A human-specific enhancer fine-tunes radial glia potency and corticogenesis.

Humans evolved an extraordinarily expanded and complex cerebral cortex, associated with developmental and gene regulatory modifications 1-3 . Human accelerated regions (HARs) are highly conserved genomic sequences with human-specific nucleotide substitutions. Although there are thousands of annotated HARs, their functional contribution to human-specific cortical development is largely unknown 4,5 . HARE5 is a HAR transcriptional enhancer of the WNT signaling receptor Frizzled8 (FZD8) active during brain development 6 . Here, using genome-edited mouse and primate models, we demonstrate that human (Hs) HARE5 fine-tunes cortical development and connectivity by controlling the proliferative and neurogenic capacity of neural progenitor cells (NPCs). Hs-HARE5 knock-in mice have significantly enlarged neocortices containing more neurons. By measuring neural dynamics in vivo we show these anatomical features correlate with increased functional independence between cortical regions. To understand the underlying developmental mechanisms, we assess progenitor fate using live imaging, lineage analysis, and single-cell RNA sequencing. This reveals Hs-HARE5 modifies radial glial progenitor behavior, with increased self-renewal at early developmental stages followed by expanded neurogenic potential. We use genome-edited human and chimpanzee (Pt) NPCs and cortical organoids to assess the relative enhancer activity and function of Hs-HARE5 and Pt-HARE5. Using these orthogonal strategies we show four human-specific variants in HARE5 drive increased enhancer activity which promotes progenitor proliferation. These findings illustrate how small changes in regulatory DNA can directly impact critical signaling pathways and brain development. Our study uncovers new functions for HARs as key regulatory elements crucial for the expansion and complexity of the human cerebral cortex.

Audiovisualization of real-time neuroimaging data.

Advancements in brain imaging techniques have significantly expanded the size and complexity of real-time neuroimaging and behavioral data. However, identifying patterns, trends and synchronies within these datasets presents a significant computational challenge. Here, we demonstrate an approach that can translate time-varying neuroimaging data into unique audiovisualizations consisting of audible representations of dynamic data merged with simplified, color-coded movies of spatial components and behavioral recordings. Multiple variables can be encoded as different musical instruments, letting the observer differentiate and track multiple dynamic parameters in parallel. This representation enables intuitive assimilation of these datasets for behavioral correlates and spatiotemporal features such as patterns, rhythms and motifs that could be difficult to detect through conventional data interrogation methods. These audiovisual representations provide a novel perception of the organization and patterns of real-time activity in the brain, and offer an intuitive and compelling method for complex data visualization for a wider range of applications.

Glioma-Induced Alterations in Excitatory Neurons are Reversed by mTOR Inhibition.

Gliomas are highly aggressive brain tumors characterized by poor prognosis and composed of diffusely infiltrating tumor cells that intermingle with non-neoplastic cells in the tumor microenvironment, including neurons. Neurons are increasingly appreciated as important reactive components of the glioma microenvironment, due to their role in causing hallmark glioma symptoms, such as cognitive deficits and seizures, as well as their potential ability to drive glioma progression. Separately, mTOR signaling has been shown to have pleiotropic effects in the brain tumor microenvironment, including regulation of neuronal hyperexcitability. However, the local cellular-level effects of mTOR inhibition on glioma-induced neuronal alterations are not well understood. Here we employed neuron-specific profiling of ribosome-bound mRNA via 'RiboTag,' morphometric analysis of dendritic spines, and in vivo calcium imaging, along with pharmacological mTOR inhibition to investigate the impact of glioma burden and mTOR inhibition on these neuronal alterations. The RiboTag analysis of tumor-associated excitatory neurons showed a downregulation of transcripts encoding excitatory and inhibitory postsynaptic proteins and dendritic spine development, and an upregulation of transcripts encoding cytoskeletal proteins involved in dendritic spine turnover. Light and electron microscopy of tumor-associated excitatory neurons demonstrated marked decreases in dendritic spine density. In vivo two-photon calcium imaging in tumor-associated excitatory neurons revealed progressive alterations in neuronal activity, both at the population and single-neuron level, throughout tumor growth. This in vivo calcium imaging also revealed altered stimulus-evoked somatic calcium events, with changes in event rate, size, and temporal alignment to stimulus, which was most pronounced in neurons with high-tumor burden. A single acute dose of AZD8055, a combined mTORC1/2 inhibitor, reversed the glioma-induced alterations on the excitatory neurons, including the alterations in ribosome-bound transcripts, dendritic spine density, and stimulus evoked responses seen by calcium imaging. These results point to mTOR-driven pathological plasticity in neurons at the infiltrative margin of glioma - manifested by alterations in ribosome-bound mRNA, dendritic spine density, and stimulus-evoked neuronal activity. Collectively, our work identifies the pathological changes that tumor-associated excitatory neurons experience as both hyperlocal and reversible under the influence of mTOR inhibition, providing a foundation for developing therapies targeting neuronal signaling in glioma.

