The neurodegenerative disorder, Alzheimer's disease, is responsible for 60 to 80% of all dementia cases.1 Neurodegeneration occurs in response to the accumulation of amyloid-β plaques and neurofibrillary tangles composed of hyperphosphorylated tau. The proteolytic processing of AβPP (amyloid β precursor protein) by β-secretase and γ-secretase releases Aβ fragments of 40 and 42 amino acid residues which miss-fold and aggregate into the pathogenic plaques.2 Similarly, proteolytic processing of phosphorylated tau releases monomers that are targeted to the 26S proteasome for degradation. However, proteasomal efficiency is poor when it comes to degrading ubiquitinated tau as these substrates lead to more neurofibrillary tangle aggregates.2 The inability to clear these accumulating plaques intracellularly results in a significant amount of oxidative and cellular stress leading to progressive neuronal loss and cognitive decline.
The Ub-proteasome system (UPS) and autophagic pathways are mechanisms that aid in the clearing of protein aggregates and dysfunctional proteins.2,3 The UPS targets misfolded proteins recognized by molecular chaperones (for example Hsp70) for degradation. Ubiquitin is conjugated to the substrate through a series of enzymatic reactions starting with the E1-activating enzyme, followed by the E2-conjugating enzyme, and subsequent transfer of ubiquitin to the target substrate by an E3-ligase. The ubiquitinated substrate can then be recognized by the 26S proteasome where it is cleaved into small peptides.
Immunocytochemistry/Immunofluorescence: beta Amyloid Antibody [NBP2-15575] - SH-SY5Y cells were fixed in 4% paraformaldehyde at RT for 15 minutes. (Green): APP protein stained by APP antibody diluted at 1:100. (Red): beta Tubulin 3/ TUJ1 protein stained by beta Tubulin 3/ TUJ1 antibody diluted at 1:200. (Blue): Hoechst 33342 staining.
The autophagy process is mediated by multiple mechanisms of targeted delivery of misfolded proteins to a lysosome for degradation. This process of delivery subdivides autophagy into chaperone mediated autophagy (CMA), microautophagy, and macroautophagy. CMA requires recognition of a specific KFERQ motif in monomeric misfolded proteins. Whereas, the macroautophagy process relies on autophagic adaptors to target bulky misfolded protein aggregates to an autophagosome for degradation.2 Initiation of an autophagosome requires the assembly of the ULK protein complex (ULK1, Atg13, and FIP200) at the phagophore (isolation membrane), leading to the activation of Beclin-1 and PI3KC3 complex. The elongation of the isolation membrane around the misfolded cargo is mediated by Atg5-Atg12-Atg16L1, which incorporate PE (phosphatidylethanolamine) and LC3-1, creating LC3-II.2,3 Completion of the autophagosome surrounding the dysfunctional proteins and fusion with lysosomes forms an autolysosome, which then degrades the autophagosome's content with lysosomal hydrolases.
In Alzheimer's disease, protein quality systems are impaired preventing adequate removal of Ab plaques and phosphorylated tau aggregates. Cecarini et al. demonstrated that the activities of the UPS and autophagy pathways were intertwined. Their work established crosstalk between systems that is responsive to Ab 42 levels, highlighting a threshold that when exceeded results in impaired proteolysis mechanisms.4 For example, excessive amounts of Aβ 42 blocked proteasome activity promoting neurodegeneration and the accumulation of immature autophagosomes, which are unable to clear the vast deposits of Aβ.4 Additionally, lysosomal dysfunction enhanced neurofibrillary tangle accumulation and neurotoxicity.3
Therapeutic strategies targeting autophagy and UPS include several small molecule inhibitors such as rapamycin, an inhibitor of mTOR. Rapamycin has been shown to enhance removal of protein aggregates and improve cognition.2,3 Protein phosphatase 2A, an inhibitor of tau phosphorylation, was shown to activate autophagy and is currently in clinical trials. Additionally, enhancing lysosomal biogenesis by inhibiting cystatin B or C stimulates clearing of autophagy cargo.3
Overall, inducing autophagy by targeting regulators of the autophagic pathway appears to show promise in ameliorating cognitive deficits and enhancing clearing of misfolded aggregates. Nevertheless, more exploration is needed to address the potential off-target effects of disrupting the distinct mechanisms of proteolytic processing.
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By Bethany Veo, PhD
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