Adenoviral and lentiviral vectors to study adult neurogenesis and cellular reprogramming

F.J. PITOSSI

Workshop; 2do. SIMPOSIO FRANCO-ARGENTINO DE NEUROCIENCIAS; 2012

Adenoviral and lentiviral vectors to study adult neurogenesis and cellular reprogramming F. J. PITOSSI Leloir Foundation-CONICET, Buenos Aires, Argentina Adenoviral and lentiviral vectors can transduce non-dividing cells such as neurons using different strategies. The adenoviral genome enters the nucleus and does not integrate into the host genome, rendering its transduction independent of the dividing state of the cell. This non-integrative property of adenoviral vectors determines that its genetic information gets diluted in each daughter cell after cell division. Lentiviral vectors, as all retroviral vectors, must integrate into the host genome in order to express its genes. Some retroviral vectors, such as the one derived from the Moloney Murine Leukemia Virus, can only enter the nucleus and integrate its genome when the nuclear pore is disrupted during cell division, rendering these vectors incapable to transduce non-dividing cells. However, lentiviral vectors posses a specific molecular mechanism to enter the nucleus of the host cell and therefore can access the host genome for integration in dividing and non-dividing cells. Since these vectors integrate their genetic information, transduced genes remain in all daughter cells after cell division. Taking advantage of their unique characteristics, they have proven to be powerful tools for the delivery of genes in the brain and in vitro. We have shown that a prenatal lypopolisaccharide (LPS) challenge could alter the brain environment of adult neural stem cells and diminish adult neurogenesis.Proliferation and total progenitor cell population remained unchanged as measured by counting the number of Nestin-positive cells. Moreover, adult microglial activation was observed only in the prenatally LPS-treated group. Cytokine expression analysis by real-time RT-PCR revealed a decrease in TGF-beta expression in the adult hippocampi of LPS-treated rats. Over-expression of TGF-beta in the adult hippocampi of prenatally LPS-treated rats using adenoviral vectors not only reverted the decrease in adult neurogenesis but also return to basal levels the performance on a behavioural task associated with adult neurogenesis (Novel object recognition).We conclude that prenatal LPS challenge decreases adult neurogenesis and novel object recognition perfomance. This effect involves long lasting changes in the local microenvironment towards a pro- inflammatory milleu. TGF-beta plays a key role in the prenatally-induced decreases in adult neurogenesis. Using lentiviral vectors expressing Oct4, Klf-4, Sox-2 and c-myc we have reprogrammed adult fibroblasts to a pluripotent state using as little as 1 infective particle/100 cells transduced.