Mouse models of aneuploidy to understand chromosome disorders.

An organism or cell carrying a number of chromosomes that is not a multiple of the haploid count is in a state of aneuploidy. This condition results in significant changes in the level of expression of genes that are gained or lost from the aneuploid chromosome(s) and most cases in humans are not compatible with life. However, a few aneuploidies can lead to live births, typically associated with deleterious phenotypes. We do not understand why phenotypes arise from aneuploid syndromes in humans. Animal models have the potential to provide great insight, but less than a handful of mouse models of aneuploidy have been made, and no ideal system exists in which to study the effects of aneuploidy per se versus those of raised gene dosage. Here, we give an overview of human aneuploid syndromes, the effects on physiology of having an altered number of chromosomes and we present the currently available mouse models of aneuploidy, focusing on models of trisomy 21 (which causes Down syndrome) because this is the most common, and therefore, the most studied autosomal aneuploidy. Finally, we discuss the potential role of carrying an extra chromosome on aneuploid phenotypes, independent of changes in gene dosage, and methods by which this could be investigated further

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-β plaques. Amyloid-β 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-β 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

Dissecting the contribution of human chromosome 21 syntenic regions to recognition memory processes in adult and aged mouse models of Down syndrome

Background: Trisomy of human chromosome 21 (Hsa21) results in a constellation of features known as Down syndrome (DS), the most common genetic form of intellectual disability. Hsa21 is orthologous to three regions in the mouse genome on mouse chromosome 16 (Mmu16), Mmu17 and Mmu10. We investigated genotype-phenotype relationships by assessing the contribution of these three regions to memory function and age-dependent cognitive decline, using three mouse models of DS, Dp1Tyb, Dp(17)3Yey, Dp(10)2Yey, that carry an extra copy of the Hsa21-orthologues on Mmu16, Mmu17 and Mmu10, respectively. Hypothesis: Prior research on cognitive function in DS mouse models has largely focused on models with an extra copy of the Mmu16 region and relatively little is known about the effects of increased copy number on Mmu17 and Mmu10 on cognition and how this interacts with the effects of aging. As aging is is a critical contributor to cognitive and psychiatric changes in DS, we hypothesised that ageing would differentially impact memory function in Dp1Tyb, Dp(17)3Yey, and Dp(10)2Yey, models of DS. Methods: Young (12-13 months and old (18-20 months mice Dp1Tyb, Dp(17)3Yey and Dp(10)2Yey mice were tested on a battery of object recognition memory test that assessed object novelty detection, novel location detection and associative object-in place memory. Following behavioral testing, hippocampal and frontal cortical tissue was analysed for expression of glutamatergic receptor proteins using standard immunoblot techniques. Results: Young (12-13 months and old (18-20 months mice Dp1Tyb, Dp(17)3Yey and Dp(10)2Yey mice were tested on a battery of object recognition memory test that assessed object novelty detection, novel location detection and associative object-in place memory. Following behavioral testing, hippocampal and frontal cortical tissue was analysed for expression of glutamatergic receptor proteins using standard immunoblot techniques. Conclusion: Our results show that distinct Hsa21-orthologous regions contribute differentially to cognitive dysfunction in DS mouse models and that aging interacts with triplication of Hsa21-orthologous genes on Mmu10

Affordable optical clearing and immunolabelling in mouse brain slices.

Traditional histological analysis is conducted on thin tissue sections, limiting the data capture from large tissue volumes to 2D profiles, and requiring stereological methods for 3D assessment. Recent advances in microscopical and tissue clearing methods have facilitated 3D reconstructions of tissue structure. However, staining of large tissue blocks remains a challenge, often requiring specialised and expensive equipment to clear and immunolabel tissue. Here, we present the Affordable Brain Slice Optical Clearing (ABSOC) method: a modified iDISCO protocol which enables clearing and immunolabeling of mouse brain slices up to 1 mm thick using inexpensive reagents and equipment, with no intensive expert training required. We illustrate the use of ABSOC in 1 mm C57BL/6J mouse coronal brain slices sectioned through the dorsal hippocampus and immunolabelled with an anti-calretinin antibody. The ABSOC method can be readily used for histological studies of mouse brain in order to move from the use of very thin tissue sections to large volumes of tissue - giving more representative analysis of biological samples, without the need for sampling of small regions only

Craniofacial dysmorphology in Down syndrome is caused by increased dosage of Dyrk1a and at least three other genes.

Down syndrome (DS), trisomy of human chromosome 21 (Hsa21), occurs in 1 in 800 live births and is the most common human aneuploidy. DS results in multiple phenotypes, including craniofacial dysmorphology, which is characterised by midfacial hypoplasia, brachycephaly and micrognathia. The genetic and developmental causes of this are poorly understood. Using morphometric analysis of the Dp1Tyb mouse model of DS and an associated mouse genetic mapping panel, we demonstrate that four Hsa21-orthologous regions of mouse chromosome 16 contain dosage-sensitive genes that cause the DS craniofacial phenotype, and identify one of these causative genes as Dyrk1a. We show that the earliest and most severe defects in Dp1Tyb skulls are in bones of neural crest (NC) origin, and that mineralisation of the Dp1Tyb skull base synchondroses is aberrant. Furthermore, we show that increased dosage of Dyrk1a results in decreased NC cell proliferation and a decrease in size and cellularity of the NC-derived frontal bone primordia. Thus, DS craniofacial dysmorphology is caused by an increased dosage of Dyrk1a and at least three other genes

Genetic dissection of triplicated chromosome 21 orthologs yields varying skeletal traits in Down syndrome model mice.

