Biomarker development for C9orf72 repeat expansion in ALS.

brain research ] (]]]]) ]]]–]]]

Available online at www.sciencedirect.com

www.elsevier.com/locate/brainres

Research Report

Biomarker development for C9orf72 repeat expansion in ALS Emily F. Mendez, Rita Sattlern Brain Science Institute and Department of Neurology, Johns Hopkins University School of Medicine, 855N Wolfe Street, Rangos 2-223, Baltimore, MD 21205, USA

art i cle i nfo

ab st rac t

Article history:

The expanded GGGGCC hexanucleotide repeat in the non-coding region of the C9orf72 gene

Accepted 16 September 2014

on chromosome 9p21 has been discovered as the cause of approximately 20–50% of familial and up to 5–20% of sporadic amyotrophic lateral sclerosis (ALS) cases, making this

Keywords:

the most common known genetic mutation of ALS to date. At the same time, it represents

C9orf72

the most common genetic mutation in frontotemporal dementia (FTD; 10–30%). Because of

ALS

the high prevalence of mutant C9orf72, pre-clinical efforts in identifying therapeutic targets

Biomarker

and developing novel therapeutics for this mutation are highly pursued in the hope of

FTD

providing a desperately needed disease-modifying treatment for ALS patients, as well as other patient populations affected by the C9orf72 mutation. The current lack of effective treatments for ALS is partially due to the lack of appropriate biomarkers that aide in assessing drug efficacy during clinical trials independent of clinical outcome measures, such as increased survival. In this review we will summarize the opportunities for biomarker development specifically targeted to the newly discovered C9orf72 repeat expansion. While drugs are being developed for this mutation, it will be crucial to provide a reliable biomarker to accompany the clinical development of these novel therapeutic interventions to maximize the chances of a successful clinical trial. This article is part of a Special Issue entitled ALS complex pathogenesis. & 2014 Elsevier B.V. All rights reserved.

1.

Introduction

The newly discovered GGGGCC hexanucleotide repeat expansion (HRE) in the non-coding region of the C9orf72 gene on chromosome 9p21 represents the most common genetic abnormality in frontotemporal dementia (FTD; 10–30%) and amyotrophic lateral sclerosis (ALS; 20–50%) (Cruts et al., 2013; DeJesus-Hernandez et al., 2011; Ling et al., 2013; Renton et al., 2011). ALS is a uniformly fatal neurologic disease with a worldwide incidence

of 2–5 per 100,000 people yearly. It is characterized by the progressive degeneration of motor neurons and interneurons in the brain and spinal cord. It is largely sporadic, occurring without a family history (90%), while familial ALS accounts for 10%. FTD is a degenerative disorder of the frontal and anterior temporal lobes and is a common form of dementia affecting individuals below the age of 65 years. The disease eventually leads to dementia and death within a median of seven years from symptom onset. FTD shares pathological hallmarks with

n Correspondence to: Johns Hopkins University, The John G. Rangos Sr. Bldg, 855 N Wolfe Street, Suite 223, Baltimore, MD 21205, USA. Fax: þ1 410 614 0659. E-mail address: [email protected] (R. Sattler).

http://dx.doi.org/10.1016/j.brainres.2014.09.041 0006-8993/& 2014 Elsevier B.V. All rights reserved.

Please cite this article as: Mendez, E.F., Sattler, R., Biomarker development for C9orf72 repeat expansion in ALS. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.09.041

2


ALS, such as the presence of cellular ubiquitinated transactivating responsive (Tar) sequence DNA binding protein (TDP-43) positive protein inclusions throughout the central nervous system (CNS) (Neumann et al., 2006; Ringholz et al., 2005). Further, it has been recognized over the last years that a large percentage of ALS patients show impairments of frontotemporal functions including cognition and behavior. At the same time, up to 15% of FTD patients develop symptoms of motor neuron degeneration. This supports not only a genetic link between these two diseases but also a pathological and mechanistic association. In addition to FTD and ALS, C9orf72 has also been associated with other neurodegenerative disorders, including Parkinson’s disease, Huntington’s disease-like syndrome as well as Alzheimer’s disease (Liu et al., 2013), supporting the importance of understanding the pathological mechanisms behind this mutation and emphasizing the need for therapeutic developments targeting the (GGGGCC)n repeat expansion and its downstream disease pathways. The currently proposed disease-causing mechanism for mutant C9orf72 include protein gain-of-function, protein loss-of-function (Ciura et al., 2013; Therrien et al., 2013), toxic RNA gain-of-function (Almeida et al., 2013; Brouwer et al., 2009; Donnelly et al., 2013; Gatchel and Zoghbi, 2005; Lagier-

Tourenne et al., 2013; Lee et al., 2013; Sareen et al., 2013; Todd and Paulson, 2010), as well as the presence of repeatassociated non-ATG (RAN) translation products (Ash et al., 2013; Mori et al., 2013b; Zu et al., 2013a) (also reviewed in (Gendron et al., 2014; Ling et al., 2013)). Given the high prevalence of this mutation, not only in ALS, but also FTD and other neurodegenerative diseases, pre-clinical efforts for therapeutic developments are ongoing and are moving rapidly towards first clinical safety trials (Donnelly et al., 2013; Fernandes et al., 2013; Lagier-Tourenne et al., 2013; Riboldi et al., 2014; Sareen et al., 2013). Clinical trials for ALS have not only failed in the past because of a lack of efficacy of the drug candidate, but also, because of the lack of an appropriate pharmacodynamic biomarker. A biomarker is a characteristic that is objectively measured as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to therapeutic intervention. The discovery of new CNS acting therapeutics has been seriously hampered by the lack of useful relevant biomarkers and/or pharmacodynamic measures of disease or drug action. The difficulty for biomarkers in the vast majority of neurologic and psychiatric diseases lies in part in the lack of availability of appropriate tissue samples. Therefore, finding peripheral

Table 1 – List of potential C9orf72 pharmacodynamic biomarkers. Readout

Detection method

Molecular biomarkers Repeat RNA Fluorescent in-situ foci hybridization (RNA FISH) Epigenetic biomarkers DNA Bisulfite methylation pyrosequencing, MSSNuPE, COBRA, MS Histone ChIP modifications Micro RNA qPCR

Biological material

References

Brain, fibroblasts, lymphoblasts, blood leukocytes

(Almeida et al., 2013; DeJesus-Hernandez et al., 2011; Donnelly et al., 2013; Lagier-Tourenne et al., 2013; Ling et al., 2013; Mizielinska et al., 2013; Renton et al., 2011; Sareen et al., 2013; Zu et al., 2013b)

Frontal cortex, cervical spinal cord, blood

(Belzil et al., 2013, 2014; Chestnut et al., 2011; Martin and Wong, 2013; Xi et al., 2013; Yang et al., 2010)

