Grant Application Form Please Complete the Following Form for IETF

Grant Application Form

Please complete the following form for IETF grant applications. This form and all the attachments below must be combined into one document before submitting electronically. Grant submissions will not be accepted otherwise.

Attachments Required 1. Specific aims of the proposal (1 page maximum). 2. Rationale of the proposal and relevance to essential tremor (1-2 pages maximum). 3. Preliminary data, if available should be incorporated into the Rationale/Relevance section. Preliminary data are not required for a proposal. However, if preliminary data are referred to in the proposal rationale, or have been used to formulate the hypotheses to be tested, such information must be formally presented in this section. 4. Research methods and procedures (1-2 pages maximum). 5. Anticipated results (half-page maximum). 6. Detailed budget and justification (1 page maximum). 7. Biographic sketch of principal investigator and all professional personnel participating in the project (standard NIH format, including biosketch and other support). 8. Copies of relevant abstracts and/or articles that have been published, are in press, or have been submitted for publication. 9. Completed conflict of interest questionnaire.

Project Title: ____Investigation of mitochondrial dysfunction in Essential Tremor______

Sponsoring Institution: __Virginia Commonwealth University______

Principal Investigator:

Last Name: ___Trimmer______First Name: __Patricia______Middle Initial: _A._

Degree(s): ___Ph.D.______Current Title/Position: _Associate Professor______

Department: Anatomy and Neurobiology and Parkinson's and Movement Disorders Center

Address: _1217 East Marshall Street, PO Box 980312______

City: __Richmond______State: ____VA______Postal Code:

____23298_____ Country______USA___E-mail address: [email protected] Phone: __804-628-5041_or

434-987-3926______Fax: _____804-827-5274______All grant applicants acknowledge that the Board of Directors of the IETF is the only entity authorized to award grants on behalf of the IETF and the amounts of and occasions for awarding such grants, if any shall be awarded at all, shall be wholly within the sole and exclusive discretion of said Board and its judgment shall be final and conclusive and not subject to review for any reason judicial or otherwise.

GrantApp5222013 PO Box 14005 | Lenexa, Kansas 66285-4005 | USA | 888.387.3667 (toll free) | 913.341.3880 (local) | essentialtremor.org Specific Aims Essential tremor (ET) is a common, insidious, progressive movement disorder characterized primarily by bilateral kinetic and postural tremor of the upper limbs1. ET interferes with daily tasks. Currently available medications are only modestly effective and have troubling side effects2. Despite the high incidence of ET, little is known about the pathophysiology, cell biology or molecular mechanisms that underlie the disorder2. Neuropathological changes observed in ET brains suggest mitochondrial dysfunction may play a role ET3, 4. The studies in this proposal will investigate the hypothesis that mitochondrial dysfunction contributes to the pathophysiology of essential tremor. We will use peripheral blood collected from ET patients and matched disease-free controls (CNT) to assess mitochondrial dysfunction. We will employ gene expression analysis and cell culture techniques to create transmitochondrial cytoplasmic hybrid (cybrid) cell lines unique to each research participant. Studies in a wide range of neurological diseases by our group and others have shown that peripheral blood mononuclear cells (PBMC) and platelets exhibit abnormalities in oxidative phosphorylation (OXPHOS) and other mitochondrial functions that are comparable to disease-related changes in the brain5-11. Documentation of a role for mitochondrial dysfunction in ET will expand opportunities to identify new treatments based on a novel, unexplored disease mechanism. In addition to the specific aims, we will cryopreserve residual ET and CNT donor PBMC, platelets and cybrid cell lines to create a valuable, renewable resource for future experiments and collaborations. Experiments in this proposal will address our overall hypothesis with the following specific aims:

Hypothesis 1: Mitochondrial gene expression will be altered in PBMC from ET patients compared to PBMC from a matched set of disease-free controls (CNT). Specific aim 1: High quality total RNA (including mitochondrial RNA) will be isolated and pre-amplified from ET and CNT donor PBMC. Quantitative real-time PCR (qPCR) will be used to assess mitochondrial DNA (mtDNA) gene expression from total RNA isolated from ET and CNT donor PBMC.

Hypothesis 2: ET, but not CNT cybrid cell lines will exhibit morphological changes in mitochondria. Specific aim 2: Create cybrid cell lines by the fusion of ET or CNT donor platelets with mtDNA-free SH-SY5Y human neuroblastoma cells. Assess morphological changes in mitochondria and autophagosomes using light microscopy (LM) and electron microscopy (EM).

Rationale and Preliminary Data Despite the fact that ET is a common neurological disorder, understanding of the underlying disease pathophysiology is lacking. ET is a complex clinical disorder that is age-related and progressive12. Many aspects of ET are consistent with its classification as a neurodegenerative disease although this categorization is controversial12, 13. Both pathological and clinical heterogeneity have complicated studies of ET and limited therapeutic development14. Multiple findings across different ET neuropathology studies, however, suggest mitochondrial dysfunction may be an unexplored ET disease pathway.

Several lines of data connect ET and mitochondrial dysfunction. PC neurons in ET exhibit torpedoes, neurofilament-packed axonal swellings that also contain cytoplasmic constituents such as mitochondria3. Torpedoes share common features with Lewy neurites (axonal swellings in Parkinson’s disease [PD]) and dystrophic neurites (axonal swellings in Alzheimer’s disease [AD]). Axonal swellings are associated with defective axonal transport that is dependent upon mitochondrial ATP to power the action of anterograde and retrograde motor proteins (kinesin and cytoplasmic dynein)15. Also, the mitochondria in torpedoes are small with a pale matrix and disordered cristae (see Figure 3 of Louis et al, 2009)3. A pale matrix (location of the most mitochondrial proteins) and disordered cristae (site of the electron transport chain protein complexes that engage in OXPHOS) are classic features of dysfunctional mitochondria.

Recent studies have reported Purkinje cell (PC) axonal swellings (torpedos), displacement and death of PCs and other degenerative changes in the cerebellum2. Mitochondrial dysfunction is associated with reduced generation of ATP, increased production of free radicals and/or altered calcium handling each of which can contribute to neuronal cell death16 such as PC loss in ET. The role of actual PC cell loss in ET is has yet to be confirmed17.

Finally, Kuo et al. (2012)4 reported decreases in autophagy, an organelle degradation system that removes protein aggregates and damaged mitochondria. Key constituents of the autophagy pathway such as microtubule-

associated protein light chain 3-II (LC3-II, an autophagosome marker) and beclin-1 (required for autophagosome formation) were significantly reduced in the cerebellum of ET cases compared to CNT4. Associated with this loss of autophagic activity was an increase in mitochondrial membrane markers suggesting reduced removal of defective mitochondria. Kuo et al (2012)4 proposed that mitochondrial accumulation in ET cerebellum requires further analysis especially of OXPHOS to determine the role of mitochondrial dysfunction in ET.

Mitochondria are complex organelles that are composed of proteins encoded by both nuclear DNA (nDNA) and mtDNA genes. Mitochondria contain multiple copies of circular mtDNA that encode 13 proteins essential for ATP production. Regulated expression of both mtDNA- and nDNA-encoded proteins in complexes I, III, IV and V is required to ensure efficient mitochondrial electron transport chain (mtETC) assembly and OXPHOS function18. Studies in a wide range of neurological diseases by our group and others have shown that PBMC and platelets exhibit abnormalities in OXPHOS and other mitochondrial functions that reflect disease-related changes in the brain6-11. As shown in Figure 1, expression of mtDNA-encoded genes for OXPHOS subunits and 12s RNA was significantly reduced in amyotrophic lateral sclerosis (ALS) patient PBMC (p=0.0001, 2way ANOVA) compared to CNT. These mtDNA-encoded genes were comparably reduced in ALS spinal cord compared to CNT (p=0.002, 2way ANOVA, Ladd et al, in preparation). In Aim 1 we will utilize these molecular techniques to determine if mtDNA gene expression is altered in ET versus CNT PBMC.

Cybrid lines (Aim 2) are being successfully used to address the role of mitochondria in neurological diseases including AD, PD, ALS and multiple sclerosis (MS) as well as other conditions such as diabetes, cancer, stem cell biology and mitochondrial disorders11, 19-26. Cybrid lines share the same host cell nuclear genetic background and environmental conditions in culture. However, each unique cybrid line expresses mtDNA from an individual donor11, 27. SH-SY5Y human neuroblastoma cells are an appropriate host because they express relevant neurotransmitters and receptors, can be differentiated into neuronal cells and are widely used to model neurological disease mechanisms28. Briefly, donated platelets from patients or CNTs are fused to SH-SY5Y cells that lack mtDNA29. Mitochondria in platelets repopulate mtDNA-free SH-SY5Y cells. Subsequent metabolic selection eliminates cybrid cells with incomplete mtDNA repopulation30. In Aim 2 cybrid lines will be created using the platelets from ET and CNT participants. We will assess mitochondrial and autophagosome morphology using LM and EM. Paired with studies of PBMC, cybrid studies will give us the opportunity to create a cell culture model of ET using a human cell line for further studies of ET pathogenesis and to develop potential new treatments. We continue to generate new data and ideas about PD pathogenesis based on PD and CNT cybrids generated in 200131. Since cybrids are created from living donors, they provide a unique insight into early stages of disease pathogenesis.

