Xiaonan Xin

Behavioral and morphological responses to cocaine require Kalirin7

BACKGROUND Long-lasting increases in dendritic spine density and gene expression in the nucleus accumbens and in the ambulatory response to cocaine occur following chronic cocaine treatment. Despite numerous reports of these findings, the molecular mechanisms leading to these morphological, biochemical, and behavioral changes remain unclear. METHODS We used mice genetically lacking Kalirin7 (Kal7(KO)), a Rho guanine nucleotide exchange factor that regulates dendritic spine formation and function. Both wild-type (Wt) and Kal7(KO) mice were given high-dose cocaine (20 mg/kg) for 4 or 8 consecutive days. Locomotor sensitization and conditioned place preference elicited by cocaine were evaluated. The nucleus accumbens core was diolistically labeled and spine density and morphology were quantified using confocal microscopy. RESULTS Cocaine increased Kalirin7 messenger RNA and protein expression in the nucleus accumbens of Wt mice. The Kal7(KO) animals showed greater locomotor sensitization to cocaine than Wt mice. In contrast, Kal7(KO) mice exhibited decreased place preference for cocaine, despite displaying a normal place preference for food. While Wt mice showed a robust increase in dendritic spine density after 4 and 8 days of cocaine treatment, dendritic spine density failed to increase in cocaine-exposed Kal7(KO) mice. Wild-type mice treated with cocaine for 8 days exhibited larger dendritic spines than cocaine-treated Kal7(KO) mice. CONCLUSIONS Kalirin7 is an essential determinant of dendritic spine formation following cocaine treatment. The absence of this single isoform of one of the many Rho guanine nucleotide exchange factors expressed in the nucleus accumbens results in enhanced locomotor sensitization and diminished place preference in response to cocaine.

Cdk5 and Trio modulate endocrine cell exocytosis

Hormone secretion by pituitary cells is decreased by roscovitine, an inhibitor of cyclin-dependent kinase 5 (Cdk5). Roscovitine treatment reorganizes cortical actin and ultrastructural analysis demonstrates that roscovitine limits the ability of secretory granules to approach the plasma membrane or one another. Trio, a multifunctional RhoGEF expressed in pituitary cells, interacts with peptidylglycine α-amidating monooxygenase, a secretory granule membrane protein known to affect the actin cytoskeleton. Roscovitine inhibits the ability of Trio to activate Rac, and peptides corresponding to the Cdk5 consensus sites in Trio are phosphorylated by Cdk5. Together, these data suggest that control of the cortical actin cytoskeleton, long known to modulate hormone exocytosis and subsequent endocytosis, involves Cdk5-mediated activation of Trio.

Cloning and characterization of novel PDE4D isoforms PDE4D6 and PDE4D7.

We report here the cloning and characterization of two novel PDE4D isoforms, PDE4D6 and PDE4D7. PDE4D6 is a supershort form and PDE4D7 a long form of PDE4D. In addition, we have identified another novel long-form variant, PDE4D8, in silico. Like other isoforms, PDE4D6 and PDE4D7 are differentially expressed. Expression of PDE4D6 is restricted to brain whereas PDE4D7 is widely expressed in many tissues. Baculovirus-expressed recombinant PDE4D6 and PDE4D7 enzymes have high affinity for cyclic AMP (cAMP) and are inhibited by rolipram. The activity of PDE4D7, not PDE4D6, is elevated by a protein kinase A (PKA)-dependent mechanism, presumably through phosphorylation of the conserved PKA site in the upstream conserved region 1 (UCR1) domain. In agreement with early reports, human PDE4D6 and PDE4D7 are localized on genomic fragments of chromosome 5. Examination of the promoter regions reveals multiple CREB binding sites upstream of the starting methionine (Met) of each gene, suggesting that the cAMP/PKA signaling pathway may regulate transcriptional expression of PDE4D6 and PDE4D7.

