Disruption of MAM integrity in mutant FUS oligodendroglial progenitors from hiPSCs.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and fatal neurodegenerative disorder, characterized by selective loss of motor neurons (MNs). A number of causative genetic mutations underlie the disease, including mutations in the fused in sarcoma (FUS) gene, which can lead to both juvenile and late-onset ALS. Although ALS results from MN death, there is evidence that dysfunctional glial cells, including oligodendroglia, contribute to neurodegeneration. Here, we used human induced pluripotent stem cells (hiPSCs) with a R521H or a P525L mutation in FUS and their isogenic controls to generate oligodendrocyte progenitor cells (OPCs) by inducing SOX10 expression from a TET-On SOX10 cassette. Mutant and control iPSCs differentiated efficiently into OPCs. RNA sequencing identified a myelin sheath-related phenotype in mutant OPCs. Lipidomic studies demonstrated defects in myelin-related lipids, with a reduction of glycerophospholipids in mutant OPCs. Interestingly, FUSR521H OPCs displayed a decrease in the phosphatidylcholine/phosphatidylethanolamine ratio, known to be associated with maintaining membrane integrity. A proximity ligation assay further indicated that mitochondria-associated endoplasmic reticulum membranes (MAM) were diminished in both mutant FUS OPCs. Moreover, both mutant FUS OPCs displayed increased susceptibility to ER stress when exposed to thapsigargin, and exhibited impaired mitochondrial respiration and reduced Ca2+ signaling from ER Ca2+ stores. Taken together, these results demonstrate a pathological role of mutant FUS in OPCs, causing defects in lipid metabolism associated with MAM disruption manifested by impaired mitochondrial metabolism with increased susceptibility to ER stress and with suppressed physiological Ca2+ signaling. As such, further exploration of the role of oligodendrocyte dysfunction in the demise of MNs is crucial and will provide new insights into the complex cellular mechanisms underlying ALS.

Spatially Self-Organized Three-Dimensional Neural Concentroid as a Novel Reductionist Humanized Model to Study Neurovascular Development.

Although human pluripotent stem cell (PSC)-derived brain organoids have enabled researchers to gain insight into human brain development and disease, these organoids contain solely ectodermal cells and are not vascularized as occurs during brain development. Here it is created less complex and more homogenous large neural constructs starting from PSC-derived neuroprogenitor cells (NPC), by fusing small NPC spheroids into so-called concentroids. Such concentroids consisted of a pro-angiogenic core, containing neuronal and outer radial glia cells, surrounded by an astroglia-dense outer layer. Incorporating PSC-derived endothelial cells (EC) around and/or in the concentroids promoted vascularization, accompanied by differential outgrowth and differentiation of neuronal and astroglia cells, as well as the development of ectodermal-derived pericyte-like mural cells co-localizing with EC networks. Single nucleus transcriptomic analysis revealed an enhanced neural cell subtype maturation and diversity in EC-containing concentroids, which better resemble the fetal human brain compared to classical organoids or NPC-only concentroids. This PSC-derived "vascularized" concentroid brain model will facilitate the study of neurovascular/blood-brain barrier development, neural cell migration, and the development of effective in vitro vascularization strategies of brain mimics.

CRISPR/Cas9 screen in human iPSC-derived cortical neurons identifies NEK6 as a novel disease modifier of C9orf72 poly(PR) toxicity.

INTRODUCTION: The most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are hexanucleotide repeats in chromosome 9 open reading frame 72 (C9orf72). These repeats produce dipeptide repeat proteins with poly(PR) being the most toxic one. METHODS: We performed a kinome-wide CRISPR/Cas9 knock-out screen in human induced pluripotent stem cell (iPSC) -derived cortical neurons to identify modifiers of poly(PR) toxicity, and validated the role of candidate modifiers using in vitro, in vivo, and ex-vivo studies. RESULTS: Knock-down of NIMA-related kinase 6 (NEK6) prevented neuronal toxicity caused by poly(PR). Knock-down of nek6 also ameliorated the poly(PR)-induced axonopathy in zebrafish and NEK6 was aberrantly expressed in C9orf72 patients. Suppression of NEK6 expression and NEK6 activity inhibition rescued axonal transport defects in cortical neurons from C9orf72 patient iPSCs, at least partially by reversing p53-related DNA damage. DISCUSSION: We identified NEK6, which regulates poly(PR)-mediated p53-related DNA damage, as a novel therapeutic target for C9orf72 FTD/ALS.

