Structured wound angiogenesis instructs mesenchymal barrier compartments in the regenerating nerve.

Organ injury stimulates the formation of new capillaries to restore blood supply raising questions about the potential contribution of neoangiogenic vessel architecture to the healing process. Using single-cell mapping, we resolved the properties of endothelial cells that organize a polarized scaffold at the repair site of lesioned peripheral nerves. Transient reactivation of an embryonic guidance program is required to orient neovessels across the wound. Manipulation of this structured angiogenic response through genetic and pharmacological targeting of Plexin-D1/VEGF pathways within an early window of repair has long-term impact on configuration of the nerve stroma. Neovessels direct nerve-resident mesenchymal cells to mold a provisionary fibrotic scar by assembling an orderly system of stable barrier compartments that channel regenerating nerve fibers and shield them from the persistently leaky vasculature. Thus, guided and balanced repair angiogenesis enables the construction of a "bridge" microenvironment conducive for axon regrowth and homeostasis of the regenerated tissue.

IL-1ß+ macrophages fuel pathogenic inflammation in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with high resistance to therapies1. Inflammatory and immunomodulatory signals co-exist in the pancreatic tumour microenvironment, leading to dysregulated repair and cytotoxic responses. Tumour-associated macrophages (TAMs) have key roles in PDAC2, but their diversity has prevented therapeutic exploitation. Here we combined single-cell and spatial genomics with functional experiments to unravel macrophage functions in pancreatic cancer. We uncovered an inflammatory loop between tumour cells and interleukin-1ß (IL-1ß)-expressing TAMs, a subset of macrophages elicited by a local synergy between prostaglandin E2 (PGE2) and tumour necrosis factor (TNF). Physical proximity with IL-1ß+ TAMs was associated with inflammatory reprogramming and acquisition of pathogenic properties by a subset of PDAC cells. This occurrence was an early event in pancreatic tumorigenesis and led to persistent transcriptional changes associated with disease progression and poor outcomes for patients. Blocking PGE2 or IL-1ß activity elicited TAM reprogramming and antagonized tumour cell-intrinsic and -extrinsic inflammation, leading to PDAC control in vivo. Targeting the PGE2-IL-1ß axis may enable preventive or therapeutic strategies for reprogramming of immune dynamics in pancreatic cancer.

ADAM17 Regulates p75NTR-Mediated Fibrinolysis and Nerve Remyelination.

We previously reported that a-disintegrin and metalloproteinase (ADAM)17 is a key protease regulating myelin formation. We now describe a role for ADAM17 during the Wallerian degeneration (WD) process. Unexpectedly, we observed that glial ADAM17, by regulating p75NTR processing, cell autonomously promotes remyelination, while neuronal ADAM17 is dispensable. Accordingly, p75NTR abnormally accumulates specifically when ADAM17 is maximally expressed leading to a downregulation of tissue plasminogen activator (tPA) expression, excessive fibrin accumulation over time, and delayed remyelination. Mutant mice also present impaired macrophage recruitment and defective nerve conduction velocity (NCV). Thus, ADAM17 expressed in Schwann cells, controls the whole WD process, and its absence hampers effective nerve repair. Collectively, we describe a previously uncharacterized role for glial ADAM17 during nerve regeneration. Based on the results of our study, we posit that, unlike development, glial ADAM17 promotes remyelination through the regulation of p75NTR-mediated fibrinolysis.SIGNIFICANCE STATEMENT The α-secretase a-disintegrin and metalloproteinase (ADAM)17, although relevant for developmental PNS myelination, has never been investigated in Wallerian degeneration (WD). We now unravel a new mechanism of action for this protease and show that ADAM17 cleaves p75NTR, regulates fibrin clearance, and eventually fine-tunes remyelination. The results presented in this study provide important insights into the complex regulation of remyelination following nerve injury, identifying in ADAM17 and p75NTR a new signaling axis implicated in these events. Modulation of this pathway could have important implications in promoting nerve remyelination, an often-inefficient process, with the aim of restoring a functional axo-glial unit.

Rac1 and Rac3 have opposite functions in Schwann cells during developmental myelination.

