SPG15 protein deficits are at the crossroads between lysosomal abnormalities, altered lipid metabolism and synaptic dysfunction.

Hereditary spastic paraplegia type 15 (HSP15) is a neurodegenerative condition caused by the inability to produce SPG15 protein, which leads to lysosomal swelling. However, the link between lysosomal aberrations and neuronal death is poorly explored. To uncover the functional consequences of lysosomal aberrations in disease pathogenesis, we analyze human dermal fibroblasts from HSP15 patients as well as primary cortical neurons derived from an SPG15 knockout (KO) mouse model. We find that SPG15 protein loss induces defective anterograde transport, impaired neurite outgrowth, axonal swelling and reduced autophagic flux in association with the onset of lysosomal abnormalities. Additionally, we observe lipid accumulation within the lysosomal compartment, suggesting that distortions in cellular lipid homeostasis are intertwined with lysosomal alterations. We further demonstrate that SPG15 KO neurons exhibit synaptic dysfunction, accompanied by augmented vulnerability to glutamate-induced excitotoxicity. Overall, our study establishes an intimate link between lysosomal aberrations, lipid metabolism and electrophysiological impairments, suggesting that lysosomal defects are at the core of multiple neurodegenerative disease processes in HSP15.

Social wasp intestines host the local phenotypic variability of Saccharomyces cerevisiae strains.

Nowadays, the presence of Saccharomyces cerevisiae has been assessed in both wild and human-related environments. Social wasps have been shown to maintain and vector S. cerevisiae among different environments. The availability of strains isolated from wasp intestines represents a striking opportunity to assess whether the strains found in wasp intestines are characterized by peculiar traits. We analysed strains isolated from the intestines of social wasps and compared them with strains isolated from other sources, all collected in a restricted geographic area. We evaluated the production of volatile metabolites during grape must fermentation, the resistance to different stresses and the ability to exploit various carbon sources. Wasp strains, in addition to representing a wide range of S. cerevisiae genotypes, also represent large part of the phenotypes characterizing the sympatric set of yeast strains; their higher production of acetic acid and ethyl acetate could reflect improved ability to attract insects. Our findings suggest that the relationship between yeasts and wasps should be preserved, to safeguard not only the natural variance of this microorganism but also the interests of wine-makers, who could take advantage from the exploitation of their phenotypic variability. Copyright © 2016 John Wiley & Sons, Ltd.

Randomised phase 3 open-label trial of first-line treatment with gemcitabine in association with docetaxel or paclitaxel in women with metastatic breast cancer: a comparison of different schedules and treatments.

BACKGROUND: This open-label study compared docetaxel/gemcitabine vs. paclitaxel/gemcitabine and a weekly (W) vs. 3-weekly (3 W) schedule in metastatic breast cancer (MBC). METHODS: Patients relapsed after adjuvant/neoadjuvant anthracycline-containing chemotherapy were randomized to: A) gemcitabine 1000 mg/m2 Day 1,8 + docetaxel 75 mg/m2 Day 1 q3W; B) gemcitabine 1250 mg/m2 Day 1,8 + paclitaxel 175 mg/m2 Day 1 q3W; C) gemcitabine 800 mg/m2 Day 1,8,15 + docetaxel 30 mg/m2 Day 1,8,15 q4W; D) gemcitabine 800 mg/m2 Day 1,15 + paclitaxel 80 mg/m2 Day 1,8,15 q4W. Primary endpoint was time-to-progression (TTP). Secondary endpoints were overall survival (OS) and overall response rate (ORR). RESULTS: Interim analysis led to accrual interruption (241 patients enrolled of 360 planned). Median TTP (months) was 8.33 (95% CI: 6.19-10.16) with W and 7.51 (95% CI: 5.93-8.33) with 3 W (p=0.319). No differences were observed in median TTP between docetaxel and paclitaxel, with 85.6% and 87.0% of patients progressing, respectively. OS did not differ between regimens/schedules. ORR was comparable between regimens (HR: 0.882; 95% CI: 0.523-1.488; p=0.639), while it was significantly higher in W than in the 3 W (HR: 0.504; 95% CI: 0.299-0.850; p=0.010) schedule. Grade 3/4 toxicities occurred in 69.2% and 71.9% of patients on docetaxel and paclitaxel, and in 65.8% and 75.2% in W and 3 W. CONCLUSIONS: Both treatment regimens showed similar TTP. W might be associated with a better tumour response compared with 3 W. TRIAL REGISTRATION: Clinicaltrial.gov ID NCT00236899.

