Reverse Signaling by Semaphorin-6A Regulates Cellular Aggregation and Neuronal Morphology.

The transmembrane semaphorin, Sema6A, has important roles in axon guidance, cell migration and neuronal connectivity in multiple regions of the nervous system, mediated by context-dependent interactions with plexin receptors, PlxnA2 and PlxnA4. Here, we demonstrate that Sema6A can also signal cell-autonomously, in two modes, constitutively, or in response to higher-order clustering mediated by either PlxnA2-binding or chemically induced multimerisation. Sema6A activation stimulates recruitment of Abl to the cytoplasmic domain of Sema6A and phos¡phorylation of this cytoplasmic tyrosine kinase, as well as phosphorylation of additional cytoskeletal regulators. Sema6A reverse signaling affects the surface area and cellular complexity of non-neuronal cells and aggregation and neurite formation of primary neurons in vitro. Sema6A also interacts with PlxnA2 in cis, which reduces binding by PlxnA2 of Sema6A in trans but not vice versa. These experiments reveal the complex nature of Sema6A biochemical functions and the molecular logic of the context-dependent interactions between Sema6A and PlxnA2

Sema6A cytoplasmic domain interacts with Abl and Mena.

<p>(<b>A</b>) Immunoprecipitations from untreated or PlxnA2-EC-treated COS-7 cells previously transfected with different combinations of myc-Sema6A, Abl-V5 and PlxnA2. Immunoprecipitations (Ip) were performed employing an anti-V5 antibody. Antibodies against V5, myc and PlxnA2 were used in the immunoblots (Ib). (<b>B</b>) Immunoprecipitations from untreated or PlxnA2-EC treated COS-7 cells previously transfected with different combinations of myc-Sema6A, Mena-V5 and PlxnA2. Immunoprecipitations (Ip) were performed employing an anti-myc antibody. Antibodies against V5, myc and PlxnA2 were used in the immunoblots (Ib). (<b>C</b>) and (<b>D</b>) Interactions were quantified by immunoblotting and densitometric analysis. Histograms represent quantification of interaction normalized to the amount of immunoprecipitated protein (Abelson-V5 for Sema6A-Abelson or Sema6A-myc for Sema6A-Mena) from three independent experiments. Students t-test *p<0,05 indicating signifiance. S6A-Abl/S6A-Abl+PlA2 <i>p</i> = 0.013, S6A-Abl-PlA2/S6A-Abl-PlA2+PlA2 <i>p</i> = 0.034. Errors bars represent standard error.</p

The nuclear receptor Nr4a1 acts as a microglia rheostat and serves as a therapeutic target in autoimmune-driven central nervous system inflammation

Microglia cells fulfill key homeostatic functions and essentially contribute to host defense within the CNS. Altered activation of microglia, in turn, has been implicated in neuroinflammatory and neurodegenerative diseases. In this study, we identify the nuclear receptor (NR) Nr4a1 as key rheostat controlling the activation threshold and polarization of microglia. In steady-state microglia, ubiquitous neuronal-derived stress signals such as ATP induced expression of this NR, which contributed to the maintenance of a resting and noninflammatory microglia phenotype. Global and microglia-specific deletion of Nr4a1 triggered the spontaneous and overwhelming activation of microglia and resulted in increased cytokine and NO production as well as in an accelerated and exacerbated form of experimental autoimmune encephalomyelitis. Ligand-induced activation of Nr4a1 accordingly ameliorated the course of this disease. Our current data thus identify Nr4a1 as regulator of microglia activation and potentially new target for the treatment of inflammatory CNS diseases such as multiple sclerosis

Cell-contraction induced by PlxnA2-Sema6A interaction depends on Abl signaling.

<p>(<b>A-F</b>) NIH3T3 cells expressing GFP (<b>A</b> and <b>D</b>), Sema6A-FL (<b>B</b> and <b>E</b>), and Sema6A-∆Abl (<b>C</b> and <b>F</b>) were treated with purified AP-Fc (AP; <b>a-c</b>) or PlxnA2-EC-Fc (PlxnA2-EC; <b>D-F</b>). Scale bar = 20 μm. (<b>G</b>) Scheme illustrates that the cell contraction is via Abl signaling. (<b>H</b>) Graph represents the contraction of the cell area in NIH3T3 transfected with GFP, Sema6A or Sema6A-∆Abl and treated with AP or PlxnA2-EC; <i>n</i> = 100–200 cells per experimental condition. Data are expressed as mean ± s.e.m; ***<i>P</i> < 0.001; Student’s <i>t</i>-test.</p

Sema6A reverse signaling is stimulated by multimerisation.

