TRIP6 functions in brain ciliogenesis.

TRIP6, a member of the ZYXIN-family of LIM domain proteins, is a focal adhesion component. Trip6 deletion in the mouse, reported here, reveals a function in the brain: ependymal and choroid plexus epithelial cells are carrying, unexpectedly, fewer and shorter cilia, are poorly differentiated, and the mice develop hydrocephalus. TRIP6 carries numerous protein interaction domains and its functions require homodimerization. Indeed, TRIP6 disruption in vitro (in a choroid plexus epithelial cell line), via RNAi or inhibition of its homodimerization, confirms its function in ciliogenesis. Using super-resolution microscopy, we demonstrate TRIP6 localization at the pericentriolar material and along the ciliary axoneme. The requirement for homodimerization which doubles its interaction sites, its punctate localization along the axoneme, and its co-localization with other cilia components suggest a scaffold/co-transporter function for TRIP6 in cilia. Thus, this work uncovers an essential role of a LIM-domain protein assembly factor in mammalian ciliogenesis.

NF-κB signaling in tanycytes mediates inflammation-induced anorexia.

OBJECTIVES: Infections, cancer, and systemic inflammation elicit anorexia. Despite the medical significance of this phenomenon, the question of how peripheral inflammatory mediators affect the central regulation of food intake is incompletely understood. Therefore, we have investigated the sickness behavior induced by the prototypical inflammatory mediator IL-1ß. METHODS: IL-1ß was injected intravenously. To interfere with IL-1ß signaling, we deleted the essential modulator of NF-κB signaling (Nemo) in astrocytes and tanycytes. RESULTS: Systemic IL-1ß increased the activity of the transcription factor NF-κB in tanycytes of the mediobasal hypothalamus (MBH). By activating NF-κB signaling, IL-1ß induced the expression of cyclooxygenase-2 (Cox-2) and stimulated the release of the anorexigenic prostaglandin E2 (PGE2) from tanycytes. When we deleted Nemo in astrocytes and tanycytes, the IL-1ß-induced anorexia was alleviated whereas the fever response and lethargy response were unchanged. Similar results were obtained after the selective deletion of Nemo exclusively in tanycytes. CONCLUSIONS: Tanycytes form the brain barrier that mediates the anorexic effect of systemic inflammation in the hypothalamus.

Identification of undescribed Relb expression domains in the murine brain by new Relb:cre-katushka reporter mice.

BACKGROUND: Noncanonical NF-κB signaling through activation of the transcription factor RelB acts as key regulator of cell lineage determination and differentiation in various tissues including the immune system. To elucidate temporospatial aspects of Relb expression, we generated a BAC transgenic knock-in mouse expressing the fluorescent protein Katushka and the enzyme Cre recombinase under control of the murine Relb promoter (RelbCre-Kat mice). RESULTS: Co-expression of Katushka and Relb in fibroblast cultures and tissues of transgenic mice revealed highly specific reporter functions of the transgene. Crossing RelbCre-Kat mice with ROSA26R reporter mice that allow for Cre-mediated consecutive ß-galactosidase or YFP synthesis identified various Relb expression domains in perinatal and mature mice. Besides thymus and spleen, highly specific expression patterns were found in different neuronal domains, as well as in other nonimmune organs including skin, skeletal structures and kidney. De novo Relb expression in the mature brain was confirmed in conditional knockout mice with neuro-ectodermal Relb deletion. CONCLUSION: Our results demonstrate the usability of RelbCre-Kat reporter mice for the detection of de novo and temporarily restricted Relb expression including cell and lineage tracing of Relb expressing cells. Relb expression during mouse embryogenesis and at adulthood suggests, beyond immunity, important functions of this transcription factor in neurodevelopment and CNS function.

Alternative NF-κB signaling controls peripheral homeostasis and function of regulatory T cells.

