Sequential CRISPR screening reveals partial NatB inhibition as a strategy to mitigate alpha-synuclein levels in human neurons.

Alpha-synuclein (αSyn) protein levels correlate with the risk and severity of Parkinson's disease and related neurodegenerative diseases. Lowering αSyn is being actively investigated as a therapeutic modality. Here, we systematically map the regulatory network that controls endogenous αSyn using sequential CRISPR-knockout and -interference screens in an αSyn gene (SNCA)-tagged cell line and induced pluripotent stem cell-derived neurons (iNeurons). We uncover αSyn modifiers at multiple regulatory layers, with amino-terminal acetyltransferase B (NatB) enzymes being the most potent endogenous αSyn modifiers in both cell lines. Amino-terminal acetylation protects the cytosolic αSyn from rapid degradation by the proteasome in a Ube2w-dependent manner. Moreover, we show that pharmacological inhibition of methionyl-aminopeptidase 2, a regulator of NatB complex formation, attenuates endogenous αSyn in iNeurons carrying SNCA triplication. Together, our study reveals several gene networks that control endogenous αSyn, identifies mechanisms mediating the degradation of nonacetylated αSyn, and illustrates potential therapeutic pathways for decreasing αSyn levels in synucleinopathies.

A subpopulation of lipogenic brown adipocytes drives thermogenic memory.

Sustained responses to transient environmental stimuli are important for survival. The mechanisms underlying long-term adaptations to temporary shifts in abiotic factors remain incompletely understood. Here, we find that transient cold exposure leads to sustained transcriptional and metabolic adaptations in brown adipose tissue, which improve thermogenic responses to secondary cold encounter. Primary thermogenic challenge triggers the delayed induction of a lipid biosynthesis programme even after cessation of the original stimulus, which protects from subsequent exposures. Single-nucleus RNA sequencing and spatial transcriptomics reveal that this response is driven by a lipogenic subpopulation of brown adipocytes localized along the perimeter of Ucp1hi adipocytes. This lipogenic programme is associated with the production of acylcarnitines, and supplementation of acylcarnitines is sufficient to recapitulate improved secondary cold responses. Overall, our data highlight the importance of heterogenous brown adipocyte populations for 'thermogenic memory', which may have therapeutic implications for leveraging short-term thermogenesis to counteract obesity.

Podocalyxin-Like Protein 1 Regulates Pluripotency through the Cholesterol Biosynthesis Pathway.

Deciphering signaling mechanisms critical for the extended pluripotent stem cell (EPSC) state and primed pluripotency is necessary for understanding embryonic development. Here, a membrane protein, podocalyxin-like protein 1 (PODXL) as being essential for extended and primed pluripotency, is identified. Alteration of PODXL expression levels affects self-renewal, protein expression of c-MYC and telomerase, and induced pluripotent stem cell (iPSC) and EPSC colony formation. PODXL is the first membrane protein reported to regulate de novo cholesterol biosynthesis, and human pluripotent stem cells (hPSCs) are more sensitive to cholesterol depletion than fibroblasts. The addition of exogenous cholesterol fully restores PODXL knockdown-mediated loss of pluripotency. PODXL affects lipid raft dynamics via the regulation of cholesterol. PODXL recruits the RAC1/CDC42/actin network to regulate SREBP1 and SREBP2 maturation and lipid raft dynamics. Single-cell RNA sequencing reveals PODXL overexpression enhanced chimerism between human cells in mouse host embryos (hEPSCs 57%). Interestingly, in the human-mouse chimeras, laminin and collagen signaling-related pathways are dominant in PODXL overexpressing cells. It is concluded that cholesterol regulation via PODXL signaling is critical for ESC/EPSC.

3D spatial genome organization in the nervous system: From development and plasticity to disease.

Chromatin is organized into multiscale three-dimensional structures, including chromosome territories, A/B compartments, topologically associating domains, and chromatin loops. This hierarchically organized genomic architecture regulates gene transcription, which, in turn, is essential for various biological processes during brain development and adult plasticity. Here, we review different aspects of spatial genome organization and their functions in regulating gene expression in the nervous system, as well as their dysregulation in brain disorders. We also highlight new technologies to probe and manipulate chromatin architecture and discuss how investigating spatial genome organization can lead to a better understanding of the nervous system and associated disorders.