A Comparative Study of the Effects of Electronic Cigarette and Traditional Cigarette on the Pulmonary Functions of C57BL/6 Male Mice.

INTRODUCTION: Electronic cigarettes (E-cigs) are in a controversial state. Although E-cig aerosol generally contains fewer harmful substances than smoke from burned traditional cigarettes, aerosol along with other compounds of the E-cigs may also affect lung functions and promote the development of lung-related diseases. We investigated the effects of E-cig on the pulmonary functions of male C57BL/6 mice and reveal the potential underlying mechanisms. METHODS: A total of 60 male C57BL/6 mice were randomly divided into four groups. They were exposed to fresh-air, traditional cigarette smoke, E-cig vapor with 12 mg/mL of nicotine, and E-cig with no nicotine for 8 weeks. Lung functions were evaluated by using quantitative analysis of the whole body plethysmograph, FlexiVent system, lung tissue histological and morphometric analysis, and RT-PCR analysis of mRNA expression of inflammation-related genes. In addition, the effects of nicotine and acrolein on the survival rate and DNA damage were investigated using cultured human alveolar basal epithelial cells. RESULTS: Exposure to E-cig vapor led to significant changes in lung functions and structures including the rupture of the alveolar cavity and enlarged alveolar space. The pathological changes were also accompanied by increased expression of interleukin-6 and tumor necrosis factor-α. CONCLUSIONS: The findings of the present study indicate that the safety of E-cig should be further evaluated. IMPLICATIONS: Some people currently believe that using nicotine-free E-cigs is a safe way to smoke. However, our research shows that E-cigs can cause lung damage regardless of whether they contain nicotine.

Spiral copropagation of two relativistic intense laser beams in a plasma channel.

The copropagation of two relativistic intense laser beams with orthogonal polarization in a parabolic plasma channel is studied analytically and numerically. A set of coupled equations for the evolution of the laser spot sizes and transverse centroids are derived by use of the variational approach. It is shown that the centroids of the two beams can spiral and oscillate around each other along the channel axis, where the characteristic frequency is determined both by the laser and plasma parameters. The results are verified by direct numerical solution of the relativistic nonlinear Schrödinger equations for the laser envelopes as well as three-dimensional particle-in-cell simulations. In the case with two ultrashort laser pulses when laser wakefields are excited, it is shown that the two wake bubbles driven by the laser pulses can spiral and oscillate around each other in a way similar to the two pulses. This can be well controlled by adjusting the incidence angle and separation distance between the two laser pulses. Preliminary studies show that externally injected electron beams can follow the trajectories of the oscillating bubbles. Our studies suggest a new way to control the coupling of two intense lasers in plasma for various applications, such as electron acceleration and radiation generation.

Cortex-wide neural dynamics predict behavioral states and provide a neural basis for resting-state dynamic functional connectivity.

Although resting-state functional magnetic resonance imaging (fMRI) studies have observed dynamically changing brain-wide networks of correlated activity, fMRI's dependence on hemodynamic signals makes results challenging to interpret. Meanwhile, emerging techniques for real-time recording of large populations of neurons have revealed compelling fluctuations in neuronal activity across the brain that are obscured by traditional trial averaging. To reconcile these observations, we use wide-field optical mapping to simultaneously record pan-cortical neuronal and hemodynamic activity in awake, spontaneously behaving mice. Some components of observed neuronal activity clearly represent sensory and motor function. However, particularly during quiet rest, strongly fluctuating patterns of activity across diverse brain regions contribute greatly to interregional correlations. Dynamic changes in these correlations coincide with changes in arousal state. Simultaneously acquired hemodynamics depict similar brain-state-dependent correlation shifts. These results support a neural basis for dynamic resting-state fMRI, while highlighting the importance of brain-wide neuronal fluctuations in the study of brain state.

High-speed light-sheet microscopy for the in-situ acquisition of volumetric histological images of living tissue.

Histological examinations typically require the excision of tissue, followed by its fixation, slicing, staining, mounting and imaging, with timeframes ranging from minutes to days. This process may remove functional tissue, may miss abnormalities through under-sampling, prevents rapid decision-making, and increases costs. Here, we report the feasibility of microscopes based on swept confocally aligned planar excitation technology for the volumetric histological imaging of intact living tissue in real time. The systems' single-objective, light-sheet geometry and 3D imaging speeds enable roving image acquisition, which combined with 3D stitching permits the contiguous analysis of large tissue areas, as well as the dynamic assessment of tissue perfusion and function. Implemented in benchtop and miniaturized form factors, the microscopes also have high sensitivity, even for weak intrinsic fluorescence, allowing for the label-free imaging of diagnostically relevant histoarchitectural structures, as we show for pancreatic disease in living mice, for chronic kidney disease in fresh human kidney tissues, and for oral mucosa in a healthy volunteer. Miniaturized high-speed light-sheet microscopes for in-situ volumetric histological imaging may facilitate the point-of-care detection of diverse cellular-level biomarkers.