Brain, blood

(Al-Chalabi et al., 2013; Belzil et al., 2013, 2014; Chestnut et al., 2011; Martin and Wong, 2013; Xi et al., 2013) (Campos-Melo et al., 2013; Chen et al., 2006; De Felice et al., 2012; Sellier et al., 2013; Toivonen et al., 2014; Williams et al., 2009)

Brain, spinal cord, muscle, blood, CSF

Imaging biomarkers PET FDG PET or PET on protein biomarker tracers MRI MRI

Brain imaging

Protein biomarkers RANT diELISA, SILK peptides

Brain, spinal cord, blood, CSF

CNS secreted proteins

CSF, blood, urine, saliva

ELISA, Western blot, mass spectrometry MRM

Brain metabolic signature

(Bowser et al., 2011; Cistaro et al., 2014a, 2014b; Davis et al., 2003; Foerster et al., 2013; Klunk et al., 2004; Lloyd et al., 2000a; Mathis et al., 2004; Turner et al., 2004, 2005; Van Laere et al., 2014) (Bede et al., 2013; Boeve et al., 2012; Bowser et al., 2011; Byrne et al., 2012; Foerster et al., 2013; Mahoney et al., 2012)

(Ash et al., 2013; Cleary and Ranum, 2014; Gendron et al., 2013a, 2013b, 2014; Mann et al., 2013; Mori et al., 2013a, 2013b; Zu et al., 2011, 2013b) (Bowser et al., 2006; Donnelly et al., 2013; Kruger et al., 2013; LagierTourenne et al., 2013; Ryberg and Bowser, 2008; Sareen et al., 2013)

FISH—fluorescent in-situ hybridization, MS-SNuPE—methylation-sensitive single-nucleotide primer extension, ChIP—chromatin immunoprecipitation, COBRA—combined bisulfite restriction analysis, PET—positron emission tomography, FDG PET—fludeoxyglucose positron emission tomography, SILK—stable isotope labeling kinetics, ELISA—enzyme-linked immunoabsorbent assay, MRM—multi-reaction monitoring, CNS—central nervous system, CSF—cerebrospinal fluid.



markers or easily accessible CNS markers that accurately reflect biology in the CNS is a major goal and critical endeavor in clinical pharmacology of CNS drug development and will not only allow for better therapeutic trial design, but also reduce the cost of clinical trials. While genetic screens can be put into place for diagnosis of C9orf72 patients, an assay to determine if a therapy is working is yet to be determined. Thus, prime candidates for pharmacodynamics biomarkers would need to be either ubiquitously expressed, so as to be detectable and alterable in skin, hair, nails, and other external tissues; secreted so as to be detectable in cerebrospinal fluid, blood, urine or saliva; of cognitive or behavioral nature, with stringent criteria for improvement; metabolic or generally detectable by advanced imaging technologies. The nature of drug delivery will determine whether a peripheral biomarker assay will be suitable. In other words, only systemic drug delivery will allow for the use of a peripheral biomarker (skin/ blood/urine), while CNS-specific deliveries, such as intrathecal injections, can only be monitored using CNS-related biomarkers (detection in CSF/brain imaging). Clinical biomarkers, such as EMG’s, brain imaging, and metabolic studies are important to assess the progression of the disease once it has onset, whereas subclinical biomarkers, such as secreted molecules are a valuable tool to assess that which is unseen. Because genetic screens are so readily available, it is possible to detect whether a person has the mutation and will develop the disease before they show any behavioral symptoms. This provides a wider open window for treatment, but would require doctors to assess whether the treatment is working in an alternate manner, independent of monitoring disease symptoms. This review will summarize possibilities for pharmacodynamic biomarker development related to the C9orf72 HRE to allow for a better clinical trial design for any upcoming therapeutic trials targeted to disease pathogenesis caused by this mutation (Table 1).

2.

Molecular biomarkers

Probably the most characterized phenomenon in C9orf72 disease is the existence of intranuclear GGGGCC repeat RNA foci visualized using RNA fluorescence in-situ hybridization (FISH) techniques (DeJesus-Hernandez et al., 2011; Gendron et al., 2014; Ling et al., 2013; Renton et al., 2011). Currently, these foci are the major cellular in vitro readout used to assess if pre-clinical therapeutic developments such as antisense oligonucleotide probes targeted to the repeat are efficacious in eliminating the repeat expansion and downstream disease phenotypes (Donnelly et al., 2013; LagierTourenne et al., 2013; Sareen et al., 2013). These foci are composed of both sense and antisense transcripts of the repeat (Lagier-Tourenne et al., 2013; Mizielinska et al., 2013; Zu et al., 2013a). Most importantly, these foci are not only detected in brain cells, but also in peripheral cells, such as skin biopsy-derived fibroblasts (Almeida et al., 2013; Donnelly et al., 2013; Lagier-Tourenne et al., 2013), lymphoblasts (Lagier-Tourenne et al., 2013) and peripheral blood leukocytes (Zu et al., 2013b), making them a potential molecular readout in clinical trials with systemic therapeutic intervention.

3

RNA foci generally appear as single dots and apart from abundance, size, shape, fluorescence intensity and colocalization with interacting proteins can be used to characterize their presence. Some of these features can easily be quantitated using advanced high-content microscopy-based imaging systems and analysis tools thus enabling the design of a semi high throughput detection assay to be used during clinical trials. Detection of RNA foci as a diagnostic marker is widely used in myotonic dystrophy 2 (DM2), a dominantly inherited disorder characterized by a CCTG repeat expansion in intron 1 of ZNF9 gene. Due to known instability of this particular expansion, RNA-FISH is performed on muscle biopsy tissue to obtain a definite diagnosis of the disease (Cardani et al., 2009). To this date, it is unknown whether muscle biopsies obtained from C9orf72 patients show RNA foci formation, but one could easily envision the use of blood leukocytes to determine whether GGGGCC repeat expansion targeted therapeutic treatment will reduce the number of RNA foci in leukocytes.

3.

Epigenetic biomarkers

Epigenetic mechanisms result in heritable changes in gene expression and function independent of changes in the DNA coding sequences. These mechanisms include DNA methylation, histone modification, chromatin remodeling as well as microRNA (miRNA) activity (Mehler, 2008). Epigenetic alterations can be caused by factors such as environmental changes or exposures to stress and epigenetic deregulation have been linked to numerous human diseases, including cancer, but also neurodevelopment and neurodegenerative disorders, including Parkinson’s disease, Huntington’s disease, fragile X syndrome, Friedreich’s ataxia, spinocerebellar ataxias as well as ALS (Al-Chalabi et al., 2013; Belzil et al., 2014; Martin and Wong, 2013; Qureshi and Mehler, 2013a; Yang et al., 2010). Interestingly, many of these neurological diseases are characterized by repeat expansions, suggesting that epigenetic processes present a significant contribution to disease pathogenesis in repeat disorders (He and Todd, 2011). Because of the increasing awareness of the role of epigenetic events and dysfunction thereof in disease progression, the development of epigenetic biomarkers to guide treatment paradigms in patients has become more prominent not only in the field of oncology, but also in neurodegenerative diseases.

