Imaging-based chromatin and epigenetic age, ImAge, quantitates aging and rejuvenation.

Biomarkers of biological age that predict the risk of disease and expected lifespan better than chronological age are key to efficient and cost-effective healthcare1-3. To advance a personalized approach to healthcare, such biomarkers must reliably and accurately capture individual biology, predict biological age, and provide scalable and cost-effective measurements. We developed a novel approach - image-based chromatin and epigenetic age (ImAge) that captures intrinsic progressions of biological age, which readily emerge as principal changes in the spatial organization of chromatin and epigenetic marks in single nuclei without regression on chronological age. ImAge captured the expected acceleration or deceleration of biological age in mice treated with chemotherapy or following a caloric restriction regimen, respectively. ImAge from chronologically identical mice inversely correlated with their locomotor activity (greater activity for younger ImAge), consistent with the widely accepted role of locomotion as an aging biomarker across species. Finally, we demonstrated that ImAge is reduced following transient expression of OSKM cassette in the liver and skeletal muscles and reveals heterogeneity of in vivo reprogramming. We propose that ImAge represents the first-in-class imaging-based biomarker of aging with single-cell resolution.

High-content analysis and Kinetic Image Cytometry identify toxicity and epigenetic effects of HIV antiretrovirals on human iPSC-neurons and primary neural precursor cells.

INTRODUCTION: Despite viral suppression due to combination antiretroviral therapy (cART), HIV-associated neurocognitive disorders (HAND) continue to affect half of people with HIV, suggesting that certain antiretrovirals (ARVs) may contribute to HAND. METHODS: We examined the effects of nucleoside/nucleotide reverse transcriptase inhibitors tenofovir disoproxil fumarate (TDF) and emtricitabine (FTC) and the integrase inhibitors dolutegravir (DTG) and elvitegravir (EVG) on viability, structure, and function of glutamatergic neurons (a subtype of CNS neuron involved in cognition) derived from human induced pluripotent stem cells (hiPSC-neurons), and primary human neural precursor cells (hNPCs), which are responsible for neurogenesis. RESULTS: Using automated digital microscopy and image analysis (high content analysis, HCA), we found that DTG, EVG, and TDF decreased hiPSC-neuron viability, neurites, and synapses after 7 days of treatment. Analysis of hiPSC-neuron calcium activity using Kinetic Image Cytometry (KIC) demonstrated that DTG and EVG also decreased the frequency and magnitude of intracellular calcium transients. Longer ARV exposures and simultaneous exposure to multiple ARVs increased the magnitude of these neurotoxic effects. Using the Microscopic Imaging of Epigenetic Landscapes (MIEL) assay, we found that TDF decreased hNPC viability and changed the distribution of histone modifications that regulate chromatin packing, suggesting that TDF may reduce neuroprogenitor pools important for CNS development and maintenance of cognition in adults. CONCLUSION: This study establishes human preclinical assays that can screen potential ARVs for CNS toxicity to develop safer cART regimens and HAND therapeutics.

Activity-dependent modulation of synapse-regulating genes in astrocytes.

Astrocytes regulate the formation and function of neuronal synapses via multiple signals; however, what controls regional and temporal expression of these signals during development is unknown. We determined the expression profile of astrocyte synapse-regulating genes in the developing mouse visual cortex, identifying astrocyte signals that show differential temporal and layer-enriched expression. These patterns are not intrinsic to astrocytes, but regulated by visually evoked neuronal activity, as they are absent in mice lacking glutamate release from thalamocortical terminals. Consequently, synapses remain immature. Expression of synapse-regulating genes and synaptic development is also altered when astrocyte signaling is blunted by diminishing calcium release from astrocyte stores. Single-nucleus RNA sequencing identified groups of astrocytic genes regulated by neuronal and astrocyte activity, and a cassette of genes that show layer-specific enrichment. Thus, the development of cortical circuits requires coordinated signaling between astrocytes and neurons, highlighting astrocytes as a target to manipulate in neurodevelopmental disorders.

Improving drug discovery using image-based multiparametric analysis of the epigenetic landscape.

