Tamara Ratovitski

Johns Hopkins University, Baltimore, MD, United States

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Publications (22)156.49 Total impact

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    ABSTRACT: Phosphorylation has been shown to have a significant impact on expanded huntingtin-mediated cellular toxicity. Several phosphorylation sites have been identified on the huntingtin (Htt) protein. To find new potential therapeutic targets for Huntington's Disease (HD), we used mass spectrometry to identify novel phosphorylation sites on N-terminal Htt, expressed in HEK293 cells. Using site-directed mutagenesis we introduced alterations of phosphorylation sites in a N586 Htt construct containing 82 polyglutamine repeats. The effects of these alterations on expanded Htt toxicity were evaluated in primary neurons using a nuclear condensation assay and a direct time-lapse imaging of neuronal death. As a result of these studies, we identified several novel phosphorylation sites, validated several known sites, and discovered one phospho-null alteration, S116A, that had a protective effect against expanded polyglutamine-mediated cellular toxicity. The results suggest that S116 is a potential therapeutic target, and indicate that our screening method is useful for identifying candidate phosphorylation sites.
    PLoS ONE 01/2014; 9(2):e88284. · 3.53 Impact Factor
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    Cell Stem Cell 08/2012; 11(13). · 25.32 Impact Factor
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    ABSTRACT: Huntington disease (HD) is a neurodegenerative disorder caused by an expansion of a polyglutamine repeat within the HD gene product, huntingtin. Huntingtin, a large (347 kDa) protein containing multiple HEAT repeats, acts as a scaffold for protein-protein interactions. Huntingtin-induced toxicity is believed to be mediated by a conformational change in expanded huntingtin, leading to protein misfolding and aggregation, aberrant protein interactions and neuronal cell death. While many non-systematic studies of huntingtin interactions have been reported, they were not designed to identify and quantify the changes in the huntingtin interactome induced by polyglutamine expansion. We used tandem affinity purification and quantitative proteomics to compare and quantify interactions of normal or expanded huntingtin isolated from a striatal cell line. We found that proteins preferentially interacting with expanded huntingtin are enriched for intrinsically disordered proteins, consistent with previously suggested roles of such proteins in neurodegenerative disorders. Our functional analysis indicates that proteins related to energy production, protein trafficking, RNA post-transcriptional modifications and cell death were significantly enriched among preferential interactors of expanded huntingtin. Expanded huntingtin interacted with many mitochondrial proteins, including AIFM1, consistent with a role for mitochondrial dysfunction in HD. Furthermore, expanded huntingtin interacted with the stress granule-associated proteins Caprin-1 and G3BP and redistributed to RNA stress granules under ER-stress conditions. These data demonstrate that a number of key cellular functions and networks may be disrupted by abnormal interactions of expanded huntingtin and highlight proteins and pathways that may be involved in HD cellular pathogenesis and that may serve as therapeutic targets.
    Cell cycle (Georgetown, Tex.) 05/2012; 11(10):2006-21. · 5.24 Impact Factor
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    ABSTRACT: Huntington disease is a genetic neurodegenerative disorder that arises from an expanded polyglutamine region in the N terminus of the HD gene product, huntingtin. Protein inclusions comprised of N-terminal fragments of mutant huntingtin are a characteristic feature of disease, though are likely to play a protective role rather than a causative one in neurodegeneration. Soluble oligomeric assemblies of huntingtin formed early in the aggregation process are candidate toxic species in HD. In the present study, we established an in vitro system to generate recombinant huntingtin in mammalian cells. Using both denaturing and native gel analysis, we have identified novel oligomeric forms of mammalian-derived expanded huntingtin exon-1 N-terminal fragment. These species are transient and were not previously detected using bacterially expressed exon-1 protein. Importantly, these species are recognized by 3B5H10, an antibody that recognizes a two-stranded hairpin conformation of expanded polyglutamine believed to be associated with a toxic form of huntingtin. Interestingly, comparable oligomeric species were not observed for expanded huntingtin shortstop, a 117-amino acid fragment of huntingtin shown previously in mammalian cell lines and transgenic mice, and here in primary cortical neurons, to be non-toxic. Further, we demonstrate that expanded huntingtin shortstop has a reduced ability to form amyloid-like fibrils characteristic of the aggregation pathway for toxic expanded polyglutamine proteins. Taken together, these data provide a possible candidate toxic species in HD. In addition, these studies demonstrate the fundamental differences in early aggregation events between mutant huntingtin exon-1 and shortstop proteins that may underlie the differences in toxicity.
