Dysregulated Purinergic Signalling in Fragile X Syndrome Cortical Astrocytes

Area of Science:

• Neuroscience
• Cellular Biology
• Biochemistry

Background:

• Fragile X syndrome (FXS) symptoms are linked to astrocyte signaling dysfunction in the cerebral cortex.
• The purinergic signaling pathway, crucial for glial and neuronal interactions, is upregulated in Fmr1 knockout (FXS) astrocytes.
• Previous studies noted heightened P2Y purinergic receptor levels, prolonged calcium release, and hyperactivity in Fmr1 knockout astrocytes.

Purpose of the Study:

• To develop a sensitive method for quantifying purinergic and pyrimidine molecules in Fmr1 knockout astrocytes.
• To compare intracellular and extracellular purine and pyrimidine concentrations between wildtype and Fmr1 knockout astrocytes.
• To investigate the role of purinergic signaling dysregulation in the cellular pathology of FXS.

Main Methods:

• Developed a hydrophilic interaction liquid chromatography-mass spectrometry protocol for purine and pyrimidine quantification.
• Compared intracellular and extracellular purinergic molecule abundance in wildtype and Fmr1 knockout mouse astrocytes.
• Assessed glycosylation of the CD39 ectonucleotidase on astrocyte membranes.

Main Results:

• Significant differences in intracellular UDP, ATP, AMP, and adenosine concentrations were observed in Fmr1 knockout astrocytes.
• Extracellular adenosine levels were significantly elevated in Fmr1 knockout astrocyte-conditioned media.
• Elevated glycosylation of the astrocyte membrane-bound CD39 ectonucleotidase was found in Fmr1 knockout cultures.

Conclusions:

• The purinergic signaling system is further dysregulated in Fmr1 knockout cortical astrocytes.
• These alterations in purinergic signaling may lead to significant changes in FXS purinergic receptor activation and cellular pathology.
• The developed LC-MS method provides a sensitive approach for studying purinergic signaling in neurological disorders.