Cerebral organoids with chromosome 21 trisomy secrete Alzheimer's disease-related soluble aggregates detectable by single-molecule-fluorescence and super-resolution microscopy.

Understanding the role of small, soluble aggregates of beta-amyloid (Aß) and tau in Alzheimer's disease (AD) is of great importance for the rational design of preventative therapies. Here we report a set of methods for the detection, quantification, and characterisation of soluble aggregates in conditioned media of cerebral organoids derived from human iPSCs with trisomy 21, thus containing an extra copy of the amyloid precursor protein (APP) gene. We detected soluble beta-amyloid (Aß) and tau aggregates secreted by cerebral organoids from both control and the isogenic trisomy 21 (T21) genotype. We developed a novel method to normalise measurements to the number of live neurons within organoid-conditioned media based on glucose consumption. Thus normalised, T21 organoids produced 2.5-fold more Aß aggregates with a higher proportion of larger (300-2000 nm2) and more fibrillary-shaped aggregates than controls, along with 1.3-fold more soluble phosphorylated tau (pTau) aggregates, increased inflammasome ASC-specks, and a higher level of oxidative stress inducing thioredoxin-interacting protein (TXNIP). Importantly, all this was detectable prior to the appearance of histological amyloid plaques or intraneuronal tau-pathology in organoid slices, demonstrating the feasibility to model the initial pathogenic mechanisms for AD in-vitro using cells from live genetically pre-disposed donors before the onset of clinical disease. Then, using different iPSC clones generated from the same donor at different times in two independent experiments, we tested the reproducibility of findings in organoids. While there were differences in rates of disease progression between the experiments, the disease mechanisms were conserved. Overall, our results show that it is possible to non-invasively follow the development of pathology in organoid models of AD over time, by monitoring changes in the aggregates and proteins in the conditioned media, and open possibilities to study the time-course of the key pathogenic processes taking place.

Patient-specific Alzheimer-like pathology in trisomy 21 cerebral organoids reveals BACE2 as a gene dose-sensitive AD suppressor in human brain.

A population of more than six million people worldwide at high risk of Alzheimer's disease (AD) are those with Down Syndrome (DS, caused by trisomy 21 (T21)), 70% of whom develop dementia during lifetime, caused by an extra copy of ß-amyloid-(Aß)-precursor-protein gene. We report AD-like pathology in cerebral organoids grown in vitro from non-invasively sampled strands of hair from 71% of DS donors. The pathology consisted of extracellular diffuse and fibrillar Aß deposits, hyperphosphorylated/pathologically conformed Tau, and premature neuronal loss. Presence/absence of AD-like pathology was donor-specific (reproducible between individual organoids/iPSC lines/experiments). Pathology could be triggered in pathology-negative T21 organoids by CRISPR/Cas9-mediated elimination of the third copy of chromosome 21 gene BACE2, but prevented by combined chemical ß and γ-secretase inhibition. We found that T21 organoids secrete increased proportions of Aß-preventing (Aß1-19) and Aß-degradation products (Aß1-20 and Aß1-34). We show these profiles mirror in cerebrospinal fluid of people with DS. We demonstrate that this protective mechanism is mediated by BACE2-trisomy and cross-inhibited by clinically trialled BACE1 inhibitors. Combined, our data prove the physiological role of BACE2 as a dose-sensitive AD-suppressor gene, potentially explaining the dementia delay in ~30% of people with DS. We also show that DS cerebral organoids could be explored as pre-morbid AD-risk population detector and a system for hypothesis-free drug screens as well as identification of natural suppressor genes for neurodegenerative diseases.

Attoampere Nanoelectrochemistry.

Electrochemical microscopy techniques have extended the understanding of surface chemistry to the micrometer and even sub-micrometer level. However, fundamental questions related to charge transport at the solid-electrolyte interface, such as catalytic reactions or operation of individual ion channels, require improved spatial resolutions down to the nanoscale. A prerequisite for single-molecule electrochemical sensitivity is the reliable detection of a few electrons per second, that is, currents in the atto-Ampere (10-18 A) range, 1000 times below today's electrochemical microscopes. This work reports local cyclic voltammetry (CV) measurements at the solid-liquid interface on ferrocene self-assembled monolayer (SAM) with sub-atto-Ampere sensitivity and simultaneous spatial resolution < 80 nm. Such sensitivity is obtained through measurements of the charging of the local faradaic interface capacitance at GHz frequencies. Nanometer-scale details of different molecular organizations with a 19% packing density difference are resolved, with an extremely small dispersion of the molecular electrical properties. This is predicted previously based on weak electrostatic interactions between neighboring redox molecules in a SAM configuration. These results open new perspectives for nano-electrochemistry like the study of quantum mechanical resonance in complex molecules and a wide range of applications from electrochemical catalysis to biophysics.

