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Aggregation of the protein tau defines tauopathies, which include Alzheimer's disease and frontotemporal dementia. Specific neuronal subtypes are selectively vulnerable to tau aggregation and subsequent dysfunction and death, but the underlying mechanisms are unknown. To systematically uncover the cellular factors controlling the accumulation of tau aggregates in human neurons, we conducted a genome-wide CRISPRi-based modifier screen in iPSC-derived neurons. The screen uncovered expected pathways, including autophagy, but also unexpected pathways, including UFMylation and GPI anchor synthesis. We discover that the E3 ubiquitin ligase CUL5 ^SOCS4 is a potent modifier of tau levels in human neurons, ubiquitinates tau, and is a correlated with vulnerability to tauopathies in mouse and human. Disruption of mitochondrial function promotes proteasomal misprocessing of tau, which generates tau proteolytic fragments like those in disease and changes tau aggregation in vitro . These results reveal new principles of tau proteostasis in human neurons and pinpoint potential therapeutic targets for tauopathies.

DNA damage and cellular metabolism exhibit a complex interplay characterized by bidirectional feedback mechanisms. Key mediators of the DNA damage response and cellular metabolic regulation include Ataxia Telangiectasia and Rad3-related protein (ATR) and the mechanistic Target of Rapamycin Complex 1 (mTORC1), respectively. Previous studies have established ATR as a regulatory upstream factor of mTORC1 during replication stress; however, the precise mechanisms by which mTORC1 is activated in this context remain poorly defined. Additionally, the activity of this signaling axis in unperturbed cells has not been extensively investigated. Here, we demonstrate that ATR promotes mTORC1 activity across various cellular models under basal conditions. This effect is particularly enhanced in cells following the loss of p16, which we have previously associated with hyperactivation of mTORC1 signaling and here found have increased ATR activity. Mechanistically, we found that ATR promotes de novo cholesterol synthesis and mTORC1 activation through the upregulation of lanosterol synthase (LSS), independently of both CHK1 and the TSC complex. Furthermore, the attenuation of mTORC1 activity resulting from ATR inhibition was rescued by supplementation with lanosterol or cholesterol in multiple cellular contexts. This restoration corresponded with enhanced localization of mTOR to the lysosome. Collectively, our findings demonstrate a novel connection linking ATR and mTORC1 signaling through the modulation of cholesterol metabolism.

Cryptococcus neoformans is an opportunistic fungal pathogen with a polysaccharide capsule that becomes greatly enlarged in the mammalian host and during in vitro growth under host-like conditions. To understand how individual environmental signals affect capsule size and gene expression, we grew cells in all combinations of five signals implicated in capsule size and systematically measured cell and capsule sizes. We also sampled these cultures over time and performed RNA-Seq in quadruplicate, yielding 881 RNA-Seq samples. Analysis of the resulting data sets showed that capsule induction in tissue culture medium, typically used to represent host-like conditions, requires the presence of either CO ₂ or exogenous cyclic AMP (cAMP). Surprisingly, adding either of these pushes overall gene expression in the opposite direction from tissue culture media alone, even though both are required for capsule development. Another unexpected finding was that rich medium blocks capsule growth completely. Statistical analysis further revealed many genes whose expression is associated with capsule thickness; deletion of one of these significantly reduced capsule size. Beyond illuminating capsule induction, our massive, uniformly collected dataset will be a significant resource for the research community.

Importance: Cryptococcus neoformans is an opportunistic yeast that kills ∼150,000 people each year. This major impact on human health makes it imperative to understand the basic biology of C. neoformans and the factors that mediate its virulence. One key virulence factor is a polysaccharide capsule that expands greatly during infection. To help define capsule synthesis and fungal biology, we provided cells with many different combinations of host-like signals and sampled the cultures over time for transcriptional analysis. The resulting time resolved data set is by far the largest gene expression resource ever produced for C. neoformans (881 RNA-seq samples), further enriched by accompanying capsule images and measurements. It revealed surprising findings, including that rich medium suppresses capsule size regardless of other signals. This landmark data resource will be enormously valuable to the research community as it continues to define the relationships between environmental signals and cryptococcal gene expression, biology, and virulence.

