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Experimental cell therapies for skeletal muscle conditions have shown little success, primarily because they use committed myogenic progenitors rather than true muscle stem cells, known as satellite cells. Here we present a method to generate in vitro-derived satellite cells (idSCs) from skeletal muscle tissue. When transplanted in small numbers into mouse muscle, mouse idSCs fuse into myofibers, repopulate the satellite cell niche, self-renew, support multiple rounds of muscle regeneration and improve force production on par with freshly isolated satellite cells in damaged skeletal muscle. We compared the epigenomic and transcriptional signatures between idSCs, myoblasts and satellite cells and used these signatures to identify core signaling pathways and genes that confer idSC functionality. Finally, from human muscle biopsies, we successfully generated satellite cell-like cells in vitro. After further development, idSCs may provide a scalable source of cells for the treatment of genetic muscle disorders, trauma-induced muscle damage and age-related muscle weakness., (© 2024. The Author(s).)

This study identified tumorigenic processes most dependent on murine heat shock protein 72 (HSP72) in the mouse mammary tumor virus-PyMT mammary tumor model, which give rise to spontaneous mammary tumors that exhibit HSP72-dependent metastasis to the lung. RNA-seq expression profiling of Hspa1a/Hspa1b (Hsp72) WT and Hsp72 -/- primary mammary tumors discovered significantly lower expression of genes encoding components of the extracellular matrix (ECM) in Hsp72 knockout mammary tumors compared to WT controls. In vitro studies found that genetic or chemical inhibition of HSP72 activity in cultured collagen-expressing human or murine cells also reduces mRNA and protein levels of COL1A1 and several other ECM-encoding genes. In search of a possible mechanistic basis for this relationship, we found HSP72 to support the activation of the tumor growth factor-β-suppressor of mothers against decapentaplegic-3 signaling pathway and evidence of suppressor of mothers against decapentaplegic-3 and HSP72 coprecipitation, suggesting potential complex formation. Human COL1A1 mRNA expression was found to have prognostic value for HER2+ breast tumors over other breast cancer subtypes, suggesting a possible human disease context where targeting HSP72 may have a therapeutic rationale. Analysis of human HER2+ breast tumor gene expression data using a gene set comprising ECM-related gene and protein folding-related gene as an input to the statistical learning algorithm, Galgo, found a subset of these genes that can collectively stratify patients by relapse-free survival, further suggesting a potential interplay between the ECM and protein-folding genes may contribute to tumor progression., Competing Interests: Declarations of interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jason E. Gestwicki has patent (related to HSP70 inhibitors) issued to Regents of the University of California. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Amyotrophic lateral sclerosis (ALS) is a degenerative motor neuron (MN) disease with severely limited treatment options. Identification of effective treatments has been limited in part by the lack of predictive animal models for complex human disorders. Here, we utilized pharmacologic ER stressors to exacerbate underlying sensitivities conferred by ALS patient genetics in induced pluripotent stem cell (iPSC)-derived motor neurons (MNs). In doing so, we found that thapsigargin and tunicamycin exposure recapitulated ALS-associated degeneration, and that we could rescue this degeneration via MAP4K4 inhibition (MAP4K4i). We subsequently identified mechanisms underlying MAP4K4i-mediated protection by performing phosphoproteomics on iPSC-derived MNs treated with ER stressors ±MAP4K4i. Through these analyses, we found JNK, PKC, and BRAF to be differentially modulated in MAP4K4i-protected MNs, and that inhibitors to these proteins could also rescue MN toxicity. Collectively, this study highlights the value of utilizing ER stressors in ALS patient MNs to identify novel druggable targets., Competing Interests: JH was a founder and consultant for Caraway Therapeutics and a founding scientific advisor for Interline Therapeutics. LR was a founder of Elevian, Rejuveron, and Vesalius Therapeutics, a member of their scientific advisory boards and a private equity shareholder. All are interested in formulating approaches intended to treat diseases of the nervous system and other tissues. He is also on the advisory board of Alkahest, a Grifols company, focused on the plasma proteome, and scientific advisory board member of ProjenX and Corsalex. None of these companies provided any financial support for the work in this manuscript. However, both ProjenX and Corsalex are focused on treating ALS, including via modulating targets that are described in this manuscript. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Watts, Giadone, Ordureau, Holton, Harper and Rubin.)

