Publications by authors named "Jelena Mojsilovic-Petrovic"

22 Publications

  • Page 1 of 1

Publisher Correction: L3MBTL1 regulates ALS/FTD-associated proteotoxicity and quality control.

Nat Neurosci 2019 Jul;22(7):1196

Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41593-019-0438-8DOI Listing
July 2019

L3MBTL1 regulates ALS/FTD-associated proteotoxicity and quality control.

Nat Neurosci 2019 06 6;22(6):875-886. Epub 2019 May 6.

Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA.

Misfolded protein toxicity and failure of protein quality control underlie neurodegenerative diseases including amyotrophic lateral sclerosis and frontotemporal dementia. Here, we identified Lethal(3)malignant brain tumor-like protein 1 (L3MBTL1) as a key regulator of protein quality control, the loss of which protected against the proteotoxicity of mutant Cu/Zn superoxide dismutase or C9orf72 dipeptide repeat proteins. L3MBTL1 acts by regulating p53-dependent quality control systems that degrade misfolded proteins. SET domain-containing protein 8, an L3MBTL1-associated p53-binding protein, also regulated clearance of misfolded proteins and was increased by proteotoxicity-associated stresses in mammalian cells. Both L3MBTL1 and SET domain-containing protein 8 were upregulated in the central nervous systems of mouse models of amyotrophic lateral sclerosis and human patients with amyotrophic lateral sclerosis/frontotemporal dementia. The role of L3MBTL1 in protein quality control is conserved from Caenorhabditis elegans to mammalian neurons. These results reveal a protein quality-control pathway that operates in both normal stress response and proteotoxicity-associated neurodegenerative diseases.
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http://dx.doi.org/10.1038/s41593-019-0384-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6588399PMC
June 2019

Poly(ADP-Ribose) Prevents Pathological Phase Separation of TDP-43 by Promoting Liquid Demixing and Stress Granule Localization.

Mol Cell 2018 09 9;71(5):703-717.e9. Epub 2018 Aug 9.

Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

In amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD), cytoplasmic aggregates of hyperphosphorylated TDP-43 accumulate and colocalize with some stress granule components, but how pathological TDP-43 aggregation is nucleated remains unknown. In Drosophila, we establish that downregulation of tankyrase, a poly(ADP-ribose) (PAR) polymerase, reduces TDP-43 accumulation in the cytoplasm and potently mitigates neurodegeneration. We establish that TDP-43 non-covalently binds to PAR via PAR-binding motifs embedded within its nuclear localization sequence. PAR binding promotes liquid-liquid phase separation of TDP-43 in vitro and is required for TDP-43 accumulation in stress granules in mammalian cells and neurons. Stress granule localization initially protects TDP-43 from disease-associated phosphorylation, but upon long-term stress, stress granules resolve, leaving behind aggregates of phosphorylated TDP-43. Finally, small-molecule inhibition of Tankyrase-1/2 in mammalian cells inhibits formation of cytoplasmic TDP-43 foci without affecting stress granule assembly. Thus, Tankyrase inhibition antagonizes TDP-43-associated pathology and neurodegeneration and could have therapeutic utility for ALS and FTD.
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http://dx.doi.org/10.1016/j.molcel.2018.07.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6128762PMC
September 2018

SAP97 Binding Partner CRIPT Promotes Dendrite Growth and .

eNeuro 2017 Nov-Dec;4(6). Epub 2017 Dec 6.

Department of Pediatrics Division of Neurology, Research Institute Children's Hospital of Philadelphia, Philadelphia, PA 19104.

The dendritic tree is a key determinant of neuronal information processing. In the motor system, the dendritic tree of spinal cord neurons undergoes dramatic remodeling in an activity-dependent manner during early postnatal life. This leads to the proper segmental spinal cord connectivity that subserves normal locomotor behavior. One molecular system driving the establishment of dendrite architecture of mammalian motor neurons relies on AMPA receptors (AMPA-Rs) assembled with the GluA1 subunit, and this occurs in an NMDA receptor (NMDA-R)-independent manner. The dendrite growth promoting activity of GluA1-containing AMPA-Rs depends on its intracellular binding partner, SAP97, and SAP97's PDZ3 domain. We show here that cysteine-rich interactor of PDZ3 (CRIPT) is a SAP97 PDZ3-domain binding partner, localizes to synapses with GluA1 and SAP97 along the dendritic tree, and is a determinant of the dendritic growth of mammalian spinal cord neurons. We further show that CRIPT has a well-conserved ortholog in the nematode, , and animals lacking CRIPT display decreased dendrite branching of the well-studied PVD neuron . The lack of CRIPT leads to a selective defect in touch perception, and this is rescued by expression of wild-type (WT) human CRIPT (hCRIPT) in the nervous system. This work brings new light into the molecular machinery that drives dendritic growth during development and may prove relevant to the promotion of nervous system plasticity following insult.
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http://dx.doi.org/10.1523/ENEURO.0175-17.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5718245PMC
July 2018

