Sodium channel-related pain disorders: From molecular mechanisms towards personalized treatment
Voltage-gated sodium channels (Navs) are crucial for pain perception. This is illustrated by several human genetic conditions leading to chronic pain or congenital painlessness. The type of mutation, its impact on neuron excitability as well as the affected Nav channel subtype delineates a complex picture of the disorders. A detailed understanding of these pain disorders, however, was precluded because of the lack of direct structural information on human Nav channels.
Very recent groundbreaking advances in cryo-electron microscopy substantially changed the view on the concept of sodium channel gating and now brings the consortium in an excellent position to revise and advance current knowledge on pain-related channelopathies. We will address the new concept of allosteric inactivation that replaces the long standing ball-and-chain model of channel inactivation, investigate sodium channel dimerization and the effect of mutations on neuronal excitability. We will interconnect these basic principles of ion-channel function to translational research in patients with neuropathic pain. The objectives will be addressed by a multidisciplinary combination of atomistic molecular dynamics simulations, high-throughput genetics, machine learning, computational drug design and electrophysiological assessment of patient-associated sodium channel mutations. Further methodologies include induced pluripotent stem cell (iPS-cell)-derived sensory neuron models, single channel gating analyses, biochemical assessments of sodium channel proteins and multimodal brain imaging studies in patients. Our long-term goal is to integrate the comprehensive data to predict clinically relevant effects of sodium channel variants and their responsiveness to analgesic drugs. The results of this team effort are likely to yield in the long-term unifying predictive models that directly link pain-associated genetic sodium channel variants with changes in molecular biophysics, altered electrophysiological function, pharmacological responsiveness and clinical phenotypes.
Research Area Translational Neurosciences- Projects
Here you find the summarys of the subprojects of the Translational Neurosciences Research Initiative.
P01 Sodium channel mutation-induced excitability changes of human nociceptors
PI: Univ.-Prof. Dr. med. Angelika Lampert, Institut für Physiologie (Neurophysiologie), RWTH Aachen
Mutations in sodium channels (Navs) linked to pain syndromes change cellular excitability, which leads to a strongly modified input into the CNS. Assessment of mutation induced effects can be achieved by heterologous expressions systems, which we aim to perform in mammalian cell lines, or with induced pluripotent stem cell (iPS-cell) derived sensory neurons. From well characterized patients (Z-project, P6) carrying disease linked mutations (identified in P2 and P3) we will generate iPS-cells and derive thereof human patient sensory neurons, which allow us to assess spontaneous activity and temperature sensitivity of patients’ cells on a multi-electrode array.
Neuropathic pain is likely to be due to aberrant activity of so called sleeping nociceptors, also called mechano-insensitive C-fibers (CMi-fibers). In patients with inherited neuropathic pain it is assumed that these fibers are hyperactive, and the reason for the patient’s pain. Although we showed that iPS-cell derived nociceptors of patients are hyperactive, the exact identity of those neurons is unclear. Here, we aim to study their specific characteristics such as conduction velocity, responsiveness to sine wave stimuli and to chemical stimuli such as solved chowage seeds. To this end, we will use our established co-culture system of iPS-cell derived nociceptors with patient-specific Schwann-cells of pain patients with sodium channel mutations and controls to mimic physiological conditions. We will perform patch clamp and set up a microfluidic system on multi-electrode arrays (in cooperation with Andreas Offenhäusser, Jülich).
We aim to correlate functions of CM- and CMi fibers identified by human microneurography (P6 and cooperation with Barbara Namer) with Nav gating in iPS-cell derived sensory neurons. We will focus on functions such as Nav slow inactivation or response to repetitive stimulation with either a sine wave or square pulses.
The response of patient derived neurons will help to identify patient specific pathomechanisms, which may correlate to the detailed small fiber investigations of this patient, as performed in P6. The effect of selected drugs which may specifically alleviate excitability changes induced by the mutation present in the iPS-cells (P4, P5), will be assessed in our system. With this approach we will have the means to identify pathomechanisms, to test patient-specific treatment and to better understand the basis of neuropathic pain.
