Comparative non-radioactive RT-PCR assay: an approach to study the neurosteroids biosynthetic pathway in humans.
Polymerase chain reaction (PCR) is a powerful tool for qualitative evaluation of nucleic acid expression. PCR has been widely applied to measure DNA and RNA messages expression. Neurosteroids synthesized in the nervous system are potent modulators of synaptic activity and have been implicated in several neuropsychiatric disorders. To examine the possibility of an altered expression of the neurosteroidogenic metabolic enzymes in neurological diseases (like Parkinson's disease, PD) we developed a comparative non-radioactive RT-PCR assay to detect the mRNA levels of the peripheral benzodiazepine receptor, the 5alpha-reductase type 1 and 3alpha-hydroxysteroid-oxidoreductase type 1 and 2 in lymphocytes obtained from PD patients. The results were compared with that obtained from simultaneous quantification of progesterone, 5alpha-dihydroprogesterone and 3alpha,5alpha-tetrahydroprogesterone in the plasma and cerebro-spinal fluid of the same individuals using a gas chromatography mass spectrometry (GC/MS) technique. We found a significant decrease of the rate-limiting enzyme 5alpha-R1 along with a significant decrease in plasma and CSF of the 3alpha,5alpha-tetrahydroprogesterone and of the 5alpha-dihydroprogesterone. Comparative RT-PCR assay, along with complimentary techniques (i.e. GC/MS), has the sensitivity, selectivity and dynamic range to allow specific and reliable quantization of the enzymes involved in the neurosteroids pathway and represent a valuable tool to assess their expression in human neuropsychiatric conditions.
Luchetti
Sabina
S
IRCCS S. Lucia Foundation, Rome, Italy.
di Michele
Flavia
F
Romeo
Elena
E
Brusa
Livia
L
Bernardi
Giorgio
G
Cummings
Brian J
BJ
Longone
Patrizia
P
D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific) / Q000235:genetics / Q000378:metabolism; D000243:Adenosine Deaminase / Q000235:genetics / Q000378:metabolism; D000328:Adult; D000368:Aged; D001773:Blood Cells / Q000378:metabolism; D015152:Blotting, Northern; D001921:Brain / Q000378:metabolism; D002460:Cell Line; D042944:Cholestenone 5 alpha-Reductase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008214:Lymphocytes / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D010300:Parkinson Disease / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D012333:RNA, Messenger / Q000096:biosynthesis*; D016601:RNA-Binding Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013256:Steroids / Q000096:biosynthesis*
D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D013256:Steroids; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific); D042944:Cholestenone 5 alpha-Reductase; C567955:ADARB1 protein, human; D000243:Adenosine Deaminase