BAFF activation of the ERK5 MAP kinase pathway regulates B cell survival

B cell activating factor (BAFF) stimulation of the BAFF receptor (BAFF-R) is essential for the homeostatic survival of mature B cells. Earlier in vitro experiments with inhibitors that block MEK 1 and 2 suggested that activation of ERK 1 and 2 MAP kinases is required for BAFF-R to promote B cell survival. However, these inhibitors are now known to also inhibit MEK5, which activates the related MAP kinase ERK5. In the present study, we demonstrated that BAFF-induced B cell survival was actually independent of ERK1/2 activation but required ERK5 activation. Consistent with this, we showed that conditional deletion of ERK5 in B cells led to a pronounced global reduction in mature B2 B cell numbers, which correlated with impaired survival of ERK5-deficient B cells after BAFF stimulation. ERK5 was required for optimal BAFF up-regulation of Mcl1 and Bcl2a1, which are prosurvival members of the Bcl-2 family. However, ERK5 deficiency did not alter BAFF activation of the PI3-kinase-Akt or NF-κB signaling pathways, which are also important for BAFF to promote mature B cell survival. Our study reveals a critical role for the MEK5-ERK5 MAP kinase signaling pathway in BAFF-induced mature B cell survival and homeostatic maintenance of B2 cell numbers

BAFF signaling in health and disease.

BAFF is a critical cytokine supporting the survival of mature naïve B cells, acting through the BAFFR receptor. Recent studies show that BAFF and BAFFR are also required for the survival of memory B cells, autoimmune B cells as well as malignant chronic lymphocytic leukaemia (CLL) cells. BAFFR cooperates with other receptors, notably the B cell antigen receptor (BCR), a process which is critical for the expansion of autoimmune and CLL cells. This crosstalk may be mediated by TRAF3 which interacts with BAFFR and with CD79A, a signalling subunit of the BCR and the downstream SYK kinase, inhibiting its activity. BAFF binding to BAFFR leads to degradation of TRAF3 which may relieve inhibition of SYK activity transducing signals to pathways required for B cell survival. BAFFR activates both canonical and non-canonical NF-κB signalling and both pathways play important roles in the survival of B cells and CLL cells

Syk tyrosine kinase is critical for B cell antibody responses and memory B cell survival

Signals from the BCR are required for Ag-specific B cell recruitment into the immune response. Binding of Ag to the BCR induces phosphorylation of immune receptor tyrosine-based activation motifs in the cytoplasmic domains of the CD79a and CD79b signaling subunits, which subsequently bind and activate the Syk protein tyrosine kinase. Earlier work with the DT40 chicken B cell leukemia cell line showed that Syk was required to transduce BCR signals to proximal activation events, suggesting that Syk also plays an important role in the activation and differentiation of primary B cells during an immune response. In this study, we show that Syk-deficient primary mouse B cells have a severe defect in BCR-induced activation, proliferation, and survival. Furthermore, we demonstrate that Syk is required for both T-dependent and T-independent Ab responses, and that this requirement is B cell intrinsic. In the absence of Syk, Ag fails to induce differentiation of naive B cells into germinal center B cells and plasma cells. Finally, we show that the survival of existing memory B cells is dependent on Syk. These experiments demonstrate that Syk plays a critical role in multiple aspects of B cell Ab responses

In vivo and ex vivo analyses of amyloid toxicity in the Tc1 mouse model of Down syndrome.

