HDAC6 inhibition as a mechanism to prevent neurodegeneration in the mSOD1 mouse model of ALS.

The loss of upper and lower motor neurons, and their axons is central to the loss of motor function and death in amyotrophic lateral sclerosis (ALS). Due to the diverse range of genetic and environmental factors that contribute to the pathogenesis of ALS, there have been difficulties in developing effective therapies for ALS. One emerging dichotomy is that protection of the neuronal cell soma does not prevent axonal vulnerability and degeneration, suggesting the need for targeted therapeutics to prevent axon degeneration.

Experimental laboratory models as tools for understanding modifiable dementia risk.

Experimental laboratory research has an important role to play in dementia prevention. Mechanisms underlying modifiable risk factors for dementia are promising targets for dementia prevention but are difficult to investigate in human populations due to technological constraints and confounds. Therefore, controlled laboratory experiments in models such as transgenic rodents, invertebrates and in vitro cultured cells are increasingly used to investigate dementia risk factors and test strategies which target them to prevent dementia.

Deep Learning-Based Identification of Intraocular Pressure-Associated Genes Influencing Trabecular Meshwork Cell Morphology.

Purpose: Genome-wide association studies have recently uncovered many loci associated with variation in intraocular pressure (IOP). Artificial intelligence (AI) can be used to interrogate the effect of specific genetic knockouts on the morphology of trabecular meshwork cells (TMCs) and thus, IOP regulation.

Design: Experimental study.

Induced pluripotent stem cell derived pericytes respond to mediators of proliferation and contractility.

Background: Pericytes are multifunctional contractile cells that reside on capillaries. Pericytes are critical regulators of cerebral blood flow and blood-brain barrier function, and pericyte dysfunction may contribute to the pathophysiology of human neurological diseases including Alzheimers disease, multiple sclerosis, and stroke. Induced pluripotent stem cell (iPSC)-derived pericytes (iPericytes) are a promising tool for vascular research.

The role of altered protein acetylation in neurodegenerative disease.

Acetylation is a key post-translational modification (PTM) involved in the regulation of both histone and non-histone proteins. It controls cellular processes such as DNA transcription, RNA modifications, proteostasis, aging, autophagy, regulation of cytoskeletal structures, and metabolism. Acetylation is essential to maintain neuronal plasticity and therefore essential for memory and learning.

Seeing Neurodegeneration in a New Light Using Genetically Encoded Fluorescent Biosensors and iPSCs.

Neurodegenerative diseases present a progressive loss of neuronal structure and function, leading to cell death and irrecoverable brain atrophy. Most have disease-modifying therapies, in part because the mechanisms of neurodegeneration are yet to be defined, preventing the development of targeted therapies. To overcome this, there is a need for tools that enable a quantitative assessment of how cellular mechanisms and diverse environmental conditions contribute to disease.

Lysosomal alterations and decreased electrophysiological activity in CLN3 disease patient-derived cortical neurons.

CLN3 disease is a lysosomal storage disorder associated with fatal neurodegeneration that is caused by mutations in CLN3, with most affected individuals carrying at least one allele with a 966 bp deletion. Using CRISPR/Cas9, we corrected the 966 bp deletion mutation in human induced pluripotent stem cells (iPSCs) of a compound heterozygous patient (CLN3 Δ 966 bp and E295K). We differentiated these isogenic iPSCs, and iPSCs from an unrelated healthy control donor, to neurons and identified disease-related changes relating to protein synthesis, trafficking and degradation, and in neuronal activity, which were not apparent in CLN3-corrected or healthy control neurons.

Single-cell eQTL mapping identifies cell type-specific genetic control of autoimmune disease.

The human immune system displays substantial variation between individuals, leading to differences in susceptibility to autoimmune disease. We present single-cell RNA sequencing (scRNA-seq) data from 1,267,758 peripheral blood mononuclear cells from 982 healthy human subjects. For 14 cell types, we identified 26,597 independent cis-expression quantitative trait loci (eQTLs) and 990 trans-eQTLs, with most showing cell type-specific effects on gene expression.

Generation of MNZTASi001-A, a human pluripotent stem cell line from a person with primary progressive multiple sclerosis.

Multiple sclerosis (MS) is a chronic autoimmune and neurodegenerative disease that results in immune cell infiltration of the central nervous system (CNS) and demyelination in young adults. Substantial progress has been made in developing disease modifying therapies for people with relapsing-remitting MS, but options remain limited for people with primary progressive MS (PPMS). PPMS accounts for ∼15% of MS diagnoses.

Image-Based Quantitation of Kainic Acid-Induced Excitotoxicity as a Model of Neurodegeneration in Human iPSC-Derived Neurons.

Excitotoxicity is a feature of many neurodegenerative diseases and acquired forms of neural injury that is characterized by disruption of neuronal morphology. This is typically seen as beading and fragmentation of neurites when exposed to excitotoxins such as the AMPA receptor agonist kainic acid, with the extent to which these occur used to quantitate neurodegeneration. Induced pluripotent stem cells (iPSCs) provide a means to generate human neurons in vitro for mechanistic studies and can thereby be used to investigate how cells respond to excitotoxicity and to identify or test potential neuroprotective agents.

Culture Variabilities of Human iPSC-Derived Cerebral Organoids Are a Major Issue for the Modelling of Phenotypes Observed in Alzheimer's Disease.

