Lysosomal Proteolysis Inhibition Selectively Disrupts Axonal Transport of Degradative Organelles and Causes an Alzheimer's-Like Axonal Dystrophy

In the hallmark neuritic dystrophy of Alzheimer's disease (AD), autophagic vacuoles containing incompletely digested proteins selectively accumulate in focal axonal swellings, reflecting defects in both axonal transport and autophagy. Here, we investigated the possibility that impaired lysosomal proteolysis could be a basis for both of these defects leading to neuritic dystrophy. In living primary mouse cortical neurons expressing fluorescence-tagged markers, LC3-positive autophagosomes forming in axons rapidly acquired the endo-lysosomal markers Rab7 and LAMP1 and underwent exclusive retrograde movement. Proteolytic clearance of these transported autophagic vacuoles was initiated after fusion with bidirectionally moving lysosomes that increase in number at more proximal axon levels and in the perikaryon. Disrupting lysosomal proteolysis by either inhibiting cathepsins directly or by suppressing lysosomal acidification slowed the axonal transport of autolysosomes, late endosomes, and lysosomes and caused their selective accumulation within dystrophic axonal swellings. Mitochondria and other organelles lacking cathepsins moved normally under these conditions, indicating that the general functioning of the axonal transport system was preserved. Dystrophic swellings induced by lysosomal proteolysis inhibition resembled in composition those in several mouse models of AD and also acquired other AD-like features, including immunopositivity for ubiquitin, amyloid precursor protein, and hyperphosphorylated neurofilament proteins. Restoration of lysosomal proteolysis reversed the affected movements of proteolytic Rab7 vesicles, which in turn essentially cleared autophagic substrates and reversed the axonal dystrophy. These studies identify the AD-associated defects in neuronal lysosomal proteolysis as a possible basis for the selective transport abnormalities and highly characteristic pattern of neuritic dystrophy associated with AD.

[1]  Qian Cai,et al.  Snapin-Regulated Late Endosomal Transport Is Critical for Efficient Autophagy-Lysosomal Function in Neurons , 2010, Neuron.

[2]  D. Walsh,et al.  Macroautophagy Is Not Directly Involved in the Metabolism of Amyloid Precursor Protein* , 2010, The Journal of Biological Chemistry.

[3]  D. Klionsky,et al.  Regulation of macroautophagy in Saccharomyces cerevisiae. , 2010, Seminars in cell & developmental biology.

[4]  D. Westaway,et al.  Lysosomal Proteolysis and Autophagy Require Presenilin 1 and Are Disrupted by Alzheimer-Related PS1 Mutations , 2010, Cell.

[5]  Takafumi Inoue,et al.  Dynein- and activity-dependent retrograde transport of autophagosomes in neuronal axons , 2010, Autophagy.

[6]  Y. Itoyama,et al.  Loss of ALS2/Alsin Exacerbates Motor Dysfunction in a SOD1H46R-Expressing Mouse ALS Model by Disturbing Endolysosomal Trafficking , 2010, PloS one.

[7]  M. Yuzaki,et al.  Reevaluation of Neurodegeneration in lurcher Mice: Constitutive Ion Fluxes Cause Cell Death with, Not by, Autophagy , 2010, The Journal of Neuroscience.

[8]  G. Bjørkøy,et al.  FYCO1 is a Rab7 effector that binds to LC3 and PI3P to mediate microtubule plus end–directed vesicle transport , 2010, The Journal of cell biology.

[9]  Kerry A. Mullaney,et al.  Alzheimer’s-related endosome dysfunction in Down syndrome is Aβ-independent but requires APP and is reversed by BACE-1 inhibition , 2009, Proceedings of the National Academy of Sciences.

[10]  Yuyu Song,et al.  Axonal Transport Defects in Neurodegenerative Diseases , 2009, The Journal of Neuroscience.

[11]  Rhiannon L. Nolan,et al.  Examination of potential mechanisms of amyloid-induced defects in neuronal transport , 2009, Neurobiology of Disease.

[12]  Jean-Pierre Julien,et al.  Real‐time imaging reveals defects of fast axonal transport induced by disorganization of intermediate filaments , 2009, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[13]  Yoshiaki Kamada,et al.  Dynamics and diversity in autophagy mechanisms: lessons from yeast , 2009, Nature Reviews Molecular Cell Biology.

[14]  L. Collinson,et al.  In vitro reconstitution of fusion between immature autophagosomes and endosomes , 2009, Autophagy.

[15]  S. Wharton,et al.  Direct evidence for axonal transport defects in a novel mouse model of mutant spastin‐induced hereditary spastic paraplegia (HSP) and human HSP patients , 2009, Journal of neurochemistry.

[16]  S. Tooze,et al.  Early endosomes and endosomal coatomer are required for autophagy , 2009, The Journal of cell biology.

[17]  A. Ballabio,et al.  Lysosomal disorders: from storage to cellular damage. , 2009, Biochimica et biophysica acta.

