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 de ric Calon, BPharm, PhD1,2* Vincent Emond, PhD,1,2 Ali H. Rajput, MD, FRCPC,3 Alex Rajput, MD, FRCPC,3 and Fre 1
Laval, Que bec City, Que bec, Canada Faculty of Pharmacy, Universite bec, Neurosciences Axis, Que bec City, Que bec, Canada Centre de Recherche du Centre Hospitalier Universitaire de Que 3 Division of Neurology, Royal University Hospital, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
2
ABSTRACT:
Essential tremor (ET) is the most prevalent adult-onset movement disorder. Despite its health burden, no clear pathognomonic sign has been identified to date because of the rarity of clinicopathological studies. Moreover, treatment options are still scarce and have not significantly changed in the last 30 years, underscoring the urgent need to develop new treatment avenues. In the recent years, leucine-rich repeat (LRR) and immunoglobulin (Ig) domain-containing Nogo receptorinteracting proteins 1 and 2 (LINGO1 and LINGO2, respectively) have been increasingly regarded as possible ET modulators due to emerging genetic association studies linking LINGO with ET. We have investigated LINGO protein and messenger RNA (mRNA) expression in the cerebellum of patients with ET, patients with Parkinson’s disease (PD), and a control group using Western immunoblotting and in situ hybridization. Protein levels of LINGO1, but not LINGO2, were significantly increased in the cerebellar cortex of ET patients compared with controls, partic-
More than 10 million Americans suffer from essential tremor (ET), which is almost 10 times more than those who have Parkinson’s disease (PD), making it the most prevalent adult-onset movement disorder.1 Despite its
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de ric Calon, Centre de Recherche du CHU *Correspondence to: Dr. Fre bec, Pavillon CHUL, Room T2-05, 2705 Laurier Blvd., Quebec, de Que QC, Canada, G1V 4G2; [email protected]
Funding agencies: Dr. Calon was supported by an International Essential Tremor Foundation grant, Canada Foundation for Innovation grant du Que bec (FRSQ) salary 10307, and a Fonds de la Recherche en Sante award. Relevant conflicts of interest/financial disclosures: Nothing to report. Full financial disclosures and author roles may be found in the online version of this article. ¶
The first two authors contributed equally to this work.
Received: 12 August 2013; Revised: 28 November 2013; Accepted: 30 December 2013 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/mds.25819
ularly in individuals with longer disease duration. Compared with controls, LINGO1 protein levels were increased in the cerebellar white matter of PD and ET patients but, for the latter, only when disease duration exceeded 20 years. However, no alteration in LINGO1 mRNA was observed between groups in either the cerebellar cortex or the white matter. We observed alterations in LINGO expression in diseased brain that seemed to progress along with the disease, being initiated in the cerebellar cortex before reaching the white matter. Because LINGO up-regulation has been identified as a potential pathological response to ongoing neurodegenerative processes, the present data suggest that LINGO1 is a potential C 2014 International Parkinson and drug target for ET. V Movement Disorder Society
K e y W o r d s : essential tremor; cerebellum; LINGO; dentate nucleus; neurodegeneration
high prevalence and consequent socioeconomic impacts, the etiopathological base of ET remains unknown. Recently, however, genetic analyses have revealed that SLC1A2 (solute carrier family 1, member 2) and LINGO (leucine-rich repeat and immunoglobulin domain-containing Nogo receptor-interacting protein) may be associated with ET. Indeed, a genome-wide association study performed in 452 ET cases and 14,394 controls has pinpointed two single-nucleotide polymorphisms in the LINGO1 gene associated with ET.3 So far, findings consistent with a genetic association between LINGO and ET have been reported in at least five populations,4-8 but not in others.9-11 Nonetheless, the relationship between LINGO1 polymorphisms and ET stood the test of a recent meta-analysis.12 In addition, the genetic variability of LINGO2 has been associated with ET and PD in North American and Asian populations.13 Despite their caveats, the results
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from these genetic association studies call for the need to decipher the role of LINGO in ET. LINGO1 and LINGO2 belong to a family of four members (LINGO1-LINGO4) expressed in the central nervous system (CNS).14,15 Given the high degree of homology between the LINGO1 and LINGO2 proteins (61%), it is conceivable that both paralogues exert similar functions. LINGO1 is a component of signaling complexes with the Nogo-66 receptor and p75 and is expressed in oligodendrocytes and neurons. As part of this complex, LINGO1 is involved in the inhibition of oligodendrocyte differentiation,16-18 axonal myelination and regeneration,18,19 and neuronal survival.20 Interestingly, the expression of LINGO1 is increased after neuronal damage, and its inhibition promotes functional recovery and axonal sprouting after spinal cord injury.21 Accordingly, a 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 PD.22,23 The role of LINGO2 is less known, but its expression is restricted to neuronal tissue.15 In summary, in vitro and in vivo data indicate that LINGO1 plays a major role in the unfortunate inhibition of neuronal regeneration in spinal cord and brain injury,24,25 suggesting that reducing LINGO1 might be of therapeutic value for neurodegenerative diseases. Interestingly, a study published earlier this year reported LINGO1 protein level alterations in patients with ET compared with controls.26 Taken together with the genetic association between LINGO and ET and the implication of LINGO in neuronal regeneration, these data imply that LINGO could play a role in ET pathophysiology. To characterize LINGO1 in the cerebellum of ET patients, our group took advantage of a well characterized series of human samples to perform postmortem analyses of LINGO1 and LINGO2 protein and RNA levels in the cerebellar cortex (Cctx), white matter (WM), and dentate nucleus (DN) of patients with ET, patients with PD, and controls.
Patients and Methods Clinicopathological Evaluation of Patients We selected 10 patients with PD, 9 patients with ET, and 16 controls based on (1) the absence of neuropathologically confirmed PD (no presence of nigral Lewy bodies) in ET brain, (2) the presence of an easily identifiable dentate nucleus in the tissue bloc, and (3) matching as close as possible for age and gender between the ET, PD, and control groups. Based on examination by neurologists, geriatricians, or internal medicine specialists, all controls had no history of neurological disease at the time of death and had normal brain histologic examination. The clinical diagno-
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sis of patients was made by one of two neurologists, as described previously, and was based on upper limb postural and/or kinetic action tremor of several years’ duration for which there was no metabolic, toxic, or other neurological cause identified.27,28 Tremor frequency and amplitude were evaluated visually, and the severity of tremor was based on the Unified Parkinson’s Disease Rating Scale (UPDRS). Although a probable or definitive Alzheimer’s disease diagnosis was ruled out by a pathologist for each individual who was included, no systematic cognitive assessment was performed. The severity of tremor was recorded at each visit and was based on visual assessment of tremor amplitude and the history of its impact on daily activities.28 Brain tissues that were used for comparison between different groups included samples from 9 patients with ET, 10 patients with PD, and 16 healthy, age-matched controls. A summary of the information is provided in Table 1, confirming homogeneity and relative equivalent tissue quality between groups.27
Handling and Processing of Tissues One-half of the brain was fixed in formalin to perform histological studies of the cerebellum to reveal cerebellar pathology, including Purkinje cell counts or infarcts. The other hemisphere was frozen at 280 C within 25 hours of death. The frozen hemisphere was cut in the frontal plane into 2-mm to 3-mm-thick slices. Horizontal slices (containing the molecular and granular layer of the Cctx and DN) were cryostatsectioned (20 lm), thaw-mounted onto SuperFrostPlus 75 3 50 mm slides (Brain Research Laboratories, Newton Heights, MA, USA), desiccated at 4 C, and stored at 280 C. The pH of cerebellar tissue was measured after homogenization in 10 volumes of unbuffered, deionized, and distilled water as an indication of tissue quality.
