Publications by authors named "Dritan Agalliu"

35 Publications

Pluripotent stem cell-derived epithelium misidentified as brain microvascular endothelium requires ETS factors to acquire vascular fate.

Proc Natl Acad Sci U S A 2021 Feb;118(8)

Ansary Stem Cell Institute, Division of Regenerative Medicine, Department of Medicine, Weill Cornell Medicine, New York, NY 10065;

Cells derived from pluripotent sources in vitro must resemble those found in vivo as closely as possible at both transcriptional and functional levels in order to be a useful tool for studying diseases and developing therapeutics. Recently, differentiation of human pluripotent stem cells (hPSCs) into brain microvascular endothelial cells (ECs) with blood-brain barrier (BBB)-like properties has been reported. These cells have since been used as a robust in vitro BBB model for drug delivery and mechanistic understanding of neurological diseases. However, the precise cellular identity of these induced brain microvascular endothelial cells (iBMECs) has not been well described. Employing a comprehensive transcriptomic metaanalysis of previously published hPSC-derived cells validated by physiological assays, we demonstrate that iBMECs lack functional attributes of ECs since they are deficient in vascular lineage genes while expressing clusters of genes related to the neuroectodermal epithelial lineage (Epi-iBMEC). Overexpression of key endothelial ETS transcription factors (, , and ) reprograms Epi-iBMECs into authentic endothelial cells that are congruent with bona fide endothelium at both transcriptomic as well as some functional levels. This approach could eventually be used to develop a robust human BBB model in vitro that resembles the human brain EC in vivo for functional studies and drug discovery.
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http://dx.doi.org/10.1073/pnas.2016950118DOI Listing
February 2021

Activation of endothelial Wnt/β-catenin signaling by protective astrocytes repairs BBB damage in ischemic stroke.

Prog Neurobiol 2021 Apr 26;199:101963. Epub 2020 Nov 26.

Department of Neurology, University of Pittsburgh, Pittsburgh, PA, USA; The Pittsburgh Institute for Neurodegenerative Diseases, University of Pittsburgh, Pittsburgh, PA, USA. Electronic address:

The role of astrocytes in dysregulation of blood-brain barrier (BBB) function following ischemic stroke is not well understood. Here, we investigate the effects of restoring the repair properties of astrocytes on the BBB after ischemic stroke. Mice deficient for NHE1, a pH-sensitive Na/H exchanger 1, in astrocytes have reduced BBB permeability after ischemic stroke, increased angiogenesis and cerebral blood flow perfusion, in contrast to wild-type mice. Bulk RNA-sequencing transcriptome analysis of purified astrocytes revealed that ∼177 genes were differentially upregulated in mutant astrocytes, with Wnt7a mRNA among the top genes. Using a Wnt reporter line, we confirmed that the pathway was upregulated in cerebral vessels of mutant mice after ischemic stroke. However, administration of the Wnt/β-catenin inhibitor, XAV-939, blocked the reparative effects of Nhe1-deficient astrocytes. Thus, astrocytes lacking pH-sensitive NHE1 protein are transformed from injurious to "protective" by inducing Wnt production to promote BBB repair after ischemic stroke.
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http://dx.doi.org/10.1016/j.pneurobio.2020.101963DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7925353PMC
April 2021

Fgfbp1 promotes blood-brain barrier development by regulating collagen IV deposition and maintaining Wnt/β-catenin signaling.

Development 2020 Aug 24;147(16). Epub 2020 Aug 24.

FIRC Institute of Molecular Oncology Foundation (IFOM), 20139 Milan, Italy

Central nervous system (CNS) blood vessels contain a functional blood-brain barrier (BBB) that is necessary for neuronal survival and activity. Although Wnt/β-catenin signaling is essential for BBB development, its downstream targets within the neurovasculature remain poorly understood. To identify targets of Wnt/β-catenin signaling underlying BBB maturation, we performed a microarray analysis that identified Fgfbp1 as a novel Wnt/β-catenin-regulated gene in mouse brain endothelial cells (mBECs). Fgfbp1 is expressed in the CNS endothelium and secreted into the vascular basement membrane during BBB formation. Endothelial genetic ablation of results in transient hypervascularization but delays BBB maturation in specific CNS regions, as evidenced by both upregulation of Plvap and increased tracer leakage across the neurovasculature due to reduced Wnt/β-catenin activity. In addition, collagen IV deposition in the vascular basement membrane is reduced in mutant mice, leading to defective endothelial cell-pericyte interactions. Fgfbp1 is required cell-autonomously in mBECs to concentrate Wnt ligands near cell junctions and promote maturation of their barrier properties Thus, Fgfbp1 is a crucial extracellular matrix protein during BBB maturation that regulates cell-cell interactions and Wnt/β-catenin activity.
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http://dx.doi.org/10.1242/dev.185140DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7473640PMC
August 2020

miR-151a enhances Slug dependent angiogenesis.

Oncotarget 2020 Jun 9;11(23):2160-2171. Epub 2020 Jun 9.

Department of Molecular Biology and Biochemistry, School of Biological Sciences, University of California, Irvine, CA 92697, USA.

MicroRNAs (miRs) are small non-coding RNAs, that modulate cognate gene expression either by inducing mRNA degradation or by blocking translation, and play crucial and complex roles in tissue homeostasis and during disease initiation and progression. The sprouting of new blood vessels by angiogenesis is critical in vascular development and homeostasis and aberrant angiogenesis is associated with pathological conditions such as ischemia and cancer. We have previously established that miR-151a functions as an onco-miR in non-small cell lung cancer (NSCLC) cells by inducing partial EMT and enhancing tumor growth. Here, we identify anti-miR-151a as a molecule that promotes endothelial cell contacts and barrier properties, suggesting that miR-151a regulates cell-cell junctions. We find that induced miR-151a expression enhances endothelial cell motility and angiogenesis and these functions depend on miR-151a-induced Slug levels. Moreover, we show that miR-151a overexpression enhances tumor-associated angiogenesis in 3D vascularized tumor spheroid assays. Finally, we verify that miR-151a is expressed in the vasculature of normal lung and NSCLC tissue. Our results suggest that miR-151a plays multi-faceted roles in the lung, by regulating multiple functions (cell growth, motility, partial EMT and angiogenesis) in distinct cell types.
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http://dx.doi.org/10.18632/oncotarget.27331DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7289531PMC
June 2020

Neuronal and glial regulation of CNS angiogenesis and barriergenesis.