In cooperation with P2 we will introduce CrispR/cas9 modification to carve out the underlying molecular mechanisms and the role of Nav subtypes. Patch-protocols will be modified according to the findings in microneurography of the specific patient (P6) and the particular current response of Nav subtypes will be assessed using patch-clamp.
This subproject will help to understand the functional mechanism underlying Nav-mutation associated excitability changes, effects on conduction velocity and how to modify those by patient-specific pharmacological approach. Parts of the project will be performed as Start-Up funding (comparable to DFG Modul Anschubförderung) by Dr. Jannis Körner.
P02 Genetics of pain-related sodium channelopathies: From genetic variants to clinical pain phenotypes
PI: Univ.-Prof. Dr. med. Ingo Kurth, Institute of Human Genetics, RWTH Aachen
Co PI: Dr. rer. nat. Natja Haag, Institute of Human Genetics, RWTH Aachen
Heritable pain-related disorders are linked to mutations in three pain-sensing voltage-gated sodium channels, NaV1.7 (SCN9A), NaV1.8 (SCN10A), and NaV1.9 (SCN11A). Patients with sodium channel mutations present with different phenotypes ranging from congenital insensitivity to pain to severe episodic pain or painful small fiber neuropathies. Although mutations in these channels are currently thought to explain a substantial proportion of heritable pain disorders, we and others only rarely identify unambiguously pathogenic mutations in these ion-channel genes, but rather variants of unclear clinical significance. According to whole-exome sequencing data from our large cohort of more than 300 patients, affected individuals show an enrichment of genetic variants in NaV channels in comparison to control cohorts. Additional patient samples recruited from the Z-project will be analyzed by whole-exome sequencing to identify additional NaV channel variants, but also to identify novel genes implicated in monogenic pain disorders. Based on this, we will assess putative functional effects of a large number of NaV channel variants by computer modelling (P3, P4) and further investigate the best candidates by biochemistry (P5) and electrophysiology in heterologous expression systems (P1, P5 for single channel recordings). This will identify variants which do not fulfill the formal criteria to be classified as causative mutation for a monogenic disorder, but with impact on NaV channel function. Based on this, the effect of selected variants on excitability and cellular function will be evaluated at a deeper mechanistic level by generating patient-derived nociceptors from iPS cells and CRISPR/Cas-based cellular models (P1). To get further insight into disease mechanisms, global changes in the transcriptome and epigenome of these cells will be addressed by RNAseq and whole-genome long read nanopore sequencing. The results will be taken back to the clinics and will be correlated with clinical findings (Z-project) and subsequent in-vivomeasurements (P6, P8). Our findings will help to understand disease signatures of genetically encoded chronic neuropathic pain.
P03 Decoding Nav dysfunction in pain at atomic resolution
PI:Jun.-Prof. Dr. J.-P. Machtens, Institute of Clinical Pharmacology RWTH Aachen University and Computational Neurophysiology Group, Institute of Complex Systems – Zelluläre Biophysik (ICS-4), Forschungszentrum Jülich
Voltage-gated sodium channels (Navs) are responsible for the depolarizing phase of the action potential with essential roles in fast electrical signaling, including signal propagation from the periphery to the central nervous system. Various pain disorders, including increased and decreased sensitivity to painful stimuli, have been linked to mutations in the genes encoding Nav1.7–Nav1.9.
Therefore, the recent determination of the first mammalian Nav structures at near-atomic resolution, including human Nav1.7, represents a breakthrough, and now enables us to link Nav structure with function using high-performance computational biology techniques.
We use state-of-the art molecular dynamics simulations to investigate the mechanisms of channel dimerization and fast-inactivation gating in Nav1.7, which both play key roles in pain-associated channel dysfunction. Furthermore, our simulations will provide a basis for state-specific drug screening. Pain-associated Nav sequence variants, identified by genetic screening and clinical investigations, will be studied to uncover their biophysical and functional consequences using molecular simulations and machine-learning techniques. These studies are expected to advance our mechanistic understanding of Nav channel function and to promote variant-specific drug development for genetically identified patients as a first step towards personalized treatment of pain disorders.
