Selected Grantee Publications
Two Neuronal Peptides Encoded from a Single Transcript Regulate Mitochondrial Complex III in Drosophila
Bosch et al., eLife. 2022.
https://www.doi.org/10.7554/eLife.82709
Transcripts with small open-reading frames (smORFs) are underrepresented in genome annotations. Functions of peptides encoded by smORFs are poorly understood. The investigators systematically characterized human-conserved smORF genes in Drosophila and found two peptides, Sloth1 and Sloth2, that are highly expressed in neurons. They showed that Sloth1 and Sloth2 are paralogs with high sequence similarity but are not functionally redundant. Loss of either peptide resulted in lethality, impaired mitochondrial function, and neurodegeneration. This work suggests the value of phenotypic analysis of smORFs using Drosophila as a model. Supported by ORIP (R24OD019847), NHGRI, and NIGMS.
Cell-Specific Regulation of Gene Expression Using Splicing-Dependent Frameshifting
Ling et al., Nature Communications. 2022.
https://www.doi.org/10.1038/s41467-022-33523-2
Precise and reliable cell-specific gene delivery remains technically challenging. Investigators report a splicing-based approach for controlling gene expression whereby separate translational reading frames are coupled to the inclusion or exclusion of mutated, frameshifting cell-specific alternative exons. Candidate exons are identified by analyzing thousands of publicly available RNA sequencing datasets and filtering by cell specificity, conservation, and local intron length. This method, which they denote as splicing-linked expression design (SLED), can be combined in a Boolean manner with such existing techniques as minipromoters and viral capsids. SLED can use strong constitutive promoters, without sacrificing precision, by decoupling the tradeoff between promoter strength and selectivity. AAV-packaged SLED vectors can selectively deliver fluorescent reporters and calcium indicators to various neuronal subtypes in vivo. The authors also demonstrate gene therapy utility by creating SLED vectors that can target PRPH2 and SF3B1 mutations. The flexibility of SLED technology enables creative avenues for basic and translational research. Supported by ORIP (T32OD011089, S10OD026859), NEI, and NIMH.
Rapid Joule Heating Improves Vitrification Based Cryopreservation
Zhan et al., Nature Communications. 2022.
https://www.doi.org/10.1038/s41467-022-33546-9
Cryopreservation by vitrification is an effective approach for long-term preservation of biosystems, but effective vitrification often requires high concentrations of cryoprotective agent (CPA), which can be toxic. The investigators described a joule heating–based platform technology for rapid rewarming of biosystems, which allows the use of low concentrations of CPA. They demonstrated the success of this platform in cryopreservation of three model systems: adherent cells, Drosophila melanogaster embryos, and rat kidney slices with low CPA concentrations. This work provides a general solution to cryopreserve a broad spectrum of cells, tissues, organs, and organisms. Supported by ORIP (R21OD028758), NIDDK, NHLBI, and NIGMS.
Promoting Validation and Cross-Phylogenetic Integration in Model Organism Research
Cheng et al., Disease Models & Mechanisms. 2022.
https://www.doi.org/10.1242/dmm.049600
Model organisms are essential for biomedical research and therapeutic development, but translation of such research to the clinic is low. The authors summarized discussions from an NIH virtual workshop series, titled “Validation of Animal Models and Tools for Biomedical Research,” held from 2020 to 2021. They described challenges and opportunities for developing and integrating tools and resources and provided suggestions for improving the rigor, validation, reproducibility, and translatability of model organism research. Supported by ORIP (R01OD011116, R24OD031447, R03OD030597, R24OD018559, R24OD017870, R24OD026591, R24OD022005, U42OD026645, U42OD012210, U54OD030165, UM1OD023221, P51OD011107), NIAMS, NIDDK, NIGMS, NHGRI, and NINDS.
Molecular and Cellular Evolution of the Primate Dorsolateral Prefrontal Cortex
Ma et al., Science. 2022.
https://www.doi.org/10.1126/science.abo7257
The dorsolateral prefrontal cortex (dlPFC) exists only in primates, lies at the center of high-order cognition, and is a locus of pathology underlying many neuropsychiatric diseases. The investigators generated single-nucleus transcriptome data profiling more than 600,000 nuclei from the dlPFC of adult humans, chimpanzees, rhesus macaques, and common marmosets of both sexes. Postmortem human samples were obtained from tissue donors. The investigators’ analyses delineated dlPFC cell-type homology and transcriptomic conservation across species and identified species divergence at the molecular and cellular levels, as well as potential epigenomic mechanisms underlying these differences. Expression patterns of more than 900 genes associated with brain disorders revealed a variety of conserved, divergent, and group-specific patterns. The resulting data resource will help to vertically integrate marmoset and macaque models with human-focused efforts to develop treatments for neuropsychiatric conditions. Supported by ORIP (P51OD011133), NIA, NICHD, NIDA, NIGMS, NHGRI, NIMH, and NINDS.
