Transcriptional and Micro-RNA Signature Profiling of Human Amyotrophic Lateral Sclerosis Muscles: a tool to identify pathogenic molecular networks and diagnostic biomarkers

Project location: ITALY, Rome
Project start date: September 2012 - Project end date: August 2014
Project number: 2012-004
Beneficiary: Università Cattolica del Sacro Cuore

Amyotrophic lateral sclerosis is a neurodegenerative disease characterised by progressive muscular paralysis reflecting degeneration of motor neurones, with an unclear etiopathogenesis and with a median survival time of about 24 months1. The vast majority of cases occurs as sporadic forms and tend to cluster in selected high risk population groups. The need for diagnostic and therapeutic tools for ALS management has been stimulating a large amount of basic and clinical researches worldwide (nearly 13000 scientific articles, 71 during the first month of 2011). An increasing number of new environmental risk factors is being recognized in the ALS epidemiological literature. This suggested that ALS represent an emerging public health issue.

This study is aimed at unraveling the molecular mechanisms occurring at the neuromuscular junction (NMJ) during ALS pathogenesis, on the basis of recent findings supporting the pressing hypothesis of muscle fibers as crucial site for basic pathogenic events 2.The study will employ microarray technology to analyze the genome-wide gene expression and micro RNA profiles in muscle specimens from sporadic ALS patients.

The project activities will be structured as follows.
- patients enrollment and sample collection
All consecutive sporadic ALS patients (predictably 25-30 patients) observed at the Department of Neurology of the UCSC-Gemelli University Hospital (Rome, IT) will be enrolled in the study, upon obtaining a written informed consent. The eligible subjects will be patients with early stage ALS. Age- and sex-matched patients referring to the same clinical department for suspect metabolic disorder not confirmed by biochemical and histopathological studies, will be recruited as un-affected control individuals (predictably 25-30 control patients). Diagnosis of ALS will be made according to revised el escorial/arlie house criteria 1. Exhaustive anamnestic and clinical information will be recorded for each individual enrolled in the project.
The tissue specimens will be immediately stored at -80°c in order to preserve RNA molecules from degradation.
- tissue processing
Each specimens will be divided into two separate fraction, one for molecular analysis (mRNA and miRNA isolation) and one to be used in cryosections for histology and immunohistochemical analysis (IHC).
- molecular analysis
The molecular profiling will be performed using the Genechip Microarray technology by Affymetrix (Santa Clara, CA, USA) to simultaneously measure the levels of miRNA and mRNA in the muscle specimens of selected ALS patients and age-matched control.
For this purpose, total RNA and miRNA will be isolated from the muscle specimens of each patient, following tissue homogenization. mRNA and miRNA will be processed and hybridized to the Affymetrix human gene 1.0 st array and to the Genechip miRNA arrays, respectively, according to the protocols provided by the manufacturer.Hybridizations will be carried out independently for each condition, according to MIAME guidelines. Gene expression will undergo statistical data analysis using ad hoc algorithms implemented in bioinformatics data analysis software packages. The results will be categorized according to their biological functions, indicated by the Gene Ontology Consortium (www.geneontology.org). Finally, the biological interpretation of the list of differentially expressed genes will be carried out using Ingenuity Pathway Analysis (IPA) (Ingenuity® Systems, www.ingenuity.com), enabling the generation of functional networks and canonical pathways that connect the differentially expressed genes. Moreover all miRNA and mRNA array data will be then imported and normalized together and analyzed by means of appropriate software, which will allow combining miRNA profiles with differential expressed mRNAs in the same patients and detect ALS-specific significant miRNAs and corresponding target genes.
- Morphological analysis
Separate aliquots of each muscle specimen will be used for histology. Cryosections will processed using standard methods for immunohistochemical analysis, in order to detect functionally significant proteins resulting from the microarray analysis in the target tissue, using confocal microscopy. This morphological assessment will also enable detecting possible differential protein expression in differently shaped/sized myofibers in each section. Moreover. Laser Capture Microdissection (LCM) technology will be employed to analyze the expression of selected mRNA and miRNA, resulting from microarray profiling, in different myofiber types.

The project activities will be performed during two years (start date: September 1st 2012, end date: August 31st 2014), according to the following timetable:
Months 1-16. patients enrolment, specimens collection;
Months 3-16: RNA and miRNA isolation and quality control, from muscle biopsies and LCM samples;
Months 17-20. Microarray analysis, data analysis and biological interpretation;
Months 21-24: data validation by qPCR using RNA and miRNA from muscles and LCM samples; morphological analysis by IHC.

This project received a grant from the Nando Peretti Foundation. It aims at unraveling at the genome-wide level the transcriptional regulation in human muscle during the early stages of ALS, prior to complete acute denervation, focusing on the molecular basis of the retrograde NMJ signaling. This would enable the identification of novel molecular networks and delineating the basis for the final demonstration of a crucial involvement of muscle in ALS pathogenesis.
Main objectives will be as follows:
- identify all human genes with altered expression in ALS human muscles;
- identify the regulation network operated by miRNAs on those genes;
- identify a panel of deregulated miRNAs and corresponding target genes
in the same target tissues, as possible molecular players of early muscle denervation and potential targets for molecular therapy. These would represent also potential biomarkers for ALS diagnosis and monitoring.
Data resulting from this study would lay the foundation for following studies focusing on new molecular targets involved in ALS.