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©2007 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