Genome-wide CRISPR screen identifies neddylation as a regulator of neuronal aging and AD neurodegeneration.

Aging is the biggest risk factor for the development of Alzheimer's disease (AD). Here, we performed a whole-genome CRISPR screen to identify regulators of neuronal age and show that the neddylation pathway regulates both cellular age and AD neurodegeneration in a human stem cell model. Specifically, we demonstrate that blocking neddylation increased cellular hallmarks of aging and led to an increase in Tau aggregation and phosphorylation in neurons carrying the APPswe/swe mutation. Aged APPswe/swe but not isogenic control neurons also showed a progressive decrease in viability. Selective neuronal loss upon neddylation inhibition was similarly observed in other isogenic AD and in Parkinson's disease (PD) models, including PSENM146V/M146V cortical and LRRK2G2019S/G2019S midbrain dopamine neurons, respectively. This study indicates that cellular aging can reveal late-onset disease phenotypes, identifies new potential targets to modulate AD progression, and describes a strategy to program age-associated phenotypes into stem cell models of disease.

Identifying novel age-modulating compounds and quantifying cellular aging using novel computational framework for evaluating transcriptional age.

The differentiation of human pluripotent stem cells (hPSCs) provides access to most cell types and tissues. However, hPSC-derived lineages capture a fetal-stage of development and methods to accelerate progression to an aged identity are limited. Understanding the factors driving cellular age and rejuvenation is also essential for efforts aimed at extending human life and health span. A prerequisite for such studies is the development of methods to score cellular age and simple readouts to assess the relative impact of various age modifying strategies. Here we established a transcriptional score (RNAge) in young versus old primary fibroblasts, frontal cortex and substantia nigra tissue. We validated the score in independent RNA-seq datasets and demonstrated a strong cell and tissue specificity. In fibroblasts we observed a reset of RNAge during iPSC reprogramming while direct reprogramming of aged fibroblasts to induced neurons (iN) resulted in the maintenance of both a neuronal and a fibroblast aging signature. Increased RNAge in hPSC-derived neurons was confirmed for several age-inducing strategies such as SATB1 loss, progerin expression or chemical induction of senescence (SLO). Using RNAge as a probe set, we next performed an in-silico screen using the LINCS L1000 dataset. We identified and validated several novel age-inducing and rejuvenating compounds, and we observed that RNAage captures age-related changes associated with distinct cellular hallmarks of age. Our study presents a simple tool to score age manipulations and identifies compounds that greatly expand the toolset of age-modifying strategies in hPSC derived lineages.

Activation of HERV-K(HML-2) disrupts cortical patterning and neuronal differentiation by increasing NTRK3.

The biological function and disease association of human endogenous retroviruses (HERVs) are largely elusive. HERV-K(HML-2) has been associated with neurotoxicity, but there is no clear understanding of its role or mechanistic basis. We addressed the physiological functions of HERV-K(HML-2) in neuronal differentiation using CRISPR engineering to activate or repress its expression levels in a human-pluripotent-stem-cell-based system. We found that elevated HERV-K(HML-2) transcription is detrimental for the development and function of cortical neurons. These effects are cell-type-specific, as dopaminergic neurons are unaffected. Moreover, high HERV-K(HML-2) transcription alters cortical layer formation in forebrain organoids. HERV-K(HML-2) transcriptional activation leads to hyperactivation of NTRK3 expression and other neurodegeneration-related genes. Direct activation of NTRK3 phenotypically resembles HERV-K(HML-2) induction, and reducing NTRK3 levels in context of HERV-K(HML-2) induction restores cortical neuron differentiation. Hence, these findings unravel a cell-type-specific role for HERV-K(HML-2) in cortical neuron development.

Lipid Deprivation Induces a Stable, Naive-to-Primed Intermediate State of Pluripotency in Human PSCs.

Current challenges in capturing naive human pluripotent stem cells (hPSCs) suggest that the factors regulating human naive versus primed pluripotency remain incompletely defined. Here we demonstrate that the widely used Essential 8 minimal medium (E8) captures hPSCs at a naive-to-primed intermediate state of pluripotency expressing several naive-like developmental, bioenergetic, and epigenomic features despite providing primed-state-sustaining growth factor conditions. Transcriptionally, E8 hPSCs are marked by activated lipid biosynthesis and suppressed MAPK/TGF-ß gene expression, resulting in endogenous ERK inhibition. These features are dependent on lipid-free culture conditions and are lost upon lipid exposure, whereas short-term pharmacological ERK inhibition restores naive-to-primed intermediate traits even in the presence of lipids. Finally, we identify de novo lipogenesis as a common transcriptional signature of E8 hPSCs and the pre-implantation human epiblast in vivo. These findings implicate exogenous lipid availability in regulating human pluripotency and define E8 hPSCs as a stable, naive-to-primed intermediate (NPI) pluripotent state.

