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UNIVERSITÀ DEGLI STUDI DI MILANO Molecular Mechanisms and Therapeutic Approaches in MN Diseases

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UNIVERSITÀ DEGLI STUDI DI MILANO Molecular Mechanisms and Therapeutic Approaches in MN Diseases UNIVERSITÀ DEGLI STUDI DI MILANO Corso di Dottorato in Ricerca biomedica integrata XXXI CICLO Molecular mechanisms and therapeutic approaches in MN diseases Tutor: Prof. Angelo Poletti Co-tutor: Prof. Mariarita Galbiati Coordinatore del Dottorato: Prof.ssa Chiarella Sforza Tesi di Dottorato di: Marco MERONI Matr.n° R11236 Anno accademico 2017 – 2018 Contents Contents 2 Contents Contents Contents .................................................................................................................................................. 2 Introduction ........................................................................................................................................ 6 Motor neuron diseases (MNDs) ...................................................................................................... 6 Chapter 1 ................................................................................................................................................. 9 Introduction ...................................................................................................................................... 10 Kennedy’s disease ......................................................................................................................... 10 Pathogenesis ................................................................................................................................. 10 Androgen receptor ........................................................................................................................ 11 Phenotype and symptoms ............................................................................................................ 15 Muscle and non-cell autonomous disease.................................................................................... 17 Diagnosis ....................................................................................................................................... 18 Therapy and symptomatic treatment ........................................................................................... 19 Experimental treatments .............................................................................................................. 19 Therapeutic potential of autophagy ............................................................................................. 20 Trehalose ....................................................................................................................................... 24 Spinal and bulbar muscular atrophy and trehalose ...................................................................... 29 Mouse models of Kennedy disease .............................................................................................. 29 Aim .................................................................................................................................................... 32 Results ............................................................................................................................................... 35 Model characterization ................................................................................................................. 35 Treatment validation .................................................................................................................... 39 Effect of trehalose or/and bicalutamide treatments on SBMA mice ........................................... 41 Morphological and biochemical analysis of skeletal muscle ........................................................ 44 ...................................................................................................................................................... 45 Analysis of the autophagic pathway in KI AR113Q mice .............................................................. 47 Discussion.......................................................................................................................................... 50 Chapter 2 ............................................................................................................................................... 53 Introduction ...................................................................................................................................... 54 Amyotrophic lateral sclerosis ........................................................................................................ 54 Pathogenesis ................................................................................................................................. 54 Phenotype and symptoms ............................................................................................................ 58 Transforming growth factor β ....................................................................................................... 59 TGFβ in motor neuron and muscle ............................................................................................... 60 TGFβ in SBMA ................................................................................................................................ 61 TGFβ in amyotrophic lateral sclerosis ........................................................................................... 61 3 Contents Aim .................................................................................................................................................... 64 Results ............................................................................................................................................... 67 TGFβ 1 gene expression ................................................................................................................ 67 TGFβ1 signaling ............................................................................................................................. 71 TGFβ 1 in ALS muscle .................................................................................................................... 75 Discussion.......................................................................................................................................... 79 Materials and methods ..................................................................................................................... 82 Bibliography ...................................................................................................................................... 90 4 Introduction Introduction 5 Introduction Introduction Motor neuron diseases (MNDs) Motor neuron diseases (MNDs) are a heterogeneous group of disorders characterized by death of motor neuron cells. MNDs occur in both adults and children. In children, MNDs are usually characterized by familial forms of the disease; symptoms can be present at birth or appear before the child learns to walk. In adults, MNDs occur more commonly in men than in women, with symptoms appearing generally after the age of 40 1. MNDs may be sporadic or familial and are clinically classified according to whether they involve upper or lower motor neurons, or both. Upper motor neuron involvement leads to positive neurological features including spasticity, brisk reflexes, clonus, and extensor plantar responses, as well as negative features such as weakness and loss. Spasticity is defined as the velocity-dependent increase in muscle tone as assessed by passive movement of the limbs. The term is used to define the stiffness and other features due to the damage of descending motor pathways in the central nervous system. This damage, frequently in the spinal cord, leads to abnormal hyper-excitability of the tonic stretch reflex. Consequently, the patient with spastic legs complains of walking difficulty, weakness, fatigue, and reduced exercise tolerance. They may also have cramps of the legs due to spontaneous clonus. Lower motor neuron loss, on the other hand, leads to muscle wasting with fasciculation, weakness, and reduced or abolished reflexes 1. Amyotrophic lateral sclerosis (ALS), also called Lou Gehrig's disease is a classical MND; it is a progressive fatal disorder that involve both upper and lower motor neurons. Symptoms usually appear first to the limbs, or swallowing muscles. Affected patients lose strength and the ability to move their arms and legs, and to hold the body upright. ALS is also characterized by other symptoms such as spasticity, spasms, muscle cramps, and fasciculations. At end stage of the disease muscles of the diaphragm and chest fail to function properly and patients lose the ability to breath without mechanical support. Usually ALS patients die from respiratory failure, within three to five years from the onset of symptoms. However, about 10 percent of affected individuals survive for ten or more years 2. Rarely ALS patient present symptoms that involve the person's personality, anyway some recent studies report evidence that some patient with ALS may develop cognitive problems involving word fluency, decision-making, and memory. ALS classical patient are people between 40 and 60 years of age, but also younger and older individuals can develop the disease. It is also known that men have a higher incidence than women. Familial forms of ALS represent only the 10 percent or less of ALS cases, with more than ten genes identified to date. 2. Progressive bulbar palsy (PBP), also called progressive bulbar atrophy, involves the brain stem, which is the bulb-shaped region containing lower motor neurons needed for swallowing, speaking, chewing, and other functions.
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