Western Immunoblotting Cctx extracts were homogenized in Tris-buffered saline and fractionated as previously described.29,30 Proteins were heated at 95 C for 5 minutes in Laemmli’s loading buffer and separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis on an 8% Tris-glycin polyacryamide gel, transferred on a polyvinylidene fluoride membrane, and blocked in 5% nonfat dry milk, 0.5% bovine serum albumin, 0.1% Tween-20 in phosphate-buffered saline, as previously described.29 Proteins were detected using anti-Lingo-1 (MilliporeUpstate, Billerica, MA, USA), anti-actin (Applied Biological Materials Inc., Richmond, BC, Canada), and anti-LINGO2 (R&D Systems, Minneapolis, MN, USA) followed by horseradish peroxidase-labeled secondary antibodies (Jackson ImmunoResearch Europe Ltd., Newmarket, UK) and chemiluminescence reagents
1997 2006 1997
2002 2008
5 6 7
8 9
1998
3
1999
1993
2
4 24
12 24 20
20
17
1,200 980
1,270 1,290 1,210
1,580
1,340
1,240
1,180
— — 1,140 1,370 1,250 1,320 1,380 1,440 1,470 1,110 1,200 1,215 1,060 1,090 1,337 1,110
73 61
80 83 59
59
45
50
58
— — — — — — — — — — — — — — — —
89 87
89 90 79
74
64
82
81
82 82 74 66 76 84 81 82 71 70 76 78 86 75 72 68
16 26
9 7 20
15
19
32
23
— — — — — — — — — — — — — — — —
3 3
4 4 4
5
3
5
3
21
27 29 20
28
30
27
No No
No No No
No
No
No
Social
No No No No No No No No No No No No No No No No
624 748
706 327 508
599
681
426
519
622 489 337 389 505 555 502 595 667 586 — — — — — —
DNDCN
424 548
424 181 344
466
445
472
417
662 300 608 463 490 310 334 395 325 284 540 336 423 501 479 474
MolCtx
695 1,050
906 717 760
913
822
1,053
1,109
1,095 811 1,155 735 1,151 490 823 1,148 933 668 1,048 605 902 823 865 695
GraCtx
125 99
171 106 155
168
185
168
187
132 114 160 197 154 94 212 186 605 185 217 112 252 218 133 113
Poly-T mRNA, MolCTx
6.39 6.31
6.69 6.17 6.31
6.43
6.75
6.64
6.39
6.07 6.26 — 6.20 6.25 5.93 6.12 6.31 6.83 6.61 6.31 — 6.07 — 6.13 6.08
Brain pH
Family History
(Continued)
ethopropazine; amantadine; bromocriptine; temazepam Trihexyphenidyl benztropine L-dopa; amantadine; bromocriptine L-dopa; bromocriptine; trihexyphenidyl; selegiline; ropinirole; amantadine L-dopa; selegiline; propranolol; amitriptyline; temazepam L-dopa; selegiline L-dopa; amantadine Bromocriptine; selegiline; L-dopa; amantadine L-dopa; selegiline
L-dopa;
Drugs and Effects on Tremor (1 or 2)
E S S E N T I A L
M F
F F F
M
F
18
19
24 24 16 24 15 5 25 18 24 23 11 20 23 11 14 23
PMI
Tremor Disease Severity/ MMSE Ethanol Onset Death Duration, y H&Y Amplitude Score Consumption
CO-1 mRNA
I N
4
F
1996
F
F F F F M M M M M F M F F F F F
2002 1988 2002 2003 2003 2005 1996 1998 1993 1996 1989 1990 1990 1990 1992 1995
Enrollment, y Sex
Ctrl 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 PD 1
Group
Brain Weight, g
Age, y
TABLE 1. Study group information
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1993 1991 1994
1996
1995
2000
2005
3 4 5
6
7
8
9
0.3
F
F
F
F
1,040
1,230
1,170
1,040
1,520 1,060 1,020
1,140 1,380
1,190
0.32
0.49
19 (6) 1249 (137) 18 (6) 1248 (151) 15 (8) 1178 (173)
21
9
24
17
24 3 6
13 15
24
PMI
—
52
71
58
57
34 50 64
65 8
60
0.11
76 (6) 82 (8) 82 (10)
88
93
79
92
63 95 74
80 78
84
—
36
22
21
35
30 45 10
15 70
24
—
3
—
2
2
1
2
2 1 2
2 4
—
27
22
No
No
No
No
—
No No Social
No No
No
372
560
538
685
395 — 424
514 529
649
MolCtx
1,006
854
966
1,273
1,118 — 1,125
828 1,124
1,285
GraCtx
136
66
114
231
164 284 186
143 285
185
Poly-T mRNA, MolCTx
6.61
6.25
6.19
6.54
6.48 6.40 6.46
6.12 6.38
6.53
Brain pH
0.82
0.4
0.21
0.59
0.06
525 (103) 433 (114) 872 (206) 193 (119) 6.24 (0.24) 562 (134) 437 (122) 931 (191) 155 (33) 6.46 (0.19) 553 (189) 502 (102) 1037 (152) 179 (75) 6.38 (0.16)
345
738
—
714
539 — —
308 677
475
DNDCN
Yes
Yes
No
Yes
Yes Yes Yes
No Yes
Family History
None for tremor Propranolol 1; clonazepam 2; L-dopa 2 Propranolol 1 None for tremor L-dopa 2; primidone 1 Propranolol 1 bromocriptine 2; L-dopa – L-dopa 2; L-dopa 2; selegiline 1 Propranolol not tolerated; amantadine 2 L-dopa 2; phenobarb 1 diazepam 1; propranolol 1
Amantadine; L-dopa; selegiline L-dopa; pramipexole
Drugs and Effects on Tremor (1 or 2)
*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.
Average (SD) Ctrl PD ET Comparison test: P value Contingency ANOVA
F F
1995 1986
F F M
F
2004
Enrollment, y Sex
Tremor Disease Severity/ MMSE Ethanol Onset Death Duration, y H&Y Amplitude Score Consumption
CO-1 mRNA
E T
10 ET 1 2
Group
Brain Weight, g
Age, y
TABLE 1. Continued
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(Lumiglo Reserve; KPL, Gaithersburg, MD, USA). Quantification was performed using a Kodak Image Station 4000MM Digital Imaging System (Carestream Health, Rochester, NY, USA). Each experiment was repeated three times. LINGO1 antibody specificity was tested by competition with Lingo 1 peptide (antibody ab25890; Abcam, Cambridge, UK) at a 60:1 ratio, which completely blocked immunostaining.
