Pinch2 regulates myelination in the mouse central nervous system.

The extensive morphological changes of oligodendrocytes during axon ensheathment and myelination involve assembly of the Ilk-Parvin-Pinch (IPP) heterotrimeric complex of proteins to relay essential mechanical and biochemical signals between integrins and the actin cytoskeleton. Binding of Pinch1 and Pinch2 isoforms to Ilk is mutually exclusive and allows the formation of distinct IPP complexes with specific signaling properties. Using tissue-specific conditional gene ablation in mice, we reveal an essential role for Pinch2 during central nervous system myelination. Unlike Pinch1 gene ablation, loss of Pinch2 in oligodendrocytes results in hypermyelination and in the formation of pathological myelin outfoldings in white matter regions. These structural changes concur with inhibition of Rho GTPase RhoA and Cdc42 activities and phenocopy aspects of myelin pathology observed in corresponding mouse mutants. We propose a dual role for Pinch2 in preventing an excess of myelin wraps through RhoA-dependent control of membrane growth and in fostering myelin stability via Cdc42-dependent organization of cytoskeletal septins. Together, these findings indicate that IPP complexes containing Pinch2 act as a crucial cell-autonomous molecular hub ensuring synchronous control of key signaling networks during developmental myelination.

Communication defects with astroglia contribute to early impairments in the motor cortex plasticity of SOD1G93A mice.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease, involving the selective degeneration of cortical upper synapses in the primary motor cortex (M1). Excitotoxicity in ALS occurs due to an imbalance between excitation and inhibition, closely linked to the loss/gain of astrocytic function. Using the ALS SOD1G93A mice, we investigated the astrocytic contribution for the electrophysiological alterations observed in the M1 of SOD1G93A mice, throughout disease progression. Results showed that astrocytes are involved in synaptic dysfunction observed in presymptomatic SOD1G93A mice, since astrocytic glutamate transport currents are diminished and pharmacological inhibition of astrocytes only impaired long-term potentiation and basal transmission in wild-type mice. Proteomic analysis revealed major differences in neuronal transmission, metabolism, and immune system in upper synapses, confirming early communication deficits between neurons and astroglia. These results provide valuable insights into the early impact of upper synapses in ALS and the lack of supportive functions of cortical astrocytes, highlighting the possibility of manipulating astrocytes to improve synaptic function.

The Pebble/Rho1/Anillin pathway controls polyploidization and axonal wrapping activity in the glial cells of the Drosophila eye.

During development glial cell are crucially important for the establishment of neuronal networks. Proliferation and migration of glial cells can be modulated by neurons, and in turn glial cells can differentiate to assume key roles such as axonal wrapping and targeting. To explore the roles of actin cytoskeletal rearrangements in glial cells, we studied the function of Rho1 in Drosophila developing visual system. We show that the Pebble (RhoGEF)/Rho1/Anillin pathway is required for glia proliferation and to prevent the formation of large polyploid perineurial glial cells, which can still migrate into the eye disc if generated. Surprisingly, this Rho1 pathway is not necessary to establish the total glial membrane area or for the differentiation of the polyploid perineurial cells. The resulting polyploid wrapping glial cells are able to initiate wrapping of axons in the basal eye disc, however the arrangement and density of glia nuclei and membrane processes in the optic stalk are altered and the ensheathing of the photoreceptor axonal fascicles is reduced.

The peptide secreted at the water to land transition in a model amphibian has antioxidant effects.

