Pleiotrophin over-expression after intrastriatal and intranigral administration of a recombinant adenoviral vector containing human pleiotrophin cDNA

TARAVINI IR; CHERTOFF M; CAFFERATA E; MURER MG; PITOSSI F; GERSHANIK O

Congreso; 11th International Congress of Parkinson's Disease and Movement Disorders; 2007

The movement disorders society

Objective: I) To generate a recombinant adenoviral vector containing human pleiotrophin (PTN) cDNA, II) to induce the over-expression of PTN in the striatum and substantia nigra pars compacta (SNpc) of normal rats by using the adenovirus vector. Background: Clinical signs of Parkinson´s disease are not evident until 50% of SNpc neurons have died, and dopamine neuron degeneration continues after diagnosis, so up to 90% dopaminergic neurons die despite treatment. Therefore, there is strong interest in research on the cause of Parkinson´s disease and on the development of novel therapeutic strategies aimed at preventing the degenerative process or restoring the structure and function of the damaged nigroestriatal system. PTN was recognized as a neurite outgrowth-promoting factor expressed during embryonic and early postnatal developmentFurthermore, PTN is expressed along axon pathways and is believed to promote neuronal differentiation and the establishment of synaptic connections. Recently, we found an over-expression of PTN in the striatum of adult rats bearing a nigroestriatal lesion (experimental parkinsonism). Other studies determined that PTN promotes survival and differentiation of dopamine neurons from embryonic stem cells. Methods and Results: We generated a recombinant adenovirus that expresses PTN. The human PTN cDNA was cloned into a shuttle vector with a human cytomegalovirus promoter and cotransfected into HEK293 cells with a plasmid containing E1- to E3-deleted type 5 adenoviral genome using a calcium phosphate coprecipitation method. The correct recombination was verified with restriction digestions of the purified viral DNA obtained by HIRT. Stocks of the adenoviral vector were obtained from large-scale preparations in HEK293 cells and then purified by double CsCl gradient ultracentrifugation. The final titer was assayed for the ability to form plaques on 293 cell monolayers and by optical absorbance (TICD50, 2.15 x 109 pfu/µl). Then, AdPTN was injected in the striatum and SNpc of normal adult rats in order to induce PTN over-expression. Seven days after injection, PTN was detected by immunohistochemistry performed on free-floating brain tissue sections. In control animals injected with an adenoviral vector expressing  beta-galactosidase (Adb-gal), PTN expression was restricted to striatal interneurons and a few neurons in the SNpc, as shown before in normal rats. AdPTN was effective in inducing PTN over-expression at the injection place and over a wide area of the adjacent striatum and SNpc. Preliminary analysis suggests that both glia and neurons were over-expressing PTN. Conclusions: We have constructed a PTN-containing adenovirus which successfully over-expressed PTN in the injection place and in adjacent areas in vivo. This adenovirus will also be useful for further studies on the effects of PTN on the differentiation and survival of other cell populations, from stem cells to transplanted dopaminergic neurons. Additionally, future experiments will increase our understanding of the functions of PTN in the adult nigrostriatal innervation, and in particular, help us to determine the potential of PTN as a preventive or restorative therapy for Parkinson´s disease.