Grant Application Form Please complete the following form for IETF grant applications. This form and all of 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 (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: Does LINGO1 cause cerebellar and tremors?

Sponsoring Institution: Centre de recherche du CHUL Centre hospitalier universitaire de Québec (CHUQ) 2705 Boulevard Laurier, Quebec, QC, Canada, G1V 4G2

Principal Investigator: Last Name: CALON First Name: Frédéric Middle Initial: Degrees: BSc (biochemistry) BPharm (pharmacist), PhD (pharmacy/Parkinson ) Current Title/Position: Professor Department: Neuroscience Address: CHU of Quebec Research Center, 2705 Laurier Blvd, CHUL T-267 City: Quebec City State: Qc Country: CANADA Zip Code: G1V 4G2 E-mail address: [email protected] Phone: (418) 525-4444 x48697 Fax: (418) 654-2761

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.

Frederic Calon IETF 2015 Does LINGO1 cause tremor? Summary (one page or less) With the support of the IETF in 2007-2008, 2009-2010 and 2012-2013, Dr Alex Rajput, a neurologist, and Dr Frederic Calon, a neuropharmacologist, have teamed up to study the neurochemistry of essential tremor (ET). Taking advantage of a movement disorder brain bank of the highest quality, we produced series of sections from patients who have died with essential tremor (ET) or Parkinson’s disease (PD), or from individuals who have died with no neurological affliction. These cerebellar samples were compared to each other to study neuropathological alterations specifically associated with ET. Thanks to IETF support, our synergistic collaboration has already led to publications on GABA receptors and LINGO1 in Brain and Movement disorders, respectively, two of the most cited journals in the field of Neurology. Having pinpointed an increase of LINGO1 as a possible signature of a neurodegenerative process in the cerebellum of ET patients, we want now to move forward and decipher the role of LINGO1 in tremor generation. LINGO1 signaling has been identified as a pathological response to lesions in the (CNS) and could cause cerebellar injury at the origin of the tremor. However, we are still unsure whether LINGO1 is a cause or a consequence of cerebellar problems underlying the generation of tremor. It is thus extremely important to determine whether this increase in LINGO1 in the cerebellum causes tremors, because if it does, drugs aimed at reducing LINGO1 could be tried in ET. Confirming that LINGO1 causes tremor would validate it as a potential drug target for ET and potentially lead to the development of new treatments. Therefore, the objective of the present grant application is to provide a clear answer to that question and to gather preliminary data that would allow us to apply to national or international funding agencies to acquire the necessary funds for developing an animal model of ET. For the millions of people suffering from ET around the world, it is essential to mode forward in deciphering the role of LINGO in ET and validate whether it is a potential drug target for ET in clinical trials.

February 2009 one-page summary Frederic Calon IETF 2015 Does LINGO1 cause Tremor? Specific aims of the proposal (1 page maximum). Specific aim #1 (year 1, months 1-6): Generation and in vitro validation of viral vectors expressing LINGO1.

Specific aim #2 (year 1, months 7-12): To determine the acute consequence of LINGO1 upregulation, we will inject viral vectors in the cerebellum of mice to induce transient overexpression of LINGO1 and investigate the following endpoints: transfection efficiency, cerebellar LINGO1 concentrations, motor behavior (including tremor), indexes of neurodegeneration (loss of Purkinje cells (PC), Aβ42 deposition, etc) and downstream molecular players.

Rationale of the proposal and relevance to essential tremor (1-2 pages maximum). More than 10,000,000 Americans suffer from essential tremor (ET), which is almost 3 times more than Alzheimer’s disease (AD) and 10 times more than Parkinson’s disease (PD), making it the most prevalent adult-onset movement disorder13,21. Formerly regarded as benign, the symptoms of ET evolve gradually and can become very disturbing for the patients14,21. Despite the health burden associated with ET, its remains poorly understood compared to PD and AD12,14,26. Even more troubling, the pharmaceutical care of ET today still relies on primidone (a prodrug of phenobarbital) and propranolol (a β-adrenergic blocker), which were respectively introduced in 1911 and 196425. This poor therapeutic arsenal stems from two main reasons: (i) the low number of potential drug targets identified in postmortem studies and (ii) the absence of reliable animals models of the pathophysiology of ET. Central to our research program is the postulate that postmortem investigations are instrumental in the development of innovative therapy for ET. Indeed, postmortem neurochemical studies have a long history of providing pivotal data for the initiation of the most successful treatments of central nervous system (CNS) diseases22. For example, current treatment for PD is based on a discovery made in the sixties showing reduced dopamine activity in the brain. Similarly, boosting cholinergic activity in the brain of AD patients aims at replacing cholinergic of the basal nucleus of Meynert, which were reported to be degenerating in AD in the mid-seventies. Despite the high prevalence of ET, few comparable postmortem studies were performed on both large and well-characterized tissue series. In addition, unlike PD or AD, there is currently no adequate animal model that can provide insights into the pathophysiology of ET and to facilitate the development of novel drugs to treat the human disease. LINGO1 upregulation in ET LINGO1 (LRR [Leucine-rich repeat] and Ig domain-containing Nogo Receptor-interacting ) belongs to a family of four members (LINGO-1 to LINGO-4) expressed in the central nervous system. LINGO1 is a component of signaling complexes with the Nogo-66 receptor and p75 and is expressed in and neurons15,16. As part of this complex, LINGO1 is involved in inhibition of differentiation, axonal myelination and regeneration, and neuronal survival2,6,15-17,20. Expression of LINGO1 is increased after neuronal damage and its inhibition promotes functional recovery and axonal sprouting after spinal cord injury7. Accordingly, reduction of LINGO1 activity was shown to improve survival, growth, and function of dopaminergic neurons both in primary cell cultures and in an animal model of PD4,7,17. In summary, in vitro and in vivo data highlight that LINGO1 plays a major role in the unfortunate inhibition of neuronal regeneration in spinal cord and brain injury11,17. Inhibition of LINGO1 as a drug target has thus received considerable interest in the fields of multiple sclerosis17, PD4 and spinal chord injury7,11. More recently, genome-wide association studies have associated single nucleotide polymorphisms (SNP) in intron 3 of the leucine-rich repeat neuronal 6A of the LINGO-1 with ET8,9,27. With the support of the IETF, we have studied the postmortem cerebellar neurochemistry of ET patients, compared to controls and to patients who died with PD. This synergistic collaboration with Dr Rajput has first led to a publication in the journal Brain showing a decrease in GABAergic receptors in the dentate nucleus of individuals with ET compared to PD patients and controls18. Based on the molecular genetic evidence cited above, we have received funding from the IETF to turn our focus onto LINGO1 and LINGO2 in ET. We have found an increase of LINGO1 without any significant changes of LINGO2 in the cerebellar cortex from individuals with ET (see appended manuscript)3, consistent with previous work in another cohort of ET subjects10. Importantly, since increased LINGO1, as discussed above, is a possible signature of CNS injury, such an increase of LINGO1 would be consistent with a neurodegenerative process in the cerebellum of ET patients. The critical question to answer now is whether LINGO1 overexpression is a treatable condition. However, at this time, we are still unsure whether LINGO1 is a cause or a consequence of cerebellar problems underlying the generation of tremor. It is thus extremely important to determine whether this increase in LINGO1 in the cerebellum causes tremors, because if it does, it emphasizes LINGO1 blockade as a potential drug target for ET. More practically, the short-term goal of the present study is to generate preliminary data essential for application to large funding agencies to fund the development of an animal model of ET, in which to test new LINGO1-based therapeutic interventions.

February 2009 1 Frederic Calon IETF 2015 Does LINGO1 cause Tremor? BDNF EGF Figure 1. A schematic summary of molecular NgR1/p75 pathways downstream of LINGO1.

LINGO1 binds to NgR, TrkB, or EGFR receptors in the EGFR TrkB CNS, leading to a downregulation of downstream signaling pathways playing a protective role in regeneration, neuronal survival and oligodendrocyte

LINGO1 differentiation and myelination. Increased LINGO1 in ET might thus contribute to cerebellar neurodegenerative processes, leading to faulty tremorogenic information going up to cerebello-thalamo-cortical (CTC) circuits. P Blocking LINGO1 action in the cerebellum might thus RhoA PI3/akt exerts a neuroprotective effect.

Abbreviations: BDNF, brain-derived neurotrophic factor; EGFR, receptor, NgR, LINGO1 inhibits nogo receptor, P75, P75 neurotrophin receptor, TrkB, neuroprotective signaling pathways tyrosine kinase receptor B.

Research methods and procedures (1-2 pages maximum).

We hypothesize that the upregulation of LINGO1 in the cerebellum induces tremor. To directly test this hypothesis, we propose to increase LINGO1 concentrations in the cerebellum using gene transfection experiments in vivo.

