Setdb1 protects genome integrity in murine muscle stem cells to allow for regenerative myogenesis and inflammation.

The histone H3 lysine 9 methyltransferase SETDB1 controls transcriptional repression to direct stem cell fate. Here, we show that Setdb1 expression by adult muscle stem cells (MuSCs) is required for skeletal muscle regeneration. We find that SETDB1 represses the expression of endogenous retroviruses (ERVs) in MuSCs. ERV de-repression in Setdb1-null MuSCs prevents their amplification following exit from quiescence and promotes cell death. Multi-omics profiling shows that chromatin decompaction at ERV loci activates the DNA-sensing cGAS-STING pathway, entailing cytokine expression by Setdb1-null MuSCs. This is followed by aberrant infiltration of inflammatory cells, including pathological macrophages. The ensuing histiocytosis is accompanied by myofiber necrosis, which, in addition to progressive MuSCs depletion, completely abolishes tissue repair. In contrast, loss of Setdb1 in fibro-adipogenic progenitors (FAPs) does not impact immune cells. In conclusion, genome maintenance by SETDB1 in an adult somatic stem cell is necessary for both its regenerative potential and adequate reparative inflammation.

Muscle stem cell niche dynamics during muscle homeostasis and regeneration.

The process of skeletal muscle regeneration involves a coordinated interplay of specific cellular and molecular interactions within the injury site. This review provides an overview of the cellular and molecular components in regenerating skeletal muscle, focusing on how these cells or molecules in the niche regulate muscle stem cell functions. Dysfunctions of muscle stem cell-to-niche cell communications during aging and disease will also be discussed. A better understanding of how niche cells coordinate with muscle stem cells for muscle repair will greatly aid the development of therapeutic strategies for treating muscle-related disorders.

Diagnostic Accuracy and Field for Improvement of Frameless Stereotactic Brain Biopsy: A Focus on Nondiagnostic Cases.

BACKGROUND: The diagnostic accuracy of frameless stereotactic brain biopsy has been reported, but there is limited literature focusing on the reasons for nondiagnostic cases. In this study, we evaluate the diagnostic accuracy of frameless stereotactic brain biopsy, compare it with the current international standard, and review the field for improvement. METHODS: This is a retrospective analysis of consecutive, prospectively collected frameless stereotactic brain biopsies from 2007 to 2020. We evaluated the diagnostic accuracy of the frameless stereotactic brain biopsies using defined criteria. The biopsy result was classified as conclusive, inconclusive, or negative, based on the pathologic, radiologic, and clinical diagnosis concordance. For inconclusive or negative results, we further evaluated the preoperative planning and postoperative imaging to review the errors. A literature review for the diagnostic accuracy of frameless stereotactic biopsy was performed for the validity of our results. RESULTS: There were 106 patients with 109 biopsies performed from 2007 to 2020. The conclusive diagnosis was reached in 103 (94.5%) procedures. An inconclusive diagnosis was noted in four (3.7%) procedures and the biopsy was negative in two (1.9%) procedures. Symptomatic hemorrhage occurred in one patient (0.9%). There was no mortality in our series. Registration error (RE) and inaccurate targeting occurred in three trigonal lesions (2.8%), sampling of the nonrepresentative part of the lesion occurred in two cases (1.8%), and one biopsy (0.9%) for lymphoma was negative due to steroid treatment. The literature review suggested that our diagnostic accuracy was comparable with the published literature. CONCLUSION: The frameless stereotactic biopsy is a safe procedure with high diagnostic accuracy only if meticulous preoperative planning and careful intraoperative registration is performed. The common pitfalls precluding a conclusive diagnosis are RE and biopsies at nonrepresentative sites.

Niche inflammatory signals control oscillating mammary regeneration and protect stem cells from cytotoxic stress.

