Presenilin-1 Mutations Reduce Cytoskeletal Association, Deregulate Neurite Growth, and Potentiate Neuronal Dystrophy and Tau Phosphorylation

Pigino, Gustavo; Pelsman, Alejandra; Mori, Hiroshi; Busciglio, Jorge

doi:10.1523/jneurosci.21-03-00834.2001

Cited by 99 publications

(83 citation statements)

References 70 publications

(77 reference statements)

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“…For heterogeneous A␤ solutions, synthetic A␤42 peptide (Bachem) was prepared as previously described (52). These heterogeneous A␤ solutions were perfused into extruded axoplasms isolated from the squid Loligo pealeii at 2-M concentration.…”

Section: Methodsmentioning

confidence: 99%

“…Proteins were separated by sodium dodecyl sulfatepolyacrylamide gel electrophorisis (SDS-PAGE) on 4-12% Bis-Tris gels (NuPage minigels, Invitrogen), using Mops Running Buffer (Invitrogen) and transferred to polyvinylidenefluoride(PVDF)membranesaspreviouslydescribed (52).Immunoblotswere blocked with 5% nonfat dried milk, in phosphate-buffered saline, pH 7.4, and probed with different polyclonal and monoclonal antibodies. Primary antibody binding was detected with horseradish peroxidase-conjugated anti-mouse and antirabbit secondary antibody (Jackson Immunoresearch) and visualized by chemiluminescence (ECL, Amersham).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Disruption of fast axonal transport is a pathogenic mechanism for intraneuronal amyloid beta

Pigino

Morfini

Atagi

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

194

172

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The pathological mechanism by which A␤ causes neuronal dysfunction and death remains largely unknown. Deficiencies in fast axonal transport (FAT) were suggested to play a crucial role in neuronal dysfunction and loss for a diverse set of dying back neuropathologies including Alzheimer's disease (AD), but the molecular basis for pathological changes in FAT were undetermined. Recent findings indicate that soluble intracellular oligomeric A␤ (oA␤) species may play a critical role in AD pathology. Real-time analysis of vesicle mobility in isolated axoplasms perfused with oA␤ showed bidirectional axonal transport inhibition as a consequence of endogenous casein kinase 2 (CK2) activation. Conversely, neither unaggregated amyloid beta nor fibrillar amyloid beta affected FAT. Inhibition of FAT by oA␤ was prevented by two specific pharmacological inhibitors of CK2, as well as by competition with a CK2 substrate peptide. Furthermore, perfusion of axoplasms with active CK2 mimics the inhibitory effects of oA␤ on FAT. Both oA␤ and CK2 treatment of axoplasm led to increased phosphorylation of kinesin-1 light chains and subsequent release of kinesin from its cargoes. Therefore pharmacological modulation of CK2 activity may represent a promising target for therapeutic intervention in AD.Alzheimer's disease ͉ Axonal transport ͉ Beta amyloid oligomer ͉ CK2 ͉ Kinesin T he complex functional changes and histopathology of Alzheimer disease (AD) make it one of the most challenging disorders faced by modern medicine. Histopathological hallmarks of AD include distinctive extracellular and intracellular aggregates of amyloid beta (A␤) and tau (1, 2). Synaptic dysfunction and axonopathy appear to be the earliest lesions in AD as corroborated by reduced immunoreactivity of synaptophysin and other synaptic markers in terminal fields of brain-affected areas (3). AD-affected neurons appear to die eventually as a consequence of synaptic dysfunction and loss, following a typical dying-back pattern of neuronal degeneration.The amyloid hypothesis (4), a dominant concept in AD research for many years, has been revised in recent years to include the notion that smaller soluble A␤ aggregates may play an early and significant role in AD. Soluble oligomers of A␤ (oA␤) have been shown to be neurotoxic both in vivo and in vitro (5-7) as well as altering synaptic structure and function (8). Moreover, oA␤ levels correlate with impairments in cognitive function, learning, and memory (9, 10), but the molecular basis of these effects are uncertain. Intracellular A␤ was first described by Wertkin et al. (11). Immunogold electron microscopy showed that intraneuronal A␤ is pre-and postsynaptically enriched in both AD human brain and AD transgenic animal models in association with dystrophic neurites and abnormal synaptic morphology (12)(13)(14). Spatial and temporal analyses of intraneuronal oA␤ accumulation show that it precedes plaque formation in both AD animal models and Down's syndrome patients, suggesting that oA␤ is an early intracellular toxic agent i...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Disruption of fast axonal transport is a pathogenic mechanism for intraneuronal amyloid beta

