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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"

Summary:

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

Principal Investigators (PIs):

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

 

 

 

 

Therapeutics

 

John K. Neubert, D.D.S., Ph.D.,University of Florida (Behavioral pain testing)

Other Significant Contributors:

Yona Levites, Ph.D., University of Florida (Therapeutics)

Robert M. Caudle, Ph.D., University of Florida (Behavioral pain testing)

 

The primary objective of this project is to design and synthesize a number of viral vectors directed at specific genes related to pain processing and then determine which of these constructs are safe and the most efficacious in doing so initially in animals.  Further, he posits that in addition to determining safety, this approach should decrease the time needed to discover novel new therapies for the treatment of TN.  Dr Golde’s team will utilize established expertise in  Adeno Associated Virus (AAV) delivery systems to evaluate the effectiveness of these vectors for reducing pain. 

Since gene therapy is a central character in this program perhaps a review of the fundamentals of gene therapy would be in order to assure that everyone has some understanding of this technology.  

Viruses are simple organisms consisting primarily of viral DNA encased in a protein coat.  In order to make a viral construct that can be used to introduce corrective DNA                                                                          

into animals/humans, it is necessary to first remove the wild-type viral DNA leaving only the protein coat.  Without its wild-type DNA, this viral protein shell can serve as a modified delivery system or a viral vector. Placing exogenous therapeutic genes into the empty protein shell creates what is termed a viral construct.  When these viral constructs are injected into the brain, spinal cord, trigeminal ganglion, etc., the therapeutic genes are released into the interior of cells through existing receptors in the cell membrane because the viral protein shell can alter its structure and lock on to these vacant receptors.  The therapeutic DNA can either integrate with the cell’s native DNA or remain as an independent strip of DNA and either way will produce the desired protein product in the injected tissue.  In short, gene therapy or delivery is the use of a viral construct to put in a copy of a gene that replaces a missing or defective gene; overproduces a protein that promotes cell growth and/or is “protective”, e.g., can control pain; and bioengineer stem cells and other types of cells to produce missing or protective proteins. 

Initially the team designed a series of experiment to evaluate best AAV serotype/rout of injection approach in order to establish the experimental paradigm for testing pain modifying genes later on.  There are a number of existing rodent models of pain. The method of measuring sensitivity to thermal stimuli in rodents - the radiant heat paw withdrawal method (Hargreaves et al., 1988) was chosen as most suitable for large throughput of various AAVs. AAVs were injected directly into sciatic nerve and gene expression was observed with immunostaining.

As shown in Fig. 1, direct sciatic nerve injection with rAAV M3 resulted in most abandoned expression of the GFP protein.

Next stage will include cloning potential pain modifier genes into the virus and deliver to rat’s sciatic nerve. Animals will then be subjected to foot sensitivity test to establish if any of the genes have profound effects on heat-induced pain.

Among genes tested are four potassium channels, six opioid receptors and secreted endogenous opioid neuropeptide and peptide hormone beta-endorphin. Current major technical obstacle is packaging these genes into the virus, due to their effects on the cells, but there are methods to overcome this challenge, that the team is implementing. Golde team obtained and characterized in vivo various AAV serotypes and promoter combinations. Potential pain modifiers will be cloned into these AAVs and most optimal packaged virus will be tested in vivo.

“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   

 

Specific Aims,

(a): to generate a pilot version of a neuro-modulation device that has been designed to encase peripheral nerve roots and to carry human cells that have been engineered to function as anti-pain generators by producing molecules with known pain inhibiting capabilities.  

(b) to test the capacity of this device, i.e., carbon nanotubes (fwCNT) loaded with human cells that have been modified to produce anti-pain molecules like transforming growth factor beta (TGF to inhibit or reduce pain transmission when encasing peripheral nerve roots. 

Progress to date: 

(a).  When fabricating the fwCNT for the TGFß-producing cells, we encountered difficulties with batch-to-batch consistency.  For example, variability in the thickness of the walls of the carbon nanotubes and proper sizing of the openings resulted in an inconsistent diffusion of TGFß out of the encasing carbon nanotubes.  At the present time we are working with our microfabrication core facility to correct this problem.

In another instance we did have some encouraging results.  In one set of experiments, we achieved good production and release of TGFß from HEK293 cells engineered to produce this cytokine that had been cultured on the chitosan-laminin matrix which is used to make the carbon nanotubes(Fig. 1).  We also observed good production and release of TGFβ when the TGFß-producing HEK293t cells were placed inside the carbon nanotubes.  Not surprisingly, variation in the production and release of TGFβ was considerable under these conditions most likely because of the aforementioned inconsistency with the construction of the carbon nanotubes.

