A transient protein folding response targets aggregation in the early phase of TDP-43-mediated disease

Abstract

Understanding the mechanisms that drive TDP-43 pathology is integral to combating neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). To address this, we sought to determine the timeline of proteomic alterations across disease course in TDP-43 proteinopathy. Using longitudinal quantitative proteomics analysis of cortex samples from the cytoplasmic TDP-43 rNLS8 mouse model of ALS and FTLD, we identified several distinct protein subsets characterized by temporal alterations in protein abundance across diverse biological pathways, including protein folding, intracellular transport, myelination, and neuronal synaptic function. Remarkably, neurons in the rNLS8 cortex elicited a transitory response primarily comprising protein-folding factors prior to and in the earliest stages of disease progression. This response included increased levels of DnaJ homolog subfamily B member 5, DNAJB5, and proof-of-concept studies showed that DNAJB5 over-expression decreased TDP-43 aggregation in cell and cortical neuron cultures. Conversely, knockout of Dnajb5 exacerbated motor impairments caused by AAV-mediated cytoplasmic TDP-43 expression in the brains and spinal cords of mice. Lastly, the late disease proteomic signatures of rNLS8 mouse cortex strongly correlated with changes in human autopsy-derived TDP-43 proteinopathy tissues, indicating commonality of disease processes. Together, these findings reveal molecular mechanisms that regulate protein levels through distinct stages of ALS and FTLD progression, and suggest that protein folding factors that combat cytoplasmic TDP-43 protein aggregation could be protective in disease.

Proteomic analysis of the TDP-43-associated insoluble fraction from NEFH-TDP-43 mouse brain suggests sustained stress granule formation, CLUH granule recruitment and impaired mitochondrial metabolism

“…Formaldehyde crosslinking and homogenization of brain tissue was adapted from previous protocols (Liu-Yesucevitz et al, 2010;San Gil et al, 2024;Sap et al, 2021). 500 µl of 0.5% formaldehyde in PBS was added to 30 mg of frozen mouse brain tissue, previously crushed under N 2 and placed on a rotating wheel for 10 mins at room temperature.…”

“…These studies identified multiple disease-related changes in biological pathways. However, these analyses were performed using soluble protein fractions (Lee et al, 2023;San Gil et al, 2024) without TDP-43 immunoprecipitation, and would therefore not accurately capture the TDP-43-associated SG proteome.…”

“…Attempts to determine the presence of TDP-43 in SG fractions isolated from tissues is limited and may lack specificity due to TDP-43 immunoprecipitation from soluble and not insoluble fractions (Montalbano et al, 2020). Proteomics analysis has been performed to observe changes in postmortem frozen motor cortex samples form ALS donors compared with healthy control samples (Lee et al, 2023) as well as longitudinal analysis in cortex samples from NEFH-TDP-43 mice (San Gil et al, 2024). These studies identified multiple disease-related changes in biological pathways.…”