Stem cells are known for their resilience and enhanced activity post-stress. The mammary gland undergoes frequent remodeling and is subjected to recurring stress during the estrus cycle, but it remains unclear how mammary stem cells (MaSCs) respond to the stress and contribute to regeneration. We discovered that cytotoxic stress-induced activation of CD11c+ ductal macrophages aids stem cell survival and prevents differentiation. These macrophages boost Procr+ MaSC activity through IL1ß-IL1R1-NF-κB signaling during the estrus cycle in an oscillating manner. Deleting IL1R1 in MaSCs results in stem cell loss and skewed luminal differentiation. Moreover, under cytotoxic stress from the chemotherapy agent paclitaxel, ductal macrophages secrete higher IL1ß levels, promoting MaSC survival and preventing differentiation. Inhibiting IL1R1 sensitizes MaSCs to paclitaxel. Our findings reveal a recurring inflammatory process that regulates regeneration, providing insights into stress-induced inflammation and its impact on stem cell survival, potentially affecting cancer therapy efficacy.

Protocol for low-input proteomic analysis of in situ fixed adult murine muscle stem cells.

Studying skeletal muscle stem cells (MuSCs) quiescence is challenging as they quickly activate within hours of isolation from muscle. Here, we present a protocol to disassociate and characterize fixed peptides from quiescent MuSCs using trapped ion-mobility time-of-flight mass spectrometry (MS). We describe steps for mouse perfusion, fluorescence-activated cell sorting preparation and sorting, protein extraction, digestion, and liquid chromatography MS analysis. This protocol can be applied to other less-abundant somatic stem cell types using mouse lines with a reporter. For complete details on the use and execution of this protocol, please refer to Zeng et al. (2022, 2023).1,2.

Restoration of CPEB4 prevents muscle stem cell senescence during aging.

Age-associated impairments in adult stem cell functions correlate with a decline in somatic tissue regeneration capacity. However, the mechanisms underlying the molecular regulation of adult stem cell aging remain elusive. Here, we provide a proteomic analysis of physiologically aged murine muscle stem cells (MuSCs), illustrating a pre-senescent proteomic signature. During aging, the mitochondrial proteome and activity are impaired in MuSCs. In addition, the inhibition of mitochondrial function results in cellular senescence. We identified an RNA-binding protein, CPEB4, downregulated in various aged tissues, which is required for MuSC functions. CPEB4 regulates the mitochondrial proteome and activity through mitochondrial translational control. MuSCs devoid of CPEB4 induced cellular senescence. Importantly, restoring CPEB4 expression rescued impaired mitochondrial metabolism, improved geriatric MuSC functions, and prevented cellular senescence in various human cell lines. Our findings provide the basis for the possibility that CPEB4 regulates mitochondrial metabolism to govern cellular senescence, with an implication of therapeutic intervention for age-related senescence.

ATAC-seq protocol for the profiling of chromatin accessibility of in situ fixed quiescent and activated muscle stem cells.

Chromatin accessibility is critical for cell identity. Conventional ATAC-seq can examine chromatin accessibility on freshly prepared muscle stem cells or satellite cells (SCs); however, isolating SCs in mice remains challenging. Here, we present a protocol to preserve the in vivo chromatin profile of SCs by applying paraformaldehyde (PFA) perfusion throughout the mouse before SC isolation. We describe steps for PFA perfusion and FACS sorting of SCs. We then detail library preparation for ATAC-seq. For complete details on the use and execution of this protocol, please refer to Dong et al.1.

Polycomb Ezh1 maintains murine muscle stem cell quiescence through non-canonical regulation of Notch signaling.

Organismal homeostasis and regeneration are predicated on committed stem cells that can reside for long periods in a mitotically dormant but reversible cell-cycle arrest state defined as quiescence. Premature escape from quiescence is detrimental, as it results in stem cell depletion, with consequent defective tissue homeostasis and regeneration. Here, we report that Polycomb Ezh1 confers quiescence to murine muscle stem cells (MuSCs) through a non-canonical function. In the absence of Ezh1, MuSCs spontaneously exit quiescence. Following repeated injuries, the MuSC pool is progressively depleted, resulting in failure to sustain proper muscle regeneration. Rather than regulating repressive histone H3K27 methylation, Ezh1 maintains gene expression of the Notch signaling pathway in MuSCs. Selective genetic reconstitution of the Notch signaling corrects stem cell number and re-establishes quiescence of Ezh1-/- MuSCs.