Small Rho GTPases such as Cdc42 and Rac1 regulate peripheral myelination during development. Deletion of Rac1 in Schwann cell conditional knockout mice causes a delay in the process of radial sorting, followed by hypomyelination as well as defective PAK1 activation and high number of immature Oct6+ Schwann cells. Rac3 has been shown to have redundant, specific and even opposite functions to Rac1 depending on the cell type, age and other factors. In neuronal cells, evidence suggests that Rac3 may oppose Rac1 by disrupting PAK1-GIT1-Paxillin signaling thus preventing cell differentiation and extension of lamellipodia. Therefore, we tested if these Rho GTPases have similar or opposite functions in Schwann cells, by deleting the genes for both proteins in mice during peripheral myelination. At P30, global deletion of Rac3 alleviates the developmental defects on axonal sorting and hypomyelination that are caused by Schwann cell conditional ablation of Rac1. Moreover, Rac3 deletion also reverses the arrest of Schwann cells at the Oct6+ stage and ameliorates the defects in PAK1 phosphorylation observed in Rac1 deficient mice. This partial rescue of the phenotype declines later on with aging. Since double transgenic animals showed dysmyelination without axonal degeneration at P60, we postulate that this deterioration is not likely due to loss of Rac3 in neurons, but it seems to be a Schwann cell-specific defect in the maintenance of myelin.

αV integrins in Schwann cells promote attachment to axons, but are dispensable in vivo.

In the developing peripheral nervous system, Schwann cells (SCs) extend their processes to contact, sort, and myelinate axons. The mechanisms that contribute to the interaction between SCs and axons are just beginning to be elucidated. Using a SC-neuron coculture system, we demonstrate that Arg-Gly-Asp (RGD) peptides that inhibit αV -containing integrins delay the extension of SCs elongating on axons. αV integrins in SC localize to sites of contact with axons and are expressed early in development during radial sorting and myelination. Short interfering RNA-mediated knockdown of the αV integrin subunit also delays SC extension along axons in vitro, suggesting that αV -containing integrins participate in axo-glial interactions. However, mice lacking the αV subunit in SCs, alone or in combination with the potentially compensating α5 subunit, or the αV partners ß3 or ß8 , myelinate normally during development and remyelinate normally after nerve crush, indicating that overlapping or compensatory mechanisms may hide the in vivo role of RGD-binding integrins.

Ablation of neuronal ADAM17 impairs oligodendrocyte differentiation and myelination.

Myelin, one of the most important adaptations of vertebrates, is essential to ensure efficient propagation of the electric impulse in the nervous system and to maintain neuronal integrity. In the central nervous system (CNS), the development of oligodendrocytes and the process of myelination are regulated by the coordinated action of several positive and negative cell-extrinsic factors. We and others previously showed that secretases regulate the activity of proteins essential for myelination. We now report that the neuronal α-secretase ADAM17 controls oligodendrocyte differentiation and myelin formation in the CNS. Ablation of Adam17 in neurons impairs in vivo and in vitro oligodendrocyte differentiation, delays myelin formation throughout development and results in hypomyelination. Furthermore, we show that this developmental defect is, in part, the result of altered Notch/Jagged 1 signaling. Surprisingly, in vivo conditional loss of Adam17 in immature oligodendrocytes has no effect on myelin formation. Collectively, our data indicate that the neuronal α-secretase ADAM17 is required for proper CNS myelination. Further, our studies confirm that secretases are important post-translational regulators of myelination although the mechanisms controlling CNS and peripheral nervous system (PNS) myelination are distinct.

Prostaglandin D2 synthase modulates macrophage activity and accumulation in injured peripheral nerves.

We have previously reported that prostaglandin D2 Synthase (L-PGDS) participates in peripheral nervous system (PNS) myelination during development. We now describe the role of L-PGDS in the resolution of PNS injury, similarly to other members of the prostaglandin synthase family, which are important for Wallerian degeneration (WD) and axonal regeneration. Our analyses show that L-PGDS expression is modulated after injury in both sciatic nerves and dorsal root ganglia neurons, indicating that it might play a role in the WD process. Accordingly, our data reveals that L-PGDS regulates macrophages phagocytic activity through a non-cell autonomous mechanism, allowing myelin debris clearance and favoring axonal regeneration and remyelination. In addition, L-PGDS also appear to control macrophages accumulation in injured nerves, possibly by regulating the blood-nerve barrier permeability and SOX2 expression levels in Schwann cells. Collectively, our results suggest that L-PGDS has multiple functions during nerve regeneration and remyelination. Based on the results of this study, we posit that L-PGDS acts as an anti-inflammatory agent in the late phases of WD, and cooperates in the resolution of the inflammatory response. Thus, pharmacological activation of the L-PGDS pathway might prove beneficial in resolving peripheral nerve injury.

Nerves and Pancreatic Cancer: New Insights into a Dangerous Relationship.