High-content phenotypic screening has become the approach of choice for drug discovery due to its ability to extract drug-specific multi-layered data. In the field of epigenetics, such screening methods have suffered from a lack of tools sensitive to selective epigenetic perturbations. Here we describe a novel approach, Microscopic Imaging of Epigenetic Landscapes (MIEL), which captures the nuclear staining patterns of epigenetic marks and employs machine learning to accurately distinguish between such patterns. We validated the MIEL platform across multiple cells lines and using dose-response curves, to insure the fidelity and robustness of this approach for high content high throughput drug discovery. Focusing on noncytotoxic glioblastoma treatments, we demonstrated that MIEL can identify and classify epigenetically active drugs. Furthermore, we show MIEL was able to accurately rank candidate drugs by their ability to produce desired epigenetic alterations consistent with increased sensitivity to chemotherapeutic agents or with induction of glioblastoma differentiation.

Emetine inhibits Zika and Ebola virus infections through two molecular mechanisms: inhibiting viral replication and decreasing viral entry.

The re-emergence of Zika virus (ZIKV) and Ebola virus (EBOV) poses serious and continued threats to the global public health. Effective therapeutics for these maladies is an unmet need. Here, we show that emetine, an anti-protozoal agent, potently inhibits ZIKV and EBOV infection with a low nanomolar half maximal inhibitory concentration (IC50) in vitro and potent activity in vivo. Two mechanisms of action for emetine are identified: the inhibition of ZIKV NS5 polymerase activity and disruption of lysosomal function. Emetine also inhibits EBOV entry. Cephaeline, a desmethyl analog of emetine, which may be better tolerated in patients than emetine, exhibits a similar efficacy against both ZIKV and EBOV infections. Hence, emetine and cephaeline offer pharmaceutical therapies against both ZIKV and EBOV infection.

Zika Virus: Origins, Pathological Action, and Treatment Strategies.

The Zika virus (ZIKV) global epidemic prompted the World Health Organization to declare it a 2016 Public Health Emergency of International Concern. The overwhelming experience over the past several years teaches us that ZIKV and the associated neurological complications represent a long-term world-wide challenge to public health. Although the number of ZIKV cases in the Western Hemisphere has dropped since 2016, the need for basic research and anti-ZIKV drug development remains strong. Re-emerging viruses like ZIKV are an ever-present threat in the 21st century where fast transcontinental travel lends itself to viral epidemics. Here, we first present the origin story for ZIKV and review the rapid progress researchers have made toward understanding of the ZIKV pathology and in the design, re-purposing, and testing-particularly in vivo-drug candidates for ZIKV prophylaxis and therapy ZIKV. Quite remarkably, a short, but intensive, drug-repurposing effort has already resulted in several readily available FDA-approved drugs that are capable of effectively combating the virus in infected adult mouse models and, most importantly, in both preventing maternal-fetal transmission and severe microcephaly in newborns in pregnant mouse models.

Repurposing of the anti-malaria drug chloroquine for Zika Virus treatment and prophylaxis.

One of the major challenges of the current Zika virus (ZIKV) epidemic is to prevent congenital foetal abnormalities, including microcephaly, following ZIKV infection of pregnant women. Given the urgent need for ZIKV prophylaxis and treatment, repurposing of approved drugs appears to be a viable and immediate solution. We demonstrate that the common anti-malaria drug chloroquine (CQ) extends the lifespan of ZIKV-infected interferon signalling-deficient AG129 mice. However, the severity of ZIKV infection in these mice precludes the study of foetal (vertical) viral transmission. Here, we show that interferon signalling-competent SJL mice support chronic ZIKV infection. Infected dams and sires are both able to transmit ZIKV to the offspring, making this an ideal model for in vivo validation of compounds shown to suppress ZIKV in cell culture. Administration of CQ to ZIKV-infected pregnant SJL mice during mid-late gestation significantly attenuated vertical transmission, reducing the ZIKV load in the foetal brain more than 20-fold. Given the limited side effects of CQ, its lack of contraindications in pregnant women, and its worldwide availability and low cost, we suggest that CQ could be considered for the treatment and prophylaxis of ZIKV.

Characterization of the Zika virus two-component NS2B-NS3 protease and structure-assisted identification of allosteric small-molecule antagonists.