Single-Cell Transcriptomic Analysis of Mononuclear Cell Populations in Skeletal Muscle.

Skeletal muscle possesses a remarkable regenerative capacity, mainly relying on a population of undifferentiated and unipotent muscle progenitors, called muscle stem cells (MuSCs) or satellite cells, and their interplay with various cell types within the niche. Investigating the cellular composition of skeletal muscle tissues and the heterogeneity among various cell populations is crucial to the unbiased understanding of how cellular networks work in harmony at the population level in the context of skeletal muscle homeostasis, regeneration, aging, and diseases. As opposed to probing the average profile in a cell population, single-cell RNA-seq has unlocked access to the transcriptomic landscape characterization of individual cells in a highly parallel manner. This chapter describes the workflow for single-cell transcriptomic analysis of mononuclear cells in skeletal muscle by taking advantage of the droplet-based single-cell RNA-seq platform, Chromium Single Cell 3' solution from 10x Genomics®. Using this protocol, we can reveal insights into muscle-resident cell-type identities, which can be exploited to study the muscle stem cell niche further.

Visualization of RNA Transcripts in Muscle Stem Cells Using Single-Molecule Fluorescence In Situ Hybridization.

Uncovering the transcriptomic signatures of quiescent muscle stem cells elicits the regulatory networks on stem cell quiescence. However, the spatial clues of the transcripts are missing in the commonly used quantitative analysis such as qPCR and RNA-seq. Visualization of RNA transcripts using single-molecule in situ hybridization provides additional subcellular localization clues to understanding gene expression signatures. Here, we provide an optimized protocol of smFISH analysis on Fluorescence-Activated Cell Sorting isolated muscle stem cells to visualize low-abundance transcripts.

Activation of Wnt/ß-catenin signaling by Zeb1 in endothelial progenitors induces vascular quiescence entry.

The establishment of a functional vasculature requires endothelial cells to enter quiescence during the completion of development, otherwise pathological overgrowth occurs. How such a transition is regulated remains unclear. Here, we uncover a role of Zeb1 in defining vascular quiescence entry. During quiescence acquisition, Zeb1 increases along with the progressive decline of endothelial progenitors' activities, with Zeb1 loss resulting in endothelial overgrowth and vascular deformities. RNA sequencing (RNA-seq) and assay for transposase-accessible chromatin sequencing (ATAC-seq) analyses reveal that Zeb1 represses Wif1, thereby activating Wnt/ß-catenin signaling. Knockdown of Wif1 rescues the overgrowth induced by Zeb1 deletion. Importantly, local administration of surrogate Wnt molecules in the retina ameliorates the overgrowth defects of Zeb1 mutants. These findings show a mechanism by which Zeb1 induces quiescence of endothelial progenitors during the establishing of vascular homeostasis, providing molecular insight into the inherited neovascular pathologies associated with human ZEB1 mutations, suggesting pharmacological activation of Wnt/ß-catenin signaling as a potential therapeutical approach.

Pre-operative cognitive burden as predictor of motor outcome following bilateral subthalamic nucleus deep brain stimulation in Parkinson's disease.