Regulatory T cells (Tregs) maintain immune homeostasis and play an important role in tissue regeneration after injury. Mutations affecting development or homeostasis of Tregs lead to immune pathologies in humans and are often fatal in mouse models. Although the pathways required for Treg development are being increasingly characterized, factors crucial for Treg homeostasis are not completely understood. Previously we have found a role for alternative NF-κB pathway in restricting T cell activation and Th17 differentiation. Here, by using the mouse model of uncontrolled alternative NF-κB signaling we identify a crucial intrinsic role of RelB signaling in regulating homeostasis and competitive fitness of Tregs. The failure of p100-/- Tregs to maintain the population of effector Tregs and efficiently suppress immune reactions results in lethal multiorgan Th1-mediated inflammation in Rag1-/- recipients. This inflammation is combined with severe lymphopenia and could be rescued by adoptive transfer of wild type Tregs. Thus in addition to its role in Th17 differentiation, RelB acts as a potent inhibitor of Treg effector functions. Our results point to RelB as a potential therapeutic target for Treg manipulation.

Cohesin-mediated NF-κB signaling limits hematopoietic stem cell self-renewal in aging and inflammation.

Organism aging is characterized by increased inflammation and decreased stem cell function, yet the relationship between these factors remains incompletely understood. This study shows that aged hematopoietic stem and progenitor cells (HSPCs) exhibit increased ground-stage NF-κB activity, which enhances their responsiveness to undergo differentiation and loss of self-renewal in response to inflammation. The study identifies Rad21/cohesin as a critical mediator of NF-κB signaling, which increases chromatin accessibility in the vicinity of NF-κB target genes in response to inflammation. Rad21 is required for normal differentiation, but limits self-renewal of hematopoietic stem cells (HSCs) during aging and inflammation in an NF-κB-dependent manner. HSCs from aged mice fail to down-regulate Rad21/cohesin and inflammation/differentiation signals in the resolution phase of inflammation. Inhibition of cohesin/NF-κB reverts hypersensitivity of aged HSPCs to inflammation-induced differentiation and myeloid-biased HSCs with disrupted/reduced expression of Rad21/cohesin are increasingly selected during aging. Together, Rad21/cohesin-mediated NF-κB signaling limits HSPC function during aging and selects for cohesin-deficient HSCs with myeloid-skewed differentiation.

Cell cycle-dependent and -independent telomere shortening accompanies murine brain aging.

Replication-based telomere shortening during lifetime is species- and tissue-specific, however, its impact on healthy aging is unclear. In particular, the contribution of telomere truncation to the aging process of the CNS, where replicative senescence alone fails to explain organ aging due to low to absent mitotic activity of intrinsic populations, is undefined. Here, we assessed changes in relative telomere length in non-replicative and replicative neural brain populations and telomerase activity as a function of aging in C57BL/6 mice. Telomeres in neural cells and sub-selected neurons shortened with aging in a cell cycle-dependent and -independent manner, with preponderance in replicative moieties, implying that proliferation accelerates, but is not prerequisite for telomere shortening. Consistent with this telomere erosion, telomerase activity and nuclear TERT protein were not induced with aging. Knockdown of the Rela subunit of NF-κB, which controls both telomerase enzyme and subcellular TERT protein allocation, did also not influence telomerase activity or telomere length, in spite of its naive up-regulation selectively under aging conditions. We conclude that telomere instability is intrinsic to physiological brain aging beyond cell replication, and appears to occur independently of a functional interplay with NF-κB, but rather as a failure to induce or relocate telomerase.

A new RelB-dependent CD117+ CD172a+ murine DC subset preferentially induces Th2 differentiation and supports airway hyperresponses in vivo.