In Situ Hybridization In situ hybridization was performed according to general methodology, as described previously.27,31 The oligonucleotide sequences we used corresponded to bases 168 through 127, 339 through 295, 1327 through 1283, and 1727 through 1683 of human Lingo-1 messenger RNA (mRNA) (NM_032808.5). In situ hybridization for housekeeping b-actin and cytochrome oxidase-1 mRNAs as well as poly-A tails (poly-T probe) was performed previously on the same series of sections.27 Oligonucleotides were labeled with 33P-dATP (PerkinElmer Inc., Waltham, MA, USA) using a three-terminal deoxynucleotidyl transferase enzyme kit (New England Biolabs, Ipswich, MA, USA). The reaction was carried out at 37 C for 60 minutes, and labeled oligonucleotides were purified with the QIAquick Nucleotide Removal Kit (Qiagen, Venlo, Netherlands). Prehybridation and hybridization conditions were performed exactly as described. Cerebellar slices were exposed to Kodak Biomax MR films for 15 days, and macroscopic optical density quantification was performed using the Kodak Image Station. The final data from each individual represent the mean of eight tissue sections. Nonspecific hybridization was considered negligible, as determined by adding a 100-fold excess of unlabeled probes.
Immunofluorescence Immunofluorescencent (IF) labeling was performed on 6-lm-thick sections of paraffin-embedded cerebellum samples. The sections were microwaved twice for 2 minutes in 0.01 M citrate buffer, pH 6.0, for antigen retrieval. Slices were then washed and pretreated with 10% normal horse serum (NHS) in 0.1 M phosphate-buffered saline (PBS) containing 0.2% Triton X-100 (TX) for 1 hour. Sections were incubated overnight at 4 C with an anti-Lingo1 antibody (1:300 dilution; Millipore-Upstate, Billerica, MA), mouse anti-Calbindin D-28K (1:500 dilution; Sigma-Aldrich, St. Louis, MO, USA), and/or mouse anti-myelin basic protein SMI99 (MBP) (1:500 dilution; Covance Research Products/Cedarlane, Burlington, Ontario, Canada) and diluted in 0.1 M PBS with 1% NHS and 0.2% TX. Sections were incubated for 2 hours with an anti-rabbit-biotin-conjugated antibody (Sigma Chemical Company) diluted in PBS containing 0.05% TX and 1% NHS. Slices were then incubated for 1
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hour with Alexa Fluor 488 streptavidin conjugate (Life Technologies, Carlsbad, CA, USA) and counterstained with 40 ,6-diamidino-2-phenylindole (DAPI) (Life Technologies, Carlsbad, CA, USA). Calbindin and MBP were detected using donkey anti-mouse secondary antibodies coupled to Alexa Fluor 555. Slices were finally incubated for 5 minutes in a 0.5% Sudan Black solution in 70% methanol and coverslipped using Mowiol anti-fade mounting medium. Two to three washes in 0.1 M PBS were included between each step. Lingo1 antibody specificity was assessed by competition with Lingo 1 peptide (ab25890; Abcam, Cambridge, UK). The antibody LINGO1 with or without LINGO1 peptide (60:1 molar ratio) was incubated for 30 minutes at room temperature. The LINGO1stained sections were examined with an i90 Nikon fluorescent microscope (Nikon Corporation, Tokyo, Japan) coupled to a Hamamatsu 1394 ORCA-285 monochrome camera (Hamamatsu Photonics, Hamamatsu, Japan) and exploited by SimplePCI software (version 5.3.0.1102; Compix Inc. Imaging Systems, Cranberry Township, PA, USA).
Statistical Analysis For LINGO1 group comparisons, because unequal variance was determined with Bartlett’s test, a Welch one-way analysis of variance followed by a Bonferroni’s multiple comparison test was performed. For LINGO2 group comparisons, normal distribution of values could not be assumed, and a nonparametric Kruskal-Wallis test was performed followed by Dunn’s multiple comparison test. Adjustments for age of death, gender, or cerebellar pH were performed using an analysis of covariance, when needed. When two groups were compared, a nonparametric MannWhitney test was used. A simple regression model was used to determine correlation and significance of the linear relationship between parameters. All statistical analyses were performed using GraphPad Prism 5 analysis software (version 5.0.1; GraphPad Software Inc., San Diego, CA, USA) and JMP (version 10.1; SAS Institute Inc., Cary, NC, USA), and P values < 0.05 were considered significant.
Results LINGO1 Expression Is Increased in the Cerebellum of ET Patients Based on the genetic evidence cited above, we hypothesized that LINGO could play a role in the degenerative changes reported in the cerebellum of ET patients.32-39 We therefore assessed the protein expression levels of LINGO1 and LINGO2 in human Cctx and WM of ET patients, PD patients. and healthy controls (Figs. 1, 2). PD patients were included to differentiate ET-specific changes (ie, kinetic tremor vs.
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FIG. 1. LINGO1 and LINGO2 expression levels are illustrated in the cerebellar cortex (Cctx) of patients with essential tremor (ET), patients with Parkinson’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 LINGO1 levels (1119%; Welch analysis of variance, P 5 0.0055; Bonferroni post-hoc test, P < 0.01) in the Cctx from ET patients, but not PD patients, compared with controls (Fig. 1A). This difference remained statistically significant after adjustment for the covariates age of death, gender, and postmortem interval using an analysis of covariance. Interestingly, the rise of LINGO1 in the Cctx of ET patients was more prominent in individuals with a long (>20-year) disease duration (1156% vs. controls; Mann-Whitney test, P 5 0.0023) (Fig. 1C). Linear correlation analyses did not show a significant relationship between disease duration and LINGO1 levels (r2 5 0.02; P 5 0.71). Because LINGO1 is also expressed in myelinating oligodendrocytes, homogenates from the WM also were analyzed. Compared with controls, the LINGO1 protein levels were found in higher concentrations in the cerebellar WM of PD patients (1113%; KruskalWallis test, P 5 0.0385) (Fig. 2A). An increase of LINGO1 in the WM was also detected in ET patients who suffered from the disease for more than 20 years (185%; Mann-Whitney test, P 5 0.0360) (Fig. 2C). In contrast, no significant difference in LINGO2 concen-
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trations was detected between groups, even in patients with longer disease duration (Fig. 1B,D, Fig. 2B,D).
LINGO Is Present Mainly in the Myelinated Fibers of the WM To investigate the localization of LINGO1 in the cerebellum, we performed IF experiments on cerebellar sections from patients with ET, patients with PD, and controls. Our results clearly show specific immunolabeling of LINGO1 in myelinated fibers of the WM, as demonstrated by (1) colocalization with MBP, a myelin marker, and (2) loss of signal by addition of the immunization peptide (Fig. 3). Some disparate staining was retrieved around calbindin-labeled Purkinje cell bodies, but no clear structure was visible (Fig. 3). However, we did not replicate previous observations of LINGO-1 enrichment in a brush pinceau structure around the axonal initial segment of Purkinje cells in ET cerebellum.26 A small staining of the DN cells was obvious in some patients but not in others, making it difficult to make a conclusion about the cellular DN localization of LINGO1. Finally, we did not observe obvious LINGO1 localization changes in ET or PD cerebellum slices compared with controls (data not shown).
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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 immunolabeling in the myelinated fibers of the cerebellum and, possibly, surrounding at least some Purkinje cells.