Specification of positional identity in forebrain organoids.

Human brain organoids generated with current technologies recapitulate histological features of the human brain, but they lack a reproducible topographic organization. During development, spatial topography is determined by gradients of signaling molecules released from discrete signaling centers. We hypothesized that introduction of a signaling center into forebrain organoids would specify the positional identity of neural tissue in a distance-dependent manner. Here, we present a system to trigger a Sonic Hedgehog (SHH) protein gradient in developing forebrain organoids that enables ordered self-organization along dorso-ventral and antero-posterior positional axes. SHH-patterned forebrain organoids establish major forebrain subdivisions that are positioned with in vivo-like topography. Consistent with its behavior in vivo, SHH exhibits long-range signaling activity in organoids. Finally, we use SHH-patterned cerebral organoids as a tool to study the role of cholesterol metabolism in SHH signaling. Together, this work identifies inductive signaling as an effective organizing strategy to recapitulate in vivo-like topography in human brain organoids.

DNA replication timing alterations identify common markers between distinct progeroid diseases.

Progeroid syndromes are rare genetic disorders that phenotypically resemble natural aging. Different causal mutations have been identified, but no molecular alterations have been identified that are in common to these diseases. DNA replication timing (RT) is a robust cell type-specific epigenetic feature highly conserved in the same cell types from different individuals but altered in disease. Here, we characterized DNA RT program alterations in Hutchinson-Gilford progeria syndrome (HGPS) and Rothmund-Thomson syndrome (RTS) patients compared with natural aging and cellular senescence. Our results identified a progeroid-specific RT signature that is common to cells from three HGPS and three RTS patients and distinguishes them from healthy individuals across a wide range of ages. Among the RT abnormalities, we identified the tumor protein p63 gene (TP63) as a gene marker for progeroid syndromes. By using the redifferentiation of four patient-derived induced pluripotent stem cells as a model for the onset of progeroid syndromes, we tracked the progression of RT abnormalities during development, revealing altered RT of the TP63 gene as an early event in disease progression of both HGPS and RTS. Moreover, the RT abnormalities in progeroid patients were associated with altered isoform expression of TP63 Our findings demonstrate the value of RT studies to identify biomarkers not detected by other methods, reveal abnormal TP63 RT as an early event in progeroid disease progression, and suggest TP63 gene regulation as a potential therapeutic target.

Mouse Rif1 is a regulatory subunit of protein phosphatase 1 (PP1).

Rif1 is a conserved protein that plays essential roles in orchestrating DNA replication timing, controlling nuclear architecture, telomere length and DNA repair. However, the relationship between these different roles, as well as the molecular basis of Rif1 function is still unclear. The association of Rif1 with insoluble nuclear lamina has thus far hampered exhaustive characterization of the associated protein complexes. We devised a protocol that overcomes this problem, and were thus able to discover a number of novel Rif1 interactors, involved in chromatin metabolism and phosphorylation. Among them, we focus here on PP1. Data from different systems have suggested that Rif1-PP1 interaction is conserved and has important biological roles. Using mutagenesis, NMR, isothermal calorimetry and surface plasmon resonance we demonstrate that Rif1 is a high-affinity PP1 adaptor, able to out-compete the well-established PP1-inhibitor I2 in vitro. Our conclusions have important implications for understanding Rif1 diverse roles and the relationship between the biological processes controlled by Rif1.

Back and forth in time: Directing age in iPSC-derived lineages.

The advent of induced pluripotent stem cells (iPSC) has transformed the classic approach of studying human disease, providing in vitro access to disease-relevant cells from patients for the study of disease pathogenesis and for drug screening. However, in spite of the broad repertoire of iPSC-based disease models developed in recent years, increasing evidence suggests that this technology might not be fully suitable for the study of conditions of old age, such as neurodegeneration. The difficulty in recapitulating late-stage features of disease in cells of pluripotent origin is believed to be a discrepancy between the fetal-like nature of iPSC-progeny and the advanced age of onset of neurodegenerative syndromes. In parallel to the issue of functional immaturity known to affect derivatives of pluripotent cells, latest findings suggest that reprogramming also subjects cells to a process of "rejuvenation", giving rise to cells that are too "young" to manifest phenotypes of age-related diseases. Thus, following the significant progress in manipulating cellular fate, the stem cell field will now have to face the new challenge of controlling cellular age, in order to fully harness the potential of iPSC-technology to advance the research and cure of diseases of the aging brain. This article is part of a Special Issue entitled SI: Exploiting human neurons.

