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Amyloid-beta Causes Autophagy Dysfunction by Inhibiting Protein Prenylation

  • Author / Creator
    Smith, Kevan Thomas
  • There is ample evidence that autophagy is affected in Alzheimer’s disease (AD) but the causes, the nature of the dysfunction and the mechanisms of autophagy impairment are unclear. Autophagy depends on vesicular trafficking and membrane fusion, events that rely on several protein complexes and small GTPases. Previously our laboratory demonstrated that a neurotoxic mechanism of amyloid-β oligomers (Aβ) is inhibition of protein prenylation. Reduced protein prenylation results in impairment of intracellular and axonal trafficking. The Rab family of small GTPases are prenylated proteins required for normal trafficking, membrane fusion and autophagy. We hypothesize that defective autophagy in AD is due to inhibited protein prenylation and restoring protein prenylation will normalize the autophagic pathway and prevent neuronal death. We performed in vitro experiments to determine the nature of autophagy dysfunction. A biomarker of autophagy is the microtubule-associated protein light chain 3-II (LC3-II), which associates with the autophagosome membrane. Neurons challenged with Aβ accumulated LC3-II when analyzed by western blot. LC3-II increase is ambiguous, since it could represent induced autophagy or blocked lysosomal degradation of LC3-II. To differentiate between these possibilities we directly examined autophagic flux by expressing mCherry-GFP-LC3 in cultured cells. Autophagic flux was decreased in cultured cells treated with Aβ, and was recovered by rescuing protein prenylation with geranylgeranylpyrophosphate. Similarly, simvastatin and psoromic acid, two agents that inhibit protein prenylation, also blocked autophagic flux in a prenylation-dependent manner. Among prenylated proteins we focus on Rab7, which is essential in autophagy progression and lysosomal biogenesis, and is altered in brains of AD patients. During autophagy, Rab7 localizes to autophagosomes together with LC3. Treatment with Aβ reduced Rab7 co-localization with LC3. Normalization of protein prenylation restored colocalization of Rab7 and LC3. Significantly, reversing autophagy dysfunction has been validated as an innovative therapeutic strategy in AD. Yet, the lack of knowledge on the nature and cause(s) of autophagy dysfunction prevents the development of selective autophagy-targeted strategies with disease-modifying value. Our work will provide an essential evidence base for potential therapeutic developments that target autophagy flux in the CNS.

  • Subjects / Keywords
  • Graduation date
    2016-06
  • Type of Item
    Thesis
  • Degree
    Master of Science
  • DOI
    https://doi.org/10.7939/R3VX06F9K
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
  • Language
    English
  • Institution
    University of Alberta
  • Degree level
    Master's
  • Department
    • Centre for Neuroscience
  • Supervisor / co-supervisor and their department(s)
    • Posse de Chaves, Elena (Pharmacology)
  • Examining committee members and their departments
    • Kar, Satyabrata (Psychiatry)
    • Colmers, William (Pharmacology)
    • Simmen, Thomas (Cell Biology)