Facial Pain Research Foundation 2017 Research Progress

Compiled and submitted April, 2018 by Douglas K. Anderson, Ph.D., Eminent Scholar Professor and Chair Emeritus, Department of Neuroscience, University of Florida College of Medicine; Director of Research and Trustee, Facial Pain Research Foundation

Trigeminal Neuralgia (TN), is a prime example of neuropathic pain which is pain caused by injury to or malfunctioning of the nervous system. TN is considered to be one of the most painful afflictions known to humans with debilitating effects on the activities of daily living of individuals with this painful condition. The cost neuropathic conditions like TN adds to the U.S. health care system is greater than $100 billion/year yet funding for research from federal agencies, primarily the National Institutes of Health (NIH) and pharmaceutical companies continues to be woefully inadequate. The meagerness of funding from traditional sources has led the growing realization that discovering the root cause of and treatment for TN and other neuropathic pain conditions is going to have to be accomplished with funding 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. Consequently, 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 six years ago, the FPRF has raised over $4 million dollars to support six separate and distinct research projects. The FPRF uses three fundamental criteria in choosing which proposals are to be considered for funding: (1) the projects are 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 are not “cut from the same cloth”, i.e., there is 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 NIH and other foundations and, in some cases, the acquisition of venture capital. Total dollars from these other sources that came to our consortium of investigators emanating, all or in part, from FPRF seed funds, is estimated to be somewhere over $30,000,000. The purpose of this yearly update is to briefly summarize these six projects and to highlight the progress that each achieved in 2017.

 

Investigating Protein-Lipid Interactions in Peripheral Nerve Myelin

Lucia Notterpek, Ph.D., Professor and Chair Department of Neuroscience University of Florida College of Medicine

Progress report (Year 3): May 2017-April 2018

Project 1: Cholesterol homeostasis in peripheral nerve myelin

Study staff: Ye Zhou, MS, graduate student

With Ye Zhou, a graduate student in Dr. Notterpek’s laboratory leading the way this group continued to make excellent progress on this project during the past year. They gave presentations based on the results at several meetings. Also they have submitted a manuscript titled “Peripheral myelin protein 22 is necessary for ABCA1-mediated cholesterol efflux’’ to PNAS Plus. However this article as written was not accepted for publication. Consequently they are in the process of adding an additional data set to this manuscript and shortly will submit the revised paper to an alternative high impact journal.

Recent publications in the literature along with data from Dr. Notterpek’s lab continue to indicate that cholesterol homeostasis is critical for myelin stability in both the central (CNS) and peripheral (PNS) nervous systems. They are focusing on mechanisms that involve cholesterol trafficking within the cells and cholesterol efflux from myelin-forming cells, including the potential exchange of cholesterol between the nerve and Schwann cells. Their studies indicate that the concentration and localization of cholesterol is tightly regulated within myelin forming cells, as in the absence of proteins that are involved in this process, both mice and humans develop neuropathies. They are also working on understanding how the correct amount of cholesterol is maintained in the myelin membrane, which is critical for membrane stability. Peripheral myelin protein 22 (PMP22), whose haploin sufficiency is linked with a compression induced neuropathy (that often is painful), is critical and necessary for assuring cholesterol transport to the membrane. Utilizing neuropathic mice with PMP22 deletion and point mutation, they discovered that PMP22 regulates cholesterol homeostasis and trafficking via the interplay with ABCA1, the major cholesterol efflux transporter. Considering the positive effects of drugs that facilitate cholesterol transport such as 2-hydroxypropyl-β-cyclodextrin (HβCD) in CNS myelination, along with the likely interaction between PMP22 and cholesterol (see below), they treated nerve explants from PMP22-deficient neuropathic mice with HβCD and found improvements in the myelination capacity of the affected cells in response to drug treatment, as compared to untreated samples. These studies will need to be repeated, but are leading us them toward potential therapeutic approaches on how abnormal levels of cholesterol in myelin could be corrected.

