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A high number of genetic ataxias are due to mutations that directly or indirectly disturb mitochondrial function (see Tables 1 and 2). Therefore, we hypothesize that these pathologies modify mitochondrial signals that could give rise to situations of excessive apoptosis, necrosis, affect the mitophagy rate or causes defects in immune intracellular repair mechanisms. These events in turn are dependent on other factors, notably metabolic and immune status of patients that could lead to different degrees of deleterious effect in diverse individuals affected by the same mutation and this would determine the severity of the pathology. Therefore we believe that an assessment of the rate of mitochondrial damage associating ROS generation with NLRP3/caspasa-1 and mitophagy activity as well as assessment of cell death may have a prognostic value helping the selection of the most appropriate therapy for patients with ataxia.

NLRP3 inflammasome activation is considered an underlying event in different metabolic diseases such as arteriosclerosis, type 2 diabetes, and metabolic syndrome. The control of such activity is now a therapeutic target in these diseases, some of them in advanced clinical trial stage (21). Interestingly, some of the metabolic diseases associated with NLRP3 inflammasome also are present in different types of ataxias. Assessing the activation rate of inflamasomas could allow the selection of patients that could benefit from therapies derived of trials of caspase-1 activity regulators that are being developed at present for the treatment of pathologies mediated by the activity dysfunction of this enzyme.

Table 1: Recessive ataxias

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The hereditary ataxias are a highly heterogenic disorder that in most of the cases affect mitochondrial function directly or indirectly. This mitochondrial dysfunction might affect cell viability with different trends depending on the genetic alteration responsible for the pathology and metabolic and immune status of the patient.


1) To develop a diagnostic platform for the study of the genes responsible of ataxias by Next Generation Sequencing (NGS)

2) To determine the responsible mutation for the genetic ataxia using this platform in a group of ataxic patients without genetic diagnosis.

3) To validate the responsible mutation by testing genetic variables (location of mutation in the gene, frequency in the reference population, segregation in other affected cases, and absence in non affected relatives) and performing functional studies of the mutated protein.

4) To assess in cultured fibroblast the alterations of mitochondrial function produced by different genetically characterized heredoataxias looking for a unifying model of ataxias.

The achievement of these objectives will allow:

1) To reduce the diagnostic pilgrimage of patients with ataxia by accomplishing a precise diagnostic of these patients in a very short time frame, with the consequent cost savings.

2) In many cases it will be possible a personalized care of the patients based on the mutated gene finding.

These objectives are realistic since:

1) We have the necessary infrastructure (a NGS platform Ion Torrent)

2) The design of the gene panel showed a high coverage (more than 90%) of the regions of the ataxia genes.

3) There are similar projects to this proposal that are currently being carried in other centers abroad (see ref. Nemeth et al., Brain Oct. 2013).



Métodos, incluyendo análisis de datos (máximo 2 páginas)

Patients, controls, sample size:

This is a prospective study that includes 48 patients with ataxia from the Neurology Outpatient Clinic of the Hospital Ramón y Cajal that have excluded non-genetic causes (infections, demyelinating diseases, vascular, toxic, vitamin deficiencies, tumors and paraneoplastic) and therefore have a presumably genetic cause. We include in this study 10 patients with already known genetic cause, to check up the quality of the mutation detection test. For mitochondrial function studies we include a control group of 30 healthy volunteers. We have chosen this sample size because HaloPlex plates have 48 wells, which allow to study the sample in 1 plate optimizing the cost of the project. With this number, you will get 2-4 groups of 5-10 cases each with different types of mutations in each group (polyglutamine, alterations in mitochondrial DNA formation, channelopathies,..) to be compared with the control group. In all cases the participants will get a Project informed consent approved by the Ethics Committee of Hospital Ramón y Cajal.

Part 1: Determination of the mutation that causes ataxia

1) Sequencing of candidate genes for ataxia in nuclear genome.

The workflow sequencing of genes includes two phases, generation of the library and proper sequencing.

