Robert Krencik

Zika virus cell tropism in the developing human brain and inhibition by azithromycin

Significance Zika virus (ZIKV) is a mosquito-borne flavivirus that has rapidly spread through the Americas and has been associated with fetal abnormalities, including microcephaly. To understand how microcephaly develops, it is important to identify which cell types of the developing brain are susceptible to infection. We use primary human tissue to show that radial glia and astrocytes are more susceptible to infection than neurons, a pattern that correlates with expression of a putative viral entry receptor, AXL. We also perform a screen of Food and Drug Administration-approved compounds, with an emphasis on drugs known to be safe in pregnancy. We identify an antibiotic, azithromycin, that reduces viral proliferation in glial cells, and compare its activity with daptomycin and sofosbuvir, two additional drugs with anti-ZIKV activity. The rapid spread of Zika virus (ZIKV) and its association with abnormal brain development constitute a global health emergency. Congenital ZIKV infection produces a range of mild to severe pathologies, including microcephaly. To understand the pathophysiology of ZIKV infection, we used models of the developing brain that faithfully recapitulate the tissue architecture in early to midgestation. We identify the brain cell populations that are most susceptible to ZIKV infection in primary human tissue, provide evidence for a mechanism of viral entry, and show that a commonly used antibiotic protects cultured brain cells by reducing viral proliferation. In the brain, ZIKV preferentially infected neural stem cells, astrocytes, oligodendrocyte precursor cells, and microglia, whereas neurons were less susceptible to infection. These findings suggest mechanisms for microcephaly and other pathologic features of infants with congenital ZIKV infection that are not explained by neural stem cell infection alone, such as calcifications in the cortical plate. Furthermore, we find that blocking the glia-enriched putative viral entry receptor AXL reduced ZIKV infection of astrocytes in vitro, and genetic knockdown of AXL in a glial cell line nearly abolished infection. Finally, we evaluate 2,177 compounds, focusing on drugs safe in pregnancy. We show that the macrolide antibiotic azithromycin reduced viral proliferation and virus-induced cytopathic effects in glial cell lines and human astrocytes. Our characterization of infection in the developing human brain clarifies the pathogenesis of congenital ZIKV infection and provides the basis for investigating possible therapeutic strategies to safely alleviate or prevent the most severe consequences of the epidemic.

Dysregulation of astrocyte extracellular signaling in Costello syndrome

Excessive HRAS signaling in Costello syndrome accelerates Ras-specific astroglial development and extracellular matrix accumulation, resulting in premature cortical circuit maturation. Perineuronal nets close the door on brain development Creating a brain is exceedingly complex, so mutations in many different genes can cause the process to go awry. Mutations in one sensitive pathway, Ras signaling, cause a family of neurodevelopmental disorders called RASopathies. Krencik et al. have used patient-derived iPSCs and mutant mice to demonstrate that the mutation in HRAS that causes one RASopathy—Costello syndrome—acts by accelerating astrocyte differentiation. The authors examined astroglial development from patient iPSCs in vitro and, similarly, astroglial development in vivo in mice carrying the same mutation as that found in Costello syndrome patients. The authors suggest that premature formation of perineuronal nets around neurons may close the experience-dependent development of the brain, producing the typical intellectual disability in Costello syndrome patients. Astrocytes produce an assortment of signals that promote neuronal maturation according to a precise developmental timeline. Is this orchestrated timing and signaling altered in human neurodevelopmental disorders? To address this question, the astroglial lineage was investigated in two model systems of a developmental disorder with intellectual disability caused by mutant Harvey rat sarcoma viral oncogene homolog (HRAS) termed Costello syndrome: mutant HRAS human induced pluripotent stem cells (iPSCs) and transgenic mice. Human iPSCs derived from patients with Costello syndrome differentiated to astroglia more rapidly in vitro than those derived from wild-type cell lines with normal HRAS, exhibited hyperplasia, and also generated an abundance of extracellular matrix remodeling factors and proteoglycans. Acute treatment with a farnesyl transferase inhibitor and knockdown of the transcription factor SNAI2 reduced expression of several proteoglycans in Costello syndrome iPSC-derived astrocytes. Similarly, mice in which mutant HRAS was expressed selectively in astrocytes exhibited experience-independent increased accumulation of perineuronal net proteoglycans in cortex, as well as increased parvalbumin expression in interneurons, when compared to wild-type mice. Our data indicate that astrocytes expressing mutant HRAS dysregulate cortical maturation during development as shown by abnormal extracellular matrix remodeling and implicate excessive astrocyte-to-neuron signaling as a possible drug target for treating mental impairment and enhancing neuroplasticity.