3.1.

DNA methylation and histone modifications

Over 20 years ago, DNA methylation was the first epigenetic marker shown to alter normal gene processing in cancer and was since used for diagnostic purposes, but also as a pharmacodynamics biomarker to monitor drug treatment (Esteller, 2008; Feinberg and Vogelstein, 1983). DNA methylation has also been reported in ALS (Chestnut et al., 2011; Martin and Wong, 2013; Yang et al., 2010) and epigenetic alterations have become even more recognized with the discovery of the C9orf72 repeat expansion. Xi and colleagues were the first to report hypermethylation of the CpG island 50 of the GGGGCC repeat in C9orf72 ALS patients, which


4


inversely correlated with disease duration, suggesting that the degree of CpG methylation associates with increased disease progression, similar to what has been reported for Friedreich’s ataxia (Evans-Galea et al., 2012; Xi et al., 2013). The authors were able to detect these epigenetic alterations not only in frontal cortex and cervical spinal cord tissue, but also in blood of ALS patients positive for the C9orf72 mutation. Interestingly, when Belzil and colleagues examined DNA methylation in cerebellar tissue of C9orf72 cases (both ALS, FTD and ALS/FTD), in which they had already identified aberrant histone modifications, only one C9orf72 FTD patient showed hypermethylation in the C9orf72 promoter region, suggesting that DNA methylation varies across brain regions and could correlate with different disease phenotypes (Belzil et al., 2013, 2014). Both, hypermethylation of CpG islands and the histone trimethylation are suggested to explain the observed reduction of C9orf72 mRNA in C9orf72 patients and treatment of C9orf72 patient-derived fibroblasts with a histone demethylation agent increased C9orf72 gene expression (Belzil et al., 2013). Most importantly, both epigenetic alterations were detectable in blood samples of patients, making the analysis of DNA hypermethylation or histone modification amenable for biomarker development, diagnostic and prognostic, as well as pharmacodynamic in combination with systemic therapeutic intervention. Similar observations have been made in lymphocytes of schizophrenic patients where the levels of H3K9 dimethylation correlated with the age of disease onset (Gavin et al., 2009), and Huntington’s disease patients aberrant epigenetic modifications (histone acetylation and methylation, function of epigenetic regulatory factor REST) where found not only in patient-derived brain tissue, but also blood (Marullo et al., 2010; Zuccato et al., 2003). Recent advances in high throughput sequencing and bioinformatics allow the investigation for more potential DNA methylation sites and subsequent screening phases can narrow down the identification of highly predictive genomic regions as candidate biomarkers, which could then be analyzed in a more quantitative matter using varying sequencing technologies, such as bisulfite pyrosequencing (Tost and Gut, 2007), MS-SnuPE (Gonzalgo and Liang, 2007), COBRA (Brena et al., 2006) or mass spectrometry (Ehrich et al., 2005). To be able to monitor histone modifications and to expand on the already identified modification sites, modified chromatin immunoprecipitation (ChIP) assays (ChIP-chip or Chip-seq) are available to perform genome-wide profiles in a high throughput fashion and can likely be adopted to routine clinical use.

3.2.

microRNAs (miRNAs)

MicroRNAs are short non-coding RNAs that regulate function and expression (both up and down-regulation) of a vast array of genes through transcriptional, post-transcriptional and epigenetic mechanisms and have been shown to play a significant role in CNS function (Gascon and Gao, 2012; Lee et al., 1993; Maciotta et al., 2013; Qureshi and Mehler, 2013b; Wightman et al., 1993). miRNAs are highly stable and show low susceptibility to degradation. They are found to be released into the circulation, including blood and cerebral spinal fluid (CSF), either associated with protein complexes or in forms of membrane-associated vesicles, (Rao et al., 2013).

Alterations in the miRNA profile have been detected in various neurological and psychiatric disorders, including Alzheimer’s disease, multiple sclerosis, schizophrenia, Parkinson’s disease, glioblastoma as well as ALS and FTD (reviewed in (Gascon and Gao, 2014; Maciotta et al., 2013; Rao et al., 2013)). Interestingly, these disease-altered profiles were often detected similarly between patient tissues and animal models, both in central and peripheral tissues, suggesting measurements of miRNA could provide a validated peripheral biomarker for neurological disease diagnosis, but also for therapeutic monitoring (Jeyaseelan et al., 2008; Liu et al., 2010). Several miRNA pathways have been identified to be dysregulated in ALS by either using a candidate approach or a broad miRNA profiling approach. For example, Williams and colleagues found that downregulation of muscle-specific miR-206, which had been shown to play a critical role in muscle re-innervation, in the SOD1mut mouse accelerated the disease progression and shortened survival (Chen et al., 2006; Toivonen et al., 2014; Williams et al., 2009). miR-9 was linked to TDP-43 and mutant SOD1 pathology in ALS, as well to neurofilament dysregulation in motor neuron disorders (Haramati et al., 2010; Li et al., 2013; Zhou et al., 2013). General miRNA profiling in patient leukocytes and spinal cord tissue revealed numerous other de-regulated miRNAs, including miR-155, which was increased in sporadic and familial ALS patients and inhibition thereof in SODmut mice lead to increased disease duration and survival (CamposMelo et al., 2013; De Felice et al., 2012; Koval et al., 2013). While it is still unknown what kind of miRNA fingerprint the C9orf72 HRE in particular could have, it is hypothesized that the observed RNA toxicity will likely lead to changes in microRNA processsing, similar to what has been reported for ALS/FTD as well as for other repeat expansion disorders, including Friedreich’s ataxia, fragile X and both myotonic dystrophies (DM1þDM2) (Bandiera et al., 2013; Campos-Melo et al., 2013; Chen-Plotkin et al., 2012; Fernandez-Costa et al., 2013; Gascon and Gao, 2014; Gong et al., 2013; Greco et al., 2012; Sellier et al., 2013). As mentioned above, microRNA profiles have been detected not only in the CNS, but also in peripheral tissues as well as CSF. Small RNAs secreted in the CSF are already being assessed as biomarkers for glioblastomas and brain injury (Rao et al., 2013). If hallmark miRNAs are found in C9orf72 HRE patients, and are confirmed to be stable and secreted in the CSF, these could prove to be a valuable readout for treatment efficacy. Similar to the above described epigenetic alterations, the detection of altered microRNA processing as a clinical routine procedure is supported by the growing sophistication of rapidly evolving quantitative detection methods to support reliable and robust sample preparation and analyses, although like every high throughput screening platform, limitations of accurate yield of extraction, purity and quantification have to be accounted for (Sandoval et al., 2013).

