Research Article Journal of Translational Science J Transl Sci, 2018 doi: 10.15761/JTS.1000247 Volume 5: 1-13 ISSN: 2059-268X Revealing the involvement of miR-376a, miR-432 and miR-451a in infantile ascending hereditary spastic paralysis by microRNA profling in iPSCs Stefania Marcuzzo 1 , Silvia Bonanno 1,2 *, Claudia Barzago 1 , Sara D’Alessandro 1,3 , Paola Cavalcante 1 , Barbara Galbardi 1,4 , Claudia Malacarne 1,5 , Michela Taiana 6 , Monica Nizzardo 6 , Stefania Corti 6 , Giulia Bechi 7 , Antonio Gambardella 8 , Silvana Franceschetti 7 , Massimo Mantegazza 7,9 , Giovanna Zorzi 10 , Renato Mantegazza 1 and Pia Bernasconi 1 1 Neurology IV – Neuroimmunology and Neuromuscular Diseases Unit, Fondazione Istituto Neurologico “Carlo Besta”, 20133 Milan, Italy 2 PhD program in Neuroscience, University of Milano Bicocca, 20126 Milan, Italy 3 Present address: ASST Papa Giovanni XXIII, 24127 Bergamo, Italy 4 Present address: San Raffaele Hospital, 20132 Milan, Italy 5 Department of Biotechnologies and Biosciences, University of Milano Bicocca, 20126 Milan, Italy 6 Dino Ferrari Center, Neuroscience Section, Department of Pathophysiology and Transplantation (DEPT), Neurology Unit, IRCCS Foundation Ca' Granda Ospedale Maggiore Policlinico, University of Milan, 20122, Milan, Italy 7 Department of Neurophysiopathology, Fondazione Istituto Neurologico “Carlo Besta”, 20133 Milan, Italy 8 Department of Medical and Surgical Sciences, Institute of Neurology, University Magna Græcia, 88100 Catanzaro, Italy 9 Institute of Molecular and Cellular Pharmacology, LabEx ICST, CNRS UMR7275 and University of Nice-Sophia Antipolis, 06560 Valbonne-Sophia Antipolis, France 10 Child Neurology Unit, Fondazione Istituto Neurologico “Carlo Besta”, 20133 Milan, Italy Abstract Infantile-onset ascending hereditary spastic paralysis (IAHSP) is a rare, early onset, autosomal recessive motor neuron disease characterized by progressive weakness and spasticity. Several mutations in the alsin 2 gene (ALS2) have been described in IAHSP patients; however, a relevant subset of patients is ALS2 mutation- negative, and pathogenic events causing the disease are unknown. Te present study aimed at better understanding the molecular mechanisms underlying motor neuron loss in IAHSP patients by identifying microRNAs (miRNAs) potentially implicated in neuronal diferentiation. Using the human induced pluripotent stem cell (iPSC) technology, we developed a patient-specifc in vitro cellular model and performed miRNome profling in fbroblasts, iPSCs and iPSCs-derived neurons obtained from an ALS2 mutation-negative IAHSP patient and a healthy control. Te selected diferentially expressed miRNAs were also analyzed in fbroblasts, iPSCs and iPSCs-derived neurons from two patients afected by other motor neuron diseases, two patients with other neurological disease, and three healthy controls. We found that miR-376a, miR-432 and miR-451a expression was altered in cell cultures obtained from the IAHSP patient compared to the other patients and controls. In addition, the hierarchical clustering analysis revealed that miR-451a was diferentially expressed in fbroblasts and iPSCs, whereas miR-376a and miR- 432 in neuronal cells. Tese results, together with the miRNA/mRNA target analysis, were indicative of a signifcant involvement of miR-451a in stem cell biology processes, and of miR-376a and miR-432 in the establishment of the neuronal phenotype. Our overall fndings identifed miR-376a, miR-432 and miR-451a as molecules involved in neuronal diferentiation, and potentially in IAHSP pathogenesis, which could provide cues for future development of patient-specifc miRNA- based therapeutic strategies for IAHSP or other motor neuron diseases. Introduction Motor neuron diseases (MNDs) are a heterogeneous group of neurological disorders characterized by a progressive and selective loss of motor neurons in the motor cortex, brainstem, and spinal cord [1], variably afected depending on the specifc MND [2]. Te spectrum of MNDs extends from childhood to adulthood and the variety of clinical manifestations refects the numerous genetic alterations identifed in patients with familial and sporadic MND [3,4]. MNDs with juvenile-onset include the infantile-onset ascending hereditary spastic paralysis (IAHSP), a rare fatal disorder characterized by weakness and spasticity due to a selective severe degeneration of the pyramidal tract, with onset at lower limbs within the frst two years of life, and slowly progressive ascending evolution [5]. Familial *Correspondence to: Silvia Bonanno, Neurology IV – Neuroimmunology and Neuromuscular Diseases Unit Fondazione Istituto Neurologico “Carlo Besta”, Via Celoria 11, 20133 Milan, Italy, E-mail: silvia.bonanno@istituto-besta.it Key words: alsin, induced pluripotent stem cells, infantile-onset ascending hereditary spastic paralysis, microRNAs, motor neuron disease Received: May 01, 2018; Accepted: May 16, 2018; Published: May 19, 2018 occurrence of the disease suggests an autosomal recessive inheritance, and several mutations in alsin 2 gene (ALS2) have been described [5,6]. Tis gene encodes for a guanine nucleotide exchange factor abundantly expressed in motor neurons, and its mutations are also responsible for juvenile primary lateral sclerosis, that typically presents later in life, and