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SummaryCentral nervous system neurons have axons that vary over 100-fold in diameter. This diversity contributes to the regulation of myelination and conduction velocity along axons, which is critical for proper nervous system function. Despite this importance, technical challenges have limited our understanding of mechanisms controlling axon diameter growth, preventing systematic dissection of how manipulating diameter affects conduction along individual axons. Here, we establish zebrafish as system to investigate the regulation of axon diameter and axonal conductionin vivo. We identify a role for importin 13b specifically in axon diameter growth, that is independent of axonal length and overall cell size. Impairment of diameter growth along individual myelinated axons reduced conduction velocity in proportion to the diameter, but had no effect on the precision of action potential propagation or ability to fire at high frequencies. This work highlights an axon diameter-specific mechanism of growth that regulates conduction speeds along myelinated axons.

Ideal therapies for regenerative medicine or healthy aging require healthy organ growth and rejuvenation, but no organ-level approach is currently available. Using Mycobacterium leprae (ML) with natural partial cellular reprogramming capacity and its animal host nine-banded armadillos, we present an evolutionarily refined model of adult liver growth and regeneration. In infected armadillos, ML reprogram the entire liver and significantly increase total liver/body weight ratio by increasing healthy liver lobules, including hepatocyte proliferation and proportionate expansion of vasculature, and biliary systems. ML-infected livers are microarchitecturally and functionally normal without damage, fibrosis, or tumorigenesis. Bacteria-induced reprogramming reactivates liver progenitor/developmental/fetal genes and upregulates growth-, metabolism-, and anti-aging-associated markers with minimal change in senescence and tumorigenic genes, suggesting bacterial hijacking of homeostatic, regeneration pathways to promote de novo organogenesis. This may facilitate the unraveling of endogenous pathways that effectively and safely re-engage liver organ growth, with broad therapeutic implications including organ regeneration and rejuvenation.

Significance Endometriosis is a chronic, incurable inflammatory disorder impacting 190 million women worldwide. Immune cells called macrophages are implicated in promoting endometriosis. Macrophages have different origins and their origin can dictate function. In this study we demonstrate that endometriotic lesion-resident macrophages are derived from the uterine lining (endometrium), the abdominal (peritoneal) cavity, and recruited bone-marrow precursors (monocytes). Endometriosis triggers continuous recruitment of monocytes that differentiate into macrophages that differ from those usually present within the peritoneal cavity. By depleting different populations, we demonstrate that endometrial macrophages are “proendometriosis” while monocyte-derived peritoneal macrophages are “antiendometriosis” acting to protect the cavity from lesion establishment. In the future, immune-based therapies may allow targeting of prodisease macrophages and/or harnessing of antiendometriosis macrophages in endometriosis., Macrophages are intimately involved in the pathophysiology of endometriosis, a chronic inflammatory disorder characterized by the growth of endometrial-like tissue (lesions) outside the uterus. By combining genetic and pharmacological monocyte and macrophage depletion strategies we determined the ontogeny and function of macrophages in a mouse model of induced endometriosis. We demonstrate that lesion-resident macrophages are derived from eutopic endometrial tissue, infiltrating large peritoneal macrophages (LpM) and monocytes. Furthermore, we found endometriosis to trigger continuous recruitment of monocytes and expansion of CCR2+ LpM. Depletion of eutopic endometrial macrophages results in smaller endometriosis lesions, whereas constitutive inhibition of monocyte recruitment significantly reduces peritoneal macrophage populations and increases the number of lesions. Reprogramming the ontogeny of peritoneal macrophages such that embryo-derived LpM are replaced by monocyte-derived LpM decreases the number of lesions that develop. We propose a putative model whereby endometrial macrophages are “proendometriosis” while newly recruited monocyte-derived macrophages, possibly in LpM form, are “antiendometriosis.” These observations highlight the importance of monocyte-derived macrophages in limiting disease progression.

