Jay Rasmussen

Amyloid polymorphisms constitute distinct clouds of conformational variants in different etiological subtypes of Alzheimer’s disease

Significance The clinical and pathological variability among patients with Alzheimer’s disease (AD) remains largely unexplained. Evidence is growing that this heterogeneity may be influenced by the heterogeneous molecular architecture of misfolded amyloid-β peptide (Aβ) in the brain. To test this hypothesis, we used unique fluorescent ligands to interrogate the molecular structure of Aβ in amyloid plaques from patients who had died with etiologically distinct subtypes of AD. We found that Aβ-amyloid plaques in the brain cluster as clouds of conformational variants that differ among certain subtypes of AD. The conformational features of AD plaques were partially transmissible to transgenic mice in a seeding paradigm, suggesting a mechanism whereby different molecular strains of Aβ propagate their features within the brain. The molecular architecture of amyloids formed in vivo can be interrogated using luminescent conjugated oligothiophenes (LCOs), a unique class of amyloid dyes. When bound to amyloid, LCOs yield fluorescence emission spectra that reflect the 3D structure of the protein aggregates. Given that synthetic amyloid-β peptide (Aβ) has been shown to adopt distinct structural conformations with different biological activities, we asked whether Aβ can assume structurally and functionally distinct conformations within the brain. To this end, we analyzed the LCO-stained cores of β-amyloid plaques in postmortem tissue sections from frontal, temporal, and occipital neocortices in 40 cases of familial Alzheimer’s disease (AD) or sporadic (idiopathic) AD (sAD). The spectral attributes of LCO-bound plaques varied markedly in the brain, but the mean spectral properties of the amyloid cores were generally similar in all three cortical regions of individual patients. Remarkably, the LCO amyloid spectra differed significantly among some of the familial and sAD subtypes, and between typical patients with sAD and those with posterior cortical atrophy AD. Neither the amount of Aβ nor its protease resistance correlated with LCO spectral properties. LCO spectral amyloid phenotypes could be partially conveyed to Aβ plaques induced by experimental transmission in a mouse model. These findings indicate that polymorphic Aβ-amyloid deposits within the brain cluster as clouds of conformational variants in different AD cases. Heterogeneity in the molecular architecture of pathogenic Aβ among individuals and in etiologically distinct subtypes of AD justifies further studies to assess putative links between Aβ conformation and clinical phenotype.

Early Increase and Late Decrease of Purkinje Cell Dendritic Spine Density in Prion-Infected Organotypic Mouse Cerebellar Cultures

Prion diseases are infectious neurodegenerative diseases associated with the accumulation of protease-resistant prion protein, neuronal loss, spongiform change and astrogliosis. In the mouse model, the loss of dendritic spines is one of the earliest pathological changes observed in vivo, occurring 4–5 weeks after the first detection of protease-resistant prion protein in the brain. While there are cell culture models of prion infection, most do not recapitulate the neuropathology seen in vivo. Only the recently developed prion organotypic slice culture assay has been reported to undergo neuronal loss and the development of some aspects of prion pathology, namely small vacuolar degeneration and tubulovesicular bodies. Given the rapid replication of prions in this system, with protease-resistant prion protein detectable by 21 days, we investigated whether the dendritic spine loss and altered dendritic morphology seen in prion disease might also develop within the lifetime of this culture system. Indeed, six weeks after first detection of protease-resistant prion protein in tga20 mouse cerebellar slice cultures infected with RML prion strain, we found a statistically significant loss of Purkinje cell dendritic spines and altered dendritic morphology in infected cultures, analogous to that seen in vivo. In addition, we found a transient but statistically significant increase in Purkinje cell dendritic spine density during infection, at the time when protease-resistant prion protein was first detectable in culture. Our findings support the use of this slice culture system as one which recapitulates prion disease pathology and one which may facilitate study of the earliest stages of prion disease pathogenesis.

