FACIAL PAIN RESEARCH FOUNDATION 2015 RESEARCH PROGRESS
Compiled and submitted April, 2016 By Douglas K. Anderson, Ph.D. Eminent Scholar Professor and Chair Emeritus Department of Neuroscience, University of Florida College of Medicine, and Director of Research and Trustee, Facial Pain Research Foundation
Trigeminal Neuralgia (TN), which is considered to be one of the most painful afflictions known to medical practice, is an example of neuropathic pain which is defined as pain caused by injury to or malfunctioning of the nervous system. Despite the debilitating effects chronic pain has on the activities of daily living of individuals dealing with painful conditions like TN and on the economics of health care (greater than $100 billion/year), research funding from traditional sources (i.e., federal agencies and pharmaceutical companies) has been and continues to be inadequate. Thus, for over a decade, there has been the growing realization that if there is to be any real progress in discovering the root cause of and treatment for TN, it is going to have to be accomplished with funds raised from the private sector.
To this end, The Facial Pain Research Foundation (FPRF) was created in January, 2011 to provide the critical elements that are necessary and sufficient to study these facial pain conditions. Accordingly, the sole mission of the FPRF is “…to establish a well-funded translational (i.e., fundamental discovery to clinical application) research continuum that is dedicated to identifying the mechanisms underlying neuropathic facial pain and to develop novel new therapeutic strategies that will permanently stop the pain of TN and related neuropathic pain syndromes”.
Since its inception a little over five years ago, the FPRF has raised $2,962,319 to support five separate and distinct research projects. In funding these projects, the FPRF adhered to three criteria: (1) the projects were novel and unique; (2) the investigator(s) responsible for each project are among the leading researchers in their respective fields; and (3) all of the projects were not “cut from the same cloth”, i.e., there was significant diversification in the research strategies among the projects. It is important to note that these FPRF funds have also served in a multiplier capacity in that some of our investigators have used FPRF support as seed funding to generate the necessary preliminary data which contributed to these investigators acquiring large grants from the National Institutes of Health (NIH). The purpose of this yearly update is to briefly summarize these five projects and some of the progress that each achieved in 2015.
Investigating Protein-Lipid Interactions in Peripheral Nerve Myelin
Lucia Notterpek, Ph.D., Professor and Chair, Department of Neuroscience, University of Florida College of Medicine
The pain of TN is thought to occur when the insulating myelin sheath of the trigeminal nerve is lost or damaged (demyelination) by the pulsations of an overlying blood vessel. Myelin is a multi-layered membrane made up of specific lipids and proteins. Due to its molecular composition, myelin functions as an insulating material in the nervous system (NS). Myelin wraps around many axons in both the peripheral (PNS) and central (CNS) nervous systems forming the myelin sheath which is instrumental in maintaining the normal conduction velocity in nerves. Consequently, an intact myelin sheath is necessary for the proper functioning of the NS. Peripheral myelin is derived from and maintained by Schwann cells. The overall goal of this project is to define specific protein-lipid interactions, with a focus on PMP22, in the establishment and maintenance of lipid membrane domains in peripheral myelin. In the absence of PMP22, myelin is prone to compression-induced damage leading to localized myelin injury. For completing the aims of the project they are using cultured cells and nerves from normal and PMP22-deficient mice and engineered molecular constructs. This project has four specific aims. Progress on each of the aims is summarized below.
Aim 1: Determine if PMP22 is critical for the trafficking and membrane association of lipid raft protein components in myelinated peripheral nerves.
While working on this aim of the project, Dr. Notterpek discovered that in the absence of PMP22, Apolipoprotein E (ApoE) was mislocalized in Schwann cells. As ApoE is critical for cholesterol trafficking, they have focused on other molecules of the lipid pathway, such as Lipin-1 (LPIN1), Low density lipoprotein receptor-related protein 1 (LRP1), and Low density lipoprotein receptor (LDLR). Molecular and biochemical studies indicate abnormal expression of these molecules in nerves of affected mice. They also found reduced ApoE secretion by mouse Schwann cells from PMP22-deficent mice. Dr. Notterpek’s current effort is focused on understanding the specific abnormalities in the protein-lipid secretory pathway in affected Schwann cells and identifying the step in the lipid secretion pathway where PMP22 plays a critical role.