<p>(<b>A</b>) Scheme representing how the induced multi-aggregation of the cytosolic domain of Sema6A activates signaling pathways. A chimeric construct was engineered by putting in frame the entire cytosolic domain of Sema6A (Sema6A-cyt) together with 3 self-aggregation FKBP domains, and GFP. The chimeric peptide targets the plasma membrane by the myristoylating sequence located at the N-terminal. The application of the drug FK1012 triggers the chemically induced multimerisation (CIM) of Sema6A-cyt via the self-aggregation of FKBP. (<b>B</b>) Cerebellar neurons transfected with MF3-GFP or MF3-Sema6A-cyt-GFP (MF3-Sema6A-cyt), and treated with 500nM FK1012 (“CIM”). GFP-positive aggregates are indicated (arrowheads). Scale bar = 20μm. (<b>C</b>) Axonal shaft of cerebellar neurons transfected with myc-Sema6A-FL or MF3-Sema6A-cyt-GFP (MF3-Sema6A-cyt), and treated with PlxnA2-EC or CIM respectively. Scale bar = 5 μm. (<b>D</b>) and (<b>E</b>) Graphs summarising the myc-Sema6A-FL aggregation in myc-Sema6A-FL-expressing neurons treated with PlxnA2-EC at different time points. The aggregation is represented as the average distance between Sema6A clusters, or the average size of Sema6A clusters; <i>n</i> = 50–100 cells per time point. Data are expressed as mean ± s.e.m; ***<i>P</i> < 0.001; one-way ANOVA followed by Bonferroni multiple comparison test. (<b>F</b>) MF3-Sema6A-cyt-expressing granular neurons were cultured on NIH3T3 cell layers, and treated with CIM. Scale bar = 50 μm. (<b>G</b>) Graph representing the axonal length of MF3-Sema6A-cyt-expressing cerebellar neurons treated with CIM for 24 and 48h; <i>n</i> = 50–100 cells per experimental condition. Data are expressed as mean ± s.e.m; ***<i>P</i> < 0.001; one-way ANOVA followed by Bonferroni multiple comparison test.</p

PlxnA2 interacts with Sema6A <i>in cis</i> and <i>in trans</i>.

<p>(<b>A</b>) Scheme summarising Sema6A-AP bindings. (<b>B-F</b>): COS-7 cells were transfected with the empty vector (mock; <b>B</b>), Sema6A-FL (<b>C</b>), PlxnA2-FL (<b>D</b>), PlxnA2-FL together with Sema6A-FL (<b>E</b>) or PlxnA2-A396E (<b>F</b>); and treated with Sema6A-AP. (<b>G</b>) Scheme summarising PlxnA2-AP bindings. (<b>H-K</b>): COS-7 cells were transfected with the empty vector (mock; <b>H</b>), Sema6A-FL (<b>I</b>), PlxnA2-FL (<b>J</b>) or PlxnA2-FL together with Sema6A-FL (<b>K</b>) and treated with PlxnA2-AP. (<b>L</b>) PlxnA2 immunoprecipitation (IP) from protein samples of COS-7 cells transfected with Sema6A-FL, PlxnA2-FL or Sema6A-FL together PlxnA2-FL. Antibodies against PlxnA2 and Sema6A were used in the immunoblots. (<b>M</b>) Sema6A IP from protein samples of COS-7 cells transfected with Sema6A-FL, PlxnA2-FL or Sema6A-FL together with PlxnA2-FL. (<b>N</b>) PlxnA2 IP from protein samples of mouse cerebellums or EGL explants. (<b>O</b>) PlxnA2 IP from protein samples of COS-7 cells transfected with PlxnA2-FL, Sema6A-∆cyt or Sema6A-∆cyt together with PlxnA2-FL. (<b>P</b>) PlxnA2 IP from protein samples of COS-7 cells transfected with PlxnA2-A396D, Sema6A-FL or Sema6A-FL together with PlxnA2-A396D. (<b>Q</b>) PlxnA2 IP from protein samples of COS-7 cells transfected with PlxnA2-FL, Sema6A-K393D or Sema6A-K393D together with PlxnA2-FL.</p

Sema6A exerts constitutive and PlxnA2-dependent cell-autonomous functions.

