Glycosylation of PrP and the transmissable spongiform encephalopathies species barrier

Transmissible spongiform encephalopathies (TSEs) are a group of fatal neurodegenerative diseases. They can be readily transmitted within a host species but are rarely transmitted between different species. This "species barrier''' effect is characterised by reduced susceptibility of exposed novel hosts and an increase in the length and variability of the incubation period. The mechanisms that underlie this barrier are yet to be fully understood; hence it is currently not possible to accurately predict the host range of a novel TSE disease. During TSE pathogenesis the host protein, PrP, misfolds and accumulates within the central nervous system. This misfolded form is denoted PrPˢᶜ to discriminate it from the normal cellular form PrPᶜ. The prion hypothesis proposes that PrPˢᶜ is the TSE infectious agent and propagates its aberrant conformation by inducing PrPᶜ to misfold. PrP is variably glycosylated at two sites in vivo, such that di-, mono- and un-glycosylated glycotypes are observed; all of these PrP glycotypes can form PrPˢᶜ. However the role of each glycotype in cross species transmission is unclear. In vitro conversion experiments have suggested that PrPᶜ's N-glycans specifically retard the cross species PrPˢᶜ seeded conversion of PrPᶜ. Thus glycosylation of PrPᶜ may inhibit cross-species TSE transmission.Previous studies have not examined the role of glycosylation of PrPᶜ in the crossspecies transmission of TSE disease. In this study glycosylation deficient transgenic mice [NPU] were challenged with three non-murine TSE agents. A barrier to crossspecies transmission was observed in normally glycosylated control mice, although a proportion of animals did develop signs of TSE disease after challenge with hamster scrapie (263K) and variant CJD (vCJD). Transgenic mice that lack both N-glycan attachment sites were resistant to cross-species TSE challenge (263K, vCJD and sporadic CJD). Moreover, absence of N-glycans at the first site only, resulted in a significant decrease in disease incidence after challenge with 263K. These data suggest that glycosylation of the first site mediates the transmission of TSE between species. However, this effect is not unique to cross-species transmission as previous reports have demonstrated that the within-species transmission of TSE is also delayed by the absence of the first N-glycan attachment site. Transgenic mice which lack IV glycosylation at the second site were shown to have a higher disease incidence than controls after cross-species challenge with 263K or sporadic CJD. Therefore, glycosylation of the second site inhibits the cross-species transmission of these strains. This effect is specific to cross-species transmission as previous murine TSE transmissions to these mice have not demonstrated an acceleration of disease. Thus the site of glycosylation determines the role of the N-glycan in cross-species transmission. Moreover, here it is shown that mice which lack the second N-glycan attachment site exhibit a reduced disease incidence after challenge with vCJD compared to control mice. Therefore, TSE strain determines the role of N-glycans in disease transmissionTo further interpret these transmission studies, the localisation of PrPᶜ in the glycosylation deficient transgenics was investigated using confocal microscopy. No significant difference in the cellular localisation of PrPᶜ was detected in mice which lack the first or second N-glycosylation sites. Therefore, the effect of N-glycan attachment at these sites on TSE transmission occurs independently of PrPᶜ's cellular location. Lower levels of anti-PrP signal were detected in the neuropil of mice that lack both N-glycan attachment sites than in control animals, suggesting a possible mechanism for the enhanced resistance of these transgenic mice to TSE challenge.Normally glycosylated PrP can be induced to adopt a misfolded conformation in vitro, by exposure to infected brain homogenate, mimicking the in vivo formation of PrPˢᶜ. Using an in vitro conversion assay, the ability of PrP derived from the glycosylation deficient transgenic to misfold was studied. PrP that lacks the first Nglycan attachment site did not misfold, suggesting a potential cause of the enhanced resistance of the transgenic mice that lack this site to TSE transmission. PrP that lacks the second N-glycan attachment site adopted a misfolded form in vitro, with efficiency equal to that of normally glycosylated PrP. Therefore the higher disease incidence observed in the transgenic mice, which lack the second PrP glycosylation site, did not occur because PrP in these animals is more readily misfolded. This suggests that other aspects of PrP biology, such as PrPˢᶜ toxicity or clearance, are influenced by glycosylation of PrP's second site and that these can alter TSE disease

Rodent Modeling of Alzheimer's Disease in Down Syndrome: In vivo and ex vivo Approaches