High-Sensitivity Dual Electrochemical QCM for Reliable Three-Electrode Measurements.

An electrochemical quartz crystal microbalance (EC-QCM) is a versatile gravimetric technique that allows for parallel characterization of mass deposition and electrochemical properties. Despite its broad applicability, simultaneous characterization of two electrodes remains challenging due to practical difficulties posed by the dampening from fixture parasitics and the dissipative medium. In this study, we present a dual electrochemical QCM (dual EC-QCM) that is employed in a three-electrode configuration to enable consequent monitoring of mass deposition and viscous loading on two crystals, the working electrode (WE) and the counter electrode (CE). A novel correction approach, along with a three standard complex impedance calibration, is employed to overcome the effect of dampening while keeping high spectral sensitivity. Separation of viscous loading and rigid mass deposition is achieved by robust characterization of the complex impedance at the resonance frequency. Validation of the presented system is done by cyclic voltammetry characterization of Ag underpotential deposition on gold. The results indicate mass deposition of 412.2 ng for the WE and 345.6 ng for the CE, reflecting a difference of the initially-present Ag adhered to the surface. We also performed higher harmonic measurements that further corroborate the sensitivity and reproducibility of the dual EC-QCM. The demonstrated approach is especially intriguing for electrochemical energy storage applications where mass detection with multiple electrodes is desired.

Dose imbalance of DYRK1A kinase causes systemic progeroid status in Down syndrome by increasing the un-repaired DNA damage and reducing LaminB1 levels.

BACKGROUND: People with Down syndrome (DS) show clinical signs of accelerated ageing. Causative mechanisms remain unknown and hypotheses range from the (essentially untreatable) amplified-chromosomal-instability explanation, to potential actions of individual supernumerary chromosome-21 genes. The latter explanation could open a route to therapeutic amelioration if the specific over-acting genes could be identified and their action toned-down. METHODS: Biological age was estimated through patterns of sugar molecules attached to plasma immunoglobulin-G (IgG-glycans, an established "biological-ageing-clock") in n = 246 individuals with DS from three European populations, clinically characterised for the presence of co-morbidities, and compared to n = 256 age-, sex- and demography-matched healthy controls. Isogenic human induced pluripotent stem cell (hiPSCs) models of full and partial trisomy-21 with CRISPR-Cas9 gene editing and two kinase inhibitors were studied prior and after differentiation to cerebral organoids. FINDINGS: Biological age in adults with DS is (on average) 18.4-19.1 years older than in chronological-age-matched controls independent of co-morbidities, and this shift remains constant throughout lifespan. Changes are detectable from early childhood, and do not require a supernumerary chromosome, but are seen in segmental duplication of only 31 genes, along with increased DNA damage and decreased levels of LaminB1 in nucleated blood cells. We demonstrate that these cell-autonomous phenotypes can be gene-dose-modelled and pharmacologically corrected in hiPSCs and derived cerebral organoids. Using isogenic hiPSC models we show that chromosome-21 gene DYRK1A overdose is sufficient and necessary to cause excess unrepaired DNA damage. INTERPRETATION: Explanation of hitherto observed accelerated ageing in DS as a developmental progeroid syndrome driven by DYRK1A overdose provides a target for early pharmacological preventative intervention strategies. FUNDING: Main funding came from the "Research Cooperability" Program of the Croatian Science Foundation funded by the European Union from the European Social Fund under the Operational Programme Efficient Human Resources 2014-2020, Project PZS-2019-02-4277, and the Wellcome Trust Grants 098330/Z/12/Z and 217199/Z/19/Z (UK). All other funding is described in details in the "Acknowledgements".

Development of a Solid and Flexible Matching Medium for Microwave Medical Diagnostic Systems.

