Basic Science and Pathogenesis

The dual leucine zipper kinase (DLK) signaling pathway regulates neural development aspects, including axonal growth, neuronal migration, and apoptosis, in diverse model organisms[1-5]. Most DLK-mutant mice exhibit perinatal mortality[6]. DLK induces JNK (c-Jun N-terminal kinase) activity in vitro. In mammals, JNK phosphorylates several target proteins, including those involved in cell movement (DCX)[7]. Studies on vitamin D treatment and primary cultures of murine neural progenitor cells have revealed an increase in DCX-expressing cells[8]. This study, we aimed to determine the effect of vitamin D administration on the expression levels of DCX and phosphorylated DCX proteins involved in development and axon growth through the DLK-MAPK signaling pathway in developing neurons and to identify other proteins that vitamin D may interact with.

10^-8M 1,25-dihydroxy vitamin D3 and ethanol treatments were administered 24 h after establishing primary cortical neuron cultures from 16-day-old rat embryos. Protein expression levels were analyzed at 24,48,72,96,120, and 144 h post-treatment. VDR-immunoprecipitation (IP) was performed 30 min after treatment to illustrate the rapid response impact.

DLK and DCX protein expression levels significantly increased 24 hours after vitamin D treatment compared to the ethanol-treated group (p <0.05 and p <0.001, respectively), while pDCX levels decreased significantly (p <0.01). At 48, 96, and 120 hours post-administration, DLK levels remained significantly elevated in the vitamin D group compared to the control group (p <0.01, p <0.05, and p <0.01, respectively), but showed significant reduction at 72 and 144 hours (p <0.01, p <0.01, respectively). At 72,96, and 120 hours, DCX protein expression was significantly lower in the vitamin D group than in the control group (p <0.001, p <0.01, and p <0.01, respectively).pDCX protein expression levels were significantly reduced at 48, 72, and 96 hours after vitamin D treatment compared to the control group (p <0.05, p <0.01, and p <0.01, respectively). At 120 hours, pDCX expression was significantly higher than in the ethanol-treated and control groups (p <0.05, p <0.05). VDR precipitated with DLK and STMN2 proteins after VDR-Ip.

The results of our study suggest that vitamin D administered to newly formed neurons may influence the levels of DLK, DCX, and p-DCX proteins, presumably performing a regulatory role at various phases of development. VDR may interact with DLK and STMN2. This study was supported by the TÜBİTAK 1002-A (Project No:123S438).