A toxic gain-of-function variant in MAPK8IP3 provides insights into JIP3 cellular roles
Wei Zhang, … , Konstantina Skourti-Stathaki, Stanley T. Crooke
Wei Zhang, … , Konstantina Skourti-Stathaki, Stanley T. Crooke
Published March 20, 2025
Citation Information: JCI Insight. 2025;10(8):e187199. https://doi.org/10.1172/jci.insight.187199.
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Research Article Cell biology Genetics Therapeutics

A toxic gain-of-function variant in MAPK8IP3 provides insights into JIP3 cellular roles

Abstract

Mitogen-activated protein kinase 8 interacting protein 3 (MAPK8IP3) gene encoding a protein called JIP3 is an adaption protein of the kinesin-1 complex known to play a role in axonal transport of cargo. Mutations in the gene have been linked to severe neurodevelopmental disorders, resulting in developmental delay, intellectual disability, ataxia, tremor, autism, seizures, and visual impairment. A patient who has a missense mutation in the MAPK8IP3 gene (c. 1714 C>T, Arg578Cys) (R578C) manifests dystonia, gross motor delay, and developmental delay. Here, we showed that the mutation was a toxic gain-of-function mutation that altered the interactome of JIP3; disrupted axonal transport of late endosomes; increased signaling via c-Jun N-terminal kinase, resulting in apoptosis; and disrupted dopamine receptor 1 signaling while not affecting dopamine receptor 2 signaling. Furthermore, in the presence of the mutant protein, we showed that an 80% reduction of mutant JIP3 and a 60% reduction of WT JIP3 by non-allele-selective phosphorothioate-modified antisense oligonucleotides was well tolerated by several types of cells in vitro. Our study identifies what we believe to be several important new roles for JIP3 and provides important insights for therapeutic approaches, including antisense oligonucleotide reduction of JIP3.

Authors

Wei Zhang, Swapnil Mittal, Ria Thomas, Anahid Foroughishafiei, Ricardo Nunes Bastos, Wendy K. Chung, Konstantina Skourti-Stathaki, Stanley T. Crooke

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Figure 7

Non-allele-selective PS-ASOs rescue patient iPSC–derived neurons from MT-JIP3–induced cell toxicity.

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Non-allele-selective PS-ASOs rescue patient iPSC–derived neurons from MT...
CRISPR control and patient iPSC–derived neurons were treated with 3 different non-allele-selective ASOs, including ASO1 (1713794), ASO2 (1713687), and ASO3 (1713510) targeting MAPK8IP3 (JIP3) for 4 days. Scrabble PS-ASO 676630 was used as a control ASO. (A) Cell viability was measured by trypan blue cell count assay and normalized to UTC of CRISPR control cells. n = 3, *P < 0.05. (B) RNA was extracted by GITC assay, and MT-MAPK8IP3 mRNA level was measured using q-PCR. Data were normalized to UTC of control cells. n = 3, *P < 0.05. (C) WT-MAPK8IP3 mRNA level was measured using q-PCR. Data were normalized to UTC of control cells. n = 3, *P < 0.05. (D) SPAG9 (JIP4) mRNA levels were measured using q-PCR. Data were normalized to UTC of control cells. n = 3. (E) CRISPR control and patient iPSC–derived neurons were treated with different concentrations of non-allele-selective ASO2 targeting MAPK8IP3 (JIP3) for 4 days. Scrabble PS-ASO 676630 was used as a control ASO. Cell viability was measured by trypan blue cell count assay and normalized to UTC of CRISPR control cells. n = 3, *P < 0.05. (F) Cell proteins were harvested after 4-day incubation of ASO2. JIP3, JIP4, and p-JNK were measured by Western blot. GAPDH was used as a loading control. (G) Expression of the MT-JIP3 in patient iPSC–derived neurons leads to increased risk of death compared with CRISPR control iPSC-derived neurons. ASO2 (5 μM) was used to treat mutation. Cells were screened using a Keyence microscope every 3 or 4 days. Viable cell numbers were manually counted in 3 representative areas from each well (center, upper center, and lower center). Cumulative risk was calculated by Cox proportional hazard regression in Prism 9.5. ****P < 0.0001. (H) 5 μM non-allele-selective ASO2 was used to treat mutations in patient iPSC neurons for 72 hours, and then 1 μM D1 agonist (SKF 81297) was added to each test group with different incubation time points. cAMP levels were measured using a cAMP-Glo Assay (Promega). Data were normalized to UTC of cells. n = 3. *P < 0.05.