LINGO1 Protein Alterations Do Not Implicate mRNA Expression Changes We further investigated whether the alterations found in LINGO1 would reflect transcription changes. As depicted above, the protein expression patterns of LINGO1 were concentrated within the Purkinje cell layer, the DN, and myelinated fibers of the WM, all accounting for only a very small parts of the cerebellum and making it technically challenging to measure their mRNA content with the available techniques, which require tissue homogenates. In situ hybridization was selected because it allows a quantitative, two-dimensional measurement of mRNA expression levels in different subregions of the cerebellum, including the cortical region, the WM, and the DN. Consistent with the IF data, LINGO1 mRNAs were mainly detected in the granular layer of the cerebellum, with a weak signal in the DN (Fig. 4A). However, there was no significant difference between the groups (Fig.
4B,C), indicating that the higher LINGO1 protein content observed in ET brains was not due to changes in mRNA expression levels.
Discussion In the current study, we conducted an analysis of LINGO1 and LINGO2 expression in the cerebellum of ET patients compared with PD patients and healthy controls. We observed that LINGO1 and, although at lower levels, LINGO2 were both expressed in the cerebellum. LINGO1 mRNA expression was mainly confined to the granular layer of the cerebellum and the DN, whereas most LINGO1-specific immunostaining was found to colocalize with the myelin-binding protein in the WM. These observations are in line with previous immunohistochemistry reports suggesting that LINGO-1 can be detected in human and rodent cerebellum,14,16,26 expressed by neurons and oligodendrocytes,16,18 but did not confirm the cellular distribution within or around Purkinje cells observed previously.14,26 However, the most striking observation reported here is a significant increase of LINGO1 protein levels in the Cctx of ET patients, compared
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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 at the mRNA level. Higher LINGO1 concentrations in both the Cctx and the WM were particularly noticeable in patients who had suffered from ET for > 20 years. Such higher levels of LINGO1 in ET are consistent with a recent report of a 50% increase in LINGO1 protein in the Cctx of ET patients compared with controls, a change not detected in the occipital brain cortex.26 A rise in LINGO1 expression may be explained by either an imbalance of post-transcriptional regulators, such as microRNAs (miRNAs), or by decreased degradation of LINGO1. Abnormal miRNA expression patterns are increasingly studied in a number of neurodegenerative disorders, including PD.40 This class of small RNAs targets mRNAs through binding to a specific miRNA binding site (seed region), resulting in the degradation or translational inhibition of the target and, thus, decreases its protein levels. Imbalances in the miRNA expression levels, therefore, could result in altered protein levels. Prediction algorithms, such as targetscan.org, indicate that there are multiple potential miRNA binding sites within the LINGO1 mRNA that are not present in the LINGO2 mRNA, which, thus, could account for the specific differences in LINGO1 expression observed in ET. The altered expression levels of LINGO1 also could be due to a decreased degradation rate in the affected brain
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regions. These observations are made in several other neurodegenerative disorders. Indeed, in PD, it has been hypothesized that failure of the protein degradation systems results in increased accumulation of ubiquitinated proteins, which form the core of Lewy bodies, one of the pathological hallmarks of PD.41 Lewy bodies are retrieved in the brainstem of a small subpopulation of ET patients.32,37,38 However, there are no indications of the presence of such structures in the cerebellum, arguing against a ubiquitin-related degradation hypothesis. Moreover, based on the high protein identity (61% identity, 77% homology) between LINGO1 and LINGO2 (NP_116197.4, NP_689783.1), it is conceivable that they are both degraded in the same manner, making it improbable that there would be degradation abnormalities of LINGO1 and not of LINGO2. It is therefore more likely that the increased expression of LINGO1 is due to post-transcriptional changes in ET. However, whether this change is causative or consequential to the disease pathogenesis remains unclear at this stage of investigation. Nevertheless, the late occurrence of the increase in LINGO during the course of the disease suggests that it is not a primary cause of ET but, rather, a faulty pathological response that develops over a long period of time. Several lines of evidence suggest that ET pathogenesis could involve a neurodegenerative process,32,42,43
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FIG. 4. Cerebellar LINGO1 messenger RNA distribution is illustrated in the control group and in patients with Parkinson’s disease (PD) and essential tremor (ET). (A) LINGO1 in situ hybridization (ISH) of human cerebellum is illustrated. An arrow indicates the dentate nucleus. (B,C) Signal quantification is illustrated in the cerebellar cortex and the cerebellar white matter in the control, PD, and ET groups.
leading to a lively debate on the issue.44-47 In this regard, mounting data suggest that increased LINGO1 expression is a marker of CNS injury, playing a vital role in vicious cycles leading to neuronal death, axon degeneration, and demyelination. First, the dysregulation of LINGO1 is found in several neurodegenerative pathologies, such as multiple sclerosis48 and PD,23 as well as in rat models of spinal cord injury49 and glaucoma.50 More specifically, it also has been shown previously that the levels of LINGO1 mRNA are upregulated in PD substantia nigra.23 Accordingly, Inoue and colleagues have demonstrated an increase in LINGO1 in the striatum of 1-methyl-4-phenyl-1,2,3,6tetrahydtopyridine (MPTP)-treated mice.23 Second, evidence suggesting that LINGO-1 and its associated pathways could be implicated in the cellular injury
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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 oligodendrocyte differentiation, axonal myelination and regeneration, 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 expression in the Cctx of ET patients, these data indicate that increased LINGO1 expression could be interpreted as an evidence of an underlying neurodegenerative 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 inhibition of LINGO1 as a drug target has already received considerable interest in the fields of multiple sclerosis,48 PD,23 and spinal cord injury.21 Moreover, several groups are studying the identification of a possible beneficial role of LINGO1 antagonists to promote neuro-restoration and remyelination.17,21,51 A LINGO1 antagonist, BIIB033, has now even reached clinical trials (ClinicalTrials.gov identifier, NCT01244139). More recently, anti-LINGO-1 Li81 antibodies have been shown to passively enter the CNS, which was sufficient to lead to strong remyelinization in rat spinal cord remyelination models.52 If LINGO1 up-regulation exerts faulty effects in ET as well, then such LINGO1 antagonists and antibodies ultimately also could be used for the treatment of ET.
Conclusion In summary, higher LINGO1 protein levels were detected in both the Cctx and WM of patients who suffered from ET for greater than 20 years. We hypothesize that these changes are due to posttranscriptional alterations, because no mRNA changes were observed. Furthermore, the present data suggest that down-regulation of LINGO in the cerebellum is a potential disease-modifying therapeutic target in ET. Acknowledgements: We thank the patients and families who generously donated tissue to our research program and the Regina Curling Classic, the Greystone Golf Classic, and RUHF, Saskatoon for their
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support of special clinics and for acquisition and maintaining the brain material.
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Mi S, Sandrock A, Miller RH. LINGO-1 and its role in CNS repair. Int J Biochem Cell Biol 2008;40:1971-1978.
Rajput AH, Adler CH, Shill HA, Rajput A. Essential tremor is not a neurodegenerative disease. Neurodegener Dis Manag 2012;2: 259-268.
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Fu Q-L, Hu B, Wu W, Pepinsky RB, Mi S, So KF. Blocking LINGO-1 function promotes retinal ganglion cell survival following ocular hypertension and optic nerve transection. Invest Ophthalmol Vis Sci 2008;49:975-985.
