Hypomorphic and dominant-negative impact of truncated SOX9 dysregulates Hedgehog-Wnt signaling, causing campomelia.

Haploinsufficiency for SOX9, the master chondrogenesis transcription factor, can underlie campomelic dysplasia (CD), an autosomal dominant skeletal malformation syndrome, because heterozygous Sox9 null mice recapitulate the bent limb (campomelia) and some other phenotypes associated with CD. However, in vitro cell assays suggest haploinsufficiency may not apply for certain mutations, notably those that truncate the protein, but in these cases in vivo evidence is lacking and underlying mechanisms are unknown. Here, using conditional mouse mutants, we compared the impact of a heterozygous Sox9 null mutation (Sox9+/-) with the Sox9+/Y440X CD mutation that truncates the C-terminal transactivation domain but spares the DNA-binding domain. While some Sox9+/Y440X mice survived, all Sox9+/- mice died perinatally. However, the skeletal defects were more severe and IHH signaling in developing limb cartilage was significantly enhanced in Sox9+/Y440X compared with Sox9+/-. Activating Sox9Y440X specifically in the chondrocyte-osteoblast lineage caused milder campomelia, and revealed cell- and noncell autonomous mechanisms acting on chondrocyte differentiation and osteogenesis in the perichondrium. Transcriptome analyses of developing Sox9+/Y440X limbs revealed dysregulated expression of genes for the extracellular matrix, as well as changes consistent with aberrant WNT and HH signaling. SOX9Y440X failed to interact with ß-catenin and was unable to suppress transactivation of Ihh in cell-based assays. We propose enhanced HH signaling in the adjacent perichondrium induces asymmetrically localized excessive perichondrial osteogenesis resulting in campomelia. Our study implicates combined haploinsufficiency/hypomorphic and dominant-negative actions of SOX9Y440X, cell-autonomous and noncell autonomous mechanisms, and dysregulated WNT and HH signaling, as the cause of human campomelia.

High-Frequency Spinal Stimulation Suppresses Microglial Kaiso-P2X7 Receptor Axis-Induced Inflammation to Alleviate Neuropathic Pain in Rats.

OBJECTIVE: Neuropathic pain poses a persistent challenge in clinical management. Neuromodulation has emerged as a last-resort therapy. Conventional spinal cord stimulation (Con SCS) often causes abnormal sensations and provides short analgesia, whereas high-frequency spinal cord stimulation (HF SCS) is a newer therapy that effectively alleviates pain without paresthesia. However, the modes of action of 10kHz HF SCS (HF10 SCS) in pain relief remain unclear. To bridge this knowledge gap, we employed preclinical models that mimic certain features of clinical SCS to explore the underlying mechanisms of HF10 SCS. Addressing these issues would provide the scientific basis for improving and evaluating the effectiveness, reliability, and practicality of different frequency SCS in clinical settings. METHODS: We established a preclinical SCS model to examine its effects in a neuropathic pain rat model. We conducted bulk and single-cell RNA sequencing in the spinal dorsal horn (SDH) to examine cellular and molecular changes under different treatments. We employed genetic manipulations through intrathecal injection of a lentiviral system to explore the SCS-mediated signaling axis in pain. Various behavioral tests were performed to evaluate pain conditions under different treatments. RESULTS: We found that HF10 SCS significantly reduces immune responses in the SDH by inactivating the Kaiso-P2X7R pathological axis in microglia, promoting long-lasting pain relief. Targeting Kaiso-P2X7R in microglia dramatically improved efficacy of Con SCS treatment, leading to reduced neuroinflammation and long-lasting pain relief. INTERPRETATION: HF10 SCS could improve the immunopathologic state in the SDH, extending its benefits beyond symptom relief. Targeting the Kaiso-P2X7R axis may enhance Con SCS therapy and offer a new strategy for pain management. ANN NEUROL 2024;95:966-983.

Fbxo9 functions downstream of Sox10 to determine neuron-glial fate choice in the dorsal root ganglia through Neurog2 destabilization.

