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.

Genetic Mapping of APP and Amyloid-ß 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 buildup of aggregated amyloid-ß (Aß) and tau proteins in the brain. Aß is produced by amyloid precursor protein (APP), a gene located on chromosome 21. People who have DS have three copies of chromosome 21 and thus also an additional copy of APP; this genetic change drives the early development of AD in these individuals. Here we use a combination of next-generation mouse models of DS (Tc1, Dp3Tyb, Dp(10)2Yey and Dp(17)3Yey) and a knockin mouse model of Aß accumulation (AppNL-F ) to determine how chromosome 21 genes, other than APP, modulate APP/Aß 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 Aß accumulation in the brain. We go on to identify a subregion of chromosome 21 that contains the gene(s) causing this decrease in Aß 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/Aß 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 in DS, compared with 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 (AD), which has been previously attributed to people with Down syndrome having three copies of the amyloid precursor protein (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; Tosh et al., 2021). 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 Aß 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.

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-ß (Aß) 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.

Endosomal structure and APP biology are not altered in a preclinical mouse cellular model of Down syndrome.

Individuals who have Down syndrome (trisomy 21) are at greatly increased risk of developing Alzheimer's disease, characterised by the accumulation in the brain of amyloid-ß plaques. Amyloid-ß is a product of the processing of the amyloid precursor protein, encoded by the APP gene on chromosome 21. In Down syndrome the first site of amyloid-ß accumulation is within endosomes, and changes to endosome biology occur early in Alzheimer's disease. Here, we determine if primary mouse embryonic fibroblasts isolated from a mouse model of Down syndrome can be used to study endosome and APP cell biology. We report that in this cellular model, endosome number, size and APP processing are not altered, likely because APP is not dosage sensitive in the model, despite three copies of App.

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 amyloid precursor protein (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 characterise 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.

The effects of Cstb duplication on APP/amyloid-ß pathology and cathepsin B activity in a mouse model.

People with Down syndrome (DS), caused by trisomy of chromosome 21 have a greatly increased risk of developing Alzheimer's disease (AD). This is in part because of triplication of a chromosome 21 gene, APP. This gene encodes amyloid precursor protein, which is cleaved to form amyloid-ß that accumulates in the brains of people who have AD. Recent experimental results demonstrate that a gene or genes on chromosome 21, other than APP, when triplicated significantly accelerate amyloid-ß pathology in a transgenic mouse model of amyloid-ß deposition. Multiple lines of evidence indicate that cysteine cathepsin activity influences APP cleavage and amyloid-ß accumulation. Located on human chromosome 21 (Hsa21) is an endogenous inhibitor of cathepsin proteases, CYSTATIN B (CSTB) which is proposed to regulate cysteine cathepsin activity in vivo. Here we determined if three copies of the mouse gene Cstb is sufficient to modulate amyloid-ß accumulation and cathepsin activity in a transgenic APP mouse model. Duplication of Cstb resulted in an increase in transcriptional and translational levels of Cstb in the mouse cortex but had no effect on the deposition of insoluble amyloid-ß plaques or the levels of soluble or insoluble amyloid-ß42, amyloid-ß40, or amyloid-ß38 in 6-month old mice. In addition, the increased CSTB did not alter the activity of cathepsin B enzyme in the cortex of 3-month or 6-month old mice. These results indicate that the single-gene duplication of Cstb is insufficient to elicit a disease-modifying phenotype in the dupCstb x tgAPP mice, underscoring the complexity of the genetic basis of AD-DS and the importance of multiple gene interactions in disease.

Genetic dissection of down syndrome-associated alterations in APP/amyloid-ß biology using mouse models.

Individuals who have Down syndrome (caused by trisomy of chromosome 21), have a greatly elevated risk of early-onset Alzheimer's disease, in which amyloid-ß accumulates in the brain. Amyloid-ß is a product of the chromosome 21 gene APP (amyloid precursor protein) and the extra copy or 'dose' of APP is thought to be the cause of this early-onset Alzheimer's disease. However, other chromosome 21 genes likely modulate disease when in three-copies in people with Down syndrome. Here we show that an extra copy of chromosome 21 genes, other than APP, influences APP/Aß biology. We crossed Down syndrome mouse models with partial trisomies, to an APP transgenic model and found that extra copies of subgroups of chromosome 21 gene(s) modulate amyloid-ß aggregation and APP transgene-associated mortality, independently of changing amyloid precursor protein abundance. Thus, genes on chromosome 21, other than APP, likely modulate Alzheimer's disease in people who have Down syndrome.