Research Methods and Procedures Research participants: ET is characterized by clinical as well as pathological heterogeneity14. It is unknown whether all clinically defined ET shares the same underlying disease pathology. For this pilot effort, we will use a research ET definition narrower than the current clinical definition. While a clinically homogeneous group does not guarantee a pathologically homogeneous group, our approach focuses on action-based bilateral upper limb tremor, and maximizes use of a sample size we can achieve within budget and time constraints. This project will focus on “classic ET,” kinetic bilateral upper extremity tremor in isolation32, 33. To further strengthen the ET group criteria, we will use ET research diagnosis of possible/probable/definite stratifications32, 34, 35. All subjects, ET or CNT, will undergo a detailed non-invasive movement disorders exam. Dr. Testa has developed a VCU IRB- approved exam protocol including tremor36, 37, dystonia assessment based on Dystonia Coalition phenotyping

experience, parkinsonism, gait and balance (ex: tandem gait) and the Montreal Cognitive Assessment. The exam is paired with a structured interview covering medical history and medications as well as potential confounders for tremor. Exams will be scored by Dr. Testa using The Essential Tremor Rating Assessment Scale37, the United Parkinson Disease Rating Scale motor section38, and the Global Dystonia Rating Scale. Exams, scores and history will be used by Dr. Testa to make study inclusion determinations. For this study, ET participants will have definite kinetic tremor of the upper extremities clinically identified as ET and not explainable by other factors such as medication side effects. ET subjects will be excluded if they have a second significant neurological diagnosis (ex: PD, post-polio syndrome) of dementia or dystonia by report or on exam. To best exclude potential dystonia, we will also exclude subjects with ET and head/neck tremor or vocal tremor. Possible ET, with mild tremor in the range of enhanced physiological tremor, will also be excluded. CNT participants will undergo the same exam and be included if there is no tremor including no possible ET, dystonia, parkinsonism and no history of other significant neurological disorder (ex: MS, dementia). All exams will be videotaped, with specific consent. For this pilot study videos are considered primary study data, plus serve as a potential quality control check available for independent review if needed. Recruitment will use the VCU Parkinson’s and Movement Disorders Center (PMDC) clinic database as well as contact with new clinic patients, our ET genetics study research database and local ET support group connections to identify potential interested participants, both ET and CNT (ex: spouse of a person with ET). Dr. Testa is one of four movement disorders neurologists at PMDC. We will also leverage ongoing work in other disorders for CNT volunteers. As ET subjects are identified, screened and enrolled, CNT subjects will be enrolled matching ET subjects in gender and age so that the ET and CNT donor ages are not significantly different (+/- 3 years). Of the people with ET in our clinic database who consented to be contacted for future research studies, 35% have definite ET without head/neck tremor, vocal tremor, dementia or other study exclusions by database and medical chart review. Our group was able to recruit matched CNT research participants for PD and ALS studies. We anticipate enrolling 10 ET and 10 CNT subjects and acquiring the PBMC and platelet samples in a timely manner.

Sample handling: Up to 50ml of sterile peripheral blood will be collected per donor in ACD tubes as described in our VCU IRB approved protocol for PBMC, platelet and cybrid work. PBMC and platelets will be separated by density gradient centrifugation within 4 hours of collection (Ficoll-Paque Premium, Beckton Dickinson). The platelet-rich serum will be reserved for use in Aim 2. A portion of the PBMC fraction will be utilized for RNA isolation (Aim 1). The remaining PBMC will be cryopreserved for future cell-based studies. Platelets (Aim 2) will be concentrated from plasma by centrifugation and fused to mtDNA-free SH-SY5Y cells to generate cybrid cell lines unique to each participant as previously described29, 30 Multiple aliquots of every cybrid line will be maintained in liquid nitrogen storage for future studies.

Specific aim 1: RNA will be isolated from 10 ET and 10 CNT PBMC samples using Qiazol Lysis Reagent following manufacturer’s instructions. The remaining DNA portion will be stored at -80oC for future studies. RNA will then be quantified and assessed for quality using a Nanodrop 2000c spectrophotometer and an Experion Electrophoresis System (BioRad). RNA will be pre-amplified using the NuGen Ovation Pico SL kit to generate sufficient complementary DNA (cDNA) for the currently proposed qPCR assays as well as future gene expression studies. The expression levels of four mitochondrial genes will be determined using a four-color multiplex real- time qPCR assay developed and optimized in our lab as previously described31, 39. The four mtDNA genes are ND2 and ND4 (complex I subunits), COX3 (a complex IV subunit) and 12s ribosomal RNA31. These four genes are distributed at intervals around the circular human mtDNA genome. Appropriate reference genes will be determined using GeNorm and relative expression of mtDNA genes will be calculated using Biogazelle qbasePLUS software.

Specific aim 2: Cybrid lines give us the opportunity to study the effects of ET patient mtDNA on the cellular and mitochondrial function in living cells. Due to the limited funds, this aim will focus on mitochondrial and autophagic vesicle morphology and function (see26, 30, 40, 41). Cybrid cell lines will be generated using platelets from the same ET and CNT blood samples used in Aim 1. Cybrid cell lines will be created by fusing a suspension of mtDNA-free SH-SY5Y cells with donor platelets and polyethylene glycol40. Each resulting cybrid line will be expanded and cryopreserved. Six to eight lines at a time will be thawed and cultured in selection medium (DMEM with dialyzed fetal calf serum but without pyruvate and uridine) to eliminate cells without complete mtDNA repopulation (6-8 weeks)30, 40. Mycoplasma-free selected ET and CNT lines will be cryopreserved in liquid nitrogen.

ET and CNT cybrid lines will be plated in multiple glass-bottom culture dishes (MatTek) to label and visualize mitochondria and autophagosomes with MitoTracker CMX-ROS and LC3-II and/or beclin-1 antibodies, respectively using our Fluoview FV1000 confocal microscope31. We will assess mitochondrial shape (fragmented or elongate) and mass from confocal images using MetaMorph software and Prism for statistics (N=50-100 cells/cybrid). The appearance and number of autophagosomes and lysosomes (labeled with LysoTrackerRed) will be quantitated from confocal images (N=50-100 cells/cybrid)31, 40. Cells from a confluent T75 flask of each cybrid line will be fixed with 2% formaldehyde and 2.5% glutaraldehyde, processed, sectioned and stained for EM by the VCU Microscopy Facility. Micrographs (N=10-25 cells/cybrid) will be collected using the VCU Microscopy Facility JEOL transmission EM. The ultrastructure of mitochondria, autophagosomes (double-membrane enclosed vesicles that contain cytoplasmic organelles, laminar or amorphous material, 0.1-0.5µm)42 and lysosomes (small, dark vesicles, 0.1µm)40, 43 will be assessed (Fig 2). We have used this type of analysis to quantitate abnormal mitochondrial and lysosome morphology in cybrids from PD, AD and ALS patients26, 31, 44, 45. To minimize the risk of mycoplasma infection, it is our practice to retrieve fresh cybrid lines every 2 months31.

Statistical analysis: Two-way ANOVAs will be used to determine statistical significance of gene expression changes generated in Aim 1 using Prism software. Prism software will also be used for t-tests and ANOVAs on the data generated in Aim 2.

Cyropreserved resources: We will store all cellular samples collected or generated in liquid nitrogen. We have been successfully storing and retrieving cryopreserved samples for over 20 years. We have also shipped cryopreserved samples of cybrid lines to international and US collaborators.

Donor Enrollment and Timeline: We already have IRB approved protocols covering all individual aspects of this study. We will create a specific consolidated protocol and consent to reduce subject burden, and submit this to the VCU IRB by April 2014 so that it will be in place prior to July 2014. We will prescreen clinical and ET genetics database subjects so subject enrollment can begin as soon as funds become available.

Anticipated Outcomes and Interpretations Specific Aim 1: We anticipate that mitochondrial gene expression will be reduced in ET compared to CNT PMBC supporting our hypothesis. Evidence of mitochondrial dysfunction in PBMC will contribute to a better understanding of the pathophysiology of ET and could lead to new therapy and biomarker development. Results from studies using ALS PBMC (Fig 1) suggest that 10 ET and 10 CNT samples are sufficient to detect a change in mtDNA gene expression. If mtDNA gene expression in ET is not significantly different from CNT, then the pathophysiology of classic ET may not be linked to altered mtDNA. This outcome is also useful information that we will report to better direct future studies on ET. However, since mitochondrial proteins are encoded by two genomes, if mtDNA gene expression is not clearly different in ET compared to CNT then mitochondrial dysfunction in ET be driven by nDNA genes. In future studies we will use the remaining RNA from this study to assess nDNA-encoded mitochondrial respiratory genes with an SABiosciences PCR array that profiles the expression of nDNA genes relevant to all five mtETC complexes.

Specific Aim 2: We anticipate that mtDNA expression will be altered in ET PBMC compared to CNT (Aim 1), and that this molecular change will be reflected in LM and EM changes in mitochondrial morphology in ET cybrid cells (Aim 2). If our studies detect abnormal mitochondrial morphology in ET cybrids then our overall hypothesis has merit. Based on observations by Kuo et al. in ET brain, we also anticipate that autophagosome expression will be reduced. If we do not detect changes in mitochondrial or autophagosome morphology, then the site of mitochondrial dysfunction in ET may be in nDNA rather then mtDNA. Further studies of mitochondrial genes in PBMC will address this issue. We will also expand our analysis to other potential changes in mitochondrial function such as oxygen utilization using our Seahorse metabolic flux analyzer31.