Development 2020 05 1;147(9). Epub 2020 May 1.

Departments of Neurology, Columbia University Irving Medical Center, New York, NY 10032, USA

Neurovascular pathologies of the central nervous system (CNS), which are associated with barrier dysfunction, are leading causes of death and disability. The roles that neuronal and glial progenitors and mature cells play in CNS angiogenesis and neurovascular barrier maturation have been elucidated in recent years. Yet how neuronal activity influences these processes remains largely unexplored. Here, we discuss our current understanding of how neuronal and glial development affects CNS angiogenesis and barriergenesis, and outline future directions to elucidate how neuronal activity might influence these processes. An understanding of these mechanisms is crucial for developing new interventions to treat neurovascular pathologies.
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http://dx.doi.org/10.1242/dev.182279DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7197727PMC
May 2020

Th17 lymphocytes drive vascular and neuronal deficits in a mouse model of postinfectious autoimmune encephalitis.

Proc Natl Acad Sci U S A 2020 03 11;117(12):6708-6716. Epub 2020 Mar 11.

Department of Neurology, Columbia University Irving Medical Center, New York, NY 10032;

Antibodies against neuronal receptors and synaptic proteins are associated with a group of ill-defined central nervous system (CNS) autoimmune diseases termed autoimmune encephalitides (AE), which are characterized by abrupt onset of seizures and/or movement and psychiatric symptoms. Basal ganglia encephalitis (BGE), representing a subset of AE syndromes, is triggered in children by repeated group A (GAS) infections that lead to neuropsychiatric symptoms. We have previously shown that multiple GAS infections of mice induce migration of Th17 lymphocytes from the nose into the brain, causing blood-brain barrier (BBB) breakdown, extravasation of autoantibodies into the CNS, and loss of excitatory synapses within the olfactory bulb (OB). Whether these pathologies induce functional olfactory deficits, and the mechanistic role of Th17 lymphocytes, is unknown. Here, we demonstrate that, whereas loss of excitatory synapses in the OB is transient after multiple GAS infections, functional deficits in odor processing persist. Moreover, mice lacking Th17 lymphocytes have reduced BBB leakage, microglial activation, and antibody infiltration into the CNS, and have their olfactory function partially restored. Th17 lymphocytes are therefore critical for selective CNS entry of autoantibodies, microglial activation, and neural circuit impairment during postinfectious BGE.
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http://dx.doi.org/10.1073/pnas.1911097117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7104239PMC
March 2020

Prophylactic TLR9 stimulation reduces brain metastasis through microglia activation.

PLoS Biol 2019 03 28;17(3):e2006859. Epub 2019 Mar 28.

Neurobiology Department, Tel Aviv University, Tel Aviv, Israel.

Brain metastases are prevalent in various types of cancer and are often terminal, given the low efficacy of available therapies. Therefore, preventing them is of utmost clinical relevance, and prophylactic treatments are perhaps the most efficient strategy. Here, we show that systemic prophylactic administration of a toll-like receptor (TLR) 9 agonist, CpG-C, is effective against brain metastases. Acute and chronic systemic administration of CpG-C reduced tumor cell seeding and growth in the brain in three tumor models in mice, including metastasis of human and mouse lung cancer, and spontaneous melanoma-derived brain metastasis. Studying mechanisms underlying the therapeutic effects of CpG-C, we found that in the brain, unlike in the periphery, natural killer (NK) cells and monocytes are not involved in controlling metastasis. Next, we demonstrated that the systemically administered CpG-C is taken up by endothelial cells, astrocytes, and microglia, without affecting blood-brain barrier (BBB) integrity and tumor brain extravasation. In vitro assays pointed to microglia, but not astrocytes, as mediators of CpG- C effects through increased tumor killing and phagocytosis, mediated by direct microglia-tumor contact. In vivo, CpG-C-activated microglia displayed elevated mRNA expression levels of apoptosis-inducing and phagocytosis-related genes. Intravital imaging showed that CpG-C-activated microglia cells contact, kill, and phagocytize tumor cells in the early stages of tumor brain invasion more than nonactivated microglia. Blocking in vivo activation of microglia with minocycline, and depletion of microglia with a colony-stimulating factor 1 inhibitor, indicated that microglia mediate the antitumor effects of CpG-C. Overall, the results suggest prophylactic CpG-C treatment as a new intervention against brain metastasis, through an essential activation of microglia.
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http://dx.doi.org/10.1371/journal.pbio.2006859DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6469801PMC
March 2019

Autoimmunity in Parkinson's Disease: The Role of α-Synuclein-Specific T Cells.

Front Immunol 2019 25;10:303. Epub 2019 Feb 25.

Department of Neurology, Columbia University Irving Medical Center, New York, NY, United States.

