Targeting miR-155 and miR-125b: A Novel Approach to Developing ALS Therapies

Amyotrophic lateral sclerosis (ALS) is a severe disease which gets worse with time and causes the breakdown of specific type of brain cells, motor neurons in both brain and spinal cord. This leads to a gradual loss of muscle function and, eventually, death. Currently, the only FDA-approved drugs for ALS, Riluzole and Edaravone, provide only modest benefits, extending life by a few months at best. There are no effective therapies that directly address the underlying causes of the disease. Micro-RNAs (miRNAs) have been found increasingly implicated in ALS due to the dysregulation of these molecules observed in ALS spinal cord and motor neurons. For example, miR-155 and miR-125b are found at higher levels in ALS patients, where they contribute to the death of motor neurons and cause inflammation. Blocking these specific miRNAs in laboratory models of ALS has shown promise, protecting motor neurons and extending survival in mice. This suggests that targeting miRNAs could be a new way to treat ALS. Developing miRNA-based therapies faces challenges, especially with delivering synthetic miRNAs or inhibitors (called antagomirs) effectively inside the central nervous system. To overcome these obstacles, researchers are focusing on small molecules that can regulate miRNA activity by interacting with their unique structures. High-throughput screening methods are being used to identify such small molecules quickly. The proposed research focuses on finding inhibitors for miR-155 and miR-125b using a step-by-step process. 1. Primary Screening with Small Molecule Microarray (SMM): One hundred thousand of small molecules will be screened to identify those that interact with miR-155 and miR-125b. MiRNAs will be labelled with fluorescent tags to establish how small molecules affect their fluorescence, indicating a potential interaction. This approach allows rapid testing of many molecules and helps identify promising candidates for further study. 2. Secondary Screening for Validation whereby two methods will confirm the findings from the primary screening: Affinity Selection Mass Spectrometry (AS-MS): This method identifies small molecules that bind to miRNAs by separating and analyzing the resulting complexes. Differential Scanning Fluorimetry (DSF): This technique measures changes in the stability of miRNA structures when small molecules bind to them. Using both methods ensures accurate results and validates the effectiveness of the identified molecules. 3. Testing for Blood-Brain Barrier (BBB) Crossing: ALS treatments must reach the brain and spinal cord, so the small molecules’ ability to cross the BBB will be evaluated using imaging tools like PET scans. These scans allow researchers to track how the molecules behave in the body and determine their potential for treating ALS. This research is unique because it explores not only individual inhibitors for miR-155 and miR-125b but also their combined effects. Targeting both miRNAs simultaneously might reduce inflammation more effectively, providing new insights into their role in ALS. If successful, this study could identify small molecules capable of regulating miRNAs and reducing neuroinflammation in ALS. These molecules could form the basis for new, innovative treatments that address the disease's root causes. Moreover, the screening techniques developed could be applied to study other miRNAs and neurodegenerative diseases, broadening the impact of the work. In summary, this research aims to tackle ALS by focusing on miR-155 and miR-125b as therapeutic targets. By identifying and testing small molecules that inhibit these miRNAs, the project hopes to create new treatments for ALS and advance understanding of miRNA biology.