Pigino

Morfini

Atagi

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

194

172

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“…ets-2, p53, and bax antibody specificity were confirmed by preabsorption of the primary antibodies with synthetic peptide, which abolished immunoreactivity. Quantitative Western blot analysis was performed as described previously (Pigino et al, 2001(Pigino et al, , 2003.…”

Section: Methodsmentioning

confidence: 99%

ets-2 Promotes the Activation of a Mitochondrial Death Pathway in Down's Syndrome Neurons

Helguera¹,

Pelsman²,

Pigino³

et al. 2005

J. Neurosci.

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Down's syndrome (DS) is characterized by mental retardation and development of Alzheimer's disease (AD). Oxidative stress and mitochondrial dysfunction are both related to neurodegeneration in DS. Several genes in chromosome 21 have been linked to neuronal death, including the transcription factor ets-2. Cortical cultures derived from normal and DS fetal brains were used to study the role of ets-2 in DS neuronal degeneration. ets-2 was expressed in normal human cortical neurons (HCNs) and was markedly upregulated by oxidative stress. When overexpressed in normal HCNs, ets-2 induced a stereotyped sequence of apoptotic changes leading to neuronal death. DS HCNs exhibit intracellular oxidative stress and increased apoptosis after the first week in culture (Busciglio and Yankner, 1995). ets-2 levels were increased in DS HCNs, and, between 7 and 14 d in vitro, DS HCNs showed increased bax, cytoplasmic translocation of cytochrome c and apoptosis inducing factor, and active caspases 3 and 7, consistent with activation of an apoptotic mitochondrial death pathway. Degeneration of DS neurons was reduced by dominant-negative ets-2, suggesting that increased ets-2 expression promotes DS neuronal apoptosis. In the human brain, ets-2 expression was found in neurons and astrocytes. Strong ets-2 immunoreactivity was observed in DS/AD and sporadic AD brains associated with degenerative markers such as bax, intracellular A␤, and hyperphosphorylated tau. Thus, in DS/AD and sporadic AD brains, converging pathological mechanisms leading to chronic oxidative stress and ets-2 upregulation in susceptible neurons may result in increased vulnerability by promoting the activation of a mitochondrial-dependent proapoptotic pathway of cell death.

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“…Procedure and reagents for CSK fractionation are described in Pigino et al (2001). Cells were rinsed in microtubule-stabilizing buffer, and the insoluble fraction was harvested in cold radioimmunoprecipitation assay buffer containing a cocktail of proteases inhibitors (Roche Complete).…”

Section: Cell Culture Cell Fractionation and Cell Extractionmentioning

confidence: 99%

“…The U-2 OS human osteosarcoma cell line (Ponten and Saksela, 1967) was grown in McCoy's medium containing 10% fetal bovine serum. Procedures for animal use were approved by the Animal Care Committee of the University of Connecticut Health Center and followed federal guidelines for research with animals.Procedure and reagents for CSK fractionation are described in Pigino et al (2001). Cells were rinsed in microtubule-stabilizing buffer, and the insoluble fraction was harvested in cold radioimmunoprecipitation assay buffer containing a cocktail of proteases inhibitors (Roche Complete).…”

mentioning

confidence: 99%

The Microtubule-associated Protein Tumor Overexpressed Gene Binds to the RNA Trafficking Protein Heterogeneous Nuclear Ribonucleoprotein A2