Fig. 1: TGFß secretion by cultured cells

Note robust secretion of TGFß by TGFß-overexpressing HEK293t cells, represented by blue bars; regular tc = regular tissue-culture, chitosan-laminin = culture dishes coated with chitosan-laminin, ensheated = HEK293t overexpressing TGFß ensheathed into chitosan-laminin, note large variation here. CTRL denotes control-transfected HEK293t cells (not detectable secreted TGFß).

Right-hand side, green bar, representing the metric for I˚ human fibroblasts, overexpressing TGFß, TGFß cDNA expression vector was transfected by electroporation (e-por).

Bars represent the mean of n=3-4 replicate experiments, error bars the SEM.

We found that TGFß produced from HEK293t was at very good levels. We also observed TGFß release from human primary dermal fibroblasts after electroporation (Fig. 1), a method adopted successfully to  transfection of primary neurons (Yeo etal.2009) Generation of lentiviral vector (pLV based vector) led to viral preps with insufficient titer, so that for now electroporation is the more effective method. We are currently addressing this issue with our viral vector core facility, which typically and routinely produces lentiviral vectors with sufficient titer so that for now electroporation is the more effective method. We are currently addressing this issue with our viral vector core facility which typically and routinely produces lentiviral vectors with sufficient titer. 

(b)  We also took this approach into live animals.  HEK293t cells transfected to overproduce TGFß were placed into fwCNTand the cell-containing devices were wrapped around the L5 dorsal root ganglia (DRG) of Sprague Dawley rats.  In a pilot experiment, we wrapped the L5 DRG in four rats (Fig. 2). These animals were sensitized by evoking inflammatory hyperalgesia by injection of complete Freunds adjuvans (CFA) into their left foot pad. One animal of four showed a significant analgesic response, upon necropsy we found that the encasement of the L5 DRG  by the cell loaded fwCNT appeared intact in this particular animal. However, the other 3 rats did not show a significant analgesic response and the fwCNT wrapping of the L5 DRG in these 3 rats appeared to be unattached and damaged.

We view this result cautiously as overall encouraging, yet in need of replication, inclusion of proper controls, and further experimental refinement. To achieve these goals, we are re-ascertaining that DRG ensheathing devices, containing TGFß secreting cells, have a sufficiently rough outer surface so that they will stick on ensheathed DRG neurons and not dislocate.

 

Fig. 2: Avoidance behavior of rats with induced inflammatory injury (CFA left foot pad) and ensheathed TGFß over-expressing HEK293t cells wrapped to their L5 DRG

Y-axis depicts withdrawal in response to von Frey filament mechanical stimulation, using automatic von Frey apparatus (Ugo Basile) and setting the pre-CFA metric at 100%. The ensheathed cells, in chitosan-laminin ensheathing device, were wrapped around the left L5 DRG, then CFA was injected into the left foot pad. On days 1, 3, 5 and 7 mechanical withdrawal was determined. Note rat 1 with significant analgesic effect. This animal showed the ensheathed device still in proximity to the left L5 DRG. In contrast, rats 2-4 did not show analgesia, they rather show a typical inflammatory injury-mediated mechanical hyperalgesia. In these animals, the ensheathing device was not in direct proximity to the left L5 DRG. 

(b): In a pilot in vivo experiment, we wrapped the L5 DRGs in 4 rats with teflon wool that we pre-coated with fwCNT. We conducted sensitization with CFA as for Aim (i). We observed an analgesic effect which was limited to a duration of 3 days, however, in all 4 animals (Fig. 3). Results are shown vs a historic control of vehicle-treated CFA-sensitized control animals that presented with a typical response.

At necropsy we had difficulty retrieving and documenting tight proximity of fwCNT-coated teflon wrapping to the L5 DRG. Rather than taking the samples for ultrastructural examination, we decided to repeat the study, aiming to provide tighter and more durable wrapping, plus inclusion of dedicated control animals. This is happening at the time of writing.

To assess cellular toxicity to trigeminal sensory neurons, acutely dissociated adult rat trigeminal ganglion (TG) neurons exposed to fwCNT-coated teflon wrapping remained alive at a rate no different from acutely dissociated cultures maintained on laminin-coated regular tissue culture dishes, using a live/dead cell staining assay. Thus, there was no detectable toxicity (not shown).

We intend to extend this encouraging finding by sampling a sufficient number of rats, dissect their TGs, and label TG neurons with live/dead fluorescent assay. In case of increased rate of dead neurons for the fwCNT-teflon exposed neurons, we will also study stress/ activation markers pERK and cFOS. Results from these studies in rat will form the basis for intended biocompatibility/tox studies in swine.

