Hawrot, James

Functional loss of TDP-43, an RNA binding protein genetically and pathologically linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), leads to the inclusion of cryptic exons in hundreds of transcripts during disease. Cryptic exons can promote the degradation of affected transcripts, deleteriously altering cellular function through loss-of-function mechanisms. Here, we show that mRNA transcripts harboring cryptic exons generated de novo proteins in TDP-43–depleted human iPSC–derived neurons in vitro, and de novo peptides were found in cerebrospinal fluid (CSF) samples from patients with ALS or FTD. Using coordinated transcriptomic and proteomic studies of TDP-43–depleted human iPSC–derived neurons, we identified 65 peptides that mapped to 12 cryptic exons. Cryptic exons identified in TDP-43–depleted human iPSC–derived neurons were predictive of cryptic exons expressed in postmortem brain tissue from patients with TDP-43 proteinopathy. These cryptic exons produced transcript variants that generated de novo proteins. We found that the inclusion of cryptic peptide sequences in proteins altered their interactions with other proteins, thereby likely altering their function. Last, we showed that 18 de novo peptides across 13 genes were present in CSF samples from patients with ALS/FTD spectrum disorders. The demonstration of cryptic exon translation suggests new mechanisms for ALS/FTD pathophysiology downstream of TDP-43 dysfunction and may provide a potential strategy to assay TDP-43 function in patient CSF.

AbstractBackgroundFrontotemporal dementia (FTD) is a rapidly progressive neurodegenerative disorder characterized by prominent atrophy of the frontal and temporal lobes and marked language and/or behavioral disturbances. Mislocalization of the TAR DNA‐binding protein 43 (TDP‐43) – characterized by both its nuclear loss and its cytosolic accumulation – occurs in up to 50% of FTD cases. Recent evidence from our group and others demonstrates that loss of TDP‐43‐dependent splicing activity occurs in FTD patient brains, is an early disease phenomenon, and contributes directly to neuronal death. Monitoring the products of TDP‐43 related mis‐splicing may, thus, yield promising biomarker candidates of early cellular dysfunction in FTD.MethodWe propose a series of proteogenomic studies in human iPSC neurons, post‐mortem brain, and patient biospecimens to identify and develop protein‐based biomarkers of RNA mis‐splicing in FTD. First, we will identify pathologic RNA splicing changes and protein products of mis‐splicing that occur in the context of TDP‐43 depletion in iPSC‐derived neurons. We will next determine which of these predicted transcripts and de novo proteins occur in post‐mortem human FTD brains, as a crucial first step in biomarker development. Finally, we will develop CSF biomarkers of FTD‐related mis‐splicing through a combination of targeted proteomics and ELISA‐based assays of lead candidate biomarkers.ResultUsing CRISPRi knockdown of TDP‐43 in i3Neurons followed by total RNAseq, we detected cryptic exon formation in STMN2 as well as hundreds of additional transcripts. We developed a proteogenomic informatic pipeline to detect de novo protein products of mis‐spliced transcripts in TDP‐43 deficient i3Neurons, revealing two in‐frame de novo peptides in the MYO18A transcript. Furthermore, we detected 38 of these mis‐spliced transcripts in ALS/FTD post‐mortem cortex, including MYO18A.ConclusionThese preliminary studies indicate that our i3Neuron CRISPRi pipeline can predict disease‐relevant markers of TDP‐43 mis‐splicing and suggest a proteogenomic workflow for development of novel protein‐based biomarkers of mis‐spliced transcripts.Together, these efforts will yield early biomarkers of TDP‐43 related cellular dysfunction that can act as surrogate measures of disease onset and progression in FTD.