Publications by authors named "Robert G Kalb"

36 Publications

Preconditioning of Caenorhabditis elegans to anoxic insult by inactivation of cholinergic, GABAergic and muscle activity.

Genes Brain Behav 2021 Apr 18;20(4):e12713. Epub 2020 Nov 18.

Department of Pediatrics, Division of Neurology, Research Institute, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.

For most metazoans, oxygen deprivation leads to cell dysfunction and if severe, death. Sublethal stress prior to a hypoxic or anoxic insult ("preconditioning") can protect cells from subsequent oxygen deprivation. The molecular mechanisms by which sublethal stress can buffer against a subsequent toxic insult and the role of the nervous system in the response are not well understood. We studied the role of neuronal activity preconditioning to oxygen deprivation in Caenorhabditis elegans. Animals expressing the histamine gated chloride channels (HisCl1) in select cell populations were used to temporally and spatially inactivate the nervous system or tissue prior to an anoxic insult. We find that inactivation of the nervous system for 3 h prior to the insult confers resistance to a 48-h anoxic insult in 4th-stage larval animals. Experiments show that this resistance can be attributed to loss of activity in cholinergic and GABAergic neurons as well as in body wall muscles. These observations indicate that the nervous system activity can mediate the organism's response to anoxia.
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http://dx.doi.org/10.1111/gbb.12713DOI Listing
April 2021

Homeostatic regulation of STING protein at the resting state by stabilizer TOLLIP.

Nat Immunol 2020 02 13;21(2):158-167. Epub 2020 Jan 13.

Department of Immunology, University of Texas Southwestern Medical Center, Dallas, TX, USA.

STING (stimulator of interferon genes) is an important innate immune protein, but its homeostatic regulation at the resting state is unknown. Here, we identified TOLLIP as a stabilizer of STING through direct interaction to prevent its degradation. Tollip deficiency results in reduced STING protein in nonhematopoietic cells and tissues, and renders STING protein unstable in immune cells, leading to severely dampened STING signaling capacity. The competing degradation mechanism of resting-state STING requires IRE1α and lysosomes. TOLLIP mediates clearance of Huntington's disease-linked polyQ protein aggregates. Ectopically expressed polyQ proteins in vitro or endogenous polyQ proteins in Huntington's disease mouse striatum sequester TOLLIP away from STING, leading to reduced STING protein and dampened immune signaling. Tollip also ameliorates STING-mediated autoimmune disease in Trex1 mice. Together, our findings reveal that resting-state STING protein level is strictly regulated by a constant tug-of-war between 'stabilizer' TOLLIP and 'degrader' IRE1α-lysosome that together maintain tissue immune homeostasis.
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http://dx.doi.org/10.1038/s41590-019-0569-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6983345PMC
February 2020

PTEN self-regulates through USP11 via the PI3K-FOXO pathway to stabilize tumor suppression.

Nat Commun 2019 02 7;10(1):636. Epub 2019 Feb 7.

Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, 77030, USA.

PTEN is a lipid phosphatase that antagonizes the PI3K/AKT pathway and is recognized as a major dose-dependent tumor suppressor. The cellular mechanisms that control PTEN levels therefore offer potential routes to therapy, but these are as yet poorly defined. Here we demonstrate that PTEN plays an unexpected role in regulating its own stability through the transcriptional upregulation of the deubiquitinase USP11 by the PI3K/FOXO pathway, and further show that this feedforward mechanism is implicated in its tumor-suppressive role, as mice lacking Usp11 display increased susceptibility to PTEN-dependent tumor initiation, growth and metastasis. Notably, USP11 is downregulated in cancer patients, and correlates with PTEN expression and FOXO nuclear localization. Our findings therefore demonstrate that PTEN-PI3K-FOXO-USP11 constitute the regulatory feedforward loop that improves the stability and tumor suppressive activity of PTEN.
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http://dx.doi.org/10.1038/s41467-019-08481-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6367354PMC
February 2019

Enhancing GABAergic Transmission Improves Locomotion in a Model of Spinal Muscular Atrophy.

eNeuro 2018 Nov-Dec;5(6). Epub 2019 Jan 2.