The recent re-emergence of Zika virus (ZIKV)1, a member of the Flaviviridae family, has become a global emergency. Currently, there are no effective methods of preventing or treating ZIKV infection, which causes severe neuroimmunopathology and is particularly harmful to the developing fetuses of infected pregnant women. However, the pathology induced by ZIKV is unique among flaviviruses, and knowledge of the biology of other family members cannot easily be extrapolated to ZIKV. Thus, structure-function studies of ZIKV proteins are urgently needed to facilitate the development of effective preventative and therapeutic agents. Like other flaviviruses, ZIKV expresses an NS2B-NS3 protease, which consists of the NS2B cofactor and the NS3 protease domain and is essential for cleavage of the ZIKV polyprotein precursor and generation of fully functional viral proteins. Here, we report the enzymatic characterization of ZIKV protease, and we identify structural scaffolds for allosteric small-molecule inhibitors of this protease. Molecular modeling of the protease-inhibitor complexes suggests that these compounds bind to the druggable cavity in the NS2B-NS3 protease interface and affect productive interactions of the protease domain with its cofactor. The most potent compound demonstrated efficient inhibition of ZIKV propagation in vitro in human fetal neural progenitor cells and in vivo in SJL mice. The inhibitory scaffolds could be further developed into valuable research reagents and, ultimately, provide a roadmap for the selection of efficient inhibitors of ZIKV infection.

Phosphatidylserine Ameliorates Neurodegenerative Symptoms and Enhances Axonal Transport in a Mouse Model of Familial Dysautonomia.

Familial Dysautonomia (FD) is a neurodegenerative disease in which aberrant tissue-specific splicing of IKBKAP exon 20 leads to reduction of IKAP protein levels in neuronal tissues. Here we generated a conditional knockout (CKO) mouse in which exon 20 of IKBKAP is deleted in the nervous system. The CKO FD mice exhibit developmental delays, sensory abnormalities, and less organized dorsal root ganglia (DRGs) with attenuated axons compared to wild-type mice. Furthermore, the CKO FD DRGs show elevated HDAC6 levels, reduced acetylated α-tubulin, unstable microtubules, and impairment of axonal retrograde transport of nerve growth factor (NGF). These abnormalities in DRG properties underlie neuronal degeneration and FD symptoms. Phosphatidylserine treatment decreased HDAC6 levels and thus increased acetylation of α-tubulin. Further PS treatment resulted in recovery of axonal outgrowth and enhanced retrograde axonal transport by decreasing histone deacetylase 6 (HDAC6) levels and thus increasing acetylation of α-tubulin levels. Thus, we have identified the molecular pathway that leads to neurodegeneration in FD and have demonstrated that phosphatidylserine treatment has the potential to slow progression of neurodegeneration.

Sip1 regulates the generation of the inner nuclear layer retinal cell lineages in mammals.

The transcription factor Sip1 (Zeb2) plays multiple roles during CNS development from early acquisition of neural fate to cortical neurogenesis and gliogenesis. In humans, SIP1 (ZEB2) haploinsufficiency leads to Mowat-Wilson syndrome, a complex congenital anomaly including intellectual disability, epilepsy and Hirschsprung disease. Here we uncover the role of Sip1 in retinogenesis. Somatic deletion of Sip1 from mouse retinal progenitors primarily affects the generation of inner nuclear layer cell types, resulting in complete loss of horizontal cells and reduced numbers of amacrine and bipolar cells, while the number of Muller glia is increased. Molecular analysis places Sip1 downstream of the eye field transcription factor Pax6 and upstream of Ptf1a in the gene network required for generating the horizontal and amacrine lineages. Intriguingly, characterization of differentiation dynamics reveals that Sip1 has a role in promoting the timely differentiation of retinal interneurons, assuring generation of the proper number of the diverse neuronal and glial cell subtypes that constitute the functional retina in mammals.

Pax6 is required for normal cell-cycle exit and the differentiation kinetics of retinal progenitor cells.

The coupling between cell-cycle exit and onset of differentiation is a common feature throughout the developing nervous system, but the mechanisms that link these processes are mostly unknown. Although the transcription factor Pax6 has been implicated in both proliferation and differentiation of multiple regions within the central nervous system (CNS), its contribution to the transition between these successive states remains elusive. To gain insight into the role of Pax6 during the transition from proliferating progenitors to differentiating precursors, we investigated cell-cycle and transcriptomic changes occurring in Pax6 (-) retinal progenitor cells (RPCs). Our analyses revealed a unique cell-cycle phenotype of the Pax6-deficient RPCs, which included a reduced number of cells in the S phase, an increased number of cells exiting the cell cycle, and delayed differentiation kinetics of Pax6 (-) precursors. These alterations were accompanied by coexpression of factors that promote (Ccnd1, Ccnd2, Ccnd3) and inhibit (P27 (kip1) and P27 (kip2) ) the cell cycle. Further characterization of the changes in transcription profile of the Pax6-deficient RPCs revealed abrogated expression of multiple factors which are known to be involved in regulating proliferation of RPCs, including the transcription factors Vsx2, Nr2e1, Plagl1 and Hedgehog signaling. These findings provide novel insight into the molecular mechanism mediating the pleiotropic activity of Pax6 in RPCs. The results further suggest that rather than conveying a linear effect on RPCs, such as promoting their proliferation and inhibiting their differentiation, Pax6 regulates multiple transcriptional networks that function simultaneously, thereby conferring the capacity to proliferate, assume multiple cell fates and execute the differentiation program into retinal lineages.