In addition to the morphophysiological changes experienced by amphibians during metamorphosis, they must also deal with a different set of environmental constraints when they shift from the water to the land. We found that Pithecopus azureus secretes a single peptide ([M + H]+ = 658.38 Da) at the developmental stage that precedes the onset of terrestrial behaviour. De novo peptide and cDNA sequencing revealed that the peptide, named PaT-2, is expressed in tandem and is a member of the tryptophyllins family. In silico studies allowed us to identify the position of reactive sites and infer possible antioxidant mechanisms of the compounds. Cell-based assays confirmed the predicted antioxidant activity in mammalian microglia and neuroblast cells. The potential neuroprotective effect of PaT-2 was further corroborated in FRET-based live cell imaging assays, where the peptide prevented lipopolysaccharide-induced ROS production and glutamate release in human microglia. In summary, PaT-2 is the first peptide expressed during the ontogeny of P. azureus, right before the metamorphosing froglet leaves the aquatic environment to occupy terrestrial habitats. The antioxidant activity of PaT-2, predicted by in silico analyses and confirmed by cell-based assays, might be relevant for the protection of the skin of P. azureus adults against increased O2 levels and UV exposure on land compared with aquatic environments.

Somuncurins: Bioactive Peptides from the Skin of the Endangered Endemic Patagonian Frog Pleurodema somuncurense.

The skin glands of amphibian species hold a major component of their innate immunity, namely a unique set of antimicrobial peptides (AMPs). Although most of them have common characteristics, differences in AMP sequences allow a huge repertoire of biological activity with varying degrees of efficacy. We present the first study of the AMPs from Pleurodema somuncurence (Anura: Leptodactylidae: Leiuperinae). Among the 11 identified mature peptides, three presented antimicrobial activity. Somuncurin-1 (FIIWPLRYRK), somuncurin-2 (FILKRSYPQYY), and thaulin-3 (NLVGSLLGGILKK) inhibited Escherichia coli growth. Somuncurin-1 also showed antimicrobial activity against Staphylococcus aureus. Biophysical membrane model studies revealed that this peptide had a greater permeation effect in prokaryotic-like membranes and capacity to restructure liposomes, suggesting fusogenic activity, which could lead to cell aggregation and disruption of cell morphology. This study contributes to the characterization of peptides with new sequences to enrich the databases for the design of therapeutic agents. Furthermore, it highlights the importance of investing in nature conservation and the power of genetic description as a strategy to identify new compounds.

dMyc is required in retinal progenitors to prevent JNK-mediated retinal glial activation.

In the nervous system, glial cells provide crucial insulation and trophic support to neurons and are important for neuronal survival. In reaction to a wide variety of insults, glial cells respond with changes in cell morphology and metabolism to allow repair. Additionally, these cells can acquire migratory and proliferative potential. In particular, after axonal damage or pruning the clearance of axonal debris by glial cells is key for a healthy nervous system. Thus, bidirectional neuron-glial interactions are crucial in development, but little is known about the cellular sensors and signalling pathways involved. In here, we show that decreased cellular fitness in retinal progenitors caused by reduced Drosophila Myc expression triggers non cell-autonomous activation of retinal glia proliferation and overmigration. Glia migration occurs beyond its normal limit near the boundary between differentiated photoreceptors and precursor cells, extending into the progenitor domain. This overmigration is stimulated by JNK activation (and the function of its target Mmp1), while proliferative responses are mediated by Dpp/TGF-ß signalling activation.

Mechanical plasticity during oligodendrocyte differentiation and myelination.

In the central nervous system, oligodendrocyte precursor cells are exclusive in their potential to differentiate into myelinating oligodendrocytes. Oligodendrocyte precursor cells migrate within the parenchyma and extend cell membrane protrusions that ultimately evolve into myelinating sheaths able to wrap neuronal axons and significantly increase their electrical conductivity. The subcellular force generating mechanisms driving morphological and functional transformations during oligodendrocyte differentiation and myelination remain elusive. In this review, we highlight the mechanical processes governing oligodendrocyte plasticity in a dynamic interaction with the extracellular matrix.

Jmy regulates oligodendrocyte differentiation via modulation of actin cytoskeleton dynamics.

