Viral-free generation and characterization of a human induced pluripotent stem cell line from dermal fibroblasts

Peripheral dermal fibroblasts (DF) from a healthy 56 year old female were obtained from the Centre for Healthy Brain Ageing (CHeBA) Biobank, University of New South Wales, under the material transfer agreement with the University of Wollongong. DFs were reprogrammed via mRNA-delivered transcription factors into induced pluripotent stem cells (iPSCs). The generated iPSCs were confirmed to be pluripotent, capable of three germ layer differentiation and are thus a useful resource for creating iPSC-derived healthy human cells of any lineage

The mRNA-based reprogramming of fibroblasts from a SOD1\u3csup\u3eE101G\u3c/sup\u3e familial amyotrophic lateral sclerosis patient to induced pluripotent stem cell line UOWi007

2020 The Authors Dermal fibroblasts were donated by a 43 year old male patient with clinically diagnosed familial amyotrophic lateral sclerosis (ALS), carrying the SOD1E101G mutation. The induced pluripotent stem cell (iPSC) line UOWi007-A was generated using repeated mRNA transfections for pluripotency transcription factors Oct4, Klf4, Sox2, c-Myc, Lin28 and Nanog. The iPSCs carried the SOD1E101G genotype and had a normal karyotype, expressed expected pluripotency markers and were capable of in vitro differentiation into endodermal, mesodermal and ectodermal lineages. This iPSC line may be useful for investigating familial ALS resulting from a SOD1 E101G mutation

Selective ferroptosis vulnerability due to familial Alzheimer's disease presenilin mutations

Mutations in presenilin 1 and 2 (PS1 and PS2) cause autosomal dominant familial Alzheimer's disease (FAD). Ferroptosis has been implicated as a mechanism of neurodegeneration in AD since neocortical iron burden predicts Alzheimer's disease (AD) progression. We found that loss of the presenilins dramatically sensitizes multiple cell types to ferroptosis, but not apoptosis. FAD causal mutations of presenilins similarly sensitizes cells to ferroptosis. The presenilins promote the expression of GPX4, the selenoprotein checkpoint enzyme that blocks ferroptosis by quenching the membrane propagation of lethal hydroperoxyl radicals. Presenilin gamma-secretase activity cleaves Notch-1 to signal LRP8 expression, which then controls GPX4 expression by regulating the supply of selenium into the cell since LRP8 is the uptake receptor for selenoprotein P. Selenium uptake is thus disrupted by presenilin FAD mutations, suppressing GPX4 expression. Therefore, presenilin mutations may promote neurodegeneration by derepressing ferroptosis, which has implications for disease-modifying therapeutics.Peer reviewe

The Ubiquitin Proteasome System Is a Key Regulator of Pluripotent Stem Cell Survival and Motor Neuron Differentiation

The ubiquitin proteasome system (UPS) plays an important role in regulating numerous cellular processes, and a dysfunctional UPS is thought to contribute to motor neuron disease. Consequently, we sought to map the changing ubiquitome in human iPSCs during their pluripotent stage and following differentiation to motor neurons. Ubiquitinomics analysis identified that spliceosomal and ribosomal proteins were more ubiquitylated in pluripotent stem cells, whilst proteins involved in fatty acid metabolism and the cytoskeleton were specifically ubiquitylated in the motor neurons. The UPS regulator, ubiquitin-like modifier activating enzyme 1 (UBA1), was increased 36-fold in the ubiquitome of motor neurons compared to pluripotent stem cells. Thus, we further investigated the functional consequences of inhibiting the UPS and UBA1 on motor neurons. The proteasome inhibitor MG132, or the UBA1-specific inhibitor PYR41, significantly decreased the viability of motor neurons. Consistent with a role of the UPS in maintaining the cytoskeleton and regulating motor neuron differentiation, UBA1 inhibition also reduced neurite length. Pluripotent stem cells were extremely sensitive to MG132, showing toxicity at nanomolar concentrations. The motor neurons were more resilient to MG132 than pluripotent stem cells but demonstrated higher sensitivity than fibroblasts. Together, this data highlights the important regulatory role of the UPS in pluripotent stem cell survival and motor neuron differentiation

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. One is the general challenge associated with using any stem cell-generated preparation for in vitro modelling (challenges amplified when using organoids compared with simpler cell culture systems). The other relates to complexities associated with defining and understanding what we mean by the term "dementia." We discuss 10 puzzles, issues, and stumbling blocks to watch for in the quest to model "dementia in a dish."The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The Australian Dementia Stem Cell Consortium has received generous start-up travel grants from the Australian NHMRC National Institute for Dementia Research. Authors have been supported by Dementia Australia Research Foundation, Yulgilbar Alzheimer’s Research Program, DHB Foundation (AP), Brain Foundation (DH, AP), the C.F. Leung Memorial Trust (AP), the University of Melbourne (AP) and Operational Infrastructure Support from the Victorian Government (DH, AP), Monash University (AG), JO and JR Wicking Trust (Equity Trustees) (ALC and AEK), University of Sydney (MV), and generous gifts from the Sinclair, Smith and Jolly families (MV). AEK is supported by a National Health and Medical Research Council (NHMRC) of Australia Boosting Dementia Research Leadership Fellowship (APP1136913). AG is supported by a NHMRC-ARC Dementia Research Development Fellowship (GNT1097461). AP is supported by an ARC Future Fellowship (FT140100047) and a NHMRC Senior Research Fellowship (1154389). LO is supported by a NHMRC of Australia Boosting Dementia Research Leadership Fellowship (APP1135720). MV is supported by a NHMRC Career Development Fellowship (APP1112813). VG is supported by Australian Research Council’s Discovery Early Career Researcher Award (DE180100775)