Pax6: a multi-level regulator of ocular development.

Eye development has been a paradigm for the study of organogenesis, from the demonstration of lens induction through epithelial tissue morphogenesis, to neuronal specification and differentiation. The transcription factor Pax6 has been shown to play a key role in each of these processes. Pax6 is required for initiation of developmental pathways, patterning of epithelial tissues, activation of tissue-specific genes and interaction with other regulatory pathways. Herein we examine the data accumulated over the last few decades from extensive analyses of biochemical modules and genetic manipulation of the Pax6 gene. Specifically, we describe the regulation of Pax6's expression pattern, the protein's DNA-binding properties, and its specific roles and mechanisms of action at all stages of lens and retinal development. Pax6 functions at multiple levels to integrate extracellular information and execute cell-intrinsic differentiation programs that culminate in the specification and differentiation of a distinct ocular lineage.

Insulin-producing cells generated from dedifferentiated human pancreatic beta cells expanded in vitro.

BACKGROUND: Expansion of beta cells from the limited number of adult human islet donors is an attractive prospect for increasing cell availability for cell therapy of diabetes. However, attempts at expanding human islet cells in tissue culture result in loss of beta-cell phenotype. Using a lineage-tracing approach we provided evidence for massive proliferation of beta-cell-derived (BCD) cells within these cultures. Expansion involves dedifferentiation resembling epithelial-mesenchymal transition (EMT). Epigenetic analyses indicate that key beta-cell genes maintain open chromatin structure in expanded BCD cells, although they are not transcribed. Here we investigated whether BCD cells can be redifferentiated into beta-like cells. METHODOLOGY/PRINCIPAL FINDING: Redifferentiation conditions were screened by following activation of an insulin-DsRed2 reporter gene. Redifferentiated cells were characterized for gene expression, insulin content and secretion assays, and presence of secretory vesicles by electron microscopy. BCD cells were induced to redifferentiate by a combination of soluble factors. The redifferentiated cells expressed beta-cell genes, stored insulin in typical secretory vesicles, and released it in response to glucose. The redifferentiation process involved mesenchymal-epithelial transition, as judged by changes in gene expression. Moreover, inhibition of the EMT effector SLUG (SNAI2) using shRNA resulted in stimulation of redifferentiation. Lineage-traced cells also gave rise at a low rate to cells expressing other islet hormones, suggesting transition of BCD cells through an islet progenitor-like stage during redifferentiation. CONCLUSIONS/SIGNIFICANCE: These findings demonstrate for the first time that expanded dedifferentiated beta cells can be induced to redifferentiate in culture. The findings suggest that ex-vivo expansion of adult human islet cells is a promising approach for generation of insulin-producing cells for transplantation, as well as basic research, toxicology studies, and drug screening.

Homozygosity mapping reveals null mutations in FAM161A as a cause of autosomal-recessive retinitis pigmentosa.

Retinitis pigmentosa (RP) is a heterogeneous group of inherited retinal degenerations caused by mutations in at least 45 genes. Using homozygosity mapping, we identified a ∼4 Mb homozygous region on chromosome 2p15 in patients with autosomal-recessive RP (arRP). This region partially overlaps with RP28, a previously identified arRP locus. Sequence analysis of 12 candidate genes revealed three null mutations in FAM161A in 20 families. RT-PCR analysis in 21 human tissues revealed high levels of FAM161A expression in the retina and lower levels in the brain and testis. In the human retina, we identified two alternatively spliced transcripts with an intact open reading frame, the major one lacking a highly conserved exon. During mouse embryonic development, low levels of Fam161a transcripts were detected throughout the optic cup. After birth, Fam161a expression was elevated and confined to the photoreceptor layer. FAM161A encodes a protein of unknown function that is moderately conserved in mammals. Clinical manifestations of patients with FAM161A mutations varied but were largely within the spectrum associated with arRP. On funduscopy, pallor of the optic discs and attenuation of blood vessels were common, but bone-spicule-like pigmentation was often mild or lacking. Most patients had nonrecordable electroretinographic responses and constriction of visual fields upon diagnosis. Our data suggest a pivotal role for FAM161A in photoreceptors and reveal that FAM161A loss-of-function mutations are a major cause of arRP, accounting for ∼12% of arRP families in our cohort of patients from Israel and the Palestinian territories.

