Role of the AAA protease Yme1 in folding of proteins in the mitochondrial intermembrane space

The vast majority of mitochondrial proteins are encoded in the nucleus and synthesized as precursor proteins on cytosolic ribosomes. After translation, these precursor proteins are imported in a largely, if not completely, unfolded state into one of the four mitochondrial subcompartments, the outer membrane, the intermembrane space, the inner membrane or the matrix. Once the precursor proteins reach their compartment of destination, they can fold into the functionally active three-dimensional native structure. Therefore, internal mitochondrial folding systems are needed in each subcompartment to assist folding of these precursor proteins upon import. Members of several “classical” chaperone families are present in the mitochondrial matrix and have been shown to support import and folding of newly imported polypeptides. However, folding of proteins in the mitochondrial intermembrane space is only poorly understood. Recently, a disulfide relay system in the intermembrane space that mediates import and folding was described, but this system is limited to proteins that form disulfide bonds. For the majority of intermembrane proteins, folding helpers that promote folding have not yet been discovered. In order to identify general folding helpers of the intermembrane space, the well studied model substrate mouse dihydrofolate reductase (DHFR) was targeted to the mitochondrial intermembrane space of S. cerevisiae and its folding analyzed. DHFR assumes its mature fold in the intermembrane space and heat shock induces DHFR aggregation. Interestingly, aggregation is counteracted by an ATP-dependent process. The i-AAA protease Yme1 that is anchored in the inner mitochondrial membrane and exposes its functional domains to the intermembrane space was able to prevent the aggregation of DHFR. A number of proteins of diverse structural and functional classes were found in the aggregate fractions of mitochondria lacking Yme1. Amongst them were factors that are involved in the establishment and maintenance of the mitochondrial ultrastructure, lipid metabolism, protein translocation and respiratory growth. Considering the diversity of the proteins affected in the absence of Yme1 and their function in mitochondria, the pleiotropic effects of the deletion of Yme1 can be readily explained. The findings of the present in vivo study confirm previous hints to a chaperone-like function of Yme1 resulting from in vitro experiments. Yme1 thus has a dual role as protease and as chaperone and occupies a key position in the protein quality control system of the mitochondrial intermembrane space

Amyloid Β-Peptide Increases Mitochondria-Endoplasmic Reticulum Contact Altering Mitochondrial Function and Autophagosome Formation in Alzheimer's Disease-Related Models.

Recent findings have shown that the connectivity and crosstalk between mitochondria and the endoplasmic reticulum (ER) at mitochondria-ER contact sites (MERCS) are altered in Alzheimer's disease (AD) and in AD-related models. MERCS have been related to the initial steps of autophagosome formation as well as regulation of mitochondrial function. Here, the interplay between MERCS, mitochondria ultrastructure and function and autophagy were evaluated in different AD animal models with increased levels of Aβ as well as in primary neurons derived from these animals. We start by showing that the levels of Mitofusin 1, Mitofusin 2 and mitochondrial import receptor subunit TOM70 are decreased in post-mortem brain tissue derived from familial AD. We also show that Aβ increases the juxtaposition between ER and mitochondria both in adult brain of different AD mouse models as well as in primary cultures derived from these animals. In addition, the connectivity between ER and mitochondria are also increased in wild-type neurons exposed to Aβ. This alteration in MERCS affects autophagosome formation, mitochondrial function and ATP formation during starvation. Interestingly, the increment in ER-mitochondria connectivity occurs simultaneously with an increase in mitochondrial activity and is followed by upregulation of autophagosome formation in a clear chronological sequence of events. In summary, we report that Aβ can affect cell homeostasis by modulating MERCS and, consequently, altering mitochondrial activity and autophagosome formation. Our data suggests that MERCS is a potential target for drug discovery in AD

Neuronal cell-based high-throughput screen for enhancers of mitochondrial function reveals luteolin as a modulator of mitochondria-endoplasmic reticulum coupling