In another set of cellular/molecular studies, they have now identified the exact amino acid motif in PMP22 that likely binds cholesterol and are in the process of finalizing this study for publication. Related to this aspect of the project, Dr. Notterpek is in discussion with a membrane biophysicist at Vanderbilt University who has an assay to monitor the incorporation of normal and cholesterol-binding mutated PMP22 into lipid micelles and are working toward coordinating and/or combining their findings with his for publication purposes. Working with another University of Florida scientist, Dr. Habibeh Khosbouei, they discovered that critical properties of the glial membrane, such as membrane resistance and capacitance, change when the levels of cholesterol are abnormal, e.g., as occurs when PMP22 is deficient. To the best of their knowledge, no one else has performed similar single cell electrophysiological recordings of Schwann cells causing great excitement in the lab because understanding how abnormal membrane-associated cholesterol levels alter the ability of myelin to protect the axon and guide membrane depolarization (neuronal activity), are critical and fundamental aspects of neurobiology. Moreover, Dr. Notterpek’s group is continuing their investigation of the impact of dietary cholesterol on peripheral myelin health using mice with different genetic backgrounds, and discovering strong impact of genotype and gender on the response of the animals to a high fat diet. These studies are at various stages of sample and data analysis which when completed will serve as the foundation for future grant applications to the NIH.

Presentations in poster format

Zhou Y, Lee S, Miles J, Tavori H, Fazio S, Notterpek L. “A novel interaction between PMP22 and ABCA1 in regulating cholesterol trafficking”. This was presented at the 2018 Neurochemistry meeting.

Bazick H, Zhou Y, Salihi Mohammed, Fritz R, Tavori H, Fazio S, Notterpek L. “Investigating the impact of high fat diet-induced dyslipidemia on neuropathic mice.” This will be presented at the 2018 peripheral nerve society meeting

Budget expenditures:

We have about $50,000 left of the total budget for this project, which I would like to extend into the 4th year to allow for Ye to finish her studies and graduate. Dr. Notterpek’s focus will have to be on getting this project funded from the NIH.

Project 2: Cholesterol homeostasis in peripheral nerve myelin, with a focus on statins

Year 1: May 2017-April 2018

Mohammed Al Salihi, MD, PhD, postdoctoral fellow is key investigator on this project

Myelin, a lipid-rich multilayered membrane made by oligodendrocytes (in CNS) or Schwann cell (in PNS), insulates axons and facilitates the transduction of neuronal signals (1). In the brain, cholesterol contained in myelin accounts for about 80% of total brain cholesterol with approximately 25% of the unesterified cholesterol of an entire adult mouse brain located in myelin, Cholesterol, therefore, plays an important role in the nervous system and is essential for brain and neuronal function (2). Statins are lipid lowering agents that are used in the treatment of dyslipidemia (abnormally elevated cholesterol or fats lipids in the blood) that contributes to the development of atherosclerosis. Thus, statins are used to reduce the risks of cardiovascular diseases due to atherosclerosis (3). According to the Centers for Disease Control and Prevention (CDC) and National Center for Health Statistics (NCHS), the usage of statins has increased from 16.3% to 23.2% between the years 2003 to 2012 in adults aged 40 and over, with Simvastatin being the most commonly used cholesterol lowering medication. The primary mechanism of action of statins is the inhibition of the HMG-CoA reductase enzyme, which is the rate limiting step in cholesterol biosynthesis (6). Despite the fact that statins have similar mechanism of action and the same effects on cholesterol profiles, they can be divided into 2 categories: Type 1, which are fungal derived statins, like Lovastatin, Pravastatin and Simvastatin; and Type 2, which include synthetically derived statins like Atorvastatin and Fluvastatin (3). There is a controversy regarding the effect of statins on peripheral nerves. Currently it is debated whether statins alleviate or aggravate neuropathic pain, and/or whether they cause the neuropathic pain. With these largely clinical observations as background, Dr. Nottterpek has developed the hypothesis that in certain individuals, statins alter the stability of myelin making it susceptible to a localized compression-induced demyelination with a subsequent painful neuropathy.

In order to test this hypothesis, they chose statins from each group and are testing their effects on cultured Schwann cells. Their results demonstrated that regarding cellular toxicity, the cells were still viable after 24h exposure to 500 uM of statins, however, morphologically they became rounded and started to lift up from the plate. In collaboration with Dr Khoshbouei’s laboratory, they were able to show that inhibiting cholesterol synthesis by statins altered the Schwann cell membrane electrical resistance, this effect being statistically significant even at low doses such as 1 and 2 μM. These studies are very novel and will likely be controversial. Nonetheless, this approach allows them to examine the impact of cholesterol lowering drugs on Schwann cell biology at a cellular detail that was previously not possible.

Dr. Al Salihi is also testing the effect of statins on myelination of neurites in vitro and has found that statins lead to the fragmentation of myelin basic protein-positive myelin segments, an effect that is reproducible. In statin treated cultures, they also detected pronounced changes in the expression of various proteins related to myelination and are now trying to understand the mechanism for these effects. Notably, when HMGCoA is inhibited by statins, some cholesterol transport proteins go down, while the levels of PMP22 robustly increase. Although at present they do not understand the mechanism for this effect, the data supports our overall hypothesis that PMP22 is critical in the maintenance of lipid homeostasis in myelin-forming glial cells.