Neurovascular dynamics of repeated cortical spreading depolarizations after acute brain injury.

Cortical spreading depolarizations (CSDs) are increasingly suspected to play an exacerbating role in a range of acute brain injuries, including stroke, possibly through their interactions with cortical blood flow. We use simultaneous wide-field imaging of neural activity and hemodynamics in Thy1-GCaMP6f mice to explore the neurovascular dynamics of CSDs during and following Rose Bengal-mediated photothrombosis. CSDs are observed in all mice as slow-moving waves of GCaMP fluorescence extending far beyond the photothrombotic area. Initial CSDs are accompanied by profound vasoconstriction and leave residual oligemia and ischemia in their wake. Later, CSDs evoke variable responses, from constriction to biphasic to vasodilation. However, CSD-evoked vasoconstriction is found to be more likely during rapid, high-amplitude CSDs in regions with stronger oligemia and ischemia, which, in turn, worsens after each repeated CSD. This feedback loop may explain the variable but potentially devastating effects of CSDs in the context of acute brain injury.

A human-specific modifier of cortical connectivity and circuit function.

The cognitive abilities that characterize humans are thought to emerge from unique features of the cortical circuit architecture of the human brain, which include increased cortico-cortical connectivity. However, the evolutionary origin of these changes in connectivity and how they affected cortical circuit function and behaviour are currently unknown. The human-specific gene duplication SRGAP2C emerged in the ancestral genome of the Homo lineage before the major phase of increase in brain size1,2. SRGAP2C expression in mice increases the density of excitatory and inhibitory synapses received by layer 2/3 pyramidal neurons (PNs)3-5. Here we show that the increased number of excitatory synapses received by layer 2/3 PNs induced by SRGAP2C expression originates from a specific increase in local and long-range cortico-cortical connections. Mice humanized for SRGAP2C expression in all cortical PNs displayed a shift in the fraction of layer 2/3 PNs activated by sensory stimulation and an enhanced ability to learn a cortex-dependent sensory-discrimination task. Computational modelling revealed that the increased layer 4 to layer 2/3 connectivity induced by SRGAP2C expression explains some of the key changes in sensory coding properties. These results suggest that the emergence of SRGAP2C at the birth of the Homo lineage contributed to the evolution of specific structural and functional features of cortical circuits in the human cortex.

Correction: Histone demethylase LSD1 restricts influenza A virus infection by erasing IFITM3-K88 monomethylation.

[This corrects the article DOI: 10.1371/journal.ppat.1006773.].

Calcineurin A gamma and NFATc3/SRPX2 axis contribute to human embryonic stem cell differentiation.

Our understanding of signaling pathways regulating the cell fate of human embryonic stem cells (hESCs) is limited. Calcineurin-NFAT signaling is associated with a wide range of biological processes and diseases. However, its role in controlling hESC fate remains unclear. Here, we report that calcineurin A gamma and the NFATc3/SRPX2 axis control the expression of lineage and epithelial-mesenchymal transition (EMT) markers in hESCs. Knockdown of PPP3CC, the gene encoding calcineurin A gamma, or NFATC3, downregulates certain markers both at the self-renewal state and during differentiation of hESCs. Furthermore, NFATc3 interacts with c-JUN and regulates the expression of SRPX2, the gene encoding a secreted glycoprotein known as a ligand of uPAR. We show that SRPX2 is a downstream target of NFATc3. Both SRPX2 and uPAR participate in controlling expression of lineage and EMT markers. Importantly, SRPX2 knockdown diminishes the upregulation of multiple lineage and EMT markers induced by co-overexpression of NFATc3 and c-JUN in hESCs. Together, this study uncovers a previously unknown role of calcineurin A gamma and the NFATc3/SRPX2 axis in modulating the fate determination of hESCs.

Glioma-Induced Alterations in Neuronal Activity and Neurovascular Coupling during Disease Progression.

