Specific disruption of astrocytic Ca2+ signaling pathway in vivo by adeno-associated viral transduction.
Astrocytes are the predominant glial-cell type in the CNS and they are known to play an active role in modulating neuronal function. Since many of the same molecules including G-protein coupled receptors (GPCRs) are expressed in both neurons and astrocytes, in vivo pharmacological manipulations to target astrocytes lack specificity. In this study, we investigated the effect of Pleckstrin Homology (PH) domain of Phospholipase C (PLC)-like protein p130 (p130PH) on Ca(2+) signaling in astrocytes in vivo. We used the serotype 2/5 recombinant adeno-associated virus (rAAV2/5) vectors to introduce p130PH fused with a tagged protein monomer red fluorescent protein at the N-terminal (i.e., transgene mRFP-p130PH). In order to selectively disrupt the Ca(2+) signaling pathway in astrocytes, the transgene was driven by a novel astrocyte-specific promoter gfaABC(1)D. Our results show that mRFP-p130PH is exclusively expressed in astrocytes with a high efficiency and a stable expression level. In vivo imaging using two-photon microscopy demonstrated reduced Ca(2+) signal in transduced astrocytes in response to ATP stimulation. As Ca(2+) signaling is a characteristic form of cellular excitability in astrocytes that can mediate chemical transmitter release and contribute to neuronal excitotoxicity, the current study provides an in vivo approach to better understand Ca(2+)-dependent gliotransmission and its involvement in glia-related diseases.
Xie
Y
Y
Dalton Cardiovascular Research Center, University of Missouri-Columbia, MO 65211, USA.
Wang
T
T
Sun
G Y
GY
Ding
S
S
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001253:Astrocytes / Q000378:metabolism*; D020013:Calcium Signaling; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics; D002478:Cells, Cultured; D000229:Dependovirus / Q000235:genetics*; D005822:Genetic Vectors; D005904:Glial Fibrillary Acidic Protein / Q000235:genetics; D008297:Male; D051379:Mice; D011401:Promoter Regions, Genetic; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D013003:Somatosensory Cortex / Q000378:metabolism; D013997:Time Factors; D014161:Transduction, Genetic; D019076:Transgenes
D002352:Carrier Proteins; D005904:Glial Fibrillary Acidic Protein; C430714:Plcl1 protein, rat; D011993:Recombinant Fusion Proteins; D000255:Adenosine Triphosphate