Macrophages are intimately involved in the pathophysiology of endometriosis, a chronic inflammatory disorder characterized by the growth of endometrial-like tissue (lesions) outside the uterus. By combining genetic and pharmacological monocyte and macrophage depletion strategies we determined the ontogeny and function of macrophages in a mouse model of induced endometriosis. We demonstrate that lesion-resident macrophages are derived from eutopic endometrial tissue, infiltrating large peritoneal macrophages (LpM) and monocytes. Furthermore, we found endometriosis to trigger continuous recruitment of monocytes and expansion of CCR2+ LpM. Depletion of eutopic endometrial macrophages results in smaller endometriosis lesions, whereas constitutive inhibition of monocyte recruitment significantly reduces peritoneal macrophage populations and increased the number of lesions. We propose a putative model whereby endometrial macrophages are pro-endometriosis whilst newly-recruited monocyte-derived macrophages, possibly in LpM form, are ‘anti-endometriosis’. These observations highlight the importance of monocyte-derived macrophages in limiting disease progression.

Pancreatic cancer (PC) patients appear to receive suboptimal care. We conducted a systematic review to identify factors that influence PC management which are amenable to quality improvement. MEDLINE, EMBASE, and the references of eligible studies were searched from 1996 to July 2014. Two authors independently selected and reviewed eligible studies. Identified factors were mapped onto a framework of determinants of care delivery and outcomes. Methodological quality of studies was assessed using Downs and Black criteria. Most of the 33 eligible studies were population-based observational studies conducted in the United States. Patient (age, socioeconomic status, race) and institutional (case volume, academic status) factors influence care delivery and outcomes (complications, mortality, readmission, survival). Two studies implemented interventions to improve quality of care (centralization to high-volume hospitals, multidisciplinary care). One study examined system determinants (referral wait times). No studies examined the influence of guideline or provider characteristics. The overall lack of health services research in PC is striking. Factors and interventions identified here can be used to plan PC quality improvement programs. Further research is needed to explore the influence of guideline and provider factors on PC management and evaluate the impact of quality improvement interventions.

The most prevalent neurological disorders of myelin include perinatal brain injury leading to cerebral palsy in infants and multiple sclerosis in adults. Although these disorders have distinct etiologies, they share a common neuropathological feature of failed progenitor differentiation into myelin-producing oligodendrocytes and lack of myelin, for which there is an unmet clinical need. Here, we reveal that a molecular pathology common to both disorders is dysregulation of activin receptors and that activin receptor signaling is required for the majority of myelin generation in development and following injury. Using a constitutive conditional knockout of all activin receptor signaling in oligodendrocyte lineage cells, we discovered this signaling to be required for myelination via regulation of oligodendrocyte differentiation and myelin compaction. These processes were found to be dependent on the activin receptor subtype Acvr2a, which is expressed during oligodendrocyte differentiation and axonal ensheathment in development and following myelin injury. During efficient myelin regeneration, Acvr2a upregulation was seen to coincide with downregulation of Acvr2b, a receptor subtype with relatively higher ligand affinity; Acvr2b was shown to be dispensable for activin receptor-driven oligodendrocyte differentiation and its overexpression was sufficient to impair the abovementioned ligand-driven responses. In actively myelinating or remyelinating areas of human perinatal brain injury and multiple sclerosis tissue, respectively, oligodendrocyte lineage cells expressing Acvr2a outnumbered those expressing Acvr2b, whereas in non-repairing lesions Acvr2b+ cells were increased. Thus, we propose that following human white matter injury, this increase in Acvr2b expression would sequester ligand and consequently impair Acvr2a-driven oligodendrocyte differentiation and myelin formation. Our results demonstrate dysregulated activin receptor signaling in common myelin disorders and reveal Acvr2a as a novel therapeutic target for myelin generation following injury across the lifespan. Electronic supplementary material The online version of this article (10.1007/s00401-018-1813-3) contains supplementary material, which is available to authorized users.

