Loss of Prohibitin Membrane Scaffolds Impairs Mitochondrial Architecture and Leads to Tau Hyperphosphorylation and Neurodegeneration

Fusion and fission of mitochondria maintain the functional integrity of mitochondria and protect against neurodegeneration, but how mitochondrial dysfunctions trigger neuronal loss remains ill-defined. Prohibitins form large ring complexes in the inner membrane that are composed of PHB1 and PHB2 subunits and are thought to function as membrane scaffolds. In Caenorhabditis elegans, prohibitin genes affect aging by moderating fat metabolism and energy production. Knockdown experiments in mammalian cells link the function of prohibitins to membrane fusion, as they were found to stabilize the dynamin-like GTPase OPA1 (optic atrophy 1), which mediates mitochondrial inner membrane fusion and cristae morphogenesis. Mutations in OPA1 are associated with dominant optic atrophy characterized by the progressive loss of retinal ganglion cells, highlighting the importance of OPA1 function in neurons. Here, we show that neuron-specific inactivation of Phb2 in the mouse forebrain causes extensive neurodegeneration associated with behavioral impairments and cognitive deficiencies. We observe early onset tau hyperphosphorylation and filament formation in the hippocampus, demonstrating a direct link between mitochondrial defects and tau pathology. Loss of PHB2 impairs the stability of OPA1, affects mitochondrial ultrastructure, and induces the perinuclear clustering of mitochondria in hippocampal neurons. A destabilization of the mitochondrial genome and respiratory deficiencies manifest in aged neurons only, while the appearance of mitochondrial morphology defects correlates with tau hyperphosphorylation in the absence of PHB2. These results establish an essential role of prohibitin complexes for neuronal survival in vivo and demonstrate that OPA1 stability, mitochondrial fusion, and the maintenance of the mitochondrial genome in neurons depend on these scaffolding proteins. Moreover, our findings establish prohibitin-deficient mice as a novel genetic model for tau pathologies caused by a dysfunction of mitochondria and raise the possibility that tau pathologies are associated with other neurodegenerative disorders caused by deficiencies in mitochondrial dynamics.     

FoxP3+ Regulatory T Cells Determine Disease Severity in Rodent Models of Inflammatory Neuropathies

Inflammatory neuropathies represent disabling human autoimmune disorders with considerable disease variability. Animal models provide insights into defined aspects of their disease pathogenesis. Forkhead box P3 (FoxP3)+ regulatory T lymphocytes (Treg) are anti-inflammatory cells that maintain immune tolerance and counteract tissue damage in a variety of immune-mediated disorders. Dysfunction or a reduced frequency of Tregs have been associated with different human autoimmune disorders. We here analyzed the functional relevance of Tregs in determining disease manifestation and severity in murine models of autoimmune neuropathies. We took advantage of the DEREG mouse system allowing depletion of Treg with high specificity as well as anti-CD25 directed antibodies to deplete Tregs in mice in actively induced experimental autoimmune neuritis (EAN). Furthermore antibody-depletion was performed in an adoptive transfer model of chronic neuritis. Early Treg depletion increased clinical EAN severity both in active and adoptive transfer chronic neuritis. This was accompanied by increased proliferation of myelin specific T cells and histological signs of peripheral nerve inflammation. Late stage Treg depletion after initial disease manifestation however did not exacerbate inflammatory neuropathy symptoms further. We conclude that Tregs determine disease severity in experimental autoimmune neuropathies during the initial priming phase, but have no major disease modifying function after disease manifestation. Potential future therapeutic approaches targeting Tregs should thus be performed early in inflammatory neuropathies.     

Mouse genetics identifies unique and overlapping functions of fibroblast growth factor receptors in keratinocytes

Fibroblast growth factors (FGFs) are key regulators of tissue development, homeostasis and repair, and abnormal FGF signalling is associated with various human diseases. In human and murine epidermis, FGF receptor 3 (FGFR3) activation causes benign skin tumours, but the consequences of FGFR3 deficiency in this tissue have not been determined. Here, we show that FGFR3 in keratinocytes is dispensable for mouse skin development, homeostasis and wound repair. However, the defect in the epidermal barrier and the resulting inflammatory skin disease that develops in mice lacking FGFR1 and FGFR2 in keratinocytes were further aggravated upon additional loss of FGFR3. This caused fibroblast activation and fibrosis in the FGFR1/FGFR2 double‐knockout mice and even more in mice lacking all three FGFRs, revealing functional redundancy of FGFR3 with FGFR1 and FGFR2 for maintaining the epidermal barrier. Taken together, our study demonstrates that FGFR1, FGFR2 and FGFR3 act together to maintain epidermal integrity and cutaneous homeostasis, with FGFR2 being the dominant receptor.     