Methodological rationale: LINGO1 is a transmembrane protein that acts through intercellular interactions into the cytoplasmic membrane (Figure 1). The full-length LINGO1 (614 aa) recombinant protein is thus difficult to produce and is unlikely to be active after an in vivo administration. We opted instead for a lentiviral-based strategy to transfect cerebellar cells in vivo. Transfection efficiency will first be confirmed in vitro and then in vivo and the impact on behavioral and molecular endpoints will be determined, as described below. This strategy will allow us to determine the impact of increased cerebellar concentration of LINGO1 for up to 6 weeks. Specific aim#1: Generation and validation in vitro of viral vectors expressing LINGO1 The complete cDNA sequence of human LINGO1 (NM_032808.6; GI:667751579, 99% amino acid identity with the murine ortholog) will be optimized for expression in the mouse and synthesized through GeneArt® gene synthesis (Life Technologies). A TOPO® cloning reaction will be used to insert the Lingo1 cDNA into a modified pLenti7.3/V5- TOPO® vector (Life Technologies). This lentiviral vector contains regulatory elements to increase gene expression and adds a C-terminal V5 tag to the protein. After sequence confirmation, constructs will be validated in neuroblastoma 2A (N2A) cells using immunohistochemistry (IHC) and Western blots (WB) analyses with anti-Lingo1 and anti-V5 antibodies. Alternatively, the CMV promoter will be substituted with the Pcp2 promoter to allow highly restricted expression in cerebellar Purkinje cells. 293FT producer cells will be cotransfected with the ViraPower Packaging mix to generate lentiviral particles that will serve to transduce N2A cells in vitro and revalidate the constructs before in vivo experiments. Escalating dose of viral vectors will be investigated to determine the best dose to be used in vivo. Specific aim#2: To determine the acute consequence of LINGO1 upregulation, viral vectors will be injected in the cerebellum of mice to induce transient overexpression of LINGO1 and investigate the following endpoints: transfection efficiency, cerebellar LINGO1 concentrations, motor behavior and fine motor skills (including tremor), indexes of neurodegeneration (loss of Purkinje cells, Aβ42 deposition, etc) and downstream molecular players. Approximately 60 adult male mice of c57bl/6 background will be used. Animals will be placed on a stereotaxic apparatus under anesthesia. The injection coordinates will be selected according to the Franklin and Paxinos atlas to target different subregions of the cerebellum. The lentivirus particles (~1.5 x 107 pfu) will be delivered using a Hamilton syringe (5 µl) connected to a hydraulic system to inject the solution at rate 0.25 µl/min. Needles will be left for an additional 5 min after completion of the injection to allow diffusion of the solution into the brain tissue. Lentivirus- mediated transgene expression normally lasts up to 6 weeks1,5. Experiment #1 (Method assessment) Groups of mice will receive injections of lenti-Lingo1 or lenti-Ctrl into the cerebellum. The cerebellar cortex, the dentate nucleus (DN) and the white matter close to the DN will be targeted. Three and 6 weeks after injection, levels of Lingo1 and other cerebellar will be investigated by WB and IHC, as well as the V5-tag to determine transfection efficiency in the subregions injected. February 2009 2 Frederic Calon IETF 2015 Does LINGO1 cause Tremor? Experiment #2 (Investigation of tremor induction) The procedures, optimal doses and time points will be dictated by the results of Experiment #1. At least 20 mice per group will be injected with lenti-Lingo1 or lenti-Ctrl (20 x 2 = 40). Behavioral endpoints will be monitored at least twice a week (see below). Molecular endpoints described below will be assessed postmortem. Assessment of motor behavior: First, measures of locomotor activity using the open field system (Lafayette Instrument, IN, USA) will be performed using a software which provides measures of distance traveled, ambulatory counts, vertical counts, resting time, ambulatory time and stereotypies. Second, fine motor skills will be assessed using challenging beam traversal and the pole test. The challenging beam traversal consists in observing the animal ease in crossing 4 x 25 cm narrowing Plexiglas beams and counting errors, number of steps made by each animal, and the time necessary to fully cross the beams. The pole test assesses the capacity of the animal to descend a 50-cm wooden pole to their home cage and is also commonly used with animal models of PD. In addition, muscle tone will be controlled with a wire suspension test, a procedure in which the mouse is suspended by its forelimbs to a stretched 2-mm diameter cable and the time that the animal remains clinging to the cable is measured. We routinely perform these tasks with the material and equipment already available in our laboratories. Future aim: Although the above tests will provide a qualitative assessment of tremor, they do not have the quantitative value of an electromyogram (EMG). To directly investigate tremor, we plan to implant mice with chronic electromyogram (EMG) electrodes in the hamstring and quadriceps muscle groups of the right hindlimb to record EMG in each of the mice during spontaneous activity before and after stereotaxic injection of LINGO vectors. Spectral analysis can be used to quantify the amplitude and frequency of tremor found in the EMGs after transfection. However, for budgetary reasons, this is beyond the scope of the present grant proposal. Tissue processing For Western blot (WB): At sacrifice, 2/3 of the animals (n=12) will be perfused with 0.9% normal saline followed by Hepes buffer (50mM, pH 7.2) containing inhibitors of proteases and phosphatases. The cerebellum will be frozen for cryostat-sectioning into coronal (12 µm) sections and dissected into cerebellar cortex or white matter samples24. For immunohistochemistry (IHC) and in situ hybridization (ISH): One-third (n=6) of animals will be perfused with with 0.9% normal saline followed by 4% paraformaldehyde (PFA) and their brain collected and postfixed in 4% PFA, cryoprotected with 20% sucrose-PBS, snap frozen at –70°C, and sectioned on a freezing microtome into coronal (25 µm) sections23,24. Assessment of changes in LINGO1 downstream signaling pathways: As introduced above, LINGO1 is CNS-specific, mostly expressed in neurons and oligodendrocytes. From a cellular perspective, it is involved in several important signaling axes driving the unfortunate inhibition of neuronal regeneration in spinal cord and brain injury17. To dissect the impact of LINGO1 upregulation, key molecular players closely associated with LINGO1 will be investigated, primarily using WB in homogenates from cerebellar cortex and white matter. When a 2D view is needed, IHC will be performed. Colocalization with neurons, astrocytes, microvessels, Purkinje cells and oligodendrocytes will be assessed using antibodies for neuronal nuclei (NeuN), glial fibrillary acidic protein (GFAP), collagen IV, calbindin, and basic protein (MBP), respectively. First, LINGO1 binds to p75/NgR1 and contributes to RhoA activation, thereby inhibiting neurite growth, oligodendrocyte differentiation, myelination and axon regeneration (Figure 1)6,7,15-17,20,27. Hence, protein levels of p75, NgR1, TROY, Rho kinase (ROCK) and RhoA will be assessed by WB/IHC after injection of viral vectors. Second, through its cytoplasmic tail, LINGO1 interacts with epidermal growth factor receptor (EGFR) to downregulate EGFR–PI3K–Akt signaling, which have been suggested to negatively impact Purkinje cell survival (Figure 1)17,27. Therefore, EGFR will be evaluated as well as other key players of the PI3K–Akt pathway using WB/IHC. Third, LINGO1 negatively interacts with members of the TRK family of receptors, thereby influencing the signaling pathways of key such as NGF, BDNF and GDNF17,27. Since this action has been particularly demonstrated with TrKB (Figure 1), BDNF levels will be measured by ELISA and TrKB with WB. Fourth, much of the work on LINGO1 has focused on its association with myelin pathology2,11,16,17,19. Hence, we will investigate key markers of oligodendrocyte maturation in vivo, MBP, myelin-associated glycoprotein (MAG) and myelin proteolipid protein (PLP) to probe for axonal degeneration/demyelination in the white matter of the cerebellum. Assessment of cerebellar neurodegeneration We will first investigate whether LINGO1 upregulation impacts Purkinje cell counts, which will be assessed by estimating the number of calbindin-positive cells using stereological procedures after performing calbindin IHC on PFA-fixed horizontal sections of the cerebellum, as described23. Immunolabeling will be revealed using Alexa Fluor- conjugated secondary antibodies (Life Technologies) and visualized using semi-motorized light/fluorescence February 2009 3 Frederic Calon IETF 2015 Does LINGO1 cause Tremor? microscopes (E800, Nikon) equipped with unbiased quantification systems (Stereoinvestigator) and high-resolution digital cameras. To further assess Purkinje cell loss, calbindin mRNA will also be assessed using in situ hybridization.

Since we have recently found a strong correlation between LINGO1 and insoluble amyloid-β42 (Aβ42) concentrations in the cerebellar cortex of ET patients (r2=0.71, P=0.005, unpublished data), we will quantify by ELISA concentrations of Aβ, which is also associated with neurodegeneration in Alzheimer’s disease24. Other markers of neurodegenerative proteinopathies such as Total tau (antibodies tau-13, HT7 and tau-5 and phosphorylated tau (antibodies AT8, AT100, AT270, CP13 and AD2) will be assessed using WB24. Each experiment will be repeated 3 times. Quantification will be performed with a KODAK 4000MM Digital Imaging System and Carestream MI software. Future aim: If the results suggest a dysfunction of Purkinje or DN cells, we plan to perform electrophysiological patch- clamp recording of these cells on cerebellar slice preparations to measure passive/active membrane intrinsic properties and synaptic activities (ex: EPSC and IPSC). This is beyond the scope of the present grant application. Data and statistical analysis For all analysis, an “n” of 12 mice will be used for a ~98% probability of detecting a 20% change if alpha is set at 0.05 and standard deviations are 15% of average. Statistical comparisons of medians between groups will be performed using a Kruskal-Wallis and/or Mann-Whitney-Wilcoxon nonparametric tests. Coefficients of correlation and significance of the linear relationship between parameters will be determined with a simple regression model. All statistical analysis will be performed using JMP Statistical Analysis Software (v 10.0) and P values under 0.05 will be considered significant. Anticipated results (half-page maximum). The proposed experiments in animals will determine whether an upregulation of LINGO1 in the cerebellum induces pathological changes, drives neurodegeneration and/or induces tremors. If our hypothesis is correct and LINGO1 transfected into the cerebellum causes tremors, we will have in hand one of the first animal models of ET, at least in a transient manner. The information gathered from the present project will also tell us whether LINGO1 is a druggable target in ET. This is not such a long term deliverable since LINGO1 antagonists are already under development to promote neurorestoration and in (ClinicalTrials.gov Identifier = NCT01864148)15,17,27, and could thus be used in ET as well. Furthermore, we expect that the data generated will be critical to secure additional funding to develop a transgenic mouse overexpressing LINGO1 in the cerebellum. The fact that the increase in LINGO1 is particularly prominent when disease duration exceeded 20 years (see appended manuscript) may suggest that the tremorogenic effect of LINGO1 may be more important on the long-term. Such a transgenic mouse could become an excellent animal model of ET. In summary, LINGO1 upregulation has been identified in the cerebellum of ET patients, and we need to know if it plays a causal role in the disease. For the millions of people suffering from ET worldwide, it is essential to move forward in deciphering the role of LINGO1 in ET. References: 1. Bissonnette, S., Muratot, S., Vernoux, N., Bezeau, F., Calon, F., Hebert, S.S. and Samadi, P., The effect of striatal pre- enkephalin overexpression in the basal ganglia of the MPTP mouse model of PD., Eur J Neurosci, 40 (2014) 2406-2416. 2. Bourikas, D., Mir, A. and Walmsley, A.R., LINGO-1-mediated inhibition of oligodendrocyte differentiation does not require the leucine-rich repeats and is reversed by p75(NTR) antagonists., Mol Cell Neurosci, 45 (2010) 363-369. 3. Delay, C., Tremblay, C., Brochu, E., Paris-Robidas, S., Emond, V., Rajput, A.H., Rajput, A. and Calon, F., Increased LINGO1 in the cerebellum of essential tremor patients., Mov Disord, 29 (2014) 1637-1647. 4. Inoue, H., et al., Inhibition of the leucine-rich repeat protein LINGO-1 enhances survival, structure, and function of dopaminergic neurons in PD models., Proc Natl Acad Sci U S A, 104 (2007) 14430-14435. 5. Jakobsson, J., Ericson, C., Jansson, M., Bjork, E. and Lundberg, C., Targeted transgene expression in rat brain using lentiviral vectors., J Neurosci Res, 73 (2003) 876-885. 6. Jepson, S., et al., LINGO-1, a transmembrane signaling protein, inhibits oligodendrocyte differentiation and myelination through intercellular self-interactions., J Biol Chem, 287 (2012) 22184-22195. 7. Ji, B., et al, LINGO-1 antagonist promotes functional recovery and axonal sprouting after ., Mol Cell Neurosci, 33 (2006) 311-320. 8. Jimenez-Jimenez, F.J., Garcia-Martin, E., Lorenzo-Betancor, O., Pastor, P., Alonso-Navarro, H. and Agundez, J.A., LINGO1 and risk for essential tremor: results of a meta-analysis of rs9652490 and rs11856808., J Neurol Sci, 317 (2012) 52-57. 9. Kuhlenbaumer, G., Hopfner, F. and Deuschl, G., Genetics of essential tremor: meta-analysis and review., Neurology, 82 (2014) 1000-1007. 10. Kuo, S.H., et al., Lingo-1 expression is increased in essential tremor cerebellum and is present in the basket cell pinceau., Acta Neuropathol, 125 (2013) 879-889. 11. Llorens, F., Gil, V. and del Rio, J.A., Emerging functions of myelin-associated proteins during development, neuronal plasticity, and neurodegeneration., FASEB J, 25 (2011) 463-475. 12. Louis, E.D., Essential tremor: evolving clinicopathological concepts in an era of intensive post-mortem enquiry., Lancet Neurol, 9 (2010) 613-622.

February 2009 4 Frederic Calon IETF 2015 Does LINGO1 cause Tremor? 13. Louis, E.D. and Ferreira, J.J., How common is the most common adult movement disorder? Update on the worldwide prevalence of essential tremor., Mov Disord, 25 (2010) 534-541. 14. Louis, E.D. and Okun, M.S., It is time to remove the ‘benign’ from the essential tremor label., Parkinsonism Relat Disord, 17 (2011) 516-520. 15. Mi, S., et al., LINGO-1 is a component of the Nogo-66 receptor/p75 signaling complex., Nat Neurosci, 7 (2004) 221-228. 16. Mi, S., et al., LINGO-1 negatively regulates myelination by oligodendrocytes., Nat Neurosci, 8 (2005) 745-751. 17. Mi, S., Pepinsky, R.B. and Cadavid, D., Blocking LINGO-1 as a therapy to promote CNS repair: from concept to the clinic., CNS Drugs, 27 (2013) 493-503. 18. Paris-Robidas, S., Brochu, E., Sintes, M., Emond, V., Bousquet, M., Vandal, M., Pilote, M., Tremblay, C., Di Paolo, T., Rajput, A.H., Rajput, A. and Calon, F., Defective dentate nucleus GABA receptors in essential tremor., Brain, 135 (2012) 105-116. 19. Pedersen, S.F., Pullman, S.L., Latov, N. and Brannagan, T.H., Physiological tremor analysis of patients with anti-myelin- associated glycoprotein associated neuropathy and tremor., Muscle Nerve, 20 (1997) 38-44. 20. Petrinovic, M.M., et al., Neuronal Nogo-A regulates neurite fasciculation, branching and extension in the developing nervous system., Development, 137 (2010) 2539-2550. 21. Rajput, A., Robinson, C.A. and Rajput, A.H., Essential tremor course and disability: A clinicopathologic study of 20 cases., Neurology, 62 (2004) 932-936. 22. Rajput, A.H., Contributions of human brain biochemical studies to movement disorders., Parkinsonism Relat Disord, 8 (2002) 425-431. 23. St-Amour, I., Bousquet, M., Pare, I., Drouin-Ouellet, J., Cicchetti, F., Bazin, R. and Calon, F., Impact of IVIg on the dopaminergic system and immune response in the acute MPTP mouse model of PD, J Neuroinflammation, 9 (2012) 234. 24. St-Amour, I., Pare, I., Tremblay, C., Coulombe, K., Bazin, R. and Calon, F., IVIg protects the 3xTg-AD mouse model of Alzheimer’s disease from memory deficit and Abeta pathology., J Neuroinflammation, 11 (2014) 54. 25. Zesiewicz, T.A., et al., Evidence-based guideline update: treatment of essential tremor: report of the Quality Standards subcommittee of the American Academy of Neurology., Neurology, 77 (2011) 1752-1755. 26. Zeuner, K.E. and Deuschl, G., An update on tremors., Curr Opin Neurol, 25 (2012) 475-482. 27. Zhou, Z.D., Sathiyamoorthy, S. and Tan, E.K., LINGO-1 and Neurodegeneration: Pathophysiologic Clues for Essential Tremor., Tremor Other Hyperkinet Mov (N Y), 2 (2012)

Detailed budget and justification (1 page maximum). Table 2 - Summary of Budget (for 1 year) Total (year 1) Key Personnel salaries Research assistant (Vincent Emond)(20%) + benefits $12089 + 1631$ Expendables (incl. Prov/FedTaxes and shipping costs) LINGO1 constructions and lentiviral particles $2800 Cell culture expendables $1000 Animals $725 Primary antibodies (8; 350$each) $2990 Histology/immunochemistry/Western blots/Elisa $3765 Total (USD) $25,000 Justification: A) Key Personnel: Vincent Emond is a highly skilled research assistant, specialized in cellular and molecular biology and working in our laboratory since November 2003. He will perform all transfection, ELISAs, Western blots and immunohistofluorescence experiments. He will be assisted by graduate students hired at the faculty of pharmacy and paid by other sources. Vincent will also perform data analysis. B) Expendables: LINGO1 constructs and lentiviral particles, cell culture reagents and materials, and animals. Primary antibodies are necessary for Western blots and immunofluorescence experiments. One Elisa kit (1275$). Histology/immunochemistry/Western reagents include secondary antibodies, revelation reagents, cost for the use of microscope, etc C) PI salaries, facilities & administrative costs are covered by Laval university and/or the research center, and health insurances costs by the Government of Quebec. Our team performs research activities in one of the most successful research centers in Canada, the Centre Hospitalier Universitaire de Québec Laval Research Center (CR-CHUQ), where more than 450 principal investigators and nearly 900 graduate students are currently working. Our laboratory is fully equipped, including for example a complete Waters HPLC system, a Synergy™ HT Multi-Detection Microplate Reader and a KODAK (Carestream) Image Station 4000 Digital Imaging Systems.

