Search

TDP-43 pathology is found in several neurodegenerative disorders, collectively referred to as “TDP-43 proteinopathies”. Aggregates of TDP-43 are present in the brains and spinal cords of >97% of amyotrophic lateral sclerosis (ALS), and in brains of ∼50% of frontotemporal dementia (FTD) patients. While mutations in the TDP-43 gene (TARDBP) are usually associated with ALS, many clinical reports have linked these mutations to cognitive impairments and/or FTD, but also to other neurodegenerative disorders including Parkinsonism (PD) or progressive supranuclear palsy (PSP). TDP-43 is a ubiquitously expressed, highly conserved RNA-binding protein that is involved in many cellular processes, mainly RNA metabolism. To investigate systemic pathological mechanisms in TDP-43 proteinopathies, aiming to capture the pleiotropic effects of TDP-43 mutations, we have further characterised a mouse model carrying a point mutation (M323K) within the endogenous Tardbp gene. Homozygous mutant mice developed cognitive and behavioural deficits as early as 3 months of age. This was coupled with significant brain structural abnormalities, mainly in the cortex, hippocampus, and white matter fibres, together with progressive cortical interneuron degeneration and neuroinflammation. At the motor level, progressive phenotypes appeared around 6 months of age. Thus, cognitive phenotypes appeared to be of a developmental origin with a mild associated progressive neurodegeneration, while the motor and neuromuscular phenotypes seemed neurodegenerative, underlined by a progressive loss of upper and lower motor neurons as well as distal denervation. This is accompanied by progressive elevated TDP-43 protein and mRNA levels in cortex and spinal cord of homozygous mutant mice from 3 months of age, together with increased cytoplasmic TDP-43 mislocalisation in cortex, hippocampus, hypothalamus, and spinal cord at 12 months of age. In conclusion, we find that Tardbp M323K homozygous mutant mice model many aspects of human TDP-43 proteinopathies, evidencing a dual role for TDP-43 in brain morphogenesis as well as in the maintenance of the motor system, making them an ideal in vivo model system to study the complex biology of TDP-43.

Abstract Amyotrophic lateral sclerosis is a complex disorder most of which is ‘sporadic’ of unknown origin but approximately 10% is familial, arising from single mutations in any of more than 30 genes. Thus, there are more than 30 familial ALS subtypes, with different, often unknown, molecular pathologies leading to a complex constellation of clinical phenotypes. We have mouse models for many genetic forms of the disorder, but these do not, on their own, necessarily show us the key pathological pathways at work in human patients. To date, we have no models for the 90% of ALS that is ‘sporadic’. Potential therapies have been developed mainly using a limited set of mouse models, and through lack of alternatives, in the past these have been tested on patients regardless of aetiology. Cancer researchers have undertaken therapy development with similar challenges; they have responded by producing complex mouse models that have transformed understanding of pathological processes, and they have implemented patient stratification in multi-centre trials, leading to the effective translation of basic research findings to the clinic. ALS researchers have successfully adopted this combined approach, and now to increase our understanding of key disease pathologies, and our rate of progress for moving from mouse models to mechanism to ALS therapies we need more, innovative, complex mouse models to address specific questions.

Summary: Amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD) is a fatal neurodegenerative disorder, and continued innovation is needed for improved understanding and for developing therapeutics. We have created next-generation genomically humanized knockin mouse models, by replacing the mouse genomic region of Sod1, Tardbp (TDP-43), and Fus, with their human orthologs, preserving human protein biochemistry and splicing with exons and introns intact. We establish a new standard of large knockin allele quality control, demonstrating the utility of indirect capture for enrichment of a genomic region of interest followed by Oxford Nanopore sequencing. Extensive analysis shows that homozygous humanized animals only express human protein at endogenous levels. Characterization of humanized FUS animals showed that they are phenotypically normal throughout their lifespan. These humanized strains are vital for preclinical assessment of interventions and serve as templates for the addition of coding or non-coding human ALS/FTD mutations to dissect disease pathomechanisms, in a physiological context.

Random mutagenesis can uncover novel genes involved in phenotypic traits. Here the authors perform a large-scale phenotypic screen on over 100 mouse strains generated by ENU mutagenesis to identify mice with age-related diseases, which they attribute to specific mutations revealed by whole-genome sequencing.

Loss-of-function mutations in a human AMPA receptor-associated protein, ferric chelate reductase 1-like (FRRS1L), are associated with a devastating neurological condition incorporating choreoathetosis, cognitive deficits and epileptic encephalopathies. Furthermore, evidence from overexpression and ex vivo studies has implicated FRRS1L in AMPA receptor biogenesis, suggesting that changes in glutamatergic signalling might underlie the disorder. Here, we investigated the neurological and neurobehavioural correlates of the disorder using a mouse Frrs1l null mutant. The study revealed several neurological defects that mirrored those seen in human patients. We established that mice lacking Frrs1l suffered from a broad spectrum of early-onset motor deficits with no progressive, age-related deterioration. Moreover, Frrs1l−/− mice were hyperactive, irrespective of test environment, exhibited working memory deficits and displayed significant sleep fragmentation. Longitudinal electroencephalographic (EEG) recordings also revealed abnormal EEG results in Frrs1l−/− mice. Parallel investigations into disease aetiology identified a specific deficiency in AMPA receptor levels in the brain of Frrs1l−/− mice, while the general levels of several other synaptic components remained unchanged, with no obvious alterations in the number of synapses. Furthermore, we established that Frrsl1 deletion results in an increased proportion of immature AMPA receptors, indicated by incomplete glycosylation of GLUA2 (also known as GRIA2) and GLUA4 (also known as GRIA4) AMPA receptor proteins. This incomplete maturation leads to cytoplasmic retention and a reduction of those specific AMPA receptor levels in the postsynaptic membrane. Overall, this study determines, for the first time in vivo, how loss of FRRS1L function can affect glutamatergic signalling, and provides mechanistic insight into the development and progression of a human hyperkinetic disorder. This article has an associated First Person interview with the first author of the paper.

