Endocrine Abstracts

SYNGR3 is involved in the re-uptake of dopamine (DA) into striatal pre-synaptic terminals. Here we elucidate the genetic control of human SYNGR3 gene (SYNGR3).

Methods and Intermediate Results: Step 1 Potential cis-acting in 2kb upstream of the transcription initiation site (TIS) of SYNGR3 were investigated using the MatInspector algorithm. Three potential Nurr1 binding sites were identified. Step 2 PCR amplification (human DNA as template) generated a 1.8Kb amplicon containing terminal 5’ Nhe1 and 3’ Xho1 sites (NheI-SYNGR3−1870/TIS−XhoI). Step 3 Cloning and subcloning of (NheI-SYNGR3−1870/TIS−XhoI) allowed this DNA fragment to be ligated into pGL3-Basic vector (promoter-less firefly luciferase vector) giving plasmid pGL3-SYNGR3−1870/TIS. Sequence of the SYNGR3−1870/TIS section was identical to that in Ensembl. Step 4 pGL3-SYNGR3−1870/TIS or pGL3-Basic vector together with pRL-TK Renilla reporter vector were transfected into SH-SY5Y cells and promoter activity determined using a dual luciferase, end-point system.

Result 1: Surprisingly, pGL3-SYNGR3−1870/TIS had less promoter activity than the basic vector (P<0.05). Re-analysis of the SYNGR3 sequence revealed a XCPE1 site between the TIS and the Translation Start Site (TSS). Step 5 Steps 2 to 4 were repeated using a longer DNA insert (SYNGR3−1870/TSS).

Result 2: Plasmid pGL3-SYNGR3-1870/TSS had greater promoter activity than the basic vector (P<0.05). Point mutation of one of the potential Nurr1 binding sites reduced promoter (P<0.05). Point mutation of all three sites reduced promoter activity to that of the basic vector.

Result 3: Gel-shift assays showed specific binding of Nurr1 to the three sites.

Result 4: Treatment of SH-SY5Y cells with Nurr1 transactivator, C-DIM12, significantly increased the cellular SYNGR3 level.

Conclusions: Nurr1 an orphan member of the endocrine nuclear receptor superfamily is involved in control of SYNGR3 expression. Increasing SYNGR3 levels via Nurr1 activation is a possible therapeutic option in Parkinson’s disease.