February 2009 5 Frederic Calon IETF 2015 Does LINGO1 cause Tremor? Annex

A) Biographic sketch of principal investigator and all professional personnel participating in the project (standard NIH format, including biosketch and other support).

B) Copies of relevant abstracts and/or articles that have been published, are in press, or have been submitted for publication. The selected paper contains the key data supporting the present grant application. Please note that our work on LINGO1 in ET cerebellum is one of the top-5 most shared articles in Movement Disorders for 2014.

Delay, C., Tremblay, C., Brochu, E., Paris-Robidas, S., Emond, V., Rajput, A.H., Rajput, A. and Calon, F., Increased LINGO1 in the cerebellum of essential tremor patients., Mov Disord, 29 (2014) 1637-1647.

C) Completed conflict of interest questionnaire.

February 2009 6 BIOGRAPHICAL SKETCH NAME POSITION TITLE Calon, Frédéric, B. Sc., B.Pharm., Ph.D. Professor of Pharmacy

EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training.)

INSTITUTION AND LOCATION DEGREE YEAR(s) FIELD OF STUDY (if applicable) UCLA (Greg Cole) Post-doc 2002-2003 Alzheimer UCLA (William Pardridge) Post-doc 2001-2002 Brain drug delivery Université Laval (Québec, Canada) Ph.D. 1998-2001 Parkinson Université Laval (Québec, Canada) B.Pharm. 1994-1997 Pharmacy Université Laval (Québec, Canada) M.Sc. 1992-1994 Parkinson Université Laval (Québec, Canada) B.Sc. 1989-1992 Biochemistry A) Positions and Honors. (list in chronological order previous positions, concluding with your present position. List any honors. Include present membership on any public or private advisory committee.) Positions and Employment Since 06/2011- Full Professor, Faculty of Pharmacy, Université Laval (Canada) Since 11/2014- Director, International associated Laboratory OptiNutriBrain (France-Canada), 2003-2011 Assistant/Associate Professor, Faculty of Pharmacy, Université Laval (Canada) Since 07/2003- Principal Investigator, Centre de recherche du CHU de Québec (CHU-Q) Since 2009 Member, l’Institut sur la nutrition et les aliments fonctionnels (INAF) Consulting Experience and Professional Membership (selected) Ad-hoc journal article peer review committees: 10-20 papers reviewed per year since 2004. Grant peer review committees: Alzheimer Association USA (2-3 grant reviews/year since 2006); Alzheimer Society Canada (53 grant reviewed from 2006 to 2011 as reviewer or chairman). Canadian Institutes of Health Research (CIHR) as external (8 since 2005) and internal (61 since 2005) board reviewer. Other national or international review board (5-10 grants/y). Professional affiliations: 1) Member of the Ordre des Pharmaciens du Québec (Pharmacy practice license #: 498100-7); 2) Member of the Society for Neuroscience (#100006813); 3) Member of the Canadian Pharmacists Association (#0043261); Honors Date Description Organisms Jul 2013 – Jun 2017 Salary Award FRQ-S Chercheur-boursier senior (244 743$) Jan 2013 Personality of the week Radio-Canada/Le Soleil (Quebec city) Dec 2012 Quebec discovery of the year Top-10 selection in the province (Quebec Science) Jul 2010- Jun 2013 Salary Award FRSQ Chercheur-boursier (204 493$/3y) July 2008 New Investigator Research Association of Faculties of Pharmacy of Canada - Award for 2008 AstraZeneca (3000$CDN) Jul 2005- Jun 2010 New Investigator award Canadian Institutes of Health Research (CIHR)– (Health Professional) Total salary: 282 500$ (5 y) April 2005 Manuscript Award IMS Health Canada (3000$CDN) Jul 2001- Jul 2005 Senior Fellowship Canadian Institutes of Health Research (CIHR) June 2002 Ph.D. thesis Distinction Gold Medal from the Governor General of Canada (best Ph.D. thesis of Laval University in 2001-02) March 2001 Manuscript Award IMS Health Canada (1500$ CDN) Jul 2001- Jul 2003 Senior post-doc fellowship CIHR (49000$can/year) (Phase 1) Jan 1998- Ph. D. Studentship Canadian Medical Research Council, Novartis and FRSQ July 2001 (Health Professional) (27100$ CDN/year) May 1992- Aug 1994 M.Sc. Studentship Canadian Network for excellence (15295$ CDN /year) Jul2005- Jun2010 New Investigator award Canadian Institutes of Health Research (CIHR)– (Health Professional) Total salary: 282 500$ (5 y) B) Selected peer-reviewed publications (from a total of 92 ; h-index = 37) (Impact factor) 1. Salem, N., Vandal, M., & Calon, F. (2014) The Benefit of Docosahexaenoic Acid for the Adult Brain in Aging and Dementia. PLEFA 92: 15-22 (2.9) 2. Vandal, M., White, P.J., Tremblay, C., St-Amour, I., Chevrier, G., Emond, V., Lefrançois, D., Virgili, J., Planel, E., Giguere, Y., Marette, A., Calon, F. (2014) Insulin reverses the high fat diet-induced increase in brain Aβ and improves memory in an animal model of Alzheimer’s disease. Diabetes 63:4291-301 (8.5). 3. Alata, W., Yue, Y., St-Amour, I., Vandal, M. & Calon, F. (2014) Human apolipoprotein E ε4 expression impairs cerebral vascularization and blood-brain barrier function in mice. J Cereb Blood Flow Metab. 35: 86-94. (5.4). 4. Conway, V., Larouche, A., Alata, W., Vandal, M., Calon, F. & Plourde M. (2014) Apolipoprotein E isoforms disrupt