As an extension to this work, in the animal models they are “walking back” from the peripheral nerves into the CNS and will begin to examine how the neuronal cell bodies and surrounding CNS microenvironment may sense/respond to ongoing chronic peripheral myelin defects. In summary, Dr. Al Salihi is having had a good start to his postdoctoral training, he learned many of the techniques used in the laboratory and he is continuing to read and learn the literature.

Budget expenditures:

We have used about $110,000 of the total $360,000 budget (with $180,000 from FPRF and $180,000 from MBI over 3 years) for this project. The expenditures include salary and supplies.

References:

  1. Snaidero N, Mobius W, Czopka T, Hekking LH, Mathisen C, Verkleij D, et al. Myelin membrane wrapping of CNS axons by PI(3,4,5)P3-dependent polarized growth at the inner tongue. Cell. 2014;156(1-2):277-90.
  2. Saher G, Stumpf SK. Cholesterol in myelin biogenesis and hypomyelinating disorders. Biochim Biophys Acta. 2015;1851(8):1083-94
  3. McFarland AJ, Anoopkumar-Dukie S, Arora DS, Grant GD, McDermott CM, Perkins AV, et al. Molecular mechanisms underlying the effects of statins in the central nervous system. Int J Mol Sci. 2014;15(11):20607-37
  4. Ovbiagele B, Schwamm LH, Smith EE, Hernandez AF, Olson DM, Pan W, et al. Recent nationwide trends in discharge statin treatment of hospitalized patients with stroke. Stroke. 2010;41(7):1508-13
  5. Qiuping Gu et al. Prescription Cholesterol-lowering Medication Use in Adults Aged 40 and Over: United States, 2003–2012. NCHC Data Brief. 2014. No. 177
  6. Schachter M. Chemical, pharmacokinetic and pharmacodynamic properties of statins: an update. Fundam Clin Pharmacol. 2005;19(1):117-25

Mapping Towards a Cure: Identification of Neurophysiologic Signatures of Trigeminal Neuralgia Pain

John K. Neubert, D.D.S., Ph.D. (Project coordinator, animal modeling)

Mingzhou Ding, Ph.D. (Human imaging)

Marcelo Febo, Ph.D. (Animal imaging)

Robert M. Caudle, Ph.D. (Therapeutics)

Todd Golde, M.D. (Gene Therapy)

University of Florida

This team of outstanding investigators at the University of Florida (UF) Colleges of Dentistry, Medicine, and Engineering and Evelyn F. and William L. McKnight Brain Institute (MBI) under the guidance of Dr. John Neubert continues to focus on identifying the neurophysiological signature of TN pain and providing promising new therapies. This group represents expertise in facial pain, functional imaging, molecular biology, pharmacology, and viral vectors.

This integrated project is investigating the cause of TN in both animals (rats) and humans. The purpose of this translational study is to determine if trigeminal injury models in rats replicates or is very similar to the neurobiology of TN in humans. Using state of the art magnetic resonance (MR) scanners and behavioral testing equipment, 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. These specific areas of the brain and spinal cord are called “signature centers of activity” that are activated by TN pain and present as targets for the new therapies their team is developing. In 2017, Dr. Neubert’s team made significant progress towards completing the goals of this project.

Identifying trigeminal nerve signaling within brainstem nerve tracts and brain regions is of utmost importance, as this will lead to novel therapies directed to relevant trigeminal targets designed to block TN pain. Dr. Neubert’s team of investigators already has access to existing therapies that they speculate will be successful in blocking TN pain and they evaluated some of these therapeutics this year (see below in Animal Studies). The human studies have identified brain regions important for both the genesis and processing of pain following a TN episode. Importantly, they have found critical brain regions that interact together in a network describe sites, interaction of networks, and inflammation.

HUMAN STUDIES

TN       Control     TN with surgery     TN without surgery     TNI       TNII      TNI/TNII

28         3                12                               16                                     17           3             8

Recruitment (Table 1)

Twenty-nine TN patients (17 females) and three age and sex matched controls have been recruited and analyzed to date. One patient had surgery and no longer felt pain and was not scanned and 6 new subjects have been scanned but not analyzed yet and are not included in this report.

 

Brain Activations during Trigeminal Pain