Glucocorticoid Receptors are Localized to Dendritic Spines and Influence Local Actin Signaling

Glucocorticoids affect learning and memory but the cellular mechanisms involved are poorly understood. The present studies tested if the stress-responsive glucocorticoid receptor (GR) is present and regulated within dendritic spines, and influences local signaling to the actin cytoskeleton. In hippocampal field CA1, 13?% of synapses contained GR-immunoreactivity. Three-dimensional reconstructions of CA1 dendrites showed that GR aggregates are present in both spine heads and necks. Consonant with evidence that GR?? mRNA associates with the translation regulator Fragile X Mental Retardation Protein (FMRP), spine GR levels were rapidly increased by group 1 mGluR activation and reduced in mice lacking FMRP. Treatment of cultured hippocampal slices with the GR agonist dexamethasone rapidly (15?C30?min) increased total levels of phosphorylated (p) Cofilin and extracellular signal-regulated kinase (ERK) 1/2, proteins that regulate actin polymerization and stability. Dexamethasone treatment of adult hippocampal slices also increased numbers of PSD95+ spines containing pERK1/2, but reduced numbers of pCofilin-immunoreactive spines. Dexamethasone-induced increases in synaptic pERK1/2 were blocked by the GR antagonist RU-486. These results demonstrate that GRs are present in hippocampal spines where they mediate acute glucocorticoid effects on local spine signaling. Through effects on these actin regulatory pathways, GRs are positioned to exert acute effects on synaptic plasticity.     

A cascade of morphogenic signaling initiated by the meninges controls corpus callosum formation

The corpus callosum is the most prominent commissural connection between the cortical hemispheres, and numerous neurodevelopmental disorders are associated with callosal agenesis. By using mice either with meningeal overgrowth or selective loss of meninges, we have identified a cascade of morphogenic signals initiated by the meninges that regulates corpus callosum development. The meninges produce BMP7, an inhibitor of callosal axon outgrowth. This activity is overcome by the induction of expression of Wnt3 by the callosal pathfinding neurons, which antagonize the inhibitory effects of BMP7. Wnt3 expression in the cingulate callosal pathfinding axons is developmentally regulated by another BMP family member, GDF5, which is produced by the adjacent Cajal-Retzius neurons and turns on before outgrowth of the callosal axons. The effects of GDF5 are in turn under the control of a soluble GDF5 inhibitor, Dan, made by the meninges. Thus, the meninges and medial neocortex use a cascade of signals to regulate corpus callosum development.     

HIV-1 Gag co-opts a cellular complex containing DDX6, a helicase that facilitates capsid assembly

To produce progeny virus, human immunodeficiency virus type I (HIV-1) Gag assembles into capsids that package the viral genome and bud from the infected cell. During assembly of immature capsids, Gag traffics through a pathway of assembly intermediates (AIs) that contain the cellular adenosine triphosphatase ABCE1 (ATP-binding cassette protein E1). In this paper, we showed by coimmunoprecipitation and immunoelectron microscopy (IEM) that these Gag-containing AIs also contain endogenous processing body (PB)-related proteins, including AGO2 and the ribonucleic acid (RNA) helicase DDX6. Moreover, we found a similar complex containing ABCE1 and PB proteins in uninfected cells. Additionally, knockdown and rescue studies demonstrated that the RNA helicase DDX6 acts enzymatically to facilitate capsid assembly independent of RNA packaging. Using IEM, we localized the defect in DDX6-depleted cells to Gag multimerization at the plasma membrane. We also confirmed that DDX6 depletion reduces production of infectious HIV-1 from primary human T cells. Thus, we propose that assembling HIV-1 co-opts a preexisting host complex containing cellular facilitators such as DDX6, which the virus uses to catalyze capsid assembly.     

A novel split kinesin assay identifies motor proteins that interact with distinct vesicle populations

Identifying the kinesin motors that interact with different vesicle populations is a longstanding and challenging problem with implications for many aspects of cell biology. Here we introduce a new live-cell assay to assess kinesin-vesicle interactions and use it to identify kinesins that bind to vesicles undergoing dendrite-selective transport in cultured hippocampal neurons. We prepared a library of "split kinesins," comprising an axon-selective kinesin motor domain and a series of kinesin tail domains that can attach to their native vesicles; when the split kinesins were assembled by chemical dimerization, bound vesicles were misdirected into the axon. This method provided highly specific results, showing that three Kinesin-3 family members-KIF1A, KIF13A, and KIF13B-interacted with dendritic vesicle populations. This experimental paradigm allows a systematic approach to evaluate motor-vesicle interactions in living cells.     

The Interaction between Polynucleotide Kinase Phosphatase and the DNA Repair Protein XRCC1 Is Critical for Repair of DNA Alkylation Damage and Stable Association at DNA Damage Sites

XRCC1 plays a key role in the repair of DNA base damage and single-strand breaks. Although it has no known enzymatic activity, XRCC1 interacts with multiple DNA repair proteins and is a subunit of distinct DNA repair protein complexes. Here we used the yeast two-hybrid genetic assay to identify mutant versions of XRCC1 that are selectively defective in interacting with a single protein partner. One XRCC1 mutant, A482T, that was defective in binding to polynucleotide kinase phosphatase (PNKP) not only retained the ability to interact with partner proteins that bind to different regions of XRCC1 but also with aprataxin and aprataxin-like factor whose binding sites overlap with that of PNKP. Disruption of the interaction between PNKP and XRCC1 did not impact their initial recruitment to localized DNA damage sites but dramatically reduced their retention there. Furthermore, the interaction between PNKP and the DNA ligase IIIα-XRCC1 complex significantly increased the efficiency of reconstituted repair reactions and was required for complementation of the DNA damage sensitivity to DNA alkylation agents of xrcc1 mutant cells. Together our results reveal novel roles for the interaction between PNKP and XRCC1 in the retention of XRCC1 at DNA damage sites and in DNA alkylation damage repair.     

