In Vivo Evidence for Lysosome Depletion and Impaired Autophagic Clearance in Hereditary Spastic Paraplegia Type SPG11

Hereditary spastic paraplegia (HSP) is characterized by a dying back degeneration of corticospinal axons which leads to progressive weakness and spasticity of the legs. SPG11 is the most common autosomal-recessive form of HSPs and is caused by mutations in SPG11. A recent in vitro study suggested that Spatacsin, the respective gene product, is needed for the recycling of lysosomes from autolysosomes, a process known as autophagic lysosome reformation. The relevance of this observation for hereditary spastic paraplegia, however, has remained unclear. Here, we report that disruption of Spatacsin in mice indeed causes hereditary spastic paraplegia-like phenotypes with loss of cortical neurons and Purkinje cells. Degenerating neurons accumulate autofluorescent material, which stains for the lysosomal protein Lamp1 and for p62, a marker of substrate destined to be degraded by autophagy, and hence appears to be related to autolysosomes. Supporting a more generalized defect of autophagy, levels of lipidated LC3 are increased in Spatacsin knockout mouse embryonic fibrobasts (MEFs). Though distinct parameters of lysosomal function like processing of cathepsin D and lysosomal pH are preserved, lysosome numbers are reduced in knockout MEFs and the recovery of lysosomes during sustained starvation impaired consistent with a defect of autophagic lysosome reformation. Because lysosomes are reduced in cortical neurons and Purkinje cells in vivo, we propose that the decreased number of lysosomes available for fusion with autophagosomes impairs autolysosomal clearance, results in the accumulation of undegraded material and finally causes death of particularly sensitive neurons like cortical motoneurons and Purkinje cells in knockout mice.     

Spatial and ecological population genetic structures within two island‐endemic Aeonium species of different niche width

The Crassulacean genus Aeonium is a well‐known example for plant species radiation on oceanic archipelagos. However, while allopatric speciation among islands is documented for this genus, the role of intra‐island speciation due to population divergence by topographical isolation or ecological heterogeneity has not yet been addressed. The aim of this study was to investigate intraspecific genetic structures and to identify spatial and ecological drivers of genetic population differentiation on the island scale. We analyzed inter simple sequence repeat variation within two island‐endemic Aeonium species of La Palma: one widespread generalist that covers a large variety of different habitat types (Ae. davidbramwellii) and one narrow ecological specialist (Ae. nobile), in order to assess evolutionary potentials on this island. Gene pool differentiation and genetic diversity patterns were associated with major landscape structures in both species, with phylogeographic implications. However, overall levels of genetic differentiation were low. For the generalist species, outlier loci detection and loci–environment correlation approaches indicated moderate signatures of divergent selection pressures linked to temperature and precipitation variables, while the specialist species missed such patterns. Our data point to incipient differentiation among populations, emphasizing that ecological heterogeneity and topographical structuring within the small scales of an island can foster evolutionary processes. Very likely, such processes have contributed to the radiation of Aeonium on the Canary Islands. There is also support for different evolutionary mechanisms between generalist and specialist species.     

Elg1, an alternative subunit of the RFC clamp loader,preferentially interacts with SUMOylated PCNA

Replication‐factor C (RFC) is a protein complex that loads the processivity clamp PCNA onto DNA. Elg1 is a conserved protein with homology to the largest subunit of RFC, but its function remained enigmatic. Here, we show that yeast Elg1 interacts physically and genetically with PCNA, in a manner that depends on PCNA modification, and exhibits preferential affinity for SUMOylated PCNA. This interaction is mediated by three small ubiquitin‐like modifier (SUMO)‐interacting motifs and a PCNA‐interacting protein box close to the N‐terminus of Elg1. These motifs are important for the ability of Elg1 to maintain genomic stability. SUMOylated PCNA is known to recruit the helicase Srs2, and in the absence of Elg1, Srs2 and SUMOylated PCNA accumulate on chromatin. Strains carrying mutations in both ELG1 and SRS2 exhibit a synthetic fitness defect that depends on PCNA modification. Our results underscore the importance of Elg1, Srs2 and SUMOylated PCNA in the maintenance of genomic stability.     