Background: Mitochondrial dysfunction is a common feature of aging, neurodegeneration, and metabolic diseases. Hence, mitotherapeutics may be valuable disease modifiers for a large number of conditions. In this study, we have set up a large-scale screening platform for mitochondrial-based modulators with promising therapeutic potential. Results: Using differentiated human neuroblastoma cells, we screened 1200 FDA-approved compounds and identified 61 molecules that significantly increased cellular ATP without any cytotoxic effect. Following dose response curve-dependent selection, we identified the flavonoid luteolin as a primary hit. Further validation in neuronal models indicated that luteolin increased mitochondrial respiration in primary neurons, despite not affecting mitochondrial mass, structure, or mitochondria-derived reactive oxygen species. However, we found that luteolin increased contacts between mitochondria and endoplasmic reticulum (ER), contributing to increased mitochondrial calcium (Ca2+) and Ca2+-dependent pyruvate dehydrogenase activity. This signaling pathway likely contributed to the observed effect of luteolin on enhanced mitochondrial complexes I and II activities. Importantly, we observed that increased mitochondrial functions were dependent on the activity of ER Ca2+-releasing channels inositol 1,4,5-trisphosphate receptors (IP3Rs) both in neurons and in isolated synaptosomes. Additionally, luteolin treatment improved mitochondrial and locomotory activities in primary neurons and Caenorhabditis elegans expressing an expanded polyglutamine tract of the huntingtin protein. Conclusion: We provide a new screening platform for drug discovery validated in vitro and ex vivo. In addition, we describe a novel mechanism through which luteolin modulates mitochondrial activity in neuronal models with potential therapeutic validity for treatment of a variety of human diseases

Multianalytical approach for the analysis of the Codices Millenarius Maior and Millenarius Minor in Kremsmuenster Abbey, Upper Austria

Abstract Two precious Carolingian manuscripts kept in the library of Kremsmuenster Abbey in Upper Austria were subject of investigation. The well-known Codices Millenarius Maior (Inv. No. CC Cim 1, Fig. 1a) and Millenarius Minor (Inv. No. CC Cim 2, Fig. 1b) were studied within the framework of the Centre of Image and Material Analysis in Cultural Heritage (CIMA) in Vienna. The manuscripts are especially famous for their precious and colorful miniatures of a very early medieval period. The aim of the work was the material identification (colors and inks) used for the make-up of the two codices in order to gain a better understanding of their evolution and their provenance. The instrumentation available in the CIMA laboratories allowed performing in situ measurements using non-destructive and non-invasive analytical methods. The investigations comprised a combination of three complementary methods: X-ray fluorescence analysis (XRF), Fourier transform infrared spectrometry in the reflection mode (rFTIR) and Raman spectrometry. In addition to the identification of the pigments and inks also a detailed characterization of the parchment concerning its manufacturing process was achieved by that combination. The identification of calcium carbonates on the surface of the parchment is an indicator for the liming or whitening of the animal skin, whereas the polishing process of the parchment surface with pumice stone, left traces of silicates, detected by rFTIR. The combination of XRF and Raman spectrometry enabled the characterization of black/brown inks in the text revealing the usage of iron gall inks. For the red inks applied for text and initials vermilion and red lead were applied in both codices. Furthermore, the pigment palette used for the illumination included: lead white, orpiment/realgar, red lead, vermilion and red iron oxides as well as azurite and indigo, together with the rather rare copper chloride hydroxide. Furthermore, in both gospels the application of metal leaves as well as powders made of silver–copper and gold–copper alloys could be determined by XRF on several folios

Mitofusin 2 knock-down enhances endoplasmic reticulum-mitochondria contact and decreases amyloid \u3b2-peptide production.

Mitochondria are physically and biochemically in contact with other organelles including the endoplasmic reticulum (ER). Such contacts are formed between mitochondria-associated ER membranes (MAM), specialized subregions of ER, and the outer mitochondrial membrane (OMM). We have previously shown increased expression of MAM-associated proteins and enhanced ER to mitochondria Ca2+ transfer in Alzheimer\u2019s disease (AD) and amyloid \u3b2- peptide (A\u3b2) related neuronal models. Here, we report that siRNA knockdown of mitofusin-2 (Mfn2), a protein that is involved in the tethering of ER and mitochondria, leads to increased contact between the two organelles. Cells depleted in Mfn2 showed increased ER to mitochondria Ca2+ transfer and longer stretches of ER forming contacts with OMM. Interestingly, increased contact resulted in decreased concentrations of intra- and extracellular A\u3b240 and A\u3b242. Analysis of \u3b3-secretase protein expression, maturation and activity revealed that the low A\u3b2 concentrations were a result of impaired \u3b3-secretase complex function. APP, BACE1 and neprilysin expression as well as neprilysin activity were not affected by Mfn2 siRNA treatment. In summary, our data show that modulation of ER-mitochondria contact affects \u3b3-secretase activity and A\u3b2 generation. Increased ER-mitochondria contact results in lower \u3b3-secretase activity suggesting a new mechanism by which A\u3b2 generation can be controlled

