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

Though a vital first step in determining whether we can help a patient is whole genome sequencing, often genomic sequencing does not answer all the questions that must be addressed. Two very common questions that we often must address are: What is the functional nature of the mutation, and what are the effects of the mutation on the functions of key cells?

The manuscript (A toxic gain-of-function variant in MAPK8IP3 provides insights into JIP3 cellular roles. JCI Insight. 2025 Mar 20;10(8). https://insight.jci.org/articles/view/187199) authored by Zhang et al. exemplifies the types of questions and experiments that we conduct to answer such questions. In this case, the mutation was in the mitogen-activated protein kinase 8 interacting protein 3 (MAPK8IP3) gene, a gene that encodes for a protein known as JIP3, but it was not clear what the effect of the mutation on the functions of JIP3 was. In short, ‘is the mutation a loss of function or a toxic gain function’ question needed to be answered. This is a vital question because the type of ASO we design varies depending on the effect of the mutation on the protein involved. However, very little was known about the functions of JIP3. So, to understand the effects of the mutation, we first needed to learn more about its cellular functions, its location in cells, and the various proteins and RNAs that it might interact with.

c-Jun N-terminal kinase-interacting protein 3 (JIP3) is a member of a family of JIP proteins and has a very close relative known as JIP4. This complicated matters because we needed to understand whether JIP4 could substitute for JIP3 for some of its functions in cells where both proteins are expressed, such as fibroblasts. (We knew that only JIP3 was expressed in neuronal cells). Since these proteins were known to be part of a signaling pathway, we first asked if JIP3 signaled via the specific pathway in which it resided, the JNK kinase pathway. This pathway, when activated, lead to the death of cells, so we had to answer this. We learned that JIP3 does, indeed, activate the JNK kinase cell death causing pathway and showed that the mutation made it even more toxic to cells. In short, the mutation causes a toxic gain of function in the JNK kinase pathway.

It was also known that JIP3 is involved in moving cargo laden endosomes (little membrane enclosed droplets that carry cargo in cells) down the cellular transport train tracks called microtubules. Once again, we showed that the mutation caused JIP3 to not function properly in this regard as another toxic gain of function. We also showed the mutation altered the interaction of JIP3 with other proteins (JIP3 does not interact with RNAs) — again, a toxic gain of function. Lastly, we showed that the mutation caused JIP3 to be mislocated in the cell.  In fibroblasts, we showed that JIP4 could sub for JIP3, but the mutation caused neurological disease because neuronal cells make primary JIP3, with minimum amounts of JIP4.  We then knew that we needed to reduce the toxic mutant protein and could design an RNase H1 ASO.

Because we had identified all the functions of JIP3, we could then show that our ASO corrected all the cellular problems, making it very likely that it would be beneficial to the patient. Since the patient was heterozygous, i.e., they had one good JIP3 gene that makes a good JIP3 protein and one bad gene that makes the toxic JIP3 protein, we knew that we needed to make an “allele-selective RNase H1 ASO” — an ASO that can selectively just reduce the toxic RNA and toxic JIP3 protein. ASO technology is the only technology capable of such specificity.  However, we also needed to know how selective the ASO needed to be for the toxic JIP3, so we lowered total JIP3 levels in neurons and found that a 10-fold selectivity should be sufficient to have a safe ASO.

Obviously, all the work done is critical, but it takes time and money. This is one reason that it may take more time to find the right ASO for some patients.

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