Rab27b regulates the release, autophagic clearance, and toxicity of alpha-synuclein

Alpha synuclein (αsyn) is the primary component of proteinaceous aggregates termed Lewy Bodies that pathologically define synucleinopathies including Parkinson’s disease (PD) and Dementia with Lewy Bodies (DLB). αSyn is hypothesized to spread through the brain in a prion-like fashion by misfolded protein forming a template for aggregation of endogenous αsyn. The release and uptake of αsyn from cell to cell are considered important processes for this prion-like spread. Rab27b is one of several GTPases essential to the endosomal-lysosomal pathway and is implicated in protein secretion and clearance but has yet to be characterized in its role in αsyn spread. In this study, we used a paracrine αsyn in vitro model to test the impact of Rab27b on αsyn release, clearance, and toxicity. shRNA-mediated knockdown (KD) of Rab27b increased αsyn-mediated paracrine toxicity. While Rab27b reduced αsyn release primarily through non-exosomal pathways, the αsyn released under KD conditions was of higher molecular weight species by size exclusion chromatography. Rab27b KD increased intracellular insoluble αsyn levels and led to an accumulation of endogenous LC3 positive puncta. Rab27b KD also decreased LC3 turnover with chloroquine treatment, indicating a defect in autophagic flux. Rab27b protein levels were increased in postmortem human brain lysates from PD and DLB subjects compared to healthy controls. These data indicate a role for Rab27b in the release, clearance, and toxicity of αsyn and ultimately in the pathogenesis of synucleinopathies.

[1]  B. Malik,et al.  Autophagic and endo-lysosomal dysfunction in neurodegenerative disease , 2019, Molecular Brain.

[2]  S. Chandra,et al.  Role of the endolysosomal system in Parkinson’s disease , 2019, Journal of neurochemistry.

[3]  D. Surmeier,et al.  Increased Lysosomal Exocytosis Induced by Lysosomal Ca2+ Channel Agonists Protects Human Dopaminergic Neurons from α-Synuclein Toxicity , 2019, The Journal of Neuroscience.

[4]  C. Chu Mechanisms of selective autophagy and mitophagy: Implications for neurodegenerative diseases , 2019, Neurobiology of Disease.

[5]  P. McLean,et al.  14-3-3 Proteins Reduce Cell-to-Cell Transfer and Propagation of Pathogenic α-Synuclein , 2018, The Journal of Neuroscience.

[6]  I. Ferrer,et al.  Transcriptional network analysis in frontal cortex in Lewy body diseases with focus on dementia with Lewy bodies , 2018, Brain pathology.

[7]  D. Alessi,et al.  LRRK2 kinase in Parkinson's disease , 2018, Science.

[8]  P. Lockhart,et al.  The emerging role of Rab GTPases in the pathogenesis of Parkinson's disease , 2018, Movement disorders : official journal of the Movement Disorder Society.

[9]  E. Masliah,et al.  Brain-derived exosomes from dementia with Lewy bodies propagate α-synuclein pathology , 2017, Acta neuropathologica communications.

[10]  E. Masliah,et al.  Brain-derived exosomes from dementia with Lewy bodies propagate α-synuclein pathology , 2017, Acta Neuropathologica Communications.

[11]  G. Cutter,et al.  Dysregulation of 14‐3‐3 proteins in neurodegenerative diseases with Lewy body or Alzheimer pathology , 2017, Annals of clinical and translational neurology.

[12]  J. Schulz,et al.  Rab7 induces clearance of α‐synuclein aggregates , 2016, Journal of neurochemistry.

[13]  Yun Gu,et al.  Rab27b is Involved in Lysosomal Exocytosis and Proteolipid Protein Trafficking in Oligodendrocytes , 2016, Neuroscience Bulletin.

[14]  Y. Ye,et al.  Unconventional secretion of misfolded proteins promotes adaptation to proteasome dysfunction in mammalian cells , 2016, Nature Cell Biology.

[15]  F. Giorgini,et al.  shRNA-Based Screen Identifies Endocytic Recycling Pathway Components That Act as Genetic Modifiers of Alpha-Synuclein Aggregation, Secretion and Toxicity , 2016, PLoS genetics.

