FACIAL PAIN RESEARCH FOUNDATION 2018 RESEARCH PROGRESS

Compiled and submitted April, 2019 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), is a prime example of neuropathic pain which is pain caused by inmalfunctioning of the nervous system and is considered to be one of the most painful disease conditions known to humans. In addition to the debilitating effects on the lives of those afflicted with neuropathic pain, the cost disorders like TN adds to the U.S. health care system exceeds $100 billion/year. Yet despite the very obvious escalating and pain fueled humanitarian and economic crises, funding targeted at discovering the basic molecular mechanisms underlying neuropathic pain conditions from traditional federal agencies, continues to be inadequate.

The meagerness of funding from these 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 eight years ago, the FPRF has raised millions of dollars to support eight 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) 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 jury to or 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 other sources that came to our consortium of investigators emanating, all or in part, from FPRF seed funds, is estimated to be somewhere over $40,000,000. The purpose of this yearly update is to briefly summarize these six projects and to highlight the progress that each achieved in 2018. I have also provided summaries of two new FPRF research projects beginning this Summer 2019.

Investigating Protein-Lipid Interactions in Peripheral Nerve Myelin

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

Progress report (Year 4): May 2018-April 2019

Project 1: Cholesterol homeostasis in peripheral nerve myelin Study staff: Ye Zhou, MS, graduate student

We made excellent progress on this project during the past year, with Ye Zhou the graduate student leading the work in the lab. Because of the great progress, Ye has successfully defended her PhD in March 2019, and will be graduating in May 2019. At the bottom of the progress report, I list publications related to this work that are now in press, or under review.

As described previously in my progress reports, the absence of functional peripheral myelin protein 22 (PMP22) is associated with shortened lifespan in rodents, and severe peripheral nerve myelin abnormalities in several species including humans. Schwann cells and peripheral nerves from PMP22 knockout (KO) mice show deranged cholesterol distribution and aberrant lipid raft morphology, supporting an unrecognized role for PMP22 in cellular lipid metabolism. To examine the mechanisms underlying these abnormalities, we studied Schwann cells and nerves from male and female PMP22 KO mice. Whole-cell current clamp recordings in cultured Schwann cells revealed increased membrane capacitance and decreased membrane resistance in the absence of PMP22, which was consistent with a reduction in membrane cholesterol. Nerves from PMP22-deficient mice contained abnormal lipid droplets, with the levels of proteins involved in cholesterol transport, apolipoprotein E (apoE) and ATP-binding cassette transporter A1 (ABCA1), highly upregulated. Despite the upregulation of ABCA1 and apoE, the absence of PMP22 resulted in reduced localization of the cholesterol transporter at the cell membrane and diminished secretion of apoE-cholesterol. In nerves from normal mice, we identified overlapping distribution of PMP22 and ABCA1 at the Schwann cell plasma membrane. Together, these results reveal a novel role for PMP22 in regulating lipid metabolism and cholesterol trafficking through functional interaction with the cholesterol efflux regulatory protein, ABCA1.

Significance of the described results (lay terms):

Understanding the subcellular events that underlie abnormal myelin formation is critical for advancing therapy development for neuropathies. PMP22 is an essential peripheral myelin protein, as its genetic abnormalities account for approximately 80% of hereditary neuropathies. Here we demonstrated that in the absence of PMP22, the cellular and electrophysiological properties of the Schwann cells plasma membrane are altered, and cholesterol trafficking and lipid homeostasis are perturbed. The molecular mechanisms for these abnormalities involve a functional interplay between PMP22, cholesterol, apoE and the major cholesterol-efflux transporter protein ABCA1. These findings establish a critical role for PMP22 in the maintenance of cholesterol homeostasis in peripheral nerves and provide new knowledge for efforts toward therapy development.

Project 2: Cholesterol homeostasis in peripheral nerve myelin, with a focus on statins Year 12: May 2018-April 2019

Mohammed Al Salihi, MD, PhD, postdoctoral fellow was the key investigator on this project, but he resigned in March 2019, and returned to training in medicine. I will be hiring a new person shortly.