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

Spinal muscular atrophy (SMA) is a neuromuscular disease characterized by degeneration of spinal motor neurons resulting in variable degrees of muscular wasting and weakness. It is caused by a loss-of-function mutation in the survival motor neuron () gene. mutants lacking recapitulate several aspects of the disease including impaired movement and shorted life span. We examined whether genes previously implicated in life span extension conferred benefits to lacking SMN. We find that reducing daf-2/insulin receptor signaling activity promotes survival and improves locomotor behavior in this model of SMA. The locomotor dysfunction in lacking correlated with structural and functional abnormalities in GABAergic neuromuscular junctions (NMJs). Moreover, we demonstrated that reduction in signaling reversed these abnormalities. Remarkably, enhancing GABAergic neurotransmission alone was able to correct the locomotor dysfunction. Our work indicated that an imbalance of excitatory/inhibitory activity within motor circuits and underlies motor system dysfunction in this SMA model. Interventions aimed at restoring the balance of excitatory/inhibitory activity in motor circuits could be of benefit to individuals with SMA.
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http://dx.doi.org/10.1523/ENEURO.0289-18.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6325564PMC
March 2019

Neuropeptide signaling regulates the susceptibility of developing C. elegans to anoxia.

Free Radic Biol Med 2019 02 6;131:197-208. Epub 2018 Dec 6.

Division of Neurology, Department of Pediatrics, Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA; Neuroscience Graduate Group, University of Pennsylvania, Philadelphia, PA 19104, USA; Department of Neurology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA.

Inadequate delivery of oxygen to organisms during development can lead to cell dysfunction/death and life-long disabilities. Although the susceptibility of developing cells to low oxygen conditions changes with maturation, the cellular and molecular pathways that govern responses to low oxygen are incompletely understood. Here we show that developing Caenorhabditis elegans are substantially more sensitive to anoxia than adult animals and that this sensitivity is controlled by nervous system generated hormones (e.g., neuropeptides). A screen of neuropeptide genes identified and validated nlp-40 and its receptor aex-2 as a key regulator of anoxic survival in developing worms. The survival-promoting action of impaired neuropeptide signaling does not rely on five known stress resistance pathways and is specific to anoxic insult. Together, these data highlight a novel cell non-autonomous pathway that regulates the susceptibility of developing organisms to anoxia.
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http://dx.doi.org/10.1016/j.freeradbiomed.2018.12.006DOI Listing
February 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 regulates behavior and expression of schizophrenia risk enriched gene sets in mouse hippocampus.

PLoS One 2018 11;13(7):e0200477. Epub 2018 Jul 11.

Feinberg School of Medicine, Department of Neurology, Northwestern University, Chicago, Illinois, United States of America.

Synapse associated protein of 97KDa (SAP97) belongs to a family of scaffolding proteins, the membrane-associated guanylate kinases (MAGUKs), that are highly enriched in the postsynaptic density of synapses and play an important role in organizing protein complexes necessary for synaptic development and plasticity. The Dlg-MAGUK family of proteins are structurally very similar, and an effort has been made to parse apart the unique function of each Dlg-MAGUK protein by characterization of knockout mice. Knockout mice have been generated and characterized for PSD-95, PSD-93, and SAP102, however SAP97 knockout mice have been impossible to study because the SAP97 null mice die soon after birth due to a craniofacial defect. We studied the transcriptomic and behavioral consequences of a brain-specific conditional knockout of SAP97 (SAP97-cKO). RNA sequencing from hippocampi from control and SAP97-cKO male animals identified 67 SAP97 regulated transcripts. As large-scale genetic studies have implicated MAGUKs in neuropsychiatric disorders such as intellectual disability, autism spectrum disorders, and schizophrenia (SCZ), we analyzed our differentially expressed gene (DEG) set for enrichment of disease risk-associated genes, and found our DEG set to be specifically enriched for SCZ-related genes. Subjecting SAP97-cKO mice to a battery of behavioral tests revealed a subtle male-specific cognitive deficit and female-specific motor deficit, while other behaviors were largely unaffected. These data suggest that loss of SAP97 may have a modest contribution to organismal behavior. The SAP97-cKO mouse serves as a stepping stone for understanding the unique role of SAP97 in biology.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0200477PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6040763PMC
January 2019

Genetic induction of hypometabolism by ablation of MC4R does not suppress ALS-like phenotypes in the G93A mutant SOD1 mouse model.

Sci Rep 2017 10 13;7(1):13150. Epub 2017 Oct 13.