[16]  R. Jahn,et al.  Functions of Rab Proteins at Presynaptic Sites , 2016, Cells.

[17]  M. Xilouri,et al.  Autophagy and Alpha‐Synuclein: Relevance to Parkinson's Disease and Related Synucleopathies , 2016, Movement disorders : official journal of the Movement Disorder Society.

[18]  B. Mollenhauer,et al.  Induction of α-synuclein aggregate formation by CSF exosomes from patients with Parkinson’s disease and dementia with Lewy bodies , 2015, Brain : a journal of neurology.

[19]  S. Dimmeler,et al.  Rab7a and Rab27b control secretion of endothelial microRNA through extracellular vesicles , 2015, FEBS letters.

[20]  B. Wang,et al.  Increased 14-3-3 phosphorylation observed in Parkinson's disease reduces neuroprotective potential of 14-3-3 proteins , 2015, Neurobiology of Disease.

[21]  Nicholas W. Wood,et al.  Loss-of-function mutations in RAB39B are associated with typical early-onset Parkinson disease , 2015, Neurology: Genetics.

[22]  P. Lockhart,et al.  Mutations in RAB39B cause X-linked intellectual disability and early-onset Parkinson disease with α-synuclein pathology. , 2014, American journal of human genetics.

[23]  C. Kyriacou,et al.  Rab11 modulates α-synuclein-mediated defects in synaptic transmission and behaviour , 2014, Human molecular genetics.

[24]  G. Braus,et al.  α-Synuclein interacts with the switch region of Rab8a in a Ser129 phosphorylation-dependent manner , 2014, Neurobiology of Disease.

[25]  M. Fukuda Rab27 Effectors, Pleiotropic Regulators in Secretory Pathways , 2013, Traffic.

[26]  C. Manzoni,et al.  Dysfunction of the autophagy/lysosomal degradation pathway is a shared feature of the genetic synucleinopathies , 2013, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[27]  Y. Tohyama,et al.  Tubulin Polymerization-promoting Protein (TPPP/p25α) Promotes Unconventional Secretion of α-Synuclein through Exophagy by Impairing Autophagosome-Lysosome Fusion* , 2013, The Journal of Biological Chemistry.

[28]  J. Schultze,et al.  X-linked dystonia parkinsonism syndrome (XDP, lubag): disease-specific sequence change DSC3 in TAF1/DYT3 affects genes in vesicular transport and dopamine metabolism. , 2013, Human molecular genetics.

[29]  P. Fraser,et al.  α-Synuclein Membrane Association Is Regulated by the Rab3a Recycling Machinery and Presynaptic Activity*♦ , 2013, The Journal of Biological Chemistry.

[30]  A. Winslow,et al.  Exosomal cell-to-cell transmission of alpha synuclein oligomers , 2012, Molecular Neurodegeneration.

[31]  E. Molnár,et al.  Molecular Mechanism of Myosin Va Recruitment to Dense Core Secretory Granules , 2012, Traffic.

[32]  J. Wuu,et al.  Upregulation of select rab GTPases in cholinergic basal forebrain neurons in mild cognitive impairment and Alzheimer's disease , 2011, Journal of Chemical Neuroanatomy.

[33]  S. Hamm-Alvarez,et al.  Rab27b regulates exocytosis of secretory vesicles in acinar epithelial cells from the lacrimal gland. , 2011, American journal of physiology. Cell physiology.

[34]  A. Al-Chalabi,et al.  Keeping up with genetic discoveries in amyotrophic lateral sclerosis: The ALSoD and ALSGene databases , 2011, Amyotrophic lateral sclerosis : official publication of the World Federation of Neurology Research Group on Motor Neuron Diseases.

[35]  B. Hyman,et al.  Heat‐shock protein 70 modulates toxic extracellular α‐synuclein oligomers and rescues trans‐synaptic toxicity , 2011, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[36]  H. Urlaub,et al.  Quantitative Analysis of Synaptic Vesicle Rabs Uncovers Distinct Yet Overlapping Roles for Rab3a and Rab27b in Ca2+-Triggered Exocytosis , 2010, The Journal of Neuroscience.