Statins are lipid-lowering agents that are used in the treatment of dyslipidemia by the inhibition of the HMG-CoA reductase enzyme, which is the rate-limiting step in cellular cholesterol biosynthesis. The influence of statins on the nervous system is controversial, particularly concerning peripheral nerves. Currently it is debated whether statins alleviate or aggravate neuropathic pain, and/or whether they cause the neuropathic pain. Based on our investigations of lipid metabolism in peripheral nerves, and these mentioned clinical observations, we hypothesize that in certain individuals, statins alter the stability of myelin making it susceptible to localized compression-induced demyelination with a subsequent painful neuropathy. Dr. Al Salihi has been testing the effects of statins on Schwann cells and on myelination of neurites in vitro, and has found that statins lead to myelin fragmentation. With Mohammed leaving the lab, we are now repeating these results and expanding the studies to more in depth analyses of myelin and Schwann cells viability. Related to the statin project, Ye is exploring the influence of exogenous cholesterol supplementation on myelinating cultures from heterozygous PMP22 knock out mice, which model compression-induced neuropathy. Quantification of internodal myelin segments and internode numbers indicate beneficial influence of cholesterol supplementation. Together, these studies will help us unravel how intracellularly made cholesterol and exogenously supplied cholesterol impact Schwann cell biology, specifically myelin formation and stability.

In a related project, we examined the influence of a neutral lipid-enriched diet on myelination in neuropathic mice. For our studies we used Trembler J (TrJ) mice that carry a spontaneous mutation in peripheral myelin protein 22 (PMP22) and model early-onset, severe neuropathy. While it is known that lipid metabolism is critical for myelination and myelin maintenance, the impact of a cholesterol-enriched diet on early-onset neuropathies has not been examined. Furthermore, the mechanisms by which dietary lipid supplementation and/or substitution (no increase in overall calorie intake) benefit nerve myelin are unclear. To address these questions, we examined the lipid profile of peripheral nerves from 6-month old TrJ mice and tested the impact of a 6-week long neutral lipid-enriched high-fat diet (HFD) on neuropathy progression in young, newly-weaned mice. Oil Red O staining showed pronounced neutral lipid accumulation in nerves from affected, neuropathic mice, along with elevated levels of key cholesterol and triglyceride transport proteins including apoE, LRP1 and ABCA1, compared with wild type (Wt). In young mice, the short-term HFD intervention increased serum cholesterol levels, without impacting triglycerides, or body and liver weights. Nerves from neuropathic TrJ mice showed improvements in the maintenance of myelinated fibers after the 6-week long dietary intervention. Concomitantly, aberrant Schwann cell proliferation was attenuated, as detected by reduction in mitotic markers and in c-Jun expression. Nerves from HFD fed TrJ mice also contained fewer macrophages, with a normalized count of inflammatory CD11b+ cells. In addition, we detected an increase in neutral lipids in the nerve endoneurium and a trend toward normalization of apoE, LRP1, and ABCA1 expression, after the HFD feeding. Together, these results demonstrate a beneficial influence of a neutral lipid-enriched diet on neuropathy progression in young TrJ mice, and support further work in investigating the potential benefits of dietary lipids on demyelinating neuropathies. In our next set of studies, we plan to further optimize the lipid-enriched diet and examine sensory deficits in the various neuropathic models, with and without dietary intervention.

Related to these studies we have the following manuscripts at various stages of publication:
1. Zhou Y, Lee S, Bazick, H, Miles J, Tavori H, Landreth GE, Fazio S, , Notterpek L. PMP22 regulates cholesterol trafficking and ABCA1-mediated cholesterol efflux. J Neuroscience, 2019 (in press).
2. Zhou Y, Lee S, Bazick, H, Miles J, Fethiere A, AISalihi M, Tavori H, Fazio S, , Notterpek L. “A neutral lipid-enriched diet improves myelination and alleviates peripheral nerve pathology in neuropathic mice. Experimental Neurology (under review)
3. Zhou Y, et al., Steatosis and cholesterol mislocalization in the liver of PMP22-deficient mice (In preparation for Journal of Lipid Research)
4. Zhou Y, et al., PMP22 senses cellular cholesterol homeostasis and regulates cholesterol subcellular trafficking via CRAC domain(In preparation for Cellular Neuroscience)
5. AlSalihi, Bazick et al., HMG-CoA reductase inhibitors and their effect on Rat Schwann cells and myelination (In preparation but still collecting data)

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

John K. Neubert, D.D.S., Ph.D.

Todd E. Golde, M.D., Ph.D.

Mingzhou Ding, Ph.D.

Robert Caudle, Ph.D.

Marcelo Febo, Ph.D.