RATIONALE: The prevalence of Alzheimer's disease is increased in people with Down syndrome. The pathology appears much earlier than in the general population, suggesting a predisposition to develop Alzheimer's disease. Down syndrome results from trisomy of human chromosome 21, leading to overexpression of possible Alzheimer's disease candidate genes, such as amyloid precursor protein gene. To better understand how the Down syndrome context results in increased vulnerability to Alzheimer's disease, we analysed amyloid-β [25-35] peptide toxicity in the Tc1 mouse model of Down syndrome, in which ~75% of protein coding genes are functionally trisomic but, importantly, not amyloid precursor protein. RESULTS: Intracerebroventricular injection of oligomeric amyloid-β [25-35] peptide in three-month-old wildtype mice induced learning deficits, oxidative stress, synaptic marker alterations, activation of glycogen synthase kinase-3β, inhibition of protein kinase B (AKT), and apoptotic pathways as compared to scrambled peptide-treated wildtype mice. Scrambled peptide-treated Tc1 mice presented high levels of toxicity markers as compared to wildtype mice. Amyloid-β [25-35] peptide injection in Tc1 mice induced significant learning deficits and enhanced glycogen synthase kinase-3β activity in the cortex and expression of apoptotic markers in the hippocampus and cortex. Interestingly, several markers, including oxidative stress, synaptic markers, glycogen synthase kinase-3β activity in the hippocampus and AKT activity in the hippocampus and cortex, were unaffected by amyloid-β [25-35] peptide injection in Tc1 mice. CONCLUSIONS: Tc1 mice present several toxicity markers similar to those observed in amyloid-β [25-35] peptide-treated wildtype mice, suggesting that developmental modifications in these mice modify their response to amyloid peptide. However, amyloid toxicity led to severe memory deficits in this Down syndrome mouse model

Mechanism and function of Vav1 localisation in TCR signalling

The antigen-specific binding of T cells to antigen presenting cells results in recruitment of signalling proteins to microclusters at the cell-cell interface known as the immunological synapse (IS). The Vav1 guanine nucleotide exchange factor plays a critical role in T cell antigen receptor (TCR) signalling, leading to the activation of multiple pathways. We now show that it is recruited to microclusters and to the IS in primary CD4+ and CD8+ T cells. Furthermore, we show that this recruitment depends on the SH2 and C-terminal SH3 (SH3B) domains of Vav1, and on phosphotyrosines 112 and 128 of the SLP76 adaptor protein. Biophysical measurements show that Vav1 binds directly to these residues on SLP76 and that efficient binding depends on the SH2 and SH3B domains of Vav1. Finally, we show that the same two domains are critical for the phosphorylation of Vav1 and its signalling function in TCR-induced calcium flux. We propose that Vav1 is recruited to the IS by binding to SLP76 and that this interaction is critical for the transduction of signals leading to calcium flux

Protein profiles in Tc1 mice implicate novel pathway perturbations in the Down syndrome brain

Tc1 mouse model of Down syndrome (DS) is functionally trisomic for ∼120 human chromosome 21 (HSA21) classical protein-coding genes. Tc1 mice display features relevant to the DS phenotype, including abnormalities in learning and memory and synaptic plasticity. To determine the molecular basis for the phenotypic features, the levels of 90 phosphorylation-specific and phosphorylation-independent proteins were measured by Reverse Phase Protein Arrays in hippocampus and cortex, and 64 in cerebellum, of Tc1 mice and littermate controls. Abnormal levels of proteins involved in MAP kinase, mTOR, GSK3B and neuregulin signaling were identified in trisomic mice. In addition, altered correlations among the levels of N-methyl-D-aspartate (NMDA) receptor subunits and the HSA21 proteins amyloid beta (A4) precursor protein (APP) and TIAM1, and between immediate early gene (IEG) proteins and the HSA21 protein superoxide dismutase-1 (SOD1) were found in the hippocampus of Tc1 mice, suggesting altered stoichiometry among these sets of functionally interacting proteins. Protein abnormalities in Tc1 mice were compared with the results of a similar analysis of Ts65Dn mice, a DS mouse model that is trisomic for orthologs of 50 genes trisomic in the Tc1 plus an additional 38 HSA21 orthologs. While there are similarities, abnormalities unique to the Tc1 include increased levels of the S100B calcium-binding protein, mTOR proteins RAPTOR and P70S6, the AMP-kinase catalytic subunit AMPKA, the IEG proteins FBJ murine osteosarcoma viral oncogene homolog (CFOS) and activity-regulated cytoskeleton-associated protein (ARC), and the neuregulin 1 receptor ERBB4. These data identify novel perturbations, relevant to neurological function and to some seen in Alzheimer's disease, that may occur in the DS brain, potentially contributing to phenotypic features and influencing drug responses

In vivo and ex vivo analyses of amyloid toxicity in the Tc1 mouse model of Down syndrome.