A wide range of genetic mouse models is available to help researchers dissect human disease mechanisms. Each type of model has its own distinctive characteristics arising from the nature of the introduced mutation, as well as from the specific changes to the gene of interest. Here, we review the current range of mouse models with mutations in genes causative for the human neurodegenerative disease amyotrophic lateral sclerosis. We focus on the two main types of available mutants: transgenic mice and those that express mutant genes at physiological levels from gene targeting or from chemical mutagenesis. We compare the phenotypes for genes in which the two classes of model exist, to illustrate what they can teach us about different aspects of the disease, noting that informative models may not necessarily mimic the full trajectory of the human condition. Transgenic models can greatly overexpress mutant or wild-type proteins, giving us insight into protein deposition mechanisms, whereas models expressing mutant genes at physiological levels may develop slowly progressing phenotypes but illustrate early-stage disease processes. Although no mouse models fully recapitulate the human condition, almost all help researchers to understand normal and abnormal biological processes, providing that the individual characteristics of each model type, and how these may affect the interpretation of the data generated from each model, are considered and appreciated.

Huntington’s disease (HD) is a monogenic fatal neurodegenerative disorder. However, there is increasing evidence that HD is a pleiotropic systemic disorder. In particular, skeletal muscle metabolism is greatly affected in HD, which in turn can have a major impact on whole-body metabolism and energetic balance. Throughout an unbiased mutagenesis approach in HD mice, we have found that Scn4a , a skeletal muscle–specific sodium channel gene, is a modifier of the disease. Mutations in Scn4a enhance HD disease progression and weight loss by accelerating muscle waste and cachexia, increasing skeletal muscle activity and energy demands. At the molecular level, Scn4a mutations activate AMP-activated protein kinase (AMPK), leading to a fibre switch towards more oxidative types. These adaptations seen in HD; Scn4a double mutant muscles are similar to those observed in healthy individuals after endurance exercise training regimes. This prompted us to assess the effects of an endurance exercise regime in HD mice, independently showing that skeletal muscle adaptations leading to the activation of AMPK are detrimental for HD pathogenesis. Although it is undeniable that physical exercise can lead to many health benefits, our work shows that, at least under certain situations such as in HD, an endurance exercise routine could be a detrimental therapeutic option.

SUMMARY Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder that results in the death of motor neurons in the brain and spinal cord. The disorder generally strikes in mid-life, relentlessly leading to paralysis and death, typically 3–5 years after diagnosis. No effective treatments are available. Up to 10% of ALS is familial, usually autosomal dominant. Several causative genes are known and, of these, mutant superoxide dismutase 1 (SOD1) is by far the most frequently found, accounting for up to 20% of familial ALS. A range of human mutant SOD1 transgenic mouse strains has been produced, and these largely successfully model the human disease. Of these, the most widely used is the SOD1 mouse, which expresses a human SOD1 transgene with a causative G93A mutation. This mouse model is excellent for many purposes but carries up to 25 copies of the transgene and produces a great excess of SOD1 protein, which might affect our interpretation of disease processes. A variant of this strain carries a deletion of the transgene array such that the copy number is dropped to eight to ten mutant SOD1 genes. This ‘deleted’ ‘low-copy’ mouse undergoes a slower course of disease, over many months. Here we have carried out a comprehensive analysis of phenotype, including nerve and muscle physiology and histology, to add to our knowledge of this ‘deleted’ strain and give baseline data for future studies. We find differences in phenotype that arise from genetic background and sex, and we quantify the loss of nerve and muscle function over time. The slowly progressive pathology observed in this mouse strain could provide us with a more appropriate model for studying early-stage pathological processes in ALS and aid the development of therapies for early-stage treatments.

Mutations in TARDBP, encoding Tar DNA binding protein-43 (TDP43), cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Attempts to model TDP43 dysfunction in mice have used knockouts or transgenic overexpressors, which have revealed the difficulties of manipulating TDP43, whose level is tightly controlled by auto-regulation. In a complementary approach, to create useful mouse models for the dissection of TDP43 function and pathology, we have identified a nonsense mutation in the endogenous mouse Tardbp gene through screening an N-ethyl-N-nitrosourea (ENU) mutant mouse archive. The mutation is predicted to cause a Q101X truncation in TDP43. We have characterised Tardbp(Q101X) mice to investigate this mutation in perturbing TDP43 biology at endogenous expression levels. We found the Tardbp(Q101X) mutation is homozygous embryonic lethal, highlighting the importance of TDP43 in early development. Heterozygotes (Tardbp(+/Q101X) ) have abnormal levels of mutant transcript, but we find no evidence of the truncated protein and mice have similar full-length TDP43 protein levels as wildtype littermates. Nevertheless, Tardbp(+/Q101X) mice have abnormal alternative splicing of downstream gene targets, and limb-clasp and body tone phenotypes. Thus the nonsense mutation in Tardbp causes a mild loss-of-function phenotype and behavioural assessment suggests underlying neurological abnormalities. Due to the role of TDP43 in ALS, we investigated potential interactions with another known causative gene, mutant superoxide dismutase 1 (SOD1). Tardbp(+/Q101X) mice were crossed with the SOD1(G93Adl) transgenic mouse model of ALS. Behavioural and physiological assessment did not reveal modifying effects on the progression of ALS-like symptoms in the double mutant progeny from this cross. In summary, the Tardbp(Q101X) mutant mice are a useful tool for the dissection of TDP43 protein regulation, effects on splicing, embryonic development and neuromuscular phenotypes. These mice are freely available to the community.