Diffusely infiltrating gliomas are known to cause alterations in cortical function, vascular disruption, and seizures. These neurological complications present major clinical challenges, yet their underlying mechanisms and causal relationships to disease progression are poorly characterized. Here, we follow glioma progression in awake Thy1-GCaMP6f mice using in vivo wide-field optical mapping to monitor alterations in both neuronal activity and functional hemodynamics. The bilateral synchrony of spontaneous neuronal activity gradually decreases in glioma-infiltrated cortical regions, while neurovascular coupling becomes progressively disrupted compared to uninvolved cortex. Over time, mice develop diverse patterns of high amplitude discharges and eventually generalized seizures that appear to originate at the tumors' infiltrative margins. Interictal and seizure events exhibit positive neurovascular coupling in uninfiltrated cortex; however, glioma-infiltrated regions exhibit disrupted hemodynamic responses driving seizure-evoked hypoxia. These results reveal a landscape of complex physiological interactions occurring during glioma progression and present new opportunities for exploring novel biomarkers and therapeutic targets.

Correction: Histone demethylase LSD1 restricts influenza A virus infection by erasing IFITM3-K88 monomethylation.

[This corrects the article DOI: 10.1371/journal.ppat.1006773.].

Histone demethylase LSD1 restricts influenza A virus infection by erasing IFITM3-K88 monomethylation.

The histone demethylase LSD1 has been known as a key transcriptional coactivator for DNA viruses such as herpes virus. Inhibition of LSD1 was found to block viral genome transcription and lytic replication of DNA viruses. However, RNA virus genomes do not rely on chromatin structure and histone association, and the role of demethylase activity of LSD1 in RNA virus infections is not anticipated. Here, we identify that, contrary to its role in enhancing DNA virus replication, LSD1 limits RNA virus replication by demethylating and activating IFITM3 which is a host restriction factor for many RNA viruses. We have found that LSD1 is recruited to demethylate IFITM3 at position K88 under IFNα treatment. However, infection by either Vesicular Stomatitis Virus (VSV) or Influenza A Virus (IAV) triggers methylation of IFITM3 by promoting its disassociation from LSD1. Accordingly, inhibition of the enzymatic activity of LSD1 by Trans-2-phenylcyclopropylamine hydrochloride (TCP) increases IFITM3 monomethylation which leads to more severe disease outcomes in IAV-infected mice. In summary, our findings highlight the opposite role of LSD1 in fighting RNA viruses comparing to DNA viruses infection. Our data suggest that the demethylation of IFITM3 by LSD1 is beneficial for the host to fight against RNA virus infection.

The spontaneous differentiation and chromosome loss in iPSCs of human trisomy 18 syndrome.

Aneuploidy including trisomy results in developmental disabilities and is the leading cause of miscarriages in humans. Unlike trisomy 21, pathogenic mechanisms of trisomy 18 remain unclear. Here, we successfully generated induced pluripotent stem cells (iPSCs) from human amniotic fluid cells (AFCs) with trisomy 18 pregnancies. We found that trisomy 18 iPSCs (18T-iPSCs) were prone to differentiate spontaneously. Intriguingly, 18T-iPSCs lost their extra 18 chromosomes and converted to diploid cells after 10 generations. fluorescence in situ hybridization analysis showed chromosome loss was a random event that might happen in any trisomic cells. Selection undifferentiated cells for passage accelerated the recovery of euploid cells. Overall, our findings indicate the genomic instability of trisomy 18 iPSCs bearing an extra chromosome 18.

Signaling networks in the control of pluripotency.

Embryonic stem cells (ESCs) are characterized by their ability of unlimited self-renewal in vitro and pluripotent developmental potential, which endows them with great values in basic research and future clinical application. However, realization of full potential of ESCs is dependent on the elucidation of molecular mechanisms governing ESCs, among which signaling pathways play critical roles. A great deal of efforts has been made in the past decades to understand what and how signaling pathways contribute to the establishment and maintenance of pluripotency. In this review, we discuss signaling networks in both mouse and human ESCs, focusing on signals involved in the control of self-renewal and differentiation. In addition, the modulation of signaling pathways by pluripotency-associated transcription factors is also briefly summarized.

Early pathogenic event of Alzheimer's disease documented in iPSCs from patients with PSEN1 mutations.

Alzheimer's disease (AD) is the most common age-related dementia characterized by progressive neuronal loss. However, the molecular mechanisms for the neuronal loss is still debated. Here, we used induced pluripotent stem cells (iPSCs) derived from somatic cells of familial AD patients carrying PSEN1 mutations to study the early pathogenic event of AD. We found that premature neuronal differentiation with decreased proliferation and increased apoptosis occured in AD-iPSC-derived neural progenitor cells (AD-NPCs) once neuronal differentiation was initiated, together with higher levels of Aß42 and phosphorylated tau. Premature neuronal differentiation in AD-NPCs was caused by PSEN1 mutations and might be correlated to multiple dysregulated processes including but not limited to Wnt-Notch pathway. Our study documented previously unappreciated early NPC dysfunction in AD-NPCs, providing valuable new insights into the early mechanisms underlying AD pathogenesis.