Human and rat brain lipofuscin proteome

The accumulation of an autofluorescent pigment called lipofuscin in neurons is an invariable hallmark of brain aging. So far, this material has been considered to be waste material without particular relevance for cellular pathology. However, two lines of evidence argue that lipofuscin may play a yet unidentified role for pathological cellular functions: (i) Genetic forms of premature accumulation of similar autofluorescent material in neuronal ceroid lipofuscinosis indicate a direct disease-associated link to lipofuscin; (ii) Retinal pigment epithelium cell lipofuscin is mechanistically linked to age-associated macular degeneration. Here, we purified autofluorescent material from the temporal and hippocampal cortices of three different human individuals by a two-step ultracentrifugation on sucrose gradients. For human brain lipofuscin, we could identify a common set of 49 (among > 200 total) proteins that are mainly derived from mitochondria, cytoskeleton, and cell membrane. This brain lipofuscin proteome was validated in an interspecies comparison with whole brain rat lipofuscin (total > 300 proteins), purified by the same procedure, yielding an overlap of 32 proteins (64%) between lipofuscins of both species. Our study is the first to characterize human and rat brain lipofuscin and identifies high homology, pointing to common cellular pathomechanisms of age-associated lipofuscin accumulation despite the huge (40-fold) difference in the lifespan of these species. Our identification of these distinct proteins will now allow research in disturbed molecular pathways during age-associated dysfunctional lysosomal degradation.     

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.     

Genome-wide association study identifies novel loci associated with circulating phospho- and sphingolipid concentrations

Phospho- and sphingolipids are crucial cellular and intracellular compounds. These lipids are required for active transport, a number of enzymatic processes, membrane formation, and cell signalling. Disruption of their metabolism leads to several diseases, with diverse neurological, psychiatric, and metabolic consequences. A large number of phospholipid and sphingolipid species can be detected and measured in human plasma. We conducted a meta-analysis of five European family-based genome-wide association studies (N = 4034) on plasma levels of 24 sphingomyelins (SPM), 9 ceramides (CER), 57 phosphatidylcholines (PC), 20 lysophosphatidylcholines (LPC), 27 phosphatidylethanolamines (PE), and 16 PE-based plasmalogens (PLPE), as well as their proportions in each major class. This effort yielded 25 genome-wide significant loci for phospholipids (smallest P-value = 9.88×10⁻²⁰⁴) and 10 loci for sphingolipids (smallest P-value = 3.10×10⁻⁵⁷). After a correction for multiple comparisons (P-value<2.2×10⁻⁹), we observed four novel loci significantly associated with phospholipids (PAQR9, AGPAT1, PKD2L1, PDXDC1) and two with sphingolipids (PLD2 and APOE) explaining up to 3.1% of the variance. Further analysis of the top findings with respect to within class molar proportions uncovered three additional loci for phospholipids (PNLIPRP2, PCDH20, and ABDH3) suggesting their involvement in either fatty acid elongation/saturation processes or fatty acid specific turnover mechanisms. Among those, 14 loci (KCNH7, AGPAT1, PNLIPRP2, SYT9, FADS1-2-3, DLG2, APOA1, ELOVL2, CDK17, LIPC, PDXDC1, PLD2, LASS4, and APOE) mapped into the glycerophospholipid and 12 loci (ILKAP, ITGA9, AGPAT1, FADS1-2-3, APOA1, PCDH20, LIPC, PDXDC1, SGPP1, APOE, LASS4, and PLD2) to the sphingolipid pathways. In large meta-analyses, associations between FADS1-2-3 and carotid intima media thickness, AGPAT1 and type 2 diabetes, and APOA1 and coronary artery disease were observed. In conclusion, our study identified nine novel phospho- and sphingolipid loci, substantially increasing our knowledge of the genetic basis for these traits.     