The transcription factor Sox10 is a key regulator in the fate determination of a subpopulation of multipotent trunk neural crest (NC) progenitors toward glial cells instead of sensory neurons in the dorsal root ganglia (DRG). However, the mechanism by which Sox10 regulates glial cell fate commitment during lineage segregation remains poorly understood. In our study, we showed that the neurogenic determinant Neurogenin 2 (Neurog2) exhibited transient overlapping expression with Sox10 in avian trunk NC progenitors, which progressively underwent lineage segregation during migration toward the forming DRG. Gain- and loss-of-function studies revealed that the temporary expression of Neurog2 was due to Sox10 regulation of its protein stability. Transcriptional profiling identified Sox10-regulated F-box only protein (Fbxo9), which is an SCF (Skp1-Cul-F-box)-type ubiquitin ligase for Neurog2. Consistently, overexpression of Fbxo9 in NC progenitors down-regulated Neurog2 protein expression through ubiquitination and promoted the glial lineage at the expense of neuronal differentiation, whereas Fbxo9 knockdown resulted in the opposite phenomenon. Mechanistically, we found that Fbxo9 interacted with Neurog2 to promote its destabilization through the F-box motif. Finally, epistasis analysis further demonstrated that Fbxo9 and probably other F-box members mediated the role of Sox10 in destabilizing Neurog2 protein and directing the lineage of NC progenitors toward glial cells rather than sensory neurons. Altogether, these findings unravel a Sox10-Fbxo9 regulatory axis in promoting the glial fate of NC progenitors through Neurog2 destabilization.

Cis-control of Six1 expression in neural crest cells during craniofacial development.

BACKGROUND: Six1 is a transcriptional factor that plays an important role in embryonic development. Mouse and chick embryos deficient for Six1 have multiple craniofacial anomalies in the facial bones and cartilages. Multiple Six1 enhancers have been identified, but none of them has been reported to be active in the maxillary and mandibular process. RESULTS: We studied two Six1 enhancers in the chick neural crest tissues during craniofacial development. We showed that two evolutionarily conserved enhancers, Six1E1 and Six1E2, act synergistically. Neither Six1E1 nor Six1E2 alone can drive enhancer reporter signal in the maxillary or mandibular processes. However, their combination, Six1E, showed robust enhancer activity in these tissues. Similar reporter signal can also be driven by the mouse homolog of Six1E. Mutations of multiple conserved transcriptional factor binding sites altered the enhancer activity of Six1E, especially mutation of the LIM homeobox binding site, dramatically reduced the enhancer activity, implying that the Lhx protein family be an important regulator of Six1 expression. CONCLUSION: This study, for the first time, described the synergistic activation of two Six1 enhancers in the maxillary and mandibular processes and will facilitate more detailed studies of the regulation of Six1 in craniofacial development.

Improved sample dispersion in cryo-EM using "perpetually-hydrated" graphene oxide flakes.

For many macromolecular complexes, the inability to uniformly disperse solubilized specimen particles within vitreous ice films precludes their analysis by cryo-electron microscopy (cryo-EM). Here, we introduce a sample preparation process using "perpetually-hydrated" graphene oxide flakes as particle support films, and report vastly improved specimen dispersion. Furthermore, we provide evidence that the presence of graphene oxide flakes in vitreous ice results in a significant reduction in electron beam-induced specimen decomposition. The new method introduced in this study incorporates hydrated graphene oxide flakes into a standard sample preparation regime, without the need for additional tools or devices, making it a cost-effective and easily adoptable alternative to currently available sample preparation approaches.

Improved sample dispersion in cryo-EM using "perpetually-hydrated" graphene oxide flakes.

For many macromolecular complexes, the inability to uniformly disperse solubilized specimen particles within vitreous ice films precludes their analysis by cryo-electron microscopy (cryo-EM). Here, we introduce a sample preparation process using "perpetually-hydrated" graphene oxide flakes as particle support films, and report vastly improved specimen dispersion. The new method introduced in this study incorporates hydrated graphene oxide flakes into a standard sample preparation regime, without the need for additional tools or devices, making it a cost-effective and easily adoptable alternative to currently available sample preparation approaches.

Neural crest stem cells and their potential therapeutic applications.