    Journal of Biological Chemistry 03/2012; 287(19):16017-28. · 4.65 Impact Factor
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    ABSTRACT: Huntington's disease (HD) is caused by a polyglutamine expansion in the Huntingtin (Htt) protein. Proteolytic cleavage of Htt into toxic N-terminal fragments is believed to be a key aspect of pathogenesis. The best characterized putative cleavage event is at amino acid 586, hypothesized to be mediated by caspase 6. A corollary of the caspase 6 cleavage hypothesis is that the caspase 6 fragment should be a toxic fragment. To test this hypothesis, and further characterize the role of this fragment, we have generated transgenic mice expressing the N-terminal 586 aa of Htt with a polyglutamine repeat length of 82 (N586-82Q), under the control of the prion promoter. N586-82Q mice show a clear progressive rotarod deficit by 4 months of age, and are hyperactive starting at 5 months, later changing to hypoactivity before early mortality. MRI studies reveal widespread brain atrophy, and histologic studies demonstrate an abundance of Htt aggregates, mostly cytoplasmic, which are predominantly composed of the N586-82Q polypeptide. Smaller soluble N-terminal fragments appear to accumulate over time, peaking at 4 months, and are predominantly found in the nuclear fraction. This model appears to have a phenotype more severe than current full-length Htt models, but less severe than HD mouse models expressing shorter Htt fragments. These studies suggest that the caspase 6 fragment may be a transient intermediate, that fragment size is a factor contributing to the rate of disease progression, and that short soluble nuclear fragments may be most relevant to pathogenesis.
    Journal of Neuroscience 01/2012; 32(1):183-93. · 6.91 Impact Factor
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    ABSTRACT: N-terminal proteolysis of huntingtin is thought to be an important mediator of HD pathogenesis. The formation of short N-terminal fragments of huntingtin (cp-1/cp-2, cp-A/cp-B) has been demonstrated in cells and in vivo. We previously mapped the cp-2 cleavage site by mass spectrometry to position Arg167 of huntingtin. The proteolytic enzymes generating short N-terminal fragments of huntingtin remain unknown. To search for such proteases, we conducted a genome-wide screen using an RNA-silencing approach and an assay for huntingtin proteolysis based on the detection of cp-1 and cp-2 fragments by Western blotting. The primary screen was carried out in HEK293 cells, and the secondary screen was carried out in neuronal HT22 cells, transfected in both cases with a construct encoding the N-terminal 511 amino acids of mutant huntingtin. For additional validation of the hits, we employed a complementary assay for proteolysis of huntingtin involving overexpression of individual proteases with huntingtin in two cell lines. The screen identified 11 enzymes, with two major candidates to carry out the cp-2 cleavage, bleomycin hydrolase (BLMH) and cathepsin Z, which are both cysteine proteases of a papain-like structure. Knockdown of either protease reduced cp-2 cleavage, and ameliorated mutant huntingtin induced toxicity, whereas their overexpression increased the cp-2 cleavage. Both proteases partially co-localized with Htt in the cytoplasm and within or in association with early and late endosomes, with some nuclear co-localization observed for cathepsin Z. BLMH and cathepsin Z are expressed in the brain and have been associated previously with neurodegeneration. Our findings further validate the cysteine protease family, and BLMH and cathepsin Z in particular, as potential novel targets for HD therapeutics.