[18]  B. Lamb,et al.  The Cleavage Products of Amyloid-β Precursor Protein Are Sorted to Distinct Carrier Vesicles That Are Independently Transported within Neurites , 2009, Journal of Neuroscience.

[19]  A. Nógrádi,et al.  Severely dystrophic axons at amyloid plaques remain continuous and connected to viable cell bodies. , 2009, Brain : a journal of neurology.

[20]  R. Kraut,et al.  The Drosophila BEACH Family Protein, Blue Cheese, Links Lysosomal Axon Transport with Motor Neuron Degeneration , 2009, The Journal of Neuroscience.

[21]  L. Goldstein,et al.  Amyloid precursor protein-induced axonopathies are independent of amyloid-beta peptides. , 2008, Human molecular genetics.

[22]  L. Gan,et al.  Cystatin C-Cathepsin B Axis Regulates Amyloid Beta Levels and Associated Neuronal Deficits in an Animal Model of Alzheimer's Disease , 2008, Neuron.

[23]  S. K. Kaasinen,et al.  Autophagy generates retrogradely transported organelles: a hypothesis , 2008, International Journal of Developmental Neuroscience.

[24]  Ralph A. Nixon,et al.  Autophagy Induction and Autophagosome Clearance in Neurons: Relationship to Autophagic Pathology in Alzheimer's Disease , 2008, The Journal of Neuroscience.

[25]  R. Nixon,et al.  Neurodegenerative lysosomal disorders: A continuum from development to late age , 2008, Autophagy.

[26]  John L Cleveland,et al.  Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes , 2008, Autophagy.

[27]  D. Holtzman,et al.  Rapid appearance and local toxicity of amyloid-β plaques in a mouse model of Alzheimer’s disease , 2008, Nature.

[28]  Xiaoxiang Zheng,et al.  Transport of autophagosomes in neurites of PC12 cells during serum deprivation , 2008, Autophagy.

[29]  D. Rubinsztein,et al.  The Itinerary of Autophagosomes: From Peripheral Formation to Kiss-and-Run Fusion with Lysosomes , 2008, Traffic.

[30]  C. Fader,et al.  Induction of Autophagy Promotes Fusion of Multivesicular Bodies with Autophagic Vacuoles in K562 Cells , 2007, Traffic.

[31]  A. Isaacs,et al.  Functional multivesicular bodies are required for autophagic clearance of protein aggregates associated with neurodegenerative disease , 2007, The Journal of cell biology.

[32]  I. Ciechomska,et al.  Non-Autophagic GFP-LC3 Puncta Induced by Saponin and Other Detergents , 2007, Autophagy.

[33]  B. Hyman,et al.  Curcumin labels amyloid pathology in vivo, disrupts existing plaques, and partially restores distorted neurites in an Alzheimer mouse model , 2007, Journal of neurochemistry.

[34]  G. Apodaca,et al.  RhoB‐Dependent Modulation of Postendocytic Traffic in Polarized Madin‐Darby Canine Kidney Cells , 2007, Traffic.

[35]  T. Noda,et al.  Dissection of the Autophagosome Maturation Process by a Novel Reporter Protein, Tandem Fluorescent-Tagged LC3 , 2007, Autophagy.

[36]  N. Emptage,et al.  The lysosome or lysosome-related organelle may serve as a Ca2+ store in the boutons of hippocampal pyramidal cells , 2007, Neuropharmacology.

[37]  Ileana M. Cristea,et al.  Induction of Autophagy in Axonal Dystrophy and Degeneration , 2006, The Journal of Neuroscience.

[38]  L. Goldstein,et al.  Axonal transport and Alzheimer's disease. , 2006, Annual review of biochemistry.

[39]  P. Hollenbeck,et al.  The axonal transport of mitochondria , 2005, Journal of Cell Science.

[40]  Masaaki Komatsu,et al.  Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice , 2005, The Journal of cell biology.

[41]  T. Ueno,et al.  Lysosomal Turnover, but Not a Cellular Level, of Endogenous LC3 is a Marker for Autophagy , 2005, Autophagy.

[42]  A. Tolkovsky,et al.  The Dynamics of Autophagy Visualised in Live Cells: from Autophagosome Formation to Fusion with Endo/lysosomes , 2005, Autophagy.

[43]  E. Masliah,et al.  Axonopathy and Transport Deficits Early in the Pathogenesis of Alzheimer's Disease , 2005, Science.

[44]  Ralph A. Nixon,et al.  Extensive Involvement of Autophagy in Alzheimer Disease: An Immuno-Electron Microscopy Study , 2005, Journal of neuropathology and experimental neurology.

[45]  Daniel J. Klionsky,et al.  Autophagy in Health and Disease: A Double-Edged Sword , 2004, Science.

[46]  S. Schmidt,et al.  Presenilin Mutations in Familial Alzheimer Disease and Transgenic Mouse Models Accelerate Neuronal Lysosomal Pathology , 2004, Journal of neuropathology and experimental neurology.