Generation of hypothalamic arcuate organoids from human induced pluripotent stem cells.

Human brain organoids represent remarkable platforms for recapitulating features of human brain development and diseases. Existing organoid models do not resolve fine brain subregions, such as different nuclei in the hypothalamus. We report the generation of arcuate organoids (ARCOs) from human induced pluripotent stem cells (iPSCs) to model the development of the human hypothalamic arcuate nucleus. Single-cell RNA sequencing of ARCOs revealed significant molecular heterogeneity underlying different arcuate cell types, and machine learning-aided analysis based on the neonatal human hypothalamus single-nucleus transcriptome further showed a human arcuate nucleus molecular signature. We also explored ARCOs generated from Prader-Willi syndrome (PWS) patient iPSCs. These organoids exhibit aberrant differentiation and transcriptomic dysregulation similar to postnatal hypothalamus of PWS patients, indicative of cellular differentiation deficits and exacerbated inflammatory responses. Thus, patient iPSC-derived ARCOs represent a promising experimental model for investigating nucleus-specific features and disease-relevant mechanisms during early human arcuate development.

Human Pluripotent Stem Cell-Derived Neural Cells and Brain Organoids Reveal SARS-CoV-2 Neurotropism Predominates in Choroid Plexus Epithelium.

Neurological complications are common in patients with COVID-19. Although severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal pathogen of COVID-19, has been detected in some patient brains, its ability to infect brain cells and impact their function is not well understood. Here, we investigated the susceptibility of human induced pluripotent stem cell (hiPSC)-derived monolayer brain cells and region-specific brain organoids to SARS-CoV-2 infection. We found that neurons and astrocytes were sparsely infected, but choroid plexus epithelial cells underwent robust infection. We optimized a protocol to generate choroid plexus organoids from hiPSCs and showed that productive SARS-CoV-2 infection of these organoids is associated with increased cell death and transcriptional dysregulation indicative of an inflammatory response and cellular function deficits. Together, our findings provide evidence for selective SARS-CoV-2 neurotropism and support the use of hiPSC-derived brain organoids as a platform to investigate SARS-CoV-2 infection susceptibility of brain cells, mechanisms of virus-induced brain dysfunction, and treatment strategies.

Human Pluripotent Stem Cell-Derived Neural Cells and Brain Organoids Reveal SARS-CoV-2 Neurotropism.

Neurological complications are common in patients with COVID-19. While SARS-CoV-2, the causal pathogen of COVID-19, has been detected in some patient brains, its ability to infect brain cells and impact their function are not well understood, and experimental models using human brain cells are urgently needed. Here we investigated the susceptibility of human induced pluripotent stem cell (hiPSC)-derived monolayer brain cells and region-specific brain organoids to SARS-CoV-2 infection. We found modest numbers of infected neurons and astrocytes, but greater infection of choroid plexus epithelial cells. We optimized a protocol to generate choroid plexus organoids from hiPSCs, which revealed productive SARS-CoV-2 infection that leads to increased cell death and transcriptional dysregulation indicative of an inflammatory response and cellular function deficits. Together, our results provide evidence for SARS-CoV-2 neurotropism and support use of hiPSC-derived brain organoids as a platform to investigate the cellular susceptibility, disease mechanisms, and treatment strategies for SARS-CoV-2 infection.

Sliced Human Cortical Organoids for Modeling Distinct Cortical Layer Formation.

Human brain organoids provide unique platforms for modeling development and diseases by recapitulating the architecture of the embryonic brain. However, current organoid methods are limited by interior hypoxia and cell death due to insufficient surface diffusion, preventing generation of architecture resembling late developmental stages. Here, we report the sliced neocortical organoid (SNO) system, which bypasses the diffusion limit to prevent cell death over long-term cultures. This method leads to sustained neurogenesis and formation of an expanded cortical plate that establishes distinct upper and deep cortical layers for neurons and astrocytes, resembling the third trimester embryonic human neocortex. Using the SNO system, we further identify a critical role of WNT/ß-catenin signaling in regulating human cortical neuron subtype fate specification, which is disrupted by a psychiatric-disorder-associated genetic mutation in patient induced pluripotent stem cell (iPSC)-derived SNOs. These results demonstrate the utility of SNOs for investigating previously inaccessible human-specific, late-stage cortical development and disease-relevant mechanisms.

