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Self-Doped and Biodegradable Glycosaminoglycan-PEDOT Conductive Hydrogels Facilitate Electrical Pacing of iPSC-Derived Cardiomyocytes.
Conductive polymers hold promise in biomedical applications owing to their distinct conductivity characteristics and unique properties. However, incorporating these polymers into biomaterials poses challenges related to mechanical performance, electrical stability, and biodegradation. This study proposes an injectable hydrogel scaffold composed of a self-doped conductive polymer, constituted of a sulfated glycosaminoglycan (GAG) with side chains of PEDOT (poly 3,4-ethylenedioxythiophene). This brush copolymer is synthesized via oxidative polymerization from an EDOT monomer grafted onto the backbone of the sulfated GAG. The GAG backbone offers biodegradability, while sulfate groups act as acidic self-doping agents. Conductive hydrogels form through oxime crosslinking, initially existing as a liquid mixture that undergoes gelation within the tissue, allowing for injectability. The conductive hydrogels show tunable stiffness and gelation kinetics influenced by both concentration and pH, and exhibit adhesive properties. They showcase dual ionic and electronic conductivity, where sulfate groups in the GAG backbone act as doping moieties, enhancing conductivity and electrical stability. These properties of conductive hydrogels are associated with the facilitation of electrical pacing of iPSC-cardiomyocytes. Furthermore, hydrogels exhibit biodegradation and show evidence of biocompatibility, highlighting their potential for diverse biomedical applications.
4D Multimaterial Printing of Soft Actuators with Spatial and Temporal Control.
Soft actuators (SAs) are devices which can interact with delicate objects in a manner not achievable with traditional robotics. While it is possible to design a SA whose actuation is triggered via an external stimulus, the use of a single stimulus creates challenges in the spatial and temporal control of the actuation. Herein, a 4D printed multimaterial soft actuator design (MMSA) whose actuation is only initiated by a combination of triggers (i.e., pH and temperature) is presented. Using 3D printing, a multilayered soft actuator with a hydrophilic pH-sensitive layer, and a hydrophobic magnetic and temperature-responsive shape-memory polymer layer, is designed. The hydrogel responds to environmental pH conditions by swelling or shrinking, while the shape-memory polymer can resist the shape deformation of the hydrogel until triggered by temperature or light. The combination of these stimuli-responsive layers allows for a high level of spatiotemporal control of the actuation. The utility of the 4D MMSA is demonstrated via a series of cargo capture and release experiments, validating its ability to demonstrate active spatiotemporal control. The MMSA concept provides a promising research direction to develop multifunctional soft devices with potential applications in biomedical engineering and environmental engineering.
Formation of seeding-competent α-synuclein aggregates in parkin-deficient iPSC-derived human neurons.
Loss-of-function mutations in PARK2 (parkin) cause early-onset familial Parkinson's disease (PD) and may also contribute to sporadic PD. While Lewy bodies, enriched in aggregated phosphorylated α-synuclein (α-Syn), are typical in PD, their presence in PARK2-mediated PD remains debated. Using human isogenic PARK2-/- induced pluripotent stem cell-derived neurons, we investigated α-Syn pathology under parkin deficiency. PARK2-/- neurons showed elevated intracellular aggregated and total α-Syn levels, increased α-Syn release, and higher levels of aggregation-inducing α-Syn seeds. These neurons also displayed more pSer129 α-Syn+ inclusions, which were further enhanced by α-Syn preformed fibril (PFF) exposure. Moreover, we identified synaptic loss in the PARK2-/- neurons, exacerbated by PFF treatment, and dysregulated Ca2+ homeostasis consistent with enhanced activity of the smooth endoplasmic reticulum Ca2+-ATPase (SERCA). Our data provide an important contribution to the debate on the role of α-Syn in the pathology of PARK2-related PD and challenge the view of PARK2-related PD as a non-synucleinopathy.
Bioactive coatings on 3D printed scaffolds for bone regeneration: Use of Laponite® to deliver BMP-2 in an ovine femoral condyle defect model.