Rbbp4 Loss Disrupts Neural Progenitor Cell Cycle Regulation Independent of Rb and Leads to Tp53 Acetylation and Apoptosis
Schultz-Rogers et al., Developmental Dynamics. 2022.
https://www.doi.org/10.1002/dvdy.467
Retinoblastoma binding protein 4 (Rbbp4) is a component of transcription regulatory complexes that control cell cycle gene expression by cooperating with the Rb tumor suppressor to block cell cycle entry. The authors used genetic analysis to examine the interactions of Rbbp4, Rb, and Tp53 in zebrafish neural progenitor cell cycle regulation and survival. Rbbp4 is upregulated across the spectrum of human embryonal and glial brain cancers, and it is essential for zebrafish neurogenesis. Rbbp4 loss leads to apoptosis and γ-H2AX in the developing brain that is suppressed by tp53 knockdown or maternal zygotic deletion. Mutant retinal neural precursors accumulate in M phase and fail to initiate G0 gene expression. Rbbp4; Rb1 double mutants show an additive effect on the number of M phase cells. The study demonstrates that Rbbp4 is necessary for neural progenitor cell cycle progression and initiation of G0, independent of Rb, and suggests that Rbbp4 is required for cell cycle exit and contributes to neural progenitor survival. Supported by ORIP (R24OD020166) and NIGMS.
Parallel Processing, Hierarchical Transformations, and Sensorimotor Associations along the “Where” Pathway
Doudlah et al., eLife. 2022.
https://www.doi.org/10.7554/eLife.78712
Visually guided behaviors require the brain to transform ambiguous retinal images into object-level spatial representations and map those representations to motor responses. These capabilities are supported by the dorsal “where” pathway in the brain, but the specific contributions of areas along this pathway have remained elusive. Using a rhesus macaque model, researchers compared neuronal activity in two areas along the “where” pathway that bridge the parieto-occipital junction: intermediate visual area V3A and the caudal intraparietal (CIP) area. Neuronal activity was recorded while the animals made perceptual decisions based on judging the tilt of 3D visual patterns. The investigators found that CIP shows higher-order spatial representations and more choice-correlated responses, which support a V3A-to-CIP hierarchy. The researchers also discovered modulation of V3A activity by extraretinal factors, suggesting that V3A might be better characterized as contributing to higher-order behavioral functions rather than low-level visual feature processing. Supported by ORIP (P51OD011106), NEI, NICHD, and NINDS.
Infection Order Outweighs the Role of CD4+ T Cells in Tertiary Flavivirus Exposure
Marzan-Rivera et al., iScience. 2022.
https://www.doi.org/10.1016/j.isci.2022.104764
The link between CD4+ T and B cells in immune responses to Dengue virus (DENV) and Zika virus (ZIKV) and their roles in cross-protection during heterologous infection are poorly known. The authors used CD4+ lymphocyte depletions to dissect the impact of cellular immunity on humoral responses during tertiary flavivirus infection in male macaques. CD4+ depletion in DENV/ZIKV–primed animals, followed by DENV, resulted in dysregulated adaptive immune responses. They show a delay in DENV-specific antibody titers and binding and neutralization in the DENV/ZIKV–primed, CD4-depleted animals but not in ZIKV/DENV–primed, CD4-depleted animals. This study confirms the role of CD4+ cells in priming an early humoral response during sequential flavivirus infections and suggests that the order of exposure affects the outcome of a tertiary infection. Supported by ORIP (P40OD012217), NIAID, and NIGMS.
Effects of Ex Vivo Blood Anticoagulation and Preanalytical Processing Time on the Proteome Content of Platelets
Yunga et al., Journal of Thrombosis and Haemostasis. 2022.
https://www.doi.org/10.1111/jth.15694
The investigators studied how various blood anticoagulation options and processing times affect platelet function and protein content ex vivo. Using platelet proteome quantification and triple quadrupole mass spectrometry, they found that anticoagulant-specific effects on platelet proteomes included increased complement system and decreased α-granule proteins in platelets from EDTA-anticoagulated blood. Heparinized blood had higher levels of histone and neutrophil-associated proteins, as well as formation of platelet–neutrophil extracellular trap interactions in whole blood ex vivo. The study indicates that different anticoagulants and preanalytical processing times affect platelet function and platelet protein content ex vivo, suggesting more rigorous phenotyping strategies for platelet omics studies. Supported by ORIP (S10OD012246), NHLBI, NCI and NEI.
Obesity Alters Pathology and Treatment Response in Inflammatory Disease
Bapat et al., Nature. 2022.
https://www.doi.org/10.1038/s41586-022-04536-0
Obesity and metabolic disease have been shown to affect immunotherapeutic outcomes. By studying classical type 2 T helper cells (TH2) in lean and obese male mouse models for atopic dermatitis, investigators found that the biologic therapies protected lean mice but exacerbated disease in obese mice. RNA sequencing and genome analyses revealed decreased activity of nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ) in TH2 cells in obese mice when compared to lean mice, indicating that PPARγ is required to prevent aberrant non-TH2 inflammation. Understanding the effects of obesity on immunological disease could inform a potential precision medicine approach to target obesity-induced immune dysregulation. Supported by ORIP (S10OD023689), NIAID, NCI, NIDDK, and NIGMS.