The Proline/Arginine Dipeptide from Hexanucleotide Repeat Expanded Inhibits the Proteasome.

eNeuro 2017 Jan-Feb;4(1). Epub 2017 Jan 31.

Division of Neurology, Department of Pediatrics, Research Institute, Children's Hospital of Philadelphia, Philadelphia, PA, 19104; Department of Neurology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104.

An intronic hexanucleotide repeat expansion (HRE) mutation in the gene is the most common cause of familial ALS and frontotemporal dementia (FTD) and is found in ∼7% of individuals with apparently sporadic disease. Several different diamino acid peptides can be generated from the HRE by noncanonical translation (repeat-associated non-ATG translation, or RAN translation), and some of these peptides can be toxic. Here, we studied the effects of two arginine containing RAN translation products [proline/arginine repeated 20 times (PR) and glycine/arginine repeated 20 times (GR)] in primary rat spinal cord neuron cultures grown on an astrocyte feeder layer. We find that PR kills motor neurons with an LD of 2 µM, but in contrast to the effects of other ALS-causing mutant proteins (i.e., SOD or TDP43), PR does not evoke the biochemical signature of mitochondrial dysfunction, ER stress, or mTORC down-regulation. PR does result in a time-dependent build-up of ubiquitylated substrates, and this is associated with a reduction of flux through both autophagic and proteasomal degradation pathways. GR, however, does not have these effects. The effects of PR on the proteasome are likely to be direct because (1) PR physically associates with proteasomes in biochemical assays, and (2) PR inhibits the degradation of a ubiquitylated test substrate when presented to purified proteasomes. Application of a proteasomal activator (IU1) blocks the toxic effects of PR on motor neuron survival. This work suggests that proteasomal activators have therapeutic potential in individuals with HRE.
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http://dx.doi.org/10.1523/ENEURO.0249-16.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5282547PMC
October 2017

Loss of RAD-23 Protects Against Models of Motor Neuron Disease by Enhancing Mutant Protein Clearance.

J Neurosci 2015 Oct;35(42):14286-306

Department of Pediatrics, Division of Neurology, Research Institute, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, Department of Neurology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104,

Unlabelled: Misfolded proteins accumulate and aggregate in neurodegenerative disease. The existence of these deposits reflects a derangement in the protein homeostasis machinery. Using a candidate gene screen, we report that loss of RAD-23 protects against the toxicity of proteins known to aggregate in amyotrophic lateral sclerosis. Loss of RAD-23 suppresses the locomotor deficit of Caenorhabditis elegans engineered to express mutTDP-43 or mutSOD1 and also protects against aging and proteotoxic insults. Knockdown of RAD-23 is further neuroprotective against the toxicity of SOD1 and TDP-43 expression in mammalian neurons. Biochemical investigation indicates that RAD-23 modifies mutTDP-43 and mutSOD1 abundance, solubility, and turnover in association with altering the ubiquitination status of these substrates. In human amyotrophic lateral sclerosis spinal cord, we find that RAD-23 abundance is increased and RAD-23 is mislocalized within motor neurons. We propose a novel pathophysiological function for RAD-23 in the stabilization of mutated proteins that cause neurodegeneration.

Significance Statement: In this work, we identify RAD-23, a component of the protein homeostasis network and nucleotide excision repair pathway, as a modifier of the toxicity of two disease-causing, misfolding-prone proteins, SOD1 and TDP-43. Reducing the abundance of RAD-23 accelerates the degradation of mutant SOD1 and TDP-43 and reduces the cellular content of the toxic species. The existence of endogenous proteins that act as "anti-chaperones" uncovers new and general targets for therapeutic intervention.
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http://dx.doi.org/10.1523/JNEUROSCI.0642-15.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4683688PMC
October 2015

Inhibition of Cytohesins Protects against Genetic Models of Motor Neuron Disease.

