Assessing the pathogenetic role of p53 activation and tRNA dysregulation to understand the pathogenesis and identify molecular therapeutics targets for Spinal Muscular Atrophy with Respiratory Distress type 1 (SMARD1)

Cariplo Foundation assigned 250.000 euro to support this project, on the scientific responsibility of Doct. Monica Nizzardo, young researcher of “Centro Dino Ferrari” at University of Milan.

This is a two-year project, started in october 2016, involving young researchers team and Neurologist equipe, including Prof. Corti and Prof. Comi at IRCCS Ca’ Granda Foundation, Ospedale Maggiore Policlinico, Milan.

Summary

-DISEASE

SMARD1 is characterized by motor neuron (MNs) degeneration, muscular atrophy, weakness and respiratory impairment which directly lead to death in childhood. Respiratory distress usually occurs within the first year of life, initially with distal, and later generalized, muscle weakness leading to early degeneration of spinal cord MNs and muscular atrophy. To date, no effective cure is available for SMARD1 and few research programs focus on analyzing the pathological mechanisms underlying the pathology. The disease results from loss of function mutations in the gene encoding immunoglobulin μ-binding protein 2 (IGHMBP2), which seems to be involved in RNA processing regulation, even if its specific functions and the reasons for selective MN death are still largely unknown. We have already reported that MNs degeneration was completely rescued by transgenic expression of wild-type IGHMBP2 both in vitro and in vivo (Link).

Recent studies showed that defects in RNA metabolic pathways are responsible for neurodegenerative disorders. Indeed, several mutations in genes involved in pre-RNA, transfer RNA metabolism, RNA stability and protein translation have been linked to motor neuron disorders.

-PROJECT

This project aims to support neurological research attempting to understand the molecular basis of MN degeneration. We will study molecular pathways involved in SMARD1 pathogenesis in order to identify novel therapeutic targets. In particular, we will focus on assessing the pathogenic role of transfer RNA and p53 pathways in SMARD1 to discover potential targets suitable for gene therapy or pharmacological treatment. Both in vitro and in vivo SMARD1 disease models, well established in our Lab, will be used. As in vivo model we will use the neuromuscular degeneration mouse model, which harbours a spontaneous homozygous mutation in the Ighmbp2 gene and develops a phenotype resembling the human disease. However, until now, since all preclinical studies on SMARD1 have been performed exclusively in the mouse model, in this project we will use a human model exploiting our experience in the stem cell field, particularly in induced pluripotent stem cells (iPSCs). Indeed, we have already successfully generated iPSCs from patients and unaffected subjects, demonstrating that these cells can differentiate into MNs which are the disease-affected cells.

We are confident that this project will have a significant impact on the neuroscience field by providing better knowledge of disease pathogenesis, with the possibility of identifying new therapeutic targets and leading to the development of novel therapeutic approaches for SMARD1.

Experimental plan

The objective of our project is to investigate a mechanistic link between transfer RNAs (tRNAs), p53 pathways deregulation and the progressive loss of MNs both in our in vitro and in vivo disease models and to identify potential related therapeutic targets.

The project will be developed as follow:

AIM 1: Evaluation of tRNA pattern dysregulation in vitro

TRNAs are the most abundant small noncoding RNAs in cells which arrive at maturity and functionality after a series of splicing events particularly crucial in neurons and MNs. In pathological conditions, it is known that the alteration in tRNA processing leads to tRNA-derived fragments accumulation, even if its contribution to the pathogenesis remain to be assessed. Moreover, biochemical investigations showed that IGHMBP2 associates physically with tRNAs and other components of the translational machinery. Here, we will analyze the presence and the pathogenetic role of tRNA fragments accumulation in SMARD1 iPSC-derived MNs. Finally, we will investigate whether these alterations are rescued in MNs derived from iPSCs genetically corrected with wild-type IGHMBP2 gene.

