Neurobiology of Aging 36 (2015) 2660.e15e2660.e20
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Serum microRNAs in sporadic amyotrophic lateral sclerosis Axel Freischmidt a, Kathrin Müller a, Lisa Zondler a, Patrick Weydt a, Benjamin Mayer b, Christine A.F. von Arnim a, Annemarie Hübers a, Johannes Dorst a, Markus Otto a, Karlheinz Holzmann c, Albert C. Ludolph a, Karin M. Danzer a, Jochen H. Weishaupt a, * a
Department of Neurology, Ulm University, Ulm, Germany Institute for Epidemiology and Medical Biometry, Ulm University, Ulm, Germany c Genomics-Core Facility, University Hospital Ulm, Center for Biomedical Research, Ulm, Germany b
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Article history: Received 26 January 2015 Received in revised form 27 April 2015 Accepted 2 June 2015 Available online 9 June 2015
MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression and specific mircoRNA “fingerprints” are thought to contribute to and/or reflect certain disease conditions. Recently, we identified surprisingly homogeneous signatures of circulating miRNAs in the serum of familial amyotrophic lateral sclerosis (ALS) patients, which were already present in presymptomatic carriers of ALS gene mutations. Here, we characterize circulating miRNAs in the serum of sporadic ALS patients. We show that, in contrast to familial ALS, miRNA signatures of sporadic ALS are highly heterogeneous suggesting a number of different etiologies. Nevertheless, 2 miRNAs, miR-1234-3p and miR-1825, could be identified to be consistently downregulated in sporadic ALS. Bioinformatic analysis revealed miRNA fingerprints resembling those of familial ALS patients and mutation carriers in 61% of sporadic ALS patients, while the remaining subgroup had clearly different miRNA signatures. These data support a higher than expected contribution of genetic factors also to sporadic ALS. Moreover, our results indicate a more heterogeneous molecular etiology of sporadic ALS compared with (mono)genic cases, which should be considered for the development of disease modifying treatments. Ó 2015 Elsevier Inc. All rights reserved.
Keywords: Amyotrophic lateral sclerosis MicroRNA Serum Biomarker Diagnosis
1. Introduction Amyotrophic lateral sclerosis (ALS) is characterized by a progressive loss of motor neurons and fatal outcome within 3e5 years (Rowland and Shneider, 2001). About 10% of ALS cases are familial (fALS) and approximately half to two-thirds thereof can be explained by mutations in known ALS genes. The remaining 90% are sporadic cases (sALS) with infrequent mutations in known ALS genes (Renton et al., 2014). Growing evidence implicates messenger RNA (mRNA) but most recently also microRNA (miRNA) dysmetabolism in ALS disease pathogenesis (Goodall et al., 2013). Twenty to 24 nucleotides mature miRNAs are mostly inhibitory post-transcriptional regulators of gene expression (Chekulaeva and Filipowicz, 2009). miRNAs are involved in the cellular response to various stressors and insults (Liu et al., 2013; Mendell and Olson, 2012; Viader et al., 2011; Zhang et al., 2013). Thereby, miRNA expression profiles can reflect * Corresponding author at: Department of Neurology, Ulm University, AlbertEinstein-Allee 11, 89081 Ulm, Germany. Tel.: þ49 (0) 731 50063073; fax: þ49 (0) 731 50063050. E-mail address: [email protected] (J.H. Weishaupt). 0197-4580/$ e see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neurobiolaging.2015.06.003
activation of specific pathogenic pathways. Dysregulation of miRNA expression has been reported, for example, in the SOD1-G93A mouse model of ALS or patient-derived tissue (Butovsky et al., 2012; Campos-Melo et al., 2013; De Felice et al., 2012; Toivonen et al., 2014). miRNAs are remarkably stable in serum and other body fluids as exosomal cargos or bound to specific proteins (Xu et al., 2012), and disease-related changes of extracellular miRNA abundance has been repeatedly shown. Although the causative involvement of these changes in disease pathogenesis is still unclear, they are at least thought to represent reactive events related to specific pathways of neurodegeneration. In the context of ALS, alterations in the peripheral miRNA abundance have been suggested to represent a systemic dysfunction of ubiquitously expressed, RNA binding proteins that are involved in ALS pathogenesis (Burgos et al., 2014; Freischmidt et al., 2013, 2014; Gandhi et al., 2013). In a recent study, we identified 30 miRNAs significantly downregulated in the serum of fALS patients that were largely independent from the underlying disease gene. Most of these miRNAs (22) were already downregulated in presymptomatic ALS mutation carriers, even years before the estimated disease onset. Moreover, downregulated miRNAs shared a common nucleotide
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consensus motif possibly indicating specific protein binding partners and downstream pathways common for different ALS genes (Freischmidt et al., 2014). In this study, we examined serum miRNA profiles of sporadic ALS patients and compared them to miRNA signatures of fALS patients (Freischmidt et al., 2014). We show that, serum miRNA profiles are far more heterogeneous in sALS than in fALS. At the same time, however, a considerable proportion of sALS patients share miRNA signatures typical for fALS patients suggesting alteration of common pathways and a high contribution of genetic factors also to sporadic ALS. 2. Material and methods 2.1. Patient cohorts and ethics statement Blood samples were drawn in accordance with the Declaration of Helsinki (World Medical Association, 1964), and study protocols were approved by the national medical ethical review boards. Participants provided prior written informed consent. ALS patients fulfilling the El-Escorial criteria for definite ALS were considered sporadic cases due to a known negative family history of the disease. Additionally, the 2 main genetic causes of familial ALS, namely mutations in the SOD1 gene and a hexanucleotide repeat expansion in C9ORF72 (DeJesus-Hernandez et al., 2011; Renton et al., 2014) were excluded by Sanger sequencing (van Es et al., 2010) or repeat-primed polymerase chain reaction (PCR) (DeJesus-Hernandez et al., 2011), respectively. sALS patients were not related to any of the control individuals. 2.2. Serum miRNA microarray analysis Individual serum samples were collected by the same center according to the same standard operating procedures. Isolation of total RNA from 8e10 mL of serum, microarray analysis using Affymetrix GeneChip miRNA 3.0 Arrays and statistical evaluation were performed exactly as recently described (Freischmidt et al., 2014). Importantly, miRNA microarrays from this study were run in one batch with fALS patients previously published (Freischmidt et al., 2014) ensuring highly comparable results. Microarray raw
