Amyotrophic lateral sclerosis (ALS), an adult-onset neurodegenerative disorder, caused by progressive motor neurons loss in the brain and spinal cord.
The pathological mechanisms of ALS are not completed understood, and there is no cure for this disease. In familial ALS, TDP43 mutation is one of the most common genetic defects.
It is well known that mislocalized cytosolic TDP-43 protein is the major component of ubiquitinated inclusions in the cytoplasm and nucleus of both neurons and glial cells in patients with familial or sporadic ALS.
Non-coding RNAs, including long non-coding RNAs (lncRNAs), have been recently recognized as important regulators of gene expression and are involved in the pathogenesis of various human neurodegenerative diseases, including ALS.
Thus, it is important to gain further knowledge about the functions and the mechanisms by which lncRNAs contribute to disease.
Using inducible pluripotent stem cells (iPSCs) with disease mutations as an in vitro differentiation model, we have characterized the role and functions of ncRNAs in multiple neurodegenerative diseases. We have identified a panel of lncRNAs highly-enriched in ALS motor neurons derived from ALS-iPSCs, among which NEAT1 are the most prominent.
Our results demonstrated that elevated expression of NEAT1 is prone to mislocalization in the cytoplasm, where it co-aggregates with TDP43 and causes motor neuron degeneration.
Also, we have shown that cytoplasmic NEAT1 can mediate liquid-liquid phase separation (LLPS) and promote TDP43 proteinopathy in ALS motor neurons. Thus, it would be of great interest to know whether other ALS-associated lncRNAs also play a role in ALS pathological phenotypes. The overall goal of this proposal is to dissect the role of the disease-associated cytosolic lncRNA in promoting TDP43 LLPS in diseased motor neurons.
Recently, it was reported that TDP43 could be mislocalized in the cytoplasm and form amyloid-like inclusions in skeletal muscle, which is similar to TDP43 proteinopathy in motor neurons (3). Since intercellular transmission of pathogenic TDP-43 is linked to ALS progression (4, 5),
we hypothesize that the TDP43 aggregates may be transmitted between motor neurons and skeletal muscle, causing TDP43 proteinopathies in the target cells.
Thus, we will develop a catalytically impaired Cas9 endonuclease-based prime editing system to create isogenic disease iPSC. We will also develop a protocol to generate 3D trunk neuromuscular organoid from ALS-iPSCs and their isogenic mutation-corrected counterparts to investigate whether lncRNA-mediated TDP43 aggregation also contributes to TDP43 proteinopathies in the 3D trunk neuromuscular organoid through various assays, such as single-cell RNA sequencing, electrophysiological and biochemical assays.
Finally, available evidence suggests that lncRNAs, such as NEAT1, may be critical for TDP43 proteinopathy and it may potentially serve as a drug target for developing a novel therapeutic strategy for ALS treatment. This is a critical and timely issue because no effective treatment to delay the progression of ALS is currently available, despite the tremendous efforts devoted to the development of therapeutics.
To explore the feasibility of the ALS-associated lncRNAs as drug targets in ALS, we will use CRISPR/ Cas gene knock out system and antisense oligonucleotides (ASOs) to target the lncRNA whose expression i strongly linked to the ALS disease phenotypes and assess whether TDP43 proteinopathy or other ALS-associated phenotypes can be ameliorated in both ALS-TDP43 iPSC-derived motor neurons and 3D trunk neuromuscular organoid.
To accomplish these goals, we will fulfill three research aims in this proposal:
Specific Aim-1. To explore the roles of the ALS-associated lncRNAs in regulating ALS pathological phenotypes through combined prime editing, 3D trunk neuromuscular organoid, and single-cell sequencing technologies.
Specific Aim-2. To investigate whether the ALS-associated lncRNAs contribute to motor neuron-to-skeletal muscle transmission of TDP43 and promote neurogenic muscle atrophy using iPSC-derived 3D trunk neuromuscular organoid.
Specific Aim-3. To explore the potential of lncRNA antisense oligonucleotides as therapeutic candidates for ALS- TDP43 proteinopathy in diseased 3D trunk neuromuscular organoid derived from iPSC.