Failed regeneration of central nervous system myelin contributes to clinical decline in neuroinflammatory and neurodegenerative diseases, for which there is an unmet therapeutic need. Here, we reveal that efficient remyelination requires death of pro-inflammatory microglia followed by repopulation to a pro-regenerative state. We propose that impaired microglia death and/or repopulation may underpin dysregulated microglia activation in neurological diseases, and reveal novel therapeutic targets to promote white matter regeneration.

Rationale: Matrix vesicles (MVs), secreted by vascular smooth muscle cells (VSMCs), form the first nidus for mineralization and fetuin-A, a potent circulating inhibitor of calcification, is specifically loaded into MVs. However, the processes of fetuin-A intracellular trafficking and MV biogenesis are poorly understood. Objective: The objective of this study is to investigate the regulation, and role, of MV biogenesis in VSMC calcification. Methods and Results: Alexa488-labeled fetuin-A was internalized by human VSMCs, trafficked via the endosomal system, and exocytosed from multivesicular bodies via exosome release. VSMC-derived exosomes were enriched with the tetraspanins CD9, CD63, and CD81, and their release was regulated by sphingomyelin phosphodiesterase 3. Comparative proteomics showed that VSMC-derived exosomes were compositionally similar to exosomes from other cell sources but also shared components with osteoblast-derived MVs including calcium-binding and extracellular matrix proteins. Elevated extracellular calcium was found to induce sphingomyelin phosphodiesterase 3 expression and the secretion of calcifying exosomes from VSMCs in vitro, and chemical inhibition of sphingomyelin phosphodiesterase 3 prevented VSMC calcification. In vivo, multivesicular bodies containing exosomes were observed in vessels from chronic kidney disease patients on dialysis, and CD63 was found to colocalize with calcification. Importantly, factors such as tumor necrosis factor-α and platelet derived growth factor-BB were also found to increase exosome production, leading to increased calcification of VSMCs in response to calcifying conditions. Conclusions: This study identifies MVs as exosomes and shows that factors that can increase exosome release can promote vascular calcification in response to environmental calcium stress. Modulation of the exosome release pathway may be as a novel therapeutic target for prevention.

Lpd is an essential, evolutionary conserved regulator of the Scar/WAVE complex during cell migration in vivo., Cell migration is essential for development, but its deregulation causes metastasis. The Scar/WAVE complex is absolutely required for lamellipodia and is a key effector in cell migration, but its regulation in vivo is enigmatic. Lamellipodin (Lpd) controls lamellipodium formation through an unknown mechanism. Here, we report that Lpd directly binds active Rac, which regulates a direct interaction between Lpd and the Scar/WAVE complex via Abi. Consequently, Lpd controls lamellipodium size, cell migration speed, and persistence via Scar/WAVE in vitro. Moreover, Lpd knockout mice display defective pigmentation because fewer migrating neural crest-derived melanoblasts reach their target during development. Consistently, Lpd regulates mesenchymal neural crest cell migration cell autonomously in Xenopus laevis via the Scar/WAVE complex. Further, Lpd’s Drosophila melanogaster orthologue Pico binds Scar, and both regulate collective epithelial border cell migration. Pico also controls directed cell protrusions of border cell clusters in a Scar-dependent manner. Taken together, Lpd is an essential, evolutionary conserved regulator of the Scar/WAVE complex during cell migration in vivo.

The pioneering cell biologist Michael Abercrombie first described the process of contact inhibition of locomotion more than 50 years ago when migrating fibroblasts were observed to rapidly change direction and migrate away upon collision. Since then, we have gleaned little understanding of how contact inhibition is regulated and only lately observed its occurrence in vivo. We recently revealed that Drosophila macrophages (haemocytes) require contact inhibition for their uniform embryonic dispersal. Here, to investigate the role that contact inhibition plays in the patterning of haemocyte movements, we have mathematically analysed and simulated their contact repulsion dynamics. Our data reveal that the final pattern of haemocyte distribution, and the details and timing of its formation, can be explained by contact inhibition dynamics within the geometry of the Drosophila embryo. This has implications for morphogenesis in general as it suggests that patterns can emerge, irrespective of external cues, when cells interact through simple rules of contact repulsion.