J Neurosci 2015 Jun;35(24):9088-105

Division of Pediatric Neurology and Department of Neurology and Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104,

Mutant genes that underlie Mendelian forms of amyotrophic lateral sclerosis (ALS) and biochemical investigations of genetic disease models point to potential driver pathophysiological events involving endoplasmic reticulum (ER) stress and autophagy. Several steps in these cell biological processes are known to be controlled physiologically by small ADP-ribosylation factor (ARF) signaling. Here, we investigated the role of ARF guanine nucleotide exchange factors (GEFs), cytohesins, in models of ALS. Genetic or pharmacological inhibition of cytohesins protects motor neurons in vitro from proteotoxic insults and rescues locomotor defects in a Caenorhabditis elegans model of disease. Cytohesins form a complex with mutant superoxide dismutase 1 (SOD1), a known cause of familial ALS, but this is not associated with a change in GEF activity or ARF activation. ER stress evoked by mutant SOD1 expression is alleviated by antagonism of cytohesin activity. In the setting of mutant SOD1 toxicity, inhibition of cytohesin activity enhances autophagic flux and reduces the burden of misfolded SOD1. These observations suggest that targeting cytohesins may have potential benefits for the treatment of ALS.
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http://dx.doi.org/10.1523/JNEUROSCI.5032-13.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4469736PMC
June 2015

GABA and glutamate are not colocalized in mossy fiber terminals of developing rodent hippocampus.

Brain Res 2012 Sep 27;1474:40-9. Epub 2012 Jul 27.

Division of Neurology, Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA.

It has been hypothesized that, in the developing rodent hippocampus, mossy fiber terminals release GABA together with glutamate. Here, we used transgenic glutamic acid decarboxylase-67 (GAD67)-GFP expressing mice and multi-label immunohistochemistry to address whether glutamatergic and GABAergic markers are colocalized. We demonstrate that in the dentate gyrus, interneurons positive for GABA/GAD are sparsely distributed along the edge of the hilus, in a different pattern from that of the densely packed granule cells. Co-staining for synaptophysin and vesicular glutamate transporter1 (VGLUT1) in postnatal day 14 brain sections from both mice and rats showed mossy fiber terminals as a group of large (2-5 μm in diameter) VGLUT1-positive excitatory presynaptic terminals in the stratum lucidum of area CA3a/b. Furthermore, co-staining for synaptophysin and vesicular GABA transporter (VGAT) revealed a group of small-sized (∼0.5 μm in diameter) inhibitory presynaptic terminals in the same area where identified mossy fiber terminals were present. The two types of terminals appeared to be mutually exclusive, and showed no colocalization. Thus, our results do not support the hypothesis that GABA is released as a neurotransmitter from mossy fiber terminals during development.
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http://dx.doi.org/10.1016/j.brainres.2012.07.042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3695404PMC
September 2012

Evaluating the role of the FUS/TLS-related gene EWSR1 in amyotrophic lateral sclerosis.

Hum Mol Genet 2012 Jul 27;21(13):2899-911. Epub 2012 Mar 27.

Department of Genetics, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305, USA.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting motor neurons. Mutations in related RNA-binding proteins TDP-43, FUS/TLS and TAF15 have been connected to ALS. These three proteins share several features, including the presence of a bioinformatics-predicted prion domain, aggregation-prone nature in vitro and in vivo and toxic effects when expressed in multiple model systems. Given these commonalities, we hypothesized that a related protein, EWSR1 (Ewing sarcoma breakpoint region 1), might also exhibit similar properties and therefore could contribute to disease. Here, we report an analysis of EWSR1 in multiple functional assays, including mutational screening in ALS patients and controls. We identified three missense variants in EWSR1 in ALS patients, which were absent in a large number of healthy control individuals. We show that disease-specific variants affect EWSR1 localization in motor neurons. We also provide multiple independent lines of in vitro and in vivo evidence that EWSR1 has similar properties as TDP-43, FUS and TAF15, including aggregation-prone behavior in vitro and ability to confer neurodegeneration in Drosophila. Postmortem analysis of sporadic ALS cases also revealed cytoplasmic mislocalization of EWSR1. Together, our studies highlight a potential role for EWSR1 in ALS, provide a collection of functional assays to be used to assess roles of additional RNA-binding proteins in disease and support an emerging concept that a class of aggregation-prone RNA-binding proteins might contribute broadly to ALS and related neurodegenerative diseases.
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http://dx.doi.org/10.1093/hmg/dds116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3373238PMC
July 2012

A yeast functional screen predicts new candidate ALS disease genes.

