Neural blastocyst complementation enables mouse forebrain organogenesis.

Genetically modified mice are commonly generated by the microinjection of pluripotent embryonic stem (ES) cells into wild-type host blastocysts1, producing chimeric progeny that require breeding for germline transmission and homozygosity of modified alleles. As an alternative approach and to facilitate studies of the immune system, we previously developed RAG2-deficient blastocyst complementation2. Because RAG2-deficient mice cannot undergo V(D)J recombination, they do not develop B or T lineage cells beyond the progenitor stage2: injecting RAG2-sufficient donor ES cells into RAG2-deficient blastocysts generates somatic chimaeras in which all mature lymphocytes derive from donor ES cells. This enables analysis, in mature lymphocytes, of the functions of genes that are required more generally for mouse development3. Blastocyst complementation has been extended to pancreas organogenesis4, and used to generate several other tissues or organs5-10, but an equivalent approach for brain organogenesis has not yet been achieved. Here we describe neural blastocyst complementation (NBC), which can be used to study the development and function of specific forebrain regions. NBC involves targeted ablation, mediated by diphtheria toxin subunit A, of host-derived dorsal telencephalic progenitors during development. This ablation creates a vacant forebrain niche in host embryos that results in agenesis of the cerebral cortex and hippocampus. Injection of donor ES cells into blastocysts with forebrain-specific targeting of diphtheria toxin subunit A enables donor-derived dorsal telencephalic progenitors to populate the vacant niche in the host embryos, giving rise to neocortices and hippocampi that are morphologically and neurologically normal with respect to learning and memory formation. Moreover, doublecortin-deficient ES cells-generated via a CRISPR-Cas9 approach-produced NBC chimaeras that faithfully recapitulated the phenotype of conventional, germline doublecortin-deficient mice. We conclude that NBC is a rapid and efficient approach to generate complex mouse models for studying forebrain functions; this approach could more broadly facilitate organogenesis based on blastocyst complementation.

The Gliopeptide ODN, a Ligand for the Benzodiazepine Site of GABAA Receptors, Boosts Functional Recovery after Stroke.

Following stroke, the survival of neurons and their ability to reestablish connections is critical to functional recovery. This is strongly influenced by the balance between neuronal excitation and inhibition. In the acute phase of experimental stroke, lethal hyperexcitability can be attenuated by positive allosteric modulation of GABAA receptors (GABAARs). Conversely, in the late phase, negative allosteric modulation of GABAAR can correct the suboptimal excitability and improves both sensory and motor recovery. Here, we hypothesized that octadecaneuropeptide (ODN), an endogenous allosteric modulator of the GABAAR synthesized by astrocytes, influences the outcome of ischemic brain tissue and subsequent functional recovery. We show that ODN boosts the excitability of cortical neurons, which makes it deleterious in the acute phase of stroke. However, if delivered after day 3, ODN is safe and improves motor recovery over the following month in two different paradigms of experimental stroke in mice. Furthermore, we bring evidence that, during the subacute period after stroke, the repairing cortex can be treated with ODN by means of a single hydrogel deposit into the stroke cavity.SIGNIFICANCE STATEMENT Stroke remains a devastating clinical challenge because there is no efficient therapy to either minimize neuronal death with neuroprotective drugs or to enhance spontaneous recovery with neurorepair drugs. Around the brain damage, the peri-infarct cortex can be viewed as a reservoir of plasticity. However, the potential of wiring new circuits in these areas is restrained by a chronic excess of GABAergic inhibition. Here we show that an astrocyte-derived peptide, can be used as a delayed treatment, to safely correct cortical excitability and facilitate sensorimotor recovery after stroke.