Perineural invasion (PNI) is defined as the presence of neoplastic cells along nerves and/or within the different layers of nervous fibers: epineural, perineural and endoneural spaces. In pancreatic cancer-particularly in pancreatic ductal adenocarcinoma (PDAC)-PNI has a prevalence between 70 and 100%, surpassing any other solid tumor. PNI has been detected in the early stages of pancreatic cancer and has been associated with pain, increased tumor recurrence and diminished overall survival. Such an early, invasive and recurrent phenomenon is probably crucial for tumor growth and metastasis. PNI is a still not a uniformly characterized event; usually it is described only dichotomously ("present" or "absent"). Recently, a more detailed scoring system for PNI has been proposed, though not specific for pancreatic cancer. Previous studies have implicated several molecules and pathways in PNI, among which are secreted neurotrophins, chemokines and inflammatory cells. However, the mechanisms underlying PNI are poorly understood and several aspects are actively being investigated. In this review, we will discuss the main molecules and signaling pathways implicated in PNI and their roles in the PDAC.

The Complex Work of Proteases and Secretases in Wallerian Degeneration: Beyond Neuregulin-1.

After damage, axons in the peripheral nervous system (PNS) regenerate and regrow following a process termed Wallerian degeneration, but the regenerative process is often incomplete and usually the system does not reach full recovery. Key steps to the creation of a permissive environment for axonal regrowth are the trans-differentiation of Schwann cells and the remodeling of the extracellular matrix (ECM). In this review article, we will discuss how proteases and secretases promote effective regeneration and remyelination. We will detail how they control neuregulin-1 (NRG-1) activity at the post-translational level, as the concerted action of alpha, beta and gamma secretases cooperates to balance activating and inhibitory signals necessary for physiological myelination and remyelination. In addition, we will discuss the role of other proteases in nerve repair, among which A Disintegrin And Metalloproteinases (ADAMs) and gamma-secretases substrates. Moreover, we will present how matrix metalloproteinases (MMPs) and proteases of the blood coagulation cascade participate in forming newly synthetized myelin and in regulating axonal regeneration. Overall, we will highlight how a deeper comprehension of secretases and proteases mechanism of action in Wallerian degeneration might be useful to develop new therapies with the potential of readily and efficiently improve the regenerative process.

Enhanced axonal neuregulin-1 type-III signaling ameliorates neurophysiology and hypomyelination in a Charcot-Marie-Tooth type 1B mouse model.

Charcot-Marie-Tooth (CMT) neuropathies are a group of genetic disorders that affect the peripheral nervous system with heterogeneous pathogenesis and no available treatment. Axonal neuregulin 1 type III (Nrg1TIII) drives peripheral nerve myelination by activating downstream signaling pathways such as PI3K/Akt and MAPK/Erk that converge on master transcriptional regulators of myelin genes, such as Krox20. We reasoned that modulating Nrg1TIII activity may constitute a general therapeutic strategy to treat CMTs that are characterized by reduced levels of myelination. Here we show that genetic overexpression of Nrg1TIII ameliorates neurophysiological and morphological parameters in a mouse model of demyelinating CMT1B, without exacerbating the toxic gain-of-function that underlies the neuropathy. Intriguingly, the mechanism appears not to be related to Krox20 or myelin gene upregulation, but rather to a beneficial rebalancing in the stoichiometry of myelin lipids and proteins. Finally, we provide proof of principle that stimulating Nrg1TIII signaling, by pharmacological suppression of the Nrg1TIII inhibitor tumor necrosis factor-alpha-converting enzyme (TACE/ADAM17), also ameliorates the neuropathy. Thus, modulation of Nrg1TIII by TACE/ADAM17 inhibition may represent a general treatment for hypomyelinating neuropathies.

Laminin 211 inhibits protein kinase A in Schwann cells to modulate neuregulin 1 type III-driven myelination.

Myelin is required for proper nervous system function. Schwann cells in developing nerves depend on extrinsic signals from the axon and from the extracellular matrix to first sort and ensheathe a single axon and then myelinate it. Neuregulin 1 type III (Nrg1III) and laminin α2ß1γ1 (Lm211) are the key axonal and matrix signals, respectively, but how their signaling is integrated and if each molecule controls both axonal sorting and myelination is unclear. Here, we use a series of epistasis experiments to show that Lm211 modulates neuregulin signaling to ensure the correct timing and amount of myelination. Lm211 can inhibit Nrg1III by limiting protein kinase A (PKA) activation, which is required to initiate myelination. We provide evidence that excessive PKA activation amplifies promyelinating signals downstream of neuregulin, including direct activation of the neuregulin receptor ErbB2 and its effector Grb2-Associated Binder-1 (Gab1), thereby elevating the expression of the key transcription factors Oct6 and early growth response protein 2 (Egr2). The inhibitory effect of Lm211 is seen only in fibers of small caliber. These data may explain why hereditary neuropathies associated with decreased laminin function are characterized by focally thick and redundant myelin.

