Lifelong choline supplementation ameliorates Alzheimer’s disease pathology and associated cognitive deficits by attenuating microglia activation

Currently, there are no effective therapies to ameliorate the pathological progression of Alzheimer's disease (AD). Evidence suggests that environmental factors may contribute to AD. Notably, dietary nutrients are suggested to play a key role in mediating mechanisms associated with brain function. Choline is a B‐like vitamin nutrient found in common foods that is important in various cell functions. It serves as a methyl donor and as a precursor for production of cell membranes. Choline is also the precursor for acetylcholine, a neurotransmitter which activates the alpha7 nicotinic acetylcholine receptor (α7nAchR), and also acts as an agonist for the Sigma‐1 R (σ1R). These receptors regulate CNS immune response, and their dysregulation contributes to AD pathogenesis. Here, we tested whether dietary choline supplementation throughout life reduces AD‐like pathology and rescues memory deficits in the APP/PS1 mouse model of AD. We exposed female APP/PS1 and NonTg mice to either a control choline (1.1 g/kg choline chloride) or a choline‐supplemented diet (5.0 g/kg choline chloride) from 2.5 to 10 months of age. Mice were tested in the Morris water maze to assess spatial memory followed by neuropathological evaluation. Lifelong choline supplementation significantly reduced amyloid‐β plaque load and improved spatial memory in APP/PS1 mice. Mechanistically, these changes were linked to a decrease of the amyloidogenic processing of APP, reductions in disease‐associated microglial activation, and a downregulation of the α7nAch and σ1 receptors. Our results demonstrate that lifelong choline supplementation produces profound benefits and suggest that simply modifying diet throughout life may reduce AD pathology.     

Alterations in Phosphorylation of Hepatocyte Ribosomal Protein S6 Control Plasmodium Liver Stage Infection

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Colonic Mucosal Proteome Signature Reveals Reduced Energy Metabolism and Protein Synthesis but Activated Autophagy during Anorexia‐Induced Malnutrition in Mice

Anorexia nervosa is an eating disorder often associated with intestinal disorders. To explore the underlying mechanisms of these disorders, the colonic proteome was evaluated during activity‐based anorexia. Female C57Bl/6 mice were randomized into three groups: Control, Limited Food Access (LFA) and Activity‐Based Anorexia (ABA). LFA and ABA mice had a progressive limited access to food but only ABA mice had access to an activity wheel. On colonic mucosal protein extracts, a 2D PAGE‐based comparative proteomic analysis was then performed and differentially expressed proteins were identified by LC‐ESI‐MS/MS. Twenty‐seven nonredundant proteins that were differentially expressed between Control, LFA, and ABA groups were identified. ABA mice exhibited alteration of several mitochondrial proteins involved in energy metabolism such as dihydrolipoyl dehydrogenase and 3‐mercaptopyruvate sulfurtransferase. In addition, a downregulation of mammalian target of rapamycin (mTOR) pathway was observed leading, on the one hand, to the inhibition of protein synthesis, evaluated by puromycin incorporation and mediated by the increased phosphorylation of eukaryotic elongation factor 2, and on the other hand, to the activation of autophagy, assessed by the increase of the marker of autophagy, form LC3‐phosphatidylethanolamine conjugate/Cytosolic form of Microtubule‐associated protein 1A/1B light chain 3 (LC3II/LC3I) ratio. Colonic mucosal proteome is altered during ABA suggesting a downregulation of energy metabolism. A decrease of protein synthesis and an activation of autophagy were also observed mediated by mTOR pathway.     

Ataxia with Vitamin E Deficiency: Refinement of Genetic Localization and Analysis of Linkage Disequilibrium by Using New Markers in 14 Families

Ataxia with vitamin E deficiency (AVED) is an autosomal recessive disease characterized clinically by neurological symptoms with often striking resemblance to those of Friedreich ataxia. This disorder has been reported previously as familial isolated vitamin E deficiency. We have mapped recently the AVED locus to a 5-cM confidence interval on chromosome 8q by homozygosity mapping in six Mediterranean families. We have now analyzed six new and two previously described families and demonstrate genetic homogeneity despite important clinical variability and wide geographic origins. Analysis of nine new tightly linked microsatellite markers, including four characterized in this study, revealed a predominant but not unique mutation in northern African populations, where this condition is more frequent. Haplotype analysis but also classical recombinations allowed us to refine the AVED position to a 1-cM interval. A YAC contig over this interval was constructed from marker STSs and YAC fingerprint data, in order to facilitate the search of the AVED gene.     

