Dysregulation of RNA processing as a driver of motor neuron dysfunction in Amyotrophic Lateral Sclerosis

Amyotrophic Lateral Sclerosis (ALS) (also known as motor neuron disease), is caused by a loss of motor nerves or neurons. These motor nerves carry signals from the brain and spinal cord to the peripheral muscles allowing everyday movements. Loss of motor nerves in these patients leads to progressive muscle paralysis and eventual death due to failure of muscles that allow us to breath. About ~10-15% of ALS cases have a family history and specific defects, called mutations, in several genes with diverse functions are known to cause ALS. However, the processes driving motor neuron loss are poorly understood. Though the cause of ALS is unknown, many of the nerves affected in these patients show a common feature. This feature involves incorrect migration of a protein called TDP43 from within the nucleus of a cell, outside into its cytoplasm. The nucleus is the command centre of the cell and contains our DNA with specific instructions on how to make different proteins, the workhorses in a cell. The cytoplasm is the gel-like liquid within the cell that contains the nucleus and has the machinery to make proteins. Each of the nucleus and the cytoplasm have defined roles that are dependent on precise availability of specific proteins. Thus, any change in the proteins' locations will interfere with the normal activity of nerve cells resulting in their death. Relocation, also called mislocalization, of TDP43 protein from the nucleus to the cytoplasm perturbs the normal function of the nerve cells causing them to die. We have developed a new way to trigger TDP43 protein mislocalization in human nerve cells in a dish. This will allow us to look at the consequences of this relocation in detail.

With the advent of a new stem cell technology called reprogramming, skin or blood cells from patients can be converted into an embryonic state. These reprogrammed cells are called induced pluripotent stem cells (iPSC) and have the potential to be converted to any type of cell in the body. Consequently, iPSC's from ALS patients can now be coaxed to become motor nerves i.e., the same kind of cells that are lost in ALS patients. This technology enables the development of human models of ALS in a dish, allowing scientists to interrogate what goes wrong within these cells to cause them to die in ALS. Using such models, we have uncovered that an important molecular process required for nerve cell survival called RNA splicing is defective in ALS.

Using the process of RNA splicing, nerve cells create many different types of proteins that are required for normal function. Incorrect execution of this process leads to the generation of ineffective proteins, which can lead to problems in the structure and function of nerve cells. In this proposal, we will use the iPSC technology to generate motor nerves and use these nerves to understand how TDP43 mislocalization causes RNA splicing defects and the role of RNA splicing in ALS. The results of this study will generate deeper insights into why ALS motor nerves die and highlight new ways to develop therapeutic drugs in our fight against ALS.

Amyotrophic Lateral Sclerosis (ALS) is a fatal disease characterized by loss of motor neurons (MNs). Understanding why MNs die is critical to develop therapeutics against ALS. Most ALS cases display nuclear loss and cytoplasmic mislocalization of the RNA-binding protein TDP43. TDP43 mislocalization is associated with splicing defects including cryptic exon inclusion (CE) in multiple genes that may have specific roles to play in MN function. However, it is not yet clear how TDP43 mislocalization leads to transcriptome-wide splicing defects and whether targeting these defects is a viable therapeutic strategy. The proposed work aims to investigate these questions by applying genomics and phenotypic assays to human induced pluripotent stem cell derived MNs.

We have developed a novel model of TDP43 proteinopathy where we can induce cytoplasmic mislocalization of the endogenous non-mutated form of TDP43, on-demand, in human MNs without any external chemical stress. We find that TDP43 mislocalization leads to cytoplasmic TDP43 aggregation, activation of apoptosis, reduced neurite complexity and soma swelling. Further, we have identified splicing defects in several genes involved in cytoskeletal integrity or neurotransmission including UNC13A and STMN2.

We will use Oxford nanopore technologies long-read sequencing to identify early and late splicing defects across the transcriptome due to TDP43 mislocalization. Next, we will deploy iCLIP and ePRINT to map the direct and indirect targets of mislocalized TDP43 in human MNs. Finally, we will target candidate mis-spliced genes in human MNs that display TDP43 mislocalization and assess the phenotypic consequences on cell survival, dendritic morphology, synaptic structure and neuronal activity.

The proposed work will shed light on the link between TDP43 mislocalization and RNA splicing, and highlight the contribution of defective splicing in specific genes to the disease-associated phenotypes in human MNs.