4.

Imaging-based biomarkers

Advanced neuroimaging techniques offer a unique noninvasive approach to serve as a diagnostic CNS biomarker for ALS, but at the same time could also be greatly suitable as a



pharmacodynamics biomarker used in clinical settings for monitoring therapeutic efficacy and also potentially to stratify patients. The wide spread of imaging techniques allows for the monitoring of brain structures, neural network connections, metabolism, as well as plasma membrane receptor distribution. The two major technologies are (1) magnetic resonance imaging (MRI) and (2) radionucleotide imaging (PET, SPECT) (reviewed in (Bowser et al., 2011; Foerster et al., 2013)). Bede and colleagues used a multiparametric MRI approach (diffusion tensor imaging (DTI) and T1-weighted voxel-based morphometry MRI) to look at mutant C9orf72specific patterns of cortical and subcortical changes (Bede et al., 2013). The results suggested a clear mutation-specific cortical and subcortical involvement reflecting the increased cognitive and behavioral impairment observed with this ALS genotype, as well as the published postmortem C9orf72 histopathology (Boeve et al., 2012; Byrne et al., 2012; Mahoney et al., 2012). These results suggest that the applied imaging approach could be a predictor to whether a C9orf72 patient will suffer cognitive decline and/or motor decline. Several independent studies were performed evaluating the metabolic signature of C9orf72-positive ALS or FTD patients using 18fluorodeoxyglucose-positron-emission tomography ([(18)F]FDG PET) (Cistaro et al., 2014b; Van Laere et al., 2014).

5

Both studies found significant hypometabolism in the limbic system and the central structures and hypermetabolism in the midbrain of C9orf72 ALS and FTD patients when compared to non-C9orf72 ALS patients (Cistaro et al., 2014b). These metabolic changes appear to be consistent with the clinical and neuropathological changes seen in C9orf72 patients when compared to non-C9orf72 ALS and/or FTD patients, again suggesting that C9orf72 patients have a unique metabolic signature, which differentiates them from other ALS and or FTD patients. One could speculate that these metabolic signatures could be used to monitor the efficacy of therapeutic intervention, which, if successful, could normalize the metabolic changes quantitated by repetitive PET scanning analysis of the individual patient. PET technology can also be developed to image diseasespecific proteins in the CNS that are altered during disease progression (see Protein Biomarkers below). Examples for successful PET imaging tracers in neurodegenerative diseases are the Pittsburgh compound B ([C11]PiB), which was used to image accumulation of beta amyloid plaques in Alzheimer’s disease patients (Klunk et al., 2004; Mathis et al., 2004) and the dopamine transporter ligand [(18)F]-FECNT, which allowed for the visualization of loss of dopamine transporters in Parkinson’s disease patients (Davis et al., 2003). Similar approaches have been taken for ALS as well, such as the

Fig. 1 – Schematic depiction of varying possibilities for mutant C9orf72-directed biomarker assay development. Biomarker readouts for C9orf72-specific therapeutics can be found at different levels of organization: systemic, cellular and molecular. Systemic: Brain imaging biomarkers, such as PET or MRI; Protein biomarkers in the CSF and/or blood, such as secreted proteins and RANT peptides; Epigenetic biomarkers in the CSF, such as miRNAs. Cellular: On the cellular level, potential biomarker readout can come from CNS cell secreted proteins or RANT, as well as secreted miRNAs, while blood cells offer the additional option of monitoring RNA foci. Molecular: This approach will only be available for peripheral cells, such as leucocytes, and offers readouts including histone modifications, DNA methylations, as well as protein dysregulation. Please cite this article as: Mendez, E.F., Sattler, R., Biomarker development for C9orf72 repeat expansion in ALS. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.09.041

6


imaging of [11C](R)-PK11195, a ligand that binds to the benzodiazepine receptor, which is upregulated in activated microglial cells (Cistaro et al., 2014a; Turner et al., 2004). Similarly, imaging benzodiazepine GABA(A) receptors in ALS patients using [(11)C]flumazenil (FMZ) revealed significant changes in extramotor brain regions when compared to healthy control subjects, supporting the clinical observations of cerebral dysfunction in ALS in addition to motor cortical deficits (Lloyd et al., 2000b; Turner et al., 2005). In a collaborative research project between our laboratory and Drs. Rothstein (Johns Hopkins University) and Gerdes (University of Montana) we are currently developing and validating a PET tracer for the astrocytic glutamate transporter GLT-1/EAAT2, which is known to be downregulated in ALS patients (Rothstein et al., 1995). While most of these protein-specific PET ligands have been used for diagnostic purposes, one could easily imagine applying them during therapeutic development to accelerate effective drug validation. As outlined below, the C9orf72 mutation has been shown to cause aberrant gene expression, hence, developing a candidate specific PET tracer to monitor the expression profile in the CNS of patients during clinical trials seems quite feasible (Fig. 1).

5.

Protein biomarkers

Probably the most commonly sought after biomarker assay consists of the quantitative analysis of proteins, which show disease-dependent aberrant expression levels. Protein-based ALS biomarker candidates can be searched for in different biological fluids including CSF, plasma, serum, urine, saliva but also in biopsied tissue samples, such as muscle, skin and nasal olfactory epithelium (Bowser et al., 2006; Hu et al., 2006; Ryberg and Bowser, 2008). With the use of varying protein detection technologies, including antibody-based Western blot analysis or quantitative ELISA and proteomics analyses based on mass spectrometry, numerous putative protein biomarkers have been identified by comparing ALS patient CSF or plasma to healthy control samples (reviewed in (Bowser et al., 2006; Kruger et al., 2013; Ryberg and Bowser, 2008)). For C9orf72-specific protein biomarkers, two observed disease phenotypes provide potential protein biomarker candidates, which could be used to monitor C9orf72-specific therapeutic efficacy: (1) aberrant expression of CNS secreted proteins and (2) accumulation of repeat-associated non-ATG translation (RANT) products.

5.1.