Genetic disruption of the small GTPase RAC1 prevents plexiform neurofibroma formation in mice with neurofibromatosis type 1

Neurofibromatosis type 1 (NF1) is a common cancer predisposition syndrome caused by mutations in the NF1 tumor suppressor gene. NF1 encodes neurofibromin, a GTPase-activating protein for RAS proto-oncogene GTPase (RAS). Plexiform neurofibromas are a hallmark of NF1 and result from loss of heterozygosity of NF1 in Schwann cells, leading to constitutively activated p21RAS. Given the inability to target p21RAS directly, here we performed an shRNA library screen of all human kinases and Rho-GTPases in a patient-derived NF1-/- Schwann cell line to identify novel therapeutic targets to disrupt PN formation and progression. Rho family members, including Rac family small GTPase 1 (RAC1), were identified as candidates. Corroborating these findings, we observed that shRNA-mediated knockdown of RAC1 reduces cell proliferation and phosphorylation of extracellular signal-regulated kinase (ERK) in NF1-/- Schwann cells. Genetically engineered Nf1flox/flox;PostnCre+ mice, which develop multiple PNs, also exhibited increased RAC1-GTP and phospho-ERK levels compared with Nf1flox/flox;PostnCre- littermates. Notably, mice in which both Nf1 and Rac1 loci were disrupted (Nf1flox/floxRac1flox/flox;PostnCre+) were completely free of tumors and had normal phospho-ERK activity compared with Nf1flox/flox ;PostnCre+ mice. We conclude that the RAC1-GTPase is a key downstream node of RAS and that genetic disruption of the Rac1 allele completely prevents PN tumor formation in vivo in mice.

Statins Disrupt Macrophage Rac1 Regulation Leading to Increased Atherosclerotic Plaque Calcification.

OBJECTIVE: Calcification of atherosclerotic plaque is traditionally associated with increased cardiovascular event risk; however, recent studies have found increased calcium density to be associated with more stable disease. 3-hydroxy-3-methylglutaryl coenzymeA reductase inhibitors or statins reduce cardiovascular events. Invasive clinical studies have found that statins alter both the lipid and calcium composition of plaque but the molecular mechanisms of statin-mediated effects on plaque calcium composition remain unclear. We recently defined a macrophage Rac (Ras-related C3 botulinum toxin substrate)-IL-1ß (interleukin-1 beta) signaling axis to be a key mechanism in promoting atherosclerotic calcification and sought to define the impact of statin therapy on this pathway. Approach and Results: Here, we demonstrate that statin therapy is independently associated with elevated coronary calcification in a high-risk patient population and that statins disrupt the complex between Rac1 and its inhibitor RhoGDI (Rho GDP-dissociation inhibitor), leading to increased active (GTP bound) Rac1 in primary monocytes/macrophages. Rac1 activation is prevented by rescue with the isoprenyl precursor geranylgeranyl diphosphate. Statin-treated macrophages exhibit increased activation of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells), increased IL-1ß mRNA, and increased Rac1-dependent IL-1ß protein secretion in response to inflammasome stimulation. Using an animal model of calcific atherosclerosis, inclusion of statin in the atherogenic diet led to a myeloid Rac1-dependent increase in atherosclerotic calcification, which was associated with increased serum IL-1ß expression, increased plaque Rac1 activation, and increased plaque expression of the osteogenic markers, alkaline phosphatase and RUNX2 (Runt-related transcription factor 2). CONCLUSIONS: Statins are capable of increasing atherosclerotic calcification through disinhibition of a macrophage Rac1-IL-1ß signaling axis.

Modulation of Drosophila post-feeding physiology and behavior by the neuropeptide leucokinin.

