CEP41 is mutated in Joubert syndrome and is required for tubulin glutamylation at the cilium

Tubulin glutamylation is a post-translational modification that occurs predominantly in the ciliary axoneme and has been suggested to be important for ciliary function. However, its relationship to disorders of the primary cilium, termed ciliopathies, has not been explored. Here we mapped a new locus for Joubert syndrome (JBTS), which we have designated as JBTS15, and identified causative mutations in CEP41, which encodes a 41-kDa centrosomal protein. We show that CEP41 is localized to the basal body and primary cilia, and regulates ciliary entry of TTLL6, an evolutionarily conserved polyglutamylase enzyme. Depletion of CEP41 causes ciliopathy-related phenotypes in zebrafish and mice and results in glutamylation defects in the ciliary axoneme. Our data identify CEP41 mutations as a cause of JBTS and implicate tubulin post-translational modification in the pathogenesis of human ciliary dysfunction.     

Impact of atypical mitochondrial cyclic-AMP level in nephropathic cystinosis

Nephropathic cystinosis (NC) is a rare disease caused by mutations in the CTNS gene encoding for cystinosin, a lysosomal transmembrane cystine/H⁺ symporter, which promotes the efflux of cystine from lysosomes to cytosol. NC is the most frequent cause of Fanconi syndrome (FS) in young children, the molecular basis of which is not well established. Proximal tubular cells have very high metabolic rate due to the active transport of many solutes. Not surprisingly, mitochondrial disorders are often characterized by FS. A similar mechanism may also apply to NC. Because cAMP has regulatory properties on mitochondrial function, we have analyzed cAMP levels and mitochondrial targets in CTNS^?/? conditionally immortalized proximal tubular epithelial cells (ciPTEC) carrying the classical homozygous 57-kb deletion (delCTNS^?/?) or with compound heterozygous loss-of-function mutations (mutCTNS^?/?). Compared to wild-type cells, cystinotic cells had significantly lower mitochondrial cAMP levels (delCTNS^?/? ciPTEC by 56%?±?10.5, P?<?0.0001; mutCTNS^?/? by 26%?±?4.3, P?<?0.001), complex I and V activities, mitochondrial membrane potential, and SIRT3 protein levels, which were associated with increased mitochondrial fragmentation. Reduction of complex I and V activities was associated with lower expression of part of their subunits. Treatment with the non-hydrolysable cAMP analog 8-Br-cAMP restored mitochondrial potential and corrected mitochondria morphology. Treatment with cysteamine, which reduces the intra-lysosomal cystine, was able to restore mitochondrial cAMP levels, as well as most other abnormal mitochondrial findings. These observations were validated in CTNS-silenced HK-2 cells, indicating a pivotal role of mitochondrial cAMP in the proximal tubular dysfunction observed in NC.     

Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease

Huntington disease is one of nine inherited neurodegenerative disorders caused by a polyglutamine tract expansion. Expanded polyglutamine proteins accumulate abnormally in intracellular aggregates. Here we show that mammalian target of rapamycin (mTOR) is sequestered in polyglutamine aggregates in cell models, transgenic mice and human brains. Sequestration of mTOR impairs its kinase activity and induces autophagy, a key clearance pathway for mutant huntingtin fragments. This protects against polyglutamine toxicity, as the specific mTOR inhibitor rapamycin attenuates huntingtin accumulation and cell death in cell models of Huntington disease, and inhibition of autophagy has the converse effects. Furthermore, rapamycin protects against neurodegeneration in a fly model of Huntington disease, and the rapamycin analog CCI-779 improved performance on four different behavioral tasks and decreased aggregate formation in a mouse model of Huntington disease. Our data provide proof-of-principle for the potential of inducing autophagy to treat Huntington disease.     

Challenges and successes in developing new therapies for hepatitis C

Hepatitis C virus (HCV) will continue to be a serious global health threat for many years to come because of the chronic nature of the infection, its high prevalence and the significant morbidity of the resulting disease. Recently, a small number of molecules have produced encouraging results in proof-of-concept clinical trials. At the same time, preclinical evidence is accumulating that development of resistance will eventually limit the efficacy of new drugs. Thus, combinations of multiple agents will be required to treat chronic HCV infection.     

Peirce on the justification of abduction

What sort of justification can be claimed for abduction? In this paper we reconstruct Peirce’s answer to this question. We show that in his early works on the logic of science Peirce provided an abductive justification of abduction, and that in his mature writings the early solution is enriched by a reference to the place abduction has in a typical scientific inquiry. Since abduction is the first stage of inquiry by which a hypothesis is suggested and which then has to be subjected to inductive testing, the fundamental abduction (ur-abduction) that justifies abduction has also to be subjected to a verification by means of a fundamental induction (ur-induction), namely that the abduction that abduction is valid is verified by an appeal to the history of science.     