During central nervous system development, oligodendrocytes form structurally and functionally distinct actin-rich protrusions that contact and wrap around axons to assemble myelin sheaths. Establishment of axonal contact is a limiting step in myelination that relies on the oligodendrocyte's ability to locally coordinate cytoskeletal rearrangements with myelin production, under the control of a transcriptional differentiation program. The molecules that provide fine-tuning of actin dynamics during oligodendrocyte differentiation and axon ensheathment remain largely unidentified. We performed transcriptomics analysis of soma and protrusion fractions from rat brain oligodendrocyte progenitors and found a subcellular enrichment of mRNAs in newly-formed protrusions. Approximately 30% of protrusion-enriched transcripts encode proteins related to cytoskeleton dynamics, including the junction mediating and regulatory protein Jmy, a multifunctional regulator of actin polymerization. Here, we show that expression of Jmy is upregulated during myelination and is required for the assembly of actin filaments and protrusion formation during oligodendrocyte differentiation. Quantitative morphodynamics analysis of live oligodendrocytes showed that differentiation is driven by a stereotypical actin network-dependent "cellular shaping" program. Disruption of actin dynamics via knockdown of Jmy leads to a program fail resulting in oligodendrocytes that do not acquire an arborized morphology and are less efficient in contacting neurites and forming myelin wraps in co-cultures with neurons. Our findings provide new mechanistic insight into the relationship between cell shape dynamics and differentiation in development.

E-cadherin-defective gastric cancer cells depend on Laminin to survive and invade.

Epithelial-cadherin (Ecad) deregulation affects cell-cell adhesion and results in increased invasiveness of distinct human carcinomas. In gastric cancer, loss of Ecad expression is a common event and is associated with disease aggressiveness and poor prognosis. However, the molecular mechanisms underlying the invasive process associated to Ecad dysfunction are far from understood. We hypothesized that deregulation of cell-matrix interactions could play an important role during this process. Thus, we focussed on LM-332, which is a major matrix component, and in Ecad/LM-332 crosstalk in the process of Ecad-dependent invasion. To verify whether matrix deregulation was triggered by Ecad loss, we used the Drosophila model. To dissect the key molecules involved and unveil their functional significance, we used gastric cancer cell lines. The relevance of this relationship was then confirmed in human primary tumours. In vivo, Ecad knockdown induced apoptosis; nonetheless, at the invasive front, cells ectopically expressed Laminin A and ßPS integrin. In vitro, we demonstrated that, in two different gastric cancer cell models, Ecad-defective cells overexpressed Laminin γ2 (LM-γ2), ß1 and ß4 integrin, when compared with Ecad-competent ones. We showed that LM-γ2 silencing impaired invasion and enhanced cell death, most likely via pSrc and pAkt reduction, and JNK activation. In human gastric carcinomas, we found a concomitant decrease in Ecad and increase in LM-γ2. This is the first evidence that ectopic Laminin expression depends on Ecad loss and allows Ecad-dysfunctional cells to survive and invade. This opens new avenues for using LM-γ2 signalling regulators as molecular targets to impair gastric cancer progression.

Dominant negative effect of polyglutamine expansion perturbs normal function of ataxin-3 in neuronal cells.

The physiological function of Ataxin-3 (ATXN3), a deubiquitylase (DUB) involved in Machado-Joseph Disease (MJD), remains elusive. In this study, we demonstrate that ATXN3 is required for neuronal differentiation and for normal cell morphology, cytoskeletal organization, proliferation and survival of SH-SY5Y and PC12 cells. This cellular phenotype is associated with increased proteasomal degradation of α5 integrin subunit (ITGA5) and reduced activation of integrin signalling and is rescued by ITGA5 overexpression. Interestingly, silencing of ATXN3, overexpression of mutant versions of ATXN3 lacking catalytic activity or bearing an expanded polyglutamine (polyQ) tract led to partially overlapping phenotypes. In vivo analysis showed that both Atxn3 knockout and MJD transgenic mice had decreased levels of ITGA5 in the brain. Furthermore, abnormal morphology and reduced branching were observed both in cultured neurons expressing shRNA for ATXN3 and in those obtained from MJD mice. Our results show that ATXN3 rescues ITGA5 from proteasomal degradation in neurons and that polyQ expansion causes a partial loss of this cellular function, resulting in reduced integrin signalling and neuronal cytoskeleton modifications, which may be contributing to neurodegeneration.