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

Dysfunction and death of motor neurons leads to progressive paralysis in amyotrophic lateral sclerosis (ALS). Recent studies have reported organism-level metabolic dysfunction as a prominent but poorly understood feature of the disease. ALS patients are hypermetabolic with increased resting energy expenditure, but if and how hypermetabolism contributes to disease pathology is unknown. We asked if decreasing metabolism in the mutant superoxide dismutase 1 (SOD1) mouse model of ALS (G93A SOD1) would alter motor function and survival. To address this, we generated mice with the G93A SOD1 mutation that also lacked the melanocortin-4 receptor (MC4R). MC4R is a critical regulator of energy homeostasis and food intake in the hypothalamus. Loss of MC4R is known to induce hyperphagia and hypometabolism in mice. In the MC4R null background, G93A SOD1 mice become markedly hypometabolic, overweight and less active. Decreased metabolic rate, however, did not reverse any ALS-related disease phenotypes such as motor dysfunction or decreased lifespan. While hypermetabolism remains an intriguing target for intervention in ALS patients and disease models, our data indicate that the melanocortin system is not a good target for manipulation. Investigating other pathways may reveal optimal targets for addressing metabolic dysfunction in ALS.
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http://dx.doi.org/10.1038/s41598-017-13304-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5640619PMC
October 2017

Dopamine induces soluble α-synuclein oligomers and nigrostriatal degeneration.

Nat Neurosci 2017 Nov 18;20(11):1560-1568. Epub 2017 Sep 18.

Neuroscience Graduate Group, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Parkinson's disease (PD) is defined by the loss of dopaminergic neurons in the substantia nigra and the formation of Lewy body inclusions containing aggregated α-synuclein. Efforts to explain dopamine neuron vulnerability are hindered by the lack of dopaminergic cell death in α-synuclein transgenic mice. To address this, we manipulated both dopamine levels and α-synuclein expression. Nigrally targeted expression of mutant tyrosine hydroxylase with enhanced catalytic activity increased dopamine levels without damaging neurons in non-transgenic mice. In contrast, raising dopamine levels in mice expressing human A53T mutant α-synuclein induced progressive nigrostriatal degeneration and reduced locomotion. Dopamine elevation in A53T mice increased levels of potentially toxic α-synuclein oligomers, resulting in conformationally and functionally modified species. Moreover, in genetically tractable Caenorhabditis elegans models, expression of α-synuclein mutated at the site of interaction with dopamine prevented dopamine-induced toxicity. These data suggest that a unique mechanism links two cardinal features of PD: dopaminergic cell death and α-synuclein aggregation.
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http://dx.doi.org/10.1038/nn.4641DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5893155PMC
November 2017

The Neuroprotective Marine Compound Psammaplysene A Binds the RNA-Binding Protein HNRNPK.

Mar Drugs 2017 Aug 7;15(8). Epub 2017 Aug 7.

Department of Pediatrics, Division of Neurology, Research Institute, Children's Hospital of Philadelphia, Room 814, 3615 Civic Center Boulevard, Philadelphia, PA 19104, USA.

In previous work, we characterized the strong neuroprotective properties of the marine compound Psammaplysene A (PA) in in vitro and in vivo models of neurodegeneration. Based on its strong neuroprotective activity, the current work attempts to identify the physical target of PA to gain mechanistic insight into its molecular action. Two distinct methods, used in parallel, to purify protein-binding partners of PA led to the identification of HNRNPK as a direct target of PA. Based on surface plasmon resonance, we find that the binding of PA to HNRNPK is RNA-dependent. These findings suggest a role for HNRNPK-dependent processes in neurodegeneration/neuroprotection, and warrant further study of HNRNPK in this context.
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http://dx.doi.org/10.3390/md15080246DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5577601PMC
August 2017

Ablation of intact hypothalamic and/or hindbrain TrkB signaling leads to perturbations in energy balance.

Mol Metab 2015 Nov 18;4(11):867-80. Epub 2015 Aug 18.

Department of Biomedical Sciences, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA.

Objective: Brain-derived neurotrophic factor (BDNF) and its receptor, tropomyosin receptor kinase B (TrkB), play a paramount role in the central regulation of energy balance. Despite the substantial body of genetic evidence implicating BDNF- or TrkB-deficiency in human obesity, the critical brain region(s) contributing to the endogenous role of BDNF/TrkB signaling in metabolic control remain unknown.

