Ataxia Telangiectasia patient-derived neuronal and brain organoid models reveal mitochondrial dysfunction and oxidative stress.

Ataxia Telangiectasia (AT) is a rare disorder caused by mutations in the ATM gene and results in progressive neurodegeneration for reasons that remain poorly understood. In addition to its central role in nuclear DNA repair, ATM operates outside the nucleus to regulate metabolism, redox homeostasis and mitochondrial function. However, a systematic investigation into how and when loss of ATM affects these parameters in relevant human neuronal models of AT was lacking. We therefore used cortical neurons and brain organoids from AT-patient iPSC and gene corrected isogenic controls to reveal levels of mitochondrial dysfunction, oxidative stress, and senescence that vary with developmental maturity. Transcriptome analyses identified disruptions in regulatory networks related to mitochondrial function and maintenance, including alterations in the PARP/SIRT signalling axis and dysregulation of key mitophagy and mitochondrial fission-fusion processes. We further show that antioxidants reduce ROS and restore neurite branching in AT neuronal cultures, and ameliorate impaired neuronal activity in AT brain organoids. We conclude that progressive mitochondrial dysfunction and aberrant ROS production are important contributors to neurodegeneration in AT and are strongly linked to ATM's role in mitochondrial homeostasis regulation.

Deconstructing heterogeneity of replicative senescence in human mesenchymal stem cells at single cell resolution.

Following prolonged cell division, mesenchymal stem cells enter replicative senescence, a state of permanent cell cycle arrest that constrains the use of this cell type in regenerative medicine applications and that in vivo substantially contributes to organismal ageing. Multiple cellular processes such as telomere dysfunction, DNA damage and oncogene activation are implicated in promoting replicative senescence, but whether mesenchymal stem cells enter different pre-senescent and senescent states has remained unclear. To address this knowledge gap, we subjected serially passaged human ESC-derived mesenchymal stem cells (esMSCs) to single cell profiling and single cell RNA-sequencing during their progressive entry into replicative senescence. We found that esMSC transitioned through newly identified pre-senescent cell states before entering into three different senescent cell states. By deconstructing this heterogeneity and temporally ordering these pre-senescent and senescent esMSC subpopulations into developmental trajectories, we identified markers and predicted drivers of these cell states. Regulatory networks that capture connections between genes at each timepoint demonstrated a loss of connectivity, and specific genes altered their gene expression distributions as cells entered senescence. Collectively, this data reconciles previous observations that identified different senescence programs within an individual cell type and should enable the design of novel senotherapeutic regimes that can overcome in vitro MSC expansion constraints or that can perhaps slow organismal ageing.

Generation of iPSC lines from hereditary spastic paraplegia 56 (SPG56) patients and family members carrying CYP2U1 mutations.

Hereditary spastic paraplegia 56 (SPG56) is an extremely rare autosomal recessive disorder caused by mutations in the CYP2U1 gene, involved in fatty acid metabolism. SPG56 causes progressive spasticity in upper and lower limbs, though due to the rarity of this subtype of spastic paraplegia, the molecular causes remain unclear and no treatment or cure exists. Here we describe the generation and validation of induced pluripotent stem cell (iPSC) lines from two unrelated patients with SPG56 and two heterozygous family members. These lines can be used to investigate the mechanisms driving progressive spasticity and evaluate the potential for gene replacement therapies.

The hallmarks of aging in Ataxia-Telangiectasia.

Ataxia-telangiectasia (A-T) is caused by absence of the catalytic activity of ATM, a protein kinase that plays a central role in the DNA damage response, many branches of cellular metabolism, redox and mitochondrial homeostasis, and cell cycle regulation. A-T is a complex disorder characterized mainly by progressive cerebellar degeneration, immunodeficiency, radiation sensitivity, genome instability, and predisposition to cancer. It is increasingly recognized that the premature aging component of A-T is an important driver of this disease, and A-T is therefore an attractive model to study the aging process. This review outlines the current state of knowledge pertaining to the molecular and cellular signatures of aging in A-T and proposes how these new insights can guide novel therapeutic approaches for A-T.

Neural Epidermal Growth Factor-Like Like Protein 2 Is Expressed in Human Oligodendroglial Cell Types.

Neural epidermal growth factor-like like 2 (NELL2) is a cytoplasmic and secreted glycosylated protein with six epidermal growth factor-like domains. In animal models, NELL2 is predominantly expressed in neural tissues where it regulates neuronal differentiation, polarization, and axon guidance, but little is known about the role of NELL2 in human brain development. In this study, we show that rostral neural stem cells (rNSC) derived from human-induced pluripotent stem cell (hiPSC) exhibit particularly strong NELL2 expression and that NELL2 protein is enriched at the apical side of neural rosettes in hiPSC-derived brain organoids. Following differentiation of human rostral NSC into neurons, NELL2 remains robustly expressed but changes its subcellular localization from >20 small cytoplasmic foci in NSC to one-five large peri-nuclear puncta per neuron. Unexpectedly, we discovered that in human brain organoids, NELL2 is readily detectable in the oligodendroglia and that the number of NELL2 puncta increases as oligodendrocytes mature. Artificial intelligence-based machine learning further predicts a strong association of NELL2 with multiple human white matter diseases, suggesting that NELL2 may possess yet unexplored roles in regulating oligodendrogenesis and/or myelination during human cortical development and maturation.