RATIONALE: The prevalence of Alzheimer's disease is increased in people with Down syndrome. The pathology appears much earlier than in the general population, suggesting a predisposition to develop Alzheimer's disease. Down syndrome results from trisomy of human chromosome 21, leading to overexpression of possible Alzheimer's disease candidate genes, such as amyloid precursor protein gene. To better understand how the Down syndrome context results in increased vulnerability to Alzheimer's disease, we analysed amyloid-β [25-35] peptide toxicity in the Tc1 mouse model of Down syndrome, in which ~75% of protein coding genes are functionally trisomic but, importantly, not amyloid precursor protein. RESULTS: Intracerebroventricular injection of oligomeric amyloid-β [25-35] peptide in three-month-old wildtype mice induced learning deficits, oxidative stress, synaptic marker alterations, activation of glycogen synthase kinase-3β, inhibition of protein kinase B (AKT), and apoptotic pathways as compared to scrambled peptide-treated wildtype mice. Scrambled peptide-treated Tc1 mice presented high levels of toxicity markers as compared to wildtype mice. Amyloid-β [25-35] peptide injection in Tc1 mice induced significant learning deficits and enhanced glycogen synthase kinase-3β activity in the cortex and expression of apoptotic markers in the hippocampus and cortex. Interestingly, several markers, including oxidative stress, synaptic markers, glycogen synthase kinase-3β activity in the hippocampus and AKT activity in the hippocampus and cortex, were unaffected by amyloid-β [25-35] peptide injection in Tc1 mice. CONCLUSIONS: Tc1 mice present several toxicity markers similar to those observed in amyloid-β [25-35] peptide-treated wildtype mice, suggesting that developmental modifications in these mice modify their response to amyloid peptide. However, amyloid toxicity led to severe memory deficits in this Down syndrome mouse model

Fully-automated μMRI morphometric phenotyping of the Tc1 mouse model of Down Syndrome

We describe a fully automated pipeline for the morphometric phenotyping of mouse brains from μMRI data, and show its application to the Tc1 mouse model of Down syndrome, to identify new morphological phenotypes in the brain of this first transchromosomic animal carrying human chromosome 21. We incorporate an accessible approach for simultaneously scanning multiple ex vivo brains, requiring only a 3D-printed brain holder, and novel image processing steps for their separation and orientation. We employ clinically established multi-atlas techniques-superior to single-atlas methods-together with publicly-available atlas databases for automatic skull-stripping and tissue segmentation, providing high-quality, subject-specific tissue maps. We follow these steps with group-wise registration, structural parcellation and both Voxel- and Tensor-Based Morphometry-advantageous for their ability to highlight morphological differences without the laborious delineation of regions of interest. We show the application of freely available open-source software developed for clinical MRI analysis to mouse brain data: NiftySeg for segmentation and NiftyReg for registration, and discuss atlases and parameters suitable for the preclinical paradigm. We used this pipeline to compare 29 Tc1 brains with 26 wild-type littermate controls, imaged ex vivo at 9.4T. We show an unexpected increase in Tc1 total intracranial volume and, controlling for this, local volume and grey matter density reductions in the Tc1 brain compared to the wild-types, most prominently in the cerebellum, in agreement with human DS and previous histological findings

Vav proteins are key regulators of Card9 signaling for innate antifungal immunity

Fungal infections are major causes of morbidity and mortality, especially in immunocompromised individuals. The innate immune system senses fungal pathogens through Syk-coupled C-type lectin receptors (CLRs), which signal through the conserved immune adaptor Card9. Although Card9 is essential for antifungal defense, the mechanisms that couple CLR-proximal events to Card9 control are not well defined. Here, we identify Vav proteins as key activators of the Card9 pathway. Vav1, Vav2, and Vav3 cooperate downstream of Dectin-1, Dectin-2, and Mincle to engage Card9 for NF-κB control and proinflammatory gene transcription. Although Vav family members show functional redundancy, Vav1/2/3(-/-) mice phenocopy Card9(-/-) animals with extreme susceptibility to fungi. In this context, Vav3 is the single most important Vav in mice, and a polymorphism in human VAV3 is associated with susceptibility to candidemia in patients. Our results reveal a molecular mechanism for CLR-mediated Card9 regulation that controls innate immunity to fungal infections