Evidence from a variety of studies implicates a role for the adaptive immune system in Parkinson's disease (PD). Similar to multiple sclerosis (MS) patients who display a high number of T cells in the brain attacking oligodendrocytes, PD patients show higher numbers of T cells in the ventral midbrain than healthy, age-matched controls. Mouse models of the disease also show the presence of T cells in the brain. The role of these infiltrating T cells in the propagation of disease is controversial; however, recent studies indicate that they may be autoreactive in nature, recognizing disease-altered self-proteins as foreign antigens. T cells of PD patients can generate an autoimmune response to α-synuclein, a protein that is aggregated in PD. α-Synuclein and other proteins are post-translationally modified in an environment in which protein processing is altered, possibly leading to the generation of neo-epitopes, or self-peptides that have not been identified by the host immune system as non-foreign. Infiltrating T cells may also be responding to such modified proteins. Genome-wide association studies (GWAS) have shown associations of PD with haplotypes of major histocompatibility complex (MHC) class II genes, and a polymorphism in a non-coding region that may increase MHC class II in PD patients. We speculate that the inflammation observed in PD may play both pathogenic and protective roles. Future studies on the adaptive immune system in neurodegenerative disorders may elucidate steps in disease pathogenesis and assist with the development of both biomarkers and treatments.
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http://dx.doi.org/10.3389/fimmu.2019.00303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6397885PMC
September 2020

A Unique Collateral Artery Development Program Promotes Neonatal Heart Regeneration.

Cell 2019 02 24;176(5):1128-1142.e18. Epub 2019 Jan 24.

Department of Biology, Stanford University, Stanford, CA 94305, USA; Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA. Electronic address:

Collateral arteries are an uncommon vessel subtype that can provide alternate blood flow to preserve tissue following vascular occlusion. Some patients with heart disease develop collateral coronary arteries, and this correlates with increased survival. However, it is not known how these collaterals develop or how to stimulate them. We demonstrate that neonatal mouse hearts use a novel mechanism to build collateral arteries in response to injury. Arterial endothelial cells (ECs) migrated away from arteries along existing capillaries and reassembled into collateral arteries, which we termed "artery reassembly". Artery ECs expressed CXCR4, and following injury, capillary ECs induced its ligand, CXCL12. CXCL12 or CXCR4 deletion impaired collateral artery formation and neonatal heart regeneration. Artery reassembly was nearly absent in adults but was induced by exogenous CXCL12. Thus, understanding neonatal regenerative mechanisms can identify pathways that restore these processes in adults and identify potentially translatable therapeutic strategies for ischemic heart disease.
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http://dx.doi.org/10.1016/j.cell.2018.12.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6435282PMC
February 2019

The Impact of Pregnancy on Hemorrhagic Stroke in Young Women.

Cerebrovasc Dis 2018 6;46(1-2):10-15. Epub 2018 Jul 6.

Department of Neurology, New York, New York, USA.

Background: Pregnancy is a sex-specific risk factor for causing hemorrhagic stroke (HS) in young adults. Unique physiological characteristics during pregnancy may alter the relative risk for HS in pregnant/postpartum (PP) women compared to HS in other young women. We compared patient characteristics and HS subtypes between young non-pregnant and PP women.

Methods: We reviewed the medical records of all women 18-45 years old admitted to our center with HS from October 15, 2008 through March 31, 2015, and compared patient characteristics and stroke mechanisms using logistic regression.

Results: Of the 130 young women with HS during the study period, 111 were non-PP women, and 19 PP women. PP women had lower proportions of vascular risk factors such as hypertension, prior stroke, and smoking, and a higher proportion of migraine (36.8 vs. 14.4%, p = 0.01). After adjusting for hypertension, smoking, migraine, prior stroke and prior myocardial infarction, PP women had lower odds of having an underlying vascular lesion (OR 0.14, 95% CI 0.04-0.44, p = 0.0009) and a higher proportion of the reversible cerebral vasoconstriction syndrome (RCVS) as cause of their HS.

Conclusions: Women with pregnancy-associated HS had fewer cerebrovascular risk factors, lower odds of having -underlying vascular lesions, and higher proportion of -migraine and RCVS compared with similar-aged non--pregnant women. Pregnancy-associated HS appears to represent a unique pathophysiological process, requiring targeted study.
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http://dx.doi.org/10.1159/000490803DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6158089PMC
June 2019

Functional morphology of the blood-brain barrier in health and disease.

Acta Neuropathol 2018 03 6;135(3):311-336. Epub 2018 Feb 6.

Department of Medicine, Section of General Pathology, University of Verona, Verona, Italy.

The adult quiescent blood-brain barrier (BBB), a structure organised by endothelial cells through interactions with pericytes, astrocytes, neurons and microglia in the neurovascular unit, is highly regulated but fragile at the same time. In the past decade, there has been considerable progress in understanding not only the molecular pathways involved in BBB development, but also BBB breakdown in neurological diseases. Specifically, the Wnt/β-catenin, retinoic acid and sonic hedgehog pathways moved into the focus of BBB research. Moreover, angiopoietin/Tie2 signalling that is linked to angiogenic processes has gained attention in the BBB field. Blood vessels play an essential role in initiation and progression of many diseases, including inflammation outside the central nervous system (CNS). Therefore, the potential influence of CNS blood vessels in neurological diseases associated with BBB alterations or neuroinflammation has become a major focus of current research to understand their contribution to pathogenesis. Moreover, the BBB remains a major obstacle to pharmaceutical intervention in the CNS. The complications may either be expressed by inadequate therapeutic delivery like in brain tumours, or by poor delivery of the drug across the BBB and ineffective bioavailability. In this review, we initially describe the cellular and molecular components that contribute to the steady state of the healthy BBB. We then discuss BBB alterations in ischaemic stroke, primary and metastatic brain tumour, chronic inflammation and Alzheimer's disease. Throughout the review, we highlight common mechanisms of BBB abnormalities among these diseases, in particular the contribution of neuroinflammation to BBB dysfunction and disease progression, and emphasise unique aspects of BBB alteration in certain diseases such as brain tumours. Moreover, this review highlights novel strategies to monitor BBB function by non-invasive imaging techniques focussing on ischaemic stroke, as well as novel ways to modulate BBB permeability and function to promote treatment of brain tumours, inflammation and Alzheimer's disease. In conclusion, a deep understanding of signals that maintain the healthy BBB and promote fluctuations in BBB permeability in disease states will be key to elucidate disease mechanisms and to identify potential targets for diagnostics and therapeutic modulation of the BBB.
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http://dx.doi.org/10.1007/s00401-018-1815-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6781630PMC
March 2018

Caveolin1 Is Required for Th1 Cell Infiltration, but Not Tight Junction Remodeling, at the Blood-Brain Barrier in Autoimmune Neuroinflammation.