Many of the neurodegenerative diseases that afflict people in later life are associated with the formation of protein aggregates. These so-called "proteinopathies" include Alzheimer's disease (AD) and Huntington's disease (HD). The insulin/insulin-like growth factor signalling (IIS) pathway has been proposed to modulate such diseases in model organisms, as well as the general ageing process. In this pathway, insulin-like growth factor binds to insulin-like growth factor receptors, such as the insulin-like growth factor 1 receptor (IGF-1R). Heterozygous deletion of Igf-1r has been shown to lead to increased lifespan in mice. Reducing the activity of this pathway had benefits in a HD C. elegans model, and some of these may be attributed to the expected inhibition of mTOR activity resulting in an increase in autophagy, which would enhance mutant huntingtin clearance. Thus, we tested if heterozygous deletion of Igf-1r would lead to benefits in HD related phenotypes in the mouse. Surprisingly, reducing Igf-1r levels led to some beneficial effects in HD females, but also led to some detrimental effects in HD males. Interestingly, Igf-1r deficiency had no discernible effects on downstream mTOR signalling in HD mice. These results do not support a broad beneficial effect of diminishing the IIS pathway in HD pathology in a mammalian system.

Tendinopathy is a tendon disorder that is caused by the failure to self-repair and has many pathological characteristics such as disorganized ECM and decreased cell viability. We have identified a possible target to combat these changes, AMPK, an energy stress sensor that was shown to maintain intracellular homeostasis. Through bulk RNA-sequencing of healthy and tendinopathic tendons from humans we have identified a novel finding of downregulation of AMPK signaling in the tendinopathic samples which suggests AMPK plays a role in tendon homeostasis. Our studies utilizing a conditional knock-out of Prkaa1 in tendon in mice showed that loss of AMPK results in degenerative ECM, impaired biomechanical properties and increased cellular senescence in Achilles tendon through the lifespan. Additionally, we found that exercise can delay senescence onset independent of AMPK. These findings highlight the importance of energy metabolism in tendon health which will assist in understanding the onset and progression of tendinopathy.

Skeletal muscle activation using optogenetics has emerged as a promising technique for inducing noninvasive muscle contraction and assessing muscle function both in vivo and in vitro. Transgenic mice overexpressing the optogenetic fusion protein, Channelrhodopsin 2-EYFP (ChR2-EYFP) in skeletal muscle are widely used; however, overexpression of fluorescent proteins can negatively impact the functionality of activable tissues. In this study, we characterized the contractile properties of ChR2-EYFP skeletal muscle and introduced the ChR2-only mouse model that expresses light-responsive ChR2 without the fluorescent EYFP in their skeletal muscles. We found a significant reduction in the contractile ability of ChR2-EYFP muscles compared with ChR2-only and WT mice, observed under both electrical and optogenetic stimulation paradigms. Bulk RNAseq identified the downregulation of genes associated with transmembrane transport and metabolism in ChR2-EYFP muscle, while the ChR2-only muscle did not demonstrate any notable deviations from WT muscle. The RNAseq results were further corroborated by a reduced protein-level expression of ion channel-related HCN2 in ChR2-EYFP muscles and gluconeogenesis-modulating FBP2 in both ChR2-EYFP and ChR2-only muscles. Overall, this study reveals an intrinsic skeletal dysfunction in the widely used ChR2-EYFP mice model and underscores the importance of considering alternative optogenetic models, such as the ChR2-only, for future research in skeletal muscle optogenetics., (© 2024 The Author(s). The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Sensory neurons specialize in detecting and signaling the presence of diverse environmental stimuli. Neuronal injury or disease may undermine such signaling, diminishing the availability of crucial information. Can animals distinguish between a stimulus not being present and the inability to sense that stimulus in the first place? To address this question, we studied Caenorhabditis elegans nematode worms that lack gentle body touch sensation due to genetic mechanoreceptor dysfunction. We previously showed that worms can compensate for the loss of touch by enhancing their sense of smell, via an FLP-20 neuropeptide pathway. Here, we find that touch-deficient worms exhibit, in addition to sensory compensation, also cautious-like behavior, as if preemptively avoiding potential undetectable hazards. Intriguingly, these behavioral adjustments are abolished when the touch neurons are removed, suggesting that touch neurons are required for signaling the unavailability of touch information, in addition to their conventional role of signaling touch stimulation. Furthermore, we found that the ASE taste neurons, which similarly to the touch neurons, express the FLP-20 neuropeptide, exhibit altered FLP-20 expression levels in a touch-dependent manner, thus cooperating with the touch circuit. These results imply a novel form of neuronal signaling that enables C. elegans to distinguish between lack of touch stimulation and loss of touch sensation, producing adaptive behavioral adjustments that could overcome the inability to detect potential threats., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Staum et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Effective tendon regeneration following injury is contingent on appropriate differentiation of recruited cells and deposition of mature, aligned, collagenous extracellular matrix that can withstand the extreme mechanical demands placed on the tissue. As such, myriad biomaterial approaches have been explored to provide biochemical and physical cues that encourage tenogenesis and template aligned matrix deposition in lieu of dysfunctional scar tissue formation. Fiber-reinforced hydrogels present an ideal biomaterial system toward this end given their transdermal injectability, tunable stiffness over a range amenable to tenogenic differentiation of progenitors, and capacity for modular inclusion of biochemical cues. Here, tunable and modular, fiber-reinforced, synthetic hydrogels are employed to elucidate salient microenvironmental determinants of tenogenesis and aligned collagen deposition by tendon progenitor cells. Transforming growth factor β3 drives a cell fate switch toward pro-regenerative or pro-fibrotic phenotypes, which can be biased toward the former by culture in softer microenvironments or inhibition of the RhoA/ROCK activity. Furthermore, studies demonstrate that topographical anisotropy in fiber-reinforced hydrogels critically mediates the alignment of de novo collagen fibrils, reflecting native tendon architecture. These findings inform the design of cell-free, injectable, synthetic hydrogels for tendon tissue regeneration and, likely, that of a range of load-bearing connective tissues., (© 2024 The Authors. Advanced Healthcare Materials published by Wiley‐VCH GmbH.)

