Research

The Role of the Lysosomal Proteases in TDP-43 Protein Homeostasis in ALS/FTD

A primary research direction in our lab focuses on Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD), devastating neurodegenerative diseases currently lacking effective disease-modifying therapies for most patients. A critical link between these conditions is the protein TDP-43. Normally found in the cell nucleus, TDP-43 pathologically mislocalizes to the cytoplasm and forms toxic aggregates in the vast majority of ALS cases (~97%) and about half of FTD cases. This disruption of TDP-43 function and location is considered a major driver of neuron death in these diseases.

The lysosome, the cell's primary recycling center, is responsible for clearing protein aggregates through processes like autophagy. Mounting evidence indicates that lysosomal function is impaired in ALS/FTD, hindering the removal of toxic TDP-43. Our research investigates the specific lysosomal enzymes, known as proteases (particularly cathepsins), responsible for breaking down TDP-43.

Key findings from our work include:

Identification of Key Proteases: Our previous research identified several specific lysosomal proteases – including Cathepsin B (CTSB), Cathepsin D (CTSD), and Asparagine Endopeptidase (AEP) – that effectively degrade TDP-43 in vitro. We generated comprehensive maps detailing precisely where these and other lysosomal proteases cleave the TDP-43 protein.  

Impact of Disease Mutations: We demonstrated that certain ALS/FTD-linked mutations in the TDP-43 gene directly impair its degradation by these lysosomal proteases. For example, the Q331K mutant TDP-43 showed significant resistance to degradation by human lysosome extracts compared to the wild-type protein. This suggests that mutations can hinder clearance, potentially creating a vicious cycle that accelerates TDP-43 accumulation.   

The Complex Interaction of Amyloid Precursor Protein (APP) and the Lysosomal Proteases in Alzheimer's Disease

The processing of Amyloid Precursor Protein (APP) into amyloid-beta (Aβ) peptides by secretase enzymes is a well-known pathway central to Alzheimer's Disease (AD). However, the routine breakdown and clearance of the full-length APP protein, particularly within the cell's recycling center – the lysosome – is less understood but critically important.   Our research confirms that APP protein is significantly enriched within the endolysosomal system of neurons. This places APP in direct contact with a variety of powerful lysosomal enzymes called cathepsins, which are responsible for its degradation. Key findings will soon be reported. 

Investigating C9ORF72-Related ALS and FTD

Our research also delves into the most frequent genetic cause of both FTD and ALS: a hexanucleotide repeat expansion (GGGGCC) in the first intron of the C9ORF72 gene. This expansion leads to the production of toxic dipeptide repeat proteins (DPRs) through repeat-associated noncanonical (RAN) translation. Understanding the mechanisms by which the repeat expansion causes pathology is crucial for developing effective gene-based therapies.  

Our work in this area includes:

Studies in Mature Brain Organoids: Building on the work of the Clelland lab investigating allele-specific excisions of C9orf72 iPSC-derived neurons (https://pubmed.ncbi.nlm.nih.gov/38621131/), we are investigating C9ORF72 edits in mature brain organoids. This advanced model system allows for the study of these genetic modifications in a more complex, 3D neural environment, providing further insight into potential therapeutic avenues.

Potential Toxins Released by C9 Astrocytes: We are also investigating the potential toxins released by C9ORF72 astrocytes. While DPRs are a specific pathology in C9ORF72 mutation carriers, studies have also indicated potential dysfunction in other cell types, such as macrophages and microglia, in mouse models. Furthermore, evidence suggests that C9ORF72-specific phenomena, including dipeptide repeat proteins and RNA foci, can be observed in the enteric nervous system and may be associated with gastrointestinal symptoms in the absence of TDP-43 aggregation. This highlights the complex, non-cell-autonomous effects that may contribute to the disease and suggests the involvement of glial cells like astrocytes in releasing toxic factors.

We hope our findings will provide significant insights into C9ORF72 gene regulation and inform gene therapy approaches, including antisense oligonucleotides (ASOs) and CRISPR gene editing. 

Autophagy and Lysosomal pH in Neurodegeneration

The accumulation of misfolded proteins is a common feature of many neurodegenerative diseases. Lysosomes play a critical role in clearing these proteins via autophagy. This degradative function relies heavily on lysosomal proteases called cathepsins.   

Disruptions to lysosomal function are strongly implicated in neurodegeneration. One critical factor is the maintenance of an acidic pH within the lysosome (typically pH 4.0-4.5), which is optimal for the activity of many cathepsins. Alterations in lysosomal pH can impair the ability of these enzymes to degrade their targets. Research, including work from our collaborators, indicates that faulty lysosomal acidification can drive the build-up of pathological proteins like TDP-43, tau, and APP in disease models. Even subtle increases in lysosomal pH can significantly reduce the activity of key cathepsins involved in degrading disease-associated proteins like TDP-43, APP and tau, potentially exacerbating disease progression. Understanding how lysosomal pH regulation and autophagy pathways are affected in disease, and how this impacts protein clearance, is crucial for identifying therapeutic targets.