Evidence for coupled transport of bicarbonate and sodium in cultured bovine corneal endothelial cells

Summary Usin gintracellular microelectrode technique, the response of the voltageV across the plasma membrane of cultured bovine corneal endothelial cells to changes in sodium and bicarbonate concentrations was investigated. (1) The electrical response to changes in [HCO ₃ ^– ] _o (depolarization upon lowering and hyperpolarization upon raising [HCO ₃ ^– ] _o ) was dependent on sodium. Lithium could fairly well be substituted for sodium, whereas potassium or choline were much less effective. (2) Removal of external sodium caused a depolarization, while a readdition led to a hyperpolarization, which increased with time of preincubation in the sodium-depleted medium. (3) The response to changes in [Na⁺] _o was dependent on bicarbonate. In a nominally bicarbonate-free medium, its amplitude was decreased or even reversed in sign. (4) Application of SITS or DIDS (10^–3 m) had a similar effect on the response to sodium as bicarbonate-depleted medium. (5) At [Na⁺] _o =151mm and [HCO ₃ ^– ] _o =46mm, the transients ofV depended, with 39.0±9.0 (sd) mV/decade, on bicarbonate and, with 15.3±5.8 (sd) mV/decade, on sodium. (6) After the preincubation of cells with lithium, replacement of Li by choline led to similar effects as the replacement of sodium by choline, though the response ofV was smaller with Li. This response could be reduced or reversed by the removal of bicarbonate or by the application of SITS. (7) Amiloride (10^–3 m) caused a reversible hyperpolarization of the steady-state potential by 8.5±2.6 mV (sd). It did not affect the immediate response to changes in [Na⁺] _o or [HCO ₃ ^– ] _o , but reduced the speed of regaining the steady-state potential after a change in [HCO ₃ ^– ] _o . (8) Ouabain (10^–4 m) caused a fast depolarization of –6.8±1.1 (sd) mV, which was followed by a continuing slower depolarization. The effect was almost identical at 10^–5 m. (9) It is suggested, that corneal endothelial cells possess a cotransport for sodium and bicarbonate, which transports net negative charage with these ions. It is inhibitable by stilbenes, but not directly affected by amiloride or ouabain. Lithium is a good substitute for sodium with respect to bicarbonate transport and is transported itself. In addition, the effect of amiloride provides indirect evidence for the existence of a Na⁺/H⁺-antiport. A model for the transepithelial transport of bicarbonate across the corneal endothelium is proposed.     

Role of ClC-5 in Renal Endocytosis Is Unique among ClC Exchangers and Does Not Require PY-motif-dependent Ubiquitylation

Inactivation of the mainly endosomal 2Cl⁻/H⁺-exchanger ClC-5 severely impairs endocytosis in renal proximal tubules and underlies the human kidney stone disorder Dent''s disease. In heterologous expression systems, interaction of the E3 ubiquitin ligases WWP2 and Nedd4-2 with a “PY-motif” in the cytoplasmic C terminus of ClC-5 stimulates its internalization from the plasma membrane and may influence receptor-mediated endocytosis. We asked whether this interaction is relevant in vivo and generated mice in which the PY-motif was destroyed by a point mutation. Unlike ClC-5 knock-out mice, these knock-in mice displayed neither low molecular weight proteinuria nor hyperphosphaturia, and both receptor-mediated and fluid-phase endocytosis were normal. The abundances and localizations of the endocytic receptor megalin and of the Na⁺-coupled phosphate transporter NaPi-2a (Npt2) were not changed, either. To explore whether the discrepancy in results from heterologous expression studies might be due to heteromerization of ClC-5 with ClC-3 or ClC-4 in vivo, we studied knock-in mice additionally deleted for those related transporters. Disruption of neither ClC-3 nor ClC-4 led to proteinuria or impaired proximal tubular endocytosis by itself, nor in combination with the PY-mutant of ClC-5. Endocytosis of cells lacking ClC-5 was not impaired further when ClC-3 or ClC-4 was additionally deleted. We conclude that ClC-5 is unique among CLC proteins in being crucial for proximal tubular endocytosis and that PY-motif-dependent ubiquitylation of ClC-5 is dispensable for this role.     