Many transgender youth seek gender affirming care, such as puberty suppression, to prolong decision-making and to align their physical sex characteristics with their gender identity. During peripubertal growth, connective tissues such as tendon rapidly adapt to applied mechanical loads (e.g., exercise) yet if and how tendon adaptation is influenced by sex and gender affirming hormone therapy during growth remains unknown. The goal of this study was to understand the how pubertal suppression influences the structural and functional properties of the Achilles tendon using an established mouse model of transmasculine gender affirming hormone therapy. C57BL/6N female-born mice were assigned to experimental groups to mimic gender-affirming hormone therapy in human adolescents, and treatment was initiated prior to the onset of puberty (at postnatal day 26, P26). Experimental groups included controls and mice serially treated with gonadotropin release hormone analogue (GnRHa), delayed Testosterone (T), or GnRHa followed by T. We found that puberty suppression using GnRHa, with and without T, improved the overall tendon load capacity in female-born mice. Treatment with T resulted in an increase in the maximum load that tendon can withstand before failure. Additionally, we found that GnRHa, but not T, treatment resulted in a significant increase in cell density at the Achilles enthesis., Competing Interests: DISCLOSURES The authors declare no conflict of interest.

Persistent inflammation has been associated with severe disc degeneration (DD). This study investigated the effect of prolonged nuclear factor κB (NF-κB) activation in DD. Using an inducible mouse model, we genetically targeted cells expressing aggrecan, a primary component of the disc extra cellular matrix, for activation of the canonical NF-κB pathway. Prolonged NF-κB activation led to severe structural degeneration accompanied by increases in gene expression of inflammatory molecules ( Il1b , Cox2 , Il6 , and Nos2 ), chemokines ( Mcp1 and Mif ), and catabolic enzymes ( Mmp3 , Mmp9 , and Adamts4 ). Increased recruitment of proinflammatory (F4/80 + ,CD38 + ) and inflammatory resolving (F4/80 + ,CD206 + ) macrophages was observed within caudal discs. We found that the secretome of inflamed caudal disc cells increased macrophage migration and inflammatory activation. Lumbar discs did not exhibit phenotypic changes, suggestive of regional spinal differences in response to inflammatory genetic overactivation. Results suggest prolonged NF-κB activation can induce severe DD through increases in inflammatory cytokines, chemotactic proteins, catabolic enzymes, and the recruitment and activation of macrophage cell populations.

Purpose of Review: Interfacial tissue exists throughout the body at cartilage-to-bone (osteochondral interface) and tendon-to-bone (enthesis) interfaces. Healing of interfacial tissues is a current challenge in regenerative approaches because the interface plays a critical role in stabilizing and distributing the mechanical stress between soft tissues (e.g., cartilage and tendon) and bone. The purpose of this review is to identify new directions in the field of interfacial tissue development and physiology that can guide future regenerative strategies for improving post-injury healing., Recent Findings: Cues from interfacial tissue development may guide regeneration including biological cues such as cell phenotype and growth factor signaling; structural cues such as extracellular matrix (ECM) deposition, ECM, and cell alignment; and mechanical cues such as compression, tension, shear, and the stiffness of the cellular microenvironment. In this review, we explore new discoveries in the field of interfacial biology related to ECM remodeling, cellular metabolism, and fate. Based on emergent findings across multiple disciplines, we lay out a framework for future innovations in the design of engineered strategies for interface regeneration. Many of the key mechanisms essential for interfacial tissue development and adaptation have high potential for improving outcomes in the clinic., (© 2024. The Author(s).)

The development of musculoskeletal tissues such as tendon, enthesis, and bone relies on proliferation and differentiation of mesenchymal progenitor cells. Gli1+ cells have been described as putative stem cells in several tissues and are presumed to play critical roles in tissue formation and maintenance. For example, the enthesis, a fibrocartilage tissue that connects tendon to bone, is mineralized postnatally by a pool of Gli1+ progenitor cells. These cells are regulated by hedgehog signaling, but it is unclear if TGFβ signaling, necessary for tenogenesis, also plays a role in their behavior. To examine the role of TGFβ signaling in Gli1+ cell function, the receptor for TGFβ, TbR2, was deleted in Gli1-lineage cells in mice at P5. Decreased TGFβ signaling in these cells led to defects in tendon enthesis formation by P56, including defective bone morphometry underlying the enthesis and decreased mechanical properties. Immunohistochemical staining of these Gli1+ cells showed that loss of TGFβ signaling reduced proliferation and increased apoptosis. In vitro experiments using Gli1+ cells isolated from mouse tail tendons demonstrated that TGFβ controls cell proliferation and differentiation through canonical and non-canonical pathways and that TGFβ directly controls the tendon transcription factor scleraxis by binding to its distant enhancer. These results have implications in the development of treatments for tendon and enthesis pathologies., (© 2024 Federation of American Societies for Experimental Biology.)