Cell Rep 2017 Nov;21(8):2104-2117

Department of Neurology, Columbia University Medical Center, New York, NY 10032, USA; Department of Pathology & Cell Biology, Columbia University Medical Center, New York, NY 10032, USA; Department of Pharmacology, Columbia University Medical Center, New York, NY 10032, USA; Columbia Translational Neuroscience Initiative, Columbia University Medical Center, New York, NY 10032, USA. Electronic address:

Lymphocytes cross vascular boundaries via either disrupted tight junctions (TJs) or caveolae to induce tissue inflammation. In the CNS, Th17 lymphocytes cross the blood-brain barrier (BBB) before Th1 cells; yet this differential crossing is poorly understood. We have used intravital two-photon imaging of the spinal cord in wild-type and caveolae-deficient mice with fluorescently labeled endothelial tight junctions to determine how tight junction remodeling and caveolae regulate CNS entry of lymphocytes during the experimental autoimmune encephalomyelitis (EAE) model for multiple sclerosis. We find that dynamic tight junction remodeling occurs early in EAE but does not depend upon caveolar transport. Moreover, Th1, but not Th17, lymphocytes are significantly reduced in the inflamed CNS of mice lacking caveolae. Therefore, tight junction remodeling facilitates Th17 migration across the BBB, whereas caveolae promote Th1 entry into the CNS. Moreover, therapies that target both tight junction degradation and caveolar transcytosis may limit lymphocyte infiltration during inflammation.
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http://dx.doi.org/10.1016/j.celrep.2017.10.094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5728697PMC
November 2017

The Wnt Inhibitor Apcdd1 Coordinates Vascular Remodeling and Barrier Maturation of Retinal Blood Vessels.

Neuron 2017 Dec 16;96(5):1055-1069.e6. Epub 2017 Nov 16.

Department of Neurology, Columbia University Medical Center, New York, NY 10032, USA; Department of Pathology & Cell Biology, Columbia University Medical Center, New York, NY 10032, USA; Department of Pharmacology, Columbia University Medical Center, New York, NY 10032, USA; Columbia Translational Neuroscience Initiative, Columbia University Medical Center, New York, NY 10032, USA. Electronic address:

Coordinating angiogenesis with acquisition of tissue-specific properties in endothelial cells is essential for vascular function. In the retina, endothelial cells form a blood-retina barrier by virtue of tight junctions and low transcytosis. While the canonical Norrin/Fz4/Lrp5/6 pathway is essential for angiogenesis, vascular remodeling, and barrier maturation, how these diverse processes are coordinated remains poorly understood. Here we demonstrate that Apcdd1, a negative regulator of Wnt/β-catenin signaling, is expressed in retinal endothelial cells during angiogenesis and barrier formation. Apcdd1-deficient mice exhibit a transient increase in vessel density at ages P10-P12 due to delayed vessel pruning. Moreover, Apcdd1 mutant endothelial cells precociously form the paracellular component of the barrier. Conversely, mice that overexpress Apcdd1 in retina endothelial cells have reduced vessel density but increased paracellular barrier permeability. Apcdd1 thus serves to precisely modulate Wnt/Norrin signaling activity in the retinal endothelium and coordinate the timing of both vascular pruning and barrier maturation.
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http://dx.doi.org/10.1016/j.neuron.2017.10.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5728434PMC
December 2017

Huntington's Disease iPSC-Derived Brain Microvascular Endothelial Cells Reveal WNT-Mediated Angiogenic and Blood-Brain Barrier Deficits.

Cell Rep 2017 05;19(7):1365-1377

Department of Biological Chemistry, University of California, Irvine, Irvine, CA 92697, USA; UCI MIND, University of California, Irvine, Irvine, CA 92697, USA; Neurobiology and Behavior, University of California, Irvine, Irvine, CA 92697, USA; Psychiatry and Human Behavior, University of California, Irvine, Irvine, CA 92697, USA; Sue and Bill Gross Stem Cell Center, University of California, Irvine, Irvine, CA 92697, USA. Electronic address:

Brain microvascular endothelial cells (BMECs) are an essential component of the blood-brain barrier (BBB) that shields the brain against toxins and immune cells. While BBB dysfunction exists in neurological disorders, including Huntington's disease (HD), it is not known if BMECs themselves are functionally compromised to promote BBB dysfunction. Further, the underlying mechanisms of BBB dysfunction remain elusive given limitations with mouse models and post-mortem tissue to identify primary deficits. We undertook a transcriptome and functional analysis of human induced pluripotent stem cell (iPSC)-derived BMECs (iBMEC) from HD patients or unaffected controls. We demonstrate that HD iBMECs have intrinsic abnormalities in angiogenesis and barrier properties, as well as in signaling pathways governing these processes. Thus, our findings provide an iPSC-derived BBB model for a neurodegenerative disease and demonstrate autonomous neurovascular deficits that may underlie HD pathology with implications for therapeutics and drug delivery.
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http://dx.doi.org/10.1016/j.celrep.2017.04.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5646270PMC
May 2017

Hello from the Other Side: How Autoantibodies Circumvent the Blood-Brain Barrier in Autoimmune Encephalitis.

Front Immunol 2017 21;8:442. Epub 2017 Apr 21.

Department of Neurology, Columbia University Medical Center, New York, NY, USA.