Biomaterial-based approaches for bone regeneration seek to explore alternative strategies to repair non-healing fractures and critical-sized bone defects. Fracture non-union occurs due to a number of factors resulting in the formation of bone defects. Rigorous evaluation of the biomaterials in relevant models and assessment of their potential to translate towards clinical use is vital. Large animal experimentation can be used to model fracture non-union while scaling-up materials for clinical use. Growth factors modulate cell phenotype, behaviour and initiate signalling pathways leading to changes in matrix deposition and tissue formation. Bone morphogenetic protein-2 (BMP-2) is a potent osteogenic growth factor, with a rapid clearance time in vivo necessitating clinical use at a high dose, with potential deleterious side-effects. The current studies have examined the potential for Laponite® nanoclay coated poly(caprolactone) trimethacrylate (PCL-TMA900) scaffolds to bind BMP-2 for enhanced osteoinduction in a large animal critical-sized bone defect. An ovine femoral condyle defect model confirmed PCL-TMA900 scaffolds coated with Laponite®/BMP-2 produced significant bone formation compared to the uncoated PCL-TMA 900 scaffold in vivo, assessed by micro-computed tomography (μCT) and histology. This indicated the ability of Laponite® to deliver the bioactive BMP-2 on the PCL-TMA900 scaffold. Bone formed around the Laponite®/BMP-2 coated PCL-TMA900 scaffold, with no erroneous bone formation observed away from the scaffold material confirming localisation of BMP-2 delivery. The current studies demonstrate the ability of a nanoclay to localise and deliver bioactive BMP-2 within a tailored octet-truss scaffold for efficacious bone defect repair in a large animal model with significant implications for translation to the clinic.
Hif-2α programs oxygen chemosensitivity in chromaffin cells.
The study of transcription factors that determine specialized neuronal functions has provided invaluable insights into the physiology of the nervous system. Peripheral chemoreceptors are neurone-like electrophysiologically excitable cells that link the oxygen concentration of arterial blood to the neuronal control of breathing. In the adult, this oxygen chemosensitivity is exemplified by type I cells of the carotid body, and recent work has revealed one isoform of the hypoxia-inducible transcription factor (HIF), HIF-2α, as having a nonredundant role in the development and function of that organ. Here, we show that activation of HIF-2α, including isolated overexpression of HIF-2α but not HIF-1α, is sufficient to induce oxygen chemosensitivity in adult adrenal medulla. This phenotypic change in the adrenal medulla was associated with retention of extra-adrenal paraganglioma-like tissues resembling the fetal organ of Zuckerkandl, which also manifests oxygen chemosensitivity. Acquisition of chemosensitivity was associated with changes in the adrenal medullary expression of gene classes that are ordinarily characteristic of the carotid body, including G protein regulators and atypical subunits of mitochondrial cytochrome oxidase. Overall, the findings suggest that, at least in certain tissues, HIF-2α acts as a phenotypic driver for cells that display oxygen chemosensitivity, thus linking 2 major oxygen-sensing systems.
FMR1 KH0-KH1 domains coordinate m6A binding and phase separation in Fragile X syndrome.
Fragile X messenger ribonucleoprotein 1 (FMR1) regulates neurodevelopment through m6A RNA interactions, yet the domain-specific roles of KH0 and KH1 in RNA binding and disease pathogenesis remain poorly understood. Using mutagenesis and AlphaFold3 structural modeling, we identify KH1 as the primary m6A-binding interface, while the KH0 domain (particularly Arg138) modulates liquid-liquid phase separation (LLPS). Pathogenic mutations in KH0 impair RNA binding and promote aberrant LLPS aggregation, whereas m6A-modified RNA suppresses LLPS formation at KH0. Structural simulations uncover synergistic interactions between KH0 and KH1 mediated by hydrophobic and electrostatic networks. These domain-specific cooperations establish a mechanistic link between m6A dysregulation, pathological phase separation, and Fragile X syndrome pathogenesis. Our findings nominate KH0 as a potential therapeutic target for RNA-driven neurodevelopmental disorders.
Consensus Recommendations for Hyperpolarized [1-13C]pyruvate MRI Multi-center Human Studies.
Magnetic resonance imaging of hyperpolarized (HP) [1-13C]pyruvate allows in-vivo assessment of metabolism and has translated into human studies across diseases at 15 centers worldwide. Consensus on best practice for multi-center studies is required to develop clinical applications. This paper presents the results of a 2-round formal consensus building exercise carried out by experts with HP [1-13C]pyruvate human study experience. Twenty-nine participants from 13 sites brought together expertise in pharmacy methods, MR physics, translational imaging, and data-analysis; with the goal of providing recommendations and best practice statements on conduct of multi-center human studies of HP [1-13C]pyruvate MRI. Overall, the group reached consensus on approximately two-thirds of 246 statements in the questionnaire, covering 'HP 13C-Pyruvate Preparation', 'MRI System Setup, Calibration, and Phantoms', 'Acquisition and Reconstruction', and 'Data Analysis and Quantification'. Consensus was present across categories, examples include that: (i) different HP pyruvate preparation methods could be used in human studies, but that the same release criteria have to be followed; (ii) site qualification and quality assurance must be performed with phantoms and that the same field strength must be used, but that the rest of the system setup and calibration methods could be determined by individual sites;(iii) the same pulse sequence and reconstruction methods were preferable, but the exact choice should be governed by the anatomical target; (iv) normalized metabolite area-under-curve (AUC) values and metabolite AUC were the preferred metabolism metrics. The work confirmed areas of consensus for multi-center study conduct and identified where further research is required to ascertain best practice.