Objective: Low back pain (LBP) is the leading cause of global disability and is thought to be driven primarily by intervertebral disc (IVD) degeneration (DD). Persistent upregulation of catabolic enzymes and inflammatory mediators have been associated with severe cases of DD. Nuclear factor kappa B (NF-κB) is a master transcription regulator of immune responses and is over expressed during inflammatory-driven musculoskeletal diseases, including DD. However, its role in triggering DD is unknown. Therefore, this study investigated the effect of NF-κB pathway over-activation on IVD integrity and DD pathology., Methods: Using skeletally mature mouse model, we genetically targeted IVD cells for canonical NF-κB pathway activation via expression of a constitutively active form of inhibitor of κB kinase B (IKKβ), and assessed changes in IVD cellularity, structural integrity including histology, disc height, and extracellular matrix (ECM) biochemistry, biomechanics, expression of inflammatory, catabolic, and neurotropic mediators, and changes in macrophage subsets, longitudinally up to 6-months post activation., Results: Prolonged NF-κB activation led to severe structural degeneration, with a loss of glycosaminoglycan (GAG) content and complete loss of nucleus pulposus (NP) cellularity. Structural and compositional changes decreased IVD height and compressive mechanical properties with prolonged NF-κB activation. These alterations were accompanied by increases in gene expression of inflammatory molecules ( Il1b, Il6, Nos2 ), chemokines ( Mcp1 , Mif ), catabolic enzymes ( Mmp3, Mmp9, Adamts4 ), and neurotrophic factors ( Bdnf , Ngf ) within IVD tissue. Increased recruitment of activated F4/80 + macrophages exhibited a greater abundance of pro-inflammatory (CD38 + ) over inflammatory-resolving (CD206 + ) macrophage subsets in the IVD, with temporal changes in the relative abundance of macrophage subsets over time, providing evidence for temporal regulation of macrophage polarization in DD in vivo, where macrophages participate in resolving the inflammatory cascade but promote fibrotic transformation of the IVD matrix. We further show that NF-κB driven secretory factors from IVD cells increase macrophage migration and inflammatory activation, and that the secretome of inflammatory-resolving macrophages mitigates effects of NF-κB overactivation., Conclusion: Overall the observed results suggest prolonged NF-κB activation can induce severe DD, acting through increases in inflammatory cytokines, chemotactic proteins, catabolic enzymes, and the recruitment and inflammatory activation of a macrophage cell populations, that can be mitigated with inflammatory-resolving macrophage secretome.

Skeletal shape depends on the transmission of contractile muscle forces from tendon to bone across the enthesis. Loss of muscle loading impairs enthesis development, yet little is known if and how the postnatal enthesis adapts to increased loading. Here, we studied adaptations in enthesis structure and function in response to increased loading, using optogenetically induced muscle contraction in young (i.e., growth) and adult (i.e., mature) mice. Daily bouts of unilateral optogenetic loading in young mice led to radial calcaneal expansion and warping. This also led to a weaker enthesis with increased collagen damage in young tendon and enthisis, with little change in adult mice. We then used RNA sequencing to identify the pathways associated with increased mechanical loading during growth. In tendon, we found enrichment of glycolysis, focal adhesion, and cell-matrix interactions. In bone, we found enrichment of inflammation and cell cycle. Together, we demonstrate the utility of optogenetic-induced muscle contraction to elicit in vivo adaptation of the enthesis.

Background: Lower preoperative pelvic obliquity (PO) and L5 tilt have been associated with good radiographic outcomes when the fusion ended short of the pelvis in children with neuromuscular scoliosis (NMS). Our purpose was to identify indications to exclude the pelvis in children with hypotonic NMS treated with growth-friendly instrumentation., Methods: This was a multicenter retrospective review. Children with spinal muscular atrophy and muscular dystrophy treated with dual traditional growing rod, magnetically controlled growing rod, or vertical expandable prosthetic titanium rib with minimum 2-year follow-up after the index surgery were identified., Results: A total of 125 patients met the inclusion criteria. Thirty-eight patients had distal spine anchors (DSAs) and 87 patients had distal pelvic anchors (DPAs) placed at the index surgery. Demographics and length of follow-up were similar between the groups but there was a greater percentage of DPA patients who were nonambulatory [79 patients (91%) vs. 18 patients (47%), P <0.0001]. Preindex radiographic measures were similar except the DSA patients had a lower PO (11 vs. 19 degrees, P =0.0001) and L5 tilt (8 vs. 12 degrees, P =0.001). Postindex and most recent radiographic data were comparable between the groups. There was no difference in the complication and unplanned returns to the operating room rates.Subanalysis of the DSA group based on ambulatory status showed similar radiographic measures except the ambulatory patients had a lower PO at all time points (preindex: 5 vs. 16 degrees, P =0.011; postindex: 6 vs. 10 degrees, P =0.045; most recent follow-up: 5 vs. 14 degrees, P =0.028). Only 1 ambulatory DSA patient had a PO ≥10 degrees at most recent follow-up compared with 6 nonambulatory DSA patients. Three (8%) DSA patients, all nonambulatory, underwent extension of their instrumentation to the pelvis., Conclusions: Pelvic fixation should be strongly considered in nonambulatory children with hypotonic NMS treated with growth-friendly instrumentation. At intermediate-term follow-up, revision surgery to include the pelvis was rare but DSAs do not seem effective at maintaining control of PO in nonambulatory patients. DSA and DPA were equally effective at maintaining major curve control, and complication and unplanned returns to the operating room rates were similar., Level of Evidence: Level III-therapeutic., Competing Interests: J.T.B. has received consulting fees from Depuy Synthes, Medtronic, and OrthoPediatrics. P.J.C. has received research grants from the Setting Scoliosis Straight Foundation and the Children’s Spine Study Group, has a patent for dynamic lung magnetic resonance imaging, and is a board or committee member of the Scoliosis Research Society, Journal of Bone and Joint Surgery American, Spine Deformity, and the Pediatric Orthopaedic Society of North America. J.T.S. has received royalties from Globus, has a patent for a hydraulic growing rod, and is a board member of the Pediatric Spine Study Group. P.D.S. has received a grant from Ipsen, has received royalties from Globus, has received consulting fees from Depuy Synthes, and has received payment for lectures from NuVasive. P.F.S. has received consulting fees from NuVasive, is a board or committee member of the Pediatric Orthopaedic Society of North America, the Scoliosis Research Society, and Journal of Children’s Orthopaedics, and has stock or stock options from Green Sun Medical. Y.L. has received a research grant from the Scoliosis Research Society, has received consulting fees from Medtronic, has received support for attending an educational event from Zimmer, and is a committee member of the Pediatric Orthopaedic Society of North America and the Scoliosis Research Society. The Pediatric Spine Study Group has received research grants from Boston Orthotics and Prosthetics, Depuy Synthes Spine, Globus Medical, Medtronic, NuVasive, nView Medical, OrthoPediatrics, Pacira, the Pediatric Spine Foundation, Stryker, and Zimmer Biomet. The remaining authors declare no conflicts of interest., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Synthetic hydrogels represent an exciting avenue in the field of regenerative biomaterials given their injectability, orthogonally tunable mechanical properties, and potential for modular inclusion of cellular cues. Separately, recent advances in soluble factor release technology have facilitated control over the soluble milieu in cell microenvironments via tunable microparticles. A composite hydrogel incorporating both of these components can robustly mediate tendon healing following a single injection. Here, a synthetic hydrogel system with encapsulated electrospun fiber segments and a novel microgel-based soluble factor delivery system achieves precise control over topographical and soluble features of an engineered microenvironment, respectively. It is demonstrated that three-dimensional migration of tendon progenitor cells can be enhanced via combined mechanical, topographical, and microparticle-delivered soluble cues in both a tendon progenitor cell spheroid model and an ex vivo murine Achilles tendon model. These results indicate that fiber reinforced hydrogels can drive the recruitment of endogenous progenitor cells relevant to the regeneration of tendon and, likely, a broad range of connective tissues., Competing Interests: The authors declare no conflict of interest., (© 2022 The Authors. Advanced Functional Materials published by Wiley‐VCH GmbH.)