A University of Florida (UF) team consisting of John K, Neubert, D.D.S.,Ph.D. (Project Leader), Mingzhou Ding, Ph.D., Robert Caudle, Ph.D., Marcelo Febo, Ph.D., and Todd Golde, M.D., Ph.D. are investigating the neurophysiological signature of trigeminal neuralgia (TN) pain and developing promising new therapies. This group represents expertise in facial pain, structural and functional neuroimaging, neurophysiology, molecular biology, pharmacology, and viral vectors. Initially, this project was developed to include both preclinical animal models of trigeminal nerve pain and human studies using imaging studies from TN subjects to investigate the cause of TN. The preclinical animal studies have yielded the novel therapeutic agent, CGRP-Botox (see Dr. Caudle’s separate report). Dr. Febo has provided additional animal imaging analyses and has identified unique connections within the brain following trigeminal nerve injury. These findings confirm unique pathways relevant to trigeminal nerve pain and can provide a foundation for studying humans using the same imaging analyses. The human studies have found critical brain regions important in transmitting pain in TN patients and we identified an exciting relationship relating to neuroinflammation, chronicity, and surgery (see below in Human Studies). These findings provide the foundation for the journal article that is in preparation and will be submitted in the next few months. We have made significant progress towards completing the goals of this project and will expand on these exciting results (see Future Directions) to provide a pathway for finding a cure for TN.

HUMAN STUDIES

Recruitment (Table 1)

TN	Control	TN with surgery	TN without surgery	TN1	TN2	TN1/TN2
42	3	20	22	28	3	11

Forty-two TN patients (25 females) and three age and sex matched controls have been recruited and data from thirty-seven TN patients have been analyzed to date. One patient who had surgery no longer felt pain and was not scanned. Two patients did not complete the scan. Data from two recently scanned subjects have not been analyzed. We are planning to recruit additional TN and control subjects (N = 10-15/group) in the next few months.

Brain Activations during Trigeminal Pain Table 1

We recorded pain ratings during the fMRI scans to allow us to uncover brain regions that become activated during a TN pain episode. There are several interesting regions that are activated (Fig. 1) including the anterior insula, caudate, putamen, thalamus, and prefrontal cortex. This is important because identification of such brain regions serves as a fundamental and necessary step towards finding central-nervous system targets for effective therapeutic interventions in TN.

Fig 1. Regions of brain activation during TN pain episode.

Neuroinflammation and TN

The pontine crossing tract (PCT) is a region of the brain that links the brainstem and left/right cerebella and is an area of interest for processing trigeminal pain. We evaluated a free water (FW) analysis of this region as a marker for TN-related neuroinflammation. As shown in Fig. 2 (left), there is a significant positive correlation between free water in the PCT and disease duration in non-surgery TN1 patients. As a comparison, we analyzed scans from subjects with temporomandibular joint disease (TMD), another chronic orofacial pain condition. We found that the increase in FW in the PCT did not correlate with disease duration in the TMD cohort (Fig. 2 (right)). This finding is important because it indicates that there may be a specific mechanism or biomarker for TN, which is not present in other chronic orofacial pain disorders. These findings suggest that in non-surgery TN1, as the disease progresses, neuroinflammation becomes more severe; this pattern is specific to TN1 and not found in TMD. This is important because the FW analysis may be used as both a diagnostic biomarker of TN and to study mechanisms related to TN.

Fig 2. Pontine Crossing Tract (PCT) free-water (FW) as function of disease duration in non-surgery TN 1 (N=12) and in TMD.

Fig 3. Different thalamus and anterior cingulate (ACC) activity produces TN pain. During the scan (A), activity from ACC to the thalamus (B) unmasked pain activity from the thalamus to the ACC (C) ignited pain.

Novel regions related to TN pain: Central Ignition via Thalamic Disinhibition

For the six TN1 patients who exhibited clear on-off pain attack patterns in the scanner, we found that specific regions of the brain (thalamus and anterior cingulate cortex) affected the pain onset. In Fig. 3A (top), the up arrows indicate pain onset, and the colored intervals define the three time periods of analysis. Immediately prior to the onset of pain attacks (Pre-pain), top-down inhibitory influence from anterior cingulate cortex (ACC) to thalamic, ACCàThalamus, was reduced relative to a baseline condition (Resting) (Fig. 3B), causing thalamic disinhibition. Concurrently, the thalamic input into ACC, an important structure of the pain matrix, was increased (Fig. 3C) immediately preceding the onset of pain attacks, a phenomenon we referred to as central ignition. These results outline a clear mechanism of TN pain and suggest a possible therapeutic approach: by enhancing the ACC top-down influence we may restore the inhibitory control over the thalamus and thereby reduce the level of pain and the frequency of pain attacks. Our simulation study shows that transcranial direct current stimulation (tDCS) applied in a M1-SO configuration may be such a therapeutic approach. We tested the efficacy of the approach in patients to date. After 20-minutes of 2 mA tDCS stimulation, both patients reported ~40% reduction in pain level (from 50 to 30). We will continue the tDCS procedure as a pilot study to investigate the utility to modulate the brain regions identified in our previous studies.

