Elisabeth Kremmer

CCR7 Coordinates the Primary Immune Response by Establishing Functional Microenvironments in Secondary Lymphoid Organs

The proper function of immune surveillance requires well-coordinated mechanisms in order to guide the patrolling immune cells through peripheral tissues and into secondary lymphoid organs. Analyzing gene-targeted mice, we identified the chemokine receptor CCR7 as an important organizer of the primary immune response. CCR7-deficient mice show severely delayed kinetics regarding the antibody response and lack contact sensitivity and delayed type hypersensitivity reactions. Due to the impaired migration of lymphocytes, these animals reveal profound morphological alterations in all secondary lymphoid organs. Upon activation, mature skin dendritic cells fail to migrate into the draining lymph nodes. Thus, in order to bring together lymphocytes and dendritic cells to form the characteristic microarchitecture of secondary lymphoid organs, CCR7 is required to rapidly initiate an adoptive immune response.

A PUTATIVE CHEMOKINE RECEPTOR, BLR1, DIRECTS B CELL MIGRATION TO DEFINED LYMPHOID ORGANS AND SPECIFIC ANATOMIC COMPARTMENTS OF THE SPLEEN

We describe the phenotype of gene-targeted mice lacking the putative chemokine receptor BLR1. In normal mice, this receptor is expressed on mature B cells and a subpopulation of T helper cells. Blr1 mutant mice lack inguinal lymph nodes and possess no or only a few phenotypically abnormal Peyers patches. The migration of lymphocytes into splenic follicles is severely impaired, resulting in morphologically altered primary lymphoid follicles. Furthermore, activated B cells fail to migrate from the T cell-rich zone into B cell follicles of the spleen, and despite high numbers of germinal center founder cells, no functional germinal centers develop in this organ. Our results identify the putative chemokine receptor BLR1 as the first G protein-coupled receptor involved in B cell migration and localization of these cells within specific anatomic compartments.

Genome-wide, large-scale production of mutant mice by ENU mutagenesis

In the post-genome era, the mouse will have a major role as a model system for functional genome analysis. This requires a large number of mutants similar to the collections available from other model organisms such as Drosophila melanogaster and Caenorhabditis elegans. Here we report on a systematic, genome-wide, mutagenesis screen in mice. As part of the German Human Genome Project, we have undertaken a large-scale ENU-mutagenesis screen for dominant mutations and a limited screen for recessive mutations. In screening over 14,000 mice for a large number of clinically relevant parameters, we recovered 182 mouse mutants for a variety of phenotypes. In addition, 247 variant mouse mutants are currently in genetic confirmation testing and will result in additional new mutant lines. This mutagenesis screen, along with the screen described in the accompanying paper, leads to a significant increase in the number of mouse models available to the scientific community. Our mutant lines are freely accessible to non-commercial users (for information, see http://www.gsf.de/ieg/groups/enu-mouse.html).

The C9orf72 GGGGCC Repeat Is Translated into Aggregating Dipeptide-Repeat Proteins in FTLD/ALS

Unusual Aggregates Several recent papers have revealed the unexpected genetic and pathological overlap between frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The most common genetic cause is the GGGGCC hexanucleotide repeat expansion upstream of the C9orf72 coding region affecting about 10% of all patients. It is currently unknown how repeat expansion might lead to neurodegeneration. C9orf72 patients show two distinct types of ubiquitinated inclusions in the central nervous system, one of which was identified as phosphorylated TDP-43 protein. However, all inclusions in the cerebellum and most inclusions in the hippocampus and neocortex lack TDP-43, and the actual disease protein is unknown. Mori et al. (p. 1335, published online 7 February; see the Perspective by Taylor) discovered that most of these characteristic inclusions contain poly-(Gly-Ala) and, to a lesser extent, poly-(Gly-Pro) and poly-(Gly-Arg) dipeptide-repeat proteins that are generated by non-ATG–initiated translation from the expanded GGGGCC repeats in three reading frames. The findings yield mechanistic insight into the pathogenesis of FTLD/ALS with C9orf72 repeat expansions and directly link this common mutation to the characteristic pathology. A new class of proteins links a common genetic mutation to the predominant pathology in certain neurodegenerative diseases. [Also see Perspective by Taylor] Expansion of a GGGGCC hexanucleotide repeat upstream of the C9orf72 coding region is the most common cause of familial frontotemporal lobar degeneration and amyotrophic lateral sclerosis (FTLD/ALS), but the pathomechanisms involved are unknown. As in other FTLD/ALS variants, characteristic intracellular inclusions of misfolded proteins define C9orf72 pathology, but the core proteins of the majority of inclusions are still unknown. Here, we found that most of these characteristic inclusions contain poly-(Gly-Ala) and, to a lesser extent, poly-(Gly-Pro) and poly-(Gly-Arg) dipeptide-repeat proteins presumably generated by non-ATG–initiated translation from the expanded GGGGCC repeat in three reading frames. These findings directly link the FTLD/ALS-associated genetic mutation to the predominant pathology in patients with C9orf72 hexanucleotide expansion.

