Gamma-Synuclein-Mediated Regulation of Norepinephrine Transporter

Summary

Principal Investigator: Anita Sidhu
Abstract: Aberrant norepinephrine [NE] neurotransmission in the human brain is linked to mood disorders, depression, drug addiction and neurodegenerative diseases. -synuclein [-Syn], a member of the synuclein family of proteins, is expressed in monoaminergic neurons, but its function in the brain is not known. We have shown that .-Syn can modulate the function and trafficking of the NE transporter [NET], through interactions with the microtubule [MT] cytoskeleton. We show here that -Syn can also regulate these NET activities. Such modulation by -Syn is unique to NET. In a rat model of depression, there is overexpression of -Syn, causing NET function and trafficking to be dysregulated and unresponsive to the effects of nocodazole [a MT destabilizing agent], probably due to tight binding of the -Syn/NET complex to the MT cytoskeleton. Chronic treatment of these animals with desipramine [a NET blocker] reduces -Syn protein expression, while increasing .-Syn levels, permitting NET to be appropriately regulated by .-Syn, with full restoration of nocodazole sensitivity. In postmortem brains from patients with depression, we also show that -Syn is overexpressed, adding clinical relevance for a role for -Syn in the genesis and maintenance of depression in humans. We hypothesize that -Syn acts as a prodepressant, and that imbalances in -Syn/.-Syn expression levels is central to the genesis of depression. When overexpressed, -Syn overrides the normative regulation of NET by .-Syn. Therefore, targeting -Syn expression levels may be key to controlling depression in humans. We will investigate here in detail the cellular and molecular mechanisms by which desipramine reduces -Syn levels in both in vitro and in vivo models, which express either -Syn alone or both -Syn and NET, in the presence or absence of .-Syn. We will also analyze the mechanisms by which other NET antidepressants decrease -Syn expression in in vitro models. Finally, we will measure neurochemical and behavioral responses to NET antidepressants in .-Syn overexpressing transgenic mice, as well as in .-Syn knock-out mice. From these studies we will be able to assess the mechanisms by which -Syn expression is regulated as well as ascertain the physiopathological relevance of sucrtain if DMI affects -Syn promoter thereby altering its expression levels, we will conduct nuclear run-on assays, using isolated nuclei. Transcription and RNA labeling will be performed for 30 min in a reaction buffer containing 5 mM each of ATP, CTP, GTP, and 32P-UTP. RNA will be isolated using Trizol reagent. Nylon membranes will be slot-blotted with 2.5 g of cDNA sequences encoding -Syn (nucleotides 49-432 of cDNA), or -actin (nucleotides 50-545 of cDNA), in separate wells. Hybridization will performed and autoradiographs will be quantified using Quantity OneTM quantitation software and -Syn levels normalized against -actin. In addition to Desipramine, we will also examine the effects of several antidepressants in T470 cells that are structurally similar and dissimilar to Desipramine, to determine if the response elicited by DMI is selective for this compound or a characteristic feature of antidepressants. Some of these antidepressants include: imipramine, reboxetine, as well as structurally un-related antidepressants, trazadone and amoxapine. Research Plan for Specific Aim 2: For these series of studies, we will use two species [and speciesappropriate non-transgenic controls] of transgenic mice: .-Syn knock-out mice expressing -Syn only and .-Syn overexpressing mice. Adult mice of bothA levels and stability, as well as their relative distribution and co-localization after treatment with the sntidepressant. The effect of other antidepressants on -Syn expression that are structurally similar and dissimilar to DMI will also be examined. Specific Aim 2: To dissect the individual participation of .-Syn and -Syn in transgenic mice [.-Syn knock-out mice and .-Syn overexpressing mice] in regulating NET function and their response to DMI. Results from these studies will help dissect the contribution of each of the synucleins on NET trafficking and cytoskeletal protein interactions, before and after treatment with desipramine. Research Plan for SA 1: Our published and preliminary results show that DMI reduces -Syn levels in the depressive rat model, WKY rat, and in endogenously expressing cells, such as the T-470 breast carcinoma cells. We will examine the molecular mechanisms by which DMI causes a reduction in -Syn levels by conducting studies in the T470 breast cancer cells which express only -Syn, but not .-Syn. Thus, time-course and dose-response studies will be followed by Western blots to analyze -Syn levels, after treatment with specific levels of DMI for specific time periods. Co-immunoprecipitation studies will be conducted to measure interactions between -Syn and microtubule binding proteins [MAPs, tau] and microtubules [tubulin]. Protein stability will be analyzed by pulse-chase labeling using [3H]methionine followed by immunoprecipitation with -Syn antibodies. mRNA levels will be assessed by real-time RTPCR and mRNA stability will be analyzed after blockade of RNA transcription by actinomycin D (10 ug/ml). To ascertain if DMI affects -Syn promoter thereby altering its expression levels, we will conduct nuclear run-on assays, using isolated nuclei. Transcription and RNA labeling will be performed for 30 min in a reaction buffer containing 5 mM each of ATP, CTP, GTP, and 32P-UTP. RNA will be isolated using Trizol reagent. Nylon membranes will be slot-blotted with 2.5 g of cDNA sequences encoding -Syn (nucleotides 49-432 of cDNA), or -actin (nucleotides 50-545 of cDNA), in separate wells. Hybridization will performed and autoradiographs will be quantified using Quantity OneTM quantitation software and -Syn levels normaecific therapy. This grant is also relevant for the ARRA in that it will immediately create 3 new professional jobs.
Funding Period: ----------------2009 - ---------------2011-
more information: NIH RePORT

