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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.

The transcriptional machinery is essential for gene expression and regulation; dysregulation of transcription can result in a range of pathologies, including neurodegeneration, cancer, developmental disorders and cardiovascular disease. A key component of RNA polymerase II-mediated transcription is the basal transcription factor IID, which is formed of the TATA box-binding protein (TBP) and 14 TBP-associated factors (TAFs), the largest of which is the TAF1 protein, encoded on the X chromosome (Xq13.1). TAF1 is dysregulated in X-linked dystonia-parkinsonism and congenital mutations in the gene are causative for neurodevelopmental phenotypes; TAF1 dysfunction is also associated with cardiac anomalies and cancer. However, how TAF1 contributes to pathology is unclear. Here, we highlight the key aspects of the TAF1 gene and protein function that may link transcriptional regulation with disorders of development, growth and adult-onset disorders of motor impairment. We highlight the need to experimentally investigate the full range of TAF1 messenger RNA variants and protein isoforms in human and mouse to aid our understanding of TAF1 biology. Furthermore, the X-linked nature of TAF1 -related diseases adds complexity to understanding phenotypes. Overall, we shed light on the aspects of TAF1 biology that may contribute to disease and areas that could be addressed for future research and targeted therapeutics., Competing Interests: We declare we have no competing interests., (© 2024 The Author(s).)

Down syndrome (DS), a prevalent cognitive disorder resulting from trisomy of human chromosome 21 (Hsa21), poses a significant global health concern. Affecting approximately 1 in 800 live births worldwide, DS is the leading genetic cause of intellectual disability and a major predisposing factor for early-onset Alzheimer's dementia. The estimated global population of individuals with DS is 6 million, with increasing prevalence due to advances in DS health care. Global efforts are dedicated to unraveling the mechanisms behind the varied clinical outcomes in DS. Recent studies on DS mouse models reveal disrupted neuronal circuits, providing insights into DS pathologies. Yet, translating these findings to humans faces challenges due to limited systematic electrophysiological analyses directly comparing human and mouse. Additionally, disparities in experimental procedures between the two species pose hurdles to successful translation. This review provides a concise overview of neuronal oscillations in human and rodent cognition. Focusing on recent DS mouse model studies, we highlight disruptions in associated brain function. We discuss various electrophysiological paradigms and suggest avenues for exploring molecular dysfunctions contributing to DS-related cognitive impairments. Deciphering neuronal oscillation intricacies holds promise for targeted therapies to alleviate cognitive disabilities in DS individuals., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Canonica, Kidd, Gibbins, Lana-Elola, Fisher, Tybulewicz and Good.)

The TATA box-binding protein-associated factor 1 (TAF1) is a ubiquitously expressed protein and the largest subunit of the basal transcription factor TFIID, which plays a key role in initiation of RNA polymerase II-dependent transcription. TAF1 missense variants in human males cause X-linked intellectual disability, a neurodevelopmental disorder, and TAF1 is dysregulated in X-linked dystonia-parkinsonism, a neurodegenerative disorder. However, this field has lacked a genetic mouse model of TAF1 disease to explore its mechanism in mammals and treatments. Here, we generated and validated a conditional cre-lox allele and the first ubiquitous Taf1 knockout mouse. We discovered that Taf1 deletion in male mice was embryonically lethal, which may explain why no null variants have been identified in humans. In the brains of Taf1 heterozygous female mice, no differences were found in gross structure, overall expression and protein localisation, suggesting extreme skewed X inactivation towards the non-mutant chromosome. Nevertheless, these female mice exhibited a significant increase in weight, weight with age, and reduced movement, suggesting that a small subset of neurons was negatively impacted by Taf1 loss. Finally, this new mouse model may be a future platform for the development of TAF1 disease therapeutics., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

TDP-43 loss of function induces multiple splicing changes, including a cryptic exon in the amyotrophic lateral sclerosis and fronto-temporal lobar degeneration risk gene UNC13A , leading to nonsense-mediated decay of UNC13A transcripts and loss of protein. UNC13A is an active zone protein with an integral role in coordinating pre-synaptic function. Here, we show TDP-43 depletion induces a severe reduction in synaptic transmission, leading to an asynchronous pattern of network activity. We demonstrate that these deficits are largely driven by a single cryptic exon in UNC13A . Antisense oligonucleotides targeting the UNC13A cryptic exon robustly rescue UNC13A protein levels and restore normal synaptic function, providing a potential new therapeutic approach for ALS and other TDP-43-related disorders., Competing Interests: Competing interests: PF, MJK and OGW have filed a patent application relating to the use of antisense oligonucleotides for the correction of cryptic splicing in UNC13A. PF is founder, advisor, and holds shares in Trace Neuroscience Inc. MJK performs consulting for and holds shares in Trace Neuroscience Inc. PH performs consulting for Trace Neuroscience Inc.