There are an estimated 6 million people with Down syndrome (DS) worldwide. In developed countries, the vast majority of these individuals will develop Alzheimer's disease neuropathology characterized by the accumulation of amyloid-β (Aβ) plaques and tau neurofibrillary tangles within the brain, which leads to the early onset of dementia (AD-DS) and reduced life-expectancy. The mean age of onset of clinical dementia is ~55 years and by the age of 80, approaching 100% of individuals with DS will have a dementia diagnosis. DS is caused by trisomy of chromosome 21 (Hsa21) thus an additional copy of a gene(s) on the chromosome must cause the development of AD neuropathology and dementia. Indeed, triplication of the gene APP which encodes the amyloid precursor protein is sufficient and necessary for early onset AD (EOAD), both in people who have and do not have DS. However, triplication of other genes on Hsa21 leads to profound differences in neurodevelopment resulting in intellectual disability, elevated incidence of epilepsy and perturbations to the immune system. This different biology may impact on how AD neuropathology and dementia develops in people who have DS. Indeed, genes on Hsa21 other than APP when in three-copies can modulate AD-pathogenesis in mouse preclinical models. Understanding this biology better is critical to inform drug selection for AD prevention and therapy trials for people who have DS. Here we will review rodent preclinical models of AD-DS and how these can be used for both in vivo and ex vivo (cultured cells and organotypic slice cultures) studies to understand the mechanisms that contribute to the early development of AD in people who have DS and test the utility of treatments to prevent or delay the development of disease

Cell models for Down syndrome-Alzheimer’s disease research

Down syndrome (DS) is the most common chromosomal abnormality and leads to intellectual disability, increased risk of cardiac defects, and an altered immune response. Individuals with DS have an extra full or partial copy of chromosome 21 (trisomy 21) and are more likely to develop early-onset Alzheimer’s disease (AD) than the general population. Changes in expression of human chromosome 21 (Hsa21)-encoded genes, such as APP, play an important role in the pathogenesis of AD in DS (DS-AD). However, the mechanisms of DS-AD remain poorly understood. To date, several mouse models with an extra copy of genes syntenic to Hsa21 have been developed to characterize DS-AD-related phenotypes. Nonetheless, due to genetic and physiological differences between mouse and human, mouse models cannot faithfully recapitulate all features of DS-AD. Cells differentiated from human induced pluripotent stem cells (iPSCs), isolated from individuals with genetic diseases, can be used to model disease-related cellular and molecular pathologies, including DS. In this review, we will discuss the limitations of mouse models of DS and how these can be addressed using recent advancements in modelling DS using human iPSCs and iPSC-mouse chimeras, and potential applications of iPSCs in preclinical studies for DS-AD

Altered Glycosylated PrP Proteins Can Have Different Neuronal Trafficking in Brain but Do Not Acquire Scrapie-like Properties

N-Linked glycans have been shown to have an important role in the cell biology of a variety of cell surface glycoproteins, including PrP protein. It has been suggested that glycosylation of PrP can influence the susceptibility to transmissible spongiform encephalopathy and determine the characteristics of the many different strains observed in this particular type of disease. To understand the role of carbohydrates in influencing the PrP maturation, stability, and cell biology, we have produced and analyzed gene-targeted murine models expressing differentially glycosylated PrP. Transgenic mice carrying the PrP substitution threonine for asparagine 180 (G1) or threonine for asparagine 196 (G2) or both mutations combined (G3), which eliminate the first, second, and both glycosylation sites, respectively, have been generated by double replacement gene targeting. An in vivo analysis of altered PrP has been carried out in transgenic mouse brains, and our data show that the lack of glycans does not influence PrP maturation and stability. The presence of one chain of sugar is sufficient for the trafficking to the cell membrane, whereas the unglycosylated PrP localization is mainly intracellular. However, this altered cellular localization of PrP does not lead to any overt phenotype in the G3 transgenic mice. Most importantly, we found that, in vivo, unglycosylated PrP does not acquire the characteristics of the aberrant pathogenic form (PrPSc), as was previously reported using in vitro models

Cathepsin B abundance, activity and microglial localisation in Alzheimer's disease-Down syndrome and early onset Alzheimer's disease; the role of elevated cystatin B

Cathepsin B is a cysteine protease that is implicated in multiple aspects of Alzheimer's disease pathogenesis. The endogenous inhibitor of this enzyme, cystatin B (CSTB) is encoded on chromosome 21. Thus, individuals who have Down syndrome, a genetic condition caused by having an additional copy of chromosome 21, have an extra copy of an endogenous inhibitor of the enzyme. Individuals who have Down syndrome are also at significantly increased risk of developing early-onset Alzheimer's disease (EOAD). The impact of the additional copy of CSTB on Alzheimer's disease development in people who have Down syndrome is not well understood. Here we compared the biology of cathepsin B and CSTB in individuals who had Down syndrome and Alzheimer's disease, with disomic individuals who had Alzheimer's disease or were ageing healthily. We find that the activity of cathepsin B enzyme is decreased in the brain of people who had Down syndrome and Alzheimer's disease compared with disomic individuals who had Alzheimer's disease. This change occurs independently of an alteration in the abundance of the mature enzyme or the number of cathepsin B+ cells. We find that the abundance of CSTB is significantly increased in the brains of individuals who have Down syndrome and Alzheimer's disease compared to disomic individuals both with and without Alzheimer's disease. In mouse and human cellular preclinical models of Down syndrome, three-copies of CSTB increases CSTB protein abundance but this is not sufficient to modulate cathepsin B activity. EOAD and Alzheimer's disease-Down syndrome share many overlapping mechanisms but differences in disease occur in individuals who have trisomy 21. Understanding this biology will ensure that people who have Down syndrome access the most appropriate Alzheimer's disease therapeutics and moreover will provide unique insight into disease pathogenesis more broadly

