Anagrafe della ricerca

Spinal and bulbar muscular atrophy (SBMA) is an X-linked late-onset and progressive neuromuscular disease caused by polyglutamine (polyQ) expansions in the androgen receptor (AR). SBMA fully manifests in males because SBMA is an androgen-dependent disease. Binding of androgens to polyQ-expanded AR triggers SBMA through the combination of toxic gain-of-function (GOF) and loss-of-function (LOF) mechanisms that lead to motor neuron loss, muscle atrophy, mild androgen insensitivity syndrome, and metabolic syndrome. Inactive AR resides in the cytosol. Androgen binding results in nuclear translocation, DNA binding, recruitment of transcription co-regulators (co-activators and co-repressors), and regulation of expression of the androgenresponsive genes. In the previous AFM funding cycle (application 22221), we showed that two specific AR coactivators, namely PRMT6 and LSD1, are aberrantly expressed in disease and contribute to polyQ-AR GOF. Our objective is to contribute to developing a therapy for SBMA patients. Here we will test the hypothesis that targeting PRMT6 and LSD1 attenuates the toxic GOF of polyQ-AR without enhancing the LOF. This hypothesis is based on our preliminary data, showing that PRMT6 enhances polyQ-AR transactivation and toxicity through direct arginine methylation of AR; LSD1 is a novel co-activator of polyQ-AR that targets specific lysine residues of AR; PRMT6 and LSD1 cooperatively enhance polyQ-AR transactivation; Knockdown of both LSD1 and PRMT6 fully suppresses polyQ-AR-induced toxicity in flies, and it attenuates phenotype in SBMA mice; and LSD1 and PRMT6 are upregulated specifically in SBMA skeletal muscle starting at the presymptomatic stage in transgenic SBMA, as well as knock-in mice and patient-derived muscle biopsies. To achieve our objective, we built up a multidisciplinary research group, which will benefit from complementary expertise, including in vivo modeling and preclinical assessment (Maria Pennuto, University of Padova, Italy), molecular/cellular biology (Manuela Basso, University of Trento, Italy), neurology (Gianni Sorarù, University of Padova, Italy), and nanoparticle technology (Valeria Chiono, Politecnico of Turin and Barbara Stella, University of Turin). To test our hypothesis, we will pursue these specific aims: Specific Aim 1: To elucidate the mechanism that leads to LSD1 and PRMT6 overexpression in SBMA muscle. We will perform a genetic LOF screening to identify the molecular players responsible for PRMT6 and LSD1 induction in the SBMA muscle. We will take advantage of a CRISPR Cas9 genetic library directed towards transcription factors in HEK293T cells. Specific Aim 2: To dissect how LSD1-mediated K demethylation enhances polyQ-AR function and toxicity. We will test the working hypothesis that LSD1 modification of specific lysines previously identified by mass spectrometry modifies AR response to androgens leading to polyQ-expanded toxic GOF. We will test this hypothesis in SBMA myotubes derived from mice (C2C12) and patients, as well as in SBMA mice. Specific Aim 3: To assess the efficacy of nanoparticle (NP)-mediated delivery of Lsd1 and Prmt6-targeting artificial miRNAs (patent) in SBMA mice. Polymeric hybrid nanoparticles will be tested on SBMA myotubes derived from mice (C2C12) and patients, as well as in SBMA mice. In vitro cell tests will assess NP biocompatibility, cell uptake, and transfection efficiency. In mouse models, we will assess the effect on survival, motor function, histopathology, and biochemistry. There is no cure to arrest or delay SBMA onset and progression. From Aim 1, we expect to identify novel molecular targets responsible for LSD1 and PRMT6 overexpression in SBMA muscle. From Aim 2, we will assess the effect of K demethylation on AR structure and function in a physiological and pathological context. From Aim 3 we will apply cutting-edge technology for translating our patented amiRs targeting LSD1 and PRMT6 and patented nanoparticles to the clinics.