Aims: Clozapine is a unique atypical anti-psychotic agent with best efficacy for treatment resistant schizophrenia compared to other agents, but with increased metabolic adverse effects. We sought to audit the prevalence of diabetes and pre-diabetes in Northland, New Zealand patients on clozapine., Method: We captured all 287 patients in Northland, New Zealand who were prescribed clozapine in September 2021 and obtained demographic, clinical and laboratory data., Results: We discovered that 26.48% had diabetes (one patient type one, 75 type two diabetes) and 14.63% had pre-diabetes that developed after a median of six years' clozapine treatment. Diabetes prevalence is approximately 6% in the general population. NZ Māori made up 65.85% of the entire cohort (35.8% of the general population) and 85.53% of the diabetes patients. NZ Europeans represented most of the remaining 30.66% on clozapine, consistent with the largely bicultural ethnic mix of our region. Māori on clozapine were younger: mean age 42 years, compared to NZ Europeans, mean age 49 years. The average BMI was 37kg/m2 for Māori, 32 for Europeans (range 21-63, SD 8); there was a moderate relationship between clozapine use and increasing BMI (correlation coefficient of 0.74). For the diabetes patients, glycaemic control was overall suboptimal with a mean Hba1c of 66mmol/mol (range 41-117)., Conclusions: Culturally appropriate, flexible and accessible services which integrate both the mental and physical health needs of Northland, New Zealand people with treatment-resistant schizophrenia on clozapine are required to reduce the 41% rate of dysglycaemia in this predominantly Māori group., Competing Interests: Nil.

Rotator cuff disease pathogenesis is associated with intrinsic (e.g., age, joint laxity, muscle weakness) and extrinsic (e.g., mechanical load, fatigue) factors that lead to chronic degeneration of the cuff tissues. However, etiological studies are difficult to perform in patients due to the long duration of disease onset and progression. Therefore, the purpose of this study was to determine the effects of altered joint loading on the rotator cuff. Mice were subjected to one of three load-dependent rotator cuff tendinopathy models: underuse loading, achieved by injecting botulinum toxin-A into the supraspinatus muscle; overuse loading, achieved using downhill treadmill running; destabilization loading, achieved by surgical excision of the infraspinatus tendon. All models were compared to cage activity animals. Whole joint function was assessed longitudinally using gait analysis. Tissue-scale structure and function were determined using microCT, tensile testing, and histology. The molecular response of the supraspinatus tendon and enthesis was determined by measuring the expression of 84 wound healing-associated genes. Underuse and destabilization altered forepaw weight-bearing, decreased tendon-to-bone attachment strength, decreased mineral density of the humeral epiphysis, and reduced tendon strength. Transcriptional activity of the underuse group returned to baseline levels by 4 weeks, while destabilization had significant upregulation of inflammation, growth factors, and extracellular matrix remodeling genes. Surprisingly, overuse activity caused changes in walking patterns, increased tendon stiffness, and primarily suppressed expression of wound healing-related genes. In summary, the tendinopathy models demonstrated how divergent muscle loading can result in clinically relevant alterations in rotator cuff structure, function, and gene expression., (© 2021 Orthopaedic Research Society. Published by Wiley Periodicals LLC.)

Architectured materials offer tailored mechanical properties but are limited in engineering applications due to challenges in maintaining toughness across their attachments. The enthesis connects tendon and bone, two vastly different architectured materials, and exhibits toughness across a wide range of loadings. Understanding the mechanisms by which this is achieved could inform the development of engineered attachments. Integrating experiments, simulations, and previously unexplored imaging that enabled simultaneous observation of mineralized and unmineralized tissues, we identified putative mechanisms of enthesis toughening in a mouse model and then manipulated these mechanisms via in vivo control of mineralization and architecture. Imaging uncovered a fibrous architecture within the enthesis that controls trade-offs between strength and toughness. In vivo models of pathology revealed architectural adaptations that optimize these trade-offs through cross-scale mechanisms including nanoscale protein denaturation, milliscale load-sharing, and macroscale energy absorption. Results suggest strategies for optimizing architecture for tough bimaterial attachments in medicine and engineering.

