Examination of potential mechanisms of amyloid-induced defects in neuronal transport

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Abstract

Microtubule-based neuronal transport pathways are impaired during the progression of Alzheimer's disease and other neurodegenerative conditions. However, mechanisms leading to defects in transport remain to be determined. We quantified morphological changes in neuronal cells following treatment with fibrils and unaggregated peptides of beta-amyloid (Aβ). Aβ fibrils induce axonal and dendritic swellings indicative of impaired transport. In contrast, Aβ peptides induce a necrotic phenotype in both neurons and non-neuronal cells. We tested several popular hypotheses by which aggregated Aβ could disrupt transport. Using fluorescent polystyrene beads, we developed experimental models of physical blockage and localized release of reactive oxygen species (ROS) that reliably induce swellings. Like the beads, Aβ fibrils localize in close proximity to swellings; however, fibril internalization is not required for disrupting transport. ROS and membrane permeability are also unlikely to be responsible for fibril-mediated toxicity. Collectively, our results indicate that multiple initiating factors converge upon pathways of defective transport.

Introduction

Alzheimer's disease (AD) is a neurodegenerative disease characterized by synaptic loss, neurofibrillary tangles, and amyloid-associated plaques. Embedded within and surrounding these plaques are dystrophic neurites, both axonal and dendritic in origin, that display swellings or spheroids filled with accumulated organelles and cytoskeleton (Gonatas et al., 1967, Probst et al., 1983, Ferrer et al., 1990, Su et al., 1993, Spires and Hyman, 2004). Protein accumulations within dystrophic neurites, which also exist in regions devoid of plaques, might reflect an impairment in bidirectional cargo transport, and are consistent with several observations that microtubule-based transport pathways are affected during the progression of AD (Benzing et al., 1993, Masliah et al., 1993, Dai et al., 2002, Cash et al., 2003, Stokin et al., 2005, Ikonomovic et al., 2007). However, pathways initiating this hypothesized breakdown in transport remain to be determined.

Considerable evidence links neurodegeneration to β-amyloid peptides of at least 42 amino acids in length (Aβ1–42), degradation-resistant variants of a self-assembling peptide cleaved from β-amyloid precursor protein (βAPP). Most familial AD (FAD) mutations occur in βAPP and related proteins, and result in increased production and pathological deposition of Aβ1–42 (Selkoe, 2001, Takahashi et al., 2002, Fernandez-Vizarra et al., 2004, Takahashi et al., 2004, Gouras et al., 2005). Animal models expressing FAD mutations replicate features of AD pathology, including increased Aβ1–42, amyloid plaques, synaptic abnormalities, and behavioral deficits (Phinney et al., 2003, Duyckaerts et al., 2008). Multiple mechanisms have been proposed by which Aβ exerts toxicity, including integrin activation, membrane depolarization, membrane permeability, excitotoxic and synaptotoxic vulnerability, and oxidative stress (Koh et al., 1990, Carette et al., 1993, Behl et al., 1994, Zhang et al., 1996, Behl and Sagara, 1997, Kawahara and Kuroda, 2000, Anderson and Ferreira, 2004, Canevari et al., 2004, Demuro et al., 2005, Lacor et al., 2007, Shankar et al., 2008). Recent studies also suggest a relationship between βAPP/Aβ and defective neuronal transport (Masliah et al., 1996, Kasa et al., 2000, Gunawardena and Goldstein, 2001, Hiruma et al., 2003, Tsai et al., 2004, Maloney et al., 2005, Stokin et al., 2005, Gotz et al., 2006, King et al., 2006, Adalbert et al., 2007, Lazarov et al., 2007), but whether and how pathways of transport impairment relate to other proposed mechanisms of amyloid toxicity require further study.

The aggregation state of neurotoxic Aβ has been vigorously debated, but requires additional assessment in the context of defective transport. Multiple studies suggest that oligomers, fibrils, and insoluble aggregates of Aβ induce a variety of neurotoxic phenotypes in vitro and in vivo (Yankner et al., 1990a, Yankner et al., 1990b, Loo et al., 1993, Pike et al., 1993, Lorenzo and Yankner, 1994, Walsh et al., 2002, Gong et al., 2003, Zou et al., 2003, Takahashi et al., 2004, Takuma et al., 2004, Tsai et al., 2004, Shankar et al., 2008). Qualitative morphological analyses of cells treated with various Aβ aggregation states reveal a disparate response, with some cells displaying neurite beading (Loo et al., 1993, Pike et al., 1993, Ivins et al., 1998), and others necrosis (Yankner et al., 1990b, Behl et al., 1994). To interpret these responses, morphological phenotypes induced by different Aβ aggregation states must be quantified, and features of each state responsible for each phenotype defined.