Aβ seeding potency peaks in the early stages of cerebral β‐amyloidosis

Little is known about the extent to which pathogenic factors drive the development of Alzheimers disease (AD) at different stages of the long preclinical and clinical phases. Given that the aggregation of the β‐amyloid peptide (Aβ) is an important factor in AD pathogenesis, we asked whether Aβ seeds from brain extracts of mice at different stages of amyloid deposition differ in their biological activity. Specifically, we assessed the effect of age on Aβ seeding activity in two mouse models of cerebral Aβ amyloidosis (APPPS1 and APP23) with different ages of onset and rates of progression of Aβ deposition. Brain extracts from these mice were serially diluted and inoculated into host mice. Strikingly, the seeding activity (seeding dose SD50) in extracts from donor mice of both models reached a plateau relatively early in the amyloidogenic process. When normalized to total brain Aβ, the resulting specific seeding activity sharply peaked at the initial phase of Aβ deposition, which in turn is characterized by a temporary several‐fold increase in the Aβ42/Aβ40 ratio. At all stages, the specific seeding activity of the APPPS1 extract was higher compared to that of APP23 brain extract, consistent with a more important contribution of Aβ42 than Aβ40 to seed activity. Our findings indicate that the Aβ seeding potency is greatest early in the pathogenic cascade and diminishes as Aβ increasingly accumulates in brain. The present results provide experimental support for directing anti‐Aβ therapeutics to the earliest stage of the pathogenic cascade, preferably before the onset of amyloid deposition.

Comparative Genomic Analysis of Mannheimia haemolytica from Bovine Sources

Bovine respiratory disease is a common health problem in beef production. The primary bacterial agent involved, Mannheimia haemolytica, is a target for antimicrobial therapy and at risk for associated antimicrobial resistance development. The role of M. haemolytica in pathogenesis is linked to serotype with serotypes 1 (S1) and 6 (S6) isolated from pneumonic lesions and serotype 2 (S2) found in the upper respiratory tract of healthy animals. Here, we sequenced the genomes of 11 strains of M. haemolytica, representing all three serotypes and performed comparative genomics analysis to identify genetic features that may contribute to pathogenesis. Possible virulence associated genes were identified within 14 distinct prophage, including a periplasmic chaperone, a lipoprotein, peptidoglycan glycosyltransferase and a stress response protein. Prophage content ranged from 2–8 per genome, but was higher in S1 and S6 strains. A type I-C CRISPR-Cas system was identified in each strain with spacer diversity and organization conserved among serotypes. The majority of spacers occur in S1 and S6 strains and originate from phage suggesting that serotypes 1 and 6 may be more resistant to phage predation. However, two spacers complementary to the host chromosome targeting a UDP-N-acetylglucosamine 2-epimerase and a glycosyl transferases group 1 gene are present in S1 and S6 strains only indicating these serotypes may employ CRISPR-Cas to regulate gene expression to avoid host immune responses or enhance adhesion during infection. Integrative conjugative elements are present in nine of the eleven genomes. Three of these harbor extensive multi-drug resistance cassettes encoding resistance against the majority of drugs used to combat infection in beef cattle, including macrolides and tetracyclines used in human medicine. The findings here identify key features that are likely contributing to serotype related pathogenesis and specific targets for vaccine design intended to reduce the dependency on antibiotics to treat respiratory infection in cattle.

Can plants serve as a vector for prions causing chronic wasting disease

Prions, the causative agent of chronic wasting disease (CWD) enter the environment through shedding of bodily fluids and carcass decay, posing a disease risk as a result of their environmental persistence. Plants have the ability to take up large organic particles, including whole proteins, and microbes. This study used wheat (Triticum aestivum L.) to investigate the uptake of infectious CWD prions into roots and their transport into aerial tissues. The roots of intact wheat plants were exposed to infectious prions (PrPTSE) for 24 h in three replicate studies with PrPTSE in protein extracts being detected by western blot, IDEXX and Bio-Rad diagnostic tests. Recombinant prion protein (PrPC) bound to roots, but was not detected in the stem or leaves. Protease-digested CWD prions (PrPTSE) in elk brain homogenate interacted with root tissue, but were not detected in the stem. This suggests wheat was unable to transport sufficient PrPTSE from the roots to the stem to be detectable by the methods employed. Undigested PrPTSE did not associate with roots. The present study suggests that if prions are transported from the roots to the stems it is at levels that are below those that are detectable by western blot, IDEXX or Bio-Rad diagnostic kits.