Glutathione Peroxidase 4 Senses and Translates Oxidative Stress into 12/15-Lipoxygenase Dependent- and AIF-Mediated Cell Death

Oxidative stress in conjunction with glutathione depletion has been linked with various acute and chronic degenerative disorders, yet the molecular mechanisms have remained unclear. In contrast to the belief that oxygen radicals are detrimental to cells and tissues by unspecific oxidation of essential biomolecules, we now demonstrate that oxidative stress is sensed and transduced by glutathione peroxidase 4 (GPx4) into a-yet-unrecognized cell-death pathway. Inducible GPx4 inactivation in mice and cells revealed 12/15-lipoxygenase-derived lipid peroxidation as specific downstream event, triggering apoptosis-inducing factor (AIF)-mediated cell death. Cell death could be entirely prevented either by alpha-tocopherol (alpha-Toc), 12/15-lipoxygenase inhibitors, or siRNA-mediated AIF silencing. Accordingly, 12/15-lipoxygenase-deficient cells were highly resistant to glutathione depletion. Neuron-specific GPx4 depletion caused neurodegeneration in vivo and ex vivo, highlighting the importance of this pathway in neuronal cells. Since oxidative stress is common in the etiology of many human disorders, the identified pathway reveals promising targets for future therapies.

ADAM10 is the physiologically relevant, constitutive α‐secretase of the amyloid precursor protein in primary neurons

The amyloid precursor protein (APP) undergoes constitutive shedding by a protease activity called α‐secretase. This is considered an important mechanism preventing the generation of the Alzheimers disease amyloid‐β peptide (Aβ). α‐Secretase appears to be a metalloprotease of the ADAM family, but its identity remains to be established. Using a novel α‐secretase‐cleavage site‐specific antibody, we found that RNAi‐mediated knockdown of ADAM10, but surprisingly not of ADAM9 or 17, completely suppressed APP α‐secretase cleavage in different cell lines and in primary murine neurons. Other proteases were not able to compensate for this loss of α‐cleavage. This finding was further confirmed by mass‐spectrometric detection of APP‐cleavage fragments. Surprisingly, in different cell lines, the reduction of α‐secretase cleavage was not paralleled by a corresponding increase in the Aβ‐generating β‐secretase cleavage, revealing that both proteases do not always compete for APP as a substrate. Instead, our data suggest a novel pathway for APP processing, in which ADAM10 can partially compete with γ‐secretase for the cleavage of a C‐terminal APP fragment generated by β‐secretase. We conclude that ADAM10 is the physiologically relevant, constitutive α‐secretase of APP.

Switch in chemokine receptor expression upon TCR stimulation reveals novel homing potential for recently activated T cells