Apolipoprotein E (APOE) is the most important susceptibility gene for late onset of Alzheimer's disease (AD), with the presence of APOE-ε4 associated with increased risk of developing AD. Here, we reprogrammed human fibroblasts from individuals with different APOE-ε genotypes into induced pluripotent stem cells (iPSCs), and generated isogenic lines with different APOE profiles. Following characterisation of the newly established iPSC lines, we used an unguided/unpatterning differentiation method to generate six-month-old cerebral organoids from all iPSC lines to assess the suitability of this in vitro system to measure APOE, β amyloid, and Tau phosphorylation levels.

Single cell eQTL analysis identifies cell type-specific genetic control of gene expression in fibroblasts and reprogrammed induced pluripotent stem cells.

Background: The discovery that somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) has provided a foundation for in vitro human disease modelling, drug development and population genetics studies. Gene expression plays a critical role in complex disease risk and therapeutic response. However, while the genetic background of reprogrammed cell lines has been shown to strongly influence gene expression, the effect has not been evaluated at the level of individual cells which would provide significant resolution.

Use of CRISPR/Cas ribonucleoproteins for high throughput gene editing of induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) have become widely used for disease modelling, particularly with regard to predisposing genetic risk factors and causal gene variants. Alongside this, technologies such as the CRISPR/Cas system have been adapted to enable programmable gene editing in human cells. When combined, CRISPR/Cas gene editing of donor-specific iPSC to generate isogenic cell lines that differ only at specific gene variants provides a powerful model with which to investigate genetic variants associated with diseases affecting many organs, including the brain and eye.

Approaches for the sensitive detection of rare base and prime editing events.

Precision chemistry entailing user-directed nucleotide substitutions and template-specified repair can be facilitated by base editing and prime editing, respectively. Recently, the diversification of adenine, cytosine, and prime editor variants obliges a considered, high-throughput evaluation of these tools for optimized, end-point applications. Herein, we outline novel, cost-effective and scalable approaches for the rapid detection of base editing and prime editing outcomes using gel electrophoresis.

Comparison of CRISPR/Cas Endonucleases for Retinal Gene Editing.

CRISPR/Cas has opened the prospect of direct gene correction therapy for some inherited retinal diseases. Previous work has demonstrated the utility of adeno-associated virus (AAV) mediated delivery to retinal cells ; however, with the expanding repertoire of CRISPR/Cas endonucleases, it is not clear which of these are most efficacious for retinal editing . We sought to compare CRISPR/Cas endonuclease activity using both single and dual AAV delivery strategies for gene editing in retinal cells.

A Simple Differentiation Protocol for Generation of Induced Pluripotent Stem Cell-Derived Basal Forebrain-Like Cholinergic Neurons for Alzheimer's Disease and Frontotemporal Dementia Disease Modeling.

The study of neurodegenerative diseases using pluripotent stem cells requires new methods to assess neurodevelopment and neurodegeneration of specific neuronal subtypes. The cholinergic system, characterized by its use of the neurotransmitter acetylcholine, is one of the first to degenerate in Alzheimer's disease and is also affected in frontotemporal dementia. We developed a differentiation protocol to generate basal forebrain-like cholinergic neurons (BFCNs) from induced pluripotent stem cells (iPSCs) aided by the use of small molecule inhibitors and growth factors.

If Human Brain Organoids Are the Answer to Understanding Dementia, What Are the Questions?

Because our beliefs regarding our individuality, autonomy, and personhood are intimately bound up with our brains, there is a public fascination with cerebral organoids, the "mini-brain," the "brain in a dish". At the same time, the ethical issues around organoids are only now being explored. What are the prospects of using human cerebral organoids to better understand, treat, or prevent dementia? Will human organoids represent an improvement on the current, less-than-satisfactory, animal models? When considering these questions, two major issues arise.

Utility of Self-Destructing CRISPR/Cas Constructs for Targeted Gene Editing in the Retina.

Safe delivery of CRISPR/Cas endonucleases remains one of the major barriers to the widespread application of genome editing. We previously reported the utility of adeno-associated virus (AAV)-mediated CRISPR/Cas genome editing in the retina; however, with this type of viral delivery system, active endonucleases will remain in the retina for an extended period, making genotoxicity a significant consideration in clinical applications. To address this issue, we have designed a self-destructing "kamikaze" CRISPR/Cas system that disrupts the Cas enzyme itself following expression.

Screening of CRISPR/Cas base editors to target the AMD high-risk Y402H complement factor H variant.

Purpose: To evaluate the efficacy of using a CRISPR/Cas-mediated strategy to correct a common high-risk allele that is associated with age-related macular degeneration (AMD; rs1061170; NM_000186.3:c.1204T>C; NP_000177.

NLRP3-Dependent and -Independent Processing of Interleukin (IL)-1β in Active Ulcerative Colitis.

A contributing factor in the development of ulcerative colitis (UC) and Crohn's disease (CD) is the disruption of innate and adaptive signaling pathways due to aberrant cytokine production. The cytokine, interleukin (IL)-1β, is highly inflammatory and its production is tightly regulated through transcriptional control and both inflammasome-dependent and inflammasome- independent proteolytic cleavage. In this study, qRT-PCR, immunohistochemistry, immunofluorescence confocal microscopy were used to (1) assess the mRNA expression of and in paired biopsies from UC and CD patient, and (2) the colonic localization and spatial relationship of NLRP3 and IL-1β in active and quiescent disease.