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

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

The Glycosylation Status of PrPC Is a Key Factor in Determining Transmissible Spongiform Encephalopathy Transmission between Species

The risk of transmission of transmissible spongiform encephalopathies (TSE) between different species has been notoriously unpredictable because the mechanisms of transmission are not fully understood. A transmission barrier between species often prevents infection of a new host with a TSE agent. Nonetheless, some TSE agents are able to cross this barrier and infect new species, with devastating consequences. The host PrP(C) misfolds during disease pathogenesis and has a major role in controlling the transmission of agents between species, but sequence compatibility between host and agent PrP(C) does not fully explain host susceptibility. PrP(C) is posttranslationally modified by the addition of glycan moieties which have an important role in the infectious process. Here, we show in vivo that glycosylation of the host PrP(C) has a significant impact on the transmission of TSE between different host species. We infected mice carrying different glycosylated forms of PrP(C) with two human agents (sCJDMM2 and vCJD) and one hamster strain (263K). The absence of glycosylation at both or the first PrP(C) glycosylation site in the host results in almost complete resistance to disease. The absence of the second site of N-glycan has a dramatic effect on the barrier to transmission between host species, facilitating the transmission of sCJDMM2 to a host normally resistant to this agent. These results highlight glycosylation of PrP(C) as a key factor in determining the transmission efficiency of TSEs between different species. IMPORTANCE The risks of transmission of TSE between different species are difficult to predict due to a lack of knowledge over the mechanisms of disease transmission; some strains of TSE are able to cross a species barrier, while others do not. The host protein, PrP(C), plays a major role in disease transmission. PrP(C) undergoes posttranslational glycosylation, and the addition of these glycans may play a role in disease transmission. We infected mice that express different forms of glycosylated PrP(C) with three different TSE agents. We demonstrate that changing the glycosylation status of the host can have profound effects on disease transmission, changing host susceptibility and incubation times. Our results show that PrP(C) glycosylation is a key factor in determining risks of TSE transmission between species

Altered hippocampal-prefrontal neural dynamics in mouse models of Down syndrome

Altered neural dynamics in the medial prefrontal cortex (mPFC) and hippocampus may contribute to cognitive impairments in the complex chromosomal disorder Down syndrome (DS). Here, we demonstrate non-overlapping behavioral differences associated with distinct abnormalities in hippocampal and mPFC electrophysiology during a canonical spatial working memory task in three partially trisomic mouse models of DS (Dp1Tyb, Dp10Yey, and Dp17Yey) that together cover all regions of homology with human chromosome 21 (Hsa21). Dp1Tyb mice show slower decision-making (unrelated to the gene dose of DYRK1A, which has been implicated in DS cognitive dysfunction) and altered theta dynamics (reduced frequency, increased hippocampal-mPFC coherence, and increased modulation of hippocampal high gamma); Dp10Yey mice show impaired alternation performance and reduced theta modulation of hippocampal low gamma; and Dp17Yey mice are not significantly different from the wild type. These results link specific hippocampal and mPFC circuit dysfunctions to cognitive deficits in DS models and, importantly, map them to discrete regions of Hsa21

Differential Regulation of Adhesion Complex Turnover by ROCK1 and ROCK2

ROCK1 and ROCK2 are serine/threonine kinases that function downstream of the small GTP-binding protein RhoA. Rho signalling via ROCK regulates a number of cellular functions including organisation of the actin cytoskeleton, cell adhesion and cell migration.In this study we use RNAi to specifically knockdown ROCK1 and ROCK2 and analyse their role in assembly of adhesion complexes in human epidermal keratinocytes. We observe that loss of ROCK1 inhibits signalling via focal adhesion kinase resulting in a failure of immature adhesion complexes to form mature stable focal adhesions. In contrast, loss of ROCK2 expression results in a significant reduction in adhesion complex turnover leading to formation of large, stable focal adhesions. Interestingly, loss of either ROCK1 or ROCK2 expression significantly impairs cell migration indicating both ROCK isoforms are required for normal keratinocyte migration.ROCK1 and ROCK2 have distinct and separate roles in adhesion complex assembly and turnover in human epidermal keratinocytes