A Role for Dendritic Translation of CaMKIIα mRNA in Olfactory Plasticity

Local protein synthesis in dendrites contributes to the synaptic modifications underlying learning and memory. The mRNA encoding the α subunit of the calcium/calmodulin dependent Kinase II (CaMKIIα) is dendritically localized and locally translated. A role for CaMKIIα local translation in hippocampus-dependent memory has been demonstrated in mice with disrupted CaMKIIα dendritic translation, through deletion of CaMKIIα 3′UTR. We studied the dendritic localization and local translation of CaMKIIα in the mouse olfactory bulb (OB), the first relay of the olfactory pathway, which exhibits a high level of plasticity in response to olfactory experience. CaMKIIα is expressed by granule cells (GCs) of the OB. Through in situ hybridization and synaptosome preparation, we show that CaMKIIα mRNA is transported in GC dendrites, synaptically localized and might be locally translated at GC synapses. Increases in the synaptic localization of CaMKIIα mRNA and protein in response to brief exposure to new odors demonstrate that they are activity-dependent processes. The activity-induced dendritic transport of CaMKIIα mRNA can be inhibited by an NMDA receptor antagonist and mimicked by an NMDA receptor agonist. Finally, in mice devoid of CaMKIIα 3′UTR, the dendritic localization of CaMKIIα mRNA is disrupted in the OB and olfactory associative learning is severely impaired. Our studies thus reveal a new functional modality for CaMKIIα local translation, as an essential determinant of olfactory plasticity

From Immunodeficiency to Humanization: The Contribution of Mouse Models to Explore HTLV-1 Leukemogenesis

The first discovered human retrovirus, Human T-Lymphotropic Virus type 1 (HTLV-1), is responsible for an aggressive form of T cell leukemia/lymphoma. Mouse models recapitulating the leukemogenesis process have been helpful for understanding the mechanisms underlying the pathogenesis of this retroviral-induced disease. This review will focus on the recent advances in the generation of immunodeficient and human hemato-lymphoid system mice with a particular emphasis on the development of mouse models for HTLV-1-mediated pathogenesis, their present limitations and the challenges yet to be addressed

Evaluation of the biocompatibility and stability of allogeneic tissue-engineered cartilage in humanized mice

International audienc

From Immunodeficiency to Humanization: The Contribution of Mouse Models to Explore HTLV-1 Leukemogenesis

The first discovered human retrovirus, Human T-Lymphotropic Virus type 1 (HTLV-1), is responsible for an aggressive form of T cell leukemia/lymphoma. Mouse models recapitulating the leukemogenesis process have been helpful for understanding the mechanisms underlying the pathogenesis of this retroviral-induced disease. This review will focus on the recent advances in the generation of immunodeficient and human hemato-lymphoid system mice with a particular emphasis on the development of mouse models for HTLV-1-mediated pathogenesis, their present limitations and the challenges yet to be addressed

Evaluation of the biocompatibility and stability of allogeneic tissue-engineered cartilage in humanized mice