A novel knockout mouse for the small EDRK-rich factor 2 (Serf2) showing developmental and other deficits.

The small EDRK-rich factor 2 (SERF2) is a highly conserved protein that modifies amyloid fibre assembly in vitro and promotes protein misfolding. However, the role of SERF2 in regulating age-related proteotoxicity remains largely unexplored due to a lack of in vivo models. Here, we report the generation of Serf2 knockout mice using an ES cell targeting approach, with Serf2 knockout alleles being bred onto different defined genetic backgrounds. We highlight phenotyping data from heterozygous Serf2+/- mice, including unexpected male-specific phenotypes in startle response and pre-pulse inhibition. We report embryonic lethality in Serf2-/- null animals when bred onto a C57BL/6 N background. However, homozygous null animals were viable on a mixed genetic background and, remarkably, developed without obvious abnormalities. The Serf2 knockout mice provide a powerful tool to further investigate the role of SERF2 protein in previously unexplored pathophysiological pathways in the context of a whole organism.

Substantially thinner internal granular layer and reduced molecular layer surface in the cerebellar cortex of the Tc1 mouse model of down syndrome - a comprehensive morphometric analysis with active staining contrast-enhanced MRI.

Down Syndrome is a chromosomal disorder that affects the development of cerebellar cortical lobules. Impaired neurogenesis in the cerebellum varies among different types of neuronal cells and neuronal layers. In this study, we developed an imaging analysis framework that utilizes gadolinium-enhanced ex vivo mouse brain MRI. We extracted the middle Purkinje layer of the mouse cerebellar cortex, enabling the estimation of the volume, thickness, and surface area of the entire cerebellar cortex, the internal granular layer, and the molecular layer in the Tc1 mouse model of Down Syndrome. The morphometric analysis of our method revealed that a larger proportion of the cerebellar thinning in this model of Down Syndrome resided in the inner granule cell layer, while a larger proportion of the surface area shrinkage was in the molecular layer.

Using mouse models to understand Alzheimer's disease mechanisms in the context of trisomy of chromosome 21.

People who have Down syndrome are at significantly elevated risk of developing early onset Alzheimer's disease that causes dementia (AD-DS). Here we review recent progress in modeling the development of AD-DS in mouse models. These studies provide insight into mechanisms underlying Alzheimer's disease and generate new clinical research questions. In addition, they suggest potential new targets for disease prevention therapies.

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.

Downregulated Wnt/ß-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/ß-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/ß-catenin. Following pathway activation, however, DYRK1A exerts the opposite effect, increasing signalling activity. In summary, we identified downregulation of hippocampal Wnt/ß-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.

Plasma biomarkers for amyloid, tau, and cytokines in Down syndrome and sporadic Alzheimer's disease.

BACKGROUND: Down syndrome (DS), caused by chromosome 21 trisomy, is associated with an ultra-high risk of dementia due to Alzheimer's disease (AD), driven by amyloid precursor protein (APP) gene triplication. Understanding relevant molecular differences between those with DS, those with sporadic AD (sAD) without DS, and controls will aid in understanding AD development in DS. We explored group differences in plasma concentrations of amyloid-ß peptides and tau (as their accumulation is a characteristic feature of AD) and cytokines (as the inflammatory response has been implicated in AD development, and immune dysfunction is common in DS). METHODS: We used ultrasensitive assays to compare plasma concentrations of the amyloid-ß peptides Aß40 and Aß42, total tau (t-tau), and the cytokines IL1ß, IL10, IL6, and TNFα between adults with DS (n = 31), adults with sAD (n = 27), and controls age-matched to the group with DS (n = 27), and explored relationships between molecular concentrations and with age within each group. In the group with DS, we also explored relationships with neurofilament light (NfL) concentration, due to its potential use as a biomarker for AD in DS. RESULTS: Aß40, Aß42, and IL1ß concentrations were higher in DS, with a higher Aß42/Aß40 ratio in controls. The group with DS showed moderate positive associations between concentrations of t-tau and both Aß42 and IL1ß. Only NfL concentration in the group with DS showed a significant positive association with age. CONCLUSIONS: Concentrations of Aß40 and Aß42 were much higher in adults with DS than in other groups, reflecting APP gene triplication, while no difference in the Aß42/Aß40 ratio between those with DS and sAD may indicate similar processing and deposition of Aß40 and Aß42 in these groups. Higher concentrations of IL1ß in DS may reflect an increased vulnerability to infections and/or an increased prevalence of autoimmune disorders, while the positive association between IL1ß and t-tau in DS may indicate IL1ß is associated with neurodegeneration. Finally, NfL concentration may be the most suitable biomarker for dementia progression in DS. The identification of such a biomarker is important to improve the detection of dementia and monitor its progression, and for designing clinical intervention studies.