Another critical question involves the localization of toxic Aβ. It is still unclear whether toxic Aβ resides intra- or extra-neuronally, or whether toxicity is induced at the soma or along neurites. Biochemical experiments in several cell lines indicate the existence of Aβ within multiple subcellular compartments (Knauer et al., 1992, Burdick et al., 1997, Cook et al., 1997, Hartmann et al., 1997, Skovronsky et al., 1998, Yang et al., 1999, Glabe, 2001, Echeverria and Cuello, 2002, Kienlen-Campard et al., 2002), but do not quantify the localization of toxic Aβ or explain the potential for differential toxicity in neuronal and non-neuronal cells. Studies in compartmentalized cultures partially address the relationship between toxicity and localization, suggesting that responses in the soma result from Aβ-induced signals initiating within neurites (Ivins et al., 1998, Song et al., 2006). These studies do not test whether Aβ internalization is necessary for toxicity.

In this study, we quantitatively tested several mechanisms of amyloid toxicity. We first determined that multiple aggregation states of Aβ induce neurotoxicity, but only fibrils, and not peptides, induce neurite swellings, indicative of defective transport. We then simulated specific features of amyloid fibrils potentially responsible for disrupting transport. Using fluorescent beads, we developed experimental models of physical blockage and oxidative stress that reliably induced neurite swellings. Though mechanisms of bead-induced toxicity do not replicate those induced by Aβ fibrils, our results reveal that multiple physiologically relevant initiators converge upon phenotypes of defective transport.

Section snippets

Cell culture

We grew Neuro-2a (N2a) mouse neuroblastoma cells (ATCC) at 37 °C/5% CO2 and passaged them in DMEM with l-glutamine (Gibco, 11965) containing 10% fetal bovine serum and penicillin/streptomycin (Gibco, 15140). Prior to imaging, we plated cells on poly-d-lysine coated glass coverslips (Carolina Supply Company, 633029), Thermanox coverslips (EMS, 72280), or poly-d-lysine coated live-imaging dishes (Mattek, P35G-020-C). To induce differentiation, cells were incubated overnight in Optimem (Gibco,

Aβ fibrils and peptides induce different modes of toxicity

To characterize the morphological response of neuronal cells to different Aβ aggregation states, we exposed N2a cells to varying doses of peptide and fibrillar Aβ for varying lengths of time, and scored neurite morphology. Unaffected neurites display a consistent diameter along the length of the neurite (Fig. 1A), while abnormal neurites display swellings (Fig. 1B) or necrosis (Fig. 1C). Quantitative analysis reveals that Aβ fibrils primarily induce swellings in a time- and dose-dependent

Discussion

We propose that there are multiple initiators of pathways that result in transport defects within neuronal processes. Experiments with both Aβ and beads revealed that swellings are a quantifiable morphological indicator associated with disrupted neuronal transport. We observe that fibrillar Aβ induces axonal and dendritic swellings, but unaggregated Aβ induces a non-specific necrotic cell response in both neuronal and non-neuronal cells. Additionally, internalization of Aβ fibrils applied

Acknowledgments

We gratefully acknowledge the assistance of Dr. Sanjay Saldanha and Dr. Stephen Adams for the design of FITC absorbance and FITC-Aβ quenching experiments, and Killian Nolan for blinded scoring of neurite swellings. Partial support for this work was provided by NIH R01GM35252 (LSBG), and F32GM071145 (SBS). LSBG is an investigator of the Howard Hughes Medical Institute.

References (111)

  • FerrariA. et al.

    beta-Amyloid induces paired helical filament-like tau filaments in tissue culture

    J. Biol. Chem.

    (2003)
  • FerrerI. et al.

    Neuronal alterations in patients with dementia: a Golgi study on biopsy samples

    Neurosci. Lett.

    (1990)
  • GalloG.

    Myosin II activity is required for severing-induced axon retraction in vitro

    Exp. Neurol.

    (2004)
  • GourasG.K. et al.

    Intraneuronal Abeta42 accumulation in human brain

    Am. J. Pathol.

    (2000)
  • GourasG.K. et al.

    Intraneuronal Abeta accumulation and origin of plaques in Alzheimer's disease

    Neurobiol. Aging

    (2005)
  • GunawardenaS. et al.

    Disruption of axonal transport and neuronal viability by amyloid precursor protein mutations in Drosophila

    Neuron

    (2001)
  • HossainS. et al.

    Binding of the Alzheimer amyloid beta-peptide to neuronal cell membranes by fluorescence correlation spectroscopy

    Exp. Mol. Pathol.

    (2007)
  • IvinsK.J. et al.

    Beta-amyloid induces local neurite degeneration in cultured hippocampal neurons: evidence for neuritic apoptosis

    Neurobiol. Dis.

    (1998)
  • Jimenez-BanzoA. et al.

    Singlet oxygen photosensitization by EGFP and its chromophore HBDI

    Biophys. J.

    (2008)
  • KaasikA. et al.

    Mitochondrial swelling impairs the transport of organelles in cerebellar granule neurons

    J. Biol. Chem.

    (2007)
  • KasaP. et al.

    Human amyloid-beta1–42 applied in vivo inhibits the fast axonal transport of proteins in the sciatic nerve of rat

    Neurosci. Lett.

    (2000)
  • KawaharaM. et al.

    Molecular mechanism of neurodegeneration induced by Alzheimer's beta-amyloid protein: channel formation and disruption of calcium homeostasis

    Brain Res. Bull.

    (2000)
  • Kienlen-CampardP. et al.