TIMP1 contributes to ovarian anomalies in both an MMP-dependent and -independent manner in a rat model

Ovulatory dysfunction occurs in women with endometriosis, yet the mechanisms are unknown. We have shown that endometriotic lesions synthesize and secrete tissue inhibitor of metalloproteinase (TIMP) 1 into the peritoneal cavity in humans and a rat model of endometriosis, where excess TIMP1 localizes in the ovarian theca in endometriosis and modulating peritoneal TIMP1 alters ovarian dynamics. Here, we evaluated whether mechanisms whereby excessive peritoneal fluid TIMP1 negatively impacts ovarian function are matrix metalloproteinase (MMP)-dependent and/or MMP-independent actions. Rats were treated with a mutated TIMP1 without MMP inhibitory function (Ala-TIMP1), wild-type TIMP1 (rTIMP1), or PBS. Rats treated with Ala-TIMP1 or rTIMP1 had fewer antral follicles, fewer new corpora lutea, and the presence of luteinized unruptured follicle syndrome compared with PBS rats. Ala-TIMP1 and rTIMP1 differentially caused downstream changes in gene expression and protein localization related to ovulation, as measured by whole-genome microarray with quantitative real-time PCR validation and immunohistochemistry. More vascular endothelial growth factor and FN were expressed and localized in ovaries of Ala-TIMP1-treated rats compared to rTIMP1- and PBS-treated rats inferring MMP-independent functions. Less caspase 3 localized in ovaries of rTIMP1 compared with the other two groups, and was thus dependent on MMP action. Furthermore, after coimmunoprecipitation, more CD63 was bound to TIMP1 in ovaries of rats treated with Ala-TIMP1 than in rTIMP1-treated rats, providing evidence for another MMP-independent mechanism of ovulatory dysfunction. We predict that MMP-dependent and MMP-independent events are involved in improper fortification of the follicular wall through multiple mechanisms, such as apoptosis inhibition, extracellular matrix components and angiogenesis. Collectively, excessive peritoneal TIMP1 causes changes in ovarian dynamics, both dependently and independently of MMP inhibition.     

Effect of aging on superovulation efficiency, aneuploidy rates, and sister chromatid cohesion in mice aged up to 15 months

Human eggs are highly aneuploid, with female age being the only known risk factor. Here this aging phenomenon was further studied in Swiss CD1 mice aged between 1 and 15 mo. The mean number of eggs ± SEM recovered from mice following superovulation peaked at 22.5 ± 3.8 eggs/oviduct in 3-mo-old females, decreasing markedly between 6 and 9 mo old, and was only 2.1 ± 0.2 eggs/oviduct by 15 mo. Measurement of aneuploidy in these eggs revealed a low rate, ～3-4%, in mice aged 1 and 3 mo, rising to 12.5% by 9 mo old and to 37.5% at 12 mo. Fifteen-month-old mice had the highest rate of aneuploidy, peaking at 60%. The in situ chromosome counting technique used here allowed us to measure with accuracy the distance between the kinetochores in the sister chromatids of the eggs analyzed for aneuploidy. We observed that this distance increased in eggs from older females, from 0.38 ± 0.01 μm at 1 mo old to 0.82 ± 0.03 μm by 15 mo. Furthermore, in 3- to 12-mo-old females, aneuploid eggs had significantly larger interkinetochore distances than euploid eggs from the same age, and measurements were similar to eggs from the oldest mice. However, the association between aneuploidy and interkinetochore distance was not observed at the oldest, 15-mo age, despite such measurements being maximal. We conclude that in aging CD1 mice, a reduction in the ovulated egg number precedes a rise in aneuploidy and, furthermore, except at very advanced ages, increased interkinetochore distance is associated with aneuploidy.     

Elucidation and chemical modulation of sulfolipid-1 biosynthesis in Mycobacterium tuberculosis

Mycobacterium tuberculosis possesses unique cell-surface lipids that have been implicated in virulence. One of the most abundant is sulfolipid-1 (SL-1), a tetraacyl-sulfotrehalose glycolipid. Although the early steps in SL-1 biosynthesis are known, the machinery underlying the final acylation reactions is not understood. We provide genetic and biochemical evidence for the activities of two proteins, Chp1 and Sap (corresponding to gene loci rv3822 and rv3821), that complete this pathway. The membrane-associated acyltransferase Chp1 accepts a synthetic diacyl sulfolipid and transfers an acyl group regioselectively from one donor substrate molecule to a second acceptor molecule in two successive reactions to yield a tetraacylated product. Chp1 is fully active in vitro, but in M. tuberculosis, its function is potentiated by the previously identified sulfolipid transporter MmpL8. We also show that the integral membrane protein Sap and MmpL8 are both essential for sulfolipid transport. Finally, the lipase inhibitor tetrahydrolipstatin disrupts Chp1 activity in M. tuberculosis, suggesting an avenue for perturbing SL-1 biosynthesis in vivo. These data complete the SL-1 biosynthetic pathway and corroborate a model in which lipid biosynthesis and transmembrane transport are coupled at the membrane-cytosol interface through the activity of multiple proteins, possibly as a macromolecular complex.