Down syndrome (DS) phenotypes result from triplicated genes, but it is generally unknown how specific three copy human chromosome 21 (Hsa21) orthologous genes or interactions between genes affect these traits. A mouse mapping panel genetically dissecting Hsa21 syntenic regions was used to investigate the contributions and interactions triplicated Hsa21 orthologous genes on mouse chromosome 16 (Mmu16). Four-month-old femurs of male and female Dp9Tyb, Dp2Tyb, Dp3Tyb, Dp4Tyb, Dp5Tyb, Dp6Tyb, Ts1Rhr, and Dp1Tyb;Dyrk1a+/+/- mice were analyzed by micro-computed tomography and 3-point bending to assess skeletal structure and mechanical properties. Male and female Dp1Tyb mice, with the entire Hsa21 homologous region of Mmu16 in three copies, display specific bone deficits similar to humans with DS and were used as a baseline comparison for the other strains in the panel. Bone phenotypes varied based on triplicated gene content, sex, and bone compartment. Three copies of Dyrk1a played a sex-specific, essential role in trabecular deficits and may interact with other genes to influence cortical deficits related to DS. Triplicated genes in Dp9Tyb and Dp2Tyb mice improved some skeletal deficits. As triplicated genes may both improve and worsen bone deficits, it is important to understand the interaction between and molecular mechanisms of skeletal alterations affected by these genes

Increased dosage of DYRK1A leads to congenital heart defects in a mouse model of Down syndrome.

Down syndrome (DS) is caused by trisomy of human chromosome 21 (Hsa21). DS is a gene dosage disorder that results in multiple phenotypes including congenital heart defects. This clinically important cardiac pathology is the result of a third copy of one or more of the approximately 230 genes on Hsa21, but the identity of the causative dosage-sensitive genes and hence mechanisms underlying this cardiac pathology remain unclear. Here, we show that hearts from human fetuses with DS and embryonic hearts from the Dp1Tyb mouse model of DS show reduced expression of mitochondrial respiration genes and cell proliferation genes. Using systematic genetic mapping, we determined that three copies of the dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1a) gene, encoding a serine/threonine protein kinase, are associated with congenital heart disease pathology. In embryos from Dp1Tyb mice, reducing Dyrk1a gene copy number from three to two reversed defects in cellular proliferation and mitochondrial respiration in cardiomyocytes and rescued heart septation defects. Increased dosage of DYRK1A protein resulted in impairment of mitochondrial function and congenital heart disease pathology in mice with DS, suggesting that DYRK1A may be a useful therapeutic target for treating this common human condition

Creation of de novo cryptic splicing for ALS and FTD precision medicine

Loss of function of the RNA-binding protein TDP-43 (TDP-LOF) is a hallmark of amyotrophic lateral sclerosis (ALS) and other neurodegenerative disorders. Here we describe TDP-REG, which exploits the specificity of cryptic splicing induced by TDP-LOF to drive protein expression when and where the disease process occurs. The SpliceNouveau algorithm combines deep learning with rational design to generate customizable cryptic splicing events within protein-coding sequences. We demonstrate that expression of TDP-REG reporters is tightly coupled to TDP-LOF in vitro and in vivo. TDP-REG enables genomic prime editing to ablate the UNC13A cryptic donor splice site specifically upon TDP-LOF. Finally, we design TDP-REG vectors encoding a TDP-43/Raver1 fusion protein that rescues key pathological cryptic splicing events, paving the way for the development of precision therapies for TDP43-related disorders

TDP-43 loss and ALS-risk SNPs drive mis-splicing and depletion of UNC13A.

Variants of UNC13A, a critical gene for synapse function, increase the risk of amyotrophic lateral sclerosis and frontotemporal dementia1-3, two related neurodegenerative diseases defined by mislocalization of the RNA-binding protein TDP-434,5. Here we show that TDP-43 depletion induces robust inclusion of a cryptic exon in UNC13A, resulting in nonsense-mediated decay and loss of UNC13A protein. Two common intronic UNC13A polymorphisms strongly associated with amyotrophic lateral sclerosis and frontotemporal dementia risk overlap with TDP-43 binding sites. These polymorphisms potentiate cryptic exon inclusion, both in cultured cells and in brains and spinal cords from patients with these conditions. Our findings, which demonstrate a genetic link between loss of nuclear TDP-43 function and disease, reveal the mechanism by which UNC13A variants exacerbate the effects of decreased TDP-43 function. They further provide a promising therapeutic target for TDP-43 proteinopathies

PolyGR and polyPR knock-in mice reveal a conserved neuroprotective extracellular matrix signature in C9orf72 ALS/FTD neurons.

Dipeptide repeat proteins are a major pathogenic feature of C9orf72 amyotrophic lateral sclerosis (C9ALS)/frontotemporal dementia (FTD) pathology, but their physiological impact has yet to be fully determined. Here we generated C9orf72 dipeptide repeat knock-in mouse models characterized by expression of 400 codon-optimized polyGR or polyPR repeats, and heterozygous C9orf72 reduction. (GR)400 and (PR)400 knock-in mice recapitulate key features of C9ALS/FTD, including cortical neuronal hyperexcitability, age-dependent spinal motor neuron loss and progressive motor dysfunction. Quantitative proteomics revealed an increase in extracellular matrix (ECM) proteins in (GR)400 and (PR)400 spinal cord, with the collagen COL6A1 the most increased protein. TGF-β1 was one of the top predicted regulators of this ECM signature and polyGR expression in human induced pluripotent stem cell neurons was sufficient to induce TGF-β1 followed by COL6A1. Knockdown of TGF-β1 or COL6A1 orthologues in polyGR model Drosophila exacerbated neurodegeneration, while expression of TGF-β1 or COL6A1 in induced pluripotent stem cell-derived motor neurons of patients with C9ALS/FTD protected against glutamate-induced cell death. Altogether, our findings reveal a neuroprotective and conserved ECM signature in C9ALS/FTD