Transcriptomic and proteomic analyses of mouse cerebellum reveals alterations in RasGRF1 expression following in vivo chronic treatment with delta 9-tetrahydrocannabinol.
We have applied transcriptomic and proteomic techniques to identify changes in the RNA and the protein levels in the mouse cerebellum after chronic treatment with Delta(9)-tetrahydrocannabinol (THC). Among approximately 14,000 transcripts in a mouse cDNA microarray library, we found 11 genes with altered expression. RasGRF1, a neuron-specific Ras guanine nucleotide exchange factor, showed a reduction both at the RNA and protein levels with a specific decrease of the protein pool associated to cell membranes. In addition, proteomic analysis on cerebellum obtained from chronically THC-treated mice detected quantitative changes of additional 27 spots, mostly in the membranous fraction. We found enrichment of alpha (Galphao, Galphaq) and beta subunits (beta4/beta2 and beta5) of guanine nucleotide-binding proteins and of two calcium-binding proteins, calretinin and hippocalcin-like protein-1. In addition, we also detected a significant increase in the membrane fraction of proteins involved in exo-endocytosis such as septins, dynamin-1, and vesicle protein sorting 29. By western blotting, we confirmed increased membrane localization of calretinin and of dynamin-1 isoforms with higher isoelectric point, indicative for an underphosphorylated state of the molecule. In conclusion, our results indicate that chronic THC modulates the expression and subcellular localization of proteins implicated in Ras signaling, calcium-buffering potential, and trafficking.
Colombo
Graziano
G
Department of Biomolecular Sciences and Biotechnology, University of Milan, Via Celoria 26, 20133, Milan, Italy.
Rusconi
Francesco
F
Rubino
Tiziana
T
Cattaneo
Angela
A
Martegani
Enzo
E
Parolaro
Daniela
D
Bachi
Angela
A
Zippel
Renata
R
D000818:Animals; D002531:Cerebellum / Q000187:drug effects* / Q000502:physiology*; D013759:Dronabinol / Q000494:pharmacology*; D015180:Electrophoresis, Gel, Two-Dimensional; D015870:Gene Expression / Q000187:drug effects; D020869:Gene Expression Profiling; D051379:Mice; D008810:Mice, Inbred C57BL; D020411:Oligonucleotide Array Sequence Analysis; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D040901:Proteomics; D011619:Psychotropic Drugs / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D020666:ras-GRF1 / Q000235:genetics* / Q000378:metabolism
D011619:Psychotropic Drugs; C513037:Rasgrf1 protein, mouse; D020666:ras-GRF1; D013759:Dronabinol