Proc Natl Acad Sci U S A 2011 Dec 7;108(52):20881-90. Epub 2011 Nov 7.

Departments of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.

Amyotrophic lateral sclerosis (ALS) is a devastating and universally fatal neurodegenerative disease. Mutations in two related RNA-binding proteins, TDP-43 and FUS, that harbor prion-like domains, cause some forms of ALS. There are at least 213 human proteins harboring RNA recognition motifs, including FUS and TDP-43, raising the possibility that additional RNA-binding proteins might contribute to ALS pathogenesis. We performed a systematic survey of these proteins to find additional candidates similar to TDP-43 and FUS, followed by bioinformatics to predict prion-like domains in a subset of them. We sequenced one of these genes, TAF15, in patients with ALS and identified missense variants, which were absent in a large number of healthy controls. These disease-associated variants of TAF15 caused formation of cytoplasmic foci when expressed in primary cultures of spinal cord neurons. Very similar to TDP-43 and FUS, TAF15 aggregated in vitro and conferred neurodegeneration in Drosophila, with the ALS-linked variants having a more severe effect than wild type. Immunohistochemistry of postmortem spinal cord tissue revealed mislocalization of TAF15 in motor neurons of patients with ALS. We propose that aggregation-prone RNA-binding proteins might contribute very broadly to ALS pathogenesis and the genes identified in our yeast functional screen, coupled with prion-like domain prediction analysis, now provide a powerful resource to facilitate ALS disease gene discovery.
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http://dx.doi.org/10.1073/pnas.1109434108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3248518PMC
December 2011

Signaling events in axons and/or dendrites render motor neurons vulnerable to mutant superoxide dismutase toxicity.

J Neurosci 2011 Jan;31(1):295-9

Division of Neurology, Department of Pediatrics, Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.

The survival of dorsal root ganglion and sympathetic neurons is promoted whether nerve growth factor (NGF) activates TrkA receptors on the cell body or the axon. Yet other aspects of neurotrophic factor actions (i.e., ability to promote axon growth, selection of neurochemical phenotype and engagement of signaling modules) differ as a function of the location of the ligand-receptor interaction. The extent to which these observations are relevant to CNS neurons is unknown. This may be particularly relevant to neurodegenerative diseases such as amyotrophic lateral sclerosis, where beneficial axon-target interactions are disturbed early in the disease process. Here we characterize the growth of pure motor neurons in compartment cultures and show that brain-derived neurotrophic factor (BDNF) stimulation of the cell body or axons/dendrites promotes survival. Expression of G37R mutant superoxide dismutase (SOD) in motor neurons will lead to death and this depends on BDNF activation of TrkB on axons and/or dendrites. BDNF action depends upon endocytosis of the BDNF-TrkB complex and de novo protein synthesis. These results highlight the importance of signaling events occurring in axons/dendrites in mutant SOD toxicity.
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http://dx.doi.org/10.1523/JNEUROSCI.4824-10.2011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3091265PMC
January 2011

FOXO3a is broadly neuroprotective in vitro and in vivo against insults implicated in motor neuron diseases.

J Neurosci 2009 Jun;29(25):8236-47

Department of Pediatrics, Division of Neurology, Abramson Research Center, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, USA.

Aging is a risk factor for the development of adult-onset neurodegenerative diseases. Although some of the molecular pathways regulating longevity and stress resistance in lower organisms are defined (i.e., those activating the transcriptional regulators DAF-16 and HSF-1 in Caenorhabditis elegans), their relevance to mammals and disease susceptibility are unknown. We studied the signaling controlled by the mammalian homolog of DAF-16, FOXO3a, in model systems of motor neuron disease. Neuron death elicited in vitro by excitotoxic insult or the expression of mutant SOD1, mutant p150(glued), or polyQ-expanded androgen receptor was abrogated by expression of nuclear-targeted FOXO3a. We identify a compound [Psammaplysene A (PA)] that increases nuclear localization of FOXO3a in vitro and in vivo and show that PA also protects against these insults in vitro. Administration of PA to invertebrate model systems of neurodegeneration similarly blocked neuron death in a DAF-16/FOXO3a-dependent manner. These results indicate that activation of the DAF-16/FOXO3a pathway, genetically or pharmacologically, confers protection against the known causes of motor neuron diseases.
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http://dx.doi.org/10.1523/JNEUROSCI.1805-09.2009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2748231PMC
June 2009

GluR1 controls dendrite growth through its binding partner, SAP97.

