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ABSTRACT: Dysregulation of glutamate handling ensuing downregulation of expression and activity levels of the astroglial glutamate transporter EAAT2 is implicated in excitotoxic degeneration of motor neurons in amyotrophic lateral sclerosis (ALS). We previously reported that EAAT2 (a.k.a. GLT-1) is cleaved by caspase-3 at its cytosolic carboxy-terminus domain. This cleavage results in impaired glutamate transport activity and generates a proteolytic fragment (CTE) that we found to be post-translationally conjugated by SUMO1. We show here that this sumoylated CTE fragment accumulates in the nucleus of spinal cord astrocytes of the SOD1-G93A mouse model of ALS at symptomatic stages of disease. Astrocytic expression of CTE, artificially tagged with SUMO1 (CTE-SUMO1) to mimic the native sumoylated fragment, recapitulates the nuclear accumulation pattern of the endogenous EAAT2-derived proteolytic fragment. Moreover, in a co-culture binary system, expression of CTE-SUMO1 in spinal cord astrocytes initiates extrinsic toxicity by inducing caspase-3 activation in motor neuron-derived NSC-34 cells or axonal growth impairment in primary motor neurons. Interestingly, prolonged nuclear accumulation of CTE-SUMO1 is intrinsically toxic to spinal cord astrocytes, although this gliotoxic effect of CTE-SUMO1 occurs later than the indirect, noncell autonomous toxic effect on motor neurons. As more evidence on the implication of SUMO substrates in neurodegenerative diseases emerges, our observations strongly suggest that the nuclear accumulation in spinal cord astrocytes of a sumoylated proteolytic fragment of the astroglial glutamate transporter EAAT2 could participate to the pathogenesis of ALS and suggest a novel, unconventional role for EAAT2 in motor neuron degeneration.
Glia 07/2011; 59(11):1719-31. · 4.82 Impact Factor
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ABSTRACT: Mature striatal medium size spiny neurons express the dopamine and cAMP-regulated phosphoprotein, 32 kDa (DARPP-32), but little is known about the mechanisms regulating its levels, or the specification of fully differentiated neuronal subtypes. Cell extrinsic molecules that increase DARPP-32 mRNA and/or protein levels include retinoic acid (RA), brain-derived neurotrophic factor, and estrogen (E(2)). We now demonstrate that RA regulates DARPP-32 mRNA and protein in primary striatal neuronal cultures. Furthermore, DARPP-32 induction by RA in vitro requires phosphatidylinositide 3-kinase, but is independent of tropomyosin-related kinase B, cyclin-dependent kinase 5, and protein kinase B. Using pharmacologic inhibitors of various isoforms of protein kinase C (PKC), we also demonstrate that DARPP-32 induction by RA in vitro is dependent on PKC zeta (PKCzeta). Thus, the signal transduction pathways mediated by RA are very different than those mediating DARPP-32 induction by brain-derived neurotrophic factor. These data support the presence of multiple signal transduction pathways mediating expression of DARPP-32 in vitro, including a novel, important pathway via which phosphatidylinositide 3-kinase regulates the contribution of PKCzeta.
Journal of Neurochemistry 07/2008; 106(2):917-24. · 4.06 Impact Factor
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ABSTRACT: Synaptic accumulation of glutamate causes neuronal death in many neurodegenerative pathologies including amyotrophic lateral sclerosis. Drugs capable of increasing glutamate uptake could therefore be therapeutically effective. We screened in a cell-based assay a library of 1040 FDA-approved drugs and nutrients for compounds that could enhance glutamate uptake. Nordihydroguaiaretic acid (NDGA), an anti-inflammatory drug that inhibits lipoxygensases, potently enhanced glutamate uptake in MN-1 cells. Given subcutaneously at 1 mg/day for 30 days in mice, NDGA increased glutamate uptake in spinal cord synaptosomes persistently throughout the treatment. However, when administered following the same regimen to the SOD1-G93A transgenic mouse model of ALS at disease onset, NDGA did not extend survival of these mice. We found that NDGA failed to sustain increased glutamate uptake in the SOD1-G93A mice despite an initial upregulation measured during the first 10 days of treatment. SOD1-G93A mice displayed a progressive increase in spinal cord expression levels of the efflux transporter P-glycoprotein beginning at disease onset. This increase was not mediated by the NDGA treatment because it was measured in untreated SOD1-G93A mice. Since P-glycoproteins control the extrusion of a broad range of toxins and xenobiotics and are responsible for drug resistance in many diseases including cancer and brain diseases such as epilepsy, we propose that the failure of NDGA in maintaining glutamate uptake upregulated in SOD1-G93A mice and its therapeutic inefficacy are due to acquired pharmacoresistance mediated by the increased expression of P-glycoprotein.
