Neuroactive steroids are endogenous neuromodulators synthesized in the brain, adrenal glands, and gonads. Systemic administration of neuroactive steroids can alter alcohol reinforcement and consumption in rodents. Neuroactive steroids that positively modulate gamma-aminobutyric acid type A receptors produce central nervous system inhibition and anxiolytic effects, which may be therapeutic during alcohol withdrawal. Pregnenolone is the precursor of all other neuroactive steroids. Recent data from our laboratories has demonstrated that systemic administration of pregnenolone decreases alcohol self-administration in alcohol-preferring (P) rats. To produce long term increases in neuroactive steroids an adeno-associated serotype 2 viral vector (AAV2) expressing the mitochondrial cytochrome P450 side chain cleavage (P450scc) enzyme was developed. P450scc initiates the neuroactive steroid synthetic pathway by converting cholesterol to pregnenolone. Overexpression of P450scc should allow us to investigate how sustained increases in steroidogenesis isolated to specific brain regions affect alcohol-related behaviors. In vitro analysis confirmed that cultured cerebral cortical neurons infected with the AAV2-P450scc vector displayed an approximate 94% increase in P450scc mRNA expression and pregnenolone levels were elevated by approximately 530% in the cell media compared to cells infected with a control vector expressing green fluorescent protein (GFP). To confirm AAV2 vector P450scc gene transduction in vivo, we stereotaxically microinjected the AAV2-P450scc or AAV2-GFP vectors bilaterally into the nucleus accumbens shell of wistar rats at a volume of 2 or 4 microL/hemisphere. One week post-injection animals were sacrificed and the nucleus accumbens was dissected to determine P450scc mRNA and protein expression. The AAV2-P450scc vector increased both P450scc mRNA and protein levels in the nucleus accumbens shell in a vector concentration dependent manner. The maximal increase in P450scc mRNA levels was 573 ± 126% (p < 0.01) while P450scc protein was increased by 172 ± 69% (p < 0.05). Studies are currently in progress to determine if the AAV2-P450scc vector alters operant ethanol self-administration, general locomotor activity, and anxiety-like behaviors. These experiments may lead to the development of new therapeutic strategies for treating alcoholism.
Development of an adeno-associated viral vector to increase neuroactive steroid production in specific brain regions
Porcu P;
2010
Abstract
Neuroactive steroids are endogenous neuromodulators synthesized in the brain, adrenal glands, and gonads. Systemic administration of neuroactive steroids can alter alcohol reinforcement and consumption in rodents. Neuroactive steroids that positively modulate gamma-aminobutyric acid type A receptors produce central nervous system inhibition and anxiolytic effects, which may be therapeutic during alcohol withdrawal. Pregnenolone is the precursor of all other neuroactive steroids. Recent data from our laboratories has demonstrated that systemic administration of pregnenolone decreases alcohol self-administration in alcohol-preferring (P) rats. To produce long term increases in neuroactive steroids an adeno-associated serotype 2 viral vector (AAV2) expressing the mitochondrial cytochrome P450 side chain cleavage (P450scc) enzyme was developed. P450scc initiates the neuroactive steroid synthetic pathway by converting cholesterol to pregnenolone. Overexpression of P450scc should allow us to investigate how sustained increases in steroidogenesis isolated to specific brain regions affect alcohol-related behaviors. In vitro analysis confirmed that cultured cerebral cortical neurons infected with the AAV2-P450scc vector displayed an approximate 94% increase in P450scc mRNA expression and pregnenolone levels were elevated by approximately 530% in the cell media compared to cells infected with a control vector expressing green fluorescent protein (GFP). To confirm AAV2 vector P450scc gene transduction in vivo, we stereotaxically microinjected the AAV2-P450scc or AAV2-GFP vectors bilaterally into the nucleus accumbens shell of wistar rats at a volume of 2 or 4 microL/hemisphere. One week post-injection animals were sacrificed and the nucleus accumbens was dissected to determine P450scc mRNA and protein expression. The AAV2-P450scc vector increased both P450scc mRNA and protein levels in the nucleus accumbens shell in a vector concentration dependent manner. The maximal increase in P450scc mRNA levels was 573 ± 126% (p < 0.01) while P450scc protein was increased by 172 ± 69% (p < 0.05). Studies are currently in progress to determine if the AAV2-P450scc vector alters operant ethanol self-administration, general locomotor activity, and anxiety-like behaviors. These experiments may lead to the development of new therapeutic strategies for treating alcoholism.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