data can be accessed at Gene Expression Omnibus (http://www. ncbi.nlm.nih.gov/geo/). GSE number is GSE52917. 2.3. Generation of heatmaps and hierarchical cluster analysis Hierarchical cluster analysis (average linkage) was performed and heatmaps were generated using the Genesis software package (http:// genome.tugraz.at/genesisclient/genesisclient/_ description.shtml). 2.4. Quantitative real-time PCR RNA isolations, reverse transcriptions and quantitative real-time PCRs (qRT-PCRs) were carried out as recently described using miScript Primer Assays (Qiagen, Hilden, Germany) as miRNA specific forward primers (Freischmidt et al., 2013, 2014). Exponential processes (Ct-values) were converted to linear comparisons relative to the control group and normalized to spiked-in miR-39-3p of Caenorhabditis elegans (Cel-miR-39-3p) using 2DDCt method. Error bars were calculated the same way (Livak and Schmittgen, 2001). Comparison of groups was performed using the 2-tailed Student t test. 3. Results 3.1. Serum miRNA profiles of sALS patients are highly heterogeneous In the discovery experiment, relative serum miRNA levels of 18 sALS patients and 16 matched healthy controls (Supplementary Table 1) were compared using Affymetrix GeneChip miRNA 3.0 Arrays. To exclude any false positive changes of miRNA abundance, we restricted our analysis to downregulated miRNAs that are not susceptible to increased hemolysis reported previously for ALS patients (Freischmidt et al., 2014; Ronnevi and Conradi, 1984). We observed a significant downregulation of only 2 miRNAs (Supplementary Table 2) in sALS patients after correction for multiple testing (false discovery rate [FDR] 0.05). The variability of miRNA alterations was higher in the ALS group compared with controls (Fig. 1). This contrasts with a set of miRNAs homogeneously downregulated in familial ALS patients and also premanifest mutations carriers, as recently reported using the identical
Fig. 1. Heterogeneous serum miRNA profiles in sALS patients. The heatmap shows downregulated serum miRNAs (uncorrected p-value 0.05) of sALS patients compared with healthy controls according to the color coding (bottom). miRNA abundances are not homogeneous across the samples. The site of onset (spinal vs. bulbar) did not have an impact on miRNA abundance. Age and gender of the individual samples as well as the fold changes of the respective miRNAs are indicated (* indicates significantly downregulated miRNAs with a false discovery rate 0.05). Abbreviations: miRNA, microRNA; sALS, sporadic amyotrophic lateral sclerosis. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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technical approach and sampling protocol by the same ALS center (Freischmidt et al., 2014). However, despite this heterogeneity, the 2 sALS-linked miRNAs that remained significantly changed after correction for multiple testing could be confirmed in an independent cohort of additional 20 sALS patients and 20 age- and gendermatched controls (Supplementary Table 3) using qRT-PCR as an alternative method (Fig. 2). We next compared the downregulation of miR-1825 and miR-1234-3p in sALS with fALS. Their relative abundance was determined in a cohort of 13 fALS patients [8 C9ORF72 and 5 SOD1 mutant patients; detailed characteristics of the cohort published previously (Freischmidt et al., 2014)] and 13 matched healthy controls by qRT-PCR. Although downregulation of miR-1825 was also evident in fALS patients and thus seems to be a common feature of ALS, significant downregulation of miR-1234-3p was restricted to the sALS cases (Fig. 2), although a nonsignificant trend (p ¼ 0.15) was observed also in the fALS samples. 3.2. Downregulation of miR-1234-3p and miR-1825 is specific for ALS To evaluate if downregulation of miR-1234-3p and miR-1825 is specific for ALS or a common feature of neurodegenerative disease condition, we also measured their relative abundance in serum samples of independent cohorts of Alzheimer’s (Supplementary Table 4) and Huntington’s disease patients (Supplementary Table 5) as well as in matched healthy controls by qRT-PCR (Fig. 2). Although both miRNAs were unaffected in Alzheimer’s disease, a significant upregulation was measured for both miR1234-3p and miR-1825 in Huntington’s disease patients, contrasting with the downregulation observed in ALS. A receiver operating characteristic analysis based on qRT-PCR data of the validation cohort (Supplementary Fig. 1) revealed an area under the curve of 0.69 (95% confidence interval: 0.52e0.86) and 0.79 (0.64e0.93) for miR-1825 and 1234-3p, respectively. miR-1234-3p thus distinguished sALS from healthy controls with high sensitivity and specificity. The combination of miR-1825 and miR-1234-3p did not substantially improve the area under the curve compared to miR-1234-3p alone (0.80; 95% confidence interval: 0.66e0.94). Levels of miR-1825 and miR-1234-3p did not
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correlate with age of onset, disease duration, or ALS-Functional Rating Scale (FRS) scores (data not shown). 3.3. Predicted mRNA targets of miR-1825 and miR-1234-3p largely overlap Although the source and mode of action of serum miRNAs is still unclear and might be different compared with cellular miRNAs, we performed an unbiased Ingenuity pathway core analysis to search for a possible functional overlap between both miRNAs downregulated in sALS. We found that, the functional network “Neurological Disease, Skeletal and Muscular Disorders, Cell-To-Cell Signaling and Interaction” was assigned to both miR1825 and miR-1234-3p. Furthermore, the predicted mRNA targets of miR-1825 and miR-1234-3p largely overlap within this network with 17 of 33 mRNAs being targeted by both miRNAs (Supplementary Fig. 2A). The most significantly predicted common targets of both miRNAs were NXPH3 and NLE1 (Supplementary Fig. 2B). Therefore, bioinformatic analysis suggests that both miRNAs that are significantly regulated in the serum of sALS are likely to be active in the same functional network at a cellular level. 3.4. A subset of sALS patients shows miRNA fingerprints similar to monogenic fALS cases In a previous study, we identified a subset of 22 miRNAs significantly downregulated in the serum of both fALS patients and preclinical mutation carriers (Freischmidt et al., 2014). Results of this previous study are highly comparable with the miRNA measurements presented in this study, as samples for both studies were collected in parallel by the same center and the miRNA microarray analysis was run in one batch. An unbiased hierarchical cluster analysis (average linkage) of microarray results from sALS patients revealed that the 22-miRNA signature identified in fALS and preclinical mutation carriers can be detected in 61% (11 of 18) of sALS patients. In contrast, the remaining 39% of sALS samples (7 of 18) are evenly distributed among healthy controls regarding the fALS-related miRNA fingerprint (Fig. 3). Thus, although serum miRNA profiles of sALS are clearly more heterogeneous when compared to samples from
Fig. 2. Validation of downregulated serum miRNAs in an independent cohort of sALS patients by quantitative reverse transcription polymerase chain reaction and comparison to fALS, Alzheimer’s disease, and Huntington disease patients. Relative abundances of miR-1234-3p and miR-1825 were determined in serum of 20 independent sALS patients and 20 additional controls, 13 fALS patients (8 C9ORF72 and 5 SOD1 mutant patients), and 13 controls, 15 Alzheimer’s disease patients and 13 controls as well as 15 Huntington’s disease patients and 15 controls. All cohorts were matched for age and gender. Ct-values were converted to linear comparisons and normalized to spiked-in Cel-miR-39-3p using 2DDCt method (bars indicate mean standard error of the mean; *p 0.05, **p 0.01 in a 2-tailed Student t test). Abbreviations: fALS, familial amyotrophic lateral sclerosis; miRNA, microRNA; n.s., not significant; sALS, sporadic amyotrophic lateral sclerosis.
