Mechanistic studies of ALS-causative mutations and RNP-focussed drug discovery using in vitro reconstitution of RNP complexes

Amyotrophic lateral sclerosis (ALS), the most common form of motor neuron disease (also known as Lou Gehrig disease), is a fatal disease of the nervous system. ALS can be inherited or have no known genetic cause, yet all patients will develop a very similar condition. In all patents, nervous cells called motor neurons, mainly located in the spinal cord, become affected and eventually die. This leads to loss of support for muscles, causing their wasting, weakness and paralysis. Two drugs currently approved for ALS extend the lifespan by a few months and are not able to cure patients. Hence, there is a clear need for new drugs to treat ALS.
ALS is characterised by abnormal metabolism of the molecular complexes in neurons composed of proteins and ribonucleic acids (RNAs), called ribonucleoprotein (RNP) complexes. Mutant, abnormally localised, or otherwise dysregulated proteins gain abnormal RNA-binding properties and structurally perturb RNP complexes leading to ALS pathology. Similarly, disease-relevant RNAs can establish unlicensed RNA-protein interactions with profound consequences for cellular RNA metabolism. We found that a specific RNA called NEAT1_2 that induces formation of RNP complexes known as paraspeckles might be an important player in ALS pathogenesis. Although absent from the neurons of healthy individuals, this RNA becomes accumulated in ALS motor neurons. In addition, many proteins that regulate NEAT1_2/paraspeckles are affected by ALS mutations. We believe it has a protective effect and helps neurons survive. However currently no NEAT1_2 targeted drugs are available.
Traditional drug discovery efforts have focussed on the protein but it has recently become possible to identify drugs which act on RNA and its complexes with proteins. More specifically, drugs which affect RNPs are currently being evaluated in patients with the fatal childhood disease spinal muscular atrophy (SMA). Recently, we have developed a system that will allow us assembling complexes similar in composition to NEAT1_2/paraspeckles, by attaching a portion of NEAT1_2 RNA to micro-beads in a multi-well test plate. This system can be used for rapid analysis of protein binding to these complexes using an ultrafast microscopic imaging analysis. In the current proposal, we aim to better understand the link between NEAT1_2/paraspeckles and ALS, and find novel small molecules that can modulate these complexes with a potential to be used in ALS research and drug discovery. Further, we will modify this system to make it suitable for finding small molecules for any ALS-relevant RNA molecule using a major ALS-causative RNA produced from C9ORF72 gene.
Our teams have significant background in RNA biology, ALS pathobiology and drug discovery as well as all the relevant laboratory tools. Therefore we are uniquely placed to use the above state-of-the-art approach we developed, to translate recent breakthroughs in our understanding of ALS pathophysiology into therapies development and ultimately, patient impact.

Amyotrophic lateral sclerosis (ALS) is an incurable neuromuscular disease and a significant unmet medical need. A significant proportion of ALS cases is caused by mutations in RNA-binding proteins (RBPs) and/or involves their functional deregulation. In particular, it is well-established that dysregulation of RNA-RBP complexes (RNPs) triggers the downstream cascade of the pathology in many ALS subtypes. We demonstrated that the stress-induced isoform of the long non-coding RNA NEAT1, NEAT1_2, that nucleates RNP granules paraspeckles, is upregulated in the surviving motor neurons in the spinal cord in all major ALS subtypes. Moreover, many protein components of NEAT1_2/paraspeckle complexes are affected by ALS-causative mutations. Positive modulation of NEAT1_2 in ALS motor neurons is expected to exert a neuroprotective effect and hence provide a therapeutic benefit. However currently no specific NEAT1_2/paraspeckle targeted therapies are available.
We have developed a scalable imaging-based platform for reconstitution of ALS-relevant RNP complexes in vitro and their quantitative analysis - "RNPs-on-beads". When functionalised with a structural motif of NEAT1_2 RNA, it can be successfully used for the reconstitution of paraspeckle-like complexes on beads ("PoBs"). We propose to validate this platform as a tool for rapid analysis of the effect of ALS-causative mutations on RNP complexes and for identification of small molecules - modulators of NEAT1_2/paraspeckles. In addition, we will test the principle suitability of our platform for RNA-focussed drug discovery aimed at other ALS-linked RNAs using C9ORF72 RNA with expanded hexanucleotide repeats and known mediators its neurotoxicity - SRSF1 and eIF4A proteins.
In summary, results of this project will lead to a better understanding of the role NEAT1_2/paraspeckles in ALS, identify new tool molecules for ALS research, and create a framework for RNP-complex focussed small molecule drug discovery in ALS.