Trisomy of human chromosome 21 enhances amyloid-ß deposition independently of an extra copy of APP.

Down syndrome, caused by trisomy of chromosome 21, is the single most common risk factor for early-onset Alzheimer's disease. Worldwide approximately 6 million people have Down syndrome, and all these individuals will develop the hallmark amyloid plaques and neurofibrillary tangles of Alzheimer's disease by the age of 40 and the vast majority will go on to develop dementia. Triplication of APP, a gene on chromosome 21, is sufficient to cause early-onset Alzheimer's disease in the absence of Down syndrome. However, whether triplication of other chromosome 21 genes influences disease pathogenesis in the context of Down syndrome is unclear. Here we show, in a mouse model, that triplication of chromosome 21 genes other than APP increases amyloid-ß aggregation, deposition of amyloid-ß plaques and worsens associated cognitive deficits. This indicates that triplication of chromosome 21 genes other than APP is likely to have an important role to play in Alzheimer's disease pathogenesis in individuals who have Down syndrome. We go on to show that the effect of trisomy of chromosome 21 on amyloid-ß aggregation correlates with an unexpected shift in soluble amyloid-ß 40/42 ratio. This alteration in amyloid-ß isoform ratio occurs independently of a change in the carboxypeptidase activity of the γ-secretase complex, which cleaves the peptide from APP, or the rate of extracellular clearance of amyloid-ß. These new mechanistic insights into the role of triplication of genes on chromosome 21, other than APP, in the development of Alzheimer's disease in individuals who have Down syndrome may have implications for the treatment of this common cause of neurodegeneration.

T1, diffusion tensor, and quantitative magnetization transfer imaging of the hippocampus in an Alzheimer's disease mouse model.

Alzheimer's disease (AD) pathology causes microstructural changes in the brain. These changes, if quantified with magnetic resonance imaging (MRI), could be studied for use as an early biomarker for AD. The aim of our study was to determine if T1 relaxation, diffusion tensor imaging (DTI), and quantitative magnetization transfer imaging (qMTI) metrics could reveal changes within the hippocampus and surrounding white matter structures in ex vivo transgenic mouse brains overexpressing human amyloid precursor protein with the Swedish mutation. Delineation of hippocampal cell layers using DTI color maps allows more detailed analysis of T1-weighted imaging, DTI, and qMTI metrics, compared with segmentation of gross anatomy based on relaxation images, and with analysis of DTI or qMTI metrics alone. These alterations are observed in the absence of robust intracellular Aß accumulation or plaque deposition as revealed by histology. This work demonstrates that multiparametric quantitative MRI methods are useful for characterizing changes within the hippocampal substructures and surrounding white matter tracts of mouse models of AD.

The integration site of the APP transgene in the J20 mouse model of Alzheimer's disease.