A cellular star atlas: using astrocytes from human pluripotent stem cells for disease studies

What roles do astrocytes play in human disease?This question remains unanswered for nearly every human neurological disorder. Yet, because of their abundance and complexity astrocytes can impact neurological function in many ways. The differentiation of human pluripotent stem cells (hPSCs) into neuronal and glial subtypes, including astrocytes, is becoming routine, thus their use as tools for modeling neurodevelopment and disease will provide one important approach to answer this question. When designing experiments, careful consideration must be given to choosing paradigms for differentiation, maturation, and functional analysis of these temporally asynchronous cellular populations in culture. In the case of astrocytes, they display heterogeneous characteristics depending upon species of origin, brain region, developmental stage, environmental factors, and disease states, all of which may render experimental results highly variable. In this review, challenges and future directions are discussed for using hPSC-derived astroglial progenitors and mature astrocytes for neurodevelopmental studies with a focus on exploring human astrocyte effects upon neuronal function. As new technologies emerge to measure the functions of astrocytes in vitro and in vivo, there is also a need for a standardized source of human astrocytes that are most relevant to the diseases of interest.

Efficient generation of region-specific forebrain neurons from human pluripotent stem cells under highly defined condition.

Human pluripotent stem cells (hPSCs) have potential to differentiate to unlimited number of neural cells, which provide powerful tools for neural regeneration. To date, most reported protocols were established with an animal feeder system. However, cells derived on this system are inappropriate for the translation to clinical applications because of the introduction of xenogenetic factors. In this study, we provided an optimized paradigm to generate region-specific forebrain neurons from hPSCs under a defined system. We assessed five conditions and found that a vitronectin-coated substrate was the most efficient method to differentiate hPSCs to neurons and astrocytes. More importantly, by applying different doses of purmorphamine, a small-molecule agonist of sonic hedgehog signaling, hPSCs were differentiated to different region-specific forebrain neuron subtypes, including glutamatergic neurons, striatal medium spiny neurons, and GABA interneurons. Our study offers a highly defined system without exogenetic factors to produce human neurons and astrocytes for translational medical studies, including cell therapy and stem cell-based drug discovery.

Zika Virus in the Human Placenta and Developing Brain: Cell Tropism and Drug Inhibition

The rapid spread of Zika virus (ZIKV) and its association with abnormal brain development constitute a global health emergency. Congenital ZIKV infection produces a range of mild to severe pathologies, including placental damage and microcephaly. However, the placenta’s role in viral transmission and the mechanisms of microcephaly have not been addressed in primary human tissues. Moreover, there is an urgent need for drugs that can prevent developmental defects following infection. Here, we identify the placental and brain cell populations most susceptible to ZIKV infection, provide evidence for a mechanism of viral entry, and show that a commonly used antibiotic protects cultured brain cells by inhibiting viral proliferation. In the early gestation placenta, the virus readily infected trophoblast subpopulations that are in direct contact with maternal blood and uterine cells, suggesting routes of ZIKV transmission to the embryo and fetus. In the brain, ZIKV preferentially infected neural stem cells, astrocytes, and microglia, whereas neurons were less susceptible to infection. These findings suggest mechanisms for microcephaly and other pathologic features of infants with congenital ZIKV infection that are not explained by neural stem cell infection alone, such as calcifications in the cortical plate and brain abnormalities caused by third trimester infection. Blocking a putative viral entry receptor, AXL, which is highly enriched in the infected placenta and brain cell types, reduced ZIKV infection of astrocytes in vitro. In a glial cell line, the macrolide antibiotic, azithromycin, inhibited viral proliferation and viral-induced cytopathic effects at clinically relevant concentrations. Our characterization of infection in primary human tissues clarifies the pathogenesis of congenital ZIKV infection and provides critical context for interpreting results from model systems. Further work on azithromycin and related compounds may yield additional therapeutic strategies to safely alleviate or prevent the most severe consequences of the epidemic.

Human astrocytes are distinct contributors to the complexity of synaptic function

Cellular components of synaptic circuits have been adjusted for increased human brain size, neural cell density, energy consumption and developmental duration. How does the human brain make these accommodations? There is evidence that astrocytes are one of the most divergent neural cell types in primate brain evolution and it is now becoming clear that they have critical roles in controlling synaptic development, function and plasticity. Yet, we still do not know how the precise developmental appearance of these cells and subsequent astrocyte-derived signals modulate diverse neuronal circuit subtypes. Here, we discuss what is currently known about the influence of glial factors on synaptic maturation and focus on unique features of human astrocytes including their potential roles in regenerative and translational medicine. Human astrocyte distinctiveness may be a major contributor to high level neuronal processing of the human brain and act in novel ways during various neuropathies ranging from autism spectrum disorders, viral infection, injury and neurodegenerative conditions.