Two factor-based reprogramming of rodent and human fibroblasts into Schwann cells.

Schwann cells (SCs) generate the myelin wrapping of peripheral nerve axons and are promising candidates for cell therapy. However, to date a renewable source of SCs is lacking. In this study, we show the conversion of skin fibroblasts into induced Schwann cells (iSCs) by driving the expression of two transcription factors, Sox10 and Egr2. iSCs resembled primary SCs in global gene expression profiling and PNS identity. In vitro, iSCs wrapped axons generating compact myelin sheaths with regular nodal structures. Conversely, iSCs from Twitcher mice showed a severe loss in their myelinogenic potential, demonstrating that iSCs can be an attractive system for in vitro modelling of PNS diseases. The same two factors were sufficient to convert human fibroblasts into iSCs as defined by distinctive molecular and functional traits. Generating iSCs through direct conversion of somatic cells offers opportunities for in vitro disease modelling and regenerative therapies.

Niacin-mediated Tace activation ameliorates CMT neuropathies with focal hypermyelination.

Charcot-Marie-Tooth (CMT) neuropathies are highly heterogeneous disorders caused by mutations in more than 70 genes, with no available treatment. Thus, it is difficult to envisage a single suitable treatment for all pathogenetic mechanisms. Axonal Neuregulin 1 (Nrg1) type III drives Schwann cell myelination and determines myelin thickness by ErbB2/B3-PI3K-Akt signaling pathway activation. Nrg1 type III is inhibited by the α-secretase Tace, which negatively regulates PNS myelination. We hypothesized that modulation of Nrg1 levels and/or secretase activity may constitute a unifying treatment strategy for CMT neuropathies with focal hypermyelination as it could restore normal levels of myelination. Here we show that in vivo delivery of Niaspan, a FDA-approved drug known to enhance TACE activity, efficiently rescues myelination in the Mtmr2-/- mouse, a model of CMT4B1 with myelin outfoldings, and in the Pmp22+/- mouse, which reproduces HNPP (hereditary neuropathy with liability to pressure palsies) with tomacula. Importantly, we also found that Niaspan reduces hypermyelination of Vim (vimentin)-/- mice, characterized by increased Nrg1 type III and Akt activation, thus corroborating the hypothesis that Niaspan treatment downregulates Nrg1 type III signaling.

α6ß1 and α7ß1 integrins are required in Schwann cells to sort axons.

During development, Schwann cells extend lamellipodia-like processes to segregate large- and small-caliber axons during the process of radial sorting. Radial sorting is a prerequisite for myelination and is arrested in human neuropathies because of laminin deficiency. Experiments in mice using targeted mutagenesis have confirmed that laminins 211, 411, and receptors containing the ß1 integrin subunit are required for radial sorting; however, which of the 11 α integrins that can pair with ß1 forms the functional receptor is unknown. Here we conditionally deleted all the α subunits that form predominant laminin-binding ß1 integrins in Schwann cells and show that only α6ß1 and α7ß1 integrins are required and that α7ß1 compensates for the absence of α6ß1 during development. The absence of either α7ß1 or α6ß1 integrin impairs the ability of Schwann cells to spread and to bind laminin 211 or 411, potentially explaining the failure to extend cytoplasmic processes around axons to sort them. However, double α6/α7 integrin mutants show only a subset of the abnormalities found in mutants lacking all ß1 integrins, and a milder phenotype. Double-mutant Schwann cells can properly activate all the major signaling pathways associated with radial sorting and show normal Schwann cell proliferation and survival. Thus, α6ß1 and α7ß1 are the laminin-binding integrins required for axonal sorting, but other Schwann cell ß1 integrins, possibly those that do not bind laminins, may also contribute to radial sorting during peripheral nerve development.

Actin polymerization is essential for myelin sheath fragmentation during Wallerian degeneration.

The mechanisms that trigger Wallerian degeneration (WD) of peripheral nerves after injury are not well understood. During the early period of WD, fragmentation of myelin into ovoid structures occurs near the Schmidt-Lantermann incisures (SLI), a noncompact region of the myelin sheath containing autotypical adherens junction. In this study, we found that new filamentous actin polymerization occurs in the SLI of mouse sciatic nerves after injury and that its inhibition prevented not only the degradation of E-cadherin in the SLI but also myelin ovoid formation. However, the inhibition of actin polymerization could not block Schwann cell dedifferentiation. The activation of Rac GTPase was observed in the distal stump of the injured nerves, and a specific Rac inhibitor, a dominant-negative Rac, and Rac1-RNA interference blocked myelin ovoid formation. Together, these findings suggest that dynamic changes in actin in the SLI are essential for initiation of demyelination after peripheral nerve injury.