The NF-κB transcription factor subunit RelB is important for the full activation of conventional dendritic cells (cDCs) during T-cell-dependent immune responses. Although the number of splenic DCs is greatly reduced in RelBnull mice, the cause and consequences of this deficiency are currently unknown. To circumvent the impact of the pleiotropic defects in RelBnull mice we used a reporter model for RelB expression (RelBKatushka mice) and conditionally deleted RelB in DCs (RelBCD11c-Cre mice). Thereby, we can show here that RelB is essential for the differentiation of a CD117+ CD172a+ cDC subpopulation that highly expresses RelB. Surprisingly, these DCs depend on p50 for their development and are negatively regulated by a constitutive p52 activation in absence of p100. The absence of p52/p100 had no influence on the homeostasis of CD117+ CD172a+ cDCs. RelB-dependent CD117+ CD172a+ DCs strongly induce the production of the type 2 cytokines IL-4 and IL-13, as well as GM-CSF from naïve Th cells. Consequently, mice lacking RelB in cDCs show an attenuated bronchial hyperresponsiveness with reduced eosinophil infiltration. Taken together, we have identified a new splenic RelB-dependent CD117+ CD172a+ cDC population that preferentially induces Th2 responses.

RelB regulates Th17 differentiation in a cell-intrinsic manner.

The role of the alternative NF-κB pathway is mainly attributed to the lymphoid organ formation and blood cancer. However, its involvement in lymphocyte differentiation is not clearly defined. Recently, we have shown that uncontrolled activation of alternative NF-κB in mice lacking the NF-κB inhibitory protein p100 (p100-/- mice) hinders plasmablast proliferation and diminishes T cell independent responses. Here we show that hyperactivation of this pathway leads to a cell-intrinsic T cell defects. p100-deficient T helper cells displayed both an activation and a proliferation defect in vitro. In addition, memory T cell formation was impaired in vivo. Moreover, p100-/- T cells failed to polarize into T helper 17 cells. This phenotype was dependent on increased RelB activation and suboptimal RORγt expression. Thus, our results demonstrate that RelB acts as a negative regulator of T cell activation and Th17 development. Targeting this pathway therefore could be beneficial in Th17-mediated pathologies.

Transcriptional Control of Synaptic Plasticity by Transcription Factor NF-κB.

Activation of nuclear factor kappa B (NF-κB) transcription factors is required for the induction of synaptic plasticity and memory formation. All components of this signaling pathway are localized at synapses, and transcriptionally active NF-κB dimers move to the nucleus to translate synaptic signals into altered gene expression. Neuron-specific inhibition results in altered connectivity of excitatory and inhibitory synapses and functionally in selective learning deficits. Recent research on transgenic mice with impaired or hyperactivated NF-κB gave important insights into plasticity-related target gene expression that is regulated by NF-κB. In this minireview, we update the available data on the role of this transcription factor for learning and memory formation and comment on cross-sectional activation of NF-κB in the aged and diseased brain that may directly or indirectly affect κB-dependent transcription of synaptic genes.

Role of nuclear factor kappa B in central nervous system regeneration.

Activation of nuclear factor kappa B (NF-κB) is a hallmark of various central nervous system (CNS) pathologies. Neuron-specific inhibition of its transcriptional activator subunit RelA, also referred to as p65, promotes neuronal survival under a range of conditions, i.e., for ischemic or excitotoxic insults. In macro- and microglial cells, post-lesional activation of NF-κB triggers a growth-permissive program which contributes to neural tissue inflammation, scar formation, and the expression of axonal growth inhibitors. Intriguingly, inhibition of such inducible NF-κB in the neuro-glial compartment, i.e., by genetic ablation of RelA or overexpression of a transdominant negative mutant of its upstream regulator IκBα, significantly enhances functional recovery and promotes axonal regeneration in the mature CNS. By contrast, depletion of the NF-κB subunit p50, which lacks transcriptional activator function and acts as a transcriptional repressor on its own, causes precocious neuronal loss and exacerbates axonal degeneration in the lesioned brain. Collectively, the data imply that NF-κB orchestrates a multicellular program in which κB-dependent gene expression establishes a growth-repulsive terrain within the post-lesioned brain that limits structural regeneration of neuronal circuits. Considering these subunit-specific functions, interference with the NF-κB pathway might hold clinical potentials to improve functional restoration following traumatic CNS injury.