Ap4b1-knockout mouse model of hereditary spastic paraplegia type 47 displays motor dysfunction, aberrant brain morphology and ATG9A mislocalization.

Mutations in any one of the four subunits (ɛ4, ß4, µ4 and σ4) comprising the adaptor protein Complex 4 results in a complex form of hereditary spastic paraplegia, often termed adaptor protein Complex 4 deficiency syndrome. Deficits in adaptor protein Complex 4 complex function have been shown to disrupt intracellular trafficking, resulting in a broad phenotypic spectrum encompassing severe intellectual disability and progressive spastic paraplegia of the lower limbs in patients. Here we report the presence of neuropathological hallmarks of adaptor protein Complex 4 deficiency syndrome in a clustered regularly interspaced short palindromic repeats-mediated Ap4b1-knockout mouse model. Mice lacking the ß4 subunit, and therefore lacking functional adaptor protein Complex 4, have a thin corpus callosum, enlarged lateral ventricles, motor co-ordination deficits, hyperactivity, a hindlimb clasping phenotype associated with neurodegeneration, and an abnormal gait. Analysis of autophagy-related protein 9A (a known cargo of the adaptor protein Complex 4 in these mice shows both upregulation of autophagy-related protein 9A protein levels across multiple tissues, as well as a striking mislocalization of autophagy-related protein 9A from a generalized cytoplasmic distribution to a marked accumulation in the trans-Golgi network within cells. This mislocalization is present in mature animals but is also in E15.5 embryonic cortical neurons. Histological examination of brain regions also shows an accumulation of calbindin-positive spheroid aggregates in the deep cerebellar nuclei of adaptor protein Complex 4-deficient mice, at the site of Purkinje cell axonal projections. Taken together, these findings show a definitive link between loss-of-function mutations in murine Ap4b1 and the development of symptoms consistent with adaptor protein Complex 4 deficiency disease in humans. Furthermore, this study provides strong evidence for the use of this model for further research into the aetiology of adaptor protein Complex 4 deficiency in humans, as well as its use for the development and testing of new therapeutic modalities.

Circumventing the packaging limit of AAV-mediated gene replacement therapy for neurological disorders.

INTRODUCTION: Gene therapy provides the exciting opportunity of a curative single treatment for devastating diseases, eradicating the need for chronic medication. Adeno-associated viruses (AAVs) are among the most attractive vector carriers for gene replacement in vivo. Yet, despite the success of recent AAV-based clinical trials, the clinical use of these vectors has been limited. For instance, the AAV packaging capacity is restricted to ~4.7 kb, making it a substantial challenge to deliver large gene products. AREAS COVERED: In this review, we explore established and emerging strategies that circumvent the packaging limit of AAVs to make them effective vehicles for gene replacement therapy of monogenic disorders, with a particular focus on diseases affecting the nervous system. We report historical references, design remarks, as well as strengths and weaknesses of these approaches. We additionally discuss examples of neurological disorders for which such strategies have been attempted. EXPERT OPINION: The field of AAV-gene therapy has experienced enormous advancements in the last decade. However, there is still ample space for improvement aimed at overcoming existing challenges that are slowing down the progressive trajectory of this field.

SMN-deficient cells exhibit increased ribosomal DNA damage.

Spinal muscular atrophy, the leading genetic cause of infant mortality, is a motor neuron disease caused by low levels of survival motor neuron (SMN) protein. SMN is a multifunctional protein that is implicated in numerous cytoplasmic and nuclear processes. Recently, increasing attention is being paid to the role of SMN in the maintenance of DNA integrity. DNA damage and genome instability have been linked to a range of neurodegenerative diseases. The ribosomal DNA (rDNA) represents a particularly unstable locus undergoing frequent breakage. Instability in rDNA has been associated with cancer, premature ageing syndromes, and a number of neurodegenerative disorders. Here, we report that SMN-deficient cells exhibit increased rDNA damage leading to impaired ribosomal RNA synthesis and translation. We also unravel an interaction between SMN and RNA polymerase I. Moreover, we uncover an spinal muscular atrophy motor neuron-specific deficiency of DDX21 protein, which is required for resolving R-loops in the nucleolus. Taken together, our findings suggest a new role of SMN in rDNA integrity.

C9ORF72-derived poly-GA DPRs undergo endocytic uptake in iAstrocytes and spread to motor neurons.