INTRODUCTION: The interrelationship between neurocognitive impairments and motor functions was observed in patients with advanced Parkinson's disease (PD). This study was conducted to identify pre-operative neurocognitive and clinical predictors of short-term motor outcome following subthalamic nucleus deep brain stimulation (STN-DBS). METHODS: All consecutive PD patients who were eligible for bilateral STN-DBS from 2009 to 2019 were evaluated before and at 1 year following surgery. Standard motor evaluation and neurocognitive tests including global cognition, memory, executive functions (attention and category fluency), confrontational speech, visuospatial abilities, and mood were conducted at baseline. The post-operative STN-DBS effects were assessed at 1 year following the surgery. Multiple regression analysis was applied to identify baseline independent predictors of post-operative STN-DBS effect. RESULTS: A total of 82 patients were analyzed. It was found that younger age at operation, higher levodopa responsiveness at baseline based on UPDRS-III total score, and better baseline verbal delayed memory and category fluency predicted post-operative motor outcome at 1 year following STN-DBS (F = 9.639, p < 0.001, R2 = .340). CONCLUSION: Our findings demonstrated the role of baseline cognitive burden, especially cognitive processes related to frontostriatal circuits, was significant clinical predictors of short-term motor outcomes following STN-DBS. Profile analysis of neurocognitive functions at baseline is recommended.

Global chromatin accessibility profiling analysis reveals a chronic activation state in aged muscle stem cells.

Regulation of chromatin accessibility is critical for cell fate decisions. Chromatin structure responds to extrinsic environments rapidly. The traditional adult stem cell isolation approach requires tissue dissociation, which triggers stem cell activation and leads to alterations in chromatin structure. To preserve the in vivo chromatin states, we utilized the PFA-perfusion-based isolation approach and characterized the DNA regulatory landscapes during muscle stem cell quiescence exit and aging. We showed that aged SCs display a chronically activated chromatin signature. Detailed analysis of the chromatin accessibility profiles identified key enhancer elements for SC quiescence. Constant activation of the enhancer elements promotes stemness and prevents SCs from differentiation, whereas genetic deletion causes cell-cycle arrest and leads to defects in activation. Our comprehensive characterization of the chromatin accessibility and transcriptomic landscapes in SC quiescence and aging broadens our understanding of these processes and identifies key distal regulatory elements for SC function.

CPEB1 directs muscle stem cell activation by reprogramming the translational landscape.

Skeletal muscle stem cells, also called Satellite Cells (SCs), are actively maintained in quiescence but can activate quickly upon extrinsic stimuli. However, the mechanisms of how quiescent SCs (QSCs) activate swiftly remain elusive. Here, using a whole mouse perfusion fixation approach to obtain bona fide QSCs, we identify massive proteomic changes during the quiescence-to-activation transition in pathways such as chromatin maintenance, metabolism, transcription, and translation. Discordant correlation of transcriptomic and proteomic changes reveals potential translational regulation upon SC activation. Importantly, we show Cytoplasmic Polyadenylation Element Binding protein 1 (CPEB1), post-transcriptionally affects protein translation during SC activation by binding to the 3' UTRs of different transcripts. We demonstrate phosphorylation-dependent CPEB1 promoted Myod1 protein synthesis by binding to the cytoplasmic polyadenylation elements (CPEs) within its 3' UTRs to regulate SC activation and muscle regeneration. Our study characterizes CPEB1 as a key regulator to reprogram the translational landscape directing SC activation and subsequent proliferation.

Deciphering the chromatin organization and dynamics for muscle stem cell function.

The chromatin landscape represents a critical regulatory layer for precise transcriptional control. Chromosome architecture restrains the physical access to the DNA elements and is one of the determinants that specifies cell identity. Adult stem cells possess the unique ability to differentiate into a specific lineage. One of the underexplored areas in skeletal muscle biology is the molecular mechanism guiding the chromatin organization changes in muscle stem cell specification, myogenic determination, and differentiation. In this review, we focus on the regulatory network guiding the progression of muscle stem cells to differentiated progeny. We summarize recent findings regarding the mechanisms directing myogenic cell fate decision and differentiation, with a particular focus on three-dimensional chromosome architecture and long noncoding RNA-associated chromatin accessibility changes.