Aim 2: Identify alterations in the lipid and cholesterol content of myelinated peripheral nerves in the absence of PMP22.
In this aim, in collaboration with Drs. Fazio and Tavori at OHSU, Dr. Notterpek’s team is analyzing nerves of normal and PMP22-deficent mice for lipids, including cholesterol, phospholipid, and triglyceride. Using histological methods, they have also identified irregular and clumped lipid distribution in the sciatic nerves of PMP22-deficient mice, including polar lipids (phospholipids) and neutral lipids (cholesterol, lipoproteins and triglycerides). An unexpected finding from this study was that the liver of PMP22-deficient mice accumulates lipids which may contribute to systemic myelin abnormalities and the progression of neuropathies.
Aim 3: Determine if the addition of exogenous cholesterol can restore functional abnormalities of PMP22-deficient Schwann cells in culture.
To begin to test cholesterol uptake mechanism in the absence of PMP22, Dr. Notterpek’s group performed an LDL uptake assay in cultured cells. Low-density lipoproteins (LDL) are the major carrier of cholesterol in the blood. As LDL is taken up by hepatic and neural tissues through LDL receptor-mediated endocytosis, it is internalized and degraded to free cholesterol and amino acids. Dr. Notterpek has indicated that these experiments are being optimized and repeated as they are important for delineating the contribution of intracellular Schwann cell-derived, and circulatory-origin, cholesterol to the observed lipid abnormalities in myelin.
Aim 4: Test if lentivirus-mediated expression of Wt-PMP22 but not CRAC-domain mutated PMP22, will rescue the myelination abnormalities in PMP22-deficient Schwann cells.
Lipids, including cholesterol move around within cells by being bound to proteins. PMP22 is an extremely hydrophobic protein and have specific motifs, called CRAC domains that in other molecules is known to bind cholesterol and assure the delivery of cholesterol to the myelin membrane. Currently it is unknown what proteins are responsible delivering cholesterol to peripheral myelin. To investigate this problem, Dr. Notterpek’s team has mutated the CRAC domain (Y153A) and the inverted highly-conserved CARC motif at aa 92-101 of PMP22, which abolishes their ability to bind cholesterol. If these domains are critical for cholesterol transport in Schwann cells then expression of the mutated forms will lead to abnormal cholesterol localization in transfected cells. Currently they are optimizing the transfection procedures so that the constructs can be analyzed.
Results reported in the following publications were supported in part by FPRF funds:
(1) Lee S, Amici S, Tavori H, Zeng WM, Freeland S, Fazio S, Notterpek L. PMP22 Is Critical for Actin-Mediated Cellular Functions and for Establishing Lipid Rafts. Journal Neuroscience 2014; 34(48):16140-16152. (Cover Article)
(2) The graduate student (Ye Zhou) working on this project just presented a poster at the American Neurochemistry meeting.
Ye Zhou, Alexander I Fethiere, Karla Arrendondo, Hagai Tavori, Sergio Fazio and Lucia Notterpek: Lipid abnormalities in mice deficient in peripheral myelin protein 22
(3) Dr. Notterpek has just completed an invited review paper for Journal of Experimental Neurology where the FPRF is acknowledged.
(4) Ye Zhou and Lucia Notterpek (2016) Promoting peripheral myelin repair. Corresponding Author – Lucia Notterpek (invited review for special edition on myelin).
