Identification of Adeno-Associated Virus Variants for Gene Transfer Into Human Neural Cell Types by Parallel Capsid Screening

Human brain cells generated by in vitrocell programming provide exciting prospects for disease modeling, drug discovery and cell therapy. These applications frequently require efficient and clinically compliant tools for genetic modification of the cells. Recombinant Adeno-associated viruses (AAVs) fulfill these prerequisites for a number of reasons, including the availability of a myriad of AAV capsid variants with distinct cell type specificity (also called tropism). Here, weharnessed a customizable parallel screening approach to assessa panel of natural or synthetic AAV capsid variants for their efficacy in lineage-related human neural cell types.We identified common lead candidates suited for the transduction of directly converted,early-stage induced neural stem cells (iNSCs), induced pluripotent stem cell (iPSC)-derived later-stage, radial glia-like neuralprogenitors,as well as differentiated astrocytic and mixed neuroglial cultures.We then selected a subsetof these candidates for functional validation in iNSCs and iPSC-derived astrocytes, usingshRNA-induced downregulation of the citrate transporter SLC25A1 and overexpression of the transcription factor NGN2 for proofs-of-concept. Our study provides a comparative overview ofthe susceptibility of different human cell programming-derived brain cell types to AAV transduction and a critical discussionof the assets and limitations of the specific AAV capsidscreening approach.

Expanding the MECP2 network using comparative genomics reveals potential therapeutic targets for Rett syndrome

Inactivating mutations in the Methyl-CpG Binding Protein 2 (MECP2) gene are the main cause of Rett syndrome (RTT). Despite extensive research into MECP2 function, no treatments for RTT are currently available. Here, we used an evolutionary genomics approach to construct an unbiased MECP2 gene network, using 1028 eukaryotic genomes to prioritize proteins with strong co-evolutionary signatures with MECP2. Focusing on proteins targeted by FDA-approved drugs led to three promising targets, two of which were previously linked to MECP2 function (IRAK, KEAP1) and one that was not (EPOR). The drugs targeting these three proteins (Pacritinib, DMF, and EPO) were able to rescue different phenotypes of MECP2 inactivation in cultured human neural cell types, and appeared to converge on Nuclear Factor Kappa B (NF-κB) signaling in inflammation. This study highlights the potential of comparative genomics to accelerate drug discovery, and yields potential new avenues for the treatment of RTT.

Oral Pathogenic Bacteria-Inducing Neurodegenerative Microgliosis in Human Neural Cell Platform

Porphyromonas gingivalis is a gram-negative bacterium found in the human oral cavity and is responsible for the development of chronic periodontitis as well as neurological diseases, including Alzheimer’s disease (AD). Given the significance of the roles of P. gingivalis in AD pathogenesis, it is critical to understand the underlying mechanisms of P. gingivalis-driven neuroinflammation and their contribution to neurodegeneration. Herein, we hypothesize that P. gingivalis produces secondary metabolites that may cause neurodegeneration through direct or indirect pathways mediated by microglia. To test our hypothesis, we treated human neural cells with bacterial conditioned media on our brain platforms and assessed microgliosis, astrogliosis and neurodegeneration. We found that bacteria-mediated microgliosis induced the production of nitric oxide, which causes neurodegeneration assessed with high pTau level. Our study demonstrated the elevation of detrimental protein mediators, CD86 and iNOS and the production of several pro-inflammatory markers from stimulated microglia. Through inhibition of LPS and succinate dehydrogenase in a bacterial conditioned medium, we showed a decrease in neurodegenerative microgliosis. In addition, we demonstrated the bidirectional effect of microgliosis and astrogliosis on each other exacerbating neurodegeneration. Overall, our study suggests that the mouth-brain axis may contribute to the pathogenesis of AD.

Expanding the MECP2 network using comparative genomics reveals potential therapeutic targets for Rett syndrome

Inactivating mutations in the Methyl-CpG Binding Protein 2 (MECP2) gene are the main cause of Rett syndrome (RTT). Despite extensive research into MECP2 function, no treatments for RTT are currently available. Here we use an evolutionary genomics approach to construct an unbiased MECP2 gene network, using 1,028 eukaryotic genomes to prioritize proteins with strong co-evolutionary signatures with MECP2. Focusing on proteins targeted by FDA approved drugs led to three promising candidates, two of which were previously linked to MECP2 function (IRAK, KEAP1) and one that was not (EPOR). We show that each of these compounds has the ability to rescue different phenotypes of MECP2 inactivation in cultured human neural cell types, and appear to act on Nuclear Factor Kappa B (NF-κB) signaling in inflammation. This study highlights the potential of comparative genomics to accelerate drug discovery, and yields potential new avenues for the treatment of RTT.Abstract Figure

Machine learning identifies candidates for drug repurposing in Alzheimer’s disease

Clinical trials of novel therapeutics for Alzheimer’s Disease (AD) have consumed a large amount of time and resources with largely negative results. Repurposing drugs already approved by the Food and Drug Administration (FDA) for another indication is a more rapid and less expensive option. We present DRIAD (Drug Repurposing In AD), a machine learning framework that quantifies potential associations between the pathology of AD severity (the Braak stage) and molecular mechanisms as encoded in lists of gene names. DRIAD is applied to lists of genes arising from perturbations in differentiated human neural cell cultures by 80 FDA-approved and clinically tested drugs, producing a ranked list of possible repurposing candidates. Top-scoring drugs are inspected for common trends among their targets. We propose that the DRIAD method can be used to nominate drugs that, after additional validation and identification of relevant pharmacodynamic biomarker(s), could be readily evaluated in a clinical trial.

Effect of amlodipine on cytosolic calcium and apoptosis in hyperglycemia induced-human neuronal cell line culture

Objective Diabetic neuropathy is complication of diabetes which is believed to be caused by improper Ca2+ (calcium) signaling and activation of apoptotic process of neuron of dorsal root ganglia. Amlodipine, antagonist Ca2+, has been already known to has neuroprotective effect in vitro or in vivo. This research investigates the effect of amlodipine in cytosolic calcium level and apoptosis in SH-SY5Y human neural cell line after exposure of chronic hyperglycemia. The study design was an experimental study using human neuronal cell line SH-SY5Y, exposed by chronic hyperglycemia for 6 days with concentration of glucose 25 µM (normoglycemia) and 50 µM (hyperglycemia), then was added amlodipine 2 µM for 30 minutes. Results In this study, hyperglycemia increased calcium concentration and caspase-3 compared with normoglycemia (p = 0.004 and p = 0.001 respectively). There was significant difference (p = 0.015) between calcium concentration in hyperglycemia induced cell line after given amlodipine 2 µM compared without amlodipine. There was significant difference (p = 0.027) between caspase 3 level in hyperglycemia induced cell line after given amlodipine 2 µM and without amlodipine. Administration of amlodipine significantly reduced cytosolic calcium and caspase-3 level in hyperglycemia induced SH-SY5Y human neural cell lines.