Single neurons and networks in the mouse claustrum integrate input from widespread cortical sources.
The claustrum is thought to be one of the most highly interconnected forebrain structures, but its organizing principles have yet to be fully explored at the level of single neurons. Here, we investigated the identity, connectivity, and activity of identified claustrum neurons in Mus musculus to understand how the structure's unique convergence of input and divergence of output support binding information streams. We found that neurons in the claustrum communicate with each other across efferent projection-defined modules which were differentially innervated by sensory and frontal cortical areas. Individual claustrum neurons were responsive to inputs from more than one cortical region in a cell-type and projection-specific manner, particularly between areas of frontal cortex. In vivo imaging of claustrum axons revealed responses to both unimodal and multimodal sensory stimuli. Finally, chronic claustrum silencing specifically reduced animals' sensitivity to multimodal stimuli. These findings support the view that the claustrum is a fundamentally integrative structure, consolidating information from around the cortex and redistributing it following local computations.
The murine ATP-binding cassette transporter C5 (Abcc5/MRP5/cMOAT) plays a role in memory consolidation, circadian rhythm regulation and glutamatergic signalling.
ATP-Binding cassette (ABC) transporters are a family of integral membrane ATPases that transport a large number of structurally unrelated compounds. The physiological role of the orphan transporter Abcc5 remains poorly understood. As previous work demonstrated that the loss of Abcc5 activity leads to elevated levels of NAAG in the brain, the impact of Abcc5 ablation was ascertained using behavioural phenotyping, circadian rhythm analysis and electrophysiological recordings of brain slices from Abcc5-/- mice and compared to wild-type littermates. Behavioural phenotyping of Abcc5-/- mice shows that the loss of murine Abcc5 activity results in profound changes in pre-pulse inhibition (PPI) as well as altered memory consolidation. Circadian measures of activity showed a delay in the timing of Abcc5-/- mice activity rhythm peak. Additionally, activity defined sleep analysis highlighted differences in sleep patterns in Abcc5-/- mice compared to wild-type controls. Patch clamp recording from pyramidal cells in the 2/3 layer of the frontal cortex showed altered synaptic AMPA/NMDA receptor current ratios and increased frequency of spontaneous excitatory postsynaptic currents (sEPSC). This study demonstrates that the loss of functional Abcc5 transporters does have behavioural consequences in mammals and alters NMDA receptor activity. These results highlight a previously unknown role of Abcc5 in the brain.
Modeling common Alzheimer's disease with high and low polygenic risk in human iPSC: A large-scale research resource.
Common forms of Alzheimer's disease (AD) are complex and polygenic. We have created a research resource that seeks to capture the extremes of polygenic risk in a collection of human induced pluripotent stem cell (iPSC) lines from over 100 donors: the IPMAR Resource (iPSC Platform to Model Alzheimer's Disease Risk). Donors were selected from a large UK cohort of 6,000+ research-diagnosed early or late-onset AD cases and elderly cognitively healthy controls, many of whom have lived through the age of risk for disease development (>85 years). We include iPSC with extremes of global AD polygenic risk (high-risk late-onset AD: 34; high-risk early-onset AD: 29; low-risk control: 27) as well as those reflecting complement pathway-specific genetic risk (high-risk AD: 9; low-risk controls: 10). All iPSC have associated clinical, longitudinal, and genetic datasets and will be available through collaboration or from cell (EBiSC) and data (DPUK) repositories.
Self-supervised predictive learning accounts for cortical layer-specificity.
The neocortex constructs an internal representation of the world, but the underlying circuitry and computational principles remain unclear. Inspired by self-supervised learning algorithms, we propose a computational theory in which layer 2/3 (L2/3) integrates past sensory input, relayed via layer 4, with top-down context to predict incoming sensory stimuli. Learning is self-supervised by comparing L2/3 predictions with the latent representations of actual sensory input arriving at L5. We demonstrate that our model accurately predicts sensory information in context-dependent temporal tasks, and that its predictions are robust to noisy and occluded sensory input. Additionally, our model generates layer-specific sparsity, consistent with experimental observations. Next, using a sensorimotor task, we show that the model's L2/3 and L5 prediction errors mirror mismatch responses observed in awake, behaving mice. Finally, through manipulations, we offer testable predictions to unveil the computational roles of various cortical features. In summary, our findings suggest that the multi-layered neocortex empowers the brain with self-supervised predictive learning.