1 long-chain fatty acids distribution in the plasma, the liver and the adipose tissue of mice. PLEFA 91: 261-7 (2.9) 5. St-Amour, I.,Paré, I., Tremblay C., Coulombe, K., Bazin R., & Calon, F. (2014) IVIg protects the 3xTg-AD mouse model of Alzheimer's disease from memory deficit and Aβ pathology. J Neuroinflammation 22: 54 (4.4) 6. Delay, C†., Tremblay, C†., Brochu, E., Paris-Robidas, S., Emond, V., Rajput A.H., Rajput A., & Calon, F. (2014) Increased LINGO1 in the cerebellum of essential tremor patients. Mov Disord, 29 1637-1647 (4.6) † co-first authors. doi: 10.1002/mds.25819 7. Ohta, Y., Tremblay C., Schneider, J., Bennett, D., & Calon, F., & Julien J.P. (2014) Interaction of Transactive Response DNA Binding Protein 43 with nuclear factor B in Mild Cognitive Impairment with Episodic Memory Deficits. Acta Neuropathologica Communications 2:37. 8. Joffre, C., Nadjar, A., Lebbadi, M., Calon F & Layé, S. (2014) n-3 LCPUFA improves cognition: the young, the old and the sick. PLEFA 91:1-20 (2.9). http://dx.doi.org/10.1016/j.plefa.2014.05.001 9. Bissonnette, S., Muratot, S., Vernoux, N., Bezeau, F., Calon, F., Hébert. S.S., Samadi, P. (2014) The effect of striatal pre-enkephalin overexpression in the basal ganglia of MPTP mouse model of Parkinson’s disease. Eur J Neurosci 40:2406-16. (3.7). 10. Do, T.M., Alata, W., Dodacki, A., Traversy, M-T., Chacun, H., Pradier, L, Scherrmann, J.M., Farinotti, R.,& Calon, F & Bourasset, F. (2014) Altered cerebral vascular volumes and solute transport at the blood-brain barriers of two transgenic mouse models of Alzheimer's disease. Neuropharmacology. 81:311-7 (3.7) 11. Petry F.R. Pelletier J., Bretteville, A., Morin, F., Calon, F., Hebert, S.S., Whittington, R.A., & Planel, E. (2014) Specifity of anti-tau antibodies when analyzing mice models of Alzheimer's disease: problems and solutions. PLoS ONE. 9:e94251 (4.3) 12. Vandal, M., Alata, W., Rioux-Perreault, C., Tremblay C., Salem Jr N., Calon, F.,† & Plourde, M.† (2014) Reduction in DHA transport to the brain of mice expressing human APOE4 compared to APOE2. J Neurochem. 129: 516-26.† co-PI. doi: 10.1111/jnc.12640 (4.3). 13. Alata, W., Paris-Robidas, S., Emond, V., Bourasset, F., & Calon F (2014) Brain Uptake of a Fluorescent Vector Targeting the Transferrin Receptor: A Novel Application of in Situ Brain Perfusion. Mol Pharm. 11: 243-53. (4.6) doi: 10.1021/mp400421a 14. St-Amour, I.,Paré, I., Alata, W., Coulombe, K., Ringuette-Ouellet, C, Drouin-Ouellet, J., Soulet, D., Bazin R., & Calon, F. (2013) Brain bioavailability of human intravenous immunoglobulin and its transport through the murine blood-brain barrier. J Cereb Blood Flow Metab. 33: 1983-92. (5.4). 15. Arsenault, D. †, Dal-Pan, A. †, Tremblay, C., Bennett, D. A., Guitton, M. J., De Koninck, Y., Tonegawa, S., & Calon, F. (2013) PAK inactivation impairs social recognition in 3xTg-AD mice without increasing brain deposition of tau and Aβ. J Neurosci 33: 10729-10740. (7.2). † co-first authors 16. St-Amour, I., Bousquet, M., Paré, I., Drouin-Ouellet, J., Cicchetti, F., Bazin R., & Calon, F. (2012) Impact of intravenous immunoglobulin on the dopaminergic system and immune response in the acute MPTP mouse model of Parkinson's disease. J Neuroinflammation 9: 234. (5.8) 17. Bories, C. †, Guitton, M. †, Julien, C., Tremblay, C., Vandal, M., Msaid, M., De Koninck Y., & Calon, F. (2012) Sex- dependent alterations of social behavior and cortical synaptic activity coincide at different ages in a model of Alzheimer’s disease. PLoS ONE 7:e46111. (4.3) † co-first authors 18. Arsenault, D., Julien, C., Chen C.T., Bazinet R.P. & Calon, F. (2012) Dietary intake of unsaturated fatty acids modulates physiological properties of entorhinal cortex neurons in mice. J Neurochem 122:427-43. (4.0). 19. Paris-Robidas, S*., Brochu, E*., Sintes, M., Emond, V., Bousquet, M., Vandal, M., Pilote, M., Tremblay, C., Di Paolo, T., Rajput A.H., Rajput A., & Calon, F. (2012) Defective GABAergic cerebellar output in essential tremor. *equal contribution. Brain. 135: 105-116. on-line Nov 26, 2011 (9.2). 20. Bousquet, M., St-Amour, I., Vandal, M., Julien, P., Cicchetti, F. & Calon, F. (2012) High-fat diet exacerbates MPTP-induced dopaminergic degeneration in mice. Neurobiol Dis 45: 529-538. -on-line Sept 24 2011- (5.1) 21. Delay, C., Calon, F., Mathews, P., & Hebert, S.S. (2011) Alzheimer-specific variants in the 3'UTR of Amyloid precursor protein affect microRNA function. Mol Neurodegener 6:70. (5.4) 22. Drouin-Ouellet, J., Gibrat, C., Bousquet, M., Calon, F., Kriz, J. & Cicchetti, F. (2011) The role of the MYD88- dependent pathway in MPTP-induced brain dopaminergic degeneration. J Neuroinflammation 8: 137. (5.8) 23. Lebbadi, M., Julien, C., .., Kang, J.X. & Calon, F. (2011) Endogenous conversion of omega-6 into omega-3 fatty acid improves neuropathology in an animal model of Alzheimer disease. J Alzheimer Dis 27: 853-869. (4.1). 24. Arsenault, D., Julien, C., & Calon, F. (2012) Chronic dietary intake of alpha-linolenic acid does not replicate the effects of DHA on passive properties of entorhinal cortex neurons. Br J Nutr. 107: 1099-1111. (3.1) 25. Tremblay, C., St-Amour, I., Schneider, J., Bennett, D. & Calon, F. (2011) Accumulation of TDP-43 in mild cognitive impairment and Alzheimer’s disease. J Neuropathol Exp Neurology 70 (9): 788-98. (5.1) 26. Lalancette-Hebert, M., Julien, C., Cordeau, P. Jr., Weng, Y.C., Bohacek, I., Calon, F.†, & Kriz J†. (2011) Accumulation of Dietary DHA in the Brain Attenuates Acute Immune Response and Development of Post- ischemic Neuronal Damage. Stroke 42: 2903-9 –on-line Aug 18 2011- (7.1) † co-senior author 27. Calon, F. (2011) Omega-3 polyunsaturated fatty acids in Alzheimer’s disease: key questions and partial answers. Current Alzheimer’s Research. 8 (5): 470-8. (4.1) 28. Paris-Robidas, S., Emond, V., Tremblay, C., Soulet, D., & Calon, F. (2011) In vivo labeling of brain capillary 2 endothelial cells following intravenous injection of monoclonal antibodies targeting the transferrin receptor. Mol Pharm. 80: 32-39. (4.7) 29. Arsenault, D., Julien, C., Tremblay, C. & Calon, F. (2011) DHA improves cognition and prevents dysfunction of entorhinal cortex neurons in 3xTg-AD mice. PLoS ONE 6: e17397. (4.3) 30. Bousquet, M., Gue, K., Emond, V., Julien, P., Kang, J. X., Cicchetti, F. & Calon, F. (2011) Transgenic conversion of omega-6 into omega-3 fatty acids in a mouse model of Parkinson’s disease. J Lipid Res 52: 263-271. (4.9) 31. Bousquet, M., Calon, F., & Cicchetti, F. (2011) Impact of omega-3 fatty acids in Parkinson's disease. Ageing Res Rev 10: 453-63. (on-line Mars 22 2011) (5.6). 32. Zhao, Y., Calon, F., Julien, C., … & Bazan, N.G. (2011) Docosahexaenoic acid-derived neuroprotectin D1 induces neuronal survival via secretase- and PPARγ-mediated mechanisms in AD models. PLoS ONE 6: e15816. (publié en ligne le 7 janvier 2011) (4.3) 33. Hamilton, L., Aumont, A., Julien, C., Vadnais, A., Calon, F., & Fernandes, K. (2010) Acceleration of aging- dependent changes in adult neural stem cell populations of the 3xTg mouse model of AD. Eur. J. Neurosci. 32: 905-920. (3.4). 34. Julien, C., Tremblay, C., Phivilay, P., Berthiaume, L., M. A., Emond, V., Julien, P. & Calon, F. (2010) High-fat diet aggravates amyloid-beta and tau pathologies in the 3xTg-AD mouse model. Neurobiol Aging 31 (1): 1516-1531 (6.0) (published on-line in October 2008) 35. Ouellet, M., Emond, V., Chen, C.T., Julien, C., Bourasset, F., Oddo, S., LaFerla, F., Bazinet, R., & Calon, F. (2009) Diffusion of docosahexaenoic and eicosapentaenoic acid through the blood-brain barrier: an in situ cerebral perfusion study. Neurochem Int 55 (2-3):476-82. (3.0) 36. Julien, C., Tremblay, C., Emond, V., Lebbadi, M., Salem, N. Jr, Bennett, D. A. & Calon, F. (2009) SIRT1 decrease parallels the accumulation of tau in AD. J Neuropathol Exp Neurology 68 (1): 48-58. (5.1) 37. Bourasset, F., Ouellet, M., Tremblay, C., Julien, C., Oddo, S., LaFerla, F., & Calon, F. (2009) Reduction of the cerebrovascular volume in a transgenic mouse model of AD. Neuropharmacology 56: 808-813. (3.7) 38. Chen, C.T., Liu, Z., Ouellet, M., Calon, F.†, & Bazinet, R., (2009) Rapid beta-oxidation of eicosapentaenoic acid in mouse brain: an in situ study. PLEFA. 80 (2-3):157-63 (2.0) † co- senior author. 39. Phivilay, A., Julien, C., Tremblay, C., Berthiaume, L., Giguère, Y., Julien, P. & Calon, F. (2009) High dietary consumption of trans fatty acids decreases brain DHA but does not alter Aβ and tau pathology in the 3xTg-AD model of Alzheimer’s disease. Neuroscience 159 (1): 296-307 (3.6) 40. Bousquet, M., St-Pierre, M., Julien, C., Salem, N., Jr., Cicchetti, F. & Calon, F. (2008) Beneficial effects of dietary omega-3 polyunsaturated fatty acid on toxin-induced neuronal degeneration in an animal model of Parkinson’s disease. FASEB J 22 (4): 1213-1225. (7.0) 41. Julien, C., Tremblay, C., …, Emond, V. & Calon, F. (2008) Decreased drebrin mRNA expression in Alzheimer disease: correlation with tau pathology. J Neurosci Res. 86 (10): 2292-302. (3.6) 42. Calon, F*. & Cole, G. M. (2007) Neuroprotective action of omega-3 polyunsaturated fatty acids against neurodegenerative : evidence from animal studies. Prostaglandins, Leukot Essent Fatty Acids. (peer- reviewed review) 77 (5-6):287-293. (2.0) (*corresponding author) (Best cited 2006-2008) 43. Tremblay, C., Pilote, M., Phivilay, A., Émond, V., Bennett, D. A., & Calon, F. (2007) Biochemical characterization of Aβ and tau pathologies in MCI and AD. J Alzheimer Dis 12 (4): 377-390. (4.1) 44. Rivest, V., Phivilay, A., Julien, C., Bélanger, S., Tremblay, C., Émond, V. & Calon, F. (2007) Novel liposomal formulation for targeted gene delivery. Pharm Res 24 (5): 981-990. (3.4) 45. Morelli, M., Di Paolo, T, Wardas, J., Calon, F., Danqing, X., & Schwarzschild, M. (2007) Role of adenosine A2A receptors in parkinsonian motor impairment and L-DOPA-induced motor complications. Prog Neurobiology 83 (5): 293-309. (13.0) 46. Samadi, P., Gregoire, L., Morissette, M., Calon, F., Hadj Tahar, A., Dridi, M., Belanger, N., Meltzer, L. T., Bedard, P. J., and Di Paolo, T. (2007) mGluR5 metabotropic glutamate receptors and dyskinesias in MPTP monkeys. Neurobiol Aging 29: 1040 – 51. (6.0). 47. Julien, C., Berthiaume, L., Hadl Tahar, A., Rajput, A. H., Bedard, P. J. Di Paolo, T., Julien, P., & Calon, F. (2006) Postmortem brain fatty acid profile of levodopa-treated Parkinson disease patients and parkinsonian monkeys. Neurochem Int 48: 404 – 414. (3.0) 48. Schapira A.H.V., Bezard E., Brotchie J., Calon F., Collingridge G.L., Ferger B., Hengerer B., Hirsch E., Jenner P., Le Novere N., Obeso J.A., Schwarzschild M.A., Spampinato U. & Davidai G. (2006) Novel pharmacological targets for the treatment of Parkinson’s disease. Nature Reviews Drug Discovery 5 (10): 845-854. (22.4) 49. Zhao, L., Ma, Q.L., Calon, F., Harris-White, M., Yang, F., Lim, G.P., Morihara, T., Ubeda, O., Ambegaokar, S., Teter, B., Frautschy, S., & Cole, G.M. (2006) Role of p21-Activated Kinase Pathway Defects in Alzheimer's Disease Cognitive Deficits. Nature Neuroscience 9: 234 – 242. (17.1) 50. Calon, F., (2006) Non-patentable drugs and the cost of our ignorance. (peer-reviewed commentary) CMAJ 174 (4): 483-4. (6.7) 51. Calon, F., Lim, G. P., Yang, F., Morihara, T., Ubeda, O., Salem, N., Jr., Frautschy, S. A., & Cole, G. M. (2005) Dietary n-3 polyunsaturated fatty acid depletion activates caspases and decreases NMDA receptor in the brain of a transgenic mouse model of Alzheimer's Disease. Eur J Neurosci 22: 617-26. (4.1). 3 52. Lim, G.P., Calon, F., Morihara, T., Yang, F., Teter, B., Ubeda, O., Salem, N. (Jr.), Frautschy, S., & Cole, G.M. (2005) A diet enriched with the omega-3 fatty acid docosahexaenoic acid (DHA) reduces amyloid burden in an aged Alzheimer mouse model. J Neurosci. 25: 3032-40. (8.1) 53. Calon, F., Lim, G.P., Yang, F., Morihara, T., Teter, B., Ubeda, O., Rostaing, P., Triller, A., Salem, N. (Jr.), Ashe, K.H., Frautschy, S., & Cole, G.M. (2004) Docosahexaenoic acid protects from dendritic pathology in an Alzheimer’s disease mouse model. 43: 633-645. (14.7) 54. Calon, F., Dridi, M., Hornykiewicz, O., Rajput, A.H., Bédard P.J., & Di Paolo, T. (2004) Increased adenosine A2A receptors in the brain of Parkinson’s disease patients with dyskinesias. Brain 127 (5): 1075-84. (9.5) 55. Calon, F., Rajput, A. H., Hornykiewicz, O., Bedard, P. J., & Di Paolo, T. (2003) Levodopa-induced motor complications are associated with alterations of glutamate receptors in PD. Neurobiol Dis 14 (3): 404-16. (4.6) 56. Calon, F., Morissette, M., Rajput, A. H., Hornykiewicz, O., Bedard, P. J., & Di Paolo, T. (2003) Changes of GABA receptors and dopamine turnover in the postmortem brains of parkinsonians with levodopa-induced motor complications. Mov Disord 18 (3): 241-53. (3.3) 57. Zhang, Y., Calon, F., Zhu, C., Boado, R. J., & Pardridge, W. M. (2003) Intravenous nonviral gene therapy causes normalization of striatal tyrosine hydroxylase and reversal of motor impairment in experimental parkinsonism. Hum Gene Ther 14 (1): 1-12. (this paper made the cover of the journal) (4.3) 58. Calon, F., & Di Paolo, T. (2002) Levodopa response motor complications--GABA receptors and preproenkephalin expression in human brain. Parkinsonism Relat Disord 8 (6): 449-54. (2.0) 59. Olivier, J. C., Huertas, R., Lee, H. J., Calon, F., & Pardridge, W. M. (2002) Synthesis of pegylated immunonanoparticles. Pharm Res 19 (8): 1137-43. (2.8) 60. Calon, F., Birdi, S., Rajput, A. H., Hornykiewicz, O., Bedard, P. J., & Di Paolo, T. (2002) Increase of preproenkephalin mRNA levels in the putamen of Parkinson disease patients with levodopa-induced dyskinesias. J Neuropathol Exp Neurol 61 (2): 186-96. (5.1) 61. Calon, F., Morissette, M., Ghribi, O., Goulet, M., Grondin, R., Blanchet, P. J., Bedard, P. J., & Di Paolo, T. (2002) Prog Neuropsychopharmacol Biol Psychiatry 26 (1): 127-38. (2.8) 62. Calon, F., Lavertu, N., Lemieux, A. M., Morissette, M., Goulet, M., Grondin, R., Blanchet, P. J., Bedard, P. J., & Di Paolo, T. (2001) Effect of MPTP-induced denervation on basal ganglia GABA(B) receptors: Correlation with dopamine concentrations and dopamine transporter. Synapse 40 (3): 225-34. (3.2) 63. Calon, F., Hadj Tahar, A., Blanchet, P. J., Morissette, M., Grondin, R., Goulet, M., Doucet, J. P., Robertson, G. S., Nestler, E., Di Paolo, T., & Bedard, P. J. (2000) Dopamine-receptor stimulation: biobehavioral and biochemical consequences. Trends Neurosci 23 (10 Suppl): S92-100. (14.8) 64. Calon, F., Grondin, R., Morissette, M., Goulet, M., Blanchet, P. J., Di Paolo, T., & Bedard, P. J. (2000) Molecular basis of levodopa-induced dyskinesias. Ann. Neurol. 47 (4 Suppl 1): S70-8. (8.0) 65. Calon, F., Morissette, M., Goulet, M., Grondin, R., Blanchet, P. J., Bedard, P. J., & Di Paolo, T. (2000) 125I-CGP 64213 binding to GABA(B) receptors in the brain of monkeys: effect of MPTP and dopaminomimetic treatments. Exp Neurol 163 (1): 191-9. (3.8) 66. Cyr, M., Calon, F., Morissette, M., Grandbois, M., Di Paolo, T., & Callier, S. (2000) Drugs with estrogen-like potency and brain activity: potential therapeutic application for the CNS. Curr Pharm Des 6 (12): 1287-312. (4.8) 67. Calon, F., Morissette, M., Goulet, M., Grondin, R., Blanchet, P. J., Bedard, P. J., & Di Paolo, T. (1999) Chronic D1 and D2 dopaminomimetic treatment of MPTP-denervated monkeys: effects on basal ganglia GABA(A)/benzodiazepine receptor complex and GABA content. Neurochem Int 35 (1): 81-91. 68. Calon, F., Goulet, M., Blanchet, P. J., Martel, J. C., Piercey, M. F., Bedard, P. J., & Di Paolo, T. (1995. Levodopa or D2 agonist induced dyskinesia in MPTP monkeys: correlation with changes in dopamine and GABAA receptors in the striatopallidal complex. Brain Res 680 (1-2): 43-52. 69. Blanchet, P. J., Calon, F., Martel, J. C., Bedard, P. J., Di Paolo, T., Walters, R. R., & Piercey, M. F. (1995) Continuous administration decreases and pulsatile administration increases behavioral sensitivity to a novel dopamine D2 agonist (U-91356A) in MPTP-exposed monkeys. J Pharmacol Exp Ther 272 (2): 854-9.