Experimental Neurology 07/2008; 213(1):229-37. · 4.70 Impact Factor
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ABSTRACT: Mature striatal medium size spiny neurons express the dopamine and cyclic AMP-regulated phosphoprotein, 32 kDa (DARPP-32), but little is known about the mechanisms regulating its levels or the specification of fully differentiated neuronal subtypes. Cell extrinsic molecules that increase DARPP-32 mRNA and/or protein levels include brain-derived neurotrophic factor (BDNF), retinoic acid, and estrogen. DARPP-32 induction by BDNF in vitro requires phosphatidylinositide 3-kinase (PI3K), but inhibition of phosphorylation of protein kinase B/Akt does not entirely abolish expression of DARPP-32. Moreover, the requirement for Akt has not been established. Using pharmacologic inhibitors of PI3K, Akt, and cyclin-dependent kinase 5 (cdk5) and constitutively active and dominant negative PI3K, Akt, cdk5, and p35 viruses in cultured striatal neurons, we measured BDNF-induced levels of DARPP-32 protein and/or mRNA. We demonstrated that both the PI3K/Akt/mammalian target of rapamycin and the cdk5/p35 signal transduction pathways contribute to the induction of DARPP-32 protein levels by BDNF and that the effects are on both the transcriptional and translational levels. It also appears that PI3K is upstream of cdk5/p35, and its activation can lead to an increase in p35 protein levels. These data support the presence of multiple signal transduction pathways mediating expression of DARPP-32 in vitro, including a novel, important pathway via by which PI3K regulates the contribution of cdk5/p35.
Journal of Biological Chemistry 04/2007; 282(10):7352-9. · 4.77 Impact Factor
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ABSTRACT: Studies of metabolism of the Alzheimer amyloid precursor protein (APP) have focused much recent attention on the biology of juxta- and intra-membranous proteases. Release or 'shedding' of the large APP ectodomain can occur via one of two competing pathways, the alpha- and beta-secretase pathways, that are distinguished both by subcellular site of proteolysis and by site of cleavage within APP. The alpha-secretase pathway cleaves within the amyloidogenic Abeta domain of APP, precluding the formation of toxic amyloid aggregates. The relative utilization of the alpha- and beta-secretase pathways is controlled by the activation of certain protein phosphorylation signal transduction pathways including protein kinase C (PKC) and extracellular signal regulated protein kinase [ERK/mitogen-activated protein kinase (MAP kinase)], although the relevant substrates for phosphorylation remain obscure. Because of their apparent ability to decrease the risk for Alzheimer disease, the effects of statins (HMG CoA reductase inhibitors) on APP metabolism were studied. Statin treatment induced an APP processing phenocopy of PKC or ERK activation, raising the possibility that statin effects on APP processing might involve protein phosphorylation. In cultured neuroblastoma cells transfected with human Swedish mutant APP, atorvastatin stimulated the release of alpha-secretase-released, soluble APP (sAPPalpha). However, statin-induced stimulation of sAPPalpha release was not antagonized by inhibitors of either PKC or ERK, or by the co-expression of a dominant negative isoform of ERK (dnERK), indicating that PKC and ERK do not play key roles in mediating the effect of atorvastatin on sAPPalpha secretion. These results suggest that statins may regulate alpha-secretase activity either by altering the biophysical properties of plasma membranes or by modulating the function of as-yet unidentified protein kinases that respond to either cholesterol or to some intermediate in the cholesterol metabolic pathway. A 'phospho-proteomic' analysis of N2a cells with and without statin treatment was performed, revealing changes in the phosphorylation state of several protein kinases plausibly related to APP processing. A systematic evaluation of the possible role of these protein kinases in statin-regulated APP ectodomain shedding is underway.