Toward point-of-care assessment of patient response: a portable tool for rapidly assessing cancer drug efficacy using multifrequency impedance cytometry and supervised machine learning.

We present a novel method to rapidly assess drug efficacy in targeted cancer therapy, where antineoplastic agents are conjugated to antibodies targeting surface markers on tumor cells. We have fabricated and characterized a device capable of rapidly assessing tumor cell sensitivity to drugs using multifrequency impedance spectroscopy in combination with supervised machine learning for enhanced classification accuracy. Currently commercially available devices for the automated analysis of cell viability are based on staining, which fundamentally limits the subsequent characterization of these cells as well as downstream molecular analysis. Our approach requires as little as 20 µL of volume and avoids staining allowing for further downstream molecular analysis. To the best of our knowledge, this manuscript presents the first comprehensive attempt to using high-dimensional data and supervised machine learning, particularly phase change spectra obtained from multi-frequency impedance cytometry as features for the support vector machine classifier, to assess viability of cells without staining or labelling.

Acceptability of Mental Health Facilities and De-addiction Centers in India.

Not much is known about disease prevalence, treatment outcomes, trained manpower, programs, and patients' awareness of diseases from South Asia, compared with the Western world. While other aspects are improving, the quantitative evaluation of awareness of diseases is lagging. Compared with other diseases, the situation for mental health disorders and addiction is worse. While no single study can fully quantify all aspects of awareness, a good starting point is to understand if increasing the number of mental health facilities is beneficial by understanding people's perception toward the likelihood of contracting various diseases, their preferred approach to treatment, and their perception of whether there are enough current facilities. We surveyed over 8000 families across several states of India and asked if they would treat a particular problem at home, visit a local healer, seek religious council, or go to a modern hospital for treatment. Our questions also included non-medical options to assess how likely people are to avoid trained medical help. We also asked people about their perceived likelihood of a family member ever suffering from (1) diarrhea, (2) high fever, (3) alcoholism, and (4) schizophrenia and other mental health problems. We reversed the order of diseases in our questions for a fraction of the population to evaluate the effect of order of questioning. Finally, we asked, if people feel they have enough local healers, religious places, general hospitals, de-addiction centers, and mental health facilities. Despite the taboo around mental health, many people claimed that their family members were unlikely to contract mental health or addiction problems, people recognized the severe paucity of mental health facilities and de-addiction centers. This raises hope for improving the mental health situation in India. We also found a significant relation between education levels and choices people make, underscoring the positive role education has in improving mental health.

Fully integrated wearable impedance cytometry platform on flexible circuit board with online smartphone readout.

We present a wearable microfluidic impedance cytometer implemented on a flexible circuit wristband with on-line smartphone readout for portable biomarker counting and analysis. The platform contains a standard polydimethylsiloxane (PDMS) microfluidic channel integrated on a wristband, and the circuitry on the wristband is composed  of a custom analog lock-in amplification system, a microcontroller with an 8-bit analog-to-digital converter (ADC), and a Bluetooth module wirelessly paired with a smartphone. The lock-in amplification (LIA) system is implemented with a novel architecture which consists of the lock-in amplifier followed by a high-pass filter stage with DC offset subtraction, and a post-subtraction high gain stage enabling detection of particles as small as 2.8  µm using the 8-bit ADC. The Android smartphone application was used to initiate the system and for offline data-plotting and peak counting, and supports online data readout, analysis, and file management. The data is exportable to researchers and medical professionals for in-depth analysis and remote health monitoring. The system, including the microfluidic sensor, microcontroller, and Bluetooth module all fit on the wristband with a footprint of less than 80 cm2. We demonstrate the ability of the system to obtain generalized blood cell counts; however the system can be applied to a wide variety of biomarkers by interchanging the standard microfluidic channel with microfluidic channels designed for biomarker isolation.