Top Publications

  1. pmc Age-dependent effects of A53T alpha-synuclein on behavior and dopaminergic function
    Adam W Oaks
    Laboratory of Molecular Neurochemistry, Department of Biochemistry and Molecular and Cellular Biology, Georgetown University Medical Center, Washington, DC, USA
    PLoS ONE 8:e60378. 2013
  2. pmc Synucleins antagonize endoplasmic reticulum function to modulate dopamine transporter trafficking
    Adam W Oaks
    Laboratory of Molecular Neurochemistry, Department of Biochemistry and Molecular and Cellular Biology, Georgetown University Medical Center, Washington, DC, USA
    PLoS ONE 8:e70872. 2013
  3. pmc Desipramine induced changes in the norepinephrine transporter, alpha- and gamma-synuclein in the hippocampus, amygdala and striatum
    ALEXIS M JEANNOTTE
    Department of Biochemistry and Molecular and Cell Biology, Georgetown University Medical Center, Georgetown University, Washington, DC 20007, United States
    Neurosci Lett 467:86-9. 2009
  4. pmc Mice expressing the A53T mutant form of human alpha-synuclein exhibit hyperactivity and reduced anxiety-like behavior
    Dianca R Graham
    Department of Biochemistry, Molecular and Cellular Biology, Georgetown University, Washington, DC 20007, USA
    J Neurosci Res 88:1777-83. 2010
  5. pmc Synuclein modulation of monoamine transporters
    Adam W Oaks
    Department of Biochemistry, Molecular, Cellular Biology and Lombardi Cancer Center, Georgetown University, Washington, DC 20007, United States
    FEBS Lett 585:1001-6. 2011
  6. pmc Hyperphosphorylated Tau in an α-synuclein-overexpressing transgenic model of Parkinson's disease
    Thomas Haggerty
    Laboratory of Molecular Neurochemistry, Department of Biochemistry and Molecular and Cell Biology, Georgetown University Medical Center, The New Research Building, Room W222, Washington, DC 20007, USA
    Eur J Neurosci 33:1598-610. 2011
  7. pmc Region-specific tauopathy and synucleinopathy in brain of the alpha-synuclein overexpressing mouse model of Parkinson's disease
    Tiffany Kaul
    Department of Biochemistry and Molecular and Cell Biology, University of California San Diego, La Jolla, California, USA
    BMC Neurosci 12:79. 2011
  8. pmc Paraquat, but not maneb, induces synucleinopathy and tauopathy in striata of mice through inhibition of proteasomal and autophagic pathways
    Jonathan Wills
    Department of Biochemistry and Molecular and Cell Biology, Georgetown University Medical Center, Washington DC, United States of America
    PLoS ONE 7:e30745. 2012