Mapping Towards a Cure: Identification of Neurophysiologic Signatures of Trigeminal Neuralgia Pain
John K. Neubert, D.D.S., Ph.D., University of Florida (Project coordinator, animal modeling)
Mingzhou Ding, Ph.D., University of Florida (Human imaging)
Marcelo Febo, Ph.D., University of Florida (Animal imaging)
Robert M. Caudle, Ph.D., University of Florida (Therapeutics)
Andrew H. Ahn, M.D., Ph.D., Eli Lilly Company, IN (Consultant)
John K, Neubert, D.D.S.,Ph.D. is a recognized expert in orofacial pain with a specific emphasis on the causes of and treatments for trigeminal neuralgia (TN). He is an associate professor in the Department of Orthodontics at the University Of Florida College of Dentistry (UFCOD). He also has an adjunct appointment in the Department of Neuroscience at the UFCOM and is on the faculty of the Evelyn F. and William L. McKnight Brain Institute of the University Of Florida (MBI). He has assembled an impressive team of co-investigators who will focus on identifying the neurophysiological signature of TN pain. To this end, Dr. Neubert planned and submitted to the FPRF for funding an ambitious project he titled “Mapping Towards a Cure: Identification of Neurophysiological Signatures of Trigeminal Neuralgia (TN) Pain.”
This complex project is designed to investigate the cause of TN in both animals (rats) and humans. The purpose of the initial component (Phase 1) of this translational study is to determine if the neurobiology of TN in rats replicates or is very similar to the neurobiology of TN in humans. Using state of the art magnetic resonance (MR) scanners, the UF team will identify and compare the individual imaging patterns caused by the activation of different areas in the brain and spinal cord in both TN patients and rats, i.e., specific areas of the brain and spinal cord that will be activated by TN pain are the so called “signature” centers of activity. Normally, a translational study is done sequentially with the animal studies preceding the human trials. However, the intellectual and technical power available at the MBI allows Dr. Neubert to run his animal and human studies simultaneously which will markedly reduce the time it takes to complete the Phase 1 component of this project. If similar imaging/activation patterns are seen in both rats and humans, then large numbers of compounds and perhaps other promising or novel therapeutic approaches can be screened in rats with the best of the best selected for inclusion in clinical trials (Phase 2). If the activation patterns are substantially different in these two species, then it becomes necessary to image additional subjects in order to strengthen the statistical power by acquiring a larger sample size. Figure 1 is an example of the type of data generated by MR scanning. In 2015, Dr Neubert’s team made significant progress towards completing the goals laid out in Phase 1.
The importance of identifying the brainstem nerve tracts responsible for activating cortical areas in TN patients cannot be overstated. Discovery of the relevant trigeminal nerve targets provides regions for treatments that can specifically block the pain of TN and eliminate or limit unwanted side effects because the treatment can be delivered locally and not systemically. Dr. Neubert’s team of investigators already has access to existing therapies that they speculate will be successful in blocking TN pain by partnering with another internationally recognized and respected investigator at UF, Dr. Todd Golde, the Director of the UF Center for Translational Research in Neurodegenerative Disease. In collaboration with Dr. Golde’s research team, they hope that shortly they will be initiating an exciting set of studies looking at therapies aimed specifically at treating trigeminal pain.
Table 1. Recruitment history of subjects.
Unable to participate
Originally Dr. Neubert proposed to scan 5-10 subjects for Phase 1 and there was some concern that he would not be able to reach this goal on his first attempt. However, due to a surprisingly large patient response, they have completed scans on eight subjects with another nine subjects being scheduled for scans. They have gained access to a large number (18) of potential subjects who have contacted Dr. Neubert but have not yet committed to having an MRI scan. Not surprising, due to the dedication of the patients suffering from TN, only 10% of subjects said they could not participate – this was mainly due to travel limitations. They were unable to reach approximately 45% of subjects that initially responded to their advertising; they are continuing to follow-up with these individuals and they encourage potential subjects to do the same with them. They have 9 subjects that are scheduled for scans in the near future.
The following images (Figure 1) show a subject’s brain activity during the scan. It is an example of one of the subjects scanned who had a spontaneous painful episode while in the scanner. The warm colors indicate that the part of the brain activates when there is more pain. This may suggest that the subject is trying to suppress the pain and cope with it in some way when pain goes up. Due to the complexity of the data set, Dr. Neubert’s team is still analyzing the results from the remaining eight subjects
Dr. Neubert’s group has completed approximately 25 high resolution fMRI scans of rats with various orofacial pain conditions. Due to the amount of data generated and complexity of the data set, they are in the progress of analyzing these scans. In a pilot study with Dr. Golde’s group, they have also used a fluorescently-labelled vector to demonstrate that they can specifically target the trigeminal system. This is important because they are confident that they have a strategy for treating trigeminal disorders, a finding they hope will allow them to enter the Phase 2 treatment studies sooner than initially thought (see below).
