Neurotox. neurotoxicity, and several pilot clinical BD-AcAc 2 trials have demonstrated cognitive improvements in HIV patients treated with GSK3 inhibitors. In addition to the GSK3 pathway, the CDK5 pathway has recently been implicated as a mediator of neurotoxicity in HIV, and HIV proteins might activate this pathway and subsequently disrupt the diverse processes that CDK5 regulates, including synapse formation and plasticity and neurogenesis. Taken together, the GSK3 and CDK5 signaling pathways are important regulators of neurotoxicity in HIV, and modulation of these factors might have therapeutic potential in the treatment of patients suffering from HIVE. In this context, the subsequent sections will focus on reviewing the involvement of the GSK3 and CDK5 pathways in neurodegeneration in HIV. studies in primary neurons and neuronal cell lines have shown that the neuroprotective effects of FGF1 and FGF2 are mediated by activation of PI3K-Akt that in turn inactivate GSK3 via phosphorylation at the Ser 9 residue [63,64]. In addition to FGF1 and FGF2, other growth factors that exert their effects via receptor tyrosine kinases also lead to inactivation of GSK3 through phosphorylation. These include growth factors such as insulin growth factor-1 (IGF-I), epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) [74,75]. To further investigate the neuroprotective effects of GSK3 regulation by FGF1 we generated lines of tg mice expressing KIAA0513 antibody the human FGF1 under a neuronal promoter (PDGF). Human FGF1 cDNA was obtained by reverse transcriptase polymerase chain reaction (RT-PCR) from human brains and cloned into PCRII vector (TA Cloning from Invitrogen, CA) and 100% fidelity of nucleotide sequence was confirmed by dideoxy sequencing. Subsequently the FGF1 cDNA fragment was subcloned into the PDGF transgene cassette. The PDGF promoter was a gift of Dr. Tucker Collins at Harvard Medical School. The final construct contains the PDGF promoter, SV40 intron, hFGFl cDNAs, and SV40 polyA (Figure 2A). Constructs were microinjected and 5 lines of BD-AcAc 2 founder BD-AcAc 2 mice were obtained. Of them, based on the levels of mRNA expression two transgenic lines (line 15 low expresser; line 12 moderate expresser) were selected. RPA and Western blot analysis showed that both lines expressed human (h)FGFl at levels comparable to the levels in the human brain (Figure 2BCF). Immunocytochemical analysis confirmed that hFGFl was primarily expressed by neurons in the neocortex, hippocampus and basal ganglia, regions selectively susceptible to the neurotoxic effects of HIV products. Both lines of hFGFl tg mice were viable, bred well and the nervous system developed normally. To determine the effects of FGF1 expression on the GSK3 signaling pathway, immunoblot analysis was performed with an antibody against phosphorylated GSK3. This showed that in the mouse line expressing moderate levels of hFGFl (line 12), levels of phosphorylated GSK3 (inactive form) were increased, while levels of pGSK3 in the low expresser line (15) were similar to nontg controls (Figure 2C). Open in a separate window Figure 2. Characterization of hFGFl tg mice, BD-AcAc 2 (a) Construct expressing hFGFl under the control of the PDGF promoter, (b) RPA analysis of FGF1 mRNA expression, (c) Immunoblot analysis of total FGF1 protein expression and inactivation of GSK3 in FGF1 tg mice, (d) Semi-quantitative analysis of hFGFl mRNA levels, (e) Semi-quantitative analysis of mFGFl mRNA levels, (e) Semi-quantitative analysis of total FGF1 protein expression by immunoblot. In order to test the hypothesis that hFGFl protects against the neurotoxic effects of HIV products, tg mice (3 mo old, 5 per group) from lines 12 and 15 received intracerebral gp120 injections (lmM, total 2l) in.