[47]  M. Futai,et al.  Lysosome and lysosome-related organelles responsible for specialized functions in higher organisms, with special emphasis on vacuolar-type proton ATPase. , 2003, Cell structure and function.

[48]  T. Ohm,et al.  Cholesterol and Tau Protein - Findings in Alzheimer’s and Niemann Pick C’s Disease , 2003, Pharmacopsychiatry.

[49]  Yasushi Hiraoka,et al.  Mutations in Dynein Link Motor Neuron Degeneration to Defects in Retrograde Transport , 2003, Science.

[50]  B. Bahr,et al.  The neuropathogenic contributions of lysosomal dysfunction , 2002, Journal of neurochemistry.

[51]  M. Mercken,et al.  Calpain Activity Regulates the Cell Surface Distribution of Amyloid Precursor Protein , 2002, The Journal of Biological Chemistry.

[52]  N. Heintz,et al.  A Novel Protein Complex Linking the δ2 Glutamate Receptor and Autophagy Implications for Neurodegeneration in Lurcher Mice , 2002, Neuron.

[53]  D. Howland,et al.  Disruption of Dynein/Dynactin Inhibits Axonal Transport in Motor Neurons Causing Late-Onset Progressive Degeneration , 2002, Neuron.

[54]  G. Schiavo,et al.  Analysis of retrograde transport in motor neurons reveals common endocytic carriers for tetanus toxin and neurotrophin receptor p75NTR , 2002, The Journal of cell biology.

[55]  S. Emr,et al.  Autophagy as a regulated pathway of cellular degradation. , 2000, Science.

[56]  Takeshi Noda,et al.  LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing , 2000, The EMBO journal.

[57]  P. Mathews,et al.  The Endosomal-Lysosomal System of Neurons in Alzheimer's Disease Pathogenesis: A Review , 2000, Neurochemical Research.

[58]  B. Deurs,et al.  Rab7: a key to lysosome biogenesis. , 2000, Molecular biology of the cell.

[59]  S. Walkley.  Cellular Pathology of Lysosomal Storage Disorders , 1998, Brain pathology.

[60]  C. Overly,et al.  Dynamic Organization of Endocytic Pathways in Axons of Cultured Sympathetic Neurons , 1996, The Journal of Neuroscience.

[61]  D. Mann,et al.  Colocalization of Lysosomal Hydrolase and β-Amyloid in Diffuse Plaques of the Cerebellum and Striatum in Alzheimer's Disease and Down's Syndrome , 1996, Journal of neuropathology and experimental neurology.

[62]  W. Dunn,et al.  Autophagy and related mechanisms of lysosome-mediated protein degradation. , 1994, Trends in cell biology.

[63]  R. Scott,et al.  Processing of the beta-amyloid precursor. Multiple proteases generate and degrade potentially amyloidogenic fragments. , 1993, The Journal of biological chemistry.

[64]  P. Hollenbeck.  Products of endocytosis and autophagy are retrieved from axons by regulated retrograde organelle transport , 1993, The Journal of cell biology.

[65]  L. Greene,et al.  Aurintricarboxylic acid rescues PC12 cells and sympathetic neurons from cell death caused by nerve growth factor deprivation: correlation with suppression of endonuclease activity , 1991, The Journal of cell biology.

[66]  R. Broadwell,et al.  Cytochemical staining of the endoplasmic reticulum and glycogen in mouse anterior pituitary cells. , 1984, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[67]  N. Shimizu,et al.  Early changes in the structure of rat sciatic nerves by ligation. , 1978, Archivum histologicum Japonicum = Nihon soshikigaku kiroku.

[68]  D. Price,et al.  THE PATHOGENESIS OF REACTIVE AXONAL SWELLINGS: ROLE OF AXONAL TRANSPORT , 1977, Journal of neuropathology and experimental neurology.

[69]  M. Ohno,et al.  Reversal of autophagy dysfunction in the TgCRND8 mouse model of Alzheimer's disease ameliorates amyloid pathologies and memory deficits. , 2011, Brain : a journal of neurology.

[70]  K. Heidenreich,et al.  Live-cell imaging of autophagy induction and autophagosome-lysosome fusion in primary cultured neurons. , 2009, Methods in enzymology.

[71]  V. Hill,et al.  Monitoring autophagy in lysosomal storage disorders. , 2009, Methods in enzymology.

[72]  T. Noda,et al.  Dynein-dependent movement of autophagosomes mediates efficient encounters with lysosomes. , 2008, Cell structure and function.

[73]  R. Nixon,et al.  Lysosomal system pathways: genes to neurodegeneration in Alzheimer's disease. , 2006, Journal of Alzheimer's disease : JAD.

[74]  D. Dickson,et al.  Ubiquitin immunoelectron microscopy of dystrophic neurites in cerebellar senile plaques of Alzheimer's disease , 2004, Acta Neuropathologica.

[75]  A. F. Soleng,et al.  A thorny question: how does activity maintain dendritic spines? , 1999, Nature Neuroscience.