Glycoproteomics Landscape of Asymptomatic and Symptomatic Human Alzheimer’s Disease Brain

Molecular changes in the brain of individuals afflicted with Alzheimer's disease (AD) are an intense area of study. Little is known about the role of protein abundance and posttranslational modifications in AD progression and treatment, in particular large-scale intact N-linked glycoproteomics analysis. To elucidate the N-glycoproteome landscape, we developed an approach based on multi-lectin affinity enrichment, hydrophilic interaction chromatography, and LC-MS–based glycoproteomics. We analyzed brain tissue from 10 persons with no cognitive impairment or AD, 10 with asymptomatic AD, and 10 with symptomatic AD, detecting over 300 glycoproteins and 1900 glycoforms across the samples. The majority of glycoproteins have N-glycans that are high-mannosidic or complex chains that are fucosylated and bisected. The Man5 N-glycan was found to occur most frequently at >20% of the total glycoforms. Unlike the glycoproteomes of other tissues, sialylation is a minor feature of the brain N-glycoproteome, occurring at <9% among the glycoforms. We observed AD-associated differences in the number of antennae, frequency of fucosylation, bisection, and other monosaccharides at individual glycosylation sites among samples from our three groups. Further analysis revealed glycosylation differences in subcellular compartments across disease stage, including glycoproteins in the lysosome frequently modified with paucimannosidic glycans. These results illustrate the N-glycoproteomics landscape across the spectrum of AD clinical and pathologic severity and will facilitate a deeper understanding of progression and treatment development.     

Functional Diversity and Evolution of the Drosophila Sperm Proteome

Spermatozoa are central to fertilization and the evolutionary fitness of sexually reproducing organisms. As such, a deeper understanding of sperm proteomes (and associated reproductive tissues) has proven critical to the advancement of the fields of sexual selection and reproductive biology. Due to their extraordinary complexity, proteome depth-of-coverage is dependent on advancements in technology and related bioinformatics, both of which have made significant advancements in the decade since the last Drosophila sperm proteome was published. Here, we provide an updated version of the Drosophila melanogaster sperm proteome (DmSP3) using improved separation and detection methods and an updated genome annotation. Combined with previous versions of the sperm proteome, the DmSP3 contains a total of 3176 proteins, and we provide the first label-free quantitation of the sperm proteome for 2125 proteins. The top 20 most abundant proteins included the structural elements α- and β-tubulins and sperm leucyl-aminopeptidases. Both gene content and protein abundance were significantly reduced on the X chromosome, consistent with prior genomic studies of X chromosome evolution. We identified 9 of the 16 Y-linked proteins, including known testis-specific male fertility factors. We also identified almost one-half of known Drosophila ribosomal proteins in the DmSP3. The role of this subset of ribosomal proteins in sperm is unknown. Surprisingly, our expanded sperm proteome also identified 122 seminal fluid proteins (Sfps), proteins originally identified in the accessory glands. We show that a significant fraction of ‘sperm-associated Sfps’ are recalcitrant to concentrated salt and detergent treatments, suggesting this subclass of Sfps are expressed in testes and may have additional functions in sperm, per se. Overall, our results add to a growing landscape of both sperm and seminal fluid protein biology and in particular provides quantitative evidence at the protein level for prior findings supporting the meiotic sex-chromosome inactivation model for male-specific gene and X chromosome evolution.     

Metabolic Enzyme Alterations and Astrocyte Dysfunction in a Murine Model of Alexander Disease With Severe Reactive Gliosis