Overall, with completion of the proposed studies we will have a better understanding of the role mitochondrial dysfunction plays in ET. This data will either confirm or disprove our theory that mtDNA abnormalities contribute to the pathophysiology of classic ET. The presence of altered mtETC gene expression in ET PBMC (Aim 1) will also provide preliminary data, which we can use to obtain additional funding. Detection of abnormal mitochondria and autophagosome morphology in ET cybrids would also support our hypothesis and augment our ability to obtain additional funding. If we obtain negative results from Aims 1 and 2, this is still reportable and interesting information at this sample size, plus further studies of nDNA-encoded mitochondrial genes will be undertaken with remaining RNA from Aim 1. Finally, we will generate a valuable research resource in the cryopreserved samples.

Future Directions The studies proposed are designed to be both informative and feasible within the allowable budget and time frame. We have chosen a specific ET research definition. Studies of mitochondrial dysfunction in samples from ET patients with head/neck tremor, clear dystonia, or other features we are excluding may or may not yield similar results to the current proposal. By carefully selecting ET patients with a distinct phenotype for this initial study, we hope to maximize the significance of our results. From work in other disorders, we feel the 20 total samples in this study will yield interpretable results. Expanding to include a wider range of ET subtypes, or comparing between disorders such as ET, PD, and ET plus PD, will require more samples. Cybrids and mtDNA expression studies from ET patients with other phenotypes would therefore be explored in a separate proposal.

The cryopreserved residual PBMC and platelets will create a renewable resource of 10 ET and 10 CNT matched human cybrid lines that can be further explored as an in vitro model to develop new therapies and improve our understanding of ET pathophysiology. Cell lines generated in this proposal are a unique resource that will be shared with other researchers and will help jump start future studies. Cryopreserved ET and CNT PBMC can be used to generate induced pluripotent stem cells (iPSC). The PMDC research labs and others have developed the ability to generate iPSC from PBMC that can be differentiated into disease-neurons for further study of ET pathophysiology, when additional funding is obtained46. RNA and DNA remaining from ET and CNT PBMC (Aim 1) will also be banked and used later to detect changes in gene expression levels in mitochondrial respiratory and biogenesis genes encoded by nDNA (see Saris et al5). This would be of interest if Aim 1 results are negative as discussed above, or if results are positive and we then want to follow up on whether there are nDNA contributions to mitochondrial dysfunction in ET. We can also use the banked PBMC RNA and DNA to conduct independent experiments and collaborate with other researchers in future investigations of splice variations and microRNA expression changes in RNA and single nucleotide polymorphisms and methylation changes in DNA depending on the results of the studies proposed her47, 48. If mitochondrial/autophagosome functions are altered in ET, then we will use cryopreserved, archived ET and CNT cybrid cell lines (Aim 2) to measure changes in OXPHOS function using our Seahorse, axonal transport after neuronal differentiation and to develop new ET specific drugs (see Cronin-Furman et al, 201331). If mitochondrial and/or autophagic function are not significantly different between ET and CNT cybrids, then mtDNA abnormalities may not play a role in ET (Aim 2). We will not be able to rule out the possibility that altered function of nDNA-encoded mitochondrial genes may be involved in the pathophysiology of ET since cybrids are created from donor mtDNA not nDNA. Future studies will specifically address this potential outcome. Cryopreserved, archived ET and CNT cybrid cell lines (Aim 2) will be used for future studies of OXPHOS function using our Seahorse, axonal transport after neuronal differentiation and therapeutic development of new ET specific drugs (see Cronin-Furman et al, 201331).

Budget and justification Personnel Effort Funds requested Patricia A. Trimmer, Ph.D., PI 5% 0 Claudia Testa, M.D., Ph.D., Co-PI 2% 0 Amy Ladd, Ph.D., Co-I 2% $1,294 Virginia Norris, M.S., B.S. 1% $ 714 Kristen Bergquist, B.S. 3% $1,473

Fringe- All Faculty and Classified Personnel 34.1% $1,187

TOTAL - Personnel $4,668

Supplies Blood sample processing, RNA extraction, pre-amplification and qPCR reagents $4,700 Cybrid creation $3,500 Cybrid selection $9,000 Electron microscopy $2,000 Antibodies, organelle markers, culture dishes, lab supplies $1,132

TOTAL- Supplies $20,332 Total costs- $25,000

Justification Dr. Trimmer, PI (Associate Professor) will have overall responsibility for both the gene expression studies and cybrid studies. She will be responsible for the light, immunofluorescence and electron microscopy morphology studies. All participants will meet regularly to ensure timely completion of the proposed research. Drs. Trimmer, Testa and Ladd will be responsible for data interpretation and manuscript preparation. Dr. Trimmer will devote at least 5% effort to this proposal with no requested salary compensation. Dr. Trimmer has extensive experience in neurodegenerative disease research, including PD, AD and ALS; this project would bring a new lab and PI into essential tremor research.

Dr. Testa, Co-PI (Associate Professor) is an expert in movement disorders neurology, clinical phenotyping and human subjects research. She will screen and enroll ET and CNT participants. She will conduct the neurological assessments of all subjects and participate in data analysis. She will devote 2% of her time to this proposal with no compensation requested.

Dr. Ladd, Co-PI (Laboratory and Research Scientist II) has extensive experience extracting and analyzing high quality RNA from human tissues. She generated the pilot data in Fig 1. She will process the ET and CNT blood samples for RNA analysis. She will devote 2% effort to this proposal.

Ms. Norris (Clinical Research Program Manager) will assist with pre-screening, scheduling, and enrolling the ET and CNT participants, including assisting with consenting subjects and managing the video camera. She will also be in charge of the blood collection. These are all work duties she regularly conducts for other studies at our center.

Ms. Bergquist (Supervisor of the Human Cell Culture Core) has supervised the use of our cybrid cell lines for 7 years. She created cybrid cell lines from the ALS and CNT patient platelets. She is qualified to create the ET and CNT cybrid lines for this proposal. She will devote 3% effort to this proposal.

Supplies- Blood collection and gene expression- ACD and centrifuges tubes, Ficoll-Pague, Qiagen RNA extraction kit, NuGen Ovation PicoSL RNA pre-amplification kit, Operon primers and probes, and BioRad multiplex PCR master mix Cybrid creation- Cell culture medium including fetal bovine serum, sterile plasticware (flasks and pipets), BioRad automated cell counting slides and fusion media including polyethylene glycol Cybrid selection- Dialyzed fetal bovine serum is required for the selection media as well as sterile flasks and pipets, cell freezing supplies like cryovials and counting slides. Electron microscopy- Fees for the processing of all ET and CNT cybrid cell lines by the VCU Microscopy Facility staff and fees for 1 hour of beam time per cybrid on the Jeol JEM-1230 TEM. Antibodies for mitochondria (Mitosciences), organelle markers (Molecular Probes), MatTek glass bottom culture dishes for confocal microscopy using our Olympus Fluoview FV1000 confocal microscope.

References 1. Zesiewicz TA, Shaw JD, Allison KG, Staffetti JS, Okun MS, Sullivan KL. Update on treatment of essential tremor. Curr Treat Options Neurol 2013;15:410-423. 2. Louis ED. Re-thinking the biology of essential tremor: from models to morphology. Parkinsonism Relat Disord 2014;20 Suppl 1:S88-93. 3. Louis ED, Yi H, Erickson-Davis C, Vonsattel JP, Faust PL. Structural study of Purkinje cell axonal torpedoes in essential tremor. Neurosci Lett 2009;450:287-291. 4. Kuo SH, Tang G, Ma K, et al. Macroautophagy abnormality in essential tremor. PLoS One 2012;7:e53040. 5. Saris CG, Horvath S, van Vught PW, et al. Weighted gene co-expression network analysis of the peripheral blood from Amyotrophic Lateral Sclerosis patients. BMC Genomics 2009;10:405. 6. Karlsson MK, Sharma P, Aasly J, et al. Found in transcription: accurate Parkinson's disease classification in peripheral blood. J Parkinsons Dis 2013;3:19-29. 7. Inarrea P, Alarcia R, Alava MA, et al. Mitochondrial Complex Enzyme Activities and Cytochrome c Expression Changes in Multiple Sclerosis. Mol Neurobiol 2013. 8. Masliah E, Dumaop W, Galasko D, Desplats P. Distinctive patterns of DNA methylation associated with Parkinson disease: Identification of concordant epigenetic changes in brain and peripheral blood leukocytes. Epigenetics 2013;8:1030-1038. 9. Pellicano C, Buttarelli FR, Circella A, et al. Dopamine transporter immunoreactivity in peripheral blood lymphocytes discriminates Parkinson's disease from essential tremor. J Neural Transm 2007;114:935-938. 10. Bogdanov M, Matson WR, Wang L, et al. Metabolomic profiling to develop blood biomarkers for Parkinson's disease. Brain 2008;131:389-396. 11. Trimmer PA, Bennett JP, Jr. The cybrid model of sporadic Parkinson's disease. Exp Neurol 2009;218:320- 325. 12. LaRoia H, Louis ED. Association between essential tremor and other neurodegenerative diseases: what is the epidemiological evidence? Neuroepidemiology 2011;37:1-10. 13. Rajput AH, Adler CH, Shill HA, Rajput A. Essential tremor is not a neurodegenerative disease. Neurodegener Dis Manag 2012;2:259-268. 14. Helmich RC, Toni I, Deuschl G, Bloem BR. The pathophysiology of essential tremor and Parkinson's tremor. Curr Neurol Neurosci Rep 2013;13:378. 15. O'Malley KL. The role of axonopathy in Parkinson's disease. Exp Neurobiol 2010;19:115-119. 16. Kawamata H, Manfredi G. Mitochondrial dysfunction and intracellular calcium dysregulation in ALS. Mech Ageing Dev 2010;131:517-526. 17. Symanski C, Shill HA, Dugger B, et al. Essential tremor is not associated with cerebellar Purkinje cell loss. Mov Disord 2014. 18. Scarpulla RC. Nucleus-encoded regulators of mitochondrial function: integration of respiratory chain expression, nutrient sensing and metabolic stress. Biochim Biophys Acta 2012;1819:1088-1097. 19. Hwang S, Kwak SH, Bhak J, et al. Gene expression pattern in transmitochondrial cytoplasmic hybrid cells harboring type 2 diabetes-associated mitochondrial DNA haplogroups. PLoS One 2011;6:e22116. 20. Silva DF, Santana I, Esteves AR, et al. Prodromal metabolic phenotype in MCI cybrids: implications for Alzheimer's disease. Curr Alzheimer Res 2013;10:180-190. 21. Silva DF, Selfridge JE, Lu J, et al. Bioenergetic flux, mitochondrial mass and mitochondrial morphology dynamics in AD and MCI cybrid cell lines. Hum Mol Genet 2013;22:3931-3946. 22. Llobet L, Gomez-Duran A, Iceta R, et al. Stressed cybrids model demyelinated axons in multiple sclerosis. Metab Brain Dis 2013;28:639-645. 23. Kaipparettu BA, Ma Y, Park JH, et al. Crosstalk from non-cancerous mitochondria can inhibit tumor properties of metastatic cells by suppressing oncogenic pathways. PLoS One 2013;8:e61747. 24. Kirby DM, Rennie KJ, Smulders-Srinivasan TK, et al. Transmitochondrial embryonic stem cells containing pathogenic mtDNA mutations are compromised in neuronal differentiation. Cell Prolif 2009;42:413-424. 25. Mimaki M, Hatakeyama H, Komaki H, et al. Reversible infantile respiratory chain deficiency: a clinical and molecular study. Ann Neurol 2010;68:845-854. 26. Swerdlow RH, Parks JK, Cassarino DS, et al. Mitochondria in sporadic amyotrophic lateral sclerosis. ExpNeurol 1998;153:135-142. 27. Swerdlow RH. Does mitochondrial DNA play a role in Parkinson's disease? A review of cybrid and other supportive evidence. Antioxid Redox Signal 2012;16:950-964.