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., (© 2024. The Author(s).)

TDP-43 pathology is found in several neurodegenerative disorders, collectively referred to as "TDP-43 proteinopathies". Aggregates of TDP-43 are present in the brains and spinal cords of >97% of amyotrophic lateral sclerosis (ALS), and in brains of ∼50% of frontotemporal dementia (FTD) patients. While mutations in the TDP-43 gene (TARDBP) are usually associated with ALS, many clinical reports have linked these mutations to cognitive impairments and/or FTD, but also to other neurodegenerative disorders including Parkinsonism (PD) or progressive supranuclear palsy (PSP). TDP-43 is a ubiquitously expressed, highly conserved RNA-binding protein that is involved in many cellular processes, mainly RNA metabolism. To investigate systemic pathological mechanisms in TDP-43 proteinopathies, aiming to capture the pleiotropic effects of TDP-43 mutations, we have further characterised a mouse model carrying a point mutation (M323K) within the endogenous Tardbp gene. Homozygous mutant mice developed cognitive and behavioural deficits as early as 3 months of age. This was coupled with significant brain structural abnormalities, mainly in the cortex, hippocampus, and white matter fibres, together with progressive cortical interneuron degeneration and neuroinflammation. At the motor level, progressive phenotypes appeared around 6 months of age. Thus, cognitive phenotypes appeared to be of a developmental origin with a mild associated progressive neurodegeneration, while the motor and neuromuscular phenotypes seemed neurodegenerative, underlined by a progressive loss of upper and lower motor neurons as well as distal denervation. This is accompanied by progressive elevated TDP-43 protein and mRNA levels in cortex and spinal cord of homozygous mutant mice from 3 months of age, together with increased cytoplasmic TDP-43 mislocalisation in cortex, hippocampus, hypothalamus, and spinal cord at 12 months of age. In conclusion, we find that Tardbp M323K homozygous mutant mice model many aspects of human TDP-43 proteinopathies, evidencing a dual role for TDP-43 in brain morphogenesis as well as in the maintenance of the motor system, making them an ideal in vivo model system to study the complex biology of TDP-43., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest. The funding institution had no role in data acquisition, analysis, or decision to publish the results., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

Down syndrome (DS) is one of the most common birth defects and the most prevalent genetic form of intellectual disability. DS arises from trisomy of chromosome 21, but its molecular and pathological consequences are not fully understood. In this study, we compared Dp1Tyb mice, a DS model, against their wild-type (WT) littermates of both sexes to investigate the impact of DS-related genetic abnormalities on the brain phenotype. We performed in vivo whole brain magnetic resonance imaging (MRI) and hippocampal 1 H magnetic resonance spectroscopy (MRS) on the animals at 3 months of age. Subsequently, ex vivo MRI scans and histological analyses were conducted post-mortem. Our findings unveiled the following neuroanatomical and biochemical alterations in the Dp1Tyb brains: a smaller surface area and a rounder shape compared to WT brains, with DS males also presenting smaller global brain volume compared with the counterpart WT. Regional volumetric analysis revealed significant changes in 26 out of 72 examined brain regions, including the medial prefrontal cortex and dorsal hippocampus. These alterations were consistently observed in both in vivo and ex vivo imaging data. Additionally, high-resolution ex vivo imaging enabled us to investigate cerebellar layers and hippocampal sub-regions, revealing selective areas of decrease and remodelling in these structures. An analysis of hippocampal metabolites revealed an elevation in glutamine and the glutamine/glutamate ratio in the Dp1Tyb mice compared to controls, suggesting a possible imbalance in the excitation/inhibition ratio. This was accompanied by the decreased levels of taurine. Histological analysis revealed fewer neurons in the hippocampal CA3 and DG layers, along with an increase in astrocytes and microglia. These findings recapitulate multiple neuroanatomical and biochemical features associated with DS, enriching our understanding of the potential connection between chromosome 21 trisomy and the resultant phenotype., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests.