Trends in the Use of Common Words and Patient-Centric Language in the Titles of Medical Journals, 1976-2015.

Importance: The language of medical research appears to be intrinsically tied to the culture of medical research and provides a unique window into broader trends in the culture of medicine. Objective: To analyze medical language from 5 premier medical journals and investigate broader changes in the culture of clinical investigation during the last 40 years. Design, Setting, and Participants: In this qualitative study using a data-driven analysis, 302 293 PubMed records were extracted from JAMA, The Lancet, Annals of Internal Medicine, the BMJ, and New England Journal of Medicine from January 1, 1976, through December 31, 2015, to identify key trends in medical language. A frequency analysis was applied across the 40-year time frame in JAMA to assess the major trends in all publication types. Patient-centered language was analyzed in clinical trials in the flanking time periods (1976-1980 and 2011-2015) across the 5 journals. Data were analyzed from November 16, 2016, through November 9, 2018. Main Outcomes and Measures: Increasing or decreasing frequency of words (monograms) and word pairs (bigrams) and the proportion of patient-centric words in journal article titles. Results: In JAMA, 50 277 articles of all publication types were included. In the frequency analysis, the most increased terms were reflective of the language of epidemiological research. The bigram analysis revealed a decline in causal language (-2.42/100 000 words to -2.03/100 000 words; false discovery rate [FDR], <0.01) and an increased description of patients in the plural form (6.92/100 000 words to 11.4/100 000 words; FDR, <0.01). A trend to separate patient from disease was observed; for example, there was a decrease in describing a patient as a diabetic (-2.21/100 000 words; FDR, <0.01) compared with a patient with diabetes. In the analysis of clinical trials in all 5 journals, 3125 titles were identified (range, 193-932 per journal). In 4 of the 5 journals, use of patient-centric keywords increased significantly (absolute increase, 18.9%-34.3%; P < .001 for 3 journals; P = .01 for 1 journal), with the New England Journal of Medicine as the exception. This finding reflects a change from shorter disease-centric titles to longer titles that describe patients with a disease. Conclusions and Relevance: Trends in medical language reflect the rise of evidence-based medicine, a shift in focus from individuals to populations, and a separation of patient and disease. Data-driven analysis of medical language provides a unique window into the changing landscape of medical culture.

Tankyrase inhibition promotes a stable human naïve pluripotent state with improved functionality.

The derivation and maintenance of human pluripotent stem cells (hPSCs) in stable naïve pluripotent states has a wide impact in human developmental biology. However, hPSCs are unstable in classical naïve mouse embryonic stem cell (ESC) WNT and MEK/ERK signal inhibition (2i) culture. We show that a broad repertoire of conventional hESC and transgene-independent human induced pluripotent stem cell (hiPSC) lines could be reverted to stable human preimplantation inner cell mass (ICM)-like naïve states with only WNT, MEK/ERK, and tankyrase inhibition (LIF-3i). LIF-3i-reverted hPSCs retained normal karyotypes and genomic imprints, and attained defining mouse ESC-like functional features, including high clonal self-renewal, independence from MEK/ERK signaling, dependence on JAK/STAT3 and BMP4 signaling, and naïve-specific transcriptional and epigenetic configurations. Tankyrase inhibition promoted a stable acquisition of a human preimplantation ICM-like ground state via modulation of WNT signaling, and was most efficacious in efficiently reprogrammed conventional hiPSCs. Importantly, naïve reversion of a broad repertoire of conventional hiPSCs reduced lineage-primed gene expression and significantly improved their multilineage differentiation capacities. Stable naïve hPSCs with reduced genetic variability and improved functional pluripotency will have great utility in regenerative medicine and human disease modeling.