The neural crest (NC) is a remarkable transient structure generated during early vertebrate development. The neural crest progenitors have extensive migratory capacity and multipotency, harboring stem cell-like characteristics such as self-renewal. They can differentiate into a variety of cell types from craniofacial skeletal tissues to the trunk peripheral nervous system (PNS). Multiple regulators such as signaling factors, transcription factors, and migration machinery components are expressed at different stages of NC development. Gain- and loss-of-function studies in various vertebrate species revealed epistatic relationships of these molecules that could be assembled into a gene regulatory network defining the processes of NC induction, specification, migration, and differentiation. These basic developmental studies led to the subsequent establishment and molecular validation of neural crest stem cells (NCSCs) derived by various strategies. We provide here an overview of the isolation and characterization of NCSCs from embryonic, fetal, and adult tissues; the experimental strategies for the derivation of NCSCs from embryonic stem cells, induced pluripotent stem cells, and skin fibroblasts; and recent developments in the use of patient-derived NCSCs for modeling and treating neurocristopathies. We discuss future research on further refinement of the culture conditions required for the differentiation of pluripotent stem cells into axial-specific NC progenitors and their derivatives, developing non-viral approaches for the generation of induced NC cells (NCCs), and using a genomic editing approach to correct genetic mutations in patient-derived NCSCs for transplantation therapy. These future endeavors should facilitate the therapeutic applications of NCSCs in the clinical setting.

Three-dimensional electron microscopy reconstruction and cysteine-mediated crosslinking provide a model of the type III secretion system needle tip complex.

Type III secretion systems are found in many Gram-negative bacteria. They are activated by contact with eukaryotic cells and inject virulence proteins inside them. Host cell detection requires a protein complex located at the tip of the device's external injection needle. The Shigella tip complex (TC) is composed of IpaD, a hydrophilic protein, and IpaB, a hydrophobic protein, which later forms part of the injection pore in the host membrane. Here we used labelling and crosslinking methods to show that TCs from a ΔipaB strain contain five IpaD subunits while the TCs from wild-type can also contain one IpaB and four IpaD subunits. Electron microscopy followed by single particle and helical image analysis was used to reconstruct three-dimensional images of TCs at ∼ 20 Å resolution. Docking of an IpaD crystal structure, constrained by the crosslinks observed, reveals that TC organisation is different from that of all previously proposed models. Our findings suggest new mechanisms for TC assembly and function. The TC is the only site within these secretion systems targeted by disease-protecting antibodies. By suggesting how these act, our work will allow improvement of prophylactic and therapeutic strategies.

Associations between the neighbourhood environment characteristics and physical activity in older adults with specific types of chronic conditions: the ALECS cross-sectional study.

BACKGROUND: Neighbourhood characteristics may influence physical activity (PA), which has positive effects on the health of older adults. Older adults with chronic conditions are less active and possibly more affected by environmental factors than their peers. Understanding neighbourhood characteristics associated with PA specific to older adults with chronic conditions is currently lacking. This cross-sectional study aimed to assess the associations between the neighbourhood environment and various forms of PA in older adults with and without visual impairment, hearing impairment, musculoskeletal disease and/or genitourinary disease. METHODS: Neighbourhood environment and PA data were collected in Hong Kong older adults (N = 909) from 124 preselected neighbourhoods stratified for walkability and socioeconomic status. Generalized linear models and zero-inflated negative binomial models with robust standard errors were used to examine associations of perceived neighbourhood environment characteristics, and the moderating effects of having specific chronic conditions, with PA outcomes. RESULTS: Thirteen perceived neighbourhood characteristics were associated with older adults' PA in the expected direction irrespective of their health condition. Nine neighbourhood characteristics had associations with PA that were dependent on hearing impairment, vision impairment, musculoskeletal disease or genitourinary disease. In general, they were stronger in participants with than without a specific chronic condition. CONCLUSIONS: Maximizing the potential for PA in older adults who have lower levels of physical functionality due to chronic conditions may require neighbourhood characteristics specific to these groups.

Phosphorylation of Sox9 is required for neural crest delamination and is regulated downstream of BMP and canonical Wnt signaling.