When naive T lymphocytes are activated and differentiate into memory/effector cells, they down‐regulate receptors for constitutive chemokines such as CXCR4 and CCR7 and acquire receptors for inflammatory chemokines such as CCR3, CCR5 and CXCR3, depending on the Th1/Th2 polarization. This switch in chemokine receptor usage leads to the acquisition of the capacity to migrate into inflamed tissues. Using RNase protection assays, staining with specific antibodies, and response to recombinant chemokines, we now show that following TCR stimulation, memory/effector T cells undergo a further and transient switch in receptor expression. CCR1, CCR2, CCR3, CCR5, CCR6 and CXCR3 are down‐regulated within 6 h, while CCR7, CCR4, CCR8 and CXCR5 are up‐regulated for 2 to 3 days. Up‐regulation of CCR7 following TCR stimulation was observed also among resting peripheral blood T cells and required neither co‐stimulation nor exogenous IL‐2. On the other hand IL‐2 down‐regulated CXCR5, up‐regulated CCR8 and facilitated the recovery of CCR3 and CCR5. Upon TCR stimulation, Th1 and Th2 cells produced comparable sets of chemokines, including RANTES, macrophage inflammatory protein‐1β, I‐309, IL‐8 and macrophage‐derived chemokine, which may modulate surface chemokine receptors and contribute to cell recruitment at sites of antigenic recognition. Altogether these results show that following TCR stimulation effector/memory T cells transiently acquire responsiveness to constitutive chemokines. As a result, T cells that are activated in tissues may either recirculate to draining lymph nodes or migrate to nearby sites of organized ectopic lymphoid tissues.

Histone methylation by the Drosophila epigenetic transcriptional regulator Ash1.

The establishment and maintenance of mitotic and meiotic stable (epigenetic) transcription patterns is fundamental for cell determination and function. Epigenetic regulation of transcription is mediated by epigenetic activators and repressors, and may require the establishment, ‘spreading’ and maintenance of epigenetic signals. Although these signals remain unclear, it has been proposed that chromatin structure and consequently post-translational modification of histones may have an important role in epigenetic gene expression. Here we show that the epigenetic activator Ash1 (ref. 5) is a multi-catalytic histone methyl-transferase (HMTase) that methylates lysine residues 4 and 9 in H3 and 20 in H4. Transcriptional activation by Ash1 coincides with methylation of these three lysine residues at the promoter of Ash1 target genes. The methylation pattern placed by Ash1 may serve as a binding surface for a chromatin remodelling complex containing the epigenetic activator Brahma (Brm), an ATPase, and inhibits the interaction of epigenetic repressors with chromatin. Chromatin immunoprecipitation indicates that epigenetic activation of Ultrabithorax transcription in Drosophila coincides with trivalent methylation by Ash1 and recruitment of Brm. Thus, histone methylation by Ash1 may provide a specific signal for the establishment of epigenetic, active transcription patterns.

Epstein–Barr virus-encoded microRNA miR-BART2 down-regulates the viral DNA polymerase BALF5

MicroRNAs (miRNAs) have been implicated in sequence-specific cleavage, translational repression or deadenylation of specific target mRNAs resulting in post-transcriptional gene silencing. Epstein–Barr virus (EBV) encodes 23 miRNAs of unknown function. Here we show that the EBV-encoded miRNA miR-BART2 down-regulates the viral DNA polymerase BALF5. MiR-BART2 guides cleavage within the 3′-untranslated region (3′UTR) of BALF5 by virtue of its complete complementarity to its target. Induction of the lytic viral replication cycle results in a reduction of the level of miR-BART2 with a strong concomitant decrease of cleavage of the BALF5 3′UTR. Expression of miR-BART2 down-regulates the activity of a luciferase reporter gene containing the BALF5 3′UTR. Forced expression of miR-BART2 during lytic replication resulted in a 40–50% reduction of the level of BALF5 protein and a 20% reduction of the amount of virus released from EBV-infected cells. Our results are compatible with the notion that EBV-miR-BART2 inhibits transition from latent to lytic viral replication.

Transcribing RNA Polymerase II Is Phosphorylated at CTD Residue Serine-7

RNA polymerase II is distinguished by its large carboxyl-terminal repeat domain (CTD), composed of repeats of the consensus heptapeptide Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Differential phosphorylation of serine-2 and serine-5 at the 5′ and 3′ regions of genes appears to coordinate the localization of transcription and RNA processing factors to the elongating polymerase complex. Using monoclonal antibodies, we reveal serine-7 phosphorylation on transcribed genes. This position does not appear to be phosphorylated in CTDs of less than 20 consensus repeats. The position of repeats where serine-7 is substituted influenced the appearance of distinct phosphorylated forms, suggesting functional differences between CTD regions. Our results indicate that restriction of serine-7 epitopes to the Linker-proximal region limits CTD phosphorylation patterns and is a requirement for optimal gene expression.