Regulation of Kalirin by Cdk5

Kalirin, one of the few Rho guanine nucleotide exchange factors (GEFs) that contains spectrin-like repeats, plays a critical role in axon extension and maintenance of dendritic spines. PC12 cells were used to determine whether Cdk5, a critical participant in both processes, regulates the action of Kalirin. Expression of Kalirin-7 in nondifferentiated PC12 cells caused GEF-activity-dependent extension of broad cytoplasmic protrusions; coexpression of dominant-negative Cdk5 largely eliminated this response. The spectrin-like repeat region of Kalirin plays an essential role in this response, which is not mimicked by the GEF domain alone. Thr1590, which follows the first GEF domain of Kalirin, is the only Cdk5 phosphorylation site in Kalirin-7. Although mutant Kalirin-7 with Ala1590 retains GEF activity, it is unable to cause extension of protrusions. Kalirin-7 with an Asp1590 mutation has slightly increased GEF activity and dominant-negative Cdk5 fails to block its ability to cause extension of protrusions. Phosphorylation of Thr1590 causes a slight increase in GEF activity and Kalirin-7 solubility. Dendritic spines formed by cortical neurons in response to the expression of Kalirin-7 with Ala1590 differ in shape from those formed in response to wild-type Kalirin-7 or Kalirin-7 containing Asp1590. The presence of Thr1590 in each major Kalirin isoform would allow Cdk5 to regulate Kalirin function throughout development.

Kalirin12 interacts with dynamin

BackgroundGuanine nucleotide exchange factors (GEFs) and their target Rho GTPases regulate cytoskeletal changes and membrane trafficking. Dynamin, a large force-generating GTPase, plays an essential role in membrane tubulation and fission in cells. Kalirin12, a neuronal RhoGEF, is found in growth cones early in development and in dendritic spines later in development.ResultsThe IgFn domain of Kalirin12, not present in other Kalirin isoforms, binds dynamin1 and dynamin2. An inactivating mutation in the GTPase domain of dynamin diminishes this interaction and the isolated GTPase domain of dynamin retains the ability to bind Kalirin12. Co-immunoprecipitation demonstrates an interaction of Kalirin12 and dynamin2 in embryonic brain. Purified recombinant Kalirin-IgFn domain inhibits the ability of purified rat brain dynamin to oligomerize in response to the presence of liposomes containing phosphatidylinositol-4,5-bisphosphate. Consistent with this, expression of exogenous Kalirin12 or its IgFn domain in PC12 cells disrupts clathrin-mediated transferrin endocytosis. Similarly, expression of exogenous Kalirin12 disrupts transferrin endocytosis in cortical neurons. Expression of Kalirin7, a shorter isoform which lacks the IgFn domain, was previously shown to inhibit clathrin-mediated endocytosis; the GTPase domain of dynamin does not interact with Kalirin7.ConclusionKalirin12 may play a role in coordinating Rho GTPase-mediated changes in the actin cytoskeleton with dynamin-mediated changes in membrane trafficking.

A Site-Specific Integrated Col2.3GFP Reporter Identifies Osteoblasts Within Mineralized Tissue Formed In Vivo by Human Embryonic Stem Cells

The use of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) for study and treatment of bone diseases or traumatic bone injuries requires efficient protocols to differentiate hESCs/iPSCs into cells with osteogenic potential and the ability to isolate differentiated osteoblasts for analysis. We have used zinc finger nuclease technology to deliver a construct containing the Col2.3 promoter driving GFPemerald to the AAVS1 site (referred to as a “safe harbor” site), in human embryonic stem cells (H9Zn2.3GFP), with the goal of marking the cells that have become differentiated osteoblasts. In teratomas formed using these cells, we identified green fluorescent protein (GFP)‐positive cells specifically associated with in vivo bone formation. We also differentiated the cells into a mesenchymal stem cell population with osteogenic potential and implanted them into a mouse calvarial defect model. We observed GFP‐positive cells associated with alizarin complexone‐labeled newly formed bone surfaces. The cells were alkaline phosphatase‐positive, and immunohistochemistry with human specific bone sialoprotein (BSP) antibody indicates that the GFP‐positive cells are also associated with the human BSP‐containing matrix, demonstrating that the Col2.3GFP construct marks cells in the osteoblast lineage. Single‐cell cloning generated a 100% Col2.3GFP‐positive cell population, as demonstrated by fluorescence in situ hybridization using a GFP probe. The karyotype was normal, and pluripotency was demonstrated by Tra1‐60 immunostaining, pluripotent low density reverse transcription‐polymerase chain reaction array and embryoid body formation. These cells will be useful to develop optimal osteogenic differentiation protocols and to isolate osteoblasts from normal and diseased iPSCs for analysis.