Dipeptide repeat (DPR) proteins are aggregation-prone polypeptides encoded by the pathogenic GGGGCC repeat expansion in the C9ORF72 gene, the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. In this study, we focus on the role of poly-GA DPRs in disease spread. We demonstrate that recombinant poly-GA oligomers can directly convert into solid-like aggregates and form characteristic ß-sheet fibrils in vitro. To dissect the process of cell-to-cell DPR transmission, we closely follow the fate of poly-GA DPRs in either their oligomeric or fibrillized form after administration in the cell culture medium. We observe that poly-GA DPRs are taken up via dynamin-dependent and -independent endocytosis, eventually converging at the lysosomal compartment and leading to axonal swellings in neurons. We then use a co-culture system to demonstrate astrocyte-to-motor neuron DPR propagation, showing that astrocytes may internalise and release aberrant peptides in disease pathogenesis. Overall, our results shed light on the mechanisms of poly-GA cellular uptake and propagation, suggesting lysosomal impairment as a possible feature underlying the cellular pathogenicity of these DPR species.

Impaired ganglioside metabolism in Huntington's disease and neuroprotective role of GM1.

Huntington's disease (HD) is a neurodegenerative disorder caused by the expansion of a polyglutamine stretch in the protein huntingtin (Htt). HD neurons are dysfunctional at multiple levels and have increased susceptibility to stress and apoptotic stimuli. We have discovered that synthesis of the ganglioside GM1 is reduced in fibroblasts from HD patients and in cell and animal models of HD, and that decreased GM1 levels contribute to heighten HD cell susceptibility to apoptosis. The apoptotic susceptibility is recapitulated through inhibition of ganglioside synthesis in wild-type striatal cells, suggesting that decreased GM1 levels might be one of the key events leading to HD pathogenesis and progression. Administration of GM1 restores ganglioside levels in HD cells and promotes activation of AKT and phosphorylation of mutant Htt, leading to decreased mutant Htt toxicity and increased survival of HD cells. Our data identify GM1 as a potential treatment for HD.

Autogenous regulation of Escherichia coli polynucleotide phosphorylase expression revisited.

The Escherichia coli polynucleotide phosphorylase (PNPase; encoded by pnp), a phosphorolytic exoribonuclease, posttranscriptionally regulates its own expression at the level of mRNA stability and translation. Its primary transcript is very efficiently processed by RNase III, an endonuclease that makes a staggered double-strand cleavage about in the middle of a long stem-loop in the 5'-untranslated region. The processed pnp mRNA is then rapidly degraded in a PNPase-dependent manner. Two non-mutually exclusive models have been proposed to explain PNPase autogenous regulation. The earlier one suggested that PNPase impedes translation of the RNase III-processed pnp mRNA, thus exposing the transcript to degradative pathways. More recently, this has been replaced by the current model, which maintains that PNPase would simply degrade the promoter proximal small RNA generated by the RNase III endonucleolytic cleavage, thus destroying the double-stranded structure at the 5' end that otherwise stabilizes the pnp mRNA. In our opinion, however, the first model was not completely ruled out. Moreover, the RNA decay pathway acting upon the pnp mRNA after disruption of the 5' double-stranded structure remained to be determined. Here we provide additional support to the current model and show that the RNase III-processed pnp mRNA devoid of the double-stranded structure at its 5' end is not translatable and is degraded by RNase E in a PNPase-independent manner. Thus, the role of PNPase in autoregulation is simply to remove, in concert with RNase III, the 5' fragment of the cleaved structure that both allows translation and prevents the RNase E-mediated PNPase-independent degradation of the pnp transcript.

Patient Preference and Physician Perceptions of Patient Preference for Oral Pharmaceutical Formulations: Results from a Real-Life Survey.

BACKGROUND: Pharmaceutical treatment prescribed according to patient preference for a formulation may have a positive impact on adherence to treatment and consequently on treatment outcomes. AIM: This study aimed at understanding patient preference for pharmaceutical formulations and attributes that trigger patient preference and physician perception of patient preference. METHODS: Between August and September 2017, gastroenterologists and patients with mild to moderate ulcerative colitis from France, Germany, Spain, and the UK participated in an online survey. The investigation was exploratory in nature, and descriptive results are presented. RESULTS: Patient (n = 380) preference appears to be driven by the appearance (format, shape, size, and color - 44%), number of units per administration (39%), and number of administrations per day (17%). Gastroenterologist (n = 159) preference is instead driven by the number of administrations per day (55%), number of units per day (26%), and tablet size (19%). Overall, 254 (67%) patients preferred a tablet formulation, 111 (29%) preferred granules, and 15 (4%) other formulations. According to gastroenterologist perception of patient preference, only 49% of patients prefer tablets, 38% prefer granules, and 13% have no preference. After switching from granules to tablets, 25% patients expressed negative feelings for granules. However, after switching from tablets to granules, 44% of patients still have positive perceptions of tablets. Among patients receiving tablets (n = 255), 18 (7%) perceived their treatment to be not at all effective versus 16 (13%) patients receiving granules (n = 125). A similar proportion of patients in the two groups perceived their treatment as extremely effective (48 vs. 46%, respectively). CONCLUSIONS: Patients generally prefer tablets. Patient and gastroenterologist perception of patient preference for different oral drug formulations is triggered by the same attributes but with inverse importance. To improve adherence, patients should be involved in the choice of the treatment formulation.