Kitamura et al. find a role for metastasis-associated macrophage (MAM) chemokine pathways CCL2–CCR2 and CCL3–CCR1 in promoting breast cancer cell pulmonary metastasis. Genetic deletion of CCR1 or CCL3 in a mouse model of breast cancer cell metastasis prevents MAM retention in the lung, reduces MAM-cancer cell interactions and reduces the number of lung metastatic foci., Pulmonary metastasis of breast cancer cells is promoted by a distinct population of macrophages, metastasis-associated macrophages (MAMs), which originate from inflammatory monocytes (IMs) recruited by the CC-chemokine ligand 2 (CCL2). We demonstrate here that, through activation of the CCL2 receptor CCR2, the recruited MAMs secrete another chemokine ligand CCL3. Genetic deletion of CCL3 or its receptor CCR1 in macrophages reduces the number of lung metastasis foci, as well as the number of MAMs accumulated in tumor-challenged lung in mice. Adoptive transfer of WT IMs increases the reduced number of lung metastasis foci in Ccl3 deficient mice. Mechanistically, Ccr1 deficiency prevents MAM retention in the lung by reducing MAM–cancer cell interactions. These findings collectively indicate that the CCL2-triggered chemokine cascade in macrophages promotes metastatic seeding of breast cancer cells thereby amplifying the pathology already extant in the system. These data suggest that inhibition of CCR1, the distal part of this signaling relay, may have a therapeutic impact in metastatic disease with lower toxicity than blocking upstream targets.

The neurodegenerative spinocerebellar ataxia type 1 (SCA1) is caused by aggregation and misfolding of the ataxin-1 protein. While the pathology correlates with mutations that lead to expansion of a polyglutamine tract in the protein, other regions contribute to the aggregation process as also non-expanded ataxin-1 is intrinsically aggregation-prone and forms nuclear foci in cell. Here, we have used a combined approach based on FRET analysis, confocal microscopy and in vitro techniques to map aggregation-prone regions other than polyglutamine and to establish the importance of dimerization in self-association/foci formation. Identification of aggregation-prone regions other than polyglutamine could greatly help the development of SCA1 treatment more specific than that based on targeting the low complexity polyglutamine region.

Anomalous expansion of a polymorphic tract in Ataxin-1 causes the autosomal dominant spinocerebellar ataxia type 1. In addition to polyglutamine expansion, requirements for development of pathology are phosphorylation of serine 776 in Ataxin-1 and nuclear localization of the protein. The phosphorylation state of serine 776 is also crucial for selection of the Ataxin-1 multiple partners. Here, we have used FRET for an in cell study of the interaction of Ataxin-1 with the spliceosome-associated U2AF65 and the adaptor 14-3-3 proteins. Using wild-type Ataxin-1 and Ser776 mutants to a phosphomimetic aspartate and to alanine, we show that U2AF65 binds Ataxin-1 in a Ser776 phosphorylation independent manner whereas 14-3-3 interacts with phosphorylated wild-type Ataxin-1 but not with the mutants. These results indicate that Ser776 acts as the molecular switch that discriminates between normal and aberrant function and that phosphomimetics is not a generally valid approach whose applicability should be carefully validated.