Variants in the ubiquitously expressed DNA/RNA-binding protein FUS cause aggressive juvenile forms of amyotrophic lateral sclerosis (ALS). Most FUS mutation studies have focused on motor neuron degeneration; little is known about wider systemic or developmental effects. We studied pleiotropic phenotypes in a physiological knock-in mouse model carrying the pathogenic FUSDelta14 mutation in homozygosity. RNA sequencing of multiple organs aimed to identify pathways altered by the mutant protein in the systemic transcriptome, including metabolic tissues, given the link between ALS-frontotemporal dementia and altered metabolism. Few genes were commonly altered across all tissues, and most genes and pathways affected were generally tissue specific. Phenotypic assessment of mice revealed systemic metabolic alterations related to the pathway changes identified. Magnetic resonance imaging brain scans and histological characterisation revealed that homozygous FUSDelta14 brains were smaller than heterozygous and wild-type brains and displayed significant morphological alterations, including a thinner cortex, reduced neuronal number and increased gliosis, which correlated with early cognitive impairment and fatal seizures. These findings show that the disease aetiology of FUS variants can include both neurodevelopmental and systemic alterations., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

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., (© 2023. BioMed Central Ltd., part of Springer Nature.)

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., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Amyotrophic lateral sclerosis is a complex disorder most of which is 'sporadic' of unknown origin but approximately 10% is familial, arising from single mutations in any of more than 30 genes. Thus, there are more than 30 familial ALS subtypes, with different, often unknown, molecular pathologies leading to a complex constellation of clinical phenotypes. We have mouse models for many genetic forms of the disorder, but these do not, on their own, necessarily show us the key pathological pathways at work in human patients. To date, we have no models for the 90% of ALS that is 'sporadic'. Potential therapies have been developed mainly using a limited set of mouse models, and through lack of alternatives, in the past these have been tested on patients regardless of aetiology. Cancer researchers have undertaken therapy development with similar challenges; they have responded by producing complex mouse models that have transformed understanding of pathological processes, and they have implemented patient stratification in multi-centre trials, leading to the effective translation of basic research findings to the clinic. ALS researchers have successfully adopted this combined approach, and now to increase our understanding of key disease pathologies, and our rate of progress for moving from mouse models to mechanism to ALS therapies we need more, innovative, complex mouse models to address specific questions., (© 2023. The Author(s).)

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., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Down syndrome (DS) phenotypes result from triplicated genes, but the effects of three copy genes are not well known. A mouse mapping panel genetically dissecting human chromosome 21 (Hsa21) syntenic regions was used to investigate the contributions and interactions of triplicated Hsa21 orthologous genes on mouse chromosome 16 (Mmu16) on skeletal phenotypes. Skeletal structure and mechanical properties were assessed in femurs of male and female Dp9Tyb, Dp2Tyb, Dp3Tyb, Dp4Tyb, Dp5Tyb, Dp6Tyb, Ts1Rhr and Dp1Tyb;Dyrk1a+/+/- mice. Dp1Tyb mice, with the entire Hsa21 homologous region of Mmu16 triplicated, display bone deficits similar to those of humans with DS and served as a baseline for 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 parameters. As triplicated genes can both improve and worsen bone deficits, it is important to understand the interaction between and molecular mechanisms of skeletal alterations affected by these genes., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

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., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

Individuals who have Down syndrome (DS) frequently develop early onset Alzheimer's disease (AD), a neurodegenerative condition caused by the buildup of aggregated amyloid-β (Aβ) and tau proteins in the brain. Aβ 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 Aβ accumulation ( App NL-F ) to determine how chromosome 21 genes, other than APP , modulate APP/Aβ 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 Aβ accumulation in the brain. We go on to identify a subregion of chromosome 21 that contains the gene(s) causing this decrease in Aβ 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/Aβ 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 Aβ 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., (Copyright © 2022 the authors.)

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., Competing Interests: F.K.W. has undertaken consultancy for Elkington and Fife Patent Lawyers unrelated to the work in the manuscript and is also a PLoS ONE Academic Editor. This does not alter our adherence to PLoS ONE policies on sharing data or materials

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., (© 2021. The Author(s).)