Antibodies against neuronal receptors and synaptic proteins are associated with autoimmune encephalitides (AE) that produce movement and psychiatric disorders. In order to exert their pathological effects on neural circuits, autoantibodies against central nervous system (CNS) targets must gain access to the brain and spinal cord by crossing the blood-brain barrier (BBB), a tightly regulated gateway formed by endothelial cells lining CNS blood vessels. To date, the pathogenic mechanisms that underlie autoantibody-triggered encephalitic syndromes are poorly understood, and how autoantibodies breach the barrier remains obscure for almost all AE syndromes. The relative importance of cellular versus humoral immune mechanisms for disease pathogenesis also remains largely unexplored. Here, we review the proposed triggers for various autoimmune encephalopathies and their animal models, as well as basic structural features of the BBB and how they differ among various CNS regions, a feature that likely underlies some regional aspects of autoimmune encephalitis pathogenesis. We then discuss the routes that antibodies and immune cells employ to enter the CNS and their implications for AE. Finally, we explore future therapeutic strategies that may either preserve or restore barrier function and thereby limit immune cell and autoantibody infiltration into the CNS. Recent mechanistic insights into CNS autoantibody entry indicate promising future directions for therapeutic intervention beyond current, short-lived therapies that eliminate circulating autoantibodies.
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http://dx.doi.org/10.3389/fimmu.2017.00442DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5399040PMC
April 2017

Endothelial Wnt/β-catenin signaling reduces immune cell infiltration in multiple sclerosis.

Proc Natl Acad Sci U S A 2017 02 30;114(7):E1168-E1177. Epub 2017 Jan 30.

Department of Developmental and Cell Biology, University of California, Irvine, CA 92697;

Disruption of the blood-brain barrier (BBB) is a defining and early feature of multiple sclerosis (MS) that directly damages the central nervous system (CNS), promotes immune cell infiltration, and influences clinical outcomes. There is an urgent need for new therapies to protect and restore BBB function, either by strengthening endothelial tight junctions or suppressing endothelial vesicular transcytosis. Although wingless integrated MMTV (Wnt)/β-catenin signaling plays an essential role in BBB formation and maintenance in healthy CNS, its role in BBB repair in neurologic diseases such as MS remains unclear. Using a Wnt/β-catenin reporter mouse and several downstream targets, we demonstrate that the Wnt/β-catenin pathway is up-regulated in CNS endothelial cells in both human MS and the mouse model experimental autoimmune encephalomyelitis (EAE). Increased Wnt/β-catenin activity in CNS blood vessels during EAE progression correlates with up-regulation of neuronal Wnt3 expression, as well as breakdown of endothelial cell junctions. Genetic inhibition of the Wnt/β-catenin pathway in CNS endothelium before disease onset exacerbates the clinical presentation of EAE, CD4 T-cell infiltration into the CNS, and demyelination by increasing expression of vascular cell adhesion molecule-1 and the transcytosis protein Caveolin-1 and promoting endothelial transcytosis. However, Wnt signaling attenuation does not affect the progressive degradation of tight junction proteins or paracellular BBB leakage. These results suggest that reactivation of Wnt/β-catenin signaling in CNS vessels during EAE/MS partially restores functional BBB integrity and limits immune cell infiltration into the CNS.
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http://dx.doi.org/10.1073/pnas.1609905114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5320985PMC
February 2017

Appearance of claudin-5 leukocytes in the central nervous system during neuroinflammation: a novel role for endothelial-derived extracellular vesicles.

J Neuroinflammation 2016 11 16;13(1):292. Epub 2016 Nov 16.

Blood-Brain Barrier Laboratory, University of Connecticut Health Center, 263 Farmington Ave., Farmington, CT, 06070, USA.

Background: The mechanism of leukocyte transendothelial migration (TEM) across the highly restrictive blood-brain barrier (BBB) remains enigmatic, with paracellular TEM thought to require leukocytes to somehow navigate the obstructive endothelial tight junctions (TJs). Transient interactions between TJ proteins on the respective leukocyte and endothelial surfaces have been proposed as one mechanism for TEM. Given the expanding role of extracellular vesicles (EVs) in intercellular communication, we investigated whether EVs derived from brain microvascular endothelial cells (BMEC) of the BBB may play a role in transferring a major TJ protein, claudin-5 (CLN-5), to leukocytes as a possible basis for such a mechanism during neuroinflammation.

Methods: High-resolution 3D confocal imaging was used to highlight CLN-5 immunoreactivity in the central nervous system (CNS) and on leukocytes of mice with the neuroinflammatory condition experimental autoimmune encephalomyelitis (EAE). Both Western blotting of circulating leukocytes from wild-type mice and fluorescence imaging of leukocyte-associated eGFP-CLN-5 in the blood and CNS of endothelial-targeted, Tie-2-eGFP-CLN-5 transgenic mice were used to confirm the presence of CLN-5 protein on these cells. EVs were isolated from TNF-α-stimulated BMEC cultures and blood plasma of Tie-2-eGFP-CLN-5 mice with EAE and evaluated for CLN-5 protein by Western blotting and fluorescence-activated cell sorting (FACS), respectively. Confocal imaging and FACS were used to detect binding of endothelial-derived EVs from these two sources to leukocytes in vitro. Serial electron microscopy (serial EM) and 3D contour-based surface reconstruction were employed to view EV-like structures at the leukocyte:BBB interface in situ in inflamed CNS microvessels.