Strain-specific modifier genes of Cecr2-associated exencephaly in mice: genetic analysis and identification of differentially expressed candidate genes

Although neural tube defects (NTDs) are common in humans, little is known about their multifactorial genetic causes. While most mouse models involve NTDs caused by a single mutated gene, we have previously described a multigenic system involving susceptibility to NTDs. In mice with a mutation in Cecr2, the cranial NTD exencephaly shows strain-specific differences in penetrance, with 74% penetrance in BALB/cCrl and 0% penetrance in FVB/N. Whole genome linkage analysis showed that a region of chromosome 19 was partially responsible for this difference in penetrance. We now reveal by genetic analysis of three subinterval congenic lines that the chromosome 19 region contains more than one modifier gene. Analysis of embryos showed that although a Cecr2 mutation causes wider neural tubes in both strains, FVB/N embryos overcome this abnormality and close. A microarray analysis comparing neurulating female embryos from both strains identified differentially expressed genes within the chromosome 19 region, including Arhgap19, which is expressed at a lower level in BALB/cCrl due to a stop codon specific to that substrain. Modifier genes in this region are of particular interest because a large portion of this region is syntenic to human chromosome 10q25, the site of a human susceptibility locus.

Protein can be taken up by damaged wheat roots and transported to the stem

Proteins of animal origin can represent a portion of the overall nitrogen (N) pool in the soil environment and there is a possibility that plants may utilize animal proteins as a N source. Using wheat (Triticum aestivum L.) we investigated if the model protein, ovalbumin was taken up into the roots and transported within the plant. In roots, ovalbumin was associated with the epidermis when no root damage was evident, but with minor root damage, it was present in intercellular spaces throughout the cortex and at the endodermis. Ovalbumin was only found in the stem when minor damage to the root system was evident. Suspension cultures of wheat protoplasts revealed that ovalbumin was not assimilated into individual plant cells. Our results suggest that ovalbumin uptake and subsequent movement in wheat is possible only after root damage has occurred. Apoplastic movement may enable animal protein to enter plant tissues above the soil level where they could be consumed by grazers.

In silico identification and high throughput screening of antigenic proteins as candidates for a Mannheimia haemolytica vaccine

This study examined the use of comparative genomic analysis for vaccine design against Mannheimia haemolytica, a respiratory pathogen of ruminants. A total of 2,341genes were identified in at least half of the 23 genomes. Of these, a total of 240 were identified to code for N-terminal signal peptides with diverse sub-cellular localizations (78 periplasmic, 52 outer membrane, 15 extracellular, 13 cytoplasmic membrane and 82 unknown) and were examined in an ELISA assay using a coupled-cell free transcription/translation system for protein expressionwith antisera from cattle challenged with serovars 1, 2 or 6 of M. haemolytica. In total, 186 proteins were immunoreactive to at least one sera type and of these, 105 were immunoreactive to all sera screened. The top ten antigens based on immunoreactivity were serine protease Ssa-1 (AC570_10970), an ABC dipeptid transporter substrate-binding protein (AC570_04010), a ribonucleotide reductase (AC570_10780), competence protein ComE (AC570_11510), a filamentous hemagglutinin (AC570_01600), a molybdenum ABC transporter solute-binding protein (AC570_10275), a conserved hypothetical protein (AC570_07570), a porin protein (AC569_05045), an outer membrane assembly protein YeaT (AC570_03060), and an ABC transporter maltose binding protein MalE (AC570_00140). The framework generated from this research can be further applied towards rapid vaccine design against other pathogens involved in complex respiratory infections in cattle.

Efficiency of protein as a nitrogen source for wheat and morphological changes in roots exposed to high protein concentrations

Rasmussen, J., Gilroyed, B. H., Reuter, T., Badea, A., Eudes, F., Graf, R., Laroche, A., Kav, N. N. V. and McAllister, T. A. 2014. Efficiency of protein as a nitrogen source for wheat and morphological changes in roots exposed to high protein concentrations. Can. J. Plant Sci. 94: 603-613. Proteins of animal origin can enter the environment through application of agricultural by-products to arable or pastured land. In this study, wheat (Triticum aestivum cv. AC Andrew) was exposed to treatments with nitrogen (N) supplied as animal protein (bovine serum albumin; BSA), inorganic N or a combination of these sources at different iso-nitrogenous concentrations. Plant growth was assessed by monitoring both wet and dry mass of shoots and data showed that protein treatments did not differ (P>0.05) from controls lacking N. Analysis of N also showed that plants supplied with protein N displayed lower N (1.2-2.4%) concentration as compared with those supplied with inorganic N (up to 12.4%) with N remaining <2.4% even when the supply of protein was increased. Root morphology was altered in plants exposed to protein N concentrations >71 mM, with the development of knob-like outgrowths with unknown function or significance. This study provides evidence that wheat plantlets grown under sterile conditions are unable to utilize BSA as efficiently as NH4NO3 as a N source, but their roots exhibit a morphological response to protein.