Variants of UNC13A, a critical gene for synapse function, increase the risk of amyotrophic lateral sclerosis and frontotemporal dementia 1-3 , two related neurodegenerative diseases defined by mislocalization of the RNA-binding protein TDP-43 4,5 . 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., (© 2022. The Author(s).)

Astrocytes play crucial and diverse roles in brain health and disease. The ability to selectively control astrocytes provides a valuable tool for understanding their function and has the therapeutic potential to correct dysfunction. Existing technologies such as optogenetics and chemogenetics require the introduction of foreign proteins, which adds a layer of complication and hinders their clinical translation. A novel technique, magnetomechanical stimulation (MMS), that enables remote and selective control of astrocytes without genetic modification is described here. MMS exploits the mechanosensitivity of astrocytes and triggers mechanogated Ca 2+ and adenosine triphosphate (ATP) signaling by applying a magnetic field to antibody-functionalized magnetic particles that are targeted to astrocytes. Using purpose-built magnetic devices, the mechanosensory threshold of astrocytes is determined, a sub-micrometer particle for effective MMS is identified, the in vivo fate of the particles is established, and cardiovascular responses are induced in rats after particles are delivered to specific brainstem astrocytes. By eliminating the need for device implantation and genetic modification, MMS is a method for controlling astroglial activity with an improved prospect for clinical application than existing technologies., (© 2021 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD) is a fatal neurodegenerative disorder, and continued innovation is needed for improved understanding and for developing therapeutics. We have created next-generation genomically humanized knockin mouse models, by replacing the mouse genomic region of Sod1 , Tardbp (TDP-43), and Fus , with their human orthologs, preserving human protein biochemistry and splicing with exons and introns intact. We establish a new standard of large knockin allele quality control, demonstrating the utility of indirect capture for enrichment of a genomic region of interest followed by Oxford Nanopore sequencing. Extensive analysis shows that homozygous humanized animals only express human protein at endogenous levels. Characterization of humanized FUS animals showed that they are phenotypically normal throughout their lifespan. These humanized strains are vital for preclinical assessment of interventions and serve as templates for the addition of coding or non-coding human ALS/FTD mutations to dissect disease pathomechanisms, in a physiological context., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

Down syndrome (DS), trisomy 21, results in many complex phenotypes including cognitive deficits, heart defects and craniofacial alterations. Phenotypes arise from an extra copy of human chromosome 21 (Hsa21) genes. However, these dosage-sensitive causative genes remain unknown. Animal models enable identification of genes and pathological mechanisms. The Dp1Tyb mouse model of DS has an extra copy of 63% of Hsa21-orthologous mouse genes. In order to establish whether this model recapitulates DS phenotypes, we comprehensively phenotyped Dp1Tyb mice using 28 tests of different physiological systems and found that 468 out of 1800 parameters were significantly altered. We show that Dp1Tyb mice have wide-ranging DS-like phenotypes, including aberrant erythropoiesis and megakaryopoiesis, reduced bone density, craniofacial changes, altered cardiac function, a pre-diabetic state, and deficits in memory, locomotion, hearing and sleep. Thus, Dp1Tyb mice are an excellent model for investigating complex DS phenotype-genotype relationships for this common disorder., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Mutations in the RNA-binding protein (RBP) FUS have been genetically associated with the motoneuron disease amyotrophic lateral sclerosis (ALS). Using both human induced pluripotent stem cells and mouse models, we found that FUS-ALS causative mutations affect the activity of two relevant RBPs with important roles in neuronal RNA metabolism: HuD/ELAVL4 and FMRP. Mechanistically, mutant FUS leads to upregulation of HuD protein levels through competition with FMRP for HuD mRNA 3'UTR binding. In turn, increased HuD levels overly stabilize the transcript levels of its targets, NRN1 and GAP43. As a consequence, mutant FUS motoneurons show increased axon branching and growth upon injury, which could be rescued by dampening NRN1 levels. Since similar phenotypes have been previously described in SOD1 and TDP-43 mutant models, increased axonal growth and branching might represent broad early events in the pathogenesis of ALS., (© 2021. The Author(s).)