In vivo imaging of optic nerve fiber integrity by contrast-enhanced MRI in mice.

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and postsynaptic projections to the visual cortex. Based on its distinct anatomical structure and convenient accessibility, it has become the favored structure for studies on neuronal survival, axonal regeneration, and synaptic plasticity. Recent advancements in MR imaging have enabled the in vivo visualization of the retino-tectal part of this projection using manganese mediated contrast enhancement (MEMRI). Here, we present a MEMRI protocol for illustration of the visual projection in mice, by which resolutions of (200 µm)3 can be achieved using common 3 Tesla scanners. We demonstrate how intravitreal injection of a single dosage of 15 nmol MnCl2 leads to a saturated enhancement of the intact projection within 24 hr. With exception of the retina, changes in signal intensity are independent of coincided visual stimulation or physiological aging. We further apply this technique to longitudinally monitor axonal degeneration in response to acute optic nerve injury, a paradigm by which Mn2+ transport completely arrests at the lesion site. Conversely, active Mn2+ transport is quantitatively proportionate to the viability, number, and electrical activity of axon fibers. For such an analysis, we exemplify Mn2+ transport kinetics along the visual path in a transgenic mouse model (NF-κB p50KO) displaying spontaneous atrophy of sensory, including visual, projections. In these mice, MEMRI indicates reduced but not delayed Mn2+ transport as compared to wild type mice, thus revealing signs of structural and/or functional impairments by NF-κB mutations. In summary, MEMRI conveniently bridges in vivo assays and post mortem histology for the characterization of nerve fiber integrity and activity. It is highly useful for longitudinal studies on axonal degeneration and regeneration, and investigations of mutant mice for genuine or inducible phenotypes.

NF-κB controls axonal regeneration and degeneration through cell-specific balance of RelA and p50 in the adult CNS.

NF-κB is dually involved in neurogenesis and brain pathology. Here, we addressed its role in adult axoneogenesis by generating mutations of RelA (p65) and p50 (also known as NFKB1) heterodimers of canonical NF-κB. In addition to RelA activation in astrocytes, optic nerve axonotmesis caused a hitherto unrecognized induction of RelA in growth-inhibitory oligodendrocytes. Intraretinally, RelA was induced in severed retinal ganglion cells and was also expressed in bystander Müller glia. Cell-type-specific deletion of transactivating RelA in neurons and/or macroglia stimulated axonal regeneration in a distinct and synergistic pattern. By contrast, deletion of the p50 suppressor subunit promoted spontaneous and post-injury Wallerian degeneration. Growth effects mediated by RelA deletion paralleled a downregulation of growth-inhibitory Cdh1 (officially known as FZR1) and upregulation of the endogenous Cdh1 suppressor EMI1 (officially known as FBXO5). Pro-degenerative loss of p50, however, stabilized retinal Cdh1. In vitro, RelA deletion elicited opposing pro-regenerative shifts in active nuclear and inactive cytoplasmic moieties of Cdh1 and Id2. The involvement of NF-κB and cell-cycle regulators such as Cdh1 in regenerative processes of non-replicative neurons suggests novel mechanisms by which molecular reprogramming might be executed to stimulate adult axoneogenesis and treat central nervous system (CNS) axonopathies.

Possibilities and limitations for high resolution small animal MRI on a clinical whole-body 3T scanner.