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Fig. 3. Sixty-one percent of sporadic amyotrophic lateral sclerosis (sALS) patients share a serum miRNA fingerprint with familial amyotrophic lateral sclerosis (fALS) patients. Heatmap shows an unbiased hierarchical cluster analysis (average linkage) of sALS patients and healthy controls based on the 22 miRNAs downregulated in both fALS patients and premanifest mutation carriers (Freischmidt et al., 2014). Eleven of 18 (61%) sALS patients cluster in one arm (green) showing similar miRNA signatures compared with genetic amyotrophic lateral sclerosis patients and mutation carriers. Seven of 18 (39%) sALS patients are scattered among the controls. Age and gender of individual probands are indicated (C ¼ healthy control; A ¼ sALS patient; * indicates sALS patients with a bulbar onset of symptoms). Abbreviation: miRNA, microRNA. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
patients with a familial background and a monogenic cause of disease, a substantial proportion of sALS cases share miRNA signatures typical for fALS. As expected, a separate analysis of the microarray data comparing the 11 sALS patients with fALS-related miRNA profiles and the 16 healthy controls confirmed the significant (FDR 0.05) downregulation of most (19 of 22) of the 22 fALS-related miRNAs (Supplementary Table 6). Altogether, 38 downregulated (FDR 0.05) miRNAs were identified in the sera of these 11 sALS patients including 5 (from a total of 8) miRNAs that were found to be exclusively downregulated in fALS patients but not in the preclinical ALS mutation carriers (Freischmidt et al., 2014). We, furthermore, compared all ALS patients with fALS-like miRNA profiles available [i.e., the 11 fALS-like sALS patients from this study together with the 9 fALS patients from our previous study (Freischmidt et al., 2014)] with the 16 healthy controls. The further increased statistical power resulted in the identification of additional significantly downregulated miRNAs (Supplementary Table 6). In contrast, not a single miRNA was significantly downregulated (FDR 0.05) when comparing the remaining 7 sALS patients without fALS-related miRNA changes with the 16 healthy controls. However, 3 miRNAs (including miR-1234-3p and miR1825) showed a nominal downregulation with an uncorrected p-value 0.05 (Supplementary Table 7).
4. Discussion Here, we present a comprehensive array-based serum miRNA analysis of sALS patients revealing a highly heterogeneous serum miRNA profile pattern. This finding contrasts with the homogeneous serum miRNA signatures observed by the identical technique and the same ALS center in familial ALS cases as well as presymptomatic mutation carriers recently (Freischmidt et al., 2014). Nevertheless, we could identify 2 mature miRNAs consistently downregulated in serum of sALS patients. We, furthermore, defined a subgroup of sALS patients who share serum miRNA fingerprints with monogenic, familial cases. We report the downregulation of miR-1234-3p and miR-1825. Both miRNAs have the potential as a diagnostic tool distinguishing sALS patients from healthy controls, at least at the level of group comparisons. However, neither levels of miR-1825 nor miR-12343p correlated with disease characteristics like age of onset, disease duration or ALS-FRS scores. The heterogeneous nature of sALS serum miRNA profiles and possibly a number of different etiologies of sALS may prevent a significant correlation of miRNA levels with clinical disease parameters. Alternatively, the downregulation of both miRNAs could be already present in the earliest stages of ALS or even be pathogenic, without further progression during the disease course.