Aberrantly expressed CNS secreted proteins

One consequence of the GGGGCC repeat formation in C9orf72 is the binding and sequestration of RNA binding proteins (RBPs) (Cruts et al., 2013; Gendron et al., 2014; Ling et al., 2013). This phenomenon has been thoroughly studied and characterized in other repeat expansion disorders, especially mytonic dystrophy 1 (DM1) (Echeverria and Cooper, 2012; Fardaei et al., 2002; Grammatikakis et al., 2010; Miller et al., 2000). Consequently, a large number of RBPs have been discovered for the C9orf72 HRE (Almeida et al., 2013;

Donnelly et al., 2013; Haeusler et al., 2014; Lagier-Tourenne et al., 2013; Lee et al., 2013; Sareen et al., 2013). The sequestration of RBPs could bring about a toxic effect in a secondhand manner. That is, these sequestered RBPs, unable to break free, cannot perform their intended function on other target RNAs, thereby propagating a misregulation of RNA processing (Polymenidou et al., 2012). This toxic RNA gain-offunction could affect expression and/or translation of a myriad of other proteins and RNAs, which has evoked transcriptome analyses of C9orf72 HRE-containing patient derived fibroblasts, human induced pluripotent stem (iPS) cells and postmortem brain tissues (Donnelly et al., 2013; Lagier-Tourenne et al., 2013; Sareen et al., 2013). Our laboratory was particularly interested in the aberrant expression of CNS secreted genes found in human postmortem C9orf72 brain tissue and C9orf72 patient-derived iPS neurons with the goal of identifying a biomarker candidate that could potentially be detected in patient CSF and possibly blood. Microarray analyses revealed several CNS genes known to be secreted that were equally aberrantly expressed in iPS neurons and brain tissue. Interestingly, when we treated iPS neurons with an antisense oligonucleotide therapeutic, this aberrant gene expression was normalized, suggesting that protein expression of these genes could serve as a potential biomarker readout in a clinical setting (Donnelly et al., 2013). Ongoing studies are aimed at validating the observed gene expression changes at the protein level in iPS neuron cell lysates as well as cell culture supernatants using ELISA and mass spectrometry techniques, such as multiple reaction monitoring (MRM) for the purposes of semi high throughput measurements of candidate proteins in human CSF (Lemoine et al., 2012).

5.2. Repeat-associated non-ATG translation (RANT) products In addition to the formation of RNA foci, repeat expansions often also lead to so called repeat-associated non-ATG translation (RAN translation or RANT). This translation occurs in the absence of the typical AUG initiation codon and was first discovered to occur across CAG expanded repeats where it leads to the formation of homopolymeric proteins (polyA, polyS, polyQ) (Zu et al., 2011). This phenomenon was soon after confirmed in other repeat expansion disorders with varying repeat nucleotide compositions, including C9orf72 ((Ash et al., 2013; Cleary and Ranum, 2014; Gendron et al., 2013b, 2014; Mori et al., 2013b; Zu et al., 2011, 2013a)). Interestingly, studies on the C9orf72 repeat expansion revealed RAN translation not only from sense strand repeats, but also from antisense strand repeats, increasing the diversity of dipeptide repeat proteins to five variants (Ash et al., 2013; Gendron et al., 2013a; Mann et al., 2013; Mori et al., 2013a; Zu et al., 2013a). Recent data support the hypothesis that these dipeptides are toxic and may therefore contribute to the disease progression in C9orf72positive patients (Kwon et al., 2014; May et al., 2014; Mizielinska et al., 2014; Zhang et al., 2014). Consequently, the presence of RANT dipeptides in CSF could provide another candidate biomarker for C9orf72 clinical trial



monitoring. Interestingly, a recent study supports this hypothesis providing evidence of the presence of GP dipeptides in CSF of ALS patients as detected with a GP dipeptidespecific ELISA assay (Su et al., 2014). Ongoing experiments in our laboratory in collaboration with Drs. Rothstein (Johns Hopkins University) and Petrucelli (Mayo Clinic Florida) are testing the efficacy of antisense oligonucleotide therapeutics on the levels of RAN dipeptides in an iPS neuron cell culture platform, validating the suitability of RANT dipeptide detection in a clinical setting for C9orf72-specific drug trials. An alternative way to monitor the formation of RANT dipeptides and the potential inhibition thereof during therapy is the use of stable isotope labeling. Stable isotope labeling kinetic (SILK) has successfully been employed to measure Aβ production in patient CSF during treatment with γ-secretase inhibitor LY450139 (Bateman et al., 2009). The advantage of this technology is that the isotope labels newly generated peptides or proteins, which is particularly important when the peptides or proteins have a high turnover rate, which would make the timing of CSF collection and protein analysis difficult and likely highly variable. It could also be of advantage if peptides or proteins are very stable and not easily degraded, which could then in turn mask any drug efficacy on the reduction of these peptides when quantified in CSF during clinical trials. Again, the use of C9orf72 iPS cells will allow for initial in vitro validation studies using SILAC (Stable isotope labeling by amino acids in cell culture) technology in combination with potential drug candidates, including antisense oligonucleotide therapy.

6.

Conclusion

The development of a pharmacodynamic biomarker acting as a surrogate for drug efficacy should become a prerequisite for the design of any future ALS clinical trials. Due to the fast progression of the disease survival time has been the current standard to evaluate therapeutic efficacy. Therefore, the availability of a biomarker that monitors response to drug treatment within a brief time period will greatly improve clinical trial design by shortening the trials, therefore reducing cost and allowing non-responding patients to enroll in another trial. In addition, especially in the case of familial ALS patients, mutation-specific drug therapy can be tested on a select patient population and negative biomarker readouts can be used to exclude drug resistant trial participants. The development of a biomarker assay to monitor C9orf72 specific drug treatment is of particular interest due to the large prevalence of the mutation not only among ALS patients, but also FTD, ALS/FTD and a growing number of other neurological disorders (Liu et al., 2013).

Conflict of interest R.S. is a co-inventor of a pending patent for “Composition of modulating C9orf72”.

7

Acknowledgments This work was supported by The Johns Hopkins University Brain Science Institute; National Institutes of Health/National Institutes of Neurological Disorders and Stroke [RO1 NS085207 (RS)]; Amyotrophic Lateral Sclerosis Association (#2094 2110) (RS); The Judith and Jean Pape Adams Charitable Foundation (RS) and The William and Ella Owens Medical Research Foundation (RS).