Coordination of neural crest cell (NCC) induction and delamination is orchestrated by several transcription factors. Among these, Sry-related HMG box-9 (Sox9) and Snail2 have been implicated in both the induction of NCC identity and, together with phoshorylation, NCC delamination. How phosphorylation effects this function has not been clear. Here we show, in the developing chick neural tube, that phosphorylation of Sox9 on S64 and S181 facilitates its SUMOylation, and the phosphorylated forms of Sox9 are essential for trunk neural crest delamination. Both phosphorylation and to a lesser extent SUMOylation, of Sox9 are required to cooperate with Snail2 to promote delamination. Moreover, bone morphogenetic protein and canonical Wnt signaling induce phosphorylation of Sox9, thereby connecting extracellular signals with the delamination of NCCs. Together the data suggest a model in which extracellular signals initiate phosphorylation of Sox9 and its cooperation with Snail2 to induce NCC delamination.

A mutation in Ihh that causes digit abnormalities alters its signalling capacity and range.

Brachydactyly type A1 (BDA1) was the first recorded disorder of the autosomal dominant Mendelian trait in humans, characterized by shortened or absent middle phalanges in digits. It is associated with heterozygous missense mutations in indian hedgehog (IHH). Hedgehog proteins are important morphogens for a wide range of developmental processes. The capacity and range of signalling is thought to be regulated by its interaction with the receptor PTCH1 and antagonist HIP1. Here we show that a BDA1 mutation (E95K) in Ihh impairs the interaction of IHH with PTCH1 and HIP1. This is consistent with a recent paper showing that BDA1 mutations cluster in a calcium-binding site essential for the interaction with its receptor and cell-surface partners. Furthermore, we show that in a mouse model that recapitulates the E95K mutation, there is a change in the potency and range of signalling. The mice have digit abnormalities consistent with the human disorder.

Structure of a type III secretion needle at 7-Å resolution provides insights into its assembly and signaling mechanisms.

Type III secretion systems of Gram-negative bacteria form injection devices that deliver effector proteins into eukaryotic cells during infection. They span both bacterial membranes and the extracellular space to connect with the host cell plasma membrane. Their extracellular portion is a needle-like, hollow tube that serves as a secretion conduit for effector proteins. The needle of Shigella flexneri is approximately 50-nm long and 7-nm thick and is made by the helical assembly of one protein, MxiH. We provide a 7-Å resolution 3D image reconstruction of the Shigella needle by electron cryomicroscopy, which resolves α-helices and a ß-hairpin that has never been observed in the crystal and solution structures of needle proteins, including MxiH. An atomic model of the needle based on the 3D-density map, in comparison with that of the bacterial-flagellar filament, provides insights into how such a thin tubular structure is stably assembled by intricate intermolecular interactions. The map also illuminates how the needle-length control protein functions as a ruler within the central channel during export of MxiH for assembly at the distal end of the needle, and how the secretion-activation signal may be transduced through a conformational change of the needle upon host-cell contact.

Suppression of apoptosis impairs phalangeal joint formation in the pathogenesis of brachydactyly type A1.

Apoptosis occurs during development when a separation of tissues is needed. Synovial joint formation is initiated at the presumptive site (interzone) within a cartilage anlagen, with changes in cellular differentiation leading to cavitation and tissue separation. Apoptosis has been detected in phalangeal joints during development, but its role and regulation have not been defined. Here, we use a mouse model of brachydactyly type A1 (BDA1) with an IhhE95K mutation, to show that a missing middle phalangeal bone is due to the failure of the developing joint to cavitate, associated with reduced apoptosis, and a joint is not formed. We showed an intricate relationship between IHH and interacting partners, CDON and GAS1, in the interzone that regulates apoptosis. We propose a model in which CDON/GAS1 may act as dependence receptors in this context. Normally, the IHH level is low at the center of the interzone, enabling the "ligand-free" CDON/GAS1 to activate cell death for cavitation. In BDA1, a high concentration of IHH suppresses apoptosis. Our findings provided new insights into the role of IHH and CDON in joint formation, with relevance to hedgehog signaling in developmental biology and diseases.