This paper reports the development of a new composite material as a matching medium for medical microwave diagnostic systems, where maximizing the microwave energy that penetrates the interrogated tissue is critical for improving the quality of the diagnostic images. The proposed material has several advantages over what is commonly used in microwave diagnostic systems: it is semi-flexible and rigid, and it can maximize microwave energy coupling by matching the tissue's dielectric constant without introducing high loss. The developed matching medium is a mirocomposite of barium titanate filler in polydimethylsiloxane (PDMS) in different weight-based mixing ratios. Dielectric properties of the material are measured using a Keysight open-ended coaxial slim probe from 0.5 to 10 GHz. To avoid systematic errors, a full dielectric properties calibration is performed before measurements of sample materials. Furthermore, the repeatability of the measurements and the homogeneity of the sample of interest are considered. Finally, to evaluate the proposed matching medium, its impact on a printed monopole antenna is studied. We demonstrate that the permittivity of the investigated mixtures can be increased in a controlled manner to reach values that have been previously shown to be optimal for medical microwave imaging (MWI) such as stroke and breast cancer diagnostic applications. As a result, the material is a good candidate for supporting antenna arrays designed for portable MWI scanners in applications such as stroke detection.

RAS-protein activation but not mutation status is an outcome predictor and unifying therapeutic target for high-risk acute lymphoblastic leukemia.

Leukemias are routinely sub-typed for risk/outcome prediction and therapy choice using acquired mutations and chromosomal rearrangements. Down syndrome acute lymphoblastic leukemia (DS-ALL) is characterized by high frequency of CRLF2-rearrangements, JAK2-mutations, or RAS-pathway mutations. Intriguingly, JAK2 and RAS-mutations are mutually exclusive in leukemic sub-clones, causing dichotomy in therapeutic target choices. We prove in a cell model that elevated CRLF2 in combination with constitutionally active JAK2 is sufficient to activate wtRAS. On primary clinical DS-ALL samples, we show that wtRAS-activation is an obligatory consequence of mutated/hyperphosphorylated JAK2. We further prove that CRLF2-ligand TSLP boosts the direct binding of active PTPN11 to wtRAS, providing the molecular mechanism for the wtRAS activation. Pre-inhibition of RAS or PTPN11, but not of PI3K or JAK-signaling, prevented TSLP-induced RAS-GTP boost. Cytotoxicity assays on primary clinical DS-ALL samples demonstrated that, regardless of mutation status, high-risk leukemic cells could only be killed using RAS-inhibitor or PTPN11-inhibitor, but not PI3K/JAK-inhibitors, suggesting a unified treatment target for up to 80% of DS-ALL. Importantly, protein activities-based principal-component-analysis multivariate clusters analyzed for independent outcome prediction using Cox proportional-hazards model showed that protein-activity (but not mutation-status) was independently predictive of outcome, demanding a paradigm-shift in patient-stratification strategy for precision therapy in high-risk ALL.

Modeling Down syndrome in cells: From stem cells to organoids.

Down Syndrome (DS) is a complex chromosomal disorder, with neurological issues, featuring among the symptoms. Primary neuronal cells and tissues are extremely useful, but limited both in supply and experimental manipulability. To better understand the cellular, molecular and pathological mechanisms involved in DS neurodevelopment and neurodegeneration, a range of different cellular models have been developed over the years including human: mouse hybrid cells, transchromosomic mouse embryonic stem cells (ESCs) and human ESC and induced pluripotent stem cells derived from different sources. All of these model systems have provided useful information in the study of DS. Furthermore, different technologies to genetically modify or correct trisomy of either single genes or the whole chromosome have been developed using these cellular models. New techniques and protocols to allow better modeling of cellular mechanisms and disease processes are being developed and the use of cerebral organoids offers great promise for future research into the neural phenotypes seen in DS.

Stroke promotes survival of nearby transplanted neural stem cells by decreasing their activation of caspase 3 while not affecting their differentiation.

Although transplantation of stem cells improves recovery of the nervous tissue, little is known about the influence of different brain regions on transplanted cells. After we confirmed that cells with uniform differentiation potential can be generated in independent experiments, one million of neural stem cells isolated from B6.Cg-Tg(Thy1-YFP)16Jrs/J mouse embryos were transplanted into the brain 24 h after induction of stroke. The lateral ventricles, the corpus callosum and the striatum were tested. Two and four weeks after the transplantation, the cells transplanted in all three regions have been attracted to the ischemic core. The largest number of attracted cells has been observed after transplantation into the striatum. Their differentiation pattern and expression of neuroligin 1, SynCAM 1, postsynaptic density protein 95 and synapsin 1 followed the same pattern observed during in vitro cultivation and it did not differ among the tested regions. Differentiation pattern of the cells transplanted in the stroke-affected and healthy animals was the same. On the other hand, neural stem cells transplanted in the striatum of the animals affected by stroke exhibited significantly increased survival rates reaching 260 ±â€¯19%, when compared to cells transplanted in their wild type controls. Surprisingly, improved survival two and four weeks after transplantation was not due to increased proliferation of the grafted cells and it was accompanied by decreased levels of activity of Casp3 (19.56 ±â€¯3.1% in the stroke-affected vs. 30.14 ±â€¯2.4% in healthy animals after four weeks). We assume that the decreased levels of Casp3 in cells transplanted near the ischemic region was linked to increased vasculogenesis, synaptogenesis, astrocytosis and axonogenesis detected in the host tissue affected by ischemia.