28. Borland MK, Trimmer PA, Rubinstein JD, et al. Chronic, low-dose rotenone reproduces Lewy neurites found in early stages of Parkinson's disease, reduces mitochondrial movement and slowly kills differentiated SH-SY5Y neural cells. Mol Neurodegener 2008;3:21. 29. Miller SW, Trimmer PA, Parker WD, Jr., Davis RE. Creation and characterization of mitochondrial DNA- depleted cell lines with "neuronal-like" properties. J Neurochem 1996;67:1897-1907. 30. Swerdlow RH, Parks JK, Miller SW, et al. Origin and functional consequences of the complex I defect in Parkinson's disease. AnnNeurol 1996;40:663-671. 31. Cronin-Furman EN, Borland MK, Bergquist KE, Bennett JP, Jr., Trimmer PA. Mitochondrial quality, dynamics and functional capacity in Parkinson's disease cybrid cell lines selected for Lewy body expression. Mol Neurodegener 2013;8:6. 32. Elble RJ. Report from a U.S. conference on essential tremor. Mov Disord 2006;21:2052-2061. 33. Testa CM. Key issues in essential tremor genetics research: Where are we now and how can we move forward? Tremor Other Hyperkinet Mov (N Y) 2013;3. 34. Shatunov A, Sambuughin N, Jankovic J, et al. Genomewide scans in North American families reveal genetic linkage of essential tremor to a region on chromosome 6p23. Brain 2006;129:2318-2331. 35. Deuschl G, Bain P, Brin M. Consensus statement of the Movement Disorder Society on Tremor. Ad Hoc Scientific Committee. Mov Disord 1998;13 Suppl 3:2-23. 36. Elble R, Bain P, Forjaz MJ, et al. Task force report: scales for screening and evaluating tremor: critique and recommendations. Mov Disord 2013;28:1793-1800. 37. Elble R, Comella C, Fahn S, et al. Reliability of a new scale for essential tremor. Mov Disord 2012;27:1567- 1569. 38. Lang AEaF, S. Assessment of Parkinson's disease. In: Munsat TL, ed. Quantification of Neurologic deficit. Boston: Butterworth-Heinemann, 1989: 285-309. 39. Keeney PM, Bennett JP, Jr. ALS spinal neurons show varied and reduced mtDNA gene copy numbers and increased mtDNA gene deletions. Mol Neurodegener 2010;5:21. 40. Trimmer PA, Borland MK, Keeney PM, Bennett JP, Jr., Parker WD, Jr. Parkinson's disease transgenic mitochondrial cybrids generate Lewy inclusion bodies. J Neurochem 2004;88:800-812. 41. Borland MK, Mohanakumar KP, Rubinstein JD, et al. Relationships among molecular genetic and respiratory properties of Parkinson's disease cybrid cells show similarities to Parkinson's brain tissues. Biochim Biophys Acta 2009;1792:68-74. 42. Boland B, Kumar A, Lee S, et al. Autophagy induction and autophagosome clearance in neurons: relationship to autophagic pathology in Alzheimer's disease. J Neurosci 2008;28:6926-6937. 43. Palay SL, Chan-Palay V. Cerebellar cortex: cytology and organization. Berlin, Heidelberg, New York,: Springer, 1974. 44. Trimmer, P.A, Tuttle JP, Sheehan JP, Bennett JP, Jr. Mechanisms of selective neuronal vulnerability to 1- methyl-4phenylpridinium (MPP+) toxicity. In: Plenum Press: 439-446. 45. Trimmer PA, Swerdlow RH, Parks JK, et al. Abnormal mitochondrial morphology in sporadic Parkinson's and Alzheimer's disease cybrid cell lines. Exp Neurol 2000;162:37-50. 46. Dowey SN, Huang X, Chou BK, Ye Z, Cheng L. Generation of integration-free human induced pluripotent stem cells from postnatal blood mononuclear cells by plasmid vector expression. Nat Protoc 2012;7:2013-2021. 47. Simon-Sanchez J, Schulte C, Bras JM, et al. Genome-wide association study reveals genetic risk underlying Parkinson's disease. Nat Genet 2009;41:1308-1312. 48. Shafi G, Aliya N, Munshi A. MicroRNA signatures in neurological disorders. Can J Neurol Sci 2010;37:177-185.

Brief Summary

Even though essential tremor (ET) is the most common movement disorder affecting as many as 10 million in the US alone, surprisingly little is known about its causes. ET is characterized by rhythmic shaking of hands as well as other parts of the body. ET is not life threatening but the inability to stop the shaking can make even simple tasks impossible. The currently available medications to suppress tremor are only modestly effective and can have troubling side effects. Based on existing published research, we suspect that there may be a loss of mitochondrial function in ET. Mitochondria are critically important structures in our cells because they produce the energy (ATP) needed for the survival of all cells, especially neurons in the brain. This proposal will explore what role loss of mitochondrial function plays in ET. We will collect blood samples from ET patients identified by a movement disorders specialist at the Virginia Commonwealth University Parkinson’s and Movement Disorders Center. We will also carefully select a comparable group of control participants, meaning people without ET or other neurological disorders. Donor blood samples will be separated into different components, including white blood cells and platelets. Some of the white blood cell sample from each donor will be used to study genes important for mitochondrial function. The remainder of the white blood cells will be frozen for future studies in our labs or by other researchers. Based on published data about ET brain cells, we suspect that ET white blood cells will have reduced levels of genes that regulate mitochondrial function. Our research group and others have shown that gene expression in blood cells mirrors changes in nerve cells in the brain. Platelets will also be used to create cell lines unique to each research participant. These unique lines are called cybrids or cytoplasmic hybrids because they are created by fusing research participant platelets (that contain mitochondria) with a human neuronal cell line (SH- SY5Y). Cybrid lines are unique because they are derived from individual living participants. For more than 20 years our research group and others have used cybrid cell lines to study disorders such as Alzheimer’s disease and Parkinson’s diseases, amyotrophic lateral sclerosis (ALS), multiple sclerosis and diabetes. By making cybrid cell lines we will generate a bank of ET and disease-free control cell lines that can be used to better understand mitochondrial function and to develop new drug treatments. The studies we have planned will be the first step in determining if mitochondrial function is abnormal in patients with ET. By the end of this funding period we will have strong evidence about whether mitochondria are abnormal in ET, have created sufficient preliminary data to apply for more funding from other groups, and have created a bank of stored blood samples and cell lines for future studies by us and other researchers interested in making progress in understanding ET.

Principal Investigator/Program Director (Last, First, Middle): Trimmer, P. A.

BIOGRAPHICAL SKETCH Provide the following information for the key personnel and other significant contributors in the order listed on Form Page 2. Follow this format for each person. DO NOT EXCEED FOUR PAGES.