Mesenchymal stem cells from preterm to term newborns undergo a significant switch from anaerobic glycolysis to the oxidative phosphorylation

We evaluated the energy metabolism of human mesenchymal stem cells (MSC) isolated from umbilical cord (UC) of preterm (< 37 weeks of gestational age) and term (≥ 37 weeks of gestational age) newborns, using MSC from adult bone marrow as control. A metabolic switch has been observed around the 34th week of gestational age from a prevalently anaerobic glycolysis to the oxidative phosphorylation. This metabolic change is associated with the organization of mitochondria reticulum: preterm MSCs presented a scarcely organized mitochondrial reticulum and low expression of proteins involved in the mitochondrial fission/fusion, compared to term MSCs. These changes seem governed by the expression of CLUH, a cytosolic messenger RNA-binding protein involved in the mitochondria biogenesis and distribution inside the cell; in fact, CLUH silencing in term MSC determined a metabolic fingerprint similar to that of preterm MSC. Our study discloses novel information on the production of energy and mitochondrial organization and function, during the passage from fetal to adult life, providing useful information for the management of preterm birth.     

FRET studies of various conformational states adopted by transthyretin

Transthyretin (TTR) is an extracellular protein able to deposit into well-defined protein aggregates called amyloid, in pathological conditions known as senile systemic amyloidosis, familial amyloid polyneuropathy, familial amyloid cardiomyopathy and leptomeningeal amyloidosis. At least three distinct partially folded states have been described for TTR, including the widely studied amyloidogenic state at mildly acidic pH. Here, we have used fluorescence resonance energy transfer (FRET) experiments in a monomeric variant of TTR (M-TTR) and in its W41F and W79F mutants, taking advantage of the presence of a unique, solvent-exposed, cysteine residue at position 10, that we have labelled with a coumarin derivative (DACM, acceptor), and of the two natural tryptophan residues at positions 41 and 79 (donors). Trp41 is located in an ideal position as it is one of the residues of β-strand C, whose degree of unfolding is debated. We found that the amyloidogenic state at low pH has the same FRET efficiency as the folded state at neutral pH in both M-TTR and W79F-M-TTR, indicating an unmodified Cys10–Trp41 distance. The partially folded state populated at low denaturant concentrations also has a similar FRET efficiency, but other spectroscopic probes indicate that it is distinct from the amyloidogenic state at acidic pH. By contrast, the off-pathway state accumulating transiently during refolding has a higher FRET efficiency, indicating non-native interactions that reduce the Cys10–Trp41 spatial distance, revealing a third distinct conformational state. Overall, our results clarify a negligible degree of unfolding of β-strand C in the formation of the amyloidogenic state and establish the concept that TTR is a highly plastic protein able to populate at least three distinct conformational states.     

Early dispersal of modern humans in Europe and implications for Neanderthal behaviour

The appearance of anatomically modern humans in Europe and the nature of the transition from the Middle to Upper Palaeolithic are matters of intense debate. Most researchers accept that before the arrival of anatomically modern humans, Neanderthals had adopted several 'transitional' technocomplexes. Two of these, the Uluzzian of southern Europe and the Chatelperronian of western Europe, are key to current interpretations regarding the timing of arrival of anatomically modern humans in the region and their potential interaction with Neanderthal populations. They are also central to current debates regarding the cognitive abilities of Neanderthals and the reasons behind their extinction. However, the actual fossil evidence associated with these assemblages is scant and fragmentary, and recent work has questioned the attribution of the Chatelperronian to Neanderthals on the basis of taphonomic mixing and lithic analysis. Here we reanalyse the deciduous molars from the Grotta del Cavallo (southern Italy), associated with the Uluzzian and originally classified as Neanderthal. Using two independent morphometric methods based on microtomographic data, we show that the Cavallo specimens can be attributed to anatomically modern humans. The secure context of the teeth provides crucial evidence that the makers of the Uluzzian technocomplex were therefore not Neanderthals. In addition, new chronometric data for the Uluzzian layers of Grotta del Cavallo obtained from associated shell beads and included within a Bayesian age model show that the teeth must date to ~45,000-43,000 calendar years before present. The Cavallo human remains are therefore the oldest known European anatomically modern humans, confirming a rapid dispersal of modern humans across the continent before the Aurignacian and the disappearance of Neanderthals.     

Ambra1 regulates autophagy and development of the nervous system

Autophagy is a self-degradative process involved both in basal turnover of cellular components and in response to nutrient starvation or organelle damage in a wide range of eukaryotes. During autophagy, portions of the cytoplasm are sequestered by double-membraned vesicles called autophagosomes, and are degraded after fusion with lysosomes for subsequent recycling. In vertebrates, this process acts as a pro-survival or pro-death mechanism in different physiological and pathological conditions, such as neurodegeneration and cancer; however, the roles of autophagy during embryonic development are still largely uncharacterized. Beclin1 (Becn1; coiled-coil, myosin-like BCL2-interacting protein) is a principal regulator in autophagosome formation, and its deficiency results in early embryonic lethality. Here we show that Ambra1 (activating molecule in Beclin1-regulated autophagy), a large, previously unknown protein bearing a WD40 domain at its amino terminus, regulates autophagy and has a crucial role in embryogenesis. We found that Ambra1 is a positive regulator of the Becn1-dependent programme of autophagy, as revealed by its overexpression and by RNA interference experiments in vitro. Notably, Ambra1 functional deficiency in mouse embryos leads to severe neural tube defects associated with autophagy impairment, accumulation of ubiquitinated proteins, unbalanced cell proliferation and excessive apoptotic cell death. In addition to identifying a new and essential element regulating the autophagy programme, our results provide in vivo evidence supporting the existence of a complex interplay between autophagy, cell growth and cell death required for neural development in mammals.