The role of host PrP in Transmissible Spongiform Encephalopathies

AbstractPrP has a central role in the Transmissible Spongiform Encephalopathies (TSEs), and mutations and polymorphisms in host PrP can profoundly alter the host's susceptibility to a TSE agent. However, precisely how host PrP influences the outcome of disease has not been established. To investigate this we have produced by gene targeting a series of inbred lines of transgenic mice expressing different PrP genes. This allows us to study directly the influence of the host PrP gene in TSEs. We have examined the role of glycosylation, point mutations, polymorphisms and PrP from different species on host susceptibility and the disease process both within the murine species and across species barriers

Downregulated Wnt/β-catenin signalling in the Down syndrome hippocampus

Pathological mechanisms underlying Down syndrome (DS)/Trisomy 21, including dysregulation of essential signalling processes remain poorly understood. Combining bioinformatics with RNA and protein analysis, we identified downregulation of the Wnt/β-catenin pathway in the hippocampus of adult DS individuals with Alzheimer’s disease and the ‘Tc1’ DS mouse model. Providing a potential underlying molecular pathway, we demonstrate that the chromosome 21 kinase DYRK1A regulates Wnt signalling via a novel bimodal mechanism. Under basal conditions, DYRK1A is a negative regulator of Wnt/β-catenin. Following pathway activation, however, DYRK1A exerts the opposite effect, increasing signalling activity. In summary, we identified downregulation of hippocampal Wnt/β-catenin signalling in DS, possibly mediated by a dose dependent effect of the chromosome 21-encoded kinase DYRK1A. Overall, we propose that dosage imbalance of the Hsa21 gene DYRK1A affects downstream Wnt target genes. Therefore, modulation of Wnt signalling may open unexplored avenues for DS and Alzheimer’s disease treatment

Genetic mapping of APP and amyloid-β biology modulation by trisomy 21

Individuals who have Down syndrome (DS) frequently develop early onset Alzheimer's disease (AD), a neurodegenerative condition caused by the build-up of aggregated amyloid-β and tau proteins in the brain. Amyloid-β is produced by amyloid precursor protein (APP), a gene located on chromosome 21. People who have Down syndrome have three copies of chromosome 21 and thus also an additional copy of APP; this genetic change drives the early development of Alzheimer's disease in these individuals. Here we use a combination of next-generation mouse models of Down syndrome (Tc1, Dp3Tyb, Dp(10)2Yey and Dp(17)3Yey) and a knockin mouse model of amyloid-β accumulation (AppNL-F ) to determine how chromosome 21 genes, other than APP, modulate APP/amyloid-β in the brain when in three copies. Using both male and female mice, we demonstrate that three copies of other chromosome 21 genes are sufficient to partially ameliorate amyloid-β accumulation in the brain. We go on to identify a subregion of chromosome 21 that contains the gene/genes causing this decrease in amyloid-β accumulation and investigate the role of two lead candidate genes Dyrk1a and Bace2 Thus an additional copy of chromosome 21 genes, other than APP, can modulate APP/amyloid-β in the brain under physiological conditions. This work provides critical mechanistic insight into the development of disease and an explanation for the typically later age of onset of dementia in people who have AD-DS, compared to those who have familial AD caused by triplication of APP Significance Statement:Trisomy of chromosome 21 is a commonly occurring genetic risk factor for early-onset Alzheimer's disease, which has been previously attributed to people with Down syndrome having three copies of the APP gene, which is encoded on chromosome 21. However, we have shown that an extra copy of other chromosome 21 genes modifies AD-like phenotypes independently of APP copy number (Wiseman et al. 2018, Brain; Tosh et al. 2021 Scientific Reports). Here, we use a mapping approach to narrow-down the genetic cause of the modulation of pathology; demonstrating that gene(s) on chromosome 21 decrease amyloid-β accumulation in the brain, independently of alterations to full-length APP or C-terminal fragment abundance and that just 38 genes are sufficient to cause this