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Interestingly, our results from sALS and fALS patients presented here and in our previous study (Freischmidt et al., 2014), respectively, indicate that downregulation of miR-1825 is a general feature of all symptomatic ALS cases whereas downregulation of miR-1234-3p is significant only in sporadic cases. Additionally, our data reveal that downregulation of serum miR-1825 and miR-12343p is specific for ALS, at least when compared with cohorts of Alzheimer’s and Huntington’s disease. We found an upregulation for both in the serum of Huntington’s disease patients without a change in Alzheimer’s disease. Previously, an upregulation of miR1825 and miR-1234-3p has been reported in different types of cancer (Haj-Ahmad et al., 2014; Hogfeldt et al., 2014; Li et al., 2013). Comparing our results to other serum miRNA studies in sALS or other neurodegenerative diseases turned out to be difficult because of different sample processing, methods, and detection limits. Ingenuity pathway analysis revealed a substantial overlap of mRNA targets of miR-1825 and miR-1234-3p and predicted NXPH3 and NLE1 as the top candidate genes regulated by both. Neither NXPH3 nor NLE1 have been implicated in ALS or neurodegeneration so far. NXPH3 encodes for a member of the neurexophilin-family of secreted neuropeptides binding neurexins involved in organization of synapses in the brain (Craig and Kang, 2007). Nxph3 knockout mice showed normal brain morphology but abnormal sensory information processing and impaired motor coordination indicating a functional role in specific neuronal circuits (Beglopoulos et al., 2005). NLE1 encodes for Notchless protein homolog 1 (Drosophila melanogaster), a direct positive regulator of the Notch signaling pathway involved in cell fate decisions (Cormier et al., 2006; Royet et al., 1998). Interestingly, a pathological Notch activation was suggested to contribute to toxicity of TAR DNA-binding protein 43 (TDP-43) in a Drosophila model of ALS (Zhan et al., 2013). Hence, although the source, target, and functional role of extracellular miRNAs remained largely elusive to date and any interpretation with regard to functional aspects of serum miRNAs has to be taken with care, our data strongly suggest to elucidate the roles of NXPH3 and NLE1 in ALS pathogenesis. In our previous study, we detected a signature of 22 miRNAs significantly downregulated in fALS and presymptomatic mutation carriers. In contrast, despite a larger sample size and identical technical procedures, only 2 miRNAs were found to be significantly downregulated in sALS in this study. This is in-line with a higher heterogeneity of molecular alterations in sALS versus fALS. However, a cluster analysis based on the 22 miRNAs reported to be downregulated in fALS and premanifest mutation carriers (Freischmidt et al., 2014), reveals a surprisingly clear-cut dichotomy: >60% (11 of 18) of our sALS samples shared a serum miRNA fingerprint with genetic cases, with the remaining 7 of 18 patients evenly distributed among control samples. As a consequence of the control-like pattern in 7 of 18 sALS patients, downregulation of the respective 22 fALS-related miRNAs was not significant when the total sALS cohort was compared with matched controls. The fALS-like serum miRNAom may indicate a (poly)genetic background conferring susceptibility for ALS. Absence of a family history for ALS in these sALS patients could be explained by reduced penetrance, de novo mutations or oligogenic inheritance (Renton et al., 2014). In contrary, absence of fALS-like miRNA patterns may mirror a higher impact of exogeneous, for example, environmental, factors and possibly a lower and/or different genetic influence in this minority of sALS patients. Interestingly, a higher than expected heritability of ALS has already been suggested by a genome-wide heritability analysis (Keller et al., 2014). Future studies will have to clarify whether the peripheral miRNA profile could be useful as an indicator of a genetic background of ALS patients independent of the presence of a positive family history.
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The molecular and pathogenic heterogeneity of the “RNA disease” ALS, which is most likely reflected by the serum miRNA profiles, might have implications and should be considered for the future development of disease modifying treatments and the planning of respective clinical trials. Disclosure statement The authors have no actual or potential conflicts of interest. Acknowledgements The authors are indebted to the patients and their families for their participation in this project. The authors are grateful to their study nurse Antje Knehr and their technicians Nadine Todt and Elena Jasovskaja as well as the Ulm Neurology biobank team for excellent patient care and technical assistance. The authors thank Dr. Michael Bonin and Dr. Michael Walter (University of Tübingen) for access to and help with IPA. This work was supported in whole or in parts by grants from the German Federal Ministry of Education and Research (STRENGTH consortium and BMBF; 01GI0704, German network for ALS research [MND-NET; 01GM1103A]), the Charcot Foundation for ALS Research (Albert C. Ludolph, Jochen H. Weishaupt), the virtual Helmholtz Institute “RNA-Dysmetabolism in ALS and FTD,” and the DFG-funded Swabian ALS registry. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.neurobiolaging. 2015.06.003. References Beglopoulos, V., Montag-Sallaz, M., Rohlmann, A., Piechotta, K., Ahmad, M., Montag, D., Missler, M., 2005. Neurexophilin 3 is highly localized in cortical and cerebellar regions and is functionally important for sensorimotor gating and motor coordination. Mol. Cell Biol. 25, 7278e7288. Burgos, K., Malenica, I., Metpally, R., Courtright, A., Rakela, B., Beach, T., Shill, H., Adler, C., Sabbagh, M., Villa, S., Tembe, W., Craig, D., Van Keuren-Jensen, K., 2014. Profiles of extracellular miRNA in cerebrospinal fluid and serum from patients with Alzheimer’s and Parkinson’s diseases correlate with disease status and features of pathology. PLoS One 9, e94839. Butovsky, O., Siddiqui, S., Gabriely, G., Lanser, A.J., Dake, B., Murugaiyan, G., Doykan, C.E., Wu, P.M., Gali, R.R., Iyer, L.K., Lawson, R., Berry, J., Krichevsky, A.M., Cudkowicz, M.E., Weiner, H.L., 2012. Modulating inflammatory monocytes with a unique microRNA gene signature ameliorates murine ALS. J. Clin. Invest. 122, 3063e3087. Campos-Melo, D., Droppelmann, C.A., He, Z., Volkening, K., Strong, M.J., 2013. Altered microRNA expression profile in amyotrophic lateral sclerosis: a role in the regulation of NFL mRNA levels. Mol. Brain 6, 26. Chekulaeva, M., Filipowicz, W., 2009. Mechanisms of miRNA-mediated post-transcriptional regulation in animal cells. Curr. Opin. Cell Biol. 21, 452e460. Cormier, S., Le Bras, S., Souilhol, C., Vandormael-Pournin, S., Durand, B., Babinet, C., Baldacci, P., Cohen-Tannoudji, M., 2006. The murine ortholog of notchless, a direct regulator of the notch pathway in Drosophila melanogaster, is essential for survival of inner cell mass cells. Mol. Cell Biol. 26, 3541e3549. Craig, A.M., Kang, Y., 2007. Neurexin-neuroligin signaling in synapse development. Curr. Opin. Neurobiol. 17, 43e52. De Felice, B., Guida, M., Guida, M., Coppola, C., De Mieri, G., Cotrufo, R., 2012. A miRNA signature in leukocytes from sporadic amyotrophic lateral sclerosis. Gene 508, 35e40. DeJesus-Hernandez, M., Mackenzie, I.R., Boeve, B.F., Boxer, A.L., Baker, M., Rutherford, N.J., Nicholson, A.M., Finch, N.A., Flynn, H., Adamson, J., Kouri, N., Wojtas, A., Sengdy, P., Hsiung, G.Y., Karydas, A., Seeley, W.W., Josephs, K.A., Coppola, G., Geschwind, D.H., Wszolek, Z.K., Feldman, H., Knopman, D.S., Petersen, R.C., Miller, B.L., Dickson, D.W., Boylan, K.B., Graff-Radford, N.R., Rademakers, R., 2011. Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron 72, 245e256. Freischmidt, A., Muller, K., Ludolph, A.C., Weishaupt, J.H., 2013. Systemic dysregulation of TDP-43 binding microRNAs in amyotrophic lateral sclerosis. Acta Neuropathol. Commun. 1, 42.