Alzheimer's Disease (AD) is a complex neurodegenerative disease resulting in progressive loss of memory, language and motor abilities caused by cortical and hippocampal degeneration. This review captures the landscape of understanding of AD pathology, diagnostics, and current therapies. Two major mechanisms direct AD pathology: (1) accumulation of amyloid β (Aβ) plaque and (2) tau-derived neurofibrillary tangles (NFT). The most common variants in the Aβ pathway in APP , PSEN1, and PSEN2 are largely responsible for early-onset AD (EOAD), while MAPT , APOE , TREM2 and ABCA7 have a modifying effect on late-onset AD (LOAD). More recent studies implicate chaperone proteins and Aβ degrading proteins in AD. Several tests, such as cognitive function, brain imaging, and cerebral spinal fluid (CSF) and blood tests, are used for AD diagnosis. Additionally, several biomarkers seem to have a unique AD specific combination of expression and could potentially be used in improved, less invasive diagnostics. In addition to genetic perturbations, environmental influences, such as altered gut microbiome signatures, affect AD. Effective AD treatments have been challenging to develop. Currently, there are several FDA approved drugs (cholinesterase inhibitors, Aß-targeting antibodies and an NMDA antagonist) that could mitigate AD rate of decline and symptoms of distress.

Cortical thickness reductions differ between individuals with psychotic disorders and comparison subjects even in early stages of illness. Whether these reductions covary as expected by functional network membership or simply by spatial proximity has not been fully elucidated. Through orthonormal projective non-negative matrix factorization, cortical thickness measurements in functionally-annotated regions from MRI scans of early-stage psychosis and matched healthy controls were reduced in dimensionality into features capturing positive covariance. Rather than matching the functional networks, the covarying regions in each feature displayed a more localized spatial organization. With Bayesian belief networks, the covarying regions per feature were arranged into a network topology to visualize the dependency structure and identify key driving regions. The features demonstrated diagnosis-specific differences in cortical thickness distributions per feature, identifying reduction-vulnerable spatial regions. Differences in key cortical thickness features between psychosis and control groups were delineated, as well as those between affective and non-affective psychosis. Clustering of the participants, stratified by diagnosis and clinical variables, characterized the clinical traits that define the cortical thickness patterns. Longitudinal follow-up revealed that in select clusters with low baseline cortical thickness, clinical traits improved over time. Our study represents a novel effort to characterize brain structure in relation to functional networks in healthy and clinical populations and to map patterns of cortical thickness alterations among ESP patients onto clinical variables for a better understanding of brain pathophysiology., Competing Interests: Declaration of Competing Interest D.O acknowledges receipt of an honorarium from Neumora Inc. for scientific presentation in 1/2022. The rest of the authors declare no competing interests., (Copyright © 2023. Published by Elsevier Inc.)

Parkinson's disease (PD) is a complex neurodegenerative disease with etiology rooted in genetic vulnerability and environmental factors. Here we combine quantitative epidemiologic study of pesticide exposures and PD with toxicity screening in dopaminergic neurons derived from PD patient induced pluripotent stem cells (iPSCs) to identify Parkinson's-relevant pesticides. Agricultural records enable investigation of 288 specific pesticides and PD risk in a comprehensive, pesticide-wide association study. We associate long-term exposure to 53 pesticides with PD and identify co-exposure profiles. We then employ a live-cell imaging screening paradigm exposing dopaminergic neurons to 39 PD-associated pesticides. We find that 10 pesticides are directly toxic to these neurons. Further, we analyze pesticides typically used in combinations in cotton farming, demonstrating that co-exposures result in greater toxicity than any single pesticide. We find trifluralin is a driver of toxicity to dopaminergic neurons and leads to mitochondrial dysfunction. Our paradigm may prove useful to mechanistically dissect pesticide exposures implicated in PD risk and guide agricultural policy., (© 2023. The Author(s).)