J Neurosci 2008 Oct;28(41):10220-33

Department of Pediatrics, Division of Neurology, Joseph Stokes Jr. Research Institute, Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.

Activity-dependent dendrite elaboration influences the pattern of interneuronal connectivity and network function. In the present study, we examined the mechanism by which the GluR1 subunit of AMPA receptors controls dendrite morphogenesis. GluR1 binds to SAP97, a scaffolding protein that is a component of the postsynaptic density, via its C-terminal 7 aa. We find that elimination of this interaction in vitro or in vivo (by deleting the C-terminal 7 aa of GluR1, GluR1Delta7) does not influence trafficking, processing, or cell surface GluR1 expression but does prevent translocation of SAP97 from the cytosol to membranes. GluR1 and SAP97 together at the plasma membrane promotes dendrite branching in an activity-dependent manner, although this does not require physical association. Our findings suggest that the C-terminal 7 aa of GluR1 are essential for bringing SAP97 to the plasma membrane, where it acts to translate the activity of AMPA receptors into dendrite growth.
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http://dx.doi.org/10.1523/JNEUROSCI.3434-08.2008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2699678PMC
October 2008

Role of GluR1 in activity-dependent motor system development.

J Neurosci 2008 Oct;28(40):9953-68

Department of Pediatrics, Division of Neurology, Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.

Activity-dependent specification of neuronal architecture during early postnatal life is essential for refining the precision of communication between neurons. In the spinal cord under normal circumstances, the AMPA receptor subunit GluR1 is expressed at high levels by motor neurons and surrounding interneurons during this critical developmental period, although the role it plays in circuit formation and locomotor behavior is unknown. Here, we show that GluR1 promotes dendrite growth in a non-cell-autonomous manner in vitro and in vivo. The mal-development of motor neuron dendrites is associated with changes in the pattern of interneuronal connectivity within the segmental spinal cord and defects in strength and endurance. Transgenic expression of GluR1 in adult motor neurons leads to dendrite remodeling and supernormal locomotor function. GluR1 expression by neurons within the segmental spinal cord plays an essential role in formation of the neural network that underlies normal motor behavior.
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http://dx.doi.org/10.1523/JNEUROSCI.0880-08.2008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3844744PMC
October 2008

Hypoxia-inducible factor-1 (HIF-1) is involved in the regulation of hypoxia-stimulated expression of monocyte chemoattractant protein-1 (MCP-1/CCL2) and MCP-5 (Ccl12) in astrocytes.

J Neuroinflammation 2007 May 2;4:12. Epub 2007 May 2.

Neurobiology Program, Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada.

Background: Neuroinflammation has been implicated in various brain pathologies characterized by hypoxia and ischemia. Astroglia play an important role in the initiation and propagation of hypoxia/ischemia-induced inflammation by secreting inflammatory chemokines that attract neutrophils and monocytes into the brain. However, triggers of chemokine up-regulation by hypoxia/ischemia in these cells are poorly understood. Hypoxia-inducible factor-1 (HIF-1) is a dimeric transcriptional factor consisting of HIF-1alpha and HIF-1beta subunits. HIF-1 binds to HIF-1-binding sites in the target genes and activates their transcription. We have recently shown that hypoxia-induced expression of IL-1beta in astrocytes is mediated by HIF-1alpha. In this study, we demonstrate the role of HIF-1alpha in hypoxia-induced up-regulation of inflammatory chemokines, human monocyte chemoattractant protein-1 (MCP-1/CCL2) and mouse MCP-5 (Ccl12), in human and mouse astrocytes, respectively.