Results: A subpopulation of leukocytes immunoreactive for CLN-5 on their surface was seen to infiltrate the CNS of mice with EAE and reside in close apposition to inflamed vessels. Confocal imaging of immunostained samples and Western blotting established the presence of CLN-5 leukocytes in blood as well, implying these cells are present prior to TEM. Moreover, imaging of inflamed CNS vessels and the associated perivascular cell infiltrates from Tie-2-eGFP-CLN-5 mice with EAE revealed leukocytes bearing the eGFP label, further supporting the hypothesis CLN-5 is transferred from endothelial cells to circulating leukocytes in vivo. Western blotting of BMEC-derived EVs, corresponding in size to both exosomes and microvesicles, and FACS analysis of plasma-derived EVs from Tie-2-eGFP-CLN-5 mice with EAE validated expression of CLN-5 by EVs of endothelial origin. Confocal imaging and FACS further revealed both PKH-67-labeled EVs from cultured BMECs and eGFP-CLN-5 EVs from plasma of Tie-2-eGFP-CLN-5 mice with EAE can bind to leukocytes. Lastly, serial EM and 3D contour-based surface reconstruction revealed a close association of EV-like structures between the marginating leukocytes and BMECs in situ during EAE.

Conclusions: During neuroinflammation, CLN-5 leukocytes appear in the CNS, and both CLN-5 leukocytes and CLN-5 EVs are detected in the blood. As endothelial cells transfer CLN-5 to leukocytes in vivo, and EVs released from BMEC bind to leukocytes in vitro, EVs may serve as the vehicles to transfer CLN-5 protein at sites of leukocyte:endothelial contact along the BBB. This action may be a prelude to facilitate TEM through the formation of temporary TJ protein bridges between these two cell types.
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http://dx.doi.org/10.1186/s12974-016-0755-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5112695PMC
November 2016

CNS autoimmune disease after infections: animal models, cellular mechanisms and genetic factors.

Future Neurol 2016 Dec;11(1):63-76

Department of Neurology, Columbia University Medical Center, 650 West 168 Street, Room 310E, New York, NY 10032, USA; Departments of Pathology & Cell Biology & Pharmacology, Columbia University Medical Center, 650 West 168 Street, Room 310E, New York, NY 10032, USA.

infections have been associated with two autoimmune diseases of the CNS: Sydenham's chorea (SC) and Pediatric Autoimmune Neuropsychiatric Disorders Associated with infections (PANDAS). Despite the high frequency of pharyngeal streptococcus infections among children, only a small fraction develops SC or PANDAS. This suggests that several factors in combination are necessary to trigger autoimmune complications: specific strains that induce a strong immune response toward the host nervous system; genetic susceptibility that predispose children toward an autoimmune response involving movement or tic symptoms; and multiple infections of the throat or tonsils that lead to a robust T17 cellular and humoral immune response when untreated. In this review, we summarize the evidence for each factor and propose that all must be met for the requisite neurovascular pathology and behavioral deficits found in SC/PANDAS.
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http://dx.doi.org/10.2217/fnl.16.4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4839655PMC
December 2016

Neurovascular and Immuno-Imaging: From Mechanisms to Therapies. Proceedings of the Inaugural Symposium.

Front Neurosci 2016 22;10:46. Epub 2016 Feb 22.

Department of Physiology and Biophysics, Keck School of Medicine, Zilkha Neurogenetic Institute, University of Southern California Los Angeles, CA, USA.

Breakthrough advances in intravital imaging have launched a new era for the study of dynamic interactions at the neurovascular interface in health and disease. The first Neurovascular and Immuno-Imaging Symposium was held at the Gladstone Institutes, University of California, San Francisco in March, 2015. This highly interactive symposium brought together a group of leading researchers who discussed how recent studies have unraveled fundamental biological mechanisms in diverse scientific fields such as neuroscience, immunology, and vascular biology, both under physiological and pathological conditions. These Proceedings highlight how advances in imaging technologies and their applications revolutionized our understanding of the communication between brain, immune, and vascular systems and identified novel targets for therapeutic intervention in neurological diseases.
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http://dx.doi.org/10.3389/fnins.2016.00046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4761864PMC
March 2016

Group A Streptococcus intranasal infection promotes CNS infiltration by streptococcal-specific Th17 cells.

J Clin Invest 2016 Jan 14;126(1):303-17. Epub 2015 Dec 14.

Group A streptococcal (GAS) infection induces the production of Abs that cross-react with host neuronal proteins, and these anti-GAS mimetic Abs are associated with autoimmune diseases of the CNS. However, the mechanisms that allow these Abs to cross the blood-brain barrier (BBB) and induce neuropathology remain unresolved. We have previously shown that GAS infection in mouse models induces a robust Th17 response in nasal-associated lymphoid tissue (NALT). Here, we identified GAS-specific Th17 cells in tonsils of humans naturally exposed to GAS, prompting us to explore whether GAS-specific CD4+ T cells home to mouse brains following i.n. infection. Intranasal challenge of repeatedly GAS-inoculated mice promoted migration of GAS-specific Th17 cells from NALT into the brain, BBB breakdown, serum IgG deposition, microglial activation, and loss of excitatory synaptic proteins under conditions in which no viable bacteria were detected in CNS tissue. CD4+ T cells were predominantly located in the olfactory bulb (OB) and in other brain regions that receive direct input from the OB. Together, these findings provide insight into the immunopathology of neuropsychiatric complications that are associated with GAS infections and suggest that crosstalk between the CNS and cellular immunity may be a general mechanism by which infectious agents exacerbate symptoms associated with other CNS autoimmune disorders.
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http://dx.doi.org/10.1172/JCI80792DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4701547PMC
January 2016

Dissecting the Role of Smooth Muscle Cells versus Pericytes in Regulating Cerebral Blood Flow Using In Vivo Optical Imaging.

Neuron 2015 Jul;87(1):4-6

Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA 92697, USA; Departments of Neurology and Pathology & Cell Biology, Columbia University Medical Center, New York, NY 10032, USA. Electronic address:

The brain regulates blood flow to match energy demand to nutrient supply. In this issue of Neuron, using in vivo optical imaging and optogenetics, Hill et al. (2015) report that arteriolar smooth muscle cells are key players in regulating cerebral blood flow in the healthy state and contribute to the "no reflow" phenomenon after ischemic stroke.
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http://dx.doi.org/10.1016/j.neuron.2015.06.024DOI Listing
July 2015

The role of angiogenesis in the pathology of multiple sclerosis.