Book Chapters (4), Published Abstracts (146), Lectures and presentations (56 since 2000)

4

C) Funding (F.Calon) Ongoing Research Support (0% overlap with the present grant application) 1) a) Canadian Institutes of Health Research (CIHR); b) PI; c) Jul 2010-Jun 2015; d) 520,000 $CDN; e) “Investigations on the role of p21-activated kinase (PAK) in Alzheimer's disease”; Objectives: Determine the role of p21-activated kinase (PAK) in Alzheimer's disease, using the 3xTg-AD animal model. – no overlap – 2) a) Canadian Institutes of Health Research (CIHR); b) PI; c) Apr 2013-Mar 2018; d) 699,329 $CDN; e) “Targeted delivery into brain capillary endothelial cells for the treatment of Alzheimer disease”; Objectives: develop vectors targeting the blood-barrier for gene therapy in CNS disorders such as Alzheimer’s disease. – no overlap – 3) a) Alzheimer’s Society Canada (CIHR); b) PI; c) Sept 2014-Aug 2016; d) 150,000 $CDN; e) “Does a thermoregulatory deficit contribute to Alzheimer's disease?”; Objectives: Investigate thermoregulation in aged transgenic mice modeling Alzheimer's disease. – no overlap – 4) a) International Consortium Neurophenol; b) main PI on the Canadian preclinical part; c) May 2012-Apr 2015; d) total=5 335 160 $CDN, my part = 95 000 $CDN; e) “Conception de produits innovants à base de raisins et de bleuets pour lutter contre le declin cognitif: projet Neurophenols”; – no overlap – 5) a) Natural Sciences and Engineering Research Council of Canada (NSERC); b) PI c) Apr 2013-Mar 2018; d) total=180,000 $CDN; e) “The role of ApoE genotype on blood-brain barrier functions”; – no overlap – 6) a) Collaborative Health Research Projects (CHRP) (CIHR/NSERC); b) main PI; c) Apr 2013-Mar 2016; d) total=5 737,204$CDN; e) “Development and bioimaging of blood-brain barrier vectorized nanoparticles”; – no overlap – 7) a) Pfizer-FRQ-S Alzheimer fund; b) main PI; c) Apr 2013-Mar 2015; d) 200 000 $CDN; e) “Augmentation de la captation cérébrale de glucose par thérapie génique: application chez un modèle animal de la maladie d'Alzheimer”; – no overlap – 8) a) Parkinson Society Canada; b) main PI; c) Sept 2014-Aug 2015; d) 45 000 $CDN; e) “Can exercise and omega-3 fatty acids synergize to restore DA neurons in PD?”; – no overlap – 9) a) L'Association des personnes concernées par le tremblement essentiel (Aptes); b) main PI; c) Sept 2014-Aug 2016; d) 30 000 Euro; e) “Is there a cerebellar accumulation of amyloid beta peptides in essential tremor?”; – no overlap – 10) Laboratoire international associé (LIA) OptiNutriBrain on nutrition and brain health, supported by INRA, Université Bordeaux, INAF, Université Laval and CHU-Q for a total of 150,000$ over 3 years. (co-main PI -50%-) – no overlap – Completed Research Support (last 3 years) 1) a) Hema-Québec- CIHR/SME Research Program; b) PI (co-PI is Dr Renée Bazin); c) Apr 2010-Mar 2014; d) total=210,000$CDN; e) “Isolation of therapeutic fractions from intravenous immunoglobulins using the triple transgenic mouse model of Alzheimer's disease”; Objectives: preclinical testing of IVIg.– no overlap – 2) a) International Essential Tremor Foundation (IETF); b) PI ; c) Aug 2012-Jul 2013; d) total=20,000$USD; e) “Postmortem investigations of LINGO in Essential Tremor patients”; – no overlap – 3) a) Talecris Biotherapeutics, Inc; b) co-PI (Main PI is Dr Renée Bazin); c) Jan 2010-Dec 2011; d) 230,000 $USD; e) “Preclinical investigation of intravenous immunoglobulin therapy in the triple transgenic mouse model of Alzheimer's disease”; – no overlap – Objectives: Preclinical study of intravenous immunoglobulin therapy in Alzheimer's disease (animal models). 4) a) Alzheimer Society Canada; b) PI; c) Sep 2010-Aug 2012; d) 150,000 $CDN; e) “TDP-43 in Alzheimer’s disease ”; no overlap. 5) a) AFMnet; b) PI, (50:50 co-PI is Dr F Cicchetti); c) June 2010-Dec 2011; d) 100,000 $CDN; e) “Efficacy of omega-3 fatty acids in an animal model of Parkinson’s disease”; – no overlap –

5 RESEARCH ARTICLE

Increased LINGO1 in the Cerebellum of Essential Tremor Patients

Charlotte Delay, PhD,1,2¶ Cyntia Tremblay, MSc,1,2¶ Elodie Brochu, MSc,1,2 Sarah Paris-Robidas, BSc,1,2 Vincent Emond, PhD,1,2 Ali H. Rajput, MD, FRCPC,3 Alex Rajput, MD, FRCPC,3 and Fred eric Calon, BPharm, PhD1,2*

1Faculty of Pharmacy, Universite Laval, Quebec City, Quebec, Canada 2Centre de Recherche du Centre Hospitalier Universitaire de Quebec, Neurosciences Axis, Quebec City, Quebec, Canada 3Division of Neurology, Royal University Hospital, University of Saskatchewan, Saskatoon, Saskatchewan, Canada

ABSTRACT: Essential tremor (ET) is the most ularly in individuals with longer disease duration. prevalent adult-onset movement disorder. Despite its Compared with controls, LINGO1 protein levels were health burden, no clear pathognomonic sign has been increased in the cerebellar white matter of PD and ET identified to date because of the rarity of clinicopathologi- patients but, for the latter, only when disease duration cal studies. Moreover, treatment options are still scarce exceeded 20 years. However, no alteration in LINGO1 and have not significantly changed in the last 30 years, mRNA was observed between groups in either the cere- underscoring the urgent need to develop new treatment bellar cortex or the white matter. We observed alterations avenues. In the recent years, leucine-rich repeat (LRR) and in LINGO expression in diseased brain that seemed to pro- immunoglobulin (Ig) domain-containing Nogo receptor- gress along with the disease, being initiated in the cerebel- interacting proteins 1 and 2 (LINGO1 and LINGO2, respec- lar cortex before reaching the white matter. Because tively) have been increasingly regarded as possible ET LINGO up-regulation has been identified as a potential modulators due to emerging genetic association studies pathological response to ongoing neurodegenerative proc- linking LINGO with ET. We have investigated LINGO pro- esses, the present data suggest that LINGO1 is a potential tein and messenger RNA (mRNA) expression in the cere- drug target for ET. VC 2014 International Parkinson and bellum of patients with ET, patients with Parkinson’s Movement Disorder Society disease (PD), and a control group using Western immuno- blotting and in situ hybridization. Protein levels of LINGO1, but not LINGO2, were significantly increased in the cere- Key Words: essential tremor; cerebellum; LINGO; bellar cortex of ET patients compared with controls, partic- dentate nucleus; neurodegeneration

More than 10 million Americans suffer from essential high prevalence and consequent socioeconomic impacts, tremor (ET), which is almost 10 times more than those the etiopathological base of ET remains unknown. who have Parkinson’s disease (PD), making it the most Recently, however, genetic analyses have revealed that prevalent adult-onset movement disorder.1 Despite its SLC1A2 (solute carrier family 1, member 2) and ------LINGO (leucine-rich repeat and immunoglobulin *Correspondence to: Dr. Fred eric Calon, Centre de Recherche du CHU de Quebec, Pavillon CHUL, Room T2-05, 2705 Laurier Blvd., Quebec, domain-containing Nogo receptor-interacting protein) QC, Canada, G1V 4G2; [email protected] may be associated with ET. Indeed, a genome-wide Funding agencies: Dr. Calon was supported by an International Essen- association study performed in 452 ET cases and tial Tremor Foundation grant, Canada Foundation for Innovation grant 14,394 controls has pinpointed two single-nucleotide 10307, and a Fonds de la Recherche en SanteduQu ebec (FRSQ) salary award. polymorphisms in the LINGO1 gene associated with ET.3 So far, findings consistent with a genetic associa- Relevant conflicts of interest/financial disclosures: Nothing to report. Full financial disclosures and author roles may be found in the online ver- tion between LINGO and ET have been reported in at sion of this article. least five populations,4-8 but not in others.9-11 Nonethe- ¶The first two authors contributed equally to this work. less, the relationship between LINGO1 polymorphisms 12 Received: 12 August 2013; Revised: 28 November 2013; Accepted: and ET stood the test of a recent meta-analysis. In 30 December 2013 addition, the genetic variability of LINGO2 has been associated with ET and PD in North American and Published online 14 February 2014 in Wiley Online Library 13 (wileyonlinelibrary.com). DOI: 10.1002/mds.25819 Asian populations. Despite their caveats, the results