ANIMAL STUDIES

Trigeminal Pain and Neuroimaging

Dr. Febo’s group has applied new analyses to the previously completed data set (~50 high-resolution fMRI scans of rats). The latest analysis compared rats with a trigeminal nerve injury (CCI) versus rats without the injury (sham). From this analysis, we found that there was connectivity in brain regions (Fig. 4), demonstrating that animals with trigeminal nerve injury show increased connections in pain sensitive structures within the brain. The different analyses used provide different ways of analyzing how these different structures interact and relate with other regions during different pain states. A number of regions were significantly different in CCI vs. Sham animals and this is important because this will allow us to compare the human and rat imaging results.

Control

Pain

Fig 4. Rat brain neural connectivity changes 2-weeks following chronic constriction injury (CCI) of the trigeminal nerve. Different analyses were completed in groups of animals that either had bilateral CCI (Pain) or sham (Control) surgeries. Significantly different regions include: dorsomedial striatum, paraventricular thalamus, posterior thalamus, ventral orbital cortex, ventral tegmental area, cerebellar nuclei. From the clustering coefficient analysis, we found these regions to be significantly different: cortical amygdala, ventromedial thalamus, anterior cingulate, ventral orbital cortex, somatosensory cortex.

For the six TN1 patients who exhibited clear on-off pain attack patterns in the scanner, we found that specific regions of the brain (thalamus and anterior cingulate cortex) affected the pain onset. In Fig. 3A (top), the up arrows indicate pain onset, and the colored intervals define the three time periods of analysis. Immediately prior to the onset of pain attacks (Pre-pain), top-down inhibitory influence from anterior cingulate cortex (ACC) to thalamic, ACCàThalamus, was reduced relative to a baseline condition (Resting) (Fig. 3B), causing thalamic disinhibition. Concurrently, the thalamic input into ACC, an important structure of the pain matrix, was increased (Fig. 3C) immediately preceding the onset of pain attacks, a phenomenon we referred to as central ignition. These results outline a clear mechanism of TN pain and suggest a possible therapeutic approach: by enhancing the ACC top-down influence we may restore the inhibitory control over the thalamus and thereby reduce the level of pain and the frequency of pain attacks. Our simulation study shows that transcranial direct current stimulation (tDCS) applied in a M1-SO configuration may be such a therapeutic approach. We tested the efficacy of the approach in patients to date. After 20-minutes of 2 mA tDCS stimulation, both patients reported ~40% reduction in pain level (from 50 to 30). We will continue the tDCS procedure as a pilot study to investigate the utility to modulate the brain regions identified in our previous studies.

FUTURE STUDIES

We plan to recruit and scan 15-20 more TN1 and control subjects. Dr. Febo’s group will continue to analyze the animal imaging data set. These results will be included in the preclinical animal imaging manuscript (in preparation)

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”)

Kim J. Burchiel, M.D., Professor, Department of Neurosurgery, Oregon Health & Science University

Scott R. Diehl, Ph.D. Professor, Department of Oral Biology, Rutgers School of Dental Medicine; Professor of Health Informatics, Rutgers School of Health Related Professions, Rutgers University

Ze’ev Seltzer, Ph.D., Professor of Pain Genetics, Faculty of Dentistry, Professor of Physiology, Faculty of Medicine, University of Toronto

As clinical expert for this project, Kim Burchiel of Oregon Health & Science University has continued to contribute new insights into the origins of Trigeminal Neuralgia (TN). New data further strengthen the view that TN is not just a single condition, with a single cause, but instead is a “heterogeneous” disorder with different causes in different patients. This means that it’s highly probable that different genes are responsible for the development of TN in different patients. To increase chances of success, strategies for finding the genes need to take into account the likelihood of such etiological heterogeneity. If TN patients can be subdivided into separate groups based on their clinical signs and symptoms that make them more likely to share the same underlying genetic mutations, this increases the chances of finding the specific genes versus when the analysis looks at all patients at once mixing up “apples and oranges.”

New findings confirm previous observations that younger patients, particularly females, develop TN without the requirement for neurovascular compression (NVC) of the trigeminal nerve. Older patients frequently do have NVC (though not always), and their prognosis from Microvascular Decompression (MVD) surgery is directly related to the severity of their compression. The size of the skull compartment that houses the trigeminal nerve (called the posterior fossa) also seems to be related to the incidence of NVC. These observations of apparent clinical subgroups will be used to perform separate analyses of TN patients that are similar to each other. For example, we will look for genes in our female subjects who do not have NVC and whose symptoms began before the age of 45. If, as we suspect, this subgroup of patients has different gene mutations causing the development of their TN than older patients without NVC our chances of finding the gene or genes causing early development of TN will be increased by focusing our search on this smaller, more homogeneous group of patients.