P04 Correcting Channel Dysfunction using variants-selective drugs
PL: Prof. Dr. rer. nat. Giulia Rossetti
Numerous studies have reported voltage-gated sodium channels (Nav) isoform channelopathies, for Nav1.7 (SCN9A), Nav1.8 (SCN10A) and Nav1.9 (SCN11A) as the primary cause of increased pain or loss-of-pain phenotypes in humans.
Significant efforts to develop compounds selective for Nav1.7, Nav1.8, Nav1.9 are currently being actively pursued by the pharmaceutical industry. Despite the recent progresses, few subtype-selective inhibitors exist only for NaV1.7 and NaV1.8 (still under clinical trial) due to the high structural conservation within the channel families: all Nav isoforms share the same overall architecture and high sequence homology.
Thanks to the recent breakthrough of the first mammalian Nav structures determination by cryo-electron microscopy, sequence variation data (Kurth P2, Rolke P6, Dohrn/Häusler Z-project) can be exploited to analyze Nav structures and dynamics and to derive functional insights in their variation-dependent gating mechanisms (Machtens P3). These will be exploited for rational drug design strategies, providing therefore the opportunity to optimize existing therapies in a personalized manner.
We will use state-of-the-art computer-assisted drug-design (CADD) combined with machine learning algorithms to identify the most promising druggable binding sites (static and transient) from molecular dynamic computer simulations (Machtens P3). A further analysis of binding sites' coevolution and network profiles will be performed to predict possible allosteric pathways and key residues affecting the gating mechanisms. The aim will be to elaborate a model connecting the druggable region of the channel to the genetic variant-dependent mechanisms regulating the gating. The model will be used to predict how the genetic variants affect the druggability of the channel and, in turn, which site can most effectively be exploited by potential drug candidates to regulate the gating mechanism in each variant. Known NaV1.7 drugs will be first used as internal probes to cross-validate the model. Next, the model will be exploited for in silicovirtual screening to identify a preliminary small library (from 10 to 20 molecules) of potential variants-selective chemical scaffolds to be tested in electrophysiological experiments (P1, P5).The mechanism of action of the successful candidates will be evaluated by computer simulations in order to expand the preliminary library of ligands to a larger data-set of lead candidate molecules.
P05 Biochemical and Functional characterization of heterologously expressed pain-related NaV mutants
PI: Prof. Dr. med. Günther Schmalzing, Institut für Pharmakologie und Toxikologie, RWTH Aachen
CO-PI: Priv.-Doz. Dr. med. Ralf Hausmann, Institut für Klinische Pharmakologie, RWTH Aachen
Chronic pain is a therapeutic challenge. Genetically encoded pain syndromes offer the chance to understand mechanisms of chronic pain on a genetic, cellular and molecular level and former studies could attribute multiple pain disorders to mutations in voltage-gated sodium channels (Dohrn et al., 2019). Human voltage-gated Na+ channels (hNaVs) are membrane proteins responsible for the rising phase of action potentials of excitable cells.
In one part of the project (principal investigator Prof. Dr. med. Günther Schmalzing) we use biochemical techniques to address the expression level, stability, oligomerization and intra-cellular trafficking of recombinant hNaV1.7 and hNaV1.8 alone or together with auxiliary proteins including hβ subunits. These techniques allow addressing effects of point mutations that map to protein regions with known or unknown functions. Together with P1 and P3 of the joint research consortium we aim to analyze the oligomeric structure of hNaV1.7 and hNaV1.8 by a combination of structure-guided targeted mutagenesis, recombinant expression, NaV solubilization and native poly-acrylamide gel electrophoresis (PAGE) (Rühlmann et al., 2019).
In another part of the project (principal investigator Priv.-Doz. Dr. med. Ralf Hausmann) we use an electrophysiological approach that allows us to address whether a particular pain-associated NaV variant affects NaV channel gating and provides novel insights into the biophysics of NaV channels in general and the mechanisms of how NaV mutants induce pain (Kaluza et al., 2018). In addition, single-channel recordings enable us to compare directly single-channel data with atomistic molecular-dynamics simulation data (P3 of the joint research consortium).