TSPYL1 as a Critical Regulator of TGFß Signaling through Repression of TGFBR1 and TSPYL2.

Nucleosome assembly proteins (NAPs) have been identified as histone chaperons. Testis-Specific Protein, Y-Encoded-Like (TSPYL) is a newly arisen NAP family in mammals. TSPYL2 can be transcriptionally induced by DNA damage and TGFß causing proliferation arrest. TSPYL1, another TSPYL family member, has been poorly characterized and is the only TSPYL family member known to be causal of a lethal recessive disease in humans. This study shows that TSPYL1 and TSPYL2 play an opposite role in TGFß signaling. TSPYL1 partners with the transcription factor FOXA1 and histone methyltransferase EZH2, and at the same time represses TGFBR1 and epithelial-mesenchymal transition (EMT). Depletion of TSPYL1 increases TGFBR1 expression, upregulates TGFß signaling, and elevates the protein stability of TSPYL2. Intriguingly, TSPYL2 forms part of the SMAD2/3/4 signal transduction complex upon stimulation by TGFß to execute the transcriptional responses. Depletion of TSPYL2 rescues the EMT phenotype of TSPYL1 knockdown in A549 lung carcinoma cells. The data demonstrates the prime role of TSPYL2 in causing the dramatic defects in TSPYL1 deficiency. An intricate counter-balancing role of TSPYL1 and TSPYL2 in regulating TGFß signaling is also unraveled.

Intrinsic and extrinsic actions of human neural progenitors with SUFU inhibition promote tissue repair and functional recovery from severe spinal cord injury.

Neural progenitor cells (NPCs) derived from human pluripotent stem cells(hPSCs) provide major cell sources for repairing damaged neural circuitry and enabling axonal regeneration after spinal cord injury (SCI). However, the injury niche and inadequate intrinsic factors in the adult spinal cord restrict the therapeutic potential of transplanted NPCs. The Sonic Hedgehog protein (Shh) has crucial roles in neurodevelopment by promoting the formation of motorneurons and oligodendrocytes as well as its recently described neuroprotective features in response to the injury, indicating its essential role in neural homeostasis and tissue repair. In this study, we demonstrate that elevated SHH signaling in hNPCs by inhibiting its negative regulator, SUFU, enhanced cell survival and promoted robust neuronal differentiation with extensive axonal outgrowth, counteracting the harmful effects of the injured niche. Importantly, SUFU inhibition in NPCs exert non-cell autonomous effects on promoting survival and neurogenesis of endogenous cells and modulating the microenvironment by reducing suppressive barriers around lesion sites. The combined beneficial effects of SUFU inhibition in hNPCs resulted in the effective reconstruction of neuronal connectivity with the host and corticospinal regeneration, significantly improving neurobehavioral recovery in recipient animals. These results demonstrate that SUFU inhibition confers hNPCs with potent therapeutic potential to overcome extrinsic and intrinsic barriers in transplantation treatments for SCI.

A method to achieve homogeneous dispersion of large transmembrane complexes within the holes of carbon films for electron cryomicroscopy.

Difficulties associated with using X-ray crystallography for structural studies of large macromolecular complexes have made single particle cryo-electron microscopy (cryoEM) a key technique in structural biology. The efficient application of the single particle cryoEM approach requires the sample to be vitrified within the holes of carbon films, with particles well dispersed throughout the ice and adopting multiple orientations. To achieve this, the carbon support film is first hydrophilised by glow discharge, which allows the sample to spread over the film. Unfortunately, for transmembrane complexes especially, this procedure can result in severe sample adsorption to the carbon support film, reducing the number of particles dispersed in the ice. This problem is rate-limiting in the single particle cryoEM approach and has hindered its widespread application to hydrophobic complexes. We describe a novel grid preparation technique that allows for good particle dispersion in the ice and minimal hydrophobic particle adhesion to the support film. This is achieved by hydrophilisation of the carbon support film by the use of selected detergents that interact with the support so as to achieve a hydrophilic and neutral or selectively charged surface.

Low-energy scanning transmission electron microscopy applied to ice-embedded biological macromolecules.