 

Fig. 3: Pilot experiment in rats indicates short-lasting analgesia associated with wrapping of the L5 DRG with fwCNT-coated teflon wool when inflicting peripheral inflammatory injury with CFA.

Teflon wool was coated with fwCNT and then wrapped around the left L5 DRG. Note reduced mechanical hyper-algesia on earlier time-points d1-3, then on d5-7 this effect wears off.  Y-axis, treatment time-line and inflammatory injury experimental detail as in Fig. 2

REFERENCE 

 Yeo M, Berglund K, Augustine G, Liedtke W. J Neurosci. 2009;29(46):14652-62. doi: 10.1523/JNEUROSCI.2934-09.2009).

SUMMARIES OF RECENTLY FPRF APPROVED PROPOSALS THAT WILL START THIS SUMMER:

Characterizing a new biological therapy for treating trigeminal neuralgia

 

Robert M. Caudle, Ph.D.Department of Oral and Maxillofacial Surgery

University of Florida College of Dentistry

Trigeminal neuralgia is a devastating disease that is poorly controlled with current therapies. Tegretol is partially effective in some patients and vascular decompression surgery provides a bit of relief for others. However, most patients do not find these therapies sufficiently effective. Because the root cause of trigeminal neuralgia is not known, it is difficult to address the underlying etiology with novel therapies. We, however, have identified neurotransmitter systems on the trigeminal neurons that transmit the pain signals from the face to the brain. The receptors in the neurotransmitter systems offer unique targets for delivering biological agents that alter the function of the neurons. Although this form of therapy does not attack the cause of the disease, we can exploit these receptors to disrupt the transmission of pain to provide relief to the patients.

We have constructed a novel biological agent that can prevent the transmission of pain signals through trigeminal neurons that express the receptors associated with a specific neurotransmitter system. Preliminary studies have demonstrated that the agent targets the appropriate neurons and that it retains its biological activity for at least 24hours following uptake by the neurons. We expect that the agent will be functional for up to three months following administration. In this project we will be scaling up production of the agent and conducting rodent studies to determine if the agent blocks pain from nerve injury as predicted. We will also evaluate the most appropriate route of administration, the dose, and the duration of action of the agent to optimize the therapy protocol. These studies are expected to provide the basis for initiating human clinical trials to develop this agent as a therapy for trigeminal neuralgia.

 

Evaluation of a cellular therapeutic for the treatment of trigeminal pain

Principal Investigators

John K. Neubert, D.D.S., Ph.D., University of Florida (Project coordinator)

Cory R. Nicholas, Ph.D., Co-Founder and Chief Scientific Officer; Neurona Therapeutics (OSC)

Neurona was conceived in 2008 to realize the therapeutic potential of neuronal cell-based therapy. The rationale for pursuing this translational effort was based on many years of rigorous scientific discovery. In the late 1990’s, co-founders John Rubenstein, MD, PhD, and Arturo Alvarez-Buylla, PhD, were among the first to demonstrate that interneurons, a type of nerve cell, in the cerebral cortex mostly originate from precursor cells in the medial ganglionic eminence (MGE), a transient structure during fetal brain development. In these early studies, MGE cells were isolated from fetal mouse brain and injected into the brains of post-natal mice. In contrast to other neural stem and progenitor cells from neighboring fetal brain regions that remained as large cellular masses post-injection, the MGE cells rapidly dispersed away from the injection site and appeared to seamlessly integrate into juvenile and adult mouse recipient brain tissue. The injected MGE cells appropriately matured into various sub-lineages of cortical interneurons that secreted GABA, the major inhibitory neurotransmitter in the central nervous system.

Based on the concept of targeted therapies, Drs. Neubert and Nicholas and have proposed a new collaborative project between the University of Florida and Neurona Theraputics to evaluate the feasibility of treating a rat model of trigeminal nerve injury-induced pain using Neurona’s human stem cells.

Aim 1 is designed to characterize the pain phenotype of the immunocompromised RNU rat, which is necessary for optimizing human cell engraftment in this study.

Aim 2 is designed to evaluate the delivery, distribution, fate, and persistence of the human interneurons at the target sites and characterize any untoward effects. Lastly, in Aim 3 is designed to determine whether the human interneuron transplants reduce trigeminal neuropathic pain. If successful, the data from these studies will support further development of Neurona’s human interneuron product candidate for the prospective treatment of patients with chronic refractory TN. These studies are important because they represent necessary preclinical safety and efficacy studies necessary for translating this treatment into patients with TN and represents a potential cure.