Methods: We assessed the importance of intact hypothalamic or hindbrain TrkB signaling in central regulation of energy balance by generating Nkx2.1-Ntrk2-/- and Phox2b-Ntrk2+/- mice, respectively, and comparing metabolic parameters (body weight, adiposity, food intake, energy expenditure and glucose homeostasis) under high-fat diet or chow fed conditions.

Results: Our data show that when fed a high-fat diet, male and female Nkx2.1-Ntrk2-/- mice have significantly increased body weight and adiposity that is likely driven by reduced locomotor activity and core body temperature. When maintained on a chow diet, female Nkx2.1-Ntrk2-/- mice exhibit an increased body weight and adiposity phenotype more robust than in males, which is accompanied by hyperphagia that precedes the onset of a body weight difference. In addition, under both diet conditions, Nkx2.1-Ntrk2-/- mice show increased blood glucose, serum insulin and leptin levels. Mice with complete hindbrain TrkB-deficiency (Phox2b-Ntrk2-/-) are perinatal lethal, potentially indicating a vital role for TrkB in visceral motor neurons that control cardiovascular, respiratory, and digestive functions during development. Phox2b-Ntrk2+/- heterozygous mice are similar in body weight, adiposity and glucose homeostasis parameters compared to wild type littermate controls when maintained on a high-fat or chow diet. Interestingly, despite the absence of a body weight difference, Phox2b-Ntrk2+/- heterozygous mice exhibit pronounced hyperphagia.

Conclusion: Taken together, our findings suggest that the hypothalamus is a key brain region involved in endogenous BDNF/TrkB signaling and central metabolic control and that endogenous hindbrain TrkB likely plays a role in modulating food intake and survival of mice. Our findings also show that female mice lacking TrkB in the hypothalamus have a more robust metabolic phenotype.
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http://dx.doi.org/10.1016/j.molmet.2015.08.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4632115PMC
November 2015

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

A Critical Role for the GluA1 Accessory Protein, SAP97, in Cocaine Seeking.

Neuropsychopharmacology 2016 Feb 7;41(3):736-50. Epub 2015 Jul 7.

Department of Psychiatry, Center for Neurobiology and Behavior, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA.

A growing body of evidence indicates that the transport of GluA1 subunit-containing calcium-permeable AMPA receptors (CP-AMPARs) to synapses in subregions of the nucleus accumbens promotes cocaine seeking. Consistent with these findings, the present results show that administration of the CP-AMPAR antagonist, Naspm, into the caudal lateral core or caudal medial shell of the nucleus accumbens attenuated cocaine priming-induced reinstatement of drug seeking. Moreover, viral-mediated overexpression of 'pore dead' GluA1 subunits (via herpes simplex virus (HSV) GluA1-Q582E) in the lateral core or medial shell attenuated the reinstatement of cocaine seeking. The overexpression of wild-type GluA1 subunits (via HSV GluA1-WT) in the medial shell, but not the lateral core, enhanced the reinstatement of cocaine seeking. These results indicate that activation of GluA1-containing AMPARs in subregions of the nucleus accumbens reinstates cocaine seeking. SAP97 and 4.1N are proteins involved in GluA1 trafficking to and stabilization in synapses; SAP97-GluA1 interactions also influence dendritic growth. We next examined potential roles of SAP97 and 4.1N in cocaine seeking. Viral-mediated expression of a microRNA that reduces SAP97 protein expression (HSV miSAP97) in the medial accumbens shell attenuated cocaine seeking. In contrast, a virus that overexpressed a dominant-negative form of a 4.1N C-terminal domain (HSV 4.1N-CTD), which prevents endogenous 4.1N binding to GluA1 subunits, had no effect on cocaine seeking. These results indicate that the GluA1 subunit accessory protein SAP97 may represent a novel target for pharmacotherapeutic intervention in the treatment of cocaine craving.
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http://dx.doi.org/10.1038/npp.2015.199DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4707820PMC
February 2016

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

Oxygen sensing neurons and neuropeptides regulate survival after anoxia in developing C. elegans.

PLoS One 2014 26;9(6):e101102. Epub 2014 Jun 26.