r e f e r e nc e s

Al-Chalabi, A., et al., 2013. Genetic and epigenetic studies of amyotrophic lateral sclerosis. Amyotroph Lateral Scler Frontotemporal Degener 14 (Suppl. 1), 44–52. Almeida, S., et al., 2013. Modeling key pathological features of frontotemporal dementia with C9ORF72 repeat expansion in iPSC-derived human neurons. Acta Neuropathol. Ash, P.E., et al., 2013. Unconventional translation of C9ORF72 GGGGCC expansion generates insoluble polypeptides specific to c9FTD/ALS. Neuron 77, 639–646. Bandiera, S., et al., 2013. Genetic variations creating microRNA target sites in the FXN 30 -UTR affect frataxin expression in Friedreich ataxia. PLoS One 8, e54791. Bateman, R.J., et al., 2009. A gamma-secretase inhibitor decreases amyloid-beta production in the central nervous system. Ann Neurol. 66, 48–54. Bede, P., et al., 2013. Multiparametric MRI study of ALS stratified for the C9orf72 genotype. Neurology 81, 361–369. Belzil, V.V., et al., 2013. Reduced C9orf72 gene expression in c9FTD/ALS is caused by histone trimethylation, an epigenetic event detectable in blood. Acta Neuropathol. Belzil, V.V., et al., 2014. Characterization of DNA hypermethylation in the cerebellum of c9FTD/ALS patients. Brain Res. Boeve, B.F., et al., 2012. Characterization of frontotemporal dementia and/or amyotrophic lateral sclerosis associated with the GGGGCC repeat expansion in C9ORF72. Brain 135, 765–783. Bowser, R., Cudkowicz, M., Kaddurah-Daouk, R., 2006. Biomarkers for amyotrophic lateral sclerosis. Expert Rev. Mol. Diagn. 6, 387–398. Bowser, R., Turner, M.R., Shefner, J., 2011. Biomarkers in amyotrophic lateral sclerosis: opportunities and limitations. Nat. Rev. Neurol. 7, 631–638. Brena, R.M., et al., 2006. Quantification of DNA methylation in electrofluidics chips (Bio-COBRA). Nat. Protoc. 1, 52–58. Brouwer, J.R., Willemsen, R., Oostra, B.A., 2009. Microsatellite repeat instability and neurological disease. BioEssays 31, 71–83. Byrne, S., et al., 2012. Cognitive and clinical characteristics of patients with amyotrophic lateral sclerosis carrying a C9orf72 repeat expansion: a population-based cohort study. Lancet Neurol. 11, 232–240. Campos-Melo, D., et al., 2013. Altered microRNA expression profile in amyotrophic lateral sclerosis: a role in the regulation of NFL mRNA levels. Mol. Brain 6, 26. Cardani, R., et al., 2009. Ribonuclear inclusions as biomarker of myotonic dystrophy type 2, even in improperly frozen or defrozen skeletal muscle biopsies. Eur. J. Histochem. 53, 107–111. Chen, J.F., et al., 2006. The role of microRNA-1 and microRNA-133 in skeletal muscle proliferation and differentiation. Nat. Genet. 38, 228–233.


8


Chen-Plotkin, A.S., et al., 2012. TMEM106B, the risk gene for frontotemporal dementia, is regulated by the microRNA-132/ 212 cluster and affects progranulin pathways. J. Neurosci. 32, 11213–11227. Chestnut, B.A., et al., 2011. Epigenetic regulation of motor neuron cell death through DNA methylation. J. Neurosci. 31, 16619–16636. Cistaro, A., et al., 2014a. Role of PET and SPECT in the study of amyotrophic lateral sclerosis. Biomed. Res. Int. 2014, 237437. Cistaro, A., et al., 2014b. The metabolic signature of C9ORF72related ALS: FDG PET comparison with nonmutated patients. Eur. J. Nucl. Med. Mol. Imaging. 41, 844–852. Ciura, S., et al., 2013. Loss of function of C9orf72 causes motor deficits in a zebrafish model of amyotrophic lateral sclerosis. Ann. Neurol. Cleary, J.D., Ranum, L.P., 2014. Repeat associated non-ATG (RAN) translation: new starts in microsatellite expansion disorders. Curr. Opin. Genet. Dev. 26C, 6–15. Cruts, M., et al., 2013. Current insights into the C9orf72 repeat expansion diseases of the FTLD/ALS spectrum. Trends Neurosci. 36, 450–459. Davis, M.R., et al., 2003. Initial human PET imaging studies with the dopamine transporter ligand 18F-FECNT. J. Nucl. Med. 44, 855–861. De Felice, B., et al., 2012. A miRNA signature in leukocytes from sporadic amyotrophic lateral sclerosis. Gene 508, 35–40. DeJesus-Hernandez, M., et al., 2011. Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron 72, 245–256. Donnelly, C.J., et al., 2013. RNA toxicity from the ALS/FTD C9ORF72 expansion is mitigated by antisense intervention. Neuron 80, 415–428. Echeverria, G.V., Cooper, T.A., 2012. RNA-binding proteins in microsatellite expansion disorders: mediators of RNA toxicity. Brain Res. 1462, 100–111. Ehrich, M., et al., 2005. Quantitative high-throughput analysis of DNA methylation patterns by base-specific cleavage and mass spectrometry. Proc. Natl. Acad. Sci. U.S.A. 102, 15785–15790. Esteller, M., 2008. Epigenetics in cancer. N. Engl. J. Med. 358, 1148–1159. Evans-Galea, M.V., et al., 2012. FXN methylation predicts expression and clinical outcome in Friedreich ataxia. Ann. Neurol. 71, 487–497. Fardaei, M., et al., 2002. Three proteins, MBNL, MBLL and MBXL, co-localize in vivo with nuclear foci of expanded-repeat transcripts in DM1 and DM2 cells. Hum. Mol. Genet. 11, 805–814. Feinberg, A.P., Vogelstein, B., 1983. Hypomethylation distinguishes genes of some human cancers from their normal counterparts. Nature 301, 89–92. Fernandes, S.A., et al., 2013. Oligonucleotide-based therapy for FTD/ALS caused by the repeat expansion: a perspective. J. Nucleic Acids 2013, 208245. Fernandez-Costa, J.M., et al., 2013. Expanded CTG repeats trigger miRNA alterations in Drosophila that are conserved in myotonic dystrophy type 1 patients. Hum. Mol. Genet. 22, 704–716. Foerster, B.R., Welsh, R.C., Feldman, E.L., 2013. 25 years of neuroimaging in amyotrophic lateral sclerosis. Nat. Rev. Neurol 9, 513–524. Gascon, E., Gao, F.B., 2012. Cause or effect: misregulation of microRNA pathways in neurodegeneration. Front. Neurosci 6, 48. Gascon, E., Gao, F.B., 2014. The emerging roles of MicroRNAs in the pathogenesis of frontotemporal dementia-amyotrophic lateral sclerosis (FTD-ALS) spectrum disorders. J. Neurogenet.