Behavior and physiology are orchestrated by neuropeptides acting as central neuromodulators and circulating hormones. An outstanding question is how these neuropeptides function to coordinate complex and competing behaviors. In Drosophila, the neuropeptide leucokinin (LK) modulates diverse functions, but mechanisms underlying these complex interactions remain poorly understood. As a first step towards understanding these mechanisms, we delineated LK circuitry that governs various aspects of post-feeding physiology and behavior. We found that impaired LK signaling in Lk and Lk receptor (Lkr) mutants affects diverse but coordinated processes, including regulation of stress, water homeostasis, feeding, locomotor activity, and metabolic rate. Next, we sought to define the populations of LK neurons that contribute to the different aspects of this physiology. We find that the calcium activity in abdominal ganglia LK neurons (ABLKs), but not in the two sets of brain neurons, increases specifically following water consumption, suggesting that ABLKs regulate water homeostasis and its associated physiology. To identify targets of LK peptide, we mapped the distribution of Lkr expression, mined a brain single-cell transcriptome dataset for genes coexpressed with Lkr, and identified synaptic partners of LK neurons. Lkr expression in the brain insulin-producing cells (IPCs), gut, renal tubules and chemosensory cells, correlates well with regulatory roles detected in the Lk and Lkr mutants. Furthermore, these mutants and flies with targeted knockdown of Lkr in IPCs displayed altered expression of insulin-like peptides (DILPs) and transcripts in IPCs and increased starvation resistance. Thus, some effects of LK signaling appear to occur via DILP action. Collectively, our data suggest that the three sets of LK neurons have different targets, but modulate the establishment of post-prandial homeostasis by regulating distinct physiological processes and behaviors such as diuresis, metabolism, organismal activity and insulin signaling. These findings provide a platform for investigating feeding-related neuroendocrine regulation of vital behavior and physiology.

Neuromedin U suppresses prolactin secretion via dopamine neurons of the arcuate nucleus.

Neuromedin U (NMU) has a precursor that contains one additional peptide consisting of 33 or 36 amino acid residues. Recently, we identified this second peptide from rat brain and designated it neuromedin U precursor-related peptide (NURP), showing it to stimulate prolactin release from the pituitary when injected via the intracerebroventricular (icv) route. Here, we examined whether NMU, like NURP, also stimulates prolactin release. Unlike NURP, icv injection of NMU significantly decreased the secretion of prolactin from the pituitary. This suppression of prolactin release by NMU was observed in hyper-prolactin states such as lactation, stress, pseudopregnancy, domperidone (dopamine antagonist) administration, and icv injection of NURP. Immunohistochemical analysis revealed that icv injection of NMU induced cFos expression in dopaminergic neurons of the arcuate nucleus, but not the substantia nigra. Mice with double knockout of NMU and neuromedin S (NMS), the latter also binding to NMU receptors, showed a significant increase of the plasma prolactin level after domperidone treatment relative to wild-type mice. These results suggest that NMU and NURP may play important reciprocal roles in physiological prolactin secretion.

Doublecortin and IGF-1R protein levels are reduced in spite of unchanged DNA methylation in the hippocampus of aged rats.

The aim of the current study was to investigate whether doublecortin (DCX), insulin-like growth factor receptor 1 (IGF-1R) and metabotropic glutamate receptor 5 (mGluR5) levels are indeed modified in the aging rat hippocampal individual subareas (rather than total hippocampal tissue as in previous reports) at the protein and mRNA level and whether the methylation status contributes to these changes. Since the aging population is not homogeneous in terms of spatial memory performance, we examined whether changes in DCX, IGF-1R and mGluR5 are linked to cognitive aging. Aged (22 months) male Sprague Dawley rats were trained in the hole-board, a spatial memory task, and were subdivided according to performance to aged impaired and aged unimpaired groups. Age- and memory performance-dependent changes in mRNA steady-state levels, protein levels and DNA methylation status of DCX, IGF-1R and mGluR5 were evaluated by RT-PCR, immunoblotting and bisulfite pyrosequencing. Extending previous findings, we detected decreased DCX protein and mRNA levels in dentate gyrus (DG) of aged animals. IGF-1 signaling is a key event and herein we show that mRNA levels for IGF-1R were unchanged although reduced at the protein level. This finding may simply reflect that these protein levels are regulated at the level of protein synthesis as well as protein degradation. We provide evidence that promoter methylation is not involved in regulation of mRNA and protein levels of DCX, IGF-1R and mGluR5 during aging. Moreover, there was no significant difference between aged rats with impaired and aged rats with unimpaired memory at the protein and mRNA level. Findings propose that changes in the abovementioned protein levels may not be relevant for performance in the spatial memory task used in aged rats.