Our dual biochemical-functional approach will allow us to address important questions such as whether a particular pain-associated NaV variant affects protein stability, dimerization or single-channel characteristics including coupled gating and activation or inactivation gating.
P06 In-depth extended phenotyping and neuromodulation using matrix stimulation
PI: Univ.-Prof. Dr. med. Roman Rolke, Department of Palliative Medicine, RWTH Aachen
Background: Sodium channels are involved in nerve fibre excitability and thus should affect perception of evoked pain both in modulating peripheral nerve endings as well as spinal signal processing. This project aims to assess the sensory and axonal profiles in patients with pain and sodium channel mutations via psychophysical testing, single nerve fibre recordings and via an intervention via electrical neuromodulation to link the function of specific sodium channel mutations to the pain processing phenotype. Patients with Nav1.7 and other sodium channel mutations with predicted functional relevance will be subject to (1) a sophisticated in-depth phenotyping of the somatosensory profile including newly developed threshold and suprathreshold electrical testing specific for axonal changes (2) olfactory testing and (3) an intervention using a matrix electrode for neuromodulation to assess the impact of sodium channel mutations on spinal pain processing and (4) in selected patients recordings of single action potentials in single nerve fibres.
Assessment aims: All selected subjects for this subject will have already undergone standard phenotyping in the central project (Z-Projekt; Dohrn/Gess) using e.g. quantitative sensory testing (QST) according to the DFNS protocol. For a subgroup of selected subjects, based on genetic testing for sodium channel mutations, electrical stimulation with specialized stimulation paradigms will be used for assessment of axonal excitability of C-fibres as read out for sodium channel function. The olfactory perception will be tested using sniffing sticks to assess stimulus response functions. A-delta fibre function will be objectively examined using pain-related evoked potentials. In selected patients the axonal excitability of single C-nociceptors will be addressed to reveal mutation specific patterns in axonal function using microneurography in co-operation with IZKF group leader PD Dr. Barbara Namer.
Intervention: Using low-frequency stimulation (LFS), all patients will receive an intervention by applying a recently developed matrix electrode with a design preferentially activating skin nociceptors. This approach tests for the impact of sodium channel mutations on spinal sensitization processes using different electrical stimulus paradigms activating specific nerve fibre subgroups. A twice daily 5-min 4 Hz LFS treatment trial describes the intervention based on a n=1 study design.
P07 Cellular and systemic effects of Nav in olfaction
PI: Dr. Markus Rothermel, Institute for Biology II – Dept. Chemosensation – AG Neuromodulation, RWTH Aachen
Loss of function of the voltage-gated sodium channel Nav1.7 causes congenital inability to experience pain in humans. Moreover, Nav1.7 is essential for odor perception in both mice and humans: absence of Nav1.7 in olfactory sensory neurons leads to synaptic signaling failure at the first synapse along the olfactory pathway, the sensory neuron-to-mitral cell synapse in the main olfactory bulb. Therefore, the olfactory bulb emerges as an ideal model system to study Nav 1.7 function. Given the striking analogy to the first synaptic pain processing stage in the spinal cord, we here aim to exploit the unconventional olfactory role of Nav 1.7 for a systematic mechanistic analysis of the channel’s cellular function. In addition to loss-of-function mutations, a variety of gain-of-function mutations for SCN9A have been identified typically causing inherited erythromelalgia, the first human pain syndrome linked to a voltage-gated sodium channel. Therefore, we aim to deter-mine whether and, if so, how such gain-of-function mutations also alter odor perception. While there are contradictory results about olfactory function gain-of-function Nav1.7 mutations in humans, there are, to our knowledge, no reports available investigating the potential influence of a gain-of-function mutation on odor perception in rodent models, which will allow for tight control of genetic variability. Here, we propose to generate novel transgenic mouse models, which will allow for specific Nav1.7 gain-of-function expression in olfactory sensory neurons. Patch-clamp recordings, high-resolution imaging in acute olfactory bulb slices as well as multiphoton in vivo recordings will provide a physiological fingerprint of synaptic signaling at the sensory neuron-to-mitral cell synapse. Furthermore, behavioral assays will test for deficits in odor-guided behaviors such as innate odor recognition and avoidance, short-term odor learning, and maternal pup retrieval. Finally, we aim to investigate the central interactions of nociception and smell and thereby plan to supplement the brain-wide human imaging data generated by Prof. Dr. Ute Habel (P8) with in vivo recordings on a cellular scale. Together, this study is designed to enhance our understanding of Nav1.7 function in sensory signaling, benefitting from the unique molecular and cellular similarities between olfaction and pain.