Amyloid β-Peptide Increases Mitochondria-Endoplasmic Reticulum Contact Altering Mitochondrial Function and Autophagosome Formation in Alzheimer’s Disease-Related Models

Recent findings have shown that the connectivity and crosstalk between mitochondria and the endoplasmic reticulum (ER) at mitochondria–ER contact sites (MERCS) are altered in Alzheimer’s disease (AD) and in AD-related models. MERCS have been related to the initial steps of autophagosome formation as well as regulation of mitochondrial function. Here, the interplay between MERCS, mitochondria ultrastructure and function and autophagy were evaluated in different AD animal models with increased levels of Aβ as well as in primary neurons derived from these animals. We start by showing that the levels of Mitofusin 1, Mitofusin 2 and mitochondrial import receptor subunit TOM70 are decreased in post-mortem brain tissue derived from familial AD. We also show that Aβ increases the juxtaposition between ER and mitochondria both in adult brain of different AD mouse models as well as in primary cultures derived from these animals. In addition, the connectivity between ER and mitochondria are also increased in wild-type neurons exposed to Aβ. This alteration in MERCS affects autophagosome formation, mitochondrial function and ATP formation during starvation. Interestingly, the increment in ER–mitochondria connectivity occurs simultaneously with an increase in mitochondrial activity and is followed by upregulation of autophagosome formation in a clear chronological sequence of events. In summary, we report that Aβ can affect cell homeostasis by modulating MERCS and, consequently, altering mitochondrial activity and autophagosome formation. Our data suggests that MERCS is a potential target for drug discovery in AD

Alterations in mitochondria-endoplasmic reticulum connectivity in human brain biopsies from idiopathic normal pressure hydrocephalus patients

Abstract Idiopathic normal pressure hydrocephalus (iNPH) is a neuropathology with unknown cause characterised by gait impairment, cognitive decline and ventriculomegaly. These patients often present comorbidity with Alzheimer’s disease (AD), including AD pathological hallmarks such as amyloid plaques mainly consisting of amyloid β-peptide and neurofibrillary tangles consisting of hyperphosphorylated tau protein. Even though some of the molecular mechanisms behind AD are well described, little is known about iNPH. Several studies have reported that mitochondria-endoplasmic reticulum contact sites (MERCS) regulate amyloid β-peptide metabolism and conversely that amyloid β-peptide can influence the number of MERCS. MERCS have also been shown to be dysregulated in several neurological pathologies including AD. In this study we have used transmission electron microscopy and show, for the first time, several mitochondria contact sites including MERCS in human brain biopsies. These unique human brain samples were obtained during neurosurgery from 14 patients that suffer from iNPH. Three of these 14 patients presented comorbidities with other dementias: one patient with AD, one with AD and vascular dementia and one patient with Lewy body dementia. Furthermore, we report that the numbers of MERCS are increased in biopsies obtained from patients diagnosed with dementia. Moreover, the presence of both amyloid plaques and neurofibrillary tangles correlates with decreased contact length between endoplasmic reticulum and mitochondria, while amyloid plaques alone do not seem to affect endoplasmic reticulum-mitochondria apposition. Interestingly, we report a significant positive correlation between the number of MERCS and ventricular cerebrospinal fluid amyloid β-peptide levels, as well as with increasing age of iNPH patients

ΕΥΨΥΧΙΑ. Mélanges offerts à Hélène Ahrweiler

Hélène Ahrweiler est l'une des grandes figures des études byzantines de ces quarante dernières années ; elle a suscité la vocation de nombreux élèves et s'est attiré l'amitié et la considération de nombreux collègues dans le monde entier. Les Mélanges qui lui sont offerts sont le reflet de cette double réussite, puisqu'ils réunissent plus d'une cinquantaine de contributions provenant des centres les plus prestigieux du byzantinisme français et international. Dans leur article, les participants aux Mélanges se font l'écho des principaux thèmes qu'Hélène Ahrweiler a développés dans ses recherches : histoire des institutions, histoire économique et sociale, idéologie politique de l'Empire, géographie historique de l'Italie au Caucase. Hélène Ahrweiler, nommée professeur à la Sorbonne en 1967, a participé à la création de l'Université de Paris I (Panthéon-Sorbonne) où, succédant à Paul Lemerle, elle a développé le Centre de recherches d'histoire et de civilisation byzantines. Par la suite, elle est devenue président de Paris I, puis recteur de l'Académie de Paris avant d'être nommée à la tête du Centre Georges Pompidou