RNF13 variants L311S and L312P associated with developmental epileptic encephalopathy alter dendritic organization in...

Developmental and epileptic encephalopathy (DEE) is a group of rare and serious neurological disorders where seizures exacerbate developmental impairment. Recently, genetic mutations in the RNF13 gene were reported to cause DEE73. Specifically, two leucines from the ubiquitin E3 ligase RNF13 are converted to serine or proline (L311S and L312P). These mutations are located within a dileucine motif, which impairs RNF13's capacity to interact with AP-3. A second motif allows RNF13 to interact with AP-1 when the dileucine sorting motif is altered. The present study demonstrates that RNF13 variants L311S and L312P are trafficked through an AP-1-dependent pathway in HeLa cells. In cultures of primary rat hippocampal neurons, the protein level of the variants is significantly higher in dendrites than for wild-type protein. L311S and L312P variants alter dendritic components similarly to an RNF13 AP-3-defective binding variant or a dominant negative for RNF13's ubiquitin ligase activity. Compared to non-transfected neurons, the variants change the distribution of EEA1-positive early endosomes throughout the dendrites. While the WT alters the distribution of lysosomes (Lamp1-positive) in dendrites, the variants only decrease their presence in proximal dendrites. Unlike the variants, RNF13 WT increases the abundance of PSD-95 in distal dendrites. Interestingly, only the variants with altered dileucine motifs decrease the total number of postsynaptic inhibitory protein Gephyrin puncta. This study reports that genetic variants L311S and L312P mainly act as a dominant negative protein. This research provides valuable insights into the dendritic defects that occur when DEE73-associated genetic variants of RNF13 are present.