Vasc Cell 2014 28;6(1):23. Epub 2014 Nov 28.

Department of Developmental and Cell Biology, University of California at Irvine, Irvine, CA 92697-2300 USA.

Angiogenesis, or the growth of new blood vessels from existing vasculature, is critical for the proper development of many organs. This process is inhibited and tightly regulated in adults, once endothelial cells have acquired organ-specific properties. Within the central nervous system (CNS), angiogenesis and acquisition of blood-brain barrier (BBB) properties by endothelial cells is essential for CNS function. However, the role of angiogenesis in CNS pathologies associated with impaired barrier function remains unclear. Although vessel abnormalities characterized by abnormal barrier function are well documented in multiple sclerosis (MS), a demyelinating disease of the CNS resulting from an immune cell attack on oligodendrocytes, histological analysis of human MS samples has shown that angiogenesis is prevalent in and around the demyelinating plaques. Experiments using an animal model that mimics several features of human MS, Experimental Autoimmune Encephalomyelitis (EAE), have confirmed these human pathological findings and shed new light on the contribution of pre-symptomatic angiogenesis to disease progression. The CNS-infiltrating inflammatory cells that are a hallmark of both MS and EAE secrete several factors that not only contribute to exacerbating the inflammatory process but also promote and stimulate angiogenesis. Moreover, chemical or biological inhibitors that directly or indirectly block angiogenesis provide clinical benefits for disease progression. While the precise mechanism of action for these inhibitors is unknown, preventing pathological angiogenesis during EAE progression holds great promise for developing effective treatment strategies for human MS.
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http://dx.doi.org/10.1186/s13221-014-0023-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4253611PMC
December 2014

Stem cell-based therapies for multiple sclerosis: recent advances in animal models and human clinical trials.

Regen Med 2014 Mar;9(2):129-32

Department of Developmental & Cell Biology, University of California, Irvine, CA 92697, USA.

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http://dx.doi.org/10.2217/rme.14.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4832963PMC
March 2014

Stepwise recruitment of transcellular and paracellular pathways underlies blood-brain barrier breakdown in stroke.

Neuron 2014 May 17;82(3):603-17. Epub 2014 Apr 17.

Department of Developmental and Cell Biology, University of California, Irvine, CA 92697, USA. Electronic address:

Brain endothelial cells form a paracellular and transcellular barrier to many blood-borne solutes via tight junctions (TJs) and scarce endocytotic vesicles. The blood-brain barrier (BBB) plays a pivotal role in the healthy and diseased CNS. BBB damage after ischemic stroke contributes to increased mortality, yet the contributions of paracellular and transcellular mechanisms to this process in vivo are unknown. We have created a transgenic mouse strain whose endothelial TJs are labeled with eGFP and have imaged dynamic TJ changes and fluorescent tracer leakage across the BBB in vivo, using two-photon microscopy in the t-MCAO stroke model. Although barrier function is impaired as early as 6 hr after stroke, TJs display profound structural defects only after 2 days. Conversely, the number of endothelial caveolae and transcytosis rate increase as early as 6 hr after stroke. Therefore, stepwise impairment of transcellular followed by paracellular barrier mechanisms accounts for the BBB deficits in stroke.
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http://dx.doi.org/10.1016/j.neuron.2014.03.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4016169PMC
May 2014

From blood to the brain: can systemically transplanted mesenchymal stem cells cross the blood-brain barrier?

Stem Cells Int 2013 12;2013:435093. Epub 2013 Aug 12.

Department of Pharmaceutical Sciences, Sue and Bill Gross Stem Cell Research Center and Chao Family Comprehensive Cancer Center, University of California, Irvine, 845 Health Sciences Road, Irvine, CA 92697, USA ; Department of Biomedical Engineering and Edwards Lifesciences Center for Advanced Cardiovascular Technology, University of California, Irvine, 845 Health Sciences Road, Irvine, CA 92697, USA.

Systemically infused mesenchymal stem cells (MSCs) are emerging therapeutics for treating stroke, acute injuries, and inflammatory diseases of the central nervous system (CNS), as well as brain tumors due to their regenerative capacity and ability to secrete trophic, immune modulatory, or other engineered therapeutic factors. It is hypothesized that transplanted MSCs home to and engraft at ischemic and injured sites in the brain in order to exert their therapeutic effects. However, whether MSCs possess the ability to migrate across the blood-brain barrier (BBB) that separates the blood from the brain remains unresolved. This review analyzes recent advances in this area in an attempt to elucidate whether systemically infused MSCs are able to actively transmigrate across the CNS endothelium, particularly under conditions of injury or stroke. Understanding the fate of transplanted MSCs and their CNS trafficking mechanisms will facilitate the development of more effective stem-cell-based therapeutics and drug delivery systems to treat neurological diseases and brain tumors.
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http://dx.doi.org/10.1155/2013/435093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3753739PMC
September 2013

The role of pericytes in blood-brain barrier function and stroke.

Curr Pharm Des 2012 ;18(25):3653-62

Departments of Neurology, University of California Irvine, USA.

Central nervous system pericytes have critical and complex inductive, structural, and regulatory roles interacting with other cell types of the neurovascular unit, especially endothelial cells and astrocytes. Pericyte-endothelial interactions are particularly prominent for blood-brain barrier (BBB) maintenance, with profound effects on basement membrane and endothelial tight junction structure and function. Under experimental conditions of hypoxia-ischemia mimicking stroke, pericytes migrate from their usual microvascular location and influence, directly or indirectly, BBB permeability. The contractile properties of pericytes provide the capacity to regulate capillary blood flow, but this may have detrimental effects on ischemic injury. Stem cell characteristics of pericytes imply an important regenerative role following stroke. Pericytes thus appear to orchestrate multiple critical functions in stroke, involving blood flow, permeability, and repair of the neurovascular unit.
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http://dx.doi.org/10.2174/138161212802002706DOI Listing
December 2012

The mouse blood-brain barrier transcriptome: a new resource for understanding the development and function of brain endothelial cells.