    Journal of Biological Chemistry 02/2011; 286(14):12578-89. · 4.65 Impact Factor
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    ABSTRACT: Huntington's disease is a progressive neurodegenerative disorder caused by a polyglutamine expansion near the N-terminus of huntingtin. The mechanisms of polyglutamine neurotoxicity, and cellular responses are not fully understood. We have studied gene expression profiles by short oligo array using an inducible PC12 cell model expressing an N-terminal huntingtin fragment with expanded polyglutamine (Htt-N63-148Q). Mutant huntingtin Htt-N63 induced cell death and increased the mRNA and protein levels of activating transcription factor 3 (ATF3). Mutant Htt-N63 also significantly enhanced ATF3 transcriptional activity by a promoter-based reporter assay. Overexpression of ATF3 protects against mutant Htt-N63 toxicity and knocking down ATF3 expression reduced Htt-N63 toxicity in a stable PC12 cell line. These results indicated that ATF3 plays a critical role in toxicity induced by mutant Htt-N63 and may lead to a useful therapeutic target.
    Brain research 07/2009; 1286:221-9. · 2.46 Impact Factor
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    ABSTRACT: Huntingtin proteolysis is implicated in Huntington disease pathogenesis, yet, the nature of huntingtin toxic fragments remains unclear. Huntingtin undergoes proteolysis by calpains and caspases within an N-terminal region between amino acids 460 and 600. We have focused on proteolytic steps producing shorter N-terminal fragments, which we term cp-1 and cp-2 (distinct from previously described cp-A/cp-B). We used HEK293 cells to express the first 511 residues of huntingtin and further define the cp-1 and cp-2 cleavage sites. Based on epitope mapping with huntingtin-specific antibodies, we found that cp-1 cleavage occurs between residues 81 and 129 of huntingtin. Affinity and size exclusion chromatography were used to further purify huntingtin cleavage products and enrich for the cp-1/cp-2 fragments. Using mass spectrometry, we found that the cp-2 fragment is generated by cleavage of huntingtin at position Arg(167). This site was confirmed by deletion analysis and specific detection with a custom-generated cp-2 site neo-epitope antibody. Furthermore, alterations of this cleavage site resulted in a decrease in toxicity and an increase in aggregation of huntingtin in neuronal cells. These data suggest that cleavage of huntingtin at residue Arg(167) may mediate mutant huntingtin toxicity in Huntington disease.
    Journal of Biological Chemistry 03/2009; 284(16):10855-67. · 4.65 Impact Factor
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    ABSTRACT: Proteolytic cleavage of mutant huntingtin may play a key role in the pathogenesis of Huntington's disease; however the steps in huntingtin proteolysis are not fully understood. Huntingtin was shown to be cleaved by caspases and calpains within a region between 460-600 amino acids from the N-terminus. Two smaller N-terminal fragments produced by unknown protease have been previously described as cp-A and cp-B. To further investigate the huntingtin proteolytic pathway, we used an inducible PC12 cell model expressing full-length huntingtin with either normal or expanded polyglutamine. This cell model recapitulates several steps of huntingtin proteolysis: proteolysis mediated by caspases within the region previously mapped for caspase cleavage, and cleavage generating two novel N-terminal fragments (cp-1 approximately 90-105 residues long and cp-2 extending beyond 115-129 epitope of huntingtin). Interestingly, the deletion of amino acids 105-114 (mapped previously as a cleavage site for cp-A) failed to affect the production of cp-1 or cp-2. Therefore, we conclude that these new fragments are distinct from previously described cp-A and cp-B. We demonstrate that cp-1 and cp-2 fragments are produced and accumulate within nuclear and cytoplasmic inclusions prior to huntingtin-induced cell toxicity, and these fragments can be formed by caspase-independent proteolytic cleavage of huntingtin in PC12 cells. In addition, inhibition of calpains leads to decreased subsequent degradation of cp-1 and cp-2 fragments, and accelerated formation of inclusions. Further delineation of huntingtin cleavage events may lead to novel therapeutic targets for HD.
