As the human life span increases, the number of people suffering from cognitive decline is rising dramatically. The mechanisms underlying age-associated memory impairment are, however, not understood. Here we show that memory disturbances in the aging brain of the mouse are associated with altered hippocampal chromatin plasticity. During learning, aged mice display a specific deregulation of histone H4 lysine 12 (H4K12) acetylation and fail to initiate a hippocampal gene expression program associated with memory consolidation. Restoration of physiological H4K12 acetylation reinstates the expression of learning-induced genes and leads to the recovery of cognitive abilities. Our data suggest that deregulated H4K12 acetylation may represent an early biomarker of an impaired genome-environment interaction in the aging mouse brain.

With the increasing interest of RNAs in regulating a range of cell biological processes, very little is known about the structure of RNAs in tissue culture cells. We focused on the 5 -untranslated region of the human immunodeficiency virus type 1 RNA genome, a highly conserved RNA region, which contains structural domains that regulate key steps in the viral replication cycle. Up until now, structural information only came from in vitro studies. Here, we developed chemical modification assays to test nucleotide accessibility directly in infected cells and viral particles, thus circumventing possible biases and artifacts linked to in vitro assays. The secondary structure of the 5 -untranslated region in infected cells points to the existence of the various stemloop motifs associated to distinct functions, proposed from in vitro probing, mutagenesis, and phylogeny. However, compared with in vitro data, subtle differences were observed in the dimerization initiation site hairpin, and none of the proposed long range interactions were observed between the functional domains. Moreover, no global RNA rearrangement was observed; structural differences between infected cells and viral particles were limited to the primer binding site, which became protected against chemical modification upon tRNA 3 Lys annealing in virions and to the main packaging signal. In addition, our data suggested that the genomic RNA could already dimerize in the cytoplasm of infected cells. Taken together, our results provided the first analysis of the dynamic of RNA structure of the human immunodeficiency virus type 1 RNA genome during virus assembly ex vivo.

The vif gene of human immunodeficiency virus type 1 (HIV-1) is essential for viral replication, although the functional target of Vif remains elusive. HIV-1 vif mutant virions derived from nonpermissive H9 cells displayed no significant differences in the amount, ratio, or integrity of their protein composition relative to an isogenic wild-type virion. The amounts of the virion-associated viral genomic RNA and tRNA 3Lys were additionally present at normal levels in vif mutant virions. We demonstrate that Vif associates with RNA in vitro as well as with viral genomic RNA in virus-infected cells. A functionally conserved lentivirus Vif motif was found in the double-stranded RNA binding domain of Xenopus laevis, Xlrbpa. The natural intravirion reverse transcriptase products were markedly reduced in vif mutant virions. Moreover, purified vif mutant genomic RNA-primer tRNA complexes displayed severe defects in the initiation of reverse transcription with recombinant reverse transcriptase. These data point to a novel role for Vif in the regulation of efficient reverse transcription through modulation of the virion nucleic acid components.

The vif gene of human immunodeficiency virus type 1 (HIV-1) is essential for the productive infection of primary blood-derived lymphocytes, macrophages, and certain human T-cell lines. It has been shown that Vif is associated with HIV-1 virions purified by sucrose density-equilibrium gradient analysis. However, the specificity of Vif incorporation into virions has not been determined. Moreover, recent studies have demonstrated that standard HIV-1 particle preparations created with sucrose density-equilibrium gradients are contaminated with cell-derived microvesicles. Here we demonstrate, as previously reported, that Vif cosediments with HIV-1 particles in sucrose density-equilibrium gradient analysis. However, we also found that, when Vif was expressed in the absence of all other HIV-1-encoded gene products and then isolated by sucrose density-equilibrium gradient centrifugation from extracellular supernatants, its sedimentation pattern was largely unaltered, suggesting that Vif can be secreted from cells. Using a newly developed OptiPrep velocity gradient method, we were able to physically separate most of the extracellular Vif from the HIV-1 virions without disrupting the infectivity of the virus. By titrating serial dilutions of purified Vif and Gag against the viral peak fraction in the OptiPrep gradient, we demonstrate that <1.0 Vif molecule per virion was present. This study shows that Vif is not significantly present in HIV-1 virions, a finding which is consistent with the idea that Vif functions predominantly in the virus-producing cells during virus assembly. The OptiPrep velocity gradient technique described here could be an easy and rapid way to purify HIV and other enveloped viruses from microvesicles and/or cell debris.