Gatchel, J.R., Zoghbi, H.Y., 2005. Diseases of unstable repeat expansion: mechanisms and common principles. Nat. Rev. Genet. 6, 743–755. Gavin, D.P., et al., 2009. Dimethylated lysine 9 of histone 3 is elevated in schizophrenia and exhibits a divergent response to histone deacetylase inhibitors in lymphocyte cultures. J. Psychiatry Neurosci. 34, 232–237. Gendron, T.F., et al., 2013a. Antisense transcripts of the expanded C9ORF72 hexanucleotide repeat form nuclear RNA foci and undergo repeat-associated non-ATG translation in c9FTD/ALS. Acta Neuropathol. Gendron, T.F., Cosio, D.M., Petrucelli, L., 2013b. c9RAN translation: a potential therapeutic target for the treatment of amyotrophic lateral sclerosis and frontotemporal dementia. Expert Opin. Ther. Targets 17, 991–995. Gendron, T.F., et al., 2014. Mechanisms of toxicity in C9FTLD/ALS. Acta Neuropathol. 127, 359–376. Gong, X., et al., 2013. MicroRNA-130b targets Fmr1 and regulates embryonic neural progenitor cell proliferation and differentiation. Biochem. Biophys. Res. Commun. 439, 493–500. Gonzalgo, M.L., Liang, G., 2007. Methylation-sensitive singlenucleotide primer extension (Ms-SNuPE) for quantitative measurement of DNA methylation. Nat. Protoc. 2, 1931–1936. Grammatikakis, I., et al., 2010. Identification of MBNL1 and MBNL3 domains required for splicing activation and repression. Nucleic Acids Res. 39, 2769–2780. Greco, S., et al., 2012. Deregulated microRNAs in myotonic dystrophy type 2. PLoS One 7, e39732. Haeusler, A.R.D., Periz, C.J., Simko, G., Shaw, E.A.J., Kim, P.G., Maragakis, M.S., Troncoso, N.J., Pandey, J.C., Sattler, A., Rothstein, R., Wang, J., J.D., 2014. C9orf72 nucleotide repeat structures initiate molecular cascades of disease. Nature. Haramati, S., et al., 2010. miRNA malfunction causes spinal motor neuron disease. Proc. Natl. Acad. Sci. U.S.A. 107, 13111–13116. He, F., Todd, P.K., 2011. Epigenetics in nucleotide repeat expansion disorders. Semin Neurol. 31, 470–483. Hu, S., Loo, J.A., Wong, D.T., 2006. Human body fluid proteome analysis. Proteomics 6, 6326–6353. Jeyaseelan, K., Lim, K.Y., Armugam, A., 2008. MicroRNA expression in the blood and brain of rats subjected to transient focal ischemia by middle cerebral artery occlusion. Stroke 39, 959–966. Klunk, W.E., et al., 2004. Imaging brain amyloid in Alzheimer’s disease with Pittsburgh Compound-B. Ann. Neurol. 55, 306–319. Koval, E.D., et al., 2013. Method for widespread microRNA-155 inhibition prolongs survival in ALS-model mice. Hum. Mol. Genet. 22, 4127–4135. Kruger, T., et al., 2013. Proteome analysis of body fluids for amyotrophic lateral sclerosis biomarker discovery. Proteomics Clin. Appl. 7, 123–135. Kwon, I., et al., 2014. Poly-dipeptides encoded by the C9ORF72 repeats bind nucleoli, impede RNA biogenesis, and kill cells. Science. Lagier-Tourenne, C., et al., 2013. Targeted degradation of sense and antisense C9orf72 RNA foci as therapy for ALS and frontotemporal degeneration. Proc. Natl. Acad. Sci. U.S.A. Lee, R.C., Feinbaum, R.L., Ambros, V., 1993. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75, 843–854. Lee, Y.B., et al., 2013. Hexanucleotide repeats in ALS/FTD form length-dependent RNA foci, sequester RNA binding proteins, and are neurotoxic. Cell Rep. Lemoine, J., et al., 2012. The current status of clinical proteomics and the use of MRM and MRM(3) for biomarker validation. Expert Rev. Mol. Diagn. 12, 333–342.



Li, Z., et al., 2013. The FTD/ALS-associated RNA-binding protein TDP-43 regulates the robustness of neuronal specification through microRNA-9a in Drosophila. Hum. Mol. Genet. 22, 218–225. Ling, S.C., Polymenidou, M., Cleveland, D.W., 2013. Converging mechanisms in ALS and FTD: disrupted RNA and protein homeostasis. Neuron 79, 416–438. Liu, D.Z., et al., 2010. Brain and blood microRNA expression profiling of ischemic stroke, intracerebral hemorrhage, and kainate seizures. J. Cereb. Blood Flow Metab. 30, 92–101. Liu, Y., et al., 2013. C9ORF72 mutations in neurodegenerative diseases. Mol. Neurobiol. Lloyd, C.M., et al., 2000a. Extramotor involvement in ALS: PET studies with the GABA(A) ligand [(11)C]flumazenil. Brain 123 (Pt 11), 2289–2296. Lloyd, C.M., et al., 2000b. Extramotor involvement in ALS: PET studies with the GABA(A) ligand [(11)C]flumazenil. Brain 123 (Pt 11), 2289–2296. Maciotta, S., Meregalli, M., Torrente, Y., 2013. The involvement of microRNAs in neurodegenerative diseases. Front. Cell Neurosci. 7, 265. Mahoney, C.J., et al., 2012. Frontotemporal dementia with the C9ORF72 hexanucleotide repeat expansion: clinical, neuroanatomical and neuropathological features. Brain 135, 736–750. Mann, D.M., et al., 2013. Dipeptide repeat proteins are present in the p62 positive inclusions in patients with frontotemporal lobar degeneration and motor neurone disease associated with expansions in C9ORF72. Acta Neuropathol. Commun. 1, 68. Martin, L.J., Wong, M., 2013. Aberrant regulation of DNA methylation in amyotrophic lateral sclerosis: a new target of disease mechanisms. Neurotherapeutics 10, 722–733. Marullo, M., et al., 2010. Analysis of the repressor element-1 silencing transcription factor/neuron-restrictive silencer factor occupancy of non-neuronal genes in peripheral lymphocytes from patients with Huntington’s disease. Brain Pathol. 20, 96–105. Mathis, C.A., Wang, Y., Klunk, W.E., 2004. Imaging beta-amyloid plaques and neurofibrillary tangles in the aging human brain. Curr. Pharm. Des. 10, 1469–1492. May, S., et al., 2014. C9orf72 FTLD/ALS-associated Gly-Ala dipeptide repeat proteins cause neuronal toxicity and Unc119 sequestration. Acta Neuropathol. Mehler, M.F., 2008. Epigenetic principles and mechanisms underlying nervous system functions in health and disease. Prog. Neurobiol. 86, 305–341. Miller, J.W., et al., 2000. Recruitment of human muscleblind proteins to (CUG)(n) expansions associated with myotonic dystrophy. EMBO J. 19, 4439–4448. Mizielinska, S., et al., 2013. C9orf72 frontotemporal lobar degeneration is characterised by frequent neuronal sense and antisense RNA foci. Acta Neuropathol. 126, 845–857. Mizielinska, S., et al., 2014. C9orf72 repeat expansions cause neurodegeneration in Drosophila through arginine-rich proteins. Science. Mori, K., et al., 2013a. Bidirectional transcripts of the expanded C9orf72 hexanucleotide repeat are translated into aggregating dipeptide repeat proteins. Acta Neuropathol. 126, 881–893. Mori, K., et al., 2013b. The C9orf72 GGGGCC repeat is translated into aggregating dipeptide-repeat proteins in FTLD/ALS. Science 339, 1335–1338. Neumann, M., et al., 2006. Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science 314, 130–133. Polymenidou, M., et al., 2012. Misregulated RNA processing in amyotrophic lateral sclerosis. Brain Res. 1462, 3–15.