Molecular basis for specific regulation of neuronal kinesin-3 motors by doublecortin family proteins.

Doublecortin (Dcx) defines a growing family of microtubule (MT)-associated proteins (MAPs) involved in neuronal migration and process outgrowth. We show that Dcx is essential for the function of Kif1a, a kinesin-3 motor protein that traffics synaptic vesicles. Neurons lacking Dcx and/or its structurally conserved paralogue, doublecortin-like kinase 1 (Dclk1), show impaired Kif1a-mediated transport of Vamp2, a cargo of Kif1a, with decreased run length. Human disease-associated mutations in Dcx's linker sequence (e.g., W146C, K174E) alter Kif1a/Vamp2 transport by disrupting Dcx/Kif1a interactions without affecting Dcx MT binding. Dcx specifically enhances binding of the ADP-bound Kif1a motor domain to MTs. Cryo-electron microscopy and subnanometer-resolution image reconstruction reveal the kinesin-dependent conformational variability of MT-bound Dcx and suggest a model for MAP-motor crosstalk on MTs. Alteration of kinesin run length by MAPs represents a previously undiscovered mode of control of kinesin transport and provides a mechanism for regulation of MT-based transport by local signals.

Rho family GTPases, Rac and Cdc42, control the localization of neonatal dentate granule cells during brain development.

The hippocampus is generally considered as a brain center for learning and memory. We have recently established an electroporation-mediated gene transfer method to investigate the development of neonatal dentate granule cells in vivo. Using this new technique, we introduced knockdown vectors against Rac1 small GTPase into precursors for dentate granule cells at postnatal day 0. After 21 days, Rac1-deficient cells were frequently mispositioned between the granule cell layer (GCL) and hilus. About 60% of these mislocalized cells expressed a dentate granule cell marker, Prox1. Both the dendritic spine density and the ratio of mature spine were reduced when Rac1 was silenced. Notably, the deficient cells have immature thin processes during migrating in the early neonatal period. Knockdown of another Rac isoform, Rac3, also resulted in mislocalization of neonatally born dentate granule cells. In addition, knockdown of Cdc42, another Rho family protein, also caused mislocalization of the cell, although the effects were moderate compared to Rac1 and 3. Despite the ectopic localization, Rac3- or Cdc42-disrupted mispositioned cells expressed Prox1. These results indicate that Rho signaling pathways differentially regulate the proper localization and differentiation of dentate granule cells.

Intermedin Enlarges the Vascular Lumen by Inducing the Quiescent Endothelial Cell Proliferation.

OBJECTIVE: Intermedin plays an important role in vascular remodeling and significantly improves blood perfusion, but the precise mechanism remains unclear. Herein, we aimed to define whether vascular lumen enlargement is responsible for the intermedin-increased blood perfusion and explore the underlying cellular and molecular mechanisms. APPROACH AND RESULTS: To study the role of intermedin, we generated the IMD-KO (Adm2-/-) mice using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) system. Intermedin significantly promoted vascular lumen enlargement in vitro (fibrin beads assay) and in vivo (murine retinas), which contributed to the improved blood perfusion in both physiological (retinal) and pathological (tumor) angiogenic models. We designed experiments to calculate the endothelial cell (EC) size and found that the lumen enlargement is because of EC proliferation but not because of a change in cell shape. ECs that construct vessel walls are considered quiescent cells because they are in a state of contact inhibition and show reduced responsiveness to VEGF (vascular endothelial growth factor). Using immunoprecipitation, Western blot assay, and fluorescent microscopy, we found that intermedin induced the formation of a signaling complex containing CRLR (calcitonin receptor-like receptor)/ß-arr1 (ß-arrestin1)/Src in ECs and promoted it internalizing into cytoplasm in a clathrin-dependent manner to activate downstream ERK1/2 (extracellular signal-regulated kinase 1/2). Importantly, this effect was not abrogated by cell-cell contacts of ECs. Through this mechanism, intermedin could reactivate the quiescent ECs to proliferate, resulting in continuous lumen expanding and a more effective blood perfusion. CONCLUSIONS: Our findings suggest a novel mechanism that may explain how quiescent ECs overcome the contact inhibition and regain the ability to proliferate for continuous vascular lumen enlargement.