Profilin 1 is required for peripheral nervous system myelination.

Myelination allows rapid saltatory propagation of action potentials along the axon and is an essential prerequisite for the normal functioning of the nervous system. During peripheral nervous system (PNS) development, myelin-forming Schwann cells (SCs) generate radial lamellipodia to sort and ensheath axons. This process requires controlled cytoskeletal remodeling, and we show that SC lamellipodia formation depends on the function of profilin 1 (Pfn1), an actin-binding protein involved in microfilament polymerization. Pfn1 is inhibited upon phosphorylation by ROCK, a downstream effector of the integrin linked kinase pathway. Thus, a dramatic reduction of radial lamellipodia formation is observed in SCs lacking integrin-linked kinase or treated with the Rho/ROCK activator lysophosphatidic acid. Knocking down Pfn1 expression by lentiviral-mediated shRNA delivery impairs SC lamellipodia formation in vitro, suggesting a direct role for this protein in PNS myelination. Indeed, SC-specific gene ablation of Pfn1 in mice led to profound radial sorting and myelination defects, confirming a central role for this protein in PNS development. Our data identify Pfn1 as a key effector of the integrin linked kinase/Rho/ROCK pathway. This pathway, acting in parallel with integrin ß1/LCK/Rac1 and their effectors critically regulates SC lamellipodia formation, radial sorting and myelination during peripheral nervous system maturation.

Methylphenidate-triggered ROS generation promotes caveolae-mediated transcytosis via Rac1 signaling and c-Src-dependent caveolin-1 phosphorylation in human brain endothelial cells.

Methylphenidate (MPH) is an amphetamine-like stimulant commonly prescribed for attention deficit hyperactivity disorder. Despite its widespread use, the cellular/molecular effects of MPH remain elusive. Here, we report a novel direct role of MPH on the regulation of macromolecular flux through human brain endothelial cells (ECs). MPH significantly increased caveolae-mediated transcytosis of horseradish peroxidase through ECs without affecting paracellular permeability. Using FRET-based live cell imaging, together with pharmacological inhibitors and lentiviral-mediated shRNA knockdown, we demonstrate that MPH promoted ROS generation via activation of Rac1-dependent NADPH oxidase (NOX) and c-Src activation at the plasma membrane. c-Src in turn was shown to mediate the phosphorylation of caveolin-1 (Cav1) on Tyr14 leading to enhanced caveolae formation and transendothelial transport. Accordingly, the inhibition of Cav1 phosphorylation by overexpression of a phosphodefective Cav1Y14F mutant or knocking down Cav1 expression abrogated MPH-induced transcytosis. In addition, both vitamin C and inhibition of NOX blocked MPH-triggered vesicular transport. This study, therefore, identifies Rac1/NOX/c-Src-dependent signaling in MPH-induced increase in transendothelial permeability of brain endothelial cell monolayers via caveolae-mediated transcytosis.

Drosophila PS2 and PS3 integrins play distinct roles in retinal photoreceptors-glia interactions.

Cellular migration and differentiation are important developmental processes that require dynamic cellular adhesion. Integrins are heterodimeric transmembrane receptors that play key roles in adhesion plasticity. Here, we explore the developing visual system of Drosophila to study the roles of integrin heterodimers in glia development. Our data show that αPS2 is essential for retinal glia migration from the brain into the eye disc and that glial cells have a role in the maintenance of the fenestrated membrane (Laminin-rich ECM layer) in the disc. Interestingly, the absence of glial cells in the eye disc did not affect the targeting of retinal axons to the optic stalk. In contrast, αPS3 is not required for retinal glia migration, but together with Talin, it functions in glial cells to allow photoreceptor axons to target the optic stalk. Thus, we present evidence that αPS2 and αPS3 integrin have different and specific functions in the development of retinal glia.

c-Src function is necessary and sufficient for triggering microglial cell activation.