    Cell cycle (Georgetown, Tex.) 10/2007; 6(23):2970-81. · 5.24 Impact Factor
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    ABSTRACT: Huntingtin (Htt) is a large protein of 3144 amino acids, whose function and regulation have not been well defined. Polyglutamine (polyQ) expansion in the N terminus of Htt causes the neurodegenerative disorder Huntington disease (HD). The cytotoxicity of mutant Htt is modulated by proteolytic cleavage with caspases and calpains generating N-terminal polyQ-containing fragments. We hypothesized that phosphorylation of Htt may modulate cleavage and cytotoxicity. In the present study, we have mapped the major phosphorylation sites of Htt using cell culture models (293T and PC12 cells) expressing full-length myc-tagged Htt constructs containing 23Q or 148Q repeats. Purified myc-tagged Htt was subjected to mass spectrometric analysis including matrix-assisted laser desorption/ionization mass spectrometry and nano-HPLC tandem mass spectrometry, used in conjunction with on-target alkaline phosphatase and protease digestions. We have identified more than six novel serine phosphorylation sites within Htt, one of which lies in the proteolytic susceptibility domain. Three of the sites have the consensus sequence for ERK1 phosphorylation, and addition of ERK1 inhibitor blocks phosphorylation at those sites. Other observed phosphorylation sites are possibly substrates for CDK5/CDC2 kinases. Mutation of amino acid Ser-536, which is located in the proteolytic susceptibility domain, to aspartic acid, inhibited calpain cleavage and reduced mutant Htt toxicity. The results presented here represent the first detailed mapping of the phosphorylation sites in full-length Htt. Dissection of phosphorylation modifications in Htt may provide clues to Huntington disease pathogenesis and targets for therapeutic development.
    Journal of Biological Chemistry 09/2006; 281(33):23686-97. · 4.65 Impact Factor
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    ABSTRACT: The activities of presenilin 1 (PS1) and 2 modulate the proteolytic processing of amyloid precursor proteins to produce Abeta1-42, and mutations in these proteins are associated with an accelerated rate of Abeta deposition. PS1 and PS2 themselves are subject to a highly-regulated endoproteolytic cleavage to generate stable 27 kDa N-terminal and 17 kDa C-terminal fragments. Here, we examined the relationship between the regulated cleavage of PS1 and the acceleration of Abeta deposition in transgenic mice that co-express Mo/Hu APPswe and varied levels mutant PS1 (A246E variant). The steady-state levels of the N- and C-terminal fragments of mutant PS1 in mice expressing low levels of mRNA were similar to that of mice expressing high levels of mRNA. Only mice expressing high levels of transgene mRNA accumulated uncleaved full-length protein. In mice co-expressing low levels of PS1A246E mRNA with Mo/Hu APPswe the age of appearance of Abeta deposits was similar to that of mice co-expressing expressing Mo/Hu APPswe with very high levels of mutant PS1. Our findings demonstrate that the levels of accumulated human PS1 N- and C-terminal fragments do not increase in proportion to the level of transgene mRNA and that similarly, the magnitude by which mutant PS1 accelerates the deposition of beta-amyloid is not proportional to the level of transgene expression.
    Neurobiology of Aging 01/2002; 23(2):171-7. · 6.17 Impact Factor
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    ABSTRACT: The introduction of two transgenes into one animal is increasingly common as transgenic experiments become more sophisticated. In this study we examine two strategies for creating double transgenic founders from a single microinjection. In the first approach, two constructs, each with its own promoter element, were coinjected into the pronucleus. In the second approach, both transgenes were cloned into one vector, separated by an internal ribosomal entry site (IRES), and placed under control of a single promoter. Both strategies save time and increase the percentage of double transgenic offspring over the standard method of mating single transgenic lines. However, despite high transgene copy numbers, the bicistronic lines did not show robust expression of either protein. Copy number and protein expression correlated much better in the coinjected lines, with expression levels in one line approaching that observed in some of our best single transgenic controls. Thus we recommend coinjection of individual plasmids for the generation of multiply transgenic founders.