Human induced pluripotent stem cells generated from epilepsy patients for use as in vitro models for drug screening.

In this paper, we describe the generation and validation of human induced pluripotent stem cell (hiPSC) lines from peripheral blood mononuclear cells (PBMCs) from 6 epilepsy patients using a non-integrative Sendai virus vector. These human cellular models will enable patient-specific drug screening to improve outcomes for individuals with this disorder.

Generation of induced pluripotent stem cell lines from peripheral blood mononuclear cells of three drug resistant and three drug responsive epilepsy patients.

Epilepsy is a common neurological disorder characterized by seizures. Unfortunately, 30-40% of all epilepsy patients are resistant to at least two or more anti-seizure medications. Attempts to treat these patients and prevent further seizures necessitates multiple drug trials for the patient. Here we describe the generation and validation of induced pluripotent stem cell (iPSC) lines from peripheral blood mononuclear cells (PBMCs) from 3 drug responsive and 3 drug resistant patients, using a non-integrative Sendai virus vector. These lines can be used to generate 2D and 3D patient-specific human cellular models that will enable personalised drug screening and pharmacogenomic studies.

Ataxia Telangiectasia iPSC line generated from a patient olfactory biopsy identifies novel disease-causing mutations.

Ataxia Telangiectasia is a rare autosomal recessive disorder caused by a mutated ATM gene. The most debilitating symptom of Ataxia Telangiectasia is the progressive neurodegeneration of the cerebellum, though the molecular mechanisms driving this degeneration remains unclear. Here we describe the generation and validation of an induced pluripotent stem cell (iPSC) line from an olfactory biopsy from a patient with Ataxia Telangiectasia. Sequencing identified two previously unreported disease-causing mutations in the ATM gene. This line can be used to generate 2D and 3D patient-specific neuronal models enabling investigations into the mechanisms underlying neurodegeneration.

Reprogramming of human olfactory neurosphere-derived cells from olfactory mucosal biopsies of a control cohort.

Human olfactory neurosphere-derived (ONS) cells are derived from the olfactory mucosa and display some progenitor- and neuronal cell-like properties, making them useful models of neurological disorders. However, they lack several important characteristics of true neurons, which can be overcome using induced pluripotent stem cell (iPSC) -derived neurons. Here we describe, for the first time, the generation and validation of an iPSC line from an olfactory biopsy from a control cohort member. This data lays the groundwork for future reprogramming of ONS cells, which can be used to generate neuronal models and compliment current ONS cell-based investigations into numerous neurological disorders.

Inhibition of the cGAS-STING pathway ameliorates the premature senescence hallmarks of Ataxia-Telangiectasia brain organoids.

Ataxia-telangiectasia (A-T) is a genetic disorder caused by the lack of functional ATM kinase. A-T is characterized by chronic inflammation, neurodegeneration and premature ageing features that are associated with increased genome instability, nuclear shape alterations, micronuclei accumulation, neuronal defects and premature entry into cellular senescence. The causal relationship between the detrimental inflammatory signature and the neurological deficiencies of A-T remains elusive. Here, we utilize human pluripotent stem cell-derived cortical brain organoids to study A-T neuropathology. Mechanistically, we show that the cGAS-STING pathway is required for the recognition of micronuclei and induction of a senescence-associated secretory phenotype (SASP) in A-T olfactory neurosphere-derived cells and brain organoids. We further demonstrate that cGAS and STING inhibition effectively suppresses self-DNA-triggered SASP expression in A-T brain organoids, inhibits astrocyte senescence and neurodegeneration, and ameliorates A-T brain organoid neuropathology. Our study thus reveals that increased cGAS and STING activity is an important contributor to chronic inflammation and premature senescence in the central nervous system of A-T and constitutes a novel therapeutic target for treating neuropathology in A-T patients.

Correction of ATM mutations in iPS cells from two ataxia-telangiectasia patients restores DNA damage and oxidative stress responses.

Patients with ataxia-telangiectasia (A-T) lack a functional ATM kinase protein and exhibit defective repair of DNA double-stranded breaks and response to oxidative stress. We show that CRISPR/Cas9-assisted gene correction combined with piggyBac (PB) transposon-mediated excision of the selection cassette enables seamless restoration of functional ATM alleles in induced pluripotent stem cells from an A-T patient carrying compound heterozygous exonic missense/frameshift mutations, and from a patient with a homozygous splicing acceptor mutation of an internal coding exon. We show that the correction of one allele restores expression of ~ 50% of full-length ATM protein and ameliorates DNA damage-induced activation (auto-phosphorylation) of ATM and phosphorylation of its downstream targets, KAP-1 and H2AX. Restoration of ATM function also normalizes radiosensitivity, mitochondrial ROS production and oxidative-stress-induced apoptosis levels in A-T iPSC lines, demonstrating that restoration of a single ATM allele is sufficient to rescue key ATM functions. Our data further show that despite the absence of a functional ATM kinase, homology-directed repair and seamless correction of a pathogenic ATM mutation is possible. The isogenic pairs of A-T and gene-corrected iPSCs described here constitute valuable tools for elucidating the role of ATM in ageing and A-T pathogenesis.