Microglial cells are the resident macrophages of the central nervous system. Their function is essential for neuronal tissue homeostasis. After inflammatory stimuli, microglial cells become activated changing from a resting and highly ramified cell shape to an amoeboid-like morphology. These morphological changes are associated with the release of proinflammatory cytokines and glutamate, as well as with high phagocytic activity. The acquisition of such phenotype has been associated with activation of cytoplasmic tyrosine kinases, including those of the Src family (SFKs). In this study, using both in vivo and in vitro inflammation models coupled to FRET-based time-lapse microscopy, lentiviruses-mediated shRNA delivery and genetic gain-of-function experiments, we demonstrate that among SFKs c-Src function is necessary and sufficient for triggering microglia proinflammatory signature, glutamate release, microglia-induced neuronal loss, and phagocytosis. c-Src inhibition in retinal neuroinflammation experimental paradigms consisting of intravitreal injection of LPS or ischemia-reperfusion injury significantly reduced microglia activation changing their morphology to a more resting phenotype and prevented neuronal apoptosis. Our data demonstrate an essential role for c-Src in microglial cell activation.

Matrix metalloproteases as maestros for the dual role of LPS- and IL-10-stimulated macrophages in cancer cell behaviour.

BACKGROUND: The interactions established between macrophages and cancer cells are largely dependent on instructions from the tumour microenvironment. Macrophages may differentiate into populations with distinct inflammatory profiles, but knowledge on their role on cancer cell activities is still very scarce. In this work, we investigated the influence of pro-inflammatory (LPS-stimulated) and anti-inflammatory (IL-10-stimulated) macrophages on gastric and colorectal cancer cell invasion, motility/migration, angiogenesis and proteolysis, and the associated molecular mechanisms. METHODS: Following exposure of gastric and colon cancer cell lines to LPS- and IL-10-stimulated human macrophages, either by indirect contact or conditioned media, we analyzed the effect of the different macrophage populations on cancer cell invasion, migration, motility and phosphorylation status of EGFR and several interacting partners. Cancer-cell induced angiogenesis upon the influence of conditioned media from both macrophage populations was assessed using the chick embryo chorioallantoic membrane assay. MMP activities were evaluated by gelatin zymograhy. RESULTS: Our results show that IL-10-stimulated macrophages are more efficient in promoting in vitro cancer cell invasion and migration. In addition, soluble factors produced by these macrophages enhanced in vivo cancer cell-induced angiogenesis, as opposed to their LPS-stimulated counterparts. We further demonstrate that differences in the ability of these macrophage populations to stimulate invasion or angiogenesis cannot be explained by the EGFR-mediated signalling, since both LPS- and IL-10-stimulated macrophages similarly induce the phosphorylation of cancer cell EGFR, c-Src, Akt, ERK1/2, and p38. Interestingly, both populations exert distinct proteolytic activities, being the IL-10-stimulated macrophages the most efficient in inducing matrix metalloprotease (MMP)-2 and MMP-9 activities. Using a broad-spectrum MMP inhibitor, we demonstrated that proteolysis was essential for macrophage-mediated cancer cell invasion and angiogenesis. CONCLUSIONS: We propose that IL-10- and LPS-stimulated macrophages distinctly modulate gastric and colorectal cancer cell behaviour, as result of distinct proteolytic profiles that impact cell invasion and angiogenesis.

A cytoskeleton symphony: Actin and microtubules in microglia dynamics and aging.