Kosturko

Maggipinto

D'Sa

et al. 2005

MBoC

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In neural cells, such as oligodendrocytes and neurons, transport of certain RNAs along microtubules is mediated by the cis-acting heterogeneous nuclear ribonucleoprotein A2 response element (A2RE) trafficking element and the cognate trans-acting heterogeneous nuclear ribonucleoprotein (hnRNP) A2 trafficking factor. Using a yeast two-hybrid screen, we have identified a microtubule-associated protein, tumor overexpressed gene (TOG)2, as an hnRNP A2 binding partner. The C-terminal third of TOG2 is sufficient for hnRNP A2 binding. TOG2, the large protein isoform of TOG, is the only isoform detected in oligodendrocytes in culture. TOG coimmunoprecipitates with hnRNP A2 present in the cytoskeleton (CSK) fraction of neural cells, and both coprecipitate with microtubule stabilized pellets. Staining with anti-TOG reveals puncta that are localized in proximity to microtubules, often at the plus ends. TOG is colocalized with hnRNP A2 and A2RE-mRNA in trafficking granules that remain associated with CSK-insoluble tissue. These data suggest that TOG mediates the association of hnRNP A2-positive granules with microtubules during transport and/or localization. INTRODUCTIONThe heterogeneous nuclear ribonucleoprotein (hnRNP) A2 is implicated in RNA processing, RNA transport, and translation regulation (Dreyfuss et al., 1993;Siomi and Dreyfuss, 1995;Hamilton et al., 1999;Kwon et al., 1999). HnRNP A2 binds to a specific cis-acting element, hnRNP A2 response element (A2RE), found in several dendritically localized mRNAs Munro et al., 1999). This interaction is necessary for transport of A2RE-mRNAs to the periphery of neural cells (Munro et al., 1999;Shan et al., 2003).A2RE-mRNAs, hnRNP A2, and other components form complexes that look by light microscopy like granules that are transported to the cell periphery by a microtubule-and kinesin-based mechanism. In cultured oligodendrocytes and neurons, granules containing A2RE-mRNAs and hnRNP A2 are associated with the cytoskeleton (CSK) . HnRNP A2 and some A2RE-mRNAs copurify with the CSK from rat brain (Hoek et al., 1998;Boccaccio et al., 1999).To identify molecular partners of hnRNP A2, we performed yeast two-hybrid analysis. One of the components identified is the tumor overexpressed gene (TOG) protein, a microtubule-associated protein (MAP) ubiquitously expressed in human tissues. TOG is a single copy gene that generates two mRNA splice variants (Nagase et al., 1995;Charrasse et al., 1998) encoding proteins that differ by a 60-aa insert near the C terminus. In dividing cells, TOG localizes with centrosome and spindle microtubules and may be necessary for microtubule rearrangements and spindle assembly (Charrasse et al., 1998;Lee et al., 2001). Orthologues of colonic and hepatic tumor overexpressed gene (ch-TOG) (Homo sapiens), Dis1p, Stu2p, XMAP215, ZYG-9, and mini spindles, in fission yeast, budding yeast, Xenopus laevis, Caenorhabditis elegans, and Drosophila melanogaster, respectively, have similar functions. TOG and its orthologues all contain multiple HEAT repeats that may serv...

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Presenilin-1 Mutations Reduce Cytoskeletal Association, Deregulate Neurite Growth, and Potentiate Neuronal Dystrophy and Tau Phosphorylation

Cited by 99 publications

References 70 publications

Disruption of fast axonal transport is a pathogenic mechanism for intraneuronal amyloid beta

Disruption of fast axonal transport is a pathogenic mechanism for intraneuronal amyloid beta

ets-2 Promotes the Activation of a Mitochondrial Death Pathway in Down's Syndrome Neurons

The Microtubule-associated Protein Tumor Overexpressed Gene Binds to the RNA Trafficking Protein Heterogeneous Nuclear Ribonucleoprotein A2

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