Methods: Primary fetal human astrocytes or mouse astrocytes generated from HIF-1alpha+/+ and HIF-1alpha+/- mice were subjected to hypoxia (<2% oxygen) or 125 muM CoCl2 for 4 h and 6 h, respectively. The expression of HIF-1alpha, MCP-1 and MCP-5 was determined by semi-quantitative RT-PCR, western blot or ELISA. The interaction of HIF-1alpha with a HIF-1-binding DNA sequence was examined by EMSA and supershift assay. HIF-1-binding sequence in the promoter of MCP-1 gene was cloned and transcriptional activation of MCP-1 by HIF-1alpha was analyzed by reporter gene assay.

Results: Sequence analyses identified HIF-1-binding sites in the promoters of MCP-1 and MCP-5 genes. Both hypoxia and HIF-1alpha inducer, CoCl2, strongly up-regulated HIF-1alpha expression in astrocytes. Mouse HIF-1alpha+/- astrocytes had lower basal levels of HIF-1alpha and MCP-5 expression. The up-regulation of MCP-5 by hypoxia or CoCl2 in HIF-1alpha+/+ and HIF-1alpha+/- astrocytes was correlated with the levels of HIF-1alpha in cells. Both hypoxia and CoCl2 also up-regulated HIF-1alpha and MCP-1 expression in human astrocytes. EMSA assay demonstrated that HIF-1 activated by either hypoxia or CoCl2 binds to wild-type HIF-1-binding DNA sequence, but not the mutant sequence. Furthermore, reporter gene assay demonstrated that hypoxia markedly activated MCP-1 transcription but not the mutated MCP-1 promoter in transfected astrocytes.

Conclusion: These findings suggest that both MCP-1 and MCP-5 are HIF-1 target genes and that HIF-1alpha is involved in transcriptional induction of these two chemokines in astrocytes by hypoxia.
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http://dx.doi.org/10.1186/1742-2094-4-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1872020PMC
May 2007

Embryonic motor neuron dendrite growth is stunted by inhibition of nitric oxide-dependent activation of soluble guanylyl cyclase and protein kinase G.

Eur J Neurosci 2007 Apr;25(7):1987-97

Department of Neurology, Children's Hospital of Philadelphia, 3615 Civic Center Boulevard, Philadelphia, PA 19104, USA.

We have examined the participation of a neuronal nitric oxide synthase (nNOS) signaling pathway in the elaboration of motor neuron dendrites during embryonic life. During chick embryogenesis, nNOS is expressed by interneurons that surround the motor neuron pools in the ventral horn. Pseudorabies virus tracing suggests that these cells, while juxtaposed to motor neurons are not synaptically connected to them. The downstream effectors, soluble guanylyl cyclase (sGC) and protein kinase G (PKG), are found in motor neurons as well as several other populations of spinal cord cells. To determine the functional significance of the nNOS/sGC/PKG signaling pathway, pharmacological inhibitors were applied to chick embryos and the effects on motor neuron dendrites monitored. Inhibition of nNOS activity led to a lasting reduction in the overall size and degree of branching of the dendritic tree. These alterations in dendritic architecture were also seen when the activity of sGC or PKG was blocked. Our results suggest that normal motor neuron dendrite elaboration depends, in part, on the activity-dependent generation of NO by ventral horn interneurons, which then activates sGC and PKG in motor neurons.
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http://dx.doi.org/10.1111/j.1460-9568.2007.05456.xDOI Listing
April 2007

Protecting motor neurons from toxic insult by antagonism of adenosine A2a and Trk receptors.

J Neurosci 2006 Sep;26(36):9250-63

Department of Neurology, Children's Hospital of Philadelphia, Joseph Stokes Jr. Research Institute, Philadelphia, Pennsylvania 19104, USA.

The death of motor neurons in amyotrophic lateral sclerosis (ALS) is thought to result from the interaction of a variety of factors including excitotoxicity, accumulation of toxic proteins, and abnormal axonal transport. Previously, we found that the susceptibility of motor neurons to excitotoxic insults can be limited by inhibiting signals evoked by brain-derived neurotrophic factor (BDNF) activation of the receptor tyrosine kinase B (TrkB). Here we show that this can be achieved by direct kinase inhibition or by blockade of a transactivation pathway that uses adenosine A2a receptors and src-family kinases (SFKs). Downstream signaling cascades (such as mitogen-activated protein kinase and phosphatidylinositol-3 kinase) are inhibited by these blockers. In addition to protecting motor neurons from excitotoxic insult, these agents also prevent toxicity that follows from the expression of mutant proteins (G85R superoxide dismutase 1; G59S p150(glued)) that cause familial motor neuron disease. TrkB, adenosine A2a receptors, and SFKs associate into complexes in lipid raft and nonlipid raft membranes and the signaling from lipids rafts may be particularly important because their disruption by cholesterol depletion blocks the ability of BDNF to render motor neurons vulnerable to insult. The neuroprotective versatility of Trk antagonism suggests that it may have broad utility in the treatment of ALS patients.
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http://dx.doi.org/10.1523/JNEUROSCI.1856-06.2006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6674510PMC
September 2006