Movement Disorders, Vol. 29, No. 13, 2014 1637 DELAY ET AL from these genetic association studies call for the need sis of patients was made by one of two neurologists, to decipher the role of LINGO in ET. as described previously, and was based on upper limb LINGO1 and LINGO2 belong to a family of four postural and/or kinetic action tremor of several years’ members (LINGO1-LINGO4) expressed in the central duration for which there was no metabolic, toxic, or nervous system (CNS).14,15 Given the high degree of other neurological cause identified.27,28 Tremor fre- homology between the LINGO1 and LINGO2 pro- quency and amplitude were evaluated visually, and the teins (61%), it is conceivable that both paralogues severity of tremor was based on the Unified Parkin- exert similar functions. LINGO1 is a component of son’s Disease Rating Scale (UPDRS). Although a prob- signaling complexes with the Nogo-66 receptor and able or definitive Alzheimer’s disease diagnosis was p75 and is expressed in oligodendrocytes and neurons. ruled out by a pathologist for each individual who As part of this complex, LINGO1 is involved in the was included, no systematic cognitive assessment was inhibition of oligodendrocyte differentiation,16-18 axo- performed. The severity of tremor was recorded at nal myelination and regeneration,18,19 and neuronal each visit and was based on visual assessment of survival.20 Interestingly, the expression of LINGO1 is tremor amplitude and the history of its impact on increased after neuronal damage, and its inhibition daily activities.28 Brain tissues that were used for com- promotes functional recovery and axonal sprouting parison between different groups included samples after spinal cord injury.21 Accordingly, a reduction of from 9 patients with ET, 10 patients with PD, and 16 LINGO1 activity was shown to improve survival, healthy, age-matched controls. A summary of the growth, and function of dopaminergic neurons both in information is provided in Table 1, confirming homo- primary cell cultures and in an animal model of geneity and relative equivalent tissue quality between PD.22,23 The role of LINGO2 is less known, but its groups.27 expression is restricted to neuronal tissue.15 In sum- mary, in vitro and in vivo data indicate that LINGO1 Handling and Processing of Tissues plays a major role in the unfortunate inhibition of One-half of the brain was fixed in formalin to per- neuronal regeneration in spinal cord and brain 24,25 form histological studies of the cerebellum to reveal injury, suggesting that reducing LINGO1 might cerebellar pathology, including Purkinje cell counts or be of therapeutic value for neurodegenerative diseases. infarcts. The other hemisphere was frozen at 280C Interestingly, a study published earlier this year within 25 hours of death. The frozen hemisphere was reported LINGO1 protein level alterations in patients 26 cut in the frontal plane into 2-mm to 3-mm-thick sli- with ET compared with controls. Taken together ces. Horizontal slices (containing the molecular and with the genetic association between LINGO and ET granular layer of the Cctx and DN) were cryostat- and the implication of LINGO in neuronal regenera- sectioned (20 lm), thaw-mounted onto SuperFrostPlus tion, these data imply that LINGO could play a role 75 3 50 mm slides (Brain Research Laboratories, in ET pathophysiology. To characterize LINGO1 in Newton Heights, MA, USA), desiccated at 4C, and the cerebellum of ET patients, our group took advant- stored at 280C. The pH of cerebellar tissue was age of a well characterized series of human samples to measured after homogenization in 10 volumes of perform postmortem analyses of LINGO1 and unbuffered, deionized, and distilled water as an indica- LINGO2 protein and RNA levels in the cerebellar cor- tion of tissue quality. tex (Cctx), white matter (WM), and dentate nucleus (DN) of patients with ET, patients with PD, and Western Immunoblotting controls. Cctx extracts were homogenized in Tris-buffered saline and fractionated as previously described.29,30 Pro- Patients and Methods teins were heated at 95C for 5 minutes in Laemmli’s loading buffer and separated by sodium dodecyl sulfate- Clinicopathological Evaluation of Patients polyacrylamide gel electrophoresis on an 8% Tris-glycin We selected 10 patients with PD, 9 patients with polyacryamide gel, transferred on a polyvinylidene fluo- ET, and 16 controls based on (1) the absence of neu- ride membrane, and blocked in 5% nonfat dry milk, ropathologically confirmed PD (no presence of nigral 0.5% bovine serum albumin, 0.1% Tween-20 in Lewy bodies) in ET brain, (2) the presence of an easily phosphate-buffered saline, as previously described.29 identifiable dentate nucleus in the tissue bloc, and (3) Proteins were detected using anti-Lingo-1 (Millipore- matching as close as possible for age and gender Upstate, Billerica, MA, USA), anti- (Applied Bio- between the ET, PD, and control groups. Based on logical Materials Inc., Richmond, BC, Canada), and examination by neurologists, geriatricians, or internal anti-LINGO2 (R&D Systems, Minneapolis, MN, USA) medicine specialists, all controls had no history of followed by horseradish peroxidase-labeled secondary neurological disease at the time of death and had nor- antibodies (Jackson ImmunoResearch Europe Ltd., mal brain histologic examination. The clinical diagno- Newmarket, UK) and chemiluminescence reagents

1638 Movement Disorders, Vol. 29, No. 13, 2014 TABLE 1. Study group information

Age, y CO-1 mRNA

Tremor Poly-T Drugs and Brain Disease Severity/ MMSE Ethanol DN- mRNA, Brain Family Effects on Group Enrollment, y Sex PMI Weight, g Onset Death Duration, y H&Y Amplitude Score Consumption DCN MolCtx GraCtx MolCTx pH History Tremor (1 or 2)

Ctrl 1 2002 F 24 — — 82 — No 622 662 1,095 132 6.07 2 1988 F 24 — — 82 — No 489 300 811 114 6.26 3 2002 F 16 1,140 — 74 — No 337 608 1,155 160 — 4 2003 F 24 1,370 — 66 — No 389 463 735 197 6.20 5 2003 M 15 1,250 — 76 — No 505 490 1,151 154 6.25 6 2005 M 5 1,320 — 84 — No 555 310 490 94 5.93 7 1996 M 25 1,380 — 81 — No 502 334 823 212 6.12 8 1998 M 18 1,440 — 82 — No 595 395 1,148 186 6.31 9 1993 M 24 1,470 — 71 — No 667 325 933 605 6.83 10 1996 F 23 1,110 — 70 — No 586 284 668 185 6.61 11 1989 M 11 1,200 — 76 — No — 540 1,048 217 6.31 12 1990 F 20 1,215 — 78 — No — 336 605 112 — 13 1990 F 23 1,060 — 86 — No — 423 902 252 6.07 14 1990 F 11 1,090 — 75 — No — 501 823 218 — 15 1992 F 14 1,337 — 72 — No — 479 865 133 6.13 16 1995 F 23 1,110 — 68 — No — 474 695 113 6.08 PD 1 1996 F 19 1,180 58 81 23 3 27 Social 519 417 1,109 187 6.39 L-dopa; ethopropazine; amantadine; bro- mocriptine; temazepam 2 1993 F 18 1,240 50 82 32 5 No 426 472 1,053 168 6.64 Trihexyphenidyl benz-

tropine L-dopa; TREMOR ESSENTIAL IN LINGO amantadine; bromocriptine 3 1998 F 17 1,340 45 64 19 3 30 No 681 445 822 185 6.75 L-dopa; bromocriptine;

Movement trihexyphenidyl; selegiline; ropinir- ole; amantadine

iodr,Vl 9 o 3 2014 13, No. 29, Vol. Disorders, 4 1999 M 20 1,580 59 74 15 5 28 No 599 466 913 168 6.43 L-dopa; selegiline; propranolol; ami- triptyline; temazepam 5 1997 F 12 1,270 80 89 9 4 27 No 706 424 906 171 6.69 L-dopa; selegiline 6 2006 F 24 1,290 83 90 7 4 29 No 327 181 717 106 6.17 L-dopa; amantadine 7 1997 F 20 1,210 59 79 20 4 20 No 508 344 760 155 6.31 Bromocriptine; selegi- line; L-dopa; amantadine 8 2002 M 4 1,200 73 89 16 3 No 624 424 695 125 6.39 L-dopa; selegiline 9 2008 F 24 980 61 87 26 3 21 No 748 548 1,050 99 6.31 1639 (Continued) 1640 AL ET DELAY TABLE 1. Continued

Age, y CO-1 mRNA Movement Tremor Poly-T Drugs and Brain Disease Severity/ MMSE Ethanol DN- mRNA, Brain Family Effects on Group Enrollment, y Sex PMI Weight, g Onset Death Duration, y H&Y Amplitude Score Consumption DCN MolCtx GraCtx MolCTx pH History Tremor (1 or 2) iodr,Vl 9 o 3 2014 13, No. 29, Vol. Disorders,

Amantadine; L-dopa; selegiline 10 2004 F 24 1,190 60 84 24 3 No 475 649 1,285 185 6.53 L-dopa; pramipexole ET 1 1995 F 13 1,140 65 80 15 2 No 308 514 828 143 6.12 No None for tremor 2 1986 F 15 1,380 8 78 70 4 No 677 529 1,124 285 6.38 Yes Propranolol 1; clona- zepam 2; L-dopa 2 3 1993 F 24 1,520 34 63 30 2 No 539 395 1,118 164 6.48 Yes Propranolol 1 4 1991 F 3 1,060 50 95 45 1 No — — — 284 6.40 Yes None for tremor 5 1994 M 6 1,020 64 74 10 2 Social — 424 1,125 186 6.46 Yes L-dopa 2; primidone 1 6 1996 F 17 1,040 57 92 35 2 No 714 685 1,273 231 6.54 Yes Propranolol 1 bromoc- riptine 2; L-dopa – 7 1995 F 24 1,170 58 79 21 1 22 No — 538 966 114 6.19 No L-dopa 2; L-dopa 2; selegiline 1 8 2000 F 9 1,230 71 93 22 2 No 738 560 854 66 6.25 Yes Propranolol not toler- ated; amantadine 2 9 2005 F 21 1,040 52 88 36 2 27 No 345 372 1,006 136 6.61 Yes L-dopa 2; pheno- barb 1 diazepam 1; propranolol 1 Average (SD) Ctrl 19 (6) 1249 (137) 76 (6) 525 (103) 433 (114) 872 (206) 193 (119) 6.24 (0.24) PD 18 (6) 1248 (151) 82 (8) 562 (134) 437 (122) 931 (191) 155 (33) 6.46 (0.19) ET 15 (8) 1178 (173) 82 (10) 553 (189) 502 (102) 1037 (152) 179 (75) 6.38 (0.16) Comparison test: P value Contingency 0.3 ANOVA 0.32 0.49 — 0.11 — — — — — 0.82 0.4 0.21 0.59 0.06

*Enrollment year corresponds to the age of death. Abbreviations: CO-1, cytochrome oxydase-1; mRNA, messenger RNA; PMI, postmortem interval; H&Y, Hoehn & Yahr scale; MMSE, Mini-Mental State Examination; DN-DCN, dentate nucleus of the deep cerebellar nuclei; GraCtx, granular layer of the cerebellar cortex; MolCtx, molecular layer of the cerebellar cortex; 1, positive; 2, negative; Ctrl, control group; F, female; M, male; PD, Parkinson’s disease group; ET, essential tremor group; SD, standard deviation; ANOVA, analysis of variance. LINGO IN ESSENTIAL TREMOR

(Lumiglo Reserve; KPL, Gaithersburg, MD, USA). hour with Alexa Fluor 488 streptavidin conjugate Quantification was performed using a Kodak Image Sta- (Life Technologies, Carlsbad, CA, USA) and counter- tion 4000MM Digital Imaging System (Carestream stained with 40,6-diamidino-2-phenylindole (DAPI) Health, Rochester, NY, USA). Each experiment was (Life Technologies, Carlsbad, CA, USA). Calbindin repeated three times. LINGO1 antibody specificity was and MBP were detected using donkey anti-mouse sec- tested by competition with Lingo 1 peptide (antibody ondary antibodies coupled to Alexa Fluor 555. Slices ab25890; Abcam, Cambridge, UK) at a 60:1 ratio, were finally incubated for 5 minutes in a 0.5% Sudan which completely blocked immunostaining. Black solution in 70% methanol and coverslipped using Mowiol anti-fade mounting medium. Two to In Situ Hybridization three washes in 0.1 M PBS were included between In situ hybridization was performed according to each step. Lingo1 antibody specificity was assessed by general methodology, as described previously.27,31 The competition with Lingo 1 peptide (ab25890; Abcam, oligonucleotide sequences we used corresponded to Cambridge, UK). The antibody LINGO1 with or with- bases 168 through 127, 339 through 295, 1327 out LINGO1 peptide (60:1 molar ratio) was incubated through 1283, and 1727 through 1683 of human for 30 minutes at room temperature. The LINGO1- Lingo-1 messenger RNA (mRNA) (NM_032808.5). In stained sections were examined with an i90 Nikon flu- situ hybridization for housekeeping b-actin and cyto- orescent microscope (Nikon Corporation, Tokyo, chrome oxidase-1 mRNAs as well as poly-A tails Japan) coupled to a Hamamatsu 1394 ORCA-285 (poly-T probe) was performed previously on the same monochrome camera (Hamamatsu Photonics, Hama- series of sections.27 Oligonucleotides were labeled matsu, Japan) and exploited by SimplePCI software with 33P-dATP (PerkinElmer Inc., Waltham, MA, (version 5.3.0.1102; Compix Inc. Imaging Systems, USA) using a three-terminal deoxynucleotidyl transfer- Cranberry Township, PA, USA). ase enzyme kit (New England Biolabs, Ipswich, MA, USA). The reaction was carried out at 37C for 60 Statistical Analysis minutes, and labeled oligonucleotides were purified For LINGO1 group comparisons, because unequal with the QIAquick Nucleotide Removal Kit (Qiagen, variance was determined with Bartlett’s test, a Welch Venlo, Netherlands). Prehybridation and hybridization one-way analysis of variance followed by a Bonferro- conditions were performed exactly as described. Cere- ni’s multiple comparison test was performed. For bellar slices were exposed to Kodak Biomax MR films LINGO2 group comparisons, normal distribution of for 15 days, and macroscopic optical density quantifi- values could not be assumed, and a nonparametric cation was performed using the Kodak Image Station. Kruskal-Wallis test was performed followed by Dunn’s The final data from each individual represent the multiple comparison test. Adjustments for age of mean of eight tissue sections. Nonspecific hybridiza- death, gender, or cerebellar pH were performed using tion was considered negligible, as determined by add- an analysis of covariance, when needed. When two ing a 100-fold excess of unlabeled probes. groups were compared, a nonparametric Mann- Whitney test was used. A simple regression model was Immunofluorescence used to determine correlation and significance of the Immunofluorescencent (IF) labeling was performed linear relationship between parameters. All statistical on 6-lm-thick sections of paraffin-embedded cerebel- analyses were performed using GraphPad Prism 5 lum samples. The sections were microwaved twice for analysis software (version 5.0.1; GraphPad Software 2 minutes in 0.01 M citrate buffer, pH 6.0, for anti- Inc., San Diego, CA, USA) and JMP (version 10.1; gen retrieval. Slices were then washed and pretreated SAS Institute Inc., Cary, NC, USA), and P val- with 10% normal horse serum (NHS) in 0.1 M ues < 0.05 were considered significant. phosphate-buffered saline (PBS) containing 0.2% Tri- ton X-100 (TX) for 1 hour. Sections were incubated Results overnight at 4C with an anti-Lingo1 antibody (1:300 dilution; Millipore-Upstate, Billerica, MA), mouse LINGO1 Expression Is Increased in the anti-Calbindin D-28K (1:500 dilution; Sigma-Aldrich, Cerebellum of ET Patients St. Louis, MO, USA), and/or mouse anti-myelin basic Based on the genetic evidence cited above, we protein SMI99 (MBP) (1:500 dilution; Covance hypothesized that LINGO could play a role in the Research Products/Cedarlane, Burlington, Ontario, degenerative changes reported in the cerebellum of ET Canada) and diluted in 0.1 M PBS with 1% NHS and patients.32-39 We therefore assessed the protein expres- 0.2% TX. Sections were incubated for 2 hours with sion levels of LINGO1 and LINGO2 in human Cctx an anti-rabbit-biotin-conjugated antibody (Sigma and WM of ET patients, PD patients. and healthy con- Chemical Company) diluted in PBS containing 0.05% trols (Figs. 1, 2). PD patients were included to differ- TX and 1% NHS. Slices were then incubated for 1 entiate ET-specific changes (ie, kinetic tremor vs.