Packaging of the human immunodeficiency virus type 1 Vif protein into virus particles is mediated through an interaction with viral genomic RNA and results in the association of Vif with the nucleoprotein complex. Despite the specificity of this process, calculations of the amount of Vif packaged have produced vastly different results. Here, we compared the efficiency of packaging of Vif into virions derived from acutely and chronically infected H9 cells. We found that Vif was efficiently packaged into virions from acutely infected cells (60 to 100 copies per virion), while packaging into virions from chronically infected H9 cells was near the limit of detection (four to six copies of Vif per virion). Superinfection by an exogenous Vif-defective virus did not rescue packaging of endogenous Vif expressed in the chronically infected culture. In contrast, exogenous Vif expressed by superinfection of wild-type virus was readily packaged (30 to 40 copies per virion). Biochemical analyses suggest that the differences in the relative packaging efficiencies were not due to gross differences in the steady-state distribution of Vif in chronically or acutely infected cells but are likely due to differences in the relative rates of de novo synthesis of Vif. Despite its low packaging efficiency, endogenously expressed Vif was sufficient to direct the production of viruses with almost wild-type infectivity. The results from our study provide novel insights into the biochemical properties of Vif and offer an explanation for the reported differences regarding Vif packaging.

Proper limb development requires concerted communication between cells within the developing limb bud. Several molecules have been identified which contribute to the formation of a circuitry loop consisting in large part of secreted proteins. The intracellular actin nucleator, Formin 1 (Fmn1), has previously been implicated in limb development, but questions remain after the identification of a Gremlin transcriptional enhancer within the 3' end of the Fmn 1 locus. To resolve this issue, a knockout mouse devoid of Fmn1 protein was created and characterized. The mice exhibit a reduction of digit number to four, a deformed posterior metatarsal, phalangeal soft tissue fusion as well as the absence of a fibula to 100% penetrance in the FVB genetic background. Importantly, this mutant allele does not genetically disrupt the characterized Gremlin enhancer, and indeed Gremlin RNA expression is upregulated at the 35 somite stage of development. Our data reveal increased Bone Morphogenetic Protein (Bmp) activity in mice which carry a disruption in Fmn1, as evidenced by upregulation of Msx1 and a decrease in Fgf4 within the apical ectodermal ridge. Additionally, these studies show enhanced activity downstream of the Bmp receptor in cells where Fmn1 is perturbed, suggesting a role for Fmn1 in repression of Bmp signaling.

Age‐associated memory decline is due to variable combinations of genetic and environmental risk factors. How these risk factors interact to drive disease onset is currently unknown. Here we begin to elucidate the mechanisms by which post‐traumatic stress disorder (PTSD) at a young age contributes to an increased risk to develop dementia at old age. We show that the actin nucleator Formin 2 (Fmn2) is deregulated in PTSD and in Alzheimer's disease (AD) patients. Young mice lacking the Fmn2 gene exhibit PTSD‐like phenotypes and corresponding impairments of synaptic plasticity, while the consolidation of new memories is unaffected. However, Fmn2 mutant mice develop accelerated age‐associated memory decline that is further increased in the presence of additional risk factors and is mechanistically linked to a loss of transcriptional homeostasis. In conclusion, our data present a new approach to explore the connection between AD risk factors across life span and provide mechanistic insight to the processes by which neuropsychiatric diseases at a young age affect the risk for developing dementia.

BackgroundRegulation of the cytoskeleton is a central feature of cell migration. The formin family of proteins controls the rate of actin nucleation at its barbed end. Thus, formins are predicted to contribute to several important cell processes such as locomotion, membrane ruffling, vesicle endocytosis, and stress fiber formation and disassociation.Methodology/Principal FindingsIn this study we investigated the functional role of Formin1-isoform4 (Fmn1-IV) by using genetically null primary cells that displayed augmented protrusive behaviour during wound healing and delayed cell spreading. Cells deficient of Fmn1-IV also showed reduced efficiency of focal adhesion formation. Additionally, we generated an enhanced green fluorescence protein (EGFP)-fused Fmn1-IV knock-in mouse to monitor the endogenous subcellular localization of Fmn1-IV. Its localization was found within the cytoplasm and along microtubules, yet it was largely excluded from adherens junctions.Conclusions/SignificanceIt was determined that Fmn1-IV, as an actin nucleator, contributes to protrusion of the cell's leading edge and focal adhesion formation, thus contributing to cell motility.