Systematic Three-Dimensional Coculture Rapidly Recapitulates Interactions between Human Neurons and Astrocytes

Summary Human astrocytes network with neurons in dynamic ways that are still poorly defined. Our ability to model this relationship is hampered by the lack of relevant and convenient tools to recapitulate this complex interaction. To address this barrier, we have devised efficient coculture systems utilizing 3D organoid-like spheres, termed asteroids, containing pre-differentiated human pluripotent stem cell (hPSC)-derived astrocytes (hAstros) combined with neurons generated from hPSC-derived neural stem cells (hNeurons) or directly induced via Neurogenin 2 overexpression (iNeurons). Our systematic methods rapidly produce structurally complex hAstros and synapses in high-density coculture with iNeurons in precise numbers, allowing for improved studies of neural circuit function, disease modeling, and drug screening. We conclude that these bioengineered neural circuit model systems are reliable and scalable tools to accurately study aspects of human astrocyte-neuron functional properties while being easily accessible for cell-type-specific manipulations and observations.

Human stem cell-derived astrocytes replicate human prions in a PRNP genotype-dependent manner

Prions are infectious agents that cause neurodegenerative diseases such as Creutzfeldt–Jakob disease (CJD). The absence of a human cell culture model that replicates human prions has hampered prion disease research for decades. In this paper, we show that astrocytes derived from human induced pluripotent stem cells (iPSCs) support the replication of prions from brain samples of CJD patients. For experimental exposure of astrocytes to variant CJD (vCJD), the kinetics of prion replication occur in a prion protein codon 129 genotype–dependent manner, reflecting the genotype-dependent susceptibility to clinical vCJD found in patients. Furthermore, iPSC-derived astrocytes can replicate prions associated with the major sporadic CJD strains found in human patients. Lastly, we demonstrate the subpassage of prions from infected to naive astrocyte cultures, indicating the generation of prion infectivity in vitro. Our study addresses a long-standing gap in the repertoire of human prion disease research, providing a new in vitro system for accelerated mechanistic studies and drug discovery.

Patient-derived iPSCs show premature neural differentiation and neuron type-specific phenotypes relevant to neurodevelopment

Ras/MAPK pathway signaling is a major participant in neurodevelopment, and evidence suggests that BRAF, a key Ras signal mediator, influences human behavior. We studied the role of the mutation BRAFQ257R, the most common cause of cardiofaciocutaneous syndrome (CFC), in an induced pluripotent stem cell (iPSC)-derived model of human neurodevelopment. In iPSC-derived neuronal cultures from CFC subjects, we observed decreased p-AKT and p-ERK1/2 compared to controls, as well as a depleted neural progenitor pool and rapid neuronal maturation. Pharmacological PI3K/AKT pathway manipulation recapitulated cellular phenotypes in control cells and attenuated them in CFC cells. CFC cultures displayed altered cellular subtype ratios and increased intrinsic excitability. Moreover, in CFC cells, Ras/MAPK pathway activation and morphological abnormalities exhibited cell subtype-specific differences. Our results highlight the importance of exploring specific cellular subtypes and of using iPSC models to reveal relevant human-specific neurodevelopmental events.

Concepts toward directing human astroplasticity to promote neuroregeneration: Promoting Neuroregeneration through Astrocytes

Astrocytes exhibit dynamic and complex reactions to various insults. Recently, investigations into the transitions that occur during cellular specification, differentiation, maturation, and disease responses have provided insights into understanding the mechanisms that underlie these altered states of reactivity and function. Here we summarize current concepts in how astrocyte state transitions, termed astroplasticity, are regulated, as well as how this affects neural circuit function through extracellular signaling. We postulate that a promising future approach toward enhancing functional repair after injury and disease would be to steer astrocytes away from an inhibitory response and toward one that is beneficial to neuroplasticity and neuroregeneration. Toward this goal, we discuss emerging biotechnological advancements, with a focus on human pluripotent stem cell bioengineering, which has high potential for effective manipulation and control of astroplasticity. Highlights include innovations in cellular transdifferentiation techniques, nanomedicine, organoid and three‐dimensional (3D) spheroid microcircuit development, and the use of biomaterials to influence the extracellular environment. Current barriers and future applications are also summarized in order to augment the design of future preclinical trials aimed toward astrocyte‐targeted neuroregeneration with a concept termed astrocellular therapeutics. Developmental Dynamics 248:21–33, 2019.