Getting to NO Alzheimer\u27s disease: neuroprotection versus neurotoxicity mediated by nitric oxide

Alzheimer\u27s disease (AD) is a neurodegenerative disorder involving the loss of neurons in the brain which leads to progressive memory loss and behavioral changes. To date, there are only limited medications for AD and no known cure. Nitric oxide (NO) has long been considered part of the neurotoxic insult caused by neuroinflammation in the Alzheimer\u27s brain. However, focusing on early developments, prior to the appearance of cognitive symptoms, is changing that perception. This has highlighted a compensatory, neuroprotective role for NO that protects synapses by increasing neuronal excitability. A potential mechanism for augmentation of excitability by NO is via modulation of voltage-gated potassium channel activity (Kv7 and Kv2). Identification of the ionic mechanisms and signaling pathways that mediate this protection is an important next step for the field. Harnessing the protective role of NO and related signaling pathways could provide a therapeutic avenue that prevents synapse loss early in disease

How a trip to Antarctica became a real-life experiment in decision-making

We were part of a group of 77 women travelling by ship to an Antarctic research station when our route was blocked by icebergs. We had to make a decision. Should we detour into rough open ocean to reach the target site, or abandon plans to visit Rothera Research Station and settle instead for a few days of exploring Antarctica\u27s calmer, protected waters? This is the story of Rothera-gate , a leadership development experience on the largest all-female expedition to Antarctica. The 2018 expedition was the culmination of a year-long strategic leadership initiative for women scientists called Homeward Bound. Men typically hold the leadership positions in STEMM (Science, Technology, Engineering, Mathematics and Medicine). In recognition of this, the Homeward Bound initiative works with women in science to enhance their opportunity to take up leadership roles globally, and contribute proactively to a sustainable world

Neuroprotection of Neuro2a cells and the cytokine suppressive and anti-inflammatory mode of action of resveratrol in activated RAW264.7 macrophages and C8-B4 microglia

Chronic inflammation is a hallmark of neurodegenerative disease and cytotoxic levels of nitric oxide (NO) and pro-inflammatory cytokines can initiate neuronal death pathways. A range of cellular assays were used to assess the anti-inflammatory and neuroprotective action of resveratrol using murine microglial (C8-B4), macrophage (RAW264.7) and neuronal-like (Neuro2a) cell lines. We examined the release of NO by Griess assay and used a Bioplex array to measure a panel of pro- and anti-inflammatory cytokines and chemokines, in response to the inflammatory stimuli lipopolysaccharide (LPS) and interferon-γ (IFN-γ). Resveratrol was a potent inhibitor of NO and cytokine release in activated macrophages and microglia. The activity of resveratrol increased marginally in potency with longer pre-incubation times in cell culture that was not due to cytotoxicity. Using an NO donor we show that resveratrol can protect Neuro2a cells from cytotoxic concentrations of NO. The protective effect of resveratrol from pro-inflammatory signalling in RAW264.7 cells was confirmed in co-culture experiments leading to increased survival of Neuro2a cells. Together our data are indicative of the potential neuroprotective effect of resveratrol during nitrosative stress and neuroinflammation

Identification and High-Resolution Imaging of alpha-Tocopherol from Human Cells to Whole Animals by TOF-SIMS Tandem Mass Spectrometry

A unique method for identification of biomolecular components in different biological specimens, while preserving the capability for high speed 2D and 3D molecular imaging, is employed to investigate cellular response to oxidative stress. The employed method enables observing the distribution of the antioxidant α-tocopherol and other molecules in cellular structures via time-of-flight secondary ion mass spectrometry (TOF-SIMS (MS1)) imaging in parallel with tandem mass spectrometry (MS2) imaging, collected simultaneously. The described method is employed to examine a network formed by neuronal cells differentiated from human induced pluripotent stem cells (iPSCs), a model for investigating human neurons in vitro. The antioxidant α-tocopherol is identified in situ within different cellular layers utilizing a 3D TOF-SIMS tandem MS imaging analysis. As oxidative stress also plays an important role in mediating inflammation, the study was expanded to whole body tissue sections of M. marinum-infected zebrafish, a model organism for tuberculosis. The TOF-SIMS tandem MS imaging results reveal an increased presence of α-tocopherol in response to the pathogen