Rotator cuff supraspinatus tendon injuries are common with high rates of anatomic failure after surgical repair. The purpose of the study was to define clinically relevant features of a mouse model of supraspinatus tendon injury to determine painful, functional, and structural outcomes; we further investigated two cell populations mediating healing using genetic lineage tracing after full detachment and repair of the supraspinatus tendon in mice. The pain was assessed using the mouse grimace scale and function by gait analysis and tensile testing. Histological and microCT analyses were used to determine enthesis/tendon and bone structure, respectively. Lineage tracing was carried out using inducible Cre lines for ScxCreERT2 (tendon cells) and αSMACreERT2 (myofibroblasts and mesenchymal progenitors). Mice only expressed pain transiently after surgery despite long-term impairment of functional and structural properties. Gait, tensile mechanical properties, and bone properties were significantly reduced after injury and repair. Lineage tracing showed relatively few Scx lin tendon cells while αSMA lin cells contributed strongly to scar formation. Despite surgical reattachment of healthy tendon, lineage tracing revealed poor preservation of supraspinatus tendon after acute injury and loss of tendon structure, suggesting that tendon degeneration is also a key impediment of successful rotator cuff repair. Scar formation after surgery is mediated largely by αSMA lin cells and results in permanently reduced functional and structural properties., (© 2020 Orthopaedic Research Society. Published by Wiley Periodicals LLC.)

Despite the negative impacts of increased ultraviolet radiation intensity on plants, these organisms continue to grow and produce under the increased environmental UV levels. We hypothesized that ambient UV intensity can generate acclimations in plant growth, leaf morphology, and photochemical functioning in modern genotypes of Coffea arabica and C. canephora . Coffee plants were cultivated for ca. six months in a mini greenhouse under either near ambient (UVam) or reduced (UVre) ultraviolet regimes. At the plant scale, C. canephora was substantially more impacted by UVam when compared to C. arabica , investing more carbon in all juvenile plant components than under UVre. When subjected to UVam, both species showed anatomic adjustments at the leaf scale, such as increases in stomatal density in C. canephora , at the abaxial and adaxial cuticles in both species, and abaxial epidermal thickening in C. arabica , although without apparent impact on the thickness of palisade and spongy parenchyma. Surprisingly, C. arabica showed more efficient energy dissipation mechanism under UVam than C. canephora . UVam promoted elevated protective carotenoid content and a greater use of energy through photochemistry in both species, as reflected in the photochemical quenching increases. This was associated with an altered chlorophyll a/b ratio (significantly only in C. arabica ) that likely promoted a greater capability to light energy capture. Therefore, UV levels promoted different modifications between the two Coffea sp. regarding plant biomass production and leaf morphology, including a few photochemical differences between species, suggesting that modifications at plant and leaf scale acted as an acclimation response to actual UV intensity.

Tendon disorders are common, affect people of all ages, and are often debilitating. Standard treatments, such as anti-inflammatory drugs, rehabilitation, and surgical repair, often fail. In order to define tendon function and demonstrate efficacy of new treatments, the mechanical properties of tendons from animal models must be accurately determined. Murine animal models are now widely used to study tendon disorders and evaluate novel treatments for tendinopathies; however, determining the mechanical properties of mouse tendons has been challenging. In this study, a new system was developed for tendon mechanical testing that includes 3D-printed fixtures that exactly match the anatomies of the humerus and calcaneus to mechanically test supraspinatus tendons and Achilles tendons, respectively. These fixtures were developed using 3D reconstructions of native bone anatomy, solid modeling, and additive manufacturing. The new approach eliminated artifactual gripping failures (e.g., failure at the growth plate failure rather than in the tendon), decreased overall testing time, and increased reproducibility. Furthermore, this new method is readily adaptable for testing other murine tendons and tendons from other animals.

Tendon disorders represent the most common musculoskeletal complaint for which patients seek medical attention; inflammation drives tendon degeneration before tearing and impairs healing after repair. Clinical evidence has implicated the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway as a correlate of pain-free return to function after surgical repair. However, it is currently unknown whether this response is a reaction to or a driver of pathology. Therefore, we aimed to understand the clinically relevant involvement of the NF-κB pathway in tendinopathy, to determine its potential causative roles in tendon degeneration, and to test its potential as a therapeutic candidate. Transcriptional profiling of early rotator cuff tendinopathy identified increases in NF-κB signaling, including increased expression of the regulatory serine kinase subunit IKKβ, which plays an essential role in inflammation. Using cre-mediated overexpression of IKKβ in tendon fibroblasts, we observed degeneration of mouse rotator cuff tendons and the adjacent humeral head. These changes were associated with increases in proinflammatory cytokines and innate immune cells within the joint. Conversely, genetic deletion of IKKβ in tendon fibroblasts partially protected mice from chronic overuse-induced tendinopathy. Furthermore, conditional knockout of IKKβ improved outcomes after surgical repair, whereas overexpression impaired tendon healing. Accordingly, targeting of the IKKβ/NF-κB pathway in tendon stromal cells may offer previously unidentified therapeutic approaches in the management of human tendon disorders., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Microfluidic sorting offers a unique ability to isolate large numbers of cells for bulk proteomic or metabolomics studies but is currently limited by low throughput and persistent clogging at low flow rates. Recently we uncovered the physical principles governing the inertial focusing of particles in high-Reynolds numbers. Here, we superimpose high Reynolds inertial focusing on Dean vortices, to rapidly isolate large quantities of young and adult yeast from mixed populations at a rate of 10 7 cells/min/channel. Using a new algorithm to rapidly quantify budding scars in isolated yeast populations and system-wide proteomic analysis, we demonstrate that protein quality control and expression of established yeast aging markers such as CalM, RPL5, and SAM1 may change after the very first replication events, rather than later in the aging process as previously thought. Our technique enables the large-scale isolation of microorganisms based on minute differences in size (±1.5 μm), a feat unmatched by other technologies.