Doublecortin marks a new population of transiently amplifying muscle progenitor cells and is required for myofiber maturation during skeletal muscle regeneration.

Muscle satellite cells are indispensable for muscle regeneration, but the functional diversity of their daughter cells is unknown. Here, we show that many Pax7(+)MyoD(-) cells locate both beneath and outside the basal lamina during myofiber maturation. A large majority of these Pax7(+)MyoD(-) cells are not self-renewed satellite cells, but have different potentials for both proliferation and differentiation from Pax7(+)MyoD(+) myoblasts (classical daughter cells), and are specifically marked by expression of the doublecortin (Dcx) gene. Transplantation and lineage-tracing experiments demonstrated that Dcx-expressing cells originate from quiescent satellite cells and that the microenvironment induces Dcx in myoblasts. Expression of Dcx seems to be necessary for myofiber maturation because Dcx-deficient mice exhibited impaired myofiber maturation resulting from a decrease in the number of myonuclei. Furthermore, in vitro and in vivo studies suggest that one function of Dcx in myogenic cells is acceleration of cell motility. These results indicate that Dcx is a new marker for the Pax7(+)MyoD(-) subpopulation, which contributes to myofiber maturation during muscle regeneration.

The serine protease inhibitor neuroserpin is required for normal synaptic plasticity and regulates learning and social behavior.

The serine protease inhibitor neuroserpin regulates the activity of tissue-type plasminogen activator (tPA) in the nervous system. Neuroserpin expression is particularly prominent at late stages of neuronal development in most regions of the central nervous system (CNS), whereas it is restricted to regions related to learning and memory in the adult brain. The physiological expression pattern of neuroserpin, its high degree of colocalization with tPA within the CNS, together with its dysregulation in neuropsychiatric disorders, suggest a role in formation and refinement of synapses. In fact, studies in cell culture and mice point to a role for neuroserpin in dendritic branching, spine morphology, and modulation of behavior. In this study, we investigated the physiological role of neuroserpin in the regulation of synaptic density, synaptic plasticity, and behavior in neuroserpin-deficient mice. In the absence of neuroserpin, mice show a significant decrease in spine-synapse density in the CA1 region of the hippocampus, while expression of the key postsynaptic scaffold protein PSD-95 is increased in this region. Neuroserpin-deficient mice show decreased synaptic potentiation, as indicated by reduced long-term potentiation (LTP), whereas presynaptic paired-pulse facilitation (PPF) is unaffected. Consistent with altered synaptic plasticity, neuroserpin-deficient mice exhibit cognitive and sociability deficits in behavioral assays. However, although synaptic dysfunction is implicated in neuropsychiatric disorders, we do not detect alterations in expression of neuroserpin in fusiform gyrus of autism patients or in dorsolateral prefrontal cortex of schizophrenia patients. Our results identify neuroserpin as a modulator of synaptic plasticity, and point to a role for neuroserpin in learning and memory.

Cell death regulates muscle fiber number.