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Mi S, Miller RH, Tang W, et al. Promotion of central nervous system remyelination by induced differentiation of oligodendrocyte precursor cells. Ann Neurol 2009;65:304-315.
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Pepinsky RB, Shao Z, Ji B, et al. Exposure levels of anti-LINGO-1 Li81 antibody in the central nervous system and dose-efficacy relationships in rat spinal cord remyelination models after systemic administration. J Pharmacol Exp Ther 2011;339:519-529.
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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 de ric Calon, BPharm, PhD1,2* Vincent Emond, PhD,1,2 Ali H. Rajput, MD, FRCPC,3 Alex Rajput, MD, FRCPC,3 and Fre 1
Laval, Que bec City, Que bec, Canada Faculty of Pharmacy, Universite bec, Neurosciences Axis, Que bec City, Que bec, Canada Centre de Recherche du Centre Hospitalier Universitaire de Que 3 Division of Neurology, Royal University Hospital, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
2
ABSTRACT:
Essential tremor (ET) is the most prevalent adult-onset movement disorder. Despite its health burden, no clear pathognomonic sign has been identified to date because of the rarity of clinicopathological studies. Moreover, treatment options are still scarce and have not significantly changed in the last 30 years, underscoring the urgent need to develop new treatment avenues. In the recent years, leucine-rich repeat (LRR) and immunoglobulin (Ig) domain-containing Nogo receptorinteracting proteins 1 and 2 (LINGO1 and LINGO2, respectively) have been increasingly regarded as possible ET modulators due to emerging genetic association studies linking LINGO with ET. We have investigated LINGO protein and messenger RNA (mRNA) expression in the cerebellum of patients with ET, patients with Parkinson’s disease (PD), and a control group using Western immunoblotting and in situ hybridization. Protein levels of LINGO1, but not LINGO2, were significantly increased in the cerebellar cortex of ET patients compared with controls, partic-
More than 10 million Americans suffer from essential tremor (ET), which is almost 10 times more than those who have Parkinson’s disease (PD), making it the most prevalent adult-onset movement disorder.1 Despite its
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de ric Calon, Centre de Recherche du CHU *Correspondence to: Dr. Fre bec, Pavillon CHUL, Room T2-05, 2705 Laurier Blvd., Quebec, de Que QC, Canada, G1V 4G2; [email protected]
Funding agencies: Dr. Calon was supported by an International Essential Tremor Foundation grant, Canada Foundation for Innovation grant du Que bec (FRSQ) salary 10307, and a Fonds de la Recherche en Sante award. Relevant conflicts of interest/financial disclosures: Nothing to report. Full financial disclosures and author roles may be found in the online version of this article. ¶
The first two authors contributed equally to this work.
Received: 12 August 2013; Revised: 28 November 2013; Accepted: 30 December 2013 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/mds.25819
ularly in individuals with longer disease duration. Compared with controls, LINGO1 protein levels were increased in the cerebellar white matter of PD and ET patients but, for the latter, only when disease duration exceeded 20 years. However, no alteration in LINGO1 mRNA was observed between groups in either the cerebellar cortex or the white matter. We observed alterations in LINGO expression in diseased brain that seemed to progress along with the disease, being initiated in the cerebellar cortex before reaching the white matter. Because LINGO up-regulation has been identified as a potential pathological response to ongoing neurodegenerative processes, the present data suggest that LINGO1 is a potential C 2014 International Parkinson and drug target for ET. V Movement Disorder Society
K e y W o r d s : essential tremor; cerebellum; LINGO; dentate nucleus; neurodegeneration
high prevalence and consequent socioeconomic impacts, the etiopathological base of ET remains unknown. Recently, however, genetic analyses have revealed that SLC1A2 (solute carrier family 1, member 2) and LINGO (leucine-rich repeat and immunoglobulin domain-containing Nogo receptor-interacting protein) may be associated with ET. Indeed, a genome-wide association study performed in 452 ET cases and 14,394 controls has pinpointed two single-nucleotide polymorphisms in the LINGO1 gene associated with ET.3 So far, findings consistent with a genetic association between LINGO and ET have been reported in at least five populations,4-8 but not in others.9-11 Nonetheless, the relationship between LINGO1 polymorphisms and ET stood the test of a recent meta-analysis.12 In addition, the genetic variability of LINGO2 has been associated with ET and PD in North American and Asian populations.13 Despite their caveats, the results
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from these genetic association studies call for the need to decipher the role of LINGO in ET. LINGO1 and LINGO2 belong to a family of four members (LINGO1-LINGO4) expressed in the central nervous system (CNS).14,15 Given the high degree of homology between the LINGO1 and LINGO2 proteins (61%), it is conceivable that both paralogues exert similar functions. LINGO1 is a component of signaling complexes with the Nogo-66 receptor and p75 and is expressed in oligodendrocytes and neurons. As part of this complex, LINGO1 is involved in the inhibition of oligodendrocyte differentiation,16-18 axonal myelination and regeneration,18,19 and neuronal survival.20 Interestingly, the expression of LINGO1 is increased after neuronal damage, and its inhibition promotes functional recovery and axonal sprouting after spinal cord injury.21 Accordingly, a 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 PD.22,23 The role of LINGO2 is less known, but its expression is restricted to neuronal tissue.15 In summary, in vitro and in vivo data indicate that LINGO1 plays a major role in the unfortunate inhibition of neuronal regeneration in spinal cord and brain injury,24,25 suggesting that reducing LINGO1 might be of therapeutic value for neurodegenerative diseases. Interestingly, a study published earlier this year reported LINGO1 protein level alterations in patients with ET compared with controls.26 Taken together with the genetic association between LINGO and ET and the implication of LINGO in neuronal regeneration, these data imply that LINGO could play a role in ET pathophysiology. To characterize LINGO1 in the cerebellum of ET patients, our group took advantage of a well characterized series of human samples to perform postmortem analyses of LINGO1 and LINGO2 protein and RNA levels in the cerebellar cortex (Cctx), white matter (WM), and dentate nucleus (DN) of patients with ET, patients with PD, and controls.
Patients and Methods Clinicopathological Evaluation of Patients We selected 10 patients with PD, 9 patients with ET, and 16 controls based on (1) the absence of neuropathologically confirmed PD (no presence of nigral Lewy bodies) in ET brain, (2) the presence of an easily identifiable dentate nucleus in the tissue bloc, and (3) matching as close as possible for age and gender between the ET, PD, and control groups. Based on examination by neurologists, geriatricians, or internal medicine specialists, all controls had no history of neurological disease at the time of death and had normal brain histologic examination. The clinical diagno-
2
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sis of patients was made by one of two neurologists, as described previously, and was based on upper limb postural and/or kinetic action tremor of several years’ duration for which there was no metabolic, toxic, or other neurological cause identified.27,28 Tremor frequency and amplitude were evaluated visually, and the severity of tremor was based on the Unified Parkinson’s Disease Rating Scale (UPDRS). Although a probable or definitive Alzheimer’s disease diagnosis was ruled out by a pathologist for each individual who was included, no systematic cognitive assessment was performed. The severity of tremor was recorded at each visit and was based on visual assessment of tremor amplitude and the history of its impact on daily activities.28 Brain tissues that were used for comparison between different groups included samples from 9 patients with ET, 10 patients with PD, and 16 healthy, age-matched controls. A summary of the information is provided in Table 1, confirming homogeneity and relative equivalent tissue quality between groups.27
Handling and Processing of Tissues One-half of the brain was fixed in formalin to perform histological studies of the cerebellum to reveal cerebellar pathology, including Purkinje cell counts or infarcts. The other hemisphere was frozen at 280 C within 25 hours of death. The frozen hemisphere was cut in the frontal plane into 2-mm to 3-mm-thick slices. Horizontal slices (containing the molecular and granular layer of the Cctx and DN) were cryostatsectioned (20 lm), thaw-mounted onto SuperFrostPlus 75 3 50 mm slides (Brain Research Laboratories, Newton Heights, MA, USA), desiccated at 4 C, and stored at 280 C. The pH of cerebellar tissue was measured after homogenization in 10 volumes of unbuffered, deionized, and distilled water as an indication of tissue quality.