Dual requirement for Pax6 in retinal progenitor cells.

Throughout the developing central nervous system, pre-patterning of the ventricular zone into discrete neural progenitor domains is one of the predominant strategies used to produce neuronal diversity in a spatially coordinated manner. In the retina, neurogenesis proceeds in an intricate chronological and spatial sequence, yet it remains unclear whether retinal progenitor cells (RPCs) display intrinsic heterogeneity at any given time point. Here, we performed a detailed study of RPC fate upon temporally and spatially confined inactivation of Pax6. Timed genetic removal of Pax6 appeared to unmask a cryptic divergence of RPCs into qualitatively divergent progenitor pools. In the more peripheral RPCs under normal circumstances, Pax6 seemed to prevent premature activation of a photoreceptor-differentiation pathway by suppressing expression of the transcription factor Crx. More centrally, Pax6 contributed to the execution of the comprehensive potential of RPCs: Pax6 ablation resulted in the exclusive generation of amacrine interneurons. Together, these data suggest an intricate dual role for Pax6 in retinal neurogenesis, while pointing to the cryptic divergence of RPCs into distinct progenitor pools.

Regulation of transcription of the RNA splicing factor hSlu7 by Elk-1 and Sp1 affects alternative splicing.

Alternative splicing plays a major role in transcriptome diversity and plasticity, but it is largely unknown how tissue-specific and embryogenesis-specific alternative splicing is regulated. The highly conserved splicing factor Slu7 is involved in 3' splice site selection and also regulates alternative splicing. We show that Slu7 has a unique spatial pattern of expression among human and mouse embryonic and adult tissues. We identified several functional Ets binding sites and GC-boxes in the human Slu7 (hSlu7) promoter region. The Ets and GC-box binding transcription factors, Elk-1 and Sp1, respectively, exerted opposite effects on hSlu7 transcription: Sp1 protein enhances and Elk-1 protein represses transcription in a dose-dependent manner. Sp1 protein bound to the hSlu7 promoter in vivo, and depletion of Sp1 by RNA interference (RNAi) repressed hSlu7 expression. Elk-1 protein bound to the hSlu7 promoter in vivo, and depletion of Elk-1 by RNAi caused an increase in the endogenous level of hSlu7 mRNA. Further, depletion of either Sp1 or Elk-1 affected alternative splicing. Our results provide indications of a complex transcription regulation mechanism that controls the spatial and temporal expression of Slu7, presumably allowing regulation of tissue-specific alternative splicing events.

Notch1 functions to suppress cone-photoreceptor fate specification in the developing mouse retina.

Notch receptor-mediated cell-cell signaling is known to negatively regulate neurogenesis in both vertebrate and invertebrate species, while being implicated in promoting the acquisition of glial fates. We studied Notch1 function directly during retinal neurogenesis by selective Cre/loxP-triggered Notch1 gene inactivation in peripheral retinal progenitor cells (RPCs) prior to the onset of cell differentiation. Consistent with its previously established role, Notch1 inactivation led to dramatic alteration in the expression profile of multiple basic helix-loop-helix transcription factors, consequently prompting premature cell-cycle exit and neuronal specification. Surprisingly, however, Notch1 inactivation led to a striking change in retinal cell composition, with cone-photoreceptor precursors expanding at the expense of other early- as well as late-born cell fates. Intriguingly, the Notch1-deficient precursors adhered to the normal chronological sequence of the cone-photoreceptor differentiation program. Together, these findings reveal an unexpected role of Notch signaling in directly controlling neuronal cell-type composition, and suggest a model by which, during normal retinogenesis, Notch1 functions to suppress cone-photoreceptor fate, allowing for the specification of the diversity of retinal cell types.