Regulatory T cells attenuate chronic inflammation and cardiac fibrosis in hypertrophic cardiomyopathy.
Hypertrophic cardiomyopathy (HCM) is a common, serious, genetic heart muscle disorder. Although the biophysical mechanisms by which gene variants in sarcomeric proteins disrupt cardiomyocyte function are largely understood, the cellular and molecular pathways leading to the complex, variable, and adverse remodeling of the non-myocyte compartment are unexplained. Here, we report that postmortem and explanted human HCM hearts exhibited chronic focal leukocyte infiltration and prominent activation of immune cells. Gene set enrichment analysis (GSEA) revealed that active immune responses were present in the mid- and late-stage HCM human hearts and in mouse hearts from several HCM mouse models. The alpha cardiac actin 1-E99K (Actc1E99K) HCM mouse model was selected for the study because it closely recapitulates the features of progressive remodeling and fibrosis seen in advanced disease in patients. Genetic depletion of lymphocytes in recombination activating gene 1-knockout (Rag-1KO) mice led to marked exacerbation of adverse cardiac remodeling in the Actc1E99K mice. Detailed characterization of cardiac regulatory T cells (Treg cells) demonstrated a time-dependent increase in Actc1E99K hearts with altered immunosuppressive profiles. Adoptive transfer of splenic Treg cells reduced cardiac fibrosis and improved systolic dysfunction in Actc1E99K mice with or without lymphocytes. In addition, low-dose interleukin-2 (IL-2)/anti-IL-2 complex (IL-2/c), which specifically induced Treg cell expansion in vivo, ameliorated cardiac fibrosis and reduced macrophage infiltration and activation in Actc1E99K mice. These data contribute to our understanding of HCM and support the use of Treg cells as a clinically testable therapeutic strategy for cardiac fibrosis in the HCM heart.
Hepcidin and Tissue-Specific Iron Regulatory Networks.
Hepcidin is primarily secreted by the liver and functions as an endocrine hormone. However, a growing number of studies show that hepcidin can also be produced locally by other cells and organs, where it acts in an autocrine/paracrine manner to mediate important iron-dependent pathways. These pathways can operate under normal homeostatic conditions or become relevant in pathophysiological conditions (inflammation, infection, cancer, liver disease, myocardial infarction, etc.). This chapter will delve into the local roles of hepcidin, highlighting its unconventional functions in barrier maintenance, host defense, growth, tissue housekeeping, and injury repair.
Widespread Changes in the Immunoreactivity of Bioactive Peptide T14 After Manipulating the Activity of Cortical Projection Neurons
Previous studies have suggested that T14, a 14-amino-acid peptide derived from acetylcholinesterase (AChE), functions as an activity-dependent signalling molecule with key roles in brain development, and its dysregulation has been linked to neurodegeneration in Alzheimer’s disease. In this study, we examined the distribution of T14 under normal developmental conditions in the mouse forebrain, motor cortex (M1), striatum (STR), and substantia nigra (SN). T14 immunoreactivity declined from E16 to E17 and further decreased by P0, then peaked at P7 during early postnatal development before declining again by adulthood at P70. Lower T14 immunoreactivity in samples processed without Triton indicated that T14 is primarily localised intracellularly. To explore the relationship between T14 expression and neuronal activity, we used mouse models with chronic silencing (Rbp4Cre-Snap25), acute silencing (Rbp4Cre-hM4Di), and acute activation (Rbp4Cre-hM3D1). Chronic silencing altered the location and size of intracellular T14-immunoreactive particles in adult brains, while acute silencing had no observable effect. In contrast, acute activation increased T14+ density in the STR, modified T14 puncta size near Rbp4Cre cell bodies in M1 layer 5 and their projections to the STR, and enhanced co-localisation of T14 with presynaptic terminals in the SN.
ER Stress and Autophagic Perturbations Lead to Elevated Extracellular α-Synuclein in GBA-N370S Parkinson's iPSC-Derived Dopamine Neurons.