Western Immunoblotting Cctx extracts were homogenized in Tris-buffered saline and fractionated as previously described.29,30 Proteins were heated at 95 C for 5 minutes in Laemmli’s loading buffer and separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis on an 8% Tris-glycin polyacryamide gel, transferred on a polyvinylidene fluoride membrane, and blocked in 5% nonfat dry milk, 0.5% bovine serum albumin, 0.1% Tween-20 in phosphate-buffered saline, as previously described.29 Proteins were detected using anti-Lingo-1 (MilliporeUpstate, Billerica, MA, USA), anti-actin (Applied Biological Materials Inc., Richmond, BC, Canada), and anti-LINGO2 (R&D Systems, Minneapolis, MN, USA) followed by horseradish peroxidase-labeled secondary antibodies (Jackson ImmunoResearch Europe Ltd., Newmarket, UK) and chemiluminescence reagents
1997 2006 1997
2002 2008
5 6 7
8 9
1998
3
1999
1993
2
4 24
12 24 20
20
17
1,200 980
1,270 1,290 1,210
1,580
1,340
1,240
1,180
— — 1,140 1,370 1,250 1,320 1,380 1,440 1,470 1,110 1,200 1,215 1,060 1,090 1,337 1,110
73 61
80 83 59
59
45
50
58
— — — — — — — — — — — — — — — —
89 87
89 90 79
74
64
82
81
82 82 74 66 76 84 81 82 71 70 76 78 86 75 72 68
16 26
9 7 20
15
19
32
23
— — — — — — — — — — — — — — — —
3 3
4 4 4
5
3
5
3
21
27 29 20
28
30
27
No No
No No No
No
No
No
Social
No No No No No No No No No No No No No No No No
624 748
706 327 508
599
681
426
519
622 489 337 389 505 555 502 595 667 586 — — — — — —
DNDCN
424 548
424 181 344
466
445
472
417
662 300 608 463 490 310 334 395 325 284 540 336 423 501 479 474
MolCtx
695 1,050
906 717 760
913
822
1,053
1,109
1,095 811 1,155 735 1,151 490 823 1,148 933 668 1,048 605 902 823 865 695
GraCtx
125 99
171 106 155
168
185
168
187
132 114 160 197 154 94 212 186 605 185 217 112 252 218 133 113
Poly-T mRNA, MolCTx
6.39 6.31
6.69 6.17 6.31
6.43
6.75
6.64
6.39
6.07 6.26 — 6.20 6.25 5.93 6.12 6.31 6.83 6.61 6.31 — 6.07 — 6.13 6.08
Brain pH
Family History
(Continued)
ethopropazine; amantadine; bromocriptine; temazepam Trihexyphenidyl benztropine L-dopa; amantadine; bromocriptine L-dopa; bromocriptine; trihexyphenidyl; selegiline; ropinirole; amantadine L-dopa; selegiline; propranolol; amitriptyline; temazepam L-dopa; selegiline L-dopa; amantadine Bromocriptine; selegiline; L-dopa; amantadine L-dopa; selegiline
L-dopa;
Drugs and Effects on Tremor (1 or 2)
E S S E N T I A L
M F
F F F
M
F
18
19
24 24 16 24 15 5 25 18 24 23 11 20 23 11 14 23
PMI
Tremor Disease Severity/ MMSE Ethanol Onset Death Duration, y H&Y Amplitude Score Consumption
CO-1 mRNA
I N
4
F
1996
F
F F F F M M M M M F M F F F F F
2002 1988 2002 2003 2003 2005 1996 1998 1993 1996 1989 1990 1990 1990 1992 1995
Enrollment, y Sex
Ctrl 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 PD 1
Group
Brain Weight, g
Age, y
TABLE 1. Study group information
L I N G O T R E M O R
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4
Movement Disorders, Vol. 00, No. 00, 2014
1993 1991 1994
1996
1995
2000
2005
3 4 5
6
7
8
9
0.3
F
F
F
F
1,040
1,230
1,170
1,040
1,520 1,060 1,020
1,140 1,380
1,190
0.32
0.49
19 (6) 1249 (137) 18 (6) 1248 (151) 15 (8) 1178 (173)
21
9
24
17
24 3 6
13 15
24
PMI
—
52
71
58
57
34 50 64
65 8
60
0.11
76 (6) 82 (8) 82 (10)
88
93
79
92
63 95 74
80 78
84
—
36
22
21
35
30 45 10
15 70
24
—
3
—
2
2
1
2
2 1 2
2 4
—
27
22
No
No
No
No
—
No No Social
No No
No
372
560
538
685
395 — 424
514 529
649
MolCtx
1,006
854
966
1,273
1,118 — 1,125
828 1,124
1,285
GraCtx
136
66
114
231
164 284 186
143 285
185
Poly-T mRNA, MolCTx
6.61
6.25
6.19
6.54
6.48 6.40 6.46
6.12 6.38
6.53
Brain pH
0.82
0.4
0.21
0.59
0.06
525 (103) 433 (114) 872 (206) 193 (119) 6.24 (0.24) 562 (134) 437 (122) 931 (191) 155 (33) 6.46 (0.19) 553 (189) 502 (102) 1037 (152) 179 (75) 6.38 (0.16)
345
738
—
714
539 — —
308 677
475
DNDCN
Yes
Yes
No
Yes
Yes Yes Yes
No Yes
Family History
None for tremor Propranolol 1; clonazepam 2; L-dopa 2 Propranolol 1 None for tremor L-dopa 2; primidone 1 Propranolol 1 bromocriptine 2; L-dopa – L-dopa 2; L-dopa 2; selegiline 1 Propranolol not tolerated; amantadine 2 L-dopa 2; phenobarb 1 diazepam 1; propranolol 1
Amantadine; L-dopa; selegiline L-dopa; pramipexole
Drugs and Effects on Tremor (1 or 2)
*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.
Average (SD) Ctrl PD ET Comparison test: P value Contingency ANOVA
F F
1995 1986
F F M
F
2004
Enrollment, y Sex
Tremor Disease Severity/ MMSE Ethanol Onset Death Duration, y H&Y Amplitude Score Consumption
CO-1 mRNA
E T
10 ET 1 2
Group
Brain Weight, g
Age, y
TABLE 1. Continued
D E L A Y A L
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(Lumiglo Reserve; KPL, Gaithersburg, MD, USA). Quantification was performed using a Kodak Image Station 4000MM Digital Imaging System (Carestream Health, Rochester, NY, USA). Each experiment was repeated three times. LINGO1 antibody specificity was tested by competition with Lingo 1 peptide (antibody ab25890; Abcam, Cambridge, UK) at a 60:1 ratio, which completely blocked immunostaining.