NAME POSITION TITLE PATRICIA A. TRIMMER, PH.D. ASSOCIATE PROFESSOR eRA COMMONS USER NAME pat5qNIH EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training.) DEGREE INSTITUTION AND LOCATION YEAR(s) FIELD OF STUDY (if applicable) Gettysburg College B.A. 1976 Biology University of Maryland at Baltimore Ph.D. 1982 Anatomy

A. Personal Statement Mitochondrial dysfunction is being increasingly recognized to play a critical role in neurodegenerative diseases like ALS, Parkinson’s, Alzheimer’s diseases as well as in other diseases like diabetes and multiple sclerosis. One way to examine mitochondrial dysfunction in human disease is analyze gene expression and morphology in postmortem tissue. Postmortem tissues have limitations because the samples are largely of late stage disease. My studies have focused on unique transmitochondrial cell lines generated by expressing patient and disease-free control donor platelet mitochondria in mitochondrial DNA-free SH-SY5Y neuroblastoma cells. Cybrid cell lines are attractive because they share the same nuclear DNA and environmental exposure in culture and differ only in the source of their mitochondrial DNA. Furthermore, cybrid lines are created from living patients and can be generated from the same patient at different stages of a disease. Recently my colleagues and I have focused on assessing the spectrum of mitochondrial and cellular changes associated with mitochondrial DNA expression changes. Disease specific human cybrid cell lines are also useful for therapy development. Since my move to Virginia Commonwealth University in June of 2010 as a member of the Parkinson’s and Movement Disorders Center (PMDC) I have become increasingly interested in Essential Tremor. I’ve seen the detrimental consequences of Essential Tremor in my husband’s grandmother (who lived to be 100) and my father-in-law who is currently 95. The opportunity to explore Essential Tremor by collaboration with Dr. Testa is a welcome opportunity. The exciting ALS data generated by Dr. Ladd suggests that we will be able to investigate the role of mitochondrial dysfunction in clinically diverse and poorly understood diseases like Essential Tremor beginning with a blood sample. The personnel in this proposal have the skills to complete this study, which will take the first step toward determining if mitochondrial dysfunction plays a role in the pathophysiology of Essential Tremor.

B. Positions and Honors Positions and Employment 1976-1982: Pre-doctoral Fellow and Teaching Assistant, Department of Anatomy, School of Medicine, University of Maryland at Baltimore, Baltimore, MD. 1982-1984: Postdoctoral Fellow, Department of Pharmacology, University of North Carolina at Chapel Hill, School of Medicine, Chapel Hill, NC. 1984-1987: Postdoctoral Fellow, Departments of Neurosurgery and Neuroscience, University of Virginia, School of Medicine, Charlottesville, VA. 1987-1993: Research Assistant Professor, Department of Neuroscience, University of Virginia, School of Medicine, Charlottesville, VA. 1993-2000: Research Assistant Professor and Core Lab Director, Department of Neurology, University of Virginia, School of Medicine, Charlottesville, VA. 2000-2010: Research Associate Professor, Department of Neurology and Core Lab Director, Neuroscience Graduate Program member, University of Virginia Udall Parkinson’s Research Center of Excellence, University of Virginia, School of Medicine, Charlottesville, VA.

PHS 398/2590 (Rev. 09/04, Reissued 4/2006) Page1 1 Biographical Sketch Format Page Principal Investigator/Program Director (Last, First, Middle): Trimmer, P. A. 2010-Present: Associate Professor, Department of Anatomy and Neurobiology, Virginia Commonwealth University, Richmond, VA 2010-Present: Associate Director for Basic Research, Parkinson’s and Movement Disorders Center, Virginia Commonwealth University, Richmond, VA

Other Professional Activities 1997-present Alzheimer’s and Related Diseases Research Award Foundation, Awards Committee member 1997-2010 Director, UVA’s Neuroscience Graduate Program Brain Awareness Program 2000 Army Neurotoxin Exposure Treatment Research Program, Peer Review Panel member 2000 NINDS Special Emphasis Panel member 2004-present Alzheimer’s Association- peer reviewer 2001 American Institute of Biological Sciences- peer reviewer 2006 Ad hoc member, NeuroA study section 2007-2008 Ad hoc member, CMND study section 2007-2012 Member, Public Education and Communication Committee, Society for Neuroscience 2010-2012 ETTN C-11 SBIR study section 2013-present NOMD study section member

Honors Best basic science presentation, World Association for Laser Therapy, Bergen, Norway 2010

C. Selected Peer-Reviewed Publications Trimmer, P. A., L. L. Phillips and O. Steward. 1991. Combination of in situ hybridization and immunocytochemistry to detect messenger RNAs in identified CNS neurons and glia in tissue culture. J.Histochem. Cytochem. 39:891-898. Steward, O., R. Tomasulo, E. Torre and P. Trimmer. 1992. Reorganization of neural connections following CNS injury: is synaptic reorganization initiated by the changes in neuronal activity which occur following injury? Brain Dysfunction 5:27-49. Trimmer, P. A. 1993. Reactive astrocytes in explant cultures of glial scars derived from lesioned rat optic nerve: an ultrastructural study. Int. J. Dev. Neurosci. 11:125-137. Swerdlow, R. H., J. K. Parks, S. Miller, J. B. Tuttle, P. A. Trimmer, J.P. Sheehan, J. P. Bennett, Jr., R. E. Davis and W. D. Parker, Jr.1996. Origin and functional consequences of the Complex I defect in Parkinson's disease. Ann. Neurol. 40:663-671. Miller, S.W., R.E. Davis, P.A. Trimmer, and W.D. Parker: Creation and characterization of mitochondrial DNA depleted cell lines with neuronal-like properties. 1996. J Neurochem. 67:1897-1907. Steward, O. and P. A. Trimmer. 1997. Genetic influences on cellular reactions to CNS injury: The reactive response of astrocytes in denervated neuropil regions in mice carrying a mutation (Wlds) that causes delayed Wallerian degeneration. J. Comp. Neurol.380:70-81. Swerdlow R.H., J.K. Parks, J.N. Davis, D.S. Cassarino, P.A. Trimmer, L.J. Currie, J. Doherty, S. Bridges, J.P. Bennett, G.F. Wooten, W.D. Parker. 1998. Matrilineal inheritance of complex I dysfunction in a multigenerational Parkinson's disease family. Annals Neurol, 44:873-881. Swerdlow, R.H., J.K. Parks, D.S. Cassarino, P.A. Trimmer, S.M. Miller, D.J. Maguire, J.P. Sheehan, R.S. Maguire, G. Pattee, V.C. Juel, L.H. Phillips, J.B. Tuttle, J.P. Bennett, Jr., R.E. Davis, and W.D. Parker, Jr. 1998. Mitochondria in sporadic amyotrophic lateral sclerosis. Exp Neurol. 153:135-142. Swerdlow, R.H., J.K. Parks, S.W. Miller, J.N. Davis II, P.A. Trimmer, J.B. Tuttle, J.P. Bennett, Jr., G.F.Wooten, R.E. Davis, and W.D. Parker, Jr. 1998. Mitochondrial dysfunction in Parkinson's disease. In: Progress in Alzheimer's and Parkinson's Diseases, Plenum Press, New York, Eds. A. Fisher, I. Hanin and M. Yoshida. p. 67-75. Sherer, T. B., P. A. Trimmer, J. K. Parks and J. B. Tuttle.2000.Mitochondrial DNA-depleted neuroblastoma (Rhoo) cells exhibit altered calcium signaling. Biochem. Biophys. Acta.1496:341-355 Trimmer, P. A., R. H. Swerdlow, J. K. Parks, P. Keeney, J. P. Bennett, Jr., S. W. Miller, R. E. Davis and W. D. Parker, Jr. 2000. Abnormal mitochondrial morphology in sporadic Parkinson’s and Alzheimer’s disease cybrid cell lines. Exp. Neurol. 162:37-50.