Resolving fundamental molecular and functional processes underlying human synaptic development is crucial for understanding normal brain function as well as dysfunction in disease. Based upon increasing evidence of species-divergent features of brain cell types, coupled with emerging studies of complex human disease genetics, we developed the first automated and quantitative high-content synaptic phenotyping platform using human neurons and astrocytes. To establish the robustness of our platform, we screened the effects of 376 small molecules on presynaptic density, neurite outgrowth, and cell viability, validating six small molecules that specifically enhanced human presynaptic density in vitro. Astrocytes were essential for mediating the effects of all six small molecules, underscoring the relevance of non-cell-autonomous factors in synapse assembly and their importance in synaptic screening applications. Bromodomain and extraterminal (BET) inhibitors emerged as the most prominent hit class and global transcriptional analyses using multiple BET inhibitors confirmed upregulation of synaptic gene expression. Through these analyses, we demonstrate the robustness of our automated screening platform for identifying potent synaptic modulators, which can be further leveraged for scaled analyses of human synaptic mechanisms and drug discovery efforts., Competing Interests: MB, GR, AN, JK, DT, SS, DL, AM, KH, KK, BC, KP, AC, LB No competing interests declared, LR Reviewing editor, eLife. LLR is a founder of Elevian, Rejuveron, and Vesalius Therapeutics, a member of their scientific advisory boards and a private equity shareholder. All are interested in formulating approaches intended to treat diseases of the nervous system and other tissues. He is also on the advisory board of Alkahest, a Grifols company, focused on the plasma proteome and brain aging. None of these companies provided any financial support for the work in this paper, (© 2023, Berryer et al.)

Aging is a complex process involving transcriptomic changes associated with deterioration across multiple tissues and organs, including the brain. Recent studies using heterochronic parabiosis have shown that various aspects of aging-associated decline are modifiable or even reversible. To better understand how this occurs, we performed single-cell transcriptomic profiling of young and old mouse brains after parabiosis. For each cell type, we cataloged alterations in gene expression, molecular pathways, transcriptional networks, ligand-receptor interactions and senescence status. Our analyses identified gene signatures, demonstrating that heterochronic parabiosis regulates several hallmarks of aging in a cell-type-specific manner. Brain endothelial cells were found to be especially malleable to this intervention, exhibiting dynamic transcriptional changes that affect vascular structure and function. These findings suggest new strategies for slowing deterioration and driving regeneration in the aging brain through approaches that do not rely on disease-specific mechanisms or actions of individual circulating factors., (© 2023. The Author(s).)

RNA sequencing (RNA-seq) is a powerful method of transcriptional analysis that allows for the sequence identification and quantification of cellular transcripts. RNA-seq can be used for differential gene expression (DGE) analysis, gene fusion detection, allele-specific expression, isoform and splice variant quantification, and identification of novel genes. These applications can be used for downstream systems biology analyses such as gene ontology or pathway analysis to provide insight into processes altered between biological conditions. Given the wide range of signaling pathways subject to chaperone activity as well as numerous chaperone functions in RNA metabolism, RNA-seq may provide a valuable tool for the study of chaperone proteins in biology and disease. This chapter outlines an example RNA-seq workflow to determine differentially expressed (DE) genes between two or more sample conditions and provides some considerations for RNA-seq experimental design., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Delivery of exogenous heat shock protein 90α (Hsp90α) and/or its induced expression in neural tissues has been suggested as a potential strategy to combat neurodegenerative disease. However, within a neurodegenerative context, a pro-inflammatory response to extracellular Hsp90α (eHsp90α) could undermine strategies to use it for therapeutic intervention. The aim of this study was to investigate the biological effects of eHsp90α on microglial cells, the primary mediators of inflammatory responses in the brain. Transcriptomic profiling by RNA-seq of primary microglia and the cultured EOC2 microglial cell line treated with eHsp90α showed the chaperone to stimulate activation of innate immune responses in microglia that were characterized by an increase in NF-kB-regulated genes. Further characterization showed this response to be substantially lower in amplitude than the effects of other inflammatory stimuli such as fibrillar amyloid-β (fAβ) or lipopolysaccharide (LPS). Additionally, the toxicity of conditioned media obtained from microglia treated with fAβ was attenuated by addition of eHsp90α. Using a co-culture system of microglia and hippocampal neuronal cell line HT22 cells separated by a chamber insert, the neurotoxicity of medium conditioned by microglia treated with fAβ was reduced when eHsp90α was also added. Mechanistically, eHsp90α was shown to activate Nrf2, a response which attenuated fAβ-induced nitric oxide production. The data thus suggested that eHsp90α protects against fAβ-induced oxidative stress. We also report eHsp90α to induce expression of macrophage receptor with collagenous structure (Marco), which would permit receptor-mediated endocytosis of fAβ., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

A hallmark of cells comprising the superficial zone of articular cartilage is their expression of lubricin, encoded by the Prg4 gene, that lubricates the joint and protects against the development of arthritis. Here, we identify Creb5 as a transcription factor that is specifically expressed in superficial zone articular chondrocytes and is required for TGF-β and EGFR signaling to induce Prg4 expression. Notably, forced expression of Creb5 in chondrocytes derived from the deep zone of the articular cartilage confers the competence for TGF-β and EGFR signals to induce Prg4 expression. Chromatin-IP and ATAC-Seq analyses have revealed that Creb5 directly binds to two Prg4 promoter-proximal regulatory elements, that display an open chromatin conformation specifically in superficial zone articular chondrocytes; and which work in combination with a more distal regulatory element to drive induction of Prg4 by TGF-β. Our results indicate that Creb5 is a critical regulator of Prg4/lubricin expression in the articular cartilage.

