Seladin-1 as a new marker for oxidative stress and degeneration in central neurons and astroglia

Seladin-1 (Sel-1) stands for Selective Alzheimer’s Disease Indicator-1. By sequence similarity, it was identified as DHCR24, a broadly expressed enzyme highly expressed in brain, that converts desmosterol to cholesterol, and conserved from plants to mammals. Its inactivating mutations are the genetic basis of desmosterolosis, a disease characterisedd by several neurologic defects. Relative to Alzheimer’s disease (AD), Seladin-1 mRNA is downregulated in the brain regions more susceptible to the AD pathology. Moreover, a relative Seladin-1 transcription fluctuation during aging in a transgenic mouse model of AD has been reported;. In brief, seladin’s role appears to be implicated with protection of cells against b amyloid (Abeta) induced toxicity and oxidative stress, suggesting a role in cell survival. Aim: This study set off to investigate the intracellular distribution of sel-1 gene product under normal conditions, or after overexpression, as well as its expression under toxic insults, using neuronal and astroglial models. Materials and Methods: Immortalised neuronal cell lines, GN11 (mouse immature,migrating neurons), GT1-7 (mouse mature hypothalamic neurons), were used; moreover primary cultures of purified mouse astrocyte type-1 cells were prepared. A custom made anti-sel-1 primary polyclonal Ab was used, along with cholesterol (filipin), ER, Golgi and mitochondrial markers. A sel-1_GFP-tagged vector was used to overexpress the protein in neurons. We used different agents to mediate toxic insults, simulating cellular oxidative stress conditions (BSO, DEM, H2O2), and the AD defects (Abeta and INFg) in neurons and astroglia. Results-Conclusion Using RT-PCR we confirmed the expression of the sel-1 gene in neurons and astroglia. Using immunofluorescence we observed a partial colocalisation of sel-1 with the Golgi and the ER, and a vesicular distribution only in the mature neurons, with the vesicles budding out of the Golgi and accumulating near the axon terminals. Overexpression of the protein in the immature GN11 neurons by transfection with Sel-1_GFP resulted in their morphological differentiation and induced a vesicular distribution similar to that of the mature neurons. Sel-1 mRNA levels quantified by Real Time PCR appear to be 100 fold higher in the mature GT1-7 neurons and astroglia, compared to the immature GN11 neurons. Moreover, sublethal toxic insults and oxidative stress significantly downregulated sel-1 mRNA in neurons and astroglial cultures; however this downregulation is accompanied with the appearance of an alternative longer splicing variant of Sel-1. Thus, we suggest this longer form to be a “stress marker” considering it became prominent upon stress induction. Moreover, this “stress marker” reached the expression levels of the normal transcript present in control samples. On the basis of sequence analysis of mouse, rat and human sel-1, we found a TATA-less CpG island that probably regulates the expression of the protein with the enzymatic activity and the cholesterol production from its precursor. The second transcript, given its apparition under cell stress, may encode a protein that plays a role in the cell response to stress. We have been working on a region that could correspond to the second promoter, and we have yet to experimentally verify all its trans acting elements. In conclusion, we hereby report a different mRNA expression of sel-1 between mature and immature neurons, and a differential protein distribution in the two cell types. Moreover we report a modification of this gene at the transcriptional level upon stress induction, both in neurons and astroglia. Thus, sel-1 is a not only a indicator of neurodegeneration due to AD pathology, but a critical cell stress marker whose precise role in cell survival is yet to be characterized. (Grants: MIUR PRIN 2003060512_001; Fondazione CARIPLO)