Targeting specificity of APOBEC-based cytosine base editor in human iPSCs determined by whole genome sequencing.

DNA base editors have enabled genome editing without generating DNA double strand breaks. The applications of this technology have been reported in a variety of animal and plant systems, however, their editing specificity in human stem cells has not been studied by unbiased genome-wide analysis. Here we investigate the fidelity of cytidine deaminase-mediated base editing in human induced pluripotent stem cells (iPSCs) by whole genome sequencing after sustained or transient base editor expression. While base-edited iPSC clones without significant off-target modifications are identified, this study also reveals the potential of APOBEC-based base editors in inducing unintended point mutations outside of likely in silico-predicted CRISPR-Cas9 off-targets. The majority of the off-target mutations are C:G->T:A transitions or C:G->G:C transversions enriched for the APOBEC mutagenesis signature. These results demonstrate that cytosine base editor-mediated editing may result in unintended genetic modifications with distinct patterns from that of the conventional CRISPR-Cas nucleases.

Chronic poly-drug administration damages adult mouse brain neural stem cells.

Cocaine and ethanol are two commonly co-abused substances; however, the neuropathology following chronic dual consumption is poorly understood. Neural stem cells (NSCs) are a subpopulation of cells within the adult brain that are integral to brain maintenance and repair making them an appealing target to reverse neurodegeneration associated with abused substances. Yet, knowledge about NSC response to chronic poly-drug administration of ethanol and cocaine is minimal. Here, we developed a novel chronic poly-drug administration paradigm of ethanol and cocaine using a transgenic mouse model to trace endogenous NSC survival and differentiation in three brain regions from both male and female mice. We report significant and distinct patterns of NSC survival and differentiation among brain regions, as well as between sexes. Additionally, poly-drug administration had synergistic effects on NSC survival. Altered cognitive and hedonic behaviors were also observed, however the extent of these behavioral changes was not proportional to the NSC changes. With this mouse model we can effectively examine cognitive and behavioral changes and correlate them with pathological changes in the brain in response to chronic poly-drug administration, which is of great value in understanding the progression of neurodegeneration in polysubstance use disorders and evaluation potential therapeutics on neuroregeneration.

Student Evaluation of Interprofessional Experiences between Medical and Graduate Biomedical Students.

BACKGROUND: Interprofessional education (IPE) has fostered increased collaboration and appreciation for different disciplines among health professionals but has yet to be established in a translational research setting. Interprofessional experiences (IPEx) implemented early in student training could increase translational research productivity. METHODS AND FINDINGS: Ten students involved in an IPE curriculum wrote autoethnographic accounts that were coded and emergent themes were grouped through constant comparative analysis. IPE led to improvements in communication, trust, appreciation, and an increased desire to seek IPE in future careers. Challenges included administrative barriers and interpersonal conflicts. CONCLUSIONS: Participants found IPE beneficial to their careers and developed a respect for each other's discipline. To implement IPE, institutions should consider possible administrative challenges and inclusion of conflict management training.

Oligodendrocyte differentiation from human neural stem cells: A novel role for c-Src.

Human neural stem cells (hNSCs) can differentiate into an oligodendrocyte lineage to facilitate remyelination in patients. Molecular mechanisms underlying oligodendrocyte fate specification remains unknown, hindering the development of efficient methods to generate oligodendrocytes from hNSCs. We have found that Neurobasal-A medium (NB) is capable of inducing hNSCs to oligodendrocyte progenitor cells (OPCs). We identified several signaling molecules are altered after cultivation in NB medium, including Akt, ERK1/2 and c-Src. While sustained activation of Akt and ERK1/2 during both NB induction and subsequent differentiation was required for OPC differentiation, c-Src phosphorylation was increased temporally during the period of NB induction. Both pharmacological inhibition and RNA interference confirmed that a transient elevation of phospho-c-Src is critical for OPC induction. Furthermore, inactivation of c-Src inhibited phosphorylation of Akt and ERK1/2. In summary, we identified a novel and critical role of c-Src in guiding hNSC differentiation to an oligodendrocyte lineage.

Zika Virus Infection of Cultured Human Fetal Brain Neural Stem Cells for Immunocytochemical Analysis.

Human fetal brain neural stem cells are a unique non-genetically modified model system to study the impact of various stimuli on human developmental neurobiology. Rather than use an animal model or genetically modified induced pluripotent cells, human neural stem cells provide an effective in vitro system to examine the effects of treatments, screen drugs, or examine individual differences. Here, we provide the detailed protocols for methods used to expand human fetal brain neural stem cells in culture with serum-free media, to differentiate them into various neuronal subtypes and astrocytes via different priming procedures, and to freeze and recover these cells. Furthermore, we describe a procedure of using human fetal brain neural stem cells to study Zika virus infection.

Spatial and Sex-Dependent Responses of Adult Endogenous Neural Stem Cells to Alcohol Consumption.