International audienceArticular cartilage (AC) has poor capacities of regeneration and lesions often lead to osteoarthritis. Current AC reconstruction implies autologous chondrocyte implantation which requires tissue sampling and grafting. An alternative approach would be to use scaffolds containing off-the-shelf allogeneic human articular chondrocytes (HACs). To investigate tolerance of allogeneic HACs by the human immune system, we developed a humanized mouse model implanted with allogeneic cartilage constructs generated in vitro. A prerequisite of the study was to identify a scaffold that would not provoke inflammatory reaction in host. Therefore, we first compared the response of hu-mice to two biomaterials used in regenerative medicine, collagen sponge and agarose hydrogel. Four weeks after implantation in hu-mice, acellular collagen sponges, but not acellular agarose hydrogels, showed positive staining for CD3 (T lymphocytes) and CD68 (macrophages), suggesting that collagen scaffold elicits weak inflammatory reaction. These data led us to deepen our evaluation of the biocompatibility of allogeneic tissue-engineered cartilage by using agarose as scaffold. Agarose hydrogels were combined with allogeneic HACs to reconstruct cartilage in vitro. Particular attention was paid to HLA-A2 compatibility between HACs to be grafted and immune human cells of hu-mice: HLA-A2+ or HLA-A2- HACs agarose hydrogels were cultured in the presence of a chondrogenic cocktail and implanted in HLA-A2+ hu-mice. After four weeks implantation and regardless of the HLA-A2 phenotype, chondrocytes were well-differentiated and produced cartilage matrix in agarose. In addition, no sign of T-cell or macrophage infiltration was seen in the cartilaginous constructs and no significant increase in subpopulations of T lymphocytes and monocytes was detected in peripheral blood and spleen. We show for the first time that humanized mouse represents a useful model to investigate human immune responsiveness to tissue-engineered cartilage and our data together indicate that allogeneic cartilage constructs can be suitable for cartilage engineering

PDZ domain-binding motif of Tax sustains T-cell proliferation in HTLV-1-infected humanized mice.

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATLL), an aggressive malignant proliferation of activated CD4+ T lymphocytes. The viral Tax oncoprotein is critically involved in both HTLV-1-replication and T-cell proliferation, a prerequisite to the development of ATLL. In this study, we investigated the in vivo contribution of the Tax PDZ domain-binding motif (PBM) to the lymphoproliferative process. To that aim, we examined T-cell proliferation in humanized mice (hu-mice) carrying a human hemato-lymphoid system infected with either a wild type (WT) or a Tax PBM-deleted (ΔPBM) provirus. We observed that the frequency of CD4+ activated T-cells in the peripheral blood and in the spleen was significantly higher in WT than in ΔPBM hu-mice. Likewise, human T-cells collected from WT hu-mice and cultivated in vitro in presence of interleukin-2 were proliferating at a higher level than those from ΔPBM animals. We next examined the association of Tax with the Scribble PDZ protein, a prominent regulator of T-cell polarity, in human T-cells analyzed either after ex vivo isolation or after in vitro culture. We confirmed the interaction of Tax with Scribble only in T-cells from the WT hu-mice. This association correlated with the presence of both proteins in aggregates at the leading edge of the cells and with the formation of long actin filopods. Finally, data from a comparative genome-wide transcriptomic analysis suggested that the PBM-PDZ association is implicated in the expression of genes regulating proliferation, apoptosis and cytoskeletal organization. Collectively, our findings suggest that the Tax PBM is an auxiliary motif that contributes to the sustained growth of HTLV-1 infected T-cells in vivo and in vitro and is essential to T-cell immortalization

CaMKIIα mRNA dendritic localization and translatability.

<p><b>A,B </b><i>In situ</i> hybridization for CaMKIIα mRNA. <b>A,</b> On a horizontal section, CaMKIIα mRNA is visible in multiple regions of the brain, particularly in the hippocampus (HC), cortex and olfactory bulb (OB). Scale bar: 500 µm. <b>B,</b> Higher magnification of the OB shows strong CaMKIIα mRNA expression in the granule cell layer (GCL) and in a diffuse staining of the external plexiform layer (EPL), where GCs extend their apical dendrites. Gl: glomerular layer. Scale bar: 25 µm. <b>C,</b> Quantification of mRNA levels in synaptosomes (SN) by quantitative PCR. CaMKIIα and PSD95 transcripts are highly elevated (7 fold) at the synaptic level, as compared to HPRT mRNA, a transcript restricted to the soma. “I” is the index of synaptic localization and represents the ratio of mRNA present in the SN fraction over the total quantity present in the OB homogenate, normalized to HPRT mRNA. Error bars represent sem (n = 3). <b>D,</b> SN were metabolically labeled with a mixture of ³⁵S-Met and ³⁵S-Cys with (lane +) or without (lane -) stimulation by 10 µM glutamate and 50 µM NMDA. Newly-synthesized proteins were detected by autoradiography (left panel) or blotted with an anti-CaMKIIα antibody (right panel). <b>Ea,b</b> Electron micrograph of a dendrodendritic synapse between a granule cell dendrite (GCd) and a mitral/tufted cell dendrite (M/Td). Ribosomes (r, arrows) are located in the GCd, at the base of, or in close proximity to, a spine. The inset displays a magnification of the boxed region showing polyribosomes with a characteristic rosette morphology. The GC is colorized in pink in b, and the blue arrow in the granule cell spine head (GSh) indicates the synapse formed onto the mitral/tufted cell dendrite (M/Td). Scale bar: 0.5 µm.</p

