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Abstract
BackgroundThe first mammalian protein histidine phosphatase (PHP) was discovered in the late 90s of the last century. One of the known substrates of PHP is ATP-citrate lyase (ACL), which is responsible - amongst other functions - for providing acetyl-CoA for acetylcholine synthesis in neuronal tissues. It has been shown in previous studies that PHP downregulates the activity of ACL by dephosphorylation. According to this our present work focused on the influence of PHP activity on the acetylcholine level in cholinergic neurons.ResultsThe amount of PHP in SN56 cholinergic neuroblastoma cells was increased after overexpression of PHP by using pIRES2-AcGFP1-PHP as a vector. We demonstrated that PHP overexpression reduced the acetylcholine level and induced cell death. The acetylcholine content of SN56 cells was measured by fast liquid chromatography-tandem mass spectrometry method. Overexpression of the inactive H53A-PHP mutant also induced cell damage, but in a significantly reduced manner. However, this overexpression of the inactive PHP mutant did not change the acetylcholine content of SN56 cells significantly. In contrast, PHP downregulation, performed by RNAi-technique, did not induce cell death, but significantly increased the acetylcholine content in SN56 cells.ConclusionsWe could show for the first time that PHP downregulation increased the acetylcholine level in SN56 cells. This might be a potential therapeutic strategy for diseases involving cholinergic deficits like Alzheimer's disease.
Highlights
The first mammalian protein histidine phosphatase (PHP) was discovered in the late 90s of the last century
We investigated the viability of SN56 cells and measured the ACh content of these cholinergic neurons after up- and downregulation of PHP
Fetal bovine serum (FBS) and Trypsin-ethylenediaminetetraacetic acid (EDTA) for detaching SN56-cells were from PAA (Marburg, Germany)
Summary
The first mammalian protein histidine phosphatase (PHP) was discovered in the late 90s of the last century. One of the known substrates of PHP is ATP-citrate lyase (ACL), which is responsible - amongst other functions - for providing acetyl-CoA for acetylcholine synthesis in neuronal tissues. It has been shown in previous studies that PHP downregulates the activity of ACL by dephosphorylation. As for instance P-selectin [7], histone H4 [8] and annexin I [9] are phosphorylated at a histidine residue and are essential for cellular signaling without knowing the kinase or phosphatase responsible for that. Three substrates have been identified for PHP i.e. ATP-citrate lyase (ACL) [10], the ß-subunit of the heterotrimeric G protein (Gß) [11] and the calciumdependent potassium channel KCa3.1 [12]
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