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

Hypoxic brain injury remains a major source of neurodevelopmental impairment for both term and preterm infants. The perinatal period is a time of rapid transition in oxygen environments and developmental resetting of oxygen sensing. The relationship between neural oxygen sensing ability and hypoxic injury has not been studied. The oxygen sensing circuitry in the model organism C. elegans is well understood. We leveraged this information to investigate the effects of impairments in oxygen sensing on survival after anoxia. There was a significant survival advantage in developing worms specifically unable to sense oxygen shifts below their preferred physiologic range via genetic ablation of BAG neurons, which appear important for conferring sensitivity to anoxia. Oxygen sensing that is mediated through guanylate cyclases (gcy-31, 33, 35) is unlikely to be involved in conferring this sensitivity. Additionally, animals unable to process or elaborate neuropeptides displayed a survival advantage after anoxia. Based on these data, we hypothesized that elaboration of neuropeptides by BAG neurons sensitized animals to anoxia, but further experiments indicate that this is unlikely to be true. Instead, it seems that neuropeptides and signaling from oxygen sensing neurons operate through independent mechanisms, each conferring sensitivity to anoxia in wild type animals.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0101102PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4072718PMC
October 2015

Genetically altering organismal metabolism by leptin-deficiency benefits a mouse model of amyotrophic lateral sclerosis.

Hum Mol Genet 2014 Sep 15;23(18):4995-5008. Epub 2014 May 15.

Division of Neurology, Department of Pediatrics, Research Institute, Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA, Neuroscience Graduate Group, Department of Neurology and

Amyotrophic lateral sclerosis (ALS) is a fatal, neurodegenerative disease that causes death of motor neurons. ALS patients and mouse models of familial ALS display organismal level metabolic dysfunction, which includes increased energy expenditure despite decreased lean mass. The pathophysiological relevance of abnormal energy homeostasis to motor neuron disease remains unclear. Leptin is an adipocyte-derived hormone that regulates whole-animal energy expenditure. Here, we report that placing mutant superoxide dismutase 1 (SOD1) mice in a leptin-deficient background improves energy homeostasis and slows disease progression. Leptin-deficient mutant SOD1 mice possess increased bodyweight and fat mass, as well as decreased energy expenditure. These observations coincide with enhanced survival, improved strength and decreased motor neuron loss. These results suggest that altering whole-body energy metabolism in mutant SOD1 mice can mitigate disease progression. We propose that manipulations that increase fat mass and reduce energy expenditure will be beneficial in the setting of motor neuron disease.
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http://dx.doi.org/10.1093/hmg/ddu214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4140473PMC
September 2014

GluA1 promotes the activity-dependent development of motor circuitry in the developing segmental spinal cord.

Ann N Y Acad Sci 2013 Mar;1279:54-9

Neuroscience Graduate Group, Department of Neuroscience, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

The neuronal dendritic tree is a key determinant of how neurons receive, compute, and transmit information. During early postnatal life, synaptic activity promotes dendrite elaboration. Spinal motor neurons utilize GluA1-containing AMPA (2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl) propanoic acid) receptors (AMPA-R) to control this process. This form of developmental dendrite growth can occur independently of N-methyl-d-aspartate receptors (NMDA-R). This review focuses on the mechanism by which the GluA1 subunit of AMPA-R transforms synaptic activity into dendrite growth, and describes the essential role of the GluA1 binding partner SAP97 (synapse-associated protein of 97 kDa molecular weight) in this process. This work defines a new mechanism of activity-dependent development, which might be harnessed to stimulate the recovery of function following insult to the central nervous system.
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http://dx.doi.org/10.1111/nyas.12053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4205102PMC
March 2013

The molecular basis of experience-dependent motor system development.

Adv Exp Med Biol 2013 ;782:23-38

Department of Neurology, Children's Hospital of Philadelphia, Perelman School of Medicine, University of Pennsylvania, 3615 Civic Center Boulevard, 19104, Philadelphia, PA, USA,

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http://dx.doi.org/10.1007/978-1-4614-5465-6_2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4530779PMC
April 2016

Stress and aging induce distinct polyQ protein aggregation states.

Proc Natl Acad Sci U S A 2012 Jun 29;109(26):10587-92. Epub 2012 May 29.

Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

Many age-related diseases are known to elicit protein misfolding and aggregation. Whereas environmental stressors, such as temperature, oxidative stress, and osmotic stress, can also damage proteins, it is not known whether aging and the environment impact protein folding in the same or different ways. Using polyQ reporters of protein folding in both Caenorhabditis elegans and mammalian cell culture, we show that osmotic stress, but not other proteotoxic stressors, induces rapid (minutes) cytoplasmic polyQ aggregation. Osmotic stress-induced polyQ aggregates could be distinguished from aging-induced polyQ aggregates based on morphological, biophysical, cell biological, and biochemical criteria, suggesting that they are a unique misfolded-protein species. The insulin-like growth factor signaling mutant daf-2, which inhibits age-induced polyQ aggregation and protects C. elegans from stress, did not prevent the formation of stress-induced polyQ aggregates. However, osmotic stress resistance mutants, which genetically activate the osmotic stress response, strongly inhibited the formation of osmotic polyQ aggregates. Our findings show that in vivo, the same protein can adopt distinct aggregation states depending on the initiating stressor and that stress and aging impact the proteome in related but distinct ways.
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http://dx.doi.org/10.1073/pnas.1108766109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3387092PMC
June 2012

Daf-2 signaling modifies mutant SOD1 toxicity in C. elegans.

PLoS One 2012 20;7(3):e33494. Epub 2012 Mar 20.

Department of Pediatrics, Division of Neurology, Children's Hospital of Philadelphia Research Institute, Philadelphia, Pennsylvania, United States of America.

The DAF-2 Insulin/IGF-1 signaling (IIS) pathway is a strong modifier of Caenorhabditis elegans longevity and healthspan. As aging is the greatest risk factor for developing neurodegenerative diseases such as Amyotrophic Lateral Sclerosis (ALS), we were interested in determining if DAF-2 signaling modifies disease pathology in mutant superoxide dismutase 1 (SOD1) expressing C. elegans. Worms with pan-neuronal G85R SOD1 expression demonstrate significantly impaired locomotion as compared to WT SOD1 expressing controls and they develop insoluble SOD1 aggregates. Reductions in DAF-2 signaling, either through a hypomorphic allele or neuronally targeted RNAi, decreases the abundance of aggregated SOD1 and results in improved locomotion in a DAF-16 dependant manner. These results suggest that manipulation of the DAF-2 Insulin/IGF-1 signaling pathway may have therapeutic potential for the treatment of ALS.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0033494PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3308959PMC
August 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

PolyQ repeat expansions in ATXN2 associated with ALS are CAA interrupted repeats.

PLoS One 2011 Mar 29;6(3):e17951. Epub 2011 Mar 29.

Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

Amyotrophic lateral sclerosis (ALS) is a devastating, rapidly progressive disease leading to paralysis and death. Recently, intermediate length polyglutamine (polyQ) repeats of 27-33 in ATAXIN-2 (ATXN2), encoding the ATXN2 protein, were found to increase risk for ALS. In ATXN2, polyQ expansions of ≥ 34, which are pure CAG repeat expansions, cause spinocerebellar ataxia type 2. However, similar length expansions that are interrupted with other codons, can present atypically with parkinsonism, suggesting that configuration of the repeat sequence plays an important role in disease manifestation in ATXN2 polyQ expansion diseases. Here we determined whether the expansions in ATXN2 associated with ALS were pure or interrupted CAG repeats, and defined single nucleotide polymorphisms (SNPs) rs695871 and rs695872 in exon 1 of the gene, to assess haplotype association. We found that the expanded repeat alleles of 40 ALS patients and 9 long-repeat length controls were all interrupted, bearing 1-3 CAA codons within the CAG repeat. 21/21 expanded ALS chromosomes with 3CAA interruptions arose from one haplotype (GT), while 18/19 expanded ALS chromosomes with <3CAA interruptions arose from a different haplotype (CC). Moreover, age of disease onset was significantly earlier in patients bearing 3 interruptions vs fewer, and was distinct between haplotypes. These results indicate that CAG repeat expansions in ATXN2 associated with ALS are uniformly interrupted repeats and that the nature of the repeat sequence and haplotype, as well as length of polyQ repeat, may play a role in the neurological effect conferred by expansions in ATXN2.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0017951PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3066214PMC
March 2011

Regulation of Foxo-dependent transcription by post-translational modifications.