Limitations: During the first year of this project Dr. Neubert’s team had issues regarding subject enrollment and staffing difficulties. They had approximately a 20% recruitment rate which indicated that they needed to increase enrollment because only about 10-20% of subjects will have a spontaneous painful episode while in the scanner. They have hired a research assistant to coordinate recruitment and have also been working with Dr. Michael Pasternak of the FPRF to help spread the word that additional subjects are needed. The FPRF has generously provided additional funding so that they can hire a full-time post-doctoral fellow to be dedicated solely to this project.
Plans for year 2:
- Continuing the Phase 1 study and recruit more subjects to scan
- Study 50-100 animals using different trigeminal injury models
- Evaluate compounds from Dr. Golde’s lab
- Test the animals using Tegretol. They recognize that this is probably everyone’s least favorite drug, but it is still effective and therefore it is necessary for them to use in their animal models as validation of the TN model.
Cell Replacement Therapy as a Treatment for Injury-Induced Neuropathic Pain
Allan Basbaum, Ph.D., FRS, Professor and Chair, Department of Anatomy, University of California San Francisco
One of the primary research tactics targeted and supported by the Facial Pain Research Foundation (FPRF) since its inception has been the development of a unique therapeutic approach which involves transplanting cells that produce a pain inhibitory neurotransmitter that can suppress the devastating pain of trigeminal neuralgia (TN) pain. The chief proponent of this approach has been and continues to be Dr. Allan Basbaum at the University California San Francisco. The following report highlights the studies carried out in his laboratory in 2015 that demonstrate how cell replacement therapy might one day be used not only to quell some common types of persistent and difficult-to-treat pain, but also to cure the conditions that give rise to them.
Dr. Basbaum’s preclinical studies using mice are directed at treating neuropathic pain. In a previous report Dr. Basbaum described his novel approach of transplanting immature precursor cells that produce a pain inhibitory transmitter from the mouse brain into the spinal cord of animals that experience pain hypersensitivity following partial nerve damage. The cells integrate into the spinal cord and serve to repair damaged circuits that characterize many neuropathic pain conditions. Comparable dysfunctional circuits likely are also major contributors to the terrible pain that is TN. One of the major questions addressed in the previous year’s studies is whether the transplanted cells function as a sophisticated pump that simply releases the inhibitory neurotransmitter randomly without a specific target or whether the grafted cells integrate and make synaptic connections with host neurons which would permit a more precise inhibitory control of pain transmission neuronal circuitry. This is an important question because if the neurotransmitter is released randomly into the milieu of the target cells, adverse side effects are more likely to occur than if the grafted cells make synaptic contact with the target cells. To answer this question, Dr. Basbaum used electrophysiological techniques to assess the functionality of the transplanted neurons and high power electron microscopy to visualize the subcellular anatomy of the transplanted cells. The transplanted cells also carried specialized markers which allowed Dr. Basbaum to identify the grafted cells and differentiate them from the host neurons. These experiments revealed a remarkable development of new synaptic connections on host neurons coming from the grafted cells. These synapses could be activated by the grafted cells which, importantly, are profoundly inhibitory, thus regulating the activity of the host nerve cells. In the coming year Dr. Basbaum will attempt to regulate pain that is associated with the trigeminal region, not only by transplanting cells into the region of the trigeminal spinal nucleus responsible for the transmission of painful sensations from the head to the brain, but also into the trigeminal ganglion itself. Although synapses are not normally present in the ganglion, the nerve cells in the ganglion express the receptors that can be accessed by the inhibitory neurotransmitter released from the transplanted cells. As targeting the ganglion is a neurosurgical procedure presently used for the management of trigeminal neuralgia, if successful, this innovative approach will introduce a new opportunity for eventual translation to the clinic.