    Biomolecular Engineering 07/2001; 17(6):157-65. · 3.17 Impact Factor
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    ABSTRACT: Mutations in superoxide dismutase 1 (SOD1) polypeptides cause a form of familial amyotrophic lateral sclerosis (FALS). In different kindreds, harboring different mutations, the duration of illness tends to be similar for a given mutation. For example, patients inheriting a substitution of valine for alanine at position four (A4V) average a 1.5 year life expectancy after the onset of symptoms, whereas patients harboring a substitution of arginine for histidine at position 46 (H46R) average an 18 year life expectancy after disease onset. Here, we examine a number of biochemical and biophysical properties of nine different FALS variants of SOD1 polypeptides, including enzymatic activity (which relates indirectly to the affinity of the enzyme for copper), polypeptide half-life, resistance to proteolytic degradation and solubility, in an effort to determine whether a specific property of these enzymes correlates with clinical progression. We find that although all the mutants tested appear to be soluble, the different mutants show a remarkable degree of variation with respect to activity, polypeptide half-life and resistance to proteolysis. However, these variables do not stratify in a manner that correlates with clinical progression. We conclude that the basis for the different life expectancies of patients in different kindreds of sod1-linked FALS may result from an as yet unidentified property of these mutant enzymes.
    Human Molecular Genetics 09/1999; 8(8):1451-60. · 7.69 Impact Factor
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    ABSTRACT: Huntington's disease (HD) is an inherited, neurodegenerative disorder caused by the expansion of a glutamine repeat in the N-terminus of the huntingtin protein. To gain insight into the pathogenesis of HD, we generated transgenic mice that express a cDNA encoding an N-terminal fragment (171 amino acids) of huntingtin with 82, 44 or 18 glutamines. Mice expressing relatively low steady-state levels of N171 huntingtin with 82 glutamine repeats (N171-82Q) develop behavioral abnormalities, including loss of coordination, tremors, hypokinesis and abnormal gait, before dying prematurely. In mice exhibiting these abnormalities, diffuse nuclear labeling, intranuclear inclusions and neuritic aggregates, all immunoreactive with an antibody to the N-terminus (amino acids 1-17) of huntingtin (AP194), were found in multiple populations of neurons. None of these behavioral or pathological phenotypes were seen in mice expressing N171-18Q. These findings are consistent with the idea that N-terminal fragments of huntingtin with a repeat expansion are toxic to neurons, and that N-terminal fragments are prone to form both intranuclear inclusions and neuritic aggregates.
    Human Molecular Genetics 04/1999; 8(3):397-407. · 7.69 Impact Factor
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    ABSTRACT: Components of the circadian system, the suprachiasmatic nucleus and the intergeniculate leaflet receive serotonin input from the raphe nuclei. Manipulations of serotonin neurotransmission disrupt cellular, electrophysiological, and behavioural responses of the circadian system to light, suggesting that serotonin plays a modulatory role in photic regulation of circadian rhythms. To study the relation between serotonin afferents and light-activated cells in the suprachiasmatic nucleus and intergeniculate leaflet, we used immunostaining for the serotonin transporter and for the transcription factor, Fos. Serotonin transporter, a plasma membrane protein located on serotonin neurons, regulates the amount of serotonin available for neurotransmission by re-accumulating released serotonin into presynaptic neurons; expression of Fos in the suprachiasmatic nucleus identifies light-activated cells involved in photic resetting of circadian clock phase. In the suprachiasmatic nucleus, immunostaining for serotonin transporter revealed a dense plexus of fibres concentrated primarily in the ventrolateral region. In the intergeniculate leaflet, serotonin transporter immunostaining identified vertically-oriented columns of fibres. Serotonin transporter immunostaining was abolished by pretreatment with the serotonin neurotoxin, 5,7-dihydroxytryptamine. Exposure to light for 30 min during the dark phase of the light cycle induced Fos expression in the ventrolateral suprachiasmatic nucleus and intergeniculate leaflet regions. In both structures the Fos-expressing cells were encircled by serotonin transporter-immunoreactive fibres often in close apposition to these cells. These results support the idea that serotonin activity plays a modulatory role in processing of photic information within the circadian system.