To generate the library we use 200 ng of DNA from each patient with the kit HaloPlex Target Enrichment System (Agilent Technologies) of 48 samples and capable of capturing 250 Kb to 2.5 Mb sequence. With the Haloplex kit the total region to capture in this experiment for subsequent sequencing is 715 Kb in size with a coverage of 99.85%, corresponding to the coding regions of candidate genes for dominant and recessive ataxias (Tables 1 and 2). The massive sequencing of genes, is performed on a system Personal Genome Machine (PGM) Ion Torrent (Life Technologies), a device based on a technology fast, simple, very efficient in terms of cost and productivity. To determine the type of chip that you need to use (314, 314, 318), it is calculate the theoretical depth of sequencing required in this experiment. We chose the chip 318 that has a capacity of 800 Mb sequence and sequencable region of this experiment is 715 Kb in size by the patient, obtaining a theoretical depth of 1118X (800/0.715). In this experiment, 5 samples can be loaded per chip, resulting in a theoretical depth of 223X (1118/5). An entire experiment is running 10 templates and 2, 318 sequencing chips. In sequencing using the Ion PGM 400™ Sequencing Kit for sequencing fragments of 400 bp average size and 318 Chip Ion v2 Kit.

2) Analysis of mitochondrial DNA Mitochondria are extracted from blood cells by differential centrifugation and later on DNA extraction with Quiagen Midi Extraction kit. The workflow massive sequencing of mitochondrial DNA from each patient includes two phases: the generation of libraries and sequencing.

To generate the library we use of 100 ng of DNA extracted from each patient. The protocol consists of four phases: 1 - mitochondrial DNA digestion each patient using the Ion Xpress ™ Plus Fragment Library Kit, 2 - ligation of the adapters ends repair and hybridization with the bar codes using the Ion Xpress Barcode Adaptors 1 - 16 kit (Life Technology), 3 - size selection of DNA fragments using E-Gel ® SizeSelect ™ 2% Agarose, and 4 - PCR amplification of DNA targets using Platinum PCR SuperMix High Fidelity kit.

The massive sequencing of genes is performed in a system Personal Genome Machine (PGM) Ion Torrent (Life Technologies). To generate the template we use the Ion OneTouch ™ Template Kit 200 v2DL, starting from 20 ul of pooled libraries each patient to a molarity of 18 pM. This phase consists of an emulsion PCR in Ion One Touch System and an enrichment of the sample in the Enrichment System.

Once generated, the template quality control is done with the Ion Sphere ™ Quality Control Kit 2.0 Qubit fluorometer (Invitrogen).

To calculate the theoretical depth sequencing necessary in this experiment, we use the 316 chip since it has a capacity of 200 Mb sequence and the region of this experiment is ~ 16 kb (mitochondrial DNA) per patient, obtaining a theoretical depth 12500X. Therefore, in this experiment can be loaded 12 samples per chip, resulting in a theoretical depth of 1000X. In sequencing using the Ion PGM ™ 200 Sequencing Kit for sequencing fragments of 200 bp average size and the chip 316

Part 2: Study of mitochondrial function

1. Obtaining cultured fibroblasts The fibroblast cultures will be obtained from skin biopsies from patients with different types of ataxia, and age-matched control subjects using the methodology previously described (Johnson et al., 1990).

Briefly, skin explants incubated at 37 ° C in Eagle's minimal essential medium (MEM) supplemented



with 10% fetal bovine serum, nonessential amino acids and antibiotics. When cells have been grown (about two weeks), they are washed with sterile PBS and 2ml of trypsine. Once dispersed, they will be grown in Petri dishes of 100-mm using the same culture medium as described above until reaching confluence. The purity of fibroblast cultures is checked by vimentin expression determined by immunocytochemistry. Before starting the various treatments, cultures will be deprived of fetal bovine serum reducing its concentration at 0.1% for 24 or 72 hours.

2. Study of mitochondrial function and generation of free radicals (ROS) Mitochondrial membrane potential will be determined in cultured fibroblasts by flow cytometry (FACS).