Finally, it is significant that a new company, Neurona, is beginning to develop these transplant approaches for the management of different neurological conditions that arise from loss of inhibition. The company’s initial objective is to treat individuals with very difficult to control seizures (epilepsy), however, chronic neuropathic pain conditions are the company’s next indication. Needless to say there will be several hurdles to cross during development, but this is exciting news and we will keep you posted as progress occurs.
“The William H. and Leila A. Cilker Genetics Research Program to Find a Cure for Trigeminal Neuralgia” (Finding the Genes that Predispose to Trigeminal Neuralgia)
Marshall Devor, Ph.D. (Project Coordinator) Professor and Chair, Department of Cell and Animal Biology, Institute of Life Sciences, Hebrew University of Jerusalem
Kim J. Burchiel, M.D. (Phenotyping and Sample Collection) Professor and Chair, Department of Neurosurgery, Oregon Health and Science University
Ze’ev Seltzer, Ph.D. (Genomics) Professor of Pain Genetics, Faculty of Dentistry, Professor of Physiology, Faculty of Medicine, University of Toronto
Scott R. Diehl, Ph.D. (Genomics Consultant) Professor, Department of Oral Biology, Rutgers School of Dental Medicine; Professor of Health Informatics, Rutgers School of Health Related Professions, Rutgers University
In recognition of their generous $500,000 matching grant to fund this project, the Trustees of the FPRF have re-named the project previously titled “Finding the Genes that Predispose to Trigeminal Neuralgia” “The William H. and Leila A. Cilker Genetics Research Program to Find a Cure for Trigeminal Neuralgia”.
The remarkable progress made in genetic analysis in the past two decades of the Human Genome Project has greatly enhanced our capacity to discover genes associated with different diseases, to reveal their function, and to develop therapeutic and/or preventive approaches precisely targeted at the specific disease. This project asks the question: Are some individuals genetically predisposed to develop trigeminal neuralgia (TN) pain? This question emanates from research which shows that although 17% of mature adults have a TN lesion (usually a vascular compression of the trigeminal nerve close to where it exits the brain) only a very small minority (0.01%) of these individuals actually experience TN pain. This disconnect between lesion and pain is an important clue suggesting that the development of pain is caused by a genetic predisposition expressed in this limited subset of individuals. Other research shows that more than one third of patients with TN do not have vascular compression of the nerve, and the rare patients with bilateral TN do not generally have vascular compression of the nerve on both sides! Further, it appears that younger patients (less than 35 years of age) are four times more likely to be women than men. All of these new findings point to the possibility of a genetic predisposition for TN.
This project was conceived with the assumption that the risk for having classical TN has a genetic basis and the study’s primary objective is to identify genetic mutation(s) (aka, sequence variant(s) or polymorphism(s)) associated with the development of TN pain. To achieve this objective, DNA has now been collected from over 600 TN patients. Analysis of the whole genome is currently being carried out on 500 of these patients using the latest in genetic sequencing technology and will be compared to matched reference controls to determine if there are candidate sequence variant(s) or polymorphism(s) present in the genome of the TN patients that are not found in the controls. Such sequence variants might cause TN pain by directly affecting the function of the proteins that these genes encode, or by altering other aspects of gene expression. Either way, assuming that genetics is indeed the basis for developing TN, there is a high probability that this project will reveal the genes and ultimately the pathophysiological mechanisms that cause certain of these lesions to be painful. With this knowledge molecular targets for treatments that will cure the pain can be identified not only for TN but perhaps also for a variety of other facial and segmental neuropathic pain conditions.
To date, nine centers, including two international centers, have actively acquired DNA samples via saliva in patients with TN1. The members of this research team have achieved their original goal of having in hand, 500 saliva samples by the end of 2015 and now all 500 samples are being sequenced and analyzed. While it is far too early to identify a gene(s) causally related to the genesis of TN pain, the sequencing procedure appears to be reliable and reproducible.