Movement Disorders, Vol. 29, No. 13, 2014 1641 DELAY ET AL

FIG. 1. LINGO1 and LINGO2 expression levels are illustrated in the cerebellar cortex (Cctx) of patients with essential tremor (ET), patients with Par- kinson’s disease (PD), and controls (C). (A,B) Western blot quantification is illustrated for LINGO1 and LINGO2 expression, respectively, in the Cctx of the control (Ctrl), PD, and ET groups. (C,D) Western blot quantification is illustrated for LINGO1 and LINGO2, respectively, in patients who had a disease duration > 20 years. OD indicates optical density. resting tremor). We have observed an increase in trations was detected between groups, even in patients LINGO1 levels (1119%; Welch analysis of variance, with longer disease duration (Fig. 1B,D, Fig. 2B,D). P 5 0.0055; Bonferroni post-hoc test, P < 0.01) in the Cctx from ET patients, but not PD patients, compared LINGO Is Present Mainly in the Myelinated with controls (Fig. 1A). This difference remained stat- Fibers of the WM istically significant after adjustment for the covariates To investigate the localization of LINGO1 in the cer- age of death, gender, and postmortem interval using ebellum, we performed IF experiments on cerebellar an analysis of covariance. Interestingly, the rise of sections from patients with ET, patients with PD, and LINGO1 in the Cctx of ET patients was more promi- controls. Our results clearly show specific immunolabel- nent in individuals with a long (>20-year) disease ing of LINGO1 in myelinated fibers of the WM, as duration (1156% vs. controls; Mann-Whitney test, demonstrated by (1) colocalization with MBP, a myelin P 5 0.0023) (Fig. 1C). Linear correlation analyses did marker, and (2) loss of signal by addition of the immu- not show a significant relationship between disease nization peptide (Fig. 3). Some disparate staining was 2 duration and LINGO1 levels (r 5 0.02; P 5 0.71). retrieved around calbindin-labeled Purkinje cell bodies, Because LINGO1 is also expressed in myelinating oli- but no clear structure was visible (Fig. 3). However, we godendrocytes, homogenates from the WM also were did not replicate previous observations of LINGO-1 analyzed. Compared with controls, the LINGO1 pro- enrichment in a brush pinceau structure around the tein levels were found in higher concentrations in the axonal initial segment of Purkinje cells in ET cerebel- cerebellar WM of PD patients (1113%; Kruskal- lum.26 A small staining of the DN cells was obvious in Wallis test, P 5 0.0385) (Fig. 2A). An increase of some patients but not in others, making it difficult to LINGO1 in the WM was also detected in ET patients make a conclusion about the cellular DN localization who suffered from the disease for more than 20 years of LINGO1. Finally, we did not observe obvious (185%; Mann-Whitney test, P 5 0.0360) (Fig. 2C). In LINGO1 localization changes in ET or PD cerebellum contrast, no significant difference in LINGO2 concen- slices compared with controls (data not shown).

1642 Movement Disorders, Vol. 29, No. 13, 2014 LINGO IN ESSENTIAL TREMOR

FIG. 2. LINGO1 and LINGO2 protein expression levels are illustrated in the cerebellar white matter (WM) of patients with essential tremor (ET), patients with Parkinson’s disease (PD), and controls (C). (A,B) Representative Western blot quantification is illustrated for LINGO1 and LINGO2 expression, respectively, in the cerebellar WM of the control (Ctrl), PD, and ET groups. (C,D) Western blot quantification is illustrated for LINGO1 and LINGO2, respectively, in patients who had a disease duration > 20 years. OD indicates optical density. Overall, our IF data are consistent with LINGO1 4B,C), indicating that the higher LINGO1 protein immunolabeling in the myelinated fibers of the cerebel- content observed in ET brains was not due to changes lum and, possibly, surrounding at least some Purkinje in mRNA expression levels. cells. Discussion LINGO1 Protein Alterations Do Not Implicate mRNA Expression Changes In the current study, we conducted an analysis of We further investigated whether the alterations LINGO1 and LINGO2 expression in the cerebellum found in LINGO1 would reflect transcription changes. of ET patients compared with PD patients and healthy As depicted above, the protein expression patterns of controls. We observed that LINGO1 and, although at LINGO1 were concentrated within the Purkinje cell lower levels, LINGO2 were both expressed in the cer- layer, the DN, and myelinated fibers of the WM, all ebellum. LINGO1 mRNA expression was mainly con- accounting for only a very small parts of the cerebel- fined to the granular layer of the cerebellum and the lum and making it technically challenging to measure DN, whereas most LINGO1-specific immunostaining their mRNA content with the available techniques, was found to colocalize with the myelin-binding pro- which require tissue homogenates. In situ hybridiza- tein in the WM. These observations are in line with tion was selected because it allows a quantitative, previous immunohistochemistry reports suggesting two-dimensional measurement of mRNA expression that LINGO-1 can be detected in human and rodent levels in different subregions of the cerebellum, includ- cerebellum,14,16,26 expressed by neurons and oligoden- ing the cortical region, the WM, and the DN. Consist- drocytes,16,18 but did not confirm the cellular distribu- ent with the IF data, LINGO1 mRNAs were mainly tion within or around Purkinje cells observed detected in the granular layer of the cerebellum, with previously.14,26 However, the most striking observa- a weak signal in the DN (Fig. 4A). However, there tion reported here is a significant increase of LINGO1 was no significant difference between the groups (Fig. protein levels in the Cctx of ET patients, compared

Movement Disorders, Vol. 29, No. 13, 2014 1643 DELAY ET AL

FIG. 3. LINGO1 protein localization is illustrated in the cerebellum. LINGO1 immunohistochemistry on cerebellar sections from patients with essential tremor. Nuclei were stained with DAPI (blue), and LINGO1 was labeled with green-fluorescent Alexa Fluor 488 streptavidin conjugate. (A-D) The colocalization LINGO1 and calbindin (red) is observed. The arrow in D indicates pericellular staining. (E-H) Colocalization of LINGO1 with MBD (red) is observed. The asterisk in H indicates a Purkinje cell. (I-L) Antibody-specific staining is observed (note that the views in D and H are at higher magnification). with PD patients and controls, that was not reflected regions. These observations are made in several other at the mRNA level. Higher LINGO1 concentrations in neurodegenerative disorders. Indeed, in PD, it has both the Cctx and the WM were particularly noticea- been hypothesized that failure of the protein degrada- ble in patients who had suffered from ET for > 20 tion systems results in increased accumulation of ubiq- years. uitinated proteins, which form the core of Lewy Such higher levels of LINGO1 in ET are consistent bodies, one of the pathological hallmarks of PD.41 with a recent report of a 50% increase in LINGO1 Lewy bodies are retrieved in the brainstem of a small protein in the Cctx of ET patients compared with con- subpopulation of ET patients.32,37,38 However, there trols, a change not detected in the occipital brain cor- are no indications of the presence of such structures in tex.26 A rise in LINGO1 expression may be explained the cerebellum, arguing against a ubiquitin-related by either an imbalance of post-transcriptional regula- degradation hypothesis. Moreover, based on the high tors, such as microRNAs (miRNAs), or by decreased protein identity (61% identity, 77% homology) degradation of LINGO1. Abnormal miRNA expres- between LINGO1 and LINGO2 (NP_116197.4, sion patterns are increasingly studied in a number of NP_689783.1), it is conceivable that they are both neurodegenerative disorders, including PD.40 This degraded in the same manner, making it improbable class of small RNAs targets mRNAs through binding that there would be degradation abnormalities of to a specific miRNA binding site (seed region), result- LINGO1 and not of LINGO2. It is therefore more ing in the degradation or translational inhibition of likely that the increased expression of LINGO1 is due the target and, thus, decreases its protein levels. Imbal- to post-transcriptional changes in ET. However, ances in the miRNA expression levels, therefore, could whether this change is causative or consequential to result in altered protein levels. Prediction algorithms, the disease pathogenesis remains unclear at this stage such as targetscan.org, indicate that there are multiple of investigation. Nevertheless, the late occurrence of potential miRNA binding sites within the LINGO1 the increase in LINGO during the course of the dis- mRNA that are not present in the LINGO2 mRNA, ease suggests that it is not a primary cause of ET but, which, thus, could account for the specific differences rather, a faulty pathological response that develops in LINGO1 expression observed in ET. The altered over a long period of time. expression levels of LINGO1 also could be due to a Several lines of evidence suggest that ET pathogene- decreased degradation rate in the affected brain sis could involve a neurodegenerative process,32,42,43

1644 Movement Disorders, Vol. 29, No. 13, 2014 LINGO IN ESSENTIAL TREMOR

underlying these neurodegenerative conditions have recently been gathered. Indeed, LINGO1 expression is increased after neuronal damage.21,23 Moreover, LINGO1 is involved in the inhibition of oligodendro- cyte differentiation, axonal myelination and regenera- tion, and neuronal survival.18 Interestingly, LINGO1 inhibition promotes functional recovery and axonal sprouting after spinal cord injury16,18,19,49; and it was shown that a reduction in LINGO1 activity improved the survival, growth, and function of dopaminergic neurons both in primary cell cultures and in an animal model of PD.23 Taken together, these results suggest that LINGO1 contributes to the inhibition of neuronal regeneration.24,25 In the context of ET and in line with our observations of increased LINGO1 expres- sion in the Cctx of ET patients, these data indicate that increased LINGO1 expression could be inter- preted as an evidence of an underlying neurodegenera- tive process in ET. Because increased LINGO1 expression is a possible signature of CNS injury, as discussed above, the increase in LINGO1 expression reported in this study would be consistent with a neurodegenerative process in the cerebellum of ET patients. It is of utmost importance to find treatments that reduce neuronal degeneration and maintain neuronal pathways and physiological circuits in ET, and our data suggest that LINGO1 is a suitable target. Incidentally, the inhibi- tion of LINGO1 as a drug target has already received considerable interest in the fields of multiple sclero- sis,48 PD,23 and spinal cord injury.21 Moreover, sev- eral groups are studying the identification of a possible beneficial role of LINGO1 antagonists to pro- mote neuro-restoration and remyelination.17,21,51 A LINGO1 antagonist, BIIB033, has now even reached FIG. 4. Cerebellar LINGO1 messenger RNA distribution is illustrated in clinical trials (ClinicalTrials.gov identifier, the control group and in patients with Parkinson’s disease (PD) and essential tremor (ET). (A) LINGO1 in situ hybridization (ISH) of human NCT01244139). More recently, anti-LINGO-1 Li81 cerebellum is illustrated. An arrow indicates the dentate nucleus. (B,C) antibodies have been shown to passively enter the Signal quantification is illustrated in the cerebellar cortex and the cere- CNS, which was sufficient to lead to strong remyelini- bellar white matter in the control, PD, and ET groups. zation in rat spinal cord remyelination models.52 If LINGO1 up-regulation exerts faulty effects in ET as 44-47 leading to a lively debate on the issue. In this well, then such LINGO1 antagonists and antibodies regard, mounting data suggest that increased LINGO1 ultimately also could be used for the treatment of ET. expression is a marker of CNS injury, playing a vital role in vicious cycles leading to neuronal death, axon Conclusion degeneration, and demyelination. First, the dysregula- tion of LINGO1 is found in several neurodegenerative In summary, higher LINGO1 protein levels were pathologies, such as multiple sclerosis48 and PD,23 as detected in both the Cctx and WM of patients who well as in rat models of spinal cord injury49 and glau- suffered from ET for greater than 20 years. We coma.50 More specifically, it also has been shown pre- hypothesize that these changes are due to post- viously that the levels of LINGO1 mRNA are up- transcriptional alterations, because no mRNA changes regulated in PD substantia nigra.23 Accordingly, Inoue were observed. Furthermore, the present data suggest and colleagues have demonstrated an increase in that down-regulation of LINGO in the cerebellum is a LINGO1 in the striatum of 1-methyl-4-phenyl-1,2,3,6- potential disease-modifying therapeutic target in ET. tetrahydtopyridine (MPTP)-treated mice.23 Second, Acknowledgements: We thank the patients and families who gen- evidence suggesting that LINGO-1 and its associated erously donated tissue to our research program and the Regina Curling pathways could be implicated in the cellular injury Classic, the Greystone Golf Classic, and RUHF, Saskatoon for their