Nuclear receptors NHR-49 and NHR-79 promote peroxisome proliferation to compensate for aldehyde dehydrogenase deficiency in C. elegans.

The intracellular level of fatty aldehydes is tightly regulated by aldehyde dehydrogenases to minimize the formation of toxic lipid and protein adducts. Importantly, the dysregulation of aldehyde dehydrogenases has been implicated in neurologic disorder and cancer in humans. However, cellular responses to unresolved, elevated fatty aldehyde levels are poorly understood. Here, we report that ALH-4 is a C. elegans aldehyde dehydrogenase that specifically associates with the endoplasmic reticulum, mitochondria and peroxisomes. Based on lipidomic and imaging analysis, we show that the loss of ALH-4 increases fatty aldehyde levels and reduces fat storage. ALH-4 deficiency in the intestine, cell-nonautonomously induces NHR-49/NHR-79-dependent hypodermal peroxisome proliferation. This is accompanied by the upregulation of catalases and fatty acid catabolic enzymes, as indicated by RNA sequencing. Such a response is required to counteract ALH-4 deficiency since alh-4; nhr-49 double mutant animals are sterile. Our work reveals unexpected inter-tissue communication of fatty aldehyde levels and suggests pharmacological modulation of peroxisome proliferation as a therapeutic strategy to tackle pathology related to excess fatty aldehydes.

Stem cell quiescence: the challenging path to activation.

Quiescence is a cellular state in which a cell remains out of the cell cycle but retains the capacity to divide. The unique ability of adult stem cells to maintain quiescence is crucial for life-long tissue homeostasis and regenerative capacity. Quiescence has long been viewed as an inactive state but recent studies have shown that it is in fact an actively regulated process and that adult stem cells are highly reactive to extrinsic stimuli. This has fuelled hopes of boosting the reactivation potential of adult stem cells to improve tissue function during ageing. In this Review, we provide a perspective of the quiescent state and discuss how quiescent adult stem cells transition into the cell cycle. We also discuss current challenges in the field, highlighting recent technical advances that could help overcome some of these challenges.

A long noncoding RNA, LncMyoD, modulates chromatin accessibility to regulate muscle stem cell myogenic lineage progression.

Epigenetics regulation plays a critical role in determining cell identity by controlling the accessibility of lineage-specific regulatory regions. In muscle stem cells, epigenetic mechanisms of how chromatin accessibility is modulated during cell fate determination are not fully understood. Here, we identified a long noncoding RNA, LncMyoD, that functions as a chromatin modulator for myogenic lineage determination and progression. The depletion of LncMyoD in muscle stem cells led to the down-regulation of myogenic genes and defects in myogenic differentiation. LncMyoD exclusively binds with MyoD and not with other myogenic regulatory factors and promotes transactivation of target genes. The mechanistic study revealed that loss of LncMyoD prevents the establishment of a permissive chromatin environment at myogenic E-box-containing regions, therefore restricting the binding of MyoD. Furthermore, the depletion of LncMyoD strongly impairs the reprogramming of fibroblasts into the myogenic lineage. Taken together, our study shows that LncMyoD associates with MyoD and promotes myogenic gene expression through modulating MyoD accessibility to chromatin, thereby regulating myogenic lineage determination and progression.

Protocol for Isolation and Characterization of In Situ Fixed Quiescent Muscle Stem Cells.

Quiescent muscle stem cells, also called satellite cells (SCs), are essential for muscle regeneration. However, quiescent SCs are quickly activated during fluorescence-activated cell sorting (FACS) isolation. Here, we present an optimized protocol to isolate quiescent muscle stem cells from fixative-perfused mice and generate high-quality cDNA libraries for RNA-sequencing analysis. Fixation preserves the signatures of quiescent muscle stem cells in vivo. Isolated SCs can be used for downstream analysis such as immunofluorescence, RNA sequencing, and mass spectrometry. For complete information on the use and execution of this protocol, please refer to Yue et al. (2020).