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-β 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-β pathology in a transgenic mouse model of amyloid-β deposition. Multiple lines of evidence indicate that cysteine cathepsin activity influences APP cleavage and amyloid-β 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-β 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-β plaques or the levels of soluble or insoluble amyloid-β42, amyloid-β40, or amyloid-β38 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., Competing Interests: F.K.W. has undertaken consultancy for Elkington and Fife Patent Lawyers unrelated to the work in the manuscript and is also a PLoS One Academic Editor. This does not alter our adherence to PLoS ONE policies on sharing data and materials.

FUsed in Sarcoma (FUS) is a multifunctional RNA binding protein (RBP). FUS mutations lead to its cytoplasmic mislocalization and cause the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Here, we use mouse and human models with endogenous ALS-associated mutations to study the early consequences of increased cytoplasmic FUS. We show that in axons, mutant FUS condensates sequester and promote the phase separation of fragile X mental retardation protein (FMRP), another RBP associated with neurodegeneration. This leads to repression of translation in mouse and human FUS-ALS motor neurons and is corroborated in vitro, where FUS and FMRP copartition and repress translation. Last, we show that translation of FMRP-bound RNAs is reduced in vivo in FUS-ALS motor neurons. Our results unravel new pathomechanisms of FUS-ALS and identify a novel paradigm by which mutations in one RBP favor the formation of condensates sequestering other RBPs, affecting crucial biological functions, such as protein translation., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).)

Aberrant microsatellite repeat-expansions at specific loci within the human genome cause several distinct, heritable, and predominantly neurological, disorders. Creating models for these diseases poses a challenge, due to the instability of such repeats in bacterial vectors, especially with large repeat expansions. Designing constructs for more precise genome engineering projects, such as engineering knock-in mice, proves a greater challenge still, since these unstable repeats require numerous cloning steps in order to introduce homology arms or selection cassettes. Here, we report our efforts to clone a large hexanucleotide repeat in the C9orf72 gene, originating from within a BAC construct, derived from a C9orf72-ALS patient. We provide detailed methods for efficient repeat sizing and growth conditions in bacteria to facilitate repeat retention during growth and sub-culturing. We report that sub-cloning into a linear vector dramatically improves stability, but is dependent on the relative orientation of DNA replication through the repeat, consistent with previous studies. We envisage the findings presented here provide a relatively straightforward route to maintaining large-range microsatellite repeat-expansions, for efficient cloning into vectors., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Research focused on Down syndrome has increased in the last several years to advance understanding of the consequences of trisomy 21 (T21) on molecular and cellular processes and, ultimately, on individuals with Down syndrome. The Trisomy 21 Research Society (T21RS) is the premier scientific organization for researchers and clinicians studying Down syndrome. The Third International Conference of T21RS, held June 6-9, 2019, in Barcelona, Spain, brought together 429 scientists, families, and industry representatives to share the latest discoveries on underlying cellular and molecular mechanisms of T21, define cognitive and behavioral challenges and better understand comorbidities associated with Down syndrome, including Alzheimer's disease and leukemia. Presentation of cutting-edge results in neuroscience, neurology, model systems, psychology, cancer, biomarkers and molecular and phar-ma-cological therapeutic approaches demonstrate the compelling interest and continuing advancement in all aspects of understanding and ameliorating conditions associated with T21., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2021 by S. Karger AG, Basel.)

The neuromuscular junction (NMJ) is the peripheral synapse formed between a motor neuron axon terminal and a muscle fibre. NMJs are thought to be the primary site of peripheral pathology in many neuromuscular diseases, but innervation/denervation status is often assessed qualitatively with poor systematic criteria across studies, and separately from 3D morphological structure. Here, we describe the development of 'NMJ-Analyser', to comprehensively screen the morphology of NMJs and their corresponding innervation status automatically. NMJ-Analyser generates 29 biologically relevant features to quantitatively define healthy and aberrant neuromuscular synapses and applies machine learning to diagnose NMJ degeneration. We validated this framework in longitudinal analyses of wildtype mice, as well as in four different neuromuscular disease models: three for amyotrophic lateral sclerosis (ALS) and one for peripheral neuropathy. We showed that structural changes at the NMJ initially occur in the nerve terminal of mutant TDP43 and FUS ALS models. Using a machine learning algorithm, healthy and aberrant neuromuscular synapses are identified with 95% accuracy, with 88% sensitivity and 97% specificity. Our results validate NMJ-Analyser as a robust platform for systematic and structural screening of NMJs, and pave the way for transferrable, and cross-comparison and high-throughput studies in neuromuscular diseases.