    Neuroscience 07/1998; 84(4):1059-73. · 3.12 Impact Factor
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    ABSTRACT: Mutations in two related genes, PS1 and PS2, account for the majority of early onset cases of familial Alzheimer's disease. PS1 and PS2 are homologous polytopic membrane proteins that are processed endoproteolytically into two fragments in vivo. In the present report we examine the fate of endogenous PS1 and PS2 after overexpression of human PS1 or PS2 in mouse N2a neuroblastoma cell lines and human PS1 in transgenic mice. Remarkably, in N2a cell lines and in brains of transgenic mice expressing human PS1, accumulation of human PS1 derivatives is accompanied by a compensatory, and highly selective, decrease in the steady-state levels of murine PS1 and PS2 derivatives. Similarly, the levels of murine PS1 derivatives are diminished in cultured cells overexpressing human PS2. To define the minimal sequence requirements for "replacement" we expressed familial Alzheimer's disease-linked and experimental deletion variants of PS1. These studies revealed that compromised accumulation of murine PS1 and PS2 derivatives resulting from overexpression of human PS1 occurs in a manner independent of endoproteolytic cleavage. Our results are consistent with a model in which the abundance of PS1 and PS2 fragments is regulated coordinately by competition for limiting cellular factor(s).
    Journal of Biological Chemistry 12/1997; 272(45):28415-22. · 4.65 Impact Factor
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    ABSTRACT: Missense mutations in two related genes, termed presenilin 1 (PS1) and presenilin 2 (PS2), cause dementia in a subset of early-onset familial Alzheimer's disease (FAD) pedigrees. In a variety of experimental in vitro and in vivo settings, FAD-linked presenilin variants influence the processing of the amyloid precursor protein (APP), leading to elevated levels of the highly fibrillogenic Abeta1-42 peptides that are preferentially deposited in the brains of Alzheimer Disease (AD) patients. In this report, we demonstrate that transgenic animals that coexpress a FAD-linked human PS1 variant (A246E) and a chimeric mouse/human APP harboring mutations linked to Swedish FAD kindreds (APP swe) develop numerous amyloid deposits much earlier than age-matched mice expressing APP swe and wild-type Hu PS1 or APP swe alone. These results provide evidence for the view that one pathogenic mechanism by which FAD-linked mutant PS1 causes AD is to accelerate the rate of beta-amyloid deposition in brain.
    Neuron 11/1997; 19(4):939-45. · 15.77 Impact Factor
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    ABSTRACT: Presenilin 1 (PS1), mutated in pedigrees of early-onset familial Alzheimer's disease, is a polytopic integral membrane protein that is endoproteolytically cleaved into 27-kDa N-terminal and 17-kDa C-terminal fragments. Although these fragments are the principal PS1 species found in normal mammalian brain, the role of endoproteolysis in the maturation of PS1 has been unclear. The present study, which uses stably transfected mouse neuroblastoma N2a cells, demonstrates that full-length polypeptides, derived from either wild-type or A246E FAD-mutant human (hu) PS1, are relatively short-lived (t1/2 1.5 h) proteins that give rise to the N- and C-terminal PS1 fragments, which are more stable (t1/2 approximately 24 h). N-terminal fragments, generated artificially by engineering a stop codon at amino acid 306 (PS1-306) of wild-type huPS1, were short-lived, whereas an FAD-linked variant that lacked exon 9 (DeltaE9) and was not endoproteolytically cleaved exhibited a long half-life. These observations suggest that endoproteolytic cleavage and stability are not linked, leading us to propose a model in which wild-type full-length huPS1 molecules are first stabilized then subsequently endoproteolytically cleaved to generate the N- and C-terminal fragments. These fragments appear to represent the mature and functional forms of wild-type huPS1.