Journal of Neurochemistry 09/2004; 90(4):1005-10. · 4.06 Impact Factor
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Troy L Carter,
Giuseppe Verdile,
David Groth, Alexey Bogush,
Stefani Thomas,
Patrick Shen,
Paul E Fraser,
Paul Mathews,
Ralph A Nixon,
Michelle E Ehrlich,
John B J Kwok,
Peter St George-Hyslop,
Peter Schofield,
Yueming Li,
Austin Yang,
Ralph N Martins,
Sam Gandy
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ABSTRACT: Presenilins are polytopic, integral proteins that control intramembranous proteolysis at the "gamma-" and "epsilon-" cleavage sites of the Alzheimer amyloid-beta precursor protein (APP) to yield amyloid-beta peptide (Abeta) and the APP intracellular domain (AICD). We have overexpressed a constitutively active, pathogenic form of PS1 (known as PS1 Delta exon 9) together with its substrate, APP-C99, in Spodoptera frugiperda (Sf9) cells. Sf9 cells have been reported to lack endogenous gamma-secretase, an unexpected finding since there exists an insect homologue of PS1. In our hands, neither intact insect cells coexpressing PS1 Delta exon 9/APP-C99 nor the aqueous homogenates of these cells displayed obvious products of the gamma- or epsilon-secretase reactions, as reported. Surprisingly, when APP-C99-expressing cells were homogenized in 3[(3-cholamidopropyl) dimethylammonio]-2-hydroxypropanesulfonic acid (CHAPSO), a detergent known to support gamma-secretase activity, subsequent incubation led to the accumulation of an AICD-like peptide (AICD-L). Aspartyl proteinase inhibitors were effective in preventing the appearance of AICD-L, but inhibitors of other classes of proteinases were ineffective. Immunoprecipitation-mass spectrometry of AICD-L revealed its identity as the minor of the two known AICDs.
Alzheimer Disease and Associated Disorders 18(4):261-3. · 2.81 Impact Factor
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Troy Carter,
Giuseppe Verdile,
D Groth, Alexey Bogush,
Stefani Thomas,
Patrick Shen,
Paul Fraser,
Paul Matthews,
Ralph Nixon,
Michelle Ehrlich,
John Kwok,
Peter St George-Hyslop,
Peter Schofeild,
Yueming Li,
Austin Yang,
Ralph Martins,
Sam Gandy
ECU Publications.
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Michelle E. Page,
Li Bao,
Pierrette Andre,
Joshua Pelta-Heller,
Emily Sluzas,
Pedro Gonzalez-Alegre, Alexey Bogush,
Loren E. Khan,
Lorraine Iacovitti,
Margaret E. Rice,
Michelle E. Ehrlich
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ABSTRACT: Early onset torsion dystonia is an autosomal dominant movement disorder of variable penetrance caused by a glutamic acid, i.e. ΔE, deletion in DYT1, encoding the protein TorsinA. Genetic and structural data implicate basal ganglia dysfunction in dystonia. TorsinA, however, is diffusely expressed, and therefore the primary source of dysfunction may be obscured in pan-neuronal transgenic mouse models. We utilized the tyrosine hydroxylase (TH) promoter to direct transgene expression specifically to dopaminergic neurons of the midbrain to identify cell-autonomous abnormalities. Expression of both the human wild type (hTorsinA) and mutant (ΔE-hTorsinA) protein resulted in alterations of dopamine release as detected by microdialysis and fast cycle voltammetry. Motor abnormalities detected in these mice mimicked those noted in transgenic mice with pan-neuronal transgene expression. The locomotor response to cocaine in both TH-hTorsinA and TH-ΔE-hTorsinA, in the face of abnormal extracellular DA levels relative to non-transgenic mice, suggests compensatory, post-synaptic alterations in striatal DA transmission. This is the first cell-subtype-specific DYT1 transgenic mouse that can serve to differentiate between primary and secondary changes in dystonia, thereby helping to target disease therapies.
Neurobiology of Disease.