Parkin-mediated monoubiquitination of the PDZ protein PICK1 regulates the activity of acid-sensing ion channels

Mutations in the parkin gene result in an autosomal recessive juvenile-onset form of Parkinson's disease. As an E3 ubiquitin-ligase, parkin promotes the attachment of ubiquitin onto specific substrate proteins. Defects in the ubiquitination of parkin substrates are therefore believed to lead to neurodegeneration in Parkinson's disease. Here, we identify the PSD-95/Discs-large/Zona Occludens-1 (PDZ) protein PICK1 as a novel parkin substrate. We find that parkin binds PICK1 via a PDZ-mediated interaction, which predominantly promotes PICK1 monoubiquitination rather than polyubiquitination. Consistent with monoubiquitination and recent work implicating parkin in proteasome-independent pathways, parkin does not promote PICK1 degradation. However, parkin regulates the effects of PICK1 on one of its other PDZ partners, the acid-sensing ion channel (ASIC). Overexpression of wild-type, but not PDZ binding- or E3 ubiquitin-ligase-defective parkin abolishes the previously described, protein kinase C-induced, PICK1-dependent potentiation of ASIC2a currents in non-neuronal cells. Conversely, the loss of parkin in hippocampal neurons from parkin knockout mice unmasks prominent potentiation of native ASIC currents, which is normally suppressed by endogenous parkin in wild-type neurons. Given that ASIC channels contribute to excitotoxicity, our work provides a mechanism explaining how defects in parkin-mediated PICK1 monoubiquitination could enhance ASIC activity and thereby promote neurodegeneration in Parkinson's disease.     

Genetic Variants of the α-Synuclein Gene SNCA Are Associated with Multiple System Atrophy

Background

Multiple system atrophy (MSA) is a progressive neurodegenerative disorder characterized by parkinsonism, cerebellar ataxia and autonomic dysfunction. Pathogenic mechanisms remain obscure but the neuropathological hallmark is the presence of α-synuclein-immunoreactive glial cytoplasmic inclusions. Genetic variants of the α-synuclein gene, SNCA, are thus strong candidates for genetic association with MSA. One follow-up to a genome-wide association of Parkinson''s disease has identified association of a SNP in SNCA with MSA.

Methodology/Findings

We evaluated 32 SNPs in the SNCA gene in a European population of 239 cases and 617 controls recruited as part of the Neuroprotection and Natural History in Parkinson Plus Syndromes (NNIPPS) study. We used 161 independently collected samples for replication. Two SNCA SNPs showed association with MSA: rs3822086 (P = 0.0044), and rs3775444 (P = 0.012), although only the first survived correction for multiple testing. In the MSA-C subgroup the association strengthened despite more than halving the number of cases: rs3822086 P = 0.0024, OR 2.153, (95% CI 1.3–3.6); rs3775444 P = 0.0017, OR 4.386 (95% CI 1.6–11.7). A 7-SNP haplotype incorporating three SNPs either side of rs3822086 strengthened the association with MSA-C further (best haplotype, P = 8.7×10⁻⁴). The association with rs3822086 was replicated in the independent samples (P = 0.035).

Conclusions/Significance

We report a genetic association between MSA and α-synuclein which has replicated in independent samples. The strongest association is with the cerebellar subtype of MSA.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00211224","term_id":"NCT00211224"}}NCT00211224. [{"type":"clinical-trial","attrs":{"text":"NCT00211224","term_id":"NCT00211224"}}NCT00211224]     

Investigations of oocyte in vitro maturation within a mouse model

This study attempted to develop a 'less meiotically competent' murine model for oocyte in vitro maturation (IVM), which could more readily be extrapolated to human clinical assisted reproduction. Oocyte meiotic competence was drastically reduced upon shortening the standard duration of in vivo gonadotrophin stimulation from 48 h to 24 h, and by selecting only naked or partially naked germinal vesicle oocytes, instead of fully cumulus enclosed oocyte complexes. With such a less meiotically competent model, only porcine granulosa coculture significantly enhanced the oocyte maturation rate in vitro, whereas no significant enhancement was observed with macaque and murine granulosa coculture. Increased serum concentrations and the supplementation of gonadotrophins, follicular fluid and extracellular matrix gel within the culture medium did not enhance IVM under either cell-free or coculture conditions. Culture medium conditioned by porcine granulosa also enhanced the maturation rate, and this beneficial effect was not diminished upon freeze-thawing. Enhanced IVM in the presence of porcine granulosa coculture did not, however, translate into improved developmental competence, as assessed by in vitro fertilization and embryo culture to the blastocyst stage.     