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Neurobiology of Aging journal homepage: www.elsevier.com/locate/neuaging
Serum microRNAs in sporadic amyotrophic lateral sclerosis Axel Freischmidt a, Kathrin Müller a, Lisa Zondler a, Patrick Weydt a, Benjamin Mayer b, Christine A.F. von Arnim a, Annemarie Hübers a, Johannes Dorst a, Markus Otto a, Karlheinz Holzmann c, Albert C. Ludolph a, Karin M. Danzer a, Jochen H. Weishaupt a, * a
Department of Neurology, Ulm University, Ulm, Germany Institute for Epidemiology and Medical Biometry, Ulm University, Ulm, Germany c Genomics-Core Facility, University Hospital Ulm, Center for Biomedical Research, Ulm, Germany b
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a b s t r a c t
Article history: Received 26 January 2015 Received in revised form 27 April 2015 Accepted 2 June 2015 Available online 9 June 2015
MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression and specific mircoRNA “fingerprints” are thought to contribute to and/or reflect certain disease conditions. Recently, we identified surprisingly homogeneous signatures of circulating miRNAs in the serum of familial amyotrophic lateral sclerosis (ALS) patients, which were already present in presymptomatic carriers of ALS gene mutations. Here, we characterize circulating miRNAs in the serum of sporadic ALS patients. We show that, in contrast to familial ALS, miRNA signatures of sporadic ALS are highly heterogeneous suggesting a number of different etiologies. Nevertheless, 2 miRNAs, miR-1234-3p and miR-1825, could be identified to be consistently downregulated in sporadic ALS. Bioinformatic analysis revealed miRNA fingerprints resembling those of familial ALS patients and mutation carriers in 61% of sporadic ALS patients, while the remaining subgroup had clearly different miRNA signatures. These data support a higher than expected contribution of genetic factors also to sporadic ALS. Moreover, our results indicate a more heterogeneous molecular etiology of sporadic ALS compared with (mono)genic cases, which should be considered for the development of disease modifying treatments. Ó 2015 Elsevier Inc. All rights reserved.
Keywords: Amyotrophic lateral sclerosis MicroRNA Serum Biomarker Diagnosis
1. Introduction Amyotrophic lateral sclerosis (ALS) is characterized by a progressive loss of motor neurons and fatal outcome within 3e5 years (Rowland and Shneider, 2001). About 10% of ALS cases are familial (fALS) and approximately half to two-thirds thereof can be explained by mutations in known ALS genes. The remaining 90% are sporadic cases (sALS) with infrequent mutations in known ALS genes (Renton et al., 2014). Growing evidence implicates messenger RNA (mRNA) but most recently also microRNA (miRNA) dysmetabolism in ALS disease pathogenesis (Goodall et al., 2013). Twenty to 24 nucleotides mature miRNAs are mostly inhibitory post-transcriptional regulators of gene expression (Chekulaeva and Filipowicz, 2009). miRNAs are involved in the cellular response to various stressors and insults (Liu et al., 2013; Mendell and Olson, 2012; Viader et al., 2011; Zhang et al., 2013). Thereby, miRNA expression profiles can reflect * Corresponding author at: Department of Neurology, Ulm University, AlbertEinstein-Allee 11, 89081 Ulm, Germany. Tel.: þ49 (0) 731 50063073; fax: þ49 (0) 731 50063050. E-mail address: [email protected] (J.H. Weishaupt). 0197-4580/$ e see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neurobiolaging.2015.06.003
activation of specific pathogenic pathways. Dysregulation of miRNA expression has been reported, for example, in the SOD1-G93A mouse model of ALS or patient-derived tissue (Butovsky et al., 2012; Campos-Melo et al., 2013; De Felice et al., 2012; Toivonen et al., 2014). miRNAs are remarkably stable in serum and other body fluids as exosomal cargos or bound to specific proteins (Xu et al., 2012), and disease-related changes of extracellular miRNA abundance has been repeatedly shown. Although the causative involvement of these changes in disease pathogenesis is still unclear, they are at least thought to represent reactive events related to specific pathways of neurodegeneration. In the context of ALS, alterations in the peripheral miRNA abundance have been suggested to represent a systemic dysfunction of ubiquitously expressed, RNA binding proteins that are involved in ALS pathogenesis (Burgos et al., 2014; Freischmidt et al., 2013, 2014; Gandhi et al., 2013). In a recent study, we identified 30 miRNAs significantly downregulated in the serum of fALS patients that were largely independent from the underlying disease gene. Most of these miRNAs (22) were already downregulated in presymptomatic ALS mutation carriers, even years before the estimated disease onset. Moreover, downregulated miRNAs shared a common nucleotide
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consensus motif possibly indicating specific protein binding partners and downstream pathways common for different ALS genes (Freischmidt et al., 2014). In this study, we examined serum miRNA profiles of sporadic ALS patients and compared them to miRNA signatures of fALS patients (Freischmidt et al., 2014). We show that, serum miRNA profiles are far more heterogeneous in sALS than in fALS. At the same time, however, a considerable proportion of sALS patients share miRNA signatures typical for fALS patients suggesting alteration of common pathways and a high contribution of genetic factors also to sporadic ALS. 2. Material and methods 2.1. Patient cohorts and ethics statement Blood samples were drawn in accordance with the Declaration of Helsinki (World Medical Association, 1964), and study protocols were approved by the national medical ethical review boards. Participants provided prior written informed consent. ALS patients fulfilling the El-Escorial criteria for definite ALS were considered sporadic cases due to a known negative family history of the disease. Additionally, the 2 main genetic causes of familial ALS, namely mutations in the SOD1 gene and a hexanucleotide repeat expansion in C9ORF72 (DeJesus-Hernandez et al., 2011; Renton et al., 2014) were excluded by Sanger sequencing (van Es et al., 2010) or repeat-primed polymerase chain reaction (PCR) (DeJesus-Hernandez et al., 2011), respectively. sALS patients were not related to any of the control individuals. 2.2. Serum miRNA microarray analysis Individual serum samples were collected by the same center according to the same standard operating procedures. Isolation of total RNA from 8e10 mL of serum, microarray analysis using Affymetrix GeneChip miRNA 3.0 Arrays and statistical evaluation were performed exactly as recently described (Freischmidt et al., 2014). Importantly, miRNA microarrays from this study were run in one batch with fALS patients previously published (Freischmidt et al., 2014) ensuring highly comparable results. Microarray raw