    Intracellular amyloid-beta 1–42, but not extracellular soluble amyloid-beta peptides, induces neuronal apoptosis

    J. Biol. Chem.

    (2002)
  • KohJ.Y. et al.

    Beta-amyloid protein increases the vulnerability of cultured cortical neurons to excitotoxic damage

    Brain Res.

    (1990)
  • NaaralaJ. et al.

    Excitatory amino acid-induced slow biphasic responses of free intracellular calcium in human neuroblastoma cells

    FEBS Lett.

    (1993)
  • PriceJ.L. et al.

    So what if tangles precede plaques?

    Neurobiol. Aging

    (2004)
  • ProbstA. et al.

    Neuritic plaques in senile dementia of Alzheimer type: a Golgi analysis in the hippocampal region

    Brain Res.

    (1983)
  • RudrabhatlaP. et al.

    Pin1-dependent prolyl isomerization modulates the stress-induced phosphorylation of high molecular weight neurofilament protein

    J. Biol. Chem.

    (2008)
  • SmithM.A. et al.

    Quantitative solubilization and analysis of insoluble paired helical filaments from Alzheimer disease

    Brain Res.

    (1996)
  • SongM.S. et al.

    Apoptosis is secondary to non-apoptotic axonal degeneration in neurons exposed to Abeta in distal axons

    Neurobiol. Aging

    (2006)
  • SuJ.H. et al.

    Identification and distribution of axonal dystrophic neurites in Alzheimer's disease

    Brain Res.

    (1993)
  • TakeuchiH. et al.

    Neuritic beading induced by activated microglia is an early feature of neuronal dysfunction toward neuronal death by inhibition of mitochondrial respiration and axonal transport

    J. Biol. Chem.

    (2005)
  • Adle-BiassetteH. et al.

    Neuronal apoptosis does not correlate with dementia in HIV infection but is related to microglial activation and axonal damage

    Neuropathol. Appl. Neurobiol.

    (1999)
  • AndersonK.L. et al.

    alpha1 Integrin activation: a link between beta-amyloid deposition and neuronal death in aging hippocampal neurons

    J. Neurosci. Res.

    (2004)
  • BehlC. et al.

    Mechanism of amyloid beta protein induced neuronal cell death: current concepts and future perspectives

    J. Neural Transm., Suppl.

    (1997)
  • BestJ.D. et al.

    Quantitative measurement of changes in amyloid-beta(40) in the rat brain and cerebrospinal fluid following treatment with the gamma-secretase inhibitor LY-411575 [N2-[(2S)-2-(3,5-difluorophenyl)-2-hydroxyethanoyl]-N1-[(7S)-5-methyl-6-ox o-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl]-L-alaninamide]

    J. Pharmacol. Exp. Ther.

    (2005)
  • BucciantiniM. et al.

    Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases

    Nature

    (2002)
  • CalderonF.H. et al.

    PC12 and neuro 2a cells have different susceptibilities to acetylcholinesterase-amyloid complexes, amyloid25–35 fragment, glutamate, and hydrogen peroxide

    J. Neurosci. Res.

    (1999)
  • CanevariL. et al.

    Toxicity of amyloid beta peptide: tales of calcium, mitochondria, and oxidative stress

    Neurochem. Res.

    (2004)
  • ChengC.L. et al.

    The effect of traumatic brain injury on the visual system: a morphologic characterization of reactive axonal change

    J. Neurotrauma

    (1988)
  • CirritoJ.R. et al.

    In vivo assessment of brain interstitial fluid with microdialysis reveals plaque-associated changes in amyloid-beta metabolism and half-life

    J. Neurosci.

    (2003)
  • CookD.G. et al.

    Alzheimer's A beta(1–42) is generated in the endoplasmic reticulum/intermediate compartment of NT2N cells

    Nat. Med.

    (1997)
  • CountermanA.E. et al.

    A physical model of axonal damage due to oxidative stress

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

    (2006)
  • De VriesL. et al.

    RGS-GAIP, a GTPase-activating protein for Galphai heterotrimeric G proteins, is located on clathrin-coated vesicles

    Mol. Biol. Cell

    (1998)
  • DixitR. et al.

    Cell damage and reactive oxygen species production induced by fluorescence microscopy: effect on mitosis and guidelines for non-invasive fluorescence microscopy

    Plant J.

    (2003)
  • DuyckaertsC. et al.

    Alzheimer disease models and human neuropathology: similarities and differences

    Acta Neuropathol.

    (2008)
  • EcheverriaV. et al.

    Intracellular A-beta amyloid, a sign for worse things to come?

    Mol. Neurobiol.

    (2002)
  • EdgarJ.M. et al.

    Oligodendroglial modulation of fast axonal transport in a mouse model of hereditary spastic paraplegia

    J. Cell Biol.

    (2004)
  • FergusonB. et al.

    Axonal damage in acute multiple sclerosis lesions

    Brain

    (1997)
  • Fernandez-VizarraP. et al.

    Intra- and extracellular Abeta and PHF in clinically evaluated cases of Alzheimer's disease

    Histol. Histopathol.

    (2004)
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