Enprofylline protects motor neurons from in vitro excitotoxic challenge.

Neurodegener Dis 2005 ;2(3-4):160-5

Children's Hospital of Philadelphia, Joseph Stokes Jr. Research Institute, PA 19104, USA.

Background: The death of motor neurons in amyotrophic lateral sclerosis (ALS) is believed to result, in part, from unrestrained activation of glutamate receptors (excitotoxicity). In some in vitro models, excitotoxic death only occurs if motor neurons develop in the presence of the growth factor, brain-derived neurotrophic factor (BDNF).

Objective: Since the increased vulnerability of motor neurons evoked by BDNF is mediated by activation of TrkB, we sought to identify pharmacological agents that can block this pathway. Adenosine receptors are known to transactivate Trk receptors, leading us to examine the effects of manipulating of adenosine receptor signaling on Trk signaling and excitotoxic sensitivity.

Methods: Spinal cord cultures were treated with adenosine receptor agonists and antagonists. The biochemical effects on Trk signaling and excitotoxic motor neuron death were examined.

Results: We show here that adenosine A(2a) antagonists can reduce activation of Trk receptors and are neuroprotective. Conversely, activating adenosine A(2a) receptors in the absence of BDNF signaling makes motor neurons vulnerable to excitotoxic challenge.

Conclusion: Selective, high-affinity adenosine A(2a) antagonists merit consideration as therapeutic agents for the treatment of ALS.
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http://dx.doi.org/10.1159/000089621DOI Listing
September 2006

Evidence that hypoxia-inducible factor-1 (HIF-1) mediates transcriptional activation of interleukin-1beta (IL-1beta) in astrocyte cultures.

J Neuroimmunol 2006 May 28;174(1-2):63-73. Epub 2006 Feb 28.

Cerebrovascular Research Group, Neurobiology Program, Institute for Biological Sciences, National Research Council of Canada, Ottawa, Canada.

Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription factor composed of HIF-1alpha and HIF-1beta subunits and involved in the regulation of gene expression in adaptive response to hypoxia. This study reports that the inflammatory cytokine interleukin-1beta (IL-1beta) shares common features of other known HIF-1alpha-regulated genes. Both human and mouse IL-1beta genes carry multiple HIF-1-binding sites in their promoter regions and are up-regulated by hypoxia and CoCl2 in human and mouse astrocytes in parallel with up-regulation of HIF-1alpha mRNA and protein. Inhibition of HIF-1alpha degradation by proteasome inhibitor, MG-132, potentiated hypoxia-induced IL-1beta release from human astrocytes, and this response was blocked in the presence of CdCl2. Mouse astrocytes with Hif1alpha+/- genotype demonstrated attenuated up-regulation of both HIF-1alpha and IL-1beta by hypoxia and CoCl2. Mutation of HIF-1-binding sites in the IL-1beta promoter abolished hypoxia-induced transactivation of the reporter gene transfected into human astrocytes. Similarly, HIF-1 binding "decoy" oligonuleotide transfected into astrocytes inhibited both hypoxia-induced transactivation of the HIF-1 reporter gene and IL-1beta secretion from transfected astrocytes. Collectively, the evidence suggests that the transcriptional activation of IL-1beta in astrocytes exposed to hypoxia occurs via HIF-1.
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http://dx.doi.org/10.1016/j.jneuroim.2006.01.014DOI Listing
May 2006

Development of rapid staining protocols for laser-capture microdissection of brain vessels from human and rat coupled to gene expression analyses.

J Neurosci Methods 2004 Feb;133(1-2):39-48

Cerebrovascular Research Group, Institute for Biological Sciences, National Research Council of Canada, 1200 Montreal Road, Building M-54, Ottawa, Ont., Canada K1A 0R6.