Fetal Alcohol Spectrum Disorders (FASD) describe ethanol-induced developmental defects including craniofacial malformations. While ethanol-sensitive genetic mutations contribute to facial malformations, the impacted cellular mechanisms remain unknown. Bmp signaling is a key regulator of epithelial morphogenesis driving facial development, providing a possible ethanol-sensitive mechanism. We found that zebrafish mutants for Bmp signaling components are ethanol-sensitive and affect anterior pharyngeal endoderm shape and gene expression, indicating ethanol-induced malformations of the anterior pharyngeal endoderm cause facial malformations. Integrating FASD patient data, we provide the first evidence that variants in the human Bmp receptor gene BMPR1B associate with ethanol-related differences in jaw volume. Our results show that ethanol exposure disrupts proper morphogenesis of, and tissue interactions between, facial epithelia that mirror overall viscerocranial shape changes and are predictive for Bmp-ethanol associations in human jaw development. Our data provide a mechanistic paradigm linking ethanol to disrupted epithelial cell behaviors that underlie facial defects in FASD.

Summary statement: In this study, we apply a unique combination of zebrafish-based approaches and human genetic and facial dysmorphology analyses to resolve the cellular mechanisms driven by the ethanol-sensitive Bmp pathway.

We recently described our initial efforts to develop a model for small cell lung cancer (SCLC) derived from human embryonic stem cells (hESCs) that were differentiated to form pulmonary neuroendocrine cells (PNECs), a putative cell of origin for neuroendocrine-positive SCLC. Although reduced expression of the tumor suppressor genes TP53 and RB1 allowed the induced PNECs to form subcutaneous growths in immune-deficient mice, the tumors did not display the aggressive characteristics of SCLC seen in human patients. Here we report that the additional, doxycycline-regulated expression of a transgene encoding wild-type or mutant cMYC protein promotes rapid growth, invasion, and metastasis of these hESC-derived cells after injection into the renal capsule. Similar to others, we find that the addition of cMYC encourages the formation of the SCLC-N subtype, marked by high levels of NEUROD1 RNA. Using paired primary and metastatic samples for RNA sequencing, we observe that the subtype of SCLC does not change upon metastatic spread and that production of NEUROD1 is maintained. We also describe histological features of these malignant, SCLC-like tumors derived from hESCs and discuss potential uses of this model in efforts to control and better understand this recalcitrant neoplasm.

Many eukaryotic viruses require membrane-bound compartments for replication, but no such organelles are known to be formed by prokaryotic viruses ^1-3 . Bacteriophages of the Chimalliviridae family sequester their genomes within a phage-generated organelle, the phage nucleus, which is enclosed by a lattice of the viral protein ChmA ^4-10 . Previously, we observed lipid membrane-bound vesicles in cells infected by Chimalliviridae , but due to the paucity of genetics tools for these viruses it was unknown if these vesicles represented unproductive, abortive infections or a bona fide stage in the phage life cycle. Using the recently-developed dRfxCas13d-based knockdown system CRISPRi-ART ¹¹ in combination with fluorescence microscopy and cryo-electron tomography, we show that inhibiting phage nucleus formation arrests infections at an early stage in which the injected phage genome is enclosed within a membrane-bound early phage infection (EPI) vesicle. We demonstrate that early phage genes are transcribed by the virion-associated RNA polymerase from the genome within the compartment, making the EPI vesicle the first known example of a lipid membrane-bound organelle that separates transcription from translation in prokaryotes. Further, we show that the phage nucleus is essential for the phage life cycle, with genome replication only beginning after the injected DNA is transferred from the EPI vesicle to the newly assembled phage nucleus. Our results show that Chimalliviridae require two sophisticated subcellular compartments of distinct compositions and functions that facilitate successive stages of the viral life cycle.