9

Qureshi, I.A., Mehler, M.F., 2013a. Epigenetic mechanisms governing the process of neurodegeneration. Mol. Aspects Med. 34, 875–882. Qureshi, I.A., Mehler, M.F., 2013b. Developing epigenetic diagnostics and therapeutics for brain disorders. Trends Mol. Med. 19, 732–741. Rao, P., Benito, E., Fischer, A., 2013. MicroRNAs as biomarkers for CNS disease. Front. Mol. Neurosci 6, 39. Renton, A.E., et al., 2011. A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron 72, 257–268. Riboldi, G., et al., 2014. Antisense oligonucleotide therapy for the treatment of C9ORF72 ALS/FTD diseases. Mol. Neurobiol. Ringholz, G.M., et al., 2005. Prevalence and patterns of cognitive impairment in sporadic ALS. Neurology 65, 586–590. Rothstein, J.D., et al., 1995. Selective loss of glial glutamate transporter GLT-1 in amyotrophic lateral sclerosis. Ann. Neurol. 38, 73–84. Ryberg, H., Bowser, R., 2008. Protein biomarkers for amyotrophic lateral sclerosis. Expert Rev. Proteomics. 5, 249–262. Sandoval, J., et al., 2013. Epigenetic biomarkers in laboratory diagnostics: emerging approaches and opportunities. Expert Rev. Mol. Diagn. 13, 457–471. Sareen, D., et al., 2013. Targeting RNA foci in iPSC-derived motor neurons from ALS patients with a C9ORF72 repeat expansion. Sci. Transl. Med. 5 (208ra149). Sellier, C., et al., 2013. Sequestration of DROSHA and DGCR8 by expanded CGG RNA repeats alters microRNA processing in fragile X-associated tremor/ataxia syndrome. Cell Rep. 3, 869–880. Su, Z., et al., 2014. Discovery of a biomarker and lead small molecules to target r(GGGGCC)-associated defects in c9FTD/ ALS. Neuron. Therrien, M., et al., 2013. Deletion of C9ORF72 results in motor neuron degeneration and stress sensitivity in C. elegans. PLoS One 8, e83450. Todd, P.K., Paulson, H.L., 2010. RNA-mediated neurodegeneration in repeat expansion disorders. Ann. Neurol. 67, 291–300. Toivonen, J.M., et al., 2014. MicroRNA-206: a potential circulating biomarker candidate for amyotrophic lateral sclerosis. PLoS One 9, e89065. Tost, J., Gut, I.G., 2007. DNA methylation analysis by pyrosequencing. Nat. Protoc. 2, 2265–2275. Turner, M.R., et al., 2004. Evidence of widespread cerebral microglial activation in amyotrophic lateral sclerosis: an [11C] (R)-PK11195 positron emission tomography study. Neurobiol. Dis. 15, 601–609. Turner, M.R., et al., 2005. Distinct cerebral lesions in sporadic and ‘D90A’ SOD1 ALS: studies with [11C]flumazenil PET. Brain 128, 1323–1329. Van Laere, K., et al., 2014. Value of 18fluorodeoxyglucosepositron-emission tomography in amyotrophic lateral sclerosis: a prospective study. JAMA Neurol 71, 553–561. Wightman, B., Ha, I., Ruvkun, G., 1993. Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans. Cell 75, 855–862. Williams, A.H., et al., 2009. MicroRNA-206 delays ALS progression and promotes regeneration of neuromuscular synapses in mice. Science 326, 1549–1554. Xi, Z., et al., 2013. Hypermethylation of the CpG island near the G4C2 repeat in ALS with a C9orf72 expansion. Am. J. Hum. Genet. 92, 981–989. Yang, Y., et al., 2010. Epigenetic regulation of neuron-dependent induction of astroglial synaptic protein GLT1. Glia 58, 277–286. Zhang, Y.J., et al., 2014. Aggregation-prone c9FTD/ALS poly(GA) RAN-translated proteins cause neurotoxicity by inducing ER stress. Acta Neuropathol.


10


Zhou, F., et al., 2013. miRNA-9 expression is upregulated in the spinal cord of G93A-SOD1 transgenic mice. Int. J. Clin. Exp. Pathol 6, 1826–1838. Zu, T., et al., 2011. Non-ATG-initiated translation directed by microsatellite expansions. Proc. Natl. Acad. Sci. U.S.A. 108, 260–265. Zu, T., et al., 2013a. RAN proteins and RNA foci from antisense transcripts in C9ORF72 ALS and frontotemporal dementia. Proc. Natl. Acad. Sci. U.S.A. 110, E4968–E4977.

Zu, T., et al., 2013b. RAN proteins and RNA foci from antisense transcripts in C9ORF72 ALS and frontotemporal dementia. Proc. Natl. Acad. Sci. U.S.A. Zuccato, C., et al., 2003. Huntingtin interacts with REST/NRSF to modulate the transcription of NRSE-controlled neuronal genes. Nat. Genet. 35, 76–83.


Identical twins with the C9orf72 repeat expansion are discordant for ALS.

ALS Caused by the C9orf72 Repeat Expansion: A Perspective.

The C9orf72 repeat expansion itself is methylated in ALS and FTLD patients.

FTD.

The C9orf72 repeat expansion disrupts nucleocytoplasmic transport.

C9orf72 repeat expansions are restricted to the ALS-FTD spectrum.

Intermediate length C9orf72 expansion in an ALS patient without classical C9orf72 neuropathology.

C9orf72 hexanucleotide repeat expansion analysis in Chinese spastic paraplegia patients.

C9ORF72 repeat expansion not detected in patients with multiple sclerosis.

Pure cerebellar ataxia linked to large C9orf72 repeat expansion.

Hippocampal sclerosis dementia with the C9ORF72 hexanucleotide repeat expansion.

Commentary: The C9orf72 Repeat Expansion Disrupts Nucleocytoplasmic Transport.

C9ORF72 hexanucleotide repeat expansion is a rare cause of schizophrenia.

FTD.

Suppression of C9orf72 RNA repeat-induced neurotoxicity by the ALS-associated RNA-binding protein Zfp106.

Searching for Grendel: origin and global spread of the C9ORF72 repeat expansion.

FTD.

FTD.

No abnormal hexanucleotide repeat expansion of C9ORF72 in Japanese schizophrenia patients.

Hypermethylation of the CpG-island near the C9orf72 G₄C₂-repeat expansion in FTLD patients.

Frontotemporal Dementia.

Altered network connectivity in frontotemporal dementia with C9orf72 hexanucleotide repeat expansion.

Amyotrophic Lateral Sclerosis with Frontotemporal Dementia in the Presence of C9orf72 Repeat Expansion-A Case Report.

Lack of c9orf72 repeat expansion in taiwanese patients with mixed neurodegenerative disorders.