Cell death can have both cell autonomous and non-autonomous roles in normal development. Previous studies have shown that the central cell death regulators grim and reaper are required for the developmentally important elimination of stem cells and neurons in the developing central nervous system (CNS). Here we show that cell death in the nervous system is also required for normal muscle development. In the absence of grim and reaper, there is an increase in the number of fibers in the ventral abdominal muscles in the Drosophila adult. This phenotype can be partially recapitulated by inhibition of cell death specifically in the CNS, indicating a non-autonomous role for neuronal death in limiting muscle fiber number. We also show that FGFs produced in the cell death defective nervous system are required for the increase in muscle fiber number. Cell death in the muscle lineage during pupal stages also plays a role in specifying fiber number. Our work suggests that FGFs from the CNS act as a survival signal for muscle founder cells. Thus, proper muscle fiber specification requires cell death in both the nervous system and in the developing muscle itself.

The Actin-Binding Protein Drebrin Inhibits Neointimal Hyperplasia.

OBJECTIVE: Vascular smooth muscle cell (SMC) migration is regulated by cytoskeletal remodeling as well as by certain transient receptor potential (TRP) channels, nonselective cation channels that modulate calcium influx. Proper function of multiple subfamily C TRP (TRPC) channels requires the scaffolding protein Homer 1, which associates with the actin-binding protein Drebrin. We found that SMC Drebrin expression is upregulated in atherosclerosis and in response to injury and investigated whether Drebrin inhibits SMC activation, either through regulation of TRP channel function via Homer or through a direct effect on the actin cytoskeleton. APPROACH AND RESULTS: Wild-type (WT) and congenic Dbn(-/+) mice were subjected to wire-mediated carotid endothelial denudation. Subsequent neointimal hyperplasia was 2.4±0.3-fold greater in Dbn(-/+) than in WT mice. Levels of globular actin were equivalent in Dbn(-/+) and WT SMCs, but there was a 2.4±0.5-fold decrease in filamentous actin in Dbn(-/+) SMCs compared with WT. Filamentous actin was restored to WT levels in Dbn(-/+) SMCs by adenoviral-mediated rescue expression of Drebrin. Compared with WT SMCs, Dbn(-/+) SMCs exhibited increased TRP channel activity in response to platelet-derived growth factor, increased migration assessed in Boyden chambers, and increased proliferation. Enhanced TRP channel activity and migration in Dbn(-/+) SMCs were normalized to WT levels by rescue expression of not only WT Drebrin but also a mutant Drebrin isoform that binds actin but fails to bind Homer. CONCLUSIONS: Drebrin reduces SMC activation through its interaction with the actin cytoskeleton but independently of its interaction with Homer scaffolds.

Repression of Mammalian Target of Rapamycin Complex 1 Inhibits Intestinal Regeneration in Acute Inflammatory Bowel Disease Models.

The mammalian target of rapamycin (mTOR) signaling pathway integrates environmental cues to regulate cell growth and survival through various mechanisms. However, how mTORC1 responds to acute inflammatory signals to regulate bowel regeneration is still obscure. In this study, we investigated the role of mTORC1 in acute inflammatory bowel disease. Inhibition of mTORC1 activity by rapamycin treatment or haploinsufficiency of Rheb through genetic modification in mice impaired intestinal cell proliferation and induced cell apoptosis, leading to high mortality in dextran sodium sulfate- and 2,4,6-trinitrobenzene sulfonic acid-induced colitis models. Through bone marrow transplantation, we found that mTORC1 in nonhematopoietic cells played a major role in protecting mice from colitis. Reactivation of mTORC1 activity by amino acids had a positive therapeutic effect in mTORC1-deficient Rheb(+/-) mice. Mechanistically, mTORC1 mediated IL-6-induced Stat3 activation in intestinal epithelial cells to stimulate the expression of downstream targets essential for cell proliferation and tissue regeneration. Therefore, mTORC1 signaling critically protects against inflammatory bowel disease through modulation of inflammation-induced Stat3 activity. As mTORC1 is an important therapeutic target for multiple diseases, our findings will have important implications for the clinical usage of mTORC1 inhibitors in patients with acute inflammatory bowel disease.