Scientific Experts

  • Tiffany Kaul
  • Adam W Oaks
  • Anita Sidhu
  • Jonathan Wills
  • Joel Credle
  • Thomas Haggerty
  • Dianca R Graham
  • ALEXIS M JEANNOTTE
  • Nicholas Marsh-Armstrong
  • David I Finkelstein
  • Jessica M Jones
  • Maya Frankfurt
  • Joel J Credle
  • Jessica Jones
  • Valeriy Duka
  • Jae Hoon Lee
  • Olga Rodriguez
  • Eliezer Masliah
  • John G McCarthy

Detail Information

Publications8

  1. pmc Age-dependent effects of A53T alpha-synuclein on behavior and dopaminergic function
    Adam W Oaks
    Laboratory of Molecular Neurochemistry, Department of Biochemistry and Molecular and Cellular Biology, Georgetown University Medical Center, Washington, DC, USA
    PLoS ONE 8:e60378. 2013
    ..These findings highlight the involvement of the synuclein family of proteins and phosphorylation of Tau in the response to dopaminergic dysfunction of the nigrostriatal pathway...
  2. pmc Synucleins antagonize endoplasmic reticulum function to modulate dopamine transporter trafficking
    Adam W Oaks
    Laboratory of Molecular Neurochemistry, Department of Biochemistry and Molecular and Cellular Biology, Georgetown University Medical Center, Washington, DC, USA
    PLoS ONE 8:e70872. 2013
    ....
  3. pmc Desipramine induced changes in the norepinephrine transporter, alpha- and gamma-synuclein in the hippocampus, amygdala and striatum
    ALEXIS M JEANNOTTE
    Department of Biochemistry and Molecular and Cell Biology, Georgetown University Medical Center, Georgetown University, Washington, DC 20007, United States
    Neurosci Lett 467:86-9. 2009
    ....
  4. pmc Mice expressing the A53T mutant form of human alpha-synuclein exhibit hyperactivity and reduced anxiety-like behavior
    Dianca R Graham
    Department of Biochemistry, Molecular and Cellular Biology, Georgetown University, Washington, DC 20007, USA
    J Neurosci Res 88:1777-83. 2010
    ..These results indicate a possible role of the A53T alpha-Syn mutation in anxiety-like and hyperactive behaviors in a PD mouse model, suggesting that these behaviors might be comorbid with this disease...
  5. pmc Synuclein modulation of monoamine transporters
    Adam W Oaks
    Department of Biochemistry, Molecular, Cellular Biology and Lombardi Cancer Center, Georgetown University, Washington, DC 20007, United States
    FEBS Lett 585:1001-6. 2011
    ....
  6. pmc Hyperphosphorylated Tau in an α-synuclein-overexpressing transgenic model of Parkinson's disease
    Thomas Haggerty
    Laboratory of Molecular Neurochemistry, Department of Biochemistry and Molecular and Cell Biology, Georgetown University Medical Center, The New Research Building, Room W222, Washington, DC 20007, USA
    Eur J Neurosci 33:1598-610. 2011
    ..The elevated state of tauopathy seen in these platelet-derived growth factor-α-synuclein mice provides further confirmation that PD may be a tauopathic disease...
  7. pmc Region-specific tauopathy and synucleinopathy in brain of the alpha-synuclein overexpressing mouse model of Parkinson's disease
    Tiffany Kaul
    Department of Biochemistry and Molecular and Cell Biology, University of California San Diego, La Jolla, California, USA
    BMC Neurosci 12:79. 2011
    ..The current studies were undertaken to analyze the distribution of tauopathy in different brain regions in a widely used mouse model of PD, the α-Syn overexpressing mouse...
  8. pmc Paraquat, but not maneb, induces synucleinopathy and tauopathy in striata of mice through inhibition of proteasomal and autophagic pathways
    Jonathan Wills
    Department of Biochemistry and Molecular and Cell Biology, Georgetown University Medical Center, Washington DC, United States of America
    PLoS ONE 7:e30745. 2012
    ....