[37]  M. Ntzouni,et al.  Cell-Produced α-Synuclein Is Secreted in a Calcium-Dependent Manner by Exosomes and Impacts Neuronal Survival , 2010, The Journal of Neuroscience.

[38]  T. Tolmachova,et al.  Rab27a and Rab27b Regulate Neutrophil Azurophilic Granule Exocytosis and NADPH oxidase Activity by Independent Mechanisms , 2010, Traffic.

[39]  Michael Wolff,et al.  Seeding induced by α‐synuclein oligomers provides evidence for spreading of α‐synuclein pathology , 2009, Journal of neurochemistry.

[40]  Brian Spencer,et al.  Inclusion formation and neuronal cell death through neuron-to-neuron transmission of α-synuclein , 2009, Proceedings of the National Academy of Sciences.

[41]  K. Kaibuchi,et al.  Anterograde transport of TrkB in axons is mediated by direct interaction with Slp1 and Rab27. , 2009, Developmental cell.

[42]  R. Beyer,et al.  Rab11a and HSP90 Regulate Recycling of Extracellular α-Synuclein , 2009, The Journal of Neuroscience.

[43]  Mark T. Handley,et al.  The Rab27 effector Rabphilin, unlike Granuphilin and Noc2, rapidly exchanges between secretory granules and cytosol in PC12 cells. , 2008, Biochemical and biophysical research communications.

[44]  Tianhong Pan,et al.  The role of autophagy-lysosome pathway in neurodegeneration associated with Parkinson's disease. , 2008, Brain : a journal of neurology.

[45]  R. Hauser,et al.  Lewy body–like pathology in long-term embryonic nigral transplants in Parkinson's disease , 2008, Nature Medicine.

[46]  S. Itohara,et al.  Rab27b is expressed in a wide range of exocytic cells and involved in the delivery of secretory granules near the plasma membrane. , 2007, Molecular biology of the cell.

[47]  G. Raposo,et al.  Rab27b Regulates Mast Cell Granule Dynamics and Secretion , 2007, Traffic.

[48]  C. Futter,et al.  Rab27b regulates number and secretion of platelet dense granules , 2007, Proceedings of the National Academy of Sciences.

[49]  C. Tanner,et al.  Projected number of people with Parkinson disease in the most populous nations, 2005 through 2030 , 2007, Neurology.

[50]  S. Lindquist,et al.  α-Synuclein Blocks ER-Golgi Traffic and Rab1 Rescues Neuron Loss in Parkinson's Models , 2006, Science.

[51]  Aled Clayton,et al.  Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological Fluids , 2006, Current protocols in cell biology.

[52]  Smita Patel,et al.  Intravesicular Localization and Exocytosis of α-Synuclein and its Aggregates , 2005, The Journal of Neuroscience.

[53]  M. Barrachina,et al.  Abnormal α-synuclein interactions with rab3a and rabphilin in diffuse Lewy body disease , 2004, Neurobiology of Disease.

[54]  H. Yokota-Hashimoto,et al.  Involvement of Rab27b in the regulated secretion of pituitary hormones. , 2002, Endocrinology.

[55]  Zhaohong Yi,et al.  The Rab27a/Granuphilin Complex Regulates the Exocytosis of Insulin-Containing Dense-Core Granules , 2002, Molecular and Cellular Biology.

[56]  T. Nozawa [Selective autophagy mechanism against Group A Streptococcus infection]. , 2018, Nihon saikingaku zasshi. Japanese journal of bacteriology.

[57]  P. Aebischer,et al.  Rab1A over-expression prevents Golgi apparatus fragmentation and partially corrects motor deficits in an alpha-synuclein based rat model of Parkinson's disease. , 2011, Journal of Parkinson's disease.

[58]  Miguel C. Seabra,et al.  Rab27a and Rab27b control different steps of the exosome secretion pathway , 2010, Nature Cell Biology.

[59]  He-Jin Lee,et al.  Assembly-dependent endocytosis and clearance of extracellular alpha-synuclein. , 2008, The international journal of biochemistry & cell biology.

[60]  V. Calabrese Projected number of people with Parkinson disease in the most populous nations, 2005 through 2030. , 2007, Neurology.

[61]  To whom correspondence should be addressed: , 2003 .