CRMP2 is hyperphosphorylated and dendritic development is impaired in primary neurons over-expressing p35 alone or in combination with HIV-gp120 protein treatment. Primary rat hippocampal neurons were infected two days after plating with p35-adv, followed by treatment with recombinant HIV-gp120 protein or vehicle control. Cells were fixed on day 4 of differentiation for double-immunolabeling analysis with antibodies against MAP2 (dendritic marker) and pSer522-CRMP2 (pSer-CRMP2). (A-C) MAP2-positive primary neurons treated with vehicle control express background levels of pSer-CRMP2. (D-F) Primary neurons infected with p35-adv display increased pSer-CRMP2 immunoreactivity and reduced MAP2-immunoreactive dendrites. (G-I) Primary neurons treated with recombinant HIV-gp120 display increased pSer-CRMP2 immunoreactivity and reduced MAP2-immunoreactive dendrites, with an accumulation of pSer-CRMP2 immunoreactivity in varicosities along the neuronal processes (arrows). (J-L) Primary neurons infected with p35-adv and treated with recombinant HIV-gp120 display increased pSer-CRMP2 immunoreactivity and extensive damage to MAP2-immunoreactive dendrites, with an abundant accumulation of pSer-CRMP2 immunoreactivity in varicosities along the neuronal processes (arrows). (M, N) Semi-quantitative image analysis of pSer-CRMP2 (M) and MAP2 (N) immunoreactivity. (O) Immunoblot analysis of total cell lysates showing increased CRMP2 phosphorylation in primary hippocampal neurons infected with p35-adv or treated with HIV-gp120 protein. (P) Semi-quantitative image analysis of fold change in CRMP2 phosphorylation at the CDK5 epitope (Ser522) in immunoblots from cells expressing p35-adv or treated with gp120. * p < 0.05 compared to vehicle-treated controls by one-way ANOVA with post-hoc Dunnett's test. Scale bar = 20 μm.