Intestinal aspartate proteases TiCatD and TiCatD2 of the haematophagous bug Triatoma infestans (Reduviidae): sequence characterisation, expression pattern and characterisation of proteolytic activity

Two aspartate protease encoding complementary deoxyribonucleic acids (cDNA) were characterised from the small intestine (posterior midgut) of Triatoma infestans and the corresponding genes were named TiCatD and TiCatD2. The deduced 390 and 393 amino acid sequences of both enzymes contain two regions characteristic for cathepsin D proteases and the conserved catalytic aspartate residues forming the catalytic dyad, but only TiCatD2 possesses an entire C-terminal proline loop. The amino acid sequences of TiCatD and TiCatD2 show 51-58% similarity to other insect cathepsin D-like proteases and, respectively, 88 and 58% similarity to the aspartate protease ASP25 from T. infestans available in the GenBank database. In phylogenetic analysis, TiCatD and ASP25 clearly separate from cathepsin D-like sequences of other insects, TiCatD2 groups with cathepsin D-like proteases with proline loop. The activity of purified TiCatD and TiCatD2 was highest between pH 2 and 4, respectively, and hence, deviate from the pH values of the lumen of the small intestine, which varied in correlation with the time after feeding between pH 5.2 and 6.7 as determined by means of micro pH electrodes. Both cathepsins, TiCatD and TiCatD2, were purified from the lumen of the small intestine using pepstatin affinity chromatography and identified by nanoLC-ESI-MS/MS analysis as those encoded by the cDNAs. The proteolytic activity of the purified enzymes is highest at pH 3 and the respective genes are expressed in the both regions of the midgut, stomach (anterior midgut) and small intestine, not in the rectum, salivary glands, Malpighian tubules or haemocytes. The temporal expression pattern of both genes in the small intestine after feeding revealed a feeding dependent regulation for TiCatD but not for TiCatD2.     

New fluorescent bile acids: synthesis, chemical characterization, and disastereoselective uptake by Caco-2 cells of 3-deoxy 3-NBD-amino deoxycholic and ursodeoxycholic acid

Deoxycholic acid (DCA), a secondary bile acid (BA), and ursodeoxycholic acid (UDCA), a tertiary BA, cause opposing effects in vivo and in cell suspensions. Fluorescent analogues of DCA and UDCA could help investigate important questions about their cellular interactions and distribution. We have prepared a set of isomeric 3α- and 3β-amino analogues of UDCA and DCA and derivatised these with the discrete fluorophore, 4-nitrobenzo-2-oxa-1,3-diazol (NBD), forming the corresponding four fluorescent adducts. These absorb in the range 465-470 nm and fluoresce at approx. 535 nm. In order to determine the ability of the new fluorescent bile acids to mimic the parents, their uptake was studied using monolayers of Caco-2 cells, which are known to express multiple proteins of the organic anion-transporting peptide (OATP) subfamily of transporters. Cellular uptake was monitored over time at 4 and 37°C to distinguish between passive and active transport. All four BA analogues were taken up but in a strikingly stereo- and structure-specific manner, suggesting highly discriminatory interactions with transporter protein(s). The α-analogues of DCA and to a lesser extent UDCA were actively transported, whereas the β-analogues were not. The active transport process was saturable, with Michaelis-Menten constants for 3α-NBD DCA (5) being K(m)=42.27±12.98 μM and V(max)=2.8 ± 0.4 nmol/(mg protein*min) and for 3α-NBD UDCA (3) K(m)=28.20 ± 7.45 μM and V(max)=1.8 ± 0.2 nmol/(mg protein*min). These fluorescent bile acids are promising agents for investigating questions of bile acid biology and for detection of bile acids and related organic anion transport processes.     

Habitat stability affects dispersal and the ability to track climate change

Habitat persistence should influence dispersal ability, selecting for stronger dispersal in habitats of lower temporal stability. As standing (lentic) freshwater habitats are on average less persistent over time than running (lotic) habitats, lentic species should show higher dispersal abilities than lotic species. Assuming that climate is an important determinant of species distributions, we hypothesize that lentic species should have distributions that are closer to equilibrium with current climate, and should more rapidly track climatic changes. We tested these hypotheses using datasets from 1988 and 2006 containing all European dragon- and damselfly species. Bioclimatic envelope models showed that lentic species were closer to climatic equilibrium than lotic species. Furthermore, the models over-predicted lotic species ranges more strongly than lentic species ranges, indicating that lentic species track climatic changes more rapidly than lotic species. These results are consistent with the proposed hypothesis that habitat persistence affects the evolution of dispersal.     