Arterial stiffening caused by deposition of calcium phosphate salts in the vessel wall is common in patients with atherosclerosis, renal failure and diabetes and contributes to high cardiovascular morbidity and mortality in these groups. Medial calcification is mediated by vascular smooth muscle cells (VSMCs) which undergo phenotypic transitions and secrete matrix vesicles (MVs) that form the first nidus for mineralization. In this study we studied the mechanisms regulating MV secretion and compared them with circulating calcifying calciprotein complexes and exosomes. Phenotypic modulation of human aortic VSMCs was induced by TGF-β1 or PDGF-BB. MVs and extracellular vesicles were isolated from cell media or platelet-free plasma, by differential ultracentrifugation and compared using Nanosight LM-10, flow cytometry, immunogold labelling, transmission electron microscopy and western blotting. We found that MVs are secreted from multivesicular bodies and enriched with the exosomal markers, CD63, CD81, CD9 and MHC I. Exosome secretion was regulated by the exosome biogenesis regulator, sphingomyelin phosphodiesterase 3 (SMPD3), and inhibition of SMPD3 prevented VSMC calcification. Treatment of VSMCs in calcifying conditions and loss of the contractile phenotype were both associated with elevated exosome secretion and increased calcification. In agreement with our in vitro data, MVB-like structures were observed in VSMCs ex vivo as shown by EM analysis of human vessel rings. Immunohistochemical staining revealed that the exosome markers, CD63 and annexin A6 were present in the extracellular (ECM) at sites of vascular calcification in vivo . Importantly, only exosomes secreted by VSMCs in calcifying conditions contained amorphous calcium phosphate crystals as detected by EDX analysis. Nanosite analysis revealed that calcifying VSMC-derived exosomes are similar in size to non-calcifying exosomes (mode 136 +/- 3.6 nm and 147+/- 5.9 nm, correspondingly) and were similar to exosomes, isolated from the blood (Mode 146+/-14 nm). Calcifying exosomes were also distinct from calcium phosphate crystals (mode 107 +/- 7 nm) and fetuin-A containing calciprotein particles (mode 182 +/- 9.3 nm). Our data indicate that vascular calcification is mediated by VSMC-derived exosomes and that loss of the contractile VSMC phenotype and mineral imbalance promote calcification by enhanced exosome secretion. Calcifying exosomes are distinct from calcium phosphate crystals or calciprotein particles, and may be presented in the circulation where their levels may be a novel indicator of vascular calcification.

Vascular calcification is a regulated pathological process similar to bone formation which is mediated by vascular smooth muscle cells (VSMCs) undergoing osteogenic transdifferentiation. Initiation of vascular calcification occurs in small membrane-bound matrix vesicles (MVs), secreted by VSMCs into the extracellular matrix however the mechanisms regulating MV biogenesis and secretion are unclear. Fetuin-A, a circulating protein abundant in MVs was used to trace the origin of VSMC-derived MVs. Alexa488-labelled fetuin-A was rapidly uptaken by human VSMCs and appeared in the late endosomal system and multivesicular bodies (MVBs) indicating that MVs are secreted from the endosomal compartment similar to exosomes; extracellular vesicles originating from MVBs and secreted by the range of cells. Biochemical analysis of MVs showed that they were enriched with exosomal markers, CD9 and CD63. Furthermore, inhibition of sphingomyelin phosphodiesterase 3 (SMPD3), which in involved in exosome biogenesis abrogated MV secretion by VSMCs confirming that MVs represent exosomes. Calcifying conditions induced exosome secretion by VSMCs and this was accompanied by elevated SMPD3 expression. Importantly, an inhibition of SMPD3 prevented VSMC calcification. Phenotypic modulation of VSMCs and loss of the contractile phenotype resulted in elevated exosome secretion while contractile VSMCs secreted significantly less exosomes. Notably, elevated extracellular calcium rapidly induced calcification of synthetic VSMCs whilst contractile VSMCs did not calcify. In agreement with this data, abundant immunohistochemical staining for CD63 was observed only in atherosclerotic human aorta in close association with calcified areas with little staining detected in the healthy vessel wall. Taken together this study demonstrates that MVs originate from MVBs and are secreted via the exosomal pathway. Loss of the contractile phenotype and mineral imbalance promote VSMC calcification by enhanced exosome secretion. Targeting the mechanisms of VSMC exosome secretion and/or their loading with calcification inhibitors may provide novel therapeutic interventions aimed for the prevention of vascular calcification.