Microglia dynamically reorganize their cytoskeleton to perform essential functions such as phagocytosis of toxic protein aggregates, surveillance of the brain parenchyma, and regulation of synaptic plasticity during neuronal activity bursts. Recent studies have shed light on the critical role of the microtubule cytoskeleton in microglial reactivity and function, revealing key regulators like cyclin-dependent kinase 1 and centrosomal nucleation in the remodeling of microtubules in activated microglia. Concurrently, the role of the actin cytoskeleton is also pivotal, particularly in the context of small GTPases like RhoA, Rac1, and Cdc42 and actin-binding molecules such as profilin-1 and cofilin. This article delves into the intricate molecular landscape of actin and microtubules, exploring their synergistic roles in driving microglial cytoskeletal dynamics. We propose a more integrated view of actin and microtubule cooperation, which is fundamental to understanding the functional coherence of the microglial cytoskeleton and its pivotal role in propelling brain homeostasis. Furthermore, we discuss how alterations in microglial cytoskeleton dynamics during aging and in disease states could have far-reaching implications for brain function. By unraveling the complexities of microglia cytoskeletal dynamics, we can deepen our understanding of microglial functional states and their implications in health and disease, offering insights into potential therapeutic interventions for neurologic disorders.

The use of an in vitro fecal fermentation model to uncover the beneficial role of omega-3 and punicic acid in gut microbiota alterations induced by a Western diet.

The influence of gut microbiota in the onset and development of several metabolic diseases has gained attention over the last few years. Diet plays an essential role in gut microbiota modulation. Western diet (WD), characterized by high-sugar and high-fat consumption, alters gut microbiome composition, diversity index, microbial relative levels, and functional pathways. Despite the promising health effects demonstrated by polyunsaturated fatty acids, their impact on gut microbiota is still overlooked. The effect of Fish oil (omega-3 source) and Pomegranate oil (punicic acid source), and a mixture of both oils in gut microbiota modulation were determined by subjecting the oil samples to in vitro fecal fermentations. Cecal samples from rats from two different dietary groups: a control diet (CD) and a high-fat high-sugar diet (WD), were used as fecal inoculum. 16S amplicon metagenomics sequencing showed that Fish oil + Pomegranate oil from the WD group increased α-diversity. This sample can also increase the relative abundance of the Firmicutes and Bacteroidetes phylum as well as Akkermansia and Blautia, which were affected by the WD consumption. All samples were able to increase butyrate and acetate concentration in the WD group. Moreover, tyrosine concentrations, a precursor for dopamine and norepinephrine, increase in the Fish oil + Pomegranate oil WD sample. GABA, an important neurotransmitter, was also increased in WD samples. These results suggest a potential positive impact of these oils' mixture on gut-brain axis modulation. It was demonstrated, for the first time, the great potential of using a mixture of both Fish and Pomegranate oil to restore the gut microbiota changes associated with WD consumption.

Calcium-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors trigger neuronal nitric-oxide synthase activation to promote nerve cell death in an Src kinase-dependent fashion.

In the retina information decoding is dependent on excitatory neurotransmission and is critically modulated by AMPA glutamate receptors. The Src-tyrosine kinase has been implicated in modulating neurotransmission in CNS. Thus, our main goal was to correlate AMPA-mediated excitatory neurotransmission with the modulation of Src activity in retinal neurons. Cultured retinal cells were used to access the effects of AMPA stimulation on nitric oxide (NO) production and Src phosphorylation. 4-Amino-5-methylamino-2',7'-difluorofluorescein diacetate fluorescence mainly determined NO production, and immunocytochemistry and Western blotting evaluated Src activation. AMPA receptors activation rapidly up-regulated Src phosphorylation at tyrosine 416 (stimulatory site) and down-regulated phosphotyrosine 527 (inhibitory site) in retinal cells, an effect mainly mediated by calcium-permeable AMPA receptors. Interestingly, experiments confirmed that neuronal NOS was activated in response to calcium-permeable AMPA receptor stimulation. Moreover, data suggest NO pathway as a key regulatory signaling in AMPA-induced Src activation in neurons but not in glial cells. The NO donor SNAP (S-nitroso-N-acetyl-DL-penicillamine) and a soluble guanylyl cyclase agonist (YC-1) mimicked AMPA effect in Src Tyr-416 phosphorylation, reinforcing that Src activation is indeed modulated by the NO pathway. Gain and loss-of-function data demonstrated that ERK is a downstream target of AMPA-induced Src activation and NO signaling. Furthermore, AMPA stimulated NO production in organotypic retinal cultures and increased Src activity in the in vivo retina. Additionally, AMPA-induced apoptotic retinal cell death was regulated by both NOS and Src activity. Because Src activity is pivotal in several CNS regions, the data presented herein highlight that Src modulation is a critical step in excitatory retinal cell death.