In this report, we applied annular bright-field and annular dark-field low-energy (30 keV) scanning transmission electron microscopy imaging to a vitreous ice-embedded biological macromolecule, T4 phage, to investigate the applicability of these methods for morphological investigation and sample screening. Multiple camera lengths were examined to find the optimal acceptance angle for both modes. Image clarity differed substantially between the modes, with the presence of ice also strongly influencing the quality of acquired micrographs. In annular dark-field mode, the proper discrimination of electrons scattered by the specimen from those scattered by the background ice was found to be difficult due to the severe overlap of the scattered electrons. The resulting micrographs lacked clarity, and the ice-embedded phage particles could only be discerned after post-processing image adjustment. However, in annular bright-field mode, despite similar overlapping of the scattered electrons, it was possible to assess the morphology and intactness of the specimen in the embedding ice, suggesting that this mode may find utility in low-energy cryo-scanning transmission electron microscopy imaging methods.

Revealing the developmental origin and lineage predilection of neural progenitors within human bone marrow via single-cell analysis: implications for regenerative medicine.

BACKGROUND: Human bone marrow stromal cells (BMSCs) are an easily accessible and expandable progenitor population with the capacity to generate neural cell types in addition to mesoderm. Lineage tracing studies in transgenic animals have indicated Nestin + BMSCs to be descended from the truncal neural crest. Single-cell analysis provides a means to identify the developmental origin and identity of human BMSC-derived neural progenitors when lineage tracing remains infeasible. This is a prerequisite towards translational application. METHODS: We attained transcriptomic profiles of embryonic long bone, adult human bone marrow, cultured BMSCs and BMSC-derived neurospheres. Integrated scRNAseq analysis was supplemented by characterization of cells during culture expansion and following provision of growth factors and signalling agonists to bias lineage. RESULTS: Reconstructed pseudotime upon the integrated dataset indicated distinct neural and osteogenic differentiation trajectories. The starting state towards the neural differentiation trajectory consisted of Nestin + /MKI67 + BMSCs, which could also be diverted towards the osteogenic trajectory via a branch point. Nestin + /PDGFRA + BMSCs responded to neurosphere culture conditions to generate a subpopulation of cells with a neuronal phenotype according to marker expression and gene ontogeny analysis that occupied the end state along the neural differentiation trajectory. Reconstructed pseudotime also revealed an upregulation of BMP4 expression during culture of BMSC-neurospheres. This provided the rationale for culture supplementation with the BMP signalling agonist SB4, which directed progenitors to upregulate Pax6 and downregulate Nestin. CONCLUSIONS: This study suggested BMSCs originating from truncal neural crest to be the source of cells within long bone marrow possessing neural differentiation potential. Unravelling the transcriptomic dynamics of BMSC-derived neural progenitors promises to enhance differentiation efficiency and safety towards clinical application in cell therapy and disease modelling.

Transplanting Human Neural Stem Cells with ≈50% Reduction of SOX9 Gene Dosage Promotes Tissue Repair and Functional Recovery from Severe Spinal Cord Injury.

Neural stem cells (NSCs) derived from human pluripotent stem cells (hPSCs) are considered a major cell source for reconstructing damaged neural circuitry and enabling axonal regeneration. However, the microenvironment at the site of spinal cord injury (SCI) and inadequate intrinsic factors limit the therapeutic potential of transplanted NSCs. Here, it is shown that half dose of SOX9 in hPSCs-derived NSCs (hNSCs) results in robust neuronal differentiation bias toward motor neuron lineage. The enhanced neurogenic potency is partly attributed to the reduction of glycolysis. These neurogenic and metabolic properties retain after transplantation of hNSCs with reduced SOX9 expression in a contusive SCI rat model without the need for growth factor-enriched matrices. Importantly, the grafts exhibit excellent integration properties, predominantly differentiate into motor neurons, reduce glial scar matrix accumulation to facilitate long-distance axon growth and neuronal connectivity with the host as well as dramatically improve locomotor and somatosensory function in recipient animals. These results demonstrate that hNSCs with half SOX9 gene dosage can overcome extrinsic and intrinsic barriers, representing a powerful therapeutic potential for transplantation treatments for SCI.