Congenital heart disease (CHD) is the most common class of human birth defects, with a prevalence of 0.9% of births. However, two-thirds of cases have an unknown cause, and many of these are thought to be caused by in utero exposure to environmental teratogens. Here we identify a potential teratogen causing CHD in mice: maternal iron deficiency (ID). We show that maternal ID in mice causes severe cardiovascular defects in the offspring. These defects likely arise from increased retinoic acid signalling in ID embryos. The defects can be prevented by iron administration in early pregnancy. It has also been proposed that teratogen exposure may potentiate the effects of genetic predisposition to CHD through gene-environment interaction. Here we show that maternal ID increases the severity of heart and craniofacial defects in a mouse model of Down syndrome. It will be important to understand if the effects of maternal ID seen here in mice may have clinical implications for women.

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.

Characterising phenotypes often requires quantification of anatomical shape. Quantitative shape comparison (morphometrics) traditionally uses manually located landmarks and is limited by landmark number and operator accuracy. Here, we apply a landmark-free method to characterise the craniofacial skeletal phenotype of the Dp1Tyb mouse model of Down syndrome and a population of the Diversity Outbred (DO) mouse model, comparing it with a landmark-based approach. We identified cranial dysmorphologies in Dp1Tyb mice, especially smaller size and brachycephaly (front-back shortening), homologous to the human phenotype. Shape variation in the DO mice was partly attributable to allometry (size-dependent shape variation) and sexual dimorphism. The landmark-free method performed as well as, or better than, the landmark-based method but was less labour-intensive, required less user training and, uniquely, enabled fine mapping of local differences as planar expansion or shrinkage. Its higher resolution pinpointed reductions in interior mid-snout structures and occipital bones in both the models that were not otherwise apparent. We propose that this landmark-free pipeline could make morphometrics widely accessible beyond its traditional niches in zoology and palaeontology, especially in characterising developmental mutant phenotypes., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

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-β accumulates in the brain. Amyloid-β 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/Aβ 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-β 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.

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., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Although many molecular mechanisms controlling developmental processes are evolutionarily conserved, the speed at which the embryo develops can vary substantially between species. For example, the same genetic program, comprising sequential changes in transcriptional states, governs the differentiation of motor neurons in mouse and human, but the tempo at which it operates differs between species. Using in vitro directed differentiation of embryonic stem cells to motor neurons, we show that the program runs more than twice as fast in mouse as in human. This is not due to differences in signaling, nor the genomic sequence of genes or their regulatory elements. Instead, there is an approximately two-fold increase in protein stability and cell cycle duration in human cells compared with mouse cells. This can account for the slower pace of human development and suggests that differences in protein turnover play a role in interspecies differences in developmental tempo., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Mutations in the RNA-binding protein FUS cause amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disease. FUS plays a role in numerous aspects of RNA metabolism, including mRNA splicing. However, the impact of ALS-causative mutations on splicing has not been fully characterized, as most disease models have been based on overexpressing mutant FUS, which will alter RNA processing due to FUS autoregulation. We and others have recently created knockin models that overcome the overexpression problem, and have generated high depth RNA-sequencing on FUS mutants in parallel to FUS knockout, allowing us to compare mutation-induced changes to genuine loss of function. We find that FUS-ALS mutations induce a widespread loss of function on expression and splicing. Specifically, we find that mutant FUS directly alters intron retention levels in RNA-binding proteins. Moreover, we identify an intron retention event in FUS itself that is associated with its autoregulation. Altered FUS levels have been linked to disease, and we show here that this novel autoregulation mechanism is altered by FUS mutations. Crucially, we also observe this phenomenon in other genetic forms of ALS, including those caused by TDP-43, VCP and SOD1 mutations, supporting the concept that multiple ALS genes interact in a regulatory network., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