Background: Successful fracture fixation depends critically on the stability of the screw-bone interface. Maximum achievable screw torque reflects the competence of this interface, but it cannot be quantified prior to screw stripping. Typically, the surgeon relies on the patients' bone mineral density and radiographs, along with experience and tactile feedback to assess whether sufficient compression can be generated by the screw and bone. However, the local bone quality would also critically influence the strength of the bone-screw interface. We investigated whether Reference Point Indentation can provide quantitative local bone quality measures that can inform subsequent screw-bone competence., Methods: We examined the associations between the maximum screw torque that can be achieved using 3.5 mm, 4.5 mm, and 6.5 mm diameter stainless steel screws at the distal femoral metaphysis and mid-diaphysis from 20 cadavers, with the femoral neck bone mineral density and the local measures of bone quality using Reference Point Indentation., Findings: Indentation Distance Increase, a measure of bone's resistance to microfracture, correlated with the maximum screw stripping torque for the 3.5 mm (p < 0.01; R = 0.56) and 4.5 mm diameter stainless steel screws (p < 0.01; R = 0.57) at the femoral diaphysis. At the femoral metaphysis, femoral neck bone mineral density significantly correlated with the maximum screw stripping torque achieved by the 3.5 mm (p < 0.01; R = 0.61), 4.5 mm (p < 0.01; R = 0.51), and 6.5 mm diameter stainless steel screws (p < 0.01; R = 0.56)., Interpretation: Reference Point Indentation can provide localized measurements of bone quality that may better inform surgeons of the competence of the bone-implant interface and improve effectiveness of fixation strategies particularly in patients with compromised bone quality., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The enthesis is an organ that connects a soft, aligned tissue (tendon/ligament) to a hard, amorphous tissue (bone) via a fibrocartilage interface. Mechanically, the enthesis sustains a dynamic loading environment that includes tensile, compressive, and shear forces. The structural components of the enthesis act to minimize stress concentrations and control stretch at the interface. Current surgical repair of the enthesis, such as in rotator cuff repair and anterior cruciate ligament reconstruction, aim to bridge the gap between the injured ends via reattachment of soft-to-hard tissues or graft replacement. In this review, we discuss the multiscale, morphological, and mechanical characteristics of the fibrocartilage attachment. Additionally, we review historical and recent clinical approaches to treating enthesis injury. Lastly, we explore new technological advancements in tissue-engineered biomaterials that have shown promise in preclinical studies., Competing Interests: The authors declare no competing financial interest.

Rotator cuff degeneration is a common affliction that results in pain and disability. Tendinopathy was historically classified with or without the involvement of the immune system. However, technological advancements in screening have shown that the immune system is both present and active in all forms of tendinopathy. During injury and healing, the coordinated effort of numerous immune cell populations work with the resident stromal cells to break down damaged tissues and stimulate remodeling. These cells deploy a wide array of tools, including phagocytosis, enzyme secretion, and chemotactic gradients to direct these processes. Yet, there remains a knowledge gap in our understanding of the sequence of critical events and regulatory factors that mediate this is process in injury and healing. Furthermore, current treatments do not specifically target inflammation at the molecular level. Typical regimens include non-steroidal anti-inflammatory drugs or corticosteroids; however, researchers have found irrevocable functional deficits following treatment, and have disputed their long-term efficacy. Therefore, developing therapeutics that specifically consider the nuances of the immune system are necessary to improve patient outcomes.

Scleraxis (Scx) is a known regulator of tendon development, and recent work has identified the role of Scx in bone modeling. However, the role of Scx in fracture healing has not yet been explored. This study was conducted to identify the role of Scx in cortical bone development and fracture healing. Scx green fluorescent protein-labeled (ScxGFP) reporter and Scx-knockout (Scx-mutant) mice were used to assess bone morphometry and the effects of fracture healing on Scx localization and gene expression, as well as callus healing response. Botulinum toxin (BTX) was used to investigate muscle unloading effects on callus shape. Scx-mutant long bones had structural and mechanical defects. Scx gene expression was elevated and bmp4 was decreased at 24 h after fracture. ScxGFP + cells were localized throughout the healing callus after fracture. Scx-mutant mice demonstrated disrupted callus healing and asymmetry. Asymmetry of Scx-mutant callus was not due to muscle unloading. Wild-type littermates (age matched) served as controls. This is the first study to explore the role of Scx in cortical bone mechanics and fracture healing. Deletion of Scx during development led to altered long bone properties and callus healing. This study also demonstrated that Scx may play a role in the periosteal response during fracture healing.-McKenzie, J. A., Buettmann, E., Abraham, A. C., Gardner, M. J., Silva, M. J., Killian, M. L. Loss of scleraxis in mice leads to geometric and structural changes in cortical bone, as well as asymmetry in fracture healing., (© FASEB.)

Piezoelectricity-driven hot-electron injectors (p-HEI) are used for self-powered monitoring of mechanical activity in biomechanical implants and structures. Previously reported p-HEI devices operate by harvesting energy from a piezoelectric transducer to generate current and voltage references which are then used for initiating and controlling the process of hot-electron injection. As a result, the minimum energy required to activate the device is limited by the power requirements of the reference circuits. In this paper we present a p-HEI device that operates by directly exploiting the self-limiting capability of an energy transducer when driving the process of hot-electron injection in a pMOS floating-gate transistor. As a result, the p-HEI device can activate itself at input power levels less than 5 nW. Using a prototype fabricated in a 0.5- [Formula: see text] bulk CMOS process we validate the functionality of the proposed injector and show that for a fixed input power, its dynamics is quasi-linear with respect to time. The paper also presents measurement results using a cadaver phantom where the fabricated p-HEI device has been integrated with a piezoelectric transducer and is used for self-powered monitoring of mechanical activity.