Scott Diehl from Rutgers University is responsible for carrying out the molecular and statistical genetic assays needed for the study. It is well established in the field of human genetics that for studies of heterogeneous human disorders such as TN having large numbers of patients and control subjects is absolutely essential for success. In the new analyses performed during the past year the number of TN patients studied has nearly doubled with total recruitment now over 1,000.

Another important advance in the current phase of this study has been use of a next generation molecular assay that provides improved coverage of variation known to exist in the human genome. The study is now using a set of DNA markers called the Precision Medicine Research Array (PMRA) that includes over 900,000 DNA polymorphisms. Polymorphisms are places in the genome where people differ in their DNA sequences that they have inherited from their parents. Using high speed computing, each these polymorphisms is tested to see if their frequency differs in TN patients versus control subjects who are also analyzed on the PMRA. Any polymorphism that is found to differ very dramatically in the patient group becomes a marker for a “candidate gene” located in the human genome near the marker that then warrants further investigation as a potential cause of TN.

Studies underway in 2019 will also use an advanced method called Next Generation Sequencing (NGS) where the entire 3.3 billion DNA letters (A, T, G and C) of the human genome are read. The initial NGS analysis will focus on a subset of 100 patients. This approach searches for very rare mutations that might cause development of TN but are so infrequent that they may never have been seen in previous human genetic study. Such extremely rare mutation are not included in the PMRA assay (since this focuses primarily on polymorphisms that are present in at least 5% of human chromosomes). Since TN occurs in only about 0.01% of the population, polymorphisms on the PMRA are unlikely to detect very rare mutations that cause TN directly – only NGS methods can do this. However, frequency differences between TN cases and controls found using PMRA data can point to which genes to focus on as most likely to contain the rare mutations that actually cause TN. The NGS technique is the “gold standard” of human genetic assays but it is currently still too costly to analyze for all of the study’s 1,000+ TN patients. As additional resources become available and as NGS costs are expected to come down in the next couple years, however, an essential future goal of the study will be to obtain NGS data on every available patient.

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

In 2018 we completed our study of the effects of cortical embryonic GABAergic inhibitory cell transplants into the region of cortex that processes the affective/emotional component of the pain experience. The studies were performed in two different neuropathic pain models in the mouse, one produced by injury to a peripheral nerve and that has some features in common with different nerve injury-induced chronic facial pain conditions and also a chemotherapy-induced neuropathic pain model produced by paclitaxel, a drug commonly used to treat cancer. What was remarkable is that the transplants, by restoring inhibitory controls, eliminated the animal’s tendency to seek a drug, namely gabapentin, which would normally alleviate their mechanical hypersensitivity. Interestingly, the transplants mimicked the effect of ablating the same region of cortex. Obviously ablation removes any hyperactive circuits, but ablation, from a clinical perspective, is a very last, obviously irreversible choice. The results of this study have been submitted for publication.

In addition to these studies, we have embarked on an extensive analysis of so-called “dark genes”, namely genes that are largely understudied, certainly in the context of pain. From a large list of genes that code for proteins that we believe could eventually be targeted by novel pharmacotherapeutics, we have identified several whose expression is dramatically altered following nerve injury. These neurons are expressed in dorsal root ganglion sensory neurons, which are homologous to the trigeminal ganglion sensory neurons, and are the first neurons in the circuits that process injury messages. After responding to the injury, these sensory neurons transmit these altered messages to the central nervous system, where they ultimately drive pain percepts. We hypothesize that dysfunction of these genes contributes to the prolonged neuropathic pains produced by nerve injury. Our ongoing studies will examine the physiological consequence of altered activity of the neurons that express these genes, and most importantly, using genetics tools, we will delete the gene from sensory neurons to assess the extent to which this manipulation alters the development of pain processing in different chronic pain models. If a particular gene deletion proves to be an important contributor to the development of nerve injury-induced persistent pain, then we will work with chemists to develop drugs that can interact with and block the function of the protein product of these genes, and effectively reduce the hyperactivity that drives the chronic pain condition.

Evaluation of adeno-associated virus (AAV) constructs directed at pain pathway targets – a rapid approach for identification of effective therapies for trigeminal neuralgia