PHS 398/2590 (Rev. 09/04, Reissued 4/2006) Page Continuation Format Page Principal Investigator/Program Director (Last, First, Middle): Trimmer, P. A. Khan S.M., D.S. Cassarino, N.N. Abramova, P.M. Keeney, M.K. Borland, P.A Trimmer, C.T. Krebs, J.C. Bennett, J.K. Parks, R.H. Swerdlow, W.D. Parker, Jr., and J.P. Bennett, Jr. 2000 Alzheimer’s disease cybrids replicate beta-amyloid abnormalities through cell death pathways. Annals Neurology 48: 148-155. Sherer, T.B., P.A. Trimmer, K. Borland, J. K. Parks, J.P. Bennett, Jr., J. B. Tuttle. 2001. Chronic reduction in complex I function alters signaling in SH-SY5Y neuroblastoma cells, Brain Res. 891(1-2): 94-105. Trimmer, P. A. 1993. Reactive astrocytes in explant cultures of glial scars derived from lesioned rat optic nerve: an ultrastructural study. Int. J. Dev. Neurosci. 11:125-137. Trimmer, P.A., Borland, M.K., Bennett, J.P., Jr., Parker, W.D., Jr., Keeney, P.M. 2004. Parkinson's disease transgenic mitochondrial cybrids generate Lewy inclusion bodies. J. Neurochem. 88:800-812. Trimmer, P.A., Keeney, P.M., Borland, M.K., Simon, F.A., Almeida, J., Swerdlow, R.H., Parks, J.P., Parker, W.D., Jr., and Bennett, J. P., Jr. 2004. Mitochondrial abnormalities in cybrid cell models of sporadic Alzheimer’s disease worsen with passage in culture. Neurobiol. Dis. 15:29-39. Trimmer, P.A. and Borland, M.K. 2005. Differentiated Alzheimer’s disease transmitochondrial cybrid cell lines exhibit reduced organelle movement. Antiox Redox Signal 7(9-10):1101-1109. Zanelli, S.A., Trimmer, P.A. and Solenski, N.J. 2006. Nitric oxide impairs mitochondrial movement in cortical neurons during hypoxia. J. Neurochem. 97:800-806. Onyango, I, Khan, S, Miller, B, Swerdlow, R, Trimmer, P, and Bennett, P Jr. 2006. Mitochondrial genomic contribution to mitochondrial dysfunction in Alzheimer’s disease. J Alzheimers Dis 9:183-193. Borland, M.K., Trimmer, P.A., Rubinstein, J.D., Keeney, P. M., Mohanakumar, K.P., Liu, L. and Bennett, J. P. 2008. Chronic low-dose rotenone reproduces Lewy neurites found in early stages of Parkinson’s disease, reduces mitochondrial movement and slowly kills differentiated SH-SY5Y neural cells. Mol Neurodegener 3:21. Borland, M.K., Mohanakumar, K.P., Rubinstein, J.D., Keeney, P.M., Xie, J., Capaldi, R., Dunham, L.D., Trimmer, P.A. and Bennett, J.P. 2009 Relationships among molecular genetic and respiratory properties of Parkinson’s disease cybrid cells show similarities to Parkinson’s brain tissues. Biochim Biophys Acta 1792:68-74. Trimmer, P.A., Schwartz, K.M., Borland, K.M., DeTaboada, L., Streeter, J. and Oron, U. 2009. Reduced axonal transport in Parkinson’s disease is restored by light therapy. Mol Neurodegener. June 17;4: 26 Keeney, P.M., Quigley, C.K., Dunham, L.D., Papageorge, C. M., Iyer, S., Thomas, R.R., Schwartz, K.M., Trimmer, P.A., Bergquist, K.E. and Bennett, Jr. J. P. 2009 Mitochondrial gene therapy augments mitochondrial physiology in a Parkinson’s disease cell model. Gene Therapy. April 19 Trimmer, P.A. and Bennett, J.P., Jr. Invited Review. 2009. The cybrid model of Sporadic Parkinson’s disease. Exp Neurol. 218:320-325. Boscolo A, Starr JA, Sanchez V, Lunardi N, DiGruccio MR, Ori C, Erisir A, Trimmer P, Bennett J, Jevtovic-Todorovic, V. 2012. The abolishment of anesthesia-induced cognitive impairment by timely protection of mitochondria in the developing rat brain: the importance of free oxygen radicals and mitochondrial integrity. Neurobiology of Disease. 45:1031-1041. Cronin-Furman, E.N., Borland, M.K., Bergquist, K.E., Bennett, J.P., Jr. and Trimmer, P.A. 2013. Mitochondrial quality, dynamics and functional capacity in Parkinson’s disease cybrid cell lines selected for Lewy body expression. Mol Neurodegener 8:6. Cronin-Furman, E. N., Barber-Singh, J., Wright, L., Yagi, T., and Trimmer, P.A. Differential effects of yeast NADH dehydrogenase (Ndi1) expression on mitochondrial function and inclusion formation in a cell culture model of sporadic Parkinson’s disease. Neurobiology of Disease, in preparation.

Research Support Funded Research Support National Institutes of Health (NINDS) 2R01 NS044372; July 2013-June 2018; competitive renewal, Matrix Metalloproteinases and Regenerative Plasticity Following Brain Trauma. Principal Investigator: Linda L. Phillips, Ph.D., PI, Patricia A. Trimmer, Ph.D., Co-Investigator, Project goal: To study how molecules present in the extracellular compartments of the brain influence synapto-dendritic plasticity. Metalloproteinases and their target matrix molecules will be examined. Fluid percussion injury (TBI) and combined percussive TBI and neuroexcitation models will be used. Measures will include anatomical, behavioral and physiological indices of recovery, as well as correlative assessment of gene expression, protein content and molecular functional activity. PHS 398/2590 (Rev. 09/04, Reissued 4/2006) Page Continuation Format Page Principal Investigator/Program Director (Last, First, Middle): Trimmer, P. A.

National Institutes of Health (NINDS) 2R01 NS057758; July 2013-June 2018; competitive renewal, New approaches to axonal protection after TBI. Principal Investigator: Thomas M. Reeves, Ph.D. PI, Patricia A. Trimmer, Ph.D., Co-Investigator, Project goal: To study immunosuppressant drugs cyclosporine-A and FK506 for improvement of axonal structure and function after TBI using electrophysiology and morphology in animals and in cell cultures/

Completed Research Industry contract with PhotoThera, Carlsbad, CA Trimmer (PI) 10/27/2011-10/26/2012 The Seahorse extracellular flux system will be used to analyze a wide range of pulsed wave parameters with 810nm laser light to obtain optimal laser conditions that stimulate mitochondrial function and increase cellular respiration in well characterized PD cybrid cell lines.

Morris K. Udall Parkinson’s Research Center of Excellence P50-NS039788 05/01/2005 to 04/30/2010, no cost extension through 04/31/2012. PI for Project 4 and the Cybrid Core Lab Director: Trimmer, Program Director- G. Frederick Wooten, M.D. The experiments in this proposal investigate consequences of altered mitochondrial function and their relevance to the pathogenic mechanisms involved in the cell death characteristic of Parkinson’s disease using transmitochondrial human cybrid cell lines containing the mitochondria from PD and disease free CNT patients. Studies focus on Lewy body formation and the role of proteolysis and disrupted proteasome function in the pathogenesis of Parkinson’s disease. This is a renewal of our Udall Research Center of Excellence that was originally funded in 1999.

Industry contract with PhotoThera, Carlsbad, CA Trimmer and Solenski, N., Co-PIs 02/01/2005 to 03/31/2007 Neuronal cell lines in culture will be exposed to low levels of laser irradiation to determine the effects on various aspects of mitochondrial function

American Parkinson’s Disease Association Trimmer (PI) 09/01/2004 to 08/31/2005 The experiments in this proposal investigate the role of protein degradation and proteasome dysfunction in the pathogenesis of Parkinson’s disease using cybrid cell lines.

Morris K. Udall Parkinson’s Research Center of Excellence P50-NS039788 08/01/1999 to 07/01/2004 PI for Project 2 and Cybrid Core Lab Director: Trimmer (Co-PI with Jeremy Tuttle on Project 2) Program Director- G. Frederick Wooten, M.D. The experiments in this proposal investigate consequences of altered mitochondrial function and their relevance to the pathogenic mechanisms involved in the cell death characteristic of Parkinson’s disease using transmitochondrial human cybrid cell lines containing the mitochondria from PD and disease.

PHS 398/2590 (Rev. 09/04, Reissued 4/2006) Page Continuation Format Page Program Director/Principal Investigator: Testa, Claudia Marie

BIOGRAPHICAL SKETCH Provide the following information for the Senior/key personnel and other significant contributors in the order listed on Form Page 2. Follow this format for each person. DO NOT EXCEED FOUR PAGES.

NAME POSITION TITLE Claudia M. Testa Associate Professor eRA COMMONS USER NAME (credential, e.g., agency login) claudiatesta EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, include postdoctoral training and residency training if applicable.) DEGREE INSTITUTION AND LOCATION MM/YY FIELD OF STUDY (if applicable) The Johns Hopkins University, Baltimore, MD BA May 1987 Biophysics Churchill College, Cambridge University, England CPGS Aug 1988 Engineering University of Michigan, Ann Arbor, MI MD, PhD May 1996 Neuroscience American Board of Psychiatry and Neurology Board May 2001 Adult Neurology Certification Feb 2011 Emory University / Atlanta Clinical and MSCR May 2012 Master of Science in Translational Science Institute Clinical Research

NOTE: The Biographical Sketch may not exceed four pages. Follow the formats and instructions below. A. Personal Statement The goals of the proposed research is to compare mitochondrial function in essential tremor versus controls, as a pilot effort to see if mitochondrial dysfunction is a player in essential tremor disease pathology as seen in other movement disorders. I have expertise in movement disorders, phenotyping, and human subjects research in neurology. I will conduct neurological examinations, characterize neurological features of essential tremor in the research cohort, and help conduct some of the analysis. I have actively participated in the research project design, including inclusion/exclusion criteria for subjects. I have experience working with biospecimen collection and data assessment as part of observational longitudinal research trials. I am part of ongoing Tremor Research Group as well as Movement Disorders Society efforts in advancing the overall field of essential tremor research.

B. Positions and Honors Positions and Employment 1996-1997 Intern, Beth Israel Hospital, Department of Internal Medicine, Boston, MA 1997-2000 Neurology Resident, Partners Neurology Program, Massachusetts General and Brigham and Women’s Hospitals, Harvard Medical School, Boston, MA 1999-2000 Chief Resident, Partners Neurology Program 2000-2002 Fellow in Movement Disorders, Department of Neurology, Emory University, Atlanta, GA 2001-2002 Associate in Neurology, Emory University 2002-2011 Assistant Professor in Neurology, Emory University, tenure track 2004-2005 Director, Movement Disorders Fellowship, Emory University 2005-2011 Medical Director, Emory Huntington Disease Center of Excellence 9/2011- Associate Professor, Neurology, Virginia Commonwealth University 9/2011- Associate Director, Clinical Care and Research, VCU Parkinson’s and Movement Disorders Center 9/2011- founder and Program Director, VCU Huntington Disease Center 2/2012- Joan Massey Clinical Chair in Parkinson’s Disease

Other Experience and Professional Memberships 2001- Huntington Study Group; 2011- Investigator member, HSG Executive Committee (elected post)

PHS 398/2590 (Rev. 06/09) Page 1 Biographical Sketch Format Page Program Director/Principal Investigator: Testa, Claudia Marie

2002- Movement Disorders Society; 2011- MDS Essential tremor task force (appointed position) 2005-07 Co-Director IBS 506R Basic Mechanisms of Neurological Diseases 2004-08 American Academy of Neurology, Neuroscience Research Prize Ad-Hoc Judge 2005- Tremor Research Group; Vice President 2013- (elected post) 2006- Society for Neuroscience, Neurobiology of Disease Workshop Organizing Committee 2008- International Essential Tremor Foundation medical advisory board member 2010- Founder, North American Essential Tremor Consortium focusing on advancing ET phenotyping/epidemiology and genetics Honors 2002 Dr. George C. Cotzias Memorial Fellowship, American Parkinson Disease Association