OBJECT: To investigate the potential of a clinical 3 T scanner to perform MRI of small rodents. MATERIALS AND METHODS: Different dedicated small animal coils and several imaging sequences were evaluated to optimize image quality with respect to SNR, contrast and spatial resolution. As an application, optimal grey-white-matter contrast and resolution were investigated for rats. Furthermore, manganese-enhanced MRI was applied in mice with unilateral crush injury of the optic nerve to investigate coil performance on topographic mapping of the visual projection. RESULTS: Differences in SNR and CNR up to factor 3 and more were observed between the investigated coils. The best grey-white matter contrast was achieved with a high resolution 3D T (2)-weighted TSE (SPACE) sequence. Delineation of the retino-tectal projection and detection of defined visual pathway damage on the level of the optic nerve could be achieved by using a T (1)-weighted, 3D gradient echo sequence with isotropic resolution of (0.2 mm)(3). CONCLUSIONS: Experimental studies in small rodents requiring high spatial resolution can be performed by using a clinical 3 T scanner with appropriate dedicated coils.

Magnetic resonance imaging of the mouse visual pathway for in vivo studies of degeneration and regeneration in the CNS.

Traditionally, depiction of isolated CNS fiber tracts is achieved by histological post mortem studies. As a tracer-dependent strategy, the calcium analog manganese has proved valuable for in vivo imaging of CNS trajectories, particularly in rats. However, adequate protocols in mice are still rare. To take advantage of the numerous genetic mouse mutants that are available to study axonal de- and regeneration processes, a MnCl2-based protocol for high-resolution contrast-enhanced MRI (MEMRI) of the visual pathway in mice acquired on a widely used clinical 3 Tesla scanner was established. Intravitreal application of MnCl2 significantly enhanced T1-weighted contrast and signal intensity along the retino-petal projection enabling its reconstruction in a 3D mode from a maximum intensity projection (MIP) calculated dataset. In response to crush injury of the optic nerve, axonal transport of MnCl2 was diminished and completely blocked proximal and distal to the lesion site, respectively. Conditions of Wallerian degeneration after acute optic nerve injury accelerated Mn2+-enhanced signal fading in axotomized projection areas between 12 and 24 h post-injury. In long-term regeneration studies 12 months after optic nerve injury, the MRI protocol proved highly sensitive and discriminated animals with rare spontaneous axonal regrowth from non-regenerating specimens. Also, structural MRI aspects shared high correlation with histological results in identical animals. Moreover, in a model of chronic neurodegeneration in p50/NF-κB-deficient mice, MnCl2-based neuron-axonal tracing supported by heat map imaging indicated neuropathy of the visual pathway due to atrophy of optic nerve fiber projections. Toxic effects of MnCl2 at MRI contrast-relevant dosages in repetitive administration protocols were ruled out by histological and optometric examinations. At higher dosages, photoreceptors, not retinal ganglion cells, turned out as most susceptible to the well-known toxicity of MnCl2. Our data accentuate in vivo MEMRI of the murine visual system as a highly specific and sensitive strategy to uncover axonal degeneration and restoration processes, even in a functional latent state. We expect MEMRI to be promising for future applications in longitudinal studies on development, aging, or regeneration of CNS projections in mouse models mimicking human CNS pathologies.

Protection of vascular smooth muscle cells by over-expressed methionine sulphoxide reductase A: role of intracellular localization and substrate availability.

Methionine sulphoxide reductase A (MSRA) that reduces methionine-S-sulphoxide back to methionine constitutes a catalytic antioxidant mechanism to prevent oxidative damage at multiple sub-cellular loci. This study examined the relative importance of protection of the cytoplasm and mitochondria by MSRA using A-10 vascular smooth muscle cells, a cell type that requires a low level of reactive oxygen species (ROS) for normal function but is readily damaged by higher concentrations of ROS. Adenoviral over-expression of human MSRA variants, targeted to either mitochondria or the cytoplasm, did not change basal viability of non-stressed cells. Oxidative stress caused by treatment with the methionine-preferring oxidizing reagent chloramine-T decreased cell viability in a concentration-dependent manner. Cytoplasmic MSRA preserved cell viability more effectively than mitochondrial MSRA and co-application of S-methyl-L-cysteine, an amino acid that acts as a substrate for MSRA when oxidized, further increased the extent of protection. This suggests an important role for an MSRA catalytic antioxidant cycle for protection of the cytoplasmic compartment against oxidative damage.