Myelin is dependent on the Charcot-Marie-Tooth Type 4H disease culprit protein FRABIN/FGD4 in Schwann cells.

Studying the function and malfunction of genes and proteins associated with inherited forms of peripheral neuropathies has provided multiple clues to our understanding of myelinated nerves in health and disease. Here, we have generated a mouse model for the peripheral neuropathy Charcot-Marie-Tooth disease type 4H by constitutively disrupting the mouse orthologue of the suspected culprit gene FGD4 that encodes the small RhoGTPase Cdc42-guanine nucleotide exchange factor Frabin. Lack of Frabin/Fgd4 causes dysmyelination in mice in early peripheral nerve development, followed by profound myelin abnormalities and demyelination at later stages. At the age of 60 weeks, this was accompanied by electrophysiological deficits. By crossing mice carrying alleles of Frabin/Fgd4 flanked by loxP sequences with animals expressing Cre recombinase in a cell type-specific manner, we show that Schwann cell-autonomous Frabin/Fgd4 function is essential for proper myelination without detectable primary contributions from neurons. Deletion of Frabin/Fgd4 in Schwann cells of fully myelinated nerve fibres revealed that this protein is not only required for correct nerve development but also for accurate myelin maintenance. Moreover, we established that correct activation of Cdc42 is dependent on Frabin/Fgd4 function in healthy peripheral nerves. Genetic disruption of Cdc42 in Schwann cells of adult myelinated nerves resulted in myelin alterations similar to those observed in Frabin/Fgd4-deficient mice, indicating that Cdc42 and the Frabin/Fgd4-Cdc42 axis are critical for myelin homeostasis. In line with known regulatory roles of Cdc42, we found that Frabin/Fgd4 regulates Schwann cell endocytosis, a process that is increasingly recognized as a relevant mechanism in peripheral nerve pathophysiology. Taken together, our results indicate that regulation of Cdc42 by Frabin/Fgd4 in Schwann cells is critical for the structure and function of the peripheral nervous system. In particular, this regulatory link is continuously required in adult fully myelinated nerve fibres. Thus, mechanisms regulated by Frabin/Fgd4-Cdc42 are promising targets that can help to identify additional regulators of myelin development and homeostasis, which may crucially contribute also to malfunctions in different types of peripheral neuropathies.

MorphoMacro for in vivo and ex vivo quantitative morphometric analysis of microglia.

Microglia cells dynamically survey the central nervous system microenvironment and, in response to tissue damage inflicted by radiation therapy, disease or infection, undergo morphological and functional changes that culminate in microglia activation. Cell shape transformation can be assessed descriptively or, alternatively, it can be quantified as a continuous variable for parameters including total cell size as well as protrusion length, ramification and complexity. The purpose of the MorphoMacro method is to quantitatively profile multiple and single microglia cells using the available ImageJ platform. This method outlines the required steps and ImageJ plugins to convert fluorescence and bright-field photomicrographs into representative binary and skeletonized images and to analyze them using the MorphoMacro software plugin for multiparametric and multilevel description of microglia cell morphology in vivo and ex vivo. Overall, the protocol provides a quantitative and comprehensive tool that can be used to identify, stratify, and monitor diverse microglia morphologies in homeostatic, different disease conditions and subsequent therapeutic monitoring.