P2X7 receptor signaling during adult hippocampal neurogenesis.

Neurogenesis is a persistent and essential feature of the adult mammalian hippocampus. Granular neurons generated from resident pools of stem or progenitor cells provide a mechanism for the formation and consolidation of new memories. Regulation of hippocampal neurogenesis is complex and multifaceted, and numerous signaling pathways converge to modulate cell proliferation, apoptosis, and clearance of cellular debris, as well as synaptic integration of newborn immature neurons. The expression of functional P2X7 receptors in the central nervous system has attracted much interest and the regulatory role of this purinergic receptor during adult neurogenesis has only recently begun to be explored. P2X7 receptors are exceptionally versatile: in their canonical role they act as adenosine triphosphate-gated calcium channels and facilitate calcium-signaling cascades exerting control over the cell via calcium-encoded sensory proteins and transcription factor activation. P2X7 also mediates transmembrane pore formation to regulate cytokine release and facilitate extracellular communication, and when persistently stimulated by high extracellular adenosine triphosphate levels large P2X7 pores form, which induce apoptotic cell death through cytosolic ion dysregulation. Lastly, as a scavenger receptor P2X7 directly facilitates phagocytosis of the cellular debris that arises during neurogenesis, as well as during some disease states. Understanding how P2X7 receptors regulate the physiology of stem and progenitor cells in the adult hippocampus is an important step towards developing useful therapeutic models for regenerative medicine. This review considers the relevant aspects of adult hippocampal neurogenesis and explores how P2X7 receptor activity may influence the molecular physiology of the hippocampus, and neural stem and progenitor cells.

Real-time Live-cell Flow Cytometry to Investigate Calcium Influx, Pore Formation, and Phagocytosis by P2X7 Receptors in Adult Neural Progenitor Cells.

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a method whereby live-cell flow cytometry is extended upon to analyze the multiple functions of P2X7 receptor activation in real-time. Using a time module installed on a flow cytometer, live-cell functionality can be assessed and plotted over a given time period to explore the kinetics of calcium influx, transmembrane pore formation, and phagocytosis. This simple method is advantageous as all three canonical functions of the P2X7 receptor can be assessed using one machine, and the gathered data plotted over time provides information on the entire live-cell population rather than single-cell recordings often obtained using technically challenging patch-clamp methods. Calcium influx experiments use a calcium indicator dye, while P2X7 pore formation assays rely on ethidium bromide being allowed to pass through the transmembrane pore formed upon high agonist concentrations. Yellow-green (YG) latex beads are utilized to measure phagocytosis. Specific agonists and antagonists are applied to investigate the effects of P2X7 receptor activity. Individually, these methods can be modified to provide quantitative data on any number of calcium channels and purinergic and scavenger receptors. Taken together, they highlight how the use of real-time live-cell flow cytometry is a rapid, cost-effective, reproducible, and quantifiable method to investigate P2X7 receptor function.

P2X7 Receptors Regulate Phagocytosis and Proliferation in Adult Hippocampal and SVZ Neural Progenitor Cells: Implications for Inflammation in Neurogenesis.

Identifying the signaling mechanisms that regulate adult neurogenesis is essential to understanding how the brain may respond to neuro-inflammatory events. P2X7 receptors can regulate pro-inflammatory responses, and in addition to their role as cation channels they can trigger cell death and mediate phagocytosis. How P2X7 receptors may regulate adult neurogenesis is currently unclear. Here, neural progenitor cells (NPCs) derived from adult murine hippocampal subgranular (SGZ) and cerebral subventricular (SVZ) zones were utilized to characterize the roles of P2X7 in adult neurogenesis, and assess the effects of high extracellular ATP, characteristic of inflammation, on NPCs. Immunocytochemistry found NPCs in vivo and in vitro expressed P2X7, and the activity of P2X7 in culture was demonstrated using calcium influx and pore formation assays. Live cell and confocal microscopy, in conjunction with flow cytometry, revealed P2X7+ NPCs were able to phagocytose fluorescent beads, and this was inhibited by ATP, indicative of P2X7 involvement. Furthermore, P2X7 receptors were activated with ATP or BzATP, and 5-ethynyl-2'-deoxyuridine (EdU) used to observe a dose-dependent decrease in NPC proliferation. A role for P2X7 in decreased NPC proliferation was confirmed using chemical inhibition and NPCs from P2X7-/- mice. Together, these data present three distinct roles for P2X7 during adult neurogenesis, depending on extracellular ATP concentrations: (a) P2X7 receptors can form transmembrane pores leading to cell death, (b) P2X7 receptors can regulate rates of proliferation, likely via calcium signaling, and (c) P2X7 can function as scavenger receptors in the absence of ATP, allowing NPCs to phagocytose apoptotic NPCs during neurogenesis. Stem Cells 2018;36:1764-1777.