Movement Disorders, Vol. 29, No. 13, 2014 1645 DELAY ET AL support of special clinics and for acquisition and maintaining the brain 23. Inoue H, Lin L, Lee X, et al. Inhibition of the leucine-rich repeat material. protein LINGO-1 enhances survival, structure, and function of dopaminergic neurons in Parkinson’s disease models. Proc Natl Acad Sci U S A 2007;104:14430-14435. References 24. Gerin CG, Madueke IC, Perkins T, et al. Combination strategies for repair, plasticity and, regeneration using regulation of gene 1. Louis ED, Ferreira JJ. How common is the most common adult expression during the chronic phase after spinal cord injury. Syn- movement disorder? Update on the worldwide prevalence of essen- apse 2011;65:1255-1281. tial tremor. Mov Disord 2010;25:534-541. 25. Llorens F, Gil V, del Rio JA. Emerging functions of myelin- 2. Yu SW, Chen CM, Chen YC, et al. SLC1A2 Variant is associated associated proteins during development, neuronal plasticity, and with essential tremor in Taiwanese population [serial online]. PLoS neurodegeneration. FASEB J 2011;25:463-475. One 2013;8:e71919. 26. Kuo SH, Tang G, Louis ED, et al. Lingo-1 expression is increased 3. Stefansson H, Steinberg S, Petursson H, et al. Variant in the in essential tremor cerebellum and is present in the basket cell pin- sequence of the LINGO1 gene confers risk of essential tremor. Nat ceau. Acta Neuropathol 2013;125:879-889. Genet 2009;41:277-279. 27. Paris-Robidas S, Brochu E, Sintes M, et al. Defective dentate 4. Thier S, Lorenz D, Nothnagel M, et al. LINGO1 polymorphisms nucleus GABA receptors in essential tremor. Brain 2012;135(pt 1): are associated with essential tremor in Europeans. Mov Disord 105-116. 2010;25:717-723. 28. Rajput A, Robinson CA, Rajput AH. Essential tremor course and 5. Tan EK, Teo YY, Prakash KM, et al. LINGO1 variant increases disability: a clinicopathologic study of 20 cases. Neurology 2004; risk of familial essential tremor. Neurology 2009;73:1161-1162. 62:932-936. 6. Vilarino-Guell C, Wider C, Ross OA, et al. LINGO1 and LINGO2 29. Tremblay C, Pilote M, Phivilay A, Emond V, Bennett DA, Calon variants are associated with essential tremor and Parkinson disease. F. Biochemical characterization of AI2 and tau pathologies in mild Neurogenetics 2010;11:401-408. cognitive impairment and Alzheimer’s disease. J Alzheimers Dis 7. Clark LN, Park N, Kisselev S, Rios E, Lee JH, Louis ED. Replica- 2007;12:377-390. tion of the LINGO1 gene association with essential tremor in a 30. Tremblay C, St-Amour I, Schneider J, Bennett DA, Calon F. Accu- North American population. Eur J Hum Genet 2010;18:838-843. mulation of transactive response DNA binding protein 43 in mild 8. Wu YW, Rong TY, Li HH, et al. Analysis of Lingo1 variant in cognitive impairment and Alzheimer disease. J Neuropathol Exp sporadic and familial essential tremor among Asians. Acta Neurol Neurol 2011;70:788-798. Scand 2011;124:264-268. 31. Julien C, Tremblay C, Bendjelloul F, Phivilay A, Coulombe MA, 9. Bourassa C V, Riviere J-B, Dion PA, Bernard G, Diab S, Panisset Emond V, Calon F. Decreased drebrin mRNA expression in Alz- M, et al. LINGO1 variants in the French-Canadian population heimer disease: correlation with tau pathology. J Neurosci Res [serial online]. PLoS One 2011;6:e16254. 2008;86:2292-2302. 10. Zuo X, Jiang H, Guo JF, et al. Screening for two SNPs of LINGO1 32. Louis ED, Faust PL, Vonsattel JP, et al. Neuropathological changes gene in patients with essential tremor or sporadic Parkinson’s dis- in essential tremor: 33 cases compared with 21 controls. Brain ease in Chinese population. Neurosci Lett 2010;481:69-72. 2007;130(pt 12):3297-3307. 11. Lorenzo-Betancor O, Samaranch L, Garcia-Martin E, et al.; Iberian 33. Erickson-Davis CR, Faust PL, Vonsattel J-PG, Gupta S, Honig LS, Parkinson’s Disease Genetics Study Group Researchers. LINGO1 Louis ED. “Hairy baskets” associated with degenerative Purkinje gene analysis in Parkinson’s disease phenotypes. Mov Disord 2011; cell changes in essential tremor. J Neuropathol Exp Neurol 2010; 26:722-727. 69:262-271. 12. Jimenez-Jimenez FJ, Garcia-Martin E, Lorenzo-Betancor O, Pastor 34. Shill HA, Adler CH, Sabbagh MN, Connor DJ, Caviness JN, Hentz P, Alonso-Navarro H, Agundez JA. LINGO1 and risk for essential JG, Beach TG. Pathologic findings in prospectively ascertained tremor: results of a meta-analysis of rs9652490 and rs11856808. J essential tremor subjects. Neurology 2008;70(16 pt 2):1452-1455. Neurol Sci 2012;317(1-2):52-57. 35. Louis ED. Essential tremor: evolving clinicopathological concepts 13. Wu YW, Prakash KM, Rong T-Y, et al. Lingo2 variants associated in an era of intensive post-mortem enquiry. Lancet Neurol 2010;9: with essential tremor and Parkinson’s disease. Hum Genet 2011; 613-622. 129:611-615. 36. Kuo S-H, Erickson-Davis CR, Gillman A, Faust PL, Vonsattel J-PG, 14. Llorens F, Gil V, Iraola S, et al. Developmental analysis of Lingo- Louis ED. Increased number of heterotopic Purkinje cells in essential 1/Lern1 protein expression in the mouse brain: interaction of its tremor. J Neurol Neurosurg Psychiatry 2011;82:1038-1040. intracellular domain with Myt1l. Dev Neurobiol 2008;68:521-541. 37. Louis ED, Vonsattel JP, Honig LS, Ross GW, Lyons KE, Pahwa R. 15. Homma S, Shimada T, Hikake T, Yaginuma H. Expression pattern Neuropathologic findings in essential tremor. Neurology 2006;66; of LRR and Ig domain-containing protein (LRRIG protein) in the 1756-1759. early mouse embryo. Gene Expr Patterns 2009;9:1-26. 38. Louis ED, Vonsattel JP, Honig LS, Lawton A, Moskowitz 16. Mi S, Miller RH, Lee X, et al. LINGO-1 negatively regulates mye- CB, Ford B, Frucht S. Essential tremor associated with patho- lination by oligodendrocytes. Nat Neurosci 2005;8:745-751. logic changes in the cerebellum. Arch Neurol 2006;63: 1189-1193. 17. Lee X, Yang Z, Shao Z, et al. NGF regulates the expression of axonal LINGO-1 to inhibit oligodendrocyte differentiation and 39. Louis ED, Vonsattel JP. The emerging neuropathology of essential myelination. J Neurosci 2007;27:220-225. tremor. Mov Disord 2008;23:174-182. 18. Jepson S, Vought B, Gross CH, et al. LINGO-1, a transmembrane 40. Harraz MM, Dawson TM, Dawson VL. MicroRNAs in Parkin- signaling protein, inhibits oligodendrocyte differentiation and mye- son’s disease. J Chem Neuroanat 2011;42:127-130. lination through intercellular self-interactions. J Biol Chem 2012; 41. Ebrahimi-Fakhari D, Wahlster L, McLean PJ. Protein degradation 287:22184-22195. pathways in Parkinson’s disease: curse or blessing. Acta Neuropa- 19. Mi S, Hu B, Hahm K, et al. LINGO-1 antagonist promotes spinal thol 2012;142:153-172. cord remyelination and axonal integrity in MOG-induced experi- 42. Axelrad JE, Louis ED, Honig LS, et al. Reduced Purkinje cell num- mental autoimmune encephalomyelitis. Nat Med 2007;13: ber in essential tremor: a postmortem study. Arch Neurol 2008;65: 1228-1233. 101-107. 20. Loov C, Fernqvist M, Walmsley AR, Marklund N, Erlandsson A. 43. Louis ED. Essential tremor. Lancet Neurol 2005;4:100-110. Neutralization of LINGO-1 during in vitro differentiation of neu- ral stem cells results in proliferation of immature neurons [serial 44. Kurtis MM. Essential tremor: is it a neurodegenerative disease? online]. PLoS One 2012;7:e29771. No. J Neural Transm 2012;119:1375-1381; discussion 1373. 21. Ji B, Li M, Wu WT, et al. LINGO-1 antagonist promotes func- 45. Bonuccelli U. Essential tremor is a neurodegenerative disease. J tional recovery and axonal sprouting after spinal cord injury. Mol Neural Transm 2012;119:1383-1377; discussion 1373. Cell Neurosci 2006;33:311-320. 46. Rajput AH, Adler CH, Shill HA, Rajput A. Essential tremor is not 22. Mi S, Sandrock A, Miller RH. LINGO-1 and its role in CNS a neurodegenerative disease. Neurodegener Dis Manag 2012;2: repair. Int J Biochem Cell Biol 2008;40:1971-1978. 259-268.

1646 Movement Disorders, Vol. 29, No. 13, 2014 LINGO IN ESSENTIAL TREMOR

47. Louis ED, Faust PL, Vonsattel JP. Purkinje cell loss is a charac- 50. Fu Q-L, Hu B, Wu W, Pepinsky RB, Mi S, So KF. Blocking teristic of essential tremor: towards a more mature understand- LINGO-1 function promotes survival follow- ing of pathogenesis. Parkinsonism Relat Disord 2012;18: ing ocular hypertension and transection. Invest Oph- 1003-1004. thalmol Vis Sci 2008;49:975-985. 48. Satoh J, Tabunoki H, Yamamura T, Arima K, Konno H. TROY 51. Mi S, Miller RH, Tang W, et al. Promotion of central nervous sys- and LINGO-1 expression in astrocytes and macrophages/microglia tem remyelination by induced differentiation of oligodendrocyte in multiple sclerosis lesions. Neuropathol Appl Neurobiol 2007;33: precursor cells. Ann Neurol 2009;65:304-315. 99-107. 52. Pepinsky RB, Shao Z, Ji B, et al. Exposure levels of anti-LINGO-1 49. Mi S, Lee X, Shao Z, et al. LINGO-1 is a component of the Li81 antibody in the central nervous system and dose-efficacy rela- Nogo-66 receptor/p75 signaling complex. Nat Neurosci 2004;7: tionships in rat spinal cord remyelination models after systemic 221-228. administration. J Pharmacol Exp Ther 2011;339:519-529.

Movement Disorders, Vol. 29, No. 13, 2014 1647