Background: Galloway-Mowat syndrome (GAMOS) is characterized by neurodevelopmental defects and a progressive nephropathy, which typically manifests as steroid-resistant nephrotic syndrome. The prognosis of GAMOS is poor, and the majority of children progress to renal failure. The discovery of monogenic causes of GAMOS has uncovered molecular pathways involved in the pathogenesis of disease., Methods: Homozygosity mapping, whole-exome sequencing, and linkage analysis were used to identify mutations in four families with a GAMOS-like phenotype, and high-throughput PCR technology was applied to 91 individuals with GAMOS and 816 individuals with isolated nephrotic syndrome. In vitro and in vivo studies determined the functional significance of the mutations identified., Results: Three biallelic variants of the transcriptional regulator PRDM15 were detected in six families with proteinuric kidney disease. Four families with a variant in the protein's zinc-finger (ZNF) domain have additional GAMOS-like features, including brain anomalies, cardiac defects, and skeletal defects. All variants destabilize the PRDM15 protein, and the ZNF variant additionally interferes with transcriptional activation. Morpholino oligonucleotide-mediated knockdown of Prdm15 in Xenopus embryos disrupted pronephric development. Human wild-type PRDM15 RNA rescued the disruption, but the three PRDM15 variants did not. Finally, CRISPR-mediated knockout of PRDM15 in human podocytes led to dysregulation of several renal developmental genes., Conclusions: Variants in PRDM15 can cause either isolated nephrotic syndrome or a GAMOS-type syndrome on an allelic basis. PRDM15 regulates multiple developmental kidney genes, and is likely to play an essential role in renal development in humans., (Copyright © 2021 by the American Society of Nephrology.)