In Situ Hybridization In situ hybridization was performed according to general methodology, as described previously.27,31 The oligonucleotide sequences we used corresponded to bases 168 through 127, 339 through 295, 1327 through 1283, and 1727 through 1683 of human Lingo-1 messenger RNA (mRNA) (NM_032808.5). In situ hybridization for housekeeping b-actin and cytochrome oxidase-1 mRNAs as well as poly-A tails (poly-T probe) was performed previously on the same series of sections.27 Oligonucleotides were labeled with 33P-dATP (PerkinElmer Inc., Waltham, MA, USA) using a three-terminal deoxynucleotidyl transferase enzyme kit (New England Biolabs, Ipswich, MA, USA). The reaction was carried out at 37 C for 60 minutes, and labeled oligonucleotides were purified with the QIAquick Nucleotide Removal Kit (Qiagen, Venlo, Netherlands). Prehybridation and hybridization conditions were performed exactly as described. Cerebellar slices were exposed to Kodak Biomax MR films for 15 days, and macroscopic optical density quantification was performed using the Kodak Image Station. The final data from each individual represent the mean of eight tissue sections. Nonspecific hybridization was considered negligible, as determined by adding a 100-fold excess of unlabeled probes.
Immunofluorescence Immunofluorescencent (IF) labeling was performed on 6-lm-thick sections of paraffin-embedded cerebellum samples. The sections were microwaved twice for 2 minutes in 0.01 M citrate buffer, pH 6.0, for antigen retrieval. Slices were then washed and pretreated with 10% normal horse serum (NHS) in 0.1 M phosphate-buffered saline (PBS) containing 0.2% Triton X-100 (TX) for 1 hour. Sections were incubated overnight at 4 C with an anti-Lingo1 antibody (1:300 dilution; Millipore-Upstate, Billerica, MA), mouse anti-Calbindin D-28K (1:500 dilution; Sigma-Aldrich, St. Louis, MO, USA), and/or mouse anti-myelin basic protein SMI99 (MBP) (1:500 dilution; Covance Research Products/Cedarlane, Burlington, Ontario, Canada) and diluted in 0.1 M PBS with 1% NHS and 0.2% TX. Sections were incubated for 2 hours with an anti-rabbit-biotin-conjugated antibody (Sigma Chemical Company) diluted in PBS containing 0.05% TX and 1% NHS. Slices were then incubated for 1
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hour with Alexa Fluor 488 streptavidin conjugate (Life Technologies, Carlsbad, CA, USA) and counterstained with 40 ,6-diamidino-2-phenylindole (DAPI) (Life Technologies, Carlsbad, CA, USA). Calbindin and MBP were detected using donkey anti-mouse secondary antibodies coupled to Alexa Fluor 555. Slices were finally incubated for 5 minutes in a 0.5% Sudan Black solution in 70% methanol and coverslipped using Mowiol anti-fade mounting medium. Two to three washes in 0.1 M PBS were included between each step. Lingo1 antibody specificity was assessed by competition with Lingo 1 peptide (ab25890; Abcam, Cambridge, UK). The antibody LINGO1 with or without LINGO1 peptide (60:1 molar ratio) was incubated for 30 minutes at room temperature. The LINGO1stained sections were examined with an i90 Nikon fluorescent microscope (Nikon Corporation, Tokyo, Japan) coupled to a Hamamatsu 1394 ORCA-285 monochrome camera (Hamamatsu Photonics, Hamamatsu, Japan) and exploited by SimplePCI software (version 5.3.0.1102; Compix Inc. Imaging Systems, Cranberry Township, PA, USA).
Statistical Analysis For LINGO1 group comparisons, because unequal variance was determined with Bartlett’s test, a Welch one-way analysis of variance followed by a Bonferroni’s multiple comparison test was performed. For LINGO2 group comparisons, normal distribution of values could not be assumed, and a nonparametric Kruskal-Wallis test was performed followed by Dunn’s multiple comparison test. Adjustments for age of death, gender, or cerebellar pH were performed using an analysis of covariance, when needed. When two groups were compared, a nonparametric MannWhitney test was used. A simple regression model was used to determine correlation and significance of the linear relationship between parameters. All statistical analyses were performed using GraphPad Prism 5 analysis software (version 5.0.1; GraphPad Software Inc., San Diego, CA, USA) and JMP (version 10.1; SAS Institute Inc., Cary, NC, USA), and P values < 0.05 were considered significant.
Results LINGO1 Expression Is Increased in the Cerebellum of ET Patients Based on the genetic evidence cited above, we hypothesized that LINGO could play a role in the degenerative changes reported in the cerebellum of ET patients.32-39 We therefore assessed the protein expression levels of LINGO1 and LINGO2 in human Cctx and WM of ET patients, PD patients. and healthy controls (Figs. 1, 2). PD patients were included to differentiate ET-specific changes (ie, kinetic tremor vs.
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FIG. 1. LINGO1 and LINGO2 expression levels are illustrated in the cerebellar cortex (Cctx) of patients with essential tremor (ET), patients with Parkinson’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 LINGO1 levels (1119%; Welch analysis of variance, P 5 0.0055; Bonferroni post-hoc test, P < 0.01) in the Cctx from ET patients, but not PD patients, compared with controls (Fig. 1A). This difference remained statistically significant after adjustment for the covariates age of death, gender, and postmortem interval using an analysis of covariance. Interestingly, the rise of LINGO1 in the Cctx of ET patients was more prominent in individuals with a long (>20-year) disease duration (1156% vs. controls; Mann-Whitney test, P 5 0.0023) (Fig. 1C). Linear correlation analyses did not show a significant relationship between disease duration and LINGO1 levels (r2 5 0.02; P 5 0.71). Because LINGO1 is also expressed in myelinating oligodendrocytes, homogenates from the WM also were analyzed. Compared with controls, the LINGO1 protein levels were found in higher concentrations in the cerebellar WM of PD patients (1113%; KruskalWallis test, P 5 0.0385) (Fig. 2A). An increase of LINGO1 in the WM was also detected in ET patients who suffered from the disease for more than 20 years (185%; Mann-Whitney test, P 5 0.0360) (Fig. 2C). In contrast, no significant difference in LINGO2 concen-
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trations was detected between groups, even in patients with longer disease duration (Fig. 1B,D, Fig. 2B,D).
LINGO Is Present Mainly in the Myelinated Fibers of the WM To investigate the localization of LINGO1 in the cerebellum, we performed IF experiments on cerebellar sections from patients with ET, patients with PD, and controls. Our results clearly show specific immunolabeling of LINGO1 in myelinated fibers of the WM, as demonstrated by (1) colocalization with MBP, a myelin marker, and (2) loss of signal by addition of the immunization peptide (Fig. 3). Some disparate staining was retrieved around calbindin-labeled Purkinje cell bodies, but no clear structure was visible (Fig. 3). However, we did not replicate previous observations of LINGO-1 enrichment in a brush pinceau structure around the axonal initial segment of Purkinje cells in ET cerebellum.26 A small staining of the DN cells was obvious in some patients but not in others, making it difficult to make a conclusion about the cellular DN localization of LINGO1. Finally, we did not observe obvious LINGO1 localization changes in ET or PD cerebellum slices compared with controls (data not shown).