For this purpose, we will use commercial kits Invitrogen that allow the identification of three specific mitochondrial markers: 1) functional mitochondria (Mitotracker deep red) 2) Total mitochondria (Mitotracker green), and 3) ROS generating mitochondria (MitoSOX ). The determinations will be carried out in cultured fibroblasts at baseline, and in a situation of oxidative stress induced by inhibiting the activity of complex I, II and III of the respiratory chain rotenone (10 uM), threnoyltryfluoacetona (TTFA, 10 uM) and antimycin A (40 ug / ml), respectively (Zhou et al., 2011). The activity of complex IV (COX) of the respiratory chain is determined as described in Rustin et al. (1994).

The determination of total glutathione levels and GSH will be done following the method described by Tietze (1969). Briefly, fibroblast cultures are washed in PBS and lysed in 100 ul of perchloric acid (PCA,

0.4N) for 30 min at 4 ◦ C, and centrifuged. The supernatants are neutralized with 4 volumes of NaH2PO4 (0.1 M), EDTA (5 mM), pH 7.5. GSH content is measured on an automatic plate reader by adding DTNB (0.6 mM), NADPH (0.2 mM) and glutathione reductase (1 U). The reaction is monitored at 412 nm for 6 minutes. In addition, glutathione determinations will be made by HPLC using the methodology described previously (Rodriguez-Martin et al., 2000).

3. Quantifying mitophagy Mitophagy rate at different culture conditions will be studied using confocal microscopy assessing the percentage of total mitochondria (Mitotracker green-positive) that colocalize with associated protein LC3 autophagy. In parallel, we will determine by Western blotting the expression of beclin 1 and ATG5.

4. Inflamasomas formation and caspase-1 activity To assess whether in fibroblasts from patients with ataxia activation induces ROS NLRP3/caspasa-1 we will study NLRP3 cellular localization by immunocytochemistry and active caspase-1 by detecting its irreversible inhibitor YVAD (biotinyl -YVAD-CMK, AnaSpec) previously added to the cultures (Herranz et al., 2012). In parallel, we will determine the rate of mitochondria producing ROS (MitoSOX) relative to caspase-1 activity by FACS.

5. Rating cell death in cultured human fibroblasts To determine DNA fragmentation characteristic of apoptosis in the cultures we will use TUNEL technique (Lopez-Toledano et al., 2004). Furthermore, we will consider the fibroblast cell cycle, and the molecules involved in cell death (Fas, FasL, Bcl-2, p53, etc.) using FACS (Sanchez Torres and Vargas, 2003). Caspase-3 and caspase-9 levels we will measure by Western blot on cultures. Necrosis rate in the cultures will be determined by assessing the lactate dehydrogenase activity (LDH).

Data analysis Data analysis will be performed at our center from basic bioinformatics (alignment detection, variants) to the filtering and selection of variants obtained clinic. The sequencing data is aligned to the reference genome UCSC hg19 in nuclear genes and mitochondrial DNA is aligned to the reference genome revised Cambridge Reference Sequence (rCRS) (GenBank accession number NC_012920) using the alignment algorithm Burrows-Wheeler Aligner (BWA). Variants are called with the Genome Analysis Toolkit (GATK) and annotated with Annovar software. Immunocytochemical analysis will be conducted using a NIKON confocal microscope coupled to C1-plus NIS program. The results will be expressed as the mean ± SEM of four independent experiments per patient, performed in triplicate or quadruplicate. The statistical analysis for immunocytochemical and biochemical studies will be performed using the Student t test or by one-way ANOVA followed by Newman-Keuls test for multiple comparisons. Differences are considered significant at p 0.05.



-Plan de trabajo y cronograma (máximo 1 página) The project will take place in the Hospital Ramón y Cajal.

The visit of patients, sampling and informed consent will be done in the Neurology Outpatient Clinic DNA extraction and sequencing of candidate genes and mitochondrial genome will be held at the Unidad Central de Apoyo para Estudios Genómicos del IRYCIS-Hospital Ramón y Cajal.

The study of mitochondrial function will be in Servicio de Neurobiología-Investigación del IRYCISHospital Ramón y Cajal.

The stages of the study are shown in Figure Annexed. A graphic timeline is also included.

No additional staff is requested in the project. There are 2 laboratory technicians to carry out the study.


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