Experimental Actinobacillus pleuropneumoniae challenge in swine: Comparison of computed tomographic and radiographic findings during disease

ABSTRACT: BACKGROUND: In pigs, diseases of the respiratory tract like pleuropneumonia due to Actinobacillus pleuropneumoniae (App) infection have led to high economic losses for decades. Further research on disease pathogenesis, pathogen-host-interactions and new prophylactic and therapeutic approaches are needed. In most studies, a large number of experimental animals are required to assess lung alterations at different stages of the disease. In order to reduce the required number of animals but nevertheless gather information on the nature and extent of lung alterations in living pigs, a computed tomographic scoring system for quantifying gross pathological findings was developed. In this study, five healthy pigs served as control animals while 24 pigs were infected with App, the causative agent of pleuropneumonia in pigs, in an established model for respiratory tract disease. RESULTS: Computed tomographic (CT) findings during the course of App challenge were verified by radiological imaging, clinical, serological, gross pathology and histological examinations. Findings from clinical examinations and both CT and radiological imaging, were recorded on day 7 and day 21 after challenge. Clinical signs after experimental App challenge were indicative of acute to chronic disease. Lung CT findings of infected pigs comprised ground-glass opacities and consolidation. On day 7 and 21 the clinical scores significantly correlated with the scores of both imaging techniques. At day 21, significant correlations were found between clinical scores, CT scores and lung lesion scores. In 19 out of 22 challenged pigs the determined disease grades (not affected, slightly affected, moderately affected, severely affected) from CT and gross pathological examination were in accordance. Disease classification by radiography and gross pathology agreed in 11 out of 24 pigs. CONCLUSIONS: High-resolution, high-contrast CT examination with no overlapping of organs is superior to radiography in the assessment of pneumonic lung lesions after App challenge. The new CT scoring system allows for quantification of gross pathological lung alterations in living pigs. However, computed tomographic findings are not informative of the etiology of respiratory disease.     

CFTR Regulates Early Pathogenesis of Chronic Obstructive Lung Disease in βENaC-Overexpressing Mice

Background

Factors determining the onset and severity of chronic obstructive pulmonary disease remain poorly understood. Previous studies demonstrated that airway surface dehydration in βENaC-overexpressing (βENaC-Tg) mice on a mixed genetic background caused either neonatal mortality or chronic obstructive lung disease suggesting that the onset of lung disease was modulated by the genetic background.

Methods

To test this hypothesis, we backcrossed βENaC-Tg mice onto two inbred strains (C57BL/6 and BALB/c) and studied effects of the genetic background on neonatal mortality, airway ion transport and airway morphology. Further, we crossed βENaC-Tg mice with CFTR-deficient mice to validate the role of CFTR in early lung disease.

Results

We demonstrate that the C57BL/6 background conferred increased CFTR-mediated Cl⁻ secretion, which was associated with decreased mucus plugging and mortality in neonatal βENaC-Tg C57BL/6 compared to βENaC-Tg BALB/c mice. Conversely, genetic deletion of CFTR increased early mucus obstruction and mortality in βENaC-Tg mice.

Conclusions

We conclude that a decrease or absence of CFTR function in airway epithelia aggravates the severity of early airway mucus obstruction and related mortality in βENaC-Tg mice. These results suggest that genetic or environmental factors that reduce CFTR activity may contribute to the onset and severity of chronic obstructive pulmonary disease and that CFTR may serve as a novel therapeutic target.     

Responses of efferent octopaminergic thoracic unpaired median neurons in the locust to visual and mechanosensory signals

Insect thoracic ganglia contain efferent octopaminergic unpaired median neurons (UM neurons) located in the midline, projecting bilaterally and modulating neuromuscular transmission, muscle contraction kinetics, sensory sensitivity and muscle metabolism. In locusts, these neurons are located dorsally or ventrally (DUM- or VUM-neurons) and divided into functionally different sub-populations activated during different motor tasks. This study addresses the responsiveness of locust thoracic DUM neurons to various sensory stimuli. Two classes of sense organs, cuticular exteroreceptor mechanosensilla (tactile hairs and campaniform sensilla), and photoreceptors (compound eyes and ocelli) elicited excitatory reflex responses. Chordotonal organ joint receptors caused no responses. The tympanal organ (Müller's organ) elicited weak excitatory responses most likely via generally increased network activity due to increased arousal. Vibratory stimuli to the hind leg subgenual organ never elicited responses. Whereas DUM neurons innervating wing muscles are not very responsive to sensory stimulation, those innervating leg and other muscles are very responsive to stimulation of exteroreceptors and hardly responsive to stimulation of proprioceptors. After cutting both cervical connectives all mechanosensory excitation is lost, even for sensory inputs from the abdomen. This suggests that, in contrast to motor neurons, the sensory inputs to octopaminergic efferent neuromodulatory cells are pre-processed in the suboesophageal ganglion.