A Role for Kalirin in the Response of Rat Medium Spiny Neurons to Cocaine

Kalirin-7 (Kal7), the major kalirin isoform in adult brain, plays a key role in the formation of dendritic spines in hippocampal/cortical neurons. Its role in the GABAergic medium spiny neurons (MSNs) of the nucleus accumbens (NAc) and striatum, the areas known to play a key role in the common reward pathway, is not as well understood. Although Kal7 expression in mouse NAc increased in response to cocaine, MSN dendritic spine density did not differ from that for the wild type in Kal7-null mice. Unlike wild-type mice, Kal7-null mice did not respond to cocaine with an increase in MSN dendritic spine density. To explore further the role of Kal7 in cocaine-induced alterations in MSN morphology, we turned to the rat. Based on immunostaining, both Kal7 and Kal12 are expressed at moderate levels in the MSNs of the NAc and striatum. Expression of Kal7 and Kal12 in MSNs of both areas increases after repeated cocaine treatments. Overexpression of Kal7 in cultured MSN neurons increases dendritic spine density, as observed in rats after long-term cocaine administration. Reducing endogenous expression of all major kalirin isoforms in cultured MSN neurons causes a decrease in total dendritic length and dendritic spine density. These data suggest that kalirin is essential for maintaining spine density in NAc MSNs under normal conditions and that Kal7 is an obligatory intermediate in the response of MSNs to repeated exposure to cocaine.

A potential translational approach for bone tissue engineering through endochondral ossification.

Bone defect repair is a significant clinical challenge in orthopedic surgery. Despite tremendous efforts, the majority of the current bone tissue engineering strategies depend on bone formation via intramembranous ossification (IO), which often results in poor vascularization and limited-area bone regeneration. Recently, there has been increasing interest in exploring bone regeneration through a cartilage-mediated process similar to endochondral ossification (EO). This method is advantageous because long bones are originally developed through EO and moreover, vascularization is an inherent step of this process. Therefore, it may be possible to effectively employ the EO method for the repair and regeneration of large and segmental bone defects. Although a number of studies have demonstrated engineered bone formation through EO, there are no approaches aiming for their clinical translation. In this study, we propose a strategy modeled after the U.S. Food and Drug Administration (FDA) aproved Autologus Chondrocyte Implantation (ACI) procedure. In its implementation, we concentrated human bone marrow aspirate via a minimally manipulated process and demonstrated the potential of human bone marrow derived cells for in vitro pre-cartilage template formation and bone regeneration in vivo.

Laser-Capture Microdissection and RNA Extraction from Perfusion-Fixed Cartilage and Bone Tissue from Mice Implanted with Human iPSC-Derived MSCs in a Calvarial Defect Model

Laser-capture microdissection (LCM) coupled to downstream RNA analysis poses unique difficulties for the evaluation of mineralized tissues. A rapid protocol was thus developed to enable sufficient integrity of bone and cartilage tissue for reliable sectioning, while minimizing RNA loss associated with prolonged decalcification and purification steps. Specifically, the protocol involves pump-assisted, cardiac perfusion-fixation with paraformaldehyde, and moderate digestion of LCM-acquired tissue with proteinase K followed by DNase treatment and separation of RNA using magnetic beads. Reverse transcription and cDNA synthesis are performed immediately after RNA purification, without need for further protein removal.