P08 Central processing of sensory input in patients with neuronal sodium channel mutations
PI: Univ-Prof. Dr. med. Ute Habel, Department of Psychiatry, Psychotherapy and Psychosomatics, RWTH Aachen
Pain research recently focused on the role of the three voltage-gated sodium channels Nav1.7, Nav1.8, and Nav1.9 that are dominantly expressed in peripheral nociceptive neurons, but manifest distinct biophysical properties. However, how the brain translates the peripheral nociceptive signaling is far from clear. This subproject proposes a systematic brain imaging approach that provides insight into which brain structures and functions are significantly involved in pain processing in inherited pain disorders associated with Nav1.7, Nav1.8, and Nav1.9 mutations.
In order to achieve this goal, we will choose pain patients identified in the consortium carrying rare sodium channel mutations (Z-project) on the basis of molecular genetic studies (P2) and clinical examinations (P6). We will adopt a standardized protocol for brain imaging, which has been established by a multi-center neuroimaging network (PING). This systematic brain imaging approach will improve data analysis reproducibility and allows us to compare with brain images obtained from other studies, such as a recent study with healthy subjects where we demonstrated functional networks that reflect pain and olfactory experience. In the present subproject, we further aim to translate this finding into patient samples and investigate the clinical relevance of the networks. Pain disorders with Nav1.7 mutation are of interest since Nav1.7 has also been shown to be the main sodium channel in olfactory sensory neurons and their processes. The noninvasive simultaneous EEG-fMRI recordings will provide both high temporal (electroencephalography, EEG) and spatial resolution (fMRI) of human brain function in responses to pain and odor stimuli. We aim to correlate our results with the clinical findings (P6, Z-project) and the results at genetic and molecular levels (P1 – P5, P7).
Z Phenotyping pain perception: the key to understanding sodium channelopathies
PI: Dr. med. Maike Dohrn, Department of Neurology, RWTH Aachen
Co. PI: Univ.-Prof. Dr. med. Martin Häusler, Department of Pediatric Neurology, RWTH Aachen
This central project opens a gate between clinical and basic research on voltage-gated sodium channel mutations that can cause both excruciating pain or insensitivity, hyper- or anosmia. In the departments of neurology and pediatric neurology, we aim to enroll and characterize affected variant carriers in three steps:
Step 1: We plan to identify 50 pediatric and 200 adult patients overall with peripheral pain disorders that might be related to mutations in voltage-gated sodium channels. For this purpose, we will study two patient groups, e.g. those with an idiopathic or familial neuropathic pain syndrome and those with a reduced pain perception. Our screening program will confirm the neuropathic character of pain and exclude an underlying large fiber degeneration.
Step 2: Candidate probands will undergo detailed clinical examinations including personal and family history, standardized neurological investigations, quantitative sensory testing (QST), pain and quality of life questionnaires, olfactory function assessment, and molecular genetic studies (P2). We expect to identify candidate sodium channel mutations in 10-30% of all pre-screened patients. We will obtain biomaterial such as blood specimens for collaborating projects and enroll mutation carriers into physiological studies such as microneurography (P6) and fMRI (P8). The mutations will be investigated on a molecular and cellular level (P1, P5) and by in silico 3D molecular dynamics modelling (P3, P4).
Step 3: Clinical, molecular genetics, and functional data will be integrated into a prospective longitudinal registry, based on which we will delineate the phenotypic range of sodium channel variants in children and adults by detailed phenotype-genotype correlations. We hereby aim to enable an easy identification of eligible patients for future investigations and an individualized drug development (P4).