Samenvatting

SMPD4 is a neutral sphingomyelinase that plays an important role in sphingolipid metabolism. Sphingolipids are a family of lipid molecules abundantly present in the brain where they play an important role in the brain development and function due to their essential cellular role as structural components of cell membranes (Ledeen 2008). SMPD4 catalyzes the hydrolysis of membrane sphingomyelin, the most abundant eukaryotic sphingolipid, to form ceramide. The generated ceramide works subsequently as a second messenger in several neurodevelopmental processes such as neuronal cell proliferation and neuronal survival (Krut O 2006).
The Mancini research group has reported that bi-allelic loss-of-function variants in the SMPD4 gene lead to a severe neurodevelopmental disorder including microcephaly, arthrogryposis, and a profound developmental delay (Magini P 2019). The first objective of this project is to study the effect of SMPD4 knockdown on the homeostasis of the nuclear envelope (NE) during mitosis, most specifically during the nuclear envelope breakdown (NEBD). This project also aims to study the effect of SMPD4 loss-of-function on neuronal cell proliferation and apoptosis, as both these processes are involved in the pathogenesis of microcephaly. Lastly, the sub-cellular localization of patient-specific missense SMPD4 mutant proteins was studied and compared to wild-type SMPD4.
Firstly, a NEBD assay was performed on SMPD4 knockdown Hela cells to observe the effect of the SMPD4 loss-of-function on the duration of the NEBD process through time-lapse confocal microscopy. Unfortunately, this project is still in progress and no conclusions can be drawn on the effect of the SMPD4 knockdown on the NEBD. Secondly, to study the effect of SMPD4 loss-of-function on neuronal cell proliferation, an EdU proliferation assay was performed on SMPD4 knockdown NSCs. From this assay, it can be concluded that SMPD4 knockdown leads to decreased proliferation in NSCs, which would explain the patients’ phenotype, as reduced proliferation of neuronal cells leads to congenital microcephaly. Next, a TUNEL apoptosis assay was performed to detect apoptotic DNA fragmentation in SMPD4 knockdown NSCs. This assay shows an increase in apoptosis in SMPD4 knockdown NSCs when compared to control NSCs, indicating that SMPD4 knockdown cells are more susceptible to apoptotic cell death. This is another neurodevelopmental process involved in the pathogenesis of microcephaly. Finally, to study the effect of patient-specific SMPD4 missense mutants on the protein’s sub-cellular localization, Hek cells transfected with the mutant SMPD4 were stained with an SMPD4, and an ER (Calnexin) or nuclear pores (mAb-414) antibody and imaged through confocal microscopy to determine the colocalization of SMPD4 with the ER and nuclear pores. This experiment showed that the SMPD4 Pro446Leu mutant results in decreased SMPD4 colocalization with the ER when compared to wild-type SMPD4, this is hypothetically caused by protein misfolding.
Together, these findings support that SMPD4 loss-of-function leads to decreased proliferation and increased apoptosis in neuronal cells, these are both processes known to be involved in the development of congenital microcephaly. Furthermore, the missense Pro446Leu mutation was observed to have an effect on the SMPD4 protein subcellular localization.