data can be accessed at Gene Expression Omnibus (http://www. ncbi.nlm.nih.gov/geo/). GSE number is GSE52917. 2.3. Generation of heatmaps and hierarchical cluster analysis Hierarchical cluster analysis (average linkage) was performed and heatmaps were generated using the Genesis software package (http:// genome.tugraz.at/genesisclient/genesisclient/_ description.shtml). 2.4. Quantitative real-time PCR RNA isolations, reverse transcriptions and quantitative real-time PCRs (qRT-PCRs) were carried out as recently described using miScript Primer Assays (Qiagen, Hilden, Germany) as miRNA specific forward primers (Freischmidt et al., 2013, 2014). Exponential processes (Ct-values) were converted to linear comparisons relative to the control group and normalized to spiked-in miR-39-3p of Caenorhabditis elegans (Cel-miR-39-3p) using 2DDCt method. Error bars were calculated the same way (Livak and Schmittgen, 2001). Comparison of groups was performed using the 2-tailed Student t test. 3. Results 3.1. Serum miRNA profiles of sALS patients are highly heterogeneous In the discovery experiment, relative serum miRNA levels of 18 sALS patients and 16 matched healthy controls (Supplementary Table 1) were compared using Affymetrix GeneChip miRNA 3.0 Arrays. To exclude any false positive changes of miRNA abundance, we restricted our analysis to downregulated miRNAs that are not susceptible to increased hemolysis reported previously for ALS patients (Freischmidt et al., 2014; Ronnevi and Conradi, 1984). We observed a significant downregulation of only 2 miRNAs (Supplementary Table 2) in sALS patients after correction for multiple testing (false discovery rate [FDR] 0.05). The variability of miRNA alterations was higher in the ALS group compared with controls (Fig. 1). This contrasts with a set of miRNAs homogeneously downregulated in familial ALS patients and also premanifest mutations carriers, as recently reported using the identical
Fig. 1. Heterogeneous serum miRNA profiles in sALS patients. The heatmap shows downregulated serum miRNAs (uncorrected p-value 0.05) of sALS patients compared with healthy controls according to the color coding (bottom). miRNA abundances are not homogeneous across the samples. The site of onset (spinal vs. bulbar) did not have an impact on miRNA abundance. Age and gender of the individual samples as well as the fold changes of the respective miRNAs are indicated (* indicates significantly downregulated miRNAs with a false discovery rate 0.05). Abbreviations: miRNA, microRNA; sALS, sporadic amyotrophic lateral sclerosis. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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technical approach and sampling protocol by the same ALS center (Freischmidt et al., 2014). However, despite this heterogeneity, the 2 sALS-linked miRNAs that remained significantly changed after correction for multiple testing could be confirmed in an independent cohort of additional 20 sALS patients and 20 age- and gendermatched controls (Supplementary Table 3) using qRT-PCR as an alternative method (Fig. 2). We next compared the downregulation of miR-1825 and miR-1234-3p in sALS with fALS. Their relative abundance was determined in a cohort of 13 fALS patients [8 C9ORF72 and 5 SOD1 mutant patients; detailed characteristics of the cohort published previously (Freischmidt et al., 2014)] and 13 matched healthy controls by qRT-PCR. Although downregulation of miR-1825 was also evident in fALS patients and thus seems to be a common feature of ALS, significant downregulation of miR-1234-3p was restricted to the sALS cases (Fig. 2), although a nonsignificant trend (p ¼ 0.15) was observed also in the fALS samples. 3.2. Downregulation of miR-1234-3p and miR-1825 is specific for ALS To evaluate if downregulation of miR-1234-3p and miR-1825 is specific for ALS or a common feature of neurodegenerative disease condition, we also measured their relative abundance in serum samples of independent cohorts of Alzheimer’s (Supplementary Table 4) and Huntington’s disease patients (Supplementary Table 5) as well as in matched healthy controls by qRT-PCR (Fig. 2). Although both miRNAs were unaffected in Alzheimer’s disease, a significant upregulation was measured for both miR1234-3p and miR-1825 in Huntington’s disease patients, contrasting with the downregulation observed in ALS. A receiver operating characteristic analysis based on qRT-PCR data of the validation cohort (Supplementary Fig. 1) revealed an area under the curve of 0.69 (95% confidence interval: 0.52e0.86) and 0.79 (0.64e0.93) for miR-1825 and 1234-3p, respectively. miR-1234-3p thus distinguished sALS from healthy controls with high sensitivity and specificity. The combination of miR-1825 and miR-1234-3p did not substantially improve the area under the curve compared to miR-1234-3p alone (0.80; 95% confidence interval: 0.66e0.94). Levels of miR-1825 and miR-1234-3p did not
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correlate with age of onset, disease duration, or ALS-Functional Rating Scale (FRS) scores (data not shown). 3.3. Predicted mRNA targets of miR-1825 and miR-1234-3p largely overlap Although the source and mode of action of serum miRNAs is still unclear and might be different compared with cellular miRNAs, we performed an unbiased Ingenuity pathway core analysis to search for a possible functional overlap between both miRNAs downregulated in sALS. We found that, the functional network “Neurological Disease, Skeletal and Muscular Disorders, Cell-To-Cell Signaling and Interaction” was assigned to both miR1825 and miR-1234-3p. Furthermore, the predicted mRNA targets of miR-1825 and miR-1234-3p largely overlap within this network with 17 of 33 mRNAs being targeted by both miRNAs (Supplementary Fig. 2A). The most significantly predicted common targets of both miRNAs were NXPH3 and NLE1 (Supplementary Fig. 2B). Therefore, bioinformatic analysis suggests that both miRNAs that are significantly regulated in the serum of sALS are likely to be active in the same functional network at a cellular level. 3.4. A subset of sALS patients shows miRNA fingerprints similar to monogenic fALS cases In a previous study, we identified a subset of 22 miRNAs significantly downregulated in the serum of both fALS patients and preclinical mutation carriers (Freischmidt et al., 2014). Results of this previous study are highly comparable with the miRNA measurements presented in this study, as samples for both studies were collected in parallel by the same center and the miRNA microarray analysis was run in one batch. An unbiased hierarchical cluster analysis (average linkage) of microarray results from sALS patients revealed that the 22-miRNA signature identified in fALS and preclinical mutation carriers can be detected in 61% (11 of 18) of sALS patients. In contrast, the remaining 39% of sALS samples (7 of 18) are evenly distributed among healthy controls regarding the fALS-related miRNA fingerprint (Fig. 3). Thus, although serum miRNA profiles of sALS are clearly more heterogeneous when compared to samples from
Fig. 2. Validation of downregulated serum miRNAs in an independent cohort of sALS patients by quantitative reverse transcription polymerase chain reaction and comparison to fALS, Alzheimer’s disease, and Huntington disease patients. Relative abundances of miR-1234-3p and miR-1825 were determined in serum of 20 independent sALS patients and 20 additional controls, 13 fALS patients (8 C9ORF72 and 5 SOD1 mutant patients), and 13 controls, 15 Alzheimer’s disease patients and 13 controls as well as 15 Huntington’s disease patients and 15 controls. All cohorts were matched for age and gender. Ct-values were converted to linear comparisons and normalized to spiked-in Cel-miR-39-3p using 2DDCt method (bars indicate mean standard error of the mean; *p 0.05, **p 0.01 in a 2-tailed Student t test). Abbreviations: fALS, familial amyotrophic lateral sclerosis; miRNA, microRNA; n.s., not significant; sALS, sporadic amyotrophic lateral sclerosis.