Given the goals that have been achieved thus far, this team of investigators has put forward a strong case for doubling the number of saliva samples collected from 500 to 1000. Such an increase in sample size would substantially enhance the power of this study in addition to strengthening the reliability of any conclusions drawn. They project that collection of an additional 500 samples could be completed by spring 2017 which appears to be an attainable goal as to date; over 600 samples have already been collected.
Novel Ways to Deliver Compounds That Can Eliminate the Pain of Trigeminal Neuralgia
Wolfgang Liedtke M.D., Ph.D., Professor, Department of Neurology, Duke University
Early in 2015, the FPRF was introduced to Dr. Wolfgang Liedtke, a remarkable clinician/scientist at Duke University with a strong interest in facial pain. Dr. Liedtke is a tenured Professor of Neurology who has built a large clinical practice in head-face pain at the Duke Neurology Clinics. He has additional appointments in Anesthesiology and Neurobiology and has received training in molecular neuroscience and physiology. He has NIH funding to support studies ongoing in his laboratory at Duke. He was the first to describe the pain-relevant TRPV4 ion channel in 2000, and has been selected a Klingenstein Fellow in neurosciences (2004), and a Harrington Discovery Institute Scholar Innovator in translational medicine (2012). He has authored/co-authored more than 100 peer-reviewed articles, many of them relating to pain, inflammation and tissue injury. Dr. Liedtke being aware of the inadequacies of the current ways (primarily systemic) drugs are given to individuals suffering with Trigeminal Neuralgia (TN) pain approached the FPRF for funds to develop novel techniques for direct anatomic targeting of specific components of the trigeminal system. The primary advantage of this approach is with delivery of the drug directly to the lesioned area, only a small fraction of the normal systemic dose of the drug will be needed thereby eliminating the unwanted side effects associated with systemic or whole body delivery of the drug. Dr. Liedtke’s proposal was reviewed by two knowledgeable scientists who recommended to the Board of Trustees (BOT) that Dr. Liedtke receive a one year $50,000 seed grant to kick start this program. The BOT approved the scientific recommendation and authorized the distribution of funds to Dr.Liedtke. The following is a brief description of his proposal.
To develop his unique delivery system, Dr Liedtke proposes to meld material science with cell engineering to create a device that will provide focused delivery of molecules or drugs that have previously shown the capacity to block or eliminate pain in other models of pain or in more primitive models of TN pain, Thus, the objective of this approach is to deliver the appropriate molecules to shut down transmission of the aberrant neuronal signals arising from injured site(s) on the trigeminal system that are responsible for producing the pain of TN. The devices are designed to be placed around peripheral trigeminal nerve branches and around the trigeminal nerve root at the point where this root exits the brainstem. The experiments described in this proposal are based on previous pioneering work by Dr. Liedtke and collaborators using a specifically-fabricated type of carbon nanomaterial, i.e., a few-walled carbon nanotubes with high electrical conductance that has been highly purified to the point that it is devoid of any neurotoxic contaminating by-products. This particular type of carbon nanomaterial is known to lower neuronal chloride ion levels by stimulating increased gene expression of KCC2, a chloride ion extruder transport molecule. Lowering intracellular chloride levels in CNS neurons will amplify the inhibitory capacity of the inhibitory neurotransmitters, GABA and glycine. In an earlier study, Dr. Liedtke and co-workers applied these ultra-pure, few-walled carbon nanotubes to cultured cortical neurons and showed that direct contact of the carbon fibers with the cultured neurons in increased levels of KCC2 in these cells causing an enhanced efflux of chloride from them. Thus, in addition to serving as a topical drug delivery vehicle, the carbon nanotubes themselves may have pain inhibiting properties.
Dr. Liedtke proposes to develop another kind of topical drug delivery vehicle. This device will be will be made of immunologically inert biocompatible polymers designed to hold human fibroblast-like cells. Using gene therapy techniques, fibroblasts (which are readily obtainable from skin) can be made to produce pain inhibiting substances like GABA or glycine effectively turning them into anti-pain generating pumps. The biocompatible polymer devices will prevent the cells from dispersing but will be made sufficiently porous to allow the pain inhibiting molecules to escape. These projects while somewhat futuristic are not science fiction and should be doable. They definitely are needed.