Motivation: Statistical and machine-learning analyses of tumor transcriptomic profiles offer a powerful resource to gain deeper understanding of tumor subtypes and disease prognosis. Currently, prognostic gene-expression signatures do not exist for all cancer types, and most developed to date have been optimized for individual tumor types. In Galgo, we implement a bi-objective optimization approach that prioritizes gene signature cohesiveness and patient survival in parallel, which provides greater power to identify tumor transcriptomic phenotypes strongly associated with patient survival., Results: To compare the predictive power of the signatures obtained by Galgo with previously studied subtyping methods, we used a meta-analytic approach testing a total of 35 large population-based transcriptomic biobanks of four different cancer types. Galgo-generated colorectal and lung adenocarcinoma signatures were stronger predictors of patient survival compared to published molecular classification schemes. One Galgo-generated breast cancer signature outperformed PAM50, AIMS, SCMGENE and IntClust subtyping predictors. In high-grade serous ovarian cancer, Galgo signatures obtained similar predictive power to a consensus classification method. In all cases, Galgo subtypes reflected enrichment of gene sets related to the hallmarks of the disease, which highlights the biological relevance of the partitions found., Availability and Implementation: The open-source R package is available on www.github.com/harpomaxx/galgo., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: There is a large variability in the recovery trajectory and outcome of first episode of psychosis [FEP] patients. To date, individuals' outcome trajectories at early stage of illness and potential risk factors associated with a poor outcome trajectory are largely unknown. This study aims to apply three separate predictors (positive symptoms, negative symptoms, and soft neurological signs) to identify homogeneous function outcome trajectories in patients with FEP using objective data-driven methods, and to explore the potential risk /protective factors associated with each trajectory., Methods: A total of 369 first episode patients (93% antipsychotic naive) were included in the baseline assessments and followed-up at 4-8 weeks, 6 months, and 1 year. K means cluster modeling for longitudinal data (kml3d) was used to identify distinct, homogeneous clusters of functional outcome trajectories. Patients with at least 3 assessments were included in the trajectory analyses (N = 129). The Scale for the Assessment of Negative Symptoms (SANS), Scale for the Assessment of Positive Symptoms (SAPS), and Neurological examination abnormalities (NEA) were used as predictors against Global Assessment of Functioning Scale (GAF)., Results: In each of the three predictor models, four distinct functional outcome trajectories emerged: "Poor", "Intermediate", High" and "Catch-up". Individuals with male gender; ethnic minority status; low premorbid adjustment; low executive function/IQ, low SES, personality disorder, substance use history may be risk factors for poor recovery., Conclusions: Functioning recovery in individuals with FEP is heterogeneous, although distinct recovery profiles are apparent. Data-driven trajectory analysis can facilitate better characterization of individual longitudinal patterns of functioning recovery., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Tyrosine kinase inhibitors (TKIs) are widely used to treat solid tumors but can be cardiotoxic. The molecular basis for this toxicity and its relationship to therapeutic mechanisms remain unclear; we therefore undertook a systems-level analysis of human cardiomyocytes (CMs) exposed to four TKIs. CMs differentiated from human induced pluripotent stem cells (hiPSCs) were exposed to sunitinib, sorafenib, lapatinib, or erlotinib, and responses were assessed by functional assays, microscopy, RNA sequencing, and mass spectrometry (GEO: GSE114686; PRIDE: PXD012043). TKIs have diverse effects on hiPSC-CMs distinct from inhibition of tyrosine-kinase-mediated signal transduction; cardiac metabolism is particularly sensitive. Following sorafenib treatment, oxidative phosphorylation is downregulated, resulting in a profound defect in mitochondrial energetics. Cells adapt by upregulating aerobic glycolysis. Adaptation makes cells less acutely sensitive to sorafenib but may have long-term negative consequences. Thus, CMs exhibit adaptive responses to anti-cancer drugs conceptually similar to those previously shown in tumors to mediate drug resistance., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Aggression and courtship behavior were examined of wild Drosophila melanogaster flies isolated from two contrasting microclimates found at Evolution Canyon in Mt. Carmel, Israel: an African-like dry tropical Slope (AS) and a European-like humid temperate Slope (ES), separated by 250 meters. Studies were carried out to ask whether behavioral differences existed between the two populations obtained from opposite slopes with divergent microclimates in Israel. First, we measured and compared intraslope aggression between same sex fly pairings collected from the same slope. Both male and female flies displayed similar fighting abilities from both slopes. ES males, however, from the humid biome, showed a tendency to lunge more per aggressive encounter, compared with AS males from the dry biome. Next, we tested interslope aggression by pairing flies from opposite slopes. ES males displayed higher numbers of lunges, and won more fights against their AS opponents. We also observed enhanced courtship performances in ES compared to AS males. The fighting and courtship superiority seen in ES males could reinforce fitness and pre-mating reproductive isolation mechanisms that underlie incipient sympatric speciation. This may support an evolutionary advantage of adaptively divergent fruit fly aggression phenotypes from different environments.

RNA sequencing (RNA-seq) is a powerful method of transcript analysis that allows for the sequence identification and quantification of cellular transcripts. RNA-seq has many applications including differential gene expression (DE) analysis, gene fusion detection, allele-specific expression, isoform and splice variant quantification, and identification of novel genes. These applications can be used for downstream systems biology analyses such as gene ontology analysis to provide insights into cellular processes altered between biological conditions. Given the wide range of signaling pathways subject to chaperone activity as well as numerous chaperone functions in RNA metabolism, RNA-seq may provide a valuable tool for the study of chaperone proteins in biology and disease. This chapter outlines an example RNA-seq workflow to determine differentially expressed (DE) genes between two or more sample conditions and provides some considerations for RNA-seq experimental design.

Balance in the transcriptome is regulated by coordinated synthesis and degradation of RNA molecules. Here we investigated whether mammalian cell types intrinsically differ in global coordination of gene splicing and expression levels. We analyzed RNA-seq transcriptome profiles of 8 different purified mouse cell types. We found that different cell types vary in proportion of highly expressed genes and the number of alternatively spliced transcripts expressed per gene, and that the cell types that express more variants of alternatively spliced transcripts per gene are those that have higher proportion of highly expressed genes. Cell types segregated into two clusters based on high or low proportion of highly expressed genes. Biological functions involved in negative regulation of gene expression were enriched in the group of cell types with low proportion of highly expressed genes, and biological functions involved in regulation of transcription and RNA splicing were enriched in the group of cell types with high proportion of highly expressed genes. Our findings show that cell types differ in proportion of highly expressed genes and the number of alternatively spliced transcripts expressed per gene, which represent distinct properties of the transcriptome and may reflect intrinsic differences in global coordination of synthesis, splicing, and degradation of RNA molecules.