronal excitability and behavioral seizure strength. Using a combination of biochemical, single-cell electrophysiology, and in vivo seizure techniques, we show that kainic acid-induced seizure induces p38-mediated phosphorylation of Thr607 in Kv4.2 in a time-dependent manner. The pharmacological and genetic disruption of this process reduces neuronal excitability and dampens seizure intensity, illuminating a cellular cascade that may be targeted for therapeutic intervention to mitigate seizure intensity and progression.

Activity-dependent isomerization of Kv4.2 by Pin1 regulates cognitive flexibility.

Voltage-gated K+ channels function in macromolecular complexes with accessory subunits to regulate brain function. Here, we describe a peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1)-dependent mechanism that regulates the association of the A-type K+ channel subunit Kv4.2 with its auxiliary subunit dipeptidyl peptidase 6 (DPP6), and thereby modulates neuronal excitability and cognitive flexibility. We show that activity-induced Kv4.2 phosphorylation triggers Pin1 binding to, and isomerization of, Kv4.2 at the pThr607-Pro motif, leading to the dissociation of the Kv4.2-DPP6 complex. We generated a novel mouse line harboring a knock-in Thr607 to Ala (Kv4.2TA) mutation that abolished dynamic Pin1 binding to Kv4.2. CA1 pyramidal neurons of the hippocampus from these mice exhibited altered Kv4.2-DPP6 interaction, increased A-type K+ current, and reduced neuronal excitability. Behaviorally, Kv4.2TA mice displayed normal initial learning but improved reversal learning in both Morris water maze and lever press paradigms. These findings reveal a Pin1-mediated mechanism regulating reversal learning and provide potential targets for the treatment of neuropsychiatric disorders characterized by cognitive inflexibility.

Pharmacological identification of cholinergic receptor subtypes: modulation of locomotion and neural circuit excitability in Drosophila larvae.

Acetylcholine (ACh) is an abundant neurotransmitter and neuromodulator in many species. In Drosophila melanogaster ACh is the neurotransmitter used in peripheral sensory neurons and is a primary excitatory neurotransmitter and neuromodulator within the central nervous system (CNS). The receptors that facilitate cholinergic transmission are divided into two broad subtypes: the ionotropic nicotinic acetylcholine receptors (nAChRs) and the metabotropic muscarinic acetylcholine receptors (mAChRs). This receptor classification is shared in both mammals and insects; however, both the pharmacological and functional characterization of these receptors within the Drosophila nervous system has lagged behind its mammalian model counterparts. In order to identify the impact of ACh receptor subtypes in regulating the performance of neural circuits within the larval CNS, we used a behavioral and electrophysiological approach to assess cholinergic modulation of locomotion and sensory-CNS-motor circuit excitability. We exposed intact and semi-intact 3rd instar larvae to ACh receptor agonists and antagonists to observe their roles in behavior and regulation of neural circuit excitability and to investigate AChR pharmacological properties in vivo. We combined this with targeted AChR RNAi-mediated knockdown to identify specific receptor subtypes facilitating ACh modulation of circuit efficacy. We identify a contribution by both mAChRs and nAChRs in regulation of locomotor behavior and reveal they play a role in modulation of the excitability of a sensory-CNS-motor circuit. We further reveal a conspicuous role for mAChR-A and mAChR-C in motor neurons in modulation of their input-output efficacy.

Considerations in repetitive activation of light sensitive ion channels for long-term studies: Channel rhodopsin in the Drosophila model.

Optogenetics is a technique used in various animal models and holds a potential for therapeutic possibilities in mammals. There are technical issues with the use of light sensitive ion channels: reproducible effects over time, controlling where the non-native proteins are targeted within the cell and changes in the biophysical properties of the cells they are expressed in. We used a variant of channel rhodopsin (ChR2-XXL) and targeted expression in neurons of larval Drosophila to investigate the acute and chronic activation, with light pulses, of the channels on synaptic function. The rhodopsin channel modifier all trans retinal (ATR) also plays a role in the sensitivity of the channel to light. Periods of acute, repetitive, and pulsatile blue light exposure over larval development produced attenuated responses. These blue light sensitive ion channels, with ATR, show accommodation and produce an electrical refractory period in inducing synaptic responses. The biological significance and aim of this study is to demonstrate that in controlling particular neurons or neuronal circuits with optogenetics, over time and throughout development, one will have to understand the dynamic nature of activating and silencing the light sensitive channels as well as the biophysical effects on neuronal activity.