 AIM 2: Assess the role of p53 activation in vitro

The related accumulation of tRNA fragments derived from aberrant tRNA processing sensitize cells to oxidative stress linked to p53 protein pathway and to the resulting cell death. Indeed, p53 is widely involved in neuronal death in response to different forms of acute insults.

Here, we aim to investigate the involvement of p53 protein in selective MNs death, assaying p53 pathway activation in SMARD1 MNs. Furthermore, we will investigate whether these alterations are rescued in MNs derived from iPSCs genetically corrected with wild-type IGHMBP2 gene. Finally, we will modulate p53 in vitro to evaluate it as potential therapeutic target.

AIM 3: Investigation on molecular targets identifies in AIM 1 and 2 as potential therapeutic targets in vivo

The most promising molecular targets among tRNA and p53 pathways will be tested in vivo.

In particular, we will overexpress or downregulate in the nmd mice the pathways found implicated in the disease pathogenesis in AIM 1 and 2. Any modification of the pathological phenotype after the treatment will be assessed. This aim will lead to the validation of novel potential therapeutic targets for SMARD1.

Dissemination plan

One of the main goal of the Research supported by Cariplo is the interaction between science world and civil society. Therefore, our project includes a defined and detailed dissemination program that is described in this session.

Our dissemination plan is designed according to Responsible Research and Innovation Principles (RRI). During the course of the project we aim to engage the public society establishing an exchange relationship, to increase scientific results availability, to provide formal and informal scientific education for young people and to take into account ethic matter in every steps of our research.

Our results will be submitted to internationally peer-reviewed journals, whenever possible we will publish under open access license in order to further increase the dissemination and the use of scientific results by all the scientific audience.

Since we will use human cells derived from patients, our lab obtained ethical approval and patient signed informed Consents related to human biological samples use for research. We will fully validate our strategy first in cell culture, and subsequently in our murine models. We will not use animal models until the in vitro data are highly successful. The in vivo part of the project has been already approved from the specific university committee and from Ministry of Health.

We identified three specific target: scientific community, patient and their families, and civil society (in particular children and families). The activities, the tools and the language will be defined in order to be appropriate for the chosen specific target.

a) Civil society

The aim is to fill the gap between science and society making research more concrete, simpler and available and will be focused on involving society in the real life of young researchers. In particular, our activities will be addressed to families with the participation to “Notte dei ricercatori” and young people creating a facebook page. We will attend to “Giornata delle Malattie Rare 2018” with a special activity planned for children of primary and pre-school.

To make our project and deliverables available, we will publish online on the Web Portal of Centro Dino Ferrari, a University Research Centre that already supports us.

b) Patient’s families

First we want to make them aware about the existence of the funded project. The interaction with patients and their family aims to establish a continuous dialog either for planning activities together or for discussing needs and obstacles.

Since SMARD1 is a rare disorder, one of the issues is to obtain biological material from patients. Thanks to the nation and international patients that we will reach with the dissemination program we expect to overcome this issues. During Famiglie SMA meetings and exchanges, we will present the scientific progress of our research and of other international groups and we will obtain a feedback from patients and their families to collect useful information that can help us to modify the research and most importantly the dissemination plan according to their needs.

c) Scientist

The aim consists in sharing project’s results and integrating different knowledge to promote collaboration among organizations of the same research field. “Young researchers network” foundation and activities will be posted in the university of Milan homepage and public talks about the specific project updates within departmental seminar program will be organized. Project results will be published in international peer-reviewed journals (open access) and presented in national and international congresses. During scientific dissemination activities we expect to create new collaborations and to exploit the idea coming from Meeting and discussion to improve our ongoing project.

Overall we expect also to sensitize the whole community towards the existence of this rare disease and the missing of a cure and of a proper assistance, hopefully increasing fundraising for Research and Patients Assistance.

Timetable and activities planned for dissemination

Preliminary data-our publications on international peer reviewed journal about SMARD1 disease