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.

Cognitive impairments in a Down syndrome model with abnormal hippocampal and prefrontal dynamics and cytoarchitecture.

The Dp(10)2Yey mouse carries a ∼2.3-Mb intra-chromosomal duplication of mouse chromosome 10 (Mmu10) that has homology to human chromosome 21, making it an essential model for aspects of Down syndrome (DS, trisomy 21). In this study, we investigated neuronal dysfunction in the Dp(10)2Yey mouse and report spatial memory impairment and anxiety-like behavior alongside altered neural activity in the medial prefrontal cortex (mPFC) and hippocampus (HPC). Specifically, Dp(10)2Yey mice showed impaired spatial alternation associated with increased sharp-wave ripple activity in mPFC during a period of memory consolidation, and reduced mobility in a novel environment accompanied by reduced theta-gamma phase-amplitude coupling in HPC. Finally, we found alterations in the number of interneuron subtypes in mPFC and HPC that may contribute to the observed phenotypes and highlight potential approaches to ameliorate the effects of human trisomy 21.

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.

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.

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.

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.

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.

C9orf72 repeat expansions cause neurodegeneration in Drosophila through arginine-rich proteins.

An expanded GGGGCC repeat in C9orf72 is the most common genetic cause of frontotemporal dementia and amyotrophic lateral sclerosis. A fundamental question is whether toxicity is driven by the repeat RNA itself and/or by dipeptide repeat proteins generated by repeat-associated, non-ATG translation. To address this question, we developed in vitro and in vivo models to dissect repeat RNA and dipeptide repeat protein toxicity. Expression of pure repeats, but not stop codon-interrupted "RNA-only" repeats in Drosophila caused adult-onset neurodegeneration. Thus, expanded repeats promoted neurodegeneration through dipeptide repeat proteins. Expression of individual dipeptide repeat proteins with a non-GGGGCC RNA sequence revealed that both poly-(glycine-arginine) and poly-(proline-arginine) proteins caused neurodegeneration. These findings are consistent with a dual toxicity mechanism, whereby both arginine-rich proteins and repeat RNA contribute to C9orf72-mediated neurodegeneration.

WNT-3, expressed by motoneurons, regulates terminal arborization of neurotrophin-3-responsive spinal sensory neurons.

Sensory axons from dorsal root ganglia neurons are guided to spinal targets by molecules differentially expressed along the dorso-ventral axis of the neural tube. NT-3-responsive muscle afferents project ventrally, cease extending, and branch upon contact with motoneurons (MNs), their synaptic partners. We have identified WNT-3 as a candidate molecule that regulates this process. Wnt-3 is expressed by MNs of the lateral motor column at the time when MNs form synapses with sensory neurons. WNT-3 increases branching and growth cone size while inhibiting axonal extension in NT-3- but not NGF-responsive axons. Ventral spinal cord secretes factors with axonal remodeling activity for NT-3-responsive neurons. This activity is present at limb levels and is blocked by a WNT antagonist. We propose that WNT-3, expressed by MNs, acts as a retrograde signal that controls terminal arborization of muscle afferents.

Valproate regulates GSK-3-mediated axonal remodeling and synapsin I clustering in developing neurons.

Valproate (VPA) and lithium have been used for many years in the treatment of manic depression. However, their mechanisms of action remain poorly understood. Recent studies suggest that lithium and VPA inhibit GSK-3beta, a serine/threonine kinase involved in the insulin and WNT signaling pathways. Inhibition of GSK-3beta by high concentrations of lithium has been shown to mimic WNT-7a signaling by inducing axonal remodeling and clustering of synapsin I in developing neurons. Here we have compared the effect of therapeutic concentrations of lithium and VPA during neuronal maturation. VPA and, to a lesser extent, lithium induce clustering of synapsin I. In addition, lithium and VPA induce similar changes in the morphology of axons by increasing growth cone size, spreading, and branching. More importantly, both mood stabilizers decrease the level of MAP-1B-P, a GSK-3beta-phosphorylated form of MAP-1B in developing neurons, suggesting that therapeutic concentrations of these mood stabilizers inhibit GSK-3beta. In vitro kinase assays show that therapeutic concentrations of VPA do not inhibit GSK-3beta but that therapeutic concentrations of lithium partially inhibit GSK-3beta activity. Our results support the idea that both mood stabilizers inhibit GSK-3beta in developing neurons through different pathways. Lithium directly inhibits GSK-3beta in contrast to VPA, which inhibits GSK-3beta indirectly by an as-yet-unknown pathway. These findings may have important implications for the development of new strategies to treat bipolar disorders.