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Fig. 3. Sixty-one percent of sporadic amyotrophic lateral sclerosis (sALS) patients share a serum miRNA fingerprint with familial amyotrophic lateral sclerosis (fALS) patients. Heatmap shows an unbiased hierarchical cluster analysis (average linkage) of sALS patients and healthy controls based on the 22 miRNAs downregulated in both fALS patients and premanifest mutation carriers (Freischmidt et al., 2014). Eleven of 18 (61%) sALS patients cluster in one arm (green) showing similar miRNA signatures compared with genetic amyotrophic lateral sclerosis patients and mutation carriers. Seven of 18 (39%) sALS patients are scattered among the controls. Age and gender of individual probands are indicated (C ¼ healthy control; A ¼ sALS patient; * indicates sALS patients with a bulbar onset of symptoms). Abbreviation: miRNA, microRNA. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
patients with a familial background and a monogenic cause of disease, a substantial proportion of sALS cases share miRNA signatures typical for fALS. As expected, a separate analysis of the microarray data comparing the 11 sALS patients with fALS-related miRNA profiles and the 16 healthy controls confirmed the significant (FDR 0.05) downregulation of most (19 of 22) of the 22 fALS-related miRNAs (Supplementary Table 6). Altogether, 38 downregulated (FDR 0.05) miRNAs were identified in the sera of these 11 sALS patients including 5 (from a total of 8) miRNAs that were found to be exclusively downregulated in fALS patients but not in the preclinical ALS mutation carriers (Freischmidt et al., 2014). We, furthermore, compared all ALS patients with fALS-like miRNA profiles available [i.e., the 11 fALS-like sALS patients from this study together with the 9 fALS patients from our previous study (Freischmidt et al., 2014)] with the 16 healthy controls. The further increased statistical power resulted in the identification of additional significantly downregulated miRNAs (Supplementary Table 6). In contrast, not a single miRNA was significantly downregulated (FDR 0.05) when comparing the remaining 7 sALS patients without fALS-related miRNA changes with the 16 healthy controls. However, 3 miRNAs (including miR-1234-3p and miR1825) showed a nominal downregulation with an uncorrected p-value 0.05 (Supplementary Table 7).
4. Discussion Here, we present a comprehensive array-based serum miRNA analysis of sALS patients revealing a highly heterogeneous serum miRNA profile pattern. This finding contrasts with the homogeneous serum miRNA signatures observed by the identical technique and the same ALS center in familial ALS cases as well as presymptomatic mutation carriers recently (Freischmidt et al., 2014). Nevertheless, we could identify 2 mature miRNAs consistently downregulated in serum of sALS patients. We, furthermore, defined a subgroup of sALS patients who share serum miRNA fingerprints with monogenic, familial cases. We report the downregulation of miR-1234-3p and miR-1825. Both miRNAs have the potential as a diagnostic tool distinguishing sALS patients from healthy controls, at least at the level of group comparisons. However, neither levels of miR-1825 nor miR-12343p correlated with disease characteristics like age of onset, disease duration or ALS-FRS scores. The heterogeneous nature of sALS serum miRNA profiles and possibly a number of different etiologies of sALS may prevent a significant correlation of miRNA levels with clinical disease parameters. Alternatively, the downregulation of both miRNAs could be already present in the earliest stages of ALS or even be pathogenic, without further progression during the disease course.