The cell adhesion molecule neuroplastin-65 is a novel interaction partner of γ-aminobutyric acid type A receptors

γ-Aminobutyric acid type A (GABA_A) receptors are pentameric ligand-gated ion channels that mediate fast inhibition in the central nervous system. Depending on their subunit composition, these receptors exhibit distinct pharmacological properties and differ in their ability to interact with proteins involved in receptor anchoring at synaptic or extra-synaptic sites. Whereas GABA_A receptors containing α1, α2, or α3 subunits are mainly located synaptically where they interact with the submembranous scaffolding protein gephyrin, receptors containing α5 subunits are predominantly found extra-synaptically and seem to interact with radixin for anchorage. Neuroplastin is a cell adhesion molecule of the immunoglobulin superfamily that is involved in hippocampal synaptic plasticity. Our results reveal that neuroplastin and GABA_A receptors can be co-purified from rat brain and exhibit a direct physical interaction as demonstrated by co-precipitation and Förster resonance energy transfer (FRET) analysis in a heterologous expression system. The brain-specific isoform neuroplastin-65 co-localizes with GABA_A receptors as shown in brain sections as well as in neuronal cultures, and such complexes can either contain gephyrin or be devoid of gephyrin. Neuroplastin-65 specifically co-localizes with α1 or α2 but not with α3 subunits at GABAergic synapses. In addition, neuroplastin-65 also co-localizes with GABA_A receptor α5 subunits at extra-synaptic sites. Down-regulation of neuroplastin-65 by shRNA causes a loss of GABA_A receptor α2 subunits at GABAergic synapses. These results suggest that neuroplastin-65 can co-localize with a subset of GABA_A receptor subtypes and might contribute to anchoring and/or confining GABA_A receptors to particular synaptic or extra-synaptic sites, thus affecting receptor mobility and synaptic strength.     

Asymmetric lipid bilayer formation stabilized by DNA at the air/water interface

The lipid bis(guanidinium)-tren-cholesterol (BGTC) is a cationic cholesterol derivative bearing guanidinium polar headgroups used for gene transfection either alone or formulated as liposomes with the zwitterionic lipid 1,2-di-[cis-9-octadecenoyl]-sn-glycero-3-phosphoethanolamine (DOPE). Previous investigations have shown its ability to strongly interact with DNA and form asymmetric lipid bilayers at the air/water interface when mixed with DOPE. Here, with a view to further investigate its physicochemical behavior, we studied the interactions of mixtures of BGTC with another zwitterionic lipid, 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine, (DMPC), with DNA at the air/water interface by using the Langmuir monolayer technique coupled with Brewster Angle Microscopy (BAM) and Polarization Modulation Infra Red Reflexion Absorption (PMIRRAS) spectroscopy and we investigate DNA–BGTC/DMPC interactions. We demonstrate that when DNA is injected into the subphase in excess compared to the positive charges of BGTC, it adsorbs to BGTC/DMPC monolayers at 20 mN/m whatever the lipid monolayer composition (1/5, 2/3 or 3/2 BGTC/DMPC molar ratio) and forms an incomplete monolayer of either isotropic or anisotropic double strands depending on the BGTC content in the monolayer. Compression beyond the collapse of some mixed DNA–BGTC/DMPC (2/3 and 3/2 molar ratio) systems leads to the formation of DNA monolayers underneath asymmetric lipid bilayers characterized by a bottom layer of BGTC in contact with DNA and a top layer mainly constituted of DMPC.     

Proteomic and functional analysis of Argonaute-containing mRNA-protein complexes in human cells

Members of the Argonaute (Ago) protein family associate with small RNAs and have important roles in RNA silencing. Here, we analysed Ago1- and Ago2-containing protein complexes in human cells. Separation of Ago-associated messenger ribonucleoproteins (mRNPs) showed that Ago1 and Ago2 reside in three complexes with distinct Dicer and RNA-induced silencing complex activities. A comprehensive proteomic analysis of Ago-containing mRNPs identified a large number of proteins involved in RNA metabolism. By using co-immunoprecipitation experiments followed by RNase treatment, we biochemically mapped interactions within Ago mRNPs. Using reporter assays and knockdown experiments, we showed that the putative RNA-binding protein RBM4 is required for microRNA-guided gene regulation.     