Stochastic trajectories of succession initiated by extreme climatic events

Deterministic or rule-based succession is expected under homogeneous biotic and abiotic starting conditions. Effects of extreme climatic events such as drought, however, may alter these assembly rules by adding stochastic elements. We monitored the succession of species composition of 30 twin grassland communities with identical biotic and abiotic starting conditions in an initially sown diversity gradient between 1 and 16 species over 13 years. The stochasticity of succession, measured as the synchrony in the development of the species compositions of the twin plots, was strongly altered by the extreme warm and dry summer of 2003. Moreover, it was independent from past and present plant diversity and neighbourhood species compositions. Extreme climatic events can induce stochastic effects in community development and therefore impair predictability even under homogeneous abiotic conditions. Stochastic events may result in lasting shifts of community composition, as well as adverse and unforeseeable effects on the stability of ecological services.     

European society of intensive care medicine study of therapeutic hypothermia (32-35°C) for intracranial pressure reduction after traumatic brain injury (the Eurotherm3235Trial)

Background

Severe malaria remains a major cause of global morbidity and mortality. Despite the use of potent anti-parasitic agents, the mortality rate in severe malaria remains high. Adjunctive therapies that target the underlying pathophysiology of severe malaria may further reduce morbidity and mortality. Endothelial activation plays a central role in the pathogenesis of severe malaria, of which angiopoietin-2 (Ang-2) has recently been shown to function as a key regulator. Nitric oxide (NO) is a major inhibitor of Ang-2 release from endothelium and has been shown to decrease endothelial inflammation and reduce the adhesion of parasitized erythrocytes. Low-flow inhaled nitric oxide (iNO) gas is a US FDA-approved treatment for hypoxic respiratory failure in neonates.

Methods/Design

This prospective, parallel arm, randomized, placebo-controlled, blinded clinical trial compares adjunctive continuous inhaled nitric oxide at 80 ppm to placebo (both arms receiving standard anti-malarial therapy), among Ugandan children aged 1-10 years of age with severe malaria. The primary endpoint is the longitudinal change in Ang-2, an objective and quantitative biomarker of malaria severity, which will be analysed using a mixed-effects linear model. Secondary endpoints include mortality, recovery time, parasite clearance and neurocognitive sequelae.

Discussion

Noteworthy aspects of this trial design include its efficient sample size supported by a computer simulation study to evaluate statistical power, meticulous attention to complex ethical issues in a cross-cultural setting, and innovative strategies for safety monitoring and blinding to treatment allocation in a resource-constrained setting in sub-Saharan Africa.

Trial Registration

ClinicalTrials.gov Identifier: NCT01255215     

Loss of the ClC-7 chloride channel leads to osteopetrosis in mice and man

Chloride channels play important roles in the plasma membrane and in intracellular organelles. Mice deficient for the ubiquitously expressed ClC-7 Cl(-) channel show severe osteopetrosis and retinal degeneration. Although osteoclasts are present in normal numbers, they fail to resorb bone because they cannot acidify the extracellular resorption lacuna. ClC-7 resides in late endosomal and lysosomal compartments. In osteoclasts, it is highly expressed in the ruffled membrane, formed by the fusion of H(+)-ATPase-containing vesicles, that secretes protons into the lacuna. We also identified CLCN7 mutations in a patient with human infantile malignant osteopetrosis. We conclude that ClC-7 provides the chloride conductance required for an efficient proton pumping by the H(+)-ATPase of the osteoclast ruffled membrane.