    Journal of Biological Chemistry 10/1997; 272(39):24536-41. · 4.65 Impact Factor
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    ABSTRACT: Mutations in the presenilin 1 (PS1) and presenilin 2 (PS2) genes can cause Alzheimer's disease in affected members of the majority of early-onset familial Alzheimer's disease (FAD) pedigrees. PS1 encodes an ubiquitously expressed, eight transmembrane protein. PS1 is endoproteolytically processed to an amino-terminal derivative (approximately 27-28 kDa) and a carboxy-terminal derivative (approximately 17-18 kDa). These polypeptides accumulate to saturable levels in the brains of transgenic mice, independent of the expression of PS1 holoprotein. We now document that, in the brains of transgenic mice, the absolute amounts of accumulated N- and C-terminal derivatives generated from the FAD-linked PS1 variants in which Glu replaces Ala at codon 246 (A246E) or Leu replaces Met at codon 146 (M146L) accumulate to a significantly higher degree (approximately 40-50%) than the fragments derived from wild-type PS1. Moreover, the FAD-linked deltaE9 PS1 variant, a polypeptide that is not subject to endoproteolytic cleavage in vivo, also accumulates in greater amounts than the fragments generated from wild-type human PS1. Thus, the metabolism of PS1 variants linked to FAD is fundamentally different from that of wild-type PS1 in vivo.
    Nature Medicine 08/1997; 3(7):756-60. · 22.86 Impact Factor
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    ABSTRACT: The NMB human neuronal cell line, transfected with a newly prepared plasmid expressing rat serotonin transporter (NMB-rSERT), shows specific [3H]5-HT uptake which is blocked by citalopram and fenfluramine (F) stereoisomers with IC50 values (1 nM. 0.5 microM (dF) and and 5 microM (IF), respectively) which are similar to those found in rat brain synaptosomes. d-Fenfluramine (0.5 and 10 microM) also stimulates tritium release from NMB-rSERT cells preloaded with [3H-]-5-HT. The d-fenfluramine-induced [3H-]5-HT release is blocked by 0.3 microM citalopram and is dependent on the density of SERT expressed per cell, but is not affected by removal of Ca++ ions from the incubation medium. Manipulation of the Na+ gradient across the plasma membrane (replacing 60 mM NaCl with an equimolar concentration of KCl or choline) also induced [3H-]5-HT release from NMB-rSERT cells, which was inhibited by 0.3 microM citalopram. These results, together with the finding that NMB-rSERT cells preloaded with 500 nM unlabelled 5-HT take up [3H-]d-fenfluramine, make NMB-rSERT cells a valuable tool for studying the transporter-mediated exchange release induced by amphetamine derivatives.
    Neuropharmacology 07/1997; 36(6):803-9. · 4.11 Impact Factor

Publication Stats

2k Citations
156.49 Total Impact Points

Institutions

  • 1997–2012
    • Johns Hopkins University
      • • Department of Psychiatry and Behavioral Sciences
      • • Department of Neurology
      • • Department of Pathology
      Baltimore, MD, United States
  • 1997–2001
    • Johns Hopkins Medicine
      • Department of Pathology
      Baltimore, MD, United States
  • 1997–1998
    • Weizmann Institute of Science
      • Department of Molecular Genetics
      Tell Afif, Tel Aviv, Israel