 

First Report

Fifth Science Meeting At

University Of Florida Orthopaedics & Sports Medicine Institute

Gainesville FL

 

 Kim Burchiel, M.D., F.A.C.S 

The Facial Pain Research Foundation convened its Fifth Scientific Meeting in Gainesville Florida March 7-8, 2019.  The FPRF has assembled a group of world-class investigators as a consortium to find a cure for Trigeminal Neuralgia.  Clinicians and scientists from the US, England, Canada, and Israel were on site, to lend their perspectives on the state of research on TN.  The agenda was ambitious, but the science and discussions lived up to the promise that we are closing in on the new knowledge that will help us find the cure.  Many new ideas were presented and as is true of high functioning groups, some of the best insights were developed during the dinners and social hours after the formal meeting. 

I will summarize some of the exciting concepts discussed at the scientific sessions, by topic.  I present not so much as a full representation of the work that is currently underway, but as an indication of the high-level science that is being devoted to finding a cure for TN.  The conference commenced with discussions of FPRF funded research, and related investigations, of which I am most familiar.

 

• Scott Diehl, Ze’ve Seltzer, and Kim Burchiel presented the latest on their project “Genes That Predispose to TN”.

 Kim Burchiel from Oregon Health & Science University discussed new insights into the origins of TN, which relate to different populations of patients with TN. Data is emerging that younger patients, particularly females, develop TN without the requirement for neurovascular compression (NVC) of the nerve.  Older patients do frequently have NVC, and their prognosis from MVD is directly related to the severity of that compression.  The size of the skull compartment that houses the trigeminal nerve (posterior fossa) seems also to be directly related to the incidence of NVC.  This new knowledge will help us direct our genetic search.

Scott Diehl from Rutgers University reviewed the latest analysis of the genetic work on TN. A number of genes are now coming into focus, all of which appear to be relevant to the development, or function, of nerves.  Now that we have identified candidate genes, the next step is to “sequence” these genes to determine if the preliminary findings stand up, and to find the exact mutations in these candidate genes.  The hope is that soon we will be able to determine the function of these genes to either discover or develop new drugs for TN, or even replace defective genes with “gene therapy”. 

• Ze’ve Seltzer from the University of Toronto gave a broad overview of the role of genetics in the development of neuropathic pain, and how findings from his studies on phantom limb pain can provide insights into the genes that predispose to TN. Some preliminary data was presented which suggests that this strategy of comparison of large data sets from patients with neuropathic pain may well provide clues to the origins of TN. 

 

• Allen Basbaum from the University of California, San Francisco discussed his work to identify novel gene targets in pain processing, what he termed the "dark genes.”

 

• Lucia Notterpek from the University of Florida presented her work on dysregulated lipid metabolism as a disease modifier in peripheral neuropathies.

 

• Wolfgang Liedtke from Duke University discussed his work on the Trigeminal nerve root as a rational target for safe and effective treatment of trigeminal nerve pain.

 

• Cory Nichols from Neurona, described the work of his company targeted on developing a human GABAergic interneuron cell therapy to treat refractory epilepsy and neuropathic pain disorders.

 

The second conference day was devoted to a range of new and innovative potential therapies for TN.

 

• Todd Golde from the University of Florida talked about the possibility of gene therapy for Trigeminal Neuralgia.

 

• Joanna Zakrzewska from the University of London, discussed what happens to our patients with TN over time.

 

• John Neubert from the University of Florida provided an overview of his FPRF project on identifying the neurophysiologic signatures of trigeminal neuralgia pain. 

 

• Mingzhou Ding from the University of Florida related his findings on trigeminal neuralgia and multimodal neuroimaging.

 

• Rob Caudle from the University of Florida presented his findings on a approach to ending neuropathic pain with a combination of substances for blocking pain transmission. A novel and unique approach using old compounds to inhibit neuropathic pain.

 

• Allan Basbaum presented an update on the work of a company he is affiliated with which is working on a genetic “switch” that can potentially selectively turn off pain generation in the trigeminal system, using a benign oral drug.

 

• Mike Iadarola from NIH expanded on the use of a compound to potentially control facial pain and plans to test for ending TN and related neuropathic pain.

 

Jerry Krbec concluded the day with a discussion on funding opportunities from other sources to support the work of the FPRF.

 

What I would like to leave you with is the sense of the awe I have for the FPRF, having taken on this mission of finding a cure for TN.  Through hard volunteer work, diligent fund raising, and the assembling of scientific expertise, the FPRF has accomplished something which in my experience is very unique.  The FPRF scientific consortium has become the “place to be” in the neuroscience of TN.  Groundbreaking work is being accomplished, and I came away from this meeting with a sense of optimism that we are close to an understanding of how we may cure TN.  In fact, the most encouraging aspect of this meeting was, to me, how many different avenues of new knowledge are opening up.  I am proud to be part of this work, and am excited to see the next levels of understanding that await!

 Kim Burchiel, M.D., F.A.C.S