Quantifying subpercent nuclear TDP-43 loss in cells and ALS cortex using junction-specific cryptic exon RT-qPCR

Area of Science:

• Neuroscience
• Molecular Biology
• Genetics

Background:

• Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are progressive neurodegenerative diseases linked to nuclear TDP-43 loss.
• A key hallmark of this dysfunction is cryptic exon (CE) splicing, often undetectable in bulk tissue due to low neuron abundance.

Purpose of the Study:

• To develop an ultrasensitive assay for quantifying STMN2 cryptic exon splicing.
• To enable precise measurement of TDP-43 dysfunction in ALS and FTD.

Main Methods:

• Developed an ultrasensitive RT-qPCR assay targeting STMN2 CE using junction-spanning primers.
• Validated the assay in differentiated SH-SY5Y neurons with TDP-43 knockdown.
• Tested assay sensitivity for detecting deficient cells.

Main Results:

• The assay demonstrated a sevenfold expansion in dynamic range.
• TDP-43 knockdown increased STMN2 CE levels 1395-fold in neurons.
• The assay detected a 42-fold CE increase in ALS motor cortex, previously missed.
• Detection limit was established at 0.16% deficient cells.

Conclusions:

• The developed RT-qPCR assay offers high sensitivity for detecting TDP-43-related cryptic exon splicing.
• This tool provides a reliable method for quantifying TDP-43 dysfunction in neurodegenerative diseases.
• Enables sensitive pharmacodynamic monitoring for future ALS-FTD therapeutic studies.