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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 immunolabeling in the myelinated fibers of the cerebellum and, possibly, surrounding at least some Purkinje cells.
LINGO1 Protein Alterations Do Not Implicate mRNA Expression Changes We further investigated whether the alterations found in LINGO1 would reflect transcription changes. As depicted above, the protein expression patterns of LINGO1 were concentrated within the Purkinje cell layer, the DN, and myelinated fibers of the WM, all accounting for only a very small parts of the cerebellum and making it technically challenging to measure their mRNA content with the available techniques, which require tissue homogenates. In situ hybridization was selected because it allows a quantitative, two-dimensional measurement of mRNA expression levels in different subregions of the cerebellum, including the cortical region, the WM, and the DN. Consistent with the IF data, LINGO1 mRNAs were mainly detected in the granular layer of the cerebellum, with a weak signal in the DN (Fig. 4A). However, there was no significant difference between the groups (Fig.
4B,C), indicating that the higher LINGO1 protein content observed in ET brains was not due to changes in mRNA expression levels.
Discussion In the current study, we conducted an analysis of LINGO1 and LINGO2 expression in the cerebellum of ET patients compared with PD patients and healthy controls. We observed that LINGO1 and, although at lower levels, LINGO2 were both expressed in the cerebellum. LINGO1 mRNA expression was mainly confined to the granular layer of the cerebellum and the DN, whereas most LINGO1-specific immunostaining was found to colocalize with the myelin-binding protein in the WM. These observations are in line with previous immunohistochemistry reports suggesting that LINGO-1 can be detected in human and rodent cerebellum,14,16,26 expressed by neurons and oligodendrocytes,16,18 but did not confirm the cellular distribution within or around Purkinje cells observed previously.14,26 However, the most striking observation reported here is a significant increase of LINGO1 protein levels in the Cctx of ET patients, compared
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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 at the mRNA level. Higher LINGO1 concentrations in both the Cctx and the WM were particularly noticeable in patients who had suffered from ET for > 20 years. Such higher levels of LINGO1 in ET are consistent with a recent report of a 50% increase in LINGO1 protein in the Cctx of ET patients compared with controls, a change not detected in the occipital brain cortex.26 A rise in LINGO1 expression may be explained by either an imbalance of post-transcriptional regulators, such as microRNAs (miRNAs), or by decreased degradation of LINGO1. Abnormal miRNA expression patterns are increasingly studied in a number of neurodegenerative disorders, including PD.40 This class of small RNAs targets mRNAs through binding to a specific miRNA binding site (seed region), resulting in the degradation or translational inhibition of the target and, thus, decreases its protein levels. Imbalances in the miRNA expression levels, therefore, could result in altered protein levels. Prediction algorithms, such as targetscan.org, indicate that there are multiple potential miRNA binding sites within the LINGO1 mRNA that are not present in the LINGO2 mRNA, which, thus, could account for the specific differences in LINGO1 expression observed in ET. The altered expression levels of LINGO1 also could be due to a decreased degradation rate in the affected brain
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regions. These observations are made in several other neurodegenerative disorders. Indeed, in PD, it has been hypothesized that failure of the protein degradation systems results in increased accumulation of ubiquitinated proteins, which form the core of Lewy bodies, one of the pathological hallmarks of PD.41 Lewy bodies are retrieved in the brainstem of a small subpopulation of ET patients.32,37,38 However, there are no indications of the presence of such structures in the cerebellum, arguing against a ubiquitin-related degradation hypothesis. Moreover, based on the high protein identity (61% identity, 77% homology) between LINGO1 and LINGO2 (NP_116197.4, NP_689783.1), it is conceivable that they are both degraded in the same manner, making it improbable that there would be degradation abnormalities of LINGO1 and not of LINGO2. It is therefore more likely that the increased expression of LINGO1 is due to post-transcriptional changes in ET. However, whether this change is causative or consequential to the disease pathogenesis remains unclear at this stage of investigation. Nevertheless, the late occurrence of the increase in LINGO during the course of the disease suggests that it is not a primary cause of ET but, rather, a faulty pathological response that develops over a long period of time. Several lines of evidence suggest that ET pathogenesis could involve a neurodegenerative process,32,42,43
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FIG. 4. Cerebellar LINGO1 messenger RNA distribution is illustrated in the control group and in patients with Parkinson’s disease (PD) and essential tremor (ET). (A) LINGO1 in situ hybridization (ISH) of human cerebellum is illustrated. An arrow indicates the dentate nucleus. (B,C) Signal quantification is illustrated in the cerebellar cortex and the cerebellar white matter in the control, PD, and ET groups.
leading to a lively debate on the issue.44-47 In this regard, mounting data suggest that increased LINGO1 expression is a marker of CNS injury, playing a vital role in vicious cycles leading to neuronal death, axon degeneration, and demyelination. First, the dysregulation of LINGO1 is found in several neurodegenerative pathologies, such as multiple sclerosis48 and PD,23 as well as in rat models of spinal cord injury49 and glaucoma.50 More specifically, it also has been shown previously that the levels of LINGO1 mRNA are upregulated in PD substantia nigra.23 Accordingly, Inoue and colleagues have demonstrated an increase in LINGO1 in the striatum of 1-methyl-4-phenyl-1,2,3,6tetrahydtopyridine (MPTP)-treated mice.23 Second, evidence suggesting that LINGO-1 and its associated pathways could be implicated in the cellular injury
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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 oligodendrocyte differentiation, axonal myelination and regeneration, 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 expression in the Cctx of ET patients, these data indicate that increased LINGO1 expression could be interpreted as an evidence of an underlying neurodegenerative 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 inhibition of LINGO1 as a drug target has already received considerable interest in the fields of multiple sclerosis,48 PD,23 and spinal cord injury.21 Moreover, several groups are studying the identification of a possible beneficial role of LINGO1 antagonists to promote neuro-restoration and remyelination.17,21,51 A LINGO1 antagonist, BIIB033, has now even reached clinical trials (ClinicalTrials.gov identifier, NCT01244139). More recently, anti-LINGO-1 Li81 antibodies have been shown to passively enter the CNS, which was sufficient to lead to strong remyelinization in rat spinal cord remyelination models.52 If LINGO1 up-regulation exerts faulty effects in ET as well, then such LINGO1 antagonists and antibodies ultimately also could be used for the treatment of ET.
Conclusion In summary, higher LINGO1 protein levels were detected in both the Cctx and WM of patients who suffered from ET for greater than 20 years. We hypothesize that these changes are due to posttranscriptional alterations, because no mRNA changes were observed. Furthermore, the present data suggest that down-regulation of LINGO in the cerebellum is a potential disease-modifying therapeutic target in ET. Acknowledgements: We thank the patients and families who generously donated tissue to our research program and the Regina Curling Classic, the Greystone Golf Classic, and RUHF, Saskatoon for their
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support of special clinics and for acquisition and maintaining the brain material.
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