MOLECULAR MECHANISMS AND THERAPEUTIC APPROACHES IN MN DISEASES

Chapter 1 Spinal and Bulbar Muscular Atrophy (SBMA or Kennedy disease) is an inherited X-linked motor neuron disease characterized by loss of lower motor neurons located in the spinal cord and brain stem resulting in bulbar and limb muscle atrophy. SBMA is due to an expansion of a CAG triplet repeat sequence in the androgen receptor (AR) gene, which codes for an elongated polyglutamine (polyQ) tract of AR protein. Sequence longer than 38Q are present in SBMA patients. It is known that the elongated polyQ tract confers a neurotoxic gain-of-function to AR, probably causing aberrant conformations of the protein, leading to protein aggregation and cell toxicity. ARpolyQ toxicity is exerted only after the interaction with androgens which induce conformational changes and AR nuclear translocation. In fact, several evidences shows that misfolded ARpolyQ exerts most of its toxicity in the nucleus. Our previous in vitro works indicate that prevention of ARpolyQ nuclear translocation and enhancement of the autophagic process could be possible approaches to counteract ARpolyQ neurotoxicity. Here, we tested in vivo a combined treatment with: a) bicalutamide, an FDA-approved antiandrogen that reduces the rate of ARpolyQ nuclear translocation, and b) trehalose, a disaccharide able to enhance ARpolyQ degradation stimulating autophagy. We took advantage of a knock in mouse model of the Kennedy disease expressing an AR with 113Q, and showing many features of the pathology. Single treatment with bicalutamide or trehalose increased AR113Q mouse survival, and the combined treatment had a synergic activity leading to a further increase in survival. Oral trehalose treatment improved mouse motor behavior measured by rotarod, without affecting the hind limb muscle force. Furthermore, bicalutamide or the combined treatment improved both muscle force and motor coordination behavior. Molecular analyses of gastrocnemius muscle indicated an increased expression of PGC1alpha mRNA, a critical regulator of mitochondrial biogenesis. Our results indicate that the combined bicalutamide and trehalose treatment could be a novel approach to counteract ARpolyQ toxicity in vivo. Chapter 2 Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disease caused by the dysfunction and loss of upper and lower motor neurons and of their target muscle cells. There is evidence showing age related gender differences in human patients and mouse models. ALS is more common in male than female, and gender influences the clinical features of the disease. Patients suffering from ALS show increased levels of transforming growth factor beta 1 (TGFβ1) in the serum and cerebrospinal fluid. Here, we tested whether altered TGFβ1 levels in ALS are associated with aberrant expression of TGFβ1 and its related signaling molecules, i.e. TGFβ-Receptor type II (TGFβ-RII) and Smads, in the spinal cord and skeletal muscle of a transgenic ALS mouse model, considering gender and disease progression. At pre-symptomatic (PS) stage TGFβ1 expression was reduced in the spinal cord of both male and female mice, while it was induced selectively in males in the skeletal muscle. At symptomatic (S) stage TGFβ1 was robustly induced both in the spinal cord and muscle of male and female mice. Similar findings were obtained in muscle biopsies derived from sporadic ALS patients. Tgfβ-RII mRNA levels were induced only in the spinal cord without gender differences. Smad2 and Smad4 gene expression were decreased in spinal cord and muscle both in male and female mice, while Smad2 protein levels were increased selectively in male muscle. SMAD3 expression was increased only in muscle. Expression of genes controlled by TGFβ1 in muscle, such as Pax7, Collagen1β1 and Fibronectin, was reduced both in male and female ALS mice at S stage independently of denervation. Our results suggest that, the expression of TGFβ1 is increased in ALS tissues, but its signaling pathway regulating the expression of downstream target genes is altered. These observations highlight TGFβ1 and its signaling as a novel therapeutic target for ALS. Furthermore, they strengthen the idea that skeletal muscle has a key role in ALS.