PLoS One 2010 Oct 29;5(10):e13741. Epub 2010 Oct 29.

Department of Anatomy, University of California San Francisco, San Francisco, California, United States of America.

The blood-brain barrier (BBB) maintains brain homeostasis and limits the entry of toxins and pathogens into the brain. Despite its importance, little is known about the molecular mechanisms regulating the development and function of this crucial barrier. In this study we have developed methods to highly purify and gene profile endothelial cells from different tissues, and by comparing the transcriptional profile of brain endothelial cells with those purified from the liver and lung, we have generated a comprehensive resource of transcripts that are enriched in the BBB forming endothelial cells of the brain. Through this comparison we have identified novel tight junction proteins, transporters, metabolic enzymes, signaling components, and unknown transcripts whose expression is enriched in central nervous system (CNS) endothelial cells. This analysis has identified that RXRalpha signaling cascade is specifically enriched at the BBB, implicating this pathway in regulating this vital barrier. This dataset provides a resource for understanding CNS endothelial cells and their interaction with neural and hematogenous cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0013741PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2966423PMC
October 2010

APCDD1 is a novel Wnt inhibitor mutated in hereditary hypotrichosis simplex.

Nature 2010 Apr;464(7291):1043-7

Department of Dermatology, Columbia University, College of Physicians and Surgeons, 630 West 168th Street, VC15 204A, New York, New York 10032, USA.

Hereditary hypotrichosis simplex is a rare autosomal dominant form of hair loss characterized by hair follicle miniaturization. Using genetic linkage analysis, we mapped a new locus for the disease to chromosome 18p11.22, and identified a mutation (Leu9Arg) in the adenomatosis polyposis down-regulated 1 (APCDD1) gene in three families. We show that APCDD1 is a membrane-bound glycoprotein that is abundantly expressed in human hair follicles, and can interact in vitro with WNT3A and LRP5-two essential components of Wnt signalling. Functional studies show that APCDD1 inhibits Wnt signalling in a cell-autonomous manner and functions upstream of beta-catenin. Moreover, APCDD1 represses activation of Wnt reporters and target genes, and inhibits the biological effects of Wnt signalling during both the generation of neurons from progenitors in the developing chick nervous system, and axis specification in Xenopus laevis embryos. The mutation Leu9Arg is located in the signal peptide of APCDD1, and perturbs its translational processing from the endoplasmic reticulum to the plasma membrane. APCDD1(L9R) probably functions in a dominant-negative manner to inhibit the stability and membrane localization of the wild-type protein. These findings describe a novel inhibitor of the Wnt signalling pathway with an essential role in human hair growth. As APCDD1 is expressed in a broad repertoire of cell types, our findings indicate that APCDD1 may regulate a diversity of biological processes controlled by Wnt signalling.
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http://dx.doi.org/10.1038/nature08875DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3046868PMC
April 2010

Myelin gene regulatory factor is a critical transcriptional regulator required for CNS myelination.

Cell 2009 Jul;138(1):172-85

Department of Neurobiology, Stanford University School of Medicine, Stanford, CA 94305-5125, USA.

The transcriptional control of CNS myelin gene expression is poorly understood. Here we identify gene model 98, which we have named myelin gene regulatory factor (MRF), as a transcriptional regulator required for CNS myelination. Within the CNS, MRF is specifically expressed by postmitotic oligodendrocytes. MRF is a nuclear protein containing an evolutionarily conserved DNA binding domain homologous to a yeast transcription factor. Knockdown of MRF in oligodendrocytes by RNA interference prevents expression of most CNS myelin genes; conversely, overexpression of MRF within cultured oligodendrocyte progenitors or the chick spinal cord promotes expression of myelin genes. In mice lacking MRF within the oligodendrocyte lineage, premyelinating oligodendrocytes are generated but display severe deficits in myelin gene expression and fail to myelinate. These mice display severe neurological abnormalities and die because of seizures during the third postnatal week. These findings establish MRF as a critical transcriptional regulator essential for oligodendrocyte maturation and CNS myelination.
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http://dx.doi.org/10.1016/j.cell.2009.04.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2757090PMC
July 2009

Motor neurons with axial muscle projections specified by Wnt4/5 signaling.

Neuron 2009 Mar;61(5):708-20

Howard Hughes Medical Institute, Kavli Institute for Brain Science, Departments of Neuroscience and Biochemistry and Molecular Biophysics, Columbia University Medical Center, New York, NY 10032, USA.

Axial muscles are innervated by motor neurons of the median motor column (MMC). In contrast to the segmentally restricted motor columns that innervate limb, body wall, and neuronal targets, MMC neurons are generated along the entire length of the spinal cord. We show that the specification of MMC fate involves a dorsoventral signaling program mediated by three Wnt proteins (Wnt4, Wnt5a, and Wnt5b) expressed in and around the floor plate. These Wnts appear to establish a ventral(high) to dorsal(low) signaling gradient and promote MMC identity and connectivity by maintaining expression of the LIM homeodomain proteins Lhx3/4 in spinal motor neurons. Elevation of Wnt4/5 activity generates additional MMC neurons at the expense of other motor neuron columnar subtypes, whereas depletion of Wnt4/5 activity inhibits the production of MMC neurons. Thus, two dorsoventral signaling pathways, mediated by Shh and Wnt4/5, are required to establish an early binary divergence in motor neuron columnar identity.
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http://dx.doi.org/10.1016/j.neuron.2008.12.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2741579PMC
March 2009