De novo Synthesis and Assembly of rRNA into Ribosomal Subunits during Cold Acclimation in Escherichia coli.

During the cold adaptation that follows a cold stress, bacterial cells undergo many physiological changes and extensive reprogramming of their gene expression pattern. Bulk gene expression is drastically reduced, while a set of cold shock genes is selectively and transiently expressed. The initial stage of cold acclimation is characterized by the establishment of a stoichiometric imbalance of the translation initiation factors (IFs)/ribosomes ratio that contributes to the preferential translation of cold shock transcripts. Whereas de novo synthesis of the IFs following cold stress has been documented, nothing was known concerning the activity of the rrn operons during the cold acclimation period. In this work, we focus on the expression of the rrn operons and the fate of rRNA after temperature downshift. We demonstrate that in Escherichia coli, rRNA synthesis does not stop during the cold acclimation phase, but continues with greater contribution of the P2 compared to the P1 promoter and all seven rrn operons are active, although their expression levels change with respect to pre-stress conditions. Eight hours after the 37°â†’10 °C temperature downshift, the newly transcribed rRNA represents up to 20% of total rRNA and is preferentially found in the polysomes. However, with respect to the de novo synthesis of the IFs, both rRNA transcription and maturation are slowed down drastically by cold stress, thereby accounting in part for the stoichiometric imbalance of the IFs/ribosomes. Overall, our data indicate that new ribosomes, which are possibly suitable to function at low temperature, are slowly assembled during cold acclimation.

SUMOylation regulates Kv2.1 and modulates pancreatic beta-cell excitability.

The covalent attachment of small ubiquitin-like modifier (SUMO) proteins regulates protein localization and function. SUMOylation has recently been shown to modulate ion-channel function; however, the extent to which this affects native currents and cellular excitability remains to be determined. The voltage-dependent K(+) (Kv) channel Kv2.1 regulates pancreatic beta-cell excitability and insulin secretion. We found that YFP-tagged SUMO1 (SUMO1-YFP) can be immunoprecipitated with Kv2.1 when these two proteins are coexpressed in HEK 293 cells. Furthermore, direct infusion of recombinant SUMO1 peptide or coexpression of SUMO1-YFP inhibited current through cloned Kv2.1 by 80% and 48%, respectively. Insulin-secreting cells express SUMO variants 1 and 3, and expression of the SUMO1-YFP in human beta-cells or insulinoma cells inhibited native Kv currents (by 49% and 33%, respectively). Inhibition of the channel resulted from an acceleration of channel inactivation and an inhibition of recovery from inactivation, resulting in the widening of beta-cell action potentials and a decreased firing frequency. Finally, these effects on channel function and excitability were augmented by the conjugating enzyme Ubc9 and rescued by the SUMO protease SENP1. Thus, protein SUMOylation can exert a strong inhibitory action on the voltage-dependent K(+) channel Kv2.1 and can regulate cellular excitability in native beta-cells.

Autogenous regulation of Escherichia coli polynucleotide phosphorylase during cold acclimation by transcription termination and antitermination.

Adaptation of Escherichia coli at low temperature implicates a drastic reprogramming of gene expression patterns. Mechanisms operating downstream of transcription initiation, such as control of transcription termination, mRNA stability and translatability, play a major role in controlling gene expression in the cold acclimation phase. It was previously shown that Rho-dependent transcription termination within pnp, the gene encoding polynucleotide phosphorylase (PNPase), was suppressed in pnp nonsense mutants, whereas it was restored by complementation with wild type allele. Using a tRNA gene as a reporter and the strong Rho-dependent transcription terminator t ( imm ) of bacteriophage P4 as a tester, here we show that specific sites in the 5'-untranslated region of pnp mRNA are required for PNPase-sensitive cold-induced suppression of Rho-dependent transcription termination. We suggest that suppression of Rho-dependent transcription termination within pnp and its restoration by PNPase is an autogenous regulatory circuit that modulates pnp expression during cold acclimation.