Altered CaMKIIα mRNA and protein synaptic localization disrupts olfactory associative learning.

<p><b>A, </b><i>In situ</i> hybridization for CaMKIIα mRNA in wild-type mice (WT, upper panels) and 3′UTR mutants (3′UTR, lower panels) shows a dramatic decrease of the dendritic staining (stars) in the mutant hippocampus (HC, left panels) and olfactory bulb (OB, right panels). EPL: external plexiform layer Scale bars: 200 µm and 50 µm, in HC and OB respectively. <b>B,</b> Quantification of the index of synaptic localization (I) of CaMKIIα and PSD95 mRNAs normalized to HPRT mRNA in WT and 3′UTR mutants. CaMKIIα mRNA index is significantly decreased in 3′UTR mutants as compared to WT (n = 3, p = 0.026) <b>C,</b> Behavioral habituation/dishabituation paradigm: both WT and 3′UTR mutants mice habituated to the habituation odor, as shown by decreased investigation time across the four trials (Hab1-Hab4) (WT: F(3,27) = 30.452, p<0.001; 3′UTR: F(3,27) = 20.469, p<0.001; ANOVA, trial effect) and discriminated. Upon presentation of the test odor (Test), the investigation time increased when compared to Hab4, indicating that the animals could discriminate the test odor from the habituation odor (WT: *p<0.00005; 3′UTR: *p<0.00005, t-test). <b>D,</b> Olfactory associative learning: mice were conditioned to associate an odor stimulus to a food reward. Success rate results are presented as the percentage of correct choice across consecutive blocks of trials (1 block = 4 trials). Conditioned WT mice learned the task as indicated by the increase in correct choice throughout blocks (F(5,49) = 3.391, p<0.01; ANOVA, block effect). In contrast, conditioned 3′UTR mutants did not show an increase in success rate across trials (F(5,54) = 1.078, p>0.1; ANOVA, block effect), showing that impaired CaMKIIα mRNA dendritic localization disrupts olfactory-associative learning. <b>E,</b> Latencies: conditioned WT mice showed a decrease in latency confirming that they learned the task (F(5,49) = 4.037, p<0.005; ANOVA, block effect). In contrast, latencies of conditioned 3′UTR mutants did not significantly change (F(5,54) = 0.635, p>0.5; ANOVA, block effect), indicating that the mutants were not able to associate odor and reward.</p

CaMKIIα expression in the OB.

<p><b>A,B,C</b> Immunostaining for CaMKIIα (red) on olfactory bulb (OB) sections. <b>A:</b> CaMKIIα immunoreactivity is present in the Granule Cell Layer (GCL) and in the External Plexiform Layer (EPL), where granule cells (GCs) extend their apical dendrites. The glomerular layer (Gl) appears unlabeled. Scale bar: 50 µm. <b>B,</b> Higher magnification of the GCL shows staining of some GCs in their cell bodies (arrow) and in their dendrites extending towards the EPL (arrowheads). Mitral cells do not express the protein (star). Scale bar: 25 µm. <b>C,</b> Magnification of the boxed region in B. Some GCs express CaMKIIα in the thin rim of cytoplasm surrounding their nuclei (arrows). Nuclei are counterstained with DAPI. Scale bar: 10 µm.</p