<p>(<b>A-F</b>) NIH3T3 cells expressing GFP alone (<b>A and B</b>), myc-Sema6A-FL (Sema6A; <b>B and E</b>), or myc-Sema6A-K393D (Sema6A-K393D; <b>C and F</b>) were treated with purified AP-Fc (AP; <b>A-C</b>) or PlxnA2-EC-Fc (PlxnA2-EC; <b>D-F</b>). Scale bar = 20 μm. (<b>G</b>) Graph represents the cell area in cells transfected with GFP, Sema6A or Sema6A-K393D and treated with AP or PlxnA2-EC; <i>n</i> = 100–200 cells per experimental condition. Data are expressed as mean ± s.e.m; ***<i>P</i> <0.001; Student’s <i>t</i>-test.</p

Sema6A signaling reduces the axonal length of granular neurons.

<p>(<b>A-C</b>) GFP-expressing granular neurons were cultured on Control cells (<b>A</b>), Sema6A Cells (<b>B</b>), PlxnA2 Cells (<b>C</b>) and PlxnA2-A396E Cells (A396E Cells, <b>D</b>). Scale bar = 20 μm. (<b>E</b>) Graph represents the axonal length of granular neurons grown on different NIH3T3 layers; <i>n</i> = 100–200 cells per experimental condition. Data are expressed as mean ± s.e.m; ***<i>P</i> < 0.001; one-way ANOVA followed by Bonferroni multiple comparison test. (<b>F</b>) Graph represents the axonal length of <i>PlxnA2</i>^<i>+/+</i> (dark columns) and <i>PlxnA2</i>^<i>-/-</i> (light columns) granular neurons grown on NIH3T3 cells. Neurons were transfected with GFP alone or together with PlxnA2-FL, and cultured on control (Ctrl) or on Sema6A cell layers; <i>n</i> = 100–200 cells per experimental condition. Data are expressed as mean ± s.e.m; ***<i>P</i> < 0.001; Student’s <i>t</i>-test. (<b>G</b>) Graph represents the axonal length of <i>Sema6A</i>^<i>+/-</i> (dark columns) and <i>Sema6A</i>^<i>-/-</i> (light columns) granular neurons grown on NIH3T3 cells. Neurons were transfected with GFP alone or together with Sema6A-FL, and cultured on control (Ctrl) or on PlxnA2 cell layers; <i>n</i> = 100–200 cells per experimental condition. Data are expressed as mean ± s.e.m; ***<i>P</i> < 0.001; Student’s <i>t</i>-test. (<b>H-J</b>) Granular neurons transfected with GFP alone (<b>H</b> and <b>I</b>) or together with Sema6A-∆cyt (<b>J</b>), and grown on control or on PlxnA2 cells. Scale bar = 20 μm. (<b>K</b>) Graph represents the axonal length of <i>Sema6A</i>^<i>+/-</i> (dark columns) and <i>Sema6A</i>^<i>-/-</i> (light columns) granular neurons. Neurons were transfected with GFP alone or together with Sema6A-∆cyt, and cultured on control (Ctrl) or on PlxnA2 cell layers; <i>n</i> = 100–200 cells per experimental condition. Data are expressed as mean ± s.e.m; ***<i>P</i> < 0.001; Student’s <i>t</i>-test.</p

Dual pathways to endochondral osteoblasts: a novel chondrocyte-derived osteoprogenitor cell identified in hypertrophic cartilage

According to the general understanding, the chondrocyte lineage terminates with the elimination of late hypertrophic cells by apoptosis in the growth plate. However, recent cell tracking studies have shown that murine hypertrophic chondrocytes can survive beyond “terminal” differentiation and give rise to a progeny of osteoblasts participating in endochondral bone formation. The question how chondrocytes convert into osteoblasts, however, remained open. Following the cell fate of hypertrophic chondrocytes by genetic lineage tracing using BACCol10;Cre induced YFP-reporter gene expression we show that a progeny of Col10Cre-reporter labelled osteoprogenitor cells and osteoblasts appears in the primary spongiosa and participates – depending on the developmental stage – substantially in trabecular, endosteal, and cortical bone formation. YFP+ trabecular and endosteal cells isolated by FACS expressed Col1a1, osteocalcin and runx2, thus confirming their osteogenic phenotype. In searching for transitory cells between hypertrophic chondrocytes and trabecular osteoblasts we identified by confocal microscopy a novel, small YFP+Osx+ cell type with mitotic activity in the lower hypertrophic zone at the chondro-osseous junction. When isolated from growth plates by fractional enzymatic digestion, these cells termed CDOP (chondrocyte-derived osteoprogenitor) cells expressed bone typical genes and differentiated into osteoblasts in vitro. We propose the Col10Cre-labeled CDOP cells mark the initiation point of a second pathway giving rise to endochondral osteoblasts, alternative to perichondrium derived osteoprogenitor cells. These findings add to current concepts of chondrocyte-osteocyte lineages and give new insight into the complex cartilage-bone transition process in the growth plate