Traceability of Marketable Japanese Shoro in New Zealand: Using Multiplex PCR To Exploit Phylogeographic Variation among Taxa in the Rhizopogon Subgenus Roseoli

Rhizopogon roseolus Corda (synonym Rhizopogon rubescens Tul.), an economically important edible mushroom associated with the Pinaceae (mostly Pinus sp.), has a global distribution resulting from the introduction of exotic trees into the Southern Hemisphere for plantation forestry. However, the marketability of R. roseolus varies with the place of origin. R. roseolus strains cultivated in New Zealand from local carpophores for the Japanese market are morphologically and biologically distinct from those produced in Japan and are consequently considered less valuable. In this study, the ITS1-5.8S-ITS2 rRNA (internal transcribed spacer [ITS]) region was used to examine the phylogenetic relationships of R. roseolus and other closely related fungi belonging to Rhizopogon subgenus Roseoli to determine the genetic basis for phenotypic differences among R. roseolus isolates from different geographic regions. Phylogenetic comparison revealed phylogeographic variation within Rhizopogon subgenus Roseoli. Collections from the United States and Europe grouped into four distinct clades. Rhizopogon roseolus isolates found in New Zealand were closely related to those from the United States, likely due to introduction of Pinus radiata from its native California in the United States. In contrast, Japanese R. roseolus isolates clustered closely with European collections. Phylogenetic differences between Japanese and New Zealand R. roseolus isolates may explain the morphological and biological properties attributed to these geographical variants. The ITS region was subsequently used to design a multiplex PCR for the simultaneous identification of Japanese and New Zealand R. roseolus isolates to track the establishment of ectomycorrhiza on P. radiata seedlings inoculated with commercially valuable R. roseolus. This diagnostic demonstrated the first fruiting of Japanese shoro cultivated on P. radiata in the Southern Hemisphere.Since the latter half of the 19th century, Northern Hemisphere species of exotic trees, in particular Pinus radiata, have been planted in the Southern Hemisphere (e.g., Chile, Argentina, South Africa, Australia, and New Zealand). Indeed, over 1,000,000 ha of exotic trees have been established in New Zealand alone (32, 9). Over 200 nonnative basidiomycete and ascomycete ectomycorrhizal (ECM) fungal species are associated with Pinus or Eucalyptus plantations in the Southern Hemisphere from the introduction of plants with intact root systems (46).The genus Rhizopogon Fries (Basidiomycota, Boletales) contains more than 100 species of hypogeous fungi (24), which form ECM associations mostly with members of the Pinaceae (44). The greatest diversity of Rhizopogon can be found in the coniferous forests of the Pacific northwestern United States (40, 30), although a number of species are known to occur in Europe (40) and Asia (3). At least four introduced species of Rhizopogon have also been reported in New Zealand, including R. clelandii Cunn., R. luteolus Fr., R. roseolus (7), and R. vinicolor A. H. Smith (5). At least two species, R. luteolus and R. roseolus, have been associated with P. radiata seedlings in forest nurseries in the North Island of New Zealand (6).In Japan, R. roseolus is known locally as shoro. Shoro is considered a delicacy (20), and its production is dependent on the collection of the carpophores in the field. In fact, only 200 years ago, shoro was the fourth most commonly consumed mushroom in Japan (34). However, the number of natural shoro has declined in the second half of the 20th century (15), resulting in its cultivation in forestry plantations since the late 1980s (50). To fulfill Japanese demand, plantations of P. radiata artificially inoculated with R. roseolus have been established in New Zealand since 1999 using spores from fruiting bodies collected locally (47). Three of the four plantations have subsequently produced fruiting bodies, but the crop has been deemed unsuitable due to consumer sensitivity in Japan to the origin of the products and doubts surrounding the authenticity of the fruiting bodies as Japanese shoro. The quality and market price of other edible fungi differ not only with species but also with their origin. In Japan, domestic Tricholoma matsutake is considered the premium source of matsutake, traded at $500/kg, while South Korean matsutake and those from China are considered less valuable ($250/kg and $100/kg, respectively) (31). Highly prized edible fungi have also been found to be contaminated with less valuable species. Tuber rufum Pico is a truffle species that is found alongside other valuable species in countries where truffles are commercially important. However, it is considered to be a poorly flavored species with no marketable value and is deemed a “contaminant” in truffières (4).Unfortunately, the taxonomy of the genus Rhizopogon is surrounded by many unresolved issues, which makes differentiation of shoro-like fungi found in different geographical locations difficult. In 1966, Smith and Zeller (40) completed the first taxonomic study of the genus Rhizopogon, dividing it into two subgenera, Rhizopogonella (subsequently transferred to Alpova [45]) and Rhizopogon. The subgenus Rhizopogon was further divided into four sections based on differences in the host plant, sporocarp morphology, and color of the peridium. All species associated with Pinus spp. were classified in Rhizopogon section Rhizopogon. Rhizopogon roseolus and R. rubescens were placed in subsection Angustispori, stirps Rubescens. A third species, Rhizopogon vulgaris Vittad., was placed in subsection Angustispori, stirps Vulgaris, due to differences in its spore morphology.Similarities between species in stirps Rubescens and stirps Vulgaris were recorded, with stirps Vulgaris considered a continuation of stirps Rubescens into the narrow-spore species. However, classification was based only on examinations of North American collections even though these species were originally described in Europe in the 19th century (40). Taxonomic reexamination of Rhizopogon using phylogenetic analyses of internal transcribed spacer (ITS) DNA sequences showed that Rhizopogon burlinghamii, R. roseolus, and R. vulgaris formed Rhizopogon section Rhizopogon clade C, separate from the other species sampled from section Rhizopogon (clades A and B). Their ITS sequences lacked insertions and deletions that are diagnostic of other section Rhizopogon clades. Rhizopogon roseolus and R. vulgaris were placed together under Rhizopogon subgenus Roseoli (13). Unfortunately, collections classified as R. rubescens were not included in the phylogenetic study. More recently, species concepts in the R. roseolus species group were examined by Martín and García (25). ITS sequence analyses separated the collections into five possible phylogenetic species.The continued taxonomic instability of Rhizopogon subgenus Roseoli has created ongoing confusion, and the correct species names are still not clear. In Japan, until now, shoro has been referred to as R. rubescens; however, R. rubescens is used widely as a synonym for R. roseolus. In this study, R. roseolus will be used to describe all collections unless specifically stated, as this taxonomic name appears to have precedence in previous phylogenetic studies of Rhizopogon (13, 19, 40) and since Mycobank (http://www.MycoBank.org/) considers R. rubescens to be a synonym of R. roseolus.Molecular diagnostic tools capable of distinguishing genetic differences in ECM fungi have been developed, allowing the differentiation of commercially important species from contaminants or similar species of less economic value. There are many studies where PCR primers designed for the amplification of the ITS region have been used to identify basidiomycetes (12, 18). Species-specific primers were created to identify and differentiate marketable boletes (28), to detect black truffle species (37), and to distinguish Asiatic black truffles from Tuber melanosporum in commercialized products (22). A multiplex PCR has also been developed to simultaneously detect different white truffle species and one of the most aggressive contaminant fungi for monitoring the persistence of a selected truffle in inoculated seedlings (1).Due to the sensitivity of consumers to the origins of shoro and the existing taxonomic complexity of the genus Rhizopogon, morphological and molecular methods were used to establish the diversity and genetic structure of Rhizopogon subgenus Roseoli. Phylogenetic relationships between shoro-like species (originally classified as R. vulgaris, R. rubescens, and R. roseolus) from different geographical locations were investigated to verify previously observed differences between shoro grown in Japan and New Zealand. A multiplex PCR was then developed for the rapid identification of ECMs and fruiting bodies grown from Japanese shoro in New Zealand to track the commercial production of this economically valuable edible fungus.     