Alexander disease (AxD) is a rare and fatal neurodegenerative disorder caused by mutations in the gene encoding glial fibrillary acidic protein (GFAP). In this report, a mouse model of AxD (GFAP^Tg;Gfap^+/R236H) was analyzed that contains a heterozygous R236H point mutation in murine Gfap as well as a transgene with a GFAP promoter to overexpress human GFAP. Using label-free quantitative proteomic comparisons of brain tissue from GFAP^Tg;Gfap^+/R236H versus wild-type mice confirmed upregulation of the glutathione metabolism pathway and indicated proteins were elevated in the peroxisome proliferator-activated receptor (PPAR) signaling pathway, which had not been reported previously in AxD. Relative protein-level differences were confirmed by a targeted proteomics assay, including proteins related to astrocytes and oligodendrocytes. Of particular interest was the decreased level of the oligodendrocyte protein, 2-hydroxyacylsphingosine 1-beta-galactosyltransferase (Ugt8), since Ugt8-deficient mice exhibit a phenotype similar to GFAP^Tg;Gfap^+/R236H mice (e.g., tremors, ataxia, hind-limb paralysis). In addition, decreased levels of myelin-associated proteins were found in the GFAP^Tg;Gfap^+/R236H mice, consistent with the role of Ugt8 in myelin synthesis. Fabp7 upregulation in GFAP^Tg;Gfap^+/R236H mice was also selected for further investigation due to its uncharacterized association to AxD, critical function in astrocyte proliferation, and functional ability to inhibit the anti-inflammatory PPAR signaling pathway in models of amyotrophic lateral sclerosis (ALS). Within Gfap⁺ astrocytes, Fabp7 was markedly increased in the hippocampus, a brain region subjected to extensive pathology and chronic reactive gliosis in GFAP^Tg;Gfap^+/R236H mice. Last, to determine whether the findings in GFAP^Tg;Gfap^+/R236H mice are present in the human condition, AxD patient and control samples were analyzed by Western blot, which indicated that Type I AxD patients have a significant fourfold upregulation of FABP7. However, immunohistochemistry analysis showed that UGT8 accumulates in AxD patient subpial brain regions where abundant amounts of Rosenthal fibers are located, which was not observed in the GFAP^Tg;Gfap^+/R236H mice.     

Does the VP1 gene of foot-and-mouth disease virus behave as a molecular clock?

Summary We have carried out a phylogenetic study of the evolution of the VP1 gene sequence from different serological types and subtypes of foot-and-mouth disease virus (FMDV). The maximum-likelihood method developed by Hasegawa and co-workers (Hasegawa et al. 1985) for the estimation of evolutionary parameters and branching dates has been used to decide between alternative models of evolution: constant versus variable rates. The results obtained indicate that a constant rate model, i.e., a molecular clock, seems to be the most plausible one. However, additional information suggests the possibility that the appearance of serotype CS has been accompanied by an episode of rapid evolution (Villaverde et al. 1991). We discuss the possibility that this evolution of RNA viruses was due to episodic positive Darwinian selection, which would have helped the new variant to escape the immunogenic pressure from the hosts. Offprint requests to: A. Moya     

The new biology of ageing

Human life expectancy in developed countries has increased steadily for over 150 years, through improvements in public health and lifestyle. More people are hence living long enough to suffer age-related loss of function and disease, and there is a need to improve the health of older people. Ageing is a complex process of damage accumulation, and has been viewed as experimentally and medically intractable. This view has been reinforced by the realization that ageing is a disadvantageous trait that evolves as a side effect of mutation accumulation or a benefit to the young, because of the decline in the force of natural selection at later ages. However, important recent discoveries are that mutations in single genes can extend lifespan of laboratory model organisms and that the mechanisms involved are conserved across large evolutionary distances, including to mammals. These mutations keep the animals functional and pathology-free to later ages, and they can protect against specific ageing-related diseases, including neurodegenerative disease and cancer. Preliminary indications suggest that these new findings from the laboratory may well also apply to humans. Translating these discoveries into medical treatments poses new challenges, including changing clinical thinking towards broad-spectrum, preventative medicine and finding novel routes to drug development.     

大鼠脊髓中的α受体在减压反射和失血性休克中的作用

大鼠脊髓蛛网膜下腔注射α激动剂可乐宁1μg,引起血压降低、心率减慢及腹腔神经节后交感神经干放电抑制。应用α阻断剂酚妥拉明阻断脊髓内源性 NE的作用,可部分抑制血压升高时反射性的心率减慢和交感神经放电抑制反应,使压力感受器反射的敏感性降低。在颈动脉放血造成不可逆性失血性休克的动物,脊髓蛛网膜下腔注射酚妥拉明可使动脉血压有一定程度的回升。以上结果表明,由脊髓α受体调制的心血管抑制效应参与减压反射以及失血性休克的发病机制。