Background: Transgenic animal models are a widely used and powerful tool to investigate human disease and develop therapeutic interventions. Making a transgenic mouse involves random integration of exogenous DNA into the host genome that can have the effect of disrupting endogenous gene expression. The J20 mouse model of Alzheimer's disease (AD) is a transgenic overexpresser of human APP with familial AD mutations and has been extensively utilised in preclinical studies and our aim was to determine the genomic location of the J20 transgene insertion. Methods: We used a combination of breeding strategy and Targeted Locus Amplification with deep sequencing to identify the insertion site of the J20 transgene array. To assess RNA and protein expression of Zbtb20, we used qRT-PCR and Western Blotting. Results: We demonstrate that the J20 transgene construct has inserted within the genetic locus of endogenous mouse gene Zbtb20 on chromosome 16 in an array , disrupting expression of mRNA from this gene in adult hippocampal tissue. Preliminary data suggests that ZBTB20 protein levels remain unchanged in this tissue, however further study is necessary. We note that the endogenous mouse App gene also lies on chromosome 16, although 42 Mb from the Zbtb20 locus. Conclusions: These data will be useful for future studies utilising this popular model of AD, particularly those investigating gene interactions between the J20 APP transgene and other genes present on Mmu16 in the mouse.

Fully-Automated µMRI Morphometric Phenotyping of the Tc1 Mouse Model of Down Syndrome.

We describe a fully automated pipeline for the morphometric phenotyping of mouse brains from µMRI data, and show its application to the Tc1 mouse model of Down syndrome, to identify new morphological phenotypes in the brain of this first transchromosomic animal carrying human chromosome 21. We incorporate an accessible approach for simultaneously scanning multiple ex vivo brains, requiring only a 3D-printed brain holder, and novel image processing steps for their separation and orientation. We employ clinically established multi-atlas techniques-superior to single-atlas methods-together with publicly-available atlas databases for automatic skull-stripping and tissue segmentation, providing high-quality, subject-specific tissue maps. We follow these steps with group-wise registration, structural parcellation and both Voxel- and Tensor-Based Morphometry-advantageous for their ability to highlight morphological differences without the laborious delineation of regions of interest. We show the application of freely available open-source software developed for clinical MRI analysis to mouse brain data: NiftySeg for segmentation and NiftyReg for registration, and discuss atlases and parameters suitable for the preclinical paradigm. We used this pipeline to compare 29 Tc1 brains with 26 wild-type littermate controls, imaged ex vivo at 9.4T. We show an unexpected increase in Tc1 total intracranial volume and, controlling for this, local volume and grey matter density reductions in the Tc1 brain compared to the wild-types, most prominently in the cerebellum, in agreement with human DS and previous histological findings.

Tc1 mouse model of trisomy-21 dissociates properties of short- and long-term recognition memory.

The present study examined memory function in Tc1 mice, a transchromosomic model of Down syndrome (DS). Tc1 mice demonstrated an unusual delay-dependent deficit in recognition memory. More specifically, Tc1 mice showed intact immediate (30sec), impaired short-term (10-min) and intact long-term (24-h) memory for objects. A similar pattern was observed for olfactory stimuli, confirming the generality of the pattern across sensory modalities. The specificity of the behavioural deficits in Tc1 mice was confirmed using APP overexpressing mice that showed the opposite pattern of object memory deficits. In contrast to object memory, Tc1 mice showed no deficit in either immediate or long-term memory for object-in-place information. Similarly, Tc1 mice showed no deficit in short-term memory for object-location information. The latter result indicates that Tc1 mice were able to detect and react to spatial novelty at the same delay interval that was sensitive to an object novelty recognition impairment. These results demonstrate (1) that novelty detection per se and (2) the encoding of visuo-spatial information was not disrupted in adult Tc1 mice. The authors conclude that the task specific nature of the short-term recognition memory deficit suggests that the trisomy of genes on human chromosome 21 in Tc1 mice impacts on (perirhinal) cortical systems supporting short-term object and olfactory recognition memory.

A genetic cause of Alzheimer disease: mechanistic insights from Down syndrome.

Down syndrome, which arises in individuals carrying an extra copy of chromosome 21, is associated with a greatly increased risk of early-onset Alzheimer disease. It is thought that this risk is conferred by the presence of three copies of the gene encoding amyloid precursor protein (APP)--an Alzheimer disease risk factor--although the possession of extra copies of other chromosome 21 genes may also play a part. Further study of the mechanisms underlying the development of Alzheimer disease in people with Down syndrome could provide insights into the mechanisms that cause dementia in the general population.