Heterogeneous splicing patterns resulting from KIF5A variants associated with amyotrophic lateral sclerosis

Area of Science:

• Neuroscience
• Genetics
• Molecular Biology

Background:

• Single-nucleotide variants (SNVs) in Kinesin Family Member 5A (KIF5A) are linked to amyotrophic lateral sclerosis (ALS).
• KIF5A is crucial for anterograde axonal transport in neurons.
• ALS-associated KIF5A SNVs are often near exon 27, potentially affecting splicing and the C-terminal cargo-binding domain.

Purpose of the Study:

• To investigate the precise splicing outcomes of KIF5A SNVs near exon 27.
• To determine if all SNVs in this region cause exon skipping.
• To elucidate the downstream effects of KIF5A SNVs on motor neuron function and survival.

Main Methods:

• In vitro minigene splicing assays in HEK293 cells.
• Splicing analysis in CRISPR-edited human stem cell-derived motor neurons.
• Splicing and pathology assessment in a KIF5A 5'ss SNV knock-in mouse model.
• Quantification of KIF5A mRNA and protein levels in different cell lines.

Main Results:

• Only 5' splice-site (5'ss) SNVs, not 3' splice-site SNVs, induced exon 27 skipping.
• Both 5'ss and 3'ss SNVs, as well as truncated KIF5A variants, severely reduced motor neuron survival and caused morphological defects.
• While KIF5A mRNA levels remained stable, KIF5A protein levels were reduced in variant lines, indicating impaired protein synthesis or stability.

Conclusions:

• KIF5A SNVs exhibit heterogeneous effects on RNA splicing but converge to reduce KIF5A protein availability.
• Reduced KIF5A protein leads to axonal transport defects and motor neuron pathology in ALS.
• This study clarifies the pathogenic mechanism of KIF5A SNVs in ALS, highlighting protein level dysregulation over splicing alone.