Heterozygous mutations in the glucocerebrosidase gene (GBA) represent the strongest common genetic risk factor for Parkinson's disease (PD), the second most common neurodegenerative disorder. However, the molecular mechanisms underlying this association are still poorly understood. Here, we have analyzed ten independent induced pluripotent stem cell (iPSC) lines from three controls and three unrelated PD patients heterozygous for the GBA-N370S mutation, and identified relevant disease mechanisms. After differentiation into dopaminergic neurons, we observed misprocessing of mutant glucocerebrosidase protein in the ER, associated with activation of ER stress and abnormal cellular lipid profiles. Furthermore, we observed autophagic perturbations and an enlargement of the lysosomal compartment specifically in dopamine neurons. Finally, we found increased extracellular α-synuclein in patient-derived neuronal culture medium, which was not associated with exosomes. Overall, ER stress, autophagic/lysosomal perturbations, and elevated extracellular α-synuclein likely represent critical early cellular phenotypes of PD, which might offer multiple therapeutic targets.
Transcriptomic profiling of purified patient-derived dopamine neurons identifies convergent perturbations and therapeutics for Parkinson's disease.
While induced pluripotent stem cell (iPSC) technologies enable the study of inaccessible patient cell types, cellular heterogeneity can confound the comparison of gene expression profiles between iPSC-derived cell lines. Here, we purified iPSC-derived human dopaminergic neurons (DaNs) using the intracellular marker, tyrosine hydroxylase. Once purified, the transcriptomic profiles of iPSC-derived DaNs appear remarkably similar to profiles obtained from mature post-mortem DaNs. Comparison of the profiles of purified iPSC-derived DaNs derived from Parkinson's disease (PD) patients carrying LRRK2 G2019S variants to controls identified significant functional convergence amongst differentially-expressed (DE) genes. The PD LRRK2-G2019S associated profile was positively matched with expression changes induced by the Parkinsonian neurotoxin rotenone and opposed by those induced by clioquinol, a compound with demonstrated therapeutic efficacy in multiple PD models. No functional convergence amongst DE genes was observed following a similar comparison using non-purified iPSC-derived DaN-containing populations, with cellular heterogeneity appearing a greater confound than genotypic background.
Cellular α-synuclein pathology is associated with bioenergetic dysfunction in Parkinson's iPSC-derived dopamine neurons.
Parkinson's disease (PD) is the second most common neurodegenerative disorder and a central role for α-synuclein (αSyn; SNCA) in disease aetiology has been proposed based on genetics and neuropathology. To better understand the pathological mechanisms of αSyn, we generated induced pluripotent stem cells (iPSCs) from healthy individuals and PD patients carrying the A53T SNCA mutation or a triplication of the SNCA locus and differentiated them into dopaminergic neurons (DAns). iPSC-derived DAn from PD patients carrying either mutation showed increased intracellular αSyn accumulation, and DAns from patients carrying the SNCA triplication displayed oligomeric αSyn pathology and elevated αSyn extracellular release. Transcriptomic analysis of purified DAns revealed perturbations in expression of genes linked to mitochondrial function, consistent with observed reduction in mitochondrial respiration, impairment in mitochondrial membrane potential, aberrant mitochondrial morphology and decreased levels of phosphorylated DRP1Ser616. Parkinson's iPSC-derived DAns showed increased endoplasmic reticulum stress and impairments in cholesterol and lipid homeostasis. Together, these data show a correlation between αSyn cellular pathology and deficits in metabolic and cellular bioenergetics in the pathology of PD.
Early deficits in an in vitro striatal microcircuit model carrying the Parkinson's GBA-N370S mutation.
Understanding medium spiny neuron (MSN) physiology is essential to understand motor impairments in Parkinson's disease (PD) given the architecture of the basal ganglia. Here, we developed a custom three-chambered microfluidic platform and established a cortico-striato-nigral microcircuit partially recapitulating the striatal presynaptic landscape in vitro using induced pluripotent stem cell (iPSC)-derived neurons. We found that, cortical glutamatergic projections facilitated MSN synaptic activity, and dopaminergic transmission enhanced maturation of MSNs in vitro. Replacement of wild-type iPSC-derived dopamine neurons (iPSC-DaNs) in the striatal microcircuit with those carrying the PD-related GBA-N370S mutation led to a depolarisation of resting membrane potential and an increase in rheobase in iPSC-MSNs, as well as a reduction in both voltage-gated sodium and potassium currents. Such deficits were resolved in late microcircuit cultures, and could be reversed in younger cultures with antagonism of protein kinase A activity in iPSC-MSNs. Taken together, our results highlight the unique utility of modelling striatal neurons in a modular physiological circuit to reveal mechanistic insights into GBA1 mutations in PD.