All individuals with Down syndrome (DS), which results from trisomy of human chromosome 21 (Ts21), present with skeletal abnormalities typified by craniofacial features, short stature and low bone mineral density (BMD). Differences in skeletal deficits between males and females with DS suggest a sexual dimorphism in how trisomy affects bone. Dp1Tyb mice contain three copies of all of the genes on mouse chromosome 16 that are homologous to human chromosome 21, males and females are fertile, and therefore are an excellent model to test the hypothesis that gene dosage influences the sexual dimorphism of bone abnormalities in DS. Dp1Tyb as compared to control littermate mice at time points associated with bone accrual (6 weeks) and skeletal maturity (16 weeks) showed deficits in BMD and trabecular architecture that occur largely through interactions between sex and genotype and resulted in lower percent bone volume in all female and Dp1Tyb male mice. Cortical bone in Dp1Tyb as compared to control mice exhibited different changes over time influenced by sex × genotype interactions including reduced cortical area in both male and female Dp1Tyb mice. Mechanical testing analyses suggested deficits in whole bone properties such as bone mass and geometry, but improved material properties in female and Dp1Tyb mice. Sexual dimorphisms and the influence of trisomic gene dosage differentially altered cellular properties of male and female Dp1Tyb bone. These data establish sex, gene dosage, skeletal site and age as important factors in skeletal development of DS model mice, paving the way for identification of the causal dosage-sensitive genes. Skeletal differences in developing male and female Dp1Tyb DS model mice replicated differences in less-studied adolescents with DS and established a foundation to understand the etiology of trisomic bone deficits., Competing Interests: Declaration of competing interest The authors declare no conflict of interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The cytoplasmic dynein motor complex transports essential signals and organelles from the cell periphery to the perinuclear region, hence is critical for the survival and function of highly polarized cells such as neurons. Dynein Light Chain Roadblock-Type 1 (DYNLRB1) is thought to be an accessory subunit required for specific cargos, but here we show that it is essential for general dynein-mediated transport and sensory neuron survival. Homozygous Dynlrb1 null mice are not viable and die during early embryonic development. Furthermore, heterozygous or adult knockdown animals display reduced neuronal growth, and selective depletion of Dynlrb1 in proprioceptive neurons compromises their survival. Conditional depletion of Dynlrb1 in sensory neurons causes deficits in several signaling pathways, including β-catenin subcellular localization, and a severe impairment in the axonal transport of both lysosomes and retrograde signaling endosomes. Hence, DYNLRB1 is an essential component of the dynein complex, and given dynein's critical functions in neuronal physiology, DYNLRB1 could have a prominent role in the etiology of human neurodegenerative diseases., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Amyotrophic lateral sclerosis (ALS) is a fatal, progressive neurodegenerative disease resulting from a complex interplay between genetics and environment. Impairments in axonal transport have been identified in several ALS models, but in vivo evidence remains limited, thus their pathogenetic importance remains to be fully resolved. We therefore analyzed the in vivo dynamics of retrogradely transported, neurotrophin-containing signaling endosomes in nerve axons of two ALS mouse models with mutations in the RNA processing genes TARDBP and FUS. TDP-43 M337V mice, which show neuromuscular pathology without motor neuron loss, display axonal transport perturbations manifesting between 1.5 and 3 months and preceding symptom onset. Contrastingly, despite 20% motor neuron loss, transport remained largely unaffected in Fus Δ14/+ mice. Deficiencies in retrograde axonal transport of signaling endosomes are therefore not shared by all ALS-linked genes, indicating that there are mechanistic distinctions in the pathogenesis of ALS caused by mutations in different RNA processing genes., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., (© 2020 Elsevier B.V. All rights reserved.)

Neurodegenerative disease encompasses a wide range of disorders afflicting the central and peripheral nervous systems and is a major unmet biomedical need of our time. There are very limited treatments, and no cures, for most of these diseases, including Alzheimer's Disease, Parkinson's Disease, Huntington Disease, and Motor Neuron Diseases. Mouse and other animal models provide hope by analysing them to understand pathogenic mechanisms, to identify drug targets, and to develop gene therapies and stem cell therapies. However, despite many decades of research, virtually no new treatments have reached the clinic. Increasingly, it is apparent that human heterogeneity within clinically defined neurodegenerative disorders, and between patients with the same genetic mutations, significantly impacts disease presentation and, potentially, therapeutic efficacy. Therefore, stratifying patients according to genetics, lifestyle, disease presentation, ethnicity, and other parameters may hold the key to bringing effective therapies from the bench to the clinic. Here, we discuss genetic and cellular humanised mouse models, and how they help in defining the genetic and environmental parameters associated with neurodegenerative disease, and so help in developing effective precision medicine strategies for future healthcare.