Laser-capture microdissection (LCM) is a technique that enables selective extraction of desired cells from heterogeneous tissues compatible with subsequent molecular analyses. The specific visualization of desired cell types prior to LCM is essential for achieving selective capture. We have developed rapid and selective staining protocols for LCM extraction of microvessels from human and rat brain. Vessels in human and rat brain sections were visualized by a 2 min exposure to fluorescein-labeled lectins Ulex Europeaus Agglutinin I (UEA I) and Ricinus Communis Agglutinin I (RCA I), respectively. Immunohistochemical staining for the endothelial-specific marker, Factor VIII-related antigen (FVIII-rAg), co-localized with that for either UEA I or RCA I, confirming the selective staining of vascular structures with these lectins. Both brain vessels and perivascular parenchyma were captured using LCM, followed by RNA isolation. RT-PCR analyses demonstrated the enrichment of LCM-captured vessels and parenchyma in FVIII-rAg and GFAP mRNA, respectively. LCM-captured human vessels also expressed the tight junction-specific gene, zonula occludens 1 (ZO-1). LCM extraction of vessels from brain sections can be used to perform molecular fingerprinting of neurovascular unit in various brain pathologies.
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http://dx.doi.org/10.1016/j.jneumeth.2003.09.026DOI Listing
February 2004

The expression and functional characterization of ABCG2 in brain endothelial cells and vessels.

FASEB J 2003 Nov 4;17(14):2085-7. Epub 2003 Sep 4.

Institute for Biological Sciences, NRC, 1200 Montreal Road Campus, Bldg. M-54, Ottawa, Ontario, Canada K1A 0R6.

Delivery of drugs to the brain is impeded by the activity of efflux pumps expressed by endothelial cells of brain vasculature. The ATP binding cassette (ABC) transporters, among which ABCB1/MDR1 P-glycoprotein and ABCC1/multidrug resistance-associated protein 1 are expressed in brain endothelial cells, participate in drug efflux properties of the blood-brain barrier (BBB). Searches of the EST (expressed sequence tags) database with the conserved ABC domain, conducted to identify other ABC transporters expressed in the BBB, recovered 15 ABC transporter sequences expressed in human brain cDNA libraries. One of these sequences, identical to ABCG2, was highly expressed in cultured human cerebromicrovascular endothelial cells and human brain tissue at both mRNA and protein levels. Overexpression of human ABCG2 in immortalized rat brain endothelial cells resulted in enhanced polarized abluminal to luminal transport of various substrates tested in the in vitro BBB model. Brain vessels extracted from tissue sections of nonmalignant human brain and glioblastoma tumors by laser capture microdissection microscopy and analyzed by real-time polymerase chain reaction showed higher expression of ABCG2 relative to ABCB1/MDR1 and ABCC1/MRP1. ABCG2 was up-regulated in both glioblastoma vessels and parenchymal tissue. These studies suggest a role for brain endothelial ABCG2 transporter in modulating drug delivery to the brain and in conferring drug resistance to glioblastomas.
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http://dx.doi.org/10.1096/fj.02-1131fjeDOI Listing
November 2003

Types of neurons and some dendritic patterns of basolateral amygdala in humans--a golgi study.

Ann Anat 2002 Jan;184(1):93-103

Institute of Anatomy, School of Medicine, Kragujevac, Serbia, Yugoslavia.

Classification of the neurons in the human basolateral amygdala is performed on preparations impregnated by the Golgi technique. Three different neuronal types are found in the nuclei of the basolateral amygdala: Type I--Pyramidal cells, with numerous dendritic spines and two subtypes (slender and squat); Type II--Modified pyramidal cells, sparsely spinous with rare dendritic spines and two subtypes (single apical and double apical) and; Type III--Non-pyramidal cells, with few dendritic spines and three subtypes (bipolar, multipolar and gliaform). The analysis of the primary dendritic branches pointed out the occasional presence of dendritic bundles (fascicular dendritic arrangement) with their predomination in the parvicellular division of the basal nucleus and paralaminar nucleus. Additionally, the presence of dendrodendritic contacts, indicated by light microscopy, was also found in the parvicellular division of the basal nucleus and especially in the paralaminar nucleus.
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http://dx.doi.org/10.1016/S0940-9602(02)80042-5DOI Listing
January 2002