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Interestingly, our results from sALS and fALS patients presented here and in our previous study (Freischmidt et al., 2014), respectively, indicate that downregulation of miR-1825 is a general feature of all symptomatic ALS cases whereas downregulation of miR-1234-3p is significant only in sporadic cases. Additionally, our data reveal that downregulation of serum miR-1825 and miR-12343p is specific for ALS, at least when compared with cohorts of Alzheimer’s and Huntington’s disease. We found an upregulation for both in the serum of Huntington’s disease patients without a change in Alzheimer’s disease. Previously, an upregulation of miR1825 and miR-1234-3p has been reported in different types of cancer (Haj-Ahmad et al., 2014; Hogfeldt et al., 2014; Li et al., 2013). Comparing our results to other serum miRNA studies in sALS or other neurodegenerative diseases turned out to be difficult because of different sample processing, methods, and detection limits. Ingenuity pathway analysis revealed a substantial overlap of mRNA targets of miR-1825 and miR-1234-3p and predicted NXPH3 and NLE1 as the top candidate genes regulated by both. Neither NXPH3 nor NLE1 have been implicated in ALS or neurodegeneration so far. NXPH3 encodes for a member of the neurexophilin-family of secreted neuropeptides binding neurexins involved in organization of synapses in the brain (Craig and Kang, 2007). Nxph3 knockout mice showed normal brain morphology but abnormal sensory information processing and impaired motor coordination indicating a functional role in specific neuronal circuits (Beglopoulos et al., 2005). NLE1 encodes for Notchless protein homolog 1 (Drosophila melanogaster), a direct positive regulator of the Notch signaling pathway involved in cell fate decisions (Cormier et al., 2006; Royet et al., 1998). Interestingly, a pathological Notch activation was suggested to contribute to toxicity of TAR DNA-binding protein 43 (TDP-43) in a Drosophila model of ALS (Zhan et al., 2013). Hence, although the source, target, and functional role of extracellular miRNAs remained largely elusive to date and any interpretation with regard to functional aspects of serum miRNAs has to be taken with care, our data strongly suggest to elucidate the roles of NXPH3 and NLE1 in ALS pathogenesis. In our previous study, we detected a signature of 22 miRNAs significantly downregulated in fALS and presymptomatic mutation carriers. In contrast, despite a larger sample size and identical technical procedures, only 2 miRNAs were found to be significantly downregulated in sALS in this study. This is in-line with a higher heterogeneity of molecular alterations in sALS versus fALS. However, a cluster analysis based on the 22 miRNAs reported to be downregulated in fALS and premanifest mutation carriers (Freischmidt et al., 2014), reveals a surprisingly clear-cut dichotomy: >60% (11 of 18) of our sALS samples shared a serum miRNA fingerprint with genetic cases, with the remaining 7 of 18 patients evenly distributed among control samples. As a consequence of the control-like pattern in 7 of 18 sALS patients, downregulation of the respective 22 fALS-related miRNAs was not significant when the total sALS cohort was compared with matched controls. The fALS-like serum miRNAom may indicate a (poly)genetic background conferring susceptibility for ALS. Absence of a family history for ALS in these sALS patients could be explained by reduced penetrance, de novo mutations or oligogenic inheritance (Renton et al., 2014). In contrary, absence of fALS-like miRNA patterns may mirror a higher impact of exogeneous, for example, environmental, factors and possibly a lower and/or different genetic influence in this minority of sALS patients. Interestingly, a higher than expected heritability of ALS has already been suggested by a genome-wide heritability analysis (Keller et al., 2014). Future studies will have to clarify whether the peripheral miRNA profile could be useful as an indicator of a genetic background of ALS patients independent of the presence of a positive family history.
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The molecular and pathogenic heterogeneity of the “RNA disease” ALS, which is most likely reflected by the serum miRNA profiles, might have implications and should be considered for the future development of disease modifying treatments and the planning of respective clinical trials. Disclosure statement The authors have no actual or potential conflicts of interest. Acknowledgements The authors are indebted to the patients and their families for their participation in this project. The authors are grateful to their study nurse Antje Knehr and their technicians Nadine Todt and Elena Jasovskaja as well as the Ulm Neurology biobank team for excellent patient care and technical assistance. The authors thank Dr. Michael Bonin and Dr. Michael Walter (University of Tübingen) for access to and help with IPA. This work was supported in whole or in parts by grants from the German Federal Ministry of Education and Research (STRENGTH consortium and BMBF; 01GI0704, German network for ALS research [MND-NET; 01GM1103A]), the Charcot Foundation for ALS Research (Albert C. Ludolph, Jochen H. Weishaupt), the virtual Helmholtz Institute “RNA-Dysmetabolism in ALS and FTD,” and the DFG-funded Swabian ALS registry. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.neurobiolaging. 2015.06.003. References Beglopoulos, V., Montag-Sallaz, M., Rohlmann, A., Piechotta, K., Ahmad, M., Montag, D., Missler, M., 2005. Neurexophilin 3 is highly localized in cortical and cerebellar regions and is functionally important for sensorimotor gating and motor coordination. Mol. Cell Biol. 25, 7278e7288. Burgos, K., Malenica, I., Metpally, R., Courtright, A., Rakela, B., Beach, T., Shill, H., Adler, C., Sabbagh, M., Villa, S., Tembe, W., Craig, D., Van Keuren-Jensen, K., 2014. Profiles of extracellular miRNA in cerebrospinal fluid and serum from patients with Alzheimer’s and Parkinson’s diseases correlate with disease status and features of pathology. PLoS One 9, e94839. Butovsky, O., Siddiqui, S., Gabriely, G., Lanser, A.J., Dake, B., Murugaiyan, G., Doykan, C.E., Wu, P.M., Gali, R.R., Iyer, L.K., Lawson, R., Berry, J., Krichevsky, A.M., Cudkowicz, M.E., Weiner, H.L., 2012. Modulating inflammatory monocytes with a unique microRNA gene signature ameliorates murine ALS. J. Clin. Invest. 122, 3063e3087. Campos-Melo, D., Droppelmann, C.A., He, Z., Volkening, K., Strong, M.J., 2013. Altered microRNA expression profile in amyotrophic lateral sclerosis: a role in the regulation of NFL mRNA levels. Mol. Brain 6, 26. Chekulaeva, M., Filipowicz, W., 2009. Mechanisms of miRNA-mediated post-transcriptional regulation in animal cells. Curr. Opin. Cell Biol. 21, 452e460. Cormier, S., Le Bras, S., Souilhol, C., Vandormael-Pournin, S., Durand, B., Babinet, C., Baldacci, P., Cohen-Tannoudji, M., 2006. The murine ortholog of notchless, a direct regulator of the notch pathway in Drosophila melanogaster, is essential for survival of inner cell mass cells. Mol. Cell Biol. 26, 3541e3549. Craig, A.M., Kang, Y., 2007. Neurexin-neuroligin signaling in synapse development. Curr. Opin. Neurobiol. 17, 43e52. De Felice, B., Guida, M., Guida, M., Coppola, C., De Mieri, G., Cotrufo, R., 2012. A miRNA signature in leukocytes from sporadic amyotrophic lateral sclerosis. Gene 508, 35e40. DeJesus-Hernandez, M., Mackenzie, I.R., Boeve, B.F., Boxer, A.L., Baker, M., Rutherford, N.J., Nicholson, A.M., Finch, N.A., Flynn, H., Adamson, J., Kouri, N., Wojtas, A., Sengdy, P., Hsiung, G.Y., Karydas, A., Seeley, W.W., Josephs, K.A., Coppola, G., Geschwind, D.H., Wszolek, Z.K., Feldman, H., Knopman, D.S., Petersen, R.C., Miller, B.L., Dickson, D.W., Boylan, K.B., Graff-Radford, N.R., Rademakers, R., 2011. Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron 72, 245e256. Freischmidt, A., Muller, K., Ludolph, A.C., Weishaupt, J.H., 2013. Systemic dysregulation of TDP-43 binding microRNAs in amyotrophic lateral sclerosis. Acta Neuropathol. Commun. 1, 42.
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