Stem cells and stroke-how glowing neurons illuminate new paths.

A reliable method of cell tracing is essential in evaluating potential therapeutic procedures based on stem cell transplantation. Here we present data collected using neural stem cells isolated from a transgenic mouse line Thy1-YFP. When transplanted into a stroke affected brain these cells give rise to neurons that express a fluorescent signal which can be used for their detection and tracing. Observed processes were compared with those taking place during normal embryonic neurogenesis as well as during in vitro differentiation. Since the same neurogenic patterns were observed, we confirm that neural stem cell transplantation fits well into the paradigm of neuronal birth and differentiation.

Neural stem cells from mouse strain Thy1 YFP-16 are a valuable tool to monitor and evaluate neuronal differentiation and morphology.

To analyse events following transplantation of stem cells in the brain robust tools for tracing stem cells are required. Here we took advantage of the mouse strain B6.Cg-Tg(Thy1-YFP)16Jrs/J (Thy1 YFP-16), where yellow fluorescent protein (YFP) is under control of the promoter of Thy1 gene. This allows visualising whole neurons, i.e. their cell body, axons and dendrites. In this work fluorescent cells were followed during embryonic development, in vitro differentiation, and after transplantation in the healthy and stroke-affected mouse brain. During embryonic development Thy1-YFP positive cells were first observed on E12.5 and subsequently located in the prosencephalon, rhombencephalon, spinal cord and peripheral nerves. Quantitative analysis by RT-PCR and immunocytochemistry revealed that Thy1-YFP positive cells during embryo development and in vitro differentiation were expressing nestin and SOX2 then MAP2, ß3-tubulin and NeuN. Thy1-YFP positive cells isolated from E14.5 represented 21.88±053% (SD) of the cultivated neurons and this remained constant along in vitro differentiation. On the other hand, proportion of Thy1-YFP positive cells reached 50% of neurons in perinatal and one month old mouse brain. Neural stem cells isolated from Thy1 YFP-16 mouse strain transplanted near hippocampus of the healthy and stroke-affected brain were distinguishable by YFP fluorescence. They differentiated into mature neurons and were detectable even 14 weeks after transplantation, the end point of our experiment. In conclusion, stem cells originating from Thy1 YFP-16 mice represent an outstanding tool to monitor neurogenesis enabling morphological analyses of new neurons and their projections, in particular after transplantation in the brain.

Nop2 is expressed during proliferation of neural stem cells and in adult mouse and human brain.

The nucleolar protein 2 gene encodes a protein specific for the nucleolus. It is assumed that it plays a role in the synthesis of ribosomes and regulation of the cell cycle. Due to its link to cell proliferation, higher expression of Nop2 indicates a worse tumor prognosis. In this work we used Nop2(gt1gaj) gene trap mouse strain. While lethality of homozygous animals suggested a vital role of this gene, heterozygous animals allowed the detection of expression of Nop2 in various tissues, including mouse brain. Histochemistry, immunohistochemistry and immunoelectron microscopy techniques, applied to a mature mouse brain, human brain and on mouse neural stem cells revealed expression of Nop2 in differentiating cells, including astrocytes, as well as in mature neurons. Nop2 was detected in various regions of mouse and human brain, mostly in large pyramidal neurons. In the human, Nop2 was strongly expressed in supragranular and infragranular layers of the somatosensory cortex and in layer III of the cingulate cortex. Also, Nop2 was detected in CA1 and the subiculum of the hippocampus. Subcellular analyses revealed predominant location of Nop2 within the dense fibrillar component of the nucleolus. To test if Nop2 expression correlates to cell proliferation occurring during tissue regeneration, we induced strokes in mice by middle cerebral artery occlusion. Two weeks after stroke, the number of Nop2/nestin double positive cells in the region affected by ischemia and the periventricular zone substantially increased. Our findings suggest a newly discovered role of Nop2 in both mature neurons and in cells possibly involved in the regeneration of nervous tissue.