Methionine sulfoxide reductase A and a dietary supplement S-methyl-L-cysteine prevent Parkinson's-like symptoms.

Parkinson's disease (PD), a common neurodegenerative disease, is caused by loss of dopaminergic neurons in the substantia nigra. Although the underlying cause of the neuronal loss is unknown, oxidative stress is thought to play a major role in the pathogenesis of PD. The amino acid methionine is readily oxidized to methionine sulfoxide, and its reduction is catalyzed by a family of enzymes called methionine sulfoxide reductases (MSRs). The reversible oxidation-reduction cycle of methionine involving MSRs has been postulated to act as a catalytic antioxidant system protecting cells from oxidative damage. Here, we show that one member of the MSR family, MSRA, inhibits development of the locomotor and circadian rhythm defects caused by ectopic expression of human alpha-synuclein in the Drosophila nervous system. Furthermore, we demonstrate that one way to enhance the MSRA antioxidant system is dietary supplementation with S-methyl-L-cysteine (SMLC), found abundantly in garlic, cabbage, and turnips. SMLC, a substrate in the catalytic antioxidant system mediated by MSRA, prevents the alpha-synuclein-induced abnormalities. Therefore, interventions focusing on the enzymatic reduction of oxidized methionine catalyzed by MSRA represent a new prevention and therapeutic approach for PD and potentially for other neurodegenerative diseases involving oxidative stress.

Identification of a new functional splice variant of the enzyme methionine sulphoxide reductase A (MSRA) expressed in rat vascular smooth muscle cells.

Reactive oxygen species contribute to ageing of the vascular system and development of cardiovascular disease. Methionine-S-sulphoxide, an oxidized form of methionine, is repaired by the enzyme methionine sulphoxide reductase A (MSRA). The enzyme, targeted to mitochondria or the cytosol by alternative splicing, is vital for oxidative stress resistance. This study was designed to examine the endogenous expression and intracellular localization of MSRA in rat aortic vascular smooth muscle cells (VSMCs). We detected robust MSRA immunoreactivity exclusively in mitochondria. Sequence analysis of msrA transcripts revealed the presence of a novel mitochondrial splice variant, msrA2a, in cultured rat VSMCs as well as in aortic tissue preparations. The enzymatic activity of a recombinant MSRA2a protein was confirmed by the reduction of methionine sulphoxide in a model substrate peptide. We conclude that multiple MSRA variants participate in the repair of oxidized proteins in VSMC mitochondria, but that other protective mechanisms may exist in the cytoplasmic compartment.

Oxidative damage, aging and anti-aging strategies.

The last two decades brought remarkable insight into the nature of normal aging in multicellular organisms. However, we are still far away from realizing extension of maximum lifespan in humans. An important modulator of lifespan is oxidative damage induced by reactive species, such as reactive oxygen species (ROS). Studies from yeast, Caenorhabditis and Drosophila primarily focused on (1) reduced generation or (2) elimination of ROS but have two principal shortcomings: (1) dietary restriction and single gene mutations are often associated with physiological impairments and (2) overexpression of components of the antioxidant system extend lifetime only under stress-induced conditions. Recent results from Drosophila indicate the involvement of an endogenous repair and elimination system for oxidatively damaged proteins in the process of aging. This system includes methionine sulfoxide reductase A (MSRA) and the carbonyl reductase Sniffer, the protein-ubiquitin ligase Parkin and the chaperone Hsp22. In this review we summarize different anti-aging strategies and discuss a synergistic interaction between protection against free radicals and specific repair/elimination of oxidative damage in lifespan extension primarily using the model system Drosophila. To achieve lifespan extension, available experiments are often methodically grouped into (1) caloric restriction, (2) single gene mutation, and (3) overexpression of genes. Here we summarize different strategies by a more causal classification: (1) prevention of ROS generation, (2) reducing free ROS level, and (3) repair and elimination of ROS-damaged proteins.