Direct electrical stimulation has been used for decades as a gold standard clinical tool to map cognitive function in neurosurgery patients ^1-8 . However, the molecular impact of electrical stimulation in the human brain is unknown. Here, using state-of-the-art transcriptomic and epigenomic sequencing techniques, we define the molecular changes in bulk tissue and at the single-cell level in the human cerebral cortex following direct electrical stimulation of the anterior temporal lobe in patients undergoing neurosurgery. Direct electrical stimulation surprisingly had a robust and consistent impact on the expression of genes related to microglia-specific cytokine activity, an effect that was replicated in mice. Using a newly developed deep learning computational tool, we further demonstrate cell type-specific molecular activation, which underscores the effects of electrical stimulation on gene expression in microglia. Taken together, this work challenges the notion that the immediate impact of electrical stimulation commonly used in the clinic has a primary effect on neuronal gene expression and reveals that microglia robustly respond to electrical stimulation, thus enabling these non-neuronal cells to sculpt and shape the activity of neuronal circuits in the human brain.

A key challenge in neuroscience is to understand the structural and functional relationships of the brain from high-dimensional, multimodal neuroimaging data. While conventional multivariate approaches often simplify statistical assumptions and estimate one-dimensional independent sources shared across modalities, the relationships between true latent sources are likely more complex - statistical dependence may exist within and between modalities, and span one or more dimensions. Here we present Multimodal Subspace Independent Vector Analysis (MSIVA), a methodology to capture both joint and unique vector sources from multiple data modalities by defining both cross-modal and unimodal subspaces with variable dimensions. In particular, MSIVA enables flexible estimation of varying-size independent subspaces within modalities and their one-to-one linkage to corresponding sub-spaces across modalities. As we demonstrate, a main benefit of MSIVA is the ability to capture subject-level variability at the voxel level within independent subspaces, contrasting with the rigidity of traditional methods that share the same independent components across subjects. We compared MSIVA to a unimodal initialization baseline and a multimodal initialization baseline, and evaluated all three approaches with five candidate subspace structures on both synthetic and neuroimaging datasets. We show that MSIVA successfully identified the ground-truth subspace structures in multiple synthetic datasets, while the multimodal baseline failed to detect high-dimensional subspaces. We then demonstrate that MSIVA better detected the latent subspace structure in two large multimodal neuroimaging datasets including structural MRI (sMRI) and functional MRI (fMRI), compared with the unimodal baseline. From subsequent subspace-specific canonical correlation analysis, brain-phenotype prediction, and voxelwise brain-age delta analysis, our findings suggest that the estimated sources from MSIVA with optimal subspace structure are strongly associated with various phenotype variables, including age, sex, schizophrenia, lifestyle factors, and cognitive functions. Further, we identified modality- and group-specific brain regions related to multiple phenotype measures such as age (e.g., cerebellum, precentral gyrus, and cingulate gyrus in sMRI; occipital lobe and superior frontal gyrus in fMRI), sex (e.g., cerebellum in sMRI, frontal lobe in fMRI, and precuneus in both sMRI and fMRI), schizophrenia (e.g., cerebellum, temporal pole, and frontal operculum cortex in sMRI; occipital pole, lingual gyrus, and precuneus in fMRI), shedding light on phenotypic and neuropsychiatric biomarkers of linked brain structure and function.

Current therapeutic strategies for Alzheimer's disease (AD) target amyloid-beta (Aβ) fibrils and high molecular weight protofibrils associated with plaques, but molecular cascades associated with AD may drive neural systems failure before Aβ plaque deposition in AD. Employing hippocampal electrophysiological recordings and dynamic calcium imaging across the sleep-wake cycle in the APP/PS1 mouse model of AD before Aβ plaques accumulated, we detected marked impairments of hippocampal systems function: In a spatial behavioral task, but not REM sleep, phase-amplitude coupling (PAC) of the hippocampal theta and gamma oscillations was impaired and place cell calcium fluctuations were hyper-synchronized with the theta oscillation. In subsequent slow wave sleep (SWS), place cell reactivation was reduced. These degraded neural functions underlying memory encoding and consolidation support targeting pathological processes of the pre-plaque phase of AD to treat and prevent hippocampal impairments.