Rheb1-mTORC1 maintains macrophage differentiation and phagocytosis in mice.

Ras homolog enriched in brain (Rheb1) is a small GTPase and is known to be a direct activator of mTORC1. Dysregulation of Rheb1 has been shown to impair the cellular-energetic state and cell homeostasis. However, the role of Rheb1 in monocytes/macrophages differentiation and maturation is not clear. Here, we investigate the role of Rheb1 in mouse myelopoiesis using a Rheb1 conditional deletion murine model. We found that the absolute number of macrophages decreased in the bone marrow (BM) of Rheb1-deficient mice. Loss of Rheb1 inhibited the monocyte-to-macrophage differentiation process. Additionally, Rheb1 deletion reduced phagocytosis ability of macrophages by inhibiting the mTORC1 signaling pathway. Furthermore, 3BDO (an activator of mTORC1) rescued the phagocytosis ability of Rheb1-deficient macrophages. Thus, Rheb1 is critical for macrophage production and phagocytosis and executes these activities possibly via mTORC1-dependent pathway.

Unmasking Proteolytic Activity for Adult Visual Cortex Plasticity by the Removal of Lynx1.

Experience-dependent cortical plasticity declines with age. At the molecular level, experience-dependent proteolytic activity of tissue plasminogen activator (tPA) becomes restricted in the adult brain if mice are raised in standard cages. Understanding the mechanism for the loss of permissive proteolytic activity is therefore a key link for improving function in adult brains. Using the mouse primary visual cortex (V1) as a model, we demonstrate that tPA activity in V1 can be unmasked following 4 d of monocular deprivation when the mice older than 2 months are raised in standard cages by the genetic removal of Lynx1, a negative regulator of adult plasticity. This was also associated with the reduction of stubby and thin spine density and enhancement of ocular dominance shift in adult V1 of Lynx1 knock-out (KO) mice. These structural and functional changes were tPA-dependent because genetic removal of tPA in Lynx1 KO mice can block the monocular deprivation-dependent reduction of dendritic spine density, whereas both genetic and adult specific inhibition of tPA activity can ablate the ocular dominance shift in Lynx1 KO mice. Our work demonstrates that the adult brain has an intrinsic potential for experience-dependent elevation of proteolytic activity to express juvenile-like structural and functional changes but is effectively limited by Lynx1 if mice are raised in standard cages. Insights into the Lynx1-tPA plasticity mechanism may provide novel therapeutic targets for adult brain disorders. SIGNIFICANCE STATEMENT: Experience-dependent proteolytic activity of tissue plasminogen activator (tPA) becomes restricted in the adult brain in correlation with the decline in cortical plasticity when mice are raised in standard cages. We demonstrated that removal of Lynx1, one of negative regulators of plasticity, unmasks experience-dependent tPA elevation in visual cortex of adult mice reared in standard cages. This proteolytic elevation facilitated dendritic spine reduction and ocular dominance plasticity in adult visual cortex. This is the first demonstration of adult brain to retain the intrinsic capacity to elevate tPA in an experience-dependent manner but is effectively limited by Lynx1. tPA-Lynx1 may potentially be a new candidate mechanism for interventions that were shown to activate plasticity in adult brain.

SERPINI1 regulates epithelial-mesenchymal transition in an orthotopic implantation model of colorectal cancer.