C. Selected Peer-reviewed Publications Testa CM, Standaert DG, Landwehrmeyer GB, Penney Jr JB, Young AB (1995) Differential expression of mGluR5 metabotropic glutamate receptor mRNA by rat striatal neurons. J Comp Neurol 354:241-252. Testa CM, Hollingsworth ZR, Shinozaki H, Penney JB, Young AB (1997) Selective metabotropic receptor agonists distinguish non-ionotropic glutamate binding sites. Brain Res 773:15-27. Testa CM, Friberg IK, Weiss SW, Standaert DG (1998) Immunohistochemical localization of metabotropic glutamate receptors mGluR1a and mGluR2/3 in the rat basal ganglia. J Comp Neurol 390:5-19. Sherer TB, Betarbet R, Testa CM, Seo BB, Richardson JR, Kim JH, Miller GW, Yagi T, Matsuno-Yagi A, Greenamyre JT (2003) Mechanism of toxicity in rotenone models of Parkinson’s disease. J Neurosci 23(34):10756-10764. Testa CM, Sherer TB, Greenamyre JT (2005) Rotenone induces oxidative stress and dopaminergic neuron damage in organotypic REsubstantia nigra cultures. Mol Brain Res 134:109-18. Sherer TB, Richardson JR, Testa CM, Seo BB, Panov AV, Yagi T, Matsuno-Yagi A, Miller GW, Greenamyre JT (2007) Mechanism of toxicity of pesticides acting at complex I: relevance to environmental etiologies of Parkinson’s disease. J Neurochem 100(6): 1469-79. Stefansson H, Steinberg S, Petursson H, Gustafsson O, Gudjonsdottir IH, Jonsdottir GA, Palsson ST, Jonsson T, Saemundsdottir J, Bjornsdottir G, Böttcher Y, Thorlacius T, Haubenberger D, Zimprich A, Auff E, Hotzy C, Testa CM, Miyatake LA, Rosen AR, Kristleifsson K, Rye D, Asmus F, Schöls L, Dichgans M, Jakobsson F, Benedikz J, Thorsteinsdottir U, Gulcher J, Kong A, Stefansson K (2009) Variant in the sequence of the LINGO1 gene confers risk of essential tremor. . Nature Genetics 41(3):277-9; doi:10.1038/ng.299 Revuelta GJ, Testa C, Greene JG (2010) Writer’s cramp: a potential early feature of Huntington’s disease. Mov Disord 30;25(6):777-8 Vilariño-Güell C, Wider C, Ross OA, Jasinska-Myga B, Kachergus J, Cobb SA, Soto-Ortolaza AI, Behrouz B, Heckman MG, Diehl NN, Testa CM, Wszolek ZK, Uitti RJ, Jankovic J, Louis ED, Clark LN, Rajput A, Farrer MJ (2010) LINGO1 and LINGO2 variants are associated with essential tremor and Parkinson disease. Neurogenetics 11(4): 401-8. She H, Yang Q, Shepherd K, Smith Y, Miller G, Testa C, Mao Z (2011) Direct regulation of complex I by mitochondrial MEF2D is disrupted in a mouse model of Parkinson disease and in human patients. J Clin Invest 121(3): 930-40. Ross OA, Conneely KN, Wang T, Vilarino-Guell C, Soto-Ortolaza AI, Rajput A, Wszolek ZK, Uitti RJ, Louis ED, Clark LN, Farrer MJ, Testa CM (2011) Genetic variants of α-synuclein do not associate with essential tremor. Movement Disorders 26(14):2552-6. Elble R, Comella C, Fahn S, Hallett M, Jankovic J, Juncos JL, Lewitt P, Lyons K, Ondo W, Pahwa R, Sethi K, Stover N, Tarsy D, Testa C, Tintner R, Watts R, Zesiewicz T (2012) Reliability of a new scale for essential tremor. Movement Disorders 27(12): 1567-9. Cloud LJ, Rosenblatt A, Margolis RL, Ross CA, Pillai JA, Corey-Bloom J, Tully HM, Bird T, Panegyres PK, Nichter CA, Higgins DS, Helmers SL, Factor SA, Jones R, Testa CM (2012) Seizures in juvenile Huntington's disease: Frequency and characterization in a multicenter cohort. Movement Disorders Nov 2. doi: 10.1002/mds.25237. Biglan KM, Zhang Y, Long JD, Geschwind M, Kang GA, Killoran A, Lu W, McCusker E, Mills JA, Raymond LA, Testa C, Wojcieszek J, Paulsen JS; PREDICT-HD Investigators of the Huntington Study Group. (2013) Refining the diagnosis of Huntington disease: The PREDICT-HD study. Front Aging Neurosci. 2013;5:12. doi: 10.3389/fnagi.2013.00012. Epub 2013 Apr 2.

PHS 398/2590 (Rev. 06/09) Page 2 Biographical Sketch Format Page Program Director/Principal Investigator: Testa, Claudia Marie Elble R, Bain P, Forjaz MJ, Haubenberger D, Testa C, Leentjens, AFG, Martinez-Martin P, Pavy-Le Traon A, Post B, Sampaio C, Stebbins GT, Weintraub D, Schrag A (2013) Task force report: Scales for screening and evaluating tremor: critique and recommendations. Mov Disord. Nov;28(13):1793-800. doi: 10.1002/mds.25648. Epub 2013 Sep 3.

D. Research Support - NIH/NORD 5 U54 NS065701 04 Role: Site PI (PI: Jinnah) 2012- Dystonia Coalition Projects A longitudinal multicenter multinational observational study to develop a better understanding of primary dystonias so the treatment of affected patients can be improve.

- Auspex Pharmaceuticals Role: Co-PI (PI: Frank) 2012- Title: A Randomized, Double-Blind, Placebo-Controlled Study of SD-809 Extended Release for the Treatment of Chorea Associated with Huntington Disease: First Time Use of SD-809 ER in HD (First-HD). Multicenter clinical trial conducted under the Huntington Study Group in US and Canada.

- Auspex Pharmaceuticals Role: Co-PI (PI: Frank) 2013- Title: An Open Label Long Term Safety Study of SD-809 ER in Patients with Chorea Associated with Huntington Disease: Alternatives for Reducing Chorea in HD (ARC-HD). Multicenter clinical trial conducted under the Huntington Study Group in US and Canada.

- CHDI Foundation Role: Site PI (PI: Landwehrmeyer) 2013- Enroll-HD A longitudinal multicenter multinational observational trial to understand the progressive phenotype of Huntington’s Disease (HD) and develop biomarkers leading to improved treatments.

Support transferred to other investigators at Emory University after 9/2011 move, work ongoing - NIH/NINDS Role: Site PI (PI: Hersch) 2008-2013 A multi-center, double-blind, placebo-controlled, high dose creatine safety, tolerability, and efficacy study in Huntington Disease (CREST-E)

- 2R24RR018827-05A1 NCRR/NIH Role: Co-PI (PI: Chan) 2009-2014 Establishment of a transgenic monkey model of Huntington’s Disease

- NIH/NINDS Role: Motor Rater (PI: Paulsen) 2010- Neurobiological Predictors of Huntington’s Disease (PREDICT-HD 2.0) A continuation of PREDICT-HD with major modifications to improve biomarker collection and encourage add- on projects that leverage PREDICT-HD 2.0 research cohort, data, and structure.

- Huntington Society of Canada Role: PI 2009- Investigator-initiated clinical trial award

Completed (partial list) - Medivation Role: Site PI (PI: Hersch) 2009-2010 A phase 3, randomized, double-blind, placebo-controlled, high dose creatine safety and efficacy study of dimebon in patients with mild-to-moderate Huntington Disease (HORIZON)

- NIH/NIA P50 AG025688 Role: Co-investigator (PI: Levey) 2005 – 2010 Emory Alzheimer’s Disease Research Center (ADRC) Co-PI role in Parkinson disease (PD) and movement disorder subject assessment and enrollment for AD/PD and MCI projects.

PHS 398/2590 (Rev. 06/09) Page 3 Biographical Sketch Format Page Program Director/Principal Investigator: Testa, Claudia Marie - NIH/NINDS 2 R01NS40068-05 Role: Motor Rater (PI: Paulsen) 2005-2010 Neurobiological Predictors of Huntington’s Disease (PREDICT-HD)

- Tremor Research Group Role: Site PI (PI: Elble) 2010-2011 Investigator-initiated award from GlaxoSmithKline Validation of the TRG Essential Tremor Rating Scale (TETRAS)

- HighQ Foundation Role: Site PI (PI - Shoulson) 2006-2011 Cooperative Huntington’s Disease Observational Trial (COHORT)

PHS 398/2590 (Rev. 06/09) Page 4 Biographical Sketch Format Page BIOGRAPHICAL SKETCH Provide the following information for the Senior/key personnel and other significant contributors in the order listed on Form Page 2. Follow this format for each person. DO NOT EXCEED FOUR PAGES.