Noncompaction of the ventricular myocardium is associated with a de novo mutation in the beta-myosin heavy chain gene

Noncompaction of the ventricular myocardium (NVM) is the morphological hallmark of a rare familial or sporadic unclassified heart disease of heterogeneous origin. NVM results presumably from a congenital developmental error and has been traced back to single point mutations in various genes. The objective of this study was to determine the underlying genetic defect in a large German family suffering from NVM. Twenty four family members were clinically assessed using advanced imaging techniques. For molecular characterization, a genome-wide linkage analysis was undertaken and the disease locus was mapped to chromosome 14ptel-14q12. Subsequently, two genes of the disease interval, MYH6 and MYH7 (encoding the alpha- and beta-myosin heavy chain, respectively) were sequenced, leading to the identification of a previously unknown de novo missense mutation, c.842G>C, in the gene MYH7. The mutation affects a highly conserved amino acid in the myosin subfragment-1 (R281T). In silico simulations suggest that the mutation R281T prevents the formation of a salt bridge between residues R281 and D325, thereby destabilizing the myosin head. The mutation was exclusively present in morphologically affected family members. A few members of the family displayed NVM in combination with other heart defects, such as dislocation of the tricuspid valve (Ebstein's anomaly, EA) and atrial septal defect (ASD). A high degree of clinical variability was observed, ranging from the absence of symptoms in childhood to cardiac death in the third decade of life. The data presented in this report provide first evidence that a mutation in a sarcomeric protein can cause noncompaction of the ventricular myocardium.     

Psychophysiological responses to stress after stress management training in patients with rheumatoid arthritis

Background

Stress management interventions may prove useful in preventing the detrimental effects of stress on health. This study assessed the effects of a stress management intervention on the psychophysiological response to stress in patients with rheumatoid arthritis (RA).

Methods

Seventy-four patients with RA, who were randomly assigned to either a control group or a group that received short-term stress management training, performed a standardized psychosocial stress task (Trier Social Stress Test; TSST) 1 week after the stress management training and at a 9-week follow-up. Psychological and physical functioning, and the acute psychophysiological response to the stress test were assessed.

Results

Patients in the intervention group showed significantly lower psychological distress levels of anxiety after the training than did the controls. While there were no between-group differences in stress-induced tension levels, and autonomic (α-amylase) or endocrine (cortisol) responses to the stress test 1 week after the intervention, levels of stress-induced tension and cortisol were significantly lower in the intervention group at the 9-week follow-up. Overall, the response to the intervention was particularly evident in a subgroup of patients with a psychological risk profile.

Conclusion

A relatively short stress management intervention can improve psychological functioning and influences the psychophysiological response to stress in patients with RA, particularly those psychologically at risk. These findings might help understand how stress can affect health and the role of individual differences in stress responsiveness.

Trial Registration

TrialRegister.nl NTR1193     

Mitochondrial haplogroups, control region polymorphisms and malignant melanoma: a study in middle European Caucasians

Background

Because mitochondria play an essential role in energy metabolism, generation of reactive oxygen species (ROS), and apoptosis, sequence variation in the mitochondrial genome has been postulated to be a contributing factor to the etiology of multifactorial age-related diseases, including cancer. The aim of the present study was to compare the frequencies of mitochondrial DNA (mtDNA) haplogroups as well as control region (CR) polymorphisms of patients with malignant melanoma (n = 351) versus those of healthy controls (n = 1598) in Middle Europe.

Methodology and Principal Findings

Using primer extension analysis and DNA sequencing, we identified all nine major European mitochondrial haplogroups and known CR polymorphisms. The frequencies of the major mitochondrial haplogroups did not differ significantly between patients and control subjects, whereas the frequencies of the one another linked CR polymorphisms A16183C, T16189C, C16192T, C16270T and T195C were significantly higher in patients with melanoma compared to the controls. Regarding clinical characteristics of the patient cohort, none of the nine major European haplogroups was associated with either Breslow thickness or distant metastasis. The CR polymorphisms A302CC-insertion and T310C-insertion were significantly associated with mean Breslow thickness, whereas the CR polymorphism T16519C was associated with metastasis.

Conclusions and Significance

Our results suggest that mtDNA variations could be involved in melanoma etiology and pathogenesis, although the functional consequence of CR polymorphisms remains to be elucidated.