An increasingly accepted concept is that the progression of colorectal cancer is accompanied by epithelial-mesenchymal transition (EMT). In our study, in order to characterize the properties of EMT in 16 colorectal cancer cell lines, the cells were first orthotopically implanted into nude mice, and the tumors in vivo, as well as cells cultured in vitro, were immunostained for EMT markers. The immunostaining revealed that seven of the cells had an epithelial phenotype with a high expression of E-cadherin, whereas other cells showed opposite patterns, such as a high expression of vimentin (CX-1, COLO205, CloneA, HCT116, and SW48). Among the cells expressing vimentin, some expressed vimentin in the orthotopic tumors but not in the cultured cells (SW480, SW620, and COLO320). We evaluated these findings in combination with microarray analyses, and selected five genes: CHST11, SERPINI1, AGR2, FBP1, and FOXA1. Next, we downregulated the expression of SERPINI1 with siRNA in the cells, the results of which showed reverse-EMT changes at the protein level and in the cellular morphology. Along with immunohistochemical analyses, we confirmed the effect of the intracellular and secreted SERPINI1 protein of SW620 cells, which supported the importance of SERPINI1 in EMT. The development of therapeutic strategies targeting EMT is ongoing, including methods targeting the transforming growth factor-ß signaling pathway as well as the Wnt pathway. SERPINI1 is an important regulator of EMT. Our findings help to elucidate the signaling pathways of EMT, hopefully clarifying therapeutic pathways as well.

Drebrin E depletion in human intestinal epithelial cells mimics Rab8a loss of function.

Intestinal epithelial cells are highly polarized and exhibit a complex architecture with a columnar shape and a specialized apical surface supporting microvilli organized in a brush border. These microvilli are rooted in a dense meshwork of acto-myosin called the terminal web. We have shown recently that Drebrin E, an F-actin-binding protein, is a key protein for the organization of the terminal web and the brush border. Drebrin E is also required for the columnar cell shape of Caco2 cells (human colonic cells). Here, we found that the subcellular localization of several apical markers including dipeptidyl peptidase IV (DPPIV) was strikingly modified in Drebrin E-depleted Caco2 cells. Instead of being mostly present at the apical surface, these proteins are accumulated in an enlarged subapical compartment. Using known intracellular markers, we show by both confocal and electron microscopy that this compartment is related to lysosomes. We also demonstrate that the enrichment of DPPIV in this compartment originates from apical endocytosis and that depletion of Rab8a induces an accumulation of apical proteins in a similar compartment. Consistent with this, the phenotype observed in Drebrin E knock-down Caco2 cells shares some features with a pathology called microvillar inclusion disease (MVID) involving both Myosin Vb and Rab8a. Taken together, these results suggest that Drebrin E redirects the apical recycling pathway in intestinal epithelial cells to the lysosomes, demonstrating that Drebrin E is a key regulator in apical trafficking in Caco2 cells.

Changes in dermal matrix in the absence of Rac1 in keratinocytes.

Keratinocytes, in response to irritants, secrete pro-inflammatory mediators which recruit and activate immune and mesenchymal cells, including fibroblasts, to repair the skin. Fibroblasts respond by synthesising collagen and promoting the crosslinking extracellular matrix (ECM). We recently showed that the deletion of Rac1 in keratinocytes causes heightened inflammation due to aberrant crosstalk with immune cells. Indeed, the skin of these mice shows a higher inflammatory response to the induction of irritant contact dermatitis (ICD), and also even to treatment with a vehicle alone, compared with controls. As inflammation is intimately linked with fibrotic disease in the skin, this raised the question as to whether this deletion may also affect the deposition and arrangement of the dermal ECM. This study assessed the effects of Rac1 deletion in keratinocytes and of the heightened inflammatory status by induction of ICD on the tissue localisation and arrangements of dermal collagen. Qualitative analysis did not reveal evidence for the formation of pathologies in the dermis. However, quantitative analysis did reveal some perturbations in the dermal matrix, namely that only the combination of the lack of Rac1 and ICD affects the architectural organisation of the dermal collagen, and that a higher inflammatory state in the tissue (i.e. when Rac1 is deleted in the keratinocytes or ICD is induced in the skin, or a combination of both) influences the diameter of the collagen fibrils. It is proposed that this increase in the diameter of collagen fibrils due to inflammation may serve as pre-fibrotic marker enabling earlier determination of fibrosis and earlier treatment. This study has revealed previously unknown effects on the ECM due to the deletion of Rac1 in keratinocytes.