The inability of either X4- or R5-using proteins to directly change levels of FHC in neurons suggests that known intracellular mediators of gp120, such as p38 and caspase-3 (Singh et al., 2005; Bardi et al., 2006), may not be directly involved in the regulation of FHC. FHC, gp120 only caused significant FHC upregulation in neuronal/glial cocultures, suggesting that glia are necessary for sustained elevation of neuronal FHC by the viral protein. Although the envelope protein induced secretion of IL-1 and TNF- in cocultures, TNF- blockade did not affect gp120-mediated induction of FHC. Conversely, studies with an IL-1 neutralizing antibody or specific IL-1 receptor antagonist revealed the primary involvement of IL-1 in gp120-induced FHC changes. Furthermore, silencing of neuronal FHC abrogates the effect of gp120 on spines, and spine density correlates negatively with FHC levels or cognitive deficit. These results demonstrate that viral and host components of HIV infection increase brain expression of FHC, leading to cellular and functional changes, and point to IL-1-targeted strategies for prevention of these alterations. SIGNIFICANCE STATEMENT This work demonstrates the key role of the cytokine IL-1 in the regulation of a novel intracellular mediator [i.e., the protein ferritin heavy chain (FHC)] of HIV-induced dendritic damage and the resulting neurocognitive impairment. This is also the first study that systematically investigates dendritic damage in layer II/III prefrontal cortex neurons of two different non-infectious models of HIV-associated neurocognitive disorders (HAND) and reveals a precise correlation of these structural changes with specific biochemical and functional alterations also reported in HIV patients. Overall, these data suggest that targeting H-1152 the IL-1-dependent FHC increase may represent a valid strategy for neuroprotective adjuvant therapies in HAND. effect of viral protein expression on dendritic branching and spines. The HIV Tg rats express seven of the nine HIV-1 proteins, including the envelope protein gp120 (Reid et al., 2001). Specifically, a 3 kbp SphICMscI fragment encompassing the 3 region of and the 5 region of was removed from pNL4-3, an infectious proviral plasmid, to make the non-infectious HIV-1clone pEVd1443 (Reid et al., 2001). Because of immunodeficiency, these animals were housed in isolation in our barrier facility. As reported in the literature, HIV Tg rats develop inflammatory and neuropathological features similar to those observed in HIV-associated neurocognitive disorders (HAND), including elevated levels of proinflammatory cytokines and behavioral problems indicative of cognitive deficits (Vigorito et al., 2007; Lashomb et al., 2009; Repunte-Canonigo et al., 2014). Moreover, this non-infectious model recapitulates other important features of cART-treated HIV patients, such as immune dysregulation and controlled viral replication (Peng et al., 2010). Finally, SD rats were used for the studies focused on gp120, as described next. Cannula implant and gp120 infusions. Male SD rats (200C400 g) were SFRP1 implanted stereotaxically, under isoflurane anesthesia, with stainless-steel cannulas placed 0.96 mm posterior and 3.18 mm lateral to bregma, 3.47 mm below the surface of the cranium at an angle of 22. Four stainless-steel screws (#0-80) were placed around the cannula, and acrylic dental cement was used to anchor the cannula. Animals were allowed 7 d to recover before receiving a bilateral infusion of 1 1 l of gp120IIIB (50 ng/l in 0.1% BSA) once daily for 7 d via a 10 l H-1152 Hamilton syringe (needle diameter, 0.48 mm; infusion rate, 0.374 l/min). Fluorescent tracers were used to assess diffusion after bilateral intracerebroventricular infusion via implanted stainless-steel cannulas and spread of the fluorescent tracers throughout the ventricular system (for representative images, see Fig. 2come from four neurons per brain (i.e., control, = 24; gp120, = 20). 0.01; red symbols denote 0.001. Neuronal cultures. Rat cortical neurons were cultured in serum-free media in the presence of a glial feeder layer, as we described previously (Shimizu et al., 2011). This culture system primarily preserves the environment because of the presence of glia; however, it still enables direct investigation of neurons that H-1152 can be separated from the glia before experiments. Selected studies were also performed in neurons grown in the absence of glia for the entire culture period, as indicated and defined as Neurobasal culture, and using previously established protocols (Sengupta et al., 2009). Neurons were typically used in weeks 2C3 of culture. For spine analyses, neurons were transfected.