Curr Drug Targets 2011 Aug;12(9):1303-10

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

The Forkhead Box O (Foxo) proteins represent an evolutionarily conserved family of transcription factors that play an important role in regulating processes including metabolism, longevity, and cell death/survival. How is it that a single transcription factor can initiate such divergent cellular responses? We will review the evidence that specific patterns of post-translational modifications play a key role in directing Foxo into various transcriptional readouts. This regulation appears to take on a two tiered regulatory model; with a group of well defined post-translational modifications regulating nuclear localization and transcriptional activity while a second set of modifications regulate the transcriptional specificity of Foxo.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3794852PMC
http://dx.doi.org/10.2174/138945011796150316DOI Listing
August 2011

A switch in retrograde signaling from survival to stress in rapid-onset neurodegeneration.

J Neurosci 2009 Aug;29(31):9903-17

University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.

Retrograde axonal transport of cellular signals driven by dynein is vital for neuronal survival. Mouse models with defects in the retrograde transport machinery, including the Loa mouse (point mutation in dynein) and the Tg(dynamitin) mouse (overexpression of dynamitin), exhibit mild neurodegenerative disease. Transport defects have also been observed in more rapidly progressive neurodegeneration, such as that observed in the SOD1(G93A) transgenic mouse model for familial amyotrophic lateral sclerosis (ALS). Here, we test the hypothesis that alterations in retrograde signaling lead to neurodegeneration. In vivo, in vitro, and live-cell imaging motility assays show misregulation of transport and inhibition of retrograde signaling in the SOD1(G93A) model. However, similar inhibition is also seen in the Loa and Tg(dynamitin) mouse models. Thus, slowing of retrograde signaling leads only to mild degeneration and cannot explain ALS etiology. To further pursue this question, we used a proteomics approach to investigate dynein-associated retrograde signaling. These data indicate a significant decrease in retrograde survival factors, including P-Trk (phospho-Trk) and P-Erk1/2, and an increase in retrograde stress factor signaling, including P-JNK (phosphorylated c-Jun N-terminal kinase), caspase-8, and p75(NTR) cleavage fragment in the SOD1(G93A) model; similar changes are not seen in the Loa mouse. Cocultures of motor neurons and glia expressing mutant SOD1 (mSOD1) in compartmentalized chambers indicate that inhibition of retrograde stress signaling is sufficient to block activation of cellular stress pathways and to rescue motor neurons from mSOD1-induced toxicity. Hence, a shift from survival-promoting to death-promoting retrograde signaling may be key to the rapid onset of neurodegeneration seen in ALS.
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http://dx.doi.org/10.1523/JNEUROSCI.0813-09.2009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3095444PMC
August 2009

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

AMPA receptor antibodies in limbic encephalitis alter synaptic receptor location.

Ann Neurol 2009 Apr;65(4):424-34

Department of Neurology, University of Pennsylvania, Philadelphia, PA 19104, USA.

Objective: To report the clinical and immunological features of a novel autoantigen related to limbic encephalitis (LE) and the effect of patients' antibodies on neuronal cultures.

Methods: We conducted clinical analyses of 10 patients with LE. Immunoprecipitation and mass spectrometry were used to identify the antigens. Human embryonic kidney 293 cells expressing the antigens were used in immunocytochemistry and enzyme-linked immunoabsorption assay. The effect of patients' antibodies on cultures of live rat hippocampal neurons was determined with confocal microscopy.

Results: Median age was 60 (38-87) years; 9 were women. Seven had tumors of the lung, breast, or thymus. Nine patients responded to immunotherapy or oncological therapy, but neurological relapses, without tumor recurrence, were frequent and influenced the long-term outcome. One untreated patient died of LE. All patients had antibodies against neuronal cell surface antigens that by immunoprecipitation were found to be the glutamate receptor 1 (GluR1) and GluR2 subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR). Human embryonic kidney 293 cells expressing GluR1/2 reacted with all patients' sera or cerebrospinal fluid, providing a diagnostic test for the disorder. Application of antibodies to cultures of neurons significantly decreased the number of GluR2-containing AMPAR clusters at synapses with a smaller decrease in overall AMPAR cluster density; these effects were reversed after antibody removal.

Interpretation: Antibodies to GluR1/2 associate with LE that is often paraneoplastic, treatment responsive, and has a tendency to relapse. Our findings support an antibody-mediated pathogenesis in which patients' antibodies alter the synaptic localization and number of AMPARs.
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http://dx.doi.org/10.1002/ana.21589DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2677127PMC
April 2009

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