Nuclear Architecture Organized by Rif1 Underpins the Replication-Timing Program.

DNA replication is temporally and spatially organized in all eukaryotes, yet the molecular control and biological function of the replication-timing program are unclear. Rif1 is required for normal genome-wide regulation of replication timing, but its molecular function is poorly understood. Here we show that in mouse embryonic stem cells, Rif1 coats late-replicating domains and, with Lamin B1, identifies most of the late-replicating genome. Rif1 is an essential determinant of replication timing of non-Lamin B1-bound late domains. We further demonstrate that Rif1 defines and restricts the interactions between replication-timing domains during the G1 phase, thereby revealing a function of Rif1 as organizer of nuclear architecture. Rif1 loss affects both number and replication-timing specificity of the interactions between replication-timing domains. In addition, during the S phase, Rif1 ensures that replication of interacting domains is temporally coordinated. In summary, our study identifies Rif1 as the molecular link between nuclear architecture and replication-timing establishment in mammals.

Retinoic Acid-Mediated Regulation of GLI3 Enables Efficient Motoneuron Derivation from Human ESCs in the Absence of Extrinsic SHH Activation.

The derivation of somatic motoneurons (MNs) from ES cells (ESCs) after exposure to sonic hedgehog (SHH) and retinoic acid (RA) is one of the best defined, directed differentiation strategies to specify fate in pluripotent lineages. In mouse ESCs, MN yield is particularly high after RA + SHH treatment, whereas human ESC (hESC) protocols have been generally less efficient. In an effort to optimize yield, we observe that functional MNs can be derived from hESCs at high efficiencies if treated with patterning molecules at very early differentiation steps before neural induction. Remarkably, under these conditions, equal numbers of human MNs were obtained in the presence or absence of SHH exposure. Using pharmacological and genetic strategies, we demonstrate that early RA treatment directs MN differentiation independently of extrinsic SHH activation by suppressing the induction of GLI3. We further demonstrate that neural induction triggers a switch from a poised to an active chromatin state at GLI3. Early RA treatment prevents this switch by direct binding of the RA receptor at the GLI3 promoter. Furthermore, GLI3 knock-out hESCs can bypass the requirement for early RA patterning to yield MNs efficiently. Our data demonstrate that RA-mediated suppression of GLI3 is sufficient to generate MNs in an SHH-independent manner and that temporal changes in exposure to patterning factors such as RA affect chromatin state and competency of hESC-derived lineages to adopt specific neuronal fates. Finally, our work presents a streamlined platform for the highly efficient derivation of human MNs from ESCs and induced pluripotent stem cells. SIGNIFICANCE STATEMENT: Our study presents a rapid and efficient protocol to generate human motoneurons from embryonic and induced pluripotent stem cells. Surprisingly, and in contrast to previous work, motoneurons are generated in the presence of retinoic acid but in the absence of factors that activate sonic hedgehog signaling. We show that early exposure to retinoic acid modulates the chromatin state of cells to be permissive for motoneuron generation and directly suppresses the induction of GLI3, a negative regulator of SHH signaling. Therefore, our data point to a novel mechanism by which retinoic acid exposure can bypass the requirement for extrinsic SHH treatment during motoneuron induction.

Programming and Reprogramming Cellular Age in the Era of Induced Pluripotency.

The ability to reprogram adult somatic cells back to pluripotency presents a powerful tool for studying cell-fate identity and modeling human disease. However, the reversal of cellular age during reprogramming results in an embryonic-like state of induced pluripotent stem cells (iPSCs) and their derivatives, which presents specific challenges for modeling late onset disease. This age reset requires novel methods to mimic age-related changes but also offers opportunities for studying cellular rejuvenation in real time. Here, we discuss how iPSC research may transform studies of aging and enable the precise programming of cellular age in parallel to cell-fate specification.

Mouse Rif1 is a key regulator of the replication-timing programme in mammalian cells.

The eukaryotic genome is replicated according to a specific spatio-temporal programme. However, little is known about both its molecular control and biological significance. Here, we identify mouse Rif1 as a key player in the regulation of DNA replication timing. We show that Rif1 deficiency in primary cells results in an unprecedented global alteration of the temporal order of replication. This effect takes place already in the first S-phase after Rif1 deletion and is neither accompanied by alterations in the transcriptional landscape nor by major changes in the biochemical identity of constitutive heterochromatin. In addition, Rif1 deficiency leads to both defective G1/S transition and chromatin re-organization after DNA replication. Together, these data offer a novel insight into the global regulation and biological significance of the replication-timing programme in mammalian cells.