Engrailed homeoprotein secretion is a regulated process

Chicken Engrailed 2 homeoprotein is transported between cells in culture. This intercellular transfer is based on unconventional secretion and internalisation mechanisms: Engrailed 2 has access to vesicles but lacks a signal sequence for secretion and is internalised by a non-endocytic process. We show that phosphorylation of a serine-rich domain within Engrailed 2 by the protein kinase CK2 specifically inhibits Engrailed 2 secretion. The availability of the serine-rich domain to CK2 is highly increased when it is displaced from its normal position to the C terminus of Engrailed 2, leading to a constitutive blockage of Engrailed 2 intercellular transfer. This indicates that intercellular transfer of Engrailed 2 is a highly regulated process.     

Role of polyamines in regulating silymarin production in Silybum marianum (L.) Gaertn (Asteraceae) cell cultures under conditions of calcium deficiency

As part of our efforts to identify the possible role of polyamines (PAs) in silymarin (Sm) production, the effects of calcium deprivation on cell growth and on endogenous PAs levels and Sm production by milk thistle (Silybum marianum (L.) Gaertn) grown in cell cultures were examined. Young cultured cells of the H2 line of S. marianum were transferred to a medium without calcium and with ethylene glycol-bis-(β-aminoethyl) ether-N,N,N′,N′-tetraacetic acid present to chelate any free calcium in order to analyze the effects of this medium on the levels of PAs and Sm produced by the cells. During the 17 days of exposure to this calcium-free medium most of the cell populations were in the G0/G1 phase (from day 7 to day 14 of culture) while PA levels underwent a progressive decline up to day 17, after which they were no longer detectable. We observed that putrescine (Put) accumulation was always lower than that observed under normal conditions. The lack of calcium in the MS medium advances the onset of the stationary phase, whose beginning is marked by an increase in the Put/spermidine (Spd) index, raising the production of Sm; the suspensions were productive for a longer time and hence produced more of the substance. Our results indicate that under stress conditions the production of Sm in young-cell suspensions of S. marianum is not associated with high levels of PAs in the medium – contrary to what one would expect – allowing us to conclude that growth inhibition appears to be the factor responsible for the maximum Sm accumulation while PAs are not directly involved in the Sm synthesis pathway by milk thistle grown in culture.