Using optogenetics to assess neuroendocrine modulation of heart rate in Drosophila melanogaster larvae.

The Drosophila melanogaster heart has become a principal model in which to study cardiac physiology and development. While the morphology of the heart in Drosophila and mammals is different, many of the molecular mechanisms that underlie heart development and function are similar and function can be assessed by similar physiological measurements, such as cardiac output, rate, and time in systole or diastole. Here, we have utilized an intact, optogenetic approach to assess the neural influence on heart rate in the third instar larvae. To simulate the release of modulators from the nervous system in response to environmental influences, we have directed expression of channel-rhodopsin variants to targeted neuronal populations to assess the role of these neural ensembles in directing release of modulators that may affect heart rate in vivo. Our observations show that the activation of targeted neurons, including cholinergic, dopaminergic, and serotonergic neurons, stimulate the release of cardioactive substances that increase heart rate after the initial activation at both room temperature and in a cold environment. This parallels previous studies suggesting these modulators play a crucial role in altering heart rate when applied to exposed hearts and adds to our understanding of chemical modulation of heart rate in intact Drosophila larvae.

The effects of potassium and muscle homogenate on proprioceptive responses in crayfish and crab.

Proprioception of limbs and joints is a basic sensory function throughout most of the animal kingdom. It is important to understand how proprioceptive organs and the associated sensory neurons function with altered environments such as increased potassium ion concentrations ([K+]) from diseased states, ionic imbalances, and damaged tissues. These factors can drastically alter neuronal activity. To assess this matter, we used the chordotonal organ in a walking leg of a blue crab (Callinectes sapidus) and the muscle receptor organ of the crayfish (Procambarus clarkii). These organs serve as tractable models for the analysis of proprioception. The preparations can help serve as translational models for these effects, which may be observed in other invertebrate species as well as mammalian species (including humans). When extracellular potassium concentration ([K+]o) is increased to 20 mM in both preparations, mixed results are observed with activity increasing in some preparations and decreasing in others after mechanical displacement. However, when [K+]o is increased to 40 mM, activity drastically decreases in all preparations. Additionally, proprioceptor sensory activity declines upon exposure to a diluted muscle homogenate, which contains a host of intracellular constituents. The robust effects of altered [K+] on proprioception in these models illuminate the potential detriments on neuronal function in cases of severe tissue damage as well as altered [K+]o.

Pharmacological identification of cholinergic receptor subtypes on Drosophila melanogaster larval heart.

The Drosophila melanogaster heart is a popular model in which to study cardiac physiology and development. Progress has been made in understanding the role of endogenous compounds in regulating cardiac function in this model. It is well characterized that common neurotransmitters act on many peripheral and non-neuronal tissues as they flow through the hemolymph of insects. Many of these neuromodulators, including acetylcholine (ACh), have been shown to act directly on the D. melanogaster larval heart. ACh is a primary neurotransmitter in the central nervous system (CNS) of vertebrates and at the neuromuscular junctions on skeletal and cardiac tissue. In insects, ACh is the primary excitatory neurotransmitter of sensory neurons and is also prominent in the CNS. A full understanding regarding the regulation of the Drosophila cardiac physiology by the cholinergic system remains poorly understood. Here we use semi-intact D. melanogaster larvae to study the pharmacological profile of cholinergic receptor subtypes, nicotinic acetylcholine receptors (nAChRs) and muscarinic acetylcholine receptors (mAChRs), in modulating heart rate (HR). Cholinergic receptor agonists, nicotine and muscarine both increase HR, while nAChR agonist clothianidin exhibits no significant effect when exposed to an open preparation at concentrations as low as 100 nM. In addition, both nAChR and mAChR antagonists increase HR as well but also display capabilities of blocking agonist actions. These results provide evidence that both of these receptor subtypes display functional significance in regulating the larval heart's pacemaker activity.