NAME POSITION TITLE Amy C Ladd, PhD Laboratory and Research Scientist II eRA COMMONS USER NAME (credential, e.g., agency login) ALADD1 EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, include postdoctoral training and residency training if applicable.) DEGREE INSTITUTION AND LOCATION MM/YY FIELD OF STUDY (if applicable) Indiana University, Bloomington, IN BS 1988-1992 Biology Purdue University, Indianapolis, IN MS 1992-1994 Biology University of Maryland, Baltimore, MD PhD 1996-2001 Pathology Virginia Commonwealth University, Richmond, Post Doctoral Training / 2003-2007 VA Molecular Pathology

A. Personal Statement During my post doctoral training in the Molecular Diagnostics Division at VCU, I spent several years assisting in the development of quality control standards for handling, banking and molecular testing of human tissues. I developed and implemented our institution’s system for cryopreservation and banking of peripheral blood from healthy and leukemic patients. I have extensive experience in extracting high quality RNA from these and other types of human tissues and performing RT-PCR and gene expression microarray experiments on these extracts. Most recently, I have been instrumental in the development of the peripheral blood bank in the VCU Parkinson’s and Movement Disorders Center where I now work. We have successfully implemented an IRB- approved protocol for collecting and cryopreserving peripheral blood for downstream molecular analyses and development of iPSC and cybrid cell lines. I have completed a pilot project looking at the expression of oxidative phosphorylation genes in ALS and control blood cells, showing that even unaffected tissues express lower levels of mitochondrial genes in ALS patients when compared to healthy controls.

B. Positions and Honors

Employment

1994 – 1996 Research Technician, Stem Cell Research Laboratory, Division of Hematology/Oncology, School of Medicine, Indiana University, Indianapolis, IN 1995 – 1996 Immunology Laboratory Teaching Assistant, Department of Biology, Purdue University, Indianapolis, IN 1996 – 2001 Graduate Student Research Assistant, Department of Pathology, University of Maryland, Baltimore, MD 2003-2007 Post Doctoral Associate, Division of Molecular Diagnostics, Department of Pathology, VCU, Richmond, VA. 2007-2011 Director, Laser Microdissection Laboratory, Department of Pathology, VCU, Richmond, VA 2007-2011 Affiliate Assistant Professor, Department of Pathology, VCU, Richmond, VA. 2011-present Laboratory and Research Scientist II, Parkinson’s and Movement Disorders Center, VCU, Richmond, VA

PHS 398/2590 (Rev. 06/09) Other Experience, Honors and Professional Memberships

2001 Graduate Merit Award for Outstanding Scholarship, Department of Pathology, University of Maryland 2005 -11 Member, of the Association for Molecular Pathologists (AMP) 2009 Ad hoc reviewer: Human Pathology, Cancer Cytopathology, Journal of Clinical Laboratory Analysis 2009 -11 Member, United States and Canadian Academy of Pathology (USCAP) 2010 -11 Steering Committee Member, Laser Technologies Applications Group, Durham, NC 2010 - Member, VCU Institutional Review Board, panel D 2012 - Member, Society for Neuroscience

B. Peer-reviewed Publications

Ladd AC, Pyatt T, Gothot A, Rice S, McMahel J, Traycoff CM, Srour EF. Orderly process of sequential cytokine stimulation is required for activation and maximal proliferation of primitive human bone marrow CD34+ hematopoietic progenitor cells residing in G0. Blood. 1997 Jul 15; 90(2): 658-68. PMID: 9226166

Traycoff CM, Orazi A, Ladd AC, Rice S, McMahel J, Srour EF. Proliferation-induced decline of primitive hematopoietic progenitor cell activity is coupled with an increase in apoptosis of ex vivo expanded CD34+ cells. Exp Hematol 1998 Jan; 26(1): 53-62. PMID: 9430514

Dumur CI, Nasim S, Best AM, Archer KJ, Ladd AC, Mas VR, Wilkinson DS, Garrett CT, and Ferreira- Gonzalez A. Evaluation of Quality Control Parameters for Microarray Gene Expression Analysis. Clinical Chemistry 2004, 50(11):1994-2002. PMID: 15364885

Dumur CI, Sana S, Ladd AC, Ferreira-Gonzalez A, Wilkinson DS, Powers CN, Garrett CT. Assessing the Impact of Tissue Devitalization Time on Genome-Wide Gene Expression Analysis in Ovarian Tumor Samples. Diagnostic Molecular Pathology 2008, 17(4):200-6. PMID: 18382347

Zhang X, Ladd AC, Budd W, Ware J, Dragoescu E, Zehner ZE. MicroRNA-17-3p is a Prostate Tumor Suppressor In Vitro and In Vivo, and is Decreased in High Grade Prostate Tumors Analyzed by Laser Capture Microdissection. Clinical & Experimental Metastasis 2009, 26(8): 965-79. PMID: 19771525

Dumur CI, Ladd AC, Wright, HV, Penberthy LT, Wilkinson DS, Powers CN, Garrett CT, DiNardo LJ. Genes Involved in Chemoradiation Therapy Response in Head and Neck Cancer. The Laryngoscope 2009, 119(1):91-101. PMID: 19117295

Ladd AC, O’Sullivan E, Lea T, Perry J, Dumur CI, Dragoescu E, Garrett CT, Powers CN. Preservation of Fine Needle Aspiration Specimens for Future use in RNA Based Molecular Testing. Cancer Cytopathology 2011, DOI: 10.1002/cncy.20130. PMID: 21287691

Rice AC, Keeney PM, Algarzae NK, Ladd AC, Thomas RR, Bennett JP. Mitochondrial DNA Copy Numbers in Pyramidal Neurons are Decreased and Mitochondrial Biogenesis Transcriptome Signaling is Disrupted in Alzheimer’s Disease Hippocampi. Journal of Alzheimer’s Disease 2013, In Press

PHS 398/2590 (Rev. 06/09) D. Research Support Ongoing Research Support

R01 CA154314-01 Chalfant (PI) 2011-2016 Evaluation of Alternative Splicing in Genes Regulating Apoptosis and Tumorigenic Capacity in Human Cancers Using LCM and RT-PCR, the goal of these studies is to examine the differential expression of splice variants of Caspase 9 and other pathway related genes in human non-small cell lung carcinoma. Role: Co-Investigator (20% TE)

Previous Research Support

R21 CA152349-01 Zehner (PI) 2011-2013 LCM Analysis and Mouse Models to Validate miRs in Prostate Tumor Progression Using LCM and RT-PCR, the goal of these studies is to examine the differential miRNA expression between human benign protastatic hyperplasia and different degrees of protastatic carcinoma. Role: Co-Investigator; my role in this research ended in late 2011 when my position at VCU changed

Non-Federal: Pharmion Corporation McCarty (PI) 2009-2011 A Phase II Study of the Use of 5-Azacytidine as Pre-Transplant Cytoreduction Prior to Allogeneic Stem Cell Transplantation for High Risk Myelodysplastic Syndromes The goal of this study was to examine the feasibility and efficacy of using the demethylating agent 5- Azacytidine prior to allogeneic stem cell transplantation in patients with high risk myelodysplastic syndrome. Role: Co-Investigator

Grant Proposal - Conflict of Interest Policy

PURPOSE The International Essential Tremor Foundation (IETF) is a nonprofit, tax-exempt organization. Maintenance of its tax- exempt status is important both for its continued financial stability and for public support.Therefore, the IRS as well as state regulatory and tax officials view the operations of the IETF as a public trust, which is subject to scrutiny by and accountable to such governmental authorities as well as to members of the public.

Consequently, there exists between the members of the Medical Advisory Board, officers and members of the IETF Board of Directors, IETF employees and the public a fiduciary duty, which carries with it a broad and unbending duty of loyalty and fidelity. The officers and members of the IETF Board of Directors and IETF employees have the responsibility of administering the affairs of the IETF honestly and prudently, and of exercising their best care, skill, and judgment for the sole benefit of the IETF. Those persons shall exercise the utmost good faith in all transactions involved in their duties, and they shall not use their positions with the IETF or knowledge gained for their personal benefit.The interests of the organization must be the first priority in all decisions and actions.

PERSONS CONCERNED This statement is directed to those persons who may submit grant proposals to the IETF Medical Advisory Board. For the purposes of this policy, a relative is any person who is related by blood or marriage, or whose relationship is similar to that of persons who are related by blood or marriage.

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AREAS IN WHICH A CONFLICT MAY ARISE NECESSITATING A REVIEW A conflict of interest, direct or indirect, may be considered to exist in some instances when individuals submit a grant:

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A conflict of interest can arise in other instances. While it is impossible to list every circumstance giving rise to a possible conflict of interest, the above will serve as a guide to the types of activities which do as well as possibly cause conflict.

Full disclosure of any situation that might raise an actual or potential conflict of interest will be reviewed by the IETF Board of Directors in order to permit an impartial and objective determination. It should be particularly noted that this disclosure relates not only to you, but also relatives.

Individuals who submit grant proposals are to read, review and sign a Conflict of Interest Questionnaire when submitting a grant.

CONFLICT OF INTEREST QUESTIONNAIRE Grant Proposal Submissions

Pursuant to the purposes and interests of the policy adopted by the IETF Board of Directors requiring disclosure of certain interests, a copy of which has been furnished to me, I hereby state that I or relatives have the following affiliations or interests and have taken part in the following transactions which, when considered in conjunction with my relationship to the International Essential Tremor Foundation (IETF) might create or be a conflict of interest. (Write “none” where applicable).

1. Advisory Board or Panel Affiliation None

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4. Financial or Material Support not otherwise listed. None

I hereby acknowledge the information given is complete and accurate to the best of my knowledge and belief. I understand that failure to accurately disclose a potential interest may cause revocation of my grant award if approved. I also understand that if any of the above circumstances change, that I am to complete a new questionnaire.

______Patricia A. Trimmer, Ph.D. ______Feb 20, 2014