Chronic alcohol abuse results in alcohol-related neurodegeneration, and critical gaps in our knowledge hinder therapeutic development. Neural stem cells (NSCs) are a subpopulation of cells within the adult brain that contribute to brain maintenance and recovery. While it is known that alcohol alters NSCs, little is known about how NSC response to alcohol is related to sex, brain region, and stage of differentiation. Understanding these relationships will aid in therapeutic development. Here, we used an inducible transgenic mouse model to track the stages of differentiation of adult endogenous NSCs and observed distinct NSC behaviors in three brain regions (subventricular zone, subgranular zone, and tanycyte layer) after long-term alcohol consumption. Particularly, chronic alcohol consumption profoundly affected the survival of NSCs in the subventricular zone and altered NSC differentiation in all three regions. Significant differences between male and female mice were further discovered.

Differential Responses of Human Fetal Brain Neural Stem Cells to Zika Virus Infection.

Zika virus (ZIKV) infection causes microcephaly in a subset of infants born to infected pregnant mothers. It is unknown whether human individual differences contribute to differential susceptibility of ZIKV-related neuropathology. Here, we use an Asian-lineage ZIKV strain, isolated from the 2015 Mexican outbreak (Mex1-7), to infect primary human neural stem cells (hNSCs) originally derived from three individual fetal brains. All three strains of hNSCs exhibited similar rates of Mex1-7 infection and reduced proliferation. However, Mex1-7 decreased neuronal differentiation in only two of the three stem cell strains. Correspondingly, ZIKA-mediated transcriptome alterations were similar in these two strains but significantly different from that of the third strain with no ZIKV-induced neuronal reduction. This study thus confirms that an Asian-lineage ZIKV strain infects primary hNSCs and demonstrates a cell-strain-dependent response of hNSCs to ZIKV infection.

Microcephaly in Zika Virus Infection.

Zika virus is a flavivirus known to cause microcephaly during development. The mechanism underlying Zika virus-induced neuropathogenesis is still poorly understood. Recent studies have utilized the cutting edge cell culture and animal model technologies to elucidate factors contributing to Zika virus-associated microcephaly. While future work is needed, current studies have suggested three main factors that contribute to Zika virus pathology: viral lineage, host immunity, and pregnancy stages. This mini review will focus on some of the recent findings that advanced our knowledge in Zika virus-associated microcephaly.

Aged dominant negative p38α MAPK mice are resistant to age-dependent decline in adult-neurogenesis and context discrimination fear conditioning.

A major aspect of mammalian aging is the decline in functional competence of many self-renewing cell types, including adult-born neuronal precursors. Since age-related senescence of self-renewal occurs simultaneously with chronic up-regulation of the p38MAPKalpha (p38α) signaling pathway, we used the dominant negative mouse model for attenuated p38α activity (DN-p38αAF/+) in which Thr180 and Tyr182 are mutated (T→A/Y→F) to prevent phosphorylation activation (DN-p38αAF/+) and kinase activity. As a result, aged DN-p38αAF/+ mice are resistant to age-dependent decline in proliferation and regeneration of several peripheral tissue progenitors when compared to wild-type littermates. Aging is the major risk factor for non-inherited forms of Alzheimer's disease (AD); environmental and genetic risk factors that accelerate the senescence phenotype are thought to contribute to an individual's relative risk. In the present study, we evaluated aged DN-p38αAF/+ and wildtype littermates in a series of behavioral paradigms to test if p38α mutant mice exhibit altered baseline abnormalities in neurological reflexes, locomotion, anxiety-like behavior, and age-dependent cognitive decline. While aged DN-p38αAF/+ and wildtype littermates appear equal in all tested baseline neurological and behavioral parameters, DN-p38αAF/+ exhibit superior context discrimination fear conditioning. Context discrimination is a cognitive task that is supported by proliferation and differentiation of adult-born neurons in the dentate gyrus of the hippocampus. Consistent with enhanced context discrimination in aged DN-p38αAF/+, we discovered enhanced production of adult-born neurons in the dentate gyrus of DN-p38αAF/+ mice compared to wildtype littermates. Our findings support the notion that p38α inhibition has therapeutic utility in aging diseases that affect cognition, such as AD.

A Screen of FDA-Approved Drugs for Inhibitors of Zika Virus Infection.

Currently there are no approved vaccines or specific therapies to prevent or treat Zika virus (ZIKV) infection. We interrogated a library of FDA-approved drugs for their ability to block infection of human HuH-7 cells by a newly isolated ZIKV strain (ZIKV MEX_I_7). More than 20 out of 774 tested compounds decreased ZIKV infection in our in vitro screening assay. Selected compounds were further validated for inhibition of ZIKV infection in human cervical, placental, and neural stem cell lines, as well as primary human amnion cells. Established anti-flaviviral drugs (e.g., bortezomib and mycophenolic acid) and others that had no previously known antiviral activity (e.g., daptomycin) were identified as inhibitors of ZIKV infection. Several drugs reduced ZIKV infection across multiple cell types. This study identifies drugs that could be tested in clinical studies of ZIKV infection and provides a resource of small molecules to study ZIKV pathogenesis.